Measurement of absolute concentration and viability of CD34+ cells in cord blood and cord blood products using fluorescent beads and cyanine nucleic acid dyes

• Published in: Cytometry (New York, N.Y.) N.Y.), 1998-06, Vol.34 (3), p.121-127
• Main Authors: Hübl, Wolfgang, Iturraspe, Jose, Martinez, Guillermo A., Hutcheson, Charles E., Roberts, Cheryl G., Fisk, Diann D., Sugrue, Michelle W., Wingard, John R., Braylan, Raul C.
• Format: Article
• Language: English
• Subjects: Antigens, CD34 - analysis; CD34; Cell Survival; cord blood; Fetal Blood - cytology; flow cytometry; Flow Cytometry - methods; Fluorescent Dyes - analysis; hematopoietic stem cells; Hematopoietic Stem Cells - cytology; Hematopoietic Stem Cells - immunology; Humans; Infant, Newborn; Leukocyte Count; Leukocytes, Mononuclear - cytology; Leukocytes, Mononuclear - immunology; Microspheres; Reproducibility of Results
• ISSN: 0196-4763; 1097-0320
• DOI: 10.1002/(SICI)1097-0320(19980615)34:3<121::AID-CYTO2>3.0.CO;2-K

Conventional flow cytometric methods for CD34+ cell counting may be affected by the high number of nucleated red blood cells or nonviable cells in cord blood and its products. We developed a simple flow cytometric no‐wash procedure that avoids these shortcomings because it provides absolute CD34+ cell counts and assesses cell viability. Samples were incubated with phycoerythrin (PE)‐labeled anti‐CD34 (Becton Dickinson Immunocytometry Systems [BD], San Jose, CA) and peridinin chlorophyll protein (PerCP)‐labeled anti‐CD45 (BD) in bead‐containing TRUCOUNT tubes (BD). After red cell lysis with a fixative‐free reagent, the impermeant nucleic acid dye YO‐PRO‐1 (Molecular Probes, Eugene, OR) was added and samples were analyzed on a single‐laser FACSCalibur (BD). A comparison with the ProCOUNT progenitor cell assay (BD) in 57 samples revealed excellent correlation of results (r = 0.98, intercept −0.2 cells/μl, slope 1.01). Precision studies conveyed coefficients of variation of 6.4 and 8.9% at concentrations of 35 and 16 CD34+ cells/μl, respectively. In untreated and leukocyte‐enriched cord blood 4.5 ± 3.8% of CD34+ cells were stained by YO‐PRO‐1, representing apoptotic or necrotic cells. In post‐thawing cryopreserved samples this number increased to 10.4 ± 5.5%. Isotype controls showed very low blank values of viable cells (0.1 ± 0.4 cells/μl, maximum 2.4) and seemed unnecessary. We found no washing‐related alteration of results in 35 samples, indicating that the method may also be performed with cell washing. Replacing YO‐PRO‐1 with TO‐PRO‐3 facilitated four‐color analysis of subpopulations of viable CD34+ cells on a FACSCalibur equipped with a second (diode) laser. We found the proposed method to be a rapid, efficient, and flexible procedure that improved validity of CD34+ cell counts. Cytometry (Comm. Clin. Cytometry) 34:121–127, 1998. © 1998 Wiley‐Liss, Inc.