Begg A C, Hofland I
Division of Experimental Therapy, The Netherlands Cancer Institute, Amsterdam.
Cytometry. 1991;12(5):445-54. doi: 10.1002/cyto.990120510.
The development of antibodies to DNA-incorporated thymidine analogs has in turn led to the development of flow cytometric techniques for rapidly measuring cell kinetics parameters. More recently, these techniques have been applied to clinical tumor material. One problem with such measurements has been the difficulty of distinguishing malignant cells from coexistent normal cells in the biopsy material. In the present study, the feasibility of selecting out the desired malignant cell population for kinetic analysis from a mixture of cells was tested in vitro. An anticytokeratin antibody was used to discriminate between a mixture of tumor cells (cytokeratin positive) and normal cells (cytokeratin negative). The cell lines chosen for the study, A549 human lung carcinoma cells and Chinese hamster ovary (CHO) cells, were pulse labeled with iododeoxyuridine (IdUrd) and sampled every hour up to 16 hours. Selecting out cells from the mixture required the application of three-color fluorescence flow cytometry, which was carried out using the fluorochromes FITC (fluorescein isothionate, green fluorescence, IdUrd-DNA antibody), PE (phycoerythrin, orange fluorescence, cytokeratin antibody), and PI (propidium iodide, red fluorescence, DNA). This allowed single laser excitation. The staining procedure involved incubation with the IdUrd antibodies (specific antibody plus FITC-conjugated second antibody) followed by the cytokeratin antibodies (specific antibody plus PE-conjugated second antibody) and lastly by the DNA stain containing RNase. Two analysis methods of the IdUrd/DNA cytograms were applied: a mid-S window analysis and a relative movement (RM) analysis. Results of the analyses for cells selected out of mixtures were compared with results of cells stained and analyzed separately. A clear separation of the two cell lines could be obtained on the basis of orange fluorescence (cytokeratin content) despite a large overlap of their DNA histograms. By gating on high or low orange fluorescence, almost pure populations of the individual cell types could be selected out for further kinetic analysis. Little difference was seen, with both the mid-S and RM analyses, between cells gated from mixtures or stained separately. It is concluded that this technique is feasible for use on clinical material, provided good cell suspensions can be obtained, leading to the hope of increasing the accuracy of kinetic measurements on human tumors.
针对掺入DNA的胸腺嘧啶类似物的抗体的发展，反过来又推动了用于快速测量细胞动力学参数的流式细胞术的发展。最近，这些技术已应用于临床肿瘤材料。此类测量的一个问题是，在活检材料中难以区分恶性细胞与共存的正常细胞。在本研究中，在体外测试了从细胞混合物中选出所需恶性细胞群体进行动力学分析的可行性。使用抗细胞角蛋白抗体区分肿瘤细胞（细胞角蛋白阳性）和正常细胞（细胞角蛋白阴性）的混合物。用于该研究的细胞系，人肺癌A549细胞和中国仓鼠卵巢（CHO）细胞，用碘脱氧尿苷（IdUrd）进行脉冲标记，并每小时取样一次，直至16小时。从混合物中选出细胞需要应用三色荧光流式细胞术，这是使用荧光染料FITC（异硫氰酸荧光素，绿色荧光，IdUrd-DNA抗体）、PE（藻红蛋白，橙色荧光，细胞角蛋白抗体）和PI（碘化丙啶，红色荧光，DNA）进行的。这允许单激光激发。染色程序包括与IdUrd抗体（特异性抗体加FITC偶联的二抗）孵育，然后与细胞角蛋白抗体（特异性抗体加PE偶联的二抗）孵育，最后与含RNase的DNA染料孵育。应用了两种IdUrd/DNA细胞图分析方法：中S期窗口分析和相对运动（RM）分析。将从混合物中选出的细胞的分析结果与单独染色和分析的细胞的结果进行比较。尽管它们的DNA直方图有很大重叠，但基于橙色荧光（细胞角蛋白含量）可以清楚地分离这两种细胞系。通过选择高或低橙色荧光，可以选出几乎纯的单个细胞类型群体用于进一步的动力学分析。对于从混合物中选出的细胞或单独染色的细胞，中S期和RM分析几乎没有差异。得出的结论是，只要能够获得良好的细胞悬液，该技术可用于临床材料，这带来了提高人类肿瘤动力学测量准确性的希望。
