Diagnosis of insulin autoimmune syndrome using polyethylene glycol precipitation and gel filtration chromatography with ex vivo insulin exchange.

CONTEXT: Insulin-binding antibodies may produce severe dysglycaemia in insulin-naïve patients ('insulin autoimmune syndrome' (IAS) or Hirata disease), while rendering routine insulin assays unreliable. OBJECTIVE: To assess the performance of clinically used insulin assays and an optimal analytical approach in the context of IAS. DESIGN: Observational biochemical study of selected patients with hyperinsulinaemic hypoglycaemia. PATIENTS: Three patients without diabetes with recurrent spontaneous hyperinsulinaemic hypoglycaemia and 'positive' insulin antibodies. MEASUREMENTS: A panel of clinically used insulin assays (Siemens ADVIA® Centaur, Siemens Immulite® 2000, DiaSorin LIAISON® XL, PE AutoDELFIA® and the Beckman Coulter Access® 2) were used before and after plasma dilution or polyethylene glycol (PEG) precipitation. Anti-insulin IgG antibodies were measured by Isletest™ -IAA ELISA. Gel filtration chromatography (GFC) was undertaken with and without preincubation of plasma with exogenous insulin. RESULTS: Dilution of IAS plasma with assay-specific buffer increased insulin recovery, supporting negative immunoassay interference by antibodies. PEG precipitation of IAS plasma decreased insulin recovery using all assays except the Immulite® 2000. GFC discriminated high molecular weight and monomeric insulin, while ex vivo addition of exogenous insulin to plasma increased insulin bound to antibody, thereby improving the sensitivity of detection of insulin immunocomplexes. CONCLUSIONS: Immunoprecipitation with PEG must be used with caution in screening for insulin-antibody complexes as results are assay dependent. GFC with addition of exogenous insulin can identify significant insulin immunocomplexes with enhanced sensitivity, with attendant greater clinical utility and avoidance of radiolabelled reagents.

A novel albumin gene mutation (R222I) in familial dysalbuminemic hyperthyroxinemia.

CONTEXT: Familial dysalbuminemic hyperthyroxinemia, characterized by abnormal circulating albumin with increased T4 affinity, causes artefactual elevation of free T4 concentrations in euthyroid individuals. OBJECTIVE: Four unrelated index cases with discordant thyroid function tests in different assay platforms were investigated. DESIGN AND RESULTS: Laboratory biochemical assessment, radiolabeled T4 binding studies, and ALB sequencing were undertaken. (125)I-T4 binding to both serum and albumin in affected individuals was markedly increased, comparable with known familial dysalbuminemic hyperthyroxinemia cases. Sequencing showed heterozygosity for a novel ALB mutation (arginine to isoleucine at codon 222, R222I) in all four cases and segregation of the genetic defect with abnormal biochemical phenotype in one family. Molecular modeling indicates that arginine 222 is located within a high-affinity T4 binding site in albumin, with substitution by isoleucine, which has a smaller side chain predicted to reduce steric hindrance, thereby facilitating T4 and rT3 binding. When tested in current immunoassays, serum free T4 values from R222I heterozygotes were more measurably abnormal in one-step vs two-step assay architectures. Total rT3 measurements were also abnormally elevated. CONCLUSIONS: A novel mutation (R222I) in the ALB gene mediates dominantly inherited dysalbuminemic hyperthyroxinemia. Susceptibility of current free T4 immunoassays to interference by this mutant albumin suggests likely future identification of individuals with this variant binding protein.

Concordance between thyroglobulin antibody assays.

BACKGROUND: Antithyroglobulin antibodies are a prevalent cause of interference in serum thyroglobulin immunoassays. Current guidelines recommend that antithyroglobulin antibodies should be measured concurrently with thyroglobulin when monitoring thyroid cancer patients post-thyroidectomy. However, the concordance between different antithyroglobulin assays has been questioned despite the availability of an international thyroglobulin antibody Reference Preparation. METHODS: Four antithyroglobulin assays currently in use in UK laboratories (Siemens Immulite(®), Brahms GmbH, PerkinElmer AutoDELFIA and Siemens ADVIA Centaur(®)) were compared in a cohort of 145 thyroid cancer patients. RESULTS: Using reference data provided by the kit manufacturer, concordance between the assays was 74%. Adjusting the cut-offs to maximize agreement increased concordance to 90%. Recovery of exogenous thyroglobulin using the Brahms Tg-plus immunoradiometric assay was neither a specific nor a sensitive test for the presence of a positive antibody result by any assay. CONCLUSIONS: Despite the availability of an international reference preparation, current antithyroglobulin assays show unacceptable variance.