IMMUNOAGGLUTINATION (ABO and Rhesus Grouping) Farah Adibah Bt Mohd Zawawi 0912598 rd 3 September 2010 INTRODUCTION One One of the the ways ways for for stude student ntss to study study and and furt furthe herr under underst stan and d the the prope propert rtie iess 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). (RBCs). The blood type that one’s belong to are inherited inherited and contributed 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
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
No Yes Yes Yes
Yes No Yes Yes
No No No Yes
Yes No Yes Yes
No Yes Yes Yes
The presence of agglutination in :
Anti-A Anti-B Anti-AB Anti-D
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 o f 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 1
Blood type B
2
A
3
O
Reason Agglutination present in the blood drop mixed with antiB. Agglutination present in the blood drop mixed with antiA. No agglutination present at all
4
A
Agglutination present in the blood drop mixed with anti-
IMMUNOAGGLUTINATION (ABO and Rhesus Grouping) Farah Adibah Bt Mohd Zawawi 0912598 rd 3 September 2010
5
B
A. Agglutination present in the blood drop mixed with antiB.
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 complementmediated 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 complementmediated 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 antiAB 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, . 5. Wikipedia n.d., Rh blood group system, Wikipedia, viewed 31 August 2010, .
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
