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Abstract: 21-hydroxylase autoantibodies (21OHAb) are the gold standard immune marker to identify patients with clinical or subclinical autoimmune Addison''s disease (AAD). No assessment of interlaboratory concordance has been made for 21OHAb measurement. Serum samples from 267 patients with primary adrenal insufficiency and from 83 healthy control subjects were distributed to four independent laboratories that determined presence and titer of 21OHAb, by using radiobinding assays with either in vitro translated 35S-radiolabelled or 125I-radiolabelled autoantigen. Cohen''s κ of inter-rater agreement ranged from 0.857 to 0.983, showing a very good concordance of the positive/negative score among the four laboratories. Passing–Bablok regression showed a good agreement of 21OHAb titers arranged by ranks, but important discrepancies emerged at the Bland–Altman plot, as the repeatability coefficient was much higher than the laboratory cut-offs, which indicates that results from different laboratories cannot be used interchangeably. A standardization international program for 21OHAb measurement is strongly needed. [Copyright &y& Elsevier]

Abstract: The aim of the workshop was to assess whether four laboratories could reproducibly measure insulin autoantibody (IAA) affinity in coded sera from non-diabetic relatives of patients with type 1 diabetes, newly diagnosed patients, and healthy blood donors, and whether combining affinity with autoantibody titer could improve concordance and performance of IAA assays. IAA affinity was measured by competitive binding using constant amounts of Tyr14A [125I] human insulin and increasing quantities of unlabeled human insulin. There was high concordance between laboratories in distinguishing high, moderate, and low affinity IAA, although IAA binding to insulin varied with assay format. Multiple islet autoantibody-positive and patient sera were identified by high affinity IAA regardless of laboratory-designated IAA status. Combining affinity and titer significantly improved sensitivity, specificity, and concordance of IAA measurement. This workshop has demonstrated that different laboratories are able to reproduce IAA affinity results and that considering IAA affinity is likely to improve the diagnostic performance of IAA assays. [Copyright &y& Elsevier]

Abstract: Islet tyrosine phosphatase 2 (IA-2) is one of the major autoantigens in type 1 diabetes. The aim of this work was to evaluate which IA-2 construct(s) among those usually employed has the highest sensitivity and specificity for detecting IA-2 autoantibodies in autoimmune diabetes and whether the combination of different IA-2 constructs into a single assay allows the detection of immunoreactivities otherwise not detectable by a single construct. For this purpose, we tested the single immunoreactivities of IA-2FL(aa 1–979), IA-2BDC(aa 256–556:630–979), IA-2IC(aa 605–979), IA-2(aa 256–760), IA-2(aa 761–928), and of 7 combinations of these fragments in the sera of 203 newly diagnosed type 1 diabetic patient (DM: 109 males,94 females, mean age 12.9 ± 7.5 years) and 43 prediabetic subject (PDM: 20 males, 23 females, mean age 10.3 ± 6.0 years) sera. IA-2IC was the single construct that showed the highest sensitivity and specificity both in DM and PDM subjects; however, all of the other IA-2 constructs investigated detected additional immunoreactivities with respect to it. The combined use into the same assay of IA-2IC, IA-2FL, and IA-2(256–760) constructs allowed detection of IA-2 Abs in additional 13.3% DM and 30.4% PDM subjects compared to the single IA-2IC construct, suggesting this methodology as a new, highly sensitive approach to the study of IA-2 autoimmunity in type 1 diabetes. [Copyright &y& Elsevier]

A total of 21,000 general population newborns (NECs) and 693 young siblings–offspring of patients with type 1A diabetes (SOCs) were class II genotyped and 293 NECs and 72 SOCs with the high-risk genotype, DR3/4, DQB1*0302 have been prospectively evaluated. Seventeen individuals who converted to persistent autoantibody positivity and two autoantibody-negative control groups (35 SOCs and 24 NECs) were typed for HLA-A class I alleles. The A1, A2 genotype was significantly increased among the autoantibody-positive subjects (47%) compared to autoantibody-negative SOCs (14%, P = 0.01) and NECs (13%, P = 0.02). Life-table analysis of DR3/4, DQB1*0302 siblings revealed a risk of 75% for development of islet autoantibodies by the age of 2 years for those with A1, A2. The HLA-A2 phenotype frequency was increased among an independent DR3/4, DQB1*0302 young diabetes cohort (64% versus 33% for autoantibody-negative NECs). These results suggest that a high incidence and early appearance of islet autoantibodies for siblings of patients with type 1A diabetes are associated with DR3/4, DQB1*0302 and potentially increased with HLA-A genotype A1, A2. [Copyright &y& Elsevier]