Comparison of a new ELISA assay with the flow cytometric assay for platelet vasodilator-associated stimulated phosphoprotein (VASP) phosphorylation in whole blood to assess P2Y12 inhibition

Abstract

Thienopyridines and other agents target the platelet P2Y(12) receptor and inhibit several platelet activities mediated by adenosine diphosphate (ADP). The measurement of vasodilator-associated stimulated phosphoprotein (VASP) phosphorylation, expressed as platelet reactivity index (PRI), mirrors the degree of P2Y(12) receptor inhibition and can detect the well-known variable response to clopidogrel. The commercially available VASP assay uses flow cytometry (FC) and requires that the test be run within 48 hours of blood collection. A new ELISA VASP assay offers the advantages of using more widely available technology and the potential to freeze and store samples before analysis. The objectives of the present study were to compare the performance of the ELISA and FC methods and to describe the relative flexibility of the ELISA-based assay. Human blood samples encompassing a wide range of levels of P2Y(12) blockade achieved in vitro by preincubation with P2Y(12) antagonists or in vivo from patients treated with clopidogrel were included, reflecting the wide spread of values reported in clinical studies. The correlation between the PRI measured by ELISA and FC was highly significant (r=0.95, p<0.001), (n=80). After the initial activation, samples were stable for at least four weeks when frozen (-20°C) prior to analysis by ELISA. Frozen samples from patients treated with clopidogrel appeared stable for up to nine weeks. Based on these results, the ELISA-based assay appears to provide a reliable and more flexible alternative to the FC method to determine P2Y(12) receptor blockade and may enable more extensive utilisation of the VASP assay in clinical studies.

Head-to-Head Comparison of Consensus-Recommended Platelet Function Tests to Assess P2Y12 Inhibition—Insights for Multi-Center Trials

“…We found their results to be strongly correlated. This conclusion is in agreement with previous studies that also found the assays to be highly concordant [10][11][12]. Indeed, Ding et al reported a correlation coefficient of 0.89 between the two VASP assays in healthy volunteers, which is very close to our coefficient (0.87).…”

The Pharmacokinetics and Pharmacodynamics of Prasugrel and Clopidogrel in Healthy Japanese Volunteers

“…The vasodilator‐stimulated phosphoprotein (VASP) phosphorylation state was determined using an enzyme‐linked immunosorbent assay (CY‐QUANT VASP/P2Y12; France) per the manufacturer's instructions 17. The platelet reactivity index (PRI) was calculated in the presence of PGE1 alone or PGE1 and ADP.…”

No association between on-treatment platelet reactivity and bleeding events following percutaneous coronary intervention and antiplatelet therapy: A post hoc analysis

“…PRU was determined as described in our prior report [9]. VASP-PRI was measured using a standardized flow cytometric assay as previously described [11]. Briefly, the PRI is the difference in VASP fluorescence intensity between resting (+prostaglandin E1 [PGE1]) and activated (+ADP) platelets, and is calculated from the median fluorescence intensity (MFI) of samples incubated with PGE1 alone or PGE1 and ADP using the following formula:…”