Flow cytometric measurement of CD62P (P-selectin) expression on platelets: a multicenter optimization and standardization effort
Flow cytometric measurement of CD62P (P-selectin) expression on platelets: a multicenter optimization and standardization effort
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DOI：
10.1111/j.1537-2995.2008.01738.x
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发表时间：
2008-07-01
期刊：
影响因子：
2.9
通讯作者：
van der Meer, Pieter F.
中科院分区：
文献类型：
--
作者：
Curvers, Joyce;de Wildt-Eggen, Janny;van der Meer, Pieter F.
背景：体外测定血小板（PLT）质量的变量之一是测定CD 62 P表达。然而，使用不同的协议，使得结果的比较几乎是不可能的。这是我们的目的是开发一个统一的CD62 P协议，将产生可比的结果在不同的laborators.Study设计和方法：固定的影响，来源和稀释的CD62 P抗体，来源的免疫球蛋白G（IgG）同种型抗体，并分析结果进行了调查。一旦确定了最佳变量，就在五个参与中心进行比较研究。在最终比较研究中，将8份分离的PLT浓缩液运送至中心，在中心根据统一方案对样本进行染色和固定。使用市售流式细胞仪（BD Biosciences和Beckman Coulter）进行分析。结果：通过使用CD 62 P和IgG单克隆抗体的单克隆，可以实现中心间的均匀性。选择使用0.5%无甲醇甲醛固定的方案。为了提高流式细胞仪之间的一致性，在电子数据分析中，BID Biosciences和Beckman Coulter流式细胞仪的同型对照阈值分别设定为0.5%和2%。在最后的比较研究中，CD62P的95%置信区间（CI）范围为8%和21%之间的新鲜和20%至40%的8天的PLT concentration.CONCLUSION：一个统一的CD62P染色协议和随后的分析可以在多个中心使用不同的流式细胞仪，产生可比的结果与可接受的95% CI。
BACKGROUND: One of the variables to determine the quality of platelets (PLTs) in vitro is measurement of CD62P expression. Different protocols are in use, however, making comparison of results virtually impossible. It was our aim to develop a uniform CD62P protocol that would yield comparable results in various laboratories.STUDY DESIGN AND METHODS: The effects of fixation, source and dilution of CD62P antibody, source of immunoglobulin G (IgG) isotypic antibody, and analysis of results were investigated. Once the optimal variables were defined, comparative studies were performed at five participating centers. In the final comparative study, eight split PLT concentrates were shipped to the centers, where samples were stained and fixed according to the uniform protocol. Analyses were performed using commercially available flow cytometers (BD Bio-sciences and Beckman Coulter).RESULTS: Uniformity between centers could be achieved by using a single clone for CD62P and IgG monoclonal antibody. A protocol was selected using fixation with 0.5 percent methanol-free formaldehyde. To increase conformity between flow cytometers, in the analysis of electronic data the thresholds of the isotypic control were set at 0.5 percent for the BID Biosciences and 2 percent for the Beckman Coulter flow cytometers. In the final comparative study, the 95 percent confidence intervals (CIs) for CD62P ranged between 8 and 21 percent in fresh and 20 to 40 percent in 8-day-old PLT concentrates.CONCLUSION: A uniform CD62P staining protocol and subsequent analysis can be used at multiple centers using different flow cytometers, yielding comparable results with acceptable 95 percent Cls.