Leers M P, Theunissen P H, Schutte T B, Ramaekers F C
Department of Pathology, De Wever Hospital, Heerlen, The Netherlands.
Cytometry. 1995 Sep 1;21(1):101-7. doi: 10.1002/cyto.990210118.
Admixture of normal and neoplastic cells is a serious problem in the evaluation of tumor cell kinetic parameters by flow cytometry, in particular for DNA diploid tumors. The admixture of non-neoplastic cells, such as stromal cells and inflammatory cells, can disturb the estimation of the proliferative tumor fraction. This problem has been addressed in fresh tumor samples by applying bivariate flow cytometric analyses for DNA and cytokeratin. We have adapted this approach for formalin-fixed and paraffin-embedded tissue samples of colorectal carcinomas. After preparation of a single cell suspension from paraffin blocks by means of an enzymatic digestion step, the cells of epithelial origin were selectively stained with a panel of subtype specific cytokeratin antibodies. DNA analysis could thus be performed on the cytokeratin-positive cells. The proliferative fractions of the paraffin-embedded samples could be compared with those of the fresh tissue samples and a very good correlation was seen between DNA indices from fresh and paraffin-embedded material. As expected, after gating on the cytokeratin-positive cells an enrichment of the S-phase fraction was seen compared with the ungated cell population. However, this enrichment was more pronounced in the cell suspensions derived from the paraffin-embedded part of the tumor compared with the fresh disaggregated, ethanol-fixed part of the tumor.
在通过流式细胞术评估肿瘤细胞动力学参数时，尤其是对于DNA二倍体肿瘤，正常细胞与肿瘤细胞的混合是一个严重问题。非肿瘤细胞（如基质细胞和炎性细胞）的混合会干扰增殖性肿瘤细胞比例的估算。通过对DNA和细胞角蛋白进行双变量流式细胞术分析，新鲜肿瘤样本中的这个问题已得到解决。我们已将此方法应用于结直肠癌的福尔马林固定石蜡包埋组织样本。通过酶消化步骤从石蜡块制备单细胞悬液后，上皮来源的细胞用一组亚型特异性细胞角蛋白抗体进行选择性染色。因此，可以对细胞角蛋白阳性细胞进行DNA分析。石蜡包埋样本的增殖比例可以与新鲜组织样本的增殖比例进行比较，并且新鲜样本和石蜡包埋样本的DNA指数之间呈现出非常好的相关性。正如预期的那样，在选定细胞角蛋白阳性细胞后，与未选定的细胞群体相比，S期细胞比例有所增加。然而，与新鲜解离的乙醇固定肿瘤部分相比，肿瘤石蜡包埋部分的细胞悬液中这种增加更为明显。
