Harmonization of glutamic acid decarboxylase and islet antigen-2 autoantibody assays for national institute of diabetes and digestive and kidney diseases consortia

Abstract

Background/rationale: Autoantibodies to islet antigen-2 (IA-2A) and glutamic acid decarboxylase (GADA) are markers for diagnosis, screening, and measuring outcomes in National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) consortia studies. A harmonization program was established to increase comparability of results within and among these studies.

Methods: Large volumes of six working calibrators were prepared from pooled sera with GADA 4.8-493 World Health Organization (WHO) units/ml and IA-2A 2-235 WHO units/ml. Harmonized assay protocols for IA-2A and GADA using (35)S-methionine-labelled in vitro transcribed and translated antigens were developed based on methods in use in three NIDDK laboratories. Antibody thresholds were defined using sera from patients with recent onset type 1 diabetes and healthy controls. To evaluate the impact of the harmonized assay protocol on concordance of IA-2A and GADA results, two laboratories retested stored TEDDY study sera using the harmonized assays.

Results: The harmonized assays gave comparable but not identical results in the three laboratories. For IA-2A, using a common threshold of 5 DK units/ml, 549 of 550 control and patient samples were concordantly scored as positive or negative, specificity was greater than 99% with sensitivity 64% in all laboratories. For GADA, using thresholds equivalent to the 97th percentile of 974 control samples in each laboratory, 1051 (97.9%) of 1074 samples were concordant. On the retested TEDDY samples, discordance decreased from 4 to 1.8% for IA-2A (n = 604 samples; P = 0.02) and from 15.4 to 2.7% for GADA (n = 515 samples; P < 0.0001).

Conclusion: Harmonization of GADA and IA-2A is feasible using large volume working calibrators and common protocols and is an effective approach to ensure consistency in autoantibody measurements.

Figure 1

Sequential steps taken in harmonization process.

Figure 2

Calibration of NIDDK calibrator samples. A, NIDDK calibrators A to F (abscissa) were tested together with the WHO reference preparation for IA-2A and GADA using the harmonized assays on five separate occasions in the Munich (open circles), Bristol (filled circles), and Denver (open triangles) laboratories to determine their WHO units/ml equivalents. The calibrated WHO units/ml for each of these measurements are shown. The median of the WHO units/ml was used to assign DK units/ml to the NIDDK calibrators. B, A set of coded samples that included 18 samples from patients with type 1 diabetes were also included in the calibration exercise. Each set was tested together with NIDDK calibrator samples on five occasions. Using calibration curves of the NIDDK calibrators, DK units/ml were assigned to each of these samples. For each laboratory, the coefficients of variation of the five replicate measurements for each of these samples are shown for IA-2A with values greater than 2.5 DK units/ml (left; n = 14 samples) and for GADA with values greater than 2.5 DK units/ml (right; n = 18 samples) for each of the laboratories. The median coefficient of variation for the sample is shown in each laboratory by the horizontal line.

Figure 3

Concordance of measurement. A, IA-2A measured in Bristol (abscissa) and Denver (ordinate axis) using the harmonized assay in 500 samples from the DASP blood donor control set (open diamonds) and 50 samples from patients with recent onset type 1 diabetes (filled squares). Thresholds of 5 DK units/ml are indicated for both laboratories, and the number of samples falling in each quadrant are indicated. B, GADA measured in Bristol (abscissa) and Denver (ordinate axis) using the harmonized assay in 974 samples representing 500 controls from the DASP blood donor control set and a second set of adult control samples (open diamonds) and 100 samples from patients with recent onset type 1 diabetes (filled diamonds). Thresholds representing the 97th percentile of the control samples are indicated for both laboratories, and the number of samples falling in each quadrant are indicated.

Figure 4

Venn diagram showing concordance of IA-2A (left panels) and GADA (right panels) in the Munich, Bristol, and Denver laboratories on control samples (upper panels; n = 500 for IA-2A, n = 974 for GADA) and patient samples (lower panels; n = 50 for IA-2A, n = 100 for GADA). Numbers refer to the number of positive samples.

Figure 5

Improved concordance of measurement of samples from the TEDDY study. Results are shown for children samples that were found to be discordant for IA-2A (upper panels, n = 24) and GADA (lower panels, n = 79) when measured in the Bristol and Denver laboratories using their local assays (left panels) and remeasured using the harmonized assays (right panels). Threshold for positivity in the respective assays are shown by the broken lines, and numbers represent the samples falling in each quadrant.