A multicolor, no-lyse no-wash assay for the absolute counting of CD34+ cells by flow cytometry.
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.
D018952:Antigens, CD34 / Q000276:immunology*; D002452:Cell Count / Q000379:methods*; D002469:Cell Separation / Q000379:methods*; D004396:Coloring Agents; D005312:Fetal Blood; D005434:Flow Cytometry / Q000379:methods*; D016650:Fluorescein-5-isothiocyanate; D019085:Fluorescent Antibody Technique, Direct / Q000379:methods; D005456:Fluorescent Dyes; D018380:Hematopoietic Stem Cell Transplantation / Q000379:methods*; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000276:immunology; D006801:Humans; D016130:Immunophenotyping / Q000379:methods; D007231:Infant, Newborn; D009696:Nucleic Acids / Q000032:analysis; D009930:Organic Chemicals
D018952:Antigens, CD34; D004396:Coloring Agents; D005456:Fluorescent Dyes; D009696:Nucleic Acids; D009930:Organic Chemicals; C461159:SYTO 13; D016650:Fluorescein-5-isothiocyanate