The Role of PFA-200 in Evaluating Platelet Dysfunction

‍PATHCHAT Edition No. 23
Please contact your local Ampath pathologist for more information.

Author: Dr. Rita Govender, MBChB (UND), DCH (SA), FC PATH (SA) CLIN HAEM (SA)

Overview of the Haemostatic System & Guide to Investigation

🔹 Haemostasis involves three stages:
1️⃣ Primary haemostasis – Formation of the platelet plug.
2️⃣ Secondary haemostasis – Stabilization of the platelet plug by cross-linked fibrin.
3️⃣ Fibrinolysis – Removal of the clot once healing is complete.

✅ Primary haemostasis is affected by:

• The vessel wall and subendothelial elements.
• Platelet function and count.
• Von Willebrand factor (vWF).

✅ Secondary haemostasis involves:

• Coagulation factor activation and fibrin stabilization.

✅ Increased fibrinolysis can lead to:

• Delayed bleeding tendencies.

Recommended First-Line Tests for Primary Haemostasis

🔹 1. Full Blood Count (FBC) & Peripheral Smear
✅ Assesses:

• Platelet count and morphology (size & granularity).
• Thrombocytosis (seen in myeloproliferative disorders).
• Thrombocytopaenia, which may cause bleeding.
• Haemoglobin levels (assessing blood loss).
• Presence of abnormal cells (suggesting haematological neoplasm).
• Signs of infection or disseminated intravascular coagulation (DIC).

🔹 2. Bleeding Time (In Vivo Test of Primary Haemostasis)
✅ Principle:

• A small incision is made on the forearm under 40 mmHg pressure.
• Time taken for bleeding to stop is recorded.

✅ Causes of Prolonged Bleeding Time (>8 min):

• Thrombocytopaenia (prolongs bleeding when platelet count is <75 × 10⁹/L).
• Platelet function defects, including:
  • Congenital causes (e.g., Glanzmann’s thrombasthenia, storage pool defects).
  • Acquired causes (e.g., drugs, uraemia, paraproteins, myelodysplastic syndromes).
• Von Willebrand Disease (VWD).
• Vascular abnormalities (e.g., Ehlers-Danlos Syndrome).
• Severe deficiencies of Factor V, Factor XI, or afibrinogenaemia.

📌 Bleeding time is affected by multiple variables (e.g., blood pressure, skin integrity) and lacks standardization. A normal bleeding time does not exclude platelet dysfunction.

PFA-200: A Modern Platelet Function Test

🔹 What is the PFA-200?

• The PFA-200 simulates platelet adhesion and aggregation under high shear stress.
• A citrated blood sample is passed through a membrane coated with platelet activators (collagen + epinephrine or collagen + ADP).
• The test measures closure time (CT), the time taken for the aperture to be occluded by a platelet plug.

✅ Advantages of PFA-200:

• Requires only a small volume of blood, making it suitable for paediatric patients.
• Unaffected by clotting factor deficiencies.
• More standardized than bleeding time tests.
• Not influenced by skin abnormalities, oedema, or fragile vessels.

Factors Affecting PFA-200 Test Results

🔹 Pre-Analytical Factors:

• Test must be performed within four hours of blood collection.
• Haematocrit <35% and platelet count <150 × 10⁹/L may affect closure times.
• Samples with HCT >50% and platelet count >500 × 10⁹/L have not been validated.
• Lipemia (fatty acids) and haemolysis can interfere with results.

Interpreting PFA-200 Results

✅ Normal Closure Times:

• Collagen/Epinephrine (Col/EPI): 82–150 seconds.
• Collagen/ADP (Col/ADP): 62–100 seconds.

✅ Abnormal Closure Times & Associated Disorders:

✔ Aspirin Effect:

• Col/EPI: Prolonged.
• Col/ADP: Normal.

✔ Von Willebrand Disease (VWD):

• Col/EPI: Prolonged.
• Col/ADP: Prolonged.

✔ Glanzmann’s Thrombasthenia (Platelet Aggregation Defect):

• Col/EPI: Prolonged.
• Col/ADP: Prolonged.

Further Testing for Abnormal PFA-200 Results

✅ If the PFA-200 test is abnormal, additional investigations are required, including:

• Complete review of medication history (including herbal, homeopathic, and occupational exposures).
• Von Willebrand Factor (vWF) antigen and multimer analysis.
• Platelet aggregometry (to assess platelet aggregation defects).
• Flow cytometry (to analyze glycoprotein receptors on platelets).
• Electron microscopy (to evaluate platelet ultrastructure).

📌 Storage Pool Disease and mild von Willebrand Disease (VWD) may have normal PFA-200 results despite platelet dysfunction.

Availability of PFA-200 Testing

🔹 Important Considerations:
✅ Testing requires a blood sample that is less than 4 hours old.
✅ Transport time should be considered before requesting the test.

Key Takeaways for Clinicians

✅ PFA-200 is a useful screening tool for platelet dysfunction and bleeding disorders.
✅ It provides a standardized alternative to the bleeding time test.
✅ Abnormal results should be followed up with detailed platelet function testing and vWF analysis.
✅ Testing requires careful pre-analytical handling and must be performed within 4 hours of collection.

References

1. Bain, B.J., Bates, I., Laffan, M.A., & Lewis, S.M. (2012). Dacie and Lewis Practical Haematology (11th edition). Churchill Livingstone.
2. British Committee for Standards in Haematology. (2011). Postgraduate Haematology: Guidelines for Laboratory Investigation of Inheritable Platelet Disorders.
3. Briggs C et al. (2014). Platelet function testing – PFA-100/200 vs. traditional aggregometry. Journal of Haematology, 165(2), 287–299.