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Thyroid function testing

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Thyroid function testing

  1. 1. Prabin Shah MSc(Biochemistry)
  2. 2. Importance of TFT 5 % of world population suffer from Thyroid problems / dysfunction Thyroid insufficiency ( due to iodine deficiency )is a worldwide problem A Major work load in Endocrine laboratory Test/s asked from newborn to elderly
  3. 3. Endocrine functions of Thyroid • Thyroid has 2 distinct endocrine systems • Secrete 2 different hormones – functionally unrelated • Iodine containing substances – Produced by Follicular cells – Triiodothyronine ( T3 ) and Thyroxine ( T4 ) • Calcitonin – by C cells
  4. 4. TFT Concerned with Disease & its effect on Production ( / ) of Iodine-↑ ↓ containing hormones Proteins which bind them Metabolic changes due to or in hormonal↑ ↓ secretion
  5. 5. Thyroid hormones- Central regulation • T3 and T4 production influenced by Thyrotrophin ( TSH ) • TSH secreted by Anterior pituitary • TSH secretion partly directly controlled by Negative feedback mechanism , + by plasma hormones and↓ – by plasma hormones.↑ • Thyrotrophin releasing hormone ( TRH ) , from Hypothalamus , acts on anterior pitutary , for rapid release of TSH ( may also its synthesis )↑
  6. 6. Hypothalamic Pituitary Thyroid Axis
  7. 7. Thyroid hormones • Synthesis of T3 and T4 is complex process • Released into circulation, transported loosely bound to plasma proteins • Major is , Thyroxine binding globulin, TBG ( migrates between α 1 and α 2 globulin ) • TBG has greater affinity for T4 , carries 50- 65 % of T4 • 2nd protein is Thyroxine-binding Prealbumin ( TBPA ) , carry only T4 , 15 – 25 % • Albumin also binds T4 , but has low affinity , minor role
  8. 8. Thyroid hormones HORMONE CIRCULATED AS CONCENTRATION % T3 FREE ( F T 3 ) 0.5 T3 TBG 50 - 60 T3 ALBUMIN MINOR T3 Prealbumin Minor T4 FREE ( F T 4 ) 0.036 – 0.056 T4 TBG 50 - 60 T4 TBPA 15 - 25 T4 ALBUMIN MINOR T3 has nearly 3 times the activity as that of T4
  9. 9. TFT- Test parameters for evaluation • T3 ( Total ) • Free T3 • T4 ( Total ) • Free T4 • TSH • Thyroglobulin ( Tg ) • Anti Thyroglobulin Antibody ( Anti Tg ) • Thyroid Peroxidase Antibody ( TPO ) • Thyroid Antibody ( Anti Tg and TPO ) • TSH receptor antibody • TBG
  10. 10. Indications for TFT • Diagnosis of thyroid disorder , with symptoms • Screening newborns , for an underactive thyroid • Diagnosis and monitoring of female infertility problems • Monitor thyroid replacement therapy , in hypothyroidism • Occasionally , to evaluate pituitary gland function • Screening adults for thyroid disorders , if recommended • Irregular menstrual cycles , Gaining weight
  11. 11. TFT One of the most common endocrine laboratory investigations requested by general clinicians To identify thyroid disease and monitor treatment So , laboratory tests have to be sensitive and accurate
  12. 12. When to test Clinical suspicion – signs and symptoms provide the best indication to request for TFT High , Intermediate and Low suspicion groups for Hyper- and Hypothyroid states
  13. 13. Thyroid function test , involves Taking good history Clinical examination Laboratory tests , usually follow , it may stop here May be , ultra sound ,( if shows large nodules) FNAC , for evaluation of thyroid nodules Thyroid scan
  14. 14. Thyroid function state • Euthyroid – state of normal thyroid function • Hyperthyroid – state of overactive thyroid • Hypothyroid – state of underactive thyroid
  15. 15. Which test should be used • In most situations , use TSH as sole test of thyroid function • TSH is most sensitive ( 98 % ) and specific ( 92 % )test • TSH can detect subclinical thyroid disease without symptoms of thyroid dysfunction. • Adding other tests is only of value in specific circumstances • When TSH is within reference range , there is 99 % likelihood that FT4 will also be within reference range • TSH and FT4 are commonly ordered tests . ( choice of TFT appears to have changed over last 10-15 years , from FT4 ( most popular ) to TSH ( now favoured)
  16. 16. When it is inappropriate to test only TSH ? • Central (secondary) hypothyroidism - This is the most significant condition in which an incorrect diagnosis of euthyroidism could be made, based on TSH alone.8 • Non compliance with replacement therapy • Early stages of therapy - During the first 2 months of treatment for hypo- or hyper-thyroidism, patients will have unstable thyroid status because TSH will not have reached equilibrium. • Acutely ill patients - TSH is altered independent of thyroid status. As a result, testing should only be performed when it is likely to have an effect on acute management. • Pregnant patients on replacement
  17. 17. T 3 • Formed from T4 , in liver • Its conc. Is a reflection of functional state of peripheral tissue • Reduced conversion seen under influence of medications and in severe non-thyroid illness • Used in diagnosis of • T3-hyperthyroidism, • Detecting early stage of hyperthyroidism, and • For indicating diagnosis of Thyrotoxicosis factitia.
  18. 18. T 4 • Main product of thyroid gland • In circulation , more than 99 % is protein-bound form. • Changes in binding protein status should be considered in thyroid assessment ( due to estrogen – containing preparations , during pregnancy , in nephrotic syndrome ) • Used for detection of • Hyperthyroidism , • Detection of primary and secondary hypothyroidism , • Monitoring of TSH-suppression therapy.
  19. 19. TSH • Formed in anterior pitutary , subjected to circardian secretion sequence • It is the central regulating mechanism for biological action of thyroid hormones • Its determination is the initial test in thyroid diagnosis • Even very slight changes in conc. Of free thyroid hormones bring much greater opposite change in TSH levels • So , is a very sensitive and specific parameter to assess thyroid function • Suitable for early detection or exclusion of disorders in central regulation circuit between Hypothalamus- Pituitary-Thyroid
  20. 20. Free T3 • F T3 is physiologically active form of T3 , which is not bound to transport proteins • Used in diagnosis of • Euthyroid , • Hyperthyroid and • Hypothyroid state • Advantage is , it is independent of changes in conc. And binding properties Of binding proteins. • So additional determination of binding parameter is not needed
  21. 21. Free T4 • F T4 is physiologically active Thyroxine component , not bound to transport proteins • Its is an important element in routine clinical diagnosis • In suspected thyroid function disorders , Free T4 and TSH are measured • Also to monitor Thyrosuppressive therapy • Advantage is , • it is independent of changes in conc. And binding properties Of binding proteins. • So additional determination of binding parameter is not needed
  22. 22. Thyroglobulin ( Tg ) • It is synthesised by Thyrocytes • Low levels of circulating Tg indicate the presence of Thyroid tissue • After successful Total thyroidectomy , Tg is not detectable in circulation • In Congenital hypothyroidism , to distinguish complete absence of thyroid and thyroid hypoplasia or other pathological condition • A marker for morphological integrity of thyroid , injury to follicle wall results in Tg↑ • To distinguish between Subacute thyroiditis and Factitious thyrotoxicosis . In latter low Tg expected due to suppression of TSH
  23. 23. Tg confirmatory test Used in combination with Tg estimation ,  To assess potential interference effects ( anti-Tg Ab or nonspecific effects in patient sample )  And so to confirm Tg result ( can also be done by Anti-Tg assay )
  24. 24. Anti-Tg ( antibody to Tg ) • ↑ Anti-Tg found in autoimmunity based thyroiditis ( 70 % sensitive ). • ↑ Anti-Tg with Anti-TPO , indicative of Hashimoto’s disease • ↑ seen in nearly 70-80 % of Hashimoto’s disease , nearly 30 % of Graves’s disease • Used to monitor • Course of Hashimoto’s thyroiditis and differential diagnosis • Additional thyroid Abs , like Anti-TPO , TSH-receptor-Abs , increase the sensitivity
  25. 25. Anti-TPO ( antibodies to thyroid peroxidase / microsomal Ab) • ↑ serum titers of Abs to TPO are found in several forms of thyroidities due to autoimmunity ( 90 % sensitive). • High titres seen in upto 90 % of chronic Hashimoto’s thyroiditis and in 70 % of Graves disease ( 75 % sensitive ). • Sensitivity can be by simultaneous determination of↑ other thyroid Abs ( anti-Tg , TSH-receptor-Ab )
  26. 26. Anti –TSHR / TSH receptor Antibody / TRAb • In diagnosis and management of Grave’s disease ( 98.8 % sensitive , 99.6 % specific ) • Majority of Abs mimic TSH action • Indications for TRAb , include A) detection or exclusion of autoimmune hyperthyroidism and its differentiation from disseminated autonomy of thyroid B) monitoring therapy of Grave’s disease and prediction of its relapse , an important decision making in therapy C) in last trimester of pregnancy , in patients with history of thyroid disease ( TRAb can cross placenta and cause neonatal thyroid disease ) D) risk assessment of neonatal hyperthyroidism
  27. 27. Limitations of TFTs Thyroid function tests are measured by immunoassays that use specific antibodies and are subject to occasional interference. Results should be interpreted in the context of the clinical picture. If the laboratory results appear inconsistent with the clinical picture, communicate this to the laboratory and request the following checks: * Confirm the specimen identity. ** Reanalyse the specimen using an alternative manufacturer’s assay. *** Analyse the specimen for the presence of a heterophilic antibody. ** When you are unsure of the relevance of a particular result, a phone call to the lab consultant can be extremely helpful
  28. 28. Thyroid function CONDITION TSH T 4 FT4 ( FTI ) Normal / Euthyroid Normal Normal Normal Hyperthyroidism Low High High Hypothyroidism ( Primary ) disease of thyroid gland High Low Low Hypothyroidism ( Secondary ) problem involving pitutary Low Low Low
  29. 29. TFT – Interpretation TSH T4 T3 INTERPRETATION High Normal Normal Mild / subclinical hypothyroidism High Low Low / Normal Hypothyroidism Low Normal Normal Mild / subclinical hyperthyroidism Low High / Normal High / Normal Hyperthyroidism Low Low / normal Low / normal Pituitary ( Secondary ) hypothyroidism , rare
  30. 30. Differential diagnosis of thyroid diseases Algorithm is available It should be combined with other diagnostic tools ( imaging , histology ) & Clinical examination ( signs and symptoms )
  31. 31. Euthyroidism “eu” in Greek , means “good” Means healthy thyroid Per definition , TSH , Total or free T4 and Total or free T3 are expected in normal range
  32. 32. Clinical relevance of Thyroid hormones General recommendations for thyroid function diagnosis • TSH – sufficient to assess function in most OP Most reliable to rule out any dysfunction • FT4 – more reliable in unstable thyroid status • TSH + FT4 – in T4 substitution therapy To check compliance with hypothyroid patients
  33. 33. TSH – initial diagnostic test • Initial test , which triggers subsequent lab testing ( imaging , histology) • Used to monitor patients under hormone substitution or suppression therapy • Being sensitive and specific , evaluates every dysfunction in central regulation of hypothalamus , pituitary and thyroid • Primary hyperthyroidism – decreased TSH • Primary hypothyroidism – increased TSH • Secondary causes and other etiologies • Must not be interpreted isolated ( along with medical history , clinical examination , other findings ) • TSH has better analytical sensitivity ( than free hormones )
  34. 34. Typical TSH findings TSH Condition Normal Exclusion of dysfunction ↓ Overt Hyperthyroidism ↓ Subclinical Hypothyroidism ↑ Overt Hypothyroidism ↑ Subclinical Hypothyroidism ↑ Neonatal Screening / Congenital Hypothyroidism
  35. 35. Hyperthyroidism - diagnosis “Hyper” , Greek word, means “Over “ Primary hyperthyroidism ( caused by thyroid itself ) - Conc. Of Total or Free T4 and Total or Free T3↑ TSH below normal Additional investigations are needed ( imaging methods )
  36. 36. T3 hyperthyroidism Compensatory mechanism to lack of Iodine ( T3 needs less I than T4 ) or Due to accelerated deiodination process T3 is ↑ T4 normal or even low
  37. 37. Grave’s disease ( hyperthyroidism ) Due to auto-Abs to TSH-receptor TSH below normal ↑ T3, T4 Anti-TSHR positive
  38. 38. Hypothyroidism , Primary “Hypo “, Greek word, means “ under “ ↓ Total or Free T4 and Total or Free T3 ↑ TSH
  39. 39. Hypothyroidism , Secondary & Tertiary Secondary – insufficient TSH productn. In Pitutary Tertiary – insufficient TRH production of Hypothalamus Both show , TSH↓ ↓ total or free T4 and total or free T3 Differentiated with TRH-stimulation test
  40. 40. Hashimoto’s thyroiditis ( hypo-) Abs against thyroid peroxidase ( Anti-TPO ) and / or Thyroglobulin ( Anti-Tg ) , cause gradual destruction of thyroid follicles Detected by the above Abs ( Anti-TPO , Anti-Tg ) in blood
  41. 41. Peripheral receptor resistance Rare , autosomal dominant Reduced end-organ response to hormones ↑ total or free T3 and total or free T4 Normal or slightly TSH↑ Common clinical feature – Goiter but absence of usual symptoms and metabolic consequences of excess hormones In fact patient stays in hypothyroid state
  42. 42. T4-T3 conversion insufficiency In liver , T4 T3 does not occur adequately→ Possibly due to Enzyme insuffiency Patient is either euthyroid or hypothyroid , Normal to TSH↑ ↑ T4 and free T4 ↓ T3 and free T3
  43. 43. Thyroid autoimmune markers and cancer markers in diagnosis Marker Normal level Increased level Decreased level Anti-TSHR No thyroid autoimmune disease Graves disease Anti-TPO Anti-Tg DO Hashimoto’s thyroiditis Thyroglobulin ( Tg ) Benign or malignant thyroid disease Goiter Graves disease Thyroid drug treatment Calcitonin Benign thyroid diseases Medullary thyroid carcinoma
  44. 44. Typical FT3 , FT4 findings FT4 FT3 ↑ ↑ Overt Hyperthyroidism N N Subclinical Hyperthyroidism ↓ ↓ , N or ↑ Overt Hypothyroidism N N Subclinical Hypothyroidism T3 / Free T3 is more sensitive indicator of developing hyperthyroidism T4 / Free T4 is a sensitive indicator of developing hypothyroidism
  45. 45. Calcitonin in TFT Diagnosis and monitoring Medullary thyroid carcinoma ( C-cell carcinoma ) Along with CEA ( for diagnostic sensitivity )↑
  46. 46. Molecular biology investigations for Thyroid disease MEN 2 point mutation of RET-proto oncogene For Hereditary / Familial medullary thyroid carcinoma ( MEN 2 ) , with corresponding familial anamnesis
  47. 47. Mutations and Thyroid diseases Mutation Disease RET-proto oncogene Familial medullary thyroid carcinoma MEN2 Thyroglobulin Hypothyroidism Gene of the nuclear thyroid hormone receptor Thyroid hormone resistence
  48. 48. Role of lab TFT most common of endocrine lab investigations Efficient Capacity to handle work load Using latest test methods ( 4 th generation , ECLIA )
  49. 49. Sample stability Stability of hormones in blood / serum At RT , 4 – 8 C , -20 C See the reagent kit insert for details
  50. 50. Interpretation of Implausible looking results Check for any lab errors Check daily IQC / other daily lab routines If needed , repeat the test/s Discuss with consultant Request a repeat sample
  51. 51. Reference ranges • To give age dependent reference ranges , from birth till adult • Ask reagent kit supplier
  52. 52. Physiological situations • Age • Circadian rhythm • Pregnancy • Iodine ingestion need to be considered
  53. 53. Specific pathological situations Thyrotropic insufficiency Resistence to thyroid hormones Malnutrition Surgery Burns
  54. 54. Influence of medications By various mechanisms
  55. 55. Pre-analytical errors Sample tube Sample quality Storage / transport Back ground noise ** Release report with foot note
  56. 56. Analytical errors • Instrument calibration • Daily maintenance of instrument • Reagent calibration • Internal quality control • Wrong sample analysed • Cross reactions • Interfering Ab’s • Ab’s against analyte
  57. 57. Post analytical errors Transcriptional error/s To use an efficient LIS
  58. 58. Role of lab -Others • Storage of primary sample , for repeat testing • Good EQAS • ILQA , if needed • Report exact TSH value • Mention Method used in lab , (in the report) • Previous reports of patient • Talk to the consultant • Ask for a repeat sample
  59. 59. Interpreting lab TFT’s A “ hidden “ health problem Many challenges remain – selection of appropriate test/s , correct interpretation of results ,  application of results in diagnosis and management

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