Flow Cytometry to Detect Human Tumor Cells in Body Fluid

描述(申请人提供)：流式细胞术已成为快速测定细胞DNA含量和人类肿瘤表型分析的重要技术。光散射和荧光检测是对人体细胞进行流式细胞术分析的两个常用参数。由于各种技术原因，光散射在确定细胞体积方面的价值有限，而电子库尔特体积测量是一种更可靠的体积测量方法。商业化的流式细胞仪都不能测量细胞体积。我们最近描述了NASA/美国癌症学会的流式细胞仪的开发和使用，该仪器能够同时测量从肿瘤分离的细胞核的电子核体积(ENV)和DNA含量。我们已经证明，通过同时测量ENV和DNA含量，我们可以区分不同人群中的正常细胞和肿瘤细胞，并识别近二倍体(非整倍体)肿瘤细胞。我们相信，该仪器可用于监测体液中肿瘤细胞的存在，如腹水、胸水、尿液和膀胱冲洗液。 目前拨款申请的具体目的是：1.开发样本制备和染色方法，用于分析已知或疑似恶性肿瘤患者的积液、尿液和膀胱冲洗液中的细胞。2.确定电子核体积、DNA含量和免疫细胞化学标记物表达的同时测量是否可以用于快速鉴定和检测这些液体样本中的肿瘤细胞。3.将流式细胞仪分析收集的数据与常规对这些标本进行细胞病理学和免疫细胞化学检查的数据进行比较，以便进行诊断。

DESCRIPTION (provided by applicant):Flow cytometry has become an important technique for rapid determination of cellular DNA content and for phenotype analysis of human tumors. Light scatter and fluorescence detection are used as two common parameters for flow cytometric analysis of human cells. For a variety of technical reasons, light scatter is of limited value in determining cellular volume as opposed to Electronic Coulter Volume measurement, which is a more reliable measure of volume. None of the commercially available flow cytometers can measure cellular volume. We have recently described the development and use of NASA/American Cancer Society flow cytometer capable of simultaneously measuring electronic nuclear volume (ENV) and DNA content of nuclei isolated from tumors. We have shown that by simultaneously measuring ENV vs. DNA content, we can discriminate between normal and tumor cells in a heterogeneous population and identify near-diploid (aneuploid) tumor cells. We believe this instrument can be used for monitoring the presence of tumor cells in body fluids such as ascites, pleural fluid, urine and in bladder washings. Specific aims of the present grant application are to: 1. Develop sample preparatory and staining methods for the analysis of cells from effusions, urine and bladder washings of patients with known or suspected malignancies. 2. Determine if simultaneous measurement of Electronic Nuclear Volume, DNA content and immunocytochemical marker expression can be used for rapid identification and detection of tumor cells in these liquid samples. 3. Compare data collected from flow cytometric analysis with that obtained from cytopathology and immunocytochemistry routinely performed on these specimens for diagnostic purpose.