Comparison of volumetric capillary cytometry with standard flow cytometry for routine enumeration of CD34+cells

BACKGROUND: This study assesses the feasibility of a new volumetric cytomet ry system for the enumeration of CD34+ cells in apheresis components, perip heral blood, and cord blood samples in routine laboratory work. This system is compared with the following flow cytometry protocols: Milan, ISHAGE, IS HAGE with 7-AAD, and flow-count fluorospheres. STUDY DESIGN AND METHODS: Correlation, linearity, and reproducibility studi es were performed for the various methods. Clonogenic cultures were perform ed, as an external control, to assess the correlation between the number of CD34+ cells per mu L and the number of colony-forming units per mu L. RESULTS: The linear regression analysis demonstrated that the five methods were comparable (R-2 ranged from 0.86 to 0.96 and slopes were close to 1). The CD34+ assay and the flow-count methods showed poor linearity for CD34cell counts below 10 cells per mu l (R-2 = 0.46 and 0.47). The reproducibil ity assay for a CD34+ count of 10 cells per mu L showed a CV of 12 percent and 25 percent for the Milan and CD34+ assay methods, respectively. The mea n CV among all five methods for the 46 evaluated samples was 20 percent. Th ere was a strong correlation between the number of CD34+ cells per mu L and colony-forming units per mu L in cord blood and apheresis samples (r = 0.7 1-0.81). CONCLUSION: The CD34+ assay is useful in CD34 enumeration in cord blood, le ukapheresis samples, and peripheral blood samples and provides comparable r esults to the Milan, ISHAGE, ISHAGE with 7-AAD, and flow-count methods. Nev ertheless, peripheral blood samples with low CD34 absolute counts (below 10 cells/mu L) should be analyzed by alternative flow cytometry protocols. Ev en though the same operator performed the study in a single laboratory, the high inter-method CV suggests that differences in sample preparation and g ating strategy are factors that increase variability. Protocols with fewer intermediate steps or fully automated protocols such as the CD34+ assay are expected to reduced intra- and inter-laboratory variability.