Regression analysis was used to evaluate the relationships between CAS, NOSPECS score, MC4-TSI level and M22-TBII levels, to reveal which of the laboratory parameters might correlate with disease activity or severity scores. activity/severity clinical activity scores (CAS), and modified NOSPECS scores were examined for a correlation with TRAb assays. Results Fifty patients (mean age, 41.3 years; 41 females) were analyzed. The mean duration of Graves’ hyperthyroidism symptom was 63 months (range, 18 to 401 months) and that of GO was 46 months (range, 18 to 240 months). All patients had been treated previously with anti-thyroid drugs for a median period of 52.3 months, and two patients underwent either radioiodine therapy or total thyroidectomy. Mean CAS and NOSPECS scores were 0.5 0.9 (standard deviation) and 4.8 3.1, respectively. Mean M22-TBII and Mc4-TSI values were 7.5 10.2 IL/L and 325.9 210.1 specimen-to-reference control ratio. TSI was significantly correlated with NOSPECS score (R = 0.479, < 0.001); however, TBII was Droxinostat not associated with NOSPECS score (= 0.097). Neither TSI nor TBII correlated with CAS (> 0.05), because GO inflammatory activity subsided in the chronic stages of GO. Conclusions In chronic-inactive GO after euthyroid restoration, GO activity score did not associate with serum levels of TRAb or TBII. However, levels of the functional antibody Mc4-TSI did correlate with GO severity. Therefore, the TSI bioassay is a clinically relevant measure of disease severity even in chronic inactive GO. Keywords: Biological assay, Chronic inactive Graves’ orbitopathy, Mc4-TSI, Thyroid-stimulating immunoglobulin, Thyrotropin-binding inhibitory immunoglobulin Graves’ orbitopathy (GO) is a component of autoimmune Graves’ hyperthyroidism, in which thyroid stimulating hormone (TSH) receptor antibody (TRAb) stimulates orbital and periorbital tissues [1]. The natural history of GO is not well understood. It is generally thought that GO has an inflammatory, active phase that subsides after 1 to 2 2 years. The average active inflammatory phase duration EPLG6 is approximately 18 months (range, 3 to 36 months) [2]. After Droxinostat the inflammation subsides, patients may suffer permanent structural changes around the eyes that require surgical repair. Currently, there are two established assays to measure TRAb: the competitive TSH-binding inhibition immunoglobulin (TBII) assay and the functional thyroid-stimulating immunoglobulin (TSI) bioassay [3,4]. The TBII assay utilizes the ability of TRAb to inhibit the binding of radiolabeled TSH to TSH-receptors. The newly-developed, third-generation TBII assay measures the inhibition in the binding of a labelled monoclonal antibody clone M22 to the TSH-receptor rather than the traditional radiolabeled TSH-TSH receptor binding [4,5]. This assay enhanced the sensitivity and specificity of earlier assays using radiolabeled TSH [6,7,8]. The TSI bioassay measures cyclic adenosine monophosphate production after TRAb binds to the TSH-receptor, thus enabling identification of functional TRAb [9,10]. The development of the Mc4-CHO cell line simplified the cell culture protocols for the TRAb bioassays. The Mc4-CHO TSI bioassay has superior diagnostic potential for differentiating Graves’ disease (GD) from painless thyroiditis [11]. Recently, several reports have focused on the relevance of TRAb, especially TSI, in untreated early stage GO [12]. Ponto et al. [13] reported that TSI levels correlated with disease activity (R = 0.89) and severity (R = 0.81) in untreated GO. Lytton et al. [12] showed a similar correlation between TSI and GO activity/severity. In previous studies we investigated whether serum TRAb in newly-diagnosed, untreated GO patients were predictive of the disease course beyond the first year after the initial diagnosis [14]. The results showed that patients with higher initial TRAb levels had a greater risk of severe disease outcomes. Likewise, we hypothesized that serum TRAb levels could provide Droxinostat important prognostic information to clinicians regarding early stage GO patients [14]. To the best of our knowledge, no prior reports of TRAb levels in chronic stage GO patients have been published. Chronic stage GO frequently imposes severe psychological, social, and economic burdens on patients because patients often experience substantial facial disfigurement including proptosis, puffy eyelids, and strabismus [1,15,16]. Although GO usually spontaneously resolves, patients often show heterogeneous clinical courses with complications such as restrictive strabismus and severe proptosis. We observed that patients who were current.