M. Safford

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.

Neutrophil maturation was studied in normal human bone marrow aspirates using multidimensional flow cytometry in comparison with morphology. The combination of the monoclonal antibodies, CD11b, CD15, and CD16, in addition to the forward and orthogonal light scattering signals permitted the isolation of neutrophilic cells from cells of other cell lineages with a purity of greater than 99%. An unexpectedly close relationship was found between the identification of neutrophil maturation by flow cytometry and morphological classification of cells sorted based on cell surface antigen expression and light scattering properties. The neutrophils could be divided into six distinct maturational stages, i.e., stage N I contained predominantly myeloblasts; stage N II, predominantly promyelocytes; stage N III, predominantly early myelocytes; stage N IV, predominantly myelocytes and metamyelocytes; stage N V, predominantly metamyelocytes and bands; and stage VI, predominantly segmented neutrophils. These data suggest that the morphologic changes during neutrophil maturation can be identified by flow cytometry using simultaneous quantitative assessment of multiple antigens in concordance with the light scattering properties of the human bone marrow cells.

Acute ethanol intoxication inhibits neutrophil delivery to sites of inflammation and, concomitantly, reduces the adhesion of neutrophils to surfaces. The effect of ethanol on several other neutrophil functions required for normal delivery are examined herein. Serum-free neutrophil suspensions showed normal resting adherence to endothelial monolayers in ethanol concentrations up to 1000 mg/dL, but when neutrophils were stimulated by 10(-6)M N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) to induce hyperadherence, ethanol induced a dose-dependent inhibition that was significant at concentrations greater than or equal to 500 mg/dL. Pretreating the endothelium with ethanol had no effect. Similarly, resting surface expression of the adhesive glycoprotein Mac-1 was unaffected by ethanol, but its up-regulation induced by fMLP was inhibited by 25.5% at 250 mg of ethanol/dL and by 52.3% at 1000 mg/dL. Release of both primary and secondary granule contents after activation showed dose-dependent inhibition, whereas resting granule content and spontaneous release were unaffected. Passive neutrophil deformability was significantly enhanced in 500 mg of ethanol/dL. Thus, ethanol affects several neutrophil delivery functions normally activated by inflammatory stimuli.

Gradual increase of CD38 on cells expressing CD34 characterizes the early cell differentiation pathway of normal human hematopoietic progenitors. In this study the coordinated expression pattern of CD34 and CD38 was assessed on leukemic blasts from bone marrow aspirates of 95 patients with newly diagnosed acute myeloid leukemia (AML). Expression was divided into six categories analogous to the differentiation pathway of normal bone marrow. The CD38 antigen was expressed on the leukemic cells of all patients and CD34+ leukemic cells were found in 79 patients (83%). In 93 patients, the leukemic cells were found along the differentiation pathway defined by CD34 and CD38. In 33 of the 93 patients, a part of the CD34+ cells did not express the CD38 antigen (categories 1 and 2). In another 33 patients, all CD34+ cells expressed CD38 (categories 3 and 4). In the remaining 27 patients, only cells were found which dimly expressed CD34 or did not express CD34 (categories 5 and 6). Of the 93 patients, 88 were treated with intensive chemotherapy according to the protocol of the German AML Cooperative Group. Of these, 21 died early and were not evaluable for treatment response. Complete remission was achieved in 14 of 22 patients (64%) in categories 1 and 2, in 19 of 26 patients (73%) in categories 3 and 4, and in 18 of 19 patients (95%) in categories 5 and 6. The event-free survival was significantly longer in patients of categories 5 and 6 compared to patients in categories 1 and 2 (p less than 0.01) and categories 3 and 4 (p less than 0.05), respectively. We conclude that in the majority of AML patients the immunophenotype of leukemic cells follows the early cell differentiation pathways defined by coordinated expression of CD34 and CD38 similar to that of normal hematopoietic progenitors. The presence of cells in the late cell differentiation stages (CD34+/-, CD38 /+) identifies patients with a higher complete remission rate and longer complete remission duration.