Observational Study
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World J Gastroenterol. Mar 21, 2015; 21(11): 3325-3329
Published online Mar 21, 2015. doi: 10.3748/wjg.v21.i11.3325
Lack of correlation between Treg quantification assays in inflammatory bowel disease patients
Gunnar Brandhorst, Darinka Todorova Petrova, Sebastian Weigand, Christoph Eberle, Nicolas von Ahsen, Jessica Schmitz, Frank Christian Schultze, Dirk Raddatz, Michael Karaus, Michael Oellerich, Philip D Walson
Gunnar Brandhorst, Darinka Todorova Petrova, Sebastian Weigand, Christoph Eberle, Nicolas von Ahsen, Department of Clinical Chemistry, University Medical Center, 37075 Goettingen, Germany
Darinka Todorova Petrova, Jessica Schmitz, Michael Oellerich, Philip D Walson, Department of Clinical Pharmacology, University Medical Center, 37075 Goettingen, Germany
Jessica Schmitz, Chronix Biomedical, 37073 Goettingen, Germany
Frank Christian Schultze, Dirk Raddatz, Clinics of Gastroenterology and Endocrinology, University Medical Center, 37075 Goettingen, Germany
Michael Karaus, Department of Internal Medicine, Evangelisches Krankenhaus Göttingen-Weende, 37075 Goettingen, Germany
Author contributions: Brandhorst G and Petrova DT contributed equally to this work; Brandhorst G, von Ahsen N, Raddatz D, Karaus M, Oellerich M and Walson PD designed the research and supervised the experiments; Weigand S and Eberle C performed the research; Brandhorst G, Petrova DT, Schmitz J and Schultze FC analyzed the data; and Brandhorst G, Petrova DT and Walson PD wrote the paper; all authors read and approved the final manuscript.
Supported by grants from Medical Faculty of the University of Goettingen, Germany, the German Research Foundation and the Open Access Publication Funds of the Goettingen University.
Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/
Correspondence to: Darinka Todorova Petrova, PhD, Department of Clinical Chemistry, University Medical Center, Robert-Koch-Str. 40, 37075 Goettingen, Germany. darinka.petrova@med.uni-goettingen.de
Telephone: +49-551-398562 Fax: +49-551-398551
Received: September 6, 2014
Peer-review started: September 7, 2014
First decision: October 14, 2014
Revised: November 3, 2014
Accepted: December 8, 2014
Article in press: December 8, 2014
Published online: March 21, 2015
Processing time: 193 Days and 20.8 Hours
Abstract

AIM: 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+CD25highCD127lowFOXP3+ 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.

CONCLUSION: 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.

Keywords: Regulatory T-cells; Inflammatory bowel disease; Crohn´s disease; Ulcerative colitis; Method comparison

Core tip: In our study neither regulatory T-cells (Tregs) percentages measured by flow cytometry defined as CD4+CD25highCD127lowFOXP3+ cells in peripheral blood mononuclear cells or by real-time PCR measured as forkhead box P3 promoter demethylation in genomic DNA correlated with self-reported inflammatory bowel disease activity. This suggests that either Treg-related immune function or the clinical scores did not accurately reflect actual disease activity. We conclude that natural and induced Tregs are not equally detected by the assays applied.