Consistency of the initial cell acquisition procedure is critical to the standardization of CD34+ cell enumeration by flow cytometry: results of a pairwise analysis of umbilical cord blood units and cryopreserved aliquots.
BACKGROUND
The CD34+ cell content is a predictive factor for engraftment and survival after umbilical cord blood (UCB) transplantation. The high variability in the CD34 assay results in different recommended cell doses for infusion across transplant centers and also limits the clinical utility of the CD34+ cell counts provided by cord blood banks (CBBs). This bi-institutional study was intended to understand the sources of this variability.


METHODS
The level of CD34 agreement between the University of Minnesota (UM) and the Madrid CBB (MCBB) was evaluated on 50 UCB units before and after cryopreservation. Two cryopreserved vials per unit were thawed and processed at both laboratories. Dual-platform ISHAGE-based flow cytometry was used for CD34 enumeration.


RESULTS
Postthaw nucleated cell recoveries were similar. However, whereas CD34+ cell enumeration before freezing was 0.35 +/- 0.22 percent, the results after thawing were 0.98 +/- 0.65 and 0.57 +/- 0.39 percent at UM and MCBB, respectively. Bland-Altman plots analysis ruled out the interchangeability of MCBB and UM CD34 values. Differences in the initial cell acquisition settings accounted for most of the CD34 discrepancy, which was no longer present after normalization of the forward scatter threshold for cell acquisition.


CONCLUSIONS
The standardization of CD34+ cell enumeration by flow cytometry is strongly reliant on a consistent initial cell acquisition procedure. The interlaboratory variation can be minimized by using frozen cell aliquots as reference samples. Both requisites should be considered for CD34 testing and UCB unit selection by regulatory institutions involved with cord blood banking and transplantation.
Flores
Ana I
AI
Madrid Cord Blood Bank and the Department of Hematology, Hospital Universitario 12 de Octubre, Madrid, Spain.
McKenna
David H
DH
Montalbán
M Angeles
MA
De la Cruz
Javier
J
Wagner
John E
JE
Bornstein
Rafael
R
D018952:Antigens, CD34 / Q000032:analysis / Q000097:blood / Q000378:metabolism*; D001772:Blood Cell Count / Q000379:methods / Q000592:standards; D002469:Cell Separation / Q000379:methods / Q000592:standards; D002470:Cell Survival; D002478:Cells, Cultured; D036101:Cord Blood Stem Cell Transplantation / Q000379:methods / Q000592:standards; D015925:Cryopreservation / Q000379:methods; D005169:Factor VIII / Q000032:analysis* / Q000378:metabolism; D005312:Fetal Blood / Q000166:cytology* / Q000378:metabolism; D005340:Fibrinogen / Q000032:analysis* / Q000378:metabolism; D005434:Flow Cytometry / Q000379:methods / Q000592:standards*; D006801:Humans; D016014:Linear Models; D016555:Matched-Pair Analysis; D015203:Reproducibility of Results
D018952:Antigens, CD34; C026912:cryoprecipitate coagulum; D005169:Factor VIII; D005340:Fibrinogen