Tests of Thyroid Function

  • Giovanni Ceccarini
  • Ferruccio Santini
  • Paolo VittiEmail author
Reference work entry
Part of the Endocrinology book series (ENDOCR)


Thyroid function testing is the most used diagnostic evaluation in endocrine practice and is used as a screening tool, to verify the clinical diagnosis of hyper and hypothyroidism, to assess adequacy of medical treatment, and in the follow up of differentiated thyroid cancer. A small fraction of T4 and T3 are present in the circulation in free or unbound form but only the free thyroid hormone fractions (FT4 and FT3) can enter cells and employ their physiological actions. Most thyroid function tests are performed on serum and are based on automated assays: currently total T4 and T3 (TT4 and TT3) concentrations are measured by competitive immunoassay methods employing immunofluorescence or chemiluminescence, but since several conditions (drugs, pregnancy, nonthyroidal illness, genetic alterations) determine binding protein abnormalities, the measurements of TT4 and TT3 as stand-alone thyroid tests is limited. The assessment of FT4 and FT3 avoids most artifacts due to abnormalities of binding proteins but is technically challenging; direct and indirect methods of measurement have been developed, and their advantages and pitfalls are discussed in this chapter. The sensitivity of the latest generation of TSH assays rarely results in false or artifactual measurements. From a clinical perspective, serum TSH is the first-line test for detecting thyroid dysfunction in ambulatory patients while FT3 measurement is usually unnecessary, since FT4 together with TSH is adequate to diagnose both overt and subclinical hypo- and hyperthyroidism. The measurement of serum thyroglobulin (Tg) in the context of the management of differentiated thyroid carcinoma and other dynamic testing and indicators of thyroid status such as urinary iodine, radioactive iodine uptake, and perchlorate discharge test are also discussed in this chapter.


Triiodothyronine (T3) Thyroxine (T4) TT4 TT3 Thyroid hormone-binding proteins FT4 measurement FT3 Measurement Free thyroid hormone tests Serum thyrotropin (TSH) Thyrotropin-releasing hormone (TRH) stimulation test Thyroglobulin (Tg) measurement Urinary iodine Perchlorate discharge test Equilibrium dialysis Ultrafiltration Free thyroid hormone index (FT4I) Radioimmunoassays (RIAs) Immunoradiometric assay Immunoenzymatic assay Chemiluminescent assay “Hook” effect Tg antibody interference Familial dysalbuminemic hyperthyroxinemia Transthyretin-associated hyperthyroxinemia Heterophilic antibodies Interference TSH reflex 


  1. Alexander EK, Pearce EN, Brent GA, Brown RS, Chen H, Dosiou C, Grobman W, Laurberg P, Lazarus JH, Mandel SJ, Peeters R, Sullivan S. 2017 guidelines of the American Thyroid Association for the diagnosis and management of thyroid disease during pregnancy and the postpartum. Thyroid. 2017;27(3):315–89.CrossRefPubMedGoogle Scholar
  2. Anderson MS, Bowers CY, Kastin AJ, Schalch DS, Schally AV, Snyder PJ, Utiger RD, Wilber JF, Wise AJ. Synthetic thyrotropin-releasing hormone. A potent stimulator of thyrotropin secretion in man. N Engl J Med. 1971;285(23):1279–83.CrossRefPubMedGoogle Scholar
  3. Baloch Z, Carayon P, Conte-Devolx B, Demers LM, Feldt-Rasmussen U, Henry JF, LiVosli VA, Niccoli-Sire P, John R, Ruf J, Smyth PP, Spencer CA, Stockigt JR. Laboratory medicine practice guidelines: laboratory support for the diagnosis and monitoring of thyroid disease. Thyroid. 2003;13:1–126.CrossRefGoogle Scholar
  4. Bartalena L, Robbins J. Thyroid hormone transport proteins. Clin Lab Med. 1993;3:583–98.CrossRefGoogle Scholar
  5. Bartalena L, Bogazzi F, Brogioni S, Burelli A, Scarcello G, Martino E. Measurement of serum free thyroid hormone concentrations: an essential tool for the diagnosis of thyroid dysfunction. Horm Res. 1996;45(3–5):142–7.CrossRefPubMedGoogle Scholar
  6. Brabant G, Brabant A, Ranft U, Ocran K, Köhrle J, Hesch RD, von zur Mühlen A. Circadian and pulsatile thyrotropin secretion in euthyroid man under the influence of thyroid hormone and glucocorticoid administration. J Clin Endocrinol Metab. 1987;65(1):83–8.CrossRefPubMedGoogle Scholar
  7. Brennan MD, Klee GG, Preissner CM, Hay ID. Heterophilic serum antibodies: a cause for falsely elevated serum thyrotropin levels. Mayo Clin Proc. 1987;62(10):894–8.CrossRefPubMedGoogle Scholar
  8. Burman KD, Bongiovanni R, Garis RK, Wartofsky L, Boehm TM. Measurement of serum T4 concentration by high performance liquid chromatography. J Clin Endocrinol Metab. 1981;53(5):909–12.CrossRefPubMedGoogle Scholar
  9. Chopra IJ, Nelson JC, Solomon DH, Beall GN. Production of antibodies specifically binding triiodothyronine and thyroxine. J Clin Endocrinol Metab. 1971;32(3):299–308.CrossRefPubMedGoogle Scholar
  10. Christofides ND, Sheehan CP. Enhanced chemiluminescence labeled-antibody immunoassay (Amerlite-MAB) for free thyroxine: design, development, and technical validation. Clin Chem. 1995;41(1):17–23.PubMedGoogle Scholar
  11. Clarke NJ, Zhang Y, Reitz RE. A novel mass spectrometry-based assay for the accurate measurement of thyroglobulin from patient samples containing antithyroglobulin autoantibodies. J Investig Med. 2012;60(8):1157–63.CrossRefPubMedGoogle Scholar
  12. Costongs GM, van Oers RJ, Leerkes B, Janson PC. Evaluation of the DPC IMMULITE random access immunoassay analyser. Eur J Clin Chem Clin Biochem. 1995;33(11):887–92.PubMedGoogle Scholar
  13. Despres N, Grant AM. Antibody interference in thyroid assays: a potential for clinical misinformation. Clin Chem. 1998;44(3):440–54.PubMedGoogle Scholar
  14. Dufour DR. Laboratory tests of thyroid function: uses and limitations. Endocrinol Metab Clin North Am. 2007;36:155–69.CrossRefGoogle Scholar
  15. Ekins R. Measurement of free hormones in blood. Endocr Rev. 1990;11(1):5–46.CrossRefPubMedGoogle Scholar
  16. Faix JD. Principles and pitfalls of free hormone measurements. Best Pract Res Clin Endocrinol Metab. 2013;27(5):631–45.CrossRefPubMedGoogle Scholar
  17. Faix JD, Miller WG. Progress in standardizing and harmonizing thyroid function tests. Am J Clin Nutr. 2016;104(Suppl 3):913S–7S.CrossRefPubMedPubMedCentralGoogle Scholar
  18. Francis Z, Schlumberger M. Serum thyroglobulin determination in thyroid cancer patients. Best Pract Res Clin Endocrinol Metab. 2008;22(6):1039–46.CrossRefPubMedGoogle Scholar
  19. Garber JR, Cobin RH, Gharib H, Hennessey JV, Klein I, Mechanick JI, Pessah-Pollack R, Singer PA, Woeber KA, American Association of Clinical Endocrinologists, American Thyroid Association Taskforce on Hypothyroidism in Adults. Clinical practice guidelines for hypothyroidism in adults: cosponsored by the American Association of Clinical Endocrinologists and the American Thyroid Association. Thyroid. 2012;22(12):1200–35.CrossRefPubMedGoogle Scholar
  20. Glinoer D. The regulation of thyroid function in pregnancy: pathways of endocrine adaptation from physiology to pathology. Endocr Rev. 1997;18:404–33.CrossRefPubMedGoogle Scholar
  21. Haugen BR, Alexander EK, Bible KC, Doherty GM, Mandel SJ, Nikiforov YE, Pacini F, Randolph GW, Sawka AM, Schlumberger M, Schuff KG, Sherman SI, Sosa JA, Steward DL, Tuttle RM, Wartofsky L. 2015 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(1):1–133.CrossRefPubMedPubMedCentralGoogle Scholar
  22. Hollander CS, Mitsuma T, Shenkman L, Woolf P, Gershengorn MC. Thyrotropin-releasing hormone: evidence for thyroid response to intravenous injection in man. Science. 1972;175(4018):209–10.CrossRefPubMedGoogle Scholar
  23. Hollowell JG, Staehling NW, Flanders WD, et al. Serum TSH, T(4), and thyroid antibodies in the United States population (1988 to 1994): National Health and Nutrition Examination Survey (NHANES III). J Clin Endocrinol Metab. 2002;87:489–99.CrossRefPubMedGoogle Scholar
  24. Hoofnagle AN, Roth MY. Improving the measurement of serum thyroglobulin with mass spectrometry. J Clin Endocrinol Metab. 2013;98(4):1343–52.CrossRefPubMedPubMedCentralGoogle Scholar
  25. Kundra P, Burman KD. The effect of medications on thyroid function tests. Med Clin North Amer. 2012;96:283–95.CrossRefGoogle Scholar
  26. Kushnir MM, Rockwood AL, Roberts WL, Abraham D, Hoofnagle AN, Meikle AW. Measurement of thyroglobulin by liquid chromatography-tandem mass spectrometry in serum and plasma in the presence of antithyroglobulin autoantibodies. Clin Chem. 2013;59(6):982–90.CrossRefPubMedPubMedCentralGoogle Scholar
  27. Letellier M, Lévesque A, Daigle F, Grant A. Performance evaluation of automated immunoassays on the Technicon Immuno 1 system. Clin Chem. 1996;42(10):1695–701.PubMedGoogle Scholar
  28. Li Calzi L, Benvenga S, Battiato S, Santini F, Trimarchi F. Autoantibodies to thyroxin and triiodothyronine in the immunoglobulin G fraction of serum. Clin Chem. 1988;34(12):2561–2.PubMedGoogle Scholar
  29. Mariotti S, Martino E, Cupini C, Lari R, Giani C, Baschieri L, Pinchera A. Low serum thyroglobulin as a clue to the diagnosis of thyrotoxicosis factitia. N Engl J Med. 1982;307(7):410–2.CrossRefPubMedGoogle Scholar
  30. Masago A, Ueda Y, Kanai H, Nagai H, Umemura S. Pituitary apoplexy after pituitary function test: a report of two cases and review of the literature. Surg Neurol. 1995;43(2):158–64.CrossRefPubMedGoogle Scholar
  31. Nelson JC, Tomei RT. Direct determination of free thyroxin in undiluted serum by equilibrium dialysis/radioimmunoassay. Clin Chem. 1988;34(9):1737–44.PubMedGoogle Scholar
  32. Nuutila P, Koskinen P, Irjala K, Linko L, Kaihola HL, Eskola JU, Erkkola R, Seppälä P, Viikari J. Two new two-step immunoassays for free thyroxin evaluated: solid-phase radioimmunoassay and time-resolved fluoroimmunoassay. Clin Chem. 1990;36(7):1355–60.PubMedGoogle Scholar
  33. Pappa T, Ferrara AM, Refetoff S. Inherited defects of Thyroid hormone binding proteins. Best Pract Res Clin Endocrinol Metab. 2015;29(5):735–47.CrossRefPubMedPubMedCentralGoogle Scholar
  34. Persani L, Terzolo M, Asteria C, Orlandi F, Angeli A, BeckPeccoz P. Circadian variations of thyrotropin bioactivity in normal subjects and patients with primary hypothyroidism. J Clin Endocrinol Metab. 1995;80:2722–8.PubMedGoogle Scholar
  35. Pierce JG. Eli Lilly lecture. The subunits of pituitary thyrotropin – their relationship to other glycoprotein hormones. Endocrinology. 1971;89(6):1331–44.CrossRefPubMedGoogle Scholar
  36. Quimby EH, Feitelberg S, Gross W. Radioactive nuclides in medicine and biology. 3rd ed. Philadelfia: Lea & Febiger; 1970.Google Scholar
  37. Refetoff S, Lever EG. The value of serum thyroglobulin measurement in clinical practice. JAMA. 1983;250(17):2352–7.CrossRefPubMedGoogle Scholar
  38. Ross DS, Burch HB, Cooper DS, Greenlee MC, Laurberg P, Maia AL, Rivkees S, Samuels M, Sosa JA, Stan MN, Walter M. 2016 American Thyroid Association guidelines for diagnosis and management of hyperthyroidism and other causes of thyrotoxicosis. Thyroid. 2016;26(10):1343–421.CrossRefPubMedGoogle Scholar
  39. Shelor CP, Dasgupta PK. Review of analytical methods for the quantification of iodine in complex matrices. Anal Chim Acta. 2011;702(1):16–36.CrossRefPubMedGoogle Scholar
  40. Shenkman L, Mitsuma T, Suphavai A, Hollander CS. Triiodothyronine and thyroid-stimulating hormone response to thyrotrophin-releasing hormone. A new test of thyroidal and pituitary reserve. Lancet. 1972;1(7742):111–2.CrossRefPubMedGoogle Scholar
  41. Smallridge RC, De Keyser FM, Van Herle AJ, Butkus NE, Wartofsky L. Thyroid iodine content and serum thyroglobulin: cues to the natural history of destruction-induced thyroiditis. J Clin Endocrinol Metab. 1986;62(6):1213–9.CrossRefPubMedGoogle Scholar
  42. Smith TJ, Hegedüs L. Graves’ Disease. New England J Med. 2016;375:1552–66.CrossRefGoogle Scholar
  43. Soldin OP, Soldin SJ. Thyroid hormone testing by tandem mass spectrometry. Clin Biochem. 2011;44:89–94.CrossRefPubMedGoogle Scholar
  44. Spencer C. Clinical utility of thyroglobulin antibody (TgAb) measurements for patients with differentiated thyroid cancers (DTC). J Clin Endocrinol Metab. 2011;96:3615–27.CrossRefPubMedGoogle Scholar
  45. Spencer CA, Lopresti JS. Measuring thyroglobulin and thyroglobulin autoantibody in patients with differentiated thyroid cancer. Nat Clin Pract Endocrinol Metab. 2008;4(4):223–33.CrossRefPubMedGoogle Scholar
  46. Spencer CA, Takeuchi M, Kazarosyan M. Current status and performance goals for serum thyrotropin (TSH) assays. Clin Chem. 1996a;42(1):140–5.PubMedGoogle Scholar
  47. Spencer CA, Takeuchi M, Kazarosyan M. Current status and performance goals for serum thyroglobulin assays. Clin Chem. 1996b;42(1):164–73.PubMedGoogle Scholar
  48. Spencer CA, Takeuchi M, Kazarosyan M, Wang CC, Guttler RB, Singer PA, Fatemi S, LoPresti JS, Nicoloff JT. Serum thyroglobulin autoantibodies: prevalence, influence on serum thyroglobulin measurement, and prognostic significance in patients with differentiated thyroid carcinoma. J Clin Endocrinol Metab. 1998;83(4):1121–7.PubMedGoogle Scholar
  49. Stanojevic M, Savin S, Cvejic D, Dukic A, Zivancevic SS. Correlation of thyroglobulin concentrations measured by radioimmunoassay and immunoradiometric assay and the influence of thyroglobulin antibody. J Immunoassay Immunochem. 2009;30:197–207.CrossRefPubMedGoogle Scholar
  50. Stockigt J. Assessment of thyroid function: towards an integrated laboratory-clinical approach. Clin Biochem Rev. 2003;24(4):109–22.PubMedPubMedCentralGoogle Scholar
  51. Stockigt JR, Lim CF. Medications that distort in vitro tests of thyroid function, with particular reference to estimates of serum free thyroxine. Best Pract Res Clin Endocrinol Metab. 2009;23(6):753–67.CrossRefPubMedGoogle Scholar
  52. Tai SS, Sniegoski LT, Welch MJ. Candidate reference method for total thyroxine in human serum: use of isotope-dilution liquid chromatography-mass spectrometry with electrospray ionization. Clin Chem. 2002;48(4):637–42.PubMedGoogle Scholar
  53. Thienpont LM, De Brabandere VI, Stöckl D, De Leenheer AP. Development of a new method for the determination of thyroxine in serum based on isotope dilution gas chromatography mass spectrometry. Biol Mass Spectrom. 1994;23(8):475–82.CrossRefPubMedGoogle Scholar
  54. Thienpont LM, Van Uytfanghe K, Van Houcke S, IFCC Working Group for Standardization of Thyroid Function Tests (WG-STFT). Standardization activities in the field of thyroid function tests: a status report. Clin Chem Lab Med. 2010;48(11):1577–83.CrossRefPubMedGoogle Scholar
  55. Thienpont LM, Van Uytfanghe K, Poppe K, Velkeniers B. Determination of free thyroid hormones. Best Pract Res Clin Endocrinol Metab. 2013;27(5):689–700.CrossRefPubMedGoogle Scholar
  56. Van Herle AJ, Uller RP, Matthews NI, Brown J. Radioimmunoassay for measurement of thyroglobulin in human serum. J Clin Invest. 1973;52(6):1320–7.CrossRefPubMedPubMedCentralGoogle Scholar
  57. Wartofsky L, Dickey RA. The evidence for a narrower thyrotropin reference range is compelling. J Clin Endocrinol Metab. 2005;90(9):5483–8.CrossRefPubMedGoogle Scholar
  58. Wilkins TA, Midgley JE, Barron N. Comprehensive study of a thyroxin-analog-based assay for free thyroxin (“Amerlex FT4”). Clin Chem. 1985;31(10):1644–53.PubMedGoogle Scholar

Copyright information

© Springer International Publishing AG, part of Springer Nature 2018

Authors and Affiliations

  • Giovanni Ceccarini
    • 1
  • Ferruccio Santini
    • 1
  • Paolo Vitti
    • 2
    Email author
  1. 1.Endocrine UnitUniversity Hospital of PisaPisaItaly
  2. 2.Department of EndocrinologyUniversity of PisaPisaItaly

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