03 September 2016

Lecture #5: THYROID FUNCTION TESTS



Laboratory evaluation of Thyroid function

1.      Test of thyroid hormone in blood

a.      Free T4 level
b.      Free thyroxine index (FT4I)
c.       Thyroid hormone–binding ratio (THBR)
d.     Total T3
e.      Free T3
f.        Reverse T3
g.      Thyroglobulin
h.      Total T4
i.        Free T3 index
j.        Thyroid hormone–binding proteins
k.      Thyroid antibodies
l.        T3 suppression
m.   TRH stimulation

2.      Evaluation of the hypothalamus–pituitary–thyroid axis

a.      Thyroid stimulating hormone

3.      Assessment of iodine metabolism

a.      Radioactive Iodine Uptake (RAIU)

4.      Estimation of gland size

a.      Iodine–123
b.      Technetium 99m pertechnetate
c.       Americium–241 Fluorescent scanning
d.     Magnetic Resonance Imaging (MRI)

5.      Thyroid biopsy

a.      Fine needle aspiration biopsy (FNAB)

6.      Observation of the effects of thyroid hormones on peripheral tissues

a.      Basal Metabolic Rate (BMR)
b.      Cardiac Muscle Contractility
c.       Photomotogram

7.      Measurement of Thyroid antibodies

a.      Thyroglobulin antibody (TgAb)
b.      Thyroid peroxidase antibody (TPO Ab)
c.       TSH receptor antibody


******  PROTEIN–BOUND IODINE (PBI)  *******

This is the organic function of blood iodine that precipitates with the serum proteins and consists of thyroxine and triiodothyronine. The PBI level in serum correlates well with the status of thyroid activity. The PBI determination is considered the most valuable method for evaluation of thyroid function.

Steps required among the common method of PBI determination:

1.      Precipitation of proteins

2.      Washing of the precipitated proteins to separate and remove the inorganic iodine

3.      Destruction of organic matter and conversion of thyroxine to inorganic iodine by:

a.      Wet ashing method using any of the following oxidizing agent

(1)   Chromic acid
(2)   Permanganate
(3)   Chloric acid

b.      Dry ashing method

4.      Quantitation of the inorganic iodide formed by its catalytic effect on the ceric arsenious acid reaction.


Chloric method – Zak et.al and Bodansky et.al

The PBI is precipitated and washed with trichloroacetic acid and digested with chloric acid and chromate to convert iodine to iodate (sodium chromate or chromic acid serves as indicator and also prevents volatilization of iodic acid). The addition of arsenious acid reduces the excess chromate to give a colorless reaction and at the same time the iodate reduced to iodide. The arsenious acid reduces ceric ammonium sulfate from a yellow to a colorless solution with the iodide acting as a catalyst.


O’neal and Sim modified method of Zak et.al. and Bodansky et.al

The above procedure was modified by using perchloric acid as a protein precipitant instead of trichloroacetic acid.


Dry ashing method:
(Foss et.al. modification of the alkaline incineration method of Barker)

The protein bound iodine is precipitated and washed by the zinc hydroxide technique; sodium carbonate is added to make the washed precipitate alkaline and the precipitate is completely dried in an oven. The protein is then incinerated in a muffled furnace to convert iodine to iodide. The ash is dissolved and the iodide present is measured using the ceric arsenious acid reaction.


******  BUTANOL EXTRACTABLE IODINE (BEI)  ******

This is more specific method for measuring thyroxine because it is less subject to interference by drugs containing organic iodine.

In this method, thyroxine and triiodothyronine are extracted from acidified serum by butanol. The extract is washed with alkali to remove inorganic iodine and iodotyrosine and then evaporated.

The subsequent steps in the oxidation and spectrophotometric determination may be carried out by method for PBI.


*******  FREE THYROXINE (FT4)  ******

The equilibrium dialysis was the first method developed to measure free T4. In this method, a serum sample was incubated in a dialysis membrane against a standard buffer until equilibrium of added T4 tracer reached the dialysate. The ratio of the counts in the dialysate to the dialysant give the dialyzable fraction or % of free T4. Recent advances in technology have allowed development of equilibrium dialysis assays that allow direct measurement of the free T4 by RIA after separating the free T4 from the protein–bound T4 with the use of dialysis cells.

Assay methodology:

1.      Chemiluminiscence
2.      RIA

Normal values:         0.8 – 2.0 ng/dl


******  TOTAL THYROXINE (T4)  ******

Elevated T4 values are characteristically seen in patients with hyperthyroidism, low levels are associated with hypothyroidism.

T4 values that do not reflect thyroid status are seen in patients with altered TBG concentration or in patients receiving medications that affect TBG binding to T4.

Total T4 is elevated in women who are pregnant, taking oral contraceptives, or are on estrogen therapy because estrogen increases hepatic production of TBG.

Total T4 is decreased in cirrhosis and nephrotic syndrome. Glucocorticoids decrease total T4 by inhibiting TSH secretion and by decreasing the concentration of TBG in serum. Dopamine therapy reduces TSH levels and therefore reduces thyroid hormone secretion.

Factors influencing decreased T4 values

1.      Androgens and other anabolic steroids
2.      Salicylates (competes in the binding sites)
3.      Anticonvulsants such as phenytoin and carbamazepine
4.      Slow induction of hepatic binding and metabolism of T4
5.      Inhibition of TSH secretion

Assay methodology:

1.      RIA
2.      Fluorometric enzyme immunoassay
3.      Fluorescence polarization immunoassay

Normal values:         4.5 – 11.0 ug/dl


******  THYROID HORMONE–BINDING RATIO (THBR)  ******

The first of the THBR test was formerly known as the resin T3 uptake (RT3U), however, in May 1987, the Nomenclature of the American Thyroid Association recommended RT3U test as well as other assays designed to estimate the distribution of thyroid hormone bound to thyroid binding proteins be termed THBR assays.

Increased thyroid–binding proteins, resulting in decreased RT3U is seen in:

1.      Pregnancy
2.      Estrogen therapy
3.      Hyperproteinemia
4.      Acute intermittent porphyria
5.      Hereditary TBG increase
6.      Acute hepatitis

Decreased thyroid–stimulating proteins, resulting in increased RT3U is seen in

1.      Nephrotic renal disease
2.      Chronic debilitating diseases
3.      Liver diseases
4.      Treatment with glucocorticoid
5.      Phenytoin and salicylates

Assay methodology:           Fluorescent polarization immunoassay

Normal values:                     0.72 – 1.24


******  FREE T4 INDEX (FT4I)  ******

The FT4I is a calculation used for the purpose of normalizing the total T4 and the THBR values in the presence of protein alterations. The calculations vary depending on the design of the THBR assay. Multiplication of the total T4 by the RT3U provides an index of effective free T4 concentration somewhat independent of changes in protein binding. It is important to note that it is not a true free T4 value but only an index of this measurement.

FT4I = T4 x THBR (THBR = T3U / normal T3U)

Normal values:         1.13 – 3.85


******  TOTAL TRIIODOTHYRONINE  ******

T3 is useful in diagnosing mild hyperthyroidism because T3 rises earlier and more markedly than does T4 in all common forms of hyperthyroidism. Clinicians typically use T3 to monitor patients receiving sodium liothyronine. This assay is of little clinical usefulness in evaluating hypothyroidism.

Assay methodology:          

1.      Solid–phase RIA
2.      Microparticle enzyme immunoassay
3.      Flourometric enzyme immunoassay

Normal values:         80 – 180 ng/dl


******  FREE T3 INDEX (FT3I)  ******

The Free T3 index is used to normalize the T3 and THBR values because T3 levels are also affected by variations in TBG concentrations.

FT3I = T3 x THBR (THBR = T3U / normal T3U)

Normal values:         20 – 63


****** FREE T3 (FT3)  ******

Free T3 is primarily used as a confirmation test in hyperthyroidism, especially in T3 thyrotoxicosis. Free T3 correlating well with total T3, may offer the advantage of not being affected by changes in TBG, but it depends on the methodology of the assay.

Assay methodology:           Solid phase RIA

Normal values:                     1.4 – 4.4 pg/ml


******  REVERSE T3 (rT3)  ******

Reverse T3 (rT3) is a metabolically inactive isomer of T3 and it is present in serum almost entirely as a result of generation from T4 in peripheral tissues. Removal of iodide from an inner phenyl ring of thyroxine produces rT3, whereas iodide removal from an outer ring produces the metabolically active T3. Elevated rT3 levels are seen in patients with severe systemic illness as a result of decreased conversion of T4 to T3 as well as inhibition of T3 degradation, rT3 is elevated in hyperthyroidism and low in hypothyroidism.

Normal values:         25 – 75 ng/dl


******  THYROID HORMONE–BINDING PROTEINS  ******

Alteration in thyroid hormone–binding proteins affects total but free concentrations of T4 and T3. Use of the estimation method, FT4I, together with a free T4 determination, demonstrates this effect. However, in patients with large changes in TBG due to such conditions as acute liver disease or congenital abnormalities, it may be necessary to directly measure binding proteins.

This is particularly useful in diagnosing familial dysalbuminemic hyperthyroxinemia. This syndrome is characterized by elevation in serum thyroxine and the free T4 index. These elevations are due to an albumin that has an abnormal binding site with a much greater affinity for T4 as compared with T3. It is important to differentiate this syndrome, a euthyroid state, from hyperthyroidism, with which it is confused.


******  THYROGLOBULIN (Tg)  ******

Thyroglobulin is the storage form of thyroid hormone precursors and is measured primarily in the monitoring of patients with follicular or papillary thyroid cancer. Injury to the thyroid gland, as occurs in these diseases, results in leakage of thyroglobulin from the follicle into the patient’s serum. It is a useful tumor marker, if present, and reflects recurrence or remission of the primary disease.

Thyroglobulin levels are increased with stimulation by TSH, thyroid stimulating immunoglobulin (TSI), TRH and HCG. Tg is the last parameter to normalize after thyrotoxicosis and is therefore useful in resolving thyroid history in the presence of normal T4 and T3 with clinical symptoms consistent with recent thyrotoxicosis. Tg may also be useful in differentiating subacute thyroiditis, in which elevated levels of Tg are present, from thyrotoxicosis factitia, which is characterized by low Tg.

Normal values:         10 – 40 ng/ml


******  THYROID ANTIBODIES  ******

Detecting the presence of thyroid antibodies in sera is important both for confirming or ruling out autoimmune disease as well as evaluating their possible interference in assays of their respective antigens. T4 and T3 autoantibodies are particularly useful in explaining free T4, T4 or T3 results, which are conflicting with clinical evaluation.

1.      Thyroid microsomal antibodies (TMAb) – are present in about 95% of patients with Hashimoto’s thyroiditis and in 85% of those with Grave’s disease.

2.      Thyroglobulin antibodies (TgAb) – are found in 60% of patients with Hashimoto’s thyroiditis and in 30% of patients with Grave’s disease.

3.      Antithyroid antibodies – titers of these antibodies are low in subacute thyroiditis although it doesn’t give exact information about thyroid function.

4.      TSH receptor antibodies (TRAb) – are of limited clinical use, although they have clarified the pathophysiology of Grave’s disease substantially.

Assay methodology:

1.      Hemagglutination assay
2.      RIA
3.      IRMA
4.      ELISA


******  T3 SUPPRESSION TEST  ******

The principle of the T3 suppression test is that when normal individuals are given thyroid hormone (T3) in quantities adequate to meet peripheral requirements, suppression of endogenous thyroid function should occur. This suppression of function, indicated by iodine uptake, is a result of decreased TS secretion.

After a baseline RAIU is determined, T3 is administered every 8 hours for 7 days. Another RAIU test is then performed. Absence of normal suppression indicates thyroid autonomy present in conditions such as Grave’s disease. Because of the limitations of this test, the T3 suppression test has been supplanted by the TRH stimulation test. There is significant risk to the patient because of the administration of potent hormones, especially to patients with thyrotoxicosis or cardiovascular disease, and because of exposing tissue to irradiation.

T3 suppression should reduce RAIU in normal subjects by at least 50%.


******  TRH STIMULATION TEST  ******

The TRH stimulation test take advantage of the extreme sensitivity of TSH secretion to the feedback mechanism of the hypothalamus–pituitary–thyroid axis. This test can detect abnormalities before thyroid hormone concentrations are outside their reference range.

It is useful in distinguishing between pituitary and hypothalamic hypothyroidism and in confirming mild hypothyroidism in patients whose free T4 and T3 results are equivocal and yet whose other clinical findings are suggestive of thyrotoxicosis.

Procedure of the test

1.      Injection of 200–500 ug of synthetic TRH after determining a baseline TSH level.

2.      Extraction of blood for TSH determination after 15, 30 and 60 minutes.

3.      A flat response indicates hyperthyroidism because elevated levels of T4 and T3 override additional stimulation of the pituitary by TRH.

4.      A flat response differentiates hypothyroidism secondary to hypopituitarism from hypothalamic hypothyroidism in which a delayed peak is seen.

5.      The augmented response seen in primary hypothyroidism provides little additional information from that derived from the combination of an elevated sTSH and decreased free T4.

6.      A flat response is also seen in hyperthyroidism since elevated endogenous levels of T4 and T3 already suppress TSH production to undetectable levels.

7.      A normal response is a two to fivefold increase over basal TSH at 15–30 minutes.


******  RADIOACTIVE IODINE UPTAKE (RAIU)  ******

In this method, the uptake by the thyroid gland 24 hours after the administration of a tracer dose of radioactive iodine is measured.

Many factors interfere with the iodine trapping mechanism of the thyroid gland:

1.      Using organic iodides used for diagnostic X–ray procedures:

a.      Bronchograms
b.      Intravenous pyelography (IVP)
c.       Lipoidal substances used in spinal conditions

2.      Used of dessicated thyroid
3.      Cortisone
4.      ACTH
5.      Antithyroid drugs
6.      Inorganic iodides

Radioactive iodine uptake measures:

1.      Ability of the gland to trap iodine.
2.      Ability of the gland to concentrate iodine
3.      Ability of the gland to convert iodine to the hormonal form.
4.      Ability of the gland to finally release it.

Increased RAIU is seen in:

1.      Hyperthyroidism
2.      Endemic goiter

Decreased RAIU is seen in

1.      Thyrotoxicosis associated with thyroiditis, thyrotoxicosis factitia, and metastases from thyroid carcinoma.

Erroneous results are recorded in:

1.      Iodine administration
2.      Thiouracil
3.      Renal disease
4.      Cardiac disease


******  RADIONUCLIDE IMAGING  ******

Radionuclide imaging is accomplished using Iodine–123 or Technetium–99m pertechnetate and is useful in determining the functional activity of the thyroid gland.

a.      Iodine–123 is administered orally in a dose of 200–300 uCi and a scan of the thyroid is obtained at 8 – 24 hours.

b.      Technetium –99 m pertechnetate is administered intravenously in a dose of 1 – 10 mCi and the scan is obtained at 30–60 minutes.

Images can be obtained with either a rectilinear scanner or a gamma camera. Radionuclide scans provide information about both the size and shape of the thyroid gland and the geographic distribution of functional activity of the gland. Functioning thyroid nodules are called “hot” nodules while the non–functioning ones are called “cold” nodules.


******  FLUORESCENT SCANNING  ******

The iodine content and an image of the thyroid gland can be obtained by fluorescent scanning without administration of a radioisotope. An external source of Americium –241 is beamed at the thyroid gland and the resulting emission of 28.5 keV x–ray from iodide ion is recorded, producing an image of the thyroid gland similar to that obtained with Iodine–123. The advantage of this procedure is that the patient receives no radioisotope and the gland can be imaged even when it is loaded with iodine. The disadvantage of this study is that it requires specialized equipment that may not be generally available.


****** THYROID ULTRASONOGRAPHY OR MAGNETIC RESONANCE IMAGING (MRI)  ******

Thyroid ultrasonography is particularly useful for measuring the size of the gland or individual nodule and for evaluating the results of therapy. It is useful also for differentiating solid from cystic lesions and to guide the operator to a deep nodule during fine needle aspiration biopsy. Thyroid ultrasonography is limited to thyroid tissue in the neck, thus, it cannot be used for susbsternal lesions.

MRI provides an excellent image of the thyroid gland, including posterior or substernal extension of a goiter or malignancy. Both transverse and coronal images of the gland can be obtained and lymph nodes as small as 1 cm can be visualized. MRI is invaluable for the demonstration of tracheal compression for a large goiter, tracheal invasion or local extension of a thyroid malignancy or metastases to local or mediastinal lymph nodes.


******  FINE NEEDLE ASPIRATION BIOPSY  ******


This is the best method for differentiation of benign from malignant thyroid disease. It is performed as an outpatient procedure and requires no preparation

Procedure:

1.      The skin over the nodule is cleansed with alcohol, and, if desired, a small amount of lidocaine can be injected intracutaneously for local anesthesia.

2.      A number 25 1½ inch needle is then inserted into the nodule and moved in and out until a small amount of bloody material is seen in the hub of the needle; the needle is then removed and with a syringe, the contents of the needle are expressed onto a clean slide.

3.      A second clean slide is placed on top of the first slide and a thin smear is obtained by drawing the slides apart quickly. It is dried and fixed then stained.


******  BASAL METABOLIC RATE (BMR)  ******

Basal metabolism refers to the heat production during complete mental and physical rest in the post–absorptive state and is a measure of the vital processes. It measures the ratio of oxygen consumption in the resting fasting state. Basal metabolism is proportional to the surface area of the body which in turn is determined by the height and weight and it varies somewhat with age and sex. Somnolent metabolic rate (SMR) – determination of metabolic rate in a sleepy state; patient is in a state of sleepiness.


******  CARDIAC MUSCLE CONTRACTILITY  ******

With echocardiography, it is relatively easy to measure such indices as the preejection period (PEP), the time from onset of the QRS complex to the opening of the aortic valve or left ventricular ejection time (LVET). These are prolonged in hypothyroidism and shortened in hyperthyroidism. Although these measurements are modified by coexistent cardiac disease, they may be the best objective tests for measuring the peripheral effects of the thyroid hormone action.





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