1 INTRODUCTION

Hematopoietic stem cell transplant (HSCT), using cytokine-mobilized peripheral blood (MPB) stem cells, harvested by apheresis, is well established as a potential curative therapy for many patients suffering with various hematologic disorders and malignancies, immune deficiencies, and congenital diseases. However, timely reconstitution of multilineage hematopoiesis in these patients requires infusion of at least 2 × 10⁶ viable hematopoietic stem/progenitor cells (HSCs)/kg patient weight. Human HSCs responsible for engraftment are contained within a population of cells that express the cell surface antigen CD34. Accurate, cross-platform methods of counting viable CD34+ cells by single platform flow cytometry based on the ISHAGE Guidelines (Keeney et al., 1998) are well established as the “Gold Standard” means to measure graft adequacy in the blood and marrow transplant setting.

The single platform ISHAGE protocol utilizes 1× NH₄Cl as a red blood cell (RBC) lysis buffer. However, this buffer is pH dependent and therefore requires daily preparation from a 10× stock. Additionally, if lysed samples are not analyzed immediately (or within 1 hr if kept on melting ice), NH₄Cl has been demonstrated to induce apoptosis/necrosis in CD34 expressing cells, thus potentially impacting the enumeration of viable CD34+ HSCs (Keeney et al., 1998). Furthermore, NH4Cl promotes further apoptosis and cell death in post-thawed samples lysed with this agent and is not recommended for use thereon. Therefore, alternative lysis reagents which are more convenient for daily use and which may better preserve CD34+ HSC viability could impact the ability to accurately enumerate viable CD34+ cells in both the “fresh” and post-thawed settings. Versalyse™ (ImmunoTech S.A.S., Marseille, France) is an alternative lysis buffer that, unlike NH₄Cl, is available in a ready-to-use format, can be stored at room temperature, and has a 90-day shelf-life after opening.

In this brief communication, we provide a preliminary evaluation of Versalyse in comparison to NH₄Cl for CD34+ HSC enumeration, as well as an assessment of post-lysis CD34+ HSC viability.

中文翻译：

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根据 ISHAGE 指南评估 Versalyse 作为替代红细胞裂解剂在评估 CD34 干细胞和祖细胞中的作用

1 简介

造血干细胞移植 (HSCT) 使用通过单采获得的细胞因子动员外周血 (MPB) 干细胞，已成为许多患有各种血液疾病和恶性肿瘤、免疫缺陷和先天性疾病的患者的潜在治疗方法。然而，在这些患者中及时重建多系造血需要输注至少 2 × 10 ^{6 个}活的造血干细胞/祖细胞 (HSC)/kg 患者体重。负责移植的人类 HSC 包含在表达细胞表面抗原 CD34 的细胞群中。根据 ISHAGE 指南（Keeney 等，  1998) 已成为衡量血液和骨髓移植环境中移植物充分性的“黄金标准”手段。

单平台 ISHAGE 方案使用 1× NH ₄ Cl 作为红细胞 (RBC) 裂解缓冲液。然而，这种缓冲液依赖于 pH 值，因此需要每天从 10× 储备液中制备。此外，如果不立即分析裂解的样品（或者如果保存在融化的冰上，则在 1 小时内），NH ₄ Cl 已被证明可诱导 CD34 表达细胞凋亡/坏死，从而可能影响活 CD34+ HSC 的计数（Keeney 等人.,  1998）。此外，NH4Cl 会促进用该试剂裂解的解冻后样品中进一步的细胞凋亡和细胞死亡，因此不建议在其上使用。因此，更方便日常使用并且可以更好地保持 CD34+ HSC 活力的替代裂解试剂可能会影响在“新鲜”和解冻后环境中准确计数活 CD34+ 细胞的能力。Versalyse™（ImmunoTech SAS，Marseille，France）是一种替代裂解缓冲液，与 NH ₄ Cl不同，它以即用型形式提供，可以在室温下储存，并且在使用后具有 90 天的保质期开幕。

在这个简短的交流中，我们提供了 Versalyse 与 NH ₄ Cl 相比对 CD34+ HSC 计数的初步评估，以及对裂解后 CD34+ HSC 活力的评估。