title: Quantitative analysis of cobblestone area-forming cells in bone marrow of patients with aplastic anemia by limiting dilution assay.
abstract: In the past, the analysis of primitive human hematopoietic progenitor cells with repopulating activity was limited by lack of appropriate in vitro assay systems. It was recently shown that cobblestone area-forming cells (CAFC) giving rise to cobblestone areas after 5 weeks in long-term marrow cultures (LTMC) represent a population of pluripotent progenitor cells with long-term marrow-repopulating activity. We have used a microtiter limiting dilution-type human LTMC system to quantitate the frequency of CAFC (week 5) in aplastic anemia (AA). In bone marrow mononuclear cells (BM-MNC) of healthy donors (n = 36) we observed a mean frequency of 84.4 CAFC per 10(5) BM-MNC (95% confidence interval limits, 66.4 to 102.4). The mean frequency of CAFC in BM of 31 AA patients was 6.6 per 10(5) BM-MNC (95% confidence interval limits, 5.3 to 7.9; n = 47). This frequency is significantly lower as compared with controls (P < .0001). The frequency of CAFC was reduced not only in pancytopenic AA patients (6.2 per 10(5) BM-MNC; P < .0001 v control), but also in patients in remission after immunosuppression (7.6; P < .0001 v control; P = .1 v pancytopenic AA patients). The CAFC frequency did not correlate with the severity or duration of the disease and did not predict response to immunosuppressive treatment. In summary, the frequency of primitive hematopoietic progenitor cells, as measured by the CAFC assay, is significantly reduced in AA. CAFC remain severely reduced even after hematologic recovery after immunosuppressive treatment. The low frequency of CAFC in remission patients is in keeping with other data pointing to a persisting defect of hematopoiesis in patients in remission after immunosuppressive treatment.
authors.affiliation: Department of Medicine III, University of Ulm, Germany.
authors.forename: H
authors.initials: H
authors.lastname: Schrezenmeier
authors.forename: M
authors.initials: M
authors.lastname: Jenal
authors.forename: F
authors.initials: F
authors.lastname: Herrmann
authors.forename: H
authors.initials: H
authors.lastname: Heimpel
authors.forename: A
authors.initials: A
authors.lastname: Raghavachar
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D000741:Anemia, Aplastic / Q000473:pathology*; D001854:Bone Marrow Cells; D002478:Cells, Cultured; D003114:Colony-Forming Units Assay / Q000379:methods*; D005260:Female; D006412:Hematopoietic Stem Cells / Q000166:cytology*; D006801:Humans; D007166:Immunosuppressive Agents / Q000627:therapeutic use; D007201:Indicator Dilution Techniques; D008297:Male; D008875:Middle Aged
chemical_list: D007166:Immunosuppressive Agents