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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,
© SUPPLEMENT TO JAPI • JANUARY 2011 • VOL. 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; Pituitary resistance to thyroid
hormone (central hyperthyroidism) TSH, unreliable.
• 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, e.g., glucocorticoids, dopamine
• Drugs, e.g., amiodarone
Problems with T4 treatment : Overdosage in treatment for fatigue or • Problems with T4 treatment : Underdosage based on misleadingly
overweight, Altered gastrointestinal absorption because of drugs or
high total T4, Altered gastrointestinal absorption because of drugs or
disease, Altered T4 clearance because of drugs, Patient compliance
disease, Altered T4 clearance because of drugs, Patient compliance
problems, Prescription error, Testing too soon after T4 dose decrease
problems, Prescription error, 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, causing
analytical artefact
complete patient evaluation including testing for antithyroid antibodies, imaging ultrasound and fine-needle
aspiration cytology.
therefore, may require estimation of free T4 levels. 1
TSH testing should be commonly carried out in the following
settings:
•
In patients presenting with suspected goitres: Serum TSH levels
must be measured.
•
As screening for congenital hypothyroidism: A heel-prick blood
specimen is used for determining serum TSH levels. This is
an established screening test for congenital hypothyroidism
and has been adopted as a routine screening measure
in many countries. The practice of routine screening for
congenital hypothyroidism by the TSH test should be more
widely adopted and continued. The low cost filterpaper TSH
methods will be available in India soon.
•
In patients with atrial fibrillation, dyslipidaemia, osteoporosis,
and infertility: Serum TSH levels should be measured at
presentation.
As screening for thyroid disorders in patients with unclear
diagnoses: Serum TSH test should be carried out in all patients
who have non-specific manifestations, are asymptomatic, and
in whom the diagnosis is not clear. The high-sensitivity TSH
test should be performed in such cases (where there’s a low
pre-test probability of the disease). The advantage of this test is
that its negative predictive value is very high and a vast majority
of the results come out negative. Measurement of serum TSH
alone can suffice during sequential follow-up visits (after the
first investigation has been carried out) in patients who have
not received treatment for thyroid disorders and for those who
may be at risk of developing thyroid dysfunction.
Important considerations for the clinician if TSH is
abnormal (Table 2)
•
In patients with abnormal TSH concentrations, a focused
history, physical examination (in particular thyroid gland
examination), repeat TSH test, serum T 3 and T 4 level
determination and occasionally imaging studies need to be
carried out.
•
It is not uncommon to see that many patients with high
TSH values are informed that they need to take thyroid
medication life-long and after having been prescribed 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
•
In patients whose TSH levels are abnormal, T3 and T4 levels
should be determined. 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. 3
Inappropriate TSH
This is a biochemical diagnosis in which elevation in circulating
FT4 and/or FT3 is associated with an “inappropriately” detectable
or elevated serum TSH concentration. If this biochemical
picture is observed then assay artefact/laboratory error should
be considered first. Once the laboratory has excluded such
explanations then the cause of “true” inappropriate TSH should
be considered. The differential diagnosis are a TSH secreting
pituitary tumour (TSH-oma) or a syndrome of thyroid hormone
resistance. The finding of an elevated serum sex hormone
binding globulin (SHBG) and circulating free a subunit may
support the diagnosis of TSH-oma, as may the finding of hyper
or hypo-secretion of other pituitary hormones. Pituitary imaging
usually confirms the diagnosis but should not be undertaken
until the appropriate biochemical confirmation has been made.
A syndrome of thyroid hormone resistance can be confirmed by
family history; sequencing of the b thyroid hormone receptor
confirms the diagnosis. When an ‘inappropriately’ detectable
or elevated serum TSH is found in association with elevated
circulating free T3 and/or T4 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 T4 and total T3 which
16
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 T3 and total
T4 may be elevated while the thyroid functional state (free T3 and
T4 levels) may be normal. Such conditions include:
1.
Hereditary abnormalities of binding proteins: These include
TBG deficiency or TBG excess, abnormal albumin levels and
abnormal transthyretin levels.
2.
Acquired deficiency of binding proteins: Conditions such
as nephrotic syndrome may cause protein loss from the
body. In severe liver disease, there’s impaired production of
proteins, and therapy with androgens or anabolic steroids
may alter the levels of carrier proteins.
3. Drug-induced alterations in T4 binding to TBG: Therapy
with salicylates, phenytoin, phenylbutazone may alter
T4-TBG binding.
4.
Presence of T4 antibodies.
The development of newer immunoassay methods for
determining free T3 and T4 has overcome many of these
problems. Radioimmunoassay measurement of total serum T4
levels is highly sensitive in reflecting the hyperthyroid (85-95%)
and the hypothyroid status (80-90%) of patients.
Total T3
Currently routine measurement of serum T3 is not carried out
(only T4 is measured) in patients suspected of having thyroid
disorders. About 25% of patients with hypothyroidism have low
normal T3 values. Free T3/ total T3 measurements, however,
should be performed in the following settings:
1.
In patients suspected of having T3 thyrotoxicosis.
2. In patients taking drugs that inhibit the peripheral
conversion of T4 to T3 (such as dexamethasone, propranolol,
propylthiouracil, amiodarone, and iodine-containing
contrast media).5
Testing both TSH and FT4
There are certain clinical situations where TSH testing
must be coupled with testing the FT4 levels. Clinical situations
where measurement of both serum TSH and FT4 is required
are principally disorders where the pituitary-thyroid axis is not
intact or is unstable. These situations include:
•
Optimising thyroxine therapy in newly diagnosed patients
with hypothyroidism.
•
Diagnosing and monitoring thyroid disorders in pregnancy.
•
Monitoring patients with hyperthyroidism in the early
months after treatment.
•
Diagnosis and monitoring treatment for central
hypothyroidism.
•
End-organ thyroid hormone resistance.
•
Sick Euthyroid State.
•
TSH-secreting pituitary adenomas.
•
Women with type I diabetes should have their thyroid
function, including serum TSH, FT4 and thyroid peroxidase
antibody status, established preconception, at booking when
pregnant and at 3 months post-partum.
© SUPPLEMENT TO JAPI • JANUARY 2011 • VOL. 59
•
Possible subclinical hypothyroidism: If screening is
performed, and a high serum TSH concentration is found,
and the FT4 is normal, the measurement should be repeated
3-6 months later, along with measurement of serum FT4,
after excluding non-thyroidal illness and drug interference.
•
Overtly hypothyroid patients (who have serum TSH greater
than 10 mU/L and low FT4 concentrations) should be treated
with thyroxine.
In patients with a high serum TSH level and normal FT4
concentrations (possible subclinical hypothyroidism), TSH
measurements and FT4 should be repeated 3 to 6 months later,
after precluding non-thyroid disorders and drug interference. In
cases of doubt in identifying specimens in which both serum TSH
and FT4 should be carried out, it is prudent to test all specimens
for TSH and FT4, rather than test for TSH alone.6
Testing TSH and FT4 and FT3
In hospital inpatient ICU: In the absence of an abnormal
thyroid gland by careful physical examination, a hospital
inpatient with a mild or moderate (<20mIU/L) increase in
serum TSH and an estimated free 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.
The same holds true for a patient with a subnormal serum
TSH and estimated free T4 and serum T3 values that are not
increased. In both cases, the great majority of patients do not
have clinically significant thyroid disease. The responsibility of
providing pertinent clinical information to help guide the lab
in selecting the most appropriate thyroid function test lies with
the requesting physician. However, if sufficient clinical details
to allow the identification of patients with subnormal TSH and
unaltered T3 and T4 values, are not available, then laboratories
should measure serum TSH and FT4 on all specimens. This is
a more prudent strategy than just measuring first-line serum
TSH, and then carrying out FT4 and FT3 estimations later when
indicated.
Considerations which
Alter Thyroid Levels
•
Binding protein abnormalities can increase totalT3 in the
absence of hyperthyroidism, notably during estrogen
treatment contraceptive pills, and pregnancy. If necessary,
a T3-uptake test or thyroxine-binding globulin measurement
can be used to calculate a free-T3 index, or a free-T3 test can
be obtained to clarify an ambiguous increased total-T3 result.
•
When hypothyroidism is suspected, a free-T4 estimate
is appropriate because total-T 3 and free-T 3 tests have
inadequate sensitivity and specificity in this setting.
•
When hyperthyroidism is suspected, the combination
of a free-T 4 estimate and a total- or free-T 3 estimate
provides the most complete assessment of the severity of
hyperthyroidism and identifies cases of “T3-toxicosis”, i.e.
a selective increase of the serum T3 concentration.
•
In some centres, free-T4 and -T3 tests are routinely used
when the TSH is increased, but in others, serum T 3
measurements are obtained only when the TSH is low and
the free T4 is within the reference interval. It is preferable
to monitor both serum free T4 and T3 in patients with low
serum TSH (other than hypothyroid patients taking T4),even
after the thyroid diagnosis is known, to establish patterns
of increasing or decreasing values over time (Table 3) (Fig.
1a, b).1,3
© SUPPLEMENT TO JAPI • JANUARY 2011 • VOL. 59 17
Table 3 : Characterization of thyroid disorders according to results of thyroid function tests
Disorder
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, ↓
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; T4 = thyroxine; T3 = triiodothyronine; FT4 = free thyroxine; Tg = thyroglobulin; TBG = thyroxine-binding
globulin; rT3 = reverse T3; 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
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, euthyroid sick; TT, thyroid in transition
Fig. 1a : Thyroid Function Test Algorithm
Thyroid Autoimmunity Thyroidspecific Autoantibodies (TPOAb,
TgAb and TRAb)
Tests for antibodies against thyroid-specific antigens, antithyroid peroxidase (TPO), thyroglobulin (Tg) and TSH receptors
are used in the diagnosis of autoimmune thyroid disorders. Over
the last five decades, antibody measurement techniques have
evolved from semi-quantitative agglutination and complement
fixation tests and whole animal bioassays to specific ligand
assays using recombinant antigens and cell culture systems
transfected with the human TSH receptor. Unfortunately, the
diagnostic and prognostic value of these thyroid autoantibody
measurements is hampered by differences in the sensitivity and
specificity of current methods. Although autoantibody tests have
inherent clinical utility in a number of clinical situations, these
tests should be selectively employed.
Thyroid peroxidase autoantibodies (TPOAb)
Originally, thyroid peroxidase autoantibodies (TPOAb) were
detected as thyroid microsomal antibodies by semi-quantitative
complement fixation and tanned erythrocyte hemagglutination
techniques and were labeled antimicrosomal antibodies (AMA).
The principal antigen in the thyroid microsomes was recently
discovered to be the thyroid peroxidase enzyme (TPO), a 100kD
Low
Subclinical Hypothyroidism
Normal
High
Hyperthyroidism
Subclinical Hyperthyroidism
Fig. 1b : Algorithm for the Diagnosis of Thyroid Dysfunction
glycosylated protein. Currently, automated tests are replacing
the older manual agglutination tests. These new tests are more
specific TPOAb immunoassays or immunometric assay methods,
and are based on purified or recombinant TPO.
Clinical Use of TPOAb Tests
An abnormal TPOAb is detected in 15 to 20 percent of
“healthy” euthyroid subjects and even higher percentages
of patients with various non-thyroid autoimmune disorders.
Approximately 70-80 % of patients with Graves’ disease and
virtually all patients with Hashimoto’s, atrophic thyroiditis
or post-partum thyroiditis have TPOAb detected. In fact,
TPOAb is implicated as a cytotoxic agent in the destructive
thyroiditic process. TPO antibodies are involved in the tissue
destructive processes associated with the hypothyroidism
observed in Hashimoto’s thyroiditis (Fig. 2). In the future,
TPOAb measurement may be used as a prognostic indicator for
thyroid dysfunction. Although the appearance of TPOAb usually
precedes the development of thyroid dysfunction, recent studies
suggest that a hypoechoic ultrasound pattern may precede a
biochemical TPOAb abnormality, as shown in Figure 2. The
paradoxical absence of TPOAb in some patients with unequivocal
TSH abnormalities likely reflects the suboptimal sensitivity and/
or specificity of current TPOAb tests or non-autoimmune thyroid
failure (atrophic thyroiditis). Although changes in autoantibody
concentrations often reflect a change in disease activity, serial
18
Prevalence of Thyroid Antidodies in the General Population
NHANES III (n =16,869)
15
14.7
10
%
Prevalence
6.9
5
5.7
3.1
0
Thayroid
Ab
Odds Ratio for Hypothyroidism
(Low TT4+ TSH > 4.5 mU/L)
TPOAb
+TgAb
TPOAb
+Alone
34.7
6.1
TgAb
Alone
0.6
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
© SUPPLEMENT TO JAPI • JANUARY 2011 • VOL. 59
both TPOAb and TgAb for a routine evaluation of thyroid
autoimmunity. According to the current guidelines, all sera
should be prescreened for TgAb by a sensitive immunoassay
method prior to Tg testing. Therefore, TgAb is primarily used
as an adjunct test for serum Tg estimation. TgAb is detected
in approximately 20% of patients with differentiated thyroid
carcinoma compared with 10% of normal subjects by the
immunoassay methods. The threshold TgAb concentration above
normal that precludes TgAb interference is either not known or
does not appear to exist. False positives may occur due to assay
artifacts or illegitimate transcription while false negatives results
may be seen in patients with metastatic disease.
TSH receptor autoantibodies (TRAb)
TSH Receptor Antibodies (TRAb) were first recognized as
long-acting thyroid stimulator (LATS) using mouse bioassays.
These autoantibodies are directed against epitopes on the
ectodomain of the TSH receptor. Methods for measuring TRAb
are even more varied than for TPOAb and TgAb. Two classes
of TRAb can be associated with autoimmune thyroid disorders
– (a) thyroid stimulating autoantibodies (TSAb) that cause
Graves’ hyperthyroidism and (b) thyroid stimulation-blocking
antibodies (TBAb) which block receptor binding of TSH. Each
class of TRAb (TSAb and TBAb) may be detected alone or in
combination in Graves’ disease and Hashimoto’s thyroiditis. The
relative concentrations of the two classes of TRAb may modulate
the severity of Graves’ hyperthyroidism and may change in
response to therapy or pregnancy. Clinical Use of TRAb Tests
TRAb tests are used in the differential diagnosis of
hyperthyroidism, the prediction of fetal and neonatal thyroid
dysfunction due to transplacental passage of maternal TRAb
and prediction of the course of Graves’ disease treated with
antithyroid drugs. Although TBII assays do not directly
measure the stimulating antibodies, these tests have comparable
diagnostic sensitivity to TSAb bioassays (70-95%) for diagnosing
Graves’ hyperthyroidism or detecting a relapse or response
to therapy. The second generation assays employing human
recombinant TSH receptor are now becoming available and
are reported to have superior diagnostic sensitivity for Graves’
disease. Current tests are manual and expensive and vary in
precision, sensitivity, specificity and reference ranges. However,
the TBII tests are important for evaluating pregnant patients
with a history of autoimmune thyroid disease, in whom there
is a risk of transplacental passage of TRAb to the infant . The
lack of specificity of the TBII methods is actually an advantage
in this clinical situation, since a TBII test will detect both the
stimulating and blocking classes of TRAb that can produce
transient hyper- or hypothyroidism, respectively, in the fetus
and newborn. TRAb plays an uncertain role in thyroid-associated
ophthalmopathy (TAO), which appears to be exacerbated by
radioiodine therapy. Since TRAb and other thyroid antibodies
levels increase acutely significantly after radioiodine therapy,
a TRAb measurement prior to radioiodine therapy may be
useful to predict risk of TAO. However, prospective studies are
needed to establish the clinical utility of TRAb measurement in
this context. Patients with very high circulating concentrations
of hCG due to choriocarcinoma or hydatiform mole, as well
as a small number of pregnant patients, may have misleading
positive results using TSAb assays.
Thyroglobulin (Tg) methods
Serum Tg measurement is used as a tumor marker in the
© SUPPLEMENT TO JAPI • JANUARY 2011 • VOL. 59 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.
19
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
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. 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.
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. 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.
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.
Women with a past history of post-partum thyroiditis
Patients on thyroxine therapy
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.
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
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.
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
Patients receiving Amiodarone and Lithium
1.
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.
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
Early TSH RIA methods had the limitation of cross-reactivity
with glycoprotein hormones (such as LH, hCG). Currently,
this problem has been almost completely overcome by using
monoclonal antibodies for TSH IMA methods. Occasionally,
however, unusual cross-reacting isoforms of TSH may be
encountered while using the current assays.
2.
Endogenous analyte antibodies
Robbins et al were the first to report an unusual thyroxine
binding globulin in the serum in 1956. Subsequently,
autoantibodies against T3, T4 and TSH have been identified
in the sera of patients with autoimmune thyroid disorders
20
© SUPPLEMENT TO JAPI • JANUARY 2011 • VOL. 59
as well as non-thyroid disorders. A number of reports have
shown interference due to T3, T4 and TSH autoantibodies
leading to anomalous free and total thyroid hormone levels
and TSH values. However, the currently used methods
rarely have this interference problem. Characteristics of
interference due to endogenous autoantibodies may lead
to falsely low or falsely high values, depending upon the
type of assay and its composition.
3.
Heterophilic antibodies
Heterophilic antibodies (particularly HAMA) may affect
IMA methods more than competitive immunoassays by
causing the formation of a bridge between the signal and
capture antibodies. This creates a false signal resulting
in a high value artifact. Moreover, the result may not be
abnormal; it may be inappropriately normal. A potential for
influencing results of neonatal screening also exists because
antibodies are able to cross the placenta. Interference due to
heterophilic antibodies can be classified into two categories:
i.
HAMA (or human anti-mouse antibodies) are relatively
weak, polyreactive, multispecific antibodies that are
frequently IgM. The presence of HAMA can alter the
total as well as free T3, T4 and TSH results due to
interference. Use of Fab fragments and heterospecies
assay configurations can be employed as approaches
to reduce this kind of interference.
ii. HAAA. Specific human anti-animal antibodies
(HAAA) are produced in response to well-defined
specific antigens after exposure to therapeutic agents
containing animal antigens (such as murine antibody)
or by coincidental immunization through workplace
contact (such as that which occurs in animal handlers).
Though assays for HAMA have been developed, there
are large inter-method differences and therefore the
reliability of these tests is questioned.
4.
Drug interferences
In vitro and in vivo effects may occur due to drug
interferences. When the specimen contains a sufficient
concentration of an interfering therapeutic or diagnostic
agent, it may lead to methodologic interference resulting
in in-vitro effects. An example is that of heparin which, in
the specimen can cause in-vitro stimulation of lipoprotein
lipase; free fatty acids are liberated that inhibit T4 binding
to serum proteins. On the other hand, when results are
altered due to administration of an interfering therapeutic
agent, then it is termed in-vivo effect. An example is that of
furosemide which competitively inhibits thyroid hormone
binding to the specimen, thereby causing an abnormal value
(low) thyroid hormone result. Interference may also be
secondary to certain pathologic conditions. For instance, in
uraemia, abnormal serum constituents such as indole acetic
acid may accumulate and cause interference. In addition,
the presence of fluorophor-related therapeutic or diagnostic
agents in the specimen may alter the results of thyroid tests
that employ fluorescent signals.1
Conclusion
•
Thyroid disorders have diverse clinical manifestations
therefore, on part of vigilant clinician every suspected case
of thyroid disease needs to be evaluated with laboratory
investigations.
•
Thereby appropriate treatment for thyroid disorders can
be instituted or conservative monitoring carried out to
anticipate potential future consequences.
•
The enhanced sensitivity and specificity of TSH assays have
greatly improved the assessment of thyroid function tests.
Since TSH levels change dynamically in response to the
alterations of T3 and T4, the approach to evaluate whether
the patient has thyroid disorder is to test the TSH levels first.
•
When hypothyroidism is suspected, a free-T4 estimate
is appropriate because total-T3 and free-T3 tests have
inadequate sensitivity and specificity in this setting.
•
When hyperthyroidism is suspected, the combination
of a free-T4 estimate and a total- or free-T3estimate
provides the most complete assessment of the severity of
hyperthyroidism and identifies cases of “T3-toxicosis”, i.e.
a selective increase of the serum T3 concentration.
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.
References