The immuassay handbook parte73

673© 2013 David G. Wild. Published by Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/B978-0-08-097037-0.00056-7

ThyroidRhys John1

Nic Christofides1

Carole A. Spencer2

David Wild2

1 This edition.2 Previous editions.

Normal Thyroid FunctionThe thyroid gland lies in the anterior neck, just below the thyroid cartilage. The principle hormone arising from the thyroid is thyroxine (T4), which is considered as a prohor-mone for the more metabolically active thyroid hormone triiodothyronine (T3). Normally, approximately 20% of T3 in the circulation originates directly from the thyroid gland secretion. Most T3 in blood is produced extrathyroi-dally from T4 by a tightly-regulated monodeiodination enzymic process, catalyzed by 5′-deiodinase enzymes. Normal thyroid hormone status is critical for the health of both children and adults. In infancy and childhood, thy-roid hormone is essential for normal physical and mental development as well as growth. In the adult, thyroid hor-mones exert a wide range of actions that control protein synthesis, oxygen consumption, heat generation, and the overall metabolic activity. It follows that thyroid hormone excess (hyperthyroidism) causes a state of hyperactivity, whereas thyroid hormone deficiency (hypothyroidism) is characterized by symptoms of slow metabolic activity such as tiredness, lethargy, and cold intolerance. However, since the signs and symptoms of both hyper- and hypothy-roidism are non-specific and often attributed to the aging process, biochemical testing is essential to determine whether any thyroid dysfunction is present.

All aspects of thyroid gland function are controlled by thyrotropin (TSH), also known as thyroid stimulating hor-mone. TSH is secreted by the anterior pituitary gland in response to the hypothalamic tripeptide, thyrotropin-releas-ing hormone (TRH). Homeostasis of the hypothalamic-pituitary-thyroid axis is achieved through negative-feedback inhibition by circulating thyroid hormone at both the hypo-thalamic and pituitary level (see Fig. 1).

Iodine is an essential element for thyroid hormone pro-duction. An adequate supply of iodine in the diet is there-fore necessary for normal thyroid function. The daily dietary intake of iodine recommended for the control of iodine deficiency disorders is 90 µg for up to 5 years, 120 µg for 6–12 years, 150 µg for adults, and 200 µg during preg-nancy and lactation. When dietary iodine is deficient, there is a compensatory thyroid gland hypertrophy (goiter).

TSH stimulates the thyroid follicular cell to trap iodine from the circulation and is transported into the follicular cell by means of the sodium iodide symporter. The iodine is then activated and incorporated into the tyrosine resi-dues of the large glycoprotein precursor molecule,

thyroglobulin (Tg) by an enzymic process catalyzed by the thyroid peroxidase enzyme, which is located at the apical membrane of the thyroid follicular cell. Iodinated Tg is stored in the colloid of thyroid follicles and serves as a storage reservoir of thyroid hormone. Under the influence of TSH, Tg is endocytosed and digested by lyzosomal enzymes to release the thyroid hormones and some undi-gested Tg into the circulation.

Calcitonin is also synthesized in the thyroid gland. See BONE METABOLISM.

People with normal thyroid function are described as euthyroid.

Clinical DisordersHYPOTHYROIDISM (THYROID HORMONE DEFICIENCY)Congenital hypothyroidism (CH) if present at birth may not be clinically obvious and will go undetected if not screened for. Without treatment, this disorder causes irre-versible brain damage, but if it is detected early and treat-ment is commenced within two weeks of age, cognitive development and IQ will be normal. In the first half of pregnancy, thyroid hormone concentrations are low in the fetus, so that the fetus is totally dependent on maternal thyroid hormone, and its supply to the fetus is controlled by the placenta and thyroid status of the mother. By mid-way through gestation, functioning of the fetal hypotha-lamic-pituitary-thyroid axis begins and is complete by delivery in the term infant. Although thyroid hormone has effects on many organ systems, the infant with CH appears normal at birth so must be protected in part by placental transfer of maternal thyroid hormone. There is increased

C H A P T E R

9.2

FIGURE 1 Control of thyroid hormone secretion (simplified).

674 The Immunoassay Handbook

intracerebral conversion of T4 to T3, resulting in increased local availability of T3 despite low serum concentrations, so that normal or near-normal cognitive outcome can occur in the most severely-affected CH infant. This is the case if therapy is started early, is adequate and maternal thyroid function was normal. Whenever fetal and mater-nal hypothyroidism occur, there is significant impairment in neurointellectual development, despite adequate ther-apy soon after birth. This can occur when there is severe iodine deficiency, potent thyrotropin receptor blocking antibodies are present, or in feto-maternal PIT 1 deficiency.

The most common cause of congenital hypothyroidism, accounting for 85% of cases is an abnormality in thyroid gland development—thyroid dysgenesis (ectopia, apla-sia, or hypoplasia)—while inborn errors of metabolism, dyshormonogenesis, account for 10–15%. Recent advances in molecular and cell biology have led to improved understanding of normal thyroid physiology and of the genes involved in thyroid gland development and disease. This has allowed the genetic defect to be identified in some very rare cases of congenital hypothyroidism. The incidence of congenital hypothyroidism is about 1 in 3000 newborns with a lower incidence in black individuals. This incidence of congenital hypothyroidism is about 4–5 times more common than phenylketonuria, for which screening programs were originally developed. There is a 2:1 inci-dence in females compared with males and there is an increased risk in Down Syndrome. Congenital hypothy-roidism is detected in many countries by screening infants a few days after birth by measuring TSH or T4 in a blood sample obtained from a heel prick and absorbed onto a filter paper with TSH screening being the most widely adopted screening procedure. TSH in neonates increases immediately after birth, but falls to normal within 24–48 h. Samples for blood spot screening should be collected opti-mally between 48 h and four days of age. This will ensure affected infants are detected early and treatment started to achieve the best possible outcome in terms of neurointel-lectual development. In infants with congenital primary hypothyroidism, blood spot TSH concentrations are usu-ally increased well above those in normal infants, so that screening with TSH is a more sensitive and specific test than screening with T4. However, the rare infant with hypothyroidism due to hypothalamic or pituitary disease (central hypothyroidism) will be missed using TSH screen-ing but these are usually detected clinically.

Infants with increased blood spot TSH concentrations should be seen immediately by a pediatrician who will start L-T4 replacement and arrange to have confirmatory serum testing performed to verify the diagnosis. In the majority of cases the infants will have a low FT4 with an increased TSH and will have a permanent hypothyroidism. Treat-ment should commence at an initial dosage of 10–15 µg/kg of L-T4 per day to rapidly increase FT4 and normalize TSH. Serum FT4/TSH should be monitored every 1–2 months in the first 6 months of life, every 3–4 months until 3 years, and every 6–12 months until growth is completed. Some infants have a transient hypothyroidism with a nor-mal FT4 and normal or slightly increased TSH on serum testing. This is relatively common in iodine-deficient areas of the world but is less common where the iodine supply is

sufficient. Other causes of transient hypothyroidism are prematurity, intrauterine exposure to maternal antithyroid drugs, maternal TRAbs, and pre- or postnatal exposure to excess iodides (povidone iodine from antiseptic swabs, or iodinated contrast media). Initial treatment in these cases will be as for any permanent cause of CH, and the tran-sient nature of the hypothyroidism can be confirmed at a later time usually after 2 years of age when L-T4 is discon-tinued and serum FT4 and TSH measured after a suitable time lapse. If the FT4 and TSH both remain normal then the patient is euthyroid and there is no need to restart L-T4. A very small number of infants, usually those who have low or very low birth weight, or are critically ill pre-term or term infants, have normal TSH concentrations on screening, but serum TSH increases during the first few weeks of life to concentrations as seen in permanent hypo-thyroidism. It is unclear whether this delayed TSH increase results from an abnormality of pituitary–thyroid feedback regulation, transient hypothyroidism, as in iodine-induced, or a mild form of permanent hypothy-roidism. Some screening programs now obtain a second specimen for screening at 2–6 weeks of age in these infants.

The main objective of screening has been achieved: the eradication of mental retardation due to congenital hypo-thyroidism; however, there remain some countries where screening is not universal.

Childhood hypothyroidism. The most common fea-ture of hypothyroidism in children and adolescents is fail-ure in growth. There is a moderate increase in weight, and bone retardation occurs. In children over 4 years of age, the most common cause of hypothyroidism is Hashimoto’s thyroiditis and occurs most commonly in association with deficiency of other endocrine glands: pancreas, adrenals, gonads, and parathyroids. In places without a congenital hypothyroid screening program, failure of hypoplastic or ectopic thyroid tissue, as growth occurs, results in cases of childhood hypothyroidism.

Primary hypothyroidism. Most cases of hypothyroid-ism in adults arise from spontaneous thyroid failure caused by autoimmune thyroid disease (Hashimoto’s thyroid-itis), which progressively destroys thyroid gland function over a period of years. The prevalence in the general popu-lation approximates to 2%. Typically, hypothyroidism presents in differing degrees ranging from mild (subclini-cal) to overt. The prevalence of this condition increases with age and is four-fold greater in women as compared to men. Thyroid failure also frequently occurs in the months or years following therapies for hyperthyroidism (surgery, radioiodine ablation, or overtreatment with antithyroid drugs). The biochemical profile of overt hypothyroidism is typically marked by an increased TSH associated with a subnormal serum free T4. The classic clinical presentation includes clinical symptoms such as tiredness and lethargy, slow mentation, intolerance of cold, increased menstrua-tion, constipation, weight gain, dry skin, edema, deafness, and loss of balance. Thyroid autoantibodies, especially thy-roid peroxidase antibodies (TPOAb), are usually detected and are good markers for the underlying autoimmune pro-cess. When a deficiency of iodine in the diet occurs a spec-trum of iodine-deficiency disorders occurs including hypothyroidism, and when the deficiency is severe, goiter, cretinism, and decreased fertility occur. In total, iodine

675CHAPTER 9.2 Thyroid

deficiency disorders are a significant public health problem affecting 38% of the world population, making it the most common cause of hypothyroidism.

Mild (subclinical) hypothyroidism. This condition is characterized by an increased serum TSH associated with a normal range free T4 and free T3. Studies suggest that the prevalence of mild hypothyroidism may be as much as 15–20% in elderly female patients. This TSH abnormality is now considered as an indicator of thyroxine deficiency, as judged by studies demonstrating abnormalities in tissue markers of thyroid hormone action and psychometric test-ing. Hence, the American Thyroid Association has recom-mended that the term ‘subclinical hypothyroidism’ be replaced by the term ‘mild hypothyroidism’. Since most cases of mild hypothyroidism (high TSH/normal T4) are secondary to Hashimoto’s thyroiditis and progress to overt hypothyroidism (high TSH/low T4) at a rate of 5% per year, physicians increasingly recognize the value of diag-nosing and treating this condition. A TPOAb measure-ment is an indicator of the severity of the underlying autoimmune condition, since the higher the TPOAb, the more rapidly the thyroid failure progresses. Both overt and mild hypothyroidism are often missed in the older patient, since the symptoms of thyroxine deficiency have an insidi-ous onset and are non-specific, such that progressive thy-roid failure is often attributed to the aging process.

Treatment to prevent progression of mild to overt hypothyroidism is agreed in patients with a serum TSH concentration greater than 10 mU/L and an increased TPOAb concentration. However in those with a serum TSH less than 10 mU/L, the benefits of treatment are less certain, given the relatively low progression of the disease. Patients with the following conditions should be consid-ered for L-T4 replacement with regular monitoring of thy-roid function:

� increased cardiovascular risk with diastolic dysfunction � hypertension � risk factors for atherosclerosis � high lipids � diabetes mellitus � smokers � symptoms of hypothyroidism � goiter � elevated TPOAb � pregnancy � infertility

Without any of these, there is no evidence for any benefit from T4 replacement, and only monitoring needs to be done. However, many clinicians believe that patients do obtain some benefit from L-T4 replacement if the TSH is above the reference range, especially when greater than 5 or 6 mU/L, and would do a trial of L-T4 replacement.

Secondary hypothyroidism, caused by the failure of hypothalamic TRH and/or pituitary TSH to stimulate the thyroid gland, accounts for <1% of all cases of thyroid fail-ure. In some cases of secondary hypothyroidism, serum TSH is not necessarily low but is often paradoxically nor-mal or slightly elevated (5–10 mU/L). It is now known that the biological activity of the TSH secreted by a damaged or understimulated pituitary is impaired and this explains the inappropriately normal TSH. Secondary hypothyroidism

may result from a variety of disease conditions or arise from hypothalamic or pituitary trauma. Usually, pituitary dysfunction is not confined to the thyroid axis but is evi-dent from deficiencies in adrenal and gonadal functions. An impaired or delayed TSH response to TRH stimula-tion (<two-fold rise) is an indicator of secondary thyroid failure.

Both primary and secondary hypothyroidism are effec-tively treated by exogenous L-T4 therapy, which serves as an adequate source of substrate for T3 production in extrathy-roidal tissues (see L-THYROXINE REPLACEMENT THERAPY).

HYPERTHYROIDISMThe main cause of hyperthyroidism in patients is Graves’ disease, with toxic multinodular goiter, silent thyroiditis, and toxic adenoma being far less prevalent, and there are other rarer causes. In the UK, Graves’ disease is the most common, but in iodine-deficient areas, toxic adenoma is more common. Hyperthyroidism is approximately 10 times more common in women than men, and in women has a prevalence of 2.5–4.7 per 1000. Graves’ disease occurs most commonly in women between 20 and 50 years of age, but toxic multinodular goiter occurs more com-monly in the over-60-year age group.

Graves’ disease is caused by thyroid stimulating auto-antibodies (TSAb) that bind to the TSH receptor and mimic the action of TSH on thyroid cells. The metabolic effects of thyroid hormone excess produce tiredness, excess sweating, and dyspnea on exertion. Most patients notice weight loss and decreased muscle mass, despite an increased appetite. Additional characteristic features include opthalmopathic manifestations including pro-truding, staring eyes (exophthalmos), and an enlarged thyroid gland.

TSAb stimulation causes excess T3 and T4 secretion, which suppresses pituitary TSH secretion to <1/100 of euthyroid levels (<0.02 mU/L), which can be detected by current TSH immunometric assays.

Graves’ disease can be treated with antithyroid drugs, such as carbimazole, propylthiouracil, or methimazole, which are typically taken for 12–24 months. If the disease recurs after cessation of the drug treatment, it may be treated by radioiodine ablation, or surgical removal of the thyroid gland.

Hyperthyroidism due to a toxic multinodular goiter (TMNG) is a condition where thyroid hypertrophy and nodularity develop, usually over a long period of time (years), often in response to iodine deficiency. As the thyroid enlarges, autonomy develops and thyroid hormone concentrations increase. There is typically an inverse relationship between thyroid size and serum TSH. However the degree of thyroid hormone excess and the magnitude of TSH suppression is usually less than in Graves’ disease. Despite a milder degree of thyrotoxico-sis, patients with TMNG tend to be older than Graves’ patients and more prone to atrial fibrillation, secondary to the exacerbation of intrinsic cardiac disease by thyroid hormone excess.

A toxic adenoma may exist for some time before hyper-thyroidism develops, with a corresponding decrease in TSH concentrations. Recent studies suggest that the


autonomy in such nodules represents constitutive activation of the TSH receptor secondary to a genetic mutation. In some cases the tumor secretes T3 predominantly causing T3-toxicosis. In this condition free T3 is elevated but free T4 is normal. It should be suspected when clinical symp-toms suggest hyperthyroidism but free T4 is within the ref-erence interval and serum TSH is suppressed. In the United States about 2% of thyrotoxic patients have T3-toxicosis and it is common in Graves’ disease and toxic multinodular goiter. T4-toxicosis, where free T4 is elevated but free T3 is normal, can also occur. Toxic adenoma is most effectively treated by surgical removal. Thyroid carcinoma is a very rare cause of hyperthyroidism, as is hyperthyroidism caused by excessive secretion of TSH from the pituitary. These lat-ter patients have normal or increased TSH concentrations in the presence of increased free thyroid hormone concen-trations, leading to the term ‘inappropriate TSH secretion’ to describe this condition. These result from either a TSH-secreting pituitary adenoma or non-tumorous TSH hyper-secretion due to thyroid hormone resistance. Thyroid hormones may be secreted by ectopic thyroid tissue in an ovarian teratoma (struma ovarii). The concentrations of thyroid hormones may increase and TSH decrease, as in thyroid tumors.

The symptoms of hyperthyroidism can result from a hypothyroid patient receiving too high a dose of T4 ther-apy. Thyrotoxicosis factitia is the name given to a condi-tion in which a formerly euthyroid person takes excessive amounts of thyroid hormones, for example in a misguided attempt to lose weight. Serum T4 and T3 concentrations are elevated, unless the patient has taken T3, in which case T4 is subnormal and T3 is increased.

Hyperthyroidism can be induced in patients with goiters if iodine is administered. This condition is known as iodine-induced hyperthyroidism or the Jod-Basedow phenomenon.

Hyperthyroidism may be caused by de Quervain’s sub-acute thyroiditis (also known as granulomatous thy-roiditis or silent thyroiditis), which is probably due to a viral infection and causes inflammation of the thyroid. The inflammation causes leakage of excess thyroid hormones into the blood from the reservoir of hormone normally held in the thyroid gland. This can result in a moderate elevation in serum thyroid hormone concentrations. Auto-immunity may play a role in the etiology of subacute thy-roiditis and thyroid autoantibodies can sometimes be detected. Patients either return to normal or pass through a euthyroid state temporarily, becoming hypothyroid for several months before finally returning to normal thyroid function. A few of these patients become hypothyroid years later.

The postpartum thyroiditis (PPT) syndrome has a similar pattern to subacute thyroiditis. Transient hyper-thyroidism occurs after delivery of the baby, followed by a period of hypothyroidism for several months and, usually, by a return to the euthyroid state. Most patients have low positive TPO antibody concentrations. Although the pres-ence of TPOAb is a risk factor for PPT, only 50% will go on to develop any thyroid dysfunction. After one period of PPT, there is an increased risk of developing the syndrome in subsequent pregnancies. Postpartum thyroiditis occurs in 5–10% of women after giving birth.

NONTOXIC GOITERSimple or nontoxic goiter is the term used to describe thy-roid enlargement without hypo- or hyperthyroidism and not due to inflammation or a tumor.

THYROID DISEASE IN PREGNANCYDuring pregnancy, several physiological changes occur, including an increase in thyroxine binding globulin pro-duction resulting in an increased total T4 in serum, and there is an increase in extra-thyroidal T4 distribution space. To maintain FT4 homeostasis an increase in T4 pro-duction occurs. In the first trimester, in response to the increasing human chorionic gonadotropin, serum TSH decreases to a nadir at around 10 weeks gestation, resulting in TSH falling below the reference range in up to 20% of women. What happens to FT4 during pregnancy is more controversial; many of the most commonly used methods show a decrease in FT4, whereas other methods do not. It is now accepted that with reliable FT4 methods, FT4 falls during pregnancy so that appropriate method-dependent reference intervals should be used.

Because there is an increased requirement for T4 during pregnancy, in those geographical locations with low or borderline iodine in the diet, iodine deficiency can result in goiter formation and relative hypothyroxinemia, affect-ing both mother and fetus. Multivitamin pills containing iodine to supply 200–250 µg/day can be taken to avoid deficiency. Even in those countries usually regarded as iodine sufficient, epidemiological surveys have revealed iodine deficiency in their population. This is exacerbated in young pregnant women who may be diet conscious and avoid iodine-supplemented salt and bread. Iodine supple-mentation before or during pregnancy will normalize thy-roid function in the mother and newborn. Iodine deficiency remains the most common treatable cause of mental retardation worldwide. There may be associated deficiencies in selenium and iron that can affect neuro-logic development of the fetus and on thyroid response to iodine therapy. In those areas of the world with severe iodine deficiency, endemic cretinism results if iodine is not added to the diet.

AUTOIMMUNE THYROID DISEASE AND PREGNANCYThyroid autoimmunity in women can be a contributory cause of infertility and also increase the risk for miscar-riage. Screening studies have shown that between 5–10% of women have thyroid autoantibodies, but normal thyroid function. With positive autoantibodies during pregnancy, there is the risk of developing maternal hypothyroidism, so regular TSH testing of the mother, with T4 treatment if hypothyroidism is found, will benefit mother and fetus.

Pre-existing hypothyroidism can be present in 2–4% of pregnant women, and it is these women who carry an increased risk of obstetric and fetal complications, and require T4 replacement. There is an increased require-ment for T4 replacement during pregnancy, and dosage needs to be increased by about 50%. A number of studies have shown that hypothyroidism in the mother (or just a


low FT4), especially in early pregnancy, can affect the fetus resulting in diminished IQ scores in the children. Current guidelines do not recommend antenatal screening for hypothyroidism in pregnancy, but mothers with known hypothyroidism should be monitored and their thyroid function maintained with increased dosage of T4.

For women who have Graves’ disease, the maternal and fetal well-being is dependent on good control of the hyper-thyroidism. Without good control, preeclampsia, fetal malformation, premature delivery, and low birth weight can result. The main aim of treatment with antithyroid drugs is to use the lowest dose to control symptoms whilst accepting slight hyperthyroidism. If hyperthyroidism is discovered in pregnancy, the causes are either Graves’ dis-ease (newly presenting or a relapse) or gestational transient thyrotoxicosis of non-autoimmune origin. TPO-Ab and TSH-receptor antibody (TRAb) should be measured, (TRAb will be present in Graves’ disease but absent in ges-tational transient thyrotoxicosis), with thyroid function tests and TRAb at six months. This will allow the diagnosis of Graves’ disease, indicate the need for fetal monitoring (for any neonatal thyrotoxicosis), and identify mothers who may develop any postpartum thyroiditis or exacerba-tion of thyrotoxicosis.

Postpartum thyroiditis is described above.

Non-Thyroidal Illness Syndrome (NTIS)During illness, either acute or chronic, changes occur in serum thyroid hormone concentrations. In mild illness, there is a reduction in T3, but in more severe illness both T3 and T4 are reduced, without an increase in TSH. Included in illness are starvation, surgical procedures, psy-chiatric conditions, myocardial infarction, bone marrow transplantation, and probably any severe illness. In the most severe forms of illness T3, T4, and TSH are reduced.

The mechanisms that give rise to serum thyroid hor-mone changes in NTIS are complex and involve the hypo-thalamus, pituitary, plasma effects, tissue uptake, intracellular deiodination, and nuclear thyroid hormone receptors and coactivators. Evidence is now accumulating that these changes occur due to a central hypothyroidism caused by a decrease in hypothalamic thyrotropin releas-ing hormone. In starvation this can be signaled by a decrease in leptin. Additionally, sepsis or trauma, while increasing the expression of type-2 deiodinase, suppresses TRH production, whilst increased cytokines may also inhibit TSH from the pituitary. The decline in serum T3 and T4 in acute illness precedes any decline in hepatic type-1 deiodinase, suggesting that an acute-phase response is the trigger to a loss of TBG or pre-albumin and also the accumulation of substances that lower thyroid hormone binding capacity.

What happens to FT4/FT3 is method dependent, but reliable methods show a decrease although this is much less than decreases seen in total T4/total T3 (TT4/TT3). In unreliable methods for FT4/FT3, there is considerable underestimate of FT4/FT3. In the recovery phase of NTIS, TSH can be transiently increased before returning to nor-mal as do FT4 and FT3.

A large proportion of patients in intensive care have various degrees of severity of NTIS. Low T3 and T4 are

strong predictors of fatal outcomes in children in Intensive Care Units and in patients with cancer and severe trauma. A markedly decreased T4 is associated with a high proba-bility of death.

Controversy surrounds the need for thyroid hormone replacement therapy in NTIS, and it is impossible to say whether replacement is beneficial or harmful. Only a large-scale prospective study could answer this question. Currently there is no convincing evidence that thyroid hormone replacement is beneficial in any patients with NTIS.

DRUG EFFECTSDrugs can have an in vitro or in vivo effect on thyroid tests and/or thyroid function.

Drugs that Affect Thyroid FunctionA wide range of drugs can cause hypothyroidism including aminoglutethimide, amiodarone, cytokines (interferon, IL-2, TNF, TGF), lithium, iodine-containing agents, and retinoids/vitamin A. Some of these drugs (amiodarone, iodine-containing agents, and cytokines) can also cause thyroiditis and hyperthyroidism in susceptible individuals. Amiodarone is an anti-arrhythmic drug that is being increasingly prescribed and has a structure similar to thy-roid hormones. It contains 2 atoms of iodine and in normal use presents the body with a large load of iodine, very much greater than normal iodine intake. It has two effects: one on thyroid function tests and one where it can induce thyroid dysfunction. Amiodarone inhibits peripheral con-version of T4 to T3 resulting in lowered FT3 and increased FT4 concentrations with normal TSH. It also inhibits both uptake of iodine in the thyroid and T4 into cells. A result-ing hypo- or hyperthyroidism can result depending on the iodine intake of the area. With high iodine intake, hypo-thyroidism chiefly occurs, whereas in iodine-deficient areas hyperthyroidism occurs more frequently than hypo-thyroidism. In patients with any underlying thyroid dys-function (multinodular goiter or subclinical Graves’ disease) the high iodine content can induce throtoxicosis, but can also cause a destructive thyroiditis. The former is easily treated with antithyroid drug but the latter is not readily responsive to the same treatment. Thyroid func-tion should be checked before starting any of these drugs as patients with any pre-treatment history of thyroid dys-function or presence of thyroid antibodies are more likely to develop thyroid dysfunction when prescribed these drugs. In the case of amiodarone, thyroid function should be checked every 6 months after starting treatment and should be continued for a year after treatment is stopped due to the long half-life of amiodarone metabolites.

Drugs that Affect Thyroid Function TestsTSHThe glucocorticoids (prednisolone, hydrocortisone, and dexamethasone) cause direct suppression of TSH, whereas high-dose salicylate or furosemide causes suppression of TSH by displacing T4 from TBG. In patients on octreo-tide or dopamine, suppression of TSH occurs as a result of


bonding to thyrotroph receptors. If receiving dopamine antagonists, amphetamines, IL-2, or theophylline, an increase in TSH occurs.

FT4

A number of drugs can increase or decrease TT4 by their effect on increasing or decreasing TBG concentrations, but are largely not seen in FT4 assays. Drugs used to control seizures (carbamazepine, phenobarbitone, and phenytoin), by increasing T4 metabolism, cause hypothyroxinemia, which is seen in TT4 and FT4 assays.

A group of drugs (amiodarone, iopanoic acid, and pro-pranolol) cause an increase in FT4 with a decrease in FT3, by inhibiting T4 to T3 conversion, without any change in TSH concentration. In patients on heparin treatment, as a result of lipoprotein lipase from vascular endothelium, an increase in non-esterified fatty acids occurs, displacing T4 from TBG and causing a transient increase in FT4. If there is delay in processing these samples, significant increases in FT4/FT3 occur as a result of continuation of this process in vitro. If patients are exposed to therapeutic or diagnostic agents containing fluorophores then these may interfere with those methods that utilize fluorescent signals.

L-THYROXINE REPLACEMENT THERAPYA normal serum TSH is now the recognized therapeutic end point for levothyroxine replacement therapy. Ameri-can guidelines recommend that this should be around 1–2 mU/L whereas UK guidelines recommend that TSH should be brought to within the reference interval. This is usually accomplished with a dose of 1.6 µg/kg/day. Nor-mally, about one-fifth of T3, the active thyroid hormone, originates directly from thyroid secretion. The remainder is produced in peripheral tissues from T4 by monodeiodin-ation. In hypothyroidism the majority of T3 must be pro-duced extrathyroidally. In hypothyroidism the endogenous T3 component is lost and it is necessary to maintain free T4 in the upper half of the normal range for TSH to be normalized. Since it takes time for the pituitary to re-equilibrate to any change in free thyroxine status, it is nec-essary to wait for 6–8 weeks after initiating L-T4 therapy or changing the L-T4 dose before checking the TSH status.

In most patients on L-T4 replacement the FT4 is toward the top end of the reference interval or slightly above, with the FT3 towards the bottom end of the reference interval. Despite this there is no benefit to be gained by using com-bined treatment of T4 and T3. Overtreatment with L-T4 is common and adverse affects on the skeleton and heart can occur. It is thought that about a fifth of patients taking L-T4 have subnormal serum TSH so are either clinically or subclinically thyrotoxic. In the elderly in particular, increased hip fracture and vertebral fracture, increased risk for atrial fibrillation or increased resting heartbeat have been documented in patients with low serum TSH (less than 0.1 mU/L). The only exception category, in whom the TSH should always be maintained suppressed (<0.02 mU/L), are patients who have undergone ablative treatment for thyroid cancer. Patients who are non-com-pliant may have normalized their serum FT4 by taking a large dose prior to their clinic visit. Non-compliance is the main reason for a TSH/ FT4 discordance in an L-T4

treated patient, but thyroid hormone resistance should also be considered in a patient if TSH does not normalize on the usual dose of L-T4. In a small number of patients, impaired bioavailability of T4 may be a factor. This is caused by inhibition of T4 intestinal uptake by foods (soy, fiber), medications (iron, calcium), malabsorption, or increased degradation (anticonvulsants, large hemangio-mas with high deiodinase activity).

ENDOGENOUS ANTIBODIES TO T3 AND T4A few patients with silent thyroiditis, primary hypothy-roidism, or Graves’ disease have antibodies to T3, T4 or both hormones in their blood. They tend to occur in patients who also have increased concentrations of thyro-globulin antibodies. If present, they can affect immunoas-says for total and free hormones by increasing the quantity of T3 or T4 antibodies. This changes the apparent concen-tration of hormone in the assay, because competitive immunoassays rely on a constant, limited amount of anti-body being present in both calibrator and sample tubes. Current labeled antibody and 2-step free T4 assays have been designed to be resistant to interference by T4 anti-bodies. Endogenous antibodies to thyroid hormones can also cause an increase in total thyroid hormone concentra-tion while the free hormones remain normal. Endogenous antibodies should be suspected when thyroid function tests are anomalous or clinical symptoms do not match the lab-oratory test results.

FAMILIAL DYSALBUMINEMIC HYPERTHYROXINEMIAFamilial dysalbuminemic hyperthyroxinemia (FDH) is a rare inherited autosomal dominant condition in which the albumin is in an altered form that has an increased affinity for T4 (and sometimes T3). In this condition the concentration of total T4 is increased although free T4 is normal. While early methods of measuring free T4 by ana-log methods gave erroneously increased results in this con-dition, current free T4 assays are variable in the effect the variant albumin has on the assay with some giving errone-ously increased results, while other free T4 assays give nor-mal results.

SYNDROMES OF RESISTANCE TO THYROID HORMONESSyndromes of resistance to thyroid hormones (RTH) are a relatively rare inherited disorder characterized by a reduced responsiveness of target tissues to thyroid hor-mone. Such patients are identified as having anomalous thyroid function test results in that they have increased circulating concentration of free T4 and free T3 in asso-ciation with non-suppressed serum TSH. In all cases higher doses of exogenous thyroid hormone are required to achieve the expected suppressive effect on pituitary TSH and on the metabolic responses in peripheral tis-sues. Although the apparent resistance to thyroid hor-mone may vary in severity it is always partial and the magnitude of resistance is dependent on the nature of


the genetic defect. This is usually but not always in the thyroid hormone receptor (TRβ) gene. More than 1000 cases have been described.

Commonly these patients have increased serum FT4 and FT3 with normal or slightly increased TSH, which responds to TRH, absence of the usual symptoms and metabolic consequences of thyroid hormone excess, and the presence of a goiter. Similar anomalous thyroid func-tion test (TFT) results are seen in patients with TSH-secreting pituitary adenoma (TSH-oma). In these patients the α-subunit is increased and sex hormone binding globu-lin (SHBG) is increased in TSH-oma, but normal in RTH. The nature of the genetic defect can be determined by DNA analysis to confirm the diagnosis of RTH, and if found, should also be tested in first degree relatives with anomalous thyroid function test results.

AnalytesTHYROTROPINThyrotropin, or thyroid stimulating hormone (TSH), is a 29 kDa glycoprotein hormone consisting of two subunits, designated α and β. The α subunit is common to TSH, luteinizing hormone (LH), follicle stimulating hormone (FSH) and human chorionic gonadotropin (hCG). Although there are some similarities between the β sub-units of these hormones, the differences between the amino acid sequences and oligosaccharides give them unique biological activities. Serum TSH is the most useful test of thyroid function and because of the log/linear rela-tionship with FT4, in the early stages of hypo- or hyper-function of the thyroid gland, TSH changes while FT4 still remains normal. TSH is therefore superior in detecting mild thyroid dysfunction.

FunctionTSH is secreted from the anterior pituitary, and it acts upon the thyroid gland, stimulating the production of T4 and T3. TSH is controlled by a negative-feedback system that maintains a constant concentration of free thyroid hormone in serum.

Reference IntervalGreat care should be taken in selecting subjects to deter-mine reference intervals for all thyroid function tests. In general, serum should be collected from at least 120 care-fully screened normal euthyroid individuals who have no detectable thyroid autoantibodies, TPOAb or TgAb (mea-sured by a sensitive immunoassay), no personal or family history of thyroid disease, no goiter and are on no medica-tion. As TSH is not normally distributed, the results should be log-transformed before determining the 95% confi-dence limits. With current sensitive immunometric assays, the lower reference (2.5 percentile) is between 0.3 and 0.4 mU/L and this appears to apply for different methods and populations studied. In the case of the upper reference (97.5 percentile) this varies between 2.1 and 7.5 mU/L, depending on the method and population studied. Various factors can account for this variable upper limit including sex, ethnicity, iodine intake, BMI, smoking and age, as well

as inclusion of subjects with mild autoimmune thyroid dis-ease. In the case of age, there appear to be two effects depending on the iodine status of the population studied. In iodine-sufficient areas TSH increases with age, whereas in iodine-insufficient areas there is no increase and may be a decline. There is also an additional factor in that each assay recognizes TSH isoforms in serum differently and this may account for a divergence of up to 1 mU/L in mea-sured TSH. In pregnancy, because of the thyrotropin sup-pression by high concentrations of hCG in the first and second trimester, the upper limit of TSH is lowered to around 2.0 mU/L. In neonates, TSH concentration is higher but falls to adult ranges as growth progresses.

0.35–4.94 mIU/L 2nd IRP 90/558(Abbott Architect)

Clinical ApplicationsIn primary hypothyroidism, the thyroid gland fails to pro-duce sufficient T4, so that TSH increases, in severe cases by several orders of magnitude. The congenital hypothy-roidism of infants can be diagnosed by measuring TSH in blood spots taken soon after birth (generally after day 3).

In secondary hypothyroidism, TSH concentration can be low but is often normal or even slightly increased, but it is associated with a low free T4 concentration.

In hyperthyroidism, T4 and T3 are increased causing TSH secretion to be suppressed. This decrease is detectable in ultrasensitive immunometric assays for TSH (see Fig. 2).

TSH immunometric assays vary in their ability to dis-criminate between normal and suppressed TSH concentra-tions. The assay sensitivity of a TSH assay gives only an indication of the assay’s ability to discriminate hyperthyroid from normal. Sensitivity is a measurement of the within-assay error at a concentration of zero, normally quoted as the concentration of TSH that is two standard deviations above zero. The minimum detection limit is a more relevant parameter than the sensitivity, being the concentration of TSH that is statistically significant from zero, using the between-assay precision profile. Another useful measure-ment is the per cent coefficient of variation (%CV) of a con-trol or patient sample pool with a TSH concentration near the lower end of the reference interval. This should be determined over a number of assays (ideally at least 20) and using several batches of reagents. However the measure that has received widespread support is the functional sensitivity, which is the concentration at which the %CV is at a certain level, e.g., 20%. It is determined by plotting precision (%CV) vs. concentration. Klee and Hay (1989) proposed five criteria that TSH assays should fulfill if they are to be used as single tests for front-line screening for thyroid dis-orders (see REFERENCES AND FURTHER READING). However, many laboratories are now using TSH and free T4 together in front-line screening as neither test is considered reliable enough to be used alone in every clinical situation.

Serum TSH usually displays a diurnal rhythm with highest concentrations during sleep, but daytime concen-trations are sufficiently stable to allow samples to be taken at any time. Similarly there is little effect on TSH concen-trations of patients on L-T4 dosage so that the time of tak-ing a blood sample is immaterial.


LimitationsIn screening a well population for thyroid disorders, TSH is the most sensitive indicator of hypo- or hyperthyroidism, and when combined with an FT4 and/or FT3, allows all subjects to be categorized as hypo-, eu- or hyperthyroid. However, in patients, TSH concentrations as well as FT4 and FT3 may be affected by the illness or the drugs the patients are taking.

� In severe non-thyroidal illness, TSH can be suppressed below the reference range. However, using third genera-tion TSH assays, the suppression due to non-thyroidal illness is not as great as that seen due to hyperthyroidism. In less sensitive assays, there may be overlap in the low-ered TSH concentrations due to non-thyroidal illness and the TSH concentration due to hyperthyroidism.

� Both dopamine and glucocorticoid, used in the emer-gency room for medical treatment, can lower TSH concentrations.

� TSH may be reduced below the reference interval due to diurnal rhythm, starvation, or depression, and in the first and second trimester of pregnancy.

� TSH on its own will miss cases of secondary hypothy-roidism as TSH may often be within the reference interval or even slightly increased (due to the secretion of a less biologically active TSH).

� TSH tends to react to T4 treatment for hypothyroidism more slowly than free T4, sometimes lagging behind by several months. TSH in patients successfully treated with T4 replacement therapy tends to be lower than normal. This is because the ratio of T4 to T3 is increased compared to the normal situation where T4 and T3 are both secreted by the thyroid. Because T3 is the most active hormone in the tissues, the overall concentration of thyroid hormones needs to be higher, and this depresses TSH secretion.

� As with all two-site immunometric assays, heterophilic antibodies can interfere with TSH assays, generally causing an erroneously increased TSH result by cross-linking the capture and labeled antibodies, but occa-sionally producing an artifactually low TSH result by preferentially binding labeled antibody. Manufactur-ers minimize this interference by the addition of non-immune serum to the assay systems, but it can still present a problem in the occasional patient.

� Current immunometric assays for TSH differ in their specificity for TSH isoforms in serum, which can account for up to 1 mU/L difference in the results when determined by different assays.

Assay TechnologyAll laboratories use immunometric assays for TSH. Because of the very wide range of concentrations that need to be measured there are several critical aspects of assay design that have to be optimized correctly, and there are significant differences in quality between the many prod-ucts on the market. All assays should have a functional sen-sitivity of 0.01–0.02 mU/L to determine the difference between TSH in normal individuals and clinical hyperthy-roid patients but also be sufficiently sensitive enough to detect subclinical hypo- and hyperthyroidism, which includes patients who are on L-T4.

Most TSH assays utilize a capture antibody immobilized onto beads, tubes, wells, or microparticles. Some assays have a single incubation format in which sample and labeled antibody are incubated together with the immobilized cap-ture antibody. Many have a dual incubation format in which the sample is incubated with the capture antibody first, fol-lowed by a washing stage before the labeled antibody is added. The principles of immunometric assays are explained in COMPETITIVE AND IMMUNOMETRIC ASSAYS.

Products commonly used to measure TSH include the Architect® (microparticle chemiluminescent-immunoassay) and ADVIA Centaur® (acridinium ester based luminescence).

Signal measurement and separation equipment such as optics and washers must be of a high specification and maintained for TSH assays. Other sources of error in TSH assays are carry-over in sample dispensers, calibration error in very low concentration calibrators, the presence of TSH in the ‘zero’ calibrator, and bias in the curve-fit at low concentrations. The solid phase must have a high capacity for the capture antibody and the tracer must have a high specific activity, to give a good signal-to-noise ratio and a linear relationship between signal and TSH concentration.

Some TSH immunometric assays are affected by inter-fering factors in a small proportion of patient samples.

FIGURE 2 High-sensitivity thyrotropin (hs-TSH).


These can non-specifically cross-link the capture and labeled antibodies. An example is ‘anti-mouse’ activity, which can cross-link mouse monoclonal antibodies. This particular form of interference can be suppressed by the presence of mouse serum in one of the assay reagents.

One of the controls included in routine assays should have a concentration close to the lower limit of the refer-ence interval, to check the clinical discrimination between hyperthyroid and normal patient samples.

Types of SampleSerum or plasma. Some assays are restricted to serum sam-ples. Screening for congenital hypothyroidism in infants is carried out using blood spots absorbed onto filter paper.

Frequency of UseVery common.

THYROXINEThyroxine (T4) or tetraiodothyronine is produced in the thyroid gland by the coupling of two di-iodinated tyrosine molecules contained within the thyroglobulin molecule (see Fig. 3).

FunctionT4 is the thyroid hormone with the highest concentration in the body. It is normally present in blood at a concentra-tion that is 50–100 times greater than that of T3. However, T3 is several times more biologically active than T4. The most important function of T4 is to serve as the precursor and primary source of T3 in the tissues through deiodin-ation. For the effects of thyroid hormones see NORMAL THY-ROID FUNCTION.

Clinical ApplicationsThere has been a gradual decline in the number of labo-ratories that offer total T4 and total T3 for the investiga-tion of thyroid dysfunction. There are three reasons for this.

First, although serum concentrations of total T4 and total T3 reflect over or under production of thyroid hor-mone by the thyroid gland, both T4 and T3 are predomi-nantly bound to binding proteins in serum, so the concentration of total T4 and total T3 will also be deter-mined by the concentration of the binding proteins in serum. In the case of total T4, approximately 99.98% is bound to plasma proteins: thyroxine binding globulin (TBG) 60–70%, transthyretin or thyroxine binding pre-albumin (TTR/TBPA) 15–30%, and albumin 10%. For T3, approximately 99.7% is bound to TBG, TTR, and

albumin. As the concentration of binding proteins can be affected by numerous conditions—genetic, non-thy-roidal illness, drugs, pregnancy—changes in these can affect serum concentrations of total T4 and total T3, which will not accurately reflect any disease activity in the thyroid gland. To improve the diagnostic accuracy of total hormone measurements, an additional test to estimate the binding proteins, usually called the thyroid hormone binding ratio test, is done, which allows a cal-culated free hormone index (FT4I or FT3I) to be reported. Although this additional test and calculation helps correct some of the abnormalities in thyroid hor-mone binding, it does not correct all and is unreliable at extremes of thyroid hormone binding concentrations. The need to carry out an additional test in combination with total thyroid hormone makes it far less attractive than measuring free T4 and free T3 directly. As it is the very small free fraction of thyroid hormone that is physi-ologically active and reflects the output of the thyroid gland, convenient FT4/FT3 methods that can be auto-mated have obvious benefits compared to measuring total hormone and carrying out a binding ratio test. The concentration of free T4/T3 in serum is independent of the concentration of thyroid hormone binding proteins, so more accurately reflects thyroid gland output than total hormone concentrations.

Second, the reference intervals of both total T4 and total T3 are relatively wide, so that subjects with milder dys-function of the thyroid gland may not have abnormal serum total T4/T3; but concentrations remain within the reference intervals, so that the sensitivity of the assays for detecting mild disease is not very high.

Third, the introduction of methods of measuring free thyroid hormone by immunoassay that can be easily auto-mated has seen their widespread adoption into the clinical chemistry laboratory and they have become the methods of choice for investigating any thyroid dysfunction. In the year 2010 in the UK, only a minority of laboratories offered total T4/totalT3 assay; the majority now offer FT4/FT3 assays. For those laboratories which participate in the United Kingdom External Quality Assessment Scheme (UKNEQAS) for thyroid hormone, for total T4 and free T4, only 3.6% measure total T4; and for total T3 and free T3, 10.1% measure total T3. It would be expected that a further decline in laboratories offering total T4/total T3 will occur.

FREE T4The concentration of free T4 is governed by the concen-tration of Total T4 and serum binding capacity (sBC) (see FREE ANALYTE IMMUNOASSAY). It is this free fraction that constitutes the biologically active fraction of T4. Free T4 assays have to determine concentrations of just a few pico-grams/mL in the presence of bound T4, which circulates at 5000 times the free T4 concentration. A conventional competitive immunoassay cannot be used, as some of the labeled T4 would bind to the binding proteins as well as to the antibody, so that the final measurement would depend on the concentration of the binding proteins. Also, anti-bodies with a high affinity for T4 could remove some of the T4 from the binding proteins, increasing the apparent free hormone concentration.

FIGURE 3 Structure of thyroxine (T4).


Most free T4 assays do not measure free T4 directly, but are indirect methods. The principles are described in FREE ANALYTE IMMUNOASSAY. Standardization of free T4 assays is against the more direct methods of equilib-rium dialysis (ED) or ultrafiltration (Thienpont et al., 2010; NCCLS guideline C-45A, Vol. 24, No 31). Eleva-tion of the ultrafiltration method to “gold standard” sta-tus has recently been questioned (Christofides and Midgley, 2009; Midgley, 2011). A method to measure free thyroxine by isotope dilution tandem mass spec-trometry has recently been developed (Soldin et al., 2005). It is of paramount importance that all methods, including ED and ultrafiltration, undergo the free hormone validity tests, especially the serum dilution test described in the FREE ANALYTE IMMUNOASSAY chapter to demonstrate that they fulfill all the free hormone validity criteria.

Free T4 assays have the practical advantage that only one initial test of thyroid function is needed instead of two (T4 and T3 uptake). More recent free T4 assays overcome most of the limitations of FTI measurements mentioned previously and which have been outlined by Midgley (2001).

Reference Interval9.0–19.0 pmol/L (Abbott Architect)0.70–1.48 ng/100 mL (Abbott Architect)

Clinical ApplicationsFree T4 is important in the investigation of all patients suspected of thyroid disorders and can be of use in monitoring patients undergoing treatment. In most cases, free T4 is increased in hyperthyroidism and decreased in hypothyroidism (see Fig. 4).

Clinical Validation of Free Thyroid Hormone (FTH) AssaysThe purpose of clinical validation of FTH assays is to pro-vide the relevant information to the user and challenge the measurement procedure.

Information for the userInformation (FTH ranges) should be provided for the euthyroid population, hypothyroidism, hyperthyroidism, pregnancy (trimester specific ranges), effects of drugs, ranges obtained in hospitalized patients, and patients who have kidney, liver, and cardiac problems. Other informa-tion that should be provided includes the effect of potential interferents such as bilirubin, hemoglobin and lipids, and data from a study that compares the FTH performance in matched serum and plasma samples and the effect of sam-ple storage (fresh, stored at 2–8 °C, –20 °C and after freeze/thaw). The performance of the assay in the more rare situ-ations of familial dysalbuminemic hyperthyroxinemia (FDH), thyroid hormone autoantibodies, and patients with heterophilic antibodies/rheumatoid factor, should also be documented by the manufacturer (and sought by the user).

Challenge of the measurement procedure (free hormone validity)Challenge of the measurement procedure to demonstrate free hormone validity is discussed in the FREE ANALYTE IMMUNOASSAY chapter.

Analytical Validation of Free Thyroid HormonesSome of the tests normally carried out as part of the ana-lytical validation of immunoassay methods cannot be used

FIGURE 4 Free thyroxine (free T4).


in FTH assays. As discussed in the FREE ANALYTE IMMUNO-ASSAY chapter, recovery (of an IRP), recovery post dilution and linearity should not be performed, as the results obtained will depend on the sBC of the samples tested.

The manufacturer should provide the user with infor-mation on precision (within-day, between-day, total imprecision), sensitivity (LoD, LoQ, and LoB)—see CLSI (previously NCCLS) guidelines CLSI EP 15-A2 and CLSI EP 17-A.

Limitations � As discussed in the chapter FREE ANALYTE IMMUNOASSAY,

there are significant methodological differences in FT4 assays resulting in numerical disagreement between methods. These differences are especially evident in sera with low T4 binding capacities.

� Some immunoassays may yield artifactually high FT4 concentrations in patients with familial dysalbumin-emic hyperthyroxinemia. In these patients the mutant albumin has an affinity towards T4 that is approxi-mately 80 times higher than normal albumin. The arti-factual elevation of FT4 may be the result of interaction of this albumin with the immunoassay reagents (e.g. analog tracer) or due to the presence of specific buffer ions (e.g., chloride ions or dyes that may be used in the assay formulations) that cause dissociation of T4 from the mutant albumin.

� In patients successfully treated with T4-replacement therapy, free T4 tends to be about 20% higher than normal. This is because the ratio of T4 to T3 is increased, compared to the normal situation where T4 and T3 are both secreted by the thyroid, and T3 is the most active hormone in the tissues. Normal concentra-tions of free T4 may be associated with undertreated patients. FT3 (and TSH) are the analytes of choice when monitoring patients on T4-replacement therapy.

� Transient hormonal abnormalities may be found in sys-temically (non-thyroidal) ill patients.

� Patients receiving heparin can have biased free T4 results as heparin stimulates the production of non-esterified fatty acids, which displace T4 from albumin. Heparin can also arise from indwelling cannulas con-taining a heparin–saline solution.

� Free T4 is normal in T3-toxicosis. � Free T4 may be increased in patients receiving treat-

ment with amiodarone. � Transient elevations in FT4 concentrations may be

observed following administration of drugs (e.g. furo-semide, ketoprofen, phenylbutazone, mefenamic acid, probenecid, sulindac, and fenclofenac) that displace T4 from their binding proteins.

� FT4 concentrations fall during the second and third tri-mester of pregnancy and thus pregnancy specific euthy-roid ranges should be established and used.

� Some patient sera containing avid autoantibodies to thyroid hormones may interfere in FT4 methods based on 1-step methodologies.

� Although manufacturers include materials to minimize interference by heterophilic antibodies there is no guarantee that all such sera will be fully corrected.

Assay TechnologyThe physico-chemical principles governing the measure-ment of free T4 have been discussed in the chapter on FREE ANALYTE IMMUNOASSAY, which includes examples of the different generations of Free T4 assays.

Types of SampleSerum or plasma.

Frequency of UseCommon.

TRIIODOTHYRONINETriiodothyronine (T3) differs from T4 in that it has three iodine atoms instead of four. It is derived primarily from T4 through deiodination outside the thyroid gland (see Fig. 5).

FunctionAlthough T3 has only 1–2% of the concentration of T4 in serum, less is bound to proteins in the serum. The concen-tration of free T3 is about one-quarter of the concentration of free T4. However, because T3 has about 4 times the bio-logical activity of T4, T3, and T4 represent similar levels of thyroid hormone activity in the blood. In tissues, primarily the liver, T4 is converted to T3, so T3 is normally considered to be the primary hormone, and T4 is a prohormone. T3 has a shorter half-life in the blood (1 day) than T4 (7 days).

T3, like T4, increases the rate of metabolic activity in many tissues in the body. It is essential for normal physical and mental development throughout childhood and for controlling the rate of metabolic activity in adults. For a description of the effects of thyroid hormones see NORMAL THYROID FUNCTION.

About 99.7% of T3 is bound to proteins in the blood but it is the minute fraction of free, unbound T3 that is bio-logically active as only the free hormone can pass into the cells in the target tissues.

Clinical ApplicationsAs is the case for total T4, only a minority of laboratories offer a total T3 assay; the assay of total T3 has been replaced by FT3 assays. See CLINICAL APPLICATIONS for total T4.

FREE T3Free T3 is a more relevant indicator of thyroid function than total T3 but until recently free T3 was difficult to measure

FIGURE 5 Triiodothyronine.


directly. The concentration of free T3 in the blood depends on the concentrations of total T3 and the thyroid hormone binding proteins, TBG, TBPA, and albumin. About 99.7% of T3 is bound (80% to TBG, 10% to TBPA, and 10% to albumin). The technical problems associated with develop-ing tests for free T3 are similar to those described for free T4 (see chapter on FREE ANALYTE IMMUNOASSAY).

Reference Interval2.63–5.7 pmol/L (Abbott Architect)1.71–3.71 pg/mL (Abbott Architect)

Clinical ApplicationsFree T3 is an important test for diagnosing or monitoring hyperthyroidism. Free T3 is not suitable for diagnosing hypothyroidism as some hypothyroid patients have reduced free T4 and elevated TSH, but a normal free T3. However, clinical studies have shown that free T3 is a bet-ter indicator of hyperthyroidism than total T3, as it is inde-pendent of thyroid hormone binding protein concentrations (see Fig. 6).

FT3 (with TSH) is the analyte of choice (rather than FT4) for monitoring patients on T4 replacement therapy.

Limitations � Free T3 is unsuitable as a test for hypothyroidism

(approximately 50% of hypothyroid patients have FT3 concentrations that lie within the euthyroid range).

� Free T3 may be normal in T4-toxicosis. � Some patient sera containing avid autoantibodies to

thyroid hormones may interfere in FT4 methods based on 1-step methodologies.

� Low concentrations of albumin cause an artificial decrease in free T3 in some labeled analog assays.

� Free T3 may be decreased in very sick patients. � Patients receiving heparin can have biased free T3

results as heparin stimulates the production of non-esterified fatty acids, which displace T3 from albumin.

Heparin can also arise from indwelling cannulas con-taining a heparin-saline solution.

� Amiodarone treatment can cause reduced free T3 concentrations.

� Transient elevations in FT3 concentrations may be observed following administration of drugs (e.g. furo-semide, ketoprofen, phenylbutazone, mefenamic acid, probenecid, sulindac, and fenclofenac) that displace T3 from their binding proteins.

� Although manufacturers include materials to minimize interference by heterophilic antibodies there is no guarantee that all such sera will be fully corrected.

Assay TechnologyThe physico-chemical principles describing the assay design of most commercial FT3 assays have been discussed in the chapter on FREE ANALYTE IMMUNOASSAY.

Types of SampleSerum or plasma.

Frequency of UseFairly common in the UK, parts of Europe and Japan. Much less common than free T4.

THYROTROPIN RECEPTOR ANTIBODIESThyrotropin (TSH) exerts its effect of stimulating the thy-roid gland by binding to receptors on thyrocyte plasma membranes to cause activation of the cAMP and phospho-lipase C signaling pathways. The TSH receptor (TSH-R) belongs to the G protein coupled class of transmembrane receptors. In serum, circulating TSH-receptor subunits are found that have been shown to be a possible antigen in the autoimmune disease process. The thyrotropin recep-tor on thyroid follicular cells is the target of autoantibod-ies, the thyrotropin receptor antibodies (TRAb). These fall into two general classes that can either stimulate

FIGURE 6 Free T3.


(TSAb) or inhibit (TBAb) thyroid hormone secretion, causing Graves’ disease or hypothyroidism respectively. In the case of Graves’ disease, stimulation of the thyrotropin receptor by TSAb causes thyroid hyperplasia and hyper-thyroidism, whereas in hypothyroidism TBAb competes with TSH for the TSH-R and blocks the biological effect of TSH. Additionally TSAb has been implicated in the pathogenesis of Graves’ ophthalmopathy.

It is thought that although TSH, TSAb, and TBAb appear to bind to different sites on the TSH receptor, TSAb and TBAb have similar affinities with overlapping epitope specificities. In autoimmune thyroid disease, both autoantibodies can be present in some patients, but in the case of Graves’ disease, TSAb predominates, and in the case of hypothyroidism, TBAb predominates. Because both TSAb and TBAb can be present in the same patient, it has been proposed that it is the relative concentrations together with receptor-binding characteristics that could account for differences in severity of the hyperthyroidism of Graves’ disease as well as the response to antithyroid drug treatment or in pregnancy.

Reference Interval<1.0 IU/L regarded as negative1.0–1.5 IU/L regarded as gray-zone>1.5 IU/L regarded as positive(BRAHMS GmbH TRAK™ assay).

Clinical ApplicationsCurrently, TRAb assays have only a very limited applica-tion in clinical practice. The diagnosis of Graves’ disease can be made in most cases simply based on the patient’s clinical presentation with biochemical confirmation of the hyperthyroidism. The autoimmune basis of Graves’ dis-ease can be established by the more easily performed and inexpensive TPO-Ab assay in the majority of patients but with the introduction of newer assays with improved sen-sitivity and ease-of-use, TRAb measurement is coming into more general use. TRAb assays can be useful for spe-cial problems, as in patients who present with initial uni-lateral exophthalmos, euthyroid Graves’ ophthalmopathy, in subclinical hyperthyroidism, in pregnancy with Graves’ disease, in hyperemesis with thyrotoxicosis or in the dif-ferential diagnosis of postpartum painless thyroiditis.

In pregnancy, in women with Graves’ disease, transpla-cental passage of TRAb from mother to fetus can result in transient neonatal hyperthyroidism. This only affects 2–10% of women with Graves’, but the severity of neona-tal hyperthyroidism is related to high concentrations of TRAb in the mother’s serum, measured late in pregnancy. TSH-receptor antibodies should be measured in the last trimester and if high, careful evaluation of the neonate is required to detect hyperthyroidism. It is recommended that all pregnant women with active Graves’ hyperthyroid-ism or who have received previous treatment, whether sur-gery or radioiodine, have TRAb measured, as TRAb can remain increased despite the hyperthyroidism being ren-dered eu- or hypothyroid and treated with L-T4.

Very rarely, in Hashimoto’s disease, blocking antibodies may cross the placenta in pregnant women and lead to a transient neonatal hypothyroidism. This should always be

considered if a neonate tests positive with an increased blood spot TSH concentration in a congenital hypothy-roid screening program and the mother is hypothyroid and on T4 replacement. The presence of TBAb in the mother and infant confirms the transient nature of the hypothy-roidism in the newborn.

Assay TechnologyThyrotropin receptor antibody can be measured in one of two ways. The specific measurement of stimulating and blocking activity requires a bioassay using intact cells. These use the cAMP second messenger system as a bio-logical endpoint to detect stimulating or blocking activity. Early assays were cumbersome and time consuming and used either human or animal cells expressing TSH recep-tors. Cells were obtained surgically from human thyroids, or animal cells from mice, guinea pig or rat FRTL-5 cell lines. Pre-extraction of the immunoglobulin from serum samples was required. Some ease of preparation was obtained by the use of endogenously-expressed or stably-transfected human TSH receptors and unextracted serum but the assays remain the province of the research labora-tory and have not come into general clinical use. TRAb bioassays use the increase in cAMP production when TSAb antibodies are present in serum, whereas TBAb are measured by inhibition of cAMP production. When both TSAb and TBAb are present together in a patient’s serum, this can make interpretation of the results difficult.

Alternatively the presence of TSH-R antibodies in serum has been identified by a receptor assay using iso-lated, solubilized or recombinant TSH receptors. TRAb can be detected in serum in assays using the inhibition of 125I-labeled TSH binding to thyroid cell membranes. Antibodies detected by the binding assays are termed thy-rotropin binding inhibitory immunoglobulins (TBII). These assays are unable to distinguish between stimulating and blocking antibody activity, but this is rarely a problem as patients present with clinical features of either hypo- or hyperthyroidism. Early assays used 125I-labeled TSH, and animal tissues as a source of the TSH receptor, but non-radioactive labels such as chemiluminescent-labeled TSH and human, recombinant TSH receptor preparations have been introduced. These newer assays are claimed to offer superior diagnostic sensitivity for Graves’ disease over ear-lier assays. A further development in assay technology has been the replacement of the labeled TSH with TSH-receptor stimulating monoclonal antibodies. With the newer assays, some degree of semi-automation or full automation has been achieved and these assays are com-mercially available. An International Standard has been introduced (TSAb WHO standard 90/672) so assays should be standardized against this standard. Most studies of thyrotropin receptor antibodies (TRAb) have used the generally available assay by competitive inhibition (TBII).

LimitationsThe TSH-binding inhibition immunoglobulin (TBII) test non-selectively detects antibodies that bind to cell mem-brane receptors for TSH, whereas Graves’ disease is caused by antibodies that not only bind but also stimulate the TSH receptors. Using the research version of the BRAHMS


TRAK assay in 86 patients with untreated Graves’ disease and 282 healthy individuals the diagnostic sensitivity was 98.8% with a specificity of 99.6%.

The measurement of TRAb has not been found to be sufficiently sensitive to predict relapse or remission in the treatment of Graves’ disease or to predict the severity and outcome of Graves’ opthalmopathy. (See Fig. 7).

Type of SampleSerum.

Frequency of UseUncommon. This test is normally only carried out in spe-cialist centers.

THYROID PEROXIDASE ANTIBODIESThe autoimmune thyroid diseases (AITD) include Graves’ disease, lymphocytic thyroiditis (Hashimoto’s thyroiditis and primary myxedema), and post-partum thyroid dysfunc-tion and are all diseases in which the immune system is pri-marily responsible for the disease process (although not necessarily the initiator). The thyroid autoantibodies to thyroid peroxidase (TPOAb) and thyroglobulin (TgAb) are a secondary response to thyroid injury. Both are polyclonal IgG antibodies and the amounts present correlate with lymphocytic infiltration of the thyroid, have complement-fixing cytotoxic activity and TPO autoantibodies correlate with thyroidal damage. The finding of TPOAb in serum indicates thyroid autoimmunity and may hence be associated with present or future occurrence of other organ-specific autoimmune diseases. Patients with eye signs typical of Graves’ ophthalmopathy but without any hyperthyroidism present are often found to have TPOAb, confirming the association with autoimmune thyroid disease despite nor-mal thyroid function. In non-immune thyroid disease e.g. thyroiditis (subacute or de Quervain’s and Riedel’s) and non-toxic goiter, although immunological disturbances occur, these are secondary to the disease process.

Reference Interval<5.61 IU/ml (Abbott Architect)

Clinical ApplicationsThe main indication to measure TPOAb is to establish autoimmunity as the basis of any thyroid dysfunction,

either Hashimoto’s or Graves’ disease, so as to distin-guish it from other forms of thyroid failure. Assays for TPOAb are more sensitive for the diagnosis of autoim-mune thyroiditis than TgAb, but with quantitative sensi-tive assay one or both antibodies are found in almost 100% of patients. TPOAb is of higher affinity and is usually present in higher concentrations than TgAb. High concentrations of thyroid antibodies confirm the diagnosis of primary autoimmune disease in patients in whom the clinical picture is unclear. Generally the higher the TPOAb concentration, the more severe the disease process, as TPOAb has been implicated as a cytotoxic agent in the destructive thyroiditic process. Once measured and found to be present, serial TPOAb concentrations do not need to be determined and have no benefit in monitoring treatment. An increased TPOAb concentration may be an indication to start treatment with thyroxine in those patients who are sub-clinically hypothyroid with an increased TSH and nor-mal FT4, as progression to overt thyroid failure occurs at the rate of 5% per year.

In women, there is a prevalence of post-partum thyroid disease of about 6% occurring around 6 months after delivery. Approximately 50% of women who are TPOAb positive early in pregnancy go on to develop some form of post-partum thyroid dysfunction, usually of a transient nature. High concentrations of TPOAb identify those women who are at risk of developing post-partum thyroid dysfunction, which may be severe enough in some women to warrant treatment. In pregnancy, the presence of TPOAb has been linked to a number of reproductive com-plications including miscarriage, infertility, IVF failure, fetal death, preeclampsia, and pre-term delivery, but whether this is a cause or effect remains to be resolved.

As AITD occurs commonly with other autoimmune dis-eases, such as insulin-dependent diabetes mellitus, which places them at increased risk of developing AITD, the presence of TPOAb is helpful in selecting which patients require monitoring of their thyroid function (see Fig. 8).

Limitations � In the normal population, the prevalence of thyroid

autoantibodies depends on the method used for their analysis. TPOAb is common in the general population and occurs more frequently in women than men, and in the elderly.

� TPOAb may be found in low concentrations in some healthy individuals with completely normal thyroid function. The significance of this is uncertain, but may

FIGURE 8 TPO antibodies.

FIGURE 7 Thyrotropin receptor antibody (TRAb).


be related to ethnic or geographic areas. A higher prev-alence of TPOAb occurs in iodine-sufficient areas (USA and Japan) than in iodine-deficient areas such as Europe.

� TPOAb can also be present in sera from patients with autoimmune disorders other than those directly related to thyroid disease, with no evidence of a thyroid dysfunction.

� In a small percentage of patients with increased serum TSH and autoimmune disease of the thyroid, TPOAb may not be detected in their sera.

Assay TechnologyOlder methods, such as hemagglutination, which has low sensitivity and is operator dependent, and immunofluores-cence, are now obsolete. These assays have been replaced by quantitative assays with better sensitivity and specific-ity, which are more precise. The principal antigen in these tests is the thyroid peroxidase (TPO) enzyme, a glycosyl-ated protein found in thyroid microsomes. They use a variety of techniques including radioimmunoassay, immu-nometric assay, and enzyme-linked immunoassays that use purified or recombinant TPO. These assays are available on most automated immunoassay analyzers, so can be done with FT4/TSH as part of thyroid function testing strategy. There is an International Reference Preparation (MRC 66/387) as calibrant, but there is wide variability in results from different TPOAb assays. Both the purity of the reagents used and the principles of the methodology contribute to these differences. There are also consider-able differences in the sensitivity (limit of detection) and reference intervals all of which contribute to the interpre-tation of whether TPOAb is present in a patient’s serum.


Frequency of UseWidely available.

THYROGLOBULIN ANTIBODIESIn Hashimoto’s thyroiditis, immunocytes invade the thy-roid gland and synthesize antibody to thyroglobulin (TgAb). Thyroglobulin is a large protein, and is the site of synthesis of T3 and T4. TgAbs belong predominantly to the immunoglobulin G (IgG) class, do not fix complement, and are not known to play a direct pathogenic role in the etiology of autoimmune thyroid disease.

Reference Interval<4.11 IU/L (Abbott Architect)

Clinical ApplicationsAs with tests for TPO antibodies, the presence of thyro-globulin antibodies indicates the autoimmune basis of Hashimoto’s thyroiditis or Graves’ disease. (see Fig. 9). However, they are less often present and less pathogenic than TPO antibodies, so this test is not as useful. Surveys

have also shown that TgAb can be present in a small per-centage of subjects without any thyroid disease, absent TPOAb in sera and normal TSH, also making it less useful. If autoimmune thyroid disease is present, the use of TgAb with TPOAb appears to offer greater diagnostic efficacy than measuring TPOAb alone. However for most laborato-ries, if a sensitive immunoassay for TPO-Ab is available, only TPO-Ab need be done.

As TgAb can interfere in the assay for thyroglobulin used for monitoring thyroid cancer patients, there is a requirement to measure TgAb in these patients and this is the main use of this assay. Approximately 20% of patients with differentiated thyroid cancer have TgAb present in their sera and, as the interference is unpredictable, current guidelines recommend screening all samples submitted for Tg assay with a sensitive TgAb assay (see THYROGLOBULIN ASSAY TECHNOLOGY). An interesting observation has been the decline seen in TgAb in cancer patients after surgery to undetectable amounts by three years after surgery. If how-ever there is a recurrence, then this decline is arrested and increasing amounts of TgAb result, and this may precede the increase in Tg, so making it an additional serial marker in monitoring tumor re-growth in these Tg cancer patients with TgAbs. It would be essential to retain the same TgAb assay if this serial TgAb were being monitored in thyroid cancer patients.

Assay TechnologyTgAb is measured by the same techniques as TPOAb (see previously).


Frequency of UseUncommon, only required when measuring Tg in thyroid cancer patients.

THYROGLOBULINThyroglobulin (Tg) is a large (660 kDa) glycoprotein, which is the site of synthesis of T3 and T4 in thyroid follicular cells. It also acts as a large storage reservoir of thyroid hormones. TSH regulates Tg in thyroid follicular cells by stimulating endocytosis at the apical membrane and proteolysis of Tg, thus releasing T4 and T3 to the circulation. Nearly all of the protein is present within the thyroid but a small amount is normally detectable in blood. Serum Tg is increased in

FIGURE 9 Tg antibodies.


many patients with thyroid disorders, such as goiter, benign thyroid nodules, thyroid adenomas, multinodular goiters, thyrotoxicosis, and in the toxic phase of thyroiditis.

Reference Interval3–40 µg/L in normal subjectsUndetectable in treated thyroid cancer patients.

Clinical ApplicationsThyroglobulin assays are now in widespread use as a tumor marker for monitoring patients with differentiated thyroid carcinoma. In patients with papillary or follicular carcinoma, and following total thyroidectomy and radioiodine ablation to remove the tumor, previously increased Tg concentra-tions will reduce to very low or undetectable levels. An increasing Tg concentration, when on a suppressive dose of thyroxine, indicates the recurrence of tumor or metastatic spread. Patients should be monitored every 6–12 months, but at more regular intervals if a detectable Tg concentration is found. This serial measurement of serum Tg in conjunc-tion with ultrasound examination has proved to be very effec-tive and is the mainstay in the detection of early tumor recurrence. Most patients are successfully free of disease after their surgery, but a significant number (approximately 15%) have recurrence of their disease. If there is recurrence it usu-ally occurs within 5 years but may be several decades later.

In comparing results from Tg measurements with whole body scans, it is important to realize that metastatic cells are unable to take up iodine and will give a negative scan result, but are capable of releasing Tg into the circulation that will give a positive Tg result. Tg assays have no place in the initial diagnosis of thyroid carcinoma, because patients with benign nodules also have increased serum Tg concentrations.

Serum Tg measurements can be of use in infants to eval-uate the cause of congenital hypothyroidism. When athy-reosis (absence or functional deficiency of the thyroid gland) occurs, serum Tg is undetectable; in defects in thy-roglobulin synthesis, inactivating mutations of the TSH receptor, and thyroid transcription factor-1, serum Tg is low or undetectable; whereas in infants with thyroid dys-genesis and other defects in thyroid hormone biosynthesis, serum Tg is normal or high. Another use has been in iden-tifying factitious hyperthyroidism due to ingestion of too much T4, when Tg is not increased, unlike true hyperthy-roidism, in which Tg is increased.

Assay TechnologyThe measurement of thyroglobulin in serum is not straightforward, as a number of factors need to be consid-ered before selecting a suitable assay. Thyroglobulin is measured by competitive and immunometric assays.

One major problem is the presence in approximately 20% of sera from thyroid cancer patients of TgAbs. These can cause interference in Tg measurement that is variable and cannot be predicted from the titer of TgAb present, and not all TgAbs will cause interference. In the case of a competitive assay, interference can cause either an under-estimate or overestimate of Tg, depending on the affinity of the TgAb and the assay reagents and also the method of separating antibody bound and free label, although most competitive assays will show an overestimate. With

immunometric assays the interference will always produce an underestimate of the patient’s Tg result. As most com-mercial Tg assays are immunometric due to ease of auto-mation, this interference severely limits the usefulness of Tg assays in thyroid cancer patients. One commercial assay claims to have minimized this interference by utiliz-ing a number of monoclonal antibodies that are directed to epitopes not involved with thyroid immunity. However, this may not remove interference in all samples.

When the same samples from TgAb-negative subjects are measured by competitive and immunometric assay, reasonable agreement is found but when TgAb positive subjects are used, a significant underestimate of Tg by immunometric assay occurs, resulting in a divergence in Tg results by competitive and immunometric assays.

If using an immunometric Tg assay, what procedure needs to be in place? Current guidelines state that a sensi-tive TgAb be done on every sample, and if present, the Tg should not be reported. For these samples, Tg can be mea-sured by a competitive assay. A number of commercial Tg immunometric assays recommend a recovery test by set-ting up an additional tube with added Tg and comparing the measured Tg to determine the recovery of Tg. If the recovery is outside certain limits, the Tg is not reported. However the use of Tg recovery has been found to be unreliable in detecting interfering antibody and should not be used to replace the measurement of TgAb.

All Tg immunoassays should be standardized against the Certified Reference Preparation CRM-457. However one manufacturer, although basing their calibration against the standard, has not opted for a one-to-one calibration, but has chosen a different ratio to maintain consistency of Tg results with a previous assay. This and other factors thus account for the large (2–3 fold) difference in Tg values from different assay systems. Some of these differences can result from the problem that different assays have in detecting Tg in cancer patients, which may be heteroge-neous compared to normal Tg. Competitive assays with polyclonal Abs have an advantage as they have broad speci-ficity, and are more likely to detect Tg isoforms than immunometric assays that use monoclonal antibodies, which do not recognize all these isoforms.

The functional sensitivity of a Tg assay is crucial as a con-sensus report found that an undetectable serum Tg mea-sured during thyroid hormone suppression of TSH is often misleading. With a cut-off of 1 µg/L, patients with metasta-ses were not being detected, but were detected after recom-binant TSH stimulation with concentrations of Tg >2 µg/L. Early Tg assays had functional sensitivities of 0.5–1.0 µg/L, close to the cut-off of 1 µg/L, whereas current commercial assays show much improved functional sensitivities ranging from 0.05–0.1 µg/L. With these improved assays, an unde-tectable Tg can be more reliably identified and with a func-tional sensitivity below 0.1 µg/L, a basal (unstimulated) Tg predicts a negative recombinant TSH stimulation test. Use of these sensitive Tg assays should obviate the need for expensive recombinant TSH stimulation of Tg testing. A consensus statement from a group of thyroid cancer special-ists proposes a surveillance guideline using TSH-stimulated Tg for patients who have undergone total or near total thy-roidectomy and 131I ablation for differentiated thyroid can-cer and have no clinical evidence of residual tumor with a serum Tg <1 µg/L during L-T4 suppression.


With the requirement to be able to identify an increase in Tg near the functional sensitivity of the assay, there is a need to maintain between-run precision at these critical concentrations, as clearly any changes in reagents or cali-bration can be crucial.

As with any tumor marker test, very high serum Tg concentrations can be seen in some patients, so providers of Tg assays should be aware of the Tg concentration above which a “high dose hook effect” occurs, as an inap-propriately low Tg will result. Manufactures of Tg assays should state the Tg concentration below which a high dose hook effect does not occur. If this is at sufficiently high a Tg concentration, then running every sample undiluted and at a set dilution will not be required. If an unexpectedly low Tg result is obtained in a patient with known metastatic disease, then an appropriate dilution of the serum should be assayed alongside an undiluted sam-ple. If the Tg is very high and a high-dose hook effect occurs, the diluted Tg (corrected for dilution) will be higher than the undiluted result.


Frequency of UseUncommon. This test is normally only carried out in spe-cialist centers.

Thyroid Testing StrategiesMost clinical biochemistry laboratories have experi-enced an inexorable increase in requests for thyroid function tests. To meet this demand, laboratories have instituted guidelines for appropriate requesting of thy-roid function tests, introduced automated procedures and adopted testing strategies. For most samples received, there is a low suspicion of a thyroid disorder and the request merely requires a rule out of any thyroid dysfunction as a cause of a patient’s illness. In this situa-tion, the ideal initial screening test would be sensitive enough to detect any degree of thyroid disorder, give a normal result when thyroid disease was not present and not be affected by a patient’s illness or prescribed drugs. There is no single test of thyroid function that is totally reliable in all these situations. Screening could be done by means of a single test, either free T4 or TSH but both have their limitations: free T4 can be lowered in non-thyroidal illness, and TSH is normal in secondary hypo-thyroidism. Both need to be followed up with additional tests, TSH if free T4 is used as a frontline screen, or free T4 if TSH is used as a frontline screen. In a small num-ber of samples, free T3 may also be required. A more satisfactory procedure is to assay both FT4 and TSH on all new requests for thyroid function tests, but assay just TSH in follow-up samples in patients who are treated for hyperthyroidism or patients who are on L-T4 for hypothyroidism. In the case of patients receiving L-T4 replacement, monitoring correct dosage only requires that a TSH assay be carried out and this is sufficient in these patients. For completeness, the autoimmune basis of any abnormal thyroid dysfunction can be documented

by assaying for TPO-Ab and TRAb. Automated immu-noassay analyzers have the ability to reflex test from one analyte to another, after appropriate decision limits have been set. This ensures that a range of thyroid function tests is performed before samples are removed from the analyzer. Using the Siemens ADVIA Centaur as an example, and FT4 and TSH as the frontline screen, the time to a FT4 and TSH result is 20 min, reflex testing to a free T3 takes 40 min. Considerable cost and time sav-ings can be made by introducing a reflex testing strategy.

With the use of a screening strategy for thyroid function testing, consideration needs to be given to a patient’s pre-vious clinical history, treatment, and drug use to be able to interpret thyroid function test results in all situations. Most assays for free T4, free T3, and TSH are sufficiently sensitive, precise, and robust to accurately predict the thy-roid status in the majority of patients. However, there are exceptions, where seemingly anomalous results are obtained, and these require careful follow up. These con-ditions include antibody interference in any of the com-monly performed assays for free T4, free T3 or TSH, thyroid hormone resistance syndromes, TSH resistance syndromes, and pituitary TSH-secreting tumors. All labo-ratories should have a protocol for dealing with these rare occurrences. In the case of antibody interference, reassay by a different methodology, preferably one that is little affected by antibody interference, is often the first investi-gation, although other methods may also be affected. Dilutions of the sample and reassay as well as polyethylene glycol precipitation of the antibody fraction and reassay may also be helpful. For follow up of the other rare condi-tions listed above, referral to a specialist endocrine center is required, where appropriate testing can be carried out. See TSH, LIMITATIONS and FREE T4, LIMITATIONS.

A typical initial thyroid function testing strategy is shown in Fig. 10. Although not shown here, free T3 can be used for following up suspected cases of hyperthyroidism, including patients with clinical symptoms of hyperthyroid-ism but normal free T4 and TSH results. Whichever labo-ratory testing strategy is used, an overall picture of thyroid hormone concentrations, consideration of the patient’s symptoms and drug use, and a record of previous treat-ment are needed before the thyroid status of a patient can be reliably determined.

GUIDELINES ON STRATEGIES (OR ALGORITHMS) FOR THYROID FUNCTION TESTSStrategies used in the diagnosis of thyroid disease invari-ably require the demonstration of abnormal concentra-tions of TSH and abnormalities (usually in the opposite direction) in thyroid hormone concentrations.

Testing strategies involve the use of a front-line “screen-ing”, using either a single analyte (e.g., TSH) or multiple tests (e.g., TSH and thyroid hormones). The strategy used is often dictated by the reimbursement authorities, the perfor-mance/validity and ease-of-use of the tests provided by the laboratory (especially following the introduction of random-access analyzers) and by the particular preference of the clinician.


HypothyroidismDiagnosisMeasurement of serum TSH, using a precise and sensitive method, is often considered sufficient as the initial test in the diagnosis of thyroid disease. If TSH is raised, measure-ment of T4 (FT4 is preferable) is required for confirmation of primary gland failure.

The use of T3 or FT3 is not recommended. It is further recommended that testing for the presence of hypothy-roidism is carried out if there is clinical suspicion (see Table 1 for clinical features) or if risk factors are present (Table 2).

The symptoms are generally related to the duration/severity of the disease, rapidity with which hypothyroidism occurs and the psychological characteristics of the patient. The signs and symptoms can include one or more of the clinical features listed (Table 1).

Monitoring of treatmentMonitoring of treatment of hypothyroidism normally involves measurement of serum TSH. This is normally required once to three times a year in patients on T4 ther-apy. If poor compliance is suspected, serum T4 (FT4) should also be measured.

Because the TSH response lags behind the FT4 response it is advisable to monitor with FT4 at the early stages of ther-apy (i.e., when trying to establish the correct T4 dosage). Users of FT4 assays should be aware that euthyroid ranges for patients on T4 therapy need to be adjusted upwards (approximately 20% higher than for the normal population). There is a school of thought that believe monitoring using FT3 is superior (or at least equal) to TSH measurement.

HyperthyroidismDiagnosisClinical suspicion (see Table 3 for clinical features) of hyper-thyroidism is normally tested by use of a highly sensitive (3rd generation) TSH. Diagnosis of hyperthyroidism is made if serum FT4 is elevated and the TSH concentration is

FIGURE 10 Typical thyroid function screening strategy (first phase).

TABLE 1 Clinical Features of Hypothyroidism

FatigueConstipationWeight gainMemory and mental impairmentDecreased mental concentrationDry or yellow skinCold intoleranceDepressionAtaxiaCoarseness or loss of hairIrregular or heavy menses and infertilityHoarsenessMyalgiasGoiterHyperlipidemiaReflex delayRelaxation phaseBradycardiaHypothermiaMyxedema fluid infiltration of tissues

TABLE 2 Risk factors for Hypothyroidism

Age over 60 yearsFemale sexPresence of goiterHistory of thyroid disease and of thyroiditisFamily history of thyroid diseaseHistory of head or neck cancerOther autoimmune diseaseDrugs (e.g., lithium, amiodarone)Dislipidemia


<0.1 mIU/L. Serum FT3 measurement is recommended if the patient has low TSH but normal FT4.

The signs and symptoms of hyperthyroidism are second-ary to the effects of excess thyroid hormone in the circula-tion. The severity of symptoms may be related to the duration of the illness, the magnitude of hormone excess as well as the age of the patient. Table 3 illustrates the spectrum of possible signs and symptoms of hyperthyroidism:

Monitoring of TreatmentMonitoring of treatment for hyperthyroidism normally involves measurement of serum FT4 every few weeks after start of treatment, until symptoms abate. Serum TSH and FT4 should then be measured once or twice a year after successful completion of treatment.

Hospitalized PatientsDiagnosis of thyroid dysfunction in hospitalized, critically-ill, patients should be based on the FT4–TSH relationship. Per-sistence of an apparent diagnostic abnormality should be con-firmed before therapy is commenced. The positive predictive value for true thyroid disease for both FT4 and TSH mea-surement could be improved by using wider reference inter-vals than for unselected populations. Measurement of FT4 is these patients is challenging, often resulting in method-spe-cific FT4 values, reflecting the biasing effect of the low sBC found in these patients (see FREE ANALYTE IMMUNOASSAY).

SummaryWhile some screening strategies may rely on one initial measurement (e.g., TSH), the common link between all the strategies is that an abnormality in only one of the factors of

the pituitary-thyroidal axis is not sufficient for diagnosing the presence of thyroid disease. Diagnosis of thyroid dis-ease can only be made if both the TSH and the thyroid hormone concentrations are abnormal (i.e., high TSH/low FT4 or low TSH/high FT4 except in rare cases). The pres-ence of an abnormal TSH with a normal FT4 is likely to be due to subclinical thyroid disease. This is an asymptomatic disease, diagnosed solely from the biochemical profile obtained. If such patients are found also to have anti-TPO antibodies then there is an increased risk of acquiring full clinically overt thyroid disease some time in the future. A school of thought exists that believes treatment of all patients with subclinical hypothyroidism is beneficial.

LIMITATION OF USING A SINGLE TSH MEASUREMENT AS THE SOLE SCREENING METHODA common strategy involves the use of a front line TSH test with the subsequent measurement, if the TSH con-centration is found to be abnormal, of thyroid hormone (free T4).

However, this strategy has a significant limitation, since the TSH measurement is not infallible and may be inap-propriately within the euthyroid range (i.e., false nega-tive), resulting in patients being misclassified as euthyroid. The TSH concentration may be inappropriately normal in certain situations (Table 4).

False positives (i.e., inappropriately abnormal concen-trations of TSH) can also occur (see Table 5).

TABLE 3 Clinical Features of Hyperthyroidism

Nervousness and irritabilityHeat intolerance and increased sweatingWeight lossFrequent bowel movementsThyroid enlargement (depending on cause)Exertional intolerance and dyspneaMental disturbances,Changes in visionPhotophobiaEye irritationDiplosisExopthalmosImpaired fertilityPalpitations and tachycardiaTremorAlterations in appetiteFatigue and muscle weaknessPretibial myxedema (with Graves’ disease)Menstrual disturbance (decreased flow)Sleep disturbance (including insomnia)Dependent lower extremity edemaSudden paralysis

TABLE 4 Situations where TSH may be Inappropriately within the Euthyroid Range

Hypothalamic/pituitary diseasePituitary resistance to thyroid hormone (extremely rare)Presence of antibodies to TSHPresence of heterophilic antibody interference or interference by rheumatoid factorsCo-existence of overt thyroid disease with other systemic illnessDrugs that may “normalize” the TSH concentrations

TABLE 5 Situations where TSH Measurement is Inappropriately Outside the Euthyroid Range

First trimester pregnancy (low TSH)TSH-secreting tumor (high TSH)Acute psychiatric illness (low)Pituitary hypothyroidism (low)Thyroid hormone resistance (high)T4 therapy (early phase, TSH is high)Antithyroid treatment (early phase, TSH low)Non-thyroidal Illness (TSH is low in the acute phase and high during recovery)Drugs (glucosteroids, dopamine, amiodarone—low TSH; amphetamines—high TSH)Interference e.g., heterophilic and rheumatoid factors (TSH may be low or high)


INTERPRETATION OF BIOCHEMICAL PROFILESTable 6 summarizes the biochemical profiles (TSH/FT4/FT3) in different clinical situations. Note, that the FT4/FT3 results obtained are assay specific.

References and Further ReadingAbalovich, M., Amino, N., Barbour, L.A., Cobin., R.H., De Groot, L.J., Glinoer, D.,

Mandel, S.J. and Stagnaro-Green, A. Management of thyroid dysfunction dur-ing pregnancy and postpartum: an Endocrine Society Clinical Practice Guideline. J. Clin. Endocrinol. Metab. 92, S1–47 (2007).

Association for Clinical Biochemistry, British Thyroid Association, British Thyroid Foundation. UK Guidelines for the Use of Thyroid Function Tests. (2006) http://www.acb.org.uk/site/guidelines.asp.

Baloch, Z., Carayon, P., Conte-Devoix, B., Demers, L.M., Feldt-Rasmussen, U., Henry, J.F., LiVosli, V.A., Nicocoli-Sire, P., John, R., Ruf, J., Smyth, P.P., Spencer, C.A. and Stockigt, J.R. Laboratory Medicine Practice Guidelines: laboratory support for the diagnosis and monitoring of thyroid disease. Thyroid 13, 3–126 (2003).

Baskin, H.J., Cobin, R.H., Duick, D.S., Gharib, H., Guttler, R.,B., Kaplan, M.M. and Segal, R.L. American Association of Clinical Endocrinologists Medical Guidelines for Clinical Practice for the Evaluation and Treatment of Hyperthyroidism and Hypothyroidism. Endocr. Pract. 8, 457–469 (2002).

Cartwright, D., O’Shea, P., Rajanayagam, O., Agostini, M., Barker, P., Moran, C., Macchia, E., Pinchera, A., John, R., Agha, A., Ross, H.A., Chatterjee, V.K. and Halsall, D.J. Familial dysalbuminaemic hyperthyroxinaemia: a persistent diag-nostic challenge. Clin. Chem. 55, 1044–1046 (2009).

Christofides, N.D. and Midgley, J.E.M. Inaccuracies in the free thyroid hormone measurement by ultrafiltration and tandem mass spectroscopy. Clin. Chem. 55, 2228–2229 (2009).

Christofides, N.D., Sheehan, C.P. and Midgley, J.E.M. One-step, labeled-antibody assay for measuring free thyroxin. I Assay development and valida-tion. Clin. Chem. 38, 11–18 (1992).

Christofides, N.D., Wilkinson, E., Stoddart, M., Ray, D.C. and Beckett, G.J. Assessment of serum thyroxine binding capacity-dependent biases in free thyroxine assays. Clin. Chem. 45, 520–525 (1999).

CLSI EP 15–A2. User Verification of Performance for Precision and Trueness; Approved Guideline – Second Edition (www.clsi.org).

CLSI EP 17-A. Protocols for Determination of Limits of Detection and Limits of Quantitation; Approved Guideline (www.clsi.org).

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TABLE 6 Summary of the Biochemical Profiles (TSH/FT4/FT3) in Different Clinical Situations. L = Low, LN = Low normal, N = Normal, SR = Slightly raised, R = Raised

FT4 FT3 TSHClinical State of Patient Diagnosis Comments

L L R Grossly hypothyroid MyxedemaL N R Hypothyroid Hypothyroid FT3 concentrations are normal in about 50%

of hypothyroidsL-LN N R Mildly hypothyroid Mild hypothyroidismN N SR Euthyroid Subclinical

hypothyroidismPatients are at risk for development of clinical hypothyroidism

L-LN N L-N-SR Hypothyroid Pituitary hypothyroidism Biologically less active TSH may be secretedN-SR N-L N-SL-SR Euthyroid NTI Abnormalities are transientN R L Hyperthyroid T3-toxicosis Occurs in early hyperthyroidism, in relapse,

after drug therapy and in areas of iodine deficiency

R R L Hyperthyroid Hyperthyroidism Classical profileR R R Hyperthyroid Pituitary hyperthyroidism Elevated alpha subunit confirms

thyrotropinomaR R N-SR Euthyroid, Hypothy-

roid in certain tissuesThyroid hormone resistance

A familial condition with T3 receptor abnormalities

R N N Euthyroid Acute psychiatric illness Returns to normal as disease process settlesN N N-SR Euthyroid Pregnant 1st trimester pregnancy often associated with

decreased TSHSR N N Euthyroid T4 therapy Higher FT4 values are often required to

achieve euthyroidismN N L Euthyroid Antithyroid treatment At early stages of treatment TSH is often

abnormalR R N Euthyroid Drugs Thyroid hormone-displacing drugsSR SR N Euthyroid Pregnant 2nd and 3rd trimester pregnancy often

associated with lower FT4 and FT3 concentrations


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The immuassay handbook parte73

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Transcript of The immuassay handbook parte73