title: Vasodilator-stimulated phosphoprotein-phosphorylation assay in patients on clopidogrel: does standardisation matter?
abstract: 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.
authors.affiliation: 3rd Medical Department, Cardiology and Emergency Medicine, Wilhelminenhospital, Vienna, Austria. matthias.freynhofer@meduniwien.ac.at
authors.forename: Matthias K
authors.initials: MK
authors.lastname: Freynhofer
authors.forename: Veronika
authors.initials: V
authors.lastname: Bruno
authors.forename: Martin
authors.initials: M
authors.lastname: Willheim
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Hübl
authors.forename: Johann
authors.initials: J
authors.lastname: Wojta
authors.forename: Kurt
authors.initials: K
authors.lastname: Huber
mesh_terms: D001800:Blood Specimen Collection / Q000592:standards / Q000706:statistics & numerical data; D015815:Cell Adhesion Molecules / Q000378:metabolism*; D002469:Cell Separation; D000077144:Clopidogrel; D003324:Coronary Artery Disease / Q000175:diagnosis / Q000188:drug therapy* / Q000453:epidemiology*; D005434:Flow Cytometry; D006801:Humans; D008840:Microfilament Proteins / Q000378:metabolism*; D015588:Observer Variation; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D015539:Platelet Activation / Q000187:drug effects; D010979:Platelet Function Tests / Q000379:methods / Q000592:standards* / Q000706:statistics & numerical data; D011446:Prospective Studies; D012015:Reference Standards; D013988:Ticlopidine / Q000008:administration & dosage / Q000009:adverse effects / Q000031:analogs & derivatives*; D014665:Vasodilator Agents / Q000378:metabolism; D000098662:Vasodilator-Stimulated Phosphoprotein
chemical_list: D015815:Cell Adhesion Molecules; D008840:Microfilament Proteins; D010750:Phosphoproteins; D014665:Vasodilator Agents; D000098662:Vasodilator-Stimulated Phosphoprotein; D000077144:Clopidogrel; D013988:Ticlopidine