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Clin Cancer Res. 2000 Feb;6(2):434-42.

Detection and analysis of cancer cells in blood and bone marrow using a rare event imaging system.

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Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA 02115, USA.


An automated rare event detection system (Rare Event Imaging System) is described for the recognition of cancer cells that appear at low frequencies (1 in 1 million) in peripheral blood (PB) or bone marrow (BM). The instrumentation includes an automated fluorescence microscope (Nikon Microphot-FXA) with a cooled charge coupled device camera and a 60-MHz Pentium personal computer. Main features of the system are rapid analysis of large microscopic fields, including a total cell count, detection of fluorescently labeled cells, and a display of digitally stored images of the detected cells. Furthermore, the X,Y coordinates of each identified object are stored and can be recalled for morphological analysis of the cell using higher magnification or different fluorescent filter sets. The preparation of the blood or BM samples for automated analysis consists of lysis of the RBCs, attachment of sample cells onto adhesion slides, fixation, and fluorescent labeling with anticytokeratin antibodies. Cytokeratin-positive cells, however, were detected in 17% of the samples from healthy blood donors using this procedure (mean number, approximately 7/10(6) mononuclear cells in positive samples). To improve the specificity of the rare event detection, a double-labeling protocol combining intracellular cytokeratin with epithelial cell adhesion molecule (Ep-CAM) (breast, ovarian, colon, and lung carcinoma antigen) or disialo-ganglioside (GD2) antigen (small cell lung carcinoma, neuroblastoma, melanoma antigen) was developed. Examples of doubly labeled cultured cells and cancer cells from breast and small cell lung cancer patients are shown. Using the double-labeling protocol, no "positive" cells were seen in samples of healthy blood donors. Automated rare event detection (cytokeratin single-staining) was applied to 355 PB, BM, and stem cell (SC) samples from breast cancer patients before autologous BM transplantation. Cytokeratin-positive cells were found in 52% of BM, 35% of PB, and 27% of SC samples at frequencies of 1-1020 positive cells/10(6) mononuclear cells, thereby establishing the efficacy of the technique in the detection of rare cancer cells in hematopoietic tissue samples of cancer patients.

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