Analysis of Marrow or PBSC for Tumor Cells

Analysis of marrow or PBSC for tumor cells is essential for assessing the efficacy of purging and for interpreting results of clinical trials. Marrow will be tested for neuroblastoma cells with monoclonal antibodies against cell surface antigens by the CHLA Immunocytology Laboratory. Immunocytology is used to assess each step of purging; details are provided below ^(7,8).

Mononuclear cells are prepared from a small aliquot of marrow by equilibrium sedimentation of cells over Ficoll-Hypaque (e.g.= 1.077). Mononuclear cells that are buoyant at the density include essentially all neuroblastoma cells, while erythrocytes and granulocytes are sedimented. This enriches for neuroblastoma cells making immunocytologic examination more sensitive.

Detection of tumor is carried out with five monoclonal antibodies that are reactive with cell surface antigens of neuroblastoma but not normal bone marrow cells (390, 459, HSAN 1.2, 14G2A and BW-575). Viable cells are incubated with a mixture of all five monoclonal antibodies (105 cells with 10 µg of each antibody in 100 µl of L 15-FCS, 30 min, room temp), washed three times, resuspended at 106 cells per ml, and cytocentrifugated onto coverslips (5 x 104 cells in 0.05 ml). After drying, cells are fixed with 2% paraformaldehyde and then sequentially incubated with 10 mM phenylhydrazine (one hour, 37°C), biotinylated goat anti-mouse immunoglobulin, and then avidin-biotin-peroxidase complexes (one hr each, room temperature; Vector Laboratories).

Bound peroxidase is visualized with diaminobenzidene/H2O2 (5 minutes, room temperature), and cells are counterstained with Mayer's hematoxylin. Cells are washed between all steps for 3 minutes with PBS.