Occult tumor cells (OTCs) shed from tumors can travel through the blood stream to anatomically distant sites and form metastatic disease, the major cause of cancer-related death in patients with solid tumors. These disseminated cells are present in circulation in extremely low concentrations and are occult to routine imaging and laboratory studies. The principal approach for detecting these cells uses immunomagnetic enrichment technology that can cause loss of OTCs with low expression of the enrichment target proteins. We have developed a novel approach, the fiber-optic array scanning technology (FAST) that enables direct detection of OTCs at very low concentrations without the need for concentration. The FAST approach uses laser printing optics for the ultra fast scanning (300,000 cells/sec) of immunofluorescently labeled cells. The key innovations of the FAST are the exceptionally large field of view (50 mm), that is enabled by an optical fiber bundle with asymmetric ends and high collection efficiency, due to a novel design of borosilicate optical fibers. The sample preparation for FAST analysis consists of the recovery of nucleated cells from whole blood with ammonium chloride lysis, deposition of 40 million nucleated cells into a large glass substrate and indirect immunoflurescent labeling of OTCs with a pan anti-cytokeratin antibody. Fluorescent emission from OTCs is detected by laser scanning at 100 scans per second by using a galvanometer-rotated mirror and a standard photomultiplier. Objects identified with FAST scanning are subsequently scanned in an automated digital microscopy using position coordinates determined in the FAST scan. OTCs are identified from these objects with automated image recognition and human review. The performance of the system was validated by spiking carcinoma cell lines (HT-29, HCT-116, SKBR-3, MCF-7) into peripheral blood samples of healthy donors. We determined that the specificity of the system is 10-5, the sensitivity is 98%, while scanning at a rate of 300,000 cells per sec. Analysis of samples from stage IV breast cancer patient is currently ongoing. The instrument and process that we implemented has a scan rate that enables the analysis of statistically relevant sample size for OTC detection without the need of using any prior enrichment procedure. Thus it will help to determine the true prognostic significance of OTC detection in the peripheral blood.

[Proc Amer Assoc Cancer Res, Volume 46, 2005]