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Hematology

Platelet Function

Overview of Platelet Function Testing

  • Definition: Platelet function tests are a group of laboratory assays used to evaluate the ability of platelets to perform their role in primary hemostasis (forming the initial platelet plug)
  • Purpose:
    • Diagnosis of Platelet Disorders: Helps identify inherited or acquired disorders of platelet function
    • Evaluation of Unexplained Bleeding: Used to investigate patients with a personal or family history of mucocutaneous bleeding (e.g., nosebleeds, easy bruising, prolonged bleeding after minor cuts)
    • Assessment of Antiplatelet Therapy: Used to monitor the effects of antiplatelet medications (e.g., aspirin, clopidogrel)
    • Preoperative Assessment: Helps assess the risk of bleeding before surgery
  • Types of Platelet Function Tests:
    • Screening Tests:
      • Platelet Function Analyzer (PFA)
    • Confirmatory Tests:
      • Platelet Aggregation Studies
      • Flow Cytometry Assays
      • Platelet Secretion Assays
      • Genetic Testing

Platelet Function Analyzer (PFA)

  • Principle:

    • The PFA-100 or PFA-200 systems are in vitro assays that simulate high-shear conditions found in small blood vessels
    • Whole blood is aspirated through a small aperture (hole) in a cartridge coated with a platelet agonist (collagen/epinephrine or collagen/ADP)
    • As the blood passes through the aperture, platelets adhere to the collagen and are activated by the agonist
    • The platelets aggregate and eventually occlude the aperture, stopping the flow of blood
    • The time it takes for the aperture to become occluded is measured and reported as the Closure Time (CT)

  • Key Components of the PFA System:

    • Instrumentation:
      • PFA-100 or PFA-200 analyzer
    • Cartridges:
      • Collagen/Epinephrine (CEPI) Cartridge: Coated with collagen and epinephrine; primarily detects aspirin-related platelet dysfunction
      • Collagen/ADP (CADP) Cartridge: Coated with collagen and ADP; detects a broader range of platelet function defects
    • Quality Control Materials:
      • Normal controls to verify instrument performance

  • Procedure:

    1. Sample Collection:
      • Collect whole blood in a sodium citrate (light blue top) tube
      • Ensure that the tube is filled to the correct volume to maintain the proper blood-to-anticoagulant ratio
    2. Sample Preparation:
      • Mix the blood sample gently but thoroughly
      • Test the sample within the specified timeframe (usually within 2-4 hours of collection)
      • Ensure that the hematocrit and platelet count are within the acceptable range for the instrument
    3. Cartridge Insertion:
      • Insert the appropriate PFA cartridge (CEPI or CADP) into the analyzer
    4. Sample Aspiration:
      • Aspirate a small amount of the whole blood sample into the analyzer
    5. Test Initiation:
      • Start the test and allow the instrument to perform the analysis
      • The analyzer will measure the time it takes for the aperture to become occluded (Closure Time)
    6. Quality Control:
      • Run control materials at regular intervals to verify instrument performance
      • Document all QC results
    7. Results:
      • The instrument reports the Closure Time (CT) in seconds for both the CEPI and CADP cartridges

  • Reporting and Interpretation:

    • Closure Time (CT):
      • Epinephrine Closure Time (Epi-CT):
      • Reference Range: Varies depending on the laboratory and the PFA instrument (e.g., 84-169 seconds)
      • Prolonged Epi-CT: Suggests aspirin-related platelet dysfunction or a severe platelet defect
      • ADP Closure Time (ADP-CT):
      • Reference Range: Varies depending on the laboratory and the PFA instrument (e.g., 71-118 seconds)
      • Prolonged ADP-CT: Suggests a platelet function defect not related to aspirin (e.g., P2Y12 inhibitors, von Willebrand disease, Glanzmann thrombasthenia)

Interpreting PFA Results:

  • What are the normal results?
    • For the CEPI test - ~84-169 Seconds
    • For the CADP test - ~71-118 Seconds
      • These ranges can vary depending on institution, so please note your institution’s specific ranges!
  • What if they are higher than the normal range?
    • This could indicate that a patient has been taking platelet inhibitors such as asprin
    • They could have Von Willebrand Disease
    • They may have Glanzmann Thrombasthenia

Factors Affecting PFA Results

  • Pre-Analytical Variables:

    • Sample Collection: Traumatic venipuncture or use of a small-gauge needle can activate platelets and affect results
    • Incorrect Blood-to-Anticoagulant Ratio: Underfilling or overfilling the collection tube can alter the test results
    • Delayed Testing: Platelet function can change over time, so the test should be performed within the specified timeframe (usually 2-4 hours of collection)
    • Storage Temperature: Improper storage temperatures can affect platelet function

  • Analytical Variables:

    • Instrument Malfunction: Ensure proper calibration and maintenance of the PFA instrument
    • Reagent Problems: Use fresh, properly stored cartridges
    • Hematocrit: Hematocrit values outside the specified range can affect the closure time
      • Low Hematocrit: Can prolong the closure time
      • High Hematocrit: Can shorten the closure time
    • Platelet Count: Platelet counts outside the specified range can affect the closure time

  • Patient-Related Variables:

    • Medications:
      • Aspirin and NSAIDs: Inhibit cyclooxygenase (COX) and impair thromboxane A2 production, leading to prolonged Epi-CT
      • P2Y12 Inhibitors: Clopidogrel, prasugrel, and ticagrelor block the ADP receptor on platelets, leading to prolonged ADP-CT and Epi-CT
    • Underlying Medical Conditions:
      • von Willebrand Disease (vWD): Deficiency or dysfunction of vWF can impair platelet adhesion and prolong closure times
      • Thrombocytopenia: Low platelet count can affect closure times

Troubleshooting Erroneous Results

  • If the PFA results are unexpected or inconsistent with the patient’s clinical presentation:

    • Check the sample for clots or hemolysis
    • Verify that the hematocrit and platelet count are within the acceptable range for the instrument
    • Repeat the test on a fresh sample
    • Review the patient’s medication list and medical history
    • Consider performing platelet aggregation studies or other specialized tests

Reflex Testing

  • If the PFA results are abnormal, additional testing may be performed to:

    • Investigate potential underlying causes of platelet dysfunction
      • Platelet Aggregation Studies: To assess platelet aggregation in response to various agonists (ADP, collagen, epinephrine, thrombin, arachidonic acid, ristocetin)
      • Flow Cytometry: To measure platelet surface markers and assess platelet activation
      • Bleeding Time: Seldom performed now a days

Key Terms

  • Platelet Function Analyzer (PFA): An in vitro assay that measures platelet function under high shear stress
  • Closure Time (CT): The time it takes for the aperture in the PFA cartridge to become occluded by a platelet plug
  • Epinephrine Closure Time (Epi-CT): Closure time using a cartridge coated with collagen and epinephrine
  • ADP Closure Time (ADP-CT): Closure time using a cartridge coated with collagen and ADP
  • Platelet Aggregation: The clumping together of platelets
  • von Willebrand Disease (vWD): An inherited bleeding disorder caused by a deficiency or dysfunction of von Willebrand factor
  • Aspirin: A medication that inhibits platelet function by inhibiting cyclooxygenase (COX)
  • P2Y12 Inhibitors: Medications that block the ADP receptor on platelets, inhibiting platelet activation and aggregation