title: Rapid discrimination of early CD34+ myeloid progenitors using CD45-RA analysis.
abstract: Mononuclear cells (MNC) isolated by density centrifugation of cord blood and healthy bone marrow, and of peripheral blood (PB) from patients treated with granulocyte-macrophage colony-stimulating factor (GM-CSF) or G-CSF after chemotherapy, were double-stained with anti CD34 monoclonal antibody (MoAb) (8G12) versus anti CD45, CD45-RB, CD45-RO, and CD45-RA, respectively, and analyzed by flow cytometry. In all specimens, CD34+ MNC co-expressed CD45 at a low level and the expression of CD45-RB was similar or slightly higher. Most CD34+ MNC were negative for CD45-RO, a weak coexpression was only seen in some bone marrow (BM) and blood samples. In contrast, CD45-RA could subdivide the CD34+ population into fractions negative, dim (+), and normal positive (++) for these subgroups, and typical staining patterns were observed for the different sources of hematopoietic cells: in BM, most CD34+ MNC were RA++. In PB, their majority was RA++ after G-CSF but RA+ or RA- after GM-CSF. In cord blood, the hematopoietic progenitors were mainly RA-/RO-. Semisolid culture of sorted CD34+ MNC showed that clusters and dispersed (late) colony-forming unit-GM (CFU-GM) originated from 34+/RA++ cells, while the 34+/RA- MNC formed compact and multicentric, both white and red colonies derived from early progenitors. Addition of 20 ng stem cell factor per milliliter of medium containing 34+/RA- cord blood MNC led to a change of many burst-forming unit-erythrocyte (BFU-E) to CFU-mix which was not, at least to this extent, seen in blood and BM. We conclude that early myeloid CD34+ cells are 45+/RA-. Because this population excludes 34+/19+ B cells and 33+ myeloid cells, both of which are RA++, two-color flow cytometric analysis using CD34 and CD45-RA facilitates the characterization and quantification of early myeloid progenitor cells.
authors.affiliation: Children's Cancer Research Institute, Vienna, Austria.
authors.forename: G
authors.initials: G
authors.lastname: Fritsch
authors.forename: P
authors.initials: P
authors.lastname: Buchinger
authors.forename: D
authors.initials: D
authors.lastname: Printz
authors.forename: F M
authors.initials: FM
authors.lastname: Fink
authors.forename: G
authors.initials: G
authors.lastname: Mann
authors.forename: C
authors.initials: C
authors.lastname: Peters
authors.forename: T
authors.initials: T
authors.lastname: Wagner
authors.forename: A
authors.initials: A
authors.lastname: Adler
authors.forename: H
authors.initials: H
authors.lastname: Gadner
mesh_terms: D000911:Antibodies, Monoclonal; D015703:Antigens, CD / Q000032:analysis*; D018952:Antigens, CD34; D016026:Bone Marrow Transplantation; D006528:Carcinoma, Hepatocellular / Q000097:blood / Q000188:drug therapy; D002478:Cells, Cultured; D003114:Colony-Forming Units Assay; D005312:Fetal Blood / Q000166:cytology / Q000276:immunology; D016179:Granulocyte Colony-Stimulating Factor / Q000627:therapeutic use; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000276:immunology; D006801:Humans; D017493:Leukocyte Common Antigens / Q000032:analysis / Q000276:immunology*; D008113:Liver Neoplasms / Q000097:blood / Q000188:drug therapy; D009000:Monocytes / Q000166:cytology* / Q000276:immunology; D009447:Neuroblastoma / Q000097:blood / Q000188:drug therapy; D011994:Recombinant Proteins / Q000627:therapeutic use; D012208:Rhabdomyosarcoma / Q000097:blood / Q000188:drug therapy; D014019:Tissue Donors
chemical_list: D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D018952:Antigens, CD34; D011994:Recombinant Proteins; D016179:Granulocyte Colony-Stimulating Factor; D017493:Leukocyte Common Antigens