he change in light transmittance and reports results as P2Y12 reaction units. In addition, the device calculates the percentage of P2Y12 inhibition, based on thrombin receptor-activating peptide -induced platelet aggregation. However, in most publications the results are expressed as PRU. rank correlation method. Differences between patients and controls were analyzed using the Mann-Whitney test, p < 0.05 was considered as significant. In the case of categorical variables differences between groups were tested with the 2 test. The results of different methods on clopidogrel-treated patients were compared to those obtained by the VASP assay by calculating coefficients of determination and diagnostic efficiencies. In the latter case the 18215015 sum of results identical by the VASP and the other investigated assay, i.e., responders by both assays plus non-responders by both assays, was divided by the number of investigated patients. DE was expressed as percentage. Results P2Y12 specific ADP aggregation test Flow cytometric analysis of vasodilator-stimulated phosphoprotein phosphorylation The VASP phosphorylation assay was carried out according to the manufacturer’s instructions using a commercially available kit from Biocytex. The test is based on the fact that in the presence of PGE1 and ADP, the extent of VASP phosphorylation is proportional to the inhibition of platelets by clopidogrel. Briefly, samples of citrateanticoagulated blood were incubated with either PGE1 or PGE1+ADP and fixed in paraformaldehyde. Platelets were then permeabilized, labeled with a CD61 phycoerythrin-labeled platelet 21346199 specific antibody and a FITC-labeled phosphorylated VASP specific mouse monoclonal antibody or a negative isotype control antibody. Samples were analyzed on a FacsCalibur flow cytometer. Geometric mean fluorescence intensity values were determined in the presence of PGE1 without or with the addition of ADP. The extent of VASP phosphorylation was expressed as platelet reactivity index calculated from the MFI values using the following formula: PRI=100/MFI Determination of reference intervals and statistical analysis Statistical analysis was performed using GraphPad Prism Software. For all methods the reference interval corresponding to the 99% central interval was determined according to the guidelines of Clinical Laboratory Standards Institute . The lowest value of the reference interval was used as the diagnostic cut-off: those patients, who were on clopidogrel therapy and had results within the reference interval were considered non-responders to the therapy. Data obtained by different methods were Chebulinic acid correlated according to Spearman’s 3 Novel P2Y12 Specific Platelet Aggregation Test doi: 10.1371/journal.pone.0069417.g001 The ratio of non-responders diagnosed by the P2Y12 specific VerifyNow P2Y12 assay was similar to that demonstrated by the conventional ADP aggregation method. Using the other P2Y12 specific method, the VASP phosphorylation test, somewhat different results were obtained. In this case the results of patients showed a wide distribution. Using the reference interval determined according to CLSI guidelines, 88.3% of the results of clopidogrel treated patients were below this limit indicating an effect specific to clopidogrel therapy. However, the wide distribution of results raised the possibility that some of these patients are not effectively protected by clopidogrel. In the past few years, a number of clinical studies involving patients on co