Enumeration of CD34-Positive Stem Cells: Evaluation and Comparison of Three Methods

Authors:
  • MBMark E. Brecher
    Mark E. Brecher
  • KGKarla Gertis
    Karla Gertis
  • AJAlan Jenkins
    Alan Jenkins
  • DNDebbie Nickischer
    Debbie Nickischer
  • JSJohn L. Schmitz
    John L. Schmitz
  • SSSara Sparks
    Sara Sparks
  • PWPatricia Wright
    Patricia Wright
  • SBStuart A. Bentley
    Stuart A. Bentley
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Abstract
Accurate enumeration of CD34+ stem cells is important in assessing the need for continued mobilization and subsequent apheresis collections. We compared two new analysis systems, ProCOUNT™ (Becton Dickinson Immunocytometry Systems) and IMAGN® 2000 STELLer™ (Biometric Imaging, Inc.) with our current (3-Color) flow cytometry-based method. The ProCOUNT™ system uses an absolute counting tube, which contains reference beads and a specific (multiple) gating strategy to determine an absolute count. The STELLer™ assay combines microvolume fluorimetry and automated analysis software to determine an absolute count. To evaluate linearity and reproducibility, peripheral blood was spiked with CD34+ cells (KG1a cell line). Three dilution series (measured at ≅0, 5, 10, 25, 50, and 100 CD34+ cells/μl) were analyzed by each method. Analysis of predicted versus actual CD34+ concentration showed excellent correlation with all methods (r2 ≥ 0.97, slope 0.98–1.04). To further assess precision, two PBSC samples, at approximately 200 and 800 CD34+ cells/μl, respectively, were analyzed 10 times by each method. Coefficients of variation for the precision analysis of these samples were 5.1%–6.4% and 5.4%–12.3%, respectively. To assess overall performance, 75 patient specimens were analyzed. Excellent correlation (r2 values of 0.89–0.98) was observed among all three methods. We conclude that the three methods provide comparable linearity and reproducibility.

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