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 Key terms: CD34; cord blood; ISHAGE; single platform; stem cellsHuman umbilical cord blood is a rich source of hematopoietic stem cells (HSC) that is being used increasingly as an alternative to bone marrow for HSC transplantation (1-3). The cellular content, in particular the number of HSC, is a principal factor in assessing the adequacy of cord blood units for clinical transplantation. HSC are contained within the CD34ϩ cell population, which represents typically less than 1% of the total leukocytes in cord blood (4). This places CD34 analysis in the category of "rareevent" analysis and has presented a challenge to the development of standardized, robust methods for the quantitation of HSC. Cord blood poses additional challenges. These include the presence of variable numbers of nucleated red blood cells (RBC; which can lead to overestimation of the leukocyte count) and the age of the samples, which is often greater than 24 h old. Increased apoptosis, cell death, and debris can potentially confound the analysis.CD34ϩ cell enumeration has relied predominantly on flow cytometric procedures (5-7). Guidelines published by the International Society for Hematotherapy and Graft Engineering (ISHAGE; 8) were a significant attempt towards standardization of the CD34 assay. These guidelines were based on a dual-platform method whereby the percentage of CD34ϩ cells was determined by flow cytometry and the leukocyte count by an automated hematology analyzer. A lyse-and-wash method for sample preparation, a gating strategy based on a forward scatter threshold, CD45 staining to define the total leukocyte population, and light scatter characteristics were recommended. Nevertheless, the use of two instruments that were used to determine the absolute CD34ϩ cell number, the potential loss of cells during the lysis and wash steps, and the

Quantitation of equilibrative, nitrobenzylthioinosine (NBMPR) sensitive (es) nucleoside transporters on blast cells isolated from patients with acute myeloblastic leukemia is useful in predicting intracellular accumulation of the antileukemic nucleoside drug, cytosine arabinoside. We previously reported the synthesis of a fluorescein‐labeled ligand for the es nucleoside transporter, 5‐(SAENTA‐x2)‐fluorescein. This paper reports the synthesis of 5‐(SAENTA‐x8)‐fluorescein in which the linkage between fluorescein and nucleoside ligand has been increased from 2 atoms to 8 atoms. This new ligand had a sixfold increase in affinity (Kd 0.9 ± 0.1 nM) as well as an 86% increase in the cell associated fluorescence output compared to its prototype 5‐(SAENTA‐x2)‐fluorescein. The fluorescence signal arising from 5‐(SAENTA‐x8)‐fluorescein specifically bound to freshly isolated and cultured leukemic myeloblasts was converted to molecules of equivalent soluble fluorescein (MESF) using standardized fluorescein microbeads and compared with the number of es nucleoside transporter sites assayed concurrently by [3H]NBMPR equilibrium binding analysis. A high correlation between the two assays was observed (r = 0.98), which enabled the cell‐bound fluorescence output of 5‐(SAENTA‐x8)‐fluorescein to be expressed in numbers of es nucleoside transporter sites per cell. The improved properties of 5‐(SAENTA‐x8)‐fluorescein over those of its prototype molecule make it a suitable reagent for flow cytometric quantitation of nucleoside transporter expression on leukemic cells isolated from patient samples. © 1993 Wiley‐Liss, Inc.

The N6-(4-nitrobenzyl) derivative of adenosine is a tight-binding inhibitor of the equilibrative inhibitor-sensitive nucleoside transporter of mammalian cells. A fluorescent ligand for this transporter has been synthesized by allowing an adenosine analogue. 5'-S-(2-aminoethyl)-N6-(4-nitrobenzyl)-5'-thioadenosine (SAENTA), to react with fluorescein isothiocyanate. The purified adduct had a SAENTA/fluorescein molar ratio of 0.92:1 calculated from its absorption spectrum. The intensity of fluorescent emission from the SAENTA-chi 2-fluorescein adduct was 30% that of fluorescein isothiocyanate (chi 2 is the number of atoms in the linkage between fluorescein and SAENTA). SAENTA-chi 2-fluorescein inhibited the influx of nucleosides into cultured leukaemic cells with an IC50 (total concentration of inhibitor producing 50% inhibition) of 40 nM. The adduct inhibited the binding of [3H]nitrobenzylthioinosine ([3H]NBMPR) with half-maximal inhibition at 50-100 nM. Mass Law analysis of the competitive-binding data suggested the presence of two classes of sites for [3H]NBMPR binding, only one of which was accessible to SAENTA-chi 2-fluorescein. Flow cytometry was used to analyse equilibrium binding of SAENTA-chi 2-fluorescein to leukaemic cells and a Kd of 6 nM was obtained. SAENTA-chi 2-fluorescein is a high-affinity ligand for the equilibrative inhibitor-sensitive nucleoside transporter which allows rapid assessment of transport capacity by flow cytometry.

The present paper reveals that a fluorescent derivative of nitrobenzylthioinosine, 5-(SAENTA-x8)-fluorescein, is a highly specific inhibitor of the neural NBTI-sensitive nucleoside transporter. 5-(SAENTA-x8)-fluorescein inhibited adenosine transport and [ Q H]NBTI binding with a K i of 4 nM in cultured chromaffin cells. Flow cytometry demonstrated that 5-(SAEN-TA-x8)-fluorescein specifically interacted with the NBTI-sensitive nucleoside transporters with high affinity (K h = 6 nM). Activation of protein kinases A and C with forskolin or nicotinic receptor agonists, respectively, resulted in 50% inhibition of the fluorescence bound to the cells. Flow cytometry will allow studying nucleoside transport in single cells from heterogeneous neural cell populations.z 1998 Federation of European Biochemical Societies.

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