Platelet function tests

When history and clinical features of the patient supports the suspicion of an IPD, and no abnormalities in plasma coagulation factors/VWD are found that could explain the bleeding tendency, platelet function tests should be ordered in a step wise fashion as described in [1,2].

Specific considerations for platelet function testing

A. Specific recommendations for patient guidance regarding infection, time period after smoking, fasting and exercise [3]

  • Patients should refrain from smoking ≥30 minutes before sampling.

  • Patients should abstain from caffeine ≥2 hours before sampling.

  • Patients should abstain from heavy exertion (≥24 hours before testing).

  • Samples should be taken fasting or after a light meal.

  • Reversible inhibitors (NSAIDs, Dipyramidole): stop ≥3 days before testing.

  • Irreversible inhibitors (ASA, thienopyridines such as clopidogrel, prasugrel, and ticlopidine): stop ≥10 days before sampling.

  • Selective serotonin reuptake inhibitors (SSRIs): stop ≥3 days before testing.

Platelet function might be influenced by surgery and pregnancy and results will be difficult to interpret. Abnormal results from these tests should thus, be repeated.

Platelet function tests performed on patients taking any of the drugs listed below should be repeated when not taking the substance for at least 10 days before sampling.

B. Recorded questionnaire on the avoidance of drugs affecting platelet function

We suggest that patients complete a questionnaire before blood sampling. For an example of medications see Appendix, Table 1.

C. Specific recommendations regarding sampling and procession of samples

  • Collect samples after a short rest (~15 minutes).

  • Use a standardized, atraumatic blood collection protocol with minimal stasis.

  • Use needles between 19 and 21 gauge to prevent vein trauma or reduced blood flow, leading to activated platelets.

  • The first 3-5 mL of blood should not be used for platelet function testing.

  • Most platelet function tests require the use of 105–109 mmol/L buffered trisodium citrate tubes.

  • The collection tubes must be filled completely to ensure the proper 9:1 ratio of blood to anticoagulant.

  • The collection tubes should be mixed gently immediately upon filling, do not subject to excessive agitation or mixing.

  • Maintain specimens at room temperature (18-24°C) only and not refrigerated or frozen.

  • Specimens should rest on the laboratory bench top for 30 minutes prior to platelet function testing.

  • Platelet function testing should be completed within 3-4 hours of collection.

First step platelet function tests

Blood smear (only in patients with thrombocytopenia)

  • To evaluate platelet size, giant platelets, grey appearance of platelets, red cell abnormalities and neutrophil inclusion bodies.

Light transmission aggregometry (LTA) in platelet-rich plasma (PRP)

  • Gold-standard test, but poorly standardized. We suggest that testing should be repeated on at least one occasion to ascertain a platelet dysfunction diagnosis. For details on recommended agonists and concentrations, see [3–5].

  • We recommend laboratories to assess the performance of new batches of agonists by comparison with a previous batch.

  • We recommend that the platelet count of PRP samples should NOT be adjusted to a standardized value with autologous PPP [4].

  • Platelet counts below 150×109/L in PRP or <75-100 in whole blood can severely influence responses to some agonists [4]. We recommend that results of such analyses should be interpreted with extreme caution.

Lumi-aggregometry [6]

  • Assessment of ATP/ADP release from platelet granules. May indicate deficiency of granules (storage pool disorder), or exocytosis pathway. For details on agonists see [7] .

Flow cytometry [8,9]

To investigate or confirm:

  • Glanzmann thrombasthenia (GT), with abnormalities in the fibrinogen receptor glycoprotein (GP) IIb/IIIa.

  • Bernard-Soulier Syndrome (BSS), with abnormalities in the VWF receptor GP Ib/IX/V.
    Note the possibility of an acquired blocking antibody against GP IIb/IIIa or GP Ib/IX.

Second-step tests

We suggest these tests may be performed only if initial testing is inconclusive, or the suspicion of a IPD persists despite normal results on first-step testing.

LTA with an expanded agonist panel [6]

Flow-cytometry with additional antibodies

  • Assessment of agonist-induced platelet activation and procoagulant platelets (to detect Scott syndrome, deficiency in phosphatidylserine exposure due to mutations in phospholipid scramblase) [8–10]. Additional surface antigen detection of GPIb/IIa, GPIV and GPVI may be considered.

Transmission electron microscopy (TEM)

  • For counting alpha- and dense granules and to detect structural abnormalities [11].