We prospectively enrolled 106 children with T1DM seen in the diabetic clinics of the King Saud Medical City, Riyadh, during the period from June 2008 through January 2010. After explaining the objectives of the study, a written informed consent was obtained from the children’s parents. A physician interviewed parents and performed physical examination for all enrolled patients. During interviews, patients and their parents were asked about persistent gastrointestinal symptoms such as diarrhea, constipation, abdominal distension, vomiting, and abdominal pain over the past year. Gender of the patients, their ages, age at onset of diabetes and duration of diabetes were recorded. Medical chart review focused on results of other antibody tests such as serum antithyroperoxidase, associated diseases, and patient’s age at onset of diabetes.
Blood samples were collected for: anti-TTG immunoglobulin subclass A (IgA) using enzyme linked immune-sorbent assay (ELISA) and endomyseal antibody (EMA) IgA subclass using indirect immunofluorescence assay, total IgA, CBC, Iron profile, glycosylated hemoglobin (HbA1C), calcium, phosphorus, albumin.
The criterion for selection of patients undergoing intestinal biopsy was any of the following:
I. Positive anti-TTG and positive EMA
II. Positive EMA alone
III. Positive anti-TTG > 50 U/ml alone
IV. Persistently positive anti-TTG at low titer 20 – 50 U/ml (two readings in 6 months)
Following endoscopy, if indicated, 6 duodenal biopsies were obtained including one from duodenal cap. Biopsies were immersed in formalin solution and examined histologically at the Department of Pathology. Formalin-fixed biopsies were stained with hematoxylin and eosin and examined under light microscopy. Biopsies were reviewed by a single pathologist and reported according to Marsh classification
]. The pathologist was blinded to clinical and endoscopic data and serologic results.
Methods of Serology tests
1) Anti -TTG IgA testing was undertaken with a commercially obtained ELISA kit (Inova Diagnostics, San Diego, California, USA). In brief, stored serum samples were thawed and diluted with horseradish peroxidase diluent and tested in duplicate at room temperature along with appropriate negative and positive controls. The optical density of each pair of duplicates was converted to an ELISA standard by reference to positive controls. An ELISA cutoff of less than 20 was considered normal and greater than 20, positive. Children with low anti-TTG titer (20–50 U/ml) had a repeat of the test after 6 months. Anti-TTG value < 20 U/ml on the second test defines transient positivity of Anti-TTG and deems intestinal biopsy unnecessary. Persistent positivity of anti-TTG at low titers was considered an indication for intestinal biopsy.
2) EMA (IgA) in serum was measured using indirect immunofluorescence assay and cryostat sections of monkey esophagus (INOVA Diagnostics Inc., San Diego, California, USA). Serum samples were incubated with substrate for 30 min in moist chamber; sections were then washed with phosphate-buffered saline and incubated for 30 min with fluorescein isothiocyanate. Finally, after washing and applying the mounting medium, sections were examined using fluorescence microscope and the results were reported by comparing with positive and negative controls which were included in every assay. The assays were performed at 3 screening dilutions of 1:5, 1:10, and 1:20. The test result was considered positive when there was a reticulated honeycomb staining of the connective tissue that surrounded the bundles of esophageal smooth muscle.
3) Total IgA: Serum level of IgA had been assayed using a nephelometric method with the aid of a BN II nephelometer (Siemens, Germany).
The study was approved by the local research and ethics committee of Children’s hospital at King Saud Medical City and had been performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki.
The data were analyzed using SPSS pc+ version 16.0 statistical software. Descriptive statistics (mean, standard deviation and proportions) were used to summarize the study variables. Student’s t-test for independent samples was used to compare the mean values of continuous study variables. The 95% confidence intervals for difference of mean were used. Chi-square test and Fisher’s exact tests were used to observe an association between the qualitative study and outcome variables. Sensitivity and specificity values were calculated to evaluate the test procedures (EMA & anti-TTG) in comparison with gold standard (Biopsy). Receiver operated characteristics (ROC) curve was used to determine the best cut-off anti-TTG value with best sensitivity and specificity to diagnose CD. A p value of less than 0.05 was considered statistically significant.