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Platelets represent a linkage among inflammation, thrombosis, and atherogenesis, and enhanced platelet activation is regarded as a risk for thrombotic disorders. The level of P-selectin expressed (CD62P) on the platelet surface is a useful marker of activated platelets (aPLT). Although CD62P has been measured briefly by flow cytometry using an anti-CD62P antibody, the assay remains imprecise and we tried to establish stable conditions for its measurement. The levels of aPLT are increased significantly by many factors, such as meals, sampling and keeping conditions, centrifugation, and the timing of fixation. For optimal results, sampling should be performed quickly in a K2-ethylenediaminetetraacetic acid (EDTA) containing a sample tube, and whole blood should be fixed with 666mmol/L formaldehyde plus 167mmol/L glyoxal for 5min. After washing with phosphate buffered saline (PBS), the fixed platelets were reacted with anti-CD62P antibody for 20min and measured by flow-cytometric detection for aPLT. The coefficient of variation of our aPLT assay was 10.4%. We also examined basic experiments to test the clinical application of our aPLT assay by monitoring aspirin therapy. The levels of aPLT after the administration of aspirin for 3 days were significantly lower than those in the group that did not receive aspirin. These results suggest that the aPLT assay is an effective analytical procedure for measuring platelet reactivity.