Platelet Function Tests

Mr. Topesh Patle

Introduction 

Platelets, also known as thrombocytes, are small, anucleate cell fragments derived from megakaryocytes in the bone marrow. They play a crucial role in hemostasis, which includes:

  1. Adhesion: Platelets adhere to exposed subendothelial collagen at sites of vascular injury via glycoprotein (GP) receptors (e.g., GP Ib-IX-V binding to von Willebrand factor).
  2. Activation: Upon adhesion, platelets become activated, undergoing morphological changes and releasing granule contents (e.g., ADP, serotonin, thromboxane A2) that amplify the hemostatic response.
  3. Aggregation: Platelets aggregate via GP IIb/IIIa receptors, which bind fibrinogen and form platelet plugs.
  4. Secretion: Activated platelets release procoagulant substances that promote fibrin formation and clot stabilization.

Platelet function can be impaired in inherited or acquired conditions, leading to bleeding disorders or thrombosis. Platelet function tests help diagnose such abnormalities and guide treatment.

Platelet Function Tests

Platelet Aggregometry

Principle:

  • Platelet aggregometry measures platelet aggregation in response to specific agonists (e.g., ADP, collagen, epinephrine, arachidonic acid).
  • Aggregation is monitored by light transmission through platelet-rich plasma (PRP) using a light transmission aggregometer (LTA).

Procedure:

  1. PRP is prepared by centrifugation of whole blood.
  2. Agonists are added to PRP, and the degree of light transmission is measured.
  3. Aggregation curves are generated to assess platelet response.

Clinical Interpretation:

  • Normal Response: Platelets aggregate in response to all agonists.
  • Defective Aggregation:
    • Absent response to ADP, epinephrine, and collagen: Glanzmann thrombasthenia (deficiency of GP IIb/IIIa).
    • Absent response to ristocetin but a normal response to other agonists: Bernard-Soulier syndrome (deficiency of GP Ib).
    • Reduced response to arachidonic acid: Aspirin therapy or cyclooxygenase (COX) pathway defects.

Platelet Function Analyzer 

Principle:

  • PFA-100 simulates primary hemostasis by measuring the time it takes for platelets in whole blood to form a plug and occlude an aperture under high shear conditions.

Procedure:

  1. Whole blood is passed through a capillary coated with collagen/ADP or collagen/epinephrine.
  2. The closure time (CT) is recorded, representing the time required to occlude the aperture.

Clinical Interpretation:

  • Prolonged CT:
    • Collagen/epinephrine prolonged, collagen/ADP normal: Aspirin therapy.
    • Both prolonged: von Willebrand disease, platelet dysfunction, or thrombocytopenia.
    • Normal CT: Normal platelet function.

Bleeding Time (BT)

Principle:

  • Bleeding time measures the time for bleeding to stop after a standardized incision on the forearm. It reflects platelet adhesion and aggregation.

Procedure:

  1. A small incision is made using a lancet.
  2. The time until bleeding ceases is recorded.

Clinical Interpretation:

  • Prolonged BT:
    • Von Willebrand disease.
    • Platelet dysfunction (e.g., uremia, aspirin use).
    • Thrombocytopenia.
  • Limitations: BT is operator-dependent and has largely been replaced by modern tests.

Ristocetin Cofactor Assay

Principle:

  • Measures the ability of von Willebrand factor (vWF) to mediate platelet agglutination in the presence of ristocetin.

Procedure:

  1. Platelet-rich plasma is mixed with ristocetin.
  2. Aggregation is measured using an aggregometer.

Clinical Interpretation:

  • Reduced Agglutination:
    • Von Willebrand disease.
    • Bernard-Soulier syndrome.

Flow Cytometry

Principle:

  • Flow cytometry detects platelet surface glycoproteins, activation markers, and granule secretion using fluorescently labeled antibodies.

Procedure:

  1. Platelets are incubated with specific antibodies against GP IIb/IIIa, GP Ib, P-selectin, or CD markers.
  2. Samples are analyzed using a flow cytometer.

Clinical Interpretation:

  • Reduced Expression:
    • GP IIb/IIIa deficiency: Glanzmann thrombasthenia.
    • GP Ib deficiency: Bernard-Soulier syndrome.
  • Increased Activation Markers:
    • Thrombotic conditions (e.g., thrombosis, myocardial infarction).

VerifyNow

Principle:

  • A point-of-care test that measures platelet aggregation in response to specific agonists (e.g., ADP, arachidonic acid).

Procedure:

  1. Whole blood is exposed to agonists in a cartridge.
  2. Optical density changes quantify platelet aggregation.

Clinical Interpretation:

  • Normal Response: Effective platelet aggregation.
  • Reduced Response:
    • Aspirin therapy (reduced response to arachidonic acid).
    • Clopidogrel therapy (reduced response to ADP).

Thromboelastography (TEG)

Principle:

  • TEG evaluates the dynamics of clot formation, stabilization, and dissolution in whole blood.

Procedure:

  1. Whole blood is placed in a cup, and a pin is inserted to measure clot formation.
  2. Parameters include clot strength (MA), reaction time (R), and clot lysis.

Clinical Interpretation:

  • Low MA: Platelet dysfunction or thrombocytopenia.
  • Prolonged R: Coagulation factor deficiency or anticoagulant therapy.

Lumi-Aggregometry

Principle:

  • Measures platelet aggregation and secretion of dense granules by detecting ATP release using luciferase.

Procedure:

  1. PRP is stimulated with agonists.
  2. ATP release is measured as luminescence.

Clinical Interpretation:

  • Reduced Secretion:
    • Storage pool defects.
    • Secretion defects.

Electron Microscopy

Principle:

  • Direct visualization of platelet morphology and ultrastructure.

Procedure:

  • Platelets are examined using transmission electron microscopy (TEM).

Clinical Interpretation:

  • Identifies structural abnormalities in inherited platelet disorders (e.g., dense granule deficiency in Hermansky-Pudlak syndrome).

Clinical Utility of Platelet Function Tests

  1. Inherited Platelet Disorders:
    • Glanzmann thrombasthenia (GP IIb/IIIa deficiency).
    • Bernard-Soulier syndrome (GP Ib deficiency).
    • Storage pool disorders (e.g., gray platelet syndrome, dense granule deficiency).
  2. Acquired Platelet Dysfunction:
    • Aspirin or antiplatelet drug therapy (e.g., clopidogrel).
    • Uremia.
    • Liver disease.
  3. Thrombotic Conditions:
    • Essential thrombocythemia.
    • Myeloproliferative disorders.
  4. Monitoring Antiplatelet Therapy:
    • Assess response to drugs like aspirin, clopidogrel, or ticagrelor.
  5. Bleeding Disorders:
    • Von Willebrand disease.
    • Disseminated intravascular coagulation (DIC).

Limitations of Platelet Function Tests

  1. Operator-Dependent Variability:
    • Tests like bleeding time are influenced by technique and are less reproducible.
  2. Environmental Factors:
    • Aggregometry is sensitive to temperature, pH, and blood handling.
  3. Specificity:
    • Some tests lack specificity for identifying the exact cause of dysfunction.
  4. Cost and Complexity:
    • Advanced tests like flow cytometry and electron microscopy are expensive and require specialized equipment.

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