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Laboratory Quality Assurance Program
Guidelines
for
Laboratory
Practice
General
Anatomic
Pathology
Chemistry
Hematology
Transfusion
Medicine
2012
Edition
Diagnostic Quality Assurance Program
College of Physicians &Surgeons of Saskatchewan
GUIDELINES FOR LABORATORY PRACTICE ‐ 2012
SUMMARY
OF
LABORATORY
GUIDELINES
CHANGES
The
following
GUIDELINES
have
been
revised,
added,
or
deleted
in
this
edition
of
the
document.
REVISED:
Performance
of
Whole
Blood
Glucose
Testing
Urinalysis
Retention
Guideline
Differential
Performance
&
Referral
Practice
Guideline
ADDED:
Reference
Textbook
List
Indirect
Platelet
Count
Procedure
for
Platelet
Estimate
Establishing
Conversion
Factor
for
PLT
Estimation
Procedure
for
WBC
Estimate
Establishing
Conversion
Factor
for
WBC
Estimation
Protocol
for
Validation
of
Linearity
on
Automated
Hematology
Analyzers
DELETED:
Laboratory
Guidelines
2012
Edition
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1
Diagnostic Quality Assurance Program
College of Physicians &Surgeons of Saskatchewan
Table of Contents
General
4
Retention
Guideline
5
Reference
Textbook
List
8
Anatomic
Pathology
10
Specimens
Exempt
from
all
Gross
&/or
Microscopic
Pathology
Laboratories
11
Surgical
Pathology
Reports
13
Removal
of
Tissue,
Blocks
or
Slides
from
the
Original
Hospital
Site
14
Performance
of
Non‐Gynecological
Cytology
16
Follow‐up
Reports
for
Gynecological
Cytology
17
Follow‐up
Program
for
Cytology
18
Chemistry
19
Estimated
Glomerular
Filtration
Rate
–
eGFR
20
Cholesterol/Triglyceride/Lipid
Testing
21
Urinalysis
22
Reporting
Sperm
in
Urine
22
Quality
Control
23
Performance
of
Whole
Blood
Glucose
Testing
24
Diagnosis
and
Monitoring
of
Thyroid
Disease
26
Presence
of
Small
Amounts
of
Albumin
29
Crosscheck/Validation
Guideline
for
Those
Facilities
with
Multiple
Chemistry
Instruments
30
Procedure/Method
Statistical
Work‐up/Validation
Study
Guidelines
31
Hematology
33
Principles
for
Hematology
Practice
34
Hematology
Films/Labelling
of
Slides
35
Morphology
of
Lymphocytes
36
Differential
Performance
and
Referral
Practice
Guideline
38
Red
Blood
Cell
Morphology
Reporting
Guideline
40
Differential
Quality
Control
42
Smudge
Cells
45
Flow
Cytometry
for
the
Diagnosis
of
Lymphoma/Leukemia
46
Malaria
47
Erythrocyte
Sedimentation
Rate
(ESR)
48
D‐dimer
49
3.2%
Na
Citrate
Anticoagulant
Recommended
vs.
3.8%
for
Coagulation
Studies
53
Vitamin
B12
&
Folate
54
Bleeding
Time
56
Semen/Sperm
Analysis
57
Crosscheck
Validation
Guideline
for
Facilities
with
Multiple
Hematology
Instruments
58
Procedure/Method
Statistical
Work‐up/Validation
Study
Guidelines
59
Protocol
for
Validation
of
Linearity
on
Automated
Hematology
Analyzers
61
Procedure
for
WBC
Estimate
64
Laboratory
Guidelines
2012
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Diagnostic Quality Assurance Program
College of Physicians &Surgeons of Saskatchewan
Establishing
Conversion
Factor
for
WBC
Estimation
66
Procedure
for
Platelet
Estimates
68
Establishing
Conversion
Factor
for
PLT
Estimation
70
Indirect
Platelet
Count
72
Transfusion
Medicine
74
Retention
of
Transfusion
Medicine
Records
75
Procedure/Method
Statistical
Validation/Work‐up
Guidelines
–
Trans.
Med.
76
Laboratory
Guidelines
2012
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Diagnostic Quality Assurance Program
College of Physicians &Surgeons of Saskatchewan
GENERAL
Laboratory
Guidelines
2012
Edition
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Diagnostic Quality Assurance Program
College of Physicians &Surgeons of Saskatchewan
RETENTION GUIDELINE
Laboratories vary in size, facility and extent of services provided. Clinical laboratories must
maintain thorough, accessible records that can demonstrate an acceptable standard of care and
compliance with the accreditation requirements.
The Laboratory Quality Assurance Program of the College of Physicians and Surgeons urges
laboratories to retain records, materials, or both for a longer period of time than specified for
educational and quality improvement needs.
Laboratories must establish policies that meet or exceed the following minimum requirements
for retention of documents and specimens as established by professional and/or regulatory
organizations.
Laboratory
Guidelines
2012
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Diagnostic Quality Assurance Program
College of Physicians &Surgeons of Saskatchewan
Laboratory
Guidelines
2012
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Diagnostic Quality Assurance Program
College of Physicians &Surgeons of Saskatchewan
Urine
- 24 hrs after
report has been
finalized
Laboratory
Guidelines
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Diagnostic Quality Assurance Program
College of Physicians &Surgeons of Saskatchewan
Microbiology
1) Manual of Clinical Microbiology 10th Edition; American Society for Microbiology; Murray,
Patrick R.
2) Bailey & Scott's Diagnostic Microbiology 12th Edition, Mosby, Inc.; Forbes, Betty A., et al.
Transfusion Medicine
1) Circular of Information, Canadian Blood Services (most recent version)
2) Canadian Standards Association Blood and Blood Component Z902 (most recent version)
4) Modern Blood Banking and Transfusion Practices 5th Edition; Harmening, Denise M.
5) Canadian Medical Association Journal Guidelines for Red Blood Cell and Plasma
Transfusion for Adults and Children; supplement to CAN MED ASSOC J 1997;
156 (11)
7) Bloody Easy 3: Blood Transfusions, Blood Alternatives and Transfusion Reactions 3rd
Edition; Ontario Regional Blood Coordinating Network
Hematology
1) Color Atlas of Hematology, Hematology and Clinical Microscopy Resource Committee,
CAP; Glassy, Eric F.
3) Hematology: Clinical Principles and Applications 3rd Edition; Rodak, Fritsma, Doig
Chemistry
1) Tietz Fundamentals of Clinical Chemistry 6th Edition, W. B. Saunders Company; Burtis,
Ashwood, Bruns
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Guidelines
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Diagnostic Quality Assurance Program
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Urinalysis
1) Graff’s Textbook of Urinalysis and Body Fluids 2nd Edition; Mundt, Shanahan
Anatomic Pathology
1) Histotechnology: A Self Instructional Text 3rd Edition; Carson & Hladik
Safety
1) Transportation of Dangerous Goods Act and Regulations
Supplement Canada Gazette, Part II [www.tc.gc.ca/eng/tdg/clear-tofc-211.htm]
Competency Evaluation
1) Canadian Society of Medical Laboratory Science
PO Box 2830 LCD 1
Hamilton, ON L8N 3N8
website www.csmls.org
Certification - Competency Profiles
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Guidelines
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Diagnostic Quality Assurance Program
College of Physicians &Surgeons of Saskatchewan
ANATOMIC
PATHOLOGY
Laboratory
Guidelines
2012
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Diagnostic Quality Assurance Program
College of Physicians &Surgeons of Saskatchewan
Irrespective of the exemptions listed below, gross &/or microscopic examinations will be
performed whenever the attending physician requests it, or at the discretion of the pathologist
when indicated by gross findings.
Laboratory
Guidelines
2012
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Diagnostic Quality Assurance Program
College of Physicians &Surgeons of Saskatchewan
Laboratory
Guidelines
2012
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Diagnostic Quality Assurance Program
College of Physicians &Surgeons of Saskatchewan
Timeliness of reports is critical to providing quality of care. Guidelines for surgical pathology
reporting include:
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Guidelines
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Diagnostic Quality Assurance Program
College of Physicians &Surgeons of Saskatchewan
Anatomic Pathologists are charged with the responsibility of keeping and guarding the integrity
of the ever-growing number of tissue containing paraffin blocks and slides derived from surgical,
cytological and autopsy diagnostic services. Documentation and maintenance (tracking) of the
continuous care shall ensure quality practice. These specimens must be maintained in orderly
files to ensure ready access. There are inevitably increasing demands for slides, blocks or tissues
to be retrieved from the original site.
*A release form must be provided and retained on file at the original institution for permanent
release.
Summary
Follow HIPA guidelines
Ensure there is sufficient material for further work-up
Indicate reason for request
Return all material as soon as possible
The lab is the custodian of tissue, blocks or slides collected.
The source of material remains the property of the patient.
In-Province Consultation
• Request may be initiated by the primary physician, surgeon or oncologist for review by local
or out-of-district pathologist.
• Request may be initiated by the original signing out pathologist who is responsible for
maintaining records and assuring return of the material.
Note: The return of materials to the original site must be documented and a consult report sent
to the original pathologist as well as the requesting pathologist.
Educational Consultation
• Requests for educational rounds should be restricted to slides; to ensure integrity of patient
property.
• Request should indicate “for rounds” and materials returned promptly to ensure ongoing
patient care.
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Guidelines
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Diagnostic Quality Assurance Program
College of Physicians &Surgeons of Saskatchewan
Research
• Regulations require pathologists to obtain patient authorization and/or an Institutional
Review Board (Ethics Committee) waiver of informed consent when using any identifiable
patient health information for research purposes.
• Requests must ensure the integrity of the patient material.
• All materials that have critical diagnostic, prognostic or medical-legal implication may be
retained at the discretion of the releasing institution.
• Return all materials as soon as possible.
References:
Guardians of the Wax…and the Patient. Editorial.; American Journal of Clinical Pathology 1995 104 p 356-7
Use of Human Tissue Blocks for research. Association of Directors of Anatomic and Surgical Pathology. Human Pathology
1996.27 p 519-520
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Guidelines
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Diagnostic Quality Assurance Program
College of Physicians &Surgeons of Saskatchewan
Some aspects of NGC require a rapid turnaround time such as FNAB performed under CT-scan
or ultrasound guidance and intra-operative cytology requests.
It is important for institutions with CT scanner facilities to be able to provide cytology service in
house. However, it is acceptable, if the lab does not have a cytology department, the
technologists in the histology lab are trained in processing the specimens. Such training is easily
obtained and can be provided by short courses provided on site and documented.
Most NGC procedures are performed on patients who are in-patient residents in a hospital/health
care institution or are required to come in for a day procedure/ambulatory care. Due to the time
factor involved in patients’ institution stay; a rapid turnaround time becomes a key factor in
availability of the service. On the other hand, the patient in an acute care setting may have an
infectious process or malignancy requiring rapid diagnosis and treatment.
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Guidelines
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Diagnostic Quality Assurance Program
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To ensure a quality cytology service, a follow-up mechanism must be in place to provide reports
to the primary and/or consulting physician.
In an attempt to eliminate the potential for “LOST – TO FOLLOW” reporting situations, the
following require follow-up letters to the primary and/or consulting physician:
1. A repeat smear was requested at the time of reporting and 3 months have lapsed since the
date of request.
2. A diagnosis is rendered requiring follow-up and none has occurred. For example:
HSIL required follow-up a.s.a.p. and if this has not occurred within 3 months, a letter
is required.
LSIL (ASCUS, AGUS) within 6 months requires a letter in 9 months.
A malignant diagnosis with no apparent followup.
3. A follow-up letter has been previously issued with no reply. These letters should be
automatically generated by the computer system and then replies must be recorded and
reviewed quarterly.
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Guidelines
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Diagnostic Quality Assurance Program
College of Physicians &Surgeons of Saskatchewan
A follow-up mechanism shall be in place to ensure that actions appropriate for abnormal findings
are implemented.
a) All patients who are reported to have a significant abnormality should be followed up by
the laboratory or other agency to which this task may be delegated, to obtain final clinical
or preferably tissue confirmation of the diagnosis.
b) Statistical data should be maintained which would include the number of cases screened
annually in each category, and all correlative follow-up data available. Discrepancies, if
any, should be included with this information.
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Guidelines
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Diagnostic Quality Assurance Program
College of Physicians &Surgeons of Saskatchewan
CHEMISTRY
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Guidelines
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Diagnostic Quality Assurance Program
College of Physicians &Surgeons of Saskatchewan
Evidence-based clinical practice guidelines suggest that an estimate of GFR (eGFR) provides the
best clinical tool to gauge kidney function. The Canadian Society of Nephrology has
recommended that laboratories report eGFR routinely for adult patients, in order to detect
chronic kidney disease.
Serum creatinine results can vary significantly and tend to be ineffective in general practice as an
early marker. 24 hr. collection for creatinine clearance is impractical and prone to error. eGFR
should be reported on outpatients over the age of 18 years. eGFR has not been validated for use
in hospitalized patients and therefore, is not recommended for reporting on inpatients.
Summary:
Reporting of eGFR is becoming the standard of care in helping identify, stage and monitor
patients with chronic kidney disease.
• eGFR is frequently used for DRUG DOSING using the Cockroft-Gault equation. eGFR-MDRD has not been
validated for this purpose.
• eGFR-MDRD assumes “steady state”. For rapidly changing kidney function, monitor serum creatinine.
(MDRD: Modification of Diet in Renal Disease)
• The reported eGFR shall be multiplied by 1.21 for patients of African descent.
NOTE: This information is intended for clinicians, patients and allied health professionals.
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Guidelines
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Diagnostic Quality Assurance Program
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The accomplishment of treatment goals also demands accurate cholesterol measurements. This
requires standardization of all cholesterol measurement for accuracy to minimize the method-
specific biases. This can be achieved ONLY by standardizing the cholesterol measurements and
ensuring accuracy that is traceable to the National Reference System for Cholesterol
(NRS/CHOL), National Cholesterol Education Program.
Clinical protocols are well established and should be followed by all testing sites.
Laboratories testing for lipids shall be capable of performing the entire profile, to include:
Cholesterol, Triglycerides, HDL and LDL, for diagnosis and assessment. All lipid
measurements should be performed by the same methodology.
Only instrumentation capable of maintaining intralaboratory precision that is less than or equal to
3 % (C.V.); and can demonstrate an accuracy bias of less than 3 % from the true value may be
used for cholesterol analysis.
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Guidelines
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Diagnostic Quality Assurance Program
College of Physicians &Surgeons of Saskatchewan
URINALYSIS
Complete urinalysis shall include microscopic examination if the following are present:
leukocyte esterase, blood, protein, turbidity and nitrites.
The microscopic examination should be completed within four hours of collection. (Note:
specimens not tested within 2 hours should be refrigerated.)
Sperm are not normally present in urine and if presence is detected, it is usually considered a
contaminant.
When sperm appear in a microscopic exam, they shall be reported as present. However, prior to
reporting, results should be discussed with the attending physician, in order to avoid errors (ie.
mislabelling, etc.).
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The purpose of QC is to detect the problems early enough to prevent their consequences.
QC emphasizes statistical control procedures, but may also include non-statistical check
procedures, such as linearity checks, reagent and calibration checks, etc.
Two or three different materials should be selected to provide concentrations that monitor
performance at different levels of medical decision-making.
For quantitative tests, the use of two levels of control material shall be run each day of use, as a
minimum.
For qualitative tests that include built-in controls, a positive and negative control shall be
performed a minimum of once per month and upon initiation of a new lot number and shipment.
For those that do not include a built-in control, known positive and negative external controls
shall be tested each day of use.
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Diagnostic Quality Assurance Program
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Glucose meters are a convenient, quick and simple means for clinicians and their patients to
obtain a blood glucose estimation. Glucose meters are not designed to diagnose diabetes and
should not be used to monitor seriously ill patients. The results from a properly used glucose
meter are accurate enough to determine insulin dosage and dietary compliance. Glucose meters
allow for frequent and rapid blood glucose estimations thereby improving the overall control of
the diabetic state. Glucose meter testing is intended to supplement rather than substitute for
clinical laboratory testing.
The basic requirements for whole blood glucose testing shall include proper training to ensure
consistent operating techniques and a quality control surveillance program to ensure proficiency.
The Clinical and Laboratory Standards Institute (CLSI) serves as the reference for this document.
All glucose meter testing, performed outside the traditional laboratory, excluding patients
performing glucose in his place of residence is required to meet the criteria of quality assurance
to ensure acceptable performance. The glucose meter is appropriate for monitoring treatment,
but not suitable for diagnostic testing (i.e. GTT) The components of glucose monitoring by
glucose meters will include, but not restricted to the following:
It is recommended that only one type of testing meter be used in a program. This is because of
potentially confusing differences among the various systems for testing and quality control
procedures.
CAUTION: very high hematocrits in newborns interfere with results, so specific instruments
shall be recommended for glucose estimations in newborns.
2. Internal QC
Daily controls ensure accurate performance of the meter. This may include:
- running controls once per day of use for each machine; alternating high and low
levels
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Guidelines
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Diagnostic Quality Assurance Program
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3. Proficiency testing
Proficiency testing is an important aspect of quality assurance and requires participation in
the recognized proficiency testing program designated/approved by the Accreditation
Program.
4. Training/Certification of Users
Training of personnel to perform glucose meter testing is critical to a successful program and
the following components are required:
establishment of a training/inservice program
initial certification on all meters in use (standardization of meters is highly
recommended).
training program of users with on-going evaluation as established by an inservice
program.
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Guidelines
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Diagnostic Quality Assurance Program
College of Physicians &Surgeons of Saskatchewan
Thyroid function tests are among the most commonly ordered laboratory tests in the province. In
the past, investigations of thyroid disease required more than one test. Sensitive thyroid
stimulating hormone (TSH) is the initial test in the diagnosis of hypothyroidism (TSH elevated
or above normal) and hyperthyroidism (TSH is suppressed or below normal). Free T4 (FT4) is
preferred over total T4 (TT4) measurement to confirm the diagnosis of hypothyroidism or
hyperthyroidism. FT4 is 0.02 – 0.04% of total T4. FT4 is the metabolically active form of TT4
and is a better indicator of thyroid status than TT4 because it is unaffected by protein binding
abnormalities such as pregnancy and oral contraceptives. Free T3 (FT3) is mainly of value in
diagnosing T3 toxicosis, in determining the T3 response to therapy, and clarifying protein binding
abnormalities. It can also be of use in the early progression of subclinical hyperthyroidism to
overt thyrotoxicosis when FT4 is normal and TSH is suppressed. FT3 is often the first to be
increased. FT3 is approximately 0.2 – 0.5% of total T3.
American Association of Clinical Endocrinologists (AACE) recommends that the TSH reference
range run from 0.3 - 3.0, versus the old range of 0.5 - 5.5. Keep in mind that there is
disagreement among practitioners.
Limitations:
1. These guidelines do not apply to neonates.
2. TSH is not reliable in the investigation of hypothalamic or pituitary disease.
3. TSH may be an unreliable indicator of thyroid status in patients with acute severe non-
thyroidal illness (e.g. CCU and ICU patients) and the test is only recommended when there
are clinical indicators of possible pre-existing thyroid disease.
4. Medications such as lithium, amiodarone, glucocorticoids, and dopamine affect TSH and
may also affect the individual’s thyroid status.
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Diagnostic Quality Assurance Program
College of Physicians &Surgeons of Saskatchewan
TSH
If TSH screen is abnormal, a FT4 will be done on the same sample reducing the need to call back
the patient for subsequent testing. If the TSH is less than 0.3mU/L a FT4 and FT3 will be done
on the same sample. Free T3 is a better indicator of the degree of thyrotoxicosis in most patients.
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Diagnostic Quality Assurance Program
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6. Subclinical hyperthyroidism: Patients with low or suppressed TSH but thyroid hormones in
the normal levels with minimal or no symptoms are followed by FT4 and/or FT3 at 6 –12
months to gauge the progression of their condition.
References:
1. HSURC guidelines. Nov 1992
2. Ontario Association of Medical Laboratories. Guidelines for the use of serum tests to detect thyroid dysfunction.
May 1987
3. Ontario Association of Medical Laboratories. Guidelines for the use of serum testing in the management of
primary hypothyroidism. June 1998
4. Alberta Medical Association. Laboratory Testing Guidelines for Investigation of Thyroid Dysfunction. May 1999
5. Protocol Steering Committee B.C. for the use of Thyroid function tests in the diagnosis and monitoring of patients
with thyroid disease. Aug 1997
6. The College of Physicians and Surgeons of Manitoba. Investigation of Thyroid Disease. February 1995
7. National Academy of Clinical Biochemistry. Laboratory Support for the Diagnosis and Monitoring of Thyroid
Disease. November 2000
8. Vanderpump MPJ, Ahlquist JAO, Franklyn JA et al 1996. Consensus statement of good practice and audit
measures in the management of hypothyroidism and hyperthyroidism. BMJ 313: 539-44.
9. Singer PA, Cooper DS, Lewy EG et al 1995. Treatment guidelines for patients with hyperthyroidism and
hypothyroidism. JAMA 273: 808-12.
10. Ladenson PW, Singer PA, Ain KB et al 2000. American Thyroid Association Guidelines for detection of thyroid
dysfunction. Arch Intern Med 160: 1573-5.
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Diagnostic Quality Assurance Program
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The Canadian Diabetes Association recommends testing for small amounts of albumin once/year
after the onset of diabetes.
The presence of small amounts of albumin in urine is detectable by dipstick methodologies, and
is approved as a screen for renal damage in the known diabetes patient.
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CROSSCHECK/VALIDATION GUIDELINE FOR THOSE FACILITIES WITH MULTIPLE CHEMISTRY/HEMATOLOGY INSTRUMENTS
PERFORMING THE SAME TEST PROCEDURE
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Diagnostic Quality Assurance Program
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PROCEDURE/METHOD STATISTICAL WORK-UP/VALIDATION STUDY GUIDELINES: CHEMISTRY/HEMATOLOGY
Qualitative Quantitative
1. Imprecision The variation in analytical results demonstrated when a particular Within run – use preferably a patient √
within run specimen of aliquot is analyzed multiple times or on multiple days. sample or pool close to the decision levels
between run Imprecision is expressed quantitatively by a statistic such as standard with a minimum of 10 data points.
deviation or coefficient of variations. Between run – 20 results from 20 separate
runs on 2 levels over a 10-day minimum
time period using appropriate QC material.
2. Patient The correlation coefficient is a means to look for a relationship, not 40 data points are recommended with a n=20 n=40
Correlation agreement, between pairs. Two methods may have a perfect minimum of 20 having 50% of the data
correlation throughout the measuring range but may not agree in points outside the reference intervals, if √ √
value (i.e. one may be double the value of the other). possible. Correlations should involve
comparison with an acceptable reference
method or laboratory.
3. Linearity (IFCC) The range of concentration or other quantity in the specimen 4 data points each in duplicate as a √
over which the method is applicable without modification (CLIS) minimum requirement, but 5 data points are
when analytical results are plotted against expected concentrations; preferred (over reportable range). Linearity
the degree to which the plot curve conforms to a straight line is a studies are expected on an initial method
measure of the system linearity. work-up and further studies as defined by
the College guidelines (i.e.
troubleshooting).
4. Reference It is common convention to define the reference range or interval of a The minimum requirement is 20 data points √
range laboratory test as the central 95% interval bounded by the 2.5 and for confirmation of an established reference
validation 97.5 percentiles of the selected patient population. Validation of an range and 120 for the establishment of a
established reference range requires a minimum of 20 samples. new reference range.
5. Accuracy Closeness of the agreement between the result of a measurement and 3 data points using acceptable reference √
the accepted reference value (true value of the analyte) material (i.e. CEQAL or CAP) 1 data point
may be acceptable for haematology
accuracy studies if related to sample
stability.
6. Sensitivity Measure of the ability of an analytical method to detect small Sensitive studies are only required for those √ √
quantities of the measured component. When concern is performance methods which have clinical relevance at When When clinically
at a very low concentration it is useful to determine the detection values close to “0” (i.e. TSH) clinically relevant
limit as influenced by imprecision. relevant
7. Specimen The conditions of handling and storage, which permits the No data generally required. As per √ √
Stability measurement and reporting of a clinically relevant result. manufacturer’s guidelines. If Storage and Storage and
manufacturer’s stability window is to be transportation transportation
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Diagnostic Quality Assurance Program
College of Physicians &Surgeons of Saskatchewan
extended a stability study is expected. dependent dependent
8. Interference The effect of any component of the sample on the accuracy of the Document the manufacturer’s interference √ √
measurement of the desired analyte. information. The method should include a Methods with Methods with
disclaimer or a process for dealing with a known known
lipemic, icteric or hemolyzed sample. interferences interferences
9. Recovery A recovery procedure involves the addition of a known amount of Recovery studies should only be necessary √
analyte to an aliquot of sample. Recovery is defined as the ratio of for those methods or analytes where Method
the amount of the analyte recovered to amount added and is given as organic extractions or equivalent are specific/organic
percentage. required as part of the methodology (i.e. extraction
Toxicology)
Work-up requirements when an instrument is moved from site “A” to site “B”: (It is assumed that the instrument has been in recent use
with acceptable performance).
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HEMATOLOGY
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Diagnostic Quality Assurance Committee
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Hematology incorporates leading edge technology to help decipher and treat troubling diseases.
As hematology has evolved, there are some generic principles that must be simply stated for
quality patient care.
Quality management practices are essential to ensuring quality care. Some of the core principles
include:
1) Hemoglobin, the single most common complex organic molecule (Hb) shall be determined
by spectrophotometric methodology.
2) WBC and platelet counts shall be tested by automated methodology as part of a CBC on
whole blood specimens. If necessary, WBC and platelet counts may be estimated on the
peripheral smear.
3) Manual PT (INR)/APTT testing shall be discontinued. Please refer to CLSI H21-A4 for
reference on collection and storage.
4) Reticulocyte Count shall be performed by automated methodology. Manual counts may used
as a QC method for automated analyzers.
5) All differential leukocyte counts shall be reported in absolute values. Reporting percentages
is optional, and would be in addition to absolute values.
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Diagnostic Quality Assurance Committee
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Unequivocal patient identification is the first step to ensuring a quality hematology slide.
With no formal standards in place for labelling slides, the basic principles require:
Unique identification of the patient (at least two identifiers)
Written instructions for labelling
Labels shall be clear and legible
Date of collection
Positive Patient ID
_______________________________ _______________________________
Last Name First Name
PHN______________________________________________________________
Date of Birth Unique ID #
Date of Slide___________________________________
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MORPHOLOGY OF LYMPHOCYTES
A variety of diseases and disorders may produce changes from the normal in numbers and/or
morphology and functions of one or more of the leukocytes. The most important feature of
variant lymphocyte morphology is the recognition of its benign nature. The pertinent fact is that
these lymphocytes are normal cells that have been altered as the result of a normal response to
stimulus. When changes in WBC’s are produced by non-malignant disorders (e.g. infections),
the cells formerly called atypical, are now referred to as reactive lymphocytes. When changes
are suspicious of being produced by malignant disorders (leukemias, lymphomas,
gammopathies) and additional investigations may be required, the cells are often referred to as
atypical and/or abnormal.
In Chronic Lymphocytic Leukemia, the lymphocytes are somewhat larger than normal, have
nuclei with clumped or condensed chromatin, and may have prominent nucleoli. The cytoplasm
may be abundant, nongranular and moderately basophilic, or it may be relatively scant.
In Lymphomas, peripheral blood involvement (i.e., abnormal circulating cells) is seen late in the
disease. Lymphoma cells can exhibit a variety of appearances and the cellular morphology is
variable and depends on the underlying type of lymphoma. These cells can exhibit variable size,
shape, nuclear, and cytoplasmic characteristics. Lymphoma cells are usually round to oval, and
can be irregular. Cell size ranges from 8 to 30 µm and the N-C ratio varies from 7:1 to 3:1. In
diffuse small lymphocytic lymphoma (the tissue equivalent of chronic lymphocytic leukemia),
the cells are generally small with round to oval nuclei, compact and coarse chromatin, and have a
scant amount of basophilic cytoplasm. They may be the same size as normal lymphocytes or
may be slightly larger. Occasionally, the nuclei exhibit an angulated appearance with slightly
more open chromatin. A small nuclear indentation may be present. Nucleoli are not seen.
Scattered prolymphocytes, which are larger cells with a centrally placed nucleus, a prominent
single nucleolus, and moderate basophilic cytoplasm, often are seen. In the small-cleaved cell
lymphomas, the cells are slightly larger than normal lymphocytes and have an angulated
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appearance. The majority of nuclei have clefts, indentations, folds, convolutions, and may even
be lobulated. The chromatin is moderately coarse and one or more nucleoli may be prominent.
Their cytoplasm is scant to moderate and basophilic. The cells in small noncleaved lymphomas
(Burkitt’s lymphoma) appear similar to L3 lymphoblasts. These cells are generally moderate in
size (10 to 25 µm) and have a round to oval nucleus with moderately coarse chromatin, and one
or more prominent nucleoli. The cytoplasm is moderate, stains dark blue, and may contain
numerous small vacuoles. Large cell lymphomas and immunoblastic lymphomas may exhibit
some of the most blast-like and abnormal morphology. These cells are large (20 to 30 µm) and
have scant to moderate amounts of deeply basophilic cytoplasm. The nuclei are generally round
to oval, but may be angulated, folded, indented, or convoluted. Nucleoli are prominent and may
be single or multiple. Vacuoles can occasionally be seen in the cytoplasm. These cells can be
easily confused with blasts. T cell lymphomas can exhibit similar morphology to any of the
above types of lymphomas. The typical appearance is a moderate-size cell with a markedly
convoluted nucleus giving a cerebriform or grooved pattern. Their chromatin is moderately
coarse and nucleoli are not apparent. The cytoplasm is generally scant and blue.
In Hairy Cell Leukemia, the abnormal lymphocytes (Hairy Cells) have scant to abundant,
agranular, light grayish-blue cytoplasm. The plasma membrane appears irregular with hair-like
or ruffled projections, which are seen more easily with phase microscopy. These cells often have
a round or oval nucleus; sometimes, the nucleus appears folded or bilobed. The chromatin is
loose and lacy, and one or two nucleoli are commonly seen.
In Sézary Syndrome, the abnormal lymphocyte is larger than normal with scanty cytoplasm, and
the nucleus is large with clefting. Nuclear folding can be so extensive as to suggest an image of
the brain, and these nuclei are thus described as cerebriform. The nuclear chromatin is fine with
little condensation. There may or may not be visible nucleoli.
References:
McTaggart Bill, SAIT Hematology Updating Correspondence Course, 5th Edition, 1993. pp. 10.
Stiene-Martin, 1998, pp. 355-356, 484-485, 490, 507-508. College of American Pathologists, Surveys, Hematology Glossary,
2001
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Avoid using the terms anisocytosis and/or poikilocytosis since they convey no specific meaning.
The numeric value is meant for internal use to indicate a significant abnormality presence. No
numeric value is reported, just the abnormality.
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The following is a list of measures undertaken by each laboratory to ensure the quality of
differential results reported on patients.
4. Differentials must be repeated if each cell does not meet the limits set out in the table below.
If the repeat is still not within the established limits, a second technologist should repeat the
differential.
5. Whenever the tech1 has concerns with a differential the rest of the CBC can be released with
a notation “Differential to follow”. The requesting physician can be invited to review the
smear if they so choose. The smear should be evaluated as soon as possible.
6. Leukocyte abnormalities seen during a smear review require a manual differential completed
regardless of the protocol for when to perform a manual differential.
7. Perform the manual differential and compare it to the automated differential using the 95%
confidence limits table. Each cell should compare within the range set.
8. Differentials between technologists should also fall within the established limits (95%
confidence limits).
9. If the manual differential performed by two technologists agrees within the established
limits, but is not in agreement with the automated differential, then the manual differential
should be reported out instead of the automated differential.
1
Tech refers to the medical laboratory technologist or combined laboratory and x-ray technologist.
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10. It is good laboratory practice to circulate unknown QA slides quarterly. The results will be
compared with peers and should be within the 95% confidence limits as set by the following
table. Any problem areas will be covered between the technologist and the supervisor.
Procedure:
2. If a 100 cell differential was performed go to the column “n=100” to determine the
acceptable range. If a 200 cell differential was done refer to column “n=200”.
E.g. you counted 32 neutrophils in a 100-cell differential.
3. Determine acceptability of the differential by checking if the number you counted for a
certain cell falls within the stated range.
E.g. Stated range is 16-35%; you are within this range.
5. Determine acceptability of each cell line by comparing automated to manual differential. For
the neutrophils/granulocytes, segmented neutrophils, band neutrophils and other neutrophil
precursors must be added together and for lymphocytes, reactive lymphocytes and
lymphocytes must be added together.
E.g. 77 segs and 15 bands = 92%
Example:
The automated or technologist differential indicated the following differential and the acceptable
range for each number was looked up in “n=100” column:
Neu: 70 acceptable range 60-79
Lymph: 15 “ “ 8-24
Mono: 7 “ “ 1-12
Eos: 3 “ “ 0-9
A 100 cell differential is completed by another technologist and based on the acceptable range
the results are as follows:
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The 95% confidence limits for various percentages of leukocytes of a given type as determined
by differential counts on stained blood smears.
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SMUDGE CELLS
Distinguished by their naked amorphous nuclear chromatin material, smudge cells were initially
described as white blood cells with broken-down nuclei in patients with chronic lymphocytic
leukemia. Subsequently, these nuclear shadows have most often been referred to as smudge
cells, but the term basket cells is used synonymously.
The mechanism is often associated primarily with traumatic disruption of cells during blood film
preparation. In the process, the cell membrane ruptures and when viewed under a microscope,
what remains looks like a smudge, hence the term, smudge cells.
Education is needed (for the ordering physicians especially) to eliminate the risk of
misinterpreting this smudge cell count as a new cell type.
Smudge cells should be reported if greater than 10 per 100 leukocytes. Report smudge cells in
absolute numbers.
*Although CLL is not often diagnosed in patients under the age of 40, patients over 30 years of age should be
considered potentially at risk. CLL is rare in patients under 30 years of age.
*In children (18 & under), the smudge cells are not counted as part of the differential. However, the presence of
smudge cells may be noted on the report.
*Smudge cells are present in those candidates for which definitive diagnostic criteria are well established. Examples
include: CLL & Acute Leukemia.
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“Flow cytometric analysis has become an acceptable medical practice in the diagnosis and
characterization of hematologic neoplasia and its role in the management of patients with these
diseases is well recognized. Despite its extraordinary power, there is great concern regarding
the inconsistent practices and wide variation in styles among laboratories involved in the flow
cytometric analysis of leukemias and lymphomas. Of particular importance are the deficiencies
in standardization and validation of procedures used in the analysis, the manner by which the
information is generated and reported to pathologists or treating physicians, and the
appropriate utililzation of this technology in patient care.” (US-Cdn Consensus
Recommendations on the Immunophenotypic Analysis of Hematologic Neoplasia by Flow
Cytometry)
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MALARIA
Malaria can be a life threatening infection and a rapid diagnosis is necessary to institute
appropriate management in a timely manner. The diagnostic information required for patient
management is best obtained by microscopy; however, a rapid test (immunologically-based) is a
very useful screening test. The Rapid Diagnostic Tests (RDT) results shall be confirmed by
microscopy.
Microscopic Diagnosis:
1. The test should be available 24/7.
2. Upon receipt of specimens at the testing laboratory, TAT is 4-6 hours.
3. The testing should be based on the protracted (20 min.) examination of four thick and
four thin blood smears.
4. Positive slides must be confirmed by a laboratory physician and/or pathologist.
5. The information in the report should include the following; a) positive or negative, b)
speciation, c) quantification of parasitemia.
6. All positive results should indicate the requirement to report Malaria cases to Public
Health and a recommendation to consult with an Infectious Disease specialist as soon as
practically possible.
7. Results must be called to the physician by the testing laboratory
8. The laboratories must participate in an external/internal quality assurance program.
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The ESR is a commonly ordered test for the assessment of inflammation and is not meant to be
used to screen an asymptomatic person for disease.
ESR is indicated in the diagnosis and therapeutic monitoring of temperal arteritis and
polymyalgia rheumatica. The ESR may be helpful in resolving conflicting clinical evidence in
patients with rheumatoid arthritis and with the evaluation and management of patients with
specific autoimmune inflammatory or infectious disorders (e.g. pelvic inflammatory disease,
bacterial endocarditis, septic arthritis and osteomyelitis).
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D-dIMER
Assessment of D-dimer for the diagnosis of Disseminated Intravascular Coagulation (DIC) and
for exclusion of Venous Thromboembolism (VTE) (Deep Vein Thrombosis and Pulmonary
Embolism).
Background:
A number of clinical trials have confirmed the utility of several different rapid D-dimer assays
together with clinical risk stratification as a safe option in excluding DVT/PE and withholding
anticoagulant therapy.
D-dimers are degradation products of fibrin that can be measured as an indicator of in-vivo
fibrinolysis. Since any physiologic or pathologic clot formation is accompanied by fibrinolysis,
D-dimers indicate the presence of clotting. This fact is exploited in two different ways in clinical
practice:
1. The presence of D-dimer is used to confirm the presence of thrombosis, e.g., DIC.
2. The absence of D-dimer can be used to rule out the presence of thrombus/thrombi, including
VTE in an appropriate clinical setting.
DIC occurs as a result of inappropriate and excessive activation of the hemostatic system; it is an
acquired syndrome characterized by the intravascular activation of coagulation with loss of
localization arising from different causes. DIC may be acute or chronic. The diagnosis of DIC
requires demonstration of abnormal laboratory results quickly so that appropriate management
can be instituted. Ideally, the test results should be available within 30 minutes of sample being
received in the laboratory.
VTE can be a difficult clinical diagnosis. Historically these disorders, when suspected, have
been confirmed using imaging techniques. Imaging studies may not be performed routinely at
all hospitals.
The sensitivity of the D-dimer assay is critically important in these tests, since the clinician is
depending on a true negative test to exclude the suspected disease. Most studies have also
shown that systematic clinical assessment of patient risk factors (or pre-test probability of
disease) and D-dimer assays is important in establishing the unlikelihood of VTE and may be
used to withhold anticoagulant therapy.
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D-dimer Assays:
In-vivo, a heterogeneous mix of fibrin degradation products of different molecular weight
usually exists. Virtually all tests use a monoclonal antibody with varying reactivity against
epitopes on the D-dimer fragment of degraded plasma.
Many methods are available but it is recommended to use the ELISA technique.
Some assays are intended for point of care use, some are qualitative, some quantitative and all
may have different units, cut off values and reference ranges. Because the tests are also used to
rule out disease, a negative result is an important result and the lower cut off value is critical,
that is, the level of D-dimer using a particular test, below which you can safely conclude that
there is not thrombosis. The cut off value may be lower than the reference range and must be
validated clinically by imaging studies and clinical assessments. This means that clinical trials
must have shown, for a particular assay, that patients with levels below this cut off value have no
evidence of thrombosis by imaging studies and have minimal likelihood of returning in
subsequent days or months with thromboembolic disease.
Clinical Assessment:
Safe and successful implementation of strategy for VTE using a rapid D-dimer test requires the
laboratory to ensure appropriate selection of an assay, a cut off value, and units of reporting. The
clinical algorithm to determine pre-test probability is utilized when requesting diagnostics testing
(D-dimer, ultrasound or CT).
1. Ensure the cut off value used for excluding DVT and/or PE has been validated in a clinical
trial using your particular assay type. Refer to the package insert and discuss with the
manufacturer of your chosen method. Semi-quantitative latex agglutination methods
commonly used for diagnosis and monitoring of DIC are not typically validated for use in
exclusion of VTE.
2. Determine whether the particular cut off value recommended can be used for all patients
suspected of VTE, or only those with certain pre-test probabilities of VTE based on clinical
assessment.
3. The CPSS recommends that all locations performing D-dimer report in FEU µg/L. Ensure
the accuracy of your calculations if you need to convert to FEU µg/L.
4. Reports should also include a critical value and a cut-off value for VTE. A comment is to be
included to explain the difference between a cut-off value and reference range.
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Examples of clinical assessment tools previously published in Canada are shown here:
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Diagnostic Algorithms
PE Probability
D-Dimer
Positive
CTPA
Negative
Negative
STOP Positive
TREAT
DVT Probability
Normal Positive
STOP TREAT
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The International Standards Committee on Thrombosis and Hemostasis, and the CLSI have
recommended guidelines to standardize whole blood collection to 3.2% sodium citrate
anticoagulant.
Several publications have indicated that the clotting times for PT and APTT are significantly
shortened with the 3.2% NaCitrate. For this reason, exchange between the two concentrations is
not acceptable. When selecting an anticoagulant for collection, it is important to note that 3.2%
NaCitrate is used for ISI assignments for thromboplastin. For these reasons, it is important for
laboratories to standardize the choice of anticoagulant for sample collection.
The anticoagulant used for coagulation assays should be 3.2% trisodium citrate. The proportion
of blood to anticoagulant volume is 9:1. Inadequate filling of the collection device will decrease
this ratio, and may lead to inaccurate results. The manufacturer recommendations should always
be followed.
If the hematocrit is very high and the usual relative volumes of blood and citrate solution are
mixed, a prolonged prothrombin time results from overcitration of the reduced proportion of
plasma in the blood sample. This means the final citrate concentration in the blood should be
adjusted in patients who have hematocrit (PCV) values above 0.55 (55%). Adjust the volume of
NaCitrate in the draw tube by applying the calculation outlined in the CLSI H21-A3. Note: To
maintain the vacuum in the NaCitrate collection tube, use a tuberculin syringe to draw out the
anticoagulant.
The anticoagulant volume may be calculated from the expression:
X = (100 – PCV) x draw volume of tube (mLs)
(595 – PCV)
References:
H21-A3 CLSI, BD Vacutainer Systems, Azko Nobel
Assessment of the influence of citrate concentration on the international Normalized Ratio (INR) determined with twelve
reagent-instrument combinations. Chantarangkul, Tripodi, Clerici, Negri, Mannucci
Effect of concentration of trisodium Citrate Anticoagulant on Calculation of the international Normalized Ratio and the
International Sensitivity Index of Thromboplastin, Duncan, Casey, Duncan, and Lloyd
The Prothrombin Time Test: Effect of Varying Citrate Concentration, Ingram, Hills.
Comparison of 3.2% vs. 3.8% Sodium Citrate Anitcoagulant Collection Tubes for Coumadin, Heparin, Abnormal and Normal
Specimens. Phillips, Sunnybrook ON
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Background:
The normal proliferation of cells depends on adequate folate and vitamin B12. Folate is
necessary for efficient thymidylate synthesis and production of DNA. B12 is needed to
successfully incorporate circulating folic acid into developing RBCs retaining folate in the RBC.
MEASURING BOTH B12 AND FOLATE LEVELS IS NOT NECESSARY IN ALL PATIENTS.
Serum B12:
The clinical indications for ordering serum B12 include:
1) Evaluation of patients with MACROCYTIC (high MCV in the CBC) anemia and the clinical
information suggesting possible B12 deficiency;
2) Evaluation of patients with psychiatric and neurologic impairment (symptoms of subacute
combined degeneration of spinal cord).
Practice Tips:
1. Persons on B12/folate supplements or other multivitamins do not require testing.
2. Lab tests are used to confirm a specific diagnosis and not as a fishing expedition.
3. Marginal B12 deficiency in any elderly patient with dementia, peripheral neurological
symptoms or impaired immunity should be taken seriously.
4. B12 should not be done on patients on oral contraceptives.
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Measuring both B12 and Folate levels is not necessary in all patients.
CBC
Normal MCV ↑
Neurological or Clinical
symptoms suggestive of B12
&/or Folate deficiency
No further testing
YES
Notes:
*B12 tests should not be done if the person is on B12
*False low B12 in pregnant women and women on oral contraceptives
Serum folates are not a useful screening tool
Folic Acid Deficiency is rare due to folate fortification in food
RBC FOLATE IS MORE INDICATIVE OF TISSUE FOLATE LEVELS
Folate tests will not be done if the person is on folate
B12 and RBC
folate
Abnormal
Normal
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BLEEDING TIME
Bleeding Time (BT) is defined as the time between the making of a small incision and the
moment the bleeding stops. It indicates how well platelets interact with blood vessel walls to
form blood clots (a test of platelet and vascular function).
Purpose:
Bleeding time is used most often to detect qualitative defects of platelets, e.g. Von Willebrand’s
disease (vWD). However, it is prolonged in many situations including vascular disorders, e.g.
Ehler-Danlos’ syndrome, pseudoxanthoma elasticum.
Issue:
The use of BT is declining and at many institutions this test has been eliminated. This is because
of three main reasons:
♦ It is neither a sensitive nor a specific test.
♦ A specific diagnosis of vWD can be made based on history and other tests.
♦ More reliable information about platelet function can be obtained by other tests.
Notes:
Use of bleeding time is not recommended (not by itself). Current practice varies and if BT is
required, the recommended method is the template device. BT is generally not recommended for
children under the age of 5.
Reference Interval:
Results are reported to the nearest half-minute. The reference range varies and is usually 1.5-9.5
minutes. The test should be discontinued if the patient hasn’t stopped bleeding by 15-20 minutes.
References:
Andrew M, Paes B, Bowker J, et al, "Evaluation of an Automated Bleeding Time Device in the Newborn,"Am J Hematol, 1990,
35(4):275-7.
Basili S, Ferro D, Leo R, et al, "Bleeding Time Does Not Predict Gastrointestinal Bleeding in Patients With Cirrhosis,"J Hepatol,
1996, 24(5):574-80.
Brown BA, Hematology: Principles and Procedures, 6th ed, Philadelphia, PA: Lea and Febiger, 1993, 267-70.
de Rossi SS and Glick MG, "Bleeding Time: An Unreliable Predictor of Clinical Hemostasis,"J Oral Maxillofac Surg, 1996,
54(9):1119-20.
George JN, Shattil SJ: The Clinical Importance of Acquired Abnormalities of Platelet Function. NEJM 324:27-29, 1991.
Gewirtz AS, Miller ML, and Keys TF, "The Clinical Usefulness of the Preoperative Bleeding Time,"Arch Pathol Lab Med, 1996,
120(4):353-6.
Henry, J. B. Clinical Diagnosis and Management by Laboratory Methods. Philadelphia: W. B. Saunders Co., 1996.
Munro J, Booth A, and Nicholl J, "Routine Preoperative Testing: A Systematic Review of the Evidence,"Health Technol Assess,
1997, 1(12):1-62.
Lewis SM, Ban BJ, Bates I. Dacie and Lewis Practical Haematology. 2006. Churchill Livingstone Elsevier, Philadelphia (PA).
Lind SE: Prolonged Bleeding Time. Am J Med 77:305-312, 1984.
Peterson P, Hayes TE, Arkin CF, et al, "The Preoperative Bleeding Time Test Lacks Clinical Benefit,"Arch Surg, 1998,
133(2):134-9.
Rodgers RP and Levin J, "A Critical Reappraisal of the Bleeding Time, "Semin Thromb Hemost, 1990, 16(1):1-20.
Triplett DA. Laboratory Evaluation of Coagulation, 1982. American Society of Clinical Pathologists Press, Chicago (IL).
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SEMEN/SPERM ANALYSIS
Semen analysis is a key component in the evaluation of male fertility, which includes more
complex testing.
The minimum WHO requirements for basic semen analysis for assuring quality of a screening
test for fertility include the evaluation of liquefaction, appearance, viscosity, volume, viability,
count, motility and morphology. For fertility purposes, a screening count is considered a useless
test.
a) Any technologist trained in counting cells (CLXT &/or MLT) may perform seminal counts in
post-vasectomy cases.
b) Tests for the evaluation of fertility shall include: sperm count, sperm motility, viability,
sperm morphology and special stains, as required, according to the WHO criteria.
When a physician specialist has experience in sperm analysis and can demonstrate
competency for fertility investigations, then fertility testing may be performed.
c) Proficiency testing is mandatory for all semen analysis testing, excluding post-vasectomy
counts.
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Work-up Definition: CLIS or International Federation of Clinical Minimum Data Requirements (where Applicability:
Element Chemistry (IFCC) appropriate):
Qualitative Quantitative
1. Imprecision The variation in analytical results demonstrated when a particular Within run – use preferably a patient √
within run specimen of aliquot is analyzed multiple times or on multiple days. sample or pool close to the decision levels
between run Imprecision is expressed quantitatively by a statistic such as standard with a minimum of 10 data points.
deviation or coefficient of variations. Between run – 20 results from 20 separate
runs on 2 levels over a 10-day minimum
time period using appropriate QC material.
2. Patient The correlation coefficient is a means to look for a relationship, not 40 data points are recommended with a n=20 n=40
Correlation agreement, between pairs. Two methods may have a perfect minimum of 20 having 50% of the data
correlation throughout the measuring range but may not agree in points outside the reference intervals, if √ √
value (i.e. one may be double the value of the other). possible. Correlations should involve
comparison with an acceptable reference
method or laboratory.
3. Linearity (IFCC) The range of concentration or other quantity in the specimen 4 data points each in duplicate as a √
over which the method is applicable without modification (CLIS) minimum requirement, but 5 data points are
when analytical results are plotted against expected concentrations; preferred (over reportable range). Linearity
the degree to which the plot curve conforms to a straight line is a studies are expected on an initial method
measure of the system linearity. work-up and further studies as defined by
the College guidelines (i.e.
troubleshooting).
4. Reference It is common convention to define the reference range or interval of a The minimum requirement is 20 data points √
range laboratory test as the central 95% interval bounded by the 2.5 and for confirmation of an established reference
validation 97.5 percentiles of the selected patient population. Validation of an range and 120 for the establishment of a
established reference range requires a minimum of 20 samples. new reference range.
5. Accuracy Closeness of the agreement between the result of a measurement and 3 data points using acceptable reference √
the accepted reference value (true value of the analyte) material (i.e. CEQAL or CAP) 1 data point
may be acceptable for haematology
accuracy studies if related to sample
stability.
6. Sensitivity Measure of the ability of an analytical method to detect small Sensitive studies are only required for those √ √
quantities of the measured component. When concern is performance methods which have clinical relevance at When When clinically
at a very low concentration it is useful to determine the detection values close to “0” (i.e. TSH) clinically relevant
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Work-up requirements when an instrument is moved from site “A” to site “B”: (It is assumed that the instrument has been in recent use with
acceptable performance).
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Purpose:
To validate the entire range through which patient values can be reported or to verify the
manufacturer’s stated linearity from the analysis of undiluted or diluted specimens for all
measured parameters.
Specimen Selection:
• For each parameter requiring linearity validation, choose a specimen with results at/near or
above the high end of the linear range published by the manufacturer of the instrument.
Ensure that there were no interfering substances noted during analysis of the specimen. This
value is equivalent to 100% while patient’s plasma or instrument diluent is equivalent to 0%.
• For WBC use a leukocytosis sample diluted in patient’s own plasma or instrument diluent.
• For PLT count use a thrombocythemic sample diluted in patient’s own plasma or instrument
diluent.
• Ensure that sufficient specimen is collected for dilution preparations and instrument
aspiration.
Procedure:
1. Verify that instrument reagents are not expired and that sufficient volume of reagents is
loaded on instrument to cycle all the prepared dilutions.
2. Check that background counts, instrument precision and calibration of the instrument are
acceptable before starting procedure.
3. Determine the volume per dilution by calculating the volume required for aspiration on the
instrument. One specimen may not provide sufficient volume. If more than one tube is
drawn, pool the tubes into one aliquot.
4. Label five clean plastic tubes 80%, 60%, 40%, 20% and 10%.
5. Prepare the dilutions using the original specimen as the 100% as shown in table provided
below. Make sure the 100% sample remains well mixed throughout the dilution preparation
process.
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6. Analyze each well-mixed dilution in triplicate on the instrument. Results with suspect
parameter flags should not be used.
7. Record all results for each parameter from all three runs on table provided.
8. Calculate Obtained Mean value for each parameter.
9. Calculate Expected value for each parameter by multiplying the Reference Mean (100%
Obtained Mean) by the multiplication factor.
10. Calculate the Difference between Obtained Mean and Expected Mean.
11. To graphically illustrate linearity, plot each parameter on linear graph paper with the
obtained mean value for the parameter on the X axis and the expected value for the
parameter on the Y axis. Draw a line through all the points on the graph. When the
obtained mean is plotted against the corresponding expected value, the plotted curve will
approximate a straight line for a linear method. Excel spreadsheet can also be used to
show linearity.
Note: Linearity is performed initially and when calibration fails to meet the laboratory’s
acceptable limits.
References:
1. Package insert from R&D Systems Inc. which sells a CBC-LINE Full Range Hematology Linearity Kit and CBC-LINE Low
Range Hematology Linearity Kit 1994
2. Shinton NK, England JM, Kennedy DA, “Guidelines for the evaluation of instruments used in Haematology laboratories” J
Clin Path 1982;35; 1095-1102
3. Package insert from STRECK Calibration Verification Assessment 2007
4. Quam EF, “Method Validation – The Linearity of Reportable Range Experiment ASCP
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WBC Linearity
WBC Results 0% 20% 40% 60% 80% 100%
(Reference)
Run #1
Run #2
Run #3
Obtained Mean
Reference Mean
X Multiplication Factor X 0.0 X 0.2 X 0.4 X 0.6 X 0.8 X 1.0
Expected Value
Difference
Allowable Evaluation Limits ± 0.4 0.4 0.4 0.4 0.4 0.4
RBC Linearity
RBC Results 0% 20% 40% 60% 80% 100%
(Reference)
Run #1
Run #2
Run #3
Obtained Mean
Reference Mean
X Multiplication Factor X 0.0 X 0.2 X 0.4 X 0.6 X 0.8 X 1.0
Expected Value
Difference
Allowable Evaluation Limits ± 0.1 0.1 0.1 0.1 0.1 0.1
HGB Linearity
HGB Results 0% 20% 40% 60% 80% 100%
(Reference)
Run #1
Run #2
Run #3
Obtained Mean
Reference Mean
X Multiplication Factor X 0.0 X 0.2 X 0.4 X 0.6 X 0.8 X 1.0
Expected Value
Difference
Allowable Evaluation Limits ± 3 3 3 3 3 3
PLT Linearity
PLT Results 0% 20% 40% 60% 80% 100%
(Reference)
Run #1
Run #2
Run #3
Obtained Mean
Reference Mean
X Multiplication Factor X 0.0 X 0.2 X 0.4 X 0.6 X 0.8 X 1.0
Expected Value
Difference
Allowable Evaluation Limits ± 10 10 10 10 10 10
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WBC Estimation is to be used for cases in which the instrument flags invalid data or suspect
WBC populations and for which you cannot obtain a valid WBC in any mode.
1. Examine the specimen for presence of a clot, strands of fibrin, or gross hemolysis before you
proceed.
2. Next examine the histograms for likely causes of interference. Histogram review may help
you decide which version to use if it is necessary to issue a partial CBC report. (If the HGB
or PLT results are critical or STAT, these should not be held back.) Most interferences are
visible in the lower region of your first WBC histogram, e.g. resistant RBC, NRBC,
megathrombocytes, clumped platelets, fibrin, etc. In some cases, the analyzer will
incorrectly include these interferences in the lymphocytes. This results in both a falsely
elevated WBC and lymphocyte count. Rule of thumb: the lowest WBC from the analyzer is
usually the most correct.
4. Two techs shall do an estimate, each using a separate slide (if a second tech is not available,
one tech can do two estimates, one on each slide)
a. Examine the smears under 10x objective for even distribution of WBC before
proceeding. If there is a ridge or layer of WBC in the tails, the estimate will be invalid.
Re-make the slide.
b. Choose an area from the tails, where the RBC are evenly and singly spread, or just
beginning to overlap. This should be the same area where you would do RBC
morphology or platelet estimation. Be aware that if the RBC/PCV are low, you must
avoid going in too deep.
c. Switch to hpf. Count the total number of WBC seen in 20 fields. Include disintegrated
cells. Do not include NRBC.
d. Divide your total by 20 to obtain the average number of WBC/hpf, to two decimal places.
Multiply the average by the conversion factor for the microscope.
Refer to conversion factor procedure for hpf.
Average your result with that of the other tech.
e. If your estimate is 0.40 or lower, just call it “less than 0.5”
f. If your estimate is over 0.40 calculate the estimate as a range of +/- 40% from this
number. Round off the results of 2.0 or lower to one decimal place; round off results
over 2.0 to the nearest whole number.
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Example 1:
Avg. number of WBC/hpf on #1 slide = 1.55 x conversion factor of 2.5 = 3.875
Avg. number of WBC/hpf on #2 slide = 2.00 x conversion factor of 2.5 = 5.00
Average estimate from two slides (3.875+5.00) ÷2=4.4375
40% Range=4.4375x4.0=1.775
Estimate =4.4375 +/- 1.775, or 2.6625 to 6.2125. Round off to whole numbers.
WBC Estimate is 3 to 6 x 10^9/L
Example 2:
Estimate is calculated as 0.30.
Estimate is 0.40 or lower, so interpret range as “less than 0.5”
Example 3:
Estimate is calculated as 0.45, range of 0.27 to 0.72.
Round off as 0.3 to 0.7.
Example 4:
Estimate is calculated to be 2.5, range of 1.5 to 3.5.
Round off as 1.5 to 4.
5. If the estimate range agrees with the analyzer WBC, report the best results from the analyzer.
Example:
Analyzer count = 5.5
Estimate from films is 4.4 +/- 40%, or 3 to 6.
Report the analyzer WBC of 5.5.
6. If the WBC estimate does not agree with the analyzer count, report the estimate as a range.
Enter a dash (-) for the WBC and append the estimate as a comment:
Examples:
WBC estimate on film appears to be less than 0.5 x 10^9/L.
WBC estimate on film appears to be 1.5 to 4 x 10^9/L.
WBC estimate on film appears to be 3 to 6 x 10^9/L.
References:
Clinical Hematology Principles, Procedures, Correlation, 2nd Edition, E. Anne Stiene-Martin et. al: J.B. Lippincott Company,
1998
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Purpose:
To provide for instruction for establishing a conversion factor for the hematology microscope by
calculating the ratio of electronic count to the number of WBC on either hpf or per oil immersion
field.
Frequency:
The conversion factor must be established once for every microscope used in hematology or
when there are major repairs or changes in the analyzer for electronic counts or a new
microscope is used for estimates.
Procedure:
Note: Total leukocyte counts can be estimated roughly either by hpf or 100x oil immersion field.
Therefore 100x oil immersion field can be substituted in procedure that states hpf.
Step: Action:
1. Perform electronic WBC count on 30 consecutive fresh patient blood samples.
Alternatively if unable to perform 30 consecutive samples it is acceptable to perform
10 samples per day on 3 consecutive days.
2. Prepare and stain one peripheral blood film for each sample.
3. For each film, under hpf microscopy, find an area where 50% of the red cells are
overlapping doubles or triplets. Then count WBC in 10 consecutive fields.
4. Divide by 10 the total number of WBC found to obtain the average number per hpf.
5. Divide the electronic WBC count by the average number of WBC per hpf.
6. Add the numbers obtained in step 5 and divide by 30 (number of observations in this
analysis) hpf.
7. Round the number calculated in step 6 to the nearest whole number to obtain an
estimation factor.
Reference:
Clinical Hematology Principles, Procedures, Correlation, 2nd Edition, E. Anne Stiene-Martin et. al: J.B. Lippincott Company,
1998.
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1. Platelet estimation is to be used for cases in which instrument flags identify invalid platelet
data, platelet clumps and for verifying platelet counts below 100x109/L. Scan the slide for
clumps under hpf. If platelet clumps are present, they cause a false decrease in platelet count.
Edit out the platelet count by replacing it with the message “Platelets clumped, results
invalid.” Do not report the MPV.
NOTE: For this purpose, platelet clumping is defined as at least 5 clumps, with at least 5
platelets per clump. Rare or occasional smaller clumps should be ignored.
2. Switch to 100x objective. Total the number of platelets in at least 10 fields. Determine the
average number per field. Multiply by the conversion factor established for your microscope.
The estimate should agree with the analyzer count +/- 40%
Example:
Average of 6.3 cells per field x conversion factor of 11 = estimate of 69 x 10^9/L.
Analyzer count of 88. Multiply by 0.6 or 1.4 for +/-40% range of 53 to 123.
Platelet estimate agrees with analyzer.
3. If your estimate disagrees with the instrument platelet count by more than 40% in the
absence of clumps:
a. Have another technologist do an estimate, when possible, to confirm the discrepency.
b. If the patient is anemic or polycythemic (RBC below 4.0 or over 6.0), the estimate may be
inaccurate. A second blood film should be prepared, (see Indirect Platelet Count
Procedure) and performed by two technologists, this may require referral.
c. Examine the platelet histograms and the blood smear for the following sources of error:
i) RBC fragments: may be small enough to be counted as platelets.
ii) Megathrombocytes: if many of the platelets on the film appear abnormally large, i.e.
approximately the size of RBC, the analyzer may not be including them in the count.
If your estimate varies from the analyzer by more than 40%, add the message
“Platelet estimate appears higher on film; megathrombocytes may be excluded from
count.” Do not comment anything if occasional large platelets are present, and your
estimate agrees within 40%.
iii) Leukocyte cytoplasmic fragments, if present in large numbers, may falsely elevate the
instrument platelet count. They appear as particles similar in size to platelets, but with
a homogeneous, rather than granular, structure.
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4. If you are reporting the platelet estimate instead of the analyzer count, append an
appropriate comment:
5. Send to Senior Technologist/Pathologist for review on first occurrence for each patient for
whom the instrument count is determined to be invalid due to RBC fragments, leukocyte
fragments, or megathrombocytes.
Reference:
Clinical Hematology Principles, Procedures, Correlation, 2nd Edition, E. Anne Stiene-Martin et. al: J.B. Lippincott Company,
1998
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Purpose:
To provide instructions for establishing a conversion factor for the hematology microscope by
calculating the ratio of electronic count to the number of platelets per oil immersion field.
Frequency:
The conversion factor must be established once for every microscope used in hematology or
when there are major repairs or changes in the analyzer for electronic counts or a new
microscope is used for estimates.
Procedure:
Step: Action:
1. Perform electronic platelet count on 30 consecutive fresh patient blood samples.
Alternatively if unable to perform 30 consecutive samples it is acceptable to perform 10
samples per day on 3 consecutive days.
2. Prepare and stain one peripheral blood film for each sample.
3. For each film, under oil immersion (100x) microscopy, find an area where 50% of the red
cells are overlapping in doubles or triplets. Then count platelets in 10 consecutive fields.
4. Divide by 10 the total number of platelets found to obtain the average number per oil
immersion field.
5. Divide the electronic platelet count by the average number of platelets per oil immersion
field.
6. Add the numbers obtained in step 5 and divide by 30 (number of observations in this
analysis) to obtain the average ratio of the platelet count to platelets per oil immersion
field.
7. Round the number calculated in step 6 to the nearest whole number to obtain an
estimation factor, the number of peripheral blood platelets represented by one platelet in
an oil immersion field.
Reference:
Clinical Hematology Principles, Procedures, Correlation, 2nd Edition, E. Anne Stiene-Martin et. al; J.B. Lippincott Company,
1998
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To determine Average, total the values in Column 4, divide by 30. Round off to the nearest
whole number to obtain the estimated factor.
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Purpose:
An indirect platelet count may be obtained by determining the ratio of platelets on a blood film
to RBC. The ratio is then used to calculate the number of platelets in comparison to the number
of RBC x 1012/L.
This method is useful for situations in which an accurate platelet count cannot be obtained from
the CBC analyzer e.g. samples with marked megathrombocytes, RBC fragments, or leukocyte
cytoplasmic fragments. It is particularly more accurate than conventional estimation when the
RBC is abnormal.
Procedure:
1. Make two good quality blood smears. Air dry and stain as for differentials. The peripheral
smears must be well made, not touching edges of slide, with rounded end and no visible tails.
3. Using a 100x oil immersion objective, count the number of platelets per 500 RBC. Two
technologists should count one slide each and the platelets counts should check within 10
platelets of each other. If the counts do not check, a third technologist counts in the same
manner.
4. Add the two agreeable counts together and multiply this result by the RBC count to obtain
the indirect platelet count in 109/L. Next calculate +/- 10 to report the result as a range rather
than an exact number.
Example: RBC = 4.10
1st count = 11 platelets per 500 RBC
2nd count = 15 platelets per 500 RBC
11+15 = 26
26 x 4.10 = 107
Add and subtract 10 to establish the reportable range.
The indirect platelet count is reported as 97 to 117 x 109/L.
5. Compare the indirect platelet count to the analyzer count. If the indirect count is within the
analyzer count +/- 30%, report the analyzer count.
6. If the indirect count is not within the analyzer count +/- 30%, report the indirect platelet
count.
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Procedure Notes:
The Indirect Platelet Count is preferable to platelet estimation when an accurate platelet count
cannot be obtained on the CBC analyzer. It is particularly more accurate than conventional
estimation when the RBC is abnormal.
References:
1. Raphael SS. Lynch’s Medical Laboratory Technology, Third Edition, Philadelphia PA: WB Saunders Company, 1976, page
1084.
2. Hematology Laboratory Safety Manual, Royal University Hospital, Saskatoon, SK 1998
3. Constantino BT. Leukocyte Cytoplasmic Fragmentation: Its Causes and Laboratory Evaluation, Canadian Journal or Medical
Laboratory Science -60, 1998, page 195
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TRANSFUSION
MEDICINE
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The most recently published standards of the Canadian Society for Transfusion Medicine are
referenced.
Each transfusion service shall have in place an established system for record-keeping that
is written and abides by the following:
DOCUMENTS
Issue Vouchers from CBS Indefinitely
Correspondence Related to Blood Components Indefinitely
Documents Related to Traceback or Lookback Indefinitely
Daily Records for issue of Blood Components/Products Indefinitely
Transfused Patient Data Indefinitely
• includes antibody investigation & resolution
• transfusion reaction investigation
Non-transfused patient data 5 years
Method Revision Indefinitely
Blood Component & Product Final Disposition Records Indefinitely
Tissue & Bone Banking Donor & Inventory Records Indefinitely
Quality Control Records 5 years
•Include reagents, serological test controls, external proficiency
testing
Utilization Reports 5 years
•Blood component product inventory
Direct & Stores Inventory 5 years
•Purchase & acquisition data
Meeting & Related Documents 5 years
Computer Program Validation & Exception List 5 years
Workload Records/Reports 3 years
In-service Education Documentation 3 years
Test & Blood Product Request Forms 13 months
SPECIMENS
Donor Segments from transfused red cells 3 weeks
Clotted and/or EDTA specimens, serum/plasma from transfused 1 to 3 weeks
patients (pre-transfusion)
Cord Blood 2 weeks
All Other Patient Specimens 1 week
Kleihauer-Betke Slides 1 year
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When initiating new methodology or implementing new testing, a validation process must be
developed.
Validation Process
• Object of the validation
Purpose
Manufacturer’s instructions/data
References
• Description
Standard Operating Procedure (SOP)
Sample quantities and types
“known” and “unknown” samples
Patient population
Test methods and conditions
Isolated testing
Parallel testing
System stress points
Testing personnel
Training plan
Training documents
Data collection/documentation required
Acceptance criteria
Reviewing personnel
• Summary
Results
Performance characteristics/limitations
Functionality
Method
SOP
Training plan and documents
Safety
Sources of error
• Conclusion
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Depending on laboratory test volumes, unknown patient samples may be obtained from another laboratory.
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Laboratory Guidelines 2012 Edition Page 80