Tanja Diana

Analytical Performance and Clinical Utility of a Bioassay for Thyroid-Stimulating Immunoglobulins

The analytical performance and the clinical utility of a thyrotropin receptor (TSHR)-stimulating immunoglobulin (TSI) bioassay were compared with those of a TSHR-binding inhibitory immunoglobulin (TBII) assay. Limits of detection (LoD) and quantitation (LoQ), assay cutoff, and the half-maximal effective concentration (EC(50)) were measured. Dilution analysis was performed in sera of hyperthyroid patients with Graves disease (GD) during antithyroid treatment (ATD). Titer was defined as the first dilution step at which measurement of TSI or TBII fell below the assay cutoff. The LoD, LoQ, cutoff, and EC(50) of the bioassay were 251-, 298-, 814-, and 827-fold lower than for the TBII assay. There were 22%, 42%, 23%, and 14% more positive samples in the TSI bioassay at dilutions of 1:3, 1:9, 1:27, and 1:81 (P < .0001), respectively. Responders to ATD demonstrated marked differences in titers compared with nonresponders. The bioassay detected lower levels of TSHR autoantibodies, and the dilution analysis provided similar predictive values of both assays in GD.

Thyroid Stimulating Antibodies Are Highly Prevalent in Hashimoto's Thyroiditis and Associated Orbitopathy

CONTEXT Thyroid-associated orbitopathy (TAO) rarely occurs in patients with Hashimotos thyroiditis (HT). OBJECTIVE There is evidence that TSH receptor stimulating antibodies (TSAb) play a role in the pathogenesis of TAO. In this report, the prevalence of TSAb in HT patients with and without TAO was studied. DESIGN This is a longitudinal observational study. SETTING The study took place in an academic joint thyroid-eye clinic. SUBJECTS A total of 1055 subjects were included. METHODS TSAb was measured with a Food and Drug Administration-cleared bioassay that uses Chinese hamster ovary cells expressing a chimeric TSH receptor and a cAMP response element-dependent luciferase. Results of TSAb activity were reported as percentage of specimen-to-reference ratio (SRR%, cutoff >140%). MAIN OUTCOME MEASURE We measured the association of TSAb with the risk of TAO in patients with HT. RESULTS Of 700 consecutive and unselected patients with HT, 44 (6%) had overt TAO. Patients with HT+TAO were older (P < .001), heavier smokers (P = .032), and clustered less with autoimmune diseases (P = .005). All healthy controls were TSAb negative. In contrast, serum was TSAb positive in 30/44 (68.2%) and 36/656 (5.5%, P < .001) patients with HT+TAO and HT, respectively. Compared to patients with HT only, serum TSAb levels were higher in HT+TAO (median SRR%, 25th and 75th percentiles): 45, 35-65 vs 192.5, 115-455.3, P < .001. Highest TSAb values were noted in patients with active and severe TAO vs those with mild and inactive TAO: 486, 392-592 vs 142, 73-192.5; P < .001. The odds ratio of TSAb positivity for the risk of TAO adjusted for gender and age was 55.9 (95% confidence interval [CI], 24.6-127, P < .0001), whereas the odds ratio per 10-fold change in TSAb SRR% (quantitative TSAb) was 133 (95% CI, 45-390, P < .0001). The area under the receiver operating characteristic curve for qualitative and quantitative TSAb was 87.2% (95% CI, 80.6-93.8) and 89.4% (95% CI, 84.1-94.7), respectively. CONCLUSIONS TSAb is strongly associated with TAO in HT and TSAb may contribute to the pathophysiology of TAO.

Analytical Performance and Validation of a Bioassay for Thyroid-Blocking Antibodies

BACKGROUND AND OBJECTIVE A cell-based bioassay for the measurement of thyroid blocking autoantibodies (TBAb) has been recently reported. The analytical performance and validation of this bioassay is assessed and described. METHODS Chinese hamster ovary cells expressing a chimeric thyrotropin receptor were treated with bovine (b) TSH and different concentrations of an immunoglobulin G (IgG) monoclonal human TBAb (K1-70). TBAb was measured as a function of luciferase activity relative to bTSH alone and expressed as percent inhibition. Results obtained in the chimeric cell line were compared with those of a wild-type cell line. Analytical performance studies were subsequently performed with the chimeric cell line only. RESULTS Immunodepletion of K1-70 IgG by using a protein G-Sepharose column showed that positive percent inhibition in the TBAb bioassay was detectable from K1-70 IgG only. The limit of blank was determined to be 12.2%. The limit of detection was 14% inhibition, equivalent to 0.4 ng/mL K1-70, while the limit of quantitation was 22% (coefficient of variation [CV] 12%) equivalent to 0.625 ng/mL K1-70. The dynamic range was between 14 ± 3.7 (mean % inhibition ± standard deviation) and 101 ± 2.6, equivalent to 0.4-10 ng/mL K1-70. The linear range was between 22 ± 2.6 and 93 ± 0.6 inhibition, equivalent to 0.625-5 ng/mL K1-70. The upper limit of the 99th percent reference range was 34% inhibition. In two laboratories, CV values for the intra- and inter-assay precisions for K1-70 ranged from 2% to 12% and from 1.7% to 14.5%, respectively. For patient sera, the CV values for the intra- and inter-assay precisions ranged from 3% to 9% and from 3% to 11%, respectively. No interference was found when follicle-stimulating hormone, luteinizing hormone, and human chorionic gonadotrophin were tested in the TBAb bioassay. The median of % inhibition values in 40 TBAb positive sera from patients with autoimmune thyroid disease were 93.5 (range 25-103) and 92 (range 64-107) for the wild type and chimeric cell lines, respectively. Further, all 40 samples of patients with various non-thyroidal autoimmune diseases were TBAb negative. CONCLUSIONS This TBAb bioassay exhibits excellent analytical performance and high level of reproducibility.

Fetal/neonatal Thyrotoxicosis in a Newborn From a Hypothyroid Woman With Hashimoto’s Thyroiditis

Context Fetal/neonatal thyrotoxicosis is a rare but potentially life-threatening condition. It is most commonly observed in poorly controlled Graves disease during pregnancy. Case Description Here we describe a fetus/newborn patient with thyrotoxicosis who was born of a woman with Hashimoto thyroiditis and levothyroxine-treated hypothyroidism. Transplacental passage of stimulating thyrotropin (TSH) receptor antibodies, which were measured by a cell-based bioassay, was the underlying mechanism of fetal/neonatal thyrotoxicosis, although the mother had no history of hyperthyroidism. Conclusion Diagnosis and management of fetal hyperthyroidism can be challenging. TSH receptor antibody testing should be considered in pregnant women with any history of autoimmune thyroid disease and symptoms of fetal hyperthyroidism.

Double-Blind, Placebo-Controlled, Randomized Trial of Selenium in Graves Hyperthyroidism

Context: Supplemental selenium (Se) may affect the clinical course of Graves disease (GD). Objective: Evaluate efficacy of add‐on Se on medical treatment in GD. Design: Double‐blind, placebo‐controlled, randomized supplementation trial. Setting: Academic endocrine outpatient clinic. Patients: Seventy untreated hyperthyroid patients with GD. Intervention: Additionally to methimazole (MMI), patients received for 24 weeks either sodium selenite 300 &mgr;g/d po or placebo. MMI was discontinued at 24 weeks in euthyroid patients. Main Outcome Measures: Response rate (week 24), recurrence rate (week 36), and safety. Results: A response was registered in 25 of 31 patients (80%) and in 27 of 33 (82%) at week 24 [odds ratio (OR) 0.93; 95% confidence interval (CI), 0.26 to 3.25; P = 0.904] in the Se (+MMI) and placebo (+MMI) groups, respectively. During a 12‐week follow‐up, 11 of 23 (48%) and 12 of 27 (44%) relapsed (OR 1.13; 95% CI, 0.29 to 2.66; P = 0.81) in the Se and placebo groups, respectively. Serum concentrations of Se and selenoprotein P were unrelated to response or recurrence rates. At week 36, 12 of 29 (41%) and 15 of 33 (45%) were responders and still in remission in the Se and placebo groups, respectively (OR 0.85; 95% CI, 0.31 to 2.32; P = 0.80). Serum levels of free triiodothyronine/free tetraiodothyronine, thyroid‐stimulating hormone receptor antibody, prevalence of moderate to severe Graves orbitopathy, thyroid volume, and MMI starting dose were significantly lower in responders than in nonresponders. A total of 56 and 63 adverse events occurred in the Se and placebo groups, respectively (P = 0.164), whereas only one drug‐related side effect (2.9%) was noted in 35 patients on placebo + MMI. Conclusions: Supplemental Se did not affect response or recurrence rates in GD.

Prevalence, Phenotype, and Psychosocial Well-Being in Euthyroid/Hypothyroid Thyroid-Associated Orbitopathy.

BACKGROUND At the onset of thyroid-associated orbitopathy (TAO), most patients are hyperthyroid, while scarce data are available on euthyroid/hypothyroid TAO. The aim of this study was to assess the prevalence, phenotype, and psychosocial burden of patients with initially euthyroid/hypothyroid TAO. METHODS The medical records of 461 consecutive and unselected patients with TAO followed at a specialized joint thyroid-eye clinic were analyzed within this retrospective cross-sectional study. Main outcome measures were the prevalence of initially eu- or hypothyroid TAO as well as ophthalmic signs and symptoms, disease-specific quality of life (QoL), work impairment, and rate of psychotherapy in initially eu-/hypothyroid versus hyperthyroid TAO. RESULTS The prevalences of eu-/hypothyroid and hyperthyroid TAO were 4.3% (n=20; [confidence interval, CI, 2.6-6.3]) and 95.7% (n=441; [9.37-9.74]), respectively. In 12 patients (2.6% [CI 1.3-4.3]), Hashimotos thyroiditis was present and in 8 (1.7% [CI 0.7-3.0]) no thyroid disease was noted at the time of inclusion. One (0.05%) patient with eu-/hypothyroid TAO and 172 (39%) with hyperthyroid TAO had clinically active TAO (p=0.001). In eu-/hypothyroid versus hyperthyroid patients, 14 (70%) versus 135 (30.6%) had a mild TAO, 6 (30%) versus 183 (64.2%) a moderate-to-severe TAO, and 0 versus 23 (5.4%) had a sight-threatening TAO (p<0.001). TAO was asymmetric in 4 (20%) eu-/hypothyroid and in 27 (6.1%) hyperthyroid patients (p=0.038). Only 5.3% versus 30.2% and 10.5% versus 44.1% of patients with eu-/hypothyroid and hyperthyroid TAO, respectively, were on sick leave (p=0.003) or work disabled (p=0.018). QoL was less impaired in eu-/hypothyroid versus hyperthyroid TAO (median visual functioning and appearance scores: 100 versus 75; p<0.001 and 81.25 versus 75; p=0.315). Of patients with eu-/hypothyroid and hyperthyroid TAO, 15% and 20.2% had psychotherapy respectively (p=0.409). Eu-/hypothyroid TAO was positively (odds ratio 7.05, p=0.060) and negatively (odds ratio: 0.09, p=0.026) associated with a unilateral involvement and thyrotropin-receptor autoantibodies respectively. CONCLUSIONS Compared with hyperthyroid TAO, QoL and working ability are less impaired in eu-/hypothyroid TAO with an often asymmetric and less severe clinical phenotype.

Decreased proportions of CD4 + IL17+/CD4 + CD25 + CD127− and CD4 + IL17+/CD4 + CD25 + CD127 − FoxP3+ T cells in children with autoimmune thyroid diseases*

Abstract Until now, altered balance of Th1 and Th2 immune cells has been postulated to play an important role in the pathogenesis of autoimmune thyroid diseases (AITD). However, recent studies on thyroid diseases have suggested a new role for Th17 cells that have been classified as a new lineage, distinct from Th1, Th2 and Treg cells. Despite wide interest, the role of Th17 cells in the pathogenesis of inflammatory and autoimmune diseases is still debated. The aim of the study was to estimate the proportions of Th17/Treg T cells in peripheral blood from patients with Graves’ disease (GD; n = 29, mean age 15.4 ± 5.1 years), Hashimoto’s thyroiditis (HT; n = 39, mean age 15.2 ± 4.1 years) and in healthy controls (n = 49, mean age 14.8 ± 3 years). Polychromatic flow cytometry and several fluorochrome-conjugated monoclonal antibodies were applied to delineate Th17 and Treg cells. The analysis of Th17/Treg T cell proportions in peripheral blood from patients with Graves’ disease revealed significantly lower ratios of CD4 + IL17+/CD4 + CD25 + CD127 − (p < 0.0021) and CD4 + IL17+/CD4 + CD25 + CD127 − FoxP3 + (p < 0.0031) than in the control group. In addition, in the case of HT, we observed a significant decrease in the ratios of CD4 + IL17+/CD4 + CD25 + CD127 − (p < 0.0001) and CD4 + IL17+/CD4 + CD25 + CD127 − FoxP3 + (p < 0.0001) T cells in comparison to healthy children. In patients with untreated GD, a statistically significant positive correlation was found between the proportions of CD4 + IL17+/CD4 + CD25 + CD127−, CD4 + IL17+/CD4 + CD25 + CD127 − FoxP3+ T cells and the TRAbs (R = 0.71, p < 0.029; R = 0.72, p < 0.026, respectively) and a positive correlation was noted between the percentage of CD4 + CD − IL − 17 + T cells and the level of TSAbs (R = 0.66, p < 0.037). We conclude that the changes in the proportion of Th17/Treg T cells in peripheral blood and their significant relationship with the level of anti-thyroid antibodies indicate an involvement of these cells in the pathogenesis of AITD.

United States and European Multicenter Prospective Study for the Analytical Performance and Clinical Validation of a Novel Sensitive Fully Automated Immunoassay for Calcitonin

BACKGROUND The objective of this study is the validation and proof of clinical relevance of a novel electrochemiluminescence immunoassay (ECLIA) for the determination of serum calcitonin (CT) in patients with medullary thyroid carcinoma (MTC) and in different diseases of the thyroid and of calcium homeostasis. METHODS This was a multicenter prospective study on basal serum CT concentrations performed in 9 US and European referral institutions. In addition, stimulated CT concentrations were measured in 50 healthy volunteers after intravenous calcium administration (2.5 mg/kg bodyweight). RESULTS In total, 1929 patients and healthy controls were included. Limits of blank, detection, and quantification for the ECLIA were 0.3, 0.5, and 1 ng/L, respectively. Highest intra- and interassay coefficients of variation were 7.4% (CT concentration, 0.8 ng/L) and 7.0% (1.1 ng/L), respectively. Medians (interval) of serum CT concentrations in 783 healthy controls were 0.8 ng/L (<0.5-12.7) and 3 ng/L (<0.5-18) for females and males, respectively (97.5th percentile, 6.8 and 11.6 ng/L, respectively). Diagnostic sensitivity and specificity were 100%/97.1% and 96.2%/96.4%, for female/males, respectively. Patients (male/female) with primary hyperparathyroidism, renal failure, and neuroendocrine tumors showed CT concentrations >97.5th percentile in 33%/4.7%, 18.5%/10%, and 8.3%/12%, females/males, respectively. Peak serum CT concentrations were reached 2 min after calcium administration (161.7 and 111.8 ng/L in males and females, respectively; P < 0.001). CONCLUSIONS Excellent analytical performance, low interindividual variability, and low impact of confounders for increased CT concentrations in non-MTC patients indicate that the investigated assay has appropriate clinical utility. Calcium-stimulated CT results suggest good test applicability owing to low interindividual variability.

Performance and Specificity of 6 Immunoassays for TSH Receptor Antibodies: A Multicenter Study

Background: The measurement of TSH receptor (TSHR) antibodies is warranted for diagnosis of Graves’ disease (GD). Objective: The performance, detection sensitivity, and specificity of 6 TSHR immunoassays were compared. Methods: Two bioassays and 4 binding assays (Kronus, Immulite, Kryptor, Dynex) were compared in a dilution study performed in patients with autoimmune thyroid disease. Both bioassays were compared to 2 binding assays using stimulatory (M22) and blocking (K1–70) monoclonal antibody (MAb) mixtures. Results: Thirty samples from stimulatory (TSAb)-positive/blocking (TBAb)-negative patients with GD were diluted serially and measured in all assays. Samples were positive until dilution 1:2,187 in the TSAb bioassay, 1:81 in the Immulite (p < 0.002 vs. bioassay) and Kronus ELISA (p = 0.039) assays, and 1:27 in the Kryptor and Dynex ELISA (p < 0.001 vs. bioassay). Ten samples from TBAb-positive/TSAb-negative patients with GD or Hashimoto’s thyroiditis were positive in all binding assays. None of the binding assays differentiated between TSAb and TBAb. Mixtures of 100% K1–70 (200 ng/mL), 80% K1–70 + 20% M22, 60% K1–70 + 40% M22, 40% K1–70 + 60% M22, 20% K1–70 + 80% M22, and 100% M22 (20 ng/mL) tested positive in both Immulite (26.4, 20.2, 15.2, 10.5, 6.3, 2.00 IU/L) and Kronus assays (27.1, 23.3, 19.3, 12.0, 5.7, 2.2 IU/L). These MAb mixtures were tested in the TBAb bioassay and showed 82, 61, 24 (negative), –26 (negative), –77 (negative), and –95% (negative) inhibition, respectively. Conclusions: The sample dilution study showed higher detection sensitivity for the TSAb bioassay, and the antibody mixture study demonstrated exclusive specificity of the bioassays over all automated and ELISA binding assays.

STIMULATORY TSH-RECEPTOR ANTIBODIES AND OXIDATIVE STRESS IN GRAVES' DISEASE.

Context We hypothesized that TSH-receptor (TSHR) stimulating antibodies (TSAbs) are involved in oxidative stress mechanisms in patients with Graves disease (GD). Methods Nicotinamide adenine dinucleotide phosphate oxidase, isoform 2 (NOX2); oxidative parameters; and oxidative burst were measured in serum, urine, and whole blood from patients with GD and control subjects. Superoxide production was investigated in human embryonic kidney (HEK)-293 cells stably overexpressing the TSHR. Lipid peroxidation was determined by immunodot-blot analysis for protein-bound 4-hydroxy-2-nonenal (4-HNE) in human primary thyrocytes and HEK-293-TSHR cells. Results Serum NOX2 levels were markedly higher in hyperthyroid untreated vs euthyroid treated patients with GD, hyperthyroid patients with toxic nodular goiter, and euthyroid healthy control subjects (all P < 0.0001). Urine oxidative parameters were increased in patients with GD vs patients with toxic goiter (P < 0.01) and/or control subjects (P < 0.001). The maximum of the zymosan A- and phorbol 12,13-dibutyrate-induced respiratory burst of leukocytes was 1.5-fold higher in whole blood from hyperthyroid patients with GD compared with control subjects (P < 0.001 and P < 0.05). Monoclonal M22 TSAbs stimulated cAMP (HEK cells) in a dose-dependent manner. M22 (P = 0.0082), bovine TSH (P = 0.0028), and sera of hyperthyroid patients with GD (P < 0.05) increased superoxide-specific 2-hydroxyethidium levels in HEK-293 TSHR cells after 48-hour incubation vs control subjects. In contrast, triiodothyronine (T3) did not affect reactive oxygen species (ROS) production. In primary thyrocytes, the 4-HNE marker was higher in patients with GD vs control subjects at 6 and 48 hours (P = 0.02 and P = 0.04, respectively). Further, after 48-hour incubation of HEK-293 TSHR cells with patient sera, 4-HNE was higher in patients with untreated GD compared with control subjects (P < 0.05). Conclusions Monoclonal M22 and polyclonal serum TSAbs augment ROS generation and/or induce lipid peroxidation.