Thyroid Function Tests

Dr. Arvind Kumar

Introduction

  • The thyroid gland plays a vital role in regulating metabolism, growth, and energy balance.

  • Thyroid Function Tests (TFTs) help evaluate how effectively the gland produces and manages thyroid hormones.

  • These tests assess hormone levels, feedback control, protein binding, and glandular activity.

  • They are essential for diagnosing hypothyroidism, hyperthyroidism, and autoimmune thyroid disorders.

  • TFTs also guide treatment decisions and help monitor therapy in thyroid patients.

  • Understanding key parameters like TSH, T3, T4, thyroglobulin, and binding indices is crucial for accurate diagnosis.

Thyroid-Stimulating Hormone

  • TSH is produced by the anterior pituitary gland.

  • It is the primary regulator of thyroid hormone production and release.

  • Even slight changes in thyroid hormone levels cause significant changes in TSH due to the sensitive negative-feedback mechanism.

  • High TSH usually indicates hypothyroidism, while low TSH indicates hyperthyroidism or thyroid hormone excess.

  • TSH is often considered the single most important initial test for thyroid assessment.

 

Thyroid Hormones

Thyroxine (T4)

  • Main hormone released by the thyroid gland.

  • Has a larger pool in circulation but lower potency.

  • Converts to T3 in peripheral tissues.

Triiodothyronine (T3)

  • More biologically active than T4.

  • Acts faster and has stronger metabolic effects.

  • Useful in diagnosing T3-toxicosis where T3 is elevated but T4 remains normal.

Free vs Total Forms

  • Free T3 and Free T4 are the physiologically active forms.

  • Total T3 and Total T4 change with protein levels in blood, so may not always reflect true thyroid status.

Difference Between T3 and T4

Feature T3 (Triiodothyronine) T4 (Thyroxine)
Iodine Content Contains 3 iodine atoms Contains 4 iodine atoms
Biological Activity More potent (3–4 times stronger) Less potent
Amount Secreted by Thyroid 20% secreted directly by thyroid 80% secreted directly by thyroid
Source Mostly formed by conversion of T4 to T3 in peripheral tissues Primarily produced by the thyroid gland
Half-Life Shorter (~1 day) Longer (~7 days)
Protein Binding Less protein-bound Highly protein-bound
Circulating Levels Low concentration in blood High concentration in blood
Speed of Action Faster action Slower action
Physiological Role Immediate metabolic effects, increases heat production and oxygen consumption Acts as a hormone reservoir and precursor for T3
Diagnostic Value Important in diagnosing T3-toxicosis Important for assessing overall thyroid hormone output
Stability in Blood Less stable due to lower binding More stable due to higher binding
Important Feature Active hormone directly acting on tissues Storage hormone providing steady supply for T3 formation

 

Carrier Proteins

Thyroid hormones travel in the bloodstream bound to specific proteins:

  • Thyroxine-Binding Globulin (TBG) – main carrier

  • Transthyretin (Pre-albumin)

  • Albumin

Functions of carrier proteins:

  • Transport thyroid hormones in blood

  • Maintain a stable hormone reservoir

  • Regulate availability of free (active) hormone

Changes in these proteins affect total T3/T4 levels but usually do not change free hormone levels.

Protein Binding Function Tests

These tests evaluate how much thyroid hormone is bound to proteins and how much is free.

Key points:

  • Only free hormones are biologically active.

  • Changes in binding proteins can falsely elevate or lower total T4/T3.

  • Assessing binding helps identify:

    • Pregnancy-related changes

    • Liver disorders

    • Nephrotic syndrome

    • Drug-induced protein changes

These tests prevent misdiagnosis when total hormones appear abnormal due to altered protein levels.

Thyroid Tests

 

Test What It Measures Clinical Use
TSH Pituitary control of thyroid First-line test
Free T4 Active thyroxine Hypo/Hyper diagnosis
Free T3 Active triiodothyronine Hyperthyroidism
Total T3/T4 Hormone + protein-bound portion Protein abnormalities
Anti-TPO / Anti-Tg / TRAb Autoimmune markers Hashimoto’s / Graves’
Thyroglobulin Thyroid tissue marker Cancer follow-up
T3 Uptake / THBR Protein binding Binding abnormalities
FTI (Index) Calculated free hormone Corrected interpretation
RAIU Iodine uptake Hyperthyroid evaluation
Ultrasound Thyroid structure Goiter, nodules

1. Thyroid-Stimulating Hormone (TSH)

Diagnostic Purpose

  • Primary screening test for thyroid disorders

  • Reflects pituitary response to circulating thyroid hormones

Clinical Significance

  • High TSH → Primary Hypothyroidism

  • Low TSH → Hyperthyroidism / thyroid hormone excess

  • Very sensitive to even minor changes in T3/T4

Reference Range

  • Adults: 0.4 – 4.0 mIU/L

  • Pregnancy 1st trimester: 0.1 – 2.5 mIU/L

Method

  • CLIA (Chemiluminescence Immunoassay)

  • ECLIA (Electrochemiluminescence)

  • ELISA

 

2. Free T4 (FT4)

Diagnostic Purpose

  • Measures biologically active thyroxine

  • Best indicator of thyroid hormone status

Clinical Significance

  • Low FT4: Hypothyroidism

  • High FT4: Hyperthyroidism, Graves’ disease

Reference Range

  • 0.8 – 2.0 ng/dL

Method

  • CLIA

  • ECLIA

  • RIA (older)

 

3. Total T4 (TT4)

Diagnostic Purpose

  • Measures hormone bound to proteins + free hormone

Clinical Significance

  • Elevated in pregnancy (↑TBG)

  • Low in protein deficiency states

  • Useful when free hormone tests are unreliable

Reference Range

  • 5.0 – 12.0 µg/dL

Method

  • Competitive immunoassays

  • CLIA, ELISA

 

4. Free T3 (FT3)

Diagnostic Purpose

  • Measures unbound, active T3

  • Important in hyperthyroidism

Clinical Significance

  • High FT3: T3-toxicosis, early hyperthyroidism

  • Low in severe illness

Reference Range

  • 2.3 – 4.2 pg/mL

Method

  • CLIA / ECLIA

  • ELISA

 

5. Total T3 (TT3)

Diagnostic Purpose

  • Measures both bound + free T3

Clinical Significance

  • Changes with protein-binding alterations

  • Elevated in hyperthyroidism

Reference Range

  • 80 – 180 ng/dL

Method

  • Competitive immunoassays (CLIA / ELISA)

 

6. Thyroid Antibody Tests

A. Anti-TPO Antibody

Purpose

  • Detects autoimmune destruction of thyroid

Clinical Significance

  • High in Hashimoto’s thyroiditis

  • High in Graves’ disease

Reference Range

  • < 35 IU/mL (positive if elevated)

Method

  • ELISA, CLIA, ECLIA

 

B. Anti-Thyroglobulin (Anti-Tg)

Purpose

  • Detects antibodies against thyroglobulin

Clinical Significance

  • Hashimoto’s thyroiditis

  • Thyroid cancer follow-up

Reference Range

  • < 40 IU/mL

Method

  • CLIA / ELISA

 

C. TSH-Receptor Antibodies (TRAb / TSI)

Purpose

  • Detects antibodies stimulating TSH receptor

Clinical Significance

  • Diagnostic test for Graves’ disease

Reference Range

  • < 1.75 IU/L

Method

  • ECLIA

  • Bioassays

 

7. Thyroglobulin (Tg)

Diagnostic Purpose

  • Protein precursor for thyroid hormone synthesis

  • Marker for thyroid cancer recurrence

Clinical Significance

  • High after thyroid damage, inflammation or cancer

  • Used to monitor thyroidectomy patients

Reference Range

  • Men: 1.4 – 29 ng/mL

  • Women: 1.5 – 38 ng/mL

Method

  • Immunoassays (CLIA / ECLIA)

  • Mass spectrometry (in specialized labs)

 

8. Reverse T3 (rT3)

Purpose

  • Inactive form of T3

  • Assesses metabolic dysfunction

Clinical Significance

  • High in Euthyroid Sick Syndrome

  • Used in critical care evaluations

Reference Range

  • 10 – 24 ng/dL

Method

  • LC-MS/MS (Highly accurate)

  • RIA

 

9. T3 Uptake (T3U) / Thyroid Hormone Binding Ratio

Diagnostic Purpose

  • Measures availability of binding sites on TBG

Clinical Significance

  • High T3U: Low TBG (liver disease, androgens)

  • Low T3U: High TBG (pregnancy, estrogen therapy)

Reference Range

  • 25 – 35%

Method

  • Competitive binding tests

  • RIA / CLIA

 

10. Free T4 Index (FTI)

Diagnosis

  • Calculated: FTI = Total T4 × T3 Uptake

Clinical Significance

  • Used when protein abnormalities alter total T4

  • Reliable substitute when free assays interfere

Reference Range

  • 4.5 – 12.0 μg/dL

Method

  • Calculated parameter based on lab values

 

11. Serum TBG (Thyroxine-Binding Globulin)

Diagnostic Purpose

  • Measures concentration of main binding protein

Clinical Significance

  • High TBG → Pregnancy, estrogen therapy

  • Low TBG → Malnutrition, nephrotic syndrome

Reference Range

  • 13 – 30 mg/L

Method

  • Immunoassays (CLIA / ELISA)

 

12. Calcitonin

Diagnostic Purpose

  • Hormone secreted by thyroid C-cells

Clinical Significance

  • High in Medullary Thyroid Carcinoma (MTC)

  • Used in screening of MEN syndrome

Reference Range

  • Men < 10 pg/mL

  • Women < 5 pg/mL

Method

  • ECLIA

  • CLIA

 

13. Radioactive Iodine Uptake (RAIU)

Diagnostic Purpose

  • Assesses iodine absorption by thyroid

Clinical Significance

  • High uptake → Graves’ disease, toxic nodular goiter

  • Low uptake → Thyroiditis, iodine overload

Reference Range

  • 6-hour uptake: 5–15%

  • 24-hour uptake: 10–30%

Method

  • Nuclear medicine scan

  • Using I-123 or I-131 isotopes

 

14. Thyroid Scan (Nuclear Imaging)

Purpose

  • Visualization of thyroid structure and function

Clinical Significance

  • Detects hot/cold nodules

  • Evaluates cancer spread

  • Differentiates Graves’ disease from thyroiditis

Method

  • Gamma camera imaging with radioactive iodine

 

15. Thyroid Ultrasound

Purpose

  • Structural imaging of thyroid gland

Clinical Significance

  • Detects nodules, cysts, goiter, thyroiditis

  • Guides FNA biopsy

Method

  • High-resolution ultrasonography

 

16. Fine Needle Aspiration (FNA)

Diagnostic Purpose

  • Cytological evaluation of thyroid nodules

Clinical Significance

  • Gold standard for diagnosing:

    • Papillary carcinoma

    • Follicular neoplasm

    • Benign nodules

    • Thyroiditis

Method

  • Ultrasound-guided aspiration

  • Cytology smear evaluation

Mixed Parameters

Mixed parameters combine two or more thyroid-related measurements to improve diagnostic accuracy.

Examples:

  • Free hormones + TSH together

  • Total T4 + binding index

  • T3 uptake + T4 levels

They help in:

  • Distinguishing true thyroid disease from protein-binding abnormalities

  • Providing a broader clinical interpretation

  • Understanding “intermediate” thyroid states

Mixed parameters are especially helpful when results are borderline or conflicting.

Calculated and Structural Parameters

These are derived or mathematically adjusted values that provide deeper insights into thyroid physiology.

Common Calculated Parameters

  • Free T4 Index (FTI) – combines total T4 and T3 uptake

  • Free T3 Index (FT3I)

  • Thyroid Hormone Binding Ratio (THBR)

  • Corrected values when free hormone assays are not available

Structural / Functional Indicators

  • Reflect thyroid gland reserve and activity

  • Help assess:

    • Thyroid gland stimulating capacity

    • Peripheral conversion of T4 to T3

    • Degree of hormone saturation on binding proteins

Calculated parameters are essential when standard free hormone assays show interference or when binding-protein abnormalities are suspected.

Clinical Significance of Thyroid Function Tests

Thyroid Function Tests (TFTs) provide essential information about the functional, biochemical, and regulatory status of the thyroid gland. Their overall clinical significance includes:

1. Diagnosis of Thyroid Disorders

TFTs are the primary tool for diagnosing:

  • Hypothyroidism (primary, secondary, subclinical)

  • Hyperthyroidism (overt & subclinical)

  • Thyroiditis (Hashimoto’s, painless, postpartum)

  • Autoimmune thyroid diseases (via antibody testing)

They help differentiate between disorders originating from the thyroid gland vs. the pituitary gland.

2. Assessment of Thyroid Hormone Levels in the Body

TFTs show how much active or inactive hormone is available, helping confirm:

  • Hormone deficiency

  • Hormone excess

  • Abnormal conversion of T4 → T3

  • Altered hormone binding due to protein abnormalities

This provides insight into actual metabolic hormone activity in tissues.

3. Evaluation of Pituitary–Thyroid Axis

TSH reflects the brain’s response to circulating thyroid hormones.
TFTs help understand:

  • Whether the pituitary gland is responding correctly

  • Feedback regulation

  • Disorders like secondary or tertiary hypothyroidism

This distinguishes central causes from thyroid gland dysfunction.

4. Monitoring of Thyroid Treatment

TFTs are essential in guiding therapy:

  • Adjusting levothyroxine dose in hypothyroid patients

  • Monitoring antithyroid drugs in hyperthyroidism

  • Evaluating response after radioactive iodine therapy

  • Assessing stability during pregnancy

They ensure proper hormone balance is maintained.

5. Detection of Autoimmune Thyroid Conditions

Antibody tests (Anti-TPO, Anti-Tg, TRAb) help:

  • Confirm autoimmune thyroiditis

  • Diagnose Graves’ disease

  • Predict risk of future thyroid dysfunction

  • Monitor remission or relapse

Autoimmunity is the most common cause of thyroid disease.

6. Evaluation of Thyroid Nodules and Cancer

Certain TFT parameters support cancer diagnosis and follow-up:

  • Thyroglobulin – tumor marker for papillary & follicular cancer

  • Calcitonin – marker for medullary thyroid carcinoma

  • Imaging tests support structural evaluation

They help detect recurrence and guide long-term management.

7. Assessment of Binding Protein Abnormalities

Tests like Total T3/T4, T3 Uptake, TBG levels, and FTI help identify:

  • Changes in thyroid hormone–binding proteins

  • Conditions like pregnancy, liver disease, nephrotic syndrome, malnutrition

  • Whether total hormone levels truly reflect thyroid status

This prevents misinterpretation due to protein disorders.

8. Differentiation of Thyrotoxicosis Causes

RAIU and thyroid scans differentiate:

  • Graves’ disease

  • Toxic nodules

  • Thyroiditis

  • Iodine-induced thyrotoxicosis

Correct diagnosis ensures appropriate treatment.

9. Evaluation During Critical Illness

TFTs help detect:

  • Euthyroid Sick Syndrome

  • Altered hormone metabolism under stress, starvation, trauma, sepsis

This prevents unnecessary thyroid treatment during severe illness.

10. Support in Pregnancy and Fertility Management

Pregnancy significantly alters thyroid physiology. TFTs help:

  • Manage maternal hypothyroidism (important for fetal brain development)

  • Diagnose hyperthyroidism

  • Monitor TSH/FT4 trimester-specific ranges

  • Evaluate recurrent miscarriages related to thyroid autoimmunity

TFTs protect both maternal and fetal health.

MCQs

1. Which hormone is most commonly measured to assess primary thyroid function?

A. T3
B. T4
C. TSH
D. TRH

2. The biologically active form of thyroid hormone is:

A. Total T4
B. Total T3
C. Free T4
D. Reverse T3

3. Elevated TSH with low T4 indicates:

A. Primary hyperthyroidism
B. Primary hypothyroidism
C. Secondary hyperthyroidism
D. Euthyroid state

4. Low TSH and low T4 suggest:

A. Primary hypothyroidism
B. Secondary hypothyroidism
C. Hashimoto’s thyroiditis
D. Graves’ disease

5. Excess production of thyroid hormone is best indicated by:

A. High TSH
B. Low TSH
C. High TRH
D. High prolactin

6. Free T3 is most useful for diagnosing:

A. Subclinical hypothyroidism
B. Early hyperthyroidism
C. Euthyroid sick syndrome
D. Normal thyroid function

7. Which test detects autoimmune thyroid disease?

A. TSH
B. Free T4
C. Anti-TPO antibodies
D. Thyroglobulin

8. Anti-TPO antibody is strongly associated with:

A. Graves’ disease
B. Hashimoto’s thyroiditis
C. Subacute thyroiditis
D. Goiter

9. Elevated T3 with normal T4 and low TSH suggests:

A. T4 toxicosis
B. T3 toxicosis
C. Hypothyroidism
D. Euthyroid sick syndrome

10. Which is the most sensitive test for early thyroid dysfunction?

A. Free T4
B. Total T4
C. TSH
D. Thyroglobulin

11. Thyroglobulin is mainly used for monitoring:

A. Graves’ disease
B. Thyroid cancer recurrence
C. Hypothyroidism treatment
D. Pituitary function

12. A high TSH and normal T4 indicate:

A. Overt hypothyroidism
B. Subclinical hypothyroidism
C. Hyperthyroidism
D. Thyroid storm

13. Low TSH and normal T4 indicate:

A. Subclinical hyperthyroidism
B. Overt hypothyroidism
C. Overt hyperthyroidism
D. Thyroiditis

14. Which test differentiates Graves’ disease from thyroiditis?

A. TSH
B. T3
C. T4
D. TSH receptor antibodies (TRAb)

15. Reverse T3 is elevated in:

A. Hyperthyroidism
B. Euthyroid sick syndrome
C. Primary hypothyroidism
D. Hashimoto’s disease

16. Thyroid-stimulating immunoglobulin (TSI) indicates:

A. Hashimoto’s
B. Graves’ disease
C. Goiter
D. Hypothyroidism

17. A patient with high free T4 and high TSH likely has:

A. Pituitary adenoma
B. Thyroid storm
C. Hashimoto’s
D. Euthyroid state

18. Which hormone provides negative feedback to TSH?

A. TRH
B. Free T4
C. Prolactin
D. ACTH

19. TSH reference range in adults is approximately:

A. 0.4–4.0 mIU/L
B. 5–10 mIU/L
C. 10–20 mIU/L
D. 20–30 mIU/L

20. A low TSH but normal T3 and T4 may occur after:

A. Pregnancy
B. Severe illness
C. Hypothyroidism
D. Pituitary hyperfunction

21. Total T4 levels depend mainly on:

A. Liver function
B. TSH secretion
C. Thyroxine-binding globulin
D. Iodine levels

22. Pregnancy increases:

A. Free T4
B. Free T3
C. TBG
D. TRH

23. In primary hyperthyroidism, free T4 levels are:

A. Low
B. Normal
C. High
D. Variable

24. The best test to monitor levothyroxine therapy is:

A. Total T3
B. Free T4
C. TSH
D. Anti-TPO

25. Thyroid peroxidase is essential for:

A. TSH secretion
B. Iodine trapping
C. Thyroid hormone synthesis
D. TBG production

26. In Hashimoto’s, TSH is typically:

A. Low
B. Normal
C. High
D. Extremely low

27. TSH receptor antibodies stimulate thyroid hormone release in:

A. Thyroiditis
B. Hashimoto’s
C. Graves’ disease
D. Endemic goiter

28. Which parameter decreases in euthyroid sick syndrome?

A. Free T4
B. Total T3
C. TSH
D. TBG

29. Thyroid-binding globulin (TBG) levels increase in:

A. Liver failure
B. Nephrotic syndrome
C. Pregnancy
D. Hyperthyroidism

30. A patient taking amiodarone may have elevated:

A. TSH
B. TBG
C. Reverse T3
D. TSI

31. TRH stimulates release of:

A. T3
B. T4
C. TSH
D. TBG

32. TSH levels are lowest in:

A. Morning
B. Afternoon
C. Midnight
D. Early morning (2–4 AM)

33. Thyroid storm typically shows:

A. High TSH
B. Extremely high T4 and T3
C. High TRH
D. Low reverse T3

34. Anti-thyroglobulin antibodies interfere with measurement of:

A. TSH
B. Thyroglobulin
C. Free T4
D. TBG

35. The first-line test in newborn thyroid screening is:

A. T4
B. T3
C. TSH
D. TRH

36. Low free T4 with normal TSH suggests:

A. Central hypothyroidism
B. Subclinical hypothyroidism
C. Hyperthyroidism
D. Euthyroid state

37. TSH levels rise when:

A. T4 increases
B. T4 decreases
C. TRH decreases
D. Iodine increases

38. A radioactive iodine uptake (RAIU) test is used to evaluate:

A. TSI levels
B. Thyroid autoantibodies
C. Thyroid function and hyperthyroidism
D. Thyroid cancer staging

39. Which TFT abnormality is seen in Graves’ disease?

A. High TSH
B. Low T4
C. High free T4, low TSH
D. Low TRAb

40. Which test is least influenced by binding protein abnormalities?

A. Total T4
B. Total T3
C. Free T4
D. Total rT3

✅ Answer Key

  1. C

  2. C

  3. B

  4. B

  5. B

  6. B

  7. C

  8. B

  9. B

  10. C

  11. B

  12. B

  13. A

  14. D

  15. B

  16. B

  17. A

  18. B

  19. A

  20. B

  21. C

  22. C

  23. C

  24. C

  25. C

  26. C

  27. C

  28. B

  29. C

  30. C

  31. C

  32. C

  33. B

  34. B

  35. C

  36. A

  37. B

  38. C

  39. C

  40. C