A Negative Thyretain TSI Bioassay Result does not Exclude the Possibility of the Presence of TSI

Comparison of a Bridge Immunoassay with Two Bioassays for Thyrotropin Receptor Antibody Detection and Differentiation

Dear Editor,

We wish to share our concern with significant aspects of the scientific design, analysis, and validity of the work “Comparison of a Bridge Immunoassay with Two Bioassays for Thyrotropin Receptor Antibody Detection and Differentiation” by S. Allelein, T. Diana, M. Ehlers, M. Kanitz, D. Hermsen, M. Schott, and G. J. Kahaly [1], which affect the reliability of the authors’ conclusions. In this publication, the authors sought to compare the FDA-cleared IMMULITE^® 2000/2000 XPi TSI immunoassay with the FDA-cleared Thyretain^TM TSI Reporter BioAssay and the non-FDA-cleared Thyretain TBI Blocking Reporter BioAssay on a set of patients, including those diagnosed with Graves’ disease (GD) and Hashimoto’s thyroiditis (HT). The authors purport to determine the presence of blocking antibodies using the non-FDA-cleared Thyretain TBI bioassay. The authors find that “[b]oth assays were highly sensitive for detecting TSHR-Ab in hyperthyroid patients with untreated GD”. Both TSI assays were positive for all ten untreated GD patients. The authors also found that both TSI assays correctly report negative results in all healthy patients. The clinical specificity of the two TSI assays was evaluated using 78 patients with HT, non-autoimmune nodular thyroid disease, and thyroid cancer. The IMMULITE TSI assay demonstrated a clinical specificity of 97.4% (76/78) compared to 93.6% (73/78) for the Thyretain TSI assay. These findings agree with previous third-party evaluations of the IMMULITE TSI assay showing 98–100% clinical sensitivity and 98.2–99.9% clinical specificity for the differential diagnosis of GD [2] [3] [4] [5] [6].

However, the authors suggest that since two treated Graves’ disease patients were negative in the Thyretain TSI assay but positive in both the IMMULITE TSI assay and the non-FDA-cleared Thyretain TBI bioassay, the IMMULITE TSI assay can be described as not specific for TSH receptor stimulating autoantibodies. The authors’ interpretation of the Thyretain TSI assay results is incorrect because of the experimental design flaws and incomplete analysis of the data. A plausible explanation of the data is that the Thyretain TSI assay results for the two treated GD patients are false negative, and the IMMULITE TSI assay results are true positive.

First, the authors do not mention the explicit warning against the use of the Thyretain TSI assay to test GD patients undergoing various drug treatments. The Thyretain TSI Reporter BioAssay package insert [7] unambiguously states that “[t]he TSI Reporter assay is intended for the qualitative detection of TSI. It is not intended for use in monitoring a patient’s treatment. The effects of various drug therapies on the performance of this Kit have not been established” [7]. Therefore, the validity of the negative Thyretain TSI results for the two treated GD patients is in question.

Second, the Thyretain TSI Reporter BioAssay manufacturer also clearly states in the package insert that “[a] negative result does not exclude the possibility of the presence of TSI. Results of the test should be interpreted in conjunction with information available from other clinical information, such as physical symptoms and thyroid hormone testing, as recommended by the American Thyroid Association (ATA)” [7]. Thus, the authors’ claim that there are no stimulating antibodies in the samples of the two treated GD patients is unfounded because it is based on negative Thyretain TSI results and ignores the manufacturer’s recommendation based on ATA guidelines to interpret the test result in conjunction with other clinical information.

Third, the authors fail to mention or consider in their analysis that the Thyretain TSI assay reports net stimulating activity as documented in previous publications by some of the authors of this study [8] [9]. Although blocking antibodies do not generate a signal in the Thyretain TSI assay, blocking antibodies, when present, interfere with that assay’s measurement of stimulating antibodies [8] [9]. As such, the assay reports “zero” net stimulating activity for samples containing equal activity of stimulating and blocking antibodies. For example, a 2017 article by Diana et al. showed that a sample with 4 ng/ml stimulating antibody M22 and 160 ng/ml blocking antibody K1–70 was negative in the Thyretain TSI assay [10]. In the absence of blocking antibodies, only 0.8 ng/ml of M22 is sufficient to generate maximal stimulating activity/signal in the Thyretain TSI assay [8]. In the presence of blocking antibody K1–70, 4 ng/ml of M22 fails to generate a signal (above the Thyretain TSI assay cutoff) and such a sample is falsely reported as TSI negative by Thyretain TSI assay [10]. Therefore, there is no reliable basis for the authors’ claim in the recent 2019 Allelein et al. [1] publication that the negative Thyretain TSI results for the two treated GD patients means the absence of stimulating antibodies. To the contrary, the positive IMMULITE TSI assay results, taken with the clinical history of the two GD patients, is more likely suggestive that the Thyretain TSI assay results are false negative and that the IMMULITE TSI results are true positive.

The two treated GD patients generated a positive result in the non-FDA-cleared Thyretain TBI assay. It is critical to note, which the authors failed to do, that this assay has not been cleared by the FDA. Moreover, the antibodies detected by the Thyretain TBI assay may be antagonist/blocking or partial (or weak) agonist/stimulating [11]. For example, if the antibodies detected by the non-FDA-cleared Thyretain TBI assay in the two treated GD patients are partial/weak agonists, that would agree with the positive results of the IMMULITE TSI assay. If the antibodies are antagonist/blocking, it would support the argument that because of the interference by blocking antibodies in measuring stimulating antibodies, the Thyretain TSI assay results for the two treated GD patients are false negative. Accordingly, both scenarios support the premise that Thyretain TSI results are false negative and IMMULITE TSI results are true positive. It is worth noting that similar to the Thyretain TSI assay, the Thyretain TBI assay reports the net activity present in a sample, as Diana et al. showed that a sample containing 120 ng/ml of blocking antibody K1–70 and 8 ng/ml stimulating antibody M22 has essentially zero net activity (24% inhibition which is below the cut-off of 40% inhibition) and is falsely reported as being negative for blocking antibodies [10].

In conclusion, the authors’ unsubstantiated assertion that the two GD patients have no stimulating antibodies is purely based on the negative Thyretain TSI results and, moreover, ignores the manufacturer’s warning against using the bioassay to monitor GD patients undergoing drug treatments and the ATA guidelines regarding the interpretation of the test results in conjunction with other clinical information. Because the Thyretain TSI assay reports net stimulating activity, relatively equal activities of simulating and blocking antibodies will produce no net stimulating activity in the bioassay leading to false negative results. It is plausible that the IMMULITE TSI results for the two GD patients are true positive, and the authors failed to produce any data that questions the specificity of the IMMULITE TSI assay.

• References

• 1 Allelein S, Diana T, Ehlers M. et al. Comparison of a bridge immunoassay with two bioassays for thyrotropin receptor antibody detection and differentiation. Horm Metab Res 2019; 51: 341-346
• 2 Tozzoli R, D'Aurizio F, Villalta D. et al. Evaluation of the first fully automated immunoassay method for the measurement of stimulating TSH receptor autoantibodies in Gravesʼ disease. Clin Chem Lab Med 2017; 55: 58-64
• 3 Autilio C, Morelli R, Locantore P. et al. Stimulating TSH receptor autoantibodies immunoassay: Analytical evaluation and clinical performance in Graves’ disease. Ann Clin Biochem 2017; 55: 172-177
• 4 Allelein S, Ehlers M, Goretzki S. et al. Clinical evaluation of the first automated assay for the detection of stimulating TSH receptor autoantibodies. Horm Metab Res 2016; 48: 795-801
• 5 Kemble DJ, Jackson T, Morrison M. et al. Analytical and clinical validation of two commercially available immunoassays used in the detection of TSHR antibodies. J Appl Lab Med 2017; 2: 345-355
• 6 Villalta D, D’Aurizio F, Da Re M. et al. Diagnostic accuracy of a new fluoroenzyme immunoassay for the detection of TSH receptor autoantibodies in Graves’ disease. Auto Immun Highlights 2018; 9: 3-8
• 7 Thyretain TSI. Reporter BioAssay Package Insert, Diagnostic Hybrids. REF PI- 3171000EN00. 2018;
• 8 Leschik JJ, Diana T, Olivo PD. et al. Analytical performance and clinical utility of a bioassay for thyroid-stimulating immunoglobulins. Am J Clin Path 2013; 139: 192-200
• 9 Li Y, Kim J, Diana T. et al. A novel bioassay for anti-thyrotrophin receptor autoantibodies detects both thyroid-blocking and stimulating activity. Clin Exp Immunol 2013; 173: 390-397
• 10 Diana T, Wüster C, Olivo PD. et al. Performance and specificity of 6 immunoassays for TSH receptor antibodies: A multicenter study. Eur Thyroid J 2017; 6: 243-249
• 11 McLachlan SM, Rapoport B. Thyrotropin-blocking autoantibodies and thyroid-stimulating autoantibodies: Potential mechanisms involved in the pendulum swinging from hypothyroidism to hyperthyroidism or vice versa. Thyroid 2013; 23: 14-24