流式细胞术测定保存血小板再表达CD62p方法的建立及应用

Flow cytometry detection of CD62p reexpression by preserved platelets and it's value in evaluation of the quality of preserved platelets

目的 ①建立并优化测定血小板再表达膜表面激活标志物CD6 2p能力的流式细胞术 (FCM) ;②测定2 2℃保存液体血小板不同保存期再表达CD6 2p的能力 ,以评价该指标在血小板质量监测以及新的保存方法研究中的应用价值。方法 ①采用浓度梯度法优化GPRP浓度条件 ,采用析因设计优化凝血酶浓度和 37℃孵育时间条件 ,寻找最佳阴、阳性对照 ;②将 13份血小板按AABB标准保存 ,并延长保存至 12d ,测定每天血小板CD6 2p再表达率 ,与其相应体内存活期作直线相关性分析。结果 ①约 1.5× 10 6个血小板内加入 2 .5mmol/LGPRP可有效防止过量凝血酶诱导的聚集反应 ,孵育条件以 1U/L凝血酶 37℃ 15min然后室温置 15min最佳。采用新鲜富含血小板血浆(FPRP)为阴性对照 ,以凝血酶激活FPRP为阳性对照效果良好。② 2 2℃保存血小板CD6 2p再表达率与其相应体内存活期呈明显正相关 ,单份相关系数 (r) 0 .91～ 0 .99,总体相关系数为 0 .99。结论 ①优化建立了一种灵敏简便 ,稳定性和重复性并好的流式细胞术方法用于测定保存血小板CD6 2p再表达率 ;②保存液体血小板CD6 2p再表达率与其相应体内存活期呈明显正相关 ,且相关性良好 ,提示CD6 2p再表达率是监测保存血小板质量的良好指标 ,同时还可能在新的保存方法研究和临床血小板功?

Objective 1) To establish and optimize a flow cytometry method for detecting CD62p reexpression by preserved platelets.2) Daily determination of the CD62p expression by platelets stored at 22℃ to assess its value in evaluation of preserved platelet quality.Methods 1) Statistic methods including factorial experiment was applied to optimize the major conditions for sample management, and the feasible negative and positive control for FCM analysis of CD62p expression were check out.2) Thirteen donors' platelets including ten PRP and three apheresis concentrations were stored in a flatbed shaker at 22±2℃ for 12 days. The CD62p expression were detected daily. The correlation between CD62p expression and in vivo residual life span of 22℃ stored platelet were analyzed.Results 1) Satisfactory conditions for analysis CD62p anew expression by FCM were obtained. 2) The CD62p anew expression by stored platelets was correlated to the in vivo residual life span of the platelets with the correlation coefficients between 0.91 and 0.99.Conclusion 1) This FCM method is simple, sensitive, stable, and easy to carry. 2) Platelet CD62p expression can be used to monitor the function of stored platelets and has the potential in the study of new platelet preservation technique.