Enumeration of CD34+ cells in cord blood: A variation on a single-platformflow cytometric method based on the ISHAGE gating strategy

Single-platform flow cytometric absolute cell counting protocols provide in creased robustness for CD34+ cell enumeration by limiting potential sources of imprecision. However, samples with any cellular fragmentation or debris , such as card blood samples, provide challenges for these assays. We descr ibe a simple, robust absolute CD34+ cell counting protocol, suitable for co rd blood, using TRUCOUNT (TM) absolute count tubes (BD Biosciences, San Jos e, CA) and a modified ISHAGE (international Society for Hemototherapy and G raft Engineering) gating strategy. An advantage of TRUCOUNT tubes is that e ach 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 exclusio n gate in the forward scatter channel reduced debris. No manual adjustment of the gating regions was required, even for samples in less than optimal c ondition. Comparison of the TRUCOUNT-ISHAGE protocol with the original dual -platform ISHAGE assay (n = 30) and the single-platform ISHAGE protocol usi ng Flow-Count (TM) Fluorospheres (Beckman Coulter, Fullerton, CA; n = 22) s howed high correlation (R-2 = 0.949 and 0.989, respectively) and no signifi cant difference or bias for samples ranging from 22 to 600 CD34+ cells per microliter. Results are presented that demonstrate the detrimental effect o f a fixative-containing lysis reagent when used in a lyse-and-wash procedur e. The TRUCOUNT-ISHAGE protocol combines the attributes of TRUCOUNT tubes a nd the ISHAGE gating strategy to provide a single-platform protocol capable of achieving readily standardization of CD34+ cell enumeration. (C) 2001 W iley-Liss, Inc.