Background: In the absence of a gold standard for hematopoietic progenitor counting, the intra-laboratory
variation between commonly used strategies for progenitor assessment was compared. Methods and Materials: We used a
pool of FITC-conjugated monoclonal antibodies (CD14, CD11b) and PE-CD34 to facilitate CD34 counting, excluding
CD14+/CD11b+ bright cells. We compared this protocol with other common methodologies, such as the single-staining
approach, known as the Milan method, and the ISHAGE multiparameter method. Results: We show that the CD14/CD11b
protocol is a valid approach to progenitor cell counting. Though different methods give different results for progenitor cell
counting, Lin’s coefficient shows high concordance among flow cytometry counts but a substantial difference with colony-
forming unit counts. Both the ISHAGE and CD14/CD11b protocol give higher counts than the Milan method.
Moreover, on revising the ISHAGE analysis, we described a rare population of cells with the CD34+CD45neg phenotype,
which could have an impact in CD34 counting. Conclusions: We have designed a valid alternative approach for hematopoietic
progenitor cell counting, and we show that different methods give different results.
Keywords: CD34, CD45, flow cytometry, ISHAGE, Milan, quality control, Hematopoietic Progenitor Cell Counting, monoclonal antibodies
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