Detection of circulating tumour cells in peripheral blood with an automated scanning fluorescence microscope

Abstract

We have developed an automated, highly sensitive and specific method for identifying and enumerating circulating tumour cells (CTCs) in the blood. Blood samples from 10 prostate, 25 colorectal and 4 ovarian cancer patients were analysed. Eleven healthy donors and seven men with elevated serum prostate-specific antigen (PSA) levels but no evidence of malignancy served as controls. Spiking experiments with cancer cell lines were performed to estimate recovery yield. Isolation was performed either by density gradient centrifugation or by filtration, and the CTCs were labelled with monoclonal antibodies against cytokeratins 7/8 and either AUA1 (against EpCam) or anti-PSA. The slides were analysed with the Ikoniscope s robotic fluorescence microscope imaging system. Spiking experiments showed that less than one epithelial cell per millilitre of blood could be detected, and that fluorescence in situ hybridisation (FISH) could identify chromosomal abnormalities in these cells. No positive cells were detected in the 11 healthy control samples. Circulating tumour cells were detected in 23 out of 25 colorectal, 10 out of 10 prostate and 4 out of 4 ovarian cancer patients. Five samples (three colorectal and two ovarian) were analysed by FISH for chromosomes 7 and 8 combined and all had significantly more than four dots per cell. We have demonstrated an Ikoniscope s based relatively simple and rapid procedure for the clear-cut identification of CTCs. The method has considerable promise for screening, early detection of recurrence and evaluation of treatment response for a wide variety of carcinomas.

Circulating tumor cells: utility for predicting response to anti-EGFR therapies?

“…The technology is still in the proof-of-principle stage but is showing promise in that it can be combined with FISH to further characterize CTCs. The range of CTC detection is similar to that of CellSearch [12].…”

Humanised IgG1 antibody variants targeting membrane-bound carcinoembryonic antigen by antibody-dependent cellular cytotoxicity and phagocytosis

“…Phagocytosis events were detected using the Ikoniscope imaging system (Ntouroupi et al, 2008). Slides are first scanned at low magnification ( Â 10) to detect green label and then revisited at high magnification ( Â 100) to identify red labelled macrophages with 'engulfed' green targets.…”

“…An alternative ADCP assay was developed using an automated microscope (Ikoniscope, Ikonisys, New Haven, CT, USA) (Ntouroupi et al, 2008). Target cells and macrophages, stained as described above, were deposited on to a poly-l-lysine coated slide, fixed with 2% formaldehyde.…”

Liquid biopsies: tumour diagnosis and treatment monitoring

“…The samples were analyzed with the Ikoniscope robotic fluorescence microscope imaging system. The results showed that the sensitivity of this method could detect less than one epithelial cell per milliliter of blood, and fluorescence in situ hybridization (FISH) could identify chromosomal abnormalities in these cells (Ntouroupi et al, 2008).…”