We also observed an association between PCA + and GADA + (OR = 19, = 0049), aTPO + (OR = 19, = 004) and HLA DQA1*0501-DQB1*0301 status (OR = 24, = 0045). 0049), aTPO + (OR = 19, = 004) and HLA DQA1*0501-DQB1*0301 status (OR = 24, = 0045). Iron deficiency anaemia (OR = 30, = 0003) and pernicious anaemia (OR = 40, 00001) were more frequent in PCA + GLPG0492 subjects. EmA-IgA + was linked to HLA DQA1*0501-DQB1*0201 + (OR = 75, = 0039), and coeliac disease was found in three individuals. No individual experienced Addison’s disease. In conclusion, GADA but not IA2A indicate the presence of thyrogastric autoimmunity in type 1 diabetes. aTPO have a female preponderance, PCA are weakly associated with HLA DQA1*0501-DQB1*0301 and GLPG0492 EmA-IgA + with HLA DQA1*0501-DQB1*0201. = 006)DQA1*0501-DQB1*0201210 (53%)103 (55%)107 (50%)n.s.DQA1*0301-DQB1*0302210 (53%)94 (50%)116 (55%)n.s.DQA1*0501-DQB1*030130 (8%)16 (9%)14 (7%)n.s. Open in a separate DLL1 windowpane The study was authorized by the Ethics Committee of the University or college Hospital of Antwerp. Informed consent was from each individual and/or parent. Methods Islet cell antibodies (ICA) were determined by indirect immunofluorescence on cryosections of human being donor pancreas (blood group O) . Antibodies to glutamic acid decarboxylase-65 (GADA) and to tyrosine phosphatase (IA2A) were determined by liquid-phase radiobinding assay using, respectively, Centricon-purified recombinant human being 35S-GAD65 and the 35S-labelled intracellular website of IA-2 as tracer . Cut-off ideals for antibody positivity were identified as the 99th percentile of antibody levels acquired in 783 non-diabetic control subjects, and amounted to 12 JDF devices for ICA, 26% tracer bound for GADA and 05% tracer bound for IA2A . These assays were validated by repeated participation in Immunology of Diabetes Workshops, and skills testing programmes of the University or college of Florida and the Louisiana State University or college. In the second option programme, our assays accomplished 100% diagnostic level of sensitivity, specificity, consistency and validity. In the combinational islet autoantibody workshop, assay GLPG0492 level of sensitivity modified for 99% specificity amounted to 73% for ICA and 85% for GADA . In the 1st IA2A proficiency programme, our method accomplished a 100% score for laboratory level of sensitivity, specificity, consistency and validity . Thyroid peroxidase antibodies (aTPO) were measured by radiobinding assay (Henningtest, Brahms, Germany; nl 100 U/ml). Thyroid function was estimated by assay of GLPG0492 TSH (nl: 047C47 mU/l) and feet4 levels (Vitros, OCD, Amersham, UK; nl: 108C219 pmol/l). Parietal cell antibodies (PCA) were recognized using indirect immunofluorescence on sections of rat gastric mucosa (Medical Diagnostics California, Carlsbad, USA; nl 1/20 dilution) . This indirect immunofluorescence (IIF) assay correlated well with the enzyme immunoassay for H+/K+ATPase antibodies (Varelisa, Pharmacia & Upjohn, GmbH, Germany; nl 10 U/ml) (= 175; = 085; 00001). Antibodies to intrinsic element (AIF) were measured by radiobinding assay (Diagnostic Products Corporation, Los Angeles, USA; nl 11). Pernicious anaemia was defined as a megaloblastic anaemia with positive AIF and/or PCA. Iron deficiency anaemia was defined as decreased haemoglobin concentration, microcytic and hypochromic indices, and decreased ferritin levels (M 20 and F 12 g/l). Anti-adrenal antibodies (AAA) and anti-endomysium IgA (EmA-IgA) were determined by indirect immunofluorescence using, respectively, unfixed freezing sections of monkey adrenal cells and sections of monkey oesophagus as antigen substrate (MeDiCa kit, Cat. no. 6001-AG and 6001-ES, respectively, nl 1/10 dilution). Quantitative dedication of 21-hydroxylase antibodies in 100 individuals using 125I-radioassay (DLD, Diagnostika GmbH, Cat. No RA007/50, Hamburg, Germany) offered the same results as those acquired by IIF. HLA-DQ (Human being Leucocyte Antigen) typing was performed as explained previously . Statistical analysis Data were analysed using SPSS (SPSS Inc., Chicago, OH, USA). Distributions of continuous data were tested for normality from the Kolmogorov Smirnov test. The unpaired 005 was regarded as significant. Results The prevalence of -cell, thyroid, gastric, adrenal and endomysial autoantibodies is definitely demonstrated in Table 1 and Fig. 1. After a disease period of 9 8 years, 77% of subjects exhibited -cell antibodies and 34% experienced thyrogastric antibodies. Stratification of individuals relating to disease duration (0C5.