CD34-positive cells and their subpopulations characterized by flow cytometry analyses on the bone marrow of healthy allogenic donors.
OBJECTIVE
Counting and separating hematopoietic stem cells from different sources has importance for research and clinical assays. Our aims here were to characterize and quantify hematopoietic cell populations in marrow donors and to evaluate CD34 expression and relate this to engraftment.


METHODS
Cross-sectional study on hematopoietic stem cell assays, using flow cytometry on donor bone marrow samples, for allogenic transplantation patients at two hospitals in São Paulo.


METHODS
Immunophenotyping of marrow cells was performed in accordance with positive findings of CD34FITC, CD117PE, CD38PE, CD7FITC, CD33PE, CD10FITC, CD19PE, CD14FITC, CD13PE, CD11cPE, CD15FITIC, CD22PE, CD61FITC and CD56PE monoclonal antibodies in CD45PerCP+ cells, searching for differentiation and maturation regions. CD34+ sorting cells were analyzed for CD38 and CD117. Rh-123 retention was done before and after sorting. Antigen expression and CD34+ cells were correlated with engraftment.


RESULTS
In region R1, 0.1% to 2.8% of cells were CD34+/CD45+ and 1.1%, CD34+/CD45-. The main coexpressions of CD45+ cells were CD38, CD22, CD19 and CD56 in R2 and CD33, CD11c, CD14, CD15 and CD61 in R3 and R4. After sorting, 2.2x10(6) CD34+ cells were equivalent to 4.9% of total cells. Coexpression of CD34+/CD38+ and CD34+/CD117+ occurred in 94.9% and 82% of events, respectively. There was a positive relationship between CD34+ cells and engraftment. More than 80% of marrow cells expressed high Rh-123. CD34+ cell sorting showed that cells in regions of more differentiated lineages retained Rh-123 more intensively than in primitive lineage regions.


CONCLUSIONS
We advocate that true stem cells are CD34+/CD45-/CD38-/low-Rh-123 accumulations.
Carvalho
Jerusa Martins
JM
Division of Hematology and Transfusion Medicine, Hospital São Paulo, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, Brazil.
Souza
Marlon Knabben de
MK
Buccheri
Valéria
V
Rubens
Cláudia Viviane
CV
Kerbauy
José
J
Oliveira
José Salvador Rodrigues de
JS
D051997:ADP-ribosyl Cyclase 1 / Q000032:analysis / Q000276:immunology; D018952:Antigens, CD34 / Q000032:analysis / Q000276:immunology; D001854:Bone Marrow Cells / Q000166:cytology / Q000276:immunology / Q000378:metabolism; D002454:Cell Differentiation; D003430:Cross-Sectional Studies; D005434:Flow Cytometry; D018380:Hematopoietic Stem Cell Transplantation; D006412:Hematopoietic Stem Cells / Q000276:immunology / Q000378:metabolism; D006801:Humans; D016130:Immunophenotyping; D017493:Leukocyte Common Antigens / Q000032:analysis / Q000276:immunology; D019009:Proto-Oncogene Proteins c-kit / Q000032:analysis / Q000276:immunology; D020112:Rhodamine 123 / Q000378:metabolism; D014019:Tissue Donors; D014184:Transplantation, Homologous
D018952:Antigens, CD34; D020112:Rhodamine 123; D019009:Proto-Oncogene Proteins c-kit; D017493:Leukocyte Common Antigens; D051997:ADP-ribosyl Cyclase 1