Application of Three-Color Flow Cytometry for the Determination of P-selection on the Platelet of Whole Blood

Objective P-selectin is known to be marker of platelet activation. We developed the technique of three-color flow cytometry determine P-selectin on the platelets whole blood and compared with the PRP method. Methods Blood was obtained from an antecubital vein through a 5ml syringe without whole blood a applying tourniquent. The first 2ml of blood was discarded. Then a 1ml blood sample was collected in 0.1ml of 3.8% sodium citrate. Within 3 min 10μl blood was added to 5μl FITC-CD42a (to label platelets), 5μl PE-CD62P (to label activation marker P-selectin) and 5μl CY-CD45 (to label WBC). After carefully mixing and 20 min of incubation at ambient temperature in the dark, the sample was fixed in formaldehyde (1%) for 10 min and washed with PBS two times. Finally the smaple was diluted to 200μl with PBS for flow cytometry. Results The results showed that the expression of P-selectin in whole blood was lower than that of the PRP. Conclusion This method would reflect very closely the physiological status of platelet activation in vivo.