Measurement of Absolute Concentration and Viability of CD34+ Cells in Cord Blood and Cord Blood Products Using Fluorescent Beads and Cyanine Nucleic Acid Dyes

Cytometry. 1998 Jun 15;34(3):121-7. doi: 10.1002/(sici)1097-0320(19980615)34:3<121::aid-cyto2>3.3.co;2-u.

Abstract

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/microl, slope 1.01). Precision studies conveyed coefficients of variation of 6.4 and 8.9% at concentrations of 35 and 16 CD34+ cells/microl, 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/microl, 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.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antigens, CD34 / analysis*
  • Cell Survival
  • Fetal Blood / cytology*
  • Flow Cytometry / methods*
  • Fluorescent Dyes / analysis*
  • Hematopoietic Stem Cells / cytology*
  • Hematopoietic Stem Cells / immunology
  • Humans
  • Infant, Newborn
  • Leukocyte Count*
  • Leukocytes, Mononuclear / cytology*
  • Leukocytes, Mononuclear / immunology
  • Microspheres
  • Reproducibility of Results

Substances

  • Antigens, CD34
  • Fluorescent Dyes