Platelet function testing: state of the art

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1 For reprint orders, please contact Review Platelet function testing: state of the art Amer M Zeidan, Peter A Kouides, Mary Ann Tara and William A Fricke CONTENTS Principles & problems Bleeding time The PFA-100 Optical aggregometry Optical lumiaggregometry Impedance lumiaggregometry Flow cytometry Aspirin & clopidogrel resistance Expert commentary Five-year view Financial disclosure Key issues References Affiliations Author for correspondence Division of Hospital Medicine, Department of Medicine, Rochester General Hospital, Rochester, NY, 14621, USA Tel.: Fax: amer.zeidan@viahealth.org KEYWORDS: aspirin resistance, bleeding time, flow cytometry, lumiaggregometry, PFA-100, platelet function testing, platelets Platelet function testing has evolved from crude tests, such as the bleeding time, to tests that permit a relatively sophisticated evaluation of platelet activity. Nonetheless, these tests are hampered by lack of specificity and sensitivity, and poor standardization of methods and techniques. The bleeding time, which has long been a staple of hemostasis testing, has been dropped from the test menu at many laboratories. In its place, tests such as the Platelet Function Analyzer-100 are increasingly used to screen patients with possible bleeding disorders. Older tests, such as platelet aggregometry and lumiaggregometry, are still used frequently because they provide insight into receptor, signaling pathway and granule release mechanisms. Flow cytometry is available in some specialized laboratories and allows for quantitative and qualititative assessment of some platelet functions, although the expense of testing is often prohibitive. Finally, the wider availability of platelet function testing has stimulated interest and demand for monitoring the effect of platelet inhibitory drugs, such as aspirin and clopidogrel. As platelet function pathways become better understood, the demand for these type of monitoring tests is likely to increase. Expert Rev. Cardiovasc. Ther. 5(5), (2007) Recognition and diagnosis of heritable platelet function disorders have lagged well behind that of plasma-based coagulation disorders. This appears to have been due, in part, to the notion that platelet function disorders are rare and, thus, unlikely to be encountered often in clinical practice. Platelet function disorders, such as Bernard-Soulier syndrome (BSS), Glanzmann s thrombasthenia (GT) and gray platelet syndrome, which seem to be the disorders most discussed in older hematology texts, are very uncommon. Furthermore, limited tests for platelet function have also contributed to their relatively infrequent diagnosis. Whereas the tests for plasma-based coagulation disorders include both global assays, such as the prothrombin time (PT) and activated partial thromboplastin time (aptt), as well as specific factor assays (e.g., factor VIII, protein C and fibrinogen), assays for platelet function are generally limited to global-type assays, such as the bleeding time, clot retraction and platelet aggregation. These tests are fairly crude, lack sensitivity and specificity and do not offer precise or detailed insight into specific platelet abnormalities. Consequently, most clinicians do not have access to the tools to diagnose platelet function, even if it is suspected. The result appears to be a continued under-diagnosis of platelet function disorders in the many patients presenting with multiple mucocutaneous bleeding symptoms who have normal von Willebrand factor (vwf) levels. Von Willebrand disease (vwd) is considered to be the most common inheritable bleeding disorder [1]. However, there is growing evidence that platelet function disorders (e.g., release defects, storage pool disorders, membrane receptor abnormalities and signal transduction defects) are relatively common heritable causes of bleeding [2]. However, their contribution to bleeding appears to vary significantly. For example, one study reported that up to 47% of women with unexplained menorrhagia may have impaired platelet aggregation in association with menorrhagia [3], while another study of patients with mucocutaneous bleeding / Future Drugs Ltd ISSN

2 Zeidan, Kouides, Tara & Fricke symptoms had a 22% prevalence of platelet function defects (another 6% of patients had a combination of vwd and platelet function abnormalities) [4]. These observations have led to a renewed interest in the assays that evaluate platelet function. Platelet function testing is hampered by many limitations in both the research and the clinical laboratory: platelets cannot be maintained in culture so they must be isolated from peripheral blood to be studied. However, the isolation process often activates the platelets or disturbs their function, which complicates interpretation of experimental results. Moreover, isolating the platelets removes them from their natural blood milieu in which they interact with plasma proteins, other blood cells and endothelial cells. The artificial media in which they are usually tested does not reflect their normal environment and may lead to results that are not consistent with their true in vivo activity. However, newer research methods have permitted increased understanding of normal platelet function [2]. With this understanding has come the recognition of the potential number and range of platelet function abnormalities and the development of newer tests to assist in their diagnosis. In this article, we will first discuss the general principles and problems related to platelet function testing and then review some of the methods used to assess platelet function. Included will be the bleeding time (BT), the Platelet Function Analyzer (PFA)-100 (Dade International Inc., FL, USA), platelet aggregation, lumiaggregation and flow cytometry. We have not included several less commonly used tests, such as thromboelastography, Plateletworks (Helena Point of Care, TX, USA), and cone and plate analyzer. Reviews of these instruments can be found in the British Journal of Haematology [2]. Finally, we have included a discussion of aspirin and clopidogrel resistance. Our goal is to inform the readers of the relative values of these methods so that they will know which tests are appropriate in the evaluation of the patient with a bleeding history and/or at risk to bleed. There are several difficulties in writing such a review: some of the tests are not widely available, and the necessary clinical correlations between test results and bleeding are either not available or are not confirmed. Nevertheless, it is our hope that with the renewed interest in platelet function disorders that most laboratories will soon be able to provide some measure of platelet function testing and that our ability to interpret the results and provide clinical guidance on the care of patients will continue to improve. Principles & problems The purpose of platelet function testing, like most tests of hemostasis, is either to diagnose a bleeding disorder in a patient who has abnormal bleeding or to assess the integrity of the hemostatic system in the hope of predicting the potential for bleeding at a subsequent invasive procedure. In addition, the wide-spread use of antiplatelet drugs has recently given rise to a new application of these tests in monitoring their effect in patients taking the drugs. The principle of essentially all platelet function testing is to assess their functional state after stimulation by a known agonist(s). BT does this via an actual cut in the patient s skin, which triggers the natural hemostatic mechanisms. If these systems are intact, the bleeding will stop within a defined period of time. The other platelet function tests stimulate platelets in vitro with various agonists and then assess their activation by measuring their extent of aggregation, release of granule contents or expression of surface markers associated with activation. Aggregation can be measured by the change in light transmission, change in electrical resistance or change in blood flow. The final phase of platelet function testing requires an interpretation of the results and, hopefully, a correlation between the results and the patient s clinical condition. Unfortunately, the notion that the results necessarily confer either normality or abnormality and that normality or abnormality connotes the presence or absence of bleeding risk is often misguided. Platelet function testing probably has as many, if not more, problems associated with it than any other laboratory test. These are due primarily to lack of standardization of technique, agonists and interpretation. Platelet aggregation, for instance, can be measured by bleeding cessation, light transmission, impedance and flow pressure, and can be conducted using whole blood (WB) or platelet-rich plasma (PRP). The source, type, and amount of agonists also vary considerably. The most commonly used agonists are collagen, ADP, epinephrine and arachidonic Percentage Trace 1 Trace 2 Trace 3 Trace :00 2:00 3:00 4:00 5:00 6:00 Time (min:s) 100 Figure 1. Simultaneous platelet aggregation (red) and ATP release (green) in response to ADP compared with a normal control aggregation (blue) and release (black) seen in whole blood lumiaggregation Expert Rev. Cardiovasc. Ther. 5(5), (2007)

3 Platelet function testing: state of the art acid, although several others are often used (FIGURE 1). The amount of each agonist used also varies among laboratories, with some using high doses of agonists that induce rapid irreversible aggregation and others using lower doses that stimulate primary and secondary waves of aggregation. It is likely that different concentrations of the same agonist may be measuring different activities. Along the same line, since there are few specific assays for constituents of the various platelet regulatory pathways, it is often not clear what is being detected by an abnormal test and interpretation of the results may be problematic in many cases. For instance, a prolonged BT probably indicates the presence of some impediment to hemostasis, but it is often not clear what that impediment is or if it is clinically significant. It has been demonstrated repeatedly that the BT has little predictive value for bleeding during subsequent invasive procedures [2] and only fair sensitivity in identifying platelet function disorders [5 7]. Similarly, there are few data on the relationship between platelet aggregation test results and clinical events. Bleeding time The BT technique, as described by Duke, has been performed for almost 100 years [8]. The technique involves making a small incision in the forearm of the patient and measuring the time until the bleeding stops. A blood pressure cuff is placed on the upper arm and inflated to, and maintained at, 40 mmhg during the procedure. Unclotted blood is gently removed from the incision by the wicking action of a piece of filter paper. As would be expected, there are numerous problems with the BT as a measure of platelet function, the most obvious being that the test requires not only adequate numbers of functioning platelets but also hemostatically stable plasma coagulation proteins, a minimum hematocrit and platelet number, and functionally intact vasculature and perivascular tissue. There are also many technical variables, such as operator technique, skin thickness, skin temperature and vascular pattern [7]. The BT has been shown to vary inversely with platelet counts less than 100,000/µl in stable patients with amegakaryocytic thrombocytopenia [9]. However, clinically significant bleeding does not typically occur until platelet counts are much lower [10]. The predictive value of the BT for procedure-related bleeding is poor [7,11,12]. The results from various studies differ somewhat but, overall, the positive predictive value of an abnormal BT is less than 10% [11]. Current practice for assessing bleeding risk prior to invasive procedures does not include a BT but relies on a convincing bleeding history instead [13]. The value of the BT in evaluating patients with a possible bleeding disorder is better but not sufficiently adequate [4,6,14]. It is almost always prolonged in BSS and GT, but less often in other platelet function disorders. Approximately 50% of patients with vwd have abnormal bleeding times; those who are affected more severely are more likely to have long BTs [4]. The sensitivity of the BT to other types of platelet function disorders (PFD) is lower. Cattaneo and Kerenyi found that less than 50% of the patients they diagnosed with a primary PFD had abnormal BTs [5,6]. Nowadays, with increasing recognition of the limitations of the BT and wider availability of other methods of platelet function testing, many hospital laboratories no longer perform it [15]. PFA-100 The PFA-100 is a device that provides a global assessment of high shear-dependent platelet function. It was developed based on the prototype instrument called the Thrombostat 4000, which was designed by Kratzer and Born [16]. In 1995, Dade/Behring (Marburg, Germany) used the same principle to develop the commercially available PFA-100 [17]. In this system, citrated blood is aspirated from the sample reservoir under vacuum through a capillary tube yielding high shear rates ( /s), corresponding to flow conditions present in small arteries and capillaries [6]. The blood is directed onto a small aperture (diameter of ~ 150 µm) in a membrane coated with platelet agonists. As the platelets begin to adhere to the membrane surrounding the aperture and aggregate, the blood flow drops and eventually stops once the aperture is completely occluded. The time to complete occlusion and cessation of flow is recorded and is termed closure time (CT). Two cartridges are currently in clinical use; the membrane of the first cartridge is coated with collagen and ADP (CADP) while the membrane of the other cartridge is coated with collagen and epinephrine (CEPI). The maximum CT is 300 s and if the flow continues beyond this limit it is termed nonclosure. This process is believed to reflect the primary hemostatic process of platelet adhesion, aggregation and secretion under high shear conditions present in small vessels. The PFA-100 end point is highly dependent on vwf interactions with glycoproteins Ib/IX/V (GPIb/IX/V) and α IIb β III (GPIIb/IIIa) present on the surface of platelets [18]. Variables affecting the PFA-100 CT In addition to drug effects, vwd, platelet receptor defects and platelet secretion and granule disorders, the PFA-100 CT results may be affected by many technical factors. These include the citrate agent used for collection of the sample, time from sample collection to testing, platelet count and hematocrit and dietary factors, such as hyperlipidemia [18]. Adults and children have similar CT reference ranges but neonates have shorter CT secondary to higher hematocrit and increased vwf ristocetin cofactor activity due to the presence of ultra large vwf [19]. CT results have minor diurnal variations with lower CTs obtained from morning-collected samples (when platelets are hyperfunctional) [20]. CT results are not significantly affected by gender, smoking or the use of oral contraceptive pills [21]. Both low-hematocrit (less than 30%) and low-platelet counts (less than ) prolong CT [22]. CT is inversely proportional to plasma vwf levels and, therefore, individuals with blood group O (who have lower vwf levels) have longer CTs, although the CT is usually within the normal range [23,24]. CT is not affected by the absence of coagulation factors, suggesting that thrombin generation and fibrin formation is not important during platelet plug formation [18,22,25]. A recent 957

4 Zeidan, Kouides, Tara & Fricke study showed that PFA-100 CT results are not affected by systemic inflammation (as reflected by elevation of C-reactive protein levels) [26]. Many drugs can affect the results of PFA-100 CT (see below). Given the many factors that may affect the PFA-100 CT, some of which are transient, repeat testing might be warranted in some cases, especially those with borderline values. CT & congenital platelet disorders As a general rule, the more severe the platelet functional defect, the more likely CT is to be prolonged. As mentioned previously, CT is very sensitive to the severe functional defects associated with GT, BSS and pseudo/platelet type vwd. In these conditions, CT is very prolonged with nonclosure by both CEPI and CADP cartridges being a typical finding [6,22,27 29]. For milder platelet functional abnormalities, which are much more common than GT and BSS, the CT is not as sensitive. The studies evaluating PFA-100 sensitivity for mild platelet defects (including primary secretion defect [PSD], storage pool diseases [SPD] and Hermansky-Pudlak Syndrome) have yielded variable results, mostly as a result of differences in study designs (e.g., sample size, prospective or retrospective case identification and selection, and variable inclusion of drug-induced platelet dysfunction, etc) [28]. Earlier studies carried out on small numbers of patients with known mild platelet functional disorders showed higher sensitivities (47 80%), with CEPI CT usually prolonged and CADP CT typically within the normal range [5,6,28 31]. A recent prospective study evaluated 144 consecutive, previously undiagnosed patients who presented with unequivocal mucocutaneous bleeding history and positive family history [4]. In this study, CT was prolonged in only 24% of patients with PSD, 61.5% of patients with type 1 vwd, and 89% of patients with both PSD and type 1 vwd. We conclude from these observations that PFA-100 CT is, at best, moderately sensitive to mild platelet functional defects and, consequently, if such abnormalities are suspected clinically, full evaluation of platelet function should proceed regardless of the PFA-100 CT results. CT & vwd CEPI and CADP CTs are usually prolonged in type 2, type 3 and most cases of type 1 vwd because of the low levels of vwf [29]. There is a strong inverse correlation between PFA-100 CT and plasma vwf levels [23]. PFA-100 CT sensitivity to vwd types 2A, 2B, 2M and 3 (excluding type 1), is more than 98%, but falls to 85 90% when type 1 is included [32]. More recent studies of previously undiagnosed patients with significant mucocutaneous bleeding found PFA-100 sensitivity for vwd to be lower than that reported in older studies (see TABLE 1) [4]. As expected, the PFA-100 is not sensitive to type 2N vwd, since the defect involves the factor VIII binding site on vwf [29]. Lack of CT prolongation in an occasional type 1 vwd is a fairly common finding and is noted more usually in the CADP CT than the CEPI CT [33]. A normal PFA-100 CT can be used with some confidence to exclude severe vwd [32]. The PFA-100 CT has been used to assess response of vwd patients to desmopressin (DDAVP). CT is sensitive to vwf levels, especially the high-molecular weight vwf multimers that are released from endothelial cells in response to DDAVP, resulting in shortening or correction of the prolonged CT in many patients after DDAVP therapy [34]. CT was affected in the same way as the vwf ristocetin cofactor levels (R:Co): normalized in patients with type 1 but not always normalized in patients with type 2 vwd [35]. Prolonged CT in patients with type 1 platelet low, type 1 platelet discordant, type 2A and type 3 vwd do not correct with vwf replacement. Fressinaud et al. have shown that in patients with type 2 or 3 vwd who were nonresponsive to desmopressin (DDAVP), vwf concentrates corrected the R:Co defects but not the CT as none of these patients had a normal platelet vwf content and the vwf concentrates did not contain the high-molecular weight vwf multimers [35]. However, none of these studies were designed to evaluate whether normalized CT correlated with improved clinical outcomes [28]. In our own experience with six patients with low R:Co and prolonged epinephrine/collagen CTs receiving DDAVP, we noted that all patients had increases of R:Co of three-, to fivefold and that five of the six had normalization of Table 1. Sensitivity of Platelet Function Analyzer-100 closure time and bleeding time in detection of von Willebrand disease in different studies. Author Number of patients Sensitivity for detection of vwd (%) Ref. with vwd BT CEPI CT CADP CT Fressinaud et al. (1998) [84] Kerenyi et al. (1999) [6] Cattaneo et al. (1999) [85] Posan et al. (2003) [31] Quiroga et al. (2004) [4] BT: Bleeding time; CADP: Collagen and ADP; CEPI: Collagen and epinephrine; CT: Closure time; vwd: von Willebrand disease. 958 Expert Rev. Cardiovasc. Ther. 5(5), (2007)

5 Platelet function testing: state of the art the CT. The one patient who did not have complete correction of the CT had a baseline CT of more than 300 s (upper limit of normal, 153 s) with R:Co of 26%. Post-DDAVP, the CT was 163 s and the R:Co was 75%. In summary, it appears that the PFA-100 may be useful in monitoring patients with moderate vwd. CT & antiplatelet agents Many drugs can affect the results of PFA-100 CT, especially antiplatelet drugs, such as aspirin and NSAIDs [25]. Aspirin affects the results in a dose-dependent fashion, usually by prolonging the CEPI CT but not the CADP CT [36]. Many studies have evaluated the PFA-100 to assess aspirin resistance (see below for detailed discussion of aspirin resistance). Clopidogrel and GPIIb/IIIa inhibitors prolong the PFA-100 CT as well [37]. CT is not affected by heparin therapy [38]. PFA-100 & prediction of surgical bleeding In a large prospective study by Koscielny et al., the authors used the PFA-100, activated partial thromboplastin time (aptt), prothrombin time (PT) and platelet count preoperatively to identify 254 of 5649 unselected patients scheduled for surgery as having either acquired (n = 182) or inherited (n = 72) impaired primary hemostasis [39 40]. The BT and vwf were also performed but only in patients with a positive bleeding history and/or evidence of impaired hemostasis (e.g., drug ingestion). CEPI CT identified 97.7% of these patients while CADP CT identified 77.7%. The overall sensitivity of the CEPI cartridge was the highest (90.8%) compared with all other screening tests (i.e., BT, aptt, PT and vwf). The positive predictive value of the CEPI cartridge was 81.8% with a negative predictive value of 93.4%. The combined use of a standardized bleeding questionnaire and the PFA-100 as a screening test enabled the detection of impaired hemostasis in almost every case with a significant reduction in cost. All patients with impaired hemostasis were initially pretreated with DDAVP. Response to DDAVP or subsequent treatments were defined as correction of any one of the abnormal PFA-100 CT results. The non-responders were additionally treated with tranexamic acid or aprotinin; those with vwd received factor VIII concentrates with vwf. Those still unresponsive to therapy received conjugated estrogens and, as a last attempt, a platelet transfusion. The administration of DDAVP led to a correction of platelet dysfunction in 229 out of the 254 patients treated (90.2%). The frequency of blood transfusion was lower, but not statistically significant (9.4 vs 12.2%; p = 0.202) in preoperatively treated patients with impaired hemostasis than in patients without impaired hemostasis. In a retrospective analysis of the study, the frequency of blood transfusion was statistically significantly higher (89.3 vs 11.3%; p < 0.001) in patients without preoperative correction of impaired hemostasis than in patients without impaired hemostasis. This study demonstrates the potential of using the PFA-100 for preoperative bleeding-risk evaluation and monitoring effects of the corrective hemostatic agents. In addition, if these results are reproduced in a prospective fashion, the PFA-100 may have value in reducing perioperative blood transfusions. PFA-100 & the BT Interest in PFA-100 has grown as a screening test for primary hemostatic function, as problems with BT have become more apparent. As previously noted in this review, the BT is an invasive, operator-dependent test that does not predict surgical bleeding risk or bleeding tendency and lacks both sensitivity and specificity [12,41]. Although the early studies showed the PFA-100 to be more sensitive for vwd than the BT, this has not been replicated in less selected patients and when studied in a prospective manner (TABLE 1) [4,31]. Consequently, we cannot support the use of the PFA-100 as an effective screening test for vwd. In patients with congenital PSD and SPD, both tests lack sensitivity [4 6]. Both tests are very sensitive to severe platelet congenital abnormalities, such as BSS, GT and pseudo/platelet-type vwd [6,22,27]. PFA-100: summary The PFA-100 is an easy, quick assay of global high sheardependent platelet function that does not require special training to perform or interpret. However, it is affected by numerous variables and has not been prospectively studied in screening for vwd. TABLE 2 summarizes the expected results of the PFA-100 CT in different hemostatic disorders. It appears to be sensitive to severe platelet defects and severe vwd but is not very sensitive to mild platelet defects or mild vwd. PFA-100 is neither specific for, nor predictive of, any particular disorder. Table 2. Summary of expected responses in PFA-100 closure time in various hemostatic defects. Hemostatic defect CEPI CT CADP CT Severe congenital platelet * P P Mild congenital platelet defects P N vwd (severe type 1 and types 2 and 3) P P vwd (mild type 1) P ( can be N) N Aspirim/nonsteroidal antiinflammatory P N drugs Clopidogrel P/N P/N α IIb β III receptor blockers P P Uremia P/N P/N Liver disease P N Clotting factor deficiency N N * Glanzmann s thrombasthenia, Bernard-Soulier syndrome, platelet-type vwd. Storage pool disorders, primary secretion defects. CADP: Collagen and adenosine-5-diphosphate; CEPI: Collagen and epinephrine; CT: Closure time; N: Normal; P: Prolonged

6 Zeidan, Kouides, Tara & Fricke Optical aggregometry A major improvement in platelet function testing was the development of an optical assay using spectrophotometry to assess platelet aggregation in PRP which was described by Gustav Born in 1962 [42].This assay soon became the gold standard for assessing platelet function defects and has been used widely to evaluate bleeding disorders [43]. The optical aggregometer uses a spectrophotometer to assess the increase in light transmission through the PRP as the platelets start aggregating and clumping together as a result of the addition of one of the various platelet agonists (chemical agents known to activate platelets). The aggregation of the platelets leaves the PRP samples less turbid, hence the other term, turbidimetry, which is used to describe this technique. The PRP is obtained via low-speed centrifugation of citrated WB for 10 min at room temperature. The remaining sample is centrifuged at 1600 g for min to yield the platelet-poor plasma supernatant that is used as a reference (control) for the assay [44]. The typical aggregation recording in response to any particular agonist involves the following phases: Shape change, as the activated platelets increase in size Primary aggregation (first wave), which is a reversible event as the platelets start clumping together Secondary aggregation (second wave), which is an irreversible event that takes place if the agonist is strong enough resulting, in turn, in the release of the platelet granular contents causing further stimulation and aggregation of platelets. In evaluating the results of such studies, attention is paid to a variety of parameters, such as the lag phase (the time from application of the agonist till the start of the aggregation), slope of the aggregation response and the maximum aggregation expressed as the percent of light transmission [45]. The agonists typically used to induce aggregation include: ADP, arachidonic acid (AA), collagen, epinephrine, ristocetin and thrombin over a range of concentrations. Optical aggregometry is believed to reflect the in vivo aggregation function of platelets. Each agonist has an established pattern of response and the specific patterns of response to stimulation with different agonists have been shown to correlate with certain platelet pathological states (TABLE 3). Most patients with vwd have normal aggregation studies; exceptions include: Patients with markedly reduced vwf (type 3 or severe type 1) have a reduced response to ristocetin Patients with dysfunctional vwf, as in type 2A, have a reduced response to ristocetin Patients with type 2B vwd who have a heightened response to lower concentrations of ristocetin [44] Despite its strengths, optical aggregometry has several problems. One of the most important of these is variation in the concentration of agonists used in the tests. For instance, some laboratories use both high and low doses of collagen and/or ADP, but not the same high and low doses. Another problem is the uncertainty regarding the sensitivity and specificity of the tests. As an example, ADP at low doses may give only primary aggregation in some people but both primary and secondary aggregation in others. Collagen at low doses also usually demonstrates biphasic aggregation but not at high doses. The clinical relevance of low-dose ADP and collagen are unclear. Epinephrine is also used by many laboratories, but it may not have true physiologic relevance. Moreover, some patients have only primary aggregation to epinephrine, although they do not have a clinical platelet-related bleeding disorder. Optical lumiaggregometry As experience using the optical aggregometry increased, it became evident that SPD can not be excluded based on normal aggregation tracings [46]. Nieuwenhuis et al. examined 106 patients with documented SPD (51 with congenital and 55 with acquired SPD, all with positive bleeding histories, prolonged BT and diminished total platelet level of ADP and serotonin) and found that 23% of all the patients (31% of congenital and 20% of acquired SPD) had normal PRP optical aggregation in responses to ADP, epinephrine and collagen [47]. As a result of these observations, the need arose for a practical assay to evaluate the platelet granular contents. The development of the lumiaggregometry by Feinman et al. in 1977 was an important milestone. This assay allowed the simultaneous measurement of platelet aggregation and ATP secretion [48]. In this instrument, platelet aggregation is determined by the usual turbidimetric method using infrared light, while simultaneously quantitating ATP secreted from platelets by luminesence produced by the firefly luciferin luciferase system. The luminescent measurement of the ATP secretion provides evidence of normal or impaired dense granule release and increases the sensitivity of the instrument for detection of SPD and secretion defects (including aspirin-induced release defects) [49]. Some of the ATP secretion abnormalities are characteristic of certain hemostatic defects and are shown in TABLE 4. Impedance lumiaggregometry In 1980, the impedance method for the evaluation of platelet aggregation in WB was described by Cardinal and Flower [50]. In 1984, Wojenski and Silver used this method to develop an automated WB lumi-aggregometry that was made available commercially (Chrono-log, Chrono-Log corp, PA, USA) [51]. In this assay, an electrode probe assembly (consisting of two precious metal wires) is inserted into a sample of anticoagulated WB and electrical resistance or impedance is measured at baseline. After the agonist is added to the sample, the activated platelets start to aggregate and accumulate on the wires increasing the impedance. The change is the impedance, which is proportional to the mass of the aggregating platelets, is measured and quantified in ohms. The sensitivity of WB lumiaggregometry is unclear compared with the optical method. Published studies on this subject suggest that they are equivalent, although no secondary wave of aggregation is usually observed and the hematocrit and platelet count appears to affect the WB method [51,52]. Our personal experience is that the optical method using PRP is more 960 Expert Rev. Cardiovasc. Ther. 5(5), (2007)

7 Platelet function testing: state of the art Table 3. Platelet aggregation responses to different agonists in selected hemostatic disorders*. ADP Epinephrine Collagen Arachidonic acid Ristocetin von Willebrand disease N N N N N,R,A,E Bernard-Soulier disease N N N N R,A Glanzmann s thrombasthenia A,R A,R A,R A,R N Storage pool disorders/ platelet secretion defects N,R N,R N N N Aspirin/ nonsteroidal anti-inflammatory drug effects N, R N, R N, R (low dose) A,R N,Q # *The responses may vary depending upon the concentration of the agonist used (see text). Usually normal unless von Willebrand factor (vwf) or ristocetin cofactor levels is very low. Type 2B and platelet-type vwd will respond to low concentrations of ristocetin. Normal initial aggregation followed by disaggregation. Second wave of aggregation may be inhibited.. # Patients taking aspirin often have an unusually jagged appearing tracing in response to ristocetin. The cause is believed to be cyclic formation and dissolution of small platelet aggregates. A: Absent; E: Enhanced; N: Normal; Q: Qualitative defect; R: Reduced. Data from [44,86]. sensitive, and we neither see the secondary wave of aggregation observed with PRP optical aggregation nor shape change. The impedance lumiaggregometry requires only 5 ml of citrated blood. Like the other methods of aggregometry reviewed, it also necessities same-day controls (often a challenging task in finding such a donor who has not ingested any aspirin products for at least a week). The same agonists used to induce aggregation in PRP optical aggregometry are used in WB impedance aggregometry with the exception of epinephrine that is not used typically in WB. WB impedance aggregometry performed on samples from patients with platelet defects (storage pool disorders, thrombasthenia or cyclooxygenase deficiency) showed the same response patterns to different aggregating agents as in PRP optical aggregometry [51,52]. In a study of 15 vwd patients using WB impedance aggregometry, the patients exhibited the same abnormalities in WB as in PRP and the combination of prolonged lag phase (> 70 s) and maximum aggregation response to ristocetin identified all the patients [53]. Optical PRP versus WB impedance lumi-aggregometry Reiss et al. compared WB impedance aggregometry with optical PRP aggregometry, and found adequate correlation between the two techniques in healthy controls when aggregation was induced with ADP or collagen [52]. Impedance WB aggregometry was, in comparison to PRP optical aggregometry, more sensitive to the aggregating effect of thrombin, ristocetin and AA, and to the inhibitory effects of prostacyclin and aspirin. WB lumiaggregometry is believed to offer several advantages over the optical PRP lumiaggregometry, including the following: Faster, since the protocol takes less than 60 min to complete compared with the time-consuming, labor-intensive optical assay; Evaluates platelets in their physiologic milieu in the presence of red and white blood cells, which are known to modulate platelet function. This is in comparison to the optical assay which evaluates platelet function in an artificial milieu devoid of red blood cells and white blood cells [54]; There is no need for centrifugation, thus avoiding injury to platelets and loss of giant thrombocytes, which may be both hypo- and hyperactive [43]; Since it is not an optical-based assay, it can theoretically be used in thrombocytopenic samples (as low as 100,000/µl) [45], as well as in lipemic and icteric specimens; It also requires a reduced amount of blood (half of the amount needed for the optical assay) making it feasible for small children and patients from whom it is difficult to draw blood; It seems to have a higher sensitivity over the optical assay for assessment of the hyperactive platelet syndrome and for the effects of antiplatelet drugs (aspirin, clopidogrel and dipyridamole) [43,52,55]. Another potential area for the use of WB impedance lumiaggregometry that still requires prospective evaluation is its ability to predict the perioperative bleeding risk. Limitations of impedance & optical lumiaggregometry Despite all the proposed advantages over the optical lumiaggregometry, the use of the WB lumiaggregometry is still limited to specialized centers. This is partly due to the fact that there are no large prospective randomized trials that validate its use and compare it to PRP aggregation studies. Both assays require careful quality control and technical expertise in performance and interpretation [56]. Both tests require careful sample collection, handling and testing within 3 h of sample collection [57]. The sensitivity of these assays is, at best, moderate. Despite the use of these assays, in addition to other assays, approximately 961

8 Zeidan, Kouides, Tara & Fricke Table 4. Platelet ATP secretion responses to different agonists in selected hemostatic disorders. ADP Epinephrine* Collagen Arachidonic acid von Willebrand disease N N N N N Bernard Soulier disease N N N N N Glanzmann s thrombasthenia A A A,R, N A,R R, N Storage pool disorders/platelet secretion defects A,R A,R A,R A,R A,R Aspirin/ nonsteroidal anti-inflammatory drugs effects A,R R N, R (low dose) A,R N *Epinephrine is not usually used in whole blood aggregation studies. ATP release in response to ristocetin is not usually assessed for clinically. A: Absent; N: Normal; R: Reduced. Data from [49,87]. Thrombin half of all the patients ( %) with definitive mucocutaneous bleeding histories and positive family history remain without a known diagnosis [4,30,31,58]. Flow cytometry Platelet function and platelet function disorders may also be evaluated by flow cytometry. In fact, flow cytometry allows for fairly precise dissection of platelet function disorders such that signal transduction, secretion and degranulation, surface and cytoskeletal structures, coagulation factor binding and microparticle formation can all be assessed [59 61]. Platelets may be assessed in PRP or WB, and they may be fixed prior to processing if the goal is to examine the baseline structural state or they may be monitored following activation to assess activation-related changes [60 61]. Secretion and degranulation have been assessed based on mepacrine uptake and release, serotonin release and granule membrane component expression [59,62 64]. Similarly, receptor quantitation or conformation can be assessed by flow cytometry. This permits identification of both quantitative and qualitative receptor abnormalities as is seen in GT and BSS [65,66]. The availability of procoagulant platelet phospholipid in activated platelets can be assessed by annexin V binding [67]. Platelet activation can also be inferred by the presence of microparticles and of platelet-monocyte and platelet granulocyte aggregates [59]. Microparticles have also been described in heparin-induced thrombocytopenia [68]. Thrombopoiesis can be measured by detecting RNA-containing platelets (reticulated platelets) with thiazole orange or auramine orange [59]. Other applications of flow cytometry include evaluating platelet functional status in myeloproliferative disorders, myelodysplastic syndromes, diabetes and atherosclerosis [59 61,69]. However, despite all of this work, the application of flow cytometry to clinical situations has been slow. This, at least in part, is due to the lack of standardization of flow testing, expense and limited clinical correlation between results and clinical events. Aspirin & clopidogrel resistance Platelet function studies have been used to monitor aspirin and/or clopidogrel effects in patients taking these drugs and to assess the response of patients with vwd and some platelet function disorders to DDAVP [35,70]. The value of monitoring for aspirin or clopidogrel effect is controversial, and numerous authors and some professional organizations have recommended against it [71 78]. Aspirin is widely used for primary and secondary prevention of MI and ischemic stroke in patients with atherothrombotic disease or who are at risk for atherothrombotic disease. Numerous studies have shown a 25% reduction in these end points in high-risk patients. However, despite its efficacy, 10 20% of patients taking aspirin develop thrombosis. Although the causes of such recurrences are likely to be multifactorial, these patients are often branded as aspirin resistant. Review of the literature shows that aspirin resistance is a poorly and variably defined concept [72]. An often used clinical definition is thrombosis in a patient who has been prescribed aspirin for long-term prophylaxis. In the laboratory, aspirin resistance is typically defined as absence of the expected platelet inhibitory effect, as detected by tests of platelet function. However, there are several possible explanations for apparent aspirin resistance that indicate that true biological resistance to aspirin is uncommon and that most aspirin resistance is due to other factors [72]. For example, several studies have concluded that most of the patients who have laboratory evidence of aspirin resistance are actually noncompliant and that, if tested after observed ingestion of aspirin, show the expected inhibition of platelet function [79]. Conversely, there is a small cohort of patients who do not consistently demonstrate the expected inhibition of platelet reactivity after taking aspirin [80]. Based on these observations, some researchers have suggested using the terms aspirin failure or aspirin unresponsiveness or subresponsiveness instead of aspirin resistance. Drug failure, defined as the failure of the drug to have the desired clinical effect, may also account for a substantial proportion of aspirin resistance. Aspirin inhibits COX-1, thereby essentially blocking the synthesis of thromboxane A 2 by platelets. It has, however, approximately 150 times less effect on COX-2, an inducible form of cyclooxygenase, which can also participate in the synthesis of thromboxane A 2 [75]. In addition, aspirin only inhibits the thromboxane pathway of platelet activation; other pathways, such as those initiated by thrombin and collagen, are generally unaffected and can bypass the aspirin blockade [74]. As a result, the pro- 962 Expert Rev. Cardiovasc. Ther. 5(5), (2007)

9 Platelet function testing: state of the art coagulant milieu created by atherosclerosis, flow turbulence, high shear forces and plaque rupture, combined with collagen exposure, thrombin generation and induction of COX 2, may well be able to overcome inhibition of thromboxane synthesis by aspirin. The laboratory definition of aspirin resistance is also problematic. Various tests, including platelet aggregation, urinary 11-dehydrothromboxane B 2, PFA-100 (CT) and the rapid platelet function assay, VerifyNow, have all been used to assess aspirin effect [76]. However, the correlation of these tests with clinical events or with each other, as well as their sensitivity and specificity, is unclear. Thus, the significance of the results is often not clear. As a result, it is uncertain which test is most appropriate to assess for aspirin effect or how to interpret the results. The final problem related to testing for aspirin effect is how to manage the patient who appears not to have the expected aspirin-induced platelet inhibition. There is little evidence to support prescribing doses of aspirin higher than 325 mg/day, and the efficacy of adding other drugs, such as clopidogrel or dipyridamole, is not yet proven in this patient population. A similar debate is ongoing regarding resistance to clopidogrel and is also unresolved [70,81,82]. There are data emerging, however, that the variability of response to clopidogrel may be related to variability of response to platelet stimulation by ADP [83]. Expert commentary The question now arises as to which of these tests is appropriate to order and in what situation. The first step in the evaluation of a patient with a possible bleeding disorder is a good personal and family bleeding history. By itself, this will eliminate a substantial number of patients from testing. The history may also help guide testing towards plasma-based coagulation disorders or towards platelet-related disorders. For plasma-based disorders, global tests, such as the prothrombin time and activated partial thromboplastin time, are indicated, followed by more specific assays of factors or other coagulation proteins as indicated. If a platelet-related disorder is suspected, testing for vwd should be carried out first, as it is probably the most common hereditary coagulation disorder [1]. The PFA-100 is a reasonable screen for vwd and for the more severe hereditary platelet function disorders, but normal CTs do not exclude either. If either the CEPI or CADP closure time is abnormal, then it is appropriate to test for both vwd and for a platelet function disorder. If the CTs are normal, then testing for vwd and a platelet function disorder is appropriate if the clinical history is convincing. However, a major problem with platelet function testing is the almost universal use of aspirin-containing medication. In our laboratory, we have had numerous instances of patients repeatedly denying exposure to aspirin or NSAIDs only to have abnormal platelet-function tests followed by admission that such drugs had been used. In fairness, some patients may not realize that these drugs, aspirin in particular, may be present in combination with other drugs or may be labeled in such a way that it is not clear that they are included in the medication. The specific platelet function testing that is performed next is really a function of the laboratory. The relative strengths and weaknesses of optical platelet aggregation and lumiaggregation have been discussed previously; both can generate data that can be used to identify platelet function disorders, although their sensitivity and specificity differ. Flow cytometry is not generally available, and the bleeding time is not useful. However, an important requirement is that a knowledgeable individual who has experience in platelet function testing review the results and provide interpretation and guidance. Simply generating numbers, whether percent aggregation or nanomoles of released ATP, even when noted as abnormal, is not adequate. The interpretation should include the results but should also indicate the type of platelet function disorder and the confidence with which the diagnosis is being made. Not infrequently, confirmatory repeat testing is appropriate. Discussion with the person ordering the test is often very helpful. Five-year view The demand for more, and better, platelet function tests is likely to increase. This will be stimulated, in part, by improved understanding of platelet function and recognition of corresponding abnormalities and, in part, by development of antiplatelet drugs that require monitoring of their effect. Global-type platelet function assays will continue to improve as they better replicate in vivo conditions. Assays specific for defined aspects of platelet function, such as receptor binding and signal transduction, will also improve. However, improvement in this area is dependent upon the availability of reagents for specific pathway components and for tests that can isolate those pathways for evaluation. To do this and continue to maintain the integrity of those pathways and their interaction with other signaling mechanisms will be key. The need to monitor drug effects on platelets will also drive the demand for platelet function testing. As the correlation between test results and clinical events is better defined and the appropriate intervention(s) when the expected drug effect is not present are determined, the laboratory will be called upon to have these tests available. However, significant challenges lie ahead. As our ability to recognize specific platelet function defects improves, we must be able to correlate those abnormalities with clinical events and establish an estimation of associated bleeding risk. Similarly, monitoring of drug effect will require understanding of the clinical implications of identifying patients resistant to antiplatelet medication. This will require very large clinical trials. Underlying all these issues is a lack of standardization. Lack of standardization of methods and reagents will limit our ability to make confirmed primary diagnoses of platelet function disorders, and interlaboratory discrepancies will remain problematic. Perhaps even more important, lack of agreement on the tests and their interpretation will interfere with our ability to identify patients resistant to the effects of antiplatelet drugs. Even when such agreement does exist, large clinical trials will be necessary to establish the clinical relevance and appropriate interventions in these cases. Financial disclosure The authors have no relevant financial interests related to this manuscript, including employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties

10 Zeidan, Kouides, Tara & Fricke Key issues There is growing evidence that platelet function disorders are relatively common heritable causes of bleeding. The principle of essentially all platelet function testing is to assess their functional state after stimulation by a known agonist(s). Platelets function testing is hampered by a lack of standardization of techniques and interpretation, difficulties in simulating the in vivo milieu and the propensity of platelets to artifactual in vitro activation. Assays for platelet function are, in general, global-type, fairly crude assays that lack sensitivity and specificity, and do not offer precise or detailed insight into specific platelet abnormalities. Bleeding time is an invasive, operator-dependent test that does not predict surgical bleeding risk or bleeding tendency and lacks both sensitivity and specificity. Its use had been abandoned by many laboratories. The Platelet Function Analyzer (PFA)-100 is a quick assay of platelet function that is easy to perform and interpret but is neither specific for, nor predictive of, any particular disorder. The PFA-100 is sensitive to severe, but not mild, platelet defects or von Willebrand disease. The optical aggregometry uses spectrophotometry to assess platelet aggregation in platelet-rich plasma and has been considered the gold standard for assessing platelet function defects. The whole blood (WB) impedance aggregometry assesses platelet aggregation by measuring the change in electrical resistance. The use of WB avoids centrifugation injury to platelets and provides faster, more accurate results of the optical aggregometry. The clinical use of flow cytometry in testing platelet function is limited by the lack of standardization, expense and limited clinical correlation between results and clinical events. 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