Usefulness of Functional Thyroid-Stimulating and Thyroid-Blocking Immunoglobulin Bioassays in an Atypical Presentation of Graves' Disease.

Background/Objective: Thyroid-stimulating hormone (TSH) receptor antibody (TRAb) is well recognized as the pathogenic antibody that causes the clinical manifestation of Graves' disease (GD). Although the majority of TRAb measured in GD is due to thyroid-stimulating immunoglobulin (TSI), there are other functional classes of TRAb, ie, thyroid-blocking immunoglobulin (TBI) and neutral antibodies, which can alter the clinical course of the disease. We present a case of a patient who demonstrated interesting coexistence of both the forms using Thyretain TSI and TBI Reporter BioAssays. Case Report: A 38-year-old woman presented with thyrotoxicosis (TSH level, 0.01 mIU/L, free thyroxine level, >7.8 ng/mL [>100 pmol/L], and free triiodothyronine level, >32.6 pg/mL [>50 pmol/L]) to her general practitioner. She was treated with 15-mg carbimazole twice daily before the dose was reduced to 10 mg. Four weeks later, she developed severe hypothyroidism, with a TSH level of 57.5 mIU/L, free thyroxine level of 0.5 ng/mL (6.7 pmol/L), and free triiodothyronine level of 2.6 pg/mL (4.0 pmol/L). Carbimazole was ceased; however, she remained severely hypothyroid, with a TRAb level of 35 IU/L. Both TSI (304% signal-to-reference ratio) and TBI (56% inhibition) were present, with predominance of the blocking form of thyroid receptor antibodies (54% inhibition). Thyroxine was commenced, and her thyroid functions remained steady and TSI became undetectable. Discussion: The results of the bioassays confirmed that both TSI and TBI can coexist in a patient and that its action changes within a short period of time. Conclusion: Clinicians and laboratory scientists should be aware of the usefulness of TSI and TBI bioassays in the interpretation of atypical presentations of GD.

Thyrotropin Receptor Autoantibody Assessment in Thyroid Eye Disease: Does the Assay Type Matter?

PURPOSE: Thyroid receptor antibodies can quantify thyroid eye disease activity, predict outcomes and aid timing of interventions. The type and generation of assay is frequently unspecified, complicating meta-analyses. To determine the clinical and biochemical relationships between a second-generation thyrotropin receptor-binding inhibition antibody (TRAb) immunoassay, detecting stimulatory and blocking antibodies, with the thyroid stimulating immunoglobulin (TSI) bridging immunoassay detecting the stimulatory component only. METHODS: Retrospective review of 100 consecutive patients attending a regional specialist service. For each patient and visit, both a TRAb and TSI were performed, and a clinical activity score (CAS) recorded. RESULTS: A significant positive correlation between TRAb and TSI (rho = 0.828, p < 0.01) but a weaker correlation between the assays and CAS (TRAb: rho = 0.439, p < 0.01; TSI: r = 0.357, p < 0.01) were found. In 10% of the episodic data, patients had a TRAb level that was disproportionately high (39.41 ± 52.84 IU/L), compared to their TSI levels (9.53 ± 12.10 IU/L) with a higher-than-average CAS (2.47 ± 1.78; range, 0-5). Within 12 months of diagnosis, a significant positive correlation between CAS and TRAb (rho = 0.503, p < 0.01) as well as between CAS and TSI (rho = 0.329, p < 0.01) were found. In patients with a diagnosis over 12 months, the correlation with CAS for both TSI and TRAb were Spearman rank correlation coefficient of 0.347 (p < 0.01) and 0.327 (p < 0.01), respectively. CONCLUSIONS: TRAb and TSI correlate strongly and to a lesser extent with the CAS. For most patients, TRAb can be replaced with the more economical TSI. TRAb also correlates better with newly diagnosed, more active patients than TSI. In a subset of patients, blocking antibodies may play a significant pathogenic role, requiring different treatment and monitoring. Further studies are required to investigate this relationship.

Thyroid-Stimulating Antibody/Thyroid-Stimulating Hormone Receptor Antibody Ratio as a Sensitive Screening Test for Active Graves' Orbitopathy.

OBJECTIVE: Graves' orbitopathy (GO), an extrathyroidal manifestation of Graves' disease, can seriously threaten a patient's quality of life. Given that immunosuppressive treatment during the early active phase of GO has been found to reduce both disease activity and severity, sensitive screening tests are needed. METHODS: The present study included 86 patients with GO, in whom serum levels of thyroid-stimulating hormone (TSH), free triiodothyronine (T3), free thyroxine, thyroid-stimulating antibody, TSH receptor antibody, thyroid peroxidase antibody, thyroglobulin, and thyroglobulin antibody were measured within 2 months before magnetic resonance imaging (MRI) for orbit assessment. RESULTS: The thyroid-stimulating antibody/TSH receptor antibody ratio was able to distinguish MRI results with a correct classification rate of 81%. When focusing on patients without T3 predominant Graves' diseases, the ratio distinguished MRI results at a rate of 92%. Receiver operating characteristic curve analysis revealed a cutoff antibody ratio of 87, which yielded a sensitivity, specificity, positive likelihood ratio, and negative likelihood ratio of 91%, 95%, 18.2, and 0.0957, respectively, for distinguished MRI results. CONCLUSIONS: The thyroid-stimulating antibody/TSH receptor antibody ratio is a highly sensitive and specific indicator for active GO, especially in patients without T3 predominance, and serves as a good screening test for active GO in primary care settings.

A rapid homogenous bioassay for detection of thyroid-stimulating antibodies based on a luminescent cyclic AMP biosensor.

Graves' disease (GD) is an autoimmune disease caused by antibodies to the thyroid stimulating hormone receptor (TSHR). The FDA-cleared Thyretain™ TSI bioassay is a highly specific method to detect thyroid stimulating antibodies (TSAb/TSI) in the blood of patients with autoimmune thyroid disease (AITD), particularly GD. To simplify the workflow of this bioassay and to support a semi-quantitative result, we have generated a stable CHO-K1 cell line expressing both a chimeric TSH receptor (TSHR-Mc4) and a luciferase-based homogeneous cAMP biosensor (GS luciferase). Here, we describe a rapid, real-time, homogenous bioassay (Turbo™ TSI Bioassay) to directly assess the functional activity of TSI and produce results in International Units of IU/L. The Turbo™ TSI bioassay works by measuring changes in the intracellular cAMP level induced by a G-protein coupled receptor (G-PCR) signaling cascade which is triggered by the binding of TSI to the TSHR. Upon binding to cAMP, the GS luciferase reporter is activated through conformational changes and generates light that can be measured in intact cells with a luminometer. The LoD and LoQ of the assay were determined to be 0.016 IU/L and 0.03 IU/L, respectively and the preliminary assay cutoff was determined to be 0.024 IU/L by ROC analysis using the Thyretain™ TSI bioassay results as reference. The analytical performance of the Turbo™ TSI bioassay is comparable to the Thyretain™ TSI bioassay as evidenced by similar EC50 values for a TSHR stimulating monoclonal antibody (M22). The specificity of the Turbo™ TSI bioassay was demonstrated by showing no response to a high concentration of a human monoclonal TSHR blocking antibody (K1-70). The precision of the assay was excellent with an overall within-laboratory precision <15% CV. When testing 198 clinical samples, the positive and negative percent agreement between the Turbo™ TSI and the Thyretain™ TSI bioassays were 98.7% and 93.5%, respectively. While both bioassays yield equivalent analytical and clinical performances, the Turbo™ TSI bioassay is much simpler to perform. It does not require cell culture, sample dilution, washing or cell lysis steps, resulting in a dramatically reduced turnaround time from about 21 h to 60 min. In addition, the same cell line showed its capability of detecting thyroid blocking antibodies (TBAb/TBI) in a competitive format. The Turbo™ TSI bioassay is user-friendly and is a very promising advancement to aid the diagnosis of autoimmune thyroid disease (AITD).

Diagnostic Utility of a New Assay for Thyroid Stimulating Immunoglobulins in Graves' Disease and Thyroid Eye Disease.

Background: The syndrome of thyrotoxicosis typically relies on radioactive iodine scans for establishing its etiology. Alternatively, the determination of thyrotropin (TSH) receptor antibodies (TRAbs) helps to distinguish Graves' disease (GD) from thyrotoxic thyroiditis. Current assays are impacted by limitations in sensitivity and/or turnaround time. Therefore, we decided to test a new assay for the detection of TSH receptor stimulating antibodies (TSAbs) and compare it with the clinically available assays. Methods: We enrolled 110 individuals in 5 cohorts: patients with incident or recurrent GD (cohort 1); patients with thyroiditis, painless or subacute (cohort 2); patients with Graves' orbitopathy/thyroid eye disease (TED) in cohort 3; patients with Hashimoto's thyroiditis (cohort 4), and a control group of normal volunteers (cohort 5). The patients were tested with the two clinically available assays: Roche Elecsys anti-TSHR assay (ROC-TBII) from Roche Diagnostics and Thyretain™ TSI Reporter BioAssay Kit (QUI-TSI) from Quidel. In addition, the samples were tested with the aequorin TSAb assay (OTS-TSI) provided by Otsuka Pharmaceutical Co., Ltd. (Tokyo, Japan). The data were collected in a cross-sectional manner before initiation of therapy. Results: We had 36 cases of GD, 17 cases of thyroiditis, 27 cases of TED, 10 cases of Hashimoto's thyroiditis, and 20 normal volunteers. OTS-TSI had 100% sensitivity and specificity in distinguishing GD from thyroiditis, identical with QUI-TSI but superior to ROC-TBII (sensitivity 86% and specificity 94.1%). OTS-TSI had 93% sensitivity and 100% specificity for the diagnosis of TED, compared with normal controls. QUI-TSI and ROC-TBII performed similarly in this analysis, demonstrating 82% sensitivity and 100% specificity. The range of detectable values for OTS-TSI was 20-29,000 mIU/L and the turnaround time was ≤6 hours, without the need for cell culture equipment. Conclusions: OTS-TSI performed excellently, though similarly to QUI-TSI, for the differential diagnosis of GD vs. thyroiditis, while being superior in that respect to ROC-TBII. Furthermore, OTS-TSI has superior sensitivity to QUI-TSI and ROC-TBII for TED diagnosis, while retaining high specificity. It has a short turnaround time and avoids the need for cell culture and sterility. Larger studies in U.S. populations are needed for its validation.

Novel mutation causing propionic acidemia associated with unexplained autoimmune thyrotoxicosis.

Propionic acidemia (PA) is a rare autosomal recessive inborn error of metabolism (IEM) with relatively higher prevalence in the United Arab Emirates (UAE). Absence of propionyl-CoA carboxylase (PCC) enzyme classically leads to acute decompensation in the early neonatal period. We report a novel homozygous frameshift variant c.2158_2159insT; p.Glu720Valfs*14 (NM_000282.3) in the last exon of the PCCA gene which led to a severe presentation of PA in a newborn Emirati female. Uniquely the diagnosis remained unclear since newborn screening revealed an isolated elevation in plasma proprionylcarnitine (C3) while urinary organic acids remained persistently negative for the classic biochemical abnormalities even during the period of critical illness. Additionally, the patient had an unexplained diagnosis of neonatal thyrotoxicosis. This case explores possible underlying causes through an extensive literature search. To date, there have been no similar reported cases in existing literature.

Diagnostic testing for Graves' or non-Graves' hyperthyroidism: A comparison of two thyrotropin receptor antibody immunoassays with thyroid scintigraphy and ultrasonography.

OBJECTIVE: Graves' disease (GD) is the most common cause of hyperthyroidism. In many cases, when the aetiological diagnosis of GD is not evident based on the clinical evaluation and thyroid function testing, it may become challenging to distinguish Graves' hyperthyroidism from other forms of thyrotoxicosis. The current study was primarly carried out to compare the diagnostic effectiveness of two TSH receptor antibody immunoassays (IMAs), ultrasonography and thyroid scintigraphy in hyperthyroidism scenario. METHODS: We retrospectively analysed consecutive patients with newly diagnosed and untreated thyrotoxicosis who underwent thyroid functional tests, both TRAb and TSI measurements, thyroid scintigraphy and ultrasonography. TRAb assessment was carried out by Kryptor® compact PLUS, while TSI by Immulite® . Echo pattern 3 corresponded to 'thyroid inferno', and the final diagnosis of GD vs non-Graves' hyperthyroidism was made according to the thyroid scan (qualitative scintigraphy). 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 all the tests. RESULTS: A total of 124 untreated hyperthyroid patients were included in our study (GD, n = 86 vs non-Graves' hyperthyroidism, n = 38). ROC curves showed that the optimal cut-off values associated with the highest diagnostic sensitivity and specificity was 0.7 IU/L for TRAb Kryptor® (93 [85.4-97.4] and 86.8 [71.9-95.5]) and 0.1 IU/L for TSI Immulite® (94.2 [86.9-98.1] and 84.2 [68.7-93.9]), respectively. For the echo pattern 3, we found a good sensitivity (92.1%) and a high PPV (95.2%) but a quite low specificity value (69.8%) and a relative low NPV (57.5%). For thyroid scintigraphy, the TcTU cut-off value of 1.3% corresponded to the best limit for sensitivity and specificity in our patients (95.3 [88.5-98.7] and 96.4 [81.6-99.4]). The Passing-Bablok regression equation and the Bland-Altman test showed a great degree of correlation and agreement existed between TRAb Kryptor® and Immulite® TSI results. CONCLUSIONS: Thyroid scintigraphy remains the most accurate method to differentiate causes of thyrotoxicosis. However, TRAb assays can be alternatively adopted in this setting, limiting the use of thyroid scintigraphy (TcTU evaluation) to TRAb-negative patients. Thyoid US is less accurate than both TRAb/TSI and thyroid scintigraphy, but the 'thyroid inferno' pattern provides a high PPV for GD.

Clinical diagnosis of Graves' or non-Graves' hyperthyroidism compared to TSH receptor antibody test.

BACKGROUND: TSH receptor antibody (TRAb) is considered the gold standard diagnostic test for the autoimmunity of Graves' disease (GD), which is commonly diagnosed clinically. AIM: To evaluate the true positive (sensitivity) and true negative (specificity) rates of clinical diagnosis of GD or non-GD hyperthyroidism compared to the TRAb test. SETTING: University teaching hospital in North West England. PARTICIPANTS: Patients in the Endocrinology service who had a TRAb measurement between December 2009 and October 2015. METHODS: Electronic patient records were studied retrospectively for a pre-TRAb clinical diagnosis of GD or non-GD hyperthyroidism. We examined descriptive statistics and binary classification tests; Fisher exact test was used to analyse contingency tables. RESULTS: We identified 316 patients with a mean age of 45 (range, 17-89) years; 247 (78%) were women. Compared to the TRAb result, clinical diagnosis had a sensitivity of 88%, specificity 66%, positive predictive value 72%, negative predictive value 84%, false negative rate 12%, false positive rate 34%, positive likelihood ratio 2.6 and negative likelihood ratio 0.2 (P < 0.0001). CONCLUSIONS: Clinicians were liable to both over- and under-diagnose GD. The TRAb test can help reduce the number of incorrect or unknown diagnoses in the initial clinical assessment of patients presenting with hyperthyroidism.

Thyroid-stimulating immunoglobulins indicate the onset of dysthyroid optic neuropathy.

PURPOSE: Recognition of dysthyroid optic neuropathy (DON) requires sensitive diagnostic tools. Clinical assessment may fail to reliably evaluate the acuteness of DON especially if signs for inflammation are missing. Aim of this cross-sectional study was to assess the relationship between thyroid-stimulating immunoglobulins (TSI) and onset of DON. METHODS: At a multidisciplinary orbital center, serum TSI levels were measured in 180 consecutive patients with thyroid eye disease (TED) and 302 healthy controls with a FDA-cleared cell-based bioassay using a chimeric TSH receptor and a CRE-dependent luciferase. RESULTS: Thirty of 180 (16.7 %) patients with TED had DON of recent onset or a past history of DON (post-DON). Optic disk swelling was present and visual-evoked potentials were pathologic in all eyes with DON of recent onset, but in one of 13 (7.7 %) with post-DON, only (p = 0.005). 19/20 (96 %) patients with DON of recent onset were TSI-positive. TSI was associated with DON of recent onset (OR: 20.96; 95 % CI 1.064-412.85, p = 0.045). All controls were TSI negative. TSI correlated with the clinical activity score (R = 0.70, p < 0.001) and higher TSI-levels were noted in active vs. inactive TED (485.1 ± 132.3 vs. 277.7 ± 143.7 %, cut-off < 140 %; p < 0.001). Six of seven (85.7 %) patients with inactive TED with recent onset DON versus one of four (25 %) with active post-DON were TSI-positive (p = 0.006). A discriminatory cut-point of 377 SRR % for TSI was determined based on a ROC analysis (sensitivity: 0.95, specificity: 0.8). CONCLUSIONS: Serum TSI levels identify patients with DON of recent onset requiring urgent therapy.

AMERICAN ASSOCIATION OF CLINICAL ENDOCRINOLOGISTS MEDICAL GUIDELINES FOR CLINICAL PRACTICE FOR THE EVALUATION AND TREATMENT OF HYPERTHYROIDISM AND HYPOTHYROIDISM.

These clinical practice guidelines summarize the recommendations of the American Association of Clinical Endocrinologists for the diagnostic evaluation of hyperthyroidism and hypothyroidism and for treatment strategies in patients with these disorders. The sensitive thyroid-stimulating hormone (TSH or thyrotropin) assay has become the single best screening test for hyperthyroidism and hypothyroidism, and in most outpatient clinical situations, the serum TSH is the most sensitive test for detecting mild thyroid hormone excess or deficiency. Therapeutic options for patients with Graves' disease include thyroidectomy (rarely used now in the United States), antithyroid drugs (frequently associated with relapses), and radioactive iodine (currently the treatment of choice). In clinical hypothyroidism, the standard treatment is levothyroxine replacement, which must be tailored to the individual patient. Awareness of subclinical thyroid disease, which often remains undiagnosed, is emphasized, as is a system of care that incorporates regular follow-up surveillance by one physician as well as education and involvement of the patient.