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. T3 and T4 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 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 in which damage may be done to thyroid, pituitary, or hypothalamus. These causes of hypothyroidism are often interrelated. Usually, the exact cause of the hypothyroidism cannot be definitively determined.

Some diseases can cause deposits of abnormal substances in the thyroid, pituitary, and/or hypothalamus, damaging these organs. These diseases are known as infiltrative processes. Amyloidosis can deposit amyloid protein. Sarcoidosis can deposit granulomas. Hemochromatosis can deposit iron. Scleroderma can cause hypothyroidism due to fibrosis of the thyroid gland.

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Often, there are not immediate symptoms of thyroid involvement with infiltrative diseases. If symptoms do develop, they are 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?

TSH and free T4 are the usual laboratory diagnostic tools in the diagnosis of hypothyroidism. In hypothyroidism due to infiltrative processes free T4 is decreased and TSH may be increased if the infiltrative process is in the thyroid. If the infiltrative process is in the pituitary or hypothalamus, TSH may not be able to increase. 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 an infiltrative process.

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. This will be true for thyroid infiltrates but not pituitary or hypothalamus infiltrates.

In a patient with unstable thyroid status, free T4 is the more reliable indicator, and, in the case of pituitary or hypothalamus infiltrates, may be the only indicator.(Table 1)

Table I.
TSH free T4
increased; more reliable instable thyroid status but will only be increased if the infiltrative process is in the thyroid gland decreased; more reliable in unstable thyroid status and may be the only indicator in pituitary or hypothalamus infiltrates

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 hypothyroidism due to an infiltrate process 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 autoantibodies 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 autoantibodies seen in autoimmune disorders; broadly reactive weak antibodies induced by infection or exposure to therapeutic monoclonal mouse antibodies (HAMA); or human anti-animal immunoglobulins produced against well-defined, specific antigens following exposure to therapeutic agents containing animal antigens 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 simple competitive binding immunoassays by forming a bridge between the capture and signal antibodies. Autoantibodies and heterophilic antibody interferences can sometimes be detected simply by using a different manufacturer’s method that employs a slightly different antibody. Test 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 as another means of identifying heterophilic interferences.

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 or 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. Physiologically, shifts toward greater total hormone binding decrease available free hormone. Theoretically, free T3 and free T4 are not affected analytically by binding. In reality, all of the free methods are binding dependent to varying degrees.

Phenytoin, carbamazepine, aspirin, and furosemide competitively inhibit hormone protein binding and acutely increase free hormone, resulting in a reduction in total hormone. Eventually, a normal equilibrium is reestablished in which free hormone levels normalize at the expense of lower 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.

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

Estrogens increase TBG, increasing 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 due to the increase in total T3 and T4 from increased TBG. However, total T4 and T3 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 total 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.

Propranolol 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 T4 and free T3 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?

Hypothyroidism symptoms in combination with abnormal thyroid function results consistent with hypothyroidism linked to testing positive for one of the infiltrative disorder would be confirmatory.

It has been suggested that the best confirmation of hypothyroidism is an evaluation of response to a trial administration of thyroxine supplement in patients with symptoms of hypothyroidism.

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?

Since infiltrative processes can compromise the pituitary or the hypothalamus, as well as the thyroid, the usual opposition of TSH and free T4 is not seen if the infiltrative process affects the pituitary or hypothalamus.

Acute or chronic nonthyroid illness has complex effects on thyroid testing. 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 result of a malfunction of the central inhibition of hypothalamic releasing hormone. The National Academy of Clinical Biochemistry guidelines for testing of hospitalized patients with nonthyroid illness are as follows:

  • 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 should have a functional sensitivity less than 0.02 mIU/mL; 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 in hospitalized patients low total T4 levels 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 in which 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 because of the thyroid stimulating activity of HCG. The decline in TSH is associated with a modest increase in free T4. 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 a value to have clinical significance, the difference should take into consideration analytical and biological variabilities. The magnitude of difference in thyroid testing values reflecting a clinical significance when monitoring a patient’s response to therapy is:

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

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 a 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, 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 and prescriptions should not be written generically, as doing so allows 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 reequilibrate to the new thyroid hormone status. Similar periods of unstable thyroid status may occur following an episode of thyroiditis.

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 result of a malfunction of the central inhibition of hypothalamic releasing hormone. The National Academy of Clinical Biochemistry guidelines for testing of hospitalized patients with nonthyroid illness are as follows