For over half a century (1), the thyrotropin (thyroid-stimulating hormone, TSH) receptor (TSHR) appeared as a central stage of the thyroid function and the main, if not the only, example of autoantigen involved in autoimmune thyroid diseases (AITDs) and recognized by pathogenic autoantibodies (TSHR antibodies, TRAbs). TRAbs were able of inducing modifications of thyroid function with a similar but prolonged mechanism (long-acting thyroid stimulator, LATS) compared to that of the natural ligand (2). These modifications are the main cause of the clinical manifestations of autoimmune hyperthyroidism or Graves’ disease (GD). In the last 60 years the detection/measurement of TRAbs was performed with biological and immunological laboratory methods, based on different assay principles.
When we consider the important role of laboratory tests, which are the cornerstone in the diagnostic pathway of any endocrine disease in the era of precision medicine, it is undeniable that until now the requests of TRAbs measurement in patients with hyperthyroidism was largely inappropriate by underutilization, due to controversies that surrounded the utility of these autoantibodies since its first reported use in clinical diagnosis (3). Recent advances and refinements of immunoassay (IMA) and bioassay methods have improved its specificity, reliability, and usability and now a wider use of the TRAb tests in clinical practice is a shared opinion.
The purpose of this review is to describe the ever-increasing diagnostic role of TRAbs in GD and other AITDs, to analyze the different methods for measuring these autoantibodies and to propose a rational approach for their use in clinical and laboratory practice.
The TSHR and the economy of thyroid gland
TSHR, member of the class A G-protein-coupled receptors with the close relatives follitropin and lutropin/choriogonadotropin receptors (LH/CGR), is essential for the function and growth of the thyroid gland and activates different signaling pathways, required for thyroid hormones synthesis and release. The TSHR structure is constituted by interplaying domains located in different sites of the thyrotroph [the extracellular leucin-rich repeat domain (LRRD) and the hinge region, the intramembrane serpentine domain (SD) and the cytoplasmic tail] (4,5). After expression on the thickness of the plasma membrane, the TSHR undergoes cleavage within the hinge region. The loss of a C-peptide leads to an extracellular A subunit (comprising the LRRD and a part of the hinge region), and a B subunit (comprising the remainder of the hinge region, the SD and the cytoplasmic tail): the shed A subunit is the autoantigen initiating and driving the autoimmune response in GD. TSHR presents intermolecular interaction partners (e.g., TSH, small molecules, G-proteins, arrestin, or autoantibodies), the last of which is of fundamental importance for the pathogenic mechanisms underlying the major TSHR-associated human pathology, the autoimmune hyperthyroidism (6-8). Autoantibodies directed against the TSHR are pathogenic for GD, other AITDs (the atrophic silent form of Hashimoto’s thyroiditis) and probably for GD-associated diseases (orbitopathy, dermopathy). Three types of TRAbs have been demonstrated, stimulating (S-TRAb or TSAb), blocking (B-TRAb or TBAb), and apoptotic antibodies (A-TRAb) (7-9) and their relative concentrations define the natural history and the clinical picture of GD (10).
Starting from the seminal experiments of Adams and Purves (2,12) (during development of a TSH assay at Otago University Medical School in Dunedin, New Zealand) 60 years ago, firstly demonstrating the presence of LATS in some thyrotoxic patients (12), the TSH receptor antibodies paved the way for the saga of the ‘receptor autoimmunity’ and for the laboratory methods for detecting and measuring autoantibodies (13).
The expanding clinical role of TRAbs in the management of hyperthyroidism
The increasing clinical relevance of TRAbs is defined by recent guidelines and surveys. Starting from the 2011 ATA guidelines (14), the clinical utility of TRAbs measurement was confined at a role of alternative way to diagnose GD…. “when a thyroid scan and uptake are unavailable or controindicated (e.g., during pregnancy and nursing)…., due to the statement:… ‘most TRAb assays are specific for GD, but thyroid stimulating immunoglobulins and first-generation thyrotropin binding inhibitor immunoglobulin assays are less sensitive….”’ than thyroid scan or RAIU. This restriction is not shared by other non-American expert endocrinologists (15,16), creating a temporarily unresolved dualism between USA and Europe.
On the basis of the continuous improvement of assay methods for TRAbs measurement, some authors underlined the high accuracy of the IMA and bioassay methods (17,18): in reference of these data, Barbesino et al. underscored the clinical utility of TRAbs measurements and a wider utilization in the management of Graves’ patients (19). Consequently, the percentage of clinicians requesting TRAb in the management of GD markedly increased in recent years worldwide: as demonstrated in a recent survey (20), with the use of a simple questionnaire, the proportion of European endocrinologists requesting TRAb measurement increased in 25 years from 38% to 85.6% (delta%: 47.6), compared to the previous survey (21); on the other hand in North America TRAbs are requested by the 54.3% of the US clinicians (22), compared with 9.1% of the previous survey (delta%: 45.2) (23) and in Asia the trend was similar (24), from 28% to 65% (delta%: 37.0) (Table 1).
As final point of this process, the recent ATA guidelines propose the use of TRAb testing as a first-line method for the determination of etiology of hyperthyroidism, “…if the diagnosis is not apparent based on the clinical presentation and initial biochemical evaluation…”. Other alternative methods are the radioactive iodine uptake (RAIU) and ultrasonography (US): the choice depends on available expertise and resources (25). It’s now possible to stay that the long run for the recognition of the TRAbs measurement as the main test for the management of hyperthyroidism has been completed.
But what are the reasons for this opinion that finally brings together endocrinologists all over the world? The first important reason is the use of TRAbs measurement not only for diagnosing GD, but also for monitoring disease activity, influencing treatment choices and improving the assessment of risk of relapse (25,26). Other reasons are surely cost, certainly lower than RAIU and US (27), the growing availability and local expertise, but in particular the improvement of the methods of detection/measurement.
The evolution of diagnostic technologies for the detection and measurement of TRAbs
In the last 60 years, a variety of laboratory methods have been proposed and employed to detect and measure TRAbs, based on two different principles: bioassays and IMAs. The first measure functional activity of TRAbs, stimulating or blocking (28) (Table 2), while IMAs measure the binding to the receptor (total TRAbs, T-TRAbs), irrespective of functional discrimination (29) (Table 3).
Until 1958, the available methods for detection of TRAbs were the bioassays, based on the original principles of Adams and McKenzie (30-32). The bioassays, at first troublesome, poorly standardized and relatively insensitive for routine diagnostic use in GD, have been later developed through three generations, based on progressive technological improvement (33). The major innovations in the bioassay detection of TRAbs were the transfection of Chinese hamster ovary (CHO) cells with luciferase reporter gene, the availability of the TSHR-LH/CGR chimeric receptor, based on the substitution of aminoacid sequence of the wild-type TSHR with a similar sequence of the rat LH/CGR (34-36), on the use of CHO cells transfected with the recombinant human TSHR, and on the cyclic nucleotide-gated calcium channel and aequorin (37). TRAb levels, measured by such methods, are highly correlated with GD activity, but their use in clinical practice needs yet optimization and harmonization (36): this is the reason of the restriction of the use to a small number of specialized laboratories all over the world.
Following early experiments demonstrating that Graves’ patient immunoglobulins inhibit the binding of radio-labeled TSH to human and guinea-pig thyroid membranes or solubilized receptors of human thyroid cells, Shewring et al. in the early 1980s firstly described a competitive radio-receptor IMA (38). The progressive improvement of the analytical schemes (receptors of different species and tissues, preparation of antigenic source, types of tracers, etc.) has brought, through three generations of methods (Table 3), to the improvement of analytical (from 1.5 to 0.8 IU/L) and clinical (from 96.4% to 97.2%) sensitivity of IMAs (Table 4). Nevertheless, these assays cannot discriminate between the different types of TRAbs, present in patients with different AITDs (39-43). Also two recent IMA methods for the measurement of TRAbs, based on a distinctive technology and assay format, have been made available in automated commercial platforms/instruments (44-46), and show a high diagnostic accuracy. These methods shows similar results in discriminating GD patients from other hyperthyroid and non-hyperthyroid patients, but still need harmonization, particularly for the use of different reference preparations (Table 5).
In all 3rd generation IMA, it’s now clear that animal (bovine, porcine) and human TSHR yielded similar functional and clinical sensitivities and specificities, as demonstrated in several experiences and studies in the last 15 years: such similarity is dependent to the identity of aminoacidic sequences in the animal and human TSHR regions for TRAb and TSH binding (47).
TRAb IMA automation passed through important breakthroughs represented by the use of new solid phases (microparticles), new tracers (fluorimetric or chemiluminescent), new assay schemes (non-competitive or two sites) and new reference preparations (NIBSC 08/204) (Table 5), that allow the reduction of assay time (minutes) and the improvement of analytical and clinical accuracy.
The role of bioassays and IMAs in the diagnostic/prognostic workup of ATDs
Defined the fundamental role of the measurement of TRAbs both in the diagnostic pathway and in the follow-up of the GD and other AITDs, the debate on which type of laboratory methods to use (IMA or bioassays) remains open and related to the different experiences of thyroidologists and researchers.
In this review a diagnostic model is proposed, which takes into account the current knowledge of commercial technologies for the measurement of TRAbs, in their most recent refinements. It is clear that both technologies have significantly improved their analytical characteristics (29,36,44,46,48): the wide spread of the measurement of these autoantibodies within the thyroid test profile and their contained cost suggest the opportunity to use the IMA methods as the first choice in the current diagnostic approaches.
With the high automated IMA technologies (now sensitive, precise, and rapid), it is possible to complete in a single run the laboratory diagnostic procedure of the thyroid diseases, also in the presence of reflex and reflective thyroid tests. Notwithstanding the belief that, despite the latest technological innovations (44), IMAs mainly detect T-TRAbs and not only the S-TRAbs and consequently their measurement is able to meet the clinical needs in the overt case of hyperthyroid symptoms (given that in most cases autoantibodies exhibit characteristics of stimulating antibodies) not only for diagnostic purposes, but also during the follow up of patients (in predicting the outcome of GD after anti-thyroid drug treatment).
Bioassays should be reserved for fine and complex diagnoses, when clinical conditions suggest the identification of the functional activities of the TRAbs, for the possible switch between B-TRAb and S-TRAb and vice versa in the same patients (49-52):
- In predicting the type of relapse of GD;
- In predicting the likelihood of fetal/neonatal hyper- or hypothyroidism;
- In evaluating the concentration of B-TRAb and S-TRAb in Hashimoto’s thyroiditis and Graves’ orbitopathy;
- In evaluating the risk of extrathyroidal manifestations of GD during pregnancy.
As a consequence of the recent quality improvement of laboratory methods, IMAs may be adopted in clinical practice for the initial diagnosis of hyperthyroidism and to follow disease activity and treatment, and bioassays may be used to assess particular type of patients with Graves’ hyperthyroidism.
Conflicts of Interest: The author has no conflicts of interest to declare.
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Cite this article as: Tozzoli R. The increasing clinical relevance of thyroid-stimulating hormone receptor autoantibodies and the concurrent evolution of assay methods in autoimmune hyperthyroidism. J Lab Precis Med 2018;3:27.