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

• Published in: Thrombosis and haemostasis Vol. 107; no. 3; p. 538
• Main Authors: Freynhofer, Matthias K, Bruno, Veronika, Willheim, Martin, Hübl, Wolfgang, Wojta, Johann, Huber, Kurt
• Format: Journal Article
• Language: English
• Published: Germany 01.03.2012
• Subjects: Blood Specimen Collection - standards; Blood Specimen Collection - statistics & numerical data; Cell Adhesion Molecules - metabolism; Cell Separation; Coronary Artery Disease - diagnosis; Coronary Artery Disease - drug therapy; Coronary Artery Disease - epidemiology; Flow Cytometry; Humans; Microfilament Proteins - metabolism; Observer Variation; Phosphoproteins - metabolism; Phosphorylation - drug effects; Platelet Activation - drug effects; Platelet Function Tests - methods; Platelet Function Tests - standards; Platelet Function Tests - statistics & numerical data; Prospective Studies; Reference Standards; Ticlopidine - administration & dosage; Ticlopidine - adverse effects; Ticlopidine - analogs & derivatives; Vasodilator Agents - metabolism
• ISSN: 0340-6245
• DOI: 10.1160/TH11-09-0623

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 (FI). 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 FI 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 FI 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 FI 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 FI after 90 minutes compared to baseline (Δ=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 FI.