Course code: PHAR-2112

Experiment NO: 01

Experiment Name: Determination of blood grouping.

Objective: To understand the basic concept of blood grouping.

Pre lab questions:

✓ What is blood grouping?
✓ What are the types of blood grouping?
✓ What are the criteria of blood donation?
✓ What is antigen?
✓ What is antibody?
✓ What is Rh factor?
✓ What is the Landstainer’s Law?

Principle: Blood grouping is the classification of blood based on the presence orabsence of two
inherited antigenic substances on the surface of red blood cells (RBCs). The ABO and Rh are the
major, clinically significant and the most important of all the blood group systems. The ABO
blood group system was first discovered by Karl Landsteiner in 1900. The human ABO blood
group system is divided into the following four major groups depending on the antigen present on
the surface of their red blood cells:

1. “A” group

2. “B” group

3. “AB” group

4. “O” group

Blood types are further organized by Rh factor:

Rh-positive: People with Rh-positive blood have Rh antigens on the surface of their red blood
cells. People with Rh-positive blood can receive Rh-positive or Rh-negative blood.

Rh-negative: People with Rh-negative blood do not have Rh antigens. People with Rh-negative
blood can receive only blood that is also Rh-negative.
Corresponding monoclonal antibodies are available which are against the antigens. The
monoclonal antibodies are –

1. Anti-A: works against Antigen A &blue in color.

2. Anti-B: works against Antigen B & yellow in color.

3. Anti-D: works against Rh.

The ABO and Rh blood grouping system is based on agglutination reaction. When red blood cells
carrying one or both the antigens are exposed to the correspondingantibodies they interact with
each other to form visible agglutination or clumping. Blood group A individuals have A antigens
on RBCs and anti-B antibodies in serum. Similarly, blood group B individuals have B antigens on
RBCs and anti-A antibodies in serum. Blood group AB individuals have both A and B antigens on
RBCs and neither anti-A nor anti-B antibodies in serum. Whereas, blood group O individuals have
neither A antigens nor B antigens, but possess both anti-A and anti-B antibodies in serum.

The individuals carrying the Rh antigen are considered to have positive blood group whereas those
individuals that lack this antigen are considered to have negative blood group.

Table no.-01:

Antigens on the surface Antibodies in the Serum ABO Blood Group

of Red Blood Cells
A Anti B A
B Anti A B
A and B Neither Anti A nor Anti B AB
Neither A nor B Anti A, Anti B, Anti AB O

Table no.-02:

ABO Blood Group Rh antigen present(Rh+) Rh antigen absence(Rh-)

A A+ A-
B B+ B-
AB AB+ AB-
O O+ O-
Together, the ABO and Rh grouping systems yield your complete blood type. There are eight
possible types: O-positive, O-negative, A-positive, A-negative, B-positive, B-negative, AB-
positive, and AB-negative.

Reagents:

➢ Anti-A

➢ Anti-B

➢ Anti-D

Equipments:

➢ 70% isopropyl alcohol.

➢ Glass slider/ petry dish.

➢ Cotton ball.

➢ Mixing stick

Procedure:

1. At first the glass slider or petry dishes are sterilized, cleaned and dried.

2. The fingertip is cleaned with 70% isopropyl alcohol.

3. Fingertip is punctured with a needle.

4. Three drops of blood fallen on a slide and marked as A, B, C.

Blood Blood
drop Blood drop drop
+ + +
Anti A Anti B Anti Rh
 Cotton batt is pressed with fingertip to stop bleeding
5. One drop of Anti-A is added with blood sample A, one drop of Anti-B is added with
blood sample B, and one drop of Anti Rh is added with blood sample C.

6. Mix each blood drop and the antiserum using a fresh mixing stick .
8. Observe agglutination in the form of fine red granules within 30 seconds. Anti Rh takes
slightly longer time to agglutinate compared to Anti A and Anti B.

Data analysis: (Troubleshooting Guide)

Sr. No Problem Possible Cause Solution

The anti-sera Ensure that the
reagents anti-sera reagents
mix with each are added
other properly onto the
1. False positive respective cavity
Result without spilling
to the sides
Incubated for a The results should
longer be read within the
time time period
Anti-sera not Ensure that the
2. No agglutination stored anti-sera is stored
Observed under proper in refrigerator
conditions (2-8oC)

Observation:

1. Sample A coagulates

2. Sample B doesn’t coagulate

3. Sample D coagulates.

Result:

Blood group is A(+ve).

Discussion:
• Knowing the process of blood grouping
• Being Conscious of blood transfusion
• Knowing the criteria of blood transfusion

Post lab questions:

✓ Why AB blood group is called Universal Recepient?
✓ Why O blood group is called Universal donar?
✓ Why agglutinins produced is persons who do not have the antigenic substance?
✓ What is Hemophilia?

Precautions:
• The fingertip is cleaned with 70% isopropyl alcohol properly.
• Mix the reagents with blood properly.
• It should be performed on a cleaned glass slide.
Experiment NO: 02

Experiment Name: Determination of clotting time.

Objective: To understand the basic concept of clotting time.

Pre lab questions:

✓ What is clotting time?
✓ What’s are the impotances of clotting time?
✓ What’s are the criteria of blood donation?
✓ What is agglutinogens?
✓ What is agglutinin?
✓ What is hemostasis?

Principle: Blood clotting is the series of reactions which involves in conversion ofliquid blood to solid. The
main and first three steps of blood clotting are:

i. Formation of prothrombin activator.

ii. Conversion of prothrombin to thrombin.

iii. The fibrin mess is formed by thrombin catalyzing its combination with fibrinogen.

These steps become activated due to extrinsic clotting factors which are available in environment. When blood
is exposed to environment from vascular system, it will form a solid clot.

Reagents:
• 70% isopropyl alcohol

Equipments:
1. Glass slider

2. Blood lancet

3. Slide

4. Filter paper

5. Cotton ball

6. Stopwatch
Procedure:

1. Prepare a clean glass slide.

2. With 70% isopropyl alcohol sterilize the finger in a circular motion starting from the side of
puncture going outwards.

3. With the blood lancet puncture the finger and transfer three separate drops of blood on the glass
slide.

4. Observe for the clot formation or coagulation by using the lancet to check for fibrin threads in the
blood every 30 seconds. This appears as a

“Threadlike” substance that clings to the tip of the lancet.

5. Record the time when the first fibrin thread was formed.

6. Record the time.

Observation:

After 5 min blood coagulation has started and whole clotting takes about 7 minutes.

Result:

Blood clotting time 5-12 minutes.

Discussion:

5-10 minutes are required for blood clotting.

Post lab questions:

✓ Why blood dose not clot inside the vessel
✓ What is haemorrhage? What are the causes of Haemorrhagic state?
✓ What are the difference between haemostasis and coagulation?

Precaution:

1. Observe the time accurately.

2. Finger should be sterilized properly.

3. The blood lancet should be sterilized.

4. The puncture should not be deep enough.

Experiment NO: 03

Experiment Name: Investigation of urine composition.

Objective: To test & find out different types of chemical constituents present urine.
Pre lab questions:
✓ What is urine?
✓ What is the composition of urine?
✓ What is the necessary urination?
Principle: Kidneys remove waste product from the blood through small filtering units called
nephrons. Each nephron consists of a ball of small blood capillaries, called a glomerulus, and a small
tube called a renal tubule. Kidneys form urine, which passes through the ureters to the bladder for
storage prior to excretion. There are three processes involved in the formation of urine:

• Filtration.
• Selective reabsorption
• Secretion.

Glomerular filtrate vs Urine:

Constituent Daily Daily Excretion

Excretion Glomerular Filtrate Urine
Water 130,000 ml 1500 ml
Sodium 20,000 mmol 150 ml
Albumin 4g (60 µmol) 0.04g (6 µmol)
Urea 900 mmol 400 mmol

Composition of Normal Urine:

Water 96%
Urea 2%
Uric acid
Creatinine
Ammonia
Sodium
Potassium 2%

Chloride
Phosphate
Sulphate
Oxalate
Reagent:

1. Silver nitrate solution.

2. Nitric acid

3. Concentrate HCl

4. Sodium hydroxide solution

5. Red litmus paper

6. Ammonium molybdate

7. Barium chloride

8. Ammonium hydroxide solution.

9. Picric acid

10. Glacial acetic acid

11 .Sodium nitro prusite

12. Benedicts Reagent

Tests:

i. To Detect Some of the Normal Inorganic Ions Present in Urine:

Experiment Observation Result

1.Test for Chlorides: White ppt of AgCl is
5ml of urine is acidified formed which is soluble Cl- is present.
with HNO3 in a test tube. in NH4OH.
AgNO3 is added drop by
drop.
2.Test for Phosphates: Heat the mixture gently.
Add 5 ml of concentrated A yellow crystalline
nitric acid + 5 ml of precipitate of
urine+ 2 ml of saturated ammonium phospho- PO4- is present.
ammonium molybdate molybdate appears.
solution
3.Test for Bicarbonate: A slight effervescence
Add 4 drops of occurs due to CO2
concentrate HCl+5 ml of evolution. Test HCO3- is present.
urine. the gas evolved with lime
water.
4.Test for Sulphates: A white precipitate of
Acidify 2 ml of urine + 1 barium sulphate is
ml dilute HCl+ formed. SO4- is present.
3 drops of 5% barium
chloride solution.
5.Test for Ammonia: Add Turning moist red litmus
1 ml of 10% sodium paper blue. NH4+ is present.
hydroxide solution +
5 ml of urine. Boil.
ii.To Detect some of the Normal Organic Constituents of Urine:

Experiment Observation Result

1.Test for Creatinine: Put a deep red color or
5 ml of urine + 4 drops of orange due to creatinine
a saturated solution of picrate Creatinine is present
picric acid + 1 ml of 10% appears
sodium hydroxide
solution.
2. Test for protein:
2/3ml of a tube is filled
with urine and upper
part is heated slowly. Turbidity doesn’t appear. Protein is not present.
Then 2/3 drops of glacial
acetic acid are added to
it.
3.Test for ketone bodies:
3ml of urine is saturated
with (NH4)2SO4. Then 3 The junction of two Ketone bodies are
drops of 5% sodium nitro liquids. present.
prusite is added and
shaken well.
Concentrated

4. Test for sugar:

5ml of Benedicts
Reagent is boiled if color Blue, green, red, yellow
doesn’t change then 3 any of this color doesn’t Sugar is not present.
drops of urine is added appear.
of it and heated again for
2 min. It is then cooled.

Result:

Provided urine from the body contains Creatinine, Ketone bodies, Cl- , NH4+, SO4-,
HCO3- , PO4-.
Post lab questions:

✓ What is the normal reaction of urine?

✓ How long do urine test results take?
✓ What can be detected in a urine test?