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 cytometry system for the enumeration of CD34+ cells in apheresis components, peripheral blood, and cord blood samples in routine laboratory work. This system is compared with the following flow cytometry protocols: Milan, ISHAGE, ISHAGE with 7-AAD, and flow-count fluorospheres.


METHODS
Correlation, linearity, and reproducibility studies were performed for the various methods. Clonogenic cultures were performed, as an external control, to assess the correlation between the number of CD34+ cells per microL and the number of colony-forming units per microL.


RESULTS
The linear regression analysis demonstrated that the five methods were comparable (R2 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 CD34+ cell counts below 10 cells per microL (R2 = 0.46 and 0.47). The reproducibility assay for a CD34+ count of 10 cells per microL showed a CV of 12 percent and 25 percent for the Milan and CD34+ assay methods, respectively. The mean CV among all five methods for the 46 evaluated samples was 20 percent. There was a strong correlation between the number of CD34+ cells per microL and colony-forming units per microL in cord blood and apheresis samples (r = 0.71-0.81).


CONCLUSIONS
The CD34+ assay is useful in CD34 enumeration in cord blood, leukapheresis samples, and peripheral blood samples and provides comparable results to the Milan, ISHAGE, ISHAGE with 7-AAD, and flow-count methods. Nevertheless, peripheral blood samples with low CD34 absolute counts (below 10 cells/microL) should be analyzed by alternative flow cytometry protocols. Even though the same operator performed the study in a single laboratory, the high inter-method CV suggests that differences in sample preparation and gating 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.
Cabezudo
E
E
Department of Criobiology and Cell Therapy, Cancer Research Institute, Duran i Reynals Hospital, Barcelona, Spain. ecabezudo@iro.es
Querol
S
S
Cancelas
J A
JA
García
J
J
D018952:Antigens, CD34 / Q000097:blood*; D001769:Blood; D001772:Blood Cell Count; D003114:Colony-Forming Units Assay; D003584:Cytological Techniques; D005240:Feasibility Studies; D005312:Fetal Blood; D005434:Flow Cytometry / Q000379:methods*; D006412:Hematopoietic Stem Cells / Q000276:immunology*; D006801:Humans; D007937:Leukapheresis; D016014:Linear Models; D012044:Regression Analysis; D015203:Reproducibility of Results
D018952:Antigens, CD34