A multicolor, no‐lyse no‐wash assay for the absolute counting of CD34+ cells by flow cytometry

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Abstract
Background We previously developed a method for counting CD34+ cells in unlysed whole blood. This method was applied to normal human bone marrow, peripheral blood after mobilization of progenitor cells, leukapheresis products, and cord blood and was validated with two different lyse‐no wash methods. However, the main advantage that we described, erythrocyte discrimination using nucleic acid staining, was also the main restriction because additional markers for the immunologic characterization of CD34+ cells cannot be included. Methods We used SYTO‐13 and fluorescein isothiocyanate (FITC)‐CD45 staining (FL1) instead of SYTO‐13 and phycoerythrin (PE)Cy5‐CD45 staining (FL3) to leave the third and fourth fluorescence parameters available for further characterization of CD34+ cells. The new method was validated by applying it to cord blood samples (n = 20). Results FITC‐CD45 antibody gave a 1.7‐fold increase in mean fluorescence intensity over SYTO‐13 alone. From absolute counts (CD34+ cells per microliter), we plotted the differences between the methods against their mean, showing that differences fell into acceptable ranges. Conclusions No‐lyse procedures may represent an advance for cell immunophenotyping and it could be applied to the measurement of additional markers. Cytometry (Clin. Cytometry) 50:249–253, 2002.