Platelet Function Assay, In Vitro


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Subject: Platelet Function Assay, In Vitro

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  • Light transmittance aggregometry (LTA) is based on platelet aggregation to ADP and other agonists and has traditionally been the most commonly used ex vivo assessment of platelet inhibition and activity. Due to the complexity of the assay and lack of standardization between institutions, LTA is a suboptimal test to monitor platelet activity in a clinical setting, and its use is increasingly limited to clinical trials.

  • Point-of-care tests for platelet reactivity have become available for the P2Y12 assay. The ADP-P2Y12 receptor plays a central role in the activation of platelets mediated through sustained activation of the GP IIb/IIIa platelet receptor. The assay measures ADP-induced platelet activation in citrated whole blood and reports results in P2Y12 reaction units (PRU). Initial versions of the assay also reported platelet reactivity to a thrombin receptor activation protein, which served as a measure of maximal platelet activation and reported a percentage of platelet inhibition. Increasingly, cardiovascular literature is defining platelet reactivity based on PRU alone and has defined a PRU of 235–240 as a threshold for increased ischemic events. Values at or above this level in patients on antiplatelet medications are referred to as “high on-treatment platelet reactivity” and may represent suboptimal dosing or intrinsic medication resistance.

  • Other in vitro assays involve (1) measuring high shear-dependent platelet function across collagen/ADP-based cartridges. It requires only 0.8 mL of blood, and its results are obtained in a few minutes. It can be performed in the laboratory or as a POC test but lacks the reproducibility of other modalities. (2) Flow cytometric analysis of the vasodilator-stimulated phosphoprotein (VASP), an intracellular marker of the residual P2Y12 reactor activity and has correlated with ischemic risk in clinical trials.


  • In vitro platelet function assays are increasingly used in cardiovascular medicine to assess adequate inhibition to reduce ischemic events, or conversely, when platelet inhibition is low enough after cessation of antiplatelet medications to allow for invasive procedures that carry substantial bleeding risk (i.e., noncardiac procedures).

  • Although no absolute consensus for a definition of a high-on treatment threshold for platelet activity exists, it is generally accepted as (1) >235–240 PRU by VerifyNow P2Y12 assay, (2) PRI >50% by VASP-P analysis, (3) >46% maximal 5 μmol/l ADP-induced aggregation, and (4) >468 arbitrary aggregation units/minute in multiplate analyzer.

  • Functional platelet assays are also utilized for

    • von Willebrand disease types 1 (results may be inconclusive in mild type 1), 2A, 2B, 2M, and 3

    • Severe functional platelet defects

    • Rapid preoperative evaluation of patients with a bleeding history

    • Useful in detecting the effect of therapy with DDAVP (desmopressin acetate)

    • To detect improved hemostasis after platelet transfusions


  • In vitro assays often do not detect mild platelet abnormalities.

  • The in vitro results have a good negative (rule out) predictive value in cases with low or intermediate suspicion for a hemostatic defect. If, however, the results with the in vitro assay are negative, but the clinical suspicion of a hemostatic defect is strong, more definitive studies are recommended (platelet aggregation assays or vWF panels [see p. 454]).

  • If the results are positive, additional studies (platelet aggregation and/or vWF panels) are recommended for a definitive diagnosis.

  • Several factors are known to effect in vitro platelet assays such as hematocrit and white cell count. Correction factors may need to be applied to ensure correct interpretation.

Suggested Readings

Bonello  L, Tantry  U, Marcucci  R Consensus and future directions on the definition of high on-treatment platelet reactivity to adenosine diphosphate. J Am Coll Cardiol.  2010;56:919–933.
Kakouros  N, Kickler  TS Hematocrit alters VerifyNow P2Y12 assay results independently of intrinsic platelet reactivity and clopidogrel responsiveness. J Thromb Haemost.  2013;1:1–9.