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Thyroglobulin and thyroperoxidase

autoantibody testing: clinical value and


methodological aspects
Thyroglobulin antibodies (Tg Abs) are circulating immunoglobulins directed against
different epitopes of the thyroglobulin molecule. Thyroid microsomal antibodies (TPO
Abs) are circulating immunoglobulins directed against a component of the smooth
endoplasmic reticulum of thyroid cells. Recently, this microsomal antigen was found to
be identical or at least to contain as main component thyroid peroxidase (TPO) (1).
Detectable levels of Tg Abs and/or TPO Abs are mainly associated with thyroid
autoimmune disorders and with thyroid cancers but low concentrations are also found in
a significant percentage of the normal population.

Part I : An introduction to thyroid


autoimmunity
While they represent a rather disparate group, thyroid autoimmune diseases are
probably caused by similar but distinct disorders of the normal immune system. These
chronic degenerative diseases may cause either primary hypothyroidism or primary
hyperthyroidism (see Table I). In mild forms, compensation mechanisms at the pituitary
level are often able to normalize the hormonal secretion of the gland. This, for example,
would explain the euthyroid goiter observed in some patients with Graves' disease.
Hashimoto's thyroiditis is usually associated with hypothyroidism but transient
hyperthyroidism or euthyroidism are not rare at early stages.

Chronic primary hyperthyroidism


Graves' disease
Chronic primary hypothyroidism
Hashimoto's thyroiditis
- juvenile lymphocytic thyroiditis
Variants :
- chronic fibrous variant

- idiopathic myxedema

- atrophic aymptomatic thyroiditis


- Schmidt's syndrome

Table I: Thyroid autoimmune diseases.

Origin of thyroglobulin and thyroperoxidase antibodies


Several researchers have attempted to identify the mechanism triggering thyroid
autoantibody production. All these studies have, until now, failed to provide a

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satisfactory answer. At this stage, it is likely that more than one mechanism is involved
and this would be consistent with antibody heterogeneity. Different groups have
suggested that there could be an antigenic stimulation at the initial step of antibody
production. For example, it has been proposed that a viral infection such as subacute
thyroiditis could induce occult antigenic changes within the thyroid. This is rather
unlikely in Graves' or Hashimoto's diseases where a vast majority of cases arise without
any recent history of thyroid infection. Similarly, almost all patients with subacute
thyroiditis recover and do not develop thyroid antibodies (6).

Recent advances in molecular biology have failed to demonstrate antigenic alterations of


the Tg molecule in thyroid autoimmune diseases. However, the antigenic stimulation
hypothesis may be valid, at least in part, for thyroid cancers. In these patients,
thyroglobulin release in the bloodstream is accelerated and iodinated forms, normally
confined to the gland, are secreted.

Another hypothesis is that a disorder in immunoregulation is at the origin of both


Graves' and Hashimoto's diseases. Some studies found alterations in the number of
natural killer cells in patients with untreated Graves' disease but no anomaly in patients
with Hashimoto's thyroiditis. Several groups have focused on the suppressor arm of the
immune system, T lymphocytes (7). Early studies were contradictory because their
authors were trying to substantiate a generalized defect of Ts cells. However, rather
than a decrease in the number of T lymphocytes or in their overall activity, it seems that
organ or antigen specific defects have to be considered. How these defects appear is
another problem. Jansson et al. (8) have found lymphocytotoxic antibodies specific for
CD8+ cells in patients with autoimmune thyroid diseases. These antibodies would in
turn, reduce the number of Ts cells and lead to autoantibody overproduction. This
diminution of CD8+ cells is observed in some patients, but not all. Other possibilities
include biochemical abnormalities inherent to the Ts cells themselves. TSH is also
thought to stimulate TPO Ab production in thyroid cells trough complex pathways
involving cyclic AMP (9).

Role of thyroglobulin and thyroperoxidase autoantibodies


in the pathogenesis of thyroid disorders
Much work has been done on antibodies binding the TSH receptor in Graves' disease
patients. The stimulating (and sometimes) blocking effects of these antibodies, their
role in the development of hyperthyroidism are well established but fall outside the
scope of this review.

In contrast, no pathological role has been found, until now, for thyroglobulin antibodies.
For thyroperoxidase antibodies, the situation is unclear. These antibodies are
complement fixing and it has been thought that they were implicated in the thyroid cell
damage characteristic of autoimmune thyroiditis. TPO antibodies are able to inhibit TPO
enzymatic activity in vitro. Thyroid peroxidase has been extensively studied by Taurog
et al. (10) and is known to catalyze iodination of tyrosine and oxidative coupling of two
diiodotyrosine residues in thyroglobulin to form thyroxin. It is reasonable to think that
blocking this mechanism will induce thyroid disorders but does not explain the cell
destruction process characteristic of thyroid autoimmunity. While there is no firm
evidence against a cytotoxic role of TPO Abs, other antibodies are certainly involved in
cell destruction, via the complement pathway (11, 12). These IgG are not well

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characterized and are found in patients with or without Tg and TPO antibodies.

Methodological aspects
Several methods have been used to detect Tg and TPO Abs since the tanned red cell
hemagglutination technique described by Boyden in 1951 for Tg Abs. They include
immunofluorescence, radioimmunoassay and ELISA. Immunofluorescence and RIA are
gradually abandoned for practical reasons while both passive hemagglutination assays
and ELISA are widely used in routine. Independently of the intrinsic quality of
commercial assays, it seems that ELISA techniques are more suitable for quantitative
determinations and may reflect more accurately the response of the autoimmune
process to treatment (20).

There has been some debate regarding the immunoglobulin classes to be detected in
these assays. Most radioimmunoassays use 125I-Protein A as radioligand and are able
to detect IgM and all IgG antibodies except IgG 3. In contrast, most ELISA methods
measure selectively IgG antibodies. This may explain some discrepancies between the
results obtained with different tests.

Another controversial issue has been the cross-reaction of Tg Abs in assays for TPO Abs.
Originally, some assays were using microsomal preparations contaminated with Tg so
that artefactual cross-reactions were indeed present. This can be prevented by adding
large amounts of Tg to the incubation buffer as it is done for passive hemagglutination
tests and for some RIA assays or by using highly purified microsomal antigen
preparations as it is done for ELISA techniques. In fact, the issue is of academic interest
but has little clinical relevance since thyroperoxidase antibodies are present much more
frequently than Tg antibodies while Tg antibodies are rarely detected in the absence of
TPO Abs (21). Moreover, as seen previously, a certain degree of cross-reaction has to be
expected due to the fact that Tg and TPO share some antigenic determinants or have
epitopes with extremely close structures.

The interference of circulating Tg in Tg Ab determination has also been observed in


several assays. It may be partially removed by heat inactivation at 56C for 30 min., a
prerequisite for passive hemagglutination assays. In ELISA tests, endogenous Tg will
lower the response but the risk of a false negative is limited to very high concentrations
of serum thyroglobulin, a very rare occurrence except in thyroid cancers where
quantitative autoantibody measurements are of little clinical relevance. The massive
release of Tg and TPO observed during subtotal thyroidectomy also causes a marked
decrease in Tg and TPO Ab concentrations (22).

Finally, major difficulties have been encountered by most groups who attempted to
correlate numerically test results obtained with different methods. Besides what has
been previously mentioned, there is here a clear unitage problem. All passive
hemagglutination kits provide results in titers while most ELISA's (or RIA's) express
results in arbitrary or international units. There is an obvious interest in standardizing all
test results in international units. This would certainly harmonize the results and ease
comparative studies but would not solve all problems. The two currently available
reference preparations (anti-Tg Abs; MRC 1st international 65/93; anti-TM/TPO Abs:
serum 66/387) are pools of pathological sera and the heterogeneity of the antibodies

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that they contain may influence assay calibration.

A last observation made by Lukinac suggests that some passive hemagglutination tests
are subject to an interference by an unknown compound present in the serum from
patients with chronic renal failure and partially removable by hemodialysis (24).

Part II: Thyroid antibodies in clinical


practice
Graves' disease
Graves' disease (syn: Basedow's disease, autoimmune thyrotoxicosis, exophtalmic
goiter) is a form of chronic hyperthyroidism characterized by one or a combination of
the following clinical signs:

hyperthyroidism with diffuse enlargement of the thyroid gland,


infiltrative ophtalmopathy (50 % of cases),
infiltrative dermopathy or pretibial myxedema (1-2 % of cases).

Common symptoms include nervousness and/or tremor, weight loss (usually in face of
an increased appetite), palpitations, heat intolerance and excessive perspiration,
emotional lability, muscle weakness and hyperdefecation.

Overt Graves' disease has obvious symptoms and is easily diagnosed when
thyrotoxicosis is accompanied by proptosis. Laboratory tests will concentrate on the
evaluation of the thyroid function in order to normalize it with antithyroid drugs
(thionamides), 131-iodine or surgery. Thyroid autoantibody testing has limited clinical
value in these cases, except for TSI Abs in women in gestational age. Tg Abs are
present in about 25 % of cases while TPO Abs are present in 90 % of cases so that
combined Tg/TPO Ab testing will identify virtually all cases.

Hashimoto's disease
Autoimmune destruction of the thyroid gland accounts for 95% of primary
hypothyroidism, of which Hashimoto's thyroiditis and idiopathic myxedema represent
the majority of cases.

Hashimoto's disease (syn. lymphocytic thyroiditis, struma lymphomatosa) is a chronic


from of hypothyroidism caused by an inflammatory infiltration and tissue damage
localized in the thyroid. Its main symptoms are the opposite of those seen in Graves'
disease: dry coarse skin, swelling of the hands, face and extremities, cold intolerance,
decreased sweating, hoarse voice, modest weight gain with anorexia, constipation ...
Weakness, fatigue, lethargy and goiter are also present. The autoimmune origin is well
established with Tg Abs and TPO Abs present in 85-100% of cases.

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The diagnosis of Hashimoto's disease is normally not difficult but two conditions deserve
a special mention:

- patients with chronic renal disease may have symptoms that mimic those of
hypothyroidism; in addition, free T4 tests are not always reliable here so that
Tg/TPO Abs testing will allow a differential diagnosis;

- de Quervain's thyroiditis (granulomatous thyroiditis or subacute thyroiditis)


is a rare disease, thought to be of viral origin and lasting from 1 to 3 months.
Most patients recover spontaneously without evidence of thyroid dysfunction.
The histologic aspect of thyroid tissue is very different compared to Hashimoto's
disease lesions. However, the clinical distinction between an Hashimoto's painful
goiter and the enlargement of the gland seen in de Quervain's thyroiditis is not
straight forward. Both Tg Ab and TPO Abs are usually low or weakly positive in
subacute thyroiditis and disappear with spontaneous healing of the gland.

In some cases, Tg and/or TPO Abs will be found together with normal or borderline TSH
and free T4. These patients will have to be monitored closely since autoimmune
thyroiditis may develop very insidiously. If antibody levels remain persistently high, the
criteria to initiate treatment will be the TSH response to TRH. As soon as this response
is impaired, treatment has to be considered.

In contrast, antibody testing, if negative, will rule out autoimmune thyroiditis for the
numerous cases of non-toxic goiters where the cause of the thyroid enlargement must
be identified (dietary goitrogens or goitrogenic drugs and chemicals).

Thyroid cancers
The determination of Tg and TPO antibodies in differentiated thyroid cancers has no
intrinsic clinical value. Tg Ab determination is of interest, as previously discussed, to
validate Tg determinations. Thyroid autoantibodies are often detected in these patients,
mainly, as expected in females and may represent either a reaction of the immune
system against the tumor cells or the coexistence of a thyroid autoimmune disorder and
of a carcinoma.

In a retrospective study of 9237 surgical specimens, Hirabayashi and Lindsay observed


that 27% female patients (vs 6% of males) had evidence of Hashimoto's disease both
inside and outside the tumor (32). Nevertheless, it has never been possible to associate
Graves' or Hashimoto's disease with an increased incidence in thyroid carcinoma.

Conclusions and perspectives


Tg and TPO Abs are routinely assayed since years. Besides specific clinical indications,
summarized in Table III, they are not, at this stage of knowledge, of particular
significance. Low concentrations in asymptomatic subjects, specially elderly women, are
not justifying any treatment.

All laboratory tests detect an heterogeneous population of antibodies, some of them


probably not involved in the development of thyroid autoimmune disorders. Even when

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they are involved, it is still unclear whether these antibodies play an active role in the
pathogenesis of thyroid autoimmunity or if they simply reflect the development of these
diseases. However, since both antibodies are directed against molecular entities playing
a key-role in the physiological secretion of thyroid hormones, it is not a surprise that
their presence at high concentrations is invariably associated with thyroid disorders.

In the view of most clinicians, assessment of TSH and thyroid hormone circulating levels
are more important than thyroid antibody determination. This is quite logical since
thyroid autoimmunity can easily be treated but not cured by supplementation of the
deficient hormonal secretion or by antithyroid drugs. However, if new treatments
become available to cure or to treat the cause i.e. a deregulation of the immune system
instead of treating the effects, the situation may entirely change.

Clinical situation Interest of Tg and TPO Ab determination and


interpretation

Clinical suspicion Absence of Tg and TPO Abs together with normal thyroid function
of Graves' disease tests rule out Graves' disease and suggest other causes
without thyroid or (diabetes, pheochromocytoma,...).
ocular signs

Eyelid changes Subnormal or elevated Tg and/or TPO Abs suggest further


suggestive of thyroid function tests and point out towards thyroid eye disease.
Graves' disease Negative findings suggest further exploration (orbital tumors).

Suspicion of Elevated Tg and/or TPO Abs point towards Hashimoto's disease


hypothyroidism in even if other thyroid tests are normal. Negative findings suggest
the adult with other goitrogenic causes (chemicals, drugs, de Quervain's
thyroid thyroiditis).
enlargement

Clinical suspicion Elevated Tg and/or TPO Abs suggest regular monitoring of


of hypothyroidism thyroid function; negative findings suggest a misdiagnosis and
in the adult other explorations (e.g. renal disease).
without thyroid
enlargement

Suspicion of Moderate level of TPO Abs confirm juvenile lymphocytic


hypothyroidism in thyroiditis.
school children

Pregnancy - Elevated TPO Abs represent a risk factor for post partum
asymptomatic thyroiditis. The test has to be performed in early pregnancy.

Pregnancy - overt Elevated TPO and Tg Abs levels establish the autoimmune origin
hyperthyroidism in of the disease.
patients without

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previous thyroid
dysfunction (rare)

Thyroid cancers Elevated Tg Abs are common in differentiated cancer. They have
no clinical value but they suggest caution in the interpretation
of serum thyroglobulin determinations.

Amiodarone Elevated TPO Abs before treatment suggests a risk of


treatment developing iodine-induced hypothyroidism so that the test should
be included in the initial laboratory workup of these patients.

Table III: Interest of Tg and TPO Ab determinations.