Gross H J, Verwer B, Houck D, Hoffman R A, Recktenwald D
Monoclonal Research Center, Becton Dickinson Immunocytometry Systems, San Jose, CA 95131-1807.
Proc Natl Acad Sci U S A. 1995 Jan 17;92(2):537-41. doi: 10.1073/pnas.92.2.537.
A flow cytometric assay was developed to detect rare cancer cells in blood and bone marrow. Multiple markers; each identified by a separate color of immunofluorescence (yellow and two shades of red), are used to reliably identify the cancer cells. Blood or bone marrow cells, which are not of interest but interfere in detecting the cancer cells, are identified by a panel of immunofluorescence markers, each of which has the same color (green). Thus, the rare cancer cells of interest are yellow and two different shades of red but not green. The requirement that the rare cancer cell be simultaneously positive for three separate colors (the specific markers) and negative for a fourth color (the exclusion color) allowed detection of as few as one cancer cell in 10(7) nucleated blood cells (a frequency of 10(-7). To test this rare-event assay prior to clinical studies, a model study was performed in which the clinical sample was simulated by mixing small numbers of cells from the breast carcinoma line BT-20 with peripheral blood mononuclear cells. We detected statistically significant numbers of BT-20 cells at mixing frequencies of 10(-5), 10(-6), and 10(-7). In control samples, no target events (BT-20) were observed when more than 10(8) cells were analyzed. For additional confirmation that the BT-20 cells in the model study were correctly identified and counted, the BT-20 cells (and only BT-20 cells) were covalently stained with a fifth fluorescence dye, 7-amino-4-chloromethylcoumarin (CMAC). CMAC fluorescence data were not used in the assay for detecting BT-20 cells. Only after the analysis using data from the specific and exclusion colors had been completed were the events identified as BT-20 cells checked for CMAC fluorescence. The putative BT-20 events were always found to be positive for CMAC fluorescence, which further increases confidence in the assay. Manual data analysis and an automated computer program were compared. Results were comparable with the manual and automated methods, but the automated "genetic algorithm" always found more BT-20 events. Cell sorting of BT-20 cells from samples that contained BT-20 at frequencies of 10(-5), 10(-6), and 10(-7) provided further evidence that these rare cells could be reliably detected. The good performance of the assay with the model system will encourage further studies on clinical samples.
开发了一种流式细胞术检测方法来检测血液和骨髓中的罕见癌细胞。使用多种标记物，每种标记物通过单独颜色的免疫荧光（黄色和两种红色色调）进行识别，以可靠地识别癌细胞。血液或骨髓细胞虽不是目标细胞但会干扰癌细胞检测，通过一组免疫荧光标记物进行识别，这些标记物均为相同颜色（绿色）。因此，感兴趣的罕见癌细胞为黄色和两种不同红色色调，但不是绿色。要求罕见癌细胞同时对三种不同颜色（特定标记物）呈阳性，而对第四种颜色（排除颜色）呈阴性，这样能够在每10⁷个有核血细胞中检测到低至一个癌细胞（频率为10⁻⁷）。为了在临床研究之前测试这种罕见事件检测方法，进行了一项模型研究，通过将少量来自乳腺癌细胞系BT - 20的细胞与外周血单个核细胞混合来模拟临床样本。在混合频率为10⁻⁵、10⁻⁶和10⁻⁷时，我们检测到了具有统计学意义数量的BT - 20细胞。在对照样本中，当分析超过10⁸个细胞时，未观察到目标事件（BT - 20）。为了进一步确认模型研究中的BT - 20细胞被正确识别和计数，将BT - 20细胞（且仅为BT - 20细胞）用第五种荧光染料7 - 氨基 - 4 - 氯甲基香豆素（CMAC）进行共价染色。CMAC荧光数据未用于检测BT - 20细胞的检测方法中。仅在使用来自特定颜色和排除颜色的数据完成分析之后，才检查被识别为BT - 20细胞的事件的CMAC荧光。推定的BT - 20事件总是被发现CMAC荧光呈阳性，这进一步增强了对该检测方法的信心。对人工数据分析和自动化计算机程序进行了比较。手动和自动化方法的结果具有可比性，但自动化的“遗传算法”总是能发现更多的BT - 20事件。从频率为10⁻⁵、10⁻⁶和10⁻⁷含有BT - 20的样本中对BT - 20细胞进行细胞分选，进一步证明了这些罕见细胞能够被可靠地检测到。该检测方法在模型系统中的良好性能将鼓励对临床样本进行进一步研究。
