Thyroid Function I Lecture Notes PDF

Thyroid Function I Cyril Mamotte Curtin Medical School Curtin University Objectives To know and understand the following: Circulating forms of thyroid hormone & thyroid transport proteins. Thyroid hormone synthesis & regulation General physiological effects of T3/T4. Causes and types of hyper an...

Thyroid Function I Cyril Mamotte Curtin Medical School Curtin University Objectives To know and understand the following: Circulating forms of thyroid hormone & thyroid transport proteins. Thyroid hormone synthesis & regulation General physiological effects of T3/T4. Causes and types of hyper and hypothyroidism Clinical features of hypo and hyperthyroidism Specialised tests: e.g. TSH receptor and TPO antibodies Context of laboratory investigations Test interpretation Complexities: Influence of illness, non equilibrium states, drugs Analytical principles and issues • Requirement of high sensitivity TSH assays • Functional sensitivity • Free thyroid hormone assays and issues Examples of Questions Clinical • Questions on interpretation of thyroid function test results (e.g. as in lecture notes) • This question is in two parts: a) Give an outline of some of the clinical features of hyper and hypothyroidism. b) • What are the causes of hyper and hypothyroidism. In context of thyroid function, what are disequilibrium states? Analytical • Why are highly sensitive (new generation) assays required for TSH? • Define functional sensitivity? • Why is it important to have highly sensitive TSH assays? • Determine the functional sensitivity for X from the following data. • What are potential sources of interference in fT4, fT3 and TSH assays? Combined analytical and clinical. • Why should thyroid function testing be avoided in the critically ill. Explain your answer. Include analytical and non-analytical aspects. • Why is it prudent to measure both TSH and thyroid hormone concentrations? Circulating Forms of T4 and T3* I- T3-TBPA: trace T3 Target Cell nucleus Thyroid Follicles T4 <1% T3 <0.5% T4-TBPA (1- 7%) T4 TGL-T3 TGL-T4 - T4-Albumin(7%) T3-Albumin(28%) •>99% of T4 and T3 are bound to transport proteins. •Free fraction which is biologically active. •Levels of transport proteins labile. •Measurement of total levels can mislead T4-TBG (72%) T3-TBG (72%) T4 T3 Target Cell nucleus T3 and T4 Hormone Synthesis – All steps stimulated by TSH OH OH a Th Pe yroi rox d ida Co se u en pli zym ng es H2N TGL I⋅ Tyrosine iodinase b I- OH I Thyroglobulin protease O I a I b a a I O H2N TGL H2N OH Storage Forms in Follicle H2N TGL I⋅ Tyrosine iodinase T3 O I OH I I OH OH I I I a H2N TGL Coupling enzymes b O I I Thyroglobulin protease I OH I b I O I a a I O H2N TGL H2N OH T4 T4, T3, rT3, deiodinases, drugs and illnesses OH I OH I b I a O I a , ss e illn H N , n ) 2 o ids M o s ic b rug cort 2 d & by uco 1 l pe 1, È lol, g y T pe o Ty opan (pr ress. St o -d I 45 % a O Mo no -d e io d OH in a OH Mo no -d e io d in a 3,3 T2 se I I b I -d o n d o i e s a n i e rT3 s), d i o tic r b s o & 2 drug ucoc 1 l pe 1, È lol, g y T pe no Ty opa (pr ress. St Mo se OH O T3 a H2N 3,5 T2 % 5 3 O T4 O I e io d in e as b I a H2N , ss e illn 3,5’ T2 O OH T4, T3 & rT3: Concentrations & Biological Activity • T4 is the predominant secretory product. • T3 has greater biological activity. *Levels are higher in children. • Deiodinase activities influenced by stress, illness and some drugs. – This can cause interpretive difficulties. • Plasma levels – T4: 60-140nM; fT4: 10-25 pM – T3: 1.1-2.7 nM; fT3: 3-8 pM Regulation of T4/T3 Secretion TRH -ve -ve Hypothalamus +ve Pituitary TSH TSH fT4, fT3 +ve Thyroid Thyroid Physiological Effects Metabolic Rate Increases Basal Metabolic Rate. Increases thermogenesis. In general, stimulates oxygen consumption by tissues with high metabolic rate (except in adult brain, testis, uterus, spleen, anterior pit, lymph nodes). Rate falls to 40% normal in absence of thyroid gland. Increases activity of Na+-K+-ATPase in many tissues. Carbohydrates Increased GIT absorption rate. Glucose levels fall and rise rapidly after a carbohydrate meal. Lipids Lowers plasma cholesterol; increases LDL-R expression. Hypothyroidism associated with decreased lipoprotein synthesis by the liver, but catabolism is also reduced. Post-heparin LPL activity diminished in deficiency. Hence may see both elevated LDL and VLDL. Cardiovascular System Increases cardiac output, cardiac rate and pulse pressure. Increased number of alpha Myosin Heavy chains (higher ATPase activity than beta; level of beta up in hypothyroidism) Physiological Effects Nervous system Important to normal brain development; increases responsiveness to catecholamines. Hypothyroidism: mentation slow. Hyperthyroidism, rapid mentation , irritability, restlessness. Thyroid hormone deficiency is an important cause of cretinism; newborn screening programmes in most developed countries. World-wide incidence 1/4000 -1/10000 Adipose Tissue Stimulates lipolysis. Bone/Muscle Potentiates influence of GH. Promotes normal growth & development. Influences expression of myosin heavy chain (MHC) in SM and CM. Relationship to effects in thyroid disease e.g. myopathy in thyroid excess, unclear. Deficiency: delays in both bone growth & epiphyseal closure. Secretion of GH also decreased. Excess or deficiency may result in muscle weakness; prolonged excess may cause severe myopathy. Hypothyroidism may be associated with elevated CK; hyperthyroidism, less often so. Hypothyroidism may be associated with elevated CK; hyperthyroidism, less often so. Other Effects Water/Salt Balance Increased thermogenesis • Activates heat-dissipation mechanisms. • Decreased peripheral resistance, increased cutaneous vasodilation, increased renal sodium and water reabsorption. • IE water retention • Expanded blood volume In hypothyroidism: chronic oedema and decreased circulating blood volume. • Increased capillary escape of albumin • Accumulation of mucopolysaccharides (hyaluronic acid/chondroitin sulphate)-hygroscopic à draws water à causing puffiness in skin • This in part explains term Myxoedema (Greek Myxos: tissue> tissue oedema). • Other skin changes: puffiness (esp. around the eyes), coarse skin rough, thick, scaly appearance; cold pale and yellowish (decreased conversion of carotene to vitamin A (carotenemia). © Springer 2009. Produced by Current Medicine Group Ltd, a part of Springer Science+Business Media © Springer 2009. Produced by Current Medicine Group Ltd, a part of Springer Science+Business Media TERMS TO KNOW Terms Normal Thyroid Function • Euthyroid Thyroid Function Tests not Normal Thyroid Function Test • Sick Euthyroid (thyroid function is ok but tests indicate otherwise) • Hyperthyroid • Hypothyroid • Subclinical hyperthyroidism (T4 and T3 are ok, but TSH is low) • Subclinical Hypothyroidism (T4 and T3 are ok, but TSH is low) Clinical Features of Thyroid Disease Hyperthyroidism •Increased BMR •Tachycardia •Heat intolerance •Diarrhoea •Weight loss •Increased appetite •Moist, velvet skin •Anxiety Hypothyroidism •Decreased BMR •Bradycardia •Cold intolerance •Constipation •Weight gain •Decreased appetite •Dry coarse skin •Lethargy •Diminished fertility & menstrual irregularities •Diminished fertility & menstrual irregularities •Galactorrhoea •Delayed growth •Hyperlipidaemia •Muscle weakness •CK levels often raised •Muscle weakness –Myopathy –CK levels may be raised (not as common as in hypothyroidism) •Thyroid storm •Myxoedema Coma Primary and Secondary and Central Causes Abnormalities in thyroid hormone levels can be due to: • Diseases of the thyroid gland. • These are known as primary (1o) diseases of the thyroid. • Primary disease because the organ involved is that which produces the hormone. • Primary disorders are more common by far. • Derangements in TRH/TSH secretion e.g. due to diseases of the hypothalamus/pituitary. These are not as common. • These are known as central/pituitary dependent causes. • Could also be termed secondary(2o) • Also very rarely, a tumour elsewhere in the body (ectopic) may produce TSH (ectopic TSH secretion). • TSH like activity of high levels of hCG • Very high concentration of hCG (eg from placental tumours) can result in hyperthyroidism Causes of Hyperthyroidism Graves Disease Most common cause. Autoimmune condition. TSH receptor antibodies; bind and stimulate the TSH receptor. Toxic adenoma Benign tumour. Toxic multinodular goiter Multifocal; autonomous secretion. Thyroiditis Inflammation of the thyroid. Leakage of thyroid hormone into circulation. Examples post-partum thyroiditis (autoimmune), toxic phase of Hashimoto’s thyroiditis (autoimmune). Subacute thyroiditis (possible viral aetiology: usually transient)-often accompanied by non-specific manifestations of inflammatory disease (fever, chills..) –May be transient. May last from weeks to months. May change to hypothyroid thyroiditis during “recovery” of the gland. Can also be cause by iodine excess in those with underlying thyroid disease* T3 toxicosis (phenomenal) Elevation of T3; normal T4 : can occur in iodine deficiency or early in Graves adenoma or multinodular goitre Causes of Hyperthyroidism and treatment Iatrogenic Drugs: Amiodarone, thyroid hormone ingestion, iodine (e.g. to subject being treated for iodine deficiency hypothyroidism) Rare causes TSH secreting pituitary tumour Ectopic TSH secreting tumour hCG secreting tumour Struma ovarii (thyroid tissue in ovarian teratoma) Treatment: Surgical, 131I therapy, carbimazole and propylthiouracil (inhibit peroxidase) T4/T3 and TSH Secretion in Graves TRH Hypothalamus +ve -ve Pituitary TSH -ve TSH or N T4, T3 +ve Thyroid In Graves disease, TSH receptor antibodies can also Stimulate the TSH receptor Causes of Hypothyroidism Hashimoto’s Disease Autoimmune thyroiditis. Most common cause. Destruction of thyroid. Positive for thyroperoxidase antibodies (anti-microsomal Ab) in 80-95% of cases. Treatment of hyperthyroidism Radio-iodine, surgical treatment, anti-thyroid drugs Thyroiditis Post-partum thyroiditis. Auto-immune in nature. Severe Iodine deficiency Particularly in mountainous regions. Worldwide, the most common cause. Congenital defects Dyshormonogenesis Inherited defects in ability to synthesise thyroid hormones. Most commonly due to failure to incorporate iodine. Agenesis/dysgenesis Absence or poorly formed thyroid gland Causes of Hypothyroidism ctd… Iatrogenic Lithium therapy. Amiodarone therapy Hypopituitarism Decreased TSH secretion. Leads to atrophy of thyroid gland. T4/T3 and TSH Secretion in Hashimoto’s TRH Hypothalamus +ve -ve Pituitary TSH -ve TSH fT4, fT3 +ve Thyroid Medical Emergencies Thyroid storm – Life threatening • Precipitated, usually in undiagnosed subjects, by major stress or illness: surgery, labour, major trauma (accident), anaesthesia – T4/T3 elevated, but apparently no greater than usual – Acute severe exacerbation of symptoms – – – – Coma may develop Tachycardia, hyperpyrexia (fever) , risk of circulatory collapse Marked anxiety/agitation Tx: symptomatic + support – Beta blockers and carbimazole (blocks T4>T3 conversion) – Glucocorticoids Myxoedema Coma – Medical emergency. – Hypothermia. Depression of respiratory centre, reduced cardiac output > cerebral hypoxia, – May develop slowly, particularly in winter, or be precipitated by illness, exposure to cold, infection, narcotics/analgesics. – Tx : fluids, thyroid hormones, glucocorticoids, avoid further heat loss Role of the Laboratory 1. Thyroid diseases are relatively common. Diagnosis on basis of clinical presentation can be difficult unless advanced. Laboratory confirmation is required. 2. Any of the clinical symptoms listed previously, particularly in combination, are valid reasons for laboratory investigations. 3. The front line tests for diagnosis is measurement of serum thyroid hormone levels (most often T4) and TSH. 4. In addition to diagnosis, the laboratory also has a role in monitoring therapeutic efficacy in the treatment of thyroid dysfunction. fT4 fT3 TSH 1o Hyperthyroidism é Or N é êê(<0.01 mU/L) 1o Hypothyroidism ê 2o Hyperthyroidism or ectopic TSH secretion é 2o or central or hypothyroidism ê Subclinical 1o Hyperthyroidism N N ê (<0.1 mU/L) Subclinical 1o Hypothyroidism N N é Subclinical 1o Hyperthyroidism N N ê (<0.1 mU/L) Subclinical 1o Hypothyroidism N N é é é é ê Or N TSH and Thyroid Gland (1o) Dysfunction Euthyroid -normal TSH Levels Hypothyroid fT4 Levels Hyperthyroid Case Example 1 80 year old patient, presents with: • Recent history of weight gain • Feeling lethargic and generally unwell • Dry skin • Feels cold….(it was middle of summer!) • Serum lipids: elevated total and LDL cholesterol – – fT4 : 5 pmol/L (10-23 pmol/L) TSH: 40 mU/L (0.4-4 mU/L) • • • • • What’s the diagnosis? The most likely disease? Graves, Toxic nodules, Pit disease etc? Any tests for autoimmune diseases? Is it a primary or secondary disorder? What is the relevance of the serum lipid results? Case Example 2 • • • 40 year old woman Anxious and “shaky” High pulse rate (i.e. tachycardia) – fT4 : 28 pmol/L (10-23 pmol/L) – TSH: <0.01 mU/L (0.4-4 mU/L) • What type of disorder is this? • Is it a primary or secondary disorder? • Is the TSH always so low? • Additional findings: TSH receptor antibody: +ve Case Example 2 • • • 40 year old woman Anxious and “shaky” High pulse rate (i.e. tachycardia) – fT4 : 28 pmol/L (10-23 pmol/L) – TSH: <0.01 mU/L (0.4-4 mU/L) • • • • • • What type of disorder is this? What’s the most likely disease? Is it a primary or secondary disorder? Is the TSH always so low? What will the TSH do once the T4 returns to normal? TSH receptor antibody: what’s this likely to be and why? TSH and T4 levels Relationship & Principles which underpin diagnosis Autonomous TSH secretion (Central, pit., or ectopic) fT4 Levels Primary Hyperthyroidism m os tc Normal om m on lin e Central or Pituitary disease Primary Hypothyroidism TSH Levels fT4 fT3 TSH 1o Hyperthyroidism é Or N é êê(<0.01 mU/L) 1o Hypothyroidism ê 2o Hyperthyroidism or ectopic TSH secretion é 2o or central or hypothyroidism ê Subclinical 1o Hyperthyroidism N N ê (<0.1 mU/L) Subclinical 1o Hypothyroidism N N é Subclinical 1o Hyperthyroidism N N ê (<0.1 mU/L) Subclinical 1o Hypothyroidism N N é é é é ê Or N Subclinical Thyroid Disease (i.e. not overt) Disease development and progression --------hypothyroidism-----------------------fT4 : 12 pmol/L (10-23 pmol/L) TSH: 3 mU/L (0.4-4 mU/L) -ve fT4 : 14 pmol/L (10-23 pmol/L) TSH: 10 mU/L (0.4-4 mU/L) fT4 : 10 pmol/L (10-23 pmol/L) TSH: 20 mU/L (0.4-4 mU/L) -ve Subclinical Thyroid Disease A biochemical definition Subclinical Hyperthyroidism* Subclinical Hypothyroidism* normal T4 & T3 and suppressed TSH normal T4 & T3 and elevated TSH Risks Risks • • • • • Further progression (5%) • General well being, depressive illnesses • Possible effects on foetal brain development in pregnancy Further progression osteoporosis atrial fibrillation thyrotoxicosis if exposed to iodine Suggested by some groups (Am College of Physicians) to screen for subclinical thyroid disease in susceptible groups: • women, those aged >50 years –the latter having a frequency of 10% for the disorder *From Stokight Clin Biochem Revs 2003;109-122 T4/T3 and TSH Secretion in Graves TRH Hypothalamus +ve -ve Pituitary TSH -ve TSH or N T4, T3 +ve Thyroid In Graves disease, TSH receptor antibodies can also Stimulate the TSH receptor T4/T3 and TSH Secretion in Hashimoto’s TRH Hypothalamus +ve -ve Pituitary TSH -ve TSH fT4, fT3 +ve Thyroid Examples of Subclinical Thyroid Diseases 1 fT4 : 28 pmol/L (10-23 pmol/L) TSH: <0.01 mU/L (0.4-4 mU/L) 2 fT4 : 22 pmol/L (10-23 pmol/L) TSH: <0.1 mU/L (0.4-4 mU/L) 3 fT4 : 10 pmol/L (10-23 pmol/L) TSH: 20 mU/L (0.4-4 mU/L) 4 fT4 : 14 pmol/L (10-23 pmol/L) TSH: 10 mU/L (0.4-4 mU/L) TSH and T4 levels Relationship & Principles which underpin diagnosis Autonomous TSH secretion (Central, pit., or ectopic) fT4 Levels Primary Hyperthyroidism mo st co mm on lin normal e fT4 low TSH Normal normal fT4 High TSH Central or Pituitary disease Primary Hypothyroidism TSH Levels fT4 Hyperthyroidism (1o) e.g. Graves, Toxic nodules é Or N fT3 é TSH êê(<0.01 mU/L) Hypothyroidism (1o) e.g. Hashimoto’s ê Hyperthyroidism (2o) e.g. ectopic TSH production é Hypothyroidism(2o) e.g. Pituitary/Hypatahamic (central) ê Subclinical 1o Hyperthyroidism N N ê (<0.1 mU/L) Subclinical 1o Hypothyroidism N N é Subclinical 1o Hyperthyroidism N N ê (<0.1 mU/L) Subclinical 1o Hypothyroidism N N é é é é ê Or N Non-equilibrium states TSH Tx Of hyperthyroidism Tx Of hypothyroidism TSH TSH is a good indicator of therapeutic efficacy in the long term. However, in the early phase of treatment for hyper and hypothyroid patients, TSH is not a good indicator of therapeutic adequacy. In hypothyroidism this can result in over-treatment and high risk of thyrotoxicosis. Monitoring of TSH is of no use in monitoring treatment of hypothyroidism due to pituitary disease. fT4 Hyperthyroidism (1o) e.g. Graves, Toxic nodules é Or N fT3 é TSH êê(<0.01 mU/L) Hypothyroidism (1o) e.g. Hashimoto’s ê Hyperthyroidism (2o) e.g. ectopic TSH production é Hypothyroidism(2o) e.g. Pituitary/Hypatahamic (central) ê Subclinical 1o Hyperthyroidism N N ê (<0.1 mU/L) Subclinical 1o Hypothyroidism N N é Early treatment phase Hyperthyroidism* N ê-êê Early treatment phase hypothyroidism* N é é é é ê Or N Conceptual consequences of Heterophilic Antibody Interference Graves fT4 fT3 TSH +ve Het Ab Interference -ve Het Ab Interference é Or N é êê(<0.01 mU/L) N or é êê(<0.01 mU/L) é é ê Or N Hashimoto’s ê Subclinical 1o Hyperthyroidism N N ê (<0.1 mU/L) N or é ê -ê ê Subclinical 1o Hypothyroidism N N é é ê Or N Early treatment phase Hyperthyroidism* N ê-êê N or é ê-êê Early treatment phase hypothyroidism* N é é ê Or N Non-thyroidal Illness (NTI) Illustrative example… TSH fT4 T4 Reference Range Serum Concentration rT3 T3 -Mild-|-Moderate-|-Severe-|------Recovery-------------- Phases of Illness Adapted from Brent GA, Heshman JM: Effects of non-thyroidal illness on thyroid function tests. In: The Thyroid Gland; A Practical Clinical Treatise. L Van Middleworth, Rd. Chicago. Year Book Medical, 1986. p86. Also as p42-6, in Tietz Textbook of Clin Chem 3rd edn, p1503 Non-thyroidal Illness (NTI) 1. Thyroid function testing should be avoided in the acutely ill. a. Influence of illness on fT3/fT4 and relationship to TSH, T4ÆT3/rT3 conversion. Changes may be appropriate to illness and treatment for thyroid dysfunction is usually not indicated. 2. Apart from the effect of acute illness on thyroid function per se, numerous medications potentially used in the acutely ill can: a. Have an effect on thyroid function (e.g. glucocorticoids decreasing basal TSH) b. Cause changes to TBG, and can cause errors in measurement of free or total thyroid hormone levels 3. Measurement of TSH is least likely to mislead than T4 or fT4 Other Tests 1. Total T4 or T3 + TBG • When fT4 and fT3 measurement might be considered unreliable. 2. Radioactive iodine uptake: Indicative of increased Thyroid hormone synthesis • Positive in Graves (homogenous scan), multinodular goitre, toxic adenoma (heterogenous scan) • Often a follow up test to establish the cause 3. TSH Receptor Antibodies • Positive in Graves • Useful if above scan is not possible, contraindicated 4. Thyroperoxidase antibodies • Hashimoto’s, Graves • Signifies susceptibility to autoimmune thyroid disease. Establishes risk of thyroid disease in response to certain medications (e.g. amiodarone, lithium) 5. Thyroglobulin: • Used as a tumor marker to monitor for recurrence in cancer of the thyroid 6. Antithyroglobulin antibodies • Assayed to check for interference in assay for Thyroglobulin Influence of Drugs II -effects on physiologyAmiadarone (anti-arrhythmic drug) Hyper or hypothyroidism both described; & reduced T4>T3 conversion also described. Lithium Hypothyroidism usually (but also reports of thyrotoxicosis). Inhibits iodine uptake and thyroid hormone release. May exacerbate or initiate thyroid disease êfT4 & T3; é TSH Other drugs Large doses of glucocorticoids can suppress TSH. Dopamine has a similar influence. ANALYTICAL ASPECTS The Importance of Functional Sensitivity for TSH Assays Sensitivity/Precision of TSH Assays Result A Result B* Result C* Ref Range fT4 20 20 20 10-23 pM fT3 7.2 7.2 7.2 3.4-6.8 pM TSH (95% CI) <0.01 <0.01 (0.008-0.012) <0.01 (0.000-0.200) 0.4-4.0 mU/L 45 year old man with some symptoms suggestive of hyperthyroidism. Does this patient have hyperthyroidism and why? *Illustrative examples only. Precision of contemporary TSH Methods i.e. in Clinical Use 50 40 30 20 10 0 0.0004 0.001 0.004 0.01 |--Hyperthyroid range--| 0.04 0.1 TSH mU/L 0.4 1 4 |--”Normal range”--| Functional sensitivity: concentration at CV of 20%. It doesn’t mean that a CV of 20% is good 10 Precision & Generation of TSH assays 50 40 4th 3rd 2nd 1st 30 20 10 0 0.0004 0.001 0.004 0.01 0.04 0.1 TSH mU/L |--Hyperthyroid range--| Adapted from Tietz Textbook of Clinical Chemistry 0.4 1 4 |--Normal range--| 10 Question The following represents the data from 10 analytical runs for TSH in mU/L The data was obtained in a MEDI 4003 class. Determine functional sensitivity of the assay. Run 1 1.03 0.96 2 1 1.2 3 4 0.96 0.68 5 0.8 1.2 6 1 1.2 7 1 1 8 1.1 1.1 9 1.0 1.0 10 0 1.75 Average 0.88 1.12 STDEV 0.34 0.29 CV 38.64 25.89 5.19 5.23 4.6 4.2 4.86 7.6 4.8 5 4.6 4.8 3.75 4.94 1.02 20.65 5.00 5.28 4.2 3.9 4.71 4.2 5.1 5 4.9 4.0 3.55 4.48 0.59 13.17 11.67 10.67 8.4 8.85 9.46 10 10 8.4 10.8 10.0 7.6 9.42 1.06 11.25 5.52 10.96 9.1 10.2 11.61 7.4 17 9 9.5 9.5 7.9 10.22 2.7 26.42 17.73 21.93 20 22.2 19.25 20.8 18 14 20 19.5 18.55 19.42 2.33 12 16.36 20.74 24 19 21.50 18 19 20 19.5 21.5 17.5 20.07 1.93 9.62 43.52 42.67 40 42.8 38.36 39 37 40 38 42.5 48 40.83 3.25 7.96 6.54 8.89 5.5 5.2 6.32 6.32 6.4 5.2 5.0 3.3 5.79 1.51 26.08 5.38 8.3 6.1 5.9 7.21 7.21 5.2 4.9 5.3 3.8 5.99 1.39 23.21 1.12 1.01 insuff insuff 0.39 0.39 3.3 0.8 0.7 insuff 1.1 1.1 100 N/A 1.06 insuff insuff 0.46 0.46 1 0.8 0.6 insuff 0.73 0.26 35.62 5.56 7.55 5.5 4.9 6.89 6.89 6.4 4.6 5.7 3.4 5.76 1.32 22.92 6.85 7.7 5.5 5.5 7.25 7.25 7.2 4.5 5.0 2.65 5.84 1.67 28.6 Precision Profile-Student Data 120 100 CV (%) 80 60 40 20 0 0 5 10 15 20 25 30 TSH Concentration mIU/L 35 40 45 Method Principles for fT4 assays Siemens Comp/Titrimetric chemiluminescent ELISA assay fT4 Protein bound T4 anti-T4 coated beads Incubation Add. = ALP labelled T4 analogue Add acridinium labelled T4 analogue Wash step Add ALP substrate (Phosphorylat ed dioxetane complex) Abbott Architect Comp/Titrimetric chemiluminescent immunoassay-2 step! fT4 Protein-bound T4 Incubation Wash removes unreacted components including Add acridinium labelled T4 analogue Wash. Removes unreacted anti-T4 coated paramagnetic particles Magnet keeps bead secure in the tube when tubes are being washed Add pre-trigger (H2O2) and trigger (NaOH) solution -> light emission Titrimetric Roche chemiluminescent fT4 assay (ELECSYS detection system) ST ST >- Ru2+ >- Ru2+ >- Ru2+ ST ST ST ST Add TPA and voltage To stimulate chemilumine scence ST ST ST ST ST ST ST ST ST ST ST ST >- Ru2+ >- Ru2+ >- Ru2+ >- Ru2+ >- Ru2+ >- Ru2+ >- Ru2+ >- Ru2+ >- Ru2+ >- Ru2+ >- Ru2+ ST ST ST ST >- Ru2+ >- Ru2+ ST ST >- Ru2+ ST Wash step removing components Not bound to ST >- Ru2+ >- Ru2+ >- Ru2+ >- Ru2+ >- Ru2+ >- Ru2+ >- Ru2+ >- Ru2+ ST ST ST ST ST Moved to measuring Cell. Capture of ST. ST by magnets >- Ru2+ >- Ru2+ >- Ru2+ ST ST ST ST T4-Biotin Binds to vacant Sites on >- Ru2+ >- Ru2+ >- Ru2+ -Sample T4 T4-Biotin 2+ 2+ >- Ru -Ru anti-T4 conjugate -Protein bound T4 Buffered diluent ST -Streptavidin coated beads (magnetized) ST ST ST ST What are possible interferences in the Roche fT4 assay and what would the effects be on the result?? High dose of biotin. Effect à Streptavidin antibodies. Effect à Anti-ruthenium antibodies à Blocking, or negatively interfering heterophilic antibodies à >- Ru2+ >- Ru2+ >- Ru2+ ST ST ST ST T4-Biotin conjugate >- Ru2+ -Ru2+ anti-T4 Buffered diluent ST -Streptavidin coated beads (magnetized) ST ST ST ST >- Ru2+ -Sample T4 -Protein bound T4 >- Ru2+ >- Ru2+ >- Ru2+ >- Ru2+ >- Ru2+ >- Ru2+ >- Ru2+ >- Ru2+ >- Ru2+ >- Ru2+ ST ST ST ST >- Ru2+ >- Ru2+ ST ST >- Ru2+ ST ST ST ST ST >- Ru2+ >- Ru2+ >- Ru2+ >- Ru2+ >- Ru2+ >- Ru2+ >- Ru2+ >- Ru2+ ST ST ST ST ST ST ST ST >- Ru2+ >- Ru2+ ST ST ST >- Ru2+ >- Ru2+ >- Ru2+ ST ST ST ST ST ST ST ST Roche Elecsys –precautions…from Kit Insert Issues with T4 and T3 assays -past, present, and likely, future- Total vs Free Hormone Levels -measurement of total levels• Previously up to late 1980s, reliable free hormone assays were not available. • Traditional approach was to measure total T4 or T3; • Interpretation of results required consideration of binding protein levels • Drugs that increase TBG – • Oestrogens, methadone, heroin, tranquilisers, clofibrate, 5-fluorouracil Drugs that decrease TBG – • Glucocorticoids, androgens, anabolic steroids. Low TBG due to illness – • Acute illness, nephrotic syndrome Inherited excess/deficient binding. – Familial dysalbuminaemic hyperthyroxinaemia – TBG variants (~40% of Indigenous Australians, lower affinity) Measurement of Total T4 assays-Influence of variant Thyroid Binding Globulin Total vs Free Hormone Levels -measurement of total levels• Some drugs (e.g. salicylate) can displaces T4/T3 from their binding sites, influencing total levels, but not the biologically free levels* • Results were therefore expressed as a free thyroid index. 1. Measure total T4/T3 2. Measure binding protein levels (either by measuring uptake or radio-labelled T3 or measuring TBG). • Express results as an index. • Does not completely compensate for binding hormone disturbances, but preferable to measurement of total levels alone. • Ideally, would measure biologically active levels, not just total levels. • fT3 and fT4 assay performance improved, in common use now, but still subject to some problems. Free T4/T3 Assays (fT4/fT3) • Numerous methods have been released to market prior to thorough testing for possible interferences. • Some believe measurement of fT4/fT3 is highly compromised particularly when patients are on some drugs. • Analytical interference in thyroid hormone assays (fT3/fT4, total T3/T4) is a complex issue, and not easy to understand; what’s important is to know that it’s an issue. • There can be significant differences in results between different methods • Measurement of both fT4 and TSH is prudent Drug Interference in free T4/T3 Assays (fT4/fT3) • Most compensate reasonably well for changes in binding protein levels. • However, the approach is still subject to interference by drugs which displace T4/T3 from binding proteins. Causes an underestimation of fT3/fT4 when on drugs such as phenytoin, carbamazepine, frusemide, salicylate. An in-vitro effect • Example of a concept for drugs that compete for binding to transport proteins Stockigt, J.R. and C.F. Lim, Medications that distort in vitro tests of thyroid function, with particular reference to estimates of serum free thyroxine. Best Pract Res Clin Endocrinol Metab, 2009. 23(6): p. 753-67. Assay Reagents T4 Drug Neat serum • Diluted serum Two-step methods, which separate a fraction of the free T4 pool from the binding proteins before the assay are considered more reliable. Heparin treatment leading to interference in fT4/fT3 assays Assays (fT /fT3) Heparin infusion4> releases lipoprotein lipase (LPL) from blood vessel wall. LPL now elevated. Blood is collected An Ex-vivo effect plasma triglycerides In the sample , LPL elevated free fatty acids + glycerol Free fatty acids displaces T4 and T3 from albumin Elevates free thyroid hormone concentrations. Exacerbated in plasma albumin is low or if plasma triglycerides are high How to approach: delay sampling in such patients and assay promptly Overview: assay Interference: fT3/fT4, and TSH •fT4/fT3 • interference by drugs or when there are changes in binding protein levels. This is method dependent •There are also examples of anti-thyroid hormone antibody interference •Heterophilic antibody interference •TSH •interference usually by heterophilic antibodies (+ve or negative), macro-TSH •TSH and fT4/fT3 •biotin, anti-streptavidin antibodies, anti-ruthenium antibodies •Safeguards •Measurement of multiple thyroid function tests helps to guard against assay interference. E.g. TSH/fT4 (or total T4). •The results of the tests should be concordant •If not, assay interferences, or rarer causes of thyroid dysfunction should be considered. •If results are anomalous (discord between TSH and fT4 of fT3) Use an alternative fT4 methods Measure total T4 to confirm. •For TSH, test for possible heterophilic antibodies (e.g. add excess mouse serum) or confirm with an alternative method. Interpretation of results –paragraph/short answer questions a) Examine the results If they fit the following patterns: i) High fT4, undetectable TSH,; ii) Low fT4, high TSH. You could say that the results are consistent with hyperthyroidism for the first case, and hypothyroidism in the second case. If it is dear that the results do not, proceed to b) below. b) State that the results don’t fit a clear case of hyper or hypothyroid disease (e.g. Graves and Hashimoto’s respectively) and what the results ought to be like for the commonest thyroid disorders: i) primary hyperthyroidism (or thyrotoxicosis): ↑fT4 or normal fT4 and ↑fT3 AND ↓TSH (<0.001) ii) primary hypothyroidism: ↓ fT4 ↑ TSH. a) and b) will show that you understand the common patterns, and what the relationships ought to be for the simplest/common causes. If they do not, consider the following possibilities. There can be several possibilities. i. ii. iii. iv. v. vi. For a low TSH (<0.01 mU/L) and normal fT4, consider T3 thyrotoxicosis Non-equilibrium or non-steady states: recent treatment for hyper or hypothyroidism. Subclinical hyper/hypothyroidism-but the patterns must fit Assay interference in fT4 (e.g. drugs) or TSH assays (heterophilic Abs)-but be specific Non-thyroidal illness. Pituitary/central disease (=pit/hypothalamic disease) Examples of Questions Clinical • Questions on interpretation of thyroid function test results (e.g. as in lecture notes) • This question is in two parts: a) Give an outline of some of the clinical features of hyper and hypothyroidism. b) • What are the causes of hyper and hypothyroidism. In context of thyroid function, what are disequilibrium states? Analytical • Why are highly sensitive (new generation) assays required for TSH? • Define functional sensitivity? • Why is it important to have highly sensitive TSH assays? • Determine the functional sensitivity for X from the following data. • What are potential sources of interference in fT4, fT3 and TSH assays? Combined analytical and clinical. • Why should thyroid function testing be avoided in the critically ill. Explain your answer. Include analytical and non-analytical aspects. • Why is it prudent to measure both TSH and thyroid hormone concentrations? Roche Elecsys Abbott Architect Assay Abbott Architect Assay- precautions for assay set up