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18TH EDITION
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Technical
Manual
18TH EDITION
E di te d b y
Christopher D. Hillyer, MD
New York Blood Center
New York, NY
AABB authors are requested to comply with a conflict of interest policy that includes disclosure of
relationships with commercial firms. A copy of the policy is located at http://www.aabb.org.
Efforts are made to have publications of the AABB consistent in regard to acceptable practices.
However, for several reasons, they may not be. First, as new developments in the practice of blood
banking occur, changes may be recommended to the Standards for Blood Banks and Transfusion
Services. It is not possible, however, to revise each publication at the time such a change is adopted.
Thus, it is essential that the most recent edition of the Standards be consulted as a reference in regard to
current acceptable practices. Second, the views expressed in this publication represent the opinions of
authors. The publication of this book does not constitute an endorsement by the AABB of any view
expressed herein, and the AABB expressly disclaims any liability arising from any inaccuracy or
misstatement.
Copyright © 2014 by AABB. All rights reserved. No part of this book may be reproduced or transmitted
in any form or by any means, electronic or mechanical, including photocopying, recording, or by any
information storage and retrieval system, without permission in writing from the Publisher.
The Publisher has made every effort to trace the copyright holders for borrowed material. If any such
material has been inadvertently overlooked, the Publisher will be pleased to make the necessary
arrangements at the first opportunity.
Cataloging-in-Publication Data
T I S W I T H T R E M E N D O U S pleasure that
I we introduce you to the 18th edition of the
AABB Technical Manual. As with all previous
adjunctive therapies that can reduce the need
for transfusion. As health-care economics join
better patient care in prompting continued
editions, this revision is based on the solid adoption of PBM, readers will find that the
foundation of knowledge gathered by past new emphasis is highly relevant to their needs.
contributors to whom we are indebted. I Other content receiving special attention
would like to especially acknowledge the tre- in this edition is that involving molecular test-
mendous contributions of Drs. Hillyer and ing. An increasing number of organizations
Grossman who have helped guide previous seek the more detailed test results possible
editions. With the 18th edition they both con- through investments in molecular technology.
clude their tenures as Associate Editors. Along Those in the workforce today need (or soon
the same lines, I want to welcome Dr. Westhoff will need) a solid understanding of the both
who has joined me in this new challenge of the theory and practice of molecular testing
providing an up-to-date comprehensive systems, which is found in this volume.
resource of information in the field of transfu- Perhaps the most obvious upgrade in the
sion medicine and cellular therapies. new edition is the relocation of the methods
This edition of the Technical Manual will from the printed pages to electronic storage
be most notable for several innovations. First, medium found inside the back cover. By mov-
the cellular therapy content has been ing the methods into the digital format, we are
expanded and reorganized to include many able for the first time to give Technical Manual
novel therapies that are moving from the users methods that are already set up as stan-
research setting into the clinical realm. In dard operating procedures (SOPs)—in a tem-
addition to updates on the sources of stem plate that reflects how procedures would
cells and the transfusion support of stem cell actually be used in real-life. Users are invited
transplantation, chapters focus on the quality to upload the methods to their facility net-
and regulatory issues associated with cord works and customize them for integration into
banking, novel stem cell therapies using non- their existing SOPs.
hematopoietic stem cells, and tissue engineer- In addition to these particular innova-
ing. The new scope will be of great value to the tions, all chapters have been revised. Some of
increasing number of professionals who now the chapter authors have added substantial
include some aspect of cellular therapy in updates in great detail to assist the reader,
their daily responsibilities. whether working at the bench or the bedside.
In a similar manner, the content on They have embraced the task of explaining the
patient blood management (PBM) reflects the issues that face all of us in the ever-changing
growing scope of what is considered PBM. The world of transfusion medicine and cellular
traditional topics covered as part of discus- therapies. We welcome your comments and
sions on blood utilization review and periop- feedback on the effectiveness of our labors.
erative blood recovery are augmented by
detailed content on anemia management, Mark K. Fung, MD, PhD
optimization of coagulation, and a host of Editor in Chief
ix
Contents
Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix
QUALI TY ISSUES
xi
xii 䡲 AA BB TECHNICAL MANUAL
4. Disaster Management . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97
Ruth D. Sylvester, MS, MT(ASCP)SBB; William FitzGerald, LTC USA (Ret);
Wendy Trivisonno; and Theresa Wiegmann, JD
Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98
Business Operations Planning. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102
Regulatory Considerations in Emergencies . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109
Testing the Disaster Plan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111
Summary of Lessons Learned from Recent Disasters . . . . . . . . . . . . . . . . . . . 112
Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112
Key Points. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113
BL OOD GROUPS
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 803
METHO DS
Methods Introduction
A P P E N DI C E S
Tania L. Motschman, MS, MT(ASCP)SBB, CQA(ASQ), Quality Director, Esoteric Business Unit, Laboratory
Corporation of America, Burlington, North Carolina; Betsy W. Jett, MT(ASCP), CQA(ASQ)CQM/OE, Vice Presi-
dent for Quality and Regulatory Affairs, New York Blood Center, New York, New York; and Susan L. Wilkinson,
EdD, MS, MT(ASCP)SBB, Interim Department Head, Analytical and Diagnostic Sciences, College of Allied
Health Sciences, and Associate Professor Emerita, University of Cincinnati, Cincinnati, Ohio
The authors have disclosed no conflicts of interest.
1
2 䡲 AABB TECHNICAL MANUAL
ment standards (ISO 9001) are generic to any mation to process managers regarding levels
industry and describe the important mini- of performance that can be used in setting pri-
mum elements of a quality management sys- orities for process improvement. Examples of
tem.13 The ISO 15189 standards are specific to quality assurance activities in transfusion
laboratory medicine.14 In addition, the Health medicine and cellular therapies include record
Care Criteria for Performance Excellence pub- reviews, monitoring of quality indicators, and
lished by the Baldrige Performance Excellence internal assessments.
Program15 provides an excellent framework for Quality management considers interrelat-
implementing quality on an organizational ed processes in the context of the organization
level. and its relations with customers and suppliers.
The AABB has defined the minimum ele- It addresses the leadership role of executive
ments that must be addressed in its quality management in creating a commitment to
system essentials (QSEs). 16 The AABB QSEs quality throughout the organization, the un-
were developed to be compatible with ISO derstanding of suppliers and customers as
9001 standards, the FDA Guideline for Quality partners in quality, the management of human
Assurance in Blood Establishments,5 and other and other resources, and quality planning.
FDA quality system approaches.17,18 The quality systems approach described
in this chapter encompasses all of these activi-
ties. It ensures application of quality principles
CO NCE P T S I N QUA LI T Y
throughout the organization and reflects the
Quality Control, Quality Assurance, changing focus of quality efforts from detec-
and Quality Management tion to prevention.
The purpose of QC is to provide feedback to Juran’s Quality Trilogy
operational staff about the state of a process
that is in progress. QC tells staff whether to Juran’s Quality Trilogy is one example of a
continue (everything is acceptable) or to stop quality management approach. This model
until a problem has been resolved (something centers around three fundamental processes
is found to be out of control). for managing quality in any organization:
Historically, transfusion services and do- planning, control, and improvement.19(p2.5)
nor centers have used many QC measures as The planning process for a new product
standard practices in their operations. Exam- or service includes activities to identify re-
ples include reagent QC; product QC; clerical quirements, develop product and process
checks; visual inspections; and measure- specifications that meet those requirements,
ments, such as temperature readings on refrig- and design the process. During the planning
erators and volume or cell counts on finished phase, the facility must perform the following
blood components. steps:
Quality assurance activities are not tied to
the actual performance of a process. Rather, 1. Establish quality goals for the project.
they include activities, such as the develop- 2. Identify the customers.
ment of documents like standard operating 3. Determine customer needs and expecta-
procedures (SOPs), to ensure consistent and tions.
correct performance of processes, training of 4. Develop product and service specifications
personnel, and qualification of materials and to meet customer, operational, regulatory,
equipment. Quality assurance activities also and accreditation requirements.
include retrospective reviews and analyses of 5. Develop operational processes for produc-
operational performance data to determine tion and delivery, including written proce-
whether the overall process is in a state of con- dures and resources requirements.
trol and to detect shifts or trends that require 6. Develop process controls and validate the
attention. Quality assurance provides infor- process in the operational setting.
CHAPTER 1 Quality Management Systems: Theory and Practice 䡲 3
The control process addresses inputs, For example, a key process for donor cen-
production, and delivery of products and ser- ters is donor selection. The “input” includes
vices to meet specifications. Process controls the individual who presents for donation and
should allow staff to recognize when things are all of the resources required for that donor’s
going wrong and to either make appropriate health screening. Through a series of activities
adjustments to ensure a product’s quality or (a process), including the verification of the
stop the process. donor’s identity, a deferral status review, a
An important goal in quality manage- mini-physical exam, and a health history
ment is to establish a set of controls that en- questionnaire, an individual is deemed an “eli-
sure process and product quality but are not gible donor.” The “output” is either an eligible
excessive. Controls that do not add value donor who can continue to the next process
should be eliminated to conserve limited (blood collection) or an ineligible donor who is
resources and allow staff to focus attention on deferred. When the selection process results in
those controls that are critical to the opera- a deferred donor, the resources (inputs) asso-
tion. ciated with that process do not continue
Statistical tools, such as process capabili- through the process but contribute to the cost
ty measurement and control charts, allow a fa- of quality. One way that donor centers at-
cility to evaluate process performance during tempt to minimize this cost is to educate po-
tential donors before screening so that those
the planning stage and in operations. These
who are not eligible do not enter the selection
tools help determine if a process is stable
process.
(ie, in statistical control) and if it is capable of
Strategies for managing a process should
meeting product and ser vice specifica-
address all of its components, including its in-
tions.19(p22.19)
terrelated activities, inputs, outputs, and re-
Quality improvement is intended to en-
sources. Supplier qualification, formal agree-
able an organization to attain higher levels of ments, supply verification, and inventory
performance by creating new or better fea- control are strategies for ensuring that the
tures that add value or by removing deficien- inputs to a process meet specifications.
cies in the process, product, or service. Oppor- Personnel training and competence assess-
t u n i t i e s t o i m p r ove m a y b e re l a t e d t o ment, equipment maintenance and control,
deficiencies in the initial planning process; management of documents and records, and
unforeseen factors discovered on implementa- implementation of appropriate in-process
tion; shifts in customer needs; or changes in controls provide assurance that the process
starting materials, environmental factors, or will operate as intended. End-product testing
other variables that affect the process. Im- and inspection, customer feedback, and
provements must be based on data-driven outcome measurement provide data to evalu-
analysis; an ongoing measurement and assess- ate product quality and improve the process.
ment program is fundamental to that process. These output measurements and quality
4 䡲 AABB TECHNICAL MANUAL
indicators are used to evaluate the effective- In service, personnel need to be able to adapt
ness of the process and process controls. a service in a way that meets customer ex-
To manage a system of processes effec- pectations but does not compromise quality.
tively, the facility must understand how its To do this, personnel must have sufficient
processes interact and what cause-and-effect knowledge and understanding of interrelated
relationships exist between them. In the donor processes to use independent judgment ap-
selection scenario, the consequences of ac- propriately, or they must have ready access to
cepting a donor who is not eligible reach into higher-level decision makers.
almost every other process in the facility. For When one designs quality management
example, if a donor with a history of high-risk systems for production processes, it is useful
behavior is not identified as such during the to think of the process as the driver, with peo-
selection process, the donated unit(s) may re- ple providing the oversight and support need-
turn positive test results for one of the viral ed to keep it running smoothly and effectively.
marker assays, triggering follow-up testing, In service, people are the focus; the underlying
look-back investigations, and donor deferral process provides a foundation that enables
and notification procedures. Components staff to deliver safe and effective services that
must be quarantined and their discard docu- meet customers’ needs in almost any situa-
mented. Personnel involved in collecting and tion.
processing the unit(s) are at risk of exposure to
infectious agents. Part of quality planning is to Quality Management as an Evolving
identify these relationships so that quick and Science
appropriate corrective action can be taken
The principles and tools used in quality man-
when process controls fail.
agement today will change as research pro-
It is important to remember that opera-
vides new knowledge of organizational behav-
tional processes include not only product
ior, technology provides new solutions, and
manufacture or service creation, but also the
the transfusion medicine and cellular thera-
delivery of a product or service. Delivery gen-
pies fields present new challenges. Periodic as-
erally involves interaction with the customer.
sessments of the quality management system
The quality of that transaction is critical to
will help identify practices that are no longer
customer satisfaction and should not be over-
effective or that could be improved through
looked in the design and ongoing assessment
the use of new technology or new tools.
of the quality management system.
to perform their newly assigned duties and ble), specimen handling, processing, and
must be deemed competent in these duties. testing.
During orientation and training, employees – Performance of instrument maintenance
should be given the opportunity to ask ques- and function checks.
tions and seek additional help or clarification. 䡲 Monitoring of the recording and reporting
All aspects of the training should be docu- of test results.
mented, and the facility trainer or designated 䡲 Review of intermediate test results or work-
facility management representative and em- sheets, QC records, proficiency testing re-
ployees should mutually agree on how em- sults, and preventive maintenance records.
ployees’ competence will be determined. 䡲 Assessment of
FDA cGMP training is required for staff – Test performance by testing previously
involved in the manufacture of blood and analyzed specimens, internal blind test-
blood products, including staff involved in col- ing samples, or external proficiency test-
lection, testing, processing, preservation, stor- ing samples.
age, distribution, and transport. 3 Likewise, – Problem-solving skills.
cGTP training is required for personnel in-
volved in similar activities for human cells, tis- A formal competency plan that includes a
sues, and cellular and tissue-based products schedule of assessments, a defined minimum
(HCT/Ps).4 Such training should provide staff for acceptable performance, and remedial
with an understanding of the regulatory basis measures is one way to ensure appropriate
for the facility’s policies and procedures as well and consistent competence assessments. As-
as the specific application of the cGMP and sessments need not be targeted to each indi-
cGTP requirements as described in the facili- vidual test or procedure performed by the em-
ty’s own written operating procedures. This ployee; instead, they can be grouped together
training should be provided periodically to en- to assess similar techniques or methods. How-
sure that personnel remain familiar with regu- ever, any test with unique aspects, problems,
latory requirements. or procedures should be assessed separately to
To ensure that skills are maintained, the ensure that staff maintain their competency to
facility should have regularly scheduled com- report test results promptly, accurately, and
petence evaluations of all staff members proficiently.1 Written tests can be used effec-
whose activities affect the quality of laboratory tively to evaluate problem-solving skills and
testing, manufacture of products, or provision cover many topics by asking one or more ques-
of products or services.1,5-10 Competence as- tions for each area to be assessed. CMS re-
sessment of management personnel should quires that employees who perform testing
also be considered. be assessed semiannually during the first
Depending on the nature of the job du- year that patient specimens are tested and an-
ties, competence assessments may include nually thereafter.1 Initial training verification
written evaluations; direct observations of ac- activities may serve as the first of these com-
tivities; reviews of work records or reports, in- petence assessments.
formation system records, and QC records; The quality oversight personnel should
testing of unknown samples; and evaluations assist in the development, review, and approv-
of employees’ problem-solving skills.5 For all al of training programs, including the criteria
testing personnel, CMS requires that each of for retraining. 5 Quality oversight personnel
the following methods be used, when applica- also monitor the effectiveness of training pro-
ble, for each test system annually1: grams and competence evaluations, and they
recommend changes as needed. In addition,
䡲 Direct observations of The Joint Commission requires analyses of ag-
– Routine patient test performance (in- gregate competence assessment data to iden-
cluding patient preparation, if applica- tify staff learning needs.9
CHAPTER 1 Quality Management Systems: Theory and Practice 䡲 9
ensure that the expectations of all parties con- tion, testing, storage, distribution, transport,
tinue to be met. Changes should be mutually and administration of blood, components, tis-
agreed upon and incorporated as needed. sues, and cellular therapy products also meet
Transfusion and cellular therapy services FDA requirements.
should maintain written contracts or agree- The facility must define acceptance crite-
ments with outside suppliers of critical mate- ria for critical supplies (see 21 CFR 210.3)3 and
rials and services, such as blood components, must develop procedures to control and pre-
cellular therapy products, irradiation, compat- vent the inadvertent use of materials that do
ibility testing, or infectious disease marker not meet specifications. Corrective action may
testing. An outside supplier may be another include returning the material to the vendor or
department within the same facility that is destroying it. Receipt and inspection records
managed independently, or it may be another enable the facility to trace materials that have
facility (eg, contract manufacturer). The con- been used in a particular process and provide
tracting facility assumes responsibility for the information for ongoing supplier qualifica-
manufacture of the product; ensuring the safe- tion.
ty, purity, and potency of the product; and en-
suring that the contract manufacturer com- Equipment Management
plies with all applicable product standards and
Equipment that must operate within defined
regulations. Both the contracting facility and
specifications to ensure the quality of blood
the contractor are legally responsible for the
components, cellular therapy products, tis-
work performed by the contractor.
sues, derivatives, and services is referred to as
It is important for the transfusion or cel-
“critical equipment” in the quality manage-
lular therapy service to participate in the eval-
uation and selection of suppliers. The service ment system. Critical equipment may include
should review contracts and agreements to en- instruments, measuring devices, and comput-
sure that all important aspects for their critical er systems (hardware and software).
Activities designed to ensure that equip-
materials and services are addressed. Exam-
ment performs as intended include qualifica-
ples of issues that could be addressed in an
tion, calibration, maintenance, and monitor-
agreement or contract include the responsibil-
ity for a product or blood sample during ship- ing. Calibration, functional and safety checks,
ment; the supplier’s responsibility to promptly and preventive maintenance should be sched-
notify the facility when changes have been uled and performed according to the manu-
made that could affect the safety of blood facturer’s recommendations and regulatory re-
components, cellular therapy products, tis- quirements of the FDA 2-4 and CMS.1 Written
sues, derivatives, or services for patients; and procedures for equipment use and control
the supplier’s responsibility to notify the facili- should comply with the manufacturer’s rec-
ty when information is discovered indicating ommendations unless an alternative method
that a product may not be considered safe, has been validated by the facility and, in some
such as during look-back procedures. instances, approved by the appropriate regula-
tory and accrediting agencies.
Receipt, Inspection, and Testing of When one selects new equipment, it is
important to consider not only the perfor-
Incoming Supplies
mance of the equipment as it will be used in
Before acceptance and use, critical materials the facility but also any issues regarding ongo-
such as reagents, blood components, cellular ing service and support by the supplier. There
therapy products, tissues, and derivatives should be a written plan for installation, oper-
should be inspected and tested (if necessary) ational, and performance qualifications.6 The
to ensure that they meet specifications for plan should provide for 1) installation accord-
their intended use. It is essential that supplies ing to the manufacturer’s specifications, 2)
used in the collection, processing, preserva- verification of the equipment’s functionality
CHAPTER 1 Quality Management Systems: Theory and Practice 䡲 11
before use by ensuring that the criteria estab- 䡲 Customer needs and expectations.
lished by the manufacturer for its intended use 䡲 Accreditation and regulatory requirements.
are met, and 3) assurance that the equipment 䡲 Specifications to be met.
performs as expected in the facility’s process- 䡲 Risk assessment.
es. After the equipment is installed, any prob- 䡲 Performance measures.
lems and follow-up actions taken should be 䡲 Nonconformance analyses.
documented. Recalibration and requalifica- 䡲 Current knowledge (eg, of other successful
tion may be necessary if repairs are made that practices).
affect the equipment’s critical operating func- 䡲 Resource needs (eg, financial, facility, hu-
tions. Recalibration and requalification should man, materials, and equipment).
also be considered when existing equipment is 䡲 Interrelationships of the new or changed
relocated. process(es) with other processes.
The facility must develop a mechanism to 䡲 Documents needed for the new or changed
uniquely identify and track all critical equip- process(es).
ment, including equipment software versions,
if applicable. The unique identifier assigned The documents developed should be re-
by the manufacturer may be used, or a unique viewed by management personnel with direct
identification code may be applied by the
authority over the process and by quality over-
transfusion or cellular therapy service or
sight personnel before implementation.
assigned through a laboratory-wide or organi-
zation-wide identification system. Maintain- Changes in policies, processes, and proce-
ing a list of all critical equipment helps in the dures should be documented, validated, re-
control function of scheduling and performing viewed, and approved. Additional information
functional and safety checks, calibrations, pre- on policies, processes, and procedures can be
ventive maintenance, and repair. The equip- found in the “Documents and Records” sec-
ment list can be used to ensure that all appro- tion later in this chapter.
priate actions have been performed and Once a process has been implemented,
recorded. Evaluating and trending equipment the facility should have a mechanism to
calibration, maintenance, and repair data help ensure that procedures are performed as de-
the facility assess equipment functionality, fined and that critical equipment, reagents,
manage defective equipment, and identify and supplies are used in conformance with
equipment needing replacement. When manufacturers’ written instructions and facili-
equipment is found to be operating outside
ty requirements. Table 1-1 lists elements that
acceptable parameters, the potential effects
constitute sound process control (among oth-
on the quality of products or test results must
er elements of a quality management system).
be evaluated and documented.
A facility using critical equipment, reagents, or
Process Management supplies in a manner that is different from the
manufacturer’s directions should validate such
Written and approved policies, processes, and use. If the activity is covered under regulations
procedures must exist for all critical functions for blood and blood components or HCT/Ps,
performed in the facility, and these functions
the facility may be required to request FDA ap-
must be carried out under controlled condi-
proval to operate at variance to requirements
tions. Each facility should have a systematic
approach for identifying, planning, and imple- (see 21 CFR 640.1202 or 21 CFR 1271.1554). If a
menting new (and making changes to existing) facility believes that changes to the manufac-
policies, processes, and procedures that affect turer’s directions would be appropriate, it
the quality of the facility’s tests, products, or should encourage the manufacturer to make
services. Such activities should include a re- such changes in the labeling (ie, the package
view of at least the following: insert or user manual).
12 䡲 AABB TECHNICAL MANUAL
The validation plan should be reviewed its and specifications supplied by the
and approved by quality oversight personnel manufacturer.
before the validation activities are carried out. 䡲 Performance qualification demonstrates
Staff responsible for carrying out the vali- that the equipment performs as expected
dation activities should be trained in the pro- for its intended use in the processes estab-
cess before the plan is executed. The results lished by the facility and that the output
and conclusions of these activities may be ap- meets the facility’s specifications. It evalu-
pended to the approved validation plan or ates the adequacy of equipment for use in a
may be recorded in a separate document. This specific process that uses the facility’s own
documentation typically contains the follow- personnel, procedures, and supplies in a
ing elements: normal working environment.
regarding general software validation princi- they must be controlled, what their require-
ples.22 ments are, and how to implement them. Rec-
ords provide evidence of what did happen
Quality Control (ie, that a process was performed as intend-
ed), and provide information needed to as-
QC testing is performed to ensure the proper
sess product and service quality. Together,
functioning of materials, equipment, and
documents and records are used by quality
methods during operations. QC performance
oversight personnel to evaluate the effective-
expectations and acceptable ranges should be
ness of a facility’s policies, processes, and
defined and be made readily available to staff
procedures. ISO 9001 provides an example of
so they will recognize, and respond appropri-
quality system documentation that includes
ately to, unacceptable results and trends. The
frequency for QC testing is determined by the the following items13:
facility in accordance with the applicable
䡲 The quality policy and objectives.
CMS, FDA, AABB, state, and manufacturer re-
䡲 A description of the interactions between
quirements. QC results should be documented
concurrently with performance.2 Records of processes.
䡲 Documented procedures for the control of
QC testing should include the following:
documents, records, and nonconforming
䡲 Identification of personnel performing the products and for corrective action, preven-
test. tive action, and internal quality audits.
䡲 Identification of reagents (including lot 䡲 Records related to the quality management
numbers and expiration dates). system, operational performance, and
䡲 Identification of equipment. product or service conformance.
䡲 Testing date and, when applicable, time. 䡲 All other “documents needed by the organi-
䡲 Results. zation to ensure the effective planning, op-
䡲 Interpretation (eg, meets or fails to meet es- eration, and control of its processes.”
tablished criteria).
䡲 Reviews. Written policies, process descriptions,
procedures, work instructions, job aids, labels,
Unacceptable QC results must be investi- forms, and records are all part of the facility’s
gated and corrective action must be imple- document management system. They may be
mented, if indicated, before the QC procedure paper based or electronic. A document man-
is repeated or the operational process is con- agement system provides assurance that doc-
tinued. If products or services have been pro- uments are comprehensive, current, and
vided since the last acceptable QC results were available and that records are accurate and
obtained, it may be necessary to evaluate the complete. A well-structured document man-
conformance of these products or services. agement system links policies, process de-
Examples of QC performance intervals for scriptions, procedures, forms, and records to-
equipment and reagents are included in Ap- gether in an organized and workable system.
pendix 1-3.
Documents
Documents and Records
Documents should be developed in a format
Documentation provides a framework for that conveys information clearly and provides
understanding and communicating about staff with the necessary instructions and
processes throughout the organization. Doc- templates for recording data. The CLSI offers
uments provide a description of or instruc- guidance regarding general levels of
tions regarding what is supposed to happen. documentation11 as well as detailed instruc-
Documents describe how processes are in- tions on how to write procedures.23 General
tended to work, how they interact, where types of documentation are described below.
CHAPTER 1 Quality Management Systems: Theory and Practice 䡲 17
some instances, it may also be important to in- The facility should maintain a record of
dicate the reason for the change. The original names; inclusive dates of employment; and
wording must not be obliterated in written corresponding signatures, identifying initials,
records; the original may be crossed out with a or identification codes of personnel autho-
single line, but it should remain legible. Write- rized to create, sign, initial, or review reports
overs and scratch-outs should not be used. and records. Magnetically coded employee
Electronic records must permit tracking of badges and other computer-related identify-
both original and corrected data and must in- ing methods are generally accepted in lieu of
clude the date and identity of the person who written signatures, provided that the badges or
made the change. There should be a process other methods meet electronic record-keeping
for controlling changes. A method for refer- requirements.
encing changes to records that is linked to the
original record and a system for reviewing Information Management
changes for completeness and accuracy are es-
sential. Audit trails for changed data in com- The quality management system should en-
puterized systems are required by the FDA.24 sure the confidentiality and appropriate use of
The following issues might be considered data and information in both oral and written
when planning record storage: communications. Privacy of patient and donor
records should be addressed to maintain the
䡲 Storage of records in a manner that protects security and confidentiality of such records.
them from damage and accidental or unau- The system should prevent unauthorized
thorized destruction or modification. access, modification, or destruction of the
䡲 Degree of accessibility of records in propor- data and information. Individuals who are au-
tion to frequency of their use. thorized to make changes to data should be
䡲 Method and location of record storage re- defined by name, code, or job responsibility.
lated to the volume of records and the Information systems should be designed with
amount of available storage space. security features to prevent unauthorized ac-
䡲 Availability of properly functioning equip- cess and use. Systems may include levels of se-
ment, such as computer hardware, and curity defined by job responsibility and re-
software to view archived records. quire the use of security codes and passwords
䡲 Documentation that all records copied, or, for paper-based systems, locked cabinets
transferred to microfiche, or converted to and keys.
digital files legitimately replace originals The integrity of data should be main-
that are stored elsewhere or destroyed. tained so that data are retrievable and usable.
䡲 Retention of original color-coded records Periodic integrity checks should be conducted
when only black-and-white reproductions to ensure that critical data have not been inad-
are available. vertently modified, been lost, or become
inaccessible. When data are sent manually or
Considerations for electronic records in- electronically from one point to another, a
clude the following: process should be in place to ensure that the
data accurately and reliably reach their final
䡲 A method of verifying the accuracy of data destination in a timely manner.
entry. Backup versions (eg, disks, tapes, or du-
䡲 Prevention of unintended deletion of data plicate hard copies) of critical data should be
or access by unauthorized persons. maintained in the event of an unexpected loss
䡲 Adequate protection against inadvertent from the storage medium. It is advisable to
data loss (eg, when a storage device is full). store backup or archived computer records
䡲 Validated safeguards to ensure that a record and databases off-site at a sufficient distance
can be edited by only one person at a time. away to ensure that disasters will not affect
䡲 Security of and access to confidential data. both the originals and the backups. The
20 䡲 AABB TECHNICAL MANUAL
backup storage facility should be secure. Envi- basis of investigations and root cause anal-
ronmental conditions in the backup storage yses.
facility should be maintained in a way that 䡲 Implement preventive actions as appropri-
protects and preserves the equipment and ate on the basis of analyses of aggregate
media for the duration of their storage. Tem- data about events and their causes.
perature and humidity should be monitored 䡲 Report to external agencies when required.
and controlled. Archival copies of computer 䡲 Evaluate effectiveness of the corrective ac-
operating systems and applications software tions and preventive actions taken.
required to view original records should be
stored in the same manner. The CLSI has published a consensus stan-
The facility should develop and maintain dard on occurrence management that ex-
alternative systems to ensure access to critical plores event management in more detail.25
data and information in the event that com- Facility personnel should be trained to
puterized data or primary sources of informa- recognize and report such occurrences. De-
tion are not available. The backup and recov- pending on the severity of an event and risk to
er y procedures for computer downtime patients, donors, and products as well as the
should be defined, and validation documenta- likelihood of recurrence, investigation of con-
tion should show that the backup system tributing factors and underlying causes may
works properly. The associated processes be warranted. The cGMP regulations require
should be tested periodically to ensure that investigation and documentation of results if a
the backup system remains effective. Special specific event could adversely affect patient
consideration should be given to staff compe- safety or the safety, purity, or potency of blood
tence and readiness to use the backup system. or components. 2 , 3 The cGTP regulations
require similar activities for deviations and
Management of Nonconforming possible product contamination or communi-
Events cable disease transmission. 4 Tools and ap-
proaches for performing root cause analysis
The quality management system should in- and implementing corrective action are dis-
clude a process for detecting, investigating, cussed in the section on “Process Improve-
and responding to events that result in devia- ment.” A summary of each event, investiga-
tions from accepted policies, processes, and tion, and any follow-up activities must be
procedures or in failures to meet require- prepared. Table 1-2 outlines suggested com-
ments, as defined by the facility, AABB stan- ponents of an internal event report.
dards, or applicable regulations.2-4 This pro- Fatalities related to blood collection or
cess should be implemented after, for transfusion or to HCT/Ps must be reported as
example, the discovery of nonconforming soon as possible to the FDA Center for Biolog-
products and services or of adverse reactions ics Evaluation and Research (CBER). [See 21
to donation, blood components, cellular ther- CFR 606.170(b) and 1271.350(a)(i), respective-
apy products, tissues, or derivatives. The facili- ly.] Instructions for reporting to CBER are
ty should define how to perform the following available in published guidance27 and on the
for nonconforming events: FDA website.28 A written follow-up report must
be submitted within 7 days of the fatality and
䡲 Document and classify occurrences. should include a description of any new pro-
䡲 Determine the effect, if any, on the quality cedures implemented to avoid recurrence.
of products or services. AABB Association Bulletin #04-06 provides ad-
䡲 Evaluate the effect on interrelated activities. ditional information on these reporting re-
䡲 Analyze the event to understand root quirements, including a form for reporting do-
causes. nor fatalities.29
䡲 Implement corrective action as appropri- Regardless of their licensure and registra-
ate, including notification and recall, on the tion status with the FDA, all donor centers,
CHAPTER 1 Quality Management Systems: Theory and Practice 䡲 21
Component Examples
blood banks, and transfusion services must components having major blood group in-
promptly report biological product devia- compatibilities.9,10
tions—and information relevant to these Hemovigilance processes also provide the
events—to the FDA2,30 using Form FDA-3486 opportunity to detect, investigate, and re-
when the event 1) is associated with manufac- spond to adverse transfusion reactions and
turing (ie, collecting, testing, processing, pack- events that result in deviations from safe blood
ing, labeling, storing, holding, or distributing); transfusion and collection practices. Adverse
2) represents a deviation from cGMP, estab- transfusion reactions and events (or incidents)
lished specifications, or applicable regula- can be reported voluntarily to the Centers for
tions or standards or that is unexpected or un- Disease Control and Prevention (CDC) Nation-
foreseen; 3) may affect the product’s safety, al Healthcare Safety Network (NHSN) Hemo-
purity, or potency; 4) occurs while the facility vigilance Module. This system was developed
had control of, or was responsible for, the through a public-private collaboration that in-
product; and 5) involves a product that has left cluded the Department of Health and Human
Services and its agencies, and the private sec-
the facility’s control (ie, has been distributed).
tor, including AABB, America’s Blood Centers,
Using the same form, facilities must also
and the American Red Cross. The AABB Center
promptly report biological product deviations
for Patient Safety, a licensed Patient Safety Or-
associated with a distributed HCT/P if the
ganization, works with hospitals to provide ad-
event represents a deviation from applicable
ditional analysis and benchmarking of hospi-
regulations, standards, or established specifi-
tal transfusion event reports, while protecting
cations that relate to the prevention of com-
data confidentiality. AABB also administers
municable disease transmission or HCT/P the AABB United States Donor Hemovigilance
contamination. This requirement pertains to Program where blood collectors can report,
events that are unexpected or unforeseeable analyze, and benchmark adverse donor reac-
but may relate to the transmission or potential tions.
transmission of a communicable disease or Each facility should track reported events
may lead to HCT/P contamination.4 More in- and look for trends. The use of classification
formation concerning biological product devi- schemes may facilitate trend analysis and typi-
ation reporting can be found on the FDA web- cally involves one or more of the following cat-
site.31 egories: the nature of the event, the process (or
There must also be a mechanism to re- procedure) in which the event occurred, the
port medical device adverse events to the FDA outcome and severity of the event, and the
and the device manufacturer. 8,32 The Joint contributing factors and underlying causes. If
Commission encourages reporting of sentinel several events within a relatively short period
events, including hemolytic transfusion reac- involve a particular process or procedure, that
tions involving the administration of blood or process or procedure should be further inves-
CHAPTER 1 Quality Management Systems: Theory and Practice 䡲 23
tigated. The most useful schemes involve the Occasionally, there may be a need for a fa-
use of multiple categories for each event, cility to deviate from approved procedures to
which allows data to be sorted in a variety of meet the unique medical needs of a particular
ways so that patterns that were previously not patient. When this situation arises, a medically
obvious can emerge. (See example in Table 1- indicated exception should be planned and
3.) Such sorting or stratification can result in approved in advance by the facility’s medical
identification of situations that require closer director. The rationale and nature of the
monitoring or problems needing corrective or planned exception should be documented.
preventive action. For smaller facilities that Careful consideration should be given to
may not have sufficient data to identify trends, maintaining a controlled process and verifying
pooling data with a larger entity (eg, the labo- the safety and quality of the resulting product
ratory or all transfusion services in a health- or service. Any additional risk to the patient
care system) or following national trends from must be disclosed.
data provided by organizations such as the
AABB, CAP, or The Joint Commission, may also Monitoring and Assessment
prove helpful. The extent of monitoring and The quality management system should de-
the length of time to monitor processes de- scribe how the facility monitors and evaluates
pends on the frequency and critical aspects of its processes. Assessments are systematic ex-
the occurrences. Reporting and monitoring of aminations to determine whether actual activ-
events are essential problem identification ities comply with planned activities, are imple-
methods for process improvement activities in mented effectively, and achieve objectives.
a quality management system. Depending on the focus, assessments can
Event: A unit of Red Blood Cells from a directed donor was issued to the wrong oncology patient. The unit
was not transfused.
Event Classification
䡲 Type of event: patient
䡲 Procedure involved: issuing products
䡲 Process involved: blood administration
䡲 Product involved: Red Blood Cells
䡲 Location: transfusion service
䡲 Other factors: directed donor
䡲 Other factors: oncology patient
Underlying Causes
䡲 Proximate cause: two patients with similar names had crossmatched blood available
䡲 Root cause: inadequate procedure for verification of patient identification during issue
Outcome
䡲 Severity: serious, FDA reportable
䡲 Patient: no harm, correct product was obtained and transfused
䡲 Product: no harm, product returned to inventory
䡲 Donor: not applicable
Successful Barriers
䡲 Problem detected during the patient identification verification step of blood administration
include: 1) evaluation of process outputs (ie, with assessment results should be reviewed by
results); 2) the activities that make up a pro- executive management.
cess as well as its outputs; or 3) a group of re-
lated processes and outputs (ie, the system). Quality Indicators
Assessments can be internal or external
Quality indicators are performance measures
and can include quality assessments, peer re-
designed to monitor one or more processes
views, self-assessments, and proficiency test-
during a defined time and are useful for evalu-
ing. Evaluations typically include comparisons
ating service demands, production, personnel,
of actual to expected results.
inventory control, and process stability. These
indicators can be process based or outcome
Internal Assessments
based. Process-based indicators measure the
Internal assessments may include evaluations degree to which a process can be consistently
of quality indicator data, targeted audits of a performed. An example of a process-based in-
single process, or system audits that are dicator is turnaround time from blood product
broader in scope and may cover a set of inter- ordering until transfusion. Outcome-based in-
related processes. These assessments should dicators are often used to measure what does
be planned and scheduled. The details of who or does not happen after a process is or is not
performs the assessments and how they are performed. The number of incorrect test result
performed should be addressed. Assessments reports is an example of such an indicator. For
should cover the quality system and the major each indicator, thresholds are set that repre-
operating systems in the donor center and sent warning limits, action limits, or both.
transfusion or cellular therapy service. These thresholds can be determined from reg-
In addition, there should be a process for ulatory or accreditation requirements, bench-
responding to the issues raised as a result of marking, or internal facility data.
the assessment, including review processes Tools frequently used for displaying qual-
and time frames. The results should be docu- ity indicator data are run charts and control
mented and submitted to management per- charts. In a run chart, time is plotted on the
sonnel who have authority over the process as- x-axis and values on the y-axis. In control
sessed as well as to executive management. charts, the mean of the data and the upper and
Management should develop corrective ac- lower control limits, which have been calculat-
tion plans with input from operational staff ed from the data, are added to the chart. Single
and quality oversight personnel for any defi- points outside the upper and lower control
ciencies noted in the assessment. Quality limits result from special causes. Statistical
oversight personnel should track progress to- rules for interpreting consecutive points out-
ward implementation of corrective actions side 1 standard deviation (SD), 2 SDs, and 3
SDs should be used to recognize a process that
and monitor them for effectiveness.
is out of control. The root cause should be de-
To make the best use of these assess-
termined, and corrective action should be ini-
ments, there should be a process to track,
tiated, if indicated.
monitor trends in, and analyze the problems
identified so that opportunities for improve-
Blood Utilization Assessment
ment can be recognized. Early detection of
trends makes it possible to develop preventive The activities of blood usage review commit-
actions before patient safety, blood, compo- tees in the transfusion setting are an example
nents, tissues, or derivatives are adversely af- of internal assessment. Guidelines are avail-
fected. Evaluation summaries provide infor- able from the AABB for both adult and pediat-
mation that is useful for addressing individual ric utilization review.34-36 Peer review of trans-
or group performance problems and ensuring fusion practices, required by the AABB, is also
the adequacy of test methods and equipment. required by The Joint Commission9 for hospi-
Any corrective or preventive action associated tal accreditation, by CMS1 for hospitals to
CHAPTER 1 Quality Management Systems: Theory and Practice 䡲 25
qualify for Medicare reimbursement, and by evolving technologies and products, such as
some states for Medicaid reimbursement. growth factors and cytokines.
Transfusion audits provide reviews of pol-
icies and practices to ensure safe and appro- External Assessments
priate transfusions and are based on measur-
External assessments include inspections, sur-
able, predetermined performance criteria.
veys, audits, and assessments performed by
(See Chapter 28.) Transfusion services should
those not affiliated with the organization, such
investigate an adequate sample of cases (eg,
as the AABB, CAP, CMS, COLA, FACT, the FDA,
5% of cases within a defined time frame or 30
The Joint Commission, or state and regional
cases, whichever is larger). Audits assess a fa-
health departments. Participation in an exter-
cility’s performance and effectiveness in the
nal assessment program provides an indepen-
following6:
dent objective view of the facility’s performance.
External assessors often bring broad-based ex-
䡲 Ordering practices.
perience and knowledge of best practices that
䡲 Patient identification.
can be shared. Such assessments are increas-
䡲 Sample collection and labeling.
ingly being performed unannounced or with
䡲 Infectious and noninfectious adverse events.
minimal notification.
䡲 Near-miss events.
In the preparation phase, there is typically
䡲 Usage and discard.
some data gathering and information to sub-
䡲 Appropriateness of use.
mit to the organization performing the assess-
䡲 Blood administration policies.
ment. To prepare, facilities can perform inter-
䡲 Ability of services to meet patient needs.
nal audits and conduct drills to ensure that
䡲 Compliance with peer-review recommen-
staff can answer questions. For most external
dations.
assessments, there is an increased emphasis
䡲 Critical laboratory results before and after
on observations of the processes and dialogue
transfusion.
with nonmanagement staff, so preparation is
key. During the assessment phase, it is impor-
One method of assessing the blood ad-
tant to know who is responsible for the asses-
ministration process is to observe a predeter-
sors or inspectors while they are in the facility.
mined number of transfusions by following a
Clear descriptions of what information can be
unit of blood as it is issued for transfusion and
given to these individuals—and in what form—
is then transfused.34
help the facility through the assessment or
Assessments of transfusion safety policy
inspection process. After the assessment,
and practice may include reviews of transfu-
identified issues should be addressed. Usually,
sion reactions and transfusion-transmitted
a written response is submitted.
diseases. The review committee may monitor
policies and practices for notifying recipients
Proficiency Testing for Laboratories
or recipients’ physicians of recalled products
and notifying donors of abnormal test results. Proficiency testing (PT) is one means for deter-
Other assessments important in transfusion mining that test systems (including methods,
practice include reviews of policies for in- supplies, and equipment) are performing as
formed consent, indications for transfusion, expected. As a condition for certification, CMS
releases of directed donor units, and outpa- requires laboratories to participate successful-
tient or home transfusions. Additional assess- ly in an approved PT program for CLIA-
ments should include, where appropriate, 1) regulated testing. When no approved PT
therapeutic apheresis; 2) use of blood recov- program exists for a particular analyte, the lab-
ery devices; 3) procurement and storage of he- oratory must have another means to verify the
matopoietic progenitor cells; 4) perioperative accuracy of the test procedure at least twice
autologous blood collection; 5) procurement annually.1 Some accrediting agencies may re-
and storage of tissue; and 6) evaluation of quire more frequent verification of accuracy.
26 䡲 AABB TECHNICAL MANUAL
PT must be performed using routine work American Society for Quality.37-39 The Joint
processes and conditions if it is to provide Commission standards for performance im-
meaningful information. PT samples should provement are outlined in Table 1-4.9,10
generally be handled and tested in the same Corrective action is defined as action tak-
way as patient or donor specimens. However, a en to address the root causes of an existing
CLIA-certified laboratory is prohibited from nonconformance or other undesirable situa-
discussing the PT or sending the samples to a tion to reduce or eliminate the risk of recur-
laboratory with a different CLIA number dur- rence. Preventive action is defined as action
ing the active survey period, even if the two taken to reduce or eliminate the potential for a
laboratories are within the same organization nonconformance or other undesirable situa-
and that would be the routine manner for han- tion to prevent occurrence. Corrective action
dling patient or donor specimens. Supervisory can be thought of as a reactive approach to ad-
review of the summary evaluation report dress the root causes of actual nonconfor-
should be documented along with investiga- mances, deviations, complaints, and process
tion and corrective action for unacceptable re- failures, whereas preventive action can be
sults. Quality oversight personnel should thought of as a proactive approach to address
monitor the PT program and verify that test the underlying causes of anticipated prob-
systems are maintained in a state of control lems identified through the analysis of data
and appropriate corrective action is taken and information.40 In contrast, remedial action
when indicated. is defined as action taken to alleviate the
symptoms of existing nonconformances or any
Process Improvement other undesirable situation.41 Remedial action,
sometimes called correction, addresses only a
Continual improvement is a fundamental goal
problem’s visible indicator and not the actual
in any quality management system. In transfu-
cause. (See comparisons in Table 1-5.) Effec-
sion and cellular therapies and clinical diag-
tive corrective and preventive action cannot
nostics, this goal is tied to patient safety goals
be implemented until the underlying cause is
and expectations for the highest quality health
determined and the process is evaluated in re-
care. The importance of identifying, investi-
lationship to other processes. Pending such
gating, correcting, and preventing problems
evaluation, it may be desirable to implement
cannot be overstated. The process of develop-
interim remedial action.
ing corrective and preventive action plans in-
volves identification of problems and their
Identification of Problems and Their
causes as well as identification and evaluation
Causes
of solutions to prevent future problems. This
process should also include evaluation of Sources of information for process improve-
near-miss events and a mechanism for data ment activities include process deviations,
collection and analysis as well as follow-up to nonconforming products and services, cus-
evaluate the effectiveness of the actions taken. tomer complaints, QC records, PT, internal au-
Statistical tools and their applications may be dits, quality indicators, and external assess-
found in publications from the AABB and the ments. Active monitoring programs may be set
䡲 The organization collects data to monitor its performance, including the following:
– Blood and blood component use.
– All confirmed transfusion reactions.
䡲 The organization compiles and analyzes data.
䡲 The organization improves performance on an ongoing basis.
CHAPTER 1 Quality Management Systems: Theory and Practice 䡲 27
up to help identify problem areas. These pro- tion is impractical, impossible, or outside the
grams should be representative of the facility boundaries of the organization. Use of the “re-
processes and consistent with organizational petitive why” prevents the mistake of inter-
goals, and they should reflect customer needs. preting an effect as a cause.
Preparation of a facility quality report at least
CAUSE-A ND-E FFECT DIAGRA M. The cause-
annually in which data from all these sources
are aggregated and analyzed can be valuable and-effect diagram, also known as the Ishika-
to identify issues for performance improve- wa or fish-bone diagram, uses a specialized
ment. form of brainstorming that breaks down prob-
Once identified, problems should be ana- lems into “bite-sized” pieces. (An example of a
lyzed to determine their scope, potential ef- cause-and-effect diagram is shown in Fig 1-1.)
fects on quality management and operational The method used in the diagram is designed to
systems, relative frequency, and extent of their focus ideas around the component parts of a
variation. Such an analysis is important to process as well as to give a pictorial represen-
avoid tampering with processes that are mere- tation of the ideas that are generated and their
ly showing normal variations or problems with interactions. When using the cause-and-effect
little effect. diagram, one looks at equipment, materials,
The underlying causes of an undesirable methods, environment, and human factors.
condition or problem can be identified by an These three tools identify both active and
individual or group. The more complex the latent failures. Active failures are those that
problem and the more involved the process, have an immediate adverse effect. Latent fail-
the greater the need to enlist a team and to for- ures are more global actions and decisions
malize the analysis. Three commonly used with potential for damage that may lie dor-
tools for identifying underlying causes in an mant and become evident only when triggered
objective manner are process flowcharting, by the presence of localized factors. The key to
use of the “repetitive why,” and the cause-and- successfully determining root causes is to
effect diagram. avoid stopping too soon or getting caught in
the trap of placing blame on an individual.
PROCESS FLOWCHART . A process flowchart
Most problems, particularly those that are
gives a detailed picture of the multiple activi-
complex, have several root causes. A method
ties and important decision points within that
that can be of use when such problems occur
process. By examining this picture, one may
is the Pareto analysis. A chart of causes, laid
identify problem-prone areas.
out in order of decreasing frequency, is pre-
REPETITIVE WHY. The “repetitive why” is pared. Those that occur most frequently are
used to work backward through the process. considered the “vital few”; the rest are consid-
One repeatedly asks “Why did this happen?” ered the “trivial many.” This method offers di-
until 1) no new information can be gleaned; 2) rection on where to dedicate resources for
the causal path cannot be followed because of maximal effect. An example of a Pareto chart is
missing information; or 3) further investiga- shown in Fig 1-2.
28 䡲 AABB TECHNICAL MANUAL
industry are finding increasing use in the tion of these principles and techniques can
health-care setting. LEAN emphasizes speed improve performance, reduce costs and waste,
and efficiency. Six Sigma emphasizes precision cut time, and eliminate non-value-added ac-
and accuracy. Six Sigma uses the data-driven tions. Additional information about both
approach to problem solving of define, mea- methods can be found on the website of the
sure, analyze, improve, and control. Applica- American Society for Quality.42
KEY POINTS
2. Customer Focus. Quality organizations should understand and meet or exceed customer
needs and expectations. These needs and expectations should be defined in a contract,
agreement, or other document developed with feedback from the customer.
3. Facilities, Work Environment, and Safety. Procedures related to general safety; biological,
chemical, and radiation safety; fire safety; and disaster preparedness are required. Space al-
location, building utilities, ventilation, sanitation, trash, and hazardous substance disposal
must support the organization’s operations.
4. Human Resources. Quality management of all personnel addresses adequate staffing levels
and staff selection, orientation, training, and competence assessment as well as specific
regulatory requirements.
5. Suppliers and Materials Management. Suppliers of critical materials and services (ie, those
affecting quality) should be qualified, and these requirements should be defined in con-
tracts or agreements. All critical materials should be qualified and then inspected and test-
ed upon receipt to ensure that specifications are met.
6. Equipment Management. Critical equipment may include instruments, measuring devices,
and computer hardware and software. This equipment must be uniquely identified and op-
erate within defined specifications, as ensured by qualification, calibration, maintenance,
and monitoring.
7. Process Management. A systematic approach to develop new, and control changes to, poli-
cies, processes, and procedures includes process validation, test method validation, com-
puter system validation, equipment validation, and QC. Validation must be planned and re-
sults reviewed and accepted.
8. Documents and Records. Documents include policies, process descriptions, procedures,
work instructions, job aids, forms, and labels. Records provide evidence that the process
was performed as intended and allow assessment of product and service quality.
9. Information Management. Unauthorized access, modification, or destruction of data and
information must be prevented and confidentiality of patient and donor records main-
tained. Data integrity should be assessed periodically and backup devices, alternative sys-
tems, and archived documents maintained.
10. Management of Nonconforming Events. Deviations from facility-defined requirements,
standards, and regulations must be addressed by documenting and classifying occurrences,
assessing effects on quality, implementing remedial actions, and reporting to external agen-
cies as required.
11. Monitoring and Assessment. Evaluation of facility processes includes internal and external
assessments, monitoring of quality indicators, blood utilization assessment, proficiency
testing, and analysis of data.
12. Process Improvement. Opportunities for improvement may be identified from deviation
reports, nonconforming products and services, customer complaints, QC records, profi-
ciency testing results, internal audits, quality indicator monitoring, and external assess-
ments. Process improvement includes determination of root causes, implementation of
corrective and preventive actions, and evaluation of the effectiveness of these actions.
REFER ENCES
1. Code of federal regulations. Title 42, CFR Part Government Printing Office, 2014 (revised an-
493. Washington, DC: US Government Print- nually).
ing Office, 2013 (revised annually). 3. Code of federal regulations. Title 21, CFR Parts
2. Code of federal regulations. Title 21, CFR Parts 210 and 211. Washington, DC: US Government
606, 610, 630, and 640. Washington, DC: US Printing Office, 2014 (revised annually).
CHAPTER 1 Quality Management Systems: Theory and Practice 䡲 31
4. Code of federal regulations. Title 21, CFR Parts 18. Code of federal regulations. Title 21, CFR Part
1270 and 1271. Washington, DC: US Govern- 820. Washington, DC: US Government Print-
ment Printing Office, 2014 (revised annually). ing Office, 2014 (revised annually).
5. Food and Drug Administration. Guideline for 19. Juran JM, Godfrey AB. Juran’s quality hand-
quality assurance in blood establishments book. 5th ed. New York: McGraw-Hill, 1999.
(July 11, 1995). Silver Spring, MD: CBER Office 20. Food and Drug Administration. Guidance for
of Communication, Outreach, and Develop- industry: Process validations: General princi-
ment, 1995. ples and practices ( January, 2011). Silver
6. Judith Levitt, ed. Standards for blood banks Spring, MD: CBER Office of Communication,
and transfusion services. 29th ed. Bethesda, Outreach, and Development, 2011.
MD: AABB, 2014. 21. Food and Drug Administration. Guidance for
7. Fontaine M, ed. Standards for cellular therapy industry: Blood establishment computer sys-
services. 6th ed. Bethesda, MD: AABB, 2013. tem validation in the user’s facility (April,
8. College of American Pathologists. Laboratory 2013). Silver Spring, MD: CBER Office of Com-
Accreditation Program checklists. Chicago: munication, Outreach, and Development,
CAP, 2013. 2013.
9. The Joint Commission. Hospital accreditation 22. Food and Drug Administration. General prin-
standards. Oakbrook Terrace, IL: Joint Com- ciples of software validation: Final guidance
mission Resources, 2014. for industry and FDA staff (January, 2002). Sil-
10. The Joint Commission. Laboratory accredita- ver Spring, MD: CBER Office of Communica-
tion standards. Oakbrook Terrace, IL: Joint tion, Outreach, and Development, 2002.
Commission Resources, 2014. 23. Clinical and Laboratory Standards Institute.
11. Clinical and Laboratory Standards Institute.
Quality Management System: Development
Quality management system: A model for lab-
and management of laboratory documents;
oratory services; approved guideline (GP26-
approved guideline. 6th ed. (GP02-A6/QMS
A4/QMS 01-A4). 4th ed. Wayne, PA: CLSI, 2011.
02-A6). Wayne, PA: CLSI, 2013.
12. Foundation for the Accreditation of Cellular
24. Code of federal regulations. Title 21, CFR Part
Therapy and the Joint Accreditation Commit-
11. Washington, DC: US Government Printing
tee of ISCT and EBMT. FACT-JACIE interna-
Office, 2014 (revised annually).
tional standards for cellular therapy product
25. Clinical and Laboratory Standards Institute.
collection, processing, and administration. 5th
Management of nonconforming laboratory
ed. Omaha, NE: FACT-JACIE, 2012.
events; approved guideline (GP32-A/QMS 11-
13. ANSI/ISO/ASQ Q9001-2008 series—quality
A). Wayne, PA: CLSI, 2007.
management standards. Milwaukee, WI: ASQ
26. Motschman TL, Santrach PJ, Moore SB. Error/
Quality Press, 2008.
14. International Organization for Standardiza- incident management and its practical appli-
tion. ISO 15189:2012. Medical laboratories— cation. In: Duckett JB, Woods LL, Santrach PJ,
Requirements for quality and competence. eds. Quality in action. Bethesda, MD: AABB,
Geneva, Switzerland: ISO, 2012. [Available at 1996:37-67.
http://www.iso.org/iso/catalogue_detail?cs- 27. Food and Drug Administration. Guidance for
number=56115 (accessed November 6, 2013).] industry: Notifying FDA of fatalities related to
15. Baldrige Performance Excellence Program. blood collection or transfusion (September,
Health care criteria for performance excel- 2003). Silver Spring, MD: CBER Office of Com-
lence. Gaithersburg, MD: National Institute of munication, Outreach, and Development,
Standards and Technology, 2013-2014 (revised 2003.
biannually). 28. Food and Drug Administration. Transfusion/
16. Quality program implementation. Association donation fatalities: Notification process for
bulletin #97-4. Bethesda, MD: AABB, 1997. transfusion related fatalities and donation re-
17. Food and Drug Administration. Guidance for lated deaths. Silver Spring, MD: Center for Bio-
industry: Quality systems approach to phar- logics Evaluation and Research, 2007. [Avail-
maceutical cGMP regulations (September, able at http://www.fda.gov/BiologicsBloodVac
2006). Silver Spring, MD: CBER Office of Com- cines/SafetyAvailability/ReportaProblem/
munication, Outreach, and Development, TransfusionDonationFatalities/default.htm
2006. (accessed August 23, 2013).]
32 䡲 AABB TECHNICAL MANUAL
29. AABB. Reporting donor fatalities. Association Medicine Section Coordinating Committee.
bulletin #04-06. Bethesda, MD: AABB, 2004. Guidelines for patient blood management and
30. Food and Drug Administration. Guidance for blood utilization. Bethesda, MD: AABB, 2011.
industry: Biological product deviation report- 36. Strauss RG, Blanchette VS, Hume H. National
ing for blood and plasma establishments (Oc- acceptability of American Association of Blood
tober, 2006). Silver Spring, MD: CBER Office of Banks Hemotherapy Committee guidelines for
Communication, Outreach, and Develop- auditing pediatric transfusion practices.
ment, 2006. Transfusion 1993;33:168-71.
31. Food and Drug Administration. Biological 37. Vaichekauskas L. You need the tools to do the
product deviations: Includes human tissue and job. In: Walters L, ed. Introducing the big Q: A
cellular and tissue-based product (HCT/P) practical quality primer. Bethesda, MD: AABB
reporting (BPDR). Silver Spring, MD: Center
Press, 2004:181-206.
for Biologics Evaluation and Research, 2010.
38. Walters L. So many tools, so little understand-
[Available at http://www.fda.gov/Biologics
ing. In: Walters L, Carpenter-Badley J, eds. S3:
BloodVaccines/SafetyAvailability/Reporta
Simple Six Sigma for blood banking, transfu-
Problem/BiologicalProductDeviations/de
fault.htm (accessed August 23, 2013).] sion, and cellular therapy. Bethesda, MD:
32. Code of federal regulations. Title 21, CFR Part AABB Press, 2007:9-24.
803. Washington, DC: US Government Print- 39. Tague NR. The quality toolbox. 2nd ed. Mil-
ing Office, 2014 (revised annually). waukee, WI: ASQ Quality Press, 2005.
33. Strong DM, AuBuchon J, Whitaker B, Kuehnert 40. Motschman TL. Corrective versus preventive
MJ. Biovigilance initiatives. ISBT Sci Ser 2008; action. AABB News 1999;21(8):5,33.
3:77-84. 41. Russell JP, Regel T. After the quality audit: Clos-
34. Shulman IA, Lohr K, Derdiarian AK, Picukaric ing the loop on the audit process. 2nd ed. Mil-
JM. Monitoring transfusionist practices: A waukee, WI: ASQ Quality Press, 2000.
strategy for improving transfusion safety. 42. American Society for Quality. Learn about
Transfusion 1994;34:11-15. quality. Milwaukee, WI: ASQ, 2013. [Available
35. Roback J, Waxman D, for the Clinical Transfu- at http://asq.org/learn-about-quality/ (ac-
sion Medicine Committee and the Transfusion cessed August 23, 2013).]
CHAPTER 1 Quality Management Systems: Theory and Practice 䡲 33
䡲 APPENDIX 1-1
Glossary of Commonly Used Quality Terms
Biovigilance Collection and analysis of adverse event data for the purpose of improving out-
comes in the use of blood products, organs, tissues, and cellular therapies.
Calibration Comparison of measurements performed with an instrument to those made
with a more accurate instrument or standard for the purpose of detecting,
reporting, and eliminating errors in measurement.
Change control Established procedures for planning, documenting, communicating, and exe-
cuting changes to infrastructure, processes, products, or services. Such proce-
dures include the submission, analysis, approval, implementation, and
postimplementation review of the change and decisions made about the change.
Formal change control provides a measure of stability and safety and avoids
arbitrary changes that might affect quality.
Control chart A graphic tool used to determine whether the distribution of data values gener-
ated by a process is stable over time. A control chart plots a statistic vs time and
helps to determine whether a process is in control or out of control according to
defined criteria (eg, a shift from a central line or a trend toward upper or lower
acceptance limits).
Design output Documents, records, and evidence in any other format used to verify that design
goals have been met. Design output should identify characteristics of a product
or service that are crucial to safety and function and to meeting regulatory
requirements. It should contain or make reference to acceptance criteria. Exam-
ples of design output include standard operating procedures; specifications for
supplies, reagents, and equipment; identification of quality control require-
ments; and results of verification and validation activities.
End-product test and Verification through observation, examination, or testing (or a combination) that
inspection the finished product or service conforms to specified requirements.
Near-miss event An unexpected occurrence that did not adversely affect the outcome but could
have resulted in a serious adverse event.
Process capability Ability of a controlled process to produce a service or product that fulfills
requirements or a statistical measure of the inherent process variability for a
given characteristic relative to design specifications. The most widely accepted
formula for process capability is Six Sigma.
Process control Activities intended to minimize variation within a process to produce a predict-
able output that meets specifications.
Qualification Demonstration that an entity is capable of fulfilling specified requirements and
verification of attributes that must be met or complied with for a person or thing
to be considered fit to perform a particular function. For example, equipment
may be qualified for an intended use by verifying performance characteristics,
such as linearity, sensitivity, or ease of use. An employee may be qualified on
the basis of technical, academic, and practical knowledge and skills developed
through training, education, and on-the-job performance.
(Continued)
34 䡲 AABB TECHNICAL MANUAL
䡲 APPENDIX 1-1
Glossary of Commonly Used Quality Terms (Continued)
Quality assurance Activities involving quality planning, control, assessment, reporting, and
improvement necessary to ensure that a product or service meets defined qual-
ity standards and requirements.
Quality control Operational techniques and activities used to monitor and eliminate causes of
unsatisfactory performance at any stage of a process.
Quality indicators Measurable aspects of processes or outcomes that provide an indication of the
condition or direction of performance over time. Quality indicators are used to
monitor progress toward stated quality goals and objectives.
Quality management The organizational structure, processes, and procedures necessary to ensure
that the overall intentions and direction of an organization’s quality program are
met and that the quality of the product or service is ensured. Quality manage-
ment includes strategic planning, allocation of resources, and other systematic
activities, such as quality planning, implementation, and evaluation.
Requirement A stated or obligatory need or expectation that can be measured or observed
and that is necessary to ensure quality, safety, effectiveness, or customer satis-
faction. Requirements can include things that the system or product must do,
characteristics that it must have, and levels of performance that it must attain.
Specification Description of a set of requirements to be satisfied by a product, material, or
process indicating, if appropriate, the procedures to be used to determine
whether the requirements are satisfied. Specifications are often in the form of
written descriptions, drawings, professional standards, and other descriptive
references.
Validation Demonstration through objective evidence that the requirements for a particular
application or intended use have been met. Validation provides assurance that
new or changed processes and procedures are capable of consistently meeting
specified requirements before implementation.
Verification Confirmation, by examination of objective evidence, that specified requirements
have been met.
CHAPTER 1 Quality Management Systems: Theory and Practice 䡲 35
䡲 APPENDIX 1-2
Code of Federal Regulations Quality-Related References
Code of Federal Regulations, Title 21
䡲 APPENDIX 1-3
Suggested Quality Control Performance Intervals for Equipment and Reagents*
Refrigerators/freezers/platelet storage
Refrigerators
䡲 Recorder Daily
䡲 Manual temperature Daily
䡲 Alarm system board (if applicable) Daily
䡲 Temperature charts (review daily) Weekly
䡲 Alarm activation Quarterly
Freezers
䡲 Recorder Daily
䡲 Manual temperature Daily
䡲 Alarm system board (if applicable) Daily
䡲 Temperature charts (review daily) Weekly
䡲 Alarm activation Quarterly
Platelet incubators
䡲 Recorder Daily
䡲 Manual temperature Daily
䡲 Temperature charts (review daily) Weekly
䡲 Alarm activation Quarterly
䡲 Ambient platelet storage Every 4 hours
Laboratory equipment
Centrifuges/cell washers
䡲 Speed Quarterly
䡲 Timer Quarterly
䡲 Function Yearly
䡲 Tube fill level (serologic) Day of use
䡲 Saline fill volume (serologic) Weekly
䡲 Volume of antihuman globulin dispensed (if applicable) Monthly
䡲 Temperature check (refrigerated centrifuge) Day of use
䡲 Temperature verification (refrigerated centrifuge) Monthly
CHAPTER 1 Quality Management Systems: Theory and Practice 䡲 37
䡲 APPENDIX 1-3
Suggested Quality Control Performance Intervals for Equipment and Reagents*
(Continued)
Equipment and Reagent Frequency of Quality Control
(Continued)
38 䡲 AABB TECHNICAL MANUAL
䡲 APPENDIX 1-3
Suggested Quality Control Performance Intervals for Equipment and Reagents*
(Continued)
Equipment and Reagent Frequency of Quality Control
Reagents
Miscellaneous
Betsy W. Jett, MT(ASCP), CQA(ASQ)CQM/OE, Vice President for Quality and Regulatory Affairs, New York
Blood Center, New York, New York; Susan L. Wilkinson, EdD, MS, MT(ASCP)SBB, Interim Department Head,
Analytical and Diagnostic Sciences, College of Allied Health Sciences and Associate Professor Emerita, Uni-
versity of Cincinnati, Cincinnati, Ohio; and Tania L. Motschman, MS, MT(ASCP)SBB, CQA(ASQ), Quality
Director, Esoteric Business Unit, Laboratory Corporation of America, Burlington, North Carolina
The authors have disclosed no conflicts of interest.
39
40 䡲 AABB TECHNICAL MANUAL
professional associations. The contents of ible power supplies and backup generators,
these regulations and guidelines are discussed should be considered to ensure that blood
in more detail in each section of this chapter. components, cellular therapy products, and
US state and local government regulations critical reagents are not compromised during
may have additional safety requirements, in- power failures. The National Electrical Code is
cluding architectural and construction safety routinely used as a national guideline for the
considerations. design of essential electrical distribution sys-
tems, with modifications approved by the local
building authority that has jurisdiction.7
FAC IL I T IE S Heating, ventilation, and air conditioning
Facility Design and Workflow must be adequate for the needs of the facility.
Environmental monitoring systems should be
Effective design and maintenance of facilities considered for laboratories that require posi-
along with the physical organization of work tive or negative air pressure differentials or
activities can help reduce or eliminate many where air filtration systems are used to control
potential hazards. Facility design, workflow, particle levels. The nationally accepted specifi-
and maintenance also affect process efficien- cations for ventilation are published by the
cy, productivity, error rates, employee and cus- American Society of Heating, Refrigerating,
tomer satisfaction, and the quality of products and Air-Conditioning Engineers.8
and services.
During the design phase for a new or ren- Housekeeping
ovated space, the location and flow of person-
nel, materials, and equipment should be con- The workplace should be kept clean and free
sidered in the context of the processes to be of clutter. Work surfaces and equipment
performed. Adequate space must be allotted should be regularly cleaned and disinfected.
for personnel movement, location of supplies Items that may accumulate dust and debris
and large equipment, and private or distrac- should not be stored above clean supplies or
tion-free zones for certain manufacturing work surfaces. Exits and fire safety equipment
tasks (eg, donor interviewing, record review, must not be blocked or obstructed in any way.
and blood component labeling). The facility Receptacles and disposal guidelines for non-
must offer designated “clean” and “dirty” spac- hazardous solid waste and biohazardous,
es and provide for controlled movement of chemical, and radiation waste should be clear-
materials and waste in and out of these areas ly delineated. Housekeeping responsibilities,
to avoid contamination. Chemical fume hoods methods, and schedules should be defined for
and biological safety cabinets (BSCs) should every work area. Written procedures, initial
training, continuing education of personnel,
be located away from drafts and high-traffic
and ongoing monitoring of housekeeping ef-
areas. The number and location of eyewash
fectiveness are essential to safe operations.
stations and emergency showers must also be
considered in planning. Staff handling hazard-
Clean Rooms
ous materials must have ready access to hand-
washing sinks. In some cases, additional spe- Clean-room facilities should be considered for
cial water sources for reagent preparation open processing activities that cannot be ac-
must be provided. The location of very heavy commodated in a BSC. Laboratories that pro-
equipment, such as irradiators, should be tak- cess cellular therapy products may choose to
en into account to ensure that the flooring has adopt clean-room specifications and mainte-
sufficient load-bearing capacity. nance practices to meet the requirements of
Laboratories must be designed with ade- the Food and Drug Administration (FDA) cur-
quate illumination, electrical power, and con- rent good tissue practice regulations.9
veniently located electrical outlets. Emergency International standards for clean rooms
backup power sources, such as uninterrupt- are published by the International Organiza-
CHAPTER 2 Facilities, Work Environment, and Safety 䡲 41
substances they are working with and where communication of the plan is acceptable for
those materials are located in the facility. This facilities with 10 or fewer employees.18
communication is achieved by means of sign-
age, labels on containers, written information, Management Controls
and training programs.
Supervisory personnel must monitor safety
practices in their areas of responsibility. Con-
Engineering Controls and PPE tinuing attention to safety issues should be ad-
If the physical workspace cannot be designed dressed in routine staff meetings and training
to eliminate the potential for exposure to haz- sessions. Periodic audits performed by a safety
ards, appropriate protective gear must be pro- professional increase safety awareness. Man-
vided. Engineering controls are physical plant agement should seek staff input on the design
controls or equipment, such as sprinkler sys- and improvement of the facility’s safety plan.
tems, chemical fume hoods, and needleless The safety program policies, procedures,
systems, that isolate or remove the hazard guidelines, and supporting references should
from the workplace. be documented in writing and made available
PPE is specialized clothing or equipment, to all personnel at risk. These documents
such as gloves, masks, and laboratory coats, should be reviewed on a regular basis and up-
worn by employees for protection against a dated as technology evolves and new informa-
tion becomes available. Work sites and safety
hazard. Employees should remove their PPE,
equipment should be inspected regularly to
such as gloves and laboratory coats, and
ensure compliance and response readiness.
should wash their hands with soap and water
Checklists may be helpful for documenting
when leaving a laboratory area. General guid-
safety inspections and assessing safety pre-
ance on the use of engineering controls and
paredness.3,4,19
PPE is included in Appendix 2-2.
Employee Health Services
Safe Work Practices
Hepatitis Prophylaxis
Employees must be trained in how to work
with hazardous materials in ways that protect All employees routinely exposed to blood must
themselves, their coworkers, and the environ- be offered hepatitis B virus (HBV) vaccine if
ment. Safe work practices are defined as tasks they do not already have HBV-protective anti-
performed in a manner that reduces the likeli- bodies (ie, anti-HBs). OSHA requires that the
hood of exposure to workplace hazards. Gen- vaccine be offered at no cost to all employees
eral recommendations for safe work practices and, if any employee refuses the vaccine, that
are included in Appendix 2-2. the refusal be documented.14
When engineering and work practice controls Employers must provide a system for monitor-
fail, employees must know how to respond ing exposure to certain substances as defined
promptly and appropriately. The purpose of in the OSHA standard if there is reason to be-
advance planning is to control a hazardous sit- lieve that exposure levels routinely exceed the
uation as quickly and safely as possible. Regu- recommended action level.20
lar testing of the emergency response plan
identifies areas for improvement and builds
Medical First Aid and Follow-Up
staff confidence in their ability to respond ef- When requested by a worker who has sus-
fectively in a real emergency. OSHA requires tained known or suspected blood exposure,
facilities with more than 10 employees to have monitoring for HBV, hepatitis C virus (HCV),
a written emergency response plan. Verbal and human immunodeficiency virus (HIV) in-
CHAPTER 2 Facilities, Work Environment, and Safety 䡲 45
fection should be provided with appropriate summaries, and supplemental records must
counseling. In some states, consent is required be preserved for at least 5 years beyond the
for this voluntary testing; rejection of offered calendar year of occurrence. Medical records
testing must be documented. The usual of employees should be preserved for the du-
schedule includes immediate tests of the ration of employment plus 30 years, with few
worker and the source of the potentially infec- exceptions.24
tious material, with follow-up testing of the
worker at intervals after exposure.13,14 All as- Latex Allergies
pects of accident follow-up should be appro-
Adverse reactions associated with latex, pow-
priately documented.
dered gloves, or both include contact dermati-
The Centers for Disease Control and Pre-
tis, allergic dermatitis, urticaria, and anaphy-
vention (CDC) has published recommenda-
laxis. Medical devices that contain latex must
tions for both pre-exposure and post-exposure
bear a caution label. The National Institute for
prophylaxis if the contaminating material is
Occupational Safety and Health (NIOSH) of-
HBV-positive or if this information is un-
fers the following recommendations to prevent
known.21 HBV immune globulin is usually giv-
these allergic reactions25:
en concurrently with HBV vaccine in cases of
penetrating injuries. When administered in
䡲 Make latex-free gloves available as an alter-
accordance with the manufacturer’s direc-
native to latex, and encourage the use of
tions, both products are very safe and carry no
latex-free gloves for activities and work en-
documented risk of infection with HBV, HCV,
vironments where there is minimal risk of
or HIV.21 Post-exposure prophylaxis for HIV is
exposure to infectious materials.
continually evolving; policies are generally
䡲 If latex gloves are used, consider providing
based on Public Health Service recommenda-
reduced-protein and powder-free gloves.
tions and current standards of practice.
䡲 Use good housekeeping practices to remove
latex-containing dust from the workplace.
Reporting Accidents and Injuries
䡲 Use work practices that reduce the chance
When an injury occurs, relevant information of reaction, such as hand washing and
should be documented, including the date, avoiding oil-based hand lotions.
time, and place where the injury occurred; the 䡲 Educate workers about latex allergy.
nature of the injury; descriptions of what hap- 䡲 Evaluate current prevention strategies.
pened from the injured person and any wit- 䡲 Periodically screen high-risk workers for la-
nesses; and first aid or medical attention pro- tex allergy symptoms.
vided. The supervisor should complete any 䡲 If symptoms develop, have workers avoid
accident reports and investigation forms re- direct contact with latex and consult a phy-
quired by the institution’s insurer and worker’s sician about allergy precautions.
compensation agencies. Employers must re-
port fatalities and injuries resulting in the hos-
FIRE PREVENTION
pitalization of three or more employees to
OSHA within 8 hours of the accident.22 Fire prevention relies on a combination of fa-
OSHA requires health service employers cility design that is based on the National Fire
with 11 or more workers to maintain records of Protection Association (NFPA) Life Safety
occupational injuries and illnesses requiring a Code, which identifies processes to maintain
level of care that exceeds the capabilities of a fire protection systems in good working order,
person trained in first aid.23 Initial documenta- and fire safe-work practices.26 The Life Safety
tion must be completed within 6 days of the Code includes both active and passive fire-
incident. Records of first aid provided by a protection systems (eg, alarms, smoke detec-
nonphysician for minor injuries, such as cuts tors, sprinklers, exit lights in corridors, and
or burns, do not need to be retained. All logs, fire-rated barriers).
46 䡲 AABB TECHNICAL MANUAL
through the body, and the length of time that checked for safety. Flexible cords should be se-
current is flowing through the body. Even low- cured to prevent tripping and should be pro-
voltage exposures can lead to serious injury.27 tected from damage from heavy or sharp ob-
jects. Flexible cords should be kept slackened
Training to prevent tension on electrical terminals, and
cords should be checked regularly for cut, bro-
Safety training should be designed to make
ken, or cracked insulation. Extension cords
employees aware of electrical hazards associ-
should not be used in lieu of permanent
ated with receptacles and connectors. This
wiring.
training should also help them recognize po-
tential problems, such as broken receptacles
Emergency Response Plan
and connectors, improper electrical connec-
tions, damaged cords, and inadequate In case of an emergency in which it is not pos-
grounding. sible to decrease the power or disconnect
equipment, the power supply should be shut
Hazard Identification and off from the circuit breaker. If it is not possible
Communication to interrupt the power supply, a nonconduc-
tive material, such as dry wood, should be
The safety plan should address the proper use
used to pry a victim from the source of cur-
of receptacles and connectors. Equipment that
rent.27 Victims must not be touched directly.
does not meet safety standards should be
Emergency first aid for victims of electrical
marked to prevent accidental use.
shock must be sought. Water-based fire extin-
guishers should not be used on electrical fires.
Engineering Controls and PPE
OSHA requires that electrical systems and
BIOSAFET Y
equipment be constructed and installed in a
way that minimizes the potential for work- The facility must define and enforce measures
place hazards. When purchasing equipment, to minimize the risk of exposure to biohazard-
the facility should verify that it bears the mark ous materials in the workplace. Requirements
of an OSHA-approved independent testing published by OSHA (Blood-borne Pathogens
laboratory, such as Underwriters Laborato- Standard) and recommendations published by
ries.28 Adequate working space should be pro- the US DHHS provide the basis for an effective
vided around equipment to allow easy access biosafety plan.13,14,16
for safe operation and maintenance. Ground-
fault circuit interrupters should be installed in Blood-Borne Pathogens Standard
damp or wet areas.
The OSHA Blood-Borne Pathogens Standard is
intended to protect employees in all occupa-
Safe Work Practices
tions where there is a risk of exposure to blood
Electrical safety practices focus on two factors: and other potentially infectious materials. It
1) proper use of electrical equipment and 2) requires the facility to develop an exposure
proper maintenance and repair of this equip- control plan and describes appropriate engi-
ment. Staff should not plug or unplug equip- neering controls, PPE, and work practice con-
ment from an electrical source with wet hands. trols to minimize the risk of exposure. The
Overloading circuits with too many devices standard also requires employers to provide
may cause the current to heat the wires to a HBV vaccinations to any staff members with
very high temperature and generate a fire. occupational exposure, provide medical fol-
Damaged receptacles and faulty electrical low-up care in case of accidental exposure,
equipment must be tagged and removed from and keep records related to accidents and ex-
service until they have been repaired and posures.
48 䡲 AABB TECHNICAL MANUAL
the area, and indicates any special protective amples of blood bank procedures for which a
equipment or work practices required. BSC could be useful. The effectiveness of the
Biohazard warning labels must be placed BSC is a function of directional airflow inward
on containers of regulated waste; refrigerators and downward through a high-efficiency fil-
and freezers containing blood or other poten- ter. Efficacy is reduced by anything that dis-
tially infectious material; and other containers rupts the airflow pattern (eg, arms moving rap-
used to store, transport, or ship blood or other idly in and out of the BSC, rapid movements
potentially infectious materials. Blood compo- behind an employee using the BSC, down-
nents that are labeled to identify their contents drafts from ventilation systems, or open labo-
and have been released for transfusion or oth- ratory doors). Care should be taken not to
er clinical use are exempted. block the front intake and rear exhaust grills.
Performance of the BSC should be certified
Engineering Controls and PPE annually.31
Injuries from contaminated needles and
OSHA requires that hazards be controlled by
other sharp objects (sometimes called
engineering or work practices whenever possi-
“sharps”) continued to be a major concern in
ble.14 Engineering controls for BSL-2 laborato-
health-care settings even after the Blood-
ries include limited access to the laboratory
Borne Pathogens Standard went into effect. In
when work is in progress and BSCs or other
2001, OSHA revised the standard to refer to
containment equipment for work that may in-
engineered sharps injury protections and
volve infectious aerosols or splashes. Hand-
needleless systems.14 The revised standard re-
washing sinks and eyewash stations must be
quires that employers implement appropriate
available. The work space should be designed
new control technologies and safer medical
so that it can be easily cleaned, and bench tops
devices in exposure control plans and that em-
should be impervious to water and resistant to
ployers solicit input from their employees to
chemicals and solvents.
identify, evaluate, and select engineering and
To help prevent exposure or cross-con-
work practice controls. Examples of safer de-
tamination, work area telephones can be
vices are needleless systems and self-sheath-
equipped with speakers to eliminate the need
ing needles in which the sheath is an integral
to pick up the receiver. Computer keyboards
part of the device.
and telephones can be covered with plastic.
Such equipment should be cleaned on a regu-
Decontamination
lar basis and when visibly soiled.
BSCs are primary containment devices Reusable equipment and work surfaces that
for handling moderate-risk and high-risk or- may be contaminated with blood require daily
ganisms. There are three types—Classes I, II, cleaning and decontamination. Obvious spills
and III—with Class III providing the highest on equipment or work surfaces should be
protection to workers. In addition to protect- cleaned up immediately; routine wipe-downs
ing personnel during the handling of biohaz- with disinfectant should occur at the end of
ardous materials, a BSC may be used to pre- each shift or a different frequency that
vent contamination of blood and cellular provides equivalent safety. Equipment that is
therapy products during open processing exposed to blood or other potentially infec-
steps. A comparison of the features and appli- tious material must be decontaminated before
cations of the three classes of cabinets is pro- it is serviced or shipped. When decontamina-
vided in Table 2-2. tion of all or a portion of the equipment is not
BSCs are not required by standard pre- feasible, a biohazard label stating which por-
cautions, but centrifugation of open blood tions remain contaminated should be at-
samples or manipulation of units known to be tached before the equipment is serviced or
positive for HBV surface antigen or HIV are ex- shipped.
50
䡲
TABLE 2-2. Comparison of Classes I, II, and III Biological Safety Cabinets*
Class I Unfiltered room air is drawn into the cabinet. Inward airflow Personal and environmental To enclose equipment (eg, centrifuges)
protects personnel from exposure to materials inside the protection or procedures that may generate aero-
cabinet. Exhaust is high-efficiency particulate air (HEPA) sols
filtered to protect the environment. It maintains airflow at a
minimum velocity of 75 linear feet per minute (lfpm) across
the front opening (face velocity).
Class II (general— Laminar flow (air moving at a constant velocity in one direc- Personal, environmental, and prod- Work with microorganisms assigned to
applies to all types of tion along parallel lines) is used. Room air is drawn into the uct protection Biosafety Levels 1, 2, or 3
Class II cabinets) front grille. HEPA-filtered air is forced downward in a laminar
flow to minimize cross-contamination of materials in the
cabinet. Exhaust is HEPA filtered.
AABB TECHNICAL MANUAL
HEPA filter to provide the downward laminar flow. Face operate because of the volume of con-
velocity = 100 lfpm. ditioned room air being exhausted
Class II, B3 Although similar in design to Type A, the system is ducted See Class II, general Allows for safe manipulation of small
and includes a negative pressure system to keep any possi- quantities of hazardous chemicals and
ble contamination within the cabinet. Face velocity = biologics
100 lfpm.
Class III Cabinet is airtight. Materials are handled with rubber gloves Maximum protection to personnel Work with Biosafety Level 4 micro-
attached to the front of the cabinet. Supply air is HEPA fil- and environment organisms
tered. Exhaust air is double HEPA filtered or may have one
filter and an air incinerator. Materials are brought in and out
of the cabinet either through a dunk tank or a double-door
pass-through box that can be decontaminated. Cabinet is
kept under negative pressure.
Facilities, Work Environment, and Safety
are not able to maintain appropriate hy- unsheathed needles, cleaning scissors, and
giene (eg, patients with tuberculosis). giving cardiopulmonary resuscitation.
In some instances, it may be necessary to
Laboratory Biosafety Precautions collect blood from donors known to pose a
high risk of infectivity (eg, collection of autolo-
Several factors need to be considered when as- gous blood or source plasma for the produc-
sessing the risk of blood exposures among lab- tion of other products, such as vaccines). The
oratory personnel. Some of these factors in- FDA provides guidance on collecting blood
clude the number of specimens processed, from such “high-risk” donors.35,36 The most re-
personnel behaviors, laboratory techniques,
cent regulations and guidelines should be con-
and type of equipment.34 The laboratory direc-
sulted for changes or additions.
tor may wish to institute BSL-3 practices for
procedures that are considered to be higher
Emergency Response Plan
risk than BSL-2. When there is doubt whether
an activity is BSL-2 or BSL-3, the safety precau- Table 2-3 lists steps to take when a spill occurs.
tions for BSL-3 should be followed. BSL-2 pre- Facilities should be prepared to handle both
cautions that are applicable to the laboratory small and large blood spills. Good preparation
setting are summarized in Appendix 2-3. for spill cleanup includes several elements:
Considerations for the Donor Room 䡲 Work areas designed so that cleanup is rela-
tively simple.
The Blood-Borne Pathogens Standard ac-
䡲 A spill kit or cart containing all necessary
knowledges a difference between hospital pa-
supplies and equipment with instructions
tients and healthy donors, in whom the preva-
for their use. It should be placed near areas
lence of infectious disease markers is
where spills are anticipated.
significantly lower. The employer in a volun-
䡲 Responsibility assigned for kit or cart main-
teer blood donation facility may determine
tenance, spill handling, record-keeping,
that routine use of gloves is not required for
and review of significant incidents.
phlebotomy as long as the following condi-
䡲 Personnel trained in cleanup procedures
tions exist14:
and procedures for reporting significant in-
cidents.
䡲 The policy is periodically reevaluated.
䡲 Gloves are made available to those who
Biohazardous Waste
want to use them, and their use is not dis-
couraged. Medical waste is defined as any waste (solid,
䡲 Gloves are required when an employee has semisolid, or liquid) generated in the diagno-
cuts, scratches, or breaks in skin; when sis, treatment, or immunization of human be-
there is a likelihood that contamination ings or animals in related research, produc-
will occur; while an employee is drawing tion, or testing of biologics. Infectious waste
autologous units; while an employee is per- includes disposable equipment, articles, or
forming therapeutic procedures; and dur- substances that may harbor or transmit patho-
ing training in phlebotomy. genic organisms or their toxins. In general, in-
fectious waste should be either incinerated or
Procedures should be assessed for risks of decontaminated before disposal in a sanitary
biohazardous exposures and risks inherent in landfill.
working with a donor or patient during the If state law allows, blood and compo-
screening and donation processes. Some tech- nents, suctioned fluids, excretions, and secre-
niques or procedures are more likely to cause tions may be carefully poured down a drain
injury than others, such as using lancets for connected to a sanitary sewer. Sanitary sewers
finger puncture, handling capillary tubes, may also be used to dispose of other potential-
crushing vials for arm cleaning, handling any ly infectious wastes that can be ground and
54 䡲 AABB TECHNICAL MANUAL
flushed into the sewer. State and local health packaged. The following disposal guidelines
departments should be consulted about laws are recommended37:
and regulations pertaining to disposal of bio-
logic waste into the sewer. 䡲 Identify biohazardous waste consistently;
Laboratories should clearly define what red seamless plastic bags (at least 2 mm
will be considered hazardous waste. For exam- thick) or containers carrying the biohazard
ple, in the blood bank, all items contaminated symbol are recommended.
with liquid or semiliquid blood are biohazard- 䡲 Place bags in a protective container with
ous. Items contaminated with dried blood are closure upward to avoid breakage and leak-
considered hazardous if there is potential for age during storage or transport.
the dried material to flake off during handling. 䡲 Prepare and ship waste transported over
Contaminated sharp objects are always con- public roads according to US Department
sidered hazardous because of the risk for per- of Transportation (DOT) regulations.
cutaneous injury. However, items such as used 䡲 Discard sharps (eg, needles, broken glass,
gloves, swabs, plastic pipettes with excess liq- glass slides, and wafers from sterile con-
uid removed, or gauze contaminated with
necting devices) in rigid, puncture-proof,
small droplets of blood may be considered
leakproof containers.
nonhazardous if the material is dried and will
䡲 Put liquids in leakproof, unbreakable con-
not be released into the environment during
tainers only.
handling.
䡲 Do not compact waste materials.
Guidelines for Biohazardous Waste
Storage areas for infectious material must
Disposal
be secured to reduce accident risk. Infectious
Employees must be trained before handling or waste must never be placed in the public trash
disposing of biohazardous waste, even if it is collection system. Most facilities hire private
CHAPTER 2 Facilities, Work Environment, and Safety 䡲 55
carriers to decontaminate and dispose of in- containing broken glass or other sharp items
fectious or hazardous waste. The facility should be disposed of using a method consis-
should disclose all risks associated with the tent with policies for the disposal of other
waste in their contracts with private compa- sharp or potentially dangerous materials.
nies. The carrier is responsible for complying
with all federal, state, and local laws for bio-
CHEMICAL SAFET Y
hazardous (medical) waste transport, treat-
ment, and disposal. One of the most effective preventive measures
that a facility can take to reduce hazardous
Treating Infectious or Medical Waste chemical exposure is to choose alternative
nonhazardous chemicals whenever possible.
Facilities that incinerate hazardous waste
When the use of hazardous chemicals is re-
must comply with EPA standards of perfor-
quired, purchasing these supplies in small
mance for new stationary sources and emis-
quantities reduces the risks associated with
sion guidelines for existing sources.38 In this
storing excess chemicals and then dealing
regulation, a hospital/medical/infectious waste
with their disposal.
incinerator is any device that combusts any
OSHA requires that facilities using haz-
amount of hospital waste or medical/infec-
ardous chemicals develop a written chemical
tious waste.
Decontamination of biohazardous waste hygiene plan (CHP) and that the plan be ac-
by autoclaving is another common method for cessible to all employees. The CHP should out-
decontamination or inactivation of blood line procedures, equipment, PPE, and work
samples and blood components. The follow- practices that are capable of protecting em-
ing elements are considered in determining ployees from hazardous chemicals used in the
processing time for autoclaving: facility.15,20 The CHP must also provide assur-
ance that equipment and protective devices
䡲 Size of load being autoclaved. are functioning properly and that criteria to
䡲 Type of packaging of item(s) being auto- determine implementation and maintenance
claved. of all aspects of the plan are in place. Employ-
䡲 Density of items being autoclaved. ees must be informed of all chemical hazards
䡲 Number of items in a single autoclave load. in the workplace and be trained to recognize
䡲 Placement of items in the autoclave to al- chemical hazards, protect themselves when
low for steam penetration. working with these chemicals, and know
where to find information about particular
It is useful to place a biologic indicator in hazardous chemicals. Safety audits and annual
the center of loads that vary in size and con- reviews of the CHP are important control steps
tents to evaluate optimal steam penetration to help ensure that safety practices comply
times. The EPA provides detailed information with the policies set forth in the CHP and that
about choosing and operating such equip- the CHP is up to date.
ment.37 Establishing a clear definition of what
For decontamination, material should be constitutes hazardous chemicals is some-
autoclaved for a minimum of 1 hour. For steril- times difficult. Generally, hazardous chemicals
ization, longer treatment times are needed. A pose a significant health risk if an employee is
general rule for decontamination is to process exposed to them or a significant physical risk,
for 1 hour for every 10 pounds of waste. Usual- such as fire or explosion, if they are handled or
ly, decontaminated laboratory wastes can be stored improperly. Categories of health and
disposed of as nonhazardous solid wastes. The physical hazards are listed in Tables 2-4 and
staff should check with the local solid waste 2-5. The NIOSH Pocket Guide to Chemical Haz-
authority to ensure that the facility is in com- ards provides a quick reference for many com-
pliance with regulations for the area. Waste- mon chemicals.39
56 䡲 AABB TECHNICAL MANUAL
Hazard Definition
The facility should identify a qualified new solvent’s hazard category (eg, corrosive or
chemical hygiene officer to be responsible for irritant). However, if the newly introduced sol-
developing guidelines for hazardous materi- vent is a suspected carcinogen and carcino-
als.20 The chemical hygiene officer is also ac- genic hazard training has not been provided,
countable for monitoring and documenting then new training must be conducted for em-
accidents and for initiating process change as ployees with potential exposure. Retraining is
needed. advisable as often as necessary to ensure that
employees understand the hazards linked to
Training the materials with which they work, particu-
larly any chronic and specific target-organ
Employees who may be exposed to hazardous health hazards.
chemicals must be trained before they begin
work in an area in which hazards exist. If a new Hazard Identification and
employee has received prior training, it may Communication
not be necessary to retrain the individual, de-
pending on the employer’s evaluation of the Hazard Communication
new employee’s level of knowledge. New em- Employers must prepare a comprehensive
ployee training is likely to be necessary regard- hazard communication program for all areas
ing such specifics as the location of each rele- in which hazardous chemicals are used to
vant material safety data sheet (MSDS), details complement the CHP and “ensure that the
on chemical labeling, PPE to be used, and site- hazards of all chemicals produced or imported
specific emergency procedures. are classified, and that information concern-
Training must be provided for each new ing the classified hazard is transmitted to em-
physical or health hazard when it is intro- ployers and employees.”15 The program should
duced into the workplace but not for each new include labeling of hazardous chemicals, in-
chemical that falls within a particular hazard structions on when and how to post warning
class.15 For example, if a new solvent is brought labels for chemicals, directions for managing
into the workplace and the solvent has hazards MSDS reports for hazardous chemicals in the
similar to existing chemicals for which training facilities, and employee training. Safety mate-
has already been conducted, then the employ- rials made available to employees should in-
er need only make employees aware of the clude the following:
CHAPTER 2 Facilities, Work Environment, and Safety 䡲 57
Hazard Definition
Combustible or flammable Chemicals that can burn (including combustible and flammable
chemicals liquids, solids, aerosols, and gases)
Compressed gases Gases or mixtures of gases in a container under pressure
Explosives Unstable or reactive chemicals that undergo violent chemical
changes at normal temperatures and pressure
Unstable (reactive) chemicals Chemicals that could be self-reactive under certain conditions
(shocks, pressure, or temperature)
Water-reactive chemicals Chemicals that react with water to release a gas that either is
flammable or presents a health hazard
䡲 The facility’s written CHP. MSDS forms typically include the follow-
䡲 The facility’s written program for hazard ing:
communication.
䡲 Identification of work areas where hazard- 䡲 Identification.
ous chemicals are located. 䡲 Hazard(s) identification.
䡲 Required list of hazardous chemicals and 䡲 Composition/information on ingredients.
the relevant MSDS. (It is the responsibility 䡲 First-aid measures.
of the facility to determine which chemi- 䡲 Fire-fighting measures.
cals may present a hazard to employees. 䡲 Accidental release measures.
䡲 Handling and storage considerations.
This determination should be based on the
䡲 Exposure controls/personal protection in-
quantity of chemical used; physical prop-
formation.
erties, potency, and toxicity of the chemi-
䡲 Physical and chemical properties.
cal; manner in which the chemical is used; 䡲 Stability and reactivity.
and means available to control the release 䡲 Toxicologic information.
of, or exposure to, the chemical.) 䡲 Ecologic information.
䡲 Disposal considerations.
Hazardous Chemical Labeling and 䡲 Transport information.
Signs 䡲 Regulatory information.
䡲 Other information.
The Hazard Communication Standard re-
quires manufacturers of chemicals and haz- At a minimum, hazardous chemical con-
ardous materials to provide the user with basic tainer labels must include the name of the
information about the hazards of these mate- chemical, name and address of the manufac-
rials through product labeling and the MSDS.15 turer, hazard warnings, symbols, designs, and
Employers are required to provide employees other forms of warning to provide visual re-
who are expected to work with these hazard- minders of specific hazards. The label may
ous materials with information about what the refer to any MSDS for additional information.
hazards of the materials are, how to read the Labels applied by the manufacturer must re-
labeling, how to interpret symbols and signs main on containers. The user may add storage
on the labels, and how to read and use the requirements and dates of receipt, opening,
MSDS. and expiration. If chemicals are aliquotted into
58 䡲 AABB TECHNICAL MANUAL
secondary containers, the secondary contain- ucts exceeds that normally found in consumer
er must be labeled with the name of the chem- applications, employees have a right to know
ical and appropriate hazard warnings. Addi- about the properties of such hazardous chemi-
tional information, such as precautionary cals. OSHA does not require or encourage em-
measures, concentration if applicable, and ployers to maintain an MSDS for nonhazard-
date of preparation, are helpful but not man- ous chemicals.
datory.
It is a safe practice to label all containers Engineering Controls and PPE
with their content, even water. Transfer con-
Guidelines for laboratory areas in which haz-
tainers used for temporary storage need not be
ardous chemicals are used or stored must be
labeled if the person performing the transfer
established. Physical facilities, and especially
retains control and intends the containers to
ventilation, must be adequate for the nature
be used immediately. Information regarding
and volume of work conducted. Chemicals
acceptable standards for hazard communica-
must be stored according to chemical compat-
tion labeling is provided by the NFPA40 and the ibility (eg, corrosives, flammables, and oxidiz-
National Paint and Coatings Association.41 ers) and in minimal volumes. Bulk chemicals
Signs meeting OSHA requirements must should be kept outside work areas. NFPA stan-
be posted in areas where hazardous chemicals dards and others provide guidelines for proper
are used. Decisions about where to post warn- storage.4,40,42
ing signs are based on the manufacturer’s rec- Chemical fume hoods are recommended
ommendations regarding the chemical haz- for use with organic solvents, volatile liquids,
ards, the quantity of the chemical in the room and dry chemicals with a significant inhalation
or laboratory, and the potency and toxicity of hazard. 4 Although constructed with safety
the chemical. glass, most fume hood sashes are not designed
to serve as safety shields. Hoods should be po-
MSDS sitioned in an area where there is minimal foot
The MSDS identifies the physical and chemi- traffic to avoid disrupting the airflow and com-
cal properties of a hazardous chemical (eg, promising the containment field.
flash point or vapor pressure), its physical and PPE that may be provided, depending on
health hazards (eg, potential for fire, explo- the hazardous chemicals used, includes chem-
sion, and signs and symptoms of exposure), ical-resistant gloves and aprons, shatterproof
and precautions for the chemical’s safe han- safety goggles, and respirators.
dling and use. Specific instructions in an indi- Emergency showers should be accessible
vidual MSDS take precedence over generic to areas where caustic, corrosive, toxic, flam-
information in the hazardous materials pro- mable, or combustible chemicals are used.4,43
There should be unobstructed access, within
gram.
10 seconds, to the showers from the areas
Employers must maintain copies of each
where hazardous chemicals are used. Safety
required MSDS in the workplace for each haz-
showers should be periodically flushed and
ardous chemical and ensure that MSDS cop-
tested for function, and associated floor drains
ies are readily accessible during each work
should be checked to ensure that drain traps
shift to employees when they are in their work
remain filled with water.
areas. When household consumer products
are used in the workplace in the same manner
Safe Work Practices
that a consumer would use them (ie, when the
duration and frequency of use, and therefore Hazardous material should not be stored or
exposure, are not greater than those that the transported in open containers. Containers
typical consumer would experience), OSHA and their lids or seals should be designed to
does not require that an MSDS be provided to prevent spills or leakage in all reasonably an-
purchasers. However, if exposure to such prod- ticipated conditions. Containers should be
CHAPTER 2 Facilities, Work Environment, and Safety 䡲 59
able to safely store the maximum anticipated cumstances of release, and mitigating fac-
volume and be easy to clean. Surfaces should tors play a role in determining the appro-
be kept clean and dry at all times. priate response. The facility’s emergency
When an employee is working with a response plan should provide guidance on
chemical fume hood, all materials should be how to determine whether a spill is inci-
kept at least 6 inches behind the face opening. dental or requires an emergency response.
The vertical sliding sash should be positioned 䡲 Emergency response releases pose a threat to
at the height specified on the certification health and safety regardless of the circum-
sticker. The airfoil, baffles, and rear ventilation stances surrounding their release. These
slot must not be blocked. Appendix 2-5 lists spills may require evacuation of the imme-
suggestions for working safely with specific diate area. The response typically comes
chemicals from outside the immediate release area by
personnel trained as emergency respond-
Emergency Response Plan ers. These spills include those that involve
immediate danger to life or health, serious
The time to prepare for a chemical spill is be- threat of fire or explosion, and high levels
fore it occurs. A comprehensive employee of toxic substances.
training program should provide each employ-
ee with all tools necessary to act responsibly at Appendix 2-7 addresses the management
the time of a chemical spill. The employee of hazardous chemical spills. Spill cleanup kits
should know response procedures, be able to or carts tailored to the specific hazards present
assess the severity of a chemical spill, know or should be available in each area. The kits or
be able to quickly look up the basic physical carts may contain rubber gloves and aprons,
characteristics of the chemicals, and know shoe covers, goggles, suitable aspirators, gen-
where to find emergency response telephone eral absorbents, neutralizing agents, a broom,
numbers. The employee should be able to as- a dust pan, appropriate trash bags or cans for
sess, stop, and confine the spill; either clean up waste disposal, and cleanup directions. Chem-
the spill or call for a spill cleanup team; and ical absorbents, such as clay absorbents or
follow procedures for reporting the spill. The spill blankets, can be used for cleaning up a
employee must know when to ask for assis- number of chemicals and thus may be easier
tance, when to isolate the area, and where to for employees to use in spill situations.
find cleanup materials. With any spill of a hazardous chemical,
Chemical spills in the workplace can be but especially of a carcinogenic agent, it is es-
categorized as follows44: sential to refer to the MSDS and contact a des-
ignated supervisor or designee trained to han-
䡲 Incidental releases are limited in quantity dle these spills and hazardous waste disposal.4
and toxicity and pose no significant safety Facility environmental health and safety
or health hazard to employees. They may personnel can also offer assistance. The em-
be safely cleaned up by employees familiar ployer must assess the extent of the employee’s
with the hazards of the chemical involved exposure. After an exposure, the employee
in the spill. Waste from the cleanup may be must be given an opportunity for medical con-
classified as hazardous and must be dis- sultation to determine the need for a medical
posed of in the proper fashion. Appendix 2- examination.
6 describes appropriate responses to inci- Another source of workplace hazards is
dental spills. the unexpected release of hazardous vapors
䡲 Releases that may be incidental or may re- into the environment. OSHA has set limits for
quire an emergency response may pose an exposure to hazardous vapors from toxic and
exposure risk to employees depending on hazardous substances.45 The potential risk as-
the circumstances. Considerations such as sociated with a chemical is determined by the
the hazardous substance properties, cir- manufacturer and listed on the MSDS.
60 䡲 AABB TECHNICAL MANUAL
ation safety training before beginning work. and control systems should be readily avail-
This training should address the presence and able in the immediate area. Blood compo-
potential hazards of radioactive materials in nents that have been irradiated are not radio-
the employee’s work area, general health pro- active and pose no threat to the staff or the
tection issues, emergency procedures, and ra- general public.
diation warning signs and labels in use.
Instruction in the following areas is also sug- Safe Work Practices
gested:
Each laboratory should establish policies and
procedures for the safe use of radioactive mate-
䡲 NRC regulations and license conditions.
rials. These policies and procedures should in-
䡲 The importance of observing license condi-
clude requirements for following general labo-
tions and regulations and of reporting vio-
ratory safety principles, appropriate storage of
lations or conditions of unnecessary expo-
radioactive solutions, and proper disposal of
sure. radioactive wastes. Radiation safety can be im-
䡲 Precautions to minimize exposure. proved with the following procedures:
䡲 Interpretation of results of monitoring de-
vices. 䡲 Minimizing the time of exposure by work-
䡲 Requirements for pregnant workers. ing as efficiently as possible.
䡲 Employees’ rights. 䡲 Maximizing the distance from the source of
䡲 Documentation and record-keeping re- the radiation by staying as far from the
quirements. source as possible.
䡲 Maximizing shielding (eg, by using a self-
The need for refresher training is deter- shielded irradiator or wearing a lead apron)
mined by the license agreement between the when working with certain radioactive ma-
NRC and the facility. terials. These requirements are usually stip-
ulated in the license conditions.
Engineering Controls and PPE 䡲 Using good housekeeping practices to min-
Although self-contained blood irradiators imize the spread of radioactivity to uncon-
present little risk to laboratory staff and film trolled areas.
badges are not required for routine operation,
blood establishments with irradiation pro- Emergency Response Plan
grams must be licensed by the NRC.48 Radioactive contamination is the dispersal of
The manufacturer of the blood irradiator radioactive material into or onto areas in
usually accepts responsibility for radiation which it is not intended—for example, the
safety requirements during transportation, in- floor, work areas, equipment, personnel cloth-
stallation, and validation of the unit as part of ing, or personnel skin. The NRC regulations
the purchase contract. The radiation safety of- state that gamma or beta radioactive contami-
ficer can help oversee the installation and vali- nation cannot exceed 2200 disintegrations per
dation processes and should confirm that minute (dpm) per 100 cm2 in the posted (re-
appropriate training, monitoring systems, stricted) area or 220 dpm/100 cm2 in an unre-
procedures, and maintenance protocols are in stricted area, such as a corridor. For alpha
place before use and that they reflect the man- emitters, these values are 220 dpm/100 cm2
ufacturer’s recommendations. Suspected mal- and 22 dpm/100 cm2, respectively.54
functions must be reported immediately so If a spill occurs, employees’ contaminated
that appropriate actions can be initiated. skin surfaces must be washed several times,
Blood irradiators should be located in se- and the radiation safety officer must be noti-
cure areas so that only trained individuals fied immediately to provide further guidance.
have access. Fire protection for the unit must Others must not be allowed to enter the area
also be considered. Automatic fire detection until emergency response personnel arrive.
CHAPTER 2 Facilities, Work Environment, and Safety 䡲 63
KEY POINTS
1. Facilities should be designed and maintained in a way that supports the work being done in
the physical space. Designing the space to accommodate planned work flow, the need to re-
strict certain areas, the movement of materials and waste, equipment location, special air-
handling requirements, and other critical aspects of the operation help ensure safety for
staff and visitors as well as the quality of products and services.
2. The facilities safety program should 1) strive to reduce hazards in the workplace, 2) ensure
that staff are trained to handle known hazards and potential risks, 3) ensure that known
hazards are clearly identified and marked, and 4) describe policies and procedures for
workplace safety and emergency response.
3. Safety programs should address fire, electrical, biological, chemical, and radioactive haz-
ards that may be found in the facility.
4. For each type of hazard, five basic elements that must be covered are 1) training; 2) hazard
identification and communication; 3) engineering controls and PPE; 4) safe work practices,
including waste disposal; and 5) an emergency response plan.
5. Management controls ensure that the safety program is implemented, maintained, and ef-
fective. Management is responsible for 1) developing and communicating the written plan,
2) ensuring implementation of the plan and providing adequate resources for this imple-
mentation, 3) providing access to employee health services for prevention strategies and
treatment of exposures, 4) monitoring compliance and effectiveness, and 5) evaluating and
improving the safety plan.
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cessed January 6, 2013).] and health for electrical trades student manu-
14. Code of federal regulations. Title 29, CFR Part al. ( January 2002) NIOSH Publication No.
1910.1030. Washington, DC: US Government 2002-123. Washington, DC: National Institute
Printing Office, 2013 (revised annually). for Occupational Safety and Health, 2002.
15. Code of federal regulations. Title 29, CFR Part 28. OSHA technical manual: TED 1-0.15A. Wash-
1910.1200. Washington, DC: US Government ington, DC: US Department of Labor, 1999.
Printing Office, 2013 (revised annually). 29. Enforcement procedures for the occupational
16. US Department of Health and Human Servic- exposure to bloodborne pathogens. Directive
es. Biosafety in microbiological and biomedi- CPL 02-02-069. Washington, DC: US Depart-
cal laboratories. 5th ed. Washington, DC: US ment of Labor, 2001.
Government Printing Office, 2009. 30. US Department of Health and Human Servic-
17. Bernard B, ed. Musculoskeletal disorders and es. Primary containment for biohazards: Se-
workplace factors: A critical review of epide- lection, installation, and use of biological safe-
miologic evidence for work-related musculo- ty cabinets. Washington, DC: US Government
skeletal disorders of the neck, upper extremity, Printing Office, 2009. [Available at http://
and low back. NIOSH publication no. 97-141. www.cdc.gov/biosafety/publications (ac-
Washington, DC: National Institute for Occu- cessed January 6, 2013).]
pational Safety and Health, 1997. 31. Richmond JY. Safe practices and procedures
18. Code of federal regulations. Title 29, CFR Part for working with human specimens in bio-
1910.38. Washington, DC: US Government medical research laboratories. J Clin Immuno-
Printing Office, 2013 (revised annually). assay 1988;11:115-19.
19. Wagner KD, ed. Environmental management 32. ATP— tested actively registered hospital disin-
in healthcare facilities. Philadelphia: WB fectant products. (March 2010) Washington,
Saunders, 1998. DC: US Environmental Protection Agency,
20. Code of federal regulations. Title 29, CFR Part 2012. [Available at http://www.epa.gov/
1910.1450. Washington, DC: US Government oppad001/chemregindex.htm (accessed Janu-
Printing Office, 2013 (revised annually). ary 6, 2013).]
66 䡲 AABB TECHNICAL MANUAL
33. Rutala WA. APIC guideline for selection and 44. Inspection procedures for 29 CFR 1910.120
use of disinfectants. Am J Infect Control 1996; and 1926.65, paragraph (q): Emergency re-
24:313-42. sponse to hazardous substance releases.
34. Evans MR, Henderson DK, Bennett JE. Poten- OSHA Directive CPL 02-02-073. Washington,
tial for laboratory exposures to biohazardous DC: Occupational Safety and Health Adminis-
agents found in blood. Am J Public Health tration, 2007.
1990;80:423-7. 45. Code of federal regulations. Title 29, CFR Part
35. Memorandum: Guideline for collection of 1910.1000. Washington, DC: US Government
blood products from donors with positive tests Printing Office, 2013 (revised annually).
for infectious disease markers (“high risk” do- 46. Cook SS. Selection and installation of self-
nors). (October 26, 1989) Silver Spring, MD: contained irradiators. In: Butch S, Tiehen A,
CBER Office of Communication, Outreach, eds. Blood irradiation: A user’s guide. Bethes-
and Development, 1989. da, MD: AABB Press, 1996:19-40.
36. Memorandum: Revision to 26 October 1989 47. Beir V. Health effects of exposure to low levels
guidelines for collection of blood or blood of ionizing radiation. Washington, DC: Nation-
products from donors with positive tests for al Academy Press, 1990:1-8.
infectious disease markers (“high-risk” do- 48. Regulatory Guide 8.29: Instruction concerning
nors). Silver Spring, MD: CBER Office of Com- risks from occupational radiation exposure.
munication, Outreach, and Development, Washington, DC: Nuclear Regulatory Commis-
1991. [Available at http://www.fda.gov/biolog sion, 1996.
icsbloodvaccines/guidancecomplianceregula 49. NCRP Report No. 115: Risk estimates for radia-
toryinformation/other recommendations for tion protection: Recommendations of the Na-
manufacturers/memorandumtobloodestab tional Council on Radiation Protection and
lishments.default.htm.] Measurements. Bethesda, MD: National
37. US Environmental Protection Agency. EPA Council on Radiation Protection and Measure-
guide for infectious waste management. EPA/ ments, 1993.
530-SW-86-014. NTIS #PB86-199130. Wash- 50. NCRP Report No. 105: Radiation protection for
ington, DC: National Technical Information medical and allied health personnel: Recom-
Service, 1986. mendations of the National Council on Radia-
38. Code of federal regulations. Title 40, CFR Part tion Protection and Measurements. Bethesda,
264. Washington, DC: US Government Print- MD: National Council on Radiation Protection
ing Office, 2013 (revised annually). and Measurements, 1989.
39. NIOSH pocket guide to chemical hazards. 51. EA-05 090. Enforcement action: Order impos-
Washington, DC: National Institute for Occu- ing increased controls (licensees authorized to
pational Safety and Health, 2010. [Available at possess radioactive material quantities of con-
http://www.cdc.gov/niosh/npg (accessed No- cern). (November 14, 2005) Rockville, MD: US
vember 8, 2010).] Nuclear Regulatory Commission, 2005.
40. NFPA 704—Standard for the identification of 52. RIS 2007-14. Fingerprinting requirements for
the hazards of materials for emergency re- licensees implementing the increased control
sponse. Quincy, MA: National Fire Protection order. (June 5, 2007) Rockville, MD: US Nucle-
Association, 2012. ar Regulatory Commission, 2007.
41. Hazardous Materials Identification System. 53. US Nuclear Regulatory Commission Regulato-
HMIS implementation manual. 3rd ed. Neen- ry Guide 8.13: Instruction concerning prenatal
ah, WI: JJ Keller and Associates, Inc., 2001. radiation exposure. Washington, DC: NRC,
42. Lisella FS, Thomasston SW. Chemical safety in 1999.
the microbiology laboratory. In: Fleming DO, 54. Nuclear Regulatory Commission regulatory
Richardson JH, Tulis JJ, Vesley D, eds. Labora- guide 8.23: Radiation surveys at medical insti-
tory safety, principles, and practices. 2nd ed. tutions. Washington, DC: NRC, 1981.
Washington, DC: American Society for Micro- 55. Code of federal regulations. Title 49, CFR Parts
biology Press, 1995:247-54. 171.22. Washington, DC: US Government
43. American national standards for emergency Printing Office, 2014 (revised annually).
eyewash and shower equipment. ANSI Z358.1- 56. Dangerous goods regulations manual. 54th ed.
2009. New York: American National Standards Montreal, PQ, Canada: International Air Trans-
Institute, 2009. port Association, 2014 (revised annually).
CHAPTER 2 Facilities, Work Environment, and Safety 䡲 67
57. United States Code. Pollution prevention act. Clinical and Laboratory Standards Institute,
42 U.S.C. §§13101 and 13102 et seq. 2011.
58. Clinical laboratory waste management. Ap-
proved guideline. 3rd ed. GP05-A3. Wayne, PA:
68 䡲 AABB TECHNICAL MANUAL
䡲 APPENDIX 2-1
Safety Regulations and Recommendations Applicable to Health-Care Settings
Agency/Organization Reference Title
䡲 APPENDIX 2-1
Safety Regulations and Recommendations Applicable to Health-Care Settings
(Continued)
Agency/Organization Reference Title
䡲 APPENDIX 2-2
General Guidelines for Safe Work Practices, Personal Protective Equipment, and
Engineering Controls
UNIFORMS AND LABORATORY COATS
Personnel should wear closed laboratory coats or full aprons over long-sleeved uniforms or gowns when they
are exposed to blood, corrosive chemicals, or carcinogens. The material of required coverings should be
appropriate for the type and amount of hazard exposure. Plastic disposable aprons may be worn over cotton
coats when there is a high probability of large spills or splashing of blood and body fluids; nitrile rubber
aprons may be preferred when caustic chemicals are poured.
Protective coverings should be removed before the employee leaves the work area and should be discarded or
stored away from heat sources and clean clothing. Contaminated clothing should be removed promptly,
placed in a suitable container, and laundered or discarded as potentially infectious. Home laundering of gar-
ments worn in Biosafety Level 2 areas is not permitted because unpredictable methods of transportation and
handling can spread contamination and home laundering techniques may not be effective.1
GLOVES
Gloves or equivalent barriers should be used whenever tasks are likely to involve exposure to hazardous materials.
TYPES OF GLOVES
The following guidelines should be used to determine when gloves are necessary1:
䡲 For donor phlebotomy when the health-care worker has cuts, scratches, or other breaks in his or her skin.
䡲 For phlebotomy of autologous donors or patients (eg, therapeutic apheresis procedures or intraoperative red
cell collection).
䡲 For persons who are receiving training in phlebotomy.
䡲 When handling open blood containers or specimens.
䡲 When collecting or handling blood or specimens from patients or donors known to be infected with a blood-
borne pathogen.
䡲 When examining mucous membranes or open skin lesions.
䡲 When handling corrosive chemicals and radioactive materials.
䡲 When cleaning up spills or handling waste materials.
䡲 When the likelihood of exposure cannot be assessed because of lack of experience with a procedure or situation.
CHAPTER 2 Facilities, Work Environment, and Safety 䡲 71
䡲 APPENDIX 2-2
General Guidelines for Safe Work Practices, Personal Protective Equipment, and
Engineering Controls (Continued)
The Occupational Safety and Health Administration (OSHA) does not require the routine use of gloves by phle-
botomists working with healthy prescreened donors or the changing of unsoiled gloves between donors if
gloves are worn.1,2 Experience has shown that the phlebotomy process is low risk because donors typically
have low rates of infectious disease markers. Also, exposure to blood is rare during routine phlebotomy, and
other alternatives can be used to provide barrier protection, such as using a folded gauze pad to control any
blood flow when the needle is removed from the donor’s arm.
Employers whose policies and procedures do not require routine gloving should periodically reevaluate the
potential need for gloves. Employees should never be discouraged from using gloves, and gloves should
always be available.
Guidelines for the safe use of gloves by employees include the following3,4:
䡲 Securely bandage or cover open skin lesions on hands and arms before putting on gloves.
䡲 Change gloves immediately if they are torn, punctured, or contaminated; after handling high-risk samples; or
after performing a physical examination (eg, on an apheresis donor).
䡲 Remove gloves by keeping their outside surfaces in contact only with outside and by turning the glove inside
out while taking it off.
䡲 Use gloves only when needed, and avoid touching clean surfaces such as telephones, doorknobs, or com-
puter terminals with gloves.
䡲 Change gloves between patient contacts. Unsoiled gloves need not be changed between donors.
䡲 Wash hands with soap or other suitable disinfectant after removing gloves.
䡲 Do not wash or disinfect surgical or examination gloves for reuse. Washing with surfactants may cause
“wicking” (ie, enhanced penetration of liquids through undetected holes in the glove). Disinfecting agents
may cause deterioration of gloves.
䡲 Use only water-based hand lotions with gloves, if needed; oil-based products cause minute cracks in latex.
Where there is a risk of blood or chemical splashes, the eyes and mucous membranes of the mouth and nose
should be protected.5 Permanent shields fixed as a part of equipment or bench design are preferred (eg,
splash barriers attached to tubing sealers or centrifuge cabinets). All barriers should be cleaned and disin-
fected on a regular basis.
Safety glasses alone provide impact protection from projectiles but do not adequately protect eyes from bio-
hazardous or chemical splashes. Full-face shields or masks and safety goggles are recommended when per-
manent shields cannot be used. Many designs are commercially available; eliciting staff input on comfort and
selection can increase use.
Masks should be worn whenever there is danger from inhalation. Simple, disposable dust masks are adequate
for handling dry chemicals, but respirators with organic vapor filters are preferred for areas where noxious
fumes are produced (eg, for cleaning up spills of noxious materials). Respirators should be fitted to their
wearers and checked annually.
(Continued)
72 䡲 AABB TECHNICAL MANUAL
䡲 APPENDIX 2-2
General Guidelines for Safe Work Practices, Personal Protective Equipment, and
Engineering Controls (Continued)
HAND WASHING
Frequent, thorough hand washing is the first line of defense in infection control. Blood-borne pathogens gen-
erally do not penetrate intact skin, so immediate removal reduces the likelihood of transfer to a mucous mem-
brane or broken skin area or of transmission to others. Thorough washing of hands (and arms) also reduces
the risks from exposure to hazardous chemicals and radioactive materials.
Employees should always wash their hands before leaving a restricted work area or using a biosafety cabinet,
between medical examinations, immediately after becoming soiled with blood or hazardous materials, after
removing gloves, or after using the toilet. Washing hands thoroughly before touching contact lenses or apply-
ing cosmetics is essential.
OSHA allows the use of waterless antiseptic solutions for hand washing as an interim method.2 These solu-
tions are useful for mobile donor collections or in areas where water is not readily available for cleanup pur-
poses. If such methods are used, however, hands must be washed with soap and running water as soon as
possible thereafter. Because there is no listing or registration of acceptable hand-wipe products similar to the
one that the Environmental Protection Agency maintains for surface disinfectants, consumers should request
data from the manufacturer to support advertising claims.
EYEWASHES
Laboratory areas that contain hazardous chemicals must be equipped with eyewash stations.3,6 Unobstructed
access within a 1- second walk from the location of chemical use must be provided for these stations. Eye-
washes must operate so that both of the user’s hands are free to hold open the eyes. Procedures and indica-
tions for use must be posted, and routine function checks must be performed. Testing eyewash fountains
weekly helps ensure proper function and flushes out stagnant water. Portable eyewash systems are allowed
only if they can deliver flushing fluid to the eyes at a rate of at least 1.5 liters per minute for 15 minutes. They
should be monitored routinely to ensure the purity of their contents.
Employees should be trained in the proper use of eyewash devices, although prevention—through consistent
and appropriate use of safety glasses or shields—is preferred. If a splash occurs, the employee should be
directed to keep his or her eyelids open and to use the eyewash according to procedures, or the employee
should go to the nearest sink and direct a steady, tepid stream of water into his or her eyes. Solutions other
than water should be used only in accordance with a physician’s direction.
After eyes are adequately flushed (many facilities recommend 15 minutes), follow-up medical care should be
sought, especially if pain or redness develops. Whether washing the eyes is effective in preventing infection
has not been demonstrated, but it is considered desirable when accidents occur.
1. Code of federal regulations. Title 29, CFR Part 1910.1030. Fed Regist 1991;56:64175-82.
2. Occupational Safety and Health Administration. Enforcement procedures for the occupational exposure to bloodborne pathogens.
OSHA Instruction CPL 02-02-069. Washington, DC: US Government Printing Office, 2001.
3. Clinical laboratory safety: Approved guideline. 3rd ed (GP17-A3). Wayne, PA: Clinical and Laboratory Standards Institute, 2012.
4. Medical glove powder report. (September 1997) Rockville, MD: Food and Drug Administration, 2009. [Available at http://www.fda.
gov/MedicalDevices/DeviceRegulationand Guidance/GuidanceDocuments/ucm113316.htm (accessed January 6, 2013).]
5. Inspection checklist: General laboratory. Chicago: College of American Pathologists, 2012.
6. American national standards for emergency eyewash and shower equipment. ANSI Z358.1-2009. New York: American National Stan-
dards Institute, 2009.
CHAPTER 2 Facilities, Work Environment, and Safety 䡲 73
䡲 APPENDIX 2-3
Biosafety Level 2 Precautions
Biosafety Level 2 precautions as applied in the blood establishment setting include at least the following1,2:
䡲 High-risk activities are appropriately segregated from lower-risk activities, and the boundaries are clearly
defined.
䡲 Bench tops are easily cleaned and are decontaminated daily with a hospital disinfectant approved by the Envi-
ronmental Protection Agency.
䡲 Laboratory rooms have closable doors and sinks. An air system with no recirculation is preferred but not
required.
䡲 Workers are required to perform procedures that create aerosols (eg, opening evacuated tubes, centrifuging,
mixing, or sonicating) in a biological safety cabinet or equivalent or to wear masks and goggles in addition to
gloves and gowns during such procedures. (Note: Open tubes of blood should not be centrifuged. If whole
units of blood or plasma are centrifuged, overwrapping is recommended to contain leaks.)
䡲 Gowns and gloves are used routinely and in accordance with general safety guidelines. Face shields or their
equivalents are used where there is a risk from splashing.
䡲 Mouth pipetting is prohibited.
䡲 No eating, drinking, smoking, applying cosmetics, or manipulating contact lenses occurs in the work area. All
food and drink are stored outside the restricted area, and laboratory glassware is never used for food or
drink. Personnel are instructed to avoid touching their face, ears, mouth, eyes, or nose with their hands or
other objects, such as pencils and telephones.
䡲 Needles and syringes are used and disposed of in a safe manner. Needles must never be bent, broken,
sheared, replaced in a sheath, or detached from a syringe before being placed in puncture-proof, leakproof
containers for controlled disposal. Procedures are designed to minimize exposure to sharp objects.
䡲 All blood specimens are placed in well-constructed containers with secure lids to prevent leaking during
transport. Blood is packaged for shipment in accordance with regulatory agency requirements for etiologic
agents or clinical specimens, as appropriate.
䡲 Infectious waste is not compacted and is decontaminated before its disposal in leakproof containers. Proper
packaging includes double, seamless, tear-resistant, orange or red bags that are enclosed in protective car-
tons. Both the cartons and the bags inside display the biohazard symbol. Throughout delivery to an incinera-
tor or autoclave, waste is handled only by suitably trained persons. If a waste management contractor is
used, the agreement should clearly define the respective responsibilities of the staff and the contractor.
䡲 Equipment to be repaired or submitted for preventive maintenance, if potentially contaminated with blood,
must be decontaminated before its release to a repair technician.
䡲 Accidental exposure to suspected or actual hazardous material is reported to the laboratory director or
responsible person immediately.
1. Clinical laboratory safety: Approved guideline. 3rd ed (GP17-A3). Wayne, PA: Clinical and Laboratory Standards Institute, 2012.
2. Fleming DO. Laboratory biosafety practices. In: Fleming DO, Richardson JH, Tulis JJ, Vesley DD, eds. Laboratory safety, principles,
and practices. 2nd ed. Washington, DC: American Society for Microbiology Press, 1995:203-18.
74 䡲 AABB TECHNICAL MANUAL
䡲 APPENDIX 2-4
Sample List of Hazardous Chemicals that May Be Encountered in a Blood Bank
Chemical Hazard
䡲 APPENDIX 2-4
Sample List of Hazardous Chemicals that May Be Encountered in a Blood Bank
(Continued)
Chemical Hazard
䡲 APPENDIX 2-5
Chemical Categories and How to Work Safely with Them
Chemical Category Hazard Precautions Special Treatment
Acids, alkalis, and corro- Irritation, severe burns, During transport, protect Store concentrated acids
sive compounds tissue damage large containers with in acid safety cabinets.
plastic or rubber bucket Limit volumes of con-
carriers. centrated acids to 1
During pouring, wear eye liter per container.
protection and chemi- Post cautions for materi-
cal-resistant-rated als in the area.
gloves and gowns as Report changes in
recommended. appearance (perchloric
Always add acid to water, acid may be explosive if
never add water to acid. it becomes yellowish or
When working with large brown) to chemical
jugs, have one hand on safety officer.
the neck and the other
hand at the base, and
position them away
from the face.
Acrylamide Neurotoxic, carcino- Wear chemically rated Store in a chemical cabi-
genic, absorbed gloves. net.
through the skin Wash hands immediately
after exposure.
Compressed gases Explosive Label contents. Transport using hand
Leave valve safety covers trucks or dollies.
on until use. Place cylinders in a stand
Open valves slowly for or secure them to pre-
use. vent tipping over.
Label empty tanks. Store in well-ventilated
separate rooms.
Do not store oxygen
close to combustible
gas or solvents.
Check connections for
leaks using soapy
water.
CHAPTER 2 Facilities, Work Environment, and Safety 䡲 77
䡲 APPENDIX 2-5
Chemical Categories and How to Work Safely with Them (Continued)
Chemical Category Hazard Precautions Special Treatment
Flammable solvents Classified according to Use extreme caution Make every attempt to
flash point—see mate- when handling. replace hazardous
rial safety data sheet, Post “No Smoking” signs materials with less haz-
classified according to in working area. ardous materials
volatility Keep a fire extinguisher Store containers larger
and solvent cleanup kit than 1 gallon in a flam-
in the room. mable solvent storage
Pour volatile solvents room or a fire safety
under a suitable hood. cabinet.
Use eye protection and Ground metal containers
chemical-resistant neo- by connecting the can
prene gloves when to a water pipe or
pouring. ground connection. If
No flame or other source the recipient container
of possible ignition is also metal, it should
should be in or near be electrically con-
areas where flammable nected to the delivery
solvents are being container during pour-
poured. ing.
Label as “flammable.”
Liquid nitrogen Freeze injury, severe Use heavy insulated The tanks should be
burns to skin or eyes gloves and goggles securely supported to
when working with liq- avoid being tipped
uid nitrogen. over.
The final container of liq-
uid nitrogen (freezing
unit) must be securely
supported to avoid
being tipped over.
78 䡲 AABB TECHNICAL MANUAL
䡲 APPENDIX 2-6
Incidental Spill Response*
Chemicals Hazards PPE Control Materials
䡲 APPENDIX 2-6
Incidental Spill Response* (Continued)
Chemicals Hazards PPE Control Materials
Flammable gases Simple asphyxiant Face shield and goggles, Hand truck (to transport
Acetylene (displaces air). neoprene boots, double cylinder outdoors if
Oxygen gases Inhaled vapors have an set of gloves, coveralls needed)
Butane anesthetic potential. with hood and feet Soap solution (to check
Propane Flammable gases pose an for leaks)
extreme fire and explo-
sion hazard.
Release can create an
oxygen-deficient
atmosphere.
Flammable liquids Vapors are harmful if Gloves (double set of 4H Absorbent material
Acetone inhaled (central ner- undergloves and butyl Absorbent boom
Xylene vous system depres- or nitrile overgloves), Absorbent pillow
Methyl alcohol toluene sants). impervious apron or Shovel or paddle (non-
Ethyl alcohol Liquid is harmful if coveralls, goggles or metal, nonsparking)
Other alcohols absorbed through the face shield, impervious Drain mat
skin. foot covers Leakproof container
Substances are extremely
flammable.
Liquid evaporates to form
flammable vapors.
Formaldehyde and Vapors are harmful if Gloves (double set of Aldehyde neutralizer or
glutaraldehyde inhaled; liquids are 4H undergloves and absorbent
4% formaldehyde harmful if absorbed butyl or nitrile over- Absorbent boom
37% formaldehyde through skin. gloves), impervious Absorbent pillow
10% formalin Substances are irritants apron or coveralls, Shovel or paddle
2% glutaraldehyde to skin, eyes, and goggles, impervious (nonsparking)
respiratory tract. foot covers Drain mat
Formaldehyde is a sus- Leakproof container
pected human carcino-
gen.
37% formaldehyde
should be kept away
from heat, sparks, and
flames.
(Continued)
80 䡲 AABB TECHNICAL MANUAL
䡲 APPENDIX 2-6
Incidental Spill Response* (Continued)
Chemicals Hazards PPE Control Materials
Mercury Mercury and mercury Gloves (double set of 4H Mercury vacuum or spill
Cantor tubes vapors are rapidly undergloves and butyl kit
Thermometers absorbed in respira- or nitrile overgloves), Scoop
Barometers tory tract, gastrointesti- impervious apron or Aspirator
Sphygmomanometers nal (GI) tract, or skin. coveralls, goggles, Hazardous waste contain-
Mercuric chloride Short-term exposure may impervious foot covers ers
cause erosion of respi- Mercury indicator powder
ratory or GI tracts, nau- Absorbent material
sea, vomiting, bloody Spatula
diarrhea, shock, head- Disposable towels
ache, or metallic taste. Sponge with amalgam
Inhalation of high con- Vapor suppressor
centrations can cause
pneumonitis, chest
pain, dyspnea, cough-
ing, stomatitis, gingivi-
tis, and salivation.
Avoid evaporation of
mercury from tiny
globules by quick and
thorough cleaning.
*This list of physical and health hazards in not intended as a substitute for the material safety data sheet (MSDS) informa-
tion. In case of a spill or if any questions arise, always refer to the chemical-specific MSDS for more complete information.
CHAPTER 2 Facilities, Work Environment, and Safety 䡲 81
䡲 APPENDIX 2-7
Managing Hazardous Chemical Spills
Actions Instructions for Hazardous Liquids, Gases, and Mercury
Deenergize. Liquids: For 37% formaldehyde, deenergize and remove all sources of igni-
tion within 10 feet of spilled hazardous material. For flammable liquids,
remove all sources of ignition.
Gases: Remove all sources of heat and ignition within 50 feet for flammable
gases.
Remove all sources of heat and ignition for nitrous oxide release.
Isolate, evacuate, and secure Isolate the spill area and evacuate everyone from the area surrounding the
the area. spill except those responsible for cleaning up the spill. (For mercury, evacuate
within 10 feet for small spills or 20 feet for large spills.) Secure the area.
Have the appropriate per- See Appendix 2-2 for recommended PPE.
sonal protective equipment
(PPE).
Contain the spill. Liquids or mercury: Stop the source of spill if possible.
Gases: Assess the scene; consider the circumstances of the release (quantity,
location, and ventilation). If circumstances indicate that it is an emergency
response release, make appropriate notifications; if the release is determined
to be incidental, contact the supplier for assistance.
Confine the spill. Liquids: Confine the spill to the initial spill area using appropriate control
equipment and material. For flammable liquids, dike off all drains.
Gases: Follow the supplier’s suggestions or request outside assistance.
Mercury: Use appropriate materials to confine the spill (see Appendix 2-6).
Expel mercury from the aspirator bulb into a leakproof container, if applicable.
Neutralize the spill Liquids: Apply appropriate control materials to neutralize the chemical (see
Appendix 2-6).
Mercury: Use a mercury spill kit if needed.
Clean up the spill. Liquids: Scoop up solidified materials, booms, pillows, and any other materi-
als. Put used materials into a leakproof container. Label the container with the
name of the hazardous materials. Wipe up residual material. Wipe the spill
area surface three times with a detergent solution. Rinse the areas with clean
water. Collect the supplies used (eg, goggles or shovels) and remove gross
contamination; place equipment to be washed and decontaminated into a
separate container.
Gases: Follow the supplier’s suggestions or request outside assistance.
Mercury: Vacuum up the spill using a mercury vacuum, or scoop up mercury
paste after neutralization and collect the paste in a designated container. Use
a sponge and detergent to wipe and clean the spill surface three times to
remove absorbent. Collect all contaminated disposal equipment and put it
into a hazardous waste container. Collect supplies and remove gross contam-
ination; place equipment that will be thoroughly washed and decontaminated
into a separate container.
(Continued)
82 䡲 AABB TECHNICAL MANUAL
䡲 APPENDIX 2-7
Managing Hazardous Chemical Spills (Continued)
Actions Instructions for Hazardous Liquids, Gases, and Mercury
Dispose. Liquids: Dispose of material that was neutralized as solid waste. Follow the
facility’s procedures for disposal. For flammable liquids, check with the facil-
ity safety officer for appropriate waste determination.
Gases: The manufacturer or supplier will instruct the facility about disposal if
applicable.
Mercury: Label with appropriate hazardous waste label and Department of
Transportation diamond label.
Report. Follow appropriate spill documentation and reporting procedures. Investigate
the spill; perform a root cause analysis if needed. Act on opportunities for
improving safety.
C h a p t e r 3
Glenn Ramsey, MD
I N T H E U N I T E D S T A T E S , when fed-
I eral laws are enacted, they are published
chronologically as statutes and placed into the
The FDA regulates drugs and medical de-
vices under the Food, Drug, and Cosmetic Act
(USC Title 21, Sections 301-399d). 5 Blood
appropriate 1 of 50 subject areas (titles) of the products are defined as drugs because they are
United States Code (USC).1 The government intended to cure, mitigate, treat, or prevent
agency responsible for the law—for blood- disease (21 USC 321).6,7 The law requires blood
related laws, the Food and Drug Administra- product manufacturers to register with the
tion (FDA)—then writes regulations (rules) to FDA, obtain biologics licenses, and follow cur-
enforce the law. Proposed rules for public rent good manufacturing practice (cGMP) reg-
comment and final rules, along with back- ulations. It also prohibits adulteration and
ground and interpretive information, are pub- misbranding, authorizes inspections, and de-
lished chronologically in the daily Federal Reg- fines civil and criminal penalties for violations.
ister.2 The current edition of all rules is collated The act controls the use of unapproved drugs
annually in the appropriate title of the Code of and devices during their investigational phas-
Federal Regulations (CFR); Title 21 addresses es and public health emergencies.
food and drugs, and Title 42 focuses on health Medical devices include instruments, in-
care.3 vitro reagents, and their parts and accessories
Some rules have associated guidance for the diagnosis and treatment of disease. The
documents to provide current thinking about FDA classifies devices as Class I, II, or III, in as-
regulatory topics. Guidance documents con- cending order of associated risk [21 USC
tain recommendations, not requirements, but 360(c)].8,9 Class I devices have no potential un-
they are usually followed by industry. Memo- reasonable risk. Class II devices must meet
randa to blood establishments were issued be- performance standards beyond basic FDA reg-
fore 1998, but some are still active references. ulations. Some Class I devices and most Class
These publications can be accessed from the II devices must be cleared by the FDA based on
FDA blood and blood products webpage.4 substantial equivalence with another device
Glenn Ramsey, MD, Medical Director, Blood Banks, Northwestern Memorial Hospital and Ann & Robert H.
Lurie Children’s Hospital of Chicago, and Department of Pathology, Feinberg School of Medicine, Northwest-
ern University, Chicago, Illinois
The author has disclosed no conflicts of interest.
83
84 䡲 AABB TECHNICAL MANUAL
already on the market. This process is called several forums are offered for input from the
510(k) clearance, referring to the applicable public and regulated groups.16 Proposed rules
section of the act describing the submission of and draft guidance documents are published
applications to the FDA for such devices [21 with an invitation for written comments,
USC 360(k)].8 Class III devices pose potential which are filed in public dockets.17 When final
unreasonable risk and require specific pre- rules are published in the Federal Register, the
market approval from the FDA, as do unprece- accompanying commentaries offer response
dented new devices before their class is deter- to key questions. The FDA also receives peti-
mined. tions to write or change regulations. Expert
opinions on current issues are sought from
several advisory committees, including the
B IOLO G IC A L P RO DU C TS
FDA’s committees on blood products and on
Biological products come from living sources cellular, tissue, and gene therapies, and the
and include blood, blood components, deriva- Department of Health and Human Services
tives, therapeutic serum, and vaccines appli- (DHHS) Committee on Blood Safety and Avail-
cable to the prevention or treatment of dis- ability. Public meetings hosted by the FDA on
ease. They are regulated by Section 351 (42 selected topics provide an additional opportu-
USC 262) of the Public Health Service Act, nity for input.
which addresses licensure, labeling, inspec- Facilities may apply to CBER for approval
tions, recalls, and penalties for violations in of exceptions to the regulations for blood
producing biological drugs. This set of regula- products or alternative procedures. These ap-
tions provides the core of federal law that is provals are periodically published, although
most specific to blood products.10 Section 361, the circumstances for these approvals may not
Regulations to Control Communicable Diseas- apply to other facilities.18
es (42 USC 264), grants the Surgeon General
inspection and quarantine powers to prevent LICENSURE AND
the spread of infectious diseases and is in-
REGISTRATION
voked in the regulation of tissues (see below).11
The FDA’s key regulations enforcing the Blood establishments are classified into three
Food and Drug Act and the Public Health Ser- categories by the FDA: 1) licensure and regis-
vice Act are in Title 21 of the CFR, Food and tration for interstate commerce, 2) registration
Drugs, and in particular, Parts 200 through 299 for manufacturers not involved in interstate
for drugs, 600 through 680 for biologicals, 800 commerce, and 3) exemption from registration
through 898 for devices, and 1270 through for transfusion services as described in the
1271 for tissues (Table 3-1).12 The FDA website regulations.19 Licensed and/or registered facil-
has links to pertinent laws in the USC and reg- ities are inspected by the FDA. For transfusion
ulations in the CFR.13 services that perform minimal manufacturing
Within the FDA, the Center for Biologics and are certified for reimbursement by the
Evaluation and Research (CBER) regulates Centers for Medicare and Medicaid Services
blood products and most other biological (CMS), the FDA accepts CMS-sanctioned labo-
therapies.14 The Center for Devices and Radio- ratory inspections and approval.20 However,
logical Health (CDRH) regulates most medical the FDA still has jurisdiction and may conduct
devices, but CBER retains primary jurisdiction its own inspections if warranted. All military
over medical devices used with blood and cel- blood facilities, including transfusion services,
lular products.15 The FDA’s Office of Regulatory register with and are inspected by the FDA.21
Affairs (ORA) has responsibility for all field op- Facilities use the Biologics License Appli-
erations, which include inspections and inves- cation (BLA, Form 356h) to apply for their
tigations. establishment and product licenses for
As part of the development process for interstate commerce. 22 Subsequent license
FDA regulations and guidance documents, amendments are based on the potential for
CHAPTER 3 Regulatory Issues In Blood Banking 䡲 85
TABLE 3-1. Regulations of Interest in Title 21 of the CFR (Food and Drugs)
adverse effects of the changes.23 Unlicensed ments for blood components (21 CFR 606).27
blood products, whether manufactured by a li- Routine inspections address the FDA’s five re-
censed or an unlicensed facility, may be quired layers of blood safety—donor screen-
shipped across state lines for a medical emer- ing, donor testing, product testing, quarantin-
gency if necessary, but this shipping should be ing, and monitor ing and investigating
unscheduled and infrequent. 24 The shipping problems. Investigators review the following
facility must retain documentation of the operational systems that create these safety
emergency for FDA review. layers: quality assurance, donor eligibility,
Facilities must register annually with the product testing, quarantine/inventory man-
FDA if they collect, manufacture, prepare, or agement, and production and processing.
process blood products (Form FDA 2830).25,26 Full inspections of all systems are desig-
Transfusion services are exempt from this re- nated Level I. After a favorable inspection pro-
quirement if they are approved for reimburse- file, facilities with four or five systems some-
ment by CMS and their preparation activities times have streamlined Level II inspections of
are basic, such as preparing Red Blood Cells three systems. (Focused inspections for prob-
from whole blood, converting unused plasma lems or fatalities need not follow these for-
to recovered plasma, pooling components, re- mats.) Within each system, the investigators
ducing leukocytes with bedside filters, or col- review standard operating procedures, per-
lecting blood only in emergencies. However, sonnel and training, facilities, equipment cali-
facilities that routinely collect blood (includ-
bration and maintenance, and records. Specif-
ing autologous units) or perform such proce-
ic requirements for individual systems and
dures as irradiation, washing, laboratory leu-
processes are discussed in detail in their re-
kocyte reduction, freezing, deglycerolization,
spective chapters of this book.
and rejuvenation must register as community
If the investigator judges that significant
or hospital blood banks. Transfusion services
objectionable practices, violations, or condi-
acting as depots for forwarding products to
tions are present under which a drug or device
other hospitals must register as distribution
is or could be adulterated or injurious to
centers. Clinical laboratories performing in-
fectious disease testing required for blood do- health, these observations are written and pre-
nors must register unless they are CMS ap- sented to the facility (Form 483). Investigators
proved. If blood irradiation is performed are instructed to seek and record the manage-
outside the blood bank or transfusion service, ment’s intentions to make corrections.25 How-
such as in a nuclear medicine department, ever, most facilities also respond in writing im-
that facility must register as well. mediately with questions or corrective actions.
Final determination that a violation has oc-
curred is made by ORA.
F D A I NS PE C T I O NS The FDA can take a number of steps in re-
New facilities are generally inspected within 1 sponse to a violation, including no action at
year of establishment by a team from CBER all. For significant violations, the FDA may is-
and ORA. Subsequent routine inspections are sue a warning letter, which is an advisory ac-
generally performed by ORA every 2 years or tion to provide the facility with the opportuni-
earlier depending on the facility’s compliance ty for voluntary compliance. Enforcement
history. actions are categorized as administrative, judi-
ORA publishes online manuals and in- cial, or recall. Administrative actions include
structions for FDA investigators for inspec- formal citations of violation and—for licensed
tions in general and for inspections of licensed facilities—suspension or revocation of a li-
and unlicensed blood banks in particular.25,26 cense. Judicial actions range from seizures of
The blueprints for blood bank inspections are products to court injunctions, civil monetary
the general regulations for cGMP and drugs penalties, and criminal prosecution. The FDA
(21 CFR 210-211) and the specific require- may also conduct recalls if necessary. ORA’s
CHAPTER 3 Regulatory Issues In Blood Banking 䡲 87
Key Regulations (21 CFR FDA Premarket Licensure, FDA Compliance Program
Type of HPC Product Jurisdiction Category except as noted) Approval, or Clearance? Manual37,38
䡲
Unmanipulated marrow Health Resources and Ser- 42 US Code 274(k) No Not applicable
vices Administration
HPCs* collected before May 25, 2005 Tissues, 21 CFR 1270 (before 1270, 1271 Subparts A-B No 7341.002A39
May 25, 2005) (see Table 3-1)
Autologous or allogeneic related- PHS Act Section 361: HCT/Ps‡ 1271.10(a)§ (must meet all No 7341.00237
donor† HPCs criteria); 1271 A-F 7342.007 Addendum40
(imported products)
Unrelated-donor peripheral blood PHS Act Section 351: 1271 A-D Planned 7341.00237
HPCs Biologics
Unrelated-donor umbilical cord cells Section 351: Biologics 1271 A-D Yes (after October 20, 2011): 7341.00237
Licensure Guidance41 licensure or IND application for
IND Guidance42 nonqualifying products
HPCs with altered characteristics or Cellular and Gene Therapy, 1271 A-D Yes: IND and BLA 7345.84843
AABB TECHNICAL MANUAL
Circular of Information for the Use of Cellular mation, are often in this category. Withdrawals
Therapy Products is jointly written by the are much more common than recalls but are
AABB and other organizations for users of not published.
these products.46 The AABB and the Founda- Recommendations for managing com-
tion for Accreditation of Cellular Therapy set mon notices have been published in the Unit-
voluntary standards and accredit facilities for ed States and Canada.50,51 Transfusion services
collection and processing of HPCs (see Chap- need to have procedures and training for rapid
ters 29 and 30 for information on these stan- responses as advised when recalls and with-
dards and accreditation procedures). drawals are received along with records of ac-
The FDA tissue regulations do not apply tions, reviews, and follow-up actions, as indi-
to facilities that receive, store, and administer cated. Accreditation requirements of the AABB
tissues but do not perform any manufacturing (Standard 7.1) and the College of American Pa-
steps. However, The Joint Commission (TJC) thologists (CAP) (Checklists TRM.42120 and
has hospital standards for handling and trac- TRM.42170) address aspects of this topic.52,53
ing tissues and investigating adverse events Most of these blood components are
(TS.03.01.01 to 03.03.01) (see Chapter 32 for transfused before a notice is received of their
information on these standards).47 nonconformance. In some recent blood guid-
ance documents on infectious diseases, the
FDA has included recommendations on
M A N AG I N G R E C A L LS A ND
whether or not to notify the recipient’s physi-
W IT H D R AWA L S cian about transfused units. In cases of possi-
The FDA’s requirements for monitoring and ble recent infectious disease exposure in
investigating problems with drugs extend to donors or transfusion recipients, the serocon-
the time after a product’s release. When blood version window periods for tests should be
banks discover after distribution that a blood consulted for scheduling prospective testing
product was in violation of rules, standards, or reviewing retrospective results, such as for a
specifications, or cGMP regulations, they must donor who has been retested since an expo-
report the problem to the FDA and their con- sure.50
signee. These problems comprise a subset of The most common reasons for BPDs and
biological product deviations (BPDs)—events blood component recalls are given in Table 3-
in which the safety, purity, or potency of a dis- 3. Look-backs investigations on units from do-
tributed blood product may be affected—all of nors found after donation to have HIV or hep-
which must be reported to the FDA.48 atitis C virus are discussed in Chapter 5.
A recall is defined as the removal or cor-
rection of a marketed product that is in viola- ME DI CAL L ABO RATO RY L AW S
tion of the law (21 CFR 7.3).49 The FDA classi-
AND REGUL ATIONS
fies recalls by severity. Most blood component
recalls are in Class III, not likely to cause ad- CMS regulates all US medical laboratories un-
verse health consequences. Class II recalls are der the Clinical Laboratory Improvement
for products that may cause temporary ad- Amendments [CLIA, 42 USC 263(a) and 42 CFR
verse effects or remotely possible serious 493] to Section 353 of the Public Health Service
problems. Class I recalls involve a reasonable Act.54,55
probability of serious or fatal adverse effects. The law and regulations establish the re-
All recalls are published by the FDA and in- quirements and procedures for laboratories to
volve about 1 in 5800 blood components is- be certified under CLIA as both a general re-
sued in the United States.50 quirement and a prerequisite for receiving
Market withdrawals are required for Medicare and Medicaid payments.
products found to be in minor violation of the To be certified, laboratories must have
law. Problems beyond the control of the man- adequate facilities and equipment, superviso-
ufacturer, such as postdonation donor infor- ry and technical personnel with training and
90 䡲 AABB TECHNICAL MANUAL
TABLE 3-3. Most Common Reasons for Biological Product Deviations and Blood Component
Recalls
Joint Commission. 60 The Joint Commission laboratory specimens and transfusion recipi-
has cooperative agreements with ASHI, CAP, ents (NPSG.01.01.01, .01.03.01), checking
and COLA to accept their laboratory accredita- blood products in the Universal Protocol pre-
tions in facility surveys.61 procedure verification process (“time-out”)
AABB and CAP can coordinate joint sur- (UP.01.01.01), and assessing transfusion appro-
veys by facilities seeking both types of accredi- priateness (MS.05.01.01).47 The Joint Commis-
tation. CMS may perform its own follow-up sion includes hemolytic transfusion reactions
surveys to validate those of the accreditation in its Sentinel Events reporting program.64
organizations. Facilities also should be familiar with all
CMS requires successful PT for ongoing relevant state and local laws and regulations,
laboratory certification of nonwaived testing.
including professional licensure requirements
Within each laboratory section, CMS regula-
for medical and laboratory personnel. In some
tions specify tests and procedures (regulated
situations, facilities providing products or ser-
analytes) that must pass approved PT if the
vices in other states must comply with local
laboratory performs them. The CMS website
has a list of approved PT providers.62 PT is dis- regulations in the customer’s location.
cussed in Chapter 1.
CO N CLUS IO N
HOS PITAL R EG UL ATI ONS AND Blood components and other blood products
ACC R E D I TAT I O N are regulated as pharmaceutical agents, and
CMS approves hospitals for Medicare reim- blood banks and transfusion services are also
bursement through state surveys or accredita- regulated as medical laboratories. These many
tion programs from The Joint Commission, the requirements make compliance complex.
American Osteopathic Association, and DNV However, close regulation underscores the rec-
Healthcare. These inspections cover CMS ognized importance to public health of safe,
regulations for blood administration and the effective blood products provided via good
evaluation of transfusion reactions [42 CFR laboratory practices using the quality manage-
482.23(c)].63 The Joint Commission has stan- ment systems described in Chapter 1 and the
dards for preventing misidentification of procedures discussed throughout this manual.
KEY POINTS
1. The FDA regulates the manufacture and use of drugs and medical devices, including blood
components, blood-derived biological products, and their ancillary testing and transfusion
equipment. The FDA website provides links to blood-related regulations in Title 21 CFR and
to explanatory guidance documents.
2. The FDA requires licensure and registration for blood manufacturers engaged in interstate
commerce and registration alone for manufacturing activities, such as irradiation or wash-
ing. Transfusion services performing only basic component preparation need not register
but must have laboratory approval from CMS.
3. Licensed and registered blood facilities are inspected by the FDA every 2 years, with a focus
on current cGMP (21 CFR 210-211) and blood component requirements (21 CFR 606). Ob-
servations of significant noncompliance or hazards are reported to the facility for its re-
sponse and correction and may result in a spectrum of sanctions, possibly including license
revocation and criminal prosecution.
4. HPCs for transplantation are regulated as tissues by the FDA, with an emphasis on prevent-
ing transmissions of infections and contaminations in autologous or closely related alloge-
neic transplants. The FDA has a licensure process for unrelated umbilical cord blood HPCs.
92 䡲 AABB TECHNICAL MANUAL
5. The FDA requires drug (and blood) manufacturers to conduct recalls or market withdrawals
when noncompliance is found after products are issued, such as for donor malaria risk or
potentially compromised component sterility. Transfusion services should have procedures
for evaluating and managing the potential impact of these recalls and withdrawals on pa-
tients transfused with such products.
6. All US medical laboratories must be approved by CMS every 2 years. Laboratory regulations
emphasize the need for adequate facilities and qualified personnel commensurate with the
complexity of testing, and they require ongoing successful performance in proficiency test-
ing by CMS-approved vendors. Laboratory approval by CMS is granted via inspections per-
formed by CMS-approved accrediting agencies or state health departments.
7. Health-care facilities also have CMS regulations for their activities, and The Joint Commis-
sion and other organizations accredit many hospitals for CMS compliance. CMS and The
Joint Commission have requirements for monitoring transfusion practices, evaluating ad-
verse transfusion reactions, and preventing mistransfusions.
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www.fda.gov/BiologicsBloodVaccines/Guid calDevices/DeviceRegulationandGuidance/
anceComplianceRegulatoryInformation/ GuidanceDocuments/ucm072753.htm (ac-
ComplianceActivities/Enforcement/Compli cessed November 16, 2013).]
CHAPTER 3 Regulatory Issues In Blood Banking 䡲 95
45. Testing HCT/P donors for relevant communi- 54. Code of federal regulations. Title 42, CFR Part
cable disease agents and diseases. Silver 493. Washington, DC: US Government Print-
Spring, MD: CBER Office of Communication, ing Office, 2013 (revised annually).
Outreach, and Development, 2013. [Available 55. United States code. Title 42, USC Part 263a.
at http://www.fda.gov/BiologicsBloodVac 56. Rauch CA, Nichols JH. Laboratory accredita-
cines/SafetyAvailability/Tissue Safety/ucm tion and inspection. Clin Lab Med 2007;27:
095440.htm (accessed November 8, 2013).] 845-58.
46. AABB, America’s Blood Centers, American As- 57. Howerton D, Anderson N, Bosse D, et al. Good
sociation of Tissue Banks, American Red laboratory practices for waived testing sites:
Cross, American Society for Apheresis, Ameri- Survey findings from testing sites holding a
can Society for Blood and Marrow Transplan- certificate of waiver under the Clinical Labora-
tation, College of American Pathologists, tory Improvement Amendments of 1988 and
Foundation for the Accreditation of Cellular recommendations for promoting quality test-
Therapy, ICCBBA, International Society for ing. MMWR Recomm Rep 2005;54(RR-13):1-
Cellular Therapy, Joint Accreditation Commit- 32.
tee, National Marrow Donor Program, and 58. Clinical Laboratory Improvement Amend-
Netcord. Circular of information for the use of ments (CLIA): How to obtain a CLIA certificate.
cellular therapy products. Bethesda, MD: (March 2006) Baltimore, MD: Centers for
Medicare and Medicaid Services, 2006. [Avail-
AABB, 2013. [Available at http://www.aabb.
able at http://www.cms.hhs.gov/CLIA/down
org/ (accessed November 8, 2013).]
loads/HowObtainCLIACertificate.pdf (ac-
47. 2012 Comprehensive accreditation manual for
cessed November 18, 2012).]
hospitals: The official handbook (CAMH).
59. Interpretive guidelines for laboratories. Ap-
Oakbrook Terrace, IL: The Joint Commission,
pendix C. Survey procedures and interpretive
2012.
guidelines for laboratories and laboratory ser-
48. Biological product deviations. Silver Spring,
vices. Baltimore, MD: Centers for Medicare
MD: CBER Office of Communication, Out-
and Medicaid Services, 2013. [Available at
reach, and Development, 2012. [Available at
http://www.cms.hhs.gov/CLIA/03_Interpre
http://www.fda.gov/BiologicsBloodVaccines/
tive_Guidelines_for_Laboratories.asp (ac-
SafetyAvailability/ReportaProblem/Biologi
cessed November 8, 2013).]
calProductDeviations/default.htm (accessed 60. List of approved accreditation organizations
November 16, 2013).] under CLIA. Baltimore, MD: Centers for Medi-
49. Code of federal regulations. Title 21, CFR Part care and Medicaid Services, 2013. [Available at
7.3. Washington, DC: US Government Printing http://www.cms.gov/Regulations-and-Guid
Office, 2014 (revised annually). ance/Legislation/CLIA/Downloads/AOList.
50. Ramsey G. Blood component recalls and mar- pdf (accessed November 8, 2013).]
ket withdrawals: Frequency, reasons, and 61. Laboratory services. Facts about the coopera-
management in the United States. Transfus tive accreditation initiative. Oakbrook Terrace,
Med Rev 2013:7:82-90. IL: The Joint Commission, 2013. [Available at
51. Recommendations for the notification of re- http://www.jointcommission.org/assets/1/
cipients of a blood component recall. (March 18/cooperative_accreditation_initiative_6_
21, 2012) St. John’s, NL and Ottawa, ON: Na- 15_11.pdf (accessed November 8, 2013).]
tional Advisory Committee on Blood and 62. CLIA-approved proficiency testing pro-
Blood Products and Canadian Blood Services, grams—2013. Baltimore, MD: Centers for
2012 [Available at http://www.nacblood.ca/re Medicare and Medicaid Services, 2012. [Avail-
sources/guidelines/recall-recipient-notifica able at http://www.cms.hhs.gov/CLIA/down
tion.html (accessed January 14, 2014).] loads/ptlist.pdf (accessed November 8, 2013).]
52. Levitt J, ed. Standards for blood banks and 63. Code of federal regulations. Title 42, CFR Part
transfusion services. 29th ed. Bethesda, MD: 482.23(c). Washington, DC: US Government
AABB, 2014. Printing Office, 2013 (revised annually).
53. College of American Pathologists Laboratory 64. Sentinel event. Oakbrook Terrace, IL: The Joint
Accreditation Program checklists. Northfield, Commission, 2012. [Available at http://www.
IL: College of American Pathologists, 2013 (re- jointcommission.org/sentinel_event.aspx (ac-
vised annually). cessed November 18, 2013).]
C h a p t e r 4
Disaster Management
Ruth D Sylvester, MS, MT(ASCP)SBB, Director, Regulatory Services, America’s Blood Centers, Washington,
District of Columbia; William FitzGerald, LTC USA (Ret), Senior Advisor, Biomedical Preparedness, American
Red Cross, Washington, District of Columbia; Wendy Trivisonno, Director, Strategic Biologic Sourcing and
Logistics, Blood Centers of America, West Warwick, Rhode Island; and Theresa Wiegmann, JD, Director of
Public Policy, AABB, Bethesda, Maryland
The authors have disclosed no conflicts of interest.
97
98 䡲 AABB TECHNICAL MANUAL
Recovery
Probability of Human Property Business Resources
Occurrence Effect Effect Effect Needed Total
High Low High Low High Low High Low High Low
5 1 5 1 5 1 5 1 5 1
External Hazards
Earthquake 5 3 4 5 4 21
Hurricane 1 1 1 2 2 7
Internal Hazards
Flooding 4 1 4 3 2 14
Workplace violence 2 5 2 4 1 14
100 䡲 AABB TECHNICAL MANUAL
be familiar with NIMS protocols so that Health and Human Services (DHHS). The
they are prepared to communicate effec- NDMS augments local medical care when
tively with responding organizations [eg, an emergency exceeds the scope of a com-
police and fire departments and the Federal munity’s health-care system. The NDMS
Emergency Management Agency (FEMA)]. consists of more than 9000 medical and
Online training courses are available on the support personnel from federal, state, and
FEMA website.6 local governments and the private sector as
䡲 National Response Framework (NRF). The well as civilian volunteers.11
NRF is a federally operated all-hazards re- 䡲 AABB Interorganizational Task Force on
sponse plan that is activated in response to Domestic Disasters and Acts of Terrorism
a presidentially declared disaster under the (AABB Disaster Task Force). The AABB
Robert T. Stafford Disaster Relief and Emer- Disaster Task Force provides support to
gency Assistance Act (amended June blood collection and transfusion facilities
2006).7,8 Under this act, the federal govern- during major emergencies, including dur-
ment, coordinated by the Department of ing NRF activation under the Stafford Act.
Homeland Security (DHS), provides per- Facilities should review the AABB Disaster
sonnel, assets, and financial support to Task Force response plan and integrate its
state and local governments that are over- response processes into their organization-
whelmed during a major disaster. The need al disaster plans.2
for blood is covered under the medical and 䡲 EMAs. EMAs are local, tribal, state, and fed-
public health section of the NRF (Emergen- eral agencies tasked with helping individu-
cy Support Function No. 8).8(p33),9 Blood col- als and organizations deal with all cycles of
lection and hospital facilities should be fa- disaster management. These agencies are
miliar with the NRF to request and receive staffed by both full-time and volunteer per-
assistance during major emergencies. sonnel (emergency managers) and use
䡲 National Terrorism Advisory System (NTAS). standard methods when responding to a
The DHS assesses threats posed by terror- disaster.11
ists and communicates those threats to the
US public, government agencies, and the Major Disaster Management Factors
private sector through the NTAS alerts.10 for Blood and Transfusion
This system replaces the color-coded
Homeland Security Advisory System In conjunction with comprehensive disaster
(HSAS) used in the past. The NTAS uses planning strategies, blood collection and hos-
only two tiers of alerts: pital facilities should consider the disaster
– Imminent Threat Alerts warn of “credi- management factors, described in this section,
ble, specific, and impending terrorist that are key for blood and blood components
threat[s] against the United States.” during all phases from collection to transfu-
– Elevated Threat Alerts warn of credible, sion.12-16
but not specific, threats against the Unit- Most of the blood and blood components
ed States. used for transfusion in the United States are
NTAS alerts are brief, clearly articulating collected, processed, tested, and stored at re-
what can be done to prevent and even, if gional blood centers and must be delivered to
possible, mitigate a threat’s impact and re- hospitals before transfusion. During an emer-
spond if necessary. Unlike the previously gency, this “just-in-time” delivery system re-
used HSAS that issued ongoing warnings, sults in the need for close coordination be-
NTAS alerts are issued for a specific period tween blood centers and the hospitals they
and expire once that period has ended. serve. This involves robust communication
䡲 National Disaster Medical System (NDMS). and information systems, transportation and
The NDMS is a cooperative asset-sharing logistics support, and critical utilities, such as
program directed by the Department of fuel and electrical power, to ensure that blood
CHAPTER 4 Disaster Management 䡲 101
can be transported and stored at required tem- outside the immediate vicinity to the affected
peratures. hospital might take 12 to 24 hours.
Emergency management personnel are Disasters resulting from the use of biolog-
often unaware of issues related to the collec- ic, chemical, radiologic, or nuclear agents may
tion, processing, storage, and distribution of result in widespread donor deferrals and the
blood and blood components and may as- potential quarantine of blood components al-
sume that blood is collected and stored direct- ready in the manufacturing process or storage
ly in hospitals. As a result, blood-related issues until the nature and effect of the agent can be
(eg, the need for transportation and logistics determined. Radiologic-nuclear attacks also
support) are often overlooked during real and would dramatically increase the demand for
simulated disasters. A continuing process of platelets, other blood components, and stem
education for emergency managers at both the cells for several weeks following an event. In
local and national levels is imperative to in- addition, in the event of an influenza pandem-
crease their awareness of issues related to ic, blood supplies may be severely strained for
blood. Blood centers should pursue active par- weeks due to high staff absenteeism and donor
ticipation in EMA planning activities and shortages as these individuals become ill, need
participate in activities of the emergency oper- to stay home to take care of loved ones, or fear
ations center to ensure continuous represen- exposure to influenza in public places. An in-
tation of blood-related issues. fluenza pandemic could also severely affect
Historically, an average of about 200 units the availability of needed supplies as manu-
of blood are needed by victims of most disas- facturers face similar absenteeism and trans-
ters. However, the public response to disasters portation difficulties.17,18
has traditionally resulted in increases in blood Hospitals and transfusion services should
donations regardless of the true medical need develop blood shortage management plans to
for transfusions. A sudden increase in unneed- maximize the probability of optimal blood dis-
ed blood donations can disrupt both local and tribution and use during a severe shortage. It is
national blood supplies. Efforts should be critical that detailed blood triage plans devel-
made to establish the medical need for blood oped by hospital transfusion services, man-
during a disaster and communicate this need agement staff, and physicians who make
to national blood organizations and the AABB transfusion decisions be available and be reg-
Disaster Task Force, blood donors, and the ularly reviewed in advance of a severe disaster.
public through a clear and consistent messag- Blood centers should work with their hospital
ing strategy.12-16 customers to ensure that they have triage
In disasters resulting in injuries that do plans in place. The Minnesota Department of
require blood transfusions to treat victims, the Health’s Minnesota Healthcare System Pre-
available local blood inventory is the primary paredness Program has developed an exten-
source for the initial treatments. Because sive set of materials to help hospitals prepare
blood collected in response to a disaster takes to respond to disasters including “When
24 to 48 hours to process, it is critical for the lo- Healthcare Resources are Scarce,” a set of
cal blood supply to remain at ample levels to guidelines to assist health-care facilities in
treat victims of potential hazards as defined in planning for events that would overwhelm the
the organizational risk assessment. The AABB system’s resources, including blood and blood
Disaster Task Force recommends maintaining components.19, 20
a 5- to 7-day supply [combined inventory at Finally, certain disasters may affect a
the blood collector and hospital(s) it serves] to blood collector’s ability to collect and process
address potential disasters.2 Efforts are made blood during the entire event, thus straining
to resupply the affected facilities if needed the local blood supply for days or weeks. For
from neighboring blood collection centers or instance, as a hurricane approaches, a blood
through national support by the AABB Disas- collector may suspend collections for a few
ter Task Force. However, delivering blood from days before and after the storm has passed,
102 䡲 AABB TECHNICAL MANUAL
resulting in a significant loss of expected col- tions have seen human and natural disasters
lections. Elective surgeries typically are post- wreak havoc in the United States and abroad.
poned during this period, helping alleviate the References, training materials, and toolkits
strain on supply. However, lessons learned with step-by-step instructions and templates
from previous disasters indicate that blood are widely available on the Internet and should
collection and transfusion facilities should be acquired and used when developing a di-
prepare for the potential acute shortage of saster plan.21-25
blood components in the days following a hur-
ricane or disaster of similar magnitude, paying Analysis of Current Situation
special attention to the supply of platelets. Fa-
The success of disaster planning depends on a
cilities should augment supplies before a pre-
risk analysis of the current situation (see sec-
dictable hazard (eg, a hurricane or severe win-
tion on “Risk Assessment”). This assessment
ter storm) and contact the AABB Disaster Task
includes determining the hazards to which an
Force for support if routine supply channels
organization is most vulnerable, the probabili-
are inadequate to bolster supplies before or af-
ty of each hazard, and the anticipated effect of
ter the event.
each event on human and physical resources
and on business operations.4,21 Effective pre-
BUS I N ES S O PE R AT IO N S vention strategies can be used for risks such as
PL AN N ING fires, power interruptions, facility security
breaches, and workplace violence. What can-
Business operations planning is generally a not be prevented should be mitigated. For ex-
three-step process: assembling a planning ample, conducting essential operations away
team; analyzing the current situation, includ- from floodplains, having multiple sources (but
ing hazards and capabilities; and developing a not necessarily multiple suppliers) of critical
plan to continue operations in an emergency. supplies, and relocating vital records (includ-
Once planning has been completed, imple- ing critical information technology resources)
mentation of the plan (which includes train- to upper floors in flood-prone areas are all mit-
ing, testing, evaluating, and revising the plan) igation strategies. Plans should be developed
can proceed.21,22 for events that have a high likelihood of occur-
ring as well as low-probability events with po-
Disaster Planning Team tentially catastrophic consequences (eg, Cate-
gory 5 hurricanes or pandemic influenza).
The first step in disaster planning is to identify
the person or team of people in an organiza-
tion who are responsible for emergency opera- Continuity of Operations Plan
tions planning. The number of people in- A Continuity of Operations Plan (COOP) is
volved will depend on the size and complexity designed to ensure continued operation of
of the operation. Assistance will be needed essential functions in the event of an emergen-
from key people, such as the organization’s ac- cy or disaster.21,25 A COOP should cover the
countant, attorney, human resources staff, and emergencies that are most likely to occur or
insurance agent. In addition, establishing a re- that could have a significant effect. A COOP
lationship with local EMAs can be vital to the should address at least the areas identified in
success of a disaster plan in an actual emer- Table 4-2. These elements are discussed in this
gency. section.
Once the responsible person has been Hospital-based blood banks and transfu-
identified or the team has been formed, a proj- sion services may be covered by the overall
ect plan should be developed to identify the hospital or laboratory COOP; however, the var-
key steps and a timeline for completing the di- ious COOPs should be reviewed to ensure that
saster plan. Business continuity planning has issues specific to blood components and
proliferated over the past 5 years as organiza- transfusion are sufficiently covered. Such
CHAPTER 4 Disaster Management 䡲 103
plans are required by the AABB in its Standards COOP Decision Trees
for Blood Banks and Transfusion Services as
COOP operations and decisions are best
well as by The Joint Commission.26,27
thought out in advance during normal opera-
tions. Flowcharts and decision trees that list
Essential Functions
options can be developed to guide leaders
Essential functions are those that must be car- during emergency operations. Such flow
ried out to ensure business continuity, such as charts and decision trees can be developed by
the storage of blood and blood components groups and refined using lessons learned from
and their transportation and delivery to hospi- exercises and real-world events. An example of
tals. Even though blood collections, compo- a decision tree developed to support blood
nent preparation, and testing may be inter- bank operations by the California Blood Bank
rupted, the continued viability of a blood Society is available on the Internet.28
center depends on its ability to provide hospi-
tal customers with blood. Blood can be im- Alternate Facilities
ported from outside the affected area to en- Alternate facilities that can be used to contin-
sure an adequate inventory until collections ue operations should be identified during the
can resume. Likewise, hospitals must be cer- planning phase of a COOP. Thought should be
tain that they can receive, store, and transfuse given to the need for equipment, supplies, in-
blood to patients in need. formation technology support, and other
104 䡲 AABB TECHNICAL MANUAL
items at the alternate location. Consideration to document the safety and availability of
should also be given to practical locations for blood components. In addition to donor rec-
alternate facilities. For example, an alternate ords, the organizational human resource files,
facility across the street will most likely be sim- legal records, payroll records, and financial
ilarly affected by an external disaster. If fund- records (such as accounts receivable and ac-
ing is not available to pay for an alternate counts payable) are critical to a business’s sur-
facility, the organization should consider es- vivability during a disaster.22,29 Records of in-
tablishing formal agreements with other orga- surance policies, bank account numbers, and
nizations in the area to provide alternate space supplies of blank checks must be available
if one facility is incapacitated. during emergency operations to ensure conti-
nuity of operations. Corporation records, stra-
Security and Safety tegic plans, and research-related records must
also be maintained. Redundancy of computer-
In a disaster, security of critical resources—
ized records should be established by periodi-
ranging from the facility and fleet to staff and
cally by backing records up on a duplicate
information systems—can become a major
server. Geographic considerations must be
concern.22 For example, crowd control may be-
taken into account in identifying off-site stor-
come an issue if large numbers of donors gath-
age locations. Not all records are maintained
er at collection locations or in the event of
electronically, so provisions to safely keep pa-
special screening requirements during a pan-
per copies of records must be considered, es-
demic influenza outbreak. In natural disasters
pecially if alternate facility operations are im-
in which fuel shortages occur, the security of
plemented.
fuel supplies can become a major challenge. A
COOP should include the measures that will
Insurance
be necessary to ensure the security of all criti-
cal resources. Adequate insurance is essential to the surviv-
Planners should review their normal op- ability of any business. The two major types of
erations security plan and determine whether insurance coverage for blood collection and
the plan adequately addresses potential transfusion facilities are property and liability.
threats and hazards to the local area. Coordi- Key provisions of these policies include valua-
nation with EMA officials can provide infor- tions of property; perils covered, such as flood-
mation on threat or risk assessments. Planners ing, wind, and power interruptions; deduct-
should consider the implementation of mea- ibles; and how to file claims. Business
sures to increase the security of their facilities, interruption insurance can cover loss of in-
staff, volunteers, and donors during changes come. Additional background information on
in the NTAS.10 relevant insurance policies is available from a
Lastly, planners should ensure that the fa- variety of resources, including the Small Busi-
cility complies with all local building codes ness Administration and insurance compa-
and applicable Occupational Safety and nies.24,30
Health Administration (OSHA) regulations.
The actions implemented should be dis- Cash Reserves
cussed with the local EMA and the insurance
The single most crucial element for the surviv-
company, and any suggestions that they pro-
ability of a business during a disaster is access
vide should be addressed.22
to cash that can provide support until normal
operations can be resumed.31,32 Cash reserves
Vital Records
equal to several months’ worth of operational
The protection of vital records is especially expenses should be accrued during normal
critical in an industry that depends on records operations.
CHAPTER 4 Disaster Management 䡲 105
delays can occur in switching to alternate can assist with obtaining 1) transportation
methods during an event, resulting in the po- support for components, critical supplies, and
tential loss of life and property. staff; 2) power restoration; 3) fuel for backup
No single method is used by organiza- generators; 4) fuel for fleet and essential staff
tions to design communications redundancy. vehicles; 5) communication support; 6) securi-
Local and state jurisdictions use different ty if staff or property is threatened; and 7) oth-
types of emergency communications equip- er utilities support (eg, telecommunications
ment (eg, fire and police personnel use radios) and Internet). Facilities should educate these
and have different local telecommunications agencies on how the blood supply operates
infrastructures (eg, cell towers) and capacities. both locally and nationally (eg, the use of
Organizations should acquire multiple redun- “just-in-time” delivery methods and the need
dant communications technologies and iden- to deliver blood from regional blood centers to
tify the order in which they should be used hospitals) and should request that they be
during an emergency. Alternative communica- deemed a critical health entity or critical infra-
tion technologies are described in the AABB structure entity in the emergency response
Disaster Handbook.2 structure.8
Blood centers and hospitals should work
COMMUNICATION WITH LOCAL, STATE, AND with the local EMA to ensure their inclusion in
NATIONAL E MERGENCY RESPONSE AGEN- local emergency communications systems.
CIES. In addition to communicating with These systems may include ongoing videocon-
routine suppliers and vendors, facilities ferences or conference calls, blast e-mail lists,
should establish and maintain relationships and 800-MHz radio networks. Furthermore,
with local, state, tribal, and national emergen- blood centers should seek admission to any pri-
cy response organizations as shown in Fig 4-1 ority networks established by local hospitals to
(see also section on Participating in Disaster improve communications during an incident.
Drills with EMAs). Periodically, management should pro-
In particular, blood collection and trans- vide informational briefings to members of the
fusion facilities should identify agencies that EMA and the public health department about
the mission of the organization, the scope of should speak with members of the media. Spe-
its operations, and the effect on local health- cific activities should take place before, dur-
care facilities if the blood center or hospital is ing, and after each interview.33 For instance,
not operational. Management should also be- written background information should be
come familiar with the NIMS and NRF docu- provided to a reporter before an interview, and
ments and should consider enrolling in FEMA a spokesperson should focus the most salient
online educational courses30 or disaster train- points of the message into “sound bites” that
ing courses provided by the state or local com- can be quoted in a story. Another useful tech-
munity. Management should request that the nique when communicating with the media is
EMA provide informational briefings to key message mapping.34 Message maps help com-
staff of the blood center or hospital. During ex- municators develop and synthesize complex
changes with the EMA, management should information by identifying three key messages
discuss resource issues (eg, transportation as- to be delivered in three short sentences with a
sistance, fuel support, storage, security, inclu- total of 27 words. This process accommodates
sion in EMA notification systems, and assign- both broadcast and print media. Most impor-
ment of a blood center or hospital liaison to tant, a spokesperson should never speak off
the EMA). Management should invite the EMA the record with a reporter. Many other tips and
to participate in its disaster exercises and ac- techniques can be used to ensure that the cor-
tively encourage the EMA to include the blood rect messages are conveyed. One resource for
center or hospital in its disaster exercise pro- such tips is the Northwest Center for Public
grams. Health Practice in collaboration with Public
National assistance during disasters (eg, Health—Seattle and King County.35
coordination of national media messages and
resupply of blood components to affected fa- Logistics
cilities) is provided through the AABB Disaster
Task Force in coordination with other national During the development of a COOP, facility
and federal organizations. Along with local planners must place heavy emphasis on the
planning efforts, blood collection and transfu- management of daily operations, including
sion facilities are encouraged to integrate the making available the supplies and equipment
task force response system into their disaster required to perform essential functions related
plans. The response system is described in the to 1) recruiting of donors, 2) collecting donated
AABB Disaster Operations Handbook on the blood, 3) manufacturing and testing blood
AABB website.2 components, and 4) delivering the components
to the hospital for transfusion to patients. Dur-
WORKING WITH PUBLIC MEDIA. Creating ing a disaster, the normal logistical systems
and disseminating a clear and consistent mes- may not be available or capable of providing
sage about the status of the local and national the necessary support. Key logistical issues to
blood supply is a critical component of an address in a COOP are described below.
ECP.12-16 Unnecessary donor surges can be pre-
vented, or donations of specific blood compo- TRANSPORTATI ON. Past disaster and terror-
nents (eg, group O red cells or platelets) can be ist events have clearly demonstrated the vul-
requested by working with public media out- nerability of transportation systems. Blood
lets to effectively convey messages about the centers should coordinate with their local
status of the blood supply following a disaster. EMAs to ensure that vehicles engaged in col-
The AABB Disaster Task Force has developed lecting or distributing blood are duly autho-
media-related resources for facilities, includ- rized as emergency support vehicles and are
ing boilerplate press releases that are available granted the appropriate clearances to operate
in the AABB Disaster Operations Handbook.2 on the roads. In addition, memoranda of un-
Only individuals who have received train- derstanding, agreements, or statements of un-
ing in risk and emergency communication derstanding should be established with the
108 䡲 AABB TECHNICAL MANUAL
appropriate EMAs regarding access to the BIOHAZ ARDS. In the aftermath of a disaster,
roads and use of law enforcement, National the disposal of biohazardous material may be-
Guard, and state militia personnel or vehicles come a serious problem for a blood center or
to deliver blood. Under the provisions of the hospital. Planners should incorporate appro-
NRF, DHHS can ask the Department of Trans- priate language in their biohazard material
portation to assist with the movement of blood disposal contracts that will ensure that ven-
by air, rail, water, and motor vehicle.8 Such re- dors establish contingency plans to meet the
quests should be directed to the AABB Disaster facility’s requirements. The blood center or
Task Force.2 hospital should coordinate with the EMA and
fire or safety agencies and should provide in-
“J U S T- I N - T I M E ” S Y S T E M S . M a n y b l o o d formation on the types of biohazardous wastes
centers and hospitals have adopted just-in- that it generates. Organizations that have irra-
time supply systems to minimize expenses as- diators should provide local fire or safety agen-
sociated with the storage of large quantities of cies with a detailed building plan that includes
supplies, maintenance of storage facilities, photographs of the ingress routes to the device
staffing, and outdated products. Blood centers and of the device as well as the manufacturer’s
and hospitals should determine the critical specifications and contact information. Affect-
items required for their business operations, ed facilities should contact the AABB Disaster
the vendor’s resupply capability (including Task Force2 if local and state EMA authorities
manufacturing schedule and transport time- cannot provide this assistance.
lines), the shelf life of each supply item, and
any storage requirements (eg, special environ- Utilities
mental factors).36
Blood centers and hospitals rely on utility ser-
STORAGE . Planners should consider the stor- vices (eg, electricity, water, fuel, sewage dis-
age capacity of their customers and identify lo- posal, telecommunications, and Internet)
cal storage companies that could provide an from a variety of sources, such as municipali-
emergency storage site. Contingency contracts ties or publicly or privately owned companies.
or contingency clauses in existing contracts Planners should establish contingency sup-
with customers and ice and dry-ice suppliers port plans with their service providers. These
should be considered. Temporary storage plans should identify emergency points of
space may be provided by commercial con- contact at the utility service provider and in-
tainers, including prevalidated refrigerated clude their telephone numbers and e-mail ad-
containers that could be placed at the blood dresses. Most utility services have priority lists
center, depending on space and electrical for service restoration during emergencies.
power. The local EMA may permit storage of During the planning process (and not during
consumables at municipal disaster supply fa- or after an emergency), planners should solicit
cilities at no cost. the support of the local EMA and hospitals to
ensure that their organization will be designat-
CONTRACTS. All vendor contracts should
ed to receive priority restoration of services.
identify the process for emergency deliveries
of supplies, equipment, and services. The ven-
Staffing
dor or supplier should have a disaster plan
that demonstrates the ability to meet a facili- Blood centers and hospitals depend on their
ty’s supply requirements in a disaster. It is ad- highly trained, competent, and motivated staff
visable to require vendors to have disaster and volunteers to be successful in their mis-
plans for their operations. Facilities might also sion of recruiting blood donors and collecting,
develop contracts containing noncompete manufacturing, testing, and distributing blood
agreements with local competitor organiza- components. Management should promote
tions for implementation during disasters. disaster preparedness to staff and volunteers
CHAPTER 4 Disaster Management 䡲 109
and should encourage them to complete fami- refresher training as required. Management
ly preparedness plans.8.22 may need to negotiate with collective bargain-
ing units to modify existing contracts.
ESSE NTIAL PERSONNEL. During the emer-
gency planning process, the management HUMAN RESOURCES ISSUES. Of impor-
team should identify staff members and vol- tance to employees—beyond the safety of
unteers (if applicable) who are responsible for loved ones and availability of food and shel-
the vital services required to maintain busi- ter—are issues such as salary continuation,
ness operations. Each department should de- flexible work schedules, implementation of
velop a “smart book” that describes its essen- telework procedures, and benefits.8,23 Because
tial functions, essential personnel who these issues are influenced by many factors,
accomplish them, vital information and rec- having a predeveloped decision matrix can be
ords, contract information, and customer in- particularly helpful during times of crisis.
formation.
Employees who are deemed essential be-
cause of their responsibilities must receive ex- REGULATORY CONSIDERATIONS IN
tensive training in the facility’s emergency EMERGENCIES
plans and participate in disaster training exer- The blood collection and transfusion commu-
cises to refine operational procedures under
nity is highly regulated, and even slight chang-
emergency conditions. Consideration should
es to processes and procedures can have far-
be given to establishing alternate work sites
reaching effects on facilities and patients,
and/or implementing telework programs to
especially during an emergency. Blood collec-
mitigate the effects of the loss of the blood
tion and transfusion facilities should carefully
center or hospital on operations. The human
consider regulatory issues in their disaster
resources department should work with man-
management plans.
agement to ensure that employee-training
plans provide for the succession of personnel
into positions held by designated essential
Federal Agencies Involved
personnel should the incumbent become un- The blood community is regulated by numer-
available. Management should also plan for ous agencies. The Center for Biologics Evalua-
housing and feeding essential personnel and tion and Research (CBER) of the Food and
providing them with fuel to help them travel to Drug Administration (FDA) has primary re-
and from work when the local infrastructure is sponsibility for ensuring the safety, purity, and
not operational.23 potency of all biologic products, including
CROSS-FUNCTIONAL TRA INING. Planners,
blood and blood components. The Center for
in close coordination with the human resourc- Devices and Radiological Health (CDRH), also
es department and, if applicable, any collec- part of the FDA, regulates medical devices,
tive bargaining units, should develop a cross- including radiation-emitting devices. The
functional training program that identifies Department of Transportation regulates the
personnel whose routine duties do not sup- movement of cargo, including blood, blood
port the facility’s essential functions during a components, and progenitor cells, through the
disaster response. These reassigned personnel transportation network. OSHA ensures the
may serve as drivers, public affairs representa- safety of the US workforce. Likewise, the mis-
tives, and recruiters or perform other nonreg- sion of the Environmental Protection Agency
ulated duties. The human resources depart- is to protect human health and the environ-
ment and management should make the ment. The Centers for Medicare and Medicaid
necessary modifications to the job descrip- Services under the Clinical Laboratory Im-
tions of the affected employees or volunteers, provement Amendments of 1988 oversees pa-
develop training plans, and conduct initial and tient (as distinct from donor) testing at blood
110 䡲 AABB TECHNICAL MANUAL
centers, hospital blood banks, and transfusion be notified regarding what tests have and have
services. not been completed on the blood.39
Consideration must be given to the regula- In very extreme circumstances, the FDA may
tions and requirements of each of these agen- issue emergency guidance to help ensure an
cies when developing emergency response adequate blood supply. For example, on Sep-
plans. Such regulatory considerations generally tember 11, 2001, the FDA issued the Policy
fall into three categories: effect on available Statement on Urgent Collection, Shipment,
blood components, effect on donors, and po- and Use of Whole Blood and Blood Compo-
tential consequences for operations. nents Intended for Transfusion to Address
Blood Supply Needs in the Current Disaster
Effects on Components in Available Situation.40 Blood collection facilities should
Inventory closely follow all developments regarding do-
nor deferrals and all emergency FDA guidance
All blood centers and hospitals must have
written procedures to follow in an emergency. during disasters.
These procedures should address the provi-
Effects on Donors
sion of emergency electrical power and con-
tinuous temperature monitoring during stor- Disasters, particularly emergencies involving
age. These procedures should address the potential infectious diseases or hazardous
potential effects of unsuitable storage condi- chemicals, can affect donor eligibility. Infec-
tions, such as extreme (high or low) tempera- tious and chemical agents should be evaluated
tures or exposure to smoke or water.37 Consid- for 1) transmissibility by blood transfusion, 2)
eration should also be given to nontraditional potential to harm recipients, and 3) potential
emergencies that may affect blood, such as a for an asymptomatic interval of infectivity or
radiologic event or exposure to a biologic or toxicity after exposure but before or after any
chemical agent.38 donor illness. If an agent is determined to be
Blood components that have potentially transmissible, a deferral period that ensures
been exposed in an event should be quaran- an adequate period of safety from infectivity or
tined, and a determination of component suit- adverse effect must be estimated, and donors
ability should be made before the components must be deferred appropriately. As with com-
are returned to inventory. When the quality, ponent suitability, CBER and the AABB Disas-
safety, purity, or potency of a component is in ter Task Force should be consulted about po-
question, CBER should be consulted. An ex- tential donor deferral issues.
ception or variance may be needed to use
components in such situations.39 In addition, Potential Consequences on
the AABB Disaster Task Force is available to Operations
assist facilities with questions about a compo-
nent’s safety, purity, or potency during a Emergencies involving power outages, floods,
disaster.2 or structural damage to facilities disrupt nor-
All blood released for use during a disas- mal operations. Disasters that do not directly
ter should be fully tested, including for infec- affect the blood center’s or hospital’s physical
tious diseases. An exception to full testing infrastructure can still disrupt operations. For
should be made in the event that blood sup- example, a pandemic influenza outbreak may
plies are exhausted, resupply is not possible, cause severe staffing shortages with up to a
and blood is needed immediately to save lives. 40% absenteeism rate.17 Collections should be
Blood-testing samples should be retained for performed only by facilities that routinely
retrospective testing after a disaster. Thor- collect blood. Facilities that collect only autol-
ough documentation of the circumstances of ogous blood should not attempt to collect allo-
the disaster is essential, and physicians should geneic blood during a disaster.
CHAPTER 4 Disaster Management 䡲 111
Adequate staffing can be challenging im- a facility can identify and correct deficiencies
mediately before, during, and after an emer- and gaps in its disaster plan.
gency while staff members tend to their fami-
lies and personal needs. Only staff trained in Internal and External Disaster Drills
regulated functions should perform these
Drills can be conducted internally (eg, a fire
functions. The FDA recommends that blood
drill or simulated hazardous spill cleanup) or
establishments have adequate backup person-
in conjunction with other organizations. Mul-
nel in the event of a disaster and that more
tiple-organization disaster drills are typically
than one backup person be trained for each
critical function. This training should be docu- conducted by local or state EMAs. Unfortu-
mented.41 Volunteers can be used to perform nately, blood-related issues are often over-
nonregulated functions, such as predonation looked in the exercise scenarios developed by
education and maintenance of the canteen. these agencies because many of the planners
Likewise, appropriate licensure is required to are unaware of how blood is collected, stored,
safely operate fleet vehicles. and distributed to hospitals. Blood collection
Emergency plans should include lists of facilities should develop relationships with
staff trained in multiple functions. For exam- EMAs, as described in the “Major Disaster
ple, staff members who have transferred from Management Factors for Blood and Transfu-
one area to another (and who have maintained sion” section.
competency in the prior area) could perform
the initial function with appropriate certifica- Participating in Disaster Drills with EMAs
tion and any necessary commercial vehicle li- The National Strategy for Homeland Security
censes. directed the establishment of a national exer-
Equipment and supplies should also be cise strategy. Homeland Security Directive 8
assessed for exposure to water, humidity, and directed the DHS to establish a National Exer-
temperature extremes. CDRH has published cise Program (NEP).43 In addition to full-scale,
an information guide about the effects of di- integrated, national-level exercises, the NEP
sasters on medical devices; it can be useful in provides tailored exercise activities that serve
planning as well as responding to disasters.42 as the primary DHS vehicle for training na-
tional leaders and staff. The NEP enhances
Records Management collaborations among partners at all levels of
Vital records must be secured and maintained. government for assigned homeland security
These documents include records of donors, missions. National-level exercises provide the
donations, manufacturing, testing, quality as- means to conduct “full-scale, full-system
surance, and product disposition. If these rec- tests” of collective preparedness, interopera-
ords are damaged or lost during a disaster, bility, and collaboration across all levels of
blood in inventory at that time may require government and the private sector.44 The
quarantine and recall. Efforts should be made program also incorporates elements to allow
to retrieve and preserve any damaged records. identification of any implications of changes
CBER should be consulted to determine the to homeland security strategies, plans, tech-
disposition of inventory when records are lost. nologies, policies, and procedures.
Blood centers and hospitals should seek
inclusion in disaster drills and exercises by con-
TE S TI N G TH E DI S AST E R PL A N
tacting their local EMAs. Participation in local,
Continual improvement of a disaster plan is state, and federal exercises increases a facility’s
critical for maintaining a high state of readi- preparedness. This participation also increases
ness for future emergencies, and participation awareness by the emergency preparedness offi-
in disaster drills is one way to achieve this goal. cials at all levels of the critical role that blood
By simulating the conditions of a real disaster, centers play in providing the blood compo-
112 䡲 AABB TECHNICAL MANUAL
nents required by victims of a disaster and the disasters in recent years. Hurricanes, tsuna-
resources that are required to do so. mis, earthquakes, tornados, fires, industrial
accidents, and terrorism have tested emergen-
Including Blood Issues in Drills cy response systems. Organizations have used
the learning points from these events to fur-
Disaster drills range from informal discussions
ther sharpen their disaster plans. Many of
about improving response methods to full-
these lessons can be found in the AABB Disas-
scale, real-time exercises that involve hundreds
ter Operations Handbook.2 Blood collection
of organizations. Each method can be used by a
and transfusion facilities are encouraged to
single organization or in conjunction with mul-
share the major learning points from real or
tiple organizations. When an organization par-
simulated disasters that they have experienced
ticipates in drills with other organizations (ie,
with AABB through the AABB National Blood
full-scale exercises), it is important for blood
Exchange at 1.800.458.9388 or by e-mail to
collection and transfusion facilities to “inject”
nbe@aabb.org. AABB is collecting such lessons
blood-related scenarios into the drills during
and sharing them with facilities around the
the planning stages. For example, drills should
world.
address 1) the need to transport blood from a
blood center to a hospital (or hospitals) to treat
victims when local roads have been damaged or CO N CLUS IO N
destroyed and 2) the effect of an unknown bio-
In the wake of disasters—such as the Septem-
logic agent release on the local blood supply,
ber 11, 2001, attacks on the World Trade Cen-
donors (eg, need to defer donors), or both. Ad-
ter and Pentagon; the rail transit bombings in
ditional information on the basic types of drills
Great Britain, Spain, and Boston; and the dev-
can be found on the FEMA Emergency Manage-
astating natural disasters that occur every
ment Institute website.45
year—organizations around the world have fo-
After-Action Review cused significant attention and resources on
strengthening their disaster plans. Given the
Perhaps the most important aspect of a well- heightened awareness of the need for organi-
executed drill or actual disaster is the after-ac- zations to be prepared, both the public and
tion review process. During this process, par- members of the media are holding organiza-
ticipants evaluate what worked well and what tions to the highest standards of excellence for
did not work well, with the aim of identifying ensuring that all necessary planning has been
lessons learned and implementing corrective done to protect both life and property from
actions to improve future responses. known threats. Blood collection and transfu-
sion facilities are not immune to this reality
S U M M A RY O F LE S S O N S and should therefore strive to maintain the
highest standards of disaster preparedness.
LEAR NED FROM RECENT
Above all else, the most important benefit of
D I S A ST E R S
careful and continual planning is assurance
Blood collection facilities and hospitals that blood and blood components are avail-
around the world have dealt with significant able to patients when and where needed.
KEY POINTS
1. Planning is the key to success during a disaster. Disaster planning is not a one-time event; it
requires continual review, exercises and practice, and learning and changing.
2. Continuity of operations planning provides a framework for facilities to maintain or restore the
critical functions necessary to collect, manufacture, store, and distribute blood components.
CHAPTER 4 Disaster Management 䡲 113
3. The cornerstone of an effective disaster plan is a thorough risk assessment of the probable
hazards and their impact on critical operations. Risks can include internal events, such as a
broken pipe that floods a laboratory, or external events, such as tornadoes and earthquakes.
4. Facilities should use the resources of the AABB Interorganizational Task Force on Domestic
Disasters and Acts of Terrorism in disaster management planning (eg, Disaster Operations
Handbook) and incorporate the task force’s national response procedures into local plans.
5. The ability to communicate during a disaster determines much of the success of a response;
facilities need well-developed and routinely tested communication plans.
6. Hospitals should have emergency plans for triage of blood components in disasters in
which demand exceeds supply (eg, a severe pandemic or nuclear or biologic attack). Blood
centers should work with their hospital customers to ensure that such plans are in place.
7. Adequate cash reserves and insurance are essential for a blood center to survive a major di-
saster.
8. Coordination with local emergency management officials before a disaster is critical to suc-
cess during that disaster.
9. Regulatory considerations during a disaster include the effects on the blood components on
the shelf, the donors in the affected area, and operations.
10. Facilities should routinely reinforce their disaster plan by conducting information sessions
with staff, volunteers and their families, suppliers, and hospital customers. The disaster
plan should be tested through participation in local or regional disaster drills.
R E F E R EN C E S
11. Public health emergency: National disaster (states, territories, tribal and local government
medical system. Washington, DC: Department jurisdictions and private sector organization).
of Health and Human Services, 2013. [Avail- Washington, DC: Federal Emergency Manage-
able at: http://www.phe.gov/emergency/pag ment Agency, 2009. [Available at http://
es/default.aspx (accessed May 27, 2013).] www.fema.gov/pdf/about/org/ncp/coop/
12. Hess JR, Thomas MJG. Blood use in war and continuity_guidance_circular.pdf (accessed
disaster: Lessons from the past century. Trans- May 27, 2013).]
fusion 2003;43:1622-33. 24. 10 things to do to prepare: A step by step guide
13. Linden JV, Davey RJ, Burch JW. The September to building your disaster preparedness plan.
11, 2001, disaster and the New York blood sup- Hartford, CT: The Hartford Financial Services,
ply. Transfusion 2002;42:1385-7. 2013. [Available at http://thehartford.com/
14. Schmidt PJ. Blood and disaster—supply and business/disaster-preparedness-planning-
demand (editorial). N Engl J Med 2002;346: guide (accessed November 8, 2013).]
617-20. 25. Emergency preparedness. Washington, DC:
15. Klein HG. Earthquake in America. Transfusion Small Business Administration, 2013. [Avail-
2001;41:1179-80. able at http://www.sba.gov/prepare (accessed
16. Glascow SM, Allard S, Doughty H, et al. Blood May 27, 2013).]
and bombs: The demand and use of blood fol- 26. Levitt J, ed. Standards for blood banks and
lowing the London bombing of 7 July 2005—a transfusion services. 29th ed. Bethesda, MD:
retrospective review. Transfus Med 2012;22: AABB, 2014.
244-50. 27. Comprehensive accreditation manual for hos-
17. Pandemic influenza update. Association Bul- pitals (CAMH). Oak Brook Terrace, IL: The
letin #09-07. Bethesda, MD: AABB, 2009. Joint Commission, 2012.
18. Pandemic influenza preparedness and re- 28. Disaster response plan. Sacramento, CA: Cali-
sponse guidance for healthcare workers and fornia Blood Bank Society, 2012. [Available at
healthcare employers. Washington, DC: Occu- http://www.cbbsweb.org/attachmnts/cbbsdi
pational Safety and Health Administration, sasterplan.pdf (accessed May 27, 2013).]
2009. [Available at http://www.osha.gov/Pub 29. Myers KN. Manager’s guide to contingency
lications/OSHA_pandemic_health.pdf (ac- planning for disasters. New York: John Wiley
cessed May 27, 2013).] and Sons, 1999.
19. Healthcare emergency preparedness. St. Paul, 30. Business insurance. Washington, DC: Small
MN: Minnesota Department of Public Health, Business Administration, 2013. [Available at
2013. [Available at http://www.health.state. http://www.sba.gov/content/business-insur
mn.us/oep/healthcare/in dex.html (accessed ance (accessed May 27, 2013).]
November 8, 2013).] 31. The conference on emergency response plan-
20. When healthcare resources are scarce. St Paul, ning for your business. Mission, KS: SkillPath
MN: Minnesota Department of Health, 2012. Seminars, 2002.
[Available at http://www.health.state.mn.us/ 32. Gannon B. The best laid plans. Presentation at
oep/healthcare/scarce/index.html (accessed America’s Blood Centers 2006 Annual Meeting.
November 8, 2013).] Houston, TX, March 4-6, 2006.
21. Open for business: A disaster planning toolkit 33. Reynolds B, Seeger M. Crisis and emergency
for the small to mid-sized business owner. risk communication (CERC). Atlanta, GA: Cen-
Tampa, FL: Institute for Business and Home ters for Disease Control and Prevention, 2012:
Safety, 2007. [Available at http://www.disaster 2-16, 157-9.
safety.org/wp-content/uploads/open-for- 34. Covello VT. Invited paper presented at the
business-english.pdf (accessed November 8, 2002 World Health Organization conference
2013).] on bio-terrorism and risk communication.
22. Ready business: Preparedness planning for Geneva, Switzerland, October 1, 2002.
your business. Washington, DC: Federal Emer- 35. Li-Vollmer M. Emergency risk communica-
gency Management Agency, 2013. [Available at tion: An online course. Seattle, WA: Northwest
http://www.ready.gov/business (accessed No- Center for Public Health Practice, University of
vember 8, 2013)] Washington School of Public Health, 2013.
23. Federal Continuity Guidance Circular 1 (CGC1). [Available at http://www.nwcphp.org/train
Continuity guidance for non-federal entities ing/opportunities/online-courses/emergen
CHAPTER 4 Disaster Management 䡲 115
Anne F. Eder, MD, PhD, Executive Medical Officer, American Red Cross, National Headquarters, Biomedical
Services Medical Office, Holland Laboratory, Rockville, Maryland; and Maria D.L.A. Muniz, MD, Clinical
Pathologist, Allegheny Health Network, Pittsburgh, Pennsylvania
The authors have disclosed no conflicts of interest.
117
118 䡲 AABB TECHNICAL MANUAL
If individuals are instructed not to donate eligibility policies on issues that are not cov-
blood for others because of their health histo- ered by regulations or standards.3,8-11 Conse-
ry, reactive test results, behavioral risks, or quently, medical decisions regarding the same
medical reasons, they may be added to a confi- issue may differ among blood centers or even
dential deferral list to prevent future dona- among physicians at the same blood center.
tions. Deferrals may be for a defined interval of Considerable variability exists in national and
time or an indefinite period, or they may be international practices for determining donor
permanent with no potential for reinstate- eligibility, which reveals the inherent uncer-
ment as a blood donor in the future.1 In addi- tainty in risk assessment.8
tion, blood centers are required to manage in- A precautionary approach attempts to
formation received after the donation that eliminate the risk of known or potential trans-
could affect the safety, quality or potency of fusion-transmitted diseases from the blood
the donated blood components and the future supply but also results in the disqualification
eligibility of the donor, and keep records of de- of large segments of the healthy population.
ferred individuals.2 Prospective donors who are deferred from
The criteria to evaluate individuals who blood donation may be disappointed, angry,
are donating blood for their own use (ie, autol- or confused about the relevance that certain
ogous donation) may be less stringent than questions have to their health or ability to do-
those for allogeneic donation. However, the fo- nate blood for transfusion to others. Blood
cus remains on providing the safest possible center staff should be able to explain the in-
blood for transfusion to the donor-patient and tended purpose of AABB and FDA require-
on evaluating the risks that the collection pro- ments as well as their center-specific eligibility
cedure poses to his or her health. screening practices.
The blood center must determine donor The most frequently asked questions
eligibility prior to collection and on the day of about federal regulations defining blood do-
collection, in accordance with federal and nor eligibility are available to the public in the
state regulations and voluntary accreditation “Questions about Blood” section of the FDA
standards. Specific criteria used to select do- website.12 Questions about the interpretation
nors are established by the Food and Drug Ad- or underlying rationale of AABB Standards for
ministration (FDA) through the Code of Feder- Blood Banks and Transfusion Services should
al Regulations (CFR), guidance documents, be directed to the AABB Standards Depart-
and memoranda to industry.2 The AABB also ment (standards@aabb.org); responses and
develops professional standards for donor se- discussion of selected issues are posted on the
lection with which accredited blood establish- AABB website.
ments must comply.1
An industry-wide effort led by AABB to
streamline and standardize donor screening SELECTION OF ALLO GENEIC
culminated with the release by the FDA, in Oc- B LO OD D ON O R S
tober 2006, of a final guidance document rec- Registration and Donor Identification
ognizing the donor history questionnaire
(DHQ) as an effective donor screening tool In the United States, blood components for al-
that provides licensed and unlicensed facilities logeneic transfusion are typically collected
with a way to meet all FDA requirements.4 The from volunteer, nonremunerated donors; oth-
DHQ is currently used by most blood centers erwise, they must be labeled as being from
in the United States.5,6 The references in this paid donors.2
chapter are to DHQ Version 1.3 (May 2008), Prospective blood donors should provide
which the FDA officially recognized as accept- an acceptable form of identification, and each
able DHQ documentation in May 2010.4,7 donor must be properly identified by the col-
Medical directors of blood collection fa- lection staff before each donation. Acceptable
cilities are responsible for determining donor forms of identification include government-
CHAPTER 5 Allogeneic and Autologous Blood Donor Selection 䡲 119
issued documents, such as a driver’s license or should explain the collection procedure to the
passport, or a blood-center-issued donor card donor in terms that the donor understands
with a unique alphanumeric code. Many and document the donor’s consent, which in-
blood collectors no longer use social security dicates that the donor has considered all the
numbers for donor identification because of educational materials and has had an oppor-
regulations restricting their use and privacy tunity to ask questions.1(p16) The donor should
concerns. be informed about possible adverse reactions
Accurate records are essential to identify to the collection procedure and the infectious
all prior donations from any given donor, in- disease tests that will be performed on his or
cluding whether the donor has ever given her donated blood components. The donor
blood under a different name, so that the link should also be informed of the notification
with all prior donations within the blood sys- process for positive test results, any reporting
tem is maintained. Accurate records are also requirements to federal or state health author-
essential to ensure that the donor can be con- ities, and the possibility of inclusion in the do-
tacted in the weeks following the donation and nor center’s deferral registry and subsequent
informed of test results or other relevant infor- deferral from future donation. The individual
mation from the current donation, if neces- should agree not to donate if his or her blood
sary. Blood centers must make reasonable at- could pose a risk to the blood supply. Donors
tempts to notify the donor within 8 weeks of should also be informed if investigational tests
the donation if any test results disqualify the or other research may be performed on sam-
individual from continued donation.2 Blood ples or information collected during the blood
centers often require donors to provide an ad- donation. Finally, the limitations of the tests to
dress and telephone number where they can detect early infections and the possibility that
be reached during this interval for counseling a test may not be performed if samples are not
or other follow-up, if necessary. Accurate do- adequate should be explained to the donor.
nation records must be kept by the donor cen- No one should donate blood for the pur-
ter for the requisite amount of time, according pose of receiving free infectious disease test-
to current regulations and standards.1,2 ing. Information about alternative HIV test
Notably, there is currently no national sites or public health options to obtain HIV
registry of deferred blood donors in the United tests should be provided to all prospective
States, and individuals who are deferred by donors.
one blood center may be eligible in another All prospective donors must be able to
blood system. The available evidence suggests give informed consent and provide an accu-
that such donor deferral registries are not nec- rate health history. They should be given an
essary because they do not contribute to blood opportunity to ask questions and they have
safety and do not prevent the release of unsuit- the right to withdraw from the process at any
able components.13 time. Blood donation centers should docu-
ment donors’ consent to have their blood col-
Educational Materials and Donor lected and tested.
Consent Blood centers must comply with applica-
ble state laws to obtain permission from par-
US blood centers provide all prospective blood ents or guardians for minors (ie, 16- or 17-year
donors with information about blood dona- olds) or legally incompetent adults.1(p16) More-
tion during the informed consent process. The over, AABB Standard 5.2.2 requires that collec-
DHQ educational materials incorporate all tion facilities have a process to provide infor-
necessary elements required by federal regula- mation about the donation process to parents
tions and AABB Standards, and blood centers or legally authorized representatives of donors
have added elements to protect both blood when parental permission is required.1(p16)
donors and transfusion recipients.1(pp15-16,60-63),4 Donor centers should also establish poli-
At each encounter, the blood center staff cies on accommodating individuals who are
120 䡲 AABB TECHNICAL MANUAL
not fluent in English or are illiterate, are vision crit measurement. These tests have potential
or hearing impaired, or have other physical implications for the potency or safety of the
disabilities. Many blood centers try to make collected component (Table 5-1). Additional
reasonable accommodations for donors’ spe- requirements apply to apheresis donors, who
cial needs. However, centers must also ensure must meet the weight and hemoglobin or he-
that the collection procedure does not pose matocrit requirements approved by the FDA
undue risk to donors or staff members and for the automated collection device.
that the informed consent process is not com- Prior to phlebotomy, the collection staff
promised. The final authority for such deci- inspect the donor’s antecubital skin, which
sions rests with the donor center’s physician must be free of lesions and evidence of injec-
who supervises donor qualification and phle- tion drug use, such as multiple needle punc-
botomy.1(p1) tures (eg, small scars lined up in “tracks”) or
sclerotic veins. Scars or pitting on the forearm
Donor Qualification by Focused
associated with frequent blood donation
Physical Examination and
should not be mistaken for evidence of injec-
Hemoglobin or Hematocrit tion drug use. Common and mild skin disor-
Measurement ders (eg, poison ivy rash) are not a cause for
Qualification procedures for blood donation deferral unless there are signs of localized bac-
include a focused physical examination (eg, terial superinfection or the condition inter-
taking the donor’s temperature and inspecting feres with proper skin disinfection in the ante-
his or her arm) and a hemoglobin or hemato- cubital site before phlebotomy.
TABLE 5-1. Physical Examination and Requirements for Allogeneic and Autologous Whole-Blood Donation
Allogeneic Autologous
AABB Reference Standard 5.4.1A; Title 21, AABB Standard 5.4.4; Title 21, CFR
CFR Part 640.3 Part 640.3
The FDA defines the minimum accept- In general, neither the FDA nor the AABB
able hemoglobin concentration for both men specifies the test method, specimen type (cap-
and women as 12.5 g/dL, and the essentially illary, venous blood), or acceptable perfor-
equivalent hematocrit requirement of 38%.2 mance characteristics for tests used for hemo-
This requirement is controversial and is peri- globin/hematocrit screening. One exception is
odically debated because normal hemoglobin that a capillary sample collected from an ear-
values are influenced by gender, race, nutri- lobe puncture is not an acceptable specimen
tional status, and other factors.14,15 In particu- for hemoglobin/hematocrit screening for allo-
lar, a single acceptance criterion for men and geneic or autologous donors.1(pp19,60) Most US
women is contentious because normal hemo- blood centers use fingerstick capillary samples
globin values for women are lower than those for hemoglobin determination. These sam-
for men. However, this requirement has re- ples tend to give slightly higher hemoglobin
mained unchanged for more than 30 years. values than venous samples.
Hemoglobin or hematocrit screening may pre- The methods to measure hemoglobin or
vent collection of blood from a donor with sig- hematocrit are generally selected for their ease
nificant anemia, which could have implica- of use in the mobile blood collection setting.
tions for the health of the donor as well as the The copper sulfate density method (Method 6-
potency of the collected component. 1) is still an acceptable screening tool in blood
A hemoglobin level lower than the 12.5 g/dL centers in the United States but has been
cutoff is the most common reason for disqual- largely replaced by methods such as spectro-
ification of potential blood donors. This single photometric measurement of hemoglobin
deferral criterion can be problematic for both with portable devices (eg, HemoCue Donor Hb
male and female donors. Some female donors Checker, HemoCue AB, Angelholm, Sweden;
with hemoglobin levels below the 12.5 g/dL DiaSpect Hemoglobin, DiaSpect Medical AB,
cutoff are actually in the normal range. In con- Uppsala, Sweden) or hematology analyzers.
trast, some male donors with a hemoglobin The point-of-care methods that use portable
level above 12.5 g/dL may, in fact, be anemic. devices yield quantitative hemoglobin results,
Furthermore, hemoglobin screening does not with a coefficient of variation (CV) of 1.5%.15 A
ensure that the donor has an adequate store of typical automated analyzer measures hemo-
iron. 16,17 Debate continues on the minimum globin levels in a venous sample with a CV
hemoglobin requirement for blood donation, 1.2%.15 Most quantitative methods currently
frequent donors’ iron status, and allowable do- in use reliably measure hemoglobin levels
nation intervals.18 within approximately 0.2 to 0.5 g/dL, and the
Donor hemoglobin screening may ensure vast majority of deferred donors have hemo-
a minimum content of hemoglobin in a unit of globin or hematocrit values near the cutoff
Red Blood Cells (RBCs), but currently neither value. For capillary-sample-based methods,
the FDA nor the AABB define potency stan- the most likely source of preanalytical error is
dards for RBC units prepared from whole- the sampling technique, and testing must be
blood collection. If a donor’s hemoglobin level performed in compliance with the manufac-
is 12.5 g/dL, a 500-mL whole-blood collection turer’s instructions.
is expected to yield about 62.5 g of hemoglobin
per unit of RBCs, but determining the final Health History Assessment—DHQ
content of hemoglobin in an RBC unit pre-
pared from whole blood is not required. AABB The AABB DHQ is now used by most blood
Standards require apheresis RBC units to be centers in the United States, although its use is
prepared using a method known to ensure a fi- not currently mandated by the FDA (Table
nal component containing a mean hemoglo- 5-2).19 Alternative procedures for collecting
bin level of 60 g of hemoglobin, with 95% of required information from blood donors are
the units sampled containing more than 50 g subject to FDA review and acceptance. In ad-
of hemoglobin.1(p27) dition, the FDA acknowledges that the DHQ
122 䡲 AABB TECHNICAL MANUAL
TABLE 5-2. Donor History Questionnaire and Eligibility Requirements for Allogeneic Blood Donations19
1. Are you feeling healthy and well today? The prospective donor shall appear to be in good health and
shall be free of major organ disease (eg, heart, liver, lungs),
cancer, or abnormal bleeding tendency, unless determined
eligible by the medical director.
Variable, temporary deferral criteria as defined by the medi-
cal director.
2. Are you currently taking an antibiotic? Variable, temporary deferral if treated for active infections,
as defined by the medical director.
No deferral for prophylactic (preventive) use of antibiotics
(eg, tetracycline for acne).
3. Are you currently taking any other medica- Variable, temporary deferral if treated for active infections.
tion for an infection?
4. Are you now taking or have you ever taken Examples:
any medications on the Medication Deferral Potent teratogens (potential for harm to unborn children):
List?
䡲 Finasteride (Proscar, Propecia), isotretinoin (Absorica,
Accutane, Amnesteem, Claravis, Myorisan, Sotret, Zena-
tane): Defer for 1 month after last dose.
䡲 Dutasteride (Avodart, Jalyn): Defer for 6 months after last
dose.
䡲 Acitretin (Soriatane): Defer for 3 years after last dose.
䡲 Etretinate (Tegison): Defer indefinitely.
TABLE 5-2. Donor History Questionnaire and Eligibility Requirements for Allogeneic Blood Donations19
(Continued)
9. In the past 8 weeks have you had any No deferral for receipt of toxoids or synthetic or killed viral,
vaccinations or other shots? bacterial, or rickettsial vaccines listed below if the donor is
symptom free and afebrile.
䡲 Anthrax, cholera, diphtheria, hepatitis A, hepatitis B, influ-
enza, Lyme disease, paratyphoid, pertussis, plague, pneu-
mococcal polysaccharide, polio (Salk/injection), Rocky
Mountain spotted fever, tetanus, or typhoid (by injection).
(Continued)
124 䡲 AABB TECHNICAL MANUAL
TABLE 5-2. Donor History Questionnaire and Eligibility Requirements for Allogeneic Blood Donations19
(Continued)
15. In the past 12 months have you come into Defer for 12 months
contact with someone else’s blood? 䡲 From the time of mucous membrane exposure to blood.
16. In the past 12 months have you had an 䡲 For nonsterile skin penetration with instruments or equip-
accidental needle-stick? ment contaminated with blood or body fluids other than
the donor’s own.
17. In the past 12 months have you had sexual
contact with anyone who has HIV/AIDS or has
had a positive test for the HIV/AIDS virus?
18. In the past 12 months have you had sexual
contact with a prostitute or anyone else who
takes money or drugs or other payment for
sex?
19. In the past 12 months have you had sexual Defer for 12 months from the time of sexual contact with an
contact with anyone who has ever used nee- individual with HIV infection or at high risk of HIV infection.
dles to take drugs or steroids, or anything not
prescribed by their doctor?
20. In the past 12 months have you had sexual
contact with anyone who has hemophilia or
has used clotting factor concentrates?
21. Female donors: In the past 12 months have
you had sexual contact with a male who has
ever had sexual contact with another male?
(Males: check “I am male.”)
22. In the past 12 months have you had sexual Defer for 12 months for sexual contact or for having lived
contact with a person who has hepatitis? with an individual who meets any of the following criteria:
23. In the past 12 months have you lived with 䡲 Has acute or chronic hepatitis B (positive hepatitis B sur-
a person who has hepatitis? face antigen test).
䡲 Has symptomatic hepatitis C.
䡲 Is symptomatic for any other viral hepatitis.
24. In the past 12 months have you had a Defer for 12 months from the time of nonsterile skin penetra-
tattoo? tion with instruments or equipment contaminated with blood
or bodily fluids other than the donor’s own, including for tat-
25. In the past 12 months have you had ear
toos or permanent makeup unless, applied by a stage-regu-
or body piercing?
lated entity with sterile needles and ink that is not reused.
26. In the past 12 months have you had or Defer for 12 months or for longest applicable period for the
been treated for syphilis or gonorrhea? following situations:
䡲 Following the completion of treatment for syphilis or gon-
orrhea.
䡲 For a reactive screening test for syphilis where no confir-
matory testing was performed.
䡲 For a confirmed positive test for syphilis (FDA reentry pro-
tocol after 1 year applies).
CHAPTER 5 Allogeneic and Autologous Blood Donor Selection 䡲 125
TABLE 5-2. Donor History Questionnaire and Eligibility Requirements for Allogeneic Blood Donations19
(Continued)
27. In the past 12 months have you been in Defer for 12 months.
juvenile detention, lockup, jail, or prison for
more than 72 hours?
28. In the past three years have you been out- Malaria:
side the United States or Canada? 䡲 Defer for 3 years after departure from malaria-endemic
area(s) for individuals(s) who have lived for longer than
5 consecutive years in a country(ies) with areas consid-
ered endemic by the Malarial Branch, Centers for Disease
Control and Prevention, US Department of Health and
Human Services.
䡲 Defer for 12 months after departure from malaria-
endemic area(s) for other individuals who have traveled to
an area where malaria is endemic.
29. From 1980 through 1996, did you spend
time that adds up to three (3) months or more
in the United Kingdom? (Review list of coun-
tries in the UK.)
30. From 1980 through 1996, were you a
member of the US military, a civilian military
employee, or a dependent of a member of the
U.S. military? Defer indefinitely for risk of vCJD, as defined in most recent
31. From 1980 to the present, did you spend FDA guidance.
time that adds up to five (5) years or more in
Europe? (Review list of countries in Europe.)
32. From 1980 to the present, did you receive
a blood transfusion in the United Kingdom?
(Review list of countries in the UK.)
33. From 1977 to the present, have you received
money, drugs, or other payment for sex? Defer indefinitely donors:
34. Male donor: From 1977 to the present, 䡲 Excluded by current FDA regulations and recommenda-
have you had sexual contact with another tions for the prevention of HIV transmission by blood and
male, even once? (Female donors: Check components.
“I am female.”) 䡲 With present or past clinical or laboratory evidence of
infection with HCV, HTLV, or HIV or as excluded by cur-
35. Have you EVER had a positive test for the rent FDA regulations.
HIV/AIDS virus? 䡲 For use of a needle to administer nonprescription drugs.
36. Have you EVER used needles to take
drugs, steroids, or anything not prescribed by
your doctor?
(Continued)
126 䡲 AABB TECHNICAL MANUAL
TABLE 5-2. Donor History Questionnaire and Eligibility Requirements for Allogeneic Blood Donations19
(Continued)
37. Have you EVER used clotting factor 䡲 Defer as directed by the medical director donors with
concentrates? abnormal bleeding tendency.
䡲 Defer for 12 months for receipt of blood, components,
human tissues, or plasma-derived clotting factor concen-
trates.
38. Have you EVER had hepatitis? Defer indefinitely for history of viral hepatitis after 11th
birthday.
39. Have you EVER had malaria? Defer for 3 years after becoming asymptomatic prospective
donors who have had a diagnosis of malaria.
40. Have you EVER had Chagas disease? Defer indefinitely for a history of babesiosis or Chagas
disease.
41. Have you EVER had babesiosis?
42. Have you EVER received a dura mater Defer indefinitely for receipt of human (cadaveric) dura
(or brain covering) graft? mater.
43. Have you EVER had any type of cancer, Variable deferral criteria, as defined by medical director.
including leukemia? Examples:
䡲 Nonhematologic cancer: Defer for 5 years after comple-
tion of treatment.
䡲 Local skin cancer, in situ cancer: No deferral if treated
completely with excision and healed.
䡲 Hematologic cancer (eg, leukemia): Defer indefinitely.
44. Have you EVER had any problems with Variable deferral criteria, as defined by medical director.
your heart or lungs?
45. Have you EVER had a bleeding condition or Variable deferral criteria, as defined by medical director.
a blood disease?
46. Have you EVER had sexual contact with Defer indefinitely, as excluded by current FDA regulations
anyone who was born in or lived in Africa? and recommendations for the prevention of HIV transmis-
sion by blood and blood components.
47. Have you EVER been in Africa?
The questions for risk of HIV group O can be deleted from
the DHQ if the blood center is using an HIV antibody test
that has been approved by FDA to include a claim for detec-
tion of HIV group O.
48. Have any of your relatives had Creutzfeldt- Defer indefinitely for family history of Creutzfeldt-Jakob
Jakob disease? disease.
vCJD = variant Creutzfeldt-Jakob disease; N/A = not applicable; HIV = human immunodeficiency virus; AIDS = acquired
immunodeficiency syndrome; FDA = Food and Drug Administration; UK = United Kingdom; HCV = hepatitis C virus; HTLV =
human T-cell lymphotropic virus.
CHAPTER 5 Allogeneic and Autologous Blood Donor Selection 䡲 127
documents contain questions related to the medical condition or history poses to the
following issues not addressed by any FDA re- blood donor and transfusion recipients.
quirement or recommendation: cancer; organ, If a potential exists for risk to the recipient
tissue, or marrow transplants; bone or skin or donor, the effectiveness and incremental
grafts; and pregnancy. However, AABB recom- benefit of screening donors by questioning
mends that blood centers implement the DHQ should be evaluated, especially in light of oth-
materials, including the following documents, er safeguards that protect the donor or other
in their entirety: transfusion practices that mitigate potential
risks to the recipient. If the blood center re-
䡲 Blood donor educational materials. ceives information after the donation that
䡲 Full-length DHQ. would have been cause for deferral had it been
䡲 User brochure, including glossary and ref- reported at the time of donation, then any
erences. subsequent actions, such as product retrieval,
䡲 Medication Deferral List. market withdrawal, or consignee notification,
should be commensurate with the potential
The use of the DHQ flow charts is option- hazard and likelihood of possible harm to the
al. All DHQ documents that the FDA has rec- recipient. The center’s approach to develop-
ognized as acceptable are available on the FDA ing donor deferral criteria should take into ac-
website. 20 The most current version is also count evidence as it becomes available to
available on the AABB website.19 modify those decisions. Some of these issues
The wording, order, and text of the DHQ that allow for medical judgment and for which
questions should not be changed. A collection questions exist in the DHQ can be explored
further with the donor, but each donor center
facility may make minor changes to the time
must develop and follow its own SOPs.3
frame of a question on the DHQ to make the
question more restrictive so that its use results
in the deferral of more donors. Blood centers B LO OD - C E NT E R - DE F I N ED
may choose to include additional questions as D O N O R E L IG IBI L I T Y C R I TE R I A
long as they place these questions at the end of
Unlike questions about potential risks to
the DHQ. The DHQ documents are intended
transfusion recipients, selection criteria di-
to be self-administered by the donor, but
rected primarily at protecting donor safety are
blood centers may choose to use direct oral
left to the discretion of the blood center’s med-
questioning, self-administration, or a combi-
ical director. Consequently, practice varies at
nation of both methods to administer the
different blood centers.3,8 AABB Standards re-
DHQ.
quires that prospective donors appear to be in
If AABB standards or FDA regulations do
good health and be free of major organ disease
not address specific medical conditions that a (eg, diseases of the heart, liver, or lungs), can-
blood center has chosen to include in the cer, or abnormal bleeding tendency, unless de-
DHQ, the blood center must develop standard termined eligible by the medical director.1(p61)
operating procedures (SOPs) for determining The rationale for each deferral for medical
the criteria for acceptance or deferral of a do- conditions should be carefully considered be-
nor. A rational approach to donor health histo- cause even temporary deferrals adversely af-
ry assessment attempts to balance the need to fect the likelihood that individuals will return
take appropriate precautions to protect the to donate blood.21 Donor centers have
blood supply with avoiding unnecessarily re- successfully eliminated some health-related
strictive policies that disqualify large segments questions and removed screening require-
of the population without contributing to ei- ments, such as assessment of pulse, without a
ther recipient or donor safety. 3 Decisions deleterious effect on donor health or dona-
about donor eligibility should be based on tion-related reactions.4,22 These findings sup-
available evidence regarding the risk that the port the conclusion that some arbitrary and
128 䡲 AABB TECHNICAL MANUAL
rigid selection criteria, ostensibly intended to period after completion of treatment provided
protect donors, are unnecessary and can be that the donor remains symptom free without
safely eliminated. relapse. The deferral period following comple-
tion of treatment for nonhematologic cancer
Cancer ranges from 1 to 5 years.24 Hematologic malig-
nancy typically results in permanent deferral
Each year in the United States, blood centers
from allogeneic blood donation, although
receive hundreds of reports of cancer in indi-
some US blood centers reportedly accept
viduals who had donated blood.
adults if they have been successfully treated
Direct transmission of cancer through
for childhood leukemia or lymphoma and
blood transfusion—although biologically
have had a long (eg, 10- to 20-year) cancer-free
plausible—has not yet been documented to
period following completion of treatment.
occur even though people with cancer fre-
These various deferral policies are currently
quently donate blood before discovering their
defensible but should be reevaluated if new in-
diagnosis. 23 A retrospective study examined
formation becomes available about the poten-
the incidence of cancer among patients in
tial for cancer transmission through blood
Denmark and Sweden who received blood
transfusion.
from donors with subclinical cancer at the
time of donation.24 Of the 354,094 transfusion
Bleeding Conditions or Blood Diseases
recipients, 12,012 (3%) were exposed to blood
components from precancerous donors, yet Bleeding conditions and blood diseases have
there was no excess risk of cancer among these the potential to affect donor safety as well as
recipients compared with recipients of blood product potency, and blood centers must de-
from donors without cancer.24 These data indi- fine their procedures for handling donors with
cate that cancer transmission by blood collect- hematologic disorders. In general, prospective
ed from blood donors with incident cancer, if donors should be evaluated for bleeding con-
it occurs at all, is so rare that it could not be de- ditions or blood diseases that 1) place the do-
tected in a large cohort of transfusion recipi- nors at risk of bleeding or thrombosis as a re-
ents that included the total blood experience sult of the collection procedure or 2) may
of two countries over several years. affect the hemostatic efficacy of their blood
In considering the future eligibility of do- and its suitability for transfusion to others.3
nors with cancer, some degree of caution is Plasma components and Cryoprecipitated
warranted to allow sufficient time for donors Antihemophilic Factor should contain ade-
to recover after chemotherapy or other treat- quate amounts of functional coagulation fac-
ment. There are currently no US federal regu- tors and should not contain significant inhibi-
lations or professional standards regarding the tory or prothrombotic factors. Similarly,
criteria that should be used to evaluate donors platelet components intended as the sole
with a history of cancer. For this reason, a source for patients should contain platelets
blood center’s medical director has consider- that have adequate function and are not irre-
able flexibility in determining donor eligibility versibly impaired by the presence of inhibitors.
policies. Individuals with a history of significant
Almost all licensed blood centers current- bleeding complications are usually counseled
ly accept donors who report localized cancers to avoid blood donation. However, screening
after treatment, with no deferral period. These donors for such a history does not prevent the
cancers include skin cancer (eg, basal cell or rare but serious thrombotic or hemorrhagic
squamous cell carcinoma) and carcinoma in complications in otherwise healthy blood
situ (eg, cervical) that have been fully excised donors.
and are considered cured. Most blood centers Individuals with hemophilia, clotting fac-
defer individuals with a history of a solid organ tor deficiencies, or clinically significant inhibi-
or nonhematologic malignancy for a defined tors—all of which are manifested by variable
CHAPTER 5 Allogeneic and Autologous Blood Donor Selection 䡲 129
bleeding tendencies—require deferral for both nosed or treated for cardiac disease. Some do-
donor safety and product potency consider- nor centers advise individuals to wait at least 6
ations. The exception is Factor XII deficiency, months after a cardiac event, procedure, or di-
which is not associated with either bleeding or agnosis. These centers then allow these indi-
thrombosis. viduals to donate if they have been asymptom-
Carriers of autosomal-recessive or sex- atic and able to perform their usual daily
linked recessive mutations in clotting factors activities during this interval.
usually are not at risk of bleeding. They typi- Causes for deferral may include recent
cally have decreased factor levels but are ac- symptoms, limitations on activity or function-
cepted by most blood centers because of the al impairment resulting from unstable angina,
normal, wide variability in clotting factor ac- recent myocardial infarction, left main coro-
tivity levels (50%-150%) compared to the nary disease, ongoing congestive heart failure,
much lower relative activity that is necessary or severe aortic stenosis.3
to maintain hemostasis (5%-30%).3 Individuals
with von Willebrand disease are typically de- Medications
ferred by most blood centers, although some
The DHQ and Medication Deferral List con-
centers may allow individuals with mild dis-
tain the requirements for deferrals for specific
ease and no history of bleeding to donate red medications as stipulated by the AABB and the
cells. Individuals taking anticoagulants to treat FDA. These requisite medication deferrals fall
or prevent venous thromboembolism are de- into five broad categories:
ferred for both donor safety and product po-
tency considerations. 1. Potent teratogens that pose potential harm
to unborn children (although there have
Heart and Lung Conditions been no documented cases of adverse fetal
Cardiovascular disease is common in the Unit- outcomes related to transfusions from
ed States, affecting an estimated 80 million (1 donors taking these medications).
in 3) adults.25 Prospective blood donors are 2. Anticoagulants and antiplatelet agents that
asked if they have ever had problems with affect component potency (plasma or
their heart or lungs as a donor safety measure, platelet components only).
but the criteria for accepting donors with a 3. Potential variant Creutzfeldt-Jakob disease
history of heart or lung disease are defined by risk from bovine insulin manufactured in the
each blood center. United Kingdom (although there have been
The collective, published experience with no documented cases of transfusion trans-
autologous donation by patients scheduled for mission from donors taking bovine insulin).
cardiac procedures has demonstrated that ad- 4. Human-pituitary-derived growth hormone,
verse effects are not more frequent than in do- which theoretically increases the risk of
nors without a history of cardiac disease.26-30 Creutzfeldt-Jakob disease (although there
Despite the relative frequency of cardiovascu- have been no documented cases of transfu-
lar disease in the adult population, vasovagal
sion transmission from donors taking these
reactions occur in only about 2% to 5% of
growth hormones).
whole-blood donations by healthy donors and
5. Antibiotics or antimicrobials to treat an
are actually more likely to occur among young,
healthy adolescents than older adults.31 infection that could be transmitted through
A rational approach to screening donors blood transfusion (excluding preventive
with a history of cardiac disease allows the ac- antibiotics for acne, rosacea, and other
ceptance of donors who are asymptomatic on chronic conditions).
the day of donation, have been medically eval-
uated, and report no functional impairment or Although blood centers may add medica-
limitations on daily activity after being diag- tions whose use requires donor deferral to the
130 䡲 AABB TECHNICAL MANUAL
Medication Deferral List, many have chosen to nized by the FDA as “acceptable” and can be
use the list as developed by the FDA and the implemented by blood establishments using
AABB or have added only a few drugs. The the corresponding full-length DHQ. 33 Two
DHQ task force has encouraged blood centers “capture questions” about new diagnoses or
to fully consider the reasons behind each local treatments since the last donation replace 17
deferral and avoid unnecessary deferral prac- previous questions about remote risks (eg,
tices.3 blood transfusion, Chagas disease, and babe-
FDA’s pregnancy risk categories, which siosis). Although the aDHQ is only somewhat
are designed to assess the benefit vs risk ratio if shorter than the DHQ and its use is restricted
drugs are taken during pregnancy, are often in- to frequent donors, it may improve donors’ ex-
appropriately used for blood donor selection. perience.
For example, categories D and X include some
commonly used drugs (eg, oral contraceptives
and anticholesterol agents) that may be con- RE CIPI ENT-SPE CIF IC
traindicated in pregnancy but pose negligible, “DESIGNATED” OR “DIRECTED”
if any risk, to any transfusion recipient. B LO OD D ON AT I O N
Local medication deferrals are often
based on concerns about the reason for the Exceptional Medical Need
potential donor’s use of the medication and In certain clinical circumstances, a patient
his or her underlying medical condition rather may benefit from blood components collected
than on any inherent threat posed by residual from a specific donor, such as 1) a patient with
medication in the collected blood compo- multiple antibodies or with antibodies to high-
nent. Most drugs used by donors pose no incidence antigens who requires units from
harm to recipients, and many factors should donors whose red cells are negative for the
be considered when evaluating the potential
corresponding antigens or 2) an infant with
risk of a drug’s use by a donor (eg, the medica-
neonatal alloimmune thrombocytopenia who
tion’s half-life, mean and peak plasma concen-
requires antigen-negative platelets from his or
tration, residual concentration in blood com-
her mother.
ponent, and dilution when transfused to a
Frequent donation by a specific donor for
recipient).
a specific patient with a medical need requires
that the donor center have a procedure that
ABBREVIATE D DHQ FOR typically calls for both a request from the pa-
FREQUE N T D O NO R S tient’s physician and approval by the donor
center’s physician. The donor must meet all
Blood centers have recognized for years that
allogeneic donor selection requirements, with
frequent and repeat donors must answer the
the exception of donation frequency, provided
same questions at every donation about re-
mote risk factors that are not likely to that they are examined and certified by a phy-
change—a situation that leaves many dedicat- sician [21 CFR 640.3(f )]. For frequent blood
ed donors dissatisfied with the donation expe- donors, the CFR allows centers to qualify a do-
rience. A small number of US blood centers nor by testing the first donation in each 30-day
have received approval from the FDA and im- period. 2 In emergency medical situations,
plemented an abbreviated DHQ (aDHQ) in blood components can be released before in-
2003 for donors who have successfully com- fectious disease test results are available pro-
pleted the full-length DHQ on at least two sep- vided that the units are labeled and managed
arate occasions and given one or more dona- in accordance with the CFR. Testing on the
tions within the past 6 months.32 units must be completed as soon as possible
The AABB aDHQ, which was developed after release or shipment, and results must be
and validated by the same task force as the promptly reported to the hospital or transfu-
full-length DHQ, has been officially recog- sion service.2
CHAPTER 5 Allogeneic and Autologous Blood Donor Selection 䡲 131
KEY POINTS
1. The DHQ and associated documents were developed by an AABB task force, and their use is
recognized by the FDA as an adequate process to determine the eligibility of volunteers for
allogeneic blood donation.
2. The most current versions of the DHQ and associated documents are available on the AABB
website, and all DHQ documents that the FDA has recognized as acceptable are available on
the FDA website.19,20
3. Prospective blood donors are informed of the risks of blood donation, clinical signs and
symptoms associated with HIV infection, behavioral risk factors for transmission of blood-
borne pathogens, and importance of refraining from blood donation if they are at an in-
creased risk of being infected.
4. To be accepted for allogeneic blood donation, individuals must feel healthy and well on the
day of donation and must meet all AABB and FDA requirements as well as medical criteria
defined by the donor center.
5. In certain clinical circumstances (eg, rare phenotype or antigen-negative Red Blood Cell
units needed), a patient may benefit from blood components collected from a specific do-
nor who may not otherwise meet all the requirements for a volunteer donor. The blood cen-
ter must have procedures and follow federal regulations for frequent donation by a specific
donor for a designated patient with a medical need.
6. The ongoing demand from patients to choose specific donors to provide blood for their
transfusions during scheduled surgeries in the absence of a defined medical need has dra-
matically decreased in the last 10 years but persists despite the lack of evidence of improved
safety with directed donations.
REFER ENCES
1. Levitt J, ed. Standards for blood banks and 5. Zou S, Eder AF, Musavi F, et al. ARCNET Study
transfusion services. 29th ed. Bethesda, MD: Group. Implementation of the uniform donor
AABB, 2014. history questionnaire across the American Red
2. Code of federal regulations. Title 21, CFR Parts Cross Blood Services: Increased deferral
600 to 799. Washington, DC: US Government among repeat presenters but no measurable
Printing Office, 2014 (revised annually). impact on blood safety. Transfusion 2007;47:
3. Eder A, Bianco C, eds. Screening blood donors: 1990-8.
Science, reason, and the donor history ques- 6. Fridey JL, Townsend M, Kessler D, Gregory K. A
tionnaire. Bethesda, MD: AABB Press, 2007. question of clarity: Redesigning the AABB
4. US Food and Drug Administration, Center for blood donor history questionnaire—a chro-
Biological Evaluation and Research. Guidance nology and model for donor screening. Trans-
fus Med Rev 2007;21:181-204.
for industry: Implementation of acceptable
7. US Food and Drug Administration. Guidance
full-length donor history questionnaire and
for industry: Revised preventive measures to
accompanying materials for use in screening
reduce the possible risk of transmission of
donors of blood and blood components. (Oc- Creutzfeldt-Jakob disease (CJD) and variant
tober 2006) Silver Spring, MD: CBER Office of Creutzfeldt-Jakob disease (vCJD) by blood and
Communication, Outreach, and Develop- blood products, (May 2010) Silver Spring, MD:
ment, 2006. [Available at http://www.fda.gov/ CBER Office of Communication, Outreach,
biologicsbloodvaccines/guidancecompliance and Development, 2010. [Available at http://
regulatoryinformation/guidances/blood/ucm www.fda.gov/downloads/biologicsbloodvac
073445.htm (accessed November 11, 2013).] cines/guidancecomplianceregulatoryinfor
CHAPTER 5 Allogeneic and Autologous Blood Donor Selection 䡲 133
nor history questionnaire. Transfusion 2006; hepatitis C virus, hepatitis B virus and human
46:1745-55. T-lymphotropic virus marker rates for directed
33. Food and Drug Administration. Guidance for versus volunteer blood donations to the Amer-
industry: Implementation of acceptable ab- ican Red Cross during 2005 to 2010. Transfu-
breviated donor history questionnaire and ac- sion 2013;53:1250-6.
companying materials for use in screening fre- 35. Brecher ME, Goodnough LT. The rise and fall
quent donors of blood and blood components. of preoperative autologous blood donation.
(May, 2013) Silver Spring, MD: CBER Office of Transfusion 2002;42:1618-22.
Communication, Outreach, and Develop- 36. Schved JF. Preoperative autologous blood do-
ment, 2013. [Available at http://www.fda.gov/ nation: A therapy that needs to be scientifical-
BiologicsBloodVaccines/GuidanceComplian ly evaluated. Transfus Clin Biol 2005;12:365-9.
ceRegulatoryInformation/Guidances/Blood/ 37. Goodnough LT. Autologous blood donation.
UCM351107.htm (accessed January 7, 2014).] Anesthesiol Clin North Am 2005;23:263-70.
34. Dorsey KA, Moritz ED, Steele WR, et al. A com-
parison of human immunodeficiency virus,
C h a p t e r 6
T H E C O L L E C T I O N O F whole blood W B CO L L E C T I ON
(WB) from the donor and subsequent
processing of the donation into components The phlebotomy staff involved in WB collec-
requires careful attention to proper tech- tion must be trained in proper collection tech-
niques to ensure the optimal care of both the niques to minimize contamination of donated
donor and the recipient. The classical, manu- units and phlebotomy-related local complica-
ally intense component-preparation methods tions, such as hematoma or nerve injury, using
are undergoing significant changes as more locally approved standard operating proce-
automation is incorporated by developers, dures. Phlebotomy must be performed only af-
from hands-off expression of components to ter the donor has been found to be eligible for
completely hands-off methods for the entire blood donation and the following items have
process. Differences are also recognized in been properly labeled using a unique dona-
methods for platelet preparation by the “buffy tion identification number (DIN) label: the
blood donor record, primary and satellite con-
coat” intermediary process and the “platelet-
tainers, and sample tubes. A final check of ap-
rich plasma” (PRP) intermediary process.
propriate labeling before phlebotomy helps
This chapter describes WB collection and
ensure that the donor history data, laboratory
component preparation and processing that
data, and other manufacturing data are associ-
may take place at the blood center or the hos-
ated with the correct blood components. The
pital-based collection facility.
Larry J. Dumont, MBA, PhD, Associate Professor, Geisel School of Medicine at Dartmouth, Lebanon, New
Hampshire; Colleen A. Aronson, MT(ASCP)SBB, Regional Director, Transfusion Services, ACL Laboratories,
Rosemont, Illinois; Mona Papari, MD, Medical Director, LifeSource, Rosemont, Illinois; and Deborah F.
Dumont, MT(ASCP)SBB, Research Technologist, Center for Transfusion Medicine Research, Dartmouth
Hitchcock, Lebanon, New Hampshire
L. Dumont has disclosed financial relationships with Advanced Preservation Technologies, New Health Sci-
ences, Fenwal, Terumo BCT, Haemonetics/Hemerus, Advanced Cell Technology, BioMed/CitraLab, and
EryDel. M. Papani, C. Aronson, and D. Dumont have disclosed no conflicts of interest.
135
136 䡲 AABB TECHNICAL MANUAL
final check may be documented by having the port and handling. PVC is rigid at room tem-
staff initial the donor record. perature and requires the addition of a plasti-
cizer such as di-(2-ethylhexyl) phthalate
Blood Containers (DEHP). DEHP not only imparts flexibility to
the PVC but also has been shown to protect
Desirable Properties
red cells against hemolysis during storage. Be-
WB collection containers must be approved by cause of concerns over possible toxicity of
the appropriate regulatory authority [eg, the DEHP, alternative plasticizers, such as butyryl-
Food and Drug Administration (FDA); Japa- trihexyl-citrate, have been made available in
nese Ministry of Health, Labor and Welfare; or some collection sets. Because of the protective
Health Canada] and must be CE marked for effect of DEHP on red cells, finding an equally
use in Europe. The containers should be ster- effective substitute for DEHP has been chal-
ile, pyrogen free, and identified by a lot num- lenging. This has recently been reviewed by
ber. They should also list an expiry date and Simmchen.1
include other label data required by regulatory Blood collection and processing sets are
authorities. generally latex free. The manufacturer’s label
The desired properties of storage contain- should be consulted to verify the absence of
ers include being easily formed and processed; latex before use in patients with a latex allergy.
flexible; kink resistant; transparent; and resis-
tant to damage throughout the anticipated Configurations, Anticoagulants, and
use, including component processing, storage, Additive Solutions
and transfusion. In addition, the container’s
material must be compatible with sterilization Collection and storage systems come in differ-
by gamma irradiation, ethylene oxide, electron ent configurations based on the intended pro-
beam, and/or steam. cessing methods (manual or automated) and
The gas (oxygen, carbon dioxide, and wa- the number and types of final components to
ter) permeability properties should be appro- be prepared from a unit of WB. Blood collec-
priate for the intended use. Gas exchange and tion systems may be intended for final
water loss before use is often limited by an processing with a classical combination of
overwrap or pouch. After removal from a manual and mechanical methods (eg, centrif-
pouch, the collection set should be used with- ugation, manual bag transfers, and manual ex-
in the time prescribed by the manufacturer. pression of contents) or may be designed for
Pouches should be labeled with the date and use in a fully automated system of blood com-
time that they are opened, and unused ponent preparation. There will be differences
containers should be stored within closed in the configurations based on the method of
pouches. choice for preparing platelets (buffy coat or
Blood containers are usually made of PRP processes). Blood collection systems may
thermoplastics, such as polyvinylchloride also include in-line filters for removal of leuko-
(PVC), ethylvinylacetate, polyolefins (polyeth- cytes from WB, Red Blood Cell (RBC) units,
ylene or polypropylene), or fluoropolymers. and/or platelets. Approved anticoagulants in-
The material formulation determines the clude anticoagulant citrate dextrose solution
physical properties that are to be matched to Formula A (ACD-A) or Formula B (ACD-B), an-
the use requirements, such as high gas perme- ticoagulant citrate phosphate dextrose solu-
ability for platelet storage or low glass transi- tion (CPD), anticoagulant citrate phosphate
tion temperature for freezing. double-dextrose solution (CP2D), and citrate
The glass transition temperature of PVC phosphate dextrose adenine solution (CPDA-
bags is about –25 C to –30 C. At and below 1) (see Tables 6-1 and 6-2). RBCs anticoagulat-
these temperatures, the container is brittle ed with CPDA-1 have a shelf life of 35 days in
and should be treated as if it were made of the United States. The use of additive solutions
glass and fragile enough to break during trans- (ASs) enables extension of RBC shelf life to
CHAPTER 6 Whole-Blood Collection and Component Processing 䡲 137
42 days in the United States and to 56 days in tween bags. This tubing is marked with repeat-
some other jurisdictions. Four ASs are current- ing serial numbers and may be sealed at
ly approved in the United States, and others several locations with either a dielectric (heat)
are approved in other regions (Table 6-3).2 sealer or a metal clip (grommet) to prepare ap-
Containers have integrally attached tub- proximately 13 to 15 segments. These seg-
ing that permits aseptic/closed transfers be- ments may be used later for ABO/Rh typing,
TABLE 6-3. RBC Additive Solutions Currently in Routine Use around the World*†
AS-1
Adsol AS-3 AS-5 AS-7
Fresensius- Nutricel Optisol SOLX
Constituents Kabi Haemon- Terumo Haemon- PAGGSM
(mM) SAGM (Fenwal) etics BCT etics MAP MacoPharma
NaHCO3 - - - - 26 - -
Na2HPO4 - - - - 12 - 16
NaH2PO4 - - 23 - - 6 8
Citric acid 2 - - 1
Na3-citrate - - 23 - - 5 -
Guanosine - - - - - - 1.4
Dextrose 45 111 55 45 80 40 47
(glucose)
Mannitol 30 41 - 45.5 55 80 55
TABLE 6-4. Use of Sterile Connecting Devices for Modification of Collection Containers6
Use Comment
To add a new or smaller needle to a blood collection If a needle is added during the procedure, an
set approved device to weld liquid-filled tubing should be
used.
To prepare components Examples include adding a fourth bag to make cryo-
precipitate, adding solution to the RBC unit, or adding
an in-line filter.
To pool blood products Appropriate use of a sterile connecting device to pool
platelets prepared from whole blood collection may
obviate potential contamination from the spike and
port entries commonly used.
To prepare an aliquot for pediatric use and divided FDA provides specific guidance if this activity is con-
units sidered to involve manufacturing of new products.
To connect additional saline or anticoagulant lines SOPs are required, although prior approval from the
during an automated plasmapheresis procedure FDA is not required.
To attach processing solutions Examples include washing and freezing RBCs.
To add an FDA-cleared leukocyte reduction filter The purpose is to prepare prestorage leukocyte-
reduced RBCs.
To remove samples from blood product containers The label may need revision if the product’s cell count
for testing is affected.
RBC = Red Blood Cell; FDA = Food and Drug Administration; SOPs = standard operating procedures.
proceed as well as during and after the collec- (shoulder side) and is often superficial. The
tion. third choice is the basilic vein, which lies on the
underside of the antecubital fossa; the basilic
Phlebotomy vein is not well anchored and may roll during
phlebotomy. Excessive probing with the needle
Vein Selection
should be avoided to prevent nerve injury.
The phlebotomist selects one arm for phlebot- Although the Occupational Safety and
omy after inspecting both of the donor’s arms. Health Administration provides an exemption
The phlebotomist checks for the presence of a from the requirements to wear gloves during
prominent, large, firm vein in the antecubital phlebotomy of volunteer blood donors, some
fossa to permit a single, readily accessible blood collection centers require their employ-
phlebotomy site that is devoid of scarring or ees to wear gloves for phlebotomy of all alloge-
skin lesions. A blood pressure cuff inflated at a neic and autologous donors.
pressure of between 40 and 60 mm Hg or a
tourniquet is applied to enlarge the vein. Pal- Disinfection Methods for Venipuncture
pating the vein is also recommended before Site
the phlebotomy site is prepared.
The first choice for the phlebotomy site is Specific instructions in the product insert for
the well-anchored median vein, which is cen- the use of approved agents should be followed
trally located in the antecubital fossa. The sec- for arm and phlebotomy site preparation
ond choice is the cephalic vein that lies laterally (Method 6-2). These methods provide surgical
CHAPTER 6 Whole-Blood Collection and Component Processing 䡲 141
cleanliness, but none of the methods can sampler that is sterile and connected to the
achieve an absolutely aseptic site. bag. If insufficient volume is available for test
Approximately 50% of donors have no samples, a second phlebotomy may be per-
bacterial colonies on culture of the venipunc- formed immediately after the blood collection.
ture site after disinfection with povidone io- Care should be taken to avoid diluting sample
dine or isopropyl alcohol plus iodine tincture. tubes.
Bacterial growth with low colony numbers (1 Blood collection containers now include
to 100) occurs in about half of the donors. safety devices, such as a sliding sheath needle
More than 100 colonies is rare (1%) in such guard, to prevent accidental needle-stick inju-
cultures.7 Bacteria residing deep within skin ries. These devices allow retraction of the nee-
layers are not accessible to disinfectants. dle into a safety guard at the completion of
Therefore, if skin tissue enters the collection blood collection. Capping of needles should
bag, it can result in contamination. In one be avoided to prevent injuries.
study, pigskin epidermal cells were detect-
able in the lavage fluid in 1 out of 150 punc- Volume of Blood Collected
tures.8 Whether human skin fragments or epi-
AABB Standards permits collection of 10.5 mL
dermal cells are carried into the bag during
of blood per kilogram of the donor’s weight for
routine blood donation has not been studied
each donation, including the blood unit and
in detail. Nonetheless, AABB Standards for
all samples for testing.4(p60) In North America
Blood Banks and Transfusion Services (Stan-
and Europe, the volume of blood collected
dards)4(p22) requires collection containers that
during routine phlebotomy is typically either
divert the first few milliliters of blood into a
450 ± 10% (405-495 mL) or 500 mL ± 10% (450-
special pouch that retains skin plugs to pre-
550 mL). The volume may be different in other
vent contamination when platelet compo-
regions and may be as low as 200 to 250 mL.
nents will be prepared from the collection.
For general blood banking applications, the
Diversion of the first aliquot of blood passing
volume of blood collected can be determined
through the needle has been shown to reduce
from the net weight in grams collected divided
the proportion of blood products containing
by the density of WB (1.053 g/mL).
viable bacteria.9
Empirical estimates may be used for the
density of RBC components as reported by
Blood Collection Process
Burstain et al.10 The theoretical density may
Method 6-3 describes steps for blood collec- also be calculated from the densities of the red
tion and sample collection for processing and cells (DRBC), density of the suspending medium
compatibility tests. The average time to collect (DS), and measured hematocrit (H, L/L): den-
500 mL of blood is less than 10 minutes. A sity of RBC component = (DRBC – DS ) × H + DS ×
draw time longer than 15 to 20 minutes may (1 – H).
not be suitable for collecting platelets or plas- Collection volumes must be within the
ma for transfusions. manufacturer’s specified range to ensure the
The collection bag should be periodically correct anticoagulant-to-WB ratio. High-vol-
mixed during the collection to ensure uniform ume allogeneic collections should be discard-
distribution of anticoagulant. Devices are ed. Low-volume allogeneic collections should
available to automatically mix the unit during be relabeled as “RBCs Low Volume” (Table 6-5).
collection. Plasma and platelets from low-volume units
Samples for donor testing may be collect- should be discarded.
ed from the diversion pouch after the pouch is Low-volume autologous collections may
isolated either by clamping or sealing the entry be retained if they are approved by a physi-
line. Samples for testing may also be aseptical- cian. Plasma from low-volume units has a
ly obtained from the collection bag with either higher citrate concentration than plasma from
an integrally attached sample container or a high-volume units and should be discarded.
142 䡲 AABB TECHNICAL MANUAL
TABLE 6-5. Weight and Volumes of Routine-Volume, Low-Volume, and Overweight Whole Blood
Collections*
The evidence indicates that the volume by hand to the gauze placed directly over the
collected does not affect in-vivo red cell sur- venipuncture site while the donor’s arm is kept
vival. Red cell recovery (average) after 21 days elevated. Pressure is applied until hemostasis
of storage in CPD was reported to be accept- is achieved, which may require more time for
able (approximately 80%) when only 300 g donors who are taking anticoagulants or anti-
(285 mL) of blood was collected.11 Recovery platelet drugs. Once hemostasis is evident, a
was more than 90% when 400 g (380 mL) of bandage or tape may be applied.
blood was collected and stored for 21 days. Postphlebotomy care includes observing
Similarly, as much as 600 g (570 mL) of blood the donor for signs or symptoms of reactions.
collected in a standard 450-mL CPD container If the donor tolerates a sitting position without
and stored for 21 days had normal in-vivo red problems, he or she may proceed to the can-
cell recovery. Undercollected units (275 mL or teen area and is encouraged to drink fluids and
290 g) in CPDA-1 stored for 35 days had a wait until being released. Local or state laws
mean 24-hour survival rate of 88%.12 These may specify the amount of time that the donor
data show that for undercollected and overcol- must wait after the donation and before leav-
lected units, variability in the amount collect- ing the collection area. The donor is encour-
ed does not adversely affect red cell storage for aged to drink more fluids and refrain from
21 to 35 days. heavy exercise for the next 4 hours, avoid alco-
Volumes of other components may also hol ingestion for several hours, and refrain
be determined gravimetrically by dividing the from smoking for 30 minutes. The donor is
net weight in grams by the appropriate density also instructed to apply local pressure to the
in g/mL. Table 6-6 provides generally accepted phlebotomy site if any bleeding recurs and to
densities and, for some, the applicable regula- call the blood center if the bleeding does not
tory documents provide a specific value.13,16 stop with pressure. A telephone number is
Often, the specific gravity (ratio of density provided so that the donor can report if he or
component to density water) is used for this she feels that the donated unit should not be
purpose, assuming that the density of water is used, has any reactions, or experiences any
1.00 g/mL. signs or symptoms of infection.
of all donors.17 Adverse reactions occur at the Red Cross also recorded a lower rate of compli-
time of donation or are reported later in about cation rates for WB collections than with
3.5% of donations. The American Red Cross apheresis methods for platelet and 2-unit RBC
observed a similar rate of 4.35% (435 per collection, with minor presyncopal reactions
10,000 donations) in 4.3 million donations in and small hematomas representing most of
2007 (see Table 6-7).18 Reactions that need the reactions. Major reactions were slightly
medical care after the donor has left the dona- more common for WB collection (7.4/10,000)
tion site occur in 1 in 3400 donors. A popula- compared with plateletpheresis (5.2/10,000)
tion-based European study found the rate of and 2-unit red cell apheresis (3.3/10,000).20
complications leading to long-term morbidity
Systemic Reactions
or disablement to be 5/100,000 donations and
2.3/100,000, respectively.19 Donor hemovigi- Vasovagal reaction complex includes dizzi-
lance programs instituted by the American ness, sweating, nausea, vomiting, weakness,
144 䡲 AABB TECHNICAL MANUAL
TABLE 6-7. Postcollection Adverse Event Rates in Calendar Year 2007 Based on 4,348,686 Whole
Blood Collections in the American Red Cross System*
Minor reactions
Presyncope 120,561 277.2
Hematoma (small) 61,029 140.3
†
Citrate (minor) 31 0.1
Other (minor) 165 0.4
Allergic (minor) 30 0.1
Subtotal 181,816 418.1
LOC and major reactions
LOC (<1 minute) 4,269 9.8
LOC (>1 minute) 591 1.4
Prolonged recovery 842 1.9
LOC with injury 438 1.0
Other (major) 82 0.2
Citrate (major)† 3 0.0
Allergic (major) 2 0.0
Subtotal 6,227 14.3
Major phlebotomy-related reactions
Hematoma (large) 387 0.9
Nerve irritation 314 0.7
Arterial puncture 449 1.0
Subtotal 1,150 2.6
All adverse events 189,193 435.1
Outside medical care 1,814 4.2
18
Adapted with permission from Benjamin et al.
*Citrate reactions after WB donations represent errors in classification.
†
95% confidence interval includes 1.0.
LOC = loss of consciousness; ND = not determined.
apprehension, pallor, hypotension, and brady- tion, intravenous fluid administration in the
cardia. In severe cases, syncope and convul- emergency room, or both. A protocol for tele-
sions may be observed. The pulse rate is often phone follow-up of donors who have experi-
low during vasovagal reactions, but the rate is enced severe reactions is helpful to assess the
often high during volume depletion. Some do- donors for any residual symptoms. Donor re-
nors with severe reactions or with prolonged actions after WB donation do not predict accu-
recovery times may need short-term observa- rately the possibility of recurrent syncope in
CHAPTER 6 Whole-Blood Collection and Component Processing 䡲 145
returning donors, although they reduce the is reduced by one-third among donors experi-
likelihood of future donations.21 encing this symptom.17
Approximately 60% of systemic reactions
occur in the canteen area.17 The main predic- Local Nerve Injury
tors of immediate and delayed vasovagal and
presyncopal reactions are young age, low esti- Nerve injuries may be unavoidable because
mated blood volume, and first-time donation nerves cannot be palpated. In 40% of cases of
status.22,23 Ingestion of antihypertensive medi- nerve injuries, phlebotomy is performed with-
cations does not appear to be a risk factor.24 out difficulty.17 Donors may complain of sen-
Approximately 15% of reactions occur away sory changes away from the phlebotomy site,
from the donation site, usually within 1 hour such as in the forearm, wrist, hand, upper arm,
of donation.17 In donors experiencing a reac- or shoulder. These injuries are usually tran-
tion, an injury to head, face, or extremity may sient, and recovery almost always occurs; how-
occur. The staff should be vigilant to detect re- ever, in 7% of injured donors, recovery may
actions early and prevent injuries as much as take 3 to 9 months.17 In severe cases, referral to
possible. Deferral strategies for young donors a neurologist may be indicated.
with estimated low blood volume (<3.5 liters)
and physiologic strategies to minimize donor Arterial Puncture
reactions in young donors are aimed at im-
Presence of bright red blood, rapid collection
proving donor safety.21 Donor education, envi-
(within 4 minutes), and a pulsating needle
ronmental controls, instructions to donors to
suggest arterial puncture. The hematoma rate
drink water right before donation, dietary in-
is higher with arterial puncture. When punc-
terventions, and muscle tension are all effec-
ture is recognized early, the needle should be
tive in reducing the rate of adverse reactions,
pulled out immediately, and local pressure
especially in young donors, in whom a 20% re-
should be applied for an extended period.
duction has been observed.25,26
In case of a vasovagal reaction, phleboto- Most donors recover quickly and completely,
my should be stopped, and the donor should but some might present with waxing and wan-
be placed in a recumbent position as soon as ing hematomas and should be evaluated for
the reaction is suspected. Applying cold wet pseudoaneurysm by ultrasound studies.
towels to the donor’s neck and shoulder area
and loosening the donor’s clothes can assist in Upper-Extremity Deep Vein Thrombosis
symptom management. Only one case of this thrombosis has been re-
Oral fluid intake before and soon after ported in the literature.17 Symptoms include
nonautomated WB donation appears to re- pain; antecubital fossa tenderness; swelling of
duce the frequency of systemic reactions.17 the arm; and a prominent, palpable, cord-like
Coffee ingestion can reduce the frequency of thickening of the thrombosed vein. Medical
reactions, but it may also reduce blood flow referral of donors who are experiencing deep
during collection because of its vasoconstric- vein thrombosis should not be delayed so that
tive effect.
treatment can begin promptly.
Bruise or Hematoma Postdonation Mortality
Both symptoms are common after phleboto-
The FDA requires blood establishments to re-
my but generally do not prevent donors from
port deaths caused by blood donation. For fis-
donating again.
cal years 2008-2012, the FDA received 50 re-
ports of postdonation fatalities for automated
Fatigue
and manual collections, of which only 11 fol-
First-time donors and females are more likely lowed WB donation. After medical review, one
to report fatigue after donation. Donor return case was ruled out, and in the remaining
146 䡲 AABB TECHNICAL MANUAL
10 cases, there was no evidence of a causal re- ther processing is removed from the platelet
lationship between blood donation and the pellet, which is held undisturbed for 30 to 60
donors’ demise.27 Most deaths that take place minutes before being resuspended.29 Leuko-
after blood donation are coincidentally related cyte reduction may occur at the PRP or final
to the donation rather than caused by it. platelet concentrate stage, depending on the
device system used. Platelet concentrates are
labeled and stored as individual units or, if
B LO O D CO M P O N E N T
cleared by regulatory authorities, pooled and
PREPARATION AND stored as transfusable doses.30
P RO C E S S I N G Compared to the PRP preparation meth-
Improvements and innovations continue to be od, the buffy coat method yields more plasma,
made to WB collection and its processing into greater red cell loss, better initial white blood
components. Apheresis is growing in impor- cell (WBC) reduction before filtration, and
tance for collection of all major components, moderate reduction in viable bacteria in the
as discussed in Chapter 7. Single WB units are platelets.
collected in plastic containers containing anti- Plastic collection containers, satellite
coagulant; typically CDP, CP2D, or CPDA-1 bags, and integrally attached tubing that are
(Methods 6-3, 6-4, and 6-5). Innovations in this hermetically sealed allow component manu-
area include scales for monitoring the collec- facturing to take place in a closed system. The
tion volume plus automatic mixing and devic- blood container should not be entered before
es to add anticoagulant at a fixed ratio as the issue except for the purposes of blood collec-
blood is withdrawn from the vein. tion or transfer of components to a different
Methods and devices are available to pro- container. The container material should not
cess WB in a variety of manners for WB leuko- have any adverse effect on the safety, purity, or
cyte reduction, separation of plasma, and sep- potency of the blood. Components prepared
with an open system require a reduction in
aration of red cells and platelets. Important
their expiration time from the time that the
secondary processing includes leukocyte re-
system was opened. The use of approved ster-
duction, irradiation to prevent graft-vs-host
ile connection devices maintains a functional-
disease (GVHD), and pathogen reduction.
ly closed system when various connections are
For preparation of platelets from WB, two
performed, such as pooling or sampling,
primary methods are available (Method 6-12):
thereby maintaining the product’s original ex-
preparation from buffy coats and preparation
piry date.
from PRP. The buffy coat method is employed
in many regions but is not currently cleared in
WB Handling Before Component
the United States. In short, non-leukocyte-
reduced WB units are first centrifuged under a
Processing—Timing and Temperatures
high g-force (ie, hard or heavy spin) and plas- The temperature conditioning and handling of
ma, and red cells and buffy coats are removed WB immediately after collection is determined
for further processing. Buffy coats from 4 or 5 by downstream processing requirements for
units are pooled with 1 unit of plasma and component preparation. In some cases, this
then centrifuged under a low g-force (ie, soft or may mean that collections at mobile blood
light spin) to separate the platelet concentrate drives or fixed collection sites should be trans-
for additional processing, such as leukocyte ported as soon as possible to the central com-
reduction. These processes are often associat- ponent preparation laboratory. With other
ed with extended holding of the WB and buffy processing methods, transportation may not
coats for 8 to 24 hours at 20 to 24 C.28 be as urgent.
Preparation of platelets from PRP begins Requirements for cooling and transporta-
with a soft spin of the WB, followed by separa- tion methods are quite variable, and the speci-
tion and hard spin of the PRP. Plasma for fur- fications of the device manufacturer should be
CHAPTER 6 Whole-Blood Collection and Component Processing 䡲 147
carefully followed. By the time phlebotomy is window for separation of plasma from WB is
completed, the blood has air cooled to about quite variable from region to region. FFP
30 C.31 If such units are left at the ambient tem- should be separated from the WB and frozen
perature, the cooling rate is quite slow and within the time frame specified in the direc-
about 6 hours more are needed for units to tions for use of the blood collection, process-
reach 25 C.31 To cool blood more rapidly, units ing, and storage system. In the United States,
are typically placed in specific storage envi- plasma that is separated from WB collection
ronments. Some centers use cooling plates and placed in the freezer within 8 hours can be
that provide rate-controlled cooling toward labeled “FFP”; if it is within 8 to 24 hours after
20 C. These cooling plates contain 1, 4-butane- collection of WB (manual or apheresis meth-
diol that has a melting temperature of 20 C and od), it can be labeled as “Plasma Frozen Within
serves as a heat absorber. With the cooling 24 Hours After Phlebotomy.” In Europe, if the
plates, about 2 hours are needed for the col- WB is refrigerated, plasma may be separated
lected blood to reach 20 C.31 within 18 hours; if the WB is held at 20 to 24 C
Many WB leukocyte reduction systems al- for platelet production, plasma can be separat-
low filtration at ambient temperature begin- ed within 24 hours, frozen, and labeled as FFP.
ning soon after collection and lasting up to 24 Shipping containers and cooling methods
hours. WB filtration may also be started and/ used to transport and hold WB and compo-
or completed at refrigerator temperatures. WB nents must be sufficiently validated according
destined for platelet preparation should not be to local and national regulations and guide-
cooled to less than 20 C. Platelets may need to lines. Validation is generally performed with
be separated from WB within 8 hours in some various quantities of units in the transport
regions or with some collection systems. Other container and a fixed amount of ice or gel-
methods are approved or are under consider- pack to determine the number of units that
ation for up to a 24-hour hold of WB at 20 to 24 can be stored and transported while maintain-
C before separation of plasma and platelets. ing the desired temperature. Portable temper-
All processes for preparing platelets by the ature monitors are available to record temper-
buffy coat methods have an extended hold of ature continuously.
WB at 20 to 24 C for approximately 2 to 24
hours following collection and before the sep- Separation by Centrifugation
aration of the buffy coat and plasma.13 The
pooled buffy coat is often held undisturbed for All current methods for separation and prepa-
approximately 2 to 18 hours at 20 to 24 C be- ration of the three major blood components—
fore separation of the platelet concentrate. RBCs, platelets, and plasma—rely on one or
The details of these hold times are gener- more centrifugation steps. As mentioned
ally adjusted to ease the operational logistics above, the first step may be a high g-force cen-
for the blood center. For example, morning trifugation followed by a low g-force step for
collections are held until the afternoon when the buffy coat platelet preparation method
plasma is separated and buffy coats prepared. (the opposite of the PRP platelet separation
The buffy coats are held overnight as either in- method) or an automated sequence of steps
dividual units or pools, and the platelets are in recently developed systems. Centrifuges
separated the following morning. Likewise, af- should be properly validated, maintained, and
ternoon collections may be held overnight, the calibrated in a way that is consistent with local
buffy coats are prepared the next morning, and regional guidelines. Centrifuge methods
and platelets are prepared in the afternoon. for blood component preparation should be
WB that will not be used to prepare plate- calibrated or checked in a systematic manner
lets should be cooled to refrigerator tempera- to verify the processing conditions.
ture as soon as possible; this is often accom- The major variables that affect the recov-
plished by placing the unit on wet ice or other ery of cells from WB by differential centrifuga-
appropriate cooling media. The allowable time tion are rotor size, centrifuge speed, duration
148 䡲 AABB TECHNICAL MANUAL
of centrifugation, and acceleration/decelera- weights, add storage solutions, clamp and seal
tion protocol. Published papers often refer to tubing, and perform other useful functions
relative centrifugal force (g-force) that is de- that assist in the consistent preparation of
rived from the radius of the centrifuge rotor blood components. Some systems also com-
and its revolutions. For a given centrifuge, the bine nearly all of the functions, including cen-
rotor size is generally not variable. Therefore, trifugation, component expression, filtration,
the other two variables (centrifuge speed and sealing, and additive addition, without relying
duration) can be altered in a stepwise fashion on operator interventions. The systems and
in a simplex strategy to determine the optimal methods used for these steps, whether
conditions for preparing PRP.32 The simplex predominantly manual or fully automated,
strategy can also be used to identify the opti- should be validated by the user.
mal conditions of centrifugation for platelet Blood component extractors are avail-
concentrates when quality control (QC) data able for making components in a semiauto-
show that the platelet counts in the platelet mated manner. After primary centrifugation,
concentrates are not satisfactory. Method 8-4 WB is placed in the extractor, and a pressure
describes a process of functional calibration of plate creates an outflow of components from
the centrifuge to maximize platelet yield. The the container. Outflow can occur from the top
device manufacturer should be consulted for or bottom of the container, depending on the
recommendations on validating the perfor- device.
mance of automated systems. When a cellular interface is detected by
On the day of preparation, some platelet an optical sensing device, outflow tubing is au-
units may contain clumps composed of plate- tomatically clamped at an appropriate level.
let aggregates.33 In routine practice, visual in- Such devices may improve the standardization
spection is adequate to determine the degree of components, but they are not widely used in
of clumping subjectively and ensure that units the United States. An automated system is
with excessive clumping are not released for available in Europe that performs multiple
labeling. Most of the clumps seen on day 0 dis- functions to prepare pooled platelet concen-
appear on day 1 of storage with continuous ag- trates derived from WB by the buffy-coat
itation, particularly those showing light to method. Steps that are automated include
moderate clumping.33 The temperature at pooling, rinsing, centrifugation, transfer, filtra-
which platelets are prepared may influence tion, and sealing.
clumping; platelets prepared at 24 C appear to
show the least amount of clumping compared DESCRIPTIONS OF MAJOR
to those prepared at less than 24 C.33 Visual in-
B LO OD CO M P O N E NTS
spections after the platelet concentrates are
prepared have shown an absence of visible red WB
cells in the vast majority of units, which im-
The minimum hematocrit of WB units in anti-
plies that the units contain fewer than 0.4 × 109
coagulant-preservative, such as CPD, is usual-
red cells. Generally, the number of red cells in
ly approximately 33%. WB is most often sepa-
a unit of platelets does not exceed 1.0 × 109.34
rated into components and is rarely used for
transfusion directly. Severe hemorrhage cases,
Blood Component Division
such as those resulting from trauma, may ben-
Following separation by centrifugation, com- efit from fresh WB transfusions when platelets
ponents must be carefully divided into sepa- are not available.35 Labile coagulation factors
rate containers for further processing. Many diminish and platelets may activate and devel-
laboratories use manual expressers for this op a storage lesion as the storage interval in-
purpose. Automated and semiautomated de- creases.
vices, available in some regions, control the WB in ACD, CPD, or CP2D has an expira-
rate of expression, monitor component tion date of 21 days when stored at 1 to 6 C; the
CHAPTER 6 Whole-Blood Collection and Component Processing 䡲 149
maximum storage time for units in CPDA-1 is fused, other components, such as platelets,
35 days. WB may be reconstituted by combin- plasma, and cryoprecipitate, should not be
ing RBCs with thawed plasma to achieve a de- prepared from low-volume units.
sired hematocrit level—for example, when RBCs may be subject to several secondary
used for neonatal exchange transfusions. Ad- processing steps, for example, leukocyte re-
ditional information about the preparation of duction, gamma or x-ray irradiation to prevent
reconstituted WB is provided in Chapter 9. GVHD, and pathogen reduction. The FDA re-
quires leukoreduced units to contain <5 × 106
RBCs WBCs per unit and retain >85% of the preleu-
RBCs in anticoagulant-preservative CPD or koreduced RBC content. The EU regulations
CPD2 have a shelf life of 21 days at 1 to 6 C with call for <1 × 106 WBCs per unit.
a hematocrit of 65% to 85%, or 35 days in Retention segments are held at the blood
CPDA-1 with a hematocrit of <80%. The addi- center for 7 days after the expiration date of
tion of additive solution (Table 6-3) reduces the unit containing red cells and at the hospi-
the hematocrit to approximately 55% to 65%. tal transfusion service for 7 days after the
Additive solutions maintain the red cells dur- transfusion. Hemolysis identified in a seg-
ing storage (for example, reduce hemolysis) ment by visual inspection often does not cor-
and extend the expiry date to 42 days or 56 relate with the presence of hemolysis in the
days, depending on regulatory approvals. He- unit. In one study, approximately three-
molysis at the end of storage should be less fourths of the visual assessments did not agree
than 1% in the United States or less than 0.8% with the hemoglobin levels measured by
in the European Union (EU). chemical methods, indicating a high false-
Hemoglobin content per unit varies be- positive rate with visual assessment.38
cause of differences between donors and pro- Visual inspection of RBC units can detect
cessing specifics. For example, more hemo- white particulate matter, abnormal color
globin is generally lost when buffy coat caused by bacterial contamination, hemoly-
preparation methods are used than with PRP- sis, and clots. Abnormal color caused by bacte-
type methods. rial contamination may be observed in the
Hemoglobin content per unit may be
bag, where some segments appear to be darker
more precisely controlled with automated col-
than others because of oxygen consumption in
lections. Total hemoglobin content is not di-
the bag. The bag content may look purple with
rectly regulated in the United States but has a
or without hemolysis, and large clots may be
lower limit of 45 g per unit or 40 g per unit for
evident. The unit can be centrifuged to facili-
leukoreduced RBCs in the EU.36 Some experts
tate inspection of the supernatant in the case
have advocated for standardizing the amount
of hemoglobin per RBC unit at 50 g.37 Depend- of suspected bacterial contamination, and vi-
ing on local practice, RBCs stored for less than sual inspection of the supernatant may reveal
7 to 10 days are issued for neonatal or pediatric murky, brown, or red fluid.39 However, visual
transfusions, and some neonatologists may inspection will not detect all contaminated
prefer RBCs without additive solutions (see units.
Chapter 23). Blood clots in RBC units are often too
RBCs that are labeled as low-volume units small to be detected by visual inspection. Clots
are made available for transfusion when 300 to are sometimes revealed during transfusion
404 mL of WB is collected into an anticoagu- when they clog the filter or in the component
lant volume calculated for 450 ± 45 mL, or laboratory when the units are filtered through
when 333 to 449 mL of WB is collected into an a leukocyte reduction filter. Units known to
anticoagulant volume calculated for 500 ± 50 have clots should not be released for transfu-
mL. Although the resulting RBCs may be trans- sion.
150 䡲 AABB TECHNICAL MANUAL
peratures <20 C.47,48 Thus, proper steps should stored at –18 C or colder. FFP that is stored at
be taken to maintain the required range of –65 C may be stored for longer than 12
temperatures during storage at the blood cen- months, but such storage requires FDA ap-
ter and during transport. proval.4(pp56-57) Rapid freezing of plasma can be
accomplished using a blast freezer, dry ice, or a
Plasma mixture of dry ice with either ethanol or anti-
freeze. Plasma should be thawed at 30 to 37 C
Plasma preparations are defined and regulated
in a waterbath or by using an FDA-cleared de-
through a dizzyingly broad combination of
vice. When a waterbath is used, the compo-
collection methods, storage temperatures,
nent should be placed in a protective plastic
freezing methods, secondary processing, tim-
overwrap. Thawing of larger units of FFP col-
ing, and storage after thawing. These specifi-
lected by apheresis may require more time.
cations are covered in an array of standards,
FFP, once thawed, has a shelf life of 24 hours at
rules, and guidelines overlaid with various re-
1 to 6 C. Thawed plasma held longer than 24
quirements of the country where the plasma is
hours must be relabeled as Thawed Plasma,
prepared and/or used. Major, although not ex-
and it can be stored for an additional 4 days at
haustive, sources of this information include
1 to 6 C.
the US Code of Federal Regulations, FDA guid-
In the past several years, many blood cen-
ance documents, the US Circular of Informa-
ters in the United States have increased the
tion,49 the AABB Standards, and EU directives.
amount of WB collected from 450 mL to 500
The definitions and requirements of the coun-
mL, resulting in a larger amount of plasma per
try where the plasma is prepared should al-
unit. A unit can contain 500 mL to 800 mL of
ways be consulted.
plasma when collection is performed by auto-
Plasma is prepared from WB collections
mated (single-donor) plasmapheresis.
and by apheresis. Plasma is generally frozen to
The Council of Europe defines “plasma,
maintain factor activity and provide an ex-
fresh frozen” as prepared from either WB or
tended shelf life. Frozen plasma is thawed for
collected by apheresis. Plasma freezing must
clinical use and may be maintained at 1 to 6 C
be initiated within 6 hours of collection, within
for some time prior to use. Frozen plasma is
18 hours if the WB is held at 1 to 6 C, or within
also the source of cryoprecipitate and cryopre-
24 hours if WB or apheresis plasma is rapidly
cipitate-reduced plasma. There are several
conditioned to 20 to 22 C following collection.
methods available for pathogen reduction of
Freezing must be completed within 1 hour to
plasma that may be applied depending on na-
less than –30 C. Plasma, fresh frozen has an ex-
tional regulatory approvals. Plasma may be
piry time of 36 months if held at less than –25 C
used for preparation of specific plasma protein
or 3 months if held at –18 C to –25 C.13 The
products through fractionation processes. The
Council of Europe does not address specific
descriptions below are based on the prevailing
thawing methods or treatment of the plasma
guidance documents and regulations of the
following thawing, including expiry dating.
FDA and Council of Europe.
AABB requires4(p17) interventions to mini-
mize the preparation of high-plasma volume
FFP
components (apheresis platelets, plasma prod-
In the United States, FFP is plasma collected ucts, and WB) for transfusion from donors who
either from a single unit WB collection or by are at risk of developing HLA antibodies.50
apheresis. FFP must be placed in the freezer These products should be collected from
within 8 hours of collection; within 6 hours if males, never-pregnant females, or parous fe-
anticoagulated with ACD; or as directed by the male donors who test negative for HLA anti-
manufacturer’s instructions for use of the bodies.
blood collection, processing, and storage sys- FFP contains normal amounts of all coag-
tem. FFP has a shelf life of 12 months when ulation factors, antithrombin, and ADAMTS13.
152 䡲 AABB TECHNICAL MANUAL
date, records for this component should be re- phosphate and 1% Triton X-100 for pathogen
tained indefinitely. Storage conditions for re- reduction. This treatment has been shown to
covered plasma are established by the frac- significantly inactivate lipid-enveloped virus-
tionator. FFP used as human plasma for es. SD plasma is manufactured in facilities that
fractionation in Europe must comply with the can manage large-scale production rather
applicable European Pharmacopoeia guide- than in blood centers. Each unit contains 200
lines. mL of plasma that is stored frozen at –18 C
with an expiration date of 12 months.56 All co-
Pathogen-Reduced Plasma agulation factors are reduced by 10% in SD
plasma, except for Factor VIII, which is re-
Plasma can be treated to inactivate microbial
duced by 20%.57 Also, SD plasma contains 50%
agents for pathogen reduction. Four such
less functional protein S in comparison to the
methods are available and in use in Europe:
nontreated FFP.58 The product is labeled with
the methylene blue, psoralen (amotosalen), ri-
the ABO blood group and, once thawed,
boflavin, and solvent/detergent treatments.
should be used within 24 hours. SD plasma is
Methylene blue (approximately 0.085 mg/
available in Europe and has been recently ap-
unit of plasma) can be added to thawed FFP,
proved in the United States.59
followed by activation using white light. After
removal of methylene blue with a filter (resid-
Cryoprecipitated Antihemophilic
ual concentration: 0.3 µmol), plasma can be
Factor
frozen. Methylene-blue-treated plasma con-
tains approximately 15% to 20% less Factor Cryoprecipitated antihemophilic factor (AHF),
VIII and fibrinogen than untreated plasma. or simply “cryoprecipitate” in Europe, is pre-
Plasma prepared from WB or by automat- pared from FFP. Cold-insoluble protein that
ed methods can be treated with 15 mL of amo- precipitates when FFP is thawed to 1 to 6 C is
tosalen per 250 mL of plasma, followed by illu- collected by centrifugation; supernatant plas-
mination with ultraviolet A light (320-400 nm) ma is transferred into a satellite container; the
with 3.0 J/cm2. After amotosalen is removed by precipitate is resuspended in a small amount
exposing treated plasma to an adsorption de- of residual plasma, generally 15 mL; and the
vice, the unit is frozen for storage at –18 C. Av- precipitate is refrozen as described in Method
erage activity values for coagulation and anti- 6-11. FFP can be thawed to prepare cryopre-
thrombotic factors are reported to be within cipitated AHF by placing the FFP in a refrigera-
reference ranges for untreated plasma. tor (at 1 to 6 C) overnight or in a circulating
Plasma prepared by apheresis or from waterbath at 1 to 6 C. An alternate method that
single WB units (volume range 170-360 mL) uses microwaves for thawing has been de-
can be treated with the Mirasol system by add- scribed. The cryoprecipitated AHF is placed in
ing 35 mL of riboflavin (vitamin B2) followed a freezer within an hour of removal from the
by illumination for 6 to 10 minutes. Immedi- refrigerated centrifuge and can be stored at
ately after illumination, the plasma can be re- –18 C for 12 months from the original collec-
leased or frozen below –30 C for 2 years. Resid- tion date. In Europe, thawing is to be per-
ual RBC levels up to 15 × 109 per liter have been formed at 2 to 6 C, and the product can be
qualified to result in successful pathogen re- stored for up to 36 months below –25 C and for
duction and leukocyte inactivation. Coagula- 3 months at –18 to –25 C.
tion and anticoagulation proteins are well pre- AABB Standards4(pp28-29) requires that cryo-
served in plasma treated with the Mirasol precipitated AHF contain at least 80 interna-
system.54,55 tional units (IU) of Factor VIII and 150 mg of
Solvent/detergent-treated plasma (SD fibrinogen per unit, although the average fi-
plasma) is prepared from a pool of plasma brinogen content is generally 250 mg.60 Euro-
from many donors (no more than 2500) that pean standards are at least 70 IU/unit of Factor
undergoes treatment with 1% tri-n-butyl VIII, 140 mg/unit of fibrinogen, and 100 IU/unit
154 䡲 AABB TECHNICAL MANUAL
of vWF. More current preparations are report- nent that contains at least 85% of the original
ed to have much higher amounts of fibrinogen red cell content. The Council of Europe’s stan-
(median, 388 mg/unit).61 Cryoprecipitated dards require a minimum of 40 g of hemoglo-
AHF also contains the vWF ristocetin cofactor bin to be present in each unit after leukocyte
activity (approximately 170 units/bag), Factor reduction.13
XIII (approximately 60 units/bag), and fibro- For WB-derived platelets, AABB Stan-
nectin. Rapid freezing of FFP is found to in- dards requires that the leukocyte reduction
crease the Factor VIII yield in cryoprecipitated process ensures that 95% of the platelet units
AHF.62 ADAMTS13 levels are normal in cryo- sampled contain <8.3 × 105 leukocytes per
precipitated AHF.63 Anti-A and anti-B are known unit, at least 75% of the units sampled contain
to be present in cryoprecipitated AHF, but the 5.5 × 1010 platelets, and at least 90% of the units
combined amount of these antibodies from sampled have a pH of 6.2 at the end of the al-
the unit of plasma is only 1.15% of the total.64 lowable storage period.4(p29) The number in the
Thawed cryoprecipitated AHF should be Council of Europe’s standards is <0.2 × 106 re-
used as soon as possible but may be held at sidual leukocytes per unit of platelets from
room temperature (20-24 C) for 6 hours as sin- WB.13
gle units or a pool prepared as a closed system In practice, approximately 1% of RBC
using an approved sterile connecting device or components do not achieve the levels of <1 ×
for 4 hours if pooling was with an open system. 106 residual leukocytes in the component.
Pooling may be accomplished with the aid of a Sickle cell trait of red cells is the most common
diluent, such as 0.9% sodium chloride (USP), to cause of filter failure. Approximately 50% of
facilitate removal of material from individual bags. the RBC units with sickle cell trait fail to filter.
At room temperature, the mean declines Although the other 50% pass through the filter,
of Factor VIII levels at 2, 4, and 6 hours are ap- the residual leukocyte content may be higher
proximately 10%, 20%, and 30%, respectively.65 than the allowable limits.67
Cryoprecipitated AHF from blood groups A and Prestorage leukocyte reduction is general-
B has higher levels of Factor VIII compared to ly performed soon after WB collection and is
always performed within 5 days of collection.
that derived from blood group O donors (about
In-line WB filters are available in collection
120 vs 80 IU per bag, respectively).66 Thawed
sets that permit preparation of LR RBCs and
cryoprecipitate should not be refrozen.
FFP. WB filters that spare platelets are now
available as well. If WB is collected without the
Granulocytes
in-line leukocyte reduction filter, a filter can be
Although it is possible to prepare granulocytes attached to the tubing by an FDA-cleared ster-
from fresh WB, current practice is to collect ile connecting device. The number of platelets
granulocytes by apheresis. Refer to Chapter 7 in LR platelet concentrates is generally lower
for information on automated collections. than in non-LR platelet concentrates.
Methods that measure residual leuko-
cytes include Nageotte hemocytometry and
B LO O D CO M P O N E N T flow cytometry (see Method 8-11). In a multi-
MODIFICATION center study, flow cytometry gave better re-
Prestorage Leukocyte Reduction by sults than Nageotte hemocytometry when
Filtration freshly prepared samples (within 24 hours)
were tested. For instance, at a concentration of
For RBCs that are leukocyte reduced, the FDA 5 white cells per µL for RBC units, the intersite
requires a residual number of <5.0 × 106 leuko- coefficient of variation was 4.9% for flow cy-
cytes per unit. The Council of Europe requires tometry and 54% for Nageotte hemocytome-
that the residual number be <1 × 106 per unit. try. In general, Nageotte hemocytometry tends
In the United States, leukocyte reduction by to underestimate the number of white cells
filtration of RBCs should result in a compo- compared to flow cytometry.68 A new semiau-
CHAPTER 6 Whole-Blood Collection and Component Processing 䡲 155
tomated methodology offers to reduce the Postwash units have a hematocrit of 51% to
technical burden associated with both Na- 53% and contain a mean of about 9.0 × 106 leu-
geotte and flow cytometry methods.69 kocytes per unit.71
Cryopreservation Platelets
RBCs Cryopreservation of platelets is not widely
available because the procedures for cryo-
Glycerol is the most commonly used cryo-
preservation are complex and not routinely
preservative agent and is added in either high
practiced at most blood centers. Several cryo-
or low concentration to RBCs within 6 days of
protectants have been described for platelet
collection. Two commonly used protocols for
cryopreservation72,73 However, 5% or 6% di-
the high-glycerol method are described in
methyl sulfoxide (DMSO) is most commonly
Methods 6-6 and 6-7.
used, mainly for autologous platelet transfu-
Frozen RBCs must be stored at tempera-
sions in patients who are refractory to alloge-
tures colder than –65 C and expire after 10
neic platelets. Cryopreserved platelets can be
years. Rare frozen units may be used beyond
stored for at least 2 years. After thawing, the
the expiration date, but only after medical re-
platelet recovery rate in vitro is about 75%,
view and approval based on the patient’s
which may be reduced further if thawed plate-
needs and availability of other rare compatible
lets are centrifuged to remove the DMSO be-
units. The units should be handled with care
fore transfusion. The in-vivo recovery rate af-
because the containers may crack if they are
ter transfusion of thawed, DMSO-reduced
bumped or handled roughly. The containers
platelets is about 35% to 42%.74 In vivo, the
can also crack during transportation.
platelets that survive after transfusion are he-
The units should be thawed at 37 C and
mostatically effective.75
generally take about 10 minutes to thaw com-
pletely. Glycerol must be removed after thaw-
Irradiation
ing and before transfusion. This removal is
generally accomplished by instruments that Cellular blood components can be irradiated
allow the addition and removal of sodium for prevention of GVHD. Frozen plasma com-
chloride solutions. In most cases, addition of ponents, such as FFP and cryoprecipitated
the glycerol and its removal (deglycerolization) AHF, are generally not irradiated because they
require the system to be opened; for this rea- are considered noncellular components. In
son, thawed and deglycerolized units can be addition, the small number of T lymphocytes
stored only for 24 hours at 1 to 6 C. The final present in the components may not survive
solution in which cells are suspended is 0.9% the freeze-thaw cycle.
sodium chloride and 0.2% dextrose. Dextrose The irradiation sources in use include
provides nutrients and has been shown to sup- gamma rays—from either cesium-137 or co-
port satisfactory posttransfusion viability for 4 balt-60 sources—and x-rays produced by radi-
days of storage after deglycerolization.70 For ation therapy linear accelerators or stand-
QC, determining the volume of red cells in the alone units. Both sources achieve satisfactory
unit after deglycerolization and examining the results in rendering T lymphocytes inactive.
last wash for hemolysis are recommended (see Freestanding instruments that allow the use of
Method 6-8). each of these sources are commercially avail-
Recently, automated addition of glycerol able for blood bank use. The US Nuclear Regu-
to RBCs and removal from RBCs in a closed latory Commission requires an increased num-
system has become possible. With this sys- ber of controls for the radioactive material
tem, glycerol is added within 6 days of WB col- license that is needed for a gamma irradiator
lection. Postthaw red cells prepared in this and its source onsite.76 The increased controls
manner are suspended in additive solution 3 are designed to reduce the risk of unauthor-
(AS-3) and can be stored for 14 days at 4 C.70 ized use of radioactive materials. The licensee
156 䡲 AABB TECHNICAL MANUAL
is required to secure any area where radioac- up to day 28 following collection. Irradiated
tive source is present, and only approved indi- cells may not be stored longer than the earlier
viduals may access the site to perform their of 14 days postirradiation or 28 days postcol-
duties. lection. Platelets may be irradiated until their
In the United States, the radiation dose to expiration date, and their postirradiation expi-
the center of the irradiation field must be at ration date is the same as the original expira-
least 25 gray (Gy) [2500 centigray (cGy)] and no tion date.
more than 50 Gy (5000 cGy).77 During dosime- Irradiation of RBCs followed by storage
try, the delivered dose of 25 Gy is targeted to does result in a decrease in the percentage of
the internal midplane of the container. More- recovery after transfusion. In addition, an in-
over, the minimum delivered dose to any por- creased efflux of potassium from red cells
tion of the blood components must be at least causes the potassium levels to rise approxi-
15 Gy in a fully loaded canister.4(p25) The Euro- mately twofold compared to nonirradiated
pean standard requires a higher dose, with no units. Platelets are not damaged by an irradia-
part of the component receiving less than 25 tion dose as high as 50 Gy.78
Gy and a maximum dose to any part of the
component of 50 Gy.13 Pooling
Each instrument must be routinely moni-
tored to ensure that an adequate dose is deliv- Platelets that are pooled have an expiration
ered to the container that houses the blood time of 4 hours from when the system was
components during irradiation. Dose map- opened for pooling. A closed system for
ping is used to monitor the instrument’s func- prestorage pooling of platelets has been li-
tion. For this purpose, irradiation-sensitive censed by the FDA. This system permits the
films or badges that monitor the delivered storage of pooled platelets for up to 5 days
dose are used for QC of the irradiators. Several from the time of WB collection. Four to six LR
systems consisting of irradiation films or or non-LR platelet units that are ABO identical
badges are commercially available.77 Verifica- can be pooled using a set consisting of a multi-
tion of the delivered dose must be performed lead tubing manifold for sterile connection. If
annually for the cesium-137 source and semi- non-LR units are pooled, they are then filtered
annually for the cobalt-60 source. For x-ray ir- as part of the pooling process. The shortest ex-
radiators, the dosimetry should be performed piration date of the pooled units determines
in accordance with the manufacturer’s recom- the expiration date of the pool.
mendations. Dose verification is also required In the United States, each pool prepared
after major repairs or relocation of the irradia- from LR platelets must have <5.0 × 106 residual
tor. For gamma irradiators, the turntable oper- leukocytes. The approved pooling set also al-
ation, timing device, and lengthening of irradi- lows sampling of the pool for detection of bac-
ation time caused by source decay should also terial contamination. A record of the unique
be monitored periodically. DIN for each individual member of the pool
Another important quality assurance step must be available. The pool must be labeled
is the demonstration that the product that was with the approximate total volume, ABO/Rh
irradiated received the desired amount of irra- type of the units in the pool, and number of
diation. For that reason, irradiation-sensitive units in the pool.
labels are used to demonstrate that irradiation Many countries in Europe prepare
of each batch of units was accomplished; this prestorage pools of buffy-coat platelets that
is a requirement in Europe.13 are preserved in platelet additive solution or in
In the United States, RBCs may be irradi- the plasma from one of the units from which
ated up to the end of their storage shelf life. platelets are prepared.79 Instruments that au-
The postirradiation expiration date is 28 days tomate the pooling process are increasingly
or the original expiration date, whichever is used in Europe. In a few countries in Europe,
earlier. In Europe, RBCs may be irradiated only systems for prestorage pooling of buffy-coat-
CHAPTER 6 Whole-Blood Collection and Component Processing 䡲 157
have been reviewed, the current donor infor- product deviations. Of these reports, 4258
mation has been compared to the previous in- (7.7%) involved QC and distribution errors.88
formation, the donor’s previous deferrals have
been examined, and all laboratory testing has
L ABE L IN G
been completed.87 Because of the limited
amount of time after collection that is avail- Blood component labeling is a highly regulat-
able for component separation, WB units may ed activity, and the documents that describe
be separated into components before all of the the requirements are listed below. Readers are
earlier processes have been completed. Sepa- advised to consult these documents and spe-
rated components are quarantined at the ap- cific national regulations for details.
propriate temperature until all of the suitabili- The FDA requirements for labeling of
ty steps have been completed and reviewed. blood and components are detailed in the
Often, physical and electronic quarantine are Guidelines for Uniform Labeling of Blood and
used simultaneously. Blood Components published in 1985.3 The
Certain blood components from previous FDA approved the International Society of
donations by donors whose more recent dona- Blood Transfusion (ISBT) 128 symbology, Ver-
tions test positive for infectious disease also sion 1.2.0, in 2000 and Version 2.0.0 in 2006.89
require quarantine and appropriate disposi- Detailed requirements are described in the
tion, as do units identified as unsuitable for Code of Federal Regulations (Title 21, CFR
transfusion because of postdonation informa- Parts 606.120, 606.121, and 606.122). AABB
tion. Other components may need to be quar- Standards requires that accredited facilities
antined so that the QC samples can be taken use ISBT 128 labels.4(p12)
and analyzed. For instance, if a sample is ob- The FDA rule that requires all blood com-
tained for bacterial contamination testing, the ponents to be labeled with a bar-coded label
component is held in quarantine for some pre- became effective on April 26, 2006. The rule re-
set amount of time and then released if the test quires that, at a minimum, the label contain
results are negative. the following bar-coded information: 1) the
A thorough understanding of the quaran- unique facility identifier (eg, registration num-
tine process is needed to prevent erroneous ber), 2) lot number relating to the donor, 3)
release of unsuitable blood components. product code, and 4) ABO group and Rh type
Components may be removed from the quar- of the donor. These pieces of information must
antine area, labeled, and released for distribu- be present in eye-readable and machine-read-
tion if all of the donor information, previous able format. The rule applies to blood centers
donor records, and current test results are sat- that collect and prepare blood components.
isfactory. Some nonconforming autologous The rule also applies to hospital transfusion
blood components may be released for autolo- services that prepare pooled cryoprecipitate
gous use only. and/or prepare divided units or aliquots of
Some blood components require emer- RBCs, platelets, and plasma for pediatric use.
gency release because they have a very short Another major part of labeling in the
storage time. Such is the case for granulocytes. United States is the information circular. The
Emergency release requires physician approv- circular must be made available to everyone
al and a label or tie tag to indicate that testing involved in the transfusion of blood compo-
was incomplete at the time of release. nents. The Circular of Information for the Use
Despite the widespread use of software to of Human Blood and Blood Components is pro-
control manufacturing processes, instances of duced by AABB, America’s Blood Centers, the
failure of quarantine and release of unsuitable American Red Cross, and the Armed Services
products continue to be reported to the FDA. Blood Program and is recognized as accept-
For instance, during fiscal year 2011, the FDA able by the FDA.49 The circular provides im-
received 54,947 reports of blood and plasma portant information about each blood compo-
CHAPTER 6 Whole-Blood Collection and Component Processing 䡲 159
nent and should be consulted for information 䡲 Double-density coding of numeric charac-
not included in this chapter. ters, which permits encoding of more infor-
Special message labels may also be af- mation in a given space.
fixed to blood component containers. The la- 䡲 Larger number of product codes, which al-
bels may include one or more of the following lows more detailed descriptions of blood
indications: 1) hold for further manufactur- components.
ing, 2) for emergency use only, 3) for autolo- 䡲 Enhanced scanning, which permits more
gous use only, 4) not for transfusion, 5) irradi- facile auditing of movements of blood com-
ated, 6) biohazard, 7) from a therapeutic ponents from one location to another.
phlebotomy, and 8) screened for special fac- 䡲 Ability to add information for autologous
tors [eg, HLA type or cytomegalovirus (CMV) donations.
antibody status]. ISBT 128 allows incorpora- 䡲 Ability to read more than one bar code with
tion of special attributes of the component, a single sweep (concatenation).
such as CMV antibody status. 䡲 Less laborious importation of “foreign” in-
Additional information on the container ventory into “own” inventory via the avail-
can be conveyed using a tie tag. Tie tags are es- ability of uniform systems for DINs and
product codes.
pecially useful for autologous and directed do-
nations. Tie tags include the patient’s identify-
In the future, ISBT 128 is expected to per-
ing information, name of the hospital where
mit information transfer by radiofrequency ID
the patient will be admitted for surgery, date of
tags or other means of electronic data trans-
surgery, and other information that may be
mission.
helpful to the hospital transfusion service.
Each component must also bear a unique
DIN that can be traced back to the blood do- QC OF BLOOD CO MPONENTS
nor. If components are pooled, a pool number
A quality management system is critical for es-
must allow tracing to the individual units with-
tablishing a current good manufacturing prac-
in a pool. tice-compliant operation (see Chapter 1). Test-
For groups planning to implement ISBT ing of blood components is necessary to
128, an important source of information is ensure the safety, purity, and potency of the
ICCBBA (formerly known as the International product and verify compliance with national
Council for Commonality in Blood Banking regulatory requirements, such as those of FDA.
Automation). This organization’s website fea- These requirements are minimum standards,
tures updates and a revised list of product and an individual manufacturer may establish
codes.90 more stringent ones. QC failures can serve as
Immediate benefits of ISBT 128 include an indicator of unexpected suboptimal re-
the following: agents or materials. Furthermore, QC data can
reveal previously unrecognized variations
䡲 Uniform labels applied on blood compo- from validated procedures and processes. A
nents manufactured by different collection timely detection system provides a proactive
centers. approach to early identification and resolution
䡲 Better traceability of components. of a manufacturing problem.
䡲 Improved self-checking features per char- Equipment QC is necessary to ensure that
acter. blood components achieve desired properties
䡲 Encoding of entire ASCII character set that in a consistent manner. Suggested QC steps for
includes alphanumeric and special charac- critical equipment in component laboratories
ters. are described in Chapter 1 and are listed in Ap-
䡲 Improved accuracy through reduction in pendix 1-3.
the number of misreads during scanning of Limitations of QC are demonstrated, for
the bar-coded information. example, in QC failures that are due to poor
160 䡲 AABB TECHNICAL MANUAL
sampling techniques and failures that may be be impractical to perform the QC steps. For ex-
ascribed to donor-related variables that can- ample, the FDA and Council of Europe do not
not be controlled. Examples of donor-associ- require QC of bedside leukocyte reduction fil-
ated variables include occult donor bactere- ters.
mia or viremia and leukocyte reduction filter A statistical process control approach has
failure resulting from the presence of sickled been suggested for QC of blood compo-
red cells. nents.15,91,92 Such an approach is expected to
National regulatory authorities provide provide a definition of product conformance
specific minimum requirements for QC testing to a standard with a given probability. This ap-
of blood products. These may differ from proach also allows a limit to be established for
country to country, and the most recent guid- nonconformance, facilitates implementation
ance documents and regulations should be re- of corrective actions, and permits QC to be in-
viewed. For certain blood components, it may dividualized for different blood components.
KEY POINTS
1. Modern blood containers are composed of soft plastic and identified by a lot number. They
should be pyrogen-free and flexible yet tough and both kink- and scratch-resistant. During
frozen storage, each plastic container has a glass transition temperature below which it be-
comes brittle and susceptible to breakage during transportation.
2. The initial 35 to 45 mL of blood drawn is allowed to collect into a diversion pouch at the col-
lection tubing. The pouch reduces bacterial contamination of collected blood by diverting
bacteria from the skin that may have otherwise entered the collection. Blood in this pouch
may be used for laboratory tests.
3. The average rate of adverse donor reactions after donation is 3.5% to 4.5%. Most reactions
are mild and require no further medical care. These reactions can be systemic (eg, fainting)
or local (eg, hematoma). About 1 in 3400 donors experience a reaction after leaving the do-
nation site and may need medical care. Deferral of low-blood-volume (less than 3.5 L) do-
nors may be helpful in reducing the risk of reactions, especially in young donors.
4. During centrifugation for component preparation, primary variables that affect cell separa-
tion and cell recovery are rotor size, centrifuge speed, and duration. The platelet-rich plas-
ma method is used in the United States for platelet concentrate preparation, and the buffy-
coat method is more common in Canada and Europe.
5. LR RBCs and platelets are not to exceed 5.0 × 106 residual WBCs per transfusion dose in the
United States or 1.0 × 106 residual WBCs per transfusion dose in Europe.
6. In plasma prepared from WB (by manual or apheresis methods) and labeled as “Plasma Fro-
zen Within 24 Hours After Phlebotomy,” the levels of all the coagulation factors are similar
to those of FFP except for some decrease in coagulation Factor VIII.
7. The radiation dose must be 25 to 50 Gy with a minimum delivered dose to any portion of the
blood components of 15 Gy in the United States. RBCs may be irradiated up until the end of
their storage shelf life; the postirradiation expiration date is 28 days after collection or the
original expiration date, whichever is sooner. The European standard requires that no part
of the component receive less than 25 Gy, a maximum dose to any part of the component of
50 Gy, and irradiation of RBCs only until day 28, with storage for no longer than 14 days after
irradiation or 28 days after collection, whichever is earliest. Platelet shelf life is not reduced
after irradiation.
8. Bar-coded and eye-readable container labels are increasingly using the ISBT symbology
(ISBT 128). ISBT 128 offers a number of advantages over the Codabar symbology, including
identification of the manufacturer throughout the world, more product codes, better accu-
CHAPTER 6 Whole-Blood Collection and Component Processing 䡲 161
racy as a result of reduced misreads during scanning, and enhanced conveyance of other la-
beling information.
9. Various approaches for QC of blood components have been promulgated by the AABB,
Council of Europe, and FDA.
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volume o f pl as ma maintain hemostatic lation factor levels in cryosupernatant pre-
function and viability. Transfusion 1994;34:44- pared from plasma treated with amotosalen
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43. Dumont LJ, AuBuchon JP, Gulliksson H, et al. 55. Rock G. A comparison of methods of pathogen
In vitro pH effects on in vivo recovery and sur- inactivation of FFP. Vox Sang 2011;100:169-78.
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laborative. Transfusion 2006;46:1300-5. position of fresh frozen plasma and virus-
44. Bertolini F, Murphy S, Rebulla P, Sirchia G. Role
inactivated therapeutic plasma prepara-
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tions: Correlation between composition and
medium. Transfusion 1992;32:152-6.
therapeutic efficacy. Thromb Res 2002;
45. Dumont LJ, Gulliksson H, van der Meer PF, et
107(Suppl 1):S3-8.
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57. Sharma AD, Sreeram G, Erb T, Grocott HP. Sol-
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vent-detergent-treated fresh frozen plas-
Collaborative on the effects of shipping plate-
ma: A superior alternative to standard fresh
lets. Transfusion 2007;47:1666-73.
46. Moroff G, George VM. The maintenance of plate- frozen plasma? J Cardiothorac Vasc Anesth
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agitation during storage. Transfusion 1990; 58. Murphy K, O’Brien P, O’Donnell J. Ac-
30:427-30. quired protein S deficiency in thrombotic
47. Gottschall JL, Rzad L, Aster RH. Studies of the thrombocytopenic purpura patients receiving
minimum temperature at which human solvent/detergent plasma exchange. Br J Hae-
platelets can be stored with full maintenance matol 2003;122:518-19.
of viability. Transfusion 1986;26:460-2. 59. Food and Drug Administration. Octaplas. Sil-
48. Moroff G, Holme S, George VM, Heaton WA. ver Spring, MD: CBER Office of Communication,
Effect on platelet properties exposure to tem- Outreach, and Development, 2013. [Available
peratures below 20 degrees C for short peri- at http://www.fda.gov/BiologicsBloodVac
ods during storage at 20 to 24 degrees C. cines/BloodBloodProducts/ApprovedProd
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49. Food and Drug Administration. Guidance for (accessed August 12, 2013).]
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tion for the use of human blood and blood cipitate and its relationship to factor VIII
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CBER Office of Communication, Outreach, and 61. Callum JL, Karkouti K, Yulia L. Cryoprecipitate:
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cryoprecipitate quality. J Clin Pathol 1985; 74. Dumont LJ, Cancelas JA, Dumont DF, et al. A
122:686-92. randomized controlled trial evaluating recov-
63. Scott EA, Puca KE, Pietz BC, et al. Analysis of ery and survival of 6% dimethyl sulfoxide-fro-
ADAMTS13 activity in plasma products using a zen autologous platelets in healthy volunteers.
modified FRETS-VWF73 assay (abstract). Transfusion 2013;53:128-37.
Blood 2005;106(Suppl):165a. 75. Lelkens CC, Koning JG, de Kort B, et al. Experi-
64. Smith JK, Bowell PJ, Bidwell E, Gunson HH. ences with frozen blood products in the Neth-
Anti-A haemagglutinins in factor VIII concen- erlands military. Transfus Apher Sci 2006;34:
trates. J Clin Pathol 1980;33:954-7. 289-98.
65. Pesquera-Lepatan LM, Hernandez FG, Lim RD, 76. Nuclear Regulatory Commission. Holders of
Chua MN. Thawed cryoprecipitate stored material licenses authorized to possess radio-
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ternative to factor VIII concentrate for con- ville, MD: Nuclear Regulatory Commission,
tinuous infusion. Haemophilia 2004;10:684- 2005. [Available at http://www.nrc.gov/read
8. ing-rm/doc-collections/enforcement/securi
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67. Schuetz AN, Hillyer KL, Roback JD, Hillyer CD.
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78. Voak D, Chapman J, Finney RD, et al. Guide-
68. Dzik S, Moroff G, Dumont L. A multicenter study
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al WBCs in WBC-reduced blood components:
ated graft-versus-host disease. Transfus Med
Nageotte hemocytometry, flow cytometry,
1996;6:261-71.
and microfluorimetry. Transfusion 2000;40:
79. Van der Meer PF, de Korte D. The buffy-coat
513-20.
method. In: Blajchman M, Cid J, Lozano M,
69. Whitley PH, Wellington M, Sawyer S, et al. A
eds. Blood component preparation from
simple, new technology for counting low levels
benchtop to bedside. Bethesda, MD: AABB
of white blood cells in blood components:
Comparison to current methods. Transfusion Press, 2011:55-81.
80. Moroff G, Friedman A, Robkin-Kline L, et al.
2012;52(Suppl):59A.
70. Valeri CR, Ragno G, Pivacek LE, et al. A multi- Reduction of the volume of stored platelet
center study of in vitro and in vivo values in concentrates for use in neonatal patients.
human RBCs frozen with 40-percent (wt/vol) Transfusion 1984;24:144-6.
glycerol and stored after deglycerolization for 81. Pisciotto P, Snyder EL, Napychank PA, Hop-
15 days at 4°C in AS-3: Assessment of RBC pro- per SM. In vitro characteristics of volume-
cessing in the ACP 215. Transfusion 2001;41: reduced platelet concentrate stored in sy-
933-9. ringes. Transfusion 1991;31:404-8.
71. Valeri CR, Pivacek LE, Cassidy GP, Ragno G. The 82. Aster RH. Effect of acidification in enhancing
survival, function, and hemolysis of human viability of platelet concentrates: Current sta-
RBCs stored at 4°C in additive solution (AS- tus. Vox Sang 1969;17:23.
1, AS-3, or AS-5) for 42 days and then bio- 83. Dumont LJ, Krailadsiri P, Seghatchian J, et al.
chemicall y modified, frozen, t h a w e d , Preparation and storage characteristics of
washed, and stored at 4°C in sodium chlo- white-cell-reduced high-concentration
ride and glucose solution for 24 hours. Trans- platelet concentrates collected by an aphere-
fusion 2000;40:1341-5. sis system for transfusion in utero. Trans-
72. Alving BM, Reid TJ, Fratantoni JC, Finlayson JS. fusion 2000;40:91-100.
Frozen platelets and platelet substitutes in 84. Dumont LJ, Beddard R, Whitley P, et al. Autolo-
transfusion medicine. Transfusion 1997;37: gous transfusion recovery of WBC-reduced
866-76. high-concentration platelet concentrates.
73. Lee DH, Blajchman MA. Novel platelet prod- Transfusion 2002;42:1333-9.
ucts and substitutes. Transfus Med Rev 1998; 85. Schoenfeld H, Muhm M, Doepfmer UR, et al.
12:175-87. The functional integrity of platelets in vol-
CHAPTER 6 Whole-Blood Collection and Component Processing 䡲 165
James W. Smith, MD, PhD, Medical Director, Oklahoma Blood Institute, Oklahoma City, Oklahoma
The author has disclosed no conflicts of interest.
167
168 䡲 AABB TECHNICAL MANUAL
Fenwal ALYX X X X
Fenwal Amicus X X X
Fenwal Autopheresis C X
Fresenius AS104 X
TerumoBCT (COBE) X X X
Spectra
TerumoBCT Spectra Optia X
TerumoBCT Trima V-4 X X X X
TerumoBCT Trima Accel X X X X
Haemonetics Cymbal X
Haemonetics MCS+ X X X
LN9000
Haemonetics MCS+ X X X
LN8150
Haemonetics PCS-2 X
*Concurrent collection refers to the ability to collect more than one type of product.
GRAN = granulocytes; PLT = plateletpheresis (single, double, triple); cRBC = concurrent* 1 unit of Red Blood Cells (RBCs);
2-RBC = double unit of RBCs; PLASMA = 1 unit of plasma; cPLASMA = concurrent plasma; V-4 = software version 4.
sion Services.1(pp62-67) The circumstances that each of which must meet minimum standards.
are unique to apheresis collection are ad- Some instruments are programmed to calcu-
dressed in the sections that follow. late the platelet yield based on the donor’s he-
matocrit, platelet count, height, and weight.
Platelets For alloimmunized patients who do not
respond to random allogeneic platelets, trans-
Apheresis is used to obtain platelets from vol-
fusions of platelets from an apheresis donor
unteer donors, patients’ family members, or
selected on the basis of a compatible platelet
donors with HLA or platelet-antigen-compati-
crossmatch or that are matched for HLA anti-
ble phenotypes. By design, apheresis proce-
gens may be the only way to achieve a satisfac-
dures are intended to collect large numbers of
tory posttransfusion platelet increment. In the
platelets from an individual, thereby providing
United States, the use of apheresis platelets
a more potent product with fewer donor expo-
has been steadily increasing over the past 25
sures for the patient. AABB Standards requires
years. It is estimated that 90% of platelets
that an apheresis platelet component contain
transfused in the United States are apheresis
at least 3 × 1011 platelets in 90% of sampled
platelets.2
units.1(pp28-29)
With newer technology and more effi-
Donor Selection and Monitoring
cient processes, higher yields of platelets may
be obtained from one donor, and the original Plateletpheresis donors may donate more fre-
apheresis unit may be split into multiple units, quently than whole-blood donors but must
CHAPTER 7 Blood Component Collection by Apheresis 䡲 169
meet all of the other criteria for whole-blood mL volume previously mentioned). The plate-
donation. The interval between donations let count of each unit should be kept on record
should be at least 2 days, and donors should but need not be written on the component la-
not undergo plateletpheresis more than twice bel. Units containing less than 3.0 × 1011 plate-
in a week or 24 times in a rolling 12-month pe- lets should be labeled with the actual platelet
riod.1(p20),3 If the donor donates a unit of whole count.3
blood or if it becomes impossible to return the It is possible to collect plasma concur-
donor’s red cells during plateletpheresis, at rently with platelets. Such collection is dis-
least 8 weeks should elapse before a subse- cussed in more detail in the “Plasma” section
quent plateletpheresis procedure unless the below.
extracorporeal red cell volume (ECV) is less Vasovagal and hypovolemic reactions are
than 100 mL. Platelets may be collected from rare in apheresis donors but may occur. Pares-
donors who do not meet these requirements thesias (tingling sensations) and other reac-
only if the component is expected to be of par- tions to citrate anticoagulant are not uncom-
ticular value to a specific intended recipient
mon. (Similar citrate toxicity reactions in
and a physician certifies in writing that the do-
recipients are discussed along with whole
nor’s health will not be compromised by the
blood transfusions in Chapter 27.) Serious re-
donation. Donors who have taken antiplatelet
actions occur less often among apheresis do-
medications that irreversibly inhibit platelet
nors than whole blood donors.4
function are deferred for specific intervals be-
fore donation (48 hours for aspirin/aspirin-
containing medications and piroxicam, 14
Laboratory Testing
days for clopidogrel and ticlopidine) because Tests for ABO group, Rh type, unexpected allo-
apheresis platelets are often the sole source of antibodies, and transfusion-transmitted dis-
platelets given to a patient.1(p61),3 eases must be performed by the collecting fa-
A platelet count is not required before the cility in the same manner as for other blood
first apheresis collection; however, triple col- components. Each unit must be tested unless
lections of platelets may not be drawn from the donor is undergoing repeated procedures
first-time donors unless a qualifying platelet to support a specific patient, in which case
count is obtained on a sample collected before testing for infectious disease markers needs to
the procedure.3 If the donation interval is less be repeated only at 30-day intervals.5
than 4 weeks, many facilities prefer that the If red cells are visible in a product, the he-
donor’s platelet count be above 150,000/µL be-
matocrit should be determined. AABB Stan-
fore plateletpheresis occurs to prevent a post-
dards state that if the component contains
donation count of less than 100,000/µL. AABB
more than 2 mL of red cells, the red cells must
Standards permits qualification of a donor
be ABO compatible with the recipient’s plasma
with a platelet count from a sample collected
and be crossmatched.1(pp37-38) In such cases, a
immediately before the procedure or one ob-
tained either before or after the previous pro- sample of donor blood is attached to the con-
cedure.1(p21) Exceptions to these laboratory cri- tainer for compatibility testing. In some in-
teria should be approved in writing by the stances, it may be desirable for the donor plas-
apheresis program physician based on docu- ma to be ABO compatible with the recipient’s
mented medical need. red cells (eg, if the recipient is a child or an
The Food and Drug Administration (FDA) ABO-mismatched allogeneic progenitor cell
specifies that the total volume of plasma col- transplant recipient). In the United States, to
lected should be no more than 500 mL (or 600 be considered leukocyte reduced, apheresis
mL for donors weighing more than 175 lb) or platelets must contain less than 5 × 106 leuko-
the volume described in the labeling of the au- cytes per unit and platelets must meet the
tomated blood cell separator device (which specifications of the apheresis device
may be more or less than the 500-mL or 600- manufacturer.3 In Europe, the guideline for
170 䡲 AABB TECHNICAL MANUAL
leukocyte-reduced components is fewer than 1. Donors must give consent for the proce-
1 × 106 leukocytes per unit. dure, and they must be observed closely
during the procedure. Emergency medical
Record-Keeping care must always be available.
2. Red-cell losses related to the procedure,
Complete records must be kept on each proce-
dure. All adverse reactions occurring during including samples collected for testing,
collection procedures (or transfusion) must be should be monitored so that no more than
documented along with the results of thor- 200 mL of red cells are removed in 8 weeks.
ough investigations. Records of all laboratory If the donor’s red cells cannot be returned
findings and collection data must be periodi- during an apheresis procedure, hemapher-
cally reviewed by a knowledgeable physician esis or whole-blood donation should be
and must be found to be within acceptable deferred for 8 weeks.
limits. FDA guidelines require a periodic re- 3. For manual collection systems, a mecha-
view of donor records to monitor platelet nism must exist to ensure safe reinfusion of
counts.3 Facilities must have policies and pro- the autologous red cells.
cedures in place to ensure that donor red cell 4. In manual procedures for donors weighing
loss during each procedure does not exceed 110 to 175 lb, no more than 500 mL of whole
acceptable limits.1(p18) blood should be removed at one time or no
more than 1000 mL during a session or
Plasma within a 48-hour period. The limits for
Apheresis devices can be used to collect plas- donors who weigh 175 lb are 600 mL and
ma for transfusion or as Source Plasma for 1200 mL, respectively. For automated pro-
subsequent manufacturing. Recently, the FDA cedures, the allowable volume has been
approved apheresis devices for the collection determined for each instrument by the
of plasma held at 1 to 6 C within 8 hours and FDA.
frozen within 24 hours after phlebotomy and 5. At least 48 hours should elapse between
plasma held at room temperature for up to 24 successive procedures. Donors should not
hours and frozen within 24 hours after phle- undergo more than two procedures within
botomy. a 7-day period.
The FDA has provided guidance with re- 6. At the time of initial plasmapheresis and at
gard to the volume of plasma that may be col- 4-month intervals for donors undergoing
lected using automated devices. A distinction serial (large-volume) plasmapheresis (donors
is made between infrequent plasmapheresis, undergoing plasmapheresis more often
in which the donor undergoes plasmapheresis than once every 4 weeks), serum or plasma
no more frequently than once every 4 weeks,
must be tested for total protein and for
and serial plasmapheresis (or Source Plasma
serum protein electrophoresis or for quan-
collection, the process to collect plasma for
titative immunoglobulins. Results must be
fractionation into plasma components), in
which the donation is more frequent than within normal limits.
once every 4 weeks. For donors in infrequent 7. A qualified licensed physician, knowledge-
plasmapheresis programs, donor selection able about all aspects of hemapheresis,
and monitoring requirements are the same as must be responsible for the program.
those for whole-blood donation. Plasma ob-
tained by these processes is intended for direct For manual collection systems, a process
transfusion. that is rarely used in the United States at pres-
For serial plasma (Source Plasma) collec- ent, requirements are outlined in the Code of
tion using either automated instruments or Federal Regulations6 and have been summa-
manual techniques, the following principles rized in previous editions of this Technical
apply6: Manual.
CHAPTER 7 Blood Component Collection by Apheresis 䡲 171
Red Cells and Multicomponent products within 8 weeks and the ECV of the
Donations procedure is <100 mL. Donors should be de-
ferred for at least 16 weeks after a double-RBC
Both AABB Standards and FDA guidance doc- donation. If an apheresis procedure is discon-
uments address the removal of red cells by au- tinued before completion and absolute red cell
tomated apheresis methods. A guidance docu- loss is <200 mL, the donor may donate again
ment issued in 2001 by the FDA finalized within 8 weeks if he or she meets all donor eli-
recommendations for the use of automated gibility criteria.
apheresis equipment to collect the following7: If a donor has a second red cell loss of
<100 mL during a subsequent donation within
䡲 Single units of RBCs and plasma. 8 weeks of a previous donation, the donor
䡲 Single units of RBCs and platelets. should be deferred for 8 weeks. If the total ab-
䡲 Single units of RBCs, platelets, and plasma. solute red cell loss within 8 weeks is >300 mL,
䡲 Double units of RBCs only. the donor should be deferred for 16 weeks
from the date of the last red cell loss. If an
The guidance document includes FDA apheresis procedure is discontinued and the
regulations requiring that equipment perform absolute red cell loss is >200 mL but <300 mL,
and be used in the manner for which it was de- the donor should be deferred for 8 weeks. If an
signed to collect or process blood and compo- apheresis procedure is discontinued and total
nents. Standard operating procedures, includ- absolute red cell loss is >300 mL, the donor
ing device manufacturers’ instructions for use should be deferred for 16 weeks.
and maintenance of current records, are de- Saline infusion is used to minimize vol-
scribed. The sections below summarize the in- ume depletion.
formation in the guidance document.
Quality-Control Issues
Donor Selection and Monitoring
The FDA has promulgated quality-control
The FDA requires that an adequate hemoglo- (QC) programs for RBC unit collection by
bin level be determined by a quantitative apheresis. There are two phases:
method for predonation hemoglobin or the
hematocrit of donors undergoing double-RBC 䡲 In Phase I QC, 100 consecutive RBC units
collection. The procedure is limited to persons are tested to determine the expected or tar-
who are larger and have a higher hematocrit get red cell volume in accordance with the
than the minimum standards for whole-blood specifications in the device operator’s man-
donations. For males, the minimum weight is ual. Target values are compared with the
130 lb, and the minimum height is 51. For fe- actual values to determine product accept-
males, the minimum weight is 150 lb, and the ability. If the QC results are satisfactory,
minimum height is 5 5. The minimum hema- which includes that at least 95% of the units
tocrit is 40% for both genders. Donors who are meet product specifications, the establish-
shorter than the minimum height or who ment may proceed to Phase II.
weigh less than the minimum weight, as estab- 䡲 Phase II QC consists of monthly testing of a
lished by the FDA in device operator manuals, representative sample of 50 units of the
should be further evaluated. These donors manufactured product from each collec-
must also meet all relevant FDA criteria for al- tion center. At least 1 unit from a single
logeneic or autologous whole-blood donation. RBC protocol or both units from a double-
Donors who have given a single unit of RBC protocol device used at the center
RBCs with platelets, plasma, or both should be should be included in the testing. At least
deferred for at least 8 weeks. The exception is 95% of the products tested should meet
when a donor serves as a plateletpheresis do- product specifications as described in the
nor or a donor of platelets with plasma by- device operator’s manual.
172 䡲 AABB TECHNICAL MANUAL
Equipment for Concurrent Plasma ified, dual-stage channel and LRS cone to con-
Collection sistently collect leukocyte-reduced platelets
(<1.0 × 106 WBCs). To increase platelet yields,
Plasma can be collected as a concurrent prod- the Trima Accel (Versions 5.0, 5.01, and 5.1)
uct during collection of apheresis platelets or uses a single-stage, doughnut-shaped channel
automated RBCs. The amount that can be col- and larger LRS cone to consistently collect leu-
lected is determined by the volume of plate- kocyte-reduced platelets.21 The Trimas use sin-
lets, red cells, or both, that are being collected gle-access kits only. The ECV of the Trimas is
and by the maximum volume that can be re- 182 to 196 mL. They are capable of collecting
moved from the donor. Equipment capable of single, double, or triple units of apheresis
concurrent plasma collection includes Hae- platelets as well as concurrent plasma and
monetics MCS+ LN9000, Haemonetics MCS+ RBCs, depending on donor size, platelet count,
LN8150, Fenwal Amicus, Fenwal ALYX, Teru- and hematocrit.21-23
moBCT (COBE) Spectra (TerumoBCT, Lake-
wood, CO), TerumoBCT Trima, and Teru- Fenwal Amicus
moBCT Trima Accel.
The Amicus is capable of collecting single,
double, or triple apheresis platelets as well as
concurrent plasma and RBCs (single-access kit
Platelets
only), depending on the donor’s size, platelet
TerumoBCT (COBE) Spectra count, and hematocrit.10,16,22,24 The Amicus
uses centrifugal force and a double compart-
The TerumoBCT (COBE) Spectra is capable of
ment belt wrapped around a spool to separate
collecting single, double, or triple apheresis the platelets. The platelets accumulate in the
platelet units as well as concurrent plasma, de- collection chamber and are transferred to the
pending on the size and platelet count of the final collection bags at the end of the proce-
donor.10,16-19 This device uses a dual-stage (dif- dure. The Amicus is capable of single- or
ferent radius) channel to collect leukocyte- double-access procedures. The ECV of the
reduced platelets. Leukocyte reduction to <5 × double-access kit is 210 mL, and that of the
106 White Blood Cells (WBCs) can be obtained single-access kit is 209 mL. The ECV of the
in approximately 85% of the collections for whole-blood bag is adjustable. Consistent leu-
software versions that are lower than 5.0.20 Use kocyte reduction is accomplished without ex-
of software versions 5.0 and 7.0 can more con- ternal filtration, and this process is approved
sistently result in the collection of products by the FDA for submission of a prior approval
containing <1.0 × 106 WBCs with the leukocyte- supplement.
reduction system (LRS), which uses a cone in
the centrifuge and saturated, fluidized, particle- Haemonetics MCS+ LN9000
bed filter technology to remove residual WBCs
leaving the second stage of the channel.10,16-19 The Haemonetics MCS+ LN9000 system is ca-
The Spectra is capable of single- or double- pable of conducting several different apheresis
access procedures. The ECV of the single- procedures, including apheresis platelet col-
access kit is 361 mL and of the double-access lection. It uses the Latham conical bowl, plas-
kit is 272 mL. The Spectra is being replaced by ma-controlled hematocrit, and plasma surge
the more efficient Trima or Trima Accel.11 technique to build up the platelets and float
them off the bowl with rapid plasma infusion.
TerumoBCT Trima and Trima Accel Although this technique results in leukocyte-
reduced platelets, the use of an inline leuko-
The TerumoBCT Trimas were designed as cyte reduction filter ensures consistency in
automated donor collection machines for leukocyte reduction.10,25-28 The LN9000 uses a
platelets, plasma, and RBCs only. The Teru- single-access kit, and the ECV ranges from 480
moBCT Trima (Version 4) uses a smaller, mod- mL (38% hematocrit) to 359 mL (52% he-
CHAPTER 7 Blood Component Collection by Apheresis 䡲 175
matocrit). The LN9000 is capable of collecting access kit.11,31 The addition of the preservative
single, double, or triple units of apheresis solution and leukocyte reduction by filtration
platelets as well as concurrent plasma, de- are performed manually and off-line after the
pending on donor size and platelet count.10,25-28 collection is complete.
KEY POINTS
1. Apheresis components must meet many of the same basic regulatory requirements (eg, do-
nor consent, storage conditions, and transportation requirements) as whole-blood-derived
components, although more specific requirements also apply to each type of apheresis-
component collection.
2. The majority of platelets collected and transfused in the United States are apheresis-derived
platelets.
3. Plasma can be collected by apheresis for transfusion or as Source Plasma for subsequent
manufacturing. The FDA provides guidance regarding the volume of plasma that may be
collected using automated devices.
4. In multicomponent donations, a variety of components or combinations of components
may be collected with apheresis technology. Regulations specific to this practice apply to
donor selection and monitoring, QC, and records.
5. Red cells can be removed concurrently with other components, or a double RBC unit may
be collected.
6. Several instruments and systems have been developed and/or adapted for apheresis collec-
tion of blood components that use different technologies. Some are appropriate for the col-
lection of only one type of component, and others can collect multiple types of compo-
nents.
7. Granulocyte collection differs from that of other components. Specific techniques should
be used and certain factors must be taken into consideration for the optimal collection of
granulocytes by apheresis.
REFER ENCES
1. Levitt J, ed. Standards for blood banks and platelets by automated methods. (December
transfusion services. 29th ed. Bethesda, MD: 17, 2007) Silver Spring, MD: CBER Office of
AABB, 2014. Communication, Outreach, and Development,
2. US Department of Health and Human Servic- 2012. [Available at http://www.fda.gov/Biolog
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47:1002-5. khardt T. Residual cell content in plasma pro-
5. Code of federal regulations. Title 21, CFR Part duced by three apheresis procedures. Transfu-
610.40. Washington, DC: US Government sion 2003;43:1522-6.
Printing Office, 2014 (revised annually). 16. Burgstaler EA, Pineda AA, Bryant SC. Prospec-
6. Code of federal regulations. Title 21, CFR Part tive comparison of plateletapheresis using
640, Subpart G. Washington, DC: US Govern- four apheresis systems on the same donors.
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7. Food and Drug Administration. Guidance for 17. Perseghin P, Mascaretti L, Riva M, et al. Com-
industry: Recommendations for collecting red parison of plateletapheresis concentrates pro-
blood cells by automated apheresis methods. duced with Spectra LRS version 5.1 and LRS
(January 30, 2001) Silver Spring, MD: CBER Of- Turbo version 7.0 cell separators. Transfusion
fice of Communication, Outreach, and Devel- 2000;40:789-93.
opment, 2001. [Available at http://www.fda. 18. Zingsem J, Glaser A, Weisbach V. Evaluation of
gov/downloads/BiologicsBloodVaccines/ a platelet apheresis technique for the prepara-
GuidanceComplianceRegulatoryInformation/ tion of leukocyte-reduced platelet concen-
Guidances/Blood/ucm080764.pdf (accessed trates. Vox Sang 1998;74:189-92.
December 10, 2013).] 19. Zingsem J, Zimmermann R, Weisbach V, et al.
8. Massey E, Paulus U, Doree C, Stanworth S. Comparison of COBE white cell-reduction and
Granulocyte transfusions for preventing infec- standard plateletapheresis protocols in the
tions in patients with neutropenia or neutro- same donors. Transfusion 1997;37:1045-9.
20. Maresh S, Randels M, Strauss R, et al. Compar-
phil dysfunction. Cochrane Database Syst Rev
ison of plateletapheresis with a standard and
2009;1:CD005341.
an improved collection device. Transfusion
9. Food and Drug Administration. Safety com-
1993;33:835-7.
munication: Boxed warning on increased mor-
21. McAteer M, Kagen L, Graminske S, et al. Trima
tality and severe renal injury, and additional
Accel improved platelet collection efficiency
warning on risk of bleeding, for use of hy-
with the merging of single stage separation
droxyethyl starch solutions in some settings.
technology with leukoreduction performance
Rockville, MD: Office of Community Outreach
of the LRS chamber (abstract). Transfusion
and Development, 2013. [Available at: http://
2002;42(Suppl):37S.
www.fda.gov/BiologicsBloodVaccines/Safety 22. Burgstaler EA, Winters JL, Pineda AA. Paired
Availability/ucm358271.htm (accessed March comparison of Gambro Trima Accel vs Baxter
30, 2014).] Amicus single-needle plateletapheresis. Trans-
10. Burgstaler EA. Current instrumentation for fusion 2004;44:1612-20.
apheresis. In: McLeod BC, Szczepiorkowski 23. Elfath MD, Whitley P, Jacobson MS, et al. Eval-
ZM, Weinstein R, Winters JL, eds. Apheresis: uation of an automated system for the collec-
Principles and practice. 3rd ed. Bethesda, MD: tion of packed RBCs, platelets, and plasma.
AABB Press, 2010:71-110. Transfusion 2000;40:1214-22.
11. Burgstaler EA. Blood component collection by 24. Yockey C, Murphy S, Eggers L, et al. Evaluation
apheresis. J Clin Apher 2006;21:142-51. of the Amicus separator in the collection of
12. Hood M, Mynderup N, Doxon L. Evaluation of apheresis platelets. Transfusion 1999;38:848
Haemonetics PCS-2 and Fenwal Auto-C plas- 25. Valbonesi M, Florio G, Ruzzenenti MR, et al.
mapheresis collection systems (abstract). Multicomponent collection (MCC) with the
J Clin Apher 1996;11:99. latest hemapheresis apparatuses. Int J Artif Or-
13. Burkhardt T, Kappelsberger C, Karl M. Evalua- gans 1999;22:511-15.
tion of a new combined centrifugation/filtra- 26. Paciorek L, Holme S, Andres M, et al. Evalua-
tion method for the collection of plasma via tion of the continuous filtration method with
plasmapheresis (abstract). Transfusion 2001; double platelet products collected on the
41(Suppl):50S. MCS+ (abstract). J Clin Apher 1998;13:87.
14. Burnouf T, Kappelsberger C, Frank K, Bur- 27. Ford K, Thompson C, McWhorter R, et al. Eval-
khardt T. Protein composition and activation uation of the Haemonetics MCS+ LN9000 to
markers in plasma collected by three apheresis produce leukoreduced platelet products (ab-
procedures. Transfusion 2003;43:1223-30. stract). J Clin Apher 1996;11:104.
178 䡲 AABB TECHNICAL MANUAL
28. Rose C, Ragusa M, Andres M, et al. Evaluation tion of rHuG-CSF: Effects on peripheral blood
of the MCS + LN9000 in-line leukoreduction counts, collection efficiency, and yield. Trans-
filter (abstract). Transfusion 1996;36(Suppl):85. fusion 2001;41:390-5.
29. Picker SM, Radojska SM, Gathof BS. Prospec- 35. Dale DC, Lises WC, Llewellyn C, et al. Neutro-
tive evaluation of double RBC collection using phil transfusions: Kinetics and functions of
three different apheresis systems. Transfus neutrophils mobilized with granulocyte colo-
Apher Sci 2006;35:197-205.
ny-stimulating factor and dexamethasone.
30. Snyder EL, Elfath MD, Taylor H, et al. Collec-
Transfusion 1998;38:713-21.
tion of two units of leukoreduced RBCs from a
36. Food and Drug Administration. Substantially
single donation with a portable multiple-com-
ponent collection system. Transfusion 2003; equiva lent 510 (k) device infor m ation,
43:1695-705. BK130065 summary. TerumoBCT Spectra Op-
31. Moog R, Frank V, Müller N. Evaluation of a tia. Silver Spring, MD: FDA, 2013. [Available
concurrent multicomponent collection sys- at:// http://www.fda.gov/BiologicsBloodVac
tem for the collection and storage of WBC- cines/BloodBloodProducts/ApprovedProd
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sion 2001;41:1159-64. formation/ucm390957.htm (accessed March
32. Nussbaumer W, Grabmer C, Maurer M, et al. 31, 2014).]
Evaluation of a new mobile two unit red cell 37. Kretschmer V, Biehl M, Coffe C, et al. New fea-
apheresis system (abstract). J Clin Apher 2006; tures of the Fresenius blood cell separator
21:20. AS104. In: Agishi T, Kawamura A, Mineshima
33. Smith JW. Automated donations: Plasma, red
M, eds. Therapeutic plasmapheresis (XII): Pro-
cells, and multicomponent donor procedures.
ceedings of the 4th International Congress of
In: McLeod BC, Szczepiorkowski ZM, Wein-
stein R, Winters JL, eds. Apheresis: Principles Apheresis of the World Apheresis Association
and practice. 3rd ed. Bethesda, MD: AABB and the 12th Annual Symposium of the Japa-
Press, 2010:125-40. nese Society for Apheresis, 3-5 June 1992, Sap-
34. Worel N, Kurz M, Peters C, Höcker P. Serial poro, Japan. Utrecht, the Netherlands: VSP BV,
granulocyte apheresis under daily administra- 1993:851-5.
C h a p t e r 8
Susan A. Galel, MD
BLOOD COMPONENTS, LIKE all United States. Initially, donors were screened
other medications in the United States, only for syphilis. In the 1960s, studies showed
are regulated by the Food and Drug Adminis- that more than 30% of patients who received
tration (FDA). The FDA requires medication multiple transfusions developed posttransfu-
manufacturers to verify the suitability of every sion hepatitis (PTH).2 Studies in the early
raw material in their products.1 For biologic 1970s found that hepatitis B virus (HBV) ac-
pharmaceuticals, the donor is the key ingredi- counted for only 25% of PTH cases.2 Both HBV
ent whose suitability must be scrutinized. and non-A, non-B (NANB) hepatitis occurred
Blood banks test a sample of blood from more frequently in recipients of blood from
each donation to identify donors and donated commercial (paid) blood donors than in recip-
components that might harbor infectious ients of blood from volunteer donors. By the
agents. This screening process is critically im- mid-1970s, implementation of sensitive tests
portant because many blood components (eg, for hepatitis B surface antigen (HBsAg) and a
red cells, platelets, plasma, and cryoprecipi- nearly universal changeover to a volunteer do-
tate) are administered intravenously to recipi- nor supply resulted in a dramatic reduction in
ents without pasteurization, sterilization, or the incidence of both HBV and NANB PTH.
other treatments to inactivate infectious Still, NANB PTH continued to occur in approx-
agents. Thus, infectious agents in a donor’s imately 6% to 10% of multitransfused pa-
blood at the time of donation that are not de- tients.2,3
tected by the screening process can be trans- In the absence of a specific test for the
mitted directly to recipients. causative agent of NANB PTH, investigators
searched for surrogate markers that could be
used to identify donations associated with
H I S TO R I C A L OVE RV I EW O F
NANB hepatitis. The presence of antibody to
BLOOD DONOR SCREENING
hepatitis B core antigen (anti-HBc) and/or the
Table 8-1 shows the progression over time of presence of elevated alanine aminotransfer-
donor testing for infectious diseases in the ase in blood donors were shown to be associ-
Susan A. Galel, MD, Associate Professor of Pathology, Stanford University School of Medicine, and Medical
Director of Clinical Services, Stanford Blood Center, Palo Alto, California
The author has disclosed a financial relationship with Roche Molecular Systems.
179
180
䡲
Year First
Implemented Screening Test Comments
2004 Sampling of platelet components to detect Testing was recommended by the AABB in 2004. Some tests are approved by FDA as quality-
bacterial contamination control tests. Since 2011, AABB has accepted only FDA-approved tests or those validated to have
equivalent sensitivity.
2006-2007 Antibody to Trypanosoma cruzi This test was approved by FDA as a donor screen late in 2006, and widespread testing was imple-
mented in 2007. The rarity of seroconversion in US residents led to endorsement in FDA 2010
guidance of one-time donor screening.
2007-2008 HBV nucleic acid test (detects HBV DNA) This test was initially implemented as part of automated multiplex assays that detect HIV RNA,
HCV RNA, and HBV DNA simultaneously. HBV DNA screening was explicitly recommended by
FDA guidance issued in October 2012.
Infectious Disease Screening
HBsAg = hepatitis B surface antigen; AIDS = acquired immune deficiency syndrome; FDA = Food and Drug Administration; HIV = human immunodeficiency virus; HTLV = human T-cell lym-
photropic virus; ALT = alanine aminotransferase; HBc = hepatitis B core antigen; HCV = hepatitis C virus; NANB = non-A, non-B; HBV = hepatitis B virus; RNA = ribonucleic acid; WNV = West
Nile virus; DNA = deoxyribonucleic acid.
䡲
181
182 䡲 AABB TECHNICAL MANUAL
ated with an increased risk of NANB PTH.4-7 results.11 Blood donated during this “window
However, concerns about the nonspecific na- period” can contain infectious HIV but is not
ture of these tests led to a delay in their imple- detected by the donor screening tests.
mentation for donor screening. The most straightforward means of pro-
The concept of surrogate testing was re- tecting the blood supply from window-period
visited in the early 1980s when concerns arose donations is to exclude potential donors with
about the transmission of AIDS by transfu- an increased likelihood of exposure to HIV.
sions before the identification of its causative The FDA initially recommended that blood
agent. In an effort to reduce the potential banks provide informational materials to do-
transmission of AIDS by transfusion, some nors listing HIV risk activities and requesting
blood banks implemented donor testing for that individuals not donate if they had en-
anti-HBc (because this antibody was highly gaged in these activities. Later, in 1990, the
prevalent in populations at increased risk of FDA recommended asking each donor directly
AIDS) or donor screening for inverted CD4/ about each risk activity. In 1992, the FDA is-
CD8 T-cell ratio (an immune abnormality sued comprehensive guidance describing this
found in both AIDS patients and those with questioning process.
the pre-AIDS lymphadenopathy syndrome).8 In the years since the discovery of HIV, the
The leaders of most blood banks, however, be- risk of transfusion-transmitted disease has
lieved that the risk of transmitting AIDS by been progressively reduced through a variety
transfusion was too low to warrant surrogate of measures:
interventions.9 After the human immunodefi-
ciency virus (HIV) was isolated and identified 1. Use of donor education and questioning to
as the causative agent of AIDS, a donor screen- minimize window-period donations and to
ing test for antibody to this agent was rapidly screen for infections for which no tests are
developed and implemented in 1985. available.
Once the HIV antibody test became avail- 2. Shortening of the window period for spe-
able and cases of HIV were recognized in both cific agents by improving and/or adding
prior donors and transfusion recipients, it be- tests to detect earlier stages of infection.
came clear that the risk of transmitting HIV via 3. Use of questions and tests that exclude
blood transfusion had been greatly underesti- donors at increased risk of blood-borne or
mated.10 The HIV experience highlighted the sexually transmitted infections.
fact that an infectious agent associated with a 4. Surveillance for transfusion-transmissible
lengthy asymptomatic carrier state could be
diseases and implementation of new donor
present in the blood supply for years without
being recognized. screening tests, when available.
In the wake of this realization, the ap-
proach to donor screening was extended be- The approach used to screen potential
yond known agents. Current donor history donors for a specific agent depends on wheth-
evaluations include screening for, and exclu- er specific risk factors are identifiable and
sion of, donors with, an increased risk of expo- whether donor screening tests are available.
sure to blood-borne or sexually transmitted Table 8-2 lists the types of screening approach-
diseases. The intention is to reduce the likeli- es used for different infectious agents.
hood that the blood supply will contain other,
as-yet-unidentified agents that are potentially DONOR SCREENING TESTS
transmissible by blood.
Rare transmissions of HIV persisted even The donor infectious disease tests required by
after implementation of donor testing due to a the FDA are specified in Title 21, Section
delay of weeks or months between the time a 610.40, of the Code of Federal Regulations
person is infected with HIV and the time the (CFR).1 The process of amending the CFR is
screening test for HIV antibody shows positive slow; therefore, the FDA initially communi-
CHAPTER 8 Infectious Disease Screening 䡲 183
cates changes in its recommendations by issu- screening tests that are currently performed by
ing guidance publications. Although FDA US blood banks.
guidance documents do not constitute legal
requirements, they define the expected stan- Logistics of Testing
dard of practice in the United States. The AABB All infectious disease testing for donor qualifi-
also issues recommendations to the blood cation purposes is performed on samples col-
banking community, and these are communi- lected at the time of donation and sent to the
cated either by Association Bulletins or by in- donor testing laboratory. In addition, platelet
clusion in AABB Standards for Blood Banks components are tested for bacterial contami-
and Transfusion Services. AABB recommenda- nation typically by the component manufac-
tions and standards do not have the force of turing facility.
law, except that one US state (California) has Laboratories that perform FDA-mandat-
incorporated some AABB standards into state ed donor testing must be registered with the
law. FDA as biologics manufacturers because this
AABB standards are often considered “qualification of raw materials” is considered
throughout the United States as defining a part of the blood component manufacturing
standard of practice in the blood banking process. The infectious disease tests and test-
community and therefore are widely imple- ing equipment used to screen donors must be
mented. Since 1985, the FDA and AABB have approved (licensed or cleared) for this purpose
issued a series of recommendations and/or by the FDA Center for Biologics Evaluation and
standards for additional screening tests in ad- Research. The tests must be performed exact-
dition to the long-standing donor screens for ly as specified in the manufacturers’ package
syphilis and HBsAg. Table 8-1 summarizes the inserts. Tests and test platforms that are ap-
chronology of changes in donor infectious dis- proved only for diagnostic use may not be used
ease testing, and Table 8-3 lists the donor for screening whole blood donors.
184 䡲 AABB TECHNICAL MANUAL
TABLE 8-3. Blood Donor Screening Tests Performed in the United States
HCV 䡲 IgG antibody to HCV ChLIA or EIA 䡲 Positive HCV RNA (FDA)†
peptides 䡲 RIBA (FDA)§
HIV-1/2 䡲 IgM and IgG antibody to ChLIA or EIA 䡲 Positive HIV RNA (FDA)†
HIV-1/2 䡲 HIV-1: IFA or Western
blot (FDA)
䡲 HIV-2: EIA (FDA)
HTLV-I/II 䡲 IgG antibody to HTLV-I/II ChLIA or EIA IFA, Western blot, RIPA, and
line immunoblot
or
*Supplemental assays with “(FDA)” are FDA-approved supplemental assays. Other supplemental assays listed are not
required but may be useful for donor counseling.
†
Positive results on some nucleic acid tests are approved by FDA as providing confirmation for reactive HBsAg, HIV anti-
body, and HCV antibody serology tests. If a nucleic acid test result is negative, serologic supplemental test(s) must be
performed.
‡
Screening for DNA or RNA in the United States is usually performed on minipools of 6 to 16 donor samples.
§
As of 2013, RIBA is not available. FDA variance may be obtained to use a second licensed screening test as an alternative.
||
T. cruzi antibody testing may be limited to one-time testing of each donor.
HBV = hepatitis B virus; ChLIA = chemiluminescent immunoassay; EIA = enzyme immunoassay; DNA = deoxyribonucleic
acid, FDA = Food and Drug Administration; Ig = immune globulin; TMA = transcription-mediated amplification; PCR = poly-
merase chain reaction; HCV = hepatitis C virus; RNA = ribonucleic acid; RIBA = recombinant immunoblot assay; HIV-1/2 =
human immunodeficiency virus types 1 and 2; IFA = immunofluorescence assay; HTLV-I/II = human T-cell lymphotropic
virus, types I and II; RIPA = radioimmunoprecipitation assay, WNV = West Nile virus; ESA = enzyme strip assay.
CHAPTER 8 Infectious Disease Screening 䡲 185
ing to that pool were considered negative for rus (WNV) activity is high in a specific geo-
HIV and HCV RNA. If a pool showed reactivity graphic area. Circulating levels of viral RNA are
on the nucleic acid test, further testing of often low during WNV infection. When donor
smaller pools and, ultimately, individual sam- samples are combined into MPs, the RNA from
ples was performed to determine which dona- a WNV-infected sample may become diluted
tion was responsible for the reactive test result. to below the detectable level. It has been esti-
Donations that were nonreactive on this addi- mated that MP-NAT screening for WNV RNA
tional testing could be released for transfu- may fail to detect 50% or more of infected do-
sion. Donations that were reactive at the indi- nations.14-16 Therefore, both the FDA and AABB
vidual sample level were considered positive have recommended ID-NAT screening for
for viral nucleic acid and could not be released WNV RNA at times of high WNV activity in a
for transfusion. region.16,17
In recent years, fully automated NAT sys-
tems have been developed. The two automat- Implications of Reactive Test Results
ed test platforms that are approved by the FDA
for donor screening use multiplex assays that A repeatedly reactive result on a screening test
detect HIV RNA, HCV RNA, and HBV DNA in (or individually reactive NAT result) typically
one reaction chamber. Reactive donations are results in mandatory discarding of the reactive
subjected to discriminatory testing to identify donation. Most blood bank computer systems
which virus is present. These systems are ap- prevent labeling and/or release of products
proved for testing of individual donations and from donations with reactive test results. A re-
pools of 6 to 16 donor samples, depending on active test result may also indicate that the do-
the platform. The availability of fully automat- nor should be prohibited from making future
ed NAT platforms raises the possibility of mov- donations, because many infections are per-
ing to routine screening of individual donor sistent. Furthermore, past donations may also
samples [individual donation (ID) screening], be considered suspect because the exact date
rather than testing of pools (MP-NAT). of onset of a donor’s infection cannot be deter-
However, it has been estimated that ID- mined.
NAT screening would minimally increase de- Both the FDA and AABB have issued rec-
tection of infected donors, whereas the associ- ommendations regarding whether reactive
ated testing cost would be significantly higher test results affect a donor’s eligibility for future
than with MP-NAT.13 Furthermore, it is not donations, components from prior donations
clear that ID-NAT screening of the entire US should be retrieved (and if so, how far back in
blood supply would be logistically feasible us- time), and patients who previously received
ing the available platforms. An additional con- components from that donor should be noti-
cern is that donors might be deferred for false- fied. These recommendations are often guided
positive results more frequently with ID-NAT by the results of supplemental or confirmatory
screening than pooled screening. testing performed on the donor sample. Many
In contrast to serologic testing policies, of these recommendations have evolved over
repeat testing is not permitted by the FDA for time.
an individually reactive NAT sample to deter- Because these recommendations are
mine whether the initially reactive result rep- complex, blood bank laboratories typically
resents a true-positive result. If an individual create checklists that list each of the actions to
(unpooled) specimen is reactive on a NAT be performed after a specific reactive test re-
screen for HIV, HCV, or HBV, the FDA requires sult is obtained. Staff use these checklists to
that the corresponding blood component be document completion of each action as they
discarded and the donor be deferred indefi- perform it.
nitely. Table 8-416-37 lists federal regulations, FDA
ID-NAT screening rather than MP-NAT guidance documents, AABB standards, and
screening is recommended when West Nile vi- AABB Association Bulletins with recommen-
TABLE 8-4. FDA, CMS, and AABB Recommendations for Blood Donor Testing and Actions Following Reactive Results*
Topics
Title 21, CFR Part 610.40 HIV-1/2, HBV, HCV, HTLV-I/II, and X
Syphilis
Title 21, CFR Part 610.41 HIV-1/2, HBV, HCV, HTLV-I/II, and X
Syphilis
Title 21, CFR Parts 610.46, 610.47, and HIV and HCV X X
610.48
Title 42, CFR Part 482.2724 HIV and HCV X X
26
CHAPTER 8
31
FDA guidance, August 2009 HIV-1 group O X X X
32 †
FDA guidance, July 2009 Parvovirus B19
33
FDA guidance, June 2005 WNV X X X
䡲
Topics
䡲
dations regarding management of blood do- fection even though the screening test results
nors with reactive test results, retrieval of other were negative.
components, and notification of prior recipi- For HIV and HCV tests, the algorithms for
ents. These regulations and recommenda- managing prior donations and recipients of
tions are described briefly below. prior donations are spelled out in Title 21, CFR
Parts 610.46 and 610.47. These requirements
Donor Eligibility are replicated in the Centers for Medicare and
Medicaid Services regulations (Title 42, CFR
FDA regulation Title 21, CFR Part 610.41, ad- Part 482.27) to ensure hospital transfusion ser-
dresses donors with reactive screening test re- vice compliance with recipient notification
sults. FDA guidance documents and AABB As- requirements. For other agents, recommenda-
sociation Bulletins contain more detailed tions for the management of previously donat-
recommendations regarding additional test- ed components may be found in the FDA
ing, donor eligibility, and donor counseling for guidance documents or AABB Association Bul-
these and other tests. Donors should be noti- letins (or both) listed in Table 8-4.
fied of any test results that affect their eligibili- In most cases, the FDA and AABB recom-
ty or that could have important implications mend retrieval and quarantine of any remain-
for their health. Blood banks should have sys- ing components from prior donations of that
tems that prevent future collections from ineli- donor. It is essential that the retrieval of in-
gible donors and the release of any compo- date components be initiated immediately af-
nents inadvertently collected from such ter the repeatedly reactive result is obtained.
individuals. This approach prevents transfusion of these
For donors deferred for reactive screening components while confirmatory testing is per-
tests, Title 21, CFR Part 610.41, provides for re- formed. The FDA requires initiation of retriev-
instatement by means of FDA-defined requali- al within 3 calendar days of a reactive HIV or
HCV test and within 1 week of reactive HBsAg,
fication algorithms. The FDA has issued guid-
anti-HBc, or anti-HTLV screening tests. If con-
ance documents (listed in Table 8-4) that
firmatory test results on the current donation
define reentry pathways for donors deferred
are negative, the FDA, in some circumstances,
for reactivity on HIV, HCV, HBsAg, anti-HBc,
permits rerelease of the prior donations. In
serologic syphilis, and HIV/HCV/HBV NAT many cases, some or all of the components
tests. Most of the pathways require that the do- from prior donations will have been trans-
nor have negative results on specified tests af- fused. For some infectious agents, the FDA
ter a defined waiting period. Blood banks that and AABB recommend that the recipients of
desire to reenter donors must follow the FDA- prior donations from confirmed positive do-
defined algorithms explicitly. nors be notified of their possible exposure to
the infectious agent.
Retrieval of Prior Donations and Recommendations for notification of re-
Notification of Prior Recipients cipients of prior donations (“look-back”) are
(“Look-Back”) usually issued by the AABB, FDA, or both at the
time a new test is implemented, but these rec-
The FDA and AABB offer guidance with regard ommendations may evolve as confirmatory
to the appropriate management of previously tests become available or medical treatments
collected blood components from donors are developed for the infection in question.
whose current donation is repeatedly reactive Look-back is required by law only for HIV and
(or, in the case of NAT, individually reactive) on HCV tests (Title 21, CFR Parts 610.46 and
an infectious disease screening test. These rec- 610.47). For an HIV look-back investigation in-
ommendations address the concern that at the volving a deceased prior recipient, the next of
time of the previous donation, the donor could kin must be notified. The CFR spells out spe-
have been in the window period of an early in- cific timelines for component retrieval and
190 䡲 AABB TECHNICAL MANUAL
recipient notification. It also specifies how far components do not appear to transmit CMV
back in time (ie, to which donations) the re- infection. Immunocompromised patients who
trieval and notification should extend. For oth- are at increased risk of transfusion-transmit-
er agents, such as WNV and T. cruzi, recom- ted disease include fetuses, low-birthweight
mendations regarding retrieval and recipient premature infants who are born to CMV-sero-
notification are included in FDA guidance negative mothers, and CMV-seronegative re-
documents and AABB Association Bulletins. cipients of solid-organ or allogeneic hemato-
Table 8-4 indicates which of these documents poietic cell transplants from seronegative
address product retrieval or recipient notifica- donors.38
tion. The majority of blood donors have had
In the absence of published guidance, it is prior exposure to CMV, indicated by the fact
not always obvious whether or when it is ap- that they have CMV antibodies. Therefore, it
propriate to notify prior recipients of their would not be possible to produce an adequate
possible exposure to infection. If there is no supply of blood if all CMV-antibody-positive
confirmatory assay available, it may be diffi- donations were discarded.
cult to determine whether a repeatedly reac- It is possible, however, to minimize CMV
tive screening test result for a donor represents transmission to patients at risk of severe CMV
a true infection. Furthermore, if there is no ef- disease, such as those described above. These
fective treatment for that infection, there may patients should be supported with cellular
be no obvious benefit to the recipient of being blood components that have a reduced risk of
told that he or she might have been exposed. transmitting CMV. These reduced-risk options
There could, however, be a public health bene- include using blood components from donors
fit from such a notification. Specifically, a re- who are CMV antibody negative or compo-
cipient who is alerted of a potential exposure nents that have been effectively leukocyte re-
can be tested and, if the results are positive, duced. The literature suggests that these two
take precautions to avoid further spread of the methods have similar but not identical effica-
infection. cy, with an estimated transmission risk by
seronegative components of 1% to 2% vs a risk
Cytomegalovirus Testing of Products of 2% to 3% with leukocyte-reduced compo-
for Immunocompromised Recipients nents.38-40 A recent study, however, found no
CMV transmissions among 100 carefully mon-
Some common infections cause relatively in-
itored allogeneic hematopoietic cell transplant
nocuous illnesses in immunocompetent indi-
recipients who received CMV-untested, leuko-
viduals but can cause severe disease in immu-
cyte-reduced components.41 Because many at-
nocompromised patients. Such is the case
risk patients receive leukocyte-reduced com-
with cytomegalovirus (CMV).
ponents and are monitored closely for CMV
CMV is a lipid-enveloped DNA virus in
infection, treated early with anti-CMV drugs,
the Herpesviridae family. Like other herpesvi-
or both, it is difficult to measure a benefit from
ruses, CMV causes lifelong infection, typically
also providing CMV-seronegative components
in a latent state, with the potential for reactiva-
to these patients.
tion. Primary CMV infection in immunologi-
cally competent individuals is mild, with
Autologous Donations
symptoms ranging from none to an infectious
mononucleosis-type syndrome. In immuno- The FDA requires infectious disease testing of
compromised patients, however, both primary autologous donations that are shipped from
infection and reactivation disease can be over- one facility to another. If the receiving facility
whelming and even fatal. CMV can be trans- does not permit autologous donations to be
mitted by blood transfusion, primarily crossed over to the general inventory, the FDA
through intact white cells contained in cellular requires testing of only the first donation in
blood components. Frozen/thawed plasma each 30-day period [Title 21, CFR Part
CHAPTER 8 Infectious Disease Screening 䡲 191
610.40(d)]. The labeling of the unit must be ing requirements vary by type of tissue. These
consistent with its testing status. Units from requirements are spelled out in Title 21, CFR
donors with repeatedly reactive tests must be Part 1271 and an August 2007 FDA guidance
labeled with biohazard labels. Some hospitals document and are summarized in Table 8-
have policies that prohibit acceptance of au- 5.43,44 The time frames for testing HCT/P do-
tologous units with positive results on some nors are also specified in these documents.
tests because there is a potential for an infec- In most cases, the samples for infectious
tious unit to be transfused to the wrong pa- disease testing must be obtained 7 days before
tient. The AABB has warned that refusal of or after the tissue donation. However, samples
test-positive units could be interpreted as vio- of peripheral blood hematopoietic progenitor
lating the Americans with Disabilities Act.42 cells or marrow may be tested up to 30 days
before donation. Autologous tissues and re-
Considerations in Testing Donors of productive tissues from sexually intimate part-
Human Cells, Tissues, and Cellular ners may be exempt from some testing re-
quirements.
and Tissue-Based Products
Blood bank laboratories that test samples
Both the questions and tests required by FDA from HCT/P donors must take care to check
to screen donors of human cells, tissues, and package inserts for HCT/P testing methods; a
cellular and tissue-based product (HCT/Ps) package insert may require a different testing
differ from those for blood donors, and screen- method for HCT/P donors than for blood
TABLE 8-5. FDA Testing Requirements for HCT/Ps (as of March 2014)
For donors of reproductive tissues, test Chlamydia trachomatis FDA-licensed, approved, or cleared diag-
for the following in nostic test
addition to the above:
Neisseria gonorrhea FDA-licensed, approved, or cleared diag-
nostic test
donors. For example, NAT for most types of dence of the infection in the donor population
HCT/P donors must be performed on individ- and the nature of the donor screening process-
ual donor samples, and MP-NAT is not permit- es in place.
ted for most HCT/P donor categories.
In some cases, FDA regulations permit Agents for Which Blood Is Tested
the use of HCT/P donations that are reactive
on infectious disease screening tests. These Transfusion transmission of HIV, HCV, and
exceptions are listed in Title 21, CFR Part HBV is now so rare that the rate of transmis-
1271.65. FDA has issued specific labeling, stor- sion cannot be measured by prospective clini-
age, and notification requirements for these cal studies. The risk can only be estimated by
tissues. theoretical modeling.
Testing of HCT/P donors for WNV RNA One theoretical source of risk is a virus
and antibody to T. cruzi is not required by FDA strain that the current test kits do not detect.
as of March 2014. However, FDA has indicated The Centers for Disease Control and Preven-
that it considers WNV to be a “relevant com- tion and the test manufacturers conduct sur-
municable disease,” and draft guidance docu- veillance for such emerging strains. Over time,
ments indicate that FDA is considering a re- the FDA has required that test manufacturers
quirement to test at least some HCT/P donors expand their detection capabilities to include
for these agents.44 new strains. A second potential cause of trans-
mission is a quarantine failure (ie, a blood
International Variations in Donor bank’s failure to quarantine a unit that tests
Testing positive). Quarantine errors are thought to be
rare in blood banks that use electronic systems
Although this chapter focuses on infectious
to control blood component labeling and re-
disease screening in the United States, the
lease because these systems are designed to
general approach to donor screening is similar
prevent the release of any unit with incom-
in other countries. However, the specific donor
plete testing or a reactive test result. Erroneous
questions and tests vary from country to coun-
releases appear to occur more frequently in
try based on the regional epidemiology of in-
fections and tests available. For example, most blood banks that rely on manual records and
countries where WNV is not endemic do not quarantine processes.45
test for this agent, although they may question The primary cause of residual transmis-
donors about travel to WNV-affected coun- sions is thought to be donations from individ-
tries. Countries where HBV is hyperendemic uals in the window period of early infection,
cannot exclude donations from individuals before test results are positive. Figure 8-1 dis-
who test positive for anti-HBc without ad- plays the sequence in which different types of
versely affecting the adequacy of their blood donor screening tests demonstrate reactivity.
supply. The AABB Standards Program Unit in Over time, the window periods have been
conjunction with the AABB Subcommittee for shortened by implementation of donor
the Evaluation of International Variances con- screening tests that detect earlier infections.
siders variance applications from facilities in However, because no test gives a positive re-
countries where national practices and avail- sult immediately after an individual acquires
able tests differ from those in the United States. an infection, a window period remains. With
NAT, the average duration of the window peri-
od is estimated to be 9.0-9.1 days for HIV and
R E SI D UA L I NF E C T I O U S R IS K S
7.4 days for HCV.13,46 The window period for
OF TRANSFUSION
HBV is longer, as discussed in the “HBV” sec-
Despite donor screening, blood components tion below.
may still transmit infections. The residual risk The likelihood that a blood donation has
of transmission varies according to the inci- been obtained from a donor in the window pe-
CHAPTER 8 Infectious Disease Screening 䡲 193
riod can be estimated mathematically using er, both of these donor populations have sig-
the incidence-window period model46: nificantly lower infection rates than the gener-
al population. The continued importance of
Probability a donation was made during using donor questioning to select donors with
the window period = length of window a low incidence of infection is explored in
period × incidence of infections in the more detail in the “HIV” section below.
donor population The current estimated risks of HIV, HCV,
and HBV transmission in donors, based on
The incidence of infections in donors can window-period and incidence calculations,
be calculated from the observed number of are shown in Table 8-6.
donors with a negative test result for one do-
nation but a positive result for a subsequent Agents for Which No Donor Screening
donation (ie, seroconverting donors). This Tests Are Available
method measures incidence rates only in re-
peat donors and does not permit assessment Essentially any infectious agent that can circu-
of the likelihood that first-time donors might late in the blood of an apparently healthy per-
be in the window period. son could be transmitted by transfusion. It is
Other methods permit measurement of impossible to estimate the risk of transmission
new infection rates in both first-time and re- for each of the infectious agents for which do-
peat donors using tests that differentiate new nors are not tested. However, transfusion-
from established infections. Such tests include transmitted infections are rarely identified.
nucleic acid tests (donor blood that contains The infections that are most likely to be recog-
HIV or HCV RNA but not antibody most nized as transmitted by transfusion are those
likely represents a very early infection) and that have a distinctive clinical presentation
“sensitive/less sensitive” antibody testing.13,46-49 and are otherwise rare in the United States.
When these alternative methods have been The likelihood that an infection will be recog-
used, new HIV and HCV infections were two to nized as transmitted by transfusion is en-
four times more common among first-time hanced if the infection is usually associated
donors than among repeat donors.46-48 Howev- with a behavioral risk that the transfusion
194 䡲 AABB TECHNICAL MANUAL
TABLE 8-6. Estimated Risks of Transfusion-Transmitted Infection in the United States Based on
Window-Period and Incidence Estimates*
recipient lacks (eg, when malaria develops in a some other countries. Pathogen reduction sys-
transfusion recipient who has not traveled tems are discussed in the “Pathogen Reduc-
outside the United States). tion Technology” section later in this chapter.
If a life-threatening agent is recognized as The AABB Transfusion-Transmitted Dis-
a potential threat to the blood supply, both the eases Committee published an extensive re-
AABB and FDA typically consider whether a view of infectious agents that are possible
donor screening question could be used to ex- threats to the blood supply.51 Potential mitiga-
clude potentially exposed donors in the ab- tion strategies were discussed for each agent,
sence of a donor screening test. Donor ques- including the documented or theoretical effi-
tioning regarding travel to and residence in cacy of pathogen reduction processes. AABB
endemic areas is currently the only means of keeps these materials up to date and adds ma-
protecting the US blood supply from malaria terials for new potential threats as they are
and variant Creutzfeldt-Jakob disease (vCJD). identified.52 The agents deemed to pose the
Most infectious agents, however, do not have highest threat from either a scientific or public
such clear geographic risk areas. In general, it perspective are briefly discussed in this chap-
is difficult to design donor questions that are ter. (See the 2009 supplement to TRANSFU-
both sensitive (ie, detect most infected indi- SION and updates on the AABB website for a
viduals) and specific (ie, exclude only infected more thorough review of these potential infec-
individuals). tious risks.51,52)
An alternative method of protecting the
blood supply from infectious agents is
pathogen inactivation or reduction. Heat SCREENING FOR SPECIFIC
inactivation, solvent/detergent treatment, AGENTS
nanofiltration, chromatography, cold ethanol HIV
fractionation, and other approaches have
been used with remarkable success to inacti- HIV-1, a lipid-enveloped, single-stranded RNA
vate or remove residual pathogens in plasma virus, was identified in 1984 as the causative
derivatives. Pathogen reduction systems for agent of AIDS, and blood donor screening for
cellular blood components are not available in antibodies to this virus was implemented in
the United States as of March 2014, although the United States in 1985. In 1992, donor
systems for platelet components are in use in screening tests were modified to include de-
CHAPTER 8 Infectious Disease Screening 䡲 195
tection of antibodies to HIV-2, a closely related dence of HIV donates blood, the likelihood
virus identified initially in West Africa. that this individual is in the window period
HIV can be transmitted sexually, paren- and that the component will transmit HIV can
terally, and from infected mothers to their in- be calculated as follows:
fants. Although heterosexual and vertical
spread of HIV predominate in some parts of Risk that the donation is in window period =
the world, new HIV cases in the United States length of window period × incidence of infec-
continue to be concentrated in men who have tion in donor population = (9.0 days/365 days/
sex with men (MSM) and individuals with year) × (1/100 person-years) = 1/4100.
high-risk heterosexual contact (defined as
contact with an individual who is HIV positive This is the likelihood that a unit from this
or in an identified risk group for HIV, such as high-risk donor would harbor HIV but be
MSM or injection drug users).53 missed by the current donor screening. This
Current donor screening for HIV includes risk is clearly much higher than the estimated
NAT testing for HIV-1 RNA and serologic test- HIV transmission risk of 1 in 1.5 million for a
ing for antibodies to HIV. The antibody tests unit of blood obtained from the current donor
approved for donor screening detect both im- population. Thus, despite the short window
mune globulin M (IgM) and IgG antibody to period with current testing, inclusion of do-
both HIV-1 and HIV-2. Some of the approved nors with a high risk of HIV would have a pro-
tests also detect antibody to the HIV-1 group foundly adverse impact on blood safety. Ac-
O, a strain of HIV-1 found primarily in Central cordingly, questioning of donors for risk and
and West Africa. If the antibody test used by a temporarily excluding those at increased risk
donor center does not have an HIV-1 group O to minimize window-period donations contin-
detection claim, the donor center must use ue to be critical for preserving blood safety.
donor questioning to exclude individuals who Despite the efficacy of current donor
have resided in, received medical treatment in, questioning approaches, there has been great
or had sex partners from HIV-1 group O en- interest in developing a more specific donor-
demic areas.31 screening algorithm for MSM that would
The average lag time from HIV-1 exposure exclude only individuals who are truly at in-
to test detection (window period) is currently creased risk of HIV. Although more specific do-
estimated to be 9-9.1 days for MP-NAT.13,46 nor screening criteria for MSM are desirable,
Based on window-period and incidence-rate the FDA states that no algorithm has yet been
calculations, the current risk of acquiring HIV developed that reliably identifies MSM whose
from transfusion is estimated to be approxi- HIV risk is as low as that of current blood
mately 1 in 1.5 million units (Table 8-6). donors.57
In the United States, blood donor screen- FDA’s rationale for permanently excluding
ing questions exclude very broadly defined MSM from donation has been less clear. Most
populations at increased risk of HIV. Given the other high-risk populations are excluded only
short delay of only days between exposure and temporarily (for 1 year after the risk activity).
detection of infection by NAT, experts have The FDA’s rationale for excluding MSM be-
questioned whether donor interviews and ex- yond 1 year appears to be based on two con-
clusion of donors with increased risk remain cerns. One concern is that there are other po-
medically necessary.54 The continued impor- tentially transfusion-transmissible infections
tance of a low-risk donor population becomes that are more prevalent in MSM populations
evident if different HIV incidence figures are (such as human herpesvirus 8, the causative
entered into the blood safety calculation. For agent of Kaposi sarcoma), and donors are not
example, HIV incidence rates as high as 1% to tested for all of these infections.54 The other
8% have been observed in some high-risk pop- concern is “quarantine release errors,” or the
ulations, such as young urban MSM.55,56 If an risk that a blood center might inadvertently
individual from a population with a 1% inci- release a unit with a positive test result.
196 䡲 AABB TECHNICAL MANUAL
However, based on current electronic controls differences in the sensitivity of various HBV as-
used by most US blood centers, release errors says and lack of certainty regarding the level of
appear to be extremely rare. virus in a blood component that is required for
Mathematical modeling suggests that the infectivity.50,59 Recent publications provide
deferral period for MSM could be shortened window-period estimates for different poten-
without a substantial increase in risk to recipi- tial infectious doses of virus (eg, 10 copies/20
ents.45 A US Department of Health and Human mL of plasma vs 1 copy/20 mL of plasma). The
Services (DHHS) advisory committee conclud- infectious window prior to a positive result on
ed in 2010 that available scientific data were the Abbott PRISM (Abbott Laboratories, Ab-
inadequate to support any specific alternative bott Park, IL) HBsAg test has been estimated to
policy.58 DHHS subsequently issued requests be 30 to 38 days.59 With the addition of HBV
for proposals to evaluate alternative ap- DNA testing in MPs of 16, the window period is
proaches to donor screening that could main- estimated to have been reduced to 18.5 to 26.5
tain the current level of blood safety. days.50 Using these MP testing estimates, US
HBV transfusion-transmission risk has been
HBV estimated to be between 1 in 843,000 dona-
HBV is a lipid-enveloped, double-stranded tions to 1 in 1.2 million donations (Table 8-6).50
DNA virus. Like HIV, HBV is transmitted par- Donor screening for HBV DNA can be of
enterally, sexually, and perinatally. Jaundice is value at a variety of points in infection. HBV
noted in only 25% to 40% of adult cases and in DNA may be detected during the infectious
a smaller proportion of childhood cases. A window period before HBsAg detection; how-
large percentage of perinatally acquired cases ever, DNA levels may be low and could be be-
result in chronic infection, but most HBV in- low the limits of detection of MP-NAT as-
fections acquired in adulthood are cleared. says.50,59 Later in infection, following the
HBV is highly prevalent in certain parts of the clearance of HBsAg, HBV NAT may detect per-
world, such as Eastern Asia and Africa, where sistent (ie, “occult” HBV) infection.60 Such in-
perinatal transmission and resultant chronic fections are interdicted in the United States by
infection have amplified infection rates in the the donor screening test for anti-HBc. HBV
population. In the United States, the incidence NAT testing can also detect acute HBV infec-
of acute HBV infection has decreased dramati- tions in individuals who have previously been
cally with the implementation of routine vac- vaccinated.61,62 Such individuals may never de-
cination programs. Perinatal screening and velop detectable HBsAg, but they may have
newborn prophylaxis have also been effective detectable DNA. The infectivity of such dona-
in reducing perinatal transmission. tions is not known because these units contain
During HBV infection, DNA and viral en- vaccine-induced antibodies to HBsAg in addi-
velope material (HBsAg) are typically detect- tion to the virus. Routine HBV DNA screening
able in circulating blood. Antibody to the core of US blood donations detects at least some of
antigen is produced soon after the appear- these infections.
ance of HBsAg, initially in the form of IgM an-
tibody, followed by IgG. As infected individuals HCV
produce antibody to the surface antigen (anti-
HBsAg), the HBsAg is cleared. HCV is a lipid-enveloped, single-stranded RNA
The FDA requires donor screening for virus. HCV was shown to be the cause of up to
HBsAg, HBV DNA, and total anti-HBc (IgM 90% of cases previously called NANB transfu-
and IgG antibody). Measurements of HBV inci- sion-related hepatitis.63 The majority of HCV
dence in donors have been complicated by the infections are asymptomatic. However, HCV
transience of HBsAg and false-positive results infection is associated with a high risk of chro-
on the HBsAg test.48,50 Published estimates of nicity, which can result in liver cirrhosis and
the infectious window have varied because of hepatocellular carcinoma.
CHAPTER 8 Infectious Disease Screening 䡲 197
by the FDA or validated to provide sensitivity do not fulfill the AABB standard for bacteria
equivalent to FDA-approved methods. None of detection.18(p11),20,73 However, two FDA-cleared
these methods is sensitive enough to detect point-of-issue assays can be used for bacteria
bacteria immediately after collection. All detection testing of platelet concentrates that
methods require a waiting time for bacteria are pooled immediately before issue.
contaminants to multiply before the compo- Since the implementation of routine bac-
nent is sampled. terial screening of apheresis platelets, the fre-
The process most commonly used in the quency of FDA-reported fatalities from con-
United States to screen apheresis platelets is a taminated apheresis platelets has declined.71
culture-based system that requires the plate- However, some contaminated apheresis plate-
let component to be stored for 24 hours before lets escape detection by this early testing, pre-
sampling. After that time, a sample is with- sumably because bacterial concentrations are
drawn and inoculated into one or more cul- below limits of detection at the time of sam-
ture bottles. The bottles are then incubated in pling; thus, septic, and even fatal, reactions do
the culture system. Some blood centers con- still occur. AABB has recommended consider-
tinue to hold the component during the first ation of policies to further reduce the risk
12 to 24 hours of culture and release it for use of bacterially contaminated platelets.75 The
only if the culture is negative at the end of that point-of-issue assays mentioned above are
time. In all cases, the culture is continued for cleared by FDA as adjunct (time-of-issue) tests
the shelf life of the unit. If the culture becomes for apheresis platelets that have been screened
positive after the component is released, the by another method. In a large clinical trial, one
blood center attempts to retrieve it. If the com- of these assays detected nine bacterially con-
ponent has not been transfused, resampling of taminated components among 27,620 aphere-
the product for culture is very informative be- sis platelets (1 in 3069 components) previously
cause approximately two-thirds of the initially screened by an early-storage culture-based as-
positive signals are determined to be caused say.76 There were also 142 false-positive results.
by either contamination of the bottle (and not As of March 2014, point-of-issue retesting of
the component) or false signals from the cul- apheresis platelets had not been widely imple-
ture system.73,74All positive cultures should be mented in the United States.
tested to determine the identity of the organ- All of the above methods provide incom-
ism. If a true-positive result is related to an or- plete assurance of bacteria detection, and
ganism that is not a skin contaminant, the do- none is practical for screening the red cell in-
nor should be notified and advised to seek ventory. Pathogen-reduction methods, which
medical consultation.19 impair proliferation of bacteria in the blood
Other methods approved in the United component, could theoretically be used to re-
States for platelet quality control testing early duce the risk of septic reactions from bacteria
in the storage period include a culture-based in blood components. Indeed, in some regions
system with a one-time-point readout and an outside the United States, pathogen reduction
optical scanning system. All of the methods has replaced bacteria detection testing for
are approved for testing leukocyte-reduced platelets, but these technologies are not
apheresis platelets, and some are approved for approved for use in the United States as of
testing pools of leukocyte-reduced, whole- March 2014.
blood-derived platelets. These methods are
not generally used for routine screening of Infections Transmitted by Insect
individual (unpooled) whole-blood-derived
Vectors
platelet concentrates. Low-technology meth-
ods for screening platelets just before issue— Until recently, malaria was the only vector-
such as visually inspecting the platelets for transmitted disease that was widely recog-
swirling or testing them for low glucose or nized as having the potential for secondary
pH—lack both sensitivity and specificity and transmission by transfusion in the United
200 䡲 AABB TECHNICAL MANUAL
States. Malaria is rare in the United States, and clinical WNV cases and animal and mosquito
the blood supply has been effectively protect- surveillance in the area.
ed by questions that exclude donors who have Two documented cases of WNV transmis-
recently traveled to or resided in malaria- sion by transfusion were traced to donations
endemic regions. In the past decade, however, screened by MP-NAT during periods when
other vector-transmitted infections have been neighboring blood collection facilities were
recognized as threats to the US blood supply, screening donors by ID-NAT.77 The most re-
and these infections are targeted by the newest cent reported transmission was traced to a do-
blood donor screening assays. nation containing WNV antibody and a low
level of virus that was not reproducibly detect-
WNV able even by ID-NAT.78 It is possible that some
transmissions have escaped recognition be-
WNV is a lipid-enveloped RNA virus in the Fla-
cause most WNV infections lack distinctive
viviridae family. First detected in the United
symptoms.
States in 1999, it subsequently spread through-
out North America, appearing in annual epi-
T. cruzi
demics every summer and autumn. Birds are
thought to be the primary reservoir for WNV, T. cruzi, the protozoan parasite that causes
with infection spreading to humans through Chagas disease, is endemic in parts of Mexico,
mosquito bites. Approximately 80% of human Central America, and South America. It is
cases are asymptomatic; 20% are associated transmitted to humans by an insect vector, the
with a self-limited febrile illness; and less than reduviid bug. Acute infection is usually self-
1% are associated with severe neuroinvasive limited but may be severe in immunocompro-
disease, such as meningoencephalitis or acute mised patients. Most infections become
flaccid paralysis. chronic but asymptomatic. Decades after the
Transfusion transmission of WNV was initial infection, 10% to 40% of infected indi-
first recognized in 2002 and traced to blood viduals develop late-stage manifestations,
donations containing viral RNA in the ab- including intestinal dysfunction or cardiac
sence of antibody. Thus NAT, rather than sero- disease, which can be fatal. Transfusion trans-
logic testing, was required to protect the blood mission of T. cruzi from the blood of chronical-
supply. Donor screening was widely imple- ly infected, asymptomatic donors has been re-
mented in 2003 using investigational NAT as- ported in endemic areas.
says. Donor tests for WNV RNA are now ap- A blood donor screening enzyme immu-
proved by FDA and required by both the FDA noassay for antibodies to T. cruzi was ap-
and AABB.17,18(pp31,32) The predominant method proved by the FDA for US use in December
of testing is in MPs. 2006. Although not initially required by the
However, as discussed in the “NAT” sec- FDA, the test was widely implemented by US
tion above, circulating levels of viral RNA are blood centers during 2007. Supplemental test-
frequently low during WNV infection, and a ing of reactive specimens using an FDA-
donor sample containing a low concentration licensed enzyme strip assay or unlicensed
of RNA may escape detection if it is diluted in a radioimmunoprecipitation assay (RIPA) is very
minipool. Therefore, both the AABB and FDA helpful for guiding donor counseling. Based
recommend testing of individual donor sam- on the results of the latter assay, about 25% of
ples, not minipools, when WNV activity is high reactive US donors appear to be truly infect-
in a particular collection region.16,17 Regional ed.79,80 All donors whose samples are reactive
WNV activity is monitored through active on the screening assay, however, must be per-
communications between neighboring blood manently deferred, regardless of the results of
collection agencies about viremic donors de- the supplemental testing, pending FDA ap-
tected as well as by public health reports of proval of a reentry algorithm.29
CHAPTER 8 Infectious Disease Screening 䡲 201
The vast majority of US donations with Reported human infections with B. microti are
reactive T. cruzi screening test results and pos- becoming more frequent. In the western Unit-
itive supplemental results are from donors ed States, Babesia infections appear to be less
born in T. cruzi-endemic areas. Other con- common, and a different species, B. duncani,
firmed-positive donors appear to have con- predominates. The vector for B. duncani has
genitally acquired infections (ie, the donor’s not been clearly defined.
mother is from a T. cruzi-endemic area), and Babesia infection is usually asymptomat-
only a small number of donor infections ap- ic, even though parasites can circulate for
pear to have been acquired from vector expo- months to years. In some individuals, however,
sure within the United States (autochthonous Babesia infection presents as a severe malaria-
cases). In the first 2 years of US donor screen- like illness that can be fatal. Immunocompro-
ing, no donor seroconversions were identi- mised, elderly, and asplenic patients are at in-
fied.79,80 In December 2010, the FDA issued creased risk of severe disease.
guidance recommending one-time screening Babesia infection is diagnosed when the
of every US donor for T. cruzi.29 intraerythrocytic parasites are seen on a blood
Before implementation of donor screen- smear. If a patient is suspected of having ac-
ing, seven cases of transfusion-transmitted quired the infection by transfusion, donors of
T. cruzi had been identified in the United the patient’s components can be recalled and
States and Canada; all of the cases with avail- tested for antibodies to Babesia using an im-
able data were linked to platelet transfusions. munofluorescence assay; the presence of
Since implementation of donor screening, high-titer antibody in the donor is suggestive
RIPA-positive donors have been identified, of recent infection. Most of the donors impli-
and recipients of their prior donations have cated in transfusion-transmitted cases have
been notified and tested. Thus far, only two been residents of endemic areas, although
prior recipients (of platelets from one donor) some were residents of nonendemic areas who
appear to have positive test results. Thus, de- were apparently exposed to Babesia during
spite reported transmission rates of 10% to travel to endemic areas.82
20% from components from infected donors Studies in Connecticut have found B. mi-
in endemic areas, no T. cruzi transmissions by croti antibodies in approximately 1% of blood
red cells in the United States have been docu- donors, suggesting that B. microti infections
mented to date. The lower infectivity of US red are highly prevalent in the state’s population
cell components compared to those in endem- and grossly underrecognized.83
ic areas may be at least partly attributable to In the absence of FDA-approved tests for
more frequent use of fresh whole blood in en- blood donor screening, there have been public
demic areas. discussions of potential interventions to re-
duce transfusion risk in the most highly en-
Babesia demic regions.81,84 Clinical trials of some inves-
tigational serologic and nucleic acid tests are
Babesia are intraerythrocytic parasites. Cases
currently under way.85
of transfusion-transmitted babesiosis are be-
ing identified with increasing frequency, and
Malaria
some have been fatal.71,81,82 There is no FDA-
approved donor screening test for this infec- Malaria is caused by an intraerythrocytic para-
tion. site of the genus Plasmodium. Infection is
Human Babesia infections are zoonotic, transmitted to humans through a mosquito
usually acquired through the bite of an infect- bite. Five species account for most human in-
ed tick. In the northeastern and Midwestern fections: P. falciparum, P. vivax, P. malariae,
United States, the most common Babesia spe- P. ovale, and P. knowlesi.
cies is B. microti. The vector is Ixodes scapular- No FDA-approved test is available to screen
is, the same tick that transmits Lyme disease. US blood donations for malaria infection.
202 䡲 AABB TECHNICAL MANUAL
increased risk of either CJD or vCJD. CJD ex- cating previous exposure. Levels of viral DNA
clusions are based on family history of the dis- during acute infection may exceed 1012 IU/mL,
ease, receipt of human growth hormone de- decreasing over weeks to months in associa-
rived from pituitary glands, or receipt of a dura tion with antibody production.
mater tissue graft. vCJD exclusions are for resi- Viral DNA, mostly at low concentrations,
dence in the United Kingdom or Europe dur- has been detected in approximately 1% of
ing specified times when BSE was endemic, re- blood donations and in essentially all lots of
ceipt of a transfusion in the United Kingdom pooled plasma derivatives. Transmission of
or France, or receipt of UK bovine insulin.95 It is parvovirus B19 by transfusion has been
thought that plasma derivative manufacturing linked only to blood components or plasma
processes remove substantial amounts of TSE products that contain high concentrations of
infectivity.95 viral DNA; only one transmission has been
documented with a product containing less
In-Process Screening for Plasma than 104 IU/mL.52
Derivatives Currently, there is no FDA-approved test
to screen fresh blood donations for parvovirus
Commercial plasma derivatives are prepared B19 infection. However, plasma-derivative
from large pools of plasma derived from thou- manufacturers require screening of incoming
sands of donors. Before the incorporation of plasma units for the presence of high-titer par-
specific pathogen-reduction processes, con- vovirus B19. This is accomplished by perform-
tamination of these large pools with viral ing NAT on pools of samples from plasma
agents was common. Today, plasma derivative units, with sensitivity adjusted to detect only
manufacturing processes incorporate meth- units with a high concentration of virus. By ex-
ods—such as prolonged heat or solvent/deter- cluding high-titer units from the plasma pools,
gent (SD) treatment—that remove or inacti- the final titer in the plasma pool is kept below
vate most known pathogens. SD treatment 104 IU/mL.
inactivates lipid-enveloped agents, such as
HIV, HCV, and HBV. Pathogen infectivity may Other Agents
also be reduced by nanofiltration, chromatog-
raphy, or cold ethanol fractionation, which are The AABB maintains a publicly accessible
used in the production of certain products. electronic resource containing expert analyses
Not all infectious agents, however, are re- of emerging infectious disease (EID) agents
moved or inactivated by these processes. that have received attention as potential
One agent that can persist in plasma- threats to the US or global blood supply.52 This
derivative products is human parvovirus B19. digital resource contains up-to-date fact
This small, nonenveloped DNA virus is ex- sheets on a variety of agents. Each fact sheet
tremely resistant to physical inactivation. includes information about clinical manifesta-
Acute infection is typically mild and self- tions and epidemiology of infection, evidence
limited; clinical manifestations include “fifth of transfusion transmissibility, and analyses
disease” (erythema infectiosum) and polyar- of the potential effectiveness of various
thropathy. Acute infection is associated with mitigation strategies (eg, donor questioning,
transient red cell aplasia that may be clinically serologic testing or NAT, or pathogen reduc-
significant in immunodeficient individuals tion). Readers are encouraged to use this rich
and those with underlying hemolytic process- resource.
es. The aplasia in immunodeficient individu- One agent that has received recent atten-
als can be prolonged. Intrauterine infection is tion is hepatitis E virus (HEV), a small, nonen-
associated with severe fetal anemia and hy- veloped, single-stranded RNA virus. HEV is
drops fetalis. thought to be primarily transmitted through
Parvovirus B19 infection is very common; food and water sources. However, recent stud-
most adults have antibodies to this agent, indi- ies have demonstrated asymptomatic viremia
204 䡲 AABB TECHNICAL MANUAL
in blood and plasma donors, and transfusion testing and bacterial testing of platelets), and
transmission has been documented.52,96 As a some PRT methods could obviate the need for
nonenveloped agent, HEV is not susceptible to irradiation, potentially offsetting some of the
SD treatment. NAT screening of donors and/or cost of PRT.
heat inactivation of plasma pools may be miti- As discussed above, PRT is now an essen-
gation strategies if this agent is deemed to tial component of the plasma-derivative man-
pose a clinically important threat. ufacturing process. SD treatment can also be
applied to pools of plasma for transfusion. An
PAT H OG E N RED UCT IO N SD-treated pooled plasma product has been
TE CH NOLO G Y approved for use in the United States.
No PRT processes are approved in the
Donor screening reduces, but cannot eliminate, United States for treatment of individual units
the infectious risks of blood transfusion. The ef- of plasma or for cellular blood components,
ficacy of blood donor testing is limited by a although some technologies are in use outside
number of factors, including the following: the United States. Processes that are available
or in development have been recently re-
1. It is not logistically feasible to test donors viewed in detail and are summarized in Table
for every infection that is conceivably 8-7.51,52,97,98
transmissible by transfusion. The benefit to be gained from PRT in the
2. For every test, there is a lag time (window United States is primarily the mitigation of
period) between when a person becomes emerging pathogens and platelet-associated
infected and when the test detects infec- bacterial sepsis. Currently, the quantifiable in-
tion. fectious risks of transfusion in the United
3. Every test has limited sensitivity (concen- States are low. Therefore, it is critically impor-
tration of the target marker that can be tant to demonstrate that PRT treatments do
detected by the test).
not introduce new hazards to patients. Rigor-
4. Developing a donor test is a long, multi-
ous preclinical and clinical studies are re-
phase process that includes identification
quired for US regulatory approval of PRT. Toxi-
of the infectious agent, selection of the type cology studies are critical because most of
of test that would be effective in interdict- these agents interact with nucleic acid, raising
ing infectious donations (eg, serology vs the theoretical potential of carcinogenicity
NAT), development of a test suitable for and mutagenicity. Treated products should be
donor screening, performance of clinical assessed for neoantigen formation and the im-
trials of the test, and regulatory approval. pact of the PRT on the final product’s clinical
During this development process, infec- efficacy. The evaluation process for PRT in
tions can be transmitted. North America was the subject of a recent con-
sensus conference.97,99
Pathogen reduction technology (PRT) Documented transmission of new infec-
provides an attractive alternative to relying on tious agents for which there are no donor
donor testing to interdict all infectious dona- screening tests could influence the risk/bene-
tions. PRT processes reduce the infectivity of fit assessment of PRTs. It is important, though,
residual pathogens in blood components. This to keep in mind that there are limits to the viral
approach could reduce the transmission of in- loads that are inactivated by these processes,
fectious agents for which there are no donor and not all agents are inactivated by PRTs.
screening tests and further reduce the residual Pooled SD plasma, for example, would have a
transmission risks of known agents. PRT could reduced risk of transmitting most infectious
theoretically enable discontinuation of some agents but a potentially increased risk of trans-
testing that is currently performed (eg, CMV mitting an agent that lacks a lipid envelope.
CHAPTER 8 Infectious Disease Screening 䡲 205
KEY POINTS
5. Based on window-period and incidence calculations, the current risk of HIV transmission
by transfusion in the United States is approximately 1 in 1.5 million units, the risk of HCV
transmission is approximately 1 in 1.1 million units, and the risk of HBV transmission is ap-
proximately 1 in 800,000 to 1 in 1.2 million units.
6. There are no donor screening tests approved by the FDA for malaria or vCJD. Donor ques-
tioning about potential exposure is the sole means of protecting the US blood supply from
these diseases.
7. AABB requires blood banks to have processes that limit and detect or inactivate bacteria in
platelet components. Pathogen reduction methods have replaced bacteria testing in some
regions outside the United States.
8. Infections transmitted to humans by vectors are increasingly recognized as a potential
source of transfusion-transmitted infection. These include WNV, T. cruzi, Babesia species,
and dengue virus.
9. PRTs may reduce the transmission of infectious agents for which there are no donor screen-
ing tests, and PRTs may further reduce the residual transmission risks of known agents. PRT-
treated plasma derivatives and pooled plasma are available in the United States. Some PRT
systems are available outside the United States for treatment of individual platelet and plas-
ma components, but as of early 2014, these were not approved for US use.
10. Blood banks must have processes in place to ensure that donations with positive test results
are not released for transfusion. In some circumstances, 1) prior donations from those do-
nors must also be retrieved and quarantined, and 2) recipients of prior donations must be
notified of their possible exposure to infection.
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39. Vamvakas EC. Is white blood cell reduction tion. Transfusion 2002;42:975-9.
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43. Code of federal regulations. Title 21, CFR Part States and dependent areas, 2011. HIV surveil-
1271. Washington, DC: US Government Print- lance report 2011:23. [Available at http://
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creases in sexually transmitted infections and and the USPHS Working Group. Guidelines for
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57. Food and Drug Administration. Blood dona- sion medicine. Transfus Med Rev 1997;11:173-
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58. US Department of Health and Human Servic- of serologic test for syphilis as a surrogate
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72. Hillyer CD, Josephson CD, Blajchman MA, et
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nents: Risks, strategies, and regulation: Joint
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3):1A-2A. 73. Ramirez-Arcos SM, Goldman M, Blajchman
62. Linauts S, Saldanha J, Strong DM. PRISM hep- MA. Bacterial infection: Bacterial contamina-
atitis B surface antigen detection of hepatits B tion, testing and post-transfusion complica-
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63. Alter HJ. Descartes before the horse: I clone, icine: Basic principles and practice. 2nd ed.
therefore I am: The hepatitis C virus in current Philadelphia: Churchill Livingstone, 2007: 639-
perspective. Ann Intern Med 1991;115:644-9. 51.
64. Smith BD, Morgan RL, Beckett GA, et al. Cen- 74. Eder AF, Kennedy JM, Dy BA, et al. Limiting
ters for Disease Control and Prevention. Rec- and detecting bacterial contamination of
ommendations for the identification of chron- apheresis platelets: Inlet-line diversion and
ic hepatitis C virus infection among persons increased culture volume improve component
born during 1945-1965. MMWR Morb Mortal safety. Transfusion 2009;49:1554-63.
Wkly Rep 2012;61:1-32. 75. AABB. Recommendations to address residual
65. Page-Shafer K, Pappalardo BL, Tobler LH, et al. risk of bacterial contamination of platelets. As-
Testing strategy to identify cases of acute hep- sociation Bulletin #12-04. Bethesda, MD:
atitis C virus (HCV ) infection and to project AABB, 2012.
HCV incidence rates. J Clin Microbiol 2008;46: 76. Jacobs MR, Smith D, Heaton WA, et al. Detec-
499-506. tion of bacterial contamination in prestorage
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culture-negative apheresis platelets on day of 86. Mali S, Steele S, Slutsker L, Arguin PM. Malaria
issue with the Pan Genera Detection test. surveillance—United States, 2007. MMWR
Transfusion 2011;51:2573-82. Surveill Summ 2009;58:1-16.
77. Centers for Disease Control and Prevention. 87. Eliades MJ, Shah S, Nguyen-Dinh P, et al. Ma-
West Nile virus transmission via organ trans- laria surveillance—United States, 2003.
plantation and blood transfusion—Louisiana, MMWR Surveill Summ 2005;54:25-39.
2008. MMWR Morb Mortal Wkly Rep 2009;58: 88. Purdy E, Perry, E, Gorlin, J. et al. Transfusion-
1263-7. transmitted malaria: Unpreventable by cur-
78. Centers for Disease Control and Prevention. rent donor guidelines? Transfusion 2003;43
Fatal West Nile virus infection after probable (Suppl):79A.
transfusion-associated transmission—Colora- 89. Gonzalez C, Layon A, Arguin P, et al. Transfu-
do, 2012. MMWR Morb Mort Wkly Rep 2013; sion-transmitted falciparum malaria from an
62:622-4. asymptomatic donor. Transfusion 2011;51
79. Otani MM, Vinelli E, Kirchhoff LV, et al. WHO (Suppl):197A.
comparative evaluation of serologic assays for 90. Mali S, Tan KR, Arguin PM. Malaria surveil-
Chagas disease. Transfusion 2009;49:1076-82. lance—United States, 2009. MMWR Morb
80. Food and Drug Administration. Blood Prod- Mortal Wkly Rep 2011;60:1-15.
ucts Advisory Committee meeting: 94th meet- 91. Spencer B, Steele W, Custer B, et al. Risk for
ing, April 1, 2009, Rockville, MD. Silver Spring, malaria in United States donors deferred for
MD: CBER Office of Communication, Out- travel to malaria-endemic areas. Transfusion
reach, and Development, 2009. [Available at 2009;49:2335-45.
http://www.fda.gov/downloads/Advisory 92. Food and Drug Administration. Guidance for
Committees/CommitteesMeetingMaterials/ industry: Recommendations for donor ques-
BloodVaccinesandOtherBiologics/BloodProd tioning, deferral, reentry and product man-
uctsAdvisoryCommittee/UCM155597.pdf (ac- agement to reduce the risk of transfusion-
cessed December 8, 2010).] transmitted malaria. (August 2013) Silver
81. Gubernot DM, Nakhasi HL, Mied PA, et al. Spring, MD: CBER Office of Communication,
Transfusion-transmitted babesiosis in the Outreach and Development, 2013. [Available
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fusion 2009;49:2759-71. BloodVaccines/GuidanceComplianceRegula
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Communication, Outreach, and Develop- blood products. (May 2010) Silver Spring, MD:
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212 䡲 AABB TECHNICAL MANUAL
97. Webert KE, Cserti CM, Hannon J, et al. Pro- 99. Klein HG, Anderson D, Bernardi MJ, et al.
ceedings of a consensus conference: Pathogen Pathogen inactivation: Making decisions
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technologies. Transfus Med Rev 2008;22:1-34. sus conference. Transfusion 2007;47:2338-47.
98. AuBuchon J, Prowse C, eds. Pathogen inactiva-
tion: The penultimate paradigm shift. Bethes-
da, MD: AABB Press, 2010.
C h a p t e r 9
Nancy M. Dunbar, MD
Nancy M. Dunbar, MD, Assistant Professor, Dartmouth-Hitchcock Medical Center, Lebanon, New Hampshire
The author has disclosed no conflicts of interest.
213
214 䡲 AABB TECHNICAL MANUAL
the hospital blood bank is considered trans- pital to allow immediate access to blood in
port, and applicable temperature require- emergency situations. Such a practice re-
ments must be met. When blood components quires that the same blood component storage
are issued from the blood bank to the patient monitoring standards be met in these other ar-
care area, maintenance of appropriate tem- eas.
perature requirements allows for the possibili- If an equipment failure occurs and pre-
ty of returning the component to inventory if it vents acceptable temperature ranges from be-
is not transfused. ing maintained, the facility should have poli-
Refrigerators, freezers, and platelet incu- cies, processes, and procedures in place to
bators for blood and blood component storage relocate blood and blood components. The
can be equipped with continuous-tempera- secondary storage location may be another
on- or off-site refrigerator or freezer, qualified
ture-monitoring devices to allow detection of
storage boxes, or coolers used with a validated
temperature deviations before products are af-
process that has been shown to maintain re-
fected. Automated electronic monitoring de-
quired storage temperatures during storage.
vices that are available include: 1) weekly pen
Because the safety, purity, potency, and quality
and chart recorders, 2) sets of hard-wired or
of the blood components could be affected by
radio-frequency temperature-recording devic-
delays in relocation to a secondary storage lo-
es, and 3) centralized temperature-monitor-
cation, it is recommended that the relocation
ing systems. Thermometers or thermocouples
occur before upper or lower acceptable storage
should be strategically placed in the equip- temperatures are exceeded. This can be ac-
ment for optimal temperature monitoring. If complished by setting the alarm points of the
an automated temperature-recording device is storage devices so that an alarm sounds before
not used, temperatures of the blood storage the unacceptable tempature limit is reached.
environment must be recorded manually ev- Some facilities may use temperature-
ery 4 hours. This requirement includes ambi- monitoring indicators for each blood compo-
ent room temperature monitoring of platelets nent container. Such indicators monitor the
that are not stored in a platelet chamber or in- liquid temperature of the immediate inner
cubator. bag, not the liquid core temperature in the
Recorded temperatures should be checked unit, which may be cooler. Policies, processes,
daily to ensure proper operation of the equip- and procedures should specify how the facility
ment and recorder. Deviations from acceptable will determine the disposition of blood com-
temperature ranges should be documented and ponents when using temperature-monitoring
explained (including any actions taken), dated, indicators.
and initialed by the person noting the devia-
tion. Specific Considerations
Most product-storage devices are equipped
with audible alarms to alert personnel that tem- Holding blood components that have been
perature ranges are approaching unacceptable dispensed from the blood bank to other hospi-
levels. Central alarm monitoring allows facili- tal areas before transfusion is considered to be
ties that do not have personnel in the vicinity “storage.” If the blood components are not
kept in a monitored device, they must be
of the equipment to alert designated staff at
stored in containers (eg, boxes or coolers) vali-
another location when an alarm is activated.
dated to maintain the correct temperature
Because platelets must be gently agitated dur-
during storage.
ing storage, typically using horizontal flatbed
or elliptical rotators, alarm systems should
Red Blood Cells
also emit alerts when the platelet agitator has
malfunctioned. RBC components are stored in plastic bags of
Transfusion services may locate blood different types with a variety of added antico-
storage refrigerators in other areas of the hos- agulants and additive solutions that modify
TABLE 9-1. Requirements for Storage, Transportation, and Expiration3(pp51-59)
Item
No. Component Storage Transport Expiration1 Additional Criteria
Whole Blood Components
1 Whole Blood 1-6 C Cooling toward 1-10 C. ACD/CPD/CP2D: 21 days
If intended for room tempera- If intended for room tempera- CPDA-1: 35 days
ture components, then store ture components, cooling
CHAPTER 9
Item
No. Component Storage Transport Expiration1 Additional Criteria
䡲
19 Apheresis Platelets 20-24 C with continuous As close as possible to 24 hours or 5 days, depending Maximum time without
gentle agitation 20-24 C2 on collection system agitation: 24 hours
20 Apheresis Platelets 20-24 C with continuous As close as possible to No change from original expi- Maximum time without
Irradiated gentle agitation 20-24 C2 ration date agitation: 24 hours
21 Apheresis Platelets Leuko- 20-24 C with continuous As close as possible to Open system: within 4 hours Maximum time without
cytes Reduced gentle agitation 20-24 C2 of opening the system agitation: 24 hours
Closed system: 5 days
22 Apheresis Platelets Platelet 20-24 C with continuous As close as possible to 5 days Maximum time without
Additive Solution Added gentle agitation 20-24 C2 agitation: 24 hours
Leukocytes Reduced
Granulocyte Components
23 Apheresis Granulocytes 20-24 C As close as possible to 24 hours Transfuse as soon as possi-
20-24 C ble; Standard 5.28.10
applies3(45)
24 Apheresis Granulocytes 20-24 C As close as possible to No change from original Transfuse as soon as possi-
Storage, Processing, Distribution, and Inventory
(Continued)
217
TABLE 9-1. Requirements for Storage, Transportation, and Expiration3(pp51-59) (Continued)
218
Item
No. Component Storage Transport Expiration1 Additional Criteria
䡲
25 Cryoprecipitated AHF –18 C Maintain frozen state 12 months from original Thaw the FFP at 1-6 C
collection Place cryoprecipitate in the
freezer within 1 hour after
removal from refrigerated
centrifuge
Plasma Components
26 Cryoprecipitated AHF (after 20-24 C As close as possible to Single unit: 6 hours Thaw at 30-37 C
thawing) 20-24 C
27 Pooled Cryoprecipitated AHF –18 C Maintain frozen state 12 months from earliest Thaw the FFP at 1-6 C
(pooled before freezing) date of collection of product Place cryoprecipitate in the
in pool freezer within 1 hour after
removal from refrigerated
centrifuge
28 Pooled Cryoprecipitated AHF 20-24 C As close as possible to Pooled in an open system: Thaw at 30-37 C
AABB TECHNICAL MANUAL
33 Plasma Frozen Within 24 –18 C or colder Maintain frozen state 12 months from collection
Hours After Phlebotomy Held
At Room Temperature Up To
24 Hours After Phlebotomy
(PF24RT24)
34 Plasma Frozen Within 24 1-6 C 1-10 C If issued as PF24RT24: Thaw at 30-37 C or using an
Hours After Phlebotomy Held 24 hours FDA-cleared device
At Room Temperature Up To
24 Hours After Phlebotomy
(after thawing)
35 Thawed Plasma5 1-6 C 1-10 C 5 days from date product was Shall have been collected
thawed or original expiration, and processed in a closed
whichever is sooner system
36 Plasma Cryoprecipitate –18 C Maintain frozen state 12 months from collection
Reduced
Storage, Processing, Distribution, and Inventory
(Continued)
219
220
Item
No. Component Storage Transport Expiration1 Additional Criteria
38 Thawed Plasma Cryoprecipi- 1-6 C 1-10 C If issued as Thawed Plasma Shall have been collected
tate Reduced Cryoprecipitate Reduced: 5 and processed in a closed
days from date product was system
thawed or original expiration,
whichever is sooner
39 Liquid Plasma 1-6 C 1-10 C 5 days after expiration of 21 CFR 610.53(c) applies
Whole Blood
40 Recovered Plasma, liquid or Refer to short supply agree- Refer to short supply agree- Refer to short supply agree- Requires a short supply
frozen ment ment ment agreement4
Tissue and Derivatives
41 Tissue Conform to source facility’s Conform to source facility’s Conform to source facility’s 21 CFR 1271.3(b),
AABB TECHNICAL MANUAL
platelet pooling system allows storage for up maintained depend on the storage container
to 5 days and the ability to perform culture- used. Hospital transfusion services must de-
based bacterial testing.17 When this system is velop policies and procedures for aliquot
used, the pool maintains the expiration date of preparation and storage that comply with
the earliest collected component in the pool. manufacturer specifications. The use of ali-
Single cryoprecipitate units are pooled af- quots for neonatal transfusion has been shown
ter thawing in a manner similar to that used to result in a decreased number of donor expo-
for platelets. The expiration time of cryopre- sures.19 The process of preparing aliquots for
cipitate pools depends on the method used for small-volume transfusion in neonates and
pooling. Cryoprecipitate pooled in an open children is discussed in greater detail in Chap-
system expires within 4 hours of pooling. ter 23. Lower-volume components (split units)
Thawed single concentrates and pooled con- may also be prepared for adult patients who
centrates using sterile connecting devices ex- require slow rates of transfusion due to con-
pire 6 hours after thawing. Thawed cryopre- cerns about fluid overload. Split units are rec-
cipitate is stored at 20 to 24 C. As an ommended when the component volume can-
alternative, the blood center may pool single not be transfused at a rate that ensures
concentrates before freezing. completion of the transfusion within 4 hours.
Reconstituted whole blood consists of
RBCs combined with ABO-compatible FFP.
DISTR IBUTION
This product can be used for neonatal ex-
change transfusion. The conventional ap- Inspection
proach is to combine group O RBCs (Rh com- Inspection is a critical control point in blood
patible with the neonate) and group AB FFP to component manufacturing and must occur
achieve a 50% ± 5% hematocrit of the final before shipping, upon receipt, and before is-
product. The volumes of the two components sue for transfusion. Proper documentation of
before pooling can be adjusted to achieve a this process includes 1) date of inspection, 2)
desired hematocrit level. Following recombi- donor identification number, 3) description of
nation, the product can be stored at 1 to 6 C for any visual abnormalities, 4) action(s) taken,
up to 24 hours. and 5) identity of the staff member performing
Current FDA uniform guidelines should
the inspection. Visible abnormalities may in-
be followed when pooled products are la-
clude discoloration of the segments, compo-
beled.18 A unique pool number should be af-
nent, or supernatant fluid or the presence of
fixed to the final container, and all units in the
visible clots, particulate matter, or other for-
pool must be documented in electronic or
eign bodies. Detection of any such abnormali-
manual records.
ties should result in product quarantine for
further investigation that may include return-
Aliquoting
ing the component to the supplier.
Patients requiring low-volume transfusions If a component is determined to be bacte-
may receive aliquots of smaller volumes de- rially contaminated, the component manufac-
rived from the original unit via an FDA-cleared turer must be notified so that an immediate
sterile connecting device or integrated transfer investigation can take place. Other compo-
bags. Available products designed for use with nents prepared from that collection should be
sterile connecting devices include transfer quarantined until the investigation is com-
packs, smaller-volume bags, and tubing with plete. If the component (or co-component)
integrally attached syringes. has been transfused, the recipient’s attending
The expiration date of the aliquot and physician should be notified, and consultation
minimum residual volumes that must be with the medical director is recommended.
CHAPTER 9 Storage, Processing, Distribution, and Inventory 䡲 225
ancies identified must be reported immediate- Final identification of the transfusion re-
ly to the supplier and resolved before the prod- cipient and blood component rests with the
uct is dispensed for transfusion. transfusionist. This individual identifies the
patient and donor unit and certifies that the
Issuance of Components identifying information on forms, tags, and la-
bels is in agreement.
Ensuring that the correct blood component is
transfused to the correct patient is paramount
Documentation
for transfusion safety. All requests for blood
components must contain two independent The patient’s medical record must include
identifiers so that the intended recipient can proper documentation of all transfusions. For
be uniquely identified. Recipient compatibility- each transfusion, this documentation must
testing records must also be reviewed. Current contain the transfusion order, consent for
testing results must be compared with histori- transfusion, component name, donation iden-
cal records, if available, and any discrepancies tification number, date and time of transfu-
must be resolved before product selection. sion, pre- and posttransfusion vital signs,
Personnel must visually inspect and doc- volume transfused, identification of the trans-
ument that the selected product is acceptable fusionist, and, if applicable, any transfusion-
for use. This inspection must include confir- related adverse events.
mation that the product does not have an ab-
normal color or appearance and that the con- Return of Blood Components and
tainer is intact. Once selected for transfusion, Reissue
the blood component must have an attached The transfusion service may receive back into
label or tie tag that contains the intended re- inventory units that meet acceptance specifica-
cipient’s two independent identifiers, dona- tions. These conditions include the following:
tion identification number, and compatibility
test result interpretation, if performed. 1. The primary container has not been opened.
At the time of issue, there must be a final 2. The component has been maintained at the
check of each unit that includes the following: appropriate temperature.
3. At least one sealed segment remains inte-
1. The intended recipient’s two independent grally attached to the container of RBCs.
identifiers, ABO group, and Rh type. 4. Documentation indicates that the compo-
2. The donation identification number, donor nent has been inspected and is acceptable
ABO group, and, if required, Rh type. for reissue.
3. The interpretation of the crossmatch test
results, if performed. Individual unit temperature indicators or
4. Special transfusion requirements (eg, cyto- temperature-reading devices can be used to
megalovirus-reduced-risk, irradiated, or determine the acceptability of products for re-
antigen-negative components), if applica- turn to inventory. Blood and blood compo-
ble. nents may also be transported or stored in
5. The expiration date and, if applicable, time. qualified containers using a validated process
6. The date and time of issue. that has been shown to maintain acceptable
temperatures for a defined interval. If time
The transfusion service must confirm that frames are used to determine the acceptability
the recipient identifying information, transfu- of a product’s return to inventory, the time
sion request, testing records, and blood com- frame must be validated by the individual fa-
ponent labeling and compatibility are accu- cility. The validation should demonstrate that
rate and in agreement. Any discrepancies for the defined period, the appropriate tem-
identified must be resolved before issue. perature of the product has been maintained.
CHAPTER 9 Storage, Processing, Distribution, and Inventory 䡲 227
Rh-compatible blood. If the patient’s ABO continued support with group O RBCs is rec-
group is unknown, group O RBCs should be is- ommended. Unexpected and significant usage
sued. of group O RBCs in the setting of massive
A more detailed discussion of emergent transfusion should be considered when deter-
transfusion that covers clinical concerns rath- mining component inventory levels. In mas-
er than inventory management can be found sive transfusion situations where large
in Chapter 15. amounts of blood may be required, policies
may be developed to provide D-positive RBCs
to select patients, such as all adult males and
Massive Transfusion postmenopausal females.
Massive transfusion can be defined as the ad- Many hospitals have developed massive-
ministration of 8 to 10 RBC units to an adult transfusion protocols to standardize the re-
patient in less than 24 hours, acute adminis- sponse to hemorrhage.21, 22 These protocols are
tration of 4 to 5 RBC units in 1 hour, or ex- designed to rapidly provide blood compo-
change transfusion of an infant. nents in a balanced ratio of plasma and plate-
To ensure the ability to accurately inter- lets to RBCs, particularly when laboratory test-
pret ABO group testing results, the patient ing is not rapid enough to guide transfusion
specimen should be obtained for testing as support. Additional studies are needed to clar-
early as possible during massive transfusion. If ify whether the use of these protocols is associ-
the patient ABO type cannot be determined, ated with improved patient outcomes.
KEY POINTS
1. Ensuring that the correct blood component is transfused to the correct patient is para-
mount for transfusion safety. It must be confirmed that the recipient’s identifying informa-
tion, transfusion request, testing records, and blood component labeling and compatibility
are accurate and in agreement. Any discrepancies identified must be resolved before com-
ponents are issued or transfused.
2. Visual inspection of the blood component is a critical control point in the manufacturing
process and must occur before shipping, upon receipt, and before issue for transfusion.
3. Refrigerators, freezers, and platelet incubators for blood component storage must be moni-
tored to ensure that proper storage conditions are maintained. Because the safety, purity,
potency, and quality of the blood components may be affected by improper storage, alarm
settings should be configured to notify necessary personnel before the upper or lower ac-
ceptable storage temperatures are exceeded.
4. Temperature requirements during transport of blood components differ from those during
storage. Blood components held outside the blood bank before transfusion are considered
to be in storage. Validated processes must ensure that acceptable storage temperatures are
maintained.
5. Acceptable time frames for returning blood components to inventory after issue should be
validated by individual facilities. Individual unit-temperature indicators or temperature-
reading devices may be used to determine component acceptability for return to inventory.
6. Thawed FFP, PF24, and PF24RT24 expire within 24 hours of thawing. These products may be
labeled as “Thawed Plasma” to allow for a 5-day shelf life if they were originally collected in
a closed system.
CHAPTER 9 Storage, Processing, Distribution, and Inventory 䡲 229
REFER ENCES
1. Food and Drug Administration. Drugs; current human RBCs frozen with 40-percent (wt/vol)
good manufacturing practice in manufacture, glycerol and stored after deglycerolization for
processing, packing, or holding. (June 19, 15 days at 4 degrees C in AS-3: Assessment of
1963) Fed Regist 1963;133:6385-7. RBC processing in the ACP 215. Transfusion
2. Code of federal regulations. Title 21, CFR Parts 2001;41:933-9.
210 and 211. Washington, DC: US Government 15. Borzini P, Mazzucco L. Platelet gels and releas-
Printing Office, 2014 (revised annually). ates. Curr Opin Hematol 2005;12:473-9.
3. Levitt J, ed. Standards for blood banks and 16. Cyr, M, Hume H, Sweeney JD, et al. Anomaly of
transfusion services. 29th ed. Bethesda, MD: the des-Arg9-bradykinin metabolism associat-
AABB, 2014.
ed with severe hypotensive reaticon during
4. Nunes E. Transport versus storage: What is the
blood transfusions: A preliminary report.
difference? AABB News 2013;15(2):4-5.
Transfusion 1999;39:1084-8.
5. Klein HG, Spahn DR, Carson JL. Red blood cell
17. Benjamin RJ, Kline L, Dy BA, et al. Bacterial
transfusion in clinical practice. Lancet 2007;
370:415-26. contamination of whole-blood-derived plate-
6. van de Watering L. Red cell storage and prog- lets: The introduction of sample diversion and
nosis. Vox Sang 2011;100:36-45. prestorage pooling with culture testing in the
7. Zimrin AB, Hess JR. Current issues to the American Red Cross. Transfusion 2008;48:
transfusion of RBCs. Vox Sang 2009;96:93-103. 2348-55.
8. Strauss RG. Data-driven blood banking prac- 18. Food and Drug Administration. Guidance: In-
tices for neonatal RBC transfusions. Transfu- dustry consensus standard for the uniform la-
sion 2000;40:1528-40. beling of blood and blood components using
9. Shrivastava M. The platelet storage lesion. ISBT 128 version 2.0.0, November 2005. (Sep-
Transfus Apher Sci 2009;41:105-13. tember 22, 2006) Silver Spring, MD: CBER Of-
10. AABB, American Red Cross, America’s Blood fice of Communication, Outreach, and Devel-
Centers, Armed Services Blood Program. Cir- opment, 2006.
cular of information for the use of human 19. Liu EA, Mannino FL, Lane TA. Prospective,
blood and blood components. Bethesda, MD: randomized trial of the safety and efficacy of a
AABB, 2013. limited donor exposure transfusion program
11. Werhli G, Taylor NE, Haines, AL, et al. Institut- for premature neonates. J Pediatr 1994;125:92-
ing a thawed plasma procedure: It just makes
6.
sense and saves cents. Transfusion 2009;49:
20. Boral LI, Dannemiller FJ, Standard W, et al. A
2625-30.
guideline for anticipated blood usage during
12. Tholpady A, Monson J, Radovancevic R, et al.
elective surgical procedures. Am J Clin Pathol
Analysis of prolonged storage on coagulation
Factor (F)V, FVII, and FVIII in thawed plasma: 1979;71:680-4.
Is it time to extend the expiration date beyond 21. Young PP, Cotton BA, Goodnough LT. Massive
5 days? Transfusion 2013;53:645-50. transfusion protocols for patients with sub-
13. Meryman HT, Hornblower M. A method for stantial hemorrhage. Transfus Med Rev 2011;
freezing and washing RBCs using a high glyc- 25:293-303.
erol concentration. Transfusion 1972;12: 22. Hendrickson JE, Shaz BH, Pereira G, et al. Im-
14556. plementation of a pediatric trauma massive
14. Valeri CR, Ragno G, Pivacek LE, et al. A multi- transfusion protocol: One institution’s experi-
center study of in vitro and in vivo values in ence. Transfusion 2012;52:1228-36.
C h a p t e r 1 0
James C. Zimring, MD, PhD, Director, Transfusion Medicine Research, Puget Sound Blood Center Research
Institute, Seattle, Washington, and Steven L. Spitalnik, MD, Professor of Pathology and Cell Biology, and
Director of Clinical Laboratories, Columbia University, New York, New York
J. Zimring has disclosed financial relationships with Immucor Inc and Haemonetics. S. Spitalnik has disclosed
no conflicts of interest.
231
232 䡲 AABB TECHNICAL MANUAL
encode their genome as RNA. In addition, be- otides comprising DNA: the purines (adenine
cause normal flora may have a substantial ef- and guanine) and pyrimidines (cytosine and
fect on human biology, DNA and RNA analysis thymine). In addition, C3 of the pentose is
may become important to monitor normal modified by a hydroxyl group [Fig 10-1(A)].
nonpathological and commensal microorgan- The individual nucleotides form DNA poly-
isms. With the possible exception of prion- mers when the phosphate group on C5 of one
associated disorders, either DNA or RNA en- nucleotide forms a covalent bond with the free
codes all known genetic material relevant to hydroxyl group on C3 of another nucleotide
transfusion medicine. [Fig 10-1(B)]. DNA molecules vary from each
other based on the sequence of nucleotides
Basic Chemistry and Structure of that are incorporated into the polymer. Each
Nucleic Acids DNA strand has a terminal 5' end that contains
DNA is a nucleic acid polymer consisting of a free phosphate (attached to C5) and a termi-
long chains of nucleotides linked together.1 nal 3' end that has a free hydroxyl group (at-
Nucleotides consist of a pentose (a carbohy- tached to C3).
drate with five carbon atoms), a phosphate The human genome consists of double-
group attached to the fifth carbon atom (C5) of stranded DNA. The bases contained within a
the pentose, and a base group attached to C1 single strand of DNA form hydrogen bonds to
[Fig 10-1(A)]. Variations in the chemistry of the complementary bases on another strand of
base group give rise to the four different nucle- DNA. In particular, thymidine (T) binds to ad-
enine (A), and guanine (G) binds to cytosine important in defining the phenotype of these
(C). When two strands have complementary cells.
sequences, they can hybridize to form a For mRNA analysis, then, the choice of
double-stranded molecule [Fig 10-1(C)]. The starting cell types is critical. Multiple manu-
two complementary strands hybridize such facturers offer reagents that simplify and expe-
that the 5' and 3' ends have opposite orienta- dite isolation of cellular DNA and/or mRNA as
tions and form a double helix in which the well as for purifying viral nucleic acids from
phosphodiester backbone is on the outside of plasma. Depending on the type of testing to be
the helix and the hydrogen-bond-paired bases performed, the quantity and quality of the nu-
are on the inside. cleic acids isolated may be important, as de-
When genes are expressed, the DNA en- termined by the relative purity of DNA or RNA
coding a given gene is transcribed into RNA and the absence of contaminating protein.
[Fig 10-1(C)]. The structure of RNA is similar to
that of DNA with the following exceptions: 1) Hybridization-Based Methods of
ribonucleotides have an additional hydroxyl Nucleic Acid Detection
group on C2 of the pentose sugar (thus form-
ing ribose), 2) uracil (U) replaces thymine (T), Before the advent of techniques that allowed
and 3) RNA coding for gene products is the amplification of a specific nucleic acid se-
typically single-stranded (although double- quence (see Polymerase Chain Reaction be-
stranded RNA has important regulatory roles). low), detection of nucleic acids depended on
Several classes of RNA exist in human hybridization-based methods. Probes with a
cells; the type described in this paragraph, particular sequence of nucleotides were syn-
which is subsequently translated into protein, thesized and labeled with one of a variety of
is messenger RNA (mRNA). When a gene is ex- detectable markers. The probe could then hy-
pressed, the transcription machinery unwinds bridize to complementary sequences of DNA
the DNA double helix, synthesizes a mRNA or RNA, allowing the detection and quantifica-
strand of complementary sequence, and rehy- tion of the corresponding complementary tar-
bridizes the DNA in its wake [Fig 10-1(C)]. In get. This could be done in the solid phase (ie,
this way, the RNA is essentially a copy of the Southern and Northern blots), the fluid phase
sequence found in the DNA. RNA is synthe- (ie, RNAse protection assay or S1 protection
sized in the 5' to 3' direction, and, thus, only a assay) or as a result of enzymatic extension (ie,
single strand of the DNA is transcribed into primer extension assay).2-5 However, compared
RNA by any given run of a polymerase. After to more recent amplification-based techniques,
synthesis in the nucleus, RNA is processed and hybridization-based methods have limited
exported to the cytoplasm, where ribosomes sensitivity and are less amenable to automa-
translate it into protein. tion. Although hybridization-based methods
are useful in basic research and still play a mi-
Isolation of Nucleic Acids nor role in some diagnostic laboratories, most
analyses of nucleic acids are performed using
The first step in most DNA and RNA analyses is
amplification-based methods.
the isolation of nucleic acids. All nucleated
cells of an individual contain identical genom-
The Polymerase Chain Reaction
ic DNA, with some notable exceptions (eg, re-
arranged genes in mature T and B cells). Ge- Nucleic acid detection and analysis were revo-
nomic DNA can be isolated from readily lutionized by the invention of the polymerase
obtainable cellular sources, such as peripheral chain reaction (PCR). PCR was the first ampli-
blood leukocytes and buccal swabs. In con- fication-based technique for generating nucle-
trast, mRNAs are distinct in different cell pop- ic acid fragments for direct analysis.6 The
ulations because their expression patterns are concept of amplification-based systems has
234 䡲 AABB TECHNICAL MANUAL
grown to include many other techniques and allow primer annealing, and 3) extension and
applications. synthesis of DNA on the primer strand. This
A PCR reaction requires 1) a DNA sample procedure continues for multiple cycles, typi-
to be analyzed; 2) gene-specific primers; 3) a cally 20 to 40, depending on the abundance of
thermostable DNA polymerase; and 4) compo- the template and the required sensitivity of the
nents that allow the DNA polymerase to enzy- assay.
matically synthesize DNA, including nucleo- An overview of the PCR process is pre-
tides (A, T, C, and G) and the proper salts and sented in Fig 10-2. This example begins with a
buffer. The PCR reaction involves repeat cycles single copy of a double-stranded DNA tem-
of heating/cooling (thermocycling), allowing plate. The DNA is denatured by heating to near
exponential DNA amplification of the frag- boiling (typically at 94-95 C), which disrupts
ment of interest. This reaction is carried out in the hydrogen bonds between complementary
a thermocycler that rapidly changes tempera- bases, thereby separating the two strands. The
ture with accuracy and precision. The thermo- temperature is then lowered (annealing reac-
cycling reaction involved is 1) heat denatur- tion) to allow gene-specific primers to anneal
ation of double-stranded DNA, 2) cooling to to their complementary target. Typically, an
A. B. C.
5’
5’ 3’ 5’ 3’
3’ 5’ 3’ 5’ 3’
5’
5’ 3’ 5’ 3’
3’ 5’ 3’ 5’ 3’
FIGURE 10-3. Potential problems in primer design that may inhibit polymerase chain reaction (PCR).
molecule can self-prime with the polymerase Using this approach, DNA extraction is sepa-
and extend the 3' end to be complementary to rate from testing, PCR reactions are assembled
the 5' end, increasing the tendency of the in one room and amplified in a second room,
primer to self-anneal. The presence of the ad- and downstream analysis (if required) is per-
ditional sequence prevents amplification of formed in a third room. There should be no
the authentic target amplicon. retrograde flow, and no materials or instru-
ments used in the amplification or analysis
Contamination between Specimens (post-PCR) rooms should make their way into
the PCR setup (pre-PCR) room. Pipette filter
One of the greatest strengths of PCR is its abili-
tips that minimize carry-over contamination
ty to amplify very small amounts of genetic
and sample aerosols are routinely used.
material. In theory, single-copy sensitivity can
Other ways to minimize potential con-
be achieved. In practice, detection of 10 copies
tamination include the addition of deoxyuri-
of DNA or fewer is not uncommon, depending
dine triphosphate (dUTP) to PCR reactions.
on the sensitivity of the assay readout. This
Polymerases incorporate dUTP in place of de-
level of sensitivity also makes the assay sus-
oxythymidine triphosphate (dTTP), and the
ceptible to false-positive results due to con-
added enzyme uracil-DNA glycosylase (UNG)
tamination from specimens being analyzed or
specifically cleaves DNA containing uracil but
amplicons generated in previous amplifica-
not normal DNA or RNA.9 Thus, adding UNG
tion reactions. Beginning with just 10 mole-
to PCR reactions destroys contaminating am-
cules of DNA, 30 rounds of amplification in a
plicons from previous amplifications but not
PCR reaction yields more than 1 × 1010 ampli-
native DNA in the specimen. The UNG is heat
cons. Thus, if only 0.0000001% of a previous
inactivated during the initial denaturation
reaction is inadvertently introduced onto a pi-
PCR step, allowing amplification of the new
pette used to set up a subsequent reaction, a
sample.
false-positive result may occur.
To minimize the possibility of contamina-
Reverse-Transcriptase PCR
tion from previously generated amplicons and
a false-positive signal, PCR laboratories rou- When amplification and analysis of mRNA is
tinely process samples in only one direction. required, a reverse-transcriptase (RT) enzyme
CHAPTER 10 Molecular Biology and Immunology in Transfusion Medicine 䡲 237
that synthesizes DNA from an RNA template is TMA plays a large role in nucleic acid test-
used.10,11 Like DNA polymerase, RT synthesizes ing for human immunodeficiency virus (HIV),
in the 5' to 3' direction and requires the an- hepatitis C virus, and West Nile virus (ie, using
nealing of a primer to initiate transcription. the Chiron/Gen-Probe system). Both tech-
When RNA is transcribed, the resulting DNA is niques use RNA as the amplification target.
a single-stranded complementary copy of the The reaction contains two primers, RT, DNA
RNA and is referred to as “complementary polymerase, RNAse H, and T7 polymerase. The
DNA” (cDNA). The cDNA is then a suitable reaction begins when a specific downstream
substrate for PCR, as described above. primer (Primer 1) hybridizes to the 3' end of
the target RNA and RT synthesizes a cDNA
Transcription-Mediated Amplification copy (Fig 10-4, Step 1). Primer 1 not only con-
and Nucleic Acid Sequence-Based tains a complementary sequence at its 3' end
Amplification that hybridizes to the target RNA, but the 5'
end also encodes a bacteriophage T7 promot-
Since the advent of PCR, several additional nu- er. The RNA template is degraded by RT in the
cleic acid amplification strategies have been TMA assay or by RNAse H in an additional step
devised. Among the most useful are two relat- with NASBA (Fig 10-4, Step 2). A second 5' up-
ed techniques, transcription-mediated ampli- stream primer (Primer 2) then binds to the
fication (TMA) and nucleic acid sequence- newly synthesized cDNA (Fig 10-4, Step 3) and
based amplification (NASBA).12,13 Although utilizes DNA polymerase to synthesize a
TMA and NASBA have some differences (de- double-stranded DNA molecule (Fig 10-4, Step
scribed in this section), they are conceptually 4). This molecule has a T7 promoter at one
similar and, thus, are described together (see end, and T7 polymerase then drives transcrip-
Fig 10-4). tion of RNA (Fig 10-4, Step 5). Numerous RNA
FIGURE 10-4. Schematic overview of transcription-mediated amplification (TMA) and nucleic acid sequence-
based amplification.
RNA = ribonucleic acid; cDNA = complementary deoxyribonucleic acid; mRNA = messenger RNA; RNAse =
ribonuclease.
238 䡲 AABB TECHNICAL MANUAL
transcripts are synthesized from a single DNA “restriction fragment length polymorphism”
template. These new RNA molecules can reen- (RFLP) analysis]. Even greater specificity can
ter the amplification cycle with Primer 2 initi- be achieved by hybridization analysis. After
ating reverse transcription, followed by RNA electrophoresis, the amplified products are
degradation and subsequent synthesis of DNA transferred to nitrocellulose paper, which is
using Primer 1 and DNA polymerase. This then hybridized with DNA probes specific for
leads to additional amplification with ongoing the amplified sequences.
cycles of transcription and template synthesis. Each of these approaches requires gel
One distinct advantage of NASBA compared to electrophoresis, which is time consuming, in-
PCR is that repeat nucleic acid denaturation is compatible with the throughput needs of
not required. Therefore, NASBA amplifies RNA donor-testing laboratories, and not easily
sequences in an isothermal reaction that does automated. Moreover, it requires opening
not require a thermocycler. tubes containing amplification reactions and
In addition to the amplification methods manipulating the products, which increases
described above, several other techniques use the risk of contamination of reagents and
nucleic acid polymerizing or ligating enzymes false-positive results in subsequent samples.
to amplify DNA and/or RNA. These include More advanced techniques for detecting
strand displacement amplification (SDA) and amplification products rely on the chemistry
the ligase chain reaction (LCR).14-16 There are of fluorescent molecules. Probes that fluoresce
also probe/signal-amplification methods, only when the correct amplicon is present are
such as the Cleavase Invader, branched DNA, included in the amplification reactions. By in-
and hybrid capture assays. Although these cluding a fluorescence spectrophotometer in
techniques have been adapted to detect the thermocycler, the generation of amplifica-
pathogens, they are not widely used in transfu- tion products can be measured at each cycle.
sion medicine and are not described further This approach, called “real-time PCR,” is high-
here.17 ly sensitive; much more quantitative than gel
electrophoresis-based methods; and, by using
Detection of Nucleic Acid multiple reporter dyes with distinct fluores-
Amplification Products cence spectra, makes detection of multiple
targets in a single reaction feasible (ie, multi-
PCR, RT-PCR, TMA, and NASBA amplify spe- plex nucleic acid amplification). In addition,
cific nucleic acid sequences. However, the am- including fluorescent probes in the reaction
plified sequences must still be detected. Tradi- allows analysis without ever opening the tube
tionally, this has been accomplished by containing the amplification products; this de-
separating the amplification reactions by gel creases the risk of laboratory contamination
electrophoresis in the presence of a fluores- with amplicons and subsequent false-positive
cent dye (eg, ethidium bromide), which makes results.
the nucleic acids visible in the gel under ultra- Two of these fluorescent methods rely on
violet light. This allows visualization of the the juxtaposition of a fluorescent molecule
bands and, if appropriate standards are in- with a quencher molecule that prevents fluo-
cluded in the gels, provides a molecular weight rescence. In the TaqMan system, a sequence-
by which one can distinguish amplicons of the specific probe has the fluorescent molecule at
correct size from cross-reactive products of one end and the quencher at the other. When
different sizes. To confirm that the amplified the probe hybridizes to its target, it essentially
nucleic acids have the correct sequence, the blocks the DNA polymerase that is synthesiz-
amplification products can be digested with ing the next round of DNA by extending from
restriction endonucleases and the sizes of the the primer. When the DNA polymerase en-
resulting molecules determined. Because the counters the hybridized probe, it degrades the
amplicon sequence is known, one can predict probe, thus separating the fluorescent and
the correct restriction sites [this is known as quencher molecules [Fig 10-5(A)]. Because
CHAPTER 10 Molecular Biology and Immunology in Transfusion Medicine 䡲 239
FIGURE 10-5. Methods of detection by sequence-specific probes during real-time polymerase chain reaction.
Pol = polymerase enzyme.
240 䡲 AABB TECHNICAL MANUAL
this occurs only when the probe hybridizes to cases, PCR-amplified material can be digested
its target, fluorescence is generated as a func- by restriction enzymes and then analyzed by
tion of amplicon generation. agarose electrophoresis to observe the result-
A second approach uses molecular bea- ing fragment sizes. RFLP analysis has low
cons that also have a fluorescent molecule on throughput and depends on the presence of a
one end of a DNA probe and a quencher mole- restriction enzyme recognition site associated
cule on the other end. In this case, the se- with the SNP in the gene of interest.
quence-specific probe is flanked by two se- Several different methods for detecting
quences that are complimentary to each other SNPs consist mostly of modifications of PCR or
and form a hairpin loop, thus juxtaposing the array technologies. With these methods, prim-
fluorescent molecule with its quencher. How- ers or probes are engineered such that hybrid-
ever, when the probe hybridizes to its target, ization depends on the presence of the correct
the hairpin loop unfolds, separating the SNP. Both multiplex PCR and DNA array sys-
quencher from the fluorescent molecule and tems can determine the genotype of blood
allowing fluorescence to occur [Fig 10-5(B)]. group antigens in individual specimens.17-19
A third approach uses two molecules that These systems offer the advantage of higher
do not fluoresce unless they are in close prox- throughput and automated readout while
imity to each other. These molecules are avoiding the need for complex interpretation.
linked to two separate DNA probes that hy- Genotyping of red cell antigens may be
bridize to adjacent sequences on the ampli- more efficient than traditional serologic typ-
con. If the amplicon is present, the probes an- ing. In addition, when a patient has received
neal in such a way that the two molecules are multiple RBC units and it is not possible to dis-
in close proximity, providing a fluorescent sig- tinguish the patient’s own red cells from trans-
nal [Fig 10-5(C)]. fused red cells, genotyping the patient’s DNA
A fourth method uses SYBR® green dye, may be the only reliable way to predict the pa-
which fluoresces when bound to double- tient’s red cell phenotype. However, occasion-
stranded DNA. SYBR® green is not sequence ally the genotype may not correlate with the
specific and, thus, detects all amplicons. How- phenotype. It is worth noting that genotyping
ever, authentic amplicons can be distin- typically focuses on known polymorphisms
guished from cross-reactive products by melt- but does not provide the entire sequence of
ing curve analysis. Because the melting curve the gene or its regulatory regions. Therefore,
is a function of amplicon size and GC content genotyping might not predict phenotype when
and the size and sequence of the correct am- 1) new, hitherto unknown, polymorphisms in
plicon is known, the melting curve can be used the coding region alter protein structure; 2)
to confirm the identity of the amplicon. new polymorphisms in the coding, promoter,
As with PCR, these fluorescent-probe or other regulatory regions prevent gene ex-
techniques can be applied to other amplifica- pression despite the presence of the correct
tion technologies, such as TMA. coding sequence; or 3) changes alter the epit-
ope (ie, modification by bacterial enzymes)
Analysis of Single-Nucleotide when epitopes depend on posttranslational
modification.
Polymorphisms
The vast majority of blood group antigens con-
PROT E IN A NA LY SI S
sist of single-nucleotide polymorphisms
(SNPs). The gene product is present in most Nucleic acid analysis, as described in the “Nu-
people, but the difference that determines the cleic Acid Analysis” section above, detects the
identity of the blood group antigen is a small presence of DNA or RNA that encodes genom-
change in sequence, often a single nucleotide. ic material and/or measures gene expression
Some SNPs destroy or create recognition at the RNA level. However, due to regulation of
sites for restriction endonucleases. In these protein translation, the presence of mRNA
CHAPTER 10 Molecular Biology and Immunology in Transfusion Medicine 䡲 241
does not always correlate with the presence of tained at the top or within the matrix due to
the encoded protein. In addition, the detec- their greater size.20 In addition to blood bank
tion of antibodies, which are proteins, cannot serology, red cells can be used to detect anti-
be determined by detecting or measuring nu- bodies against other antigens by linking these
cleic acids. Accordingly, measuring protein- particular antigens to the red cell surface. Ag-
protein and/or protein-carbohydrate interac- glutination-based assays can also be engi-
tions provides relevant biological information neered using particles other than red cells,
in transfusion medicine that may not be ob- such as latex beads. Each of these techniques
tainable by nucleic acid analysis. uses the same basic principles and has broad
applicability in clinical diagnostic testing.
Fluid-Phase Assays (Agglutination- Although agglutination reactions are very
Based Methods) sensitive and easy to perform, the formation of
agglutinates depends on the proper stoichio-
Depending on antibody isotype, immunoglob- metric ratio of antibody to antigen. When the
ulins have 2 to 10 antigen-binding sites per ratio of antigen to antibody is in a range such
molecule. Each antibody can bind more than that agglutination readily occurs, it is referred
one target molecule, allowing antibodies to to as the “zone of equivalence.” In this situa-
cross-link antigens present in multiple copies tion, each arm of the antibody binds to a dif-
on particles, such as red cells or beads. This ferent particle, and a network (or lattice) of
cross-linking can aggregate particles that have linked particles results in agglutination [Fig
the relevant antigen on their surface, a process 10-6(B)]. A false-negative result is generated if
known as “agglutination.” Agglutination is an the stoichiometric ratio is outside the zone of
old, but generally reliable, serologic method equivalence at either extreme.
for detecting antibody-antigen interactions A prozone effect can occur when such a
and is used extensively in transfusion medi- high concentration of antibody is present that
cine. the likelihood of an antibody being able to
Agglutination can be detected by several bind to two separate particles or red cells is di-
methods. The antigen copy number and den- minished [Fig 10-6(A)]. This is seen most com-
sity varies depending on the blood group. Ag- monly with non-red-cell-based agglutination
glutination is used for serological crossmatch- assays, such as the rapid plasma reagin screen-
ing (donor red cells incubated with recipient ing test for syphilis serology. Although prozone
plasma or serum), screening for unexpected effects are unusual in classical red cell serolo-
antibodies (reagent red cells of known blood gy, they have been observed when titers of red
group antigen composition incubated with cell antibodies are very high. In particular, dis-
patient plasma or serum), and blood group crepant reverse ABO typing due to prozone ef-
antigen phenotyping of the donor or recipient fects has been reported.21,22 Diluting the se-
(test red cells incubated with monoclonal anti- rum being tested and using EDTA containing
bodies or reagent-quality antisera of known diluents decreases the likelihood of prozone
specificity). effects. One reason why prozone effects are
Because red cells are easily visible due to uncommon in red cell serology is that the use
their red color, several systems were devised to of antihuman globulin (AHG) largely over-
detect agglutination. These include 1) tube comes these problems. However, a secondary
testing in which agglutination is visually de- “prozone-like” effect occurs if there is inade-
tected by the adhesion of red cells to one an- quate washing before adding AHG.23 In this
other in the postcentrifuge pellet, 2) passive case, residual immune globulin G (IgG) in so-
agglutination in microtiter plates where agglu- lution binds the AHG and competes for AHG
tination is visualized by the spread pattern of binding to IgG on the red cells. This may occur
red cells in individual wells, and 3) gel testing with very high titers of blood group antibodies
in which unagglutinated red cells pass through or even in the presence of highly increased lev-
a matrix but agglutinated complexes are re- els of polyclonal antibodies, as in hypergam-
242 䡲 AABB TECHNICAL MANUAL
FIGURE 10-6. Effects of relative concentrations of antigen and antibody on the outcome of agglutination
reactions.
FIGURE 10-7. Schematic representation of (A) phenotyping red cells and (B) detecting antibodies by solid-
phase assay.
well (positive reaction). A positive reaction in- antibodies against platelet antigens using the
dicates the presence of the antigen on the red approaches described above.24
cells being tested.
Enzyme-Linked Immunosorbent Assay
S O L I D - PH A S E A S S AY S F O R D E T E C T I N G
A N T I B O D I E S TO R E D C E L L A N T I G E N S . An- Enzyme-linked immunosorbent assay (ELISA),
tigen-coated particles, consisting of red cells also referred to as “enzyme immunoassay,”
or red cell fragments, are coated onto microti- can detect antibodies and antigens. A signal is
ter plate wells [Fig 10-7(B)]. Patient serum is generated via an enzyme linked to a secondary
then added to each well, followed by incuba- antibody or antigen that converts a substrate
tion and then washing. If the patient serum to a measurable product (eg, a color change or
has antibodies against the red cell antigens a chemiluminescent reaction). For this reason,
coated on the well, then the antibodies bind to ELISAs have considerable signal amplification
the red cells or their fragments. Indicator red and are significantly more sensitive than fluid-
cells coated with antihuman IgG are then add- phase agglutination or SPRCAs. In most cases,
ed. A positive reaction is demonstrated by dif- ELISAs use purified or recombinant antigens
fuse adherence of the indicator red cell to the or antibodies, depending on the analyte de-
well, whereas a negative reaction is demon- tected. However, intact red cells can be used to
strated by clustering of indicator cells in a but- screen for red cell antibodies, referred to as the
ton. “enzyme-linked antiglobulin test.”25
Solid-Phase Assays for Platelet Testing DETECTION OF ANTI BODIES BY ELISA (IN-
DIRECT ELISA). To detect antibodies against
Solid-phase red cell adherence (SPRCA) tech- a given antigen, the antigen is coated onto mi-
nology has also been adapted to detect anti- crotiter plate wells [Fig 10-8(A)]. The test sam-
gens on platelets, such as HPA-1a, as well as ple is then added, incubated, and washed. If
244 䡲 AABB TECHNICAL MANUAL
FIGURE 10-8. Schematic representation of (A) indirect enzyme-linked immunosorbent assay (ELISA), (B)
sandwich ELISA, and (C) competitive ELISA.
antibodies against the antigen are present, the target without interfering with each other.
they bind to the antigen-coated well and the Typically, this is accomplished by monoclonal
bound antibodies are detected by incubating antibodies. Microtiter plate wells are coated
with anti-Ig (eg, anti-IgG) linked to an enzyme with one antibody, the “capture antibody” [Fig
(eg, alkaline phosphatase or horseradish per- 10-8(B)]. The specimen is then incubated in
oxidase). After further washing, enzyme sub- the well; if the antigen is present, it binds to
strate is added and is converted to a detectable the solid-phase antibody coated in the well.
color if enzyme is present. Quantification is The plate is then washed and incubated with
performed using a standard curve and a spec- the second antibody, which is linked to a re-
trophotometer to measure the absorbance at porter enzyme (the “conjugate”). Because the
the wavelength appropriate for that enzyme/ second antibody is specific for the target anti-
substrate product. In some cases, samples gen, it binds to the well only if antigen was
may need to be diluted to ensure that they bound to the capture antibody. After addition-
yield absorbance values in the linear range of al washing, enzyme substrate is added and is
the assay. converted to a detectable color if enzyme is
present.
DETECTION OF ANTI GENS BY SANDWICH
ELISA. In sandwich ELISA, two separate anti- DETECTION OF ANTIGENS BY COMPETI-
bodies are used that bind different epitopes on TIVE ELISA. Competitive ELISA is similar to
the same target antigen; the antibodies bind indirect ELISA in that the target antigen is
CHAPTER 10 Molecular Biology and Immunology in Transfusion Medicine 䡲 245
FIGURE 10-10. Immune globulin (Ig) isotypes (A), IgG subclasses, and their relative activation of complement
and binding to Fc-gamma receptors (FcRs) (B).
248 䡲 AABB TECHNICAL MANUAL
FIGURE 10-11. Mechanisms of red cell destruction by antibody binding. Upon binding (A), an immune globulin
G (IgG) represents a ligand for Fc-gamma receptors (FcRs) on phagocytes (B). If the red cell avoids FcR-
mediated phagocytosis, opsonization may be increased by activation of complement with deposition of C3b (C).
if the combined opsonization of FcR binding and C3b is not sufficient to mediate clearance, completion of the
complement cascade may lead to insertion of the membrane attack complex (MAC) into the red cell surface,
resulting in lysis (D). In reality, these processes likely occur simultaneously, with the ultimate outcome being
the aggregate effect of competing pathways.
CR = complement receptor.
plement. The complement system consists of Once activated, the complement system
a cascade of proteases that, once activated, provides at least two distinct mechanisms of
amplifies the initial signal, leading to the pro- target destruction. The first involves target op-
duction of a large number of effector mole- sonization by complement components. Dur-
cules. Although there are several complement- ing early events in complement activation, C3
activation pathways, this discussion focuses covalently attaches to the antigen surface by
on the “classical pathway” initiated by Fc re- thioester bonds, providing multiple copies of
gions. C3b, which are recognized by the complement
IgM is highly efficient in activating com- receptor 1 (CR1) and CRIg on phagocytes.
plement. However, to avoid indiscriminant ac- When a phagocyte encounters a C3b-coated
tivation, IgM undergoes a conformational shift molecule, it ingests and destroys it. C3b also
after binding antigen, thereby exposing com- rapidly degrades sequentially into iC3b, C3c,
plement-binding sites in the heavy-chain con- and C3dg. Because C3dg is recognized by CR2,
stant region. This interaction is so potent that, which is not on phagocytes, complement can
in theory, a single antigen-bound IgM is suffi- degrade past the point of promoting phagocy-
cient to lyse a target. In contrast, IgG does not tosis. In the second mechanism, downstream
require a conformational change to bind com- of C3 activation, the cascade assembles the
plement, but complement activation requires membrane attack complex (MAC). The MAC
clustered binding of multiple IgG molecules to consists of complement proteins C5b-C9 ar-
the same target. This prevents indiscriminate ranged into a structure resembling a hollow
activation of complement by unbound circu- tube inserted into the membrane of the target
lating IgG. cell. This nonselective channel between the
250 䡲 AABB TECHNICAL MANUAL
inside of a target cell and its external environ- The term “hemolysis” in this context can
ment results in osmotic lysis of the target (if it cause confusion for health-care providers not
is sensitive to osmotic shock). accustomed to blood bank terminology be-
cause they typically think of hemolysis as the
Specific Outcomes of MAC Assembly, rupture of red cells within the circulation (ie,
C3 Opsonization, and Fc Opsonization intravascular hemolysis). In contrast, in extra-
of Antibody-Coated Red Cells vascular hemolysis, the red cells hemolyze in
the digestive compartment (ie, lysosomes) of
The effector mechanisms induced by antibody
binding have different effects on bacteria, vi- phagocytes. This is a very important distinc-
ruses, particles, and various human tissues. In tion because phagocytes in the RES consume a
general, once an IgG antibody binds to a red substantial number of senescent, autologous
cell, the target cell may undergo FcR-mediated red cells each day in the normal process of red
phagocytosis by phagocytes (Fig 10-11). If the cell turnover. Thus, red cell consumption in
antibody initiates the complement cascade, this fashion uses a pathway that evolved spe-
C3b deposition on the red cell surface contrib- cifically to break down and recycle red cell
utes to opsonization, leading to phagocytosis contents (eg, hemoglobin and iron) in a man-
mediated by CR1 and CRIg. Finally, if comple- ner that avoids tissue damage.
ment activation is complete, MAC insertion This does not mean, however, that extra-
causes red cell lysis. The relative contributions vascular removal of antibody-coated red cells
of each pathway vary based on the relative is biologically equivalent to clearance of nor-
amounts of antibody isotype and subclass and mal, senescent red cells. On the contrary,
on the nature of the antigen (eg, antigen densi- DHTRs can cause substantial morbidity and
ty or linkage to cytoskeleton). The sections be- occasional mortality. Indeed, in murine mod-
low describe what is known about these pro- els of incompatible transfusion, rapid clear-
cesses with regard to red cell destruction and ance of antibody-coated red cells induces
the clinical manifestations of hemolysis. systemic inflammation and cytokine storm.
Nonetheless, extravascular hemolysis is dis-
Extravascular Hemolysis tinctly different from intravascular hemolysis
Consumption of antibody- and/or C3b-bound in which red cell contents are directly released
red cells by phagocytes in the reticuloendothe- into the circulating blood.
It is not clear why some red cell antibod-
lial system (RES; predominantly in the spleen
and liver) is referred to as “extravascular” he- ies preferentially promote opsonization and
molysis because the red cells are destroyed phagocytosis instead of osmotic lysis by the
outside of their normal compartment, the in- MAC. Substantial evidence indicates that the
travascular space. This process is also com- antibody type and/or the topographical ar-
monly referred to as a “delayed hemolytic rangement of the target antigen on the red
transfusion reaction” (DHTR) because it often cells are important. In addition, although
occurs days after transfusion in contrast to complement may be activated, the aggregate
“intravascular” hemolysis (see below), which opsonization of red cells by C3b and antibody
is recognized acutely during the transfusion may result in phagocytosis before MAC-
[eg, an acute hemolytic transfusion reaction induced lysis occurs. Consistent with these ex-
(AHTR)]. The delayed kinetics of DHTRs are planations is the observation that extravascu-
due to the milder clinical manifestations of ex- lar hemolysis is typically induced by IgG red
travascular hemolysis and/or the need for the cell antibodies, whereas intravascular hemoly-
implicated antibodies to develop or increase sis is typically induced by IgM red cell antibod-
their titer before the onset of significant red ies. The latter is much more efficient at fixing
cell destruction. complement and promoting MAC formation.
CHAPTER 10 Molecular Biology and Immunology in Transfusion Medicine 䡲 251
KEY PO I NT S
1. Hybridization-based methods can be used to detect genes, gene products, and polymor-
phisms. However, compared to amplification-based methods, straight hybridization meth-
ods lack sensitivity.
2. Amplification-based methods (PCR, TMA, NASBA, SDA, and LCR) are highly sensitive but
are also susceptible to problems with contamination and/or inhibitors due to the nature of
geometric amplification involved.
3. The presence of nucleic acids predicts, but does not always equate with, expression of the
corresponding protein or antigen structure.
4. Analysis of protein expression detects the actual gene product(s). Therefore, it does not suf-
fer from the problems of nucleic acid testing, which relies on predicting protein expression.
5. Protein analysis is less sensitive than nucleic acid testing because no amplification is in-
volved, but protein analysis is also less susceptible to contamination and inhibition, which
can lead to false-positive and false-negative results.
6. Methods of detecting protein suffer from nonamplification-based artifacts (ie, heterophilic
antibodies, prozone effects, and hook effects) that can lead to erroneous results.
7. There can be substantial variability in detecting antigens and antibodies based on different
methods.
8. Immunoglobulins can cause destruction of red cells by several different mechanisms, based
largely on the antigen recognized and the antibody structure.
CHAPTER 10 Molecular Biology and Immunology in Transfusion Medicine 䡲 253
9. Most IgG antibodies that cause red cell destruction induce extravascular hemolysis by pro-
moting consumption of red cells by phagocytes (through Fc receptors and/or complement-
based opsonization). This typically presents as a delayed hemolytic transfusion reaction.
10. IgM antibodies (and in some rare cases IgG) that cause red cell destruction typically induce
intravascular hemolysis through complement activation to the membrane attack complex.
This typically presents as an acute hemolytic transfusion reaction.
11. Despite the serious nature of hemolysis due to incompatible transfusion, many antibodies
to red cell antigens are clinically insignificant and do not result in destruction of red cells.
Incompatibility is best avoided whenever possible, but when compatible blood is not avail-
able, incompatible RBC units can be used when the antigens are known to be clinically in-
significant. Such maneuvers must be carefully considered on a-case-by-case basis and with
extensive communication with the clinicians who are requesting the blood products.
REF EREN CE S
1. Alberts B, Bray D, Lewis J, et al. Molecular biol- 10. Temin HM, Mizutani S. RNA-dependent DNA
ogy of the cell. 3rd ed. New York: Garland Sci- polymerase in virions of Rous sarcoma virus.
ence, 1994:98-105. Nature 1970;226:1211-13.
2. Southern EM. Detection of specific sequences 11. Baltimore D. RNA-dependent DNA poly-
among DNA fragments separated by gel elec- merase in virions of RNA tumour viruses. Na-
trophoresis. J Mol Biol 1975;98:503-17. ture 1970;226:1209-11.
3. Alwine JC, Kemp DJ, Stark GR. Method for de- 12. Compton J. Nucleic acid sequence-based am-
tection of specific RNAs in agarose gels by plification. Nature 1991;350:91-2.
transfer to diazobenzyloxymethyl-paper and 13. Kwoh DY, Davis GR, Whitfield KM, et al. Tran-
hybridization with DNA probes. Proc Natl scription-based amplification system and de-
Acad Sci U S A 1977;74:5350-4. tection of amplified human immunodeficien-
4. Melton DA, Krieg PA, Rebagliati MR, et al. Effi- cy virus type 1 with a bead-based sandwich
cient in vitro synthesis of biologically active hybridization format. Proc Natl Acad Sci U S A
RNA and RNA hybridization probes from plas- 1989;86:1173-7.
mids containing a bacteriophage SP6 promot- 14. Walker GT, Fraiser MS, Schram JL, et al. Strand
er. Nucleic Acids Res 1984;12:7035-56. displacement amplification—an isothermal,
5. Berk AJ, Sharp PA. Sizing and mapping of early
in vitro DNA amplification technique. Nucleic
adenovirus mRNAs by gel electrophoresis of
Acids Res 1992;20:1691-6.
S1 endonuclease-digested hybrids. Cell 1977;
15. Walker GT, Little MC, Nadeau JG, Shank DD.
12:721-32.
Isothermal in vitro amplification of DNA by a
6. Mullis KB, Faloona FA. Specific synthesis of
restriction enzyme/DNA polymerase system.
DNA in vitro via a polymerase-catalyzed chain
Proc Natl Acad Sci U S A 1992;89:392-6.
reaction. Methods Enzymol 1987;155:335-50.
16. Wu DY, Wallace RB. The ligation amplification
7. Masukawa A, Miyachi H, Ohshima T, et al.
[Monitoring of inhibitors of the polymerase reaction (LAR)—amplification of specific DNA
chain reaction for the detection of hepatitis C sequences using sequential rounds of tem-
virus using the positive internal control]. plate-dependent ligation. Genomics 1989;4:
Rinsho Byori Jap 1997;45:673-8. 560-9.
8. Al-Soud WA, Radstrom P. Purification and 17. Denomme GA, Van Oene M. High-throughput
characterization of PCR-inhibitory compo- multiplex single-nucleotide polymorphism
nents in blood cells. J Clin Microbiol 2001;39: analysis for red cell and platelet antigen geno-
485-93. types. Transfusion 2005;45:660-6.
9. Pang J, Modlin J, Yolken R. Use of modified nu- 18. Bugert P, McBride S, Smith G, et al. Microarray-
cleotides and uracil-DNA glycosylase (UNG) based genotyping for blood groups: Compari-
for the control of contamination in the PCR- son of gene array and 5'-nuclease assay tech-
based amplification of RNA. Mol Cell Probes niques with human platelet antigen as a mod-
1992;6:251-6. el. Transfusion 2005;45:654-9.
254 䡲 AABB TECHNICAL MANUAL
19. Hashmi G, Shariff T, Seul M, et al. A flexible ar- by a simple modification. Vox Sang 1982;42:
ray format for large-scale, rapid blood group 157-9.
DNA typing. Transfusion 2005;45:680-8. 24. Procter JL, Vigue F, Alegre E, et al. Rapid
20. Langston MM, Procter JL, Cipolone KM, Stron- screening of platelet donors for PIA1 (HPA-1a)
cek DF. Evaluation of the gel system for ABO alloantigen using a solid-phase microplate im-
grouping and D typing. Transfusion 1999;39: munoassay. Immunohematology 1998;14:141-
300-5. 5.
21. Voak D. Observations on the rare phenome- 25. Leikola J, Perkins HA. Enzyme-linked antiglob-
non of anti-A prozone and the non-specific ulin test: An accurate and simple method to
blocking of haemagglutination due to C1 com- quantify red cell antibodies. Transfusion 1980;
plement fixation by IgG anti-A antibodies. Vox 20:138-44.
Sang 1972;22:408-19. 26. Kindt TJ, Osborne BA, Goldsby RA. Kuby im-
22. Judd WJ, Steiner EA, O’Donnell DB, Oberman munology. 6th ed. New York: WH Freeman and
HA. Discrepancies in reverse ABO typing due Company, 2007.
to prozone. How safe is the immediate-spin 27. Murphy K, Travers P, Walport M. Janeway’s im-
crossmatch? Transfusion 1988;28:334-8. munobiology. 7th ed. New York: Garland Sci-
23. Salama A, Mueller-Eckhardt C. Elimination of ence, 2008.
the prozone effect in the antiglobulin reaction
C h a p t e r 1 1
Christine Lomas-Francis, MSc, FIBMS, Technical Director, Laboratory of Immunohematology and Genomics,
New York Blood Center, New York, New York
The author has disclosed no conflicts of interest.
255
256 䡲 AABB TECHNICAL MANUAL
aids understanding of the molecular aspects of lele name is italicized—for example, RHD for
individual blood groups. the gene encoding the RhD protein. The name
usually is not italicized when the word “gene”
or “allele” follows the name: for example,
BASIC PRINCIPLES OF
“RHD gene,” or “RHD allele.” The Internation-
GENE T IC S al Society of Blood Transfusion (ISBT) Working
Gregor Mendel established the basic tech- Party on Red Cell Immunogenetics and Blood
niques of genetic analysis when, in 1865, he Group Terminology5,6 has developed allele ter-
published his classic breeding experiments minology for use in transfusion medicine. For
with pea plants. Mendel’s observations led him alleles encoding polymorphic common anti-
to conclude that there is a “factor” or unit of gens, the name is based on the ISBT antigen
inheritance, now known as a gene, that is name: eg, FY*01 or JK*02 refer to the alleles en-
passed from one generation to another ac- coding the Fya and Jkb antigens, respectively.
cording to two simple rules: the principles of Alternatively, where letters are commonly
independent segregation and independent as- used, symbols such as FY*A or JK*B are accept-
sortment (see “Inheritance of Genetic Traits” able. A genotype may also be written as the
section below). italicized antigen, eg, Fya or Jkb when the geno-
Cytology studies, toward the end of the type has been inferred from testing by hemag-
19th century showed that each living cell has a glutination.
characteristic set of chromosomes in the nu- Chromosomes are the gene-carrying
cleus. In the early part of the 20th century, it structures that are visible during nuclear divi-
was realized that chromosomes carry genes. sion in the nucleus of the cell; they contain the
Biochemical studies showed that the chromo- genetic material (DNA) necessary to maintain
somal material is primarily made up of nucleic the life of the cell and the organism. A human
acids and associated proteins.1,2 somatic cell contains 46 chromosomes that
Many excellent texts offer a greater in- make up 23 pairs; each pair has one paternally
sight into classical genetics.4 The fundamental and one maternally derived chromosome. In
principles of genetics outlined in this chapter males and females, 22 of the pairs are homolo-
are intended to serve as a review of inheritance gous chromosomes (a pair of chromosomes in
and expression of blood group antigens. which males and females carry equivalent
genes) and are referred to as the autosomes
Genes (Alleles) and Chromosomes (any chromosome that is not a sex chromo-
some). The remaining pair is nonhomologous
A gene is a segment of deoxyribonucleic acid and consists of the sex chromosomes that de-
(DNA) that encodes a particular protein. A termine a person’s sex (gender). The sex-deter-
gene is the basic unit of inheritance of any mining chromosomes of the male are X and Y,
trait (defined as a genetically determined whereas females have two X chromosomes.
characteristic or condition), including blood The karyotype represents the chromosome
group antigens, that is passed from parents to complement of a person; this is written as
offspring. Genes are arranged on chromo- “46,XY” and “46,XX” for a normal male and fe-
somes with each gene occupying a specific lo- male, respectively. The hereditary information
cation known as the gene locus. A locus may carried by the chromosomes is passed from a
be occupied by one of several alternative parent cell to a daughter cell during somatic
forms of the gene called alleles. For example, cell division and from parents to offspring
the gene that encodes the protein carrying the (children) by the gametes during reproduc-
Jka antigen is an alternative form (allele) of the tion.
one that encodes the Jkb antigen. The terms Chromosomes are best studied during
“gene” and “allele” can be used interchange- cell division (mitosis; see “Mitosis” section be-
ably. Based on internationally accepted gene low) when they become discrete structures in
and allele terminology, the written gene or al- the nucleus and can be visualized by various
CHAPTER 11 Blood Group Genetics 䡲 257
long arm is numbered 1p1 or 1q1, respectively. chromosomes, which would be incompatible
With greater resolution, there is further with life.
distinction into subbands (eg, 1p11 and 1p12) Meiosis ensures genetic diversity through
that, in turn, can be subdivided (eg, 1p11.1 two mechanisms: independent assortment
and 1p11.2). Genes can be individually and crossing-over. Through independent as-
mapped to a specific band location (Fig 11-2), sortment, each daughter cell randomly re-
and the chromosomal location of the genes ceives either maternally or paternally derived
encoding the 34 red cell systems5-9 are shown homologous chromosomes. Chromosomal
in Table 11-1. crossing-over involves exchange of genetic
material between homologous chromosome
Cell Division pairs. Such shuffling of genetic material en-
sures diversity and produces genetically
As a cell divides, the chromosomes replicate unique gametes that fuse to produce a unique
and each daughter cell receives a full comple- zygote.
ment of genetic material. In somatic cells, this
occurs through mitosis; in reproductive cells a X Chromosome Inactivation
similar process called “meiosis” takes place. A (Lyonization)
feature common to both types of cell division
is that, before the start of the process, each Females have two copies of X-borne genes in
chromosome replicates to form two identical their somatic cells, while males have only one
daughter chromatids attached to each other copy of X-borne genes. Because most X-borne
through the centromere (Fig 11-1). genes do not have a homolog on the Y chro-
mosome, there is a potential imbalance in the
Mitosis dosage of X-borne genes between males and
females. This difference is compensated for by
Somatic cells divide for growth and repair by X chromosome inactivation, (also called ly-
mitosis (Fig 11-3). Through this process, a sin- onization), a process through which most of
gle cell gives rise to two daughter cells with the genes on one of the two X chromosomes in
identical sets of chromosomes. The daughter each female somatic cell are inactivated at a
cells, like the parent cell, are diploid (2N); that very early stage of embryonic development.17
is, they contain 46 chromosomes in 23 pairs It is a matter of chance whether the maternal
and have all the genetic information of the or paternal X chromosome is inactivated in
parent cell. any one cell, but once inactivation has oc-
curred, all descendants of that cell will have
Meiosis the same inactive X chromosome. Some X-
borne genes escape inactivation; the first gene
Meiosis occurs only in germ cells that are in- found to escape inactivation was XG, the gene
tended to become gametes (sperm and egg encoding the antigens of the Xg blood group
cells). Somatic cells are diploid (2N), whereas system. Like XG, most of the genes that escape
gametes are haploid [having half the chromo- inactivation are located on the extreme tip of
somal complement of somatic cells (1N)]. Mei- the short arm of the X chromosome, but sever-
osis is a process of cell division and replication al are clustered in regions on the short and
that leads to the formation of haploid gametes. long arms of the chromosome.18(pp359-370),19
During meiosis, diploid cells undergo DNA The XK gene, which encodes the Kx blood
replication, followed by two cycles of cell divi- group system, is the only other X-borne gene
sion to produce four haploid gametes (see Fig known to encode red cell antigens. Changes or
11-4). Because sperm and egg cells fuse at fer- deletions in XK result in McLeod phenotype
tilization, the gametes must be haploid. If each red cells that lack Kx antigen and have reduced
gamete carried a diploid (2N) set of 46 chro- expression of Kell antigens.20,21 The XK gene,
mosomes, the resulting zygote would have 92 unlike XG, is subject to X chromosome inacti-
CHAPTER 11 Blood Group Genetics 䡲 259
(Continued)
260 䡲 AABB TECHNICAL MANUAL
Dombrock (014) DO 12p12.3 Do glycoprotein; ART 4 Doa, Dob, Gya, Hy, Joa +
(ART4) [CD297] 3 more
[Gy(a–)]
Colton CO 7p14.3 Aquaporin 1 (AQP1) Coa, Cob, Co3, Co4
(015) (AQP1) [Co(a–b–)]
Landsteiner- LW 19p13.2 LW glycoprotein Intra- LWa, LWab, LWb
Wiener (ICAM4) cellular adhesion mole- [LW(a–b–)]
(016) cule 4 (ICAM4)
[CD242]
Chido/ Rodgers CH/RG (C4A, 6p21.32 Complement compo- Ch1, Ch2, Rg1 + 6
(017) C4B) nent: more
C4A; C4B [Ch–Rg–]
H H 19q13.33 Fucosyltransferase, H
(018) (FUT1) carbohydrate [CD173] [Bombay (Oh)]
Kx XK Xp21.1 XK glycoprotein Kx
(019) (XK) [McLeod phenotype]
Gerbich (020) GE 2q14.3 Glycophorin C (GPC) Ge2, Ge3, Ge4, and 8
(GYPC) [CD236] more
Glycophorin D (GPD) [Leach phenotype]
Cromer (021) CROM 1q32.2 DAF Cra, Tca, Tcb, Tcc, Dra,
(CD55) [CD55] Esa, IFC, and 11 more
[Inab phenotype]
Knops KN 1q32.2 CR1 Kna, Knb, McCa, Sla, Yka,
(022) (CR1) [CD35] and 4 more
Indian IN 11p13 Hermes antigen [CD44] Ina, Inb, and 2 more
(023) (CD44)
Ok OK 19p13.3 Neurothelin, basigin Oka, OKGV, OKGM
(024) (BSG) [CD147]
Raph RAPH (CD151) 11p15.5 Tetraspanin MER2
(025) [CD151] [Raph–]
JMH JMH 15q24.1 Semaphorin 7A JMH and 5 more
(026) (SEMA7A) [CD108] [JMH–]
I GCNT2 6p24.2 Glucosaminyltransfer- I
(027) (IGNT) ase, carbohydrate [I– or i adult]
Globoside (028) GLOB 3q26.1 Transferase, carbohy- P
(B3GALNT1) drate [P–]
(Gb4, globoside)
CHAPTER 11 Blood Group Genetics 䡲 261
vation, with the result that a female who is a Genotype and Phenotype
carrier (a person who carries one gene for a re-
The genotype of a person is the complement
cessive trait and one normal gene) of a gene
of genes inherited from his or her parents; the
that is responsible for the McLeod phenotype
can have a dual population of Kx– (McLeod term is frequently also used to refer to the set
phenotype) and Kx+ (non-McLeod) red cells. of alleles at a single gene locus. The phenotype
Flow cytometry, using selected Kell antibodies, is the observable expression of the genes in-
shows the weakening of Kell antigens on red herited by a person and reflects the biologic
cells of the McLeod phenotype and demon- activity of the gene(s). Thus, the presence or
strates two red cell populations in carrier fe- absence of antigens on the red cells, as deter-
males. This mixed-cell population reflects the mined by serologic testing, represents the phe-
randomness of whether the maternal or pater- notype. The presence or absence of antigens
nal X chromosome is inactivated in any single on the red cells predicted by DNA-based test-
somatic cell lineage. ing represents the genotype. Sometimes the
262 䡲 AABB TECHNICAL MANUAL
result in group A red cells, A/B would result in said to be “heterozygous.” For example, a per-
group AB red cells, B/B and B/O would result in son with K–k+ red cells is homozygous at the
group B red cells, and O/O would result in KEL locus for the allele (KEL*02) encoding the
group O red cells. k antigen. A person who is heterozygous for
When identical alleles for a given locus KEL*01 and KEL*02 (KEL*01/02 genotype),
are present on both chromosomes, the person would have red cells that are K+k+.
is said to be “homozygous” for the particular Antigens that are encoded by alleles at the
allele. A person who is hemizygous for an al- same locus are said to be “antithetical” (mean-
lele has only a single copy of an allele instead ing “opposite”); thus, K and k are a pair of anti-
of the customary two copies; an example is the thetical antigens. It is incorrect to refer to red
deletion of one RHD in a D-positive pheno- cells that are K–k+ or Kp(a–b+) as being homo-
type. When different (ie, not identical) alleles zygous for the k or Kpb antigen; rather, it
are present at a particular locus, the person is should be said that the cells have a double
264 䡲 AABB TECHNICAL MANUAL
dose of the antigen and that they are from a population had genetic uniformity. It is not yet
person who is homozygous for the allele. understood what, if any, evolutionary advan-
Genes are allelic whereas antigens are anti- tages were derived from the extensive poly-
thetical. morphism displayed by red cell antigens, but
The quantity of antigen expressed (anti- many publications associate resistance to, or
gen density) is influenced by whether a per- susceptibility for, a particular disease with a
son is heterozygous or homozygous for an al- particular blood type.22
lele; the antigen density is generally greater The difference between two alleles is the
when a person is homozygous. In some blood result of a permanent change in the DNA. An
group systems, this difference in antigen den- event that leads to the production of an altered
sity is manifested by antibodies giving stron- gene and a new allele or polymorphism that
ger reactions with cells that have a double did not exist in the biologic parents is referred
dose of the antigen. Red cells with the Jk(a+b–) to as a “mutation.” The mutation rate of ex-
phenotype, encoded by a JK*A/A genotype,
pressed genes resulting in a new phenotype
have a double dose of the Jka antigen and often
has been estimated to be less than 10-5 (<1 in
are more strongly reactive with anti-Jka than
100,000) in humans, and a mutation has to oc-
those that are Jk(a+b+) and have a single dose
cur in the germ cells (gametes) to be inherited.
of the antigen. Similarly, M+N– red cells tend
A mutation can occur spontaneously or
to be more strongly reactive with anti-M than
be brought about by agents such as radiation
are M+N+ red cells. Antibodies that are weakly
(eg, ultraviolet rays or x-rays) or chemicals. A
reactive may not be detected if they are tested
with red cells expressing a single dose of the mutation may occur within a gene or in the
antigen. This observable difference in strength intergenic regions. It may be silent—that is,
of reaction, based on homozygosity or hetero- have no effect on the encoded protein—or it
zygosity for an allele, is termed the “dosage ef- may alter the gene product and potentially
fect.” cause an observable effect in the phenotype.
In the context of an allele that encodes a pro-
Polymorphism tein that carries red cell antigens, any geneti-
cally induced change must be recognized by a
For blood group genetics, polymorphism re-
specific antibody before an allele can be said
fers to the occurrence in the population of ge-
to encode a new antigen.
nomes with allelic variation (two or more
Numerous genetic events that generate
alleles at one locus) producing different phe-
red cell antigens and phenotypes have been
notypes, each with appreciable (greater than
identified. The event can occur at the level of
1%) frequency. Some blood group systems (Rh
the chromosome (eg, deletion or transloca-
and MNS for example) are highly polymorphic
tion of part of a chromosome), the gene (eg,
and have many more alleles at a given locus
than other systems such as Duffy, and Colton.5 deletion, conversion, or rearrangement), the
An allele that is polymorphic in one popula- exon (eg, deletion or duplication), or the nu-
tion is not necessarily polymorphic in all pop- cleotide (eg, deletion, substitution, or inser-
ulations; for example, the FY allele associated tion). The mechanism that has given rise to
with silencing of Fyb in red cells (FY*02N.01) is most diversity in the human genome is single
polymorphic in populations of African ethnici- nucleotide polymorphism (SNP)—that is, a
ty with a prevalence of greater than 70%, but single nucleotide change in the DNA.23-25 Ac-
this allele is not found in other populations. A cordingly, the majority of polymorphic blood
gene polymorphism may represent an evolu- group antigens are the result of SNPs.3,26,27 DNA
tionary advantage for a population, and a analysis to predict the red cell phenotype is
polymorphic population is likely to adapt to discussed in more detail in the section on
evolutionary change more rapidly than if the “Blood Group Genomics.”
CHAPTER 11 Blood Group Genetics 䡲 265
INHERITANCE OF GENETIC
TR AI TS
A genetic trait is the observed expression of
one or more genes. The inheritance of a trait
(and red cell antigens) is determined by
whether the gene responsible is located on an
autosome or on the X chromosome (sex-
linked) and whether the trait is dominant or
recessive.
FIGURE 11-7. Autosomal dominant inheritance of the ABO alleles. Based on the ABO groups of his children, I-1
would be expected to have a B/O rather than a B/B genotype (showing that the B allele is dominant over O)
because two of his children (II-6 and II-7) are group O and must have inherited an O allele from their father (I-1)
in addition to the O allele inherited from their mother (I-2). Similarly, II-2 and II-3 are B/O, based on the ABO
type of their children, showing the dominance of B over O.
CHAPTER 11 Blood Group Genetics 䡲 267
FIGURE 11-8. Autosomal recessive inheritance. The offspring of II-3, the Lu(a–b–) proband, and II-4, his
Lu(a+b+) wife, demonstrate that Lu is recessive to Lua and Lub and that the presence of the silent Lu allele is
masked by the product of Lua or Lub at the phenotype level.
268 䡲 AABB TECHNICAL MANUAL
expressed by all males who carry the gene for and is inherited in a sex-linked dominant
the trait. The most striking feature of both manner. The first indication that the Xga anti-
dominant and recessive X-linked inheritance gen is X-borne came from the observation that
is that there is no male-to-male transmission; the prevalence of the Xg(a–) and Xg(a+) phe-
that is, the trait is never transmitted from fa- notypes differed noticeably between males
ther to son. and females; the Xga antigen has a prevalence
of 89% in females and only 66% in males.5
Sex-Linked Dominant Inheritance Figure 11-9 shows the inheritance of the
Xga antigen in a three-generation family. In
A trait encoded by an X-borne allele that has
generation I, the father (I-1) is Xg(a+) and has
sex-linked dominant inheritance is expressed
transmitted Xga to all his daughters but to
by hemizygous males and by both heterozy-
none of his sons. His eldest daughter (II-2), for
gous and homozygous females. A male passes
example, must be heterozygous for Xga/Xg; she
his single X chromosome to all of his daugh-
received the allele encoding the Xga antigen
ters and all daughters will express the condi-
from her Xg(a+) father and a silent allele, Xg,
tion or trait. When a female is heterozygous for
from her Xg(a–) mother. II-2 has transmitted
an allele that encodes a dominant trait, each of
Xga to half her children, regardless of whether
her children, whether male or female, has a
they are sons or daughters.
50% chance of inheriting the trait. When a fe-
male is homozygous for an X-borne allele with
Sex-Linked Recessive Inheritance
dominant inheritance, the encoded trait is ex-
pressed by all her children. A trait encoded by an X-borne recessive allele
The Xga antigen (Xg blood group system) is carried, but not expressed, by a heterozy-
is encoded by an allele on the X chromosome gous female. A male inherits the trait from his
FIGURE 11-9. Sex-linked dominant inheritance. The Xga antigen is encoded by an allele on the tip of the short
arm of the X chromosome. This family demonstrates the sex-linked dominant inheritance of the Xga antigen.
CHAPTER 11 Blood Group Genetics 䡲 269
mother, who is usually a carrier (or could be Mutations in XK result in red cells with the
homozygous for the trait if she is the offspring McLeod phenotype; such red cells lack Kx and
of a male who expresses the trait and a carrier have reduced expression of Kell antigens
female). An affected male transmits the trait to (McLeod syndrome). McLeod syndrome is as-
all of his daughters, who in turn transmit the sociated with late-onset clinical or subclinical
trait to approximately half of their sons. There- myopathy, neurodegeneration, and central
fore, the prevalence of the expression of an X- nervous system manifestations as well as with
borne recessive trait is much higher in males acanthocytosis and, frequently, compensated
than in females. A carrier female who mates
hemolytic anemia. More than 30 different XK
with a male lacking the trait transmits the trait
gene mutations associated with a McLeod
to one-half of her daughters (who will also be
phenotype have been found. Different XK
carriers) and to one-half of her sons (who will
be affected). If mating is between an affected mutations appear to have different clinical ef-
male and a female who lacks the trait, all of the fects and may account for the variability in the
sons will lack the trait and all of the daughters prognosis.28 Sequencing of XK to determine
will be carriers. If an X-borne recessive trait is the specific type of mutation in individuals
rare in the population, the trait is expressed al- with McLeod phenotypes has clinical prognos-
most exclusively in males. tic value. McLeod syndrome is an X-linked re-
The XK gene encodes the Kx protein and cessive condition and, as demonstrated by the
demonstrates X-borne recessive inheritance. family in Fig 11-10, is found only in males.
FIGURE 11-10. Sex-linked recessive inheritance. This family demonstrates that a sex-linked trait that is
recessive in females will be expressed by any male who inherits the trait. Homozygosity for such a trait is
required for it to be expressed in females. The trait skips one generation and is carried through females.
270 䡲 AABB TECHNICAL MANUAL
The Principles of Independent ing B antigens) does not influence the inheri-
Segregation and Independent tance of another allele (eg, an M allele, on
Assortment chromosome 4, encoding M antigens). This is
demonstrated by the family in Fig 11-11.
The passing of a trait from one generation to
the next follows certain patterns or principles. Linkage and Crossing-Over
The principle of independent segregation re-
fers to the separation of homologous chromo- Linkage is the physical association between
somes and their random distribution to the two genes that are located on the same chro-
gametes during meiosis. Only one member of mosome and are inherited together. Examples
an allelic pair is passed on to the next genera- include RHD and RHCE encoding the antigens
tion, and each gamete has an equal probability of the Rh system, which are both on chromo-
of receiving either member of a parental ho- some 1 and are linked loci that do not assort
mologous allelic pair; these chromosomes are independently.
randomly united at fertilization and thus seg- Crossing-over is the exchange of genetic
regate independently from one generation to material between homologous chromosome
the next. The family in Fig 11-11 demonstrates pairs (Fig 11-4). In this process, a segment
the independent segregation of the ABO alleles from one chromatid (and any associated
on chromosome 9. genes) changes places with the corresponding
The principle of independent assort- part of the other chromatid (and its associated
ment states that alleles determining various genes); the segments are rejoined, and some
traits are inherited independently from each genes will have switched chromosomes. Thus,
other. In other words, the inheritance of one crossing-over is a means to shuffle genetic ma-
allele (eg, a B allele, on chromosome 9, encod- terial. Because crossing-over can result in new
FIGURE 11-11. Independent segregation and independent assortment are illustrated by the inheritance of blood
group alleles in one family. Parental ABO alleles were randomly transmitted (independent segregation), and each
child has inherited a different combination. The family also illustrates that the alleles encoding antigens of the
ABO and MNS blood group systems are inherited independently from each other.
CHAPTER 11 Blood Group Genetics 䡲 271
gene combinations on the chromosomes in- first recognized example of autosomal linkage
volved, it is also referred to as recombination, in humans and is explained in Fig 11-13.
and the rearranged chromosomes can be re- Although crossing-over occurs readily be-
ferred to as recombinants. Crossing-over and tween distant genes, rare examples of recom-
recombination, using chromosome 1 as an ex- bination have been documented for genes that
ample, are explained in Fig 11-12. are very closely linked or adjacent on a chro-
Two gene loci carried by the same chro- mosome. Such genes include those encoding
mosome that are not closely linked are re- the MN (GYPA) and Ss (GYPB) antigens on
ferred to as being syntenic. For example, the chromosome 4 and are reviewed by Dan-
loci for RH and FY, both located on chromo- iels.18(pp96-142)
some 1, are syntenic because the distance be-
tween them (RH on the short arm and FY on
Linkage Disequilibrium
the long arm) is great enough for them to un-
dergo crossing-over and to assort indepen- Genes at closely linked loci tend to be inherit-
dently. ed together and constitute a haplotype (a
The frequency of crossing-over involving combination of alleles at two or more closely
two genes on the same chromosome is a mea- linked loci on the same chromosome). The al-
sure of the distance [measured in centimor- leles encoding the MNS antigens are inherited
gans (cM)] between them; the greater the dis- as four haplotypes: MS, Ms, NS, or Ns. Because
tance between two loci, the greater the linked genes do not assort independently, the
probability that crossing-over (and recombi- antigens encoded by each of these haplotypes
nation) will occur. In contrast, genes located have a different prevalence in the population
very close together (linked) tend to be trans-
than would be expected by random assort-
mitted together with no recombination. The
ment. If M and S were not linked, the expected
degree of crossing-over between two genes
prevalence for M+ and S+ in the population
can be calculated by analyzing pedigrees of
families informative for the genes of interest would be 17% (from frequency calculations),
and observing the extent of recombina- whereas the actual or observed prevalence
tion.The traditional method of linkage analysis (obtained from testing and analyzing families)
requires the use of lod (logarithm of the odds) of the MS haplotype is 24%.18(pp96-142) This con-
scores.29 Linkage analysis was the basis stitutes linkage disequilibrium, which is the
through which chromosomes were mapped tendency of specific combinations of alleles at
and the relative position and distance between two or more linked loci to be inherited togeth-
genes established. Linkage between Lutheran er more frequently than would be expected by
(LU) and ABH secretion (SE or FUT2) was the chance.
FIGURE 11-12. Crossing-over and recombination. In the diagram, chromosome 1 is used as an example. The
very closely linked RH genes, RHD and RHCE, are located near the tip of the short arm of chromosome 1. The
loci for FY and KN are on the long arm of the chromosome and are not linked. During meiosis, crossing-over
occurs between this homologous chromosome pair, and portions of the chromosome break and become
rejoined to the partner chromosome. Crossing-over of the long arm of chromosome 1 results in recombination
between the loci for FY and KN such that the gene encoding the Fyb antigen now travels with a gene that encodes
the Sl(a–) phenotype of the Knops system.
272 䡲 AABB TECHNICAL MANUAL
FIGURE 11-13. Linkage between LU and SE. I-2 is homozygous for Lub and se and must transmit these alleles to
all her offspring. I-1 is doubly heterozygous (Lua/Lub and Se/se). He has transmitted Lub with Se and Lua with se,
showing linkage between Lu and Se. Several such informative families would need to be analyzed to statistically
confirm linkage.
Gene Interaction and Position Effect DCe is the haplotype, and the RHD allele is in
cis to the RHCE*Ce allele.
Alleles that are carried on the same chromo-
The expression of red cell antigens may
some are referred to as being in cis position be modified or affected by gene or protein
whereas those on opposite chromosomes of a interactions that manifest primarily as re-
homologous pair are in trans position. Alleles duced antigen expression. One example in
that are in cis and linked are always inherited which the haplotype on one chromosome af-
together on the same chromosome, whereas fects the expression of the haplotype on the
genes in trans segregate independently. paired chromosome is commonly referred to
Historically, the Rh blood group system as the “position effect” and can be observed
was used to explain the meaning of cis and with Rh antigen expression. When a Ce haplo-
trans. For example, the DCe/DcE genotype was type (note the absence of RHD) is in trans to a
described as having C and e alleles in cis in the D antigen-encoding haplotype, the expression
DCe haplotype, with c and E alleles in cis in the of D is dramatically reduced and a weak D
partner DcE haplotype, whereas C and E and phenotype can result. When the same D-en-
also c and e are in opposing haplotypes and are coding haplotype is inherited with either ce or
in trans. In this alignment, C and e, for exam- cE, D antigen is normally expressed. The cause
of this reduced antigen expression is not
ple, are always inherited together, but C and E
known, but may involve differences in gene
are not. The preceding explanation, which im-
expression levels or altered assembly of pro-
plies that one gene encodes C and c antigens
teins in the membrane. In the presence of the
while another linked gene encodes E and e an- Kell system antigen Kpa, expression of other
tigens, was based on the Fisher-Race theory of Kell system antigens encoded by the same al-
three genes at the RH locus. In contrast, mo- lele is suppressed to varying degrees (cis-mod-
lecular analysis indicates that only one gene ifier effect). This is best observed in persons
(RHCE), with four alleles (RHCE*Ce, RHCE*cE, who have a silenced K0 (a Kellnull gene) in trans.
RHCE*ce, and RHCE*CE), encodes one protein The amino acid change that results in the ex-
that carries the CcEe antigens. Thus, for Rh, pression of Kpa adversely affects trafficking of
CHAPTER 11 Blood Group Genetics 䡲 273
the Kell glycoprotein to the red cell surface, so the high-prevalence antigen Wrb to the Diego
that the quantity of Kpa-carrying Kell glycopro- blood group system took many years because
tein that reaches the red cell surface is greatly the only red cells that lacked Wrb were rare
reduced. MNS null phenotypes [En(a–) and MkMk] and
Suppressor or modifier genes affect the variants, yet Wra, the antigen that is antitheti-
expression of another gene, or genes. For ex- cal to Wrb was clearly independent of MNS.
ample, KLF1, located on chromosome This anomaly was resolved when it was under-
19p13.3-p13.12, encodes erythroid Krüppel- stood that GPA (or more precisely, amino acids
like factor, which is a transcription factor es- 75 to 99), which carries MN antigens, must be
sential for terminal differentiation of red cells. present in the red cell membrane for Wrb ex-
Singleton et al30 first discovered that heterozy- pression. The Wra/Wrb polymorphism is en-
gosity for nucleotide changes in KLF1 is re- coded by DI and carried on band 3, whereas
sponsible for the dominant Lu(a–b–) pheno- GPA is the product of GYPA, a gene that is in-
type,5 which is also known as the In(Lu) dependent of DI. An absence of RhD and RhCE
phenotype. This heterozygosity is character- protein (Rhnull) results in red cells that lack LW
ized by reduced expression of antigens in the antigens and lack or have reduced expression
Lutheran system (Lumod) and for P1, Inb and of U and S or s antigens, again demonstrating
AnWj antigens. the interaction in the membrane of the prod-
In kind, the transcription factor GATA-1, ucts of two or more independent blood group
encoded by the X-borne GATA-1 gene, is es- genes.
sential for erythroid and megakaryocyte differ- The sequential interaction of genes at
entiation. Changes in this gene were associat- several loci is required for the expression of
ed with the X-linked type of Lumod that initially ABO, H, Lewis, and I antigens on red cells and
presented as an Lu(a–b–) phenotype.31 Sero- in secretions. These antigens are carbohydrate
logic differentiation of these Lumod phenotypes determinants carried on glycoproteins or gly-
from the true Lu(a–b–) (Lunull) phenotype can colipids, and the genetics of these antigens is
be challenging, yet has clinical value. Now that more complex than that of protein-based anti-
the molecular basis of these phenotypes is un- gens. The carbohydrate antigens are carried
derstood, sequencing of the relevant genes can on oligosaccharide chains that are assembled
be used to make the distinction. by the stepwise addition of monosaccharides.
In the past, independent, unidentified ABO genes and the genes encoding the other
modifier or regulator genes were postulated to carbohydrate-based antigens do not encode
be the basis of several null or variant pheno- membrane proteins but do encode an enzyme,
types when a silent or inactive (nonfunction- a glycosyltransferase, that catalyzes the se-
ing) gene was not evident. For example, the quential transfer of the appropriate immuno-
regulator type (as opposed to the amorph dominant monosaccharide. Each monosac-
type) of Rhnull was shown through family stud- charide structure is transferred by a separate
ies to result from a gene not at the RH locus. glycosyltransferase, such that two genes are re-
This phenotype is now known to result from quired for a disaccharide, three for a trisaccha-
various silencing changes in RHAG, a gene lo- ride, and so on. An inactivating change at one
cated on chromosome 6 (and thus indepen- locus can prevent or modify the expression of
dent of RH). RHAG encodes the Rh-associated the other gene products. The product encoded
glycoprotein RhAG, which is required in the by the H gene is the biosynthetic precursor for
red cell membrane for Rh antigen expression. A and B antigen production; if the H gene is si-
Similarly, molecular analysis indicates that the lenced, A or B antigens cannot be produced. A
modifying gene that causes the Rhmod pheno- mutated A or B allele may result in a glycosyl-
type is a mutated RHAG.5 transferase that is inactive or that causes more
Several red cell antigens require the inter- or less antigen to be expressed. Details on the
action of the products of two or more indepen- biosynthesis of the ABO, H, Lewis, and I anti-
dent genes for their expression. Assignment of gens may be found in Chapter 12.
274 䡲 AABB TECHNICAL MANUAL
Allele (Gene) Frequency blood group genetics, and its use is demon-
strated below. In a population of European
The allele frequency is the proportion of one
ethnicity, the frequencies of the two alleles en-
allele relative to all alleles at a particular gene
coding K or k can be determined as follows:
locus in a given population at a given time.
This frequency can be calculated from the Frequency of the K allele = p
prevalence of each phenotype observed in a Frequency of the k allele = q
population. The sum of allele frequencies at Frequency of the KK genotype = p2
any given locus must equal 100% (or 1 in an al- Frequency of the Kk genotype = 2pq
gebraic calculation) in the population sample
Frequency of the kk genotype = q2
tested. The genotype frequency in a popula-
tion is the number of individuals with a given The K antigen is expressed on the red cells of
genotype divided by the total number of indi- 9% of people of European ethnicity; there-
viduals in population. fore:
p2 + 2pq = the frequency of people who carry K
and are K+
The Hardy-Weinberg Equilibrium
Thus, p2 + 2pq = 0.09
Gene frequencies tend to remain constant
from generation to generation in any relatively q2 = 1 – (p2 + 2pq) = the frequency of people
large population unless they are influenced by who carry kk and are K–
factors such as selection, mutation, migration, q2 = 1 – 0.09
or nonrandom mating, any of which would
q2 = 0.91
have to be rampant to have a discernible ef-
fect. According to the principles proposed by q= 0.91
the British mathematician Hardy and the Ger-
q = 0.95 = the frequency of k
man physician Weinberg, gene frequencies
reach equilibrium. This equilibrium can be ex-
pressed in algebraic terms by the Hardy-Wein- Because the sum of the frequencies of both al-
berg formula or equation: leles must equal 1.00:
p+q=1
p2 + 2pq + q2 = 1 p=1–q
If two alleles, classically referred to as A p = 1 – 0.95
and a, have gene frequencies of p and q, the p = 0.05 = the frequency of K
homozygotes and heterozygotes are present in
the population in the following proportions:
Once the allele frequencies for K and k have
been calculated, it is possible to calculate the
AA = p2 Aa = 2pq aa = q2 percentage of k+ (both K+k+ and K–k+) and K+
(both K+k– and K+k+) people:
In such a two-allele system, if the gene
frequency for one allele, say p, is known, q can Prevalence of k+ = 2pq + q2
be calculated by p + q = 1. = 2(0.05 × 0.95) + (0.95)2
The Hardy-Weinberg equation permits
the estimation of genotype frequencies from = 0.9975 × 100
the phenotype prevalence in a sampled popu- = a calculated preva-
lation and, reciprocally, allows the determina- lence of 99.75% (the
tion of genotype frequency and phenotype observed prevalence
prevalence from the gene frequency. The of the k+ phenotype is
equation has a number of applications in 99.8%)
276 䡲 AABB TECHNICAL MANUAL
TABLE 11-2. Allele Frequencies of K and k Calculated Using Direct Counting (assuming the absence
of null alleles)
K+k– 2 4 4
K+k+ 88 176 88 88
K–k+ 910 1820 0 1820
Totals 1000 2000 92 1908
Allele frequency 0.046 0.954
A random sample of 1000 people tested for K and k antigens, have a total of 2000 alleles at the Kk locus
because each person inherits two alleles, one from each parent. Therefore, the two persons with a K+k– phe-
notype (each with two alleles) contribute a total of four alleles. To this are added 88 K alleles from the K+k+
group, for a total of 92 K alleles or an allele frequency of 0.046 (92 ÷ 2000). The frequency of the k allele is
0.954 (1908 ÷ 2000).
CHAPTER 11 Blood Group Genetics 䡲 277
greatest number of alleles (greatest polymor- population genetics but also for monitoring
phism) have the highest power of discrimina- chimerism after marrow transplantation.37,38
tion and are the most useful for determining STR analysis also has been used to monitor pa-
relationships. Blood is a rich source of inherit- tients for graft-vs-host disease after organ
ed characteristics that can be detected, includ- transplantation, particularly after a liver trans-
ing red cell, HLA, and platelet antigens. Red plant.39
cell and HLA antigens are easily identifiable, In a case of disputed paternity, if an al-
are polymorphic, and follow Mendelian laws leged father cannot be excluded from paterni-
of inheritance. The greater the polymorphism ty, the probability of his paternity can be
of a system, the less chance there is of finding calculated. The calculation compares the
two people who are identical. The extensive probability that the alleged father transmitted
polymorphism of the HLA system alone allows the paternal obligatory genes with the proba-
the exclusion of over 90% of falsely accused bility that any other randomly selected man
men in cases of disputed paternity. from the same racial or ethnic group transmit-
Serologic methods of identity testing have ted the genes. The result is expressed as a like-
been surpassed and replaced by DNA-based lihood ratio (paternity index) or as a percent-
assays32 (referred to as DNA fingerprinting, age. The AABB has developed standards and
DNA profiling, or DNA typing) that were pio- guidance documents for laboratories that per-
neered by Jeffreys and colleagues.33,34 Tandem- form relationship testing.40
ly repeated sequences of DNA of varying
lengths occur predominantly in the noncoding
B LO OD G RO U P G E N E M A P PI N G
genomic DNA, and they are classified into
groups depending on the size of the repeat re- Gene mapping is the process through which a
gion. The extensive variation of these tandem- gene locus is assigned to a location on a chro-
ly repeated sequences between individuals mosome. The initial mapping of blood group
makes it unlikely for the same number of re- genes was accomplished by testing many fam-
peats to be shared by two individuals, even if ilies for selected red cell antigens. Pedigrees
these individuals are related. Minisatellite were analyzed for evidence of recombination
[also referred to as variable number of tandem between the genes of interest to rule out or es-
repeats (VNTR)] loci have tandem repeat units tablish linkage of a blood group with another
of nine to 80 base pairs, whereas microsatellite marker, with a known chromosomal location.
[also referred to as short tandem repeat [STR)] The gene encoding the antigens of the
loci consist of two to five base pair tandem re- Duffy blood group system was the first to be
peats.35 Microsatellites and minisatellites are assigned to a chromosome, by showing that
reviewed by Bennett.36 the gene is linked to an inherited deformity of
Assays for VNTR and STR sequences in- chromosome 1. More recently, recombinant
volve the electrophoretic separation of DNA DNA methods were used to establish the phys-
fragments according to size. DNA profiling in- ical locations of genes, and today, with se-
volves amplification of selected, informative quencing of the human genome, determining
VNTR and STR loci using locus-specific oligo- the location of a gene involves a computer da-
nucleotide primers with the subsequent mea- tabase sequence search. The Human Genome
surement of the size of the PCR products. Project (http://www.ornl.gov/sci/tech resourc
Hundreds of STR loci have been mapped es/Human_Genome/home.shtml) has result-
throughout the human genome, and many ed in construction of a physical gene map in-
have been applied to identity testing. Analysis dicating the position of gene loci and the dis-
of different STR loci (usually at least 12) is used tance between loci is expressed by the number
to generate a person’s DNA profile that is virtu- of base pairs of DNA.
ally guaranteed to be unique to that person (or Currently, 34 blood group systems are rec-
to two identical twins). DNA fingerprinting is a ognized by the ISBT.6 The genes for all of them
powerful tool not only for identity testing and have been cloned and assigned to their respec-
278 䡲 AABB TECHNICAL MANUAL
tive chromosomes (see Table 11-1). Tradition- have two distinct populations of cells (red cells
ally, genes were mapped to chromosomes ac- and leukocytes), that of their true genetic type
cording to the metaphase banding patterns and that of their twin. The percentage of the
produced through staining with Giemsa (G two cell lines in each twin tends to vary; the
banding) or quinacrine (Q banding). Other major cell line is not necessarily the autolo-
methods used in chromosome mapping have gous cell line, and the proportions of the two
included deletion mapping (partial or total cell lines may change throughout life. Chime-
loss of a chromosome related to the presence ric twins have immune tolerance; they do not
or absence of a gene), somatic cell hybridiza- make antibody against the A or B antigens that
tion (based on the fact that human-rodent hy- are absent from their own red cells but are
brid cell lines randomly shed human chromo- present on the cells of the engrafted twin. This
somes, permitting the association of a trait tolerance extends beyond red cells to negative
with the presence or absence of a chromo- mixed lymphocyte cultures and the mutual ac-
some), in-situ hybridization (use of fluores- ceptance of skin grafts.
cent DNA probes with a preparation of intact In twin chimeras, the dual cell popula-
chromosomes), and chromosome walking (a tion is strictly confined to blood cells. Tetraga-
technique to clone a gene from its known clos- metic or dispermic chimeras present chime-
est markers). Details on gene mapping proce- rism in all tissues and are more frequently
dures are beyond the scope of this chapter, but identified because of infertility than because
reviews are available.7 of mixed populations of red cells. The mecha-
Advances in genetic technology and in- nism(s) leading to the development of tetraga-
formation generated through the Human Ge- metic chimeras are unknown, but they arise
nome Project make it feasible to construct a through the fertilization of two maternal nu-
physical gene map of the absolute positions of clei by two sperm followed by fusion of the two
gene loci in which the distance between loci is zygotes and development into one person
expressed by the number of base pairs of DNA. containing two cell lineages.
More commonly, chimeras occur through
medical intervention and arise from the trans-
CHIMERISM
fer of actively dividing cells, such as through
The observation that a sample gives mixed- hematopoietic transplantation.41 However,
field agglutination is not unusual in a labora- chimerism may be more prevalent than once
tory. Often this is the result of artificially in- thought. DNA analysis to confirm the Rh-neg-
duced chimerism through the transfusion of ative status of Northern European blood do-
donor red cells or a stem cell transplant. On nors identified a donor with a dual red cell
rare occasions, the observation of mixed-field population of which 95% was Rh negative and
agglutination identifies a true chimera, that is, 5% was Rh positive. The donor was confirmed
a person with a dual population of cells de- to be a chimera. Chimerism was found to be
rived from more than one zygote. Indeed, the the cause of a discrepancy observed when
first example of a human chimera was a female ABO was determined, and news headlines
blood donor discovered through mixed-field were made by a case of disputed maternity
agglutination during antigen typing. Most hu- when a woman was falsely excluded as the
man chimeras can be classified as either twin mother of her children because of chime-
chimeras or tetragametic (dispermic) chime- rism.42,43
ras. Chimerism is not a hereditary condition.41
Twin chimerism occurs through the for-
B LO OD G RO U P T E R M I N OLO G Y
mation of placental blood vessel anastomo-
ses, which results in the mixing of blood be- Antigens were originally named using an al-
tween two fetuses. This vascular bridge allows phabetical (eg, A/B, C/c) notation or they were
hematopoietic stem cells to migrate to the named after the proband whose red cells car-
marrow of the opposite twin. Each twin may ried the antigen or who made the first known
CHAPTER 11 Blood Group Genetics 䡲 279
antibody (eg, Duclos). A symbol with a super- Thus, 001001 identifies the A antigen and
script letter (eg, Lua, Lub; Jka, Jkb) was used, and 004001 identifies the D antigen. Alternatively,
a numerical (eg, Fy3, Jk3, Rh32) terminology the sinistral zeros may be omitted so that the A
was introduced. In blood group systems, anti- antigen becomes 1.1 and the D antigen be-
gens are named using more than one scheme comes 4.1. Each system also has an alphabeti-
(eg, the Kell blood group system: K, k, Jsa, Jsb, cal abbreviation (Table 11-1); thus KEL is the
K11, K17, TOU). ISBT symbol for the Kell system, the Rh ISBT
In 1980, the ISBT established its Working system symbol is RH, and an alternative name
Party on Terminology for Red Cell Surface An- for the D antigen is RH1. This alphanumeric
tigens. The working party was charged to de- terminology, which was designed primarily for
velop a uniform nomenclature that would be computer use, is not ideal for everyday com-
“both eye and machine readable” and “in munication. To achieve uniformity, a recom-
keeping with the genetic basis of blood mended list of user-friendly alternative names
groups.” A blood group system consists of one was compiled.45
or more antigens under the control of a single The ISBT working party meets periodical-
gene locus or of two or more homologous ly to assign names and numbers to newly
genes that are so closely linked that virtually discovered antigens. For terminology criteria;
no recombination occurs between them. tables listing the systems, antigens, and phe-
Thus, each blood group system is genetically notypes; and other information see ISBT Red
independent from every other blood group Cell Immunogenetics and Blood Group Termi-
system. The failure of an antibody to be reac-
nology web resources.6 A comprehensive re-
tive with red cells of a particular null pheno-
view of the terminology and its usage is found
type is not sufficient for assignment of the cor-
in Garratty et al.45
responding antigen to a system. Some null
The working party is charged to develop,
phenotypes are the result of inhibitor or modi-
maintain, and monitor a terminology for
fying genes that may suppress the expression
blood group genes and their alleles.6 The ter-
of antigens from more than one system [eg,
minology takes into account the guidelines for
the Rhnull phenotype lacks not only Rh anti-
human gene nomenclature published by the
gens but also LW system antigens, Fy5 antigen
Human Genome Organization (HUGO), which
(Duffy system), and sometimes U (MNS sys-
tem) antigen]. Similarly, a blood group antigen is responsible for naming genes based on the
must be shown to be inherited through family International System for Human Gene No-
studies, or the expression of the antigen must menclature.46 For information regarding the
be demonstrated to be associated with a varia- current status of gene and allele terminology
tion in the nucleotide sequence of the gene see ISBT Red Cell Immunogenetics and Blood
controlling the system, to be assigned antigen Group Terminology web resources.6 An exam-
status by the ISBT terminology working party. ple of the traditional and current ISBT termi-
A blood group antigen must be defined sero- nology as it applies to alleles, genotypes, phe-
logically by an antibody; a polymorphism that notypes, and antigens is shown in Table 11-3.
is detectable only by DNA analysis and for
which there is no corresponding antibody can- B LO OD G RO U P G E NO M I C S
not be called a blood group antigen.
The working party established a terminol- As discussed in earlier sections of this chapter,
ogy consisting of uppercase letters and Arabic the antigens expressed on red cells are the
numerals to represent blood group systems products of genes and can be detected directly
and antigens.6,44 Each system can also be iden- by hemagglutination techniques (as long as
tified by a set of numbers (eg, ABO system = relevant antisera are available). Their detec-
001; Rh system = 004). Similarly, each antigen tion is an important aspect of the practice of
in the system is assigned a number (eg, A anti- transfusion medicine because an antigen can,
gen = 001; B antigen = 002; D antigen = 001). if it is introduced into the circulation of an
280 䡲 AABB TECHNICAL MANUAL
individual who lacks that antigen, elicit an im- sulting proteins differ by one amino acid, me-
mune response. thionine at residue 48 for S and threonine for s
It is the antibody from such an immune (designated c.143T>C p.Met48Thr). As a re-
response that causes problems in clinical sult, assay design and interpretation are fairly
practice, such as patient/donor blood transfu- straightforward for the prediction of most phe-
sion incompatibility, maternal-fetal incompat- notypes.
ibility, and the reason why antigen-negative However, detailed serologic and molecu-
blood is required for safe transfusion in these lar studies have shown that there are far more
patients. Hemagglutination is simple, quick, alleles than phenotypes for some systems, es-
and relatively inexpensive. When carried out pecially ABO and Rh. More than 100 different
correctly, it has a specificity and sensitivity alleles encoding the glycosyltransferases re-
that is appropriate for most testing. However, sponsible for the four ABO types have been
hemagglutination has limitations; for exam- identified, and a single nucleotide change in
ple, it is difficult and often impossible to ob- an A or B allele can result in an inactive trans-
tain an accurate phenotype for a recently ferase and a group O phenotype (see Chapter
transfused patient or to type red cells that are 12). Testing for the common Rh antigens D,
coated with IgG, and some typing reagents are C/c, and E/e is uncomplicated for most popu-
in short supply or not available. Because the lations, but antigen expression is more com-
genes encoding the 34 known blood group sys- plex in some ethnic groups. There are more
tems have been cloned and sequenced and the than 200 RHD alleles encoding weak D or par-
molecular bases of most blood group antigens tial D phenotypes, and more than 100 RHCE
and phenotypes are known, DNA-based meth- alleles encoding altered, or novel, hybrid Rh
ods (genotyping) are increasingly being used proteins, some of which result in weakened
as an indirect method to predict a blood group antigen expression (see Chapter 13). RH geno-
phenotype. This approach has introduced typing, particularly in minority populations,
blood group genomics, often referred to as requires sampling of multiple regions of the
“molecular immunohematology,” into the gene(s) and algorithms for interpretation.
practice of transfusion medicine. Prediction of The basis of DNA assays is the amplifica-
a blood group antigen by testing DNA is sim- tion of a target gene sequence through the
ple and reliable for the majority of antigens be- polymerase chain reaction (PCR), followed by
cause most result from SNPs that are inherited manual, semi-automated, or automated
in a straightforward Mendelian manner. For downstream analysis. Commonly used meth-
example, the antithetical antigens S and s arise ods are sequence-specific PCR (SSP-PCR) and
from GYPB alleles that differ by one nucleo- allele-specific PCR (AS-PCR). For manual
tide—143T for S and 143C for s—and the re- methods, gel electrophoresis is used to sepa-
CHAPTER 11 Blood Group Genetics 䡲 281
rate the PCR products for fragment size deter- DNA-Based Assays to Predict the Red
mination. As an alternative, the assay may in- Cell Phenotype: Recently Transfused
clude digestion of the PCR products with a Patients
restriction fragment length polymorphism
(RFLP), followed by electrophoresis and visu- In patients receiving chronic or massive trans-
alization of the fragments. Semi-automated fusions, the presence of donor red cells often
approaches include real-time PCR using fluo- makes typing by hemagglutination inaccurate.
rescent probes with quantitative and qualita- Time-consuming and cumbersome cell sepa-
tive automated read-out. Manual methods are ration methods that are often unsuccessful in
labor-intensive, and each assay is performed isolating and typing the patient’s reticulocytes
separately on each sample. Automated DNA can be avoided when DNA typing is used. PCR-
arrays allow for higher throughput with a larg- based assays mostly use DNA extracted from
er number of target alleles in the PCR reaction, WBCs isolated from a sample of peripheral
which makes possible the determination of blood. Interference from donor-derived DNA
numerous antigens in a single assay. Most is avoided by targeting and amplifying a region
available platforms are based on fluorescent of the gene that is common to all alleles so that
bead technology or mass spectrometry. Rou- the minute quantity of donor DNA is not de-
tine ABO and RhD testing is not currently tected. This approach makes possible reliable
available on most automated platforms be- blood group determination with DNA pre-
cause the expression of these antigens is com- pared from a blood sample collected after
plex and further development is required. transfusion. DNA isolated from a buccal smear
To resolve discrepancies and identify new or urine sediment is also suitable for testing. In
alleles, specialty referral laboratories use transfusion-dependent patients who produce
methods that are similar to those used for alloantibodies, an extended antigen profile is
high-resolution HLA typing, ie, gene-specific important to determine additional blood
amplification of coding exons followed by se- group antigens to which the patient can be-
quencing, or gene-specific cDNA amplifica- come sensitized.
tion and sequencing. These methods are used In the past, when a patient with autoim-
to investigate new alleles and resolve serologic mune hemolytic anemia was transfused be-
and molecular discrepancies. The application fore the patient’s red cell phenotype for minor
of these methods has been reviewed by several antigens was established, time- and resource-
groups.47-49 consuming differential allogeneic absorptions
were required to determine the presence or
Clinical Application of the Prediction absence of alloantibodies underlying the auto-
antibody. Establishing the patient’s most prob-
of Blood Groups by DNA Analysis
able phenotype through DNA-based assays
A major use of DNA-based assays is to predict makes it possible to match the antigen profile
the red cell phenotype of a fetus, or of a patient of the absorbing red cells to that of the patient,
who has been transfused, or when red cells are thereby reducing the number of cell types re-
coated with IgG. Additional applications in- quired for absorption. This approach also al-
clude the resolution of discrepancies in the lows matching of the antigen profile of the do-
ABO and Rh systems and identification of the nor to that of the patient for the most clinically
molecular basis of unusual serologic results. significant, common antigens (eg, Jka, Jkb; S, s)
DNA analysis also affords the capability of dis- when transfusion is required. This matching
tinguishing alloantibodies from autoantibod- avoids the use of “least incompatible” blood
ies. This section gives an overview of some of for transfusion, and allows transfusion of units
the major applications of DNA-based analysis that are “antigen-matched for clinically signifi-
that are currently employed in patient and do- cant blood group antigens” to prevent delayed
nor testing. These, and additional clinical ap- transfusion reactions and circumvent addi-
plications, are summarized in Table 11-4. tional alloimmunization.
282 䡲 AABB TECHNICAL MANUAL
TABLE 11-4. Applications of DNA-Based Assays for Patient and Donor Testing
䡲 When antibody is not available (eg, anti-Doa, -Dob, -Jsa, -V, -VS).
䡲 Help identify alloantibody when a patient’s type is antigen-positive and a variant phenotype is possible (eg,
anti-D in a D-positive patient, anti-e in an e-positive patient).
䡲 When the patient’s red cells are coated with immunoglobulin (DAT+).
– When direct agglutinating antibodies are not available.
– When the antigen is sensitive to the IgG removal treatment (eg, antigens in the Kell system are denatured
by EDTA-glycine-acid elution).
– When testing requires the indirect antiglobulin test and IgG removal techniques are not effective at
removing cell-bound immunoglobulin.
– When antisera are weakly reactive and reaction is difficult to interpret (eg, anti-Doa, anti-Dob, anti-Fyb).
䡲 To detect weakly expressed antigens (eg, Fyb with the FyX phenotype); where the patient is unlikely to make
antibodies to transfused antigen-positive RBCs.
䡲 Aid in the resolution of complex serologic investigations, especially those involving high-prevalence anti-
gens when reagents are not available.
䡲 Identify if a fetus is or is not at risk for hemolytic disease of the fetus and newborn.
– Predict if the partner of a prospective mother with anti-D is homozygous or heterozygous for RHD.
䡲 When antibody is weak or not available (eg, anti-Doa, -Dob; -Jsa, -Jsb; -V/VS).
䡲 Type donors for reagent red cells for antibody screening cells and antibody identification panels (eg, Doa,
Dob, Jsa, V, VS).
䡲 Determine zygosity of donors on antibody detection/identification reagent panels, especially D, S, Fya, and
Fyb.
CHAPTER 11 Blood Group Genetics 䡲 283
DNA-Based Assays to Predict the Red patients may make alloantibodies to these an-
Cell Phenotype: When Red Cells Are tigens, autoantibody production with Rh-
Coated with IgG related specificity is prevalent, and distin-
guishing between the two is critical for safe
In patients with or without autoimmune he- transfusion practice to avoid hemolytic trans-
molytic anemia, the presence of immunoglob- fusion reactions.50,51 Delayed hemolytic trans-
ulin bound to the red cells [positive result on fusion reaction in particular, places patients
direct antiglobulin testing (DAT)] often makes with SCD at risk for life-threatening anemia,
antigen typing results by serologic methods in- pain crisis, acute chest syndrome, and/or
valid. Certain methods, such as treatment of acute renal failure. Patients may also experi-
the red cells with chloroquine diphosphate or ence hyperhemolysis, in which hemoglobin
EDTA-glycine acid (EGA) may be employed to levels drop below pretransfusion levels due to
remove the red-cell-bound IgG. These meth- bystander hemolysis of the patients’ own anti-
ods are not always successful, the antigen of gen-negative red cells. RH genotyping has re-
interest may be denatured by the treatment vealed that many of these patients have vari-
(eg, EGA destroys antigens of the Kell blood ant RHD and/or RHCE alleles that encode
group system), and direct agglutinating anti- amino acid changes in Rh proteins that result
bodies for the antigen of interest may not be in altered or partial antigens. For details on
available. DNA testing allows determination of RHD and RHCE alleles that encode partial an-
an extended antigen profile to select antigen- tigen, refer to Chapter 13.
negative red cell units for transfusion. Reports of autoantibodies to Jka and Jkb
are not uncommon. With the discovery of vari-
DNA-Based Assays to Distinguish ant JK alleles that encode partial Jka and Jkb an-
Alloantibody from Autoantibody tigens, it is probable that some previously
identified autoantibodies were alloantibodies
When an antibody specificity is found in a pa-
(see Chapter 14). DNA analysis for JK variants
tient whose red cells express the correspond-
is helpful to clarify the situation. As in other
ing antigen, it is essential to know whether the
blood group systems, Kidd system genetic di-
antibody is an allo- or autoantibody and a
versity is higher in populations of African an-
DNA-based investigation is helpful for transfu-
cestry.
sion management. If DNA typing predicts the
red cells to be antigen positive, further investi-
DNA-Based Testing in Prenatal
gation by high-resolution gene sequencing
Practice
should be considered because the sample may
have a novel amino acid change in the protein DNA-based testing has affected prenatal prac-
carrying the blood group antigen. These novel tice in the areas that are discussed below.
amino acid changes result in new epitopes and Hemagglutination, including antibody titers,
altered (weakened or partial) expression of the gives only an indirect indication of the risk and
conventional antigen. severity of hemolytic disease of the fetus and
This situation is especially relevant in pa- newborn (HDFN). Antigen prediction by DNA-
tients with sickle cell disease (SCD) or thalas- based assays can be used to identify the fetus
semia who require long-term transfusion sup- who is not at risk of HDFN (ie, who is predicted
port and are at risk of alloimmunization that is to be antigen-negative) so that the mother
often complicated by the presence of autoanti- need not be aggressively monitored. Testing of
bodies. In patients of African ancestry who fetal DNA should be considered when a moth-
have SCD, partial expression of common Rh er's serum contains an IgG alloantibody that
antigens (D, C, c, and e) is prevalent. Such pa- has been associated with HDFN and the fa-
tients frequently present with a combination ther's antigen status for the corresponding an-
of anti-D, -C, and -e and yet their red cells type tigen is heterozygous or indeterminable, or he
serologically as D+, C+, and e+. Although such is not available for testing.
284 䡲 AABB TECHNICAL MANUAL
To Identify a Fetus at Risk for Anemia of are not favored because of their more invasive
the Neonate nature and associated risk to the fetus. A non-
invasive sample source is the cell-free fetal
The first application of DNA-based assays for DNA that is present in maternal plasma as ear-
the prediction of blood group phenotype oc- ly as 5 weeks of gestation; the amount of DNA
curred in the prenatal setting and was report- increases with gestational age and reliable re-
ed by Bennett et al52 who tested fetal DNA for
sults in DNA-based assays are obtained start-
the presence of RHD. Because of the clinical
ing at about 15 weeks of gestation (sometimes
significance of anti-D, RHD is probably the
earlier, depending on the gene of interest).54,55
most frequent target gene, but DNA-based as-
These assays are particularly successful for D
says can be used to predict the antigen type of
typing because the D-negative phenotype in
the fetus for any antigen if the molecular basis
the majority of samples is due to the absence
is known. When the implicated IgG antibody
of the RHD gene.
in the maternal circulation is not anti-D, it is
Testing for the presence or absence of a
prudent, when possible, to also test the fetal
gene is less demanding than testing for a sin-
DNA for RHD to preempt unnecessary re-
gle gene polymorphism or SNP to predict, for
quests for D– blood for intrauterine transfu-
example, the K/k antigen status. Cell-free fetal
sion; this is particularly relevant to avoid the
DNA from the maternal plasma is routinely
use of rare r'r' or r''r'' blood when anti-c or
tested in Europe for the presence of a fetal
anti-e is the implicated antibody.
RHD gene to eliminate the unnecessary
PCR analyses for the prediction of fetal D
administration of antepartum RhIG to the ap-
phenotype are based on detecting the pres-
proximately 40% of D-negative women who
ence or absence of specific portions of RHD. In
populations of European ancestry, the molec- are carrying a D-negative fetus. In the United
ular basis of the D-negative phenotype is usu- States, due to intellectual property ownership,
ally associated with deletion of the entire RHD, testing of cell-free fetal DNA is limited to wom-
but several other molecular bases have been en with active anti-D and is available only
described. In populations of Asian ancestry commercially.
15% to 30% of D-negative people have an in-
tact but inactive RHD, while others with red DNA-Based Testing for D in Pregnant
cells that are nonreactive with anti-D have the Women
Del phenotype. Approximately a quarter of D- Serologic typing for D cannot easily distin-
negative people of African ethnicity have an guish women whose red cells lack some epi-
RHD pseudogene (RHD), which does not en- topes of D (partial D) and are at risk for D im-
code the D antigen, and many others have a munization, from those with a weak D
hybrid RHD-CE-D gene (eg, the rS phenotype). phenotype who are not at risk for D immuni-
Predicting the D type by DNA analysis requires zation. Red cells with partial D type as D+,
probing for multiple nucleotide changes. The some in direct tests and others by indirect
choice of assays depends upon the patient’s tests. These women might benefit from receiv-
ethnicity and the degree of discrimination de- ing RhIG prophylaxis if they deliver a D+ fetus.
sired. Establishing the fetal KEL genotype is RHD genotyping can distinguish weak D from
also of great clinical value in determining partial D to guide RhIG prophylaxis and blood
whether a fetus is at risk for severe anemia, be- transfusion recommendations.
cause the strength of the mother's anti-K anti-
body often does not correlate with the severity
DNA-Based Testing of Paternal Samples
of the infant’s anemia. The same is true for
anti-Ge3.53 The father’s red cells should be tested for the
Amniocytes, harvested from amniotic flu- antigen corresponding to the antibody in the
id, are the most common source of fetal DNA. maternal plasma. If the red cells are negative,
Chorionic villus sampling and cordocentesis the fetus is not at risk. If the father is positive
CHAPTER 11 Blood Group Genetics 䡲 285
for the antigen, zygosity testing can determine and have the potential to be used for mass
whether the father is homozygous or heterozy- screening of donors. This practice not only in-
gous for the gene encoding the antigen, partic- creases the antigen-negative inventory by ex-
ularly when there is no allelic antigen or no an- panding combinations of the minor antigens
tisera to detect the allelic product. and some high-prevalence antigens, but also
Zygosity testing of paternal samples is allows consideration of the possibility of pro-
most often performed when testing for possi- viding donor components that are DNA
ble HDFN due to anti-D or anti-K. If the pater- matched to the patient’s type. Although DNA
nal red cells are K+ and the mother has anti-K, methods are not yet licensed by the Food and
they can be tested serologically for expression Drug Administration for the labeling of donor
of the allelic k antigen. However, many centers units in the United States, DNA testing is a
do not have a licensed reagent available and valuable screening tool and only negative test
genetic counselors often request DNA testing. results need to be confirmed using a licensed
If the paternal red cells are K–, the maternal reagent when one is available.
anti-K is mostly likely the result of immuniza-
tion through transfusion. DNA-Based Testing to Confirm D Type
For maternal anti-D, DNA testing of RHD of Donors
zygosity is the only method to determine the
paternal gene copy number. Several different Donor centers must test donors for weak D to
genetic events cause a D-negative phenotype, avoid labeling a product as D-negative that
and multiple assays must be conducted to ac- might result in anti-D in response to trans-
curately determine RHD zygosity, especially in fused RBCs. Some donor red cells with very
minority ethnic groups. If the father is RHD weak D expression (weak D type 2, and espe-
homozygous, all of his children will be D+ and cially those with the Del phenotype) are not
any pregnancy in his partner needs to be mon- typed as D+ using current methods and are la-
itored. If the father is heterozygous, the fetus beled as D–. The prevalence of weak D red cells
has a 50% chance of being at risk. The D type not detected by serologic reagents is approxi-
of the fetus should be determined to prevent mately 0.1% (but may vary depending on the
invasive and unnecessary testing so the moth- test method and population). Although the
er need not be aggressively monitored or re- clinical significance has not been established,
ceive immune-modulating agents. donor red cells with weak D expression have
been associated with alloimmunization. RHD
DNA-Based Testing for Antigen- genotyping would improve donor testing by
Negative Blood Donors confirming D– phenotypes,56 but a high-
throughput and cost-effective platform is not
DNA-based typing to predict donor antigen yet available.
profiles in the search for antigen-negative
units is now a standard procedure for blood Discrepancies between Serologic
centers, especially when suitable antibodies (Phenotype) and DNA (Genotype)
are not available. Because red cell typing for Testing
Dombrock antigens is notoriously difficult,
one of the most frequent approaches is to type Differences between serologic and DNA test-
for Doa and Dob. Many other antibody specific- ing results do occur and must be investigated.
ities are unavailable for mass donor screening. Often, these discrepancies lead to interesting
These specificities include anti-Hy, -Joa, -Jsa, discoveries such as the presence of a novel al-
-Jsb, -CW, -V, and -VS. Even specificities consid- lele or genetic variant, particularly when peo-
ered to be common, such as anti-S and anti- ple of diverse ethnicities are tested. Causes of
Fyb, are not always readily available. discrepancies include recent transfusions,
DNA arrays can be used to screen for mul- stem cell transplantation, and natural chime-
tiple minor antigens in a single assay format rism. Stem cell transplantation and natural
286 䡲 AABB TECHNICAL MANUAL
chimerism also may cause results of testing not expressed on the red cells because of a
DNA from somatic cells to differ from results mutation that silences the gene. Such changes
of testing DNA from extracted from peripheral result in discrepancies in the typing of patients
WBCs. Thus, when using DNA testing, it is im- and donors. Homozygosity (or compound het-
portant to obtain an accurate medical history. erozygosity) for a silenced gene results in a
Many genetic events can cause apparent dis- null phenotype and most null phenotypes
crepant results between hemagglutination and have more than one molecular basis.5
DNA test results and the genotype does not al- With donor typing, the presence of a
ways predict the phenotype (see Table 11-5 for grossly normal gene whose product is not ex-
examples).3,5,27 pressed on the red cell surface results in the
donor being falsely typed as antigen-positive.
Silenced or Nonexpressed Genes Although this situation means loss of an anti-
gen-negative donor, it does not jeopardize the
DNA testing interrogates a single SNP or a few safety of blood transfusion. However, if a
SNPs associated with antigen expression and grossly normal gene is detected in a patient
cannot sample every nucleotide in the gene. but the gene is not expressed, the patient
Although a gene may be detected by DNA test- could produce an antibody if he or she re-
ing, there are times when the gene product is ceives a transfusion of antigen-positive blood.
TABLE 11-5. Examples of Some Molecular Events Where Analysis of Gene and Phenotype Do Not
Agree
To avoid misinterpretation, routine as- the FyX phenotype in people of European an-
says must include appropriate tests to detect a cestry is as high as 2%, and the allele has been
change that silences gene expression if preva- found in persons of African ancestry also. Si-
lent in the population tested. Silenced alleles lencing mutations associated with the loss of
can be specific to a particular ethnic group. Kidd antigen expression occur more often in
For example, in the Duffy blood group system, people of Asian ancestry, whereas nucleotide
a single nucleotide change (–67T>C) within changes encoding amino acid changes that
the promoter region (GATA box) of FY prevents weaken Kidd expression occur in people of Af-
transcription of FY*A and/or FY*B in red cells rican ethnicity.
but not in other tissues. Although silencing of The routine detection of some blood
FY*A is rare, silencing of FY*B is frequent in group polymorphisms by DNA analysis is
persons of African ethnicity where homozy- complex and not practical at this time. This in-
gosity for the –67T>C change in FY*B results in cludes situations where 1) a large number of
the Fy(a–b–) phenotype, which has a preva- alleles encode one phenotype (eg, ABO, Rh,
lence of 60% or higher. To ensure accuracy, and null phenotypes in many blood group sys-
testing for the GATA box mutation must be in- tems), 2) a phenotype results from alleles with
cluded in typing for Duffy in persons of African a large deletion (eg, GE:–2,3 and GE:–2,–3,–4),
ethnicity. or 3) a phenotype results from hybrid alleles
When the assay is used to predict the (eg, in the Rh and MNS systems). In addition,
presence or absence of D antigen, particularly there is a high probability that not all alleles in
in populations of African ancestry, it is essen- all ethnic populations are known.
tial to include a test for the complete but inac- Blood group genomics has become an es-
tive RHD pseudogene (RHD), which has a sential component of the practice of transfu-
37-bp sequence duplication. If the assay tests sion medicine.57 Genomics has provided a
for GYPB*S (S antigen), additional testing greater understanding of genetic blood group
should be performed to detect a C>T change at variants, including the complexity of the
nucleotide 230 in GYP*B exon 5 or a change in molecular basis of Rh variants, such as those
intron 5 (+5g>t); both changes prevent expres- that encode the Hr–hrS– and hrB–HrB–
sion of S antigen when testing persons of Afri- phenotypes58,59 and the associated partial Rh
can ancestry. Homozygosity, or compound antigens that are a daily challenge for the man-
heterozygosity, for the 230T, or the +5g>t agement of patients with SCD.50,51 RH genotyp-
change, results in the S–s–U+W phenotype. ing expands and extends matching for Rh in
Other common causes of discrepancies this patient population. High-throughput plat-
include the presence in the sample of an forms provide a means to test relatively large
altered FY*B allele that encodes an amino acid numbers of donors, thereby opening up the
change causing an FyX phenotype with greatly possibility of changing the way antigen-nega-
reduced expression of Fyb antigen. The red tive blood is provided to patients to prevent
cells type as Fy(b–) with most serologic re- immunization or to eliminate transfusion re-
agents. The prevalence of the allele encoding actions for those who are already immunized.
KEY PO I NT S
1. Genetics is the study of heredity, that is, the mechanisms by which a particular characteris-
tic, such as a blood group, is passed from parents to offspring.
2. A gene is a segment of DNA and is the basic unit of inheritance; it occupies a specific loca-
tion on a chromosome (the gene locus). Alleles are alternative forms of a gene at the same
gene locus (eg, alleles JK*A and JK*B encode the Jka and Jkb antigens, respectively).
288 䡲 AABB TECHNICAL MANUAL
3. A human somatic (body) cell is diploid, containing 46 chromosomes in 23 pairs: 22 pairs are
alike (homologous) in males and females and are termed autosomes. The remaining pair is
the sex chromosomes: X and Y for males, or two X chromosomes for females.
4. Somatic cells divide for growth and repair by mitosis. Mitosis replicates the chromosomes
and produces two identical nuclei in preparation for cell division. The new cells are diploid,
like the parent cell, and have all the genetic information of the parent cell.
5. Meiosis is the process by which germ cells divide to become gametes (sperm and egg cells);
diploid cells undergo DNA replication and two divisions to form four gametes, each of
which is haploid and has half the chromosomal complement of the parent somatic cell.
6. The term “genotype” traditionally refers to the complement of genes inherited by each per-
son from his or her parents; the term is also used to refer to the set of alleles at a single gene
locus. Whereas the genotype of a person is his or her genetic constitution, the phenotype is
the observable expression of the genes and reflects the biologic activity of the gene(s). Thus,
the presence or absence of antigens on the red cells, as determined by serologic testing, rep-
resents the phenotype.
7. When identical alleles for a given locus are present on both chromosomes, a person is ho-
mozygous for the particular allele, whereas when nonidentical alleles are present at a par-
ticular locus, the person is heterozygous. Antigens encoded by alleles at the same locus are
said to be antithetical. Thus, genes are allelic but not antithetical, whereas antigens are anti-
thetical but not allelic.
8. The expression of blood group antigens on the red cell may be modified or affected by gene
interaction. Homozygosity (or compound heterozygosity) for a silenced gene results in a
null phenotype and most null phenotypes have more than one molecular basis.
9. A blood group system consists of one or more antigens under the control of a single gene lo-
cus (eg, KEL encodes the Kell blood group antigens) or of two or more homologous genes
(eg, RHD and RHCE encode the Rh blood group antigens) so closely linked that virtually no
recombination occurs between them; thus, each blood group system is genetically indepen-
dent. Currently, 34 blood group systems are recognized.
10. The genes encoding the 34 blood group systems have been sequenced and the molecular
bases of most antigens and phenotypes are known so that DNA-based methods (genotyp-
ing) can be used to predict a blood group phenotype.
11. DNA-based assays (blood group genotyping) have major applications in patient and donor
testing. They can be used to predict the red cell phenotype of a fetus, or of a patient who was
transfused, or when red cells are coated with IgG; they can be used to resolve ABO and Rh
discrepancies and to identify the molecular basis of unusual serologic results. DNA analysis
aids in distinguishing alloantibodies from autoantibodies and is being applied to high-
throughput screening of donors.
REFER ENCES
1. Brown TA. Introduction to genetics: A molecu- in medicine. 6th ed. Philadelphia: Saunders,
lar approach. London: Garland Science, 2011. 2004.
2. Clark DP, Russell LD. Molecular biology: Made 5. Reid ME, Lomas-Francis C, Olsson ML. Blood
simple and fun. St. Louis: Cache River Press, group antigen factsbook. 3rd ed. San Diego:
2010. Academic Press, 2012.
3. Reid ME, Denomme GA. DNA-based methods 6. International Society of Blood Transfusion.
in the immunohematology reference laborato- Committee on Terminology for Red Cell Sur-
ry. Transfus Apher Sci 2011;44:65-72. face Antigens. Blood group terminology. Am-
4. Nussbaum RL, Thompson MW, McInnes RR, sterdam: ISBT, 2013 [Available at http://www.
Willard HF. Thompson & Thompson genetics isbtweb.org/working-parties/red-cell-immu
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33. Jeffreys AJ, Wilson V, Thein SL. Hypervariable http://www.genenames.org/ (accessed August
‘minisatellite’ regions in human DNA. Nature 12, 2013).]
1985;314:67-73. 47. Avent ND. Large scale blood group genotyp-
34. Jeffreys AJ, Wilson V, Thein SL. Individual-spe- ing. Transfus Clin Biol 2007;14:10-15.
cific ‘fingerprints’ of human DNA. Nature 48. Monteiro F, Tavares G, Ferreira M, et al. Tech-
1985;316:76-9. nologies involved in molecular blood group
35. National Institute of Standards and Technolo- genotyping. ISBT Science Series 2011;6:1-6.
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dem repeat DNA internet database. Gaithers- sisted laser desorption/ionization, time of
burg, MD: NIST, 2013. [Available at http:// flight mass spectrometry-based blood group
www.cstl.nist.gov/div831/strbase/index.htm genotyping – the alternative approach. Trans-
(accessed August 12, 2013).] fus Med Rev 2013;27:2-9.
36. Bennett P. Demystified ... microsatellites. Mol 50. Chou ST, Westhoff CM. The role of molecular
Pathol 2000;53:177-83. immunohematology in sickle cell disease.
37. Khan F, Agarwal A, Agrawal S. Significance of Transfus Apher Sci 2011;44:73-9.
chimerism in hematopoietic stem cell trans- 51. Noizatt-Pirenne F, Tournamille C. Relevance of
plantation: New variations on an old theme. RH variants in transfusion of sickle cell pa-
Bone Marrow Transplant 2004;34:1-12. tients. Transfus Clin Biol 2011;18:527-35.
38. Thiede C, Bornhauser M, Ehninger G. Evalua- 52. Bennett PR, Le Van Kim C, Colin Y, et al. Prena-
tion of STR informativity for chimerism test- tal determination of fetal RhD type by DNA
ing—comparative analysis of 27 STR systems amplification. N Engl J Med 1993;329:607-10.
in 203 matched related donor recipient pairs. 53. Pate LL, Myers J, Palma J, et al. Anti-Ge3
Leukemia 2004;18:248-54. causes late-onset hemolytic disease of the
39. Domiati-Saad R, Klintmalm GB, Netto G, et al. newborn: The fourth case in three Hispanic
Acute graft versus host disease after liver families. Transfusion 2013;53:2152-7.
transplantation: Patterns of lymphocyte chi- 54. Daniels G, Finning K, Martin P, Soothill P. Fetal
merism. Am J Transplant 2005;5:2968-73. blood group genotyping from DNA from ma-
40. Mount M, ed. Standards for relationship test- ternal plasma; an important advance in the
ing laboratories. 11th ed. Bethesda, MD: AABB, management and prevention of haemolytic
2014. disease of the fetus and newborn. Vox Sang
41. Bluth MH, Reid ME, Manny N. Chimerism in 2004;87:225-32.
the immunohematology laboratory in the mo- 55. Clausen FB, Christiansen M, Steffensen R, et
lecular biology era. Transfus Med Rev 2007; al. Report of the first nationally implemented
21:134-46. clinical routine screening for fetal RHD in D–
42. Wagner FF, Frohmajer A, Flegel WA. RHD posi- pregnant women to ascertain the requirement
tive haplotypes in D negative Europeans. BMC for antenatal RhD prophylaxis. Transfusion
Genet 2001;2:10. 2012;52:752-8.
43. Cho D, Lee JS, Yazer MH, et al. Chimerism and 56. Wagner FF. RHD PCR of D-negative blood do-
mosaicism are important causes of ABO phe- nors. Transfus Med Hemother 2013;40:172-81.
notype and genotype discrepancies. Immuno- 57. Hillyer C, Shaz B, Winkler A, Reid ME. Integrat-
hematology 2006;22:183-7. ing molecular technologies for red blood cell
44. Daniels GL, Anstee DJ, Cartron J-P, et al. Blood typing and compatibility testing into blood
group terminology 1995. ISBT Working Party centers and transfusion services. Transfus Med
on Terminology for Red Cell Surface Antigens. Rev 2008;22:117-32.
Vox Sang 1995;69:265-79. 58. Pham B-N, Peyrard T, Tourret S, et al. Anti-HrB
45. Garratty G, Dzik WH, Issitt PD, et al. Terminol- and anti-hrB revisited. Transfusion 2009;49:
ogy for blood group antigens and genes: His- 2400-05.
torical origins and guidelines in the new mil- 59. Reid ME, Hipsky CH, Velliquette RW, et al.
lennium. Transfusion 2000;40:477-89. Molecular background of RH in Bastiaan, the
46. HGNC searches. Cambridge, UK: HUGO Gene RH:-31,-34 index case, and two novel RHD al-
Nomenclature Committee, 2013 [Available at leles. Immunohematology 2012;28:97-103.
C h a p t e r 1 2
Laura Cooling, MD, MS, Associate Professor, Department of Pathology, and Associate Medical Director, Trans-
fusion Medicine, University of Michigan, Ann Arbor, Michigan
The author has disclosed no conflicts of interest.
291
292 䡲 AABB TECHNICAL MANUAL
The ABO system contains four major ABO to the subterminal galactose of the H antigen
phenotypes: A, B, O, and AB. The four pheno- to form A antigen. In group B individuals, an
types are determined by the presence or ab- 13 galactose is added to the same subter-
sence of two antigens (A and B) on red cells minal galactose to form B antigen. In group AB
(see Table 12-1). The ABO system is also char- individuals, both A and B structures are syn-
acterized by the presence or absence of natu- thesized. In group O individuals, neither A nor
rally occurring antibodies, termed “isohemag- B antigens are synthesized as a result of a mu-
glutinins,” directed against missing A and B tation in the ABO gene.7,10 As a consequence,
antigens. As shown in Table 12-1, an inverse group O individuals express only H antigen. A
reciprocal relationship exists between the and B antigens are also absent in the very rare
presence of A and/or B antigens on red cells Bombay phenotype because of the absence of
and the presence of anti-A, anti-B, or both, in the H-antigen precursor (see section below on
sera. For example, group O individuals, who the H system).
lack A and B antigens on red cells, possess As terminal motifs, the A and B antigens
both anti-A and anti-B. It is believed that the can be displayed on a number of oligosaccha-
immunizing sources for such naturally occur- ride scaffolds that differ in their size, composi-
ring antibodies are gut and environmental tion, linkages, and tissue distribution. On red
bacteria, such as the Enterobacteriaceae, which cells, ABH sites are present on N-linked (65%-
have been shown to possess ABO-like struc- 75%) and O-linked (5%-15%) glycoproteins,
tures on their lipo-polysaccharide coats.8,9 polyglycosylceramides (10%-15%), and sim-
pler glycosphingolipids (Fig 12-2). ABO anti-
Biochemistry gens are subclassified by the carbohydrate se-
quence immediately upstream of the ABO
The A and B antigens are defined by three sug- motif. In humans, ABH is expressed predomi-
ar terminal epitopes on glycolipids and glyco- nantly on four different oligosaccharide back-
proteins.7 As shown in Fig 12-1, H antigen is bones (see Table 12-2). On human red cells,
characterized by a terminal 12 fucose; it is the majority of endogenous ABH antigen syn-
the immediate biosynthetic precursor for both thesized is present on Type 2 chain structures.
A and B antigens and is required for their ex- The ability to synthesize and use different
pression. In group A individuals, an N-acetyl- carbohydrate chains is genetically determined
galactosamine is added, in an 13 linkage, and can contribute to antigenic differences in
European African
Anti-A Anti-B A1 Cells B Cells O Cells ABO Group Ethnicity Ethnicity
0 0 + + 0 O 45 49
+ 0 0 + 0 A 40 27
0 + + 0 0 B 11 20
+ + 0 0 0 AB 4 4
0 0 + + + Bombay* Rare Rare
*H null phenotype (see section on H antigen).
+ = agglutination; 0 = no agglutination.
CHAPTER 12 ABO, H, and Lewis Blood Groups 䡲 293
FIGURE 12-2. Schematic representation of the red cell membrane showing antigen-bearing glycosylation of
proteins and lipids.
GPI = glycosylphosphatidylinositol. (Courtesy of ME Reid)
A epitope GalNAc1-3(Fuc1-2)Gal1-R
Type 1 A GalNAc1-3(Fuc1-2)Gal1-3GlcNAc1-3-R
Type 2 A GalNAc1-3(Fuc1-2)Gal1-4GlcNAc1-3-R
Type 3 A GalNAc1-3(Fuc1-2)Gal1-3GalNAc1-3(Fuc1-2)Gal1-4GlcNAc1-3-R
(repetitive A)
Type 4 A GalNAc1-3(Fuc1-2)Gal1-3GalNAc1-4Gal1-4Gal1-4Glc-Cer
(globo-A)
*Underlined sequences denote the critical differences between Type 1, 2, and 4 chains. Linkages and anomery ( or
linked) of the A antigen galactose are in bold. Bracketed sequences denote repetitive A antigen characteristic of Type 3 chain
A antigen.
Cer = ceramide; Fuc = fucose; Gal = galactose; GalNAc = N-acetyl-galactosamine; Glc = glucose; GlcNAc = N-acetyl-glucosa-
mine; R = upstream oligosaccharide.
CHAPTER 12 ABO, H, and Lewis Blood Groups 䡲 295
tions. 18 A plethora of mutations associated 22% to 35% of individuals with A2B possess an
with weak A and B subgroups has been de- alloanti-A1 in their sera. Because the A2 pheno-
scribed. As an example, group A 2 (a weak A type reflects the inefficient conversion of HA
subgroup) is commonly the result of a nucleo- antigen, A2 red cells have increased reactivity
tide deletion and frameshift, resulting in an with the anti-H lectin Ulex europaeus. Enzyme
enzyme with an additional 21 amino acids at studies comparing A1 and A2 glycosyltransfer-
the C-terminus of the molecule.10,18 ase activity show that the A1 enzyme is 5 to 10
The O allele is an amorph, encoding a times more active than the A2 enzyme, result-
nonfunctional enzyme. The group O pheno- ing in quantitative and qualitative differences
type, therefore, is an autosomal recessive trait, in A antigen expression.7,10 The latter includes
representing inheritance of two nonfunction- the synthesis of unusual Type 3 and Type 4
al ABO genes. Overall, more than 50 O alleles chain A antigen on A1 red cells that is not pres-
have been identified.7,18 The two most com- ent on A2 or weaker A subgroups.10,11
mon O alleles (O01 and O02) contain a nucleo- In addition to A2 , several weaker A sub-
tide deletion and frameshift, leading to a trun- groups have been described (eg, A3, Ax, Am, and
cated, 117-amino-acid protein. Another Ael). The extremely weak A (and B) subgroups
common O allele is O03 (or O 2 ), a group of are infrequently encountered and are usually
nondeletional O alleles that contains a muta- recognized by apparent discrepancies be-
tion at amino acid 268 (Gly268Arg), which is a tween red cell (forward) and serum (reverse)
critical residue for donor binding (UDP-galac- grouping results. Most weak A and B sub-
tose or UDP-N-acetylgalactosamine). One groups were originally described before the
German study found that O03 and a related al- advent of monoclonal typing reagents and
lele (Aw08) were responsible for 25% of all ABO were based on reactivity with human poly-
typing discrepancies caused by reverse-group- clonal anti-A, anti-B, and anti-A,B reagents.
ing problems in healthy donors.19 It was specu- Weak A subgroups are frequently nonreactive
lated that weak anti-A and anti-B could reflect with human polyclonal anti-A (see Table 12-3)
weak residual glycosyltransferase activity; and can show variable reactivity with human
however, a later study was unable to demon- polyclonal anti-A1, anti-A,B, and murine
strate A antigen or enzyme activity in individu- monoclonal antibodies (not shown).10,13,18 The
als with the O03 allele.20 degree of reactivity with commercial murine
monoclonal reagents is clone dependent;
ABO Subgroups however, most commercially available anti-A
agglutinates A3 red cells. Because of the recip-
ABO subgroups are phenotypes that differ in rocal relationship between H and synthesis of
the amount of A and B antigen carried on red A and B antigens, nearly all weak A and B sub-
cells and present in secretions. In general, A groups have higher H expression.7 In clinical
subgroups are more common than B sub- practice, it is seldom necessary to identify a
groups. Clinically, the two most common sub- patient’s specific A or B subgroup.
groups encountered are A1 and A2. A1 repre- When performed, classification of weak A
sents the majority of group A donors (80%) subgroups is typically based on the following:
and is characterized by approximately 1 mil-
lion A antigen epitopes per red cell. A2 is the 1. Degree of red cell agglutination by anti-A
second most common subgroup (20%) and and anti-A1.
possesses only one fifth (2.2 × 105) the number 2. Degree of red cell agglutination by human
of A antigen sites as A1. Both A1 and A2 are and some monoclonal anti-A,B.
strongly agglutinated by reagent anti-A in rou- 3. Degree of H antigen (anti-H lectin and
tine direct testing. A1 can be distinguished Ulex europaeus) expression.
from A2 by the lectin Dolichos biflorus, which 4. Presence or absence of anti-A1 (Method 2-
agglutinates A1 red cells but not A2 red cells. In 9).
addition, 1% to 8% of individuals with A2 and 5. Presence of A and H in saliva.
296 䡲 AABB TECHNICAL MANUAL
A1 4+ 0 4+ 0 0 4+ 0 A&H
A2 4+ 0 4+ 2+ 0/2+* 4+ 0 A&H
mf mf
A3 2+ 0 2+ 3+ 0/2+* 4+ 0 A&H
Ax 0/ 0 1-2+ 4+ 0/2+ 4+ 0 H
Ael 0 0 0 4+ 0/2+ 4+ 0 H
B 0 4+ 4+ 0 4+ 0 0 B&H
mf mf
B3 0 1+ 2+ 4+ 4+ 0 0 B&H
Bx 0 0/ 0/2+ 4+ 4+ 0 0 H
B(A) /2+ †
4+ 4+ 0 4+ 0 0 B&H
*The occurrence of anti-A1 is variable in these phenotypes.
†
Most often detected with anti-A clones containing the MHO4 clone.
1+ to 4+ = agglutination of increasing strength; = weak agglutination; mf = mixed-field agglutination; 0 = no agglutination.
Chemically, acquired B is the result of that contain EDTA prevents complement ac-
deacetylation of the A antigen’s N-acetyl- tivation and hemolysis.
galactosamine, yielding a B-like galactosamine
sugar.23,24 In patients’ samples, acquired B is Anti-A,B
often present in the setting of infection by gas-
Sera from group O individuals contain an anti-
trointestinal bacteria. Many enteric bacteria
body known as “anti-A,B” because it is reactive
possess a deacetylase enzyme capable of con-
with both A and B red cells. Such anti-A and
verting A antigen to a B-like analog.24 Identifi-
anti-B reactivity cannot be separated by differ-
cation of the acquired B phenotype can also be
ential adsorption, suggesting that the anti-
influenced by reagent pH and specific mono-
body recognizes a common epitope shared by
clonal anti-B typing reagents. 22 In the past,
the A and B antigens.7 Saliva containing secret-
anti-B reagents containing the ES-4 clone were ed A or B substance can inhibit the activity of
associated with an increased incidence of ac- anti-A,B against both A and B red cells.
quired B.
To resolve a patient’s true red cell type Anti-A1
and confirm the presence of acquired B, red
cells should be retyped using a different Anti-A1 is present as an alloantibody in the se-
monoclonal anti-B or acidified (pH 6.0) hu- rum of 1% to 8% of A2 individuals and 22% to
man anti-B. Acidified human anti-B does not 35% of A2B individuals. Anti-A1 is sometimes
react with acquired B antigen. The ability of present in the sera of individuals with other
monoclonal anti-B to recognize acquired anti- weak A subgroups. Group O serum contains a
B should be noted in the manufacturer’s insert. mixture of anti-A and anti-A1.24 Anti-A1 can
cause ABO discrepancies during routine test-
ABO Antibodies ing and lead to incompatible crossmatches
with A1 and A1B red cells. Anti-A1 is usually of
Anti-A and Anti-B IgM isotype, reacting best at room tempera-
Immune globulin M (IgM) is the predominant ture or below, and is usually considered clini-
isotype found in group A and group B individ- cally insignificant. Anti-A1 is considered clini-
uals, although small quantities of IgG antibody cally significant if reactivity is observed at
can be detected. In group O serum, IgG is the 37 C.24 Group A2 patients with an anti-A1 that
major isotype for anti-A and anti-B. As a con- is reactive at 37 C testing should be transfused
sequence, hemolytic disease of the fetus and with group O or A2 red cells only; group A2B pa-
newborn (HDFN) is more common among the tients should receive group O, A2, A2B, or B red
offspring of group O mothers than of mothers cells.
with other blood types because IgG can cross
the placenta but IgM cannot. Routine Testing for ABO
Both IgM and IgG anti-A and anti-B pref- Donor blood samples are routinely typed for
erentially agglutinate red cells at room tem- ABO at the time of donation and on receipt of
perature (20 to 24 C) or cooler, and both can Red Blood Cell (RBC) units in the hospital
efficiently activate complement at 37 C. The transfusion service (confirmatory typing). Re-
complement-mediated lytic capability of cipient samples are typed before transfusion.
these antibodies becomes apparent if serum ABO grouping requires both antigen typing of
testing includes an incubation phase at 37 C. red cells for A and B antigen (red cell grouping
Hemolysis caused by ABO antibodies should or forward type) and screening of serum or
be suspected when either the supernatant se- plasma for the presence of anti-A and anti-B
rum is pink to red or the cell button is smaller isoagglutinins (serum grouping or reverse
or absent. Hemolysis must be interpreted as a type). Both red cell and serum or plasma
positive result. The use of plasma for testing or grouping are required for donors and patients
of reagent red cells suspended in solutions because each grouping serves as a check on
298 䡲 AABB TECHNICAL MANUAL
the other. Reverse or serum grouping is not re- identified in a patient, it may be necessary to
quired in two circumstances: 1) for confirma- transfuse group O red cells pending an investi-
tion testing of labeled, previously typed donor gation. It is important to obtain a sufficient
red cells and 2) in infants younger than 4 pretransfusion blood sample from the patient
months of age. As previously discussed, isoag- to complete any additional studies that may be
glutinins are not present at birth and develop required.
only after 3 to 6 months of age.
Commercially available anti-A and anti-B Red Cell Testing Problems
for red cell typing are extremely potent and ag-
glutinate most antigen-positive red cells di- ABO testing of red cells may give unexpected
rectly, even without centrifugation. Most results for many reasons, including the follow-
monoclonal typing reagents have been formu- ing:
lated to detect many weak ABO subgroups (see
manufacturers’ inserts for specific reagent 1. Weak ABO expression that results from
characteristics). Additional reagents (anti-A1 inheritance of a weak ABO subgroup.
and anti-A,B) and special techniques to detect Some patients with leukemia and other
weak ABO subgroups are not necessary for malignancies can also show weakened
routine testing but are helpful for resolving ABO expression.25
ABO-typing discrepancies. 2. Mixed-field agglutination with circulating
In contrast to commercial ABO typing re- red cells of more than one ABO group fol-
agents, human anti-A and anti-B in the sera of lowing out-of-group red cell transfusion
patients and donors can be relatively weak, re- or hematopoietic progenitor cell (HPC)
quiring incubation and centrifugation. Tests transplantation (eg, group O to group A).
for serum grouping, therefore, should be per- Mixed-field agglutination is also present
formed using a method that adequately de- in some ABO subgroups (A3), blood
tects human anti-A and anti-B. Several meth- group chimerism in fraternal twins, and
ods are available for determining ABO group, very rare cases of mosaicism arising from
including slide, tube, microplate, and column dispermy.
agglutination techniques. 3. Neutralization of anti-A and anti-B typing
reagents by high concentrations of A or B
ABO Discrepancies blood group substance in serum, result-
Table 12-1 shows the results and interpreta- ing in unexpected negative reactions with
tions of routine red cell and serum tests for serum- or plasma-suspended red cells.
ABO. A discrepancy exists when the results of 4. Spontaneous agglutination or autoagglu-
red cell tests do not agree with those of serum tination of serum- or plasma-suspended
tests, usually due to unexpected negative or red cells caused by heavy coating of red
positive results in either the forward or reverse cells by potent autoagglutinins.
typing (see Table 12-3). ABO discrepancies 5. Nonspecific aggregation of serum- or
may arise from intrinsic problems with either plasma-suspended red cells caused by
red cells or serum or from technical errors in abnormal concentrations of serum pro-
performing the test (see Table 12-4 and section teins or infused macromolecular solu-
on Resolving ABO Discrepancies). tions.
When a discrepancy is encountered, the 6. False-positive reactions caused by a pH-
discrepant results must be recorded, but inter- dependent autoantibody, a reagent-
pretation of the ABO group must be delayed dependent antibody (eg, EDTA or para-
until the discrepancy has been resolved. If the ben), or rouleaux.
specimen is from a donor unit, the unit must 7. Anomalous red cell grouping resulting
be quarantined and cannot be released for from acquired B, B(A), or A(B) pheno-
transfusion. When an ABO discrepancy is types.
CHAPTER 12 ABO, H, and Lewis Blood Groups 䡲 299
Category Causes
Leukemia/malignancy
Transfusion
Transplantation
Transplantation
Acquired B antigen
B(A) phenomenon
Out-of-group transfusion
Transplantation
Fetomaternal hemorrhage
ABO subgroup
Hypogammaglobulinemia
Transplantation
Cold alloantibody
Transplantation
the patient’s medical record for conditions, false-positive reactions caused by autoanti-
medications, or recent transfusions that may bodies can be eliminated by washing red cells
have contributed to the conflicting test results with warm saline (Method 2-17). Autoaggluti-
(Method 2-4). Samples with apparent weak or nation caused by IgM can also be inhibited or
missing ABO antigens and/or antibodies may dispersed by incubating red cells in the pres-
require tests using methods that enhance ence of either dithiothreitol or 2-aminoethyl-
antigen-antibody binding, including incubat- isothiouronium bromide (Method 3-16).
ing red cells at 4 C (Method 2-5), using These reagents reduce the disulfide bonds on
enzyme-treated red cells (Method 2-6), and IgM molecules, decreasing their polyvalency
conducting adsorption and elution studies and ability to directly agglutinate red cells.
(Method 2-7). In some instances, it may be
necessary to test for the secretion of ABO anti-
gens in saliva (Method 2-8). Patients with sus- T HE H SYS TE M
pected B(A), acquired B, or A(B) phenotypes H antigen is ubiquitously expressed on all red
should be retested using different monoclonal cells except the rare Bombay phenotype. Be-
and human polyclonal reagents. cause H antigen serves as the precursor to
ABO discrepancies caused by unexpected both A and B antigens, the amount of H anti-
serum reactions are not uncommon. Com- gen on red cells depends on an individual’s
monly encountered causes of serum-group- ABO type. H antigen is highly expressed on
ing discrepancies include cold autoantibodies, group O red cells because group O individuals
rouleaux, cold-reacting alloantibodies (eg, lack a functional ABO gene. In group A and B
anti-M), and weak A subgroups with an anti- individuals, the amount of H antigen is con-
A1. To resolve an ABO discrepancy caused by siderably less because H is converted to the A
an anti-A1 in a group A individual, red cells and B antigens, respectively. The amount of H
should be tested with Dolichos biflorus lectin, antigen on red cells, based on agglutination
which agglutinates group A 1 but not A 2 and with the anti-H lectin Ulex europaeus, is repre-
weaker A subgroups. The presence of an anti- sented thus: O>A2>B>A1>A1B. H antigen is
A1 should be confirmed by testing serum present on HPCs, red cells, megakaryocytes,
against A 1 , A2, and O red cells (Method 2-9). and other tissues.2,3,28 H antigen has been im-
Reverse-grouping problems resulting from plicated in cell adhesion, normal hematopoi-
either a cold alloantibody (Method 2-10) or etic differentiation, and several malignan-
autoantibody can be identified with a room- cies.6,7,29
temperature antibody detection test and a di-
rect antiglobulin test. Techniques to identify Biochemistry and Genetics
ABO antibodies in the presence of cold auto-
antibodies include testing at 37 C without cen- H antigen is defined by the terminal disaccha-
trifugation (Method 2-11) and cold autoad- ride fucose 12 galactose. Two different fu-
sorption (Method 4-5). Serum or plasma cosyltransferase (FUT) enzymes are capable of
properties can induce rouleaux formation that synthesizing H antigen: FUT1 (H gene) and
resembles agglutination with A1 and B red FUT2 (secretor gene). FUT1 specifically fu-
cells. Saline replacement or saline dilution cosylates Type 2 chain oligosaccharides on red
(Method 3-7) can be used to distinguish rou- cell glycoproteins and glycolipids to form Type
leaux from agglutination and identify ABO an- 2 chain H. In contrast, FUT2 or secretor recog-
tibodies. nizes Type 1 chain precursors to form Type 1
Cold autoantibodies can cause autoag- chain H and Leb antigens in secretions (Fig 12-
glutination of red cells and unexpected reac- 3).10 Secretion of Type 1 chain ABH antigens in
tions during red cell typing. Red cells heavily saliva and other fluids requires a functional
coated with autoantibodies can spontaneous- FUT2 gene. FUT2 is not expressed in red cells
ly agglutinate and cause false-positive reac- but is expressed in salivary glands, gastrointes-
tions in tests with anti-A and anti-B. Usually, tinal tissues, and genitourinary tissues.1,7,10
302
䡲
AABB TECHNICAL MANUAL
FIGURE 12-3. Synthesis of Type 1 chain ABH and Lewis antigens by Secretor (FUT2), Lewis (FUT3), and ABO enzymes. Type 2 chain ABH antigen synthesis is also shown
for comparison.
Fuc = fucose; Gal = galactose; GalNAc = N-acetylgalactosamine; GlcNAc = N-acetylglucosamine; R = other upstream carbohydrate sequence. Reproduced with permission
from Cooling.30
CHAPTER 12 ABO, H, and Lewis Blood Groups 䡲 303
Type 1 chain ABH antigen present on red cells least one functional secretor gene (Se). The red
is passively adsorbed from circulating glyco- cells from H-deficient secretors lack serologi-
lipid antigen present in plasma (see “Lewis cally detectable H antigen but can carry small
System” section).24 amounts of A and/or B antigen because, unlike
persons with classic Bombay phenotype, para-
Null Phenotypes Bombay persons express Type 1 chain ABH an-
Bombay (Oh ) Phenotype tigens in their secretions and plasma (Method
2-8).24 Type 1 chain A or B antigen in plasma is
Originally described in Bombay, India, the Oh then passively adsorbed onto red cells, result-
or Bombay phenotype is a rare, autosomal re- ing in weak A or B antigen expression. Para-
cessive phenotype characterized by the ab- Bombay can also occur in group O individuals,
sence of H, A, and B antigens on red cells and as evidenced by trace Type 1 chain H on their
in secretions. Genetically, Oh individuals are red cells and in their secretions. Red cells from
homozygous for nonfunctional H (hh) and se- para-Bombay individuals are designated “Ah,”
cretor (sese) genes, resulting in a complete ab- “Bh,” and “ABh.”
sence of Type 1 and Type 2 chain H, A, and B. In laboratory testing, red cells from para-
Oh red cells type as H negative with the anti-H Bombay individuals may (or may not) have
lectin Ulex europaeus and monoclonal anti-H. weak reactions with anti-A and anti-B re-
Because these individuals lack a functional agents. In some cases, A and B antigens may
secretor gene necessary for Leb synthesis, Oh be detected only after adsorption and elution.
individuals also type as Le(b–) (see “Lewis Para-Bombay red cells are nonreactive with
System” section). Genotyping studies have de- anti-H lectin, monoclonal anti-H, and human
scribed a wide range of inactivating mutations anti-H from Oh individuals. The sera of para-
in both the H and secretor genes in Oh individ- Bombay individuals contain anti-H, anti-HI,
uals.10,18 The Oh phenotype is also present in
or both and, depending on their ABO type,
leukocyte adhesion deficiency 2 (LAD2) due to
anti-A and anti-B.13,24
a mutation in the GDP-fucose transporter
gene.25
Anti-H
Because they lack all ABH antigens, Oh in-
dividuals possess natural isohemagglutinins to Alloanti-H (Bombay and Para-Bombay)
A, B, and H (see Table 12-1). In routine ABO
typing, these individuals initially type as group The anti-H in Bombay and para-Bombay phe-
O. The Oh phenotype becomes apparent dur- notypes is clinically significant and associated
ing antibody-detection tests with group O red with acute hemolytic transfusion reactions.
cells, which are rich in H antigen. The anti-H The antibody is predominantly of IgM isotype
present in Oh individuals strongly agglutinates and exhibits a broad thermal range (4 to 37 C)
all group O red cells and sometimes demon- with all red cells except Oh red cells. As with
strates in-vivo hemolysis. The Oh phenotype anti-A and anti-B, alloanti-H is capable of acti-
can be confirmed by demonstrating an ab- vating complement and causing red cell he-
sence of H antigen on red cells and the pres- molysis.
ence of an anti-H in serum that is reactive with
group O red cells but not with Oh red cells from Autoanti-H and Autoanti-HI
other individuals. Autoantibodies to H and HI antigens can be
encountered in healthy individuals. When
Para-Bombay Phenotype present, these autoantibodies are most com-
Individuals with the para-Bombay phenotype mon in A1 individuals, who have very little H
are H-deficient secretors.7,10 Genetically, these antigen on their red cells. Autoanti-H and
individuals are homozygous for a nonfunc- autoanti-HI are usually of IgM isotype and are
tional H gene (hh), but they have inherited at reactive at room temperature.
304 䡲 AABB TECHNICAL MANUAL
phenotype, are present during pregnancy. Fi- typing as Le(a–b–) with human Lewis antibod-
nally, anti-Leb can demonstrate ABO specifici- ies (see above).
ty (anti-LebH, anti-ALeb, and anti-BLeb), and is
preferentially reactive with Le(b+) red cells of a Disease Associations
specific ABO group.24,28 Anti-LebH, the most
The Leb and H antigens are receptors for Heli-
common reactivity, is more strongly reactive
cobacter pylori, a gram-negative bacterium
with Le(b+) group O and A2 red cells than with
implicated in gastritis, peptic ulcer disease,
group A1 and B red cells, which have low H an- gastric carcinoma, mucosa-associated lym-
tigen levels. Anti-LebL is strongly reactive with phoma, and idiopathic thrombocytopenia. Leb
all Le(b+) red cells, regardless of ABO group. and Type 1 H antigens are also receptors for
Most examples of Lewis antibodies are sa- noroviruses, common causes of acute gastro-
line agglutinins that are reactive at room tem- enteritis. An Le(a–b–) phenotype is associated
perature. Unlike ABO, the agglutination is rela- with increased susceptibility to infections by
tively fragile and easily dispersed, requiring Candida and uropathogenic Escherichia coli,
gentle resuspension after centrifugation. Ag- cardiovascular disease, and possibly de-
glutination is sometimes observed after 37 C creased graft survival after renal transplanta-
incubation, but the reaction is typically weaker tion.6,25
than that at room temperature. On occasion,
Lewis antibodies can be detected in the anti-
human globulin (AHG) phase. Such detection I AND i ANTIGENS
may reflect either IgG or bound complement The I and i antigens are ubiquitous, structural-
(if polyspecific AHG reagent is used). Very ly related antigens present on all cell mem-
rarely, Lewis antibodies can cause in-vitro he- branes. The minimum epitope common to
molysis. Hemolysis occurs more often when both i and I is a terminal repeating lactos-
fresh serum containing anti-Lea or anti-Leab is amine (Gal14GlcNAc) or Type 2 chain pre-
tested, particularly against enzyme-treated red cursor. The minimum i antigen is a linear,
cells. nonbranched structure containing at least two
successive lactosamine structures.14 The I anti-
Transfusion Practice gen is a polyvalent, branched glycan derived
In general, Lewis antibodies are not consid- from the i antigen (Fig 12-4). Both i and I serve
ered clinically significant. Red cells that are as substrate and scaffold for the synthesis of
compatible in tests at 37 C, regardless of Lewis ABO, Lewis X [Gal1-4(Fuc1-3) GlcNAc],
phenotype, are expected to have normal in- and other Type 2 chain antigens.1,2,14 On red
vivo survival. It is not necessary to transfuse cells, i and I antigens are present on N-linked
antigen-negative RBCs in most patients. Un- glycoproteins and glycolipids.
like ABO antigens, Lewis antigens are extrinsic
glycolipid antigens that are readily eluted and
Phenotypes
shed from transfused red cells within a few Two phenotypes are recognized according to
days of transfusion.25 Furthermore, Lewis anti- the presence or absence of I antigen: I and i (I–).
gens in transfused plasma can neutralize Lew- The i phenotype is characteristic of neonatal
is antibodies in the recipient. For these rea- red cells, whereas I+ is the common pheno-
sons, hemolysis is rare following transfusion of type in adults. With increasing age, there is a
either Le(a+) or Le(b+) red cells. gradual increase in I antigen accompanied by
Lewis antibodies are not a cause of HD- a reciprocal decrease in i antigen; most chil-
FN.13 Lewis antibodies are predominantly of dren develop an adult I+ phenotype by age 2.14
IgM isotype and do not cross the placenta. In An increase in i antigen can occur in people
addition, Lewis antigens are poorly expressed with chronic hemolytic disorders and is a sign
on neonatal red cells, with many newborns of stressed erythropoiesis.32
CHAPTER 12 ABO, H, and Lewis Blood Groups 䡲 307
FIGURE 12-4. Structure of I gene (GCNT2) (above) and synthesis of I antigen from i antigen (below).
Gal = galactose; GlcNAc = N-acetylglucosamine; R = other upstream carbohydrate sequence.
Two genetic disorders are associated with ed with abnormal glycosylation, chronic he-
an increase in i antigen.14 The iadult phenotype molysis, splenomegaly, and erythroid multi-
(I–i+) is an autosomal recessive phenotype nuclearity.
caused by mutations in the I gene (GCNT2 or
IGnT). In populations of Asian ancestry, the Genetics
i adult phenotype can be associated with con- The I gene (GCNT2) encodes a 16 N-acetyl-
genital cataracts. Increased i antigen levels are glucosaminyltransferase that converts the lin-
also present in people with congenital dys- ear i antigen into the branched I antigen.14,18
erythropoietic anemia Type 2 (also known as The gene resides on chromosome 6p24 and
hereditary erythroblastic multinuclearity with contains five exons, including three tissue-
positive acidified serum lysis test). The latter specific exons (exons 1A, 1B, and 1C). As a re-
is a genetic defect in Golgi transport associat- sult, three slightly different mRNA transcripts
308 䡲 AABB TECHNICAL MANUAL
can be synthesized, depending on which exon group O and group A2 red cells, which are rich
1 is used. in H antigen, than with group A1 red cells. Anti-
In the iadult phenotype without cataracts, IH is suspected when serum from a group A in-
there is a mutation in exon 1C that is specific dividual directly agglutinates all group O red
for I antigen synthesis in red cells. As a conse- cells but is compatible with most group A
quence, I antigen is missing on red cells but is donor blood tested. Other examples of com-
still synthesized in other tissues that use either plex reactivity include anti-IA, -IP1, -IBH, and
exon 1A or exon 1B. In the iadult phenotype with -ILebH.24
cataracts, there is a loss of I antigen synthesis
Anti-i
in all tissues caused by either gene deletion or
mutations in exons 2 and 3. Autoanti-i is a relatively uncommon cold ag-
glutinin in sera from healthy individuals. Like
Antibodies anti-I, anti-i is primarily of IgM isotype but is
weakly reactive at 4 to 10 C. Anti-i is most
Anti-I
strongly reactive with cord and iadult red cells
Anti-I is common in the serum of healthy indi- and more weakly reactive with I+ adult red
viduals. Anti-I is usually of IgM isotype and is cells (Table 12-6). Patients with infectious
strongly reactive at 4 C with titers of <64. Sam- mononucleosis often have transient but po-
ples with higher titers may also be detectable tent anti-i. As with anti-I, complex reactivity
at room temperature. Anti-I is identified by can sometimes occur (eg, anti-iH).
strong reactions with adult red cells but weak
Cold Agglutinin Syndrome
or no agglutination with cord red cells (see Ta-
ble 12-6). Anti-I can be enhanced by 4 C incu- Autoanti-I and autoanti-i are pathologically
bation, the presence of albumin, or use of significant in cold agglutinin syndrome (CAS)
enzyme-treated red cells. An alloanti-I can be and mixed-type autoimmune hemolytic ane-
seen in the iadult phenotype. mia. In those disorders, autoanti-I (or anti-i)
Some examples of anti-I can demon- behaves as a complement-binding antibody
strate complex reactivity and are more strong- with a high titer and wide thermal range. Pri-
ly reactive with red cells of specific ABO, P1, or mary CAS occurs with lymphoproliferative dis-
Lewis phenotypes. Many of those antibodies orders (eg, Waldenström macroglobulinemia,
appear to recognize branched oligosaccha- lymphoma, and chronic lymphocytic leuke-
rides that have been further modified to ex- mia). A potent autoanti-I can also occur in the
press additional blood group antigens. Anti-IH setting of infection. Mycoplasma pneumoniae
is commonly present in the serum of A1 indi- infections are a common cause of autoanti-I
viduals. Anti-IH is more strongly reactive with and can be accompanied by a transient hemo-
TABLE 12-6. Comparative Serologic Behavior of the I/i Blood Group Antibodies with Saline Red Cell
Suspensions
4C I adult 4+ 0-1+
i cord 0-2+ 3+
i adult 0-1+ 4+
22 C I adult 2+ 0
i cord 0 2-3+
i adult 0 3+
CHAPTER 12 ABO, H, and Lewis Blood Groups 䡲 309
TABLE 12-7. Phenotypes and Prevalence in the P1PK and GLOB Group
+ + 0 + None P1 79 94
0 + 0 + P1* P2 21 6
variants.34 Analogous to the ABO system, the Pk antigen, the ultimate precursor of all globo-
rare p and Pk phenotypes are associated with type glycosphingolipids. To make the Pk anti-
the presence of naturally occurring antibodies gen, 1,4 galactosyltransferase 1 (A4GALT1)
against missing antigens (anti-P1, anti-P, and adds a terminal galactose, in an 14 linkage,
anti-PP1Pk). to CDH. The Pk antigen can then serve as a
substrate for 1,3 N-acetylgalactosaminyl-
Biochemistry transferase I (B3GALNACT1), which adds a
The synthesis of the Pk, P, and P1 antigens 13 N-acetylgalactosamine to the terminal
proceeds through the stepwise addition of galactose of Pk (Gb3) to form P antigen. In
sugars to lactosylceramide, a ceramide dihex- some cells, including red cells, the P antigen is
ose (CDH). (See Fig 12-5 and Table 12-8.) The further elongated to form additional, globo-
first step in this process is the synthesis of the family antigens, such as Luke (LKE), Type 4
GlcCer
(CDH)
A4GALT1
Lc3 Pk
(Gb3)
B3GalNAcT1
A4GALT1
Paragloboside P NOR
(PG, nLc4) (Gb4)
A4GalT1
B3GalT5
X2 SPG P1 Gb5
ST3GAL2 FUT1/2
FIGURE 12-5. Synthesis of P1, Pk, P, and related glycosphingolipid antigens. Carbohydrate structures are
shown in Table 12-8.
CHAPTER 12 ABO, H, and Lewis Blood Groups 䡲 311
CDH Gal1-4Glc1-1Cer
Gb4, P GalNAc1-3Gal1-4Gal1-4Glc1-1Cer
Gb5 Gal1-3GalNAc1-3Gal1-4Gal1-4Glc1-1Cer
NOR1 Gal1-4GalNAc1-3Gal1-4Gal1-4Glc1-1Cer
Globo-H Fuc1-2Gal1-3GalNAc1-3Gal1-4Gal1-4Glc1-1Cer
LKE NeuAc2-3Gal1-3GalNAc1-3Gal1-4Gal1-4Glc1-1Cer
NOR2 Gal1-4GalNAc1-3Gal1-4GalNAc1-3Gal1-4Gal1-4Glc1-1Cer
nLc4, PG Gal1-4GlcNAc1-3Gal1-4Glc1-1Cer
P1 Gal1-4Gal1-4GlcNAc1-3Gal1-4Glc1-1Cer
SPG NeuAc2-3Gal1-4GlcNAc1-3Gal1-4Glc1-1Cer
X2 GalNAc1-3Gal1-4GlcNAc1-3Gal1-4Glc1-1Cer
Sialosyl-X2 NeuAc2-3GalNAc1-3Gal1-4GlcNAc1-3Gal1-4Glc1-1Cer
*Glycosphingolipid family. Note: Neolacto are type 2 chain glycosphingolipids.
CDH = ceramide dihexose or lactosylceramide; Cer = ceramide; Gal = galactose; GalNAc = N-acetylgalactosamine; Glc = glu-
cose, GlcNAc = N-acetylglucosamine; NeuAc = N-acetylneuramanic acid (sialic acid); PG = paragloboside; SPG = sialosyl-
paragloboside.
triggers zoonotic illness capable of causing aplastic crisis. P1, Pk, P, and LKE antigens can
bacterial meningitis.33 Pk expression may also all serve as receptors for P-fimbriated uro-
modulate host resistance to human immuno- pathogenic E. coli, a cause of chronic urinary
deficiency virus.39 The P antigen is a receptor tract infections.33 LKE is a common oncofetal
for parvovirus B19, the etiologic agent of ery- marker present on tumors and pluripotent
thema infectiosum (fifth disease). In some pa- embryonic, mesenchymal, and very small em-
tients, B19 can cause a transient anemia or bryonic-like stem cells.4,34
KEY PO I NT S
1. The antigens of the ABO, H, Lewis, I, and P blood group systems are defined by small car-
bohydrate epitopes on glycoproteins and glycolipids. They are widely distributed on many
tissues, including embryonic stem cells, and are considered histo-blood-group antigens.
2. The ABO system contains four major ABO phenotypes: A, B, O, and AB. The four phenotypes
are determined by the presence or absence of two antigens (A and B) on red cells. An inverse
reciprocal relationship exists between the presence of A and B antigens on red cells and the
presence of anti-A, anti-B, or both, in sera.
3. ABO grouping requires both antigen typing of red cells for A and B antigen (red cell group-
ing or forward type) and screening of serum or plasma for the presence of anti-A and anti-B
isoagglutinins (serum grouping or reverse type).
4. H antigen is ubiquitously expressed on all red cells, except the rare Bombay phenotype.
5. H antigen is the precursor to both A and B antigens; thus, the amount of H antigen on red cells
depends on the person’s ABO group. H antigen is highly expressed on group O red cells be-
cause group O persons lack a functional ABO gene. In group A and B persons, the amount of H
antigen is considerably less because H is converted to the A and B antigens, respectively.
6. Lewis antigens are not synthesized by red cells but are passively adsorbed onto red cell
membranes from a pool of soluble Lewis glycolipid present in plasma.
7. The three common Lewis phenotypes indicate the presence or absence of Lewis and secre-
tor enzymes.
8. With increasing age, there is a gradual increase in I antigen accompanied by a reciprocal de-
crease in i antigen. Most children develop an adult I+ phenotype by age 2.
9. Autoanti-I and autoanti-i are pathologically significant in cold agglutinin syndrome and
mixed-type autoimmune hemolytic anemia.
10. More than 99% of donors have the P1 (P1+) or P2 (P1–) phenotype. Both phenotypes synthe-
size Pk and P antigens and differ only in the expression of the P1 antigen.
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C h a p t e r 1 3
The Rh System
Gregory A. Denomme, PhD, FCSMLS(D), Director of Immunohematology and Transfusion Services, Diagnos-
tic Laboratories, BloodCenter of Wisconsin, Milwaukee, Wisconsin, and Connie M. Westhoff, PhD, SBB,
Director of Immunohematology, Genomics, and Rare Blood, New York Blood Center, New York, New York
G. Denomme disclosed no conflicts of interest. C. Westhoff has disclosed financial relationships with Novar-
tis, BioArray, and Grifols.
317
318 䡲 AABB TECHNICAL MANUAL
sodium dodecylsulfate polyacrylamide gel type, using a single term (Table 13-2). In the
electrophoresis led to the discovery that Rh modified Wiener’s nomenclature, “R” indi-
proteins have a molecular weight of 30,000 to cates that D is present, and a subscripted
32,000 kDa.8-10 N-terminal amino acid se- number or letter indicates the C/c and E/e an-
quencing of Rh was accomplished in the late tigens: R1 for Ce, R2 for cE, Ro for ce, and RZ for
1980s.11 The findings led to the cloning of the CE. In addition, “r” indicates haplotypes that
RHCE gene by Cartron12 and colleagues in lack D, and the C/c and E/e antigens are indi-
1990 and of RHD in 1992.13,14 The different cated using superscripted symbols: r for Ce, r
RHCE alleles responsible for the C or c and E or for cE, and ry for CE (Table 13-2).
e antigens were determined in 1994.15 The International Society of Blood Trans-
fusion (ISBT) Working Party on Terminology
for Red Cell Surface Antigens adopted six-digit
T E R M I N O LO G Y
numbers to indicate red cell antigens. The first
Early Rh nomenclature reflects the differences three numbers represent the system, and the
in opinion concerning the number of genes remaining three digits refer to the antigenic
that encode DCE antigens. The Fisher-Race specificity; the Rh system was assigned Num-
terminology was based on the premise that ber 004. In 2008, the ISBT committee recog-
three closely linked genes, C/c, E/e, and D, were nized the antigens of the Rh-associated glyco-
responsible. In contrast, the Wiener nomen- protein (RHAG) as a new blood group system
clature (Rh-Hr) was based on the belief that a (Number 30).17
single gene encoded several blood group fac-
tors. There are actually two genes, RHD and
RH GENES AND R h PROT EINS
RHCE, as proposed by Tippett.16
The Fisher-Race CDE terminology is often Two genes (RHD and RHCE) are closely linked
preferred for written communication, but a on chromosome 1 and encode 416 amino ac-
modified version of Wiener’s nomenclature ids. One gene encodes the D antigen, and the
makes it possible to identify the Rh antigens other encodes CE antigens in four combina-
present on one chromosome, that is, a haplo- tions (ce, cE, Ce, or CE) [Fig 13-1(A)]. Each
320 䡲 AABB TECHNICAL MANUAL
Rh positive
DCe R1 42 17 70
DcE R2 14 11 21
Dce R0 4 44 3
DCE RZ <0.01 <0.01 1
Rh negative
ce ra 37 26 3
Ce r 2 2 2
cE r 1 <0.01 <0.01
CE ry <0.01 <0.01 <0.01
FIGURE 13-1. (A) RHD and RHCE genes. The inverted gene orientation, the antigens they encode, and the
deletion of RHD that results in the D-negative red cell phenotype are shown. (B) Predicted 12-transmembrane
domain model of the RhD and RhCE proteins. The amino acid differences between RhD and RhCE are shown as
grey circles. The zigzag lines represent the location of possible palmitoylation sites. Positions 103 and 226 in
RhCE critical for C or c and E or e expression are indicated as open circles.18
CHAPTER 13 The Rh System 䡲 321
gene has 10 exons, and the two genes share New antigens may result from single nucleo-
97% identity. The two proteins created by tide polymorphisms (SNPs) to major gene re-
these genes differ by 32 to 35 amino acids [Fig arrangements. For example, the genetic ex-
13-1(B), shown as circles on RhD], depending changes between RHD and RHCE can create
on whether D is compared to C or c. hybrid proteins that express an RhD protein
The last decade has witnessed the devel- with a portion of RhCE, or vice versa. Rear-
opment of an abundance of information on rangements are common and thought to be fa-
the genetic diversity of the RH locus, and the cilitated by the inverted orientation of the RH
antigens identified by molecular testing have genes [Fig 13-1(A)].18 The structure promotes
far exceeded the number of antigens identified hairpin loop formation and subsequent genet-
by serology. More than 275 RHD and 50 RHCE ic exchange via template conversion; one
alleles have been documented. A directory of member acts as a donor template during repli-
RHD alleles is maintained on a Rhesus-data- cation but remains unchanged in the process.
base,19 and RHCE and RHD alleles are listed on The donated region can span several base
the National Center for Biotechnology Infor- pairs, single exons, or multiple exons.
mation human blood group mutation web-
site.19,20 The ISBT Working Party on Red Cell
Immunogenetics and Blood Group Terminolo- A NT I G E N S
gy maintains, names, and catalogs new al- Routine donor and patient typing tests only for
leles.21 D. Testing for other common Rh antigens is
Most D-negative (Rh-negative) pheno- performed primarily to resolve or confirm an-
types are the result of complete deletion of the tibody identification. Exceptions include pa-
RHD gene [Fig 13-1(A)]. These phenotypes tients receiving chronic transfusion, such as in
provide the immunological rationale for why some sickle cell disease (SCD) programs, to
the transfusion of Rh-positive blood to a D- provide antigen-matched donor units to mini-
negative individual often results in produc- mize alloimmunization.
tion of anti-D. The immunogenicity of a pro-
tein correlates with the degree of foreignness Rh Phenotypes
to the host, and the large number of amino
acid differences in D explains why exposure Testing of red cells with the five principal Rh
can result in a potent immune response. antisera has been used to predict the RH geno-
RHCE [found in all but rare D– – individu- type (Table 13-3). Some haplotypes are more
als, where the dashes represent missing anti- common in certain ethnic groups. The fre-
gens] encodes both C/c and E/e antigens on a quencies of the predicted RH genotypes are
single protein. C and c antigens differ by four uncertain (eg, the frequencies of RoRo vs Ror in
amino acids, but only the serine to proline at persons of African ancestry), and frequencies
position 103 is predicted to be extracellular are more uncertain in people of mixed ethnici-
[Fig 13-1(B)]. The E and e antigens differ by ty. Serologic testing cannot determine whether
one amino acid, a proline to alanine at amino red cells are from a homozygous (D/D) or het-
acid position 226, located on the fourth extra- erozygous (D/–) person because anti-D sel-
cellular loop of the protein. The RH genes and dom shows any difference in reactivity be-
proteins shown in Fig 13-1(B) are typical of the tween red cells with a single or a double dose
majority of individuals. Commercial antibody of D antigen. RHD zygosity can be determined
reagents are available to detect the expression by DNA molecular testing for the presence of a
of the principal Rh antigens—D, C, c, E, and e RHD deletion or an inactive RHD.18
(Table 13-3). The Rh haplotype influences the level of
The five principal antigens are responsi- D antigen expression. Less D is expressed in
ble for the majority of Rh incompatibilities, al- the presence of C, a phenomenon called the
though the Rh system is more complex, with “Ceppellini effect.”22 Red cells from a DCe/DCe
61 antigens identified to date (Table 13-1). (R1R1) individual express significantly fewer D
322 䡲 AABB TECHNICAL MANUAL
TABLE 13-3. Results of Tests with Five Principal Rh Antisera with Phenotype and Predicted RH
Genotype
Antisera
Predicted Alternative
Anti-D Anti-C Anti-E Anti-c Anti-e Phenotype Genotype Genotype
Rh positive*
+ + 0 + + D, C, c, e R1r R1R0
DCe/ce DCe/Dce
R0 r
Dce/Ce
+ + 0 0 + D, C, e R1R1 R1 r
DCe/DCe DCe/Ce
+ + + + + D, C, c, E, e R1R2 R1 r
DCe/DcE DCe/cE
R2 r
DcE/Ce
RZ r
DCE/ce
R0 RZ
Dce/DCE
+ 0 0 + + D, c, e R0 r R0 R0
Dce/ce Dce/Dce
+ 0 + + + D, c, E, e R2 r R2 R0
DcE/ce DcE/Dce
R0 r
Dce/cE
+ 0 + + 0 D, c, E R2 R2 R2 r
DcE/DcE DcE/cE
+ + + 0 + D, C, E, e R1 RZ RZ r
DCe/DCE DCE/Ce
+ + + + 0 D, C, c, E R2 RZ RZ r
DcE/DCE DCE/cE
+ + + 0 0 D, C, E RZ RZ RZ r y
DCE/DCE DCE/CE
CHAPTER 13 The Rh System 䡲 323
TABLE 13-3. Results of Tests with Five Principal Rh Antisera with Phenotype and Predicted RH
Genotype (Continued)
Antisera
Predicted Alternative
Anti-D Anti-C Anti-E Anti-c Anti-e Phenotype Genotype Genotype
Rh negative†
0 0 0 + + c, e rr
ce/ce
0 + 0 + + C, c, e r r
Ce/ce
0 0 + + + c, E, e rr
cE/ce
0 + + + + C, c, E, e rr
Ce/cE
0 y 1 y 2 y
*Rare genotypes (R r , R r , and R r ) not shown (prevalence of <0.01%).
†
Rare genotypes (rry, rry, rry, and ryry) not shown (prevalence of <0.01%).
antigen sites than red cells from a DcE/DcE encode proteins with amino acid changes
(R2R2) individual. Therefore, it is important to have been reported. These alleles can cause
choose a consistent haplotype when titrating numerous variations in the expression of D,
anti-D in the antenatal setting because signifi- and red cells with some form of altered D ex-
cantly different titers can be obtained. pression are encountered in routine transfu-
sion practice. An estimated 1% to 2% of indi-
D Antigen viduals of European ethnicity carry RHD
alleles that encode altered D antigens, and the
The D antigen is composed of numerous epit- incidence in individuals of African ethnicity is
opes (designated by “epD”) that were original- higher. Altered D is organized into four groups:
ly defined by antibodies from D-positive peo- weak D, partial D (including category D), Del,
ple who made anti-D. Subsequent studies with and nonfunctional RHD.24-27
monoclonal antibodies defined 30 or more
epitopes designated as “epD1” to “epD9.”23 WEA K D. Traditionally, weak D red cells were
Each epitope has additional subdivisions (eg, defined as having a reduced amount of D anti-
epD6.1). D epitopes are highly conformational gen (formerly called “Du”) that required an in-
and consist of more than simple linear amino direct antiglobulin test (IAT) for detection.
acid residues. Amino acid changes in intracel- However, the number of samples identified as
lular regions of the protein may alter D epit- being weak D depended on the typing reagent
opes. and method used, which have changed over
the years. In 1999, Wagner and Flegel proposed
D-Positive (Rh-Positive) Phenotypes a system to classify altered D red cells on the
basis of their nucleotide substitutions (re-
Most D-positive red cell phenotypes have a viewed in Flegel and Denomme28). Weak D
conventional RhD protein [Fig 13-1(B)]. How- types are the result of an SNP that encodes a
ever, more than 275 different RHD alleles that single amino acid change predicted to be
324 䡲 AABB TECHNICAL MANUAL
located in the intracellular or transmembrane protein resulting from regions of RhD joined to
region of the protein, rather than on the outer RhCE can result in the loss of D epitopes and
surface of the red cell (Fig 13-2). The amino also generate new antigens. For example, DVI
acid changes may affect the insertion of the red cells carry the BARC antigen. A few partial
protein in the membrane and, thus, reflect the D phenotypes are the result of multiple nucle-
reduced number of D antigen sites on the red otide changes. Some partial D types are de-
cells. tected only by the IAT. In contrast to weak D
Uniquely different SNPs cause weak D types, partial changes are predicted to be lo-
Types 1-84.19 The most common is weak D cated on the exterior membrane surface (Fig
Type 1, which has a valine-to-glycine amino
13-2).29
acid substitution at position 270. Types 1, 2,
and 3 represent approximately 90% of the D EL . Red cells that express extremely low lev-
weak D types in persons of European ethnici- els of D antigen that cannot be detected by
ty.25 Weak D types can be further weakened routine serologic methods are designated as
when C is present in trans to a weak D type (eg, “D-elution” or Del. Red cells have enough D an-
r in trans with weak D Type 2). tigens to adsorb and elute anti-D. Del cells are
PARTIAL D. Red cells with “category D” have
found in 10% to 30% of D-negative people of
historically been classified by epitope studies. Asian ancestry and result from several differ-
Category D individuals type as D positive, but ent RHD mutations that severely reduce RhD
can make anti-D when they are exposed to expression in the membrane. Del cells are
conventional D antigen. Category D pheno- much less common in individuals of European
types are now classified as partial D. The ma- ancestry (0.027%) and carry different
jority of partial D phenotypes are due to hy- nucleotide substitutions than those of Asian
brid genes in which portions of RHD are ancestry.26,27 Del red cells can usually be char-
replaced by corresponding portions of RHCE acterized by RHD genotyping and careful ad-
(Fig 13-3). The novel sequences of the hybrid sorption-elution studies.
FIGURE 13-2. Structural models of weak D and partial D. The locations of amino acid changes are shown as
solid circles in the plasma membrane or on the interior. Weak D Types 1 and 2 (shown by the arrows) are found
in approximately 80% of people with weak D phenotypes. Partial D types are encoded by single amino acid
changes that are generally present on the exterior of the cell.
CHAPTER 13 The Rh System 䡲 325
FIGURE 13-3. RHD and RHCE genes. The 10 exons of RHD and RHCE are depicted as white and grey boxes, re-
spectively. Also shown are some examples of RHD encoding partial D and of RHCE with mutations often found in
people of African ethnicity. These mutations complicate transfusions in patients with sickle cell disease.
NONFUNCTI ONAL RHD. RHD genes that (Table 13-4). DHAR and Crawford phenotypes
cannot produce a full-length polypeptide are can cause D typing discrepancies. Less dra-
deemed nonfunctional and have been given matic are changes in Rhce (RHCE) proteins
allele designations.21 that mimic a D-epitope structure encoded by
D EPITOPES ON RHCE (RHCE). Expression alleles designated as “ceRT ” and “ceSL.” 30,31
of D epitopes by the protein product of the The red cells are weakly reactive with some,
RHCE gene, in the absence of RHD, further but not all, monoclonal anti-D. Most impor-
complicates serologic determination of D sta- tantly, individuals with DHAR and Crawford lack
tus. Several Rhce (RHCE) proteins have D-spe- RhD and can be sensitized to D.32,33
cific amino acids result in epitopes that are re-
ELEVATED D. Several rare deletion pheno-
active with some monoclonal anti-D. They are
more often found in a specific population. Ex- types, designated as “D– –,” “Dc–,” and “DCw–,”
amples include DHAR, which is found in indi- have an enhanced expression of D antigen and
viduals of German ancestry, and Crawford (ce- no, weak, or altered C/c and E/e antigens.34
CF), found in individuals of African ancestry. These variants are the converse of partial D
These two examples are notable because the (discussed above) and result from the replace-
red cells show strong reactivity with some ment of portions of RHCE by RHD. The addi-
monoclonal reagents but are nonreactive with tional RHD sequences in RHCE result in the
others, including polyclonal anti-D reagents additional expression of (hybrid) D along with
TABLE 13-4. Reactivity of FDA-Licensed Anti-D Reagents with Some D Variant Red Cells
326
Crawford
DVI DBT DHAR (Whites) (Blacks)
䡲
Reagent IgM Monoclonal IgG IS/AHG* IS/AHG* IS/ AHG* IS/AHG* ceRT ceSL
because no IAT is performed, the results of fusion should be based on the patient popula-
positive rosetting tests (to detect fetomaternal tion, risk of immunization to D, and limited
hemorrhage) must be carefully evaluated; ma- supply of D-negative blood components.
ternal weak D types that are reactive only in These policies should address what should be
the IAT phase have a false-positive rosette test done when a partial D type is found before the
result. patient makes anti-D. Anti-D is a clinically sig-
nificant antibody, and preventing immuniza-
D Typing Discrepancies tion in females of childbearing potential is im-
portant to avoid the complications of HDFN.
D typing discrepancies should always be in- For other patients, the complications of anti-D
vestigated and resolved (see “Resolving D Typ- are less serious, and the decision to transfuse
ing Discrepancies” below). An Rh-negative Rh-positive or Rh-negative blood should take
blood transfusion is an appropriate option for into consideration dependence on D-negative
patients needing immediate transfusion, but a blood transfusions for future bleeding epi-
thorough clerical and serologic investigation sodes and a possible increased risk of multiple
should be performed. RHD genotyping is also blood group antibodies in addition to anti-D.46
useful to resolve D typing discrepancies.45 Not all D-negative patients make anti-D
Because donor centers test for weak D, a when they are exposed to D-positive RBCs.
donor who is correctly classified as Rh positive The incidence in D-negative hospitalized pa-
may be classified as Rh negative as a recipient. tients switched to D-positive blood compo-
This discrepancy should not be considered nents is much lower, than anticipated.47 AABB
problematic but, rather, should be communi-
Standards requires that transfusion services
cated to the patient and health-care staff and
must have policies that address the adminis-
be noted in the patient’s medical record.
tration of D-positive red cells to D-negative
patients and the use of RhIG, which is a hu-
Clinical Considerations man blood product that is not entirely without
The long history of transfusing patients who risk.38(p37)
have weak D red cells with D-positive RBCs
suggests that weak D Types 1, 2, and 3, which G Antigen
are present in the majority of people of Euro-
The G antigen is found on red cells possessing
pean ancestry with weak D, are unlikely to
C or D and maps to the 103Ser residue on RhD,
make anti-D. People with these weak D types
RhCe, and RhCE (Fig 13-1). Antibodies to G ap-
can therefore safely receive D-positive blood.
pear as anti-D plus anti-C and cannot be sepa-
The less common weak D Types 11 and 15
rated. However, the antibody can be adsorbed
have been reported to make anti-D, suggesting
by either D–C+ or D+C– red cells. The presence
that they have altered D epitopes.3 Empirical
of anti-G can explain why a D-negative person
data are needed to determine the risk of pro- who was transfused with D– (C+) blood or a D-
duction of anti-D in people with other weak D negative woman who delivered a D– (C+) child
types. and subsequently appeared to have made
Unfortunately, serologic reagents cannot anti-D. Anti-D, -C, and -G can be distinguished
typically be used to distinguish people with by adsorption and elution studies.48 These
partial D that is reactive only with enhanced
analyses are not usually necessary in the pre-
methods and techniques from D-positive indi-
transfusion setting. However, it is important to
viduals. Many partial D red cells (eg, DIIIa, the
provide RhIG prophylaxis to pregnant women
most common partial D type in people of Afri-
who have anti-G only.
can ancestry) type as strongly D positive in di-
rect tests and are recognized only after the pa-
C/c and E/e Antigens
tients produce anti-D.
Policies regarding D typing procedures The common RHCE alleles encode the princi-
and selection of blood components for trans- pal C or c and E or e antigens. However, more
CHAPTER 13 The Rh System 䡲 329
than 50 different RHCE alleles are known, and cells type as C positive, but these patients can
many are associated with altered or weak ex- make apparent anti-C or anti-Ce (rhi) when
pression of the principal antigens and, in some they are stimulated.
cases, loss of high-prevalence antigens.37 Par- In individuals of African ancestry, altered
tial C and many partial e antigens are well rec- C expression most often results from the in-
ognized, and the majority are reported in indi- heritance of a RHDIIIa-CE(4-7)-D hybrid and
viduals of African ethnicity. less often of a RHD-CE(4-7)-D hybrid.37 These
hybrids do not encode D antigen, but they do
Compound Antigens (ce, Ce, cE, and CE) encode C antigen on a hybrid background that
differs from the normal background (Fig 13-3).
Compound antigens define epitopes that de- The gene has an incidence of approximately
pend on conformational changes that result 20% in people of African ancestry. It is inherit-
from amino acids associated with both C/c ed with an RHCE allele designated as
and E/e. These antigens were previously “RHCE*ceS” that encodes altered e antigen and
referred to as “cis products” to indicate that the a V–VS+ phenotype.50 The expressed product
antigens were expressed from the same haplo- of the hybrid RHDIIIa-CE(4-7)-D linked to
type. However, it is now known that these anti- RHCE*ceS is referred to as the “(C)ceS” or “rS”
gens are expressed on a single protein. These haplotype. Red cells with the rS haplotype of-
antigens are shown in Table 13-5 and include ten type as strongly C-positive with monoclo-
ce or f, Ce or rhi, cE or Rh27, and the uncom- nal reagents, and the presence of the altered C
mon CE or Rh22. is undetected.
Patients with these types of red cells fre-
Altered or Variant C and e Antigens quently make alloantibodies with C-like, and
occasionally e-like, specificities that may
Nucleotide changes in RHCE result in quanti-
appear to be autoantibodies. The red cells
tative and qualitative changes in C/c or E/e
may also lack the high-prevalence hrB antigen
antigen expression; altered C and altered e are
(hrB–).51,52 Altered or partial e expression is as-
encountered most frequently. In persons of
sociated with other RHCE*ce alleles.37,53 These
European ancestry, altered C is associated with
alleles are found primarily in people of African
amino acid changes on the first extracellular
ethnicity; some examples are shown in Fig 13-
loop of RhCe and the expression of CW
3. Some people lack the high-prevalence hrS
(Gln41Arg) or CX (Ala36Thr) antigens. Altered antigen (hrS–). Anti-hrS is a clinically signifi-
C is also associated with changes that result in cant antibody and has caused transfusion fa-
the expression of the novel antigens JAHK talities.53 Not all red cells designated as hrB– or
(Ser122Leu) and JAL (Arg114Trp). These red hrS– by serologic testing are compatible with
antibodies produced by patients with altered
or partial e expression.
TABLE 13-5. Compound Rh Antigens on Rh An additional complication is that altered
Proteins RHCE*ce is often inherited with a partial RHD
(eg, DIII, DAU, or DAR).54 As discussed above,
Compound Present on Red
Antigen Cells with These
patients with partial D red cells are at risk of
Designation Rh Protein Haplotypes producing anti-D.
Most Rh antibodies are IgG, although some High-Protein Reagents and Controls
sera may have an IgM component. Typically, Some Rh reagents for use in slide, rapid tube,
Rh antibodies do not activate complement, al- or microplate tests contain high concentra-
though rare exceptions have been reported. As tions of protein (20% to 24%) and other mac-
a result, in transfusion reactions involving Rh romolecular additives. These reagents are pre-
antibodies, hemolysis is primarily extravascu- pared from pools of human sera and give
lar rather than intravascular. reliable results; however, high-protein levels
Most Rh antibodies have the potential to and macromolecular additives may cause
cause clinically significant HDFN and transfu- false-positive reactions (see “Causes of False-
sion reactions. Anti-c, which is clinically the Positive and False-Negative Rh Typing Results”
most important Rh antibody after anti-D, may below). These reagents must be used accord-
cause severe HDFN. Anti-C, -E, and -e do not ing to the manufacturers’ instructions and with
often cause HDFN, and when they do, it is usu- the appropriate controls. False-positive results
ally mild. The reactivity of Rh antibodies is en- could cause a D-negative patient to receive D-
hanced by enzyme treatment of red cells, and positive blood and become immunized. If red
332 䡲 AABB TECHNICAL MANUAL
cells exhibit aggregation in the control test, the Causes of False-Positive and False-
results of the test are not valid. Negative Rh Typing Results
Low-Protein Reagents and Controls False-positive typing results can be caused by:
Most Rh reagents in routine use are low-pro- 1. Immunoglobulin coating of the cells as a
tein reagents formulated predominantly with result of warm or cold autoagglutinins.
IgM monoclonal antibodies. Spontaneous ag- Wash the red cells several times and retest
glutination causing a false-positive result can them with low-protein reagents by direct
occur, although this happens much less fre- methods. If an IAT is required, IgG coating
quently than with high-protein reagents. A on the red cells can be removed by treating
negative result from a test that was performed
the cells with glycine/EDTA (Method 2-21)
concurrently with a similar reagent serves as a
or chloroquine (Method 2-20) and retest-
control. For example, for ABO and Rh typing,
ing.
the absence of agglutination by anti-A or anti-B
2. Induction of rouleaux by serum factors that
serves as a negative control for spontaneous
aggregation. For red cells that show agglutina- can be eliminated by thoroughly washing
tion with all reagents (eg, type AB or D+), a the red cells and retesting.
control performed as described by the reagent 3. Use of the wrong reagent.
manufacturer is required (with the exception 4. Contamination with reagent from another
of donor retyping). In most cases, a suitable vial.
control is a suspension of the patient’s red cells 5. Nonspecific aggregation of the red cells due
with autologous serum or 6% to 10% albumin. to some component of the reagent other
Indirect antiglobulin testing is not valid for red than the antibody (ie, a preservative, antibi-
cells with a positive direct antiglobulin test otic, or dye).
(DAT) result unless a method is used to re- 6. Testing of polyagglutinable red cells agglu-
move the IgG antibody. Positive and negative tinated with reagents that contain human
controls should be tested, and the positive serum.
control cells should have a single dose of the
antigen or be known to show weak reactivity. False-negative typing results can be caused by:
KEY POINTS
1. The Rh system is highly immunogenic, complex, and polymorphic. More than 50 Rh anti-
gens have been characterized, although the five principal antigens—D, C, c, E, and e—are
responsible for the majority of clinically significant antibodies.
2. “Rh positive” and “Rh negative” refer to the presence or absence, respectively, of the D anti-
gen.
3. Contemporary Rh terminology distinguishes between antigens (such as D and C), genes
(such as RHD and RHCE), alleles (such as RHCE*ce and RHCE*Ce), and proteins (such as
RhD and Rhce).
4. Most D-negative (Rh-negative) phenotypes result from complete deletion of the RHD gene.
Exposure of D-negative individuals to RhD often results in the development of anti-D.
5. RHCE encodes both C/c and E/e antigens on a single protein. C and c differ by four amino
acids, whereas E and e differ by one amino acid.
6. Routine donor and patient Rh typing procedures test only for D. Testing for other common
Rh antigens is used to resolve or confirm antibody identification and, for many SCD trans-
fusion programs, or for other patients receiving chronic transfusions to match patients and
donors for D, C, and E.
7. Weak D phenotypes are defined as having a reduced amount of D antigen and are detected
by IAT. Weak D usually results from amino acid changes that impair the insertion of the pro-
tein in the membrane. Many different mutations cause weak expression of D.
8. RHD genotyping can identify those pregnant females and blood transfusion recipients with
a serologic weak D phenotype who can be managed safely as Rh positive.
9. Most anti-D reagents approved by the FDA combine a monoclonal IgM (that is reactive at
room temperature for routine testing) and a monoclonal or polyclonal IgG (that is reactive
by IAT for the determination of weak D). The exception is anti-D for column agglutination
testing, which contains only IgM. These reagents may show different reactivity with red cells
that have weak D, partial D, or D-like epitopes.
334 䡲 AABB TECHNICAL MANUAL
10. When determining the D type of a patient, an IAT for weak expression of D is not necessary
except when testing the red cells of an infant born to a mother at risk of D immunization.
Rh-negative donors must be tested by a method that detects weak D.
11. Most Rh antibodies are IgG, although some may have an IgM component. With rare excep-
tions, Rh antibodies do not activate complement and, thus, cause primarily extravascular
rather than intravascular hemolysis. Antibodies almost always result from red cell immuni-
zation through pregnancy or transfusion.
REFER ENCES
1. Rosenfeld R. Who discovered Rh? A personal the human blood group RhD polypeptide.
glimpse of the Levine-Wiener argument. Proc Natl Acad Sci U S A 1992;89:10925-9.
Transfusion 1989;29:355-7. 14. Arce MA, Thompson ES, Wagner S, et al. Mo-
2. Levine P, Stetson RE. An unusual case of intra- lecular cloning of RhD cDNA derived from a
group agglutination. JAMA 1939;113:126-7. gene present in RhD-positive, but not RhD-
3. Flegel WA. Homing in on D antigen immuno- negative individuals. Blood 1993;82:651-5.
genicity. Transfusion 2005;45:466-8. 15. Simsek S, de Jong CAM, Cuijpers HTM, et al.
4. Mollison PL, Hughes-Jones NC, Lindsay M, Sequence analysis of cDNA derived from retic-
Wessely J. Suppression of primary RH immu- ulocyte mRNAs coding for Rh polypeptides
nization by passively-administered antibody: and demonstration of E/e and C/c polymor-
Experiments in volunteers. Vox Sang 1969;16: phism. Vox Sang 1994;67:203-9.
421-39. 16. Tippett P. A speculative model for the Rh blood
5. Freda V, Gorman J, Pollack W. Rh factor: Pre- groups. Ann Hum Genet 1986;50 (Pt 3):241-7.
vention of isoimmunization and clinical trials 17. Daniels G, Castilho L, Flegel WA, et al. Interna-
in mothers. Science 1966;151:828-30. tional Society of Blood Transfusion Committee
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13. Le Van Kim C, Mouro I, Cherif-Zahar B, et al. 23. Scott ML, Voak D, Liu W, et al. Epitopes on Rh
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CHAPTER 13 The Rh System 䡲 335
24. Ye L, Wang P, Gao H, et al. Partial D phenotypes 40. Wagner T, Kormoczi GF, Buchta C, et al. Anti-D
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D(el) and weak D phenotypes in Chinese. Vox Transfusion 2000;40:428-34.
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28. Flegel WA, Denomme GA. Allo- and autoanti- 44. Lacey PA, Caskey CR, Werner DJ, Moulds JJ.
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29. Wagner FF, Frohmajer A, Ladewig B, et al. Transfusion 1983;23:91-4.
Weak D alleles express distinct phenotypes. 45. Flegel WA, Denomme GA, Yazer MH. On the
Blood 2000;95:2699-708. complexity of D antigen typing: A handy deci-
30. Wagner FF, Ladewig B, Flegel WA. The RHCE sion tree in the age of molecular blood group
allele ceRT: D epitope 6 expression does not diagnostics. J Obstet Gynaecol Can 2007;29:
require D-specific amino acids. Transfusion 746-52.
46. Schonewille H, van de Watering LM, Brand A.
2003;43:1248-54.
Additional red blood cell alloantibodies after
31. Chen Q, Hustinx H, Flegel WA. The RHCE al-
blood transfusions in a nonhematologic allo-
lele ceSL: The second example for D antigen
immunized patient cohort: Is it time to take
expression without D-specific amino acids.
precautionary measures? Transfusion 2006;
Transfusion 2006;46:766-72.
46:630-5.
32. Beckers EA, Porcelijn L, Ligthart P, et al. The
47. Frohn C, Dumbgen L, Brand J-M, et al. Proba-
RoHAR antigenic complex is associated with a
bility of anti-D development in D–patients re-
limited number of D epitopes and alloanti-D
ceiving D+ RBCs. Transfusion 2003;43:893-8.
production: A study of three unrelated persons 48. Issitt PD, Anstee DJ. Applied blood group se-
and their families. Transfusion 1996;36:104-8. rology. 4th ed. Durham, NC: Montgomery Sci-
33. Westhoff CM. Review: The Rh blood group D entific Publications, 1998.
antigen: Dominant, diverse, and difficult. Im- 49. Singleton BK, Green CA, Avent ND, et al. The
munohematol 2005;21:155-63. presence of an RHD pseudogene containing a
34. Daniels G. Human blood groups. 2nd ed. Cam- 37 base pair duplication and a nonsense mu-
bridge, MA: Blackwell Science, 2002. tation in Africans with the Rh D-negative
35. Race RR, Sanger R. Blood groups in man. 6th blood group phenotype. Blood 2000;95:12-18.
ed. Oxford: Blackwell, 1975. 50. Daniels GL, Faas BH, Green CA, et al. The VS
36. Colin Y, Cherif-Zahar B, Le Van Kim C, et al. and V blood group polymorphisms in Afri-
Genetic basis of the RhD-positive and RhD- cans: A serologic and molecular analysis.
negative blood group polymorphism as deter- Transfusion 1998;38:951-8.
mined by Southern analysis. Blood 1991;78: 51. Reid ME, Storry JR, Issitt PD, et al. Rh haplo-
2747-52. types that make e but not hrB usually make VS.
37. Reid ME, Lomas-Francis C, Olsson ML. The Vox Sang 1997;72:41-4.
blood group antigen factsbook. 3rd ed. San Di- 52. Vege S, Westhoff CM. Molecular characteriza-
ego, CA: Academic Press, 2012. tion of GYPB and RH in donors in the Ameri-
38. Levitt J, ed. Standards for blood banks and can Rare Donor Program. Immunohematol
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AABB, 2014. 53. Noizat-Pirenne F, Lee K, Pennec PY, et al. Rare
39. Schmidt PJ, Morrison EC, Shohl J. The antige- RHCE phenotypes in black individuals of Afro-
nicity of the Rh o (D u ) blood factor. Blood Caribbean origin: Identification and transfu-
1962;20:196-202. sion safety. Blood 2002;100:4223-31.
336 䡲 AABB TECHNICAL MANUAL
54. Westhoff CM, Vege S, Halter-Hipsky C, et al. 64. Polin H, Danzer M, Hofer K, et al. Effective mo-
DIIIa and DIII Type 5 are encoded by the same lecular RHD typing strategy for blood dona-
allele and are associated with altered RHCE*ce tions. Transfusion 2007;47:1350-5.
alleles: Clinical implications. Transfusion 65. Chou St, Westhoff CM. The role of molecular
2010;50:1303-11. immunohematology in sickle cell disease.
55. Ness PM. To match or not to match: The ques- Transfus Apher Sci 2011;44:73-9.
tion for chronically transfused patients with 66. Fasano RM, Monaco A, Meier ER, et al. RH ge-
sickle cell anemia. Transfusion 1994;34:558-60. notyping in a sickle cell disease patient con-
56. Vichinsky EP, Luban NL, Wright E, et al. Pro- tributing to hematopoietic stem cell trans-
spective RBC phenotype matching in a stroke plantation donor selection and management.
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center transfusion trial. Transfusion 2001;41: 67. Tilley L, Green C, Poole J, et al. A new blood
1086-92. group system, RHAG: Three antigens resulting
57. Chou ST, Jackson T, Vege S, et al. High preva- from amino acid substitutions in the Rh-asso-
lence of red blood cell alloimmunization in ciated glycoprotein. Vox Sang 2010;98:151-9.
sickle cell disease despite transfusion from Rh- 68. Dahl KN, Parthasarathy R, Westhoff CM, et al.
Protein 4.2 is critical to CD47-membrane skel-
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58. Reid ME, Rios M, Powell VI, et al. DNA from
69. Nicolas V, Le Van Kim C, Gane P, et al.
blood samples can be used to genotype pa-
RhRhAG/ankyrin-R, a new interaction site be-
tients who have recently received a transfu-
tween the membrane bilayer and the red cell
sion. Transfusion 2000;40:48-53.
skeleton, is impaired by Rh(null)-associated
59. Pirelli KJ, Pietz BC, Johnson ST, et al. Molecular
mutation. J Biol Chem 2003;278:25526-33.
determination of RHD zygosity: Predicting risk 70. Food and Drug Administration. Draft Guid-
of hemolytic disease of the fetus and newborn ance: Recommended methods for blood
related to anti-D. Prenat Diagn 2010;12-13: grouping reagents evaluation. Silver Spring,
1207-12. MD: CBER Office of Communication, Out-
60. Matheson KA, Denomme GA. Novel 3 rhesus reach, and Development, 1992. [Available at
box sequences confound RHD zygosity assign- http://www.fda.gov/downloads/Biologics
ment. Transfusion 2002;42:645-50. BloodVaccines/GuidanceComplianceRegula
61. Lo YM, Corbetta N, Chamberlain PF, et al. toryInformation/Guidances/Blood/ucm
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serum. Lancet 1997;350:485-7. 71. Judd WJ, Moulds M, Schlanser G. Reactivity of
62. Van der Schoot CE, Soussan AA, Koelewijn J, et FDA-approved anti-D reagents with partial D
al. Non-invasive antenatal RHD typing. Trans- red blood cells. Immunohematol 2005;21:146-
fus Clin Biol 2006;13:53-7. 8.
63. Gassner C, Doescher A, Drnovsek TD, et al. 72. Denomme GA, Dake LR, Vilensky D, et al. Rh
Presence of RHD in serologically D–, C/E+ discrepancies caused by variable reactivity of
individuals: A European multicenter study. partial and weak D types with different sero-
Transfusion 2005;45:527-38. logic techniques. Transfusion 2008;48:473-8.
C h a p t e r 1 4
Jill R. Storry, PhD, FIBMS, Associate Professor, Region Skane Office of Medical Services, Department of Clini-
cal Immunology and Transfusion Medicine, Lund, Sweden.
The author has disclosed no conflicts of interest.
337
TABLE 14-1. Clinical Significance of Antibodies to Blood Group Antigens
338
ISBT System No. of Hemolytic Transfusion Reaction (HTR), Acute (AHTR), or Hemolytic Disease of the Fetus and Newborn
No. Name Antigens Delayed (DHTR) (HDFN)
䡲
002 MNS 46 Rare examples of anti-M and -N active at 37 C cause AHTRs Anti-S, -s, -U, and some other antibodies cause severe
and DHTRs; anti-S, -s, -U, and some other antibodies may HDFN; anti-M rarely causes severe HDFN.
cause AHTRs and DHTRs.
003 P1PK 3 Only very rare examples active at 37 C cause AHTRs and No.
DHTRs.
004 Rh 55 Rh antibodies can cause severe AHTRs and DHTRs Anti-D can cause severe HDFN (see Chapter 22).
(see Chapter 13).
005 Lutheran 20 Anti-Lua and -Lub have caused mild DHTRs; anti-Lu8 has No.
caused AHTRs.
006 Kell 35 Kell antibodies can cause severe AHTRs and DHTRs. Anti-K can cause severe HDFN.
AABB TECHNICAL MANUAL
007 Lewis 6 Anti-Lea and -Leb are not generally considered to be clinically No.
significant.
008 Duffy 5 Anti-Fya, -Fyb, and -Fy3 cause AHTRs and DHTRs; anti-Fy5 Anti-Fya and -Fyb cause HDFN.
causes DHTRs.
009 Kidd 3 Anti-Jka is a common cause of DHTRs; anti-Jka and -Jk3 also No. Anti-Jka does not usually cause HDFN.
cause AHTRs.
010 Diego 22 One anti-Dia caused DHTR, but there is little evidence; anti-Dib, Anti-Dia, -Dib, -Wra, and -Wrb plus some others have caused
DHTRs, and anti-Wra cause HTRs. severe HDFN.
014 Dombrock 8 Anti-Doa and -Dob cause AHTRs and DHTRs. No.
015 Colton 4 Anti-Coa causes AHTRs and DHTRs; anti-Cob and -Co3 have Anti-Coa has caused severe HDFN, and -Co3 has caused mild
caused mild HTRs. HDFN.
018 H 1 Anti-H in Bombay phenotype can cause severe intravascular Anti-H in Bombay phenotype has the potential to cause
HTRs; anti-HI in para-Bombay is not usually clinically signifi- severe HDFN (see Chapter 12).
cant (see Chapter 12).
019 Kx 1 Anti-Kx and -Km in McLeod syndrome has caused severe Antibodies found only in males.
HTRs.
020 Gerbich 11 No. Three examples have been reported of anti-Ge3 causing
CHAPTER 14
HDFN.
024 Ok 3 Anti-Oka is very rare and no cases of HTR have been reported. No.
Other Blood Groups
026 JMH 6 One example has been reported of anti-JMH causing AHTR. No.
䡲
(Continued)
339
340
䡲
ISBT System No. of Hemolytic Transfusion Reaction (HTR), Acute (AHTR), or Hemolytic Disease of the Fetus and Newborn
No. Name Antigens Delayed (DHTR) (HDFN)
028 Globoside 1 Globoside has caused intravascular HTRs. No, but anti-PP1Pk is associated with a high rate of spontane-
ous abortion.
032 JR 1 Mild DHTRs and one case of AHTR have been reported to be Two examples of severe HDFN due to anti-Jra have been
AABB TECHNICAL MANUAL
033 Lan 1 Mild to severe HTR due to anti-Lan has been reported. Anti-Lan is generally not a cause of HDFN, although cases of
mild HDFN have been reported.
034* Vel 1 Severe AHTR and mild to severe DHTR due to anti-Vel have Anti-Vel is generally not a cause of HDFN, although cases of
been reported. severe HDFN have been reported.
The MNS Glycoproteins and the Genes TABLE 14-2. Frequencies of Some Phenotypes
that Encode Them of the MNS System
FIGURE 14-1. Model of two proposed membrane complexes containing Band 3 and Rh proteins: 1) containing tetramers of Band 3 and heterotrimers of RhD, RhCE, and
RhAG, and linked to the spectrin matrix of the cytoskeleton through Band 3, protein 4.2, and ankyrin; and 2) containing Band 3, RhD, and RhCE, and linked to the
spectrin/actin junction through glycophorin C (GPC), p55, and protein 4.1 and through Band 3 and adducin.
CHAPTER 14 Other Blood Groups 䡲 343
FIGURE 14-2. GYPA, GYPB, and the hybrid GYPB–A–B gene responsible for GP.Mur, and a representation of the
proteins they encode, showing the regions of proteins encoded by the various exons.
= pseudoexon not represented in the mRNA or the encoded protein.
Other MNS Antigens and Antibodies individuals who lack all or part of GPA have
caused severe HTRs and HDFN.
The other MNS antigens are either of high or
low prevalence in most populations. The simi-
larity of sequence between certain regions of T HE LUT H E R A N SY S TE M
GYPA and GYPB may occasionally lead to
Lutheran is a complex system consisting of 20
GYPA pairing with GYPB during meiosis. If re-
antigens, including four antithetical pairs: Lua/
combination then occurs, either by crossing
Lub (His77Arg); Lu6/Lu9 (Ser275Phe); Lu8/
over or by a less well-defined mechanism
called “gene conversion,” a hybrid gene can be Lu14 (Met204Lys); and Aua/Aub (Thr529Ala).16
formed consisting partly of GYPA and partly of The other Lutheran antigens are highly preva-
GYPB. A large variety of these rare hybrid lent in all populations tested. Lua (LU1) has a
genes exists, and they give rise to low-preva- prevalence of about 8% in people of European
lence antigens and, in the homozygous state, or African ethnicity but is rare elsewhere; its
to phenotypes that lack high-prevalence anti- antithetical antigen, Lub (LU2), is common
gens.10 Red cells of some of the phenotypes re- everywhere. In the other Lutheran polymor-
sulting from hybrid genes react with an anti- phism, Aua and Aub have a prevalence of
body called “anti-Mia.” The phenotypes were around 80% and 50%, respectively, in people of
grouped together as the Miltenberger series, European ancestry.
but this classification is obsolete because the Lutheran antigens are destroyed by treat-
hybrids are now well defined.14 ment of the red cells with trypsin or -chymo-
One example that is relatively common in trypsin, whereas papain and ficin have little ef-
Southeast Asia is the hybrid gene that is re- fect. Most Lutheran antibodies are not reactive
sponsible for the GP.Mur (previously Mi.III) with red cells treated with the sulfhydryl re-
phenotype. The hybrid gene is mostly GYPB, agents 2-aminoethylisothiouronium bromide
but a small region of GYPB encompassing the (AET) or dithiothreitol (DTT), which reduce
3 end of the pseudoexon and the 5 end of the the disulfide bonds of the immunoglobulin su-
adjacent intron has been replaced by the perfamily (IgSF) domains (Method 3-18).
equivalent region from GYPA. This means that The Lutheran antigens are located on a
the defective splice site in GYPB is now re- pair of glycoproteins that span the membrane
placed by the functional splice site from GYPA, once, have five extracellular IgSF domains, and
and the new, composite exon is expressed in differ by the length of their cytoplasmic do-
the mRNA and represented in the protein.10 mains as a result of alternative RNA splicing.
This provides an unusual amino acid se-
The isoform with the longer cytoplasmic do-
quence that is immunogenic and represents
main interacts with spectrin of the membrane
the antigen Mur. The amino acid sequence
skeleton.17 The location of the Lutheran anti-
that results from the junction of exons B3 and
gens on the IgSF domains is shown in Fig 14-3.
A3 gives rise to Hil and MINY (Fig 14-2).
The Lutheran glycoproteins are adhesion mol-
Mur antigen is rare in people of European
and African ethnicity but has a prevalence of ecules that bind isoforms of laminin that con-
about 7% in people of Chinese and 10% in tain -5 chains. Laminin is a glycoprotein of
people of Thai ancestry. Anti-Mur has the po- the extracellular matrix, and Lutheran-laminin
tential to cause severe HTRs and HDFN. In interactions may play a role in the migration of
Hong Kong and Taiwan, anti-Mur is the most mature erythroid cells from the marrow to the
common blood group antibody after anti-A peripheral blood at the latest stages of erythro-
and -B. In Southeast Asia, it is important that poiesis. Upregulation of Lutheran glycopro-
red cells for antibody detection include a Mur+ teins on red cells of patients with sickle cell
sample.15 disease could play a part in adhesion of these
Antibodies to regions of GPA with the ge- cells to the vascular endothelium and the re-
neric name Ena that may be made by very rare sultant crises of vascular occlusion.17
CHAPTER 14 Other Blood Groups 䡲 345
TH E K E L L A N D K X S Y S T EMS
The antigen often referred to as “Kell,” but cor-
rectly named “K” or “KEL1,” is the original an-
tigen of the Kell system and the first blood
group antigen to be identified following the
discovery of the antiglobulin test in 1946. Its
FIGURE 14-3. Diagram of the two isoforms of the
antithetical antigen, k or KEL2, was identified
Lutheran glycoprotein, showing the five extracellular
3 years later. The Kell system now consists of
immunoglobulin superfamily domains and the
35 antigens numbered from KEL1 to KEL38, of
location of the Lutheran antigens on these domains,
which three are obsolete.21,22 The Kell system
the single membrane-spanning domain, and the
cytoplasmic domains. includes six pairs (K/k, Jsa/Jsb, K11/K17, K14/
K24, VLAN/VONG, and KYO/KYOR) and one
triplet (Kpa/Kpb/Kpc) of Kell antithetical anti-
gens. Initially, most antigens joined the Kell
The extremely rare Lunull phenotype aris- system through genetic associations observed
es from homozygosity for inactive LU gene.18 in family studies. These associations have now
The red cells lack any expression of Lutheran been confirmed by DNA sequencing of the
antigens, and individuals with this phenotype KEL gene.
346 䡲 AABB TECHNICAL MANUAL
The Kell Glycoprotein and the KEL TABLE 14-3. Frequencies of Some Kell
Gene Phenotypes
Kell Antigens
K has a prevalence of about 9% in people of
FIGURE 14-4. Diagram of the Kell and Xk proteins European ancestry and about 1.5% in people
linked by a single disulfide bond. The Xk protein has of African ancestry. It is rare in East Asia (Table
cytoplasmic N- and C-terminal domains and 10 14-3). The k antigen is highly prevalent in all
membrane-spanning domains. The Kell glycoprotein populations. K and k result from a single nu-
has a large, folded, extracellular, C-terminal domain cleotide polymorphism (SNP) in exon 6, which
and an intracellular N-terminal domain. encodes Met193 in K and Thr193 in k. Asn191
CHAPTER 14 Other Blood Groups 䡲 347
is N-glycosylated in the product of the k but Kell antigens are resistant to papain, ficin,
not the K allele. trypsin, and -chymotrypsin but are de-
Kpa (KEL3) is found in about 2% of people stroyed by a mixture of trypsin and -chymo-
of European ancestry and is not present in trypsin. They are also destroyed by DTT and
people of African or Japanese ancestry (Table AET (see above) and by EDTA glycine.
14-3); Kpb (KEL4) has high prevalence in all
populations. Whereas 2.3% of people of Euro- Kell Antibodies and Their Clinical
pean ancestry are Kp(a+) only 1.2% of K+ per- Significance
sons of that same ancestry are Kp(a+). Nine
percent of people of European ancestry are K+, Kell antibodies are usually IgG, predominantly
but only 2.7% of Kp(a+) people from the same IgG1.13 They should be considered potentially
population are K+. This strong allelic associa- clinically significant from the perspective of
tion has been confirmed by family studies. causing severe HDFN and HTRs. Patients with
Only one example of the KKpa haplotype has Kell antibodies should be transfused with anti-
been found.24 Kpc (KEL21), an antigen with gen-negative blood whenever possible.
very low prevalence, is the product of another Anti-K is the most common immune red
allele of Kpa and Kpb. KEL alleles encoding the cell antibody outside the ABO and Rh systems;
three Kp antigens differ by single base substi- one-third of all non-Rh red cell immune anti-
tutions within codon 281 (exon 8): Kpa, TGG, bodies are anti-K. An antiglobulin test is usual-
Trp281; Kpb, CGG, Arg281; and Kpc, CAG, ly the method of choice for detecting anti-K,
Gln281. The mutation associated with Kpa ex- although occasional samples may agglutinate
pression appears to reduce the quantity of Kell red cells directly. Most anti-K appears to be in-
glycoprotein in the red cell membranes, giving duced by blood transfusion. Because anti-K
rise to a slight reduction in expression of Kell can cause severe HDFN, it is usual practice in
antigens in Kpa/Kpa homozygotes but a more some countries for girls and women of child-
obvious weakening of Kell antigens in individ- bearing potential to receive only K– red cells.
uals who are heterozygous for Kpa and the null Anti-K, -k, -Kpa, -Kpb, -Jsa, -Jsb, -Ku, -Ula, -K11,
allele K0. -K19, and -K22 are all reported to have caused
Jsa (KEL6) is almost completely confined severe HDFN. Anti-K, -k, -Kpb, -Jsa, -Jsb, -Ku,
to people of African ethnicity. The prevalence and -K19 have all been implicated in acute or
of Jsa in African Americans is about 20% (Table delayed HTRs.
14-3). Jsb (KEL7) is highly prevalent in all popu- The pathogenesis of HDFN caused by
lations, and Js(a+b–) has not been found in anti-K differs from that resulting from anti-D.
persons of non-African ethnicity. Jsa represents Anti-K HDFN is associated with lower concen-
Pro597; Jsb, Leu597. trations of amniotic fluid bilirubin than anti-D
K17 (Wka) (Ala302) has a prevalence of HDFN of comparable severity. Postnatal hy-
0.3% in English donors; K11 (Val302), its anti- perbilirubinemia is not prominent in infants
thetical antigen, has very high prevalence. K14 with anemia caused by anti-K. There is also re-
(Arg180) and K24 (Pro180) are very high- and duced reticulocytosis and erythroblastosis in
very low-prevalence antigens, respectively. the anti-K disease compared with anti-D
VLAN and VONG are low-prevalence antigens HDFN. These symptoms suggest that anti-K
associated with Arg248Gln and Arg248Trp, re- HDFN is associated with a lower degree of he-
spectively. molysis and that fetal anemia in anti-K HDFN
The presence of the low-prevalence anti- results predominantly from a suppression of
gens Ula, KYO, and K23 and the absence of the erythropoiesis. The Kell glycoprotein appears
high-prevalence antigens K12, K13, K18, K19, on erythroid progenitors at a much earlier
K22, TOU, RAZ, KALT, KTIM, KUCI, KANT, stage of erythropoiesis than do Rh antigens.
KASH, KELP, KETI, and KHUL result from sin- Consequently, anti-K probably facilitates
gle amino acid substitutions in the Kell glyco- phagocytosis of K+ erythroid progenitors at an
protein. early stage of development by macrophages in
348 䡲 AABB TECHNICAL MANUAL
the fetal liver, before the erythroid cells pro- dopeptidases that process a variety of peptide
duce hemoglobin.25 hormones. Although the function of the Kell
Antibodies mimicking Kell specificities glycoprotein is not known, it is enzymatically
have been responsible for severe AIHA. Pres- active and can cleave the biologically inactive
ence of the autoantibody is often associated peptide big-endothelin-3 to create the biologi-
with apparent depression of all Kell antigens. cally active vasoconstrictor endothelin-3. Con-
Although most examples of anti-K are stimu- sequently, Kell might play a role in regulating
lated by pregnancy or transfusion, a few cases vascular tone, but there is no direct evidence
of apparently non-red-cell immune anti-K for this.28 No obvious pathogenesis is associat-
have been described. In some cases, the anti- ed with the K0 phenotype.
bodies were found in untransfused, healthy, In addition to erythroid cells, Kell anti-
male blood donors; in others, microbial infec- gens may be present on myeloid progenitor
tion was implicated as an immunizing agent. cells, and Kell glycoprotein has been detected
in testis, lymphoid tissues, and with Xk protein
Null (K0) and Mod Phenotypes in skeletal muscle.
Like most blood group systems, Kell has a null
Kx Antigen (XK1), McLeod Syndrome,
phenotype (K0) in which none of the Kell anti-
gens is expressed and the Kell glycoprotein and McLeod Phenotype
cannot be detected in the membrane. Immu- Kx is the only antigen of the Kx blood group
nized K0 individuals may produce anti-Ku system. It is located on a polytopic protein that
(anti-KEL5), an antibody that is reactive with spans the red cell membrane 10 times and is
all cells except those of the K0 phenotype. Ho- linked to the Kell glycoprotein by a single di-
mozygosity for a variety of nonsense, mis- sulfide bond (Fig 14-4). Xk protein is encoded
sense, and splice-site mutations have been as- by the XK gene on chromosome Xp21.1.
sociated with K0 phenotype.26 McLeod syndrome is a very rare X-linked
Kmod red cells have only very weak expres- condition that develops almost exclusively in
sion of Kell antigens, and individuals with this males and is associated with acanthocytosis
phenotype are homozygous (or doubly hetero- and a variety of late-onset muscular, neurolog-
zygous) for missense mutations, resulting in ic, and psychiatric symptoms. It results from
single-amino-acid substitutions within the hemizygosity for inactivating mutations and
Kell glycoprotein.27 Some Kmod individuals deletions of the XK gene.29 McLeod syndrome
make an antibody that resembles anti-Ku but is associated with McLeod phenotype, in
differs in being nonreactive with Kmod red cells. which Kell antigens are expressed weakly and
Other phenotypes in which Kell antigens have Km (KEL20) as well as Kx are absent. When
substantially depressed expression result from transfused, people with McLeod phenotype
Kpa/K0 heterozygosity (see above), absence of without chronic granulomatous disease (CGD)
Xk protein (see below), and absence of the produce anti-Km only, which is compatible
Gerbich antigens Ge2 and Ge3, which are lo-
with both McLeod and K0 phenotype red cells.
cated on the glycophorins C and D. The reason
The function of the Xk-Kell complex is not
for this phenotypic association between Kell
known, but Xk has structural resemblance to a
and Gerbich is not known, although Kell glyco-
family of neurotransmitter transporters.
proteins, Xk, and glycophorins C and D could
Deletion of part of the X chromosome
all be located within the same membrane pro-
that includes XK may also include CYBB, ab-
tein complex (Fig 14-1).
sence of which is responsible for X-linked
CGD. When transfused, CGD patients with
Functional Aspects
McLeod syndrome usually produce anti-Kx
The Kell protein has structural and sequence plus anti-Km, making it almost impossible to
homology with a family of zinc-dependent en- find compatible donors. It is recommended,
CHAPTER 14 Other Blood Groups 䡲 349
therefore, that transfusion of males with CGD The coding region for the Fy allele in peo-
and McLeod syndrome be avoided. ple of African ancestry is identical to that of
the Fyb allele, which encodes Asp42. Fy pro-
duces no Duffy glycoprotein and no Fyb anti-
THE DUF FY SY STE M
gen in red cells because of an SNP in the pro-
The Duffy system officially includes five anti- moter region of DARC. This mutation disrupts
gens that reside on a glycoprotein known as the binding site for the erythroid-specific
the Duffy antigen receptor for chemokines GATA-1 transcription factor and prevents ex-
(DARC). The DARC gene consists of two exons, pression of the gene in erythroid tissue.30 Duffy
with exon 1 encoding only the first seven ami- glycoprotein is present on many cells through-
no acids of the Duffy glycoprotein.21,22 DARC is out the body; thus, Fy(a–b–) people of African
on chromosome 1q23.2. ethnicity lack Duffy glycoprotein on their red
cells but not on cells from other tissues. This
Fya (FY1) and Fyb (FY2) explains why they do not make anti-Fyb and
only very rarely make anti-Fy3 or anti-Fy5 (see
In people of European and Asian ancestry, the below). Although the GATA-1 binding site mu-
Duffy polymorphism consists of two antigens, tation in people of African ancestry has been
Fya and Fyb, giving rise to three phenotypes: found only in Duffy genes with the Fyb se-
Fy(a+b–), Fy(a+b+), and Fy(a–b+) (Table 14-4). quence, the mutation has been detected in
The Fya and Fyb alleles represent an SNP in silent Fya alleles in Papua New Guinea and
exon 2 of DARC that encodes Gly42 and Asp42, Brazil.
respectively, in the N-terminal extracellular Fyx is a weak form of Fyb that results from
domain of the glycoprotein (Fig 14-5). Fya and an Fyb allele with a missense mutation encod-
Fyb are very sensitive to most proteolytic en- ing an Arg89Cys substitution in a cytosolic do-
zymes, including bromelin, -chymotrypsin, main of the glycoprotein (Fig 14-5). Fyb anti-
ficin, papain, and pronase, but are not de- gen may be undetected in some samples of
stroyed by trypsin. People of African ethnicity
anti-Fyb, although the antigen can be detected
have a third allele, Fy, that is more abundant
by adsorption/elution.
than Fya and Fyb. Fy produces no Duffy glyco-
protein on red cells and, hence, neither Fya nor
Fy3, Fy4, Fy5, and Fy6
Fyb. Individuals who are homozygous for Fy
have the red cell phenotype Fy(a–b–), whose Very rare people of non-African ancestry with
prevalence varies from about 70% in African Fy(a–b–) red cells are homozygous for inacti-
Americans to 100% in residents of Gambia (Ta- vating mutations in their DARC genes. These
ble 14-4). individuals have no Duffy glycoprotein on
European or
Phenotype Asian Ethnicity African Ethnicity Whites Blacks Japanese
people of Japanese ethnicity results from het- HUT11 has been detected on endothelial cells
erozygosity for a dominant inhibitor gene, of the vasa recta, the vascular supply of the re-
named In(Jk) in analogy with the In(Lu) domi- nal medulla, but it is not present in renal tu-
nant inhibitor of Lutheran and other antigens. bules.
Very weak expression of Jka and/or Jkb can be Normal red cells are rapidly lysed by 2M
detected on In(Jk) red cells by adsorption/elu- urea because urea transported into the cells
tion tests. makes them hypertonic and they burst as a re-
sult of the osmotic influx of water. Because of
Kidd Antibodies and Their Clinical the absence of the urea transporter, Jk(a–b–)
Significance cells are not hemolyzed by 2M urea, and this
Anti-Jka and –Jkb are not common antibodies can be used as a method for screening for Jk(a–
and are generally found in antibody mixtures. b–) donors.39
They are usually IgG1 and IgG3, but some are The Jk(a–b–) phenotype is not associated
partly IgG2, IgG4, or IgM. About 50% of anti-Jka with any clinical defect, although two unrelat-
and –Jkb bind complement.13 ed Jk(a–b–) individuals had a mild urine-
Kidd antibodies are often difficult to de- concentrating defect.40
tect. Some agglutinate antigen-positive cells
directly, but the reactions are usually weak. T HE D IE GO S Y ST EM
Generally, an antiglobulin test is required, and
use of enzyme-treated cells may be necessary Band 3, the Red Cell Anion Exchanger
to detect weaker antibodies.
Kidd antibodies may cause severe acute The 22 antigens of the Diego system are locat-
HTRs. They are also a very common cause of ed on Band 3, the common name for the red
delayed HTRs, probably because they are of- cell anion exchanger or solute carrier family
ten not detected in pretransfusion testing due 4 A1 (SLC4A1).41 Band 3 is a major red cell
to their tendency to drop to low or undetect- membrane glycoprotein with approximately
able levels in plasma. Anti-Jk3 can also cause 106 copies per red cell. Band 3 has a transmem-
acute or delayed HTRs. Despite their hemolyt- brane domain that traverses the membrane
ic potential, Kidd antibodies only very rarely about 14 times, with an N-glycan on the fourth
cause severe HDFN. extracellular loop. Band 3 also has a long cyto-
Kidd antibodies have been implicated in plasmic N-terminal domain that interacts with
acute renal transplant rejection, suggesting the membrane skeleton proteins ankyrin,
that Kidd antigens can behave as histocom- 4.1R, and protein 4.2 and functions as a bind-
patibility antigens.37 ing site for hemoglobin (Figs 14-1 and 14-7).
The short cytoplasmic C-terminal domain
The Kidd Glycoprotein in Urea binds carbonic anhydrase II.
Transportation Band 3 in red cells has at least two major
The Kidd antigens are located on a red cell functions: the rapid exchange of HCO3– and Cl–
urea transporter, also known as “human urea ions, which are important in CO2 transport,
transporter 11” (HUT11 or UT-B1).38 When red and attachment of the red cell membrane to
cells approach the renal medulla, which con- the cytoskeleton.9 Tetramers of Band 3 form
tains a high concentration of urea, the urea the core of the Band 3/Rh ankyrin macrocom-
transporter permits rapid uptake of urea and plex of red cell membrane proteins, which
prevents the cells from shrinking in the hyper- could function as a gas channel for O2 and
tonic environment. As the red cells leave the CO2.8 Band 3 is also a component of the junc-
renal medulla, urea is transported rapidly out tional complex that links the red cell mem-
of the cells, preventing the cells from swelling brane to the membrane skeleton via glycopho-
and carrying urea away from the kidney. rin C (Fig 14-1).9 The Band 3 gene (SLC4A1)
CHAPTER 14 Other Blood Groups 䡲 353
TABLE 14-6. Phenotypes of the Dombrock System and Their Approximate Frequencies
Frequency (%)
Do(a+b–) + – + + + 18 11
Do(a+b+) + + + + + 49 44
Do(a–b+) – + + + + 33 45
Gy(a–) – – – – – Rare 0
Hy– – + w
+w – – 0 Rare
DOYA– – – +w
+w
+w Rare Rare
nase. They are also sensitive to the disulfide- 14-8).45 Colton antibodies are usually IgG and
bond-reducing agents AET and DTT. reactive by an IAT, although agglutinating IgM
Dombrock antibodies are usually IgG and anti-Coa has been found. Colton antibodies
reactive by an IAT. They are in short supply and have been implicated in severe HDFN and
are often of poor quality, with very weak reac- HTRs. Colton antigens are resistant to proteo-
tivity. Screening for Dombrock-compatible lytic enzymes.
donors, therefore, is best performed by molec-
ular genetics (SNP testing).
T HE L AN D ST E IN E R - W IE N ER
Anti-Doa and -Dob have been responsible
S Y S TE M
for acute and delayed HTRs. There is little in-
formation regarding the clinical significance of LWa (LW5) and LWb (LW7; Gln100Arg) are anti-
other Dombrock antibodies. No Dombrock thetical antigens of high and low prevalence,
antibody has caused HDFN. respectively.41 Anti-LWab is reactive with all red
cells except those of the extremely rare LW-
TH E CO LTO N S Y S TE M null phenotype and Rhnull cells, which are also
LW(a–b–). LW antigens are expressed more
Coa (CO1; Ala45) is a high-prevalence antigen; strongly on D+ than D– red cells and more
Cob (CO2; Val45), its antithetical antigen, has a strongly on umbilical cord red cells than on
prevalence of about 8% in people of European those of adults. LW antigens are unaffected by
ancestry but is less common in other ethnic treatment of the red cells with papain, ficin,
groups.41 Anti-Co3 is reactive with all red cells trypsin, or -chymotrypsin but are destroyed
except those of the extremely rare Co(a–b–) by pronase. Disulfide-bond-reducing agents
phenotype that results from various inactivat- (AET and DTT) either destroy or greatly reduce
ing mutations. Co4 (Gln47) is a high-preva- LWa or LWab (LW6) on red cells.
lence antigen whose presence is required for The LW glycoprotein is intercellular adhe-
the expression of Coa due to the proximity of sion molecule-4 (ICAM-4), an IgSF adhesion
the polymorphism.44 The Colton antigens are molecule. ICAM-4 binds integrins on macro-
located on aquaporin-1, a water channel (Fig phages and erythroblasts, and it is probably
356 䡲 AABB TECHNICAL MANUAL
FIGURE 14-8. A three-dimensional model of aquaporin-1, showing the six membrane-spanning domains as
cylinders. The first extracellular loop is glycosylated and contains the Coa/Cob polymorphism. The third
extracellular loop and first intracellular loop contain alanine (A)-proline (P)-asparagine (N) motifs and form a
channel in the membrane through which water molecules pass.
the red cells from the plasma. Ch1 to Ch6, Rg1, stroyed by trypsin treatment of red cells.
and Rg2 each have a prevalence greater than Whereas Ge2 and Ge4 are also sensitive to pa-
90%; WH has a prevalence of about 15%. A pain, Ge3 is resistant to papain treatment.
complex relationship exists between the nine Consequently, papain-treated red cells can be
determinants and SNPs in C4A and C4B, the used for distinguishing anti-Ge2 from anti-
genes encoding the C4 chains. The expres- Ge3 in the absence of the very rare Ge:–2,3,4
sion of Chido/Rodgers on red cells is destroyed phenotype red cells.
by treatment of the cells with proteolytic en- Gerbich antibodies may be IgM and di-
zymes, such as papain or ficin. rectly agglutinating, but most are IgG and re-
No Chido/Rodgers antibodies are known quire an IAT for detection. They are not gener-
to have caused HTRs or HDFN, and antigen- ally considered to be clinically significant and
negative blood is not required for transfusion. have not caused HTRs. However, anti-Ge3 has
Chido/Rodgers antibodies are generally IgG. caused HDFN that tends to manifest 2 to 4
Detection of these antibodies with native red weeks after birth. Some autoantibodies with
cells usually requires an IAT, but they often specificities resembling anti-Ge2 or -Ge3 have
directly agglutinate red cells coated artificially been responsible for AIHA.
with C4d. Binding of Chido/Rodgers antibod-
ies to red cells is readily inhibited by plasma
from Ch/Rg+ individuals; this is a useful aid to T HE CROM ER SY ST E M
identification of these antibodies (Method 3- The 18 Cromer antigens are located on the
17). complement-regulatory glycoprotein, decay
accelerating factor (DAF or CD55).3,47 They in-
TH E G ER B I C H S Y S TE M clude the antithetical antigens Tca (Arg52), Tcb
(Leu52), Tcc (Pro52), WESa (Arg82), and WESb
The Gerbich system consists of six highly prev- (Leu82). Tca and WESb have high prevalence,
alent antigens—Ge2, Ge3, Ge4, GEPL, GEAT, and Tcb, Tcc, and WESa, have low prevalence,
and GETI—and five antigens with very low although both Tcb and WESa are present in ap-
prevalence—Wb, Lsa, Ana, Dha, and GEIS. proximately 0.5% of people of African ancestry
These antigens are located on the sialoglyco- and WESa is present in 0.6% of people of Finn-
proteins glycophorin C (GPC), glycophorin D ish ancestry. Other antigens—Cra, Dra, Es, IFC,
(GPD), or both. These two glycoproteins are UMC, GUTI, SERF, ZENA, CROV, CRAM, and
produced by the same gene, GYPC, by initia- CROZ, CRUE, and CRAG—have high preva-
tion of translation at two different sites on the lence.
mRNA. GPD lacks the N-terminal 21 amino ac- Anti-IFC is the antibody made by individ-
ids of GPC. GPC and GPD are part of the junc- uals with the very rare Cromer-null phenotype
tional complex of membrane proteins. Their
(Inab phenotype), and it is reactive with all red
C-terminal cytoplasmic domains interact with
cells, apart from those of individuals with the
the membrane skeleton through 4.1R, p55,
and adducin and serve as an important link
between the membrane and its skeleton.9,45 TABLE 14-7. Phenotypes Lacking High-
GPC appears to be exploited as a receptor Prevalence Gerbich Antigens and the
by some strains of the malarial parasite P. falci- Antibodies that May Be Produced
parum. There are three types of “Gerbich-neg-
ative” phenotypes (Table 14-7). The first of Phenotype Antibodies
these phenotypes, Ge:–2,–3,–4, is the true null
Ge:–2,3,4 (Yus type) Anti-Ge2
in which both GPC and GPD are absent from
the red cells, and the cells are elliptocytic. In Ge:–2,–3,4 (Ge type) Anti-Ge2 or -Ge3
the other phenotypes, Ge:–2,3,4 and Ge:–2, Ge:–2,–3,–4 (Leach Anti-Ge2, -Ge3, or -Ge4
–3,4, GPD is absent and an abnormal form of type)
GPC is present. Ge2, Ge3, and Ge4 are de-
358 䡲 AABB TECHNICAL MANUAL
Inab phenotype. Cromer antigens are readily TABLE 14-8. Approximate Frequencies of
destroyed by -chymotrypsin treatment of red Knops Antigens in Two Populations
cells but not by papain, ficin, or trypsin treat-
ment. The disulfide-bond-reducing agents Frequency (%)
AET and DTT reduce antigen expression only
Antigen Whites Blacks
slightly.
CD55 helps protect the red cells from lysis Kna KN1 99 100
resulting from autologous complement by in- b
Kn KN2 6 0
hibiting the action of C3-convertases. Inab-
a
phenotype red cells do not undergo undue he- McC KN3 98 94
molysis, however, because of the activity of Sl1 (Sla) KN4 98 60
another complement regulatory glycoprotein,
Yka KN5 92 98
CD59. CD55 and CD59 are both linked to the
b
red cell membrane by a glycosylphosphati- McC KN6 0 45
dylinositol (GPI) anchor. Pathological levels of Sl2 KN7 0 80
hemolysis occur in paroxysmal nocturnal he-
moglobinuria, which is associated with a clon- Sl3 KN8 100 100
al defect in GPI biosynthesis and the absence KCAM KN9 98 20
of both CD55 and CD59 in affected red cells.
Cromer antibodies are not usually con-
sidered to be clinically significant because tigens are generally resistant to papain and fi-
there is no firm evidence that any of them has cin, although this may depend on the antibod-
caused an HTR, and the evidence from func- ies used, and are destroyed by trypsin or -
tional cellular assays is equivocal. No Cromer chymotrypsin treatment. They are also de-
antibodies have been implicated in HDFN, stroyed, or at least weakened, by AET and DTT.
and they are probably sequestered by high lev- CR1 appears to be involved in the roset-
els of CD55 in the placenta. Cromer antibodies ting of red cells that is associated with severe P.
are usually IgG and require an IAT for detec- falciparum malaria. The McCb and Sl2 alleles,
tion. They are inhibited by serum or concen- present almost exclusively in individuals of Af-
trated urine from antigen-positive individuals rican ancestry, may confer a degree of protec-
and are removed from serum by platelet con- tion from the parasite. This might explain the
centrates. very strong difference in the prevalence of
some antigens, especially Sl1, McCb, Sl2, and
TH E K N O P S S Y S TE M KCAM, among populations of European and
African ethnicity (Table 14-8).
The nine antigens of the Knops system are lo- Knops antibodies are not clinically signif-
cated on the complement-regulatory glyco- icant and can be ignored when selecting blood
protein complement receptor 1 (CR1 or for transfusion.5 They are usually difficult to
CD35).48 All are polymorphic, although Kna, work with, often making it difficult to distin-
McCa, Sl1, and Yka have relatively high preva- guish antigen-negative cells from those with
lence (Table 14-8). weak expression. They are generally IgG and
Kna/Knb represent Val 1561Met, McCa/ reactive only by an IAT.
b
McC , Lys1590Glu, Sl1/Sl2, and Arg1601Gly. Sl3
requires the presence of Ser1610 and Arg1601
T HE I N D I A N S Y S TE M
(Sl2) for expression. Absence of KCAM results
from an Ile1615Val substitution. An apparent The low-prevalence antigen Ina (Arg46) and its
null phenotype, the Helgeson phenotype, indi- antithetical antigen Inb (Pro46) plus two other
cates very low levels of red cell CR1 and very high-prevalence antigens, INFI and INJA, are
weak expression of Knops antigens. Knops an- located on CD44, the predominant cell surface
CHAPTER 14 Other Blood Groups 䡲 359
receptor for the glycosaminoglycan hyaluro- dividuals were CD151 deficient and had end-
nan, a component of the extracellular matrix.41 stage renal failure, sensorineural deafness, and
AnWj (901009), an antigen with very high prev- pretibial epidermolysis bullosa, suggesting
alence, may also be located on or associated that CD151 is essential for the proper assem-
with CD44, but the evidence is incomplete. In- bly of basement membranes in kidney, inner
dian antigens have reduced expression on red ear, and skin.50 MER2-negative individuals
cells with the In(Lu) phenotype, and AnWj is with anti-MER2 but only single amino acid
virtually undetectable on In(Lu) cells. Ina and substitutions in CD151 do not have these
Inb are sensitive to treatment of red cells with symptoms.
proteolytic enzymes—papain, ficin, trypsin, - MER2 antigen is resistant to treatment of
chymotrypsin—and are also destroyed by the red cells with papain but is destroyed by tryp-
disulfide-bond-reducing agents AET and DTT. sin, -chymotrypsin, and pronase and by AET
AnWj, however, is resistant to all these en- and DTT. MER2 antibodies react in an IAT.
zymes but shows variable outcomes with re- There is no evidence that anti-MER2 is clini-
ducing agents. cally significant.
Anti-Ina and -Inb often agglutinate red
cells directly, but the reaction is usually en-
hanced by an IAT. Indian antibodies are not T HE J O H N M I LTO N HAG E N
generally considered to be clinically signifi- S Y S TE M
cant, although there is one report of anti-Inb
This system consists of six antigens with very
causing an HTR. AnWj, however, has caused
high prevalence—JMH, JMHK, JMHL, JMHG,
severe HTRs, and In(Lu) red cells should be se-
JMHM, and JMHQ—on the semaphorin glyco-
lected for transfusion.5
protein CD108 (Sema7A). Anti-JMH is typically
produced by individuals with an acquired loss
TH E O K SY ST E M of CD108. This most often occurs in elderly pa-
tients and is associated with a weakly positive
Oka, OKGV, and OKVM have very high preva-
lence and are located on the IgSF molecule direct antiglobulin test result. The absence of
CD147 or basigin, which has two IgSF do- the other JMH antigens results from different
mains. Oka is resistant to proteolytic enzymes missense mutations in SEMA7A.51 JMH anti-
and disulfide-bond-reducing agents. Only two gens are destroyed by proteolytic enzymes and
alloanti-Oka antigens and a single example disulfide-bond-reducing agents. They are not
each of anti-OKGV and -OKVM are known; all detected on cord red cells.
are reactive by an IAT.49 In-vivo survival tests JMH antibodies are usually reactive in an
and cellular functional assays with one anti- IAT. They are not generally considered to be
Oka have suggested that it could be clinically clinically significant, although one example
significant, but no clinical information exists. was implicated in an acute HTR.
TH E R A PH S Y S TE M T HE G IL L S Y S T EM
MER2 (RAPH1), which is located on the tet- GIL antibodies detect a very high-prevalence
raspanin CD151, was initially defined by antigen, GIL, located on aquaporin 3 (AQP3), a
mouse monoclonal antibodies that recognized member of the aquaporin superfamily of wa-
a quantitative polymorphism, and about 8% of ter and glycerol channels (like the Colton anti-
the population has undetectable levels of gen).52 AQP3 enhances the permeability of the
MER2 on their mature red cells. Alloanti-MER2 red cell membrane by glycerol and water.
was found in three Israeli Jews originating GIL antigen is resistant to proteolytic en-
from India who had a RAPH-null phenotype zymes and disulfide-bond-reducing agents.
resulting from a single nucleotide deletion that GIL antibodies are reactive by an IAT. Anti-GIL
led to a premature stop codon. These three in- has not been implicated in HTRs or HDFN,
360 䡲 AABB TECHNICAL MANUAL
although monocyte monolayer assays have have the potential to cause a positive cross-
suggested a potential to cause accelerated de- match.
struction of GIL+ red cells.
T HE JR SY ST E M
TH E RH AG SY ST E M
The high-prevalence antigen Jra has been pro-
The four antigens of the RHAG system are lo- moted to a new blood group system, JR, fol-
cated on the Rh-associated glycoprotein lowing the independent findings of two groups
(RhAG), which is also described in Chapter demonstrating that the Jr(a–) phenotype was
13.53 RhAG is closely associated with the Rh due to inactivating nucleotide changes in
protein in the membrane as part of the Band ABCG2.56,57 The gene encodes ABCG2, a multi-
3/Rh/ankyrin macrocomplex (Fig 14-1). Ola is pass membrane protein family member of the
very rare, and homozygosity for the allele en- adenosine triphosphate (ATP)-binding cas-
coding Ola is associated with an Rhmod pheno- sette transporters that is broadly distributed
type. Duclos and DSLK have high prevalence, throughout the body. Jra has long been associ-
and absence of these antigens is associated ated with drug resistance in cancer and resis-
with an aberrant U (MNS5) antigen. RHAG4 is tance to xenobiotics, and it might be impor-
a low-prevalence antigen whose antibody was tant for porphyrin homeostasis.58
associated with a single case of severe HDFN.3 The Jr(a–) phenotype is present predomi-
nantly in people of Japanese ancestry. Jra anti-
gen is resistant to proteolytic enzymes and
disulfide-bond-reducing agents. Anti-Jra is re-
TH E F O R S S Y S T EM active by an IAT and has caused HTR. It is not
usually implicated in HDFN, although one
FORS is a new blood group system consisting
case has been reported.
of a single antigen, Forssman glycosphingolip-
id antigen (FORS1). The presence of FORS1 on
human erythrocytes is unusual and was shown
to be the result of an enzyme-activating amino T HE L AN S Y ST E M
acid substitution arising from a missense mu-
Lan, another high-prevalence antigen, was
tation in the human Forssman synthase gene
also elevated to a new blood group system fol-
GBGT1. FORS1 was demonstrated biochemi-
lowing the discovery that it was carried on
cally on the red cells of two blood donors from ABCB6, another ATP-binding cassette trans-
different families with the Apae phenotype, porter molecule on the erythrocyte mem-
which was first described in 1987.54,55 Apae had brane.59 Unlike Jra, Lan is not associated with
been previously thought to constitute a sub- any single geographic or ethnic group, and this
group of A in the ABO system but has now is mirrored by the diversity of mutant alleles in
been shown to be based on the presence of the Lan– individuals studied. ABCB6 is associ-
FORS1 antigen on red cells. Forssman syn- ated with porphyrin transport and was
thase adds a terminal 3--N-acetylgalactos- thought to have an important role in heme
amine to its globoside acceptor. FORS1 is not synthesis.60 However, the existence of ABCB6-
usually present on the red cells of primates but deleted individuals indicates that there may be
is highly expressed on the red cells and uroepi- compensation by other transporters in the ab-
thelia of lower mammals, such as dogs and sence of ABCB6.
sheep. As with other carbohydrate blood Lan antigen is expressed variably on red
group antigens, naturally occurring antibodies cells in different individuals but is resistant to
to FORS1 are present in all human sera, with proteolytic enzymes and disulfide-bond-
the exception of rare FORS1+ individuals, and reducing agents. Anti-Lan is reactive by an IAT
CHAPTER 14 Other Blood Groups 䡲 361
and has been implicated in HTR but not gen- Blood Group Collections
erally in HDFN.
Although many antigens belong to blood
group systems, others have not been shown to
TH E VE L SY ST E M belong to a system. These are mostly antigens
with either very high or very low prevalence.
Vel is a high prevalence blood group antigen
Some of them are included in blood group col-
that has been shown to depend on the pres-
lections that contain two or more antigens that
ence of small integral protein 1 (SMIM1), a
are related serologically, biochemically, or ge-
protein of unknown function newly discovered
netically but do not fit the criteria for system
on the erythrocyte surface.60-62 Absence of Vel status.1
antigen in the vast majority of individuals re- The high-prevalence antigen ABTI is sero-
gardless of ethnic background, is due to a 17- logically related to Vel. However, it has been
base pair deletion in SMIM1, which results in excluded from SMIM1 by sequencing analysis
the absence of the protein at the cell mem- and thus remains in a collection. Like Vel, ABTI
brane. expression differs substantially and it is gener-
Vel antigen expression is generally weak ally expressed only weakly on cord red cells.
on cord RBCs and differs substantially from ABTI is resistant to treatment of red cells with
one individual to another. Patterns of expres- proteolytic enzymes or disulfide-bond-reduc-
sion are a consequence both of zygosity for the ing agents. Anti-ABTI has not caused HDFN
17-bp deletion and also for a SNP in a GATA-1 and clinical data are limited.
transcription factor site in intron 2.62 Serologi- The Cost collection contains Csa and Csb,
cal expression is not affected by protease treat- antithetical antigens with relatively high and
ment although sensitivity to reducing agents low prevalence, respectively. These antigens
such as 0.2 M DTT is variable. Anti-Vel are of- are serologically related to those of the Knops
ten a mixture of IgG and IgM, readily activate system but do not appear to be located on
complement and have been implicated in CR1. Cost antibodies are not clinically signifi-
mild to severe HTRs, although HDFN is rare. cant.
Era and Erb are antithetical antigens with
ANTIGENS THAT DO NOT very high and low prevalence, respectively.
Anti-Er3 is produced by individuals with Er(a–
BELO NG TO A BLOOD GROU P
b–) red cells. There is no evidence that Er anti-
SY S TE M bodies are clinically significant.
Carbohydrate antigens of the Ii and GLOB
CD59—A Potential New Blood Group collections are described in Chapter 12.
System Carbohydrate antigens associated with
An antibody to a high-prevalence antigen de- MNS antigens that are not encoded by GYPA or
tected in the plasma of a transfused CD59- GYPB are included in a seventh collection,
deficient child was shown to be specific for MNS CHO. These antigens have been shown to
CD59.63 The antibody was readily inhibited be due to altered glycosylation of the O-linked
sugars on GPA and GPB.2
with soluble protein. Sequence analysis of
samples from the family revealed that the par-
ents (first-degree cousins) were heterozygous
High-Prevalence Antigens (901 Series)
and the child homozygous for a silencing mu- The 901 series of the ISBT classification con-
tation in CD59. The antibody was an IgG anti- tains six antigens (Table 14-9): five have a
body, and although the child’s RBCs had been prevalence well in excess of 99%, whereas Sda
weakly DAT-positive following transfusion, in- has a prevalence of about 91%. All are inherit-
compatible blood was well-tolerated. Thus, ed, and none is eligible to join a system.1 All six
CD59 has been proposed as a blood group sys- antigens are resistant to papain, trypsin, -
tem but is as yet, unconfirmed. chymotrypsin, and AET treatment of the red
362 䡲 AABB TECHNICAL MANUAL
TABLE 14-9. Antigens of the 901 Series of High-Frequency Antigens and Their Clinical Significance
cells, and all except AnWj and Sda are ex- HLA-B7; Bgb, HLA-B17 (B57 or B58); and Bgc,
pressed strongly on cord cells. HLA-A28 (A68 or A69, which cross-reacts with
Sda represents carbohydrate structures on HLA-A2). Many individuals, however, do not
red cells, and the product of the Sda gene is express Bg antigens on their red cells, despite
probably a (1,4)N-acetylgalactosaminyltrans- having the corresponding HLA antigens on
ferase. The strength of Sda on red cells is highly their lymphocytes.
variable, and Sda is not detected on cord red There are a few reports of Bg antibodies
cells. Agglutination of Sd(a+) red cells has a causing HTRs.64 These antibodies are some-
characteristic “mixed-field” appearance of ag- times present as contaminants in reagents.
glutinates and free red cells; when viewed mi- HLA antigens on red cells are not destroyed by
croscopically, the agglutinates are refractile. papain, ficin, pronase, trypsin, -chymotryp-
Anti-Sda is inhibited by urine from Sd(a+) sin, AET, or DTT. They can be stripped from
individuals (Method 3-19), and by guinea pig red cells with chloroquine (Method 2-20) or
urine. EDTA/glycine-HCl (Method 2-21).
more, discrete mutations in KLF1 appear to Although mentioned above in the “Lu-
give rise to different phenotypes; for example, theran System” section, it is worth repeating
the change of Glu325Lys does not result in the that a mutation in GATA-1 resulted in the X-
In(Lu) phenotype but is associated with severe linked Lu(a–b–) phenotype in one family.20 It is
congenital dyserythropoietic anemia. These likely that additional mutations in these and
red cells demonstrate weakened expression of other erythroid-specific transcription factors
antigens in the Colton (AQP1), Cromer (DAF), will be identified as the causes of altered blood
and LW (ICAM-4) blood group systems.66 group antigen expression.
KEY POINTS
1. Of 339 recognized antigen specificities, 297 belong to 1 of 34 blood group systems repre-
senting either a single gene or two or three closely linked homologous genes. Some groups
of antigens that are not eligible to join a system are classified together as collections. Anti-
gens not classified in a system or collection have either low or high prevalence and make up
the 700 and 901 series, respectively.
2. M and N are antithetical, polymorphic antigens. M, N, S, s, and ‘N’ are all destroyed by treat-
ment of the red cells with papain, ficin, bromelin, or pronase, although this effect with S and
s is variable. M and N—but not S, s, or ‘N’—are destroyed by trypsin treatment.
3. Anti-M is relatively common, and anti-N is quite rare. Most anti-M and -N are not clinically
significant. When M or N antibodies active at 37 C are encountered, antigen-negative or
compatible red cells should be provided. Anti-S, -s, and -U are generally IgG antibodies that
are active at 37C. They have been implicated in HTRs and severe and fatal HDFN.
4. The antigen often referred to as “Kell” is correctly named “K” or “KEL1”; its antithetical anti-
gen is k or KEL2.
5. Because Kell antibodies can cause severe HDFN and HTRs, patients with Kell antibodies
should be transfused with antigen-negative blood whenever possible. Anti-K is the most
common immune red cell antibody not in the ABO and Rh systems.
6. In people of European or Asian ancestry, the Duffy polymorphism consists of two antigens,
Fya and Fyb, and three phenotypes, Fy(a+b–), Fy(a+b+), and Fy(a–b+). Fya and Fyb are very
sensitive to most proteolytic enzymes. In people of African ancestry, a third allele, Fy, may
result in neither Fya nor Fyb. Individuals who are homozygous for Fy have the red cell pheno-
type Fy(a–b–).
7. Anti-Fya (common) and anti-Fyb (rare) are generally detected by an IAT and may cause acute
or delayed HTRs that are usually mild, although some have been fatal.
8. Kidd antigens are resistant to proteolytic enzymes, such as papain and ficin.
9. Kidd antibodies anti-Jka and -Jkb are not common, are generally present in antibody mix-
tures, and are often difficult to detect. An IAT is usually required, and use of enzyme-treated
cells may be necessary to detect weaker antibodies. Kidd antibodies may cause severe acute
HTRs and are a common cause of delayed HTRs.
10. The 22 antigens of the Diego system are located on Band 3, the red cell anion exchanger.
Anti-Dia and -Wra can cause severe HDFN. Anti-Wra can also cause HTRs.
REF EREN CE S
1. Daniels GL, Fletcher A, Garratty G, et al. Blood on terminology for red cell surface antigens.
group terminology 2004: From the Interna- Vox Sang 2004;87:304-16.
tional Society of Blood Transfusion committee
364 䡲 AABB TECHNICAL MANUAL
2. Storry JR, Castilho L, Daniels G, et al. Interna- 16. Crew VK, Green C, Daniels G. Molecular bases
tional Society of Blood Transfusion Working of the antigens of the Lutheran blood group
Party on Red Cell Immunogenetics and Blood system. Transfusion 2003;43:1729-37.
Group Terminology: Berlin report. Vox Sang 17. Eyler CE, Telen MJ. The Lutheran glycoprotein:
2011;101:77-82. A multifunctional adhesion receptor. Transfu-
3. Storry JR, Castilho L, Daniels G, et al. Interna- sion 2006;46:668-77.
tional Society of Blood Transfusion Working 18. Karamatic Crew V, Mallinson G, Green C, et al.
Party on Red Cell Immunogenetics and Termi- Different inactivating mutations in the LU
nology: Cancun report. Vox Sang 2014 (in press). genes of three individuals with the Lutheran-
4. International Society of Blood Transfusion. null phenotype. Transfusion 2007;47:492-8.
Blood group terminology. Amsterdam: ISBT, 19. Singleton BK, Burton NM, Green C, et al. Mu-
2013. [Available at http://www.isbtweb.org/ tations in EKLF/KLF1 form the molecular ba-
working-parties/red-cell-immunogenetics- sis of the rare blood group In(Lu) phenotype.
and-blood-group-terminology (accessed No- Blood 2008;112:2081-8.
vember 19, 2013).] 20. Singleton BK, Roxby DJ, Stirling JW, et al. A
5. Poole J, Daniels G. Blood group antibodies and novel GATA1 mutation (Stop414Arg) in a fami-
their significance in transfusion medicine. ly with the rare X-linked blood group Lu(a–b–)
Transfus Med Rev 2007;21:58-71. phenotype and mild macrothrombocytic
6. Reid ME, Lomas-Francis C, Olsson ML. The thrombocytopenia. Br J Haematol 2012;161:
blood group antigen factsbook. London: Aca- 139-42.
21. Westhoff CM, Reid ME. Review: The Kell,
demic Press, 2012.
Duffy, and Kidd blood group systems. Immu-
7. Daniels G. Human blood groups. 3rd ed. Ox-
nohematol. 2004;20:37-49.
ford: Wiley Blackwell, 2013.
22. Lee S, Zambas ED, Marsh WL, Redman CM.
8. Bruce LJ, Beckmann R, Ribeiro ML, et al. A
Molecular cloning and primary structure of
Band 3-based macrocomplex of integral and
Kell blood group protein. Proc Natl Acad Sci
peripheral proteins in the RBC membrane.
U S A 1991;88:6353-7.
Blood 2003;101:4180-8.
23. Kormoczi GF, Scharberg EA, Gassner C. A novel
9. Mohandas N, Gallagher PG. Red cell mem-
KEL*1,3 allele with weak Kell antigen expres-
brane: Past, present, and future. Blood 2008;
sion confirming the cis-modifier effect of
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KEL3. Transfusion 2009;49:733-9.
10. Blumenfeld OO, Huang CH. Molecular genet-
24. Daniels G, Hadley A, Green CA. Causes of fetal
ics of the glycophorin gene family, the anti-
anemia in hemolytic disease due to anti-K.
gens for MNSs blood groups: Multiple gene re- Transfusion 2003;43:115-16.
arrangements and modulation of splice site 25. Lee S, Russo DC, Reiner AP, et al. Molecular de-
usage result in extensive diversification. Hum fects underlying the Kell null phenotype. J Biol
Mutat 1995;6:199-209. Chem 2001;276:27281-9.
11. Sim BK, Chitnis CE, Wasniowska K, et al. Re- 26. Lee S, Russo DC, Reid ME, Redman CM. Muta-
ceptor and ligand domains for invasion of tions that diminish expression of Kell surface
erythrocytes by Plasmodium falciparum. Sci- protein and lead to the Kmod RBC phenotype.
ence 1994;264:1941-4. Transfusion 2003;43:1121-5.
12. Mayer DC, Cofie J, Jiang L, et al. Glycophorin B 27. Lee S, Debnath AK, Redman CM. Active amino
is the erythrocyte receptor of Plasmodium fal- acids of the Kell blood group protein and mod-
ciparum erythrocyte-binding ligand, EBL-1. el of the ectodomain based on the structure of
Proc Natl Acad Sci U S A 2009;106:5348-52. neutral endopeptidase 24.11. Blood 2003;102:
13. Klein HG, Anstee DJ. Mollison’s blood transfu- 3028-34.
sion in clinical medicine. 12th ed. Oxford: Wi- 28. Danek A, Rubio JP, Rampoldi L, et al. McLeod
ley-Blackwell, 2014. neuroacanthocytosis: Genotype and pheno-
14. Tippett P, Reid ME, Poole J, et al. The Milten- type. Ann Neurol 2001;50:755-64.
berger subsystem: Is it obsolescent? Transfus 29. Tournamille C, Colin Y, Cartron JP, Le Van KC.
Med Rev 1992;6:170-82. Disruption of a GATA motif in the Duffy gene
15. Broadberry RE, Lin M. The incidence and sig- promoter abolishes erythroid gene expression
nificance of anti- “Mia” in Taiwan. Transfusion in Duffy-negative individuals. Nat Genet 1995;
1994;34:349-52. 10:224-8.
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30. Inoue H, Kozlowski SD, Klein JD, et al. Regulat- sickle cell adhesion and vaso-occlusion in vi-
ed expression of renal and intestinal UT-B urea vo. Blood 2007;110:2708-17.
transporter in response to varying urea load. 46. Storry JR, Reid ME, Yazer MH. The Cromer
Am J Physiol Renal Physiol 2005;289:F451-F8. blood group system: A review. Immunohema-
31. Hadley TJ, Peiper SC. From malaria to chemo- tology 2010;26:109-18.
kine receptor: The emerging physiologic role 47. Moulds JM. The Knops blood-group system: A
of the Duffy blood group antigen. Blood 1997; review. Immunohematology 2010;26:2-7.
89:3077-91. 48. Smart EA, Storry JR. The OK blood group sys-
32. Horuk R, Chitnis CE, Darbonne WC, et al. A re- tem: A review. Immunohematology 2010;26:
ceptor for the malarial parasite Plasmodium 124-6.
vivax: The erythrocyte chemokine receptor. 49. Karamatic Crew V, Burton N, Kagan A, et al.
Science 1993;261:1182-4. CD151, the first member of the tetraspanin
33. Daniels G. The molecular genetics of blood
(TM4) superfamily detected on erythrocytes,
group polymorphism. Hum Genet 2009;126:
is essential for the correct assembly of human
729-42.
basement membranes in kidney and skin.
34. Hadley TJ, Lu ZH, Wasniowska K, et al. Post-
Blood 2004;104:2217-23.
capillary venule endothelial cells in kidney ex-
50. Seltsam A, Strigens S, Levene C, et al. The mo-
press a multispecific chemokine receptor that
is structurally and functionally identical to the lecular diversity of Sema7A, the semaphorin
erythroid isoform, which is the Duffy blood that carries the JMH blood group antigens.
group antigen. J Clin Invest 1994;94:985-91. Transfusion 2007;47:133-46.
35. Daniels G, Bromilow IM. Essential guide to 51. Roudier N, Ripoche P, Gane P, et al. AQP3 defi-
blood groups. 2nd ed. Oxford: Wiley Blackwell, ciency in humans and the molecular basis of a
2010. novel blood group system, GIL. J Biol Chem
36. Holt S, Donaldson H, Hazlehurst G, et al. Acute 2002;277:45854-9.
transplant rejection induced by blood transfu- 52. Tilley L, Green C, Poole J, et al. A new blood
sion reaction to the Kidd blood group system. group system, RHAG: Three antigens resulting
Nephrol Dial Transplant 2004;19:2403-6. from amino acid substitutions in the Rh-asso-
37. Sands JM. Molecular mechanisms of urea ciated glycoprotein. Vox Sang 2010;98:151-9.
transport. J Membr Biol 2003;191:149-63. 53. Svensson L, Hult AK, Stamps R, et al. Forssman
38. Heaton DC, McLoughlin K. Jk(a–b–) red blood expression on human erythrocytes: Biochemi-
cells resist urea lysis. Transfusion 1982;22:70-1. cal and genetic evidence of a new histo-blood
39. Sands JM, Gargus JJ, Frohlich O, et al. Urinary group system. Blood 2013;121:1459-68.
concentrating ability in patients with Jk(a–b–) 54. Stamps R, Sokol RJ, Leach M, et al. A new vari-
blood type who lack carrier-mediated urea ant of blood group A. Apae. Transfusion 1987;
transport. J Am Soc Nephrol 1992;2:1689-96. 27:315-18.
40. Byrne KM, Byrne PC. Review: Other blood 55. Saison C, Helias V, Ballif BA, et al. Null alleles of
group systems—Diego, Yt, Xg, Scianna, Dom- ABCG2 encoding the breast cancer resistance
brock, Colton, Landsteiner-Wiener, and Indi-
protein define the new blood group system Ju-
an. Immunohematology 2004;20:50-8.
nior. Nat Genet 2012;44:174-7.
41. Wagner FF, Poole J, Flegel WA. Scianna anti-
56. Zelinski T, Coghlan G, Liu XQ, Reid ME. ABCG2
gens including Rd are expressed by ERMAP.
null alleles define the Jr(a–) blood group phe-
Blood 2003;101:752-7.
42. Reid ME. Complexities of the Dombrock blood notype. Nat Genet 2012;44:131-2.
group system revealed. Transfusion 2005;45: 57. Robey RW, To KK, Polgar O, et al. ABCG2: A
92S-9S. perspective. Adv Drug Deliv Rev 2009;61:3-13.
43. Arnaud L, Helias V, Menanteau C, et al. A func- 58. Helias V, Saison C, Ballif BA, et al. ABCB6 is dis-
tional AQP1 allele producing a Co(a–b–) phe- pensable for erythropoiesis and specifies the
notype revises and extends the Colton blood new blood group system Langereis. Nat Genet
group system. Transfusion 2010;50:2106-16. 2012;44:170-3.
44. Daniels G. Functions of red cell surface pro- 59. Krishnamurthy P, Schuetz JD. The role of
teins. Vox Sang 2007;93:331-40. ABCG2 and ABCB6 in porphyrin metabolism
45. Zennadi R, Moeller BJ, Whalen EJ, et al. Epi- and cell survival. Curr Pharm Biotechnol 2011;
nephrine-induced activation of LW-mediated 12:647-55.
366 䡲 AABB TECHNICAL MANUAL
60. Ballif BA, Helias V, Peyrard T, et al. Disruption CD59, detected in a CD59-deficient patient.
of SMIM1 causes the Vel– blood type. EMBO Transfusion 2014 (in press).
Mol Med 2013;5:751-61. 64. Nance ST. Do HLA antibodies cause hemolytic
61. Storry JR, Joud M, Christophersen MK, et al. transfusion reactions or decreased RBC sur-
Homozygosity for a null allele of SMIM1 de- vival? Transfusion 2003;43:687-90.
fines the Vel-negative blood group phenotype. 65. Borg J, Papadopoulos G, Georgitsi M, et al.
Nat Genet 2013;45:537-41. Haploinsufficiency for the erythroid transcrip-
62. Cvejic A, Haer-Wigman L, Stephens JC, et al. tion factor KLF1 causes hereditary persistence
SMIM1 underlies the Vel blood group and in- of fetal hemoglobin. Nat Genet 2010;42:801-5.
fluences red blood cell traits. Nat Genet 2013; 66. Arnaud L, Saison C, Helias V, et al. A dominant
45:542-5. mutation in the gene encoding the erythroid
63. Anliker M, von Zabern I, Höchsmann B, et al. A transcription factor KLF1 causes a congenital
new blood group antigen is defined by anti- dyserythropoietic anemia. Am J Hum Genet
2010;87:721-7.
C h a p t e r 1 6
Identification of Antibodies to
Red Cell Antigens
Phyllis S. Walker, MS, MT(ASCP)SBB, retired from Reference Laboratory, Blood Centers of the Pacific-Irwin
Center, San Francisco, California, and Janis R. Hamilton, MS, MT(ASCP)SBB, Manager, Reference Laboratory,
American Red Cross Blood Services, Detroit, Michigan
P. Walker has disclosed no conflict of interest. J. Hamilton has disclosed a financial relationship with Bio-Rad
Laboratories, Inc.
391
392 䡲 AABB TECHNICAL MANUAL
gen, the patient’s ethnic origin may provide phenomenon is known as “dosage.” Antibod-
clues to the specificity of the antibody. Table ies in the Rh, MNS, Duffy, and Kidd systems
16-1 lists some rare blood types that are more most commonly demonstrate dosage. Reagent
commonly associated with certain ethnic red cells should be refrigerated when not in
groups.4 use, and they should not be used for antibody
detection after their expiration date.
A N A LY T I C A L PHA SE O F
Antibody Identification Panels
A N TI B O DY ID E N T I F I C ATI O N
Identification of an antibody to red cell anti-
Specimen Requirements
gen(s) requires testing the serum against a
Either serum or plasma may be used for anti- panel of selected red cell samples (typically 8-
body detection and identification; however, 14 samples) with known antigenic composi-
plasma may not be suitable for detecting com- tion for the major blood groups. Usually, the
plement-activating antibodies. Depending on red cell samples are obtained from commer-
the test methods used, a 5-mL to 10-mL ali- cial suppliers, but institutions may assemble
quot of whole blood usually contains enough their own panels using red cells from local
serum or plasma for identifying simple anti- sources. Except in special circumstances, pan-
body specificities; more whole blood may be el cells are group O, thereby allowing the se-
required for complex studies. When autolo- rum of any ABO group to be tested.
gous red cells are studied, the use of samples Each reagent red cell of the panel is from a
anticoagulated with EDTA avoids problems as- different donor. The reagent red cells are se-
sociated with the in-vitro uptake of comple- lected so that if one takes all of the examples of
ment components by red cells, which may oc- red cells into account, a distinctive pattern of
cur when clotted samples are used. positive and negative reactions exists for each
of many antigens. To be functional, a reagent
Reagents and Test Methods red cell panel must make it possible to identify
with confidence those clinically significant al-
Antibody Detection Red Cells loantibodies that are most commonly encoun-
Group O red cells suitable for pretransfusion tered, such as anti-D, -E, -K, and -Fya. The phe-
antibody screening are commercially available notypes of the reagent red cells should be
and offered as sets of either two or three re- distributed so that single specificities of the
agent single-donor red cells. Pooled red cells common alloantibodies can be clearly identi-
for antibody detection are usually obtained fied and most others can be excluded. Ideally,
from two different donors and may be used patterns of reactivity for most examples of sin-
only when donor serum is tested. Each labora- gle alloantibodies should not overlap with any
tory should decide whether to use two or three other (eg, all of the K+ samples should not be
reagent donor red cells for antibody-detection the only ones that are also E+). Also, it is im-
testing. All reagent red cells licensed by the portant to include reagent red cells with dou-
Food and Drug Administration (FDA) for this ble-dose antigen expression to detect com-
purpose must express the following antigens: mon antibodies that frequently show dosage.
D, C, E, c, e, M, N, S, s, P1, Lea, Leb, K, k, Fya, Fyb, Commercial panels are accompanied by a
Jka, and Jkb. Three-cell antibody detection sets sheet that lists the phenotypes of the reagent
usually offer red cells from presumed homozy- red cells. The combination of reagent red-cell
gous donors with double-dose expression for samples varies from lot to lot, so it is essential
the following common antigens: D, C, E, c, e, to use the correct phenotype sheet when inter-
M, N, S, s, Fya, Fyb, Jka, and Jkb. Some weakly re- preting panel results. Commercial reagent red
active antibodies are reactive only with red cells for tube testing are diluted to a 2% to 5%
cells from donors who are homozygous for the suspension in a preservative solution that can
genes encoding the antigens; this serologic be used directly from the bottle. Washing the
394 䡲 AABB TECHNICAL MANUAL
Phenotype Population
At(a–) Blacks
Cr(a–) Blacks
DISK– Dutch>Europeans>any
En(a–) Finns>Canadians>English>Japanese
Es(a–) Mexicans, South Americans, Blacks
GUTI– Chileans
hrs– Blacks
Hy– Blacks
In(b–) Indians>Iranians>Arabs
Jk(a–b–) Polynesians>Finns>Japanese>any
Jo(a–) Blacks
Js(b–) Blacks
k– Whites>any
K12– Whites
K14– French-Cajuns
K22– Israelis
KCAM– Blacks>any
Kn(a–) Whites>Blacks>any
CHAPTER 16 Identification of Antibodies to Red Cell Antigens 䡲 395
Phenotype Population
Kp(b–) Whites>Japanese
Lan– Whites>Japanese>Blacks>any
Lu(a–b–) Any
Lu20– Israelis
Lu21– Israelis
MAM– Arabs>any
MAR– Finns>any
McC(a–) Blacks>Whites>any
Oh (Bombay) Indians>Japanese>any
Ok(a–) Japanese
P– Japanese>Finns>Israelis>any
PEL– French-Canadians
k
PP1P – Swedes>Amish>Israelis>Japanese>any
Sl(a–) Blacks>Whites>any
Tc(a–b+c-) Blacks
Tc(a–b–c+) Whites
SERF– Thais
UMC– Japanese
Vel– Swedes>any
WES(b–) Finns>Blacks>any
Yk(a–) Whites>Blacks>any
Yt(a–) Arabs>Jews>any
Used with permission from Reid, Lomas-Francis, and Olsson.4
Any = may be found in any population; > = more prevalent than.
396 䡲 AABB TECHNICAL MANUAL
grading agglutination.) Positive reactions are tained with the test serum. If there is an anti-
compared to the antigen patterns of the panel gen pattern that matches the test serum pat-
cells to help assign specificity. A single alloan- tern exactly, this pattern most likely identifies
tibody usually produces a clear pattern with the specificity of the antibody in the serum. If
antigen-positive and antigen-negative re- there are remaining specificities that were not
agent red cells. For example, if a serum sample excluded, additional testing is needed to elimi-
is reactive only with red cell samples 3 and 5 of nate the possibilities and confirm the suspect-
the reagent red cell panel shown in Table 16-2, ed specificity. This process requires testing of
anti-E is very likely present. Both reactive red the serum with additional selected red cells.
cells express the antigen, and all nonreactive Although the exclusion (rule-out) ap-
cells lack it. When there is no discernible pat- proach often identifies simple antibodies, it
tern to explain the reactivity, possible explana- should be considered only as a provisional
tions include multiple antibodies, dosage, and step, particularly if the rule out was based on
variations in antigen expression. These factors the lack of reactivity with red cells that have
are discussed in more detail later in this chap- weaker expression of the antigen (eg, cells
ter. Negative reactions are important in anti- from heterozygous donors).
body identification because they allow tenta-
tive exclusion of antibodies to antigens SELECTED CELLS. Selected cells are red cells
expressed on the nonreactive red cells. Exclu- that have been chosen because they express
sion of antibodies is an important step in the some specific antigens and lack others. Select-
interpretation process and must be performed ed red cells with different antigen combina-
to ensure proper identification of all of the an- tions can be used to confirm or rule out the
tibodies present. presence of antibodies. For example, if a pat-
tern of reactive red cells fits anti-Jk a exactly,
EXCLUSION, “ RULE OUT,” OR “ CROSS but anti-K and anti-S were not excluded, the
OUT.” A widely used first approach to the in- serum should be tested with selected red cells.
terpretation of panel results is to exclude spec- Ideally, red cells with the following phenotypes
ificities on the basis of nonreactivity of the pa- should be chosen: Jk(a–), K+, S–; Jk(a–), K–, S+;
tient’s serum with red cells that express the and Jk(a+), K–, S–. The reaction pattern with
antigen. Such a system is sometimes referred these red cells should both confirm the pres-
to as a “cross-out” or “rule-out” method. Once ence of anti-Jka and include or exclude anti-K
results have been recorded on the work sheet, and anti-S. Whenever possible, selected red
the antigen profile of the first nonreactive red cells should have a strong expression of the
cell is examined. If an antigen is present on the antigen being tested (ie, from homozygous do-
red cell sample and the serum was not reactive nors or red cells with double-dose expression).
with it, the presence of the corresponding an- Such red cells help ensure that nonreactivity
tibody may tentatively be excluded. Many with the selected red cell indicates the absence
technologists actually cross out such antigens of the antibody and not that the antibody was
from the list at the top of the panel sheet to fa- too weak to be reactive with a selected red cell
cilitate the process. After all of the antigens on that had a weak expression of the antigen.
the list for that red cell have been crossed out,
the same process is performed with the other PROBABILIT Y. To ensure that an observed
nonreactive red cells; additional specificities pattern of reactions is not the result of chance
are then excluded. In most cases, this process alone, conclusive antibody identification re-
leaves a group of antibodies that have not quires the serum to be tested against a suffi-
been excluded. cient number of reagent red cell samples that
Next, the red cells that are reactive with lack—and express—the antigen that corre-
the serum are evaluated. The pattern of reac- sponds with the antibody’s apparent specifici-
tivity for each specificity that was not excluded ty. A standard approach (which is based on
is compared to the pattern of reactivity ob- Fisher’s exact method) has been to require that
TABLE 16-2. Example of a Reagent Red Cell Panel for Antibody Identification
Cell Rh-hr MNS Kell P Lewis Duffy Kidd Others Cell Results
w a a a b a b a b
D C E c e f C V M N S s K k Kp Js P1 Le Le Fy Fy Jk Jk 37 C AHG
CHAPTER 16
1 + + 0 0 + 0 0 0 + 0 0 + 0 + 0 0 + 0 + + + 0 + Bg(a+) 1
2 + + 0 0 + 0 + 0 + + + 0 0 + 0 0 + 0 0 0 0 + 0 2
3 + 0 + + 0 0 0 0 0 + 0 + 0 + 0 0 0 + 0 0 + + + 3
4 0 + 0 + + + 0 0 + 0 + + 0 + 0 0 + 0 + + 0 + 0 4
5 0 0 + + + + 0 0 0 + + + 0 + 0 0 + 0 + 0 + 0 + 5
6 0 0 0 + + + 0 0 + 0 + 0 + + 0 0 + 0 + + 0 0 + 6
7 0 0 0 + + + 0 0 + + + + 0 + 0 0 + 0 + 0 + + 0 7
8 + 0 0 + + + 0 + 0 + 0 0 0 + 0 0 + 0 0 0 0 0 + 8
9 0 0 0 + + + 0 0 + + + + + 0 0 0 0 + 0 + 0 + + 9
10 0 0 0 + + + 0 0 + 0 0 + + + + 0 + 0 0 0 + + + Yt(b+) 10
Identification of Antibodies to Red Cell Antigens
11 + + 0 0 + 0 0 0 + + 0 + 0 + 0 0 + 0 + 0 + 0 + 11
AC AC
+ indicates presence of antigen, 0 indicates absence of antigen, AC = autocontrol, AHG = antihuman globulin.
䡲
399
400 䡲 AABB TECHNICAL MANUAL
three antigen-positive red cell samples are re- ed under the same conditions as serum and
active and that three antigen-negative red cell reagent red cells, is an important part of anti-
samples are not reactive for each specificity body identification. The autocontrol is not the
identified.13 In some cases, the use of two reac- same as or equivalent to a DAT (Method 3-14).
tive and two nonreactive red cell samples is Incubation and the presence of enhancement
also an acceptable approach for antibody con- reagents may cause reactivity in the autocon-
firmation.5,14 When that approach is not possi- trol that is only an in-vitro phenomenon. If the
ble, a more liberal approach (which is derived autocontrol is positive in the antiglobulin
from calculations by Harris and Hochman15) phase, a DAT should be performed. If the DAT
allows the minimum requirement for a proba- result is negative, antibodies to an enhance-
bility (p) value of 0.05 to be met with two re- ment medium constituent or autoantibodies
active and three nonreactive red cell samples that are reactive only in the enhancement me-
or with one reactive and seven nonreactive red dium should be considered. Warm autoanti-
cell samples (or the reciprocal of either combi- bodies and cold autoantibodies, such as anti-I,
nation). Comparative p-value calculations are -IH, or -Pr, may be reactive in an IAT when cer-
shown in Table 16-3. Additional details on cal- tain enhancement media are used; therefore,
culating probability may be found in the sug- testing should be repeated in another medi-
gested readings list at the end of this chapter. um. If the DAT result is positive, it must be in-
The possibility of false-negative results with terpreted with careful attention to the transfu-
antigen-positive red cells must be considered sion history. Autoantibodies or drugs could
as well as that of unexpected positive results
explain a positive DAT result; however, if the
(ie, caused by the presence of an additional
patient has an alloantibody and was recently
antibody or an error in the presumptive anti-
transfused with blood that expressed the
body identification).
corresponding antigen, the circulating donor
red cells could be coated with alloantibody,
Autologous Control
resulting in a positive DAT result associated
The autologous control (autocontrol), in with a clinically significant delayed transfu-
which serum and autologous red cells are test- sion reaction.
5 3 2 0.100 0.035
6 4 2 0.067 0.022
6 3 3 0.050 0.016
7 5 2 0.048 0.015
7 4 3 0.029 0.008
8 7 1 0.125 0.049
8 6 2 0.036 0.011
8 5 3 0.018 0.005
8 4 4 0.014 0.004
CHAPTER 16 Identification of Antibodies to Red Cell Antigens 䡲 401
There are situations, however, where the that no single method is optimal for detecting
genotype of a person may not predict the red all antibodies. Any laboratory that performs
cell phenotype. Mutations that inactivate gene antibody detection or identification should
expression or rare new alleles may not be iden- use routine methods and have access to some
tified by the specific assay performed. The alternative approaches.
genotype obtained from DNA isolated from When a pattern of weak reactions fails to
leukocytes and other hematopoietic cells may indicate specificity or the presence of an anti-
differ from that of other tissues in people with body is suspected but cannot be confirmed,
a history of transplantation.19 the use of enhancement techniques or testing
When DNA testing is used as a tool in an- of panel cells treated with enzymes or chemi-
tibody identification, the predicted red cell cals may be helpful. An autocontrol should al-
phenotype should be used judiciously. If a ways be included with each technique.
sample appears to contain an antibody speci-
ficity when the predicted phenotype of the pa- LISS and PEG
tient is antigen positive, the apparent antibody
should be further investigated. This discrepan- LISS and PEG techniques (Methods 3-4 and 3-
cy may indicate that the patient’s red cells do 5) are used to enhance reactivity and reduce
not actually express the antigen due to a gene incubation time. LISS may be used to suspend
mutation not detected by the assay. Alterna- test red cells for use in tube or column aggluti-
tively, the patient might have an altered or par- nation tests or as an additive medium for tube
tial antigen due to an additional gene poly- or solid-phase tests. Care should be taken to
morphism. It is important to remember that follow the instructions in the manufacturer’s
the antibody in the sample could be, in fact, an product insert closely to ensure that the ap-
alloantibody. propriate proportion of serum to LISS is
achieved. Commercially prepared LISS addi-
tives or PEG additives may contain additional
Complex Antibody Problems
enhancing agents. Because LISS and PEG en-
Not all antibody identifications are simple. hance autoantibodies, their use may compli-
The exclusion procedure does not always lead cate alloantibody identification in samples
directly to an answer, and additional testing that also contain autoantibodies.20,21
may be required. When an antibody screen or
incompatible crossmatch detects an unex- Enzymes
pected antibody, the next step may be to deter-
mine whether the antibody is an autoantibody Ficin and papain are the most frequently used
or alloantibody. An autocontrol, which may enzymes for complex antibody identification.
not have been performed in the initial testing, They destroy or weaken antigens, such as M,
can start the identification process. Figure 16- N, Fya, Fyb, Xga, JMH, Ch, and Rg (Table 16-4).
1 shows some approaches to identifying anti- Antibodies to these antigens are nonreactive
bodies in a variety of situations when the auto- with treated red cells. Conversely, ficin-treated
control is negative, and Fig 16-2 shows some and papain-treated red cells show enhanced
approaches to identifying antibodies when the reactivity with other antibodies (eg, Rh, P, I,
autocontrol is positive. Kidd, and Lewis). Additional enzymes that are
commonly used in immunohematology labo-
ratories include trypsin, -chymotrypsin, and
Selected Serologic Procedures
pronase. Depending on the enzyme and meth-
Many techniques and methods are used in od used, other antigens may be altered or de-
complex antibody identification. Some of the stroyed. Antigens that are inactivated by one
methods described in this chapter are used proteolytic enzyme may not be inactivated
routinely by many laboratories; others are by other enzymes. The clinical significance
used selectively and may apply only in special of antibodies that are reactive only with
circumstances. It is important to remember enzyme-treated cells is questionable; such
CHAPTER 16
Identification of Antibodies to Red Cell Antigens
䡲
“enzyme-only” antibodies may not have clini- 5% saline suspension of red cells and incubat-
cal significance.22 ing the mixture for 60 minutes at 37 C. Periodic
In addition to enhancing the reactivity of mixing during the incubation promotes con-
certain antibodies, enzyme techniques may be tact between the red cells and the antibodies.
used to separate mixtures of antibodies. For It is helpful to remove the serum before wash-
example, if a serum sample contains anti-Fya ing the cells for an IAT because the standard
and anti-Jka, many of the red cell samples on three to four washes may be insufficient to re-
the initial panel would be reactive. Then, if a move all of the unbound immunoglobulin if
panel of enzyme-treated red cells were tested, increased amounts of serum are used. More
the anti-Jk a reactivity would be enhanced, than four washes are not recommended be-
whereas the anti-Fy a reactivity would be de- cause bound antibody molecules may dissoci-
stroyed. Procedures for the preparation and ate. Increasing the serum-to-red-cell ratio is
use of proteolytic enzymes are given in not appropriate for tests using LISS or com-
Methods 3-8 to 3-13. mercial PEG, which may contain LISS. Tests
performed in a low-ionic-strength medium
Temperature Reduction require specific proportions of serum and
additive.
Some antibodies (eg, anti-M, -N, -P1, -Le a,
-Leb, and -A1) react better at room temper- Increased Incubation Time
ature or below, and their specificity may be ap-
parent only at a temperature below 22 C. An For some antibodies, a 15-minute incubation
autocontrol is especially important for tests at period may not be sufficient to achieve equi-
low temperatures because many sera also librium; therefore, the reactions may be nega-
contain anti-I or other cold-reactive autoanti- tive or weak, particularly in saline or albumin
bodies. media. Extending the incubation time to be-
tween 30 and 60 minutes for albumin or saline
Increased Serum-to-Cell Ratio tests often improves the reactivity and helps
clarify the pattern of reactions. Extended incu-
Increasing the volume of serum incubated bation may be contraindicated when LISS or
with a standard volume of red cells may en- PEG is used. If the incubation period exceeds
hance the reactivity of antibodies that are the recommended times for these methods,
present in low concentrations. One acceptable the reactivity may be diminished or lost. Care
procedure involves mixing four volumes must be taken to use all reagents according to
(drops) of serum with one volume of a 2% to the manufacturer’s directions.
406 䡲 AABB TECHNICAL MANUAL
agents, such as 2-aminoethylisothiouronium 6. Destroying selected red cell antigens for use
bromide (AET), 2-mercaptoethanol (2-ME), or in antibody investigations (eg, antigens in
DTT, can be used to weaken or destroy anti- the Kell, Dombrock, Cartwright, LW, and
gens in the Kell system and some other anti- Knops systems) (Method 3-18).
gens (Method 3-18).30,31 ZZAP reagent, which
contains proteolytic enzyme and DTT, dena- Adsorption
tures antigens that are sensitive to DTT (eg, all
Kell system antigens) as well as antigens that Antibody can be removed from a serum sam-
are sensitive to enzymes (Method 4-8).32 Gly- ple by adsorption onto red cells that express
cine-HCl/EDTA treatment of red cells destroys the corresponding antigen. After the antibody
Bg and Kell system antigens as well as the Era attaches to the membrane-bound antigens
antigen (Methods 2-21 and 4-2).33 Chloroquine and the serum and cells are separated, the spe-
cific antibody remains attached to the red
diphosphate can be used to weaken the ex-
cells. It may be possible to harvest the bound
pression of Class I HLA antigens (Bg antigens)
antibody by elution or examine the adsorbed
on red cells.34 Chloroquine treatment also
serum for antibody(ies) remaining after the
weakens some other antigens, including Rh
adsorption process.
antigens (Method 2-20).
Adsorption techniques are useful for the
following purposes:
Sulfhydryl Reagents
Sulfhydryl reagents, such as DTT and 2-ME, 1. Separating multiple antibodies present in a
can be used to cleave the disulfide bonds that single serum.
join the monomeric subunits of the IgM pen- 2. Removing autoantibody to permit the
tamer. Intact 19S IgM molecules are cleaved detection or identification of underlying
into 7S Ig subunits, which have altered sero- alloantibodies.
logic reactivity.35 The interchain bonds of 7S Ig 3. Removing unwanted antibody (often anti-
monomers are relatively resistant to such A, anti-B, or both) from serum that contains
cleavage. Sulfhydryl reagents (DTT and 2-ME an antibody that is suitable for reagent use.
as well as AET) can also be used to cleave di- 4. Confirming the presence of specific anti-
sulfide bonds that are responsible for the con- gens on red cells by their ability to remove
formation of certain blood group antigens antibody of corresponding specificity from
and, therefore, are used to destroy certain red previously characterized serum.
cell antigens. 5. Confirming the specificity of an antibody
Uses of sulfhydryl reagents include the by showing that it can be adsorbed onto red
following: cells of only a particular blood group phe-
notype.
1. Determining the Ig class of an antibody
(Method 3-16). Adsorption serves different purposes in
2. Identifying antibodies in a mixture of IgM different situations; no single procedure is sat-
and IgG antibodies, particularly when an isfactory for all purposes (Methods 4-5, 4-8,
agglutinating IgM antibody masks the pres- 4-9, and 4-10). A basic procedure for antibody
ence of IgG antibodies. adsorption can be found in Method 3-20. The
3. Determining the relative amounts of IgG usual serum-to-cell ratio is one volume of se-
and IgM components of a given specificity rum to an equal volume of washed, packed red
(eg, anti-A or -B). cells. To enhance antibody removal, a larger
4. Dissociating red cell agglutinates caused by volume of red cells increases the proportion of
IgM autoantibodies (Method 2-18). antigen. The incubation temperature should
5. Dissociating IgG antibodies from red cells be that at which the antibody is optimally re-
using a mixture of DTT and proteolytic active. Pretreating red cells with a proteolytic
enzyme (ZZAP reagent) (Method 4-8). enzyme may enhance antibody uptake and
408 䡲 AABB TECHNICAL MANUAL
reduce the number of adsorptions required to for eluting warm-reactive auto- and alloanti-
remove an antibody completely. Because en- bodies. Commercial kits are also available for
zymes destroy some antigens, antibodies di- performing elution. (See Table 17-2 for a list of
rected against those antigens are not removed elution methods and their uses, advantages,
by enzyme-treated red cells. To ensure that an and disadvantages.)
adsorption process is complete (ie, that no un- Elution techniques are useful for the fol-
adsorbed antibody remains), it is essential to lowing:
confirm that the adsorbed serum is nonreac-
tive with a reserved sample of the adsorbing 1. Investigation of a positive DAT result
red cells that was not used for adsorption. Ad- (Chapter 17).
sorption requires a substantial volume of red 2. Concentration and purification of antibod-
cells, and vials of reagent red cells are usually ies, detection of weakly expressed antigens,
not sufficient. Blood samples from donor units and identification of multiple antibody
or staff members are the most convenient specificities. Such studies are used in con-
sources. junction with an appropriate adsorption
When separating mixtures of antibodies, technique, as described below and in
the selection of red cells of the appropriate Method 2-7.
phenotype is extremely important. If one or 3. Preparation of antibody-free red cells for
more antibodies have been previously identi- phenotyping or autologous adsorption
fied, red cells that express the corresponding studies. Procedures used to remove cold-
antigens can be used to remove the known an- and warm-reactive autoantibodies from red
tibodies and leave the unknown antibody(ies) cells are discussed in Methods 4-5 and 4-8.
in the adsorbed serum. For example, if a per-
son who types K+k–, Fy(a–b+) has produced Technical factors that influence the out-
anti-k, it may be necessary to adsorb the anti-k come of elution procedures include the follow-
onto K–k+, Fy(a–b+) reagent red cells to re- ing:
move the anti-k. Then, the adsorbed serum
can be tested with common K–k+, Fy(a+b–) 1. Incomplete washing. Sensitized red cells
red cells to detect possible anti-Fy a. should be thoroughly washed before an
elution to prevent contamination of the
Elution eluate with unbound residual antibody. Six
Elution dissociates antibodies from sensitized washes with saline are usually adequate,
red cells. Bound antibody may be released by but more washes may be needed if the
changing the thermodynamics of antigen- serum contains a high-titer antibody (the
antibody reactions, neutralizing or reversing considerations in Item 3 below should be
forces of attraction that hold antigen-antibody kept in mind). To confirm the efficacy of the
complexes together, or disturbing the struc- washing process, supernatant fluid from
ture of the antigen-antibody binding site. The the final wash should be tested for antibody
usual objective is to recover bound antibody in activity and found to be nonreactive.
a usable form. 2. Binding of protein to glass surfaces. If an
Various elution methods have been de- eluate is prepared in the test tube that was
scribed in the literature. Selected procedures used during the sensitization or washing
are given in Methods 4-1 through 4-4. No sin- phases, antibody that nonspecifically binds
gle method is best for all situations. Heat or to the test tube surface may dissociate dur-
freeze-thaw elution techniques are usually re- ing the elution. Similar binding can also
stricted to the investigation of HDFN caused occur from a whole blood sample when a
by ABO incompatibility because these elution patient has a positive DAT result and has
procedures rarely work well for other antibod- free antibody in the serum. To avoid such
ies. Acid or organic solvent methods are used contamination, red cells used to prepare an
CHAPTER 16 Identification of Antibodies to Red Cell Antigens 䡲 409
eluate should be transferred to a clean test will contain only that antibody. Both the eluate
tube before washing and then to another and adsorbed serum can be used for further
clean tube before the elution procedure is testing. Unmodified red cells are generally
initiated. used for adsorptions when subsequent elu-
3. Dissociation of antibody before elution. IgM tions are being prepared.
antibodies, such as anti-A or anti-M, or
low-affinity IgG may spontaneously disso- Titration
ciate from the red cells during the wash
The titer of an antibody is usually determined
phase. To minimize the loss of bound anti-
by testing serial twofold dilutions of the serum
body, cold (4 C) saline or wash solution pro-
with selected red cells. Results are expressed as
vided by the manufacturer should be used
the reciprocal of the highest serum dilution
for washing.
that shows macroscopic agglutination. Titra-
4. Incorrect technique. Such factors as incom-
tion values can provide information about the
plete removal of organic solvents or failure
relative amount of antibody present in a se-
to correct the tonicity or pH of an eluate
rum sample or the relative strength of antigen
may cause the reagent red cells used to test
expression on red cells.
the eluate to hemolyze or appear “sticky.”
Titration studies are useful for the follow-
The presence of stromal debris may inter-
ing purposes:
fere with the reading of test results. Careful
technique and strict adherence to proce-
dures should eliminate such problems. 1. Prenatal studies. When the antibody has a
5. Instability of eluates. Dilute protein solu- specificity that is known to cause HDFN or
tions, such as those obtained by elution the antibody’s clinical significance is
into saline, are unstable. Eluates should be unknown, the results of titration studies
tested as soon after preparation as possible. may contribute to the decision about per-
Alternatively, bovine albumin may be forming additional procedures (eg, Doppler
added to a final concentration of 6% w/v, sonography or amniocentesis). (See Chap-
and the preparation may be frozen during ter 22 and Method 5-3.)
storage. Eluates can also be prepared in 2. Antibody identification. Some antibodies
antibody-free plasma, 6% albumin, or a that agglutinate virtually all reagent red cells
similar protein medium. When commercial may give an indication of specificity by dem-
elution kits are used, the manufacturer’s onstrating reactivities of different strengths
instructions for preparation and storage with different red cell samples in titration
should be followed. studies. For example, potent undiluted auto-
anti-I may be reactive with both adult and
umbilical cord blood red cells, but titration
Combined Adsorption-Elution
studies may reveal reactivity with adult I+
Combined adsorption-elution tests can be cells at a higher dilution than with cord
used to separate a mixture of antibodies in a blood I– red cells. The reactivity of most
single serum sample, detect weakly expressed antibodies weakens progressively with serial
antigens on red cells, or help identify weakly dilutions (ie, a 2+ reaction becomes 1+ in the
reactive antibodies. The process consists of next dilution), and weak antibodies (<1+)
first incubating serum with selected red cells may lose their reactivity when diluted. How-
and then eluting antibody from the adsorbing ever, some antibodies that have weak reac-
red cells. tions when undiluted continue to react at
Care must be taken when selecting the dilutions as high as 1 in 2048. Such anti-
adsorbing cells to separate a mixture of anti- bodies include anti-Ch, -Rg, -Csa, -Yka, -Kna,
bodies. The cells should express only one of -McCa, and -JMH. When weak reactions are
the antigens corresponding to an antibody in observed in an IAT, titration studies may
the mixture so that the eluate from the cells be performed to determine whether the
410 䡲 AABB TECHNICAL MANUAL
Expression Antigens
orate, thus producing misleading antibody serve which antigens the reactive red cells
identification results. have in common. For example, if all of the red
The pH or other characteristics of the cells reactive at room temperature are P1+ but
storage medium can affect the rate of antigen the anti-P1 pattern is not complete, the anti-
deterioration.36,37 For example, Fya and Fyb an- body could be anti-P1 that is not reactive with
tigens may weaken when the red cells are red cells with a weaker expression of the anti-
stored in a medium with low pH and low ionic gen. (Sometimes, such red cells are designated
strength. Thus, certain antibodies may dem- on the panel sheet as “+w.”) In this case, it
onstrate differences in reactivity with red cells might be helpful to use a method that enhanc-
from different manufacturers if the suspend- es anti-P1, such as testing at a colder tempera-
ing media are different. ture.
The age and nature of the specimen must If all of the reactive red cells are Jk(b+) but
be considered when red cells are typed. Anti- not all Jk(b+) red cells are reactive, the reactive
gens on red cells from clotted samples tend to red cells might be Jk(a–b+) with a double-dose
deteriorate more quickly than antigens on red expression of the antigen. In this case, en-
cells from donor units that are collected in ci- hancement techniques, such as enzymes,
trate anticoagulants, such as acid-citrate-dex- LISS, or PEG, might help demonstrate reactivi-
trose or citrate-phosphate-dextrose. Red cells ty with all of the Jk(b+) red cells. Typing the pa-
in donor units collected in approved anticoag- tient’s red cells to confirm that they lack the
ulants usually retain their antigens throughout corresponding antigen is also very helpful.
the standard shelf life of the blood component. Finally, the presence of some antigens
EDTA samples up to 14 days old are suitable in common may suppress the expression of
for antigen typing.38 However, the manufactur- certain antigens. This suppression can cause
er’s instructions should be consulted when weak antibodies to be missed or certain cells
commercial typing reagents are used. to be unexpectedly nonreactive when a sus-
pected antibody fails to show reactivity with all
No Discernible Specificity antigen-positive cells. For example, In(Lu) is
Factors other than variation in antigen expres- known to suppress the expression of Lutheran
sion may contribute to difficulty in interpret- antigens, P1, In b, and AnWj. Similarly, Kp a is
ing results of antibody identification tests. If known to weaken the expression of Kell anti-
the reactivity with the serum is very weak, the gens. (See Chapter 14 for a more detailed dis-
pattern of reactivity and cross-out process cussion.)
have excluded all likely specificities, or both,
INHERENT VARI ABILIT Y. Neb u lo u s re a c -
alternative approaches to interpretation should
tion patterns that do not appear to fit any par-
be used.
ticular specificity are characteristic of certain
PRESENCE OF ANTIGENS IN COMMON. In- antibodies, such as anti-Bga, -Kna, -McCa, -Sla,
stead of excluding antibodies to antigens on -Yka, -Csa, and -JMH. Antigens corresponding
nonreactive red cells, it might be helpful to ob- to these antibodies vary markedly in their
412 䡲 AABB TECHNICAL MANUAL
expression on red cells from different individ- the results of testing performed with a single
uals. For example, the expression of Knops panel of reagent red cells. Perhaps the easiest
blood group antigens shows marked differenc- way to identify multiple antibodies is to deter-
es between individuals of either African or Eu- mine the phenotype of the patient’s pretrans-
ropean ethnicity, and this difference in expres- fusion autologous red cells and then use a se-
sion is caused by variations in the CR1 copy lected cell panel to identify or exclude all
numbers on the red cells.39 Rarely, a pattern of common antibodies to the red cell antigens
reactive and nonreactive red cells cannot be that the patient lacks. (See the discussion on
interpreted because the typing result for a re- selected cells earlier in this chapter.) The
agent red cell is incorrect or the reagent red presence of multiple antibodies may be sug-
cell has a positive DAT result. If the red cell gested by a variety of test results, such as the
sample is from a commercial source, the man- following:
ufacturer should be notified immediately of
the discrepancy. 1. The observed pattern of reactive and nonre-
active red cells does not fit a single antibody.
UNLISTED ANTIGENS. Sometimes, a serum
When the exclusion approach fails to indi-
reacts with an antigen that is not routinely list- cate a specific pattern, it is helpful to deter-
ed on the antigen profile supplied by the re- mine whether the pattern matches two
agent manufacturer—Ytb is an example. Even combined specificities. For example, if the
though serum studies yield clearly reactive reactive red cells on the panel in Table 16-2
and nonreactive test results, anti-Ytb may not are numbers 3, 5, 6, 9, and 10, none of the
be suspected. In such circumstances, it is use- specificities remaining after crossing out
ful to ask the manufacturer for additional phe- fits a pattern exactly. However, if both E and
notype information. If only one cell is unex- K are considered together, a pattern is dis-
pectedly reactive, this reaction is most likely cerned, with cells 3 and 5 showing reactivity
caused by an antibody to a low-prevalence an- because of anti-E, and cells 6, 9, and 10
tigen. These antibodies are discussed in more because of anti-K. If the reaction pattern
detail later in this chapter. does not fit two combined specificities, the
ABO T YPE OF RED CELLS TESTED. A serum
possibility that more than two antibodies
sample may be reactive with many or all of the are present must be considered. The more
group O reagent red cells but not with red cells antibodies a serum sample contains, the
of the same ABO group as the autologous red more complex identification and exclusion
cells. Such a reaction occurs most frequently become, but the basic process remains the
with anti-H, anti-IH, or anti-LebH. Group O and same.
A2 red cells have more H antigen than A1 and 2. Reactivity occurs at different test phases.
A1B red cells, which express very little H. (See When reactivity occurs at several phases,
Chapter 12 for more information.) Thus, sera each phase should be analyzed separately.
containing anti-H or anti-IH are strongly reac- The pattern at room temperature may indi-
tive with group O reagent red cells, whereas cate a different specificity from the pattern
autologous A 1 or A 1 B red cells or donor red at the AHG phase. It is also helpful to look
cells used for crossmatching may be weakly re- for variations in the strength of the reac-
active or nonreactive. Anti-LebH is strongly re- tions at each phase of testing. Table 16-6
active with group O, Le(b+) red cells but weak- provides information about the character-
istic reactivity of several antibodies.
ly reactive or nonreactive with Le(b+) red cells
3. Unexpected reactions occur when attempts
from A1 or A1B individuals.
are made to confirm the specificity of a sus-
pected single antibody. If a serum suspected
Multiple Antibodies
to contain anti-e is reactive with some e-
When a serum sample contains two or more negative cells, another antibody may be
alloantibodies, it may be difficult to interpret present or the suspected antibody may not
CHAPTER 16 Identification of Antibodies to Red Cell Antigens 䡲 413
Anti-M IgG > IgM Most Most Rare Sensitive Resistant Rare Rare
Anti-N IgM > IgG Most Most Rare Sensitive Resistant No Rare
Anti-S IgG > IgM Most Most Variable Resistant Yes Yes
Anti-s IgG > IgM Most Variable Resistant Yes Yes
Anti-U IgG Most Resistant Resistant Yes Yes
Anti-P1 IgM Most Most Resistant Resistant No Rare
(IgG rare)
Anti-D IgG > IgM Some Some Most Resistant Resistant Yes Yes
(IgA rare)
Anti-C IgG > IgM Some Some Most Resistant Resistant Yes Yes
Anti-E IgG > IgM Some Some Most Resistant Resistant Yes Yes
Anti-c IgG > IgM Some Some Most Resistant Resistant Yes Yes
Anti-e IgG > IgM Some Some Most Resistant Resistant Yes Yes
a
Anti-Lu IgM > IgG Most Most Resistant or Variable Rare No
weakened
Anti-Lub IgG > IgM Some Most Resistant or Variable Mild Yes
weakened
Anti-K IgG > IgM Some Most Resistant Sensitive Yes Yes
Anti-k IgG > IgM Most Resistant Sensitive Yes Yes
a
Anti-Kp IgG Most Resistant Sensitive Yes Yes
Anti-Kpb IgG > IgM Most Resistant Sensitive Yes Yes
Anti-Jsa IgG > IgM Most Resistant Sensitive Yes Yes
b
Anti-Js IgG Most Resistant Sensitive Yes Yes
a
Anti-Le IgM > IgG Most Most Most Most Resistant Resistant No Rare
Anti-Leb IgM > IgG Most Most Most Most Resistant Resistant No No
a
Anti-Fy IgG > IgM Most Sensitive Resistant Yes Yes
b
Anti-Fy IgG > IgM Most Sensitive Resistant Yes Yes
a
Anti-Jk IgG > IgM Most Resistant Resistant Yes Yes
Anti-Jkb IgG > IgM Most Resistant Resistant Yes Yes
a
Anti-Di IgG Most Resistant Resistant Yes Yes
b
Anti-Di IgG Most Resistant Resistant Yes Yes
414 䡲 AABB TECHNICAL MANUAL
TABLE 16-6. Serologic Reactivity of Some Common Blood Group Antibodies (Continued)
AHG = antiglobulin reagent; DTT = dithiothreitol; HDFN = hemolytic disease of the fetus and newborn; HTR = hemolytic
transfusion reaction; Ig = immunoglobulin.
be anti-e. Testing a panel of selected e-neg- c. Type the patient’s red cells for com-
ative red cells may help identify an addi- mon red cell antigens, and eliminate
tional specificity. from consideration specificities that
4. No discernible pattern emerges. When vari- correspond to antigens on the patient’s
able reaction strengths are observed and autologous red cells. This step may not
dosage or other variations in antigen be possible if the patient has been
strength do not provide an explanation, transfused recently or has had a posi-
additional approaches and methods of test- tive DAT result.
ing are needed. Some helpful steps include d. Use a method to inactivate certain
the following:
antigens on the reagent cells. Enzyme
a. If strongly positive results are
treatment renders cells negative for
obtained, use the exclusion method
with nonreactive cells to eliminate such antigens as Fya and Fyb (see Table
some specificities from initial consid- 16-4).
eration. e. Use adsorption or elution methods to
b. If weak or questionable positive results separate antibodies (Methods 3-20, 4-1,
are obtained, test the serum against and 4-2).
cells with a strong expression of the f. Enhance antibody reactivity by using a
antigen that corresponds with any sus- more sensitive method (eg, PEG,
pected specificity, and combine this enzymes, increased incubation time,
approach with methods that enhance or increased serum-to-cell ratio; see
the reactivity. Methods 3-5 and 3-8 through 3-13).
CHAPTER 16 Identification of Antibodies to Red Cell Antigens 䡲 415
however, weak sensitization and mixed-field antigen; however, other possible explanations
agglutination can be difficult to differentiate. If include that the red cells may be ABO incom-
a pretransfusion specimen is not available, it patible, have a positive DAT result, or be poly-
may be helpful to use red cell separation pro- agglutinable.
cedures to isolate autologous red cells for test- If an antibody in the serum of a pregnant
ing or perform a DNA-based genotype, as de- woman is suspected of reacting with a low-
scribed earlier in this chapter. Performing a prevalence antigen, testing the father’s red
DAT on autologous red cells, testing the post- cells with maternal plasma (if the plasma is
transfusion serum with DAT-negative autolo- ABO compatible) can predict the possibility
gous red cells, or both may help distinguish an that the fetal red cells also carry the paternal
autoantibody from an alloantibody. If a DAT antigen and are incompatible with the mater-
result from autologous red cells is negative, the nal antibody, even if the antibody specificity is
reactivity is consistent with an alloantibody. If unknown. If a newborn has a positive DAT re-
the posttransfusion serum is reactive with sult, testing the mother’s serum or an eluate
DAT-negative autologous red cells, the reactiv- from the infant’s red cells against the father’s
ity is consistent with an autoantibody (Chap- red cells can implicate an antibody to a low-
ter 17 and Fig 16-2). prevalence antigen as the probable cause.
That test can be performed only if the mother’s
Antibodies to Low-Prevalence Antigens plasma is ABO compatible with the father’s red
cells or if the eluate from the infant’s red cells
If a serum sample is reactive only with a single
does not contain anti-A or -B that would be re-
donor or reagent red cell sample, an antibody
active with the father’s red cells. Some refer-
to a low-prevalence antigen should be sus-
ence laboratories do not attempt to identify
pected. To identify such an antibody, one can
antibodies to low-prevalence antigens be-
test the serum with a panel of reagent red cells
that express low-prevalence antigens. Alterna- cause the antibodies are not clinically mean-
tively, the one reactive red cell sample can be ingful. Antibodies may be identified when
tested with known antibodies to low-preva- time permits and suitable reagents are avail-
lence antigens. Unfortunately, sera that con- able.
tain antibodies to low-prevalence antigens
often contain multiple antibodies to low-prev- Antibodies to Reagent Components and
alence antigens. Although low-prevalence an- Other Anomalous Serologic Reactions
tigens are rare by definition, antibodies that Antibodies to a variety of drugs and additives
recognize some of them are much less rare. can cause positive results in antibody detec-
Many antibodies to low-prevalence antigens tion and identification tests. The mechanisms
are reactive only at temperatures below 37 C are probably similar to those discussed in
and therefore have doubtful clinical signifi- Chapter 17. Most of these anomalous reac-
cance. To confirm the suspected specificities, tions are in-vitro phenomena and have no
one may need the expertise and resources of a clinical significance in transfusion therapy,
reference laboratory. other than causing laboratory problems that
If an antibody to a low-prevalence anti- delay transfusions. The reactions rarely cause
gen is suspected, transfusion should not be erroneous interpretations of ABO typing that
delayed while identification studies are per- could endanger the patient. For a more de-
formed. Because antisera to type donor units tailed discussion, see the suggested reading by
for low-prevalence antigens are rarely avail- Garratty.
able, it is usually necessary to rely on the cross-
match to avoid transfusion of antigen-positive ROULEAUX. Rouleaux are aggregates of red
units. When the serum is reactive with only cells that often look like a stack of coins when
one donor unit or reagent red cell, the most viewed microscopically. Rouleaux formation is
likely cause is an antibody to a low-prevalence an in-vitro phenomenon produced by abnor-
CHAPTER 16 Identification of Antibodies to Red Cell Antigens 䡲 417
mal serum protein concentrations. It may be the antigen, but the DAT result should be neg-
difficult to detect antibodies by direct aggluti- ative.
nation in a test serum that contains rouleaux-
RE D-CELL-REL ATE D ANOMA LIE S. The age
producing proteins; however, it is not a prob-
lem in an IAT, where the serum is washed of red cells can cause anomalous serologic re-
away. The saline replacement technique can actions. Antibodies exist that are reactive only
be used to detect direct-agglutinating antibod- with stored red cells. Such antibodies can
ies in the presence of rouleaux (Method 3-7). cause agglutination of reagent red cells by all
techniques, and the reactivity may be en-
PRESERVATIVE SOLUTIONS. A n t i b o d i e s hanced with enzyme-treated red cells. Such re-
that are reactive with an ingredient in the solu- activity is not affected by washing the red cells,
tion used to preserve reagent red cells (eg, and the autocontrol is usually nonreactive. No
chloramphenicol, neomycin, tetracycline, hy- reactivity is seen when freshly collected red
drocortisone, EDTA, sodium caprylate, or vari- cells (ie, from freshly drawn donor or autolo-
ous sugars) may agglutinate red cells suspend- gous blood samples) are tested.
ed in that solution. The autologous control is
often nonreactive unless a suspension of au- Patients with a Positive Autocontrol
tologous red cells is prepared with the manu-
Reactivity of a patient’s serum with the autolo-
facturer’s red cell diluent or a similar preserva-
gous cells may indicate the presence of warm or
tive. Such reactions can often be avoided by
cold autoantibodies, antibodies to certain
washing the reagent red cells with saline be-
drugs, alloantibodies to transfused red cells if
fore testing. Adding the medium to the autolo-
the patient was recently transfused, or antibod-
gous control and converting a nonreactive test
ies to a constituent of the test medium. When
to a reactive test can often confirm the role of
the autocontrol is positive, a DAT should be
the preservative. In some cases, however,
performed (Chapter 17 and Fig 16-2).
washing the reagent red cells does not circum-
vent the reactivity, and the resolution may be NO RECENT TRANSFUSIONS. If the reactivi-
more complex. ty in the serum occurs at room temperature or
below, the cause is often anti-I or another cold
ENHA NCEME NT MEDIA . Antibodies that are
autoagglutinin. If the reactivity occurs in the
reactive with ingredients in other reagents,
AHG phase, the reactivity is usually associated
such as commercially prepared LISS additives
with a positive DAT result and possible auto-
or albumin, can cause agglutination in tests
antibodies. If, in addition, the serum is reac-
that use reagent red cells, donor red cells, au- tive with all cells tested, autoadsorption or
tologous red cells, or all three. Ingredients that other special procedures may be necessary to
have been implicated include parabens (in determine whether there are underlying allo-
some LISS additives), sodium caprylate (in antibodies that are masked by the autoanti-
some albumins), and thimerosal (in some bodies. If the serum is nonreactive or shows
LISS/saline preparations). Antibody to ingre- only weak reactivity, an eluate may demon-
dients in enhancement media may be suspect- strate more potent autoantibody reactivity.
ed if the autologous control is positive but the (See “Cold Autoantibodies” and “Warm Auto-
DAT result is negative. Omitting the enhance- antibodies” sections below and Chapter 17 for
ment medium usually circumvents the reac- a more detailed discussion.)
tivity.
In some cases, antibodies to reagent in- RE CENT TRANSFUSIONS. If the autocontrol
gredients show blood group specificity (eg, is positive in the AHG phase, there may be an-
paraben-dependent anti-Jka, paraben-depen- tibody-coated donor red cells in the patient’s
dent antibody to Rh protein, or caprylate-de- circulation, resulting in a positive DAT result
pendent autoanti-e).40-42 The autocontrol may that may show mixed-field reactivity. An elu-
be reactive if the patient’s own red cells express tion should be performed, especially when
418 䡲 AABB TECHNICAL MANUAL
tests on serum are inconclusive. For example, COLD AUTOANTIBODIES. Potent cold auto-
a recently transfused patient may have a posi- antibodies that are reactive with all red cells,
tive autocontrol and serum that is weakly reac- including the patient’s own, can create special
tive with most but not all Fy(a+) red cells. It problems—especially when the reactivity per-
may be possible to confirm anti-Fya specificity sists at temperatures above room temperature.
in an eluate because more antibody is often Cold autoantibodies may be benign or patho-
bound to donor red cells than is free in the logic. (See Chapter 17 for a more detailed dis-
plasma. Furthermore the preparation of an el- cussion.)
uate concentrates the antibody. It is rare for There are different approaches to testing
transfused red cells to make the autocontrol sera with potent cold agglutinins. To detect
positive at other test phases, but this can oc- underlying and potentially clinically signifi-
cur, especially with a newly developing or cant antibodies, methods that circumvent the
cold-reacting alloantibody. cold autoantibody can be used. Procedures for
Some patients may form warm autoanti- the detection of alloantibodies in the presence
bodies following red cell transfusion; there- of cold-reactive autoantibodies include the
fore, if the positive DAT test does not have a following:
mixed-field appearance—and especially if the
serum is reactive with all red cells—then the 1. Prewarming techniques in which red cells
possibility of an autoantibody should be con- and serum are prewarmed to 37 C sepa-
sidered. Detection of alloantibodies in sam- rately before they are combined (Method 3-
ples with autoantibody may require allogeneic 6).
adsorptions (Method 3-20). If the patient’s 2. The use of anti-IgG rather than polyspecific
phenotype is known for the common red cell AHG reagent.
antigens, an allogeneic adsorption may re- 3. Cold autoadsorption of the patient’s serum
quire only one adsorption using a reagent cell with autologous red cells to remove autoan-
that is matched for the antigens that the pa- tibodies but not alloantibodies.
tient lacks. If the patient’s phenotype is un- 4. Adsorption with rabbit erythrocytes or rab-
known, it is necessary to perform multiple al- bit erythrocyte stroma.
logeneic adsorptions using a combination of WARM AUTOANTIBODIES. Pa t i e n t s w i t h
reagent cells, each of which lacks some of the warm-reactive autoantibodies in their sera
common red cell antigens. Cells that are ho- create a special challenge because the anti-
mozygous for the common red cell antigens, body is reactive with virtually all red cells test-
such as R1 (DCe), R2 (DcE), and r (ce), are fre- ed. The majority of warm autoantibodies are
quently used. The adsorbed serum is tested IgG, although some warm autoantibodies are
with reagent cells to rule out the common an- IgM. IgM warm autoantibodies are unusual,
tibodies that correspond to the antigens that but they have caused severe (often fatal) auto-
are missing from the adsorbing cell. For exam- immune hemolytic anemia.43 If a patient with
ple, if the adsorbing cell types R1 (DCe), K–, warm autoantibodies requires a transfusion, it
Fy(a+b–), Jk(a–b+), and S–s+, the adsorbed is important to detect any underlying clinically
plasma could be tested for anti-c, anti-E, anti- significant alloantibodies that the autoanti-
K, anti-Fy b, anti-Jk a , and anti-S. It is never bodies may mask. (For more information, see
possible, however, to rule out antibodies to Chapter 17 and Methods 4-8 through 4-10.)
high-prevalence antigens when allogeneic ad- The reactivity of most warm-reactive autoanti-
sorption techniques are used. bodies is greatly enhanced by certain methods
If the DAT result does have a mixed-field such as PEG and enzymes and, to a lesser ex-
appearance and the serum is reactive with all tent, by LISS. It is often helpful to omit the en-
cells tested, a transfusion reaction caused by hancement media when testing sera that con-
an alloantibody to a high-prevalence antigen tain warm autoantibodies. If such tests are
should be considered (Fig 16-2). nonreactive, the same procedure can be used
CHAPTER 16 Identification of Antibodies to Red Cell Antigens 䡲 419
for compatibility testing without the need for clinical significance. Table 16-6 summarizes
adsorptions. the expected reactivity and clinical signifi-
cance of commonly encountered alloantibod-
Frequency of Antibody Testing ies. For some antibodies, little or no data exist,
After a clinically significant antibody has been and the decision about clinical significance
identified, antigen-negative Red Blood Cell must be based on the premise that clinically
(RBC) units must be selected for all future significant antibodies are those that are active
transfusions, even if the antibodies are no lon- at 37 C, in an IAT, or both.
ger detectable.17(p37) In addition, an AHG cross- Certain laboratory tests have been used to
match must be performed. It is rarely neces- predict the clinical significance of antibodies.
sary to repeat the identification of known The monocyte monolayer assay, which quanti-
antibodies. AABB Standards for Blood Banks fies phagocytosis, adherence of antibody-coat-
and Transfusion Services states that in patients ed red cells, or both, can be used to predict the
with previously identified antibodies, testing in-vivo clinical significance of some antibod-
methods should be used that identify addi- ies. The test for antibody-dependent cellular
tional clinically significant antibodies.17(p36) cytotoxicity, which measures lysis of antibody-
Each laboratory should define and validate coated red cells, and the chemiluminescence
methods for the detection of additional anti- assay, which measures the respiratory release
bodies in these patients. of oxygen radicals after phagocytosis of anti-
body-coated red cells, have been helpful in
Immunohematology Reference predicting in-vivo antibody reactivity—partic-
Laboratories ularly for predicting the severity of HDFN. For
cold-reactive antibodies, in-vitro thermal am-
When antibody problems cannot be resolved
plitude studies may predict the likelihood of
or rare blood is needed, a reference laboratory
in-vivo hemolysis.44
can provide consultation and assistance
In-vivo tests may also be used to evaluate
through its access to the American Rare Donor
the significance of an antibody. The most
Program (ARDP). (See Method 3-21.)
common technique is a red cell survival study
in which radiolabeled, antigen-positive red
P O S TA N A LY TI C A L cells (usually labeled with 51Cr) are infused into
CO NS I D E R AT IO N S : SE L E C T I NG the patient. After a specified period has
BLOOD FOR TRANSFUSION elapsed, a sample of blood from the patient is
After an antibody has been identified, it is im- tested for radioactivity. With this technique, it
portant to determine its clinical significance. is possible to measure the survival of 1 mL or
Antibodies that are reactive at 37 C, in an IAT, less of infused cells. Another in-vivo tech-
or both are potentially clinically significant. nique, flow cytometry, can also be used to
Antibodies that are reactive at room tempera- measure the survival of infused red cells, but a
ture and below are usually not clinically signif- larger aliquot of red cells (about 10 mL) is usu-
icant; however, there are many exceptions. For ally required. Interpretation of in-vivo survival
example, anti-Vel, -P, and -PP1P k (-Tja) may be test results is complicated by the fact that
reactive only at cold temperatures yet may small aliquots of incompatible red cells may
cause red cell destruction in vivo. Anti-Ch, have a faster rate of destruction than an entire
anti-Rg, and many of the Knops and Cost anti- transfused RBC unit. Comparison with docu-
bodies have little or no clinical significance mented cases in the literature and consulta-
despite their reactivity in an IAT. Reported tion with a reference laboratory should pro-
experience with examples of antibodies with vide guidance about previous examples of
the same specificity can be used in assessing the similar specificities.
420 䡲 AABB TECHNICAL MANUAL
Phenotyping Donor Units bodies from two different sources, but others
consider this step unnecessary—especially
Whenever possible, RBC units selected for
when potent reagents are available and an
transfusion to a patient with potentially clini-
AHG crossmatch will be performed. Different
cally significant antibodies should be tested
lots of antibody from the same manufacturer
and found negative for the corresponding an-
and even different reagents from different
tigen(s). Even if the antibodies are no longer
manufacturers may have been prepared from
detectable, all subsequent RBC transfusions to
that patient should lack the antigen to prevent the same source material because manufac-
a secondary immune response. The transfu- turers often share these resources.
sion service must maintain records of all pa- If a donor unit is tested for selected anti-
tients in whom clinically significant antibodies gens and labeled by the blood center, the use
have been previously identified, and an AHG of licensed (commercial) reagents, if available,
crossmatch procedure is required if the serum is required. If no licensed reagent is available,
contains—or has previously contained—a the unit may be labeled with appropriate
clinically significant antibody.17(pp37-38,75) wording (eg, “Tested and found negative for
A potent example of the antibody should XX antigen using unlicensed typing re-
be used to identify antigen-negative blood. Of- agents”).47 Except for results of ABO and D typ-
ten, the antibody is a commercial antiserum, ing, there is no requirement that the hospital
but to save expensive or rare reagents, units repeat testing of donor minor antigen typing if
can be tested first for compatibility with the the results are attached to the units on the la-
patient’s serum. Then, the absence of the anti- bel or by a tag. Minor antigen typing results on
gen in compatible units can be confirmed with packing slips or not physically attached to the
commercial reagents. If the antibody is unusu- donor unit should be confirmed by the hospi-
al and commercial antiserum is not available, tal if the unit is used for clinical care.
a stored sample from the sensitized patient
can be used to select units for transfusion at a Antigen-Negative Blood vs Crossmatch
later time, especially if the patient’s later sam- for Compatibility
ples lose reactivity. If a patient’s serum is used
as a typing reagent, the antibody should be For certain antibodies, typing the donor units
well characterized and retain its reactivity after may not be necessary, and the patient’s serum
storage. Appropriate negative and weak-posi- can be used to select serologically compatible
tive controls (eg, from heterozygous donors) RBC units. This is especially true for antibod-
should be used at the time of the testing. The ies that characteristically are reactive below
following criteria, established by the FDA for 37 C (eg, anti-M, -N, -P1, -Lea, -Leb, and -A1)
licensing some reagents, should be used as and that do not ordinarily produce a second-
guidelines for human-source reagents used in ary immune response following the transfu-
lieu of commercial reagents45,46: sion of antigen-positive RBC units.
It can sometimes be a best practice to
1. Anti-K, -k, -Jka, -Fya, and –Cw: dilution of 1:8 provide phenotypically matched, antigen-neg-
must produce at least a 1+ reaction. ative RBC units as a prophylactic measure
2. Anti-S, -s, -P1, -M, -I, -c (saline), -e (saline), when the patient has no detectable antibody.
and -A1: dilution of 1:4 must produce at When a patient of the R1R1 phenotype produc-
least a 1+ reaction. es anti-E, some serologists suggest that RBC
3. Most other specificities: undiluted must units should be negative for both the E and c
produce at least a 2+ reaction. antigens. This recommendation is based on
the assumption that the stimulus to produce
When selecting units for patients with anti-E may also have stimulated anti-c or anti-
clinically significant antibodies, some serolo- cE that remains undetected by routine tests.48
gists recommend typing the units with anti- Similarly, for an R2R2 patient with demonstra-
CHAPTER 16 Identification of Antibodies to Red Cell Antigens 䡲 421
ble anti-C, the use of e-negative donor blood nic composition of the donor population, if
may be considered. available.
When a patient has potent warm autoan- When units of rare (<1 in 5000) or uncom-
tibody and compatibility cannot be demon- mon (<1 in 1000) phenotypes are needed, the
strated by routine testing or when an antibody ARDP can be very helpful. This program,
has not been specifically demonstrated but which is accessible only to personnel from an
cannot be conclusively excluded, it may be accredited reference laboratory, can identify
prudent to select RBC units that are phenotyp- blood suppliers that are known to have poten-
ically matched for clinically significant anti- tially compatible units (usually frozen RBC
gens. units) and donors who may be eligible to do-
nate (Method 3-21).
When Rare Blood Is Needed Family members are another potential
source of rare blood donors. The absence of
Rare blood includes units that are negative for high-prevalence antigens is usually associated
high-prevalence antigens or are negative for a with the inheritance of the same rare recessive
combination of common antigens. When a pa- blood group gene from each heterozygous
tient has multiple antibodies, it can be helpful parent. Children from the same parents have
to determine the prevalence of compatible do- one chance in four of inheriting the same two
nors. To calculate this prevalence, one must rare genetic mutations, making siblings much
multiply the prevalence of donors who are more likely than the general population to
negative for one antigen by the prevalence of have the rare blood type. In most cases, blood
donors who are negative for each of the other from the patient’s parents, children, and half of
antigens. For example, if a serum contains the patient’s siblings express only one rare
anti-c, anti-Fya, and anti-S and if the preva- gene. If transfusion is essential and there is no
lence of antigen-negatives are c-negative = alternative to transfusing incompatible blood,
18%, Fy(a–) = 34%, and S-negative = 45%, then these heterozygous (single-dose) donors are
the prevalence of compatible units is 0.18 × preferable to random donors. For infants with
0.34 × 0.45 = 0.028, or 2.8%. If the patient is HDFN resulting from multiple antibodies or
group O, the prevalence of group O donors an antibody to a high-prevalence antigen, the
(45%) is factored into the calculation as fol- mother (if she is ABO compatible) is often the
lows: 0.028 × 0.45 = 0.013, or 1.3%. logical donor.
If any of these antibodies is present alone, If the clinical situation allows, autologous
finding compatible blood is not very difficult; RBC transfusions should be considered for pa-
but the combination requires a large number tients with rare phenotypes who are expected to
of units to provide one compatible unit. The need rare blood in the future. For some patients
calculation above uses the prevalence in pop- with multiple antibodies who are not able to
ulations of European ethnicity, and prevalence donate autologous units, it may be necessary to
may be different in populations of non- determine whether any of the antibodies is less
European ethnicity. In calculating the proba- likely to cause red cell destruction and, in a crit-
bility of compatible donors, one should use ical situation, to give blood that is incompatible
the prevalence that corresponds with the eth- for that particular antigen.
KEY POINTS
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saline. Immunohematol 1993;9:15-18. sociated autoanti-Jka antibodies: Three exam-
26. Morton JA, Pickles MM, Terry AM. The Sda ples detected using commercially prepared
blood group antigen in tissues and body fluids. low-ionic strength saline containing parabens.
Vox Sang 1970;19:472-82. Transfusion 1982;22:31-5.
27. O’Neill GJ, Yang SY, Tegoli J, et al. Chido and 41. Judd WJ, Storry JR, Amnesley TD, et al. The
Rodgers blood groups are distinct antigenic first example of a paraben-dependent anti-
424 䡲 AABB TECHNICAL MANUAL
body to an Rh protein. Transfusion 2001;41: Harmening DM. Modern blood banking and trans-
371-4. fusion practices. 6th ed. Philadelphia: FA
42. Dube VE, Zoes C, Adesman P. Caprylate-de- Davis, 2012.
pendent auto-anti-e. Vox Sang 1977;33:359-63. Issitt PD, Anstee DJ. Applied blood group serology.
43. Arndt PA, Leger RM, Garratty G. Serologic find- 4th ed. Durham, NC: Montgomery Scientific
ings in autoimmune hemolytic anemia associ- Publications, 1998.
ated with immunoglobulin M warm autoanti- Judd WJ, Johnson S, Storry J. Judd’s methods in
bodies. Transfusion 2009;49:235-42. immunohematology. 3rd ed. Bethesda, MD:
44. Petz LD, Garratty G. Immune hemolytic ane- AABB Press, 2008.
mias. 2nd ed. Philadelphia: Churchill Living- Kanter MH. Statistical analysis. In: Busch MP,
stone, 2004. Brecher ME, eds. Research design and analy-
45. Food and Drug Administration. 7342.001: In- sis. Bethesda, MD: AABB, 1998:63-104.
spection of licensed and unlicensed blood Klein HG, Anstee DJ. Mollison’s blood transfusion in
banks, brokers, reference laboratories, and clinical medicine. 12th ed. Oxford: Wiley-
contractors. Silver Spring, MD: CBER Office of Blackwell, 2014.
Compliance and Biologics Quality, 2010:50-3. Levitt J, ed. Standards for blood banks and transfu-
[Available at http://www.fda.gov/downloads/ sion services. 29th ed. Bethesda, MD: AABB,
BiologicsBloodVaccines/GuidanceComplian 2014.
ceRegulatoryInformation/ComplianceActivi Lomas-Frances C, DePalma H. 2007 Rock Øyen
ties/Enforcement/CompliancePrograms/ Symposium. DNA-based assays for patient
UCM337001.pdf (accessed November 20, testing: Their application, interpretation, and
2013).] correlation of results. Immunohematology
46. Code of federal regulations. Title 21, CFR 2008;24:180-90.
660.25, 660.26. Washington, DC: US Govern- Menitove JE. The Hardy-Weinberger principle:
Selection of compatible blood based on math-
ment Printing Office, 2014 (revised annually).
47. Shirey RS, Edwards RE, Ness PM. The risk of ematic principles. In: Fridey JL, Kasprisin CA,
Chambers LA, Rudmann SV, eds. Numbers for
alloimmunization to c (Rh4) in R1R1 patients
blood bankers. Bethesda, MD: AABB, 1995:
who present with anti-E. Transfusion 1994;
111.
34:756-8.
Reid ME, Lomas-Francis C, Olsson M. The blood
group antigen factsbook. 3rd ed. London:
SUG G ES TE D R EA DI N GS Elsevier Academic Press, 2012.
Rolih S. A review: Antibodies with high-titer, low-
Dake L, ed. Standards for immunohematology ref- avidity characteristics. Immunohematology
erence laboratories. 8th ed. Bethesda, MD: 1990;6:59-67.
AABB, 2013. Rudmann SV, ed. Serologic problem-solving: A sys-
Daniels G. Human blood groups. 3rd ed. Hoboken, tematic approach for improved practice.
NJ: Wiley-Blackwell, 2013. Bethesda, MD: AABB Press, 2005.
Daniels G, Poole J, deSilva M, et al. The clinical sig- Weisbach V, Kohnhauser T, Zimmermann R, et al.
nificance of blood group antibodies. Transfus Comparison of the performance of microtube
Med 2002;12:287-95. column systems and solid-phase systems and
Engelfriet CP, Overbeeke MA, Dooren MC, et al. Bio- the tube low-ionic-strength solution additive
assays to determine the clinical significance of indirect antiglobulin test in the detection of
red cell antibodies based on Fc receptor- red cell alloantibodies. Transfus Med 2006;16:
induced destruction of red cells sensitized 276-84.
with IgG. Transfusion 1994;34: 617-26. Westhoff C. 2007 Rock Øyen Symposium. Potential
Garratty G. In-vitro reactions with red blood cells of blood group genotyping for transfusion
that are not due to blood group antibodies: A medicine practice. Immunohematology 2008;
review. Immunohematology 1998; 14:1-11. 24:190-5.
C h a p t e r 1 7
test
I fit to performing a DAT (or autocontrol) as part
THE DIRECT ANTIGLOBULIN
(DAT) is a simple test used to determine of routine pretransfusion testing. The predic-
if red cells have been coated in vivo with im- tive value of a positive DAT result is 83% in a
munoglobulin (Ig), complement, or both. The patient with hemolytic anemia, but only 1.4%
DAT is used primarily for the investigation of in a patient without, hemolytic anemia.1
hemolytic transfusion reactions, hemolytic Small amounts of IgG and complement
disease of the fetus and newborn (HDFN), au- that are lower than the detection limit of rou-
toimmune hemolytic anemia (AIHA), and tine testing techniques appear to be present
drug-induced immune hemolysis. A positive on all red cells. Using sensitive testing tech-
DAT result may or may not be associated with niques, 5 to 90 IgG molecules/red cell2 and 5 to
immune-mediated hemolysis. As shown in Ta- 40 C3d molecules/red cell3 have been detected
ble 17-1, there are many causes of a positive in healthy individuals. Depending on the tech-
DAT result. nique and reagents used, the DAT can detect
100 to 500 molecules of IgG/red cell and 400 to
1100 molecules of C3d/red cell. Positive DAT
TH E DAT
results are reported in 1:1000 to 1:14,000 blood
The DAT should be performed on every pa- donors and 1% to 15% of hospital patients.4
tient in whom the presence of hemolysis has These large differences in incidence are proba-
been established to distinguish immune from bly related to the different DAT techniques
nonimmune hemolytic anemia. The DAT used.
should also be performed when a positive au- Most blood donors with a positive DAT re-
tocontrol is found in antibody identification sult appear to be perfectly healthy, and most
studies (see Chapter 16), but there is no bene- patients with positive DAT results have no
Regina M. Leger, MSQA, MT(ASCP)SBB, CMQ/OE(ASQ), Research Associate, American Red Cross Blood Ser-
vices, Southern California Region, Pomona, California
The author has disclosed no conflicts of interest.
425
426 䡲 AABB TECHNICAL MANUAL
and anti-C3b,-C3d reagents are currently li- results can be obtained if the washed red cells
censed. The red cells need to be washed to re- are allowed to sit before they are tested with
move free plasma globulins and complement; anti-IgG or if the reading of the results is de-
otherwise, the antiglobulin reagent can be layed. Some anticomplement reagents, in con-
neutralized, leading to a false-negative result. trast, demonstrate stronger reactivity if cen-
The saline used for washing the red cells trifugation is delayed for a short time after the
should be at room temperature; washing red reagent has been added. When the DAT result
cells with warm (eg, 37 C) saline can result in is positive with both anti-IgG and anti-C3, the
the loss of red-cell-bound, low-affinity IgG. red cells should be tested with an inert control
The red cells should be tested immediately af- reagent (eg, 6% albumin or saline). Lack of ag-
ter washing to prevent false-negative results glutination of the red cells in the control re-
due to the elution of IgG. agent provides some assurance that the test
Although any red cells may be tested, results are accurately interpreted. If the con-
EDTA-anticoagulated blood samples are pre- trol is reactive, the DAT result is invalid [see
ferred. The EDTA prevents in-vitro fixation of sections below on warm AIHA (WAIHA) and
complement by chelating the calcium that is cold agglutinin disease (CAD)]. Reactivity with
needed for C1 activation. If red cells from a this control reagent can indicate spontaneous
clotted blood sample have a positive DAT re- agglutination caused by heavy coating of IgG
sult due to complement, the results should be or rare warm-reactive IgM, or it can indicate
confirmed on red cells from freshly collected IgM cold autoagglutinins that were not disso-
blood kept at 37 C or an EDTA-anticoagulated ciated during routine washing.
specimen if these results are to be used for di-
agnostic purposes. Evaluation of a Positive DAT Result
The DAT can be initially performed with a
A positive DAT result alone is not diagnostic of
polyspecific antihuman globulin (AHG) reagent
hemolytic anemia. Understanding the signifi-
that is capable of detecting both IgG and C3d
cance of this positive result requires knowl-
(see Method 3-14). If the results are positive,
edge of the patient’s diagnosis; recent drug,
tests with monospecific reagents (anti-IgG and
pregnancy, and transfusion history; and the
anticomplement) need to be performed to ap-
presence of acquired or unexplained hemolyt-
propriately characterize the immune process
ic anemia. Dialogue with the attending physi-
involved and determine the diagnosis. Be-
cian is important. Clinical considerations to-
cause polyspecific reagents are usually blend-
gether with laboratory data should dictate the
ed and testing conditions for optimally detect-
extent to which a positive DAT result is evalu-
ing IgG and C3d on red cells may differ, some
ated.
laboratories perform the DAT initially with
anti-IgG and anti-C3d reagents separately. If
Patient History
the polyspecific reagent is polyclonal, pro-
teins other than IgG or C3d (eg, IgM, IgA, or The following situations may warrant further
other complement components) can occa- investigation of a positive DAT result.
sionally be detected; however, specific re-
agents to distinguish these other proteins by 1. Evidence of in-vivo hemolysis (ie, red cell
serologic techniques are not readily available. destruction). If a patient with anemia who
If umbilical cord blood samples are to be test- has a positive DAT result shows evidence of
ed, it is appropriate to use anti-IgG only be- hemolysis, testing to evaluate a possible
cause HDFN results from fetal red cell sensiti- immune etiology is appropriate. Reticulocy-
zation with maternally derived IgG antibody tosis; spherocytes observed on the peripheral
and complement activation rarely occurs.4 blood film; hemoglobinemia; hemoglobin-
It is important to follow the reagent man- uria; decreased serum haptoglobin; and
ufacturer’s instructions and recognize any elevated levels of serum unconjugated
product limitations. False-negative or weaker (indirect) bilirubin or lactate dehydroge-
428 䡲 AABB TECHNICAL MANUAL
䡲 Serum test results are negative or inconclu- only at the antiglobulin phase. If an IgM anti-
sive for a patient who has been recently body is being investigated or suspected, how-
transfused. ever, centrifugation and reading after the 37 C
䡲 HDFN is suspected but no alloantibodies incubation should be performed. Technical
were detected in the maternal plasma. considerations for elution are discussed in
Chapter 16.
Performing an elution routinely on the red In cases of hemolytic transfusion reac-
cells of all patients who have a positive DAT re- tions or HDFN, specific antibody (or antibod-
sult is not recommended. The majority of pre- ies) is usually detected in the eluate that may
transfusion patients with a positive DAT result or may not be detectable in the serum. For
have a nonreactive eluate that is often associat- transfusion reactions, newly developed anti-
ed with an elevated serum globulin level.7-9 bodies that are initially detectable only in the
Elution frees antibody from sensitized red eluate are usually detectable in the serum after
cells and recovers antibody in a usable form. about 14 to 21 days.18 If the eluate is nonreac-
Multiple elution methods have been described tive and a non-group-O patient has received
and reviewed.15 Many laboratories use com- plasma containing anti-A or anti-B (as a result
mercial acid elution kits, primarily for ease of of the transfusion of group O platelets, for ex-
use and decreased exposure to potentially ample) and the recipient appears to have im-
harmful chemicals; these kits are suitable to mune hemolysis, the eluate should be tested
recover antibody in most cases. False-positive against A1 and/or B cells. It may be appropriate
eluate results associated with high-titer anti- to test the eluate against red cells from
bodies have been reported when the low ionic recently transfused donor units, which could
wash solution supplied with the commercial have caused immunization to a rare antigen.
acid eluates was used.16 Because no single elu- For cases of HDFN when no maternal anti-
tion method is ideal in all situations, an alter- body has been detected and paternal red cells
native elution method (eg, an organic solvent) are ABO incompatible with maternal plasma,
may be used in some high-complexity refer- testing an eluate prepared from the infant’s red
ence laboratories when a nonreactive acid elu- cells with the paternal red cells may detect a
ate result is not in agreement with clinical da- maternally derived antibody to a low-preva-
ta.17 lence antigen,
Table 17-2 lists the uses of some common When the eluate reacts with all cells test-
elution methods. Typically, eluates are tested ed, autoantibody is the most likely explana-
tion, especially if the patient has not been characteristic features of this rare type of he-
transfused recently. However, if the patient has molysis are hemoglobinemia, and, when the
been recently transfused, an antibody to a plasma hemoglobin level exceeds the renal
high-prevalence antigen should be consid- threshold, hemoglobinuria. Conversely and
ered. When no unexpected antibodies are more commonly, extravascular hemolysis re-
present in the serum and the patient has not sults when macrophages in the spleen and liv-
been transfused recently, no further serologic er phagocytose red cells completely or partial-
testing of an autoantibody detected only in the ly (producing spherocytes) or destroy red cells
eluate is necessary. by cytotoxic events, resulting in an increase in
The patient’s complete history, including serum bilirubin. This distinction is a simplifi-
the presence of potential passive antibodies, cation, however, because hemoglobin can also
needs to be reviewed when the serologic test be released into the plasma following extravas-
results are evaluated. If both the serum and el- cular destruction if hemolysis is brisk.
uate are nonreactive, there is evidence of im- Immune hemolytic anemias can be clas-
mune hemolysis, and the patient has received sified in various ways. One classification sys-
a drug reported to have caused immune-me- tem is shown in Table 17-3. The AIHAs are sub-
diated hemolysis, testing to demonstrate drug- divided into the major types: WAIHA, CAD,
related antibodies should be considered (see mixed- or combined-type AIHA, and paroxys-
“Laboratory Investigation of Drug-Induced mal cold hemoglobinuria (PCH). Not all cases
Immune Hemolysis” section below). fit neatly into these categories. Table 17-4
shows the typical serologic characteristics of
AUTO I M M U N E H E M O LY T I C the AIHAs. Drugs (discussed in the “Drug-In-
ANEMIA duced Immune Hemolytic Anemia” section
below) may also induce immune hemolysis;
Immune-mediated hemolysis is the shorten- the effects of drug-induced autoantibodies are
ing of red cell survival as a result of an immune serologically indistinguishable from WAIHA.
response. If the marrow is able to adequately
compensate, the reduced red cell survival may Warm Autoimmune Hemolytic
not result in anemia. Immune-mediated he- Anemia
molysis is only one cause of hemolytic anemia,
and many causes of hemolysis are unrelated to The majority of AIHA cases are caused by
immune reactions. warm-reactive autoantibodies that are opti-
The serologic investigations carried out in
the blood bank do not determine whether a
patient has a “hemolytic” anemia. The diagno-
sis of hemolytic anemia rests on clinical find- TABLE 17-3. Classification of Immune
ings and laboratory data, such as hemoglobin Hemolytic Anemias
or hematocrit values; reticulocyte count; red Autoimmune hemolytic anemia (AIHA)
cell morphology; bilirubin, haptoglobin, and
LDH levels; and, sometimes, red cell survival Warm AIHA
studies. The serologic findings help determine Cold agglutinin disease
whether the hemolysis has an immune basis
Mixed-type AIHA
and, if so, what type of immune hemolytic
anemia is present. This is important because Paroxysmal cold hemoglobinuria
the treatment for each type is different. Alloimmune hemolytic anemia
In some cases, the destruction of red cells
takes place in the intravascular space with the Hemolytic transfusion reaction
release of free hemoglobin into the plasma. Hemolytic disease of the fetus and newborn
The red cells are ruptured following activation
Drug-induced immune hemolytic anemia
of the classical complement cascade. The
CHAPTER 17 DAT/Immune Hemolysis 䡲 431
Serum IAT, 35% aggluti- IgM agglutinating IgG IAT-reactive Negative routine
nate untreated red antibody, titer antibody plus IgM IAT result, IgG
cells at 20 C 1000 (60%) at agglutinating anti- biphasic hemolysin
4 C, reactive at body reactive at in Donath-Land-
30 C 30 C steiner test
AIHA = autoimmune hemolytic anemia; WAIHA = warm AIHA; CAD = cold agglutinin disease; PCH = paroxysmal cold
hemoglobinuria; DAT = direct antiglobulin test; IgG = immunoglobulin G; IAT = indirect antiglobulin test.
mally reactive with red cells at 37 C. The auto- of autoantibody exceeds the available binding
antibody is usually IgG, but it can be IgM or sites on the patient’s red cells. The DAT result
IgA. in such cases is usually strongly positive.
Autoantibody in the serum typically is re-
Serologic Characteristics active against all cells by an IAT. Approximately
60% of patients with WAIHA have serum anti-
The DAT result may be positive due to IgG plus
bodies that react with untreated saline-
complement (67% of cases), IgG without com- suspended red cells. When tested with PEG,
plement (20%), or complement without IgG enzyme-treated red cells, column agglutina-
(13%).4 Performing an elution at initial diagno- tion, or solid-phase methods, more than 90%
sis and/or during pretransfusion testing is use- of these sera can be shown to contain autoan-
ful to demonstrate that the IgG coating the pa- tibody. Agglutination at room temperature is
tient’s red cells is autoantibody. present in about one-third of patients with
Typically in WAIHA, the eluate is reactive WAIHA, but these cold agglutinins have nor-
with virtually all red cells tested, and reactivity mal titers at 4 C and are nonreactive at 30 C
is enhanced in tests against enzyme-treated and 37 C. Thus, these cold agglutinins are non-
red cells, with polyethylene glycol (PEG) en- pathogenic and the patient does not have CAD
hancement, or in column agglutination and in addition to WAIHA.4
solid-phase tests. The eluate usually has no se- An unusual subcategory of WAIHA is as-
rologic activity if the only protein coating the sociated with IgM agglutinins in the plasma
red cells is complement. that are reactive at 37 C.4,19 This type of WAIHA
If the autoantibody has been adsorbed by is characterized by severe hemolysis, and the
the patient’s red cells in vivo, the serum may prognosis for these patients can be poor. The
not contain detectable free antibody. The se- red cells are typically spontaneously aggluti-
rum contains free antibody when the amount nated in the DAT; that is, the washed red cells
432 䡲 AABB TECHNICAL MANUAL
are reactive with all reagents tested, including When the DAT result is positive due to
a control, such as 6% albumin (see “Serologic IgG, antiglobulin-reactive typing reagents can-
Problems” section below). Complement is not be used unless the red-cell-bound IgG is
usually detected on the red cells; IgG or IgM first removed (see Methods 2-20 and 2-21). An
may or may not be detected. IgM agglutinins alternative is to use low-protein antisera (eg,
are often detected in an eluate (eg, acid) when monoclonal reagents) that do not require an
it is inspected for agglutination after the 37 C antiglobulin test (refer to the manufacturer’s
incubation and before the antiglobulin test is instructions for the detection of spontaneous
conducted. Some serum IgM warm autoagglu- agglutination). It is helpful to know which of
tinins may be difficult to detect; some are en- the common red cell antigens are lacking on
hanced in the presence of albumin or at low the patient’s red cells to predict which clinical-
pH. Optimal reactivity of the agglutinin some- ly significant alloantibodies the patient may
times occurs between 20 C and 30 C rather have produced or may produce in the future.
than at 37 C. These antibodies have low titers Antigens absent from autologous cells could
at 4 C, usually <64, which easily differentiates well be the target of present or future alloanti-
this IgM warm antibody from those in CAD. To bodies.
prevent misinterpretation of titration results, The presence of autoantibody in the se-
titrations at different temperatures (eg, 37 C, rum increases the complexity of the serologic
30 C, room temperature, and 4 C) need to be evaluation and the time needed to complete
carried out with separate sets of tubes to avoid pretransfusion testing. If a patient who has
carry-over agglutination.4,19 warm-reactive autoantibodies in the serum
needs a transfusion, it is important to deter-
Serologic Problems mine whether alloantibodies are also present.
Some alloantibodies may make their presence
Warm autoantibodies can cause technical dif-
known by reacting more strongly or at differ-
ficulties during red cell testing. Spontaneous
ent phases than the autoantibody, but quite
agglutination can occur if the red cells are
often, routine testing may not suggest the exis-
heavily coated with IgG and the reagent con-
tence of masked alloantibodies.21,22
tains a potentiator, such as albumin. This has
Methods to detect alloantibodies in the
been observed when high-protein Rh typing
presence of warm-reactive autoantibodies are
sera are used. If the control reagent provided
used to attempt to remove, reduce, or circum-
by the manufacturer for these antisera is reac-
vent the autoantibody. Antibody detection
tive, the typing is invalid. IgG can less com-
methods that use PEG, enzymes, column ag-
monly cause spontaneous agglutination in
glutination, or solid-phase red cell adherence
lower protein reagents (eg, monoclonal typing
usually enhance autoantibodies. Antibody de-
sera); this reactivity is often weaker or more
tection tests using low-ionic-strength saline
fragile than true agglutination and may not be
(LISS) or saline tube methods may not detect
detected by a 6% albumin control.20
autoantibodies but they do detect most clini-
Warm-reactive IgM agglutinins can also
cally significant alloantibodies. Other proce-
cause spontaneous agglutination, resulting in
dures involve adsorption; two widely used ad-
ABO and Rh typing problems and/or reactivi-
sorption approaches are discussed below.
ty with the negative control reagent for the
DAT.19 In these cases, treatment with dithio-
Adsorption with Autologous Red Cells
threitol (DTT) or 2-mercaptoethanol (2-ME)
(Method 2-18) to disrupt the IgM agglutinin is In a patient who has not been transfused re-
required to accurately interpret typing and cently, adsorption with autologous red cells
DAT results. When the spontaneous agglutina- (autologous adsorption; see Method 4-8) is the
tion is disrupted, the control reagent is nonre- best way to detect alloantibodies in the pres-
active. ence of warm-reactive autoantibodies. Only
CHAPTER 17 DAT/Immune Hemolysis 䡲 433
autoantibodies are removed, and alloantibod- the alloantibody in the adsorbed serum. The
ies, if present, remain in the serum. adsorbing red cells must not have the antigens
Autologous adsorption typically requires against which the alloantibodies are reactive.
some initial preparation of the patient’s red Because alloantibody specificity is unknown,
cells. At 37 C, in-vivo adsorption has occurred, red cells of different phenotypes are usually
and all antigen sites on the patient’s own red used to adsorb several aliquots of the patient’s
cells may be blocked. A gentle heat elution at serum.
56 C for 3 to 5 minutes can dissociate some of Given the number of potential alloanti-
the bound IgG. This can be followed by treat- bodies, the task of selecting the red cells may
ment of the autologous red cells with proteo- appear formidable. However, red cell selection
lytic enzymes to increase their capacity to ad- is based only on those few antigens for which
sorb autoantibody. Treatment of the red cells alloantibodies of clinical significance are like-
with ZZAP, a mixture of papain or ficin and ly to be present. These include the common
DTT, accomplishes both of these actions in Rh antigens (D, C, E, c, and e), K, Fya and Fyb,
one step. It is proposed that the sulfhydryl Jka and Jkb, and S and s. Red cell selection is
component makes the IgG molecules more made easier by the fact that some of these an-
susceptible to the protease and dissociates the tigens can be destroyed by appropriate pre-
antibody molecules from the cell.23 Multiple treatment (eg, with enzymes or ZZAP) before
sequential autologous adsorptions with new use in adsorption procedures (see Table 16-4).
aliquots of red cells may be necessary if the se- Antibodies to high-prevalence antigens can-
rum contains high levels of autoantibody. not be excluded by allogeneic adsorptions be-
Once autoantibody has been removed, the ad- cause the adsorbing red cells are expected to
sorbed serum is tested for alloantibody reac- express the antigen and adsorb the alloanti-
tivity. body along with autoantibody.
Autologous adsorption is not recom- When the patient’s phenotype is not
mended for patients who have been trans- known, group O red cell samples of three dif-
fused within the last 3 months because a blood ferent Rh phenotypes (R1R1, R2R2, and rr)
sample may contain some transfused red cells should be selected (see Method 4-9). One sam-
that might adsorb alloantibody. Red cells nor- ple should lack Jka and another, Jkb. As shown
mally survive for about 110 to 120 days. In pa- in Table 17-5, ZZAP or enzyme pretreatment of
tients with AIHA, autologous and transfused the adsorbing red cells reduces the phenotype
red cells can be expected to have shortened requirements. Untreated red cells may be
survival. However, determining how long used, but the adsorbing red cells must include
transfused red cells remain in circulation in at least one sample that is negative for the S, s,
patients who need repeated transfusions is not Fya, Fyb, and K antigens in addition to the Rh
feasible. It has been demonstrated that very and Kidd requirements stated above.
small amounts (<10%) of antigen-positive red If the patient’s phenotype is known or can
cells are capable of removing alloantibody re- be determined, adsorption with a single sample
activity in in-vitro studies.24 Therefore, it is of red cells may be possible. Red cells can be
recommended to wait for 3 months after selected that match the patient’s phenotype or
transfusion before performing autologous ad- at least match the Rh and Kidd phenotypes if
sorptions. ZZAP treatment is used. For example, if a pa-
tient’s phenotype is E– K– S– Fy(a–) Jk(a–),
untreated adsorbing red cells need to lack all
Adsorption with Allogeneic Red Cells
five antigens, but enzyme-treated red cells
The use of allogeneic red cells for adsorption only need to be E– K– Jk(a–) and ZZAP-treated
(allogeneic adsorption) may be helpful when red cells only need to be E– Jk(a–). Adsorption
the patient has been recently transfused or in- using untreated red cells in the presence of
sufficient autologous red cells are available. PEG (Method 4-10) or LISS25,26 are modifica-
The goal is to remove autoantibody and leave tions that have been used to decrease the
434 䡲 AABB TECHNICAL MANUAL
incubation time for adsorptions and increase autoantibody may have an unusual specificity
efficiency. that is not reactive with the red cells used for
adsorption. For example, autoantibodies with
Testing of Adsorbed Serum Kell, LW, or EnaFS specificity are not removed
by ZZAP-treated red cells (see Table 16-4 for a
In some cases, each aliquot of serum may
list of antigens altered by various agents). The
need to be adsorbed two or three times to re-
possibility that the sample contains an auto-
move the autoantibody. The fully adsorbed ali- or alloantibody to a high-prevalence antigen
quots are then tested against reagent red cells should always be considered when adsorption
known to either lack or carry common anti- fails to remove the reactivity.
gens of the Rh, MNS, Kell, Duffy, and Kidd Autoantibodies sometimes have patterns
blood group systems (eg, antibody detection of reactivity that suggest the presence of allo-
cells). If an adsorbed aliquot is reactive, the ali- antibody. For example, the serum of a D– pa-
quot should be tested to identify the antibody. tient may have apparent anti-C reactivity. The
Adsorbing several aliquots with different red anti-C reactivity may reflect warm-reactive au-
cell samples provides a battery of potentially toantibody even if the patient’s red cells lack C.
informative specimens. For example, if the ali- The apparent alloanti-C would, in this case, be
quot adsorbed with Jk(a–) red cells subse- adsorbed by C– red cells, both autologous and
quently is reactive only with Jk(a+) red cells, allogeneic. This is unlike the behavior of a true
the presence of alloanti-Jka can be inferred alloanti-C, which would be adsorbed only by
confidently. C+ red cells. In one study, the serum adsorbed
Occasionally, autoantibody is not re- with autologous red cells often retained auto-
moved by three sequential adsorptions. Addi- antibodies that mimicked alloantibodies in
tional adsorptions can be performed, but the addition to the true alloantibody(ies) present,
performance of multiple adsorptions has the whereas serum adsorbed with allogeneic red
potential to dilute the serum. If the adsorbing cells most often contained only alloantibod-
cells do not appear to remove the antibody, the ies.27 This reflects an inefficiency of autologous
CHAPTER 17 DAT/Immune Hemolysis 䡲 435
such red cells survive longer than the patient’s adsorptions. The ability to implement such a
own red cells.4 In the absence of hemolysis or protocol depends on the ability of the transfu-
evidence of compromised survival of trans- sion service, and more often the blood suppli-
fused cells, autoantibody specificity is not im- er, to maintain an adequate inventory of phe-
portant. However, donor units that are nega- notyped units to meet the antigen-matching
tive for the antigen may be chosen because needs.
this is a simple way to circumvent the autoan- In recent years, molecular technologies
tibody and detect potential alloantibodies. have been applied to red cell genotyping for
If the autoantibody shows broader reac- patients with warm autoantibodies to deter-
tivity—reacting with all cells but showing mine which alloantibodies the patient can
some relative specificity (eg, preferentially re- make. DNA tests are attractive for determining
acting with e+ red cells)—whether to transfuse the predicted phenotype of patients with a
blood lacking the corresponding antigen is de- positive DAT (IgG) result because IgG is not al-
batable. It may be undesirable to expose the ways successfully removed and some red cell
patient to Rh antigens absent from autologous antigens are sensitive to IgG removal treat-
cells, especially D and especially in females of ment.35-37 Recent transfusions do not interfere
childbearing potential, merely to improve se- with molecular testing. It must be remem-
rologic compatibility testing results with the bered that genotyping may not accurately pre-
autoantibody. (For example, when a D– pa- dict the phenotype if uncommon or rare si-
tient has autoanti-e, available e– units are like- lencing mutations are present or the patient
ly to be D+; D–e– units are extremely rare.) has received a stem cell transplant.
Referral of the sample for molecular investiga- Some experts propose that an electronic
tion to determine the risk for allo- or autoanti- crossmatch can be safely used for patients
body production will aid in decision making in with autoantibodies when the presence of
these complex cases and potentially improve common, clinically significant alloantibodies
patient care. has been excluded.38,39 This approach circum-
Some laboratories use the adsorbed se- vents the need to issue units that are labeled
rum to screen and select nonreactive units “incompatible”; however, as discussed above,
(units that are antigen-negative for clinically this practice can also lead to a false sense of se-
significant alloantibodies, if detected) for curity.
transfusion. Other laboratories do not perform Although resolving serologic problems for
a crossmatch with the adsorbed serum be- these patients is important, delaying transfu-
cause all units will be incompatible in vivo due sion in the hope of finding serologically com-
to the autoantibody. Issuing a unit that is sero- patible blood may, in some cases, cause
logically compatible with adsorbed serum greater danger to the patient. Only clinical
may provide some assurance that the correct judgment can resolve this dilemma; therefore,
unit has been selected and avoid incompati- dialogue with the patient’s physician is impor-
bility because of additional antibodies (eg, tant.
anti-Wra), but this practice can also provide a
false sense of security about the safety of the Transfusion of Patients with Warm-
transfusion for these patients. Reactive Autoantibodies
A transfusion management protocol us-
ing prophylactic antigen-matched units for Patients with warm-reactive autoantibodies
patients with warm autoantibodies, where fea- may have no apparent hemolysis or may have
sible, in combination with streamlined ad- life-threatening anemia. Patients with little or
sorption procedures has been described.34 The no evidence of significant hemolysis tolerate
same antigens for the commonly occurring, transfusion quite well. The risk of transfusion
clinically significant antibodies (D, C, E, c, e, K, is somewhat increased in these patients be-
Fya, Fyb, Jka, Jkb, and S and s) are taken into ac- cause of the difficulties with pretransfusion
count, as discussed in the previous section on testing. The duration of survival of the trans-
CHAPTER 17 DAT/Immune Hemolysis 䡲 437
fused red cells is about the same as that of the tination, and concentrated eluates are all
patient’s own red cells. methods that have been used to detect lower
In patients with active hemolysis, transfu- levels of red cell-bound IgG.40
sion may increase hemolysis, and the trans- Anti-IgG, anti-C3d, and the combined
fused red cells may be destroyed more rapidly anti-C3b,-C3d reagents are the only licensed
than the patient’s own red cells. This is related products available in the United States for use
to the increased red cell mass available from with human red cells. AHG reagents that react
the transfusion and the kinetics of red cell de- with IgA or IgM are available commercially but
struction.4 Destruction of transfused cells may probably have not been standardized for use
increase hemoglobinemia and hemoglobin- with red cells in agglutination tests. They must
uria. Disseminated intravascular coagulation be used cautiously, and their hemagglutina-
can develop in patients with severe posttrans- tion reactivity must be carefully standardized
fusion hemolysis. by the user.4 In countries outside the United
The transfusion of patients with AIHA is a States, AHG reagents for the detection of IgM
clinical decision that should be based on the and IgA in tube tests or column agglutination
balance between the risks and clinical need. tests may be available.
Transfusion should not be withheld solely be-
cause of serologic incompatibility. The vol- Cold Agglutinin Disease
ume transfused should usually be the smallest
amount required to maintain adequate oxygen CAD, which is less common than WAIHA, is
delivery and not necessarily the amount re- the hemolytic anemia that is most commonly
quired to reach an arbitrary hemoglobin level.4 associated with autoantibodies that react pref-
The patient should be carefully monitored erentially in the cold. CAD occurs as an acute
throughout the transfusion. or chronic condition. The acute form is often
secondary to Mycoplasma pneumoniae infec-
DAT-Negative AIHA tion. The chronic form is often seen in elderly
patients and is sometimes associated with
Clinical and hematologic evidence of WAIHA lymphoma, chronic lymphocytic leukemia, or
is present in some patients whose DAT result is Waldenström macroglobulinemia. Acrocyano-
negative. The most common causes of AIHA sis and hemoglobinuria may occur in cold
associated with a negative DAT result are red- weather. CAD is often characterized by aggluti-
cell bound IgG below the detection threshold nation, at room temperature, of red cells in an
of the antiglobulin test, red-cell-bound IgM EDTA specimen, sometimes to the degree that
and IgA that are not detectable by routine AHG the red cells appear to be clotted.
reagents, and low-affinity IgG that is washed
off the red cells during the washing phase for Serologic Characteristics
the DAT.4,40
Nonroutine tests can be applied in these Complement is the only protein detected on
situations. Unfortunately, these assays require red cells in almost all cases of CAD. If the red
standardization and many have a low predic- cells have been collected properly and washed
tive value. One of the easier tests is for low- at 37 C, there will be no immunoglobulin on
affinity antibodies. Washing with ice-cold (eg, the cells and no reactivity in the eluate. If other
4 C) saline or LISS may help retain antibody on proteins are detected, a negative control for
the cells; a control (eg, 6% albumin) is neces- the DAT (eg, 6% albumin or saline) should be
sary to confirm that cold autoagglutinins are tested to ensure that the cold autoagglutinin is
not causing the positive results.4,40 Comple- not causing a false-positive result. The cold-re-
ment fixation antibody consumption assay, active autoagglutinin is usually IgM, which
enzyme-linked antiglobulin test, radiolabeled binds to red cells in the lower temperature of
anti-IgG, flow cytometry, solid-phase, direct the peripheral circulation and causes comple-
PEG test, direct Polybrene test, column agglu- ment components to attach to the red cells. As
438 䡲 AABB TECHNICAL MANUAL
the red cells circulate to warmer areas, the IgM test with 6% bovine albumin to determine
dissociates but the complement remains. whether autoagglutination persists. If the con-
IgM cold-reactive autoagglutinins associ- trol test result is nonreactive, the results ob-
ated with immune hemolysis usually react at tained with anti-A and anti-B are usually valid.
30 C, and 60% have a titer of 1000 when test- If autoagglutination still occurs, it may be nec-
ed at 4 C.4 If 22% to 30% bovine albumin is in- essary to treat the red cells with sulfhydryl re-
cluded in the test system, pathologic cold ag- agents. Because cold-reactive autoagglutinins
glutinins will react at 30 C or 37 C.4 On are almost always IgM and sulfhydryl reagents
occasion, pathologic cold agglutinins have a denature IgM molecules, reagents (such as 2-
lower titer (ie, <1000), but they have a high ME or DTT) can be used to abolish autoagglu-
thermal amplitude (ie, reactive at 30 C with or tination (see Method 2-18). Treating the red
without the addition of albumin). The thermal cells with ZZAP reagent, as in the preparation
amplitude of the antibody has greater signifi- for adsorptions, can also be used (see Method
cance than the titer. Hemolytic activity against 4-8).
untreated red cells can sometimes be demon- When the serum agglutinates group O re-
strated at 20 C to 25 C. Except in rare cases agent red cells, ABO serum tests are invalid.
with Pr specificity, enzyme-treated red cells Repeating the tests using prewarmed serum
are hemolyzed in the presence of adequate and group A1, B, and O red cells and allowing
complement. the red cells to “settle” after incubation at 37 C
To determine the true thermal amplitude for 1 hour (instead of centrifuging the sample)
or titer of the cold autoagglutinin, the speci- often resolves any discrepancy (see Method 2-
men is collected and maintained strictly at 37 11). By eliminating the centrifugation step, in-
C until the serum and red cells are separated to terference by cold-reactive autoantibodies
avoid in-vitro autoadsorption. Alternatively, might be avoided. Weak anti-A and/or -B in
plasma can be used from an EDTA-anticoagu- some patients’ sera may not react at 37 C. Al-
lated specimen that has been warmed for 10 to ternatively, adsorbed serum (either autoad-
15 minutes at 37 C (with repeated mixing) and sorbed or adsorbed with allogeneic group O
then separated from the cells, ideally at 37 C. red cells) can be used. Rabbit erythrocyte stro-
This process should release autoadsorbed an- ma should not be used for ABO serum tests be-
tibody back into the plasma. cause anti-B and anti-A1 may be removed.41,42
In chronic CAD, the IgM autoagglutinin is
usually a monoclonal protein with kappa light Detection of Alloantibodies in the
chains. In the acute form induced by Myco- Presence of Cold-Reactive Autoantibodies
plasma or viral infections, the antibody is
Cold-reactive autoagglutinins rarely mask
polyclonal IgM with normal kappa and lamb-
clinically significant alloantibodies if serum
da light-chain distribution. Rare examples of
tests are conducted at 37 C and IgG-specific
IgA and IgG cold-reactive autoagglutinins have
reagents are used for the antiglobulin phase.
also been described.4
The use of potentiators (eg, albumin or PEG) is
not recommended because they may increase
Serologic Problems
the reactivity of the autoantibodies. In rare in-
Problems with ABO and Rh typing and other stances, it may be necessary to perform autol-
tests are not uncommon. Often, it is only nec- ogous adsorption at 4 C (see Method 4-5).
essary to maintain the blood sample at 37 C Achieving the complete removal of potent
immediately after collection and to wash the cold-reactive autoagglutinins is very time con-
red cells with warm (37 C) saline before test- suming and usually unnecessary. Removal of
ing. Alternatively, an EDTA sample can be sufficient cold autoagglutinins may be facili-
warmed to 37 C for about 10 minutes, after tated by treating the patient’s cells with en-
which the red cells are washed with warm sa- zymes or ZZAP before adsorption. One or two
line. It is helpful to perform a parallel control cold autologous adsorptions should remove
CHAPTER 17 DAT/Immune Hemolysis 䡲 439
FIGURE 17-1. Proposed unifying theory of drug-induced antibody reactions (based on a cartoon by Habibi as
cited by Garratty28). The thicker lines represent antigen-binding sites for the Fab region of the drug-induced
antibody. Drugs (haptens) bind loosely or firmly to cell membranes, and antibodies may be made to 1) the drug
[producing in-vitro reactions typical of a drug adsorption (penicillin-type) reaction]; 2) membrane components
or mainly membrane components (producing in-vitro reactions typical of autoantibody); or 3) part-drug, part-
membrane components (producing an in-vitro reaction typical of the so-called immune complex
mechanism).28(p55)
442 䡲 AABB TECHNICAL MANUAL
䡲 A drug (or metabolite) must be present in chronic lymphocytic leukemia, is the most
vitro for the antibody in the patient’s serum commonly used drug to produce drug-inde-
to be detected. Antibodies may cause he- pendent antibodies and AIHA.49
molysis, agglutination, and/or sensitization
of red cells in the presence of the drug. Non-Immunologic Protein Adsorption
䡲 The patient need only take a small amount
The positive DAT result associated with some
of the drug (eg, a single dose).
䡲 Acute intravascular hemolysis with hemo-
drugs is caused by modification of the red cell
globinemia and hemoglobinuria is the usu- membrane by the drug and is independent of
al presentation. Renal failure is quite com- antibody production. Hemolytic anemia asso-
mon. ciated with this mechanism is rare.
䡲 Once antibody has been formed, severe he- Cephalosporins (primarily cephalothin)
molytic episodes may recur after exposure are the drugs with which positive DAT results
to very small quantities of the drug. and NIPA were originally associated. In vitro,
red cells coated with cephalothin in pH 9.8
On occasion, it appears that a patient’s se- buffer and incubated with normal plasma ad-
rum contains an “autoantibody” in addition to sorb albumin, IgA, IgG, IgM, C3, and other
a drug antibody reacting in the presence of the proteins in a nonimmunologic manner. For
drug. Rather than a true autoantibody, it is be- this reason, the indirect antiglobulin test result
lieved that this reactivity is due to the presence with virtually all plasma will be positive. Other
of circulating drug or drug plus antibody com- drugs that cause NIPA and a positive DAT re-
plexes.50,53 In these cases, an eluate is usually sult include diglycoaldehyde, cisplatin, oxali-
nonreactive when the drug is not present in platin, and beta-lactamase inhibitors (clavu-
the system. However, in some cases involving lanic acid, sulbactam, and tazobactam).12
piperacillin, the eluate reacts while the patient NIPA should be suspected when a pa-
is still taking the drug. A sample collected sev- tient’s plasma/serum and most normal plas-
eral days after the drug has been discontinued ma/sera are reactive in an indirect antiglobu-
will be nonreactive. A true warm autoantibody lin test with drug-treated red cells but the
is expected to be reactive in an eluate prepared eluate from the patient’s red cells is nonreac-
from the patient’s red cells, and autoantibody tive with the drug-treated cells.
in the serum persists. Consequently, DIIHA
caused by piperacillin can be misdiagnosed as Laboratory Investigation of Drug-
WAIHA, especially if the eluate reacts. Differ- Induced Immune Hemolysis
entiation of warm-reactive autoantibody from
The drug-related problems that are most com-
drug-induced immune hemolytic anemia is
monly encountered in the blood bank are
important for clinical management.53
those associated with a positive DAT result and
a nonreactive eluate. Recent red cell transfu-
Drug-Independent Antibodies:
sions and/or dramatic hemolysis may result in
Autoantibody Production
a weak DAT result by the time hemolysis is sus-
Some drugs induce autoantibodies that ap- pected. When other, more common causes of
pear serologically indistinguishable from immune-mediated hemolysis have been ex-
those of WAIHA. Red cells are coated with IgG, cluded and a temporal relationship exists be-
and the eluate as well as the serum react with tween the administration of a drug and the
virtually all cells tested in the absence of the hemolytic anemia, a drug antibody investiga-
drug. The antibody has no direct or indirect in- tion should be pursued.
vitro interaction with the drug. The prototype The patient’s serum should be tested for
drug for such cases is methyldopa, which is unexpected antibodies by routine procedures.
now used much less frequently than in the If the serum does not react with untreated red
past. Currently, fludarabine, used to treat cells, the tests should be repeated with the
444 䡲 AABB TECHNICAL MANUAL
drug(s) suspected of causing the problem. Drug-treated red cells should always be
Some drug formulations contain inert ingredi- tested with saline and normal serum (or plas-
ents (eg, pill or capsule forms), and other ma) as negative controls. This approach en-
drugs are combinations of two drugs (eg, sures that the observed reactivity with the
piperacillin plus tazobactam). Although it patient’s serum/plasma is appropriately inter-
would seem logical to test the patient’s serum preted. Antibodies reactive with red cells treat-
with the actual drug that the patient received, ed with some drugs (eg, beta-lactams and plat-
inert ingredients or drug combinations can inums) have been detected in the plasma from
make preparation of drug-treated red cells dif- blood donors and patients without hemolytic
ficult or make the results confusing. It is pref- anemia thought to be due to environmental
erable to test serum using pure drug formula- exposure. Therefore, misinterpretation of re-
tions as well as separate components of activity in a patient’s serum is possible.51-52
combination drugs. Whenever possible, a positive control
If the drug has already been reported to should be tested with drug-treated red cells.
cause hemolytic anemia, testing methods may Negative results of a patient’s serum and elu-
be described in the case reports. Far more drug ate without a positive control can only be in-
antibodies are detected by testing serum in the terpreted as showing that antibodies to that
presence of drug; therefore, when a previous
drug were not detected. The drug may or may
report of antibodies to a drug is not available,
not be bound to the test red cells.
an initial screening test can be performed with
If the drug in question is known to cause
a solution of the drug at a concentration of ap-
NIPA, the patient’s serum and the controls
proximately 1 mg/mL in phosphate-buffered
(negative and positive) should also be tested at
saline (see Method 4-13). Serum, rather than
a dilution of 1 in 20. Normal sera at this dilu-
plasma, is the preferred specimen for testing
tion do not usually contain enough protein for
for hemolysis to be observed; this also allows
for the addition of fresh normal serum (as a NIPA to be detected.
source of complement) to the test system. The An immune response may be caused by a
addition of the fresh complement increases metabolite of a drug rather than the drug itself.
the sensitivity of the test for the detection of If the clinical picture is consistent with im-
in-vitro hemolysis resulting from complement mune-mediated hemolysis and the above tests
activation. are noninformative, it may be helpful to test
If these tests are not informative, at- metabolites of the parent drug that are present
tempts can be made to coat normal red cells in the serum or urine of an individual who is
with the drug. The patient’s serum and an elu- taking that drug.54 Antibodies to some nonste-
ate from the patient’s red cells can be tested roidal antiinflammatory drugs have required
against the drug-treated red cells (see Method testing in the presence of metabolite.55 The
4-12). This is the method of choice when ceph- metabolism and half-life of the drug deter-
alosporins (except for ceftriaxone) are thought mines when the drug metabolite should be
to be implicated. Results that are definitive for collected. Pharmacology information for the
a drug-induced positive DAT result are reactiv- metabolite(s) detectable in serum or urine and
ity of the eluate with drug-treated red cells and to previous reports for the drug under investi-
absence of reactivity with untreated red cells. gation should be consulted.
KEY POINTS
1. The DAT is used to determine whether red cells have been coated in vivo with immunoglob-
ulin, complement, or both. The DAT is used primarily for the investigation of hemolytic
transfusion reactions, HDFN, AIHA, and drug-induced immune hemolysis.
2. The DAT should be used to determine whether a hemolytic anemia has an immune etiology.
CHAPTER 17 DAT/Immune Hemolysis 䡲 445
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haemolysis: An 18-year study of 865 cases re- 55. Johnson ST, Fueger JT, Gottschall JL. One cen-
ferred to a regional transfusion centre. Br Med ter’s experience: The serology and drugs asso-
J 1981;282:2023-7. ciated with drug-induced immune hemolytic
46. Shulman IA, Branch DR, Nelson JM, et al. Au- anemia—a new paradigm. Transfusion 2007;
toimmune hemolytic anemia with both cold 47:697-702.
448 䡲 AABB TECHNICAL MANUAL
䡲 APPENDIX 17-1
Drugs Associated with Immune Hemolytic Anemia
Drug Method of Detection
Aceclofenac +Drug
Acetaminophen + Drug
Acyclovir DT
Aminopyrine DT
Amoxicillin DT
Amphotericin B + Drug
Ampicillin DT + Drug
Antazoline + Drug
Azapropazone AA DT
Butizide + Drug
Carbimazole AA DT + Drug
Carbromal DT
Cefamandole DT
Cefazolin DT
Cefixime DT + Drug
Cefotaxime DT + Drug
Cefoxitin AA DT + Drug
Ceftazidime AA DT + Drug
Ceftizoxime DT + Drug
Ceftriaxone + Drug
Cefuroxime DT
Cephalexin DT
Cephalothin DT + Drug NIPA
Chloramphenicol AA DT
Chlorpromazine AA + Drug
Chlorpropamide + Drug
Cimetidine DT +Drug
CHAPTER 17 DAT/Immune Hemolysis 䡲 449
䡲 APPENDIX 17-1
Drugs Associated with Immune Hemolytic Anemia (Continued)
Drug Method of Detection
Ciprofloxacin +Drug
Cisplatin DT +Drug NIPA
Cladribine AA
Clavulanate NIPA
Cyanidanol AA DT + Drug
Cyclofenil AA + Drug
Cyclosporin DT
Diclofenac AA DT + Drug
Diethylstilbestrol + Drug
Diglycoaldehyde NIPA
Dipyrone DT + Drug
Erythromycin DT
Etodolac + Drug
Fenoprofen AA + Drug
Fluconazole DT + Drug
Fludarabine AA
Fluorescein DT + Drug
Fluorouracil + Drug
Furosemide + Drug
Hydralizine DT
Hydrochlorothiazide DT + Drug
Hydrocortisone DT + Drug
9-Hydroxy-methyl-ellipticinium + Drug
Ibuprofen +Drug
Imatinib mesylate DT
Insulin DT
Isoniazid DT + Drug
Levodopa AA
Levofloxacin DT +Drug
Mefenamic acid AA
(Continued)
450 䡲 AABB TECHNICAL MANUAL
䡲 APPENDIX 17-1
Drugs Associated with Immune Hemolytic Anemia (Continued)
Drug Method of Detection
Mefloquine DT + Drug
Melphalan + Drug
6-Mercaptopurine DT
Methadone DT
Methotrexate AA DT + Drug
Methyldopa AA
Nabumetone +Drug
Nafcillin DT
Naproxen + Drug
Penicillin G DT
Phenacetin + Drug
Phenytoin DT
Piperacillin DT + Drug
Probenicid + Drug
Procainamide AA
Propyphenazone + Drug
Pyrazinamide DT + Drug
Pyrimethamine DT
Quinidine DT + Drug
Quinine + Drug
Ranitidine DT + Drug
Rifabutin + Drug
Rifampicin DT + Drug
Stibophen + Drug
Streptomycin AA DT + Drug
Sulbactam NIPA
Sulfamethoxazole + Drug
CHAPTER 17 DAT/Immune Hemolysis 䡲 451
䡲 APPENDIX 17-1
Drugs Associated with Immune Hemolytic Anemia (Continued)
Drug Method of Detection
Sulfasalazine + Drug
Sulfisoxazole +Drug
Sulindac AA DT + Drug
Suprofen AA + Drug
Tazobactam NIPA
Teicoplanin AA + Drug
Teniposide AA + Drug
Tetracycline DT
Ticarcillin AA DT
Tolbutamide DT
Tolmetin AA + Drug
Triamterene DT + Drug
Trimethoprim + Drug
Vancomycin + Drug
Zomepirac AA + Drug
AA = drug-independent autoantibody; DT = testing with drug-treated red cells; + Drug = testing in the presence of drug;
NIPA = nonimmunologic protein adsorption.
C h a p t e r 1 8
Brian R. Curtis PhD, D(ABMLI), MT(ASCP)SBB, Director, Platelet and Neutrophil Immunology Lab, Blood-
Center of Wisconsin, Milwaukee, Wisconsin
B. Curtis has disclosed a financial relationship with Gen-Probe Incorporated.
453
454 䡲 AABB TECHNICAL MANUAL
frequency antigens designated “a” and the Glanzmann thrombasthenia, in which platelet
lower-frequency antigens designated “b.”4 GPIIb/IIIa is absent or dysfunctional due to in-
HPAs for which antibodies against only one of herited mutations in ITGA2B and/or ITGB3.7
the two antithetical antigens have been de- Patients with Glanzmann thrombasthenia
tected are labeled with a “w” for “workshop,” who are exposed to normal platelets by trans-
such as HPA-6bw. fusion or pregnancy can make isoantibodies
against GPIIb/IIIa.
Platelet Alloantigens on GPIIb/IIIa GPIIb/IIIa is the most abundantly ex-
pressed (approximately 80,000 molecules/
HPA-1a is the platelet alloantigen that was dis-
platelet) glycoprotein complex on the platelet
covered first and is most familiar.5 Originally
membrane, making it highly immunogenic.
named “Zwa” and more commonly referred to
Antibodies against HPA-1a account for the
as “PlA1,” it is expressed on GPIIIa, the -sub-
vast majority (>80%) of the HPA-specific plate-
unit of the integrin GPIIb/IIIa (2b/3) com-
plex. let antibodies detected in the sera of alloim-
Integrins are a broadly distributed family munized people of European ancestry. HPA-1a
of adhesion molecules consisting of an and a antibodies are produced by the 2% of individu-
chain held together by divalent cations in a als with the platelet type HPA-1b/1b. Antibod-
heterodimeric complex.6 Integrins are essen- ies specific for HPA-1b are commonly detected
tial for platelet adhesion and aggregation be- in patients with posttransfusion purpura
cause they serve as receptors for ligands, such (PTP).
as fibrinogen, collagen, fibronectin, von Wille- Twenty of the 33 HPAs are carried by GPI-
brand factor (vWF), and other extracellular Ib (6) and GPIIIa (14). Like HPA-1a/1b, the
matrix proteins. HPA-4a/4b antigens are also expressed on
Binding of fibrinogen by GPIIb/IIIa re- GPIIIa and have been implicated in fetal and
sults in platelet aggregation, which leads to the neonatal alloimmune thrombocytopenia
formation of the “platelet plug” to stop bleed- (FNAIT), PTP, and platelet transfusion refrac-
ing. The importance of GPIIb/IIIa in hemosta- toriness. The low-frequency HPA-4b antigen is
sis is demonstrated by the serious bleeding more common in populations of Japanese and
that occurs in patients with the rare disorder Chinese ancestry.
456 䡲 AABB TECHNICAL MANUAL
The HPA-3a/3b antigens are expressed on cause of Bernard Soulier syndrome (BSS). BSS
GPIIb, but despite the high rate of incompati- is a disorder characterized by prolonged
bility for both antigens in the general popula- bleeding time, thrombocytopenia, and the
tion, detection of HPA-3 antibodies is uncom- presence of “giant platelets” that affects ap-
mon. Some HPA-3 antibodies are difficult to proximately 1 in 1 million people.7,15 BSS pa-
detect in monoclonal antigen capture assays, tients whose platelets are devoid of GPIb/V/IX
such as the modified antigen capture enzyme- can produce antibodies when they are ex-
linked immunosorbent assay (MACE) and posed to the protein complex on normal plate-
monoclonal antibody immobilization of plate- lets through transfusions or pregnancy.
let antigens (MAIPA), in which GPIIb is ex-
tracted from platelets with detergents that can Platelet Alloantigens on GPIa/IIa
denature the antigenic epitopes recognized by
The integrin GPIa/IIa, also known as integrin
HPA-3 antibodies.8,9 X-ray crystallography
21, is a major collagen receptor on platelets.
studies could not determine the portion of the The GPIa protein carries the HPA-5a/5b anti-
domain of GPIIb where the HPA-3 antigens are gens. Antibodies against HPA-5 antigens are
located, which might explain the difficulties the second most frequently detected, after
with detection of HPA-3 antibodies.10 anti-HPA-1a, in patients with FNAIT and are
An additional 17 different low-frequency also frequently detected in patients with PTP.
platelet antigens are expressed on either GPIIb About 3000 to 5000 molecules of the GPIa/IIa
or GPIIIa (Table 18-1). These antigens were all heterodimeric complex are expressed on
discovered in cases of FNAIT when specific an- platelets, and higher expression correlates
tibodies in maternal sera were detected that with the presence of both HPA-5b and threo-
were reactive only with the fathers’ GPIIb/IIIa. nine at amino acid 807 in GPIa. 16 HPA-13bw,
The vast majority of these antigens are private -18bw, and -25bw are low-frequency antigens
antigens restricted to the single families in that are also expressed on GPIa and have all
which they were discovered. HPA-6bw and been implicated in FNAIT. Interestingly, the
HPA-21bw are exceptions, having antigen fre- HPA-13bw polymorphism has been reported
quencies of 1% and 2%, respectively, in people to cause functional defects that reduce platelet
of Japanese ancestry, and HPA-9bw has been responses to collagen-induced aggregation
implicated in several cases of FNAIT.11-14 and spreading on collagen-coated surfaces.4
ed in sera from patients with immune platelet Although platelets are often transfused
refractoriness.17-19 without regard to ABO compatibility, the use of
mismatched platelets frequently results in
Other Antigens on Platelets lower posttransfusion recovery rates.23,24 In
some cases, high-titer immunoglobulin G (Ig
ABO and Other Blood Groups G) A,B antibodies in blood group O recipients
Most of the ABO antigen on platelets is carried are reactive with transfused platelets carrying
on saccharides attached to the major platelet large amounts of A or B antigens, resulting in
membrane glycoproteins (Table 18-2). GPIIb platelet transfusion refractoriness.21 Recovery
and platelet endothelial cell adhesion mole- of transfused platelets can also be influenced
cule 1 (PECAM-1/CD31) carry the largest by the transfusion of group O platelets to
group A recipients. Anti-A and/or anti-B in the
amounts of A and B antigens.20 Platelet A and B
donor plasma might be reactive with soluble A
antigen levels are quite variable from individu-
or B in the recipient plasma to form immune
al to individual, with 5% to 10% of non-group-
complexes that bind to transfused (and autol-
O individuals expressing extremely high levels
ogous) platelets via FcRIIa, thereby influenc-
of A1 or B on their platelets.20,21 These “high ex- ing the survival of the transfused platelets.25
pressers” have highly active glycosyltransfer- Clinical trials comparing ABO-identical to
ases, which are much more efficient at attach- -unmatched platelets in patients with cancer
ing A or B antigens.20 who require multiple platelet transfusions
Interestingly, although individuals with have suggested that rates of refractoriness are
the subgroup A2 red cell phenotype express significantly higher when unmatched compo-
lower levels of A on their red cells than A1 indi- nents are used.22,25 Although other red cell an-
viduals, they do not express detectable A anti- tigens (eg, Lea, Leb, I, i, P, Pk, and Cromer) are
gens on their platelets. As a result, A2 platelets also present on platelets, there is no evidence
have been successfully transfused to group O that these antigens significantly reduce plate-
patients, even those with high-titer anti-A.22 let survival in vivo.26,27
ABO Same as for red cells GPIIb, IIIa, IV, Ia/ Multiple ABO Multiple
IIa, GPIb/V/IX,
CD31
HLA-A, B, Same as for leukocytes Class I HLA Multiple MHC Multiple
and C
GPIV 90-97% (Africans) CD36 Tyr325Thr* CD36 1264T>G*
90-97% (Asians) Pro90Ser* 478C>T*
99.9% (Caucasians) Exons 1-3 del
GPVI N/A GPVI N/A GP6/N/A N/A
*ABO saccharides are attached to platelet GPs during their glycosylationj.
†
Only the most common changes are shown.
‡
Only the most common mutations are shown.
N/A = not applicable.
458 䡲 AABB TECHNICAL MANUAL
transfusions is very small, this possibility mother as washed platelet products.50 Once
should be investigated when most of the the diagnosis of FNAIT has been made in a
crossmatches are positive or HLA-matched family, subsequent fetuses are at risk. Antena-
transfusions fail. If platelet-specific antibodies tal treatment with IVIG with or without ste-
are present, donors of known platelet antigen roids has proven to be an effective means of
phenotype or family members, who may be reducing fetal thrombocytopenia and prevent-
more likely to share the patient’s phenotype, ing intracranial hemorrhage.51 (For a more in-
should be tested. Platelet crossmatching or depth discussion of FNAIT, see Chapter 22.)
HPA genotyping should be considered for pa-
tients who do not respond to ABO- and HLA- Posttransfusion Purpura
compatible platelets.
PTP is a rare syndrome characterized by the
development of dramatic, sudden, and self-
Fetal and Neonatal Alloimmune
limiting thrombocytopenia 5 to 10 days after a
Thrombocytopenia
blood transfusion in patients with a previous
FNAIT (also known as neonatal alloimmune history of sensitization by pregnancy or trans-
thrombocytopenia and abbreviated as “NATP,” fusion.52 Coincident with the thrombocytope-
“NAT,” “NAIT,” or “NIT”) is a syndrome involv- nia is the development of a potent platelet-
ing immune destruction of fetal platelets by specific alloantibody, usually anti-HPA-1a, in
maternal antibody that is analogous to red cell the patient’s serum. Other specificities have
destruction in hemolytic disease of the fetus been implicated; these are almost always asso-
and newborn. During pregnancy, a mother ciated with antigens on GPIIb/IIIa. PTP differs
may become sensitized to an incompatible fe- from transfusion reactions caused by red cell
tal platelet antigen inherited from the father of antibodies in that the patient’s own antigen-
the fetus. IgG specific for the platelet antigen negative platelets as well as any transfused
crosses the placenta, causing thrombocyto- antigen-positive platelets are destroyed. The
penia. pathogenesis of autologous platelet destruc-
FNAIT is the most common cause of se- tion in PTP is not fully understood; however,
vere fetal/neonatal thrombocytopenia, and af- mounting evidence suggests that distinct
fected infants are at risk of major bleeding platelet autoantibodies transiently arise, along
complications, especially intracranial hemor- with the alloantibodies, and cause both autol-
rhage. The most commonly implicated plate- ogous and transfused antigen-negative plate-
let antigen incompatibility in FNAIT is for let destruction.52 These panreactive autoanti-
HPA-1a, but all HPAs identified to date have bodies often target the same glycoprotein that
been implicated.48 A serologic diagnosis of expresses the HPA against which alloantibod-
FNAIT may be made by 1) testing maternal se- ies were made.
rum for platelet antibodies using assays that Platelet antibody assays usually reveal an
can differentiate platelet-specific from non- antibody in the serum with HPA-1a specificity.
platelet-specific reactivity and 2) performing Genotyping documents the absence of HPA-1a
platelet genotyping on parental deoxyribonu- or other platelet-specific antigens. Plasma ex-
cleic acid (DNA).49 Demonstration of both a change—once the treatment of choice for
platelet-specific (HPA) antibody in the mater- PTP—has now largely been supplanted by
nal serum and the corresponding incompati- IVIG as first-line therapy. Transfusion of anti-
bility for the antigen in the parental platelet gen-negative platelets may be of value during
types confirms the diagnosis. the acute phase of PTP; however, such plate-
Treatment of acutely thrombocytopenic lets have reduced in-vivo survival due to de-
newborns includes the administration of in- struction by the aforementioned platelet auto-
travenous immune globulin (IVIG) with or antibodies.53
without antigen-compatible platelet transfu- Following recovery, future transfusions
sions that are sometimes provided by the should be from HPA-la-negative donors if
462 䡲 AABB TECHNICAL MANUAL
possible. Washed red cells may offer some pro- it may develop in up to 5% of patients treated
tection against recurrence; however, this is with unfractionated heparin. Low-molecular-
controversial and there is at least one report of weight heparin is less likely to be associated
PTP being precipitated by a frozen deglycero- with HIT than unfractionated heparin.
lized red cell transfusion.54 Moreover, accord- A reduction in baseline platelet count by
ing to recent data reported from the Serious 30% to 50% occurs generally within 5 to 14
Hazards of Transfusion surveillance program, days after primary exposure to heparin and
the frequency of PTP has rather dramatically sooner if the patient has been exposed to hep-
decreased coincidently with the introduction arin within the last 3 months. The platelet
of leukocyte-reduced blood products.55 Al- count is often less than 100,000/µL but usually
though no data have been reported to explain recovers within 5 to 7 days upon discontinua-
this trend, use of leukocyte-reduced products tion of heparin. More than 50% of patients
may also be of benefit in reducing the risk of with HIT develop thrombosis, which can occur
PTP recurrence. in the arterial, venous, or both systems.61 Pa-
tients may develop stroke, myocardial infarc-
tion, limb ischemia, deep venous thrombosis,
Drug-Induced Thrombocytopenia
or ischemia of other organs. The thrombotic
Thrombocytopenia caused by drug-induced complications may lead to limb amputation or
platelet antibodies is a recognized complica- prove fatal. Because the rate of HIT-associated
tion of drug therapy. Drugs commonly impli- thrombosis is so substantial, it is of critical im-
cated include quinine, sulfa drugs, vancomy- portance to discontinue heparin therapy when
cin, GPIIb/IIIa antagonists, and heparin.56,57 a diagnosis of HIT is suspected. Moreover,
Both drug-dependent and non-drug-depen- strong consideration should be given to using
dent antibodies may be produced. Non-drug- an alternative (nonheparin) anticoagulant (eg,
dependent antibodies, although stimulated by a direct thrombin inhibitor) to prevent throm-
drugs, do not require the continued presence bosis.61
of the drug to be reactive with platelets and are The mechanism of HIT includes forma-
serologically indistinguishable from other tion of a complex between heparin and plate-
platelet autoantibodies. let factor 4 (PF4), a tetrameric protein released
Although several mechanisms for drug-
from platelet granules. Antibodies (IgG, IgA,
induced antibody formation have been de-
and some IgM) are produced to the complex,
scribed, most clinically relevant drug-depen-
and IgG in the complex attaches secondarily to
dent platelet antibodies are thought to result
platelet receptor FcRIIa via its Fc, resulting in
when a drug interacts with platelet membrane
platelet activation with subsequent thrombin
glycoproteins, inducing conformational chang-
generation. The antibody may also bind to
es recognized by drug-dependent antibod-
ies.58,59 These antibodies can cause a thrombo- complexes formed at other sites, notably on
cytopenia of sudden and rapid onset that endothelial cells and monocytes. Thus, HIT
usually resolves within 3 to 4 days after the might involve activation and damage not only
drug is discontinued. of platelets but also of endothelium and
Among the drug-induced immune re- monocytes/macrophages, causing increased
sponses to platelets, those triggered by expo- susceptibility to thrombosis. This understand-
sure to heparin have particular clinical impor- ing of the mechanism of HIT antibodies is ex-
tance because of both the widespread use of ploited by enzyme-linked immunosorbent as-
this anticoagulant and the devastating throm- say (ELISA) tests in which microtiter wells are
botic complications associated with the hepa- coated with the complexes of PF4 and heparin
rin-induced thrombocytopenia (HIT) syn- (or heparin-like molecules) rather than with
drome.60 The incidence of HIT is unknown, but the platelets themselves.62
CHAPTER 18 Platelet and Granulocyte Antigens and Antibodies 䡲 463
Platelet Genotyping most often detected in the eluates, they are in-
frequently detected (in approximately 17% of
Genotyping for the SNPs in the genes encod-
cases) in the plasma. Patients with ITP may
ing HPA can be performed by any of the myri-
have antibodies that are reactive with one or
ad molecular methods available. Polymerase
several GP targets.61
chain reaction (PCR) followed by restriction
fragment length polymorphism analysis and
Testing for Drug-Dependent Platelet
allele-specific PCR are two methods that have
been used successfully.67 These techniques are Antibodies
reliable, but they are also laborious and time Any platelet serology test that is used to detect
consuming. Higher-throughput methods have platelet-bound Ig can be modified to detect
been developed, such as real-time PCR and al- platelet drug-dependent antibodies. Each pa-
lele-specific fluorescent probes.67 tient serum or plasma sample must be tested
against normal platelets in the presence and
Testing for Platelet Autoantibodies absence of the drug. Moreover, at least one
Numerous assays have been developed to de- normal control serum sample should be tested
tect platelet autoantibodies in patients with with and without the drug to control for the
ITP. Although many tests are quite sensitive, nonspecific antibody binding that might be
particularly in detecting cell-surface, platelet- induced by the drug’s presence. A positive con-
associated Igs, none has been sufficiently spe- trol serum sample that is known to be reactive
cific to be particularly useful in either the diag- with the drug being assayed should be tested
nosis or management of ITP. The American with and without the drug to complete the
Society of Hematology practice guidelines for evaluation. A positive result shows that the se-
ITP state that serologic testing is unnecessary, rum is positive (or more positive) against nor-
assuming that the clinical findings are com- mal platelets in the presence of the drug vs
patible with the diagnosis.71 However, platelet without the drug and that the drug did not
antibody tests may be helpful in the evaluation nonspecifically cause a positive result with
of patients suspected of having ITP when non- normal serum controls. Flow cytometry is the
immune causes may be present. The goal of most sensitive and most commonly used
serologic testing in ITP is to detect autoanti- method to detect both IgG and IgM drug-
body bound to the patient’s own platelets with dependent antibodies.49,73 Limitations to
or without demonstration of similar reactivity detection of drug-dependent platelet antibod-
in the patient’s plasma. ies include that 1) for many drugs, the optimal
Newer assays are designed to detect im- concentration for antibody detection has not
munoglobulin binding to platelet-specific epi- been determined and hydrophobic drugs are
topes found on platelet GPIIb/IIIa, GPIa/GPIIa, difficult to solubilize; 2) the presence of non-
and/or GPIb/IX complexes. These solid-phase, drug antibodies can mask the antibodies; and
GP-specific assays appear to have improved 3) a patient may be sensitized to a drug metab-
specificity in distinguishing ITP from nonim- olite and not the native drug.
mune thrombocytopenia, but this benefit is Assays for heparin-dependent antibodies
often balanced by a decrease in sensitivity.65,72 include the PF4 ELISA, which involves the ad-
One commercially available test uses eluates dition of the patient’s serum diluted at a 1:50
prepared from the patient’s washed platelets.65 ratio alone and in the presence of high-dose
The eluates are tested against a panel of (100 U/mL) heparin to plastic microplate wells
monoclonal-antibody-immobilized platelet to which bound complexes of PF4 and heparin
glycoprotein complexes, and platelet antibod- or heparin-like molecules (eg, polyvinyl sulfo-
ies are detected using an enzyme-linked anti- nate) are affixed.62 Heparin-dependent anti-
human Ig. In the indirect phase of the assay, bodies bind to the complexes and are detected
patient plasma is tested against the same gly- via enzyme-conjugated antihuman Ig. An op-
coprotein panel. Although autoantibodies are tical density value, generally above 0.4, in the
466 䡲 AABB TECHNICAL MANUAL
PF4-heparin well that can be inhibited by ized and given human neutrophil alloantigen
high-dose heparin confirms the presence of (HNA) designations by the Granulocyte Anti-
heparin-dependent antibodies. Although IgG gen Working Party of the ISBT (Table 18-5).76
antibodies are the most clinically relevant an- This nomenclature system follows a similar
tibodies, a few patients with HIT appear to convention to that used for HPA nomencla-
have only IgM or IgA antibodies. PF4 ELISAs ture. Several of the antigens on granulocytes
are available in two forms: those that detect are shared with other cells and are not granu-
but do not differentiate IgG, IgM, and IgA hep- locyte specific.
arin-dependent antibodies, and those that de-
tect only IgG. HNA
The 14C-serotonin release assay (SRA) is a
functional assay that uses washed platelets for Antigens on FcRIIIb
detection of heparin-dependent antibodies.74
Normal, fresh platelets are incubated with The first granulocyte-specific antigen detected
14
C-serotonin, which is taken up into their was NA1, later named “HNA-1a.” Three alleles
dense granules. The platelets are then ex- of HNA-1 have now been identified—HNA-1a,
posed to the patient’s serum in the presence of HNA-1b, and HNA-1c—and are located on the
low (0.1 U/mL) and high (100 U/mL) concen- protein FcRIIIb (CD16b).77 FcRIIIb is a GPI-
trations of heparin. Release of at least 20% of linked protein receptor for the Fc of IgG and is
the radioactivity at the low dose of heparin and present only on the surfaces of neutrophils.
inhibition of this release below 20% at the high Neutrophils express 100,000 to 200,000 mole-
dose confirms the presence of heparin-depen- cules of FcRIIIb, but there are rare individuals
dent antibodies. (approximately 0.1%) whose neutrophils ex-
Other functional tests used to detect hep- press no FcRIIIb (CD16 null) and who can
arin-dependent antibodies include the hepa- produce antibodies that are reactive with
rin-induced platelet-aggregation test and the FcRIIIb when they are exposed to it through
heparin-induced platelet-activation test. The transfusion or pregnancy.78,79 Antibodies to
PF4 ELISA and the SRA are both more sensitive HNA-1a and -1b have been implicated in
and specific than the platelet-aggregation test TRALI, NAN, and AIN.77
for the detection of heparin-dependent plate-
let antibodies in patients for whom there is a Antigens on CD177
clinical suspicion that these antibodies are
present. However, in asymptomatic patients HNA-2 (previously known as “NB1”) is not an
receiving heparin or who have not yet received alloantigen because HNA-2 antibodies are iso-
the drug, neither test is sufficiently predictive antibodies that recognize common epitopes
of HIT to warrant its use in screening.75 on CD177 protein, which is missing from the
neutrophils of immunized individuals. Neu-
trophils from approximately 3% to 5% of peo-
GRA NU LO C Y T E A NT I GE N S AN D ple lack expression of CD177 on neutrophils.77
ANTIBODIES Lack of CD177 is thought to be caused by a
Antibodies against granulocyte (neutrophil) messenger ribonucleic acid splicing defect
antigens are implicated in the following clini- that results in a truncated protein that cannot
cal syndromes: neonatal alloimmune neutro- be expressed.80 Interestingly, CD177 is ex-
penia (NAN), transfusion-related acute lung pressed only on a subpopulation of neutro-
injury (TRALI), immune neutropenia after phils in CD177-positive individuals.81 The pro-
HPC transplantation, refractoriness to granu- portion of the CD177-positive neutrophil
locyte transfusion, and chronic benign auto- population ranges from 0% to 100%.82 CD177
immune neutropenia of infancy (AIN). To is expressed only on neutrophils and belongs
date, nine neutrophil antigens carried on five to the Ly-6/uPAR/snake-toxin family of
different glycoproteins have been character- proteins.81
CHAPTER 18 Platelet and Granulocyte Antigens and Antibodies 䡲 467
occasionally develop in women after pregnan- in the children of women who lack the
cy, and HNA-3a antibodies are the most fre- FcRIIIb protein. Neutropenia in NAN can oc-
quent cause of fatal TRALI.89 HNA-3b antibod- casionally be life-threatening because of in-
ies are rarely detected, but several have been creased susceptibility to infection.95 Manage-
found during screening of the serum of mul- ment with antibiotics, IVIG, granulocyte
tiparous blood donors. colony-stimulating growth factor, and/or plas-
ma exchange may be helpful.
Antigens on CD11a and CD11b
TRALI
HNA-4a and HNA-5a, both high-prevalence
antigens, are present on monocytes and lym- TRALI is an acute, often life-threatening reac-
phocytes as well as granulocytes. HNA-4a is tion characterized by respiratory distress,
carried on the CD11b/18 (Mac-1, CR3, m2) hypo- or hypertension, and noncardiogenic
glycoprotein.77 CD11b/18 plays a role in neu- pulmonary edema that occurs within 6 hours
trophil adhesion to endothelial cells and of a blood component transfusion.96 TRALI has
phagocytosis of C3bi opsonized microbes. been the most common cause of transfusion-
There is some evidence showing that alloanti- related death for more than 10 years.97 In
bodies against HNA-4a interfere with CD11b/ TRALI, the causative antibodies are most often
18-dependent neutrophil adhesion and en- found in the plasma of the blood donor. When
hance neutrophil respiratory burst.90 Antibod- these antibodies are transfused, they cause ac-
ies against HNA-4a have been implicated in tivation of primed neutrophils that are seques-
NAN, and autoantibodies against CD11b/18 tered in the lungs of certain patients. The acti-
have also been described.91,92 vated neutrophils undergo oxidative burst,
HNA-5a is carried on CD11a/18 (LFA-1, releasing toxic substances that damage pul-
L2) glycoprotein.93 CD11a/18, like CD11b/ monary endothelium and resulting in capillary
18, plays a role in neutrophil adhesion to en- leak and pulmonary edema. Class I and II HLA
dothelial cells. Antibodies that are reactive and HNA antibodies have all been implicated
with HNA-5a have been found in a chronically in TRALI. However, in a recent large clinical
transfused patient with aplastic anemia and study of TRALI, HNA and Class II HLA anti-
have also been reported to be associated with bodies, but not Class I HLA antibodies, were
NAN.94 The patient who made the original significantly associated with TRALI.98 (For a
HNA-5a antibody experienced prolonged sur- more in-depth discussion of TRALI, see Chap-
vival of an HLA nonidentical skin graft that ter 27.)
was attributed to the HNA-5a antibody.93
Autoimmune Neutropenia
Other Neutrophil Antigens
Autoimmune neutropenia may occur in adults
Neutrophils do not express ABH or other red or in infants. When present in adults, it may be
cell group antigens, but they do express mod- idiopathic or be secondary to such diseases as
est amounts of Class I and II HLA only upon rheumatoid arthritis or systemic lupus erythe-
activation. matosus or to bacterial infections.99 In autoim-
mune neutropenia of infancy, usually occur-
Immune Neutrophil Disorders ring in children between the ages of 6 months
and 2 years, the autoantibody has neutrophil
Neonatal Alloimmune Neutropenia
antigen specificity (usually HNA-1a or -1b) in
NAN is caused by maternal antibodies against about 30% of the patients. The condition is
antigens on fetal neutrophils; the most fre- generally self-limiting, with recovery usually
quent specificities are against HNA-1a, HNA- occurring in 7 to 24 months, and is relatively
1b, and HNA-2 antigens. NAN may also occur benign and manageable with antibiotics.87
CHAPTER 18 Platelet and Granulocyte Antigens and Antibodies 䡲 469
Testing for Granulocyte Antibodies ture for 30 minutes, and washed in EDTA and
and Antigens phosphate-buffered saline. Neutrophil-bound
antibodies are then detected with fluorescein
Granulocyte antibody testing is technically isothiocyanate-labeled antihuman IgG or IgM
complex and labor intensive. The inability to with either a fluorescence microscope or a
maintain the integrity of granulocytes for test- flow cytometer.89,100 A combination of aggluti-
ing that are stored at room temperature, in re- nation and immunofluorescence tests is bene-
frigerated conditions, or by cryopreservation ficial.79,88 Other methods include chemilumi-
requires that cells be isolated from fresh blood nescence, SPRCA, and the monoclonal
on each day of testing. This demands that antibody immobilization of granulocyte anti-
readily available blood donors typed for the
gens (MAIGA) assay, which is similar to the
various granulocyte antigens be available.
MAIPA assay, but uses monoclonal antibodies
Class I HLA antibodies that are often present
to capture the various glycoproteins that ex-
in patient sera complicate detection and iden-
press HNA. The MAIGA assay is used to differ-
tification of granulocyte antibodies. For these
entiate between HLA- and HNA-specific anti-
reasons, it is critical that granulocyte antibody
bodies.
and antigen testing be performed by an expe-
rienced laboratory using appropriate controls. HNA TYPING. As with HPA, typing for HNA is
largely performed using molecular methods to
GRANULOCYTE AGGLUTINATION TE ST . This
detect the allelic variants that determine the
was one of the first tests developed for the de-
antigens. Any methods used in HPA typing can
tection of granulocyte antibodies. It is typically
be applied to HNA typing with simple modifi-
performed by overnight incubation of small
cations to the primer and probe sequences.
volumes of isolated fresh neutrophils with the
Readers are referred to several publications on
patient’s serum in a microplate. The wells are
this subject.101,102 Because the splicing defect
viewed under an inverted phase microscope
that results in CD177 deficiency is not known,
for neutrophil agglutination or aggregation.
typing for HPA-2/CD177 requires testing for
GRA NULOCYTE IMMUNOFLUORESCENCE CD177 on freshly isolated neutrophils using
TE ST . This test also requires fresh target cells specific monoclonal antibodies and the granu-
that are incubated, usually at room tempera- locyte immuno fluorescence test.
KEY POINTS
1. Platelets express a variety of antigenic markers on their surfaces. Some of these antigens,
such as ABH and HLA, are shared with other cells, whereas HPAs are essentially platelet spe-
cific. There are currently 33 HPAs carried on six platelet glycoproteins.
2. HLA sensitization is the most common immune cause of platelet refractoriness and can be
diagnosed by the demonstration of significant levels of antibodies to HLA Class I in a
patient’s serum. When antibodies to HLA antigens are demonstrated, a widely used
treatment approach is to supply apheresis platelets from donors whose Class I HLAs are
similar to those of the patient. HLA-matched platelets should be irradiated to prevent
transfusion-associated GVHD.
3. Compared with HLA matching for platelet-refractory patients, crossmatching can be both
more convenient and cost effective. It avoids exclusion of HLA-mismatched but compatible
donors and has the added advantage of selecting platelets when the antibodies involved are
directed at platelet-specific rather than HLA antigens.
4. Although blood group antibodies (anti-A and -B) and platelet-specific antibodies can be re-
sponsible for poor responses to platelet transfusion, they are less commonly implicated in
the refractory state than HLA antibodies.
470 䡲 AABB TECHNICAL MANUAL
5. Sensitization to HPA is the most common cause of FNAIT, a syndrome involving immune
destruction of fetal platelets by maternal antibody. Platelet-specific antibodies are also in-
volved in PTP, a rare syndrome characterized by severe thrombocytopenia that occurs 5 to
10 days after a blood transfusion. The most commonly implicated antibody in both condi-
tions is anti-HPA-1a. Serologic testing using intact platelets and antigen capture assays to-
gether with HPA genotyping is used to confirm both of these diagnoses.
6. Autoantibodies directed against platelet antigens may result in ITP. Chronic ITP, which is
most common in adults, is characterized by an insidious onset and moderate thrombocyto-
penia that may be present for months to years before diagnosis. Females are twice as likely
to be affected as males. The goal of serologic testing in ITP is to detect autoantibody bound
to the patient’s own platelets.
7. Granulocyte (neutrophil) antigens are implicated in the clinical syndromes NAN, TRALI,
immune neutropenia after hematopoietic progenitor cell transplantation, refractoriness to
granulocyte transfusion, and chronic benign autoimmune neutropenia of infancy.
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474 䡲 AABB TECHNICAL MANUAL
Robert A. Bray, PhD, Professor of Pathology, and Co-director, Histocompatibility and Molecular Immunoge-
netics Laboratory, Department of Pathology, Emory University, Atlanta, Georgia; Marilyn S. Pollack, PhD, Pro-
fessor of Pathology, University of Texas Health Science Center, and Director, University Health System
Histocompatibility and Immunogenetics Laboratory, San Antonio, Texas; and Howard M. Gebel, PhD, Profes-
sor of Pathology, and Co-director, Histocompatibility and Molecular Immunogenetics Laboratory, Depart-
ment of Pathology, Emory University, Atlanta, Georgia
The authors have disclosed no conflicts of interest.
475
476 䡲 AABB TECHNICAL MANUAL
sion reactions (FNHTRs), transfusion-related Class II genes is a group of non-HLA genes that
acute lung injury (TRALI), and transfusion- code for molecules that include the comple-
associated GVHD (TA-GVHD). ment proteins C2, Bf, C4A, and C4B; a steroid
Interest in HLA polymorphisms has ex- enzyme (21-hydroxylase); and a cytokine (tu-
tended beyond their role as transplantation mor necrosis factor). This non-HLA region is
antigens. Studies correlating them with dis- often referred to as “MHC Class III” even
ease susceptibility and disease resistance be- though it does not contain any HLA genes.
gan soon after serologic techniques for HLA
Class I typing were developed. Historically, Organization of HLA Genetic Regions
HLA antigen typing had been of value in rela-
tionship assessments and forensic investiga- The HLA Class I region contains (in addition to
tions. However, with the development of de- the classical genes HLA-A, HLA-B, and HLA-C)
oxyribonucleic acid (DNA) typing, molecular other gene loci designated HLA-E, HLA-F,
methods replaced the mixed leukocyte culture HLA-G, HLA-H, HFE, HLA-J, HLA-K, HLA-L,
(MLC) as the preferred method to select MICA, and MICB. The latter genes encode
matched donor-recipient pairs for HPC trans- nonclassical, or Class Ib, HLA proteins, which
plantation. Molecular HLA testing also fos- have limited polymorphism and low levels of
tered a resurgence in the investigation of dis- expression.4 Some Class Ib genes express non-
ease associations that permitted the analysis functional proteins or no proteins whatsoever.
of peptide-binding restriction parameters Genes that are unable to express a functional
needed for effective vaccine development and protein product are termed “pseudogenes”
provided a more accurate tool for anthropo- and presumably represent an evolutionary
logic population studies. Because of the poly- dead end. In contrast, other nonclassical HLA
morphic nature of the HLA genes, a complex proteins that are expressed have been associ-
nomenclature was developed (and is continu- ated with a variety of functions. For example,
ally evolving) to define unique allele sequenc- HLA-E is associated with the surveillance sys-
es based on the relationship of each allele’s tem of one subset of natural killer cells. HLA-G
protein sequence to the serologic specificity of is expressed by the trophoblast and may be in-
the corresponding antigen.1,2 volved in the development of maternal im-
mune tolerance of the fetus. Hereditary hemo-
chromatosis (HH), an iron overload disorder
G E N E T I C S OF T HE M H C with a 10% carrier frequency in people of
Class I and II HLA antigens are cell-surface gly- Northern European ancestry, is associated
coproteins that are products of closely linked with two missense mutations in a Class I-like
genes mapped to the p21.3 band on the short gene.5 The gene that causes HH was initially
arm of chromosome 6 (Fig 19-1). That genom- named “HLA-H”; however, the HLA-H desig-
ic region is called the “MHC” and is usually in- nation had already been assigned to an HLA
herited en bloc as a haplotype. Each of the Class I pseudogene by the World Health Orga-
many loci has multiple alleles with codomi- nization (WHO) Nomenclature Committee.6
nant expression of the products from each The gene that is responsible for HH is now
chromosome. With the exception of immuno- called “HFE.” Additional Class I-like genes that
globulin (Ig) idiotypes, the HLA system is the code for molecules, such as CD1, are also lo-
most polymorphic genetic system described in cated outside the MHC. These molecules pres-
humans. ent nonprotein antigens (such as lipids) to T
The genes HLA-A, HLA-B, and HLA-C cells.
encode the corresponding Class I A, B, and C The genomic organization of the MHC
antigens. The genes HL A-DRB1, -DRB3, Class II (HLA-D) region is more complex. An
-DRB4, -DRB5; HLA-DQA1, -DQB1; and HLA- MHC Class II molecule consists of a noncova-
DPA1, -DPB1 encode the corresponding Class lent complex of two structurally similar
II antigens. Located between the Class I and chains: the -chain and the -chain. Both of
CHAPTER 19
The HLA System
䡲
FIGURE 19-1. The HLA complex is located on the short arm of chromosome 6. The centromere is to the top left of the figure, the telomere to the bottom right. The organi-
zation of the Class I, II, and III regions is shown.3
477
478 䡲 AABB TECHNICAL MANUAL
these chains are encoded within the MHC. The the phenotype represents the combined ex-
polymorphism of HLA Class II molecules re- pression of both haplotypes. However, to de-
sults from differences in both the -chain and fine haplotypes, parents (and possibly other
the -chain; this polymorphism depends on family members) must also be phenotyped to
the Class II isoform. For example, with HLA- determine which alleles are inherited together.
DR, the -chain is essentially monomorphic, Figure 19-2 illustrates inheritance of haplo-
but the -chain is very polymorphic. Multiple types.
loci code for the - and -chains of the Class II
MHC proteins. Finding HLA-Identical Siblings
Different haplotypes have different num-
A child inherits one copy of chromosome 6
bers of Class II genes and pseudogenes. The
from each parent; hence, one MHC haplotype
proteins coded by DRA1 and DRB1 result in
is inherited from each parent. Because each
HLA-DR1 through HLA-DR18 antigens. The
parent has two different copies of chromo-
products of DRA1 and DRB3 (if present) ex-
some 6, four different combinations of haplo-
press HLA-DR52; those of DRA1 and DRB4 (if types are possible in the offspring (assuming
present) express HLA-DR53; and those of that no recombination occurs). The inheri-
DRA1 and DRB5 (if present) express HLA- tance pattern is important in predicting
DR51. The HLA-DQ1 through DQ9 antigens whether family members will be compatible
are expressed on the glycoproteins coded by donors for transplantation. The chance that
DQA1 and DQB1 in the DQ cluster. Many of two siblings will be genotypically HLA identi-
the other genes of the DQ cluster are probably cal is 25%. The chance that any one patient
pseudogenes. A similar organization is found with “n” siblings will have at least one HLA-
in the HLA-DP gene cluster. identical sibling is 1 – (3/4)n. Having two sib-
Although not generally considered part of lings provides a 44% chance, and having three
the HLA system, the MHC Class III region con- siblings provides a 58% chance, that one sib-
tains four complement genes with alleles that ling will be HLA identical. Moreover, each time
are typically inherited together as a unit, a new sibling is tested, that new sibling has
termed a “complotype.” More than 10 different (only) a 25% chance of being a match, no mat-
complotypes are inherited in humans. Two of ter how many siblings have previously been
the Class III genes, C4A and C4B, encode for tested.
variants of the C4 molecule and antigens of the
Chido/Rodgers blood group system. These Absence of Antigens
variants have distinct protein structures and
functions; the C4A molecule (if present) car- Before the advent of molecular-based HLA
ries the Rg antigen, and the C4B molecule (if typing, the absence of an antigen in serologic
present) carries the Ch antigen. Both of these phenotyping results was attributed to homo-
antigens are adsorbed onto the red cells of in- zygosity at a locus (eg, inheritance of A1 from
dividuals who possess the gene(s). both parents, which in reality represented only
an apparent absence of the antigen as a result
Patterns of Inheritance of limitations of phenotyping methods) or to a
null (nonexpressed) allele. With DNA sequenc-
Although MHC organization is complicated, ing and other molecular HLA typing methods,
its inheritance follows the established princi- homozygosity can now be presumed with a
ples of Mendelian genetics. Every person has higher degree of confidence. However, homo-
two different copies of chromosome 6 and zygosity still can be proven only through fami-
possesses two HLA haplotypes, one from each ly studies or methods permitting hemizygous
parent. The expressed gene products consti- typing (ie, typing of an individual haplotype).
tute the phenotype, which can be determined A null allele is characterized by one or more
by typing for HLA antigens or alleles. Because substitutions that are within or outside the
HLA genes are autosomal and codominant, gene’s coding region and that prevent expres-
CHAPTER 19 The HLA System 䡲 479
FIGURE 19-2. The designations a/b and c/d represent paternal and maternal HLA haplotypes, respectively.
Except for crossovers, the HLA complex is transmitted en bloc from parent to offspring.
sion of a functional protein at the cell surface. DR antigens to recombination (see below).
Such inactivation of a gene may be caused by The HLA-A, HLA-B, and HLA-DR loci are close
nucleotide substitutions, deletions, or inser- together, with 0.8% crossover between the A
tions that lead to a premature cessation in the and B loci and 0.5% between the B and DR loci.
antigen’s synthesis. In the absence of a family Crossovers between the HLA-B and HLA-C loci
study, a phenotyping study revealing a single or between the HLA-DR and the HLA-DQ loci
allele at any locus offers only presumptive evi- are extremely rare, whereas crossovers be-
dence for homozygosity. In this situation, the tween the DQ and DP loci are relatively com-
allele should be listed only once because it is mon.7,8 In family studies and relationship eval-
unknown whether that allele is present twice uations, the possibility of recombination
(a true homozygote) or there is another allele should always be considered.
not detected by the available method.
Linkage Disequilibrium
Crossovers
The MHC system is so polymorphic that the
The genes of the HLA region occasionally number of possible unique HLA phenotypes is
demonstrate chromosome crossover, in which theoretically greater than that of the global hu-
segments containing linked genetic material man population. Moreover, new HLA alleles
are exchanged between the two chromosomes are constantly being discovered and character-
during meiosis or gametogenesis. The recom- ized. As of March 2014, 2579 HLA-A alleles,
binant chromosomes are then transmitted as 3285 HLA-B alleles, 1512 DRB1 alleles, and 509
new haplotypes to the offspring. Crossover fre- DQB1 alleles had been identified.9 However,
quency is related partly to the physical dis- because HLA genes are inherited as an entire
tance between the genes and partly to the re- chromosome, in reality, many HLA haplotypes
sistance or susceptibility of specific A, B, and are overrepresented compared with what
480 䡲 AABB TECHNICAL MANUAL
FIGURE 19-3. Stylized diagram of Class I and Class II major histocompatibility complex molecules showing
and polypeptide chains, their structural domains, and attached carbohydrate units.
antigens is more restricted than that of Class I nor leukocytes and the duration of storage.11
antigens. Class II antigens are expressed con- Soluble HLA in blood components may be in-
stitutively on B lymphocytes, monocytes, mac- volved in the immunomodulatory effect of
rophages, dendritic cells, intestinal epitheli- blood transfusion.
um, and early hematopoietic cells. There is
also constitutive expression of Class II anti- Configuration
gens on some endothelial cells, especially
A representative three-dimensional structure
those lining the microvasculature. However, in
of Class I and Class II molecules can be ob-
general, endothelium, particularly that of larg-
tained by x-ray crystallographic analysis of pu-
er blood vessels, is negative for Class II antigen
rified HLA antigens (see Fig 19-4). The outer
expression, although Class II antigen expres-
domains, which contain the regions of amino
sion can be readily induced (for instance, by
acid variability and the antigenic epitopes of
interferon- during immune activation). Rest-
the molecules, form a structure known as the
ing T lymphocytes are negative for Class II an-
“peptide-binding groove.” Alleles that are de-
tigen expression but can become positive
fined by polymorphisms in the HLA gene se-
when activated.
quences encode unique amino acid sequences
Soluble HLA Class I and Class II antigens
and therefore form unique binding grooves,
shed from cells are present in blood and body
each of which is able to bind peptides of differ-
fluids and may play a role in modulating im-
ent sequences. The peptide-binding groove is
mune reactivity.10 Levels of soluble HLA in-
critical for the functional aspects of HLA mole-
crease with infection [including with human
cules (see “Biologic Function” section below).
immunodeficiency virus (HIV)], inflammato-
ry disease, and transplant rejection, but HLA
Nomenclature for HLA Antigens
levels decline with progression of malignancy.
Levels of soluble HLA in blood components An international committee sponsored by the
are proportional to the number of residual do- WHO establishes the nomenclature of the HLA
482 䡲 AABB TECHNICAL MANUAL
FIGURE 19-4. Ribbon diagram of HLA Class I and Class II molecule. Note the peptide in the groove of each
molecule.
system. This nomenclature is updated regular- resent a single specificity to be “split” into
ly to incorporate new HLA alleles.2 HLA anti- specificities that were serologically (and, later,
gens are designated by a number following the genetically) distinct. The designation for an in-
letter that denotes the HLA series (eg, HLA-A1 dividual antigen that was split from an earlier
or HLA-B8). Previously, antigenic specificities recognized antigen often includes the number
that were not fully confirmed carried the prefix of the parent antigen in parentheses [eg, HLA-
“w” (eg, HLA-Aw33) for “workshop.” When the B44 (12)].
antigen’s identification became definitive, the In addition to “splits,” certain apparently
WHO Nomenclature Committee dropped the distinct HLA antigens may have other epitopes
“w” from the designation. (The Committee in common. Antibodies that are reactive with
meets regularly to update nomenclature by these shared determinants often cause cross-
recognizing new specificities or genetic loci.) reactions in serologic testing. The collective
The “w” prefix is no longer applied in this term for a group of HLA antigens that exhibit
manner and is now used only for the following: such cross-reactivity is “cross-reactive group”
1) Bw4 and Bw6, to distinguish such “public” (CREG).
antigens (see “‘Public’ Antigens” section be-
low) from other B locus alleles; 2) all serologi- “Public” Antigens
cally defined C locus specificities, to avoid
In addition to splits and CREGs, HLA proteins
confusion with components of the comple-
have reactivity that is common to many differ-
ment system; and 3) Dw specificities that were
ent HLA specificities. Called “public” antigens,
defined by mixed leukocyte reactions but are
these common amino acid sequences appear
now known to be caused by HLA-DR, HLA-DQ,
to represent the less variable portion of the
and HLA-DP polymorphisms. The numeric
HLA molecule. Two well-characterized public
designations for the HLA-A and HLA-B speci-
antigens, HLA-Bw4 and HLA-Bw6, are present
ficities were assigned according to the order of
in almost all HLA-B molecules.12 The HLA-A
their discovery.
locus molecules A23, A24, A25, and A32 also
have a Bw4-like epitope.
Splits and Cross-Reactive Groups
Public antigens are clinically important
Refinement of serologic methods permitted because patients exposed to them through
antigens that were previously believed to rep- pregnancy, transfusion, or transplantation can
CHAPTER 19 The HLA System 䡲 483
make antibodies to these antigens if the pa- the typing was determined by molecular tech-
tients do not express the epitopes themselves. niques).
A single antibody, when directed against a A similar system is used for naming Class
public antigen, can resemble multiple discrete I alleles. The name of the locus—for example,
alloantibodies, and this has significant conse- “HLA-B”—is followed by an asterisk and then
quences for identifying compatible donors for by several digits separated by colons. The first
transplantation and platelet transfusion. two digits correspond to the antigen’s serologic
specificity in most cases. The next group of
Nomenclature for HLA Alleles digits makes up the code for a unique amino
acid sequence in exons 2 and 3, with numbers
Because nucleotide sequencing is used to in-
vestigate the HLA system, increasing numbers being assigned in the order in which the
of HLA alleles are being identified, many of DNA sequences were determined. Therefore,
which share a common serologic phenotype. B*27:04 represents the HLA-B locus, has a se-
The minimum requirement for designation of rologic specificity of B27, and was the fourth
a new allele is the sequence of exons 2 and 3 unique sequence 2 and 3 allele described in
for HLA Class I and exon 2 for HLA Class II this family (see Table 19-1). A third “place” in
(DRB1). These exons encode the variable ami- the allele name is added for alleles that differ
no acids that confer HLA antigen specificity. only by synonymous (“silent”) nucleotide sub-
A uniform nomenclature has been adopt- stitutions in exons 2 and 3 for Class I or in exon
ed that takes into account the locus, major se- 2 for Class II. For example, A*01:01:02 differs
rologic specificity, and allele group deter- from A*01:01:01 only in that the codon for iso-
mined by molecular typing techniques. For leucine in position 142 is ATT instead of ATC. A
example, although many alleles have been se- fourth “place” in the allele name can be added
quenced only for exon 2, nucleotide sequenc- for alleles that differ only in sequences within
ing has identified at least 165 unique amino introns or in 3 or 5 untranslated regions. Fi-
acid sequence variants (alleles) of HLA-DR4 as nally, the nomenclature accommodates al-
of March 2014. The first HLA-DR4 variant is leles with null or low expression or other char-
designated “DRB1*04:01,” indicating the locus acteristics by the addition of an “N” or “L,”
(DR), protein (1 chain), major serologic spec- respectively, or another letter as appropriate to
ificity (04 for HLA-DR4), and sequence 2 varia- the end of the allele name. The other official
tion allele number (variant 01). The asterisk expression modifiers are as follows: S (secret-
indicates that an allele name follows (and that ed, not on cell surface), Q (expression level
questionable), A (unknown but aberrant ex- peptide in the context of the specific Class I
pression, perhaps null), and C (cytoplasmic molecule displaying it, then TCR binding acti-
expression only). The last two have not been vates the cytotoxic properties of the T cell,
used to date. which attacks the cell, characteristically elicit-
ing an inflammatory response. The presenta-
Biologic Function tion of antigen by Class I molecules is especial-
ly important in host defense against viral
The essential function of the HLA system is
pathogens and malignant transformation. Tu-
self/nonself discrimination, which is accom-
mor cells that do not express Class I antigens
plished by the interaction of T lymphocytes
escape this immune surveillance.
with peptide antigens presented by HLA pro-
teins. T lymphocytes interact with peptide an-
Role of Class II Molecules
tigens only when the T-cell receptor (TCR) for
antigen engages both an HLA molecule and Like Class I molecules, Class II molecules are
the antigenic peptide contained within the synthesized in the endoplasmic reticulum, but
TCR’s peptide-binding groove. This limitation peptide antigens are not inserted into the pep-
is referred to as “MHC restriction.”13 tide-binding groove there. Instead, an invari-
In the thymus, T lymphocytes with TCRs ant chain (Ii) is inserted as a place holder. The
that bind to a self HLA molecule are selected Class II-invariant chain complex is transport-
(positive selection), with the exception of ed to an endosome, where the invariant chain
those with TCRs that also bind to a peptide de- is removed by a specialized Class II molecule
rived from a self-antigen, in which case the T called “DM.” The DM locus is also localized to
lymphocytes are deleted (negative selection). the MHC. A Class II antigenic peptide is then
Some self-reactive T cells escape negative se- inserted into the peptide-binding groove.
lection. If not functionally inactivated (for in- Peptide antigens that fit into the Class II
stance, by the mechanism of anergy), such peptide-binding groove are typically 12 to 25
self-reactive T cells may become involved in amino acids in length and are derived from
an autoimmune process. proteins that are taken up by the cell through
endocytosis (of exogenous proteins). Exoge-
Role of Class I Molecules nous proteins, which may be normal self-pro-
Class I molecules are synthesized, and peptide teins or proteins derived from pathogens, such
antigens are inserted into the peptide-binding as bacteria, are degraded to peptides by en-
groove, in the endoplasmic reticulum. Peptide zymes in the endosomal pathway. Class II mol-
antigens that fit into the Class I peptide-bind- ecules are then transported to the cell surface,
ing groove are typically eight or nine amino ac- where the molecules are available to interact
ids in length and are derived from proteins with CD4-positive T lymphocytes, which se-
made by the cell (endogenous proteins). Such crete immunostimulatory cytokines in re-
endogenous proteins—which may be normal sponse. That mechanism is especially impor-
self-proteins; altered self-proteins, such as tant for the production of antibodies.
those in cancer cells; or viral proteins, such as
those in virus-infected cells—are degraded in I D E NT I F I C AT IO N O F HL A
the cytosol by a large multifunctional protease ANTIGENS AND ALLELES
(LMP) and are transported to the endoplasmic
reticulum by a transporter associated with an- Methods for the identification of HLA antigens
tigen processing (TAP). The LMP and TAP and alleles fall into two categories: 1) molecu-
genes are both localized to the MHC. lar (DNA based) and 2) serologic (antibody
Class I molecules are transported to the based). Historically, cell-based assays were
cell surface, where the molecules are available also used.
to interact with CD8-positive T lymphocytes. If Detailed procedures for commonly used
the TCR of a CD8 cell can bind the antigenic assays are provided in the ASHI Laboratory
CHAPTER 19 The HLA System 䡲 485
Manual from the American Society for Histo- DR16), whereas high-resolution typing distin-
compatibility and Immunogenetics.14 De- guishes individual alleles (eg, DRB1*01:01:01
pending on the clinical situation, a particular from DRB1*01:02:01). Several PCR-based
HLA antigen/allele detection or typing meth- methods have been developed; three general
od may be preferable (see Table 19-2). approaches are described below.
lar DNA sequence.16 The sequence-specific ficient antibody has bound to the lymphocyte
method requires the performance of multiple membranes, the complement cascade is acti-
PCR assays in which each reaction is specific vated through the membrane attack complex,
for a particular allele or group of alleles. The leading to lymphocytotoxicity. Damage to the
amplified alleles are directly visualized after cell membrane can be detected by the addi-
agarose gel electrophoresis. Because SSPs have tion of dye. Cells that have no attached anti-
such specific targets, the amplified material body, activated complement, or damage to the
indicates the presence of the allele or alleles membrane keep the vital dyes from penetrat-
that have that sequence. The pattern of ing; however, cells with damaged membranes
positive and negative PCR amplifications is allow the dye to enter. The cells are examined
examined to determine the actual HLA for dye exclusion or uptake under phase con-
allele(s) present. Primer pair sets are commer- trast microscopy. If a fluorescent microscope is
cially available that can determine a complete available, fluorescent vital dyes can also be
HLA-A, -B, -C, -DR, -DQA1, -DQB1, and -DPB1 used.
allele-level phenotype. Because HLA-Class II antigens (DR, DQ,
and DP) are expressed on B cells and not on
SEQUENCE-BASED T YPING. High-resolution resting T cells, typing for these antigens usual-
nucleic acid sequencing of HLA alleles gener- ly requires manipulation of the initial lympho-
ates allele-level sequences. Sequence-based cyte preparation to yield an enriched B-cell
typing (SBT) can be used to characterize new population before testing.
allele(s). Although SBT is considered the “gold The interpretation of serologic reactions
standard” for HLA typing, ambiguities often requires skill and experience. The extreme
occur when two different base pairs at the polymorphic nature of the HLA system; varia-
same position can result in two different possi- tion in antigen frequencies among different
ble combinations of alleles. These ambiguities racial groups; reliance on biologic antisera and
occur because SBT evaluates both maternal living target cells; and complexities introduced
and paternal HLA genes (haplotypes) simulta- by splits, CREGs, and “public” antigens all con-
neously, and which haplotype to assign each tribute to difficulties in accurate serologic HLA
base pair to is not always certain. New tech- typing. Given these problems, most US labora-
niques for high-resolution SBT allow separa- tories now rely primarily on DNA-based meth-
tion of HLA haplotypes before sequencing, ods for HLA typing. However, although the
thereby avoiding such ambiguities. more common “null alleles,” such as DRB4*01:
03:01:02N and C*04:09N, can now be identified
Serologic (Lymphocytotoxicity) Assays by routine molecular typing methods, rare null
alleles may not be as easily identified. It is im-
The microlymphocytotoxicity test can be used
portant to stay current on the ever-changing
to detect HLA-A, -B, -C, -DR, and -DQ anti-
array of expressed and nonexpressed HLA
gens. Lymphocytes are used for testing be-
polymorphisms. A very useful resource is the
cause they are readily obtained from anticoag-
International Immunogenetics Project’s IMGT/
ulated peripheral blood and are a more
HLA Database.2,9
reliable target than granulocytes. Lymphocytes
obtained from lymph nodes or spleen may
Cellular Assays
also be used. HLA-typing sera are obtained
primarily from multiparous women. Some Historically, the MLC [also called “mixed leu-
mouse antihuman HLA monoclonal antisera kocyte reaction” (MLR)] and primed lympho-
are also available. cyte typing (PLT) assays were used to detect
HLA sera of known specificities are placed genetic differences in the Class II region. In the
in different wells of a microdroplet test plate. A MLC, mononuclear cells from different indi-
suspension of lymphocytes is added to each viduals are cultured together; the ability of one
well. Rabbit complement is then added; if suf- population (the responder) to recognize the
CHAPTER 19 The HLA System 䡲 487
HLA-D (combined DR, DQ, and DP) antigens/ tients for platelet refractoriness, and investi-
alleles of the other (the target) as foreign can gating FNHTR or TRALI cases. The PRA “HLA
be detected by measuring the proliferation of antibody screen” not only detects the presence
the responders. In PLT, reagent cells previously of cytotoxic HLA antibodies but can often also
stimulated by specific Class II mismatched identify the specificity of those antibodies.
types allow the identification of those types by Although they have long been the gold
their accelerated proliferative responses to standard for antibody identification, comple-
stimulator cells sharing the original mis- ment-dependent cytotoxic assays are not opti-
matches. mal tests. Their sensitivity and specificity are
These classical cellular assays have been marginal, but more importantly, most cytotox-
largely replaced by molecular typing methods ic assays cannot identify all of the possible
for HLA antigens. However, these assays are HLA antibody specificities in highly sensitized
still used in some laboratories to monitor im- patients whose cells are reactive with 80% or
mune function, assess relative functional more of test panel cells or in patients with
compatibility, or select a partially mismatched Class II antibodies.
unrelated donor for HPC transplantation.
Solid-Phase Assays
CROSSM ATCHING AND The current approach to identify HLA antibod-
DE TE CT ION OF HL A ies (especially for highly sensitized solid-organ
ANTIBODIES transplant candidates) relies on the use of
beads or microparticles (ie, solid-phase meth-
Serologic Assays odology) coated either with clusters of HLA
The same microlymphocytotoxicity testing Class I or Class II antigens (ie, an HLA pheno-
used for serologic HLA typing can be used to type) or with recombinantly expressed, indi-
test serum specimens for antibodies against vidually purified HLA antigens (single antigen
selected target cells. This type of testing is beads).17 Antibody binding is detected by
referred to as “lymphocyte crossmatching.” staining with a fluorescently labeled antihu-
Crossmatching consists of incubating serum man globulin (AHG). Flow cytometry and
from a potential recipient with lymphocytes microarray methods are more sensitive than
(unfractioned or separated into T and B lym- lymphocytotoxicity or ELISA testing and focus
phocytes) from prospective donors. A varia- on the detection of IgG antibodies. The use of
tion of the microlymphocytotoxicity test uses single-antigen bead assays are of particular
an antiglobulin reagent, which increases assay importance for highly sensitized patients
sensitivity. Flow cytometry is yet another where multiple HLA antibody specificities
crossmatch method with even greater sensitiv- cannot be reliably distinguished and identified
ity than the antiglobulin-enhanced cross- with either cell-based cytotoxic assays or sol-
match. id-phase assays using clusters of HLA mole-
Testing the patient’s serum against a pan- cules.
el of 30 to 60 (or more) different target cells The clinical significance of the low-level
was historically used to assess the extent of antibodies that are detectable by the more
HLA alloimmunization. A positive result was sensitive solid-phase assays and that may not
target cell death. The percentage of panel cells cause a positive cytotoxicity or flow-cytomet-
that died after reacting with the cytotoxic anti- ric crossmatch is controversial. Newer adapta-
bodies in patient serum was referred to as the tions of the solid-phase technology can deter-
“panel-reactive antibody” (PRA) level in that mine whether these antibodies do or do not fix
patient. Determination of cytotoxic PRA was complement. However, use of this method is
(and may still be) useful for following patients also controversial because the significance of
awaiting deceased-donor solid-organ trans- noncomplement-fixing antibodies is also un-
plantation, conducting the work-up of pa- known.
488 䡲 AABB TECHNICAL MANUAL
Approaches such as HLA Matchmaker Cases of TRALI have been reported that
can be referred to as “pull” approaches, where- appear to be caused by donor antibodies
in donors are selected (or pulled into) the pro- against Class I or Class II antigens in recipi-
cess. In contrast, antibody specificity identifi- ents. Because Class II antigens are not ex-
cation strategies can be considered “push” pressed on resting neutrophils, an alternate
approaches that exclude (push away) inappro- explanation for activation of neutrophils is re-
priate donors. Either approach has a similar quired in these instances. One hypothesis is
net effect: identification of donors who have that Class II antigens on the recipient’s pulmo-
the greatest likelihood of optimizing post- nary macrophages are targeted by the comple-
transfusion platelet counts. ment-activating antibodies. The subsequent
As an alternative approach, HLA-alloim- release of cytokines and chemokines results in
munized patients often respond to cross- the recruitment and activation of neutrophils
match-compatible platelets selected by using in the lungs.22 Yet another explanation is that
patient serum and samples of apheresis plate- activated neutrophils, which can express HLA
lets in a platelet antibody assay. Crossmatch- Class II antigens in response to a patient’s in-
ing techniques may assess compatibility for flammatory state, could be further activated
both HLA and platelet-specific antibodies.20 with the binding of donor anti HLA Class II an-
Histocompatible platelet components are dis- tibodies, resulting in TRALI.
cussed further in Chapter 18.
Chimerism and TA-GVHD
Febrile Nonhemolytic Transfusion “Chimerism” refers to the presence of two cell
Reactions populations, such as transfused or transplant-
HLA, granulocyte, and platelet-specific anti- ed donor cells in a recipient, in an individual.
bodies have been implicated in the pathogen- Persistent chimerism after blood transfusion
esis of FNHTRs. The recipient’s antibodies, re- may lead to the development of TA-GVHD in
acting with transfused antigens, elicit the the recipient. The development of TA-GVHD
release of cytokines (eg, interleukin-1) that are depends on the following factors: 1) the degree
capable of causing fever. Serologic investiga- to which the recipient is immunocompro-
tion, if undertaken, may require multiple tech- mised, 2) the number and viability of lympho-
niques and target cells from a number of dif- cytes in the transfused component, and 3) the
ferent donors (see Chapter 27). degree of HLA similarity between the donor
and recipient. The development of TA-GVHD
TRALI with the use of fresh blood components from
blood relatives has highlighted the pathogenic
In TRALI (a potentially fatal transfusion reac- role of the HLA system.
tion that is recognized with increasing fre- Figure 19-5 illustrates the conditions for
quency), acute noncardiogenic pulmonary increased risk of TA-GVHD. The parents have
edema develops in response to transfusion one HLA haplotype in common. Each child,
(see Chapter 27). The pathogenesis of TRALI therefore, has one chance in four of inheriting
appears to reflect the presence of HLA or neu- the same haplotype from each parent, and
trophil antibodies in donor blood. Studies child 1 is homozygous for the shared parental
have shown that up to one-third of blood com- HLA haplotype. Transfusion of blood from
ponents can contain detectable amounts of child 1 to an unrelated recipient with different
HLA antibodies.21 If present, such antibodies haplotypes would have no untoward conse-
can be reactive with and fix complement to the quences. If, however, child 1 were a directed
recipient’s granulocytes, leading to severe cap- donor for a relative who was heterozygous for
illary leakage and pulmonary edema. Rarely, that haplotype (eg, one of the parents or child
the recipient’s HLA antibodies are reactive 3), the recipient’s body would fail to recognize
with transfused leukocytes from the donor. the antigens on the transfused lymphocytes as
490 䡲 AABB TECHNICAL MANUAL
FIGURE 19-5. HLA haplotypes in a family at risk for transfusion-associated graft-vs-host disease (GVHD). In
contrast to the family shown in Fig 19-2, each parent shares a common HLA haplotype, HLA-A1,B8,DR17. Child
1 is homozygous for the haplotype shared by the parents and by child 3. The lymphocytes of child 1 are capable
of producing posttransfusion GVHD if they are transfused to either parent or to child 3.
foreign and would not eliminate them. The been postulated that scleroderma is a form of
donor cells would recognize the recipient’s GVHD resulting from chimeric cells derived
other haplotype as foreign and would become from fetal cells transferred across the placenta
activated, proliferate, and attack the host. during pregnancy.25 Furthermore, the persis-
To avoid this situation, it is recommended tence of donor lymphocytes originally present
that all cellular components from blood rela- in and transplanted with a solid-organ al-
tives be irradiated before transfusion. Other lograft has been documented to cause fatal
specially chosen donor units, including HLA- GVHD in recipients of these organs.26
matched platelets, may also present an in-
creased risk of TA-GVHD. Rarely, TA-GVHD Hemolytic Transfusion Reactions
has occurred after the transfusion of blood HLA incompatibility has rarely been implicat-
from an unrelated donor, usually within popu- ed in shortened red cell survival in patients
lations with relatively limited genetic diversi- with antibodies to HLA antigens, such as Bga
ty, such as in Japan. (B7), Bgb (B17-B57 or B58), and Bgc (A28-A68 or
Chimerism is proposed to be responsible A69). These antigens are expressed, although
for the maintenance of tolerance in some or- weakly, on red cells. Such incompatibility may
gan transplant recipients as well as for the not be detected by conventional pretransfu-
maintenance of HLA sensitization.23,24 It has sion testing.
CHAPTER 19 The HLA System 䡲 491
positive B-cell crossmatch is significant when deceased-donor renal allografts were 21.6
caused by donor-specific HLA Class I or Class years and 13.8 years, respectively.38
II antibodies. The significantly better graft survival rate
Serum from a patient awaiting deceased- for recipients of living- vs deceased-donor re-
donor kidney transplant surgery is tested at nal allografts, even when donors and recipi-
regular intervals for the degree of alloimmuni- ents are completely unrelated, coupled with
zation by determining the percent PRA as well inadequate numbers of deceased-organ do-
as the specificities of the detected antibodies. nors has led to a relatively new practice, kid-
If an antibody with a defined HLA specificity is ney paired donation (KPD).39 Recently, KPDs
identified in a recipient, a common practice is have been facilitated through local and na-
to avoid donors who express the correspond- tional registries, which permit patients with
ing HLA antigen(s). Such antigens are deemed ABO- or HLA-incompatible potential living
“unacceptable.” A more recent approach is the donors to exchange these donors for the do-
use of solid-phase assays to identify HLA anti- nors of other patients in the same situation. As
bodies and unacceptable antigens and then a simple example, a blood group A transplant
calculate the patient’s PRA (cPRA) using a da- candidate with an incompatible blood group B
tabase of more than 12,000 HLA-typed do- potential living kidney donor could exchange
nors.36 Frozen serum samples used for period- that donor for the incompatible blood group A
ic PRA testing are often stored so that living kidney donor of a blood group B trans-
“historic” samples with the highest PRA can be plant candidate. Patients with HLA-incompat-
used in addition to the preoperative sample ible potential donors have similar possibilities
for pretransplantation crossmatching. for donor exchange, and multiple “pairs” can
Prospective crossmatching is often not be involved in one continuous exchange
performed for recipients who are conclusively (chain) process.
devoid of HLA antibodies (ie, cPRA = 0%). The introduction of altruistic donors (ie,
Prompt transplantation with reduced cold- individuals who choose to donate a kidney
ischemia time for the renal allograft may pro- without having a specific intended recipient)
vide greater benefit to the patient than pro- can significantly expand KPD options. Briefly,
spective crossmatching, provided that 1) a the altruistic donor donates a kidney to a pa-
very sensitive method for antibody detection, tient with an incompatible potential living do-
such as flow cytometry or microarrays, has nor who then donates to a different recipient
been used, and 2) it is certain that the patient with an incompatible donor, starting a “chain”
has had no additional sensitizing event (ie, im- with the possibility of a large number of living-
munizations or transfusions in the 2 weeks be- donor transplants. One chain of 10 trans-
fore or at any time after that serum was plants was recently reported.40 Currently, there
screened).37 is a movement to establish a national KPD pro-
The approach to kidney transplants with gram.
living donors is different. In the past, when
several prospective living donors were being Other Solid-Organ Transplants
considered, MLC testing of recipients and do-
nors was sometimes performed, but this is For liver, heart, lung, and heart/lung trans-
rarely (if ever) the case today. HLA matching of plants, ABO compatibility remains the prima-
recipients with kidney donors (both living and ry immunologic concern for donor selection,
deceased) contributes to long-term allograft and determining pretransplant ABO compati-
survival by decreasing the likelihood of chron- bility between the donor and recipient is re-
ic rejection. According to the Scientific Regis- quired. However, it has been shown that young
try for Transplant Recipients, recent 1-year pediatric heart or liver transplant recipients,
graft survival rates from living and deceased who have low levels of ABO isoagglutinins,
renal donors were 96.5% and 91.7%, respec- have successful outcomes with ABO-incom-
tively, and the half-lives of living-donor and patible hearts or livers.41,42 Although it is not a
CHAPTER 19 The HLA System 䡲 493
KEY POINTS
1. Genes encoded by the major histocompatibility complex (or HLA complex in humans) are
critical components of the immune system and play a major role in distinguishing self from
nonself.
2. HLA genes are located within multiple loci on chromosome 6. Each locus is extremely poly-
morphic.
3. HLA genes encode multiple Class I (eg, HLA-A, -B, and -C) and Class II (eg, HLA-DR, -DQ,
and -DP) cell-surface proteins.
4. Class I proteins are expressed ubiquitously, but Class II proteins have restricted tissue distri-
bution.
CHAPTER 19 The HLA System 䡲 495
5. Everyone inherits a set of HLA genes from his or her mother and father, referred to as a “ma-
ternal haplotype” and “paternal haplotype,” respectively.
6. Together, the maternal and paternal haplotypes are referred to as a “genotype.” The cell-sur-
face expression of proteins encoded by these HLA genes is referred to as a “phenotype.”
7. Class I and Class II HLA proteins are strongly immunogenic and can induce an immune re-
sponse, eg, formation of HLA antibodies.
8. Donor-directed HLA antibodies are associated with graft dysfunction and/or loss.
9. Solid-phase assays (eg, flow cytometry and Luminex) have become the gold standard for de-
tecting and identifying HLA antibodies.
10. Identification of donor-directed HLA antibodies can be used to perform a virtual (in-silico)
crossmatch.
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20. Friedberg RC. Independent roles for platelet 33. Standards for accredited laboratories. Mt Lau-
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21. Bray RA, Harris, SB, Josephson CD, et al. Unap- term graft survival is improved in cadaveric re-
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23. Starzl TE, Demetris AJ, Murase N, et al. Chime- 36. Cecka JM. Calculated PRA (CPRA): The new
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41. Daebritz SH, Schmoeckel M, Mair H, et al.
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29. Kurtzberg J, Laughlin M, Graham ML, et al.
42. Heffron T, Welch D, Pillen T, et al. Successful
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ABO-incompatible pediatric liver transplanta-
stem cells for transplantation into unrelated
tion utilizing standard immunosuppression
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30. Aversa F, Tabilio A, Velardi A. Treatment of
Liver Transpl 2006;12:972-8.
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stem cells from related donors with one fully icance of HLA matching in cardiac transplan-
mismatched HLA haplotype. N Engl J Med tation. J Heart Lung Transplant 1999;18:226,30.
1998;339:1186-93. 44. Thorsby E. Invited anniversary review: HLA as-
31. Bryan CF, Winklhofer FT, Murillo D, et al. Im- sociated diseases. Hum Immunol 1997;53:1-
proving access to kidney transplantation with- 11.
out decreasing graft survival: Long-term out- 45. Pile KS. HLA and disease associations. Pathol-
comes of blood group A2/A2B deceased donor ogy 1999;31:202-12.
kidneys in B recipients. Transplantation 2005; 46. Howell WM, Jones DB. The role of human leu-
80:75-80. kocyte antigen genes in the development of
32. Tyden G, Donauer J, Wadstrom J, et al. Imple- malignant disease. J Clin Pathol Mol Pathol
mentation of a protocol for ABO-incompatible 1995;48:302-6.
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rience with 60 consecutive transplantations. sis for the association of the 8.1 ancestral hap-
Transplantation 2007;83:1153-5. lotype (A1, B8, DR3) with multiple immuno-
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pathological diseases. Immunol Rev 1999;167: 52. Hill AV. The immunogenetics of resistance to
257-74. malaria. Proc Assoc Am Physicians 1999;111:
48. Pelin Z, Guilleminault C, Risch N, et al. 272-7.
HLADQB1*0602 homozygosity increases rela- 53. Slingluff CL Jr, Yamshchikov G, Neese P, et al.
tive risk for narcolepsy but not for disease se- Phase I trial of a melanoma vaccine with
verity in two ethnic groups. US Modafinil in gp100(280-288) peptide and tetanus helper
Narcolepsy Multicenter Study Group. Tissue peptide in adjuvant: Immunologic and clinical
Antigens 1998;51:96-100. outcomes. Clin Cancer Res 2001;7:3012-24.
49. Cinova J, Palova-Jelinkova L, Smythies LE, et 54. Hughes DA, Vilar FJ, Ward CC, et al. Cost-ef-
al. Gliadin peptides activate blood monocytes
fectiveness analysis of HLA B*5701 genotyping
from patients with celiac disease. J Clin Immu-
in preventing abacavir hypersensitivity. Phar-
nol 2007;27:201-9.
50. Van Gaalen FA, van Aken J, Huizinga TW, et al. macogenetics 2004;14:335-42.
Association between HLA class II genes and 55. Chung W-H, Hung S-I, Chen YT. Human leu-
autoantibodies to cyclic citrullinated peptides kocyte antigens and drug hypersensitivity.
(CCPs) influences the severity of rheumatoid Curr Opin Allergy Clin Immunol 2007;7:31723.
arthritis. Arthritis Rheum 2004;50:2113-21. 56. Haldane JBS. The estimation and significance
51. Mayr A, Schlosser M, Grober N, et al. GAD au- of the logarithm of a ratio of frequencies. Ann
toantibody affinity and epitope specificity Hum Genet 1955;20:309-11.
identify distinct immunization profiles in chil- 57. Woolf B. On estimating the relation between
dren at risk for type 1 diabetes. Diabetes 2007; blood groups and disease. Ann Hum Genet
56:1527-33. 1955;19:251-3.
C h a p t e r 2 0
Hemotherapy Decisions
and Their Outcomes
Theresa Nester, MD, Associate Medical Director, Puget Sound Blood Center, and Associate Professor of Labo-
ratory Medicine, University of Washington, Seattle, Washington; Shweta Jain, MD, Transfusion Medicine Fel-
low, Puget Sound Blood Center, Seattle, Washington; and Jessica Poisson, MD, Director of Transfusion
Services, USC Medical Center, Los Angeles, California
The authors have disclosed no conflicts of interest.
499
500 䡲 AABB TECHNICAL MANUAL
that a hemoglobin threshold of 7 g/dL was ap- many patients may limit their reserve. So what
propriate for all critically ill patients, except should the indication be for RBC transfusion
perhaps those with unstable cardiac condi- in the setting of acute anemia? A recent meta-
tions. Similarly, a 2008 systematic review of the analysis of 19 RCTs evaluated the effects of dif-
critical care literature determined that in most ferent RBC transfusion thresholds on clinical
cohort studies in 272,586 patients, RBC trans- outcomes between 1956 to 2011, a period
fusions were an independent predictor of during which the definitions of liberal vs re-
death, infection, multiorgan dysfunction, and strictive RBC transfusion strategies varied.6,7
acute respiratory distress syndrome.2 Of the 6264 patients in these studies, most
Most randomized, controlled trials (RCTs) were in surgical or intensive care units. The
conducted to date have demonstrated that a lower transfusion threshold (hemoglobin =
restrictive RBC transfusion strategy is not infe- 7.0-10.0 g/dL, and most commonly 7.0-8.0 g/dL)
rior to a liberal transfusion strategy. A recent was not inferior to the higher hemoglobin level
RCT that evaluated transfusion strategies in (9.0-13.3 g/dL, and most commonly 9.5-10.0
patients with severe upper gastrointestinal g/dL). In the lower threshold groups, fewer
bleeding showed a superior outcome with a RBC units were transfused without an adverse
restrictive strategy for patients with cirrhosis impact on mortality, morbidity, or time to
or Child-Pugh Class A or B disease.3 Because of functional recovery. There were also no signifi-
such evidence, many studies now compare the cant differences in the incidence of major
risk of anemia to that of transfusion and the complications, such as stroke, pulmonary ede-
degree to which each risk affects clinical out- ma, or infection. Caution must be used in ex-
comes. tending these findings to all patient popula-
The appropriate RBC transfusion thresh- tions because some high-risk groups (eg,
old has become a central point of discussion. patients with acute brain injury or renal fail-
The hemoglobin level in any individual patient ure) were not adequately represented.
only tells a portion of the story and, by itself, Although the findings of this meta-analy-
does not reflect the compensatory mecha- sis suggest that a restrictive transfusion strate-
nisms used to respond to the anemia. Stable gy is appropriate for many hospitalized, stable
tissue oxygenation may be maintained by patients, many clinicians still pause at the
increased cardiac output (depending on the thought of withholding transfusion from a pa-
coronary arteries’ ability to dilate) and in- tient with coronary artery disease. In the past,
creased oxygen extraction, which result in low- large observational studies gave conflicting re-
er venous oxygen content.4 Clinicians are ap- sults regarding whether a higher or lower red
propriately looking for ways to monitor a cell transfusion threshold was beneficial.8-10
patient’s response to anemia before deciding More recently, an RCT known as “FOCUS” in-
to administer a transfusion. At the same time, cluded 2016 patients (average age = 82 years)
hospital-utilization and blood-management with risk factors for, or a history of, cardiovas-
committees look for guidance on the levels of cular disease who required hip surgery.11 The
hemoglobin, below which to consider transfu- results showed that the restrictive strategy (he-
sion in a given patient population. moglobin <8 g/dL) was not inferior to a liberal
Parallel to the RCTs performed thus far is transfusion strategy (hemoglobin <10 g/dL)
the observation that patients without evidence with respect to mortality, morbidity, or func-
of cardiovascular disease who refuse transfu- tion. The primary outcome of death or inabili-
sion on religious grounds and undergo elective ty to walk independently across the room at 60
surgery tolerate hemoglobin reductions of 6 to days was similar in the two groups (34.7% in
7 g/dL with only a minimal rise in periopera- the restrictive vs 35.2% in the liberal groups).
tive mortality risk.5 Rates of in-hospital cardiac events or death as
Although many otherwise-healthy adults well as other complications were also similar.
can tolerate significant anemia equivalent to a The 1999 multicenter Transfusion Re-
halving of their red cell mass, comorbidities in quirements in Critical Care (TRICC) trial in 838
CHAPTER 20 Hemotherapy Decisions 䡲 501
critically ill patients, including 326 with ath- moglobin level <6 g/dL almost always requires
erosclerotic disease but not acute, unstable a transfusion, whereas a level >10 g/dL rarely
myocardial conditions showed that mortality does so. Of course, most patients have a hemo-
at 30 days was no different in the subgroup globin level between these limits, and many
with a primary or secondary diagnosis of car- have one or more comorbidities that affect
diac disease between the two strategies their tolerance of anemia. The assemblage of
(hemoglobin <7 g/dL vs <10 g/dL).1 Cardiac experience and knowledge must be blended
events (pulmonary edema and myocardial in- into the art of medicine to address this need.
farction) were more frequent in the liberal In addition to symptoms, research continues
strategy group, although there were no signifi- to identify consistent signs, such as venous ox-
cant differences in rates of these events during ygen saturation, that reflect how an individual
the 48 hours prior to death in the patients who patient is tolerating anemia.13
died. The subgroup analysis showed a trend
toward increased mortality in patients with Transfusion in Patients with
ischemic heart disease in the restrictive arm, Hemorrhagic Shock
leading the investigators to suggest that pa-
tients with active coronary ischemic syn- A brief history of the changes in transfusion
dromes may need a higher transfusion thresh- support over the last 40 years for patients ex-
old than 7 g/dL. periencing hemorrhagic shock can be re-
The FOCUS and TRICC trials account for viewed elsewhere.14 Currently, in the civilian
60% of the available data on the risk of myo- setting, such patients represent 2% to 3% of all
cardial infarction following restrictive vs liber- trauma admissions. Signs of acute blood loss
al RBC transfusion strategies in patients who in such patients include tachycardia, hypoten-
have coronary artery disease. An analysis of sion, increased respiratory rate, and mental
combined data from these trials and six small- status changes (Table 20-1). When these
er trials did not find an elevated risk [risk ratio symptoms are not addressed quickly, the mor-
(RR) = 0.88; 95% confidence interval (CI) = tality rate in patients experiencing hemorrhag-
0.38, 2.04] for myocardial infarction using a re- ic shock ranges from 40% to 70%.
strictive strategy.12 The statistical power of the Before 1995, aggressive resuscitation us-
combined data was sufficient to detect moder- ing crystalloid and RBCs was the standard of
ate to large differences in risk of myocardial in-
farction, but the analysis could have missed a
twofold higher risk of myocardial infarction TABLE 20-1. Signs and Symptoms of Acute
with a restrictive transfusion strategy. In light Blood Loss
of the available data, the Clinical Transfusion Tachycardia
Medicine Committee of the AABB published
guidelines suggesting that transfusion should Palpitations
be considered for hospitalized patients with Cooling of extremities
preexisting cardiovascular disease who have
Pallor
symptoms or a hemoglobin level of 8 g/dL or
less.12 Hypotension
For patients with acute coronary syn- Reduced arterial pressure
drome, no clinical trials have compared re-
strictive and liberal transfusion strategies. For Reduced central venous (jugular) pressure
this reason, the development of a guideline Acidosis
based on hemoglobin levels is difficult for pa-
Increased respiratory rate
tients with this diagnosis.
The most appropriate hemoglobin Decline in urinary output
threshold for transfusion is a patient-specific, Mental status changes
and even situation-specific, parameter. A he-
502 䡲 AABB TECHNICAL MANUAL
care in civilian hospitals. In the late 1990s, infusion for low fibrinogen levels, although not
trauma surgeons started to recognize the po- widely emphasized in these protocols, has also
tential deleterious effects of too much crystal- been established.18
loid, including increased risk of acute respira-
tory distress syndrome, multiple-organ failure, Transfusion in Patients with Chronic
and abdominal compartment syndrome. Anemia
Gradually, and after new data became avail-
able from the military experience supporting Transfusion is much less commonly indicated
combat casualties in the Iraq and Afghanistan when anemia has persisted for weeks or
wars, a new approach to hemorrhagic shock, months because compensatory mechanisms
damage control resuscitation, was adopted. have had time to work than in patients with
This approach included the early transfusion anemia of more recent onset. These longer-
of plasma and platelets in addition to RBCs term anemias are usually best treated by ad-
while minimizing crystalloid use. More em- dressing their etiologies, such as providing
phasis was also placed on addressing the lethal supplementation to treat a nutritional defi-
triad of acidosis, hypothermia, and coagulopa- ciency (eg, of iron) or reducing the rate of au-
thy early in patient resuscitation. toimmune hemolysis. Congenital hemoglo-
Since publication in 2007 of a seminal binopathies, such as sickle cell disease, are
study involving 252 military personnel with treated according to disease-related protocols
combat casualties and using almost a 1:1 ratio for purposes that are not necessarily related to
of plasma to RBCs, many publications from ci- oxygen delivery. Hypoproliferative anemias
vilian hospitals have echoed the idea that us- secondary to chemotherapy or end-stage renal
ing an increased ratio of plasma or platelets to disease are often treated with marrow stimu-
RBCs can improve outcomes.15 In 2012, the lants, such as recombinant erythropoietin.
Prospective, Observational, Multi-Center Mas- Any of these diseases could conceivably re-
sive Transfusion Study evaluated 905 patients quire a transfusion if patient symptomatology
in 10 Level I US civilian trauma centers who requires more rapid reversal than would be
survived for 30 minutes after admission and possible by treating the underlying mecha-
received at least 1 RBC unit within the first 6 nisms, but transfusion is usually considered
hours and at least three other blood products only as a last resort in these patients.
within 24 hours of admission.16 An increased Some patients become transfusion de-
ratio of plasma to RBCs or platelets to RBCs pendent because of their inability to create
was independently associated with decreased and maintain an adequate red cell mass. These
6-hour mortality. Furthermore, patients with patients often “declare” the hemoglobin at
plasma to red cell ratios less than 1:2 during which their symptoms are best controlled. The
the first 6 hours were three to four times more symptomatology reported by patients with
likely to die than patients with ratios of 1:1 or chronic anemia does not generally correlate
higher. well with laboratory values in different pa-
The Army Surgeon General has estab- tients but often corresponds well with these
lished a clinical policy requiring transfusions values in an individual over time.
of Fresh Frozen Plasma (FFP), platelets, and
RBCs in a 1:1:1 ratio for patients injured in Selected Clinical Issues
combat who require a massive transfusion of 6
Use of Whole Blood
whole blood platelet units or 1 apheresis plate-
let unit following transfusion of 6 RBC units. Whole blood provides oxygen-carrying capaci-
This emphasis on addressing coagulopathy ty, stable coagulation factors (concentrations
early in a massive resuscitation pushes labora- of Factors V and VIII decrease during storage),
torians to develop faster ways of evaluating co- and blood volume expansion. Thus, it is po-
agulation results to provide optimal transfu- tentially useful for patients with concomitant
sion support.17 The benefit of cryoprecipitate red cell and volume deficits, such as actively
CHAPTER 20 Hemotherapy Decisions 䡲 503
bleeding patients, and helps support coagula- tional studies.23,24 At least one of the studies
tion if coagulation factor consumption is the was difficult to interpret because of clinical
primary cause of coagulopathy.19 However, differences in the two groups of patients.25 The
whole blood is rarely available for allogeneic authors of an earlier meta-analysis that fo-
transfusion. Component therapy has evolved cused on homogeneous subgroups concluded
as an improved way to expand the blood com- that the available data were inadequate to
ponent supply to meet the demand. Whole prove causality between older stored blood
blood is mostly commonly used in the United components and increased morbidity and
States today for autologous transfusion. The mortality.26
ABO type of transfused whole blood must be Three larger RCTs have been undertaken
identical to that of the recipient. to clarify the issue. The Age of Red Blood Cells
in Premature Infants trial included 377 very
Duration of Storage low-birthweight premature infants requiring
transfusion. Transfusions of fresh RBC units
With the rapid disappearance of 2,3-diphos-
phoglycerate (DPG) from stored red cells and (average age = 5.1 days) vs standard-issue RBC
the concomitant increase in hemoglobin’s af- units (average age = 14.6 days) were compared,
finity for O2, a concern is that RBC units stored with rates of major neonatal morbidities as the
for more than 1 to 2 weeks may not provide the primary outcome measure and the rate of no-
intended increased availability of O2 at the tis- socomial infection as a secondary outcome. In
sue level, at least for the first 12 to 24 hours af- this study, there were no clinically meaningful
ter transfusion.20,21 Beyond 7 to 10 days of stor- or statistically significant differences in either
age, the P50 of hemoglobin decreases from 27 outcome.27 The Red Cell Storage Duration
to 16 mmHg, markedly shifting the dissocia- Study randomly assigned patients aged at least
tion curve to the left. 12 years who required complex cardiac sur-
There are other known changes to red gery to receive RBCs less than 11 days old or
cells that occur with storage over time, some- greater than 20 days old throughout their peri-
times referred to collectively as the “storage le- operative course.28 The primary outcome mea-
sion.” These changes include shape changes, sure is an assessment of clinical outcomes us-
such as decreased deformability of the red cell, ing the Multiple Organ Dysfunction Score. One
and decreased adenosine triphosphate. In ad- ongoing RCT, the Age of Blood Evaluation
dition, a loss of nitric oxide occurs within a few study, involves critically ill patients who re-
hours after collection.22 Whether these chang- quire transfusion; this prospective random-
es are clinically important is unclear. ized trial will compare all-cause mortality at 90
The corollary question regarding whether days in patients receiving RBCs less than 8
the age of blood matters for clinical outcomes days old vs standard-issue RBC units.29
is an important and controversial one. A re- Certainly, if the storage lesion is detri-
cent meta-analysis that evaluated both obser- mental to a patient’s clinical course, research
vational studies and RCTs in which death was must determine whether and, if so, why cer-
the primary outcome showed that transfu- tain patient populations are more affected
sions of older RBC units (>21 days old) were than others. With the tenuous balance that al-
associated with a significantly increased risk of ready exists between the safety and supply of
death [odds ratio (OR) = 1.16, 95% CI = 1.07, this human-derived resource, having fewer
1.24]. The majority of studies in this analysis blood components available to support pa-
were observational, and the three RCTs ana- tients may prove more detrimental than hav-
lyzed had only small numbers of patients. ing stored blood components available. Deter-
Therefore, the conclusion that transfusions of mining a better way to store RBCs—one that
older blood components caused the increased prevents the storage lesion—seems to be a
mortality is weakened by the confounding and prudent approach. Opportunities to improve
unintentional bias that arises with observa- RBC storage, prevent cell damage, and
504 䡲 AABB TECHNICAL MANUAL
improve the preservation of 2,3-DPG continue components should have their white cells
to be pursued.30 reduced.32 Although initially introduced in
several European countries to decrease the
ABO Matching transmission of prions, universal leukocyte re-
duction is most often used to reduce the risk of
Although matching patient and donor ABO
postoperative infection and improve post-
types ensures compatibility in that blood
transfusion survival by reducing transfusion-
group system, inventory considerations may
related immunomodulation (TRIM).
suggest that an ABO-compatible rather than
The benefit of removing leukocytes for
an ABO-identical RBC unit should be trans-
patients who will be multitransfused and/or
fused (Table 20-2). Although a compatible but
transplanted is clearly evident in terms of re-
nonidentical RBC unit introduces some isoag-
duced rates of alloimmunization, episodes of
glutinins directed against the recipient’s A
refractoriness to platelet transfusion, and fe-
and/or B antigens, the small amount of plas-
brile reactions.33-36 However, universal imple-
ma in an additive system or packed RBC unit
mentation of leukocyte reduction may not
(or even multiple units) is insufficient to cause
cause a measureable drop in febrile reaction
hemolysis.
rates, given the proportion of patients who are
Components containing larger amounts
susceptible to these reactions.37 A positive
of plasma, such as platelets (see below) or
clinical impact of universal leukocyte reduc-
whole blood units, raise the potential for iso-
tion has been difficult to identify and was not
agglutinins to cause hemolysis. Whole blood,
seen in the only large-scale, prospective RCT
when used, would be transfused to an ABO- of its implementation.38 Even in more defined
identical recipient because of this issue. In the
situations, such as cardiac surgery, prospective
setting of ABO-incompatible heart transplan-
trials have reached contradictory conclusions,
tation in an infant, the transfusion service may including when they were performed by the
be asked to plasma-reduce or wash an RBC same research team.39-42 The many retrospec-
unit, largely because the original protocol tive analyses ascribing benefits to leukocyte
called for this modification rather than be- reduction have often been limited by con-
cause solid evidence indicates that incompati- founders.43,44 However, some support strong
ble passive ABO isoagglutinins can impair a arguments that removing leukocytes does lead
new graft.31 to reduced lengths of stay, reduced infection
rates, and improved outcomes.45,46 Other po-
Leukocyte Reduction tential concerns associated with the presence
The most appropriate use of leukocyte reduc- of leukocytes in blood components, such as
tion remains controversial. Ardent proponents increased red cell adhesive properties, raise
on both sides argue about whether all cellular new questions about their impact.47 For a thor-
ough examination of this complex subject, the
TABLE 20-2. Selection of ABO-Compatible Red reader is referred to several excellent meta-
Blood Cell Units analyses and other publications.48-51
Many studies have addressed the possi-
Recipient Blood Compatible Red Blood bility that leukocyte reduction can reduce the
Group Cell Units* incidence of clinical outcomes due to TRIM,
but the results have been contradictory.50 One
A A, O
hypothesis was that the savings resulting from
B B, O reduced immunomodulation could offset the
AB AB, A, B, O
costs of leukocyte reduction, but this was not
demonstrable in a large prospective RCT.42
O O Nonetheless, several countries maintain a leu-
*Red Blood Cells prepared as additive system or “packed” kocyte-reduced blood supply, and the need for
units. leukocyte reduction remains controversial.43
CHAPTER 20 Hemotherapy Decisions 䡲 505
that the number of units transfused using this Incompatible RBC Transfusion
approach to a group of patients could be re-
Occasionally, transfusion of RBCs may be nec-
duced, thus reaping benefits for inventory
essary when no serologically compatible units
management as well as donor exposure rates.63 are available for a patient. This most often oc-
Issues that remain to be resolved for this ap- curs in patients with autoantibodies that typi-
proach include the following: cally are reactive with red cells from all donors;
however, as long as the presence of alloanti-
䡲 Whether the approach could be imple- bodies can be ruled out, the transfused cells
mented successfully for all patients in an should survive as long as autologous cells.
institution. The key point in ensuring a safe and suc-
䡲 Whether blood collection establishments cessful transfusion in such a situation is the
would be able to provide the needed hemo- exclusion of the presence of alloantibodies.
globin content for all RBC units. Because this may be difficult and the benefi-
䡲 Whether the approach could be sustained cial effects of the transfusion may be tempo-
in an era when most units are produced by rally limited, a conservative transfusion strate-
apheresis, with a standardized hemoglobin gy is usually recommended for patients with
content. autoimmune hemolytic anemia. Determina-
tion of the patient’s phenotype before transfu-
Emergency Transfusion sion simplifies subsequent investigations of
the presence of alloantibodies. (Chapters 15-
The unexpected need to transfuse possibly a
17 contain a more complete discussion of this
large amount of RBCs may require application issue.)
of alternative procedures. Release of group O Other situations in which all units appear
RBCs or antigen-negative, uncrossmatched to be incompatible include the presence of al-
units may be necessary if waiting to complete loantibodies to high-frequency antigens and/
standard pretransfusion testing routines could or multiple antibody specificities. If serologic
induce anemic morbidity (see Chapter 15). testing fails to resolve the problem or the prob-
An emergency-release protocol might be lem is identified but sufficient time is not
integrated into one that allows rapid provision available to acquire compatible units, consul-
of a large number of RBC units to accommo- tation between the transfusion service physi-
date the needs of patients who are bleeding cian and the patient’s clinician is advised to
rapidly. Elements of such a block-release sys- weigh the risks and benefits of transfusion and
tem might include completing the paper work to consider which alternative therapies are
for multiple units in advance and prepackag- suitable. If the need is sufficiently urgent,
ing these units to facilitate immediate delivery ABO-compatible but crossmatch-incompati-
to the patient. The need for and components ble RBCs may need to be used.
of such a system vary according to the types of Depending on the alloantibody’s specific-
ity or possible specificities that have not been
patients served by the institution and the lo-
ruled out, incompatible RBC transfusion does
gistics of blood delivery. The expectations of
not always result in immediate hemolysis, and
clinicians (particularly surgeons, anesthesiol-
the incompatible cells may remain in the cir-
ogists, and emergency physicians) for rapid culation long enough to provide therapeutic
delivery of large volumes of RBCs should be benefit.64
discussed with them so that appropriate re- If time permits and equipment is avail-
quirements can be met reliably. In evaluating able, the survival of a radiolabeled aliquot of
these clinical needs, attention should be given the incompatible RBCs can be determined.
not just to the time required to issue RBCs However, this is beyond the capability of most
from inventory to the laboratory but also for laboratories and is rarely needed. An in-vivo
the units to reach the patient. crossmatch can be performed by cautiously
CHAPTER 20 Hemotherapy Decisions 䡲 507
hemorrhage with patients’ platelet counts.71 For patients who are already bleeding or
The researchers were unable to identify a are about to undergo a hemostatic challenge,
platelet count threshold below which the risk such as a surgical procedure, attempts are
of hemorrhage increased rapidly. However, made to keep the platelet count higher, usually
this paper was often cited as the source of the in the vicinity of 50,000/µL.79 Although there
dictum that patients’ platelet counts should be are no trials to document that this is the most
kept above 20,000/µL. In the 1960s, when this appropriate level, it has become generally ac-
study was conducted, aspirin was commonly cepted as adequate.80 An even higher target, up
used to treat fever, pain, and transfusion reac- to 100,000/µL, is often used for intracerebral,
tions among patients with neutropenia. But pulmonary, and ophthalmic hemorrhage. This
with current knowledge about the platelet dys- is to provide a greater “cushion” to ensure ade-
function induced by aspirin and associated quate hemostasis in these vital and suscepti-
changes in hematology practice, the study re- ble organs even if the platelet count should
sults are less applicable today (Table 20-4).73 drop. Higher cut points might also be used in
Over the last decade, several prospective massive transfusion or disseminated intravas-
studies using either historical or randomized cular coagulation (DIC), where the platelet
count may drop rapidly.
control groups have documented the success-
The role of anemia should also be consid-
ful application of 10,000/µL as a prophylactic
ered in the prevention or treatment of hemor-
transfusion threshold in inpatients.67,74-76
rhage. In a 2001 study in healthy volunteers, a
These results parallel the finding that stool
reduction in hematocrit from 41% to 35% re-
blood loss does not accelerate until a platelet
sulted in almost a doubling of the bleeding
count of approximately 5000/µL is reached.77
time, whereas a decrease in the platelet count
Many institutions have now adopted 10,000/ by one-third had no effect.81 Traditionally, this
µL as their standard prophylactic platelet result has been attributed to the rheologic
transfusion threshold, but others have opted properties of flowing blood. Given their larger
to combine laboratory data with the patient’s size and higher density, red cells tend to occu-
clinical status to determine the most appropri- py the central (axial) portion of the blood flow,
ate transfusion point72 (Table 20-4). Further- pushing platelets to the periphery in proximity
more, because platelets adsorb circulating with the vessel wall. This makes teleologic
thrombopoietin (TPO) and higher platelet sense because it is only along the vessel wall
counts are associated with lower levels of free that a rent in the endothelium can occur
TPO,78 maintaining patients at a lower platelet where the platelets would then perform their
count has been suggested to lead to shorter in- hemostatic functions.
tervals of thrombocytopenia, which is a poten- Recent research has focused on elucidat-
tial additional benefit. ing the mechanisms of red cell and platelet
communication, which might also explain the taking aspirin has been associated with in-
hemostatic effect of a higher hematocrit. creased blood component usage.87-92 The pro-
These newer studies have shown, for example, phylactic use of platelets (and plasma) after
that the red cell membrane augments throm- cardiac surgery has been shown to be neither
bin generation,82 adenosine diphosphate re- necessary nor beneficial, but a patient experi-
leased from red cells may be a chemical mes- encing excessive blood loss postoperatively
senger for platelet activation,83 and red cell whose heparin level has been reversed may
phosphatidyl serine expression might be an- benefit from a dose of platelets even before his
other pathway for thrombin generation. Re- or her postoperative platelet count is known.
gardless of the postulated mechanism, correc- Patients treated with irreversible platelet an-
tion of anemia may be an additional tool to tagonists (eg, clopidogrel) during cardiac cath-
use for bleeding prevention, particularly in pa- eterization and who proceed directly to cardi-
tients with thrombocytopenia. In patients with ac surgery may need one or more doses of
uremia, RBC transfusion or the administra- platelets (as well as increased RBC transfu-
tion of erythropoietin to increase the hemato- sions) because their own platelets are no lon-
crit improves hemostasis similarly.84 Thus, al- ger functional.93,94 Antiplatelet therapies con-
though the patient’s cardiovascular system tinue to be used in catheterization because
might be tolerating the anemia associated they appear to improve outcomes even if the
with chemotherapy (or hemorrhage), the he- patient requires immediate surgery.95 The ef-
mostatic system may not.85 fect of these drugs can be antagonized through
pharmacologic intervention, including the use
Transfusion to Correct of desmopressin acetate.96
Thrombocytopathy Given the high proportion of patients
treated with aspirin for a variety of reasons,
Patients whose platelets are not able to com- patients who experience bleeding while tak-
plete all of the complex metabolic steps neces- ing aspirin may be encountered frequently. Al-
sary for activation, granular release, and aggre- though the daily consumption of 81 mg aspi-
gation may have an increased likelihood of rin for cardiac prophylaxis is unlikely to
bleeding and/or an inability to respond to contribute to hemostatic difficulties, some pa-
hemorrhage appropriately. These abnormali- tients are hyperresponders to aspirin and their
ties may be congenital (eg, Glanzmann throm- bleeding times may be dramatically extended.
basthenia) or acquired as the result of disease Platelet transfusions are occasionally request-
(eg, myelodysplasia) or drug treatment (eg, ed to correct the effect of aspirin. In these cas-
with aspirin or glycoprotein IIb/IIIa antago- es, only a small proportion—approximately
nists). In addition, patients who have recently 20%—of circulating platelets need to be func-
undergone extracorporeal circulation (eg, dur- tional to correct the deficiency.97 Therefore,
ing cardiac bypass surgery) may have platelet neither achievement of donor platelet levels of
counts that appear to be adequate for hemo- 50,000/µL nor a full therapeutic dose of plate-
stasis but, in fact, are dysfunctional due to pro- lets is required in most patients.
longed exposure to roller pumps and foreign Patients with significant renal disease (ie,
surfaces, leading to partial activation and de- a creatinine level exceeding 3 mg/dL) may also
granulation.86 have dysfunctional platelets due to the uremic
In all of these circumstances, the decision environment. Transfusion of platelets to these
whether to transfuse platelets probably needs patients is of little value, however, because
to be based on the patient’s clinical status they, too, rapidly succumb to the same meta-
rather than his or her platelet count. Patients bolic derangement. Desmopressin acetate (1-
undergoing surgery while still under the effect deamino-8-D-arginine vasopressin, or DDAVP)
of previously ingested aspirin do not necessar- treatment is usually recommended to aug-
ily bleed more than other patients, although ment the responsiveness of these platelets.98
clinician knowledge that the patient has been Alternatively, cryoprecipitate may provide the
510 䡲 AABB TECHNICAL MANUAL
increased amount of von Willebrand factor smallest number of platelets that would need
(vWF) that is believed to be helpful in activat- to be transfused if patients received just
ing these platelets if tachyphylaxis to multiple enough platelets to achieve the desired target
doses of desmopressin acetate precludes fur- level when they reached the transfusion
ther treatment.99 Dialysis to decrease the ure- threshold.101 This analysis argued for the use of
mia is often indicated in clinical scenarios small therapeutic doses administered more
where optimal platelet function is desired. frequently. The use of larger units in a clinical
study resulted in longer intertransfusion inter-
Dosage vals, although this temporal increase was
smaller than the increase in the number of
The amount of platelets considered a thera- platelets transfused.102 In a paired study of
peutic dose remains undecided, but recent re- marrow transplant patients, larger units pro-
search is elucidating this issue.100 Much early vided the expected lengthening of intertrans-
research was performed using platelet units fusion intervals and, unexpectedly, resulted in
with significantly lower platelet counts than both count increments and corrected count
units currently being produced. Initial platelet increments (CCIs) that were higher than those
separation efficiencies were significantly lower achieved with smaller units.103
than those achieved through evolutionary im- The Strategies for Transfusion of Platelets
provements in both component production study104 was a multicenter, prospective RCT
processes and larger whole-blood collections. designed to show that a lower platelet dose for
Accordingly, many blood centers now report prophylactic transfusions was not inferior to
mean contents that are 20% to 40% above the the standard dose; the primary outcome was
required minimum of 5.5 × 1010 platelets per incidence of WHO Grade 2 (or higher) bleed-
unit derived from whole blood. As a result, ing. The trial enrolled patients undergoing he-
fewer units need to be pooled to obtain the matopoietic stem cell transplantation and
same platelet dose. At the same time, accep- nontransplant patients with chemotherapy-
tance of lower platelet counts in patients has induced thrombocytopenia. The experimen-
facilitated the progressive reduction in the tal arm received low-dose prophylactic plate-
standard dose from 10 to 8, 6, or even 4 units let transfusions (1.5-2.9 × 1011 platelets per
per pool (or transfusion). transfusion, defined as half the standard dose)
The amount of platelets that can be col- and the control arm received a standard dose
lected by apheresis also merits consideration. (3.0-6.0 × 1011 platelets per transfusion). Al-
The standard of 3.0 × 1011 apheresis platelets though sample size calculations indicated that
per unit is the amount that could be practically approximately 270 patients would be neces-
collected with early instruments and may also sary in each treatment arm, the study was
have accounted for the maximum collection stopped after enrolling 130 patients based on a
time that donors would tolerate. These apher- preestablished safety threshold because the
esis units yielded an increment similar to the cumulative incidence of Grade 4 bleeding ex-
transfusion of a pool of 6 to 8 units of whole- ceeded 5% between the two study arms. The
blood-derived platelet components. Today, the main trends of this study did not support the
increased efficiency of apheresis instruments hypotheses that patients in the low-dose arm
allows the collection of two or even three times would require fewer products and have a
the standard quantity. Although blood centers shorter duration of thrombocytopenia.
derive a significant economic boost from split- Another recent multicenter, prospective,
ting platelet apheresis units, whether patients RCT, the platelet dose (PLADO) study,105 did
are better served by receiving larger platelet proceed to completion. Patients with hypo-
units remains to be determined. proliferative thrombocytopenia secondary to
The question of what the optimal platelet chemotherapy for hematologic malignancies
dose is has been approached in several ways. A or undergoing either autologous or allogeneic
mathematical model was used to calculate the stem cell transplantation were randomly
CHAPTER 20 Hemotherapy Decisions 䡲 511
assigned to a prophylactic platelet transfusion based on content and blood volume,108 can
dose of 1.1 (low dose), 2.2 (medium dose), or provide a yardstick to determine whether spe-
4.4 × 1011/m2 platelets (high dose). The pa- cial efforts, such as selection of antigen-
tients were transfused with their assigned dose negative units to combat alloimmunization,
prophylactically for morning platelet counts of may be helpful.
10,000/µL. Additional platelet transfusions
could be given for active bleeding or planned Unit Type and Age
invasive procedures. The primary endpoint
was the percentage of patients in each group The types of platelet units used for routine
with at least one episode of WHO Grade 2 or transfusion varies by institution. Currently,
higher bleeding. Of the 1272 patients who re- about 90% of platelet transfusions in the Unit-
ceived at least one platelet transfusion, the pri- ed States are derived from apheresis collec-
mary endpoint was achieved in 71%, 69%, and tions, and the usage of these platelets has in-
70% in the low-, medium-, and high-dose creased in a steady, linear manner for the past
groups, respectively. These differences were two decades.109 Local pricing policies and the
not statistically significant. Those in the low- preference of transfusion services to avoid the
dose arm did receive an average of one more extra processing steps associated with whole-
platelet transfusion, however. blood-derived units are probably significant
The lifespan of transfused platelets ap- factors in this choice (Table 20-6). Some in-
pears to be abnormally short in patients with vitro measures have identified a larger platelet
thrombocytopenia. Although autologous storage lesion in platelets produced via the
platelets stored for 5 or 7 days and then re- platelet-rich plasma (PRP) method than by
infused to healthy people usually survive for apheresis,110 and one paired reinfusion study
more than 5 days, the lifespan of platelets in has confirmed these findings.111 However, the
patients with severe thrombocytopenia may radiolabeled autologous recovery and survival
be only 2 days. This shortened lifespan may be rates of the two types of platelets do not ap-
attributable to the fixed loss of platelets of ap- pear dissimilar even after 7 days of stor-
proximately 7100/µL per day from circulation age.112,113 Platelets derived from whole-blood
for maintenance of normal hemostasis.106 buffy coats may have the advantages of re-
When the patient’s platelet count is low (and, duced activation during centrifugal prepara-
of course, still subject to daily reductions due tion steps, but these are currently not available
to senescence), this obligatory loss represents in the United States, although they are proba-
a large proportion of circulating platelets and bly the most widely used platelet component
leads to the need for transfusions every 2 to 3 around the world. From a clinical efficacy
days even in patients achieving good incre- standpoint, all three platelet preparations
ments from transfusions.107 More RCTs similar yield good clinical results.
to the PLADO study are needed to clarify All platelet unit types accumulate a stor-
which dosage provides optimal hemostasis. age lesion over time that leaves them less re-
As with adult RBC transfusions, the size of sponsive to physiologic agonists. Typically,
the patients (and their spleens) are usually not these platelets also bear markers of platelet ac-
considered in selecting the platelet unit dose tivation, although none of these markers has
to be transfused. One of the dose studies men- proven to be a useful predictor of recovery or
tioned above attempted to set the dose based survival after transfusion.114-117 Studies in
on patient weight, and the outcomes of the which radiolabeled platelets at the limit of the
two studies may be helpful in determining the storage period are reinfused are commonly re-
clinical importance of such a strategy. The pa- quired for FDA licensure of new techniques of
tient’s size and the content of the unit are rou- collecting or storing platelets. Both recovery
tinely assessed through the CCI (Table 20-5). and survival appear to become shorter with in-
This measure, or a similar approach of calcu- creased storage time in such radiolabeling
lating the recovery rate of transfused platelets studies. However, not all series of clinical
512 䡲 AABB TECHNICAL MANUAL
CCI
CI (per L) × BSA
CCI =
unit content
Platelet Recovery
CI × patient mass (kg) × blood volume (estimated at 75 mL/kg) × 100
Platelet recovery (%) =
unit content
Sample Calculations
Patient mass: 80 kg blood volume = 80 kg × 75 mL/kg = 6000 mL
Patient BSA: 2.0 m2 (determined from a table or nomogram, available in many textbooks)
Pretransfusion platelet count: 5000/L
CI = 20,000/L
Posttransfusion platelet count: 25,000/L
Platelet count in unit: 1.5 × 106/L
Volume of unit: 267 mL unit content = 4.0 × 1011 platelets
TABLE 20-6 Characteristics of Platelet Unit Types Available in the United States
Whole-Blood Derived
Prestorage
Platelet Unit Characteristic Individual Unit Pooled* Apheresis
observations have shown a decrement in CCI out-of-group transfusions, possibly due to the
with increased storage time, perhaps due to stimulation of higher isoagglutinin titers in the
limited study sizes and the inherent variability recipients.127 Failure to achieve expected plate-
between patients and outcomes of transfu- let increments with an out-of-group transfu-
sions at different points during the course of sion should prompt a trial of ABO-identical
illness.118,119 Therefore, platelet-transfusion transfusions, particularly if the patient is not
policies in most institutions call for the most alloimmunized to platelet-specific or HLA an-
efficient use of the short-lived and scarce re- tigens (Fig 20-1).128
source by transfusing the most appropriate An out-of-group platelet transfusion,
units that are closest to their outdate. Some such as of a group O unit to a group A recipi-
patients may appear to be more sensitive to ent, results in the transfusion of about 300 mL
the storage lesion than others and may benefit of plasma containing isoagglutinins that are
from receipt of platelets that have been stored directed against antigens present on the recip-
for less time; this can only be determined em- ient’s red cells. Although it is not standard
pirically. practice to transfuse a group O plasma unit to
a group A recipient, this practice is common
Out-of-Group Transfusions for platelet transfusions.80 Many patients show
no signs or symptoms of the mismatch, al-
The importance of providing platelet transfu- though the majority may have a transiently
sions using units of the same ABO group as positive direct antiglobulin test result that, at
that of the recipient is an unresolved issue. least, causes some immunohematologic con-
There are several points to consider: 1) the fusion regarding its cause.129
presence of ABH antigens on platelets that A high titer of isoagglutinins in a platelet
could be targeted by the recipient’s isoaggluti- unit can cause hemolysis of recipient red cells
nins,120 2) the presence of plasma in the unit that may be clinically significant and even fa-
that could lead to hemolysis of the recipient’s tal. This outcome may be more likely in situa-
red cells, and 3) the potential that the incom- tions where the recipient is smaller (and, thus,
patibility could have immunologic effects that the volume transfused represents a greater
could affect patient outcomes. Typically, the proportion of the recipient’s plasma volume),
presence of ABO-incompatible donor red cells when apheresis units are used (and all the
in the platelet product is not a significant con- plasma comes from the same donor and is not
cern because of their very low levels. “diluted” by plasma of lower isoagglutinin ti-
A recently published systematic review121 ters from other units in the pool or neutralized
showed that ABO-identical and ABO-compati- by the presence of soluble ABO antigens in the
ble platelet transfusions (eg, group A platelets other units), and in patients undergoing multi-
in an AB recipient) result in a higher CCI than ple transfusions.130 The risk of hemolysis with
ABO-incompatible platelet transfusions (eg, apheresis platelet units is in the range of
group O platelets in a non-group O recipient). 1:3000 to 1:10,000.131,132 Whether ABO-compat-
Whether the benefits of transfusing ABO- ible but nonidentical plasma poses a risk
identical or -compatible platelets also trans- through circulating immune complexes has
lates into improved clinical outcomes in terms been considered.133
of decreased bleeding severity or need for Different approaches may be used to pre-
transfusions is not clear from the existing data. vent acute hemolysis when incompatible
These data do indicate that a subset of recipi- plasma must be transfused as part of a platelet
ents with a higher isoagglutinin titer, particu- transfusion. One approach is to limit the
larly anti-A, have poorer recovery after trans- amount of plasma being transfused by centri-
fusion with a high-ABH-antigen-expression fuging the unit and expressing off the majority
unit, especially if the platelets comes from an of the plasma shortly before transfusion (see
A1 donor.122-126 Any diminution in response ap- Method 6-13). This reduces the potential se-
pears to be more pronounced with repeated verity of any hemolysis but may not prevent it
514 䡲 AABB TECHNICAL MANUAL
and, in any case, does result in the loss of some that these effects might be mediated by the
proportion of the unit’s platelet content. An formation of circulating immune complex-
approach that achieves a similar result is to es.133 For example, minimization of exposure
use platelet additive solutions, which allow to incompatible plasma was associated with
platelets to be stored in electrolyte solutions improved survival probability in marrow
that replace up to 65% of plasma, reducing the transplant recipients in one retrospective
amount of incompatible isoagglutinins. An- analysis,135 and another retrospective study
other approach is to avoid out-of-group trans- suggested that such a strategy reduced in-hos-
fusions of units having a dangerously high titer pital mortality after cardiac surgery by two-
of isoagglutinins. This approach usually in- thirds.136 This finding was not replicated in a
volves identifying units with amounts of anti-A subsequent, larger study, however.137 There is
above a particular threshold, often a titer of also some suggestion that transfusion with
200. This approach lacks a solid evidence base, ABO-incompatible platelets may hasten the
its outcomes vary by method,134 and it does not development of alloimmunization and plate-
identify all potentially harmful units. 135 Avoid- let refractoriness138 and may reduce survival
ance of out-of-group plasma or amelioration after marrow transplantation.139
of the situation through volume reduction is Any delayed immunologic impact of the
especially important for pediatric recipients, transfusion of mismatched plasma with plate-
in whom the volume transfused is relatively lets remains to be fully elucidated, although
greater, and in neonates, where hyperbilirubi- avoidance of out-of-group transfusion when
nemia may have particularly adverse conse- possible would obviate this concern. Weighed
quences. against the practical issue of a human-derived
The possibility exists that the transfusion therapeutic product that expires in 4 to 5 days
of incompatible plasma may also have other, issuing a platelet unit with incompatible plas-
less-immediate but still untoward effects on ma generally carries a lower risk than with-
recipients’ immune systems. It is postulated holding transfusion.
CHAPTER 20 Hemotherapy Decisions 䡲 515
bodies, however, may still require lymphocyto- Dealing with Platelet Refractoriness
toxicity testing in some circumstances.
Responses to platelet transfusion are most of-
Before the ready availability of such test-
ing, the detection or supposition of immuno- ten quantitated using the CCI 1 hour after
logic platelet refractoriness led to a call for transfusion (Table 20-5).151 However, a sample
HLA-matched platelets.144,145 Even a large do- collected 10 minutes after transfusion yields
nor registry may not be able to provide a com- similar information and may be easier to ob-
pletely matched unit for a given patient; many tain routinely.152 The CCI calculation is based
“HLA-matched” units actually carry antigens on the count increment (count increment =
against which the patient may have an alloan- posttransfusion count – pretransfusion count),
tibody. (The presence of serologic cross-reac- platelet content of the unit (expressed as × 10-11),
tive groups prevents recognition of some anti- and size of the patient [expressed as body sur-
gens as foreign but, at the same time, reduces face area (BSA) in m2]. For an adult patient
the range of donors whose platelets are truly with a BSA of 2.0 m2 whose platelet count rose
compatible.) Platelet crossmatching is another from 5,000/µL to 25,000/µL after a platelet
approach that can be utilized, with the lack of transfusion containing 4.0 × 1011 platelets, the
in-vitro reactivity used as a predictor of good CCI would be:
in-vivo compatibility. This technique is often
still used when alloimmunization is directed at (count increment BSA)/unit content =
a platelet antigen because few blood-collec- (20,000/µL × 2.0 m2)/4.0 = 10,000
tion agencies have phenotyped their donors
for these antigens. However, neither approach Units (m2/µL) are usually omitted when
can guarantee a good result more than 50% to reporting the result. A CCI above 7500 is con-
80% of the time, although a “good” HLA match sidered evidence of a successful transfusion;
(A or BU grade) provides the best prediction of two transfusions with CCIs below 7500 within
transfusion success.146 An alternative, simpler an hour after transfusion are evidence of re-
approach that parallels the practice with pa- fractoriness. A similar approach is used to
tients who are alloimmunized against red cell compare the recovery rate of platelets to the
antigens is the selection of platelet units that expected rate.108
lack the antigen(s) against which the recipient Occasionally, despite diligent efforts, no
is immunized and those from cross-reacting compatible platelets can be found to transfuse
groups (Fig 20-1).147 to a patient with platelet refractoriness. Alter-
Antibodies provoked by or directed at a native measures can be tried to stem or fore-
wide variety of drugs can lead to thrombocyto- stall hemorrhage, but these are of unpredict-
penia.148 These drug-dependent platelet anti- able benefit. Administration of antifibrinolytic
bodies (DDPAs) can lead to mild or profound agents, such as -aminocaproic acid (Amicar),
thrombocytopenia and to refractoriness to either intravenously or, in the case of gingival
transfused platelets.149 Although often thought bleeding, as a mouthwash may allow the clot
of as rare, DDPAs can be caused by many that is formed to be sustained.108
drugs and are present in many patients. For Curiously, administration of intravenous
example, in one study, approximately 10% of Ig (IVIG), an effective therapy for autoimmune
patients receiving gentamicin had a drop in thrombocytopenia, appears to be of little ben-
their platelet counts, and almost half of these efit in alloimmune refractoriness. IVIG can
patients (5.9% overall) had an antibody that yield increased increments shortly after trans-
interacted with platelets in the presence of fusion, but the durability of the response is
gentamicin.150 When refractoriness to platelet limited and, by 24 hours after transfusion, the
transfusion cannot be explained by the pa- patient usually returns to his or her baseline
tient’s condition or alloimmunization to HLA level.108 The claim that platelets are accumu-
or platelet antigens, consideration should be lating at sites of bleeding even though they are
given to the presence of DDPAs. not detected in circulation via a platelet count
CHAPTER 20 Hemotherapy Decisions 䡲 517
increment has not been substantiated and is study of 54 patients with TTP found no in-
unlikely to be true.108,153-155 Some experts have creased frequency of neurologic events or
advocated administration of platelets by slow death in patients who received platelet trans-
drip, perhaps after making aliquots of a thera- fusions.154 Other, well-designed trials are need-
peutic dose and administering it over 4 to 12 ed to refute or confirm the belief that platelet
hours. This approach has the benefit of allow- transfusion is contraindicated in TTP.
ing the clinician to feel that “something” is be- Platelet transfusion is also usually avoid-
ing done while minimizing the demand on the ed in patients with heparin-induced thrombo-
transfusion service inventory; however, the cytopenia (HIT), especially the immunologic
predicted benefit to the patient is based pri- (Type II) form, to forestall the development of
marily on anecdotal experience. limb- and life-threatening thromboses.162
warfarin reversal in the absence of intracranial associations, including the American Society
hemorrhage, the panel did not develop recom- of Anesthesiologists and the College of Ameri-
mendations due to insufficient data. The panel can Pathologists, recommend correction of the
recommended against plasma infusion in situ- overcoagulation by plasma transfusion prior
ations that might result in prophylactic plasma to surgery or to facilitate thrombosis only for
infusion, such as acute pancreatitis or critical- an INR of approximately 1.5 to 2.0.171,172
ly ill nonsurgical noncardiac patients. In these The British Committee for Standards in
types of patients where coagulopathy or Haematology noted, similarly, the limited
bleeding is absent, the risk of lung injury and utility of attempting to correct mild overcoag-
mortality outweigh any perceived benefit.165 A ulation.173 Their guidelines for correcting
more recent review echoes the conclusion that excessive anticoagulation and those of the
prophylactic plasma infusion may not provide American College of Chest Physicians174 (Ta-
benefit.168 ble 20-7) notably avoid calling for the use of
plasma in lieu of administration of vitamin K
Common Clinical Scenarios and the until very abnormal INRs and bleeding are en-
Limitations of Laboratory Tests to countered. Administration of vitamin K can re-
Support Transfusion Decisions verse the effects of warfarin promptly (within
6-24 hours) without complicating the reestab-
Many studies have shown that abnormal coag- lishment of healthy levels of anticoagula-
ulation test results do not predict an increased tion.175
risk of hemorrhage.169 Standard coagulation Prothrombin complex concentrates (PCCs)
screening tests are poor predictors of hemor- can also be used to rapidly correct the effects
rhage risk, in part because they are inordinate- of warfarin.176 Success has been reported from
ly sensitive to mild deficiencies of multiple combining the transfusion of 2 units of plasma
procoagulants.170 As a result, the test results with three-factor PCCs, or by using a four-fac-
become abnormal at factor levels that are not tor PCC.177 This is discussed more fully in the
clinically associated with bleeding. Additional “PCC” section below.
confusion arises from the fact that the critical With the approval of new anticoagulants
thresholds in these studies, 1.3 times the up- that act at different points of the coagulation
per limit of normal or 1.5 times the midpoint system, questions about reversal have arisen
of the reference range (usually almost the when emergent surgery is required or bleeding
same number), usually fall at about an inter- occurs. The direct thrombin inhibitors and
national normalized ratio (INR) of 1.5 to 2.0. Factor Xa inhibitors function as their names
Because the usual target for oral anticoag- indicate and offer the benefits of short half-
ulation therapy is an INR of 2.0 to 3.0, some cli- lives and a return to baseline coagulation lev-
nicians assume that patients with an INR close els in approximately 24 hours with adequate
to 2.0 require correction before surgery. How- renal function upon cessation of the drug.
ever, although reducing coagulation in pa- However, there is currently no approved rever-
tients with an INR of 2.0 prevents their con- sal agent. Case reports indicate that attempted
genital risk factor for hypercoagulability (eg, reversal with plasma in bleeding patients was
Factor V Leiden) or acquired abnormality (eg, not successful, and more evidence on the utili-
prosthetic cardiac valve) from triggering un- ty of PCCs and prohemostatic therapies is nec-
wanted clotting, this does not mean that nor- essary.178 Laboratory tests to determine the
mal, physiologic triggers of the clotting sys- amount of drug circulating are still in develop-
tem will be unable to cause an appropriate ment.
thrombotic sequence. This distinction is im- Patients with cirrhosis commonly have
portant to make when deciding whether a pa- coagulation abnormalities due to their syn-
tient requires correction of a slightly abnor- thetic difficulties. They also often experience
mal coagulation level. The guidelines for gastrointestinal bleeding due to increased por-
plasma administration of several professional tal vein pressure. However, much more fre-
CHAPTER 20 Hemotherapy Decisions 䡲 519
quent hemorrhage and an inability to clot Less activity may be expected in the procoagu-
might be expected in these patients based on lant system of a patient with cirrhosis, but, at
their prothrombin times (PTs) only. The reason the same time, there will be less of an
why people with cirrhosis do not bleed abnor- opposing force from the coagulation-control
mally—and why intricate pro- and antithrom- system.179 The PT and partial thromboplastin
botic balances are inherent in their clotting time do not reflect thrombin generation in
system—was recently elucidated by demon- these patients in a way that predicts bleeding.
stration that thrombin generated in vitro in Until a more accurate predictive test becomes
plasma from healthy individuals and people available, one role of the transfusion medicine
with cirrhosis was the same, but only after the physician will continue to be to help other cli-
addition of thrombomodulin, which activates nicians understand why patients with severe
protein C, to the test system.179 liver disease often do not require heroic efforts
As the liver’s synthetic production de- (or heroic amounts of plasma) to completely
clines, the amount of procoagulants circulat- normalize their coagulation system.
ing in plasma decreases, but so does the level What happens if an attempt is made to
of certain anticoagulants, such as protein C. correct the patient’s PT before a procedure?
520 䡲 AABB TECHNICAL MANUAL
Often, the answer is “surprisingly little.” The manifest can be much more difficult, and in-
authors of one study noted that the mean re- creased transfusion of other components may
duction in INR was only 0.03 per unit of plas- be needed. Although the early use of non-RBC
ma transfused.180 Another study showed that components reduces mortality risk,16 this ap-
the PT decreased to the normal range in less proach fails to take into consideration each
than 1% of plasma recipients who had mildly patient’s situation; direct involvement of a
abnormal PTs before transfusion, and the dif- transfusion medicine specialist can best guide
ference in the upper limit of normal was re- hemotherapy in these complex, rapidly evolv-
duced by half in only 14.5%.181 The mean de- ing situations. Such consultation also takes
crease in PT was only 0.2 second, and there into account other important factors, such as
was no correlation between PT abnormality reduced patient temperature and the presence
and subsequent RBC transfusion. This study of acidosis that decrease the in-vivo activity of
also revealed that only 1 in 10 patients receiv- the coagulation system significantly.183
ing plasma had their PT rechecked within 8 Rapid whole-blood testing techniques
hours—despite the fact that the ordering clini- have become integrated into many centers’
cian thought that the correction was clinically transfusion protocols for massively bleeding
important and the expected shortening of PT patients. Common viscoelastic coagulation
occurred infrequently. The small corrections testing (VCT) platforms include thromboelas-
in PT/INR may reflect the fact that these re- tography and rotational thromboelastometry.
ductions have an exponential relationship, These tests measure the speed and strength of
rather than a linear relationship, to the pro- clot formation. The benefits of these tests
portion of coagulation factors in circulation include that they generate initial results within
(Fig 20-2). 15-20 minutes and can be used to assess fibri-
In cases of rapid bleeding, a more proac- nolysis. Current challenges, however, involve
tive approach may be beneficial. The altera- lack of standardization of results, correlation
tions that occur in a massive transfusion with standard coagulation tests, or training in
situation are a combination of dilutional coag- interpreting the results.184
ulopathy, injury-driven factor consumption, A recent meta-analysis of RCTs on TEG-
and activation of fibrinolysis.182 Correction of a based treatment in massive transfusion in-
true coagulopathy after it becomes clinically cluded nine studies of cardiac surgery and one
7
6
5
4
INR
3
2
1
0
0 20 40 60 80 100 120
% Factor Levels
FIGURE 20-2. Exponential relationship of international normalized ratio (INR) to % factor levels.
(Used with permission from Wayne L. Chandler, MD, Houston Methodist Hospital.)
CHAPTER 20 Hemotherapy Decisions 䡲 521
liver transplant study in 776 patients.185 The a change in the PT because the relationship
authors found no difference in mortality be- between factor activity and PT is more expo-
tween TEG-guided therapy and conventional nential than linear.179,188
practice. There were moderate decreases in A usual dose of plasma is 10-20 mL/kg.
bleeding and numbers of patients transfused This dose is expected to increase the level of
with both FFP and platelets in patients under- coagulation factors by 20% immediately after
going TEG-guided treatment; however, the infusion. Precise prediction of the amount of
heterogeneity of the studies limited the ability plasma needed to be transfused to correct a
to draw definitive conclusions. VCT use in particular coagulopathy is not currently possi-
trauma appears to predict the need for mas- ble. Thus, posttransfusion repetition of the co-
sive transfusion and risk of mortality; however, agulation test that prompted the transfusion is
most data come from observational studies, warranted.179,181
and this issue would benefit from additional When attempting to correct a coagulopa-
RCT evidence.186 thy with plasma transfusion, the biological
half-life of procoagulants must also be consid-
Dose and Timing of Plasma ered. Factor VII has the shortest half-life in
Transfusion vivo (approximately 5 hours). If a transfusion
Although the level of coagulation factors nor- raises the patient’s activity of this factor from
mally varies widely between 50% and 150% of 30% to 45% 5 hours later, for example, the
the activity of circulating clotting factors, nu- activity level will be halfway back to the
merous publications show that people have steady-state level for that patient (ie, to 37%).
the ability to form clots with significantly low- Additional correction attempts must now
er levels of clotting factors.187 For single factor change the factor concentration in an en-
deficiencies, such as deficiencies of Factors larged plasma volume, and multiple plasma
VIII or IX, 30% activity is often needed for he- transfusions can produce pulmonary edema
mostasis. In patients with multiple factor defi- through fluid overload. In addition, if correc-
ciencies, such as after trauma, factor levels tion is truly required before a hemostatic chal-
closer to 40% may be needed for hemostasis lenge, such as major surgery, the plasma
(see Fig 20-2 and Table 20-8). In addition, the should be infused shortly before the procedure
further the patient’s procoagulant activity is for the benefit to be incurred at the time of the
from the normal range, the easier it is to effect hemostatic challenge.
DIC or ongoing, high-volume hemorrhage, it with or without the use of antifibrinolytic ther-
may be wise to initiate cryoprecipitate transfu- apy. Note that bleeding from the urinary tract
sion (or at least prepare the component) as the is a relative contraindication to antifibrinolytic
critical point of 100 mg/dL is approached (eg, therapy. States of systemic fibrinolysis can oc-
at approximately 120 mg/dL). cur with medical conditions such as wide-
Although the dosage of cryoprecipitate is spread amyloidosis or as a result of chemo-
often stated in tens of units (eg, 10, 20, or 30 therapy with L-asparaginase, typically used to
units), the dosage required to achieve the de- treat acute lymphoblastic leukemia.
sired effect is readily calculated. This calcula- Although no longer considered for rou-
tion is based on the difference between the tine use due to the availability of concentrated
current and desired (usually 200 mg/dL) con- plasma derivatives of copurified Factor VIII
centration of fibrinogen, a projection of the and vWF, cryoprecipitate can be used as a
patient’s plasma volume [as (1 – hematocrit) × source of vWF for patients with von Wille-
0.7 dL/kg × body mass, or 30 dL if the patient’s brand disease (vWD). The most common form
weight is unknown], and the usual fibrinogen of vWD is Type 1, which results in a deficiency
content of cryoprecipitate (250 mg/unit): of a normal protein. Type 1 vWD most often
can be managed with administration of des-
Dose (units) = [desired fibrinogen increment mopressin. Patients with Types 2 and 3 vWD
(mg/dL) × plasma volume]/250 mg/unit need to have their bleeding managed with an
exogenous source of vWF. See Table 20-9 for
Although the small volume (5-15 mL) of general factor replacement guidelines for the
each cryoprecipitate unit facilitates rapid treatment of vWD.
thawing, the pooling of units (usually includ- Cryoprecipitate can also be applied topi-
ing rinsing the bags to maximize recovery) can cally on surfaces where suturing is ineffective
be time consuming. Planning ahead for rapid to achieve hemostasis and that are slow to
use later in case a patient needs a massive generate clot, such as the raw surface of trau-
transfusion, for example, can certainly facili- matically injured liver.199 When applied simul-
tate patient care. Some facilities have chosen taneously with calcium and thrombin (usually
to produce pools of cryoprecipitate using ster- using two syringes), a thick, gelatinous mass
ile techniques before freezing the component. quickly forms over the area and quells the
This product has a higher fibrinogen content bleeding. This approach can also be used to
and a shorter time to issue when needed. bind together structures, such as in surgery in
Dysfibrinogenemias are rare congenital the inner ear. Cryoprecipitate is not typically
abnormalities that result in dysfunctional fi- requested from the transfusion service in cur-
brinogen molecules. These molecules can lead rent practice because products that have both
to an increased risk of thrombosis, bleeding, or lyophilized plasma and thrombin are avail-
both, although they can also have no clinical able commercially.200 Similarly, virus-inactivat-
manifestations.195 Patients may require admin- ed fibrinogen concentrates are available that
istration of cryoprecipitate to correct the are more readily stored (lyophilized) and pack-
deficiency. Some degree of dysfunctional fi- aged for simple use (discussed below).201
brinogen is also produced in damaged or re-
generating livers and in neonates, but this
GRANULOCY T E TRANSFUSION
rarely results in the need to transfuse normal
fibrinogen.196 The development of apheresis technology sev-
States of localized fibrinolysis can be in- eral decades ago allowed the collection of
duced during surgeries that disrupt the pros- granulocytes (neutrophils) to transfuse to pa-
tatic bed, urothelium, or salivary gland tissue tients with neutropenia and serious infections.
because plasminogen activators are produced Most of these patients were not able to mount
by these tissues. In these cases, cryoprecipitate an appropriate response because of marrow
infusions may be warranted to stop bleeding hypoplasia (eg, due to chemotherapy). The
524 䡲 AABB TECHNICAL MANUAL
TABLE 20-9. General Factor Replacement Guidelines for Treatment of Hemophilia A and von
Willebrand Disease
Hemophilia*
Severe epistaxis, oral mucosal 20-30 10-15 20-30 1-2
bleeding†
Hemarthrosis, hematoma, persistent 30-50 15-25 30-50 1-3
hematuria,‡ gastrointestinal bleed-
ing, retroperitoneal bleeding
Trauma without signs of bleeding, 40-50 20-25 40-50 2-4
tongue/retropharyngeal bleeding†
Trauma with bleeding, surgery, 100 50 100 10-14
intracranial bleeding§
von Willebrand Disease¶
Major surgery 50 40-60 daily
Minor surgery 30 30-50 daily or every other day
Dental extractions 30 20-30, single dose 12 hours
Spontaneous bleeding 30 20-30, single dose
*Data from US Pharmacopeia.197 Dosing intervals based on a half-life of Factor VIII over 8-12 hours (2-3 doses/day) and
half-life of Factor IX over 18-24 hours (1-2 doses/day). Maintenance doses of one-half the initial dose (as shown) may be
given at these intervals. The dosing frequency depends on the severity of bleeding, with more frequent dosing used for seri-
ous bleeding.
†
In addition to antifibrinolytics.
‡
Painless spontaneous hematuria usually requires no treatment. Increased oral or intravenous fluids are necessary to main-
tain renal output.
§
Factor may be administered continuously. Following the initial loading dose, a continuous infusion at a dose of 3 IU/kg per
hour is given. Subsequent doses are adjusted according to measured plasma factor levels.
¶
Concentrates labeled in terms of the ratio of von Willebrand factor to ristocetin cofactor. The recommended doses for
adults, number of infusions, and target plasma levels are the same as those for Factor VIII.198
therapy was also administered to patients with ery of granulocyte production, differentiation,
congenital neutrophil dysfunction [eg, chronic and function [such as granulocyte colony-
granulomatous disease (CGD)]. Not all pa- stimulating factor (G-CSF) and granulocyte/
tients benefited from this approach, however, macrophage colony-stimulating factor], the
and only modest improvements in outcome frequency of granulocyte transfusion fell to
were noted in a majority of—but not all— very low levels.209,210 This low utilization was
trials.202-206 The published literature offers re- also prompted by the recognition that the
views of this and other topics related to granu- amount of granulocytes that could be collect-
locyte transfusion therapy.207,208 ed from a donor, even one mobilized with pri-
With the development of more potent an- or administration of a corticosteroid, on the
timicrobial drugs and the availability of cyto- order of 1 × 1010, was only 1% to 10% of what a
kines that promote more rapid marrow recov- healthy marrow would produce each day in re-
CHAPTER 20 Hemotherapy Decisions 䡲 525
sponse to a serious infection. Trials whose au- granulocytes generated either through an
thors reported success in using granulocyte apheresis procedure or from whole-blood-
transfusion therapy generally transfused high- derived buffy coats. The latter can provide
er doses of granulocytes, but these higher only a limited number of cells and has not
yields were very difficult to obtain. been found to be as clinically efficacious as
More recently, the concept of granulocyte apheresis.218 Today, potent antibiotics are used
transfusion therapy using components with much more commonly than granulocyte
higher, “more therapeutic” content has gener- transfusions in neonates. IVIG may also be giv-
ated renewed attention to this component. en because premature infants may have hypo-
Despite the availability of newer antimicrobial gammaglobulinemia,219 although IVIG may in-
agents, infection remains a common problem crease these infants’ susceptibility to other
in patients undergoing rigorous chemothera- potentially fatal infections.220
py regimens, especially those culminating in Patients with severe neutropenia (abso-
hematopoietic stem cell transplantation, and lute neutrophil count <500/µL) are consid-
more rigorous T-cell depletion schemes in al- ered for granulocyte therapy if the infection
logeneic hematopoietic transplantation that cannot be controlled with appropriate bacteri-
lead to an increased frequency of serious and cidal antimicrobials and the neutropenia is
fatal infections, usually due to yeasts and temporary, meaning that recovery of endoge-
fungi.211,212 Case reports of success in treating nous production in a few days is likely. Patients
patients with CGD with fungal infections dur- with CGD are usually considered for granulo-
ing transplantation have also rekindled inter- cyte transfusions if they have deep-seated ab-
est in granulocyte transfusion.213,214 scesses and/or fungal infections that are not
With the administration to donors of 8 mg responding to antimicrobial therapy.
dexamethasone orally and 5 µg/kg G-CSF sub- Components must be ABO compatible if
cutaneously 8 to 16 hours before apheresis, the granulocyte collection contains 2 mL or
yields of up to 1011 granulocytes (or more) have more of red cells, as is often the case. If the pa-
been reported.215 A clinical trial to document tient requires CMV-reduced-risk transfusions,
the effectiveness of these more potent compo- the donor should be CMV seronegative be-
nents is under way through the Transfusion cause leukocyte reduction cannot be used on
Medicine Hemostasis Clinical Trials Network these components. If the recipient is alloim-
of the National Heart, Lung and Blood Insti- munized to HLA antigens, HLA-compatible
tute. This use of G-CSF is not approved by the donors need to be found or the transfused
FDA, and donor informed consent should be cells could be cleared rapidly and possibly
obtained before using this approach. In addi- cause untoward reactions without achieving
tion, the suggestion that corticosteroid admin- their intended purpose. If patients are at risk
istration may lead to posterior subcapsular of transfusion-associated graft-vs-host dis-
cataracts has prompted some clinicians to ease, as is usually the case for patients with
consider avoiding the dexamethasone admin- neutropenia, units may be gamma irradiated.
istration to donors even though this decreases Donors often undergo infectious disease
yield by one-quarter.216 The potential utility of testing when they present for predonation
prophylactic granulocyte transfusions remains stimulation to facilitate rapid release of the
unclear at present, again owing to dosage con- collected component following documenta-
siderations.217 tion of ABO/Rh type. Granulocyte units should
Neonates born preterm or after pro- be stored for the shortest period possible at
longed premature rupture of membranes are room temperature without agitation and ad-
at increased risk of bacterial sepsis. Their neu- ministered through regular blood component
tropenia is related to a temporary limitation in filters (eg, 170 microns). Drugs known to inter-
the production of neutrophils from a marrow act with granulocytes, such as amphotericin,
primed to produce red cells. Some of these pa- are best given at times remote from the granu-
tients have been treated with transfusions of locyte transfusions (ie, 12 hours before or
526 䡲 AABB TECHNICAL MANUAL
FDA approval did not include patients who coagulant deficiency and achievement of he-
had experienced acute thromboembolic mostasis can be challenging.237,238 However,
events, myocardial infarction, DIC, stroke, un- rFVIIa is less effective in patients with signifi-
stable angina, or severe peripheral vascular cant acidosis or hypothermia.
disease within 3 months before administra- In a registry that collected reports of ad-
tion. For this reason, the package insert indi- verse events after use of rFVIIa, the drug was
cates that the product may not be suitable in listed as a contributing cause of death for a
patients who have experienced thromboem- large proportion of patients who died after its
bolic events in the prior 3 months. In patients administration.239 However, in a review of 35
with a history of HIT who are subsequently placebo-control trials involving 4468 people, a
shown to be negative for HIT on enzyme- significant increase in arterial thrombosis, but
linked immunosorbent assay, exposure is like- not venous thrombosis, was attributed to
ly to be safe because the heparin will be gone rFVIIa use.240 A recent Cochrane analysis iden-
by the time the HIT antibody is recalled. How- tified 29 RCTs with 4290 patients who were
ever, use is contraindicated in patients with treated off label with rFVIIa in a variety of clin-
known HIT. The reduced capacity of a cirrhotic ical situations. The meta-analysis included
liver to clear the circulating byproducts of the separate analyses of 16 studies of prophylactic
coagulation cascade, such as D-dimers, may use and 13 of therapeutic use in addition to an
lead to DIC or other derangements of the co- analysis of all of the trials. The authors found
agulation system. Therefore, the use of PCC in no effect on mortality in the prophylactic
patients with liver disease remains risky.173 group and a trend toward decreased mortality
in the therapeutic group. Bleeding and trans-
Recombinant Factor VIIa fusion rate outcomes favored rFVIIa use, but
The production of the activated form of coagu- these data were only reported in the smaller
lation Factor VII via recombinant Factor VIIa studies, so these findings are of uncertain val-
(rFVIIa) has led to the exploration of this drug ue. Of concern was a trend toward increased
as a means of addressing a wide variety of thromboembolic events in the therapeutic
hemorrhagic conditions. The drug was devel- group. When all studies were analyzed togeth-
oped and is approved for use in bypassing an- er, a statistically significant increase in arterial
tibodies against Factor VIII in patients with he- thromboembolic events was found (RR 1.45;
mophilia A and achieving hemostasis without 95% CI 1.02 to 2.05).241 Given the open ques-
needing to achieve hemostatic levels of Factor tion of whether any adverse events are related
VIII in the face of potent antibodies. It can also to unwanted clotting and the exclusion of pa-
be used to address congenital deficiency of tients with a propensity to clot, including
Factor VII.235 those with atherosclerotic disease, from many
Beyond these applications, rFVIIa been of the trials, it may be prudent to refrain from
considered for use in a wide variety of condi- employing rFVIIa in patients who might be at
tions that are not related to hemophilia or Fac- increased risk of unwanted thrombosis.
tor VII deficiency in which bleeding is difficult A substantial stumbling block in the use
to control or is threatening the patient’s life. of rFVIIa has been its cost. If it is effective in
For example, rFVIIa has been used to counter- substantially reducing hemorrhage or morbid-
act the effect of warfarin by replacing the inac- ity, then its use might be cost-effective. Anoth-
tive factor (produced in the presence of the vi- er approach is to adopt a standardized rather
tamin K antagonist) with active rFVIIa that can than weight-based dosing practice, which re-
immediately stimulate the downstream proco- duces consumption of the drug and appears to
agulants and produce fibrin.236 The use of provide similar clinical results.242 Clinical con-
rFVIIa has also received much attention in sultation by a transfusion medicine specialist
trauma treatment, liver transplantation, and may be very helpful in guiding the use of this
massive transfusion, where correction of pro- product.243
CHAPTER 20 Hemotherapy Decisions 䡲 529
gens, the large pool sizes used to manufacture inhibition is greatly accelerated by heparin,
these products reduces this variability. Hyper- which induces a change in the polypeptide’s
immune globulins are manufactured from conformation; this is why antithrombin is
samples provided by donors chosen for their known as “heparin cofactor.”
higher titers of activity against selected agents, Patients who are congenitally deficient in
such as hepatitis A or B virus, tetanus, rabies, antithrombin have an increased risk of devel-
varicella-zoster virus, or CMV, but are usually oping thrombosis.250 Acquired deficiency of
available only in the intramuscular form. antithrombin can also occur due to reduced
The products may, on occasion, convey antithrombin synthesis (as in hepatic dis-
sufficient isoagglutinins or red cell alloanti- ease), increased antithrombin loss (as in a ne-
bodies of a particular specificity (usually anti- phrotic syndrome), increased antithrombin
D) to cause a positive direct antiglobulin test breakdown (eg, due to L-asparaginase treat-
result in the recipient, but clinically detectable ment), or increased antithrombin consump-
or significant hemolysis following use of these tion (eg, in DIC, trauma, or surgery). The ad-
products is rare.248 Administration of hyperim- ministration of heparin also accelerates the
mune anti-D to an Rh-positive patient for metabolism of antithrombin and can lead to a
treatment of ITP may induce a life-threatening relative resistance to heparin.250
or fatal acute hemolysis, and the product car- Although antithrombin is stable in frozen
ries a black-box warning to remind physicians or thawed plasma, antithrombin deficiencies
of this potential outcome. are usually addressed through the infusion of
antithrombin concentrate. Congenital anti-
Antithrombin thrombin deficiency is rare. However, anti-
Antithrombin circulates in plasma as a serine thrombin has been used in the treatment of
protease inhibitor that inactivates the serine sepsis and DIC to stem thrombotic complica-
proteases of the coagulation system, including tions and in patients being heparinized for
thrombin and Factors IXa, Xa, XIa, and XIIa, by extracorporeal circulation who do not
covalent binding at their serine active site.249 experience the expected effects of heparin
The ability of antithrombin to accomplish this treatment.
532 䡲 AABB TECHNICAL MANUAL
KEY POINTS
5. Data on using blood components to reverse the effects of newer antiplatelet agents and an-
ticoagulants are very limited, as are the data on using clotting factor concentrates.
6. In the setting of hemorrhagic shock, the current data support the transfusion of platelets,
plasma, and cryoprecipitate early in the resuscitation effort. An increased ratio of these
products to RBCs, often referred to as a 1:1:1 protocol, has been shown to decrease mortality
in one large prospective cohort study and a series of retrospective studies.
7. For patients without underlying cardiac disease, the data support using an RBC transfusion
threshold of 7 to 8 g/dL of hemoglobin. For patients with underlying cardiac disease, cur-
rent guidelines recommend a threshold of 8 g/dL of hemoglobin or less. Data are lacking on
the appropriate transfusion threshold in patients with acute coronary syndrome; therefore,
a transfusion trigger cannot be established in this setting at this time. The decision to trans-
fuse RBCs should also take into account the patient’s clinical situation and response to
anemia.
8. Although prophylactic platelet transfusions during aplasia are common practice, it is uncer-
tain whether a prophylactic vs a therapeutic transfusion approach is optimal.
9. The most common prophylactic transfusion threshold is 10,000 platelets/µL. The current
standard prophylactic dose of platelets of 3 to 4 × 1011 for an adult may be halved without
risk of bleeding.
10. When platelets bearing ABO antigens that are foreign to the recipient are transfused, the ef-
fect on the platelet count may be blunted. When plasma in platelet units contains antibod-
ies against A and/or B antigens expressed on the recipient red cells, hemolysis may occur,
with a 1:3000 to 1:10,000 risk from apheresis platelets. Steps are often taken to limit the
amount of incompatible plasma transfused or to avoid high-titer units, especially for pedi-
atric patients, when time permits.
11. Alloimmunized platelet refractory patients may be supported by transfusion of platelets
from donors lacking the targeted epitopes by either phenotyping the donor (unit) or provid-
ing HLA-matched units to patients with HLA alloimmunization.
12. There are very limited data to suggest a benefit in transfusing plasma in settings other than
intracranial hemorrhage after anticoagulation with vitamin K antagonists or massive trans-
fusion.
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540 䡲 AABB TECHNICAL MANUAL
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sion 2012;52:20S-9S. coagulants in patients with major bleeding. J
169. Segal JB, Dzik WH, Transfusion Medicine/He- Thromb Thrombolysis 2013;35:391-8.
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invasive procedures: An evidence-based re- 180. Holland LL, Foster TM, Marlar RA, Brooks JP.
view. Transfusion 2005;45:1413-25. Fresh frozen plasma is ineffective for correct-
170. Burns ER, Goldberg SN, Wenz B. Paradoxic ef- ing minimally elevated international normal-
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Pathol 1993;100:94-8. bin time and bleeding in patients with mild
171. American Society of Anesthesiologists Task coagulation abnormalities. Transfusion 2006;
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194. Bolton-Maggs PHB. The rare inherited coagu- bling M. Biologic and clinical effects of granu-
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196. Martinez J. Disorders of fibrinogen. In: Hoff- tern Med 1986;104:619-32.
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197. United States Pharmacopoeial Convention.
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cessful treatment of invasive aspergillosis in
198. Mannucci PM. How I treat patients with von
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199. Lupinetti FM, Stoney WS, Alford WC Jr, et al.
ulating factor-mobilized granulocytes, and li-
Cryoprecipitate-topical thrombin glue. Initial
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214. Bielorai B, Toren A, Wolach B, et al. Successful
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treatment of invasive aspergillosis in chronic
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201. Kozek-Langeneker S, Sorensen B, Hess J, fusions followed by peripheral blood stem cell
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15:R239. ministration of G-CSF and dexamethasone for
202. Graw RG Jr, Herzig G, Perry S, Henderson IS. the mobilization of granulocytes in normal
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203. Fortuny IE, Bloomfield CD, Hadlock DC, et al. 216. Ghodsi Z, Straus RG. Cataracts in neutrophil
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patients with acute non-lymphocytic leuke- roids. Transfusion 2001;41:1464-8.
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204. Higby DJ, Yates JW, Henderson ES, Holland JF. efficacy of prophylactic granulocyte transfu-
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205. Vogler WR, Winton EF. A controlled study of 218. Vamvakas EC, Pineda AA. Meta-analysis of
the efficacy of granulocyte transfusions in pa- clinical studies of the efficacy of granulocyte
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219. Jenson HB, Pollock BH. The role of intrave- young children with factor VIII (FVIII) defi-
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1998;22:50-63. centrates given to prevent bleeding and bleed-
220. Weisman LE, Weisman E, Lorenzetti PM. High ing-related complications in people with he-
intravenous doses of human immune globulin mophilia A or B. Cochrane Database Syst Rev
suppress neonatal Group B streptococcal im- 2006;19:CD003429.
munity in rats. J Pediatr 1989;115:445-9. 234. KCentra package insert. Kankakee, IL: CSL
221. Foster PR. Prions and blood products. Ann Behring GmbH, 2013.
Med 2000;32:501-13. 235. Mariana G, Testa MG, Di Paolantonio T, et al,
222. Berger A, Doerr HW, Scharrer I, Weber B. Fol- ad hoc study group. Use of recombinant, acti-
low-up of four HIV-infected individuals after vated factor VII in the treatment of congenital
administration of hepatitis C virus and GBV- factor VII deficiencies. Vox Sang 1999;77:131-6.
C/hepatitis G virus-contaminated intravenous 236. Deveras RA, Kessler CM. Reversal of warfarin-
immunoglobulin: evidence for HCV but not induced excessive anticoagulation with re-
GBH-C.HGV transmission. J Med Virol 1997; combinant human factor VIIa concentrate.
53:25-30. Ann Intern Med 2002;137:884-8.
223. Report of the tribunal of inquiry into the Blood 237. Levy M, Peters M, Buller HR. Efficacy and safe-
Transfusion Service Board. Ireland: Govern- ty of recombinant factor VIIa for treatment of
ment Publications, 1996. severe bleeding. Crit Care Med 2005;33:883-90.
224. Power JP, Davidson F, O’Riordan J, et al. Hepa- 238. Niemann CU, Behrends M, Quan D, et al. Re-
combinant factor VIIa reduces the transfusion
titis C infection from anti-D immunoglobulin.
requirements in liver transplant patients with
Lancet 1995;346:372-3.
high MELD scores. Transfus Med 2006;16:93-
225. Yap PL. Intravenous immunoglobulin and
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hepatitis C virus: An overview of transmission
239. O’Connell KA, Wood SS, Wise RP, et al. Throm-
episodes with emphasis on manufacturing da-
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ta. Clin Ther 1996;18(Suppl B):43:58.
binant human factor VIIa. JAMA 2006;295:293-
226. Doweiko JP, Nompleggi DJ. The role of albu-
8.
min in human physiology and pathophysiolo-
240. Levi M, Levy JH, Andersen HF, Truloff D. Safety
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of recombinant activated factor VII in ran-
enter Enteral Nutr 1991;15:476-83. domized clinical trials. N Engl J Med 2010;
227. Vermeulen LC Jr, Ratko TA, Erstad BL, et al. A
363:1791-800.
paradigm for consensus: The University Hos- 241. Simpson E, Lin Y, Stanworth S, et al. Recombi-
pital consortium guidelines for the use of al- nant factor VIIa for the prevention and treat-
bumin, nonprotein colloid and crystalloid so- ment of bleeding in patients without haemo-
lutions. Arch Intern Med 1995;155:373-9. philia. Cochrane Database Syst Rev 2012;3:
228. Vincent JL, Dubois MJ, Navickis RJ, Wilkes CD005011.
MM. Hypoalbuminemia in acute illness: Is 242. Goodnough LT, Lublin DM, Zhang L, et al.
there a rationale for intervention? A meta- Transfusion medicine service policies for re-
analysis of cohort controlled trials. Ann Surg combinant factor VIIa administration. Trans-
2003;237:319-34. fusion 2004;44:1325-31.
229. Perel P, Roberts I, Ker K. Colloids versus crys- 243. Mathew P, Simon TL, Hunt KE, Crookston KP.
talloids for fluid resuscitation in critically ill How we manage requests for recombinant fac-
patients. Cochrane Database Syst Rev 2012;6: tor VIIa (NovoSeven). Transfusion 2007;47:8-
CD000567. 14.
230. Brecher ME, Owen HG, Bandarenko N. Alter- 244. Bromberg ME. Immune thrombocytopenic
natives to albumin: Starch replacements for purpura: The changing therapeutic landscape.
plasma exchange. J Clin Apher 1997;12:146-53. N Engl J Med 2006;355:164-5.
231. Bork K. Pruritis precipitated by hydroxyethyl 245. Durabi K, Abdel-Wahab O, Dzik WH. Current
starch: A review. Br J Dermatol 2005;152:3-12. usage of intravenous immune globulin and
232. Manco-Johnson MJ, Abshire TC, Brown D, et the rationale behind it: Massachusetts General
al. Initial results of a randomized, prospective Hospital data and a review of the literature.
trial of prophylaxis to prevent joint disease in Transfusion 2006;46:741-53.
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246. Eijkhout HW, van Der Meer JW, Kallenberg CG, 249. Griffin JH. Control of coagulation reactions. In:
et al for the Inter-University Working Party for Beutler E, Lichtman MA, Coller BS, et al, eds.
the Study of Immune Deficiencies. The effect Williams’ hematology. 6th ed. New York: Mc-
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noglobulin on the incidence of recurrent in- 250. Bucur SJ, Levy JH, Despotis GJ, et al. Uses of
fections in patients with primary hypogam- antithrombin III concentrate in congenital
maglobulinemia: A randomized, double- and acquired deficiency states. Transfusion
blind, multicenter crossover trial. Ann Intern 1998;38:481-98.
Med 2001;135:165-74. 251. White B, Perry D. Acquired antithrombin defi-
247. Nydegger UE. Immunoglobulins. In: Simon ciency in sepsis. Br J Haematol 2001;112:26-31.
TL, Snyder EL, Solheim BG, et al, eds. Rossi’s 252. Kolarich D, Turecek PL, Weber A, et al. Bio-
principles of transfusion medicine. 4th ed. chemical, molecular characterization, and gly-
Bethesda, MD: AABB Press, 2009:260-72. coproteomic analyses of 1-proteinase inhibi-
248. Schwartz J, Spitalnik S, Grima KM. Severe he- tor products used for replacement therapy.
molysis following administration of Rho(D) Transfusion 2006;46:1959-77.
immune globulin in an ITP patient associated
with anti-C. Blood 2006;107:2585.
C h a p t e r 2 1
Administration of Blood
Components
T H E S A F E A D M I N I S T R A T I O N of Recipient Consent
blood and its components requires mul-
tidisciplinary collaboration among clinical The AABB Standards for Blood Banks and
and ancillary services and clinicians. Policies Transfusion Services states, “The blood bank or
and procedures should be developed with in- transfusion service medical director shall par-
put from transfusionists, the transfusion ser- ticipate in the development of policies, pro-
vice, surgeons, anesthesiology care providers, cesses, and procedures regarding recipient
primary-care physicians, and transport per- consent for transfusion.”1(p44) Recipient in-
sonnel. The transfusionist typically provides formed consent should address indications
the last line of defense in the detection of er- for; risks, benefits, and possible side effects of;
rors before the transfusion commences. All and alternatives to transfusions of allogeneic
personnel involved in the chain of preparing, blood components. Some state laws require
delivering, and administering a transfusion certain additional elements in the patient con-
should be given appropriate training to ensure sent.
the provision of the safest transfusion possible The patient has the right to choose or re-
for patients. fuse a transfusion and must have an opportu-
nity to ask questions of a learned professional
before providing consent. Documentation of
EV E N TS A ND CO N S I D E R AT I O N S the consent process must be entered into the
BEFORE DISPENSING patient’s medical record. Some facilities re-
CO MPO N EN TS quire an institution-approved signed consent
form to document that the consent process
Before a transfusion begins, thoughtful con-
has occurred and the risks/benefits of transfu-
sideration, planning, and preparation are re-
sion were discussed with the patient or legal
quired.
Kim Maynard, BSN, RN, OCN, Breast Coordinator (former Transfusion Safety Officer, Transfusion Medicine
Service), Dartmouth-Hitchcock Medical Center, Lebanon, New Hampshire
The author has disclosed no conflicts of interest.
545
546 䡲 AABB TECHNICAL MANUAL
representative. Each institution needs to have tient with renal or cardiopulmonary disease
a process for recording a patient’s refusal to re- may require a slower infusion rate to prevent
ceive blood or blood components in the pa- fluid overload, for example.
tient’s medical record. Institutional policies A patient with an elevated temperature
should identify health-care providers who are may destroy cellular components at an in-
allowed to obtain consent and the length of creased rate.3 Moreover, if the patient presents
time for which that consent remains valid. with an elevated temperature before the trans-
Consent for transfusion must be ob- fusion, it may be difficult to determine later
tained from patients who have the requisite whether this was caused by a transfusion reac-
capacity to make such decisions. If a patient is tion. Administration of an antipyretic should
unable to give consent, a legally authorized be considered in such cases.
representative or surrogate may do so (de-
pending on local and state laws). If no one is Medical Order
available to provide consent and the need for
transfusion is considered a medical emergen- Blood Component
cy, the blood component may be administered A licensed provider writes two orders for the
based on the doctrine of implied consent. In- components to be administered. The first or-
dividual state and local laws governing re- der requests that compatible crossmatch (if
quirements for implied consent may vary, but appropriate) unit(s) be prepared for the pa-
the emergent need for transfusion should be tient and notes special processing require-
carefully documented in the medical record.2 ments. The second order explains to the trans-
fusionist how to administer the component,
Patient Education and History including the transfusion rate. Both of these
The transfusionist should educate the patient orders should specify:
about reporting any symptoms that may be in-
dicative of a reaction and how long the trans- 䡲 Patient name and other independent iden-
fusion will take. The patient’s questions should tifier (eg, date of birth or medical record
be answered before the transfusion is started. number).
It is important to collect a history from 䡲 Component [eg, Red Blood Cells (RBCs) or
the patient before the component is ordered to apheresis platelets] to prepare or adminis-
assess whether the patient is at increased risk ter.
of a transfusion reaction. This history includes 䡲 Special processing required (eg, leukocyte
previous transfusions and any adverse reac- reduction, irradiation, or washing).
tions. If the patient has had previous reactions 䡲 Number of units or volume to administer.
to transfusions, the medical team should de- 䡲 Date and time for the infusion.
termine whether the patient needs to receive 䡲 Flow rate or period for administering the
medications before the transfusion or wheth- component.
er special processing of the component is indi-
cated to mitigate the risk of an adverse reac- Note: It is appropriate for the rate and du-
tion. ration of the transfusion (eg, not to exceed 4
hours from the time that the container is en-
Baseline Assessment of Recipient tered until completion)4 to be described in a
policy approved by the hospital’s medical staff.
A baseline physical assessment should include
vital signs. Many institutions also routinely Laboratory Testing
measure oxygen saturation using oximetry.
The assessment should include pretransfusion After receiving an order from a licensed pro-
symptoms, such as shortness of breath, rash, vider, the transfusion service ensures that
pruritus, wheezing, and chills, as a basis for compatible components are provided. To issue
comparison after the transfusion starts. A pa- blood components for a specific patient (ex-
CHAPTER 21 Administration of Blood Components 䡲 547
cept in the case of an emergency transfusion), peripheral intravenous line, implanted port, or
the laboratory must collect a pretransfusion peripherally inserted central catheter (PICC)]
blood sample. Typically, the sample is ob- is acceptable for the infusion of blood compo-
tained within 3 days of transfusion, with the nents.
draw date considered to be day 0.1(p36) Institu- Acceptable intravenous catheter sizes for
tional policies may vary regarding the sample use in transfusing cellular blood components
outdate. If the patient has not had a transfu- range from 22 to 14 gauge.6 A 20- to 18-gauge
sion or been pregnant in the prior 3 months, intravenous catheter is suitable for the general
the sample may be acceptable for longer than adult population and provides adequate flow
3 days for testing purposes. rates without excessive discomfort to the pa-
According to The Joint Commission, con- tient. When an infant or a toddler is trans-
tainers used for blood specimens must be la- fused, a 24- to 22-gauge intravenous catheter
beled in the presence of the patient.5 The sam- may be suitable but requires infusion through
ple must be labeled at the patient’s side with at a syringe (see Chapter 23).7 The flow rate may
least two unique identifiers (eg, the patient’s need to be adjusted depending on the catheter
name, date of birth, or identification number). size.
The identification of the person collecting the There is an increased possibility of hemo-
sample and the date on which the sample was lysis when red cells are transfused rapidly us-
collected must be traceable.1(p35) ing handheld syringes through 23-gauge or
Pretransfusion testing of the recipient’s smaller needles.8 With the use of smaller cath-
blood sample, including for unexpected anti- eters, blood dilution and a pump are helpful to
bodies to red cell antigens, is described in de- administer the unit to prevent slow flow rates
tail in Chapters 15 and 16. The time interval that lead to clogging of the intravenous cathe-
from sample receipt to availability of the re- ter. Units suspended in a preservative solution,
quested component can vary greatly. One such as Adsol, usually do not require addition-
reason is that availability depends on the in- al dilution. It is important that the infusion
ventory of components maintained by the line be patent before the component is re-
transfusing facility, and some components ceived at the patient’s bedside.
may need to be obtained from an external sup-
plier. Furthermore, if the test results from the Prophylactic Medications Given
pretransfusion sample show that the patient Before Transfusion
has clinically significant red cell antibodies,
Although antipyretics (eg, acetaminophen)
obtaining corresponding antigen-negative or
were commonly ordered in the past to reduce
crossmatch-compatible units may require ad-
the risk of febrile nonhemolytic transfusion re-
ditional time. Moreover, some components re-
actions, indications for their use are contro-
quire thawing, pooling, relabeling, or other
versial.9 Some providers use antipyretics in a
preparation before release. All of these factors
prophylactic manner, some wait to use them
necessitate timely communication between
until the patient has experienced at least one
laboratory and transfusing personnel. For ex-
febrile transfusion reaction, and others believe
ample, components that are pooled or require
that prophylactic use of antipyretics may mask
thawing may have a shortened shelf life after
the elevated temperature that results from a
being prepared (4-24 hours); transfusionists
transfusion reaction. Evidence supporting any
should be aware of the decreased time avail-
of these approaches is limited.9-12 Some experts
able to complete a transfusion of such compo-
recommend that “in the absence of definitive
nents.1(pp51-59)
evidence-based studies, pretransfusion medi-
cation to prevent transfusion reactions should
Venous Access
not be encouraged.”13
The transfusionist must determine whether In a Cochrane review14 of studies on pre-
the patient’s venous access [with a central line, medication to prevent allergic reactions and
548 䡲 AABB TECHNICAL MANUAL
ence of clots or clumps, or loss of bag integ- radiofrequency identification devices, biomet-
rity. ric scanning, mechanical or electronic locks
䡲 Identification of patient and unit. The pa- that prevent access to bags assigned to other
tient’s two independent identifiers (eg, patients, and handheld computers suitable for
name and identification number) must transferring blood request and administration
match the information on both the label on data from the patient’s bedside to the transfu-
the unit or attached tag and the medical or- sion service information system in real time.
der. The requirements of the institution for Each system provides a method to bring staff
patient identification must be satisfied. In- toward self-correction during the proce-
stitutions often require, for example, an ad- dure.27,28 Studies show that rates of positive re-
ditional check to verify that the unit was se- cipient identification can be increased by such
lected for the patient whose sample was systems. However, none of these systems ne-
collected for crossmatch. gates the need for good quality management,
䡲 Medical order. The transfusionist should such as adequate standard operating proce-
verify that the component matches the unit dures, regular training, periodic competency
called for in the medical order and that any assessment, and system monitoring.
special processing in the order was per-
formed. In particular, the transfusionist Infusion Sets
should ensure that leukocyte reduction or
Components must be administered through
irradiation was performed if ordered.
special IV tubing with a filter designed to re-
䡲 Blood type. The patient’s ABO group (and
move blood clots and particles that are poten-
Rh type if required) should be compatible
tially harmful to the patient. Standard blood
with that of the unit. Interpretation of
administration tubing typically has a 170- to
crossmatch tests (if performed) is also veri-
260-micron (macroaggregate) filter, but this
fied.
micron size is not mandated or required. The
䡲 Donation identification number.
tubing can be primed with either 0.9% sodium
䡲 Expiration date (and time, if applicable).
chloride or the component itself. The manu-
The unit should not be transfused if the ex-
facturer’s instructions should be reviewed for
piration date or time has passed.
proper use. The IV setup should have a mecha-
nism that allows bypass of the blood IV admin-
The transfusion should be withheld if any
istration tubing to start 0.9% sodium chloride
discrepancy or abnormality is found.
in the event of a reaction. A suggested mecha-
Proper bedside identification of the recip-
nism is to have a “Y” port or three-way stop-
ient is the final step to prevent the administra-
cock close to the infusion site that allows for
tion of an incorrect blood component to a pa-
the administration of 0.9% sodium chloride.
tient. Although individuals often worry about
the possibility of exposure to infectious agents
Microaggregate Filters
from transfusion, equal concern should focus
on the inadvertent transfusion of incompati- Microaggregate filters are not used for routine
ble blood. Approximately 1 in every 19,000 blood administration. These second-genera-
units of RBCs is transfused to the wrong pa- tion filters were originally developed to re-
tient each year; 1 in 76,000 transfusions results move leukocytes and to complement or
in an acute hemolytic transfusion reaction, replace the clot screen in the 1970s.29 They
and 1 in 1.8 million units of transfused RBCs have since been replaced by more efficient
results in death from an acute hemolytic trans- leukocyte reduction filters.30 Microaggregate
fusion reaction.26 filters have a screen filter depth of 20 to 40
To prevent the potentially fatal conse- microns and retain fibrin strands and clumps
quences of misidentification, specific systems of dead cells. Red cells, which are 8 microns in
have been developed and marketed, including diameter, can flow through the filters. Micro-
identification bracelets with bar codes and/or aggregate filters are typically used for the
552 䡲 AABB TECHNICAL MANUAL
Component First 15 Minutes After 15 Minutes Special Considerations ABO Compatibility Filter
Red Blood Cells 1-2 mL/min As rapidly as tolerated; Infusion duration should Whole blood: ABO identical In-line (170-260 micron)
(RBCs) (60-120 mL/hour) approximately 4 mL/ not exceed 4 hours. RBCs: ABO compatible with Leukocyte reduction if
minute or 240 mL/hour For patients at risk of fluid recipient’s plasma indicated
overload, may adjust flow Crossmatch required
rate to as low as 1 mL/kg/
hour.
Platelets 2-5 mL/min 300 mL/hour or as toler- Usually given over Crossmatch not required In-line (170-260 micron)
(120-300 mL/hour) ated 1-2 hours ABO/Rh compatibility pref- Leukocyte reduction if
For patients at risk of fluid erable but not required indicated
overload, use slower flow
rate (see under RBCs) May be HLA matched
Plasma 2-5 mL/min As rapidly as tolerated; Thaw time may be needed Crossmatch not required In-line (170-260 micron)
AABB TECHNICAL MANUAL
(120-300 mL/hour) approximately 300 mL/ before issue ABO compatibility with
hour For patients at risk of fluid recipient red cells
overload, use slower flow
rate (see under RBCs)
Granulocytes 1-2 mL/min 120-150 mL/hour or as Over approximately 2 Crossmatch required In-line (170-260 micron)
(60-120 mL/hour) tolerated hours ABO/Rh compatibility No leukocyte reduction fil-
Infuse as soon as possi- required ter or depth-type micro
ble after collection/release May be HLA matched aggregate filters
of product; irradiate
Cryoprecipitated AHF As rapidly as tolerated Infuse as soon as possi- Crossmatch and ABO com- In-line (170-260 micron)
ble after thawing; pooling patibility not required
is preferred.
CHAPTER 21 Administration of Blood Components 䡲 555
Once the patient is stable, the transfusion units are transfused within 4 hours of the start
service should be notified of a suspected of the initial transfusion. Therefore, if more
transfusion reaction, and institutional policy than 1 unit can be infused in 4 hours, blood
should be followed for returning the compo- administration tubing sets may be used for
nent bag and/or to order the laboratory stud- more than one component.
ies needed to evaluate the reaction.
UNIQUE TRANSFUSION
Completing the Transfusion
S ET T IN G S
The patient is assessed at the completion of
See Chapter 23 for information about transfu-
the transfusion, and his or her vital signs are
sion in pediatric and neonatal patients.
obtained. The bag and tubing are discarded in
a biohazard container if the transfusion was
Operating Room and Trauma: Rapid
uneventful.
Because patients can experience transfu- Infusions
sion reactions several hours to days after the If components need to be administered rapid-
transfusion is complete, clinical staff should ly, the use of pressure infusion, large-bore ad-
continue to monitor the patient periodically ministration tubing, and 8-Fr intravenous
for 4 to 6 hours after the end of the transfusion catheters can decrease the infusion time with-
to detect febrile or pulmonary reactions that out inducing hemolysis.37,38 Specific tubing
may be associated with blood administration. sets are designed for rapid blood administra-
If the patient is not under direct clinical super- tion with appropriate filters. Flow rates as fast
vision after a transfusion, clinical staff should as 10 to 25 mL/second (600-1500 mL/minute)
provide written instructions to the patient and have been reported with such tubing. Howev-
caregiver regarding signs and symptoms to re- er, at such rapid infusion rates, steps should be
port and a phone number to call or a person to taken to avoid hypothermia. Furthermore,
contact should a reaction occur later. when multiple units are infused through the
The transfusion should be documented in same tubing, the flow rate may decrease ap-
the patient’s medical record. At a minimum, preciably.
AABB Standards requires documentation of Hypocalcemia has been noted with rapid
the following1(p45): transfusions. This is usually transient and de-
pendent on the amount and rate of citrate in-
1. Transfusion order. fused. Calcium replacement may be adminis-
2. Recipient consent. tered based on the patient’s ionized serum
3. Component name. calcium level and the rate of citrate adminis-
4. Donation identification number. tration.39
5. Date and time of transfusion. Transfusion-associated hyperkalemic car-
6. Pre- and posttransfusion vital signs. diac arrest has been reported with rapid ad-
7. Volume transfused. ministration of RBCs. It may develop with rap-
8. Identification of the transfusionist. id RBC administration even with a modest
9. Transfusion-related adverse events. transfusion volume of between 1 (in a neo-
nate) and 54 units. Contributing factors are ac-
If additional units are transfused, the in- idosis, hypoglycemia, hypocalcemia, and hy-
stitution’s guidelines and/or manufacturer’s pothermia at the time of cardiac arrest.40
recommendations should be consulted to de- If components are urgently needed and a
termine whether the same blood administra- delay in transfusion could be detrimental to
tion tubing may be used for subsequent units. the patient, the transfusion service should
If there are no contraindications from the have a process to provide components before
manufacturer, institutions frequently allow the all pretransfusion compatibility testing is
tubing to be reused as long as subsequent completed. In such cases, uncrossmatched
556 䡲 AABB TECHNICAL MANUAL
units are released with a signed statement one. The disadvantage is that there is no
from the requesting physician indicating that trained assistant available in the event of a se-
the clinical situation requires urgent release vere adverse reaction. Issues to consider when
before the completion of testing. If compo- preparing for a transfusion in the home in-
nents in the transfusion service inventory are clude the following:
not immediately accessible to a trauma unit or
operating room, a supply of group O red cells 䡲 Availability of a competent adult in the
may be maintained at these sites. The transfu- home to assist in patient identification and
sion service must ensure proper storage of to summon medical assistance if needed.
components at these satellite storage sites. 䡲 A mechanism to obtain immediate physi-
cian consultation.
Out-of-Hospital Transfusion 䡲 A telephone to contact emergency person-
Transfusing blood in a nonhospital setting re- nel and easy access for emergency vehicles.
䡲 Documentation of prior transfusions unac-
quires a well-planned program that incorpo-
rates all the relevant aspects of the hospital companied by adverse reactions.
䡲 The ability to properly dispose of medical
setting and emphasizes safety consider-
ations.41,42 An outpatient surgery center, oncol- waste.
ogy clinic, or dialysis center is likely to be able
to provide medical assistance in a timely man- CO N C LUS IO N S
ner in the event of an adverse reaction. Medi-
cal staff availability, medications, and equip- Transfusion of blood components and the cre-
ment to handle adverse reactions must be ation of blood administration procedures and
arranged for in advance. Staff should be com- policies should be patient centric. Following
petent in performing blood administration these policies helps the transfusionist quickly
procedures and patient monitoring. Blood ad- recognize and report suspected transfusion re-
ministration outside the hospital should be actions. Close monitoring and early interven-
performed by personnel with substantial ex- tion can make a critical difference in patient
perience in blood administration in this set- outcomes. The transfusion service should pe-
ting. riodically audit the blood administration pro-
Transfusion in the home generally allows cess to identify instances of nonconformance,
close monitoring of the transfusion event be- analyze their causes, and institute corrective
cause the personnel-to-patient ratio is one to actions.
KEY POINTS
1. Blood administration involves the process of informed consent, preparation of the recipi-
ent, administration of the appropriate product to the correct recipient, and careful observa-
tion of the recipient during and after the transfusion for any adverse reaction. All steps must
be appropriately documented in the patient’s medical record.
2. A licensed care provider should initiate requests for blood administration with an order for
the appropriate blood component and an order for the administration of the component(s).
3. The recipient should be informed of and educated about the upcoming administration of
the blood component so that he or she can give informed consent for the transfusion.
4. A baseline assessment of the recipient should be performed for subsequent comparison.
5. Just before the planned administration, the transfusionist must verify appropriate venous
access, administration of any prophylactic medications required, and availability of re-
quired equipment (eg, blood warmer, infusion pump, pressure devices, and emergency
equipment).
CHAPTER 21 Administration of Blood Components 䡲 557
6. Institutions should identify appropriate blood and blood component issue and delivery
mechanisms to ensure that the transfusionist receives the components in a timely manner.
7. Transfusion services should ensure that other departments are aware of the requirement for
returning components if a transfusion is delayed.
8. Before a transfusion is initiated, verification of recipient and component identification
should be performed. The following items should be verified: 1) the appearance of the unit,
2) identity of the recipient and unit, 3) medical order, 4) blood type, and 5) expiration date/
time of the component.
9. Components must be administered through the appropriate infusion sets and filter if indi-
cated. No solution other than 0.9% sodium chloride injection, USP, should be administered
through the same tubing unless the tubing has been flushed with 0.9% sodium chloride,
USP, immediately before and after the transfusion.
10. The infusion should start slowly at approximately 2 mL per minute for the first 15 minutes.
During this time, the transfusionist should remain near the patient. If no sign of reaction
appears, the infusion rate can be increased. The transfusionist monitors the patient
throughout the infusion and stops the infusion in the event of an adverse reaction.
11. Infusions must be completed within 4 hours. After completion, the transfusionist takes the
patient’s vital signs. If the patient will not be under direct clinical supervision after the
transfusion, the patient and caregiver should receive instructions regarding signs and
symptoms to report and whom to report these reactions to.
12. The following information, at a minimum, about the transfusion must be documented in
the patient’s medical record: 1) the transfusion order, 2) patient consent for transfusion, 3)
name of component, 4) donation identification number, 5) date and time of infusion, 6)
pre- and posttransfusion vital signs, 7) volume transfused, 8) identity of the transfusionist,
and 9) any adverse reaction.
REFER ENCES
1. Levitt J, ed. Standards for blood banks and 6. De la Roche MR, Gauthier L. Rapid transfusion
transfusion services. 29th ed. Bethesda, MD: of packed red blood cells: Effects of dilution,
AABB, 2014. pressure, and catheter size. Ann Emerg Med
2. Stowell CP, Sazama, K, eds. Informed consent 1993;22:1551-5.
in blood transfusion and cellular therapies: 7. Barcelona SL, Vilich F, Cote CJ. A comparison
Patients, donors, and research subjects. of flow rates and warming capabilities of the
Bethesda, MD: AABB Press, 2007. level 1 and rapid infusion system with various-
3. Klein H, Anstee D. Mollison’s blood transfu- size intravenous catheters. Anesth Analg 2003;
sion in clinical medicine. 12th ed. Oxford: Wi- 97:358-63.
ley-Blackwell, 2014. 8. Miller MA, Schlueter AJ. Transfusions via
4. AABB, the American Red Cross, America’s hand-held syringes and small-gauge needles
Blood Centers, and the Armed Services Blood as risk factors for hyperkalemia, Transfusion
Program. Circular of information for the use of 2004;44:373-81.
human blood and blood components. Bethes- 9. Kennedy LD, Case LD, Hurd DD, et al. A pro-
da, MD: AABB, 2013. [Available at http:// spective, randomized, double-blind controlled
www.aabb.org (accessed November 26, 2013).] trial of acetaminophen and diphenhydramine
5. The Joint Commission. National patient safety pretransfusion medication versus placebo for
goals effective January 1, 2013. Oakbrook Ter- the prevention of transfusion reactions. Trans-
race, IL: The Joint Commission, 2013. [Avail- fusion 2008;48:2285-91.
able at http://www.jointcommission.org/as 10. Ezidiegwu CN, Lauenstein KJ, Rosales LG, et
sets/1/18/NPSG_Chapter_Jan2013_HAP.pdf al. Febrile nonhemolytic transfusion reac-
(accessed November 26, 2013).] tions: Management by premedication and cost
558 䡲 AABB TECHNICAL MANUAL
implications in adult patients. Arch Pathol Lab other vascular access devices. J Infus Nurs
Med 2004;128:991-5. 2007;30:341-4.
11. Geiger TL, Howard SC. Acetaminophen and 23. Frelich R, Ellis MH. The effect of external pres-
diphenhydramine premedication for allergic sure, catheter gauge, and storage time on he-
and febrile nonhemolytic transfusion reac- molysis in RBC transfusion. Transfusion
tions: Good prophylaxis or bad practice? 2001;41:799-802.
Transfus Med Rev 2007;21:1-12. 24. Wong KF. Virtual blood bank. J Pathol Inform
12. Wang SE, Lara PN Jr, Lee-Ow A, et al. Acet- 2011;2:6.
aminophen and diphenhydramine as premed- 25. Lum G, D’Amarino MJ. Use of Laboratory ro-
ication for platelet transfusions: A prospective bots for transport and delivery of blood prod-
randomized double-blind placebo-controlled ucts. Lab Med 2009;40:517-22.
trial. Am J Hematol 2002;70:191-4. 26. Vamvakas EC, Blajchman MA. Transfusion re-
13. Tobian AR, King K, Ness PM. Prevention of fe- lated mortality: The ongoing risks of allogene-
brile nonhemolytic and allergic transfusion re- ic blood transfusion and the available strate-
actions with pretransfusion medication: Is this gies for their prevention. Blood 2009;113:3406-
evidence-based medicine? Transfusion 2008; 17.
48:2274-6. 27. Pagliaro P, Rebulla P. Transfusion recipient
14. Marti-Carvajal AJ, Sola I, Gonzalez LE, et al. identification. Vox Sang 2006;91:97-101.
Pharmacological interventions for the preven- 28. Koshy R. Navigating the information technolo-
tion of allergic and febrile non-haemolytic gy highway: Computer solutions to reduce er-
transfusion reactions. Cochrane Database Syst rors and enhance patient safety. Transfusion
Rev 2010;(6):CD007539. 2005;45(Suppl 4):189S-205S.
15. Patterson BJ, Freedman J, Blanchette V, et al. 29. Wortham ST, Ortolano GA, Wenz B. A brief his-
Effect of premedication guidelines and leuko-
tory of blood filtration: Clot screens, microag-
reduction on the rate of febrile nonhaemolytic
gregate removal, and leukocyte reduction.
platelet transfusion reactions. Transfus Med
Transfus Med Rev 2003;17:216-22.
2000;10:199-206.
30. Lane TA. Leukocyte reduction of cellular blood
16. Goss JE, Chambers CE, Heupler FA, et al. Sys-
components: Effectiveness, benefits, quality
temic anaphylactoid reactions to iodinated
control, and costs. Arch Pathol Lab Med 1994;
contrast media during cardiac catheterization
118:392-404.
procedures: Guidelines for prevention, diag-
31. Zoon KC, Jacobson ED, Woodcock J. Hypoten-
nosis, and treatment. Cath Cardiovasc Diagn
sion and bedside leukocyte reduction filters.
1995;34:99-104.
17. Tramer MR, von Elm E, Loubeyre P, Hauser C. Int J Trauma Nurs 1999;5:121-2.
Pharmacological prevention of serious ana- 32. Dickson DN, Gregory MA. Compatibility of
phylactic reactions due to iodinated contrast blood with solutions containing calcium. S Afr
media: Systematic review. Br Med J 2006;333: Med J 1980;57:785-7.
675-81. 33. The Joint Commission. Comprehensive ac-
18. Boyan CP, Howland WS. Cardiac arrest and creditation manual for hospitals. Oakbrook
temperature of bank blood. JAMA 1963;183: Terrace, IL: The Joint Commission, 2013.
58-60. 34. Bradbury M, Cruickshank JP. Blood transfu-
19. Donham JA, Denning V. Cold agglutinin syn- sion: Crucial steps in maintaining safe prac-
drome: Nursing management. Heart Lung tice. Br J Nurs 2000;9:134-8.
1985;14:59-67. 35. Perkins HA, Payne R, Ferguson J, Wood M.
20. Iserson KV, Huestis DW. Blood warming: Cur- Nonhemolytic febrile transfusion reactions:
rent applications and techniques. Transfusion Quantitative effects of blood components with
1991;31:558-71. emphasis on isoantigenic incompatibility of
21. Hirsch J, Menzebach A, Welters ID, et al. Indi- leukocytes. Vox Sang 1966;11:578-600.
cators of erythrocyte damage after microwave 36. Oldham J, Sinclair L, Hendry C. Right patient,
warming of packed red blood cells. Clin Chem right blood, right care: Safe transfusion prac-
2003;49:792-9. tice. Br J Nurs 2009;18:312, 314, 316-20.
22. Houck D, Whiteford J. Improving patient out- 37. Iserson KV, Knauf MA. Confirmation of high
comes: Transfusion with infusion pump for blood flow rates through 150 micron filter/
peripherally inserted central catheters and highflow tubing. J Emerg Med 1990;8:689-91.
CHAPTER 21 Administration of Blood Components 䡲 559
38. Floccare DJ, Kelen GD, Altman RS, et al. Rapid 40. Smith HM, Farrow SJ, Ackerman JD, et al. Car-
infusion of additive red blood cells: Alternative diac arrests associated with hyperkalemia dur-
techniques for massive hemorrhage. Ann ing red blood cell transfusion: A case series.
Emerg Med 1990;19:129-33. Anesth Analg 2008;106:1062-9.
39. Denlinger JK, Nahrwold ML, Gibbs PS, Lecky 41. Fridey JL. Practical aspects of out-of-hospital
JH. Hypocalcaemia during rapid blood trans- transfusion. Am J Clin Pathol 1997;107 (Suppl
fusion in anaesthetized man. Br J Anaesth 1):S64-71.
1976;48:995-1000. 42. Evans CS. Out-of-hospital transfusion. Trans-
fusion 1997;37:756-67.
C h a p t e r 2 2
Melanie S. Kennedy, MD, Clinical Associate Professor Emeritus, Attending Physician, Transfusion Medicine,
Wexner Medical Center, Department of Pathology, The Ohio State University, Columbus, Ohio; Meghan Del-
aney, DO, MPH, Medical Director, Red Cell Genomics, Puget Sound Blood Center, Medical Director, Blood
Bank, Seattle Children’s Hospital, Assistant Professor, University of Washington, Seattle, Washington; Scott
Scrape, MD, Assistant Professor, Director, Transfusion Medicine, Wexner Medical Center, Department of
Pathology, The Ohio State University, Columbus, Ohio
The authors have disclosed no conflicts of interest.
561
562 䡲 AABB TECHNICAL MANUAL
the calculated dose to the right of the decimal the mother, but the titer is rarely greater than 4
point is <0.5 of a vial, it should be rounded and thus poses no risk to the fetus. Occasion-
down to the next whole number plus one vial ally, the DAT result may be positive in a new-
(Table 22-1). In the above example, the dose to born with no evidence of hemolysis. About
be given is two vials. Additional examples are 10% of the 28-week gestation dose will be pres-
as follows: ent at delivery (the half-life of IgG is 25 days).
Examples: This anti-D is not active immunization, so
postpartum RhIG should be given if the new-
1.6 vials calculated = born is D positive.
2 (round up) + 1 (add 1) = 3 RhIG is entirely IgG, whereas active im-
munization has an IgM component. Thus, new
1.4 vials calculated = anti-D produced by the mother can often be
1 (round down) + 1 (add 1) = 2 detected in the saline phase and can be com-
pletely or partially inactivated by 2-mercapto-
Postpartum RhIG should be given to the
ethanol or DTT treatment, whereas RhIG can-
mother within 72 hours of delivery. If prophy-
not. In addition, passively acquired anti-D
laxis is delayed, the ACOG recommends that
rarely achieves a titer above 4. Antibody titers
treatment still be administered. If the D type of
do not correlate with the effectiveness of the
the newborn is unknown or undetermined (eg,
RhIG or the amount of FMH.
for a stillborn infant), RhIG should be admin-
The mechanism of action of RhIG has not
istered.
been completely elucidated. Current evidence
Depending on the preparation, RhIG can
shows that D-positive red cells are opsonized
be given by intramuscular (IM) or intravenous
by RhIG and removed by macrophages, which
(IV) injection. If IM preparations are used,
release cytokines that result in immunomodu-
multiple doses are given in different sites or at
lation.27 The number of IgG molecules known
different times within 72 hours. Multiple doses
to prevent immunization is much smaller than
of the IV preparation may be administered ac-
the D antigen sites on red cells.
cording to the instructions in the package in-
sert.
ABO HEM O LY TIC DISEASE
Serology and Mechanism
Because of the use of RhIG, ABO incompatibil-
Administration of RhIG during pregnancy may ity is now the most common cause of HDFN.
produce a positive antibody screening result in HDFN is triggered when naturally occurring
Dose
Notes:
1. Based on a maternal blood volume of 5000 mL.
2. 1 vial of 300 g (1500 IU) is needed for each 15 mL of fetal red cells or 30 mL of fetal whole blood.
CHAPTER 22 Perinatal Issues in Transfusion Practice 䡲 567
IgG anti-A,B in a group O mother is transport- man platelet antigen HPA-1a, which is present
ed across the placenta and bound to fetal red in about 98% of the US population.29 About
cells expressing A or B antigens. Destruction of 10% of cases are caused by anti-HPA-5b, 4% by
fetal red cells rarely leads to severe anemia be- anti-HPA-1b, 2% by anti-HPA-3a, and 6% by
cause fetal ABO antigens are poorly developed other antibodies. The incidence of affected
and antibody is neutralized by tissue and solu- pregnancies is approximately 1 per 1500 to
ble antigens. Also, ABO HDFN has no comple- 2000.30
ment-mediated hemolytic mechanism. If an In about 25% of FNAIT cases, the platelet
umbilical cord DAT result is negative, ABO antibody develops during the first pregnancy
HDFN is unlikely even if the mother has the and that fetus is affected. The maternal anti-
corresponding ABO antibody(ies). After birth, body has been detected as early as 17 weeks’
hyperbilirubinemia can be successfully treat- gestation and the fetus may develop thrombo-
ed with phototherapy in most cases; in rare sit- cytopenia as early as 20 weeks’ gestation. How-
uations, exchange transfusions may be re- ever, the disease is often not discovered until
quired. birth, when the newborn presents with pete-
Group A and B infants of group O mothers chiae, ecchymoses, or intracranial hemor-
are more severely affected by ABO HDFN. In rhage. Intracranial hemorrhage occurs in 10%
populations of European or Asian ancestry, to 30% of infants and 50% of fetuses with
group A infants are most commonly affected; FNAIT.29 The greatest risk of hemorrhage oc-
in populations of African ancestry, group B in- curs when the fetal platelet count is less than
fants are most likely to be affected. The overall 50,000/µL. The response of the fetal hemato-
incidence of ABO HDFN is higher in people of poietic system to FNAIT is variable and may
African than European ancestry.8(p529) In pa- include compensatory extramedullary hema-
tients with severe disease, the DAT result is topoiesis. In rare cases, hydrops fetalis devel-
nearly always positive.28 ops. Fetal anemia without red cell incompati-
If ABO HDFN is ruled out, antibodies bility can also occur.
against low-prevalence red-cell antigens in- A history of giving birth to a newborn with
herited from the father should be suspected. thrombocytopenia can alert the obstetrician
Testing the eluate from cord blood or maternal to a potentially affected pregnancy. The preg-
serum (if ABO compatible) against the father’s nant woman and the father should be typed
red cells with an antiglobulin technique is of- for platelet antigens, and the woman should
ten diagnostic. be screened for the alloantibody. DNA testing
of the father can determine the zygosity of the
antigen involved.31
IMM U NE T HRO M B O C Y T OP E N I A The DNA genotype of the fetus can be de-
Maternal IgG antibodies to platelets can cross termined as early as 11 to 13 weeks’ gestation.
the placenta and cause severe thrombocyto- Assessment of the fetus should begin at or be-
penia. Two categories of immune thrombocy- fore 20 weeks’ gestation, when severe throm-
topenia are recognized: FNAIT and ITP. The bocytopenia and hemorrhage can occur.
diagnostic distinction between them is impor- When cordocentesis is used to determine the
tant for therapy selection. platelet count, irradiated, CMV-reduced-risk,
antigen-negative platelets should be used.
FNAIT If needed, platelet transfusion should be
given to the fetus to treat thrombocytopenia
FNAIT is caused by antibodies that are specific and avoid hemorrhage. Many blood suppliers
for platelet antigens inherited from the father have identified donors who are negative for
that are are absent in the mother. Platelet anti- human platelet alloantigen 1a, the most
gens represent specific polymorphisms in widely implicated platelet antibody, and can
platelet membrane glycoproteins. Approxi- prepare suitable platelets for IUT. The mother
mately 80% of FNAIT cases are caused by hu- is negative for the implicated alloantigen and
568 䡲 AABB TECHNICAL MANUAL
can be a donor if her platelets are washed. bocytopenia in a pregnant woman is common
When platelet transfusion is needed urgently and is rarely associated with ITP. However, ITP
and maternal platelets are unavailable, un- may cause thrombocytopenia in both the
selected platelets may be used.32 mother and fetus. Fortunately, only about 10%
The mother is given IVIG after the fetus is of newborns with ITP have platelet counts
determined to be affected. The dose is usually <50,000/µL, and only 1% to 2% have a high risk
1 g/kg each week. In a retrospective review, of hemorrhage.36
IVIG treatment alone appeared to be as safe A pregnant woman with thrombocytope-
and effective as cordocentesis with platelet nia or a preexisting diagnosis of ITP should be
transfusion.33 However, IVIG treatment failures tested for serum platelet autoantibody. A neg-
may occur and can necessitate fetal platelet ative result generally indicates that the throm-
count monitoring by cordocentesis and re- bocytopenia was caused by other conditions
quire platelet transfusions.34 The goal of both and suggests that the fetus or neonate is not at
transfusion and IVIG treatment is to avoid risk. In contrast, a pregnant woman with sig-
hemorrhage. Ultrasound monitoring of the fe- nificant thrombocytopenia and petechiae or
tus to detect hemorrhage is not recommend- other evidence of hemorrhage caused by auto-
ed because intracranial hemorrhage usually antibody should undergo similar treatment to
indicates permanent brain damage. Before that used in nonpregnant women with ITP.
vaginal delivery, the fetal platelet count should Prednisone at a dose of 1 to 2 mg/kg is
be >50,000/µL. usually effective. However, with persistent ma-
After birth, the infant’s platelet count may ternal thrombocytopenia, IVIG 1 g/kg/day for
decrease in the first few hours to days. In 2 to 3 2 to 5 days is indicated. Although the maternal
weeks, when the antibody has been cleared, platelet count is often monitored in these cas-
the infant’s platelet count returns to normal. es, it does not correlate with the newborn’s
The presence or absence of severe thrombocy- platelet count.36 Fetal blood sampling to deter-
topenia and intracranial hemorrhage in the mine platelet count is not usually recom-
firstborn correlates with the outcomes of sub- mended because the risk of morbidity and
sequent pregnancies.35
mortality from cordocentesis is greater than or
equal to the risk of severe bleeding in utero or
ITP
at delivery. However, if fetal sampling is per-
Autoantibodies against platelets, which are re- formed, a platelet transfusion should be ad-
active with the mother’s own platelets as well ministered at the same time. Platelet transfu-
as donor or fetal platelets, are another cause of sions may be needed in about 15% of
fetal and neonatal thrombocytopenia. Throm- newborns.36
KE Y POI N T S
1. HDFN is caused by maternal antibodies that are specific to a paternal red cell antigen. The
maternal IgG antibody is transported across the placenta, where it destroys the fetal red cells.
2. Some antibodies, such as anti-I, -P1, -Lea and -Leb, can be ignored. The most common clini-
cally significant antibodies are anti-D, -K, and -c.
3. Molecular typing of fetal DNA can be performed on maternal plasma early in the second tri-
mester.
4. The recommended titer method is saline AHG with 60-minute incubation at 37 C. Other
methods, such as those using albumin AHG or gel, may result in higher titers that may lead
to misinterpretation by the obstetrician.
5. For IUT, the blood should be irradiated, CMV reduced-risk, hemoglobin S negative, group O
(in most cases), and less than 7 days old.
CHAPTER 22 Perinatal Issues in Transfusion Practice 䡲 569
6. The rosette test is a sensitive method for detecting fetomaternal hemorrhage of ap-
proximately 10 mL or more. Flow cytometry can precisely measure hemoglobin F and/
or D-positive red cells.
7. The calculated RhIG dose should be rounded up if the number to the right of the decimal
point is 0.5 or rounded down if the number is <0.5. In either case, a vial should be added to
the result.
8. Despite the prevalence of ABO HDFN, severe anemia rarely occurs. After birth, hyperbiliru-
binemia can usually be treated with phototherapy alone.
9. In fetal/neonatal alloimmune thrombocytopenia, the platelet antibody may develop at
around 17 weeks of gestation in the first pregnancy and fetal thrombocytopenia may devel-
op as early as 20 weeks. Irradiated, CMV-reduced-risk, antigen-negative platelets should be
given to treat thrombocytopenia and avoid hemorrhage.
R E F E R EN C E S
1. Bowman JM, Pollock JM, Penston LE. Fetoma- globulin in humans. Int Immunol 2001;13:993-
ternal transplacental hemorrhage during 1002.
pregnancy and after delivery. Vox Sang 1986; 11. Dennery PA, Seidman DS, Stevenson DK. Neo-
51:117-21. natal hyperbilirubinemia. N Engl J Med 2001;
2. Sebring ES, Polesky HF. Fetomaternal hemor- 344:581-90.
rhage: Incidence, risk factors, time of occur- 12. Moise KJ Jr. Management of rhesus alloimmu-
rence, and clinical effects. Transfusion 1990; nization in pregnancy. Obstet Gynecol 2008;
30:344-57. 112:164-76.
3. Bowman JM. The prevention of Rh immuniza- 13. Kanra T, Erdem G, Tekinalp G, et al. Further
tion. Transfus Med Rev 1988;2:129-50. hemolytic disease of the newborn caused by
4. Bowman JM. Controversies in Rh prophylaxis: anti-M. Am J Hematol 1996;53:280-1.
Who needs Rh immune globulin and when 14. Wagner FF, Flegel WA. RHD deletion occurred
should it be given? Am J Obstet Gynecol 1985; in the Rhesus box. Blood 2000;95:3662-8.
151:289-94. 15. Akolekar K, Finning K, Kuppusamy R, et al, Fe-
5. Klein HG, Anstee DJ. The Rh blood group sys- tal RHD genotyping in maternal plasma at 11-
13 weeks of gestation. Fetal Diagn Ther 2011;
tem. In: Mollison’s blood transfusion in clinical
29:301-6.
medicine. 12th ed. Oxford: Wiley-Blackwell,
16. Hilden JO, Backleman K, Nilsson J, Ernerudh J.
2014:167-213.
Flow-cytometric quantitation of anti-D anti-
6. van der Schoot CE, Martine Tax GH, et al. Pre-
bodies. Vox Sang 1997;72:172-6.
natal typing of Rh and Kell blood group system
17. Schonewille H, Klumper FJCM, Watering
antigens: The edge of a watershed. Transfus LMG, et al. High additional maternal red cell
Med Rev 2003;17:31-44. alloimmunization after Rhesus- and K-
7. Vaughan JI, Manning M, Warwick RM, et al. In- matched intrauterine intravascular transfu-
hibition of erythroid progenitor cells by anti- sions for hemolytic disease of the fetus. Am J
Kell antibodies in fetal alloimmune anemia. N Obstet Gynecol 2007;196:143.e1-6.
Engl J Med 1998;338:798-803. 18. Rodunovic N, Lockwood CJ, Alvarez M, et al.
8. Klein HG, Anstee DJ. Haemolytic disease of the The severely anemic and hydropic isoimmune
fetus and newborn. In: Mollison’s blood trans- fetus: Changes in hematocrit associated with
fusion in clinical medicine. 12th ed. Oxford: intrauterine death. Obstet Gynecol 1992;79:
Wiley-Blackwell, 2014:499-548. 390-3.
9. Pollock JM, Bowman JM. Anti-Rh(D) subclass- 19. Schwartz J, Winters JL, Padmanabhan A, et al.
es and severity of Rh hemolytic disease of the Guidelines on the use of therapeutic apheresis
newborn. Vox Sang 1990;59:176-9. in clinical practice—Evidence-based ap-
10. Firan M, Rawdon R, Radu C, et al. The MHC proach from the Writing Committee of the
class I-related receptor, FcRn, plays an essen- American Society for Apheresis. The Sixth Spe-
tial role in the maternal transfer of gamma- cial Issue. J Clin Apher 2013;28:145-284.
570 䡲 AABB TECHNICAL MANUAL
20. Margulies M, Voto LS, Mathet E. High dose in- globulin test-negative neonates. Pediatrics
travenous IgG for the treatment of severe Rhe- 2002;110:127-30.
sus alloimmunization. Vox Sang 1991;61:181- 29. Davoren A, Curtis BR, Aster RH, McFarland JG.
9. Human platelet antigen-specific alloantibod-
21. American Academy of Pediatrics Subcommit- ies implicated in 1162 cases of neonatal allo-
tee on Hyperbilirubinemia. Management of immune thrombocytopenia. Transfusion
hyperbilirubinemia in the newborn 35 or more 2004;44:1220-5.
weeks gestation. Pediatrics 2004;114:297-316. 30. Williamson LM, Hackett G, Rennie J, et al. The
22. Domen RE. Policies and procedures related to natural history of fetomaternal alloimmuniza-
weak D phenotype testing and Rh immune tion in the platelet-specific antigen HPA-1a
globulin administration. Arch Pathol Lab Med (PlA1, Zwa) as determined by antenatal screen-
2000;124:1118-21. ing. Blood 1998;92:2280-7.
23. Flegel, WA. How I manage patients and donors 31. Radder CM, Brand A, Kanhai HH. A less inva-
with weak D phenotype. Curr Opin Hematol sive treatment strategy to prevent intracranial
2006;13:476-83. hemorrhage in fetal and neonatal alloimmune
24. Prevention of Rh D alloimmunization. ACOG thrombocytopenia. Am J Obstet Gynecol 2001;
practice bulletin #4. Washington, DC: Ameri- 185:683-8.
can College of Obstetricians and Gynecolo- 32. Kiefel V, Bassler D, Kroll H, et al. Antigen-posi-
gists, 1999. tive platelet transfusion in neonatal alloim-
25. Radel DJ, Penz CS, Dietz AB, Gastineau DA. A mune thrombocytopenia (NAIT). Blood 2006;
combined flow cytometry-based method for 107:3761-3.
fetomaternal hemorrhage and maternal D. 33. Van den Akker ESA, Oepkes D, Lopriore E, et al.
Transfusion 2008;48:1886-91. Noninvasive antenatal management of fetal
26. Sandler SG, Delaney M, Gottschall JL, College and neonatal alloimmune thrombocytopenia:
of American Pathologists Transfusion Medi- Safe and effective. Br J Obstet Gynaecol 2007;
cine Resource Committee. Proficiency tests re- 114:469-73.
veal the need to improve laboratory assays for 34. Silver RM, Porter TF, Branch DW, et al. Neona-
fetomaternal hemorrhage for Rh immunopro- tal alloimmune thrombocytopenia: Antenatal
phylaxis. Transfusion 2013;53:2098-102. management. Am J Obstet Gynecol 2000;182:
27. Branch DR, Shabani F, Lund N, Denomme GA. 1233-8.
Antenatal administration of Rh-immune glob- 35. Birchall JE, Murphy MF, Kroll H. European col-
ulin causes significant increases in the immu- laborative study of the antenatal management
nomodulary cytokines transforming growth of feto-maternal alloimmune thrombocytope-
factor- and prostaglandin E2. Transfusion nia. Br J Haematol 2003;122:275-88.
2006;48:1316-22. 36. Webert KE, Mittal R, Sigouin C, et al. A retro-
28. Herschel M, Karrison T, Wen M, et al. Isoim- spective 11-year analysis of patients with idio-
munization is unlikely to be the cause of he- pathic thrombocytopenic purpura. Blood
molysis in ABO-incompatible but direct anti- 2003;102:4306-11.
C h a p t e r 2 3
TR A N S F U S I O N P R A C T I C E I N n e o - T R A N SF U SI O N I N IN FAN TS
natal and pediatric patients differs from YO U N G E R TH A N 4 M ON TH S
that in adults.1 The differences are related to
physiologic changes occurring during the Considerations for Component
transition from fetus to adolescent. Blood Preparation and Therapy
volume, hematologic values, immune system The fact that patients younger than 4 months
maturity, and physiologic responses to hypo- have small blood/plasma volumes and imma-
volemia and hypoxia are variable in this het- ture organ system functions necessitates spe-
erogeneous population, contributing to the cial approaches to component therapy. This is
complexity and intricacies of pediatric trans- especially important for very-low-birthweight
fusion practice. (VLBW) infants (<1500 g) and extremely low-
This chapter discusses neonatal and pedi- birthweight infants (<1000 g).
atric transfusion practice during two distinct
periods: infancy from birth to 4 months, and Fetal and Neonatal Physiology Affecting
infancy after 4 months and childhood. The pe-
Transfusion Practice
diatric practices addressed include 1) small-
volume component preparation, 2) transfu- Healthy full-term neonates have a mean cord
sion indications for blood components, 3) blood hemoglobin level of 16.9 ± 1.6 g/dL,
transfusion administration and exchange whereas that of preterm neonates is 15.9 ±
transfusion, 4) transfusion support in specific 2.4 g/dL. The hemoglobin concentration nor-
diseases, 5) rationale for special processing of mally declines during the first few weeks of
blood components, and 6) pediatric massive life, resulting in the physiologic anemia of in-
transfusion protocols (MTPs). fancy in newborns and physiologic anemia of
Cassandra D. Josephson, MD, Director of Transfusion, Tissue, and Apheresis Services, Children’s Healthcare of
Atlanta, and Associate Professor, Pathology and Pediatrics, Emory University School of Medicine, and Erin
Meyer, DO, MPH, Associate Director of Transfusion, Tissue, and Apheresis Services, Children’s Healthcare of
Atlanta, and Assistant Professor of Pathology and Laboratory Medicine, Emory University School of Medicine,
Atlanta, Georgia
C. Josephson has disclosed financial relationships with Immucor and Octapharma. E. Meyer has disclosed no
conflicts of interest.
571
572 䡲 AABB TECHNICAL MANUAL
prematurity in preterm infants.2 Both anemias nates receive multiple transfusions, they have
are considered self-limited and are usually tol- proportionately lower levels of circulating fetal
erated without harmful effects. hemoglobin and an increase in adult hemo-
The rate of decline in hemoglobin levels is globin levels.
a function of gestational age at birth. At 4 to 8
weeks after birth, hemoglobin decreases to as Erythropoietic Response
low as 8.0 g/dL in preterm infants weighing
The EPO response in newborns differs from
1000 to 1500 g and 7.0 g/dL in neonates weigh-
that in adults and older children. In the latter
ing less than 1000 g at birth.3 The physiologic
groups, oxygen sensors in the kidney recog-
decrease in hemoglobin concentration is due
nize decreases in oxygen delivery, resulting in
to several factors: 1) a decrease in erythropoie-
the release of EPO into the circulation. In con-
tin (EPO) resulting in diminished red cell pro-
trast, in the fetus, this sensor is located in the
duction, 2) a decrease in survival of fetal red
liver and is less sensitive, resulting in reduced
cells, and 3) an increasing blood volume due to
EPO production in the face of hypoxia (hypo-
rapid growth. Reduced EPO production re-
responsiveness). This response likely occurs to
sults from increased oxygen delivery to tissues
prevent polycythemia of the fetus in the hy-
because of increased pulmonary blood flow, poxic intrauterine environment.
elevated arterial pO2 levels, and increased red Although EPO production eventually
cell 2,3-diphosphoglycerate (2,3 DPG) and he- shifts from the liver to the kidney, most prema-
moglobin A levels. ture infants produce the smallest amount of
EPO for any degree of anemia.6 As an alterna-
Clinical Considerations Related to tive to transfusion, the use of recombinant hu-
Infant Size and Blood Volume man EPO has been shown to reduce donor ex-
Blood volumes of pediatric patients vary with posures in premature infants and minimize
body weight. A full-term newborn has a blood the severity of anemia.7 As compliance with
volume of approximately 85 mL/kg compared strict transfusion threshold criteria has in-
to 100 mL/kg in a preterm newborn. Due to creased, clinicians have decreased their phle-
the small total blood volumes of preterm in- botomy rates in VLBW infants, reducing the
fants (100 mL or less), blood banks must be ca- rates of iatrogenically induced anemia and
pable of providing appropriately sized blood numbers of transfusions. Thus, in most cases,
components. this approach achieves the same goal as EPO
Many factors, including iatrogenic blood therapy (ie, decreases the use of transfusions
loss from repeated phlebotomy, lead to fre- and the number of donor exposures) without
expensive pharmacotherapy and its risk of ad-
quent transfusions. Hypovolemia is not toler-
verse effects.
ated well in newborns because their left ven-
tricular stroke volume decreases without an
increase in heart rate when >10% of their
Cold Stress
blood volume is lost. Thus, newborns must Hypothermia in the neonate can trigger or ex-
physiologically increase their peripheral vas- aggerate a number of responses, including 1)
cular resistance with decreasing cardiac out- an increase in metabolic rate; 2) hypoglyce-
put to maintain systemic blood pressure. Ulti- mia; 3) metabolic acidosis; and 4) potential
mately, poor tissue perfusion and oxygenation apneic events that may lead to hypoxia, hypo-
occur, resulting in metabolic acidosis.4 tension, and cardiac arrest. In-line blood
Although transfusion may be required, it warmers are required for all Red Blood Cell
is neither necessary nor acceptable to replace (RBC) exchange transfusions to combat the ef-
the amount of blood lost mL for mL; rather, fects of hypothermia. A radiant heater should
transfusions can be administered to maintain never be used to warm the blood being trans-
a target hemoglobin level in certain clinical sit- fused due to the risk of hemolysis. Further-
uations.5 When ill preterm and full-term neo- more, to prevent hemolysis in neonates under-
CHAPTER 23 Neonatal and Pediatric Transfusion Practice 䡲 573
TABLE 23-1. Transfusion Guidelines for RBCs in Infants Younger than 4 Months23,26
1. Hematocrit <20% with low reticulocyte count and symptomatic anemia (tachycardia, tachypnea, poor
feeding).
d. With significant tachycardia or tachypnea (heart rate >180 beats/minute for 24 hours, respiratory
rate >80 beats/minute for 24 hours).
e. With significant apnea or bradycardia (>6 episodes in 12 hours or 2 episodes in 24 hours requir-
ing bag and mask ventilation while receiving therapeutic doses of methylxanthines).
f. With low weight gain (<10 g/day observed over 4 days while receiving 100 kcal/kg/day).
infants younger than 4 months because of factant therapy, nitric oxide therapy, high-
their immature immunologic status. frequency ventilators, and compliance with
Multiple observational studies have transfusion practice guidelines. These advanc-
shown that alloimmunization to red cell anti- es have substantially decreased the number of
gens is rare during the neonatal period.9,29,30 RBC transfusions administered in this popula-
For this reason, repeated typing and screening, tion. Most neonatal transfusions are now given
which are required for adults and children old- to VLBW infants.32
er than 4 months, is unnecessary in younger
infants and contributes to significant iatrogen- Aliquoting for Small-Volume
ic blood loss. Also, the transfusion service Transfusion
should avoid transfusing any components that
The purpose of creating small-volume aliquots
may passively transfer unexpected alloanti-
is to limit donor exposures, prevent circulatory
bodies or ABO-incompatible antibodies to re-
overload, and potentially decrease donor-
cipients.31
related risks.33-37 Several technical approaches
can be used to accomplish these goals and
Components for Neonatal Transfusion
minimize blood wastage.38
Advances in neonatology now permit the sur- Small-volume RBC transfusion aliquots
vival of extremely premature infants with sur- are commonly made with a multiple-pack
576 䡲 AABB TECHNICAL MANUAL
system.38,39 Quad packs, employed mostly by from a single unit.38 However, blood compo-
blood centers, are produced from a single unit nents may still be wasted when used in ali-
of whole blood that is diverted into a primary quots that are larger than the dose selected for
bag with three integrally attached smaller each patient based on body weight.
bags. The plasma is then separated and divert- Hospital transfusion services that have a
ed into one bag during component prepara- sterile connection device have multiple addi-
tion. The remaining red cells are drawn into tional options to produce aliquots, such as
the smaller bags as needed for transfusion. transfer packs [eg, PEDI-PAK system (Genesis
Each of the smaller units has the same expira- BPS, Hackensack, NJ) Fig 23-2], small-volume
tion date as the original unit because the sys- bags, or tubing with integrally attached syring-
tem’s original seal has remained intact and a es.38 Syringe sets (Fig 23-3) offer the greatest
“closed system” is maintained. accuracy for obtaining the desired volume to
A hospital transfusion service can then re- be transfused based on volume-per-weight
move (either by heat sealer or metal clips) each calculations.38 Some syringe sets have an at-
aliquot as needed. For hospital transfusion tached 150-micron in-line filter for use during
services without a sterile connection device the aliquoting process so that, when issued by
the blood bank, the cells are ready to be placed
(Fig 23-1), this method provides three aliquots
on a syringe pump without further manipula-
tion of the component at the bedside. This
process eliminates the need for the nurse to
transfer blood from the pack to a syringe at the
bedside for delivery by a syringe pump. Re-
moving this additional step reduces the risk of
contamination, mislabeling, or damage to the
unit that results in blood loss or spillage.38
Reducing donor exposures is more readily
accomplished by this technique, which en-
ables a recipient to receive multiple small-vol-
ume transfusions from a single unit until it
reaches its expiration date.40,41 Many hospital
transfusion services assign a single unit of
RBCs to one or more infants based on their
weight.34-40
Once an aliquot is produced at either the
blood center or hospital blood bank, it must be
labeled with the expiration date and the origin
and disposition of each smaller unit must be
recorded. Aliquot expiration dates vary from
institution to institution, and local standard
operating procedures should always be fol-
lowed.
FIGURE 23-2. Diagram of PEDI-PAK (reproduced with permission of Genesis BPS, Hackensack, NJ).
nine and mannitol in AS and its relation to re- extended-storage media present no major risk
nal toxicity. Moreover, mannitol is a potent when used for small-volume transfusions.43
diuretic with effects on fluid dynamics that However, for infants with renal or hepatic
can result in fluctuations in the cerebral blood insufficiency, it is recommended that AS solu-
flow of preterm infants. tion be removed from RBC units, particularly if
Most evidence suggests that small-vol- multiple transfusions from the same unit are
ume transfusions (5 to 15 mL/kg) containing expected. The safety of AS-preserved RBCs in
AS are safe for this patient population. Specifi- trauma-related massive transfusions, cardiac
cally, when AS-1 and AS-3 were compared, no surgery, or exchange transfusions is unknown
harmful effects were observed in neonates re- in this population. Therefore, AS-preserved
ceiving small-volume, simple transfusions.40-42 RBC units should be used with caution in
These transfusions were as effective as CPDA-1 these settings.21,42-45
RBCs in increasing hemoglobin levels in recip-
RBC Age
ients. Luban and colleagues used theoretical
calculations in a variety of clinical settings to The age of RBC units and its impact on patient
demonstrate that red cells preserved in outcomes has become a concern, although
FIGURE 23-3. Syringe with filter (reproduced with permission from Charter Medical, Ltd, Winston-Salem, NC).
578 䡲 AABB TECHNICAL MANUAL
clinical confirmation of the basis for this con- Specific Indications for RBCs
cern is controversial. A randomized controlled
In neonates, symptomatic anemia is the major
trial conducted in Canada, Age of Red Blood
indication for simple transfusion. Specifically,
Cells in Premature Infants (ARIPI), randomly a venous hemoglobin of <13 g/dL in the first 24
assigned low birthweight infants to be trans- hours of life necessitates clinical consideration
fused with RBCs that were 7 days old or less of an RBC transfusion.48 RBC transfusions are
(mean = 5.1 days, n = 188) or with standard- also considered when approximately 10% of a
issue RBCs divided into aliquots and stored for sick neonate’s blood volume has been re-
2 to 42 days (mean = 14.6 days, n = 189).46 The moved or lost. When 10 mL/kg of RBCs with a
primary composite endpoints included necro- hematocrit of >80% are transfused, the expect-
tizing enterocolitis (NEC), intraventricular ed increase in hemoglobin concentration in a
hemorrhage (IVH), and bronchopulmonary neonate is approximately 3 g/dL. A similar vol-
dysplasia. The ARIPI trial found no differences ume of RBCs with AS usually has a hematocrit
in the primary endpoints between infants in of 65%, and its transfusion results in a project-
the two arms, suggesting that in the study pop- ed posttransfusion hemoglobin increase of
ulation, the age of RBCs does not affect these <3 g/dL (see Table 23-2 for blood component
dosing recommendations and expected re-
common morbidities of prematurity.
sults).49
ARIPI’s external validity has been ques-
Two randomized controlled trials, the
tioned because of the study's liberal transfu-
Premature Infants in Need of Transfusion
sion strategy, use of SAG-M units, and average
(PINT) and its follow up study [PINT Follow-
duration of blood storage. These practices do up Outcomes Study (PINTOS)] as well as a
not reflect the transfusion practices or storage University of Iowa study compared the out-
solution and ages of RBCs used in many cen- comes of restrictive (Hb = 7 g/dL) vs liberal
ters in the United States.47 Thus, it has not RBC transfusion triggers (Hb = 10 g/dL) in
been firmly established that there is a causal VLBW infants.50-52 The Iowa trial revealed a
relationship between morbidities and transfu- lower rate of transfusion events (3.3 vs 5.2;
sion of older RBC units in premature infants.47 p=0.025) with the restrictive strategy com-
TABLE 23-2. Blood Components and Dosing of Small Volumes in Neonatal and Pediatric Patients49
pared to the liberal strategy.52 However, rates of residual bilirubin. In addition, in antibody-
periventricular leukomalacia and death were mediated hemolytic processes, exchange
higher in the restrictive arm. The PINT study therapy removes both free antibody and anti-
found no significant difference between the two body-coated red cells, replacing them with
arms, which had the same thresholds as the antigen-negative red cells.
Iowa study, in the composite endpoint of death Exchange transfusion needs to be per-
or any of bronchopulmonary dysplasia, reti- formed before the development of kernicterus.
nopathy of prematurity (Stage >3), or brain in- In full-term infants, kernicterus rarely develops
jury (periventricular leukomalacia, intracranial at bilirubin levels less than 25 mg/dL. However,
hemorrhage Grade 4, or ventriculomegaly).50,52 in ill VLBW infants, kernicterus can occur at bil-
The PINTOS study revealed that at 18 to 24 irubin levels as low as 8 to 12 mg/dL.54
months after birth, infants in the PINT study’s A double-volume exchange transfusion
restrictive arm had more neurodevelopmental (two 85-mL/kg transfusions for full-term in-
impairments than those in the liberal arm.50,51 fants and two 100-mL/kg transfusions for
In summary, these studies indicated that VLBW infants) removes approximately 70% to
maintenance of higher hemoglobin levels in 90% of the circulating red cells and approxi-
low birthweight infants may provide long- mately 50% of the total bilirubin.55 However,
term neurologic protection.50-52 Therefore, after the first exchange transfusion, bilirubin
whether a restrictive or liberal RBC transfusion levels may rise again because of a re-equilibra-
strategy should be adopted in this population tion of the extravascular tissue and plasma bil-
is not clear and requires further prospective irubin, which may necessitate another ex-
randomized controlled trial data. The Transfu- change transfusion.56
sion of Premature Infants Study is currently Occasionally, exchange transfusion is
being conducted in the United States.53 used to eliminate toxins, drugs, or chemicals
administered to the mother near the time of
Exchange Transfusion for delivery. Exchange transfusion is also used
Hyperbilirubinemia when toxic doses have been administered to
the infant or accumulate at high levels in the
Exchange transfusion in neonates involves re- infant as a result of prematurity and/or an in-
placement of one or two whole-blood vol- born error of metabolism.57,58
umes. The primary purpose of this therapy is
to treat excessively high levels of unconjugated Exchange Transfusion
bilirubin (hyperbilirubinemia). In high con-
centrations, bilirubin may cross the blood- COMPONENT CHOICE AND PHYSIOLOGIC
brain barrier; concentrate in the basal ganglia EFFECTS. Typically, RBCs are resuspended in
and cerebellum of preterm and full-term in- ABO-compatible thawed Fresh Frozen Plasma
fants; and cause irreversible damage, known (FFP) for an exchange transfusion. No single
as “kernicterus,” to the central nervous sys- method of combining components has been
tem. Preterm and full-term infants are suscep- shown to be superior to another. Most often,
tible to hyperbilirubinemia because their im- RBCs <5 to 7 days old and stored in CPDA-1
mature liver conjugates bilirubin poorly and are used to avoid high levels of potassium and
their incompletely developed blood-brain bar- to maximize red cell survival.59 When using AS-
rier allows bilirubin transit. Phototherapy (use RBC units, some blood banks elect to remove
of fluorescent ultraviolet lights) is the current the additive-containing plasma to reduce the
treatment of choice for hyperbilirubinemia; volume transfused.
exchange transfusion is reserved for patients Most transfusion services provide RBC
who fail phototherapy. units that are hemoglobin S negative, cyto-
Two critical objectives of exchange trans- megalovirus (CMV) reduced-risk (leukocyte
fusions are the removal of unconjugated bili- reduced and/or CMV seronegative), and irra-
rubin and maximization of albumin binding of diated. Irradiation should be performed just
580 䡲 AABB TECHNICAL MANUAL
before the exchange to prevent potentiation of container. A standard filter and in-line blood
the potassium storage lesion. Some experts warmer are recommended.
recommend washing or removing the super- With both techniques, the absolute maxi-
natant of red cells that have been irradiated to mum volume of each withdrawal and infusion
avoid the complications of hyperkalemic car- depend on the infant’s body weight and hemo-
diac arrhythmias.60 dynamic status. Usually, no more than 5 mL/
The glucose load administered during ex- kg body weight or 5% of the infant’s blood vol-
change transfusion can be high in some cases, ume is removed and replaced during a 3- to 5-
which stimulates the infant’s pancreas to re- minute cycle.59 The exchange transfusion
lease insulin and results in rebound hypogly- should not be performed rapidly because sud-
cemia. Therefore, infant plasma glucose levels den hemodynamic changes may affect cere-
should be monitored during the first few hours bral blood flow and shift intracranial pressure,
following exchange transfusion. contributing to IVH.61 A total double-volume
exchange transfusion typically takes 90 to 120
VOLUME A ND HE MATOCRIT CONSIDER-
ATIONS. A double-volume exchange in neo- minutes.59
nates rarely necessitates the infusion of more
than 1 RBC unit. The unit’s hematocrit should Platelet Transfusion Support
be approximately 45% to 60%, and the unit Mild-to-moderate thrombocytopenia (plate-
should have sufficient plasma (based on esti- let count <150,000/µL) is the most common
mated blood volume) to provide clotting fac- hemostatic abnormality in ill preterm and full-
tors.60 If the neonate’s condition requires a term infants, and it affects approximately 20%
higher postexchange transfusion hematocrit, a of infants in neonatal intensive care units.62
small-volume RBC transfusion may be given The causes of thrombocytopenia include im-
or a unit with a higher hematocrit can be used paired platelet production, increased platelet
for the initial exchange transfusion. The re- destruction, abnormal platelet distribution,
constituted blood should be well mixed to sus-
and/or platelet dilution secondary to massive
tain the intended hematocrit throughout the
transfusion. The most common cause is in-
exchange. The infant’s hematocrit and biliru-
creased destruction of platelets that is general-
bin can be measured by removing the last ali-
ly associated with a variety of self-limited con-
quot of the exchange unit.
ditions.
VA SCUL AR ACCESS. Umbilical venous cath-
eters are used for exchange transfusions in Indications
preterm and full-term infants just after birth. If Most platelet transfusions in preterm and full-
umbilical venous catheters are not available,
term infants are performed to treat platelet
small saphenous catheters may be used.
counts less than 50,000/µL in the presence of
TECHNIQUES. Two exchange-transfusion active bleeding.63
techniques are commonly employed: isovolu- Prophylactic platelet transfusions in this
metric and manual push-pull. In isovolumet- population are controversial (see Table 23-3
ric exchange transfusion, two catheters of for transfusion indications and thresholds).27,64
identical size provide vascular access. The Unlike adult patients who rarely have severe
catheters allow simultaneous withdrawal and bleeding complications until platelet counts
infusion of blood and are regulated by a single decline to less than 10,000/µL, preterm infants
peristaltic pump. The umbilical vein is typical- with other complicating illnesses may bleed at
ly used for infusion, and the umbilical artery is higher platelet counts. This increased risk may
used for withdrawal. The manual push-pull be attributable to 1) lower concentrations of
technique uses a single vascular access portal plasma coagulation factors, 2) circulation of
with a three-way stopcock attached to the unit an anticoagulant that potentiates thrombin
of blood, the patient, and a graduated discard inhibition, 3) intrinsic or extrinsic platelet
CHAPTER 23 Neonatal and Pediatric Transfusion Practice 䡲 581
TABLE 23-3. Transfusion Guidelines for Platelets in Neonates and Older Children23,26
With Thrombocytopenia
1. Platelet count 5,000 to 10,000/μL with failure of platelet production.
2. Platelet count <30,000/μL in neonate with failure of platelet production.
3. Platelet count <50,000/μL in stable premature infant:
a. With active bleeding, or
b. Before an invasive procedure, with failure of platelet production.
4. Platelet count <100,000/μL in sick premature infant:
a. With active bleeding, or
b. Before an invasive procedure in patient with DIC.
Without Thrombocytopenia
1. Active bleeding in association with qualitative platelet defect.
2. Unexplained excessive bleeding in a patient undergoing cardiopulmonary bypass.
3. Patient undergoing ECMO with:
a. A platelet count of <100,000/μL, or
b. Higher platelet counts and bleeding.
DIC = disseminated intravascular coagulation; ECMO = extracorporeal membrane oxygenation.
because it is unnecessary and harmful to the lants (proteins C and S) and the non-vitamin-
platelets.62,63 K-dependent antithrombin protein are at low
In addition, when platelets are stored in a levels at birth. In spite of these issues, the pro-
syringe, the pH has been shown to decrease coagulant and anticoagulant systems are usu-
rapidly, a potential problem for an already ill ally in balance in healthy newborns, so spon-
and acidotic recipient.68-70 Therefore, when taneous bleeding and thrombosis are rare (see
volume reduction in an open system is used Table 23-4).72 However, the reserve capacity for
and the product is placed in a syringe, the pro- both systems is limited. Therefore, serious
cessing should be done as close to the time of bleeding may occur in sick premature infants
issuance as possible and the product should during the first week of life. Cryoprecipitate
be infused within 4 hours of processing. and FFP can be transfused to treat bleeding or
clotting complications, such as disseminated
Plasma Transfusion Support to intravascular coagulation (DIC).73,74
Enhance Hemostasis
FFP
Infants must synthesize their own coagulation
factors because significant amounts are not FFP is frequently used to replace coagulation
transplacentally transferred from the mother. factors in preterm and full-term infants, par-
Furthermore, infants are unable to produce ticularly if multiple factor deficiencies are
normal levels of these proteins in the early present, such as hemorrhagic disease of the
postnatal period. newborn or vitamin K deficiency (Table 23-5).
Physiologically, low levels of vitamin K- The usual dose of FFP is 10 to 15 mL/kg, which
dependent factors (Factors II, VII, IX, and X) is expected to increase all factor activity levels
and contact factors (Factor XI, Factor XII, by 15% to 20% unless there is a marked con-
prekallikrein, and high-molecular-weight ki- sumptive coagulopathy.64,68
ninogen) contribute to altered coagulation test To limit donor exposure for each recipient
results (see Table 23-4).71,72 Also, the naturally while minimizing plasma wastage, blood can
occurring vitamin K-dependent anticoagu- be collected into a system with multiple, inte-
TABLE 23-4. Screening Laboratory Tests for Hemostasis: Neonates vs Adults (reproduced with
permission)71
TABLE 23-5. Transfusion Guidelines for Plasma Products in Neonates and Older Children23,26
grally attached bags that create ready-to- creased or dysfunctional fibrinogen (congeni-
freeze aliquots.26 Once thawed, the aliquots tal or acquired) or Factor XIII deficiency. Cryo-
can be subdivided further for several patients precipitate is usually given in conjunction with
if they can be used within a 24-hour period. platelets and FFP to treat DIC in newborns.
FFP for infants must be ABO compatible Typically, 1 unit is sufficient to achieve hemo-
and free of clinically significant and unexpect- static levels in an infant.
ed antibodies. Transfused antibodies can ABO-compatible cryoprecipitate is pre-
reach high concentrations in infants and chil- ferred because transfusion of a large volume of
dren with very small plasma volumes. A com- ABO-incompatible cryoprecipitate may result
mon practice at some institutions is to use in a positive DAT result and, in very rare cases,
group AB FFP because a single unit can pro- a mild hemolysis.75,76
vide multiple small-volume doses for several Cryoprecipitate transfusion is not recom-
neonates. mended for patients with Factor VIII deficien-
cy because the standard therapy is to treat this
Cryoprecipitated Antihemophilic Factor
condition with recombinant Factor VIII prod-
Cryoprecipitate transfusions are primarily ucts or virus-inactivated, monoclonal-anti-
used to treat conditions resulting from de- body-purified, plasma-derived products.73,77
584 䡲 AABB TECHNICAL MANUAL
Furthermore, cryoprecipitate should be used at risk of TA-GVHD, and the components must
only to treat von Willebrand disease if plasma- be obtained from CMV-seronegative donors to
derived, virus-inactivated concentrates con- prevent virus transmission if the recipient is
taining von Willebrand factor are not available. CMV seronegative.28(p40) Granulocytes must
See Table 23-5 for other guidelines regarding also be ABO compatible with the red cells of
cryoprecipitate use.64 the recipient infant because of the significant
red cell content in these components.28(p37)
Granulocyte Transfusion Support Many institutions also provide D-compatible
Indications components to decrease Rh alloimmuniza-
tion.
The role of granulocyte transfusion for sepsis Intravenous immune globulin (IVIG) in
in neonates is unclear, and this treatment is the treatment of early neonatal sepsis has also
rarely used. It is important to establish the fol- been studied, but there is a lack of agreement
lowing factors before the transfusion of granu- on its routine application to this patient popu-
locytes: 1) strong evidence of bacterial or fun- lation.78,80-85
gal septicemia; 2) absolute neutrophil count
less than 500/µL, chronic granulomatous dis- Transfusion Administration
ease, or leukocyte adhesion deficiency; and 3)
diminishing storage pool (such that 7% of nu- Vascular Access
cleated cells in the marrow are granulocytes Vascular access is the most difficult aspect of
that are metamyelocytes or more ma- transfusion administration in patients young-
ture).15,78,79 (See Table 23-6 for guidelines on er than 4 months, particularly in preterm in-
granulocyte transfusion support.) fants who require long-term or continuous in-
travenous infusions. The umbilical vein is
Components and Dose most frequently cannulated after birth to ad-
Granulocyte concentrates are produced by minister fluids and transfusions and to moni-
standard apheresis techniques or by pooling tor central venous pressure.86 Vascular cathe-
buffy coats from whole blood. A typical dose ters (24-gauge) and small needles (25-gauge)
for infants is 10 to 15 mL/kg body weight, generally can be safely used for RBC transfu-
which is approximately 1 × 109 to 2 × 109 poly- sions without causing hemolysis if constant
morphonuclear cells/kg.15,78 Treatment should flow rates are applied. The outcomes of trans-
be administered daily until an adequate neu- fusions using smaller-gauge needles and cath-
trophil count is achieved and/or the patient eters have not been evaluated.
shows clinical improvement.
According to AABB Standards, these com- Pumps and Warming
ponents must be irradiated when the patient is
When administered slowly, small-volume
transfusions typically do not require a blood
warmer; however, control of the rate and vol-
TABLE 23-6. Transfusion Guidelines for ume transfused is important. Electromechani-
Granulocytes in Neonates and Older cal syringe delivery pumps administer the
Children23,26 blood at a constant rate and are able to provide
1. Neonates or children with neutropenia or adequate control. These pumps cause mini-
granulocyte dysfunction with bacterial sepsis mal hemolysis and may even be used with in-
and lack of responsiveness to standard ther- line leukocyte-reduction filters.87,88 Although
apy. there are several devices that can be used to
transfuse blood components, it is important to
2. Neutropenic neonates or children with fungal
test and validate the mechanical system cho-
disease not responsive to standard therapy.
sen for blood component administration.
CHAPTER 23 Neonatal and Pediatric Transfusion Practice 䡲 585
Filters and Transfusion Sets above 65%, viscosity increases and oxygen
transport decreases. However, in neonates, the
All blood component transfusions require a
exponential rise in viscosity can occur at a he-
standard filter (150 to 260 microns), even if the
matocrit as low as 40%.90 Congestive heart fail-
components have undergone leukocyte reduc-
ure can result because infants have limited ca-
tion before storage or at the bedside.75 Micro-
pability to increase their cardiac output to
aggregate filters (20 to 40 microns) are occa-
compensate for hyperviscosity. Central ner-
sionally used for simple transfusions because
vous system abnormalities, pulmonary and re-
of their small priming volume. However, he-
nal failure, and NEC can occur from the resul-
molysis may occur when stored RBCs are ad-
tant decreased blood flow.
ministered through these filters using negative
A partial exchange is used to normalize
pressure.89
the hematocrit to between 55% and 60% and
The plastic tubing in the administration
improve tissue perfusion while maintaining
sets can add significant amounts of dead-
blood volume. This exchange is accomplished
space volume to the transfusion and may need
by removing whole blood and replacing it with
to be accounted for when preparing a transfu-
normal saline or other crystalloid solutions.
sion dose. Pediatric infusion sets created for
Plasma is not used to replace whole blood be-
platelets and other small-volume components
cause NEC has been reported as a complica-
have less dead space than standard sets.
tion of plasma transfusion.91
The formula below can be used to ap-
Administration Rates
proximate the volume of replacement fluid re-
A lack of clinical studies and evidence-based quired and the volume of whole blood that
practices related to blood transfusions in neo- must be withdrawn for the partial exchange:
nates has led to variability both in rates of
transfusion and in devices used among insti- (Blood (Observed Hct –
tutions. The rate of RBC and other blood com- Volume of volume) × Desired Hct)
replacement =
ponent administration is dictated by the clini- Observed Hct
fluid
cal needs of the pediatric patient. Despite
concerns from neonatologists that rapid blood
infusion rates may adversely affect intravascu-
ECMO
lar volume and electrolyte levels, an increased
risk of IVH in these small and fragile patients ECMO is a prolonged treatment where blood is
has not been clearly demonstrated. Therefore, removed from the patient’s venous circulation,
administering a simple transfusion over 2 to 4 circulated through a machine to remove CO2
hours is adequate in nonemergent situations. and replenish O2, and then returned to the pa-
However, in states of shock or severe bleeding, tient. ECMO has been successfully used since
a rapid infusion is often required. the early 1980s to provide gas exchange inde-
pendently of a patient’s lungs. ECMO allows
Unique Therapies and Situations in patients to recover without exposure to aggres-
Neonates sive ventilator support that can cause baro-
trauma and permanent lung damage and to
Polycythemia
maintain the circulation of oxygenated blood
Neonatal polycythemia is defined as a venous during cardiac surgery or in patients with dis-
hematocrit >65% or a hemoblogin >22 g/dL at ease when other forms of treatment fail.92,93 In
any time during the first week after birth. Ap- neonates and children, ECMO has become a
proximately 5% of all newborns develop poly- lifesaving advance treatment of meconium as-
cythemia, and this risk may be higher in small- piration syndrome, persistent pulmonary hy-
for-gestational-age neonates and infants of pertension of the newborn, congenital dia-
diabetic mothers. Once the hematocrit rises phragmatic hernia, and respiratory failure due
586 䡲 AABB TECHNICAL MANUAL
Cardiac arrest 5-10 min 2 units RBCs O-neg RBCs <14 days, AS
ECMO circuit disruption 5-10 min 2 units RBCs O-neg RBCs <14 days, AS
Neonate transferred for 1-2 hours 2 units RBCs O-neg RBCs <10 days,
ECMO 1 unit FFP AB plasma CPD or CPDA
1 unit platelets
Cardiac ICU 30-60 min 2 units RBCs Type specific <7 days, AS
Gradual respiratory or Hours to days 2 units RBCs Type specific <10 days, CPD
cardiac failure on con-
ventional support
ECMO = extracorporeal membrane oxygenation; RBCs = Red Blood Cells; AS = additive solution; FFP = Fresh
Frozen Plasma; CPD = citrate-phosphate-dextrose; CPDA = citrate-phosphate-dextrose-adenine; ICU = inten-
sive care unit.
CHAPTER 23 Neonatal and Pediatric Transfusion Practice 䡲 587
TABLE 23-8. Transfusion Guidelines for RBCs in Patients Older than 4 Months23,26
TABLE 23-9. Irradiation Guidelines for Neonates and Older Children Who Require Cellular Blood
Components23,26
1. Premature infants weighing <1200 g at birth.
2. Any patient with:
a. Known or suspected cellular immune deficiency.
b. Significant immunosuppression related to chemotherapy or radiation treatment.
3. Any patient receiving:
a. Components from blood relatives.
b. HLA-matched or crossmatched platelet components.
CHAPTER 23 Neonatal and Pediatric Transfusion Practice 䡲 591
unwashed RBCs or platelet products procured not been well defined (ie, 1:1:1 or 2:1:1). How-
from the mother of an infant is strongly dis- ever, studies indicate that an MTP in the pedi-
couraged.26 Maternal cells must be washed for atric setting is feasible not only for providing
effective treatment of hemolytic disease of the rapid and balanced blood product support but
newborn and neonatal alloimmune thrombo- also for decreasing the risk of thromboembolic
cytopenia when maternal RBCs and platelets events.131-133 Furthermore, when Hendrickson
are transfused. et al examined the impact of coagulopathy in
102 pediatric trauma patients, they found that
abnormal prothrombin time, activated partial
Massive Transfusion in the Pediatric thromboplastin time, and platelet count at
Setting emergency room admission were strongly as-
Trauma is the leading cause of death in in- sociated with mortality (p = 0.005, 0.001, and
fants, children, and young adults aged 1 to 21 <0.0001, respectively).134 These investigators
years. Although trauma rarely leads to hemor- did not examine the effect of MTP resuscita-
rhagic shock and massive transfusion, resusci- tion on coagulopathy and mortality, which
tation after trauma can be challenging. may provide insights into further optimiza-
The evidence to support pediatric MTP is tion of MTPs. Although adult studies have
limited, but several pediatric institutions use demonstrated benefit from MTP, the role of
MTPs to improve the outcomes of patients the MTP and the optimal ratio of blood com-
who have experienced trauma. The appropri- ponents still needs to be defined in the pediat-
ate ratios of RBCs to plasma and platelets have ric setting.131-133
KEY PO I NT S
1. RBCs are the most frequently transfused blood product in children. Frequent blood loss, in-
cluding iatrogenic losses from repeated phlebotomy, contribute to the need for RBC trans-
fusions in the neonatal population.
2. Symptomatic anemia and/or a target hemoglobin level is a preferred strategy for neonatal
RBC transfusion rather than a milliliter-for-milliliter replacement of due to blood losses.
3. A full-term newborn has a blood volume of approximately 85 mL/kg whereas a preterm in-
fant has a total blood volume of 100 mL/kg.
4. In infants <4 months of age initial patient testing must include ABO and D typing of their
red cells and a screen for unexpected red cell antibodies, using either plasma or serum from
the infant or mother. During any one hospitalization, crossmatch compatibility testing and
repeat ABO and D typing may be omitted as long as all of the following criteria are met: the
antibody screen is negative; transfused red cells are group O, ABO-identical, or ABO-com-
patible; and transfused cells are either D negative or the same D type as the patient. Testing
the infant’s reverse type for anti-A and/or anti-B is not necessary.
5. Small-volume simple RBC (no matter what the storage solution) transfusions when admin-
istered slowly have been shown to have little effect on serum potassium concentrations in
infants less than 4 months of age despite elevated potassium levels in the plasma of stored
RBCs.
6. During component preparation if aliquots are made with a sterile docking device, it is con-
sidered a “closed system” and the original units expiration date can be used for the new ali-
quot product.
7. Dosing of RBC units with additive solutions such as AS-1, AS-3, and AS-5 should not exceed
10-15 mL/kg for neonates.
592 䡲 AABB TECHNICAL MANUAL
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2013. 90. Lindemann R, Haga P. Evaluation and treat-
76. Bandarenko N, King K, eds. Blood transfusion ment of polycythemia in the neonate. In:
therapy: A physician’s handbook. 11th ed. Christiansen RD, ed. Hematologic problems of
Bethesda, MD: AABB, 2014 (in press). the neonate. Philadelphia: WB Saunders, 2000:
77. Pressey JG, Manno CS. Therapy for hemophilia 171-83.
and von Willebrand disease. In: Herman JK, 91. Black VD, Rumack CM, Lubchenco LD, Koops
Manno CS, eds. Pediatric transfusion therapy. BL. Gastrointestinal injury in polycythemic in-
Bethesda, MD: AABB Press, 2002:355-82. fants. Pediatrics 1985;76:225-31.
78. Price TH. The current prospects for neutrophil 92. Kevy SV. Extracorporeal therapy for infants
transfusion for the treatment of granulocyto- and children. In: Petz LD, Swisher SN, Klein-
penic infected patients. Transfus Med Rev man S, et al, eds. Clinical practice of transfu-
2000;14:2-11. sion medicine. 3rd ed. New York: Churchill
79. Marfin AA, Price TH. Granulocyte transfusion Livingstone, 1996:733-55.
therapy. J Intensive Care Med 2013 (in press). 93. Bartlett RH, Andrews AF, Toomasian JM, et al.
80. Christensen RD, Bradley PP, Rothstein G. The Extracorporeal membrane oxygenation for
leukocyte left shift in clinical and experimen- newborn respiratory failure: Forty-five cases.
tal neonatal sepsis. J Pediatr 1981;98:101-5. Surgery 1982;92:425-33.
81. Sweetman RW, Cairo MS. Blood component 94. Friedman DF, Montenegro LM. Extracorporeal
and immunotherapy in neonatal sepsis. Trans- membrane oxygenation and cardiopulmonary
fus Med Rev 1995;9:251-8. bypass. In: Hillyer CD, Strauss RG, Luban NLC.
82. Rosenthal J, Cairo MS. Neonatal myelopoiesis Handbook of pediatric transfusion medicine.
and immunomodulation of host defenses. In: London, United Kingdom: Elsevier Academic
Petz LD, Swisher SN, Kleinman S, et al, eds. Press, 2004:181-9.
Clinical practice of transfusion medicine. 3rd 95. Sharon BI. Management of congenital hemo-
ed. New York: Churchill Livingstone, 1996:685- lytic anemias. In: Simon TL, Dzik WH, Snyder
703. ES, et al, eds. Rossi’s principles of transfusion
83. Jenson HB, Pollack BH. The role of intravenous medicine. 4th ed. Bethesda, MD: AABB/Wiley-
immunoglobulins for the prevention and Blackwell, 2009:448-69.
treatment of neonatal sepsis. Semin Perinatol 96. Cohen AR, Norris CF, Smith-Whitley K. Trans-
1998;22:5063. fusion therapy for sickle cell disease. In: Capon
84. Sandberg K, Fasth A, Berger A, et al. Preterm SM, Chambers LA, eds. New directions in pe-
infants with low immunoglobulin G levels diatric hematology. Bethesda, MD: AABB,
have increased risk of neonatal sepsis but do 1996:39-88.
not benefit from prophylactic immunoglobu- 97. Adams DM, Schultz WH, Ware RF, Kinney TR.
lin G. J Pediatr 2000;137:623-8. Erythrocytapheresis can reduce iron overload
85. Hill HR. Additional confirmation of the lack of and prevent the need for chelation therapy in
effect of intravenous immunoglobulin in pre- chronically transfused pediatric patients. J Pe-
vention of neonatal infection (editorial). J Pe- diatr Hematol Oncol 1996;18:3-7.
diatr 2000;137:595-7. 98. Adams RJ, McKie VC, Hsu L, et al. Prevention
86. Ehrenkranz RA. The newborn intensive care of a first stroke by transfusions in children with
unit. In: Oski FA, ed. Principles and practice of sickle cell anemia and abnormal results on
596 䡲 AABB TECHNICAL MANUAL
125. Bradley MT, Milam JD, Anderson DC. Use of 130. Schoenfeld H, Muhn M, Doepfmer UR, et al.
deglycerolized red blood cells to prevent post- The functional integrity of platelets in volume-
transfusion infection with cytomegalovirus in reduced platelet concentrates. Anesth Analg
neonates. J Infect Dis 1984;150:334-9. 2005;100:78-81.
126. Gilbert GL, Hayes K, Hudson H, et al. Preven- 131. Dehmer JJ, Adamson MD. Massive transfusion
tion of transfusion-associated cytomegalovi- and blood product use in the pediatric trauma
rus infection in infants by blood filtration to patient. Semin Pediatr Surg 2010;19:286-91.
remove leukocytes. Lancet 1989;i:1228-31. 132. Hendrickson JE, Shaz BH, Pereira G, et al. Im-
127. Strauss RG. Selection of white cell-reduced
plementation of a pediatric trauma massive
blood components for transfusions during
transfusion protocol: One institution’s experi-
early infancy. Transfusion 1993;33:352-7.
128. Fergusson D, Hebert PC, Lee SK, et al. Clinical ence. Transfusion 2011;52:1228-36.
outcomes following institution of universal 133. Chidester SJ, Williams N, Wang W, Groner JI. A
leukoreduction of blood transfusions in pre- pediatric massive transfusion protocol. J Trau-
mature infants. JAMA 2003;289:1950-6. ma Acute Care Surg;73:1273-7.
129. Moroff G, Friedman A, Robkin-Kline L, et al. 134. Hendrickson JE, Shaz BH, Pereira G, et al. Co-
Reduction of the volume of stored platelet agulopathy is prevalent and associated with
concentrates for use in neonatal patients. adverse outcomes in transfused pediatric
Transfusion 1984;24:144-6. trauma patients. J Pediatr 2012;160:204-9.
C h a p t e r 2 4
Mary Ghiglione, RN, MSN, MHA, National Director, Patient Blood Management, Accumen, Inc, San Diego,
California, and Kathleen E. Puca, MD, MT(ASCP)SBB, Medical Director, BloodCenter of Wisconsin, Milwau-
kee, Wisconsin
The authors have disclosed no conflicts of interest.
599
600 䡲 AABB TECHNICAL MANUAL
of banked blood in the treatment of exsangui- sons for PBM include the risk of clerical errors,
nating wounds suffered in the armed conflicts errors in patient identification, the threat of a
of the same period. Yet transporting the blood new transfusion-transmitted pathogen, and
was difficult, and battlefield surgeons devel- the mounting evidence that transfusions are
oped techniques to treat casualties when no associated with adverse patient outcomes.
transfusion was possible. Other potential drivers of PBM include in-
A second example is the influence of Je- creasing patient demand for improved quality
hovah’s Witnesses, who cite Biblical passages in health care, the projected shrinking of the
as the foundation for their refusal of blood eligible donor base, and increasing health-care
transfusion. By the middle of the 20th century, costs.9,10
blood transfusion had become universally ac-
cepted as a medical treatment for a wide range Risks of Transfusion
of indications. Jehovah’s Witness patients had
to seek out those few physicians and hospitals The infectious risks of transfusion that became
that would provide medical and surgical care so widely known in the late 20th century,
without blood transfusion. These surgeons and which are now rare, as well as unknown
hospitals became increasingly utilized by Je- emerging pathogens continue to be a concern.
hovah’s Witnesses and other patients, and blood Improvements in donor screening, develop-
conservation programs began to flourish. With ment of effective tests, and research into
an emphasis on an individualized, patient- pathogen inactivation technology have all
centric approach, these programs led the way played a role in reducing the known infectious
toward the PBM movement seen today. disease risks of transfusion. Currently, the risk
Although the focus is frequently on sur- of either hepatitis C virus transmission or hu-
gery, PBM encompasses the entire scope of a man immunodeficiency virus transmission is
patient’s hospital experience. It includes: less than 1 in 1,000,000 donations, and the risk
of hepatitis B is less than one in 300,000.11
1. Efforts to identify and manage anemia and The noninfectious risks of transfusion,
bleeding risks before any treatment begins. including hemolytic transfusion reactions,
2. Use of blood-sparing surgical techniques transfusion-related acute lung injury, transfu-
and intraoperative blood recovery meth- sion-associated circulatory overload, and
ods. allergic reactions are more common than in-
3. Adjunctive strategies during intensive care fectious adverse events and often go unrecog-
unit (ICU) and postoperative care that nized. Chapter 27, Noninfectious Complica-
decrease the need for transfusion. tions of Blood Transfusion, discusses the
4. Blood utilization review and feedback to diagnosis, etiology, treatment, and prevention
ordering physicians. of these risks in detail.
5. PBM education of all health-care providers An emerging body of evidence indicates
involved in patient care. that transfusion may not provide the expected
therapeutic outcomes long assumed by both
The scope of PBM activities is discussed clinicians and patients. This is especially the
in detail in the section on Basic Elements of a case in stable, nonbleeding patients.12 Ran-
Patient Blood Management Program. domized controlled trials assessing the effica-
cy of RBC transfusion have shown that restric-
tive transfusion strategies for nonbleeding
TH E RAT I O NA LE F O R PAT IE N T
patients have no negative effect, and may even
BL O O D MANA G E ME NT
improve outcomes for some patients.8,13-16
Blood transfusion became universally accept- Recent research is identifying a link be-
ed as a mainstream medical therapy long be- tween allogeneic transfusion and worse pa-
fore all the risks and complications associated tient outcomes. Although many of these stud-
with it were recognized. The compelling rea- ies on blood transfusion are observational,
CHAPTER 24 Patient Blood Management 䡲 601
they have enrolled large numbers of patients communication, or their physician’s demean-
across many different specialties (eg, cardiolo- or, some patients trust that the provider who
gy, surgery, ICU, gastroenterology). Such stud- orders the transfusion is acting in their best in-
ies have repeatedly concluded that a strong as- terest. Too often, patients do not ask for, or are
sociation exists between RBC transfusion and not provided with, information on alternative
worse patient outcomes.5,8,9,17 treatment options before receiving a transfu-
Several studies have shown the risk of sion.28
nosocomial infection in ICU, surgical, and Allogeneic transfusion is considered a
trauma patients to be two to four times higher therapeutic intervention and, as with other
in transfused patients than in nontransfused medical interventions, requires the patient to
patients.5 In addition, the risk is dose-depen- provide informed consent.30,31 In order for the
dent (ie, the more blood the patient receives, patient to be adequately “informed,” the con-
the greater the risk of adverse events).18,19 RBC sent discussion needs to include information
transfusion has been associated, in a dose- about the risks and benefits not only of the
related fashion, with higher rates of cancer re- transfusion option, but of the alternatives to
currence, stroke, myocardial infarctions, renal transfusion and the refusal of treatment as
complications, acute lung injury and respira- well.32 The PBM approach, which encompass-
tory arrest, longer ICU and hospital stays, and es all the options, provides an excellent avenue
mortality both in the hospital and the long for open communication, joint consideration
term (5 years).5,17,20-24 of treatments, and the potential for improved
These deleterious effects on clinical out- patient outcomes and satisfaction.
comes are not solely related to allogeneic RBC PBM strategies are a necessity for patients
transfusions. One single-center study of more for whom blood transfusion is not an option
than 800 critically ill patients demonstrated due to medical status, cultural influences, reli-
that 1) transfusion was independently associ- gious beliefs, or unavailability of compatible
ated with the risk of acute lung injury and 2) blood. From the patient’s perspective, the ra-
the risk was higher with the transfusion of tionale for PBM is the ability to make a more
plasma-rich blood components (platelets and informed choice, having access to a higher
plasma) than with transfusion of RBC units.25 quality of care, and potentially experiencing
Plasma transfusion in critically ill patients has less risk.
shown little benefit and has been associated
with worse patient outcomes.26,27 The Physician’s Perspective
Thus, a growing body of literature strong-
After “first do no harm,” a physician’s primary
ly suggests that for the nonbleeding patient
responsibility is to ensure effective treatment
the benefit of transfusion is outweighed by un-
with minimal risk. Clinicians intend to do
expected risks and worse outcomes. The risks
what is best for their patients. But too often
can be reduced only by a shift from the more
they take blood transfusion for granted, as-
conventional transfusion practice (where
suming that benefits will be achieved and per-
transfusion is often a default decision) to a
ceiving the risks to be minimal.
PBM approach to transfusion practice (where
Medical school curricula devote a bare
the decision to transfuse is an option consid-
minimum of time for education on the risks of,
ered after evaluation of the risks, benefits, and
indications for, and alternatives to the use of
alternatives specific to each patient).
blood.33,34 Physicians often learn transfusion
practices based on the norms and standards
The Patient’s Perspective
developed years ago. It is difficult enough for
Despite advances in safety of the blood supply, them to keep up to date with current research
patients are concerned about needing a blood and best practices in their own specialty, let
transfusion during their treatment.28,29 Wheth- alone to keep abreast of developments in
er due to their underlying illness, inadequate transfusion medicine.
602 䡲 AABB TECHNICAL MANUAL
Studies show a wide variation in transfu- blood- and transfusion-related activities for
sion practice. Whether or not a patient re- surgical patients ranged from $522 to $1183
ceives a blood transfusion is often influenced per unit transfused. They also found that total
by the hospital where the patient is admitted annual blood expenditures correlated not with
or by the physician providing the clinical care, the volume of surgeries performed, but with
rather than by the patient’s clinical condi- the transfusion rate. The authors concluded
tion.3,35-38 that reducing the proportion of patients who
Transfusion based on physician behavior receive transfusions, the number of RBC units
can be improved with the implementation of transfused per patient, or both can be an im-
evidence-based clinical practice guidelines portant cost-saving strategy for hospitals.
and computer “dashboards” identifying best In a model simulation, Zilberberg and
practices linked to blood utilization and pa- Shorr40 showed that universal adoption of a re-
tient outcomes. Providing individual physi- strictive transfusion therapy (hemoglobin less
cians with their usage data in comparison to than 7 g/dL) in all ICU patients in US hospitals
that of their colleagues can be a powerful tool could potentially result in cost savings of near-
for process improvement. ly $1 billion annually. Even more important,
Audits and evaluation of transfusion the simulation indicated that this strategy
practices, along with analysis of blood utiliza- could improve quality of care and patient out-
tion data, are tools hospitals can apply to iden- comes by preventing nearly 40,000 transfu-
tify best practices. From these tools, guide- sion-attributable complications.
lines, algorithms, and protocols can be A recent blood utilization project involv-
developed and implemented across a service ing 464 US hospitals identified opportunities
line in an effort to assist individual physicians for significant reductions in blood utilization
in meeting quality standards and improving and costs.3 Several elements of a PBM program
patient care. were noted as important steps for hospital
Thus, a PBM approach benefits not only leaders to consider.
patients, but the physicians who treat them. Appropriate reimbursement for health-
Physicians who use the patient-centric ap- care supplies and services, including blood
proach of PBM will gain a reputation for better and transfusion, is a growing concern for hos-
patient outcomes, shorter lengths of stay, and pitals. Medicare reimbursements have failed
reduced costs. In some facilities, blood utiliza- to keep pace with increased costs, which have
tion data are included on a physician “score- exacerbated the revenue pressures. The Cen-
card,” acknowledging those physicians whose ters for Medicare and Medicaid Services (CMS)
transfusion practice complies with the hospi-
structure for outpatient reimbursement iden-
tal’s guidelines.
tified that in 2013, payments for the more fre-
quently transfused blood components would
The Hospital’s Perspective decrease.41 An understanding of all the con-
Economic Pressures tributing factors to the cost of transfusion (in-
cluding reimbursement) is vital for hospitals to
Due to the economic pressures facing hospi- develop. The economic pressures can be an
tals today, it is estimated that by 2020 one in additional incentive for reducing unnecessary
three hospitals will close or reorganize into a transfusions and improving blood utilization.
different type of health-care service.39 Oppor-
tunities to reduce costs are eagerly sought by Requirements for Accreditation
hospital administrators, and reducing blood
usage is no exception. The Joint Commission promotes several stan-
The acquisition cost of an RBC unit is only dards related to hospital blood administration42
a fraction of the total cost of that unit to the and requires hospitals to review the use of
hospital. Using activity cost-based modeling, blood and blood components in an effort to
Shander et al2 reported that the total cost for monitor and improve practices. The Joint
CHAPTER 24 Patient Blood Management 䡲 603
Commission also has developed PBM Perfor- one transfusion in a 10-year period.48 Although
mance Measures, which include seven areas data are limited, it is estimated that patients
for improvement.43 Although they are not uni- who are 65 or older receive 55% to 60% of RBC
versally practiced, the measures can help hos- transfusions.47 Similar population demograph-
pitals to monitor blood usage. ics are expected in Germany, where the pre-
Both AABB and the College of American dicted shortfall in the blood supply could be
Pathologists (CAP) promote standards for 47% by 2020.49
transfusion service laboratories. These re- Blood availability may also be affected by
quirements address more specific, technical changes in donor eligibility and storage of
issues in quality testing and patient safety re- blood units. A better understanding of iron de-
lated to blood administration.44,45 Often, it is pletion in frequent blood donors has prompt-
the responsibility of the transfusion service to ed a discussion of increased donor hemoglo-
ensure that requirements related to transfu- bin requirements and prolonged donation
sion are met—even when the requirements in-
intervals.50,51 If ongoing trials confirm a rela-
volve activities outside the purview of the lab-
tionship between the age of stored blood units
oratory staff.
and increased morbidity and mortality follow-
For instance, the CAP requirement
ing transfusion in acutely ill adult patients,
TRM.41025-Transfusionist Training Phase II
blood inventory management will change.52-56
requires all personnel who administer blood
Any shortening of the shelf-life of the RBC
components to be trained in proper identifica-
unit could put more pressure on diminishing
tion of transfusion recipients. AABB Standards
for Blood Banks and Transfusion Services re- blood supplies and donor recruitment efforts
quires a peer-review program for monitoring may not be able to make up for the loss.57 Im-
and addressing transfusion practices in all cat- plementing PBM strategies to reduce the need
egories of blood and blood components.45(p91) for allogeneic blood and making sure only ap-
Both require collaboration of other hospital propriate transfusions of the correct dose are
departments for compliance. With the multi- given are two ways to support the available
disciplinary, team-oriented approach of PBM, blood supply for a community.
an additional benefit to hospitals may be in-
creased ease of compliance, as well as more BAS IC ELE MENTS OF A PBM
readily accepted and implemented corrective PROG R A M
actions and process improvements.
As noted earlier in the section on Definition
The Community’s Perspective and Scope of PBM, several PBM strategies can
be implemented to decrease the use of alloge-
Initiatives to limit the use of blood also benefit
neic blood transfusions. They encompass all
the community by supporting a safe and ade-
aspects of the patient’s evaluation and clinical
quate blood supply for those who need it. The
increasing age of the population and the di- management. A PBM approach is typically im-
minishing donor pool may have significant plemented in elective surgical patients in the
impacts on blood availability. In the next 10 to preoperative, intraoperative, and postopera-
20 years, it is expected that the number of indi- tive phases of care as outlined in Table 24-158
viduals in the United States who are more than and in the following sections. PBM strategies
65 years of age will increase at a faster rate are also relevant to medical patients. Although
than those aged 16 to 64, and make up 20% of any one of the strategies used individually will
the population.46,47 be successful in decreasing the use of alloge-
A recent review of data from the Health neic blood transfusion, they are most effective
and Retirement Study reported that 31% of when combined and individualized for the
Americans over the age of 65 received at least specific patient.59
604 䡲 AABB TECHNICAL MANUAL
Anemia is a common condition and is es- The use of ESAs to lower transfusion rates in
timated to be as high as 75% depending on the patients undergoing elective, orthopedic sur-
patient’s underlying conditions, reason for gery is well established.71 However, due to the
surgery, and definition of anemia used.62,63 risk of thromboembolic events, tumor growth,
Anemia has been independently associated and death associated with ESAs, the Food and
with increased perioperative mortality and Drug Administration (FDA) has restricted its
morbidity in patients undergoing joint re- use, recommending more conservative dosing
placement and cardiac surgery.62,64 In a retro- and strict monitoring.72
spective study of noncardiac surgery patients
65 years or older, 30-day postoperative mortal- Addressing Bleeding Risk
ity and cardiac event rates increased by 1.6%
for every percentage-point decrease from the A second important aspect of PBM is minimiz-
normal hematocrit range.65 Even mild anemia ing the risk of bleeding. Assessing a patient’s
has been shown to be an independent risk fac- risk for bleeding in relation to the type and
tor for adverse outcomes in patients undergo- complexity of surgery and the presence of any
ing colon surgery.66 pre-existing anemia and/or coagulopathy can
If anemia is detected, the initial test re- help formulate an individualized plan. Estab-
sults may suggest possible etiologies.67 Addi- lishing protocols for discontinuation or dose
tional laboratory testing such as creatinine, adjustment of antiplatelet and anticoagulant
reticulocyte count, iron and iron-binding ca- drugs is an important function of a PBM
pacity, ferritin, vitamin B12, and folate may program and an essential preoperative plan-
help in the diagnosis. Several published guide- ning tool.
lines can assist the PBM program in develop- Published guidelines such as those devel-
ing algorithms for identifying and treating oped by the Society for Thoracic Surgery and
anemia in medical and preoperative pa- the American College of Chest Physicians can
tients.67-70 Whenever possible, an elective, provide a basis for development of such proto-
high-blood-loss surgery should be delayed un- cols.73-75 Protocols help practitioners to opti-
til the anemia is explained and appropriately mize the patient’s hemostasis before an elec-
treated. tive surgery while minimizing risk for
Pharmacologic agents for anemia may in- thrombotic events.
clude iron (both oral and intravenous prepara- Herbal supplements such as garlic, gink-
tions), folic acid, vitamin B12, or erythropoie- go, and ginseng, have also been known to in-
sis-simulating agents (see Appendix 24-1). The crease bleeding risk. Patients should be asked
choice of therapeutic agent should be guided about their use and ingestion should be dis-
by the underlying etiology of the anemia, the continued before surgery. Readers are direct-
patient’s other medical conditions, and avail- ed to more focused reviews on the effect of
able time to treat before any surgery. herbal supplements on coagulation.76,77
Intravenous (IV) iron replacement using
iron sucrose, ferric gluconate, or iron dextran Preoperative Autologous Blood
has been shown to quickly correct iron defi- Donation
ciency anemia and may be the preferred route
over oral preparations in patients scheduled Historically, preoperative autologous blood
for surgery or in the postoperative period. The donation (PAD) had been the primary means
efficacy of IV iron in treating anemia has been to reduce the use of allogeneic blood. With
consistently proven in reducing the need for PAD the patient donates his/her own blood,
allogeneic blood transfusions in surgical pa- ideally 4 to 6 weeks, before surgery. The goal of
tients. the preoperative donation is for regeneration
Erythropoietic stimulating agents (ESAs) of the patient’s red cell mass and return of the
are also effective in increasing the hemoglo- patient’s hemoglobin to the precollection val-
bin level if sufficient iron stores are present. ue before surgery.
606 䡲 AABB TECHNICAL MANUAL
With increasing safety of the blood sup- The ANH-collected whole blood is typi-
ply, the number of autologous units collected cally stored at room temperature (for up to
in the United States has declined.1,78 Other fac- 8 hours) and is often reinfused near the end of
tors contributing to the decline include high the procedure or whenever transfusion is
wastage of PAD blood (45% or more is discard- needed.87(p22) An added value of ANH is that
ed),79,80 higher risk for preoperative anemia af- platelets and coagulation factors remain viable
ter donation,80,81 errors related to production when the product is stored at room tempera-
and handling of these units,83,84 and increasing ture. Although ANH is considered a relatively
acquisition costs that may not be reimburs- safe procedure, reports on the clinical efficacy
able. and benefits are mixed.88 In some studies, the
In addition, a systematic review showed use of ANH resulted in lower allogeneic trans-
that patients in a PAD program had a higher fusion rates, fewer units transfused, and de-
likelihood of receiving transfusions (allogeneic creased complications,89,90 while others failed
and/or autologous) compared to those pa- to show any benefit.91,92
tients who did not participate in PAD because ANH is more likely to be of benefit in pa-
their hemoglobin did not completely recover tients undergoing high-blood-loss procedures
before surgery.82 Therefore, patients in PAD (1500 mL or greater) who have preoperative
programs incur a higher overall risk to their hemoglobin levels of 12 g/dL or higher. Poten-
health with a greater likelihood of being trans- tial contraindications for ANH include active
fused. cardiac ischemia, restrictive or obstructive
Despite these concerns, PAD may be a pulmonary disease, renal failure, hemoglobin-
reasonable option for patients with rare blood opathy associated with hemolysis, and known
types or multiple red cell alloantibodies or for coagulation abnormalities.93 Planning and co-
patients who refuse allogeneic blood. In these ordination by the surgical team are important
situations, advanced planning and evaluation for ANH to be effective.
of the patient are crucial before PAD is at-
tempted. In order to mitigate the anemia in- Intraoperative Blood Recovery
duced by PAD, the collection needs to occur at
least 4 to 6 weeks before surgery (earlier if Intraoperative blood recovery is another com-
there are freezing capabilities for the units) to mon PBM strategy. Use of recovered shed
allow sufficient time for recovery of the pa- blood is efficacious in several procedures,
tient’s red cell mass.85 such as cardiothoracic, orthopedic, neurolog-
ic, vascular, and trauma surgery. Reduction in
Intraoperative Strategies allogeneic RBC transfusions by 38%, with an
average savings of 0.68 unit of allogeneic RBCs
Acute Normovolemic Hemodilution per patient has been reported.94
Acute normovolemic hemodilution (ANH) is Intraoperative blood recovery involves
the removal of whole blood from the patient collecting shed blood from the surgical site,
immediately before or shortly after the begin- centrifuging it, and washing it with normal sa-
ning of the surgical procedure into a standard line. During the centrifugation and washing
blood bag containing anticoagulant. Crystal- process, plasma, platelets, red cell stroma,
loid or colloid solutions or both are reinfused contaminants, and anticoagulant are removed.
to maintain adequate circulatory volume. The washed red cells are transferred to a sepa-
ANH lowers the patient’s hematocrit, improves rate bag, and then administered to the same
blood fluidity, and increases cardiac output patient. Ideally, washed recovered blood should
and organ blood flow, offsetting the decline in have a hematocrit of at least 45% to 55%.
oxygen capacity of the diluted blood.86 Any Concerns regarding the safety of blood re-
blood lost from the patient during the surgery covery often surround its use in cancer and
is then diluted and has a reduced red cell con- obstetric surgeries. The concern is that un-
tent. wanted material and cells (eg, tumor cells,
CHAPTER 24 Patient Blood Management 䡲 607
bacteria, and amniotic fluid) from the surgical lection and Administration and other AABB
field may end up in the washed product and publications.58,87,101-103
be re-infused to the patient. However, the use
of double suction setup and leukocyte reduc- Anesthesia and Surgical Techniques
tion filters has been shown to reduce these
Minimizing intraoperative bleeding is critical
risks.95,96 In addition, reviews do not support
for reducing the need for allogeneic transfu-
the theoretical concern for increased risk of ei-
sion. Obvious measures include meticulous
ther amniotic fluid emboli or cancer recur-
surgical technique and rapid and rigorous
rence, although the quality of the studies has
control of bleeding. Proper positioning of the
been questioned.97,98 Prospective, appropriate-
patient can reduce blood loss and is guided by
ly powered studies are needed to define the
two basic principles—elevating the surgical
risks and benefits of recovered blood in these
site above the level of the heart and avoiding
controversial settings.
compression of venous drainage of the surgi-
A recent novel approach for blood recov-
cal field.104 The deliberate reduction of mean
ery in surgeries involving cardiopulmonary
arterial pressure, known as “controlled or de-
bypass (CPB) is the Hemobag modified ultra-
liberate hypotension,” in selected patients can
filtration system (Global Blood Resources,
reduce blood flow to the surgical site and
LLC, Somers, CT). After completion of CPB
thereby decrease blood loss. However, the
and decannulation, residual whole blood from
benefits must be balanced against potential
the CPB circuit, which can be up to 2 liters, is
risk for organ ischemia.59,105
ultrafiltered and hemoconcentrated. Excess
Maintaining normothermia is important
water and inflammatory mediators are re-
for optimal coagulation and hemostasis. Ef-
moved, resulting in a whole blood product
forts to keep the patient warm by using warm
with hematocrit of approximately 50%. In con-
IV fluids, air warming blankets and by keeping
trast to washed, recovered blood, blood pro-
the surgical suite warmer can help to avoid hy-
cessed with the ultrafiltration system contains
pothermia and thus decrease surgical blood
platelets, plasma proteins, and coagulation
loss. Optimal fluid management, including
factors that have been preserved and concen-
choice of infusion fluids, volume, and timing
trated.99,100 The use of this device should theo-
of administration, can also affect surgical
retically reduce exposure to allogeneic blood
blood loss and transfusion requirements.
more than washed, recovered blood, especially
Modern devices for tissue dissection, such as
exposure to plasma and platelet transfusions;
harmonic scalpels, water jet dissectors, and
however, the clinically relevant impact of this
electrocautery with argon beam coagulation
newer technique remains unknown.
can minimize tissue damage and provide bet-
Quality control and quality management
ter hemostasis at the incision site. Other mea-
programs for autologous blood products pre-
sures influencing surgical blood loss include
pared by intraoperative blood recovery devices
use of tourniquets, direct control of bleeding,
are not yet as well developed and accepted
infiltration of vasoconstrictors into the surgi-
among hospitals as other aspects of transfu-
cal wound, choice of ventilation patterns, and
sion medicine. Significant opportunity exists
choice of anesthesia.59,104-106
in hospitals for collaboration between the
transfusion service, surgery, perfusion, anes-
Point-of-Care Testing and Transfusion
thesia, and nursing departments in the devel-
Algorithms
opment of blood recovery programs. Guide-
lines and regulatory requirements for Point-of-care testing (POCT) has several ad-
establishing, enhancing, and maintaining vantages over conventional laboratory testing
quality and safety for these particular transfu- in that it can 1) be utilized whenever necessary,
sion practices are described in the AABB Stan- 2) provide more rapid turnaround time, and 3)
dards for Perioperative Autologous Blood Col- use minimal sample volumes for testing. Use
608 䡲 AABB TECHNICAL MANUAL
ing, making postoperative blood recovery un- botomy. Routine, standing orders are a com-
necessary.116 In addition, unwashed shed mon practice. Laboratory testing should be
blood is less desirable because it has a hema- ordered when clinically justified and the re-
tocrit ranging from 20% to 30% and contains sults are likely to change clinical management.
activated clotting and complement factors, in- When ordered, collection for laboratory test-
flammatory mediators, cytokines, and fat par- ing should be consolidated to minimize the
ticles that can increase the risk for febrile reac- number of tubes collected. Another approach
tions.120,121 is the use of pediatric or small volume tubes
For patients with substantial postopera- for sample collection. This strategy can reduce
tive blood loss, improved product quality and blood loss from laboratory testing by up to
safety—hematocrit ranging from 60% to 80% 70%.126,127 Smaller sample volumes, often less
with removal of contaminants—can be than 0.5 mL, can also be obtained with the use
achieved by using devices that wash and con- of POCT devices.
centrate the postoperative wound drainage Even the process of drawing the blood
blood. Maintaining competency of nursing can be a significant cause of blood loss. Pa-
staff and higher cost for these devices may be tients with invasive lines (eg, central venous
disadvantages for some hospitals. However, catheters or arterial catheters) have a three-
when compared to allogeneic blood transfu- fold increase in phlebotomy volume com-
sions, use of postoperative washed products in pared to patients without such catheters. The
joint replacement surgery is potentially com- ease with which samples can be obtained and
parable in cost.122 the added requirement to discard the first few
mLs (up to 10 mL) each time the line is ac-
cessed can contribute to greater blood loss.128
Limiting Phlebotomy-Related Blood Such catheters should be discontinued as
Loss soon as they are no longer required. Orders for
specimens drawn from these catheters should
Minimizing the impact of phlebotomy on the be consolidated to minimize the amount of
development of iatrogenic anemia is a key blood otherwise discarded. Use of a device for
strategy for reducing transfusion requirement. blood sampling whereby the initial volume of
Iatrogenic anemia related to routine laborato- blood can be reinfused into the patient instead
ry testing is common. In a study of 145 West- of discarded has shown reduction in mean
ern European ICUs, blood loss from phleboto- phlebotomy volume by 50%, higher hemoglo-
my averaged 41.1 mL per day per patient,123 bin levels, and fewer RBC transfusions even
which could result in the loss of nearly a unit of when a restrictive transfusion practice is im-
blood for an ICU stay of 1-2 weeks. A US retro- plemented.127,128
spective database study of over 17,000 patients
with acute myocardial infarction who were not
anemic on admission showed that patients Increased Tolerance of Anemia and
who developed moderate to severe anemia
Transfusion Thresholds
had experienced nearly 100 mL higher mean
blood losses from phlebotomy over the course A patient’s response to anemia is highly indi-
of their hospitalization than those who did not vidualized and dependent on his or her ability
develop anemia.124 In a single-center retro- to maintain adequate oxygen delivery to the
spective study of patients with prolonged ICU tissues. Tolerance is dependent on the pa-
stays, after day 21 the odds of being transfused tient’s volume status, his or her physiologic re-
was independently associated with phleboto- serve (including cardiac, pulmonary, and renal
my volume.125 function), and the dynamics of the anemia.
Reducing the quantity and frequency of The response becomes one of the most impor-
laboratory testing is a logical measure to re- tant factors in determining the need for trans-
duce the amount of blood loss related to phle- fusion.129,130
610 䡲 AABB TECHNICAL MANUAL
Patients with chronic anemia due to blood volume and shifts in fluid between fluid
chronic renal failure or slow gastrointestinal compartments. In the nonbleeding patient,
bleeding often have physiologically adapted to single-unit RBC transfusion followed with
a lower hemoglobin level by increasing their clinical reassessment is advocated.134,135 Often
cardiac output, heart rate, or stroke volume. a single RBC unit provides an adequate in-
Rapid blood loss from surgical bleeding or crease in hemoglobin and relieves the patient’s
trauma often results in patients displaying he- symptoms. The potential impact of imple-
modynamic instability, shock, and other menting a single-unit transfusion policy can
symptoms that require more rapid volume re- be significant. Based on a transfusion thresh-
placement. old of 8 g/dL of hemoglobin and adoption of a
Increasing a patient’s tolerance for lower single-unit strategy, one center predicted that
hemoglobin includes increasing oxygen deliv- half an RBC unit or more could be saved per
ery or decreasing oxygen consumption, which patient.136 When combined with strict adher-
can limit the need for RBC transfusion. Strate- ence to a restrictive transfusion threshold, a
gies to optimize the patient’s hemodynamic single-unit transfusion policy can have an
status and oxygenation may include maintain- even greater impact.
ing normovolemia with intravenous fluids, use
of appropriate vasopressor agents, use of sup-
plemental oxygen or mechanical ventilation,
adequate pain control and/or sedation, main- BL O O D UT I L IZ A T I O N REV IE W
taining normothermia, and avoidance and AND CHA NGING PHYSICIAN
prompt treatment of any infections.131 BEH AV IO R
Incorporating evidence-based transfu-
sion protocols may further aid in reducing un- Changing the behavior of physicians whose
necessary transfusions. Historically, hemoglo- transfusion practices are embedded in tradi-
bin levels of 10 g/dL or less have been used as tion and habit can be challenging. The ele-
the threshold for transfusion. Recent random- ments required to create a culture change in
ized control trials involving nonbleeding criti- physician transfusion practice include: 1) a de-
cally ill patients, post-cardiac-surgery patients, sire for change, 2) the provision of a new be-
elderly hip fracture patients with cardiac dis- havior practice, 3) a perception of the new
ease, and patients with upper gastrointestinal practice as safe and straightforward, and 4) a
bleeding have established evidence-based presentation of change that is nonthreatening
transfusion thresholds. In all these trials, the to autonomy.119
use of a more restrictive transfusion threshold More and more hospital administrators
(eg, hemoglobin of 7 to 8 g/dL) has been are recognizing that the use of blood products
shown to decrease RBC transfusions without may worsen patient outcomes and that the
increasing morbidity or mortality.8,132 In cost of allogeneic blood is significant. Thus, a
addition, an arbitrary hemoglobin level or desire for change is already under way. Strate-
“threshold” should not be the sole driver for gies focused on education of appropriate
transfusion. Transfusion decisions should be transfusion practice, providing sound alterna-
individualized and based not only on the he- tives to transfusion, and communication and
moglobin level but also on the patient’s clini- feedback by respected colleagues or “champi-
cal signs and symptoms of anemia and their ons” are fundamental elements of change in
ability to tolerate and compensate for the ane- transfusion practices. Building a team of dedi-
mia.133 cated stakeholders and champions as the net-
Traditionally, physicians have ordered 2 work for change management is crucial for
units for RBC transfusion, a practice that success of any change and a key element for a
evolved without clear rationale. A unit of blood successful PBM program.
can have varying effects on hemoglobin and Studies have demonstrated the ability of
hematocrit, depending on the patient’s total several interventions for changing transfusion
CHAPTER 24 Patient Blood Management 䡲 611
practice. Although a systematic review did not quality improvement are powerful tools to
find one intervention more effective than an- change transfusion practice. Providing physi-
other, the authors concluded that even simple cians with outcome data in relationship to
interventions appear to be effective.137 Inter- their transfusion rates for a surgical procedure
ventions can be broadly grouped into: 1) edu- or specific treatment as well as comparison to
cation, 2) adoption of guidelines, 3) reminders, their peers and best practices are likely to mo-
and 4) audits with feedback. tivate changes. Providing physicians with reg-
Education can be in the form of sched- ular reports helps to maintain awareness and
uled conferences or one-on-one teaching. encourages physicians’ interest in looking for
One-on-one education with physicians, al- additional strategies to reduce avoidable
though more time consuming, is likely to have transfusions.142
a more sustained effect. The educational con- A recent initiative to improve transfusion
tent needs to be evidence-based, provided by practice has been the employment of transfu-
respected colleagues or opinion leaders, and sion safety officers or patient blood manage-
be ongoing. Providing repeated sessions and ment coordinators. As change agents they can
easy access to educational information, such provide regular education to all staff involved
as on the hospital website, can help support in transfusion practice, increase awareness of
physicians as they consider changing their and access to transfusion alternatives, develop
practices. a network of caregivers and “champions” for
Evidence-based transfusion guidelines the promotion of optimal transfusions, and
are the basis for improving appropriateness in provide utilization reports for continuous im-
transfusion. Introduction of the guidelines provement. Working with the local “champi-
needs to be combined with education and re- on,” these coordinators can be instrumental in
minders such as posters and pocket guide- changing the culture and reducing allogeneic
lines. Issuing a memo of the transfusion guide- transfusions.143
lines to physicians without follow-up typically
fails to reduce blood usage.138 Transfusion
guidelines can be reinforced when they are Medical Education
used with computerized provider order entry
(CPOE) systems. Incorporating decision sup- Education is an integral part of any PBM initia-
port tools into CPOE transfusion order sets tive. Because behavioral change is sometimes
and requiring physicians to document the in- difficult to achieve, repetition and reinforce-
dication when outside of the guidelines can be ment over time are typically required for suc-
effective in improving practice.139,140 cess. Because PBM is multidisciplinary, differ-
Interventions to change transfusion prac- ent audiences may respond to different
tice go hand in hand with auditing or monitor- approaches.
ing of transfusion practice. Audits that occur at Educational delivery options may in-
the time of the transfusion order or during the clude journal club, grand rounds, webinars,
24 hours after the transfusion and are accom- online courses, guest lecturers, conference at-
panied with education provide a clear oppor- tendance, one-to-one instruction, self-study,
tunity for changing behavior.141 Prospective and blood usage review feedback. Provision of
audits (approval before issuing the product) continuing education credits for educational
performed manually require resources and activities can be an effective motivator for par-
can be time consuming. The implementation ticipation.
of CPOE can provide a more practical ap- Although ordering physicians and nurs-
proach to monitoring each transfusion order. ing staff need the greatest understanding of
Chapter 28 provides a detailed discussion of PBM, other groups of hospital personnel also
blood utilization auditing. benefit from educational opportunities. For
Blood utilization reports that incorporate instance, information technology and finan-
benchmarking with the goal of continuous cial personnel may not need to know the most
612 䡲 AABB TECHNICAL MANUAL
technical details, but they do need to under- TABLE 24-2. General Strategy for Implementing
stand the intent, benefits, and outcomes of a PBM Program
PBM efforts so they can effectively support
those efforts. 1. Education
a. A knowledgeable advocate
b. Executives
c. Core group (pharmacy, nursing, blood
PRO GR AM DEVE LO P MEN T bank)
Successful implementation of a PBM program d. Any department involved in PBM activi-
requires planning, education, and teamwork. ties (ordering clinicians, finance, informa-
It is important to first analyze the current tion technology)
transfusion practices within the hospital. A
needs assessment can help to evaluate the 2. Buy-in from executives
current behaviors and understand the hospital a. “C” suite (CEO, COO, CFO, CMO)
culture and readiness for change. b. Medical executive committee
Demonstrating the clinical benefits as c. Blood utilization/transfusion committee
well as potential cost savings with a PBM pro- d. Surgical services committee
gram is key to ensuring support and buy-in at
all levels. Care must be taken to ensure that 3. Business proposal
clinical staff understand that the primary ob- a. Background/current environment
jective of a PBM program is to improve patient b. Program description
outcomes, even though hospital administra- c. Financial aspects
d. Risk/benefit analysis
tors may also focus on the cost savings.
e. Summary/conclusion
The specific activities that are carried out
as part of PBM program implementation can
vary by institution, and are defined by the ex- 4. Teamwork
ecutive management team of the facility. a. Broad-based stakeholder group of those
affected by PBM program (multidisci-
Guidance is available from a variety of profes-
plinary emphasis, identification of con-
sional organizations. The Society for the Ad-
cerns, details to be addressed, enhanced
vancement of Blood Management offers Ad-
buy-in)
ministrative and Clinical Standards for Patient
b. Smaller, more focused steering commit-
Blood Management Programs, which outlines
tee (baseline usage data, relationship to
12 standards related to the activities of a for-
strategic plan, implementation steps,
mal, comprehensive, organization-wide PBM
timeline, policy review, monitor progress
program.144 A PBM program can be designated
checks)
as an activity level 1, 2, or 3 program as out- c. Effective meetings
lined in AABB Standards for a Patient Blood d. Milestone celebrations
Management Program.145(pp2-3) The responsibil-
ities for oversight and monitoring at each ac- 5. Evaluation
tivity level are described in Appendix 24-2. a. Possible improvements/lessons learned
An effective PBM program involves a mul- b. Study of metrics/outcome data
tidisciplinary effort with input and participa- c. Analysis/report of program success
tion from administration, physician and d. Possible program expansion
nursing leadership, laboratory, transfusion
medicine, ethics, registration, surgery, finance,
and technology personnel. Identifying cham-
pions who are willing to be spokespersons and menting a PBM program is summarized in Ta-
drivers of the program is essential when start- ble 24-2. More detailed descriptions of PBM
ing a program. A general strategy for imple- program implementation are available.146,147
CHAPTER 24 Patient Blood Management 䡲 613
KEY PO I NT S
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69. Goodnough LT, Maniatis A, Earnshaw P, et al. fus Med 2006;16:307-11.
81. Lee GC, Cushner FD. The effects of preopera-
Detection, evaluation, and management of
tive autologous donations on perioperative
preoperative anemia in the elective orthopae-
blood levels. J Knee Surg 2007;20:205-9.
dic surgical patient: NATA guidelines. Br J An-
82. Carless P, Moxey A, O’Connell D, Henry D. Au-
aesthesia 2011;106:13-22.
tologous transfusion techniques: A systematic
70. Patel MA, Carson JL. Anemia in the preopera-
review of their efficacy. Transfus Med 2004;
tive patient. Med Clin North Am 2009;93: 1095-
14:123-44.
1104.
83. Goldman M, Remy-Prince S, Trepanier A, De-
71. Weber EW, Slappendel R, Hemon Y, et al. Ef-
cary F. Autologous donation error rate in Cana-
fects of epoetin alfa on blood transfusions and
da. Transfusion 1997;37:523-7.
postoperative recovery in orthopaedic sur-
84. Domen RE. Adverse reactions associated with
gery: The European Epoetin Alfa Surgery Trial autologous blood transfusion: Evaluation and
(EEST). Eur J Anaesthesiol 2005;22:249-57. incidence at a large academic hospital. Trans-
72. Food and Drug Administration. FDA drug fusion 1998;38:301-6.
safety communication: Erythropoiesis-stimu- 85. Singbartl G. Preoperative autologus blood do-
lating agents (ESAs) Procrit, Epogen, and nation – part I. Only two clinical parameters
Aranesp. Silver Spring, MD: FDA, 2010. [Avail- determine efficacy of the autologous prede-
able at: http://www.fda.gov/Drugs/DrugSafe posit. Minerva Anestesiol 2007;73:143-51.
ty/PostmarketDrugSafetyInformationforPa 86. Messmer K, Kreimeier U, Intaglietta M. Pres-
tientsandProviders/ucm200297.htm (accessed ent state of intentional hemodilution. Eur Surg
March 24, 2014).] Res 1986;18:254-63.
73. Holbrook A, Schulman S, Witt DM, et al. Anti- 87. Puca KE, ed. Standards for Perioperative autol-
thrombotic Therapy and Prevention of ogous blood collection and administration.
Thrombosis. 9th ed. American College of 5th ed. Bethesda, MD: AABB, 2013.
Chest Physicians Evidence-Based Clinical 88. Segal JB, Blasco-Colmenares E, Norris EJ,
Practice Guidelines. Chest 2012; 141(2)(Sup- Guallar E. Preoperative acute normovolemic
pl):e152S–e184S. hemodilution: A meta-analysis. Transfusion
74. Ferraris VA, Brown JR, Despotis GJ, et al. 2011 2004;44:632-44.
update to the Society of Thoracic Surgeons 89. Jarnagin WR, Gonen M, Maithel MD, et al. A
and the Society of Cardiovascular Anesthesiol- prospective randomized trial of acute normo-
CHAPTER 24 Patient Blood Management 䡲 617
boelastography for management of life-threat- 129. Lelubre C, Vincet JL. Red blood cell transfu-
ening postinjury coagulopathy. Transfusion sion in the critically ill patient. Ann Intensive
2012;52:23-33. Care 2011;1:43.
116. Henry DA, Carless PA, Moxey AJ, et al. Anti-fi- 130. Madjdpour C, Sphan DR, Weiskopf RB. Ane-
brinolytic use for minimising perioperative al- mia and perioperative red blood cell transfu-
logeneic blood transfusion. Cochrane Data- sion: A matter of tolerance. Crit Care Med
base Syst Rev 2011;(3):CD001886. 2006;34(Suppl):S102-S108.
117. Goodnough LT, Shander A. Current status of 131. Physiology of anemia and oxygen transport.
pharmacologic therapies in patient blood In: Seeber P, Shander A. Basics of blood man-
management. Anesth Analg 2013;116:15-34. agement. 1st ed. Malden, MA: Wiley-Blackwell,
118. Spotnitz W, Burks S. Hemostats, sealants, and 2007:9-20.
adhesives: Components of the surgical tool- 132. Carson JL, Carless PA, Hebert PC. Transfusion
box. Transfusion 2008;48:1502-16. thresholds and other strategies for guiding al-
119. Spotnitz W, Burks S. State of the art review: He- logeneic red blood cell transfusion. Cochrane
mostats, sealants, and adhesives II: Update as Database Syst Rev 2012;(4):CD002042.
well as how and when to use the components 133. Carson J, Grossman BJ, Kleinman S, et al. Red
of the surgical toolbox. Clin Appl Thromb He- blood cell transfusion: A clinical practice
most 2010;16:497-514. guideline from the AABB. Ann Intern Med
120. Munoz M, Garcia-Vallejo JJ, Ruiz MD, et al. 2012;157:49-58.
Transfusion of postoperative shed blood: Lab- 134. Administrative and clinical standards for pa-
oratory characteristics and clinical utility. Eur tient blood management programs. Engle-
Spine J 2004;13(Suppl 1):5107-13. wood, NJ: Society for the Advancement of
121. Sinardi D, Marino A, Chillemi S, et al. Compo-
Blood Management, 2010.
sition of the blood sampled from surgical
135. Napolitano LM, Kurek S, Luchette FA, et al.
drainage after joint arthroplasty: Quality of re-
Clinical practice guideline: Red cell transfu-
turn. Transfusion 2005;45:202-7.
sion in adult trauma and critical care. Crit Care
122. Rao VK, Dyga R, Bartels C, Waters JH. A cost
Med 2009;37:3124-57.
study of postoperative cell salvage in the set-
136. Ma M, Eckert K, Ralley F, Chin-Yee I. A retro-
ting of elective primary hip and knee arthro-
spective study evaluating single-unit red blood
plasty. Transfusion 2012;52:1750-60.
cell transfusions in reducing allogeneic blood
123. Vincent JL, Baron JF, Reinhart K, et al. Anemia
exposure. Transfus Med 2005;15:307-12.
and blood transfusion in critically ill patients.
137. Tinmouth A. Reducing the amount of blood
JAMA 2002;288:1499-507.
124. Salisbury AC, Reid KJ, Alexander KP, et al. Diag- transfused by changing clinicians’ transfusion
nostic blood loss from phlebotomy and hospi- practices. Transfusion 2007;47:132S-136S.
tal-acquired anemia during acute myocardial 138. Tinmouth A, MacDougall L, Fergusson D, et al.
infarction. Arch Intern Med 2011;171:1646-53. Reducing the amount of blood transfused. A
125. Chant C, Wilson G, Friedrich JO. Anemia, systematic review of behavioral interventions
transfusion, and phlebotomy practices in criti- to change physicians’ transfusion practices.
cally ill patients with prolonged ICU length of Arch Intern Med 2005;165:845-52.
stay: A cohort study. Crit Care 2006;10:R140. 139. Lam H-TC, Schweitzer SO, Petz MH, et al. Ef-
126. Sanchez-Giron F, Alvarez-Mora F. Reduction of fectiveness of a prospective physician self-au-
blood loss from laboratory testing in hospital- dit transfusion-monitoring system. Transfu-
ized adult patients using small-volume (pedi- sion 1997;37:577-84.
atric) tubes. Arch Pathol Lab Med 2008;132: 140. Fernandez Perez ER, Winters JL, Gajic O. The
1916-19. addition of decision support into computer-
127. Fowler RA, Berenson M. Blood conservation in ized physician order entry reduces red blood
the intensive care unit. Crit Care Med 2003; cell transfusion resource utilization in the in-
31(Suppl):S715-S720. tensive care unit. Am J Hematol 2007;82:631-3.
128. Mukhopadhyay A, Yip HS, Prabhuswamy D, et 141. Damiani G, Pinnarelli L, Sommella L, et al. Ap-
al. The use of a blood conservation device to propriateness of fresh-frozen plasma usage in
reduce red blood cell transfusion require- hospital settings: A meta-analysis of the im-
ments: A before and after study. Crit Care pact of organization interventions. Transfu-
2010;14:R7. sion 2010;50:139-44.
CHAPTER 24 Patient Blood Management 䡲 619
142. Toy P. Effectiveness of transfusion audits and 145. Holcomb J, ed. Standards for a patient blood
practice guidelines. Arch Pathol Lab Med 1994; management program. Bethesda, MD: AABB,
118:435-7. 2014.
143. Brevig J, McDonald J, Zelinka ES, et al. Blood 146. Thomas J. Building a business case. In: Puca K,
transfusion reduction in cardiac surgery: Mul- Johnson ST, eds. Transfusion medicine’s
tidisciplinary approach at community hospi- emerging positions: Transfusion safety officers
tal. Ann Thorac Surg 2009;87:532-9. and patient blood management coordinators.
144. Freedman J, Luke K, Escobar M, et al. Experi- Bethesda, MD: AABB Press, 2013:77-90.
ence of a network of transfusion coordinators 147. Ozawa S, Thorpe E, Valenti J, Waters JH. Devel-
for blood conservation [Ontario Transfusion opment of a blood management program. In:
Coordinators (OnTraC)]. Transfusion 2008; Waters JH, ed. Blood management: Options
48:237-50. for better patient care. Bethesda, MD: AABB
Press, 2008:33-82.
䡲 APPENDIX 24-1
620
Intravenous (IV) Iron Iron high-molecular- Used to treat iron defi- Iron is an essential ele- 䡲 Blood loss is a major cause of iron defi-
Therapy weight dextran; iron low- ciency and iron-defi- ment of hemoglobin and ciency.
molecular-weight dex- ciency anemia. the actual site of oxygen 䡲 Even under the best of circumstances,
tran; iron gluconate; iron binding. It helps trans- oral iron is not well tolerated, and
sucrose; iron carboxy- port oxygen to the tis- patients are often noncompliant due to
methyl dextran. sues. gastrointestinal (GI) symptoms.1
䡲 IV iron given concurrently with ESAs
There is substantial evi- provides better response than oral iron
dence that IV iron is and allows lowering of ESA dose in cer-
effective in treating iron- tain patient populations.1
deficiency anemia in 䡲 High-molecular-weight dextran is asso-
many chronic condi- ciated with serious side effects.1
tions.
Erythropoietic Stimulat- Epoetin alfa: darbepoetin 䡲 Approved for treatment 䡲 ESAs stimulate differ- 䡲 Response seen by day 5 to 7 in iron-
ing Agents (ESAs) alfa. of anemia resulting from entiation of stem cells replete patients.
AABB TECHNICAL MANUAL
chronic kidney failure, into immature red cells; 䡲 One unit equivalent hematocrit increase
chemotherapy, certain increase rate of mitosis by day 7; 3-5 units by day 28.
treatments for human and release of reticulo- 䡲 Darbepoetin is an alternative used in
immunodeficiency virus, cytes into the circula- renal and oncology settings.2,3
and also to reduce the tion; and induce
number of blood transfu- hemoglobin forma-
sions during and after tion.
certain elective noncar- 䡲 A glycoprotein hor-
diac, nonvascular sur- mone produced in the
geries (hemoglobin kidney, a growth factor
>10 g/dL but 13 g/dL). for red cell precursors
in marrow.
䡲 Synthetic erythropoie- 䡲 Society for Thoracic Surgery 2011
tin produced by recom- guidelines report “It is reasonable to
binant DNA technology. use preoperative erythropoietin plus
iron, given several days before cardiac
surgery, for preoperative anemia, in
candidates who refuse transfusion (eg,
Jehovah’s Witnesses), or in patients
who are at high risk for postoperative
anemia.”4
䡲 Black box warning due to increased risk
for death, myocardial infarction, stroke,
venous thromboembolism, thrombosis
of vascular access, and tumor progres-
sion or recurrence.5
Antifibrinolytics Epsilon-aminocaproic 䡲 Enhances hemostasis 䡲 Blocks fibrinolysis by 䡲 Reduces blood loss and transfusion
CHAPTER 24
acid (EACA) associated with surgical inhibiting activation of requirements in cardiac and joint
complications following plasminogen to plas- replacement surgeries.8
heart surgery; orthope- min, which is responsi- 䡲 Rapid intravenous administration
Tranexamic acid (TA)6 dic surgery; some hema- ble for limiting and should be avoided; may induce hypo-
(Similar to EACA, but 10 tologic disorders dissolving clots. tension, bradycardia, and/or arrhyth-
times as potent) associated with throm- 䡲 TA provides equivalent mia.
bocytopenia; and mas- antifibrinolytic effect as 䡲 Use with caution in hematuria of upper
sive traumatic bleeding EACA but at only 1/10 urinary tract origin, unless the possible
in the presence of fibri- the concentration and benefits outweigh risk.
nolysis. has a slower renal 䡲 Contraindicated in patients with dis-
Patient Blood Management
(Continued)
623
䡲 APPENDIX 24-1
624
Concentrates (Continued) treatment of bleeding human plasma. gic and hypersensitivity reactions.18
only in patients with con- 䡲 Precursor to fibrin; 䡲 Thromboembolic events have been
genital fibrinogen defi- thrombin converts reported.18
ciency.18 fibrinogen to fibrin to 䡲 Use of fibrinogen concentrate in obstet-
form soluble fibrin clot, ric hemorrhage and cardiac surgery has
which is then stabilized been reported but is considered off-
by activated Factor XIII. label and future studies are needed to
prove efficacy.12
Other pharmaceuticals Desmopressin (DDAVP) 䡲 Useful in patients with 䡲 Raises circulating Fac- 䡲 Responsiveness to DDAVP in mild
that can help with hemo- synthetic analog of mild hemophilia A or tor VIII and vWF; hemophilia A and type 1 VWD is usu-
stasis vasopressin19 Type I vWD (DDAVP unidentified effect on ally confirmed by elective challenge
is contraindicated in platelets and endothe- (trial).
Type 2B and platelet- lium.12 䡲 Should be used with caution in patients
type VWD). 䡲 Synthetic analog of the with fluid or electrolyte imbalance, as
natural pituitary hor- with cystic fibrosis; patients may
AABB TECHNICAL MANUAL
human thrombin); fibrin is not effective. The types thrombin, which speeds
sealants, polyethylene of bleeding where topical clot formation.
glycol polymer sealants; agents may be used
synthetic adhesives include: diffuse raw sur-
face bleeding, oozing
venous-type bleeding,
bone bleeding, and nee-
dle-hole bleeding.
Pharmaceuticals used Oxytocin 䡲 Obstetric hemorrhage. Stimulates uterine con- Oxytocin is the standard treatment for
Patient Blood Management
primarily in obstetrics to 䡲 FDA-approved to treat tractions. nary, cardiac, renal, and hepatic dis-
control bleeding uterine atony that has ease.
(Continued) not responded to con- 䡲 Asthma is a relative contraindica-
ventional treatment. tion.26,28
Misoprostol 䡲 Obstetric hemorrhage. Synthetic prostaglandin. Works most effectively when used with
䡲 Used to treat uterine other medications listed in this table
atony. (synergistic action).26,27
Other drugs that help Proton pump inhibitors 䡲 Peptic ulcer. Reduces incidence of Proton pump inhibitors are more effec-
with hemostasis 䡲 Upper GI hemorrhage. upper GI rebleeding tive than H2-antagonists in preventing
after sclerotherapy by persistent or recurrent bleeding from
increasing pH to above peptic ulcer, although this advantage
4, which is necessary seems to be more evident in patients
for clot formation and not having adjunct sclerosis therapy.29
stabilization.
AABB TECHNICAL MANUAL
Octreotide 䡲 Variceal hemorrhage. 䡲 An octapeptide that Octreotide and somatostatin are at least
䡲 Acute non-variceal upper mimics natural soma- as effective as the conventional vasoac-
GI bleeding. tostatin pharmacologi- tive drugs and balloon tamponade in the
cally. treatment of variceal hemorrhage with
䡲 Long-acting analog of the advantage of fewer side effects.30
somatostatin (has a
much longer half-life —
approximately 90 min-
utes, compared to 2 to
3 minutes for soma-
tostatin).
䡲 Reduces splanchnic
blood flow via multifac-
torial mechanism.
䡲 Most effective when
combined with sclero-
therapy.
䡲 Eliminates vasospasm
complications seen
with vasopressin.
*Drugs approved for use in the United States as of April 2014. The table is intended to provide general information. Professionals seeking additional information and individuals seeking
personal medical advice should consult a qualified physician.
1. Goodnough LT, Shander A. Current status of pharmacologic therapies in patient blood management. Anesth Analg 2013;116:15-34.
2. Goodnough LT, Monk TG, Andriole GL. Erythropoietin therapy. N Engl J Med 1997;336:933-8.
3. Ross SD, Allen IE, Henry DH, et al. Clinical benefits and risk associated with epoetin and darbepoetin in patient with chemotherapy-induced anemia: A systematic review of the literature. Clin Ther
CHAPTER 24
2006;28:801-31.
4. Ferraris VA, Brown JR, Despotis GJ, et al. Society of Thoracic Surgeons Blood Conservation Guideline Task Force; Society of Cardiovascular Anesthesiologists Special Task Force on Blood Transfusion; Inter-
national Consortium for Evidence Based Perfusion. 2011 update to the Society of Thoracic Surgeons and the Society of Cardiovascular Anesthesiologists blood conservation clinical practice guidelines. Ann
Thorac Surg 2011;91:944-82.
5. Epogen (epoetin alfa) injection for intravenous or subcutaneous use package insert. Thousand Oaks, CA: Amgen, 2012. [Available at: http://pi.amgen.com/united_states/epogen/epogen_pi_hcp_english.pdf
(accessed on March 10, 2013).]
6. Cyklokapron tranexamic acid tablets and tranexamic acid injection package insert. New York, NY: Pfizer, 2014.
7. Use of desmopressin, antifibrinolytics, and conjugated estrogens in hemostasis. In: Goodnight SH, Hathaway WE. Disorders of hemostasis and thrombosis a clinical guide. 2nd ed. New York: McGraw-Hill,
2001:528-42.
8. Henry DA, Carless PA, Moxey AJ, et al. Anti-fibrinolytic use for minimising perioperative allogeneic blood transfusion. Cochrane Database Syst Rev 2011;(3):CD001886.
9. Ipema HJ, Tanzi M. Use of topical tranexamic acid or aminocaproic acid to prevent bleeding after major surgical procedures. Ann Pharmacother 2012;46:97-107.
10. Holbrook A, Schulman S, Witt DM, et al. Evidence-based management of anticoagulant therapy. Antithrombotic therapy and prevention of thrombosis. 9th ed. American College of Chest Physicians evidence-
Patient Blood Management
13. Garcia DA, Baglin TP, Weitz JI, et al. Parenteral anticoagulants. Antithrombotic therapy and prevention of thrombosis. 9th ed. American College of Chest Physicians evidence-based
clinical practice guidelines. Chest 2012;141(Suppl):e24S-e43S.
14. Kcentra (Prothrombin Complex Concentrate, Human). Silver Spring, MD: Food and Drug Administration, 2013. [Available at: http://www.fda.gov/BiologicsBloodVaccines/BloodBloodProducts/ApprovedProd
ucts/LicensedProductsBLAs/FractionatedPlasmaProducts/ucm350130.htm (accessed April 1, 2014).]
(Continued)
627
628
䡲 APPENDIX 24-1
Pharmacologic Therapies for Supporting Patient Blood Management* (Continued)
䡲
15. Holland L, Warkentin TE, Refaai M, et al. Suboptimal effect of a three-factor prothrombin complex concentrate (Profilnine-SD) in correcting supratherapeutic international normalized ratio due to warfarin
overdose. Transfusion 2009;49:1171-7.
16. Siegal DM, Garcia DA, Crowther MA. How I treat target-specific oral anticoagulant-associated bleeding. Blood 2014;123:1153-8.
17. Simpson E, Lin Y, Stanworth S, et al. Recombinant factor VIIa for the prevention and treatment of bleeding in patients without haemophilia. Cochrane Database Syst Rev 2012;(3):CD005011.
18. RiaSTAP, Fibrinogen Concentrate (Human) for Intravenous Use, Lyophilized Powder for Reconstitution. Package insert. Kankakee, IL: CSL Behring, 2011. [Available at http://labeling.cslbehring.com/PI/US/Ri-
aSTAP/EN/RiaSTAP-Prescribing-Information.pdf (accessed on April 6, 2014).]
19. Desmopressin acetate injection package insert. Irvine, CA: Sicor Pharmaceuticals, 2014.
20. Salzman EW, Weinstein MJ, Weintraub RM, et al. Treatment with desmopressin acetate to reduce blood loss after cardiac surgery: A double-blind randomized trial. N Engl J Med 1986;314:1402-6.
21. Cattaneo M, Harris AS, Stromberg U, et al. The effect of desmopressin on reducing blood loss in cardiac surgery: A meta-analysis of double-blind, placebo-controlled trials. Thromb Haemost 1995;74:1064-
70.
22. Crescenzi G, Landoni G, Biondi-Zoccai G, et al. Desmopressin reduces transfusion needs after surgery: A meta-analysis of randomized clinical trials. Anesthesiology 2008;109:1063-76.
23. Mongan PD, Hosking MP. The role of desmopressin acetate in patients undergoing coronary artery bypass surgery: A controlled clinical trial with thromboelastographic risk stratification. Anesthesiology
1992;77:38-46.
24. Laupacis A, Fergusson D. Drugs to minimize perioperative blood loss in cardiac surgery: Meta-analyses using perioperative blood transfusion as the outcome. The International Study of Peri-operative Trans-
fusion (ISPOT) investigators. Anesth Analg 1997;85:1258-67.
25. Spotnitz WD. Hemostats, sealant, and adhesives: A practical guide for the surgeon. The American Surgeon 2012;78:1305-21.
26. Esler MD, Douglas J M. Planning for hemorrhage: Steps an anesthesiologist can take to limit and treat hemorrhage in the obstetric patient. Anesthesiol Clin North Am 2003;21:127- 44.
AABB TECHNICAL MANUAL
27. Soltani H, Hutchon DR, Poulose TA. Timing of prophylactic uterotonics for the third stage of labour after vaginal birth. Cochrane Database Syst Rev 2010;(8):CD006173.
28. Shields L. Uterotonic agents fact sheet. Obstetric hemorrhage toolkit. Stanford, CA: California Maternal Quality Care Collaborative, 2010. [Available at http://www.cmqcc.org/resources/934/download (ac-
cessed January 1, 2013).]
29. Gisbert JP, González L, Calvert X, et al. Proton pump inhibitors versus H2-antagonists: A meta-analysis of their efficacy in treating bleeding peptic ulcers. Aliment Pharmacol Ther 2001;15:917-26.
30. Abid S, Jafri W, Hamid S, et al. Terlipressin vs. octreotide in bleeding oesophageal varices as an adjuvant therapy with endoscopic band ligation: A randomized double-blind placebo-controlled trial. Am J Gas-
troenterol 2009;104:617-23.
CHAPTER 24 Patient Blood Management 䡲 629
䡲 APPENDIX 24-2
Responsibilities for Activity Levels 1, 2, and 3 PBM Programs*
Activity Activity Activity
Item Responsibility Level 1 Level 2 Level 3
Christopher A. Tormey, MD, Assistant Professor of Laboratory Medicine, Yale University School of Medicine,
New Haven, and Medical Director, Transfusion Service, VA Connecticut Healthcare System, West Haven, Con-
necticut and Jeanne E. Hendrickson, MD, Assistant Professor of Laboratory Medicine, Yale University School
of Medicine, and Associate Medical Director, Transfusion Medicine/Apheresis Service, Yale-New Haven Hos-
pital, New Haven, Connecticut
C. Tormey has disclosed no conflicts of interest. J. Hendrickson has disclosed a financial relationship with
Terumo BCT.
The views expressed in this article are those of the authors and do not necessarily reflect the position or policy
of the Department of Veterans Affairs or the United States government.
631
632 䡲 AABB TECHNICAL MANUAL
TABLE 25-1. Compatibility for HSCT by ABO Blood Group of the Donor and the Recipient
incompatible red cells that may be safely in- used to exchange incompatible recipient red
fused, with currently acceptable volumes cells with donor-compatible red cells. In pa-
ranging from 10 to 20 mL. As suggested by tients believed to be at particularly high risk of
some, the recipient’s antibody titer may also the passenger lymphocyte syndrome (usually
be used in guiding facility policy.2 In the ab- based on the type of posttransplant immuno-
sence of red cell depletion or cryopreserva- suppression), prophylactic erythrocytaphere-
tion, plasmapheresis of the recipient immedi- sis may be warranted before transplantation.8
ately before graft infusion may be warranted to
reduce the circulating titer of ABO antibodies. Bidirectional ABO Incompatibility
The second challenge, the continuous
In bidirectional ABO incompatibility, compli-
production of antibodies against the A and/or
cations arising from both major and minor
B antigens of engrafted donor red cells and
erythroid precursors, can continue for up to 3 ABO-incompatible HSCT can occur in the re-
to 4 months after HPC infusion. As a result, cipient. To prevent these complications, the
erythropoiesis is often delayed, with red cell processing of HPC grafts might include the re-
engraftment potentially occurring >40 days af- duction of both red cell and plasma content.
ter transplantation. Time to red cell engraft- The posttransplant period can be complicated
ment may be even further prolonged with the by the onset of hemolysis within days or weeks
use of reduced-intensity or nonmyeloablative (as occurs in minor incompatibility), delayed
conditioning regimens.5,7 In severe cases, pure engraftment of red cells, and/or, in some cas-
red cell aplasia (PRCA) can develop.5 There- es, PRCA (as occurs in major incompatibility).
fore, HSCT involving major ABO incompatibil-
ity may render some recipients transfusion Incompatibility Related to Non-ABO
dependent for several months following trans- Antigens
plantation. Fortunately, major incompatibility Red cell antigens of other blood group systems
tends not to affect engraftment or the produc- can present challenges similar to the ones de-
tion of other myeloid-lineage cells. scribed for ABO-incompatible transplants.
Overall, these incompatibilities are generally
Minor ABO Incompatibility less frequently encountered, but the presence
Analogous to red cell depletion in major in- of red cell antibodies in the patient (more
compatibility, plasma depletion of the graft common) and/or donor (less common) re-
product can significantly decrease donor allo- quires attention and approaches that are simi-
antibodies in minor ABO incompatibility. lar to those outlined above for ABO incompati-
However, even if isoagglutinins are not com- bility. Special consideration of graft donor
pletely removed, the ensuing hemolysis is typ- selection may also be needed when reduced-
ically mild and self-limited.4 A more significant intensity or nonmyeloablative conditioning
problem with minor-incompatible HSCT re- regimens are used in alloimmunized recipi-
sults from the rapid generation of anti-A and/ ents, and cognate graft donor antigen/recipi-
or -B by donor lymphocytes against residual ent alloantibody pairs should be avoided if at
recipient red cells. This so-called “passenger all possible.
lymphocyte syndrome” occurs approximately
5 to 16 days after infusion of HPCs. The patient B LO OD CO M P O N E NT
experiences acute, immune-mediated hemo- CO N SI D E R AT I ON S
lysis that can result in morbidity and even
mortality. Fortunately, in most cases, the he-
Selection of Blood Components
molysis is not severe and eventually subsides
with the clearance of recipient red cells.4 If the The selection of appropriate blood compo-
hemolysis is severe and potentially life threat- nents for recipients of allogeneic HSCT is
ening, therapeutic erythrocytapheresis can be particularly problematic. When determining
634 䡲 AABB TECHNICAL MANUAL
transfusion requirements for an allogeneic stage organ damage, or HSCT recipients in the
HSCT recipient, it is vital that the blood bank postoperative stage.11,12
or transfusion service keep detailed records of The mechanisms underlying anemia and
the patient’s pretransplant ABO group and RBC transfusion dependence in HSCT are suf-
ABO antibody titers and the donor’s ABO ficiently exceptional to warrant brief further
group. It is also imperative to determine the discussion. After intensive chemotherapy, se-
stage of the transplant (ie, preparative period, rum erythropoietin (EPO) levels increase rap-
immediate posttransplant period, or posten- idly and peak in the first week after treat-
graftment period with absence of recipient red ment.13-15 Although recipients of autologous
cells and/or antibodies).9 Recommendations HSCT maintain adequate EPO levels through-
for optimal component selection at each point out the posttransplantation period, allogeneic
in the transplant process are shown in Table recipients do not.16-20 Allogeneic HSCT recipi-
25-2. ents experience a prolonged period of inap-
With regard to Rh(D), the recipient can propriately low endogenous EPO levels, which
continue to receive the type of components often necessitate prolonged RBC transfusion
transfused before the transplantation as long support. Interestingly, mixed results have been
as the HSCT donor and recipient match. How- obtained by the various researchers who have
ever, if the recipient is Rh negative and the do- examined recombinant EPO dosing after allo-
nor is Rh positive or vice versa, only Rh-nega-
geneic HSCT.13-20 Larger studies are necessary
tive Red Blood Cell (RBC) units should be
to determine whether EPO is capable of reduc-
transfused to prevent alloimmunization to the
ing the RBC transfusion burden and prevent-
highly immunogenic D-antigen. Given the
ing the complications of HSCT, such as PRCA.
small risk of D alloimmunization from red
It is noteworthy that the US Food and Drug
cells contained in Rh-positive platelet units,
Administration has recently strengthened its
Rh-negative platelets may be preferred if the
warnings about EPO use after some studies
blood supply allows this. However, it has re-
showed that EPO treatment had adverse ef-
cently been suggested that this practice be re-
fects (decreased survival and/or tumor pro-
evaluated.10
gression) in some patients with cancer.16 Al-
RBC Support though no studies have specifically linked EPO
use to poor outcomes in HSCT patients, clini-
The majority of HSCT recipients require trans- cians should be mindful of the potential risks
fusion support in the peritransplant period, of EPO usage.
regardless of incompatibilities or blood group
antigen mismatches. Symptomatic anemia is Platelet Support
one of the most common indications for RBC
transfusion in HSCT recipients. In the absence Platelet recovery after allogeneic HSCT has
of symptomatic anemia, a patient’s status and been studied extensively. Several factors are
underlying comorbid conditions can be used strongly associated with the rate at which pa-
to determine whether RBC transfusion may be tients become platelet transfusion indepen-
warranted. Because specific RBC transfusion dent, including: 1) the relationship between
thresholds in HSCT populations are lacking, the donor and the recipient (ie, whether they
clinicians can rely on general RBC transfusion are related vs unrelated), 2) conditioning regi-
guidelines. For instance, a threshold hemoglo- men used, 3) presence of graft-vs-host disease
bin level of 7 g/dL is likely appropriate for (GVHD) or cytomegalovirus (CMV) infection,
most stable, nonpostoperative adult HSCT re- and 4) HPC CD34+ cellular source/dose.17-23 To
cipients. However, a slightly higher threshold summarize broadly, the findings of several
of 8 g/dL is likely appropriate for adults with studies suggest that slower platelet engraft-
preexisting heart disease, those at risk of end- ment tends to be more common in patients
TABLE 25-2. Transfusion Support for Patients Undergoing HSCT by Type of ABO Incompatibility and Stage of Transplant
*Due to the short shelf life and limited availability of group AB platelets, it may not always be possible to provide fully matched ABO platelet products as recommended in the table. Therefore,
blood banks and transfusion services may consider providing ABO-mismatched platelets that have been volume reduced to diminish their plasma content.
RBCs = Red Blood Cells.
635
636 䡲 AABB TECHNICAL MANUAL
receiving grafts from unrelated donors, who sions, 2) the transfusion “threshold,” 3) the ap-
have higher-grade GVHD, or who have pre- propriate transfusion dose, and 4) HLA or
transfusion viral infections.17-20 There is also platelet antibodies.
increasing evidence to suggest that the source
of an allogeneic transplant—such as cord Prophylactic vs Therapeutic Platelet
blood HPCs [HPCs(C)] vs HPCs from apheresis Transfusions
[HPC(A)]—is predictive of time to platelet en-
graftment. Several studies have shown that the Platelet transfusions are frequently adminis-
median time to platelet engraftment for tered for prophylaxis of bleeding, rather than
HPC(C) transplants is, on average, significant- to treat acute hemorrhage, in HSCT recipients.
ly longer than for recipients of HPC(A) or HPCs Although this has been a traditional manage-
from marrow.20-23 Thus, longer-term depen- ment approach for many years, several recent
dence on platelet transfusion is more likely for studies have questioned the utility and clinical
individuals in any of the above categories. benefit of this practice. Two studies, both pub-
One major consideration for platelet lished by Wandt and colleagues,27,28 examined
transfusion in HSCT recipients is compatibili- the assumption that prophylactic platelet
ty. Plasma (including the significant volume of transfusion could be replaced by therapeutic
plasma in platelets) contains variable amounts transfusion in recipients of autologous trans-
of isoagglutinins. Although transfusion of lim- plants. The researchers found that although
ited quantities of ABO-incompatible plasma patients assigned to a therapeutic (rather than
and platelets is common in routine transfu- prophylactic) transfusion regimen had some
sion, this practice cannot be readily applied to bleeding, there was no evidence of life-threat-
the recipients of allogeneic HSCT without ening hemorrhage. Moreover, therapeutic in-
careful consideration.24 In ABO-incompatible terventions resulted in dramatic reductions in
HSCT, the plasma-containing components platelet usage. However, Stanworth and
should be compatible with both the donor and colleagues29 concluded, based on a recently
the recipient whenever possible. Recommen- published trial, that prophylactic platelet
dations for component selection are more ful- transfusions were more effective in preventing
ly described in Table 25-2. hemorrhage in patients with hematologic ma-
In addition to the risk of hemolysis, some lignancies than in a nontransfused control
researchers have found other morbidities as- population. Therefore, questions still remain
sociated with ABO-incompatible platelet regarding the nature of appropriate, evidence-
transfusions. For instance, one pediatric study based prophylactic transfusion strategies for
showed that such transfusions, when com- patients with thrombocytopenia. The results
bined with the use of melphalan, correlated of the studies conducted to date and future tri-
with the development of hepatic venoocclu- als will help shape prophylactic platelet trans-
sive disease, possibly resulting from the bind- fusion practice for HSCT recipients.30
ing of A- and/or B-antigens expressed on the
surface of hepatic endothelial cells.25 There- Platelet Transfusion Threshold
fore, some centers have an institutional policy
requiring the transfusion of ABO-identical The acceptable threshold for prophylactic
RBCs and platelets only to HSCT recipients; at transfusion of platelets has been studied ex-
least one group has noted that this policy may tensively for >20 years. One of the earliest es-
be associated with improved patient survival.26 tablished thresholds to prevent spontaneous
There is an obvious need for additional pro- hemorrhage was a platelet count of <20,000/µL
spective studies to address this issue. for patients undergoing chemotherapy/HSCT.31
In the context of platelet transfusion Over time and as clinical trial data have ac-
support in HSCT recipients, the following ad- crued, studies have consistently revealed that
ditional areas are discussed more extensively a platelet count <10,000/µL in uncomplicated
below: 1) prophylactic vs therapeutic transfu- thrombocytopenia (ie, in patients without co-
CHAPTER 25 HSCT Recipient Transfusion Support 䡲 637
existing conditions such as fever, bleeding, or of the evaluation and treatment of platelet re-
bacteremia/sepsis) is a reasonable threshold fractoriness driven by HLA and HPA antibod-
to prevent increased bleeding or hemorrhage- ies, see Chapters 18 and 19.
related morbidity.30,32-35 However, a study by In addition to leading to potential prob-
Nevo and colleagues36 raises questions about lems with platelet transfusion, recipient allo-
this threshold. In this study, HSCT recipients antibodies against HPA or HLA could delay
who received transfusions at a platelet count platelet or white cell engraftment in some
<10,000/µL had significantly increased non- transplant settings. This is analogous to what
hemorrhagic mortality rates and reduced sur- occurs in the red cell lineage with major ABO-
vival compared to those infused at counts mismatched transplants. The issue of HPA and
<20,000/µL. Although changes in the 10,000/ HLA matching in HSCT donor-recipient pairs
µL threshold should not be based on this study has also been raised by several groups because
alone, it is imperative that data continue to be of concerns that mismatches between donors/
collected to examine the appropriateness of recipients for HPA (which may serve as minor
this target platelet count and, as noted previ- histocompatibility antigens) may increase the
ously, whether prophylactic platelet transfu- risk of GVHD, although two studies have found
sions are necessary at any platelet count.30 otherwise.41,42
treatment for patients with intractable bleed- the clinical efficacy of transfused granulocytes
ing. in the setting of HSCT, a multicenter random-
Another possible hemostasis therapy aris- ized controlled trial is currently under way.49
es from the fact that GVHD often results in the The results of this study will be helpful in un-
depletion of Factor XIII, increasing the risk and derstanding the potential benefit of granulo-
severity of gastrointestinal hemorrhage.45 The cyte infusion for severely ill HSCT recipients.
high concentration of Factor XIII in cryopre-
cipitate makes cryoprecipitate a potentially
useful therapy for GVHD-related bleeding.
Formal studies are required to determine the S PE C I A L P ROC ES SI N G O F
efficacy of this approach. B LO OD CO M P O N E NTS F O R
H S C T R E C I PI E N TS
Transfusion Support for Autologous
The irradiation of cellular blood products (eg,
Transplant Recipients
RBCs, platelets, and granulocytes) is intended
Because recipients of autologous transplants to inhibit the proliferation of donor lympho-
are not exposed to foreign graft products from cytes, thereby preventing transfusion-associ-
donors, virtually all of the concerns regarding ated GVHD, a reaction that is almost uniformly
major/minor incompatibility (and their im- fatal.9,50 Although it is generally accepted that
pact on transfusion support) are not applica- HSCT recipients require irradiated compo-
ble to this patient group. Before, during, and nents during, and for at least 1 year after,
after HSCT, autologous recipients should be transplantation, it is unclear whether these pa-
supported by transfusions of blood compo- tients require irradiated components after this
nents as needed and in compliance with stan- time. Despite the absence of evidence indicat-
dard protocols that are applicable to any trans- ing that irradiation is essential after this time
fusion recipient. However, because of their
has elapsed, many institutions provide irradi-
underlying immunosuppression, autologous
ated products indefinitely to HSCT recipients.
recipients may require specialized products or
This is likely a prudent policy given the poten-
components involving additional processing
tial for lifelong immunosuppression associat-
steps. Such issues and concerns (applicable to
ed with HSCT and the possibility of relapse of
both autologous and allogeneic transplant re-
cipients) are discussed in greater detail below. the malignancy for which the patient initially
underwent the HSCT.
Blood banks and transfusion services
must rigorously ensure irradiation of compo-
PAT I E N TS W I T H N E U T RO PEN I A nents transfused to HSCT recipients. Part of
A N D I N F E C T IO N T H AT I S this vigilance is the development of systems or
U N R ES P O N S IV E TO policies to identify patients who require irradi-
ANTI MIC RO BIA L T H E R A P Y ated products. One approach to help reduce
the possibility of inadvertently releasing a
Traditionally, infusions of fresh granulocytes
have been used to treat severe, antibiotic- nonirradiated unit for transfusion to an HSCT
refractory bacterial or fungal infections in pa- recipient is to require universal irradiation of
tients with absolute neutrophil counts less selected blood components. In some institu-
than 500/µL.46,47 (For a more in-depth discus- tions, all platelet products are irradiated upon
sion of granulocyte therapy, see Chapter 18.) receipt from the regional blood center. Al-
To date, one study has demonstrated that neu- though this strategy may not be practical for
trophil transfusions can be efficacious in treat- other components, such as RBCs, additional
ing infection in HSCT recipients with neutro- approaches may be developed, such as irradi-
penia; however, this Phase I/II study included ation of all components issued to in- and out-
only 11 participants.48 To more fully establish patient hematology/oncology wards.
CHAPTER 25 HSCT Recipient Transfusion Support 䡲 639
above 50,000/µL (to prevent cerebrovascular and supporting such patients can be a signifi-
bleeding).58,59 Such results may also be impor- cant challenge for the transfusion service.
tant in HPC donor selection for non-HLA- The flow of information from the trans-
matched, nonmyeloablative transplants. An plant center to local providers is rarely perfect;
additional consideration is to perform red cell with increased complexity of care, critical data
exchange transfusion before transplantation can be overlooked. Some institutions have cre-
with a goal of <30% to 45% hemoglobin S to ated a process to deal with such situations. For
prevent vasoocclusive events resulting from example, the nationally networked Depart-
granulocyte colony-stimulating factor admin- ment of Veterans Affairs medical center data-
istration.60 base can be consulted to detect any ABO or Rh
Beta-thalassemia major and severe aplas- discrepancies and determine whether a partic-
tic anemia may be cured by matched sibling ular patient has ever had an alloantibody de-
HSCT in childhood, yet prior transfusion ex- tected at another network hospital. If a trans-
posure may adversely influence engraftment fusion service or blood bank does have an
and outcomes in subsequent transfusions.61-63
interconnected hospital laboratory informa-
Potential reasons for this association include
tion system, such databases can be useful for
humoral or cellular immunization to trans-
gathering information on transplant recipi-
fused antigens (whether they are defined or
ents.
other minor histocompatibility antigens) or
Regardless of the availability of an inter-
iron overload. Early transplantation for eligible
connected information system, the transfu-
patients may decrease the risk of graft rejec-
sion service should consider developing a pol-
tion, and careful attention to iron status at the
time of transplantation is also recommended. icy with its hematology-oncology colleagues
The intensity of conditioning regimens is also that clearly outlines the approach to patients
likely a key factor in outcomes of HSCT in pa- who receive their HSCT at an outside facility.
tients with beta-thalassemia major or severe This policy should call for communicating the
aplastic anemia, and their long-term risk/ben- following important information to a patient’s
efit ratios deserve careful consideration. usual health-care facility after that patient re-
turns from an off-site transplant facility: 1)
whether the transplant was autologous or allo-
IN F OR MATI O N PO RTA B IL I T Y geneic; and, 2) if the transplant was allogeneic,
FOR HSCT RECIPI ENTS the ABO type of the donor, and any auto- or al-
Many patients undergo HSCT far from their loantibodies developed by the patient during
usual medical institution. Before transplanta- care at that facility. In some cases, if such in-
tion procedures, all of the patient’s transfusion formation is not completely communicated,
history (including red cell, platelet, or leuko- the transfusion service can contact the trans-
cyte alloantibody testing history) should be plant center and obtain the required informa-
communicated to the transplanting facility. tion at the time of the patient’s first posttrans-
Eventually, these patients may return for fol- plant visit. A portable worksheet with these
low-up care to their primary care physicians, data should also be made available to patients.
KEY POINTS
1. Allogeneic HSCT recipients face distinct transfusion challenges because of their immuno-
suppression, preexisting diseases, and potential for changes in their expressed blood group
systems.
2. ABO compatibility is not critical in the selection of potential HSCT donors because pluripo-
tent and early-committed HPCs lack ABO antigens. ABO incompatibility does, however, in-
fluence transfusion decisions during the peritransplant period.
CHAPTER 25 HSCT Recipient Transfusion Support 䡲 641
3. ABO incompatibilities, which are present in 20% to 40% of donor/recipient pairs, fall into
three categories: 1) incompatible in the major crossmatch, 2) incompatible in the minor
crossmatch, and 3) bidirectionally incompatible. Such incompatibilities have the potential
to produce acute and, in the case of major incompatibility, ongoing intravascular hemolysis
and pure red cell aplasia. They can be partially mitigated by red cell and/or plasma deple-
tion of the graft before infusion.
4. Management approaches to transfusion are similar for adult and pediatric HSCT recipients;
however, indications for transplantation, stem cell source, and donor selection may be sub-
tly different.
5. When transfusion requirements for allogeneic HSCT recipients are determined, it is vital
that the blood bank or transfusion service keep detailed records of the recipient’s pretrans-
plant ABO group and the donor’s ABO group. It is also imperative to determine the stage of
the transplant.
6. Platelet concentrates are most frequently transfused to HSCT recipients to prevent an acute
hemorrhage. A platelet count threshold of 10,000/µL in uncomplicated thrombocytopenia
is widely accepted as safe for allogeneic recipients.
7. Cellular blood components are irradiated to inhibit the proliferation of donor lymphocytes,
thereby preventing transfusion-associated GVHD. Many services provide irradiated compo-
nents indefinitely to HSCT recipients.
8. RBC and platelet components that have been leukocyte reduced before storage are general-
ly considered to be equivalent to CMV-seronegative units in terms of CMV transmission
risk. Some studies, however, suggest that seronegative units may be marginally safer.
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marrow transplantation for sickle cell anemia. transplants for acquired aplastic anaemia: A
Blood 1995;85:879-84. report from the Aplastic Anaemia Working
59. McPherson ME, Anderson AR, Haight AE, et al. Party of the European Bone Marrow Trans-
Transfusion management of sickle cell pa- plant Group. Bone Marrow Transplant 1994;
tients during bone marrow transplantation 13:233-7.
C h a p t e r 2 6
Therapeutic Apheresis
Robertson D. Davenport, MD
THERAPEUTIC APHERESIS IS the stances in the blood, the volume of blood pro-
treatment of diseases through removal cessed, and the equilibrium between blood
or extracorporeal manipulation of blood com- and the substance’s extravascular volume of
ponents or specific blood substances. It is dis- distribution. The procedure is most efficient at
tinct from blood component collection by the beginning because the amount of the com-
apheresis, which is covered in Chapter 7. Stan- ponent removed decreases exponentially with
dards and guidelines for therapeutic aphere- time (Fig 26-1). Continued production or mo-
sis, clinical privileging, and documentation are bilization from the extravascular space will re-
provided by AABB and The American Society sult in a less-than-expected reduction. A 1.0
for Apheresis (ASFA).1-4 plasma volume exchange typically removes
approximately two-thirds of a substance if it
does not move significantly from the extravas-
P R I N C IP L E S A N D M O D A L I T IE S
cular to the intravascular space.
The goal of therapeutic apheresis is to remove Continuous flow centrifuge apheresis de-
a pathologic element from blood or to modu- vices have a rotating channel designed to in-
late cellular function by manipulation such as troduce whole blood at one site, and the blood
through extracorporeal photopheresis (Table elements subsequently separate by density as
26-1), with or without replacement of the re- blood flows through the channel. The resulting
moved element. Apheresis can be performed layers of plasma, platelets, leukocytes, or red
manually, but automated techniques are faster cells can be removed selectively. The compo-
and more efficient. In therapeutic plasma ex- nent to be removed is diverted into a collec-
change (TPE), 1.0 or 1.5 plasma volumes are tion bag, while the remaining blood compo-
typically processed. Larger volume procedures nents are mixed with the replacement fluid
can increase the risk of coagulopathy, citrate and returned to the patient. The device con-
toxicity, or electrolyte imbalance, depending trols the flow rates of blood withdrawal, anti-
on the replacement fluid. The effectiveness of coagulant solution and replacement fluid in-
apheresis in removing pathologic substances fusion, as well as centrifuge speed to achieve
depends on the concentration of those sub- optimal separation.
Robertson D. Davenport, MD, Medical Director, Blood Bank and Transfusion Service, and Associate Professor,
University of Michigan Health System, Ann Arbor, Michigan
The author has disclosed no conflicts of interest.
645
646 䡲 AABB TECHNICAL MANUAL
FIGURE 26-1. Theoretical removal of a substance by apheresis. For a substance that is strictly intravascular,
36.8% of the initial concentration remains after a single blood volume exchange. However, if the substance is
also present in the extravascular space with a total volume of distribution equal to twice the blood volume,
60.6% will remain after a single blood volume has been processed.
CHAPTER 26 Therapeutic Apheresis 䡲 647
Typical Course
(Number of
Indication Modifying Conditions Category treatments)
Antiglomerular basement membrane Dialysis dependent and no DAH III Daily or QOD
disease (Goodpasture syndrome) (7-10)
DAH I
Dialysis independence I
Aplastic anemia; pure red cell aplasia Aplastic anemia III Daily or QOD
(variable)
Pure red cell aplasia III
Autoimmunic hemolytic anemia: Severe WAIHA III Daily or QOD
WAIHA; cold agglutinin disease (variable)
Severe cold agglutinin disease II
Burn shock resuscitation III Once
Cardiac transplantation Desensitization, positive cross- III Daily or QOD
match due to donor specific HLA (variable)
antibody
Antibody-mediated rejection III
Catastrophic antiphospholipid II Daily (3-5)
syndrome
Chronic focal encephalitis (Rasmussen III QOD (3-6)
encephalitis)
Chronic inflammatory demyelinating I 2-3/week
polyradiculoneuropathy (variable)
Coagulation factor inhibitors Alloantibody IV
Autoantibody III Daily (variable)
Cryoglobulinemia Symptomatic/severe I QOD (3-8)
Dermatomyositis or polymyositis IV
Dilated cardiomyopathy, idiopathic NYHA II-IV III QOD (5)
CHAPTER 26 Therapeutic Apheresis 䡲 649
Typical Course
Number of
Indication Modifying Conditions Category treatments)
Typical Course
(Number of
Indication Modifying Conditions Category treatments)
Typical Course
(Number of
Indication Modifying Conditions Category treatments)
neuropathy (AIDP and CIDP), antiglomerular clude renal transplantation with presensitiza-
basement membrane antibody disease, and tion, and antibody-mediated organ transplant
myasthenia gravis. Examples of conditions in rejection.
which the goal is to remove an alloantibody in-
652 䡲 AABB TECHNICAL MANUAL
In myeloma with hyperviscosity, the goal patients, 43% in the TPE group and none in the
is to remove an excessive paraprotein (M pro- control group recovered renal function. An-
tein). Measurement of plasma viscosity may other randomized clinical trial showed no
not be useful in guiding therapy in some pa- benefit of TPE in a composite outcome mea-
tients because plasma viscosity may not corre- sure of death, dialysis dependence, and glo-
late with symptoms. Normal plasma viscosity merular filtration rate.9 However, biopsy con-
ranges from 1.4 to 1.8 centipoise (cP). Because firmation of the renal diagnosis was not
most patients are not symptomatic until the required in this trial. Similarly, a retrospective
plasma viscosity is more than 4.0 or 5.0 cP, pa- cohort study showed no benefit of TPE in ei-
tients with mild elevations may not require ther reducing mortality or preserving renal
treatment. In general, hyperviscosity becomes function.10 If TPE is to be undertaken, biopsy
a concern when M protein concentrations confirmation of cast nephropathy may be ad-
reach 3 g/dL for IgM, 4 g/dL for IgG, and 6 g/dL visable.
for IgA.6 Patients receiving rituximab (anti- Diseases in which immune complexes
CD20) therapy for IgM myeloma may experi- may be pathogenic and can be removed by
ence a transient increase in M protein levels. apheresis include rapidly progressive glomer-
Patients with pretreatment IgM greater than ulonephritis, cryoglobulinemia, and vasculi-
5 g/dL are at particular risk of developing tis. Other indications include conditions treat-
symptomatic hyperviscosity.7 ed by removal of protein-bound drugs or
There are conflicting data regarding the toxins or high-concentration lipoproteins.
efficacy of TPE for treatment of acute renal In TTP, a deficiency of the vWF-cleaving
failure in myeloma. A randomized controlled metalloprotease ADAMTS-13 results in accu-
trial of TPE vs. conventional care showed no mulation of high-molecular-weight vWF mul-
difference in mortality or renal function at 6 timers with subsequent intravascular platelet
months.8 However, among dialysis-dependent activation and platelet-rich thrombi in the
CHAPTER 26 Therapeutic Apheresis 䡲 653
capacity. Indications for cytapheresis are listed transcranial Doppler imaging, transfusion re-
in Table 26-4. duces the risk of stroke.28,29 Chronic red cell ex-
In acute leukemia, high blast counts (typ- change, typically every 4 to 6 weeks, can be ef-
ically >100,000/L) can result in microvascu- fective in normalizing cerebral blood flow
lar stasis with headache, mental status chang- while minimizing iron overload associated
es, visual disturbances, or dyspnea. The with simple transfusions. Red cell exchange
leukocyte count at which a patient may be- may have a role in other sickle cell syndromes,
come symptomatic is variable. Typically, pa- including priapism, multiorgan failure, hepat-
tients with acute or chronic lymphocytic leu- ic/splenic sequestration, intrahepatic cho-
kemia tolerate higher cell counts than patients lestasis. In addition, the technique may be
with myelogenous leukemia, and cytapheresis used for prevention of iron overload, and pre-
may not be required. Cytapheresis commonly vention or management of vaso-occlusive
results in a less-than-predicted reduction in pain crisis.
leukocyte count, despite excellent collection, RBCs for replacement must be ABO com-
because of mobilization and re-equilibration patible and negative for known clinically sig-
of cells from extravascular sites. Myelogenous nificant alloantibodies. For sickle cell disease,
leukemia cells commonly have a higher densi- the RBCs should be matched for C, E, and K, if
ty than lymphocytic cells and can be difficult possible.30 It is desirable to use relatively fresh
to separate from red cells. Use of hydroxyethyl units to maximize posttransfusion red cell
starch enhances red cell sedimentation by survival. Units containing either citrate-phos-
rouleaux formation and can improve the effi- phate-dextrose-adenine (CPDA)-1 or additive
ciency of cytapheresis in acute myelogenous solutions (AS) may be used. It is desirable that
leukemia. Massive thrombocytosis, typically all units used in a given procedure contain the
>1,000,000/L, can occur in essential throm- same anticoagulant solution so that they have
bocythemia, polycythemia vera, or as a reac- similar hematocrits. Chronic red cell ex-
tive phenomenon. Such patients may be at risk change may carry a lower risk of alloimmuni-
of thrombosis or hemorrhage. Reduction in zation than simple transfusion in sickle cell
platelet count is commonly less than predicted disease.31
because of mobilization of platelets to the pe-
ripheral blood, primarily from the spleen. ECP
Red cell exchange is most commonly per-
formed in the setting of sickle cell disease ECP is a specialized procedure in which the
(SCD). The goal is both to reduce the burden of buffy-coat layer is collected from peripheral
hemoglobin S and to provide donor red cells blood, treated with 8-methoxypsoralen and ul-
containing hemoglobin A. Acute chest syn- traviolet A light, and re-infused into the pa-
drome is a serious complication of SCD, pre- tient. The treatment causes cross-linking of
senting as dyspnea, chest pain, and cough, of- leukocyte DNA, which prevents replication
ten accompanied by fever, leukocytosis, and induces apoptosis. The procedure was de-
decreasing hematocrit, and pulmonary infil- veloped for the treatment of cutaneous T-cell
trates. Respiratory failure can develop, and lymphoma, although it is increasingly used for
death occurs in about 3% of cases.25 Red cell other indications (Table 26-5). ECP has com-
exchange is indicated for progressive infil- plex immunomodulatory effects, including in-
trates and hypoxemia refractory to conven- duction of monocyte differentiation to den-
tional therapy and simple transfusion.26,27 A dritic cells, alteration of T-cell subsets, and
common goal is to reduce hemoglobin S to changes in cytokine production profiles.32 ECP
less than 30% with a final hematocrit not to ex- may be effective in acute and chronic skin
ceed approximately 30%. Red cell exchange graft-vs-host disease (GVHD), although the
may also be indicated for prevention of stroke role in non-skin GVHD is less well defined.33
in sickle cell anemia. For patients with elevat- ECP for solid organ transplant rejection
ed cerebral blood flow velocity determined by has been best studied in cardiac and lung
CHAPTER 26 Therapeutic Apheresis 䡲 655
Dermatomyositis or Leukocytapheresis IV
polymyositis
Lymphocytapheresis III
RCE = red cell exchange; HSCT = hematopoietic stem cell transplantation; HPC = hematopoietic progenitor cells.
656 䡲 AABB TECHNICAL MANUAL
transplantation. In a randomized clinical trial, approved by the Food and Drug Administra-
prophylactic photopheresis in conjunction tion (FDA).
with previous generation immunosuppres- In the two FDA-approved LDL apheresis
sion (not including calcinurin inhibitors or devices, selective removal of LDL can be ac-
mycophenolate) resulted in fewer rejection complished by passing heparinized plasma
episodes, decreased HLA antibodies, and re- over a dextran sulfate column or beads coated
duced coronary artery intimal thickness, but with anionic polyacrylate ligands, or by pre-
no difference in time to first episode, inci- cipitation of heparin-LDL complexes in acidi-
dence of hemodynamic compromise, or sur- fied plasma. Because LDL production contin-
vival at 6 or 12 months.34,35 In recurrent cardiac ues, LDL apheresis treatments must be
rejection, photopheresis may decrease severi- repeated, typically at 2-week intervals, indefi-
ty of rejection and allow for reduced dosage of nitely. There is evidence that LDL apheresis re-
duces the incidence of major coronary events
immunosuppressives.36 ECP may have a role in
and stroke.39 In addition, atherosclerotic
the setting of cardiac rejection with hemody-
plaque regression can occur in some pa-
namic compromise and for bronchiolitis oblit-
tients.40 Secondary effects of LDL apheresis
erans syndrome status after lung transplanta-
that may be beneficial include reduction in
tion.37,38
levels of C-reactive protein, fibrinogen, tissue
factor, and soluble adhesion molecules.41,42
Selective Adsorption
LDL apheresis may also be used in the treat-
There are currently few established indica- ment of primary or recurrent FSGS, although
tions for selective adsorption of plasma pro- the mechanism of action has not been well de-
teins (Table 26-6) and few devices have been fined.43
CHAPTER 26 Therapeutic Apheresis 䡲 657
Treatment
Indication Modifying Conditions Modality Category
IgG can be selectively removed by passing an alternative mechanism has been proposed
plasma over a column of staphylococcal pro- in ITP.44 Staphylococcal protein A adsorption
tein A bound to silica. The putative mecha- treatment can be performed manually or in
nism of action is the removal of pathogenic au- conjunction with automated TPE. Affinity col-
toantibodies or immune complexes, although umns containing anti-IgG or ABO blood group
658 䡲 AABB TECHNICAL MANUAL
substances have been tested in clinical trials, TABLE 26-7. Reported Frequency of Adverse
but are not currently approved for use in the Reactions to Apheresis*
United States.
Reaction Frequency (%)
causes, such as pulmonary edema, pulmo- When plasma is exposed to foreign sur-
nary embolism, air embolism, obstruction of faces of plastic tubing or filtration devices, the
the pulmonary microvasculature, anaphylac- kinin system can be activated, resulting in pro-
tic reactions, and transfusion-related acute duction of bradykinin. Infusion of plasma con-
lung injury (TRALI).49 Hemothorax or hemo- taining bradykinin can cause abrupt hypoten-
pericardium resulting from vascular erosion sion. Patients taking angiotensin-converting
due to a central venous catheter is rare, but enzyme (ACE) inhibitors are more susceptible
when it occurs it is typically unsuspected, and to the hypotensive reactions because the
may be fatal.50 Pulmonary edema that results drugs block enzymatic degradation of bradyki-
from volume overload or cardiac failure is usu- nin.53 Hypotensive reactions are more likely
ally associated with dyspnea, an increase in di- during selective adsorption procedures be-
astolic blood pressure, and characteristic chest cause the devices expose plasma to a very
radiograph findings. Acute pulmonary edema large surface area. Because some ACE inhibi-
may also arise from damage to alveolar capil- tors have a long duration of action, stopping
lary membranes secondary to an immune re- the drug only the day before the procedure
action or to vasoactive substances in plasma may not be sufficient to prevent a reaction.
or colloid solutions prepared from human Intensive TPE without plasma replace-
plasma. Predominantly ocular reactions (peri- ment causes depletion of coagulation factors.
orbital edema, conjunctival swelling, and tear- A 1.0 plasma volume exchange will typically
ing) have occurred in donors sensitized to the reduce coagulation factor levels by 25% to
ethylene oxide gas used to sterilize disposable 50%, though Factor VIII levels are less affect-
plastic apheresis kits. 51,52 ed.54 Levels of fibrinogen, a large molecule
Hypotension during apheresis can be a without extravascular distribution, are re-
sign of citrate toxicity; hypovolemia; or a vaso- duced by about 66%. If the patient has normal
vagal, allergic, drug, or transfusion reaction. hepatic synthetic function, coagulation factor
Hypovolemia can occur early in a treatment of levels typically return to near normal within 2
a small patient when the return fluid consists days. Thus, many patients can tolerate TPE ev-
of the saline used to prime the apheresis cir- ery other day for 1 or 2 weeks without develop-
cuit. Vasovagal reactions are characterized by ing significant coagulopathies that require
bradycardia and hypotension. Such reactions plasma replacement.
usually respond well to a fluid bolus and plac- Bleeding as a consequence of coagula-
ing the patient in the Trendelenburg position. tion factor depletion is rare but has been re-
When hypotension occurs during plasma ported. For patients at risk, plasma may be
or red cell exchange, a transfusion reaction used for replacement at the end of the proce-
such as TRALI, acute hemolysis, bacterial con- dure. Apheresis can also cause thrombocyto-
tamination, or anti-IgA-related anaphylaxis penia, particularly with HPC collection. Inten-
should be considered. Hypotension is more sive TPE can cause hypogammaglobulinemia.
frequent in children, the elderly, neurology pa- Serum levels of IgG and IgM recover to about
tients, anemic patients, and those treated with 40% to 50% of the preapheresis level at 48
intermittent-flow devices that have large ex- hours.54 The absolute immunoglobulin level at
tracorporeal volumes. Continuous-flow devic- which a patient becomes at risk of infections
es typically do not have large extracorporeal has not been established.
volumes but can produce hypovolemia if re- Albumin-bound drugs are removed by
turn flow is inadvertently diverted to a waste TPE. This can result in subtherapeutic levels of
collection bag, either through operator over- medications unless dosage adjustments are
sight or device malfunction. Hypovolemia may made. High-molecular-weight biologicals
also be secondary to inadequate volume or such as IVIG, antithymocyte globulin, and
protein replacement. During all procedures, it monoclonal antibodies have a long intravas-
is essential to maintain careful and continuous cular half-life and are readily removed by
records of the volumes removed and returned. apheresis. TPE shortly after administration of
660 䡲 AABB TECHNICAL MANUAL
such drugs should be avoided because it may The choice of placement site for a central
significantly impair their effectiveness. In ad- catheter is influenced by the expected dura-
dition, intensive apheresis reduces the con- tion of treatment. Subclavian or internal jugu-
centration of potentially diagnostic plasma lar access is generally preferable for treat-
constituents, so blood for testing should be ments lasting up to several weeks. Femoral
drawn before initiating a course of treatment. access should be used only temporarily be-
Collapsed or kinked tubing, malfunction- cause of the higher risk of infection. Patients
ing pinch valves, or improper threading of tub- requiring long-term treatment usually have a
ing may damage red cells in the extracorporeal tunneled catheter. With proper care, tunneled
circuit. Instrument-related hemolysis has catheters can be used for prolonged periods.
been reported in 0.06% of therapeutic aphere- Good catheter care is very important to
sis procedures.45 Hemolysis can also occur maintain patency and prevent complications.
with the use of incompatible replacement flu- Catheters need to be flushed regularly. Hepa-
ids such as 5% dextrose solution (eg, D5W rin or 4% trisodium citrate is usually placed in
used to dilute 25% albumin) or ABO-incom- each lumen after each use to prevent occlu-
patible plasma. The operator should carefully sion by clots. If a port becomes clotted, instil-
observe plasma collection lines for pink dis- lation of a fibrinolytic agent such as urokinase
coloration suggestive of hemolysis. Other or recombinant tissue plasminogen activator
types of equipment failure such as problems may restore patency. Routine dressing care is
essential to prevent insertion site infections.
with the rotating seal, leaks in the plastic, and
An arteriovenous (AV) fistula may also be used
roller pump failure are rare.
for apheresis, but apheresis personnel should
Fatalities during apheresis are rare. Death
be suitably trained before attempting to access
has been reported in 0.006% to 0.09% of thera-
an AV fistula.
peutic procedures.45,55 Most fatalities are at-
Venous access devices may cause further
tributable to underlying medical conditions.
vascular damage, sometimes resulting in
thrombosis. Infrequently, they may result in
severe complications such as pneumothorax
VAS C U L A R AC C E S S or perforation of the heart or great vessels.
Other complications include arterial puncture,
Therapeutic apheresis requires good vascular deep hematomas, and arteriovenous fistula
access to achieve adequate flow rates. Periph- formation. Bacterial colonization often com-
eral access generally requires at least a 17- plicates long-term placement and may lead to
gauge needle for blood withdrawal and at least catheter-associated sepsis, especially in pa-
an 18-gauge catheter for return. Patients with- tients who are receiving immunosuppressants.
out adequate peripheral veins or who require Inadvertent disconnection of catheters may
multiple frequent procedures may require produce hemorrhage or air embolism.
central venous catheters. Central venous cath-
eters for apheresis must have rigid walls to ac-
commodate the negative pressure generated
in the withdrawal line. Peripherally inserted PAT IE N T EVA L U A T IO N
central catheters (PICC lines) typically do not All patients should be evaluated by a physician
support the high flow rates used in apheresis familiar with apheresis before treatment be-
and should not be used. At least two lumens gins. The indication, type of procedure, fre-
are required for continuous-flow procedures, quency and number of treatments, and the
although single-lumen catheters can be used goal or endpoint should be documented in the
for intermittent-flow procedures. An implant- patient’s medical record. The nature of the
ed subcutaneous port may be an option for procedure, the expected benefits, the possible
some patients requiring long-term treatment, risks, and the available alternatives must be
such as chronic red cell exchange. explained to the patient, and the patient’s con-
CHAPTER 26 Therapeutic Apheresis 䡲 661
KEY PO I NT S
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32. Bladon J, Taylor PC. Extracorporeal photo- apheresis reduces P-Selectin, CRP and fibrino-
pheresis: A focus on apoptosis and cytokines. gen—possible important implications for im-
J Dermatol Sci 2006;43:85-94. proving atherosclerosis. Ther Apher Dial 2006;
33. Del Fante C, Scudeller L, Viarengo G, et al. Re- 10:219-23.
sponse and survival of patients with chronic 43. Muso E, Mune M, Yorioka N, et al. Beneficial
graft-versus-host disease treated by extracor- effect of low-density lipoprotein apheresis
poreal photochemotherapy: A retrospective (LDL-A) on refractory nephrotic syndrome
study according to classical and National Insti- (NS) due to focal glomerulosclerosis (FGS).
tutes of Health classifications. Transfusion Clin Nephrol 2007;67:341-4.
2012;52:2007-15. 44. Silverman GJ, Goodyear CS, Siegel DL. On the
34. Barr ML, Baker CJ, Schenkel FA, et al. Prophy- mechanism of staphylococcal protein A im-
lactic photopheresis and chronic rejection: Ef- munomodulation. Transfusion 2005;45:274-
fects on graft intimal hyperplasia in cardiac 80.
transplantation. Clin Transplant 2000;14:162- 45. McLeod BC, Sniecinski I, Ciavarella D, et al.
6. Frequency of immediate adverse effects asso-
35. Barr ML, Meiser BM, Eisen HJ, et al. Photo- ciated with therapeutic apheresis. Transfusion
pheresis for the prevention of rejection in car- 1999;39:282-8.
664 䡲 AABB TECHNICAL MANUAL
46. Norda R, Berseus O, Stegmayr B. Adverse 51. Leitman SF, Boltansky H, Alter HJ, et al. Aller-
events and problems in therapeutic hema- gic reactions in healthy plateletpheresis do-
pheresis. A report from the Swedish registry. nors caused by sensitization to ethylene oxide
Transfus Apher Sci 2001;25:33-41. gas. N Engl J Med 1986;315:1192-6.
47. Haddad S, Leitman SF, Wesley RA, et al. Place- 52. Purello D’Ambrosio F, Savica V, Gangemi S, et
bo-controlled study of intravenous magne- al. Ethylene oxide allergy in dialysis patients.
sium supplementation during large-volume Nephrol Dial Transplant 1997;12:1461-3.
leukapheresis in healthy allogeneic donors. 53. Owen HG, Brecher ME. Atypical reactions as-
Transfusion 2005;45:934-44. sociated with use of angiotensin-converting
48. Marques MB, Huang ST. Patients with throm- enzyme inhibitors and apheresis. Transfusion
botic thrombocytopenic purpura commonly 1994;34:891-4.
develop metabolic alkalosis during therapeu- 54. Orlin JB, Berkman EM. Partial plasma ex-
tic plasma exchange. J Clin Apher 2001;16:120- change using albumin replacement: Removal
4. and recovery of normal plasma constituents.
49. Askari S, Nollet K, Debol SM, et al. Transfu- Blood 1980;56:1055-9.
sion-related acute lung injury during plasma 55. Kiprov DD, Golden P, Rohe R, et al. Adverse re-
exchange: Suspecting the unsuspected. J Clin actions associated with mobile therapeutic
Apher 2002;17:93-6. apheresis: Analysis of 17,940 procedures. J Clin
50. Duntley P, Siever J, Korwes ML, et al. Vascular Apher 2001;16:130-3.
erosion by central venous catheters. Clinical
features and outcome. Chest 1992;101:1633-8.
C h a p t e r 2 7
Noninfectious Complications of
Blood Transfusion
Catherine A. Mazzei, MD, Medical Director, American Red Cross, Northern California Blood Services Region,
Oakland, California; Mark A. Popovsky, MD, Associate Clinical Professor, Harvard Medical School, Boston,
Massachusetts, and Vice President and Chief Medical Officer, Haemonetics Corporation, Braintree, Massa-
chusetts; and Patricia M. Kopko, MD, Clinical Professor of Pathology, University of California, San Diego, Cali-
fornia
C. Mazzei and P. Kopko have disclosed no conflicts of interest. M. Popovsky has disclosed a financial relation-
ship with Haemonetics Corporation.
665
666 䡲 AABB TECHNICAL MANUAL
Therapeutic/Prophylactic
Type Incidence Etiology Presentation Diagnostic Testing Approach
Fatal HTR: sites), back pain, pain along Repeat patient ABO, pre- and Analgesics (may need mor-
1 in 1.8 million infusion vein, anxiety posttransfusion sample phine)
Further tests as indicated to Pressors for hypotension
define possible incompatibil- (low-dose dopamine)
ity Hemostatic components
Further tests as indicated to (platelets, cryoprecipitate, or
detect hemolysis (LDH, FFP) for bleeding
bilirubin, etc)
Febrile, nonhemo- 0.1 to 1% with uni- Accumulated cyto- Fever, chills/rigors, head- Rule out hemolysis (DAT, Leukocyte-reduced blood
lytic versal leukoreduction kines in platelet unit ache, vomiting inspect for hemoglobinemia, Antipyretic premedication
Antibody to donor repeat patient ABO) (acetaminophen, no aspirin)
WBCs Rule out bacterial
contamination
WBC antibody screen†
Urticarial 1:100-1:33 Antibody to donor Urticaria, pruritis, flushing Rule out hemolysis (DAT, Antihistamine, treatment or
(1%-3%) plasma proteins inspect for hemoglobinemia, premedication (PO or IV)
Noninfectious Complications of Blood Transfusion
(Continued)
667
TABLE 27-1. Categories of Adverse Transfusion Reactions and Their Management* (Continued)
668
Therapeutic/Prophylactic
Type Incidence Etiology Presentation Diagnostic Testing Approach
䡲
Anaphylactic 1:20,000-1:50,000 Antibody to donor Hypotension, urticaria, bron- Rule out hemolysis (DAT, Trendelenburg (feet-up) posi-
plasma proteins chospasm (respiratory dis- inspect for hemoglobinemia, tion
(includes IgA, tress, wheezing), local edema, repeat patient ABO) Fluids
haptoglobin, C4) anxiety Anti-IgA Epinephrine (adult dose: 0.2-
Cytokines IgA, quantitative 0.5 mL of 1:1000 solution SC or
IM; in severe cases, 1:10,000
IV, initial rate 1mcg/minute)
Antihistamines, corticosteroids,
beta-2 agonists
IgA-deficient blood components
TRALI 1:1,200-1:190,000 WBC antibodies in Hypoxemia, respiratory fail- Rule out hemolysis (DAT, Supportive care until recovery
donor (occasionally ure, hypotension, fever, inspect for hemoglobinemia, Deferral of implicated donors
in recipient), other bilateral pulmonary edema repeat patient ABO)
WBC-activating Rule out cardiogenic
AABB TECHNICAL MANUAL
Hypotension asso- Dependent on clinical Inhibited metabolism Flushing, hypotension Rule out hemolysis (DAT, Withdraw ACE inhibition
ciated with ACE setting of bradykinin with inspect for hemoglobinemia, Avoid albumin volume replace-
inhibition infusion of brady- repeat patient ABO) ment for plasmapheresis
kinin (negatively Avoid bedside leukocyte filtra-
charged filters) or tion
activators of
prekallikrein
Circulatory over- <1% Volume overload Dyspnea, orthopnea, cough, Chest X-ray Upright posture
load tachycardia, hypertension, Rule out TRALI Oxygen
headache IV diuretic (furosemide)
Phlebotomy (250-mL incre-
ments)
Nonimmune hemo- Rare Physical or chemical Hemoglobinuria, hemoglobi- Rule out patient hemolysis Identify and eliminate cause
lysis destruction of blood nemia (DAT, inspect for hemoglobi-
(heating, freezing, nemia, repeat patient ABO)
hemolytic drug or Test unit for hemolysis
Noninfectious Complications of Blood Transfusion
solution added to
blood)
Air embolus Rare Air infusion via line Sudden shortness of breath, X-ray for intravascular air Place patient on left side with
䡲
Therapeutic/Prophylactic
Type Incidence Etiology Presentation Diagnostic Testing Approach
䡲
Hypocalcemia (ion- Dependent on clinical Rapid citrate infusion Paresthesia, tetany, Ionized calcium PO calcium supplement for mild
ized calcium; citrate setting (massive transfusion arrhythmia Prolonged Q-T interval on symptoms during therapeutic
toxicity) of citrated blood, electrocardiogram apheresis procedures
delayed metabolism Slow calcium infusion while
of citrate, apheresis monitoring ionized calcium lev-
procedures) els in severe cases
Hypothermia Dependent on clinical Rapid infusion of cold Cardiac arrhythmia Central body temperature Employ blood warmer
setting blood
Delayed (>24 hours) Transfusion Reactions—Immunologic
Alloimmuni- 1:100 (1%) Immune response to Positive blood group Antibody screen Avoid unnecessary transfusions
zation, red cell foreign antigens on antibody screening test DAT Leukocyte-reduced blood
antigens RBCs
Alloimmuni- 1:10 (10%) WBCs and Platelet refractoriness, Platelet antibody screen Avoid unnecessary transfusions
AABB TECHNICAL MANUAL
zation, HLA anti- platelets (HLA) delayed hemolytic reaction, HLA antibody screen Leukocyte-reduced blood
gens hemolytic disease of the
newborn
Hemolytic 1:2500-11,000 Anamnestic immune Fever, decreasing Antibody screen Identify antibody
response to red cell hemoglobin, new DAT Transfuse compatible RBCs as
antigens positive antibody screening Tests for hemolysis (visual needed
test, mild jaundice inspection for hemoglobine-
mia, LDH, bilirubin, urinary
hemosiderin as clinically
indicated)
Graft-vs-host Rare Donor lymphocytes Erythroderma, maculopapu- Skin biopsy Corticosteroids, cytotoxic
disease engraft in recipient lar rash, anorexia, nausea, HLA typing agents
and mount attack on vomiting, diarrhea, hepatitis, Molecular analysis for Irradiation of blood components
host tissues pancytopenia, fever chimerism for patients at risk (including
components from related
donors and HLA-selected com-
ponents)
CHAPTER 27
Posttransfusion Rare Recipient platelet Thrombocytopenic Platelet antibody screen and IVIG
purpura antibodies (apparent purpura, bleeding 8-10 days identification HPA-1a-negative platelets
alloantibody, usually after transfusion Plasmapheresis
anti-HPA-1a) destroy
autologous platelets
Delayed (>24 hours) Transfusion Reactions—Nonimmunologic
Iron overload Typically after >100 Multiple transfusions Diabetes, cirrhosis, cardiomy- Serum ferritin Iron chelators
RBC units with obligate iron opathy Liver enzymes
load in transfusion- Endocrine function tests
dependent patient
*For platelet refractoriness, see chapter on platelet and granulocyte antigens and antibodies; for septic transfusion reactions, see chapter on transfusion-transmitted diseases. For a recent
summary of transfusion reactions, see Popovsky.4
†
Blood group antibody screening test.
AHTR = acute hemolytic transfusion reaction; HTR = hemolytic transfusion reaction; DIC = disseminated intravascular coagulation; DAT = direct antiglobulin test; IV = intravenous; Hb =
hemoglobin; LDH = lactate dehydrogenase; CRYO = cryoprecipitated antihemophilic factor; FFP = fresh frozen plasma; WBC = white blood cell; PO = by mouth; SC = subcutaneous; IM =
intramuscular; IgA = immunoglobulin A; ACE = angiotensin-converting enzyme; TRALI = transfusion-related acute lung injury; RBC = Red Blood Cell; IVIG = intravenous immunoglobulin;
HPA = human platelet antigen.
Noninfectious Complications of Blood Transfusion
䡲
671
672 䡲 AABB TECHNICAL MANUAL
sepsis and TRALI. Hemolysis may not be de- tors of complement, and IgG antibodies, when
tected immediately and does not occur in sep- present at sufficient concentrations and of the
sis and TRALI. Respiratory difficulty is not typ- relevant subclass, may activate complement as
ically a symptom of an AHTR. Immediate well.
treatment requirements for acute hemolysis Complement activation involves C3
are identical to those for sepsis—stop the cleavage with the ensuing production of C3a,
transfusion and maintain hemodynamic sta- an anaphylatoxin, which is released into the
bility—and can be instituted while the diagno- plasma, and C3b, which coats the red cells. If
sis is being determined. complement activation proceeds to comple-
The patient’s underlying disease process tion, which is characteristic of ABO antibodies,
can make the diagnosis of any AHTR extremely a membrane attack complex is assembled on
difficult. Patients with glucose-6-phosphate the red cell surface, and intravascular lysis oc-
dehydrogenase (G6PD) deficiency, autoim- curs. C5a, an anaphylatoxin that is 100 times
mune hemolytic anemia, or sickle cell disease more potent than C3a, is produced as part of
present particularly complicated situations this hemolysis. C3a and C5a promote the re-
when symptoms such as fever and hypoten- lease of histamine and serotonin from mast
sion occur. In these patients, autoantibodies cells, leading to vasodilation and smooth-
and multiple alloantibodies delay the serolog- muscle contraction, particularly of bronchial
ic diagnosis of an AHTR and make identifica- and intestinal muscles. C3a and C5a are recog-
tion of the responsible antibody a challenge. nized by many other cell types as well, includ-
Acute hemolysis may also result from nonim- ing monocytes, macrophages, endothelial
mune mechanisms, as described in the “Non- cells, platelets, and smooth muscle, and are in-
immune-Mediated Hemolysis” section below. volved in the production and release of cyto-
kines, leukotrienes, free radicals, and nitric ox-
ide. The end result may include wheezing,
Pathophysiology flushing, chest pain or tightness, and gastroin-
testinal symptoms. These symptoms may also
The interaction of preformed antibodies with be caused by release of bradykinin and norepi-
red cell antigens is the immunologic basis for nephrine caused by antigen-antibody com-
AHTRs. The most severe reactions are associ- plex stimulation.
ated with transfusions of red cells that are ABO Phagocytosis of IgG-coated red cells leads
incompatible with the recipient, resulting in to cytokine release, which plays a role in pro-
acute intravascular destruction of the trans- ducing the effects of acute hemolysis.8 Inter-
fused cells. Transfusion of ABO-incompatible leukin-8 (IL-8), which activates neutrophils,
plasma, as can occur with apheresis platelets, and tumor necrosis factor alpha (TNF),
may also cause hemolysis of the patient’s red which activates the coagulation cascade, have
cells. Although this form of acute hemolysis is also been found after in-vitro incubation of
not usually clinically significant or character- incompatible group O whole blood and group
ized by typical hemolytic symptoms, it can be A or B red cells.9 Other cytokines involved in
severe if donors have high titers of ABO anti- the pathogenesis of AHTRs include IL-1, IL-6,
bodies. Although rare, the most common cir- and monocyte chemoattractant protein 1
cumstance is when group O platelets from do- (MCP-1). In a mouse model of HTR,
nors with high titers of anti-A are transfused to transfusion of incompatible red cells resulted
group A recipients.6,7 in very high plasma levels of MCP-1 and IL-6
When preformed immunoglobulin M and lower levels of TNF.10
(IgM) or IgG antibodies recognize correspond- If complement activation does not pro-
ing red cell antigens, complement activation ceed to completion, which typically happens
may occur, resulting in intravascular hemoly- with non-ABO antibodies, the red cells can un-
sis, hemoglobinemia, and, eventually, hemo- dergo extravascular hemolysis where cells
globinuria. IgM antibodies are strong activa- coated with C3b and/or IgG are rapidly
674 䡲 AABB TECHNICAL MANUAL
removed from the circulation by phagocytes. dence of ABO HTR to be 1:80,000 and the risk
In extravascular hemolysis, the consequences of a fatal ABO HTR to be 1 in 1.8 million.11 Of
of complement activation, including release of the transfusion-related fatalities reported to
anaphylatoxins and opsonization of red cells, the Food and Drug Administration (FDA) from
may still have adverse effects. 2007 to 2011, 23% (in 50 patients) were caused
Coagulation abnormalities associated by HTRs.1
with AHTRs may be caused by various mecha-
nisms. The “intrinsic” pathway of the clotting Treatment
cascade may be activated by antigen-antibody
Prompt recognition of an AHTR and immedi-
interaction, resulting in activation of Factor
ate cessation of the transfusion are crucial.
XII, also known as “Hageman factor.” Activa-
The unit of blood should be returned to the
tion of the Hageman factor can result in hypo-
blood bank for a transfusion reaction investi-
tension through its effect on the kinin system.
gation. The infusion of saline should be con-
The kinin system produces bradykinin, which
tinued to maintain venous access, treat hypo-
in turn increases vascular permeability and
tension, and maintain renal blood flow, with a
causes vasodilation. Activated complement,
goal of a urine flow rate >1 mL/kg/hour. Saline
TNF, and IL-1 may increase the expression of
infusion alone may not be adequate therapy,
tissue factor. Tissue factor, expressed by leuko-
and the urine output must be carefully moni-
cytes and endothelial cells, can activate the
tored so as not to cause volume overload in the
“extrinsic” pathway and is associated with the
patient. Studies have concluded that low-dose
development of DIC. DIC is an often life-
dopamine may improve renal function initial-
threatening consumptive coagulopathy. Its
ly, but no solid evidence exists that it can pre-
characteristics include microvascular thrombi
vent renal failure.12 However, these studies,
formation with ischemic organ and tissue
which did not include patients with AHTRs,
damage; consumption of platelets, fibrinogen,
did show a 25% increase in urine output in the
and coagulation factors; and activation of fi-
acute setting. Thus, low-dose dopamine (1-5
brinolysis with resultant production of fibrin-
µg/kg/minute) may still have a role in treating
degradation products. The end result of these
the complications of an AHTR, but no recom-
activations can vary from generalized oozing
mendations regarding its use may be made
to uncontrolled bleeding.
based on published evidence at this time.
Shock may be a component of DIC. Hypo-
The addition of the diuretic furosemide
tension, caused by the release of vasoactive
(40-80 mg intravenously in adults, 1-2 mg/kg
amines, kinins, and other mediators, produc-
in children) promotes increased urine output
es a compensatory vasoconstrictive response
and further enhances renal cortical blood
that further aggravates organ and tissue dam-
flow.13 If urine output remains diminished af-
age. Renal failure may occur as well. Free he-
ter a liter of saline has been infused, acute tu-
moglobin impairs renal function, but compro-
bular necrosis may have occurred, and the pa-
mised renal cortical supply is thought to be the
tient may be at risk of developing pulmonary
major contributing factor in renal failure. In
edema. At this point, a nephrologist should be
addition, antigen-antibody complex deposi-
consulted for further management of the pa-
tion, vasoconstriction, and thrombi formation
tient. Oliguric renal failure may be complicat-
contribute to the development of renal vascu-
ed by hyperkalemia and subsequent cardiac
lar compromise.
arrest; therefore, these patients should be
monitored with telemetry. Metabolic acidosis
Frequency
and uremia often necessitate the institution of
The frequency of AHTRs is not easy to deter- dialysis.
mine. The authors of a review article based on DIC is an equally serious component of
data from several surveillance systems esti- an AHTR. DIC is extremely difficult to treat
mated the risk of clinical or laboratory evi- and may be the first indication that hemolysis
CHAPTER 27 Noninfectious Complications of Blood Transfusion 䡲 675
Treatment Treatment
Hemolysis of nonimmune etiology may cause If transfusion-related sepsis is suspected, the
symptoms whose severity depends on the de- transfusion should be stopped immediately
gree of hemolysis and amount of component and supportive care of the patient should be
transfused. In all cases, the transfusion should initiated. Broad-spectrum antibiotics may be
be discontinued and appropriate care should indicated. (See Chapter 8 for a more detailed
be administered. (See the earlier section on discussion of bacterial contamination of
the treatment of AHTRs for details on manag- transfused blood, including frequency data
ing hypotension and declining renal function.) and prevention strategies.)
CHAPTER 27 Noninfectious Complications of Blood Transfusion 䡲 677
recipient. Although IgA deficiency is present in amine, may be administered. Once the symp-
approximately 1:700 people of European an- toms have dissipated, the transfusion may be
cestry, only a small percentage of these people resumed and a laboratory work-up need not
ever make antibodies against IgA. Those who be initiated.30
do are divided into two groups based on IgA If the symptoms do not subside—or, in
levels and the type of antibody formed. People the case of severe urticaria or urticaria accom-
with absolute IgA deficiency (<0.05 mg/dL) panied by hypotension if dyspnea, significant
may form class-specific antibodies that are of- edema, or gastrointestinal symptoms do not
ten associated with anaphylactic reactions. subside—the transfusion must be stopped and
Those with decreased but detectable amounts the reaction promptly treated. Severe urticari-
of IgA, or relative IgA deficiency, can form sub- al reactions may require treatment with meth-
class-specific antibodies (eg, anti-IgA1 or anti- ylprednisolone (125 mg intravenously) or
IgA2) that typically result in less severe reac- prednisone (50 mg orally). Once a severe reac-
tions.26 tion or developing anaphylaxis is identified,
Although precautions should be taken action should be promptly initiated to main-
when transfusing an IgA-deficient patient, it tain oxygenation and stabilize hypotension.
must be kept in mind that the majority of ATRs Epinephrine (1:1000) may be administered in-
are caused by allergens to substances other tramuscularly or subcutaneously at an adult
than IgA.26 Other well-known triggers include dose ranging from 0.2 to 0.5 mL or a pediatric
patient antibodies against haptoglobin,27 peni- dose of 0.01 mL/kg. If symptoms persist, the
cillin, and the C4 determinant of comple- dose may be repeated every 5 to 15 minutes up
ment.28 Patients taking ACE inhibitors can ex- to three times, unless palpitations, extreme
perience transfusion reactions that are thought anxiousness, or tremors occur. If the patient is
to result from dual actions on bradykinin: inhi- unconscious or in shock, epinephrine may be
bition 1) of its catabolism by the ACE inhibitor given intravenously at a dilution of 1:10,000
and 2) of bradykinin by low levels of prekalli- (100 µg/mL) and an initial rate of 1 µg/minute.
krein activity in plasma protein fraction. Such patients ideally receive cardiac monitor-
ing because of the epinephrine’s arrhythmic
Frequency potential.
ATRs are quite common, with an overall fre- Supplemental oxygen should be adminis-
quency of approximately 1% to 3% of transfu- tered, and the airway should be maintained.
sions. ATRs also represent 12% to 33% of all Hypotensive patients should be placed in the
transfusion reactions.29,30 Urticaria is relatively Trendelenburg position and supported with
common, whereas anaphylactic reactions oc- crystalloids. If bronchospasm is present, respi-
cur much less often. As with any ATR, anaphy- ratory symptoms may not respond to the epi-
laxis occurs most commonly during the trans- nephrine, and addition of a beta-2 agonist or
fusion of plasma or platelets; it has an aminophylline may be required. Patients who
incidence of 1:20,000 to 1:50,000 transfu- do not respond, possibly because of the use of
sions.29,31 Of the transfusion-associated fatali- a beta-adrenergic blocker or an ACE inhibitor,
ties reported to the FDA from 2007 to 2011, 6% may respond to the addition of glucagon as a
(in 12 patients) were caused by anaphylaxis.1 1-mg bolus intravenously or by continuous in-
fusion.23,32
Treatment
Prevention
Urticaria is the only transfusion reaction in
which the administration of the component Premedication with an antihistamine (25 to 50
may be routinely resumed after prompt treat- mg diphenhydramine) 30 minutes before
ment. When a patient develops symptoms, the transfusion may be helpful in patients with
transfusion should be paused so that an anti- a history of multiple or severe urticarial trans-
histamine, typically 25 to 50 mg diphenhydr- fusion reactions. Routine prophylaxis may also
680 䡲 AABB TECHNICAL MANUAL
be beneficial for patients undergoing multiple to the signs and symptoms traditionally asso-
transfusions, as in plasma exchange; however, ciated with TRALI, there is a growing apprecia-
routine antihistamine premedication of all pa- tion that the disorder can be associated with a
tients receiving a transfusion has not been dramatic transient neutropenia or leukope-
shown to decrease the risk of ATR.33 If antihis- nia.36
tamines are not sufficient, prednisone (20-50 All plasma-containing components, in-
mg orally) or parenteral steroids may be cluding whole blood, RBCs, platelets, cryopre-
of benefit. For patients whose reactions are se- cipitated AHF, and fresh frozen plasma (FFP),
vere, unrelenting, and unresponsive to pre- have been implicated in TRALI. Transfusion
medication, washing red cells or platelets may volumes as small as 15 mL have led to TRALI.
be considered. Plasma reduction or replace- TRALI is a form of acute lung injury (ALI).
ment in platelet products may also be benefi- The American-European Consensus Conference37
cial. Alternatively, the administration of de- defined ALI as acute hypoxemia with a PaO2/
glycerolized RBCs has met with some success FiO2 ratio of 300 mm Hg and bilateral pulmo-
when reactions have occurred even after RBCs nary edema on frontal chest radiograph. The
were washed with 2 L of saline. Canadian Consensus Conference38 relied on
The plasma used for transfusions for pa- this definition of ALI when creating its diag-
tients with diagnosed IgA deficiency who pro- nostic criteria for TRALI: 1) ALI with hypox-
duce anti-IgA should be from IgA-deficient do- emia and PaO2/FiO2 300 or SpO2 <90% on
nors (<0.05 mg/dL). If the local blood center room air, 2) no preexisting ALI before transfu-
cannot provide these components, they may sion, 3) onset of symptoms within 6 hours of
be available through the American Rare Donor transfusion, and 4) no temporal relationship
Program (see Method 3-13). Other cellular with an alternative risk factor for ALI. The pan-
components (RBCs and platelets) can be de- el also defined “possible TRALI” using the
pleted of plasma proteins through washing. same criteria as for TRALI, with the exception
IgA deficiency without the presence of anti-IgA that possible TRALI occurs in the setting of an
or without a history of an anaphylactoid/ alternative risk factor for ALI.
anaphylactic reaction does not warrant the Although the lung injury in ALI is usually
use of IgA-deficient or plasma-depleted com- irreversible, the lung injury in TRALI is most
ponents. Intravenous immune globulin (IVIG) often transient. Approximately 80% of pa-
products from various manufacturers have tients with TRALI improve within 48 to 96
different IgA amounts. The manufacturer hours. The remaining 20% of patients who do
should be contacted to obtain detailed infor- not improve rapidly have either a protracted
mation about a product and lot number.34 Au-
clinical course or a fatal outcome. In one of the
tologous donation programs may also be con-
largest studies of TRALI, 100% of the patients
sidered for patients with IgA deficiency once
required oxygen support and 72% required
they have recovered.
mechanical ventilation.39
TRALI
Differential Diagnosis
Presentation The three main conditions that need to be dis-
Clinical signs and symptoms of TRALI typical- tinguished from TRALI are 1) anaphylactic
ly include fever, chills, dyspnea, cyanosis, hy- transfusion reactions, 2) TACO, and 3) transfu-
potension, and new-onset bilateral pulmonary sion-related sepsis. In anaphylactic transfu-
edema.35 An increase in blood pressure fol- sion reactions, bronchospasm, laryngeal ede-
lowed by hypotension is not uncommon. ma, severe hypotension, erythema (often
TRALI can be life threatening or fatal. Symp- confluent), and urticaria are prominent symp-
toms arise within 6 hours of transfusion, with toms. Fever and pulmonary edema are not as-
most cases becoming evident within 1 to 2 sociated with anaphylactic reactions. The clin-
hours after the end of transfusion. In addition ical presentation of TACO is very similar to that
CHAPTER 27 Noninfectious Complications of Blood Transfusion 䡲 681
of TRALI, with respiratory distress, tachypnea, sion, and it predisposes the recipient to devel-
and cyanosis as the most prominent features. op TRALI if a second event is experienced. The
The key distinction between TACO and TRALI infusion of BRMs or antibodies is the second
is that the pulmonary edema in TACO is car- event. These stimuli, which ordinarily do not
diogenic, whereas it is noncardiogenic in activate neutrophils, activate the primed neu-
TRALI. High fever with hypotension and vas- trophils in the pulmonary microvasculature,
cular collapse are prominent features of trans- which results in pulmonary endothelial dam-
fusion-related sepsis. Respiratory distress is age, capillary leakage, and pulmonary edema.
infrequently associated with these reactions.
With rapid onset of respiratory distress, in ad- Frequency
dition to TACO and TRALI, coincident myocar-
Although the true incidence of TRALI is un-
dial infarction and pulmonary embolus as well
known, TRALI is the leading cause of transfu-
as other possible causes of ALI should be con-
sion-related mortality reported to the FDA,
sidered.
with more than 34 cases reported in 2007.41 Re-
cent efforts to decrease the amount of trans-
Pathophysiology
fusable plasma collected from female donors
The precise mechanism of lung injury in appear to be decreasing the number of TRALI
TRALI has not been determined. TRALI has fatalities in the United States.1
been associated with the infusion of antibod- In 2006, the year before many blood cen-
ies to leukocyte antigens and of biologic re- ters implemented measures to reduce the risk
sponse modifiers (BRMs).39 Infusions of these of TRALI from plasma transfusions, 35 fatal
antibodies or BRMs are thought to initiate a cases of TRALI, 22 of which were associated
sequence of events that results in cellular acti- with transfusion of FFP, were reported to the
vation and damage of the basement mem- FDA. Since 2008, the year after many blood
brane. Pulmonary edema occurs secondary to centers implemented such measures, the
leakage of protein-rich fluid into the alveolar numbers of TRALI fatalities and of TRALI fatal-
space. ities associated with FFP transfusions reported
BRMs accumulate in some cellular com- to the FDA have decreased each year.
ponents during storage. BRMs enhance the
polymorphonuclear cell oxidative burst, are Treatment
soluble in chloroform, and consist of a mixture
Treatment of TRALI consists of respiratory and
of lysophosphatidylcholines.40
circulatory support. Treatment should be as
Antibodies to HLA Class I antigens, HLA
intensive as dictated by the clinical picture.
Class II antigens, and human neutrophil anti-
Oxygen supplementation with or without me-
gens (HNAs) have been associated with TRALI.
chanical ventilation is required in almost all
These antibodies can be formed after exposure
cases. Pressor agents may be needed to sup-
to foreign antigens via pregnancy, transfusion,
port blood pressure. Diuretics are not indicat-
or transplantation. In the majority of TRALI
ed because TRALI is not related to volume
cases with antibodies, the source of the anti-
overload. Administration of corticosteroids
body is the donor, not the recipient.
has not been shown to improve the clinical
A two-event model of the mechanism of
outcome in TRALI or acute respiratory distress
TRALI has been hypothesized.40 In the first
syndrome.42
event, generation of biologically active com-
pounds activates pulmonary vascular endo-
Prevention
thelial cells and primes neutrophils, resulting
in sequestration of neutrophils in the pulmo- Strategies to reduce the risk of TRALI are com-
nary microvasculature. This first event can plicated by a number of factors. Although
result from a variety of physiologic stressors, approximately 10% of blood donations con-
including sepsis, surgery, and massive transfu- tain HLA and/or HNA antibodies, TRALI
682 䡲 AABB TECHNICAL MANUAL
occurs in fewer than 1:1000 transfusions.39,40 central venous pressure; dyspnea; orthopnea;
There is no mechanism to identify which pa- new ST-segment and T-wave changes on elec-
tients are at risk for developing TRALI. In 2013, trocardiogram; elevated serum troponin; and
AABB announced that a new standard in the likely elevated brain natriuretic peptide (BNP),
29th edition of Standards for Blood Banks and a cardiac marker.45 Increased blood pressure
Transfusion Services would require that plas- characterized by a widening of the pulse pres-
ma products and whole blood collected for sure is characteristic of TACO. Radiographs
transfusion be from male donors, never-preg- show a widened cardiothoracic ratio.
nant female donors, or females who have been
tested since their last pregnancy and found to Differential Diagnosis
be negative for HLA antibodies.5
In 2014 the AABB issued Association Bul- TACO is frequently confused with TRALI be-
letin 14-02, providing guidance on how to im- cause both types of reactions produce pulmo-
nary edema. It is possible for TACO and TRALI
plement the new TRALI risk reduction require-
to occur concurrently in the same patient. The
ment. In this bulletin, AABB outlined a
timelines and the clinical presentation are
number of considerations for implementation
similar, but hypertension is a constant feature
of TRALI risk reduction requirements in plas-
of TACO, whereas it is only an infrequent and
ma components and whole blood intended for
transient manifestation of TRALI. Further-
transfusion.43
more, rapid improvement with diuresis or ino-
Although the measures required by AABB
tropic agents is consistent with TACO.
and described in Association Bulletin 14-02
In congestive heart failure, BNP levels are
are expected to reduce the risk of TRALI, it is
elevated. Several studies have shown that a
important to recognize they do not eliminate
posttransfusion-to-pretransfusion BNP ratio
TRALI because they do not decrease the risk of
of 1.5 with a posttransfusion level at least 100
TRALI from RBCs, platelet concentrates, or
pg/mL as a cutoff yielded a sensitivity and
cryoprecipitate. HLA antibody screening ad-
specificity greater than 80% in TACO.46 Howev-
dresses only the risk of TRALI from HLA anti-
bodies. A practical test to screen the blood er, a recent study in the intensive care setting
supply for HNA antibodies is not available. In found that BNP was only of moderate value for
addition, none of these risk-reduction mea- distinguishing TACO from TRALI.47 With rapid
sures addresses the risk of TRALI from BRMs. onset of respiratory distress, possible causes of
ALI, such as coincident myocardial infarction,
pulmonary embolus, and others, should be
TACO
considered in addition to TACO and TRALI.
Presentation
Frequency
It is well known that transfusion can precipi-
tate acute pulmonary edema caused by vol- The incidence of TACO is unknown, but sever-
ume overload, but only recently has this prob- al studies suggest that TACO is much more
lem been recognized as an important common than previously known. In the gener-
complication. Patients older than 70 years and al population, one group found TACO in 1:707
infants are at greatest risk, although all trans- recipients of RBCs, and 20% of the affected pa-
fusion recipients are susceptible to some de- tients received a single unit of RBCs. In studies
gree.44 Whereas large volumes of components of older orthopedic patients undergoing hip or
and nonblood fluids are most frequently im- knee replacement, TACO was found in 1% and
plicated, modest volumes can also precipitate 8%, respectively.48,49 In one report from the
TACO. High flow rates are frequently cofactors. Quebec hemovigilance network, the incidence
TACO has no diagnostic signs or symp- was 1:5000 components, and 1.3% of the cases
toms. Within 1 to 2 hours of transfusion, pa- resulted in death.50 In the critical care setting,
tients may develop any or all of the following: 1:356 units transfused resulted in TACO.51
gallop; jugular venous distension; elevated From 2007 to 2011, 15% of the transfusion-
CHAPTER 27 Noninfectious Complications of Blood Transfusion 䡲 683
associated fatalities reported to the FDA (in 32 who lose blood rapidly may have preexisting
patients) were a consequence of TACO.1 or coexisting hemostatic abnormalities or de-
Although RBCs are most commonly asso- velop them during resuscitation. Hemostatic
ciated with TACO, a recent prospective surveil- abnormalities may include dilutional coagu-
lance study found that FFP was a frequent lopathy, DIC, and liver and platelet dysfunc-
cause of this complication.52 In this study, 4.8% tion.
of patients developed TACO with a prevalence
rate of 1.5%. Of the 24 reactions, 14 (58%) oc- Citrate Toxicity
curred in the intensive care unit.
PATHOPHYSI OLOGY AND MA NIFESTA-
TIONS. Plasma, whole blood, and platelets
Treatment
contain citrate as an anticoagulant. When
As soon as symptoms suggest TACO, the trans- large volumes of these components are trans-
fusion should be stopped. The symptoms fused rapidly, particularly in the presence of
should be treated by placing the patient in a liver disease, plasma citrate levels may rise,
seated position, providing supplementary oxy- binding calcium and ionized calcium and re-
gen, and reducing the intravascular volume sulting in hypocalcemia. In patients with a
with diuretics. If symptoms persist in con- normally functioning liver, citrate is rapidly
firmed TACO, administration of additional di- metabolized; thus, these symptoms are tran-
uretics or therapeutic phlebotomy in 250-mL sient.53 Hypocalcemia is more likely to cause
increments is appropriate. manifestations in patients who are hypother-
mic or in shock.
Prevention A decrease in ionized calcium levels in-
creases neuronal excitability, which in the con-
In the absence of ongoing and rapid blood scious patient leads to symptoms of perioral
loss, components should be administered and peripheral tingling, shivering, and light-
slowly, particularly in patients at risk of TACO headedness, followed by a diffuse sense of vi-
(ie, pediatric patients, patients with severe bration, muscle cramps, fasciculations, spasm,
anemia, and patients with congestive heart and nausea. In the central nervous system, hy-
failure). Rates of 2 to 4 mL/minute and 1 mL/ pocalcemia is thought to increase the respira-
kg of body weight per hour are the most fre- tory center’s sensitivity to carbon dioxide,
quently cited, despite a paucity of data on ap- causing hyperventilation. Because myocardial
propriate infusion rates. Total fluid input and contraction is dependent on the intracellular
output must be monitored. movement of ionized calcium, hypocalcemia
depresses cardiac function.54
Complications of Massive
Transfusion TREATMENT AND PREVENTI ON. Unless the
patient has a predisposing condition that hin-
The potential complications of massive trans- ders citrate metabolism, hypocalcemia caused
fusion, usually defined as the receipt of more by citrate overload during massive transfusion
than 10 RBC units within 24 hours, include can usually be treated by slowing the infusion.
metabolic and hemostatic abnormalities, im- Calcium replacement should be considered
mune hemolysis, and air embolism. Metabolic when the calcium concentration falls to below
abnormalities can depress ventricular func- 50% of its normal value or the symptoms of
tion. Hypothermia from refrigerated blood, ci- hypocalcemia are evident.55
trate toxicity, and lactic acidosis from under-
perfusion and tissue ischemia, which are often Hyperkalemia and Hypokalemia
complicated by hyperkalemia, can contribute
to this effect. Although metabolic alkalosis PATHOPHYSIOLOGY. When RBCs are stored
caused by citrate metabolism may occur, it is at 1 to 6 C, the intracellular potassium gradual-
not likely to be clinically significant. Patients ly leaks into the supernatant plasma or addi-
684 䡲 AABB TECHNICAL MANUAL
tive solution. Although the concentration in enzymatic activity is reduced as the core body
the supernatant may be high (see Chapter 6) temperature lowers if a blood warmer is not
because of the small volume, the total extra- used. Mortality rates associated with hemo-
cellular potassium load is less than 0.5 mEq for static abnormalities range from 20% to 50%.60
fresh RBC units and only 5 to 7 mEq for units at The high rate of mortality results from hypo-
expiration. These potassium concentrations thermia, metabolic acidosis, and coagulopa-
rarely cause problems in the recipient because thy.61
rapid dilution, redistribution into cells, and ex- Studies of military and civilian trauma pa-
cretion blunt the effect. However, hyperkale- tients demonstrated a progressive increase in
mia can be a problem in patients with renal the incidence of microvascular bleeding
failure, premature infants, and newborns re- (MVB) characteristic of a coagulopathy with
ceiving large transfusions, such as in cardiac increasing transfusion volumes that typically
surgery or exchange transfusion; otherwise, occurs after replacement of two to three blood
hyperkalemia is typically a transient effect volumes (20 to 30 units).62,63 Although platelet
during very rapid transfusions. counts, coagulation parameters, and levels of
Hypokalemia occurs more frequently selected clotting parameters correlate with the
than hyperkalemia after transfusion because volume transfused, contrary to expectations
potassium-depleted donor red cells reaccu- from a simple dilutional model, the relation-
mulate this ion intracellularly, and citrate me- ship is marked by tremendous variability.
tabolism causes further movement of potassi- Moreover, there is frequently discordance be-
um into the cells in response to the tween the laboratory assessment and the clini-
consumption of protons. Catecholamine re- cal evidence of bleeding. It has been suggested
lease and aldosterone-induced urinary loss that the platelet deficits play a more important
can also trigger hypokalemia in the setting of role in causing the bleeding than the coagula-
massive transfusion.56 tion deficiencies.
MVB typically occurs when the platelet
TREATMENT A ND PRE VENTION. No treat- count falls below 50,000 to 60,000/µL. Howev-
ment or preventive strategy for hypokalemia er, no simple relationship can be determined
and hyperkalemia is usually necessary, provid- between a patient’s coagulation test results
ed that the patient is adequately resuscitated and the onset of bleeding. The etiology of
from the underlying condition that required bleeding (elective surgery vs massive trauma)
massive transfusion.57 For infants receiving may play a role as well.64
small-volume transfusions infused slowly, Subsequent studies have refined these
units may be used safely until the expiration observations. Significant platelet dysfunction
date.58 Although washing of RBC units results has been demonstrated in massively trans-
in very low levels of potassium, there is no evi- fused patients.65,66 Low fibrinogen and platelet
dence that this is indicated for routine RBC counts are better predictors of hemostatic fail-
transfusions, even in patients with impaired ure than elevations of prothrombin time (PT)
renal function.59 and partial thromboplastin time (PTT), sug-
gesting that consumptive coagulopathy is an
Hemostatic Abnormalities in Massive important factor in MVB in addition to dilu-
Transfusion tion.63 Similarly, Harke and Rahman67 showed
that the degree of platelet and clotting abnor-
PATHOPHYSIOLOGY. Coagulopathy can oc- malities correlates with the length of time that
cur in massive transfusion, particularly when the patient is hypotensive, suggesting that
the lost blood is initially replaced with RBCs shock is the most important cause of DIC. In
and asanguineous fluids. Coagulopathy in aggregate, Collins68 concluded that “coagulop-
massive transfusion is frequently ascribed to athy in heavily transfused patients was due to
the dilution of platelets and clotting factors as hypoperfusion, not transfusion.” More recent-
patients lose hemostatically active blood, and ly, more powerful hemostatic assays have been
CHAPTER 27 Noninfectious Complications of Blood Transfusion 䡲 685
introduced that may be more predictive of mophilia A or B. However, the off-label use of
blood component requirements.69 rFVIIa is growing in several areas, including to
These data may not be generalizable to treat hemorrhagic bleeding in trauma and sur-
patients undergoing massive transfusion in gery.72,73 The recombinant product works by
the controlled setting of the operating room, targeting the site of tissue damage, where it
where hypotension caused by volume loss is binds to tissue factor. This complex activates
prevented. In this context, coagulation factor Factor X to Factor Xa and, ultimately, Factor
levels may have priority over platelet prob- IXa. In patients with hemophilia, rFVIIa by-
lems. Murray and colleagues64 documented passes the need for Factor VIII or Factor IX
that excessive bleeding in elective surgery pa- through the activation of the small amounts of
tients transfused with more than one blood Factor X on activated platelet surfaces at the
volume (RBCs and crystalloid) corresponded site of injury. Studies of the efficacy of rFVIIa
to a prolongation in PT and PPT compared to have produced mixed results.74 In the absence
patients with normal hemostasis. of definitive data, transfusion services should
establish guidelines on the reasonable use of
TREATMENT AND PREVENTION. The dilu- rFVIIa. In contrast, antifibrinlytics may have a
tional model of coagulopathy in massive role in controlling massive bleeding from trau-
transfusion suggests that prophylactic re- ma. The 2010 CRASH-2 study concluded that
placement of hemostatic components based tranexamic acid should be given as early as
on the volume of RBCs or whole blood trans- possible in the trauma patient.75
fused prevents the development of a bleeding
diathesis. No specific regimen has yet been
shown to be superior to any other in prospec-
Air Embolism
tive studies, and this is a controversial topic.70
According to AABB guidelines, there are insuf- Air embolism can occur if blood in an open
ficient data to recommend for or against a 1:1 system is infused under pressure or if air en-
RBC to plasma transfusion ratio in massive ters a central catheter while containers or
transfusion.71 blood administration sets are being changed.
Previously, the predominant thinking was Air embolism has been reported in association
that the replacement of platelets and coagula- with intraoperative and perioperative blood
tion factors in the massively transfused surgi- recovery systems that allow air into the blood
cal and trauma patient should be based on the infusion bag. The minimum volume of air em-
identification of a specific abnormality using bolism that is potentially fatal for an adult is
platelet counts, international normalized ra- approximately 100 mL.76 Symptoms include
tio, activated PTT, and fibrinogen levels. Fre- cough, dyspnea, chest pain, and shock.
quent monitoring of these laboratory values If air embolism is suspected, the patient
serves to avoid overuse of platelets and plasma should be placed on the left side with the head
products (FFP and Cryoprecipitated AHF) by down to displace the air bubble from the pul-
anticipating the specific components needed monic valve. Aspiration of the air is sometimes
while avoiding dilutional coagulopathy. It is attempted. However, proper use and inspec-
imperative that the laboratory provide results tion of infusion pumps, equipment for blood
of these tests rapidly. Intraoperative and post- recovery or apheresis, and tubing couplers are
operative laboratory testing, such as thrombo- still essential to prevent this complication.
elastography, may be useful.
Duffy, Kell, and MNS systems, in order of de- quire transfusion of antigen-negative RBCs.
creasing frequency.78(pp358-89) Many institutions have programs to provide at
In a DHTR/DSTR, antibodies may be least partially phenotypically matched blood
found in the serum, on transfused red cells, or for patients with sickle cell disease and some
both. Routine antibody screening and anti- other patients who have developed multiple
body identification should be possible. If alloantibodies. Patients with sickle cell disease
transfused red cells are still present, the DAT may develop a complication known as “sickle
result may be positive. When the DAT result is cell HTR,” where autologous and allogeneic
positive, an eluate should be performed and cells are destroyed (see Chapter 23).
the antibody identified. If a segment from the
unit is available, antigen typing may confirm Refractoriness to Platelet Transfusions
the diagnosis.
See Chapter 18.
Frequency
TA-GVHD
As with AHTRs, the estimated rate of DHTRs
varies widely from study to study. Some of this Presentation
variation is the result of the practice of consid-
The clinical manifestations of TA-GVHD typi-
ering DSTRs and DHTRs as one category. Also,
cally begin 8 to 10 days after transfusion, al-
improvements in laboratory techniques have
though symptoms can occur as early as 3 days
contributed to the increased number of DSTRs
and as late as 30 days. Signs and symptoms in-
detected. It is known that these reactions oc-
cur much more frequently than AHTRs, with clude a maculopapular rash, fever, enterocoli-
estimates of approximately 1:2500 for either tis with watery diarrhea, elevated liver func-
type of delayed reaction and a frequency that tion test results, and pancytopenia. The rash
is twice as high for DSTRs.79 These reactions begins on the trunk and progresses to the
are likely to be greatly underrecognized be- extremities. In severe cases, bullae may
cause most patients do not undergo red cell develop.81
antibody screening following transfusion.80 Unlike GVHD after allogeneic marrow
transplantation, TA-GVHD leads to profound
Treatment marrow aplasia, with a mortality rate higher
than 90%. The time course of the reaction is
The treatment of DHTRs consists of monitor-
very rapid; death typically occurs within 1 to 3
ing the patient and providing appropriate sup-
weeks of the first symptoms.
portive care. The most frequent therapy is
correction of the anemia by transfusing anti-
Differential Diagnosis
gen-negative RBCs as needed. When a DSTR is
identified by the laboratory, the patient’s phy- Because the clinical manifestations of TA-
sician and the transfusion service director GVHD appear several days after a transfusion,
should be notified so that unrecognized he- it may be difficult to associate the patient’s
molysis may be appropriately identified and symptoms with the transfusion. The symp-
treated. toms can easily be attributed to other condi-
tions, including drug reactions and viral ill-
Prevention ness. In cases of TA-GVHD, a skin biopsy
DHTRs/DSTRs caused by known antibody reveals a superficial perivascular lymphocytic
specificities can be prevented by the transfu- infiltrate, necrotic keratinocytes, compact or-
sion of antigen-negative RBCs. It is essential to thokeratosis, and bullae formation. Molecular
obtain prior transfusion records for the recipi- techniques, including HLA typing, cytogenet-
ent because alloantibodies may have been ics, and chimerism assessment, can be used to
identified that are no longer detectable but re- make the diagnosis.82
688 䡲 AABB TECHNICAL MANUAL
Well-documented indications
Intrauterine transfusions
Prematurity, low birthweight, or erythroblastosis fetalis in newborns
Congenital immunodeficiencies
Hematologic malignancies or solid tumors (neuroblastoma, sarcoma, Hodgkin disease)
Peripheral blood stem cell/marrow transplantation
Components that are crossmatched, HLA matched, or directed donations (from family members or other
related donors)
Fludarabine therapy
Granulocyte components
Potential indications
Other malignancies, including those treated with cytotoxic agents
Donor-recipient pairs from genetically homogeneous populations
Usually not indicated
Patients with human immunodeficiency virus
Full-term infants
Nonimmunosuppressed patients
the disorder may be more common than previ- patients with previously normal platelet
ously believed.87 Hospitals that participated in counts and no other significant medical ab-
SHOT reported 44 cases of PTP in 8 years. normalities, it can be a challenge in patients
Patients typically present with wet purpura with multiple medical problems. Platelet se-
and thrombocytopenia 9 days (range = 1-24), rology studies may aid in the diagnosis.
on average, after a transfusion.88 The thrombo-
cytopenia is often profound, with platelet Pathophysiology
counts of <10,000/µL. Bleeding from mucous
The pathogenesis of PTP is related to the pres-
membranes and the gastrointestinal and uri-
ence of platelet-specific alloantibodies in a pa-
nary tracts is common. Mortality rates in large
tient who has previously been exposed to
case series range from 0 to 12.8%, primarily
platelet antigens via pregnancy or transfusion.
due to intracranial hemorrhage.67,68
The female-to-male ratio of affected patients
PTP has most commonly been associated
is 5 to 1. Antibodies against human platelet an-
with transfusions of RBCs or whole blood;
tigen 1a (HPA-1a), located on glycoprotein
however, the disorder has also been associated
IIIa, are identified in about 70% of PTP cases.
with transfusions of platelets or plasma.
Antibodies to HPA-1b, other platelet antigens,
and HLA antigens have also been implicated
Differential Diagnosis
in PTP.88
The differential diagnosis of PTP includes con- The reason for the concomitant destruc-
sideration of other causes of thrombocytope- tion of autologous platelets in this disorder is
nia, such as autoimmune thrombocytopenic unknown; three theories have been advanced.
purpura, thrombotic thrombocytopenic pur- According to the first theory, immune com-
pura, heparin-induced thrombocytopenia, plexes bind to platelets through the Fc recep-
DIC, and drug-induced thrombocytopenia. Al- tor, causing destruction of platelets.89 The sec-
though the diagnosis of PTP can be obvious in ond theory posits that the patient’s platelets
690 䡲 AABB TECHNICAL MANUAL
absorb a soluble platelet antigen from donor hemosiderin and ferritin. Transferrin becomes
plasma, making them susceptible to immune saturated after the administration of 10 to 15
destruction. According to the third theory, the RBC units to a nonbleeding patient.90 As iron
platelet alloantibody has autoreactivity that accumulates in the reticuloendothelial system,
develops when a patient is re-exposed to a liver, heart, spleen, and endocrine organs, tis-
foreign platelet-specific antigen.88 The third sue damage leading to heart failure, liver fail-
theory currently has the most support. ure, diabetes, and hypothyroidism may occur.
The use of leukocyte reduction filters may Patients who are chronically transfused for
decrease the incidence of PTP. In the three diseases such as thalassemia, sickle cell dis-
years prior to the implementation of universal ease, and other chronic anemias are at greatest
leukocyte reduction, in the United Kingdom, risk for iron overload. Prevention of the accu-
there were 10.3 cases of PTP per year, com- mulation of these toxic levels of iron by reduc-
pared to 2.3 cases per year after universal leu- ing the body’s iron stores through the use of
kocyte reduction (p<0.001).87 iron chelators is therefore extremely impor-
tant. A cumulative dose of 50 to 100 RBC units
Treatment can cause significantly greater morbidity and
Because the duration of thrombocytopenia in mortality than the underlying anemia.91
untreated patients is about 2 weeks, it can be Iron chelators bind to iron in the body
difficult to assess the effectiveness of therapies and tissues and help remove it through the
for PTP. Steroids, whole-blood exchange, and urine and/or feces. The development of iron-
plasma exchange have all been used to treat chelating drugs, such as parenteral deferox-
PTP. The current treatment of choice for PTP is amine and the oral agent deferiprone, greatly
IVIG.88 Patients respond within 4 days, on av- reduced the complications of iron overload in
erage, and some respond within hours. chronically transfused patients, leading to im-
proved quality of life. Deferiprone is more ef-
Prevention fective than deferoxamine in reducing myo-
cardial siderosis.92
PTP typically does not recur with subsequent The newest agent, deferasirox, has a
transfusions. However, there are four case re- much longer half-life than deferoxamine and
ports of PTP recurrence. Therefore, for pa- may be administered in one daily oral dose. Al-
tients with previously documented PTP, efforts though in-vivo studies are in the early stages,
should be made to obtain components from deferasirox has similar rapid access to intracel-
antigen-matched donors. Autologous dona- lular iron stores in cultured myocytes as defer-
tions and directed donations from antigen- iprone.93 Unlike deferiprone, which has occa-
matched donors and family members may sionally been associated with agranulocytosis,
also be appropriate. Because PTP has also oc- the most frequent side effects of deferasirox
curred after transfusions of deglycerolized re- are transient gastrointestinal distress and
juvenated or washed RBCs, such manipula- mildly increased creatinine levels that are rare-
tions are not indicated for the prevention of ly of clinical significance. Deferasirox treat-
recurrence.88 ment for a median of 2.7 years showed efficacy
in children and adults with sickle cell disease,
Iron Overload
resulting in a significant dose-dependent de-
A unit of RBCs contains approximately 250 mg cline in their serum ferritin levels without pro-
of iron. The average rate of excretion of iron is ducing any new adverse events.94 Lower doses
approximately 1 mg per day. As red cells are of deferasirox have also been used to reduce
destroyed, the majority of the released iron iron overload in non-transfusion-dependent
cannot be excreted and is stored in the body as patients with minimal side effects.94
CHAPTER 27 Noninfectious Complications of Blood Transfusion 䡲 691
E-mail fatalities2@fda.hhs.gov
Telephone/voicemail 240-402-9160
Fax 301-827-6748, Attn: CBER Fatality Program Manger
Express mail US Food and Drug Administration
CBER Office of Compliance and Biologics Quality
Document Control Center
10903 New Hampshire Avenue
WO71, G112
Silver Spring, MD 20993-0002
FDA = Food and Drug Administration; CBER = Center for Biologics Evaluation and Research.
FAT A L IT Y REP O R T IN G formation for the FDA. The report should con-
REQ UI REME N TS tain the patient’s medical records, including
laboratory reports, and the autopsy results
When the death of a patient results from a re-
when available. The patient’s underlying ill-
action to or complication of a transfusion, cur-
ness may make determination of the cause of
rent good manufacturing practice regulations
require that the fatality be reported to the FDA death difficult. If there is any clinical suspicion
by the facility that performed the compatibili- that the transfusion may have contributed to
ty testing.95 The director of the Office of Com- the patient’s death, that possibility should be
pliance and Biologics Quality at the FDA Cen- investigated. Most transfusion-associated fa-
ter for Biologics Evaluation and Research must talities are caused by acute hemolysis, TRALI,
be notified as soon as possible by telephone, or TACO. Investigations of these cases must at-
express mail, or facsimile or electronic mail, tempt to rule out laboratory, transfusion ser-
followed by the submission of a written report vice, or blood administration errors.96
within 7 days. Table 27-3 lists the contact in-
KEY POINTS
1. The blood supply is safer today than at any time in history. Hemovigilance and blood man-
agement programs decrease the risk of noninfectious complications of transfusion.
2. Many transfusion reactions have signs or symptoms that may be present in more than one
type of reaction. Early recognition of the reaction, prompt cessation of the transfusion, and
further evaluation are key to the successful resolution of a reaction.
3. Acute intravascular hemolytic reactions are often caused by sample or patient misidentifi-
cation and are thus, for the most part, preventable.
4. Allergic reactions range from urticaria (hives) to anaphylaxis. Patients experiencing severe
reactions should be checked for IgA deficiency and transfused accordingly.
5. Recognition of TRALI requires diagnosis by exclusion. Most patients recover from TRALI
with supportive care.
6. TACO can be confused with TRALI because both feature pulmonary edema. TACO should
be suspected in nonbleeding patients and those with congestive heart failure.
692 䡲 AABB TECHNICAL MANUAL
REF EREN CE S
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lection and transfusion: Annual summary for tions. In: Popovsky MA, ed. Transfusion reac-
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fice of Communication, Outreach, and Devel- 1-51.
opment, 2013. [Available at http://www.fda. 9. Davenport RD, Strieter RN, Standiford TJ, et al.
gov/BiologicsBloodVaccines/SafetyAvailabili Interleukin-8 production in red cell incompat-
ty/ReportaProblem/TransfusionDonationFa ibility. Blood 1990;76:2439-42.
talities/ucm346639.htm (accessed March 17, 10. Hod EA, Cadwell CM, Liepkalms JS, et al. Cyto-
2014).] kine storm in a mouse model of IgG-mediated
2. The National Healthcare Safety Network hemolytic transfusion reactions. Blood 2008;
(NHSN) manual: Biovigilance component. 112:891-4.
(June 2011) Atlanta, GA: Centers for Disease 11. Vamvakas EC, Blajchman MA. Transfusion-re-
Control and Prevention, 2011. [Available at lated mortality: The ongoing risks of allogene-
http://www.cdc.gov/nhsn/PDFs/Biovigi ic blood transfusion and the available strate-
lance/BV-HV-protocol-current.pdf (accessed gies for their prevention. Blood 2009;113:3406-
December 3, 2013).] 17.
3. Eder AF, Chambers LA. Noninfectious compli- 12. Friedrich JO, Adhikari N, Herridge MS, et al.
cations of blood transfusion. Arch Pathol Lab Meta-analysis: Low-dose dopamine increases
Med 2007;131:708-18. urine output but does not prevent renal dys-
4. Jenner PW, Holland PV. Diagnosis and man- function or death. Ann Intern Med 2005;142:
agement of transfusion reactions. In: Petz LD, 510-24.
ed. Clinical practice of transfusion medicine. 13. Ludens JH, Hook JB, Brody MJ, et al. Enhance-
3rd ed. New York: Churchill Livingstone, 1996: ment of renal blood flow by furosemide.
905-29. J Pharmacol Exp Ther 1968;163:456-60.
5. Levitt J, ed. Standards for blood banks and 14. Goldfinger D. Acute hemolytic transfusion re-
transfusion services. 29th ed. Bethesda, MD: actions—A fresh look at pathogenesis and
AABB, 2014. considerations regarding therapy. Transfusion
6. Josephson CD, Mullis NC, Van Demark C, et al. 1977;17:85-98.
Significant numbers of apheresis-derived 15. Toh CH, Dennis M. Disseminated intravascu-
group O platelet units have “high-titer” anti- lar coagulation: Old disease, new hope. BMJ
A/A,B: Implications for transfusion policy. 2003;327:974-7.
Transfusion 2004;44:805-8. 16. Norman KE. Alternative treatments for dis-
7. Sapatnekar S, Sharma G, Downes KA, et al. seminated intravascular coagulation. Drug
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plasma: A survey of 3156 North American lab- 30. Vamvakas EC. Allergic and anaphylactic reac-
oratories. Arch Pathol Lab Med 2007;131:909- tions. In: Popovsky MA, ed. Transfusion reac-
16. tions. 4th ed. Bethesda, MD: AABB Press,
18. Dubey A, Verma A, Sonker A, et al. Transfusion 2012:99-147.
medicine illustrated. Sudden increased inci- 31. Stainsby D, Jones H, Asher D, et al. Serious
dence of transfusion reactions reported from a hazards of transfusion: A decade of hemovigi-
ward: Root cause analysis. Transfusion 2009; lance in the UK. Transfus Med Rev 2006;20:
49:409-10. 273-82.
19. Brand A. Passenger leukocytes, cytokines, and 32. Brown SGA, Mullins RJ, Gold MS. 2. Anaphy-
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20. Oberman HA. Controversies in transfusion 33. Kennedy LD, Case LD, Hurd DD, et al. A pro-
medicine: Should a febrile transfusion re- spective, randomized, double-blind controlled
sponse occasion the return of the blood com- trial of acetaminophen and diphenhydramine
ponent to the blood bank? Con. Transfusion pretransfusion medication versus placebo for
1994;34:353-5. the prevention of transfusion reactions. Trans-
21. Ezidiegwu CN, Lauenstein KJ, Rosales LG, et fusion 2008;48:2285-91.
al. Febrile nonhemolytic transfusion reactions. 34. Sandler SG. How do I manage patients sus-
Management by premedication and cost im- pected of having had an IgA anaphylactic
plications in adult patients. Arch Pathol Lab transfusion reaction? Transfusion 2006;46:10-
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22. Wang, Rachel R. Effects of prestorage vs 35. Popovsky MA, Haley NR. Further characteriza-
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23. Tang AW. A practical guide to anaphylaxis. Am 2000;16:157-9.
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24. Savage, WJ, Tobian AA, Fuller AK, et al. Allergic crease in neutrophil count in transfusion-re-
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1716-22. American-European consensus conference on
25. Savage WJ, Savage JH, Tobian AA, et al. Allergic ARDS. Definitions, mechanisms, relevant out-
agonists in apheresis platelet products are as- comes, and clinical trial coordination. Am J
sociated with allergic transfusion reactions. Respir Crit Care Med 1994;149:818-24.
Transfusion 2012;52:575-81 38. Kleinman S, Caulfield T, Chan P, et al. Toward
26. Selective deficiency of IgA. Atlanta, GA: Na- an understanding of transfusion-related acute
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article/001476.htm (accessed December 3, 39. Popovsky MA, Moore SB. Diagnostic and
2013).] pathogenetic considerations in transfusion-
27. Shimada E, Odagiri M, Chaiwong K, et al. De- related acute lung injury. Transfusion 1985;25:
tection of Hpdel among Thais, a deleted allele 573-7.
of the haptoglobin gene that causes congenital 40. Silliman CC, Boshkov LK, Mehdizadehkashi Z,
haptoglobin deficiency. Transfusion 2007;47: et al. Transfusion-related acute lung injury:
2315-21. Epidemiology and a prospective analysis of
28. Lambin P, Le Pennec PY, Hauptmann G, et al. etiologic factors. Blood 2003;101:454-62.
Adverse transfusion reactions associated with 41. Kopko PM, Popovsky MA. Transfusion-related
a precipitating anti-C4 antibody of anti-Rodg- acute lung injury. In: Popovsky MA, ed. Trans-
ers specificity. Vox Sang 1984;47:242-9. fusion reactions. 4th ed. Bethesda, MD: AABB
29. Domen RE, Hoeltge GA. Allergic transfusion Press, 2012:191-215.
reactions: An evaluation of 273 consecutive re- 42. Steinberg KP, Hudson LD, Goodman RB, et al.
actions. Arch Pathol Lab Med 2003;127:316-20. Efficacy and safety of corticosteroids for per-
694 䡲 AABB TECHNICAL MANUAL
95. Code of federal regulations. Title 21, CFR Part (September 22, 2003) Silver Spring, MD: CBER
606.170(b). Washington, DC: US Government Office of Communication, Outreach, and De-
Printing Office, 2011 (revised annually). velopment, 2003.
96. Guidance for industry: Notifying FDA of fatali-
ties related to blood collection or transfusion.
C h a p t e r 2 8
Approaches to Blood
Utilization Auditing
Alan Tinmouth, MD, FRCPC, MSc, Head, General Medicine and Transfusion Medicine, The Ottawa Hospital,
and Scientist, University of Ottawa Centre for Transfusion Research, Clinical Epidemiology Program, Ottawa
Hospital Research Institute, Ottawa, Ontario, Canada, and Simon Stanworth, FRCP, FRCPath, DPhil, Consul-
tant Haematologist, Oxford University Hospitals NHS Trust, and Honorary Senior Clinical Lecturer, University
of Oxford, Oxford, United Kingdom
A. Tinmouth is supported by a Research Award from the Department of Medicine, The Ottawa Hospital; and
has disclosed a financial relationship with Amgen. S. Stanworth has disclosed no conflicts of interest.
697
698 䡲 AABB TECHNICAL MANUAL
Factors that help ensure high levels of partici- trends in the total number of blood units
pation in national audits in England are: transfused, perhaps to control ordering or in-
ventory management, requires a different pro-
1. The audits contribute to “Quality Account” cess from that required to limit inappropriate
reports required of National Health Service transfusion requests. In all instances, the
(NHS) hospitals (Health Act 2009). transfusion service (technologists and/or phy-
2. Data are used by the Care Quality Commis- sicians) and the institutional transfusion med-
sion, an independent regulator of all health icine or blood utilization committee must
and social care services in England. work together to perform the appropriate au-
3. NHS hospitals participating in a national dit(s) and follow-up.
audit may receive a discount from the NHS
Litigation Authority, which manages negli-
R ATIONALE FOR MONITO RING
gence and other claims against the NHS in
England.
B LO OD U T I LI Z AT I O N
4. The National Blood Transfusion Committee The primary motivation for monitoring blood
oversees, promotes, and supports all utilization is to identify instances when blood
national audits. product utilization is less than optimal. Inter-
ventions can then be implemented to change
In a similar vein, The Joint Commission transfusion practice. Therefore, audits or mon-
announced a certification program starting in itoring must be combined with a recognized
2014 to further recognize accredited hospitals process to effectively provide feedback on the
that implement system-wide measures to im- findings to the appropriate health-care profes-
prove blood utilization. sionals.
A number of different methods can be Improving or ensuring optimal use of
employed to meet the requirements to moni- blood transfusions is important for several
tor or audit blood transfusion use. However, reasons. Blood is a biologic agent associated
the general objective of all blood utilization with many possible adverse events, including
programs is to ensure the appropriate use of both infectious and noninfectious complica-
blood components. Alternative or comple- tions.5,6 Many of these complications are well
mentary goals may include reducing inappro- known, others are not usually recognized by
priate use and, as a result, reducing costs. physicians or the general population, and
Historically, audits of blood use have concen- some potential complications are still not fully
trated on controlling or reducing the total understood. Unnecessary complications from
number of blood components transfused and/ inappropriate blood transfusions need to be
or individual “overtransfusions.” This focus avoided. In addition, blood components are a
was based on the assumption that inappropri- scarce and expensive resource. Transfusing a
ate use consisted primarily of overtransfusion. single unit of Red Blood Cells (RBCs) has been
Unnecessary transfusions result in unneces- estimated to cost from $400 to $760 when all
sary costs and adverse events. However, with the associated costs are included, and many
increased attention on limiting patients’ expo- regions have experienced shortages.6-8
sure to blood components, undertransfusion Through the careful monitoring of blood
may be a concern. In addition, the dose of utilization, instances of inappropriate blood
blood transfusion requests, which may result component use can be identified and correc-
in overuse or underuse, might also need to be tive actions can be taken. If a “real-time” or
assessed in audits. However, neither of these prospective audit system is used, then a mem-
issues has historically been a major focus of ber of the transfusion medicine service can in-
the monitoring of blood transfusion utiliza- tervene, which may result in a change in the
tion.4 transfusion request before the issue of a blood
The methods used to audit blood use de- unit. A retrospective review does not alter the
pend on the desired objectives. Monitoring current transfusion episode but it does identi-
CHAPTER 28 Approaches to Blood Utilization Auditing 䡲 699
fy issues that can be addressed through inter- (usually within 24 hours); this has been termed
ventions designed to change future transfu- a “concurrent review” because it still affords
sion practice. the ability to provide timely feedback on indi-
A variety of interventions have been used vidual transfusion episodes.9 More remote ret-
to change transfusion practice (Table 28-1). rospective audits allow for the review of aggre-
One of the most common interventions for gate transfusion data, which can then be
change is audit and feedback, which is defined analyzed in several ways. Reviews of individual
by the Cochrane Effective Practice and Organi- and aggregate transfusion data are not mutu-
zation of Care Group as “any summary of clini- ally exclusive. Because they may serve differ-
cal performance of health care over a specified ent functions or purposes, they can, in fact, be
period of time.” However, there is less under- complementary.
standing of the effective elements of audit and
feedback or how the feedback should be struc- Prospective (“Real-Time”) Audits
tured to produce changes in behavior in differ-
ent settings. Following the delivery of an In a prospective audit, individual transfusion
intervention, ongoing monitoring allows for requests are reviewed in “real time” (ie, before
assessment of the sustained effectiveness of the issue of the blood component). An elec-
the intervention and need for ongoing or addi- tronic review using a computer-based algo-
tional intervention. rithm and/or a manual review by technolo-
In summary, audits serve the dual func- gists is undertaken whereby the request is
tion of 1) identifying areas of concern in the compared to local transfusion audit criteria.
use of blood products and 2) monitoring inter- Clinical data (eg, hematocrit and indication
ventions or changes in the identified areas. for RBC transfusion) are required to perform
the review. Ideally, this information is obtained
from the clinical staff as part of the transfusion
T Y PE S OF TRANSFUSION
request process10,11 or pretransfusion blood
AUDI TS values are automatically retrieved from the
Audits of transfusion practice can examine in- laboratory information system.12,13 With com-
dividual blood transfusion requests and/or ag- puter provider/physician order entry (CPOE),
gregate transfusion data. Reviews of individual the institutional guidelines can be incorporat-
requests can be performed either prospective- ed into the request process.11 Laboratory staff
ly in real time or retrospectively. Reviews of in- can also obtain these data from the laboratory
dividual transfusions may also be performed information system,14 although this is more
(retrospectively) very shortly after the event labor intensive and difficult to implement
TABLE 28-1. Use of Interventions to Change Transfusion Practice with Different Types of Audits
Individual education/feedback ++ ++ +
Group education/feedback/teaching – – ++
Guideline dissemination + + +
Incorporation of reminders/guidelines into ++ ++ +
transfusion request form or computerized
order entry system
++ = very complementary to audit type; + = can be complementary to audit type; – = less complementary or not complemen-
tary to audit type.
700 䡲 AABB TECHNICAL MANUAL
29
Joshi 1997 Retrospective audit, guidelines RBCs 20.7% 50% - -
(Continued)
701
TABLE 28-2. Studies of the Effectiveness of Prospective and Retrospective Audits in Changing Transfusion Practice (Continued)
702
Study Interventions Components Transfusions Patients Transfused per Patient Units Transfused
proves the chance of changing the physician’s implementation of massive transfusion proto-
future transfusion behavior. cols) or communication with the transfusion
As with the prospective audit, the concur- service might not be readily recalled in retro-
rent review can be conducted by a computer spective audits.
algorithm or manually using transfusion audit
criteria. The review may occur at the time of Retrospective Audits
the transfusion or shortly after the request is
filled. Requests that do not meet the audit cri- Retrospective audits of blood utilization are
teria are flagged for subsequent review by a se- commonly performed by hospital teams or
nior technologist or transfusion medicine phy- blood transfusion services. The results should
sician. When performing the review, the be reviewed by the hospital’s transfusion med-
transfusion medicine physician may have ac- icine committee. The frequency of such audits
cess to additional laboratory results that help varies from one institution to another. Com-
to determine the appropriateness of each re- puterized systems may facilitate ongoing utili-
quest. However, the reviewer must also con- zation review across many specialties.11 Unlike
sider the fact that these additional results were prospective and concurrent reviews, retro-
not available to the ordering physician at the spective audits can be used to examine aggre-
time he or she made the request. Because con- gate transfusion data and trends in transfusion
current audits do not delay the filling of trans- utilization. The results may be informative for
fusion requests, these audits can assess urgent understanding wider differences in transfu-
transfusions and use stricter criteria for appro- sion practice across different specialty groups.
priateness than prospective audits. Individual transfusion requests can also be
Following the identification of an inap- reviewed for appropriateness as part of a retro-
propriate transfusion request, the ordering spective audit, but doing so may be cumber-
physician may be contacted by telephone or e- some unless the data are collected prospec-
mail to discuss the case. The less immediate tively or the laboratory information system
nature and ability to use written communica- can link data on transfusion events and labo-
tion may reduce any animosity from the order- ratory results.
ing physician and result in a more meaningful Retrospective reviews can analyze data in
dialogue. Shanberge and colleagues35 report- a variety of ways. The simplest analysis is an
ed a 77% reduction in the number of Fresh examination of the total numbers of blood
Frozen Plasma units transfused after the intro- components transfused and patients trans-
duction of a concurrent audit system com- fused. However, these data might show con-
bined with a new guideline and an education siderable temporal variability that limits
program. meaningful analysis. Further analysis is re-
Conducting concurrent audits is time quired to make observations regarding
consuming, and a transfusion medicine physi- differences in blood utilization. The mean or
cian usually needs to review inappropriate median number of units transfused per hospi-
transfusion requests and follow up with the re- talized patient and/or procedure provides a
questing physicians.13 For these reasons, per- more meaningful summary of blood compo-
forming a concurrent audit of all transfusion nent use. Simply dividing the total number of
requests may not be feasible. Using selective units transfused by the total number of pa-
audits, as discussed for prospective audits, is tients who received a transfusion is not an ap-
an option to reduce the workload involved. propriate statistical analysis; any observed
Concurrent audits may be particularly relevant changes in total blood component use, partic-
for certain patient groups, such as patients ularly during a short period, could be skewed
treated for trauma or others in the emergency by a small number of patients who required a
department, where the urgent need for blood large number of units. The proportion of pa-
precludes a prospective audit, and accurate tients transfused should also be determined
time lines (key to determining the appropriate and can be further examined by procedure or
704 䡲 AABB TECHNICAL MANUAL
clinical specialty. All of these analyses com- goal is to increase the uptake of research re-
prise the minimal set required to monitor dif- sults and/or best practices into daily prac-
ferences in blood utilization. Additional analy- tice.39
ses could include assessments of appropriate The process of closing the gap between
and inappropriate transfusion rates, although knowledge and action involves first distilling
these analyses require collecting additional knowledge, usually in the form of guidelines.
clinical data and reviewing individual transfu- National or international guidelines can sim-
sion requests, which may be labor-intensive ply be adopted; however, local development of
exercises. guidelines with the involvement of local stake-
Utilization trends by individual product, holders may increase adherence to guide-
individual physician, or clinical service can be lines.40 Audits then serve the purpose of identi-
analyzed. For health-care institutions with fying gaps between the guidelines (best
multiple sites, similar clinical services at practices) and current practice (action). When
different sites can also be compared. These important gaps are identified through audits,
additional layers of analysis may provide im- interventions can be developed to target prac-
portant information to identify variations in tice changes. To improve the chance of achiev-
practice and detect inappropriate transfusion ing meaningful and lasting changes in prac-
practices. These areas could then be targeted tice, interventions should be carefully chosen
by interventions to improve transfusion prac- and designed.
tice. Unfortunately, the process of identifying
In general, a retrospective review is less the best interventions has not been well de-
labor intensive than a prospective or concur- fined. Ideally, the facilitators of and barriers to
rent review, particularly because it involves change are first identified so that any selected
less immediate attention from a technologist
intervention targets these identified fac-
or transfusion medicine physician. The
tors.41,42 Concurrent,13 retrospective,37 and
amount of time required to perform the review
prospective21 audits have been effective indi-
depends on the amount of detail desired.
vidual interventions in changing transfusion
The use of retrospective audits and the
practice. However, other interventions, includ-
provision of dedicated feedback to clinicians
ing dissemination of guidelines,11,20,28,30,32-35 ed-
have been effective in reducing the total num-
ucation,15,20,24,30,33,36 introduction of new trans-
ber of units transfused,20,24,29-33 number of units
fusion request forms, and computer order
transfused per patient,30-32,37,38 proportion of
entry,24,30,32 have been commonly used in con-
patients transfused,26,28,36 and number of inap-
propriate transfusions24,29,34 (Table 28-2). The junction with audits.
long-term durability of these changes in trans-
fusion practice following the introduction of a EF FE CTIVE N ES S OF
retrospective audit has not been evaluated. M ON I TO R I N G A N D
I NT E RV E N T IO N S TO C H A N G E
I N TE RVE NT I O NS TO C H A N G E TRANSF USION PRAC TICE
TR A N SF U SI O N P R ACT I C E Most published studies on auditing efforts
As previously described, the results of transfu- have shown that prospective and retrospective
sion audits should ideally be used in conjunc- audits reduce either the total amount of blood
tion with interventions to effect change and transfused or the proportion of inappropriate
improve transfusion practice. Thus, audits transfusions (Table 28-2). These reductions
should be considered a critical component of a occurred in studies that used a concurrent au-
larger process to optimize the utilization of dit alone,13 a prospective audit alone,21 or a ret-
blood components. This process can be more rospective audit and feedback alone.37 The
widely conceived as dissemination, knowledge prospective audit study showed a reduction in
transfer, or implementation work, where the the utilization of RBCs and frozen plasma but
CHAPTER 28 Approaches to Blood Utilization Auditing 䡲 705
not platelets.21 The retrospective audit study of individual transfusions by providing data on
showed a reduction in the utilization of RBCs trends in the use of transfusions.
but not frozen plasma or platelets.37 The re- Lists of the steps for implementing pro-
sults from these two studies might suggest that spective, concurrent, and retrospective re-
prospective and retrospective audits used in views are provided in Tables 28-3, 28-4, and
conjunction with other interventions are more 28-5, respectively. A transfusion request form
effective in improving blood utilization. How- or order entry system incorporating guidelines
ever, the poor designs of the studies (almost all and/or clinical information (see sample in Ap-
of which were uncontrolled, single-center, be- pendix 28-1) can aid in identifying inappropri-
fore-and-after studies) that examined inter- ate transfusion requests through either pro-
ventions to change transfusion practice and spective or concurrent audits.46 Similarly
the possibility of publication bias (eg, because laboratory information systems and comput-
the results of studies in which an intervention er algorithms can be used to screen transfu-
did not result in an improvement in transfu- sions for appropriateness in all types of au-
sion practice were not published) do not allow dits.13,47 Reviews may be more frequent in
the true or relative effectiveness of individual, larger transfusion services or less frequent in
or combinations of, interventions to be deter- smaller hospitals. The retrospective data can
mined.43,44 allow smaller hospitals to compare their trans-
fusion practices (eg, number of units trans-
fused per patient by diagnosis-related group)
SE L E C T I N G A N AUD I T P RO C E S S with those of other similar institutions.
TO MO NITO R T R A N SF U S I ON S
How transfusions should be monitored de- CO N CLUS IO N S
pends on a number of factors that are specific
Auditing transfusion practice is a required
to each institution.45 The first step in deter-
function for all transfusion services. Transfu-
mining how to monitor transfusions is to de-
sion audits provide information on levels of
cide which type of audits to use. The prospec-
compliance with standards and frequency of
tive and concurrent audits serve identical
unnecessary transfusions. Audits combined
purposes and cannot be used jointly to assess with guidelines are clearly essential to evaluate
an individual transfusion episode. Prospective transfusion practices at individual institutions,
audits require the greatest amount of time and they permit the identification of subopti-
from laboratory staff and transfusion medi- mal transfusion practices.
cine physicians, including 24-hour coverage In general, the findings of most audits
and support from the laboratory information continue to show unnecessary use of blood
system. These requirements may present diffi- outside established guidelines. The translation
culties for smaller hospitals and busy transfu- of research findings into hospital practice is
sion medicine services, respectively. In gener- often slow and haphazard, and this applies as
al, concurrent reviews are more practical in much to transfusion as to other branches of
smaller hospitals or hospitals with limited re- health care. Many interventions are undertak-
sources for laboratory staff or transfusion en by hospitals to change their transfusion
medicine physicians because transfusions can practices, but there are real uncertainties
be reviewed in the 12 to 24 hours following the about their effectiveness and durability.
transfusion episode. If a universal prospective There is a need for research that defines
audit of all blood components is not feasible, the determinants of appropriate transfusion
then the prospective audit could be limited to behavior to better guide the design and selec-
particular components (eg, intravenous im- tion of interventions that can produce opti-
mune globulin or recombinant Factor VIIa) or mal changes in transfusion practice. Ideally,
specific clinical areas. Retrospective reviews the selection of audits and interventions
supplement prospective or concurrent audits should be based on an assessment of local
706 䡲 AABB TECHNICAL MANUAL
TABLE 28-3. Steps for Implementing a Prospective Audit System to Monitor Blood Component
Utilization
1. The extent and frequency of the audit process is determined.
䡲 The audit may be performed on all blood components or only on specific blood products.
䡲 Areas with urgent needs for transfusion (eg, emergency or operating rooms) may be excluded from the
audit to avoid delays in transfusion for their patients.
䡲 To limit resource demands, a selective audit may be used. For example, transfusion requests may be
audited only from a single ward, selected on a rotating basis, because of its high transfusion volume or a
perceived problem in its transfusion practice.
䡲 Similarly, audits may be performed only during specific hours to limit off-hour work by laboratory staff
and transfusion medicine physicians.
2. The requirements for clinical information at the time the transfusion requests are met.
䡲 This may be part of the request form (Appendix 28-1) or computer order entry.
3. Criteria for appropriate or inappropriate indications are developed for transfusions.
䡲 Audit criteria should be less stringent than optimal transfusion guidelines to reduce audit workload and
conflicts with requesting physicians.
䡲 Audit criteria should be developed in conjunction with a transfusion medicine committee and various
medical specialists who use significant amounts of blood products to increase acceptance of transfusion
audits.
4. Transfusion requests are screened by laboratory staff, transfusion nurses, or a computer algorithm to iden-
tify inappropriate requests.
5. For all inappropriate transfusion requests, the requesting physician is contacted by the transfusion medi-
cine service.
䡲 The requesting physician is usually contacted by a transfusion medicine physician or senior technologist.
䡲 A predefined mechanism is developed to override/bypass a review if blood is required urgently and to
avoid unacceptable delays in filling a transfusion request.
6. Any decisions to change the transfusion request are made in conjunction with the ordering physician.
TABLE 28-4. Steps for Implementing a Concurrent Audit System to Monitor Blood Component
Utilization
1-4. The same as for the Prospective Audit.
5. All inappropriate transfusion requests are subsequently reviewed by senior laboratory staff or a transfu-
sion medicine physician who discusses the transfusion request with the requesting physician.
䡲 Contact with the requesting physician is made within 24 hours of the transfusion so that the physician
remembers the clinical details. This time frame is optimal for providing feedback and potentially chang-
ing future transfusion behavior.
䡲 Contact can be made by telephone or electronically.
CHAPTER 28 Approaches to Blood Utilization Auditing 䡲 707
TABLE 28-5. Steps for Implementing a Retrospective Audit System to Monitor Blood Component
Utilization
1. Determine the extent and frequency of the audit reviews.
䡲 The frequency of the data reviews needs to be based on the volume of transfusions and resources avail-
able. Records should not be reviewed more than 6 months after transfusions were administered.
䡲 The components to be reviewed need to be determined and should include all conventional components
and frequently transfused blood derivatives.
2. Determine the outcomes.
䡲 Totals for the numbers of transfusions and patients transfused are the simplest data to collect but pro-
vide a limited understanding of, and ability to monitor, changes in transfusion utilization. Providing data
on utilization by patient (ie, mean or median number of units per patient) and proportion of patients
transfused provides a greater understanding of utilization.
䡲 The appropriateness of transfusions can also be reported if clinical and/or laboratory data are available.
These can be aggregate data from prospective or concurrent reviews, or the laboratory information sys-
tem can link laboratory data to data on transfusion events.
3. Review the audit data.
䡲 Audit data should be reviewed by the transfusion medicine service and the transfusion medicine commit-
tee.
䡲 Data may be analyzed on individual physicians, departments, or diagnoses.
䡲 Data may be compared within the institution or with data from similar institutions to evaluate overall utili-
zation rates for blood components.
䡲 Data should be shared with relevant departments and physicians.
4. Determine whether any additional interventions to optimize transfusion practice are warranted.
K EY PO I NTS
1. Auditing the use of blood components is a necessary function for all transfusion medicine
services and is required by AABB and The Joint Commission. The main purpose of auditing
blood utilization is to assess the appropriate use of components and help optimize their
use.
2. Different forms of audits (prospective, concurrent, or retrospective) may be used depending
on the objectives of the audit and the resources available. In all cases, providing feedback to
the users, using other interventions, or both are necessary to effect change in transfusion
practice.
3. Prospective audits require the review of transfusion requests before issue of components,
which permits the opportunity to intervene and stop or change an inappropriate transfu-
sion request. Individual transfusion requests are reviewed using prespecified audit criteria
(eg, guidelines).
708 䡲 AABB TECHNICAL MANUAL
RE FE RE N CES
1. Mintz PD. Quality Assessment and improve- patients at four hospitals. Transfusion 2010;50:
ment of blood transfusion practice. In: Mintz 753-65.
PD, ed. Transfusion therapy: Clinical princi- 9. Becker J, Shaz B for the Clinical Transfusion
ples and practice. 3rd ed. Bethesda, MD: AABB Medicine Committee and the Transfusion
Press, 2011:813-36. Medicine Section Coordinating Committee.
2. Comprehensive accreditation manual for hos- Guidelines for patient blood management and
pitals (CAMH): The official handbook. Oak- blood utilization. Bethesda, MD: AABB, 2011.
Brook Terrace, IL: The Joint Commission, 10. Haspel RL, Uhl L. How do I audit hospital
2014. blood product utilization? Transfusion 2012;
3. Levitt J, ed. Standards for blood banks and 52:227-30.
11. Baer VL, Henry E, Lambert DK, et al. Imple-
transfusion services. 29th ed. Bethesda: AABB,
menting a program to improve compliance
2014.
with neonatal intensive care unit transfusion
4. Saxena S, Wehrli G, Makarewicz K, et al. Moni-
guidelines was accompanied by a reduction in
toring for underutilization of RBC compo-
transfusion rate: A pre-post analysis within a
nents and platelets. Transfusion 2001;41:587-
multihospital health care system. Transfusion
90. 2011;51:264-9.
5. Popovsky M, ed. Transfusion reactions. 4th ed. 12. Yazer MH, Waters JH. How do I implement a
Bethesda, MD: AABB Press, 2012. hospital-based blood management program?
6. Tinmouth AT, Fergusson DA, Chin-Yee IH, et Transfusion 2012;52:1640-5.
al. Clinical consequences of red cell storage in 13. Cohn CS, Welbig J, Bowman R, et al. A data-
the critically ill. Transfusion 2006;46:2014-27. driven approach to patient blood manage-
7. Amin M, Fergusson D, Wilson K, et al. The so- ment. Transfusion 2014;54:316-22.
cietal unit cost of allogenic red blood cells and 14. Sarode R, Refaai MA, Matevosyan K, et al. Pro-
red blood cell transfusion in Canada. Transfu- spective monitoring of plasma and platelet
sion 2004;44:1479-86. transfusions in a large teaching hospital re-
8. Shander A, Hofmann A, Ozawa S, et al. Activi- sults in significant cost reduction. Transfusion
ty-based costs of blood transfusions in surgical 2010;50:487-92.
CHAPTER 28 Approaches to Blood Utilization Auditing 䡲 709
15. Tavares M, DiQuattro P, Nolette N, et al. Re- 28. Hameedullah, Khan FA, Kamal RS. Improve-
duction in plasma transfusion after enforce- ment in intraoperative fresh frozen plasma
ment of transfusion guidelines. Transfusion transfusion practice—Impact of medical au-
2011;51:754-61. dits and provider education. J Pak Med Assoc
16. Rothschild JM, McGurk S, Honour M, et al. As- 2000;50:253-6.
sessment of education and computerized de- 29. Joshi G, McCarroll M, O’Rourke P, et al. Role of
cision support interventions for improving quality assessment in improving red blood cell
transfusion practice. Transfusion 2007;47:228- transfusion practice. Ir J Med Sci 1997;166:16-
39. 19.
17. Yeh CJ, Wu CF, Hsu WT, et al. Transfusion audit 30. Morrison JC, Sumrall DD, Chevalier SP, et al.
of fresh-frozen plasma in southern Taiwan. The effect of provider education on blood uti-
Vox Sang 2006;91:270-4. lization practices. Am J Obstet Gynecol 1993;
18. Rehm JP, Otto PS, West WW, et al. Hospital- 169:1240-5.
wide educational program decreases red 31. Brandis K, Richards B, Ghent A, et al. A strategy
blood cell transfusions. J Surg Res 1998;75:183- to reduce inappropriate red blood cell transfu-
6. sion. Med J Aust 1994;160:721-2.
19. Cheng G, Wong HF, Chan A, et al. The effects of 32. Rosen NR, Bates LH, Herod G. Transfusion
a self-educating blood component request therapy: Improved patient care and resource
form and enforcements of transfusion guide- utilization. Transfusion 1993;33:341-7.
lines on FFP and platelet usage. Queen Mary 33. Ayoub MM, Clark JA. Reduction of fresh frozen
Hospital, Hong Kong. British Committee for plasma use with a simple education program.
Standards in Hematology (BCSH). Clin Lab
Am Surg 1989;55:563-5.
Haematol 1996;18:83-7. 34. Giovanetti AM, Parravicini A, Baroni L, et al.
20. Solomon RR, Clifford JS, Gutman SI. The use of
Quality assessment of transfusion practice in
laboratory intervention to stem the flow of
elective surgery. Transfusion 1988;28:166-9.
fresh-frozen plasma. Am J Clin Pathol 1988;
35. Shanberge JN. Reduction of fresh-frozen plas-
89:518-21.
ma use through a daily survey and education
21. Hawkins TE, Carter JM, Hunter PM. Can man-
program. Transfusion 1987;27:226-7.
datory pretransfusion approval programmes
36. Handler S. Does continuing medical educa-
be improved? Transfus Med 1994;4:45-50.
tion affect medical care. A study of improved
22. Littenberg B, Corwin H, Gettinger A, et al. A
transfusion practices. Minn Med 1983;66:167-
practice guideline and decision aid for blood
80.
transfusion. Immunohematology 1995;11:88-
37. Lam HT, Schweitzer SO, Petz L, et al. Are retro-
94.
23. Tuckfield A, Haeusler MN, Grigg AP, et al. Re- spective peer-review transfusion monitoring
duction of inappropriate use of blood prod- systems effective in reducing red blood cell
ucts by prospective monitoring of transfusion utilization? Arch Pathol Lab Med 1996;120:
request forms. Med J Aust 1997;167:473-6. 810-16.
24. Arnold DM, Lauzier F, Whittingham H, et al. A 38. Lam HT, Schweitzer SO, Petz L, et al. Effective-
multifaceted strategy to reduce inappropriate ness of a prospective physician self-audit
use of frozen plasma transfusions in the inten- transfusion-monitoring system. Transfusion
sive care unit. J Crit Care 2011;26:636. 1997;37:577-84.
25. Rubin GL, Schofield WN, Dean MG, et al. Ap- 39. Graham ID, Logan J, Harrison MB, et al. Lost in
propriateness of red blood cell transfusions in knowledge translation: Time for a map? J Con-
major urban hospitals and effectiveness of an tin Educ Health Prof 2006;26:13-24.
intervention. Med J Aust 2001;175:354-8. 40. Harrison MB, Graham ID, Fervers B. Adapting
26. Capraro L, Syrjala M. Advances in cardiac sur- knowledge to a local context. In: Straus S, Tet-
gical transfusion practices during the 1990s in roe J, Graham ID, eds. Knowledge translation
a Finnish university hospital. Vox Sang 2001; in health care. Oxford: Blackwell Publishing,
81:176-9. 2009;73-82.
27. Tobin SN, Campbell DA, Boyce NW. Durability 41. Cabana MD, Rand CS, Powe NR, et al. Why
of response to a targeted intervention to modi- don’t physicians follow clinical practice guide-
fy clinician transfusion practices in a major lines? A framework for improvement. JAMA
teaching hospital. Med J Aust 2001;174:445-8. 1999;282:1458-65.
710 䡲 AABB TECHNICAL MANUAL
42. Legare F, O’Connor AM, Graham ID, et al. Pri- 45. Qu L, Kiss JE. Blood utilization review. In: Sax-
mary health care professionals’ views on barri- ena S, ed. The transfusion committee: Putting
ers and facilitators to the implementation of patient safety first. 2nd ed. Bethesda, MD:
the Ottawa Decision Support Framework in AABB Press, 2013:75-91.
practice. Patient Educ Couns 2006;63:380-90. 46. Hannon T, Gross I. Transfusion guidelines: De-
43. Wilson K, MacDougall L, Fergusson D, et al. velopment and impact on blood management.
The effectiveness of interventions to reduce In: Saxena S, ed. The transfusion committee:
physician’s levels of inappropriate transfusion: Putting patient safety first. 2nd ed. Bethesda,
What can be learned from a systematic review MD: AABB Press, 2013:121-44.
of the literature. Transfusion 2002; 42:1224-9. 47. Audet AM, Goodnough LT, Parvin CA. Evaluat-
44. Tinmouth A, MacDougall L, Fergusson D, et al. ing the appropriateness of red blood cell
Reducing the amount of blood transfused: A transfusions: The limitations of retrospective
systematic review of behavioral interventions medical record reviews. Int J Qual Health Care
to change physicians’ transfusion practices. 1996;8:41-9.
Arch Intern Med 2005;165:845-52.
CHAPTER 28 Approaches to Blood Utilization Auditing 䡲 711
䡲 APPENDIX 28-1
Transfusion Order Form in Use at St. Vincent Indianapolis Hospital Since 2001
Used with permission from Hannon T, Gross I. Transfusion guidelines: Development and impact on blood management. In:
Saxena S, ed. The transfusion committee: Putting patient safety first. 2nd ed. Bethesda, MD: AABB Press, 2013:121-44.
C h a p t e r 2 9
Scott A. Koepsell, MD, PhD, Assistant Professor, Department of Pathology and Microbiology, University of
Nebraska Medical Center, Omaha, Nebraska; Eapen K. Jacob, MD, Assistant Professor, Department of Labora-
tory Medicine and Pathology, Mayo Clinic, Rochester, Minnesota; and David H. McKenna Jr, MD, Associate
Professor, Department of Laboratory Medicine and Pathology, University of Minnesota Medical School, Min-
neapolis, Minnesota
S. Koepsell and E. Jacob have disclosed no conflicts of interest. D. McKenna has disclosed a financial relation-
ship with Novartis, Inc.
713
714 䡲 AABB TECHNICAL MANUAL
TABLE 29-1. Diseases Treated with are more common in a pediatric population,
Autologous or Allogeneic Transplantation whereas the number of patients with a clonal
disorder in their marrow or a hematologic ma-
Hematologic Cancers lignancy is greater in adults. Ultimately, the
decision to perform HSC transplantation re-
Leukemia
quires a complex integration of many vari-
Lymphoma ables. These variables include patient goals,
prognosis, disease progression, previous ther-
Myeloma
apy, age, availability of a suitable HSC source
Marrow Failure States/Clonal Disorders of (ie, marrow, mobilized peripheral blood, or
Marrow UCB), and type of transplant (ie, autologous vs
allogeneic, and myeloablative vs nonmyeloab-
Aplastic anemia
lative).
Fanconi anemia
Autologous Transplantation
Pure red cell aplasia
In general, autologous HSC transplantation is
Amegakaryocytosis
used for hematopoietic rescue after high-dose
Paroxysmal nocturnal hemoglobinuria antineoplastic therapy. The antitumor effect of
the transplant comes solely from the chemo-
Myelofibrosis
therapy and radiotherapy used during the con-
Myelodysplasia ditioning phase of transplantation. For older
patients who are not traditional candidates for
Inborn Errors of Metabolism/Congenital Immuno-
autologous transplantation or patients with
deficiency
other significant morbidities, reduced-intensi-
Mucopolysaccharidoses ty induction chemotherapy can broaden the
clinical utility of HSC transplantation.
Leukodystrophies Donor requirements for autologous
Osteopetrosis transplants are based on the donor’s disease
state. The patient must be healthy enough to
Severe combined immunodeficiency syndrome undergo mobilization (as described later in
Wiskott-Aldrich syndrome this chapter) and procurement of HSCs either
by peripheral blood apheresis collection or
Pediatric Cancers marrow aspiration. Significant levels of prior
Wilms tumor chemotherapy, radiation, or ongoing marrow
disease involvement may make the mobiliza-
Neuroblastoma tion and collection of HSCs unfeasible due to
Ewing sarcoma the reduced quality or number of HSCs.
Eligibility requirements are not mandated
Medulloblastoma by the Food and Drug Administration (FDA)
Rhabdomyosarcoma for autologous HSC transplantation [Title 21,
Code of Federal Regulations (CFR) Part
Hemoglobinopathies 1271.90], so the use of screening question-
Thalassemia
naires to identify relevant communicable dis-
ease is not required. However, a general health
Sickle cell disease assessment is needed per AABB Standards for
Cellular Therapy Product Services (CT Stan-
Autoimmune Diseases
dards).1 AABB CT Standards also requires labo-
Solid Tumors ratory testing for human immunodeficiency
virus (HIV) 1/2; hepatitis B virus; hepatitis C
CHAPTER 29 Hematopoietic Stem Cells 䡲 715
virus; syphilis; human T-cell lymphotropic vi- for which there is an FDA licensed screening
rus, Types I and Type II (HTLV-I/II); and cyto- test [Title 21, CFR Part 1271.3(r)] include HIV,
megalovirus (CMV) because the autologous hepatitis B and C, human transmissible spon-
products are cryopreserved and stored with giform encephalopathy, Treponema pallidum,
other products, so the presence of these virus- HTLV-I/II, and CMV. Donor questionnaires
es would pose a contamination risk. have been developed to assist with screening6
and medical record review for these diseases
Allogeneic Transplantation using FDA guidance documents.7
Indications for allogeneic HSC transplantation In the United States, infectious disease
vary. However, in general, allogeneic trans- testing for HSC donors must be performed in a
plantation is used to treat malignant condi- Clinical Laboratory Improvement Act certified
tions only when both the induction antineo- laboratory as mandated by the FDA. If screen-
plastic therapy and the transplanted cells, due ing or testing detects a risk of a relevant com-
to the graft-vs-neoplasm (GVN) effect, are municable disease, the potential HSC donor is
therapeutic. For patients with an inborn error considered ineligible. All parties (the donor,
of metabolism, congenital immunodeficiency, the recipient, and their physicians) are in-
or other diseases and conditions in which formed of the donor’s eligibility status, and a
germline mutations are present in the patient’s risk-benefit analysis is performed to deter-
cells, allogeneic HSC transplants offer thera- mine whether the donor’s HSCs should be
peutic benefit by helping to replace the defi- used. If a decision is made to proceed with a
cient cellular machinery. transplant of the ineligible donor’s HSCs, the
In the allogeneic setting, screening and urgent medical need, as defined by the FDA
infectious disease testing are mandated in the [Title 21, CFR Part 1271.3(u)], is documented.
United States to determine whether the trans- Depending on the institution’s accrediting
plantation of mobilized peripheral blood or body and the circumstances, such as when the
UCB poses a risk of transmission of a relevant HSCs are from a related first- or second-degree
communicable disease to the recipient [Title ineligible donor, the requirement for docu-
21 CFR 1271.3(r)]. This screening and testing mentation of an urgent medical need may
includes the administration of a screening vary. Finally, in addition to screening and test-
questionnaire, a physical examination, a re- ing for infectious diseases, the donor’s medical
view of the relevant medical records, and ap- evaluation is used for determining whether
plicable testing [Title 21, CFR Parts 1271.3(s) the donor’s health is adequate to allow that do-
and 21 CFR 1271 Subpart C]. Although marrow nor to undergo the HSC mobilization and pro-
products are administered under Sections 375 curement process (described below).
and 379 of the Public Health Services Act, mar-
row screening and testing are very similar to Histocompatibility
mobilized peripheral blood and UCB screen-
ing and testing because the administration of In addition to infectious diseases, donor char-
all of these products is governed by the stan- acteristics that may affect transplant out-
dards of various accrediting bodies, such as comes include histocompatibility with the
the AABB, the Foundation for the Accredita- recipient, gender, age, parity, and ABO com-
tion of Cellular Therapy (FACT),4 and the Na- patibility. Of these characteristics, histocom-
tional Marrow Donor Program (NMDP).5 patibility is the most important. In general, if a
For UCB transplantation, the screening healthy HLA-matched related donor is avail-
and testing process is performed on the moth- able, that donor is selected instead of an HLA-
er and her samples (see Chapter 30). Relevant matched unrelated donor. However, recent
communicable diseases that can be transmit- data have shown that in patients with acute
ted by transplanted HSCs to the recipient or to myeloid leukemia or myelodysplastic syn-
the people who handle the components and drome, an HSC transplant from an HLA-
716 䡲 AABB TECHNICAL MANUAL
matched, unrelated donor may be as effective of predicting graft failure based on the pres-
as a transplant from a sibling.8,9 ence of DSAs, screening HSC transplant candi-
Major histocompatibility antigens were dates for DSAs may become more routine as
first studied in depth in various animal models more data emerge on this topic.14
for skin transplantation.10(pp97-111) In humans, UCB has several unique characteristics
these are HLA antigens and are categorized with regard to HLA matching. The level of HLA
into Class I (HLA-A, -B, and -C) and Class II matching required for UCB is less stringent
(HLA-DR, -DQ, and -DP). These highly poly- than that required for marrow and mobilized
morphic molecules are essential in determin- peripheral blood HSCs. Data on UCB indicate
ing graft survival as well as the likelihood that that matching at 4 of 6 loci is sufficient for
the recipient will develop graft-vs-host dis- HLA-A and -B at the antigen level and for HLA-
ease (GVHD). As molecular techniques have DRß1 at the allele level, provided that a suffi-
supplanted serologic methods, the resolution cient cell dose is achieved.15 As the number of
has improved and the number of antigens that mismatched alleles increases (from one to
can be compared has increased. Matching at
two), a higher total nucleated cell (TNC) dose
the antigen level has been replaced by allele-
is needed to overcome their deleterious effect,
level matching for the final selection of periph-
including the use of combined double-cord
eral blood and marrow HSC components for a
transplants.16 In addition, early evidence indi-
given recipient.
cates that noninherited, maternal HLA may be
HLA matching in HSC transplantation
has been reviewed in detail elsewhere and is permissive when considering donor-recipient
summarized here only briefly.11 HLA matching mismatches.17 Unit-to-unit matching of 4 of 6
has an important impact on outcomes, espe- loci in the setting of double-cord blood trans-
cially in low-risk patients. Allogeneic trans- plantation is also performed at various institu-
plantation using either mobilized peripheral tions; however, firm evidence to support this
blood or marrow HSCs with high-resolution practice is not available at this time.
(allele-level) mismatching at HLA-A, -B, -C,
and -DR1 is associated with a 5% to 10% de- Other Donor Characteristics
crease in survival with each mismatch.12 The For marrow and mobilized peripheral blood
results are similar, although the evidence is a HSC donors, other factors that may have a
bit less clear, for HSC grafts with mismatches positive effect on transplant outcomes include
in Class II HLA-DQ and -DP. For mobilized
male gender, younger age, nulliparity, ABO
peripheral blood HSC grafts, allele-level mis-
and CMV status matching, and greater size of
matching is probably as detrimental to surviv-
the donor relative to the recipient. Other than
al as antigen-level mismatching, although the
HLA, only donor age appears to be associated
data come from smaller studies. Many centers
with survival.11 ABO incompatibility has been
now match at high resolution for 8 to 10 loci
(HLA-A, -B, -C, -DR, and -DQ). High-resolu- reviewed extensively. ABH antigens found on
tion 8/8 and 10/10 matched transplants of red cells, platelets, and neutrophils would sug-
HSCs from unrelated donors are at least in part gest that ABO incompatibility would affect
responsible for the improvement in outcomes outcomes. However, inconsistent results on
of matched unrelated transplants compared to outcomes—such as survival, nonrelapse mor-
matched related donors.12 tality, GVHD, and graft failure—of both major
Not surprisingly, HSC transplants in re- and minor incompatible transplants have
cipients who have antibodies against donor been observed.18 The risk of delayed red cell
HLA [known as donor-specific antibodies engraftment, pure red cell aplasia, increased
(DSAs)] have adverse outcomes.13 The level at transfusion requirements, and both immedi-
which DSAs have a significant impact is less ate and delayed hemolysis is increased in ma-
clear, as is the appropriate course of action jor ABO-incompatible allogeneic HSC trans-
when DSAs are detected. Despite the difficulty plantation.
CHAPTER 29 Hematopoietic Stem Cells 䡲 717
Other characteristics that are specific to cluding T-cell depletion, use of antithymocyte
UCB HSC donation and may affect outcomes globulin, and pharmacologic measures.22
include issues related to maternal history, unit The GVN effect is also thought to be driv-
collection, processing, and unit storage.19 en by donor lymphocytes. In recent years, mo-
Characteristics of the UCB unit have been uti- bilized peripheral blood has greatly outpaced
lized in a scoring system, the “Cord Blood Ap- marrow as an HSC source due to its easier col-
gar,” to determine the utility of the unit after lection and the belief that GVN effect may be
consideration of the TNC count, CD34 positiv- improved. The first randomized controlled tri-
ity, colony-forming-unit count, mononuclear al comparing marrow and mobilized peripher-
cell content, and volume.20 al blood HSC grafts in unrelated donor mye-
loablative transplantations found that chronic
GVHD, but not acute GVHD, risk was higher in
DE TE RMI N AT ION OF GRAF T patients who received a peripheral blood HSC
SOURCE transplant.23
The choice of the source of HSCs for allogeneic
transplantation is determined by several vari- Kinetics
ables, including the availability of an ade- The kinetics of engraftment are affected by
quately matched donor. As described above, many variables, such as degree of HLA match-
marrow and mobilized peripheral blood prod- ing, dose of HSCs, and use of granulocyte
ucts in general need a higher level of HLA colony-stimulating factor (G-CSF). In addition,
matching than UCB units. For this reason, a the characteristics of the stem cell source may
UCB transplant may be the best alternative play a role. For instance, stem cells in UCB ap-
when only 1 and 2 allele-mismatched donor pear to have a greater regenerative capacity
units are available. In addition, for some cen- than those in marrow and peripheral blood.24
ters, the relatively rapid availability of UCB In general, the rate of engraftment is predicted
units compared to marrow and peripheral by the number of progenitor cells in each graft.
blood products plays a substantial role in graft This is greatest in mobilized peripheral blood,
choice in patients with an immediate need for followed by marrow, UCB, and other HSC
transplantation. A typical unrelated marrow or sources.
peripheral blood donor search may take In a meta-analysis of studies that com-
months, whereas a search for UCB takes sever- pared related transplantation with donor-mo-
al days to a few weeks.21 bilized peripheral blood and marrow, the me-
dian time to neutrophil engraftment was 14 vs
GVHD and GVN Effect 21 days and to platelet engraftment was 14 vs
22 days, respectively.25 These findings have
GVHD and GVN effect, although linked in been corroborated by a more recent meta-
terms of their cause, have opposite outcomes. analysis of studies of allogeneic related trans-
In GVHD, donor lymphocytes attack host-re- plantation in which mobilized peripheral
cipient tissues in the skin, lung, liver, gastroin- blood vs marrow HSC engraftment was 15 vs
testinal tract, and other organs. GVHD may be 21 days for neutrophils and 13 vs 21 days for
categorized as acute or chronic, and it occurs platelets, respectively.26 The more rapid en-
in more than 40% of individuals who receive graftment of peripheral blood compared to
an allogeneic HSC transplant. GVHD is associ- marrow HSCs was also seen in a study of unre-
ated with significant morbidity and mortality. lated allogeneic transplantation in which me-
Not surprisingly, the risk of GVHD is related to dian neutrophil engraftment occurred at 15
the graft source. Consequently, GVHD risk in- and 19 days, respectively, and median platelet
creases as the lymphocyte content of the HSC engraftment occurred at 20 and 27 days, re-
product increases. Various techniques have spectively.27 These findings were confirmed in
been used to decrease the risk of GVHD, in- the randomized controlled trial of Anasetti
718 䡲 AABB TECHNICAL MANUAL
and colleagues23 in which the relative differ- Transplant physicians face a complex
ences in time to engraftment was 5 days short- choice when determining which donor and
er for neutrophils and 7 days shorter for plate- stem cell source is best for their patients based
lets. A comparison of adult unrelated marrow on numerous factors, including the patient’s
to UCB HSC grafts showed that engraftment disease, disease stage, age, and comorbidities.
occurred earlier with both neutrophils (medi- As more data are collected, the choice of HSC
an of 18 days for matched marrow, 20 for mis- graft may evolve.
matched marrow, and 27 for mismatched
UCB) and platelets (median of 29 days for CO L L E C T I ON / S O U RC E S O F
matched marrow and mismatched marrow, H SC S
and 60 days for mismatched UCB).28 In the set-
ting of reduced-intensity UCB transplantation, Regardless of the source of HSCs, standards of
median neutrophil engraftment times ap- both FACT and AABB require all institutions
proaching those of mobilized peripheral blood that collect HSCs to have a procedure in place
and marrow HSC transplants have been ob- to obtain informed consent from the donor or
served.24 the donor’s representative, as dictated by local
laws.1(pp16-17,20-21),4 The informed consent pro-
Survival cess should include providing information to
the donor regarding the risks/benefits of the
The most important outcome measure for any procedure, the tests performed on the donor
transplant procedure is patient survival. For that are designed to protect the recipient, al-
recipients of transplants from matched related ternative collection methods, and protection
donors, mobilized peripheral blood may offer of their health information. In addition, the
an advantage in overall and disease-free donor should be given the opportunity to ask
survival over marrow HSC grafts, at least in re- questions as well as to refuse donation. The
cipients with late-stage hematologic malig- risks that are specific to each collection proce-
nancies.25 In recipients of myeloablative trans- dure are discussed below.
plants from unrelated donors for hematologic Another requirement for all facilities that
malignancies, a recent randomized controlled collect HSCs is to provide donor access to
trial indicated that marrow and mobilized pe- medical care based on the risks and clinical
ripheral blood sources have equivalent effects situation associated with each type of dona-
on survival, with marrow grafts associated tion. Specifically, procedures should be in
with less chronic GVHD but more graft fail- place to provide medical or emergency care to
ure.23 donors who experience adverse effects. Clear-
Based on these findings, the rapid in- ance from the donor’s physician for HSC col-
crease in the use of mobilized peripheral blood lection should be documented.
relative to marrow in recent years may change.
This may not be true, however, in nonmye- Marrow HSC Collection
loablative patients or transplant recipients at In addition to undergoing relevant donor
high risk of infection or graft failure. For pedi- screening, infectious disease testing, and HLA
atric transplant recipients, marrow may actu- compatibility testing, marrow donors must
ally be preferred over mobilized peripheral also be physically suitable for donation. Mar-
blood, although reports differ.29,30 In addition, row harvest is an invasive procedure per-
when mismatched marrow and mismatched formed under sterile conditions in the operat-
UCB were compared, there was no difference ing room under anesthesia. Therefore, the
in the rate of overall mortality in adults with donor must be able to tolerate the type of an-
leukemia; however, recipients of matched esthesia required to successfully perform the
marrow HSC grafts did have a lower overall harvest. Another consideration is the donor’s
mortality rate.28 medical history. Autologous donors and some
CHAPTER 29 Hematopoietic Stem Cells 䡲 719
allogeneic donors may have had previous radi- have significant decreases in their hemoglobin
ation therapy to the pelvis, which may limit concentrations after the procedure. As a result,
the amount of marrow available for harvest in almost all marrow donors donate autologous
the posterior iliac crest. Similarly, previous Red Blood Cells (RBCs) before the procedure,
chemotherapy may limit the number of nucle- and 76% of donors receive at least 1 autolo-
ated cells that can be aspirated from the mar- gous RBC unit during or shortly after marrow
row space. For autologous donors, a signifi- harvest.31 If the donor requires an allogeneic
cant tumor burden in the marrow space is a RBC or platelet transfusion before or during
contraindication for collection of HSCs by the procedure, the units should be irradiated
marrow harvest because the graft would be to prevent viable leukocytes from these blood
contaminated by tumor cells. products from contaminating the graft. Mar-
Physically, the donor must be able to tol- row donors should be made aware that they
erate the volume loss associated with marrow might need to undergo a transfusion as part of
harvest, which means that young or small do- the informed consent process.
nors may not be suitable. The Be The Match
Registry limits the volume collected from a Peripheral Blood HSC Collection
marrow donor to 20 mL/kg.5 Typically, the vol-
ume of the harvest requested is dictated by the Pharmacologic methods for mobilizing HSCs
recipient’s weight, with a minimum of 2.0 to from the marrow into the peripheral circula-
3.0 × 108 nucleated cells/kg needed to facilitate tion combined with apheresis technology have
efficient engraftment. Thus, during harvest, made peripheral HSC collection the most
checking the TNC count midway through the common procedure for HSC donation.32 Be-
procedure can help with estimating the total cause peripheral collection of HSCs requires
volume needed. Alternatively, CD34 quantita- only vascular access, most apheresis proce-
tion midway through the procedure or on the dures used to collect HSCs are performed on
final product for QC may also be performed, an outpatient basis, with minimal side effects.
depending on the institution’s policies. However, donors with poor vascular access or
The marrow harvest technique varies who may need a number of apheresis proce-
considerably depending on institutional prac- dures for the collection of a sufficient number
tice. In general, an 11- to 14-gauge needle on a of HSCs may require the placement of a cen-
syringe flushed with anticoagulant is inserted tral line, which imposes an additional risk.
into the posterior iliac crest, and approximate- AABB CT Standards requires that the place-
ly 5 mL of marrow is aspirated. The needle and ment of any central line be confirmed before
syringe are then rotated to a different vector, HSC collection is initiated.1(p48)
and the aspiration is repeated. Vigorous aspi- HSCs can be mobilized into the peripher-
ration is avoided to prevent significant periph- al circulation using a variety of chemothera-
eral blood contamination of the product. The peutic agents, hematopoietic growth factors,
aspirated marrow is collected into a large col- or receptor antagonists. For most healthy allo-
lection bag containing anticoagulant and me- geneic HSC donors, sufficient numbers of HSCs
dia and/or an infusible-grade electrolyte solu- can be mobilized with the administration of
tion. The process is repeated utilizing different hematopoietic growth factor, often G-CSF,
bone sites until the collection volume target, alone. G-CSF is administered once per day at a
based on TNC count or donor volume limit, is dose of 5 to 20 µg/kg, and doses are often
reached. rounded to the nearest vial size.33 Total white
Serious complications of marrow harvest cell count and CD34 percentage can be moni-
are rare. However, minor complications, such tored to determine the optimal collection time,
as pain at the site of harvest, fatigue, insomnia, which is usually 3 to 4 days after initiation of
nausea, dizziness, and anorexia, occur fre- G-CSF treatment. The side effects of G-CSF,
quently but resolve in most donors by 1 month which are common and mild, include bone
after the procedure.31 Marrow donors often pain, myalgia, headache, insomnia, flu-like
720 䡲 AABB TECHNICAL MANUAL
be useful when storage space is limited. Be- dure originally described by Pablo Rubinstein
cause apheresis instruments collect mononu- of the New York Placental Blood Program.38
clear cells efficiently, with very little red cell Briefly, the thawing process involves slow, se-
content, peripheral blood-derived HSCs gen- quential addition of a wash solution (eg, 10%
erally do not require red cell depletion. dextran followed by 5% albumin), transfer into
Buffy coat concentration of marrow in- an appropriately sized bag for centrifugation,
volves centrifugation and harvesting of the and resuspension of cell pellet(s) before deliv-
white cell fraction and can be performed with ery to the patient care unit for infusion. Many
an apheresis or cell-washing device. Manual laboratories perform two centrifugation steps,
centrifugation may be used when product vol- removing the supernatant from the first spin
ume is too low for apheresis or cell washing and centrifuging that portion a second time
devices. Buffy coat preparation is usually used before combining the two cell pellets. This ap-
to reduce the unit volume for cryopreservation proach optimizes cell recovery.39
or as a method of red cell reduction before fur- Marrow harvest typically involves se-
ther manipulation (eg, immunomagnetic se- quential filtration in the operating room or the
lection). laboratory to remove bone spicules, aggre-
The thawing procedure for all HSCs, re- gates, and debris. Opinions regarding the use
gardless of source, is similar. Although this of standard blood filters upon infusion of
procedure is straightforward, it should be HSCs vary, however. Whether to use a standard
done carefully because frozen plastic contain- blood filter (>170 microns) is up to the individ-
ers are prone to break for a variety of reasons.37 ual cell processing laboratory and/or trans-
The product should be handled with care plant center. If an institution opts to use a
while it is verified to determine the product’s standard blood filter, the laboratory should
identity and ensure the integrity of the bag. validate its filtration process.
The product is then placed into a clean or ster-
ile plastic bag and submerged in a 37 C water-
bath. If the freezing bag breaks, the product SPECIALIZED CELL-
may be recovered using this approach, but a PROC ESSING METHODS
risk-benefit discussion with the patient’s phy- Specialized cell-processing methods are used
sician should take place to determine the to optimize product purity and potency be-
course of the patient’s care. yond levels obtained through routine meth-
Gentle kneading allows the thaw proce- ods. Several of these methods, which require
dure to proceed relatively quickly while pre- unique reagents and instrumentation, are dis-
venting recrystallization and consequent cell cussed in other chapters. For this reason, the
damage/death. A hemostat should be used to descriptions of these methods in this chapter
prevent loss of the product if the bag breaks, are brief and focus on their application to
and the contents should be aseptically divert- HSCs.
ed into a transfer bag. A sample should also be
sent for culture. Elutriation
Washing the HSCs removes lysed red
cells, hemoglobin, and cryoprotectant [di- Counter-flow centrifugal elutriation is a spe-
methyl sulfoxide (DMSO)]. Although UCB is cialized method that separates cell popula-
typically red-cell depleted before cryopreser- tions based on two physical characteristics—
vation, it remains the primary HSC product size and density (sedimentation coefficient). A
that is routinely washed. This practice is centrifuge is used to separate the cell popula-
changing, however, and Chapter 30 discusses tions of a cell product based on density alone.
alternative approaches to UCB preparation for However, if fluid/media is passed through the
infusion. Historically, most institutions based chamber housing the cells in the direction that
their UCB processing methodology, including is opposite (counterflow) to the centrifugal
the thawing/washing procedure, on the proce- force, adjustment of flow rate and/or centrifu-
722 䡲 AABB TECHNICAL MANUAL
gation speed allows the separation of cell pop- FLT-3 ligand, and thrombopoietin along with
ulations based on size as well. Through this novel and/or proprietary ingredients. The me-
process, cells with “signature” size/density dia, culture vessels, and culture duration used
profiles can be separated from the rest of the vary from protocol to protocol.
cells. Historically, this method was used for
T-cell depletion of HSC grafts. In more recent
CRYO PRESERVATION
years, the method has been used to enrich
monocytes for preparation of dendritic-cell Methods for cryopreservation must be used
vaccines. because HSCs may need to be stored for weeks
to years prior to being transplanted.40 Most
Cell-Selection Systems cell-processing laboratories use the cryopro-
tectant DMSO, usually at 10% final concentra-
Immunomagnetic cell selection systems
tion, and a source of plasma protein for cryo-
incorporating monoclonal-antibody-based
preservation of HSCs. DMSO is a colligative
technologies to target cell-surface antigens
cryoprotectant; it diffuses rapidly into the cell,
(eg, CliniMACS system, Miltenyi Biotec Ber-
reducing the osmotic stress on the cell mem-
gisch, Gladbach, Germany) have become a
brane. DMSO prevents dehydration injury by
widely used method of cell depletion/enrich-
moderating the nonpenetrating extracellular
ment at many institutions. These methods in-
solutes that form during ice formation. It also
volve the isolation of the cell type of interest by
slows extracellular ice crystal formation. Some
either positive selection (target cells retained)
laboratories add hydroxyethyl starch (HES),
or negative selection (target cells depleted).
which allows the use of a decreased concentra-
Monoclonal antibodies (eg, anti-CD34 for HSC
tion of DMSO (eg, 5% DMSO and 6% HES).
isolation) are coupled to 50-nm ferromagnetic
HES is a nonpenetrating (extracellular),
particles. Magnetically labeled target cells are
macromolecular cryoprotectant. This high-
retained in the process as the cell suspension
molecular-weight polymer likely protects the
passes through a column in which a magnetic
cell by forming a glassy shell, or membrane,
field is generated. Unlabeled cells pass
around the cell, retarding the movement of
through the column and are collected in a neg-
water out of the cell and into the extracellular
ative-fraction bag. Target cells are then re-
ice crystals.
leased from the column by removal of the
The HSCs may be frozen at a controlled
magnetic field from the column, which allows
rate or a noncontrolled rate, in which the HSC
passage of the cells into a separate collection
product is simply transferred into a freezer bag
bag.
and placed in a –80 C mechanical freezer. Con-
trolled-rate freezing is favored in the clinical
Cell Expansion
laboratory setting, and it utilizes computer
Because the dose of nucleated, CD34+, and programming to incrementally decrease HSC
colony-forming cells has a positive correlation product temperature in a closely monitored
with patient outcomes, much effort has been fashion. The controlled rate freezing protocols
focused on ex-vivo expansion of HSCs and used vary from institution to institution.
progenitors. It is thought that successful ex- In general, the HSC product is placed in
pansion enhances hematopoietic engraftment the chamber and initially cooled at a rate of
while reducing transfusion dependence, risk 1 C/minute. When the temperature decreases
of infection, and duration of hospitalization. to approximately –14 C to –24 C, the HSC
In recent years, UCB has become the focus of product begins to transition from a liquid to a
expansion trials due both to the higher prolif- solid. At this time, the freezer undergoes a pe-
erative and self-renewal capacity of HSCs from riod of supercooling to counteract the latent
UCB and the limit on cell quantity in a UCB heat of fusion that is released by the phase
collection. Most expansion cultures contain a change. Following solidification of the HSC
cytokine cocktail that includes stem cell factor, product, cooling proceeds at the rate of 1 C/
CHAPTER 29 Hematopoietic Stem Cells 䡲 723
minute until the product has reached –60 C. At marrow, peripheral blood, or UCB.42-45 Howev-
this point, the product is cooled at a controlled er, the similar correlation between results and
rate determined by the institution until it engraftment speed and likelihood as well as
reaches –100 C. Following both controlled-rate the more rapid availability of results have
and noncontrolled-rate freezing, the HSC made CD34+ cell enumeration the accepted,
product is transferred to a storage freezer. An albeit surrogate, QC test for graft potency. The
increasing number of laboratories store HSCs clonogenic assay is still useful, despite difficul-
in the vapor phase of liquid nitrogen (LN2) at ties in standardization, especially for HSCs
temperatures below –150 C; however, some that are stored for a long time (eg, UCB bank-
laboratories do store cells in the liquid phase ing).46
of LN2.
blood products than marrow products and for DMSO to culture dishes suppressed colony-
products with higher concentrations of HSCs. forming units. However, these studies were
Cryopreserved products are shipped in performed on fresh cells, and studies of the ef-
dry shippers charged with LN2. These contain- fects of DMSO on HSCs that have been previ-
ers maintain temperatures below –150 C for up ously cryopreserved are limited. The possible
to 2 weeks and their temperatures are continu- functional defect due to DMSO coupled with
ously monitored.1(p35) If the products are the not-infrequent need to hold clinical prod-
shipped to a noncontiguous facility or on pub- ucts (because of patient-care-related issues)
lic roads, an appropriately labeled outer con- raise concern about the possibility of cell inju-
tainer must be used to further protect the ry from the infusion of thawed, unwashed HSC
product during transport and shipping.4 De- products.
pending on the mode of transport (eg, air or The patient’s vital signs should be
ground), additional federal government re- checked before infusion, immediately after in-
quirements must be met. If products are fusion, and 1 hour after infusion, at a mini-
shipped abroad, international requirements mum. All of the monitoring information
must be met. If high-dose conditioning thera- should be captured on the accompanying in-
py has been given to the recipient, shipment fusion form. When completed, this form
using a qualified courier is required.4 The should be returned to the laboratory. If an ad-
product should not be x-rayed; instead, it verse reaction occurs, more frequent monitor-
should be manually inspected, if necessary. ing is required.
Appropriate records must accompany the Reactions associated with HSC infusion
product. may be very similar to those that occasionally
The receiving institution must have pro- occur with blood transfusion (ie, allergic, he-
tocols in place for receipt and inspection of the molytic, and febrile reactions and those due to
product for acceptability for transplant.1(pp36-37),4 microbial contamination). However, some re-
actions may be less likely depending on the
cell-processing technique used (eg, red cell re-
PATIENT CARE
duction, plasma reduction, postthaw wash-
Once an HSC product is ready for infusion, it ing, or dilution). Reactions often attributed to
should be delivered to the patient care unit DMSO (eg, nausea, vomiting, cough, and
without delay. After the physician approves the headache) are less common with infusions of
product for infusion and proper identification smaller volumes and/or washed/diluted prod-
procedures are carried out, the product is in- ucts.52,53 HSC products are usually well tolerat-
fused by intravenous (IV) drip directly into a ed, however. Because the possibility of a severe
central line, typically without a needle or reaction does exist, aggressive IV hydration
pump. Some institutions use a standard blood (eg, 2 to 6 hours before and 6 hours after infu-
filter at the bedside. To maximize cell dose, the sion, with the use of diuretics as needed) and
product bag and IV tubing may be flushed use of prophylactic antiemetics, antipyretics,
with sterile saline after the bag empties. Sterile and antihistamines may be warranted.
saline also may be added directly to the bag if The transplant physician and the medical
the flow rate becomes too slow. director of the cell therapy laboratory should
HSC products are usually infused as be notified immediately of an unexpected or
quickly as the patient can tolerate, particularly moderate-to-severe reaction. An investigation
for thawed cells that have not been washed or should begin and include laboratory testing
diluted, to lessen the DMSO toxicity to the (eg, direct antiglobulin test, antibody titer,
cells. Although Rowley and Anderson51 con- Gram’s stain, or culture) that targets the signs/
cluded that DMSO is not toxic to HSCs at clini- symptoms of the patient.
cally relevant concentrations (ie, 5% or 10%) at Data on clinical outcomes (eg, engraft-
either 4 C or 37 C for up to 1 hour of incuba- ment) and adverse events should be reviewed
tion, they also noted that the addition of 1% regularly and discussed with the institutional
CHAPTER 29 Hematopoietic Stem Cells 䡲 725
quality management group. Quarterly reviews product and requires licensing or an exemp-
are reasonable for engraftment analysis. The tion from licensing from the FDA as part of an
medical director’s review should include as- investigational new drug (IND) application.
sessment of the HSC product’s quality indica- HSCs from unrelated donors facilitated
tors (eg, dose, viability, and colony-forming through the Be The Match Registry may be ad-
units), associated deviations, and presence of ministered under their IND (BB-IND 6821) or
infusion reactions, with a focus on potential an institutionally held IND. Similarly, HSCs
laboratory-related component affecting any can now be obtained from FDA-licensed UCB
less-than-optimal outcome. sources or administered under an institutional
IND.
OTH E R R E G UL ATO RY
CO NS I DE RAT IO N S CO N CLUS IO N
The relevant regulations with regard to HSC The indispensable, lifesaving role of HSCs in
collection are discussed earlier in this chapter. medicine has been established, especially for
In general, HSCs that are minimally manipu-
patients with hematologic disorders. As the
lated and collected for transplantation in an
understanding of HSC biology increases and
autologous fashion or transplanted to a first-
the ability to engineer HSC grafts expands, the
or second-degree relative are regulated solely
clinical applications of HSCs will likely contin-
under Section 361 of the Public Health Service
Act and are subject to the jurisdiction of the ue to grow. Along with the fast-paced growth
Center for Biologics Evaluation and Research in the use of HSCs, emerging novel technolo-
of the FDA. If HSCs are manufactured in a way gies and approaches to manipulate HSCs are
that alters their relevant biological characteris- adding to the challenge of ensuring that HSCs
tics (eg, if they are genetically modified, ex- continue to serve as a safe and effective cellu-
panded ex vivo, or combined with a drug) or lar therapy product for patient use. Addressing
the cells are intended for transplantation into this challenge will require regulatory agencies
a non-first- or second-degree relative, then the and accrediting bodies to continue to update
HSC product is subject to regulation under Ti- and modify their applicable rules, regulations,
tle 21, CFR Part 1271, as a drug and/or biologic and standards.
KEY PO I NT S
1. Hematopoietic stem cells (HSCs) derived from the patient being treated (autologous) or a
donor (allogeneic) can be used to treat a variety of malignant and nonmalignant conditions.
2. Autologous HSCs in general are used to rescue the marrow function of a patient undergoing
high-dose chemotherapy and/or radiotherapy.
3. In addition to rescuing the marrow function of a patient undergoing high-dose chemother-
apy and/or radiotherapy, allogeneic HSCs also have graft-vs-neoplasm effect and/or the
ability to replace defective cellular machinery.
4. Regardless of the HSC donor source, AABB CT Standards requires laboratory testing for hu-
man immunodeficiency virus, Types 1/2; hepatitis B virus; hepatitis C virus; syphilis; hu-
man T-cell lymphotrophic virus, Types I and II; and cytomegalovirus.
5. Screening and testing for infectious diseases in allogeneic HSC donors is mandated by the
Food and Drug Administration (FDA), and when a risk for a communicable disease is dis-
covered, the donor is ineligible (but may still donate if there is urgent medical need).
6. Allogeneic HSC donors are chosen with regard to their histocompatibility with the recipient.
ABO-Rh compatibility between the donor and recipient is not necessary.
726 䡲 AABB TECHNICAL MANUAL
7. HSCs can be obtained by aspiration of marrow, collection of umbilical cord blood, or by pe-
ripheral blood mobilization followed by collection with an apheresis machine.
8. HSCs usually require minimal manipulation, and can be stored following cryopreservation
with the cryoprotectant, dimethyl sulfoxide.
9. Specialized HSC manipulation techniques can be used to reduce the HSC product volume,
lysed cells, red cells, and cryoprotectant depending on the recipient’s clinical needs.
10. Quality control (QC) is essential for providing a safe and efficacious HSC product. Common
QC testing includes cells counts (CD34+ cell enumeration, total nuclear cell count), micro-
bial contamination testing, and viability testing.
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C h a p t e r 3 0
Aleksandar M. Babic, MD, PhD, Medical Director, and Donna M. Regan, MT(ASCP)SBB, Executive Director, St.
Louis Cord Blood Bank and Cellular Therapy Laboratory, SSM Cardinal Glennon Children’s Hospital, St. Louis,
Missouri
The authors have disclosed no conflicts of interest.
729
730 䡲 AABB TECHNICAL MANUAL
CBBs in Dusseldorf and Milan opened shortly Engaging pregnant women and making
thereafter. Recent reports indicate that more arrangements before they give birth to donate
than 600,000 unrelated UCB units are banked their infant’s UCB is critical for achieving the
worldwide for potential clinical use, and near- desired results. Early education of pregnant
ly 30,000 unrelated UCB transplants have been women allows for more complete medical
performed to date.19 In 2011 alone, the World screening, comprehensive donor protection,
Marrow Donor Association reported that 4093 availability of adequate supplies to collect
UCB products were shipped to hospitals for UCB, and acquisition of truly informed con-
transplantation to unrelated patients in 47 sent from the woman. Recruitment by a public
countries; in comparison, 3,743 marrow grafts CBB typically begins with education of the
were performed in that year.19 In addition, a physician/midwife by the CBB and the distri-
number of private CBBs have been established bution of informational materials to obstet-
worldwide for families to bank the UCB of rics offices or prenatal class staff. This ap-
their infants for future use by family members. proach enlists support for UCB donation while
Two of the largest private CBBs have recently providing information to obstetric staff. This
reported that they have stored UCB stem cells information empowers staff to introduce the
from 700,000 newborns, and each of these idea of UCB donation to pregnant women and
CBBs has distributed approximately 250 UCB respond to their initial inquiries while refer-
products for traditional transplantation and ring more detailed questions to CBB person-
regenerative applications. nel.
The sections of this chapter describe the The informational materials that CBBs
current generally accepted practices for UCB distribute to obstetrics office and prenatal
banking (primarily from a public CBB’s per- class staff about UCB donation address basic
spective), including donor-related issues, col- information, such as the name of the CBB,
lection and processing methods, and storage whether the CBB is public or private, the cost
and shipment as well as recommended trans- (or lack thereof) of donation, names of partici-
plant center-related activities for product pating hospitals, medical uses of UCB, and
preparation and infusion. The chapter contin- how UCB is collected. The materials also dis-
ues with a brief discussion of UCB banking cuss the risks of UCB donation to the mother
economics and how inventories can be creat- and infant and whether the donated unit will
ed to meet the needs of a diverse patient popu- be available for the donating family’s use.
lation and address cell dose limitations while Within an informational packet, a CBB may in-
continuing to serve as cost-effective means of clude the health history questionnaire, which
providing access to therapy in an “affordable
is used to solicit information about the preg-
health care” environment. The chapter finish-
nancy, risk factors of the parents, and medical
es with an overview of standards and regula-
history of first-degree family members.
tions.20
The CBB explains to potential donors the
need for their written permission (informed
DONOR-REL ATED ISSUES consent) to collect and store the UCB for later
use in transplantation or research. Participat-
Recruitment ing mothers must understand that their blood
Most women agree to donate the UCB of their will be drawn and tested for certain infections,
infants when they become aware that the UCB such as hepatitis and human immunodefi-
that is normally discarded as medical waste ciency virus, to reduce the risk of transmission
can be recovered and used to save the life of of disease through the transplantation of their
another individual. However, a woman’s ability infant’s UCB. The CBB emphasizes that all in-
to donate UCB may be limited by the availabil- formation and test results obtained during the
ity of a CBB servicing the area in which she process are held strictly confidential to pro-
gives birth. tect the identity and privacy of donors and
CHAPTER 30 Umbilical Cord Blood Banking 䡲 731
that the family will not be approached to do- UCB. A CBB may use a single consent form
nate more cells after the infant is delivered. covering all activities. Presented in the prena-
The role of the pregnant woman’s physi- tal period, a single consent process affords the
cian cannot be understated. During pregnan- pregnant woman adequate time to seek infor-
cy, a woman trusts her physician to provide re- mation and consider her options under low-
liable care and advice. Therefore, a woman’s stress circumstances. Other CBBs may use a
decision to donate or store her UCB can be as phased consent process that permits collec-
simple as acceptance of a recommendation of- tion and, if the resulting harvest meets criteria
fered by her physician. In addition, a woman’s for banking, allows the bank to approach the
decision to donate may be influenced by re- mother later for permission to screen, process,
cruitment materials designed to appeal to all test, and store the product. This latter process
ethnic groups. facilitates collection from mothers who have
In spite of broad awareness campaigns not previously been introduced to UCB
and recruitment efforts, some women may not banking.
be aware of UCB donation or may not have Criteria have been suggested that protect
registered to participate when they arrive at a the woman’s ability to make an informed deci-
hospital to give birth. For this reason, informa- sion during childbirth. The Advisory Council
tional material on UCB donation should also on Blood Stem Cell Transplantation (ACBSCT)
be available in the labor and delivery area. The has recommended that each CBB develop a
extent to which women not previously in- policy on informed consent that considers the
formed about UCB donation can be recruited stage of labor and stress of the woman, the
at this stage (ie, delivery) varies. However, in amount of counseling on UCB donation that
most cases, pregnant women have been previ- she has received, and the amount of time
ously informed of the option of UCB donation available for an adequate discussion of UCB
but simply have not registered or completed donation.28,29 Final judgment regarding the
the necessary documentation. woman’s ability to give intralabor consent
Pregnant women may also be solicited for should rest with the obstetric staff.
UCB donation by private CBBs. For a fee, these In October 2011, the FDA classified UCB
banks will arrange for UCB to be collected and banking as a manufacturing rather than an in-
held in long-term storage for use only by that vestigational activity when a CBB distributes
family. These CBBs provide written or video products for specified indications. If not li-
information to pregnant women that is specif- censed, manufactured UCB products may still
ic to their program. be distributed for nonlicensed (clinical re-
search) applications as an investigational new
Consent drug (IND).
Consent must be obtained from the pregnant
Health History and Medical Evaluation
woman for UCB collection, processing, test-
ing, storage, and medical use.21-26 Although the It is incumbent on the CBB to provide a safe
UCB actually belongs to the newborn infant, product by minimizing the transmission of ge-
consent is obtained from the mother because netic or infectious diseases. A donor-eligibility
her infant cannot provide it and testing of her determination, based on donor screening and
blood for transmissible diseases is required. testing for relevant communicable disease
Consent from the father is not necessary and agents and diseases, is required for all donors
does not increase the safety of the UCB for of cells or tissue, including UCB cells. A robust
transplantation.27 medical health history designed to solicit
Although consent to collect UCB must be information on exposures to infectious diseas-
obtained before delivery of the infant, CBBs es and history of symptoms indicating genetic
use different approaches to obtain consent for disorders is obtained by interviewing the
further manufacturing, testing, and use of mother and reviewing her medical record. The
732 䡲 AABB TECHNICAL MANUAL
the CBB medical director and approved by its additional means to assess completion of col-
quality control unit.22,24(pp1-2,71-72) lection.
Once the collection is complete, the tub-
ing is “stripped,” forcing the blood inside the
U C B CO L L E C T I O N
tubing into the collection bag and allowing it
The methods to collect UCB consist of cleans- to mix with the anticoagulant. This can be ac-
ing of the cannulation site and aseptic collec- complished most easily with a specialized
tion. Typically, a sterile collection bag contain- blood-banking tool (ie, a tube stripper), al-
ing citrate-phosphate-dextrose (CPD) solution though newer bags include a filtered vent to
anticoagulant is used, although acid-citrate- eliminate the need for additional equipment.
dextrose and lyophilized heparin are also ac- The UCB unit can then be packed, stored, and
ceptable anticoagulants for UCB collection. transported to the cell-processing laboratory
After delivery, the umbilical cord is in a temperature-controlled environment.
clamped, cut, and separated from the infant.
The clamping must be timed so as not to inter- In-Utero Collection
fere with routine delivery practice. UCB can be In-utero collection is generally performed by
collected before (in utero) or after (ex utero) the obstetrician or nurse/midwife after the
the placenta has been delivered. There are ad- newborn has been delivered and assessed and
vantages and disadvantages to each method, the umbilical cord has been clamped and cut.
but neither appears to be better overall.31 A If the well-being of the newborn and/or moth-
brief discussion of the two methods is provid- er is in question, the collection is not attempt-
ed below. ed. If a decision to collect the UCB is made,
Regardless of whether the UCB is collect- care must be taken to maintain sanitary condi-
ed in vivo vs ex vivo, the process of UCB collec- tions. Unless a sterile bag or extension set is
tions is essentially identical. As in whole-blood used, the traditional supplies are not sterile
collection, the venipuncture needle is at- and inadvertent misplacement could contam-
tached to the collection bag. After an appropri- inate the surgical field.
ate umbilical vein is identified, the site is For logistical reasons, it is advisable to
cleansed. Typically, this involves wiping the have preassembled collection kits available for
cord with isopropanol and then scrubbing it in-utero collection. An instructional packet
with a broad-spectrum topical microbicide may be included in these kits. This packet
(eg, povidone iodine) for at least 30 seconds. might contain, for example, a donor informa-
Alternatively, a few CBBs have chosen to use a tion form; description of the collection proce-
broad-spectrum antimicrobial formulation of dure; list of collection kit contents; maternal
2% chlorhexidine gluconate/70% isopropyl al- medical history form; consent form(s); and
cohol in place of traditional iodophors. Subse- packaging, storage, and shipping instructions.
quently, a hemostat is placed on the tubing a Collection kits also include items such as one
few inches from the needle. The hemostat is or two collection bags, antimicrobial supplies,
opened once the vein has been accessed, al- tubing closures, appropriate sample tubes for
lowing blood to flow into the bag. Removal of infectious disease testing, sample tube labels,
the hemostat before puncturing the vein may product labels, biohazard and other appropri-
allow air to contaminate the tubing. While the ate stickers (eg, for indicating temporary stor-
UCB is being collected (a 3- to 5-minute pro- age temperatures), and a secondary specimen
cess), the UCB should be gently mixed with the and product bag. In some cases, the hospital’s
anticoagulant to prevent clotting. The umbili- maternal label may be used to identify mater-
cal cord appears collapsed when the collection nal tubes and UCB products because these la-
is complete. Placement of the bag on a labora- bels include two forms of identification and
tory scale during collection allows the collec- precautions are taken to protect donor confi-
tor to monitor the volume and provides an dentiality on these labels.
734 䡲 AABB TECHNICAL MANUAL
Validated containers and/or processes are tion, such as application of pressure to the
used to ship the UCB. Acceptable tempera- cord (“milking”), can increase the collection
tures during shipping can range from cold to volume. Caution must be used to prevent ma-
ambient, depending on the shipping distance ternal contamination and lysis of cells.
and conditions, and are defined based on sta- Because blood clotting readily occurs
bility studies. Temperature during transport during UCB collection, which adversely affects
may be monitored depending on the risk to the volume and number of cells collected, it is
the UCB involved in the shipping process. important to minimize delays during the col-
The primary advantage of in-utero com- lection process. Wong and colleagues hypoth-
pared to ex-utero collection is the substantial- esized that a higher incidence of macroscopic
ly lower cost because dedicated CBB collection clots in ex-utero collections resulted in the col-
personnel are not required. As a result, the lection of lower nucleated cell and CFU
only costs are for collection and shipping sup- counts.34
plies. In addition, several studies have suggest- Because it occurs outside the delivery
ed that in-utero collection methods may result room with all of its activity, ex-utero collection
in the collection of greater volumes of cells may inherently provide better process control
and higher counts of nucleated and CD34+ (ie, a lower risk of microbial contamination
cells and colony-forming units (CFUs).32,33 and labeling errors), but it raises the costs as-
In addition, the initial costs and efforts sociated with collection. In addition, a lack of
associated with education and training of the availability of collectors on all shifts may limit
physician/midwife collectors need to be con- the opportunity for a facility to participate in
sidered. However, once obstetrics practice UCB collection. Some investigators have ques-
group members are trained, their hands-on tioned these points.35 Because data demon-
experience should improve the quality of their strate that in-utero and ex-utero collection
collections. In-service and educational ses- methods are equivalent, the choice of UCB
sions can provide refresher courses and dem- collection method should be based on the
onstrate appreciation of clinicians’ support for needs and capabilities of each UCB collection
UCB collection. Subsequently, a program of program.31
continued competency assessments, collec-
tion-site visits, and regular communication
U C B P RO C ES S IN G
can be utilized to maintain high-quality UCB
collections. Methods
In the early 1990s, UCB units were often stored
Ex-Utero Collection
in an unmanipulated state without volume re-
Ex-utero collections are often performed by duction (ie, red cells and/or excess plasma
dedicated UCB collection staff. The advantag- were not removed before storage) in an effort
es of this approach over in-utero collection are to conserve the number of stem cells in the
its use of standardized collection methods, product.18,36 Concern about infusion-related
limited personnel involvement, and greater complications associated with red cell incom-
control. patibility, free hemoglobin, dimethyl sulfoxide
After delivery, the cord is clamped at a (DMSO), and logistical issues surrounding
time that does not interfere with the physi- limited storage capacity motivated an evalua-
cian’s routine practice. The placenta is imme- tion of processing methods to minimize the
diately taken by CBB staff to a suitable loca- red cell content and size of the final UCB unit
tion, where it is suspended in a device to allow while limiting stem cell loss.
collection of blood by gravity. The collection At present, a variety of processing tech-
proceeds as described above. The placenta niques for volume reduction and removal of
and cord are more accessible in the ex-utero red cells are employed, and the majority of
setting and, therefore, the use of manipula- these methods involve sedimentation, centrif-
CHAPTER 30 Umbilical Cord Blood Banking 䡲 735
ugation, and/or filtration. The most common cells from UCB. The technology is adapt-
means of reducing red cell content is the use of able to a large-scale processing environ-
sedimenting agents, such as hydroxyethyl ment. The SEPAX machine, which is essen-
starch (HES), gelatin, polygeline, and dex- tially composed of a centrifuge with piston
tran.37,39 One of the earliest methods for pro- position sensors surrounding the chamber
cessing UCB utilizing HES was developed by and pneumatic pump system, is operated
Pablo Rubinstein.39 Modifications of his meth- using computer-controlled protocols to
od have been commonly used by other pro- achieve component separation. Consum-
grams.40 An alternative preparation method able kits consist of a large syringe-type bar-
involves density separation by layering UCB rel separation chamber with bags and tub-
cells over Percoll or Ficoll-Hypaque. This ing connected via a stopcock assembly.
method ultimately results in a mononuclear Biosafe’s SEPAX system received FDA clear-
cell-enriched product essentially devoid of red ance in January 2007 and a European CE
cells.41 Also, utilization of commercially avail- mark of approval in 2001.
able leukocyte reduction filters and semiauto- 䡲 The AXP (AutoXpress Cord Blood Process-
mated methods results in similar in-vitro cell ing System), manufactured by ThermoGen-
recovery to the standard HES method.37,42 esis (Rancho Cordova, CA), is an automat-
Initially, UCB was processed with other ed, functionally closed system that harvests
HSC products in laboratories that were part of stem-cell-rich buffy coat from UCB. The
a research facility, hospital transfusion ser- system reduces a unit of UCB to a precise
vice, or blood center. However, the methodol- volume chosen by the operator and selec-
ogy for processing UCB products has evolved tively isolates mononuclear cells. The sys-
to involve dedicated processing laboratories tem includes the AXP device, a docking sta-
designed to comply with current good manu- tion, a processing set, and XpressTRAK
facturing practice (cGMP) by using more stan- software. The AXP system received 510(k)
dardized processing techniques for volume clearance from the FDA in October 2007.
reduction, removal of red cells, and cryo- 䡲 PrepaCyte-CB, a UCB-processing system
preservation. manufactured by CytoMedical Design
An important driving force behind most Group (St. Paul, MN), received FDA 510(k)
significant recent changes in UCB processing clearance in January 2009. PrepaCyte-CB is
was a set of recommendations made by FDA as a functionally closed sterile bag system
part of its process for the submission of bio- composed of three integrally attached pro-
logics license applications (BLAs). These rec- cessing and storage bags containing the
ommendations provide guidance on how to PrepaCyte-CB separation solution. While
provide assurances of the safety, purity, poten- isolating nucleated cells, PrepaCyte-CB re-
cy, and effectiveness of UCB products. These moves most red cells and nucleated red
recommendations have compelled the indus- cells from the final processed UCB unit.
try to standardize its manufacturing processes The system requires plasma extractors and
by using reagents that have been approved for a standard laboratory centrifuge to sedi-
human use and systems and supplies that ment and concentrate desired cells.
have been cleared by the FDA. To date, the fol-
lowing systems have been approved by the Each of these methods has advantages
FDA for the processing of UCB: and disadvantages with respect to cell recov-
ery, red cell removal, processing time, and
䡲 Automation technology incorporated into cost. Each laboratory must therefore validate
the SEPAX Cord Blood Processing System and determine which processing method is
manufactured by Biosafe SA (Lake Geneva, most appropriate for its situation. Cellular
Switzerland). This technology offers a function, product integrity, and safety must be
closed and sterile processing system that considered when developing a validation plan.
harvests nucleated cells and enriches stem Nucleated and CD34+ cell recovery, viability,
736 䡲 AABB TECHNICAL MANUAL
potency (eg, ability to form CFUs), and sterility Cryopreservation and Long-Term
testing (before and after cryopreservation) are Storage
typical validation performance measures. Fac-
Successful outcomes of UCB transplantation
tors to consider when choosing a processing
hinge on the CBB’s ability to preserve the in-
method include workload, processing time,
tegrity of UCB over substantial lengths of time.
cost, maximization of storage time, and pro-
Freezing and storage methods must be robust
duction efficiency (number of UCB units pro- enough to ensure that the quality of the UCB
cessed per staff member per day). unit is maintained for many years.
The most common method of UCB cryo-
Quality Control Testing and preservation consists of freezing the cells in a
Characterization cryogenic bag in the presence of 10% DMSO as
Quality control (QC) testing to assess the ade- a cryoprotectant.43-45 One of the main advan-
quacy of the product and process is essential. tages of using cryogenic bags over freezing vi-
QC testing is usually performed on receipt als is the ability to perform post-thaw confir-
matory HLA typing and QC testing using an
(prior to manipulation) of a UCB unit and be-
integrally attached tubing segment. Both
fore cryopreservation. Typical QC assays in-
AABB and the Foundation for the Accredita-
clude counts of nucleated cells (white cells
tion of Cellular Therapy (FACT) mandate the
plus nucleated red cells), mononuclear cells
use of cryogenic bags with attached segments
(monocytes plus lymphocytes), and CD34+
for the freezing of UCB.22,24(p53)
cells; hematocrit; CFU assay; and viability and It has been well established that slow
sterility (aerobic, anaerobic, and fungal) test- cooling in a programmable controlled-rate
ing. All testing methods must be validated for freezing device at a rate of 1 C/minute results
their intended use. in adequate recovery of CD34+ cells/human
UCB products used for allogeneic trans- progenitor cells.44,45 Such instruments com-
plant must be tested for HLA Class I and Class pensate for the heat of fusion phase, mitigate
II antigens, including HLA-A, -B, and -DRB1 process variability, and limit cell damage. As
loci; HLA-C and HLA-DQB typing is recom- with all critical processes involved in collect-
mended.22 In this setting, ABO/Rh typing is ing and storing UCB, the freezing process must
performed primarily to assist clinicians with be validated and procedures must be in place
red cell and plasma product support after in- that govern the use and operation of the con-
fusion. Table 30-1 lists these basic tests. trolled-rate freezer (or equivalent), expected
Test Method(s)
cooling rates and freezing curve parameters, and storage conditions. The CBB’s UCB pro-
endpoint freezing temperature, addition of gram must address individual products before
cryoprotectant, final cell and cryoprotectant distribution (lot release testing) and demon-
concentrations, and storage temperature. Be- strate the stability of the drug’s active ingredi-
cause equipment failures can occasionally oc- ents, used in determining expiration dates.
cur, simplified passive freeze methods can also
be validated to provide satisfactory cryo- Post-Thaw Stability and Other Testing
preservation of human stem cell products.46,47
Both AABB and FACT require cryopre- Accreditation organizations require UCB
served UCB to be stored at <–150 C.22,24(p54) products to have integrally attached segments
Once frozen, UCB units are typically trans- that are representative of the UCB product and
ferred into a monitored liquid-nitrogen (LN2) used to verify the results of HLA typing.24(p78)
storage container and either overwrapped and One of these segments can be used for confir-
immersed in liquid (at –196 C) or in vapor matory testing—a process in which product
phase to minimize the potential for cross- identity is confirmed and potency is evaluated
contamination during long-term storage. before the UCB unit is released to a transplant
To ensure the safety and stability of the facility. Extended HLA typing, viability, poten-
stored UCB units, control measures should be cy, or stability testing can be performed using
established for product security and segrega- other replicate aliquots of the UCB unit (reten-
tion, storage container monitoring, inventory tion samples). Progenitor assays, CD34+ cell
control, and duration of storage. Storage con- enumeration, viability testing, or others tests
tainers must be located in a secure area to pre- performed on segment material before distri-
vent unauthorized access. For units in which bution may be useful in determining the prod-
transmissible disease screening and testing re- uct’s potency. Traditionally performed as an
sults are positive or the testing is incomplete, internal QC process, the results of these tests
there must be a designated quarantine storage are not released due to their lack of demon-
area or process to prevent accidental release. strated correlation with engraftment. Howev-
To ensure that temperature and LN2 levels are er, transplant centers, knowing that these re-
continuously maintained, a monitoring sys- sults are available, are requesting them from
tem with local and remote alarm capabilities CBBs when determining which UCB units to
must be in place. Alarm limits should be set to select. Accordingly, studies are under way to
allow adequate time for staff to respond to no- assist with interpretation of the variable re-
tifications and react before inventory is com- sults obtained when testing is performed on
promised. All UCB products and reference the limited material in these samples.
sample storage locations should be cataloged
using an inventory-management system that S HI P M E N T
allows for rapid retrieval. The duration of stor-
age and product expiration dates should be With a number of commercial carriers avail-
defined in operating procedures, even when able, UCB can be routinely transported to pro-
expiry dates have yet to be determined. cessing laboratories or transplant facilities
Studies of the long-term effects of ultra- within hours to a few days. It is important to
low-temperature storage of UCB units have ensure that UCB units and products are prop-
shown that retention of viability and/or prolif- erly packaged and shipped to prevent damage
erative function of UCB cells frozen in the liq- or deterioration during shipment. This pro-
uid phase of LN2 remains acceptable for at cess is the responsibility of the CBB. Validated
least 23 years.48,49 However, each CBB must es- packaging and shipping procedures should
tablish and maintain a written testing program demonstrate that acceptable temperatures
designed to assess the integrity, potency, safe- and unit integrity are maintained.24(p35) Each
ty, and stability of drug products manufac- CBB needs to define the shipping conditions
tured under its facility-specific manufacturing (temperature, type of shipping container, and
738 䡲 AABB TECHNICAL MANUAL
packaging material) for the transport of its creating an ultracold environment. FACT re-
fresh and frozen UCB products. quires that LN2 dry shippers be validated to
Shipping methods must also be designed maintain temperature of <–150 C for at least 48
to protect the safety of the personnel in- hours beyond the time of delivery to the trans-
volved.22,50 The FDA, International Air Trans- plant facility.22
port Association (IATA), US Department of For a dry shipper to be fully effective, it
Transportation (DOT), AABB, and FACT have must be charged or filled with LN2 24 hours be-
established packaging and labeling require- fore the estimated time of release from the
ments for the shipping of biologics.22,24,51-53 processing laboratory. This allows for com-
IATA requires that shipping containers be able plete absorption of the LN2 into the wall of the
to withstand extreme external temperature shipping container. Improperly prepared dry
variability and that primary outer containers shippers present a risk of LN2 leakage, and
be leakproof, constructed to resist breakage, CBBs may be subject to civil/criminal penal-
and durable enough to withstand pressure ties from US DOT in the event of a spill.
changes and falls. In addition, CBBs must Dry shippers are vulnerable to incident
package UCB in a secondary inner container, handling and can be compromised if they are
such as a plastic resealable bag, with enough mistreated or positioned incorrectly. This will
absorbent material to contain the contents of result in warm conditions and thawing of the
the product in the event of a leak or break.22,52 product, rendering it unusable for the clinical
procedure. To minimize the risk of product
Shipping Fresh UCB loss, shipping instructions must be clear and
the conditions of transport, particularly tem-
The requirements for the transport of freshly
collected UCB are not well defined, and each perature, must be continuously monitored.
facility must establish transport temperature
criteria and acceptable limits that are com- Transport Labeling and Record
mensurate with the risks involved. Available Requirements
options include transport at room tempera- AABB and FACT require continuous monitor-
ture or use of insulated, precooled stabilizing ing of the temperature during shipment,
packs. Wada and colleagues observed a 1% which is accomplished through the use of a
drop in viability for every 4-hour increase in data logger.22,24(p35) According to AABB and
transport time for recently collected UCB units FACT, the shipping container must include the
that were shipped at ambient temperature.54 name, address, and telephone number of the
However, a series of studies has shown that shipping and receiving institutions; the phras-
UCB can be preserved in CPD for up to es “medical specimen,” “do not irradiate” (if
48 hours before processing and retains satis- applicable), and “do not x-ray”; and biohazard
factory cell recovery rates and progenitor con- labels (as appropriate).22,24(p68) Biological prod-
tent.55-58 ucts must be packaged and shipped in compli-
ance with all applicable governmental regula-
Shipping Cryopreserved Units tions. Transportation records that identify the
An advantage of selecting a frozen UCB prod- shipping facility, date and time the unit was
uct over a fresh marrow or apheresis product is shipped and received, courier, and contents of
that the frozen UCB product can be shipped each shipping container should be main-
and secured at the transplant facility before tained.22
the recipient is conditioned for transplant.
Cryopreserved products are shipped to trans- R E CE I P T OF U CB F OR
plant facilities in portable LN2 “dry” shipping
T R A N SP L AN TAT I O N
containers to maintain the products in a fro-
zen state. Insulated containers are used that UCB transplantation is a coordinated effort
allow LN2 to be absorbed into the vessel wall, potentially involving several entities—the reg-
CHAPTER 30 Umbilical Cord Blood Banking 䡲 739
istry, such as the National Marrow Donor Pro- the CBB’s shipper validation should be re-
gram (NMDP), CBB, and cell-processing labo- quested.
ratory/clinical team at the transplant center. The identity of the UCB unit should be
The process is initiated by the placement of a verified at the time of inspection, before the
reservation of or order for a UCB unit, usually unit is placed in storage. The product label
by the clinical program’s transplant coordina- should indicate the appropriate minimum
tor, based on the institutional algorithm or partial label requirements—unique product
guidelines. The coordinator may rely on the identifier (ie, unit number) and proper prod-
laboratory or medical director to answer ques- uct name. All other product information
tions related to a prospective UCB unit, in- should be attached to the product on a tie tag
cluding those associated with technical issues and/or in the accompanying paper work.
and donor medical history. It is advisable to Whether affixed or attached, the CBB paper
initiate involvement of the cell-processing lab- work that accompanies the UCB and the docu-
oratory early in the unit-selection process.59 ments generated by the transplant program/
Once the unit’s quality is confirmed and it coordinator should be compared. Any incon-
is approved for shipment, the coordinator sistencies should be immediately and appro-
should notify the cell-processing laboratory of priately investigated.
the impending arrival of a UCB unit, including The unit information, including donor
any special handling requirements (eg, size medical history and infectious disease testing
and dimensions of product canister or indica- results, should again be reviewed for com-
pleteness. If there are any comments not pre-
tions of risk factors requiring quarantine).
viously communicated to the receiving labora-
When the unit is received at the laboratory, it
tory, particularly any related to the donor’s
should be carefully unpacked and examined to
medical history, they should be referred to the
verify its labeling and integrity before it is
receiving laboratory’s medical director. The
placed into the LN2 storage tank. The dry ship-
medical director should determine whether
per may be weighed to check for excessive LN2
the information is significant and requires any
loss (in general, greater than 10 pounds).
further action and/or notification of the trans-
Because both AABB and FACT standards
plant physician. If any infectious disease test-
require continuous temperature monitoring of
ing is found to be incomplete or the results are
cryopreserved products during shipment, positive, the unit should be placed into quar-
temperature-monitoring devices must be in- antine storage, and the appropriate personnel
cluded in the shipment. Upon the unit’s arriv- (eg, laboratory supervisor, medical/laboratory
al, the technologist should confirm that the director, coordinator, and/or transplant physi-
UCB remained within the acceptable tempera- cian) should be notified. The product label/tie
ture range during shipment. If the tempera- tag must be updated accordingly (eg, to in-
ture-monitoring device displays a digital tem- clude a biohazard label) and a special medical
perature, the temperature when the unit is release form may need to be completed as
unpacked should be recorded. If the device well.
does not show a temperature or the data can- Any further testing deemed necessary by
not be downloaded, the CBB should forward a the transplant center (eg, additional HLA or
copy of the data when they become available. viability testing or CFU count) should be
If the device for continuous temperature performed as soon as possible on an integrally
monitoring cannot be located, a temperature- attached segment, if one accompanies the
measuring device from the transplant center unit. If a unit’s identity is in question and an
should be inserted for several hours to ensure integrally attached segment is not available, a
that the shipper can maintain an acceptable rapid (Class I serologic) HLA test may be per-
temperature. The CBB should be notified that formed along with the standard product test-
no temperature-monitoring device was pres- ing on the thawed product (ie, on the day of
ent in the shipment, and documentation of the transplant procedure).60
740 䡲 AABB TECHNICAL MANUAL
Instructions for handling and preparing should not be used with red cell-replete prod-
frozen UCB products are provided by the UCB ucts according to recent FACT requirements.
manufacturer in the shipment. However, the The traditional washing method original-
receiving laboratory may use alternative thaw- ly described in 1995 is recommended if the
ing methods based on its own validated proce- product’s exposure to DMSO, free hemoglobin,
dures, clinical protocol, or physician prefer- and volume approach critical limits for recipi-
ence. ents, particularly pediatric patients or those
with underlying cardiac, pulmonary, or sensi-
tizing conditions.39 More recently, a dilution or
TH AW ING AND WAS H ING OF
simple reconstitution approach has gained
UCB support, primarily due to concerns about cell
Coordination of the transplant event requires loss during the wash step.63,64 Both methods
consistent communication among all mem- are initiated in similar fashion:
bers of the clinical team. In the days before the
planned transplantation, the coordinator 䡲 The UCB product is carefully removed from
should verify the infusion date with the clini- the storage tank and a thorough inspection
cal team, and both the coordinator and labora- is performed to evaluate the container’s in-
tory should again review all relevant records. tegrity. Verification of the product’s identity
Subsequently, the patient care unit should be on its label is conducted by two technolo-
contacted at least 1 day before the day of gists.
transplant to schedule an infusion time. Given 䡲 The unit is sealed in a clean or sterile trans-
the wide variety of types of products received parent bag (ie, a plastic zipper bag) and
from an increasing number of CBBs, the most submerged in a 37 C waterbath of clean or
appropriate thawing procedure must be deter- sterile water or saline. Gentle kneading of
mined based on the transplant center’s vali- the UCB bag during thawing helps acceler-
dated method or CBB’s recommendations. ate the thawing process while preventing
There may be considerations if the product recrystallization and consequential cell
was manufactured to reduce red cell and plas- damage or death. If a leak is discovered af-
ma content (RBC reduced) or to reduce plas- ter initiation of the thaw procedure, the site
ma content only (red cell replete) before cryo- of the container break is determined and a
preservation. The choice of the thawing hemostat is employed or the product is po-
procedure may be further affected by the re- sitioned to prevent further escape of the
quirements of the clinical protocol in which cellular material. The contents can then be
the patient is enrolled. aseptically transferred into a transfer bag
Currently, three different practices for under a biological safety cabinet.40
preparing UCB products for infusion are avail- 䡲 The thaw solution is used for product dilu-
able: the traditional thaw-and-wash method, tion and should be prepared in advance. It
the thaw-and-dilution technique, and the bed- typically contains 10% dextran and a pro-
side thaw method.61,62 tein source, usually human serum albumin
Despite the potential advantage of the in a final concentration of approximately
bedside product preparation method in mini- 2.5% to 4.2%. The supplementation of the
mizing potential cell loss from postthaw ma- thaw solution with protein (albumin) has
nipulation, this approach is not recommended been proven to restore osmolarity and ex-
because of the difficulty of rescuing the prod- tend cell viability.39 Subsequently, a volume
uct at the bedside if the bag’s integrity is com- of solution at least equal to the volume of
promised and the adverse effect of prolonged the UCB product is gradually added to the
exposure of thawed cells to DMSO if the infu- bag while it is gently mixed. The product
sion is delayed. Furthermore, this method and solutions are drained into a labeled
lacks the capacity for process control and transfer bag and left to equilibrate for 5
product assessment. Finally, this method minutes. At this stage, if the product is to be
CHAPTER 30 Umbilical Cord Blood Banking 䡲 741
reconstituted or diluted only, samples are similar to those for infusions of other HSC
removed for product testing. products.65
䡲 If the product is to be washed, the labeled Once the UCB product has been thawed
transfer bag is centrifuged at 400-600 × g for and washed, it should be delivered to the pa-
15 minutes at 10 C. The supernatant is ex- tient care unit without delay. Subsequently, a
pressed, leaving behind a pellet of washed form acknowledging the receipt of the UCB
UCB cells. If it is the policy of the transplant should be signed by a nurse, who then notifies
laboratory to centrifuge the supernatant, the patient’s physician of the UCB unit’s arriv-
the second labeled transfer bag is spun at al. After the physician approves the infusion
400 × g for 15 minutes at 10 C, the superna- and proper identification procedures are fol-
tant is again expressed, and the two cell lowed, the unit is infused by intravenous (IV)
pellets are combined into one labeled bag. drip or syringe push directly into a central line
䡲 The UCB cells are resuspended in a volume without a needle or a pump. Some institutions
of thaw solution that is appropriate for the use a standard blood filter at the bedside. If a
weight of the patient while additional con- filter is used in the laboratory after the thaw/
sideration is given to the potential for fluid wash, a second standard blood filter at the
overload. bedside is not needed.
䡲 Some laboratories filter the resuspended The thawed UCB must be transfused as
product with a standard blood filter (at 170- soon as clinically possible. Timely infusion is
260 microns). most likely to be challenging with UCB that
䡲 Samples are removed for QC tests, such as has not been washed or diluted. Although
Rowley and Anderson66 concluded that DMSO
total nucleated cell (TNC), CD34+, and
is not toxic to HSCs at clinically relevant con-
CD3+ cell counts; microbial culture (bacte-
centrations (ie, 5% or 10%) at either 4 C or 37 C
ria, fungus); confirmatory ABO/Rh typing;
for up to 1 hour of incubation, they also noted
CFU assay; and viability testing. Final vol-
that the addition of 1% DMSO to culture dish-
ume, cell dose, and cell recovery are deter-
es suppresses CFU assay results. It is impor-
mined. After completion of paper work and
tant to note that these studies were performed
labeling, the unit can be released for infu-
on fresh cells.67 Studies of the effects of DMSO
sion).58 on HSCs that have been previously cryopre-
served are limited. However, some investiga-
It is anticipated that thawing procedures
tors have noted similar suppression of colony
based on the type of product processed will be formation when thawed UCB samples are not
standardized through the collaboration of washed and are immediately placed into CFU
UCB bankers and transplanters. culture.67 Thus, the possible functional defect
due to DMSO coupled with the not infrequent
INFUSION OF UCB need to hold clinical products raises the con-
cern that cell injury could occur with thawed
To facilitate the product infusion procedure, it UCB, particularly when cells are not washed or
is necessary to maintain good communication diluted.
between the patient and the physician over- The unit bag and IV tubing should be
seeing the infusion. Hence, it is a good practice flushed with sterile saline after the unit bag
to again describe the general process, includ- empties to maximize cell dose. Sterile saline
ing graft selection, cell processing, infusion, may be added directly to the unit bag if the
potential side effects/adverse reactions, and flow rate becomes unusually slow. Because the
plans for premedication. This conversation final volume of a UCB unit is relatively low
should take place on, or shortly before, the day (roughly 60 to 100 mL with the thaw/wash
of the transplant procedure. A procedure for methods described above), the infusion rate is
infusion is outlined below.50 The general ap- slow (5-10 mL/minute) and the infusion pro-
proach and potential adverse reactions are cess is typically completed within 15 to 30
742 䡲 AABB TECHNICAL MANUAL
minutes of the unit’s receipt in the patient care action exists, aggressive IV hydration is recom-
unit. mended (eg, for 2 to 6 hours before and 6
It is recommended that the patient’s vital hours after infusion with diuretics as needed).
signs be checked before infusion, immediate- In addition, administration of prophylactic an-
ly after infusion, and 1 hour after infusion at a tiemetics, antipyretics, and antihistamines is
minimum. Should an adverse reaction occur, suggested.
more frequent monitoring is recommended.
The transplant physician and the medical di-
ECO N O M I C I S S U ES
rector of the cell therapy laboratory should be
notified immediately of an unexpected or The establishment of a diverse and sustainable
moderate to severe reaction. A prompt investi- inventory from which approximately 1% of
gation should include confirmation of product products are distributed for clinical use is an
identity, a product inspection, and the initia- expensive undertaking for a CBB. One of the
tion of any appropriate laboratory testing (eg, main reasons for this selectivity in product uti-
direct antiglobulin test, antibody titers, Gram’s lization is the strong association of successful
stain, or culture). All of the monitoring results outcomes of UCB transplantation with the
should be captured on the accompanying in- grade of the HLA match and cell dose.73 As a
fusion form, which should be returned to the result, UCB selection has recently been direct-
laboratory. Serious adverse reactions must ed toward utilization of products with high
then be communicated as appropriate to the TNC content. This finding further complicates
NMDP, distributing CBB, and/or the IND ap- the strategy of banking products from donors
plication or license holder for reporting to the who are not well represented in registries due
FDA. to the reduced volume and cellular content in
Reactions associated with UCB infusion the collections from these populations.
may be very similar to those that occasionally To limit the negative economic impact of
occur with blood transfusion (ie, allergic, he- these trends, CBBs must expand their collec-
molytic, or febrile reactions and those due to tion efforts to increase the proportion of units
microbial contamination). However, with with higher TNC counts.74 In addition, ex-
combinations of the various processing meth- panding the number of collection sites to al-
ods for UCB (eg, red cell depletion, plasma re- low broader participation of donors could
duction, and postthaw wash step), some types support diversification efforts. Improving the
of reactions to UCB (ie, those attributed to red efficiency of the collection process in estab-
cell antigens and plasma proteins) may be less lished centers could increase the number of
likely to occur with UCB than other blood products eligible for further manufacture and
transfusions. Likewise, if the UCB has under- their likelihood of selection while offsetting
gone red cell depletion and/or wash steps, re- the associated costs related to education, staff-
nal failure due to infusion of red cells and free ing, and transportation. Collaborating to iden-
hemoglobin should occur less often than with tify alternative uses for clinical-grade products
infusions of HSCs from other sources. Reac- could also help CBBs achieve self-sufficiency.
tions (ie, nausea, vomiting, coughing, and The transition to a full cGMP setting has
headache) often attributed to DMSO should been accompanied by spending money to ex-
be less common with UCB infusions as pand UCB inventories. The facility used to
well.68,69 Bacterial contamination, which oc- manufacture human cells, tissues, and cellu-
curs in up to 5% of collections, is unusual in lar and tissue-based products (HCT/Ps) must
released UCB units because CBBs exclude be maintained in a clean, sanitary, and orderly
these units from their inventories.70-72 manner to prevent the introduction, transmis-
UCB infusions are usually very well toler- sion, or spread of communicable diseases, as
ated; if reactions occur, they are typically mild described in Title 21, CFR Part 1271.190(b). To
and readily managed by the clinical team.65 minimize the risk of product contamination,
However, because the possibility of a severe re- manufacturers (CBBs) may upgrade their facil-
CHAPTER 30 Umbilical Cord Blood Banking 䡲 743
ities to offer International Organization for search received a contract to collect data on
Standardization Class 5 processing areas, de- all allogeneic (related and unrelated) HSC
fine robust cleaning and disinfection plans, transplantations performed in the United
enhance environmental monitoring to ensure States and those that were completed with
continued control of the manufacturing areas, products procured through the program
and develop comprehensive stability plans to but performed outside the United States.
establish expiry dates of products manufac- 䡲 Solicitation and subsequent contractual
tured under these conditions. Facility retrofit- agreements with CBBs to collect and store
ting, process redesign, and hiring of additional at least 150,000 new UCB units that meet
staff to accommodate upgraded activities in- specific criteria comprise the National Cord
crease the fixed cost of UCB manufacturing. Blood Inventory (NCBI). CBBs with a con-
Whereas pharmaceutical drug manufac- tract from the NCBI also provide UCB units
turers can recover the cost of research and de- that do not meet these criteria for research
velopment along with a substantial profit mar- studies.
gin from the consumer, it is improbable that 䡲 Requirement for the US Government Ac-
CBBs will be able to incorporate increasing countability Office to report on efforts to
costs of UCB product manufacture into the increase UCB unit collection for the NCBI.
amounts invoiced to clinical programs for 䡲 Establishment of the ACBSCT to make rec-
product acquisition. Doing so may encourage ommendations to the Secretary of DHHS.
transplant centers to pursue alternative means
of treatment for their patients, a strategy that This legislation not only validated UCB as
would be detrimental to the survival of UCB a credible therapeutic option but also provid-
manufacturers. Therefore, public CBBs must ed financial support to CBBs for the creation of
be innovative and resourceful in all aspects of larger inventories. However, federal funding
their business strategy. does not cover the costs associated with man-
ufacturing and is limited in terms of appropri-
The Stem Cell Therapeutic and ations. In the 2010 reauthorization, Congress
Research Act challenged CBBs to expand their collection
while lowering costs and improving the effi-
The Stem Cell Therapeutic and Research Act of ciency of UCB unit collection without decreas-
2005 was passed by Congress and signed by ing the quality of the UCB units collected. In
President George W. Bush in December 2005 addition, CBBs are required to demonstrate
as Public Law 109-129. In October 2010, Presi- ongoing measurable progress toward achiev-
dent Obama signed the Stem Cell Therapeutic ing self-sufficiency in their collection and
and Research Reauthorization Act of 2010 banking operations.
(Public Law 111-264). The implementation of
both acts is managed by the Health Resources
and Services Administration (HRSA) of the US R E G U L AT I O NS A ND
Department of Health and Human Services S TA N D A R D S
(DHHS). Provisions in these acts that are spe-
FDA
cific to UCB include:
In 1997, the FDA announced a novel, risk-
䡲 The CW Bill Young Cell Transplantation based, tiered approach to the regulation of so-
Program, intended to increase the numbers matic cells and tissues.75 The approach out-
of UCB units and establish an outcomes da- lined in the FDA’s proposed approach to regu-
tabase to collect data to characterize and lation of cellular and tissue-based products
refine all aspects of UCB transplantation. (February 27, 1997) was followed by the good
Through the Stem Cell Therapeutic Out- tissue practice (GTP) regulations of 2004
comes Database, the Center for Interna- “requiring cells and tissues to be handled ac-
tional Blood and Marrow Transplant Re- cording to procedures designed to prevent
744 䡲 AABB TECHNICAL MANUAL
contamination and preserve [cell and] tissue tial risk, are required to comply with the re-
function and integrity.”51 This historic docu- quirements for donor testing and screening,76
ment has set the framework for cell therapy establishment registration,77 and the current
laboratories by identifying the agency’s expec- GTP regulations.51
tations regarding the prevention of disease Since 2004, CBBs have been required to
transmission and laboratory process controls. register with the FDA as manufacturers of
The GTP requirements are intended to 1) pre- UCB51 and demonstrate compliance with
vent unknowing use of contaminated tissue at
GTPs. For CBBs that manufacture and/or store
risk of transmitting infectious disease, 2) pre-
more than minimally manipulated products
vent improper processing of tissue in ways
(eg, UCB that is activated, expanded, or genet-
that could cause damage or risk of contamina-
tion, and 3) ensure the safety and efficacy of ically modified) or combine UCB with non-
products that are more than minimally manip- tissue components, the FDA requires submis-
ulated.75,76 Based on these principles, all hu- sion of an IND application and adherence to
man cellular and tissue-based products in- licensure application requirements. Table 30-2
tended for use in unrelated recipients, summarizes the FDA’s regulations governing
regardless of degree of manipulation or poten- HCT/Ps, including UCB.
TABLE 30-2. Summary of FDA Regulations Regarding Human Cells, Tissues, and Cellular and
Tissue-Based Products (HCT/Ps) and Hematopoietic Progenitor Cells-Cord (HPC-Cs)75
HCT/Ps that are or will be regu- 䡲 All allogeneic, unrelated HCT/Ps derived from cord and peripheral blood
lated as biological products 䡲 HCT/Ps that are more than minimally manipulated (eg, expanded, acti-
vated, or genetically modified).
䡲 HCT/Ps that are combined with a drug, device, or biological product.
䡲 HCT/Ps that are intended for nonhomologous use (eg, for cardiac
repair).
HPC-Cs that are currently subject 䡲 Allogeneic, unrelated, and minimally manipulated cells intended for use
to biologics license application in unrelated donor HPC transplantation procedures in conjunction with
(BLA) requirements an appropriate preparative regimen for hematopoietic and immunologic
reconstitution in patients with disorders affecting the hematopoietic sys-
tem that are inherited, acquired, or result from myeloablative treatment.
䡲 FDA intends to grant licensure to products manufactured according to
recommended establishment and process controls and that comply with
all applicable regulatory requirements.
HPC-C that is currently subject to 䡲 When no satisfactory alternative treatment is available, HPC-Cs that are
investigational new drug require- not FDA licensed and are:
ments 1. Allogeneic, unrelated, and minimally manipulated.
2. Intended for use in unrelated donor HPC transplantation procedures
in conjunction with an appropriate preparative regimen for hemato-
poietic and immunologic reconstitution in patients with disorders
affecting the hematopoietic system that are inherited, acquired, or
result from myeloablative treatment.
3. Manufactured in a US cord blood bank and intended to be used in
the United States.
䡲 Manufactured in a US cord blood bank before BLA approval and do not
meet licensing requirements.
䡲 Prospectively manufactured in the United States and for which there is
no satisfactory alternative.
FDA = Food and Drug Administration.
CHAPTER 30 Umbilical Cord Blood Banking 䡲 745
Until 2011, minimally manipulated UCB may be units from UCB establishments in
intended for use in unrelated recipients was which a BLA is pending; units that do not meet
not licensed. However, the FDA has long con- licensing requirements, or units that come from
sidered licensure as a way of improving the non-US CBBs that have chosen not to apply
safety and quality of UCB by instituting re- for licensure. Under such circumstances, the
quirements that would be legally enforce- FDA requires submission of an IND application.
able.51 The FDA believed that compelling clini- FDA published the guidance for such applica-
cal safety and efficacy data existed to support tions as companion documents to the BLA.80
the development of product standards as an In March 2014, FDA updated the 2009
approach toward licensure. In 1998, the FDA BLA and 2011 IND guidance documents. The
issued a request for the public to submit com- BLA guidance document was updated to ex-
ments regarding establishment controls, pro- pand the indications for use, clarify the type of
cess controls, and product standards for UCB clinical data required in the application, and
manufacturers.78 After reviewing the submit- replaced the term “HPC-C” with “HPC, Cord
ted information, the FDA determined that suf- Blood.” Similar revisions were made in the
ficient data did exist to support the develop- IND guidance document, in addition to clari-
ment of processing and product standards for fying that a table of contents is required for all
granting licensure. IND applications.
In October 2009, the FDA published its fi- In summary, the FDA regulates HPCs,
nal guidance, which described ways to assist HPC-C blood for unrelated allogeneic use as a
UCB manufacturers in submitting a BLA.79 drug under the Food Drug and Cosmetic Act
Originally published with indications restrict- and as biological products under the Public
ed to “hematopoietic reconstitution in pa- Health Services Act. Therefore, regulations ap-
tients with hematologic malignancies, certain plicable to HPC-Cs include:
lysosomal storage and peroxisomal enzyme
deficiency disorders, primary immunodefi- 1. Title 21, CFR Part 201 and 610 Subpart G –
ciency diseases, bone marrow failure, and beta Labeling.
thalassemias,” the guidance was modified by 2. Title 21, CFR Part 202: Prescription drug
the FDA in 2011 to broaden the indications to advertising.
“use in unrelated donor hematopoietic pro- 3. Title 21, CFR Parts 210 and 211: cGMP.
genitor cell transplantation procedures in con- 4. Title 21, CFR Part 600: Biological products,
junction with an appropriate preparative regi- general.
men for hematopoietic and immunologic 5. Title 21, CFR Part 601: Licensing.
reconstitution in patients with disorders af- 6. Title 21, CFR Part 610: General biological
fecting the hematopoietic system that are in- products standards.
herited, acquired, or result from myeloablative 7. Title 21, CFR Part 1270 (Section 351 of the
treatment.” This guidance applies to hemato- Public Health Services Act): HCT/Ps with
poietic progenitor cells (HPCs), UCB [HPCs- systemic effect intended for use in unre-
cord (HPC-Cs)] manufactured by US UCB lated persons.
establishments and non-US UCB establish- 8. Title 21, CFR 1271 (Section 361 of the Public
ments that distribute UCB in the United Health Services Act): HCT/P registration
States. For establishments that manufacture and listing, donor eligibility, and GTP.
UCB products for autologous use or use by a
first- or second-degree blood relative, the FDA In the event that one regulation is in con-
encourages the same manufacturing recom- flict with another, the one that is more specifi-
mendations and applicable regulations be fol- cally applicable to UCB supersedes the one
lowed.20 that is more general.
The FDA recognizes that there will be cir- In combination, standards regulating bio-
cumstances in which the best available UCB logic drugs are akin to those for pharmaceuti-
unit for a patient is not a licensed unit. There cal manufacturing, and their application to a
746 䡲 AABB TECHNICAL MANUAL
cellular therapy product, such as UCB, is chal- ment, materials, records, storage, distribution,
lenging. In contrast to pharmaceutical drugs, quality audits, quality plans, errors/deviations,
which are chemicals, are manufactured in tab- labeling, personnel, equipment, validation,
let lots, and can be terminally sterilized, UCB outcome reviews, and adverse event reporting.
products are composed of viable cells, are In 2009, the ACBSCT recommended that both
manufactured in single-unit lots, and must be AABB and FACT be recognized as accreditation
processed aseptically. Recognizing the value organizations for the NCBI. Both organiza-
and unique nature of each UCB product, FDA tions incorporated specific standards to en-
has been very cautious in regulating UCB to sure that CBBs comply with the contracts held
avoid preventing patients from having access with HRSA.
to existing or prospectively manufactured In a post-licensure era, it is expected that
products. At the time of this writing, the FDA UCB banking will continue to evolve to bal-
has approved the BLAs of five CBBs in the ance the creation of large inventories of safely
United States (see Table 30-3). manufactured products that not only meet de-
mands for cell dose and matching from clini-
AABB and FACT cians, but also target new populations and ap-
The AABB and FACT are the two professional plications for use of this abundantly available
organizations that have established standards resource. Currently, there are several ongoing
for HPCs, including UCB, in the United clinical trials involving HSCs and HPC expan-
States.22,24 AABB has incorporated the require- sion approaches to overcome limited per-unit
ments for UCB processing facilities in its Stan- cell doses and the lengthy time required to
dards for Cellular Therapy Product Services.24 achieve an absolute neutrophil count of 500/µL;
FACT and NetCord combined their efforts to UCB-derived natural killer, T-regulatory, and
establish a unique set of standards for UCB-re- dendritic cells for immunotherapeutic treat-
lated activities.22 Both AABB and FACT have ments; and UCB-derived mesenchymal stem
created standards that align with the FDA’s or stromal cells to enhance engraftment and
GMP and GTP requirements and center on regenerative applications. However, optimal
quality systems essentials. These standards methods for production and clinical applica-
cover donor suitability, collection, processing bility of these new products have yet to be es-
controls, document controls, facility manage- tablished.
KEY POINTS
1. UCB stem cells have a higher proliferative capacity and immune tolerance than stem cells
from adult sources, as demonstrated by the decreased incidence of GVHD in UCB recipients
despite the less stringent HLA-matching requirements for UCB transfusion. These features
allow the use of more flexible matches with UCB, which provide the ability to support pa-
tients with rare HLA types and permit matches across ethnic lines.
2. UCB has evolved from being considered biologic waste to acceptance as a viable source of
HSCs. More than 30,000 unrelated donor UCB transplant procedures have been performed,
and there are an estimated 600,000 UCB units banked worldwide for use as an HPC source.
In 2008, UCB surpassed marrow as the most frequently utilized source of donor cells for un-
related transplantation.
3. Engaging pregnant women and arranging donation of their infant’s UCB in advance is criti-
cal for achieving the best results. Early education allows for more complete medical screen-
ing, comprehensive donor protection, availability of adequate supplies, and acquisition of
thorough informed consent.
4. UCB can be collected before (in utero) or after (ex utero) the placenta has been delivered.
There are procedural and economic advantages and disadvantages to each method. When
performed properly, both are acceptable for providing high-quality collections.
5. Current methods of manufacturing UCB products involve reduction of red cell and plasma
fractions before cryopreservation. Methods generally involve sedimentation and/or centrif-
ugation and may be performed manually or with automated devices that have been cleared
for use by the FDA.
6. Transplant centers must determine the most appropriate thawing procedure for the prod-
ucts received based on the manufacturer’s instructions, red cell volume, patient-specific
variables, and their own validated method. Current practices for preparing UCB for infusion
consist of bedside thawing, the traditional thaw-and-wash method, and a thaw-and-recon-
stitution technique.
7. Following the announcement of a tiered approach to regulating UCB in 1997, the FDA pub-
lished a guidance document in 2009 defining a process by which CBBs can submit a BLA to
the FDA for UCB. Through this process, five public CBBs have been authorized to manufac-
ture HPC products from UCB for allogeneic use.
8. The clinical utility of several cell types within UCB (eg, mesenchymal stem and immune
cells) is being investigated for nonhematopoeitic applications, immunomodulation, and re-
generative medicine.
9. To ensure their economic sustainability, CBBs must adapt to recent trends in product selec-
tion, improve collection efficiency, collaborate to identify alternative uses for clinical-grade
products, and successfully transition to a full cGMP and licensure environment.
R E F E R EN C E S
1. Barker JN, Davies SM, DeFor T, et al. Survival and umbilical cord blood transplants in chil-
after transplantation of unrelated donor um- dren with acute leukemia. Blood 2001;97:2962-
bilical cord blood is comparable to that of hu- 71.
man leukocyte antigen-matched unrelated 3. Eapen M, Rubinstein P, Zhang MJ, et al. Out-
donor bone marrow: Results of a matched- comes of transplantation of unrelated donor
pair analysis. Blood 2001;97:2957-61. umbilical cord blood and bone marrow in chil-
2. Rocha V, Cornish J, Sievers EL, et al. Compari- dren with acute leukaemia: A comparison
son of outcomes of unrelated bone marrow study. Lancet 2007;369:1947-54.
748 䡲 AABB TECHNICAL MANUAL
4. Laughlin MJ, Eapen M, Rubinstein P, et al. Out- 15. Barker JN, Krepski TP, DeFor TE, et al. Search-
comes after transplantation of cord blood or ing for unrelated donor hematopoietic stem
bone marrow from unrelated donors in adults cells: Availability and speed of umbilical cord
with leukemia. N Engl J Med 2004;351:2265-75. blood versus bone marrow. Biol Blood Marrow
5. Rocha V, Labopin M, Sanz G, et al. Transplants Transplant 2002;8:257-60.
of umbilical-cord blood or bone marrow from 16. Gluckman E, Broxmeyer HA, Auerbach AD, et
unrelated donors in adults with acute leuke- al. Hematopoietic reconstitution in a patient
mia. N Engl J Med 2004;351:2276-85. with Fanconi's anemia by means of umbilical-
6. Brunstein CG, Eapen M, Ahn KW, et al. Re- cord blood from an HLA-identical sibling.
duced-intensity conditioning transplantation N Engl J Med 1989;321:1174-8.
in acute leukemia: The effect of source of un- 17. Broxmeyer HE. Biology of cord blood cells and
related donor stem cells on outcomes. Blood future prospects for enhanced clinical benefit.
2012;119:5591-8. Cytotherapy 2005;7:209-18.
7. Broxmeyer HE, Hangoc G, Cooper S, et al. 18. Rubinstein P, Rosenfield RE, Adamson JW, Ste-
Growth characteristics and expansion of hu- vens CE. Stored placental blood for unrelated
man umbilical cord blood and estimation of bone marrow reconstitution. Blood 1993;81:
its potential for transplantation in adults. Proc 1679-90.
Natl Acad Sci U S A 1992;89:4109-13. 19. World Marrow Donor Association. Accredited
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43. Institute of Medicine. Cord blood: Establishing
31. Lasky LC, Lane TA, Miller JP, et al. In utero or ex
a national hematopoietic stem cell bank pro-
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32. Surbek DV, Schonfeld B, Tichelli A, et al. Opti- 44. Fraser JK, Cairo MS, Wagner EL, et al. Cord
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vs after placenta delivery. Bone Marrow Trans- J Hematother 1998;7:521-61.
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33. Solves P, Moraga R, Saucedo E, et al. Compari- PA, et al. Optimal cryopreservation of human
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2003;31:269-73. 46. McCullough J, Haley R, Clay M, et al. Long-
34. Wong A, Yuen PM, Li K, et al. Cord blood col- term storage of peripheral blood stem cells
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Levels of nucleated cells, haematopoietic pro- nitrogen technique compared with cells fro-
genitor cells, leukocyte subpopulations and zen and stored in a mechanical freezer. Trans-
macroscopic clots. Bone Marrow Transplant fusion 2010;50:808-19.
2001;27:133-8. 47. Antoniewicz-Papis J, Lachert E, Wozniak J, et
35. Solves P, Moraga R, Mirabet V, et al. In utero or al. Methods of freezing cord blood hematopoi-
ex utero cord blood collection: An unresolved etic stem cells. Transfusion 2014;54:194-202.
question. Transfusion 2003;43:1174-6. 48. Kobylka P, Ivanyi P, Breur-Vriesendorp BS.
36. Kurtzberg J, Laughlin M, Graham ML, et al. Preservation of immunological and colony-
Placental blood as a source of hematopoietic forming capacities of long-term (15 years)
750 䡲 AABB TECHNICAL MANUAL
cryopreserved cord blood cells. Transplanta- units shipped from a single cord blood bank.
tion 1998;65:1275-8. Transfusion 2003;43:1285-95.
49. Broxmeyer HE. Will iPS cells enhance thera- 62. Hahn T, Bunworasate U, George MC, et al. Use
peutic applicability of cord blood cells and of nonvolume-reduced (unmanipulated after
banking? Cell Stem Cell 2010;6:21-4. thawing) umbilical cord blood stem cells for
50. Chrysler G, McKenna D, Schierman T, et al. allogeneic transplantation results in safe en-
Umbilical cord blood banking. In: Broxmeyer graftment. Bone Marrow Transplant 2003;32:
HE, ed. Cord blood: Biology, immunology, 145-50.
banking, and clinical transplantation. Bethes- 63. Barker JN, Abboud M, Rice RD, et al. A “no-
da, MD: AABB Press, 2004:219-57. wash” albumin-dextran dilution strategy for
51. Food and Drug Administration. Current good cord blood unit thaw: High rate of engraftment
tissue practice for human cell, tissue and cel- and a low incidence of serious infusion reac-
lular and tissue based products establish- tions. Biol Blood Marrow Transplant 2009;
ments; Inspection and enforcement. Fed Reg- 15:1596-602.
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52. IATA. International Air Transport Association of cord blood thawing methods on cell recov-
(IATA) dangerous goods regulations. 44 ed. Ge- ery, potency, and infusion. Transfusion 2010;
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53. Code of federal regulations. Title 49 CFR, Sub- 65. McKenna D, McCullogh J. Umbilical cord
part C, 171-177. Washington, DC: US Govern- blood infusions are associated with mild reac-
ment Printing Office, 2014 (revised annually). tions and are overall well-tolerated. Cytothera-
54. Wada RK, Bradford A, Moogk M, et al. Cord py 2003;5:438.
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blood through the Hawaii Cord Blood Bank poietic progenitor cells. Bone Marrow Trans-
and store the units at the Puget Sound Blood plant 1993;11:389-93.
Center. Transfusion 2004;44:111-18. 67. Laroche V, McKenna DH, Moroff G, et al. Cell
55. Hubel A, Carlquist D, Clay M, McCullough J. loss and recovery in umbilical cord blood pro-
Short-term liquid storage of umbilical cord cessing: A comparison of postthaw and post-
blood. Transfusion 2003;43:626-32. wash samples. Transfusion 2005;45:1909-16.
56. Hubel A, Carlquist D, Clay M, McCullough J. 68. Davis JM, Rowley SD, Braine HG, et al. Clinical
Cryopreservation of cord blood after liquid toxicity of cryopreserved bone marrow graft
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57. Hubel A, Carlquist D, Clay M, McCullough J. 69. Stroncek DF, Fautsch SK, Lasky LC, et al. Ad-
Liquid storage, shipment, and cryopreserva- verse reactions in patients transfused with
tion of cord blood. Transfusion 2004;44:518- cryopreserved marrow. Transfusion 1991;31:
25. 521-6.
58. Solomon M, Wofford J, Johnson C, et al. Fac- 70. Bornstein R, Flores AI, Montalban MA, et al. A
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ment before cryopreservation. Transfusion achieves sufficient cell levels for transplanta-
2009;50:820-30. tion in most adult patients. Stem Cells 2005;
59. McCullough J, McKenna D, Kadidlo D, et al. Is- 23:324-34.
sues in the quality of umbilical cord blood 71. M-Reboredo N, Diaz A, Castro A, Villaescusa
stem cells for transplantation. Transfusion RG. Collection, processing and cryopreserva-
2005;45:832-41. tion of umbilical cord blood for unrelated
60. McCullough J, McKenna D, Kadidlo D, et al. transplantation. Bone Marrow Transplant
Mislabeled units of umbilical cord blood de- 2000;26:1263-70.
tected by a quality assurance program at the 72. Lecchi L, Ratti I, Lazzari L, et al. Reasons for
transplantation center. Blood 2009;114:1684-8. discard of umbilical cord blood units before
61. Nagamura-Inoue T, Shioya M, Sugo M, et al. cryopreservation. Transfusion 2000;40:122-4.
Wash-out of DMSO does not improve the 73. Barker JN, Scaradavou A, Stevens CE. Com-
speed of engraftment of cord blood transplan- bined effect of total nucleated cell dose and
tation: Follow-up of 46 adult patients with HLA match on transplantation outcome in
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1061 cord blood recipients with hematologic hematopoietic stem/progenitor cell products;
malignancies. Blood 2010;115:1843-9. request for comments. Fed Regist 1998;63:
74. Bart T, Boo M, Balabanova S, et al. Impact of 2985.
selection of cord blood units from the United 79. Guidance for industry: Minimally manipulat-
States and Swiss registries on the cost of bank- ed, unrelated allogeneic placental/umbilical
ing operations. Transfus Med Hemother 2013; cord blood intended for hematopoietic recon-
40:14-20. stitution for specified indications. (October
75. Food and Drug Administration. Proposed 2014). Silver Spring, MD: CBER Office of Com-
approach to regulation of cellular and tissue- munication, Outreach, and Development,
based products. CBER Office of Communica- 2009. [Available at http://www.fda.gov/down
tion, Outreach, and Development, 1997. loads/BiologicsBloodVaccines/Guidance
[Available at http://www.fda.gov/downloads/ ComplianceRegulatoryInformation/Guidanc
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UCM062601.pdf (accessed December 6, 80. Food and Drug Administration. Guidance for
2013).] industry and FDA staff: IND applications for
76. Food and Drug Administration. Eligibility de- minimally manipulated, unrelated allogeneic
termination for donors of human cells, tissues, placental/umbilical cord blood intended for
and cellular and tissue-based products; Final hematopoietic and immunologic reconstitu-
Rule. Fed Regist 2004;69:29786-834. tion in patients with disorders affecting the
77. Food and Drug Administration. Human cells, hematopoietic system. (March 2014) Silver
tissues, and cellular and tissue-based prod- Spring, MD: CBER Office of Communication,
ucts; establishment registration and listing; fi- Outreach, and Development, 2014. [Available
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78. Food and Drug Administration. Request for cines/GuidanceComplianceRegulatoryInfor
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C h a p t e r 3 1
Tissue-Derived Non-Hematopoietic
Stem Cell Sources for Use in
Cell-Based Therapies
Yameena Jawed, MD, Postdoctoral (Research) Fellow, Indiana Center for Vascular Biology and Medicine, Indi-
ana University School of Medicine; Brian Johnstone, PhD, Preclinical Director, Center for Regenerative Medi-
cine, Director, Cardiovascular Ischemia and Vasculogenesis Core, and Associate Research Professor, Indiana
University School of Medicine; Sreedhar Thirumala, PhD, Senior Scientist, Cook General BioTechnology, LLC;
and Keith March, MD, PhD, Director, Indiana Center for Vascular Biology and Medicine, Director, Vascular
and Cardiac Center for Adult Stem Cell Therapy, and Professor of Medicine, Physiology, and Biomedical Engi-
neering, Indiana University School of Medicine, Indiana University, Indianapolis, Indiana
The authors have disclosed no conflicts of interest.
753
754 䡲 AABB TECHNICAL MANUAL
hypotheses of “paracrine support” are dis- from genetically nonidentical donor tissue in
cussed in detail below. This new understand- carefully controlled environments with estab-
ing has led to a widespread adoption of the lished controls to ensure quality and safety.
more general phrase “cell-based therapy” to Much research has focused on the ability
encompass the panoply of beneficial effects of of these regenerative therapies to replace or-
treatment with stem cells from a variety of gan transplants. In 2006, an article was pub-
sources—effects that are independent of sta- lished on what was labeled the “first artificial-
ble tissue engraftment and tissue regenera- ly grown organ.”5 In this study, scientists were
tion. In practice, though, the term “regenera- able to produce sections of a bladder and pro-
tive medicine” is still widely used, especially by vide some degree of urinary continence to pa-
the lay public, to refer to any stem cell-based tients with spina bifida. Currently, most re-
treatment. search relates to tissue and organ progenitor
Strictly speaking, regenerative medicine cells that either increase or support the func-
is simply defined as the “process of replacing tionality of preexisting tissues or induce new
or regenerating human cells, tissues or organs organ structures to form in situ, a process
to restore or establish normal function.”3 The technically termed “organ reconstruction.”
origins of cellular therapy date back to 1968 The reconstruction of bioengineered organs or
with the first bone marrow transplant of he- even for a regenerative therapy treatment po-
matopoietic stem cells to restore the hemato- tentially requires a massive number of stem
poietic system in dysfunctional or ablated cells. For many stem-cell-mediated therapies,
marrow.4 From these origins, there has been the number of cells needed to effectively treat
great progress in identifying many different a patient greatly surpasses the number of cells
sources of potentially therapeutic stem and available from donors.
progenitor cells in the adult system. These
findings have also been translated to early-
M S C S O U RC E S
stage clinical trials to test the effectiveness of
these approaches in a number of diseases that Tissue sources of stem cells used for cell-based
are not currently addressed by traditional therapies are varied and cover nearly every or-
pharmacologic therapies or surgery. gan system in the body. Based on the develop-
Treatment with both autologous and allo- mental stage of the donor, these cells can be
geneic stem and progenitor cells has been classified as embryonic, fetal, or adult. Embry-
evaluated. Autologous cell therapy has the ad- onic stem cells are primordial cells isolated
vantage of involving fewer potential regulatory from the eight-cell blastula that has the capac-
hurdles and, in some cases, such as recon- ity to form any cell type found in the human
structive surgery, is arguably within the tradi- body. Stem cells isolated from postnatal or re-
tional scope of the practice of medicine and, productive tissues are known as “adult stem
therefore, outside regulatory oversight. Autol- cells.” Most of these cells were discovered and
ogous cell-based therapies, although attractive defined based on such attributes as the ability
and perhaps more tractable than allogeneic to proliferate for many generations in culture
cell-based therapies, are practical only when as well as their intrinsic property of differenti-
the source of cells is abundant and their ex- ation in response to environmental or chemi-
traction is without significant morbidity or en- cal stimuli. The ability to differentiate along
hanced mortality. For these reasons, autologous multiple lineages (eg, mesodermal, ectoder-
therapy has been limited to cells from marrow mal, and endodermal) is important. The hall-
and adipose tissues; however, the former is marks of pluripotentiality and multipotentiali-
mostly composed of hematopoietic cells, ty distinguish stem cells from progenitor cells;
whereas the latter is made up of approximately the latter have undergone partial differentia-
15% to 30% MSCs on isolation.4,5 Allogeneic tion in situ and, thus, are committed to differ-
therapies, however, usually require the mass entiate terminally into a single cell type (eg,
production of stem cells originally obtained endothelial progenitor cells or preadipocytes,
CHAPTER 31 Tissue-Derived Stem Cells 䡲 755
which form endothelial cells and adipocytes, from the nondesirable cell types (such as leu-
respectively). kocytes and endothelial cells) and then ex-
The most studied adult multipotent cells panded under conditions that are permissive
are mesoderm-derived MSCs, alternatively for maintaining stem-cell-like properties. Be-
termed “multipotent stromal cells,” which cause of the cost and time required to generate
originate from the stromal compartment of sufficient numbers of qualified cells under
skeletal muscle, reproductive organs (umbili- current good manufacturing practice (cGMP)
cal cord and uterus), amnion, marrow, and ad- conditions, the effort is only economically
ipose tissues. The recent advances in tech- practical when culture expansion yields suffi-
niques to reprogram terminally differentiated cient numbers of MSCs at lower passages to
cells to induce pluripotency (termed “induced treat many hundreds, if not thousands, of pa-
pluripotent stem cells”) have opened up a vast tients.
source of stem cells from nonembryonic tis- Most adult tissues contain low percentag-
sues. Induced pluripotent cells present excit- es of MSCs that are not available in sufficient
ing new opportunities for cell-based therapies abundance, with the exception of adipose tis-
and are covered in detail in Chapter 33. Mar- sues. BM-MSCs constitute only a small frac-
row-derived MSCs (BM-MSCs), which are the tion (approximately 1 in 3.4 × 104 cells) of cells
nonhematopoietic cells isolated from marrow in adult marrow.28 Although the frequency of
stroma, are also discussed in detail in Chapter MSCs in fetal tissues may be higher, these tis-
33 and are only briefly discussed here, mostly sues are comparatively quite small and limited
to compare them with MSCs derived from oth- in availability, and their use is associated with
er tissues and to discuss their current thera- ethical concerns when their collection in-
peutic uses. volves fetal destruction.29 Examples of MSCs
BM-MSCs were the first nonhematopoiet- found in low numbers in source tissues but
ic stem cells discovered and, due to having that are highly expandable are muscle-derived
been studied in great detail, are considered the MSCs (M-MSCs), cord blood MSCs (CB-
archetypal MSCs. Because the numbers and MSCs), umbilical cord MSCs (UC-MSCs), pla-
quality of MSCs decline with the aging of do- cental MSCs (Pl-MSCs), endometrial lining
nors and due to the invasiveness of the proce- MSCs (EL-MSCs), and dental pulp MSCs (DP-
dures required to procure marrow, other MSCs).
sources of MSCs were sought. Perhaps the Adipose-derived stem cells (ASCs), which
most primitive adult MSCs are those obtained are phenotypically similar to BM-MSCs, are
from umbilical cord tissue, umbilical cord more than 500-fold more prevalent per gram
blood, amnion, and amniotic fluid.6-10 The of fat than per gram of marrow.30 Most patients
dental pulp of the third molar is another have excess fat that can be easily and safely ex-
source of MSCs.11 It has been recently recog- tracted for processing to isolate ASCs. This
nized that adipose tissue is a rich and abun- property makes adipose tissue an ideal source
dant source of cells with stem cell properties.12 of MSCs for either autologous or allogeneic
Additional sources of MSCs are placenta and applications. ASCs are perhaps the only type of
uterine endometrial cells shed during men- MSC available in numbers that are sufficient
ses.13,14 Each of these tissue-specific MSCs is for immediate or “point-of care” use in thera-
discussed in more detail below. peutic applications without the need for cul-
MSCs from different tissue sources differ ture expansion.31-33 Extraction of adipose tissue
with respect to prevalence, proliferative capac- is accomplished with minimally invasive tech-
ity, immunophenotype, and differentiation niques, such as lipoaspiration, with little asso-
potential (Table 31-1), which may be impor- ciated morbidity even on removal of tissue
tant variables governing the clinical utility of volumes as large as 1-3 liters. Thus, the
each MSC type. In the cases of low abundance procurement and use of ASCs is a uniquely
in source tissues or difficulty of obtaining tis- practical approach to point-of-care autolo-
sue specimens, MSCs must be first isolated gous applications.
TABLE 31-1. Comparison of MSCs Derived from Different Tissue Sources
756
Frequency Cell
Study (Colony- Proliferation
(Reference Forming Potential in
䡲
Number) MSC Source Units) Vitro Senescence Multilineage Differentiation Potential Immunophenotype
19 BM + + +++ MSCs have higher osteogenesis and neu- CD106 +++ HLA-ABC ++++ CD146 +
rogenesis than BM-MSCs.
20 Pl + +++ + Osteogenesis and chondrogenesis are CD49d ++
BM + + +++ comparable but Pl-MSCs do not maintain CD49d +
adipogenesis as readily as BM-MSCs.
21 SV +++ +++ + Adipogenesis is highest in SV-MSCs and VEGFR-2 ++ CD10 +
PM +++ +++ + AT-MSCs. Chondrogenesis is superior in VEGFR-2 +++ CD10 +++
SkM +++ +++ +++ SV-MSCs. BM-MSCs, SV-MSCs, and PM- VEGFR-2 ++ CD10 +
AT +++ ++ ++ MSCs have the highest osteogenic poten- VEGFR-2 + + CD10 +
BM + +++ + tial. VEGFR-2 ++ CD10 +
22 DP +++ ++ + All dental cells express higher levels of CD106 ++
23 PD ++ ++ + neuronal markers than BM-MSCs. PD- CD106 +
BM + + +++ MSCs express higher levels of tendon- CD106 +++
ED +++ +++ + specific markers, whereas DP-MSCs retain CD106 +
weaker and chondrogenic and adipogenic
potential compared with BM-MSCs.
24 AF + +++ + Osteogenesis and adipogenesis are CD44 + CD105 +
BM +++ + +++ similar. CD44 ++ CD105 ++
25 DP All MSCs undergo osteogenesis, chondro- All MSCs express CD44, CD105, and CD90. All
UC genesis, and adipogenesis. BM-MSCs and are negative for CD34 and CD45.
AT AT-MSCs show a loss of osteogenic differ-
CHAPTER 31
Periadventitial cells (called “pericytes”), sue rescue and repair as well as to modulate
which support the integrity of microvessels the immune system.45 The demonstrated abili-
(capillaries and arterioles), have been recently ty of MSCs to effect change in disease models
discovered to possess multilineage potential.34 and patients in the absence of long-term en-
Traktuev et al highlighted the perivascular lo- graftment and differentiation in numbers re-
cation and presumptive prepericyte or peri- quired to account for damaged tissue replace-
cyte function of MSCs within adipose tissue.35 ment can be most easily explained by
The extension of this finding to recognize the paracrine mechanisms. Thus, exogenously ad-
perivascular location of MSCs throughout the ministered MSCs home to the site of injury or
body led to the current hypothesis that peri- disease through signals that are primarily in-
cytes are the source of all MSCs; this can ex- flammatory and reside temporarily at the site
plain both the high phenotypic similarity of where they secrete paracrine factors with pro-
MSCs from different tissue sources as well as survival, antiinflammatory, and repair-induc-
the broad distribution of these cells, in associ- ing properties before clearance from the sys-
ation with blood vessels, throughout the tem.46
body.35-37 It has also been proposed that the bi- Although the evidence supports the pre-
ological function of perivascular MSCs is inti- dominant function of secreted factors in the
mately related to systemic circulation in that diminution of injury or disease, there is also
once activated, they either secrete trophic fac- evidence that cell-mediated contact may be an
tors that promote endogenous repair into the important immunomodulator during the peri-
blood or may even mobilize to home to in- od of residency of MSCs at the target site.47 The
jured tissues, where they provide structural concept of paracrine support by MSCs was
units for repair through differentiation.38 first directly demonstrated in diseases of pe-
ripheral tissues and the central nervous sys-
tem by treatment of disease models with the
P RO PE RT I E S O F C LI N I C A L
cocktail of factors present in media condi-
RELEVANCE
tioned by the growth of BM-MSCs and ASCs.48
MSCs display several key complementary In another early demonstration, ablating pro-
properties that are of potential clinical use: duction of a single factor (hepatocyte growth
immunomodulation, paracrine effects, and factor) by stable small interfering ribonucleic
differentiation. Immunomodulatory func- acid knockdown abolished the salutary effects
tions include immunosuppression or immune of ASCs in a mouse hind-limb ischemia model
stimulation, depending on the signals in the of peripheral vascular disease.46
microenvironment.39 In the laboratory envi-
ronment, MSCs can be induced to differenti-
I SO L AT I O N A N D E X PAN SI O N
ate into endothelial cells, neural cells, astro-
cytes, cardiomyocytes, skeletal myocytes, Obtaining sufficient numbers of MSCs com-
chondrocytes, adipocytes, osteocytes, and monly requires their extensive expansion in
other cells that are developmentally derived cell culture. The isolation and expansion of
from the endoderm and exoderm.40-43 The low MSCs from marrow aspirate is covered in de-
immunogenicity of MSCs also makes them a tail in Chapter 33. Liposuction surgery is a
relatively safe allogeneic cell source in com- well-tolerated and safe procedure for produc-
parison with certain other cell types, including ing a large amount of lipoaspirate that can be
embryonic stem cells.44 There is evidence from processed to yield ASCs.
preclinical and, in some cases, clinical studies The method of ASC isolation in different
to suggest that each of these properties con- laboratories varies subtly, but it usually in-
tributes to the therapeutic efficacy of MSCs. volves enzymatic dissociation from adipocytes
The paracrine effects of MSCs are increas- and extracellular matrix, filtration to remove
ingly recognized as an important, if not the particulates, and low-speed centrifugation to
primary, mechanism of action to promote tis- separate adipocyte and nonadipocyte cells.
CHAPTER 31 Tissue-Derived Stem Cells 䡲 759
Following surgical removal of subcutaneous “Wharton’s jelly.” Stromal cells associated with
fat, lipoaspirate samples are washed exten- the matrix include specialized fibroblast-like
sively in phosphate-buffered saline to deplete cells, mast cells, and MSCs.52,53 Methods for
erythrocytes and then treated by continuous isolation of UC-MSCs are varied and differ be-
agitation with collagenase Type I solution for tween laboratories; however, in all cases, the
30 minutes to 1 hour at 37 C. It is important vein and arteries are first removed, followed by
that the collagenase also contain neutral pro- processing of the remaining cord tissue into
teases, presumably to liberate cells from the smaller fragments (reviewed by Can and
extracellular matrix fragments; these cells are colleagues54). In some protocols, the interior of
removed by subsequent filtration. The solu- the cord is scraped to remove the Wharton’s
tion is neutralized with an equal volume of jelly before mechanical disruption. The pieces
serum-containing medium and centrifuged at are placed in an enzymatic solution of collage-
low speed (approximately 300 × g for 10 min- nase (typically Type I) and, depending on the
utes). The buoyant adipocytes migrate in op- protocol, other proteolytic enzymes to digest
position to centrifugal force. The sedimenting the matrix and release the stromal cells. Vari-
cells are a heterogenous mixture termed the ous digestion times, from 30 minutes to 16
“stromal-vascular fraction” (SVF), which con- hours, have been employed, depending on the
tains variable proportions of endothelial cells, enzymes used and their concentration.53
pericytes, ASCs, and resident leukocytes (pri- Alternative protocols involve the conduct
marily monocytes and macrophages).47-50 The of explant culture with the cord fragments.56
cell pellet can then be resuspended in an 160 For protocols involving enzymatic digestion,
mM NH4Cl solution (for 10 minutes at room the homogenate is filtered through a 100-mi-
temperature) to lyse erythrocytes, and the sus- cron filter to remove debris. UC-MSCs from
pension is passed through a 100-micron cell the total cell population can be selected by ei-
strainer to remove debris. After an additional ther fluorescence-activated cell sorting or cul-
low-speed centrifugation, the cells are resus- ture at low density to identify colony-forming
pended in growth media, such as DMEM/F12 cells. Typical mesenchymal cell surface mark-
with 10% fetal bovine serum, for counting and ers, such as CD90, CD105, and CD73, are ex-
are then placed in tissue culture flasks and pressed by UC-MSCs; however, the level of ex-
grown under typical conditions of 5% CO2 at pression varies depending on the isolation
37 C. method used.56
Enrichment of ASCs occurs through plat- Isolation of amnion-derived Pl-MSCs is
ing on uncoated tissue-culture plastic in the accomplished by first removing the chorion,
presence of a rich medium that is not permis- followed by digesting them at 37 C with either
sive for leukocyte and endothelial cell attach- dispase II (2.4 U/mL for 1 hour) or trypsin-
ment; thus, the latter cells are removed with a EDTA (various concentrations and incubation
media change after overnight culture. After an times have been used) to release the epithelial
initial lag phase, ASCs proliferate with dou- cells. The Pl-MSCs are then released through
bling times of 36 to 48 hours, depending on the subsequent digestion with collagenase (0.75
growth medium used. The cell surface marker mg/mL) and deoxyribonuclease (0.075 mg/
profile of ASCs is altered with their expansion. mL).57 Human chorionic Pl-MSCs are liberated
For example, the CD34 marker expression of similarly after removal of the surrounding lay-
cells in culture decreases rapidly and becomes ers and treatment with 2.4 U/mL dispase II at
unmeasurable after two or three passages.34,36 37 C, followed by 1 to 3 hours of treatment with
Conversely, the CD105 and CD166 markers, collagenase II (270 U/mL) or collagenase A
which are expressed at low levels in fresh iso- (0.83 mg/mL). For both chorionic and amni-
lates, increase during culture.51 onic Pl-MSCs, the liberated cells are centri-
The umbilical cord consists of two arter- fuged at 200 × g for 10 minutes to obtain the
ies and a vein supported by a surrounding ma- cell pellet, which is then resuspended, count-
trix of gelatinous connective tissue known as ed, and cultured.13 An alternative to digesting
760 䡲 AABB TECHNICAL MANUAL
70
60
50
Number of Trials
40
30
20
10
0
Disease Category
FIGURE 31-2. Diseases for which mesenchymal stem cell therapy is under investigation (as of March 2014).
CNS = central nervous system.
CHAPTER 31 Tissue-Derived Stem Cells 䡲 763
(13 trials), Type 1 diabetes (10 trials), and lu- clinical trial of SVF administration in the treat-
pus (4 trials). ment of symptomatic, nonrevascularizable
The potential of MSC treatment for modi- ischemic myocardium. The POSEIDON trial
fying these diseases is supported by a substan- evaluated both allogeneic and autologous BM-
tial volume of literature demonstrating the MSCs as a therapy for ischemic cardiomyopa-
immunomodulatory effects of these cells in thy and found that both cell types were safe
preclinical studies of animal diseases. Admin- when delivered directly into the myocardi-
istration of MSCs significantly reduced the um.86 Two Phase II randomized, double-blind,
incidence and severity of GVHD after trans- placebo-controlled trials of intramyocardially
plantation of major-histocompatibility-com- injected autologous BM-MSCs for heart failure
plex-mismatched blood and tissues in animal have been recently initiated in Europe and the
models.74-77 These preclinical results have been United States to confirm the positive effects of
translated into seminal early trials and then these cell treatments observed in early open-
successful Phase II and III clinical trials with label Phase I/II trials.87,88
BM-MSCs for prevention and treatment of Diseases of peripheral limb vascular in-
acute and chronic GVHD after allogeneic he- sufficiency are also under clinical investiga-
matopoietic stem cell transplantation.78-80 The tion as targets for MSC therapy. In six patients
success of the Phase II and III trials led to regu- with Buerger disease, 24 weeks of treatment
latory approval of the first licensed stem cell with multiple intramuscular ASC injections
therapy, Prochymal (Osiris Therapeutics), in had positive clinical outcomes, including de-
North America for treatment of refractory creased pain.89 Intramuscular injection of
GVHD. This therapy is also available in ASCs also reduced symptoms of diabetic foot
Canada. and atherosclerotic obliterans.90 A random-
The discovery in 1998 that MSCs differen- ized, placebo-controlled trial of BM-MSC in-
tiate into cartilage under the influence of jected directly into the affected limb of
transforming growth factor-beta ushered in a patients with critical limb ischemia demon-
new era of investigations of MSC performance strated safety as well as indices of efficacy.91
in a multitude of chondrogenic conditions.81 Central nervous system trials involving
At the time of publication, 28 clinical studies MSC treatment have been dominated by in-
were evaluating the use of MSCs to treat osteo- vestigations of the treatment of ischemic
arthritis and rheumatoid arthritis. Similarly, stroke (nine trials).92 Additional diseases for
clinical trials have demonstrated that MSCs which MSCs are being evaluated include Al-
can be used successfully to repair various bone zheimer disease, Parkinson disease, spinal
defects, including critical size defects in the cord injury, amyotrophic lateral sclerosis, and
long bone, hard palate reconstruction, and multiple sclerosis.93-96 The safety and efficacy
calvarial defects.82-84 of intravenous autologous BM-MSC infusion
in patients with severe middle cerebral artery
Cardiovascular and Cerebral Diseases ischemic stroke was evaluated in a 5-year fol-
low-up study in which the mortality rate of the
MSCs are being investigated for stand-alone treated group was lower than that of the
treatment of ischemic heart diseases as well as control group, and no major side effects were
in combination with coronary artery bypass observed during the follow-up period.97
surgery and left ventricular device implanta-
tion. Early open-label, uncontrolled trials of Inflammation Modulation and Barrier
MSCs and ASCs have had safety-related pri-
Repair
mary endpoints. The APOLLO trial demon-
strated that the intracoronary delivery of au- The antiinflammatory and proendothelial sur-
tologous SVF to patients with acute ST vival activities of MSCs suggest that they might
segment elevation myocardial infarction was have therapeutic utility for diseases involving
safe.85 The complementary PRECISE trial was a systemic inflammation and associated endo-
764 䡲 AABB TECHNICAL MANUAL
thelial barrier dysfunction, such as sepsis, tients with biliary cirrhosis also showed that
acute lung injury, and pancreatitis. Related po- the treatment was safe and improved liver
tentially therapeutic properties in this context function.109 Autologous BM-MSCs were ad-
include immunomodulation; differentiation ministered to 53 patients with liver failure due
into lung epithelial cells; secretion of antimi- to complications of hepatitis B infection, and
crobial peptides; as well as other cytoprotec- patient responses were evaluated at early and
tive factors affecting endothelium, such as ke- late intervals.110 Within 3 weeks of the infu-
ratinocyte growth factor and angiopoietin.98-101 sions, there was a significant improvement in
Preclinical studies in murine models have liver function in treated patients compared to
shown that MSCs can be beneficial in acute the 105 matched patients admitted in the
lung injury induced by lipopolysaccharide, same time frame. Although indices of safety at
pneumonia, or systemic sepsis.102 In one study, the early and late time points (approximately
ASC therapy decreased oxidative stress and 3.5 years) were no different in the treatment
minimized ischemia/reperfusion-induced dam- and control groups, the benefit of the treat-
age to the rat lung.103 Phase I trials of both BM- ment did not persist at the late time point.
MSCs and ASCs to treat acute respiratory dis-
tress syndrome are under way. Intravenous
UC-MSC therapy has also been shown to alle- CU R R E N T R E S E A RCH A N D
viate fibrosis, improve histologic scores, and DEVE LOP MEN T : FO C U S O N
decrease inflammatory cytokine levels in rats C E L L C U LTU R E A N D H A N D L I N G
with chronic pancreatitis.104 In the context of the positive results and lack of
apparent safety concerns from numerous clin-
Gastrointestinal Diseases ical studies conducted on MSCs to date, there
Intestinal diseases are an additional category are many issues that must be routinely
of disorders for which MSC treatment is cur- addressed before regulatory approval and
rently undergoing investigation. The largest widespread adoption of MSCs in the clinical
number of trials (11) address Crohn’s disease. setting. For many culture protocols, animal-
In a Phase I trial of 10 patients with fistulizing derived supplements, such as bovine serum
Crohn’s disease, local injections of BM-MSCs (which is also commonly included in preserva-
resulted in complete fistula closure in seven tion media), have historically been used as a
patients and partial closure in three.105 In 2012, source of growth factors and to facilitate post-
a Phase III trial investigating the safety and ef- thaw cell culture. However, the use of bovine
ficacy of allogeneic ASCs for the treatment of serum and the xenogeneic proteins it contains
complex perianal fistulas in patients with could stimulate immune reactions in the host.
Crohn’s disease was initiated.106 In addition, bovine serum must be obtained
Treatment of end-stage, chronic liver dis- from sources that minimize the risk of trans-
eases, primarily cirrhosis, using MSCs is un- mitting prions and other as-yet-unidentified
der active investigation. A Phase I/II clinical zoonotic diseases.111,112 Furthermore, DMSO’s
study, in which eight patients with end-stage toxicity at concentrations currently used to
liver disease were treated with predifferentiat- cryopreserve MSCs along with the undesirable
ed autologous BM-MSCs injected into either osmotic shock to the cells during its addition
the portal vein or peripheral vein, showed sig- and removal may render DMSO undesirable to
nificant improvement in liver function and preserve MSCs intended for clinical use.
clinical parameters.107 A placebo-controlled Considerable effort has been invested in
trial of 30 patients with decompensated liver identifying substitutes for bovine serum and
cirrhosis demonstrated that systemic infu- DMSO; both of these agents must be depleted
sions of UC-MSCs were safe and improved liv- by washing the cells upon thawing and before
er function at 1 year.108 An open-label trial of injection.69-73 For example, there has been con-
peripheral venous delivery of UC-MSCs to pa- siderable effort directed at developing serum-
CHAPTER 31 Tissue-Derived Stem Cells 䡲 765
free, or at least animal-serum-free, cryopreser- and has been calculated to occur at a frequen-
vation media containing cell protectants oth- cy of 10-9.128 There has been at least one report
er than DMSO.113,114 Strategies for eliminating of MSC transformation in vivo to form tu-
or reducing the exposure of MSCs to DMSO mors.19 These studies emphasize the need to
and technologies to remove DMSO from thoroughly test highly expanded MSCs to be
thawed cells have been proposed in the litera- used in clinical trials for genetic alterations.
ture but rarely implemented in clinical set- These results also may suggest that, whenever
tings. possible, lower-passage MSCs, or ASCs and
An important consideration that has re- umbilical MSCs, should be used because these
strained efforts to alter the growth conditions cells require less expansion due to their much
of MSCs is that such modifications might alter greater availability after their initial recovery
the phenotype, genetic stability, or subsequent from tissue.
biological properties of MSCs.115-118 Alterations Tumorigenesis may also be promoted by
that could influence these properties are not the recruitment of MSCs to the tumor stroma.
limited to serum components because micro- There are conflicting reports regarding wheth-
nutrient levels can also influence genomic sta- er human MSCs support tumor formation. A
bility and biological properties.119 A particular number of investigations have shown that
concern regarding the modification of media MSCs enhance tumor growth, whereas others
constituents is that much of the preclinical have reported that MSCs are associated with
and clinical data that underlie our knowledge tumor suppression.130-141 These discrepant re-
regarding the potential benefits and safety of sults may be due to the different experimental
MSCs may not be directly relevant to cells systems used to assess tumor promotion. In
grown in medium containing bovine serum addition, the conditions for each MSC prepa-
substitutes.120 The absence of continuity be- ration might have differed in substantial ways
tween the extensive amount of previous pre- that influenced the outcomes.
clinical data on MSC safety and efficacy may Given this still-evolving understanding, it
increase the regulatory burden for obtaining is important to assess each MSC preparation
licensing to market MSCs as drugs. for tumorigenic potential and carefully con-
In addition to addressing issues of manu- sider the exclusion of patients with active or
facturing and storage, the adequate demon- recent cancer diagnoses in the context of clini-
stration that exogenously administered MSCs cal applications.
possess an exceedingly low potential to form
tumors or promote endogenous tumor forma-
CO N C LUS IO N S A N D F U T U R E
tion has been required to obtain regulatory ap-
DIRE CTIONS
proval for MSCs. Published reports indicate a
lack of consensus on the tumor-formation po- It is evident that the field of cell-based thera-
tential of multipotent MSCs. Long-term cul- pies holds great promise and is on the cusp of
tures of MSCs have been reported to be associ- widespread commercialization, which will
ated with genomic instability, resulting in the have an extensive impact on the health-care
accumulation of genetic alterations that have field in the coming years. Today, the potential
the potential to become manifest as malignant market value of stem cell therapy has been es-
transformation.121-123 Conversely, other studies timated at $5.1 billion with adult stem cells
have found no indication of chromosomal ab- holding a majority share (more than 80%) of
errations with extended cultures of MSCs and the market.142
ASCs.124-127 These opposing findings may be An important concern revolves around
due to subtle differences in the MSC cultures the potential expense of both autologous and
examined, culture conditions, or methods for allogeneic cell-based therapies. Much emphasis
detecting genetic abnormalities. over the last several years has focused on off-the-
Regardless, tumor formation resulting shelf allogeneic cell therapy products, which are
from MSC or ASC treatment is indeed very rare considered to be more suitable for clinical
766 䡲 AABB TECHNICAL MANUAL
adoption and successful commercialization. Finally, the successful use and ultimate
However, allogeneic therapies may be chal- potential for widespread adoption of these
lenging to produce cost-effectively due to the therapies will be affected by regulatory re-
complexities involved in their manufacturing quirements for the production and marketing
and storage. For allogeneic use, cells from a sin- of any cell-based therapies, including issues
gle qualified source must be massively expanded ranging from product characterization to safe-
and stored for a long time to treat large num-
ty testing and clinical trial design. As the field
bers of patients. Because successful commer-
continues to mature, the regulatory pathway
cialization is heavily influenced by costs, the
and its challenges are becoming progressively
manufacturing and banking of these massive
number of cells in a safe, robust, and cost- refined. It is hoped that this evolution will con-
effective manner while preserving key thera- tinue to lead to the establishment of a better-
peutic properties will be critical. These param- defined path for permitting the field to test the
eters are significantly affected by the source of promise of cell therapy more efficiently and
material, isolation and manufacturing meth- meet the expected future demands for these
ods, safety, banking, shipping, and tracking. treatments.
KEY POINTS
1. Multipotent mesenchymal stem cells (MSCs) can be harvested from a diverse array of tis-
sues, including marrow, adipose tissue, reproductive organs, placenta, dental pulp, and
skeletal muscle.
2. The hallmarks of pluripotentiality and multipotentiality distinguish stem cells from progen-
itor cells.
3. The beneficial properties of these therapeutic cells appears to be more related to transitory
effects produced by secreted substances or brief contact with target cells rather than direct
tissue replacement.
4. The key complementary properties that are of potential clinical use are immunomodula-
tion, paracrine effects, and differentiation.
5. The method of MSC isolation usually involves enzymatic dissociation, filtration, and low-
speed centrifugation.
6. Early clinical experience suggests that cellular therapies are safe and effective; however, lon-
ger-term studies are required to fully assess the durability of these effects and the potential
for tumorigenicity.
7. Key to future commercial success will be developing well-characterized and consistent
therapeutic cell products through establishing strict controls for manufacturing, storage,
and supply processes.
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col 2009;116:166-72. Marrow Transplant 2000;25:1299-301.
59. Kanafi MM, Pal R, Gupta PK. Phenotypic and 72. Higman MA, Port JD, Beauchamp NJ Jr, Chen
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800.
60. Tirino V, Paino F, De Rosa A, Papaccio G. Iden-
73. Alessandrino P, Bernasconi P, Caldera D, et al.
tification, isolation, characterization, and
Adverse events occurring during bone marrow
banking of human dental pulp stem cells.
or peripheral blood progenitor cell infusion:
Methods Mol Biol 2012;879:443-63.
Analysis of 126 cases. Bone Marrow Transplant
61. Gronthos S, Arthur A, Bartold PM, Shi S. A
1999;23:533-7.
method to isolate and culture expand human
74. Polchert D, Sobinsky J, Douglas G, et al. IFN-
dental pulp stem cells. Methods Mol Biol
gamma activation of mesenchymal stem cells
2011;698:107-21.
for treatment and prevention of graft versus
62. Stangel-Wojcikiewicz K, Jarocha D, Piwowar
host disease. Eur J Immunol 2008;38:1745-55.
M, et al. Autologous muscle-derived cells for 75. Sudres M, Norol F, Trenado A, et al. Bone mar-
the treatment of female stress urinary inconti- row mesenchymal stem cells suppress lym-
nence: A 2-year follow-up of a Polish investiga- phocyte proliferation in vitro but fail to pre-
tion. Neurourol Urodyn 2014 (in press). vent graft-versus-host disease in mice.
63. Ott HC, Davis BH, Taylor DA. Cell therapy for J Immunol 2006;176:7761-7.
heart failure—Muscle, bone marrow, blood, 76. Christensen ME, Turner BE, Sinfield LJ, et al.
and cardiac-derived stem cells. Semin Thorac Mesenchymal stromal cells transiently alter
Cardiovasc Surg 2005;17:348-60. the inflammatory milieu post-transplant to
64. Wu X, Wang S, Chen B, An X. Muscle-derived delay graft-versus-host disease. Haematologi-
stem cells: isolation, characterization, differ- ca 2010;95:2102-10.
entiation, and application in cell and gene 77. McGuirk JP, Weiss ML. Promising cellular ther-
therapy. Cell Tissue Res 2010;340:549-67. apeutics for prevention or management of
65. Gharaibeh B, Lu A, Tebbets J, et al. Isolation of graft-versus-host disease (a review). Placenta
a slowly adhering cell fraction containing stem 2011;32(Suppl 4):S304-10.
cells from murine skeletal muscle by the pre- 78. Lim JH, Lee MH, Yi HG, et al. Mesenchymal
plate technique. Nat Protoc 2008;3:1501-9. stromal cells for steroid-refractory acute graft-
66. George B. Regulations and guidelines govern- versus-host disease: A report of two cases. Int J
ing stem cell based products: Clinical consid- Hematol 2010;92:204-7.
erations. Perspect Clin Res 2011;2:94-9. 79. Prasad VK, Lucas KG, Kleiner GI, et al. Efficacy
67. Thirumala S, Goebel WS, Woods EJ. Clinical and safety of ex vivo cultured adult human
grade adult stem cell banking. Organogenesis mesenchymal stem cells (prochymal) in pedi-
2009;5:143-54. atric patients with severe refractory acute
770 䡲 AABB TECHNICAL MANUAL
epithelial protein permeability in cultured hu- parison of functional characteristics for cell
man alveolar type II cells by secretion of an- lines cultured in medium without and with fe-
giopoietin-1. J Biol Chem 2010;285:26211-22. tal calf serum. J Cancer Res Clin Oncol 2005;
101. Lee JW, Fang X, Gupta N, Serikov V, Matthay 131:377-84.
MA. Allogeneic human mesenchymal stem 112. Lange C, Cakiroglu F, Spiess AN, et al. Acceler-
cells for treatment of E. coli endotoxin-in- ated and safe expansion of human mesenchy-
duced acute lung injury in the ex vivo perfused mal stromal cells in animal serum-free medi-
human lung. Proc Natl Acad Sci U S A 2009; um for transplantation and regenerative
106:16357-62. medicine. J Cell Physiol 2007;213:18-26.
102. Hayes M, Curley G, Laffey JG. Mesenchymal 113. Woods EJ, Perry BC, Hockema JJ, et al. Opti-
stem cells—A promising therapy for acute re- mized cryopreservation method for human
spiratory distress syndrome. F1000 Med Rep dental pulp-derived stem cells and their tis-
2012;4:2. sues of origin for banking and clinical use.
103. Sun CK, Yen CH, Lin YC, et al. Autologous Cryobiology 2009;59:150-7.
transplantation of adipose-derived mesenchy- 114. Thirumala S, Gimble JM, Devireddy RV. Cryo-
mal stem cells markedly reduced acute isch- preservation of stromal vascular fraction of
emia-reperfusion lung injury in a rodent mod- adipose tissue in a serum-free freezing medi-
el. J Transl Med 2011;9:118. um. J Tissue Eng Regen Med 2010;4:224-32.
104. Zhou CH, Li ML, Qin AL, et al. Reduction of fi- 115. Bieback K, Ha VA, Hecker A, et al. Altered gene
brosis in dibutyltin dichloride-induced chron- expression in human adipose stem cells cul-
ic pancreatitis using rat umbilical mesenchy- tured with fetal bovine serum compared to hu-
mal stem cells from Wharton’s jelly. Pancreas man supplements. Tissue Eng Part A 2010;16:
2013;42:1291-302. 3467-84.
105. Ciccocioppo R, Bernardo ME, Sgarella A, et al. 116. Abdelrazik H, Spaggiari GM, Chiossone L,
Autologous bone marrow-derived mesenchy- Moretta L. Mesenchymal stem cells expanded
mal stromal cells in the treatment of fistulising in human platelet lysate display a decreased
Crohn’s disease. Gut 2011;60:788-98. inhibitory capacity on T- and NK-cell prolifer-
106. TiGenix. Cx601 Phase III in perianal fistulas ation and function. Eur J Immunol 2011;41:
enrolls first patients. Leuven, Belgium: TiGe 3281-90.
nix, 2012. [Available at http://www.tigenix. 117. Goedecke A, Wobus M, Krech M, et al. Differ-
com/en/page/4/company (accessed Decem- ential effect of platelet-rich plasma and fetal
ber 9, 2013).] calf serum on bone marrow-derived human
107. Kharaziha P, Hellstrom PM, Noorinayer B, et al. mesenchymal stromal cells expanded in vitro.
Improvement of liver function in liver cirrho- J Tissue Eng Regen Med 2011;5:648-54.
sis patients after autologous mesenchymal 118. Tonti GA, Mannello F. From bone marrow to
stem cell injection: A phase I-II clinical trial. therapeutic applications: Different behaviour
Eur J Gastroenterol Hepatol 2009;21:1199-205. and genetic/epigenetic stability during mes-
108. Zhang Z, Lin H, Shi M, et al. Human umbilical enchymal stem cell expansion in autologous
cord mesenchymal stem cells improve liver and foetal bovine sera? Int J Dev Biol 2008;
function and ascites in decompensated liver 52:1023-32.
cirrhosis patients. J Gastroenterol Hepatol 119. Arigony AL, de Oliveira IM, Machado M, et al.
2012;27(Suppl 2):112-20. The influence of micronutrients in cell culture:
109. Wang L, Li J, Liu H, et al. A pilot study of umbil- A reflection on viability and genomic stability.
ical cord-derived mesenchymal stem cell Biomed Res Int 2013;2013:597282.
transfusion in patients with primary biliary 120. Krampera M, Galipeau J, Shi Y, et al. Immuno-
cirrhosis. J Gastroenterol Hepatol 2013;28 logical characterization of multipotent mesen-
(Suppl 1):85-92. chymal stromal cells—The International Soci-
110. Peng L, Xie DY, Lin BL, et al. Autologous bone ety for Cellular Therapy (ISCT ) working
marrow mesenchymal stem cell transplanta- proposal. Cytotherapy 2013;15:1054-61.
tion in liver failure patients caused by hepatitis 121. Rubio D, Garcia-Castro J, Martin MC, et al.
B: Short-term and long-term outcomes. Hepa- Spontaneous human adult stem cell transfor-
tology 2011;54:820-8. mation. Cancer Res 2005;65:3035-9.
111. Bruserud O, Tronstad KJ, Berge R. In vitro cul- 122. Wang Y, Huso DL, Harrington J, et al. Out-
ture of human osteosarcoma cell lines: A com- growth of a transformed cell population de-
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rived from normal human BM mesenchymal chymal stromal cells. Molecular Can 2010;
stem cell culture. Cytotherapy 2005;7:509-19. 9:129.
123. Rosland GV, Svendsen A, Torsvik A, et al. Long- 133. Prantl L, Muehlberg F, Navone NM, et al. Adi-
term cultures of bone marrow-derived human pose tissue-derived stem cells promote pros-
mesenchymal stem cells frequently undergo tate tumor growth. Prostate 2010;70:1709-15.
spontaneous malignant transformation. Can- 134. Shinagawa K, Kitadai Y, Tanaka M, et al. Mes-
cer Res 2009;69:5331-9. enchymal stem cells enhance growth and me-
124. Bernardo ME, Zaffaroni N, Novara F, et al. Hu- tastasis of colon cancer. Int J Cancer 2010;
man bone marrow derived mesenchymal stem 127:2323-33.
cells do not undergo transformation after 135. Khakoo AY, Pati S, Anderson SA, et al. Human
long-term in vitro culture and do not exhibit
mesenchymal stem cells exert potent antitu-
telomere maintenance mechanisms. Cancer
morigenic effects in a model of Kaposi’s sarco-
Res 2007;67:9142-9.
ma. J Exp Med 2006;203:1235-47.
125. Avanzini MA, Bernardo ME, Cometa AM, et al.
136. Zhu Y, Sun Z, Han Q, et al. Human mesenchy-
Generation of mesenchymal stromal cells in
the presence of platelet lysate: A phenotypic mal stem cells inhibit cancer cell proliferation
and functional comparison of umbilical cord by secreting DKK-1. Leukemia 2009;23:925-33.
blood- and bone marrow-derived progenitors. 137. Cousin B, Ravet E, Poglio S, et al. Adult stromal
Haematologica 2009;94:1649-60. cells derived from human adipose tissue pro-
126. Tarte K, Gaillard J, Lataillade JJ, et al. Clinical- voke pancreatic cancer cell death both in vitro
grade production of human mesenchymal and in vivo. PloS One 2009;4:e6278.
stromal cells: Occurrence of aneuploidy with- 138. Maestroni GJ, Hertens E, Galli P. Factor(s) from
out transformation. Blood 2010;115:1549-53. nonmacrophage bone marrow stromal cells
127. Grimes BR, Steiner CM, Merfeld-Clauss S, et inhibit Lewis lung carcinoma and B16 melano-
al. Interphase FISH demonstrates that human ma growth in mice. Cell Mol Life Sci 1999;55:
adipose stromal cells maintain a high level of 663-7.
genomic stability in long-term culture. Stem 139. Lu YR, Yuan Y, Wang XJ, et al. The growth in-
Cells Dev 2009;18:717-24. hibitory effect of mesenchymal stem cells on
128. Prockop DJ, Brenner M, Fibbe WE, et al. Defin- tumor cells in vitro and in vivo. Cancer Biol
ing the risks of mesenchymal stromal cell ther- Ther 2008;7:245-51.
apy. Cytotherapy 2010;12:576-8. 140. Dasari VR, Kaur K, Velpula KK, et al. Upregula-
129. Rodriguez R, Rubio R, Masip M, et al. Loss of tion of PTEN in glioma cells by cord blood
p53 induces tumorigenesis in p21-deficient
mesenchymal stem cells inhibits migration via
mesenchymal stem cells. Neoplasia 2009;11:
downregulation of the PI3K/Akt pathway. PloS
397-407.
One 2010;5:e10350.
130. Galie M, Konstantinidou G, Peroni D, et al.
141. Secchiero P, Zorzet S, Tripodo C, et al. Human
Mesenchymal stem cells share molecular sig-
nature with mesenchymal tumor cells and fa- bone marrow mesenchymal stem cells display
vor early tumor growth in syngeneic mice. On- anti-cancer activity in SCID mice bearing dis-
cogene 2008;27:2542-51. seminated non-Hodgkin’s lymphoma xeno-
131. Lin G, Yang R, Banie L, et al. Effects of trans- grafts. PloS One 2010;5:e11140.
plantation of adipose tissue-derived stem cells 142. Mason C, Brindley DA, Culme-Seymour EJ, et
on prostate tumor. Prostate 2010;70:1066-73. al. Cell therapy industry: Billion dollar global
132. Kucerova L, Matuskova M, Hlubinova K, et al. business with unlimited potential. Regen Med
Tumor cell behaviour modulation by mesen- 2011;6:265-72.
C h a p t e r 3 2
Lance D. Trainor, MD, Divisional Medical Director, LabCorp, Dublin, Ohio, and Rita A. Reik, MD, Chief Medi-
cal Officer, OneBlood, Lauderhill, Florida
The authors have disclosed no conflicts of interest.
773
774 䡲 AABB TECHNICAL MANUAL
about the donor’s travel and medical history cessing, human tissue allografts may be com-
and any high-risk behaviors, 2) available medi- bined with other biocompatible agents to
cal records, 3) the autopsy report (if an autopsy achieve desired handling and functional char-
was performed), 4) a thorough physical assess- acteristics. Bone allografts, due to their hard
ment of the donor to identify evidence of high- and rigid nature, can be precisely machined
risk behaviors or active communicable disease, for compatibility with surgical instrumenta-
5) the suitability of any blood sample collected tion.
for infectious disease testing, and 6) the cir- Autografts are tissues implanted into the
cumstances of death. A history of high-risk ac- individual from whom they were removed. For
tivity that increases the possibility of transmis-
example, bone that has been surgically re-
sion of disease leads to the rejection of the
moved from a patient’s ilium can be shaped to
donor. Allografts, similar to blood products,
desired dimensions and implanted into the
are released for transplantation only after the
donor has been tested for relevant communi- vertebral disk space of the same patient. The
cable diseases and the results are determined autograft bone promotes fusion of adjacent
to be acceptable (see Table 32-1). vertebrae, providing stability and relief from
the pain of degenerative disc disease or trau-
Background and Definitions ma. Advantages of autografts include the elim-
ination of communicable disease transmis-
Human tissue banking in the United States sion risk and the ready availability of graft
started more than 60 years ago at the US Navy
material. Disadvantages include morbidity as-
Tissue Bank. The use of tissue allografts, now
sociated with the additional surgical proce-
common, has evolved into a highly innovative
dure for the patient, including pain and poten-
and continuously changing field. Collabora-
tion between tissue bank scientists and end- tial surgical-site infection. In addition, the
user surgeons has produced a wide variety of quality (eg, strength) and quantity of autolo-
life-saving and life-enhancing tissue grafts. gous tissue may not be adequate for the in-
Allografts are tissues transferred between tended use, and removal of the patient’s tissue
individuals of the same species. Allografts may may adversely affect function at the site from
be processed to remove cells or carefully pre- which it was removed.
served to maintain cellular viability. They can Isografts, which are tissues transferred be-
be derived from a single tissue or multiple tis- tween genetically identical individuals, such
sues acting as a functional unit. During pro- as identical twins, are uncommonly used.
Xenografts are tissues transplanted from steady, controlled rate. The use of cryoprotec-
one species to another; a growing number of tants, such as glycerol or dimethyl sulfoxide,
medical products are being developed from minimizes cell damage caused by cell shrink-
highly processed nonhuman animal tissues. age and intracellular ice formation during
freezing.
Tissue Processing Refrigerated storage can preserve cellular
viability in osteochondral allografts for which
After tissue has been procured from a de-
preservation of living chondrocytes is essen-
ceased donor, it is processed using aseptic
tial. These grafts, which consist of an intact ar-
technique in a controlled environment in a fa-
ticular surface with associated soft tissue and
cility designed to prevent contamination or
bone, are used for treating traumatic and de-
cross-contamination. Similar to good manu-
generative joint conditions. Refrigeration is
facturing practice, good tissue practice (GTP)
not suitable for long-term storage of allografts.
requires the facility to establish and maintain
controls over temperature, humidity, ventila-
Clinical Uses of Allografts
tion, and air filtration. Thorough cleaning and
disinfection of processing rooms and equip- Allografts are used in a variety of surgical pro-
ment are required to ensure a proper environ- cedures. Cadaveric human bone can be used
ment. to replace bone lost to degenerative disease,
Tissues are debrided of extraneous soft trauma, or malignancy. Allogeneic human
tissue and cut to specification. In some cases, bone has unique healing characteristics, in-
more than 100 grafts can be produced from a cluding osteoconductive and osteoinductive
single donor. Precision bone grafts, including a properties. In vivo, it acts as a scaffold that al-
growing array of spinal implants, are crafted lows recipient capillary growth into the graft
using sophisticated, computer-aided cutting (osteoconductivity) and provides stimulation
devices to meet the exacting specifications re- for the production of new bone (osteoinduc-
quired by surgical instrumentation; precisely tivity) by exposing the patient’s osteogenic
milled allografts allow surgeons to operate progenitor cells to bone morphogenetic pro-
more quickly and efficiently. teins (BMPs), growth factors in bone that in-
During processing, various solutions, in- duce new bone formation. The result is creep-
cluding antibiotics, alcohols, and surfactants, ing substitution, in which bone remodeling
may be used to reduce or eliminate bacterial occurs through osteoclastic resorption of the
contamination and remove extraneous lipids implanted tissue and osteoblastic generation
and other biologic material. Depending on the of new bone.
type of graft, graft sterilization may or may not A variety of human tissues are used for
be possible; grafts containing viable cells or a transplantation.4 Selected clinical uses are list-
fragile matrix cannot be subjected to steriliza- ed in Table 32-2. Bone-tendon (Achilles ten-
tion methods without destruction of cellular don) or bone-ligament-bone (patellar-tibia
or matrix integrity. Ionizing radiation is the ligament) grafts are routinely used for anterior
most frequently used sterilization technique. cruciate ligament repair. The implanted ten-
Ethylene oxide and proprietary methods of tis- don or ligament spans the joint space, and the
sue sterilization may also be used by tissue bone provides an anchor into the femur and/
processors. or tibia, restoring joint stability. Crushed bone
Several methods of tissue preservation subjected to acid demineralization can be
are available for long-term storage of human used alone or suspended in a biologically
allografts, including freezing and lyophiliza- compatible carrier and applied to exposed
tion (freeze drying). Both processes destroy bone surfaces. BMPs in demineralized bone
cell viability. Tissue integrity can be enhanced stimulate osteogenesis, fusion of adjacent
through cryopreservation, a process in which bones, and healing. Cadaveric skin can be
tissues are frozen in a protective medium at a used as a temporary wound dressing for severe
776 䡲 AABB TECHNICAL MANUAL
burns, protecting underlying tissues from de- cilities. Advances in screening, testing, and
hydration and environmental pathogens. Hu- processing methods continue to improve the
man skin can be processed to remove cellular safety profiles of human tissue products.
elements, producing an acellular collagen ma-
trix that provides a scaffold for revasculariza-
R E G U L AT I O NS A ND
tion and cellular incorporation in soft tissue
reconstructive surgery. In addition, donor tis- S TA N D A R D S
sues can be used to treat a variety of ocular The Food and Drug Administration (FDA) reg-
conditions. Thinning, scarring, or clouding of ulates the activities of tissue banks and tissue
the cornea may be treated by corneal trans- distribution intermediaries, which are estab-
plantation. Sclera and amnion-derived al- lishments or persons engaged in the recovery,
lografts can be used to treat glaucoma, scleral screening, testing, labeling, processing, stor-
ulcers, and traumatic injuries. age, and/or distribution of human tissues for
Although bone and soft tissue allografts clinical use, under Title 21, Parts 1270 and
may provoke a recipient immune response, 1271 of the Code of Federal Regulations
such responses are not usually clinically signif- (CFR).3 These entities manufacture what the
icant, presumably due to a lack of residual cel- FDA classifies as human cells, tissues, and cel-
lular material in processed grafts.5 Therefore, it lular and tissue-based products (HCT/Ps). Ex-
is not necessary to match most allografts to the amples of common tissue products are listed
recipient’s HLA or ABO type. Possible excep- in Table 32-3.
tions include cryopreserved veins and arteries, The three rules of 21 CFR Part 1271 con-
for which some clinicians request ABO com- cern 1) registration of tissue bank establish-
patibility, and frozen, unprocessed bone al- ments, 2) donor eligibility, and 3) GTP related
lografts containing marrow or red cells. Devel- to handling HCT/Ps. Compliance with current
opment of Rh(D), Fy(a), and Jk(b) antibodies GTP regulations is required of tissue banks and
in recipients following transplantation of un- tissue distribution intermediaries to control
processed bone allografts has been document- contamination and cross-contamination that
ed.6 If unprocessed bone is to be used for an may lead to transmission of disease. Tissue-
Rh(D)-negative female of childbearing poten- dispensing institutions, such as hospitals, den-
tial, her future offspring may be at risk of he-
tal offices, and surgical centers that provide
molytic disease of the fetus and newborn. Rh
and use tissue within their own facility are not
Immune Globulin prophylaxis should be con-
subject to this oversight except under certain
sidered if the Rh type of the red cells or marrow
circumstances (ie, redistribution of allografts
contained in the allograft is positive or un-
or autografts to affiliated institutions located
known.
at a different address or attempts to sterilize
autografts).
Disease Transmission through Tissue
For a hospital–based tissue-dispensing
Transplantation service, regulatory oversight is governed by
Rare, sporadic transmissions of infectious dis- voluntary accrediting organizations. Title 21,
eases, including human immunodeficiency vi- CFR Parts 1270 and 1271 do not apply to facili-
rus (HIV), hepatitis C virus (HCV), hepatitis B ties that only receive, store, and dispense tis-
virus (HBV), and Creutzfeldt-Jakob disease sue, and engage in no further manufacturing
(CJD), have been documented following tissue or redistribution of the tissue product. Howev-
implantation.7 In addition, bacterial and fun- er, The Joint Commission, AABB, College of
gal infections from allografts have resulted in American Pathologists (CAP), Association of
morbidity and death. Potential sources of periOperative Registered Nurses (AORN),
contaminating agents include donor flora, AATB, and Eye Bank Association of America
premortem infection, and environmental (EBAA) all publish standards that apply to the
contaminants in recovery and processing fa- practices of tissue-dispensing services.8-13 The
778 䡲 AABB TECHNICAL MANUAL
TABLE 32-3. Examples of Common Cell and are safe, available, and of high quality. AATB’s
Tissue Products8,14 standards pertain to institutional and quality
program requirements; donor authorization/
Amniotic membrane informed consent; donor screening and test-
Bone and demineralized bone matrix ing; and tissue recovery, processing, release,
and distribution.12 EBAA’s scope encompass-
Bone marrow
es all aspects of eye banking.13 Accreditation
Bone paste, powder, or putty by these organizations is based on verified
Cancellous chips compliance with established standards and
periodic inspections. Both organizations
Cardiac (heart) valves serve as scientific and educational resources
Cartilage for the donation and transplantation com-
munities. A tissue-dispensing service may
Cornea
find AATB and EBAA accreditation of a sup-
Dermal matrix plier to be valuable in assessing that suppli-
Dura mater er’s qualifications.
Embryos
H O SPI TA L T I SS U E S E RVI C E S
Fascia
There is no requirement for a tissue-dispens-
Hematopoietic stem cells
ing service to be managed in any particular de-
Ligaments partment or by any specific individual. The
Meniscus AABB supports a tissue-dispensing service
within the transfusion service, which has ex-
Ocular tissues
pertise in providing human-derived products
Oocytes that are perishable, potentially infectious, and
sometimes in short supply and that require
Pericardium
temperature-controlled storage. The tasks of
Peripheral blood stem cells ordering, receiving, storing, distributing (issu-
Sclera ing), tracking, and tracing products as well as
investigating adverse events, including com-
Semen and sperm plaints, recalls, and look-back investigations,
Skin are activities that are common to the transfu-
sion service and tissue-dispensing service.
Tendons
Umbilical cord blood stem cells Responsibility for Hospital-Based
Vascular grafts (veins and arteries) Tissue Services
The Joint Commission requires that organiza-
tions assign oversight responsibility for their
location of a tissue-dispensing service in a tissue program, use standardized procedures
hospital or medical facility and the scope of its in tissue handling, maintain traceability of all
responsibilities dictate which standards are tissues, and have a process for investigating
applicable. All of these standards are updated and reporting adverse events. Either a central-
regularly; therefore, a periodic review of the ized or decentralized process is permitted to
most recent versions is required to guarantee manage these activities. In either model, des-
ongoing compliance. ignated oversight is required to coordinate tis-
The AATB and EBAA are voluntary accred- sue-related activities and ensure standardiza-
iting organizations dedicated to ensuring that tion of practices throughout the organization.
human tissues intended for transplantation The Joint Commission standards apply to
CHAPTER 32 Tissue Services in the Hospital 䡲 779
human and nonhuman cellular-based trans- dures to receive or monitor evidence of com-
plantable and implantable products, including pliance or noncompliance, such as reviewing
both tissue allografts and certain medical de- FDA warning letters and tissue allograft recalls
vices, as classified by the FDA. or market withdrawals. Accreditation by AATB
and/or EBAA may be desirable.
Written Standard Operating Qualification information for each sup-
Procedures plier should be reviewed and approved annu-
ally by the hospital tissue service. During these
Hospital tissue services must have written pro- reviews, the performance of suppliers in meet-
cedures (printed or electronic) for all functions ing the transplant facility’s needs should be
pertaining to the acquisition, receipt, storage, evaluated. Each year, the tissue service should
issuance, and tracing of tissue grafts as well as determine whether the supplier remains regis-
procedures for investigating adverse events tered with the FDA; whether AATB and/or
and handling recalls. Manufacturers’ instruc- EBAA accreditation is current can also be con-
tions for handling tissues should be incorpo- firmed. FDA web postings should be reviewed
rated into standard operating procedures for information related to closures, recalls, or
(SOPs). When the blood bank or transfusion MedWatch reports. FDA inspection reports on
service is responsible for tissue, the AABB re- tissue processors can be requested from the
quires the medical director to approve all FDA through the Freedom of Information Act.
medical and technical policies and procedures Complaints from transplanting surgeons con-
pertaining to tissue.9(p2) cerning the supplier’s tissue should be re-
viewed along with any reports of infections
Tissue Supplier Qualifications and that might have been caused by transplanted
Certification allografts. Further use of a particular tissue
supplier should depend on approval by the tis-
In contrast to blood banks, tissue processors
sue service’s director. Hospital management
and distributors often specialize in providing
may consider establishing a committee of in-
particular types of products (grafts). As a re-
ternal stakeholders, including transplant phy-
sult, a hospital tissue-dispensing service may
sicians, to provide oversight in the approval of
need to acquire human tissue products from
tissue suppliers.
more vendors than the number of vendors
from which a transfusion service commonly
Inspection of Incoming Tissue
obtains blood products.
Tissue suppliers should be selected based Allografts
on their ability to reliably provide high-quality Before being placed in storage, tissue allografts
tissues that meet expectations for availability, must be inspected upon receipt from a tissue
safety, and effectiveness. The tissue service supplier to ensure that the packaging remains
should establish minimal criteria for the quali- intact and the label is complete, appears to be
fication of prospective suppliers. According to accurate, and is adequately affixed and legible
The Joint Commission, tissue supplier require- before acceptance into inventory. The incom-
ments must include evidence of current FDA ing inspection results should be recorded
registration and state licensure if such licen- along with the date, time, and name of the staff
sure is required. A written process for review person who conducted the inspection.
and approval of suppliers is expected and The Joint Commission requires that hos-
should contain the elements listed in Table 32- pitals verify package integrity and ensure that
4. A list of approved suppliers that includes the transport temperature was controlled and
documentation of each supplier’s qualifica- acceptable. Inspection of the shipping con-
tions, certifications, and appropriate licenses tainer for evidence of residual coolant (eg, wet
or permits should be developed and main- ice for refrigerated grafts or dry ice for frozen
tained. Tissue services should establish proce- grafts) may be useful to determine that the re-
780 䡲 AABB TECHNICAL MANUAL
Criterion Documentation/Performance
erators and freezers is required. Room-tem- records of these steps. The records should be
perature storage equipment need only be accurate, legible, and indelible. They must
monitored if this is required by the allograft’s identify the staff who have handled the tissue
package insert. Storage equipment should and the dates when the tissue was handled as
have functional alarms and emergency back- well as the time when the tissue was accepted,
up capability. Lyophilized tissues whose pack- prepared, or processed. The records should be
age insert instructions specify ambient tem-
detailed and provide a clear history of all ac-
perature or colder storage can tolerate a very
tions performed. Documentation of the tissue
broad range of temperatures and their temper-
atures do not require monitoring. supplier, unique numeric or alphanumeric
Storage SOPs should address steps to be identifier(s) of the allograft, its expiration date,
taken in case of excursions from allowable and the recipient’s name must be maintained
temperature limits or in the event of equip- for all tissue grafts used.
ment or power failure. Emergency backup al- Tissue service records need to permit bi-
ternatives, including arrangements for tempo- directional traceability of all tissues from the
rary storage, should be described in written donor and tissue supplier to the recipient(s) or
procedures. other final disposition, including the discard
of tissue because of damage to package integ-
Tissue Allograft Traceability and rity, opening of the tissue package in the oper-
Record Keeping ating room without use, or expiration. Records
Proper management of human allografts re- should be retained for 10 years, or longer if re-
quires that the hospital tissue service docu- quired by state or federal law, after distribu-
ment all of the steps taken in tissue handling tion, transplantation, discard, or expiration
as they occur and maintain comprehensive (whichever occurs last).
782 䡲 AABB TECHNICAL MANUAL
Tissue usage information cards or other State health departments have lists of
systems supplied with the allograft by the tis- communicable diseases that must be reported
sue bank must be completed and returned to when they are newly diagnosed. A new diagno-
the tissue source facility. This information sis of HIV or viral hepatitis in a tissue allograft
helps maintain the traceability of the allograft recipient where the allograft is suspected as a
and expedites market withdrawals or recalls possible source must be reported to the state
when necessary. The tissue supplier may also department of health by the patient’s physi-
use this information to better understand al- cian or the director or medical director of the
lograft utilization, obtain positive or negative hospital tissue service. An epidemiologic in-
feedback, and better meet the needs and ex- vestigation may be needed to establish wheth-
pectations of customers. er the tissue allograft was the source of the re-
cipient’s infection. Early notification of the
Recognizing and Reporting Adverse state department of health can result in timely
Events Possibly Caused by Allografts assistance with the investigation.
Use of human-derived medical products, such
as tissue allografts, carries risks that must be
Recalls and Look-Back Investigations
balanced with clinical benefits. Human al- A tissue product recall or market withdrawal
lografts have been associated with bacterial, occurs when a tissue allograft is determined by
viral, fungal, and prion transmissions. In addi- the tissue supplier to be compromised or po-
tion, allografts may have structural defects tentially infectious. The supplier may recall all
that sometimes lead to unsuccessful outcomes of the tissues from a specific donor or process-
as a result of donor selection or processing fac- ing lot, sequester tissues in inventory, and no-
tors. tify hospitals to which the allografts were
Hospital tissue services are required to shipped. The hospital may be instructed by
have procedures to investigate in a timely way the supplier to quarantine the allografts in in-
any infections suspected to be caused by a tis- ventory, identify recipients, and/or notify the
sue allograft. The Joint Commission requires transplanting surgeon(s) of the recall. Sur-
that allograft-transmitted infections and other geons should evaluate the circumstances of
severe adverse events be immediately report- the recall and notify the recipient as appropri-
ed by the hospital to the tissue supplier. ate that a tissue graft was recalled.
Transplant surgeons play a critical role in Look-back investigation can be triggered
the identification of allograft-associated ad- when a tissue donor is subsequently found to
verse outcomes and need to immediately noti- have been infected with HIV, human T-cell
fy the hospital tissue service when they sus- lymphotropic virus Types I or II, HBV, HCV, or
pect such events. Prompt notification of other infectious disease known to be transmit-
adverse events enables the hospital tissue ser- ted by tissue grafts. Because most tissues are
vice to investigate the cause, report the issue procured from deceased donors, infectious
to the tissue supplier, and institute corrective disease testing of subsequent donor blood
action, including sequestration of any other samples is not possible. Look-back investiga-
suspect allografts. The investigation of infec-
tions involving tissue grafts are uncommon.
tions and other adverse events requires coop-
eration between the tissue-dispensing service, Tissue Autograft Collection, Storage,
clinicians, and tissue supplier. Consultation
and Use
with the hospital infection-control depart-
ment or an infectious disease specialist may Surgical reconstruction using the patient’s
be beneficial. Early notification can prevent an own tissues often has advantages over the use
untoward outcome for other recipients of al- of cadaveric tissue. These advantages include
lografts from an implicated donor. ready availability, faster incorporation, appro-
CHAPTER 32 Tissue Services in the Hospital 䡲 783
priate size or shape, and relative safety from sion support before, during, and after trans-
viral disease transmission. plantation.
A bone flap removed during decompres- OPOs evaluate potential donors, obtain
sive craniectomy is a commonly used example authorization (consent), and prepare organs
of tissue autograft use. In this procedure, a sec- for transportation. The United Network for Or-
tion of skull is excised by the neurosurgeon to gan Sharing (UNOS) coordinates the US organ
reduce intracerebral pressure caused by brain transplant system. The evolving UNOS guide-
swelling due to trauma, stroke, or surgery. After lines for organ allocations are designed to
removal, the skull fragment is rinsed, achieve equitable distribution of life-saving
packaged, frozen, and stored for future reim- organs. Factors such as severity of recipient ill-
plantation during a procedure known as “cra- ness, geographic proximity, and donor-recipi-
nioplasty.” ent compatibility are considered. UNOS pro-
Written procedures should address the vides detailed policies and other relevant
collection, microbial testing, packaging, stor- information on its website.15
age, and issuance of tissue autografts for reim- Depending on the organ, ABO and/or
plantation. Bacterial testing by obtaining ap- HLA compatibility may be important factors
propriate cultures should be performed after for transplantation success. ABO antigens ex-
surgical removal and before packaging. Auto- pressed on the vascular endothelium of organs
grafts should not be collected from patients constitute strong histocompatibility barriers.
with systemic infections or if the tissue is in Major incompatibility between donor ABO an-
tigens and recipient plasma can result in acute
close proximity to an infected area. Autografts
humoral rejection of the transplanted organ
may be stored at the medical facility where
and threaten a successful outcome. As a result,
they are collected or at an off-site, FDA-regis-
UNOS requires two separate determinations
tered tissue bank. Procedural recommenda-
of ABO type for both 1) transplant candidates
tions have been published by the AATB and
prior to listing their needs on the Organ Pro-
AORN.
curement and Transplantation Network wait-
ing list and 2) donors prior to making an organ
TR ANSF USION SERVICE available for transplant. ABO-incompatible or-
SU P P O RT F O R O RG A N gan transplants are sometimes performed fol-
TR A N SP L AN TAT I O N lowing a conditioning regimen that may in-
clude plasma exchange.
Organ transplantation relies on the coordinat- Services that may be required by a trans-
ed activities of organ-procurement organiza- plant program include provision of cytomega-
tions (OPOs) and the hospital transplant team. lovirus-reduced-risk components, blood irra-
A successful transplant program requires an diation, massive transfusion support, ABO
interdisciplinary team that has well-defined subgroup typing, and immunohematology ref-
policies, procedures, and communication erence testing. Transfusion services should
pathways. The transfusion service provides understand and address such expectations of
appropriate compatibility testing and transfu- the transplant team.
KEY POINTS
1. Surgical use of human allografts has grown dramatically in recent years, with more than
2 million tissue grafts used annually in the United States. The majority of these grafts are ob-
tained from deceased human donors who meet stringent screening requirements similar to
those applied to blood donors.
2. Not all tissue allografts are sterile. Depending on the type of allograft, sterilization may not
be possible because it could jeopardize the viability of the cellular elements or fragile matrix
784 䡲 AABB TECHNICAL MANUAL
of the graft and adversely affect in-vivo performance. Methods of sterilization include the
use of ionizing radiation or ethylene oxide and some proprietary processes and techniques.
3. Rare examples of HIV, HCV, HBV, CJD, and bacterial and fungal disease transmission from
allografts have been documented. Therefore, like blood components, allografts are released
for use only after infectious disease testing has been completed and the results deemed ac-
ceptable.
4. In general, bone and soft tissue allografts do not need to be matched for HLA or ABO type.
5. Hospital-based tissue services are not subject to FDA regulatory oversight if their activities
are limited to receiving, storing, and dispensing tissue to a requesting surgeon. However,
The Joint Commission, AABB, CAP, AORN, AATB, and EBAA publish standards that apply to
such services.
6. Tissue banks are engaged in the recovery, screening, testing, labeling, processing, storage,
and distribution of human tissues. They are regulated by the FDA under the Title 21, CFR
Parts 1270 and 1271 as manufacturers of HCT/Ps.
7. No regulatory or standard-setting organization requires a hospital tissue-dispensing service
to be managed by a particular department or individual. However the AABB favors a cen-
tralized model for managing this activity within the transfusion service.
REFER ENCES
1. American Association of Tissue Banks. About pitals: The official handbook. Oakbrook Ter-
AATB. McLean, VA: AATB, 2013. [Available at race, IL: The Joint Commission, 2014:TS1.
http://www.aatb.org/About-AATB (accessed 9. Levitt J, ed. Standards for blood banks and
December 8, 2013).] transfusion services. 29th ed. Bethesda, MD:
2. Eastlund DT, Eisenbrey AB for the Tissue Com- AABB, 2014.
mittee. Guidelines for managing tissue al- 10. College of American Pathologists. Standards
lografts in hospitals. Bethesda, MD: AABB, for laboratory accreditation. Northfield, IL:
2006. CAP, 2012.
3. Code of federal regulations. Title 21, CFR Parts 11. Association of Perioperative Registered Nurs-
1270 and 1271. Washington, DC: US Govern- es. Perioperative standards and recommended
ment Printing Office, 2014 (revised annually). practices. Denver, CO: AORN, 2013.
4. Woll JE, Smith DM. Bone and connective tis- 12. Dock N, Osborne J, Brubaker S, et al. Stan-
sue. Clin Lab Med 2005;25:499-518.
dards for tissue banking. 13th ed. McLean, VA:
5. Malinin TI. Preparation and banking of bone
American Association of Tissue Banks, 2012.
and tendon allografts. In: Sherman OH,
13. Eye Bank Association of America. Medical
Minkoff J, eds. Arthroscopic surgery. Balti-
standards. Washington, DC: EBAA, 2011.
more, MD: Williams and Wilkins, 1990:65-86.
14. Food and Drug Administration. FDA regula-
6. Cheek RF, Harmon JV, Stowell CP. Red cell allo-
tion of human cells, tissues, and cellular and
immunization after a bone allograft. Transfu-
sion 1995;35:507-9. tissue based products (HCT/Ps) product list.
7. Eastland T, Warwick, RM. Diseases transmit- Rockville, MD: Food and Drug Administration,
ted by transplantation of tissue and cell al- 2013. [Available at www.fda.gov/Biologics
lografts. In: Warwick E, Brubaker SA, eds. Tis- BloodVaccines/TissueTissueProducts/Regula
sue and cell clinical use: An essential guide. tionofTissues/ucm150485.htm (accessed No-
West Sussex, United Kingdom: Blackwell, vember 13, 2013).]
2012:72-113. 15. United Network for Organ Sharing. Richmond,
8. The Joint Commission. Transplant safety. In: VA: UNOS, 2013. [Available at www.unos.org
Comprehensive accreditation manual for hos- (accessed December 8, 2013).]
C h a p t e r 3 3
Mickey B. C. Koh, MD, PhD, Director, Stem Cell Transplantation, St. George’s Hospital and Medical School,
London, United Kingdom, Medical Director, Cell Therapy Facility, Health Sciences Authority, Singapore;
Edward R. Samuel, PhD, MSc, Clinical Scientist and Senior Research Associate, Department of Haematology,
University College London Medical School, Royal Free Campus, London, United Kingdom; and Garnet Suck,
PhD, MSc, Division Director, Productions, Institute for Transfusion Medicine, German Red Cross Blood Dona-
tion Service North-East nonprofit GmbH, Berlin, Germany
The authors have disclosed no conflicts of interest.
785
786 䡲 AABB TECHNICAL MANUAL
of graft-vs-host disease (GVHD), which is also quality-assurance systems embedded into the
mediated by T-cells, was protective and re- processes. In addition, the stringent donor-
duced the chance of relapse.1 selection processes, widespread use of auto-
In addition to HSCs and their differentiat- mation, use of mandatory release criteria for
ed progeny, the marrow contains nonhemato- blood units, and requirements of traceability
poietic cells, often referred to as “stromal and hemovigilance of blood banks have been
cells,” which include mesenchymal stem cells incorporated into cellular immunotherapy.
(MSCs). In addition to providing stromal sup-
port for hematopoietic cells, MSCs give rise to Donor Lymphocyte Infusion: A Post-
structural components, such as bone, carti- HSCT Treatment
lage, tendon, and fat. Their wide-ranging and
potent differentiation properties have generat- Although allogeneic HSCT can achieve long-
ed considerable clinical interest in their use for lasting cures in many patients, relapse still oc-
cell therapy. MSCs also appear to exert immu- curs in some patients and is often associated
nosuppressive effects and are therefore of in- with a poor prognosis.2 A second HSCT is pos-
terest as immunomodulators. sible but is often associated with high toxicity
Self-renewing pluripotent stem cells are and morbidity risk, especially if the relapse oc-
rare and difficult to isolate. The demonstration curs within a year of the first transplant.
that pluripotent stem cells can be derived
from differentiated cells, including skin fibro- Clinical Efficacy of Donor Lymphocyte
blasts and blood, has transformed the field of Infusions
stem cell therapy. The use of these cells has the
potential to have myriad applications in all ar- The discovery that T cells are one of the main
eas of medicine. drivers of the GVT or GVL response led to the
development of donor leukocyte (lymphocyte)
infusions (DLIs) after HSCT. One of the key ob-
IM MUNE CELLS FOR CLINICAL servations in the development of DLIs was that
THERAPY patients with chronic myeloid leukemia (CML)
Cellular immunotherapy is the exploitation of that relapsed after HSCT could achieve long-
immune cells to target and treat diseases. This lasting remissions with the administration of
approach is most established in cancer pa- DLIs.3
tients, where these novel treatment modalities Lymphocytes for DLI are collected from a
are used to overcome the limitations and tox- donor via leukapheresis using a process simi-
icities of chemotherapy and radiotherapy. lar to peripheral blood stem cell collections,
Considerable progress in cellular process- except that prestimulation with a colony-stim-
ing technology has allowed for advanced and ulating factor or mobilizing agent is not usual-
complex immune-cell manipulations. It is now ly required. The leukapheresis product is usu-
possible to characterize immune cells with ally then aliquoted into doses starting from as
high precision, isolate specific cell types with low as 1 106 CD3-positive cells/kg recipient
high purity, and engineer and amplify them ex weight, followed by half- or 1-log increments.
vivo in sophisticated culture systems that The initial dose is often infused fresh, and ali-
comply with good manufacturing practice quots for subsequent doses are cryopreserved.
(GMP) requirements. The adoption of auto- The efficacy of DLIs is dependent on the
mation has also allowed for more reproducible type and aggressiveness of the underlying dis-
immune cell expansion. ease and the disease burden at the time of re-
Blood banks have been instrumental in lapse. It has been demonstrated that patients
these advances in cellular immunotherapy with relapsed CML benefit most from DLIs
due to their preexisting infrastructure, which with an 80% complete response rate for those
includes the GMP-compliant processing of in cytogenetic relapse; patients in hematologic
blood units for transfusion and meticulous relapse respond less well.3 In comparison, only
CHAPTER 33 Blood/Marrow-Derived Non-HPC and Immune Cells 䡲 787
15% to 40% of patients with relapsed acute fications include the use of CD8-depleted and
myeloid leukemia (AML) respond to DLIs, and alloantigen-depleted DLIs and the introduc-
patients with acute lymphocytic leukemia tion of a “suicide” gene, such as the Herpes
(ALL) have virtually no response to DLI. The simplex virus-tyrosine kinase gene, into the T
lack of efficacy of DLI in acute leukemias com- cells to selectively eliminate them if GVHD de-
pared to CML is thought to be related to a lack velops.4
of antigen expression on tumor cells and the A group of researchers at University Col-
more rapid proliferative kinetics associated lege London Hospital has also pioneered a
with acute leukemias.4 strategy of using a dose-escalating DLI
The starting dose for DLIs is often in the regimen to treat residual or progressive dis-
order of 1 × 106 CD3-positive cells/kg.5 Lower ease or mixed donor chimerism after reduced-
starting doses have been administered, espe- intensity, T-cell-depleted transplantation.5 This
cially in the unrelated transplant setting. Re- strategy has resulted in an impressive 70%
sponse is usually not immediate and can take response rate in patients with low-grade lym-
as long as 3 months. For this reason, subse- phomas or Hodgkin disease.
quent doses of DLIs are often administered as
far apart as 3 months and in incremental doses New Directions in DLI Applications:
half to 1 log apart so that responses can be ad- Targeted and Antigen-Specific T-Cell
equately gauged.3 Therapy
There is no exact correlation between
dose and response. Patient response can be Adoptive immunotherapeutic approaches em-
difficult to predict because it depends on the ploying more targeted or antigen-specific T-
disease, stage of relapse, patient factors, HLA cell populations have been used successfully
matching, and donor characteristics.6 in the treatment of hematologic malignancies
and solid tumors as well as viral infections af-
Complications of DLIs ter HSCT. To develop immunotherapies using
cytotoxic T lymphocytes (CTLs; T cells ex-
The major complication of DLI is GVHD, pressing CD8), potential target antigens on tu-
which is caused by alloreactive donor T cells mor cells or viruses must first be identified.
attacking healthy host cells. In early studies, These antigens must be capable of providing
up to 50% to 90% of patients developed GVHD epitopes for specific immune responses and
after DLI. However, more recently, the rate of be present in sufficient quantity and duration
GVHD following DLI has declined due to an to engage responder T cells.4
improved understanding of the biology of DLIs A step forward in efficiently isolating
and predictive risk factors for GVHD in this T cells was achieved with the development of
setting. The GVHD that occurs after DLI can be the major histocompatibility complex (MHC)
very severe and require systemic immune sup- multimer method. In this method, high-affini-
pression, which can lead to significant mor- ty binding of MHC molecules to the T-cell re-
bidity and mortality as a result of opportunis- ceptor is achieved through oligomerization of
tic infections. MHC molecules (multimers) as ligands.7 This
Another major complication of DLI is the method allows specific detection and isola-
development of marrow aplasia, which is tion of antigen-specific T cells through fluores-
thought to be due to the immune-mediated cence-activated cell sorting in a flow cytome-
destruction of host hematopoeisis.6 ter or the use of closed magnetic selection
The variable results in the initial studies systems, such as the automated CliniMACS in-
of DLIs in patients with frank disease relapse strument (Miltenyi Biotec, Bergisch Gladbach,
together with the treatment’s potential toxici- Germany). The multimer method has recently
ties have resulted in crucial modifications to been improved with the invention of the novel
this T-cell therapy to increase its GVT activity streptamer technology, which has entered
while minimizing its side effects. These modi- clinical testing.8,9
788 䡲 AABB TECHNICAL MANUAL
in 5 of 19 patients with AML. These results of- nential NK-cell expansion, for example, was
fer another proof of the ability of NK cells to achieved with the EBV-transformed lympho-
separate GVL from GVHD with the aim of ex- blastoid cell line or the leukemic cell line K562,
ploiting the former while minimizing the lat- engineered to present both IL-15 and the co-
ter. As in the haploidentical transplant setting, stimulatory molecule CD137 (4-1BB) on the
the effect was more marked when KIR-ligand surface (K562-CD137/IL-15). These protocols
mismatched (indicating alloreactive) donors are undergoing clinical testing.16,19
were used. Automated cell processing within biore-
In clinical protocols involving NK cells, ef- actors, such as Wave™ (GE Healthcare Life Sci-
fectors are enriched by magnetic CD3 deple- ences, Freiburg, Germany) or G-Rex (Wilson
tion followed by CD56 selection or through Wolf Manufacturing, New Brighton, MN), pro-
combined CD3 (T-cell) and CD19 (B-cell) de- vides a developing platform for the mass pro-
pletion using the CliniMACS (Miltenyi Bio- duction of NK cells in less time with better re-
tech) to reduce the risk of T-cell-mediated producibility.16 These methods are also used
GVHD and EBV infections.16 for other immune effector therapies. Further-
more, shipment of fresh NK cells under IL-2
Ex-Vivo Expansion protection is feasible without the need for a
controlled incubator environment (37 C and
An alternative method to in-vivo expansion of
5% CO2) and facilitates international distribu-
NK cells is expansion of NK cells in long-term,
tion of such products.20
GMP-compatible culture systems that gener-
NK-cell therapy products are generally
ate large numbers of highly cytotoxic effectors. safe and well tolerated, especially when in-
This method has been established for clinical vivo injections of IL-2 are not required. How-
“off-the-shelf” production of the highly cyto- ever, NK-cell products are not entirely risk free.
toxic NK cell line NK-92 [Conkwest (Del Mar, Two cases of significant hemolytic anemia af-
CA) proprietary line].17,18 In contrast, GMP- ter treatment with minor ABO-mismatched
compatible expansion of primary NK cells has NK-cell-enriched products have been report-
remained challenging, although promising ed that were presumably due to the presence
protocols are under development. of isoagglutinin-producing passenger B lym-
New cell-culture media, including serum- phocytes in the NK-cell product.21
free media, are now available. Furthermore,
novel growth factors, such as IL-15, are being Cytokine-Induced Killer Cells:
used instead of the traditional IL-2 to further
Polyclonal T Cells with NK-Cell Features
optimize culture conditions with efficient bi-
asing of proliferation of the NK-cell popula- The traditional LAK cell culture protocol has
tion within LAK cultures after long-term ex- been further developed to generate a popula-
pansion.19 tion of rapidly expanding polyclonal T cells
Highly purified NK cells are more chal- known as “cytokine-induced killer” (CIK)
lenging to expand. However, purified NK cells cells.22 Culture conditions for CIK expansion
can be grown on feeder cell sources of either are designed to obtain an increased propor-
autologous or allogeneic origin. An elegant tion of superior cytotoxic CD56+CD3+, double-
way to obtain autologous feeder cells is to re- positive T cells or NK-like T cells. The genera-
tain the nonselected, NK-cell-negative frac- tion of such CD56+ T cells is promoted in this
tion after a magnetic isolation procedure. Fol- procedure through the initial addition of gam-
lowing irradiation of this by-product to inhibit ma interferon (IFN; 1000 U/mL). The T-cell-
unwanted cell proliferation during culture, activating, anti-CD3 monoclonal antibody
these cells can be seeded as a feeder layer to OKT3 (50 ng/mL) and IL-2 (300 U/mL) are
support NK-cell stimulation and proliferation. added 24 hours later, and the cultures are
Other protocols have been established that in- maintained under permanent IL-2-stimula-
volve the use of allogeneic cell sources. Expo- tion for 21 to 28 days.23 The heterogeneous CIK
790 䡲 AABB TECHNICAL MANUAL
population at the end of culture comprises a of eliciting full T-cell activation upon engage-
very small proportion (about 2%) of NK cells. ment of costimulatory molecules (eg, B7:CD28
Most of the cells (>90%) in the culture are T engagement) and mediate cytotoxic responses
cells, of which about 35% express the double- against a broad range of malignancies. Partic-
positive phenotype CD3+CD56+. ularly attractive is the intrinsic potential of
DCs to gain immunologic memory for com-
Characteristics of CIK Cells bating subsequent tumor invasions.27
CIK cells combine features of NK-cell Defining DCs and Their Subsets
(CD56+CD3–) and T-cell (CD56–CD3+) effectors.
The different pathways involved in CIK cyto- Unlike T cells, no single cell surface marker is
toxicity are under active investigation. Similar exclusively expressed on DCs that can be used
to NK cells, CIK cells have cytolytic potential to define these cells. Instead, a combination of
that is immediate and independent of antigen morphology and various surface markers is
priming.24 However, studies have shown that used, including the expression of MHC Class II
target cell recognition requires direct MHC antigens and absence of markers that define
Class I engagement, a mechanism that is not other lineages, such as CD3 and CD19. DCs
yet fully understood. As with NK cells, the criti- also express a variety of adhesion molecules,
cal role of the activating receptor NKG2D in tu- including CD11a (lymphocyte function-asso-
mor lysis has been demonstrated for targets, ciated antigen 1); the intercellular adhesion
such as myeloma cells, that express its ligands molecule 1 family; and costimulatory mole-
MIC A/B or ULBPs. cules, such as CD80 and CD86. Two additional
markers of mature DCs in humans are CD83
Clinical Trials and CMRF-44.28
In the blood, DCs are present in different
Results from clinical trials using autologous or subsets that are distinguished by CD303
allogeneic CIK cells in a variety of hematologic (BDCA2, CLEC4C; plasmacytoid DC), CD1C
malignancies and solid tumors have been re- (or BDCA1; myeloid DC), and CD141 (BDCA3
ported. Overall, CIK cell therapy was well toler- or thrombomodulin; myeloid DC) markers.
ated and associated with a low risk of GVHD. 25 Different functions could be attributed to
Results for cancer clearance and increased these subsets, with CD141-positive myeloid
survival varied. Patients with a lower tumor DCs playing a role in eliciting CD8 T-cell re-
burden, such as those who had undergone sponses.29
HSCT, are probably the most likely to benefit The rarity of DCs has made it difficult to
from CIK effectors. In this sense, CIK cells may study them, although they were first described
be used as an alternative to DLI. Strategies to more than 30 years ago.26 The ability to gener-
increase the cytolytic potential of CIK cells, in- ate large numbers of DCs from CD34+ marrow
cluding co-administration of IL-2 and engi- precursors or CD14+ monocytes in vitro has
neered CARs, are currently being investigated. expanded the field of DC-based cellular im-
munotherapy.
DCs in Immunotherapy
Expansion Protocols
DCs play a critical role in the regulation of the
adaptive immune response. DCs have been Initial tissue-culture protocols for the genera-
called “sentinels of the immune system,” and tion of DCs (the first-generation DC vaccines)
they possess the ability to elicit a primary im- used a combination of granulocyte macro-
mune response in resting naïve T lympho- phage colony-stimulating factor (GM-CSF)
cytes. Naïve CD8+ T cells, for example, differ- with IL-4 for the stimulation of monocytes in
entiate into CTLs through interaction with peripheral blood mononuclear cells (PBMCs)
cognate antigens presented by mature DCs on to differentiate into DCs. However, the result-
their MHC Class I receptors.26 DCs are capable ing DC vaccines usually contained a mixture of
CHAPTER 33 Blood/Marrow-Derived Non-HPC and Immune Cells 䡲 791
immature or only partially mature DCs and of- to GM-CSF. PAP is an immunogenic prostate
ten other additional cell types. Since then, re- antigen whereas GM-CSF is an immune-cell
finements of protocols (known as “second- activator. Each dose is manufactured by ob-
generation” DC expansion protocols) have taining a patient’s T cells via leukapheresis and
been described using several new cytokine/ shipping them to the company to manufacture
GM-CSF combinations that include IFN, tu- the vaccine. After this process, the patient’s
mor necrosis factor (TNF), or IL-15.29 One such own cells are reinfused into the patient to treat
cytokine cocktail consists of TNF, IL-1, IL-6, the prostate cancer. Provenge is administered
and prostaglandin 2 for additional DC stimu- intravenously in a three-dose schedule given
lation. More recently, “third-generation” DC at about 2-week intervals.
vaccines have been developed that are polar- The effectiveness of Provenge was studied
ized toward a cytotoxic immune response (in- in 512 patients with metastatic prostate cancer
nate and Type 1 immunity) and are currently that was refractory to hormone treatment
being tested in clinical trials.29,30 in a randomized, double-blind, placebo-
In-vitro manipulation of DCs includes controlled, multicenter trial.33 The median
loading (pulsing) with tumor recognition mol- survival duration for patients receiving
ecules, such as tumor-antigen-derived pep- Provenge treatments was 25.8 months com-
tides, recombinant tumor antigens, or tumor- pared to 21.7 months for those who did not re-
derived ribonucleic acid or deoxyribonucleic ceive the treatment.
acid. Furthermore, DC tumor-hybrid vaccines
have been created with the potential to acti- MSCs
vate a combined CD4+ and CD8+ T-cell re-
MSCs are a rare subset of cells of nonhemato-
sponse through simultaneous presentation of
poietic origin that were first discovered in 1968
several tumor antigens, including a direct fu-
by Friedenstein as an adherent fibroblast-like
sion of DCs to leukemic blasts, which are easi-
population after isolation from the marrow,
ly obtainable from patients with leukemia.31,32
However, release of suppressive factors, such where they represent 0.001% to 0.01% of total
as TGF, by the tumor cell fused in the hybrid cells.34 It was subsequently shown that MSCs
may limit the vaccine’s efficacy.31 could be isolated from various tissues, includ-
ing amniotic fluid, adipose tissue, umbilical
Cancer Vaccine Approved by Food and cord blood, dental pulp, muscle connective
tissue, and placenta, and could be rapidly ex-
Drug Administration
panded in vitro, offering the potential for use
One of the successes of cellular therapy has in clinical cellular therapy.35 The rarity of MSCs
been in the treatment of prostate cancer. A has meant that the majority of research to date
first-generation DC-based vaccine, sipuleucel- has relied on isolating them from the marrow
T (Provenge; APC 8015; Dendreon Corpora- or adipose tissue before expansion in culture,
tion, Seattle, WA), was approved by the US a prerequisite for clinical-scale therapies.
Food and Drug Administration (FDA) based on MSCs are often referred to as “multipotent”
a 4-month improvement in survival compared due to their capacity for differentiation into
to placebo in the pivotal randomized clinical cells of mesodermal lineage, including osteo-
trial of men with castration-resistant metastat- blasts, chondrocytes, adipocytes, and, under
ic prostate cancer.33 This is an autologous cel- specific conditions, several nonmesodermal
lular immunotherapy designed to stimulate a cell types, including neurons.36 It is this capac-
patient’s own immune system to respond ity for multipotency that has generated a great
against the prostate cancer. Sipuleucel-T con- deal of interest in using MSCs for tissue engi-
sists of autologous PBMCs that are enriched neering and regenerative medicine.
for antigen-presenting DCs that have been ac- Expanded MSC cultures do not all possess
tivated ex vivo with a recombinant fusion pro- the same level of multipotency. Although a low
tein, prostatic acid phosphatase (PAP), fused frequency of self-renewing progenitors has
792 䡲 AABB TECHNICAL MANUAL
been identified among marrow-derived MSCs, including GVHD, autoimmunity, and solid-or-
it remains unclear whether MSCs from other gan-transplant rejection. The suppressive
tissues share these properties. This has result- properties displayed by culture-expanded
ed in the use of the term “multipotent MSCs” MSCs are promoted through the expression of
as an alternative to “MSCs” because “multipo- indolamine 2,3-dioxygenase and other effector
tent MSCs” does not imply that the cells have molecules, many of which are augmented by
“stem-cell-like” properties or self-renewal IFN- stimulation.41,42
ability without differentiation (Table 33-1).37
The defining characteristics of MSCs have Isolation and Expansion of MSCs
often differed among investigators, which
prompted the publication of the minimal cri- The isolation and initial expansion of MSCs is
teria for the definition of the phenotypic and performed in three distinct phases over a peri-
functional properties of MSCs by the Interna- od of 3 to 4 weeks:
tional Society of Cellular Therapy (ISCT).38 The
ISCT guidelines for defining MSCs are listed in 1. Isolation and seeding.
Table 33-2. 2. Cell passaging.
MSCs have been identified as modulators 3. Final harvest.
of several effector functions and immune re-
sponses through their interaction with both The clinical production of MSCs for ther-
the innate and adaptive immune systems. apeutic use is carried out in large tissue-cul-
They play a key role in the support of hemato- ture flasks, although MSC production can be
poiesis, contributing to the maintenance of industrialized in 25,000-cm2 culture chambers
the highly specialized microenvironment of
or CellSTACKsTM (Sigma-Aldrich Corning, New
the marrow through the regulation of HSC
York, NY). Marrow aspirates/harvests remain
numbers and control of their maturation and
the most common starting material for the iso-
differentiation.39 MSCs have been shown to be
lation of MSCs. Following harvest, marrow is
capable of exerting a profound immunosup-
pressive effect on both polyclonal and anti- collected into preservative-free heparin be-
gen-specific T-cell responses through induc- fore transportation to the laboratory, where
tion by cellular or humoral stimuli. They the first step is the isolation of bone-marrow
largely have no immunologic restriction, mononuclear-cell (BMMNC) fraction by light-
meaning that they can be used without HLA density centrifugation. At this stage, MSCs are
matching.40 The immunoregulatory functions isolated either by their ability to adhere to tis-
of MSCs, including the suppression and inhi- sue culture plastic, as described below, or are
bition of T-, B-, and NK-cell functions and DC directly purified using monoclonal antibodies
activity, therefore offer a promising option for to CD105, CD146, or CD271 by immunomag-
the treatment of immune-mediated disorders, netic or flow-cytometric sorting.43
TABLE 33-2. Criteria for Identification of MSCs Adherent cells consistently achieve 80%
confluence after 9 to 14 days, at which point
䡲 Adherence to tissue culture plastic the cells are passaged following enzymatic dis-
䡲 Phenotype (levels of expression) sociation from tissue culture flasks with tryp-
sin. Passaging of cell cultures once they have
Positive (95% +) Negative (2% +)
achieved confluence is often a prerequisite
䡲 CD73 䡲 CD45
䡲 CD90 䡲 CD34 when an attempt is made to propagate cells in
䡲 CD105 䡲 CD14 sufficient quantity for therapeutic use. Howev-
䡲 CD11b er, continual passage of MSCs can lead to rep-
䡲 CD79 licative exhaustion and alterations in pheno-
䡲 CD19 type, which may play a role in modifying their
䡲 HLA-DR regenerative and immune-suppressive prop-
䡲 Demonstration of in-vitro differentiation into erties or potentially limiting their survival and
osteoblasts, adipocytes, chondrocytes function in vivo.44 The malignant transforma-
tion potential of MSCs can be eliminated by
MSCs = mesenchymal stem cells.
reducing cell passaging, although it has also
been reported that MSCs can be safely ex-
panded in vitro until the 25th passage.45 MSCs
are harvested and can be cryopreserved at the
For isolation by plastic adherence,
recommended therapeutic dose or adminis-
BMMNCs are seeded onto tissue-culture flasks
tered “fresh.”
at a density ranging from 0.5 × 105 to 5 × 105
Immunophenotyping by flow cytometry
BMMNCs per cm.2 The BMMNCs are cultured is an essential part of the quality assurance/
in alpha-modified minimum essential medi- quality control process of manufacturing us-
um supplemented with 10% fetal bovine se- ing the cell-surface markers CD73, CD90, and
rum (FBS). Alternative MSC expansion proto- CD105. Differentiation of MSCs into a specific
cols have also been described using human lineage often requires the addition of a com-
platelet lysate autologous serum as an alterna- plex array of growth factors that are selected
tive source of serum and growth-factor-sup- based on the desired mature cell phenotype.
plemented, serum-free media.35 Standardization of MSC isolation and ex-
Despite the prescreening of FBS for use in pansion is now gathering pace among the cell
clinical studies, its application in clinical- therapy community as MSCs are increasingly
grade cellular therapies still raises concerns used in clinical trials and manufactured under
because, theoretically, it is a putative source of GMP conditions. The technology for culturing
prions and virus transmission. These concerns large numbers of expanded MSCs for clinical
make the future use of serum-free media in all use has also undergone significant develop-
clinical products an attractive option. ment. Devices, such as The Quantum Cell Ex-
The propagation of adherent cells is pansion System (Terumo BCT, Lakewood, CO),
maintained by the removal of nonadherent a closed automated hollow fiber bioreactor
cells on the third day and their subsequent culture system with disposable sets, are now
commercially available and designed to repro-
feeding every 3 to 5 days with fresh culture me-
ducibly grow adherent cells in GMP environ-
dium. Cultures are reviewed daily for:
ments while minimizing both space and oper-
ator variability.
䡲 Adherence of cells to flasks.
䡲 Shape of adherent cells (round vs fibrocytic
Clinical Applications of MSCs
appearance).
䡲 Confluence of cells. The main indications for use of MSCs are in
䡲 Amount of debris in medium as an indica- the treatment of GVHD, Crohn disease, cardio-
tion of medium change. vascular disorders, multiple sclerosis, spinal
794 䡲 AABB TECHNICAL MANUAL
cord injury, liver disease, diabetes mellitus, Optimal therapeutic doses have not been
and various bone and cartilage defects. MSCs defined, although clinical responses have been
can be used without HLA matching because documented with infusions of MSCs as low as
they do not induce GVHD and are not rejected 0.8 × 105/kg. However, a Phase III, industry-led
by recipient T cells. These characteristics favor clinical trial examining the transfusion of high
banking of cryopreserved MSCs from third- doses of MSCs (2 × 106/kg) for the treatment of
party donors. Such banked MSCs can be re- steroid-refractory GVHD showed no increase
leased and shipped as required for multiple in overall complete response rate.44
clinical applications as “off-the-shelf” prod- The clinical use of MSCs for tissue regen-
ucts. eration has received the most interest for
One of the earliest studies that illustrated cardiovascular repair.50 Many trials have had
the regenerative properties of MSCs involved encouraging results in the improvement of
their use in treating three children with osteo- cardiac function after injection of autologous-
genesis imperfecta. 46 This disease is caused by marrow-derived MSCs. Adipose-tissue-derived
a deficiency in the production of Type I colla- MSCs are also currently being investigated as
gen and results in defective connective tissue part of two industry-led clinical trials, APOLLO
formation, leading to multiple fractures, bony and PRECISE, exploring their safety, feasibility,
deformities, and shortened stature. Following and efficacy in patients with either acute or
the infusion of unmanipulated allogeneic chronic myocardial infarction.51
marrow, MSCs from within the graft migrated Although the therapeutic effects of MSCs
to the bone and gave rise to osteoblasts, where in cardiac repair have been acknowledged in
the scientific community, several issues re-
their presence correlated with an improve-
main unresolved, including the optimal dose
ment in bone structure and function. The
and the mechanism of improvement in cardi-
same group of researchers has since modified
ac function. The differentiation of MSCs into
their treatment protocol and demonstrated its
cardiomyocytes remains controversial, and
safety and efficacy after administering two
the literature in this field is still unclear and
doses of marrow-derived, ex-vivo-expanded
contradictory. There have been a number of
MSCs following standard marrow transplanta-
recent publications providing evidence that in
tion in patients with this disease.47 vitro, MSCs may be induced to exhibit cardio-
The therapeutic role of MSCs in HSCT has myocyte-like features, but the exact culture
been largely targeted toward alleviation of conditions are not clear. A number of com-
GVHD following transplantation. However, the pounds are involved in their generation
codelivery of MSCs at the time of transplanta- through modulation of signaling pathways.
tion has received attention because of their There has been no direct evidence of cardio-
potential role in graft tolerance and engraft- myocyte differentiation in vivo following ad-
ment. Marrow-derived MSCs have been used ministration of MSCs for cardiac repair, but
successfully for the treatment of steroid-re- functional improvement has been observed.
fractory, severe, acute GVHD, for which there These observations in preclinical and human
is currently no established definitive therapy. trials have led to the hypothesis that the im-
The clinical benefits of infusing MSCs to treat provements are related to the paracrine ef-
GVHD were first demonstrated when haploi- fects of transplanted cells, which induce myo-
dentical MSCs were administered in two trans- cardial repair by releasing signals, including
fusions to a 9-year-old boy with treatment-re- cytokines and chemokines, into the surround-
sistant, Grade IV acute GVHD of the gut.48 This ing tissue rather than generation of de-novo
has triggered great interest over the past de- cardiomyocytes.50-52
cade, and subsequent Phase I/II trials have The multipotency of MSCs has also be-
demonstrated both the safety and efficacy of come attractive to industry, and two commer-
the use of MSCs in HSCT in the setting of ste- cially driven clinical trials involve third-party
roid-refractory GVHD.49 MSCs manufactured by Osiris Therapeutics
CHAPTER 33 Blood/Marrow-Derived Non-HPC and Immune Cells 䡲 795
Inc. (Columbia, MD) for use in patients with these capabilities was called into question
GVHD or Crohn’s disease and mesenchymal when Gurdon55 demonstrated that the nuclei
precursor cells manufactured by Mesoblast of differentiated cells actually retain all of the
Ltd. (New York, NY) for the treatment of car- genetic information in pluripotent stem cells.
diovascular, orthopedic, and neurologic dis- The cloning of Dolly the sheep, for example,
ease. Although the clinical efficacy of such showed that the genome of even fully special-
MSC products has yet to be fully evaluated in ized cells remains genetically totipotent; that
many of the proposed disease indications, is, the genome can support the development
their application as cellular therapies contin- of an entire organism.
ues to evolve rapidly. The next key finding was that manipula-
tion of transcription factor expression can
MSCs and Tissue Engineering change a cell’s fate.56 Experiments revealed
The use of MSCs in tissue engineering has de- that lineage-associated transcription factors
veloped rapidly over the last few years with the can change cell fate when these factors are ec-
advent of scaffolds to improve the outcomes of topically expressed in certain heterologous
stem cell applications by offering more precise cells. Lineage-associated transcription factors
targeting of MSCs combined with the possibil- help establish and maintain cellular identity
ity of making them biodegradable, depending during development by driving the expression
on the therapy. Scaffolds can be used in a of cell-type-specific genes while suppressing
number of ways: 1) seeding of MSCs onto scaf- lineage-inappropriate genes.
folds in vitro and implantation after a short in- The third contributory result was ob-
cubation period, 2) MSC seeding and subse- tained from the Nobel Prize-winning work of
quent differentiation into lineage-specific cell Yamanaka,57 who screened a pool of 24 pluri-
types in short-term culture (1-2 weeks) before potency-associated candidate genes for fac-
implantation, and 3) induction of differentia- tors. He determined that a core set of four
tion in culture before seeding onto scaffolds transcription factors comprising Klf4, Sox2,
and implantation shortly afterwards. c-Myc, and Oct4 was sufficient to produce in-
In 2008, the first fully tissue-engineered duced pluripotent stem cells (iPSCs). This re-
bronchial transplant was reported. 53 In this search was initially conducted in murine mod-
case, a nonimmunogenic, decellularized piece els, but the approach was also successful with
of human donor trachea was reseeded with adult human skin fibroblasts. Many other in-
autologous marrow-derived MSCs. The MSCs vestigators since then have demonstrated that
were differentiated into chondrocytes in vitro this cocktail of genes can be used to derive
before surgical implantation to replace a bron- iPSCs from other somatic cell populations, in-
chus that had been previously damaged by tu- cluding keratinocytes, neural cells, and stom-
berculosis infection. The paucity of donor or- ach and liver cells.
gans has limited the use of this approach. As a Now that iPSCs can be generated, the pri-
result, the use of biosynthetic nanocomposite mary question is whether iPSCs and ESCs are
material seeded with MSCs has emerged as a molecularly and functionally equivalent.
therapeutic option for tracheal replacement.54 Comparisons of their genome-wide-expres-
sion patterns and global histone modifications
have demonstrated a high degree of similarity
IND UCED PLURIP OTE N T ST EM
between ESCs and iPSCs. However, unlike
CELLS
ESCs, iPSCs are derived from somatic tissues
The archetypical pluripotent cell is the embry- and therefore do not raise the ethical issues as-
onic stem cell (ESC), which is derived from sociated with ESCs.58 The ability to obtain
embryos and is capable of long-term self- starting material from skin, blood, and umbili-
renewal and differentiation into all cells and cal cord immediately opens up the horizon for
tissues in the human body. The uniqueness of translational clinical applicability.59
796 䡲 AABB TECHNICAL MANUAL
GMP facilities, and commercial companies. The question that arises next is how the
Harmonization in the terminology of cell-ther- issuance and inventories of these biologics will
apy products is being undertaken by the Cellu- be managed in the local hospital environment.
lar Therapy Coding and Labeling Group, a The hospital pharmacy is the natural domain
technical subgroup of ICCBBA that promotes for medicinal products oversight. However, the
the use of ISBT 128 (the information technolo- unique qualities of cell therapy products ren-
gy standard for transfusion medicine and der them more suitable for the sphere of activ-
transplantation) labeling. This harmonization ity and concern of the blood bank and GMP
will, of course, facilitate the transportation of processing facility.
such products across national borders. In ad- Most importantly, the maturation of the
dition, the Alliance for Harmonization of Cell cell-therapy field and its clinical applicability
Therapy Accreditation consists of representa- in many fields of medicine will mean that
tives from a variety of organizations involved more patients will be treated with this novel
therapeutic option. Because living cells are of-
in HSCT and cell therapy. Its primary aim is to
ten administered to patients, short-term as
create a single set of quality, safety, and profes-
well as longer-term side effects and potential
sional requirements for cellular therapy.
toxicities should be systematically monitored.
Cell therapy products need to be gov-
Such monitoring systems have been in place
erned under suitable regulatory frameworks,
for pharmaceuticals (pharmacovigilance pro-
including national ones that cover cells, tis-
grams) and blood (hemovigilance programs),
sues, and organs. Such national regulatory
and these programs could be extended to cel-
frameworks for cell-therapy products are not lular therapy products (cellulo-vigilance pro-
in existence worldwide but will be important grams).
as the field develops and the need grows for
cross-border transportation of cells.
Cellular products can be classified as ei- CO N CLUS IO N
ther substantially or minimally manipulated. The history of cell therapy has seen great ad-
Substantially manipulated cell-therapy prod- vances of late with the entrance into the mar-
ucts are treated as biologics. In the United ket of the first FDA-approved therapeutic can-
States, these are known as “351” human cells, cer vaccine, Provenge, as well as the dramatic
tissues, and cellular and tissue-based products successes of antigen-specific T-cell therapy in
and are regulated by the Center for Biologics hematologic diseases. These successes will
Evaluation and Research of the FDA. In Eu- certainly spur continuing interest in the devel-
rope, substantially manipulated products are opment of clinical trials. The attraction of an
known as advanced therapeutic medicinal “off-the shelf,” third-party product is that it
products (ATMPs). They are manufactured in will increase the number of patients who can
accordance with national legislation and over- be treated and heighten interest in the inter-
sight by the European Medicines Agency. national transportation of such products.
KEY POINTS
1. Hematopoietic stem cell transplantation (HSCT) is well established for the treatment of he-
matologic diseases. A graft-vs-tumor (GVT) effect is largely mediated by cytotoxic immune
effector cells, such as T cells and natural killer (NK) cells.
2. Recent technological advances allow good manufacturing practice (GMP)-compliant isola-
tion, characterization, expansion, and modification of immune effector cells for application
as cellular therapeutics in clinics.
3. Donor lymphocyte infusion (DLI) has demonstrated considerable benefit as post-HSCT
treatment in relapsed patients, particularly in chronic myeloid leukemia (CML). These are
798 䡲 AABB TECHNICAL MANUAL
obtained from leukapheresis and constitute mainly T cells. A major side effect is graft-vs-
host disease (GVHD) as a consequence of a healthy host cell attack by alloreactive T cells.
4. NK cells are innate immune cells, do not require antigen priming, and do not induce GVHD.
Current promising developments to improve clinical outcome include treatment with allo-
reactive NK cells, mismatched for their inhibitory receptors, as well as in-vivo or ex-vivo ac-
tivation and expansion of NK cells.
5. Cytokine-induced killer cells (CIK) cells or NK-like T cells are expanded in long-term cul-
tures. Trials with CIK cell therapy have thus far demonstrated good tolerability with a low
risk of GVHD and some efficacy. They may be applied as an alternative to DLI.
6. Dendritic cells (DCs) play a key role in regulating the adaptive immune response to an anti-
gen-specific manner. A DC-based vaccine, sipuleucel-T (Provenge) is approved by the US
Food and Drug Administration as an autologous immunotherapy against prostate cancer.
7. Mesenchymal stem cells (MSCs) possess potent differentiation properties (multipotency)
and have sparked considerable clinical interest. They are generally isolated from marrow or
adipose tissue and expanded in vitro. They can give rise to structural components, such as
bone, cartilage, tendon, and fat, and appear to exert immunosuppressive functions. Indica-
tions for applications of MSCs include GVHD, Crohn disease, cardiovascular disorders, cord
injury, liver disease, diabetes mellitus and bone and cartilage defects.
8. Promising therapeutic effects of autologous MSCs in regenerative cardiac repair have been
reported although the potential for differentiation of MSCs into cardiomyocytes remains
controversial and more results are needed.
9. Third-party MSCs can be applied “off-the-shelf” without HLA matching and are not reject-
ed by recipient T cells.
10. The field of stem cell therapy has been transformed with the discovery that self-induced
pluripotent stem cells (iPSCs) can be generated from differentiated cells, including kerati-
nocytes, neural cells, and stomach and liver cells. The use of iPSCs involves considerably
fewer ethical issues compared to that of embryonic stem cells.
11. iPSCs can potentially be applied in a wide range of medical conditions and for in-vitro gen-
eration of hematopoietic cells. Techniques for large-scale GMP-compliant manufacturing
of iPSCs are being developed.
12. Cellular products are classified as either minimally or substantially manipulated, with a
more stringent regulatory framework for production of the latter in the US and Europe.
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Index
803
804 䡲 AABB TECHNICAL MANUAL
with clinically significant antibodies, 274, phenotyping, 281, 329-330, 379, 420,
379, 419-421 588, 654
for exchange transfusions, 574, 579-580 traceability of, 225
for HSC transplantation, 633-634 transporting, 100-101, 146-147, 213-214,
for intrauterine transfusions, 564 215-220, 225
for massive transfusions, 386 volume reduction of, 157, 223, 513-514,
of platelets, 375, 459-461, 513-514, 581-582 581-582, 590
rare types, 421 washed, 223, 590-591, 639
of red cell components, 375, 378-379, 504 Blood containers
for red cell exchange, 654 additive solutions in, 136-137, 138
in urgent situations, 228, 385-386 for aliquots, 576
in warm autoimmune hemolytic anemia, anticoagulant-preservative solutions in,
435-436 136, 137
Blood components. See also specific configurations of, 136-137, 139
components diversion pouch on, 139, 141
administration of (See Blood modification by sterile connecting device,
administration) 139, 140
aliquoting, 224, 575-576, 583 properties of, 136
bacterial contamination of, 198-199, 224 Blood donation. See Blood collection; Donors
CMV-reduced-risk, 190, 525, 564, 639 Blood exposure, 44-45, 47-48, 49, 53, 124
costs of, 602 Blood group genomics, 278-287
cryopreservation of, 155 for antigen-negative blood donors, 285
density of, 143 to confirm D type of donors, 285
expiration of, 213, 215-220, 551 discrepancies between phenotype and
identification of, 139-140, 226, 384-385, genotype, 240, 285-287, 402
549, 550, 551 to distinguish alloantibody from
inspection of, 148, 149, 224, 226, 385, 549, autoantibody, 283
550-551 to predict phenotype of recently transfused
irradiated, 155-156, 222, 590, 688, 689 patients, 240, 281
issuing, 226, 384-385, 549-550 to predict phenotype when red cells are
labeling, 139, 158-159, 226, 384-385 coated with IgG, 283, 401-402, 436
leukocyte reduction of, 154-155, 222-223, in prenatal practice, 283-285
504-505, 590 Blood group systems. See also specific blood
monitoring use of, 697-707 groups
ordering, 226, 367-368, 546, 699-700, 703- clinical significance of antibodies in, 338-
704 340, 413-414
pooling, 156-157, 223-224 defined, 279
preparation and processing of, 146-148, genetics of, 255-279
221-224 chimerism, 278
quality control of, 159-160 gene mapping, 259-261, 277-278
quarantine of, 157-158, 189, 224 inheritance patterns, 265-273
rare, 421 population genetics, 274-275
receiving into inventory, 225 principles of, 256-258, 261-264
records of, 384-385 relationship testing, 276-277
regulations regarding, 83 terminology for, 278-279, 280
retrieval of prior donations, 187-188, 189- Blood loss
190 phlebotomy-related, 609
return and reissue of, 226-227, 550 signs and symptoms of, 501
selection of (See Blood component transfusion for, 501-502
selection) Blood management. See Patient blood
shortages of, 101-102 management
storage of, 213-214, 215-220, 221, 503-504 Blood order schedules, 227
testing Blood pressure
ABO and Rh, 225-226, 297-298, 378 of donors, 120
infectious disease, 182-191, 192-204 hypotension
INDEX 䡲 809
in cold agglutinin disease, 308-309, 392, 439 Immunomodulation, 504, 758, 762-763, 764
disease associations with, 307, 392 In-vivo testing, 419, 506-507
genetics of, 260, 307-308 Incarceration, donor history of, 125
phenotypes, 306-307 Incidence, defined, 274
transfusion practice with, 309 Incinerators, hospital/medical/infectious
Iatrogenic anemia, 609 waste, 55
ICAM-4, 355-356 Incubation times, 405
Identification Incubators, platelet, 36, 214
of blood components Independent assortment, 270
before administration, 226, 551 Independent segregation, 270
before issue, 226, 549-550 Indian system, 260, 339, 358-359
donation identification number, 135, Indirect antiglobulin test, 372, 373-374
139-140, 159 Induced pluripotent stem cells, 755, 795-796
labeling, 158-159, 384-385 Infants. See Neonates; Pediatric patients
of donors, 118-119, 139 Infection, in transplant patients, 638, 782
of equipment, 11 Infectious disease screening
errors in, 551, 675 approaches to, 183
of personnel, 19 of autologous donations, 190-191
of persons issuing blood, 385 for Babesia, 201
of phlebotomists, 370, 547 for bacterial contamination, 198-199
of problems and solutions, 26-29 for chikungunya virus, 202
of recipients, 226, 368-370, 384-385, 547, for CMV, 190
549, 550, 551 for dengue virus, 202
of umbilical cord blood, 739 for HBV, 196
Idiopathic thrombocytopenia, 463, 465, 517, for HCV, 196-197
568 for HEV, 203-204
IgA, 247, 248, 678-679, 680 historical overview of, 179-182
IgE, 247, 248 for HIV, 194-196
IgG, 247, 248, 249, 573 in HSC transplantation donors, 191-192,
IgG-coated cells (check cells), 376 714-715
IgM, 246, 248 for HTLV, 197
cold-reactive autoagglutinins, 437-439 international variations in, 192
in complement activation, 249 logistics of, 183
dispersing autoagglutination caused by, for malaria, 199-200, 201-202
301, 438 nucleic acid testing, 185-186
in infants, 573 for parvovirus B19, 203
in intravascular hemolysis, 251 for prions, 202-203
structure of, 247 reactive test results in, 186-190
Immediate-spin crossmatch, 380 residual infectious risks of transfusion, 192-
Immune complexes, 652 194
Immune thrombocytopenic purpura, 463, 465, serologic testing, 185
517, 568 for syphilis, 198
Immunity in tissue transplantation, 774
antibodies in, 246-248 for Trypanosoma cruzi, 200-201
complement in, 248-250 for umbilical cord blood, 732
extravascular hemolysis in, 250 in the United States, 184
Fc receptors in, 248, 249, 250 for West Nile virus, 200
in infants, 573 Infectious diseases
intravascular hemolysis in, 251 emerging agents, 203, 204
Immunoglobulin products, 526, 529-531, 532 safety precautions for, 47-55
Immunoglobulins, 246-248. See also specific screening for (See Infectious disease
immunoglobulins screening)
Immunohematology reference laboratories, transmitted by plasma derivatives, 203
419 transmitted by tissue transplantation, 777,
Immunomagnetic cell separation, 722 782
822 䡲 AABB TECHNICAL MANUAL
PBSC (peripheral blood stem cells). See Personal protective equipment, 44, 70-71
Peripheral blood HSCs for biosafety, 52-53
PCC. See Prothrombin complex concentrates for chemical safety, 58
PCR. See Polymerase chain reaction gloves, 45, 53, 70-71
PEDI-PAK system, 576, 577 Personnel
Pediatric patients (older than 4 months). See accidents and injuries in, 45, 49
also Neonates blood exposure in, 44-45, 47-48, 49
ABO antigens and antibodies in, 293, 300 competency assessment of, 7-8
CMV prevention in, 589-590 contact list of, 105
Lewis antigens in, 305 disaster plans for, 108-109
pretransfusion testing in, 300, 587 essential, 105, 109
thrombocytopenia in, 581 hepatitis prophylaxis for, 44
transfusions in identification of, 19
aliquoting for small volumes, 224, 575- latex allergies in, 45
576 orientation program for, 7-8
of CMV-reduced-risk components, 590 protective equipment for, 44, 52-53, 70-71
of Cryoprecipitated AHF, 578, 583 records, 19
of FFP, 578, 583, 589 safety monitoring programs for, 44
of granulocytes, 584, 589 selection of, 7
in HSC transplantation patients, 639- staffing plan, 9, 108-109, 110-111
640 training (See Training)
of irradiated components, 590 PF4 ELISA, 465-466
of leukocyte-reduced components, 590 pH, 406, 411
massive, 591 pH meters, 37
of platelets, 578, 581, 582, 589, 590 Phenotype. See also specific blood groups
of RBCs, 578, 586-589 calculations for, 274, 421
with sickle cell disease, 587-588, 639 defined, 261-262
syringe infusion pumps for, 549 and genetic mutations, 264
with thalassemia, 588-589, 640 and genotypes, 240, 261-262, 285-287, 402
vascular access for, 547 nomenclature for, 280
of volume-reduced components, 590 prevalence of, 274
of washed components, 590-591 rare, 394-395, 421
Pedigrees, 265, 266 Phenotyping
Peer review, 24-25, 697-707 antigen-matching, 281, 329-330, 379, 588,
PEG. See Polyethylene glycol 654
Penicillin, 442 autologous red cells, 401-402
Performance improvement standards, 26 with DNA-based assays, 280-281, 282
Periadventitial cells, 758 for antigen-negative donors, 282, 285
Pericytes, 758 to confirm D type of donors, 285, 330
Peripheral blood HSCs to distinguish alloantibody from
allogeneic, 715-717 autoantibody, 282, 283
autologous, 714-715 in prenatal practice, 283-285, 330
collection of, 718, 719-720 in recently transfused patients, 240, 281,
cryopreservation of, 722-723 282, 330
donor eligibility of, 714-717 when red cells are coated with IgG, 282,
engraftment kinetics of, 717-718 283, 401-402, 436
infectious disease testing on donors of, 191, donor units, 420
714-715 solid-phase assays for, 242-243
infusion of, 724-725 Phlebotomy. See also Blood collection
mobilization of, 719-720 adverse reactions to, 142-146
patient survival with, 718 blood loss due to, 609
processing, 720-722 for collection of blood samples, 368, 370
quality control of, 723 disinfection methods for, 140-141
regulations for, 87, 88, 89, 725 of donors, 140-146
shipping and transport of, 723-724 vein selection for, 140
828 䡲 AABB TECHNICAL MANUAL
Red cell antibodies. See also specific blood for quality systems, 1-2
groups for radioactive materials, 60-61
with autoantibodies, 432-435 recalls and withdrawals, 89, 90
and with HDFN, 338-340, 347-348, 413-414, safety, 39-40, 60-61, 68-69
562 for tissue, 777-778
and with hemolytic transfusion reactions, Reissuing blood products, 227-228, 550
338-340, 413-414, 686-687 Rejuvenated RBCs, 216
clinical significance of, 338-340, 347-348, Relationship testing, 276-277, 493
376, 392, 413-414, 419 Relative risk, 494
defined, 391 Remedial action, 26, 27
detection of (See Antibody detection) Renal failure, 652, 674
disease associations with, 392 Reports
distinguishing alloantibodies from of adverse events related to tissue grafts,
autoantibodies, 283 782
dosage effect of, 264, 393, 410 facility quality, 27
effect of DTT on, 413-414 of fatalities, 20, 45, 87, 145, 691
effect of enzymes on, 413-414 of injuries, 45, 87
to high-prevalence antigens, 392-393, 394- internal event, 20, 21-22
395, 415-416, 564 Requests for transfusion, 367-368, 383, 546,
high-titer, low-avidity, 409-410 699-704
identification of (See Antibody Requirement, defined, 34
identification) Respiratory distress, 658-659, 680-681
low-affinity, 437 Restriction fragment length polymorphism
to low-prevalence antigens, 416 analysis, 238
multiple, 412, 414 Retrospective audits, 699, 701-702, 703-704,
naturally occurring, 391, 392 707
nonhemolytic, 251-252 Reverse transcriptase PCR, 236-237
in selection of units, 379, 419-421 RFLP. See Restriction fragment length
serologic reactivity of, 413-414 polymorphism analysis
in sickle cell disease, 329-330, 379, 588 Rh Immune Globulin, 565-566
in tissue transplant patients, 777 antepartum administration of, 564, 565
Red cell exchange, 646, 653-654, 655, 675 in antibody identification problems, 392
Red cell losses, in apheresis, 170, 171 dosage for, 565-566
Red cell reduction, 720-721 positive DAT after, 428
Red cell substitutes, 507 postpartum administration of, 565-566
Reference laboratories, 419 serology and mechanism of, 566
Refrigerators, 36, 214 use in platelet transfusions, 515
Regenerative medicine, 753-754. See also Stem Rh system, 317-333
cells antibodies of, 331, 338
Registration antigens of, 259, 318-319, 321-330
of donors, 118-119 C/ c and E/ e, 328-330
of facilities, 84, 86 D, 323-328
Regulatory issues, 83-91 G, 328
for biological products, 84, 85 characterization of, 317, 319
for blood-related devices, 83-84, 87 clinical considerations for, 328
for cellular therapy products, 760, 796- ethnic differences in, 318-319, 320, 321,
797 325, 326, 327
for emergencies, 109-111 genes and proteins of, 259, 272, 273, 319,
FDA inspections, 86-87 320, 321, 325
hospital regulations and accreditation, 91, genotypes, 321, 322-323
603-604 haplotypes in, 319, 320, 321-322
for HSCs, 87-89, 725, 743-746 phenotypes, 321-323
licensure and registration, 84, 86 RhAG, 261, 331, 340, 360
medical laboratory laws and regulations, Rhnull, 273, 331
89-91 terminology for, 318-319, 319, 320
834 䡲 AABB TECHNICAL MANUAL
Sexual contacts, of blood donors, 124, 125, 126 Source Plasma, 170
Sharps injuries, 49, 141 Specific gravity, of blood cells and
Shipping components, 143
blood components, 146-147, 150-151, 213- Specification, defined, 34
214, 215-220, 225 Spills
containers for, 38, 225, 724-725, 738 blood, 53, 54
in disaster planning, 107-108 chemical, 59, 78-82
frozen products, 225, 724, 738 radioactive, 62
hazardous materials, 63 “Splits,” 482
HSCs, 723-724, 734, 737-738 SPRCA. See Solid-phase red cell adherence
labeling, 738 testing
monitoring temperature during, 213-214, SSOP. See Sequence-specific oligonucleotide
724-725, 738, 739, 779-780 probes
MSCs, 761-762 SSP. See Sequence-specific primers
plasma derivatives, 220 Staffing, 9, 108-109, 110-111
samples, 63 Standard Operating Procedures. See
tissue, 220, 779-780 Procedures
Shock, 501-502, 674 Standard precautions, 48
Short tandem repeat analysis, 277 Standards
Showers, emergency, 58 for biosafety, 47
Siblings, 266, 478 for performance improvement, 26
Sickle cell disease for quality management, 1- 2
alloimmunization in, 329-330, 379, 588, for tissue transplantation, 778-779
639 Staphylococcal protein A absorption, 657
blood selection in, 379, 587-588, 654 Stem Cell Therapeutic and Research Act, 743
delayed transfusion reactions in, 588, 687 Stem cells
genotyping for, 283, 330 adipose-derived, 755, 758-759, 762, 794
in HSCT patients, 639 embryonic, 795
in pediatric patients, 587-588, 639 hematopoietic
red cell exchange in, 587, 654 collection of, 718-720, 733-734
separation of transfused from autologous cryopreservation of, 722-723, 736-737
cells in, 401 donors of, 714-717
transfusion in, 379, 587-588 infusion of, 724-725
Side effects. See Adverse reactions irradiation of, 638
Signs, safety, 46, 48-49, 57, 58 processing, 720-722
Silent mutations, 264, 267, 286-287 quality control of, 723, 736
Single nucleotide polymorphisms, 240, 264 regulation of, 87, 88, 89, 725
Sipuleucel-T, 791 shipping and transport of, 723-724, 737-
Six Sigma, 28-29 738
Skeletal repair, with MSCs, 763 sources of, 717-720
Skin appearance, in donors, 120 storage of, 736-737
Skin grafts, 123, 775, 777 thawing, 721
Solid-phase red cell adherence testing washing, 721, 740-741
for detection of HLA antibodies, 487 induced pluripotent, 755, 795-796
for detection of platelet antibodies, 463-464 mesenchymal
for phenotyping red cells, 242-243 autologous vs allogeneic use, 754
for platelet crossmatching, 460 future directions for, 765-766
for pretransfusion testing, 377, 396 identification criteria for, 793
Soluble substances, 406 isolation and expansion of, 758-760,
Solutions 792-793
additive, 136-137, 138, 576-577 multipotentiality of, 754-755, 791-792
anticoagulant-preservative, 136, 137 properties of, 756-757, 758, 786, 791-792
intravenous, 552 research and development on, 764-765
Solvent/detergent-treated plasma, 153, 203, shipping, 761-762
204, 205 sources of, 754-758
836 䡲 AABB TECHNICAL MANUAL