Fig. 3

Principle of iFISH. a Combined in situ phenotypic immunostaining and karyotypic FISH demonstrate that among 3 CD45 negative non-hematopoietic cells, immunostaining of tumor biomarker (CK18 in this study) alone indicates that Cell 1 and 2 respectively have high and low CK18 expression, and Cell 3 has no visible CK18 detected; whereas image of FISH alone performed with CEP of chromosome (chromosome 8 in this study) shows that Cell 1 and 3 are abnormally triploid, and Cell 2 are diploid. Merged iFISH image demonstrates that all of Cell 1–3 are CTCs. Cell 1 has triploid chromosome 8 with strong CK18 expression; Cell 2 possesses disomy of chromosome 8 with low CK18 expression; and Cell 3 shows triploid chromosome 8 with negative CK18 expression. b Diverse tumor biomarker-iFISH, including CA19-9, CK18, EpCAM, and HER2-iFISH are illustrated. Experimental protocol of SE-iFISH was previously published [7]. Briefly, 6–8.5 ml peripheral blood, collected into a tube containing acid citrate dextrose anti-coagulant (Becton–Dickinson, Franklin Lakes, NJ, USA), were subjected to centrifuging to remove plasma, followed by centrifuging again on the top of non-hematopoietic cell separation matrix to remove RBCs. Remaining WBCs were incubated with anti-WBC immunomagnetic beads, and subsequently loaded on the separation matrix, then spun down. Cell pellet thoroughly mixed with the cell fixative was applied on the formatted and coated CTC slide. The air dried samples were subjected to FISH probe hybridization and antibody staining performed with Alexa Fluor 594 conjugated monoclonal anti-CD45 and Alexa Fluor 488 conjugated with the indicated antibody [56], followed by image collection and analysis