Thyroglobulin 

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Thyroglobulin testing is primarily used as a tumor marker to evaluate the effectiveness of treatment for differentiated thyroid cancer and to monitor for recurrence.

The reference range of thyroglobulin in euthyroid persons without thyroglobulin antibody (TgAb) using CRM-457 standards is 3-40 ng/mL in countries with adequate iodide intake. In about 8% of the general population, thyroglobulin values are less than 10 ng/mL. In newborn babies, the thyroglobulin level may be as high as 36-48 ng/mL up to 48 hours after birth. [1, 2, 3]

Increased thyroglobulin levels are found in the following conditions:

Untreated and metastatic differentiated thyroid cancers (papillary and follicular; not medullary or anaplastic or other rarer types)

Metastases after initial treatment

Hyperthyroidism; this is not always correlated with increased thyroxine

Subacute thyroiditis

Some cases of benign adenoma

Decreased thyroglobulin levels are found in the following conditions:

Thyrotoxicosis factitia

Goitrous hypothyroidism in infants

Thyroglobulin is measured from the serum after drawing a 5-mL venous blood sample using aseptic precautions and following standard protocol, which includes the following:

Explanation of the purpose of the test and collection procedure

Confirming the indication and assessment of the signs and symptoms of thyroid disease

Noting the use of medications, if any

Advice for overnight fasting prior to collection

Interpretation, counseling, treatment, monitoring, and follow-up by the referring physician

Although it is usually ordered as a single test, thyroglobulin may also be part of the complete thyroid panel, which consists of of thyroid hormone levels and antibody titers.

Thyroglobulin (Tg) is a large, iodinated, glycosylated protein with a molecular mass of 660 kDa. About 70% of the iodide in thyroglobulin exists in the inactive precursors monoiodotyrosine (MIT) and diiodotyrosine (DIT), while 30% is in the iodothyronyl residues thyroxine (T4) and triiodothyronine (T3). Of the weight of thyroglobulin, 8%-10% is carbohydrate, while 0.2%-1% is iodide, depending on the amount of iodine in the diet.

Iodine, which is necessary for the bioactivity of thyroid hormones, is a rare element that is a scarce component of soil; consequently, the normal human diet is iodine-poor. In the average adult, the recommended daily iodine allowance is approximately 150 µg. The recommended amount in pregnant and lactating women is slightly higher. This can usually be achieved without iodine supplementation aside from iodated or iodized salt. The total-body iodine reserve is approximately 20-30 mg.

Consumed iodine is absorbed as iodide in the gut; it is then concentrated in the thyroid gland against a strong electrochemical gradient. This process is energy-dependent and linked to the Na-K ATPase–dependent thyroidal iodide transporter, whose activity, in conjunction with thyrotropin or thyroid-stimulating hormone (TSH), controls the ratio of iodide in the thyroid gland to serum iodide.

In animals, this ratio ranges from 500:1 in the setting of chronic TSH stimulation to 5:1 or less following hypophysectomy. In humans with a normal diet, this ratio is 25:1. The ratio of T4 to T3 is approximately 7:1 when iodine supplies are sufficient. This ratio decreases in iodine deficiency, as does the ratio of DIT to MIT.

Thyroglobulin is thus a very large prohormone—the precursor of T3 and T4, the circulating and actively functioning thyroid hormones. The thyroid synthesizes thyronine from tyrosine, a process which takes place in thyroglobulin. Thyroglobulin contains 2 subunits, which together contain 115 tyrosine residues, each of which is a potential site for iodination. The entire thyroglobulin molecule contains about 5000 amino acids, and it provides the conformation required for tyrosyl coupling and iodide organification necessary for the formation of diamino acid thyroid hormones. It is synthesized in the basal portion of the thyroid follicular cell and moves to the lumen, where it is a storage form of T3 and T4 in the colloid.

Several weeks’ worth of these hormones is contained within a healthy thyroid gland. However, upon TSH stimulation, the colloid re-enters the cell within minutes, and the phosphorylase activity significantly increases. Thyroglobulin is hydrolyzed by various acid proteases and peptidases into its constituent amino acids, including T3 and T4, which are discharged from the basal portion of the cell.

Serum thyroglobulin is affected by iodide availability and intake; therefore, thyroglobulin reference ranges are determined according to geographical indices of iodide levels.

In selecting subjects for the normal cohort for thyroglobulin reference-range determinations, persons with a goiter, a history of cigarette smoking, personal or family history of thyroid disease, the presence of thyroid autoantibodies (TgAb and or thyroid peroxidase antibodies-TPOAb), and a serum TSH level of less than 0.5 mIU/L or greater than 2 mIU/L should be excluded.

Thyroglobulin is produced by the normal thyroid gland, as well as the thyroid gland affected by Graves disease, thyroiditis, and differentiated thyroid carcinoma (including the two most common subtypes—papillary and follicular). In persons with cancer, thyroglobulin levels are increased in the circulation, usually above 100 ng/mL.

Thyroglobulin testing is primarily used as a tumor marker to evaluate the effectiveness of treatment for differentiated thyroid cancer and to monitor for recurrence. It is not helpful for detecting thyroid cancer per se.Specifically, thyroglobulin estimation by means of stimulation following both surgery and I-131 ablation is a good prognostic indicator of disease status. [4] Following successful treatment, thyroglobulin levels are found to decrease; they rise upon recurrence. The test is also used in the workup of hyperthyroidism and hypothyroidism. Interestingly, in children with normal thyroid function, thyroglobulin may be used to estimate both deficient and excess thyroid intake. [5]

Although a baseline thyroglobulin level prior to a total thyroidectomy or radioiodine thyroid ablation can be taken, it is only to determine if the tumor is producing thyroglobulin. Since thyroglobulin is produced by differentiated thyroid cancer, which is usually diagnosed with fine-needle aspiration cytology (FNAC), a preoperative thyroglobulin level is not essential except as an additional point of reference.

In the first few weeks after surgery, the serum thyroglobulin levels depend on the completeness of the surgery, the degree of leakage from the surgical margins, and whether thyroid hormone has been administered to prevent the expected rise of TSH. This last factor is usually a confounding factor in determining the actual thyroglobulin levels.

After complete removal of the gland by surgery or radioactive iodine, L-thyroxin (L-T4) is usually given to combat the resultant hypothyroidism, and serum thyroglobulin levels decrease, with a half-life of approximately 2-4 days. Instead of a single reading, the pattern of change in serum thyroglobulin values in patients receiving L-T4 treatment (hence low TSH) is a better indicator of tumor burden. However, because TSH stimulates serum thyroglobulin more than 10-fold, TSH-stimulated serum thyroglobulin measurements are more sensitive for detecting disease confined to the neck than serum thyroglobulin levels measured during TSH suppression. Poorly differentiated metastatic tumors have blunted (< 3-fold increase) TSH-stimulated serum thyroglobulin responses.

In order to reconcile these seemingly disparate issues of thyroid hormone replacement following the therapeutic destruction of the thyroid gland, a recombinant form of TSH is now available to directly stimulate thyroglobulin production in a patient receiving L-T4 and natural TSH suppression. This is preferable to withholding thyroid hormone replacement in order to stimulate TSH production, and hence detection of thyroglobulin, as was done in the past, when the patient had to suffer symptoms of hypothyroidism for several weeks. If thyroxine is withheld postoperatively, at least 6 weeks should elapse before testing for elevated thyroglobulin levels.

An isolated thyroglobulin reading, whether normal or elevated, does not by itself suggest a recurrence or poor prognosis for thyroid cancer; changes in levels over time are more important when monitoring for recurrence. Serial thyroglobulin testing should be performed at the same laboratory, since variations between test methods at different laboratories are possible.

The serum thyroglobulin test results may need to be confirmed with a radioactive iodine (123) scan; if any residual thyroid tissue or recurrence of thyroid cancer is detected, radioactive iodine (131) may be given for thyroid ablation.

After testing, the patient may resume (or start) thyroid medication and continue all normal activity. Monitoring must continue at regular intervals, as deemed appropriate by the treating physician, to detect metastatic thyroid cancer. This interval may range from a few weeks to a few months after surgery and yearly thereafter.

The following are factors that interfere with the measurement of thyroglobulin levels:

The presence of autoantibodies to thyroglobulin, leading to decreased values; for this reason, thyroglobulin antibody tests are always recommended at the same time as the thyroglobulin test

High thyroglobulin levels in newborns, which fall to normal adult values age 2 years

Lack of sensitivity and specificity of the test

The accuracy of the thyroglobulin assay depends primarily on the specificity of the antibody used and the absence of antithyroglobulin autoantibodies. Thyroid antibodies are also known as thyroid autoantibodies, antithyroid antibodies, antimicrosomal antibodies, thyroid microsomal antibodies, thyroperoxidase antibodies, antithyroperoxidase, antithyroglobulin antibodies, thyroglobulin antibodies, and thyroid-stimulating immunoglobulin.

These antibodies may appear in some individuals and are not affected by behavioral or lifestyle changes. A quarter of patients with well-differentiated thyroid carcinoma have autoantibodies, which decrease the value of thyroglobulin assay. Thyroglobulin autoantibodies also exist in 12.5% of the general population. With successful treatment of the thyroid cancer, thyroiditis, or autoimmune thyroid disease, autoantibodies disappear over time.

A thyroglobulin estimation is necessarily accompanied by a measurement of thyroid antibodies, without which the values obtained may prove misleading. Thyroid antibodies that have to be measured in certain types of thyroid testing include the thyroid peroxidase antibody (TPOAb), thyroglobulin antibody (TgAb), and thyroid-stimulating hormone receptor antibody (TRAb). In case of differentiated thyroid cancer, only the thyroglobulin antibody needs to be measured.

The most frequently used method for measuring thyroglobulin is enzyme-linked immunosorbent assay (ELISA). A detailed description of the contents of the thyroglobulin ELISA kit (EIA-3377), instructions for its use, and interpretation of results are outlined in the kit and are not mentioned in detail here.

Thyroglobulin ELISA EIA-3377

Highly specific anti–human-thyroglobulin antibodies are bound to microwells. Thyroglobulin, if present in diluted serum or plasma, binds to the respective antigen. Washing of the microwells removes unspecific serum and plasma components. Horseradish peroxidase (HRP) conjugated anti–human-thyroglobulin antibodies immunologically detects the bound thyroglobulin, forming an antibody/thyroglobulin/conjugate complex. Washing of the microwells removes unbound conjugate.

An enzyme substrate in the presence of bound conjugate hydrolyzes to form a blue color. The addition of an acid stops the reaction, forming a yellow end-product. The intensity of this yellow color is measured photometrically at 450 nm. The amount of color is directly proportional to the concentration of thyroglobulin present in the original sample.

Other testing methods

Other methods commonly adopted for thyroid testing include the following:

Enzyme multiplied immunoassay technique (EMIT)

Microparticle capture enzyme immunoassay (MEIA)

Clone enzyme donor immunoassay (CEDIA)

Fluorescence polarization immunoassay (FPIA)

Radioimmunoassay (RIA)

Immunoradiometric assay (IRMA)

Immunochemiluminometric assay (ICMA)

Immunoassay techniques such as RIA and IRMA are very sensitive and specific but have now been superseded by nonradioactive labels as in EMIT, FPIA, and ICMA, as they are not associated with concerns regarding nuclear waste disposal.

EMIT is a homogeneous competitive immunoassay in which wash steps are not necessary to separate antibody-bound hormone from nonbound substances or labeled from unlabeled hormone. It is based on the principle of competition between the hormone in the patient’s sample and reagent-labeled hormone for the specific antibody. The labeled hormone is used for detection.

In FPIA, fluorophore-labeled hormone competes with the patient’s hormone for antibody in the homogeneous system. Antibody-bound labeled hormone rotates slowly, emitting lower-energy polarized light. The highest polarized light emitted by the sample or standard (with no hormone) is measured. This test has good sensitivity and specificity and moderate complexity. Compared with RIA, fewer safety regulations need to be followed.

In ICMA, peroxidase-labeled antibody binds with the patient hormone or antigen to form a complex. The addition of luminal or acridinium esters substrate forms an oxidized product that is fluorescent (emits light) for a short time and can be measured by a luminometer.

P. Anthony Weil. The Diversity of the Endocrine System. Harper’s Illustrated Biochemistry. 28th Edn. 436-438.

Frances Fischbach. Chemistry Studies-Thyroid Function Tests. A Manual of Laboratory Diagnostic Tests. 6th Edn. 482-498.

Pagana KD, Pagana TJ. Mosby’s Diagnostic and Laboratory Test Reference. 8th Edn. 916-918.

Lee JI, Chung YJ, Cho BY, Chong S, Seok JW, Park SJ. Postoperative-stimulated serum thyroglobulin measured at the time of 131I ablation is useful for the prediction of disease status in patients with differentiated thyroid carcinoma. Surgery. 2013 Jun. 153(6):828-35. [Medline].

Zimmermann MB, Aeberli I, Andersson M, Assey V, Yorg JA, Jooste P, et al. Thyroglobulin is a sensitive measure of both deficient and excess iodine intakes in children and indicates no adverse effects on thyroid function in the UIC range of 100-299 µg/L: a UNICEF/ICCIDD study group report. J Clin Endocrinol Metab. 2013 Mar. 98(3):1271-80. [Medline].

Jayita Poduval, MS, MBBS, DNB(ENT), DORL Assistant Professor, Department of ENT, Pondicherry Institute of Medical Sciences, India

Disclosure: Nothing to disclose.

Eric B Staros, MD Associate Professor of Pathology, St Louis University School of Medicine; Director of Clinical Laboratories, Director of Cytopathology, Department of Pathology, St Louis University Hospital

Eric B Staros, MD is a member of the following medical societies: American Medical Association, American Society for Clinical Pathology, College of American Pathologists, Association for Molecular Pathology

Disclosure: Nothing to disclose.

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