The thyroid gland produces hormones that are essential for normal body metabolism. Blood testing is now commonly available to determine the adequacy of the levels of thyroid hormones. These blood tests can define whether the thyroid gland's hormone production is normal, overactive, or underactive.

1. Thyroid function test

2. ANATOMY
The thyroid gland is a butterfly-shaped endocrine
gland .
 Located on either side of the trachea
below the larynx.
Weights about 30g in adults

3. PHYSIOLOGY
Gland is highly vascular
Composed of large number of follicles and
parafolliular cells
Secrete 3 hormones
• Thyroxine(T4) secreted by follicular cells
• Triiodothyronine(T3)
• Thyrocalcitonin -by parafollicular cells

4. BIOSYNTHESIS OF THYROID
HORMONESteps in hormone synthesis and secretion:
Iodide Trapping
Oxidation Reaction
Organification Reaction
Coupling Reaction
Hormone Release

5. REGULATION

6. Concept of FT3 and FT4
 Out of the total T3 and T4 in circulation, most of
it remains bound to thyroid binding globulin ,
prealbumin and albumin.
 Only about 0.05% of each T3 and T4 remains free in
circulation. This is FT3 and FT4.
 These are better indicators for thyroid function than
total T3 and Total T4. (total=bound+free)
 For example in pregnancy, level of thyroid binding
globulin rises; hence though total T3 and total T4
remains same, level of FT3 and FT4 decreases.

8. THYROID FUNCTION TEST
The main objectives :
• To assess the functional status of the gland
• To characterise the anatomical features of the thyroid gland,
and
• To possibly evaluate the cause for the thyroid dysfunction

9. It is emphasised that a single thyroid function test is not
absolute in diagnostic accuracy and it must be thus a
careful selection of such tests so that their combination
can give comprehensive data that would enhance the
diagnostic accuracy

10. On the basis of the functions of the gland
I.Tests based on primary function of thyroid
• Radioiodine “uptake” studies and ‘turnover’ (RAI or
RIU)studies
• PBI¹³¹ in serum
• T3-suppression test
• TSH-stimulation test
• TRH-stimulation test.
CLASSIFICATION

11.  II.Tests measuring blood levels of thyroid
hormones
• Serum PBI and BEI
• Circulating T3 and T4 level
• Circulating TSH level
• In vitro resin-uptake of T3
• Plasma tyrosine level

12.  III.Tests based on metabolic effects of thyroid
hormones
• BMR
• Serum cholesterol level
• Serum creatine level
• Serum uric acid
• Serum CK enzyme.
 IV.“Scanning” of thyroid gland
 V.Immunological tests to detect autoimmune
diseases of thyroid gland
• Agar gel diffusion test (precipitation test)
• TRCH test: Tanned red cells haemagglutination test
• Complement fixation test

13. TESTS BASED ON PRIMARY FUNCTION
OF THYROID
(a)Radioactive “Uptake” Studies
• Iodine plays a key role in the metabolism of the thyroid
gland.
• I¹³¹ “tracer” is most commonly used for thyroid function
studies.
• Recently, 99m Tc has also been used as it behaves like
iodine and has added advantage of lower radiation dose
to the patient.

14. Procedure
• Dose of I ¹³¹ = 10μci given orally.
• Thyroid accumulation of radio-I ² is measured
externally over the gland.
• Radio-iodine uptake of the gland reflects the iodine-
“trapping” ability
• Thyroid uptake of I 131 is routinely measured 24-
hours after the administration of oral dose.
• “Turnover” is faster in ‘active’ and hyperfunctioning
gland and slower in underactive hypofunctioning
gland.

15.  Normal range:
20 to 40 per cent
Interpretations
• An abnormally high RAI uptake is usually consistent
with hyperthyroid state.
• In endemic goitre and some cases of non-toxic
sporadic goitre also may be high.
• Abnormally low thyroid uptake is characteristic
of hypothyroidism.

16. Urinary excretion of I ¹³¹ and “T” Index:
• Renal excretion of I¹³¹ an indirect evidence of thyroid
function.
• Proportion of the administered dose excreted is inversely
proportional to thyroid uptake. If uptake is “more”, less of
I¹³¹ will be excreted and vice versa.
• 24 hours urine is collected accurately and radioactivity
is measured.
 Normal range:
It is 30 to 60 percent of the administered dose

17.  “T”-index
• Activity is measured in urine sample after 0 to 8 hours,0 to
24 hours and 0 to 48 hours.
 ‘T’-index is calculated as follows:
T = 0-8hrs excretion expressed as percent×100
(0-24hrs excretion in%)×(0-48hrs excretion in%)
Normal value of “T”
2.5 to 12
 Interpretations
• A ‘T’-index > 17 indicates hyperfunctioning of the gland.
• A “T”-index < 2.5 indicates hypothyroidism.

18. Thyroid “Clearance’’ Rate
The amount of I¹³¹ that is accumulated in thyroid over a fixed
interval, in relation to the mean plasma concentration of I¹³¹
Hence, Thyroid Clearance rate =
Thyroid I¹³¹ accumulation rate
Plasma I¹³¹ concentration.
This gives a direct index of thyroid activity with regard to
I² accumulation.

19.  Normal value:
60 ml/mt.
Interpretations
•Clearance rate is high with thyroid hyperfunction.
•The value is also high when “intrathyroidal iodine pool”
is small.
•Lower values are indicative of hypothyroid status

20. b)Serum PBI¹³¹
Administered I¹³¹ accumulates in the thyroid gland and
appears as “labelled” hormone bound to proteins.Normally
it is a slow process.
In hyperthyroidism,level of protein-bound radioactivity
increases in plasma,which can be measured accurately by
a scintillation counter. The result is expressed
as“conversion ratio”, which indicates the proportion of the
total plasma radioactivity at 24 hrs.
 Normal value:35 per cent.

21. Interpretations
o In hyperthyroidism:
• It is usually greater than 50 percent.
• It is of no value in the assessment of patients who have
been treated for hyperthyroidism, as high values may persist
for long time after such treatments.
• PBI¹³¹ is found to be elevated in 50 per cent of the patients
with Hashimoto’s thyroiditis.

22. (c)T3-Suppression Test
1.After a 24 hrs RIU studies and obtaining the basal value and
serum T4 values, 20µg of T3 four times daily is given for 7
to 10 days (or alternatively 25µg three times a day for 7 days)
2.RIU is repeated after T3 administration and serum T4 values
are also determined.

23. Interpretations
• A suppression is indicated by the 24 hrs RIU falling to < 50 per
cent of the “initial” uptake (as exogenousT3 suppresses TSH)
and total T4 to approx 2µg/100ml or less
• Non suppression indicates autonomous thyroid
function.
• In Graves’ disease, no change seen as the action is due to
LATS (long-acting thyroid stimulator)and is not under control of
hypothalamopituitary axis.
Use
To differentiate borderline high normal from primary
hyperthyroidism (Graves’ disease)

24. d)TSH-Stimulation Test
• Following completion of 24 hrs RIU studies, 3 injections of
TSH, each 5 USP units are given at 24 hrs intervals.
• 24-hour thyroidal RIU is measured after 42 hours after the
final TSH dose.
Interpretations
• In primary hypothyroidism: There is failure of stimulation of the
gland.
• In secondary hypothyroidism: There is stimulation of the gland
showing increase RIU.
Use
The test is useful in differentiating primary hypothyroidism from
secondary hypothyroidism.

25. TRH-Stimulation Test
With the availability of synthetic TRH, which is a
tripeptide, suitable for human use, it is now
possible to assess the functional integrity of
thyrotropic cells or the factors that influence the
secretory response.
Procedure
200 to 400µg of TRH is administered IV and
blood samples at 0, 20,40 and 60 minutes are analysed for
TSH content.

26. Interpretations
• Peak response in normal is about 4 times elevation
of TSH levels at 20 and 40 minutes sample as compared
to basal TSH level.
• In primary hypothyroidism:The response will be
exaggerated and prolonged.
• In secondary hypothyroidism:The response will
be blunted.
• In tertiary hypothyroidism,i.e. hypothalamic in origin, the
increase in TSH is delayed
 Use
Currently this test is used to locate the site of pathological
lesion for hypothyroid states

27. TESTS MEASURING BLOOD LEVELS OF
THYROID HORMONES
a)Serum PBI and BEI Levels:
• It is indirect measure of thyroid hormones.
• It is useful where isotope techniques are not
available.
Disadvantage
• Technically time consuming lengthy procedure
• Also measures non-hormonal I 2 and iodotyrosines.
 Normal value:
Ranges from 4.0 to 8.0µg%

28. Interpretations
• More than 95 per cent of hyperthyroidism cases show
greater than 8.0µg%
• 87 per cent of hypothyroidism cases show value below
3µg%

29. Precautions and Limitations
o Easily affected by I2 contamination both exogenous and
endogenous.
• Exogenous: To eliminate exogenous contamination, all
glass wares and syringes should be iodinefree.
• Endogenous: Iodides, iodine containing drugs and I2-
containing radiological contrast media can give false high
results.
o The test is also affected by “trace” elements and chemicals
that interfere iodine-reduction reaction.
o Values are also affected by alterations in serum TBG
level.Increased serum TBG gives higher values whereas
decreased TBG gives lower values

30. Serum TBG may be increased in:
•Pregnancy
•Oestrogen therapy
•On oral contraceptive pills.
Serum TBG may be decreased in:
•Hypoproteinaemic states
•Nephrotic syndrome
•Androgen therapy and anabolic drugs like danazol
•Dicoumarol therapy
•Inherited TBG deficiency

31. o Certain drugs may give misleading results by
competing with T4 for Protein binding sites, e.g.
phenytoin sodium, salicylates, etc
Serum BEI: Butanol extractable iodine involves
extraction of serum with n-butanol and subsequent
washing of the extracts with alkaline solution.
This removes the inorganic iodine and iodotyrosines.
Interpretations
• In normal: Value ranges from 3.5 to 7.0µg%.
• In hyperthyroidism: Values are more than 10µg%.

32. (b)Serum T4 Levels
Most commonly used methods are:
• Competitive protein binding assay (CPBA)
• Radioimmunoassay (RIA)
• ELISA technique.
Interpretations
Normal range of serum T4 is 4.0 to 11.0µg%.
• In hyperthyroidism:The value is usually more than
12.0µg%
• In hypothyroidism:Less than 2.5µg%

33. (c)Effective Thyroxine Ratio (ETR)
This integrates into a single procedure the measurement of
total serum thyroxine and also binding capacity of thyroid
hormone proteins.
• ETR provides the most reliable single test of thyroid
function
 Advantage: It is not affected by oral
contraceptives,pregnancy, excess iodine or any
other drugs

34. d)Serum T3 Level
Radioimmune assay is the method of choice for
measurement of serum T3 level.
 Normal Range and Interpretations
• Normal value: 100 to 250 ng% (μg%).
• Values in females tend to be slightly on higher side than
compared to males.
• In hyperthyroidism: It is usually more than 350 ng%
• In hypothyroidism: Less than 100 ng%. It may be
useful test for hyperthyroidism, but it is less useful for
diagnosis of hypothyroidism

35. (e)Serum TSH Level
• Measurement of serum TSH also provides a very
sensitive index of thyroid function.
• By radioimmunoassay
• Normal range is 0 to 3μu/ml-1.6μu/ml
• Diagnosis of primary hypothyroidism.(TSH↑)

36. (f)In vitro I¹³¹-T3 uptake by resin/red cells(Hamolsky
et al1957):
Method
1.A known amount of I¹³¹ -T3 is added to a standard
volume of serum from a patient
2.The amount of I¹³¹ -T3 which binds to the serum
proteins varies inversely with the endogenous
thyroid hormones already bound to serum proteins (TBG).
3.Residual free I¹³¹ -T3 is then adsorbed by
resin/sponge/sephadex/red cells, which is removed
from the sample and then the adsorbed/bound I¹³¹ is
measured.
This method thus gives the measure of T4 binding in the serum
and not the actual level of thyroid hormones

37. Interpretations
•In normal subjects: The value is 21 to 35 per cent.
•In hyperthyroidism:The resin uptake in
hyperthyroidism will be more, greater than 35 per
cent.
•In hypothyroidism:The reverse will occur.
•Resin uptake of I¹³¹ -T3 also gets influenced by
drugs,hormones, pregnancy, etc.

38. g)Plasma Tyrosine Level
Rivlin et al (1965) studied plasma tyrosine level in
normal subjects and in thyroid disorders.
Interpretation
• Normal level: It was found to be from 11.8 +
0.4μg/ml.
• In hyperthyroidism: Plasma tyrosine level
was found to be elevated in more than 70
percent cases.

39. o Increased tyrosine level in hyperthyroidism:
Its Mechanism:
It is suggested that excess thyroid hormones has
inhibitory effect on hepatic and tissue tyrosine
transaminase, as a result tyrosine catabolism is
reduced and thus increasing plasma tyrosine level.
• In hypothyroidism: The decreased level of
plasma tyrosine was observed (average
9.8μg/ml)

40. TESTS BASED ON METABOLIC
EFFECTSOF THYROID HORMONES
These tests are of much use where facilities for isotope
techniques are not available.
(a)BMR:
The test is helpful in diagnosis and is of particular value in
assessing the severity and prognosis.

41. Interpretations
• A BMR between: 5 per cent and +20 per cent is
considered as normal.
• In euthyroid states:–10 to +10 per cent of normal
• In hyperthyroidism:+50 per cent to +75 percent is
usually found.
• In hypothyroidism: Value below –20 per cent
is suggestive (usually –30 per cent to –60 percent seen in
hypothyroid states).

42. (b)Serum Cholesterol Level
• It is useful in assessment of hypothyroidism,where it
is usually high.
• Baron has shown that 90 per cent of
hypothyroidism cases have serum cholesterol
greater than 260 mg%. In hypothyroidism, the
synthesis of cholesterol is impaired, but its
catabolism is reduced more, leading to high
cholesterol level

43. c)Serum Creatine Level
• Griffiths advocated the estimation of serum
creatine level for diagnosis of hyperthyroidism, who
considered a serum level greater than 0.6 mg% is
diagnostic.
• He compared serum creatine with BMR.
• He considered a normal serum creatine and normal
BMR excludes thyroid dysfunction and held that when
symptoms of thyroid disorders is present, a raised serum
creatine is highly significant even though BMR is normal.

44. d)Serum Uric Acid Level
Serum uric acid has been found to be increased in
myxoedematous males and postmenopausal
women,ranging from 6.5 to 11.0 mg%.
(e)Serum CK Level
Serum CK level are often raised in
hypothyroidism and in thyrotoxic
myopathy

45. f)Hypercalcaemia
It is very rarely found in severe thyrotoxicosis; there is
an increased turnover of bone, probably due to direct
action of thyroid hormones

46. THYROID SCANNING
• Scintiscans provide visualisation of the distribution of radioactive I ² in
the gland and also permits characterization of its anatomical features.
• The scan also permits functional classification of nodules as:–
 ‘Hot’ or ‘warm’: Areas of increased uptake. Hot nodules
suggest-increased thickness of the gland due to adenoma or
carcinoma.
 ‘Cold’ nodules:Which are due to reduced/or absent uptake. It
may be due to cysts, haemorrhagic nodules

47. • Scanning also provides useful information regarding
size, shape, position of the gland.
• Facilitates identification and localisation of func-
tioning thyroid tissues in “ectopic” or ‘Metastatic’sites,
e.g. in lungs and bones

48. Use of 99m technetium pertechnate:
Recently, 99m technetium pertechnate has been used.
It has similar properties as I ²
 Advantages
• Radiation effect is low
• Has very short half-life of 6 hours
• Virtual absence of Particulate radiations.

49. IMMUNOLOGICAL TESTS FOR
THYROID FUNCTIONS
I.Determination of Antithyroid Autoantibodies
• Antithyroid autoantibodies are found in a variety of thyroid disorders
as well as, in other autoimmune diseases and certain malignancies
• These autoantibodies are directed against several thyroid
components and thyroid hormone antigens

50. They are:
•Thyroglobulin (Tg)
•Thyroid microsomal antigen
•TSH receptor
•A non-thyroglobulin (non-Tg) colloid antigen
•Thyroid stimulating hormone (TSH) and
•Thyroxine (T4).

51. • Only anti-Tg (antithyroglobulin)and antimicrosomal
autoantibodies are commonly used in evaluating thyroid status
and function
• Anti-Tg autoantibodies are directed against thyro-globulin (Tg), a
major constituent of thyroid colloid.
• Several different techniques are available and used in clinical
laboratory to detect and quantify Tg-autoantibodies in blood

52. They are mainly:
• Agargel diffusion precipitation
• Tanned red cells haemagglutination test
(TRCHTest)
• Enzyme-linked immunoabsorbent assay (ELISA)
• Immunofluorescence of tissue sections
• Radioimmunoassay (RIA) method.
Most widely used method is based on
haemagglutination

53. o Tanned Red Cells Haemagglutination Test
(TRCH Test)
Principle:
In TRCH test, an aliquot of patient’s serum is mixed with
erythrocytes that have been treated/coatedwith tannic acid
and then quoted with purified human Tg-antigen.
When antibodies, if present in patient’s serum, com- bine
with tanned red cells coated with antigen, agglutination
occurs which is visible as a ‘carpet’ at the bottom.Lack of
agglutination is indicated by setting of the cells at the
bottom as a compact button or ring.

54.  Procedure
• Prior to testing, patient’s serum is inactivated at 56°C× for ½ hour
Note:Heating is important for inactivation of complement and
thyroid binding globulin (TBG), which other-wise would interfere
with the assay.
• A dried perspex tray with wells is taken. Serial
double dilutions of the patient’s inactivated serum is made to
establish Tg-antibody titre.
• A suspension of tanned-red cells coated with Tg-antigen is put
in each well.
• Tray is shaken and then kept in 4°C undisturbed for overnight.
• Reading is taken next morning.

55.  Interpretation
• Titres are usually considered negative at less than1 in 10 dilution
ratio.
• The reported result is the highest dilution that causes agglutination.
• The test is not highly specific
• Reactivity occurs more frequently in Hashimoto’s thyroiditis.
• In Grave’s disease (thyrotoxicosis) a high titre can be seen.
• Low titres may also be found in patients with non-toxic goitre,thyroid
carcinoma and pernicious anaemia

56. ELISA and RIA methods:
• These methods have been developed for measuring
anti-Tg antibodies.
• More sensitive and specific .
• Some assays also allow identification of
subclasses of Tg-antibodies.

57. II.Determination of Antimicrosomal Antibodies
Antimicrosomal antibodies are directed against a
protein component of thyroid cells microsomes. These
antibodies can be measured using:
• Complement fixation test (CFT)
• Immunofluorescence of tissue sections
• Passive haemagglutination test similar to TRCH
• ELISA techniques
• Radioimmunoassays (RIA)

58.  a)Tanned Red Cells Haemagglutination Test
—Using Microsomal Antigen
Tanned erythrocytes agglutination method uses red cells
coated with tannic acid and with microsomal
antigen isolated from human hyperplastic thyroid glands.
 Interpretation
• Positive reactivity occurs in nearly all adult patients with Hashimoto’s
thyroiditis and in Grave’s disease.
• Low titres may seen in normal asymptomatic individuals.

59. b)Complement Fixation Test (CFT)
CFT is used also in clinical laboratory but not routinely
as compared to TRCH test.
Limitations of anti-microsomal assays:
• Limited availability of human thyroid tissue
• Contamination of microsomal preparations with Tg.
• Presence of irrelevant thyroid antigens and auto-
antibodies.

60. III.Newer Tests
Recently the following newer techniques have been put
forward:
• Determination of antithyroid peroxidase antibody
(anti-TPo antibodies)
• Determination of thyrotropin-receptor
antibodies(Trab)

61. (a)Determination of Antithyroid Peroxidase
Antibody(Anti-TPO Antibody)
• In recent years, TPO has been identified and claimed as the
main and possibly the only autoimmune component of
microsomes.
• Its purification by using affinity chromatography
• Production by recombinant technology
• ELISA and RIA methods for measuring anti-TPO antibodies
• Methods are easy to perform, provide greater sensitivity and
specificity as compared to TRCH Tests, and can be used for
“screening”
• A suitable “immunometric assay” has been developed

62. Immunometric Assay
Principle:
Immunometric assay is based on competitive inhibition of
the binding of radio iodinated TPO to an anti-TPO
monoclonal antibody coated onto plastic tubes.
 Advantages
• Easy to perform
• Assay is rapid (only 2 hours incubation period
isrequired).
Result: The antibody concentration is expressed as
units/ml.

63. Interpretation
• In normal healthy persons:The mean anti-TPO
activity in serum is 69 + 15 units/ml.
• Detectable concentration of anti-TPO antibodies are
observed in nearly all patients with Hashimoto’s
thyroiditis, spontaneous adult myxoedema (idiopathic
primary type) and in a majority of patients with
Grave’s disease.
• The frequency of detectable anti-TPO autoanti-
bodies found in normals and non thyroid cases is
similar

64. (b)Determination of Thyrotropin-Receptor
Antibodies(TRAb)
• Thyrotropin-receptor antibodies (TRAb) are group of
related immunoglobulins (Igs) that bind to thyroid cell
membranes at or near the “TSH receptor” site.
• These antibodies have recently been demonstrated
frequently in patients with Grave’s disease and also in
other thyroid autoimmune disorders

65. Types of receptor antibodies:
1.Thyrotropin binding inhibitory immuno-
globulins (TBI)
2.Thyroid stimulating immunoglobulins (TSIgs).
 Their presence is determined by either:
(i) radioreceptor assays; (ii) bioassays

66. 1.Thyrotropin-binding Inhibitory Immunoglobulins(TBI)
• Determined by direct radio receptor assay.
• The method assesses the capacity of Igs to inhibit the
binding of radioisotope labelled TSH to its receptors in
thyroid membrane.
• In this method, detergent-solubilised porcine TSH-
receptors and125 I-labelled TSH are used.
• The ability of a purified fraction of serum Igs to
displace125I-labelled TSH from the receptors is
measured.

67. Interpretation
• Normal -only less than 10 per cent inhibition.
• This method detects over 85 per cent of patients with
Grave’s disease

68. 2.Thyroid Stimulating Immunoglobulins (TSIgs)
• In vitro bioassay utilised. The method assesses the
capacity of the Igs (antibodies) to stimulate a functional
activity of the thyroid gland such as adenyl cyclase
stimulation leading to increase in cyclic-AMP formation.
• Measurement of increase in cyclic-AMP level
can be done using human thyroid slices, frozen human
thyroid cells culture or a cloned line of thyroid follicular
cells.

69. Interpretations
• In normal:Range is 70 to 130 per cent
• TSIgs have been detected in patients with untreated
Grave’s disease. It has been claimed to be highly
sensitive and specific technique in diagnosing Grave’s
disease.
• TSIgs measurement has also been found to be useful for
predicting relapse or remission in hyperthyroid patients.
• Useful for predicting the development of neonatal
hyperthyroidism

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