Study type: Blood collected in a lavender-top [EDTA] tube for platelet count; lavender-top [EDTA] or blue-top [3.2% sodium citrate] tube for platelet function test by aggregrometry; lavender-top [EDTA], pink-top [K₂EDTA] or yellow-top [ACD Solution B] tube for platelet receptor flow cytometry. The laboratory should be consulted regarding recommended collection containers and order of draw when multiple tubes will be drawn with platelet function studies; related body system: Circulatory/Hematopoietic and Immune systems. For the platelet count: the specimen should be mixed gently by inverting the tube 10 times. The specimen should be analyzed within 24 hr when stored at room temperature or within 48 hr if stored at refrigerated temperature. If it is anticipated the specimen will not be analyzed within 24 hr, two blood smears should be made immediately after the venipuncture and submitted with the blood sample. For platelet function studies: the laboratory should be consulted prior to specimen collection for special instructions regarding specimen stability and transportation instructions.

What Are Platelets and Why Are They Important?

Platelets are nonnucleated, cytoplasmic, round or oval disks formed by budding off of large, multinucleated cells (megakaryocytes) that move throughout the body via the circulatory system. Platelets have an essential function in coagulation, hemostasis, and blood thrombus formation. The three stages in the process of primary hemostasis include the three “A”s:

• Adhesion: Endothelial injuries initiate a vascular spasm that results in vasoconstriction in the damaged area. Shear stress is the frictional force that blood flow exerts on the blood vessel wall, a mechanical factor involved in circulation that influences the activation of platelets. Additionally, collagen in the endothelial extracellular matrix releases substances that cause circulating platelets to adhere to the damaged site. The adhered platelets form a temporary platelet plug, mediated by the binding of von Willebrand factor (vWF) to Gp Ib-IX-V receptors in the platelet membrane. A complex signaling process, very simply described here, occurs between hemodynamic shear forces, agonists, receptors, and other protein mediators (e.g., cytokines and chemokines). An agonist is a substance that binds to a specific receptor site to stimulate a specific response. ADP, a platelet agonist, is involved in the conformational changes in shape that platelets undergo as they release a number of procoagulant factors from storage granules, including thromboxane A2, a very potent platelet agonist.
• Activation is triggered by adhesion: The procoagulant factors enter the circulation and activate other platelets. Newly activated platelets also release factors to stimulate further platelet activation, and the cycle continues in a positive feedback loop. More and more activated platelets become adhered to collagen fibers on the damaged endothelium.
• Aggregation: The binding of fibrinogen to the activated platelet alphaIIbbeta3 receptor is a critical step in platelet aggregation. Activated platelets clump via extended pseudopods at the site of vessel injury. Tissue factor and Factor VIIa initiate the activation of coagulation factors that promotes the process of secondary hemostasis. Platelets can also aid in the initiation phase of coagulation by providing binding sites for prothrombin and Factor XI. The generation of thrombin during secondary hemostasis amplifies further platelet activation. Glycoprotein IIb/IIIa receptors on the activated platelets bind fibrinogen or vWF in cross-links, causing the aggregated platelets to form a stabilized plug. For additional information related to activation of the coagulation factors in secondary hemostasis, refer to the studies titled “Coagulation Factors” and “Fibrinogen.”

Evaluating Platelet Count

Thrombopoiesis or platelet production is reflected by the measurement of the immature platelet fraction (IPF). This parameter can be correlated to the total platelet count in the investigation of platelet disorders. A low platelet count with a low IPF can indicate a disorder of platelet production (e.g., drug toxicity, aplastic anemia, or bone marrow failure of another cause), whereas a low platelet count with an increased IPF might indicate platelet destruction or abnormally high platelet consumption (e.g., mechanical destruction, disseminated intravascular coagulation, idiopathic thrombocytopenic purpura [ITP], thrombotic thrombocytopenic purpura).

Thrombocytosis is an increase in platelet count. In reactive thrombocytosis, the increase is transient and short-lived, and it usually does not pose a health risk. One exception may be reactive thrombocytosis occurring after coronary bypass surgery. This circumstance has been identified as an important risk factor for postoperative infarction and thrombosis. The term thrombocythemia describes platelet increases associated with chronic myeloproliferative disorders.

Thrombocytopenia describes platelet counts of less than 140 × 10³/microL. Decreased platelet counts occur whenever the body’s need for platelets exceeds the rate of platelet production; this circumstance will arise if production rate decreases or platelet loss increases. Platelet counts may also be decreased in the presence of autoantibodies as seen in refractoriness to platelet transfusions, posttransfusion purpura, or neonatal alloimmune thrombocytopenia. For additional information regarding platelet antibodies, refer to the study titled, “Platelet Antibodies.” The severity of bleeding is related to platelet count as well as platelet function. Platelet counts can be within normal limits, but the patient may exhibit signs of internal bleeding; this circumstance usually indicates an anomaly in platelet function.

When abnormal platelet findings are flagged by an automated cell counter, the situation must be investigated; this usually begins with a review of a peripheral blood smear or a platelet estimate. Abnormally large or giant platelets may result in underestimation of automated counts by 30% to 50%. Giant platelets can be as large as a normal red blood cell (RBC). A normal RBC is 7–88 micrometers, and a normal platelet is 1.5–3 micrometers. Giant platelets are associated with relatively rare platelet disorders, e.g., inherited disorders such as Bernard-Soulier and May-Hegglin or a condition known as immune thrombocytopenia (previously referred to as immune thrombocytopenic purpura). Underestimation of the platelet count may also be caused by platelet clumping, which can be caused by a traumatic venipuncture or the presence of EDTA-dependent antibodies that interfere with GP IIb/IIIa platelet receptors. In both cases, the problem may be solved by recollecting the specimen. Additionally, in the case of clumping, the re-draw should be performed using a collection container with a different anticoagulant, e.g., sodium citrate (blue top). The laboratory’s platelet estimate procedure may include a mathematical calculation to approximate the platelet count from a platelet estimate as well as making an assessment of platelet characteristics that indicate a normal review:

• the number of platelets in each field is consistent
• platelet size and shape are fairly consistent from field to field
• the platelet estimate and automated platelet count correspond within an acceptable limit

The platelet count may also be affected by administration of heparin that is associated with two types of thrombocytopenia: Type I heparin-induced thrombocytopenia (HIT) is believed to occur as the result of an interaction between heparin and circulating platelets. Type I HIT causes a mild thrombocytopenia soon after administration of heparin, usually occurs in patients who have not been previously received heparin, and resolves in a few days whether the heparin therapy continues or is stopped. Type II HIT, believed to be the result of an immune-mediated response, also occurs in patients who have not been previously treated with heparin, begins later than type I HIT unless the patient is sensitized from a previous exposure, and can result in a 50% or greater decrease in platelet count. Functional assays for HIT II antibodies are available but not routinely requested. The risk of thrombosis is high with type II HIT, and administration of heparin should be stopped in patients who develop type II HIT.

Evaluating Platelet Size and Shape

Platelet size, reflected by mean platelet volume (MPV), and cellular age are inversely related; that is, younger platelets tend to be larger. An increase in MPV indicates an increase in platelet turnover. Therefore, in a healthy patient, the platelet count and MPV have an inverse relationship. Abnormal platelet size may also indicate the presence of a disorder. MPV and platelet distribution width are both increased in ITP. MPV is also increased in May-Hegglin anomaly, Bernard-Soulier syndrome, myeloproliferative disorders, hyperthyroidism, and pre-eclampsia. MPV is decreased in Wiskott-Aldrich syndrome, septic thrombocytopenia, and hypersplenism. Platelet size and shape may be evaluated by manually performing a “platelet estimate” as described in the previous section subtitled, “Evaluating Platelet Count.”

Platelet Function Tests

Platelet dysfunction can be inherited or acquired. Common causes of acquired platelet dysfunction include medications such as aspirin, clopidogrel, and nonsteroidal anti-inflammatory drugs or systemic disorders such as myeloproliferative neoplasms, systemic lupus erythematosus, and uremia. A combination of tests that evaluate platelet function (adhesion, aggregation, flow cytometry) and genetic testing can be used to identify the presence and type of dysfunction and to differentiate between inherited and acquired dysfunction. The bleeding time and clot retraction tests have largely been replaced by

• Platelet function tests that demonstrate platelet adhesion and aggregation when a blood sample is exposed to various agonists known to induce aggregation, e.g., ADP, arachidonic acid, collagen, epinephrine, ristocetin, thrombin, thromboxane) and
• Flow cytometry studies that determine the presence and quantity of platelet receptors and activation markers on the platelet surface.

A variety of laboratory methods and specimen types are employed to evaluate platelet aggregation; some methods require platelet-rich plasma, while others are performed using whole blood specimens. Many of the aggregation systems use natural platelet agonists and high shear flow conditions to simulate the normal physiological environment of circulating platelets. Platelet aggregation can be measured in a patient specimen by “time to closure” or from aggregometers using optical or impedance detection:

• Endpoint closure time (e.g., PFA-100®)—As aggregation takes place, the time to closure (blockage of an aperture in the test system) is measured.
• Optical (e.g., CHRONO-LOG®, PlateletWorks®, VerifyNow®)—As aggregation takes place, a decrease in optical density occurs, and the amount of light transmitted increases. The change in density creates an agonist-specific aggregation pattern and reaction time. A second form of optical aggregometry is lumi-aggregometry, which is a combination of spectrophotometric and fluorescent measurement.
• Impedance (e.g., CHRONO-LOG®, Multiplate® Analyzer)—A baseline measurement of electrical current between two electrodes is taken before the agonist is added. Once the agonist is added, platelets aggregate on both electrodes, reducing/impeding the magnitude of the current in direct proportion to the degree of aggregation.

Tests That Measure Response to Therapeutics, e.g., Aspirin, P2Y12 Inhibitors, and Glycoprotein (GP) IIb/IIIa Inhibitors

Antiplatelet drugs prevent platelets from clumping and forming clots. These tests are mainly used to identify patients who are nonresponders to drug therapy and therefore may not be good candidates to undertake a specific or combined approach to antiplatelet therapy. The use of platelet function testing to guide the dosing or withdrawal (e.g., establishing time frames for withholding before surgery) of therapeutics is not generally recommended at this time by the American Heart Association (AHA) or the Choosing Wisely Campaign (an initiative of the American Board of Internal Medicine Foundation) due to the lack of strongly supportive data that clearly identify its clinical utility in this regard. According to current AHA guidelines, platelet function testing may be considered for guidance on the timing of cardiac surgery in some patients (e.g., those who have recently received P2Y12 receptor inhibitors or who have ongoing dual antiplatelet therapy). There are current AHA recommendations for the timing of withdrawal from aspirin; guidelines may vary by provider and facility.

Currently, there are three classes of antiplatelet agents: aspirin (acetylsalicylic acid), P2Y12 inhibitors, and GP IIb/IIIa inhibitors.

Aspirin blocks production of thromboxane A2, thereby blocking platelet aggregation. The test uses an agonist to activate platelets in the patient sample. Platelet function is measured by the amount of activated platelet GP IIb/IIIa receptors that bind to fibrinogen-coated microparticles. If aspirin has had the expected antiplatelet effect, aggregation will be reduced.

Thienopyridines are a class of P2Y12 inhibitors (e.g., clopidrogrel [Plavix]), prasugrel (Effient), ticlopidine (Ticlid), and ticagrelor (Brilinta®) whose antiplatelet effect is achieved by inhibiting ADP-mediated platelet activation. P2Y12 inhibitors irreversibly inhibit the binding of ADP to the P2Y12 receptor on the platelet surface, which has the effect of also disrupting platelet degranulation and inhibiting other receptors down the line.

The GP IIb/IIIa inhibitors block platelet aggregation by preventing fibrinogen and vWF from binding to the IIb/IIIa platelet receptor, which disrupts the eventual formation of a stable clot.

Genetic Testing

Genetic test methods to identify drug-related defects in platelet function are mainly used to identify patients who are nonresponders to drug therapy and therefore may not be good candidates to undertake a specific or combined approach to antiplatelet therapy. The use of genetic testing to guide the dosing of antiplatelet therapeutics is not recommended at this time by the American Heart Association (AHA) or the Choosing Wisely Campaign due to the lack of strongly supportive data that clearly identify its clinical utility in this regard.

Next-generation sequencing is used to identify more than 30 genetic variants associated with significant defects in platelet function. Drugs such as clopidogrel (Plavix), abciximab (ReoPro), eptifibatide (Integrilin), and tirofiban block platelet receptor sites and inhibit platelet function. Aspirin also can affect platelet function by the irreversible inactivation of a crucial cyclooxygenase enzyme. Medications such as clopidogrel and aspirin are prescribed to prevent heart attack, stroke, and blockage of coronary stents. Studies have confirmed that up to 30% of patients receiving these medications may be nonresponsive.

The metabolism of many commonly prescribed medications is driven by the cytochrome P450 (CYP450) family of enzymes. Genetic variants can alter enzymatic activity that results in a spectrum of effects ranging from the total absence of drug metabolism to ultrafast metabolism. Impaired drug metabolism can prevent the intended therapeutic effect or even lead to serious adverse drug reactions. Poor metabolizers are at increased risk for drug-induced adverse effects due to accumulation of drug in the blood, while ultra-rapid metabolizers require a higher than normal dosage because the drug is metabolized over a shorter duration than intended. Other genetic phenotypes used to report CYP450 results are intermediate metabolizer and extensive metabolizer. CYP2C19 is a gene in the CYP450 family that metabolizes drugs such as clopidogrel. Genetic testing can be performed on blood samples submitted to a laboratory. Testing for the most common genetic variants of CYP2C19 is used to predict altered enzyme activity and anticipate the most effective therapeutic plan. The test method commonly used is polymerase chain reaction. Counseling and informed written consent are generally required for genetic testing.

Knowledge of genetics assists in identifying those who may benefit from additional education, risk assessment, and counseling. Genetics is the study and identification of genes, genetic mutations, and inheritance. For example, genetics provides some insight into the likelihood of inheriting a tendency to abnormally metabolize drugs. Some conditions are the result of mutations involving a single gene, whereas other conditions may involve multiple genes and/or multiple chromosomes. Further information regarding inheritance of genes can be found in the study titled “Genetic Testing.”

Platelet Transfusion Reaction—Related to the Therapeutic Use of Platelets in Some IgA-Deficient Individuals

IgA is primarily involved in immune health maintenance of the mucosa that lines the gastrointestinal, genitourinary, and respiratory systems. IgA deficiency is the most common deficiency of the five main immunoglobulin classes (IgA, IgD, IgE, IgG, and IgM). An IgA deficiency-mediated transfusion reaction is a relatively rare type of anaphylaxis. It has been observed when an IgA-deficient recipient is transfused with a platelet product containing anti-IgA antibodies. The reaction usually occurs within 1 hour of transfusion. A reaction does not occur in all patients with an IgA deficiency and can occur along a spectrum ranging from no symptoms or adverse reaction to full-blown, life-threatening anaphylaxis. Commonly transfused blood products, such as platelets, contain small amounts of plasma. Antibodies to IgA and other immunoglobulins are present in the plasma unless they are sourced from

• an autologous platelet-rich plasma donation (the donated product is collected from the patient and then administered to the patient in special applications, e.g., orthodontics, orthopedics, wound healing)
• a donation from an IgA antibody–deficient donor
• platelet units that have been specially treated with a saline wash and centrifugation process. Large/national blood centers (e.g., American Red Cross) maintain a registry of rare donors and may be able to access IgA-deficient donors.