Rapid Enrichment and Detection of Melanoma Cells from Peripheral Blood Mononuclear Cells by a New Assay Combining Immunomagnetic Cell Sorting and Immunocytochemical Staining

Abstract

Commonly used methods for detection of melanoma cells in blood, including RT-PCR and immunocytochemistry, display only a limited sensitivity and specificity. Reliable detection of less than one melanoma cell per ml of blood is hardly possible using these methods. To obtain greater sensitivity so that a single melanoma cell in up to 25 ml of blood can be detected (5 × 10⁷ peripheral blood mononuclear cells, or PBMC), we developed a new assay for combined enrichment and immunocytochemical detection of disseminated melanoma cells from PBMC of patients with malignant melanomas. Melanoma cells are directly magnetically labeled using colloidal superparamagnetic microparticles approximately 60 nm in diameter conjugated to the anti-melanoma monoclonal antibody 9.2.27, with no reactivity to normal cells in blood. Magnetically labeled melanoma cells are enriched from PBMC by magnetic cell separation and detected by a new approach for immunocytochemical staining with monoclonal mouse anti-melanoma antibodies (antiMelanA and HMB-45). The efficiency of this assay was demonstrated in a model system in which 5–500 tumor cells from the melanoma cell line SKMEL-28 were seeded into PBMC samples from healthy donors containing 5 × 10⁷ leukocytes. Mean recovery of the seeded tumor cells was 47.4 ± 13.99% (n = 15). Applying the assay to 20–50 ml blood samples of patients with stage III-IV malignant melanomas, we were able to detect melanoma cells in two of eight patients (25%).