3. Introduction
The laboratory diagnostic process to obtain a
result can be divided into three phases: the pre-
analytical, analytical and post-analytical phases .
4. The pre-analytical phase is defined as the period
from the physician’s indication of the test up to
the laboratory analysis of the biological material
The importance of this phase is also supported
by many publications mentioning the fact that
up to 46 – 68 % of erroneous results are caused
by failure to follow or respect the pre-analytical
phase rules
5. The pre-analytical phase is followed by the
analytical phase, involving the sample analysis
itself. Each laboratory must have an established
quality control system to ensure the validity of
the issued results
6. The analytical phase ends with the post-
analytical phase, defined as the period from
obtaining the lab result to its hand-over to the
physician
7. It is necessary to keep in mind that biological
samples constitute a risk of infection, and
therefore personal protective equipment
(rubber gloves, protective coat) should be used
for work with biological material (material
collection, lab work with the sample). In
addition, a face mask and safety goggles must
be used for highly infectious samples such as
HIV or hepatitis C
8. THYR0ID FUNCTION TEST
INTRODUCTION :
The main objectives for the laboratory
procedures in evaluation of thyroid diseases are:
• 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. TESTS BASED ON PRIMARY FUNCTION
OF THYROID
(a) Radioactive “Uptake” Studies Iodine plays a
key role in the metabolism of the thyroid gland.
I 131 “tracer” is most commonly used for
thyroid function studies because of low cost,
easy availability, and convenient shelf life.
10. Short lived isotopes of iodine like I132 and I123
are preferred for use in paediatric practice and
in pregnant and lactating women. Recently,
99mTc has also been used as it behaves like
iodine and has added advantage of lower
radiation dose to the patient.
11. Procedure
Dose of I131 = 10 μci given orally. Thyroid
accumulation of radio-I2 is measured externally
over the gland. Radioiodine uptake of the gland
reflects the iodine-“trapping”ability.
12. Thyroid uptake of I131 is routinely measured 24-
hours after the administration of oral dose,
although 4-hour uptake or 48-hour uptake are
also measured when rapid turnover or delayed
uptake situation is expected. “Turnover” is faster
in ‘active’ and hyperfunctioning gland and
slower in underactive hypofunctioning gland
13. 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, but not specific since subacute
thyroiditis and administration of large doses of I2
and thyroid hormones may also lower the I131
uptake of the gland.
14. Urinary excretion of I131 and “T” Index: Renal
excretion of I131 is 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 per cent of the
administered dose.
15. “T”-index Activity is measured in urine sample after
0 to 8 hours, 0 to 24 hours and 0 to 48 hours.
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
16. 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
mid-way in that time period provides the index of
rate at which the thyroid gland is handling I131.
(Rationale is This gives a direct index of thyroid
activity with regard to I2 accumulation.
• Normal value: 60 ml/mt. similar to the concept of
renal clearance.)
17. Interpretations
• Clearance rate is high with thyroid
hyperfunction, the value has been distinctly high
with no overlap.
• The value is also high when “intrathyroidal
iodine pool” is small.
• Lower values are indicative of hypothyroid
status
18. (b)Serum PBI131 Administered I131
accumulates in the thyroid gland and appears as
“labelled” hormone bound to proteins. Normally
it is a slow process, but in hyperthyroidism, level
of protein-bound radioactivity increases in
plasma, which can be measured accurately by a
scintillation counter. The result is conveniently
expressed as “conversion ratio”, which indicates
the proportion of the total plasma radioactivity
at 24 hrs. • Normal value: 35 per cent.
19. Interpretations
• In hyperthyroidism: It is usually greater than 50 per cent
• It is of no value in the assessment of patients who have
been treated for hyperthyroidism, either surgically or
with radioactive I2, 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, when the thyroid
uptake is usually normal or low, a combination of findings
which is very suggestive of this condition. The reason for
these discrepancies is that PBI131 is not a measure of
plasma thyroxine concentration
20. (c) T3-Suppression Test
1. After a 24 hrs RIU studies and obtaining the
basal value and serum T4 values, 20 μg of T3
daily is given for 7 to 10 days (or alternatively 25
μg three times a day for 7 days).
2. RIU is repeated after T3 administration and
serum T4 values are also determined four times
21. Interpretations
• A suppression is indicated by the 24 hrs RIU
falling to < 50 per cent of the “initial” uptake (as
exogenous T3 suppresses TSH) and total T4 to
approx 2 μg/100 ml 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
22. (d)TSH-Stimulation Test
1. Following completion of 24 hrs RIU studies, 3
injections of TSH, each 5 USP units are given at
24 hrs intervals.
2. 24-hour thyroidal RIU is measured after 42
hours after the final TSH dose.
23. Interpretations
• In primary hypothyroidism: There is failure of
stimulation of the gland.
• In secondary hypothyroidism: There is
stimulation of the gland showing increase RIU
24. The test is useful in differentiating primary
hypothyroidism from secondary
hypothyroidism.
25. (e) 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
26. Procedure
200 to 400 μg of TRH is administered IV and
blood samples at 0, 20, 40 and 60 minutes are
analysed for TSH content.
27. 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.
29. (a) Serum PBI and BEI Levels:
Chemical estimation of Protein bound I2 is used
for long time as a test for thyroid function.
• It is indirect measure of thyroid hormones.
• It is useful where isotope techniques are not
available.
Normal value: Ranges from 4.0 to 8.0 μg%
30. Interpretations
• More than 95 per cent of hyperthyroidism cases
show greater than 8.0 μg%
• 87 per cent of hypothyroidism cases show value
below 3 μg%
• Care should be taken to interpret values between
4.0 and 5.0 μg%.
31. (b) Serum T4 Levels
Most commonly used methods are:
• Competitive protein binding assay (CPBA)
• Radioimmunoassay (RIA)
• ELISA technique.
32. Interpretations
• Normal range of serum T4 is 4.0 to 11.0 μg%.
• In hyperthyroidism: The value is usually more
than 12.0 μg%
• In hypothyroidism: Less than 2.5 μg%.
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. At
the present time, the ETR provides the most
reliable single test of thyroid function available
which can be readily carried out on a sample of
serum and only requires radioisotope laboratory
34. (d) Serum T3 Level
Radioimmune assay is the method of choice for
measurement of serum T3 level. CPBA is not
good and accurate as T3 has very low affinity for
TBG.
35. 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 than350ng%
• In hypothyroidism: Less than 100 ng%. It may be
useful test for hyperthyroidism, but it is less useful
for diagnosis of hypothyroidism.
36. (e) Serum TSH Level
Measurement of serum TSH also provides a very
sensitive index of thyroid function. By
radioimmunoassay, the normal range is 0 to 3
μu/ml average be in 1.6 μu/ml. It is of particular
value in the diagnosis of primary
hypothyroidism.
37. (f) In vitro I131-T3 uptake by resin/red cells
(Hamolsky et al 1957):
Method
1. A known amount of I131-T3 is added to a
standard volume of serum from a patient
2. The amount of I31-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.
38. Interpretations
• In normal subjects: The value is 21 to 35 per cent.
• In hyperthyroidism: Saturation of binding of TBG
with endogenous T4 and T3 is greater than normal,
hence little of tracer I131-T3 can bind to TBG and more
I131-T3 will be free to be adsorbed by resin/sponge.
The resin uptake in hyperthyroidism will be more,
greater than 35 per cent.
• In hypothyroidism: The reverse will occur. The
proportion of I131-T3 taken up by the resin is inversely
reduced and less than 21 per cent.
39. (g) Plasma Tyrosine Level
Rivlin et al (1965) studied plasma tyrosine level in
normal subjects and in thyroid disorders.
Interpretations
• Normal level: It was found to be from 11.8 + 0.4
μg/ml.
• In hyperthyroidism: Plasma tyrosine level was
found to be elevated in more than 70 per cent cases.
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 catabolis
is reduced and thus increasing plasma tyrosine level.
40. In hypothyroidism: The decreased level of
plasma tyrosine was observed (average 9.8
μg/ml).
41. TESTS BASED ON METABOLIC EFFECTS
OF 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.
At least two estimations consecutively after
proper sedation and physical/mental rest will be
helpful.
42. 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 per cent
is usually found.
• In hypothyroidism: Value below –20 per cent is
suggestive (usually –30 per cent to –60 per cent
seen in hypothyroid states).
43. (b) Serum Cholesterol Level
It is useful in assessment of hypothyroidism, where
it is usually high. Not of much value in
hyperthyroidism, though it is usually low. Baron has
shown that 90 per cent of hypothyroidism cases have
serum cholesterol greater than 260 mg%. He found
poor correlation with severity as judged by BMR. In
hypothyroidism, the synthesis of cholesterol is
impaired, but its catabolism is reduced more, leading
to high cholesterol level.
44. (c) Serum Creatine Level
Griffiths advocated the estimation of serum
creatine level for diagnosis of hyperthyroidism,
who considered a serum level greater than
0.6mg% is diagnostic. anormal 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.
45. (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%.
46. (e) Serum CK Level
Serum CK level are often raised in
hypothyroidism but the estimation does not
help in diagnosis. CK levels are also raised in
thyrotoxic myopathy.
47. (f) Hypercalcaemia
It is very rarely found in severe thyrotoxicosis;
there is an increased turnover of bone, probably
due to direct action of thyroin hormones.It is
very rarely found in severe thyrotoxicosis; there
is an increased turnover of bone, probably due
to direct action of thyroin hormones.
48. THYROID SCANNING
Scintiscans provide visualisation of the
distribution of radioactive I2 in the gland and
also permits characterization of its anatomical
features
49. Use of 99m technetium pertechnate
: Recently, 99m technetium pertechnate has been
used. It has similar properties as I2. Thyroid
follicles ‘trap’ pertechnate ions, similar to I2.
Advantages
• Radiation effect is low
• Has very short half-life of 6 hours
• Virtual absence of Particulate radiations
50. 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. They are:
• Thyroglobulin (Tg)
• Thyroid microsomal antigen
• TSH receptor
• A non-thyroglobulin (non-Tg) colloid antigen
• Thyroid stimulating hormone (TSH) and
• Thyroxine (T4).
51. Several different techniques are available and
used in clinical laboratory to detect and quantify
Tg-autoantibodies in blood.
They are mainly:
• Agar gel diffusion precipitation .
• Tanned red cells haemagglutination test (TRCH
Test)
• Enzyme-linked immunoabsorbent assay
(ELISA)
• Immunofluorescence of tissue sections
• Radioimmunoassay (RIA) method.
52. Practical Implications of
Immunological Tests
Thyroid autoantibodies detection is of
importance in diagnosis of the following
conditions:
• In nodular goitres, detection of thyroid
autoantibodies in high titres make the possibility
of goitres being due to carcinoma less likely.
• Primary hypothyroidism can be differentiated
from obesity and other hypometabolic states.
53. • Autoimmune thyroiditis diagnosis is
confirmed.
• In differential diagnosis of endocrine
exophthalmos other ocular lesions can be
excluded.
• Serological tests may provide choice of line of
treatment in patients with Grave’s disease.
54. Procedure
• Prior to testing, patient’s serum is inactivated
at 56°C × for ½ hour.
55.
56. Interpretation
• Titres are usually considered negative at less than
1 in 10 dilution ratio.
• The reported result is the highest dilution that
causes agglutination (carpet of red cells at bottom
of the well).
• The test is not highly specific and about 5 to 10
per cent of the normal population may have a low
titre of Tg-autoantibodies with no symptoms of
the disease.
57. • Reactivity occurs more frequently in Hashimoto’s
thyroiditis. It is positive in very high titre in more
than 85 per cent of the patients.
• In Grave’s disease (thyrotoxicosis) a high titre even
greater than 1600 are common in more than 30 per
cent of patients.
• Positive responses with high titre also observed
in spontaneous adult myxoedema (primary) in
more than 45 per cent of cases. In another 30 per
cent cases titres may be low but positive.
58. ELISA and RIA methods: These methods have
been developed for measuring anti-Tg
antibodies. Correlate well with agglutination
tests but are generally more
59. sensitive and specific for thyroid autoimmune
diseases. Some assays also allow identification
of subclasses of Tgantibodies. The clinical
significance of these subclasses is still not clear.
60. Adrenal Cortex Hormones
Cortisol
Total Serum Cortisol
This test is used mainly to diagnose Cushing’s
syndrome, in which the fluctuations in diurnal
cortisol levels are
suppressed. However, this test is less reliable than
the 24-hr urinary free cortisol test. The test is made
on serum or
plasma collected between 8 and 9 a.m. or as a daily
profile, always before meals, and haemolysis should
be prevented.
The patient must rest for at least 30 minutes before
collection
Adrenal Cortex Hormones
61. Elevated cortisol levels are tested for the following
diagnoses: burns, Crohn’s diseases, Cushing’s
disease, Cushing’s syndrome, ectopic ACTH
production, eclampsia, severe hepatopathies,
hyperpituitarism, hypertension, hyperthyroidism,
severe infections, obesity, osteoporosis, acute
pancreatitis, pregnancy (elevated CBG, cortisol
levels may be up to 2.5 times higher in the third
trimester), severe renal diseases, shock conditions,
increased stress (heat, cold, traumatic
or mental stress), surgery, virilization
62. Reduced cortisol levels are typical of the following
diagnoses: Addison’s disease, adrenal insufficiency,
adrenogenital syndrome
(congenitaladrenalhyperplasia),
hypophysectomy, hypopituitarism, hypothyroidism,
hepatopathy, rheumatoid arthritis, and following
administration of dexamethasone and derivative
preparations, ketoconazole, morphine, the inhalation of
glucocorticoids for asthma bronchiale treatment and
grapefruit juice ingestion.
63. Urinary Free Cortisol
Free cortisol is unconjugated cortisol filtered
through the glomeruli to the urine. Free cortisol
represents only about 5% of the total circulating
cortisol, but the amount filtered to the urine
accurately follows the secretory pattern of the
adrenal cortex. This test is important because 24-
hour urine collection eliminates the influence of
diurnal variations.
The main indication for the assay is Cushing’s
syndrome and the differential diagnosis of
obesity; the assay is not suitable for a diagnosis
of Addison’s disease.
64. The patient should not be subject to physical
strain or stress. Urine should be collected in
a glass or plastic container (refrigerated
container), and urine preservation with boric acid
(10 g in a 3-litre container) or acetic acid (33%) is
advisable. If the patient is catheterized, the
collection bag must be placed on ice and
emptied into a refrigerated container every hour
65. CRH Test
Indications: Cushing’s syndrome – to distinguish
between a primary (peripheral) and a secondary
(central) origin.
To determine cortisol and ACTH, 1 ml of
anticoagulated blood with K2EDTA is taken (the
patient must rest for at least 2 hours before
sampling)..
66. Following the determination of basal values, 100 μg
(human) CRH is administered intravenously.
Samples should then be taken at intervals of 15, 30,
45 and 60 minutes
Elevated ACTH with normal cortisol levels is
indicative of an adrenal tumour.
67. ACTH (Synacthen) Stimulation Test
This is a diagnostic test to evaluate the adrenal
cortex function used for suspected cortisol
production deficiency.
The administration of exogenous ACTH
stimulates the adrenal cortex. The test points to
the secretory capacity of the adrenal corte
68. Insulin Test
Hypoglycaemia is a strong stress impulse for
ACTH and STH secretion. Blood for blood sugar,
cortisol, ACTH and STH
assays is taken in the morning. I.v. insulin is
administered: 0.05 – 0.1 U/kg of body weight
for suspected hypothalamic-
-pituitary insufficiency, 0.15 U/kg for an
anticipated normal response, 0.20 – 0.30 U/kg
in obese patients with Cushing’s
syndrome.
69. Blood is taken in 30, 60, 90 and 120 minutes.
Evaluation: Blood sugar in healthy people drops
under 2.2 mmol/l and cortisol rises over 320
nmol/l, or reaches a value 1.5 times higher than
the baseline value. ACTH reaches at
least twice as high against the baseline value.
Contraindications for the test include ICHS,
cerebrovascular , gestosis or epilepsy
70. REFRANCES
CLINICAL BIOCHEMISTRY
Basic Concept of Clinical Biochemistry
Kalpana Luthra
Associate Professor
Department of Biochemistry
All India Institute of Medical Sciences New Delhi
– 110 029
Textbook of Medical Biochemistry
