Anthony Nolan Research Institute, London, United Kingdom.
UCL Cancer Institute, University College London, London, United Kingdom.
PLoS One. 2020 Oct 23;15(10):e0240190. doi: 10.1371/journal.pone.0240190. eCollection 2020.
Allogeneic haematopoietic cell transplantation (HCT) is a curative therapy for severe haematological disorders. However, it carries significant risk of morbidity and mortality. To improve patient outcomes, better graft selection strategies are needed, incorporating HLA matching with clinically important graft characteristics. Studies have shown that the cellular content of HCT grafts, specifically higher ratios of T regulatory (Tregs)/T cells, are important factors influencing outcomes when using adult peripheral blood mobilised grafts. So far, no equivalent study exists in umbilical cord blood (CB) transplantation due to the limitations of cryopreserved CB samples.
To establish the most robust and efficient way to measure the Treg content of previously cryopreserved CB units, we compared the enumeration of Treg and CD3+ cells using flow cytometry and an epigenetic, DNA-based methodology. The two methods were assessed for their agreement, consistency and susceptibility to error when enumerating Treg and CD3+ cell numbers in both fresh and cryopreserved CB samples.
Epigenetic enumeration gave consistent and comparable results in both fresh and frozen CB samples. By contrast, assessment of Tregs and CD3+ cells by flow cytometry was only possible in fresh samples due to significant cell death following cryopreservation and thawing.
Epigenetic assessment offers significant advantages over flow cytometry for analysing cryopreserved CB; similar cell numbers were observed both in fresh and frozen samples. Furthermore, multiple epigenetic assessments can be performed from DNA extracted from small cryopreserved CB segments; often the only CB sample available for clinical studies.
同种异体造血细胞移植（HCT）是治疗严重血液系统疾病的一种有治愈可能的疗法。然而，它存在着显著的发病率和死亡率风险。为了改善患者的预后，需要更好的移植物选择策略，将 HLA 配型与具有临床重要性的移植物特征相结合。研究表明，HCT 移植物的细胞含量，特别是 T 调节（Treg）/T 细胞的更高比例，是使用成人外周血动员移植物时影响结果的重要因素。到目前为止，由于冷冻保存的 CB 样本的限制，在脐带血（CB）移植中还没有等效的研究。
为了建立一种最稳健和有效的方法来测量以前冷冻保存的 CB 单位的 Treg 含量，我们比较了使用流式细胞术和基于表观遗传学的 DNA 方法对 Treg 和 CD3+细胞的计数。评估了这两种方法在计数新鲜和冷冻 CB 样本中 Treg 和 CD3+细胞数量时的一致性、一致性和对误差的敏感性。
表观遗传计数在新鲜和冷冻 CB 样本中均给出了一致和可比的结果。相比之下，由于冷冻保存和解冻后细胞大量死亡，流式细胞术评估 Tregs 和 CD3+细胞仅可在新鲜样本中进行。
与流式细胞术相比，表观遗传学评估在分析冷冻 CB 方面具有显著优势；在新鲜和冷冻样本中观察到相似的细胞数量。此外，从冷冻 CB 小片段中提取的 DNA 可以进行多次表观遗传学评估；这通常是临床研究中唯一可用的 CB 样本。
