Comparative quantification of umbilical cord blood CD34+ and CD34+ bright cells using the ProCount-BD and ISHAGE protocols.
The total number of CD34+ cells is the most relevant clinical parameter when selecting human umbilical cord blood (HUCB) for transplantation. The objective of the present study was to compare the two most commonly used CD34+ cell quantification methods (ISHAGE protocol and ProCount - BD) and analyze the CD34+ bright cells whose 7-amino actinomycin D (7AAD) analysis suggests are apoptotic or dead cells. Twenty-six HUCB samples obtained at the Placental Blood Program of New York Blood Center were evaluated. The absolute numbers of CD34+ cells evaluated by the ISHAGE (with exclusion of 7AAD+ cells) and ProCount (with exclusion of CD34+ bright cells) were determined. Using the ISHAGE protocol we found 35.6 +/- 19.4 CD34+ cells/microL and with the ProCount method we found 36.6 +/- 23.2 CD34+ cells/microL. With the ProCount method, CD34+ bright cell counts were 9.3 +/- 8.2 cells/microL. CD34+ bright and regular cells were individually analyzed by the ISHAGE protocol. Only about 1.8% of the bright CD34+ cells are alive, whereas a small part (19.0%) is undergoing apoptosis and most of them (79.2%) are dead cells. Our study showed that the two methods produced similar results and that 7AAD is important to exclude CD34 bright cells. These results will be of value to assist in the correct counting of CD34+ cells and to choose the best HUCB unit for transplantation, i.e., the unit with the greatest number of potentially viable stem cells for the reconstitution of bone marrow. This increases the likelihood of success of the transplant and, therefore, the survival of the patient.
Pranke
P
P
Laboratório de Hematologia, Faculdade de Farmácia, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brasil. ppranke@adufrgs.ufrgs.br
Hendrikx
J
J
Alespeiti
G
G
Nardi
N
N
Rubinstein
P
P
Visser
J
J
D018952:Antigens, CD34 / Q000097:blood*; D001771:Blood Banks; D001772:Blood Cell Count / Q000379:methods*; D002470:Cell Survival; D003114:Colony-Forming Units Assay / Q000379:methods*; D003609:Dactinomycin / Q000031:analogs & derivatives; D005312:Fetal Blood / Q000166:cytology*; D005434:Flow Cytometry; D005456:Fluorescent Dyes; D006412:Hematopoietic Stem Cells / Q000166:cytology*; D006801:Humans; D015203:Reproducibility of Results
D018952:Antigens, CD34; D005456:Fluorescent Dyes; D003609:Dactinomycin; C025942:7-aminoactinomycin D