Prediction of Impending Type 1 Diabetes through Automated Dual-Label Measurement of Proinsulin:C-Peptide Ratio

Abstract

BackgroundThe hyperglycemic clamp test, the gold standard of beta cell function, predicts impending type 1 diabetes in islet autoantibody-positive individuals, but the latter may benefit from less invasive function tests such as the proinsulin:C-peptide ratio (PI:C). The present study aims to optimize precision of PI:C measurements by automating a dual-label trefoil-type time-resolved fluorescence immunoassay (TT-TRFIA), and to compare its diagnostic performance for predicting type 1 diabetes with that of clamp-derived C-peptide release.MethodsBetween-day imprecision (n = 20) and split-sample analysis (n = 95) were used to compare TT-TRFIA (AutoDelfia, Perkin-Elmer) with separate methods for proinsulin (in-house TRFIA) and C-peptide (Elecsys, Roche). High-risk multiple autoantibody-positive first-degree relatives (n = 49; age 5–39) were tested for fasting PI:C, HOMA2-IR and hyperglycemic clamp and followed for 20–57 months (interquartile range).ResultsTT-TRFIA values for proinsulin, C-peptide and PI:C correlated significantly (r2 = 0.96–0.99; P<0.001) with results obtained with separate methods. TT-TRFIA achieved better between-day %CV for PI:C at three different levels (4.5–7.1 vs 6.7–9.5 for separate methods). In high-risk relatives fasting PI:C was significantly and inversely correlated (rs = -0.596; P<0.001) with first-phase C-peptide release during clamp (also with second phase release, only available for age 12–39 years; n = 31), but only after normalization for HOMA2-IR. In ROC- and Cox regression analysis, HOMA2-IR-corrected PI:C predicted 2-year progression to diabetes equally well as clamp-derived C-peptide release.ConclusionsThe reproducibility of PI:C benefits from the automated simultaneous determination of both hormones. HOMA2-IR-corrected PI:C may serve as a minimally invasive alternative to the more tedious hyperglycemic clamp test.

A composite immune signature parallels disease progression across T1D subjects

“…We selected a broad range of assays to test ( Table 1). This included low-dimension assays selected to assess expected features of disease progression, including proinsulin/C-peptide ratio (18,31), a marker of pancreatic islet β cell dysfunction; a demethylated insulin DNA assay measuring β cell death (19); antigen-specific CD8 + T cell frequency and phenotype as measured by qDot multimer assay (16); and a transcriptional signature of regulatory T cells that had previously been identified to discriminate between subjects with and without T1D (17). Higher-dimension (genome-scale) assays were also included to identify data-driven features of disease progression.…”

“…The Treg transcript assay was performed using the NanoString platform as described previously (14,17), with samples for the Treg assay sorted concomitantly with the cell subset RNA-Seq sample set. The proinsulin/C-peptide assay was conducted using a trefoil time-resolved fluorescence immunoassay (31), adapted to an AutoDelfia automatic system (PerkinElmer) (18). The demethylated insulin assay was conducted using the RainDance droplet digital PCR platform (RainDance Technologies).…”

“…To this end, we established a stringent, well-defined, and collaborative assay evaluation and data analysis method. The assays selected for study included measures that were hypothesized to directly relate to T1D pathogenesis; these included antigen-specific CD8 + T cell frequencies (16), a Treg transcriptional signature previously associated with T1D (17), the ratio of proinsulin to C-peptide (18), and a measure of demethylated insulin DNA in serum (19). Other selected assays showed prior utility in understanding T1D pathogenesis.…”

SLC30A8 polymorphism and BMI complement HLA-A*24 as risk factors for poor graft function in islet allograft recipients

“…Assays and outcome measures Plasma C-peptide and proinsulin (to correct for proinsulin interference in the C-peptide assay) were measured by time-resolved fluorescence immunoassay on AutoDELFIA (PerkinElmer, Turku, Finland) [23,24]. HbA 1c levels were determined by HPLC (Tosoh Bioscience, Tessenderlo, Belgium), and lymphocyte subclasses by an Epics XL flow cytometer (Beckman Coulter, Miami, FL, USA).…”

Cause or effect? A review of clinical data demonstrating beta cell dysfunction prior to the clinical onset of type 1 diabetes

“…PI/C ratios above the 66th percentile were associated with a risk of progression of 50% for multiple Ab+ relatives and 68% for islet tyrosine phosphatase (IA2)+ relatives vs. a risk of 13% for those with lower ratios [62]. Further analyses suggested that fasting PI/C ratios may perform even better for T1D risk prediction when adjusted for insulin sensitivity [63]. Among Ab+ relatives in the TrialNet PTP study in serum samples obtained ∼12 months before T1D diagnosis higher PI/C ratios were associated with increased odds of T1D progression, even after adjustment for age, sex, and BMI-Z scores [64].…”