Thyroid Function Tests: Clinical Significance of T3, T4, and TSH. Anti-Thyroid Peroxidase (TPO) Test. Free Thyroid Profile Tests (Free T3, T4, and TSH Tests). IVth Semester, Unit V. HSBTE DMLT

Thyroid function tests

Thyroid function tests are a set of blood tests that are used to evaluate the function of the thyroid gland. The thyroid is a small, butterfly-shaped gland located in the neck. The thyroid gland regulates metabolism by producing and releasing hormones, primarily thyroxine (T4) and triiodothyronine (T3). Additionally, the pituitary gland in the brain produces thyroid-stimulating hormone (TSH), which stimulates the thyroid gland to produce T4 and T3. Thyroid function tests include measurements of Thyroid-Stimulating Hormone (TSH), Free Thyroxine (FT4) Free Triiodothyronine (FT3), Thyroxine (T4), Triiodothyronine (T3), Thyroid Antibodies, and Reverse Triiodothyronine (rT3).

Clinical Significance of T3, T4, and TSH: Measurement of levels of thyroid hormones (T3 and T4) and thyroid-stimulating hormone (TSH) assess thyroid gland function. These tests are used to diagnose and manage thyroid disorders. The following are the clinical significance of T3, T4, and TSH:

1. Thyroid-Stimulating Hormone (TSH) Clinical Significance:

Elevated TSH levels indicate hypothyroidism, a condition where the thyroid gland is underactive and not producing enough thyroid hormones (T3 and T4).

Low TSH levels may suggest hyperthyroidism, where the thyroid is overactive, leading to increased production of T3 and T4.

TSH is often the first test ordered to assess overall thyroid function.

2. Thyroxine (T4) and Triiodothyronine (T3) Clinical Significance: T4 and T3 are the primary thyroid hormones responsible for regulating metabolism, energy production, and overall growth and development. Total T4 and T3 include both free and protein-bound forms of these hormones. Free T4 (FT4) and Free T3 (FT3) represent the unbound, biologically active forms of these hormones.

Abnormal T4 and T3 levels are indicative of thyroid dysfunction:

· High T4 and T3 levels may suggest hyperthyroidism.

· Low T4 and T3 levels may indicate hypothyroidism.

· Thyroid dysfunction is called T3 thyrotoxicosis.

3. Interplay between TSH, T4, and T3 Clinical Significance: TSH, T4, and T3 levels are interrelated in a feedback loop called the hypothalamus-pituitary-thyroid axis.

High TSH with low T4 or T3 suggests primary hypothyroidism (thyroid gland dysfunction).

Low TSH with high T4 or T3 indicates primary hyperthyroidism (overactive thyroid gland).

TSH is often used as a screening tool, while T4 and T3 provide more specific information about thyroid hormone levels.

4. Autoimmune Thyroid Conditions Clinical Significance:

Measurement of thyroid antibodies, such as anti-thyroid peroxidase (TPO) and anti-thyroglobulin antibodies, helps diagnose autoimmune thyroid disorders like Hashimoto's thyroiditis and Graves' disease.

Elevated antibody levels indicate an immune system attack on the thyroid gland.

5. Pregnancy and Thyroid Function Clinical Significance:

Thyroid function is critical during pregnancy for the proper development of the fetus.

Abnormal thyroid function during pregnancy may lead to complications, such as developmental issues and miscarriage.

6. Monitoring Thyroid Replacement Therapy Clinical Significance:

TSH, T4, and T3 levels are monitored in individuals receiving thyroid hormone replacement therapy (e.g., levothyroxine).

Regular testing ensures that hormone levels are within the therapeutic range.

Anti-Thyroid Peroxidase (TPO) Test

The Anti-Thyroid Peroxidase (TPO) test is a blood test used to measure the presence of antibodies against thyroid peroxidase in the body. Thyroid peroxidase is an enzyme found in the thyroid gland. It is essential for the production of thyroid hormones, thyroxine (T4), and triiodothyronine (T3). The presence of anti-TPO antibodies indicates an autoimmune response against the thyroid gland.

Purpose of the Anti-Thyroid Peroxidase (TPO) Test: The test is primarily used to diagnose autoimmune thyroid disorders, such as Hashimoto's thyroiditis and to a lesser extent, Graves' disease. These conditions are characterized by the immune system mistakenly attacking the thyroid gland. It leads to inflammation and potential disruption of thyroid function.

Interpretation of Results

1. Normal Results: A normal result indicates the absence or very low levels of anti-TPO antibodies in the blood.

2. Abnormal Results (Positive): Elevated levels of anti-TPO antibodies suggest an autoimmune attack on the thyroid gland. This finding is often associated with conditions such as Hashimoto's thyroiditis.

Clinical Significance:

1. Hashimoto's thyroiditis: This is the most common cause of hypothyroidism (underactive thyroid). It is characterized by the presence of anti-TPO antibodies. Over time, the immune system damages the thyroid gland. This leads to a decline in thyroid hormone production.

2. Graves’ Disease: Anti-TPO antibodies may also be present in Graves' disease. Graves' disease is an autoimmune condition causing hyperthyroidism (overactive thyroid).

3. Diagnosis: The presence of anti-TPO antibodies helps in confirming the diagnosis of autoimmune thyroiditis.

4. Monitoring: In individuals with known autoimmune thyroid conditions, the Anti-TPO test is used to monitor the progression of the disease or the effectiveness of treatment.

Determination of T3, T4, and TSH

Method: RIA Test (Radioimmune assay)

Determination of T3 (RIA Test)

Principal: I125 labeled T3 is used in solid phase radioimmune assay of T3. A blocking agent is also used to free T3 from carrier protein. I125 labeled T3 competes with T3 inpatient sample serum for antibodies. A mixture of the sample and I125 labeled T3 is incubated for 2 hours at 37 degrees C. The supernatant is separated after incubation. Isolated antibody bound with I125 labeled T3 is measured by using a gamma counter.

Procedure: Collect sample serum. The sample may be stored at 2 to 8 degrees C for seven days OR The sample may be stored at -20 degrees C for 2 months. The sample should be at room temperature before the RIA test.

Use the following labeled tubes

1. T: Polystyrene tube for control total t3.

2. NSB (Non-Specific Binding Tube): Polystyrene tube for blank.

3. A to F coated tubes: For calibrators.

4. Tube X1 and X2 coated tubes: For Specimen.

Fill all tubes as per the below table

Incubate all tubes at 37 degrees C for 2 hours. Separate supernatant and pass through absorbent paper for 2 to 3 minutes. Count for one minute in (CPM: Count per minute) in gamma counter. Not down maximum CPM in calibrator tube. It is maximum bound (MB).

Calculation: Determine net count using the formula CPM-NSB. Determine the percent bound for the calibrator and patient sample serum by using the formula

Net count/MB count X 100.

Plot a standard curve for serum T3. Determine the T3 value using a graph.

Determination of T4 (RIA Test)

Principal: I125 labeled T4 is used in solid phase radioimmune assay of T4. A blocking agent is also used to free T4 from carrier protein. I125 labeled T4 competes with T4 in the patient sample serum for antibodies. Mixture of the sample and I125 labeled T4 is incubated for 2 hours at 37 degrees C. The supernatant is separated after incubation. Isolated antibody bound with I125 labeled T3 is measured by using a gamma counter.

Procedure: Collect sample serum. The sample may be stored at 2 to 8 degrees C for seven days Or sample may be stored at -20 degrees C for 2 months. The sample should be at room temperature before the RIA test.

Use the following labeled tubes

1. T: Polystyrene tube for control total T4.

2. NSB (Non-Specific Binding Tube): Polystyrene tube for blank.

3. A to F coated tubes: For calibrators.

4. Tube X1 and X2 coated tubes: For Specimen.

Fill all tubes as per the below table

Incubate all tubes at 37 degrees C for 2 hours. Separate supernatant and pass through absorbent paper for 2 to 3 minutes. Count for one minute in (CPM: Count per minute) in gamma counter. Not down maximum CPM in calibrator tube. It is maximum bound (MB).

Calculation: Determine net count using the formula CPM-NSB. Determine the percent bound for the calibrator and patient sample serum by using the formula

Net count/MB count X 100.

Plot a standard curve for serum T4. Determine T4 value using graph.

Determination of TSH

Principle: The solid phase immunoradiometric assay (IRMA) method is used to determine TSH. Monoclonal and polyclonal anti-TSH antibodies are used. One I125 labeled anti-TSH polyclonal antibody in liquid phase and monoclonal anti-TSH antibodies immobilized to the polystyrene tube wall.

• · Patient serum TSH is entrapped in between monoclonal antibody and polyclonal anti-TSH.

• · Unbound I125 labeled anti-TSH is removed from the tube by decanting.

• · The radioactivity inside the tube is directly proportional to the TSH concentration inside the tube.

• · TSH concentration in the sample is calculated by comparing patient serum CPM with CPM obtained from the calibrator.

Procedure: Collect sample serum. The sample may be stored at 2 to 8 degrees C for seven days. The sample may be stored at -20 degrees C for 2 months. The sample should be at room temperature before the RIA test.

Use the following labeled tubes

1. T: Polystyrene tube for control total TSH.

2. NSB (Non-Specific Binding Tube): Polystyrene tube for blank.

3. A to F coated tubes: For calibrators.

4. Tube X1 and X2 coated tubes: For Specimen.

Fill all tubes as per the below table

Incubate all tubes at 37 degrees C for 2 hours. Separate supernatant and pass through absorbent paper for 2 to 3 minutes. Count for one minute in (CPM: Count per minute) in gamma counter. Not down maximum CPM in calibrator tube. It is maximum bound (MB).

Calculation: Determine net count using the formula CPM-NSB. Determine the percent bound for the calibrator and patient sample serum by using the formula

Net count/MB count X 100.

Plot a standard curve for serum TSH. Determine the TSH value using a graph.

Free Thyroid Profile Tests (Free T3, T4 and TSH Test)

A Free Thyroid Profile Test is a blood test that measures the levels of various hormones produced by the thyroid gland. These hormones include thyroxine (T4), triiodothyronine (T3), and thyroid-stimulating hormone (TSH).

TSH is produced by the pituitary gland and stimulates the thyroid gland to produce T4 and T3. The Portion of T4 and T3 hormones that are not bound to proteins in the blood is called Free Thyroid hormones. Free T4 (FT4) and Free T3 (FT3) tests measure the levels of these hormones that are available for the body to use.

The Free Thyroid Profile Test measures the levels of free thyroxine (FT4), free triiodothyronine (FT3), and thyroid-stimulating hormone (TSH) in the blood.

Determination of Free Thyroid Profile Tests (Free T4, T3)

Principle: T3 and T4 are present in the blood as free T4 and Free T3 and bind with thyroid binding proteins (TBP). They remain in equilibrium inside blood circulation.

Procedure: The equilibrium dialysis method is used to determine FT4 and FT3.

· Undiluted serum sample is dialyzed. Dialysate contains FT4 and FT3. It is analyzed by using an immune assay.

· Adjust the serum sample pH 7.5. Incubate at 37 degrees C for 30 minutes. Carry out ultra-filtration centrifugation at 37 degrees C for 30 minutes. Ultra filtrate is analyzed for T4/T3.

Dr Pramila Singh