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Enumerating regulatory T cells in cryopreserved umbilical cord blood samples using FOXP3 methylation specific quantitative PCR

Duggleby, Richard C; Tsang, Hoi Pat; Strange, Kathryn; McWhinnie, Alasdair; Lamikanra, Abigail A; Roberts, David J; Hernandez, Diana; ... Danby, Robert D; + view all (2020) Enumerating regulatory T cells in cryopreserved umbilical cord blood samples using FOXP3 methylation specific quantitative PCR. PLoS ONE , 15 (10) , Article e0240190. 10.1371/journal.pone.0240190. Green open access

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Abstract

Background 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. Study design and methods 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. Results 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. Conclusion 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.

Type: Article
Title: Enumerating regulatory T cells in cryopreserved umbilical cord blood samples using FOXP3 methylation specific quantitative PCR
Location: United States
Open access status: An open access version is available from UCL Discovery
DOI: 10.1371/journal.pone.0240190
Publisher version: https://doi.org/10.1371/journal.pone.0240190
Language: English
Additional information: Copyright: © 2020 Duggleby et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Keywords: Science & Technology, Multidisciplinary Sciences, Science & Technology - Other Topics, EXPRESSION, PREDICTION, POTENCY, GRAFTS, TREGS
UCL classification: UCL
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Medical Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Medical Sciences > Cancer Institute
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Medical Sciences > Cancer Institute > Research Department of Haematology
URI: https://discovery.ucl.ac.uk/id/eprint/10179641
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