BACKGROUND: This 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.METHODS: Quantitative 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.RESULTS: The 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 33 ± 24 CTCs per 10 mL of blood and with a diameter of 14 to 20 μm (vs 8‐12 μm 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.CONCLUSIONS: This 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. © 2015 American Cancer Society.