Single-platform flow cytometric absolute cell counting protocols provide increased robustness for CD34+ cell enumeration by limiting potential sources of imprecision. However, samples with any cellular fragmentation or debris, such as cord blood samples, provide challenges for these assays. We describe a simple, robust absolute CD34+ cell counting protocol, suitable for cord blood, using TRUCOUNT absolute count tubes (BD Biosciences, San Jose, CA) and a modified ISHAGE (International Society for Hematotherapy and Graft Engineering) gating strategy. An advantage of TRUCOUNT tubes is that each tube is supplied with a known number of lyophilized fluorescent beads. The method includes no-wash fixative-free ammonium chloride red blood cell lysis and the viability dye, 7-amino actinomycin D, to exclude dead cells. The threshold was set on CD45 expression in the FL1 channel and an exclusion gate in the forward scatter channel reduced debris. No manual adjustment of the gating regions was required, even for samples in less than optimal condition. Comparison of the TRUCOUNT-ISHAGE protocol with the original dual-platform ISHAGE assay (n = 30) and the single-platform ISHAGE protocol using Flow-Count Fluorospheres (Beckman Coulter, Fullerton, CA; n = 22) showed high correlation (R(2) = 0.949 and 0.989, respectively) and no significant difference or bias for samples ranging from 22 to 600 CD34+ cells per microliter. Results are presented that demonstrate the detrimental effect of a fixative-containing lysis reagent when used in a lyse-and-wash procedure. The TRUCOUNT-ISHAGE protocol combines the attributes of TRUCOUNT tubes and the ISHAGE gating strategy to provide a single-platform protocol capable of achieving readily standardization of CD34+ cell enumeration.
Copyright 2001 Wiley-Liss, Inc.