MEASUREMENT OF ABSOLUTE CONCENTRATION AND VIABILITY OF CD34(-BLOOD AND CORD-BLOOD PRODUCTS USING FLUORESCENT BEADS AND CYANINE NUCLEIC-ACIDDYES() CELLS IN CORD)

Conventional flow cytometric methods for CD34(+) cell counting may he affected by the high number of nucleated red blood cells or nonviable cells in cord blood and its products. We developed a simple Row cytome tric no-wash procedure that avoids these shortcomings because it provi des absolute CD34(+) cell counts and assesses cell viability, Samples were incubated with phycoerythrin (PE)-labeled anti-CD34 (Becton Dicki nson Immunocytometry Systems [BD], San lose, CA) and peridinin chlorop hyll protein (PerCP)-labeled anti-CD45 (BD) in bead-containing TRUCOUN T tubes (BD), After red cell lysis with a fixative-free reagent, the i mpermeant 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 c ells/mu l, slope 1.01), Precision studies conveyed coefficients of var iation of 6.4 and 8.9% at concentrations of 35 and 16 CD34(+) cells/mu l, respectively, In untreated and leukocyte-enriched cord blood 4.5 /- 3.8% of CD34(+) cells were stained by YO-PRO-1, representing apopto tic or necrotic cells. In post-thawing cryopreserved samples this numb er increased to 10.4 +/- 5.5%, Isotype controls showed very low blank values of viable cells (0.1 +/- 0.4 cells/mu l, maximum 2.4) and seeme d unnecessary. We found no washing-related alteration of results in 35 samples, indicating that the method may also be performed with cell w ashing, Replacing YO-PRO-1 with TO-PRO-3 facilitated four-color analys is of subpopulations of viable CD34(+) cells on a FACSCalibur equipped with a second (diode) laser, We found the proposed method to be a rap id, efficient, and flexible procedure that improved validity of CD34() cell counts. (C) 1998 Wiley-Liss, Inc.