Section 1Objective:Platelet function testing includes platelet aggregometry and measurement of other platelet activation and function.Among them,whole blood flow cytometry is a very powerful but relatively new laboratory technique,which has several advantages:minimal volumes required,minimal sample manipulation,platelets in their physiological milieu.As a specific platelet surface marker,P-selectin mediates the induction of inflammation by platelets and leukocytes(including monocytes and neutrophils)aggregation.This study aims to investigate the methodology of platelet surface P-selectin from the aspects of sampling,activation and flow cytometry,and to further explore whether P-selectin on platelet surface can be a good indicator of anti-platelet therapy.Methods:A 2.7 mL blood sample was collected using a 21-gauge needle from a peripheral venous puncture of healthy subj ects.Variable-controlling approach was adopted in the pretreatment.Different whole blood dilution factors,agonists of different types and concentration gradients,and different flow cytological parameters were set to compare the expression of P-selectin on the platelet surface using flow cytometry.Results:1.Diluting the sample 10 times after blood collection reduces platelet aggregation;2.P-selectin expression showed a good positive correlation with ADP concentration,and P-selectin expression reaches the maximum at ADP concentration of 20μM.3.The sheath fluid flow rate has an effect on the test results.4.As the storage time increases,the expression level of P-selectin on the platelet surface increases significantly.Conclusion:Appropriate sample dilution should be performed immediately after blood collection.Samples should be treated with ADP at a final concentration of 20μM and should be collected with lower sheath fluid flow rate.Section 2Objective:To prevent stent restenosis after percutaneous coronary stenting,patients are required to receive dual antiplatelet therapy.However,due to the heterogeneity in the response to antiplatelet drugs in patients after surgery,platelet function testing is required to guide antiplatelet medication.Berberine,as an alkaloid,has been shown to have anti-inflammatory effects.This study intends to use commercial platelet detection tools and flow cytometry to detect P-selectin to evaluate the anti-platelet function of berberine.Methods:This study was a single-center,randomized,open-label,controlled,dose-escalating,parallel-group study of 11 patients with stable coronary artery disease or unstable angina after elective PCI for more than 8 weeks but less than or equal to 40 weeks.Patients receiving dual antiplatelet therapy(aspirin and clopidogrel)were assessed by means of VASP,VerifyNow-P2Y12 and flow cytometry of surface P-selectin after taking antiplatelet drugs at 4/8/12 weeks of berberine treatment to explore the antiplatelet effect of berberine.Results:Compared with the control group,there was no significant difference in the expression of platelet surface P-selectin,PRI(measured by VASP),PRU and inhibition rate(measured by VerifyNow-P2Y12).Long-term treatment with berberine shows no obvious adverse effects.Conclusion:This study is not sufficient to demonstrate that berberine inhibits platelet aggregation through the P2Y12 pathway or achieves anti-inflammatory effects by reducing P-selectin expression. |