TSH Receptor Antibodies (TRAb)
Thyroid Peroxidase Antibodies (TPOAb)
Genetic testing for FMTC and MEN 2
The effectiveness of laboratory thyroid tests is of major relevance, as is their appropriate use in a given clinical situation. In the daily practice many generalists and various specialists depend on reliable diagnosis of a thyroid disorder or its definitive exclusion. Fortunately, thyroid tests have considerably improved making this option generally possible.
Standards of laboratory practice have been set forward by the relevant societies including the American Thyroid Association, the European Thyroid Association, and also the German Endocrine Society and guidelines for thyroid testing have been issued in order to assure analytical and clinical performance goals for patients as well as to achieve a cost effective use of these techniques. Despite their undisputed usefulness such guidelines may hardly substitute for personal experience and expertise of the thyroid specialist which may dictate deviation from rules in certain situations.
In the present report we will give an update on thyroid tests that reflects the vast experience in dealing with thyroid problems at our university hospital and provide critical comments on practical aspects of the usage of various diagnostic tests.
In this respect it is important to keep in mind that Germany still is a country with moderate iodine deficiency.
First, the patient who on clinical grounds is highly likely to have thyroid disease, second, the entirely asymptomatic patient with a low a priori probability of thyroid disease and third, the patient in whom thyroid testing is severely hampered by interfering factors, such as intake of various drugs, and/or the presence of nonthyroidal illness (NTI).
With a thyroid disease being clinically unlikely, screening of serum basal thyrotropin (TSH) levels may be the single most effective means of excluding thyroid dysfunction. Indeed, this is the approach we favor.
In case of substantial clinical suspicion use of a combination of thyroid in vitro parameters seems to be more appropriate, i.e.
Experience in our own laboratory with more than 12 000 samples per year has shown that functional assay sensitivity is the most important performance criterion and should be assessed independently in each laboratory. In addition, control samples with TSH concentrations of about 0.1 mU/l, 0.3 mU/l and 4.0 mU/l should be included in every run in order to early recognize imprecision of TSH determinations. It is the manufacturer|s and the laboratory|s responsibility, to correctly define analytical and clinical performance of a TSH assay.
| · | Functional sensitivity <0.1 mU/l |
| · | Cross reactivity with other glycoprotein hormones (LH, FSH, HCG) <0.01 % |
| · | Parallel course of sample dilutions and the standard curve. |
| · | Full recovery of a TSH standard added to patient's sample. |
| · | Estimates of WHO- or Medical Research Council (MRC) standards ±5% of the expected values. |
| · | Clear-cut separation of euthyroid and hyperthyroid patients (with an overlap of less than 1%). |
| · | Lack of interference with heterophile antibodies. |
| · | Absense of a high dose "hook" effect at TSH concentrations of <300 mU/l |
When these quality measures are operative retesting of an inappropriate TSH value has been a rare necessity in our experience. Nonspecific interference will best be detected in a situation where examination of a patient is done by a thyroid expert who should notice apparent discrepancies between the presentation of the patient and results of in vitro tests. In case of discrepant laboratory results we recommend to retest the sample and to check for parallelity with standard curve in diluted samples. If necessary, a new specimen from the patient should be collected and TRH stimulation should be performed. When using this approach, retesting the sample in a different type of TSH assay and the need for checking for the presence of heterophile antibodies is hardly ever necessary. In our setting it is important to note that low normal or suppressed TSH values may be frequently found in patients with secondary hypothyroidism due to pituitary abnormalities. In such situations clinical information is particularly helpful to distinguish primary hyperthyroidism with suppressed TSH from secondary hypothyroidism with inappropriately low TSH values.
Suspicion of secondary hypothyroidism is an important condition where screening for thyroid dysfunction by basal TSH alone may be inappropriate or misleading. In a considerable portion of patients TSH values may be found within the normal range despite manifest hypothyroidism as evidenced by decreased FT4 values. The condition can only reliably be diagnosed by interpreting TSH in relation to free thyroid hormone concentrations (FT4). Since determination of TSH alone is unable to distinguish between subclinical and overt thyroid dysfunction, each abnormal TSH value has to be followed or accompanied by assessment of peripheral hormone status.
TRH testing has become unneccessary in most thyroid disorders except for hypothalamic or pituitary diseases.
| · | Severe nonthyroidal illness (NTI) |
| · | Binding protein abnormalities and variations |
| · | Presence of antibodies to thyroid hormones |
| · | Drugs (e.g. furosemid, salicylates, heparin, barbiturates) |
As a rule, thyroid hormone measurements should always be interpreted in connection with serum TSH. Interpretation is straight forward in most cases except for severely ill patients with NTI syndrome and rarely for patients with TSH producing pituitary adenoma or thyroid hormone resistance (Table 3).
| · | subclinical hyperthyroidism |
| · | secondary hyperthyroidism |
| · | severe nonthyroidal illness (NIT) |
| · | drug interference (dopamine, high doses of glucocorticoids) |
| ELEVATED TSH IN THE ABSENCE OF OVERT HYPOTHYROIDISM | |
| · | subclinical hypothyroidism |
| · | thyroid hormone resistance |
| · | TSH producing pituitary adenoma |
| · | Recovery from nonthyroidal illness (NTI) |
Thyroid hormone resistance is characterized by a clinically euthyroid or hyperthyroid state, abnormally elevated free thyroid hormone concentrations, and normal or even increased TSH values. Hyperthyroidism in the presence of an increased or normal TSH is also found in secondary hyperthyroidism due to a TSH producing pituitary adenoma. Differential diagnosis can be done by TRH stimulation which can significantly increase TSH values only in thyroid hormone resistance and by determination of free alpha-subunit which is characteristically elevated in TSH-producing adenomas.
Table 4 shows the results of in vitro thyroid testing in a patient with TSH producing pituitary adenoma and with thyroid hormone resistance syndrome. Thyroid Autoantibodies
| Diagnosis | total T4
(nmol/l) |
FT4
(pmol/l) |
total T3
(nmol/l) |
TSH
(mU/l) |
TSH after TRH
(mU/l) |
|
|
|
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| Patient U. R. | TSH producing
pituitary adenoma |
191 | 42 | 4.39 | 6.0 | 8.0 |
| Patient C. K. | thyroid hormone
resistance syndrome |
214 | 44 | 3.50 | 4.8 | 44 |
Figure 2 depicts a scheme of the proposed diagnostic strategy in hyperthyroidism and the role of autoantibody determination. The incidence of detectable TRAb in the sera of patients with untreated active Graves' hyperthyroidism tends to considerably decline during the course of antithyroid drug therapy with after one year of treatment about half of the patients converting to negative antibody status (Table 5). Despite of their pathogenetic relevance and demonstration of treatment associated changes, use of TRAb determination has been disappointing with respect to prediction of remission or relapse of hyperthyroidism after withdrawal of antithyroid medication. A great number of retrospective and prospective studies have addressed this problem. From a clinical point of view we can briefly summarize that only persistence of high antibody titers is a rather specific indicator of persistence or imminent relapse of Graves' hyperthyroidism.
| View figure 1 | View figure 2 |
| Graves' Disease | ||
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N = 111 | 89% |
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N = 73 | 56% |
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| Hashimoto thyroiditis and primary myxedema | N = 37 | 8% |
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| Autonomously functioning thyroid nodules | N = 275 | 1% |
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| Nontoxic goiter | N = 108 | 8% |
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| Healthy controls | N = 40 | 0% |
It is important to note that TSH receptor antibodies do pass the placenta barrier and the presence of markedly elevated antibody titres in the blood of the mother indicates a risk of the newborn to develop intrauterine and/or neonatal hyperthyroidism.
Although TRAb are very useful in the clinical setting, their measurement has some limitations that should not be overlooked. These relate to the use of non-human thyroid tissue and to assessment of receptor binding instead of functional activity at the receptor. The latter may be particularly relevant for prognostic implications of antibody determination, because we could clearly demonstrate dissociation of TSH binding inhibiting antibodies and T3- release stimulating antibodies in some patients with Graves' disease during the course of antithyroid therapy. Similarly, with the use of more advanced techniques based on expression of recombinant human TSH receptor in stably transfected cell lines we observed a discordant (positive/negative) behaviour of binding inhibiting and stimulatory activities at recombinant TSH receptor in about one third of patients that have been treated up to 6-12 months with antithyroid drugs. This discrimination is not possible with the TRAb assay, in which both types of antibodies give a positive result. In patients with untreated Graves' disease, stimulatory and receptor binding activities were correlated (Figure 5).
| View figure 5 |
TPO antibodies are found to be present in about 90% of patients with autoimmune thyreoiditis (Hashimoto thyroiditis and primary myxedema) and about 75% of patients with active Graves' disease. Interestingly, TPOAb can be detected also in patients with non-immune thyroid diseases or even in older persons without evidence of thyroid disorders. This limited specificity of TPOAb determination may be overcome by higher cut-off values, which result, however, in a reduced sensitivity in the diagnosis of clinical autoimmune thyroid disease (Table 6). TPOAb may also be detected in association with other autoimmune disorders like insulin dependent diabetes mellitus (IDDM), Addisons' disease or systemic lupus erythematodes.
| 100 mU/l | >200 mU/l | >500 mU/l | ||
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| Grave's Disease | ||||
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n = 51 | 76% | 67% | 53% |
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n = 75 | 68% | 60% | 47% |
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| Hashimoto thyroiditis and primary myxedama | n = 35 | 89% | 77% | 63% |
| Autonomously functioning nodules | n = 51 | 16% | 10% | 6% |
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| Nontoxic goiter | n = 41 | 2% | 0% | 0% |
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| Healthy controls | ||||
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n = 36 | 0% | 0% | 0% |
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n = 33 | 12% | 6% | 3% |
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Modern immunoassay techniques have nowadays mostly replaced RIA methods, but still encounter a variety of methodological problems: Tg levels may vary among different methods used by as much as 65%. This may be improved in the future by adoption of a universal standard preparation. Major differences that are presumably related to different specificities of the anti-Tg antibodies used in various methods may still remain.
Owing to lack of appropriate standardization, values that have been reported for sensitivity may not be directly comparable. As with the TSH assay the best way would be to use functional sensitivity defined as the lowest Tg- concentration that is detectable with an interassay-CV below 20%. The functional sensitivity should be below 1 ng/ml.
Good assay precision is also important for measuring high serum Tg concentrations. When very high serum concentrations of Tg (>1000 ng/ml) are being measured, a high dose "hook" effect may occur. Testing the serum in a 1:10 dilution may identify this problem. Tg antibodies which are present in about 2030% of patients with thyroid carcinoma, still constitute a major problem of interference in Tg assays. Therefore Tg measurement routinely must include validation by recovery of an external addition of Tg. Although recovery studies are useful to identify problems with interference, demonstration of intact recovery does not necessarily guarantee correctness of the value. Determination of TgAb may be of additional value in this setting. Interpretation of Tg-values should be made in appreciation of the clinical presentation of the patient.
Calcitonin is available as a sensitive and specific tumor marker in the diagnosis of medullary thyroid carcinoma. Sensitivity of assay procedures has recently been improved. As for Tg, the results from different methods are not directly comparable so as to require separate standardization by each laboratory. Elevation of calcitonin almost always indicates the presence of medullary thyroid carcinoma, except for slightly elevated levels that are found in other neuroendocrine tumors, such as carcinoid, lung cancer or in pheochromocytoma and in patients with renal failure. The sensitivity of calcitonin measurement is improved by stimulation with pentagastrin. Figure 6 shows an example of a patient with MEN 2A before and after thyroidectomy for medullary thyroid carcinoma.As for thyroglobulin assays, a high dose hook effect may occur at very high concentrations of calcitonin.
| View figure 6 |
It should be noted that genetic testing is cost effective as compared to biochemical evaluation by calcitonin testing, particularly as subjects with no mutation being detectable can be excluded from long-term biochemical surveillance programmes. Generally, validation of the result by a second independent sample is required. Patients should best be treated at a specialized center. Table 7 briefly summarizes the experience gained at our institution with this novel test in MEN 2 patients.
| Mutation
(Affected codon and Change in Nucleotide Sequence) |
No. of
Affected Families |
No. of
Affected Family Members |
MTC | Pheo | pHPT | |
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| MEN 2A | 634 TGS -> CGC | 4 | 12 | 10 | 9 | 2 |
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| MEN 2A | 634 TGC -> TAC | 3 | 16 | 14 | 4 | 1 |
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| MEN 2A | 611 TGC -> TAC | 1 | 2 | 2 | 1 | 0 |
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| MEN 2A | 620 TGC -> CGC | 1 | 1 | 1 | 1 | 0 |
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| MEN 2A | 790 TTG -> TTC | 1 | 6 | 4 | 3 | 0 |
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| MEN 2B | 918 ATG -> ACG | 3 | 3 | 3 | 2 | 0 |
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Out of 68 patients with an apparent sporadic form of medullary thyroid carcinoma, 3 cases turned out to be familiar disease by genetic testing. Thus, genetic screening is recommended in each patient with medullary thyroid carcinoma.
The documentation of a thyroid ultrasonography examination should include:
| View figure 7 | View figure 8 | View figure 9 |
Scintigraphy with 99m-Technetium is indicated in case of:
Szintigraphy with 123-Iodine is indicated:
Scintigraphy with 131-Iodine is indicated:
Indications for fine needle aspiration generally include:
| · | Any nodule exceeding 1.0 cm in size, except for scintigraphically documented autonomously functioning nodules which are known to rarely harbor carcinoma. Smaller lesions may in most cases be sufficient to be followed up by ultrasonography after 3-6 months, thereafter at yearly intervals. In that case the delay in diagnosis of a differentiated thyroid microcarcinoma would not adversely affect the prognosis of the patient. |
| · | Rapidly growing focal lesions. |
| · | Suspicion of an intrathyroidal metastasis of a non-thyroidal tumor. |
| · | Purulent thyroiditis |
| · | Subacute thyroiditis de Quervain if diagnosis is ambiguous |
| · | Chronic autoimmune thyroiditis in case of a suspicion of lymphoma |
| · | Aspiration of cysts. |
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