At a Glance

The thyroid gland produces two related hormones, thyroxine (T4) and triiodothyronine (T3), which play a critical role in thermogenic and metabolic homeostasis. T4 and T3 are normally synthesized and released in response to a combined hypothalamic pituitary signal mediated by thyroid stimulating hormone (TSH) from the anterior pituitary and thyrotropin releasing hormone from the hypothalamus. There is a negative feedback from thyroid hormone concentration, primarily T3, to TSH production causing total T4, total T3, free T4, and free T3 concentrations to move in opposition to TSH concentration.

Hypothyroidism is a condition in which the thyroid gland is functionally inadequate. Causes of hypothyroidism include autoimmune disorders, such as Hashimoto’s disease (also known as Hashimoto’s thyroiditis), atrophic thyroiditis, and postpartum thyroiditis; iodine deficiency, the most common cause of hypothyroidism in underdeveloped areas; congenital defects; medications or treatments that can result in hypothyroidism; central hypothyroidism in which the thyroid is not stimulated by the pituitary or hypothalamus; and infiltrative processes that may damage the thyroid, pituitary, or hypothalamus. These different causes of hypothyroidism are often interrelated. Usually, the exact cause of the hypothyroidism cannot be definitively determined.

Hashimoto’s disease, also known as chronic lymphocytic thyroiditis, is the most common thyroid disease in the United States. In Hashimoto’s disease, the immune system attacks the thyroid gland, resulting in inflammation that can cause an under active thyroid gland. In response to the decreased thyroid hormone production by the thyroid gland, the anterior pituitary gland increases its production of TSH. This can cause the thyroid to enlarge. Such an enlargement is known as a goiter.

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The trigger for the immune system attack on the thyroid is not known. Speculation about the trigger includes trauma; environmental exposures, such as cigarette smoke; a genetic flaw; or virus or bacterium, although infection as a trigger is now thought of as less likely. Hashimoto’s disease is most common in women between 40 and 60 years of age and there is a genetic predisposition. The incidence of the disease is high in the Japanese population, most likely as a result of genetic factors and a diet high in iodine.

Often, there are no symptoms associated with Hashimoto’s disease for many years, and the condition remains undiagnosed until an enlarged thyroid gland or routine blood tests reveal a problem. If symptoms do develop, they are either related to the increased pressure in the neck caused by the goiter or the usual symptoms of hypothyroidism, which include fatigue, cold intolerance, weight gain, depression, and dry skin.

What Tests Should I Request to Confirm My Clinical Dx? In addition, what follow-up tests might be useful?

Antithyroid antibodies are often helpful in the diagnosis of an autoimmune thyroid disorder. There are three different antithyroid antibodies: thyroperoxidase antibody (TPOAb), an antibody to a follicular enzyme involved in oxidation and organification of iodine; thyroglobulin antibody (TgAb), an antibody to thyroglobulin, the protein made up of the essential amino acid tyrosine to which the iodine is attached; and TSH receptor inhibiting immunoglobulin, which competes with TSH for receptor binding sites but does not activate them.

If an antithyroid antibody is present, it is often indicative of a prior attack on thyroid tissue. Antithyroid antibodies are the most specific test for Hashimoto’s disease, but they are not present in all cases and they can sometimes be found in patients without an autoimmune thyroid problem. Usually, a combination of TPOAb and TgAb testing is used to add more specificity.

TSH and free T4 are the usual laboratory diagnostic tools in the diagnosis of hypothyroidism. In hypothyroidism due to Hashimoto’s disease, free T4 is decreased and TSH is increased in an effort to provide the body with adequate free T4. T3 is not generally reliable in the diagnosis of hypothyroidism. Measuring TSH, free T4, or other analytes will not identify the cause of the hypothyroidism as Hashimoto’s disease.

In a patient with stable thyroid status, TSH is the more sensitive test in the diagnosis of hypothyroidism, since the relationship between TSH and free T4 is log/linear. Intraindividual variation for free T4 is quite small, so any small deficiency of free T4 will be sensed by the anterior pituitary relative to the individual’s set point and cause an amplified, inverse response in TSH.

In a patient with unstable thyroid status, free T4 is the more reliable indicator; however, it is possible for high TSH stimulation to keep T4 levels within normal limits for quite some time.(Table 1)

Table I.
TSH Free T4 Antithyroid antibodies
Increased, in combination with decreased free T4diagnostic of primary hypothyroidism Decreased, in combination with increased TSH diagnostic of primary hypothyroidism Present, indicative of autoimmune thyroid disease but not always present; TPOAb and TgAb are often used in combination

Are There Any Factors That Might Affect the Lab Results? In particular, does your patient take any medications – OTC drugs or Herbals – that might affect the lab results?

Interferences may obscure the diagnosis of Hashimoto’s disease or complicate the monitoring of the effectiveness of thyroid replacement therapy.

Most thyroid testing is performed by either immunoassay, in which labeled and unlabeled ligands compete for a limited number of antibody sites, or immunometric assays, in which an antibody is bound to a solid surface rather than an antibody. Cross reactivity of auto-antibodies or heterophilic antibodies can affect diagnostic accuracy of competitive binding-based tests.

The term heterophilic antibodies is often loosely applied to relatively weak antibodies with multiple activity sites, known as auto-antibodies, seen in auto immune disorders; broadly reactive antibodies induced by infections or exposure to therapy containing monoclonal mouse antibodies (HAMA); or human anti-animal immunoglobulins produced against well defined, specific antigens following exposure to therapeutic agents containing animal antigen or by coincidental immunization through exposure to animal antigens.

The latter, Human Anti-Animal Antibodies (HAAA), are strong reactors. HAMA and HAAA affect immunometric assays more than they affect simple competitive immunoassays. In immunometric assays HAMA and HAAA can form a bridge between the capture and signal antibodies. Auto-antibodies and heterophilic antibody interferences can sometimes be detected by simply using a different manufacturer’s method that employs a slightly different antibody. Tests in which dilutions are acceptable, such as total T4, total T3, or TSH, but not free T4 or free T3, may be checked for linearity of response to help identify heterophilic antibody interference.

Most circulating thyroid hormones are bound to protein. Only that hormone that is free is biologically active. Variations in binding protein will cause variations in concentrations of total hormones. In general, serum TSH is less affected by binding issues than T3 and T4, and T4 is bound more tightly than T3. T3 and T4 circulate in the body bound to thyroid binding globulin (TBG); transthyretin, formally known as thyroxine binding prealbumin; and serum albumin. Physiological shifts toward greater total hormone binding will decrease available free hormone. Theoretically, free T3 and free T4 are not affected analytically by binding, but in reality, all of the free methods are binding dependent to varying degrees.

Phenytoin, carbamazepine, aspirin, and furosemide compete with thyroid hormone for protein binding sites and, thus, acutely increase free hormones and reduce total hormones. Eventually, a normal equilibrium is reestablished where free levels normalize at the expense of total levels.

Heparin stimulates lipoprotein lipase, liberating free fatty acids, which inhibit total T4 protein binding and elevate free T4.

Free fatty acids are known to affect some methods.

Estrogens increase TBG, increasing total thyroid hormones.

Liver disease, androgens, and nephrotic syndrome decrease TBG, decreasing total thyroid hormones.

Indole acetic acid, which accumulates in uremia, may interfere with thyroid binding.

Pregnancy is associated with lower albumin levels. Therefore, albumin-dependent methods are not suitable for accessing thyroid status during pregnancy.

TSH levels decline in the first trimester of pregnancy partly because of the increase in total T3 and T4 from increased TBG. Total T3 and T4 are also increased in the first trimester by increased Human Chorionic Gonadotropin (HCG), which is structurally and, to some extent, functionally similar to TSH.

Glucocorticosteroids can lower T3 and inhibit TSH production. This interaction is of particular concern in sick, hospitalized patients in whom the elevated TSH in primary hypothyroidism may be obscured.

Propanolol has an inhibitory effect on T4 to T3 conversion. Eighty percent of T3 is produced enzymatically in nonthyroid tissue by 5 monodeiodination of T4.

Free T3 and free T4 are often method dependent.

Methods that use fluorescent tags may be affected by the presence of fluorophore-related therapeutic or diagnostic agents.

What Lab Results Are Absolutely Confirmatory?

If there is a question about the cause of a goiter associated with hypothyroidism, a fine needle aspiration examined cytologically can confirm the presence of autoimmune thyroiditis.

It has been suggested that the best confirmation of hypothyroidism, in general, is an evaluation of response to a trial administration of thyroid supplementation in patients with symptoms of hypothyroidism.

Lone TSH testing may not be predictive of autoimmune disorders in which TSH may be normal, elevated, or depressed.

What Tests Should I Request to Confirm My Clinical Dx? In addition, what follow-up tests might be useful?

Up to 20% of patients with autoimmune hypothyroidism have antibodies against TSH receptor, which prevent binding with TSH. TSH receptor blocking antibodies are difficult to analyze.

Are There Any Factors That Might Affect the Lab Results? In particular, does your patient take any medications – OTC drugs or Herbals – that might affect the lab results?

It is important that Hashimoto’s disease be treated appropriately. If replacement therapy is inadequate, the thyroid gland will continue to enlarge and cholesterol levels may increase. Such hypercholesterolemia is generally seen as an increase in low density lipoprotein, which places the patient at greater risk of atherosclerosis. Hypothyroidism can also lead to an enlarged heart and, in rare cases, heart failure. Hashimoto’s disease is also associated with an increased rate of birth defects, if inadequately treated. If replacement therapy is too strong, symptoms of hyperthyroidism can develop, placing excessive strain on the heart and increasing the risk of osteoporosis.

Since Hashimoto’s disease is a disorder of the immune system, patients with the disorder have a statistically increased risk of developing other autoimmune disorders, such as insulin dependent diabetes, rheumatoid arthritis, pernicious anemia, Addison’s disease, early menopause, vitiligo, thrombocytopenic purpura, or lupus erythematosus.

A combination of high free T4 and high TSH may be an indication of therapeutic noncompliance. Acute ingestion of missed levothyroxine (L-T4) just prior to a clinic visit will raise the free T4 but fail to normalize the TSH because of a “lag effect”. Free T4 is the short-term indicator, whereas TSH is a long-term indicator. Since TSH is the long-term indicator, it is not influenced by time of L-T4 ingestion.

When testing free T4, the daily dose of L-T4 should be withheld until after sampling, as free T4 is significantly increased above baseline for up to 9 hours after ingesting L-T4. Ideally, L-T4 should be taken prior to eating, at the same time each day, and at least 4 hours apart from other medications. Many medications and even vitamins and minerals can influence L-T4 absorption. L-T4 should not be taken with iron supplements.

Patients should not switch from brand to brand of L-T4 and prescriptions should not be written generically, as doing so will allow brand to brand switches. Although stated concentrations of L-T4 may be the same, slight variations exist between pharmaceutical manufacturers in terms of bioavailability. Also, medication storage recommendations should be scrupulously followed. Medication should be stored away from humidity, light, and increased temperatures. When ordering medication it is best to avoid the summer for shipping.

TSH or free T4 levels may be diagnostically misleading during transition periods of unstable thyroid function. Often these transition periods occur in the early phase of treating hyper- or hypothyroidism or changing the L-T4 dose. It takes 6-12 weeks for pituitary TSH secretion to re-equilibrate to the new thyroid hormone status. Similar periods of unstable thyroid status may occur following an episode of thyroiditis.

TSH or free T4 levels may be diagnostically misleading in cases of abnormalities in hypothalamic or pituitary function in which the usual negative feedback is not seen and TSH may remain within normal ranges.

Free T4 and TSH have reduced specificity in hospitalized patients with nonthyroid illness. Most hospitalized patients have low serum total T3 and free T3. These abnormalities are seen with both acute and chronic nonthyroid illness and are thought to be the malfunction of central inhibition of hypothalamic releasing hormone. The National Academy of Clinical Biochemistry guidelines for testing of hospitalized patients with nonthyroid illness include:

  • Acute or chronic nonthyroid illness has complex effects on thyroid function testing. Whenever possible, diagnostic testing should be deferred until the illness has resolved, except in cases in which there is a suggestion of presence of thyroid dysfunction.
  • Physicians should be aware that some thyroid tests are inherently not interpretable in severely ill patients or patients receiving multiple medications.
  • TSH in the absence of dopamine or glucocorticoid therapy is the more reliable test.
  • TSH testing in the hospitalized patient should have a functional sensitivity of less than 0.02 mIU/L. Otherwise, sick, hyperthyroid patients with profoundly low TSH cannot be differentiated from patients with mild transient TSH suppression caused by nonthyroid illness.
  • An abnormal free T4 in the presence of serious somatic disease is unreliable. In hospitalized patients, abnormal free T4 testing should reflex to total T4. If both free T4 and total T4 are abnormal in the same direction, a thyroid condition may exist. Discordant free T4 and total T4 abnormalities are more likely the result of illness, medication, or a testing artifact.
  • Total T4 abnormalities should be considered in conjunction with the severity of the patient illness. A low T4 in patients not in intensive care is suspicious of hypothyroidism, since low total T4 levels in nonthyroid illness in hospitalized patients are most often seen in sepsis. If a low total T4 is not associated with an elevated TSH and the patient is not profoundly sick, hypothyroidism secondary to pituitary or hypothalamic deficiency should be considered.
  • Reverse T3 formed by the loss of an iodine group from T4 where the position of the iodine atoms on the aromatic ring is reversed is rarely helpful in the hospital setting, because paradoxically normal or low values can result from impaired renal function and low binding protein concentrations.

Trimester specific reference ranges should be used in pregnancy.

During pregnancy, estrogens increase TBG to 2-3 times prepregnancy levels. This shifts binding such that total T3 and total T4 are approximately 1.5 times nonpregnant levels at 16 weeks gestation.

TSH is also altered during pregnancy. TSH is decreased in the first trimester due to the thyroid stimulating activity of HCG. The decline in TSH is associated with a modest increase in free T4 from the increased TBG. In approximately 2% of pregnancies the increase in free T4 leads to a condition known as gestational transient thyrotoxicosis. This condition may be associated with hyperemesis.

In the second and third trimester, free hormone levels decrease 20-40% below reference ranges.

Pregnant patients receiving L-T4 replacement may require increased dose to maintain a normal TSH and free T4.

TSH has a very short half-life of 60 minutes and is subject to circadian and diurnal variation peaking at night and reaching a nadir between 10 AM and 4 PM. T4 has a much longer half-life of 7 days.

It should be noted that there is a continuous decrease in the TSH/free T4 ratio from mid-gestation through completion of puberty. In adulthood, TSH increases in the elderly. Age related reference ranges, or at least ratio adjusted reference ranges, should be used for these analytes.

For a change in analyte value to have clinical significance, the difference should take into consideration analytical and biological variabilities. The magnitude of difference in thyroid testing values reflecting clinical significance when monitoring a patient’s response to therapy are:

  • T4 28 nmol/L (2.2 μg/dL)
  • free T4 6 pmol/L ( 0.5 ng/dL)
  • T3 0.55 nmol/L (35 ng/dL)
  • free T3 1.5 pmol/L (0.1 ng/dL)
  • TSH 0.75 mIU/L