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LEARNING OBJECTIVES
Upon completion of this article, the reader will be able to:
- Describe different methods for platelet function testing.
- Describe clinical features of platelet disorders.
- Identify the effect of antiplatelet drugs and laboratory testing available.
Platelets are small nonnucleated blood cells that have a vital role in hemostasis and thrombosis and are produced in the bone marrow from megakaryocytes.
Accurate measurement of platelet function is important for identifying patients with platelet dysfunction. Platelet function testing is also increasingly important for evaluating and monitoring antiplatelet therapies such as aspirin and clopidogrel.
Platelets promote hemostasis by four interconnected mechanisms: adhesion to the sites of vascular injury; cellular activation and release of granule contents; aggregation, recruitment and amplification to form a hemostatic platelet plug; and providing a procoagulant phospholipid surface.
Platelet function tests may be recommended for those who bruise easily, have excessive bleeding, or take medications after a stroke or heart attack that can alter platelet function; to detect resistance to aspirin or clopidogrel; and before certain surgeries.
PLATELET FUNCTION TESTING METHODOLOGIES
Complete blood count (CBC) and peripheral blood smear
Investigation of platelet disorders should start with a measurement of the platelet count and review of peripheral smear morphology. However, caution must be exercised in interpreting platelet function testing because abnormal results are often observed in thrombocytopenic patients; distinction of an abnormal result caused by intrinsic platelet dysfunction from thrombocytopenia alone may not be possible.
Platelet function tests
For nearly a century the bleeding time test was the only platelet screening function test available. The test, which involves cutting the skin and measuring the time it takes for the bleeding to stop, is fraught with variability, and many laboratories no longer use it.
Newer, semi-automated whole blood platelet function screening assays are gaining popularity as an initial screen for platelet function. Most of these assays use small, stand-alone devices that measure platelet function in whole blood and can be used in laboratories that otherwise could not perform these studies.
These assays include the PFA-100 (Platelet Function Analyzer, Siemens USA), the VerifyNow (Accumetrics), the Plateletworks (Helena), and the IMPACT (Diamed). The thromboelastograph (TEG) (Haemonetics), while not a new technology, has been adapted recently for use in the clinical coagulation laboratory. It measures a combination of coagulation, platelet function, and fibrinolysis.1
PFA-100 (Platelet Function Analyzer) is a device that measures shear-induced, platelet-related primary hemostasis in a citrated whole blood specimen. The PFA-100 measures the blockage of an aperture in a membrane coated with collagen and adensophine diphosphate (ADP) by platelets, mimicking damaged endothelium. It is fairly adept at screening patients for mild congenital bleeding disorders. It has been used to detect aspirin resistance, but the results with clopidogrel are variable and it is not recommended for this purpose. PFA-100 results can be affected by low platelet counts and low hematocrits, but they are not affected by heparin. The PFA-100 is included in recently published Clinical and Laboratory Standards Institute (CLSI) guidelines for platelet testing.
VerifyNow, a rapid platelet function assay, is an automated turbidimetric whole blood assay designed to assess platelet aggregation, based on the ability of activated platelets to bind fibrinogen. Agonist-activated platelets and fibrinogen-coated polystyrene microparticles agglutinate in whole blood in proportion to the number of available platelet GP IIb/IIIa receptors, which makes this device appropriate for the measurement of antiplatelet drug (e.g., aspirin and clopidogrel) effect rather than screening for intrinsic platelet dysfunction.
Platelet Aggregation measures the ability of agonists (e.g., ADP, epinephrine, collagen, arachidonic acid, ristocetin) to cause in vitro platelet activation and platelet-platelet binding (Figure 1). As such, platelet aggregation is often useful to distinguish intrinsic platelet disorders involving surface glycoproteins, signal transduction, and platelet granules. Platelet aggregation studies can be performed in whole blood by an impedance technique or in platelet-rich plasma by a turbidimetric technique, often called light transmission aggregometry. Many factors can affect the platelet aggregation results, such as thrombocytopenia, thrombocytosis, processing temperature, stirring rate and processing time. Before testing, patients should discontinue, if possible, any medication, such as aspirin or nonsteroidal anti-inflammatory agents. The laboratory performance of platelet aggregation has been highly varied, and recent CLSI and North American guidelines have been published in an attempt to standardize testing.2,3
Figure 1. Diagram of typical platelet aggregation results in normal individuals compared to patients with Glanzmann’s Thrombasthenia and Bernard-Soulier syndrome
Other specialized testing
Used only at specialized centers, flow cytometry is used to study platelet structure and function. Flow cytometric analysis is based on the detection of cell surface proteins with fluorescently labeled antibodies. It is used in the detection of platelet activation and to diagnose deficiencies of platelet surface glycloproteins. These methods can be combined with the use of platelet agonists to measure dense granules, as well as release, aggregation, microparticle formation, and platelet procoagulant activity.
Electron microscopy can be used for the ultrastructural evaluation of platelets, particularly in patients with suspected storage pool disorders (SPDs) in which a decrease or absence of dense bodies is observed. Giant platelet disorders also have characteristic electron microscopic findings.
High-pressure liquid chromatography is an additional technique to study the presence of storage and release defects by measuring the ratio of ADP to adenosine triphosphate (ATP) within platelets. Dense granule defects can also be studied by lumiaggregometry, where ATP released from dense granules is measured by a firefly luciferin-luciferase reagent simultaneously with aggregation. Future platelet studies will likely include investigation of platelet signaling pathways, because many platelet disorders appear to involve dysregulation of signaling pathways. Genomic approaches will also be used to identify specific inherited platelet defects.4
CLINICAL FEATURES OF PLATELET DISORDERS
In a patient with a bleeding diathesis, a detailed personal and family bleeding history should be obtained before beginning a laboratory evaluation of platelet function. Bleeding disorders caused by coagulation proteins, fibrinolysis, or the vascular system should be excluded. The history should include the age of onset and a duration assessment, as well as bleeding problems, including whether the bleeding is spontaneous or associated with trauma or surgery.
Platelet-mediated bleeding disorders typically manifest with a mucocutaneous bleeding pattern involving small vessels.4 Ecchymosis, petechiae, purpura, epistaxis, and gingival bleeding are commonly observed. This is in contrast to coagulation protein disorders in which deep-tissue bleeding and hemarthroses are more common. Von Willebrand disease (VWD), an abnormality of von Willebrand factor (VWF), has bleeding symptoms similar to platelet dysfunction, and evaluation for VWD should be included in the initial evaluation of a possible platelet disorder.
Many drugs and foods, such as caffeine and garlic, can affect platelet function; therefore a complete drug and dietary history should be obtained. It is important to remember that aspirin, an irreversible inhibitor of platelet function, is an ingredient in many over-the-counter and prescription medications, such as cold and flu remedies. The clinical history should include investigation of systemic disorders, such as renal disease, hepatic failure, connective tissue disorders, myeloproliferative disorders, myelodysplastic disorders, malignancy, and cardiovascular disease, as platelet dysfunction is associated with many of these diseases. In addition, some specific clinical features such as albinism, deafness, nephritis, and susceptibility to infections may help in the differential diagnosis of the inherited platelet disorders.
Glycoprotein disorders
Glanzmann’s thrombasthenia (GP IIb/IIIa deficiency) is a congenital deficiency or dysfunction of GP IIb/IIIa
(αIIb/β3 integrin)
, the fibrinogen receptor responsible for mediating platelet aggregation that manifests in lifelong mucocutaneous bleeding. Many different mutations of both the GP IIb and GP IIIa have been described. Often, mutations of one subunit prevent the formation of the entire complex on the platelet surface, and it is common to detect neither GP IIb nor GP IIIa on the surface of the platelet.5
The CBC in individuals with Glanzmann’s thrombasthenia is usually normal, with an abnormal PFA-100. Absent or decreased aggregation response will be seen upon addition of ADP, collagen, epinephrine and arachidonic acid aggregating agents, whereas the ristocetin-induced aggregation is normal (Figure 1). This finding is virtually diagnostic of Glanzmann’s thrombasthenia, but the disorder can be confirmed by platelet flow cytometry, crossed immunoelectrophoresis of platelet membrane proteins, or genetic analysis.
Bernard-Soulier syndrome (GP Ib/V/IX deficiency)is a congenital deficiency of the platelet glycoprotein glycoprotein Ibα/Ibβ/V/IX receptor, the surface receptor responsible for mediating VWF-induced platelet adhesion and aggregation. Most of the genetic defects are due to mutations of the GPIba gene, but may also be due to defects of the GPIbβ or GPIX genes.The disorder is associated with severe, lifelong mucocutaneous bleeding with thrombocytopenia.
Individuals with Bernard-Soulier syndrome typically have moderately severe thrombocytopenia (40,000 to 100,000/ul) with uniformly large, granulated platelets. Normal platelet aggregation is noted with exposure to ADP, collagen, epinephrine, and arachidonic acid, but aggregation is characteristically absent with the addition of ristocetin or botrecetin, and the PFA-100 test is abnormal, as seen in Figure 1. The glycoprotein abnormality can be confirmed with flow cytometry or crossed immuno-electrophoresis, where a combined lack of GP Iba, GPIbβ and GPIX is identified.5
Other glycoprotein disorders include collagen receptor disorders, ADP receptor disorders and Platelet-Type VWD (also called Pseudo-VWD), in which an unusual gain-of-function abnormality of GP Ib leads to increased binding of platelet GP Ib/V/IX to von Willebrand factor. 4
PLATELET RELEASE DEFECTS
Storage pool disorders (alpha and dense granule disorders)
Platelet storage pool disorders are the result of a deficiency of alpha and/or dense granules. Between 10% and 18% of patients with congenital platelet dysfunction have storage pool disorders.
Dense granule storage pool disorders (δ-SPD) can be seen as a singular clinical entity or as part of other hereditary disorders, such as Chediak-Higashi, Hermansky-Pudlak syndrome, thrombocytopenia with absent radii (TAR syndrome), or Wiskott-Aldrich syndrome. Patients with δ-SPD have a bleeding diathesis of variable severity characterized by mucocutaneous bleeding and peri-operative bleeding. The platelet count is normal in patients and the peripheral smear shows platelets with apparently normal granule staining. A decreased aggregation response to ADP, epinephrine, arachidonic acid, and collagen is seen in approximately 75% of patients with δ-SPD showing a primary wave of aggregation with an absent to decreased secondary wave. The PFA-100 may be abnormal in some but not all patients. Decreased ATP release is observed by lumi-aggregometry, and decreased mepacrine dense granule uptake is observed by flow cytometry. Ultrastructural abnormalities in δ-SPD show absent to decreased dense granules.5
The alpha storage pool disorder δ-SPD leads to gray platelet syndrome (GPS) with large (mean 13 fL) platelets devoid of alpha granules, giving them a ghostly gray color on the peripheral smear. The disorder is characterized by mild, lifelong bleeding symptoms. Some patients may have marrow fibrosis, pulmonary fibrosis, and splenomegaly. Platelet aggregation studies may be normal for ADP and epinephrine, but are often abnormal for thrombin and collagen. Flow cytometry studies have shown increased surface P-selectin, but decreased alpha granule P selectin. By electron microscopy, platelets are enlarged and alpha granules are not present; however, dense granules, mitochondria, and lysosomes appear normal.4
Signal transduction disorders
Platelet signal transduction is a complex and incompletely understood process, so defects of signal transduction are a poorly defined group of disorders. However, they may constitute a significant percentage of patients with abnormal secondary wave of aggregation and decreased granule release in which alpha and dense granules are not deficient. These disorders include defects of the platelet cyclooxygenase, and phospholipase C pathways, including phospholipase C activation, calcium mobilization, pleckstrin phosphorylation, and tyrosine phosphorylation. Abnormalities of the GTP-binding proteins that link surface receptors and intracellular enzymes, such as Gaq, Gai2, and Gas, also have been described.
In general, patients with signaling defects show decreased primary aggregation and decreased granule release without granule deficiency. Identification of the exact defect requires detailed biochemical and genetic studies, not available in most laboratories.
Disorders of platelet procoagulant activity (Scott syndrome)
Scott syndrome is a rare congenital platelet functional disorder. It is due to defective “flip” of anionic phospholipids, such as phosphatidyl serine, to the outer table of the platelet membrane during platelet activation. Patients with this syndrome will have normal platelet aggregation studies but abnormal platelet procoagulant activity and decreased microparticle formation.
Myosin heavy chain disorders
Mutations in the MYH9 gene encoding for the nonmuscle myosin heavy chain IIA result in a spectrum of macrothrombocytopenia disorders with neutrophil inclusions referred to as MYH9 disorders. Clinically, MYH9 disorders are characterized by a mild bleeding diathesis with large platelets and neutrophils with Dohle-like inclusions, a ribonucleoprotein complex composed of aggregates of myosin IIA protein MYH9 mRNA, and clusters of ribosomes. There are variable clinical findings of nephritis, sensorineural hearing loss, and cataracts. There is a genotype-phenotype correlation, with MYH9 mutations in the motor head domain of myosin IIA having severe macrothrombocytopenia and deafness, nephritis, and cataracts, while mutations in the tail domain have mild macrothrombocytopenia and low risk of nonhematologic complications. Immunostains of the peripheral smear with an antibody against human nonmuscle myosin heavy chain IIA will show abnormal subcellular localization associated with the neutrophil inclusions. Platelet aggregation and platelet function screening studies are normal, attesting to the increased functionality of the larger platelets. 6
LABORATORY TESTING FOR ANTIPLATELET DRUG EFFECT
Antiplatelet therapy with aspirin and clopidogrel (Plavix®) decreases the incidence of secondary myocardial infarction, stroke, peripheral artery disease, and cardiovascular death in vascular patients. Aspirin inhibits platelet function by acetylating cytoplasmic cyclooxygenase, and clopidogrel (Plavix) blocks the membrane adenosine diphosphate (ADP) receptor P2Y12. While they are effective as a class of therapy, many patients continue to experience adverse events despite therapy with these agents.
Aspirin inhibits platelet aggregation to the agonist arachidonic acid, and clopidogrel inhibits aggregation to ADP. Many patients have been described with a suboptimal response to these drugs, with demonstration of residual platelet reactivity (RPR), sometimes called drug “resistance.” There have been many laboratory techniques devised to measure the effect of antiplatelet drugs, including light transmittance aggregometry, whole blood impedance aggregometry, PFA-100, Accumetrics VerifyNow, urine-based 11-dehydro thromboxane B2, and VASP phosphorylation. However, there is little standardization of functional testing for RPR. Meta-analyses have demonstrated suboptimal response to aspirin in 27.1% (95% CI 21.5-32.6) of patients, with a suboptimal response to clopidogrel in 22% (95% CI 15-29) of cases. There is little agreement among the laboratory tests, with only 2% of subjects identified as suboptimal response by all.
Suboptimal response is more than a laboratory phenomenon, as meta-analyses have correlated aspirin clopidogrel RPR in vascular disease patients to increased risk of stroke, acute myocardial infarction, and cardiovascular death. Indeed, RPR has been consistently linked to adverse outcomes despite poor laboratory test agreement.
Testing strategies for clopidogrel have included both functional and genomic assays. The best documented genomic association for clopidogrel is CYP2C19 *2, which leads to poor clopidogrel metabolism to the active form and has been shown to be associated with RPR and major adverse cardiovascular events.7 A genomic profile may identify some proportion of risk, but this may change over time with the clinical scenario and other drug therapy, so platelet function testing is likely to provide more complete therapeutic information. At present, there is little evidence to guide therapy in patients with RPR. Current efforts are directed toward the possibilities of dual antiplatelet drug therapy; alternative P2Y12 antagonists, such as prasugrel and ticagrelor; or the addition of antithrombotic drugs, such as dipyridamole or cilostazole. Answers and recommendations for an appropriate testing and therapeutic regimen are anticipated upon completion of clinical trials.3,7
The adequate function of platelets is crucial for normal hemostasis. Bleeding disorders can be associated with platelet dysfunction or abnormal platelet counts. The laboratory evaluation of all of these disorders should start with measurement of the platelet count and a platelet function screening test. A careful and thorough patient history is essential, especially in exclusion of drugs that can interfere with platelet function. Use of appropriate platelet functional tests and other ancillary tests may be needed to positively diagnose many platelet disorders. Repeat testing is often necessary due to variability of platelet functional test results. The laboratory may also be involved in the adequacy assessment of antiplatelet drugs; however, a lack of gold-standard tests and guidance regarding treatment alterations requires further study before clinical implementation.
Dr. Kottke-Marchant is Chair, Pathology and Laboratory Medicine Institute and Section Head of Hemostasis and Thrombosis in the Clinical Pathology Department at Cleveland Clinic.
She also is Chair, Department of Pathology at the Cleveland Clinic Lerner College of Medicine at Case Western Reserve University and Adjunct Professor,
Dept. of Biomedical Engineering, at Case Western Reserve University. Her current research is on hemostasis, particularly genetic determinants of aspirin and clopidogrel response.
She is the author and co-author of over 250 articles, abstracts, and chapters, and is Past President of the International Society for Laboratory Hematology (ISLH).
References
- Harrison P, Mumford A. Screening tests platelet function: Update on their appropriate uses for diagnostic testing. Semin Thromb Hemost. 2009;35:150-157.
- Christie DJ, Avari T, Carrington LR, et al. Platelet function testing by aggregometry: Approved Guideline. CLSI Guideline H58-A (ISBN 1-56238-683-2). Clinical and Laboratory Standards Institute, Wayne, PA. 2008.
- Hayward CP, Moffat KA, Rab A, et al. Development of North American consensus guidelines for medical laboratories that perform and interpret platelet function testing using light transmission aggregometry. Am J Clin Pathol. 2010;134:955-963.
- Kottke-Marchant K. Platelet Testing (Chapt. 7), Platelet Disorders (Chapt. 14) and Antiplatelet Agents (Chapt. 25). In: Kottke-Marchant K, ed. An Algorithmic Approach to Hemostasis Testing. Northfield, IL: CAP Press, 2008.
- Nurden AT, Nurden P. Congenital disorders associated with platelet dysfunction. Thromb Haemost. 2008;99:253-263.
- Althus K, Greinacher A. MYH9-related platelet disorders. Semin Thromb Hemost. 2009;35:189-203.
- Ahmad T, Voora D, Becker RC. The pharmacogenetics of antiplatelet agents: towards personalized therapy? Nat Rev Cardiol. 2011;8:560-571.
