title: Lack of correlation between Treg quantification assays in inflammatory bowel disease patients.
abstract: OBJECTIVE
  To compare the number of regulatory T-cells (Tregs) measured by flow cytometry with those obtained using a real-time quantitative PCR (qPCR) method in patients suffering from inflammatory bowel disease (IBD).
  
  
  METHODS
  Tregs percentages obtained by both flow cytometry and qPCR methods in 35 adult IBD patients, 18 out of them with Crohn´s disease (CD) and 17 with ulcerative colitis (UC) were compared to each other as well as to scores on two IBD activity questionnaires using the Harvey Bradshaw Index (HBI) for CD patients and the Simple Colitis Clinical Activity Index (SCCAI) for UC patients. The Treg percentages by flow cytometry were defined as CD4(+)CD25(high)CD127(low)FOXP3(+) cells in peripheral blood mononuclear cells, whereas the Treg percentages by qPCR method were determined as FOXP3 promoter demethylation in genomic DNA.
  
  
  RESULTS
  We found an average of 1.56% ± 0.78% Tregs by using flow cytometry, compared to 1.07% ± 0.53% Tregs by using qPCR in adult IBD patients. There were no significant correlations between either the percentages of Tregs measured by flow cytometry or qPCR and the HBI or SCCAI questionnaire scores in CD or UC patients, respectively. In addition, there was no correlation between Treg percentages measured by qPCR and those measured by flow cytometry (r = -0.06, P = 0.73; Spearman Rho). These data suggest that, either Treg-related immune function or the clinical scores in these IBD patients did not accurately reflect actual disease activity. Until the cause(s) for these differences are more clearly defined, the results suggest caution in interpreting studies of Tregs in various inflammatory disorders.
  
  
  CONCLUSIONS
  The two methods did not produce equivalent measures of the percentage of total Tregs in the IBD patients studied which is consistent with the conclusion that Tregs subtypes are not equally detected by these two assays.
authors: Gunnar Brandhorst, Darinka Todorova Petrova, Sebastian Weigand, Christoph Eberle, Nicolas von Ahsen, Department of Clinical Chemistry, University Medical Center, 37075 Goettingen, Germany.
authors: Gunnar
authors: G
authors: Brandhorst
authors: Gunnar Brandhorst, Darinka Todorova Petrova, Sebastian Weigand, Christoph Eberle, Nicolas von Ahsen, Department of Clinical Chemistry, University Medical Center, 37075 Goettingen, Germany.
authors: Darinka Todorova
authors: DT
authors: Petrova
authors: Gunnar Brandhorst, Darinka Todorova Petrova, Sebastian Weigand, Christoph Eberle, Nicolas von Ahsen, Department of Clinical Chemistry, University Medical Center, 37075 Goettingen, Germany.
authors: Sebastian
authors: S
authors: Weigand
authors: Gunnar Brandhorst, Darinka Todorova Petrova, Sebastian Weigand, Christoph Eberle, Nicolas von Ahsen, Department of Clinical Chemistry, University Medical Center, 37075 Goettingen, Germany.
authors: Christoph
authors: C
authors: Eberle
authors: Gunnar Brandhorst, Darinka Todorova Petrova, Sebastian Weigand, Christoph Eberle, Nicolas von Ahsen, Department of Clinical Chemistry, University Medical Center, 37075 Goettingen, Germany.
authors: Nicolas
authors: N
authors: von Ahsen
authors: Gunnar Brandhorst, Darinka Todorova Petrova, Sebastian Weigand, Christoph Eberle, Nicolas von Ahsen, Department of Clinical Chemistry, University Medical Center, 37075 Goettingen, Germany.
authors: Jessica
authors: J
authors: Schmitz
authors: Gunnar Brandhorst, Darinka Todorova Petrova, Sebastian Weigand, Christoph Eberle, Nicolas von Ahsen, Department of Clinical Chemistry, University Medical Center, 37075 Goettingen, Germany.
authors: Frank Christian
authors: FC
authors: Schultze
authors: Gunnar Brandhorst, Darinka Todorova Petrova, Sebastian Weigand, Christoph Eberle, Nicolas von Ahsen, Department of Clinical Chemistry, University Medical Center, 37075 Goettingen, Germany.
authors: Dirk
authors: D
authors: Raddatz
authors: Gunnar Brandhorst, Darinka Todorova Petrova, Sebastian Weigand, Christoph Eberle, Nicolas von Ahsen, Department of Clinical Chemistry, University Medical Center, 37075 Goettingen, Germany.
authors: Michael
authors: M
authors: Karaus
authors: Gunnar Brandhorst, Darinka Todorova Petrova, Sebastian Weigand, Christoph Eberle, Nicolas von Ahsen, Department of Clinical Chemistry, University Medical Center, 37075 Goettingen, Germany.
authors: Michael
authors: M
authors: Oellerich
authors: Gunnar Brandhorst, Darinka Todorova Petrova, Sebastian Weigand, Christoph Eberle, Nicolas von Ahsen, Department of Clinical Chemistry, University Medical Center, 37075 Goettingen, Germany.
authors: Philip D
authors: PD
authors: Walson
mesh_terms: D000328:Adult; D018791:CD4 Lymphocyte Count / Q000379:methods*; D003093:Colitis, Ulcerative / Q000175:diagnosis / Q000276:immunology*; D003424:Crohn Disease / Q000175:diagnosis / Q000276:immunology*; D019175:DNA Methylation; D005260:Female; D005434:Flow Cytometry; D051858:Forkhead Transcription Factors / Q000032:analysis / Q000235:genetics; D005819:Genetic Markers; D006801:Humans; D053645:Interleukin-2 Receptor alpha Subunit / Q000032:analysis; D053699:Interleukin-7 Receptor alpha Subunit / Q000032:analysis; D008297:Male; D008875:Middle Aged; D011237:Predictive Value of Tests; D011401:Promoter Regions, Genetic; D060888:Real-Time Polymerase Chain Reaction; D015203:Reproducibility of Results; D011795:Surveys and Questionnaires; D050378:T-Lymphocytes, Regulatory / Q000276:immunology*; D055815:Young Adult
chemical_list: C418974:FOXP3 protein, human; D051858:Forkhead Transcription Factors; D005819:Genetic Markers; C508017:IL2RA protein, human; D053645:Interleukin-2 Receptor alpha Subunit; D053699:Interleukin-7 Receptor alpha Subunit
keywords: Crohn´s disease; Inflammatory bowel disease; Method comparison; Regulatory T-cells; Ulcerative colitis