Laboratory Evaluation of Thyroid Function

14 SUPPLEMENT TO JAPI JANUARY 2011 VOL. 59 Laboratory Evaluation of Thyroid Function Shashank R Joshi Introduction The past Over the past five decades, improvements in the sensitivity and specificity of thyroid test methodologies have dramatically impacted the clinical strategies for detecting and treating thyroid disorders. In the 1950s, only one thyroid test was available - an indirect estimate of the serum total (free + protein-bound) thyroxine (TT4) concentration, using the protein bound iodine (PBI) technique. Since 1970, technological advances in radioimmunoassay (RIA) and immunometric assay (IMA) methodologies have progressively improved the specificity and sensitivity of the methods. The present scenario Currently, thyroid testing is performed on serum specimens using either manual or automated methods employing specific antibodies. Methodology is still evolving as performance standards are established by the professional organizations and new technology and instruments are developed by manufacturers. A multitude of tests are currently available for testing thyroid function: Serum-based methods are available for measuring both total (TT4 and TT3) and free (FT4 and FT3) thyroid hormone concentrations. In addition, measurements can be made of the thyroid hormone binding proteins, Thyroxine Binding globulin (TBG), Transthyretin (TTR)/Prealbumin (TBPA) and Albumin, as well as for the pituitary thyroid stimulator, thyrotropin (thyroid stimulating hormone, TSH) and the thyroid hormone precursor protein, Thyroglobulin (Tg). The recognition of autoimmunity as the leading cause of thyroid dysfunction, has led to the development and incorporation of tests to determine thyroid autoantibodies thyroid peroxidise antibodies (TPOAb), thyroglobulin antibodies (TgAb), and TSH receptor antibodies (TRAb). The current review will cover the current status and limitations of the thyroid testing methods most commonly Table 1 : Reference values of thyroid function test Test Range TSH 0.5-4.7mU/L T3 0.92-2.78nmol/L FT3 0.22-6.78 pmol/l T4 58-140 nmol/l FT4 10.3-35pmol/L Note Where possible manufacturers reference ranges should be confirmed locally using an adequate population size of at least 120 ambulatory subjects. For TSH, reference ranges should be established using specimens collected between 0800h and 1800h and using 95% confidence limits from log transformed data. Since TSH, free and total thyroid hormones change during pregnancy, trimester related reference ranges should be available with data generated locally or contrywise. 2 Department of Endocrinology, Grant Medical College and Sir JJ Group of Hospitals, Endocrinologist, Lilavati and Bhatia Hospital, Mumbai. used in clinical practice and as recommended by the global and Indian guidelines. 1 The Value of Laboratory Testing in Thyroid Diseases The value of clinical diagnosis in thyroid dysfunction is limited because clinical manifestations of the disease vary considerably; patients may present with diverse characteristics of the disease along with differing severity levels and non-specific signs and symptoms. Physicians do consider and rule out thyroid dysfunction more frequently than they establish a diagnosis of thyroid disorder. Considering this scenario, many patients with thyroid disorders will remain undiagnosed if laboratory evaluation of only those patients with clearly suggestive signs and symptoms of thyroid dysfunction is performed. It, therefore, becomes imperative to implement routine laboratory screening to identify such patients, so that appropriate treatment for thyroid disorders can be instituted or conservative monitoring carried out to anticipate potential future consequences. Reference values of laboratory tests of thyroid function (Table 1). The enhanced sensitivity and specificity of TSH assays have greatly improved the assessment of thyroid function tests. Since TSH levels change dynamically in response to the alterations of T3 and T4, the approach to evaluate whether the patient has thyroid disorder is to test the TSH levels first. The sensitive immunoradiometric assays (IRMA) for TSH are sensitive enough to distinguish between the lower limit of the reference range or suppressed values of TSH that are seen in thyrotoxicosis. Extremely sensitive TSH assays are now available; the 4 th /5 th generation assays can detect TSH levels as low as 0 0.004mU/L. However, for practical purposes, TSH values of 0.1mU/L are considered sufficient. If TSH levels are found to be abnormal, then circulating T3 and T4 levels should be estimated. Although radioimmuno assays are widely available to measure total T3 and T4, these are highly protein bound and several factors can influence their levels. Hence it is important to measure free or unbound T3 and T4 levels. Testing for thyroid dysfunction TSH is the first test to perform serum TSH level remains the single best test of thyroid function. Thyroid-stimulating hormone testing is the preferred approach because: 1. TSH is central to the negative-feedback system 2. Small changes in serum thyroid function cause logarithmic amplification in TSH secretion 3. The most advanced (third-generation) chemiluminescent TSH assays can now detect both elevation and significant lowering of TSH levels, and are capable of reliably measuring values <0.1mU/L, thus aiding detection of subclinical thyrotoxicosis. A normal TSH value is a sufficient indicator to stop further testing of thyroid function in most cases. However, in cases suggestive of possible hypothalamic pituitary disease (central), a free-t 4 level estimation is desirable. In such patients, TSH levels may not reliably indicate the regulation of T 4 replacement, and,

SUPPLEMENT TO JAPI JANUARY 2011 VOL. 59 15 TSH below normal Primary hyperthyroidism Pituitary/hypothalamic disease with central hypothyroidism (TSH unreliable) Prolonged thyrotroph cell suppression after recent hyperthyroidism in euthyroid or hypothyroid patient Old age Drugs, e.g., glucocorticoids, dopamine Problems with T 4 treatment : Overdosage in treatment for fatigue or overweight, Altered gastrointestinal absorption because of drugs or disease, Altered T 4 clearance because of drugs, Patient compliance problems, Prescription error, Testing too soon after T 4 dose decrease Many severe systemic illnesses (Sick Enthyrid State) therefore, may require estimation of free T 4 levels. 1 TSH testing should be commonly carried out in the following settings: In patients presenting with suspected goitres: Serum TSH levels must be measured. As screening for congenital hypothyroidism: A heel-prick blood specimen is used for determining serum TSH levels. This is an established screening test for congenital hypothyroidism and has been adopted as a routine screening measure in many countries. The practice of routine screening for congenital hypothyroidism by the TSH test should be more widely adopted and continued. The low cost filterpaper TSH methods will be available in India soon. In patients with atrial fibrillation, dyslipidaemia, osteoporosis, and infertility: Serum TSH levels should be measured at presentation. As screening for thyroid disorders in patients with unclear diagnoses: Serum TSH test should be carried out in all patients who have non-specific manifestations, are asymptomatic, and in whom the diagnosis is not clear. The high-sensitivity TSH test should be performed in such cases (where there s a low pre-test probability of the disease). The advantage of this test is that its negative predictive value is very high and a vast majority of the results come out negative. Measurement of serum TSH alone can suffice during sequential follow-up visits (after the first investigation has been carried out) in patients who have not received treatment for thyroid disorders and for those who may be at risk of developing thyroid dysfunction. Important considerations for the clinician if TSH is abnormal (Table 2) In patients with abnormal TSH concentrations, a focused history, physical examination (in particular thyroid gland examination), repeat TSH test, serum T 3 level determination and occasionally imaging studies need to be carried out. It is not uncommon to see that many patients with high TSH values are informed that they need to take thyroid medication life-long and after having been prescribed T 4, no further workup or explanation is undertaken. In patients with goitrous changes or the presence of thyroid nodules, TSH concentration may be in the normal range because of an unaltered thyroid function. This warrants Table 2 : Some causes of abnormal serum TSH concentrations TSH above normal Primary hypothyroidism Pituitary thyrotroph adenoma; Pituitary resistance to thyroid hormone (central hyperthyroidism) TSH, unreliable. Generalized thyroid hormone resistance Thyrotoxicosis from overly rapid correction of severe hypothyroidism with parenteral T 4 Old age Drugs, e.g., amiodarone Problems with T 4 treatment : Underdosage based on misleadingly, high total T 4 Altered gastrointestinal absorption because of drugs or disease, Altered T 4 clearance because of drugs, Patient compliance problems, Prescription error, Testing too soon after T 4 dose increase Recovery phase after severe systemic illness (Sick Enthyrid State) Combination of pulsatile TSH secretion and analytical precision limits Combination of pulsatile TSH secretion and analytical precision limits Antibody in patient serum against antibody in TSH assay, causing analytical artefact complete patient evaluation including testing for antithyroid antibodies, imaging ultrasound and fine-needle aspiration cytology. In patients whose TSH levels are abnormal, T 3 levels should be determined. Free T 3 level estimation is preferred over total T 3 estimation because these hormones are extensively (>99%) bound to plasma proteins and only the unbound forms are active. 3 Inappropriate TSH This is a biochemical diagnosis in which elevation in circulating FT4 and/or FT3 is associated with an inappropriately detectable or elevated serum TSH concentration. If this biochemical picture is observed then assay artefact/laboratory error should be considered first. Once the laboratory has excluded such explanations then the cause of true inappropriate TSH should be considered. The differential diagnosis are a TSH secreting pituitary tumour (TSH-oma) or a syndrome of thyroid hormone resistance. The finding of an elevated serum sex hormone binding globulin (SHBG) and circulating free a subunit may support the diagnosis of TSH-oma, as may the finding of hyper or hypo-secretion of other pituitary hormones. Pituitary imaging usually confirms the diagnosis but should not be undertaken until the appropriate biochemical confirmation has been made. A syndrome of thyroid hormone resistance can be confirmed by family history; sequencing of the b thyroid hormone receptor confirms the diagnosis. When an inappropriately detectable or elevated serum TSH is found in association with elevated circulating free T 3 and/or T 4 concentrations, the TSH is termed inappropriate. Such cases may occur due to assay artefacts or laboratory errors and this should be considered first. However, if on repeat determination, TSH is still found to be inappropriate, other common explanations for apparent elevation of FT4 should be considered. These include the presence of binding protein abnormalities (such as familial dysalbuminaemic hyperthyroxinaemia) or assay dependent antibody interference in the measurements of FT4, FT3 or TSH. To distinguish between TSHomas and thyroid hormone resistance, estimations of SHBG, a subunit and other anterior pituitary hormones may be carried out. Total T4 Several laboratories measure the total T 4 and total T 3 which

16 SUPPLEMENT TO JAPI JANUARY 2011 VOL. 59 is not a true reflection of the thyroid status of an individual. This is because thyroid hormones circulate in the body largely in the inactive form, bound to carrier proteins (thyroid binding globulin (TBG), transthyretin and albumin) while only the small unbound fraction is metabolically active. Moreover, in some clinical conditions, particularly those in which there is an alteration of the amount of carrier proteins, the total T 3 and total T 4 may be elevated while the thyroid functional state (free T 3 and T 4 levels) may be normal. Such conditions include: 1. Hereditary abnormalities of binding proteins: These include TBG deficiency or TBG excess, abnormal albumin levels and abnormal transthyretin levels. 2. Acquired deficiency of binding proteins: Conditions such as nephrotic syndrome may cause protein loss from the body. In severe liver disease, there s impaired production of proteins, and therapy with androgens or anabolic steroids may alter the levels of carrier proteins. 3. Drug-induced alterations in T4 binding to TBG: Therapy with salicylates, phenytoin, phenylbutazone may alter T4-TBG binding. 4. Presence of T4 antibodies. The development of newer immunoassay methods for determining free T3 and T4 has overcome many of these problems. Radioimmunoassay measurement of total serum T4 levels is highly sensitive in reflecting the hyperthyroid (85-95%) and the hypothyroid status (80-90%) of patients. Total T3 Currently routine measurement of serum T3 is not carried out (only T4 is measured) in patients suspected of having thyroid disorders. About 25% of patients with hypothyroidism have low normal T3 values. Free T3/ total T3 measurements, however, should be performed in the following settings: 1. In patients suspected of having T3 thyrotoxicosis. 2. In patients taking drugs that inhibit the peripheral conversion of T4 to T3 (such as dexamethasone, propranolol, propylthiouracil, amiodarone, and iodine-containing contrast media). 5 Testing both TSH and FT4 There are certain clinical situations where TSH testing must be coupled with testing the FT4 levels. Clinical situations where measurement of both serum TSH and FT4 is required are principally disorders where the pituitary-thyroid axis is not intact or is unstable. These situations include: Optimising thyroxine therapy in newly diagnosed patients with hypothyroidism. Diagnosing and monitoring thyroid disorders in pregnancy. Monitoring patients with hyperthyroidism in the early months after treatment. Diagnosis and monitoring treatment for central hypothyroidism. End-organ thyroid hormone resistance. Sick Euthyroid State. TSH-secreting pituitary adenomas. Women with type I diabetes should have their thyroid function, including serum TSH, FT4 and thyroid peroxidase antibody status, established preconception, at booking when pregnant and at 3 months post-partum. Possible subclinical hypothyroidism: If screening is performed, and a high serum TSH concentration is found, and the FT4 is normal, the measurement should be repeated 3-6 months later, along with measurement of serum FT4, after excluding non-thyroidal illness and drug interference. Overtly hypothyroid patients (who have serum TSH greater than 10 mu/l and low FT4 concentrations) should be treated with thyroxine. In patients with a high serum TSH level and normal FT4 concentrations (possible subclinical hypothyroidism), TSH measurements and FT4 should be repeated 3 to 6 months later, after precluding non-thyroid disorders and drug interference. In cases of doubt in identifying specimens in which both serum TSH and FT4 should be carried out, it is prudent to test all specimens for TSH and FT4, rather than test for TSH alone. 6 Testing TSH and FT4 and FT3 In hospital inpatient ICU: In the absence of an abnormal thyroid gland by careful physical examination, a hospital inpatient with a mild or moderate (<20mIU/L) increase in serum TSH and an estimated free T 4 (by either a free-t 4 test or a free-t 4 index) within the health-related reference interval can usually be followed without treatment and re-evaluated later. The same holds true for a patient with a subnormal serum TSH and estimated free T 4 and serum T 3 values that are not increased. In both cases, the great majority of patients do not have clinically significant thyroid disease. The responsibility of providing pertinent clinical information to help guide the lab in selecting the most appropriate thyroid function test lies with the requesting physician. However, if sufficient clinical details to allow the identification of patients with subnormal TSH and unaltered T3 and T4 values, are not available, then laboratories should measure serum TSH and FT4 on all specimens. This is a more prudent strategy than just measuring first-line serum TSH, and then carrying out FT4 and FT3 estimations later when indicated. Considerations which Alter Thyroid Levels Binding protein abnormalities can increase totalt 3 in the absence of hyperthyroidism, notably during estrogen treatment contraceptive pills, and pregnancy. If necessary, a T 3 -uptake test or thyroxine-binding globulin measurement can be used to calculate a free-t 3 index, or a free-t 3 test can be obtained to clarify an ambiguous increased total-t 3 result. When hypothyroidism is suspected, a free-t 4 estimate is appropriate because total-t 3 and free-t 3 tests have inadequate sensitivity and specificity in this setting. When hyperthyroidism is suspected, the combination of a free-t 4 estimate and a total- or free-t 3 estimate provides the most complete assessment of the severity of hyperthyroidism and identifies cases of T 3 -toxicosis, i.e. a selective increase of the serum T 3 concentration. In some centres, free-t 4 and -T 3 tests are routinely used when the TSH is increased, but in others, serum T 3 measurements are obtained only when the TSH is low and the free T 4 is within the reference interval. It is preferable to monitor both serum free T 4 and T 3 in patients with low serum TSH (other than hypothyroid patients taking T 4 ),even after the thyroid diagnosis is known, to establish patterns of increasing or decreasing values over time (Table 3) (Fig. 1a, b). 1,3

SUPPLEMENT TO JAPI JANUARY 2011 VOL. 59 17 Table 3 : Characterization of thyroid disorders according to results of thyroid function tests Disorder TSH T 4 T 3 FT 4 Tg TBG rt 3 ATPO ATG TBII TSI TBA Primary hypothyroidism N or N or N N or N or N or n n or Transient neonatal hypothyroidism N or N N N n Hashimoto thyroiditis hypothyroidism N or N or N or N or N n or n n or Graves disease N n or Neonatal Graves disease N n or n or n or TSH deficiency N or N n N n n n Thyroid dishormonogenesis N, N n N n n n or Thyroid hormone resistance N or N n N n n n TSH-dependent hyperthyroidism N n N n n n T 4 protein-binding abnormalities [*] N V V N N V + V n N n n n Nonthyroidal illness V N or V N N N or n N n n n Subacute thyroiditis [ ] or or or or or N or n n n n n TSH = thyroid-stimulating hormone; T 4 = thyroxine; T 3 = triiodothyronine; FT 4 = free thyroxine; Tg = thyroglobulin; TBG = thyroxine-binding globulin; rt 3 = reverse T 3 ; ATPO = antithyroidperoxidase; ATG = antithyroglobulin; TBII = TSH-binding inhibiting immunoglobulin; TSI = thyroidstimulating immunoglobulin; TBA = TSH receptor-blocking antibody; N = normal; n = negative; V = variable. * The spectrum of binding protein abnormalities includes increased or decreased TBG binding, increased or decreased transthyretin binding, and albumin binding. Subacute thyroiditis involves a transient period of hyperthyroidism followed by a transient hypothyroid state. (Reprinted from Fisha DA (ed) : Disorders of Thyroid Function, Quest Diagnostic Manual. 3rd Editor, p 268.) High TSH Primary Central Subclinical * * Normal Subclinical Hyperthyroidism Inappropriate TSH secretion Overt Hyperthyroidism Low Low Free T 4 High * ES, euthyroid sick; TT, thyroid in transition Fig. 1a : Thyroid Function Test Algorithm Thyroid Autoimmunity Thyroidspecific Autoantibodies (TPOAb, TgAb and TRAb) Tests for antibodies against thyroid-specific antigens, antithyroid peroxidase (TPO), thyroglobulin (Tg) and TSH receptors are used in the diagnosis of autoimmune thyroid disorders. Over the last five decades, antibody measurement techniques have evolved from semi-quantitative agglutination and complement fixation tests and whole animal bioassays to specific ligand assays using recombinant antigens and cell culture systems transfected with the human TSH receptor. Unfortunately, the diagnostic and prognostic value of these thyroid autoantibody measurements is hampered by differences in the sensitivity and specificity of current methods. Although autoantibody tests have inherent clinical utility in a number of clinical situations, these tests should be selectively employed. Thyroid peroxidase autoantibodies (TPOAb) Originally, thyroid peroxidase autoantibodies (TPOAb) were detected as thyroid microsomal antibodies by semi-quantitative complement fixation and tanned erythrocyte hemagglutination techniques and were labeled antimicrosomal antibodies (AMA). The principal antigen in the thyroid microsomes was recently discovered to be the thyroid peroxidase enzyme (TPO), a 100kD TSH High Normal Low Free T4 Free T4 Low Normal Normal High Subclinical Hyperthyroidism Subclinical Hyperthyroidism Fig. 1b : Algorithm for the Diagnosis of Thyroid Dysfunction glycosylated protein. Currently, automated tests are replacing the older manual agglutination tests. These new tests are more specific TPOAb immunoassays or immunometric assay methods, and are based on purified or recombinant TPO. Clinical Use of TPOAb Tests An abnormal TPOAb is detected in 15 to 20 percent of healthy euthyroid subjects and even higher percentages of patients with various non-thyroid autoimmune disorders. Approximately 70-80 % of patients with Graves disease and virtually all patients with Hashimoto s, atrophic thyroiditis or post-partum thyroiditis have TPOAb detected. In fact, TPOAb is implicated as a cytotoxic agent in the destructive thyroiditic process. TPO antibodies are involved in the tissue destructive processes associated with the hypothyroidism observed in Hashimoto s thyroiditis (Fig. 2). In the future, TPOAb measurement may be used as a prognostic indicator for thyroid dysfunction. Although the appearance of TPOAb usually precedes the development of thyroid dysfunction, recent studies suggest that a hypoechoic ultrasound pattern may precede a biochemical TPOAb abnormality, as shown in Figure 2. The paradoxical absence of TPOAb in some patients with unequivocal TSH abnormalities likely reflects the suboptimal sensitivity and/ or specificity of current TPOAb tests or non-autoimmune thyroid failure (atrophic thyroiditis). Although changes in autoantibody concentrations often reflect a change in disease activity, serial

18 SUPPLEMENT TO JAPI JANUARY 2011 VOL. 59 Prevalence of Thyroid Antidodies in the General Population NHANES III (n =16,869) 15 14.7 10 % Prevalence 5 0 Thayroid Ab Odds Ratio for (Low TT4+ TSH > 4.5 mu/l) Fig. 2 : Thyroid Autoantibody Prevalence and Associations with (Reprinted from Hollowell JG, Staehling NW, Hannon WH, Flanders WD, Gunter EW, Spencer CA, and Braverman LE. Serum thyrotropin, thyroxine, and thyroid antibodies in the United States population (1988 to 1994): NHANES III. 2002;J Clin Endocrinol Metab 2002,87:489-99) thyroid autoantibody measurements are not recommended for monitoring treatment for AITD. The prevalence of TPOAb is higher in patients with non-thyroid autoimmune diseases such as type 1diabetes and pernicious anemia. Aging is also associated with higher prevalence of TPOAb that parallel the increased prevalence seen in both subclinical (mild) and clinical hypothyroidism. A euthyroid subject with detectable TPOAb is at increased risk of development of hypothyroidism. Detectable level of TPOAb typically precedes the development of an elevated TSH and is therefore a risk factor for hypothyroidism. Moreover, reproductive complications (such as miscarriage, infertility, IVF failure, fetal death, pre-eclampsia, preterm delivery and post-partum thyroiditis and depression) have been associated with the presence of TPOAb. The enhanced sensitivity and specificity of the TPO immunoassay methods make them a more cost-effective option over the older semi-quantitative AMA agglutination tests, since they obviate the need for additional TgAb measurements in the routine diagnosis of autoimmune thyroid disorders. 7 Thyroglobulin autoantibodies (TgAb) Antithyroglobulin autoantibodies (TgAb) were the first thyroid antibodies to be recognized to circulate in patients with autoimmune thyroid disorders. The first TgAb methods were based on tanned red cell hemagglutination. Subsequently, methodologies have evolved in parallel with TPOAb methodology from semi-quantitative techniques, to more sensitive ELISA and RIA methods and more recently chemiluminescent immunoassays. Unfortunately, the intermethod variability of current TgAb assays is even greater than that of the TPOAb tests discussed above. Clinical Use of TgAb Tests Auto antibodies against Tg are encountered in autoimmune thyroid conditions, usually in association with TPOAb. However, the recent NHANES III study found that 3 % of subjects with no risk factors for thyroid disease had detectable TgAb without TPOAb. In these subjects with only TgAb detected, no association with TSH abnormalities was found so that the clinical significance of an isolated TgAb abnormality remains to be established. This suggests that it is unnecessary to measure 6.9 TPOAb +TgAb 5.7 TPOAb +Alone 3.1 TgAb Alone 34.7 6.1 0.6 both TPOAb and TgAb for a routine evaluation of thyroid autoimmunity. According to the current guidelines, all sera should be prescreened for TgAb by a sensitive immunoassay method prior to Tg testing. Therefore, TgAb is primarily used as an adjunct test for serum Tg estimation. TgAb is detected in approximately 20% of patients with differentiated thyroid carcinoma compared with 10% of normal subjects by the immunoassay methods. The threshold TgAb concentration above normal that precludes TgAb interference is either not known or does not appear to exist. False positives may occur due to assay artifacts or illegitimate transcription while false negatives results may be seen in patients with metastatic disease. TSH receptor autoantibodies (TRAb) TSH Receptor Antibodies (TRAb) were first recognized as long-acting thyroid stimulator (LATS) using mouse bioassays. These autoantibodies are directed against epitopes on the ectodomain of the TSH receptor. Methods for measuring TRAb are even more varied than for TPOAb and TgAb. Two classes of TRAb can be associated with autoimmune thyroid disorders (a) thyroid stimulating autoantibodies (TSAb) that cause Graves hyperthyroidism and (b) thyroid stimulation-blocking antibodies (TBAb) which block receptor binding of TSH. Each class of TRAb (TSAb and TBAb) may be detected alone or in combination in Graves disease and Hashimoto s thyroiditis. The relative concentrations of the two classes of TRAb may modulate the severity of Graves hyperthyroidism and may change in response to therapy or pregnancy. Clinical Use of TRAb Tests TRAb tests are used in the differential diagnosis of hyperthyroidism, the prediction of fetal and neonatal thyroid dysfunction due to transplacental passage of maternal TRAb and prediction of the course of Graves disease treated with antithyroid drugs. Although TBII assays do not directly measure the stimulating antibodies, these tests have comparable diagnostic sensitivity to TSAb bioassays (70-95%) for diagnosing Graves hyperthyroidism or detecting a relapse or response to therapy. The second generation assays employing human recombinant TSH receptor are now becoming available and are reported to have superior diagnostic sensitivity for Graves disease. Current tests are manual and expensive and vary in precision, sensitivity, specificity and reference ranges. However, the TBII tests are important for evaluating pregnant patients with a history of autoimmune thyroid disease, in whom there is a risk of transplacental passage of TRAb to the infant. The lack of specificity of the TBII methods is actually an advantage in this clinical situation, since a TBII test will detect both the stimulating and blocking classes of TRAb that can produce transient hyper- or hypothyroidism, respectively, in the fetus and newborn. TRAb plays an uncertain role in thyroid-associated ophthalmopathy (TAO), which appears to be exacerbated by radioiodine therapy. Since TRAb and other thyroid antibodies levels increase acutely significantly after radioiodine therapy, a TRAb measurement prior to radioiodine therapy may be useful to predict risk of TAO. However, prospective studies are needed to establish the clinical utility of TRAb measurement in this context. Patients with very high circulating concentrations of hcg due to choriocarcinoma or hydatiform mole, as well as a small number of pregnant patients, may have misleading positive results using TSAb assays. Thyroglobulin (Tg) methods Serum Tg measurement is used as a tumor marker in the

SUPPLEMENT TO JAPI JANUARY 2011 VOL. 59 19 management of patients with differentiated thyroid carcinomas (DTC). Current Tg methods are based either on IMA or RIA techniques. There is a trend for non-isotopic IMA methods to replace RIA methods because IMA methods are easier to automate, have shorter turn around times, wider working ranges and use reagents with a longer shelf life. 4 Thyroid Function Tests in Special Patient Populations Patients with atrial fibrillation, hyperlipidaemia, osteoporosis, infertility Patients presenting with atrial fibrillation, hyperlipidemia, subfertility and osteoporosis, should undergo serum TSH estimations as assessment of thyroid function because: Atrial fibrillation may be secondary to thyrotoxicosis in about 5-10% of patients. Osteoporosis may be secondary to hyperthyroidism and can be corrected by treating the underlying cause. Both hyper as well as hypothyroidism may be contributing factors in menstrual cycle disorders, fetal loss and infertility. Women with type 1 diabetes Type 1 diabetes in women raises their likelihood of developing post-partum thyroid dysfunction by three times. Women with type 1 diabetes should have their thyroid function (including TSH, FT4 and thyroid peroxidise antibody status) assessed at preconception, at the time of registration for pregnancy and at three months post-partum. Women with a past history of post-partum thyroiditis In women with post-partum thyroiditis, there is an increased long-term risk of developing hypothyroidism and its recurrence in subsequent pregnancies. Therefore, all women with a history of post-partum thyroiditis should be recommended to have a yearly thyroid function test, and also prior to and at 6 to 8 weeks after their subsequent pregnancies. Patients with diabetes The frequency of patients with type 1 diabetes and asymptomatic thyroid dysfunction is high. These patients should have a yearly thyroid function test. In patients with type 2 diabetes, thyroid function should be assessed at diagnosis, however, annual thyroid function assessment may not be recommended. Down syndrome and Turner s syndrome Patients of Down syndrome as well as Turner s syndrome are recommended to undergo thyroid function assessment annually, keeping in mind the high incidence of hypothyroidism seen in these patients. Patients receiving Amiodarone and Lithium Therapy with amiodarone is associated with iodide-induced thyroid dysfunction (hypothyroidism or hyperthyroidism) because of the presence of 75 mg iodine per each 200 mg tablet. Patients on amiodarone treatment should have thyroid function assessment at the time of beginning of amiodarone therapy and thereafter every 6 months during treatment and till 12 months after cessation of therapy. Lithium therapy (for bipolar disorder) is associated with mild to overt hypothyroidism in up to 34% to 16% of patients respectively, which can occur abruptly even many years after cessation of therapy. Thyrotoxicosis can also occur due to long-term treatment with lithium but is relatively rare. Therefore, all patients on lithium therapy should have a thyroid function assessment before commencement of treatment and thereafter every 6-12 months during lithium therapy. Post neck irradiation Patients who undergo surgery or external radiation therapy of the neck, or both, for head and neck cancer (including lymphoma) have a high incidence (up to 50%) of hypothyroidism. The incidence is particularly high in patients who undergo surgery and receive high doses of radiation because the effect is dose-dependent. The onset of overt hypothyroidism due to surgery or irradiation is gradual and may precede subclinical hypothyroidism for many years. In such patients, thyroid function assessment should be carried out annually. Following destructive treatment for thyrotoxicosis by either radioiodine or surgery Patients treated with radioiodine or those who undergo thyroidectomy should be screened indefinitely for the development of hypothyroidism or recurrence of hyperthyroidism. Assessment of thyroid function in these patients should be done four to eight weeks after treatment, followed by quarter yearly assessments for the subsequent year and annually thereafter. Treatment of thyrotoxicosis with anti-thyroid drugs Antithyroid drugs used in the management of thyrotoxicosis, carbimazole and propylthiouracil, decrease thyroid hormone secretion. Thyroid function assessment should be carried out every 1-3 months to determine whether stable hormonal concentrations have been reached when antithyroid therapy is instituted and annually thereafter if long-term treatment is used. Patients on thyroxine therapy In patients undergoing thyroxine therapy regardless of the cause, long-term follow-up with annual measurements of serum TSH are recommended. This helps to check compliance, verify the dosage and take account of variations in dosage requirements due to concomitant medications. In pregnant women, the dose may need to be increased by a minimum of 50 µg per day to maintain normal serum TSH levels. The TSH levels should be tested in each trimester. 8 Interferences with Thyroid Test Methodologies There are four categories of interferences in competitive immunoassays (IMA) as well as non-competitive IMAs: 1. Cross reactivity interferences 2. Endogenous analyte antibodies 3. Heterophilic antibodies 4. Drug interactions 1. Cross reactivity interferences Early TSH RIA methods had the limitation of cross-reactivity with glycoprotein hormones (such as LH, hcg). Currently, this problem has been almost completely overcome by using monoclonal antibodies for TSH IMA methods. Occasionally, however, unusual cross-reacting isoforms of TSH may be encountered while using the current assays. 2. Endogenous analyte antibodies Robbins et al were the first to report an unusual thyroxine binding globulin in the serum in 1956. Subsequently, autoantibodies against T3, T4 and TSH have been identified in the sera of patients with autoimmune thyroid disorders

20 SUPPLEMENT TO JAPI JANUARY 2011 VOL. 59 as well as non-thyroid disorders. A number of reports have shown interference due to T3, T4 and TSH autoantibodies leading to anomalous free and total thyroid hormone levels and TSH values. However, the currently used methods rarely have this interference problem. Characteristics of interference due to endogenous autoantibodies may lead to falsely low or falsely high values, depending upon the type of assay and its composition. 3. Heterophilic antibodies Heterophilic antibodies (particularly HAMA) may affect IMA methods more than competitive immunoassays by causing the formation of a bridge between the signal and capture antibodies. This creates a false signal resulting in a high value artifact. Moreover, the result may not be abnormal; it may be inappropriately normal. A potential for influencing results of neonatal screening also exists because antibodies are able to cross the placenta. Interference due to heterophilic antibodies can be classified into two categories: i. HAMA (or human anti-mouse antibodies) are relatively weak, polyreactive, multispecific antibodies that are frequently IgM. The presence of HAMA can alter the total as well as free T3, T4 and TSH results due to interference. Use of Fab fragments and heterospecies assay configurations can be employed as approaches to reduce this kind of interference. ii. HAAA. Specific human anti-animal antibodies (HAAA) are produced in response to well-defined specific antigens after exposure to therapeutic agents containing animal antigens (such as murine antibody) or by coincidental immunization through workplace contact (such as that which occurs in animal handlers). Though assays for HAMA have been developed, there are large inter-method differences and therefore the reliability of these tests is questioned. 4. Drug interferences In vitro and in vivo effects may occur due to drug interferences. When the specimen contains a sufficient concentration of an interfering therapeutic or diagnostic agent, it may lead to methodologic interference resulting in in-vitro effects. An example is that of heparin which, in the specimen can cause in-vitro stimulation of lipoprotein lipase; free fatty acids are liberated that inhibit T4 binding to serum proteins. On the other hand, when results are altered due to administration of an interfering therapeutic agent, then it is termed in-vivo effect. An example is that of furosemide which competitively inhibits thyroid hormone binding to the specimen, thereby causing an abnormal value (low) thyroid hormone result. Interference may also be secondary to certain pathologic conditions. For instance, in uraemia, abnormal serum constituents such as indole acetic acid may accumulate and cause interference. In addition, the presence of fluorophor-related therapeutic or diagnostic agents in the specimen may alter the results of thyroid tests that employ fluorescent signals. 1 Conclusion Thyroid disorders have diverse clinical manifestations therefore, on part of vigilant clinician every suspected case of thyroid disease needs to be evaluated with laboratory investigations. Thereby appropriate treatment for thyroid disorders can be instituted or conservative monitoring carried out to anticipate potential future consequences. The enhanced sensitivity and specificity of TSH assays have greatly improved the assessment of thyroid function tests. Since TSH levels change dynamically in response to the alterations of T3 and T4, the approach to evaluate whether the patient has thyroid disorder is to test the TSH levels first. When hypothyroidism is suspected, a free-t4 estimate is appropriate because total-t3 and free-t3 tests have inadequate sensitivity and specificity in this setting. When hyperthyroidism is suspected, the combination of a free-t4 estimate and a total- or free-t3estimate provides the most complete assessment of the severity of hyperthyroidism and identifies cases of T3-toxicosis, i.e. a selective increase of the serum T3 concentration. References 1. Spencer C. Thyroid Function Tests: Assay of Thyroid Hormones and Related Substances, www.thyroidmanager.com. 2010. 2. Supit EJ, Peiris AN. Interpretation of Laboratory Thyroid Function Tests: Selection and Interpretation. Southern Medical Journal. 2002;95:481-85. 3. Werner SC, Ingbar SH. Werner & Ingbar s The Thyroid: a fundamental and clinical text, 9th edition. Lippincott Williams & Wilkins. 4. Mascarenhas JMA. RxPG AIPG 2004 Book, 2006. 5. Daniels GH, Amiodarone-Induced Thyrotoxicosis, The J Clinical Endo & Metab 2000;86:3-8. 6. Walfish PG. Triiodothyronine and thyroxine interrelationships in health and disease. Can Med Assoc J 1976;115:338 42. 7. Düsünsel R, Poyrazoglu HM, Gündüz Z et al. Evidence of central hypothyroidism in children on continuous ambulatory peritoneal dialysis. Adv Perit Dial. 1999;15:262-8. 8. UK Guidelines for the Use of Thyroid Function Tests, 2006.