BACKGROUNDThis　study　was　aimed　at　establishing　a　sensitive　and　specific　isolation,　characterization,　and　enumeration　method　for　living　circulating　tumor　cells　(CTCs)　in　patients　with　colorectal　carcinoma.　METHODSQuantitative　isolation　and　characterization　of　CTCs　were　performed　through　a　combination　of　immunomagnetic　negative　enrichment　and　fluorescence-activated　cell　sorting.　Isolated　CTCs　were　identified　by　immunofluorescence　staining.　The　viability　and　purity　of　the　sorted　cells　were　determined　by　flow　cytometry.　Blood　samples　spiked　with　HCT116　cells　(range,　3-250　cells)　were　used　to　determine　specificity,　recovery,　and　sensitivity.　The　method　was　used　to　enumerate,　characterize,　and　isolate　living　CTCs　in　10　mL　of　blood　from　patients　with　colorectal　carcinoma.　RESULTSThe　average　recovery　of　HCT116　cells　was　61%　or　more　at　each　spiking　level,　and　the　correlation　coefficient　was　0.992.　An　analysis　of　samples　from　all　18　patients　with　colorectal　carcinoma　revealed　that　94.4%　were　positive　for　CTCs　with　an　average　of　3324　CTCs　per　10　mL　of　blood　and　with　a　diameter　of　14　to　20　m　(vs　8-12　m　for　lymphoma).　All　patients　were　CD47(+),　with　only　4.3%　to　61.2%　being　CD44(+).　The　number　of　CTCs　was　well　correlated　with　the　patient　TNM　stage　and　could　be　detected　in　patients　at　an　early　cancer　stage.　The　sorted　cells　could　be　recultured,　and　their　viability　was　preserved.　CONCLUSIONSThis　method　provides　a　novel　technique　for　highly　sensitive　and　specific　detection　and　isolation　of　CTCs　in　patients　with　colorectal　carcinoma.　This　method　complements　the　existing　approaches　for　the　de　novo　functional　identification　of　a　wide　variety　of　CTC　types.　It　is　likely　to　help　in　predicting　a　patient＇s　disease　progression　and　potentially　in　selecting　the　appropriate　treatment.　Cancer　2015;121:3036-3045.　(c)　2015　American　Cancer　Society.