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Cover Story
By Catherine Hammett-Stabler, PhD, DABCC
(Editor's note: The Learning Scope offers a unique opportunity for clinical laboratory personnel to earn continuing education credit. Simply by reading this peer-reviewed article and sending your answers to the questions that follow to the American Society for Clinical Laboratory Science (ASCLS), you can receive 0.1 CEU, or 1 contact hour. The credit is issued through ASCLS' PACE program. Additional details concerning the CEU offer are listed to the left of the answer sheet on page 17.)
At her annual physical examination, a 48-year-old hospital administrator stated that she had experienced increasing fatigue and some memory loss over the last year. Increasingly dry skin had prompted her to see a dermatologist earlier. She attributed these changes to a new air conditioning system in the administration area when she realized she was frequently cold and wore her suit jacket more often.
Her weight has remained stable this year, but she stated she has always had difficulty maintaining her weight. Her physician added luteinizing hormone (LH), follicle-stimulating hormone (FSH) and thyroid stimulating hormone (TSH) to a laboratory request. The LH and FSH, measured to assess her menopausal status, were within the reference ranges, but the TSH was elevated.
There are several processes that govern and regulate thyroid function. In recent years, several groups have released guidelines or recommendations for testing algorithms that should be used to evaluate thyroid dysfunction and for monitoring during treatment. Before discussing these recommendations, we will review the basics of thyroid function and some of the more common forms of dysfunction.
Thyroid Function
The thyroid regulates metabolism through the synthesis and secretion of triiodothyronine (T3) and thyroxin (T4). These two hormones regulate many metabolic functions including carbohydrate, protein, lipid metabolism and oxygen consumption. The two thyroid hormones, T3 and T4, are synthesized in the follicular cells of the thyroid. The synthesis and release of these hormones is controlled by close interaction between the hypothalamus, the pituitary and the thyroid. The hypothalamus synthesizes and secretes thyrotropin-releasing hormone (TRH), which binds to receptors on the thyrotropic cells of the anterior pituitary to initiate production and secretion of thyroid stimulating hormone (TSH, thyrotropin).
Meanwhile in the thyroid, a series of steps occur that involves 1) iodine uptake and binding to thyroglobulin, 2) intramolecular rearrangement of iodotyrosyls and, 3) storage of thyroglobulin (Tg)-bound T3 and T4.
When T3 or T4 is needed, TSH released from the pituitary stimulates the cells of the thyroid and increases the synthesis and release of T3 and T4. Additionally, the T4 released can undergo peripheral conversion to T3 in the pituitary and hypothalmus (as well as in other tissues). Triiodothyronin then acts locally on the pituitary cells (negative feedback) to inhibit TSH secretion. Upon release, 99.7 percent of the T3 and 99.97 percent of the T4 is protein bound for transport in plasma. The carrier proteins include thyroid-binding globulin (TBG), transthyretin (thyroid-binding prealbumin) and albumin. The remaining nonprotein bound, or free, T3 and T4 (fT3 and fT4) account for the observed biological activity.
Table 1: THYROID DYSFUNCTION
Causes of hyperthyroidism Symptoms of hypersecretion
Graves' Disease nervousness, palpitations,
Thyroid adenoma fatigue, heat intolerance,
Thyroiditis weight loss, dyspnea, sweating
subacute thyroiditis
silent thyroiditis
Toxic multinodular goiter Signs of hypersecretion
Ectopic thyrotoxicosis tachycardia, tremor,
Thyroid carcinoma thyroid enlargement, lid
TSH hypersecretion retraction, hyperactivity
Trophoblastic tumors
Exogenous thyrotoxicosis
Causes of hypothyroidism Symptoms of hyposecretion
Primary hypothyroidism dry skin, cold intolerance, voice
Chronic autoimmune thyroiditis change, paresthesia, weakness
Radioiodine therapy and fatigue, weight gain,
Thyroidectomy impaired memory
Defective thyroid hormone
Biosynthesis defect
Congenital defect Signs of hyposecretion
Iodine deficiency coarse hair and skin, cold skin,
Antithyroid drugs periorbital edema, slow reflex
Hypothyrotropic hypothyroidism relaxation
TSH or TRH deficiency
Resistance to thyroid hormone
Thyroid Dysfunction and Diseases
Reports by the U.S. Clinical Preventive Services and the Canadian Task Force on the Periodic Health Examination have indicated that thyroid disease accounts for considerable morbidity in both countries.
In the United States the prevalence of total thyroid disease is estimated to be 1 percent-4 percent in adolescents and adults. Yearly, approximately 0.05 percent-0.1 percent of U.S. adults will be diagnosed with hyperthyroidism, while 0.08 percent-0.2 percent will be diagnosed with hypothyroidism. In both countries the incidence of hypothyroidism is slightly higher in women of age 60 and older.
Hyperthyroidism
Hyperthyroidism includes several disorders (Table 1), all of which are characterized by an increase in the concentrations of circulating thyroid hormones (T3 or T4). Although this increase in thyroid hormones leads to the signs and symptoms (Table 1) found in patients with the disease, these symptoms are not unique and patients will not present with all of the symptoms listed.
The most common form of hyperthyroidism is that associated with Graves' disease. This syndrome occurs across the population but is higher in women between the ages of 30-60 years. Most patients with Graves' disease will have thyrotoxicosis (increased thyroid hormone concentrations), a diffusely enlarged thyroid gland and exophthalmus.
Graves' disease is an autoimmune disorder caused by IgG autoantibodies that bind to and activate the TSH receptors (they mimic TSH). Hyperthyroidism from causes other than Graves' disease in- clude hyperfunctioning solitary thyroid adenoma, TSH producing adenoma, silent thyroiditis (including lymphocytic and postpartum disorders), thyroid carcinoma and ingestion of excessive thyroid hormones.
In hyperthyroidism, concentrations of both the free and total hormones are increased. Occasionally only the T3 (free and total) is increased. This condition is known as T3 toxicosis. The increased concentrations of the hormones lead to increased metabolic activity in tissues and increased sensitivity of tissues to catecholamines. Effects of the hormones on the heart may be significant with tachycardia, arrhythmias and systolic hypertension observed.
Hypothyroidism
Hypothyroidism results from the under-secretion of thyroid hormones. Again, symptoms (Table 1) are nonspecific and include fatigue, cold intolerance, slowing of mental and physical performance, impaired memory, dry skin and paresthesias.
Hypothyroidism may be congenital or acquired, either primary or secondary. Congenital hypothyroidism occurs if the volume of the thyroid gland is insufficient or if there is an inborn defect in thyroid hormone synthesis or metabolism. Primary hypothyroidism occurs when damage to the thyroid gland leads to decrease synthesis of the hormones.
In the United States, the most common cause of hypothyroidism in adults is chronic autoimmune thyroiditis--with incidence highest in women age 40 years and older. It is found as both an atrophic (nongoitrous) form and a goitrous form (Hashimoto's disease).
In either case, the disease is thought to result from both antibody- and cell-mediated thyroid destruction. Several of the autoantibodies identified in sera or thyroid tissue from patients with this disorder include those which inhibit TSH action or receptor-binding, those which inhibit the action of thyroid peroxidase and those which stimulate thyroid growth. Previous radioiodine therapy and iodine deficiency also contribute significantly to the world-wide incidence. Secondary hypothyroidism is rare and occurs with pituitary or hypothalamic disease.
In congenital and primary hypothyroidism the decreased plasma concentrations of T3 and T4 lead to an increased release of TSH. In secondary hypothyroidism, T3 and T4 are decreased but TSH is within the reference range or slightly elevated. A hypothalamic cause is determined when the TSH increases after administration of TRH.
As one can see there are many types of and causes of overt thyroid disease. In addition to the conditions described above, other factors influence thyroid function and hormone action. Many factors such as other hormones, stress, illness and drugs affect thyroid hormone synthesis. The effects of other hormones may be quite complicated. For example, estrogens increase TBG thus increasing total T3 and T4, whereas, androgens have the opposite effect. In pregnancy, the placenta produces nonthyroid hormones, which also stimulates synthesis of TBG.
Many forms of stress, such as fasting or malnutrition, illness, surgery, decrease T4 to T3 conversion (hence a decrease in T3) and decrease synthesis of TSH and the thyroid hormone binding proteins. Additionally, the normal transport and metabolism of thyroid hormones may be altered in chronic or severe illness. In severe illness, fT4 concentrations usually remain normal but total T4 concentrations decrease. This decrease may be due to inhibitors that prevent binding of T4 to the binding proteins. Pituitary secretion of TSH is also effected in that secretion remains within normal limits for about 85 percent of patients despite the decreases in T3 and T4. During the recovery phase T4 and TSH concentrations rise concomitantly and TSH may even exceed the expected reference range.
In renal failure, iodine clearance and excretion is decreased leading to an increase in the iodine pool, but since the kidney is a site of T4 to T3 conversion, there will be a decrease in T3. In nephrotic syndrome, binding proteins may be lost.
For some patients, the interaction of drugs with the binding proteins or the action of some drugs on the synthesis of TRH, TSH and the thyroid hormones may yield unexpected results. Table 2, while not a complete list, identifies some of the more commonly encountered drugs that may lead to confusing results and to repeated testing when a laboratory error is suspected. While some of these interactions are well known, others may be unsuspected.
Table 2: Drugs Affecting TSH and freeT4
TSH increased - amiodarone, carbamazepine
(with valproic acid) , clomiphene, ferrous
sulfate, iodides, lithium, lovastatin,
metoclopramide, morphine, phenytoin,
prazosin, prednisone, propranolol, valproic
acid
TSH decreased - anabolic steroids, aspirin,
carbamazepine, danazol, dopamine
(levodopa), glucocorticoids (corticosteroids),
nifedipine, somatostatin
free T4 increased - amiodarone, aspirin,
carbamazepine, furosemide, phenytoin,
propranolol, tamoxifen, valproic acid
free T4 decreased anabolic steroids,
carbamazepine, clofibrate, corticosteroids,
lithium, estrogen, phenobarbital, phenytoin
(from Young DS, Effects of Drugs on Clinical Laboratory Tests, 4th Edition. AACC Press)
Testing Methodologies and Strategies
In the 1950's, thyroid function was crudely "measured" in the laboratory using protein bound iodine (PBI). The development of radioimmunoassay (RIA) in the 1960's revolutionized testing. The evolution of automated, robust nonisotopic assays for TSH, total T4, total T3, and fT4 continues to improve our ability to biochemically confirm thyroid dysfunction. Occasionally, other tests such as reverse T3, fT3, antithyroid antibodies, or thyroglobulin are needed, but for the remainder of this discussion we will focus on the more commonly used tests.
TSH
Perhaps the biggest strides have been made in the assays for TSH measurement. TSH assays have been classified into categories based upon assays of analytical sensitivity and functional sensitivity.
Early first-generation TSH radioimmunoassays were capable of differentiating euthyroid from hypothyroid, but, they had poor analytical and functional sensitivity. Analytical sensitivity pertains to the ability of the assay to differentiate between 0 and the lowest standard. Often analytical sensitivity is determined by repeated measurement of a hormone free sample, such as a zero calibrator. Functional sensitivity is defined as the lowest level of TSH detected with an interassay coefficient of variation of 20 percent and is determined by performing precision profiles over several weeks (and preferably lots).
The next generation of TSH assays was developed as specific TSH monoclonal and polyclonal antibodies became available. Typically, most second and third generation assays are based on immunometric assays (IMA).
An IMA differs from RIA in that two TSH antibodies are used. The first TSH antibody, often bound to a solid phase, recognizes the TSH ß-chain and is used to extract the TSH from the sample. The second TSH antibody is labeled and binds to a different portion of the TSH molecule. Because the molecule being measured is bound "between" the two antibodies, these assays are often referred to as sandwich assays.
The second generation TSH assays, defined as having functional sensitivity of 0.1-0.2 mU/L at 20 percent, can distinguish between ambulatory patients who are euthyroid versus hyperthyroid. They have limited sensitivity in distinguishing between mildly subnormal 0.01-0.1mU/L and the low thyrotoxic values. Third generation assays have a functional sensitivity of 0.01-0.02 mU/L and can distinguish the mildly depressed TSH levels of sick hospitalized or thyroxine-treated patients from the profoundly depressed values of hyperthyroidism. Most third generation assays use a chemiluminescent label.
When considering a TSH assay for use, clinical laboratories should verify the analytical performance claims (sensitivity/generation) of the manufacturer by conducting an in-house precision profile study.
Many of us have found some assays advertised to be third generation are not able to consistently achieve the precision requirements described above. Addition-ally, studies have shown that even with the "best" of assays, precision may deteriorate over time as calibration ages, detection devices need adjusting, etc. As a result, the precision profile, or at least an abbreviated version, should be repeated periodically so that performance expectations can be consistently met.
T3 andT4
Most laboratories use one of the nonisotopic immunoassays for the measurement of total T3 and T4 in serum. Because both hormones are tightly bound to their respective carrier proteins, one of the first steps in any of these assays is the addition of barbital buffers or blocking agents to displace bound hormone and/or inhibit further binding. The polyclonal or monoclonal antibodies used in these assays must exhibit high-specificity in order to distinguish between two molecules differing by one iodine atom. Both heterogeneous and homogeneous methods are available and most are automated.
Three methods are used for the measurement of fT3 or fT4 in serum: equilibrium dialysis, two-step immunoextraction methods and analog methods. Equilibrium dialysis is considered to be, by many, the gold standard for the measurement of free hormones. It is however technically demanding and very few laboratories use this method.
Two-step and analog methods compare well when testing fT4 concentrations in ambulatory patients (including pregnant patients with mild TBG elevations). Either method may be used to accurately diagnose thyroid disease in these patients; however, problems occur when testing several groups of patients particularly with some of the analog methods.
Inaccurate measurements may be ob-served when testing samples from patients with TBG excess or deficiency, hypoalbuminemia, dysalbuminemia and T4 autoantibodies. When testing severely ill, euthyroid patients in whom the total T4 is depressed, equilibrium dialysis may reveal a normal or high fT4 concentration whereas the alternate methods may give low values. Whichever technique is chosen, it is important the laboratory scientist understand the limitations of the assay to assist physicians interpreting the data.
Table 3: Resources of Guidelines, Recommendations and Consensus Statements
1990 American Thyroid Association (ref. 1)
1995 American Association of Clinical Endocrinologists (www.aace.com)
1995 National Academy of Clinical Biochemistry (Clin Chem 42 (1), 1996)
1996 Royal College of Physicians of London, Society for Endocrinology
(ref 5)
1997 Alberta Clinical Practice Guidelines Advisory Committee (www.amda.ab.ca)
1997 United States Clinical Preventive Services (cpmcnet.columbia.edu)
Current Recommendations
Multiple groups (Table 3) have issued recommendations, guidelines and consensus statements regarding thyroid function testing. All of these experts agree with the earlier recommendation of the American Thyroid Association (ATA) that screening of the general population should not be done.
Screening is recommended only for newborns and those considered being at risk of developing disease. There is a slight difference between the definition each group assigns to "increased risk," but generally includes patients who have a strong family history of thyroid disease, patients with other autoimmune disorders and patients receiving amiodarone or lithium.
Screening may be appropriate for some elderly patients and some postpartum women 4-8 weeks after delivery. Each group recommends that the physician have a specific thyroid disease in mind when selecting tests.
Because of the impact of many pathological processes on thyroid hormone synthesis, it is recommended that testing be deferred until the acute illness is resolved. If testing is necessary, interpretation of data may be difficult and confusing.
First, as discussed above, many of the fT4 assays used do not give accurate results under these conditions. The low fT4 values obtained with many methods reflect the impairment of serum thyroid hormone binding due to non-thyroidal illness. Second, transient abnormalities in TSH values may be encountered in response to treatment with glucocorticoid drugs, dop-amine, or other drugs. In these situations, TSH concentrations may be low in the acute phase of glucocorticoid drug treatment and elevated during the recovery phase.
The recommended testing strategy begins with a sensitive TSH--either a good second or true third generation assay. This one test has been consistently shown to be useful in assessing thyroid function in ambulatory patients who do not have pituitary or neuropsychiatric disease.
If the TSH concentration is within the laboratory reference range, the patient is considered euthyroid and no further testing is needed. Suppressed or increased TSH concentrations suggest thyroid dysfunction and warrant additional laboratory testing such as a fT4 or T3. Performing the testing in this fashion--an initial screening test followed in sequence by additional tests as warranted--has proven efficient and cost effective. Multiple schemes have been published and, when developed jointly between the laboratory and physicians, work well.
After the Diagnosis
Much attention is given in the literature to the interpretation of screening algorithms and while such testing does contribute to our volumes, quite a bit occurs after the diagnosis has been made. After a diagnosis of hyperthyroidism is made, radioiodine therapy, antithyroid drug therapy or thyroidectomy may be used to lower hormone levels. If monitoring is necessary during the early phase of antithyroid therapy, fT4 and fT3 are better indicators of the acute response to treatment than TSH because of the lag in the pituitary reset of TSH secretion. During this period TSH levels may remain depressed even if there is rapid development of a hypothyroid state. After the patient has regained a stable thyroid status, TSH will again become an optimal test for uncovering any subtle hormone excess or deficiency.
For patients with hypothyroidism, if monitoring is necessary in the early phases of replacement therapy, fT4 is the optimal test since serum TSH concentrations remain high, again due to a lag in pituitary reset of TSH secretion. After the patient has received a stable T4 dose for at least 2 months, TSH again becomes the optimal test because the therapeutic end-point for replacement therapy is a normal-range TSH concentration. The lag in pituitary reset can be useful diagnostically since the TSH provides a more integrated estimate of circulating fT4 status over time, much as a hemoglobin A1C concentration provides integrated information on glucose status.
The ATA recommends TSH concentrations be monitored 2-3 months after the initiation of replacement therapy and whenever dose or formulation is changed. After the first year monitoring should be done yearly or in response to changes in clinical presentation or drug therapy.
A serum TSH may be useful in identifying compliance problems since poorly compliant patients may take enough thyroxine to raise their T4 concentrations to normal for their doctor's appointment. However, elevated TSH concentrations will persist because of inconsistent drug therapy. Keep in mind that elevated or mildly elevated TSH concentrations may also indicate suboptimal T4 replacement. A high-normal fT4 may be required for some patients to normalize TSH. The higher fT4 compensates for the inability to produce T3, which contributes to some extent to the feedback regulation of TSH.
Recommended Reading
1. Davey R. Thyroxine, Thyrotropin and Age in a Euthyroid Hospital Patient Population. Clin Chem. 1997; 43:2143-8.
2. Davey, R.X., Clark, M.I., Webster, A. R. Thyroid Function Testing Based on Assay of Thyroid Stimulating Hormone: Assessing an Algorithm's Reliability. MJA. 1996; 164:329-32.2.
3. Klee, G.G., Hay, I. D. Biochemical Thyroid Function Testing. Mayo Clin Proc. 1994; 69: 469-71.
4. Laurberg, P., et al. Iodine Intake and the Pattern of Thyroid Disorders: A Comparative Epidemiological Study of Thyroid Abnormalities in the Elderly in Iceland and in Jutland, Denmark. J Clin Endocrinol Metab. 1998;83:765-9.
5. Nicoloff, J.T., Spencer, C.A. The Use and Misuse of the Sensitive Thyrotropin Assays. J Clin Endocrinol Metab. 1990; 71:553-8.
6. Rae, P., et al. Assessment of Thyroid Status in Elderly People. BMJ. 1993; 307:177-80.
7. Spencer, C.A., et al. Interlaboratory/Intermethod Differences in Function-al Sensitivity of Immunometric Assays of Thyrotropin (TSH) and Impact on Reliabil-ity of Measurement of Subnormal Concentrations of TSH. Clin Chem. 1995; 41:367-74.
8. Surks, M. I., et al. American Thyroid Association Guidelines for Use of Laboratory Tests in Thyroid Disorders. JAMA 1990;263:1529-32.
9. Vanderpump, M.P.J., et al. Consensus Statement for Good Practice and Audit Measures in the Management of Hypothyroidism and Hyperthyroidism. BMJ. 1996; 313: 539-44.
10. Weetman, A. P. Hypothyroidism: Screening and Subclinical Disease. BMJ. 1997; 314: 1175-78.
Dr. Hammett-Stabler is assistant professor in the Department of Pathology, Immunology and Laboratory Medicine at the University of Florida, and associate medical director of the clinical chemistry section of the Core Labora-tory, Shands at UF, Gainesville, FL.
THYROID TESTING RELATED WEBSITES
Guidelines for the Use of Serum Tests to Detect Thyroid Dysfunction--
http://www.tantech.com/oaml/tsh.html
Thyroid Tests--http://www.interscilsa.com/book/indi29.html#282
Gland Central Thyroid Library http://www.glandcentral.com/resources /library.html
The Learning Scope Objectives and Questions
OBJECTIVES
1. Describe the mechanisms involved in thyroid hormone synthesis.
2. List the most common causes of hyperthyroidism and hypothyroidism.
3. Describe the current recommendation regarding the screening for thyroid disease.
QUESTIONS
1. The thyroid regulates metabolism through the synthesis and secretion of:
a. TRH
b. TSH
c. T3 and T4
d. TBG
2. Which of the following forms of thyroxin acts at the target tissue?
a. free T4
b. thyroglobulin -T4
c. albumin -T4
d. TBG -T4
3. The most common form of hyperthyroidism, Graves' disease, is characterized by all but which of the following.
a. lgG autoantibodies
b. thyrotoxicosis
c. decreased T4
d. exophthalmos
4. Which of the following is increased in hypothyroidism?
a. T4
b. TBG
c. rT3
d. TSH
5. All but which of the following factors possibly contribute to the effect of severe illness on thyroid hormone synthesis?
a. decreased syntheses of TSH
b. inhibition of T4 protein interaction
c. increased synthesis of albumin
d. alteration of hormone transport
6. Which of the following techniques is most commonly used for the measurement of TSH concentrations?
a. radioimmunoassay
b. enzyme immunoassay
c. immunometric assay
d. equilibrium dialysis
7. Which of the following methods is considered the gold standard for the measurement of free T4?
a. immunometric assay
b. high performance
chromatography
c. radioimmunoassay
d. equilibrium dialysis
8. Screening for thyroid disease is recommended for which of the following groups?
a. all female patients under age 30
b. women in the first trimester of pregnancy
c. critically ill elderly patients
d. newborns
9. Which of the tests listed below is recommended as the first test when screening patients for thyroid dysfunction?
a. TSH
b. fT4
c. T4
d. T3
10. A scheme introduced in the early 1990's to classify TSH assays is based on?
a. analytical sensitivity
b. manufacturer's claims
c. functional sensitivity
d. immunoassay type
11. Laboratories can verify functional sensitivity by
a. repeated measurements of the zero calibrator
b. performing precision profiles
c. calling the manufacturer
d. performing daily QC
12. When a hospitalized patient's TSH and fT4 do not agree (both are decreased), which of the following are possible explanations?
a. the effect of drug(s)
b. sick euthyroid syndrome
c. an analogue method is used for the fT4
d. all of the above
13. Which of the following acts in the pituitary to inhibit TSH secretion?
a. T4
b. T3
c. TBG
d. TEG
14. Which of the following is NOT a carrier protein for the thyroid hormones?
a. albumin
b. thyroid-binding globulin
c. transthyretin
d. transcortin
15. If monitoring is necessary in the early stages of disease treatment
a. rT3 should be used for patients with hypothyroidism.
b. fT4 should be used for patients with hyperthyroidism.
c. TSH should be used for patients with hypothyroidism.
d. TSH should be used for all patients.
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