Reproducibility and standardized reporting of the vasodilator-stimulated phosphoprotein phosphorylation assay

Abstract

The platelet vasodilator-stimulated phosphoprotein (VASP) phosphorylation test is a new platelet function assay used to determine responsiveness to clopidogrel. We investigated if a commercially available VASP assay has a good reproducibility when conducted with half or quarter of the recommended volume of reagents. Secondly, we hypothesized that the platelet reactivity indexes (PRI) in the VASP assay, calculated with three possible measurements of fluorescence intensities (FI): mean, geometric mean or median, are not equal. The VASP assay was performed in 280 patients on clopidogrel therapy and 35 volunteers without clopidogrel therapy. The VASP assay had an excellent reproducibility even if half or quarter of the volume of reagents recommended by the manufacturer were used: there was a high correlation between analyses with full vs. half and full vs. quarter volumes of reagents (r = 0.94; r = 0.93; p < 0.001, respectively). The PRI index was 4% higher when calculated with the geometric mean FI or the median FI (83%) as compared to the mean FI (80%; p < 0.001) in healthy volunteers without clopidogrel therapy. The PRI was 6% higher when calculated with the geometric mean FI or the median FI (52%) as compared to the mean FI (49%; p < 0.01) in patients on clopidogrel therapy. In conclusion, using half of the volume of reagents may constitute a cost-saving strategy for any laboratory performing the assay. Secondly, differences in calculation of PRI (mean, geometric mean or median fluorescence intensities) significantly contribute to the variability of results and reported cut-off values of the PRI. This variability can be easily avoided to facilitate comparison of data between different laboratories.