Crissman J D, Zarbo R J, Niebylski C D, Corbett T, Weaver D
Henry Ford Hospital, Harper Hospital, Detroit, Michigan.
Mod Pathol. 1988 May;1(3):198-204.
DNA quantitation of solid tumors requires either nuclear extraction or disaggregation into single cells for flow cytometric (FCM) analysis. We investigated methods for disaggregating colon carcinomas comparing mechanical and enzymatic techniques in fresh tumors and compared these to extraction of nuclei from paraffin blocks. Mechanical dissociation resulted in the highest yield of tumor cells, especially tumor cells with abnormal DNA content. In addition, mechanically disaggregated intact cells, particularly when dual labeled with cytokeratin, produced optimum DNA histograms. Cytokeratin staining allowed gating on epithelial cell populations and excluded contaminating diploid inflammatory and stromal cells, improving our ability to calculate synthesis phase fractions (SpF). Human colon tumor nuclei extracted from paraffin-embedded tissue resulted in greater variation of DNA histograms and less sensitivity in identifying aneuploid cells compared to mechanically dissociated intact cells from fresh tumors. The quality of the histograms and the ability to identify aneuploid cell populations were improved by modifying the standard "Hedley" technique by additional enzyme incubation. Mechanical dissociation of fresh tumors was the preferred technique for FCM DNA analysis. Nuclear extraction from paraffin blocks resulted in some loss of sensitivity in identifying aneuploid cell lines and decreased ability to measure SpF.
实体瘤的DNA定量分析需要进行细胞核提取或将其分解为单细胞以进行流式细胞术（FCM）分析。我们研究了新鲜肿瘤中机械和酶解技术相结合的结肠癌分解方法，并将其与石蜡块中细胞核的提取方法进行了比较。机械解离产生的肿瘤细胞产量最高，尤其是DNA含量异常的肿瘤细胞。此外，机械解离的完整细胞，特别是用细胞角蛋白进行双重标记时，能产生最佳的DNA直方图。细胞角蛋白染色可对上皮细胞群体进行门控，并排除污染的二倍体炎症和基质细胞，提高了我们计算合成期分数（SpF）的能力。与新鲜肿瘤中机械解离的完整细胞相比，从石蜡包埋组织中提取的人结肠肿瘤细胞核导致DNA直方图的变化更大，识别非整倍体细胞的敏感性更低。通过额外的酶孵育改进标准的“Hedley”技术，可提高直方图的质量和识别非整倍体细胞群体的能力。新鲜肿瘤的机械解离是FCM DNA分析的首选技术。从石蜡块中提取细胞核在识别非整倍体细胞系时会导致一些敏感性损失，并降低测量SpF的能力。
