Cell surface phenotypes of 113 B lineage acute lymphocytic leukemia (ALL) cases, defined by the presence of HLA-DR and at least one B-cell- specific antigen (either CD19, CD20, or CD22), were compared with antigen-defined stages of normal B lymphocyte development. The cases were first evaluated for expression of HLA-DR, CD19, CD34, CD10, CD20, and CD22 by indirect one-color immunofluorescence. Pairwise comparisons of cell surface marker expression were performed for each leukemic sample: no correlations were observed for paired antigen expression on the leukemic samples using antigens expressed either early or late during normal B lymphoid development. Complete immunophenotypes of the cases were then compared with normal B-cell developmental stages. Sixteen different complete immunophenotypes were observed on the leukemias that were not found in normal marrow; at least 78% of the cases demonstrated such “asynchronous” combinations of B lymphoid- associated differentiation antigens. Several samples were subsequently studied by two-color immunofluorescence, and the presence of doubly labeled cells with “asynchronous” antigen combinations was confirmed. These results indicate that the majority of B lineage leukemias exhibit “developmental asynchrony,” as compared with normal marrow B cells. The data further suggest that ALL cases do not accurately represent cells arrested at the stage where the leukemogenic event occurred. Rather, ALL appears to be a disease in which there may be maturation of leukemic blasts; but this maturation is “asynchronous” when compared with the normal developmental process.

Multiparameter flow cytometry was applied on normal human bone marrow (BM) cells to study the lineage commitment of progenitor cells ie, CD34+ cells. Lineage commitment of the CD34+ cells into the erythroid lineage was assessed by the coexpression of high levels of the CD71 antigen, the myeloid lineage by coexpression of the CD33 antigen and the B-lymphoid lineage by the CD10 antigen. Three color immunofluorescence experiments showed that all CD34+ BM cells that expressed the CD71, CD33, and CD10 antigens, concurrently stained brightly with anti-CD38 monoclonal antibodies (MoAbs). In addition, the CD38 antigen was brightly expressed on early T lymphocytes in human thymus, characterized by CD34, CD5, and CD7 expression. Only 1% of the CD34+ cells, 0.01% of nucleated cells in normal BM, did not express the CD38 antigen. The CD34+, CD38- cell population lacked differentiation markers and were homogeneous primitive blast cells by morphology. In contrast the CD34+, CD38 bright cell populations were heterogeneous in morphology and contained myeloblasts and erythroblasts, as well as lymphoblasts. These features are in agreement with properties expected from putative pluripotent hematopoietic stem cells; indeed, the CD34 antigen density decreased concurrently with increasing CD38 antigen density suggesting an upregulation of the CD38 antigen on differentiation of the CD34+ cells. Further evidence for a strong enrichment of early hematopoietic precursors in the CD34+, CD38- cell fraction was obtained from culture experiments in which CD34+ cell fractions with increasing density of the CD38 antigen were sorted singularly and assayed for blast colony formation. On day 14 of incubation, interleukin-3 (IL-3), IL-6, and GM-CSF, G-CSF, and erythropoietin (Epo) were added in each well. Twenty-five percent of the single sorted cells that expressed CD34 but lacked CD38 antigen gave rise to primitive colonies 28 to 34 days after cell sorting. The ability to form primitive colonies decreased rapidly with increasing density of the CD38 antigen. During 120 days of culture, up to five sequential generations of colonies were obtained after replating of the first-generation primitive colonies. This study provides direct evidence for the existence of a single class of progenitors with extensive proliferative capacity in human BM and provides an experimental approach for their purification, manipulation, and further characterization.

The expression of two epitopes on glycophorin A (GPA) during erythroid development was examined on normal human bone marrow using quantitative flow cytometry. The highly correlated binding of two monoclonal antibodies, one sensitive and the other insensitive to glycosylation, indicated that the two epitopes were coordinately expressed during erythroid development. Both antigens reached a maximum expression during the early normoblast stage and were maintained at a constant amount per cell throughout further maturation to erythrocytes. These data suggest that glycosylation of GPA, as detected by antibodies recognizing blood group (M) and (N) antigens, does not increase during erythroid maturation.

A panel of B lymphoid-reactive monoclonal antibodies was used to analyze the phenotypic changes that accompany B lymphocyte development in normal human bone marrow. The B lymphoid cells were identified using light scattering and the expression of CD19 on a flow cytometer. Quantitative three-color immunofluorescence was then used to correlate other cell surface antigens on these cells identified as B lymphoid in normal marrow. CD10 and CD20 identified almost exclusive populations and provided a convenient means of discriminating between the less and more mature B lineage cells. The CD10+ cells could be further subdivided using CD34. The population of CD19+, CD10+, CD34+ cells comprised only 0.6% of marrow cells, but these contained the majority of terminal deoxynucleotidyl transferase (TdT+) cells. In the assessment of class II antigens, HLA-DR was expressed on all B lineage cells whereas HLA-DP preceded HLA-DQ in appearance during the developmental process. Among the later antigens expressed on B lineage cells, cell surface IgM, CD20, and HLA-DQ were expressed at essentially the same time. Cell surface CD10 was lost at the time when CD21 and CD22 were acquired on the cell surface. These data illustrate that multiparameter flow cytometry can be used to define a continuous progression of stages of B lymphocyte development based on cell surface antigen expression even though these cells represent a minor fraction of normal marrow cells.

The low frequency of plasma cells and the lack of specific cell surface markers has been a major obstacle for a detailed characterization of plasma cells in normal human bone marrow. Multiparameter flow cytometry enabled the identification of plasma cells in normal bone marrow aspirates. The plasma cells were located in a unique position in the correlation of forward light scattering, orthogonal light scattering, and immunofluorescent-labeled CD38. The identity of the sorted cell populations was confirmed by microscopic examination of Wright's stained slides and slides stained for cytoplasmic immunoglobulin using polyclonal antibodies reactive with light chains; ie, anti-kappa fluorescein isothiocyanate and anti lambda phycoerythrin (PE). The purity of the sorted plasma cells was greater than 97% (n = 4). The average frequency of plasma cells in normal bone marrow aspirates was low--0.25% of the nucleated cells (n = 7)--but surprisingly consistent between individuals (SD = .05; range 0.14% to 0.30%). A detailed analysis showed two distinct populations of plasma cells: (1) A population relatively smaller by forward light scattering expressed CD22, CD35, and sigE and was identified as early plasma cells (ie, lymphoplasmacytoid), and (2) a population larger by forward light scattering lacked these markers and was identified as mature plasma cells. The antigenic profile of the normal plasma cells was determined in two-color immunofluorescence studies. The expression of cell surface immunoglobulin G (IgG), IgA, IgE, IgD, IgM, and the cell surface antigens CD10, CD11b, CD13, CD11c, CD14, CD15, CD16, CD19, CD22, CD20, CD33, CD35, CD45, and HLA-DR was determined on the plasma cells. A significant heterogeneity in cell surface antigen expression was observed within the plasma cell population. Unexpectedly, myeloid- specific cell surface antigens such as CD33 and CD13 and the early B- cell antigen identified by CD10 were expressed on a proportion of plasma cells. These observations imply that the association of myeloid and early B-cell markers described in multiple myeloma may not be associated with the neoplasia but is a normal phenomenon.