The assessment of nonviable haematopoietic cells by Annexin V staining method in flow cytometry has recently been published by Duggleby et al. Resulting in a better correlation with the observed colony formation in methylcellulose assays than the standard ISHAGE protocol, it presents a promising method to predict cord blood potency. Herein, we applied this method for examining the parameters during processing which potentially could affect cord blood viability. We could verify that the current standards regarding time and temperature are sufficient, since no significant difference was observed within 48 hours or in storage at up to . However, the addition of DMSO for cryopreservation alone leads to an inevitable increase in nonviable haematopoietic stem cells from initially 14.8% ± 4.3% to at least 30.6% ± 5.5%. Furthermore, CFU-assays with varied seeding density were performed in order to evaluate the applicability as a quantitative method. The results revealed that only in a narrow range reproducible clonogenic efficiency (ClonE) could be assessed, giving at least a semiquantitative estimation. We conclude that both Annexin V staining method and CFU-assays with defined seeding density are reliable means leading to a better prediction of the final potency. Especially Annexin V, due to its fast readout, is a practical tool for examining and optimising specific steps in processing, while CFU-assays add a functional confirmation. 1. Introduction Since its first application in 1989 by Gluckman et al. [1], transplant using cord blood (CB) as an alternative stem cell source to bone marrow has been well established in clinical practice for the treatment of blood-related diseases. As of today, the enumeration of haematopoietic stem cells (HSCs) by detecting the expression of the surface marker CD34 in flow cytometry following the protocol of the International Society of Hematotherapy and Graft Engineering (ISHAGE) is the most common used technique to predict the potential quality of a unit [2]. Although this analysis allows an immediate readout and the amount of infused CD3 cells in most cases correlates well with the chances of engraftment in the patients [3], exceptions occur in which the ability of the transplant to reconstitute the patients, immune system, the so-called potency, is lower than expected and might result in failure of engraftment [4]. This might rely on different composition of the unit in terms of short-term and long-term haematopoietic stem/progenitor cells, with only the later being responsible for permanent reconstitution of the
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