Assay for the determination of human carcinoma cells in circulating blood

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Abstract
Methods have been developed in an in vitro system (1) of assessing the number of disseminated tumor cells in peripheral blood and (2) of enriching tumor cells from peripheral blood samples for further characterization. Cells from three human carcinoma lines (E 14, ChaGo, and LEDWiDr) were mixed with leukocytes from normal individuals in various ratios. The proportions of tumor cells were determined by a quantitative assay using 3H-thymidine, 3H-leucine, and 3H-galactose incorporation. Determination of tumor cell proportions with this method was most accurate in the range of 5×104 to 5×103 tumor cells mixed with a constant number (5×105) of lymphocytes. It was possible to separate 75Se-labeled tumor cells from 51Cr-labeled blood leukocytes by centrifugation in isopyknic Percoll density gradients. These cells were mixed at different ratios and subjected to Percoll gradient centrifugation. By this approach as few as 5×103 tumor cells could by identified in the presence of 5×107 leukocytes, representing a ratio of 1:10,000. Percoll centrifugation did not damage the tumor cells. In blood cells from two lung cancer patients with lung metastases the incorporation of 3H-thymidine and 3H-galactose was significantly enhanced compared with that in blood cells from patients with primary lung tumors and in cells from normal individuals. This difference became even more apparent when metabolically-labeled blood cells were subsequently separated by Percoll gradient centrifugation.