Removal of neuroblastoma cells from bone marrow by a direct monoclonal antibody rosetting technique

A one-step direct monoclonal antibody rosetting technique is described for removal of neuroblastoma cells from bone marrow. Two monoclonal antibodies (MoAbs) (BW 575, BW 625) were directly coupled to ox red blood cells by use of CrCl3. The IgG1 antibody BW 575 detects a 95-kD neuroblastoma cell-associated glycoprotein and the IgG3 antibody BW 625 recognizes the ganglioside GD 2. After coupling MoAbs to the erythrocytes, specific strong and stable rosettes were formed with neuroblastoma cells and effectively separated from mononuclear cells using density gradient centrifugation. A total of 1.5% IMR5 neuroblastoma cells were reliably removed from mononuclear cells beyond the limit of detection (less than 0.01%) as judged by tetanus toxin labeling. No impairment of stem cell growth (CFU-GM, BFU-E, CFU-GEMM, CFU-M) was observed. Recovery rate of mononuclear cells ranged between 35 and 69%. A red blood cell/nucleated cell ratio more than 50:1 resulted in increased loss of mononuclear cells and a ratio less than 30:1 in incomplete neuroblastoma cell removal. Using indirect rosettes the purging efficacy was lower and the mononuclear cell loss higher. We conclude that the direct monoclonal antibody rosetting technique may be a technically simple and effective alternative purging method for neuroblastoma patients, which is applicable even in cases demonstrating weak expression of one antigen.