Clin Lab Med 24 (2004) 19–28

Thyroid disease: pathophysiology and

diagnosis
Laurence M. Demers, PhD, DABCC, FACB
Department of Pathology and Medicine, The M. S. Hershey Medical Center,
The Pennsylvania State University, 500 University Drive, Hershey, PA 17033, USA

The thyroid is a butterfly-shaped gland that is located in the front of the

neck just above the trachea; it weighs approximately 15 to 20 g in the adult
human. The thyroid produces and releases into the circulation at least two
potent hormones, thyroxine (T4) and triiodothyronine (T3), that influence
basal metabolic processes or enhance oxygen consumption in nearly all
body tissues. Thyroid hormones also influence linear growth; brain function,
including intelligence and memory; neural development; dentition; and bone
development [1–3].
The thyroid gland produces T4 and T3 by using iodide that is obtained
from dietary sources or from the metabolism of thyroid hormones and other
iodinated compounds. About 100 lg of iodide is required on a daily basis
to generate sufficient quantities of thyroid hormone. Dietary ingestion of
iodide in the United States ranges between 200 lg/day and 500 lg/day and
varies geographically; ingestion is greater in the western part of the United
States than in the eastern states. The specialized thyroid epithelial cells of
the thyroid gland are equipped with a Na/I symporter that helps concentrate
iodide at 30 to 40 times the level in plasma to ensure adequate amounts for
the synthesis of thyroid hormone. The iodide that is trapped by the thyroid
gland is oxidized to iodine by the enzyme, thyroid peroxidase. The iodine
undergoes a series of organic reactions within the thyroid with tyrosine
residues on thyroglobulin protein to produce intermediary compounds,
monoiodotyrosine (MIT) and diiodotyrosine (DIT). Tetraiodothyronine or
T4 is synthesized in thyroglobulin by the coupling of two DIT molecules. T3
is produced from the coupling of a MIT molecule and a DIT molecule or by
59-monodeiodination of T4 by way of an iodothyronine 59-monodeiodinase
type 1 enzyme that is located in the thyroid, pituitary, liver, and kidney; the
conversion of T4 toT3 also can take place in these tissues. T4 and T3 that are

E-mail address: lmd4@psu.edu

0272-2712/04/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved.
doi:10.1016/j.cll.2004.01.002
20 L.M. Demers / Clin Lab Med 24 (2004) 19–28

stored in the thyroglobulin of the thyroid gland are released into the
circulation through the action of pituitary-derived thyrotropin after the
protein is endocytosed at the apical surface of the thyroid epithelial cell
and hydrolyzed by intracellular lysosomes.
A normal individual produces approximately 90 to100 lg of T4 and 30 to
35 lg of T3 on a daily basis. It was estimated that about 80% of the T3 is
derived from peripheral metabolism (59-monodeiodination) of T4 and only
about 20% is secreted directly from the thyroid gland itself. On a weight
basis, T3 is about three to five times more potent as a thyroid hormone than
T4; it is believed to be the biologically active form of the hormone.
Thyroid gland function and hormone synthesis and release are regulated
by thyrotropin that is secreted by thyrotroph cells that are located in the
anterior pituitary gland. The pituitary secretion of thyrotropin is influenced,
in turn, by the releasing factor, thyrotropin-releasing hormone (TRH) that
is produced in the hypothalamus (Fig. 1). The secretion of thyrotropin and
TRH is regulated by negative feedback from thyroid hormone, pre-
dominantly T3, from the circulation or T3 that is produced locally from
intracellular conversion of T4 to T3. When circulating thyroid hormone
levels are elevated, the synthesis and secretion of serum thyrotropin is
blunted. In contrast, when circulating levels of T4 and T3 are low, serum
thyrotropin levels are increased in a compensatory fashion. The geometric
mean level of serum thyrotropin in normal individuals is approximately

Fig. 1. Regulation of thyroid gland function.

 L.M. Demers / Clin Lab Med 24 (2004) 19–28 21

1.5 lU/mL as recently reported in the National Health and Nutrition

Examination Survey (NHANES) III study. When hypothalamic-pituitary
function is intact, serum thyrotropin levels are markedly suppressed (to
\0.05 lU/mL) in patients who have hyperthyroidism and elevated
circulatory levels of serum T4, whereas a marked increase in thyrotropin
([5 lU/mL) occurs in patients who have hypothyroidism and low blood
levels of serum T4. The mechanism through which thyrotropin interacts with,
and binds to, the thyroid gland is well-understood. Thyrotropin binds to
a specific membrane receptor that is located on the surface of the thyroid
epithelial cell and activates the cell signaling mechanisms through the
enzyme, adenylate cyclase, that is located in the plasma membrane. Activa-
tion of adenylate cyclase increases intracellular cyclic adenosine mono-
phosphate levels that, in turn, stimulate additional intracellular signaling
events that lead to thyroid hormone formation and secretion.
T4 and T 3 circulate bound primarily to carrier proteins. T4 binds strongly
to thyroxine binding globulin (TBG, 75%) and weakly to thyroxine-
binding prealbumin (TBPA, transthyretin, 20%) and albumin (5%). T3
binds tightly to TBG and weakly to albumin with little binding to TBPA.
The geometric mean for serum T4 in normal individuals is approximately 8
lg/dL, whereas the mean serum T3 level is approximately 130 ng/dL. Under
normal protein-binding conditions, all but 0.03% of serum T4 and 0.3%
of serum T3 is protein bound. Only a small amount of unbound (or free)
T4 (approximately 2 ng/dL) and T3 (approximately 0.3 ng/dL) circulates
in a free state. The free concentration is considered to be responsible for
the biologic effects of the thyroid hormones. Physiologic situations are
associated with a change in the serum concentration of these thyroid-
binding proteins (eg, pregnancy, nonthyroidal illness, ingestion of drugs
that affect the level or affinity of these binding proteins). Under these
circumstances, the serum concentrations of total T4 and total T3 change
in parallel to the changes that occur in the thyroid hormone-binding
proteins; however, the serum concentrations of free T4 and free T3 remain
normal and the individual remains euthyroid. In contrast, the serum
concentration of free T4 and free T3 are increased in hyperthyroidism and
decreased in hypothyroidism.

Thyroid function testing

Serum-based tests are available by immunoassay for measuring the
concentration of thyroid hormones in the circulation, including total T4
(TT4), total T3, free T4 (FT4), and free T3 (FT3) hormone. In addition, direct
measurement of the thyroid hormone–binding plasma proteins, thyroxine
binding globulin (TBG), transthyretin/prealbumin, and albumin also are
available. The thyroid test measurement that has the greatest usefulness for
evaluating patients who are suspected of having thyroid disease is the third
22 L.M. Demers / Clin Lab Med 24 (2004) 19–28

generation thyrotropin assay. Most current third generation thyrotropin

assays with a functional sensitivity of 0.02 lU/mL or less (interassay
imprecision \20%) easily can distinguish hyper- and hypothyroidism from
euthyroidism and may differentiate the patient who suffers from the
‘‘euthyroid sick syndrome’’ from true hyperthyroidism with a suppressed
thyrotropin level. Other methods in thyroid testing include the measurement
of thyroid gland autoantibodies, including anti thyroid peroxidase (TPOab),
antithyroglobulin, and antibodies against the thyrotropin receptor. All of
these test methods are available on automated immunoassay instruments
that are located in most hospital and reference laboratories with tight
(\10%) method between run coefficients of variation.

Screening for and managing thyroid disease

Measuring the level of thyrotropin usually is selected for thyroid disease
screening in an at-risk ambulatory population. Current third generation
thyrotropin tests have sufficient sensitivity and specificity to cover the entire
spectrum of thyroid disease, ranging from overt hypothyroidism to overt
hyperthyroidism. A thyrotropin-alone first test strategy has been used by
many primary care physicians and seems to work well in an outpatient,
ambulatory population. A thyrotropin-centered strategy in a hospital
setting can be fraught with complications and be diagnostically misleading
because of the high prevalence of severe nonthyroidal illnesses (NTI) and
the use of medications that can affect the metabolism and the action of
thyroid hormones. Assessment of thyroid status in pregnancy is affected by
a thyrotropin-centered test strategy because the blood levels of serum
thyrotropin and serum FT4 may be altered from the physiology of
pregnancy itself. In general, the thyrotropin-centered test strategy for
screening seems to be satisfactory in an ambulatory setting and is the most
popular approach taken by primary care practitioners. Serum thyrotropin
screening of the neonatal population for hypothyroidism is an accepted
practice in Europe and is being considered in the United States; however,
TT4 still remains the initial screening test for congenital hypothyroidism in
this country.
When an abnormal serum thyrotropin result is obtained, the recom-
mended reflex strategy is to obtain a serum FT4 level. Under most cir-
cumstances, the serum FT4 level, whether obtained as an indirect estimate
test as performed on most automated instruments or as measured directly by
equilibrium dialysis, shows an inverse correlation to the serum thyrotropin
level. Discordance between serum FT4 and serum thyrotropin, however, is
not uncommon, even in patients who do not have a significant nonthyroidal
illness. Usually, it is caused by the presence of subclinical disease that may
be referred to as ‘‘mild thyroid disease.’’ In this situation, an abnormal
serum thyrotropin result is observed in the presence of a normal free T4.
 L.M. Demers / Clin Lab Med 24 (2004) 19–28 23

When the converse situation occurs (ie, when an abnormal serum FT4 result
is associated with a normal serum thyrotropin), an abnormality in the
protein binding of thyroid hormone usually is the cause.
The measurement of thyroid hormones in patients who have unstable
thyroid disease and are under the care of a physician frequently produces
discordant thyroid test results as well. Patients who are being treated with
L-thyroxine (LT-4) replacement therapy for hypothyroidism can show
a discordance between serum thyrotropin and FT4 during the early phase
of treatment or when changing the dose of LT-4. Changes in serum FT4
usually precede changes in serum thyrotropin by several weeks because
usually it takes about 8 weeks for the pituitary-thyroid axis to equilibrate
and adjust. In the case of patients who are being treated for hyperthyroid-
ism, the suppressed serum thyrotropin may not normalize for several
months; the serum FT4 level normalizes much sooner and is a better marker
to gauge effectiveness of the therapy that is used in the first few months of
treating hyperthyroidism.

Reference intervals for thyroid function tests

The NHANES III report brought into focus the need to reevaluate the
reference range for thyroid function tests, particularly thyrotropin, in all
populations and at all ages. The median serum thyrotropin level in the total
population was 1.49 lU/mL, which is considerably less than the 4.5 lU/mL
upper limit of normal that is reported by most laboratories. Several
endocrinologists recently proposed a re-evaluation of the upper reference
limit; they suggested that a serum thyrotropin level that was greater than
3.0 lU/mL may not be ‘‘normal,’’ regardless of the serum FT4 result.
Patients in the subclinical category of hypothyroidism could fit into this
range. The NHANES III report also documented the significant increase in
serum thyrotropin that starts around 50 years of age in a disease-free
population. The median level of serum thyrotropin increases to 1.60 lU/mL
after age 50, 1.79 lU/mL after age 60, 1.98 lU/mL after age 70, and 2.08
lU/mL after age 80. Thus, it was suggested that age-related reference ranges
be considered for individuals who are older than age 50 (Table 1).

Table 1
Relative thyroid hormone reference ranges in adults
Method Reference range, conventional (International) Units
Thyrotropin 0.4–4.5 lU/mL (0.4–4.5 mU/L)
Total T4 4.0–12.0 lg/dL (51–154 nmol/L)
Free T4 0.7–1.8 ng/dL (9.0–23.2 pmol/L)
Total T3 100–200 ng/dL (1.54–3.08 nmol/L)
Free T3 208–596 pg/dL (3.2–9.2 pmol/L)
24 L.M. Demers / Clin Lab Med 24 (2004) 19–28

Thyroid function testing in elderly patients

There is an increased prevalence of a low or high serum thyrotropin level
(with normal FT4 results) in elderly patients compared with younger
individuals. Age-adjusted reference intervals for thyroid hormones should
be considered by clinical laboratories, particularly for the elderly. Some
elderly patients can have mild thyroid disease with the symptoms masked by
the normal aging process. Diseases, such as Hashimoto’s thyroiditis, are
encountered with increasing prevalence in the elderly and usually are
associated with a high serum thyrotropin level and a increased TPOab titer.
An unexplained, increased serum thyrotropin level should suggest the mea-
surement of an anti-TPO antibody titer. A low serum thyrotropin level is
a frequent finding in the elderly but may be a transient phenomenon that
results from a systemic illness. Nevertheless, it is a persistent finding in
approximately 2% of otherwise healthy, older individuals who have no
other apparent evidence of thyroid dysfunction.

Diagnosis of hypothyroidism
The most common cause of hypothyroidism is a disorder that is intrinsic
to the thyroid gland itself and is known as primary hypothyroidism.
Hypothyroidism leads to a hypometabolic state; generalized symptoms of
thyroid hormone deficiency include fatigue, lethargy, cold intolerance,
slowed speech and intellectual functions, slowed reflexes, periorbital edema,
and a thickened, dry coarse skin. Primary hypothyroidism is more prevalent
in women and is caused by the loss of functional thyroid tissue that results
from infiltrative diseases to the thyroid (viral and bacterial), autoimmune
thyroiditis, thyroid surgery, radiation injury to the neck or defects in thy-
roid hormone synthesis cause by iodine deficiency, antithyroid drugs, cer-
tain medications (eg, steroids), or a congenital defect in one or more of
the enzymes that catalyze the synthesis or release of thyroid hormone.
Secondary hypothyroidism is less common and can occur in patients who
have a deficiency in pituitary hormone secretion due to pituitary infarction
or disease or as a result of hypothalamic disease and a deficiency in TRH
release. Because of the negative feedback relationship between thyroid
hormone and the hypothalamic-pituitary axis, an increased serum level of
thyrotropin occurs in most patients who have true primary hypothyroidism
(see Fig. 1). These considerations suggest that serum thyrotropin should be
measured first in patients who are suspected of hypothyroidism; this should
be followed by reflex testing to a serum FT4 if the thyrotropin result exceeds
the upper limit of normal. The concomitant finding of a decrease in serum
FT4 and an increase in serum thyrotropin usually confirms the diagnosis of
primary hypothyroidism that is caused by thyroid gland failure. An increase
in the serum thyrotropin level, together with a normal or low-normal serum
FT4, suggests that the patient may be at an early stage in the development of
 L.M. Demers / Clin Lab Med 24 (2004) 19–28 25

primary hypothyroidism; this situation has been called ‘‘subclinical

hypothyroidism’’ or ‘‘mild thyroid failure.’’ The suspicion of primary
thyroid failure may be suggested by a history of radioiodine (131-I) therapy,
thyroid surgery, ingestion of antithyroid drugs, or a family history of thyroid
disease. An increased titer of antithyroid autoantibodies frequently is
observed in the serum of patients who have Hashimoto’s thyroiditis or post-
partum thyroiditis.
Distinct from primary hypothyroidism is the finding of a subnormal
serum FT4 concentration with a normal or subnormal serum thyrotropin
level. This biochemical picture suggests that the patient may have
hypothyroidism secondary to a decrease in thyrotropin secretion. This
combination of results suggests a diagnosis of hypopituitarism and may
warrant a detailed work-up of the hypothalamic-pituitary axis. Secondary
hypothyroidism that is caused by a disorder within the hypothalamic-
pituitary axis is rare in comparison with the hypothyroidism that results
from primary thyroid gland failure. In this secondary form of thyroid
dysfunction, the decrease in pituitary thyrotropin secretion generally is
associated with deficiencies in other pituitary hormones.

Diagnosis of hyperthyroidism
A detailed history and physical examination often can suggest that
a patient has hyperthyroidism, a hypermetabolic state that is associated with
the increased availability and production of thyroid hormone. The term
‘‘thyrotoxicosis’’ has been used to denote the excess of thyroid hormone
in this condition that causes a wide constellation of symptoms, including
nervousness, heart palpitations, rapid pulse, fatigability, muscle weakness,
weight loss, diarrhea, heat intolerance, sweating, variable eye changes, and
gland enlargement. The most common cause of primary hyperthyroidism is
Graves disease, an autoimmune disease that afflicts 0.4% of the United
States population with a 5:1 ratio favoring women. Graves’ disease is
characterized by the production of antibodies to the thyrotropin receptor
that produces an autonomous state of stimulation to the thyroid gland.
Primary hyperthyroidism also can be caused by the presence of a diffuse and
multinodular goiter or by the development of a solitary adenoma. Less
common causes of hyperthyroidism include acute or subacute thyroiditis,
thyroid cancer, iatrogenic intake of thyroid medications or iodide, or drug-
induced thyrotoxicosis. T3 thyrotoxicosis is an uncommon thyrotoxic
condition that affects 5% of previously treated hyperthyroid patients;
they present with normal thyroxine levels but elevated T3 levels and all the
symptoms and signs of thyrotoxicosis. Secondary causes for hyperthyroid-
ism are rare; however, it can be caused by a thyrotropin-secreting pituitary
adenoma. The diagnosis of hyperthyroidism usually can be made by the
finding of an abnormally high serum level of FT4 with a suppressed level of
26 L.M. Demers / Clin Lab Med 24 (2004) 19–28

serum thyrotropin (\0.05 lU/mL) as a result of negative feedback in-

hibition at the level of the pituitary. Serum FT4 levels are elevated in all but
a low percentage (\5%) of untreated ambulatory hyperthyroid patients.
Because the serum concentration of total thyroid hormone is affected by
changes in the serum concentration of binding proteins, the serum FT4 level
should be measured in conjunction with a serum thyrotropin level to
confirm the diagnosis. The serum thyrotropin in the true hyperthyroid
patient is typically suppressed to undetectable levels (\0.05 lU/mL; normal,
0.4–4.5 lU/mL). The combination of an increase in serum FT4 and un-
detectable serum thyrotropin usually establishes the diagnosis of hyperthyroid-
ism. An occasional hyperthyroid patient may have an undetectable serum
thyrotropin with a serum FT4 concentration within the normal range. In
these patients, the finding of a high serum FT3 may suggest the diagnosis of
hyperthyroidism as a result of T3 thyrotoxicosis.
A serum thyrotropin level may be suppressed or undetectable in some
individuals who have normal serum FT4 and FT3 levels. In the absence of
drugs that suppress thyrotropin secretion, these patients could have mild
hyperthyroidism, often termed ‘‘subclinical hyperthyroidism.’’ Other
patients who have clinical signs of hyperthyroidism may have high levels
of serum FT4 and FT3 with normal or increased levels of serum thyrotropin.
This form of hyperthyroidism could be caused by an increase or auto-
nomous secretion of thyrotropin from a pituitary tumor.

Thyroid function testing and the effect of medications

Several medications can cause in vivo and in vitro effects on thyroid
function tests that can lead to misinterpretation of laboratory test results
and an incorrect diagnosis.

In vivo effects
In general, the serum level of thyrotropin is affected less by medications
than by effects on the thyroid hormone concentrations themselves. For
example, estrogen-induced elevations in TBG produce elevated TT4 levels
but have little or no effect on the serum thyrotropin concentration because
the regulation of pituitary thyrotropin secretion by FT4 is independent of
binding-protein effects.
Glucocorticoids in large and chronic doses can decrease the serum T3
level by inhibiting the peripheral conversion of T4 to T3 and by inhibiting
pituitary thyrotropin secretion. High doses of propranolol, a medication
that is used frequently to treat manifestations of thyrotoxicosis, also has
an inhibitory effect on T4 to T3 conversion. Propranolol that is given to
individuals who do not have thyroid disease can provoke an increase in
thyrotropin as a result of the impaired conversion of T4 to T3.
Iodide that is contained in solutions that are used to sterilize the skin
and in radiopaque dyes and contrast media that are used in coronary
 L.M. Demers / Clin Lab Med 24 (2004) 19–28 27

angiography and CT scans, can cause hyper- and hypothyroidism in

susceptible individuals. In addition, the iodide-containing antiarrhythmic
drug, amiodarone, that is used to treat heart disease, has complex effects on
thyroid gland function that can induce hypothyroidism or hyperthyroidism
in susceptible patients who have positive thyroid autoantibodies. L-T4–
treated patients who take amiodarone may have disproportionately elevated
serum thyrotropin levels relative to their serum FT4 concentration.

In vitro effects
Intravenous heparin administration, through in vitro stimulation of
lipoprotein lipase, can liberate free fatty acids that inhibit T4 binding to
serum proteins and falsely elevate the serum FT4 level. In certain pathologic
conditions, such as uremia, abnormal serum constituents, such as indole
acetic acid, may accumulate and interfere with thyroid hormone binding.
Some thyroid test methods that use fluorescence detection may be sensitive
to the presence of fluorophore-containing therapeutic or diagnostic agents
that are used in radiology.

Thyroid function testing and nonthyroidal illness

Patients who are seriously ill have abnormalities in their thyroid test
results, often without evidence of thyroid dysfunction. These abnormalities
are seen with acute and chronic critical illnesses. The terms ‘‘NTI,’’ ‘‘euthyroid
sick syndrome,’’ and ‘‘low-T3 syndrome’’ are frequently used to describe this
subset of patients. The spectrum of changes in thyroid test results relates to the
severity and stage of illness, technical factors that affect the methods, and, in
some cases, the medications that are given to these patients.
Thus, the laboratory diagnosis of thyroid disease can be extremely
difficult to make in very ill patients (ie, individuals hospitalized for various
NTIs). Changes in thyroid hormone secretion, distribution, and metabo-
lism, as well as subnormal concentrations of the serum thyroxine-binding
proteins have been described in sick, hospitalized patients who did not have
true evidence of primary thyroid disease. Thus, individuals who have
a severe illness can have abnormal thyroid function test results although
they may not have a specific thyroid problem. In general, the more severe
the systemic illness, the greater the degree of abnormality in thyroid func-
tion test results. Abnormal circulating levels of thyroid hormone or serum
thyrotropin in sick patients may reflect adaptations to the catabolic state;
similar changes also occur during fasting and typically revert to normal
when the patient recovers from the underlying systemic illness.
It has been established that serum FT4 and thyrotropin measurements
have reduced specificity for detecting thyroid dysfunction when patients
have a severe NTI. Generally, it is recommended that thyroid function
testing of hospitalized patients be limited to those who have significant
clinical symptoms or a history of thyroid dysfunction.
28 L.M. Demers / Clin Lab Med 24 (2004) 19–28

Serum thyrotropin levels remain within normal limits in most patients

who have NTIs, provided that no dopamine or glucocorticoid therapy is
being administered. In acute NTI there may be a mild, transient decrease in
serum thyrotropin into the 0.05 to 0.1 lU/mL range, followed by a rebound
to mildly elevated values during recovery. In the hospital setting, it is critical
to use a thyrotropin assay with a functional sensitivity at or below 0.02
lU/mL. Without this level of sensitivity, sick hyperthyroid patients who
have profoundly low serum thyrotropin values (\0.002–0.05 lU/mL)
cannot be discriminated reliably from patients who have a transient mild
thyrotropin suppression (0.05–0.1 lU/mL) that is caused by NTI. Minor
elevations in thyrotropin are less diagnostic for hypothyroidism in the
hospital setting. Sick hypothyroid patients often exhibit the combination of
a low T4 and elevated thyrotropin level ([20 lU/mL).

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