Enumeration of CD34-positive hematopoietic progenitor cells by flow cytometry: comparison of a volumetric assay and the ISHAGE gating strategy.

In order to optimize peripheral blood stem cell (PBSC) collection for transplantation, absolute CD34 counts are necessary to determine the exact time-point for sufficient leukapheresis. In an effort to establish and to validate a rapid and reproducible assay for PBSC enumeration, different recommendations for selection of monoclonal antibodies, lysis techniques, analysis parameters and gating strategies were developed. In this methodical study, two gating strategies for PBSC enumeration were compared, in order to validate the accuracy of PBSC counting in peripheral blood and apheresis products. Gating strategy I was performed using volumetric flow cytometry and reference beads whereas gating strategy II was done according to the ISHAGE guidelines. The highly standardized volumetric assay seems to be superior to the more 'expert-reliant' ISHAGE procedure requiring more 'manual work' by the cytometrist.