Detection and Quantification of Small Numbers of Circulating Tumour Cells in Peripheral Blood Using Laser Scanning Cytometer (LSC®)

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Abstract
The detection of circulating tumour cells disseminated from solid tumours requires extremely sensitive methods. Molecular genetic methods, which are most sensitive, are not applicable to solid tumours because no tumour-specific genetic markers are available. Detection of disseminated tumour cells by immunocytochemistry is time-consuming, whereas fluorimetry is fast and quantitative. The laser scanning cytometer (LSC®) provides an automated microscopic procedure for screening up to 5×104 cells in suitable time. Using this system together with an enrichment procedure which allows up to ten thousand-fold enrichment, we have quantified minimal numbers of tumour cells. In a model system, breast cancer cell line cells diluted into peripheral blood mimicked seeding of tumour cells into the periphery. After staining with fluorochrome-conjugated anti-epithelial antibody, slides were screened for positive events directly or after enrichment with antibody-coated magnetic beads. One positive cell was unequivocally detectable in 104 cells and 50 out of 60 tumour cells were reliably recovered from a 20 ml blood volume, equal to 1–2 cells per 107, after magnetic bead enrichment. This method allows quantitation of tumour cells in peripheral blood and bone marrow in reasonable time and will, for the first time, enable extensive investigation of the seeding behaviour of tumours.