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Review Article

Assessment of Thyroid Function: Towards


an Integrated Laboratory - Clinical Approach
Jim Stockigt
Department of Endocrinology and Diabetes and Ewen Downie Metabolic Unit, and Monash University Department of
Medicine, Alfred Hospital, Prahran, VIC 3181, Australia
For correspondence: Prof J Stockigt e-mail: jrs@netspace.net.au

Abstract
Laboratory assessment of thyroid function is now often initiated with a low pre-test probability, by clinicians who may not have
a detailed knowledge of current methodology or testing strategies. Skilled laboratory staff can significantly enhance the choice
of appropriate tests and the accuracy of clinical response; such involvement requires both appropriate training and relevant
information from the clinician. Measurement of the serum thyroid stimulating hormone (TSH) concentration with an assay of
adequate sensitivity is now the cornerstone of thyroid function testing; for untreated populations at risk of primary thyroid dysfunction, a normal TSH concentration rules out an abnormality with a high degree of certainty. However, in several
important situations, most notably pituitary abnormalities and early treatment of thyroid dysfunction, serum TSH can give a
misleading indication of thyroid status. An abnormal TSH concentration alone is never an adequate basis for initiation of
treatment, which should be based on the typical relationship between trophic and target gland hormones, based on serum TSH
and an estimate of serum free thyroxine (T4). Six basic assumptions, some clinical, some laboratory-based, need to be
considered, together with the relevant limiting conditions, for reliable use of this relationship. Current methods of free T4
estimation remain imperfect, especially during critical illness. Diagnostic approach differs significantly between initial
diagnosis and follow-up of treated thyroid dysfunction. In some situations, serum triiodothyronine (T3) is also required, but
serum T3 lacks sensitivity for diagnosis of hypothyroidism, and has poor specificity during non-thyroidal illness. Where assay
results are anomalous, most atypical findings can be resolved by attention to the clinical context, without further investigation.
(Clin Biochem Rev 2003; 24:110-23)

Introduction
This review summarizes principles for the appropriate use of
laboratory assays in the diagnosis and follow-up of thyroid
disorders. Symptoms, physical signs, imaging techniques and
cytological examination will not be considered in detail,
although it is self-evident that laboratory results should be
interpreted in this broader context. The recent monograph
Laboratory support for the diagnosis of thyroid disease
from the National Academy of Clinical Biochemistry, USA,
should be consulted for detailed guidelines on the
preparation, laboratory use and application of current thyroid
1
assays. References for specific points in this review are cited
2
at www.thyroidmanager.org.

It is now well known that the presentations of thyrotoxicosis


and hypothyroidism are so diverse (Table 1) that it is difficult
to rule out these conditions clinically, or to make a conclusive
diagnosis until the disorder is far advanced. Any of the
presentations summarized in Table 1 is reason to seek
confirmatory clinical features, and to measure serum TSH,
using an assay sufficiently sensitive to clearly separate the
suppressed values characteristic of thyrotoxicosis from the
lower limit of the normal reference interval.
Apart from using laboratory tests when thyroid dysfunction is
suspected, or in groups with an increased risk of thyroid
dysfunction (Table 2), there are several situations where
routine testing is appropriate. First, neonatal screening for
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Stockigt J

Table 1. Diverse clinical presentations of thyrotoxicosis and hypothyroidism

Common to Both

Thyrotoxicosis

Hypothyroidism

Classical presentations
Goitre
Postpartum
Menstrual disturbance
Neonatal
Incidental finding

Heart failure
Arrhythmia
Eye disease
Anxiety state
Weight loss
Diarrhoea
Apathetic hyperthyroidism
Myopathy
Periodic paralysis
Dermopathy
Itch
Thyroid storm

Anaemia
Constipation
Depression
Dementia
Myalgia
Nerve entrapment
Hyperlipidaemia
Hypoventilation
Galactorrhoea
Infertility
Puberty, precocious or delayed
Delayed growth
Hypothermia, coma

congenital hypothyroidism is widely established. Second,


recommendations from the American College of Physicians
(Table 3) suggest that thyroid dysfunction is sufficiently
common in women over 50 to justify routine testing at
presentation for medical care (case-finding); the majority of
abnormal findings in this group will identify subclinical
rather than overt dysfunction (see below). Third, the finding
of significant intellectual impairment in the offspring of
3
women who were mildly hypothyroid early in pregnancy
may justify routine testing of thyroid function, either before
conception, or as early as possible in pregnancy.
The application of population reference intervals to
individuals can obscure rather than clarify the diagnosis of
minimal or mild thyroid dysfunction. Serial assessment of
thyroid function shows that individuals remain close to a
4
particular set-point for TSH and T4. Such studies suggest
that potentially important variations from the individual
set-point can still fall within the population norm. This issue
is most relevant for serum TSH values in the upper tail of the
logarithmically distributed normal range, especially in the
assessment of optimal replacement therapy, or diagnosis of
mild thyroid failure. Notably, TSH values of 2-4 mU/L, still
within the reference interval, are associated with an increased
5
prevalence of positive thyroid peroxidase (TPO) antibody.
In practice, individualized interpretation of laboratory data
remains difficult unless there is information on set-point prior
to assessment for possible thyroid dysfunction.
Current laboratory techniques make the diagnosis and
follow-up of thyroid disorders straightforward in the large

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majority of patients, but, in a small minority, problems due to


assay artefacts or atypical clinical presentations can lead to
misdiagnosis, inappropriate treatment, or unnecessary further
investigation. Such cases emphasize the importance of
continuing review of diagnostic methodology by careful
clinical correlation.
Terminology
The distinction between overt and subclinical
hypothyroidism or thyrotoxicosis is based on whether an
abnormal serum TSH concentration is associated with
abnormal levels of the circulating thyroid hormones, T3 and
T4, or whether serum TSH alone is abnormal. The
terminology mild thyroid failure rather than subclinical
hypothyroidism is gaining support, based on evidence that
potentially important tissue abnormalities can occur during
progressive thyroid failure before the serum T4 concentration
becomes clearly subnormal. It should be noted that the more
frequently thyroid function is tested in the absence of clinical
features, the greater the proportion of results with serum TSH
as the sole abnormality.
The terminology of thyroid antibody assays can be
ambiguous. The terms microsomal and TPO antibody refer to
the same moiety, which is the most sensitive marker of
immune thyroid damage, usually associated with the
lymphocytic infiltration that is most extreme in Hashimotos
thyroiditis. The more sensitive and specific radioimmunoassay
for TPO antibody has now superseded microsomal antibody
techniques. Anti-thyroglobulin assays are less relevant for the
diagnosis of immune thyroid disease, but are crucial for the

Assessing Thyroid Function

Table 2. Groups at increased risk of thyroid dysfunction


General
Positive family history
Previous postpartum thyroid dysfunction
Positive thyroid peroxidase antibody
Previous thyroid disease or surgery
Women over age 55
Origin from areas of endemic iodine deficiency
Very low birth weight premature infants
Associated diseases
Diabetes mellitus
Any autoimmune disease
Other endocrine deficiencies
Down syndrome
Turner syndrome
Thalassaemia major
Pituitary or hypothalamic abnormality
Severe head injury
Recent Cushing's syndrome
Therapy
Pituitary surgery or irradiation
Head and neck irradiation
Radical laryngeal/pharyngeal surgery
Treatment of growth hormone deficiency
Cytotoxic therapy
Lithium
Amiodarone
Exposure to iodine excess, eg contrast agents
Interferon , interferon
Interleukin 2
Therapeutic monoclonal antibodies
Granulocyte colony stimulating factor

Table 3. Case Finding / Screening for Thyroid Disease

valid interpretation of serum thyroglobulin assays, for


example in the follow-up of differentiated thyroid cancer.
Measurement of TSH receptor antibody (TRAb) identifies
the probable causative agent in Graves disease, generally by
measuring the extent to which a test serum inhibits binding of
labelled TSH to a receptor preparation. Such assays do not
distinguish between stimulatory and inhibitory activity.
Prevalence of Thyroid Dysfunction
The Whickham study, from an iodine replete region in
Northern England, showed a prevalence of 1.9-2.7% overt
thyrotoxicosis and 1.4-1.9% overt hypothyroidism in women,
with progressive increase with age; prevalence in males was
5
10-fold lower. Estimates of subclinical hypothyroidism were
4-5 fold higher, with about 10% of women over 50 showing
an increase in serum TSH, again with progressive increase
with age. Further studies from the UK, USA and Australian
data from the Busselton study suggest a similar
6,7
prevalence.
The 20 year Whickham follow-up showed that the likelihood
of women developing hypothyroidism rose with age and was
increased about 8-fold if either TPO antibody was positive or
serum TSH increased in the initial study; this risk rose to
almost 40-fold if both were abnormal. The likelihood of
eventual hypothyroidism increased progressively for initial
TSH values over 2 mU/L, values still well within the
reference interval.
Thyroid dysfunction is also common when younger women
are tested post-partum. A Perth study found abnormal thyroid
function in 11.5% of women tested 6 months after delivery,
with TSH values over 4.8 mU/L in 6%, of whom almost 90%
had positive TPO antibody, indicating an autoimmune
8
abnormality. In about half the untreated women with initial
TSH elevation, TSH remained increased 30 months after
delivery, consistent with other studies that show an increased

Recommendations of American College of Physicians, 1998 (modified from reference 20)


It is reasonable to screen women older than 50 years of age for unsuspected thyroid dysfunction, using a
sensitive TSH assay.
Free T4 should be measured when the TSH level is undetectable or >10 mU/L. An undetectable TSH level
and an elevated free T4 level identify overt thyrotoxicosis. A TSH >10 mU/L and a low free T4 identify overt
hypothyroidism. Patients with either of these conditions are likely to benefit from treatment.
There is evidence that subclinical thyrotoxicosis and hypothyroidism have
adverse effects (Table 4), but therapeutic benefit has not yet been conclusively demonstrated.

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Stockigt J

prevalence of late hypothyroidism after postpartum


dysfunction.
Findings from one region do not necessarily apply in other
populations, because of ethnic differences or environmental
variations such as iodine intake. For example, in Hong Kong,
where iodine intake is marginally deficient, only 1.2% of
Chinese women aged over 60 years had serum TSH values
>5 mU/L, with a comparable prevalence of suppressed
values indicating possible thyrotoxicosis. In general,
hypothyroidism is more common with abundant iodine
intake, with goitre and subclinical thyrotoxicosis more
common with low iodine intake. Hence, optimal strategies
for thyroid testing may vary between regions.

All current methods of measuring TSH, free T4 or free T3 in


serum, whether by immunoassay or immunometric
techniques, are comparative, i.e. they depend on the
assumption that the unknown sample and the assay standards
are identical in all measured characteristics other than the
concentration of analyte. A result will be spurious when this
condition is not fulfilled, for example when a sample shows
anomalous binding of tracer to serum proteins or antibodies,
or non-specific interference, either with the system that
separates bound from unbound tracer, or the assay signal.
Heterophilic antibodies remain a potential cause of spurious
assay results and there are currently no techniques that
conclusively rule out this type of interference.
Measurement of Serum TSH

Accuracy and Sensitivity of Clinical and Laboratory


Assessment
To place laboratory testing in perspective we need to
consider the sensitivity and accuracy of clinical assessment
for thyroid dysfunction. Studies of patients evaluated in
primary care show that clinical acumen alone lacks sensitivity
and specificity. In two Scandinavian studies of over 3000
unselected patients who were assessed by both clinical and
laboratory criteria, a thyroid disorder was not suspected by
primary care physicians in over 90% of those who tested
positive, even when clinical features were apparent in
9,10
retrospect. Further, in up to one-third of patients evaluated
for suspected thyroid dysfunction by specialists, laboratory
results led to revision of the clinical assessment.
There are some dissenting opinions on the relative value of
clinical and laboratory evaluation of thyroid function. Some
have expressed the view that the clinical criteria are being
sidelined, while biochemical assessments are lacking in
specificity. This point is generally made by considering TSH
and free T4 measurements individually, rather than in the
trophic hormone - target gland relationship that is the
cornerstone of endocrine diagnosis (see below).
Thyrotoxicosis and hypothyroidism can each have important
consequences before the usual clinical features appear
(Table 4). It may be no more valid to consider these
diagnoses only when typical symptoms or signs appear, than
to wait for the thirst and polyuria before considering the
possibility of diabetes!
While laboratory tests facilitate early diagnosis before
clinical features are obvious, increased sensitivity carries the
price of decreased diagnostic specificity. It remains difficult
to distinguish spurious results from those that indicate mild
dysfunction, especially in the presence of associated illness,
where abnormal TSH and free T4 results lack specificity.
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Secretion of TSH from the anterior pituitary is regulated by


negative feedback from the serum free thyroid hormone
concentrations. Immunometric TSH assays that use two
antibodies against different epitopes of TSH show greatly
improved assay sensitivity. Serum TSH can be precisely
measured to at least 0.03 mU/L, so that the lowest
concentrations in normal subjects are clearly distinguishable
from those found in thyrotoxicosis. However, assay
specificity is not perfect, and false-positive detectable serum
TSH is still found in occasional patients with definite
thyrotoxicosis. The serum TSH response to changes in serum
free T4 is logarithmic; a two-fold change in free T4 induces
inverse 10-100 fold changes in TSH. This feedback
amplification of the serum TSH response as the serum free
T4 increases or decreases, accounts for the fact that serum
TSH can fall outside the reference interval several years
before there is a diagnostic change in serum free T4.
Typical values for the lower reference limit for TSH are
0.3-0.5 mU/L, with upper limits of 4-5 mU/L, but the mean
and median values are in the range 1-1.5 mU/L because of the
logarithmic distribution. The terminology for subnormal
serum TSH values needs to be clarified. Values associated
with thyrotoxicosis, either overt or subclinical are
suppressed, (ie <0.03 mU/L) and need to be distinguished
from subnormal-detectable values in the range
0.05-0.4 mU/L that do not indicate thyrotoxicosis.
Subnormal-detectable values are common in patients with
goitre, only a few of whom develop thyrotoxicosis. Notably,
during severe illness, serum TSH is often subnormal without
indicating any persistent abnormality of thyroid function.
Suppressed TSH values <0.03 mU/L can occur during
critical illness without indicating any intrinsic abnormality of
thyroid function and transient increases to above normal can
occur during the recovery phase.

Assessing Thyroid Function

Table 4. Importance of Subclinical Thyroid Dysfunction21


Subclinical thyrotoxicosis
(suppressed TSH, normal freeT4, free T3 )
Progression to overt thyrotoxicosis
Exposure to iodine may precipitate severe
thyrotoxicosis
Threefold increased risk of atrial fibrillation
after 10 years
Osteoporosis risk is increased
Subclinical hypothyroidism
(Mild thyroid failure)
(increased TSH, normal free T4)
Non specific symptoms may improve with
treatment
Progression to overt hypothyroidism
(~ 5% per year)
Adverse effect on foetal brain development
in pregnancy
Adverse effects on vascular compliance
Independent risk factor for atherosclerotic
disease?
Beneficial effect of treatment on lipids?
Increased prevalence of depressive illness?

Estimation of Serum Free T4


There have been many approaches to the estimation of free
thyroid hormone concentrations in serum; some ingenious
11
approaches have been of questionable validity. Some free
T4 methods have been marketed before they have been
rigorously assessed, so that unexpected interferences may
only be noted after methods have been used for some time.
Currently available assays compensate well when changes in
total T4 and T3 are due to altered concentration of thyroid
binding globulin (TBG), but no current method reflects the in
vivo concentration of free hormone in undiluted serum.
Equilibrium dialysis is often considered the reference
method, but it is also subject to error, especially as a result of
generation of fatty acids during sample incubation, and
under-estimation of the effect of competitors that displace T4
and T3 from binding proteins in vivo (see below). Two-step
methods that separate a fraction of the free T4 pool from the
binding proteins before the assay step are generally least
prone to analytical artefacts.
Numerous medications can displace T4 and T3 from TBG
(Table 5), but it is technically difficult to get an accurate
reflection of these effects with current free T4 methods that
use diluted samples. Competitors are usually less
protein-bound than T4 itself, so that with progressive

Table 5. Medications that influence thyroid hormone or


TSH levels
Inhibit pituitary TSH secretion
Dopamine, dobutamine, glucocorticoids, octreotide
Iodine load increases thyroid hormone
synthesis
Contrast agents, amiodarone, topical preparations
Impair thyroid hormone release
Iodine excess, lithium, glucocorticoids,
aminoglutethimide
Inhibit T4-T3 5' deiodination
Amiodarone, glucocorticoids, beta blockers *
Contrast agents, e.g. iopanoic acid, ipodate
Augment abnormal immune function
Interleukin 1, interferon , interferon
Monoclonal antibody therapy
Modify binding of T4, T3 to plasma proteins
a. Increase concentration of T4 binding
globulin
Estrogen, heroin, methadone
Clofibrate, 5-fluorouracil, perphenazine, tamoxifen
b. Decrease concentration of T4 binding
globulin
Glucocorticoids, androgens, l-asparaginase
c. Displace T4 and T3 from binding proteins
Furosemide, salicylates, phenytoin,
carbamazepine
Non-steroidal antiinflammatory agents #
Heparin
Displace T4 from tissue pool
Oral cholecystographic agents,
some alkylating agents
Modify thyroid hormone action
Amiodarone, phenytoin
Increase clearance of T4,T3
Barbiturates, phenytoin, carbamazepine, rifampicin
Sertraline?, fluoxetine?, dothiepin?
Impair absorption of ingested T4
Aluminium hydroxide, ferrous sulfate,
cholestyramine, calcium carbonate
Colestipol, sucralfate, soya preparations,
kayexalate
* those with membrane-stabilizing effect, e.g.
propranolol
# some members of the group
In vitro effect of in vivo heparin administration

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dilution, the free concentration of competitor declines before


the free T4 concentration. If T4, with a free fraction of about
1:4000 in undiluted serum, is compared with a drug that has
a free fraction in serum of 1:50, progressive dissociation will
sustain the free T4 concentration at 1:100 dilution, while the
free drug concentration decreases markedly after a dilution of
only 1:10. This difference leads to an under-estimate of free
T4 after sample dilution, as demonstrated by comparing the
T4-displacing effect of frusemide in three commercial free
T4 assays; the effect of frusemide was least obvious in the
method with highest sample dilution (Figure 1). Because of
this effect, the apparent free T4 concentration in diluted
samples will be an under-estimate in the presence of high
therapeutic concentrations of drugs such as phenytoin,
carbamazepine, frusemide, mefenamic acid (Ponstan) and
11
salicylate.

Figure 1. Effect of addition of frusemide to normal serum on


estimates of free T4 using three commercial free T4 methods
that involve varying degrees of sample dilution. The effect of
the competitor is progressively obscured with increasing
sample dilution. Redrawn from reference 22.
In contrast to such under-estimates of free T4, heparin has the
opposite effect to increase the apparent free T4 concentration,
12
due to an in vitro artefact of sample storage. In the presence
of a normal serum albumin concentration, non-esterified fatty
acid (NEFA) concentrations >3 mmol/L will increase free T4
by displacement from TBG, but such concentrations are
uncommon in vivo. However, serum NEFA may increase to
these levels during storage or incubation of samples from
heparin-treated patients, as a result of heparin-induced lipase
activity (Figure 2). This effect is accentuated if serum
triglyceride concentrations are high, serum albumin
o
concentration is low, or incubation at 37 C is prolonged;
under these conditions doses of heparin as low as 10 units
may produce this artefact, low molecular weight heparin
preparations have a similar effect.
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Figure 2. Mechanism of heparin-induced increase in


9
apparent serum free T4. Heparin acts in vivo (left) to
liberate lipoprotein lipase from vascular endothelium. Lipase
acts in vitro to increase the concentration of non-esterified
fatty acids; concentrations above 2-3 mmol/L displace of T4
and T3 from TBG. This effect is accentuated by low serum
albumin and high triglyceride concentrations and by sample
incubation at 37C.

The fallibility of current free T4 methodology is demonstrated


by a study of bias in nine commonly used commercial
methods in relation to an equilibrium dialysis reference
13
method, see below. Hence it is absolutely essential, especially
in pregnant women and in patients with an
associated illness, to interpret results only in relation to a
13
specific method. It is becoming clear that, in numerous
clinical situations, free T4 estimation does not serve as a
robust, reliable index of thyroid function. Despite the
theoretical attraction of measuring the concentration of free
or biologically active hormone, it remains uncertain whether
current free T4 methodology is any improvement over an
uncontentious measurement of total T4. The limitations of free
T4 methodology are most evident where the diagnosis of
thyroid dysfunction is clinically and analytically most
difficult. For this reason, it is important that total T4 methods
be retained for reference.
The TSH-T4 Relationship
Whatever strategy is used for first-line testing, a sensitive
serum TSH assay and an estimate of serum free T4 are both
necessary for definitive assessment of thyroid status. As
shown in Figure 3, the common types of thyroid dysfunction
can be identified in a single sample from characteristic
diagonal deviations in the normal free T4-TSH relationship.

Assessing Thyroid Function

Table 6. The free T4 -TSH relationship in the assessment


of thyroid status.
Assumptions and Limiting Conditions

Figure 3. The relationship between serum TSH and total free


T4 concentrations in normal subjects (N) and in various
typical abnormalities of thyroid function: primary
hypothyroidism (A); central or pituitary-dependent hypothyroidism (B); thyrotoxicosis due to autonomy or abnormal
thyroid stimulation (C); and TSH-dependent thyrotoxicosis
or generalised thyroid hormone resistance (D).
Note that linear free and total T4 responses correspond to
logarithmic TSH changes. Findings at A and C represent
primary thyroid abnormalities, while results in areas B and D
suggest a primary pituitary abnormality. Results in the
intermediate areas are most often due to non-steady state
sampling conditions, or an altered T4 -TSH relationship.

The figure shows primary hypothyroidism due to target gland


failure (high serum TSH, low free T4 : A), failure of TSH
secretion (both low: B), autonomous or abnormally stimulated
target gland function (high free T4, suppressed TSH: C), and
primary excess of TSH, or thyroid hormone resistance (both
high: D). Abnormal results that fall outside these areas
suggest that some other factor has disturbed the TSH-free T4
relationship. (The link between components of other
feedback systems can also be applied to the investigation of
hypogonadism, glucocorticoid and mineralocorticoid
abnormalities, hypoglycaemia, hypercalcaemia etc.)
The diagnostic validity of this relationship depends on a
number of assumptions and limiting conditions (Table 6). It
is notable that only the last three of these assumptions can be
validated in the laboratory; the first three must be verified
clinically. The first assumption, steady-state conditions,
should be questioned when associated illness or medications
perturb the pituitary-thyroid axis; the large difference
between the half-lives of TSH (one hour) and T4 (one week)

1.

Steady-state conditions
(N.B. difference in half-life of TSH and T4 )
Acute effects of medications
Early response to therapy
Evolution of transient thyroid dysfunction
Recovery from severe illness

2.

Normal trophic-target hormone


relationship
Alternative thyroid stimulators
Immunoglobulins
Chorionic gonadotrophin
Medications
T3 , triiodothyroacetic acid
Glucocorticoids
Dopamine
Amiodarone
Recent thyrotoxicosis
Recent longstanding hypothyroidism (Figure 5)
Variable individual setpoint
TSH receptor mutations

3.

Tissue responses proportional to serum


free T4 concentration
T3 excess
Hormone resistance syndromes
Slow onset/offset of thyroid hormone action
Drug effects ? (amiodarone, phenytoin)

4.

Accurate estimate of active hormone


concentration
Alternative agonist in excess (e.g.T3)
Changes in serum binding proteins
TSH of altered biologic activity
(pituitary disorders)
Spurious assay results
TSH
Heterophilic antibodies
Free T4
Circulating inhibitors of
binding
Heparin artefact (Figure 2)
Rheumatoid factor

5.

Appropriate reference ranges


Influence of age
Medications
Associated illness
Nutrition

6.

Adequate assay sensitivity


Poor precision at TSH detection limit.

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Table 7. Indications for measurement of serum free T3


Required
Potential thyrotoxicosis with suppressed TSH
and normal serum free T4.
During antithyroid drug therapy to identify
persistent isolated T3 excess.
Diagnosis of amiodarone-induced thyrotoxicosis.
Useful
Early recurrence of thyrotoxicosis.
Extent of T3 excess during suppressive T4 therapy
or after T4 overdose
Not required or misleading
Diagnosis of hypothyroidism
During critical illness
During routine T4 replacement
Screening of asymptomatic subjects
Monitoring of T3 treatment

Clinical Applications
Application of diagnostic strategy will differ depending on
the test group, i.e. testing of untreated subjects in whom
clinical features suggest thyroid dysfunction, screening or
case finding in at risk groups, evaluation of the response to
treatment, or assessment when associated illness or drug
therapy are likely to complicate both clinical and laboratory
assessment.
Untreated Subjects
Assessment of untreated subjects now commonly begins with
measurement of TSH alone, with free T4 and/or free T3
added only if TSH is abnormal, or if an abnormality of TSH
secretion is suspected. According to this algorithm, free T4
should be measured to distinguish between overt and
subclinical hypothyroidism when serum TSH is elevated,
while a suppressed or subnormal TSH level should be
followed by assay of both free T4 and free T3 to distinguish
subclinical from overt thyrotoxicosis and to identify T3
toxicosis.

accounts for many transient nondiagnostic abnormalities, especially


during critical illness. In several situations
T3 as well as T4 is an important, or
dominant, determinant of thyroid status.
The relevance of serum free T3
measurement is summarized in Table 7.
Note that serum free T3 is of little value in
monitoring T3 treatment because of wide
variations that depend on the interval
between dosage and sampling.
The TSH First Testing Strategy
Either serum TSH or free T4 can be used
for initial screening and case finding, but
TSH gives better first line testing than free
T4, at slightly higher cost. Because thyroid
gland abnormalities are 10-20 times more
common than variations due to pituitary dysfunction, TSH
changes can generally be regarded as giving an inverse
reflection of thyroid status. An algorithm for the
assessment of thyroid function based on initial
measurement of TSH is shown in Figure 4. However, there
are important situations in which TSH alone can give a
misleading or ambiguous assessment of thyroid status (Table
8), despite the high negative predictive value of a
normal serum TSH concentration in ruling out primary
hypothyroidism or thyrotoxicosis.
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Clin Biochem Rev Vol 24 November 2003

Figure 4. Algorithm for the assessment of thyroid function


based on initial assay of serum TSH. Abnormal TSH values
lead to further assays as shown. Assays of free T4 are always
required if pituitary dysfunction is suspected, during the
early treatment of thyroid dysfunction, during critical illness
and with the use of drugs that influence the pituitary-thyroid
axis. (* Further testing is indicated if pituitary dysfunction is
known or suspected, during critical illness and in the first
6-12 months of treatment for thyroid dysfunction).

Assessing Thyroid Function

Table 8. Serum TSH alone can give a false or uncertain indication of thyroid status.
Condition

TSH

Free T4

Free T3

N-H

L-N
L-N

L
L

N,H

Thyrotoxicosis
Subclinical
Early Treatment

U
U

N
H-N-L

N
H-N-L

Hypothyroidism
Subclinical
Early Treatment

H
H

N
L-N

TSH Assay Artefact


Euthyroid subject
Thyrotoxic subject

H
L-N-H

N
H

Medications
Dopamine
Glucocorticoids

L
L

N
N

Critical illness
Euthyroid subject
Hypothyroid subject

U,L
N

L,N
L,N

Abnormal TSH secretion


Thyrotoxic
Central TSH excess
Hypothyroid
Pituitary-hypothalamic disturbance
Very low birth weight premature infants
Euthyroid
Thyroid hormone resistance

N
H

____________________________________________
U: undetectable TSH <0.03 mU/L; L: low; N: normal; H: high.

Response to Treatment
In patients with newly treated thyrotoxicosis, TSH may
remain suppressed for several months after normalisation of
serum free T4 and free T3; serious over-treatment may result
if TSH alone is used for adjustment of antithyroid drug
dosage. Furthermore, during drug treatment, thyrotoxicosis
may persist due solely to T3 excess. Hence, reassessment of
serum free T4 and free T3 levels is recommended after about
3-4 weeks drug treatment of thyrotoxicosis to allow
appropriate dose adjustment. During long-term treatment,
TSH generally gives a reliable guide to optimal drug dosage.
Similarly, during long-term replacement or suppressive
therapy with T4, serum TSH is the best single index of

appropriate dosage. Optimal replacement is generally


reflected by a low-normal TSH value of about 1 mU/L, often
with a slightly increased level of serum free T4. However,
during the early phase of treatment of hypothyroidism, free
T4 should also be measured because TSH may remain
inappropriately elevated for many months after normalisation
of T4 (Figure 5). In general, serum TSH should initially be
checked after 2-3 months, but need only be checked
annually after the first year of treatment.
During TSH suppressive therapy with T4, for example in the
management of differentiated thyroid cancer, periodic
assessment of free T4 and free T3 in addition to TSH, is
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have a definite place in the assessment of potential difficulties


with various free T4 estimates, particularly during critical
illness, as demonstrated by a key study of severely ill
14
euthyroid patients (Figure 6). Various free T4 assays gave
widely discrepant method-dependent abnormal free T4
estimates, while the total T4 assay suggested that the
majority of these patients remained euthyroid, with
subnormal serum TSH, probably attributable to glucocorticoid
14
treatment. Free T4 methods that were influenced by
albumin binding of tracer tended to give subnormal
estimates, while equilibrium dialysis and related methods
appeared to be vulnerable to the heparin-NEFA artefact, with
a trend towards high free T4 estimates.

Figure 5. Serial changes in serum free T4 and TSH in


response to T4 replacement in a patient with longstanding
severe untreated primary hypothyroidism without evidence
of pituitary enlargement or tumour. Normalization of serum
TSH lagged 7-10 months behind normalization of serum free
T4.
appropriate to limit the degree of thyroid hormone excess,
because over-treatment can have important adverse effects on
the cardiovascular system and on bone density. In the
treatment of hypothyroidism due to pituitary or hypothalamic
disease, serum TSH is of no value in assessing T4 dosage,
which should be judged from clinical response and serum
free T4.
Difficult Diagnostic Situations
Especially in hospital practice, interpretation of thyroid
function is often compromised by associated illness or by
medications. There is a high prevalence of abnormal serum
free T4 or TSH values in patients with acute medical or
psychiatric illness, but when TSH and free T4 are considered
together, as in Figure 3, few of these abnormalities indicate
true thyroid dysfunction. Clinical assessment of thyroid
status is difficult in the face of associated disease and some
have advocated widespread laboratory testing. However,
because of low specificity, opinion has moved away from
routine testing during critical illness unless there is a clinical
indication.
During any severe illness, one or more of the assumptions
outlined in Table 6 may not be valid, for example when there
are wide fluctuations from the steady state due to acute
inhibition of TSH secretion or abnormally rapid T4
clearance. Serum free T4 estimates are prone to multiple
method-dependent interferences, for example due to heparin
(see above Figure 2). Measurement of total T4 continues to
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There are important issues in selecting a panel of thyroid tests


that will best serve a particular clinical population. For
example, the multiple effects of severe illness on free T4
estimates may be of minor importance for a laboratory that
serves predominantly ambulatory patients. A different assay
profile will be required in a laboratory that needs to exclude
thyroid dysfunction during critical illness or in pregnancy.
Effects of Medications on Serum T4 and TSH
The multiple effects of medications on the pituitary-thyroid
axis are summarized in Table 5. Effects on serum TSH are
generally physiological, whereas most effects on free T4
estimates are methodological. Notably, lithium and
iodine-rich compounds, in particular amiodarone, can cause
thyroid function to become abnormal. Amiodarone is the
15
most complex and difficult drug that affects thyroid status.
There may be poor correlation between circulating thyroid
hormone levels and clinical manifestations in amiodaroneinduced thyroid dysfunction, because of interaction of this
drug or its metabolites with thyroid hormone receptors. In
iodine-replete regions the predominant amiodarone-induced
thyroid abnormality is hypothyroidism, which is especially
prevalent in those with associated autoimmune thyroiditis.
Amiodarone causes two forms of thyrotoxicosis, one due
directly to iodine excess and the other attributed to a unique
type of thyroiditis. Benign euthyroid hyperthyroxinaemia
occurs in up to 25% of treated patients, who show increased
serum concentrations of free T4, with normal TSH and
normal or low concentrations of free T3.
Lithium, used in the management of bipolar illness, has
multiple effects on the pituitary-thyroid axis, the most
important being inhibition of hormone release. Lithium can
exacerbate, or may initiate autoimmune thyroid disease with
development of goitre and eventual hypothyroidism; there
are also some reports of lithium-induced thyrotoxicosis.
Serum TSH, free T4 and free T3 assays generally give a true
index of thyroid status during lithium treatment.

Assessing Thyroid Function

Figure 6. Free T4 estimates by six


different kit methods in euthyroid
patients
after
bone
marrow
transplantation. Therapy included heparin
and glucocorticoids. Mean values are
normalized to 100%, with reference
limits shown by the boxes. A high
proportion of free T4 estimates are
abnormal, either increased or decreased,
depending on the method. Serum total T4
remained normal in 19 of the 20 study
subjects, while serum TSH was
subnormal in 11, independent of the
method. Redrawn from reference 14.

Phenytoin commonly results in subnormal serum total T4,


with an apparent lowering of free T4, due predominantly to
the dilution-related artefact described above, without the
anticipated increase in TSH. Such findings are hard to
distinguish from central hypothyroidism due to pituitary
deficiency, but the major discrepancy is probably a
methodological artefact related to underestimation of free T4
16
in diluted serum samples. The assessment of patients who
have had pituitary surgery and are also taking phenytoin,
remains very difficult.
Antibody Measurements

17

In subclinical hypothyroidism, the presence of TPO


antibodies indicates a 4-5-fold increase in the chance of
developing overt hypothyroidism. The presence of this
antibody also indicates an increased likelihood of postpartum
thyroiditis or amiodarone-induced hypothyroidism. The
finding of persistently positive thyrotropin receptor antibody
(TRAb) is useful in indicating that apparent remission of
Graves disease is unlikely to be sustained. TRAb
measurement can also indicate the possibility of neonatal or
intrauterine thyrotoxicosis in the infant of a mother with
autoimmune thyroid disease and may also define the
aetiology of atypical eye disease.

Thyroglobulin

18

Serum thyroglobulin concentrations should always be


interpreted in relation to the prevailing level of TSH, which
is responsive to alterations in thyroid hormone dosage. In the
long-term follow-up of differentiated thyroid cancer, an
undetectable serum thyroglobulin concentration in the
presence of high serum TSH indicates effective ablation of
differentiated thyroid tissue, benign and malignant. Such a
finding may justify less rigorous long-term T4-induced
suppression of TSH. Thyroglobulin is undetectable in
thyrotoxicosis factitia, and generally extremely high in
subacute thyroiditis and in amiodarone-induced thyrotoxicosis
due to thyroiditis.
There are still unsolved technical problems in the
optimisation of thyroglobulin assays. Major issues relate to
assay standardization, interference from endogenous
anti-thyroglobulin antibodies and heterophilic antibodies, as
well as inherent problems of assay sensitivity.
The Laboratory-Clinical Interface
The diverse clinical presentations of thyroid dysfunction
mandate laboratory requests from clinicians who may be
unfamiliar with the interpretation of current assays, or with
effects that interfere with these techniques. Comments from
the laboratory can improve clinical response; the quality of
this assistance depends on both the training and experience of
the reporter and the available clinical information.
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Table 9. Clinical correlation is required for interpretation of laboratory results #

Clinical context

Assay Results
Free T4

Free T3

Pregnancy
L*
L,N
Antithyroid drug treatment, initial months
H,N,L
H,N,L
Recent T4 therapy for hypothyroidism
N
N
Hypothyroidism, appropriate T4 dose
H
N
Hypothyroidism, intermittent compliance
H,N
Appropriate T4 suppressive therapy
H
N

Excessive T3 treatment
L
H,N
Recombinant TSH, suppressive T4
H
N
Hypopituitarism
L
Phenytoin
L*
Critical illness
L*
L
Heparin effect in critical illness
L,N,H*
L
Recovery phase of critical illness
L,N
Drugs that inhibit T4&T3 binding to TBG
L*
L*
Amiodarone effect in euthyroid subject
H
L
Acute T4 overdose
HH
H,N
___________________________________________________________
U undetectable; L low; N normal; H high;
* effect dependent on assay method
# typical reference intervals are shown in Table 10.
depends on interval between dosage and sampling

The competitive binding assays that are used for thyroid


diagnosis were developed about 30 years ago with in house
reagents. Optimal diagnostic technology was initially
available only to specialists and results were often slow.
Sophisticated standardised reagents and automated
instrumentation (eg solid phase antibodies, magnetic
separation systems, chemiluminescent detection systems)
have now replaced these early methods; results are rapidly
available to a wide range of practitioners. Non-specialist
users of endocrine assays are most likely to benefit from
laboratory-based assistance in the interpretation of results,
but with assay automation, laboratorians have become more
distant from the bedside. As clinicians receive less assistance,
they provide progressively less relevant information and vice
versa. Laboratorians, in turn may see results that are
uninterpretable or ambiguous unless the relevant clinical
information is available (Table 9). In these situations, the
possibility of an assay artefact would need to be considered,
but review of the clinical context often resolves an apparently
anomalous result, so that assay validity is affirmed and
unnecessary further investigation is avoided.

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Clin Biochem Rev Vol 24 November 2003

TSH
N
U
H
N
H
U,L
U
HH
L,N
L,N
L
L
H
L,N
N
N

Discordant or Anomalous Results


After review of the clinical context, the following steps are
helpful in evaluating anomalous thyroid results:
a.
Review of the medication history.
b.
Confirmation of serum TSH by an alternative
method that identifies the degree of TSH
suppression.
c.
Follow-up sampling to establish whether the
abnormality is transient or persistent.
d
An alternative estimation of serum free T4,
avoiding one-step methods that are known to
frequently give spurious results, especially
during critical illness.
e.
Measurement of serum total T4 to establish
whether the serum free T4 estimate is
disproportionately high or low, due to a
pre-analytical or method-dependent artefact.
f.
Evaluation of the sample for possible heterophilic
antibody interference; note that there are no
established criteria that rule out such interference.
g.
Investigation of propositus and family members for
evidence of unusual binding abnormalities or
hormone resistance.

Assessing Thyroid Function

Table 10. Typical Reference Ranges for Serum Thyroid


Hormones and TSH*

References
1.

Hormone

Reference Range

Total thyroxine (T4)

60-140 nmol/L

Free T4

10-25 pmol/L

Total triiodothyronine (T3)

1.1-2.7 nmol/L

Free T3

3-8 pmol/L

TSH

0.4-4.0 mU/L

* Ranges should be determined for the particular


methods used in each laboratory.
Higher values in childhood.

Clinical Feedback, Quality Assurance and Cost


Effectiveness
Clinical feedback will remain a key aspect of quality
assurance in laboratory testing. While assay precision or
reproducibility can be evaluated from the laboratory,
diagnostic accuracy requires clinical correlation.
The majority of thyroid diagnosis is now quite
straightforward, but it is easy to underestimate problems that
remain, since they represent only a small fraction of the total.
Despite the elegance and ingenuity of current assay
techniques, diagnostic inaccuracy of immunoassays still
wastes substantial resources and studies of "cost
effectiveness" do not evaluate the human costs that result
from unnecessary further testing, false alarms and
19
inappropriate management.
Some thyroid disorders, notably papillary, follicular and
medullary carcinomas require follow-up by tumour marker
assays over many years. Changes in thyroglobulin and
calcitonin assay methodology made without clinical
consultation, in particularly changes made before the lower
limits of detection have been critically defined, (i.e. apparent
increase in sensitivity with loss of specificity), can give a
false and at times disastrous impression of reactivation of
disease.
Footnote: Typical reference intervals for common thyroid
assays are given in Table 10.

2.

3.

4.

5.

6.

7.

8.

9.

10.

11.
12.

13.

National Academy of Clinical Biochemistry.


Laboratory Medicine Practice Guidelines.
Laboratory Support for the Diagnosis of
Thyroid Disease. Thyroid 2003;13:1-126 and
www.nacb.org/lmpg/thyroid_LMPG_word.stm
For references to specific points in this text see:
Thyroid Disease Manager Chapter 6b. Clinical
Strategies for Thyroid Function Testing. www.
thyroidmanager.org
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