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

Autor: Duggleby RC; Anthony Nolan Research Institute, London, United Kingdom.; UCL Cancer Institute, University College London, London, United Kingdom., Tsang HP; National Health Service Blood and Transplant, Oxford, United Kingdom.; Nuffield Division of Clinical Laboratory Sciences, Radcliffe Department of Medicine, University of Oxford, Oxford, United Kingdom., Strange K; Anthony Nolan Research Institute, London, United Kingdom.; UCL Cancer Institute, University College London, London, United Kingdom., McWhinnie A; Anthony Nolan Research Institute, London, United Kingdom., Lamikanra AA; National Health Service Blood and Transplant, Oxford, United Kingdom.; Nuffield Division of Clinical Laboratory Sciences, Radcliffe Department of Medicine, University of Oxford, Oxford, United Kingdom., Roberts DJ; National Health Service Blood and Transplant, Oxford, United Kingdom.; Nuffield Division of Clinical Laboratory Sciences, Radcliffe Department of Medicine, University of Oxford, Oxford, United Kingdom., Hernandez D; Anthony Nolan Research Institute, London, United Kingdom.; UCL Cancer Institute, University College London, London, United Kingdom., Madrigal JA; Anthony Nolan Research Institute, London, United Kingdom.; UCL Cancer Institute, Royal Free NHS Trust, London, United Kingdom., Danby RD; Anthony Nolan Research Institute, London, United Kingdom.; UCL Cancer Institute, University College London, London, United Kingdom.; Department of Haematology, Oxford University Hospitals NHS Foundation Trust, Oxford, United Kingdom.
Jazyk: angličtina
Zdroj: PloS one [PLoS One] 2020 Oct 23; Vol. 15 (10), pp. e0240190. Date of Electronic Publication: 2020 Oct 23 (Print Publication: 2020).
DOI: 10.1371/journal.pone.0240190
Abstrakt: 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.
Competing Interests: The authors have declared that no competing interests exist.
Databáze: MEDLINE
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