临床HSPCs移植参考的CD34+细胞检测方法的依据探讨修改.docVIP

临床HSPCs移植参考的CD34检测方法的 一、欧洲临床细胞分析事务委员会1998年推荐的方法 (检测实验室采用) 以下是其发表文章的摘要译文： 对造血干/祖细胞(HSPCs)移植可靠评价推动了以CD34特征分子为基础的流式细胞测定的发展。虽然流式细胞测定低含量细胞偏差较大，但技术的进展促进了测定的准确性和精度的提高。在此回顾报道的文献，我们推荐如下的HSPCs检测： 1）Ⅰ/Ⅲ型CD34膜单抗；2）除小细胞如血小板、未裂解红细胞以及细胞碎片等；3）进行CD45膜标记复染，HSPCs的；4）在流式结果分析时，表达CD45（即弱阳性）侧向角也就是中-CD45dim部分，CD34+HPC的。5）统计的细胞应包含CD34弱阳性和强阳性细胞；6）扣除阴性对照；7）100个CD34+细胞，10%的精度； 原文： Cytometry. 1998 Jun 15;34(3):128-42. Flow cytometric enumeration of CD34+ hematopoietic stem and progenitor cells. European Working Group on Clinical Cell Analysis. Gratama JW, Orfao A, Barnett D, Brando B, Huber A, Janossy G, Johnsen HE, Keeney M, Marti GE, Preijers F, Rothe G, Serke S, Sutherland DR, Van der Schoot CE, Schmitz G Department of Clinical and Tumor Immunology, Daniel den Hoed Kliniek, Rotterdam, The Netherlands. gratama@immh.azr.nl Abstract The need for a rapid and reliable marker for the engraftment potential of hematopoietic stem and progenitor cell (HPC) transplants has led to the development of flow cytometric assays to quantitate such cells on the basis of their expression of CD34. The variability associated with enumeration of low-frequency cells (i.e., as low as 0.1% or 5 cells/microl) is exceedingly large, but recent developments have improved the accuracy and precision of the assay. Here, we review and compare the major techniques. Based on the current state of the art, we recommend 1) bright fluorochrome conjugates of class II or III monoclonal antibodies (mAbs) that detect all glycoforms of CD34, 2) use of a vital nucleic acid dye to exclude platelets, unlysed red cells, and debris or use of 7-amino actinomycin D to exclude dead cells during data acquisition, 3) counterstaining with CD45 mAb to be included in the definition of HPC, 4) during list mode data analysis, Boolean gating to resolve the CD34+ HPCs from irrelevant cell populations on the basis of the low levels of CD45 expression and low sideward light-scatter signals of HPCs, 5) inclusion of CD34dim and CD34bright populations in