Development of automated quantitative multiparameter immunocytochemical profiling of circulating tumor cells (CTCs) in breast cancer (BC).

e21124 Background: The currently accepted definition of a CTC is a nucleated cell that is positive by qualitative fluorescence for EpCAM, expresses cytokeratin, and is negative for the pan-hematopoietic marker CD45. Such approach has limitations such as operator bias, difficulty distinguishing true signal from autofluoresence or filter bleed-through, and most importantly, it cannot differentiate expression levels that are low, intermediate or high. This is a problem because CTCs are in fact heterogeneous with respect to phenotypic expression of various markers. Methods: Peripheral blood samples were subjected to our previously described, negative depletion technology. Cytospins of enriched samples were stained with 3 to 5 antibodies conjugated to various Alexa Fluor dyes plus a nuclear dye and then analyzed with both confocal microscopy and a traditional epifluorescence microscope equipped with a Nuance (CRi) multispectral imaging camera and associated software. This imaging system allows not only fluores...