LABORATORY TESTS FOR EVALUATION OF THYROID DISORDERS Maryam

LABORATORY TESTS FOR EVALUATION OF THYROID DISORDERS Maryam Tohidi Anatomical & clinical pathologist Research Institute for Endocrine Sciences

v v v - Maryam Tohidi / RIES - (15 -25 gr), (12 -20 gr), … 2 lobes connected by isthmus, located anterior to the trachea. Each lobe have about 20 -40 follicles with central lumens containing: Colloid (a protein reach material) Thyroid hormones Thyroglobulin (Tg) Other glycoproteins. Thyroid cells: Foillicular epithelial cells & para-follicular C cells Other non- parenchymal elements (Fibeoblasts, endothelial cells, lymphocytes, …)

Maryam Tohidi / RIES

T 3 RECEPTORS The effects of free thyroid hormones are mediated by T 3 receptors located in the nucleous of cells. v 4 isoforms of T 3 receptor: 1, 2, 1 & 2. v 1 & 1 on most tissues v Maryam Tohidi / RIES v 1 : widely expressed and especially high expression in cardiac and skeletal muscles v 1: predominately expressed in brain, liver and kidney v 2 is unique to the pituitary & some regions of hypothalamus (negative feed back)

STEPS OF THYROID HORMONE SYNTHESIS & SECRETION Maryam Tohidi / RIES 1 - Active transport of inorganic iodide into the cells by sodium- iodide symporter in the baso-lateral membrane (role of TSH & serum iodide level) 2 - Iodination of tyrosine residues on Tg (a large glycoprotein synthesized by RER) to form MIT & DIT (oxidative reaction by TPO) 3 - Coupling of iodotyrosine molecules within Tg to T 4 & T 3 (oxidative reaction by TPO) 4 - Proteolysis (lysosomal degredation) of Tg with release of free iodotyrosine, T 4 & T 3 & secretion of iodothyronin in to the circulation

Maryam Tohidi / RIES

Maryam Tohidi / RIES

BOUND VERSUS FREE THYROID HORMONES 100% of circulating T 4 is of thyroidal origin v About 50% of T 4 is monodeiodinated to T 3 (5’ position) & 40% to reverse T 3 (5 position inner ring) v 20%of T 3 is of thyroidal origin & 80% produced enzymatically in non- thyroidal tissue (liver & kidney) by 5’monodeiodination v T 4: 70% bound to TBG, 20% transthyretin (prealbumin), 10% albumin v T 3: mostly bound to TBG v Free T 4: 0. 03 % of total T 4 v Free T 3: 0. 3% total T 3 v Free hormones are biologically active. v Maryam Tohidi / RIES

ALTERATIONS IN TBG v ü ü ü Maryam Tohidi / RIES ü Increase: Pregnancy Genetic Acute & chronic hepatitis idiopathic Drugs: (Estrogens, OCP, Tamoxifen, Clofibrate, methadone, Heroin, Phenothiazines) Decrease: Liver failure Nephrotic Syn. Malnutrition idiopathic Genetic : complete or partial deficiency Drugs: (Androgens, Glucocorticoids, Phenytoin, High doses of salicylates, including aspirin)

TRH Serum TRH assay is not useful for evaluation & diagnosis of thyroid disorders because: - It is difficult to develop a specific Ab for immunoassay. - Low concentration in the peripheral circulation. Maryam Tohidi / RIES v

TRH STIMULATION TEST An intravenous bolus of 200– 400 µg TRH is injected. Peak TSH secretion occurs 15 min after administration of exogenous TRH. Failure of TSH to rise means truly suppressed TSH level. The availability of the fourth generation of TSH assay (ICMA) has rendered the TRH stimulation test obsolete. Maryam Tohidi / RIES

THYROID DISEASES v Thyroid dysfunctions: Hyperthyroidism v Hypothyroidism v Thyroid enlargement or nodules Non-neoplastic lesions hyperplasia, thyroiditis v Neoplastic lesions - Benign - Malignant v Maryam Tohidi / RIES v (Benign nodular goiter,

THYROID FUNCTIONAL STATE Euthyroid state v Subclinical hypothyroidism v Overt hypothyroidism v Subclinical hyperthyroidism v Overt hyperthyroidism v Maryam Tohidi / RIES

LABORATORY TESTS FOE EVALUATION OF THYROID DISORDERS TSH T 4 T 3 Free T 4 Free T 3 resin uptake Anti- TPO Anti- Tg Thyrogolbuline TSH receptor Ab-blocking Ab TSH receptor Ab TSI (previously known as thyroidstimulating immunoglobulin) Maryam Tohidi / RIES

TSH A glycoprotein secreted by the pituitary v alpha subunit (LH, FSH, h. CG) & beta subunit v - - Immunoassay for TSH: First generation (1965) Second generation(1980 s- IRMA), sensitivity 0. 1 -0. 2 µIU/L Third generation, sensitivity 0. 005 µIU/L Maryam Tohidi / RIES v

ELEVATED SERUM TSH LEVEL Hypothyroidism causes: TSH-secreting pituitary tumor Thyroid hormone resistance Due to laboratory interference Maryam Tohidi / RIES Rare (most common cause)

SUPPRESSED SERUM TSH LEVEL Thyrotoxicosis Maryam Tohidi / RIES * After treatment of hyperthyroidism (because TSH can remain suppressed for several months) During the first trimester of pregnancy (due to h. CG secretion) In response to certain medications (e. g. , high doses of glucocorticoids or dopamine). Factitious thyrotoxicosis

ASSAYS FORT 3 & T 4 & FREE HORMONES Immunoassay for Total forms v Free hormones: - Physical separation of the unbound hormone fraction by ultracentrifugation or equilibrium dialysis - Immunoassay methods affected by illness or changes in binding proteins that are more resolved now) v Maryam Tohidi / RIES

T 3 R-UPTAKE TEST Maryam Tohidi / RIES The test is conducted by adding a known, excess amount of 125 I labeled T 3 to a measured volume of serum. The added radiolabeled T 3 binds to and saturates the unoccupied TBG sites and does not displace the more tightly bound T 4. The excess, unbound 125 I T 3 is then separated from the serum bound 125 I T 3 by adding an adsorbent, such as ion exchange resin beads and centrifuging. The serum is decanted, the beads are washed and then counted in a scintillation counter.

Hyperthyroid cases exhibit a decreased concentration of unoccupied TBG sites and an increased T 3 resin uptake. Hypothyroid patients have increased unoccupied TBG sites and decreased T 3 resin uptake. Maryam Tohidi / RIES

Maryam Tohidi / RIES

FREE THYROXINE INDEX (FT 4 I) Calculation of Free T 4 Index = Total T 4 x T 3 uptake. example: T 4 = 13. 0, T-uptake= 29% FT 4 I: 13. 0 X 29%= 3. 77 (1. 33 -4. 68) TSH= 3. 2 (0. 3 -4. 5) Maryam Tohidi / RIES T 4= 4. 8 T- Uptake= 26% FT 4 I: 4. 8 X 26%= 1. 25 (1. 33 -4. 68) TSH= 5. 6 (0. 3 -4. 5)

THYROID FUNCTIONAL STATES Symptoms TSH (m. IU/L) free T 4 yes > 10. 0 decreased Subclinical hypothyroid no 4. 5 -10. 0 normal Euthyroid no 0. 45 -4. 5 normal Subclinical hyperthyroid no 0. 1 -0. 45 normal Thyrotoxicosis yes < 0. 10 increased Maryam Tohidi / RIES Overt hypothyroid

PRIMARY HYPOTHYROIDISM TSH: v T 4: v T 3: N or v FT 4: v T 3 RU: v FT 4 I: v anti TPO: N or v anti Tg: N or v Maryam Tohidi / RIES

HASHIMOTO, S THYROIDITIS TSH: v T 4: N or v T 3: N or v FT 4: N or v anti TPO: v anti Tg: v Maryam Tohidi / RIES

GRAVES’ DISEASE TSH: v T 4: v T 3: v FT 4: v T 3 RU: v FT 4 I: v anti TPO: v anti Tg: v Tg: (Not needed for diagnosis & management) v Maryam Tohidi / RIES

NON- THYROIDAL ILLNESS Maryam Tohidi / RIES Abnormal thyroid tests without underlying thyroid disorders. v TSH: Variable v T 4: N or v T 3: v FT 4: Variable v anti TPO: negative v anti Tg: negative

CASE 1 A 32 y/o woman (4. 5 -12. 0) T 3: 251 ng/m. L (60 -220) TSH: 2. 48 µIU/m. L (0. 28 - 4. 5) T-Uptake: 29 % (25 -35) FT 4 I= (13. 8 * 29)/100 = 4. 0 (1. 33 -4. 68) Maryam Tohidi / RIES T 4: 13. 8 µg/d. L

CASE 2 A 32 y/o woman (4. 5 -12. 0) T 3: 251 ng/m. L (60 -220) TSH: 0. 18 µIU/m. L (0. 28 - 4. 5) T-Uptake: 37 % (25 -35) FT 4 I= (13. 8 * 37)/100 = 5. 11 (1. 33 -4. 68) Maryam Tohidi / RIES T 4: 13. 8 µg/d. L

CASE 3 A 58 y/o woman (4. 5 -12. 0) T 3: 85 ng/m. L (60 -220) TSH: 8. 43 µIU/m. L (0. 28 - 4. 5) T-Uptake: 28 % (25 -35) FT 4 I= (6. 2 * 28)/100 = 1. 74 (1. 33 -4. 68) Anti- TPO= 356 IU/m. L (up to 40) Maryam Tohidi / RIES T 4: 6. 2 µg/d. L

CASE 4 A 73 y/o man (4. 5 -12. 0) T 3: 62 ng/m. L (60 -220) TSH: 23. 7 µIU/m. L (0. 28 - 4. 5) T-Uptake: 23 % (25 -35) FT 4 I= (4. 1 * 23)/100 = 0. 94 (1. 33 -4. 68) Anti- TPO= 356 IU/m. L (up to 40) Maryam Tohidi / RIES T 4: 4. 1 µg/d. L

CASE 5 A 46 y/o man (4. 5 -12. 0) T 3: 283 ng/m. L (60 -220) TSH: < 0. 005 µIU/m. L T-Uptake: 35 % (0. 28 - 4. 5) (25 -35) FT 4 I= (14. 9 * 35)/100 = 5. 22 (1. 33 -4. 68) Anti- TPO= 85 IU/m. L (up to 40) Maryam Tohidi / RIES T 4: 14. 9 µg/d. L

THYROGLOBULIN v v Maryam Tohidi / RIES v Normal individulas: up to 30 ng/m. L (different expected values in various kits) Reflects thyroid mass, thyroid injury & TSH stimulation Increased in Graves disease, thyroiditis & large nodular goiter. Factitious thyrotoxicosis: undetectable Tg level. Clinically indicated only in follow- up of well differentiated thyroid cancers.

Maryam Tohidi / RIES Thyroglobulin (h. Tg) reflects thyroid mass Normal

THYROGLOBULIN AS A TUMOR MARKER For Maryam Tohidi / RIES well differentiated thyroid cancers (PTC & FTC) When Anti- Tg is not positive. Case presentation

TRAB (STIMULATING & BLOCKING) Mainly thyroid stimulating Ab (TSAb) in Graves, disease v Target of TSAb: TSH receptor v TSAb mimic the action of TSH v Thyroid blocking Ab is also present in Graves, disease but do not cause symptoms v TRAb in 85 % of cases of Graves, disease Clinical utility ? ? ? v TRAb assay used to predict the outcome of drug treated patients (lower titer before treatment, better chance for remission after 6 -12 months of therapy) v TRAb assay for prediction of thyroid dysfunction in neonates of mothers with Graves, disease. v Maryam Tohidi / RIES

Thank you for your attention Maryam Tohidi / RIES