Diagnosis of insulin autoimmune syndrome using polyethylene glycol precipitation and gel filtration chromatography withex vivoinsulin exchange

Abstract

SummaryContextInsulin‐binding antibodies may produce severe dysglycaemia in insulin‐naïve patients (‘insulin autoimmune syndrome’ (IAS) or Hirata disease), while rendering routine insulin assays unreliable.ObjectiveTo assess the performance of clinically used insulin assays and an optimal analytical approach in the context of IAS.DesignObservational biochemical study of selected patients with hyperinsulinaemic hypoglycaemia.PatientsThree patients without diabetes with recurrent spontaneous hyperinsulinaemic hypoglycaemia and ‘positive’ insulin antibodies.MeasurementsA 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.ResultsDilution 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.ConclusionsImmunoprecipitation 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.

Assessment and Management of Anti-Insulin Autoantibodies in Varying Presentations of Insulin Autoimmune Syndrome

“…Low insulin recovery following PEG precipitation using an immunoassay suggested IA. GFC with and without addition of exogenous human insulin showed predominantly high molecular weight (HMW) insulin immunoreactivity, confirming IAS ( 6 ). IAs were positive by ELISA and RIA, the latter indicating a high insulin-binding capacity.…”

“…In this series, the first clue to IAS came from high insulin concentrations and insulin/C-peptide molar ratios in samples drawn during hypoglycemia. Immunoassay results were shown to be nonlinear to dilution at presentation (linearity improving following plasma exchange) and to underestimate MS-detected insulin in neat plasma, consistent with assay interference due to the IA competing with the immunoassay antibodies for insulin-binding sites ( 6 ). Consistent with previous observations ( 28–31 ), immunoassay C-peptide concentrations, in the five patients in whom they were measured, were reported at hundreds to thousands of picomoles per liter, concurrent with hypoglycemia.…”

“…Plasma insulin and C-peptide were measured using immunoassay platforms approved for clinical use. PEG precipitation studies were performed as previously published ( 6 ), with analyte recovery taken to be the PEG supernatant insulin concentration expressed as a percentage of insulin measured in matched saline-diluted samples. GFC was performed as previously described ( 6 ).…”

“…Anti–insulin antibody (IA) assays are not standardized and yield variable, qualitative, or semiquantitative results ( 5 ); moreover, detection of IA does not prove the presence of circulating insulin-antibody complexes ( 6 ). Methods currently used to confirm hormone-antibody complexes include precipitation with polyethylene glycol (PEG), which is not specific ( 7 ), and gel filtration chromatography (GFC), which may be used in conjunction with ex vivo addition of insulin to enhance sensitivity ( 6 ). Mass spectrometry (MS) methods now offer quantification of insulin ( 8 ) that is more robust in the face of antihormone antibody interference than immunoassay ( 9 ).…”

Insulin autoimmune syndrome in an occidental woman: a case report and literature review

“…Church and cols. demonstrated that immunoprecipitation with polyethylene glycol (PEG) must be used with caution in screening for insulinantibody complexes as gel filtration chromatography (GFC) with addition of exogenous insulin enhances sensitivity for the identification of insulin immunocomplexes (16). It is noteworthy that we did not consider the possibility of laboratory analytical interference, and we did not test heterophile antibodies.…”