An immunological enrichment method for epithelial cells from peripheral blood

摘要: Abstract The ability of primary tumours to metastasize accounts for the majority cancer deaths. emergence circulating carcinoma cells in peripheral blood is supposed be an indicator cell spread. We have focused on this phenomenon order develop a sensitive technique enriching epithelial derived basis two-layer density gradient and subsequent immune magnetic sorting. Epithelial are possess cytoskeleton containing assembly intermediate filaments. During carcinogenesis these filaments do not undergo modifications antibody binding epitopes such as occur protein domains surface markers. developed which form single band. This was demonstrated by recovery experiments using [3H]thymidine-labelled showed were enriched within first step factor 20. In second MACS system applied. Cells stained with preformed FITC-conjugated mouse anti-human cytokeratin bound rat anti-mouse coupled superparamagnetic particles (immune paramagnetic separation complex; IPSC) subjected high fields. two-step procedure confirmed dispersing 50 5 × 105, 106, 107, 108, 109 leucocytes. Specific IPSC flow cytometry, confocal laser, fluorescent electron microscopy. specificity method further proved dual staining PSA prostatic separated from vitro. By means double-step it possible isolate up 15–20 out originally suspended into 107 human represented enrichment between 20 000 200 000, depending initial number. immunologically captured can used cytogenetic investigation situ dybridization (ISH) and/or polymerase chain reaction (PCR) detect specific gene aberrations. combined buoyant capable detecting free blood.