Alfano Martina e Aragno Elena
Introduction
The TSH receptor is a member of the superfamily of receptors with seven transmembrane domains linked to G proteins. Activation of G proteins by the hormone receptor complex results in stimulation of cAMP production by adenylate cyclase and inositol phosphate turnover by phospholipases. Site-directed mutagenesis has shown that the 3-dimensional receptor structure is important for the interaction with TSH and/or TRAbs (Fig1).
There are two broad types of TRAb measured by either bioassay or receptor assay. Receptor, or TSH Binding Inhibitory Immunoglobulin (TBII) assays do not measure biologic activity directly but assess whether the specimen contains immunoglobulins that can block the binding of TSH to an in vitro receptor preparation. TSH stimulating antibodies (TSAb) appear to bind the N-terminal portion of the extracellular domain and mimic the actions of TSH by inducing post-receptor signal transduction and cell stimulation. In contrast, the C-terminal region is more important for TSH receptor blocking antibodies (abbreviated TBAb or TSBAb) which block stimulation by either TSAb or TSH, causing hypothyroidism.
It is well-established that the hyperthyroidism of Graves’ disease (or Basedow’s disease) is caused by generation of TSH-receptor stimulating autoantibodies (fig. 2), but many patients also harbor TSH receptor antibodies (TRAb) that do not stimulate the receptor or even block the receptor.
Graves’ disease is a common autoimmune disorder with a series of clinical manifestations. Classical signs and symptoms which have defined the disease are hyperthyroidism, diffuse hyperactive goitre, and ophthalmopathy. A less common manifestation is localized dermopathy (pretibial myxoedema), and rarely thyroid acropachy with peripheral swellings may occur. The combination of manifestations varies among patients, and occasionally it may be difficult to classify patients as having Graves’ disease or not.
It has now been shown that the lack of correlation between TRAb levels and the clinical status of patients is largely because of circulating TRAb’s that are heterogeneous. The fact that TRAb heterogeneity can coexist within an individual patient and change over time is one reason why it has been difficult to develop
diagnostically accurate TRAb tests. Indeed, the clinical presentation of Graves’ patients who exhibit both TSAb and TBAb/TSBAb will likely depend on the relative concentration and affinity of the predominant antibody. A shift from stimulating to blocking TRAb may explain the spontaneous remission of Graves’ disease during pregnancy as well as radioiodide induction of transient hypothyroidism. It is important to note that bioassays that use cell preparations to measure the biologic effects of TRAb (stimulation, inhibition of TSH activity or growth) can detect functional changes in TRAb heterogeneity. In contrast, the receptor, or TSH Binding Inhibitory Immunoglobulin (TBII) type of assays, which are used by many clinical laboratories, merely measure the ability of a serum or IgG preparation to block the binding of a TSH preparation and do not measure the biological response.
TSH Receptor Antibody (TRAb) Tests
There are two currently available methods for measuring TRAb:
1. Biological assays measure the ability of TRAb to stimulate or inhibit TSHR activity. They measure the production of cAMP when sera containing TRAb are exposed to TSHR on cell preparations such as FRTL-5 or CHO. Therefore, they are able to differentiate between TSAb and TBAb. These tests are not routinely available in all countries, and are still most often used in a research setting.
2. Receptor or TSH binding inhibition tests (TBII) do not measure stimulatory activity directly but detect factors in the serum specimen that block the binding of a labeled TSH preparation to an in-vitro TSH receptor preparation. These tests are the more commonly used TRAb assays in clinical laboratories, and are freely available commercially for clinical use.
• The tests of 1 ° Generation are RIA assays competitive in the liquid phase.
In a reaction well is put together in the liquid phase a porcine TSH receptor to compete with the patient's serum containing TRAb and TSH of porcine origin labeled with iodine 125. The separation between free / bound is achieved by precipitation with PEG (a macromolecule capable of binding the antigen / antibody complex that are formed and precipitating).
• The tests of 2 ° Generation are dosages in the solid phase, in RIA or ELISA (tubes or microplates).The principle of the assay is competition between TRAb in a serum sample and labeled bovine or porcine TSH for binding to human recombinant TSH receptors immobilized on coated tubes.
• The tests of 3 ° Generation are dosages in the solid / liquid phase, in ELISA.
The principle of the assay is competition between TRAb in a serum sample and a human labeled thyroid-stimulating monoclonal antibody (M22) for binding to porcine TSH receptor.
All methods are competitive and therefore the value of TRAb in serum is inversely proportional to the signal of the labeled. ( FIG.3)
In recent years the evolution of the methods has seen an improvement in terms of sensitivity and specificity, tanks to the scheme of the assay in solid phase and the use of material of human origin with greater affinity for the receptor.
While first-generation TBII assays using porcine cells and bovine labelled TSH had a sensitivity of only 50–80%, second-generation assays using recombinant human
TSHR are said to be 90–99% sensitive and 95–100% specific. Third-generation assays using human monoclonal TSHR stimulating antibodies are said to be even better with improved sensitivities (97%) compared to second generation assays (94%) .
Clinical performance of TRAb measurements
In order to evaluate the clinical efficacy of 2° and 3° generation TRAb assays, first we report the data of a work that show results of TRAb measurements in patients with various thyroid abnormalities diagnosed without the use of TRAb measurements, and discuss whether such measurements give useful information.
TRAb was measured by a 2° generation method. The Fig. 4 shows the results of TRAb measurements in patients with various newly diagnosed thyroid disorders and in healthy controls. The horizontal dotted line indicates the distinction between the values of TRAb positive and negative (cut-off 1.0 IU / L). It can be seen as patients with Grave’s disease have almost all of a value above the cut-off while the healthy controls, as well as patients with non-toxic goitre, are all negative TRAb (except one). In patients with autoimmune hypothyroidism, characterized by the presence of thyroglobulin autoantibody (TgAb) and thyroperoxidase autoantibody (TPOAb), there is a low percentage of TRAb (probably type blockers). In summary this study shows an excellent specificity of the method with the ability to discriminate between patients with GD from healthy ones.
In another study were evaluated basic and clinical performance of a new 3° generation, rapid, and fully automated electrochemiluminescence immunoassay Elecsys Anti-TSHR (Elecsys TRAb). Clinical performance of the assay was evaluated with Graves' disease patients, autoimmune thyroiditis and normal subjects; the results were compared with a radio-receptor assay 1° generation.
The figures 5 and 6 show TRAb values in serum from unselected cohorts of patients with newly diagnosed Graves’ disease, Graves' disease patients treated with antithyroid drugs (metimazole) in remission, patients with autoimmune thyroiditis, and healthy controls. The dotted horizontal line indicates the distinction between TRAb positive and negative test results with the two methods. The Fig.5 shows how patients with GD all have a value of TRAb positive, while the healthy controls have all TRAb absent (<0.3 IU / L). In Fig. 6 with the 1° generation method, some patients with GD have a negative value TRAb, and healthy controls, although negative, have low levels of TRAb. The results of Elecsys TRAb measurements in these cohorts of patients show a higher sensitivity and specificity to discriminate these patients than Riazen TRAb.
Clinical Uses of TRAb Measurements
The present study focuses on the use of TRAb measurements for differential diagnosis by the new methods. TRAb should be measured with a high quality assay in all patients with newly diagnosed hyperthyroidism. This is to diagnose Graves’ disease and also to obtain a pretreatment status for future evaluation of remission in Graves’ disease.
TRAb measurement in Graves’ disease during therapy with antithyroid drugs has been discussed in a recent study. Disappearance of TRAb during therapy assists importantly in diagnosing remission. Furthermore, persistent high TRAb is useful for identification of patients who do not enter remission. Such patients may develop a syndrome of persistent thyroid drive, and they often respond unsatisfactorily to antithyroid drug therapy.
TRAb measurement may be useful for diagnosing Graves’ ophthalmopathy in euthyroid patients.
Transplacental passage of TRAb causes fetal or neonatal thyrotoxicosis in 1–5% of pregnancies in women with current or past GD. There is general agreement that TRAb measurements can be used to predict fetal and/or neonatal thyroid dysfunction in pregnant women with a previous history of autoimmune thyroid disease (AITD). High levels of TRAb in the mother during the third trimester of pregnancy suggest a risk of thyroid dysfunction in the offspring. It has a clear role in evaluation of risk for fetal or neonatal hyperthyroidism.
It is not find indication for routine measurement of TRAb in hypothyroid patients. Rarely transplacental passage of blocking TRAb from a levothyroxine substituted mother with autoimmune hypothyroidism may lead to transient neonatal hypothyroidism. The hypothyroidism would be detected by neonatal screening for hypothyroidism and treated. Subsequent measurement of TRAb in the mother and the newborn will reveal the nature of the disorder, and levothyroxine should be gradually withdrawn in the infant after some months. This is a very rare event, calculated to occur in one out of 180,000 births.
Conclusions
TRAb measurements using modern 2nd-3rd generation receptor assays are increasingly more freely available, quickly done and cheap (certainly in high volume laboratories).They offer a greater advantage in terms of higher sensitivity and specificity, logistical considerations and cost savings.
The new 3° generation assay has a high sensitivity for detecting Graves' disease and a specificity for discriminating from other thyroid diseases and healthy subjects, which is in accordance to the some of different non automated TRAb 2° generation assay based on the human recombinant TSH receptor.
Furthermore, newer automated 3rd generation assays provide excellent sensitivity and specificity with high PPV and NPV in subjects with biochemical hyperthyroidism and may represent the future technology for rapid fully automated TRAb detection.
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