title: Analytical evaluation of a real-time PCR-based DNA demethylation assay to assess the frequency of naturally occurring regulatory T cells in peripheral blood.
abstract: OBJECTIVE
  Regulatory T cells (Tregs) which may indicate operational tolerance provide a promising biomarker for individualization of immunosuppression. Naturally thymus-derived Tregs (nTregs) represent the major suppressive phenotype and can be identified by their demethylation status in the Tregs Specific Demethylated Region (TSDR) of the Forkhead-Box-P3 (FOXP3) gene using quantitative PCR (qPCR).
  
  
  METHODS
  The analytical performance of a TSDR demethylation qPCR assay was assessed in whole blood of healthy individuals (HI) and kidney transplant recipients (KTR). The assay was compared to conventional flow cytometry and the agreement of results between two laboratories using a comparable qPCR protocol was assessed. In addition, the effect of gender, age, and medications was investigated.
  
  
  RESULTS
  Within and between series imprecision was <20% (n=6). Whole blood samples are suitable for analysis within 3days when stored at room temperature; both whole blood and DNA samples - within 12months when frozen at -80°C. A significant correlation between the qPCR results and flow cytometry was lacking both with samples from HI and KTR. qPCR results between laboratories showed a bias of 76% but correlated well (r=0.645; p=0.0002, n=29). nTregs determined by qPCR were significantly (p<0.05) higher in HI (0.73%±0.23%, n=60) than in KTR (0.45%±0.21%, n=60) and in female HI (1.0%±0.27%, n=30) than in male HI (0.45%±0.23%, n=27). No effect of drugs or age was observed.
  
  
  CONCLUSIONS
  The qPCR assay for nTregs provides reproducible results and is of sufficient quality for working with patient samples although inter-laboratory differences can be encountered due to a lack of method standardization. It was confirmed that gender-specific reference ranges are required.
authors.affiliation: Central Institute for Clinical Chemistry and Laboratory Medicine, Klinikum Stuttgart, Germany.
authors.forename: Maria
authors.initials: M
authors.lastname: Metzker
authors.affiliation: Central Institute for Clinical Chemistry and Laboratory Medicine, Klinikum Stuttgart, Germany*. Electronic address: m.shipkova@klinikum-stuttgart.de.
authors.forename: Maria
authors.initials: M
authors.lastname: Shipkova
authors.affiliation: Department of Clinical Chemistry, University Medicine Goettingen, Germany.
authors.forename: Nicolas
authors.initials: N
authors.lastname: von Ahsen
authors.affiliation: Department of Clinical Chemistry, University Medicine Goettingen, Germany.
authors.forename: Rainer
authors.initials: R
authors.lastname: Andag
authors.affiliation: Central Institute for Clinical Chemistry and Laboratory Medicine, Klinikum Stuttgart, Germany.
authors.forename: Manabu
authors.initials: M
authors.lastname: Abe
authors.affiliation: Department of Neurology, Klinikum Stuttgart, Germany.
authors.forename: Ortrun
authors.initials: O
authors.lastname: Canzler
authors.affiliation: Central Institute for Clinical Chemistry and Laboratory Medicine, Klinikum Stuttgart, Germany.
authors.forename: Corinne
authors.initials: C
authors.lastname: Klett
authors.affiliation: Central Institute for Transfusion Medicine, Klinikum Stuttgart, Germany.
authors.forename: Simone
authors.initials: S
authors.lastname: Leicht
authors.affiliation: Department of Nephrology and Transplant Center, Klinikum Stuttgart, Germany.
authors.forename: Christoph
authors.initials: C
authors.lastname: Olbricht
authors.affiliation: Central Institute for Clinical Chemistry and Laboratory Medicine, Klinikum Stuttgart, Germany.
authors.forename: Eberhard
authors.initials: E
authors.lastname: Wieland
mesh_terms: D016022:Case-Control Studies; D019175:DNA Methylation; D005260:Female; D006801:Humans; D007166:Immunosuppressive Agents / Q000097:blood; D008297:Male; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D050378:T-Lymphocytes, Regulatory / Q000166:cytology*; D016559:Tacrolimus / Q000097:blood
chemical_list: D007166:Immunosuppressive Agents; D016559:Tacrolimus
keywords: Biomarkers; Flow cytometry; Immune tolerance; Kidney transplantation; Methylation; Polymerase chain reaction; Reference values; Reproducibility of results