Table 1: Thyroid panel. Result (reference interval) TSH 89.5 miu/l ( ) Total T4 5.2 µg/dl ( ) T3 uptake 39% (22-35)

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1 Introduction Thyroid disease is the second most common endocrine disorder (behind diabetes), and its prevalence increases with increasing age. The incidence of newly diagnosed thyroid cancer is increasing at a rate faster than that of most other malignancies. Laboratory tests are important for recognizing thyroid disease, and for monitoring patients with thyroid dysfunction and thyroid cancer. Like all laboratory tests, particularly those measured by immunoassays, thyroid-related tests have a number of limitations that can produce misleading results in certain clinical situations. This module will review a number of cases that highlight the use of thyroid tests as well as their limitations, and will stress areas where the laboratory can provide critical information to the patient s health care provider to allow for proper utilization and interpretation of various thyroid-related tests. Case 1 A 29-year-old woman presented to her primary care physician complaining of fatigue, weight gain, and difficulty concentrating at work. She has been healthy for most of her life and has no family history of significant medical problems. Physical examination revealed a goiter that was nontender. Her physician ordered a panel of thyroid tests; results are as follows: Table 1: Thyroid panel. Test Result (reference interval) TSH 89.5 miu/l ( ) Total T4 5.2 µg/dl ( ) T3 uptake 39% (22-35) Free T4 0.4 ng/dl ( ) Question #1 Her primary care provider calls you to ask what these results mean. What would you tell him? Should all of these tests have been ordered? Thought-provoking Question #1 The markedly elevated thyroid stimulating hormone (TSH) and low free thyroxine are consistent with the clinical picture suggesting hypothyroidism. The total thyroxine (T4) and tri-iodothyronine (T3) uptake results, changing in opposite directions, suggest the presence of increased thyroxine-binding globulin, which can be due to a number of conditions as well as medications. Performing measurement of both total T4 (and its related test, T3 uptake) and free T4 is not recommended for routine use. Page 1 of 10

2 Discussion There are a number of tests for thyroid function that are available for laboratories to offer clinicians. The most common are TSH, T4 and T3, free T4 and T3, and estimates of thyroid binding proteins. In the circulation, over 99% of both T4 and T3 are bound to proteins, predominantly thyroxine binding globulin (TBG). The bound forms are not felt to be physiologically active; it is believed that free thyroid hormones are responsible for all hormone actions and, in turn, provide feedback control on production of TSH. Guidelines for use of thyroid function tests recommend TSH as the initial test for evaluation of thyroid function. 1-3 TSH is typically the most sensitive test of thyroid function, since there is an inverse logarithmic relationship between TSH and free thyroid hormone levels, so that a slight change in thyroid function causes a much greater magnitude change in TSH. 1,2 In addition, almost all thyroid dysfunction is due to thyroid disease (rather than pituitary or hypothalamic dysfunction), so that TSH becomes abnormal much earlier than do tests of thyroid hormones. High TSH is typically seen in hypothyroidism, and low TSH in hyperthyroidism. There is some disagreement about upper reference limits for TSH in nonpregnant adults. The National Academy of Clinical Biochemistry (NACB) recommends that reference limits be determined in individuals with no history or family history of thyroid disease, and who are free of antithyroid antibodies (which are commonly found in the general population). 1 Using this approach, upper reference limits are typically around 2.5 miu/l. While some laboratories have adopted such reference limits, this classifies a relatively large number of individuals as having impaired thyroid function. In the most detailed natural history study, women having both elevated TSH (>6 miu/l) and positive thyroid antibodies developed hypothyroidism at a rate of 4% per year, and after 20 years of follow-up, only half had become hypothyroid. 4 The American Thyroid Association (ATA) guidelines, however, point out that adverse effects of hypothyroidism on the heart typically do not occur until TSH values are greater than 10 miu/l. 3 In addition, there is some evidence that an increase in TSH levels with increasing age (particularly in those over age 80-85) is not associated with thyroid dysfunction that benefits from treatment. 5,6 Thus, most laboratories still use reference intervals derived from the central 95% of the population, with upper reference limits typically around miu/l. Since free thyroid hormones are the active forms, guidelines recommend free T4 evaluation to determine the level of thyroid function; free T3 is less commonly abnormal in hypothyroidism. 1-3 Because total thyroid hormones are also affected by changes in binding protein levels, total T4 is less sensitive and specific than free T4 for determination of thyroid function in most settings. 1 Free T4 can be either directly measured by one of a variety of assay techniques (which are still referred to by some as free T4 estimates, 1 since they are affected by some important clinical conditions and medications 7 ) or estimated from total T4 and measurement or evaluation of the level of TBG. In normal individuals, a relatively common proportion of TBG binding sites are occupied by thyroid hormone; TBG is most commonly evaluated by determining the ability of the serum to bind labeled thyroid hormone, either directly or by determining the amount of the labeled hormone that did not bind to TBG and is then bound to an added resin. The results of such thyroid hormone uptake tests and total T4 (or T3) are used to estimate free T4. Page 2 of 10

3 Guidelines have recommended that laboratories and clinicians consider performing TSH as an initial screen for thyroid function, with reflex performance of direct or estimated free T4 if TSH is abnormal. Based on these guidelines, you develop a new panel termed thyroid panel which includes measurement of TSH and reflex measurement of free thyroxine. You present this panel to your clinical colleagues, and it is approved by the medical staff as a more cost-effective way to evaluate thyroid function. In addition to educational efforts to make health care providers aware of the new panel in your laboratory newsletter, you also put order comments on TSH, free T4, and total T4 tests in the physician order entry software, making providers aware of the new panel and the recommendation to use it rather than individual tests. Case 2 A primary care provider consults you about test results for his patient, a 35-year-old man who had presented to him stating that his wife had insisted on him seeing the doctor, since he wanted to now sleep with the windows open in the winter. He had also lost about 15 pounds over the past 3 months despite no change in exercise or food intake. The provider had ordered the new thyroid panel on his patient, with the following results: Test Result (reference interval) TSH <0.01 miu/l ( ) Free T4 1.6 ng/dl ( ) Question #2 What advice would you give your colleague? Are there other tests that may be of help in this setting? Short Answer to Question #2 The patient s symptoms and suppressed TSH result strongly suggest hyperthyroidism. A number of patients who are hyperthyroid, particularly those with Graves disease, will have normal free T4 but elevated free T3; you recommend measurement of free T3. The clinician orders the free T3 and receives the result which is elevated at 5.7 ng/ml (reference interval ) the following day. Discussion Hyperthyroidism is also a relatively common condition, and like hypothyroidism its incidence increases with increasing age, although not as dramatically as hypothyroidism. While Graves disease is the most common cause overall, the rising incidence in older individuals is mostly due to toxic multinodular goiter. 8 There is actually poor correlation between degree of thyroid hormone abnormality and symptoms of hyperthyroidism, with older patients often having less typical symptoms. 8 Page 3 of 10

4 Generally, TSH is also the most sensitive test for diagnosis of hyperthyroidism. While most individuals with hyperthyroidism have increases in both free T4 and free T3, both early and late in the course many individuals will only have elevation of free T3. 8 While guidelines from the NACB recommend measurement of total T3, 1 the ATA and American Association of Clinical Endocrinologists (AACE) guidelines only state that since free T3 measurements are less widely validated than those of free T4, measurement of total T3 is frequently preferred. 8 In the author s institution, measurements of free T3 have been used exclusively (instead of total T3) since the 1990 s with no clinical evidence to indicate issues with their performance. In a study using fresh frozen CAP proficiency serum samples, there was little difference in the frequency of analytical bias between total and free T3 tests. 9 In following patients with hyperthyroidism, free T4 and T3 often return to normal significantly sooner than does TSH. The ATA/AACE guidelines recommend that laboratories consider reflex testing of free T4 and T3 (either direct measurement or estimates) in all patients with suppressed TSH to reduce the need for repeat blood draws. 8 Based on these recommendations, you return to the medical staff with a suggested modification to the thyroid panel reflex testing; the laboratory will perform reflex free T4 on all patients with high TSH, and reflex free T4 and free T3 on all patients with suppressed TSH. This change is approved by the medical staff. You update your order comments in the laboratory information system and also put out a brief information item in the laboratory newsletter. Case 3 You are called by one of the obstetrics nurse practitioners. She has a 27-year-old woman who is in her second trimester of pregnancy and has a history of hypothyroidism due to Hashimoto thyroiditis, diagnosed at age 16. She has been following the patient with TSH measurements and had increased her patient s thyroid hormone dosage as pregnancy progressed as recommended in the guidelines. At the most recent visit, she also ordered free T4 measurement, with the following results: Test Result (reference interval) TSH 1.7 miu/l ( ) Free T4 0.5 ng/dl ( ) Question #3 Are these reference intervals appropriate in pregnancy? Short Answer to Question #3 Because of changes in thyroid function that occur during pregnancy, guidelines have recommended establishing or using pregnancy-specific reference limits for TSH (Table 2) that vary by trimester. 10,11 Page 4 of 10

5 Table 2: ATA/AACE Trimester-specific TSH reference intervals. 11 Trimester First Second Third Reference Interval (miu/l) Discussion Thyroid hormone is critical to normal fetal development, and maternal hypothyroidism (or inadequately treated maternal hypothyroidism) can lead to impaired brain development in babies. While routine screening for thyroid disease during pregnancy is not recommended, testing is needed in women with known thyroid disease or in those with signs or symptoms of thyroid disease that develop during pregnancy. Guidelines have recommended establishing or using pregnancy-specific reference limits for TSH (Table 2) that varies by trimester. 10,11 In women with hypothyroidism, the target level for TSH is 2.5 miu/l, 10 much lower than the upper reference limit for nonpregnant individuals. However, it is often difficult for both reference and hospital-based laboratories to have different reference intervals for TSH on laboratory reports for pregnant women, since information on pregnancy status is not often known by the laboratory. It is often easier to use recommendations from professional societies with specific laboratory clients, namely those known to care for pregnant women. One approach would be to create a new panel termed thyroid panel, pregnancy which has different reference limits based on trimester. You discuss this with the medical staff, who agree to implement this panel as well. You send a letter to all of the providers who provide care for pregnant women to advise them of the change and request that they utilize this panel to make interpretation of results on their patients easier. Table 2: ATA/AACE Trimester-specific TSH reference intervals. 11 Trimester First Second Third Reference Interval (miu/l) Pregnancy is also one of the situations that can cause unreliable measurements of thyroid hormones (both total and free T4 and T3). TBG increases to a variable extent during pregnancy, 12 causing a variable increase in total T4 (and T3). Despite this variable increase, some guidelines have suggested that a target value for T4 should be 50% higher than that in nonpregnant individuals, since the average increase in TBG during pregnancy is 50%, 10,11 but this recommendation can lead to misinterpretation of thyroid status. 12 Free T4 measurements are often significantly decreased in normal pregnant women, although the decrease is method-dependent. 13,14 Clinical guidelines do not recommend use of free T4 measurement during pregnancy for this reason. 10,11 Free T4 calculated from total T4 and T3 uptake appears to be more reliable in pregnant women, but still not as reliable as TSH. 10,11 Question #4 What other situations can produce unreliable thyroid function tests? Page 5 of 10

6 Short Answer to Question #4 Other common situations that cause unreliable thyroid hormone measurements are conditions that change TBG levels or hormone binding to TBG, autoimmune thyroid disease, and acute illness, 7 as summarized in Table 3. Discussion In addition to pregnancy, TBG is increased by estrogen use, selective estrogen receptor modifiers (SERM) such as tamoxifen, opiates, methadone, and acute and chronic hepatitis. Low TBG is less common, but can occur in protein losing conditions such as nephrotic syndrome, cirrhosis, and congenital deficiency, and induced by glucocorticosteroids and androgens. Such conditions will affect total T4 and T3 measurements, but do not affect free T4 or T3. Altered binding protein affinity for thyroid hormones occurs in acute illness, phenytoin use, and intravenous heparin or high-dose furosemide. These conditions will decrease total T4, but have variable effects on free thyroid hormone levels that are method dependent. While some guidelines have recommended using estimates of free T4 based on total T4 and T3 uptake measurements, these do not seem to be any more reliable in that setting. 15 Acute illness also decreases peripheral conversion of T4 to T3, causing disproportionate decreases in T3. Thyroid hormone antibodies occur in a significant minority of patients with autoimmune thyroid disease. 16 These antibodies commonly cause variably increased results, often with marked elevation in total thyroid hormone measurements and lesser increases (or no increase) in measured free thyroid hormone levels. Table 3: Factors affecting thyroid hormone measurement. Increased TBG Decreased TBG Factor Test Results Examples Decreased TBG affinity Impaired conversion of T4 to T3 Thyroid hormone autoantibodies Increased total T4, T3 Decreased total T4, T3 Decreased total T4, T3; method dependent changes in free T4, T3 (sometimes increased, sometimes decreased) Decreased total, free T3 Variable increases in total T4 and/or T3, lesser effects on free T4 and/or T3 Pregnancy, acute and chronic hepatitis, estrogen, tamoxifen, opiates, methadone Nephrotic syndrome, cirrhosis, congenital deficiency, androgens, glucocorticoids Acute illness, phenytoin, furosemide, heparin Acute illness Hashimoto thyroiditis TSH measurements are less commonly unreliable, but are frequently affected by acute illness and pituitary disease. In acute illness, a variety of factors seem to affect TSH production that may either increase or decrease levels. In one study of acutely ill individuals subsequently found to be euthyroid, TSH varied between 0.1 and 20 miu/l. 17 In patients with pituitary disease, an abnormal form of TSH is produced that has reduced biological Page 6 of 10

7 activity, but which is measured in immunoassays, resulting in normal or mildly increased serum TSH values in most patients. 18 Because of this, TSH levels cannot be used to monitor persons with hypothyroidism due to pituitary disease. In this situation, measurement of free T4 is the preferred test for following treatment. Case 4 A surgeon calls you about one of his patients, a 25-year-old woman who has a history of Hashimoto thyroiditis and presented with a nontender nodule in her neck. On physical examination, she had palpable cervical adenopathy and a 3 cm nodule in her thyroid, which by fine-needle aspiration was papillary carcinoma. He had ordered a preoperative thyroglobulin level, which was undetectable. He thinks this result is clinically unlikely, and would like an explanation on why the laboratory would produce such an unreliable result. Question #4 What would you say to the surgeon? Short Answer Question #4 While thyroglobulin is produced by almost all thyroid cancers, there is limited or no utility of preoperative measurement of thyroglobulin. The most common cause of falsely low thyroglobulin results is antibodies to thyroglobulin, which are seen primarily in individuals with a history of thyroiditis, as in this patient. For follow-up testing after treatment of her thyroid cancer, measurement of thyroglobulin by mass spectrometry is likely to have no interference from thyroglobulin antibodies. Discussion Thyroglobulin is the major protein secreted by thyroid cells into colloid, and is the storage form of thyroid hormones. It is specific to thyroid tissue, and is found in the circulation in trace amounts, in proportion to the mass of the thyroid and in response to damage to the thyroid gland. It is also produced by differentiated (papillary and follicular) thyroid cancers and is commonly used as a tumor marker in patients at high risk for residual or recurrent thyroid carcinoma after treatment of the primary tumor. It is not recommended for evaluation of patients who present with thyroid nodules, however, and testing should not be performed until three to four weeks after surgery. 19 An important consideration in interpreting levels is that reference intervals are based on individuals who have thyroid glands; after total thyroidectomy, especially if remnant thyroid tissue is ablated with radioactive iodine, thyroglobulin should be undetectable, not within the reference interval derived from healthy individuals. Since there is little clinical utility to measuring thyroglobulin in other settings, laboratory reports should indicate that detectable thyroglobulin in an individual with no remaining normal thyroid tissue strongly suggests residual thyroid carcinoma. One limitation of thyroglobulin testing is that thyroglobulin production is TSH dependent, as is the growth of differentiated thyroid carcinoma. Individuals with high risk of recurrence are often given higher doses of thyroid hormone to keep TSH suppressed, which can also inhibit thyroglobulin production by residual thyroid cancer. An undetectable thyroglobulin in a Page 7 of 10

8 person with suppressed TSH is therefore not a reliable indicator of absence of residual disease. High risk patients are often given recombinant TSH injections, with thyroglobulin and TSH measured at baseline and at two and four days after initial TSH administration, so it is not uncommon to see several thyroglobulin requests within a few days in thyroid cancer patients. If thyroglobulin remains undetectable at days three and five after the first administration of recombinant TSH, residual thyroid cancer is unlikely, since less than 1% of thyroid cancers do not produce thyroglobulin. Thyroglobulin is normally measured by noncompetitive immunometric assays, which use two antibodies to detect thyroglobulin. Antibodies to thyroglobulin, which are commonly found in the population and appear even more commonly in persons with thyroiditis and/or thyroid cancer, lead to falsely low results (often undetectable) in immunometric assays. Assays differ in their ability to detect thyroglobulin antibodies, so that a sensitive assay should be used in patients with thyroid cancer. Guidelines recommend routine measurement of thyroglobulin antibodies at the same time as thyroglobulin in all patients with thyroid cancer. 1 In the past few years, assays utilizing mass spectrometry of peptides derived from thyroglobulin have become available from a number of reference laboratories. Such assays seem to eliminate the interference from thyroglobulin antibodies. 20 A common approach to evaluating patients is to measure thyroglobulin and thyroglobulin antibodies; detectable thyroglobulin indicates residual thyroid tissue. Undetectable thyroglobulin with negative thyroglobulin antibodies (both before and after recombinant TSH in high risk patients) indicates no residual thyroid tissue or thyroid cancer. Undetectable thyroglobulin with positive thyroglobulin antibodies would be followed by measurement of thyroglobulin by mass spectometry. Based on the facts you learned in this case, and the fact that your institution has had a recent increase in thyroid cancer cases diagnosed, you decide to develop another new panel for thyroglobulin testing, called thyroglobulin for thyroid cancer monitoring. It includes measurement of thyroglobulin by immunometric assay and thyroglobulin antibodies, and in any antibody-positive sample with undetectable thyroglobulin, measurement of thyroglobulin by mass spectrometry as a reflex test. This panel is also approved by your medical staff. You also add an order comment on your current thyroglobulin test indicating that, if ordered for monitoring patients with thyroid cancer, to consider ordering the panel instead. Key Points TSH is the most important thyroid function test, and can be used as the primary test for evaluation of most patients with suspected or known thyroid disease. However, TSH cannot be used to evaluate thyroid function in persons with pituitary disease; free T4 should be used instead. Measured or estimated free T4 is an important additional test in evaluating patients with abnormally high TSH, while free T4 and free T3 are both important in evaluating patients with suppressed TSH. A number of common conditions and medications can cause misleading thyroid function tests; the most common are acute illness, pregnancy, and pituitary disease. Special considerations should exist for evaluating thyroid function tests in these settings. Page 8 of 10

9 Thyroglobulin is an important test for monitoring patients after treatment for differentiated thyroid cancer, and should be evaluated in light of thyroglobulin antibody levels; in antibody-positive individuals, measurement of thyroglobulin by mass spectrometry appears reliable. In patients at high risk for recurrence who have suppressed TSH as part of management, repeat measurement of thyroglobulin following recombinant TSH measurement is appropriate. Key Go To References Resources 1. Demers L, Spencer C. Laboratory medicine practice guidelines: laboratory support for the diagnosis and monitoring of thyroid disease. Thyroid. 2003;13: Dufour D. Laboratory tests of thyroid function: uses and limitations. Endocrinol Metab Clin North Am. 2007;36: Haugen B, Alexander E, Bible K, et al American Thyroid Association management guidelines for adult patients with thyroid nodules and differentiated thyroid cancer: The American Thyroid Association guidelines task force on thyroid nodules and differentiated thyroid cancer. Thyroid. 2016;26: Demers L, Spencer C. Laboratory medicine practice guidelines: laboratory support for the diagnosis and monitoring of thyroid disease. Thyroid. 2003;13: Garber J, Cobin R, Gharib H, et al. Clinical practice guidelines for hypothyroidism in adults: cosponsored by the American Association of Clinical Endocrinologists and the American Thyroid Association. Endocr Pract. 2012;18: Jonklaas J, Bianco A, Bauer A, et al. Guidelines for the treatment of hypothyroidism: prepared by the American Thyroid Association task force on thyroid hormone replacement. Thyroid. 2014;24: Vanderpump M, Tunbridge W, French J, et al. The incidence of thyroid disorders in the community: a twenty-year follow-up of the Whickham Survey. Clin Endocrinol (Oxf). 1995;43: Kahapola-Arachchige K, Hadlow N, Wardrop R, et al. Age-specific TSH reference ranges have minimal impact on the diagnosis of thyroid dysfunction. Clin Endocrinol (Oxf). 2012;77: Bremner A, Feddema P, Leedman P, et al. Age-related changes in thyroid function: a longitudinal study of a community-based cohort. J Clin Endocrinol Metab. 2012;97: Dufour D. Laboratory tests of thyroid function: uses and limitations. Endocrinol Metab Clin North Am. 2007;36: Page 9 of 10

10 8. Bahn R, Burch H, Cooper D, et al. Hyperthyroidism and other causes of thyrotoxicosis: management guidelines of the American Thyroid Association and the American Association of Clinical Endocrinologists. Endocr Pract. 2011;17:e2-e Steele BW, Wang E, Klee GG, et al. Analytic bias of thyroid function tests: analysis of a College of American Pathologists fresh frozen serum pool by 3900 clinical laboratories. Arch Pathol Lab Med. 2005;129: De Groot L, Abalovich M, Alexander EK, et al. Management of thyroid dysfunction during pregnancy and postpartum: an Endocrine Society Clinical Practice Guideline. J Clin Endocrinol Metab. 2012;97: Stagnaro-Green A, Abalovich M, Alexander E, et al. Guidelines of the American Thyroid Association for the diagnosis and management of thyroid disease during pregnancy and postpartum. Thyroid. 2011;21: Midgley J, Hoermann R. Measurement of total rather than free thyroxine in pregnancy: the diagnostic implications. Thyroid. 2013;23: Anckaert E, Poppeb K, Van Uytfanghec K, et al. FT4 immunoassays may display a pattern during pregnancy similar to the equilibrium dialysis ID LC/tandem MS candidate reference measurement procedure in spite of susceptibility towards binding protein alterations. Clin Chim Acta. 2010;411: Lee R, Spencer C, Mestman J, et al. Free T4 immunoassays are flawed during pregnancy. Am J Obstet Gynecol. 2009;200:260,e1-e Faix JD, Rosen HN, Velazquez FR. Indirect estimation of thyroid hormone-binding proteins to calculate free thyroxine index: comparison of nonisotopic methods that use labeled thyroxine ("T-uptake"). Clin Chem. 1995;41: Despres N, Grant A. Antibody interference in thyroid assays: a potential for clinical misinformation. Clin Chem. 1998;44: Spencer C, Eigen A, Shen D, et al. Specificity of sensitive assays of thyrotropin (TSH) used to screen for thyroid disease in hospitalized patients. Clin Chem. 1987;33: Persani L. Central hypothyroidism: pathogenic, diagnostic, and therapeutic challenges. J Clin Endocrinol Metab. 2012;97: Haugen B, Alexander E, Bible K, et al American Thyroid Association management guidelines for adult patients with thyroid nodules and differentiated thyroid cancer: The American Thyroid Association guidelines task force on thyroid nodules and differentiated thyroid cancer. Thyroid. 2016;26: Netzel B, Grebe S, Leon B, et al. Thyroglobulin (Tg) testing revisited: Tg assays, TgAb assays, and correlation of results with clinical outcomes. J Clin Endocrinol Metab. 2015;100:e1074-e83. Page 10 of 10

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