Prabin Shah MSc(Biochemistry)

Importance of TFT5 % of world population suffer from Thyroid

problems / dysfunctionThyroid insufficiency ( due to iodine

deficiency )is a worldwide problemA Major work load in Endocrine laboratoryTest/s asked from newborn to elderly

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

TFTConcerned with Disease & its effect on Production ( ↑ / ↓ ) of

Iodine-containing hormonesProteins which bind themMetabolic changes due to ↑ or ↓ in hormonal

secretion

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 )

Hypothalamic Pituitary Thyroid Axis

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

Thyroid hormonesHORMONE

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

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

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

TFTOne 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

When to testClinical suspicion – signs and symptoms

provide the best indication to request for TFTHigh , Intermediate and Low suspicion

groups for Hyper- and Hypothyroid states

Thyroid function test , involvesTaking good historyClinical examinationLaboratory tests , usually follow , it may

stop hereMay be , ultra sound ,( if shows large

nodules)FNAC , for evaluation of thyroid nodulesThyroid scan

Thyroid function state• Euthyroid – state of normal thyroid function

• Hyperthyroid – state of overactive thyroid

• Hypothyroid – state of underactive thyroid

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)

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

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.

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.

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

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

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

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

Tg confirmatory testUsed 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 )

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

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 )

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

Limitations of TFTsThyroid 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

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

TFT – Interpretation TSH T4 T3 INTERPRETATI

ON

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

Differential diagnosis of thyroid diseasesAlgorithm is availableIt should be combined with other diagnostic

tools ( imaging , histology ) &Clinical examination ( signs and symptoms )

Euthyroidism “eu” in Greek , means “good”Means healthy thyroidPer definition , TSH , Total or free T4 and

Total or free T3 are expected in normal range

Clinical relevance of Thyroid hormones General recommendations for thyroid function diagnosis

• TSH – sufficient to assess function in most OPMost reliable to rule out any dysfunction• FT4 – more reliable in unstable thyroid status• TSH + FT4 – in T4 substitution therapyTo check compliance with hypothyroid patients

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 )

Typical TSH findingsTSH Condition

Normal Exclusion of dysfunction

↓ Overt Hyperthyroidism

↓ Subclinical Hypothyroidism

↑ Overt Hypothyroidism

↑ Subclinical Hypothyroidism

↑ Neonatal Screening / Congenital Hypothyroidism

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 normalAdditional investigations are needed

( imaging methods )

T3 hyperthyroidism Compensatory mechanism to lack of Iodine

( T3 needs less I than T4 ) orDue to accelerated deiodination processT3 is ↑T4 normal or even low

Grave’s disease ( hyperthyroidism )Due to auto-Abs to TSH-receptorTSH below normal↑ T3, T4Anti-TSHR positive

Hypothyroidism , Primary“Hypo “, Greek word, means “ under “↓ Total or Free T4 and Total or Free T3↑ TSH

Hypothyroidism , Secondary & TertiarySecondary – insufficient TSH productn. In

PitutaryTertiary – insufficient TRH production of

HypothalamusBoth show , ↓ TSH↓ total or free T4 and total or free T3Differentiated with TRH-stimulation test

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

Peripheral receptor resistanceRare , autosomal dominantReduced end-organ response to hormones↑ total or free T3 and total or free T4Normal or slightly ↑ TSHCommon clinical feature – Goiter but absence

of usual symptoms and metabolic consequences of excess hormones

In fact patient stays in hypothyroid state

T4-T3 conversion insufficiencyIn liver , T4 → T3 does not occur adequatelyPossibly due to Enzyme insuffiencyPatient is either euthyroid or hypothyroid , Normal to ↑ TSH↑ T4 and free T4↓ T3 and free T3

Thyroid autoimmune markers and cancer markers in diagnosis

Marker Normal level Increased level

Decreased level

Anti-TSHR No thyroid autoimmune disease

Graves disease

Anti-TPOAnti-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

Typical FT3 , FT4 findingsFT4 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

Calcitonin in TFTDiagnosis and monitoring Medullary thyroid

carcinoma ( C-cell carcinoma )Along with CEA ( for ↑ diagnostic sensitivity )

Molecular biology investigations for Thyroid diseaseMEN 2 point mutation of RET-proto

oncogene

For Hereditary / Familial medullary thyroid carcinoma ( MEN 2 ) , with corresponding familial anamnesis

Mutations and Thyroid diseasesMutation Disease

RET-proto oncogene Familial medullary thyroid carcinoma MEN2

Thyroglobulin Hypothyroidism

Gene of the nuclear thyroid hormone receptor

Thyroid hormone resistence

Role of labTFT most common of endocrine lab

investigationsEfficient Capacity to handle work loadUsing latest test methods ( 4 th generation ,

ECLIA )

Sample stability Stability of hormones in blood / serumAt RT , 4 – 8 C , -20 CSee the reagent kit insert for details

Interpretation of Implausible looking resultsCheck for any lab errorsCheck daily IQC / other daily lab routinesIf needed , repeat the test/sDiscuss with consultantRequest a repeat sample

Reference ranges• To give age dependent reference ranges ,

from birth till adult• Ask reagent kit supplier

Physiological situations• Age • Circadian rhythm• Pregnancy • Iodine ingestion

need to be considered

Specific pathological situations Thyrotropic insufficiencyResistence to thyroid hormonesMalnutrition SurgeryBurns

Influence of medicationsBy various mechanisms

Pre-analytical errorsSample tubeSample quality Storage / transportBack ground noise

** Release report with foot note

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

Post analytical errorsTranscriptional error/sTo use an efficient LIS

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

Interpreting lab TFT’sA “ hidden “ health problemMany challenges remain – selection of appropriate test/s , correct interpretation of results , application of results in diagnosis and

management