ReviewTSH receptor autoantibody immunoassay in patients with Graves' disease: Improvement of diagnostic accuracy over different generations of methods. Systematic review and meta-analysis
Introduction
The thyrotropin receptor (TSHR) is a major autoantigen in autoimmune hyperthyroidism and specific autoantibodies acting as TSHR agonists (TRAb) are pathogenic (i.e. responsible for clinical manifestations) and are the diagnostic hallmark of Graves' disease (GD) [1]. Measurement of TRAb plays a crucial role in the differential diagnosis of hyperthyroidism, which has important therapeutic and prognostic implications [2].
In recent years there has been significant progress in elucidating the TSH receptor structure and the functional activities of TRAb [3] and in developing advanced techniques for their measurement [4]. After the Adams' historical discovery of TRAb (at that time called first long-acting thyroid stimulator — LATS) as a cause of hyperthyroidism [5] and identification of LATS as an immunoglobulin [6], until the early 1970s the only available methods for detection of TRAb were in vivo cell-based bioassays [7].
Following early seminal experiments demonstrating that Graves' immunoglobulins can inhibit the binding of radio-labeled TSH to human [8], guinea-pig thyroid membranes [9], or solubilized receptors of human thyroid [10], and that porcine thyroid may provide equivalent responses to human thyroid [11], Rees Smith and Hall in the early 1980s for the first time described a competitive receptor immunoassay [12]. Further modifications of the analytical procedure (use of receptors of different species and tissues, different types of preparation of antigenic source, washing procedures, types of tracer, etc.) and the commercial availability of reagents, have made this assay the method of choice for TRAb measurement in most clinical laboratories [13]. These methods, based on the principle of the inhibition of 125I-TSH binding (radio-receptor assay) or enzyme-labeled-TSH binding (enzyme-receptor assay) and diffused in the clinical laboratories for 20 years, were defined as ‘liquid phase’ 1st generation (1G) immunoassays. Despite their high specificity (99.2%, range: 97.5–100%), these assays did not show a similar diagnostic sensitivity (79.8%, range: 52–94%) [14], [15], [16], [17], [18], [19], [20], [21], [22], [23]. As a consequence, a significant proportion (6–48%; mean, 20.2%) of clinically hyperthyroid GD patients were defined TRAb negative by 1G methods. The differences in the results obtained may depend on the different types of patients studied, the analytical methods used, the source of TSHR (recombinant human or purified porcine) and the assay procedure (times of incubation, positivity thresholds, reference values).
In order to increase the sensitivity of TRAb assay, in the late 1990s, 2nd generation (2G) immunoassays using monoclonal antibodies (moAb), recombinant human [18] or native purified porcine [24] TSHR immobilized on plastic surface and bovine TSH labeled with 125I [18], acridinium ester [18] or with biotin–streptavidin–peroxidase [19] have been made available. Several studies have shown that the clinical sensitivity of these assays increased, with only a little decrease in specificity. Subsequently, the 2nd generation (2G) ‘solid-phase’ commercial immunoassays were divided in two types, the porcine (p2G) and the human (h2G) TRAb assays. In Europe for a long time the recombinant human TSHR-based 2G assays have been considered the gold standard with the highest diagnostic accuracy.
In 2003 a new moAb (M22) with stimulating activity was described by Sanders [25] and subsequently a new method for measuring TRAb was proposed [26], in which the moAb M22 (labeled with biotin to TSHR-coated ELISA plate wells) substituted the bovine or porcine TSH used in previous ‘liquid-phase’ and ‘solid-phase’ TRAb assays. The method was called manual 3rd generation assay (m3G). Five years ago, the first fully automated electrochemiluminescence immunoassay [27] and, more recently, a second fully automated M22-based fluoro-enzymatic immunoassay [28] became commercially available and these were defined as automated 3rd generation assay (a3G).
In the course of the development of these three generation TRAb assays, the analytical and functional sensitivities continuously increased [28], [29], despite the use of different reference preparations and calibrators (MRC B65/122 for 1G and NISBC 90/672 for 2G and 3G, respectively). Their analytical sensitivity improved from about 3 IU/L in the ‘liquid phase’ assay, to about 1.5 IU/L in the solid-phase TSH-based assay, and to about 0.8 IU/L in the manual or automated solid-phase M22-based assay [29].
Consequently, a higher diagnostic accuracy for GD was expected, and demonstrated in single experimental studies. To our knowledge, a systematic review of the diagnostic accuracy of recent TRAb assay is lacking in the literature. In the present review we report a meta-analysis of the most relevant published reports, and discuss the role of TRAb measurement in the diagnosis of GD in light of the results observed.
Section snippets
Methods
We performed a systematic review of English articles using MEDLINE database and the search terms: TSH receptor antibody assay, TSH receptor antibody tests, and human Graves' disease, from 1990 to January 2012. The aim of this work was to evaluate the diagnostic accuracy of available commercial methods for TRAb quantitation, based on 2G and 3G immunoassay principle, in adult untreated Graves' disease. Early reports involving 1st generation immunoassays were not included in the meta-analysis, but
Results
The 21 included studies involved a total number of 3081 patients and 3795 controls: 1451 patients and 1819 controls for the 2G group (Table 1) and 1630 patients and 1976 controls for the 3G group (Table 2).
Discussion
Within the last two decades great efforts have been made to improve the 1st generation methods for TRAb measurement, based on competitive binding of patients TRAb and labeled bovine TSH to a soluble porcine TSHR [45]; after the cloning of human TSHR, 2nd generation TRAb assays with high diagnostic accuracy become available. More recently, a completely new method for manual TRAb measurements (3rd generation) has been described, based on the binding of a labeled human monoclonal antibody, termed
Take-home messages
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TSH receptor is a major autoantigen in autoimmune hyperthyroidism.
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TSH receptor autoantibodies are the main diagnostic hallmark of Graves' disease.
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2nd- and 3rd-generation immunoassays for TRAb measurement have sensitivity and specificity closed to 100% in the diagnosis of Graves' disease.
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The rapid and inexpensive TRAb immunoassay in automated analytical platforms should be widely used to screen hyperthyroid patients at an early stage of the disease.
References (53)
- et al.
The role of TSH receptor antibodies in the management of Graves' disease
Eur J Intern Med
(2011) - et al.
Clinical review about TRAb assay's history
Autoimmun Rev
(2010) - et al.
Bioassays for TSH-receptor autoantibodies: an update
Autoimmun Rev
(2010) Thyrotropin-receptor antibodies in thyroid diseases: advances in detection techniques and clinical applications
Clin Chim Acta
(2000)- et al.
Human monoclonal thyroid stimulating autoantibody
Lancet
(2003) - et al.
TSH receptor antibodies
Thyroid
(2007) - et al.
Insight into thyroid-stimulating autoantibody interaction with the thyrotropin receptor N-terminus based on mutagenesis and re-evaluation of ambiguity in the region of the receptor crystal structure
Thyroid
(2011) - et al.
Abnormal responses in the assay of thyrotropin
Proc Univ Otago Med Sch
(1956) - et al.
Characterization of the long-acting thyroid stimulator of Graves' disease
Proc Natl Acad Sci U S A
(1964) - et al.
Thyroid-stimulating immunoglobulins in Graves' disease
Lancet
(1974)