Quantitative Determination of TSH

Prepared by

Hassan Megahed Hassan

Faculty of Science - B.S.U

 1 Quantitative Determination of TSH

Thyroid Gland

Hormonal control
In this lab
we will
study

Determination of TSH

Case study
 1 Thyroid Gland

• The thyroid is one of the glands that make up the endocrine system.

• The thyroid makes three hormones: Thyroxin (T4), triiodothyronine

(T3) and calcitonin.

• (T4) & (T3) are peptide hormones formed from iodine added to

amino acid Tryrosine to form → Mono and Di-iodo tyrosine.

• only a small amount of T3 in the blood comes from the thyroid.

• Most T3 is made from T4. (T4 deiodinzed then convert to T3)

 2 Hormonal control Feed back mechanism (- &+)

• Thyroid-releasing hormone (TRH) produced by +

hypothalamus which stimulate Thyroid-stimulating

hormone (TSH), made by the pituitary gland in the brain,

regulates thyroid hormone production (T3, T4 ).

+
• In long lope negative feed back mechanism

- Stimulus from Thyroid gland to hypothalamus.

• In short lope feed back mechanism
- Stimulus from Thyroid gland to pituitary gland .
 2 Hormonal control

• In long lope negative feed back mechanism

• Low level of (T3, T4 ) cause increase of (TRH & TSH ).

• High level of (T3, T4 ) cause decrease of (TRH & TSH ).

• In short lope negative feed back mechanism

• Low level of (T3, T4 ) cause increase of (TSH ).

• High level of (T3, T4 ) cause decrease of (TSH ).

• Tests to evaluate thyroid function(TFT) include the following: (TSH), (T4) and (T3).

3 Quantitative Determination of TSH

A Purpose of TSH test

• Evaluating of thyroid function (TFT) and how well the thyroid is working.

• To diagnose & help find the cause of thyroid disorders as hyperthyroidism & hypothyroidism.

Primary hypothyroidism. (Thyroid) disorder “Cancer”

• Helps to differentiate among: Secondary hypothyroidism. (Pituitary) disorder “Tumor “

Tertiary hypothyroidism. (Hypothalamus) disorder

 B Principle of the TSH test

• By: Sandwich ELISA.

B Principle of the TSH test

TMB with H2O2

1ry A.b TSH 2ry A.b with HRP Stop solution

Primary antibody = Capture antibody = Mouse monoclonal antibodies (Mab).

Secondary antibody = Detector antibody = Secondary anti-TSH antibody = probe A.b.

TMB with H2O2 (oxidizing agent) Stop solution : H2SO4 (2N)

C Procedure of TSH test

Pipet 50 µl standard, Wash x 4(300 µl) Pipet 100 µl Stop Solution

control or sample

- Pipet 100 µl TSH-HRP Tracer - Pipet 100 µl an TMB

Read at 450/630 nm
- Incubate 60 min. at RT. - Incubate 20 min. at RT.
 12 strips = each strip contain 8 wells

96 well = 12 * 8
strip wells

Strip Well
-The polystyrene wells are coated
with captured antibody against TSH.
- Standards, controls and patient
samples are added to the wells. A CH

Standards (μL mL) Controls B CL

C S
A= 0 C H=7.9 ± 1.2
D
B= 0.3 CL=0.3± 0.1
E
C= 1 F
D= 3 G
E= 6
F= 15
IF sample more than 30 In this case you make sample dilution
G= 30
- The HRP labeled anti-TSH is added
A CH
to the wells (at R.T).
B CL
- 50μL added and incubate it for 1hr. S
C
D
E
F
G
- Wash 4 times by washing solution
A CH
“phosphate buffer saline”(1X).
B CL
300μL. C S

D
-NOTE: washing solution
E
“phosphate buffer saline”(20X). F
G
TO make ”(1X) take 1ml of washing

solution and add it to 19 ml dist.

Water.
- A solution of TMB “bonded to
H2O2” is added to wells cause the A CH
B CL
development of a blue color.
C S
- 100μL added and leave it for 20
D
min. in dark condition.
E
F
G

The intensity of the color is proportional to the amount of TSH present in the sample.
- The color development is
A CH
stopped by addition of Stop B CL

C S
solution (100μL H2SO4 “2N”),
D
causing the blue color to change E

to yellow. F
G

The intensity of the color is proportional to the amount of TSH present in the sample.
D Calculation of results

- Standard curves are constructed for each assay by plotting absorbance value against the concentration
of each standard.
- The TSH concentrations of patient samples are then read from the standard curve.

TSH concentration(mIU/L)
E Normal values

Normal TSH levels typically fall between 0.4 and 4.0 milliunits per liter (mIU/L), according to the

American Thyroid Association (ATA).

F Abnormal values

Note: An abnormal TSH indicates an excess or deficiency in the thyroid hormone in the body, but

it does not indicate the reason why so usually followed by additional testing to investigate the cause

of the increase or decrease.

 Causes of low level of TSH Causes of high level of TSH

• An overactive thyroid gland (hyperthyroidism) • An underactive thyroid gland (hypothyroidism)

high T3 and T4 as Graves disease (autoimmune disorder) as Hashimoto thyroiditis
• Excessive amounts of thyroid hormone medication • Increased TSH concentration is observed in:
taken for an underactive (or removed) thyroid g. primary hypothyroidism
• Insufficient anti-thyroid medication in a person • TSH-producing pituitary tumor and in newborn
treated for hyperthyroidism. • A person with hypothyroidism is receiving too
• Damage to the pituitary gland. much thyroid medication.
• thyroid cancer • A rare inherited disorder is present in which the
• Decreased TSH concentration is observed in: 1ry body and/or pituitary do not respond normally to
hyper-thyroidism, secondary and tertiary thyroid hormones, resulting in high TSH despite
hypothyroidism. clinically normal thyroid function
Graves disease Symptoms

• Enlargement of thyroid gland (goiter)

• Bulging of eyes (Exophthalmos)

• Anxiety or nervousness

• Increased appetite, despite having weight loss

Hashimoto thyroiditis Symptoms

• Enlargement of thyroid gland (goiter)

• Weight gain

• Increased sensitivity to cold

 4 Case study

A. A 50 year old housewife complains of progressive weight gain of 20 pounds in 1 year,

fatigue, postural dizziness, loss of memory, slow speech, deepening of her voice, dry skin,

constipation, and cold intolerance.

“ CBC and differential WBC are normal. The serum T4 concentration is 3.8 ug/dl, the serum

TSH is 23.0 mIU/ml, and the serum cholesterol is 255 mg/dl”.

1. What is the likely diagnosis? Hypothyroidism (Hashimoto thyroiditis).

2. What are the most likely causes? Pituitary tumor, Autoimmune thyroid disease …. etc.
 4 Case study

B. A 35 year old nurse complained of nervousness, weakness, and palpitations with exertion
for the past 6 months. Recently, she noticed excessive sweating and wanted to sleep with
fewer blankets than her husband. She had maintained a normal weight of 120 pounds but
was eating twice as much as she did 1 year ago. Menstrual periods have been regular but
there was less bleeding.
Serum T4=15.6 ug/dl and serum T3=185 ng/dl.
1. What is the level of thyroid function in this patient? Hyperthyroid.
2. Are additional diagnostic tests necessary to define the level of thyroid function and if so
which one(s)? Yes, TSH
# best wishes