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Review Article
Introduction This is an open access article distributed under the terms of the
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Among all endocrine disorders, thyroid dysfunction License, which allows others to remix, tweak, and build upon the
is possibly most common endocrine disorder barring work non-commercially, as long as the author is credited and the
new creations are licensed under the identical terms.
obesity. Almost one-third of the world’s population
lives in areas of iodine deficiency.[1,2] In recent studies in For reprints contact: reprints@medknow.com
Indian school children and adults, goiter rate was 15.5% How to cite this article: Garg MK, Mahalle N, Hari Kumar K.
and 9.6%; hypothyroidism was present in 7.3% and 21% Laboratory evaluation of thyroid function: Dilemmas and pitfalls. Med
and hyperthyroidism in 0.3% and 0.6%, respectively. J DY Patil Univ 2016;9:430-6.
430 © 2016 Medical Journal of Dr. D.Y. Patil University | Published by Wolters Kluwer - Medknow
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monoiodothyronine, diiodothyronine, triiodothyronine All these advances and high prevalence of thyroid disorders
(T3), and thyroxin (T4). The thyroid gland is only source implies that thyroid function tests (TFTs) are performed
of T4, whereas T3 is secreted from thyroid gland and commonly. Most of the times, interpretation of TFT is
also generated in peripheral tissue by deiodinase enzyme easy, indicating euthyroidism (normal FT4 and TSH),
system. The same system also generates an inactive hypothyroidism (low FT4 or FT3 with high TSH), or
form of T3 — reverse T3. Thyroid hormones circulate thyrotoxicosis (high FT4 or FT3 with low TSH). However,
as protein-bound form (thyroid binding globulin [TBG], the normal ranges reflect two standard deviations around
transthyretin [TTR]/prealbumin [TBPA], and albumin) the mean. Hence, 2.5% of the population may show minor
and free form.[2] abnormalities on both side of normal range in spite of being
euthyroid. Sometimes interpretation becomes difficult when
Evaluation of thyroid functions consist of anatomical there is an alteration in relation between thyroid hormones
(ultrasonography, scintigraphy); physiological (total and TSH. These pitfalls in investigations will cause dilemma
T4 [TT4], total T3 [TT3], free thyroxine [FT4], free in physicians and patients mind alike. Variation or errors
triiodothyronine [FT3], thyrotropin [thyroid-stimulating in hormonal evaluation can be preanalytical, analytical,
hormone [TSH]] and thyroglobulin [Tg]); immunological and postanalytical. Out of these, we will discuss pitfalls in
(TPOAb, TSH receptor antibodies [TRAb], and anti Tg preanalytical and analytical factors.
antibodies [TgAb]); and pathological assessment (fine needle
aspiration). Preanalytical variations
The normal range of various thyroid hormones is given
Hormonal Tests in Table 1. Preanalytical variations are related to age,
pregnancy, medications, systemic, and genetic diseases.
Overview
In an ambulatory patient, TFTs have limited preanalytical
Over last 50 years, laboratory evaluation of thyroid disorder interferences [Table 2]. Physiological variables such as
has undergone sea of change. There has been gradual age, pregnancy, TSH/FT4 relationship, and biological
improvement in sensitivity and specificity of diagnostic tests differences can affect TFTs evaluation. The hypothalamo-
for thyroid. In the 1950s, only indirect estimate of the TT4 pituitary-thyroid unit matures from fetal life until the end
(free and protein-bound) concentration, using the protein-
bound iodide technique was available. The development
Table 1: Normal ranges of thyroid function tests
of competitive immunoassays in the early 1970s and more
recently, noncompetitive immunoradiometric assay methods Parameter Normal range
Total T4
have progressively improved the specificity and sensitivity
Adult (µg/dL) 5-12
of thyroid hormone testing. Presently, serum-based tests are Neonate (µg/dL) 6.4-12.6
available for measuring the concentration of both the total Total T3
(T4 and T3) and free (FT4 and FT3) thyroid hormones in the Adult (ng/dL) 70-190
circulation by radioimmunoassay and chemiluminescence Neonate (ng/dL) 100-470
assay.[7] In addition, measurements of the thyroid hormone TSH
Adult (mIU/L) 0.4-4.2
binding plasma proteins are available.[8] Improvements in the
Neonate (mIU/L) 1.0-39.0
sensitivity of assays to measure the pituitary TSH from first
First trimester (mIU/L) 0.1-2.5
generation assays to fifth generation assays with sensitivity Second trimester (mIU/L) 0.2-3
to detect TSH levels as low as <0.004 mIU/l, allow TSH to Third trimester (mIU/L) 0.3-3
be used for detecting both hyper- and hypo-thyroidism. As Free T4
a result, TRH stimulation test and T3 suppression test have Neonate (ng/dL) 0.9-2.2
become obsolete. Furthermore, measurement of the thyroid Adolescents (ng/dL) 0.8-2.0
Adults (ng/dL) 0.8-1.8
gland precursor protein, Tg as well as the measurement of
Free T3
calcitonin in serum, have become important tumor markers
Neonates (pg/mL) 2.4-4.2
for managing patients with differentiated and medullary Adolescents (pg/mL) 2.9-5.1
thyroid carcinomas (MTCs), respectively. Furthermore, Adults (pg/mL) 2.3-4.2
there has been development of more sensitive and specific Thyroglobulin (ng/mL) 5-25
tests for TPOAb, TgAb, and TRAb. Current thyroid tests are TBG (mg/L) 12-30
usually performed on serum by either manual or automated Prealbumin (mg/dL) 15.7-29.6
TSH: Thyroid stimulating hormone, TBG: Thyroid binding globulin, T4: Thyroxin,
methods that employ specific antibodies.[9] T3: Triiodothyronine
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of puberty.[10] Both TSH and FT4 concentrations are higher cut-offs for T4, T3 and lower cut-offs for TSH are suggested
in children, especially in the 1st week of life and throughout during pregnancy, which should be standardized in local
the 1st year.[11] Failure to recognize this could lead to missing laboratory. In patients with unstable thyroid function, such
and/or under-treating cases of congenital hypothyroidism. as the first trimester of pregnancy, during the early course of
Age-related normal reference limits should be used for all treatment for hypo- or hyper-thyroidism FT4 measurement
TFTs.[12] Furthermore, with every decade there is rise of TSH is a more reliable indicator of thyroid status than TSH.
values as well as increased variability of TSH around mean; Normally, there is inverse linear correlation between FT4
however, the clinical significance of this is unclear as both and TSH but each individual has a genetically determined
raised and suppressed TSH in elderly have been shown to FT4 set-point for HPT axis.[18]
be associated with increased cardiovascular morbidity.[13,14]
During pregnancy owing to the increased estrogen levels Another important source of preanalytical variation is
mean TBG concentration increases to 2-3 times the medications.[19] Drugs can cause both in vitro as well as
prepregnancy level by 20 weeks of gestation. It results in a in vivo effects on TFTs estimation. Drugs such as estrogen
shift in the TT4 and TT3 reference range to approximately can increase TBG levels leading to falsely high TT4 levels
1.5 times the nonpregnant level by 16 weeks of gestation.[15] though free thyroid hormone levels and TSH stay normal.
Furthermore, during pregnancy there is rise in human Glucocorticoids can lead to suppressed TSH levels and
chorionic gonadotropin (HCG) levels which cross-react reduced conversion of T4 to T3 leading to lower T3 levels.
partly with TSH receptor leading to mildly suppressed levels. Metformin and dopamine too suppresses TSH secretion.[20]
The peak rise in HCG and nadir in serum TSH level occurs Propranolol also inhibits deiodinase enzyme leading to
together at about 10-12 weeks of gestation.[16,17] Thus, higher lower T3 levels and may be associated with a mild increase in
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TSH because of low T3 levels. Iodine and iodine-containing associated with low TBG levels due to drugs or genetic
drugs such as amiadarone can affect TFTs and cause both mutation in TBG gene. Inappropriately, normal TSH with
hyperthyroidism as well as hypothyroidism.[21] Lithium can low T4 is also a feature of central hypothyroidism.[19]
also cause both hypothyroidism as well as hyperthyroidism.[22]
Drugs such as phenytoin, carbamazepine, furosemide, and Increased levels of thyroid hormone and TSH are
heparin may displace free thyroid hormones from TBG commonly found in patients with hypothyroidism, who
leading to elevated free hormone levels. Many drugs increase is noncompliant with therapy and consume medicine just
the metabolism of thyroxin and may increase requirements before the test. However, similar results can be observed
such as phenytoin, carbamazepine, rifampicin, imatinib, in rare conditions such as thyroid hormone resistance
and sunitinib.[19] syndrome and TSH-secreting pituitary tumor. These two
conditions can be differentiated by clinical feature, pituitary
Furthermore, patients who are critically sick suffer from imaging, measurement of serum α-subunit, sex hormone
nonthyroidal illness syndrome (NTIS) also previously called binding globulin, measurement of thyroid hormones in
sick euthyroid syndrome, which is commonly associated relatives, and genetic testing.[26]
with low T3 levels and as clinical condition worsens even
T4 levels also go down.[23] TSH in the absence of dopamine A rare condition Allen Herndon Dudley syndrome due to
or glucocorticoid administration is more reliable test for mutation in monocarboxylate transporter-8 gene, which is
NTIS patients. Diagnosis of NTIS should be based on the required for thyroid hormone transportation into various
clinical condition of the patient if patient is not critically cells, leads to raised T3, low T4, and normal or elevated TSH
sick low FT4 levels are unlikely, and one may be dealing levels. A similar hormonal profile with raised T3, low T4,
with hypothyroid case.[24] In nephrotic syndrome, there is and normal TSH levels has been reported in patients with
increases loss of T4 which is bound to albumin and TBG, resistance to thyroid hormone due to mutation in thyroid
may lead to low T4 with normal or raised TSH.[25] receptor-α.[27] Rarely, serum and urinary measurement of
monoiodothyronine and diiodothyronine is used to detect
Noncompliant patients may exhibit discordant serum TSH rare genetic condition - iodotyrosine deiodinase deficiency,
and FT4 values (high TSH/high FT4) because of persistent which can also have raised T4, normal/low T3, and normal
disequilibrium between FT4 and TSH. Noncompliant TSH levels.[26]
patients may consume thyroxin intermittently leading
to normal or near normal T4, T3 levels but persistently Analytical variations
raised TSH values. Noncompliance can be evaluated by For estimation of TFTs, serum is preferred specimen and
simultaneous oral and intravenous administration of the ideally whole blood samples should be allowed to clot for
thyroxin labeled with two different iodine isotope tracers. more than 30 min and then centrifuged and separated.
Normally, approximately 80% of the T4 and 95% of the T3 Serum can be stored at 4-8°C for up to 7 days. Storage
administered orally are absorbed. Patients with absorption at −20°C is recommended if the assay is to be delayed
defects due to interfering substances such as cholestyramine, for more than 1 week. Collection of serum in barrier gel
calcium, iron, or small bowel bypass or resection, celiac tubes does not affect the results of most TFTs. Generally,
disease, helicobacter pylori infection, atrophic gastritis, thyroid hormones are quite stable whether stored at room
achlorhydria, and isolated thyroxin absorption defect can temperature, in refrigerator or frozen. Furthermore, TSH
be evaluated by the administration of a single oral dose of and T4 in dried whole blood spots used to screen for neonatal
100 µg of L-T3 or 1 mg of levothyroxine (L-T4), followed hypothyroidism are also stable for months when stored
by their measurement in blood sampled at various intervals with a desiccant. Similarly, hemolysis, hyperlipidemia, and
and values plotted on graph and compared.[25] hyperbilirubinemia do not produce interference in hormone
estimation by different assays.[18]
In addition to pregnancy and neonatal period, increased TBG
levels due to systemic diseases such as acute intermittent Equilibrium dialysis is gold standard for measurement of
porphyria, acute hepatitis, biliary cirrhosis, HIV infection, free hormone assay. However, this assay is complex and
and hereditary TBG excess will lead to increased TT4 not widely available. Commonly used methods to measure
and normal TSH levels. A genetic mutation in albumin free hormones are one-step or two-step method. Two-step
(familial dysalbuminemic hyperthyroxinemia) and TTR method is more reliable than one-step method. Hence,
(TTR associated hyperthyroxinemia) which avidly bind to measurement of free thyroid hormone may vary from assay
T4 - also alters the relation between T4 and TSH similarly. In methods. It is advisable to generate normative data for free
contrast, low T4 and normal TSH can be found in conditions thyroid hormone in local laboratory with particular assay
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21. Martino E, Bartalena L, Bogazzi F, Braverman LE. The effects 28. Loh TP, Kao SL, Halsall DJ, Toh SA, Chan E, Ho SC, et al.
of amiodarone on the thyroid. Endocr Rev 2001;22:240-54. Macro-thyrotropin: A case report and review of literature. J Clin
22. Lazarus JH. The effects of lithium therapy on thyroid and Endocrinol Metab 2012;97:1823-8.
thyrotropin-releasing hormone. Thyroid 1998;8:909-13. 29. Plebani M. Errors in clinical laboratories or errors in laboratory
23. Warner MH, Beckett GJ. Mechanisms behind the non-thyroidal medicine? Clin Chem Lab Med 2006;44:750-9.
illness syndrome: An update. J Endocrinol 2010;205:1-13. 30. American Thyroid Association Guidelines Task Force, Kloos RT,
24. Stockigt JR. Guidelines for diagnosis and monitoring of thyroid Eng C, Evans DB, Francis GL, Gagel RF, et al. Medullary
thyroid cancer: Management guidelines of the American Thyroid
disease: Nonthyroidal illness. Clin Chem 1996;42:188-92.
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25. Morris JC. How do you approach the problem of TSH elevation
31. American Thyroid Association (ATA) Guidelines Taskforce on
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Thyroid Nodules and Differentiated Thyroid Cancer, Cooper DS,
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Doherty GM, Haugen BR, Kloos RT, Lee SL, et al. Revised
26. Gurnell M, Halsall DJ, Chatterjee VK. What should be done American Thyroid Association management guidelines for
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27. van Mullem AA, Chrysis D, Eythimiadou A, Chroni E, 32. Bahn RS, Burch HB, Cooper DS, Garber JR, Greenlee MC,
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Commentary
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