Tembhare Prashant, Badrinath Yajamanam, Ghogale Sitaram, Subramanian Papagudi Ganesan
Hematopathology Laboratory, Advanced Centre for Treatment Research and Education in Cancer, Tata Memorial Centre, Navi Mumbai, India.
Curr Protoc Cytom. 2017 Apr 3;80:6.38.1-6.38.15. doi: 10.1002/cpcy.15.
The clinical use of flow cytometric DNA ploidy assay has been extended towards stratifying the risk of diseases, such as monoclonal gammopathies or B cell acute lymphoblastic leukemia, and to detect circulating tumor cells, both of which require detection of minute cell populations. This unit describes a protocol for determining DNA ploidy in fixed samples with simultaneous surface immunophenotyping. It is an easy method for simultaneous 6- to 8-color immunophenotyping and DNA content analysis using FxCycle Violet (FCV; DAPI) dye. This protocol is a one-step modification of routine multicolor immunophenotyping that includes surface staining followed by fixation and then DNA staining with FCV. It utilizes mature lymphocytes from the sample as an internal control for determination of DNA index. It is a sensitive method that allows DNA-ploidy determination and cell cycle analysis in a rare tumor population as low as 100 events, as well as DNA ploidy determination in various subsets of hematopoietic cells in the same sample based on their immunophenotype. © 2017 by John Wiley & Sons, Inc.
流式细胞术DNA倍体分析的临床应用已扩展到对疾病风险进行分层，如单克隆丙种球蛋白病或B细胞急性淋巴细胞白血病，以及检测循环肿瘤细胞，这两者都需要检测微小细胞群体。本单元描述了一种在固定样本中同时进行表面免疫表型分析来确定DNA倍体的方案。这是一种使用FxCycle Violet（FCV；DAPI）染料同时进行6至8色免疫表型分析和DNA含量分析的简便方法。该方案是对常规多色免疫表型分析的一步改进，包括表面染色、固定，然后用FCV进行DNA染色。它利用样本中的成熟淋巴细胞作为确定DNA指数的内部对照。这是一种灵敏的方法，可在低至100个事件的罕见肿瘤群体中进行DNA倍体测定和细胞周期分析，还可根据免疫表型对同一样本中造血细胞的各个亚群进行DNA倍体测定。© 2017约翰威立国际出版公司
