REVIEWPlatelet function analysis
Introduction
Human platelets are small and discoid in shape, with dimensions of approximately 2.0–4.0 by 0.5 μm, and a mean volume of 7–11 fl.1 They are the second most numerous corpuscle in the blood normally circulating at between 150–450 × 109/l. Platelets are anucleated cells derived from megakaryocytes and typically circulate for 10 days.1 Their shape and small size enables the platelets to be pushed to the edge of vessels, placing them in the optimum location required to constantly survey the integrity of the vasculature. Platelets are also surprisingly multifunctional and are involved in many pathophysiological processes including haemostasis and thrombosis, clot retraction, vessel constriction and repair, inflammation including promotion of atherosclerosis, host defence and even tumour growth/metastasis (Fig. 1). Although, any test(s) of platelets could therefore potentially measure any one or more of these vital processes, the majority of available tests focus only on those function(s) involved directly in haemostasis.
Upon vessel wall damage, platelets undergo a highly regulated set of functional responses including adhesion, spreading, release reactions, aggregation, exposure of a procoagulant surface, microparticle formation and clot retraction (Fig. 2). All of these platelet responses function to rapidly form a haemostatic plug that occludes the site of damage to prevent blood loss.[2], [3] When there is a defect in any of these functions and/or platelet number, haemostasis is usually impaired and there may be an associated increased risk of bleeding. In contrast, a marked increase in platelet number or reactivity can lead to inappropriate thrombus formation. Arterial thrombi can also develop within atherosclerotic lesions resulting in stroke and myocardial infarction, two of the major causes of morbidity and mortality in the western world.4 Anti-platelet therapy can therefore be beneficial in the treatment and prophylaxis of arterial thrombotic conditions, but must be carefully administered without increasing the risk of bleeding to an unacceptable level.1
The main use of platelet function tests has been traditionally to identify the potential causes of abnormal bleeding,5 to monitor pro-haemostatic therapy in patients with a high risk of bleeding and to ensure normal platelet function either prior to or during surgery.[6], [7] However, they are increasingly being utilised to monitor the efficacy of anti-platelet therapy and to potentially identify platelet hyperfunction to predict thrombosis.[6], [8] For a full list of potential clinical uses of platelet function tests see Table 1.
Section snippets
History of platelet function testing
Platelets were first described by the remarkably early observations of Bizzozero in the late 1800s.9 Not only did he identify platelets as distinct corpuscles within human blood but he observed them forming thrombi within damaged areas of vessel wall using real-time microscopy. Today, modern imaging methods are utilised to study in detail the same real time interactions of platelets with the vessel wall and dynamics of thrombus formation.10
Table 2 illustrates a list of traditional tests of
Quality control, blood sampling and anticoagulation
Platelet function testing presents many challenges in ensuring that accurate and meaningful results are obtained. Firstly, unlike with coagulation tests, there are no widely available internal or external quality control materials available. Most assays are performed on fresh blood and so many laboratories either establish normal ranges using control volunteer blood and/or assay known normal samples in parallel to ensure that each test/reagent is viable. Many platelet function tests such as
Initial approach to diagnosing platelet dysfunction
When investigating a suspected bleeding disorder, it is essential to obtain a detailed clinical and family history and perform a physical examination before choosing the laboratory tests to be undertaken.[3], [27] In particular a recent drug history is critically important and patients should be advised to discontinue where possible any drug that could interfere with platelet function. Some drugs may cause a transient haemostatic defect, so repeat or deferred testing is often necessary. Other
The bleeding time
The bleeding time (BT), developed by Duke in 1910,34 was the first in vivo test of platelet function and was still regarded as the most useful test of platelet function until the early 1990s.12 The bleeding time is the time taken for bleeding to stop after an incision is made into the skin, usually into the anterior surface of forearm (Fig. 3). The test has been refined and standardised particularly with the use of a sphygmomanometer cuff and a spring-loaded template device to make standard
Platelet aggregation
Platelet aggregometry was developed in the early 1960s and soon became regarded as the “gold standard” of platelet function testing.37 This is still the most widely used test for identifying and diagnosing platelet function defects and can be performed within commercially available multi-channel aggregometers. Whole blood aggregometers using impedance technology are also available and are sometimes combined with luminometry to simultaneously measure dense granular ADP release. However, in most
The platelet function analyser – PFA-100®
The PFA-100® is a relatively simple bench top instrument that simulates high shear platelet function within disposable test cartridges (Fig. 4).[17], [38], [39] Citrated blood is aspirated under constant negative pressure from the sample reservoir through a capillary and a microscopic aperture (147 μm) cut into a membrane. The membrane is coated with either collagen/epinephrine (CEPI) or collagen/ADP (CADP). The presence of these platelet activators and the high shear rates (5000–6000 s−1)
The cone and plate(let) analyser
Another test that has recently been developed is based upon the adhesion of platelets to the extracellular matrix using whole blood exposed to high shear. The original The cone and plate(let) analyser (CPA) device tested whole blood platelet adhesion and aggregation on a plate coated with extracellular matrix (ECM).[19], [51] The CPA has now been developed into a fully automated commercial instrument called the IMPACT® (Diamed, Switzerland) (see Fig. 5) and now utilises polystyrene plates
The ultegra rapid platelet function assay – RPFA®
The Ultegra-RPFA® (Accumetrics, Inc, San Diego, California) is a turbidimetric based optical detection system (see Fig. 6) that measures platelet induced aggregation as an increase in light transmittance.[23], [52] This modified platelet aggregometry device was originally developed as a near patient testing instrument in order to provide a simple and rapid functional means of monitoring anti-Gp IIb/IIIa therapy with various anti-platelet drugs (e.g. abciximab).52 The disposable cartridges
Hemostasis analysis system®
Hemodyne have developed and refined a Hemostasis Analysis System® (HAS) over the last 7 years (Fig. 7). This instrument is based upon the original technique developed by Carr.21 The HAS measures platelet contractile force (PCF®), clot elastic modulus (CEM®) and thrombin generation time (TGT®) in a sample (700 μl) of clotting whole blood. PCF® is the force generated by platelets during clot retraction. In this instrument a small sample of whole blood is trapped between two parallel surfaces.
Plateletworks®
Platelets within whole blood, when stimulated by agonists forms aggregates with a reduction in the number of free platelets. By counting the number of platelets in a control sample (usually EDTA) and within a sample that has been activated with a classical platelet agonist it is possible to assess the degree of platelet aggregation that can occur in a given sample. The Plateletworks® aggregation kits (Helena Biosciences) are simply based upon comparing platelet counts within a baseline EDTA
Flow cytometry
There is no doubt that one of the biggest advances in platelet function analysis has been the application of flow cytometry.[25], [62] This technique however, requires access to an expensive instrument and specialised training to perform. A number of largely research applications continue to evolve into clinically useful tests particularly as many of the reagents, antibodies and dyes are now commercially available.11 A list of currently available diagnostic flow cytometry assays are shown in
Conclusions
The last guidelines for platelet function testing were written in the late 1980s.12 Given the advances in this field and the introduction of potentially useful additions to our existing portfolio of platelet function tests, work is currently in progress to rewrite these guidelines. Nevertheless, the incorporation of new platelet function tests into the routine laboratory has already begun and a number of new approaches have been suggested.[5], [29], [40] Flow cytometric-based platelet function
Acknowledgements
The author thank Dr. David Keeling and Professor Steve Watson for carefully reading the manuscript. I also thank Professor Sam Machin for providing Fig. 3.
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