Platelet Aggregation

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Platelet Aggregation Catherine P.M. Hayward* and Karen A. Moffat†

* Departments of Pathology and Molecular Medicine and Medicine, McMaster University, Hamilton, ON, Canada, †Department of Medicine, McMaster University, Hamilton, ON, Canada

OVERVIEW OF METHODS FOR THE EVALUATION OF PLATELET AGGREGATION 609 Born Turbidometric Aggregometry With Platelet-Rich Plasma 609 Whole Blood Aggregometry 613 Measurement of Dense Granule Release With Aggregometry 615 PREPARATION OF SAMPLES FOR PLATELET AGGREGATION TESTS 616 ANALYTICAL CONSIDERATIONS AND POTENTIAL INTERFERENCES FOR AGGREGATION TESTS 618 QUANTITATIVE ENDPOINTS OF AGGREGATION AND RELEASE ASSAYS 619 AGGREGATION INTERPRETATION IN THE ASSESSMENT OF BLEEDING DISORDERS 620 QUALITY EVALUATION OF PLATELET AGGREGATION TESTS 623 REFERENCES 623

OVERVIEW OF METHODS FOR THE EVALUATION OF PLATELET AGGREGATION The first descriptions of measuring platelet function by light transmittance aggregometry (LTA) were independently published by Born and O’Brien in 1962.1,2 Over the next 20 years, methods were developed to evaluate dense granule adenosine triphosphate (ATP) release with LTA and to test aggregation in whole blood (WBA) by measuring the changes in electrical impedance that occur with platelet accumulation on electrodes during aggregation.3–6 More recently, flow cytometry (Chapter 35) and platelet counting methods (that measure the loss of single platelets after an agonist is added) have been applied to measure platelet aggregation responses for research purposes. Fifty years after LTA was developed, it remains the most popular method for evaluating platelet aggregation, and like WBA, its main use is for the assessment of bleeding disorders.7–10 This chapter provides an overview of platelet aggregation and its use for bleeding disorder assessment. The use of platelet aggregometry for monitoring antiplatelet therapy is discussed in Chapter 36. Recent surveys of diagnostic and research laboratory practices have identified the need to standardize and improve platelet aggregation test practices.7–10 When LTA and WBA are performed for the assessment of bleeding disorders, a panel of agonists is used to assess platelet aggregation responses to different agonist stimulation pathways, and to assess platelet agglutination mediated by von Willebrand factor (VWF).11,12 Table 34.1 summarizes information from recent guidelines on the agonists that are commonly used for LTA and WBA, Platelets. https://doi.org/10.1016/B978-0-12-813456-6.00034-5 Copyright © 2019 Elsevier Inc. All rights reserved.

including the recommended concentrations for aggregation testing.13–17 Aggregation testing can also be assessed without an added agonist although spontaneous aggregation is uncommon and its evaluation does not have a proven role in bleeding disorder assessments. Some instruments (e.g., a LumiAggregometer, Chrono-Log Corporation, Haverston, PA) allow LTA or WBA to be tested simultaneously with measurements of platelet dense granule ATP release,3,5 as discussed later in this chapter. Because many aspects of aggregation testing lack standardization,7–10,18,19 guideline recommendations have been developed for application to laboratory practice,14–18 including the control of preanalytical, analytical, and postanalytical variables, and the interpretation of aggregation test findings.

Born Turbidometric Aggregometry With Platelet-Rich Plasma Principle of Turbidometric Aggregometry Turbidometric aggregometry, or LTA, is based on the methods that were originally published by Born and O’Brien.1,2 The test monitors agonist-induced changes in the turbidity (or optical density) of a platelet suspension while stirring the sample in a clear receptacle at 37°C.1,2 The test is commonly done using an aggregometer and platelets suspended in plasma (plateletrich plasma [PRP]), although it is also possible to test suspensions of washed or gel-filtered platelets for research purposes. Recent studies have also explored testing platelet aggregation responses on certain automated coagulation analyzers.20,21

Settings and Endpoints Used for Turbidometric Aggregometry Before LTA is performed on an aggregometer, the respective limits for no aggregation (0%) and 100% aggregation are set on the instrument, using the PRP suspension and the subject’s autologous platelet-poor plasma (PPP) (or buffer if testing washed or gel-filtered platelets).1,2 After prewarming the sample, an agonist or an agglutinating agent (such as ristocetin) is added, followed by monitoring for changes in the turbidity of the stirred sample using an optical system.1,2 Aggregation instruments are typically set to test aggregation at body temperature (37°C) while mixing the sample at a low shear force, typically using a magnet and a stir bar, set to turn at 1000 revolutions per minute (rpm; recommendations vary by instrument manufacturer).

Proteins That Influence Turbidometric Aggregometry Findings Platelet aggregation induced by an activating agonist, at low shear, is mediated by interactions between residues in the fibrinogen γ-chain and integrin αIIbβ3 (glycoprotein [GP] IIb-IIIa (see Chapter 12).22,23 Although studies of knockout

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TABLE 34.1 Summary of the More Commonly Used Agonists for Platelet Aggregation Studies Aggregation Method Agonist

Light Transmission

Whole Blood (Impedance)

Recommendation on use of single/multiple concentrations 0.5–10 μM, 5 μM to starta 2 μMc Test higher concentration(s) 2.0–10 μMb if abnormal with the lower concentrationa,b,c

5–20 μMa

ADP

Epinephrine

Anticoagulant

0.5–10 μM, 5 μM to starta 5 μMc 5–10 μMb

Not recommendeda

Test with a single concentration as testing much higher concentrations is generally not helpfulb Test higher concentration(s) if abnormal with the lower concentrationc Collagen (type 1 fibrillary) 1–5 μg/mL, 2 μg/mL 1–5 μg/mLa to starta,c Test higher concentration(s) Concentration that if abnormal with the detects lower concentrationa,b,c abnormalities from NSAIDb a

c

a

0.5–1.0 mM

Thromboxane analog 1–2 μMa, 1 μMc U46619 1 μMb Test at a single concentrationb Test higher concentration(s) if abnormal with 1μM concentrationc

Not provided

10 0.25 mg/mL

0.8–1.5 mg/mLa 1.2–1.5 mg/mLb 1.2 mg/mLc Test higher concentration, if 2 mg/mLc agglutination absent with 1.2 mg/mLc

1.0 mg/mLa

Test lower concentration, if agglutination induced with 1.2 mg/mLc High dose

In citrated PRP, the aggregation response to epinephrine always occurs in two phases, as illustrated in Fig. 34.1, unless there is no response or absent secondary aggregation. The initial, and more limited, first wave of aggregation is followed by a faster, more extensive phase of aggregation that requires thromboxane generation and is accompanied by release of platelet secretory granule contents, including dense granule ADP and ATP. The ADP released by platelets augments the secondary phase of

0

0.6 mg/mL 0.5–0.6 mg/mLb 0.5–0.7 mg/mLc a

Most laboratories assessing platelet aggregation responses use samples collected into a weak calcium chelator, typically 3.2% or 3.8% buffered sodium citrate, at a 9:1 ratio of blood to anticoagulant (the more commonly used 3.2% concentration contains 109 mM of the dihydrate form of trisodium citrate Na3C6H5O7 2H20).7 Aggregation responses are higher with 3.2% than 3.8% sodium citrate.29 The citrate anticoagulant reduces the extracellular ionized calcium concentration, which increases aggregation responses to some commonly used weak agonists, including adenosine diphosphate (ADP) and epinephrine.11,30–32

Turbidometric Aggregation Findings With Different Agonists

Arachidonic acid 0.5–1.6 mM , 1 mM Test at a single 0.5–1.64 mMb a,b concentration Test higher concentration(s) if abnormal with 1 mM concentrationc

Ristocetin Low dose

mice indicate that plasma vitronectin and other αIIbβ3 ligands also influence the extent and stability of turbidometric aggregation responses to activating agonists,22–24 the influence of these proteins on human platelet aggregation responses is unknown. When the aggregation test is done using ristocetin (to induce VWF binding to platelets), the initial phase reflects agglutination mediated by VWF binding to GPIb-IX-V,25,26 and this is followed by platelet activation and αIIbβ3-dependent platelet aggregation.27,28

a

20 30

Notes: This summary of published recommendations shows data that were reproduced, with permission, from the Clinical and Laboratory Standards Institute guideline,a the North American guidelineb and the guideline from the International Society on Thrombosis and Haemostasisc that are listed below. a Christie DJ, Avari T, Carrington LR, et al. Clinical and Laboratory Standards Institute (CLSI). Platelet function testing by aggregometry; approved guideline. CLSI document H58-A (ISBN 1-56238-683-2) [database on the Internet], vol. 28(31). Clinical and Laboratory Standards Institute, Wayne, PA, USA; 2008. p. 1–45. b Hayward CP, Moffat KA, Raby A, et al. Development of North American consensus guidelines for medical laboratories that perform and interpret platelet function testing using light transmission aggregometry. Am J Clin Pathol 2010;134:955–63. c Cattaneo M, Cerletti C, Harrison P, Hayward CP, Kenny D, Nugent D, Nurden P, Rao AK, Schmaier AH, Watson SP, Lussana F, Pugliano MT, Michelson AD. Recommendations for the Standardization of Light Transmission Aggregometry: A Consensus of the Working Party from the Platelet Physiology Subcommittee of SSC/ISTH. J Thromb Haemost 2013. https://doi.org/10.1111/jth.12231.

Percent

610

40 50 60

Aggregation Epinephrine

70

Collagen ATP release Collagen Epinephrine

80 90 100 2:00

4:00 6:00 Time (min:s)

8:00

Fig. 34.1 Patterns of normal light transmission platelet aggregation and dense granule ATP release. The lumiaggregometer tracing shows the typical patterns of normal aggregation (shown as percent values) and dense granule ATP release (lower tracings) with epinephrine (a weak agonist) and collagen (a strong agonist). With collagen, ATP release and aggregation begin at the same time. With epinephrine, the aggregation response is biphasic, and ATP release begins at the start of the secondary aggregation wave (which is at the 2min time point in the tracing). This figure also illustrates that it takes longer to reach the maximal aggregation with epinephrine than with collagen.

Platelet Aggregation

aggregation with epinephrine and other weak agonists and, accordingly, secondary aggregation can be absent or reduced with weak agonists in subjects with severe dense granule deficiency unless exogenous ADP is added.33–35 However, absent secondary aggregation can be a normal variant when LTA is tested with epinephrine using platelet count-adjusted, citrated PRP.36 In citrated plasma, biphasic LTA responses can follow stimulation by other weak agonists (e.g., ADP) that do not trigger dense granule release until aggregation occurs. Depending on the volume of agonist added to the test (which should not exceed 10% of final volume), a slight change in light intensity may be evident after adding an agonist, before shape change or aggregation begins. While two waves of aggregation occur when citrated PRP is tested with epinephrine (Fig. 34.1), even when added at very high concentrations, the visualization of two waves for other weak agonists can require titration of the agonist because the primary and secondary waves often appear fused. Fig. 34.2 shows two waves with 2.5 μM ADP and fused primary and secondary aggregation waves with 5.0 μM ADP.

Stability and Timing of Turbidometric Aggregation Responses When aggregation is performed with low concentrations of a weak agonist (e.g., 2.5 μM ADP, as in Fig. 34.2), some reversal of aggregation may occur, which is referred to as deaggregation

611

or disaggregation.37 With deaggregation, the amount of aggregation at the end of the test (which is called the final aggregation) is lower than the maximal aggregation. Deaggregation can accompany reduced aggregation although it can be a normal finding with some agonists, tested at low concentrations. Fig. 34.3 shows an example of mild deaggregation with 2.5 and 5.0 μM ADP due to an aspirin-induced defect; the deaggregation in this case is abnormal at the higher ADP concentration. Extensive deaggregation is uncommon, and it usually accompanies reduced maximal aggregation. Fig. 34.4 shows deaggregation with 5.0 μM ADP due to combined therapy with clopidogrel and aspirin. Fig. 34.5 shows deaggregation with a number of agonists, due to acquired defects from a bone marrow disorder. To evaluate the stability of aggregation, LTA and WBA are typically monitored for a number of minutes after maximal aggregation is achieved. Aggregation needs to be monitored longer with epinephrine (e.g., 10 min compared to 5 min for other agonists), given the slower response.14,36,38 Guidelines recommend completing aggregation tests within 4 h of sample collection, although 2 h may be preferable.29

Mechanisms of Turbidometric Aggregation Responses to Different Agonists With collagen, the responses evident in aggregation tracings reflect a complex series of events.39 First, platelets adhere to

Fig. 34.2 Examples of normal platelet aggregation responses, evaluated by optical or impedance endpoints. The tracings show aggregation responses for the same healthy control (color-coded by agonist, as indicated; AA indicates arachidonic acid, U46619 indicates the thromboxane A2 analog U46619). Aggregation is evident with all agonists except the lower concentration of ristocetin that detects gain-offunction defects. The optical (light transmittance) aggregation tracings show responses to 2.5 and 5.0 μM ADP, 1.25 and 5.0 μg/mL Horm collagen, 1.6 mM arachidonic acid, 1.0 μM U46619 and 0.5 and 1.25 mg/mL ristocetin. With ADP, the response to 2.5 μM ADP is biphasic, whereas primary and secondary waves are fused at the higher concentration. With collagen, there is an obvious reduction in the light transmission after adding the agonist, due to shape change, and aggregation is more rapid with the higher concentration. Maximal aggregation responses to agonists are sustained, without evidence of deaggregation. Whole blood impedance aggregation (measured in Ohms, simultaneous with ATP release, with 5.0 μM ADP, 1.25 and 5.0 μg/mL Horm collagen, 1.0 mM arachidonic acid, 1.0 μM U46619, and 0.25 and 1.0 mg/mL ristocetin) also detects responses to agonists but not shape change. The ATP release with 1.0 mg/mL ristocetin (which is not normally assessed when testing whole blood aggregation) illustrates that the agglutination results in platelet activation.

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Fig. 34.3 Typical platelet aggregation abnormalities induced by aspirin. The light transmission aggregation tracings (color-coded by agonist) show the responses to 2.5 and 5.0 μM ADP, 1.25 and 5.0 μg/mL Horm collagen, 1.6 mM arachidonic acid (AA), 1 μM thromboxane A2 analog U46619 (U46619), and 0.5 and 1.25 mg/mL ristocetin. With aspirin, there is absent aggregation with arachidonic acid but normal aggregation with U46619. Aspirin also impairs aggregation with the lower concentration of collagen and reduces secondary aggregation with ADP (leading to some deaggregation, which is not normal with 5 μM ADP) and epinephrine (not shown). Aspirin can also reduce the maximal aggregation with ristocetin.

Fig. 34.4 Typical platelet aggregation abnormalities induced by combined aspirin and clopidogrel therapy. The light transmission aggregation tracings (agonists indicated) in this figure were kindly provided by Dr. Kandice Kottke-Marchant, Cleveland Clinic, USA. The responses to 5.0 μM ADP and 1.0 mM arachidonic acid (AA), each tested in duplicate, illustrate the reduced aggregation with arachidonic acid from aspirin and the markedly impaired response to ADP, with striking deaggregation, from clopidogrel.

Fig. 34.5 Acquired platelet function abnormalities in a subject with a myelodysplastic syndrome, associated with acquired bleeding problems. The light transmission aggregation tracings (color-coded by agonist; AA indicates arachidonic acid, U46619 indicates thromboxane A2 analog U46619) show the responses to 2.5 and 5.0 μM ADP, 1.25 and 5.0 μg/mL Horm collagen, 1.6 mM arachidonic acid, 1 μM U46619, and 0.5 and 1.25 mg/mL ristocetin. Aggregation with the higher concentration of collagen is normal, whereas the response to 1.25 mg/mL ristocetin is reduced and it is markedly impaired with ADP, the lower concentration of collagen, arachidonic acid, and U46619. Deaggregation is evident after the initial responses to U46619 and 5.0 μM ADP. The subject had a myelodysplastic syndrome that was complicated by development of bleeding problems that could not be explained by thrombocytopenia. Platelet dense granules were normal.

Platelet Aggregation

and are activated by the collagen fibrils, leading to shape change.11 The shape change is associated with an initial reduction in light transmittance that is followed by rapid increases in light transmittance from platelet aggregation (Figs. 34.1 and 34.2). With collagen and other strong agonists, dense granule secretion also occurs but is independent of platelet aggregation (e.g., Fig. 34.1), and this explains why platelet release can be normal when platelet aggregation is absent with strong agonists, as in Glanzmann thrombasthenia.40 Many agonists used to study aggregation (e.g., ADP, epinephrine, the thromboxane A2 analog U46619, collagen) activate platelets through direct binding interactions with an external platelet membrane receptor11 (Chapter 18). However, arachidonic acid is added as a substrate, which platelets convert to thromboxane A2, which in turn stimulates thromboxane receptors in order to initiate platelet aggregation11 (Fig. 34.2). As this conversion of arachidonic acid to thromboxane A2 requires the platelet enzymes cyclooxygenase-1 (COX-1) and thromboxane synthase, aggregation responses to arachidonic acid are inhibited by aspirin and other nonsteroidal anti-inflammatory drugs (NSAIDs) that block COX-1.11 Aggregation responses to arachidonic acid can also be impaired by congenital defects in COX-1 or thromboxane synthase.11 Accordingly, it can be helpful to compare aggregation responses to arachidonic acid and the thromboxane A2 analog U46619 to decide if there is evidence of an aspirin-like defect (Fig. 34.2 shows normal responses, whereas Fig. 34.3 shows the hallmark features of an NSAID-induced defect: impaired aggregation with arachidonic acid but not with the thromboxane analog). It is important to recognize that other disorders can impair arachidonic acid responses, including congenital thromboxane receptor defects (these impair aggregation responses to both arachidonic acid and thromboxane analog), dense granule deficiency, and platelet secretion defects from a variety of inherited platelet disorders including those caused by RUNX1 mutations (these may impair aggregation with other agonists, including thromboxane analog U46619—see Fig. 34.6).11,41 Combined treatment with aspirin (Chapter 50) and clopidogrel inhibits the ADP receptor P2Y12 (Chapter 51), in addition to COX-1, and accordingly, reduces maximal aggregation with ADP and arachidonic acid and leads to more extensive ADP deaggregation (Fig. 34.4) than observed with aspirin alone (Fig. 34.3). While some platelet function disorders impair aggregation responses to many but not all agonists (examples are shown in Figs. 34.5 and 34.6), congenital and acquired Glanzmann thrombasthenia cause profound aggregation defects,40 typically absent aggregation with all agonists except ristocetin (Fig. 34.7). However, some aggregation may be present when the αIIbβ3 deficiency or dysfunction is incomplete, as in some variant forms of Glanzmann thrombasthenia.42 Another important issue to consider is that the aggregation responses to some agonists, including collagen and epinephrine, are dependent on positive feedback from thromboxane A2 generation, and from the ADP released from dense granules (this is illustrated in the aspirin-induced defect shown in Fig. 34.3).11,31,33,34,41 Most aggregation assays are designed to detect reduced rather than increased aggregation, given that loss of function is seen much more frequently than gain-of-function defects.43–48 Nonetheless, ristocetin-induced platelet aggregation (RIPA) is routinely used in diagnostic aggregation studies to assess both gain-of-function and loss-of-function defects44–48 affecting VWF binding to GPIb-IX-V; these interactions can be induced by ristocetin or high shear force (Chapter 10). For RIPA, two concentrations of ristocetin are tested: (1) a low concentration that causes minimal or no platelet agglutination unless there is a gain-of-function defect from type 2B or platelet-type von Willebrand disease (VWD); and (2) a high concentration that triggers

613

rapid agglutination of normal platelets, mediated by VWF binding to platelet GPIb-IX-V.45–47 RIPA often produces a “saw tooth” pattern in turbidometric aggregation tracings (see Figs. 34.2 and 34.3). Fig. 34.2 shows normal RIPA, whereas Fig. 34.8 compares abnormal RIPA due to type 2B VWD (upper panel) and type 2A VWD (lower panel). Fig. 34.9 shows the gainof-function defect in RIPA for another type 2B VWD subject, whose plasma (unlike normal plasma) increased RIPA with the lower concentration of ristocetin when mixed with control PRP. In contrast, the loss-of-function defect in type 2A VWD (Fig. 34.7, lower panel) delays aggregation with the higher concentration of ristocetin. As the agglutination response to ristocetin (Fig. 34.2) is usually followed by platelet activation and aggregation mediated by αIIbβ3,44,48 a biphasic response is sometimes evident, as illustrated in Figs. 34.8 and 34.9. Reduced or delayed RIPA can reflect VWF deficiency or dysfunction (Fig. 34.8, lower panel), Bernard-Soulier syndrome (GPIb-IX-V deficiency or dysfunction) (Fig. 34.10 and Chapter 48)47,49 but most often, reduced RIPA is seen in combination with other aggregation abnormalities due to aspirininduced (e.g., Fig. 34.3) and other platelet function defects (Figs. 34.5 and 34.6, lower panel),36,40 including Glanzmann thrombasthenia (Chapter 48; a severe dysfunction, or absence, of αIIbβ3) (Fig. 34.7, upper panel). With Glanzmann thrombasthenia, there is no aggregation following ristocetin-induced agglutination. Some laboratories test platelet responses to additional agonists, including: thrombin receptor-activating peptides (TRAP); calcium ionophore A23187; antibodies that activate platelets through Fc receptors (e.g., antibodies associated with heparin-induced thrombocytopenia); and collagen-related peptide (CRP).

Whole Blood Aggregometry WBA4–6,12,50–54 is evaluated by monitoring changes in electrical impedance (in Ohms). In WBA assays, a monolayer of platelets first attaches to the electrodes prior to aggregation.4–6,12,50–55 Then, after an agonist is added, aggregating platelets attach to the monolayer of platelets on the electrode.4,12,50 Although LTA and WBA endpoints are quite different, their tracings show some similar features, as illustrated in Fig. 34.2, which shows LTA and WBA results from the same control subject. Depending on the volume of agonist added, there is sometimes a small reduction in impedance before WBA and ATP release are evident (e.g., Fig. 34.2). Some laboratories use only WBA for the assessment of bleeding disorders, whereas others that evaluate WBA also test LTA. The usefulness of WBA to assess bleeding disorders and platelet function has been less certain because the literature is not as extensive as it is for LTA. Recent studies have directly compared the diagnostic usefulness of WBA compared to LTA for the assessment of bleeding disorders, mainly using the Multiplate whole blood aggregometer (DiaPharma Group, West Chester, OH)56,57 (discussed later). Despite some similarities between WBA and LTA, it is important to recognize that there are some inherent differences. For example, biphasic aggregation is more readily detected by LTA, and release sometimes appears to precede aggregation in WBA, but not LTA, assays. The instrument manual for the lumiaggregometer recommends performing WBA with a larger receptacle and stir bar, and a higher mixing speed (1200 compared to 1000 rpm) than used for LTA, and it also recommends routine simultaneous measurement of ATP release with WBA. Although measuring ATP release with ristocetin is not recommended for routine WBA tests, Fig. 34.2 illustrates that the agglutination phase of RIPA is accompanied by platelet activation and ATP release.

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Fig. 34.6 Platelet aggregation abnormalities in several cases with a hereditary platelet function disorder due to a RUNX1 mutation. Light transmission aggregation tracings are shown (color-coded by agonist; evaluated using platelet count-adjusted platelet rich plasma; AA indicates arachidonic acid, U46619 indicates the thromboxane A2 analog U46619). The upper panel shows results for an individual who had mild thrombocytopenia and a low platelet count sample that showed reduced maximal aggregation with collagen and U46619 but normal findings with AA and ristocetin. The lower panel shows results for an unrelated individual with a different RUNX1 haploinsufficiency mutation, who was not thrombocytopenic and had reduced maximal aggregation with collagen, AA, U46619, and ristocetin.

The recommended concentrations of some agonists differs for WBA and LTA assays (Table 34.1).12–16 All blood cells are present in samples tested by WBA. Both the platelet count (even within the normal range) and leukocyte count influence the WBA responses measured by electrical impedance.58 When WBA is tested with collagen, neutrophils and monocytes, along with platelets, attach to the electrodes; whereas leukocyte attachment to electrodes is minimal when ADP aggregation is tested with whole blood.51,53 Unlike LTA, WBA is performed after diluting whole blood samples 1:1 with physiological saline so it requires less blood than PRP aggregation.12 Undiluted blood can be tested by WBA if the diluted sample platelet count will be less than 100  109 platelets/L.12 Accordingly, WBA requires less blood than PRP aggregation. For aggregation tests on subjects with macrothrombocytopenia, WBA assesses the functions of all blood platelets, including the largest forms that are lost when

PRP is prepared by centrifugation.12 Unlike LTA, WBA is not affected by lipemia.12 The findings for WBA and LTA with some agonists differ because WBA tests aggregation responses at platelet counts that are much lower than in PRP assays. Accordingly, WBA is not used to assess aggregation responses to epinephrine because some healthy subjects show no aggregation (Table 34.1).13,54,55 However, with LTA, epinephrine aggregation can also be absent when the PRP contains less than 250  109 platelets/L.38 The inability to use some agonists for WBA could affect the diagnostic utility of WBA57,58 because weak agonists such as epinephrine are very helpful to detect LTA abnormalities from common platelet function disorders.36,43 While much of the published data on WBA for bleeding disorders used Chrono-Log instruments, the Multiplate instrument detects aggregation abnormalities from some platelet function disorders,12,57–60 but not as well as LTA.56,57

Platelet Aggregation

615

Congenital GT

34

100 90 80 70 60 50 40 30 20 10 0 –10

Percent

1.25 mg/mL Ristocetin

Collagen AA U46619

0

30

60

90

120

150

180

210

240

270

300

Time (s) Acquired GT: inhibitor detection by mixing studies 100 90 80

Collagen Control AA plasma

70 Percent

60 50 40 30 20 10

Collagen

0 AA

–10 0

30

60

90

120

150

180

210

Time (s)

Measurement of Dense Granule Release With Aggregometry The release of platelet dense granule contents can be monitored as part of WBA or LTA assays (Figs. 34.1 and 34.2).3,5,11,12,61–63 The methods for assessing platelet dense granule release have been recently reviewed.63 The most commonly used method is a bioluminescent assay that uses D-luciferin and firefly luciferase to measure ATP release in response to agonist stimulation, usually simultaneously with aggregation.3,7,8,12,62,63 The test principle, which is illustrated in Fig. 34.11, is as follows: platelet activation stimulates the release of dense granule ATP. The released ATP combines with the added D-luciferin, and in the presence of luciferase, it undergoes conversion to inorganic pyrophosphate and the intermediate luciferyl adenylate and then combines with oxygen to generate the final reaction products: adenosine monophosphate, oxyluciferin plus light.12,63 The emitted light is quantified using an ATP standard to calibrate each sample. As the luciferase reagent is quite labile at 37°C, ATP release testing needs to be done by carefully controlled procedures, usually adding the reagent before rather than after aggregation to reduce some of the variability. Nonetheless, the amount of ATP release still shows considerable variability, which limits the diagnostic usefulness of measuring ATP release.64,65 Other methods that can be used to quantify dense granule release with aggregation include serotonin release assays63 (because serotonin is stored in platelet dense granules; see Chapter 19). Serotonin release into plasma (or into the supernatant of washed platelets) can be measured by biochemical

240

270

Patient plasma 300

Fig. 34.7 Platelet aggregation abnormalities associated with congenital and acquired Glanzmann thrombasthenia. Light transmission aggregation tracings are shown (color-coded by agonist; evaluated using platelet count-adjusted platelet rich plasma; AA indicates arachidonic acid, U46619 indicates the thromboxane A2 analog U46619). The upper panel shows findings for congenital Glanzmann thrombasthenia (GT), manifested by a reduced response to 1.25 mg/mL ristocetin (as agglutination was not followed by aggregation) and absent aggregation with other agonists (1.25 μg/mL collagen, 1.6 mM arachidonic acid, and 1.0 μM U46619). Shape change with collagen is preserved. The lower panel (which shows responses to 1.6 mM arachidonic acid and 5 μg/mL collagen) illustrates transfer of GT-like defect to normal platelets by plasma from an individual with acquired GT (with documented αIIbβ3 auto-antibodies). The normal control plasma did not inhibit aggregation of normal platelets.

assays, including radioactive methods that require a preincubation of PRP with radioactive serotonin before testing aggregation and measuring how much radioactive serotonin was released into the plasma after aggregation.61,63,66 When normal platelets are tested with weak agonists, such as epinephrine, dense granule release is delayed until the secondary phase of aggregation begins (Fig. 34.1). However, with strong agonists, such as collagen or thrombin, there is simultaneous aggregation and secretion (Fig. 34.1 illustrates this for collagen).11,33 ATP release with collagen is impaired by aspirin, whereas ATP release by thrombin is preserved. At low concentrations, strong agonists may fail to induce dense granule secretion.11 Dense granule release is typically assessed with a panel of agonists (e.g., thrombin, collagen, epinephrine, thromboxane analog U46619, and arachidonic acid).11,63 When thrombin is used to measure ATP release with LTA and WBA, aggregation is not measured because a clot forms. Compared to aggregation endpoints, dense granule release endpoints show much greater variability61,63–67 and this limits their usefulness to detect impaired platelet function from a bleeding disorder.65 Despite this limitation, dense granule release assays are often used to determine if reduced aggregation is associated with impaired dense granule release and/or if there is a defect in dense granule release despite normal aggregation findings, as discussed later.11,16,63,68 However, release assays are not a suitable replacement for aggregation tests because some platelet function disorders impair aggregation but not dense granule release.65

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Percent

Type 2B von Willebrand disease

5 mg/mL collagen 100 5 mM ADP 90 80 1.25 mg/mL ristocetin 70 1 mM U46619 60 2.5 mM ADP 0.5 mg/mL ristocetin 50 40 30 1.25 mg/mL collagen 20 10 1.6 mM AA 0 –10 0

20

40

60

80

100 120 140 160 180 200 220 240 260 280 Time (s)

Percent

Type 2A von Willebrand disease

100 90 80 70 60 50 40 30 20 10 0 –10 0

5 mg/mL collagen 1.25 mg/mL collagen

1.25 mg/mL ristocetin

0.5 mg/mL ristocetin 30

60

90

120

150 180 Time (s)

210

240

270

300

Fig. 34.8 Examples of gain-of-function and loss-of-function platelet aggregation defects identified by ristocetin. The panels show abnormal ristocetin-induced platelet agglutination (evaluated by light transmission aggregation, using platelet count-adjusted platelet-rich plasma) from type 2B (upper and middle panel) and type 2A (lower panel) von Willebrand disease. Type 2B von Willebrand disease is associated with a gain-of-function response to 0.5 mg/mL ristocetin, without other aggregation abnormalities (upper panel shows responses to 0.5 and 1.25 mg/mL ristocetin, 2.5 and 5.0 μM ADP, 1.25 and 5.0 μg/mL collagen, 1.0 μM thromboxane A2 analog U46619, and 1.6 mM arachidonic acid [AA]). Unlike normal plasma, type 2B von Willebrand disease plasma induces increased aggregation with ristocetin when added to normal platelets, as illustrated in the middle plasma. In contrast, loss of function from type 2A von Willebrand disease impairs the agglutination response to 1.25 mg/mL ristocetin (in this case, manifested by a delayed response) but not responses to other agonists (e.g., collagen).

Some caution is warranted about the simultaneous measurement of ATP release and aggregation. The commercial D-luciferin/luciferase reagent, which contains magnesium, can potentiate sub-maximal PRP and whole blood aggregation responses of sodium citrate-anticoagulated samples.69,70 Like magnesium, the reagent can falsely normalize LTA findings for some canine and some human platelet disorders (an example of the reagent normalizing the impaired epinephrine aggregation response of Quebec platelet disorder is shown in Fig. 34.12), although such potentiation appears uncommon among human platelet disorders.70,71 With Quebec platelet disorder, the potentiating effects are evident with both native and platelet count adjusted PRP70 and could cause false negative aggregation findings. WBA and LTA reference intervals for tests done with added D-luciferin/luciferase reagent should not be applied to aggregation tests performed without this reagent.

PREPARATION OF SAMPLES FOR PLATELET AGGREGATION TESTS Before blood samples are collected for aggregation studies (including samples from healthy controls), the list of the subject’s current medications should be reviewed to minimize

interference from aspirin and other NSAIDs. It is reasonable to have subjects rest prior to drawing the sample because exercise can affect some aggregation responses, as can smoking and some dietary practices (e.g., taking fish oil supplements).13,72–77 Many laboratories do not require subjects to fast prior to having a sample drawn for aggregation testing because eating a light meal before testing has minimal effects, and lipemic samples are uncommon. Samples collected in the morning to midday show similar findings, and many laboratories avoid collecting samples at other times of the day because of diurnal variation in some aspects of platelet function.77,78 LTA and WBA are usually performed using samples collected by venipuncture (via a needle or a butterfly cannula collection device) and gently mixed with 3.2% (105–109 mM) or 3.8% (0.129 M) sodium citrate anticoagulant (9 volumes whole blood to 1 volume anticoagulant). Samples are commonly collected using vacuum-evacuated or screw-capped plastic collection tubes that cause minimal platelet activation or platelet loss from adhesion, and the harvested PRP should be kept in tubes that limit platelet activation and loss.14,19,79 Recent studies have explored the use of alternative anticoagulants to sodium citrate, to preserve platelet function beyond the usual 3–4 h limit for aggregation and release assays.50,80–82 However, none of the published studies evaluated the

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34

Fig. 34.9 Example of a mixing study to evaluate if a gain-of-function platelet aggregation defect with ristocetin is due to a defect in von Willebrand factor. The panels show abnormal ristocetin-induced platelet agglutination (evaluated by light transmission aggregation, using platelet count-adjusted platelet-rich plasma and 0.5 and 1.25 mg/mL ristocetin) due to type 2B von Willebrand disease, including the results of mixing studies (lower panel). Unlike adding pooled normal plasma (PNP), adding type 2B von Willebrand disease platelet poor plasma (PPP) to normal platelet rich plasma (PRP), transfers the gain-of-function defect (lower panel).

suitability of alternative anticoagulants (that do not chelate calcium) for detecting impaired aggregation from bleeding disorders. It is common practice to collect a sample for a complete blood count determination when drawing samples for LTA or WBA. The blood count information is useful to determine whether the subject has thrombocytopenia and/or very large platelets, and ideally this should be determined before centrifuging a sample collected for LTA15 or diluting a sample for WBA. To preserve platelet function, one should handdeliver samples for aggregation testing to the laboratory, and not use pneumatic tube transport systems.83 Because platelet function is impaired by changes in sample pH,29 care should be taken to sustain a normal blood pH during sample collection and processing; typically, the pH is maintained using a buffered anticoagulant and capped tubes (to minimize exposure to air that lowers the pH) until aggregation is performed.13,15 For WBA by lumiaggregometry, the instrument manufacturer recommends a rest period of 20–30 min

after drawing the sample before testing responses to ADP, to limit effects of any ADP released from red cells during sample collection. Similarly, for LTA, a brief rest period of about 15 min is recommended, before and after the centrifugation steps to isolate PRP,15 although the rest steps do not affect some agonist responses. PRP for aggregation and dense granule release testing is prepared by a low-speed centrifugation, often for 10 min.15 The relative centrifugal force (RCF; g force) used affects the numbers and size of platelets in the harvested PRP (more platelets are lost when higher speed centrifugation is used), and the degree of red cell contamination.84,85 This has led some experts to recommend a 10-min centrifugation at 200–250 g for PRP preparation.85 The dimensions of the tube used to hold the sample will affect the g force during centrifugation, so it is important that laboratories validate their procedure. Caution is required when preparing PRP from subjects with macrothrombocytopenia because large platelets are removed at a lower RCF than normal platelets.15

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Fig. 34.10 Platelet aggregation abnormalities due to Bernard-Soulier syndrome. The light transmission aggregation tracing (color-coded by agonist) are shown for platelet-rich plasma. The upper panel shows LTA responses for a child with Bernard-Soulier syndrome with macrothrombocytopenia and a severe GPIb-IX-V deficiency. There is absent aggregation with 1.25 mg/mL ristocetin but normal aggregation with other agonists (results shown for two concentrations of ristocetin and ADP, 5 μg/mL Horm collagen, and 1 μM thromboxane A2 analog U46619).

D-luciferin

+ ATP Firefly luciferase Mg++

Luciferyl adenylate + PPi

O2

There is no widely accepted lower limit for PRP platelet counts for aggregation testing, and low platelet count samples can be used to diagnose some thrombocytopenic disorders with aggregation defects (e.g., Bernard-Soulier syndrome, type 2B VWD) (Table 34.2). Accordingly, laboratories should not refuse to test low platelet count samples and at the very least, they should test aggregation with ristocetin to help assess for Bernard-Soulier syndrome and for type 2B and platelet type VWD.

Firefly luciferase

Oxyluciferin + AMP + H2O + CO2 + light Fig. 34.11 Principle of dense granule release ATP assays. The reagent for measuring ATP release contains D-luciferin, firefly luciferase, and magnesium. Platelet activation induced by an agonist leads to release of dense granule ATP. The released ATP combines with Dluciferin, and in the presence of firefly luciferase and magnesium, the intermediate luciferyl adenylate and inorganic phosphate are formed. Next, the luciferyl adenylate combines with oxygen, leading to the formation of oxyluciferin, adenosine monophosphate, water, and carbon dioxide plus light. The emitted light is quantified as the assay endpoint.

Once PRP is harvested for LTA, the remaining sample is often centrifuged at 1500 g to obtain autologous PPP for setting the LTA baseline and for adjusting the PRP to a standardized platelet count, if desired.15 Because cold exposure significantly alters platelet function, aggregation samples should be kept at room temperature until just before testing, and WBA and LTA should be assessed after a brief preincubation at 37°C, typically for 1–3 min, before testing aggregation responses at body temperature.15 The recommended preincubation time should be validated to bring the sample to body temperature before an agonist is added.15 LTA can be evaluated using native PRP (used without a platelet count adjustment) or PRP that is first adjusted to a standardized platelet count by adding autologous PPP.36,86–88 Both sample types have been validated for bleeding disorder evaluations by LTA.36,43 The responses to some weak agonists (e.g., ADP and epinephrine) are different for native and platelet count-adjusted samples,36,86–88 as further discussed in section “Aggregation interpretation in the assessment of bleeding disorders.”

ANALYTICAL CONSIDERATIONS AND POTENTIAL INTERFERENCES FOR AGGREGATION TESTS Manufacturer recommendations for aggregometry instrument performance should be considered because the procedures for operating the instrument, setting the baseline, deflecting for maximal aggregation, and verifying baseline stability before adding an agonist vary by instrument, as do the dimensions of the aggregation cuvettes and stir bars. Most diagnostic aggregometer instruments use software that generates reports of the quantitative data (e.g., extent of maximal aggregation, sometimes final aggregation, lag, and slope) in addition to tracings of the aggregation response. LTA is normally monitored for approximately 5 min after adding an agonist.13,15,38 Epinephrine responses are slower, often requiring monitoring for 10 min to ensure that maximal aggregation is achieved and recorded. Because LTA is performed using an optical detection principle, improper sample collection, platelet activation and medical conditions that change the optical characteristics of plasma have the potential to modify LTA findings. Accordingly, caution is advised when samples show signs of platelet activation (e.g., loss of swirling when PRP is gently pipetted up and down), evidence of visible hemolysis (which could have activated platelets and desensitized aggregation responses to ADP), significant leukocyte or red cell contamination, or appear icteric or lipemic.89 There are significantly more red cells in PRP samples prepared at 150 g compared to 200–250 g, which can reduce the maximal aggregation achieved.29 It is considered good practice to validate the PRP preparation procedure by checking the appearance of the PRP and the leukocyte, red cell and platelet counts of PRP samples. To avoid significant preanalytical artifacts from time-dependent changes in platelet function, laboratories should complete aggregation testing

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619

34 Fig. 34.12 A comparison of light transmission aggregation reference intervals for maximal aggregation, evaluated for native and platelet count-adjusted platelet-rich plasma. The findings by agonist (AA indicates arachidonic acid, U46619 indicates thromboxane A2 analog U46619) for native and platelet count-adjusted samples are similar except platelet count-adjusted samples show greater variation in responses to weak agonists (ADP and epinephrine), and native samples show greater variation with 0.5 mg/mL ristocetin. (Reproduced with permission from Castilloux JF, Moffat KA, Liu Y, Seecharan J, Pai M, Hayward CP. A prospective, cohort study of light transmission platelet aggregometry for bleeding disorders: Is testing native platelet rich plasma noninferior to testing platelet count adjusted samples? Thromb Haemost 2011;106:675–82.)

TABLE 34.2 A Suggested Approach for Testing Aggregation Responses of Platelet-Rich Plasma Samples With Low Platelet Counts Platelet-Rich Plasma Platelet Count (× 109/L)a Agonist

≤80

>80–≤100

>100–≤140

>140–250

Ristocetin 0.5–0.6 mg/mL

Test all samples and if the aggregation response is increased, evaluate for possible type 2B and platelet-type von Willebrand disease

Ristocetin 1.2–1.5 mg/mL

Test all samples and review findings from the von Willebrand disease screens to exclude von Willebrand disease. If the aggregation response is reduced or absent, evaluate for Bernard-Soulier syndrome

Collagen high concentration (e.g., 5 μg/mL of type I collagen)

Interpret findings with caution

ADP 2–10 μM

Omit

The reduced sample platelet count will likely influence the findings. If possible, use reference limits for low platelet count samples to interpret

Thromboxane A2 analog U46619 1 μM

Omit

Arachidonic acid 0.5–1.6 mM

Omit

Epinephrine 5–10 μM

Omit

The reduced sample platelet count will likely influence the findings. If possible, use reference limits for low platelet count samples to interpret

a

This table was reproduced with permission and modified from Hayward CP, Moffat KA, Pai M, et al. An evaluation of methods for determining reference intervals for light transmission platelet aggregation tests on samples with normal or reduced platelet counts. Thromb Haemost 2008;100:134–45.

within 4 h of sample collection for LTA13,15,29 and within 3 h of sample collection for WBA.90

QUANTITATIVE ENDPOINTS OF AGGREGATION AND RELEASE ASSAYS The evaluation of aggregation findings requires a qualitative review of the aggregation tracing and a quantitative analysis of the maximal aggregation (MA) responses with evaluated agonists.11–16 Additional parameters that can be assessed include: shape change (for LTA);91 final aggregation; the amount of deaggregation (i.e., the difference between the maximal and final aggregation); the slope of the aggregation response; and the area under the aggregation curve, which reflects the rapidity and extent of maximal aggregation and deaggregation.13,15 In

practice, most abnormalities that impair aggregation and/or cause deaggregation reduce the maximal aggregation achieved. Most aggregation and dense granule release parameters do not have a Gaussian distribution (i.e., the data do not show a symmetrical, bell-shaped distribution).38,67 Accordingly, the 95% reference interval limits for quantitative analysis of LTA and WBA should be estimated by nonparametric statistical methods, using a minimum of 40 unique donor samples to limit false positives and false negatives.14,38,67,91 Because many clinical laboratories retest some controls and face challenges in obtaining samples from large numbers of healthy controls, laboratories should use statistical methods for reference interval determination that accommodate data with variable numbers of repeat measures for subjects.14,38,92 For all quantitative results that are reported, reference intervals must be established

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Percent

Quebec platelet disorder 5 µg/mL collagen

100 90 80 70 60 50 40 30 20 10 0 –10

1.25 mg/mL ristocetin

5 µM ADP 1.6 mM AA 1.25 µg/mL collagen 2.5 µM ADP 0.5 mg/mL ristocetin 1 µM U46619 20

40

60

80

100

120

140

160

180

200

220

240

260

280

Time (s) 110 100 90

100 µM epinephrine + luciferase reagent

Percent

80 70

6 µM epinephrine + luciferase reagent

60 50 40 30

overlapping responses to 6 and 100 µM epinephrine (no luciferase reagent)

20 10 0 –10 –20 0

50

100

150

200

250

300

350

400

450

500

550

Time (s) Fig. 34.13 Platelet aggregation abnormalities due to Quebec platelet disorder. The light transmission aggregation tracings (color-coded by agonist) for a person with Quebec platelet disorder show responses to multiple agonists (AA indicates arachidonic acid, U46619 indicates thromboxane A2 analog U46619), evaluated with platelet count-adjusted platelet-rich plasma (PRP). The only abnormalities found were reduced aggregation and deaggregation with U46619 (which was not seen in other affected family members) and minimal aggregation with 6 and 100 μM epinephrine (lower panel; also evident with native PRP [not shown]), unless the reagent for measuring dense granule ATP release was added, as reported in: Hayward CP, Moffat KA, Castilloux JF, Liu Y, Seecharan J, Tasneem S, Carlino S, Cormier A, Rivard GE. Simultaneous measurement of adenosine triphosphate release and aggregation potentiates human platelet aggregation responses for some subjects, including persons with Quebec platelet disorder. Thromb Haemost 2012;107(4):726–34.

for each laboratory’s procedure, instrument, and reagent combination.13–15 Validation of reference intervals for new reagent lots can be done with a smaller number of samples. The reference interval used should be sample-type specific, given that maximal aggregation responses to some agonists (e.g., ADP, epinephrine, and ristocetin) differ between native and platelet count-adjusted PRP, tested by otherwise identical procedures on common samples (Fig. 34.13).The common practice for reference interval determination is to use a representative population, of males and females, that are not taking drugs that inhibit platelet function, given that males and females show similar results by LTA and WBA assays.93 As platelet function is different in neonates compared to adults (see Chapter 25),94,95 adult aggregation reference intervals are considered acceptable for children older than neonates,14 as discussed later. Uncertainties exist about whether there is a maximal acceptable platelet count for LTA and WBA. For WBA, the sample platelet counts influence the measured aggregation, with low platelet count samples showing reduced responses.52,93,96–98 While some have suggested testing undiluted blood when the platelet count is low, to improve detection of platelet function abnormalities, there is need for further validation of WBA using undiluted samples. When PRP is collected from blood with normal platelet counts, the PRP platelet count shows little

relationship to maximal aggregation.36,52,86–88,93 When LTA is evaluated for thrombocytopenic individuals, with native PRP containing <200–250  109 platelets/L, there is a relationship between the maximal aggregation response and the sample platelet count.38 To compensate for this, regression analysis of diluted healthy control samples has been used to derive reference intervals for PRP with <250  109 platelets/L and guide which agonists to test, based on the expected responses for samples with similar platelet counts38 (updated recommendations are outlined in Table 34.2). Aggregation and dense granule release tests show some within and between subject variability that is greater for release than aggregation endpoints and affects reference intervals.36,61,64,65 Although most aggregation findings are confirmed when tests are repeated, repeat testing is usually advised to confirm findings and to exclude a preanalytical or analytical artifact.

AGGREGATION INTERPRETATION IN THE ASSESSMENT OF BLEEDING DISORDERS LTA and WBA should be reported with (1) an overall interpretative comment, (2) a list of the agonists tested and their final concentrations in the test, and (3) the quantitative information recorded on the aggregation responses (e.g., maximal

Platelet Aggregation

aggregation with each agonist, as a minimum), accompanied by validated reference intervals. Many laboratories use adult aggregation reference intervals for children older than neonates, based on published data.94,95 Neonatal platelet function is discussed in more detail in Chapter 25. Sample-type specific patterns and reference intervals need to be considered for a proper evaluation of aggregation findings. Laboratories should be aware that LTA findings for native and platelet count-adjusted PRP differ significantly with some weak agonists, such as ADP and epinephrine (Fig. 34.13).36,86–88 Furthermore, absent secondary aggregation with epinephrine is an abnormal finding for native PRP, whereas it can be a normal variant for platelet count-adjusted PRP36,86–88 or reflect a platelet function disorder, such as Quebec platelet disorder (Fig. 34.12). Although native PRP shows less variability than platelet count-adjusted PRP in LTA assays with ADP and epinephrine (and accordingly, narrower reference intervals; Fig. 34.13), platelet count-adjusted samples show less variable responses to ristocetin than native PRP.36 However, LTA with platelet count-adjusted PRP provides more sensitive detection

621

of abnormal responses to weak agonists, such as epinephrine36 (Fig. 34.14, which summarizes the likelihood, as an odds ratio, of detecting an abnormality with individual agonists or an agonist panel using either native or platelet count-adjusted PRP). Impaired aggregation with multiple agonists, with both sample types, is quite predictive of a bleeding disorder36 (Fig. 34.14). Similar likelihood analyses were recommended to guide the interpretation of ATP release findings, but with the variability in ATP release endpoints, the results are not predictive of a bleeding disorder.65 Table 34.3 (which was adapted from the North American consensus guidelines14) summarizes patterns of abnormal aggregation findings and the potential causes to consider. If the sample tested had a low platelet count, the results should first be evaluated for abnormalities with ristocetin that would suggest either type 2B VWD (Fig. 34.8), Bernard-Soulier syndrome (Fig. 34.9), or platelet type VWD. If aggregation is strikingly impaired or absent with all agonists except ristocetin, Glanzmann thrombasthenia (Fig. 34.7) should be considered as a potential cause because variants with thrombocytopenia

Fig. 34.14 The likelihood (as an odds ratio) of detecting impaired platelet aggregation from a bleeding disorder using native or platelet count-adjusted platelet-rich plasma samples. The findings for native (N) or platelet count-adjusted samples (A) illustrate that both sample types detect impaired platelet function from bleeding disorders. Results for the entire panel (shown at top) illustrate that multiple abnormal agonist responses are more predictive of a bleeding disorder. Among agonists (AA indicates arachidonic acid, U46619 indicates thromboxane A2 analog U46619) epinephrine, U46619, and the lower concentrations of collagen and ADP are more likely to detect abnormalities from common aggregation defects. Abnormalities with collagen are more frequently seen with platelet countadjusted samples although native samples are also helpful to detect platelet function disorders. (The data shown are from a prospective cohort study and were reproduced, with permission, from Castilloux JF, Moffat KA, Liu Y, Seecharan J, Pai M, Hayward CP. A prospective, cohort study of light transmission platelet aggregometry for bleeding disorders: Is testing native platelet rich plasma noninferior to testing platelet count adjusted samples? Thromb Haemost 2011;106:675–82.)

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TABLE 34.3 Patterns of Abnormal Platelet Aggregation and Potential Causesa Aggregation Findings

Potential Causes

Aggregation absent or markedly reduced with arachidonic acid but normal with thromboxane A2 analog U46619. Aggregation also reduced with lower concentrations of collagen and there is absent secondary aggregation with epinephrine Aggregation is present with only ristocetin (or is markedly impaired with all agonists except ristocetin). Platelet count and size may be normal, or there may be an associated macrothrombocytopenia

These abnormalities are typical of an aspirin-like defect, which can be caused by drugs that inhibit cyclooxygenase-1 and, less commonly, by inherited defects in thromboxane generation

Aggregation absent with high concentrations of ristocetin and there is thrombocytopenia and very large platelets Aggregation is present but reduced with high concentrations of ristocetin, without an associated thrombocytopenia Aggregation is abnormally increased with low concentrations of ristocetin. Thrombocytopenia and platelet clumping may be present

Aggregation is abnormal with multiple agonists and is markedly reduced with ADP, with significant deaggregation Aggregation tests show abnormalities with two or more agonists that differ from the abnormalities described above Abnormalities with only one agonist (excluding collagen or ristocetin)

Aggregation is normal with all agonists but is markedly reduced with collagen, tested at low and high concentrations Normal aggregation findings

This type of abnormality suggests possible Glanzmann thrombasthenia, due to inherited or acquired defects in integrin αIIbβ3. Gain-of-function defects in this receptor should be considered if macrothrombocytopenia is present Possible Bernard-Soulier syndrome, which can be caused by inherited or acquired abnormalities (e.g., from autoantibodies) in the GPIb-IX-V. A von Willebrand factor deficiency should be excluded Possible von Willebrand disease or a defect in the GP Ib-IX-V. Ristocetininduced platelet aggregation abnormalities usually reflect a significant deficiency or dysfunction of von Willebrand factor The findings suggest a gain-of-function defect in platelet—von Willebrand factor interactions, possibly due to type 2B or platelet-type von Willebrand disease. A false positive should be considered if there is borderline increased aggregation. The findings of the von Willebrand disease screen, including multimers, should be reviewed These abnormalities suggest a possible defect in the platelet ADP receptor P2Y12, which can be inherited or drug-induced (e.g., from clopidogrel or prasugrel). The drug history should be reviewed These types of findings are common and suggest a platelet function disorder is present. Often these types of abnormalities are associated with defects in dense granule secretion and, less commonly, dense granule deficiency Nondiagnostic findings that could represent a false positive. If the abnormality is seen only with epinephrine, the possibility of Quebec platelet disorder should be considered, particularly if there is a history of delayed bleeding A platelet collagen receptor defect, involving glycoprotein VI or α2β1, should be considered There are some platelet function disorders that do not impair aggregation findings (e.g., mild dense granule deficiency and some dense granule release defects, Scott syndrome)

a

This table was modified and reproduced, with permission, from Hayward CP, Moffat KA, Raby A, et al. Development of North American consensus guidelines for medical laboratories that perform and interpret platelet function testing using light transmission aggregometry. Am J Clin Pathol 2010;134:955–63.

have now been reported (reviewed in Refs. 42, 95). If the findings are abnormal with multiple agonists but are not consistent with Glanzmann thrombasthenia, many different conditions, including acquired defects (e.g., Fig. 34.6) and other inherited platelet disorders, need to be considered (Table 34.3). If the platelet count of the evaluated subject is normal, the aggregation findings should be evaluated for “hallmark patterns” (summarized in Table 34.3) that suggest either an aspirin-like defect (Fig. 34.3; markedly reduced aggregation with arachidonic acid but normal aggregation with a thromboxane analog, accompanied by absent secondary responses to epinephrine and reduced aggregation with low concentrations of collagen), Glanzmann thrombasthenia (Fig. 34.7; aggregation is absent or markedly reduced with all agonists except ristocetin), VWD (Fig. 34.8; aggregation is abnormal only with ristocetin), or a P2Y12 defect (aggregation is markedly reduced and shows significant deaggregation with ADP, similar to the abnormalities induced by clopidogrel shown in Fig. 34.5).14,68 However, the most common finding is an abnormality that does not fit with one of these patterns, that often reflects a platelet secretion or activation defect, with or without dense granule deficiency.14,33–35,68,99–102 Some disorders (e.g., Quebec platelet disorder, Fig. 34.12) have aggregation findings that could reflect platelet dysfunction due to a variety of causes or that are nondiagnostic (e.g., absent secondary aggregation with epinephrine and/or an aggregation abnormality with only one agonist).68,103 Some subjects with Quebec platelet disorder have additional

abnormalities that could be confused with other disorders, such as platelet secretion defects (e.g., impaired aggregation responses to ADP and/or collagen and sometimes impaired aggregation with the thromboxane A2 analog U46619, as illustrated in Fig. 34.12) although release is normal in Quebec platelet disorder.70,103 Some aggregation defects from congenital disorders are rare (e.g., markedly reduced maximal aggregation and deaggregation with ADP, due to P2Y12 deficiency). The ADP aggregation responses of congenital P2Y12 defects look very similar to abnormalities from clopidogrel therapy (Fig. 34.5), and they are associated with abnormal responses to other agonists (e.g., low concentrations of collagen) that rely on ADP feedback through P2Y12.104 Some defects have been described for genetically modified animals (e.g., absent shape change with ADP in P2Y1 deficient animals105) but have not yet been reported to occur in humans. The agonist panel selected for testing can influence the detection of some aggregation abnormalities and the ability to distinguish between some causes of abnormalities.43 Table 34.4 illustrates these issues, with important practice points, based on case findings. If aggregation is normal but dense granule release is impaired with multiple agonists including thrombin, a further evaluation for dense granule deficiency should be considered given that dense granule deficiency has a relatively high prevalence, approaching that of VWD. We recommend testing for platelet dense granule deficiency, regardless of aggregation findings as we have observed

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TABLE 34.4 A Summary of Aggregation Findings and Practice Points for Some Typical Cases of Platelet Function Disorders Reason for Evaluation and Maximal Aggregation Finding Case 1 Very Large Bruises, Heavy Periods

Case 2 Large Bruises, Often Without Trauma and Serious Bleeding With Surgery

Case 3 Relatives With Serious Bleeding Problems Including Joint Bleeds

ADP, 2.5 and 5.0 μM Horm Collagen, 1.25 and 5.0 μg/mL Epinephrine, 6 μM

Normal

Reduced maximal aggregation

Normal

Reduced maximal aggregation

Reduced maximal aggregation with only the lower concentration

Normal

Normal

Absent secondary aggregation

Arachidonic acid, 1.6 mM Thromboxane A2 analog U46619, 1 μM Ristocetin 0.5 and 1.25 mg/mL Final diagnosis, based on aggregation findings and other tests Practice points

Reduced maximal aggregation

Reduced maximal aggregation

Reduced maximal aggregation, for both native and adjusted plateletrich plasma samples. (Relatives also had absent secondary aggregation with epinephrine) Normal

Reduced maximal aggregation with significant deaggregation

Reduced maximal aggregation with significant deaggregation

Normal

Normal

Normal

Normal

Impaired platelet function with multiple agonists due to dense granule deficiency

Platelet function defect, undetermined type

Quebec platelet disorder, based on genetic tests. Note that the aggregation findings were nondiagnostic

Laboratories can confuse these abnormalities with an aspirin-like defect particularly if they do not assess the aggregation response to thromboxane analog: in this case, the abnormalities with thromboxane analog exclude an aspirin-like defect. The other strategy that is often helpful to make the correct diagnosis is to test for dense granule deficiency and/or measure dense granule release: in this case, there was impaired release (with strong and weak agonists) and markedly reduced numbers of dense granules per platelet

The findings exclude an aspirin-like defect and suggest a platelet function defect of undetermined cause. Dense granule deficiency was excluded by electron microscopy

Aggregation testing is not sufficiently sensitive to detect all platelet function disorders. A single agonist abnormality is a nondiagnostic finding. Reference ranges differ for native and platelet count-adjusted samples, particularly with weak agonists, e.g., epinephrine

Agonist

many individuals with reduced platelet dense granule counts (median; range: 2.8; 1.4–4.6 dense granules/platelet; reference interval cutoff: <4.9) have nondiagnostic aggregation findings. Additionally, some individuals with confirmed dense granule deficiency have nondiagnostic dense granule ATP release findings.65

QUALITY EVALUATION OF PLATELET AGGREGATION TESTS Because LTA and WBA are performed with freshly prepared samples, it is not possible to provide external controls for these assays.19,106,107 Accordingly, the evaluation of a healthy control sample, in parallel with patient sample(s), is considered the important, minimal quality control.14,15,19 Modified LeveyJennings charts, which plot data from different subjects, can be useful to track aggregation test data for healthy controls over time. The tracking can also be useful when comparing findings for current and new lots of agonists before a switchover occurs. Some organizations now offer external quality assurance for platelet aggregation tests, either as postanalytical interpretation exercises or as additives for a control sample before performing testing.

REFERENCES 1. Born GV. Aggregation of blood platelets by adenosine diphosphate and its reversal. Nature 1962;194:927–9. 2. O’Brien JR. Platelet aggregation. Part II. Some results of a new method. J Clin Pathol 2009;15:452–5. 3. Feinman RD, Lubowsky J, Charo I, Zabinski MP. The lumiaggregometer: a new instrument for simultaneous measurement of secretion and aggregation by platelets. J Lab Clin Med 1977;90(1):125–9. 4. Cardinal DC, Flower RJ. The electronic aggregometer: a novel device for assessing platelet behavior in blood. J Pharmacol Methods 1980;3(2):135–58. 5. Ingerman-Wojenski CM, Silver MJ. A quick method for screening platelet dysfunctions using the whole blood lumi-aggregometer. Thromb Haemost 1984;51(2):154–6. 6. Sweeney JD, Labuzzetta JW, Michielson CE, Fitzpatrick JE. Whole blood aggregation using impedance and particle counter methods. Am J Clin Pathol 1989;92(6):794–7. 7. Cattaneo M, Hayward CP, Moffat KA, Pugliano MT, Liu Y, Michelson AD. Results of a worldwide survey on the assessment of platelet function by light transmission aggregometry: a report from the platelet physiology subcommittee of the SSC of the ISTH. J Thromb Haemost 2009;7(6):1029. 8. Moffat KA, Ledford-Kraemer MR, Nichols WL, Hayward CP. Variability in clinical laboratory practice in testing for disorders of platelet function: results of two surveys of the North American

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9.

10.

11. 12. 13.

14.

15.

16.

17. 18. 19. 20. 21. 22. 23.

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