Platelet Function Tests

Platelet function tests assess how platelets adhere, release granule contents and aggregate in response to agonists. They are used when bleeding is out of proportion to the platelet count, when the platelet count, PT, aPTT and fibrinogen are normal, and when the effect of antiplatelet drugs must be assessed.

The main methods are light transmission aggregometry (the reference method), lumi aggregometry with ATP release, the platelet function analyser closure time, whole blood impedance aggregometry, flow cytometry for glycoproteins and activation markers, point-of-care P2Y12 and aspirin reactivity assays, and electron microscopy.

Inherited platelet function disorders are uncommon and usually mild, apart from Glanzmann thrombasthenia and Bernard-Soulier syndrome. Acquired dysfunction from aspirin, other antiplatelet drugs, non-steroidal anti-inflammatory drugs, uraemia, liver disease, myeloid neoplasms, paraproteinaemia and cardiopulmonary bypass is far more frequent. Light transmission aggregometry is labour intensive and operator dependent, and abnormal results are interpreted with the clinical history and the pattern of agonist responses, not a single value.

Indications.

• Lifelong mucocutaneous bleeding (epistaxis, menorrhagia, easy bruising, bleeding after dental procedures) with a normal screen and normal von Willebrand assays.
• A family history of a bleeding disorder.
• Unexplained bleeding with antiplatelet therapy.
• Large platelets on the film.
• Preoperative assessment when the history is suggestive.
• Assessment of response to P2Y12 inhibitors in selected patients.

Specimen.

• Citrated blood (3.2% sodium citrate, 9 parts blood to 1 part citrate), collected by a clean, atraumatic venepuncture with a needle of 21 gauge or wider. The first tube is discarded when drawing several samples.
• Kept at room temperature. Never refrigerate, since cooling activates platelets.
• Tested between 30 minutes and 4 hours after collection.
• No aspirin for 7 to 10 days, no non-steroidal anti-inflammatory drugs for 3 days, and no P2Y12 inhibitor for 7 to 14 days when the test is for diagnosis of an inherited disorder. Caffeine, alcohol, fatty meals and some foods and herbal products (garlic, ginger, ginkgo, fish oil) should be avoided on the morning of testing.
• A platelet count of at least 100 x10⁹/L is needed for reliable aggregometry. Interpretation is limited under about 50 x10⁹/L.

Light transmission aggregometry. Platelet rich plasma is prepared by slow centrifugation and platelet poor plasma by fast centrifugation, which sets the 100% light transmission baseline. Agonists are added with stirring at 37 °C and the rise in light transmission is recorded for 5 to 10 minutes. Results are reported as maximum aggregation (percentage), with shape change, lag phase, slope and the presence of a secondary wave. Typical agonists and final concentrations are:

• ADP 2.5 to 10 micromol/L, with low doses showing a primary and secondary wave.
• Adrenaline 5 to 10 micromol/L.
• Collagen 1 to 5 microg/mL.
• Arachidonic acid 1 to 1.5 mmol/L.
• Ristocetin 1.2 mg/mL (high dose) and 0.5 to 0.7 mg/mL (low dose).
• Thrombin receptor activating peptide, and the thromboxane mimetic U46619.

Normal maximum aggregation with strong agonists is usually over 60 to 70%. Reference ranges are method specific and are set locally from healthy controls.

Lumi aggregometry. Luciferin luciferase detects ATP released from dense granules at the same time as aggregation, so storage pool disorders can be separated from release defects.

Platelet function analyser closure time. Citrated whole blood is aspirated at a high shear rate through an aperture in a membrane coated with collagen and adrenaline or collagen and ADP. Closure time is the time for the aperture to occlude. Typical ranges are about 80 to 160 seconds for collagen and adrenaline, and about 60 to 120 seconds for collagen and ADP, reagent specific.

It is prolonged by von Willebrand disease, severe platelet function defects, aspirin (collagen and adrenaline cartridge only), a platelet count under about 100 x10⁹/L and a haematocrit under about 0.30. It is a poor screen for mild disorders, and a normal result does not exclude them.

Flow cytometry. Surface glycoproteins (GPIIb/IIIa as CD41 and CD61, GPIb-IX-V as CD42a and CD42b), activation markers (P-selectin as CD62P, CD63), dense granule uptake of mepacrine, and the vasodilator stimulated phosphoprotein phosphorylation index as a measure of P2Y12 inhibition.

Point-of-care reactivity assays. Whole blood optical or impedance assays report P2Y12 reaction units and aspirin reaction units. High on treatment reactivity is conventionally over about 208 P2Y12 reaction units and over about 550 aspirin reaction units. Cut offs are assay specific, and routine monitoring does not improve outcome in most patients.

Differentiating pre-analytical drug effect. Many apparent defects reflect recent aspirin or non-steroidal drug use. A repeat test after a clean drug free interval, with a history confirmed by drug assay or urinary thromboxane where needed, separates this from inherited disease.

Critical findings and findings needing immediate action.

• Absent aggregation consistent with Glanzmann thrombasthenia in a patient who needs surgery or has active bleeding needs urgent haematology planning. Platelet transfusion risks alloimmunisation against GPIIb/IIIa, so human leucocyte antigen matched platelets, recombinant activated factor VII or antifibrinolytic therapy are considered.
• Bernard-Soulier syndrome with bleeding at delivery or surgery needs the same planning.
• Any suspected inherited disorder before a planned procedure needs specialist review in advance.

Interferences and pitfalls.

• Platelet activation during collection, cooling, delayed testing, traumatic venepuncture and underfilled tubes cause false abnormal results.
• Lipaemic or haemolysed samples distort the optical signal.
• A low platelet count, or an abnormal platelet size, limits reliability.
• Concurrent drugs (aspirin, non-steroidal anti-inflammatory drugs, selective serotonin reuptake inhibitors, some antibiotics, antihistamines, tricyclic antidepressants, valproate) lower aggregation.
• Healthy people can show reduced response to adrenaline or ADP without bleeding, so one weak agonist response is not diagnostic.
• Methods and reagents differ between laboratories, and the reference ranges are local.
• Closure time is normal in many mild disorders, and cannot exclude them.
• Responses can be reduced by anaemia, and by a haematocrit over 0.50 in whole blood methods.

Special groups.

• Neonates: platelets are hyporeactive to several agonists in the first weeks, and results need age specific interpretation. Testing needs small volumes and is difficult.
• Children: the volume of blood needed is large, so flow cytometry and genetic testing are often used first.
• Pregnancy: aggregation is largely normal or increased, and ristocetin response rises with von Willebrand factor.
• Chronic kidney disease: uraemic platelet dysfunction improves with dialysis, desmopressin and correction of anaemia.
• Older adults: usually on antiplatelet drugs, so the drug history is essential.

Follow up tests.

• Full blood count and film, and mean platelet volume.
• Von Willebrand screen and clotting screen.
• Flow cytometry for platelet glycoproteins, and electron microscopy of platelets.
• Genetic panel (ITGA2B, ITGB3, GP1BA, GP1BB, GP9, MYH9, RUNX1, NBEAL2, HPS genes).
• Urea and creatinine, liver function tests, and serum protein electrophoresis.
• Bone marrow examination when a neoplasm is suspected.