Measurement of absolute concentration and viability of CD34+ cells in cord blood and cord blood products using fluorescent beads and cyanine nucleic acid dyes.
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.
Hübl
W
W
Department of Pathology, University of Florida College of Medicine, Gainesville, USA.
Iturraspe
J
J
Martinez
G A
GA
Hutcheson
C E
CE
Roberts
C G
CG
Fisk
D D
DD
Sugrue
M W
MW
Wingard
J R
JR
Braylan
R C
RC
D018952:Antigens, CD34 / Q000032:analysis*; D002470:Cell Survival; D005312:Fetal Blood / Q000166:cytology*; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes / Q000032:analysis*; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000276:immunology; D006801:Humans; D007231:Infant, Newborn; D007958:Leukocyte Count; D007963:Leukocytes, Mononuclear / Q000166:cytology* / Q000276:immunology; D008863:Microspheres; D015203:Reproducibility of Results
D018952:Antigens, CD34; D005456:Fluorescent Dyes