Laboratory
assays

Upon vessel injury, the aggregation of platelets at the site of injury results in the formation of a plug to prevent blood loss until other coagulation and repair mechanisms can be activated and achieve haemostasis. Platelets promote haemostasis by first adhering to the site of vascular injury, releasing haemostatic signals, aggregating to form the platelet plug and providing a phospholipid surface on which other haemostatic processes depend.^1,2

Receptors expressed on the platelet surface react with extracellular matrix proteins exposed at the site of vascular injury. von Willebrand factor (VWF) facilitates platelet adhesion via the glycoprotein Ib/IX/V (GPIb/IX/V) complex on the platelet surface and the glycoprotein IIb/IIIa (GPIIb/IIIa; integrin α_IIb/β₃) complex mediates adherence to fibrin or fibronectin associated with the vascular wall. The subsequent release of adenosine diphosphate (ADP) induces a conformational change in the GPIIb/IIIa fibrinogen receptor complex, which in turn initiates platelet aggregation and the recruitment of additional platelets to form the platelet plug. Platelet aggregation can also be stimulated by epinephrine, thrombin and collagen. Once activated, platelet membrane phospholipids provide a binding site for phospholipid-dependent coagulation factors.²

A deficiency in the number or functionality of platelets can lead to spontaneous and/or trauma-induced bleeding. Bleeding disorders attributable to platelet or VWF disorders typically result in a mucocutaneous bleeding pattern, in contrast to coagulation factor deficiencies that typically result in deep tissue bleeding and haemarthroses. Platelet function assays are useful not only to diagnose bleeding diatheses associated with platelet dysfunction, but also to monitor the effect of anti-platelet medications. A combination of different methodologies and conditions is necessary to fully characterise platelet (dys)function in a particular patient.^1,2

The most widely used types of assays for screening for platelet defects and for assessing platelet function are discussed below.

PFA tests the ability of citrated whole blood to clot in response to contact with structures and substances, intended to simulate a damaged blood vessel, under shear stress conditions. Samples are drawn through a capillary tube that terminates at a collagen-coated membrane with a standardised pore size in the presence of ADP or epinephrine. The time required to close the membrane pore with a platelet plug and block blood flow is measured and is proportional to platelet function. PFA is highly sensitive to von Willebrand disease, but less so for platelet disorders, and can therefore miss the presence of a mild platelet disorder.³

Differences in closure time between the ADP- or epinephrine-based assay provide some information about whether a platelet dysfunction is attributable to an inherent deficiency (prolonged closure time in the presence of ADP ± epinephrine) or anti-platelet therapy (prolonged in the presence of epinephrine). This assay has been shown to be more sensitive than traditional bleeding time assays for the diagnosis of platelet function deficiencies, however it is fairly non-specific since many variables may affect platelet function.^1,2 In vivo bleeding time remains a popular screening method in German-speaking countries, although it is used less frequently elsewhere (e.g., in the UK and North America).⁴

LTA using citrated platelet-rich plasma (PRP) is applied as a diagnostic test for platelet defects or to monitor anti-platelet treatment. Although developed in the 1960s, LTA continues to serve as the gold standard for the assessment of platelet aggregation in response to different agonists.

The optical density of PRP decreases when platelets aggregate following the addition of a platelet agonist, resulting in an increased level of light transmission. Both the rate and maximum level of aggregation can be measured using a photometer, with 0% transmittance representing the PRP alone and 100% transmittance representing a platelet-poor control plasma. Reaction kinetics can then be visualised using appropriate software.

Agonists assessed represent different in vivo platelet activation pathways and include ADP, arachidonic acid, collagen, epinephrine, thrombin receptor activating peptide (TRAP), thromboxane A2 mimetic U46619, calcium ionophore A23187 and the antibiotic ristocetin. With the exception of ristocetin, all of these agents act in a GPIIb/IIIa-dependent manner. Ristocetin induces binding of VWF to the platelet GPIb/IX/V complex and induces platelet aggregation even in the absence of a functional GPIIb/IIIa complex.

Although widely used as a clinical laboratory assay to diagnose platelet function disorders, the sensitivity of assay components to both patient- and laboratory-related variables requires that LTA be performed by staff experienced in both the method and the interpretation of results. Variants on the LTA method have been developed that circumvent some of the challenges. Impedance whole blood aggregometry, as the name suggests, assesses the ability of platelets in whole blood samples to adhere to the surface of a set of electrodes in response to a surface receptor agonist. Aggregation is measured as a function of electrical impedance. The use of whole blood may not only better represent in vivo physiological environment than PRP, but the lack of manipulation also avoids inadvertent artificial activation of platelets in the sample. Point-of-care versions of this assay are also available, further reducing the need for specialised laboratory services.^1,2

Lumi-aggregometry uses a modified luciferin-luciferase system to measure ATP secretion from platelet-dense granules in PRP, washed platelets or whole blood. Released ATP following platelet activation by thrombin and collagen oxidises a luciferin-luciferase reagent to generate luminescence that is proportional to the amount of ATP present. A lumi-aggregometer with a photomultiplier is used for detection of the resulting luminescence. Results are compared against an ATP release standard provided by the manufacturer. This method can be used to assess specific deficiencies in the number and content of dense granules.¹ 

Flow cytometry can be used to assess both the physical and antigenic properties of platelets. Monoclonal antibodies that recognise specific antigens and are conjugated to fluorescent markers identify the presence, density, binding and activation state of specific receptors or other antigens on the platelet surface. By using different fluorochromes, multiple antigens can be assessed simultaneously. While flow cytometry can be applied to small volumes of whole blood, even those with low platelet counts, the method requires staff experienced in both the method and the interpretation of results.^1,2

1. Paniccia R, Priora R, Liotta AA, Abbate R. Platelet function tests: a comparative review. Vasc Health Risk Manag 2015;11:133-48.
2. Kottke-Marchant K, Corcoran G. The laboratory diagnosis of platelet disorders. Arch Pathol Lab Med 2002;126:133-46.
3. Harrison P. Assessment of platelet function in the laboratory. Hamostaseologie 2009;29(1):25-31.
4. Streif W, Oliveri M, Weickardt S et al. Testing for inherited platelet defects in clinical laboratories in Germany, Austria and Switzerland. Results of a survey carried out by the Permanent Paediatric Group of the German Thrombosis and Haemostasis Research Society (GTH). Platelets 2010;21(6):470-8.
5. Nichols WL, Hultin MB, James AH, et al. von Willebrand disease (VWD): evidence-based diagnosis and management guidelines, the National Heart, Lung, and Blood Institute (NHLBI) Expert Panel report (USA). Haemophilia 2008;14:171-232.
6. Keesler DA, Flood VH. Current issues in diagnosis and treatment of von Willebrand disease. Res Pract Thromb Haemost 2018;2:34-41.
7. Sharma R, Flood VH. Advances in the diagnosis and treatment of Von Willebrand disease. Hematology Am Soc Hematol Educ Program 2017;2017:379-84.
8. James AH, Eikenboom J, Federici AB. State of the art: von Willebrand disease. Haemophilia 2016;22 Suppl 5:54-9.
9. Bodo I, Eikenboom J, Montgomery R, et al. Platelet-dependent von Willebrand factor activity. Nomenclature and methodology: communication from the SSC of the ISTH. J Thromb Haemost 2015;13:1345-50.