Lack of correlation between Treg quantification assays in inflammatory bowel disease patients

AIM:To compare the number of regulatory T-cells( Tregs) measured by flow cytometry with those obtained using a real-time quantitative PCR(q PCR) method in patients suffering from inflammatory bowel disease(IBD).METHODS:Tregs percentages obtained by both flow cytometry and q PCR 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 q PCR 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 q PCR in adult IBD patients.There were no significant correlations between either the percentages of Tregs measured by flow cytometry or q PCR and the HBI or SCCAI questionnaire scores in CD or UC patients,respectively.In addition,there was no correlation between Treg percentages measured by q PCR 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 resultssuggest 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.