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Clinical Pathology

Total Thyroxine (T4)

By Dayyal Dg.Twitter Profile | Updated: Sunday, 14 April 2024 22:01 UTC
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Doctor performing routine medical checkup.
Doctor performing routine medical checkup. Freepik / @freepik

Thyroxine, also recognized as T4, is a vital hormone produced by the thyroid gland. It plays a pivotal role in regulating metabolic processes and fostering growth throughout the body.

The presence of T4 in the bloodstream can be categorized into two distinct forms:

  • Total T4: This encompasses the entire quantity of thyroxine present in the blood. It includes both the 'bound' T4, which is attached to proteins, and the 'free' T4. The bound T4 is inactive as it is attached to proteins, preventing it from entering body tissues.
  • Free T4: This refers exclusively to the unbound portion of T4 that is not attached to proteins. Free T4 is the metabolically active form of the hormone, capable of entering body tissues to exert its effects.

A T4 test is a diagnostic tool that measures the concentration of T4 in your blood. Abnormal levels, either too high or too low, can be indicative of thyroid disease. The measurement of Free T4 is particularly crucial as it reflects the active form of thyroxine hormone available for tissue uptake, thereby providing a more accurate reflection of thyroid function.

The quantification of total serum thyroxine, which encompasses both the free and protein-bound forms, is typically achieved through the application of competitive immunoassay techniques. The standard range for adults is established between 5.0 and 12.0 μg/dl.

It is common practice to conduct a combined analysis of either total or free thyroxine alongside Thyroid Stimulating Hormone (TSH) levels. This combined approach offers the most comprehensive evaluation of thyroid functionality.

Causes of Increased Total T4

  1. Hyperthyroidism: Primary hyperthyroidism is suggested by the concurrent rise in both T4 and T3 levels, accompanied by a decrease in TSH.
  2. Augmented Thyroxine-Binding Globulin: An increase in TBG concentration leads to a decrease in free hormone levels, which in turn stimulates the release of TSH from the pituitary, restoring the free hormone concentration to normal. The reverse process occurs if the concentration of binding proteins decreases. In both scenarios, the level of free hormones remains within the normal range, while the concentration of total hormone undergoes changes. Consequently, estimating only the total T4 concentration can lead to misinterpretation of results in situations that alter the concentration of TBG.
  3. Factitious Hyperthyroidism
  4. Tumors Secreting TSH in the Pituitary Gland

Causes of Decreased Total T4

  1. Primary Hypothyroidism: Primary hypothyroidism is characterized by a decrease in T4 levels coupled with an increase in TSH.
  2. Secondary or Pituitary Hypothyroidism
  3. Tertiary or Hypothalamic Hypothyroidism
  4. Hypoproteinemia: This condition, exemplified by the nephrotic syndrome, can lead to a decrease in total T4.
  5. Pharmaceutical Influence: Certain drugs, such as oestrogen and danazol, can contribute to reduced total T4 levels.
  6. Severe Non-Thyroidal Illness

Free Thyroxine (FT4)

Free thyroxine (FT4) represents a minuscule fraction of total T4, remains unbound to proteins, and is the metabolically active variant of the hormone. It accounts for approximately 0.05% of total T4. The standard range is 0.7 to 1.9 ng/dl. The concentrations of free hormones (FT4 and FT3) align more accurately with the metabolic state than total hormone levels, as they remain unaffected by fluctuations in TBG concentrations.

The assessment of FT4 proves beneficial in scenarios where the total T4 level is likely to be modified due to changes in TBG level, such as during pregnancy, intake of oral contraceptives, or in the presence of nephrotic syndrome.

Total and Free Triiodothyronine (T3)


  1. Diagnosis of T3 Thyrotoxicosis: A condition characterized by hyperthyroidism with diminished TSH and elevated T3, and normal T4/FT4 levels is referred to as T3 thyrotoxicosis.
  2. Early Detection of Hyperthyroidism: In the initial stages of hyperthyroidism, total T4 and free T4 levels remain within the normal range, but T3 levels are elevated.

A low T3 level does not contribute significantly to the diagnosis of hypothyroidism as it is observed in approximately 25% of healthy individuals.

For routine evaluation of thyroid function, TSH and T4 are measured. T3 is not routinely estimated due to its very low normal plasma levels.

The standard T3 level is 80-180 ng/dl.

Free T3: The measurement of free T3 provides accurate values in patients with altered serum protein levels, such as during pregnancy, intake of estrogens or oral contraceptives, and in the presence of nephrotic syndrome. It represents 0.5% of total T3.

Thyrotropin Releasing Hormone (TRH) Stimulation Test


  1. Confirmation of Secondary Hypothyroidism Diagnosis
  2. Evaluation of Suspected Hypothalamic Disease
  3. Suspected Hyperthyroidism

This test is not frequently used in current times due to the availability of sensitive TSH assays.


  • A baseline blood sample is collected for the estimation of basal serum TSH level.
  • TRH is administered intravenously (200 or 500 μg), followed by the measurement of serum TSH at 20 and 60 minutes.


  1. Normal Response: A rise of TSH > 2 mU/L at 20 minutes, followed by a slight decline at 60 minutes.
  2. Exaggerated Response: A further significant rise in an already elevated TSH level at 20 minutes, followed by a slight decrease at 60 minutes; observed in primary hypothyroidism.
  3. Flat Response: No response; observed in secondary (pituitary) hypothyroidism.
  4. Delayed Response: TSH is higher at 60 minutes compared to its level at 20 minutes; seen in tertiary (hypothalamic) hypothyroidism.

Antithyroid Antibodies

Box 1: Thyroid autoantibodies
  • Useful for diagnosis and monitoring of autoimmune thyroid diseases.
  • Antimicrosomal or antithyroid peroxidase antibodies: Hashimoto’s thyroiditis
  • Anti-TSH receptor antibodies: Graves’ disease

In thyroid disorders such as Hashimoto’s thyroiditis and Graves’ disease, various autoantibodies, including TSH receptor, antimicrosomal, and antithyroglobulin, are detected. In almost all patients with Hashimoto’s disease, antimicrosomal (also known as thyroid peroxidase) and anti-thyroglobulin antibodies are observed. TSH receptor antibodies (TRAb) are primarily tested in Graves’ disease to predict the outcome post-treatment.

Radioactive Iodine Uptake (RAIU) Test

This direct test evaluates the trapping of iodide by the thyroid gland (through the iodine symporters or pumps in follicular cells) for thyroid hormone synthesis. Patients are administered a tracer dose of radioactive iodine (either 131I or 123I) orally. This is followed by the measurement of the amount of radioactivity over the thyroid gland at intervals of 2 to 6 hours and again at 24 hours. RAIU directly correlates with the functional activity of the thyroid gland. The normal RAIU is about 10-30% of the administered dose at 24 hours, but this varies according to geographic location due to differences in dietary intake.

Causes of Increased Uptake

Hyperthyroidism due to Graves’ disease, toxic multinodular goiter, toxic adenoma, TSH-secreting tumor.

Causes of Decreased Uptake

Hyperthyroidism due to administration of thyroid hormone, factitious hyperthyroidism, subacute thyroiditis.


RAIU is most useful in the differential diagnosis of hyperthyroidism by distinguishing causes into those due to increased uptake and those due to decreased uptake.

Thyroid Scintiscanning

An isotope (99mTc-pertechnetate) is administered and a gamma counter assesses its distribution within the thyroid gland.


  • Differential diagnosis of high RAIU thyrotoxicosis:
    • Graves’ disease: Uniform or diffuse increase in uptake
    • Toxic multinodular goiter: Multiple discrete areas of increased uptake
    • Adenoma: Single area of increased uptake
  • Evaluation of a solitary thyroid nodule:
    • ‘Hot’ nodule: Hyperfunctioning
    • ‘Cold’ nodule: Non-functioning; about 20% of cases are malignant.

The interpretation of thyroid function tests is shown in Table 1.

Table 1: Interpretation of thyroid function tests
Test resultsInterpretations
TSH Normal, FT4 Normal Euthyroid
Low TSH, Low FT4 Secondary hypothyroidism
High TSH, Normal FT4 Subclinical hypothyroidism
High TSH, Low FT4 Primary hypothyroidism
Low TSH, Normal FT4, Normal FT3 Subclinical hyperthyroidism
Low TSH, Normal FT4, High FT3 T3 toxicosis
Low TSH, High FT4 Primary hyperthyroidism

Neonatal Screening for Hypothyroidism

During the neonatal phase, a deficiency in thyroid hormones can lead to severe mental impairment, a condition known as cretinism. This can be averted through prompt detection and intervention. The Thyroid Stimulating Hormone (TSH) level is typically assessed using dry blood spots on filter paper or cord serum, collected between the 3rd and 5th days of life. An elevated TSH level is indicative of hypothyroidism. For infants diagnosed with hypothyroidism, a Radioactive Iodine Uptake (RAIU) scan using 123I should be performed to differentiate between thyroid agenesis and dyshormonogenesis.


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