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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Model study detecting breast cancer cells in peripheral blood mononuclear cells at frequencies as low as 10(-7).
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Model study detecting breast cancer cells in peripheral blood mononuclear cells at frequencies as low as 10(-7).

机译：模型研究以低至10（-7）的频率检测外周血单个核细胞中的乳腺癌细胞。

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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.
机译：开发了一种流式细胞术检测血液和骨髓中的稀有癌细胞。多个标记；每一种都由一种单独的免疫荧光颜色（黄色和两个红色阴影）标识，用于可靠地识别癌细胞。不需要的但干扰癌细胞检测的血液或骨髓细胞由一组免疫荧光标记物鉴定，每种标记物具有相同的颜色（绿色）。因此，感兴趣的稀有癌细胞是黄色和两种不同的红色阴影，而不是绿色。稀有癌细胞同时对三种不同颜色（特定标记）呈阳性，而对第四种颜色（排除颜色）呈阴性的要求允许在10（7）个有核血细胞中检测到少至一个癌细胞（一种频率10（-7）。为了在临床研究之前测试这种罕见事件测定，进行了模型研究，其中通过将乳腺癌行BT-20的少量细胞与外周血单核细胞混合来模拟临床样品在混合频率为10（-5），10（-6）和10（-7）的情况下，我们检测到具有统计学意义的BT-20细胞数量，而在对照组样品中，没有观察到目标事件（BT-20）。超过10（8）个细胞进行了分析。为进一步确认模型研究中的BT-20细胞已正确识别和计数，将BT-20细胞（仅BT-20细胞）用第五种荧光染料共价染色， 7-氨基-4-氯甲基香豆素（CMAC）。没有CMAC荧光数据用于检测BT-20细胞的分析中。仅在使用来自特定颜色和排除颜色的数据完成分析之后，才将确定为BT-20细胞的事件检查为CMAC荧光。总是发现推定的BT-20事件对CMAC荧光呈阳性，这进一步增加了测定的信心。比较了手动数据分析和自动计算机程序。结果与手动和自动方法相当，但是自动“遗传算法”总是发现更多的BT-20事件。从包含BT-20的样品中以10（-5），10（-6）和10（-7）的频率对BT-20细胞进行细胞分选提供了进一步的证据，表明可以可靠地检测到这些稀有细胞。模型系统的良好分析性能将鼓励对临床样品进行进一步的研究。

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