|Year : 2016 | Volume
| Issue : 4 | Page : 430-436
Laboratory evaluation of thyroid function: Dilemmas and pitfalls
MK Garg1, Namita Mahalle2, K.V. S. Hari Kumar3
1 Department of Endocrinology, Military Hospital, Shillong, Meghalaya, India
2 Department of Biochemistry, Deenanath Mangeshkar Hospital and Research Center, Erandawane, Pune, Maharashtra, India
3 Department of Endocrinology, CH WC, Chandimandir, Panchkula, Haryana, India
|Date of Web Publication||12-Jul-2016|
K.V. S. Hari Kumar
Department of Endocrinology, CH WC, Chandimandir, Panchkula, Haryana
Source of Support: None, Conflict of Interest: None
Among all endocrine disorders, thyroid dysfunction is possibly most common endocrine disorder barring obesity. This implies that thyroid function tests (TFT) are routinely ordered for laboratory test for its evaluation. Furthermore, recently laboratory values of thyroid function, thyroid-stimulating hormone (TSH), free thyroxine (FT4), and free triiodothyronine (FT3), have gathered importance in view of rapidly changing cut-offs for treatment of thyroid disorders during pregnancy. Most of the times, interpretation of TFT is easy, indicating euthyroidism (normal FT4 and TSH), hypothyroidism (low FT4 or FT3 with high TSH), or thyrotoxicosis (high FT4 or FT3 with low TSH). However, the normal ranges reflect two standard deviations around the mean. Hence, 2.5% of the population may show minor abnormalities on both side of normal range in spite of being euthyroid. Sometimes interpretation becomes difficult when there is an alteration in relation between thyroid hormones and TSH. These pitfalls in investigations will cause dilemma in physicians and patients mind alike. Problems in hormonal evaluation can be preanalytical, analytical, and postanalytical. In an ambulatory patient, TFTs have limited preanalytical interferences such as age, pregnancy, medications, genetic mutations, systemic diseases, and critical illnesses. Analytical errors occur due to heterophile antibodies and macro-TSH. Postanalytical errors include wrong entry of the result, mistakes in the units of the parameter checked and failure to identify the normal data.
Keywords: Hypothyroidism, thyroid function tests, thyroxine, triiodothyronine
|How to cite this article:|
Garg M K, Mahalle N, Hari Kumar K. Laboratory evaluation of thyroid function: Dilemmas and pitfalls. Med J DY Patil Univ 2016;9:430-6
| Introduction|| |
Among all endocrine disorders, thyroid dysfunction is possibly most common endocrine disorder barring obesity. Almost one-third of the world's population lives in areas of iodine deficiency., In recent studies in Indian school children and adults, goiter rate was 15.5% and 9.6%; hypothyroidism was present in 7.3% and 21% and hyperthyroidism in 0.3% and 0.6%, respectively. Prevalence of antithyroid peroxidase antibodies (TPOAb) was around 3.7% among children and adolescents and 13.3% among adults., It is believed that in India alone there are 42 million patients suffering from thyroid disorders. All this data implies that thyroid disorders are commonly present in population, and their evaluation routinely requires laboratory evaluation of thyroid function. In fact, subclinical hypothyroidism and subclinical hyperthyroidism are only laboratory diagnosis where patient usually has no symptoms. Furthermore, recently laboratory values of thyroid function have gathered importance in view of rapidly changing cut-offs for the treatment of thyroid disorders during pregnancy.
Iodine is the main constituent of thyroid hormones, which enters thyroid gland as inorganic or organic form through sodium iodide symporter. In thyroid gland, iodine get organized by peroxidase enzymes at cell colloid interface and forms iodothyronines — monoiodothyronine, diiodothyronine, triiodothyronine (T3), and thyroxin (T4). The thyroid gland is only source of T4, whereas T3 is secreted from thyroid gland and also generated in peripheral tissue by deiodinase enzyme system. The same system also generates an inactive form of T3 — reverse T3. Thyroid hormones circulate as protein-bound form (thyroid binding globulin [TBG], transthyretin [TTR]/prealbumin [TBPA], and albumin) and free form.
Evaluation of thyroid functions consist of anatomical (ultrasonography, scintigraphy); physiological (total T4 [TT4], total T3 [TT3], free thyroxine [FT4], free triiodothyronine [FT3], thyrotropin [thyroid-stimulating hormone [TSH] and thyroglobulin [Tg]); immunological (TPOAb, TSH receptor antibodies [TRAb], and anti Tg antibodies [TgAb]); and pathological assessment (fine needle aspiration).
| Hormonal Tests|| |
Over last 50 years, laboratory evaluation of thyroid disorder has undergone sea of change. There has been gradual improvement in sensitivity and specificity of diagnostic tests for thyroid. In the 1950s, only indirect estimate of the TT4 (free and protein-bound) concentration, using the protein-bound iodide technique was available. The development of competitive immunoassays in the early 1970s and more recently, noncompetitive immunoradiometric assay methods have progressively improved the specificity and sensitivity of thyroid hormone testing. Presently, serum-based tests are available for measuring the concentration of both the total (T4 and T3) and free (FT4 and FT3) thyroid hormones in the circulation by radioimmunoassay and chemiluminescence assay. In addition, measurements of the thyroid hormone binding plasma proteins are available. Improvements in the sensitivity of assays to measure the pituitary TSH from first generation assays to fifth generation assays with sensitivity to detect TSH levels as low as <0.004 mIU/l, allow TSH to be used for detecting both hyper- and hypo-thyroidism. As a result, TRH stimulation test and T3 suppression test have become obsolete. Furthermore, measurement of the thyroid gland precursor protein, Tg as well as the measurement of calcitonin in serum, have become important tumor markers for managing patients with differentiated and medullary thyroid carcinomas (MTCs), respectively. Furthermore, there has been development of more sensitive and specific tests for TPOAb, TgAb, and TRAb. Current thyroid tests are usually performed on serum by either manual or automated methods that employ specific antibodies.
All these advances and high prevalence of thyroid disorders implies that thyroid function tests (TFTs) are performed commonly. Most of the times, interpretation of TFT is easy, indicating euthyroidism (normal FT4 and TSH), hypothyroidism (low FT4 or FT3 with high TSH), or thyrotoxicosis (high FT4 or FT3 with low TSH). However, the normal ranges reflect two standard deviations around the mean. Hence, 2.5% of the population may show minor abnormalities on both side of normal range in spite of being euthyroid. Sometimes interpretation becomes difficult when there is an alteration in relation between thyroid hormones and TSH. These pitfalls in investigations will cause dilemma in physicians and patients mind alike. Variation or errors in hormonal evaluation can be preanalytical, analytical, and postanalytical. Out of these, we will discuss pitfalls in preanalytical and analytical factors.
The normal range of various thyroid hormones is given in [Table 1]. Preanalytical variations are related to age, pregnancy, medications, systemic, and genetic diseases. In an ambulatory patient, TFTs have limited preanalytical interferences [Table 2]. Physiological variables such as age, pregnancy, TSH/FT4 relationship, and biological differences can affect TFTs evaluation. The hypothalamo-pituitary-thyroid unit matures from fetal life until the end of puberty. Both TSH and FT4 concentrations are higher in children, especially in the 1st week of life and throughout the 1st year. Failure to recognize this could lead to missing and/or under-treating cases of congenital hypothyroidism. Age-related normal reference limits should be used for all TFTs. Furthermore, with every decade there is rise of TSH values as well as increased variability of TSH around mean; however, the clinical significance of this is unclear as both raised and suppressed TSH in elderly have been shown to be associated with increased cardiovascular morbidity., During pregnancy owing to the increased estrogen levels mean TBG concentration increases to 2-3 times the prepregnancy level by 20 weeks of gestation. It results in a shift in the TT4 and TT3 reference range to approximately 1.5 times the nonpregnant level by 16 weeks of gestation. Furthermore, during pregnancy there is rise in human chorionic gonadotropin (HCG) levels which cross-react partly with TSH receptor leading to mildly suppressed levels. The peak rise in HCG and nadir in serum TSH level occurs together at about 10-12 weeks of gestation., Thus, higher cut-offs for T4, T3 and lower cut-offs for TSH are suggested during pregnancy, which should be standardized in local laboratory. In patients with unstable thyroid function, such as the first trimester of pregnancy, during the early course of treatment for hypo- or hyper-thyroidism FT4 measurement is a more reliable indicator of thyroid status than TSH. Normally, there is inverse linear correlation between FT4 and TSH but each individual has a genetically determined FT4 set-point for HPT axis.
Another important source of preanalytical variation is medications. Drugs can cause both in vitro as well as in vivo effects on TFTs estimation. Drugs such as estrogen can increase TBG levels leading to falsely high TT4 levels though free thyroid hormone levels and TSH stay normal. Glucocorticoids can lead to suppressed TSH levels and reduced conversion of T4 to T3 leading to lower T3 levels. Metformin and dopamine too suppresses TSH secretion. Propranolol also inhibits deiodinase enzyme leading to lower T3 levels and may be associated with a mild increase in TSH because of low T3 levels. Iodine and iodine-containing drugs such as amiadarone can affect TFTs and cause both hyperthyroidism as well as hypothyroidism. Lithium can also cause both hypothyroidism as well as hyperthyroidism. Drugs such as phenytoin, carbamazepine, furosemide, and heparin may displace free thyroid hormones from TBG leading to elevated free hormone levels. Many drugs increase the metabolism of thyroxin and may increase requirements such as phenytoin, carbamazepine, rifampicin, imatinib, and sunitinib.
Furthermore, patients who are critically sick suffer from nonthyroidal illness syndrome (NTIS) also previously called sick euthyroid syndrome, which is commonly associated with low T3 levels and as clinical condition worsens even T4 levels also go down. TSH in the absence of dopamine or glucocorticoid administration is more reliable test for NTIS patients. Diagnosis of NTIS should be based on the clinical condition of the patient if patient is not critically sick low FT4 levels are unlikely, and one may be dealing with hypothyroid case. In nephrotic syndrome, there is increases loss of T4 which is bound to albumin and TBG, may lead to low T4 with normal or raised TSH.
Noncompliant patients may exhibit discordant serum TSH and FT4 values (high TSH/high FT4) because of persistent disequilibrium between FT4 and TSH. Noncompliant patients may consume thyroxin intermittently leading to normal or near normal T4, T3 levels but persistently raised TSH values. Noncompliance can be evaluated by simultaneous oral and intravenous administration of the thyroxin labeled with two different iodine isotope tracers. Normally, approximately 80% of the T4 and 95% of the T3 administered orally are absorbed. Patients with absorption defects due to interfering substances such as cholestyramine, calcium, iron, or small bowel bypass or resection, celiac disease, helicobacter pylori infection, atrophic gastritis, achlorhydria, and isolated thyroxin absorption defect can be evaluated by the administration of a single oral dose of 100 µg of L-T3 or 1 mg of levothyroxine (L-T4), followed by their measurement in blood sampled at various intervals and values plotted on graph and compared.
In addition to pregnancy and neonatal period, increased TBG levels due to systemic diseases such as acute intermittent porphyria, acute hepatitis, biliary cirrhosis, HIV infection, and hereditary TBG excess will lead to increased TT4 and normal TSH levels. A genetic mutation in albumin (familial dysalbuminemic hyperthyroxinemia) and TTR (TTR associated hyperthyroxinemia) which avidly bind to T4 - also alters the relation between T4 and TSH similarly. In contrast, low T4 and normal TSH can be found in conditions associated with low TBG levels due to drugs or genetic mutation in TBG gene. Inappropriately, normal TSH with low T4 is also a feature of central hypothyroidism.
Increased levels of thyroid hormone and TSH are commonly found in patients with hypothyroidism, who is noncompliant with therapy and consume medicine just before the test. However, similar results can be observed in rare conditions such as thyroid hormone resistance syndrome and TSH-secreting pituitary tumor. These two conditions can be differentiated by clinical feature, pituitary imaging, measurement of serum α-subunit, sex hormone binding globulin, measurement of thyroid hormones in relatives, and genetic testing.
A rare condition Allen Herndon Dudley syndrome due to mutation in monocarboxylate transporter-8 gene, which is required for thyroid hormone transportation into various cells, leads to raised T3, low T4, and normal or elevated TSH levels. A similar hormonal profile with raised T3, low T4, and normal TSH levels has been reported in patients with resistance to thyroid hormone due to mutation in thyroid receptor-α. Rarely, serum and urinary measurement of monoiodothyronine and diiodothyronine is used to detect rare genetic condition - iodotyrosine deiodinase deficiency, which can also have raised T4, normal/low T3, and normal TSH levels.
For estimation of TFTs, serum is preferred specimen and ideally whole blood samples should be allowed to clot for more than 30 min and then centrifuged and separated. Serum can be stored at 4-8°C for up to 7 days. Storage at −20°C is recommended if the assay is to be delayed for more than 1 week. Collection of serum in barrier gel tubes does not affect the results of most TFTs. Generally, thyroid hormones are quite stable whether stored at room temperature, in refrigerator or frozen. Furthermore, TSH and T4 in dried whole blood spots used to screen for neonatal hypothyroidism are also stable for months when stored with a desiccant. Similarly, hemolysis, hyperlipidemia, and hyperbilirubinemia do not produce interference in hormone estimation by different assays.
Equilibrium dialysis is gold standard for measurement of free hormone assay. However, this assay is complex and not widely available. Commonly used methods to measure free hormones are one-step or two-step method. Two-step method is more reliable than one-step method. Hence, measurement of free thyroid hormone may vary from assay methods. It is advisable to generate normative data for free thyroid hormone in local laboratory with particular assay method. The thyroid hormone estimation, in most of the laboratories currently, is done by the chemiluminescence method. This modality has the least interference and the normative data used from the population is derived using the same test method.
Another important issue is the presence of heterophile antibodies in serum which can lead to falsely high or low TFTs. Heterophile antibodies are antibodies induced by external antigens (heterophile antigens) that cross-react with self-antigens. The best-known heterophile antibodies are human anti-mouse antibodies (HAMA), which can react with the mouse monoclonal antibodies that are used in many immunometric assays, such as in TSH estimation where if they are present they may lead to erroneously high or low values of TSH. Manufacturers are currently employing various approaches to deal with the HAMA issue with varying degrees of success, including the use of chimeric antibody combinations and blocking agents to neutralize the effects of HAMA on their methods.,
Macro-TSH is a rare condition where serum contains antibodies against TSH (anti-TSH Ig) which binds to TSH and neutralizes its activity, but leaves open epitope to interact with assay antibodies leading to spuriously high value. This can be detected by:
- Linearity test: Serum is tested with serial dilution. In normal subjects, it shows a linear pattern (decreasing TSH concentration with increasing dilution), but in the presence of heterophile antibodies or macro-TSH it shows nonlinear pattern (increase in TSH concentration with dilution).
- Polyethylene glycol (PEG) precipitation: Same amount of PEG is added to serum of patient and normal subject. It is centrifuged, and TSH is measured again. In normal subjects, the decrease in TSH is proportional to the dilution, whereas in patients TSH levels decrease drastically due to the precipitation of antibodies.
- TSH sequestration test: This is done to detect the presence of anti-TSH antibodies. Patient's serum is mixed with serum of hypothyroid patient with known high TSH in 1:1 ration and incubated for 4 h. TSH is re-measured. In the absence of anti-TSH antibodies, the TSH values will be average of these two. However, in presence of anti-TSH antibodies this value will decrease, because of sequestration of TSH by anti-TSH antibodies.
- Gel filtration chromatography: High molecular weight of anti-TSH antibodies can be demonstrated by gel filtration chromatography revealing TSH peak fraction belongs to molecular weight of immunoglobulin.
Postanalytic errors are relatively less in frequency when compared with the preanalytical and analytical variations. This is subdivided into the intra-laboratory (procedures done after conducting the test) and departmental (analysis of the result by the user). The sequential steps in the postanalytic phase includes generation of the report, delivery of the report to the concerned department, review of the result by the treating doctor, interpretation of the result, and carrying out appropriate therapy based on the test results. The errors that may happen during postanalytical phase have been summarized in the box no 1.
Postanalytical errors in interpretation of thyroid function tests
- Intra-laboratory phase:
- Wrongful data entry — Reporting a correct value in different patient.
- Wrong entry of the units — mg/L instead of mg/dL.
- Data entry errors — 20 instead of 2.0.
- Normal range not derived from the local population.
- Postlaboratory phase:
- Delayed access of the test results due to excessive turnaround time.
- Underutilization of the test results for lack of understanding.
- Failure to interpret correctly (high T4 is normal in pregnancy).
- Failure to understand the physiology (delay in TSH rise after antithyroid drugs).
| Other Tests|| |
Calcitonin is secreted by “C” cells of thyroid. Currently, it is measured with chemiluminescent immunometric assays, which is highly specific and not affected by interfering substance such as procalcitonin, which is raised in many physiological and pathological conditions. Calcitonin level is affected by age and sex. Serum calcitonin levels are <40 ng/L in children <6 months of age, <15 ng/L in children between 6 months and 3 years, and <10 ng/L above 3 years and adults. It is raised in MTC. It is used to screen multiple endocrine neoplasia 2, planning treatment, and follow-up after treatment for MTC. Mild elevation of calcitonin has been observed in 3-10% normal adults, C-cell hyperplasia, autoimmune thyroiditis, chronic renal failure, and mastocytosis. Elevated calcitonin levels may also occur from nonthyroidal neuroendocrine neoplasms and heterophile antibodies.
Tg is a protein exclusively produced by thyroid follicular cells. Most laboratories currently use immunometric assays to measure serum Tg. Immunometric assays are prone to interference from Tg autoantibodies, which commonly cause falsely low serum Tg. Serum Tg levels are elevated in most of thyroid diseases and are insensitive in the diagnosis of thyroid dysfunction. However, its utility is to detect functioning thyroid tissue after total thyroidectomy for thyroid cancer and the diagnosis of thyrotoxicosis fictitia. Fecal thyroxin estimation can also help in the diagnosis of thyrotoxicosis fictitia. Tg levels basal and stimulated should be undetectable after treatment for thyroid cancer. Serum Tg levels <0.5 ng/ml after recombinant TSH stimulation commensurate with 99% chances of disease-free state. However, poorly differentiated thyroid cancer may still exist with low serum Tg levels. A cut-off of <1.0 ng/ml levels of serum Tg on thyroxin suppressive dose and >2.0 ng/ml after TSH stimulation has been suggested for persistent disease.
Measurement of TPOAb will be useful in the diagnosis of autoimmune thyroid disease. Measurement of anti-TRAb can be useful to diagnosis in suspected thyrotoxicosis during pregnancy to differentiate gestational thyrotoxicosis and Grave's disease to predict neonatal thyrotoxicosis in mother's with present or past Grave's disease. Anti-TgAb is measured in all patients with thyroid cancer to avoid misinterpretation of high Tg levels during follow-up.
| Conclusion|| |
Usually, interpretation of TFTs is straight forward, but knowledge of commonly occurring pitfalls is essential to avoid misinterpretation and mismanagement of the case. For example, only emphasis on raised TSH may lead to the prescription of thyroxin in a case of resistance of thyroid hormone, and avoidance of thyroxin in patients treated with radioiodine with low TSH and T4. It is prudent to interpret TFT in relation to the clinical condition of patients than merely treating the laboratory report.
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Conflicts of interest
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[Table 1], [Table 2]