Comparison of ISHAGE protocol CD34 cell enumeration with a lineage negative backgating technique

Background: CD34⁺ cell enumeration in PBSC apheresis products has become the standard for assessing graft hematopoietic potential. Methods: An in-house, three color, lineage negative-gating technique [University of Nebraska Medical Center (UNMC) protocol] for CD34 cell enumeration was compared with the ISHAGE protocol over 100 apheresis products. Cell doses determined by both methods were compared with each other and to colony-forming units-granulocyte/macrophage (CFU-GM) assay results. Results: Overall, the assays compared well with each other for samples with CD34 cell doses >0.2 × I0⁶/kg (r values >0.8). The ISHAGE method showed a constant negative bias, with a mean of 38% in comparison to the UNMC protocol, which was more linear at lower cell doses. Both assays showed similar correlation with CFU-GM doses after log conversion (UNMC, r=0.915; ISHAGE, r=0.917). When comparing integer values, however, the ISHAGE method correlated with CFU-GM only in the high dose range (CFU-GM>2 × 10⁴/kg), while the UNMC method correlated across the entire measured range of CFU-GM doses. Finally, an inter-technologist gating reproducibility study (n=6) yielded a 23% coefficient of variation (CV) for the ISHAGE method and a 7% CV for the UNMC method, when the same two sets of CD34 histograms were analyzed to calculate cell dose. Discussion: In this study the lineage negative protocol (UNMC) had a larger dynamic range, correlated better with CFU-GM results and showed better inter-technologist reproducibility than the ISHAGE method.