Open Access

Journal of Endocrine Disorders

Review Article

Assessment of Thyroid Function

Jun-Guan TAN1 and Tar-Choon AW2*
Abstract
1
Department of Laboratory Medicine, Khoo Teck Puat
Hospital, Singapore Thyroid disorders are among the most common endocrine disorders and
2
Department of Laboratory Medicine, Changi General accounts for a significant amount of Thyroid Function Tests (TFT) ordered.
Hospital, Singapore To interpret TFT effectively a firm appreciation of thyroid pathophysiology
2
Department of Medicine, Yong Loo Lin School of is required. Almost all the thyroid hormones are bound by thyroid binding
Medicine, National University of Singapore proteins with small quantities of free hormones that are the active moieties.
*Corresponding author: Tar-Choon AW, Department Levels of thyroid hormones are affected by alterations in their binding protein
of Laboratory Medicine, Changi General Hospital, concentrations and thus their use has been surpassed by the availability of free
Singapore hormone tests. Small changes in thyroid hormones exert a much larger inverse
change in Thyroid Stimulating Hormone (TSH) levels. Thus TSH will better reflect
Received: June 23, 2018; Accepted: July 27, 2018; thyroid dysfunction compared to thyroid hormones except when the pituitary-
Published: August 03, 2018 thyroid axis is deranged or when thyroid function is unstable (e.g. immediately
after commencing/altering therapy and in critical inter-current illness). Thyroid
testing in sick individuals is to be avoided since TFT may be abnormal without
intrinsic thyroid disease. TSH is the best initial screening test. For diagnosis free
thyroxine (fT4) is required for confirmation, identifying subclinical dysfunction
and quantifying disease severity. Thyroid antibodies aid in differential diagnosis
- anti-thyroid peroxidase for autoimmune thyroiditis and anti-TSH receptor for
Graves’ disease. Thyroglobulin (Tg) serves as a tumor marker in the follow-up
of differentiated thyroid cancer. As anti-thyroglobulin (Tg-Ab) is quite prevalent
in thyroid cancer patients, the presence of Tg-Ab will falsely lower Tg readings.
Thus concomitant Tg-TgAb measurements are needed; for Tg-Ab positive
patients Tg-Ab may serve as a surrogate tumor marker. In pregnancy the
normal adult reference intervals must be replaced by trimester-specific ranges
for fT4 and TSH as well as the use of thyroid antibody testing where appropriate.
When thyroid tests are at variance with the clinical picture rarer entities (e.g.
thyroid hormone resistance, pituitary TSH-secreting adenoma) as well as assay
artifacts and antibody interferences have to be considered.
Keywords: Thyroid Function; TSH; Free T4; Thyroid Antibodies

Abbreviations appropriately investigated with imaging) will only be covered briefly

where relevant. The availability of the new Thyroid Stimulating
TFT: Thyroid Function Tests; TRH: Thyrotropin Releasing Immunoglobulin (TSI) assay, recent acknowledgement of high
Hormone; TSH: Thyroid Stimulating Hormone; T4: Thyroxine; T3: dose biotin interference on thyroid tests, and increase in gestational
Tri-Iodo Thyronine; FT4: Free T4; FT3: Free T3; TT4: Total T4; TT3: TSH thresholds will be considered. Each common thyroid test will
Total T3; Tg: Thyroglobulin; Tg-Ab: Anti-Thyroglobulin; TPO-Ab: be reviewed followed by their clinical use in the areas of screening,
Anti-Thyroid Peroxidase; TSI: Thyroid Stimulating Immunoglobulin; diagnosis, and monitoring therapy.
TRAb: TSH Receptor Antibodies; HCG: Human Chorionic
Gonadotropin; GD: Graves’ Disease; NTI: Non-Thyroidal Illness; Brief Pathophysiology
MNG: Multi-Nodular Goiter; TSHoma: TSH-Secreting Pituitary Interpretation of thyroid function tests requires a firm
Adenoma; RTH: Thyroid Hormone Resistance understanding of thyroid physiology [5]. Thyrotropin Releasing
Introduction Hormone (TRH) from the hypothalamus stimulates the release of
TSH from the anterior pituitary gland. Thereafter, TSH regulates the
Thyroid disorders are the fifth most common endocrine production of thyroid hormone, thyroxine (T4) and triiodothyronine
disorder in adults in the United States (US) after diabetes, obesity (T3), by the thyroid gland. The bulk of thyroid hormones
and dyslipidemia, osteoporosis, and erectile dysfunction [1]. (approximately 85%) is secreted as T4, while a smaller fraction (15%)
Consequently, thyroid function tests are very commonly ordered [2]. is secreted as T3. The thyroid hormones are highly protein bound,
In fact the annual volume of Thyroid Stimulating Hormone (TSH) mainly by thyroid binding globulin (TBG) and to a lesser extent
tests ordered is 59 million in the US [3] and another 10 million in the transthyretin and serum albumin. It is the non-protein-bound or free
United Kingdom [4]. T4 (fT4) and free T3 (fT3) that are the biologically active forms of
This review provides an update on laboratory testing of thyroid thyroid hormone. While T3 is the active moiety much of its serum
dysfunction (hypothyroidism and hyperthyroidism). Structural concentration derives from the peripheral conversion of T4 to T3 by
disorders such as goiter, nodules, and cancer (which are more deiodinases. FT3 and fT4 exert negative feedback on the hypothalamus

J Endocr Disord - Volume 5 Issue 1 - 2018 Citation: Jun-Guan TAN and Tar-Choon AW. Assessment of Thyroid Function. J Endocr Disord. 2018; 5(1):
ISSN : 2376-0133 | www.austinpublishinggroup.com 1030.
Tar-Choon et al. © All rights are reserved
Tar-Choon AW Austin Publishing Group

and pituitary. Their relationship to TSH is inversely log-linear such concentration is inversely proportional to the unoccupied antibody-
that minor changes in the pituitary-thyroid axis produce a large binding sites. Unlike TSH, the reference interval for fT4 shows a
change in TSH concentrations [6-8]. Each individual has a unique wider spread of between 9.5-25.0 pmol/L on different assay systems.
TSH-fT4 set point [9]. In early thyroid dysfunction fT4 remains One must be cognizant of some causes of misleading fT4 results
normal while TSH may be decreased (subclinical hyperthyroidism) especially autoantibody interferences [26]. Although rare, familial
or increased (subclinical hypothyroidism) [10]. dysalbuminemic hyperthyroxinemia is an autosomal dominant
condition with an albumin possessing greatly enhanced affinity for
Analytes
T4 (and occasionally T3) resulting in spuriously high free (and total)
Despite progress in immunoassay technology all laboratory thyroid hormones [27].
tests, including thyroid function, are still plagued by reports of false
positive or negative results [11]. Sources of interferences include Total T4 and total T3
serum proteins (e.g. rheumatoid factor, binding proteins), heterophile Radioimmunoassays for tT4 and tT3, widely used in the 70s, have
(anti-animal) antibodies, drugs and their metabolites [12], and other been largely replaced by automated non-isotopic immunoassays [13].
cross-reacting substances (autoantibodies) with assay components With the progress and wide availability of fT3 and fT4 assays, tT3
[13]. Only the clinician can suspect such interferences and alert the and tT4 now play a secondary role and are largely used to confirm
laboratory to investigate the discordance between test results and doubtful free thyroid hormone results [18]. As the concentration of
the patient’s condition. Close rapport and communication between tT4 is 10-fold higher than tT3, its measurement is more reliable. TT3
the clinic and laboratory is vital for an efficient and seamless thyroid and tT4 concentrations are also impacted by variations in thyroid
testing service. binding proteins. In pregnancy, thyroid binding globulin increases
up to 2.5 fold that of antenatal value and stays constant till term [28]
TSH
while albumin concentration decreases by 25% due to increase in
TSH is a large 28 to 30-kDa dimeric glycopeptide hormone that plasma volume. In critically ill patients, decreases in thyroid binding
shares a common 92 amino acid alpha subunit with human chorionic proteins also contribute to a fall in tT4 [29]. In theory, the diagnostic
gonadotrophin (hCG), follicle-stimulating hormone and luteinizing accuracy of total hormone measurements would be equivalent to free
hormone, but it has a unique 118 amino acid beta chain. With the hormone tests if patients have similar binding protein concentrations.
introduction of highly sensitive TSH immunometric assays capable This free-total thyroid hormone discrepancy is particularly noticeable
of detecting TSH <0.01mIU/L in the 90s [6,14-15], TSH is now the at the high and low ends of hormone concentrations and also in
most widely ordered thyroid function test and is recommended hospitalized patients [30].
for first line screening [16]. This development has also led to the
decline in the measurement of TSH release following thyrotropin- Anti-TPO (TPO-Ab)
releasing hormone stimulation [17]. While all analytical platforms Anti-TPO is a common autoantibody against Thyroid Peroxidase
will not produce exactly equivalent TSH values, the reference range (TPO). Historically, anti TPO were detected as thyroid microsomal
for TSH in healthy iodine-replete subjects is generally quite similar antibodies using agglutination or immuno-fluorescence methods. It
at between 0.4-4.0 mIU/L even in different geographic and ethnic was only in 1985 that TPO was recognized as the target antigen of
groups. However, TSH alone is not sufficient for assessing subclinical thyroid autoantibodies [31]. Thereafter, radioimmunoassay [32] and
disease and states with deranged pituitary-thyroid axis such as central chemiluminescent assays [33] for TPO-Ab became available. Eighty
hypothyroidism, hospitalized patients or in the early phases of percent of Graves’ disease patients have high levels of anti-TPO
therapy (antithyroid drugs or thyroid hormone replacement) [18]. In antibodies [34], while positive anti-TPO Ab is detected in over 90%
these circumstances concomitant fT4 is required [19]. In comparison of patients with autoimmune thyroid disease [35]. In the community,
to other thyroid function parameters TSH is least susceptible to the prevalence of TPO-Ab is 11-12 % euthyroid subjects [32,33].
autoantibody interferences [20]. However, metformin (a commonly
Anti-TSH receptor (Anti-TSHR)
used drug in type 2 diabetes) can depress TSH levels [21].
Anti-TSHR or TSH Receptor antibodies (TRAbs) are antibodies
Free T3 and T4 that bind to the TSH Receptor. TRAbs may be stimulatory or
As the biologically active forms of thyroid hormones, fT4 and inhibitory; distinguishing between them is unnecessary as it is evident
fT3 are considered to be more sensitive and meaningful indicators from their clinical presentation. In Graves’ disease (GD) stimulatory
of thyroid disease [18] than total T3 (tT3) and total T4 (tT4). Since TRAbs bind to and activate the TSH receptor [36] causing production
fT3 and fT4 exist in minute concentrations (pico-molar) compared of excess thyroid hormones. The fully automated Roche Elecsys TRAb,
to tT3 and tT4 (nano-molar), free hormones are harder to measure a 27 minute assay, became available in 2008 [37-38]. Sensitivity and
accurately [3]. Traditionally fT3 and fT4 were measured directly by specificity of TRAbs in the differential diagnosis of GD is excellent,
equilibrium dialysis and ultrafiltration, but their tedium and expense with sensitivity and specificity above 90% [39]. In subclinical
have confined them to the realms of research. The widespread use disease, the more specific thyroid stimulating immunoglobulins
of free thyroid hormones began with automation and advent of (TSI) might improve diagnosis. Algorithms incorporating TSI have
non-isotopic assays [22,23]. The current available free hormone shown faster diagnosis for GD and cost savings [40]. However, TSI
tests are indirect assays as they use a specific high-affinity antibody bioassays are cumbersome, time-consuming and unsuitable for
to extract free thyroid hormones from serum [24,25]. Thereafter, routine use in clinical laboratories. This changed in 2016 when FDA
the antibody-containing bound thyroid hormone is separated from approved the fully automated TSI on the Siemens Immulite 2000
serum prior to incubation with a labeled probe. The free hormone [41]. As stimulatory antibodies account for the majority of TRAbs,

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Tar-Choon AW Austin Publishing Group

concordance between TRAbs and TSI should be similar. A recent magnetically captured and a chemiluminescent signal generated;
study indeed demonstrated equivalent performance between the luminescence is inversely proportional to fT4 in the sample. High
Roche TRAb and Siemens TSI in patients with untreated GD [42]. levels of exogenous biotin in the patient sample will saturate the
Hence, the new TSI may add limited value to the already widely streptavidin reagent and inhibit the proper formation of the antibody
available Roche TRAbs besides the TSI involves a longer assay time complex, resulting in a low signal. In non-competitive immunoassays
(60 mins). The clinical utility of TRAbs [39] include predicting low signal translates into spuriously low analyte concentration, while
short-term relapse of GD after antithyroid drug therapy but are less in competitive immunoassays low signal will result in falsely high
effective in predicting long-term relapse or remission. In pregnant concentrations of analyte [51].
women with GD, a negative TRAb are unlikely to result in neonatal
The combination of low TSH, high fT4 and high TRAb
thyrotoxicosis while high titers of TRAb need close monitoring. GD
results engendered by biotin will be translated as biochemical
patients with ophthalmopathy have high TRAb levels; positive TRAb
hyperthyroidism. However, this biotin effect on TFT is seen in
in unilateral proptosis favors a diagnosis of GD, but TRAbs are unable
patients prescribed high doses of biotin. Patients with inborn error
to predict the course of ophthalmopathy or its response to treatment.
of metabolism such as Biotin-Thiamine-responsive Basal Ganglia
Anti-Thyroglobulin (Tg-Ab) Disease (BTBGD) or biotin cycle defects (biotinidase deficiency
Tg-Ab on its own adds limited value to the diagnosis of and multiple carboxylase deficiency) are prescribed megadoses of
autoimmune thyroid disease with anti-TPO more sensitive and 10-15mg/kg biotin per day [51]. Factitious Graves’ disease was also
specific for Hashimoto thyroiditis [43], and TRAbs more useful for reported in a patient with multiple sclerosis prescribed 300 mg biotin
GD. The clinical utility of Tg-Ab is to ensure the reliability of Tg daily [52]. It is thus important, for the ordering doctor and the scientist
results in the setting of DTC. In fact for Tg-Ab positive DTC, Tg-Ab to be aware about their assay methodology [53]. The Roche package
may serve as a surrogate tumor marker instead of Tg [44,45]. insert clearly states that biotin interference is only encountered in
patients consuming more than 5 mg biotin per day. Although there
Thyroglobulin (Tg) exist dedicated biotin supplement available for purchase over the
Tg, a dimeric protein produced by the thyroid gland, is counter or internet, most adult multivitamin supplements on the
undetectable in patients with differentiated thyroid cancer (DTC) market contain less than 1mg of biotin per tablet and so will not cause
after thyroidectomy or radioiodine ablation. With improved assay any assay interference. Hence in the usual clinical practice, the issue
sensitivity Tg is a valuable tumor marker for the follow-up of DTC of biotin induced assay interference is unlikely to be encountered.
[46]. However, the concomitant presence of thyroglobulin antibodies When clinically discordant thyroid results are found, a detailed
(Tg-Ab) in patient sera will result in the under-estimation of the true history (including supplements) will rule out biotin interference.
Tg result [47]. As the prevalence of Tg-Ab is 25% in DTC patients This interference can be confirmed by re-testing samples on biotin-
[48] and 10% in the general population [43], it would be prudent for free automated immunoassays. Another method of overcoming such
the lab to provide the Tg-Ab titers together with Tg for a meaningful interference is biotin neutralization. A simple method of sample
interpretation of all Tg results [49]. pretreatment using streptavidin reagents to neutralize biotin has been
recently described [54].
The Biotin Effect
Clinical Evaluation
Many automated immunoassays make use of biotin-labelled
antigen or antibody [50]. Biotin’s natural affinity with streptavidin Screening
enables the biotinylated antigen-antibody complex to bind to The purpose of screening is early detection of asymptomatic
the streptavidin with very high avidity and specificity. By coating disease and to improve clinical outcomes. The US National Health
streptavidin onto a solid phase (coated tube or micro particles) it can Screening program, NHANES 2007-2012, shows a prevalence of 7.1%
capture and bind the biotinylated antibody (e.g. TRAb) or biotinylated for thyroid dysfunction - subclinical hyperthyroidism 3.1%, clinical
antigen (e.g. TSH, fT4). In one popular automated system (Roche) hyperthyroidism 0.3%, subclinical hypothyroidism 3.5%, and clinical
TSH is a non-competitive immunoassay. A monoclonal biotinylated hypothyroidism 0.2% respectively [55]. Despite the substantial
TSH-specific antibody and a monoclonal ruthenium-labelled TSH- burden of thyroid disorders, screening asymptomatic adults remains
specific antibody reacts with TSH in the patient’s serum to form controversial. The American Thyroid Association, American Academy
a sandwich complex. Following addition of streptavidin–coated of Clinical Endocrinology, American Academy of Family Physicians
microparticles, the sandwich complex is bound onto this solid and American College of Physicians recommend screening while the
phase through the interaction of biotin and streptavidin. The TSH- Royal College of Physicians London and the United States Preventive
antibody-microparticle complex is separated, washed and bound to Services Task Force are against it [56]. For screening asymptomatic
a magnetic electrode. When a voltage is applied a chemiluminescent ambulatory subjects, TSH alone is sufficient. When TSH exceeds the
signal is generated and this signal is proportional to the sample TSH reference range, the laboratory may provide reflex testing instead of
concentration. In the Roche system, fT4 and TRAb are competitive repeat testing at another occasion. In view of significant clustering of
immunoassays. A monoclonal ruthenium-labelled T4-specific thyroid disease in families [57], women over 50 and the elderly [58],
antibody extracts fT4 in the patient’s serum. Thereafter biotinylated-T4 screening may also be done on these at-risk populations. If abnormal,
competes for unoccupied binding sites on the ruthenium-T4-Ab. thyroid antibodies (TPO-Ab) may be used to assess risk for future
The biotin-T4-ruthenium-T4-Ab sandwich complex binds onto the thyroid dysfunction. However, cognizance must be given to the
streptavidin–coated micro particle solid phase through the interaction extraneous effects of various conditions, especially medications, on
of biotin and streptavidin. This entire antibody-hapten complex is thyroid test results [12,56].

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Diagnosis of thyroid conditions

In symptomatic or patients suspected of thyroid dysfunction, the
concurrent use of fT4 and TSH is preferred [59]. Thereafter, further
testing (thyroid antibodies or radionuclide uptake studies) may be
undertaken.
Suspected hypothyroidism: A first line thyroid panel comprising
of fT4 and TSH should be done see Figure 1. TSH in overt primary
hypothyroidism is often greater than 15.0mIU/L with corresponding
low fT4 levels. In subclinical hypothyroidism TSH levels are typically
between 4.0-10.0 mIU/L with normal fT4 levels. The most frequent
cause of hypothyroidism is Hashimoto’s thyroiditis. A proper
history is necessary to elicit other causes such as previous treatment
(thyroidectomy, radioiodine ablation, and neck irradiation) and
current medications (e.g. lithium, amiodarone and interferon-α) Figure 1: Flow Chart for Suspected Hypothyroidism.
[12,56]. FT3 or tT3 has no clinical utility in the evaluation of
hypothyroidism as their levels remain in the normal range until quite
late due to hyper-stimulation of the remaining functioning thyroid
tissue by TSH and up-regulation of type 2 iodothyronine deiodinases
[60]. Deranged thyroid function tests, notably a low T3 or fT3, is
frequently encountered in hospitalized patients with non-thyroidal
inter-current illnesses or non-thyroidal illness (NTI) [61,62]. The NTI
syndrome is characterized by a combination of abnormalities in the
pituitary-thyroid axis, thyroid hormone binding proteins, and thyroid
hormone action without any intrinsic pituitary or thyroid disease.
Hence a low fT3 is neither specific nor sensitive for hypothyroidism,
and adds little value in the diagnosis of hypothyroidism. Thyroid
function tests should not be ordered in sick hospital inpatients unless
absolutely necessary.
Thyroid antibodies can be used to assess the risk of developing
overt hypothyroidism in females with TSH >6mIU/L - 4.3 % per year Figure 2: Flow Chart for Suspected Hyperthyroidism.

in those with elevated TPO-Ab versus 2.1% in those with normal

TPO-Ab [63]. In addition, women with normal TSH (2.5-4.0 mIU/L) goiter (MNG), whilst autonomously functioning thyroid adenoma
and TPO-Ab positivity also progressed to subclinical and overt or thyroiditis are less common [67]. Eye signs and smooth goiters
hypothyroidism [64]. When fT4 is decreased in the face of low or are suggestive of GD. TRAbs are useful in the diagnosis of GD when
normal TSH, consider central or secondary hypothyroidism [56]. the presentation is atypical. Radionuclide scanning may be done if
Such patients may have a history of post-partum hemorrhage, head the diagnosis is still uncertain. There will be a diffuse uptake in GD,
injury, neurosurgery, short stature, amenorrhea, and infertility. A while in toxic MNG the thyroid gland uptake will be focal. Subacute
full pituitary panel (growth hormone, prolactin, follicle-stimulating thyroiditis may present with pain (localized or radiating to the jaw)
hormone, luteinizing hormone, testosterone, adrenocorticotrophic and elevated C-reactive protein and erythrocyte sedimentation rate,
hormone) will be necessary to assess for concomitant hypopituitarism while drug-induced thyroiditis are usually painless [68]. In subacute
from pituitary or hypothalamic tumors; imaging (CT or MRI) will be or drug-induced thyroiditis the radionuclide uptake will be reduced
required for their delineation (Figure 1). or absent.
Suspected hyperthyrodism: Hyperthyroidism refers to the For subclinical hyperthyroidism, fT4 is normal but TSH is
inappropriately high synthesis and secretion of thyroid hormones suppressed. In a subset of patients with T3-toxicosis, only fT3 is
by the thyroid gland, while the term ‘‘thyrotoxicosis’’ refers to a elevated while fT4 is normal and TSH suppressed (<0.01mU/L) or
clinical state that results from inappropriately high thyroid hormone undetectable. These changes may represent the earliest stages of
action in tissues [59]. Symptoms and signs include heat intolerance, disease or that caused by an autonomously functioning thyroid
nervousness, hyperactivity, tremor, weakness, diarrhea and increased nodule [46]. Unless T3-toxicosis is suspected there is no need to order
appetite. Elderly subjects may display a more sedate, apathetic fT3 or tT3.
presentation with weight loss and cardiac symptoms such as heart
In patients with elevated fT4, but normal or raised TSH, the
failure [65,66].
possibility of rare disorders such as TSH-secreting pituitary adenoma
For suspected hyperthyroidism, a thyroid panel comprising (TSHoma) or thyroid hormone resistance (RTH) must be considered.
TSH and fT4 is sufficient (see Figure 2). In overt hyperthyroidism The prevalence of TSHoma has been reported to be 2.8 per million
TSH is often <0.01mIU/L while fT4 is elevated. The most frequent [69]. Some TSHoma are polymorphous and may co-secrete growth
cause of hyperthyroidism is GD followed by toxic multinodular hormone and prolactin. In TSHoma, patients exhibit more classical

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Tar-Choon AW Austin Publishing Group

symptoms of thyrotoxicosis. Biochemically, the TSH α-subunit and therapies (radioiodine ablation, thyroidectomy) and consideration
Sex-Hormone Binding Globulin (SHBG) are increased with blunted of non-thyroidal illness, will usually uncover the underlying cause.
TSH response to TRH stimulation. Resistance to thyroid hormone Reassessment of thyroid function and exclusion of assay artifacts
(RTH) is caused by a mutation in the thyroid hormone receptor β should be undertaken next. Lastly, consider the possibility of
gene [70]. Three quarters of RTH are autosomal dominant. In RTH, rare disorders. With good knowledge of laboratory science and
the TSH response to TRH stimulation is normal or exaggerated while appreciation of medical context, users will be able to interpret thyroid
the TSH α-subunit and SHBG are normal (Figure 2). function tests successfully.
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J Endocr Disord - Volume 5 Issue 1 - 2018 Citation: Jun-Guan TAN and Tar-Choon AW. Assessment of Thyroid Function. J Endocr Disord. 2018; 5(1):
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