Curvers Joyce, de Wildt-Eggen Janny, Heeremans Judith, Scharenberg John, de Korte Dirk, van der Meer Pieter F
The Sanquin Blood Bank Southeast Region, Sanquin Blood Supply Foundation, Nijmegen, Location Maastricht, the Netherlands.
Transfusion. 2008 Jul;48(7):1439-46. doi: 10.1111/j.1537-2995.2008.01738.x. Epub 2008 May 13.
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.
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 Biosciences and Beckman Coulter).
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 BD 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.
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 CIs.
体外测定血小板（PLT）质量的变量之一是测量CD62P表达。然而，目前使用的方案各不相同，这使得结果的比较几乎不可能。我们的目标是开发一种统一的CD62P方案，该方案能在各个实验室产生可比的结果。
研究了固定、CD62P抗体的来源和稀释度、免疫球蛋白G（IgG）同型抗体的来源以及结果分析的影响。一旦确定了最佳变量，就在五个参与中心进行了比较研究。在最后的比较研究中，将八份分装的血小板浓缩物运送到各中心，按照统一方案对样本进行染色和固定。使用市售流式细胞仪（BD生物科学公司和贝克曼库尔特公司）进行分析。
通过使用单一克隆的CD62P和IgG单克隆抗体，可以实现各中心之间的一致性。选择了一种使用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，BD生物科学公司流式细胞仪的同型对照阈值设定为0.5%，贝克曼库尔特公司流式细胞仪的同型对照阈值设定为2%。在最后的比较研究中，新鲜血小板中CD62P的95%置信区间（CI）在8%至21%之间，8天龄血小板浓缩物中CD62P的95%置信区间在20%至40%之间。
一种统一的CD62P染色方案及后续分析可在多个使用不同流式细胞仪的中心使用，产生具有可接受的95%CI的可比结果。
