INTRODUCTION — Platelets play a key role in both hemostasis and thrombosis. Accordingly, not only is accurate measurement of platelet function critical for identifying patients with platelet dysfunction or hyperfunction, but it also is becoming increasingly important for the monitoring of modern antiplatelet therapy.

The history of platelet function testing began over 100 years ago when platelets were first identified as distinct circulating cells, rather than fragments, and it was found that they were vital for both hemostasis and thrombosis [1]. Despite these early observations, for the majority of the 20^th century the only means of assessing platelet function were a small number of fairly unreliable tests (eg, manual platelet count, inspection of the peripheral blood smear, bleeding time).

A major problem concerning the testing of platelet function is the difficulty in simulating hemostasis in vitro. In addition, platelets are sensitive to manipulation, and are prone to artifactual in vitro activation. Early attempts to simulate hemostasis in vitro included methods in which platelets were counted before and after exposure to foreign surfaces (eg, glass columns) or thrombus formation was monitored within closed plastic tube loops. Many of these early tests remained as research tools because of their technical difficulty, and were thus restricted to specialized centers.

The ability to test platelet function in the routine laboratory improved with the introduction of platelet aggregometry [2]. Remarkably, further advances in laboratory platelet analysis have lagged behind both the functional and molecular analysis of the coagulation/fibrinolytic system. The table (table 1) illustrates the small number of tests that were available up to the late 1980s. The availability of newer and more reliable platelet function analyzers, flow cytometry, and molecular biological techniques is changing our approach to platelet function analysis. Another table (table 2) lists a number of relatively new platelet function tests that are currently available.

This topic review will present a brief history of platelet function testing, and will review major advances in the analysis of platelet function.

Related issues are discussed in separate topic reviews:

●Inherited and acquired disorders of platelet function – (see "Congenital and acquired disorders of platelet function")

●Automated hematology instrumentation – (see "Automated hematology instrumentation")

●Resistance to aspirin and clopidogrel – (see "Nonresponse and resistance to aspirin" and "Clopidogrel resistance and clopidogrel treatment failure")

PLATELET COUNTING METHODS

Manual platelet counting — The platelet count continues to be used as a first-line test of platelet function. Before the introduction of automated cell counters, the platelet count was performed manually. In fact, the manual count, using phase contrast microscopy, is still the gold standard international reference method [3,4]. However, this procedure is imprecise, with typical coefficients of variation (CV) in the range of 15 to 25 percent, making this method totally impractical for calibrating automated cell counters [5].

Automated cell counter methods — Although modern automated cell counters are rapid, precise, and reproducible, they tend to overestimate the platelet count in samples that contain cellular debris (eg, thalassemia, thrombotic thrombocytopenic purpura, leukemia). Conversely, in patients with large platelets, as in immune thrombocytopenia (ITP) [6], the platelet count is underestimated [7]. (See "Automated hematology instrumentation", section on 'Platelet count and size'.)

An accurate estimation of the peripheral blood platelet count, especially in patients with extreme degrees of thrombocytopenia, is becoming extremely relevant, given that the platelet transfusion threshold is now set at 10,000/microL and that certain degrees of thrombocytopenia may initiate different treatment decisions [6,8-12].

Optical counting methods — Advances in commercial analyzers have led to the development of optical counting methods, in which platelets are identified by their light scattering properties or via fluorescence following addition of a suitable dye [13,14]. These methods increase the accuracy of the count, as compared with impedance methods, as both normal and large sized platelets are easily discriminated from noise, cellular debris, and other cell populations. (See "Automated hematology instrumentation", section on 'Cell counting by light scattering'.)

Flow cytometric methods — Optical counters agree more closely with a flow cytometric procedure. In this latter method, platelets are identified via analysis of samples incubated with a fluorescent monoclonal antibody directed against an antigen on the surface of the platelet (eg, anti-CD61 and anti-CD41a) [5,15,16]. The platelet count is derived by dividing the number of fluorescent platelets counted by the number of red blood cell events (RBC ratio) and then multiplying this ratio by the RBC count provided by a suitably calibrated impedance analyzer.

This method is independent of both pipetting and dilution artifacts, provided that sufficient platelets are counted and that samples are optimally diluted to insure that cells are counted one by one, rather than in groups of two or more. The derived platelet counts are not only highly accurate, but are also precise, with typical coefficients of variation (CVs) of less than 5 percent [5,15]. Given the clear superiority of this method to the manual count, the ICSH/ISLH have proposed that the method could become the new international reference method [17].

The immunoplatelet CD61 method is also available on an automated cell counter, the Abbott CELL DYN 4000 [16,18,19]. (See "Automated hematology instrumentation", section on 'Immunologic counting of platelets' and "Automated hematology instrumentation", section on 'Abbott'.)

In a comparative study, the optical and CD61 immunoplatelet methods were found to be in good agreement for platelet counts in the range of 25,000 to 547,000/microL [16]. However, for platelet counts <25,000/microL, the optical method tended to overestimate the platelet count (figure 1). The lowest absolute platelet count which these methods could reliably distinguish from true zero was 1730/microL for the optical method and 20 and 9/microL for the anti-CD41a and the anti-CD61 immunoplatelet methods, respectively.

Given these improvements, increasing the accuracy of platelet counting in the ranges encountered in patients with severe thrombocytopenia (ie, platelet counts <20,000/microL) might result in further reduction in the platelet transfusion threshold to as low as 5000/microL, without an associated increase in bleeding risk. This could result in further cost savings by decreasing the frequency of unnecessary platelet transfusions [20].

However, it has been suggested that the majority of full blood counters overestimate platelet counts in patients with severe thrombocytopenia and that, as a result, patients may be under-transfused [21,22]. Further work is in progress to study the full impact of inaccurate platelet counting in thrombocytopenic patients.

Platelet count ratio method for platelet function testing — When platelets within anticoagulated whole blood samples are stimulated by agonists (eg, adenosine diphosphate [ADP], epinephrine, ristocetin), they form aggregates, with a resulting reduction in the number of free platelets. (See 'Platelet aggregometry' below.)

By counting the number of platelets in a control sample and a sample that has been activated by ADP, it is possible to assess platelet function via a platelet count ratio (ie, the control platelet count divided by the platelet count after addition of an appropriate agonist). This ratio correlates well with the results of platelet aggregometry [23], and can be performed on available automated cell counters, using commercially available reagents (eg, Plateletworks).

A whole blood platelet function assay using the ICHOR Point of Care Hematology Counter has been developed to count samples pre- and post-activation [24]. This instrument provides a means of assessing both platelet count and function within an acute care environment [25].

Platelet counting can also be performed before and after exposure of samples to foreign surfaces (eg, glass columns).

PLATELET AGGREGOMETRY

Classical platelet aggregometry — Turbidimetric platelet aggregometry, developed in the 1960s, revolutionized the ability to identify and diagnose alterations in platelet function [2], and soon became the "gold standard" for platelet function testing. In this technique, blood is centrifuged at sufficiently low force to obtain platelet-rich plasma (PRP), which is stirred in a cuvette at 37ºC between a light source and a measuring photocell. Upon addition of an agonist, such as adenosine diphosphate (ADP), platelets aggregate; the resultant increased transmission of light (ie, reduced turbidity of the PRP) is detected and recorded as a function of time after addition of the reagent. These changes reflect the formation of relatively large platelet aggregates, which are in turn derived from microaggregates.

The use of a panel of platelet agonists (eg, collagen, ADP, epinephrine, ristocetin) at a range of concentrations triggers classical platelet responses, including shape change as well as primary and secondary aggregation. The recorded response depends upon the normal functioning of the platelet, the presence of inhibitors of platelet function, as well as the concentration of agonist, facilitating detection of classical platelet disorders based upon the pattern of aggregation [26]. A number of classical examples are shown on the accompanying figure and table (figure 2 and table 3).

A great deal of information can be obtained via aggregometry, although the test is labor intensive, requires careful quality control, and a fair degree of technical expertise in its performance and interpretation. Although modern aggregometers with multi-channel capability, computer control, and the ability to measure ATP release via luminescence have improved the technology, the test is currently limited to specialized laboratories and is not well standardized [27-29].

There are a number of efforts in progress to improve standardization, including new guidelines from various societies including the International Society on Thrombosis and Haemostasis (ISTH) [30].

Comparative testing suggests that turbidimetric aggregometry underestimates both the degree and duration of inhibition of platelet fibrinogen receptor glycoprotein (GP) IIb/IIIa by anti-GPIIb/IIIa therapy (eg, abciximab) [31]. (See 'Whole blood aggregometry' below.)

Evidence has also been accumulating that turbidimetric aggregometry is not sensitive enough to monitor the formation of platelet micro-aggregates or for monitoring platelet function [32,33]. For example, it has been suggested that aggregates comprised of less than 100 platelets cannot be detected by the turbidimetric technique [34]. Aggregometry is thus relatively insensitive for either detecting preexisting aggregates or monitoring the early phase of aggregation. This may be particularly important for studying aggregates in patients with platelet hyperfunction.

As standard aggregometry testing generally requires high volumes of anticoagulated blood, researchers have shown that the test can also be performed within 96-well plates. Mixing of samples is possible using appropriate shaking devices that induce vortices within each well. The advantage of this approach is that substantially lower blood/PRP volumes are required and that replicates, controls, and dose-response curves using ranges of classical agonists can easily be performed. Data capture using laboratory plate readers to measure light absorbance allows all results to be easily processed and calculated automatically [35,36].

Inclusion of aggregometry within automated clotting analyzers not only facilitates in-depth platelet investigations but also decreases the amount of time required to perform testing in a standardized fashion [37].

Whole blood aggregometry — Measurement of platelet aggregation within whole blood by electrical impedance was developed in response to the difficulties inherent in aggregometry noted above [38]. In this method, whole blood is stirred at 37ºC between two platinum wire electrodes set at a fixed distance [39]. The electrode becomes covered with platelets, with further adhesion of platelet aggregates after addition of agonists. The mass of platelets sticking onto the electrodes changes the impedance with time, which is monitored on a recorder.

Comparative studies indicate that this method gives results similar to those obtained via classical turbidimetric aggregometry, and outperforms turbidimetric methods when monitoring anti-platelet therapy [23,31,40]. Similar to conventional aggregometry, whole blood aggregation impedance methods are relatively insensitive to the presence of smaller platelet aggregates. Within-donor variation tends to be slightly higher than with conventional aggregometry and the test still requires technical expertise and is relatively expensive. A multichannel (5 channels) impedance-based analyzer (Multiplate system) has become available and includes disposable cuvettes and standardized reagents [41].

This method is proving to be useful for diagnosing platelet defects and monitoring aspirin and clopidogrel and other P2Y₁₂ inhibitors, and is in widespread use in Europe [42].

Light scattering methods — Particle size analysis by flow cytometry is very accurate, not only for counting platelets, but also for monitoring formation of small (and large) aggregates during the early aggregation phase [43]. Continuous measurement of platelet aggregation is not possible with this method alone, and disaggregation can occur during dilution procedures if the samples are not appropriately handled.

An aggregometer (PA-200, Kowa Ltd) has been developed that uses a combination of laser light scattering and aggregometry to monitor the continuous formation of platelet microaggregates [44,45]. The test is sensitive enough to detect aggregates containing only two or three platelets. The method employed by this apparatus is based on the observation that the intensity of light scatter is proportional to the size of the aggregates formed. This is accomplished by focusing on a limited area of the platelet-rich plasma in the cuvette and detecting high intensity light scatter at 90 degrees, via a series of lenses.

Platelet aggregation assessed in this manner correlates well with conventional aggregometry in samples showing gross changes in light transmission. However, the light scatter technique can detect small aggregates (the primary aggregation response) that are not detectable via light transmission in the conventional aggregometer; the larger aggregates (the secondary aggregation response) are detected by both methods. The instrument not only measures both the size and number of platelet aggregates but also uses more physiologic concentrations of agonists, at concentrations which may be reduced by up to three orders of magnitude, compared with those used in light transmission techniques.

This technology may prove useful for detecting platelet hyperreactivity in a variety of disease states (eg, acute coronary syndromes, stroke, and thrombotic thrombocytopenic purpura) in response to low concentrations of various agonists. As an example, spontaneous formation of small aggregates (<100 platelets) has been observed in patients with acute coronary syndromes, but not in controls [46]. Further, upon agonist stimulation the EC50 for epinephrine-induced primary aggregation was 50 times lower in these patients than in controls, while the EC50 for secondary aggregation was only two times lower. This observation suggests that platelet hypersensitivity is only detectable by primary aggregation of microaggregates in these patients.

The VerifyNow Assay — This test was previously known as the Ultegra Rapid Platelet Function Assay (RPFA).

Data from recent clinical trials (eg, EPIC, EPILOG) have suggested that >80 percent of platelet receptors need to be blocked in order to achieve significant clinical efficacy with antiplatelet agents [47]. Monitoring of receptor blockade is thus becoming crucial in order to ensure optimal dosing and maximal clinical benefit for individual patients at risk of developing ischemic complications (eg, during percutaneous coronary intervention) [48]. (See "Early trials of platelet glycoprotein IIb/IIIa receptor inhibitors in coronary heart disease", section on 'The platelet glycoprotein IIb/IIIa receptor'.)

Previously employed tests such as radiolabeled antibody binding assays, aggregometry, and flow cytometry, although suitable, are time consuming, expensive, and require a fair degree of technical expertise and quality control [49]. Given the increased use of both oral and infused GPIIb/IIIa antagonists, there exists a demand for a simple, cheap, and rapid method that could be used at the bedside and clinic in order to monitor anti-platelet therapy in a standardized fashion [50-52].

A modified platelet aggregometry device has been developed in order to provide a simple and rapid functional means of monitoring anti-GPIIb/IIIa therapy (eg, abciximab) [53]. The VerifyNow GPIIb/IIIa Assay is based upon the principle that fibrinogen-coated beads will agglutinate in whole blood in proportion to the number of available platelet GPIIb/IIIa receptors. A disposable cartridge employed for this test contains fibrinogen-coated beads and a platelet activator (TRAP – Thrombin Receptor Activating Peptide) within reaction wells [52]. A citrated whole blood sample is inserted into the cartridge within the instrument; TRAP activates the platelets, resulting in GPIIb/IIIa exposure and binding of the fibrinogen-coated beads to the platelet receptors that are not blocked with the drug in question; results (percent inhibition) are available within a few minutes.

Comparative evaluations demonstrate that the instrument mirrors both platelet aggregometry and the degree of GPIIb/IIIa blockade [51,52]. There are two other cartridges (VerifyNow Aspirin and VerifyNow P2Y₁₂) available for monitoring either aspirin or P2Y₁₂ inhibition, respectively. The aspirin and P2Y₁₂ cartridges contain arachidonic acid and ADP as agonists, respectively. (See "Nonresponse and resistance to aspirin", section on 'Observations'.)

THE IN VIVO BLEEDING TIME — The bleeding time invented by Duke in 1910 [54] was the first in vivo test of platelet function. Although the test has been refined by the Ivy technique [55,56], it is poorly reproducible, invasive, insensitive, and time consuming [57]. It is impractical to use serially or during surgical procedures, and requires experienced operators to judge the subjective endpoint of the test.

●A normal bleeding time does not predict the safety of surgical procedures, nor does an abnormal bleeding time predict for excessive surgical bleeding. It is not recommended as a preoperative screening test. (See "Preoperative assessment of hemostasis", section on 'PFA-100 and bleeding time'.)

●Despite the above, the bleeding time is still occasionally used in some institutions as a first-line clinical screening test, and it can be useful for identifying patients with severe hemostatic defects (eg, von Willebrand disease, Glanzmann thrombasthenia) [58,59]. (See "Congenital and acquired disorders of platelet function", section on 'Bleeding time'.)

The clear advantage of the bleeding time as a platelet function test is that it studies natural hemostasis, does not require expensive specialized equipment, and is not prone to anticoagulation artifacts. However, given the many problems with the method, the introduction of reliable in vitro tests that simulate primary hemostasis might eventually result in the phasing out of the bleeding time.

INSTRUMENTS THAT SIMULATE PLATELET FUNCTION IN VITRO — Platelet aggregometry does not mimic the physiological processes of platelet adhesion, activation, and aggregation that occur during hemostasis in vivo. A number of investigators have developed other tests in an attempt to mimic or simulate the processes that occur in vivo during vessel wall damage. Accordingly, many platelet adhesion tests have been developed to study shear-induced platelet activation [60,61].

Clot signature analyzer — An instrument called the hemostatometer was developed for testing global hemostasis [62]. The methodology involves punching holes within a tube that contains flowing non-anticoagulated blood under controlled conditions. Consequent platelet activation results in the formation of a primary hemostatic plug with clot stabilization via fibrin formation. Alternatively, the blood sample can be exposed to a small amount of collagen, which results in thrombus formation and complete stenosis of the tube.

A commercial form of this instrument, called the clot signature analyzer (CSA), measures both hemostatic plug formation in the "punch" and collagen channels within a disposable plastic cassette [63,64], providing a global assessment of hemostasis under controlled conditions. The formation of the hemostatic plugs within both channels is shear-dependent (>10,000/second in the punch channel and 6200/second in the collagen channel) and relies upon functioning GPIb, von Willebrand factor (VWF), and GPIIb/IIIa. The test is applicable to detecting abnormalities in both primary hemostasis (ie, platelet activation) and coagulation.

Thrombotic status analyzer — The thrombotic status analyzer (TSA) measures both thrombotic and thrombolytic activities within non-anticoagulated blood samples [65]. The test involves drawing whole blood through a capillary tube. The resulting hemodynamic forces induce platelet activation and capillary tube occlusion. After stabilization and upon compression of the platelet-rich thrombus, thrombolysis can also be measured. The test is capable of detecting defective or excessive platelet function and monitoring both anti-platelet and thrombolytic therapy.

The platelet function analyzer — The principle behind this platelet function analyzer (PFA-100/200), originally developed in 1985 [66], is to expose platelets within citrated whole blood to high shear (5000 to 6000/second) within a capillary tube and monitor the drop in flow rate as the platelets form a hemostatic plug within a very small aperture (150 microns) at the center of a membrane coated with collagen and either adenosine diphosphate (ADP) or epinephrine [67,68]. The test records a parameter called the closure time (CT).

The test is simple, rapid, does not require specialist training, and is a potential screening tool for assessing hemostatic abnormalities [69,70]. It is particularly advantageous in the pediatric setting [71]. The test has replaced the in vivo bleeding time in some institutions [72]. The PFA-100 can also serve as a screening tool for assessing platelet dysfunction [69,73]. As with the in vivo bleeding time, this test is sensitive to both the platelet count and hematocrit [74,75]; it is therefore recommended that a complete blood count be performed along with each test.

Although abnormal closure times are an indication of platelet dysfunction, they are not specific for any disorder, and should not be used for general screening purposes without knowledge of other variables that influence the test (eg, VWF levels, complete blood count, other platelet function disorders) [76-78]. This test also has limited value in the diagnosis of patients presenting with mucocutaneous bleeding, due to its low sensitivity for the detection of primary secretion defects and storage pool disease [79].

Use in von Willebrand disease — Data confirm that the above testing system is highly dependent on VWF and is useful for evaluating patients with von Willebrand disease (VWD) as well as monitoring therapy with desmopressin (DDAVP) [80,81] (see "Clinical presentation and diagnosis of von Willebrand disease", section on 'Laboratory testing'). However, a number of limitations have been noted:

●The test appears to be coagulation factor independent, as blood from patients with hemophilia A or B, factor VII deficiency, or afibrinogenemia all exhibit normal closure times in the instrument.

●Comparison studies have shown that the PFA-100 is more sensitive (>70 percent) than the bleeding time (20 to 30 percent) in detecting all subtypes of VWD [82]. The exception is type 2N VWD, in which the hemostatic defect resides in the factor VIII binding site on VWF [83]. (See "Classification and pathophysiology of von Willebrand disease".)

●Factor VIII concentrates containing VWF, or even VWF concentrates, often fail to correct the closure time in patients with type 3 VWD, suggesting that either platelet VWF is critical in the test or that the PFA-100 is extremely sensitive to the absence of the highest molecular weight multimers in these concentrates [80,81].

Use in platelet secretion defects — This test may also be useful for the diagnosis and monitoring of patients with platelet secretion defects [84]. The collagen/epinephrine cartridge appears to be not only more sensitive to these defects but also gives a prolonged closure time (CEPI-CT) in subjects who have ingested aspirin [70,85,86], and may identify patients who are aspirin insensitive. The closure time using the collagen/adenosine diphosphate cartridge (CADP-CT) is normal in subjects taking aspirin [86]; thus, performance of both tests may allow differentiation between an aspirin-like defect and other platelet function disorders [87].

The INNOVANCE PFA P2Y cartridge gives prolonged CTs in patients with both congenital severe and moderate defects of the platelet P2Y₁₂ receptor [88,89].

If the screening test obtained by the PFA-100 is normal, it is highly likely that the patient has no severe primary platelet function defect, and other platelet tests would not be required. However, normal results may not always exclude milder platelet function defects (eg, storage and release defects). Therefore, if the clinical suspicion of a platelet defect is high, further diagnostic tests should always be performed [90].

Monitoring antiplatelet drugs — The CEPI-CT (but not the CADP-CT) can be used to monitor aspirin therapy, which results in prolongation of the CT, but abnormal results can sometimes be corrected by high VWF levels [91]. Both CEPI-CT and CADP-CT are not consistently prolonged in samples from patients receiving P2Y₁₂ inhibitors. Given the lack of sensitivity of the existing cartridge formulations to detect P2Y₁₂ inhibition, the INNOVANCE PFA P2Y cartridge was developed [92].

The PFA-100 may also be useful for monitoring other anti-platelet drugs, such as GPIIb/IIIa antagonists.

Use in anesthesia — The use of this testing in anesthesia is discussed separately. (See "Anesthesia for dialysis patients", section on 'Management of bleeding diathesis (elective surgery)'.)

Cone and plate(let) analyzer — This test of platelet function is based upon the adhesion of platelets to extracellular matrix (ECM) under flow conditions [93-95]. The system consists of a cone and plate device in which a blood sample is exposed to a plate under arterial flow conditions for a few minutes. Platelet adhesion and aggregation on the surface of the plate are monitored by an image analyzer. The interaction of platelets with the substrate is totally dependent upon presence of GPIIb/IIIa, GPIb, and plasma VWF.

The CPA, either as a semi- or fully automated system, has been used to monitor defects in primary hemostasis, anti-GPIIb/IIIa therapy, hyperreactive platelets, to monitor thrombocytopenic patients for bleeding risk, and for early diagnosis and treatment monitoring in patients with thrombotic thrombocytopenic purpura [93,95,96]. The main advantages of this system are that it uses a small volume of blood (150 to 250 microL), yields results in 5 to 15 minutes, and exhibits a close resemblance to in vivo physiological conditions.

The test is now commercially available as the IMPACT (immediate microscopic platelet adhesion cone and plate technology) device and uses disposable polystyrene plates. The test measures platelet adhesion and aggregation using 130 microL of whole blood that has been pipetted onto the polystyrene plate and exposed to a shear rate of 1800/second for two minutes. Surface bound platelets are imaged and quantified by image analysis software after washing and staining. The test displays two parameters: surface coverage (SC) and average size (AS). The SC is a measure of the percentage of the plate covered by platelets, and the AS is the means size of surface bound objects. The test is apparently sensitive to afibrinogenemia, VWD, Glanzmann thrombasthenia, and Bernard-Soulier syndrome. However, widespread experience with this device is limited.

Microfluidic devices — Researchers have developed a number of microfluidic devices that measure real time thrombus formation in whole blood. Several multimodal approaches to investigate platelet function under flow within microfluidic devices appear promising in clinical settings [97]. One example is the Total Thrombus formation analysis system (T-TAS) [98]. This a flow-microchip chamber with thrombogenic surfaces that generates images of thrombi in real time that imitate vessel wall injury and produce quantitative data.

PLATELET-MEDIATED THROMBIN GENERATION — Estimates suggest that platelets can accelerate thrombin generation by five to six orders of magnitude. The mechanisms by which platelets accomplish this include the following:

●Blood coagulation occurs more efficiently on cell surfaces. In particular the interaction between platelets and clotting factors occurs with many different proteins and at many different levels of the coagulation cascade.

●The exposure of negatively charged phospholipids during platelet activation (platelet factor 3) provides a surface on which both the tenase and prothrombinase complexes can generate thrombin. (See "Overview of hemostasis", section on 'Multicomponent complexes'.)

●Thrombin, which converts fibrinogen to fibrin, is a potent activator of platelets.

●Platelets from patients with the rare congenital platelet defect Scott Syndrome (a defect in the scramblase TMEM16F) cannot expose negatively charged phospholipids or generate procoagulant microvesicles and therefore exhibit significantly reduced thrombin generation [99].

●Anti-GPIIb/IIIa antagonists (eg, 7E3 and RGDS) inhibit thrombin generation [100]. As platelets from patients with Glanzmann thrombasthenia, who have congenital defects in GPIIb/IIIa, also exhibit reduced thrombin generation, GPIIb/IIIa appears to influence the conversion of prothrombin to thrombin. This is probably mediated by direct binding of prothrombin on both resting and activated platelets [101].

Accordingly, measurement of thrombin generation in the presence of platelets provides a simple assessment of an important physiologic function of platelets. Although most assays have been based upon coagulation [102], use of thrombin specific chromogenic and fluorescent substrates has resulted in the development of instruments to measure this activity [103,104].

Hemostatus device — Point of care measurements of the prothrombin time, activated partial thromboplastin time, and the platelet count have all been used to identify patients who are at risk of bleeding. (See "Approach to the adult with a suspected bleeding disorder", section on 'Laboratory evaluation'.)

A point of care instrument has been developed to assess platelet function by analysis of the effects of platelet activating factor (PAF) on the kaolin-activated clotting time [105,106]. This test measures the effect of four different concentrations of PAF on the shortening of the clotting time. The hemostatus test accurately identifies a bleeding tendency, especially when platelet counts are <70,000/microL. The test is insensitive to aspirin and GPIb function, but is sensitive to abnormalities in GPIIb/IIIa. Thus the test may be useful for monitoring GPIIb/IIIa inhibitors and identifying patients with Glanzmann thrombasthenia [107].

Instruments measuring physical properties of the clot — The balance among clot formation, retraction, and lysis reflects the ability of the hemostatic plug to perform its hemostatic function. Thus, by measuring various properties of the clot during hemostasis one can potentially detect a number of acquired and congenital platelet abnormalities.

The process of clot retraction may be measured either in whole blood or platelet rich plasma by incubation in the presence of calcium in glass tubes and either calculating differences in volumes pre- and post- clotting or by visual assessment of the clot with time. Abnormal clot retraction may reflect deficiencies in platelet number, platelet glycoproteins, release of activating agents, or fibrinogen levels. A number of instruments have been developed that can accurately measure various physical properties of the clot with time. Two types of commercial instruments will be discussed here: thromboelastography (TEG), which measures clot formation dynamically in whole blood; and the Hemodyne device, which measures global platelet function. (See 'Thromboelastography (TEG) and ROTEM' below and 'Platelet contractile force' below.)

Thromboelastography (TEG) and ROTEM — Thromboelastography (TEG) tests both platelet function and coagulation by assaying several parameters of clot formation dynamically in whole blood (figure 3). There are several devices available commercially to measure TEG at the bedside or point of care. While the specifics of the devices vary, they all measure the same general parameters. As examples:

●The TEG and the related rapid TEG (r-TEG) devices monitor the interaction of platelets within the fibrin mesh of the clot during clot formation and lysis over time. The physical property of the clot is measured by use of a cylindrical cup that holds a whole blood sample at 37ºC and is oscillated to and fro with a rotation cycle of 10 seconds. As the clot forms, the torque of the rotating cup is transmitted to an immersed pin. The degree of pin rotation is converted to an electrical signal via a transducer and monitored via a chart recorder. The strength of the developing clot increases the magnitude of the output, whereas during clot lysis, the bonds between the cup and the pin are broken, and the signal decreases. The forces that are generated are used to measure the clotting time, kinetics of clot initiation, clot strength, and clot lysis over time (table 4).

●The ROTEM (Rotational Thromboelastometry) device is an adaptation of the TEG in which the cup remains stationary and the pin rotates directly in the sample. Results obtained are essentially identical to the TEG.

TEG has been used successfully as a point-of-care test within surgical departments (especially in trauma and orthotopic liver transplantation) and to predict for thromboembolic events in surgical patients. The usefulness of TEG in general hematologic practice remains uncertain.

The indications and use of this testing is discussed in a 2018 Guideline from the British Society of Haematology [108]. Additional details of this testing and discussions of its use in specific clinical settings is described in separate topic reviews. (See "Coagulopathy in trauma patients", section on 'Thromboelastography' and "Intraoperative transfusion of blood products in adults", section on 'Use of a transfusion algorithm or guideline' and "Postpartum hemorrhage: Medical and minimally invasive management", section on 'Viscoelastic testing' and "Anesthesia for the patient with liver disease", section on 'Coagulation management'.)

Of note, the TEG instrument may be insensitive to samples from patients who have taken aspirin [109-111]. However, the TEG PlateletMapping assay can overcome the limitations of thrombin-generated viscoelastic testing to reliably and accurately measure the ability of platelets to participate in clot formation and therefore measure the effects of antiplatelet drugs [112].

Platelet contractile force — Platelet contractile force (PCF) is a clinically useful and sensitive measure of global platelet function. During platelet activation, contractile forces within the cytoskeleton mediate both shape change and pseudopod formation. The platelets then adhere to the fibrin mesh via surface receptors (eg, GPIIb/IIIa) on the pseudopods. Once the clot is fully formed, the platelets begin a process of clot retraction, placing the entire clot under stress, resulting in the collapse of the clot and reduction in size.

The Hemodyne instrument, based on a previously described technique [113], facilitates a direct measurement of these forces. The test is performed by placing a small sample (800 microL) of either citrated whole blood or platelet rich plasma within a sample cup between two parallel plates. Upon addition of thrombin or other agonists, platelets within the sample attach to the plates and extend their pseudopodia along polymerizing strands of fibrin. Once the fibrin/platelet network is sufficiently formed, platelet contractile forces begin to transmit outwards to the plates and are detected by a transducer, the output of which is proportional to the generated force. Two key properties are measured simultaneously:

●Platelet contractile force (PCF) – The force exerted by activated platelets on the fibrin network

●Elastic modulus (EM) – The elastic modulus (clot rigidity), a measure of the physical structure of the fibrin/cellular network [114]

PCF is decreased in thrombocytopenia, various acquired and congenital platelet abnormalities (eg, uremia, Glanzmann thrombasthenia, during cardiopulmonary bypass) [115], and GPIIb/IIIa inhibitors [116]. Clot EM is influenced by fibrinogen concentration and is measurable in the absence of platelets. It may also be sensitive to clotting factor deficiencies, anticoagulant effects, and in multiple myeloma. Overall, low PCF values appear to predict a bleeding risk, while high PCF/EM values may predict a thrombotic tendency (eg, coronary artery disease) [117].

MEASUREMENT OF PLATELET ACTIVATION — A number of tests have been developed to measure reactivity of circulating platelets and/or to show that platelets have been activated in vivo. The difficulty with these methods is the prevention of artifactual platelet activation during the processing of the blood sample. The earliest measurements included the detection of platelet aggregates within blood samples [118,119], measuring threshold aggregation responses to adenosine diphosphate (ADP) or arachidonate, and testing for the presence of spontaneous aggregation.

Soluble activation markers — Another method of assessing platelet activation is measurement of platelet release products within platelet poor plasma, some of which, however, are not specific to platelets. In these methods, blood samples are usually collected into tubes containing inhibitors of platelet activation (eg, theophylline and PGE₁).

The most useful tests are assays for platelet-specific proteins, such as platelet factor 4 and beta thromboglobulin. These proteins are released from the alpha granules upon platelet activation and aggregation, although careful venipuncture and blood handling are essential to avoid artifactually high levels. The ratio of these two proteins often indicates if there has been a problem with artifactually induced activation.

Other platelet activation assays have included thromboxane B₂ measurement (a metabolite of thromboxane A₂) within plasma. Measurement of the stable urinary thromboxane metabolite 11-dehydrothromboxane B₂ can also be used to monitor aspirin therapy (AspirinWorks test). Uptake and release of radiolabeled serotonin from the dense granules is also a traditional test for assessing storage and release defects. These assays have a number of disadvantages, including false elevation of levels due to sample handling, insensitivity, need for radioactive materials, and high cost.

Soluble P-selectin is also derived from activated platelets and can be measured as a marker of platelet activation in platelet poor plasma [120]. P-Selectin or CD62p is an alpha granular membrane protein, which is expressed only on the surface of activated platelets and can be detected by flow cytometry. Other soluble markers now include soluble or sCD40L, GPV, and GPVI. (See 'Flow cytometric analysis of platelets and activation markers' below.)

The soluble protein is derived by enzymatic cleavage of the membrane bound form. In vivo studies have demonstrated that degranulated platelets rapidly lose their surface P-Selectin and continue to circulate and function [121]. Although soluble P-selectin is specific for platelet activation in normal subjects, some may also be derived from endothelial activation/damage [120].

Flow cytometric analysis of platelets and activation markers — Whole blood flow cytometry is a very powerful but relatively new laboratory technique for assessing platelet activation and function [122,123]. Flow cytometry can be used to measure activated platelets, platelet hypo- or hyper-reactivity, platelet-leukocyte aggregates, platelet microparticles, and platelet turnover. These assays may be of particular use in identifying patients at risk for thrombosis. Specific flow cytometric assays have also been developed for monitoring anti-GPIIb/IIIa antagonist therapy, as well as diagnosing congenital deficiencies in platelet glycoproteins, storage pool disease, and heparin-induced thrombocytopenia.

The use of whole blood in these assays has several advantages over either purified platelets or platelet rich plasma [122,124]:

●Platelets are analyzed in the presence of leukocytes and erythrocytes and only small quantities of blood are required (2 to 5 microL). Providing the venipuncture technique is well standardized, the minimal manipulation of fresh samples results in very little artifactual in vitro activation.

●It is possible to study platelets from patients with thrombocytopenia.

●In vivo activation state and dose response to classical agonists (eg, thrombin, TRAP, ADP) can be studied by these extremely sensitive assays.

Platelet activation markers that are commonly measured include exposure of granule membrane markers (eg, CD62p, CD63, LAMP-1 and CD40L), activation dependent changes in the conformation of the GPIIb/IIIa complex (eg, PAC-1, Ligand Induced Binding site (LIBS), and Receptor Induced Binding site (RIBS) antibodies), binding of secreted proteins (eg, thrombospondin [TSP] and Multimerin), and exposure of phosphatidyl serine (factor Va or VIIIa binding, Annexin-V, Lactadherin).

As discussed above, surface expressed CD62p (P-selectin) is rapidly cleaved to release soluble P-selectin into the plasma [121]. Thus, the levels of CD62p on the platelet surface may not be a true reflection of the in vivo activation state of platelets and should be complemented by measurements of all other markers including soluble P-selectin. A full spectrum of all activation markers should be measured, especially since the time frame of expression for each marker is unknown.

Activated platelets have been demonstrated in a wide variety of disorders, including acute coronary syndromes [122,125,126], stroke [127,128], antiphospholipid syndrome [129], and during angioplasty [130]. The study of platelet activation markers thus has the potential to optimize antiplatelet therapy (eg anti-GPIIb/IIIa drugs) and to be used as a predictor of thrombotic events after invasive techniques (eg, angioplasty) [130].

One of the problems with measuring the activation status or reactivity of circulating platelets is that the clinical picture is incomplete, since fully activated platelets within thrombi and clots are unlikely to circulate. Measurement of platelet-leukocyte complexes may be a more sensitive marker of in vivo platelet activation. By using a double labeling technique, it is possible to study aggregates of platelets with both neutrophils and monocytes by flow cytometry [131]. When platelets become activated they can bind to monocytes and neutrophils by a number of different receptor-ligand pairs or bridges (eg, CD62p – PSGL1, CD36 – TSP – CD36, Mac1 – Fg – GPIIb/IIIa, and CD40L – CD40). Increased numbers of circulating platelet-leukocyte complexes have been demonstrated in patients with myocardial infarction, unstable and stable angina [125], and during cardiopulmonary bypass.

Circulating monocyte-platelet complexes may be a relatively stable and a more sensitive marker than other activation dependent markers (eg, P-selectin). The platelets induce tissue factor expression after binding to monocytes, perhaps increasing a prothrombotic tendency in such conditions as meningococcal sepsis [132], sickle cell disease [133], preeclampsia, and acute coronary syndromes.

Flow cytometric analysis of platelets can also be used to count platelets accurately (see 'Platelet counting methods' above), diagnose various glycoprotein deficiencies (eg, Bernard-Soulier syndrome and Glanzmann thrombasthenia) and to study platelets from patients with defective aggregation, secretion, microparticle generation, or procoagulant activity [133-135].

By using the dye Mepacrine in conjunction with platelet activation in vitro it is possible to diagnose dense granule deficiency and storage pool disease (both storage and release defects) in a simple and reproducible assay [136,137]. This assay is simpler than either the luminescence or high-performance liquid chromatography (HPLC) assays that measure intracellular or released ADP [138].

Flow cytometry of vasodilator-stimulated phosphoprotein phosphorylation now also provides a simple means to monitor P2Y₁₂ inhibition [139].

Reticulated platelets — Nucleic acid-specific dyes can be used to measure young platelets that contain residual RNA. These young platelets are analogous to red cell reticulocytes and have thus been termed reticulated platelets. Although there is evidence to support that the youngest platelets are being measured [140,141], it is possible that the dense granular component of the larger young platelets is also contributing to the signal [142,143]. Assays can now be optimized in order to control for this problem [144]. (See "Automated hematology instrumentation", section on 'Reticulated platelets'.)

The assay for reticulated platelets is a simple, noninvasive method of assessing platelet turnover or thrombopoiesis in different types of thrombocytopenia, is a simple predictor of bone marrow recovery after transplantation, and can be used to monitor the response to therapy with growth factors such as thrombopoietin [145]. Additional clinical studies will need to be performed before the assay may be reliably used as a predictor of thrombosis in patients with myeloproliferative syndromes, such as essential thrombocythemia [146].

The test is now available as an additional fully automated complete blood count parameter called the Immature Platelet Fraction (IPF) that uses IPF master software within the Sysmex XE or XN series analyzers [147,148].

SUMMARY — In addition to the evaluation of platelet numbers and morphology by examination of the peripheral blood smear, a number of tests of platelet function are available to the clinician (table 1 and table 2). These are described in the text and include the following major tests:

●Manual and automated platelet counting (figure 1), including presence of reticulated (young) platelets (see 'Manual platelet counting' above and 'Automated cell counter methods' above)

●Platelet aggregometry (figure 2) (see 'Platelet aggregometry' above)

●In vivo bleeding time (see 'The in vivo bleeding time' above)

●The platelet function analyzer (see 'The platelet function analyzer' above)

●Platelet mediated thrombin generation (see 'Platelet-mediated thrombin generation' above)

●Thromboelastography (figure 3 and table 4) (see 'Thromboelastography (TEG) and ROTEM' above)

●Measurement of platelet activation (see 'Measurement of platelet activation' above)