Purification of common acute lymphoblastic leukemia antigen positive cells from normal human bone marrow

Abstract

Mononuclear cells expressing the common acute lymphoblastic leukemia antigen (CALLA) were purified from normal adult human bone marrow, where they constitute a small fraction of the total population. This was accomplished by a two-step purification from Ficoll-Hypaque-isolated mononuclear cells. Isolated mononuclear cells were first labeled with a mixture of monoclonal antibodies (MoAb) specific for myeloid and erythroid precursor cells, and immune rosettes were then formed with sheep erythrocytes coated with rabbit anti-mouse antibodies (R/M-SRBC). Sedimentation through Ficoll-Hypaque then eliminated the majority of mature myeloid cells. The second step consisted of labeling the remaining rosette-negative cells with CALLA-specific MoAb and purifying CALLA+ cells by fluorescence activated cell sorting. Alternatively, CALLA+ cells were purified in a second R/M-SRBC rosette sedimentation step. The purified CALLA+ cells, which morphologically were medium to large lymphoid cells, were subsequently studied using dual fluorescence techniques to identify surface markers as well as intracytoplasmic staining to detect terminal deoxynucleotidyl transferase enzyme (TdT) and intracytoplasmic mu. While the CALLA+ cell suspensions contained very few mature myeloid cells or T lymphocytes, the finding that 5% to 11% of them were cyto-mu+ and 13% to 22% expressed the B1 differentiation antigen clearly indicated that at least some of these cells were B cell precursors. Because 48% to 63% of the cells were TdT+ and practically all of them expressed Ia antigen, it appears that these cells are a mixture of very early lymphoid precursor cells as well as more differentiated pre-B cells. The phenotype of these normal cells is very similar to that of common ALL cells. Differences in the surface marker phenotypes between adult and fetal CALLA+ cells that have previously been purified were also identified.