Chapter 5 .2 ThyroidFunction test For medical laboratory

Editor's Notes

• #6: The thyroid gland lies in the anterior neck, anterior and lateral to the upper trachea. The thyroid gland is a small tissue situated in the neck just below the larynx (voice box). Its hormones increase the basal metabolic rate and are necessary for proper growth and development. The thyroid also excretes calcitonin, a hormone that participates in the regulation of plasma Ca2+ concentration by inhibiting bone resorption.
• #7: The gland has two lateral lobes connected by an isthmus. The isthmus crosses the trachea anteriorly at about the level of the second tracheal cartilage. A pyramidal lobe may be present at the isthmus, extending upwards towards the hyoid. The lateral lobes lie against the trachea and esophagus inferiorly and against the cricoid and thyroid cartilages superiorly. The thyroid gland is enclosed within the pretracheal fascia. In practice this means that as the trachea moves, the thyroid will move also. The trachea and thyroid move up during swallowing.
• #8: The circulating thyroid hormones, thyroxine (3, 5, 3’, 5’ -tetraiodothyronine T4 and 3, 5, 3’- triiodotyrosine, T3) whose structures are shown are iodinated derivatives of the amino acid tyrosine. Their immediate precursors are monoiodotyrosine (MIT) and diiodotyrosine (DIT), but these latter compounds do not appear to any significant extent in plasma except under rare pathologic conditions. Neither MIT nor DIT has any hormonal activity.
• #9: The various events occurring in the synthesis and release of thyroid hormones occur in five steps. In step 1, the entry of inorganic iodide ion into the thyroid cell is facilitated by an active transport system that can be inhibited by anions such as ClO4-, SCN-, or high concentrations of I-. The I- is trapped once it enters the thyroid gland and does not diffuse out readily. In step 2, the I- oxidized with the help of a peroxidase enzyme system to an active state, presumably I2, which prepares it for step 3. The activated iodine reacts in step 3 with tyrosine residues in the protein, thyroglobulin, a large glycoprotein present in the thyroid cells. The products are MIT and DIT residues attached to thyroglobulin, with the iodine in the 3' position in MIT and in the 3, 5- positions in DIT. The MIT and DIT residues are oxidatively coupled In step 4 by an enzyme system to form T3 and T4 residues attached to thyroglobulin; two DIT residues are condensed to form T4, whereas the coupling of MIT and DIT yields T3.
• #10: The thyroid gland develops early in the fetus with a follicular structure and a colloid region. These are the sites for the synthesis of thyroid hormones. The thyroid gland is unusual among endocrine glands in that a hormone precursor is stored extracellularly. The follicular cells synthesize thyroglobulin which is released into the colloid. In the colloid iodine is added. The hormones tetraiodothyronine (thyroxine) and triiodothyronine are released to pass into the capillary bed and into the circulation.
• #11: T4 is the principal iodinated hormone secreted by the thyroid; peripheral tissues convert T4 to T3, the active hormonal entity that enters tissue cells. About 70% of the plasma T3 is derived from the tissue deiodination of T4, while the rest is of thyroidal origin. A reverse T3 (rT3) in which the iodine molecules are in the 3, 3’, 5’ position instead of the 3, 5, 3’ position of T3, is formed to a minor extent during the deiodination process. Reverse T3 has no hormonal activity and its determination is of limited usefulness. The thyroglobulin with its mixture of iodinated tyrosyl and thyronine residues is stored in the thyroid follicles until the signals for release are given. In step 5, some of the stored iodinated thyroglobulin is degraded by a cellular protease to yield a mixture of free MIT, DIT, T3, and T4. MIT and DIT are enzymatically deiodinated, and the I- is recycled within the gland (step 6). T3 and T4 are poorly soluble in plasma and are transported primarily by a thyroid-binding globulin (TBG), a plasma protein that carries the majority (70 to 75%) of circulating T4 and T3. The remaining 25 to 30% of T4 is transported by albumin and pre-albumin, but T3 has no affinity for prealbumin and circulates only with TBG and albumin. TBG has a single binding site for T4 or T3, but in normal individuals only about 30% of the binding sites are occupied. A very low concentration of free (unbound) T4 and T3 exists in plasma in equilibrium with the protein-bound forms. Only the free hormone (FT4 and FT3) can bind to receptors on cell surfaces.
• #12: The releasing hormone is termed TRH and is made by the hypothalamus. It stimulates release of TSH from the anterior portion of the pituitary gland. TSH stimulates the thyroid gland to make T4 (and some T3). The action of thyroid hormones are: increases metabolic rate of cells, ATP and heat production, breakdown of liver glycogen and are calorigenic, or causes energy usages.
• #13: The hypothalamus releases thyroid releasing hormone (TRH) in response to low levels of thyroid hormones. TRH binds to receptors in the anterior pituitary which result in production and release of thyrotropin/ thyroid stimulating hormone (TSH). TSH binds to receptors in the follicular cells of the thyroid causing the follicular cells to synthesize thyroglobulin which is released into the colloid. In the colloid iodine is added. The hormones tetraiodothyronine (thyroxine) and triiodothyronine are released to pass into the capillary bed and into the circulation. This is the typical positive feedback mechanism. High levels of thyroid hormones feedback to the hypothalamus and pituitary to inhibit production and release of TRF and TSH. This is negative feedback. Thyroid hormone release continues in this cycle of positive and negative feedback to maintain homeostasis and normal levels of hormones. The plasma concentration of thyroid hormones in normal individuals is kept relatively constant by a sensitive negative feedback control by T4 on pituitary release of TSH. The hypothalamus, with its releasing hormone (TSH), and the thyroid gland, with its secretion of T4, interact in a dynamic fashion to regulate T4 (and T3) levels. Thus the evaluation of thyroid status is not a simple procedure because it does not depend solely on the measurement of circulating thyroid hormones.
• #14: The serum concentrations of both T4 and T3 are usually elevated in hyperthyroidism, with the few exceptions in which only the T3 may be abnormal. Both T4 and T3 levels are increased in conditions that raise the plasma concentration of TBG (pregnancy, taking of birth control pills or estrogens). There is no increase in the basal metabolic rate, however, because the extra T4 and T3 are bound to the newly synthesized TBG as equilibrium between the free and bound hormones is maintained. The person is euthyroid (normal thyroid function) be cause the level of free hormone, the biologically active entity, remains essentially unchanged. Free T4 or Free T4 Index determined from T3 Uptake test can help to determine when TBG is elevated.
• #15: The picture and description about Grave’s disease, explains the most common cause of hyperthyroidism. Goiter is a term for enlarged thyroid. Exophthalmos is the term for bulging eyes. Heat intolerance means that the patient feels excessively warm and body temperature may be slightly elevated. Psychological and behavioral symptoms such as anxiety also occur.
• #16: The main difference with secondary hyperthyroidism compared with Grave’s disease is that 2nd hyperthyroidism is caused by pituitary disease.
• #17: Causes include autoimmune diseases that destroy the functional cells of the thyroid gland in adults as well as infants born without a functional thyroid gland. Screening of newborns for hypothyroidism: In some states, it is customary to measure serum T4 and to check all low or questionable values with a TSH test; an increased TSH confirms a diagnosis of hypothyroidism. In other states, the TSH assay may be performed first, and elevated values confirmed by a decreased T4 concentration. It is important to make the diagnosis early and institute treatment with T3­ or T4 before there is arrested development. A serum T4 < 6.0 g/dL should be confirmed by finding serum TSH> 30U/mL and vice versa. The serum concentrations of T4 and T3 are usually decreased in hypothyroidism, but there may be some overlap with the reference range in mild cases. Their concentrations are also lowered when the TBG level has been decreased by disease (nephrosis, in which there is loss of TBG and the other binding proteins into the urine, and in liver disease, in which there is decreased synthesis of TBG), by medications that reduce the synthesis of TBG (androgens, anabolic steroids) or compete with T4 and T3 for binding sites (aspirin, phenytoin, tolbutamide and others). The lowering of plasma total T4 and T3 concentration accompanying a decrease in TBG concentration has no effect on the basal metabolic rate. The TBG level is decreased and therefore the protein bound T4 and T3 are decreased. But Free T4 is normal so this is not a true hypothyroidism. Some reference laboratories are able to measure free T4 to determine this. Otherwise, correction formulas for calculating free T4 Index are available.
• #18: This condition can result from disease of the pituitary such as following childbirth, particularly if extensive blood loss and shock occurred; such as following cerebral vascular accident/stroke or tumors.
• #19: Chemiluminescent immunoassay and radioimmunoassay are the common methods for thyroid hormone but RIA is the historical method.
• #20: This same method may be used for total T4 or T3. The labeled tracer is different (either T4 or T3). First, the patient sample has a protein denaturant added that detaches thyroid hormones from their binding proteins. In competitive radioimmunoassay a hormone acts as an antigen in the system. The test kit provides a predetermined amount of 125I labeled-T4 (or T3) and antibody to T4 or T3. The labelled-T4 (or T3) competes with the patient sample T4 (or T3) for the antibody. After equilibration the antibody - bound hormone (labeled and unlabeled) is separated from the unbound. The detection system (gamma radiation counter) determines the amount of reagent antibody needed to bind to a known amount of labeled T4 (or T3). This relates inversely to the amount of patient T4 (or T3)in the sample. Concentration of the patient T4 (T3) is then determined.
• #22: The T3 uptake (T3U) test provides an indirect estimate of the binding capacity of the plasma thyroid-binding proteins (TBG or TBG + albumin + prealbumin, depending on the conditions of the assay). This is a test for TBG levels. The term T3 in the name of the test refers to the reagent used. Serum is incubated with 125I-T3; the labeled hormone becomes firmly attached to all unoccupied binding sites on the TBG. The excess, unattached 125I-T3 is removed by the addition of a suitable, solid adsorbent (frequently a resin, but other materials including antibody bound to a solid matrix are sometimes used) and counted in counts/ minute (cpm). T3U is the percent of labeled hormone taken up by the adsorbent and is inversely related to the unoccupied binding sites on TBG. cpmr is the counts/ minute from the reference and cpmp is the counts per minute of the patient sample. A serum of known T3U is used for calibration. T3U measurements are gradually being replaced by direct, immunoassay measurement of TBG. T3U values are usually calculated as follows: %T3U = (cpmp /cpmR) x % T3UR To determine the Free T4 Index (estimate of Free T4) use the formula: FTI = T3 uptake X T4 concentration
• #23: Free thyroxine levels are measured typically with an immunoassay methodology. A major component of this procedure involves separating the trace amounts of free thyroxine from protein bound thyroxine. Separation is typically achieved by ultracentrifugation using a millipore filter prior to analysis with immunoassay. Fluorescent or chemiluminescent immunoassays are popular methods to analyze free thyroxine levels.
• #26: Patient results should be compared with the appropriate reference ranges to interpret if hyper- or hypothyroidism exists in comparison with other relevant results such as TSH In some countries, such as USA, the TSH assay has become more sensitive and reliable than the Free T4 or total T4 test so it is used to screen for hyper or hypothyroidism. Therefore, it is important to keep in mind the expected TSH results in primary and secondary hyper and hypothyroidism when interpreting results.