Thyroid function testing

1. Prabin Shah MSc(Biochemistry)

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. 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. 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. 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. Hypothalamic Pituitary Thyroid
Axis

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
•2
nd
protein is Thyroxine-binding Prealbumin ( TBPA ) ,
carry only T4 , 15 – 25 %
•Albumin also binds T4 , but has low affinity , minor
role

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. 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. 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. 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. 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. 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. Thyroid function state
•Euthyroid – state of normal thyroid function
•Hyperthyroid – state of overactive thyroid
•Hypothyroid – state of underactive thyroid

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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. Differential diagnosis of thyroid
diseases
Algorithm is available
It should be combined with other diagnostic tools
( imaging , histology ) &
Clinical examination ( signs and symptoms )

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. 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. 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. Typical TSH findings
TSH Condition
Normal Exclusion of dysfunction
↓ Overt Hyperthyroidism
↓ Subclinical Hypothyroidism
↑ Overt Hypothyroidism
↑ Subclinical Hypothyroidism
↑ Neonatal Screening / Congenital
Hypothyroidism

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. 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. Grave’s disease ( hyperthyroidism )
Due to auto-Abs to TSH-receptor
TSH below normal

↑
T3, T4
Anti-TSHR positive

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

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. 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. 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. 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. 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. 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. Calcitonin in TFT
Diagnosis and monitoring Medullary thyroid
carcinoma ( C-cell carcinoma )
Along with CEA ( for diagnostic sensitivity )
↑

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. 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. 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. Sample stability
Stability of hormones in blood / serum
At RT , 4 – 8 C , -20 C
See the reagent kit insert for details

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. Reference ranges
•To give age dependent reference ranges , from birth
till adult
•Ask reagent kit supplier

52. Physiological situations
•Age
•Circadian rhythm
•Pregnancy
•Iodine ingestion
need to be considered

53. Specific pathological situations
Thyrotropic insufficiency
Resistence to thyroid hormones
Malnutrition
Surgery
Burns

54. Influence of medications
By various mechanisms

55. Pre-analytical errors
Sample tube
Sample quality
Storage / transport
Back ground noise
** Release report with foot note

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. Post analytical errors
Transcriptional error/s
To use an efficient LIS

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. 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