Aim: To determine the blood group of given blood

sample(your own blood)

Materials/ Equipments/ Chemicals

The kit contains Antisera A, Antisera B, Anti D Monoclonal.

Antisera D is colourless and is obtained from monkeys.

Principle:
The surfaces of red blood cells membrane contain a variety of genetically determined antigens called
isoantigens or agglutinogens, while the plasma contains antibodies called as agglutinins. To determine
the blood group of a person, their cells are made to react with commercially available antisera containing
known agglutinins. The slide is then observed under the microscope or sunlight to detect the presence or
absence of clumping and hemolysis
(agglutination) of red blood cells which occur as a result of antigen-antibody reaction.
 Blood grouping is done on the basis of agglutination. Agglutination means the collection of separate
particles like RBCs into clumps or masses. Agglutination occurs if an antigen is mixed with its
corresponding antibody which is called isoagglutinin, i.e. occurs when A antigen is mixed with anti-A or
when B antigen is mixed with anti-B.

The RBC of the ‘O’ group has no antigen and so agglutination does not occur with any other group of
blood. So, ‘O’ group blood can be given to any blood group persons and the people with this blood group
are called ‘universal donors’.

Plasma of AB group blood has no antibody. This does not cause agglutination of RBC from any other
group of blood. People with AB groups can receive blood from any blood group. So, people with this
blood group are called ‘universal recipients’.

Blood with antigen B and A antibody belongs to the ‘B’ group. And blood having antigen A belongs
to ‘A’ group which has B antibody in the serum.

Theory:
Blood is fluid connective tissue and the most crucial component of the circulatory system. In a
healthy person, approximately 5 liters of blood circulates throughout their body. It consists of
erythrocytes, leukocytes and platelets suspended in plasma along with millions of different molecules with
their own specific roles and functions. Even though components of blood are the same for all humans,
there are various blood types. In fact, more than 40 blood groups are identified but all of them are not
clinically significant. The two most important group systems examined are the ABO-system and the
Rhesus system.

ABO BLOOD GROUPING :

● The ABO blood group system is the most important blood type system.
● Determination of ABO blood groups depends upon the immunological reaction between
antigen and antibody. Antigens are also called agglutinogens because of their capacity to
cause agglutination of RBCs.
● Karl Landsteiner discovered the ABO Blood Group System in 1901 and was awarded the
Nobel Prize in Physiology or Medicine in 1930. Adriano Sturli and Alfred von Decastello
who were working under Landsteiner discovered type AB a year later in 1902.
● Janský is credited with the first classification of blood into the four types (A, B, AB, O).
● Reuben Ottenberg successfully transfused blood between two people in New York. He was
the first person to record pre-transfusion testing for blood compatibility in a clinical setting.
Later in 1954 he was the first to be awarded with the Karl Landsteiner Award.
● ABO grouping is based on the presence or absence of two surface antigens on the RBCs
namely A and B, so blood is divided into four groups: ‘A, B, AB and ‘O’ group.

Rh GROUPING:
● In 1940, Landstenier and Wiener discovered the presence of Rh antigen on human RBCs. The
Rh system is named after the Rhesus monkey, in which it was first discovered.
● The Rh antigen is observed on the surface of RBCs of the majority (nearly 80 per cent) of
humans.
● D is the most predominant Rh antigen.
● The term Rh positive and Rh negative is commonly used with reference to presence or
absence of D(Rh) antigen.
● When introduced into D ( Rh) negative individuals, it stimulates production of anti-D
antibodies. Therefore, the Rh group should also be matched before transfusions.
● A special case of Rh incompatibility (mismatching) has been observed between the Rh-ve
blood of a pregnant mother with Rh+ve blood of the foetus. Rh antigens of the foetus do not
get exposed to the Rh-ve blood of the mother in the first pregnancy as the two bloods are well
separated by the placenta. However, during the delivery of the first child, there is a possibility
of exposure of the maternal blood to small amounts of the Rh+ve blood from the foetus.
● In such cases, the mother starts preparing antibodies against Rh antigen in her blood. In case
of her subsequent pregnancies, the Rh antibodies from the mother (Rh-ve) can leak into the
blood of the fetus (Rh+ve) and destroy the fetal RBCs. This could be fatal to the foetus or
could cause severe anemia and jaundice to the baby. This condition is called erythroblastosis
foetalis.

MNS ANTIGEN SYSTEM:

● The MNS antigen system, first described by Landsteiner and Levine in 1927 is based on two genes:
Glycophorin A and Glycophorin B.
● The blood group is under control of an autosomal locus on chromosome 4 and also under control of a
pair of codominant alleles LM and LN. Anti-M and Anti-N antibodies are usually IgM types and
rarely, associated with transfusion reactions.

BOMBAY BLOOD GROUP:

● The Bombay Blood or hh blood group is a rare blood phenotype first discovered in Mumbai (then
called Bombay). It was discovered in 1952 by Dr Y.M. Bhende.
● The Bombay Blood group is a rare blood group, phenotypes of this group lacking H antigen on the red
cell membrane and have anti-H in the serum. It fails to express any A, B or H antigen on their red
cells or other tissues. In simpler terms, it is quite an abstract type of blood group.
● The Bombay Blood Group occurs in those individuals who have inherited two recessive strains of the
H gene. Such individuals do not produce the H carbohydrate.

Procedure:
1. First, we took a glass slide (which had 12 depressions) and cleaned it by using ethanol.
2. We unpacked the Monoclonal Antibodies (A,B&D)kit.
3. Then we wiped our ring finger or middle finger using alcohol swabs (70% ethanol) and waited for it to
get dried.
4. After all this we pricked our fingertips using Lancet from a distance.
5. As blood started flowing out, it was allowed to fall on the three circles of the glass slide, by gently
pressing the fingertip
6. Now with the help of a dropper Antisera-A, Antisera-B, and Antisera-D were added in the first, second
and third circle respectively in a sequential order
7. Mixed the blood samples and the antiserum using three different ends of the glass slide or by using
three separate toothpicks for each antisera and waited for one minute to observe the result.
8. In order to stop flow of the blood from the pricked part we applied pressure over there using alcohol
swabs.

Observation:

Result:
Discussion:
Blood typing is the test to determine the blood group of a person. If you need a Blood transfusion or plan
to donate Blood, this test is required. Because not all Blood types are compatible, knowing one's Blood
Group is crucial. Receiving Blood that isn't compatible with one's Blood type can result in a hazardous
immunological reaction. Knowing our Blood Group allows us to choose the right Blood for transfusion in
the event of an emergency or an accident. A Blood transfusion from the incorrect Blood group might be
fatal.

The antigens are both the source of the issue and the solution. Because these compounds cause the body's
immune system to react, it’s critical to check compatibility before receiving Blood or donating organs. If a
person with Blood Group B receives Blood from a person with Blood Group A, the recipient's body will
recognize the A-type antigens as alien bodies and reject the transfusion. Because group AB includes and
recognizes both types of antigens, it can. accept Blood from any type, but group O can only accept Blood
from type O. The Rh-factor, which is highly immunogenic, follows the same rule. Antibodies developed
by Rh-negative moms after having an Rh-positive baby may harm an Rh-positive baby during the second
pregnancy, resulting in severe anemia. As a result, it's not unexpected that numerous documents, from
university applications to work ID cards, seek Blood group information.

Clinical significance:
The importance of blood grouping is in- ● Blood
transfusion for treatment purposes.
● Determination of Rh incompatibility between the mother and the child
● Choice of a donor in tissue/organ transplantation
● Genetic Studies
● Medical legal use - Any red stain on a clothing maybe claimed to be blood by a supposed victim
● Susceptibility to disease - The people of blood type O are more susceptible to peptic ulcer. Blood
type A is more commonly seen in carcinoma of stomach and to some extent in diabetes mellitus
● Paternity disputes - The ABO, Rh and other grouping systems can be used to settle cases of
disputed paternity. Antigens A and B are dominant, whereas O is recessive. It is possible to prove
that a person could not have been the father, but not that he was/is the father.

Precaution:
1.The slides must be dry, dust-free, and grease free.
2.Identify and mark all slides, containers and test tubes clearly and legibly. Double check every step of the
procedure.
3.The droppers used should be different for all the three different antisera bottles.
4.While mixing the antisera with the blood don't forget to use separate toothpicks to mix them.
5.Drop the antiserum from a distance so that the tip of the dropper doesn't touch the sample.
6.Discard the alcohol swabs, Lancet, cotton balls, and toothpick properly after use.
7.Incinerate the needle in the incinerator.