Clinical Chemistry

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Clinical Chemistry

Authors: Adrenal - Renu Virk, M.D., Cardiac - Larry H. Bernstein, M.D., Thyroid - Nat Pernick, M.D. (see Reviewers page)
Last revised: 10 March 2011
Copyright: (c) 2007-2011, PathologyOutlines.com, Inc.
Disclaimer: This information is intended for pathologists and laboratory personnel, who understand that test interpretation must be made in the context of clinical history using reasonable medical judgment.

Table of contents

Primary references

Index (table of contents in alphabetical order)

A-F: ACTH stimulation, adrenal insufficiency, aldosterone, aspartate aminotransferase (AST), calcitonin, calcium stimulation test, captopril suppression test, CK-MB, CRH stimulation, fludrocortisone suppression test, free thyroxine index

G-L: hyperaldosteronism, hypercortisolism, hyperthyroidism, hypothyroidism, insulin induced hypoglycemia,

M-Z: metyrapone stimulation, nonthyroidal illness, pentagastrin stimulation test, reverse T3, saline suppression test, screening-adult thyroid disorders, screening-neonatal hypothyroidism, screening-pregnancy, T3 receptors, T3 uptake, thyroglobulin, thyroglobulin antibody, thyroid binding globulin, thyroid physiology, thyroid stimulating hormone (TSH), thyroid stimulating hormone receptor antibody, thyroperoxidase antibody, thyrotropin releasing hormone (TRH), thyroxine (T4), thyroxine-free (free T4), triiodothyronine (T3), triiodothyronine-free (free-T3), urinary iodide

Primary references

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American Journal of Clinical Pathology, (AJCP)

Archives of Pathology and Laboratory Medicine (Archives)

Biomed Center

Henry’s Clinical Diagnosis and Management by Laboratory Methods (2006, 21st ed)

Websites: ARUP Laboratories, Specialty Laboratories

Journal search terms: each disease entity listed

Please refer to these primary references for more detailed discussions and additional images

Thyroid related tests

Calcitonin

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Also known as thyrocalcitonin

Physiology: (1) preprocalcitonin (141 amino acids) is produced by parafollicular C cells of thyroid gland; (2) peptide cleavage leads to procalcitonin (116 amino acids), which contains immature calcitonin (33 amino acids); (3) peptidylglycine-amidating mono-oxidase enzyme produces mature calcitonin (32 amino acids)

Function: calcitonin inhibits osteoclasts from resorbing bone, leading to decreased serum calcium (antagonized by parathyroid hormone); levels are controlled by serum calcium

Diagrams: calcitonin production; calcitonin and parathyroid hormone

Methodology: immunometric assays using monoclonal antibodies to N terminal region and C terminal region; results vary by methodology (Tumori 2003;89:566, Clin Chem Lab Med 2007;45:662)

Indications: (a) medullary thyroid carcinoma-to detect residual tissue or metastases after thyroidectomy (get baseline prior to surgery and also measure 6 months post surgery; long doubling times predict good prognosis (J Clin Endocrinol Metab 2005;90:6077); (b) may help monitor other neuroendocrine carcinomas that produce calcitonin

Limitations: medullary thyroid carcinoma is rarely calcitonin negative (World J Surg Oncol 2006;4:97); high levels in newborns should be repeated several weeks later to see if they drop; run pentagastrin stimulation test if calcitonin value > 10 ng/L but not clearly diagnostic

Reference ranges: adults: < 10 ng/L (pg/ml) indicates no medullary thyroid carcinoma

Suggested reference ranges for Advantage ® system: (Clin Chem 2004;50:1828)

Children less than 6 months: < 40 ng/L; 6 months to 3 years: < 15 ng/L; older children same as adults

Adult women: < 5 ng/L; adult men: <12 ng/L

High values: medullary thyroid carcinoma (> 100 ng/L), C cell pathology (in MEN2 patients), neuroendocrine tumors, hypergastrinemia, hypercalcemia

References: Wikipedia

Calcium stimulation test for calcitonin

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A provocative test to expose C cell abnormalities, since it increases calcitonin at all stages of medullary thyroid carcinoma

Indications: has been replaced by genetic testing; may be less sensitive than pentagastrin stimulation test

Procedure: give 2.5 mg/kg of calcium gluconate over 30 seconds, collect blood at time 0, 1, 2 and 5 minutes to determine calcitonin levels

Interpretation: rise in calcitonin > 100 ng/L suggests early medullary thyroid carcinoma

Free thyroxine index

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Also called T7 index (since measurement involves T3 and T4)

Indications: quantitate pituitary resistance to thyroid hormone (J Clin Endocrinol Metab 2000;85:2299); previously the primary method to estimate free T4 (now measure free T4 directly) or screen for thyroid dysfunction (now use TSH)

Calculation: FTI = total T4 x T3 uptake / mean T3 uptake of reference laboratory, or total T4 x serum binding correction factor

Interpretation: usually increased during hyperthyroidism, decreased during hypothyroidism; attempts to correct for changes in total thyroxine caused by changes in thyroid binding globulin

Limitations: nonspecific, low results also associated with non-thyroidal illness; free T4 is more accurate in women taking oral contraceptives (Clin Nucl Med 1981;6:168) and in general

Hyperthyroidism

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See also Hyperthyroidism in Thyroid chapter

Hyperfunction of gland (usually elevated levels of free T3/T4, but see subclinical hyperthyroidism below)

Thyrotoxicosis refers to clinical effects of free thyroid hormone; source may not be thyroid gland

May be masked by nonthyroidal illness syndrome (J R Soc Med 2003;96:185)

Diagnosis: high free T4; TSH may be low (primary hyperthyroidism) or high (secondary hyperthyroidism); in subclinical hyperthyroidism, T3 and T4 are normal but TSH is low

Primary hyperthyroidism: intrinsic thyroid gland abnormality producing excess T4; high free T4, low TSH, normal TRH stimulation test

Secondary hyperthyroidism: abnormal TRH stimulation test (problem is at level of pituitary, including TSH producing tumors), high TSH, high free T4

Subclinical hyperthyroidism: low TSH (< 0.1 µIU/ml), normal T3 and T4 (Eur J Endocrinol 2005;152:1), no clinical hyperthyroidism; may be due to exogenous thyroid (Hormones (Athens) 2006;5:119); patients have increased risk of atrial fibrillation, cardiac death and osteoporosis

T3 hyperthyroidism: 1-4% of hyperthyroid patients; low TSH, high free T3, normal free T4; associated with early treatment of hyperthyroidism with antithyroid drugs

T4 hyperthyroidism: high T4, normal T3; due to primary hyperthyroidism causes, also iodine, amiodarone (Archives 2003;127:e275), pregnancy (2%)

Causes: Graves’ disease (85%-circulating autoantibodies to TSH receptor); overdose of exogenous thyroid hormone, hyperfunctioning multinodular goiter or thyroid adenoma, thyroiditis, struma ovarii, iodide ingestion, choriocarcinoma/hydatidiform mole, maternal Graves’ disease, pituitary adenoma; table of causes

Treatment: beta blockers for symptoms, thionamide-type drugs to block new hormone synthesis, iodine to block release of T4/T3

References: Am Fam Physician 2005;72:623, Med J Aust 2004;180:186, eMedicine #1, #2

Hypothyroidism

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See also Hypothyroidism in Thyroid chapter

May initially have transient hyperthyroidism

May cause macrocytic, nonmegaloblastic anemia with normal RDW

Primary hypothyroidism: due to destruction or ablation of thyroid gland (surgery, radiation, Hashimoto’s thyroiditis, developmental), interference with thyroid hormone synthesis (idiopathic, genetic [J Med Genet 2005;42:379], drugs [lithium, iodide, methimazole, PTU]), iodine ingestion (Intern Med 2007;46:391) or iodine deficiency; T4 is low causing high TSH

Other causes: chronic renal failure has normal TSH, low T3 and T4 (Nucl Recept 2005;3:1), bromide intoxication (AJCP 1988;89:802)

Secondary hypothyroidism: pituitary disorder causes reduced TSH secretion

Tertiary disease: hypothalamic lesion causes reduced TRF secretion

Subclinical hypothyroidism: high TSH, normal T3 and T4; no clinical symptoms of hypothyroidism (Am Fam Physician 2005;71:1763)

Cretinism: hypothyroidism developing during infancy/childhood; may be due to maternal hypothyroidism (maternal T3/T4 crosses placenta and is critical to fetal brain development before fetal thyroid gland develops); now rare due to newborn testing and iodine supplementation (Pediatrics 2006;117:2290)

Myxedema: chronic hypothyroidism in older child or adult

References: Wikipedia, eMedicine #1; #2

Nonthyroidal illness affecting thyroid function tests

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Also called euthyroid sick syndrome or low T4 syndrome

Seriously ill patients (sepsis, myocardial infarction-Angiology 2002;53:699) may have abnormal thyroid function tests without thyroid pathology

Due to reduced serum iodothyronine deiodinases (J Clin Endocrinol Metab 2003;88:3202)

Often caused by malnourishment (JPEN J Parenter Enteral Nutr 2006;30:415)

Initially these patients have low total T3 and free T3; later thyroid binding globulin is disrupted causing low total T4

TSH is initially low to low-normal, then rises to normal or above normal as illness resolves, then returns to normal levels

TSH is suppressed by glucagon, dopamine and high doses of corticosteroids, which complicates interpretation

Common in medical intensive care unit (44%), and associated with poorer prognosis (Metabolism 2007;56:239)

Diagrams: in critical illness, inhibition of 5’ deiodinase causes reduced T3 and increased reverse T3 levels

References: J Clin Endocrinol Metab 1999;84:151, eMedicine

Pentagastrin stimulation test

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Indications: detect residual medullary thyroid carcinoma or other parafollicular C cell (thyroid gland)l pathology (Surgery 1999;126:1089, J Clin Endocrinol Metab 2004;89:515); intraoperative testing may predict lateral neck lymph node involvement (Surgery 2007;141:166)

Note: also measures gastric gastrin release and resulting pH changes (Urol Int 2003;70:178)

Methodology: give 0.5 µg/kg body weight of pentagastrin IV over 5 minutes, collect blood at time 0, 1, 2, 5 and 10 minutes and measure calcitonin levels

Interpretation: 50-100 ng/L indicates possible medullary thyroid carcinoma or other pathology; >100 ng/L indicates probable medullary thyroid carcinoma (J Clin Endocrinol Metab 2007;92:450)

Baseline or post-pentagastrin values > 10 ng/L indicates C cell pathology in MEN2 or residual tissue in medullary thyroid carcinoma patients after surgery

Limitations: elevated calcitonin after testing in patients with pseudohypoparathyroidism (Exp Clin Endocrinol Diabetes 2002;110:86), neuroendocrine carcinoma of foregut (Langenbecks Arch Surg 2000;385:398)

Adverse effects: substernal discomfort,nausea, vomiting, metallic taste, abdominal cramping, flushing,warmth, urgency, esophageal spasm, tachycardia, headache (Clin Chem 2002;48:1505)

Reverse T3

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Major metabolite of T4; 40% of T4 is 5 deiodinated (by 5-deiodinase) peripherally to form reverse T3

No biologic activity

Half life is 4 hours (short)

Bound to thyroid binding globulin

Wilson’s syndrome: questionable entity with no supporting publications (to my knowledge) of reverse T3 dominance causing hypothyroidism and low body temperature

Indications: not routinely measured; serum levels correlate with severity of hyperemesis gravidarum (Arch Gynecol Obstet 2000;264:57)

High levels: nonthyroidal illness (have reduced total serum T3), healthy newborns (J Clin Invest 1975;55:1137), hyperthyroidism, use of amiodarone, propranolol or estrogen, pregnancy (J Clin Endocrinol Metab 1977;44:660), fasting (Thyroid 1992;2:11)

Screening-adult thyroid disorders (not cancer)

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See also screening-pregnancy below

Incidence: neonatal hypothyroidism occurs in 1 per 3 to 5,000 births, increased risk (up to 1 per 1,000 births) with multiple pregnancies (J Clin Endocrinol Metab 2007 May 8; [Epub ahead of print], Eur J Endocrinol 2005;153:765) and in iodine-deficient areas (Pediatr Int 2007;49:76); transient hypothyroidism of prematurity is associated with chorioamnionitis (J Perinat Med 2005;33:514)

Rarely due to maternal autoimmune thyroid disease (Hormones (Athens) 2003;2:113)

Low maternal free T4 in first trimester, even without symptoms, may cause neurodevelopmental difficulties in newborns (Pediatrics 2006;117:161)

Symptoms (if no laboratory screening): hypothermia, constipation, jaundice, poor feeding, hoarse cry, macroglossia, hypoactivity (Bosn J Basic Med Sci 2005;5:26); also extrathyroidal congenital abnormalities (Horm Res 2007;68:272)

Indications: routinely performed on all newborns; screening prevents severe mental retardation if treatment begins within 1 month of birth; mild hypothyroidism may cause cardiovascular mortality or damage to offspring of pregnant women

Methodology: use dry blood spots or cord serum to measure both T4 and TSH

If measure only T4 (checking for low values), have high false positive rate due to prematurity or congenital absence of thyroid binding globulin, and miss patients with elevated TSH but normal T4

If measure only TSH (checking for values of 10 mIU/L or higher), have false positives from premature or severely stressed infants and miss infants with hypothalamic or pituitary disease

Algorithm (flowchart):

Very low birth weight infants (J Med Screen 2005;12:166) and dizygotic twins (An Pediatr (Barc) 2006;65:129) should be retested at 2 weeks and 4-6 weeks to detect late onset hypothyroidism

TSH less than 10 mIU/L - no further action for newborns

TSH 10-20 mIU/L - retest at 2-6 weeks

TSH > 20 mIU/L - endocrine workup needed

Treatment: thyroxine (not mixed with soy [Arch Dis Child 2004;89:37, Endocr Pract 2001;7:193] or iron [South Med J 1997;90:637], which inhibit absorption) and close followup by endocrinologist (J Perinat Med 2005;33:543); may be able to discontinue if clinical and laboratory values are normal after 2-3 years of followup (Zhejiang Univ Sci B 2005;6:1206; treatment usually associated with normal subsequent growth (Pediatr Int 2007;49:443) but intellectual defects may persist (J Clin Endocrinol Metab 2006;91:418)

References: Pediatrics 2006;117:2290, J Pediatr Endocrinol Metab 2006;19:1291, J Clin Endocrinol Metab 2006;91:3370, eMedicine

Screening-pregnancy related thyroid disorders

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Overt thyroid disease is present in 1% of pregnant women, 2-3% have subclinical hypothyroidism, 10-15% have positive anti-thyroid antibodies

All pregnant and lactating women require additional iodine intake; monitor with urinary iodine concentration

Hypothyroidism should be corrected before initiation of pregnancy and euthyroidism maintained throughout (Ther Drug Monit 2006;28:431); however low T4 with normal TSH may not affect perinatal outcome (Obstet Gynecol 2007;109:1129)

Therapy also recommended for subclinical maternal hypothyroidism, although risk to fetus is unknown

Maternal hyperthyroidism (Graves’ disease-Nat Clin Pract Endocrinol Metab 2007;3:470) can cause fetal hyperthyroidism (due to transplacental TSH-receptor antibodies); also associated with maternal hypothyroidism due to anti-thyroid drugs

Maternal autoimmune thyroid disease is associated with increased risk of miscarriage (J Coll Physicians Surg Pak 2006;16:468); these women should have TSH monitored for increased levels

Postpartum thyroiditis occurs in 2-5% (Obstet Gynecol 2006;108:1283)

Thyroid function screening recommended for women with history of thyroid disease (Gynecol Endocrinol 2007;23:138), family history, goiter, thyroid antibodies or autoimmune disorders, signs/symptoms suggestive of thyroid disease, type I diabetes, prior head and neck radiation, history of miscarriage or preterm delivery (J Clin Pathol 2005;58:449)

However, up to 1/3 of women with hypothyroidism would be missed by targeted screening (J Clin Endocrinol Metab 2007;92:203)

References: J Clin Endocrinol Metab 2007 Jun 3; [Epub ahead of print], National Guideline Clearinghouse

T3 receptors

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4 isoforms: alpha1, alpha2, beta1, beta2

Alpha1, Alpha2 and Beta1 receptors are present in most tissue, bind T3 to promote protein synthesis

Beta2 receptor is unique to anterior pituitary gland, hypothalamus and developing ear; mediates negative feedback of TSH (Mol Endocrinol 2005;19:1529); mutations cause thyroid hormone resistance with varying degrees of hypothyroidism

Alpha2 receptor is inhibitory

References: Colorado State University, OMIM 190120 (alpha1), 190160 (beta)

T3 or T4 uptake

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Also called T3 or T4 resin uptake or Thyroid Hormone Binding Ratio (THBR)

Measures proteins, not thyroid hormones

Relative measurement of unoccupied thyroid hormone binding sites on circulating proteins, including thyroid binding globulin (TBG)

Should be reported as a ratio to normal serum

Indications: previously used to calculate free thyroxine index (total T4 x T3 uptake), an estimate of free T4 (now free T4 is ordered directly); attempts to correct for changes in thyroid binding globulin due to liver disease, protein losing states, pregnancy or various drugs

Method: add labeled T3 (or T4) plus resin (a secondary binder) to serum; amount of labeled T3 bound to resin is measured by chemiluminescence; more labeled T3 is bound to resin if more thyroid binding globulins are occupied by thyroid hormone

Recommended to be based on the ratio between absorbent counts divided by the total minus absorbent counts, rather than the ratio between absorbent counts and total counts (see guideline 12)

Interpretation: high with hyperthyroidism, low with hypothyroidism

Limitations: invalid if other proteins or immunoglobulins compete with TBG, including familial dysalbuminemic hyperthyroxinemia (J Clin Endocrinol Metab 2000;85:2786, Thyroid 2004;14:155)

Note: thyroid uptake is also a nuclear medicine scan using I-131 or technetium (reference)

Note: T3 uptake in red blood cells is related to clinical depression (Physiol Res 2006;55:195)

Thyroglobulin

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Prohormone of T3 and T4 synthesized by thyroid follicular cells and cosecreted into serum with T3 and T4

Serum level is affected by thyroid mass, thyroid injury and TSH receptor stimulation

Tyrosine residues of thyroglobulin are iodinated with thyroid peroxidase to produce mono- and diiodotyrosyls, which combine to form T3 (mono+di) and T4 (di+di)

Thyroglobulin releases T3 and T4 into circulation via lysosomal degradation

Indications: used primarily to monitor recurrence of well differentiated thyroid cancer after total thyroidectomy or I-131 treatment (Endocr Regul 2006;40:53); also to diagnose thyroid dysgenesis in congenital hypothyroidism (J Med Genet 2005;42:379) or to distinguish subacute thyroiditis (elevated levels) from factitious thyrotoxicosis (undetectable levels despite elevated T3 and T4 levels)

Methodology: immunoassay

Monitoring recurrence of well differentiated thyroid carcinoma (J Clin Endocrinol Metab 2005;90:5047)

- give patient recombinant human TSH, and measure thyroglobulin 72 hours after last dose

- rise of serum thyroglobulin above 2 ng/ml (if had remnant ablation and receiving hormone suppressive therapy) correlates with presence of recurrent disease

- can confirm recurrent disease by whole body iodine scan

- well differentiated tumors typically have 10x rise in thyroglobuln

- poorly differentiated tumors may not concentrate iodide, may have blunted response to TSH stimulation

- can also monitor change of thyroglobulin level when receiving T4 treatment post-thyroidectomy

Limitations: 20% with thyroid cancer have antithyroglobulin antibodies which interfere with assay (falsely lower serum concentration by binding to thyroglobulin and making it “invisible” to the assay; should determine presence of antibodies when measure thyroglobulin level for tumor monitoring; if antibodies are present, PCR may be useful to measure thyroglobulin; also interference by heterophile antibodies (J Clin Endocrinol Metab 2003;88:3069)

Reference range: < 30 ng/ml (45 pmol/L) (chart)

High values: Graves’ disease, thyroiditis, nodular goiter, well differentiated thyroid carcinoma

Low levels: factitious thyrotoxicosis (surreptitious use of thyroid hormone-levels are undetectable although T3 or T4 are elevated)

Thyroglobulin antibody

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See Stains chapter for immunohistochemistry using Thyroglobulin antibody

Indications: a marker of autoimmune thyroid disease (note: other thyroid antibodies are to TSH and thyroperoxidase); also ordered with serum thyroglobulin, since it may interfere with thyroglobulin assay

Modest correlation with presence of anti-thyroperoxidase antibodies (Autoimmunity 2006;39:497)

Methodology:

(a) immunoassays now most common

(b) quantitative passive hemagglutination: formerly most common, uses chromic chloride hemagglutination or tanned red blood cells, 80% with Hashimoto’s thyroiditis have titers > 1:1000; also present, at lower titers, in Graves’ disease (60%), thyroid carcinoma (30%), pernicious anemia, Sjogren’s syndrome, normals (3-18%)

(c) indirect immunofluorescence microscopy (uncommon): uses monkey thyroid gland tissue; patterns are floccular, dull colloid spaces but bright peripheral fluorescence or diffuse bright uniform colloid staining in ground-glass pattern; 5-8% are false positives with positive immunofluorescence attributed to CA2 (second colloid antigen) but are negative for anti-thyroglobulin or anti-thyroperoxidase by other methods

Concordance between automated methods needs improvement (Clin Chim Acta 2007;376:88)

Interpretation: present in patients with Hashimoto’s thyroiditis, other autoimmune disease, also women with no clinical disease; values < 4 .0 IU/ml are unlikely to interfere with thyroglobulin assay

Thyroid binding globulin (TBG)

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Deficiency does not cause thyroid disease by itself

Increases/decreases cause increase/decrease in total T3 and T4; however free T3 and T4 are unchanged

Physiology: produced in liver; major carrier protein for T3 and T4; has highest affinity, but lowest concentration of the carrier proteins

Other thyroid carrier proteins are transthyretin and albumin, which have lower affinity but higher concentration

Indications: interpreting T3 and T4 levels that do not match clinical findings or other laboratory results (example - total T3 and T4 levels are very low, TSH is normal, free T3 and T4 are normal, patient is clinically euthyroid)

Methodology: chemiluminescence immunoassay

Reference range: 13-39 µg/dL (150-360 nmol/L) (chart)

High values: inherited abnormalities, drugs (clofibrate, estrogens, 5-FU, heroin/methadone), hepatitis, pregnancy, idiopathic

Low values: inherited abnormalities, drugs (androgens, glucocorticoids), liver failure, malnutrition, nephrotic syndrome, idiopathic

References: eMedicine (TBG deficiency), OMIM 314200

Thyroid physiology

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Theodor Kocher (Swiss surgeon) won Nobel Prize in 1909 for his work on thyroid physiology (award presentation speech)

Hypothalamus secretes thyrotropin releasing hormone (TRH), which acts on anterior pituitary to secrete thyroid stimulating hormone (TSH, thyrotropin), which increases thyroid follicular cell update of iodide and stimulates synthesis of T3 and T4; free T3 and free T4 act as negative feedback on hypothalamus and pituitary

Thyroid cells actively take up iodide, then iodinate tyrosine residues on thyroglobulin, then form mono- and di-iodotyrosine, which are coupled to form T3 and T4; thyroglobulin undergoes proteolysis to release free iodotyrosine, T4 and T3; iodotyrosines are deiodinated to reuse the iodide; T4 is deiodinated to T3

Thyroid follicles contain colloid, composed primarily of thyroglobulin, the storage site for T3 and T4

T3 formation: 80% from deiodination of T4 in nonthyroid tissue, 20% produced in thyroid gland from coupling of mono-iodotyrosine plus di-iodotyrosine (i.e. tyrosine plus 3 iodides) and deiodination of T4

T4 is formed from two di-iodotyrosine molecules (i.e. tyrosine plus 4 iodides)

70% of T4 is bound to thyroxine binding globulin (TBG), 20% to transthyretin (formerly called prealbumin), 10% to albumin

T3 primarily binds to TBG, but affinity is only 10% that of T4 to TBG

Free components of T4 and T3 are metabolically active - 0.03% of T4 and 0.3% of T3

Interpretation of thyroid function tests may be difficult if (a) abnormal protein binding proteins, (b) nonthyroidal illness, (c) acute psychiatric illness or mediations that affect thyroid hormone binding or the hypothalamic-pituitary-thyroid axis

Diagrams: chemistry of T3 and T4

References: Wikipedia, Physiol Rev. 2001;81:1097, T3/T4 production and negative feedback, ThyroidManager.org

Thyroid stimulating hormone (TSH)

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Also called thyrotropin

Promotes increased uptake of iodide into thyroid follicular cells by active transport, synthesis of thyroglobulin, formation of T3 and T4, release of T3 and T4 into circulation

Composed of alpha subunit (same sequence as LH, FSH, hCG) and beta unit (differs from other hormones)

Indications: (1) initial screening test for hyper- or hypothyroidism (AJCP 1996;105:11), unless (a) hypothalamic-pituitary-thyroid axis is not intact due to disease or medication (b) there is thyroid hormone resistance, or (c) there was recent therapy for hyper- or hypothyroidism; for these cases, measure free T4 and possibly also free T3 or total T4

(2) annual screening for patients receiving thyroid hormone replacement therapy (wait 6 weeks to retest due to altered dose)

(3) monitor patients with thyroid carcinoma and hormone suppressive therapy (target is 0.5 to 0.10 µU/mL if low risk and < 0.1 µU/mL if high risk

(4) screening of elderly (low yield for other populations) and pregnant women

First generation - RIA; not useful for detecting hyperthyroidism

Third generation - uses chemiluminescent label; detects TSH of .005 mIU/L; American Thyroid Association recommends using assays that have a coefficient of variation of 20% or less for interassay precision

Fourth generation - not widely available yet; more sensitive than third generation, but not needed for most clinical applications

TSH within reference range excludes thyroid dysfunction, but may have normal function with abnormal TSH (low TSH in elderly, clinically euthyroid, normal T4)

Interpretation (serum):

< 0.01 mIU/L - severe thyrotoxicosis (suggestive of Graves’ disease); also nonthyroidal illness plus dopamine or somatostatin

0.01 to 0.1 mIU/L - mild or subclinical hyperthyroidism or acute nonthyroidal illness; also hypothyroidism due to pituitary disorders, glucocorticoids

0.5 to 2.0 mIU/L - target range for replacement thyroxine in primary hypothyroidism

0.5 to 5.0 mIU/L - standard reference range; may include hyperthyroid patients with TSH producing tumors or pituitary resistance to thyroid hormone; some recommend lowering upper limit to 2.5 mIU/L (J Clin Endocrinol Metab 2005;90:5483)

> 5.0 mIU/L - hypothyroidism; also clinically normal patients with antibodies to animals (Postgrad Med J 2006;82:e27) or other interfering substances (Clin Chem 2006;52:541, Clin Chem 2004;50:946)

Nonthyroidal illness: low TSH acutely, high TSH with resolution

Pregnancy: low TSH in first trimester due to TSH-like effects of hCG

Third trimester amniotic fluid (Bayer ADVIA Centaur): reference range is 0.04 to .51 mIU/mL, median 0.10 mIU/mL (AJCP 2007;128:158)

Notes: (a) Henry does not believe reference range should be age adjusted (excluding children), because mild abnormalities may be associated with increased mortality

(b) within the reference range, increasing TSH concentrations are associated with increasing cardiovascular risk parameters (Thyroid 2007;17:243)

References: Clin Chem 2005;51:1480 (reference range)

Thyroid stimulating hormone receptor antibody

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Also called TRAb (TSH Receptor Antibody)

Previously called thyroid stimulating immunoglobulin or long acting thyroid stimulators (LATS)

Present in 85-95% with Graves’ disease

Either stimulating (causing hyperthyroidism), blocking (usually no clinical impact) or no effect

Antibodies cross placenta and may cause thyroid dysfunction in newborns

Indications: not for routine clinical testing; to confirm Graves’ disease in difficult cases, monitor hormone replacement therapy, diagnose neonatal thyrotoxicosis

Methodology: thyrotropin binding inhibiting immunoglobulin technique, RIA

Reference ranges: negative - 0-9%, indeterminate - 10-15%, positive - 16% or more

Interpretation: positive results are consistent with autoimmune disease; low antibody titer before drug therapy for Graves’ disease is a good prognostic factor for remission after drug therapy (Horm Metab Res 2004;36:92) or relapse (Horm Metab Res 2007;39:56), predicts risk of thyroid dysfunction in newborns of mothers with Graves’ disease

References: J Clin Invest 2005;115:1972, Specialty Laboratories

Thyroperoxidase antibody (Anti-TPO)

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Associated with Hashimoto’ thyroiditis (90%) and Graves’ disease (75%), although also present in women with no clinical disease

Antibody is directed against antigen within microsomal fraction of thyroid epithelial cells

Methodology: ELISA, RIA, IRMA; previously indirect immunofluorescence microscopy with substrate of air dried human or cryostat monkey tissue, looking for cytoplasmic but not nuclear staining of thyroid follicular cells

Concordance between automated methods needs improvement (Clin Chim Acta 2007;376:88)

Interpretation: does not predict relapse of Graves’ disease after carbimazole treatment withdrawal (Thyroid 2006;16:1041)

DD: MELAS syndrome (Pediatr Neurol 2007;36:414)

Thyrotropin releasing hormone (TRH)

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Also called thyrotropin releasing factor

Produced by hypothalamus; enhances TSH synthesis and stimulates its secretion; also affects prolactin, and other pituitary hormones to a lesser extent

Also produced in pancreatic beta cell, where it colocalizes with insulin, highest levels in neonates (Acta Biomed 2007;78 Suppl 1:216)

Feedback inhibition due to circulating T3 and T4

Regulated also by leptin, which mediates food appetite

Resistance occurs rarely (OMIM 188545)

Methodology: can measure by RIA but specimen must be frozen

TRH stimulation test: (1) measure baseline TSH, (2) give IV bolus of TRH, (3) measure TSH (should rise within 5 minutes, peak in 20 minutes); "fold increase" is a better measure of response than absolute increase

Interpretation:

Exaggerated - primary hypothyroidism

Suppressed - hyperthyroid patients (including Graves’ disease), also multinodular goiter, renal failure, Cushing’s syndrome, depression, drugs

No response - pituitary disorder (may not be necessary for diagnosis, J Clin Endocrinol Metab 2003;88:5696)

Thyroxine (total T4)

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Note: in this section, “T4” means total T4, unless stated otherwise

Indications: TSH is a more sensitive and specific screening test for thyroid dysfunction than total T4, but total T4 may be helpful in interpreting TSH results

Physiology: usual daily production is 110 nmol/day; half life is 7 days

100% produced in thyroid

50% of T4 is 5’ deiodinated to form T3, 40% is 5 deiodinated to form reverse T3 via 5-deiodinase

Higher T4/T3 ratio when abundant iodide

70% of T4 is bound to thyroxine binding globulin, 20% to transthyretin (thyretin), 10% to albumin; up to 5% to IgM, IgG and lipoproteins

< 1% of T4 is free, but this form is active; protein bound T4 is inactive and does not enter cells

Altered protein-binding characteristics:

(a) account for more abnormal T4 values than actual thyroid dysfunction

(b) may be due to drugs that increase protein binding and cause increased T4; patient may be clinically euthyroid; recommend measuring free T4

(c) may be due to familial dysalbuminemic hyperthyroxinemia (increased albumin binding of T4), clinically euthyroid (J Pediatr Endocrinol Metab 2002;15:801)

(d) patient may be euthyroid with high T4 but peripheral resistance (Best Pract Res Clin Endocrinol Metab 2006;20:529)

Infants: TSH surge at birth causes increased T4 levels (Thyroid 1999;9:71)

Thyroxine administration: T4 levels usually constant

T3 administration: does not affect T4 levels

Laboratory testing: immunoassay

Interpretation: primary hypothyroidism - low T4 and high TSH

primary hyperthyroidism - high T4 and T3 and low TSH

T4 thyrotoxicosis - high T4, low/normal T3; due to iodine, beta blockers, amiodarone, steroids or nonthyroidal illness

T3 thyrotoxicosis - low/normal T4, high T3, low TSH

Severe nonthyroidal illness - low T3, low T4 - associated with poor prognosis

Euthyroid hyperthyroxinemia - high T4, normal TSH, otherwise euthyroid; due to increased binding proteins associated with estrogens (pregnancy) or liver disease, psychiatric illness (Acta Psychiatr Scand 2006;114:132), familial dysalbuminemia; recommended to measure free T4, which correlates better with thyroid functional status

Reference range: serum - 5-12.5 µg/dL (lower in children, upper limit is higher in pregnancy, chart); amniotic fluid (Bayer ADVIA Centaur) - not detectable (AJCP 2007;128:158)

Note: within laboratory variation is frequently high (Archives 2005;129:318)

Conversion factor: multiply by 12.87 to convert from µg/dL to nmol/L (conversion chart)

Thyroxine-free (free T4)

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Physiology: biologically active fraction of T4 in circulating blood (0.03% of total T4)

Patients appear to have a genetically determined free T4 setpoint with significant individual variation; initially, TSH secretion shows marked changes in response to small free T4 changes

Previously estimated by free thyroxine index

Indications: evaluate thyroid function; free T4 together with TSH is most accurate assessment of thyroid status; free T4 is better than total T4 since (a) free T4 reflects biologically active hormone, (b) free T4 is not affected by changes in thyroid binding proteins or their binding characteristics

Methodology: high-affinity hormone antibody, immunoassay or equilibrium dialysis

High affinity hormone antibody

- Used on most automated platforms

- Measures fractional occupancy of hormone antibody-binding sites

- Free hormone concentration is inversely proportional to number of unoccupied antibody binding sites

- Quantify using hormone labeled with fluorescence or chemiluminescence

- Convert to free hormone levels using calibrators

Diagram: 2 step competitive binding technique

Immunoassay:

- commonly used

- affected by changes in binding protein concentration or nonthyroidal illness (Clin Chem 2007;53:985)

Equilibrium dialysis:

- not affected by changes in binding protein concentration or nonthyroidal illness

- time consuming / not practical for most laboratories

- dialyze sample in nonprotein buffer against ultrafiltratable membrane, until free T4 reaches equilibrium on both sides of membrane, then analyze T4 in protein-free solution

Reference ranges:

serum: 0.7-1.7 ng/dl (up to 2.2 ng/dl at 0-30 days of life)

amniotic fluid (Bayer ADVIA Centaur): less than 0.10 to 0.77 ng/dL (1.29-9.93 pmol/L, AJCP 2007;128:158)

Limitations: (a) results of high affinity hormone antibody or immunoassay may be spurious if binding globulin abnormality, T3 or T4 autoantibodies (J R Soc Med 2003;96:50), pregnancy, nonthyroidal illness, carbamazepine or phenytoin, premature infants (J Perinatol. 2004;24:640); recommended to use equilibrium dialysis methodology

(b) within laboratory variation is frequently high (Archives 2005;129:318)

Interpretation:

increased - Graves’ disease, toxic multinodular goiter or toxic adenoma, iatrogenic/factitious; transient increase in subacute thyroiditis or Hashimoto’s thyroiditis; rarely increased in thyroid cancer, secondary to amiodarone, secondary to pituitary disease

decreased - primary, secondary or tertiary hypothyroidism, tissue resistance to thyroid hormone

Triiodothyronine (total T3)

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Physiology: usual daily production is 50 nmol/day

80% from deiodination of T4 in nonthyroid tissue, 20% arises in thyroid gland from coupling of mono-iodotyrosine plus di-iodotyrosine (i.e. tyrosine plus 3 iodides) and deiodination of T4

This conversion from T4 is blocked by glucocorticoids, amiodarone and propranolol

Metabolized by conjugation with sulfate and conversion to triiodothyroacetic acid (triac)

More T3 is free than T4 (.3% vs. .03%); free T3 is active, protein bound form is inactive and does not enter cells

Values usually parallel T4, but not are usually measured

With nonthyroidal illness (myocardial infarction), T3 levels may be low, although TSH normal

Low T3 syndrome is a strong predictor of death in cardiac patients (Circulation 2003;107:708)

Indications: to diagnose T3 thyrotoxicosis (5% of hyperthyroidism, more common with iodine deficiency), particularly if T4 is normal or mildly elevated

Methodology: competitive immunoassay using enzymes, fluorescence or chemiluminescence

Reference range: serum - 60-160 µg/dL (0.9 to 2.5 nmol/L) ??, higher upper limit in pregnancy

Interpretation:

May be normal in hyperthyroidism if nonthyroidal illness or taking drugs (amiodarone or propranolol) that reduce T4 to T3 conversion

T3 gives clinicians a sense of the severity and recovery from hyperthyroidism, because it increases more and decreases faster than T4

Note: T3 normal in 15-30% of hypothyroid cases, so not diagnostic; depressed only if severely hypothyroid (T4 < 2 µg/dL, 32 nmol/L)

Limitations: difficult to interpret if receiving thyroid preparations containing T3

Conversion factor: convert from ng/dL to nmol/L by multiplying by 0.0154 (conversion chart)

Triiodothyronine-free (free T3)

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Active component of T3; about .3% of total serum T3 is free

Reference range (adults): 2.4-4.2 pg/mL

Urinary Iodide

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Physiology: required for thyroid hormone production and euthyroid state; most ingested iodide is rapidly excreted in urine, thus serves as estimate of recent dietary iodine intake

Methodology: mass spectrometry, digestion methods using colorimetry

Indications: estimate dietary iodine (1/3 of world’s population has iodine deficiency disorders); often used in epidemiology studies

Reference range: > 100 µg/L is not associated with iodine deficiency or goiter; 50-99 µg/L associated with mild deficiency, goiter in 5-20%, 20-49 µg/L associated with moderate deficiency, goiter in 2-30%, <20 µg/L associated with severe deficiency, goiter in >30%

End of Clinical Chemistry chapter