流式细胞术联合测定保存血小板表达的磷脂酰丝氨酸和CD62p

Flow Cytometry Combined Assay for Phosphatidylserine and CD62p Expressed by Preserved Platelets

人类血小板通过不同的技术进行离体保存后 ,其特性改变差异较大。建立适用于各种保存血小板特性分析的流式细胞术 (FCM)对保存血小板的质量监测和新保存方法研究有重要意义。本研究通过对FCM分析方法主要条件的优化评估 ,建立了联合测定血小板膜表面包括磷脂酰丝氨酸在内的多参数FCM定量分析方法。在标本制作中 ,血小板不需要洗涤即可直接进行标记 ,减少了血小板离心洗涤过程中的激活。除了FCM常用的同型对照外 ,文中还将新鲜富含血小板血浆 (FPRP)、凝血酶激活FPRP和液氮处理FPRP作为血小板标准阴性、阳性对照 ,直接应用于FCM分析 ,且效果良好。该方法中Gly Pro Arg Pro乙酸盐 (GPRP)的选择应用 ,既防止了血小板聚集和纤维蛋白的形成 ,同时可稳定血小板 ,减少制备过程中人为激活 ,克服了在血小板、Ca2 + 和血浆共存条件下FCM定量分析血小板的困难 ,尤其是为深低温处理血小板包括PS在内的多参数分析提供了良好的方法基础。研究表明该方法标本处理简单 ,结果分析简便、准确 ,重复性好。作者还通过低温、低渗或激活剂的方法制备了几种膜表面分子标记显著不同的血小板应用于FCM分析 ,不仅证实了所建立的FCM分析方法在各种不同膜表面分子标记的血小板分析中的实用性 ,又表明了文中制备的这 4种不同膜表面分?

Human platelets have distinct characters when preserved by different methods. A efficient flow cytometric assay for different preserved platelets expression of CD62p and phosphatidylserine(PS) is in dire need. Efficient flow cytometric assay for CD62p and PS expressed by preserved platelets was established and the major conditions were optimized. The platelets need not to be washed to wipe off plasma and can be labelled diredtly during the sample preparation. It is efficient for flow cytometric analysis when fresh platelet riched plasma(FPRP) was set as negative control, thrombin actived FPRP, and liquid nitrogen treated FPRP were set as positive control respectively. Gly Pro Arg Pro acetate salt (GPRP) was applied to prevent platelets aggregation and fibrin formation, stabilize platelets and minimize the artifical platelets activation. This is also the key to conquer difficulty of flow cytometric quantitive analysis when platelet, Ca 2+ and plasma coexist. This flow cytometric method is specially suitable for the multi parameter assay including PS expression for cryopreserved platelets. Minimal sample manipulation, no fixation, and GPRP application resulted in minor artefacts and good sample stability. Results suggested, this flow cytometric assay for preserved platelets is simple and efficient. In addition, the author prepared four different methods treated platelets that can be easily distinguished through this flow cytometric assay. It not only makes sure the practicability of this flow cytometric assay, but also suggests the value of the treated platelets applied in preserved platelets flow cytometric assay.