Thyroid function testing

Introduction

Thyroid hormones thyroxine (T4) and tri-iodothyronine (T3) are produced, stored, and secreted by the thyroid gland. These hormones, particularly T3, play a major role in multiple biological and metabolic processes. They act by binding to thyroid receptors that are distributed in almost every organ. Typically, this process regulates gene transcription and the subsequent production of various proteins that are involved in development, growth, and cellular metabolism. [1] Thyroid function tests (TFTs) are the most commonly used endocrine test. [Patient information]

Thyroid hormone production

Thyroid hormone production is regulated by the hypothalamus and pituitary gland. Hypothalamic thyrotropin-releasing hormone (TRH) stimulates pituitary thyrotropin (TSH) synthesis and secretion. In turn, TSH stimulates production and release of T4 and T3 from the thyroid gland. Once released, T4 and T3 exert a negative feedback mechanism on the production of TRH and TSH. [1] Hypothalamic-pituitary-thyroid axisFrom the collection of Dr Sheikh-Ali

The protein thyroglobulin (Tg) is produced and used by the thyroid gland to produce T4 and T3. T3 is the biologically active form of thyroid hormone whereas T4 is considered a prohormone to T3. The thyroid gland produces 100% of circulating T4 but only 20% of circulating T3. The remaining 80% of T3 is produced by the conversion of T4 to T3 in the peripheral tissues. Acute illnesses, as well as certain drugs, may inhibit the process of converting T4 to T3 and, therefore, affect their serum levels.

Thyroid hormone-binding proteins

T3 and T4 circulate in peripheral blood bound to proteins (thyroxine-binding globulin [TBG], pre-albumin [transthyretin], and albumin). Approximately, 99.98% of T4 and 97% of T3 are protein-bound. Only the unbound or 'free' portion, free T3 (FT3) and free T4 (FT4), are active. Therefore, any changes in the quantity or quality of thyroid-binding proteins will produce changes in circulating thyroid hormone levels. [1]

Indications for TFTs: general considerations

According to population studies in the US and the UK, the prevalence of overt hypothyroidism varies from 0.1% to 2%, and of subclinical hypothyroidism from 4% to 10% of adults, with possibly a higher frequency in older women. In the US, the prevalence of hyperthyroidism is approximately 1.2% (0.5% overt and 0.7% subclinical); the most common causes include Graves' disease, toxic multinodular goitre, and toxic adenoma. [2] [3]

In the US, the American Thyroid Association suggested that all adults should have serum TSH concentration measured at 35 years of age and every 5 years thereafter. [4] However, the US Preventive Services Task Force found that evidence was insufficient to recommend for or against routine screening for thyroid disease in adults. [5] In the UK, screening the healthy adult population for thyroid disease is not currently practised. [6] [UK guidelines for the use of thyroid function tests]

Screening may be appropriate in people at higher risk of developing thyroid dysfunction. Screening and further surveillance should be considered in patients: [7] [8] [9] [10]

• With a goitre

• Who have had surgery or radiotherapy affecting the thyroid gland

• Who have pituitary or hypothalamic disease, surgery, or irradiation

• With diabetes mellitus type 1

• With Addison's disease

• With first-degree relative with autoimmune thyroid disease

• With vitiligo

• With pernicious anaemia

• With leukotrichia (prematurely grey hair)

• With psychiatric disorders

• Receiving medications and iodine-containing compounds (e.g., amiodarone hydrochloride, radiocontrast agents, expectorants containing potassium iodide, kelp, interferon alpha, and tyrosine-kinase inhibitors, most notably sunitinib).

• With Down's or Turner's syndrome.

Screening for thyroid dysfunction during pregnancy

In pregnancy, oestrogen levels increase and thyroid-binding globulin concentrations rise, which leads to an increase in T4 and T3. In the first trimester, serum TSH also falls due to the effect of human chorionic gonadotrophin (hCG), which may be associated with a slight and transient increase in FT4. These changes are small and in most of the pregnant women, FT4 concentrations remain within the normal range for non-pregnant women. [11] In the second and third trimesters, FT4 and FT3 decrease, sometimes below the non-pregnant women's reference level. There is insufficient evidence to recommend for or against screening in pregnant women.

Universal screening compared with case finding for detection and treatment of thyroid hormonal dysfunction during pregnancy did not result in a decrease in adverse outcomes. [12] However, the Endocrine Society recommends screening groups at high risk for thyroid dysfunction - that is, women with: [13]

• History of hyperthyroid or hypothyroid disease, postpartum thyroiditis, or thyroid lobectomy

• Family history of thyroid disease

• Goitre

• Thyroid antibodies (when known)

• Symptoms or clinical signs suggestive of thyroid under-function or over-function, including anaemia, elevated cholesterol, or hyponatraemia

• Type 1 diabetes

• Other autoimmune disorders

• History of therapeutic head or neck irradiation

• Prior history of miscarriage or preterm delivery

• Infertility: should have screening with TSH as part of infertility work-up.

TFTs should be monitored closely in pregnant women with hypothyroidism because thyroxine replacement often needs to be increased by 30% to 50% during the first trimester. Thyroxine dosage should be titrated to maintain serum TSH concentrations of 0.1 to 2.5 mIU/mL in the first trimester and 0.2 to 3 mIU/mL in the second and third trimester. [12] [14]

TFTs

TSH assay

• A serum TSH assay is the test of choice to screen for thyroid function disorders in the absence of hypothalamic or a pituitary pathology. [4] [15] [16] [17] In most reference laboratories, the normal range for TSH is 0.45 to 4.5 mIU/L. [18] [19] TSH is sensitive to any change in the plasma concentration of thyroid hormones. [20] TSH may require an average of 6 to 8 weeks to adjust to changes in thyroid hormone levels. Therefore, it is recommended to check TSH levels 6 to 8 weeks after thyroxine adjustment or any antithyroid drug treatment. A subnormal TSH level should trigger the measurement of FT4. If this is not elevated, FT3 should be measured to identify cases of T3-thyrotoxicosis. Suppressed or elevated TSH confirms presence of thyroid dysfunction but not its cause.

Free T4 (FT4) and Free T3 (FT3) assays

• FT4 assay is the test of choice to evaluate an abnormal TSH level. It is used in preference to a total T4 assay. A free T3 assay would be the preferred test over a total T3 assay; however, some commercially available free T3 assays are variable and unreliable. Free T3 should be measured in evaluating patients with thyrotoxicosis, and when the FT4 is not elevated in the presence of a subnormal TSH. FT4 and FT3 assays are a good measure of thyroid gland output and are independent of thyroid hormone-binding protein concentrations. Typical normal range for FT4 is 12 to 30 picomol/L (0.9 to 2.3 nanogram/dL) and for FT3 is 2 to 7 picomol/L (230 to 420 picogram/dL). [1]

Total T4 and total T3 assays

• Previously, before improved FT4 and FT3 assays, total T4 and total T3 assays were ordered to evaluate an abnormal TSH assay. However, total T4 and total T3 levels can be affected by changes in the levels of circulating thyroid hormone-binding protein levels. They measure both free and protein-bound hormones. Normal range for total T4 is 206 to 309 nanomol/L (5.5 to 12.5 microgram/dL) and normal range for total T3 is 0.92 to 2.76 nanomol/L (60 to 180 nanogram/dL).

• Conditions associated with elevated total T4 and total T3 levels secondary to increased TBG levels include pregnancy, oestrogen use, liver diseases (e.g., hepatitis), drug use (e.g., tamoxifen or methadone), or rarely, hereditary TBG excess. [21] [22] Other rare conditions resulting in elevated total T4 and total T3 levels are increased albumin or transthyretin binding.

• Conditions associated with decreased total T4 and total T3 levels secondary to decreased TBG levels include androgen excess, glucocorticoid excess, nephrotic syndrome, hereditary TBG deficiency, and drug use (e.g., niacin or danazol). [22] [23] [24]

• Illness, starvation, and poor nutrition may also decrease total T4 and total T3 levels by decreasing albumin and transthyretin levels and possibly interfering with the binding capacity of the carrier proteins.

Thyroid autoantibodies

• TSH-receptor antibodies (TRAb) are not routine tests but may be of use in selected cases where diagnosis is equivocal. The results are useful in identifying thyroid disease aetiology. TRAb can be either stimulatory or blocking to the TSH receptor. Thyroid-stimulating immunoglobulin (TSI) is an example of a stimulatory TRAb and is usually elevated in Graves' disease.

• Thyroid peroxidase antibodies (TPOAb) are also helpful in identifying thyroid disease aetiology. TPOAb are usually present in Hashimoto's disease and other autoimmune thyroid diseases.

• Tg antibody test is used primarily to help diagnose autoimmune conditions involving the thyroid gland.

Tg assay

• Usually ordered for surveillance in patients with differentiated thyroid cancer when the patient does not have Tg autoantibodies in the serum.

• In addition, it may be ordered when investigating the underlying cause of hyperthyroidism. Tg is usually elevated in primary hyperthyroidism and thyroiditis but not in factitious thyrotoxicosis (excessive use of thyroid hormone medication causing thyrotoxicosis).

• An increase in serum Tg occurs in 33% to 88% of patients who undergo thyroid fine needle biopsy (FNB). Serum Tg concentrations typically return to baseline about 2 to 3 weeks after FNB. The degree of increase in serum Tg after FNB is highly variable (ranging from 35% to 341%) and not a predictor of whether the biopsied nodule is benign or malignant. [25]

Radioactive iodine uptake (RAIU) and scan

• Usually ordered in the setting of thyrotoxicosis to help identify the underlying aetiology. It measures the amount of radioactive iodine (usually I-123) that is taken up by the thyroid gland. High uptake may indicate hyperthyroidism. The increased uptake may be diffuse and homogeneous as seen in Graves' disease, or take on the appearance of hot nodules, as seen in multinodular toxic goitre. Low uptake may indicate thyroiditis or factitious thyrotoxicosis in the appropriate clinical setting.

• RAIU cannot be performed in certain patients (e.g., pregnant or nursing women or iodine-contaminated patients); in such cases, serum TRAb measurement is helpful in identifying Graves' disease.

TRH stimulation test

• Used to evaluate TSH response to TRH stimulation in the setting of central hypothyroidism. It may also help differentiate TSH secretory tumour from resistance to thyroid hormone syndrome (RTH). In RTH, the TSH response is normal. TRH stimulation test is not a specific test.

Calcitonin

• Calcitonin is usually a marker of medullary thyroid cancer. [26] However, calcitonin levels may also be increased, although infrequently, in other clinical conditions such as C-cell hyperplasia, pulmonary and pancreatic neuroendocrine tumours, renal failure, and hypergastrinaemia (use of proton-pump inhibitors). [27]

• A single, unstimulated calcitonin measurement can be used in the initial work-up of thyroid nodules. [27] However, this practice is not done routinely in the US.

Low TSH - associated with a high FT4 and/or FT3

• Suggestive of hyperthyroidism. The most common causes include Graves' disease, toxic thyroid adenoma, and toxic multinodular goitre. Radioactive iodine uptake (RAIU) helps to differentiate between these conditions. In cases of Graves' disease, RAIU is diffusely increased. For toxic nodules: uptake is increased in the area of a single nodule, or in multiple areas in cases of toxic multinodular goitre; the remaining thyroid tissue uptake is suppressed. Furthermore, in Graves' disease, thyroid stimulating immunoglobulins (TSI) are present in about 90% of patients, though usually not required for diagnosis.

• In subacute or granulomatous thyroiditis, RAIU is low. Patients with subacute thyroiditis have elevated thyroid hormone levels initially, secondary to excessive release of stored T4 and T3 from the thyroid gland. Later, thyroid hormone levels decrease below normal, before returning to normal when inflammation subsides.

• Other causes include factitious thyrotoxicosis (caused by excessive use of thyroid hormone medication), in which case thyroglobulin levels and RAIU are low, or iodine-induced hyperthyroidism (e.g., following use of amiodarone or exposure to radiocontrast agents), where RAIU is also low. Differentiating causes of low TSH and high free T4 (FT4) and/or free T3 (FT3)From the collection of Dr Sheikh-Ali

Low TSH - associated with a low FT4 and/or FT3

• Suggests secondary (central) hypothyroidism, which is associated with pituitary or hypothalamic dysfunction. TSH can be low, normal, or slightly elevated. Evaluation for deficiencies in other pituitary hormones should be obtained before imaging (i.e., pituitary MRI). Hormone tests should include: ACTH with cortisol, FSH, LH, estradiol (female), testosterone (male), prolactin, GH, and insulin-like growth factor 1 (IGF1). For this condition, thyroid replacement therapy is monitored by checking the levels of FT4 and FT3. [28]

• Other causes of these results include non-thyroid illness (sick euthyroid syndrome) where abnormalities in thyroid tests secondary to acute systemic illness are observed with no true thyroid dysfunction. TSH can be normal or low followed by rebound elevation during recovery from acute illness. [18] FT4 can be normal, low, or high. FT3 is usually low secondary to decreased conversion of T4 to T3. Thyroid hormone replacement is not recommended in this condition. [29] [30]

• In the second and third trimesters of pregnancy, FT4 and FT3 decrease, sometimes below the non-pregnant woman's reference level. Differentiating causes of low TSH and low free T4 (FT4) and/or free T3 (FT3)From the collection of Dr Sheikh-Ali

Low TSH - associated with a normal FT4 and/or FT3

• In the absence of non-thyroidal illness or relevant drug therapy, these results suggest subclinical (or mild) hyperthyroidism. In this case RAIU can be slightly elevated or normal.

• In non-thyroid illness (sick euthyroid syndrome), TSH can be normal or low followed by rebound elevation during recovery from acute illness. [18] FT4 can be normal, low, or high. FT3 is usually low secondary to decreased conversion of T4 to T3.

• The following drugs may cause these results: dopamine, dopaminergic agonists, glucocorticoids, cytokines, or octreotide, because they inhibit pituitary TSH secretion. Similar results may also occur following exposure to radiocontrast agents. [31] [32]

• Recent treatment of hyperthyroidism with antithyroid medication may also cause these results. It may take up to 6 to 8 weeks for TSH to adjust after initiating therapy. [33]

• In the first trimester of pregnancy, serum TSH falls due to the effect of human chorionic gonadotrophin. This may be associated with a slight and transient increase in FT4. [11] Differentiating causes of low TSH and normal free T4 (FT4) and/or free T3 (FT3)From the collection of Dr Sheikh-Ali

High TSH - associated with a high FT4 and/or FT3

• If this result is found then assay artifact/laboratory error should be considered first. Changing laboratory method may help in identifying the problem. [34]

• If assay results are correct, the major diagnoses are a TSH-secreting pituitary tumour (TSH-oma) or a syndrome of resistance to thyroid hormone. The finding of an elevated serum sex hormone-binding globulin (SHBG) and circulating free alpha subunit may support the diagnosis of TSH-oma, as may the finding of hyper- or hyposecretion of other pituitary hormones. [35] Pituitary imaging (MRI) usually confirms the diagnosis but should not be undertaken until the appropriate biochemical confirmation has been made. RAIU shows diffusely increased uptake.

• Thyroid hormone resistance syndrome can be confirmed by positive family history, absence of adenoma on pituitary MRI, and normal levels of serum alpha subunit glycoprotein. By contrast, with a TSH-oma where TSH production is autonomous, T4 or T3 administration eventually suppresses the high TSH in thyroid hormone resistance syndrome. [36] [37] [38]

• Thyroxine replacement therapy (for possible hypothyroidism) taken within a few hours of TFT can raise FT4 levels. However, FT3 is almost always normal in these situations. [39]

• Abnormal thyroid function has been found in patients with acute psychiatric disorders. [40] Differentiating causes of high TSH and high free T4 (FT4) and/or free T3 (FT3)From the collection of Dr Sheikh-Ali

High TSH - associated with a low FT4 and/or FT3

• Suggests primary hypothyroidism. Underproduction of the thyroid hormones (T4 and T3) may occur with autoimmune thyroiditis (Hashimoto's disease), which is the most common cause of primary hypothyroidism. More than 90% of patients with Hashimoto’s thyroiditis have positive TPOAb.

• Other causes include thyroidectomy or radioactive iodine treatment of the thyroid without adequate thyroid hormone replacement.Differentiating causes of high TSH and low free T4 (FT4) and/or free T3 (FT3)From the collection of Dr Sheikh-Ali

High TSH - associated with a normal FT4 and/or FT3

• Subclinical (or mild) hypothyroidism occurs when TSH is above reference range with a normal FT4 and FT3. The risk of progression to overt hypothyroidism is 2% to 5% per year. [41] The risk is higher in patients with positive TPOAb. [42] The decision to treat these patients is controversial. Generally, thyroxine replacement is not recommended when TSH is below 10 mIU/L. [43] TSH and FT4 should be repeated at 6- to 12-month intervals to monitor for improvement or worsening in thyroid status in untreated patients. [41]

• Differentials include the recovery from non-thyroid illness (sick euthyroid syndrome). TSH can be normal or low followed by rebound elevation during recovery from acute illness. [18]

• Other differentials include poor adherence to thyroxine replacement therapy or its mal-absorption: for example, in coeliac sprue, or as a result of interference from other co-administered medications, such as calcium carbonate, ferrous sulphate, and colestyramine.

• Concomitant medication that promotes increased metabolism of thyroid hormone (e.g., rifampicin [rifampin], phenytoin, carbamazepine, barbiturates) can also cause these results. [18]

• In addition, problems with assay procedures (e.g., interference of abnormal antibodies in serum) can cause false elevation in TSH. Changing the laboratory method may help identify the problem. [34] Differentiating causes of high TSH and normal free T4 (FT4) and/or free T3 (FT3)From the collection of Dr Sheikh-Ali

Normal TSH - associated with a low FT4 and/or FT3

• These results may occur following secondary (central) hypothyroidism, which is associated with pituitary or hypothalamic dysfunction. TSH can be low, normal, or slightly elevated. Evaluation for deficiencies in other pituitary hormones should be obtained before imaging (i.e., pituitary MRI). Hormone tests should include: ACTH with cortisol, FSH, LH, estradiol (female), testosterone (male), prolactin, GH, and (insulin-like growth factor 1 (IGF1). For this condition, thyroid replacement therapy is monitored by checking the levels of FT4 and FT3. [28]

• Other causes include drug use (e.g., phenytoin, rifampicin [rifampin], carbamazepine, barbiturates) and assay error when interfering substances are present.Differentiating causes of normal TSH and low free T4 (FT4) and/or free T3 (FT3)From the collection of Dr Sheikh-Ali

Drug effects

• Many commonly used medications affect thyroid function. [43] Therefore, the possible effect of these drugs both on the results of TFTs and on the effectiveness of treatment should always be considered in decisions regarding patient care.

  • Iodine, amiodarone, or lithium increase or decrease thyroid hormone secretion.

  • Dopamine and its agonists, as well as glucocorticoids, cytokines, or octreotide, decrease TSH secretion.

  • Rifampicin (rifampin), phenytoin, carbamazepine, or barbiturates increase hepatic metabolism.

  • Carbimazole, propylthiouracil, or lithium decrease thyroid hormone synthesis.

  • Beta-blockers, glucocorticoids, amiodarone, propylthiouracil, or radiocontrast dyes impair T4 to T3 conversion.

  • Furosemide, NSAIDs, mefenamic acid, carbamazepine, or beta-blockers displace T4/T3 from plasma proteins.

  • Oestrogens, tamoxifen, heroin, methadone, or raloxifene increase thyroxine binding globulin (TBG), total T4, and total T3 levels.

  • Androgens, anabolic steroids, or glucocorticoids decrease TBG, total T4, and total T3 levels.

  • Colestyramine, aluminium hydroxide, ferrous sulphate, sucralfate, calcium carbonate, or proton-pump inhibitors impair absorption of thyroxine.

  • Interleukin-1, interferon-alfa, interferon-beta, and TNF-alpha are associated with risk of autoimmune thyroid dysfunction.

  • Amiodarone may also modify thyroid hormone action.Drug effects on the thyroidFrom the collection of Dr Sheikh-Ali