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© SUPPLEMENT TO JAPI • JANUARY 2011 • VOL. 59

Laboratory Evaluation of Thyroid Function
Shashank R Joshi

Introduction

used in clinical practice and as recommended by the global and
Indian guidelines.1

ver 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 Value of Laboratory Testing in
Thyroid Diseases

The past

O

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
TSH
T3
FT3
T4
FT4

Range
0.5 -4.7mU/L
0.92-2.78nmol/L
0.22-6.78 pmol/L
58-140 nmol/L
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.

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 4th/5th
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-T4 level estimation is desirable. In such patients, TSH levels
may not reliably indicate the regulation of T4 replacement, and,

Free T3 and T4 level estimation is preferred over total T3 and T4 estimation because these hormones are extensively (>99%) bound to plasma proteins and only the unbound forms are active. Testing too soon after T4 dose decrease problems.g. a subunit and other anterior pituitary hormones may be carried out. Altered T4 clearance because of drugs. serum T 3 and T 4 level determination and occasionally imaging studies need to be carried out. These include the presence of binding protein abnormalities (such as familial dysalbuminaemic hyperthyroxinaemia) or assay dependent antibody interference in the measurements of FT4. T3 and T4 levels should be determined. repeat TSH test. A syndrome of thyroid hormone resistance can be confirmed by family history. Such cases may occur due to assay artefacts or laboratory errors and this should be considered first. 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. The advantage of this test is that its negative predictive value is very high and a vast majority of the results come out negative. Once the laboratory has excluded such explanations then the cause of “true” inappropriate TSH should be considered. imaging ultrasound and fine-needle aspiration cytology. the TSH is termed ‘inappropriate’. • In patients with atrial fibrillation. Important considerations for the clinician if TSH is abnormal (Table 2) • In patients with abnormal TSH concentrations. • 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 T4. and in whom the diagnosis is not clear. The differential diagnosis are a TSH secreting pituitary tumour (TSH-oma) or a syndrome of thyroid hormone resistance. 59 15 Table 2 : Some causes of abnormal serum TSH concentrations TSH below normal • Primary hyperthyroidism • Pituitary/hypothalamic disease with central hypothyroidism (TSH unreliable) • • • • • • TSH above normal • Primary hypothyroidism • Pituitary thyrotroph adenoma. as may the finding of hyper or hypo-secretion of other pituitary hormones. • In patients with goitrous changes or the presence of thyroid nodules. Altered T4 clearance because of drugs. TSH is still found to be inappropriate. The high-sensitivity TSH test should be performed in such cases (where there’s a low pre-test probability of the disease).© SUPPLEMENT TO JAPI • JANUARY 2011 • VOL. The practice of routine screening for congenital hypothyroidism by the TSH test should be more widely adopted and continued. amiodarone Problems with T4 treatment : Overdosage in treatment for fatigue or • Problems with T4 treatment : Underdosage based on misleadingly overweight. 1 TSH testing should be commonly carried out in the following settings: • In patients presenting with suspected goitres: Serum TSH levels must be measured. estimations of SHBG.. may require estimation of free T4 levels. Total T4 Several laboratories measure the total T4 and total T3 which . causing analytical artefact complete patient evaluation including testing for antithyroid antibodies. This warrants • In patients whose TSH levels are abnormal. osteoporosis. Pituitary imaging usually confirms the diagnosis but should not be undertaken until the appropriate biochemical confirmation has been made. other common explanations for apparent elevation of FT4 should be considered. Prescription error. This is an established screening test for congenital hypothyroidism and has been adopted as a routine screening measure in many countries. e. To distinguish between TSHomas and thyroid hormone resistance. Testing too soon after T4 dose increase Many severe systemic illnesses (Sick Enthyrid State) • 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. sequencing of the b thyroid hormone receptor confirms the diagnosis. The finding of an elevated serum sex hormone binding globulin (SHBG) and circulating free a subunit may support the diagnosis of TSH-oma. e. Altered gastrointestinal absorption because of drugs or high total T4. If this biochemical picture is observed then assay artefact/laboratory error should be considered first.. • As screening for congenital hypothyroidism: A heel-prick blood specimen is used for determining serum TSH levels. Pituitary resistance to thyroid hormone (central hyperthyroidism) TSH. dyslipidaemia. a focused history. glucocorticoids. 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.g. Prescription error. Patient compliance problems. if on repeat determination. Patient compliance disease. However. • Generalized thyroid hormone resistance Prolonged thyrotroph cell suppression after recent hyperthyroidism • Thyrotoxicosis from overly rapid correction of severe hypothyroidism in euthyroid or hypothyroid patient with parenteral T4 Old age • Old age Drugs. The low cost filterpaper TSH methods will be available in India soon. Altered gastrointestinal absorption because of drugs or disease. dopamine • Drugs. therefore. no further workup or explanation is undertaken. TSH concentration may be in the normal range because of an unaltered thyroid function. FT3 or TSH. are asymptomatic. unreliable. 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. physical examination (in particular thyroid gland examination). When an ‘inappropriately’ detectable or elevated serum TSH is found in association with elevated circulating free T3 and/or T4 concentrations.

It is preferable to monitor both serum free T4 and T3 in patients with low serum TSH (other than hypothyroid patients taking T4). it is prudent to test all specimens for TSH and FT4. • End-organ thyroid hormone resistance. a T3-uptake test or thyroxine-binding globulin measurement can be used to calculate a free-T3 index. Radioimmunoassay measurement of total serum T4 levels is highly sensitive in reflecting the hyperthyroid (85-95%) and the hypothyroid status (80-90%) of patients. These situations include: • Optimising thyroxine therapy in newly diagnosed patients with hypothyroidism. • Monitoring patients with hyperthyroidism in the early months after treatment. In patients with a high serum TSH level and normal FT4 concentrations (possible subclinical hypothyroidism). free-T4 and -T3 tests are routinely used when the TSH is increased. at booking when pregnant and at 3 months post-partum.e. Moreover. 2. rather than test for TSH alone. • In some centres. 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. transthyretin and albumin) while only the small unbound fraction is metabolically active. In both cases. then laboratories should measure serum TSH and FT4 on all specimens. in some clinical conditions. 4. particularly those in which there is an alteration of the amount of carrier proteins. This is because thyroid hormones circulate in the body largely in the inactive form. the total T3 and total T4 may be elevated while the thyroid functional state (free T3 and T4 levels) may be normal. 1a. bound to carrier proteins (thyroid binding globulin (TBG). Considerations which Alter Thyroid Levels • Binding protein abnormalities can increase totalT3 in the absence of hyperthyroidism. • Women with type I diabetes should have their thyroid function. • When hyperthyroidism is suspected. after excluding non-thyroidal illness and drug interference. 59 • Possible subclinical hypothyroidism: If screening is performed. there’s impaired production of proteins. a selective increase of the serum T3 concentration. if sufficient clinical details to allow the identification of patients with subnormal TSH and unaltered T3 and T4 values. b). notably during estrogen treatment contraceptive pills. This is a more prudent strategy than just measuring first-line serum TSH. propranolol. however. Total T3 Currently routine measurement of serum T3 is not carried out (only T4 is measured) in patients suspected of having thyroid disorders. including serum TSH. but in others.even after the thyroid diagnosis is known. • TSH-secreting pituitary adenomas.1. • Diagnosis and monitoring treatment for central hypothyroidism. • Overtly hypothyroid patients (who have serum TSH greater than 10 mU/L and low FT4 concentrations) should be treated with thyroxine. a free-T4 estimate is appropriate because total-T 3 and free-T 3 tests have inadequate sensitivity and specificity in this setting. FT4 and thyroid peroxidase antibody status. The same holds true for a patient with a subnormal serum TSH and estimated free T4 and serum T3 values that are not increased. Hereditary abnormalities of binding proteins: These include TBG deficiency or TBG excess. In cases of doubt in identifying specimens in which both serum TSH and FT4 should be carried out. • Diagnosing and monitoring thyroid disorders in pregnancy. phenylbutazone may alter T4-TBG binding. phenytoin. abnormal albumin levels and abnormal transthyretin levels. In patients taking drugs that inhibit the peripheral conversion of T4 to T3 (such as dexamethasone. About 25% of patients with hypothyroidism have low normal T3 values. after precluding non-thyroid disorders and drug interference. and therapy with androgens or anabolic steroids may alter the levels of carrier proteins. or a free-T3 test can be obtained to clarify an ambiguous increased total-T3 result. the measurement should be repeated 3-6 months later. • When hypothyroidism is suspected. serum T 3 measurements are obtained only when the TSH is low and the free T4 is within the reference interval.16 is not a true reflection of the thyroid status of an individual. TSH measurements and FT4 should be repeated 3 to 6 months later. Presence of T4 antibodies. are not available.6 Testing TSH and FT4 and FT3 In hospital inpatient ICU: In the absence of an abnormal thyroid gland by careful physical examination. established preconception.or free-T 3 estimate provides the most complete assessment of the severity of hyperthyroidism and identifies cases of “T3-toxicosis”. However. © SUPPLEMENT TO JAPI • JANUARY 2011 • VOL. 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. Free T3/ total T3 measurements. 3. the combination of a free-T 4 estimate and a total. i. and then carrying out FT4 and FT3 estimations later when indicated. the great majority of patients do not have clinically significant thyroid disease. In severe liver disease. amiodarone. should be performed in the following settings: 1. The development of newer immunoassay methods for determining free T3 and T4 has overcome many of these problems. propylthiouracil. 2. a hospital inpatient with a mild or moderate (<20mIU/L) increase in serum TSH and an estimated free T4 (by either a free-T4 test or a free-T4 index) within the health-related reference interval can usually be followed without treatment and re-evaluated later. Acquired deficiency of binding proteins: Conditions such as nephrotic syndrome may cause protein loss from the body. In patients suspected of having T3 thyrotoxicosis. to establish patterns of increasing or decreasing values over time (Table 3) (Fig.3 .5 Testing both TSH and FT4 There are certain clinical situations where TSH testing must be coupled with testing the FT4 levels. Such conditions include: 1. • Sick Euthyroid State. and a high serum TSH concentration is found. If necessary. and the FT4 is normal. Drug-induced alterations in T4 binding to TBG: Therapy with salicylates. and pregnancy. along with measurement of serum FT4. and iodine-containing contrast media).

† Subacute thyroiditis involves a transient period of hyperthyroidism followed by a transient hypothyroid state. 2). TT. Quest Diagnostic Manual. Currently. TPO antibodies are involved in the tissue destructive processes associated with the hypothyroidism observed in Hashimoto’s thyroiditis (Fig. 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). Tg = thyroglobulin.© SUPPLEMENT TO JAPI • JANUARY 2011 • VOL. These new tests are more specific TPOAb immunoassays or immunometric assay methods. 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. and ↑ albumin binding. (Reprinted from Fisha DA (ed) : Disorders of Thyroid Function. Although the appearance of TPOAb usually precedes the development of thyroid dysfunction. 1a : Thyroid Function Test Algorithm Thyroid Autoimmunity Thyroidspecific Autoantibodies (TPOAb. ↓ or ↑ ↑ ↑ N N ↑ or ↓ TBG N N N N N N N rT3 ↓ ↓ ↓ ↑ ↑ ↓ ↑ ATPO N or ↑ N ↑ ↑ n or ↑ n n ATG N or ↑ N ↑ ↑ n or ↑ N N TBII N or ↑ ↑ n or ↑ ↑ ↑ n n TSI n n n ↑ ↑ n n TBA n or ↑ ↑ n or ↑ n or ↑ n or ↑ n n N N V+ N N ↑ ↑ V N or ↑ ↑ or ↓ n n n n n N N N N n n n n n n n n n n n n n n n n TSH = thyroid-stimulating hormone. ATG = antithyroglobulin. 59 17 Table 3 : Characterization of thyroid disorders according to results of thyroid function tests Disorder Primary hypothyroidism Transient neonatal hypothyroidism Hashimoto thyroiditis hypothyroidism Graves’ disease Neonatal Graves’ disease TSH deficiency Thyroid dishormonogenesis TSH ↑ ↑ ↑ ↓ ↓ N or ↓ ↑ T4 ↓ ↓ N or ↓ ↑ ↑ ↓ ↓ T3 N or ↓ ↓ N or ↓ ↑ ↑ ↓ ↓ FT4 ↓ ↓ N or ↓ ↑ ↑ ↓ ↓ Thyroid hormone resistance TSH-dependent hyperthyroidism T4 protein-binding abnormalities[*] Nonthyroidal illness Subacute thyroiditis[†] N or ↑ ↑ N V ↓ or ↑ ↑ ↑ V N or ↓ ↑ or ↓ ↑ ↑ V ↓ ↑ or ↓ ↑ ↑ N V ↑ or ↓ Tg N or ↓ N or ↓ N or ↓ ↑ ↑ ↓ N. rT3 = reverse T3. antithyroid peroxidase (TPO). 1b : Algorithm for the Diagnosis of Thyroid Dysfunction glycosylated protein. increased or decreased transthyretin binding. Over the last five decades. N = normal. and are based on purified or recombinant TPO. Unfortunately. these tests should be selectively employed. TBII = TSH-binding inhibiting immunoglobulin. 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). euthyroid sick. thyroid in transition Fig. recent studies suggest that a hypoechoic ultrasound pattern may precede a biochemical TPOAb abnormality. T3 = triiodothyronine. Although autoantibody tests have inherent clinical utility in a number of clinical situations. 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. 3rd Editor. Thyroid peroxidase autoantibodies (TPOAb) Originally. In fact. ATPO = antithyroidperoxidase. TPOAb is implicated as a cytotoxic agent in the destructive thyroiditic process. The principal antigen in the thyroid microsomes was recently discovered to be the thyroid peroxidase enzyme (TPO). Although changes in autoantibody concentrations often reflect a change in disease activity. thyroglobulin (Tg) and TSH receptors are used in the diagnosis of autoimmune thyroid disorders. V = variable. serial . * The spectrum of binding protein abnormalities includes increased or decreased TBG binding. p 268. TSI = thyroidstimulating immunoglobulin. automated tests are replacing the older manual agglutination tests. FT4 = free thyroxine. atrophic thyroiditis or post-partum thyroiditis have TPOAb detected. n = negative. TgAb and TRAb) Tests for antibodies against thyroid-specific antigens. as shown in Figure 2. T4 = thyroxine. TPOAb measurement may be used as a prognostic indicator for thyroid dysfunction.) High Primary Hypothyroidism • ES • TT TSH • Subclinical Hyperthyroidism • ES • TT TSH • Inappropriate TSH secretion • ES • TT Normal • Central Hypothyroidism • ES Low • TT Low • Subclinical Hypothyroidism • ES* • TT* High Free T4 • ES • TT Low Overt Hyperthyroidism Free T4 Normal Free T4 Normal Hypothyroidism High * ES. In the future. Approximately 70-80 % of patients with Graves’ disease and virtually all patients with Hashimoto’s. a 100kD Low Subclinical Hypothyroidism Normal High Hyperthyroidism Subclinical Hyperthyroidism Fig. the diagnostic and prognostic value of these thyroid autoantibody measurements is hampered by differences in the sensitivity and specificity of current methods. TBA = TSH receptor-blocking antibody. TBG = thyroxine-binding globulin.

2 : Thyroid Autoantibody Prevalence and Associations with Hypothyroidism (Reprinted from Hollowell JG. However. However. TRAb plays an uncertain role in thyroid-associated ophthalmopathy (TAO). Staehling NW.5 mU/L) TPOAb +TgAb TPOAb +Alone 34. the TBII tests are important for evaluating pregnant patients with a history of autoimmune thyroid disease. 2002. pre-eclampsia. According to the current guidelines. Gunter EW.7 10 % Prevalence 6. Thyroglobulin (Tg) methods Serum Tg measurement is used as a tumor marker in the . the intermethod variability of current TgAb assays is even greater than that of the TPOAb tests discussed above. Detectable level of TPOAb typically precedes the development of an elevated TSH and is therefore a risk factor for hypothyroidism.  Clinical Use of TgAb Tests Auto antibodies against Tg are encountered in autoimmune thyroid conditions. which appears to be exacerbated by radioiodine therapy. 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.7 6. a TRAb measurement prior to radioiodine therapy may be useful to predict risk of TAO. Hannon WH. these tests have comparable diagnostic sensitivity to TSAb bioassays (70-95%) for diagnosing Graves’ hyperthyroidism or detecting a relapse or response to therapy. since they obviate the need for additional TgAb measurements in the routine diagnosis of autoimmune thyroid disorders. In these subjects with only TgAb detected. may have misleading positive results using TSAb assays.869) 15 14. A euthyroid subject with detectable TPOAb is at increased risk of development of hypothyroidism. IVF failure. 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.  Clinical Use of TRAb Tests TRAb tests are used in the differential diagnosis of hyperthyroidism. as well as a small number of pregnant patients. sensitivity. Current tests are manual and expensive and vary in precision. reproductive complications (such as miscarriage. Therefore. Each class of TRAb (TSAb and TBAb) may be detected alone or in combination in Graves’ disease and Hashimoto’s thyroiditis.7 3. TgAb is primarily used as an adjunct test for serum Tg estimation. usually in association with TPOAb. all sera should be prescreened for TgAb by a sensitive immunoassay method prior to Tg testing. This suggests that it is unnecessary to measure © SUPPLEMENT TO JAPI • JANUARY 2011 • VOL. prospective studies are needed to establish the clinical utility of TRAb measurement in this context.1 0 Thayroid Ab Odds Ratio for Hypothyroidism (Low TT4+ TSH > 4. The threshold TgAb concentration above normal that precludes TgAb interference is either not known or does not appear to exist. the recent NHANES III study found that 3 % of subjects with no risk factors for thyroid disease had detectable TgAb without TPOAb. since a TBII test will detect both the stimulating and blocking classes of TRAb that can produce transient hyper. in the fetus and newborn. 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. 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. to more sensitive ELISA and RIA methods and more recently chemiluminescent immunoassays.6 Fig. respectively. thyroxine. Serum thyrotropin.18 Prevalence of Thyroid Antidodies in the General Population NHANES III (n =16. Patients with very high circulating concentrations of hCG due to choriocarcinoma or hydatiform mole. fetal death. False positives may occur due to assay artifacts or illegitimate transcription while false negatives results may be seen in patients with metastatic disease. Subsequently. However. specificity and reference ranges. 59 both TPOAb and TgAb for a routine evaluation of thyroid autoimmunity. The first TgAb methods were based on tanned red cell hemagglutination. The lack of specificity of the TBII methods is actually an advantage in this clinical situation. preterm delivery and post-partum thyroiditis and depression) have been associated with the presence of TPOAb.87:489-99) thyroid autoantibody measurements are not recommended for monitoring treatment for AITD. The second generation assays employing human recombinant TSH receptor are now becoming available and are reported to have superior diagnostic sensitivity for Graves’ disease. 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.9 5 5. 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.7 Thyroglobulin autoantibodies (TgAb) Antithyroglobulin autoantibodies (TgAb) were the first thyroid antibodies to be recognized to circulate in patients with autoimmune thyroid disorders.or hypothyroidism. methodologies have evolved in parallel with TPOAb methodology from semi-quantitative techniques. Spencer CA. TgAb is detected in approximately 20% of patients with differentiated thyroid carcinoma compared with 10% of normal subjects by the immunoassay methods. Unfortunately. Although TBII assays do not directly measure the stimulating antibodies.1 TgAb Alone 0. Methods for measuring TRAb are even more varied than for TPOAb and TgAb. Since TRAb and other thyroid antibodies levels increase acutely significantly after radioiodine therapy. Moreover.J Clin Endocrinol Metab 2002. in whom there is a risk of transplacental passage of TRAb to the infant . and thyroid antibodies in the United States population (1988 to 1994): NHANES III. and Braverman LE. no association with TSH abnormalities was found so that the clinical significance of an isolated TgAb abnormality remains to be established. infertility. Flanders WD.

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

3. In addition. Poyrazoglu HM. References .115:338–42. Moreover. Peiris AN. • Thereby appropriate treatment for thyroid disorders can be instituted or conservative monitoring carried out to anticipate potential future consequences. in the specimen can cause in-vitro stimulation of lipoprotein lipase. 2010. Werner & Ingbar’s The Thyroid: a fundamental and clinical text. the combination of a free-T4 estimate and a total. For instance. Thyroid Function Tests: Assay of Thyroid Hormones and Related Substances. 5. 1999. Werner SC. Southern Medical Journal. there are large inter-method differences and therefore the reliability of these tests is questioned. www. it may be inappropriately normal. Use of Fab fragments and heterospecies assay configurations can be employed as approaches to reduce this kind of interference. An example is that of heparin which. A potential for influencing results of neonatal screening also exists because antibodies are able to cross the placenta. On the other hand. the approach to evaluate whether the patient has thyroid disorder is to test the TSH levels first. 1. 2. Adv Perit Dial.15:262-8. Interpretation of Laboratory Thyroid Function Tests: Selection and Interpretation. Though assays for HAMA have been developed. Interference due to heterophilic antibodies can be classified into two categories: i. 59 as well as non-thyroid disorders. Ingbar SH.thyroidmanager. UK Guidelines for the Use of Thyroid Function Tests.1 Conclusion • Thyroid disorders have diverse clinical manifestations therefore. Gündüz Z et al. thereby causing an abnormal value (low) thyroid hormone result. on part of vigilant clinician every suspected case of thyroid disease needs to be evaluated with laboratory investigations. 8. Drug interferences In vitro and in vivo effects may occur due to drug interferences. Mascarenhas JMA. 2002. Interference may also be secondary to certain pathologic conditions. 2006.com. a free-T4 estimate is appropriate because total-T3 and free-T3 tests have inadequate sensitivity and specificity in this setting. Characteristics of interference due to endogenous autoantibodies may lead to falsely low or falsely high values. 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. a selective increase of the serum T3 concentration. multispecific antibodies that are frequently IgM. in uraemia. When the specimen contains a sufficient concentration of an interfering therapeutic or diagnostic agent.86:3-8.or free-T3estimate provides the most complete assessment of the severity of hyperthyroidism and identifies cases of “T3-toxicosis”. it may lead to methodologic interference resulting in in-vitro effects. • When hyperthyroidism is suspected. polyreactive. depending upon the type of assay and its composition. 7. Supit EJ. Spencer C. the presence of fluorophor-related therapeutic or diagnostic agents in the specimen may alter the results of thyroid tests that employ fluorescent signals. 4. Düsünsel R. 3. An example is that of furosemide which competitively inhibits thyroid hormone binding to the specimen. 6. HAAA. Evidence of central hypothyroidism in children on continuous ambulatory peritoneal dialysis. • When hypothyroidism is suspected. The J Clinical Endo & Metab 2000. when results are altered due to administration of an interfering therapeutic agent. abnormal serum constituents such as indole acetic acid may accumulate and cause interference. 2006.95:481-85. T4 and TSH results due to interference. The presence of HAMA can alter the total as well as free T3. Walfish PG. Triiodothyronine and thyroxine interrelationships in health and disease. HAMA (or human anti-mouse antibodies) are relatively weak. 9th edition. Amiodarone-Induced Thyrotoxicosis. A number of reports have shown interference due to T3. then it is termed in-vivo effect. Since TSH levels change dynamically in response to the alterations of T3 and T4. i. Daniels GH. However. This creates a false signal resulting in a high value artifact.20 © SUPPLEMENT TO JAPI • JANUARY 2011 • VOL. free fatty acids are liberated that inhibit T4 binding to serum proteins. 4. RxPG AIPG 2004 Book. ii. Lippincott Williams & Wilkins. the currently used methods rarely have this interference problem. Can Med Assoc J 1976. the result may not be abnormal. • The enhanced sensitivity and specificity of TSH assays have greatly improved the assessment of thyroid function tests. 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). T4 and TSH autoantibodies leading to anomalous free and total thyroid hormone levels and TSH values.e.