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

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.

SUBMITTER: Duggleby RC

PROVIDER: S-EPMC7584164 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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

Duggleby Richard C RC Tsang Hoi Pat HP Strange Kathryn K McWhinnie Alasdair A Lamikanra Abigail A AA Roberts David J DJ Hernandez Diana D Madrigal J Alejandro JA Danby Robert D RD

PloS one 20201023 10

<h4>Background</h4>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 ...[more]

PMID: 33095809

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Project description:BackgroundFor hallux rigidus, dorsal cheilectomy remains a treatment option even with advances in interposition techniques and devices. Cheilectomy aims to alleviate dorsal impingement and improve pain and function as well as range of motion. Cryopreserved umbilical cord allograft, with properties to mitigate inflammation and scar formation, has theoretical benefit for improving outcomes following cheilectomy. In this first prospective randomized and blinded cheilectomy study reported, we aimed to compare outcomes between cheilectomy alone and cheilectomy with umbilical cord allograft.MethodsPatients were randomized to cheilectomy alone (CA) or cheilectomy with cryopreserved umbilical cord (ie, amniotic membrane-umbilical cord [AM-UC]). Patients were evaluated with American Orthopaedic Foot & Ankle Society (AOFAS), Foot Function Index (FFI), and visual analog scale (VAS) pain outcomes collected preoperatively and at 6 months and 1 year postoperatively. In addition, radiographic range of motion data were collected using stress radiographs. Fifty-one patients (26 AM-UC, 25 CA) completed the study, with 5 bilateral surgeries in the AM-UC group and 2 in the CA group, totaling 31 and 27 feet, respectively.ResultsThe AM-UC group had statistically significant improved AOFAS and FFI scores at 1 year compared with the CA group, but there was no difference at 6 months. There was no significant difference between groups for VAS-pain scores at any time point, but overall VAS-pain improved in both groups from preoperative values. There was no significant difference in range of motion (total arc) between groups and changes in range of motion (total arc) in both groups from preoperative to 1 year postoperative were small.ConclusionWe present the results of the first randomized and blinded prospective study of cheilectomy surgery patients. When appropriately selected, cheilectomy remains a good option for patients with symptomatic hallux rigidus. Cryopreserved umbilical cord is a potential adjuvant to cheilectomy, with 1-year results showing improvements in functional outcome scores.Level of evidenceLevel II, prospective comparative study.

Project description:Preterm delivery is the leading cause of neonatal morbidity and mortality. Two-thirds of preterm deliveries are idiopathic. The initiating molecular mechanisms behind spontaneous preterm delivery are unclear. Umbilical cord blood DNA samples are an easy source of material to study the neonatal state at birth. DNA methylation changes can be exploited as markers to identify spontaneous preterm delivery. To identify methylation differences specific to idiopathic preterm delivery, we assessed genome-wide DNA methylation changes in 24 umbilical cord blood samples (UCB) using the 450 K Illumina methylation array. After quality control, conclusions were based on 11 term and 11 idiopathic preterm born neonates. The differentially methylated positions (DMPs) specific for preterm/term delivery, neonatal sex, use of oxytocin and mode of initiation of labor were calculated by controlling the FDR p value at 0.05.The analysis identifies 1855 statistically significant DMPs between preterm and term deliveries of which 508 DMPs are also attributable to clinical variables other than preterm versus term delivery. 1347 DMPs are unique to term vs preterm delivery, of which 196 DMPs do not relate to gestational age as such. Pathway analysis indicated enrichment of genes involved in calcium signalling, myometrial contraction and relaxation pathways. The 1151 DMPs that correlate with advancing gestational age (p?<?0.05) include 161 DMPs that match with two previously reported studies on UCB methylation. Additionally, 123 neonatal sex specific DMPs, 97 DMPs specific to the induction of labour and 42 DMPs specific to the mode of initiation of labor were also identified.This study identifies 196 DMPs in UCB DNA of neonates which do not relate to gestational age or any other clinical variable recorded and are specific to idiopathic preterm delivery. Furthermore, 161 DMPs from our study overlap with previously reported studies of which a subset is also reported to be differentially methylated at 18 years of age. A DMP on MYL4, encoding myosin light chain 4, is a robust candidate for the identification of idiopathic preterm labour as it is identified by all 3 independent studies.

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

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

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