Platelet Function Testing

Cynthia S Johns, MSA, MT(ASCP)SH

Normal hemostasis consists of a delicate balance between the blood vessels, platelets, and coagulation proteins, a relationship known as the triad of hemostasis. While each component of the triad plays a very specific role in the hemostatic process, they all work together to preserve the integrity of the fluid state of the blood. The platelets contribution to the maintenance of a normal hemostatic system has long been recognized. Platelets are the bodys first (primary) line of defense against blood loss whenever the vasculature has been compromised, and both normal (or near normal) platelet number and function are essential for this process to occur. Consequently, whenever a patients clinical condition includes symptoms associated with a defect in primary hemostasis such as easy bruising, menorrhagia or epistaxis (nosebleeds), a platelet abnormality should be considered and investigated. Platelet disorders can be divided into two categories: quantitative and qualitative. Quantitative defects are abnormalities in platelet number, whereas qualitative defects are abnormalities in platelet function. Since thrombocytopenia is the most common cause of platelet-related bleeding1, a platelet count and evaluation of a peripheral blood smear should always be the first step in the investigative process of a defect of primary hemostasis. Only after an adequate number of platelets has been confirmed should platelet function studies be considered. The Anatomy of the Platelet Platelets are the smallest formed components of human blood, ranging in size from 2-4 microns.2 Platelets are not considered to be true cells due to their lack of a nucleus. Platelets are cytoplasmic fragments of the megakaryocyte, with a maturation time of 4-5 days. Platelets normally live about 10 days in the peripheral circulation. Based on evaluation by electron microscopy, the platelet may be divided into three distinct anatomic zones (Table 1). The platelet is surrounded by a zone of membranes, which is responsible for adhesion and aggregation. The plasma membrane, platelet membrane, and a

Volume 18, Number 4 July-August 2004

Objective: The reader will be able to identify the common methods to evaluate platelet function.

CLINICAL HEMOSTASIS REVIEW

3176 S. Peoria Court, Aurora, CO 80014 www.esoterix.com EDITOR Dorothy M. Adcock, MD

EDITORIAL ASSISTANT Monica Thibault

submembrane area are contained within this zone. The plasma membrane surrounds the surface of the platelet and contains mucopolysaccharides that adsorb procoagulant plasma proteins such as fibrinogen, factor V, factor VIII, factor XI and factor XII. The purpose of adherence to the platelet surface keeps coagulation proteins localized to the site of the vessel injury so they can participate in the coagulation process, resulting in fibrin formation. The platelet membrane is made up of a system of microtubules that provide support and structure to the platelet. The dense tubular system extends throughout the interior of the platelet and provides an expansion of the surface area of the platelet.3 It supplies a calcium storage pool and contains phospholipids. Phospholipids are directly responsible for the formation of prostaglandins, which play a major role in mediating platelet aggregation. (Figure 1). The dense tubular system is also the site of prostaglandin systhesis.3 The submembrane section of the platelet membrane contains receptor glycoproteins (GP) or integrins such as GPIb, GPIIb, GPIIIa, and GP IX. Glycoproteins provide the major points of attachment for fibrinogen, von Willebrand factor (vWF) and other soluble ligands that are necessary for platelet adhesion and aggregation (Figure 2).

Figure 1. Platelet Biochemistry

Clinical Hemostasis Review is published by Esoterix and is circulated to selected physicians. Copyright 2004. Esoterix is a leading laboratory services company providing esoteric testing in numerous disease corridors. The opinions expressed in the articles are those of the author(s) and do not necessarily reflect the opinions or recommendations of the advertisers, editors, or publisher. The publisher reserves copyright and renewal on all published material and such material may not be reproduced in whole or in part without written permission from the publisher. Consult the full prescribing information on any drugs or devices discussed. All correspondence should be directed to the attention of the Editor, Clinical Hemostasis Review, 3176 S. Peoria Ct, Aurora, CO 80014.

Platelet Biochemistr y
Phospholipid

Ar achadonic Acid Cyclooxygenase (inhibited by aspir in)

Pr ostaglandin G 2 (PGG 2)

Pr ostacyclin PGI 2 (inhibits aggr egation)

Thr omboxane A2 (stimulates aggr egation)

CLINICAL HEMOSTASIS REVIEW / JULY-AUGUST 2004

Figure 2. Platelet Adhesion and Aggregation

A d h e s io n a n d A g g re g a tio n
IIb/IIIa IIb/IIIa

F IB

PL
FI B
P latelet
Ib/IX Ib/IX

F IB

C oag P roteins (F X )

vW F
Ia/IIa E ndothelial C ell

vW F
E ndothelial C ell

C ollagen

The second described anatomic zone of the platelet is the cytoskeleton, which is made up of microtubules and microfilaments. These serve to maintain the discoid shape of the platelet and provide contractile capabilities to aid in the release mechanism of the platelet. Finally, the third anatomic region is the platelet organelle zone and this consists mainly of the alpha granules and dense bodies, but also contains lysosomes, peroxisomes, mitochrondria and glycogen granules. The organelle zone is responsible for storage and release of the platelets contents, which contribute to platelet aggregation. Alpha granules are found in abundance and contain many procoagulant proteins necessary for the hemostatic process. Dense bodies are fewer in number, and store and release smaller non-protein molecules. See Table 1. Table 1. Platelet Anatomic Zones
Zone Membrane Primary Structures Plasma membrane Platelet membrane Cytoskeleton Organelle Submembrane Microfilaments Alpha Granules Components Mucopolysacchrides for absorption of procoagulant plasma proteins Microtubules and microfilaments for structure and support Receptor glycoproteins (GP) Albumin Alpha-2 antiplasmin C1 esterase inhibitor Platelet Factor 4 (PF4) Low affinity PF4 Beta thromboglobulin Platelet derived growth factor Fibrinogen Factor V Fibronectin vWF antigen ATP ADP Serotonin Calcium Function Platelet adhesion and aggregation

Maintain shape, provide contraction Primary and secondary platelet aggregation, platelet adhesion

Dense Bodies

Provide energy source for platelet, platelet adhesion.

CLINICAL HEMOSTASIS REVIEW / JULY-AUGUST 2004

Platelet Physiology The platelets contribution to the hemostatic mechanism is through adhesion to the site of injury and aggregation with one another, a process known as primary hemostasis. In their normal circulating state, platelets are discoid in shape with a smooth surface. This makes the platelet non-adherent to the endothelium or other platelets. When endothelial cells become damaged, platelets come in contact with exposed collagen and other adhesive proteins and undergo a conformational, or shape change. They become spherical or an irregular globulin form by extending pseudopods of varying size and length to spread out over the exposed subendothelium. Endothelial cells also release cytokines and other substances that cause the platelets to become stimulated. Once stimulated, platelets respond quickly by adhering to the site of injury to form a platelet plug or microthrombus over the damaged area of the vessel. The platelets also release the contents of their organelles, a stage referred to as the release reaction of the platelets. The substances secreted by platelets stimulate additional platelet activation and aggregation and the formation of a primary hemostatic platelet plug. Simultaneously, the blood coagulation proteins are activated, culminating with thrombin generation and the deposition of fibrin, which stabilizes the platelet plug.

Platelet Function Testing Platelet function studies measure and/or monitor the platelets ability to adhere and aggregate. These tests have historically presented a challenge for the clinical laboratory due to the lack of reliable, accurate and easy to perform testing procedures. In the past, platelet function has been assessed by the bleeding time and by platelet aggregation studies. In the laboratory, the bleeding time has been utilized to measure platelet adhesion, while aggregation studies have been used to observe the release reaction and the platelets ability to stick to each other in response to external stimulating substances (agonists). However, both methods are time consuming, cumbersome, prone to operator-specific variables and lack correlation to bleeding risk.4,5 Since evaluation of platelet function can be of critical importance in the hemostatic management of a patient, especially for those undergoing surgical procedures where there is a history of clinically significant bleeding,6 improved ability to assess platelet function in a timely and efficient manner is essential. Bleeding Time The bleeding time is defined as the time it takes for a standardized skin wound to stop bleeding. When vessel injury is induced by a standardized cutting implement, platelets adhere, aggregate, and form a hemostatic platelet plug. The bleeding time measures the ability of platelets to arrest bleeding and, therefore, is a measure of both platelet number and function. Capillary contractility and both the intrinsic and extrinsic systems of coagulation function in a minor capacity in the bleeding time. The bleeding time assesses in vivo platelet function and when applied in the appropriate clinical setting is useful as a screening procedure to detect both congenital and acquired disorders of platelet function.1 While the methodology for performing bleeding times has improved over the years with the availability of devices to standardize the size and depth of the incisions, the bleeding time remains a test with considerable lack of precision and questionable correlation with clinically significant patient conditions.7 See Table 2. Table 2. Bleeding Time Methods
Method Duke Ivy Mielke Template (Simplate, Surgicut) Device Lancet Lancet Template Disposable template devices Incision Type Puncture, variable in depth Puncture, variable in depth Incision, 1 mm x 9 mm Incision, approximately 1 mm x 5 mm Location of Testing Earlobe Forearm with standardized pressure Forearm with standardized pressure Forearm with standardized pressure

JULY-AUGUST 2004 / CLINICAL HEMOSTASIS REVIEW 4

There are many factors that influence the results of the bleeding time. Environmental factors such as the condition of the skin (temperature, failure to bleed, subcutaneous bleeding) and patient age contribute to the variability of the results. Other factors include the length and/or depth of the puncture, the orientation of the puncture (horizontal vs. vertical), excessive wiping of the puncture site, and variations in the determination of the endpoint, decrease the bleeding times precision and accuracy and make it less useful as a screening test to predict possible bleeding abnormalities. Bleeding time reference values range from approximately 2.5 to 9.5 minutes but vary with the direction, length and depth of the incision. Due to variations in technique and patient population, it is recommended that each laboratory establish its own reference values. Prolonged bleeding times are found in the following situations: marked thrombocytopenia6 (platelet count less than 50,000 per mL) acquired or inherited platelet dysfunction following administration of aspirin or aspirin-containing drugs following administration of other drugs that inhibit platelet function such as non-steroidal anti-inflammatory drugs (NASIDs) and antihistamines If the incision fails to bleed or if a small vein is cut, the bleeding time of the incision should be disregarded and the test repeated. In older patients, bleeding may only occur subcutaneously. Obtaining an accurate bleeding time in these patients may not be possible. The bleeding time increases in proportion to the decrease in platelet count. The use of aspirin, aspirincontaining drugs, non-steroidal anti-inflammatory drugs and antihistamines causes a prolonged bleeding time. The patient should be instructed not to take any aspirin, drugs containing aspirin, or other drugs known to inhibit platelet function for at least one week before the test is performed. The bleeding time should be measured as part of a diagnostic work-up of a qualitative platelet disorder. It is not useful as a presurgical screening test for individuals without a history of bleeding,2 or for the evaluation of a coagulation factor deficiency. Platelet Aggregation Platelets function in primary hemostasis by forming an initial platelet plug at the site of vascular injury. This process occurs partly through the ability of platelets to bind to one another. Platelet aggregation studies observe the ability of platelets to adhere to one another in response to stimulation by an exogenous substance (agonist), as opposed to platelet adhesion, or the ability of the platelets to stick to a surface. Substances that can induce platelet aggregation include collagen, adenosine diphosphate (ADP), epinephrine, thrombin, serotonin, arachidonic acid, snake venoms, antigen-antibody complexes, soluble fibrin monomer complexes, and fibrin(ogen) degradation products (FDP). The antibiotic ristocetin induces in vitro platelet agglutination rather than aggregation. External aggregating agents used in platelet aggregation testing induce platelet aggregation, cause platelets to release endogenous ADP, or both. Platelet aggregation can be studied through utilization of a platelet aggregometer, a photo-optical or electrical impedence instrument connected to a chart recorder, and can be performed on platelet-rich plasma or whole blood anticoagulated with sodium citrate. When the platelet is at rest, it circulates in a discoid shape. Immediately upon stimulation, the platelet undergoes a shape-change to a more swollen spherical shape. This process takes approximately 3 seconds. Within approximately the next 10 seconds, the granules centralize and the cytoskeleton begins to contract and release the contents of the granules. When these platelet substances have been expelled into the surrounding area, aggregation occurs and platelet clumps are formed. These stages can be observed on a normal aggregation tracing (Figure 3). Platelet Rich Plasma Method Platelet-rich plasma, which is turbid in appearance, is placed in a cuvette, warmed to 37C in the heating block of the instrument, and stirred by a small magnetic bar. Baseline light transmittance through the platelet-rich plasma is recorded. The addition of an aggregating agent causes the formation of larger platelet aggregates with a corresponding increase in light transmittance, due to a clearing in the platelet-rich plasma. The change in light transmittance is converted to electronic signals and recorded as a tracing by the chart recorder. Whole Blood Method Whole blood is diluted in saline to form a suspension. Parallel electrodes coated with collagen are immersed in the suspension and a small current applied. As platelets aggregate in response to the addition of an agonist, they collect on the surface of the electrode, thereby altering the strength of the current. The change in current is measured by the aggregometer, converted to electronic signals and recorded on the chart in a tracing similar to that seen with platelet-rich plasma aggregation.8 Whole blood platelet aggregation has an advantage over

CLINICAL HEMOSTASIS REVIEW / JULY-AUGUST 2004

traditional PRP methods since sample preparation and manipulation are not required. Platelet Aggregation Testing Platelet aggregation can be difficult to perform due to a variety of pre-analytic variables that can be encountered. For example, a clean venipuncture is crucial. Hemolyzed samples should not be utilized because red blood cells contain ADP, which can prematurely activate platelets. Specimens from fasting patients are preferred for testing since lipemic samples may obscure changes in optical density during platelet aggregation. All aggregation studies should be performed within 3-4 hours of sample collection in order to maintain the viability of the platelets. Therefore, testing must occur in close proximity to the patient and cannot be delayed or deferred once the blood sample has been collected. It is essential that the patient refrain from taking any anti-inflammatory or other drugs known to interfere with platelet function for at least one week prior to testing as these drugs inhibit the platelets release reaction, giving abnormal platelet aggregation results. Marked thrombocytopenia can make the aggregation responses difficult to interpret. Platelet aggregation occurs as a two-step process, known as primary and secondary waves of aggregation (Figure 3). The primary wave of aggregation is observed when platelets adhere to one another in the presence of an agonist such as ADP, epinephrine or ristocetin. Secondary aggregation is characterized as that aggregation which occurs after the platelets have been stimulated to secrete the substances contained in their organelles. It should be noted that some agonists will stimulate primary aggregation and some will stimulate secondary aggregation. Others will stimulate both primary and secondary aggregation, yielding a biphasic aggregation curve. In addition, different concentrations of the same agonist can produce varying patterns of primary and secondary aggregation. For example, low concentrations of ADP induce biphasic aggregation (i.e., both a primary and a secondary wave of aggregation); very low concentration of ADP induce a primary wave followed by disaggregation; and high concentrations of ADP induce a single, broad wave of aggregation. A biphasic aggregation response to ADP will not be seen in patients with platelet release disorders. Patients with Glanzmanns thrombasthenia show incomplete aggregation with ADP regardless of the final concentration. Figure 3. Platelet Aggregation Tracing

Biphasic Platelet Aggregation Curve

Primary Wave
% Transmittance

Secondary Wave

100 Time (minutes)

Platelet aggregation induced by collagen is characterized by a lag period before aggregation occurs, followed by only a single wave of aggregation. A biphasic agglutination response is seen with the antibiotic ristocetin; however, often only a single broad wave of agglutination will occur. Platelet aggregation induced with arachidonic acid causes a rapid secondary wave of aggregation. Biphasic aggregation is observed with epinephrine; however many healthy patients only produce a primary wave of aggregation with epinephrine.9 The aggregating agent thrombin induces a biphasic wave of aggregation. Platelet aggregation induced by serotonin normally produces a primary wave of aggregation with a maximum of 10% to 30% transmittance followed by disaggregation.9

CLINICAL HEMOSTASIS REVIEW / JULY-AUGUST 2004

In patients with severe von Willebrand disease, agglutination to ristocetin is characteristically absent. Decreased to normal agglutination to ristocetin can be seen in patients with mild von Willebrand disease. Correction of the abnormal ristocetin agglutination curves can be seen by the addition of normal, platelet-poor plasma to the patients platelet-rich plasma. Abnormal ristocetin-induced platelet agglutination may also occur in patients with Bernard-Soulier syndrome, platelet storage pool defects, and idiopathic thrombocytopenic purpura (ITP). In evaluating patients with suspected platelet disorders, the aggregating agents most commonly used are ADP in varying concentrations, collagen, epinephrine, and ristocetin. Aspirin, aspirin compounds, and non-steroidal anti-inflammatory drugs inhibit the secondary wave of aggregation by inhibiting the release reaction of the platelet. Reduced or absent aggregation as well as disaggregation curves may be observed in patients taking medication containing aspirin. Other medications and/or substances have also been identified as inhibiting platelet function, such as ibuprofen, red wine and a variety of herbs. Patients should be questioned carefully about possible ingestion of these substances prior to interpreting abnormal aggregation results. Lumiaggregation The lumiaggregometer is an instrument that can detect both platelet aggregation and dense granule secretion at the same time from a whole blood sample.10 During whole blood platelet aggregation, firefly luciferin is added to the sample. When ATP is released from the dense granules following platelet stimulation, it acts upon the luciferin to form luciferase and the amount of luminescence measured. The pattern observed includes both the platelet aggregation and ATP release. The advantage of the lumiaggregometer is that it is possible to characterize the response of the platelets by both the pattern of aggregation and a more specific indicator of the platelet release function. Platelet Function Analyzer The PFA 100 Platelet Function Analyzer (Dade Behring, Marburg Germany) is a new method for assessment of platelet dysfunction.11 The system utilizes test cartridges containing membranes coated with collagen/ epinephrine or collagen/ADP to stimulate platelet aggregation. The system monitors platelet adhesion and aggregation on the membrane as whole blood is aspirated under controlled flow conditions through a microscopic aperture cut into the membrane. The time required for the platelet plug to occlude the aperture is an indicator of the platelet function in the sample.6 Studies indicate that the PFA-100 System is a reliable and cost-effective tool for detecting certain bleeding disorders such as von Willebrand disease and aspirin-induced bleeding.12 The Clot Signature Analyzer (CSA) (Xylum) can measure platelet hemostasis time (PHT) and collagen induced thrombus formation (CITF).13 Whole, non-anticoagulated blood flows through a tubing system containing two holes in separate locations. Downstream pressure is measured dynamically within the tubing as the blood is flowing. As platelets begin to aggregate and plug the holes, downstream pressure is increased. The time required for the pressure to increase to a predetermined level is measured and is a direct reflection of platelet function.13 The VerifyNowUltegra System (Accumetrics, San Diego, CA) is a turbidimetric based optical detection system that measures platelet induced aggregation as an increase in light transmittance. The system consists of an instrument, a disposable assay device and controls. The assay device contains reagents based on microbead agglutination technology and contains a lyophilized preparation of human fibrinogen-coated beads, platelet agonist, preservative and buffer. The patient sample is anticoagulated whole blood, which is automatically dispensed from the blood collection tube into the assay device by the instrument, with no blood handling required by the user.14 Plateletworks (Helena Laboratories, Beaumont, TX) is a point-of-care system that provides rapid platelet function information by calculating the percentage of platelet aggregation by measuring a change in the baseline platelet count compared to the platelet count of blood that is added to an agonist tube. If platelets are functioning normally, they will aggregate when added to the agonist tube, become separated from the non-aggregated platelets, and show a marked decrease in the platelet count.15 These newest analyzers have provided another avenue for the evaluation of the variety of physiologic functions exhibited by stimulated platelets. When combined with other testing methodologies and clinical conditions, they can provide valuable information regarding the patients hemostatic status.

CLINICAL HEMOSTASIS REVIEW / JULY-AUGUST 2004

References Bick RL. Platelet function defects: A clinical review. Semin Thromb Hemost 1992; 18:167-85. Kottke-Marchant K; Corcoran G; The Laboratory Diagnosis of Platelet Disorders; Arch Patholo Lab Med. 2002126:133-146. George JN, Coleman RW. Platelets: Overview of Platelet Structure and Function. In Hemostasis and Thrombosis. Basic Principles and Clinical Practice. 4th Ed. Coleman RW, Hirsh J, Marder Vj, er.al. Ed., Lippincott Williams and Wilkins, Philadelphia, 2001, 381-86. Rodgers RPC, Levin J. A critical reappraisal of the bleeding time. Semin Thromb Hemost, 1990, 16:6-20. Lind DE. The bleeding time does not predict surgical bleeding, Blood, 1991, 77:2547-52. Kindu SK, er al. Characterization of an In-Vitro Platelet Function Analyser, PFA-100TM. Clin. Appl Thrombosis/ Hemostasis, 2:4:241-49,1996. Goodnight SH, Hathaway WE. Disorders of Hemostasis and Thrombosis. McGraw-Hill, New York, NY 2001. Chrono-Log Corporation Product Information, Havertown, PA, 2000. Triplett DA, et al. Platelet Function; Laboratory Evaluation and Clinical Application. American Society of clinical Pathologists, Chicago, IL 1978. White MM, Foust JT, Mauer AM, Robertson JT, Jennings LK. Assessment of lumiaggregometry for research and clinical laboratories. Thrombo Haemost. 1992:67:572-577. Mammen EF, Comp PC, Gosselin R, er. al. PFA-100TM System: A new method for assessment of platelet dysfunction. Semin Thromb Hemost, 1998, 24:195-202. PFA-100 Platelet function Analyzer News Release, Dade Behring Inc., Deerfield, IL, August 12,1999. Li C. The Xylum Clot Signature Analyzer; a novel dynamic measure of global hemostasis testing. Presented at the International Society on Thrombosis and Haemostasis Seminar, Washington D.C., August 17,1999. VerifyNowTM Ultegra System Product Information, Accumetrics, San Diego, CA 92121 PlateletworksTM Product Information, Helena Laboratories, Beaumont, TX.

CLINICAL HEMOSTASIS REVIEW / JULY-AUGUST 2004

Self Assessment
Platelet Function Testing
1. Platelets directly participate in which of the following coagulation mechanisms? a. Extrinsic coagulation pathway b. Primary hemostasis c. Secondary hemostasis d. Intrinsic coagulation pathway The primary wave of platelet aggregation is due to which of the following? a. Secretion of the contents of the alpha granules b. Ability of the platelet to contract c. Ingestion of aspirin or aspirin-containing compounds d. Stimulation by an agonist Ristocetin-induced platelet aggregation may be abnormal in which of the following? a. von Willebrand disease b. Bernard-Soulier syndrome c. Platelet storage pool defects d. All of the above The plasma membrane plays a role in platelet adhesion and aggregation due to the presence of: a. Microtubules b. Glycoproteins c. Organelles d. Lysosomes Prolonged bleeding times can be found in: a. Inherited platelet dysfunction b. Platelet counts less than 50,000 per ml c. Following aspirin ingestion d. All of the above The platelet-rich plasma method for performing platelet aggregation is based on which of the following? a. Increase in strength of a current across an electrode b. Increase in the amount of light reflected off of platelet aggregates c. Increase in light transmittance as platelet aggregates form d. Increase in the size of platelet aggregates

Volume 18, Number 4 Self Assessment Registration Form

Record your answers below by circling the correct answer for each question. Participation is confidential. 1. 2. 3. 4. 5. 6. a a a a a a b b b b b b c c c c c c d d d d d d

2.

3.

Participants must receive a score of 80% to receive credit. This form must be filled out completely. Please print. Category Applied For: ASCLS P.A.C.E. AMTIE CEU State ______

4.

Name_________________________ Title___________________________ Work Phone _______________________ Street_________________________

5.

City__________________________ State__________ Zip____________ SS#__________________________ Licensure Type_________________ Licensure Number______________ Were objectives clearly stated and met? yes no Was text material well organized and informative? yes no Were table/figures useful and clear? yes no Did self assessment questions reflect objectives and text? yes no To earn continuing education credit, answer the questions in the Self Assessment by recording your answers on the Registration Form. Complete the Registration Form, and mail it or a photocopy with your check for $15.00 (for processing and issuing your credit certificate), make check payable to:Esoterix Coagulation. Mail to: CHR Self Assessment 3176 South Peoria Court Aurora, CO 80014

6.

CLINICAL HEMOSTASIS REVIEW / JULY-AUGUST 2004