Isolation of circulating cancer cells from whole blood by immunomagnetic cell enrichment and unenriched immunocytochemistry in vitro

Purpose: We improved tumor cell detection compared with currently available immunocytochemical methods by immunomagnetic cell enrichment. Materials and Methods: Two methods of immunomagnetic enrichment using antibody coated magnetic beads were tested and compared with unenriched immunocytochemistry, including positive selection of epithelial cells with the antiepithelial antibody BER-EP4 and depletion of mononuclear cells with the anti-leukocyte antibody CD45. Various numbers of tumor cells from the 4 tissue culture cell lines DU 145, RT-4, KTCTL-2 and KTCTL-30 obtained from urological tumors were added to whole blood and mononuclear cells were isolated by density centrifugation. After incubation of the cell suspensions with beads cell separation was done in a magnetic field. After centrifugation on glass slides immunocytochemical staining for cytokeratin was performed. A total of 96 experiments were completed and negative controls were obtained. Results: The number of tumor cells detected by positive selection and depletion was significantly higher compared with immunocytochemistry (Wilcoxon test p <0.01). Mean enrichment factor and tumor cell recovery rates were 12.9% and 43.5% for positive selection, and 9.4% and 32.6% for depletion, respectively (p <0.05). With 1 tumor cell suspended in up to 30 ml. full blood unenriched immunocytochemistry failed to detect cancer cells, whereas positive selection revealed epithelial cells in 12 of 14 cases (85.5%) and depletion in all 14 (p <0.05). No false-positive results were observed. Conclusions: Compared with unenriched immunocytochemistry immunomagnetic enrichment significantly improves the detection of epithelial cells added to blood. A significant advantage was observed for positive selection. Immunomagnetic enrichment may be important for clinical practice in the future.