Laboratory Evaluation of Thyroid Function - Journal of the ...

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 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

© SUPPLEMENT TO JAPI • JANUAry 2011 • VOL. 59 15 

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 and T level 

3 4 

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 T4, 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 below normal TSH above normal 

• Primary hyperthyroidism • Primary hypothyroidism 

• Pituitary/hypothalamic disease with central hypothyroidism (TSH 

unreliable) 

• Prolonged thyrotroph cell suppression after recent hyperthyroidism 

in euthyroid or hypothyroid patient 

• Old age • Old age 

• Drugs, e.g., glucocorticoids, dopamine • Drugs, e.g., amiodarone 

• 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 T4 • Problems with T treatment : Overdosage in treatment for fatigue or • Problems with T treatment : Underdosage based on misleadingly 

4 4 

, overweight, Altered gastrointestinal absorption because of drugs or high total T Altered gastrointestinal absorption because of drugs or 

4 

disease, Altered T clearance because of drugs, Patient compliance disease, Altered T clearance because of drugs, Patient compliance 

4 4 

problems, Prescription error, Testing too soon after T dose decrease problems, Prescription error, Testing too soon after T dose increase 

4 4 

• 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, 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 and T levels 

3 4 

should be determined. Free T and T level estimation is 

3 4 

preferred over total T and T estimation because these 

3 4 

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 β 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


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 and total 

3 

T may be elevated while the thyroid functional state (free T and 

4 3 

T levels) may be normal. Such conditions include: 

4 

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 (


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, ↓ 

or ↑ 

N ↑ n N n n n 

Thyroid hormone resistance N or ↑ ↑ ↑ ↑ ↑ N ↑ n N n n n 

TSH-dependent hyperthyroidism ↑ ↑ ↑ ↑ ↑ N ↑ n N n n n 

T protein-binding abnormalities 4 [*] 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 

Hypothyroidism 

• ES 

• TT 

• Central 


• ES 

• TT 

• Subclinical 


• ES* 

• TT* 

• Inappropriate TSH 

secretion 

• ES 

• TT 

Normal • ES 

• TT 

• Subclinical 

Hyperthyroidism 

• ES 

• TT 

Overt 

Hyperthyroidism 

Low 

Low Free T4 * ES, euthyroid sick; TT, thyroid in transition 

High 

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 

Hypothyroidism Hyperthyroidism 

Subclinical Hypothyroidism 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


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 Hypothyroidism 

(Low TT4+ TSH > 4.5 mU/L) 

Fig. 2 : Thyroid Autoantibody Prevalence and Associations with 

Hypothyroidism (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


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


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.

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