Vasodilator-stimulated phosphoprotein-phosphorylation assay in patients on clopidogrel: Does standardisation matter?

The vasodilator-stimulated phosphoprotein-phosphorylation (VASP-P) flow-cytometric assay is mainly used in clinical trials to measure thienopyridine effects. However, there are remarkable differences in the reported optimal cut-offs, ranging from 48-61% platelet reactivity index (PRI). We therefore investigated whether a lack of standardisation might explain the differences in the cut-offs. We measured VASP-P in 62 individuals. PRI was calculated using the mean, geometric mean and median fluorescence intensities (Fl). Stability of the blood-samples (time-to-assay, 0-2 days) and stability of the processed samples (0-120 minutes) within the recommended time-span were tested. Time-to-assay significantly influenced the PRI (p<0.001): the PRI from mean Fl after two days was lower compared to values on day 1 (52 +/- 22.9 vs. 57.7 +/- 24.1, p<0.001). The PRI from the geometric mean Fl after two days was lower compared to day 0 as well as day 1 (51.3 +/- 23 vs. 58.2 +/- 24.2 and vs. 59.1 +/- 23.7, both p<0.001). The PRI from median Fl was stable over time (day 0: 59.1 +/- 25%, day 1: 59.7 +/- 24.1% and day 2: 56.4 +/- 23.9%, all p=ns). Furthermore, the lag time of the processed samples significantly altered the PRI (all p<0.001) with a maximum difference for PRI based on geometric mean Fl after 90 minutes compared to baseline (Delta=3.92%PRI, p<0.001). The differences in the reported cut-offs might be explained by a lack of standardisation. More precise standardisation is inevitable, as the PRI significantly depends on the method of calculation, the time-to-assay as well as on the lag time after processing. Tolerably stable results were obtained for the PRI from the median Fl.