30 Disorders of thyroid function

Lothar Thomas

30.1 Physiological function of the thyroid

Thyroid hormones influence numerous physiological and biochemical events in the cell. Throughout the body, they are the main regulators of the basal metabolic rates and influence the metabolism of every class of foodstuff. They regulate the growth and differentiation of cells and are especially active in liver, heart, kidney, skeleton, gastrointestinal system, and the central nervous system. However, each tissue responds in a characteristic fashion. Thyroid hormones influence intermediary metabolic processes such as the activities of other hormones or the metabolism of drugs. They are the main regulators of obligate heat production in homothermous creatures due to their effect on the basal metabolic rate and they stimulate respiration. Thyroid hormones influence cellular functions in the long term range via nuclear receptors and time enhanced via extra nuclear binding proteins /1/.

30.1.1 Hypothalamic-pituitary-thyroid axis

The hypothalamic-pituitary-thyroid axis is a classic system sub serving control of the hormone secretion by the thyroid /2, 3/:

• Thyrotropin releasing hormone (TRH) at hypothalamic level
• Thyroid stimulating hormone (TSH) at pituitary level
• Tetraiodothyronine (thyroxine, T4), triiodothyronine (T3), and reverse T3 (rT3) at peripheral cellular level.

Refer to Fig. 30.1-1 – Hypothalamic-pituitary-thyroid axis

Pulsatile and non pulsatile secretion of TRH by the hypothalamus leads to the release of TSH from the pituitary, which in turn stimulates the thyroid to release the pro hormone T4 into the circulation. T4 is converted peripherally into metabolically active T3 and metabolically inactive rT3. T3 and T4 feedback inhibition controls TRH and TSH secretion.

Whereas all of the circulating T4 is secreted directly by the thyroid, the same is true for only 20% of the circulating T3. The other 80% results from the peripheral mono iodination of T4 to T3. Most circulating T4 and T3 is bound to plasma proteins such as thyroxine binding globulin (TBG), thyroxine binding pre albumin (transthyretin), and albumin. Only about 0.03% of T4 and 0.3% of T3 exist in free form (FT4 and FT3). Only FT4 and FT3 are metabolically active.

The serum levels of thyroid hormones are generally maintained within a narrow range by means of feedback inhibition of hypothalamic TRH and pituitary TSH. Circulating thyroid hormone is registered by the cells of the para ventricular nucleus of the hypothalamus, where T4 is converted to T3 by mono iodination. If there is a decrease in intracellular T3, TRH is released into the hypothalamic pituitary circulation and stimulates anterior pituitary thyrotropic cells to produce TSH. TSH levels increase in the plasma and the thyroid responds by releasing T4 and T3. The resulting increase in the concentration of thyroid hormones exerts feedback inhibition on the secretion of TRH and TSH.

The feedback control mechanism is dependent upon the presence of thyroid hormone receptors, which bind to the promoters of TRH and TSH subunit genes and regulate their expression. In the presence of their ligand, T3, thyroid hormone receptors mediate ligand dependent repression of the transcription of these genes. In the absence of T3, the transcription rate is not simply returned to baseline, but ligand independent over expression occurs. This results in a disproportionate increase in TSH with a log-linear relationship between the concentration of TSH and FT3 or, for example, FT4.

Refer to:

• Fig. 30.1-2 – Inverse log-linear relationship between FT4 and TSH
• Fig. 30.1-3 – Typical patterns of T4, T3 and TSH in postpartum thyroiditis.

In a system like this, small linear decreases in thyroid hormone serum level lead to an exponential increase in TSH and small increases in thyroid hormones lead to a sharp decline in TSH. This is why TSH determination is much more sensitive than thyroid hormone measurement for diagnosing thyroid dysfunction. Early measurement of TSH can detect minor changes in T4 and T3 levels that are not yet clinically apparent.

FT4 concentration within the reference interval and in association with:

• Elevated TSH level indicates latent (subclinical) hypothyroidism
• Decreased TSH level indicates latent (subclinical) hyperthyroidism.

The hypothalamic-pituitary-thyroid axis responds relatively slowly. In a hypothyroid patient receiving L-thyroxine treatment, for example, it takes 4–6 weeks for TSH to return from elevated level to normal. It also takes several months for low TSH level to return to the reference interval in patients treated for hyperthyroidism. These situations as well as extra thyroidal factors such as critical illness, medication use, abnormal T4 and T3 protein binding, or dysproteinemia can result in discordance between TSH levels and concentrations of T3 and T4.

30.1.2 Thyroid disorders

In many cases the disorder is characterized by the inability of the thyroid gland to respond to pituitary TSH stimulation and produce normal amounts of thyroid hormones. Most commonly primary hypothyroidism is caused by destruction of the thyroid gland (Hashimoto thyroiditis, chronic lyphocytic thyroiditis). Clinical findings are cold intolerance, dry skin, constipation and with short stature in children. Thyroid disorders are the second most common type of endocrine disorders worldwide, second only to diabetes mellitus. For instance, the prevalence of overactive thyroid gland (hyperthyroidism) in women is 2% while the prevalence of underactive thyroid gland (hypothyroidism) is 1%. The prevalence is 5–10 times lower in men.

Hyperthyroidism and hypothyroidism are classified as:

• Primary: due to thyroid disease or thyroid dysfunction mediated by autoantibodies
• Secondary: due to pituitary dysfunction or thyroid involvement in systemic disease
• Tertiary: due to dysfunction of the hypothalamic-pituitary-thyroid axis.

According to the clinical symptoms and laboratory results /4/:

• Subclinical means in the first instance no more than that the result of thyroid stimulating hormone (TSH) has changed but that thyroid hormones remain normal. TSH is the main indicator of thyroid dysfunction.
• Overt describes functional disorders of the thyroid of endogenous or exogenous causes and levels of TSH, free T4 or free T3 outside of their reference intervals.

30.1.2.1 Hypothyroidism

Hypothyroidism is a term for any condition in which the tissues of the body are expressed to a not physiological concentration of thyroid hormones. Causes of hypothyroidism are deficiency of thyrotropin releasing hormone (TRH), deficiency of TSH, thyroid destruction, and thyroid deficiency. Hypothyroidism is characterized by the inability of the thyroid gland to respond to pituitary TSH stimulation and to produce normal amounts of thyroid hormones.

Clinical findings: cold intolerance, dry skin, and constipation. Primary hypothyroidism is associated with short stature in children (refer to chapter 35). Hypothyroidism results from autoimmune destruction of the thyroid gland mediated by autoantibodies caused by Hashimoto thyroiditis or chronic lymphocytic thyroiditis which modulate the formation of autoantibodies, primarily anti-thyroperoxidase and anti-thyroglobulin are determined. In secondary and tertiary hypothyroidism, collectively known as central hypothyroidism the underlying cause is dysfunction of the pituitary gland or the hypothalamus.

Laboratory findings:

• Decreased FT4 and/or FT3 and normal TSH (84% of cases) in central hypothyroidism.
• Decreased FT4 and/or FT3 and elevated TSH in distruction of the thyroid gland.
• Primary hypothyroidism is characterized by the inability of the thyroid gland to respond to pituitary TSH stimulation.

30.1.2.2 Hyperthyroidism

Hyperthyroidism is a term for any condition in which the tissues of the body are expressed to a supraphysiologic concentration of thyroid hormones. This is the case in Graves’ disease, which is also called diffuse toxic goiter /60/.

Clinical picture: sweating, palpitations, nervousness, tachycardia, irritability, insomnia, frequent stools, weight loss despite a good appetite. Physical examination shows mild proptosis, stare, lid lag, a smooth, diffuse, nontender goiter.

Laboratory findings: decrease in TSH and increase in FT4 and/or FT3.

30.1.2.3 Thyroid nodules and thyroid enlargement

Thyroid enlargement and thyroid nodules are common in the general population in Germany (prevalence 20–36%) and with an increase. According to the SHIP (study of health in Pomerania) in people of 20 to 79 years of age every second of the investigated population has thyroid nodules. Reasons are deficiency of iodine and an improved sonographic examination.

In almost all cases of nodules and enlargement, the disorders are caused by dietary iodine deficiency with subsequent low iodine content of the thyroid (refer to Section 10.11 – Iodine). A compensatory increase in the synthesis of paracrine and autocrine growth factors modulated by TSH leads to thyroid hypertrophy. In Germany, 10–20% of goiters and thyroid nodules are functionally autonomous and account for half of all cases of hyperthyroidism /5/.

Thyroid nodules caused by clonal aberrations are a second group of thyroid disorders. The frequency increases with age and nodules can be detected on ultrasound in approximately 50% of individuals over the age of 60 years. Most, however, are benigne. In the case of nodules with a diameter of greater than 1 cm, functional autonomy and medullary thyroid cancer must be ruled out by determining TSH and calcitonin and performing a fine needle aspiration biopsy.

Unless treated promptly, maternal hypothyroidism due to iodine deficiency or other causes can result in congenital hypothyroidism (cretinism) in the newborn. Neonatal screening for hypothyroidism by means of TSH determination is mandatory in many countries.

Disorders of thyroid function may represent adaptive responses to physiological states or be due to primary thyroid disease, systemic non-thyroidal illness, or dysfunction of the hypothalamic-pituitary-thyroid axis.

Adaptive physiological responses occur in the thyroid during childhood, old age, and pregnancy (Tab. 30.1-1 – Thyroid function and physiological states).

Reactive thyroid dysfunction in systemic disease is often due to a dysfunction of the hypothalamic-pituitary-thyroid axis (Tab. 30.1-2 – Adaptive response to non-thyroidal illness).

Primary functional disorders of the thyroid (hyper- and hypothyroidism) are common all over the world. Their incidence is dependent on a number of factors, the most important of which is inadequate iodine intake. In areas with high iodine supply, hypothyroidism is more common than hyperthyroidism, whereas hyperthyroidism dominates in areas with mild and moderate iodine supply. Primary hyperthyroidism and hypothyroidism can be divided into a number of nosological subtypes with different etiology, clinical presentation, prognosis, and outcome of therapy (Tab. 30.1-3 – Primary thyroid disorders associated with hyper-, hypo- and euthyroidism).

Overt thyroid dysfunction denotes the presence of disease with clinical symptoms. Latent or subclinical disease is defined biochemically because clinical symptoms are absent /6, 7/. Refer to Tab. 30.1-4 – Subclinical thyroid function disorder.

Reference intervals for TSH and thyroid hormones in pregnant women are depending on the country /8/. Refer to Tab. 30.1-5 – Reference intervals for TSH and thyroid hormones in pregnant women.

Diagnosis of functional disorders of the thyroid is based on the sensitivity of the hypothalamic-pituitary-thyroid axis and are generally manifested by serum TSH changes and additional findings:

• Overproduction of T4 by the thyroid or increased peripheral conversion of T4 to T3; FT4 and/or FT3 are elevated in serum (hyperthyroidism)
• Reduced production of T4 by the thyroid; FT4 and/or FT3 are decreased in serum (hypothyroidism)
• Insufficient peripheral conversion of T4 to T3 (low T3 syndrome) in severe systemic non-thyroidal illness; serum FT4 normal and FT3 decreased or low normal.

Important exceptions are:

• Critically ill patients with hyperthyroidism may not show an adequate increase in FT3 despite clear FT4 elevation, due to a low T3 syndrome
• The conversion of T4 to T3 may be increased while T4 production by the thyroid is normal or decreased; serum FT4 level is low or low normal and FT3 is normal or near the upper reference interval value. This situation can occur temporarily following hyperthyroidism. Due to persistent activation of T4 deiodinase, the increased conversion of T4 to T3 continues (hyperconversion thyrotoxicosis).

30.1.2.4 Goiter

A goiter is an abnormal enlargement of the thyroid gland. The presence of a goiter does not necessarily mean that the thyroid gland is malfunctioning. A goiter indicates there is a condition present which is causing the thyroid to grow abnormally. Most common factors causing thyroid gland to enlarge are:

• Iodine deficiency. A lack of dietary iodine is the main cause of goiters in main parts of the world. The initial iodine deficiency may be made even worse by a diet high in hormone-binding foods such as cabbage, broccoli and cauliflower. The urinary iodine excretion is an estimate of the grade of endemic goiter (Tab. 30.1-6 – Severity of endemic goiter).
• Graves’ disease; autoantibodies directed against thyroid tissue stimulate the production of excess thyroxine
• Hashimotos thyroiditis; autoimmune disease resulting in underactive thyroid
• Multinodular goiter; several fluid-filled or solid nodules have developed in both sides of the thyroid
• Solitary thyroid nodules
• Thyroid cancer
• Pregnancy; human chorionic gonadotropin (hCG), may cause the thyroid gland to enlarge slightly
• Inflammation can cause over- or underproduction of thyroid hormones. The inflamed thyroid causes pain and swelling.

30.1.2.5 Thyroid nodules

Thyroid nodules have become relatively common (about 50% of the population) in medical practice and the prevalence increases with age /56/. In adults the risk of malignancy is 5% to 15%. Fine-needle aspiration cytology (FNA) of thyroid nodules is a useful diagnostic tool. The Bethesda system for Reporting Thyroid Cytopathology (BSRTC) uses 6 categories for thyroid cytology reporting (1, nondiagnostic; 2, benigne; 3, atypia of undetermined significance; 4, follicular neoplasm; 5, suspicion for neoplasm; 6, malignant). About 70% of FNAs are categorised as Bethesda 2 /57/.

30.1.2.6 Chronic thyroiditis

Chronic thyroiditis is one of the most prevalent autoimmune diseases and is associated with antibodies to thyroid peroxidase (TPOAb) and thyroglobulin (TgAb). In some patients TSH-receptor autoantibodies (TRAb) with stimulating (TSAb) and blocking activity (TBAb) were also reported. In a study /58/. TRAb were tested with ELISA using a 2.5 IU/L cut off for positivity. TRAb positivity was found in 13% patients with chronic thyroiditis and hypothyroidism.

30.1.2.7 Medication effects on the thyroid

Medications are known to adversely affect thyroid function /11/. Refer to Tab. 30.1-7 – Medication effects on the thyroid.

30.1.3 Diagnosis of thyroid disorders

Medications are known to adversely affect thyroid function /11/. Refer to Tab. 30.1-7 – Medication effects on the thyroid.

Functional diagnosis of the thyroid

In the majority of patients, a diagnosis can be made based on a selection of adequate laboratory tests aimed at the detection or exclusion of a thyroid disease or thyroid dysfunction. The selection depends on the clinical concerns. One must differentiate between methods aimed to assess /9/: a) The function of the thyroid gland, b) the classification of a thyroid disease, and c) the hypothalamic-pituitary-thyroid axis. Laboratory tests for assessing thyroid gland function are: Thyroxine (T4); usually determined as free T4 (FT4); Triiodothyronine (T3); determined as total or, more commonly, free T3 (FT3).

Functional tests for assessing the thyroid diseases are autoantibodies

Tests are: anti-TPOAb, anti-TgAb, TSH receptor antibodies, and inflammation markers e.g., CRP.

Functional test for assessing hypothalamic-pituitary-thyroid axis

Thyroid Stimulating Hormone (TSH)

Diagnosis of hyperfunctional (autonomous) hot nodules of the thyroid

The prevalance is very low. The degree of malignancy in Germany is 1.3% /59/. The determination of calcitonin is recommended by the German Society of Endocrinology in patients with hot nodules, because 0.5% of C-cell carcinoma are detected.

Diagnosis of hypofunctional autonomous cold nodules of the thyroid

The degree of malignancy in Germany is 1.3% /59/.

Recommended diagnostic approaches to the different clinical questions /10/ are shown in:

• Fig. 30.1-4 – Laboratory diagnosis for suspected hyperthyroidism
• Fig. 30.1-5 – Etiological clarification of primary hyperthyroidism
• Fig. 30.1-6 – Laboratory diagnosis for suspected hypothyroidism
• Fig. 30.1-7 – Etiological clarification of primary hypothyroidism.

The recommendation of the thyroid section of the German Endocrine Society are listed in Tab. 30.1-6 – Severity of endemic goiter.

The terms euthyroidism, hyperthyroidism, and hypothyroidism describe the biological effects of thyroid hormones in the peripheral tissues but do not always describe the functional status of the thyroid.

When diagnosing thyroid disorders and dysfunction, it is important to follow the recommendations provided by the relevant national and international societies and organizations.

30.1.3.1 Diagnostic approach using TSH and FT4

Thyroid status can be accurately screened with thyroid-stimulating hormone (TSH), and additional testing such as free thyroxine (FT4) is required only when TSH is abnormal. An inverse relationship usually exists between TSH and FT4, and findings can be interpreted as follows /6, 7, 11/:

• An increase in TSH and a decrease in FT4 confirms the diagnosis of overt hypothyroidism caused by thyroid gland failure
• An increase in TSH together with a normal FT4 indicates latent (subclinical) hypothyroidism
• A decrease in TSH below 0.1 mIU/L and an increase in FT4 and/or FT3 confirms the diagnosis of overt hyperthyroidism caused by thyroid gland failure
• A TSH level in the range of 0.5 to 0.1 mIU/L and an increase in FT4 and /or FT3 indicates latent (subclinical) hyperthyroidism
• A normal or subnormal TSH with a subnormal FT4 suggests that the patient may have hypothyroidism secondary to a decrease in TSH secretion: this combination of results suggests a diagnosis of hypopituitarism and may warrant an examination of hypothalamic-pituitary-thyroid axis.

30.1.3.2 Discordance between TSH and FT4

In hospitalized patients a diagnostic approach that focuses on TSH alone can be misleading, due to the effects of non-thyroidal illness or drugs that affect the metabolism of T4 and T3 and the secretion of TSH. This often may lead to confusing results.

Discordance between the levels of TSH and thyroid hormones can occur:

• In results indicating latent (subclinical) thyroid disorder, in which the TSH level is abnormal but FT4 is normal
• If thyroid hormone protein binding is abnormal. In this case, TSH is normal and TT4 (and TT3 less frequently) outside the reference interval; depending on the method used, FT4 and FT3 are usually normal
• In hypothyroid patients receiving treatment with L-thyroxine, in the initial phase of treatment or following dose adjustment (Tab. 30.1-8 – Initial investigations in suspected thyroid disease). Because it takes 8 weeks to establish a new equilibrium in the hypothalamic-pituitary-thyroid axis, the change in FT4 precedes the change in TSH by several weeks.
• Following treatment for hyperthyroidism; FT4 returns to normal much more quickly than TSH
• In patients with unstable thyroid disease. The FT4 level changes much more quickly than that of TSH.
• In the elderly; compared to younger individuals, they more often have slightly increased or decreased TSH levels with normal FT4 and T3 (FT3)
• If the conversion of T4 to T3 is disrupted by drugs such as amiodarone, propylthiouracil, dexamethasone, iodine containing contrast medium, or, to a lesser extent, propranolol
• In heparinized patients; an increase in the release of non esterified free fatty acids results in increased levels of FT4 and FT3.

30.1.3.3 Investigation of the cause thyroid disorder

Investigations for evaluation the cause of thyroid disorders are:

• Thyroglobulin antibodies (anti-TgAb), thyroid peroxidase antibody (anti-TPOAb), TSH receptor antibodies (TRAb)
• Thyroglobulin
• Excretion of iodine in urine
• Thyroid scan
• Fine-needle biopsy.

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30.2 Thyroid-stimulating hormone (TSH)

Provided hypothalamic and anterior pituitary function are normal, the serum TSH level reflects the availability of thyroid hormones in the tissues. The TSH secretion changes logarithmically with arithmetic changes in serum concentrations of free T4 (Fig. 30.1-2 – Inverse log-linear relationship between FT4 and TSH). Therefore, alterations in serum FT4 that are within the reference interval will cause increases or decreases in serum TSH that are likely to be outside its reference interval /1/. TSH circulates in the blood as various isoforms that differ from pituitary TSH.

30.2.1 Indication

• Differentiation of euthyroidism from all forms of hyperthyroidism, provided a high sensitivity immunoassay (detection limit < 0.01 mIU/L) is used /2/
• Screening for primary hyperthyroidism
• Detection of subclinical hyperthyroidism or subclinical hypothyroidism (in conjunction with FT4)
• Monitoring of hyperthyroidism treatment in cases of treatment induced hypothyroidism; in the first few months and always in conjunction with FT4 determination
• Detection of thyroid hormone resistance (in conjunction with FT4)
• Screening for neonatal hypothyroidism
• Screening for primary hypothyroidism
• Assessment of thyroid function in non-thyroidal illness (in conjunction with FT4).

TRH test

• Unclear cases where concurrent thyroid disorder is suspected (patients with severe non-thyroidal illness)
• In individual cases of suspected latent (subclinical) hypothyroidism or hyperthyroidism
• Hypothalamic or pituitary disorders.

30.2.2 Method of determination

Immunoassay

Two-site (sandwich) immunoassays (enzyme, fluorescence, luminescence, or immunometric) are used /3/. The assays are calibrated against the WHO second IRP 80/558. Four generations of TSH assays with different detection limits exist (Tab. 30.2-1 – Generation nomenclature for TSH assays). Most laboratories use third generation assays.

Neonatal TSH screening

In the first 1–2 days after birth, a capillary blood sample from the heel is collected on filter paper. After the blood has been allowed to dry for one hour, the filter paper is sent to the laboratory for analysis. TSH is determined from 3–8 mm disks of filter paper following elution. Although the TSH concentration in congenital hypothyroidism is usually > 15–20 mIU/L, the assay should have a detection limit of < 5 mIU/L.

TRH test

Three test procedures exist.

Intravenous: administration of 200 μg (400 μg) TRH in adults or 7 μg/kg body weight in children. Blood collection after 30 min. to determine TSH concentration in response to stimulation.

Nasal: nasal application of 2 mg TRH. Blood can be collected for TSH determination for up to two hours following TRH application, after which time plateau levels are reached.

Oral: administration of 40 mg TRH. Blood is collected 3–4 hours after TRH stimulation.

Regardless of which TRH test is used, blood must always be collected before TRH administration to determine the basal TSH concentration. The diagnostic significance of the results of the TRH test is shown in Tab. 30.2-2 – Diagnostic assessment of the TRH test.

30.2.3 Specimen

• Serum: 1 mL
• 1 blood spot (neonatal TSH screening)

30.2.4 Reference interval

Refer to Tab. 30.2-3 – Reference intervals for TSH.

30.2.5 Clinical significance

The main clinical scenarios for measurement of serum TSH are screening for thyroid dysfunction. However the TSH concentration is dependent on variables. In iodine sufficient geographical regions the reference interval for serum TSH in adults ages 20–59 years is 0.40–4.0 mIU/L. Age, sex, ethnicity, iodine intake, reproductive status, body mass index, assay specific properties are independent factors that modify TSH level. Because of clinical experiences a TSH level of ≥ 10 mIU/L is generally be considered elevated and may be indicative of subclinical hypothyroidism or overt hypothyroidism and levels below 0.40 mIU/L may be indicative of subclinical hyperthyroidism. Throughout the 24 hour cycle females have significant higher TSH levels than males, the magnitude of these differences did not exceed 0.1 mIU/L /4/.

30.2.5.1 Low TSH

The serum TSH level is a sensitive indicator for assessing the biological effects of T4 and T3. Current recommendations suggest that tests with a detection limit of less than 0.01 mIU/L should be used. Measured values with this limit are highly accurate and can be used to reliably assess the TSH threshold value of 0.10 mIU/L that indicates to the clinician that further investigation of hyperthyroidism is required.

In outpatients without clinical signs of hyperthyroidism or hypothyroidism, no further investigations are necessary if the TSH level is within the reference interval.

Values of 0.01–0.39 mIU/L may indicate subclinical or overt hyperthyroidism /5/ and FT4 and FT3 should also be determined in all cases, because low TSH values are often transitory, especially in the range of 0.1 to 0.4 mIU/L. Some healthy elderly people might have low serum TSH level without identifiable thyroid disease. In these individuals, a low serum TSH might be due to a change in the set point of the hypothalamic-pituitary-thyroid axis with ageing /1/. Values of > 10 mIU/L may indicate subclinical or overt hypothyroidismn /6/.

Causes of low serum TSH levels that are not indicative of subclinical hyperthyroidism are shown in Tab. 30.2-4 – Causes of low TSH not associated with subclinical hypothyroidism.

The behavior of TSH in thyroid disease and non-thyroidal illness is shown in Tab. 30.2-5 – TSH in thyroid disease and non-thyroidal illness. The relationship between TSH and T4 is shown in Fig. 30.2-1 – Relationship between T4 and TSH.

Third generation TSH assays with a detection limit of less than 0.01 mIU/L must be used /7/:

• To distinguish between subclinical hyperthyroidism (TSH ≥ 0.1 mIU/L) and overt hyperthyroidism (TSH below 0.01 mIU/L)
• To accurately detect TSH secreting pituitary tumors (TSH ≥ 0.01 mIU/L)
• To monitor suppression therapy (TSH below 0.01 mIU/L)
• To differentiate between non-thyroidal illness (TSH ≥ 0.01 to 0.39 mIU/L) and overt hyperthyroidism (TSH below 0.01 mIU/L)
• During treatment with glucocorticoids, amiodarone, and with dopamine agonists (TSH ≥ 0.01 mIU/L).

30.2.5.2 Elevated TSH

Latent (subclinical) hypothyroidism is defined as TSH level of 4.0–10.0 mIU/L and normal FT4 concentration /6/. The presence of thyroid antibodies predicts the risk of progression from subclinical to overt hypothyroidism. Subclinical and overt hypothyroidism are associated with increased risk of coronary heart disease, particularly with TSH levels of ≥ 10 mIU/L. Overt hypothyroidism is defined as TSH level ≥ 20 mIU/L and decreased FT4 concentration. Hypothyroidism is associated with dyslipidemia, especially increased total cholesterol and LDL cholesterol.

30.2.6 Comments and problems

Quality criteria are required for TSH assays; the most important criteria are listed in Tab. 30.2-6 – Quality criteria for TSH assays.

Reference reagent

The recombinant human TSH reference reagent rTSH 94/674 was created under the auspices of the WHO. It contains 6.70 mIU rTSH per ampoule and is recommended to calibrate TSH assays.

Many manufacturers do not use the WHO reference reagent because there are big changes in the reference range compared to ranges used until now. The IFCC Committee for Standardization of Thyroid Function developed a global harmonization approach for TSH measurements. Reference interval measurements were made using a unique calibrator. The reference intervals showed improved uniformity using the new calibrator /8/.

Method of determination

Heterophilic antibodies reacting with anti-mouse antibodies can interfere in two site (sandwich) immunoassays by forming a linkage between the capture and detection antibody, resulting in a falsely high TSH concentration. Interference by heterophilic antibodies is usually eliminated by adding serum from another animal species. For example, if the antibody is a mouse antibody, bovine serum is added, which contains antibodies that bind to and block the heterophilic mouse antibodies.

An alternative method is to create a chimeric antibody. This is a human antibody with a variable region (Fab) of mouse origin and an Fc fragment of human origin. This prevents the binding of anti-mouse antibody directed against the Fc fragment. The use of these tests leads to fewer false TSH results.

TSH levels may be decreased in two-site immunometric assays using biotin streptavidin technology.

Explanation: in a sandwich assay TSH is sandwiched between two different antibodies: one is labeled with the a signal to be measured (antibody labeled with biotin). The other one, named the capture antibody will allow the separation of the immune complexes on a solid phase mediated by streptavidin. The higher the concentration of TSH, the higher the signal linked to the solid phase will be. At the end of the incubation not solid phase bound reagent is separated from the reaction milieu. Biotin prevents the binding of the immune complex to streptavidin and will result in a false depressed TSH level in the non competitive sandwich assay. This is the case in patients with high dose of biotin therapy (300 mg/day) e.g., in patients with inherited metabolic disease like propionic acidemia, biotinidase deficiency or in patients with parenteral nutrition /9/.

Influence factors

TSH exhibits a peak between 2:00 a.m. and 4:00 a.m. and a nadir between 4:00 p.m. and 8:00 p.m. /10/. The circadian variation in TSH is absent in infants younger than one month old and in patients with Sheehan’s syndrome. Sleep deprivation and short term untreated hypothyroidism affect nocturnal TSH surge. Circadian TSH rhythm is not affected by treatment of hypothyroidism with either thyroxine or T3 /4/.

Stability

In serum at 4 °C or 18–22 °C for four days, longer if deep-frozen /11/.

References

1. Cooper DS, Biondi B. Subclinical thyroid disease. The lancet 2012; 379: 1142–54.

2. Ladenson PW, Singer PA, Ain KB, Bagchi N, Bigos T, Levi EG, et al. American Thyroid Association guidelines for detection of thyroid dysfunction. Arch Int Med 2000; 160: 1573–5.

3. Spencer CA, Takeuchi M, Kazarosyan M, MacKenzie F, Beckett GF, Wilkinson E. Interlaboratory/intermethod differences in functional sensitivity of immunometric assays of thyrotropin (TSH) and impact on reliability of measurement of subnormal concentrations of TSH. Clin Chem 1995; 41: 367–74.

4. Ehrenkranz J, Bach PR, Snow GL, Schneider A, Lee JL, Ilstrup S, et al. Circadian and circannual rhythms in thyroid hormones: determining the TSH and free T4 reference intervals based upon time of day, age, and sex. Thyroid 2015; 25: 954–61.

5. Bondi B, Cooper DS. Subclinical hyperthyroidism. N Engl J Med 2018; 378: 2411–9.

6. Schübel J, Feldkamp J, Bergmann A, Drossard W, Voigt K. Latent hypothyoidism im adults. Dtsch Arztebl 2017; 114: 430–8.

7. Beckett G, MacKenzie F. Thyroid guidelines – are thyroid-stimulating hormone assays fit for purpose? Ann Clin Biochem 2007; 44: 203–8.

8. Thienpoint LM, van Uytfanghe K, De Grande LAC. Reynders D. Das B, Faix JD, Mac Kenzie F, et al. Harmonization of serum thyroid stimulating hormone measurements paves the way for the adoption of a more uniform reference interval. Clin Chem 217; 63: 1248–60.

9. Piketty ML, Polak M, Flechtner I, Gonzales-Briceno L, Souberbielle JC. False biochemical diagnosis of hyperthyroidism in streptavidin-biotin-based immunoassays: the problem of biotin intake and related interferences. Clin Chem Lab Med 2017; 55: 780–8.

10. Russell W, Harrison RF, Smith N, Darzy K, Shalet S, Weetman AP, Ross RJ. Free triiodothyronine has a distinct circadian rhythm that is delayed but parallels thyrotropin levels. J Clin Endocrinol Metab 2008; 93: 2300–6.

11. Koliakos G, Gaitatzi M, Grammaticos P. Stability of serum TSH concentration after non refrigerated storage. Panminerva Medica 1999; 41: 99–101.

12. Referenzbereiche für Kinder und Erwachsene. Elecsys Schilddrüsen-Tests. Edition Roche Diagnostics, 2004.

13. Hollowell JG, Staehling NW, Flanders WD, Hannon WH, Gunter EW, Spencer CA, Braverman LE. Serum TSH, T4 and thyroid antibodies in the United States population (1988 to 1994): National Health and Nutrition Examination Survey (NHANES III). J Clin Endocrinol Metab 2002; 87: 489–99.

14. Kratzsch J, Fiedler GM, Leichtle A, Brügel M, Buchbinder S, Otto L, et al. New reference intervals for thyrotropin and thyroid hormones based on National Academy of Clinical Biochemistry criteria and regular ultrasonography of the thyroid. Clin Chem 2005; 51: 1480–6.

15. Chatterjee S, O’Malley BP, Price DE, Fielding AM, Aitken R. Low but detectable serum thyroid-stimulating hormone concentrations in ambulant subjects not receiving thyroxine. Ann Clin Biochem 2003; 40: 639–42.

16. Boelaert K. The association between serum TSH concentration and thyroid cancer. Endocrine-related cancer 2009; 16: 1065–72.

17. Haymart MR, Repplinger DJ, Leverson GE, Elson DF, Sippel SJ, Jaume JC, et al. High serum thyroid stimulating hormone level in thyroid nodule patients is associated with greater risks of differentiated thyroid cancer and advanced tumor stage. J Clin Endocrinol Metab 2008; 93: 809–14.

18. Leger J, Olivieri A, Donaldson M, Torresani T, Krude H, van Vliet G, et al. European Society for Paediatric Endocrinology Consensus Guidelines on Screening, Diagnosis, and Management of Congenital Hypothyroidism. J Clin Endocrinol Metab 2014; 99: 363–84.

19. Marucci G, Faustini-Fustini M, Righi A, Pasquini E, Frank G, Agati R, Foschini MP. Thyrotropin-secreting pituitary tumours: significance of atypical adenomas in a series of 10 patients and association with Hashimoto thyroiditis as a cause of delay in diagnosis. J Clin Pathol 2009; 62: 455–9.

20. Alexander EK, Pearce EN, Brent GA, Brown RS, Chen H, Dosiou C, et al. 2017 Guidelines of the American THyroid Association for the Diagnosis and Management of Thyroid Disease During Pregnancy and the Postpartum. Thyroid 2017; 27: 315–89.

21. Rotondi M, Leporati P, La Manna A, Pirali B, Mondello T, Fonte R, et al. Raised serum TSH levels in patients with morbid obesity: is it enough to diagnose subclinical hypothyroidism? Eur J Endocrinol 2009; 160: 403–8.

22. Heinze HG. Diskrepanz zwischen peripheren Schilddrüsenhormonwerten und TSH-Spiegel. Dtsch Med Wschr 1995; 120: 1639–40.

23. Feldkamp J, Röher HD, Scherbaum WA. Rezidivprophylaxe und medikamentöse Therapiestrategien nach Operation an der Schilddrüse. Dtsch Ärztebl 1998; 95: A-2324–8.

24. National Academy of Clinical Biochemistry 2004. www.nacb.org/Impg/thyroid_Impg_pub.stm

25. Arafah BM. Increased need for thyroxine in women with hypothyroidism during estrogen therapy. N Engl J Med 2001; 344: 1743–9.

26. Bschor T, Bauer M. Schilddrüsenfunktion bei Lithiumbehandlung. Nervenarzt 1998; 60: 189–95.

27. Somwaru LL, Arnold AM, Joshi N, Fried LP, Cappola AR. High frequency of and factors associated with thyroid hormone over-replacement and under-replacement in men and women aged 65 and over. J Clin Endocrinol Metab 2009; 94: 1342–5.

28. Hallengren B, Lantz M, Andreasson B, Grennert L. Pregnant women on thyroxine substitution are often dysregulated in early pregnancy. Thyroid 2009; 19: 391–4.

29. Yandell SD, Harvey WC, Fernandes RJ, Barr PW, Feldman M. Radioiodine studies, low serum thyrotropin, and the influence of statin drugs. Thyroid 2008; 18: 1039–42.

30. Mills F, Jeffery J, Mackenzie P, Cranfield A, Ayling RA. An immunoglobulin G complexed form of thyroid-stimulating hormone (macro thyroid-stimulating hormone) is a cause of elevated serum thyroid-stimulating hormone concentration. Ann Clin Biochem 2013; 50: 416–20.

31. Marqusee E, Haden ST, Utinger RD. Subclinical thyrotoxicosis. Endocrin Metab Clin North Am 1998; 27: 37–49

30.3 Thyroid hormones

TSH determination is the primary investigation used to diagnose disorders of thyroid function. The TSH level provides an assessment of the biological effects of triiodothyronine (T3) in the tissues while the concentrations of thyroxine (T4) and T3 are indicators of thyroid secretory function. Hyperthyroidism is characterized by excessive secretion of T4 and/or T3 while hypothyroidism is caused by a deficiency of these hormones. In subclinical hyperthyroidism or hypothyroidism, the TSH level is abnormally low or high, while the concentrations of T4 or T3 are still within the reference interval.

T4 and T3 circulate in the plasma as free (unbound) hormones or bound to one of the following binding proteins: thyroxine binding globulin (TBG), transthyretin (also known as thyroxine binding pre albumin), and albumin. The protein bound and unbound forms are together referred to as “total” hormone (TT4, TT3) whereas the unbound hormone is referred to as “free” hormone (FT4 and FT3). The free hormone is the biologically active form and correlates most closely with thyroid function.

The kinetics of T3 metabolism differ from those of T4 because, compared to T4, it has a 10–15 times lower affinity for TBG. As a result, only 0.02–0.03% of T4 exists in free form while 0.2–0.3% of T3 is unbound. Approximately 75% of T4 is bound to TBG while 10–20% is bound to transthyretin and albumin respectively. T3 shows a similar binding behavior, however, a smaller proportion is bound to transthyretin. Less than 5% of each hormone is bound to lipoproteins. If there is a change in the concentration of TBG, a temporary shift from bound to free hormone occurs. However, because of the fixed set point in the hypothalamic-pituitary-thyroid axis, the concentration of free hormone quickly returns to its previous value. When free hormone is metabolized, bound hormone dissociates immediately from the binding protein because it is bound non covalently. Dissociation also occurs when serum is diluted in vitro, resulting in a decrease in the concentration of free hormone /1/.

T4 can be seen as a pro hormone of T3 because the effects of both thyroid hormones at molecular level in the hypothalamus and peripheral tissues are mediated by nuclear T3 receptors only. All T4, but only 15–20% of T3, is secreted by the thyroid. The remaining T3 is produced from T4 in the liver, kidneys, and other peripheral organs.

The daily production rate of T3 is approximately half that of T4 and the serum level of FT3 is 3–4 times lower than that of FT4 (5 compared to 20 fmol/L). The conversion of T4 to T3 is catalyzed by deiodinase type 1 in the peripheral tissues and deiodinase type 2 in the hypothalamic-pituitary system. Because the Michaelis constant of deiodinase type 2 is 1,000 times lower than that of deiodinase type 1, it can be assumed that more T4 is converted to T3 per cell in the pituitary than in the peripheral tissues. Circulating T4 has a half life of 6.7 days, while the half life of T3 is only 0.75 days. T3 exhibits a circadian rhythm while T4 does not /2/.

Based on the fact that the free hormone fraction is the biologically active fraction, FT4 and FT3 determinations are usually used to assess thyroid function.

30.3.1 Free T4 (FT4), total T4 (TT4)

Approximately 100% of circulating T4 is secreted by the thyroid.

30.3.1.1 Indication

Free thyroxine

• Further investigation in patients with TSH levels outside the reference interval
• Suspected thyroid disorder in critically ill patients (in conjunction with TSH determination)
• Suspected latent (subclinical) hypothyroidism or hyperthyroidism when TSH is elevated or reduced
• Conditions that suggest deregulation of the hypothalamic-pituitary-thyroid axis
• Initially after treatment of hyperthyroidism (as the secretion of TSH may be suppressed during weeks to months after treatment)
• Monitoring of dose titration in L-thyroxine treatment.

Total thyroxine (TT4)

• Discordance between TSH and FT4 results.

30.3.1.2 Method of determination

Serum T4 and T3 levels reflect not only hormone production, but also the concentrations of thyroid hormone binding proteins. In the presence of elevated levels of binding proteins, TT4 and TT3 are usually increased but FT4 and FT3 levels remain normal.

TT4

Principle: competitive immunoassay with enzyme, luminescent, and fluorescent labels. In the heterogenous competitive assay, the T4 of the sample and labeled T4 (e.g., enzyme labeled T4) compete for the limited quantity of anti-T4 antibodies of the test reagent. The higher the T4 level in the patient sample, the lower the amount of enzyme labeled T4 is antibody bound. Patient T4 antibody complexes and enzyme labeled T4 antibody complexes bind to a second, solid phase bound capture antibody. Substrate is added to measure the activity of the enzyme. The amount of capture antibody bound enzyme labeled T4 is inversely proportional to the concentration of T4 in the sample. The same principle is used for the determination of T3.

FT4

FT4 measurement methods are classified as direct or indirect /25/.

• Direct methods employ physical separation of FT4 from the protein-bound thyroxine using e.g., equilibrium dialysis or filtration techniques. Equilibrium dialysis is the most reliable method (reference method). The method separates FT4 from protein bound thyroxine before direct measurement and uses undiluted serum as a retentive. The FT4 is then measured, usually by liquid chromatography with tandem mass spectrometry.
• Indirect methods are used in clinical laboratories and measure FT4 on automated immunoassay platforms, which utilize different competitive assay formats to selectively measure FT4 without disrupting the protein bound thyroxine. The principle of one step analog immunoassay is as follows: a chemically modified T4 analog replaces labeled T4 as competitive molecule. The T4 analog binds with a high avidity to the thyroxine antibody but with a lower avidity as native serum thyroxine to the binding proteins of the serum. For standardization of FT4 immunoassays refer to Ref. /25/.

30.3.1.3 Specimen

Serum: 1 mL

30.3.1.4 Reference interval

Refer to Tab. 30.3-1 – Reference intervals for T4 and FT4.

30.3.1.5 Clinical significance

After diagnosis of TSH, the result of FT4 is the basic parameter used to assess thyroid hormone secretion since it reflects hormone production by the thyroid directly. The clinical assessment of FT4 and TT4 is shown in Tab. 30.3-2 – Assessment of FT4 and TT4 levels.

Because FT4 is not affected by inter individual and intra individual variations in the concentrations of binding proteins, it has a higher diagnostic significance than TT4. FT4 represents the metabolically reactive fraction of serum T4 as well as current hormone production (thyroid secretion, extra thyroidal conversion of T4 to T3) and elimination (extravascular transport, metabolism, renal elimination).

FT4 has better discriminatory power than TT4 at the limits of the reference interval, which is why TT4 determination is only necessary in certain patients, to distinguish between thyroid related and non-thyroidal elevations, for example.

Primary hyperthyroidism must not be diagnosed on the basis of elevated FT4 and/or FT3 unless TSH is also suppressed (< 0.01 mIU/L).

Subclininical hypothyroidism is not diagnosed on the basis of low FT4 unless TSH is significantly elevated. An excessive TSH increase and low FT4 indicates overt hypothyroidism.

If there is discordance between FT4 and TSH values in a patient with non-thyroidal illness (NTI), TT4 should also be determined to establish whether a thyroid disorder is present. A thyroid disorder is more likely if FT4 and TT4 behave similarly. Discordance between FT4 and TT4 is usually caused by NTI /6/.

Tab. 30.3-3 – Diseases and conditions associated with changes in FT4 and/or TT4 lists some conditions and diseases associated with changes in FT4 and T4 levels.

30.3.1.6 Comments and problems

Method of determination

Immunoassays from 9 manufacturers were compared with a candidate reference measurement procedure (cRMP) based on equilibrium dialysis isoptope dilution-mass spectrometry (ED-ID-MS) for determining FT4 and FT3. For FT4 (FT3) the mean bias of 2 (4) assays was up to minus 42% (minus 30%). After recalibration to the cRMP eliminated assay specific biases approximately half of the manufacturers matched the internal quality control targets within about 5%; however, within run instability was observed /7/.

Dilution of serum samples leads in most immunoassays to a reduction in the FT4 concentration compared to equilibrium dialysis. At a dilution factor of 10, measured values are reduced by 20–80% /8/.

Individual variation

Throughout the course of a year, the FT4 concentration can vary by ± 25% and the TSH level can vary by ± 50%. This is due to seasonal variation in the pituitary set point for TRH secretion /9/.

Autoantibodies against T4

Thyroid hormone autoantibodies are directed against T4 and T3 and interfere with the determination of FT4 and FT3 in 1.2% of cases /10/. They interfere with competitive solid phase immunoassays in which an inverse relationship exists between the FT4 concentration of the sample and labeled T4.

In the absence of interfering factors, T4 from the sample and labeled T4 from the test reagent compete for the limited number of T4 specific antibodies bound to the solid phase. In the presence of T4 autoantibodies, however, some of the T4 and labeled T4 may bind to the autoantibody rather than to the solid phase. This results in a falsely low measurement signal that indicates an increased T4 concentration in the sample.

Stability

In serum and plasma at room temperature (22 °C) for 24–48 h; deep-frozen for more than a year.

30.3.2 Total T3 (TT3), free T3 (FT3)

Approximately 80% of circulating T3 is produced by peripheral conversion of T4 and only 20% is secreted directly by the thyroid gland. In plasma, T3 is bound primarily to TBG and, with lower affinity, to transthyretin. FT3 has five times the metabolic activity of FT4, while rT3 has less than 5% of the metabolic activity of FT4.

30.3.2.1 Indication

• Suspicion of T3 hyperthyroidism in cases with suppressed TSH and normal FT4 concentration
• Further evaluation of patients with subclinical hyperthyroidism
• Suspicion of non-thyroidal illness
• Prognostic assessment of the treatment of patients with Graves’ disease
• Evaluation of the severity of primary hypothyroidism
• Monitoring of T3 replacement therapy

30.3.2.2 Method of determination

Refer to T4/FT4.

30.3.2.3 Specimen

Serum: 1 mL

30.3.2.4 Reference interval

Refer to Tab. 30.3-4 – Reference intervals for TT3 and FT3.

30.3.2.5 Clinical significance

Approximately 99.8% of T3 in the serum is bound to proteins and is determined as TT3. Only 0.2–0.3% exists in free, metabolically active form (FT3). Because T3 binds to proteins with 10 times lower affinity than T4, the determination of TT3 and FT3 are of roughly equal diagnostic value /11/.

However, because its protein binding affinity is 10 times lower than that of T4, T3 is affected to a much lesser degree by changes in protein binding capacity. As a result, FT3 does not have the same importance compared to TT3 that FT4 has compared to TT4.

The advantages of determining FT3 rather than TT3 are limited and the secretory performance of the thyroid can demonstrated just as reliably by TT3 as by FT3.

The TT3 and FT3 levels reflect the conversion of T4 to T3 in the tissues, and to a lesser extent, thyroid secretion of T3, which accounts for only 20% in euthyroid individuals. This is not the case in autoimmune hyperthyroidism. In Graves’ disease, regardless of whether thyrotoxicosis is caused by T4 or by T3 or T4-T3, 33 ± 6% of the T3 is secreted by the thyroid /12/.

The conversion rate may be reduced, leading to reduced TT3 and FT3 concentrations:

• In the critically ill, in non-thyroidal illness, or low T3 syndrome, due to inhibition of deiodinase
• Due to drugs such as glucocorticoids, propranolol, and amiodarone
• In the elderly, in whom mild hyperthyroidism can therefore be missed.

In iodine deficiency, a compensatory increase in TT3 and FT3 can occur. TT3 and FT3 are also of limited value in the diagnosis of hypothyroidism, since the levels of both are maintained within the lower reference interval by decreased secretion of T4 with subsequent conversion to T3.

TT3 or FT3 determination is clinically relevant:

• In patients with subclinical hyperthyroidism who have suppressed TSH level and normal FT4 concentration; these patients are at increased risk for the development of overt hyperthyroidism
• For the detection of isolated T3 over secretion (T3 thyrotoxicosis), which accounts for up to 10% of all cases of hyperthyroidism.
• In critical ill patients with non-thyroidal illness TT3 and to a lesser extent FT3 levels are decreased; in critical ill patients with hyperthyroidism therefore TT3 and FT3 concentration may be minimally increased or normal
• In the early stage of thyroid hyper function, especially in the case of autonomy
• For diagnosing recurrent hyperthyroidism, since T3 elevation may be an early symptom
• To exclude factitious hyperthyroidism caused by L-thyroxine treatment.

Progressive central fat accumulation in individuals with a BMI greater than 25 kg/m² is associated with an increase in FT3. The FT4 to FT3 ratio also correlates with waist circumference, blood pressure, fasting plasma glucose, cholesterol and LDL cholesterol concentration /13/.

The clinical significance of TT3 and FT3 levels is shown in Tab. 30.3-5 – Assessment of TT3 and FT3 levels.

30.3.2.5.1 Levothyroxine treatment

Levothyroxine (LT4), the linchpin of thyroid hormone replacement therapy, is a very commonly prescribed drug. In the Rhineland Study 23% of the participants stated that they were taking LT4 /28/. Among these participants overtreatment (TSH concentration < 0.26 mU/L) and undertreatment (TSH concentration > 4.27 mU/L) were studied. Among these participants 18% were overtreated and 4% were undertreated. Participants over 70 years of age and above were four times as likely to be overtreated.

30.3.2.6 Low T3 syndrome

The evaluation of thyroid function in critical illness remains complex because the changes occur at all levels of the hypothalamic-pituitary-thyroid axis. During illness, a decrease in T3 and pulsatile TSH release and increase in rT3 occur. This constellation of findings is termed the euthyroid sick syndrome or non-thyroidal illness or the low T3 syndrome /14/. Low T3 syndrome is the most common manifestation in non-thyroidal illness and due to inhibition of 5’deiodinase which catalyzes the conversion of T3 from T4. A study /15/ showed that low T3 syndrome was highly prevalent in chronic kidney disease (CKD). Furthermore, serum T3 levels were associated with the severity of CKD even in the normal TSH level (Tab. 30.3-6 – Prevalence of low T3 in chronic kidney disease).

30.3.2.6.1 T3 and FT3 for treatment monitoring

If thyroid hormone overdosage is suspected during L-thyroxine treatment for goiter or following thyroid surgery, FT3 can be determined in addition to TSH.

During the initial phase of L-thyroxine treatment for hypothyroidism, the FT3 concentration is lower on average than the FT4 concentration. The FT3 level is important in terms of preventing overdosage if TSH declines to the lower reference interval value when the thyroxine dose is increased. The FT3 should remain within the reference interval.

At the start of thyrostatic therapy for hyperthyroidism, the FT3 concentration increases due to a compensatory increase in the production of T3, which contains one less iodine atom per molecule than T4. Blood collection should take place at least 12 hours, but preferably 24 hours, after the last dose.

30.3.2.7 Comments and problems

Reference interval

Depending on the study, FT3 and TT3 levels are 10–50% lower in older than in younger individuals. These lower values, which are thought to result from decreased conversion of T4 to T3, are observed in men from the age of 60 years; in women, levels decline progressively from the age of 70 years.

Method of determination

Refer to TT4 and FT4 and to Ref. /24/.

Dilution of the sample

Dilution disrupts the free to bound ratio equilibrium of T4.

Circadian rhythm

Like TSH, FT3 exhibits a circadian rhythm. However, the peak and nadir are reached 0.5–2.5 hours later than those of TSH /2/.

Questionable high free T4 (FT4 ) concentration

In euthyroid patients by history and examination, and despite normal TSH persistently elevated free T4 (FT4) is measured. In this situation the laboratorians chose to repeat FT4 measurement on a different assay platform to evaluate the presence of an assay-specific interfering substance. When the second immunoassay also generates a higher than expected result, a variant TT4-binding protein affecting both assays is suspected. Because one-step automated FT4 immunoassays use a competitive format, decreased signal intensity is assumed to reflect the displacement of TT4 in the assay reagent by endogenous free hormone. In patients having a mutation in albumin consistent with the autosomal dominant condition familial dysalbuminemic hyperthyroxinemia (FDH) the mutation leads to increased T4 binding affinity to albumin, resulting in a higher TT4 “set point” in the hypothalamic-pituitary-thyroid axis to maintain the FT4 within the normal range. One step immunoassays are prone to false increases in measured FT4 in patients with FDH given that the mutant albumin binds with excess affinity to the labeled TT4 analog that is added to the patient's serum /26, 27/.

30.3.2.8 Reverse T3 (rT3)

When there is a reduced need for active thyroid hormone, the body can regulate the biotransformation of T4 into T3 by promoting the mono deiodination of T4 to inactive 3,3’,5’-triiodothyronine (rT3) (Fig. 30.7-5 – Pathways of thyroid hormone metabolism). The plasma rT3 concentration indirectly reflects the conversion of T4 to T3 in the tissues. It is also an indirect measure of the metabolism of thyroid hormones to diiodothyronine.

30.3.2.9 Indication

Determination of the cause of an inexplicably low FT4, T3, or FT3 concentration.

30.3.2.10 Method of determination

Radioimmunoassay

30.3.2.11 Specimen

Serum: 1 mL

30.3.2.12 Reference interval

Conversion: μg/L × 1.54 = nmol/L

30.3.2.13 Clinical significance

In severe non-thyroidal illness, the decline in TT3 or FT3 is associated with an almost identical increase in rT3. Low T3 syndrome occurs during the neonatal period, in seriously ill adults, during fasting and low carbohydrate intake, in liver disease, and as an effect of various drugs such as corticosteroids, antiarrhythmics, and beta blockers. A decline in TT4 is seen only in very severe non-thyroidal illness and FT4 is normal or even increased for a long time. rT3 can therefore be seen as a regulator of T4 deiodination /16/. Refer to Tab. 30.1-2 – Adaptive response to non-thyroidal illness.

The ratio of T3 to rT3 can be of value in identifying clinical deterioration in severe non-thyroidal illness and in monitoring treatment outcome. At present, measurement of the rT3 concentration does not have a role in routine thyroid investigations.

References

1. Roelfsema F, Veldhuis JD. Thyrotropin secretion patterns in health and disease. Endocrine Rev 2013; 34: 619–57.

2. Russell W, Harrison RF, Smith N, Darzy K, Shalet S, Weetman AP, Ross RJ. Free triiodothyronine has a distinct circadian rhythm that is delayed but parallels thyrotropin levels. J Clin Endocrinol Metab 2008; 93: 2300–6.

3. Liappis N, Schlebusch H, von Perjés M, Berg I. Referenzwerte für die Konzentration des freien Thyroxins, des freien Trijodthyronins und des Thyroxin-bindenden Globulins im Blutserum euthyreoter Kinder. Klin Pädiat 1991; 203: 113–5.

4. Referenzbereiche für Kinder und Erwachsene. Elecsys Schilddrüsen-Tests. Edition Roche Diagnostics, 2004.

5. Midgley JEM. Direct and indirect free thyroxine assay methods: theory and practice. Clin Chem 2001; 47: 1353–63.

6. Glinoer D. The regulation of thyroid function during normal pregnancy: importance of the iodine nutrition status. Best Practice & Research Clinical Endocrinol & Metab 2004; 18: 133–52.

7. Thienpont LM, van Uytfanghe K, Beastall G, Faix JD, Ieiri T, Miller WG, et al. Report of the IFCC working group for standardization of thyroid function tests; part 2: free thyroxine and free triiodothyronine. Clin Chem 2010; 56: 912–20.

8. Sapin R. Serum thyroxine binding capacity-dependent bias in five free thyroxine immunoassays: assessment with serum dilution experiments and impact on diagnostic performance. Clin Biochem 2001; 34: 367–71.

9. Andersen S, Pedersen KM, Bruun NH, Lauberg P. Narrow individual variants in serum T4 and T3 in normal subjects: a clue to the understanding of subclinical thyroid disease. J Clin Endocrinol Metab 2002; 87: 1068–72.

10. Gledenning P, Siriwardhana D, Hoad K, Musk A. Thyroxine autoantibody interference is an uncommon cause of inappropriate TSH secretion using the Immulite 2000 assay. Clin Chim Acta 2009; 403: 136–8.

11. Klee GG. Clinical usage recommendations and analytic performance goals for total and free triiodothyronine measurements. Clin Chem 1996; 42: 155–9.

12. Woeber KA. Triiodothyronine production in Grave’s hyperthyroidism. Thyroid 2006; 16: 687–90.

13. De Pergola G, Ciampolillo A, Paolotti S, Trerotoli P, Georgino R. Free triiodothyronine and thyroid stimulating hormone are directly associated with waist circumference, independently of insulin resistance, metabolic parameters and blood pressure in overweight and obese women. Clin Endocrinol 2007; 67: 265–9.

14. The National Academy of Clinical Biochemistry. Laboratory Medicine Practice Guidelines for the Diagnosis and Monitoring of Thyroid Disease. NACB edition.

15. Song SH, Kwak IS, Lee DW, Kang YH, Seong EY, Park JS. The prevalence of low triiodothyronine according to the stage of chronic kidney disease in subjects with a normal thyroid-stimulating hormone. Nephrol Dial Transplant 2009; 24: 1534–8.

16. Vogeser M, Jacob K. Measurement of free triiodothyronine in intensive care patients – comparison of two routine methods. Eur J Clin Chem Clin Biochem 1997; 35: 873–5.

17. Kratzsch J, Fiedler GM, Leichtle A, Brügel M, Buchbinder S, Otto L, et al. New reference intervals for thyrotropin and thyroid hormones based on National Academy of Clinical Biochemistry criteria and regular ultrasonography of the thyroid. Clin Chem 2005; 51: 1480–6.

18. Grüning T, Zöphel K, Wunderlich G, Franke WG. Influence of female sex hormones on thyroid parameters determined in a thyroid screening. Clin Lab 2007; 53: 547–53.

19. Arafah BM. Increased need for thyroxine in women with hypothyroidism during estrogen therapy. N Engl J Med 2001; 344: 1743–9.

20. Kahric-Janicic N, Soldin SJ, Soldin OP, West T, Gu J, Jonklaas J. Tandem mass spectrometry improves the accuracy of free thyroxine measurements during pregnancy. Thyroid 2007; 17: 303–11.

21. Fischer DA. Euthyroid low thyroxine (T4) and triiodothyronine (T3) states in prematures and sick neonates. Pediat Clin North Am 1990; 37: 1297–1312.

22. Centanni M, Gargano L, Canettieri G, Viceconti N, Franchi A, Delle Fave G, et al. Thyroxine in goiter, Helicobacter pylori infection, and chronic gastritis. N Engl. J Med 2006; 354; 1787–95.

23. Kahaly GJ, Dietlein M, Gärtner R, Mann K, Dralle H. Amiodaron und Schilddrüsenfunktion. Dt Ärztebl 2007: 104: A 3550–5.

24. Uy HL, Reasner II CA. Elevated thyroxine levels in a euthyroid patient. Postgrad Med 1994; 96: 195–202.

25. Kratzsch J, Baumann NA, Ceriotti F, Lu ZX, Schott M, van Herwaarden AE, et al. Global FT4 immunoassay standardization: an expert opinion review. Clin Chem Lab Med 2021; 59 (6): 1013–23.

26. Crawford AR. Commentary on questionable high free T4 concentrations: when confirming against an alternative method is not enough. Clin Chem 2022; 68 (9): 1132.

27. Nerenz RD. Commentary on questionable high free T4 concentrations: when confirming against an alternative method is not enough. Clin Chem 2022; 68 (9): 1133.

28. Jongejan AN, Stingl RM, de Rijke YB, Peeters RP, Breteler RM, de Vries FM. Over- and undertreatment with Levothyroxin: Findings of the population based Rhineland study. Dtsch Arztebl 2023; 120: 731–8.

30.4 Thyroxin-binding globulin (TBG)

TBG is the most important plasma transport protein for thyroid hormones. It is a 54 kDa glycoprotein, consisting of 395 amino acids and four asparagine linked oligosaccharide chains. The carbohydrate component is thought not to influence immunogenicity and T4 binding. The TBG gene is located on the long arm of the X chromosome and is composed of five exons.

With the exception of its functions as a storage and transport protein for T4 and T3 and an inhibitor of their renal elimination, little is known about the physiological role of TBG /1/.

30.4.1 Indication

• Unexplained discordance between TSH and TT4 and/or FT4 serum levels.
• Unexplained discordance between TT4 and FT4 serum levels.
• Markedly increased or decreased serum TT4.
• Suspected congenital TBG deficiency.

30.4.2 Method of determination

Radioimmunoassay or enzyme immunoassay.

30.4.3 Specimen

Serum: 1 mL

30.4.4 Reference interval

Conversion: mg/L × 17 = nmol/L

30.4.5 Clinical significance

TBG determination may be necessary to assess thyroid function if there is discordance between the levels of TSH and TT4 or FT4.

The assessment criterion used in this case is the ratio of T4 to TBG (Tab. 30.4-1 – Assessment of thyroid function using the T4/TBG ratio).

Hepatic synthesis of TBG is increased during pregnancy or by the use of estrogen containing oral contraceptives.

Congenital TBG disorders include partial or complete TBG deficiency and TBG excess. Complete deficiency, caused by a nucleotide deletion at codon 352 has been described in a number of Japanese families. The TBG molecule is truncated and retained within the endoplasmic reticulum. The incidence of TBG deficiency in the Japanese population is estimated to be 0.09% /2/.

References

1. Horn K, Kubiczek T, Pickardt CR, Scriba PC. Thyroxine-binding globulin (TBG): preparation, radioimmunoassay and clinical significance. Klin Wschr 1977; 55: 881–94.

2. Noguchi T, Miki T, Takamatsu J, Nakajima T, Kumahara Y. Mass screening for complete deficiency of thyroxine-binding globulin in adult Japanese by comprehensive health examination. Internal Medicine 1996; 35: 266–9.

30.5 Thyroglobulin

Thyroglobulin (Tg) is a water soluble glycoprotein that consists of two subunits, each with a molecular weight of 300 kDa (Section 28.22 – Thyroglobulin). It is the most important precursor in the biosynthesis of thyroid hormone. Of the 134 tyrosine residues in the Tg molecule, less than one-fifth are iodized. Following its synthesis, Tg is stored in the thyroid follicles. TSH stimulates the release of Tg into the circulation, where it has a half life of 3–65 hours and from where it is subsequently eliminated /1/.

Each Tg subunit has only two autoantigenic epitopes. The autoantibodies produced consist mainly of IgG type. In Hashimotos thyroiditis, the predominant autoantibody is IgG₂, whereas in Graves’ disease, thyroid cancer, and non-thyrotoxic goiter, the predominant autoantibody type is IgG₄.

Papillary and follicular thyroid carcinomas belong to the differentiated thyroid carcinoma family and account for many endocrine cancer cases. Traditionally, modest differentiated cancers are treated by total thyroidectomy followed by radioiodine therapy and, with the resultant elimination of benign residual sources of thyroglobulin secretion, the stage was set for using serum thyroglobulin measurement as a pivotal tool in differentiated thyroid cancer follow-up /2/.

The prognostic role of thyroglobulin in predicting recurrence in differentiated thyroid cancer patients treated by lobectomy is controversial. Circulating thyroglobulin was found non-reliable in assessing early response and predicting recurrence in patients with hemithyroid ectomy, especially those with a low initial classification according to the American Thyroid Classification. In a study circulating thyroglobulin was found non-reliable in assessing early response and predicting recurrence in patients with hemithyroidectomy, especially those with a low initial classification of the American Thyroid Association /2/. Small thyroglobulin concentrations produced by persistent or recurrent cancer tissue may be easily obscured in the presence of a thyroid lobe producing significant thyroglobulin amounts, considering that the remaining thyroid lobe produces about half of the thyroglobulin produced by the intact thyroid gland (i.e., 10–40 ng/mL) /2/.

References

1. Schott M, Seissler J, Scherbaum WA. Diagnostik bei autoimmunen Schilddrüsenerkrankungen. J Lab Med 2006; 34: 254–7.

2. Giovanella L, Ceriani L, Garo ML. Is thyroglobulin a reliable biomarker of differentiated thyroid cancer in patients treated by lobectomy? A sytematic review and metaanalysis. Clin Chem Lab Med 2022; 60 (7): 1091–1100.

30.6 Autoimmune thyroid diseases

Autoimmune endocrine disease of the thyroid can result in either underactivity or over activity of the gland and may be associated with autoimmune disease of other endocrine glands such as the adrenals, pancreatic islet cells, and ovaries. The most common autoimmune endocrine diseases are type 1 diabetes (T1D) and autoimmune thyroid diseases (AITD). The characteristic features of both disorders are T cell infiltration of target organs and autoantibody production, leading to organ dysfunction and destruction. Often, a patient may have more than one autoimmune endocrine disease at the same time. This is known as polyglandular autoimmune syndrome. T1D and AITD are the most commonly associated diseases. Up to 20% of patients with T1D have thyroid autoantibodies and half of these will develop AITD in addition. Conversely, 2.3% of children with AITD have islet cell autoantibodies /1/.

T1D and AITD share specific HLA associations:

• A positive association of Graves’ disease with DR3, but a negative association with DR5
• A positive association of Graves’ disease with DQA1*0501 in linkage disequilibrium with DQB1*0201 in DR3 haplotypes
• A positive association of Hashimoto’s thyroiditis with HLA DQB1*301.

Autoimmune thyroid diseases, including Graves’ disease and Hashimotos’ thyroiditis, are archetypal organ-specific autoimmune diseases. Graves’ disease is the common cause of hyperthyroidism and is characterized by the presence of anti-thyrotropin receptor antibody (TRAb), Hashimotos thyroiditis is the most common cause of hypothyroidism and is characterized by the presence of anti-thyroglobulin antibody and/or anti-thyroid peroxidase antibody and thyroid tissue destruction by infiltrating lymphocytes. Thyroid hyper function or hypofunction is the result of hereditary factors, inadequate iodine intake, pregnancy, radiotherapy, viral infection, surgery, invasive diseases, or autoimmunity. The term AITD refers to both autoimmune hyperthyroidism (Graves’ disease) and autoimmune hypothyroidism (Hashimotos thyroiditis).

According to a systematic review of the literature, the incidence of AITD per 100,000 individuals varied /2/:

• The reported incidence of autoimmune hypothyroidism varied between 2.2/100,000/year (males) and 498.4/100,000/year (females).
• The reported incidence for autoimmune hyperthyroidism ranged from 0.70/100,000/year (black males) to 99/100,000/year (Caucasian females).

The prevalence of hypothyroidism has been found to be between 0 and 7.8/1,000 men and between 2.0 and 19.4/1,000 women /2/.

The three most important antigens involved in thyroid autoimmunity are:

• Thyroglobulin (Tg), against which thyroglobulin antibodies (anti-TgAb) are produced
• Thyroid peroxidase (TPO), against which thyroid peroxidase antibodies (anti-TPOAb) are produced
• The TSH receptor (TR), against which TSH receptor antibodies (TRAb) are produced.

Thyroid autoantibodies of any kind inform the physician concerning the etiology of the thyroid disease, but these measurements do not substitute for a thorough history and physical examination and measurements of TSH, FT4 and FT3. Anti-TPOAb and/or anti-TgAb are common in patients with Hashimotos’ thyroiditis and in patients with Graves’ disease, but are not diagnostic of Graves’ disease. If the diagnosis of Graves’ disease is questionable in the setting of hyperthyroidism, a measurement of TRAb can be helpful /3/.

The prevalence of autoantibodies in the healthy population and in patients with autoimmune thyroid disease shows geographical variation. Some of this variation is explained by differences in detection limit and cutoff value (30–100 kIU/L) used when determining anti-TgAb and anti-TPOAb. Thus, according to a clinical study /4/, the prevalence of autoimmune thyroid disease was 0.2% in men and 2% in women, while the prevalence of subclinical autoimmune thyroid disease determined on the basis of anti-TgAb and anti-TPOAb was 10 times higher.

Other investigators have found that although 98% of serum samples that are positive for anti-TgAb are also positive for anti-TPOAb, only 65% of anti-TPOAb-positive sera also show an increased anti-TgAb concentration /5/. Therefore, samples should only be screened for anti-TgAb if Tg determination is undertaken in patients with differentiated thyroid cancer (Section 28.22 – Thyroglobulin).

Low concentrations of anti-TgAb and anti-TPOAb are present in the healthy population. However, these autoantibodies belong to the group of natural antibodies that have low affinity for a broad spectrum of antigens.

Diagnostic algorithms are shown in:

• Fig. 30.6-1 – Diagnosis of autoimmune thyroiditis
• Fig. 30.6-2 – Diagnosis of Graves’ disease.

30.6.1 Thyroglobulin antibodies (anti-TgAb)

Thyroglobulin (Tg) is a water soluble glycoprotein that consists of two subunits, each with a molecular weight of 300 kDa (Section 28.22 – Thyroglobulin)). It is the most important precursor in the biosynthesis of thyroid hormone. Of the 134 tyrosine residues in the Tg molecule, less than one-fifth are iodized. Following its synthesis, Tg is stored in the thyroid follicles. TSH stimulates the release of Tg into the circulation, where it has a half life of 3–65 hours and from where it is subsequently eliminated /4/.

Each Tg subunit has only two auto antigenic epitopes. The autoantibodies produced consist mainly of IgG type. In Hashimotos thyroiditis, the predominant autoantibody is IgG₂, whereas in Graves’ disease, thyroid cancer, and non-thyrotoxic goiter, the predominant autoantibody type is IgG₄.

30.6.1.1 Indication

• Screening of serum from patients with differentiated thyroid cancer in which Tg determination is undertaken
• Monitoring of differentiated thyroid cancer after therapeutic intervention
• Suspected autoimmune thyroiditis with negative anti-TPOAb.

30.6.1.2 Method of determination

Immunoassay

Enzyme immunoassay (EIA), immunochemiluminescence assay (ICMA), or immunometric assay (IMA), calibrated against the MRC (Medical Research Council) reference preparation 65/93 with 1,000 MRC units per ampoule.

30.6.1.3 Specimen

Serum: 1 mL

30.6.1.4 Reference interval

Up to 60 kIU/L /5/ or 100 kIU/L /6/, depending on the manufacturer of the assay.

30.6.1.5 Clinical significance

In a study of the Danish population /5/ using a cutoff value of 60 kIU/L, the prevalence of anti-TgAb was 7.7% in male aged 60–65 years. In female aged 18–22 years the prevalence was 9.1% and increased to 20% in the age of 60–65 years. In regions with adequate iodine intake, normal anti-TPOAb with increased anti-TgAb rarely indicated a functional thyroid disorder, unless thyroid cancer was present.

30.6.1.5.1 Differentiated thyroid cancer

Anti-TgAb have been found to be prognostic in patients with thyroid differentiated cancer, with failing antibody levels associated with low risk of recurrent disease, whereas rising or continously positive antibodies indicated risk for recurrent cancer /7/. They also have been found to predict risk of cancer in thyroid nodules.

The Tg concentration is used to monitor differentiated thyroid cancer after treatment /5/. However, no or only low thyroglobulin (Tg) is measured due to elevated anti-TgAb levels or due to undetected anti-TgAb (i.e., lack of analytical sensitivity of the assay) in 15–30% of patients /8/. Using immunometric assays for the determination of Tg the anti-TgAb interfere with the Tg assay by measuring a falsely low Tg concentration (Section 28.22 – Thyroglobulin). Increased Tg levels due to residual metastatic disease may be overlooked in these patients.

30.6.1.5.2 Autoimmune thyroiditis

Increased anti-TgAb concentrations are measured in 12–30% of patients with autoimmune hyperthyroidism and up to 60–80% of patients with autoimmune thyroiditis (Tab. 30.6-1 – Thyroglobulin antibodies in different disorders) /9/. Because their incidence is much lower than that of anti-TPOAb, anti-TgAb determination has a minor role in the diagnosis and monitoring of autoimmune thyroiditis. However, since the prevalence of isolated anti-TgAb positivity in autoimmune thyroiditis is thought to be approximately 6%, some investigators caution against omitting anti-TgAb determination /10/.

30.6.1.6 Comments and problems

Guidelines recommend that anti-TgAb should be measured concurrently with thyroglobulin when monitoring thyroidectomized thyroid cancer patients to alert the laboratory to the possibility of assay interference /11/.

Method of determination

Despite the availability of an international reference preparation, current anti-TgAb assays show unacceptable variance. Concordance between the assays was only 74% /12/.

30.6.2 Thyroid peroxidase antibodies (anti-TPOAb)

Thyroid peroxidase (TPO) is a membrane bound heme protein with a molecular weight of approximately 100 kDa that catalyzes thyroid hormone synthesis on the apical membrane of follicular cells, where it exists as a dimeric enzyme linked by disulfide bonds. In thyroid hormone synthesis, TPO is involved in the iodination of tyrosine residues and the oxidative coupling of two tyrosine residues to thyroglobulin. It requires iodide and H₂O₂ in order to initiate hormone synthesis. The function of TPO is described in detail in Ref. /13/. TPO can be purified following trypsin treatment of thyroid microsomes. In the past, microsomes were used as antigens to detect TPOAb, which is why the term “microsomal antibodies” is used in older literature.

30.6.2.1 Indication

Recommended indications are /5/:

• TSH increase of unknown etiology
• Goiter of unknown etiology
• Evaluation of suspected polyglandular autoimmune disease
• Family evaluation in established cases of autoimmune thyroid disease
• Risk assessment for the development of hypothyroidism during treatment with drugs affecting thyroid (e.g., lithium salts, amiodarone, interferon-alpha, and interleukin 2)
• Risk assessment for the development of hypothyroidism in Down’s syndrome
• Risk assessment for the development of a functional thyroid disorder during pregnancy and postpartum thyroiditis
• Screening for latent (subclinical) hypothyroidism prior to in vitro fertilization
• Prior to treatment of subclinical hypothyroidism.

30.6.2.2 Method of determination

Immunoassays

Enzyme immunoassay (EIA), immunochemiluminescence assay (ICMA), or immunometric assay (IMA), calibrated against the MRC (Medical Research Council) reference preparation 65/93 with 1,000 MRC units per ampoule.

30.6.2.3 Specimen

Serum: 1 mL

30.6.2.4 Reference interval

Up to 60 kIU/L /5/ or 100 kIU/L /6/, depending on the manufacturer.

30.6.2.5 Clinical significance

Using a cutoff value of 60 kIU/L, the prevalence of anti-TPOAb in the Danish population was 11.3% in men aged 60–65 years and ranged from 12.3% in women aged 18–22 years to 29.7% in women aged 60–65 years /5/. The prevalence was 5.8% in euthyroid blood donors and greater than 98% in autoimmune thyroiditis /5/. TPO is a primary antigen in the initiation of an immune response in the thyroid. Patients with increased anti-TPOAb levels have a significantly increased risk of developing latent or overt hypothyroidism.

High concentrations of anti-TPOAb (> 2,000 kIU/L) were exclusively found in persons with HLA-DR3 or HLA-DR5, the same haplotypes as those associated with thyroid autoimmune diseases /14/.

Tab. 30.6-2 – TPOAb in thyroid diseases describes the behavior of anti-TPOAb in thyroid disorders.

30.6.2.6 Comments and problems

Method of determination

A comparative study of eight kits for anti-TPOAb determination with isotopic, enzymatic and luminescent tracers and with recombinant highly purified TPO showed approximately 6% positive results in control sera. The majority of assays demonstrated a good diagnostic performance /15/.

In autoimmune thyroid disease, anti-TPOAb react mainly with two immunodominant regions (IDR) of TPO, A and B (IDR-A and IDR-B). Some antibodies, however, bind to antigenic determinants outside these regions. Around half of anti-TPOAb bind to IDR-B while the rest react with IDR-A or antigens outside the IDRs. The corresponding distribution in healthy individuals is unknown. It is thought that IDR-A and IDR-B are associated with forbidden detrimental reactions while the non-A and non-B regions are part of the natural antigen reservoir.

30.6.3 TSH receptor antibodies (TRAb)

There are two types of TRAb; thyroid stimulating antibodies (TSAb) and TSH-blocking antibodies (TBAb) /26/. TSAb stimulate thyroid gland and cause Graves’ hyperthyroidism. TBAb block TSH stimulation of thyroid gland and cause hypothyroidism. Both TSAb and TBAb block TSH binding to thyroid cells as TSH receptor antibodies (TRAb).

TRAb has been measured by different assay methods and given various names. Among them, TSAb have been measured as TRAb to diagnose Graves’ disease.

TSAb is measured as a stimulator assay, and TBAb as a TSH blocking assay. It has been generally believed that Graves’ patients have TSAb but do not have TBAb, and that blocking antibody (TBAb) positive patients with hypothyroidism have TBAb but do not have TSAb. But TBAb positive patients with hypothyroidism and Graves’ patients with hyperthyroidism may have both TBAb and TSAb /26/.

30.6.3.1 Indication

TRAb measurements as either TSAb or TBII have the following clinical applications /29/:

• Diagnosis of Graves’ disease in the clinical setting of thyrotoxicosis, diffuse goiter, opthalmopathy (exophthalmos) and dermatopathy (pretibial myxedema)
• Differentiating of Graves’ disease from other forms of hyperthyroidism
• Follow-up of treated Graves’ disease
• In early pregnancy to differentiate gestational thyrotoxicosis from Graves’ disease
• Differentiation the transient hyperthyroid phase of postpartum thyroidism from Graves’ disease
• In cases where the cause of thyrotoxicosis is not clearly apparent.

30.6.3.2 Method of determination

Two different principles are used to assess the level of autoantibodies directed against the TSH receptor (TSHR):

• In the assay binding of serum TRAb to TSHR coated ELISA plate wells is inhibited by a TSHR human monoclonal autoantibody M22 labelled with peroxidase and a signal is then developed by addition of a colour-geneating substrate. The concentration of TRAb in test samples is inversely proportional the signal in the assay /27, 29/.
• Bioassays e.g., TSHR stimulating immunoglobulin (TSI) bioassay. This method is based on the ability of autoantibodies similar to TSH to induce the second messenger cAMP in cell lines /28/.

30.6.3.3 Specimen

Serum: 1 mL

30.6.3.4 Reference interval

30.6.3.5 Clinical significance

Hyperthyroidism

TRAb are autoantibodies against membrane receptors and are much more prevalent in hyperthyreosis than other thyroid autoantibodies (anti-TPOAb, anti-TgAb). TRAb are stimulatory and the determination has few potential clinical applications. They inform the clinician concerning the etiology of hyperthyroid disease and differentiate Graves’ disease from other forms of hyperthyroidism, even though the diagnosis of Graves’ disease is easily confirmed in most patients with measurements low TSH level, an elevated FT4 concentration and the clinical setting of diffuse goiter, exophthalmos and dermatopathy. In early Graves’disease, if FT4 levels are normal, FT3 is useful /31/.

Detection of TRAbs may have the most value in complex clinical settings such as /3/:

• Differentiating Graves’ disease from non autoimmune cause (e.g., toxic multinodular goiter) of hyperthyroidism
• Predicting the risk of recurrent Graves’ disease following therapy with antithyroid drugs
• In pregnancy including of postpartum period, and the risk neonatal thyroid dysfunction /24/.
• In patients with transient thyrotoxicosis (TSH level below 0.1 mIU/L, and without clinical symptoms) TRAk measured as thyroid stimulating immunoglobulin (TSI) can be twice the upper reference value. Spontaneous resolution occurred at a mean of 5.5 weeks /35/.

Refer to Tab. 30.6-3 – Thyroglobin receptor antibodies in thyroid diseases.

Hypothyroidism

For the detection of autoimmune thyroid disease, either Hashimoto thyroiditis or Graves’ disease, based on the robustness of the current assays and their availability, anti-TPOAb and anti-TgAb can be considered as the first-line tests, especially since this strategy may also be more cost-effective than measuring TRAb /3/.

In patients with transient thyrotoxicosis (TSH level below 0.1 mIU/L, and without clinical symptoms) TRAb measured as thyroid stimulating immunoglobulin (TSI) can be twice the upper reference value. Spontaneous resolution occurred at a mean of 5.5 weeks /35/.

30.6.3.6 Comments and problems

Third generation TRAb assays have no advantages over second generation assays in the diagnosis or follow-up of patients with Graves’ disease. Both second and third generation assays exhibit high inter method variability, which may lead to misinterpretation of results, especially in the follow-up of pregnant women with recent onset of Graves’ disease. For this group, use of the second-generation assay is recommended until the outcome of calibration against the NIBSC standard is known /27/.

Anti-TPO serum samples in the range of 30–198 U/mL are stable after 12 years storage at –80 °C with an estimated nonsignificant average percentage deviation of +2.2% /37/.

30.6.3.7 T4 and T3 antibodies

The presence of T4 or T3 autoantibodies should be considered if there is discordance between the TSH level and the concentrations of FT4 or FT3, and the clinical symptoms do not correspond with the thyroid hormone concentrations. The prevalence is dependent on the immunoassay used and was 0.05% in the samples used in a study /36/.

References

1. Huber A, Menconi F, Corathers S, Jacobson EM, Tomer Y. Joint genetic susceptibility to type 1 diabetes and autoimmune thyroiditis: from epidemiology to mechanisms. Endocrine Reviews 2008; 29: 697–725.

2. McGrogan A, Seaman HE, Wright JW, de Vries CS. The incidence of autoimmune thyroid disease: a systematic review of the literature. Clin Endocrinol 2008; 69: 687–96.

3. Giannone M, Dalla Costa M, Sabbadin C, Garelli S, Salva M, Masiero S, et al. TSH-receptor autoantibodies in patients with chronic thyroiditis and hypothyroidism. Clin Chem Lab Med 2022; 60 (7): 1020–30.

4. Schott M, Seissler J, Scherbaum WA. Diagnostik bei autoimmunen Schilddrüsenerkrankungen. J Lab Med 2006; 34: 254–7.

5. Pedersen IB, Knudsen N, Jorgensen T, Perrild H, Ovensen L, Laurberg P. Thyroid peroxidase and thyroglobulin autoantibodies in a large survey of populations with mild and moderate iodine deficiency. Clin Endocrinol 2003; 58: 36–42.

6. Okosieme OE, Evans C, Moss L, Parkes AB, Kuvera LD, Premawardhana E, et al. Thyroglobulin antibodies in serum of patients with differentiated thyroid cancer: relationship between epitope specificities and thyroglobulin recovery. Clin Chem 2005; 51: 729–34.

7. Kim ES, Lim DJ, Baek KH, Lee JM, Kim MK, Kwon HS, et al. Thyroglobulin antibody is associated with increased cancer risk in thyroid nodules. Thyroid 2010; 20: 885–91.

8. Dufour DR. Thyroglobulin antibodies – failing the test. J Endocrinol Metab 2011; 96: 1276–8.

9. Saravanan P, Dayan CM. Thyroid autoantibodies. Endocrin Metab Clin North Am 2001; 30: 315–37.

10. Tozzoli R, Villalta T, Kodermaz G, Bagnasco M, Tonutti E, Bizzaro N. Autoantibody profiling of patients with autoimmune thyroid disease using a new multiplexed immunosaasay. Clin Chem Lab Med 2006; 44: 837–42.

11. Perros PE, ed. British Thyroid Association, Royal College of Physicians. Guidelines for the management of thyroid cancer. 2 nd edn. Royal College of Physicians, 2007.

12. Taylor KP, Parkington D, Bradbury S, Simpson HL, Jefferies SJ, Halsall D. Concordance between thyroglobulin antibody assays. Ann Clin Biochem 2011; 48: 367–9.

13. Ohtaki S, Nakagawa H, Nakamura M, Kotani T. Thyroid peroxidase: experimental and clinical integration. Endocrine J 1996; 43: 1–14.

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15. D’Herbimez M, Sapin R, Gasser F, Schlienger JL, Wemeau JL. Concordance of eight kits for antithyroid peroxidase autoantibodies determination. Clin Chem Lab Med 2000; 38: 561–6.

16. Cooper DS. Subclinical hypothyroidism. N Engl J Med 2001; 345: 260–5.

17. Targher G, Chonchol M, Zoppini G, Salvagno G, Pichiri I, Franchini M, et al. Prevalence of thyroid autoimmunity and subclinical hypothyroidism in persons with chronic kidney disease not requiring chronic dialysis. Clin Chem Lab Med 2009; 47: 167–71.

18. Meloni A, Mandas C, Jores RD, Congia M. Prevalence of autoimmune thyroiditis in children with celiac disease and effect of gluten withdrawal. Pediatrics 2009; 155: 51–5.

19. Schmidt M, Voell M, Rahlff I, Dietlein M, Kobe C, Faust M, et al. Long-term follow-up of antithyroid peroxidase antibodies in patients with chronic autoimmune thyroiditis (Hashimoto’s thyroiditis) treated with levothyroxine. Thyroid 2008; 18: 755–60.

20. Vestgaard M, Ringholm Nielsen L. Rasmussen AK, Damm P, Mathiesen ER. Thyroid peroxidase antibodies in pregnant women with type 1 diabetes: impact on thyroid function, metabolic control and pregnancy outcome. Acta Obstet et Gynecol 2008; 87: 1336–42.

21. Stagnaro-Green, Abalovich M, Alexander E, Azizi F, Mestman J, Negro R, Nixon A, et al. Guidelines of the American Thyroid Association for the diagnosis and management of thyroid disease during pregnancy and postpartum. Thyroid 2011; 21: 1081–1121.

22. Wesseloo R, Kamperman AM, Bergink V, Pop VJM. Thyroid peroxidase antibodies during early gestation and the subsequent risk for first-onst postpartum depression: a prospective study. J Affect Disord 2018; 225: 399–403.

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30.7 Biochemistry and physiology of thyroid function

30.7.1 Thyroid hormone synthesis

The thyroid gland produces T4 and T3 using iodide obtained from the diet or from the metabolism of thyroid hormones /1/. At least 100 μg of iodide is required per day. The iodide ions are transported via the blood to the thyroid, where thyroid hormones are synthesized in the following steps:

• Active uptake of iodide ions into the thyroid follicular cells across the basolateral cell membrane by the sodium-iodide symporter. The iodide ion is concentrated by a factor of 30–40 in the cytoplasm of the thyroid follicular cell and then released across the apical cell membrane by an ion transporter into the colloid of the thyroid follicle.
• Oxidation of the iodide ions to iodine and iodination of tyrosine residues in immature thyroglobulin molecules, which contain 134 tyrosine residues (iodization). This takes place extra cellularly, with the involvement of a H₂O₂ generating system in a reaction catalyzed by TPO. The iodine then undergoes a series of biochemical reactions that yield the intermediate products 3-monoiodotyrosine (MJT) and diiodotyrosine (DJT).
• T4 is produced in the thyroglobulin molecule by the coupling of two DIT molecules, while T3 is produced by the coupling of MIT with DIT or the 5’-monodeiodination of T4 catalyzed by type 1,5’-deiodinase, which is found in the thyroid, pituitary, liver, and kidney. T4 and T3 produced in the thyroid remain attached to mature thyroglobulin molecules, which are packaged into vesicles and transported to the colloid of the thyroid follicle. T3 and T4 is stored in the thyroid follicles in form of sterically levorotatory, metabolically active substances (LT3 and LT4).

Refer to Fig. 30.7-1 – Thyroidal iodine metabolism and hormone synthesis.

T4 and T3 (plus a small amount of thyroglobulin) are released into the circulation by reverse endocytosis into capillaries surrounding the thyroid follicular cells. Approximately 10 μg of T3 and 100 μg of T4 are released into the circulation daily. T3 has a biological half life of 19 hours whereas T4 has a half life of 190 hours. Approximately 25 μg of T3 is produced daily by the conversion of T4 to T3. Metabolically inactive reverse T3 (rT3) is also produced in the peripheral tissues when metabolically active LT4 is converted to LT3.

99.5% of circulating thyroid hormone is bound to transport proteins. T3 and T4 binding has the following advantages:

• Only minimal loss of thyroid hormone through the kidneys
• Presence of a large thyroid hormone pool with maintenance of a constant hormone concentration
• Even supply to all tissues.

Drugs and other hormones also affect thyroid hormone synthesis and binding affinity. Changes in the total thyroid hormone concentration in the blood (especially T4) can therefore occur in the absence of thyroid dysfunction. Approximately 0.3% of T3 occurs as FT3 and 0.03% of T4 occurs as FT4.

30.7.2 Thyroid hormone action in the tissues

In order to exert their effects on the tissues, thyroid hormones first have to bind to cellular hormone receptors /2/. These receptors are located on the cell membrane, in the mitochondria, in the nucleus, and in the form of cytoplasmic binding proteins. Approximately 15% of the T3 in hepatocytes is bound to the cell membrane, 10–15% to the mitochondria, and 50% to cytoplasmic binding proteins. These proteins are thought to act as a reserve for T4 and T3, controlling the amount of hormone available for the nucleus and mitochondria.

Refer to Fig. 30.7-2 – Cellular binding sites of T3 and T4.

For thyroid hormones to exert their biological functions at nuclear receptors, T4 must first be converted to T3. Conversion of T4 to T3 takes place /3/:

• In the peripheral tissues, catalyzed by 5’-deiodinase type 1 (D1). In autoimmune hyperthyroidism, the relative proportion of T3 to T4 is increased due to TSH receptor stimulation or thyroid stimulating immunoglobulin. This is partly due to the promotion of the iodotyrosine coupling reaction that produces T3 and partly due to the activation of D1 in the thyroid, which leads to increased intrathyroidal conversion of T4 to T3. In non-thyroidal illness, D1 activity is reduced, which results in low T3 (low T3 syndrome).
• In the brain, catalyzed by 5’-deiodinase type 2 (D2). The function of D2 is to maintain a constant T3 concentration in special tissues such as the central nervous system, even in the face of iodine deficiency or hypothyroidism.
• In the skin and placenta, catalyzed by 5’-deiodinase type 3 (D3). This deiodinase transforms T4 into inactive reverse T3 (rT3). During pregnancy, thyroid hormone metabolism is altered by increased degradation of T4 to rT3.

The actions of thyroid hormones on a target cell are mediated by the transportation of T4 and T3 into the cytoplasm and the subsequent conversion of T4 to T3, catalyzed by 5’-deiodinase. T3 enters the nucleus and binds to the thyroid hormone receptor. Thyroid hormone receptors are proteins that act as nuclear transcription factors. Following T3 binding, they regulate gene transcription in the nucleus, thereby mediating actions of T3 such as the synthesis of mitochondrial enzymes.

Refer to Fig. 30.7-3 – Regulation of mitochondrial enzyme induction by T3 via the nuclear receptor.

Thyroid hormone receptors have a central DNA binding domain as well as a T3 binding site and bind to thyroid response elements (TRE) of target genes /4/. TREs are located in the regulatory region of genes and control the transcription of genes that are regulated by T3. The increase in alkaline phosphatase that occurs in autoimmune hyperthyroidism is a clinical marker of the effect of T3 that reflects transcriptional regulation: it results from the actions of T3 on liver and bone.

Refer to Fig. 30.7-4 – Effect of T3 on gene transcription.

The thyroid hormone receptor is regulated by the genes, TR-α and TR-β, which are located on chromosomes 3 and 17. More than 30 mutant forms of TR-β are known. These mutations are responsible for thyroid hormone resistance syndrome, in which TR is unable to bind T3 effectively. This results in reduced transcription of genes that are activated physiologically by T3.

30.6.3 Thyroid hormone metabolism

Quantitatively T4 is the main synthetic product of the thyroid follicular cells and T3 is the metabolically active form of thyroid hormone in the peripheral tissues /3/. The most important reactions in metabolic activation and inactivation are ring deiodination reactions. Metabolically active T3, for example, is produced mainly by outer ring deiodination of T4 in peripheral tissues. T3 and T4 are inactivated by inner ring deiodination.

Refer to Fig. 30.7-5 – Pathways of thyroid hormone metabolism.

Thyroid hormone is inactivated in the following reactions:

• Inner ring deiodination e.g., of T3 to 3,3’-diiodothyronine by various deiodinases, which are integral membrane proteins that require thiols as co factors and contain selenocysteine residues
• Esterification of the phenolic OH group with sulfate by sulfotransferases. Sulfation (e.g., conjugation of a sulfo group to the phenolic OH group of thyroid hormones), accelerates the deiodination of various iodothyronines by deiodinases and causes irreversible hormone inactivation.
• Etherification of the phenolic OH group with glucuronic acid by UDP-glucuronyltransferases. Glucuronidized forms of thyroid hormones are excreted in the bile or feces and are intermediate products of enterohepatic circulation.

30.7.4 Hypothalamic-pituitary-thyroid system

The hypothalamus and anterior pituitary control the concentration of FT4 and FT3 in the circulation /6/. When FT3 or FT4 contacts the para ventricular nucleus (FT4 is converted to FT3 by a 5’-deiodinase type 2), the intracellular concentration of FT3 increases, which leads to a decline in the secretion of TRH into the hypothalamic-pituitary-thyroid axis. TRH is a tripeptide amide (pGlu-His-ProNH₂) that regulates the synthesis, release and biological activity of TSH.

Hypophysiotropic TRH neurons secrete TRH into the pericapillary space of the external zone of the median eminence for conveyance to anterior pituitary thyrotrophs. Under basal conditions, the activity of the hypophysiotropic TRH neurons is regulated by the negative feedback effects of TSH, to ensure stable circulating thyroid concentrations. However, the set point for negative feedback regulation by TSH is influenced by other hormonal and neuronal inputs on the hypophysiotropic TRH neurons /5/.

TSH is released from anterior pituitary thyrotrophs following TRH binding to its receptors. An influx of Ca²⁺ into the cell occurs, which leads to activation of the calcium-phosphatidyl cascade. As a result, the α- and β-subunits of TSH are synthesized and glycosylated and TSH is secreted. Glycosylation is very important for the biological activity of TSH.

TSH released into the circulation binds to the TSH receptors of the thyroid follicle. The actions of TSH at its receptor in the thyroid follicle are mediated by TSH receptor (TSHR)-G protein coupled synthesis of intracellular cyclic adenosine mono phosphate.

Refer to Fig. 6.2-4 – Gs-protein-mediated signal transmission of the parathyroid hormone sensing receptor.

TSH binding to its receptors initiates the following:

• The cAMP regulatory cascade, which has a positive effect on the growth of thyroid follicular cells and thyroid hormone secretion
• The phospholipase C-diacylglycerol regulatory cascade, which requires a TSH concentration that is 5–10 times higher for activation. This pathway, which generates the intracellular signals myo-inositol 1,4,5 triphosphate (1,4,5 PIP3) and diacylglycerol, is important for iodination and hormone synthesis.

30.7.5 Biochemistry of TSH

The approximate molecular weight of TSH is 28 kDa, but its circulating forms are heterogeneous and have a higher molecular weight due to varying degrees of post translational glycosylation and sialylation of the α- and β-chains, which in turn determine the half life and biological activity /7/. Sialylation of the α-chain increases the biological activity of TSH, while sialylation of the β- chain reduces it. Lowest TSH levels are measured in the late afternoon; levels increase throughout the evening and reach a maximum during the late evening and night. The TSH concentration declines again during sleep. TSH secretion is partially basal (non pulsatile) and partially pulsatile /8/. In relation to the distribution volume, the daily basal secretion is 12.5 ± 1.1 mU/L and the daily pulsatile secretion is 18.1 ± 1.7 mU/L.

Subclinical hypothyroidism

Subclinical hypothyroidism in younger population is a cardiovascular risk factor such as elevated LDL cholesterol and hypertension /9/. In individuals above the age of 65 in whom the serum TSH levels are elevated but who do not manifest other clinical signs and symptoms of disease the question arises of whether TSH elevations have the same clinical and metabolic implication as in younger patients. One possibility is that a decrease in deiodinase type 2 activity give rise to a higher TSH level at any serum level of FT4. Because it is the deiodinase type 2 that gives rise to most serum FT3, this would also explain the age related increase in TSH and decline in serum FT3.

30.7.6 Biochemistry of the TSH receptor

The TSH receptor has an important role in controlling the growth of the thyroid and regulating hormone synthesis /7/. Mutations in the TSH receptor gene can lead to increased or decreased receptor activity. The P162A and I167N mutations are associated with loss of function; the receptor remains deactivated. Mutations that lead to continuous activation of the receptor, on the other hand, are associated with increased function. This is the case for gain-of-function mutations of the TSH receptor that produce the toxic adenoma or multinodular goiter phenotype. Germ line loss-of-function mutations of the TSH receptor are associated with hypothyroidism and TSH resistance.

References

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5. Burch HB. Drug effects on the thyroid. New Engl J Med 2019; 381: 749–61.

6. Winter WE, Signorio MR. Review: Molecular thyroidology. Ann Clin Lab Sci 2001; 31: 221–44.

7. Roelfsema F, Veldhuis JD. Thyrotropin secretion patterns in health and disease. Endocrine Rev 2013; 34: 619–57.

8. Soboll S. Thyroid hormone action on mitochondrial energy transfer. Biochim Biophys Acta 1993; 1144: 1–16.

9. Klein I. Subclinical hypothyroidism – just a high serum thyrotropin (TSH) concentration or something else? J Clin Endocrinol Metab 2013; 98: 508–10.

30.8 Thyroid cancer

Lothar Thomas

Cancer of the thyroid is not common but should be considered whenever a lump in the thyroid is found. The forms of thyroid cancer are differentiated by some features:

• papillary and follicular cancers: These cancers are often remarkably indolent, tending to metastize to local lymph nodes
• medullary cancer: These carcinomas are differentiated tumors accounting for about 3% of thyroid cancers
• undifferentiated thyroid cancers: lymphoproliferative tumors and other cancers can metatasize to the thyroid.

Early diagnosis of thyroid cancer is hampered by the inability of fine-needle biopsy /1/. Approximately one third of cases are not accurately classified while preoperative cancer staging detects lymph nodal involvement in only half of cases /2/.

Thyrotropin receptor mRNA, a surrogate marker for circulating cancer cells was an opinion for early detection of malignancy from peripheral blood. In a study /2/ the authors investigated if TSH mRNA can be detected in low sample volumes by employing an ultrasensitive method-droplet digital PCR. Thyrotropin receptor mRNA was detectable in 4 mL sample volumes, with the test having good specificity (80%) but modest diagnostic accuracy (68.1%).

References

1. Hambleton, Kandil E. Appropriate and accurate diagnosis of thyroid nodules: a review of tyroid fine-needle aspiration. Int J Clin Exp Med 2013; 6: 413–22.

2. Milijus JRJ, Zmrzijak UP. Detecting thyrotropin receptor mRNA from peripheral blood of patients with differentiated thyroid cancer rules out non-aggressive cases. Laboratory Medicine 2024. doi: 10.1177/00045632241228217.