Evaluation of analytic and clinical performance of two immunoassays for detecting thyroid‐stimulating receptor antibody in the diagnosis of Graves’ disease

Abstract Objective To evaluate the analytical and clinical performance of two immunoassays for diagnosis of Graves’ disease (GD), the Immulite thyroid‐stimulating immunoglobulin (TSI), and Elecsys Anti‐TSH receptor (TSHR) assay. Methods Precision and analytical measurement range were assessed using pooled samples of patients. The comparison between the two methods was evaluated using 579 clinical samples, and receiver operating characteristic (ROC) curves were drawn using the final diagnosis as reference. Clinical sensitivity and specificity, accuracy, positive predictive value (PPV), and negative predictive value (NPV) were calculated for the two tests. Results The repeatability and intermediate imprecision coefficient of variation (CV%) of the TSI assay were 3.8% and 4.1% at 0.95 IU/L, and 3.5% and3.6% at 19.5 IU/L, respectively. The assays were linear over a range 0.27–38.5 IU/L. There was a high correlation between the quantitative results of the two methods (correlation coefficient r = 0.930). The cut‐off value obtained by ROC analysis for TSI assay was 0.7 IU/L with sensitivity of 93.7% and specificity of 85.1%. An overall qualitative agreement of 91.5% between two methods was observed. Among 44 patients with discordant qualitative results, the TSI assay provided more satisfactory results consistent with clinical diagnoses. Conclusion The TSI assay showed excellent analytical performance and provided a high PPV for GD.


| INTRODUC TI ON
Graves' disease (GD) is an autoimmune thyroid disease that usually affects multiple tissues and organs. 1 Epidemiological studies indicate that the incidence of GD is ~20-40 cases per 100,000 population per year. 2 The most common clinical feature of GD is hyperthyroidism. Thyroid-stimulating hormone (TSH) receptor (TSHR) autoantibodies (TRAbs) are pathognomonic of GD, detected in the serum of approximately 98% of patients with untreated GD. 3 According to TRAbs functional features, three varieties are recognized in patients: thyroid-stimulating immunoglobulin (TSI), thyroidstimulating blocking antibody, and cleavage/neutral antibodies. 3,4 The pathogenesis of GD is associated with TSI, which binds to the N-terminus of TSHR extracellular domain and leads to stimulation of the thyroid gland independent of the normal feedback-regulated TSH. 5 Differentiate between TRAbs types and its heterogeneous molecular is essential for GD diagnosis. 3 Although the diagnosis of GD is mainly based on the clinical characteristics of hyperthyroidism, including sweating and palpitations, as well as the specific features of GD, such as orbital lesions and thyroid enlargement, TSI may be found before autoimmune thyrotoxicosis becomes biochemically or clinically manifest. 6 Therefore, the specific and sensitive detection of TSI from other types of TRAbs is becoming increasingly important for the diagnostic accuracy of the Graves' hyperthyroidism and of the extrathyroidal manifestations of GD. In the last 50 years, immunoassays have been used to detect TRAbs and now display excellent analytical and clinical performance in most laboratories, but have a drawback in that they cannot differentiate the types of TRAbs. 7 However, a developed reagent of TSI, the Immulite TSI assay (Siemens Healthcare) has been newly licensed in China market in April 2020. The new assay employs a pair of recombinant human TSHR constructs in a sandwich format that can directly measure TSI with a noncompetitive immunoassay. 8,9 The analytical and clinical performance of this newly direct detection of the TSI has been rarely evaluated based on the Chinese population thus far. Therefore, the aim of the present study was to evaluate the analytical performance and the diagnostic efficacy of the TSI assay, in comparison with that of Elecsys TSHR autoantibody (Anti-TSHR) assay developed by Roche Diagnostics in a large cohort of serum samples obtained from Chinese patients.

| Patients
In total, 559 patients were recruited from the Endocrine Clinic,

| Overview of the immulite TSI and elecsys anti-TSHR
The Immulite TSI assay is a chemiluminescence immunoassay with

| Precision evaluation
Repeatability and intermediate imprecision were analyzed according to the Clinical Laboratory Standard Institute (CLSI) guideline EP15-A3. 11 A total of 25 replicates in 5 days were assayed by two concentrations of patient pooled samples.

| Linearity evaluation
The linearity was determined by serially diluting serum samples with a high concentration clinical sample into a low-level sample according to the CLSI EP6-A. 12 And two replicates in 1 day were tested at five levels. Statistical linearity was established when none of the nonlinear terms in second-and third-order polynomial models was statistically significant. 12 For any sample determined to be statistically nonlinear, the amount of nonlinearity should be less than the manufacturer suggested target (15% or 0.5 IU/L, whichever is greater) to be considered nominally linear.

| Comparative evaluation
The values measured by the TSI assay were compared with those assayed by Anti-TSHR assay (Roche, Diagnostics, Mannheim, Germany) according to the CLSI EP9-A2. 13 The qualitative results of two assays determined by the claimed clinical decision point proposed by the manufacturer were compared, and the overall percent consistency was analyzed according to the CLSI EP12-A2. 14 For inconsistent cases, the electronic medical records, including demographic characteristics, laboratory data, and thyroid ultrasound results, were investigated. The performance of the two methods in GD diagnosis and the consistency of the two methods between GD group and other thyroid diseases group were also evaluated. The diagnostic efficacy was evaluated using a receiver operating characteristic (ROC) curve analysis.

| Statistical analysis
Statistical analyses were performed using MedCalc Software (ver- as almost perfect agreement. 15 All the results of statistical analyses were considered significant when p values were <0.05.

| Precision and linearity evaluation
The repeatability for TSI assay and Anti-TSHR assay in the low-level sample was 3.8% and 7.0%, while it was 3.5% and 1.7% in the highlevel sample, respectively. The intermediate imprecision of the two methods was 4.1% and 7.8% at the low level, 3.6% and 2.0% at the high level ( Figure 1). In addition, all the validated coefficients of variation were below the CVs claimed by the manufacturer. The results of the precision validation were shown in Table 2.
The analytical measurement range of TSI assay and Anti-TSHR  Table 3.

| Comparison of the TSI assay and the anti-TSHR assay
Among the 559 clinical samples tested in this study, the quantitative comparison between the TSI assay and the Anti-TSHR assay was evaluated by the Pearson correlation analysis (Figure 3). There was a high correlation between the results of two assays with a slope  Among 166 GD patients and 393 patients with other thyroid diseases, the Bland-Altman analysis showed a good relationship between TSI assay and the Anti-TSHR assay, respectively (Figure 4).

| ROC analysis
ROC curve analysis has been performed for TSI assay and Anti-TSHR  Table 6, the TSI assay showed a relatively high positive predictive value (PPV) and negative predictive value (NPV) in identifying GD, 94% and 86%, respectively.

| DISCUSS ION
In this study, we evaluated the analytic performance of the Immulite TSI system. The precision and analytical measurement range were evaluated according to CLSI guidelines, and the results of the TSI method were compared with those of the anti-TSHR. Compared with the anti-TSHR, the TSI method has better precision. In the linear evaluation, it is observed that the two detection systems have similar nonlinearity with low value and high deviation. With the increase in analyte concentration, a parabolic linear reaction appeared, and the trinomial curve was the best fitting model, which was also reported in the previous study. 16 There was a high correlation between the results of the Anti-TSHR and the TSI assay (r = 0.930), and the slope was TA B L E 3 The linearity of the Immulite TSI assay and Elecsys Anti-TSHR assay   while the TSI assay specifically detects TSI. In our study, no significant difference was observed between the Anti-TSHR and the TSI assay.
Both the Anti-TSHR and the TSI assay were proved to be highly accurate according to the ROC curve, but the latter had a slightly higher sensitivity than the Anti-TSHR assay, which confirm what has been proved in another study. 17 Also the diagnostic sensitivity of TSI assay for GD resulted higher than the ELiA™-TSH-R assay, which is a new third-generation automatic fluorescence enzyme immunoassay for TRAbs. 18 In all probability, this is associated with the lower analytical sensitivity of the TSI assay and the innovative technology used, which allows to measure TSI through a double epitope recognition.

| CON CLUS ION
The Immulite TSI assay specifically measured the pathogenic antibody of GD and exhibited excellent analytical performance. The Immulite TSI assay may be of great value for the clinical diagnosis of GD.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no competing interests.

AUTH O R CO NTR I B UTI O N S
All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission. Honorarium: We would like to thank all the nursing staff at the Huashan Hospital who helped recruit patients into the study and for collecting samples throughout.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data generated or analyzed during this study are included in this published article and information about experimental sessions and results is available from the corresponding author on reasonable request.

F I G U R E 5 ROC curves for the Immulite TSI assay and Elecsys
Anti-TSHR assay TA B L E 6 Diagnostic accuracy of the two tests in discriminating untreated GD patients and non-GD patients in our cohort (n = 494)