Thyroid function tests
Thyroid function tests are a series of blood tests designed to evaluate the performance of the thyroid gland, a butterfly-shaped endocrine organ in the neck that produces hormones regulating metabolism, energy levels, and growth.[1] These tests primarily measure levels of thyroid-stimulating hormone (TSH) from the pituitary gland and thyroid hormones such as thyroxine (T4) and triiodothyronine (T3), helping to diagnose conditions like hypothyroidism (underactive thyroid) and hyperthyroidism (overactive thyroid).[2] They are essential for screening asymptomatic individuals, confirming clinical suspicions of thyroid dysfunction, and monitoring treatment efficacy in patients with known disorders.[3] The cornerstone of thyroid function testing is the TSH assay, which serves as an initial screening tool due to its sensitivity in detecting subtle changes in thyroid activity.[1] Elevated TSH levels typically indicate primary hypothyroidism, where the thyroid fails to produce sufficient hormones, while low TSH suggests hyperthyroidism or, less commonly, pituitary dysfunction.[2] If TSH is abnormal, further tests measure free T4 (the unbound, active form of thyroxine) to assess hormone availability; low free T4 with high TSH confirms hypothyroidism, whereas high free T4 with low TSH points to hyperthyroidism.[1] Total T4 and total T3 tests quantify both bound and unbound hormones but are less precise due to influences from binding proteins, making free hormone measurements preferable in most cases.[2] Additional tests, such as thyroid antibody assays, identify autoimmune causes of dysfunction, including anti-thyroid peroxidase antibodies in Hashimoto's thyroiditis or thyroid-stimulating immunoglobulins in Graves' disease.[1] T3 levels are particularly useful for confirming hyperthyroidism when T4 is normal, as T3 is the more potent hormone.[2] Non-blood tests like the radioactive iodine uptake (RAIU) scan complement these by measuring the thyroid's iodine absorption, aiding differentiation between causes of hyperthyroidism such as Graves' disease (high uptake) or thyroiditis (low uptake).[2] Factors like medications (e.g., biotin supplements, estrogens) or pregnancy can interfere with results, necessitating careful interpretation by healthcare providers.[3] Overall, these tests enable precise diagnosis and management, with normal ranges varying by age, sex, and laboratory standards.[1]Fundamentals of Thyroid Function Tests
Purpose and Clinical Indications
Thyroid function tests are a group of blood tests designed to measure levels of thyroid hormones, such as thyroxine (T4) and triiodothyronine (T3), along with thyroid-stimulating hormone (TSH), to assess the thyroid gland's production and regulation of these hormones essential for metabolism, growth, and development.[4] These tests evaluate the thyroid's overall activity and help distinguish between normal (euthyroid) states and various thyroid disorders.[5] The primary clinical indications for ordering thyroid function tests include screening for hypothyroidism and hyperthyroidism in individuals with suggestive symptoms like fatigue, weight changes, or palpitations, or in those with risk factors such as family history or autoimmune conditions.[6] They are also used to monitor treatment efficacy in patients on thyroid hormone replacement therapy, such as levothyroxine for hypothyroidism, ensuring hormone levels remain within therapeutic ranges.[7] Additionally, these tests aid in evaluating structural abnormalities like goiters or thyroid nodules to determine if they are associated with functional changes, and in assessing pituitary function for secondary hypothyroidism where TSH production is impaired.[8] Common disorders linked to these indications encompass primary hypothyroidism (e.g., Hashimoto's thyroiditis), secondary hypothyroidism due to pituitary issues, hyperthyroidism from Graves' disease, and subacute thyroiditis.[4] According to guidelines from the American Thyroid Association (ATA), TSH measurement serves as the first-line screening test for thyroid dysfunction in outpatient settings due to its high sensitivity for detecting early changes in thyroid hormone levels.[2] The ATA recommends routine screening beginning at age 35 years and every five years thereafter for adults at average risk, with more frequent testing in high-risk groups such as pregnant individuals or those with a personal history of thyroid disease.[9] Similar recommendations from the British Thyroid Association emphasize TSH combined with free T4 (FT4) for initial evaluation in suspected cases.[4] Despite their utility, thyroid function tests have limitations in that they primarily indicate the presence of dysfunction but do not identify the underlying etiology, necessitating complementary approaches like thyroid ultrasound, antibody testing, or biopsy for comprehensive diagnosis.[4] Factors such as non-thyroidal illness, medications, or pregnancy can influence results, requiring clinical correlation.[7]Laboratory Methods and Assay Principles
Thyroid function tests rely on immunoassay techniques to quantify key analytes such as thyroid-stimulating hormone (TSH), thyroxine (T4), and triiodothyronine (T3). These methods have evolved from the historical radioimmunoassay (RIA), which used radiolabeled antigens like iodine-125 for detection, to safer and more sensitive approaches including enzyme-linked immunosorbent assay (ELISA) and chemiluminescent immunoassay (CLIA).[10] ELISA employs enzyme-conjugated antibodies to produce a colorimetric signal, while CLIA generates light through chemiluminescent reactions, enabling automated, high-sensitivity measurements in clinical laboratories.[11] For enhanced precision in free hormone assessment, liquid chromatography-tandem mass spectrometry (LC-MS/MS) is utilized, particularly as a reference method when combined with separation techniques.[12] The principles underlying these assays vary by analyte. Competitive binding assays are standard for total hormones (total T4 and total T3), where endogenous hormone competes with a labeled tracer for limited antibody binding sites, inversely correlating signal intensity with concentration.[10] Free T4 and free T3 measurements involve physical separation of unbound fractions via equilibrium dialysis or ultrafiltration, followed by detection to avoid overestimation due to protein binding influences.[10] TSH assays typically use a two-site sandwich format, in which one antibody captures the antigen and a second labeled antibody detects it, producing a directly proportional signal.[10] Standard units for reporting results include milli-international units per liter (mIU/L) for TSH, micrograms per deciliter (μg/dL) or nanomoles per liter (nmol/L) for total T4 and total T3, and nanograms per deciliter (ng/dL) or picomoles per liter (pmol/L) for free T4 and free T3.[12] Serum is the preferred sample matrix over plasma, as anticoagulants in plasma can alter hormone binding and assay performance.[5] Fasting is not routinely required, and samples maintain stability when processed promptly, with refrigerated storage viable for 24–48 hours and frozen aliquots suitable for extended periods up to months.[10] As of 2025, automation in high-throughput analyzers has revolutionized thyroid testing by integrating CLIA platforms for rapid, simultaneous multi-analyte processing with reduced manual error.[11] The International Federation of Clinical Chemistry (IFCC) continues to drive standardization through its Committee for Standardization of Thyroid Function Tests (C-STFT), implementing reference measurement procedures and calibration protocols to harmonize results across global laboratories and minimize inter-method variability.[12] Assay interferences pose significant challenges to accuracy. Heterophile antibodies, such as human anti-mouse antibodies, can bridge capture and detection reagents in sandwich immunoassays, yielding falsely elevated TSH or thyroglobulin levels; blocking agents are commonly added to mitigate this.[10] Biotin supplementation interferes with streptavidin-biotin detection systems in many CLIA assays, causing erroneous high or low readings for T4, T3, and TSH; strategies include biotin-free assay designs or advising patients to discontinue supplements 24–48 hours prior to testing.[11]| Immunoassay Method | Principle | Key Applications | Advantages |
|---|---|---|---|
| Radioimmunoassay (RIA) | Competitive binding with radiolabeled tracer | Historical use for total T4, total T3, TSH | High sensitivity but limited by radiation safety concerns[5] |
| Enzyme-Linked Immunosorbent Assay (ELISA) | Enzyme-linked antibody producing colorimetric signal | Thyroid autoantibodies, some hormone assays | Cost-effective, non-radioactive[10] |
| Chemiluminescent Immunoassay (CLIA) | Chemiluminescent signal from label-antibody reaction | TSH, total/free T4/T3 in automated systems | High throughput, sensitivity <0.02 mIU/L for TSH[12] |
| Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) | Ionization and mass detection post-chromatographic separation | Free T4/T3 reference measurement | Superior specificity, minimal interference[10] |
Thyroid-Stimulating Hormone (TSH)
Historical Development
The recognition of thyroid-stimulating hormone (TSH), a pituitary-derived factor essential for thyroid gland stimulation, emerged in the early 20th century through experiments demonstrating that anterior pituitary extracts could induce thyroid hypertrophy in hypophysectomized animals. In 1927, Philip E. Smith reported that bovine pituitary extracts restored thyroid function in hypophysectomized tadpoles, marking the initial identification of a thyroid stimulator, later termed TSH.[13] By the 1930s, further purification of TSH from bovine pituitaries confirmed its role, though early bioassays relied on labor-intensive animal-based measurements of thyroid response.[14] The development of radioimmunoassay (RIA) in the 1960s revolutionized TSH measurement by enabling direct quantification in human serum. In 1963, Robert D. Utiger and colleagues established an RIA for human TSH using radioimmunoprecipitation techniques, achieving sensitivities around 2.0 mIU/L sufficient for detecting elevated levels in primary hypothyroidism but not for euthyroid or hyperthyroid states.[15] This innovation built on the foundational RIA work of Rosalyn Yalow and Solomon Berson, who received the 1977 Nobel Prize in Physiology or Medicine for developing the technique to measure peptide hormones like insulin. In 1975, the World Health Organization (WHO) established the first International Reference Preparation (IRP) for human TSH (coded 68/38), calibrated via immunoassay and bioassay to standardize global measurements; this was updated periodically, with the second IRP (80/558) in 1983.[16] During the 1970s and 1980s, concerns over radioisotope safety prompted a shift from RIA to non-isotopic assays, including enzyme-linked and chemiluminescent methods. The introduction of immunoradiometric assays (IRMA) in the early 1980s, leveraging monoclonal antibodies, enhanced sensitivity to approximately 0.1 mIU/L (second-generation assays), allowing better discrimination of thyroid dysfunction.[17] From the 1990s onward, advancements in automated immunoassay platforms yielded third-generation assays with functional sensitivities of 0.01 mIU/L and fourth-generation assays reaching 0.001–0.004 mIU/L, facilitating detection of subclinical thyroid disease.[18] A pivotal milestone in TSH-related testing was the development of recombinant human TSH (rhTSH) in the 1990s, approved by the U.S. Food and Drug Administration in 1998 for stimulating thyroglobulin production in thyroid cancer follow-up without requiring thyroid hormone withdrawal. Initial studies in 1997 demonstrated rhTSH's efficacy in elevating TSH levels safely via intramuscular injection, transforming diagnostic protocols for differentiated thyroid cancer.[19] These historical advances, from bioassays to ultrasensitive immunoassays, have progressively refined TSH testing's clinical utility.[17]Measurement Standards and Units
Current thyroid-stimulating hormone (TSH) assays are primarily automated immunochemiluminometric assays (ICMAs) classified into generations based on functional sensitivity, which determines the lowest TSH concentration measurable with acceptable precision (coefficient of variation ≤20%). Third-generation assays achieve a functional sensitivity of ≤0.01 mIU/L, enabling reliable discrimination between euthyroid and hyperthyroid states.[10] Ultrasensitive fourth-generation assays offer enhanced sensitivity around 0.001 mIU/L, improving detection in subclinical conditions and research applications.[17] Calibration of TSH assays relies on traceability to World Health Organization (WHO) International Reference Preparations (IRPs) to ensure global standardization. The current reference is the 4th International Standard (IS) for TSH, human, for immunoassay (coded 81/615), established in 2023 with an assigned potency of 11.7 mIU per ampoule, replacing the 3rd IS (81/565) from 1993 which had a potency of 11.5 mIU per ampoule and replaced the 2nd IS (80/558) from 1983.[20][21] This standard facilitates consistent potency assignment across commercial kits through bioassay and immunoassay comparisons in international collaborative studies. The standard unit for TSH measurement is milli-international units per liter (mIU/L), reflecting the biological activity defined by the WHO IRP. This unit is equivalent to micro-international units per milliliter (μIU/mL), with a conversion factor of 1:1, ensuring compatibility across laboratory reporting systems.[22] Quality control in TSH assays emphasizes precision and accuracy, with intra-assay variability (within-run coefficient of variation) typically below 5% and inter-assay variability (between-run) under 10% at clinically relevant concentrations.[10] Proficiency testing programs, such as those from the College of American Pathologists (CAP) and External Quality Assessment Schemes (EQAS), monitor laboratory performance by distributing control samples and evaluating agreement against peer results to maintain reliability.[23] Harmonization efforts, led by organizations like the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC), minimize lab-to-lab differences in TSH measurements, which can otherwise vary by up to 20-50% due to method-specific biases.[12] Physiological variations necessitate age-, pregnancy-, and ethnicity-specific adjustments in interpreting measurements, as TSH levels rise with age, decline in early pregnancy due to hCG influence, and differ across ethnic groups (e.g., lower in Black populations compared to White or Hispanic).[24] For specialized applications, such as monitoring differentiated thyroid cancer, the recombinant human TSH (rhTSH) stimulation test uses Thyrogen (thyrotropin alfa) at a dose of 0.9 mg administered intramuscularly on two consecutive days to elevate TSH without thyroid hormone withdrawal.[25] This protocol enhances thyroglobulin detection and radioiodine uptake while preserving patient quality of life.[26]Clinical Interpretation and Reference Ranges
The reference range for serum TSH in healthy adults is typically 0.4 to 4.0 mIU/L, though this can vary slightly by laboratory assay and population characteristics.[27] These ranges are lab-specific and often age-adjusted; in elderly individuals over 70 years, the upper limit may extend to approximately 6.0 mIU/L due to a rightward shift in the TSH distribution curve.[28] During pregnancy, reference ranges are lower and trimester-specific: 0.1 to 2.5 mIU/L in the first trimester, 0.2 to 3.0 mIU/L in the second, and 0.3 to 3.0 mIU/L in the third, reflecting physiological changes in thyroid demand.[29] Clinical interpretation of TSH levels serves as the primary screening tool for thyroid dysfunction, with patterns guiding diagnosis when correlated with free thyroxine (FT4) levels.[2] Elevated TSH above 4.0 mIU/L, particularly when exceeding 10 mIU/L, indicates primary hypothyroidism due to insufficient thyroid hormone production, prompting confirmation with low FT4.[30] Conversely, suppressed TSH below 0.1 mIU/L suggests hyperthyroidism from excess thyroid hormone inhibiting pituitary secretion, also confirmed by elevated FT4 or total T4.[2] Normal TSH within the reference range, even with suggestive symptoms, may necessitate further evaluation with FT4 or thyroid antibodies if clinical suspicion persists.[27] Subclinical thyroid dysfunction represents milder abnormalities where TSH deviates from normal but FT4 remains within reference limits.[31] Subclinical hypothyroidism is defined by TSH between 4.0 and 10 mIU/L with normal FT4, often linked to early autoimmune thyroiditis and carrying risks for progression to overt disease.[31] Subclinical hyperthyroidism features TSH below 0.1 mIU/L with normal FT4, associated with potential cardiovascular and bone effects, though management depends on symptoms and comorbidities.[27] In central hypothyroidism, arising from pituitary or hypothalamic disorders, TSH levels are low or inappropriately normal despite low FT4, contrasting with the elevated TSH of primary hypothyroidism.[2] This pattern requires imaging or additional pituitary testing for confirmation.[30] Dynamic testing, such as thyrotropin-releasing hormone (TRH) stimulation, was historically used to assess pituitary reserve by measuring TSH response to TRH injection but is now rarely employed due to the sensitivity of modern TSH assays.[32] Recovery of TSH from suppression after hyperthyroidism treatment can also indicate restored euthyroidism.[2] Several factors influence TSH interpretation, including diurnal variation with peak levels occurring at night and nadir in the afternoon, which may affect timing of sampling.[33] Non-thyroidal influences, such as acute illness, can transiently alter TSH without true dysfunction.[31] Certain medications may also impact levels, underscoring the need for clinical context in evaluation.[27]Circulating Thyroid Hormones
Total Thyroxine (T4)
Total thyroxine (T4), also known as total T4, refers to the total concentration of thyroxine in serum, encompassing both the protein-bound and unbound (free) fractions.[2] The thyroid gland primarily secretes T4, accounting for approximately 90% of its hormone output, with the remainder being triiodothyronine (T3).[34] As the major circulating thyroid hormone, T4 serves as a prohormone that is largely converted to the more biologically active T3 in peripheral tissues through deiodination.[34] This conversion underscores T4's role in reflecting overall thyroid secretory activity, though its levels are significantly influenced by variations in thyroid hormone binding proteins.[2] Total T4 is measured in serum using immunoassays, such as radioimmunoassay (RIA) or chemiluminescent immunoassay (CLIA), which detect the hormone through antibody-antigen interactions.[5] The typical reference range for total T4 is 4.5-12.0 μg/dL (58-155 nmol/L), with slight variations between laboratories and populations; females often exhibit marginally higher values than males.[5] For unit conversion, total T4 in nmol/L is calculated by multiplying the value in μg/dL by 12.87.[10] In clinical practice, total T4 levels are elevated in hyperthyroidism due to increased thyroid secretion and in conditions of thyroxine-binding globulin (TBG) excess, such as during pregnancy or with estrogen therapy.[2] Conversely, low total T4 indicates hypothyroidism from reduced thyroid output or TBG deficiency, as seen in certain genetic disorders or androgen excess.[35] However, total T4 measurements are limited by their sensitivity to alterations in binding proteins; for instance, estrogen-induced increases in TBG can elevate total T4 without reflecting true hyperthyroidism, necessitating complementary tests like TSH for accurate interpretation.[36]Free Thyroxine (FT4)
Free thyroxine (FT4) represents the unbound, biologically active fraction of thyroxine (T4) in serum, constituting approximately 0.03% of total circulating T4 and serving as the primary form available for diffusion into cells to mediate thyroid hormone effects.[12] Measurement of FT4 typically employs two main approaches: the analog method, an indirect two-site immunoassay that estimates the free hormone fraction without physical separation and is the most commonly used technique in clinical laboratories due to its speed and automation, and equilibrium dialysis, a direct method that physically separates unbound FT4 from protein-bound forms using a semipermeable membrane, often coupled with immunoassay or mass spectrometry for detection and regarded as the reference standard for accuracy despite its higher cost and longer processing time.[12][5][37] Reference ranges for FT4 vary by assay method, population, and laboratory but generally fall between 0.8 and 1.8 ng/dL (equivalent to 10.3 to 23.2 pmol/L).[12][38] To convert FT4 values from ng/dL to pmol/L, multiply by 12.87.[39] Compared to total T4 assays, FT4 measurement offers the advantage of being largely independent of variations in thyroid hormone binding proteins such as thyroxine-binding globulin (TBG), providing a more reliable indicator of thyroid status in conditions like pregnancy, liver disease, or estrogen therapy where binding protein levels fluctuate.[12][5] Clinically, FT4 is essential for confirming abnormalities detected by TSH screening; for instance, elevated FT4 alongside suppressed TSH indicates overt hyperthyroidism, while low FT4 with elevated TSH signifies overt hypothyroidism, guiding diagnosis and therapy monitoring in primary thyroid disorders.[12] Potential pitfalls in FT4 interpretation include assay interferences from heterophile antibodies or biotin supplementation, which can lead to falsely elevated or depressed results in analog methods, and discrepancies during critical illness where nonthyroidal illness syndrome may lower FT4 levels independently of thyroid function.[40] Certain drugs, such as heparin, which displaces T4 from binding proteins via non-esterified fatty acids, or phenytoin, which accelerates hormone metabolism, can artifactually alter FT4 measurements, necessitating alternative approaches like index calculations in select cases.[40][12]Total Triiodothyronine (T3)
Total triiodothyronine (T3) represents the overall concentration of T3 in serum, encompassing both protein-bound and unbound fractions. Approximately 80% of circulating T3 arises from the peripheral deiodination of thyroxine (T4) by type 1 and type 2 deiodinases, mainly in the liver, kidney, and skeletal muscle, while the remaining 20% is directly secreted by the thyroid gland.[41] As the principal active form of thyroid hormone, T3 binds to nuclear receptors in target tissues to mediate genomic effects on metabolism, thermogenesis, and cellular differentiation. Its shorter half-life of about 1 day, compared to T4's approximately 7 days, enables quicker adjustments in hormone action in response to physiological needs.[42] Measurement of total T3 employs competitive immunoassays, such as chemiluminescent immunoassay (CLIA) or radioimmunoassay (RIA), analogous to those for total T4, with results reported in nanograms per deciliter (ng/dL) or nanomoles per liter (nmol/L). The adult reference range is typically 80-180 ng/dL (1.2-2.8 nmol/L), though values may vary slightly by laboratory and age.[43] To convert total T3 from ng/dL to nmol/L, multiply the value by 0.0154.[44] In clinical practice, total T3 testing aids in diagnosing and evaluating hyperthyroidism, where elevations are more pronounced and occur earlier than T4 changes, particularly in T3 toxicosis with normal total T4 levels. Conversely, total T3 remains within normal limits in early or mild hypothyroidism due to preferential preservation of T3 production, rendering it less sensitive for detecting hypothyroid states until advanced stages.[45][46] Total T3 concentrations are influenced by alterations in binding proteins and deiodinase activity; for instance, pregnancy elevates total T3 through increased thyroxine-binding globulin (TBG) synthesis, raising bound hormone levels without affecting free T3. T4 serves as the primary precursor for this peripheral T3 generation.[47]Free Triiodothyronine (FT3)
Free triiodothyronine (FT3) represents the unbound, biologically active fraction of triiodothyronine (T3), comprising approximately 0.3% of total circulating T3 and serving as the primary mediator of thyroid hormone effects on target tissues.[48] Unlike total T3, which includes protein-bound forms, FT3 directly reflects the hormone available for cellular uptake and action, making it a key indicator of thyroid hormone bioactivity.[2] FT3 is typically measured using automated immunoassays, such as chemiluminescent or enzyme-linked methods, though these are less precise than those for free thyroxine (FT4) owing to FT3's lower serum concentrations (around 3-5 pg/mL).[49] For enhanced accuracy, particularly in research or complex cases, liquid chromatography-tandem mass spectrometry (LC-MS/MS) following ultrafiltration is employed as a reference method, offering superior specificity by isolating the free fraction from binding proteins.[49] Adult reference ranges for FT3 generally fall between 2.3 and 4.2 pg/mL (or 3.5 to 6.5 pmol/L), with conversion from pg/mL to pmol/L achieved by multiplying by 1.54.[50] Clinically, FT3 measurement aids in evaluating active thyroid status, particularly when elevated in T3-predominant hyperthyroidism, such as certain cases of Graves' disease where FT3 rises disproportionately to FT4 due to preferential T3 secretion or enhanced deiodination.[51] In non-thyroidal illness syndrome, FT3 levels are often low, similar to total T3, reflecting reduced peripheral conversion but helping differentiate from primary hypothyroidism.[52] Its advantages include better assessment of tissue hormone availability compared to total T3, proving useful in scenarios like amiodarone therapy—where FT3 may decrease due to inhibited deiodinase activity—or pregnancy, where altered binding proteins necessitate monitoring the free fraction for accurate interpretation.[53][54] Despite these benefits, FT3 assays exhibit high variability across methods and laboratories, with immunoassays prone to overestimation at low levels and poor correlation to clinical outcomes in some settings.[49] Consequently, FT3 is not recommended for routine thyroid screening, where TSH and FT4 remain the preferred initial tests due to their stability and reliability.[2]Thyroid Hormone Binding Proteins
Thyroxine-Binding Globulin (TBG)
Thyroxine-binding globulin (TBG) is a glycoprotein synthesized primarily in the liver and serves as the major transport protein for thyroid hormones in the bloodstream.[55] It is encoded by the SERPINA7 gene located on the long arm of the X chromosome at Xq22.2. TBG exhibits high affinity for thyroxine (T4) and triiodothyronine (T3), binding approximately 75% of circulating T4 and 75% of T3, thereby maintaining a reservoir of hormones and regulating their delivery to tissues.[55] TBG levels are measured using direct immunoassays, such as radioimmunoassay or enzyme-linked immunosorbent assay (ELISA), or indirectly calculated from total T4 concentrations and thyroid hormone uptake tests.[56] The reference range for serum TBG in adults is typically 15-30 mg/L, though values can vary slightly by laboratory and population.[57] TBG concentrations are regulated by hepatic synthesis and influenced by hormonal and pathological factors. Estrogens, including those elevated during pregnancy or from oral contraceptive use, increase TBG production and prolong its half-life through enhanced sialylation, often raising levels by 2- to 3-fold.[55] Conversely, androgens and anabolic steroids suppress TBG synthesis, while conditions such as chronic liver disease impair production due to reduced hepatic function, and nephrotic syndrome causes urinary loss of TBG leading to decreased serum levels.[55][58] Alterations in TBG levels significantly affect total thyroid hormone measurements but do not alter free hormone concentrations or clinical thyroid status. Excess TBG, as seen in estrogen-related states, elevates total T4 and T3 while free T4, free T3, and thyroid-stimulating hormone (TSH) remain normal, potentially mimicking hyperthyroidism if only total levels are assessed.[55] TBG deficiency, conversely, lowers total T4 and T3 but preserves normal free hormone levels and euthyroid function, avoiding misdiagnosis of hypothyroidism.[35] Genetic variants in the SERPINA7 gene cause X-linked inherited TBG deficiencies, classified as complete (TBG-C) or partial (TBG-P), affecting up to 1 in 4,000 males.[59] These mutations, often point substitutions or deletions, result in absent or reduced TBG function, leading to low total T4 and T3 but normal free hormones and no requirement for thyroid hormone replacement, as patients remain euthyroid.[59][60] TBG testing is indicated in cases of suspected binding protein abnormalities, such as discrepancies between total and free thyroid hormone levels, family history of inherited TBG variants, or conditions known to alter binding protein concentrations like pregnancy or nephrotic syndrome.[35]Transthyretin and Albumin
Transthyretin (TTR), also known as prealbumin, is a secondary carrier protein for thyroid hormones, binding approximately 15-20% of circulating thyroxine (T4) with a lower affinity compared to thyroxine-binding globulin (TBG).[55] It is synthesized primarily in the liver and choroid plexus, with normal serum reference levels ranging from 20 to 40 mg/dL.[55][61] TTR exists as a tetramer and facilitates the transport of T4, though its binding capacity for triiodothyronine (T3) is minimal.[55] Albumin, the most abundant serum protein at concentrations around 4 g/dL, serves as another auxiliary binder, accounting for about 5% of T4 and approximately 20% of T3, despite its low affinity for these hormones.[55] Despite this lower affinity—roughly 1,000 times weaker than that of TBG—albumin's high plasma concentration enables it to contribute significantly to the total hormone pool as a low-affinity reservoir.[55][62] Together, TTR and albumin act as buffers against rapid fluctuations in free thyroid hormone levels, providing a secondary pool that stabilizes availability during physiological stress.[55] TTR plays a particularly vital role in transporting thyroid hormones across the blood-brain barrier and within cerebrospinal fluid, where it is the predominant binder.[63][64] Levels of TTR decrease in conditions such as malnutrition and inflammation, reflecting its status as a negative acute-phase reactant.[65] Similarly, hypoalbuminemia occurs in chronic illnesses, liver failure, and nephrotic syndrome, leading to mildly reduced total T4 concentrations without typically altering free T4 levels.[66][67] Direct measurement of TTR and albumin is rarely performed in routine thyroid function testing, as their primary clinical relevance emerges through indirect effects on total thyroid hormone levels in systemic diseases like non-thyroidal illness syndrome.[67][68] Genetic mutations in the TTR gene can lead to familial transthyretin amyloidosis, where variant proteins exhibit altered thyroxine binding affinity, potentially disrupting hormone transport and contributing to systemic complications.[69][70]Assessments of Hormone Binding and Availability
Thyroid Hormone Uptake (T Uptake)
The thyroid hormone uptake test, also known as T3 resin uptake (T3RU), is an indirect assay that measures the available binding sites on thyroid hormone-binding proteins in serum, primarily thyroxine-binding globulin (TBG).[10] The principle involves adding a fixed amount of radiolabeled triiodothyronine (T3), typically with iodine-125 or iodine-131, to the patient's serum sample. The labeled T3 competes with endogenous thyroid hormones for binding to serum proteins; unbound T3 is then adsorbed onto an anion-exchange resin or similar matrix, and the percentage of radioactivity bound to the resin is quantified as the uptake value. This uptake is inversely proportional to the number of unsaturated binding sites on TBG, providing an estimate of binding protein capacity.[10][71] Results are reported as a percentage uptake, with a typical reference range of 25-35% in euthyroid individuals with normal TBG levels, though slight variations exist across laboratories.[10][72] The test's utility lies in estimating TBG saturation; elevated uptake (>35%) indicates reduced TBG availability or increased unsaturated sites, often due to low TBG states such as those induced by androgens or nephrotic syndrome, while low uptake (<25%) suggests high TBG levels, as seen with estrogen administration or pregnancy.[10][71] Developed in the 1950s using radiolabeled T3 partitioned between serum and resin, it served as a key tool in the pre-free hormone assay era to adjust total thyroid hormone measurements for binding protein variations.[10] Today, it is less commonly used, largely supplanted by direct free thyroxine (FT4) immunoassays, but remains relevant in calculating the free thyroxine index (FTI) as total T4 multiplied by the normalized T3 uptake value to approximate free hormone levels.[10][17] Despite its historical role, the T3 uptake test has limitations, including interference from non-TBG binders like transthyretin and albumin, which can alter results without reflecting true thyroid function.[10] It is not a direct measure of thyroid hormone levels or function and requires interpretation alongside total T4 to avoid misdiagnosis, particularly in conditions with extreme binding abnormalities.[10][71]Protein Binding Function Tests
Protein binding function tests evaluate the capacity and integrity of thyroid hormone binding proteins, particularly thyroxine-binding globulin (TBG), to assess alterations in hormone-protein interactions that may affect total thyroid hormone measurements without reflecting true glandular dysfunction. These assays provide direct or indirect measures of binding protein saturation and function, complementing total hormone levels in scenarios where binding abnormalities are suspected.[5] Direct immunoassays for TBG quantify the protein concentration in serum using techniques such as radioimmunoassay or enzyme-linked immunosorbent assay (ELISA), offering a precise estimate of TBG levels typically ranging from 12 to 20 mg/L in adults. These methods involve competitive binding of labeled TBG to specific antibodies, with separation of bound and free fractions to determine concentration, and are particularly useful for identifying inherited or acquired TBG excess or deficiency.[5][73][74] T4 binding capacity tests, including saturation analysis, measure the maximum binding sites available on TBG and other proteins by adding exogenous radiolabeled T4 to saturate unoccupied sites, followed by quantification of bound hormone via equilibrium dialysis or chromatography. For instance, in saturation methods, the added (125)I-T4 primarily binds to TBG, with results expressed as binding capacity in mcg T4/dL, helping to reveal reduced capacity in conditions like liver disease or nephrotic syndrome.[75][76][77] Indexed T3 or T4 assays adapt principles similar to thyroid hormone uptake tests but incorporate the patient's endogenous T3 or T4 levels rather than an added tracer, providing a ratio such as T4/TBG to estimate binding saturation and correlate with free hormone availability. This approach, exemplified by the T4/TBG ratio, adjusts for variations in binding protein concentration and has shown strong correlation with direct free T4 measurements in clinical studies.[78] In clinical practice, these tests are applied to confirm euthyroid hyperthyroxinemia, where elevated total T4 occurs due to increased TBG binding capacity—such as in pregnancy or estrogen therapy—while free hormone and TSH remain normal, thus avoiding misdiagnosis of hyperthyroidism. They also aid in differentiating binding protein defects, like TBG deficiency, from primary thyroid disorders by demonstrating normal or abnormal binding function despite discrepant total hormone levels.[79][80][81] Emerging research-oriented tests, such as gel filtration chromatography and electrophoresis, enable detailed profiling of binding proteins by separating serum components based on size or charge, allowing visualization of abnormal binding patterns like those in familial dysalbuminemic hyperthyroxinemia. For example, thyroxine-binding protein electrophoresis (T4BPE) uses radiolabeled T4 to identify variant binders, providing insights into rare inherited abnormalities not detectable by routine assays.[75][82][83] These tests offer advantages in resolving diagnostic ambiguities arising from binding alterations, enabling precise differentiation between euthyroid states with abnormal total hormones and genuine thyroid dysfunction, which is critical in complex cases like non-thyroidal illness. However, pitfalls include interference from paraproteins or autoantibodies, which can falsely elevate or depress binding measurements in immunoassays, and their limited routine use due to the availability of more direct free hormone assays.[62][79][80]Derived and Calculated Parameters
Free Thyroxine Index (FTI)
The free thyroxine index (FTI), also known as the T7 index, is a calculated parameter used to estimate the concentration of free thyroxine (FT4) in the blood by correcting total thyroxine (T4) levels for variations in thyroid hormone binding proteins.[5] The formula for FTI is typically total T4 (in μg/dL) multiplied by the T3 resin uptake (T3RU) expressed as a ratio to the midpoint of the normal range (e.g., 1.0 for normal binding).[5] This indirect method was developed to approximate unbound, biologically active T4 without direct measurement.[84] The primary purpose of FTI is to provide a more reliable indicator of thyroid function than total T4 alone, particularly when binding protein levels fluctuate, as it normalizes for these changes to better reflect free hormone availability.[2] The typical reference range for FTI is 4.5–12.0 μg/dL, which aligns closely with total T4 ranges but accounts for binding normalization.[85] Historically, FTI served as a standard test from the 1960s through the pre-1980s era, when direct FT4 assays were not widely available, offering a practical way to assess thyroid status amid technological limitations in hormone measurement.[86] However, with the advent of accurate direct immunoassay techniques for FT4 in the 1980s, FTI has become obsolete in most clinical laboratories and is rarely used today.[84] In interpretation, FTI values parallel those of direct FT4: elevated levels suggest hyperthyroidism, while low levels indicate hypothyroidism, providing improved discrimination over total T4 in conditions with altered binding.[5] It is particularly useful in thyroxine-binding globulin (TBG) abnormalities, such as during pregnancy, where total T4 may be elevated due to increased TBG but FTI remains normal, avoiding misdiagnosis.[87] A variant, the free T3 index (FTI3), applies a similar calculation—total T3 multiplied by T3 uptake—to estimate free triiodothyronine levels and correct for binding variations in T3 assessments.[5] Key limitations of FTI include its assumption of constant non-TBG binding contributions (e.g., from transthyretin and albumin), which can lead to inaccuracies, and its tendency to exaggerate deviations in states of extreme binding alterations.[5] Additionally, FTI performs poorly in severe non-thyroidal illness, where hormone binding dynamics are disrupted beyond simple TBG effects, making it unreliable as a standalone diagnostic tool.[84]Advanced Structure Parameters (SPINA-GT, SPINA-GD, and Others)
Advanced structure parameters, derived from the Structure Parameter Inference Approach (SPINA), offer model-based estimates of thyroid secretory capacity and peripheral deiodinase activity, enabling a deeper analysis of thyroid homeostasis beyond routine hormone assays. These parameters utilize cybernetic models of the hypothalamic-pituitary-thyroid axis to quantify functional properties of the gland and periphery, incorporating TSH and thyroid hormone concentrations. SPINA-GT was introduced in 1999 as a systems-theoretic method, while SPINA-GD followed in 2002, with subsequent refinements integrating protein binding and kinetic data for enhanced accuracy through 2016.[88] These parameters are mainly used in research and specialized diagnostics, not routine clinical testing.[88] SPINA-GT quantifies the thyroid's maximum secretory capacity for thyroxine, calculated asSPINA\text{-}GT = \frac{\beta_{T} \cdot TT4 \cdot (D_{T} + TSH)}{\alpha_{T} \cdot TSH}
where \beta_{T} is the total deiodination rate constant (typically 0.1), D_{T} is the pituitary deiodinase activity (1 mU/L), \alpha_{T} is the pituitary sensitivity to thyroxine (1 × 10^{-10} L/nmol), TT4 is total thyroxine, and TSH is thyroid-stimulating hormone. This parameter is expressed in units of pmol/s, with a normal range of 1.4–8.7 pmol/s. It provides insight into the gland's ability to produce T4 under TSH stimulation and is particularly useful in primary thyroid disorders, where low values indicate impaired secretion, such as in hypothyroidism.[88] SPINA-GD measures the summed activity of peripheral 5'-deiodinases responsible for T4-to-T3 conversion, given by
SPINA\text{-}GD = \frac{\beta_{31} \cdot TT3}{1 + K_{M1} / FT4}
where \beta_{31} is the clearance rate constant for T3 (0.26), K_{M1} is the Michaelis-Menten constant (5 nmol/L), TT3 is total triiodothyronine, and FT4 is free thyroxine. This parameter is expressed in units of nmol/s, with a normal range of 20–60 nmol/s. This index reflects extrathyroidal thyroid hormone activation and is reduced in conditions like non-thyroidal illness syndrome (sick euthyroid state), aiding differential diagnosis by highlighting deiodinase dysfunction.[88] Additional parameters include the TSH index, a logarithmic adjustment of TSH based on free hormone levels to assess pituitary reserve; the total thyroxine-stimulating index (TTSI), which evaluates overall TSH secretory drive; the T4 free index variant (TFQI), a feedback quantile-based measure of thyroid control; and the reconstructed set point, denoting the equilibrium threshold for pituitary TSH response. These are employed in research for dissecting central versus peripheral contributions to thyroid dysregulation and in software tools for automated computation.[88] Clinically, these parameters support differential diagnosis, such as identifying low SPINA-GT in hypothyroid states or low SPINA-GD in euthyroid illness, and are calculated via dedicated programs. Their key advantage is delivering quantitative, integrated evaluations of glandular, pituitary, and peripheral thyroid functions, improving precision in challenging scenarios over isolated tests. Validation through in vivo studies confirms their specificity for primary disorders and reliability across populations.[88]