Effective purging of bone marrow by a combination of immunorosette depletion and complement lysis.

The effectiveness of a simple immunorosette technique for the depletion of common acute lymphatic leukemic (cALL) blasts from autologous bone marrow transplants was studied. Erythrocytes were sensitized with tetramolecular complexes consisting of rat anti-mouse IgG1 monoclonal antibodies (McAbs) that crosslink two different mouse McAbs. One of the McAbs was directed against glycophorin A, and the other was directed against marker glycoproteins of B cells and their precursors (CD9, CD10, CD19, or CD22). Immunorosettes were formed by addition of the sensitized erythrocytes to the cALL+ cells. After density-gradient separation of immunorosettes from mixtures of cALL+/terminal deoxynucleotidyl transferase-positive (TdT+) leukemic blasts and mononuclear bone marrow cells, nearly a 2-log depletion of leukemic cells was measured by flow cytometry. Clonogenic assays with two cALL+B-cell lines (Ros-17 and Nalm-16) were performed to compare the efficacy of complement-mediated cell lysis, immunorosette depletion, and a combination of both procedures. Complement-mediated cytotoxicity with the three McAbs in combination with baby rabbit complement yielded a 1- to 2-log cell kill. Immunorosette depletion resulted in a 3-log reduction of clonogenic units. Sequential application of the two methods (immunorosette depletion with CD19 McAb followed by a complement lysis with CD9 and CD10 McAbs) led to superior results in causing a 4- to 5-log purging effect. These purging procedures did not cause a loss of normal myeloid (granulocyte-macrophage colony-forming units, CFU-GM) or erythroid (erythroid burst-forming units, BFU-e) progenitors from the bone marrow. This study indicates that the combination of the two methods results in a highly efficient purging procedure for the removal of cALL+ cells from autologous bone marrow cells.