IMMUNOAGGLUTINATION (ABO and Rhesus Grouping)

Farah Adibah Bt Mohd Zawawi

0912598
rd
3 September 2010

INTRODUCTION

One of the ways for students to study and further understand the properties of
antibodies/immunoglobulin and their reaction with antigens is by performing the blood test. This
test is done to determine one’s blood group. A blood group is a way of classifying the blood
based on the presence of absence of antigens on the membranes of human red blood cells
(RBCs). The blood type that one’s belong to are inherited and contributed from both parents.
There are about 30 blood group systems available. However, the 2 most common ones are the
ABO and the Rh blood group systems.

The ABO blood group system is the most significant blood group system considering its
role in human blood transfusion. The anti-A and anti-B antibodies are usually IgM antibodies
produced in the early lives by susceptibility to environmental substances e.g. food and bacteria.
All humans and lots of primates can be sorted for the ABO blood group. A, B, AB and O are the
four principal types, with 2 antigens (A and B) and two antibodies respectively (antibody A and
antibody B).

As for Rh blood group system, next to ABO, is the most clinically important in blood
transfusion. It is also the most polymorphic of the human blood groups i.e. comprising of at least
45 independent antigens. The most vital ones are D, C, c, E and e. However, the D antigen is the
most immunogenic of all and thus, the commonly-used terms Rh factor, Rh positive and Rh
negative only do refer to the D antigen. It is crucial to take note on ones’ Rh blood group;
besides its importance in blood transfusion, it is as well a relevant cause of hemolytic disease of
the newborn.
 IMMUNOAGGLUTINATION (ABO and Rhesus Grouping)
Farah Adibah Bt Mohd Zawawi
0912598
rd
3 September 2010

OBJECTIVES

1. To understand the basis of specificity of antigen-antibody binding

2. To understand the basic principles involved in determining the blood group e.g.
agglutination

3. To further understand the importance of blood compatibility in blood transfusion

4. To understand the cause of one important immunoagglutination disease ; the hemolytic

disease of the newborn

APPARATUSES AND CHEMICALS:

1. Anti Sera (A, B, AB, D)

2. Alcohol swab

3. Blood lancet

4. Labeled white ceramic tile

5. Toothpicks

PROCEDURES

1. An alcohol swab was used to clean the third finger. The one of the left hand was
preferred as the left hand makes less contact with the environment as compared with the
right one.

2. The finger was dried with a clean cotton wool and then firm strokes were applied on the
finger to stimulate blood circulation.

3. The ball of the finger was punctured with a sterile lancet. Then, a gentle pressure was
applied on the finger.

4. Four drops of blood were put on the respective square of the tile. The finger was then
wiped with the alcohol swab.
 IMMUNOAGGLUTINATION (ABO and Rhesus Grouping)
Farah Adibah Bt Mohd Zawawi
0912598
rd
3 September 2010

5. A drop of Anti-Sera (A<B<AB and D) was put beside the blood drops and each of the
blood drop together with the respective Anti-Serum were mixed using clean toothpicks.
This was also followed by gentle rocking of the tile.

6. The result was observed and recorded.

7. The whole procedure was repeated for the rest of the group members present.

RESULT
 IMMUNOAGGLUTINATION (ABO and Rhesus Grouping)
Farah Adibah Bt Mohd Zawawi
0912598
rd
3 September 2010

Stude
nt
1 2 3 4 5
The
presence of
agglutination
in :
Anti-A No Yes No Yes No
Anti-B Yes No No No Yes
Anti-AB Yes Yes No Yes Yes
Anti-D Yes Yes Yes Yes Yes

DISCUSSION:

Generally, agglutination indicates the presence of blood group antigens in the blood. It
specifically implies that the blood has reacted with a certain antibody and therefore is not
compatible with the anti-serum/blood containing that kind of antibody. If no agglutination occurs
in the blood, means that the blood does not have the antigens binding to that particular antibody
present in the anti-serum/blood.

I. ABO Blood Group

Based on the results above, it could be observed that:

Student Blood type Reason

1 B
2 A
3 O
4 A
Agglutination present in the blood drop mixed with anti-
B.
Agglutination present in the blood drop mixed with anti-
A.
No agglutination present at all
Agglutination present in the blood drop mixed with anti-
 IMMUNOAGGLUTINATION (ABO and Rhesus Grouping)
Farah Adibah Bt Mohd Zawawi
0912598
rd
3 September 2010

A.
5 B Agglutination present in the blood drop mixed with anti-
B.

Student 1 and 5 belong to the blood group B, indicating that they have B antigens on the
surface on their RBCs. These B antigens form antigen-antibody complex with the anti-B
antibody due to the high specificity of the epitope of the B antigen with the anti-B antibody. Due
to this matter, it is important to take note that it is the A antibodies that are present in the blood
plasma of these students. A antibodies do not bind with the naturally-present B antigens on the
RBCs membrane thus no harm would have been caused to the RBCs i.e. the complement-
mediated lysis of the RBCs are prevented.

Student 2 and 4 belong to the blood group A, indicating that they have A antigens on the
surface of their RBCs. These A antigens form antigen-antibody complex with the anti-A
antibody due to the high specificity of the epitope of the A antigen with the anti-A antibodies.
Due to this matter, it is important to take note that it is the B antibodies that are present in the
blood plasma of these students. B antibodies do not bind with the naturally-present A antigens on
the RBCs membrane thus no harm would have been caused to the RBCs i.e. the complement-
mediated lysis of the RBCs are prevented.

Student 3 belong to the blood group O, indicating that neither A or B antigens are present
on the surface of their RBCs. This is why their blood reacts with neither anti-A, anti-B or anti-
AB anti-sera. Due to this matter, it is important to take note that both A and B antibodies are
present in the blood plasma of this student. A and B antibodies would just form antigen-antibody
complexes with foreign antigens i.e. exogenous A and B antigens. Since people with O blood
group do not produce A and B antigens, their blood would not be rejected when it is given to
others of different blood group types. As a consequence, in blood transfusion, they are assumed
as ‘Universal donors’. In spite of this, they can only receive O blood type.

Another blood group to be mentioned here is the AB blood group. Those with AB blood
group have both A and B antigens on the membrane of their RBCs. Both antigens would form
 IMMUNOAGGLUTINATION (ABO and Rhesus Grouping)
Farah Adibah Bt Mohd Zawawi
0912598
rd
3 September 2010

antigen-antibody complexes with their respective antibodies (A antigen with A antibody, B

antigen with B antibody). Due to this matter, it is important to note that both A and B antibodies
are not present in the blood plasma of that particular person. Their blood does not distinguish
against any other ABO types. Those of AB blood group are called the ‘Universal receivers’ but
their blood will be agglutinated if given to those from other blood group types (A,B,O) because
they have both (A,B) antigens.

In this practical however, the blood of those with A and B blood group types showed
agglutination when reacted with anti-AB antiserum due to the fact that this antiserum contains
both A and B antibodies. Thus, the antigens that present in both blood group types would bind to
the respective antibody according to their specificity.

II. Rh Blood Group System

The results have shown that the blood of all five students agglutinated in the presence of
anti-D antiserum thus indicating that they are of Rh positive type. The inference that could be
made from this observation is that all 5 students have the Rh factor/ most immunogenic D
antigen on the membrane of their RBCs. The agglutination occurs because the immunogenic D
antigen reacted with the anti-D antibodies (Rho(D) Immunoglobulin). This particular antibody
does not present in the blood plasma of those Rh positive blood group. A person with Rh positive
blood can receive blood from a person with Rh negative blood without any problems.

As for persons with Rh negative blood group, they do not have D antigen on the surface
of their RBCs. No agglutination would occur consequentially. They can develop anti-D
antibodies in the blood plasma if they receive blood from person with Rh positive blood, whose
D antigens are able to trigger the production of Rh antibodies. Mothers of Rh negative would be
developing anti-D antibodies on account of previously carrying Rh positive fetus. The anti-D
antibodies would be affecting the next and coming fetuses of Rh positive thus killing them i.e.
the fetuses’ RBCs would be lysed and destroyed.
 IMMUNOAGGLUTINATION (ABO and Rhesus Grouping)
Farah Adibah Bt Mohd Zawawi
0912598
rd
3 September 2010

In transfusion medicine, a successful transfusion would be that both ABO and Rh blood
groups are compatible between the donors and patients. If they are not compatible, agglutination
would occur and the result could be fatal. The clumped RBCs would clog the blood vessels and
interrupt the systemic blood circulation. The agglutinated RBCs could also be lysed and its
contents leaked out of the body. The hemoglobin present in RBCS is toxic outside the cell.

Some measures that are/could be taken in order to further increase the efficiency of the
results obtained are:

1. Sterilising the table with 70% ethanol in order to eliminate any possible source of
contamination
2. Putting on gloves. Also to eliminate any possible source of contamination from the bare
hands.
3. If possible, the first blood drop derived is not used for this blood test. This is because the
first blood drop could contain any foreign materials already present at that particular
finger. This could affect the results.
4. Once all the blood drops have been obtained, the antisera are put onto the blood drops
and then being mixed altogether instantaneously. This is to avoid the drying up of the
reaction mixture which in turn can cause aggregation of cells. Subsequently, false results
are obtained.

CONCLUSION

In conclusion, agglutination occurs as a result of the presence of antibody with specific

binding with the antigens present in the RBCs. This binding resulted in an immunologic response
in which the incompatible RBCs would subsequently be destroyed. This is actually the
foundation principle used in blood test. It is also important in medicinal therapeutic purposes.
The examples are blood transfusion as well in the prevention of the hemolytic disease of the
newborn. Thus, it is vital for everyone to know his/her own blood group, in case of any
emergencies where quick blood transfusion is needed and also in the case of pregnant mothers
(the hemolytic disease of the newborn).
 IMMUNOAGGLUTINATION (ABO and Rhesus Grouping)
Farah Adibah Bt Mohd Zawawi
0912598
rd
3 September 2010

REFERENCES

1. Avent, ND & Reid, ME 2000, ‘The Rh blood group system: a review’, Blood, vol. 95, no.
2, p. 2.
2. Bloodindex n.d., Blood Grouping-Methods & Procedure, Bloodindex, viewed 31 August
2010,< http://www.bloodindex.org/blood_grouping_methods_procedure.php>.
3. Home Health UK n.d., Blood Group Test ABO and Rhesus (D), Home Health UK,
viewed 31 August 2010, < http://www.homehealth-uk.com/medical/bloodgroup.html>.
4. Wikipedia n.d., ABO blood group system, Wikipedia, viewed 31 August 2010,
<http://en.wikipedia.org/wiki/ABO_blood_group_system>.
5. Wikipedia n.d., Rh blood group system, Wikipedia, viewed 31 August 2010,
<http://en.wikipedia.org/wiki/Rh_blood_group_system>.

KULLIYAH OF PHARMACY

DEPARTMENT OF BASIC MEDICAL SCIENCE

YEAR 2 SEMESTER 1

IMMUNOLOGY (PHM 2132)

 IMMUNOAGGLUTINATION (ABO and Rhesus Grouping)
Farah Adibah Bt Mohd Zawawi
0912598
rd
3 September 2010

IMMUNOAGGLUTINATION (ABO and Rhesus

Grouping)

STUDENT NAME : FARAH ADIBAH BT MOHD ZAWAWI

MATRIC NUMBER : 0912598

SIGNATURE :

DATE OF EXPERIMENT : 25th AUGUST 2010

DATE OF SUBMISSION : 3rd SEPTEMBER 2010

TIME OF SUBMISSION : 12.30pm

NAME OF LECTURERS : DR. ELAMIN, DR. KHIN MA MA