Platelet Function Testing: Update and Future Directions

Abstract

Platelets play a key role in maintaining normal hemostasis and are also recognized as partners in the development of arterial thrombosis. Today, platelet function testing is used for very different clinical purposes; first, for investigation of platelet dysfunction in acute bleeding and diagnosis of platelet disorders in patients with long-lasting bleeding tendency, and second, for testing the efficacy of antiplatelet therapy in patients with increased thromboembolic risk. Moreover, it has been discussed whether platelet function testing can be used for prediction of bleeding risk (e.g., prior to major surgery). Ever since light transmission aggregometry was introduced, laboratories around the world have worked on testing platelet function, and during the last decades a wide range of new methods has emerged. Besides the clinical utility of platelet function testing, the present review summarizes the test principles and advantages and disadvantages of the different methods, depending on the purpose for which it is to be used. A critical step in investigation of platelet function is the preanalytical factors that can substantially affect test results. Therefore, this review also provides an overview of preanalytical variables that range from patient-related factors such as smoking, coffee, and exercise prior to blood sampling to selection of anticoagulant, needle gauge, and time from blood sampling to analyses. Finally, this review outlines further perspectives on platelet function testing for clinical practice and for research purposes.

Keywords

platelets - platelet activation - platelet function tests - platelet disorders - preanalytical

Since the beginning of the 20th century, it has been evident that platelets are crucial for maintaining normal hemostasis.[1] [2] Despite their humble size and relatively short half-life of 7 to 10 days, platelets are involved in numerous different pathophysiological processes, including bleeding, thrombosis,[3] inflammation,[4] angiogenesis,[5] wound healing, and tumor growth and metastasis.[6] [7] In the case of hemorrhage, both normal concentration and function of platelets are essential to restore hemostasis. However, due to their thrombotic capacity, platelets are double-edged swords as they also play a key role in atherothrombosis and thus are important targets for prevention of arterial thrombotic events.

Primary Hemostasis

Vascular lesions trigger vasoconstriction primarily mediated by endothelin-1, which is synthesized by the damaged endothelium. Simultaneously, subendothelial collagen is exposed and von Willebrand factor (VWF) and adenosine triphosphate (ATP) are released from endothelial cells.[8] [9] Subsequently, platelets adhere to the endothelium by a shear-dependent interaction with VWF and subendothelial collagen, leading to platelet activation.[10] [11] First, this results in shape change of the platelets into a multi-pseudopodal form, leading to an increased surface area of the platelets. Second, cytoplasmic granules are secreted from the platelets. The cytoplasmic platelet granules contain, among other things, serotonin, platelet-activating factor, and adenosine diphosphate (ADP), with the latter being an essential physiological agonist. ADP binds to the platelet membrane receptors P2Y1 and P2Y12 and thereby aids in platelet aggregation. Moreover, activated platelets synthesize thromboxane A2 from arachidonic acid; a reaction catalyzed by the enzyme cyclooxygenase 1. Thromboxane A2 is released from the platelet phospholipid membrane and further stimulates activation of new platelets and promotes platelet aggregation.[12] [13] The primary platelet plug is then formed by means of fibrinogen, which interconnects platelets via the glycoprotein (GP) IIb/IIIa receptors. Finally, the frail primary platelet plug is stabilized when fibrinogen is converted to fibrin by secondary hemostasis with thrombin being a pivotal player.[14]

Clinical Application of Platelet Function Testing

A global evaluation of primary hemostasis can be attempted by measuring bleeding time. This method was introduced in 1910 by Duke[15] and further refined by the Ivy technique.[16] Measurement of bleeding time is quite simple and encompasses physiological hemostasis including the vessel wall endothelium. However, determination of bleeding time has low reproducibility, is invasive, and is not sensitive to mild platelet defects. Moreover, test results do not correlate with bleeding tendency.[17] Due to these limitations, measurement of bleeding time has been replaced by other ex vivo platelet function tests.[18]

Platelet function testing by light transmission aggregometry (LTA) was introduced in the 1960s by Born and O'Brien,[19] [20] and LTA revolutionized the diagnostic workup of platelet disorders. This assay remains the gold standard, but since the late 1980s, several new methods have been introduced.

Platelet function testing is indicated in different clinical situations: in the patient with acute bleeding, in the diagnosis of hereditary platelet function disorders, and in testing the efficacy of antiplatelet therapy. Some platelet function tests are both specific and sensitive enough for investigation of platelet disorders, while others have low sensitivity and are only marketed with cartridges containing selected agonists at fixed concentrations, making them only applicable as a screen of platelet function or for evaluating the efficacy of antiplatelet therapy.

To provide an overview of platelet function tests of today, this review summarizes the currently used assays including their advantages and disadvantages and clinical utility. Moreover, the review discusses the preanalytical variables that are important to consider when investigating platelet function. Finally, future directions for platelet function testing for clinical purposes or for research are outlined.

Platelet Function Tests

General Considerations

As described, activation of a resting platelet through one or more pathways results in multiple processes including adhesion to endothelia, aggregation to other platelets, and degranulation, which are mediated by surface receptors and downstream signaling. Therefore, when attempting to evaluate platelet function ex vivo, the outcome of the platelet function test depends on which pathways are activated and which processes (e.g., aggregation or surface receptor expression) are registered. The laboratory tests used today reflect different aspects of platelet function; however, no available test mirrors all parts of the entire process. Particularly, the vascular contribution to platelet activation and aggregation is not assessed with current methods. An overview of today's most widely used tests for determination of platelet function is presented in [Table 1], including the principle, clinical use, advantages, and disadvantages.

Table 1 An overview of analytical principles, indications, and advantages and disadvantages of the most commonly used platelet function tests

Apparatus	Measurement principle	Indication	Advantages	Disadvantages
Platelet Aggregation Profiler: PAP8/PAP4-Aggregometer	•  Turbidimetric-based optical detection of changes in light transmission in platelet-rich plasma	•  Platelet function disorders •  Effect of antiplatelet therapy •  Forecast bleeding risk	•  Gold standard •  Wide range of agonists in different concentrations	•  Time consuming •  Large sample volume •  Requires sample preparation •  Insensitive to some storage pool deficiencies •  Sensitive to thrombocytopenia •  and lipidemia
Chronolog-log Multiplate Analyzer	•  Changes in electrical impedance between two electrodes in whole blood	•  Platelet function disorders •  Effect of antiplatelet therapy •  Forecast bleeding risk	•  Wide range of agonists in different concentrations Chrono-Log: can also determine concurrently ATP release when using the luciferin-luciferase reagent	•  Insensitive to some storage pool deficiencies •  Strongly correlated to platelet count
Platelet Function Analyzer: Innovance PFA-100/200	•  Time to thrombus formation in high-shear blood flow through a collagen-coated membrane in whole blood	•  Rapid exclusion of severe platelet dysfunction and severe von Willebrand disease Effect of antiplatelet therapy	•  Point-of-care instrument •  High-shear-induced platelet aggregation •  No sample preparation •  Low sample volume	•  Insensitive to storage pool deficiencies •  Dependent on hematocrit levels and von Willebrand factor •  Sensitive to thrombocytopenia
VerifyNow	•  Turbidimetric-based optical detection of changes in light transmission through whole blood	•  Effect of antiplatelet therapy	•  Point-of-care instrument •  Fast •  No sample preparation	•  Expensive •  Sensitive to thrombocytopenia, lipidemia, anemia, and fibrinogen levels
IMPACT: Cone and Plate(Let) Analyzer	•  Quantification of platelets adhered to a plate under arterial flow conditions using whole blood	•  Platelet function disorders •  Effect of antiplatelet therapy	•  Shear-induced platelet aggregation •  Fast •  No sample preparation •  Low sample volume	•  Expensive •  Dependent on von Willebrand factor
PlateletWorks	•  Single platelet impedance counting in native tube and after addition of agonists	•  Effect of antiplatelet therapy	•  Fast •  No sample preparation •  Low sample volume	•  Time-dependent (analysis within 10 minutes) •  Few studies on clinical outcomes
Flow cytometry	•  Fluorophore-labeled antibodies against surface glycoproteins and surface-bound activating markers in whole blood	•  Platelet function disorders •  Assessment of platelet activation state	•  Independent of thrombocytopenia •  Sensitive to storage pool deficiencies	•  Sensitive to autoantibodies •  Time consuming •  Requires highly specialized operators
ROTEM Platelet	•  Impedance aggregometry in whole blood	•  Effect of antiplatelet therapy	•  Point-of-care instrument •  Rapid •  Portraits global clot formation	•  Lack of clinical studies
TEG Platelet mapping	•  Calculation of difference between maximal hemostatic activity and AA/ADP-activated coagulation in whole blood	•  Effect of antiplatelet therapy	•  Point-of-care instrument •  Rapid •  Portraits global clot formation	•  Lack of clinical studies

Abbreviations: AA, arachidonic acid; ADP, adenosine diphosphate; ROTEM, rotational thromboelastometry; TEG, thromboelastography.

Platelet activation ex vivo is initiated by adding agonists to the patient sample. Addition of an agonist activates the platelet by binding to their relevant receptor. The level of aggregation induced by different agonists is used to distinguish between platelet disorders or to quantify their severity. The aggregation agonists most frequently used are listed in [Table 2]. When investigating the effect of antiplatelet medication, an agonist directed toward the obstructed receptor is used. If evaluating the effect of aspirin, arachidonic acid is used as an agonist, whereas a combination of ADP and prostaglandin E₁ is used for clopidogrel efficacy testing.

Table 2 An overview of the most used agonists for dynamic platelet function tests

Agonist	Target
Collagen	Glycoprotein (GP) VI and GPIa/IIa receptors
Adenosine diphosphate (ADP)	P2Y1 and P2Y12 receptors
Thrombin-related activating peptide (TRAP)	PAR1 and PAR4 thrombin receptors
Arachidonic acid	The cyclooxygenase pathway
Epinephrine	α2-receptor
Ristocetin	von Willebrand factor binding to GPIb/V/IX at high concentrations; abnormal binding at low concentrations in type 2B von Willebrand disease

Source: Modified from Higgins et al.[72]

Platelet function can be assessed in either whole blood or in platelet-rich plasma (PRP). Analyzing platelet aggregation in anticoagulated whole blood has the advantage of saving time because of the reduced sample processing. Furthermore, whole blood can be considered a more physiological environment, allowing a more realistic depiction of platelet aggregation potential; however, platelet–vessel wall interaction is not assessed in either matrix. Conversely, by analyzing platelet function in PRP there is also a risk of introducing a source of error due to platelet activation or loss of platelet subpopulations during processing, especially centrifugation.[21]

Light Transmission Aggregometry

Employing LTA, platelet aggregation is evaluated by measuring changes in light transmission after addition of platelet agonists to PRP. Prior to analysis, the maximum light transmittance through the sample (100%) is determined in platelet-poor plasma (PPP) from the patient, thereby adjusting the measurement for other absorbent components in plasma. After this initial baseline establishment, PRP is added to the test chamber with subsequent addition of an agonist. Light transmittance is then recorded continuously as it gradually increases as a result of platelet aggregation. Results are reported as the maximum percentage of aggregation in relation to light transmittance through PPP.[22]

Platelet aggregation measured by LTA can be induced by numerous agonists and different concentrations of agonists.[18] [23] This enables evaluation of many aspects of the aggregation process. Also, both the two-phased aggregation and the disaggregation can be evaluated visually. LTA is considered the gold standard of platelet aggregation analysis, and its correlation to stent thrombosis, ischemic cardiac events, and bleeding risk has been well documented,[23] although evaluation of antiplatelet medication and thrombotic risk is not as well substantiated as the use of LTA is for investigation of bleeding disorders.

Because LTA is the most widely used platelet aggregation assay, several attempts have been made to standardize methodology between laboratories. However, due to the absence of any international standard reagents, this issue remains unsolved.[24] Another drawback of LTA is the use of PRP. The lack of other blood components and the low shear conditions of the test lead to a setting that differs from the physiological platelet activation and aggregation in response to vessel-wall damage.[25] LTA is also sensitive to low platelet count. Furthermore, LTA analysis requires a relatively large blood volume due to the production of both PRP and PPP, especially if several agonists are used. Lastly, the test is time-consuming and requires skilled staff both for analysis performance and result interpretation.

Modification of LTA for the Automated Laboratory

The above-mentioned obstacles in LTA analysis have facilitated the development of more manageable, high-throughput assays requiring smaller blood volumes. These tests represent modified LTA tests and require the same sample preparation as LTA, but they are either applied to a standard 96-well plate or assessed by some automated hemostasis analyzers. In the first version, PRP is added to the plate in which wells are either precoated with agonists or contain an agonist solution. Following PRP addition, the plate is stirred leading to platelet aggregation and subsequent change in light absorbance. Results are reported as percentage of aggregation identical to traditional LTA, but instead of measuring light transmittance, it measures light absorption.[26] [27] This approach maintains the flexibility of LTA but enables rapid evaluation of different activation pathways in several patients at once. Furthermore, it only requires standard ELISA (enzyme-linked immunosorbent assay)-based equipment, but still experienced personnel have to be employed to perform the testing and interpretation of the results.

Platelet aggregation testing by light transmission has recently also been introduced on automated coagulation analyzers (e.g., Sysmex the CS2000, 2500, and 5100 series).[28] For all these methods, preparation of PRP is still needed; however, the automated method has the advantage of auto-pipetting of both PRP and reagents, thus demanding less manual handling than traditional aggregometers. The method has been tested in healthy individuals and individuals suspected of bleeding disorders with good correlation to other methods,[29] [30] [31] but further clinical validation is still awaited.

Whole Blood Aggregometry

The first instrument to allow evaluation of platelet aggregation in whole blood was the Chrono-log,[32] first introduced in 1980 and employing multiple-electrode aggregometry.[33] This device introduced measurement of platelet aggregation by changes in impedance, and besides, this method can also determine concurrently ATP release when using the luciferin-luciferase reagent.[34] The Chrono-log was subsequently supplemented by the semi-automated instrument Multiplate analyzer.[35] This impedance-based analysis enables determination of platelet aggregation in unprocessed blood. Whole blood or diluted whole blood is added to a test cell containing two electrodes. Upon addition of an agonist, the platelets aggregate and adhere to the electrodes resulting in increasing impedance (or resistance) between the electrodes, which is continually registered and converted to arbitrary aggregation units (AU). Platelet aggregation is usually reported as area under the curve in AU*min as suggested by the manufacturer,[36] but the maximum aggregation amplitude (measured in AU) and slope of the aggregation curve designated as aggregation velocity (AU/min) are also registered.[37]

The major advantage of whole blood aggregometry is the ability to analyze platelets in a fairly natural environment. Furthermore, the method requires a smaller sample volume than LTA, is less time-consuming, and easier to perform than LTA because of the omitted sample (PRP, PPP) preparation. The test result is not influenced by lipidemia or bilirubinemia, but is, however, influenced by platelet count.[38] On the other hand, some methods are very expensive (disposable cartridges) or have limited breadth of analysis (i.e., standard Multiplate test panel).

Platelet Function Analyzer

The platelet function analyzer (PFA 100/200) was introduced in 1995 as a refinement of the Thrombostat 4000 test.[33] Like impedance aggregometry, it is capable of assessing platelet aggregation in whole blood. At analysis, blood is aspirated through a small perforation in a membrane creating a high shear stress. The membrane is coated with aggregation agonists, being with the two main ones epinephrine and collagen (CEPI cartridge) or ADP and collagen (CADP cartridge).[39] The agonists, along with the high sheer stress, activate platelets on their way through the membrane resulting in aggregation and adhesion leading to thrombus formation and blockage of the perforation. The time to membrane closure is measured and reported as the closure time (CT) in seconds.

PFA 100/200 provides a global function test of hemostasis with the ability to detect or exclude severe forms of von Willebrand disease, platelet disorders, and platelet-affecting medication, primarily aspirin. The test is highly sensitive to VWF levels, since the thrombus formation is greatly dependent on VWF especially because of the high shear stress condition.[40] Furthermore, the analysis is also sensitive to platelet count and hematocrit, but due to the global test of platelets (and VWF) it is not able to distinguish between specific disorders.[21] Moreover, the CT may be prolonged due to intake of certain foods or supplements and over-the-counter medications.

VerifyNow

VerifyNow is a point-of-care instrument on which platelet function can be tested in whole blood by a turbidimetric-based optical detection. The blood sample is loaded on to the instrument without any preceding processing. The whole blood is drawn into wells containing both fibrinogen-coated beads and aggregation agonist. Light is transmitted through the well and as platelets adhere and aggregate onto the fibrinogen-coated beads, the light transmission increases.[41]

This device is specifically designed to determine the effect of antiplatelet therapy (aspirin or P2Y12 inhibitors) through the affinity for fibrinogen and is not suitable for other purposes. It is frequently used in an acute setting since it does not require any processing of the sample, making the test easy to perform without the involvement of specialized laboratories. However, VerifyNow may not be as sensitive to the decreased effect of aspirin as other methods, e.g., LTA,[42] and clinical validation of the manufacturer's cut-off limits is lacking.

IMPACT: Cone and Plate(Let) Analyzer

The cone and plate(let) analyzer is a point-of-care instrument designed to evaluate platelet function ex vivo. This is obtained by measuring platelet adhesion to a thrombogenic surface under high shear stress. The thrombogenic surface is a polystyrene surface on which plasma proteins are immobilized. Whole blood is added to the surface and high shear stress is impressed to the platelets by a rotating cone. This stimulates platelets to adhere to the plasma proteins (in particular fibrinogen and VWF) on the stationary plate. The plate is subsequently washed and stained with the May–Grunwald stain. After staining, the platelet aggregates are measured by employing an image analyzer for macroscopic evaluation, and additionally the results are expressed as percentage coverage of the plate and average aggregate size.[43]

The obvious advantage of this method is the mimicking of the physiological conditions involved in primary hemostasis. Furthermore, no blood processing is needed, and it is fully automated and requires only a low sample volume. The method is, however, highly dependent on plasma proteins, hematocrit, and platelet count.[21] [33] [41]

PlateletWorks

The PlateletWorks investigates platelet aggregation in whole blood and is developed mainly for quick investigation of antiplatelet medication effects.[44] Blood is drawn directly into a tube with added agonist and, additionally, into a tube without agonist. Platelet count has to be performed within 10 minutes in both tubes using an impedance cell counter. As the impedance platelet counter registers only single platelets and not platelet aggregates due to its set size threshold, it returns a lower platelet count in the agonist tube compared to the agonist-free tube. Percent aggregation is then calculated based on the difference in platelet count between the two tubes. The analysis is easy to perform as it requires minimal sample handling and can in principle be conducted employing any impedance counter. However, it is critical that the sample is analyzed within 10 minutes to obtain correct results.[45] Correlation between PlateletWorks and other aggregation-based methods such as LTA and impedance aggregometry is moderate.[45] [46] [47] Furthermore, the method has not been clinically validated to a great extent.

Flow Cytometry

Some platelet function disorders are difficult to detect with the methods described previously. In particular, storage-pool deficiencies are difficult to identify by means of routine laboratory testing, hence, calls for more esoteric testing. Flow cytometry can identify both lack of surface proteins and granular insufficiencies caused by either granule deficiency or defects in granule release.[48] This method employs fluorophore-tagged antibodies directed against various platelet surfaces or intracellular markers. The blood sample is incubated with antibodies and the platelets are directed past a laser beam which activates the fluorophore. Fluorescence is detected for each individual platelet, and the result is expressed in either mean fluorescence intensity or percentage of fluorescence-positive platelets. The method allows for designing panels with several fluorophores, facilitating the measurements of multiple platelet markers at once. Surface markers can be measured on the resting platelet (e.g., GPIb for Bernard–Soulier syndrome) or after activation with various agonists.[49] When analyzing granular capacity, permeabilization of the membrane prior to fluorophore labeling is necessary to evaluate intracellular components. Flow cytometric analysis of platelet aggregation has also been described.[50]

Flow cytometric analysis of platelet function is highly sensitive for decreased expression of surface and intracellular proteins, and it allows detection of multiple activation markers in one sample. As platelets are counted individually, the assay is not influenced by platelet count like LTA or impedance aggregometry, and it can be performed on blood volumes as low as 1 mL.[51] Furthermore, panels can be designed individually, allowing a high degree of freedom in both research and clinical use. Disadvantages are the high cost of instrumentation and labor-heavy procedure, and the analysis demands skilled personnel to an even higher degree than LTA. Furthermore, interpretation of results can be challenging.

Thromboelastometry and Thromboelastography

Standard ROTEM and TEG protocols are unable to test platelet function. However, platelet aggregation tests are available to both ROTEM and TEG. An aggregometer is available as an add-on device to ROTEM, which allows measurement of platelet aggregation in whole blood based on impedance aggregometry. By adding arachidonic acid and ADP as agonists, the platelet module can evaluate the effect of antiplatelet drugs. The TEG platelet mapping system[52] also provides an opportunity to measure the effect of antiplatelet therapy.[53] First, maximal hemostatic activity is determined by a kaolin-activated test. The effect of antiplatelet therapy is evaluated by adding reptilase, coagulation factor XIIIa, and arachidonic acid or ADP to whole blood. The inhibition of platelet activation is then calculated using the result of the kaolin-activated test as a reference. However, clinical implementation for test for platelet function by ROTEM or TEG is still a long time coming.

Preanalytical Considerations

For all dynamic platelet function tests, precautions should be taken to avoid undue preanalytical activation of platelets in the sample, as this will cause spurious test results. A multitude of both patient-related and sampling-related factors influence preanalytical platelet activation as illustrated in [Fig. 1]. Furthermore, platelets are sensitive to handling, and stability ex vivo is short. All these factors can induce preanalytical errors. Besides, other factors such as choice of anticoagulant, route of transport, buffers, and temperature during measurement induce variation to the results and should therefore be standardized. Thus, laboratories performing platelet function analyses should carefully consider sources of preanalytical variation and should develop standardized laboratory protocols covering these sources of variation. This section outlines the most important contributors to preanalytical variation in platelet function assays.

Fig. 1 Factors contributing to preanalytical variation and/or preanalytical error in platelet function testing.

Patient-Related Factors

Circadian rhythm,[54] coffee consumption,[55] smoking,[56] exercise,[57] [58] food intake,[59] and medications such as nonsteroidal anti-inflammatory agents (NSAIDs)[60] or dietary supplements such as fish oil[61] are all factors which influence platelet activation. Ideally, therefore, the blood sample should be obtained during morning hours in a patient who is fasting, has refrained from coffee, tobacco, and exercise on the morning of blood sampling, and has paused relevant medications, including over-the-counter medications and dietary supplements, during the last 10 to 14 days. In practice, it is difficult to control all these factors, and it is not possible in the acute setting. However, information regarding those patient-related factors should be collected to assist in interpreting tests results, and in elective patients, medications, NSAIDs, and fish oil should be paused. Platelet count should always be measured to assist interpretation, as some platelet function methods are dependent on this parameter.

Blood Sampling

Many factors can activate platelets during blood sampling. Endothelial damage during venipuncture will expose collagen and tissue factor and cause release of platelet-activating factors from the endothelium. High shear stress and vibration also activate platelets.[62] Hemolysis induces release of Ca²⁺ and ADP from erythrocytes. Therefore, it is recommended that blood for platelet function analysis is obtained with smooth venipuncture under minimal tourniquet pressure using a 19 to 21 gauge needle and a short connecting tube.[63] [64] Some guidelines recommend that the first few mL is discarded; the rationale behind this is to avoid endothelial-derived activating substances in the tube and, if a system with pre-evacuated tubes is used, to fill the dead space in connecting and avoid air mixing into the tube which interferes with the blood:anticoagulant ratio.[64]

Choice of Tubes and Anticoagulant

Blood should be drawn into tubes with a nonactivating surface, e.g., polypropylene.[64] Tubes should contain anticoagulant to avoid thrombin generation and subsequent platelet activation. Different anticoagulants can be used for analysis, most commonly citrate (a Ca²⁺ chelator), hirudin (a direct thrombin inhibitor), or heparin (an indirect coagulation factor Xa and thrombin inhibitor). The choice of anticoagulant, however, will influence the results significantly, depending on the method and the parameters measured.[65] [66] [67] [68] In general, the use of citrate has been shown to yield lower overall aggregation values than, e.g., hirudin or heparin.[65] [66] [67] [68] Thus, reference intervals established using one type of anticoagulated tube are not transferable to other types. Ethylenediaminetetraacetic acid (EDTA) may cause conformational changes of the GPIIb/IIIa complex and thereby impair fibrinogen binding on the platelet surface, and this anticoagulant should therefore be avoided, as reviewed by Mannuß.[69]

Transport and Storage Conditions

It is recommended to transport samples upright and avoid pneumatic tube systems or other transport systems which expose the sample to high mechanical forces.[62] If use of a pneumatic tube system is necessary for logistic reasons, it is important to perform a thorough local validation as previous studies have suggested that use of pneumatic tube systems may affect platelet function testing.[62] [70] [71]

Studies investigating the effect of storage time on platelet function test results generally find a significant increase in platelet activation during the first 30 minutes after blood sampling.[49] [65] [66] [68] Therefore, it is recommended that the blood sample is left resting for a minimum of 30 minutes before analysis. Stability varies between methods, but in general samples for platelet function testing have relatively short stability (usually 2–4 hours). This may further be influenced by the choice of anticoagulant, with longer stability described in heparinized blood than in citrated blood.[65] [67] [68] The manufacturer's instructions should be followed, or stability should be verified locally. Finally, storage temperature can influence results. Kaiser et al found that storage at both 4°C and 37°C affected stability negatively; thus, it is recommended to store samples at room temperature.[65]

Conclusion and Perspectives

Since the development of LTA 60 years ago, options for platelet function testing have expanded widely. There is currently a variety of platelet function assays available for the diagnosis of bleeding disorders, assessment of the patient with acute bleeding, and investigation of antiplatelet medication effects. They range from easy to use, point-of-care assays to sophisticated flow cytometry protocols which have been revolutionary in providing detailed analysis of surface-bound markers of platelet activation and aiding in the diagnosis of rare platelet disorders.

At present, platelet function analyses are mainly available at specialized laboratories. The development of methods with a higher degree of automation or higher throughput, e.g., the modified 96-well assay or LTA on automated coagulation analyzers, will hopefully make platelet function tests available for more patients worldwide in the future. Another important issue for future research and development is the standardization of methods, both to minimize preanalytical variation and to enable comparison across laboratories. Furthermore, in recent years, the contribution of platelets to a range of physiological and pathophysiological conditions other than bleeding and thrombosis has been explored, e.g., inflammation, angiogenesis, and tumor growth and metastasis. The clinical value of platelet function testing for diagnosis and prognosis in these conditions remains to be elucidated.

Conflict of Interest

J.A.H. has no conflicts of interest to disclose. J.B.L. and A.-M.H. have no conflicts of interest pertaining to the present article, but have the following general disclosures within the past 36 months: J.B.L. has received speaker's fees from Bristol-Myers Squibb and travel support from Bayer. A.-M.H. has received speaker's fee from CSL Behring and an unrestricted research grant from CSL Behring.

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Publication History

Article published online:
16 November 2022