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

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.

Cell tracking velocimetry as a tool for defining saturation binding of magnetically conjugated antibodies

“…Separation of these cells is particularly difficult not only because of the very low percentage of stem cells present but also because of variation in expression levels of the stem cell marker, CD34 (29,30). Preliminary results suggest that optimal magnetic labeling of CD34 1 cells from umbilical cord blood requires at least 15 ml/10 6 cells a-PE-MACS when the cells are saturated with a-CD34-PE (unpublished results).…”

Absolute Values of Dendritic Cell Subsets in Bone Marrow, Cord Blood, and Peripheral Blood Enumerated by a Novel Method

“…The use of TruCount tubes loaded with beads (BDIS) decreased both the inter-and intralaboratory variability when 60 specimens from HIV type 1-infected donors were shipped to five laboratories. The acquisition of cellular events along with TruCount beads has been demonstrated to be superior to standard methods in enumerating rare cells, as well as for CD34 + progenitor cells in cord blood [48] and in mobilized leukopheresis products [49] with less variability between data analyzed by different operators. The absolute DC numbers obtained by our lyse/no wash method appear to be in range with some other flow cytometric DC enumeration studies [44,45].…”

Death induction by CD99 ligation in TEL/AML1-positive acute lymphoblastic leukemia and normal B cell precursors

“…Absolute cell counting was performed using a method published previously with Trucount tubes (BD Biosciences) [30]. Briefly, a fixed aliquot of cell suspension was collected from cultures, transferred into Trucount tubes, and diluted with 200 l PBS.…”