BLOOD GROUPING Institute of Lifelong Learning, University of Delhi
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2 LIFE SCIENCES (LAB MANUAL) 2
3 LIFE SCIENCES (LAB MANUAL) BLOOD GROUPING Aim To determine A, B and O blood group in human blood. Learning Objectives To know about various types of blood groups and the importance of matching the blood groups of the recipient and donor in blood transfusion cases. Materials / Requirements Microscope Chemicals / solutions: Antiserums Anti A, Anti B and Rh, and alcohol Glassware: Sterilized pricking needle, glass slides, dropper, and cotton. Theoretical aspects of the experiment In 1900, Landsteiner first reported the presence of two blood antigens, A and B on the surface of human blood cell. Based on this, he divided human red blood cells into three groups. These were A, B and O. Now, there are 30 more antigens reported on the surface of red blood cells. These form the basis of blood groups. In blood transfusion cases, blood group of recipient and donor must be matched. Importance of this experiment If improperly matched, the recipient s system will produce antibodies causing clumping of transferred cells, blocking circulation through capillaries and produce serious and even fatal results. ABO blood groups are determined by a gene I (Isoglutinin). There are 4 alleles of this gene viz. IA, IB, IC. Proteins produced by A and B alleles are antigens. Individual with blood group type A have Antigen A on the surface of RBC and antibodies to type B blood in plasma. Blood group B have Antigen B on blood cells and antibodies to type A blood in their plasma. Blood group AB has Antigens for both A and B on the surface of blood cells but no antibodies for either type A or type B in their plasma. Blood groups O have no antigen on their blood cells but have antibodies to both A and B in their plasma. People with type AB blood group can receive blood of any type and therefore are called UNIVERSAL RECEPIENT. People with blood group O can donate blood to any individual and therefore are called UNIVERSAL DONOR. 3
4 LIFE SCIENCES (LAB MANUAL) If a transfusion is made between an incompatible donor and recipient, the recipient s blood undergo cascade of reactions of antigens on its cell and antibodies in plasma which produce clumping that clogs capillaries. Other cells burst releasing hemoglobin that can crystallize in kidneys and cause renal failure. Rh compatibility Rhesus monkey was the first in which it was discovered and because of this is called rhesus or Rh factor. Rh is made up of Rh+ve individuals who can make Rh antigens and Rh-ve individuals who can t make this antigen. At present six Rh agglutinogens are known and of these Rh anti-d is of clinical importance. If Rh positive blood is given to Rh negative person, anti Rh factor develops in the blood in days. As a result, the second transfusion of Rh positive blood to this person causes hemoagglutination. The Hemolytic Disease of new born infants called erythroblastosis foetalis results from Rh incompatibility between Rh-ve mother and Rh+ve fetus. Rh+ blood from fetus enters mother s system during birth causing her to produce Rh anti bodies. First child is usually not affected. However, subsequent pregnancy with Rh+ve fetus causes a massive secondary reaction of maternal immune system to prevent hemolytic Disease of New Born (HDN). Rh-ve mothers are given Rh antibodies during first pregnancy with an Rh +ve fetus and also subsequent Rh +ve fetuses. Preparation for the experiment Solutions/buffers/media: Preparation of antiserum is not required as these come in the form of small kits, which can be purchased and are ready for use. Precision/care is required during the use and storage of the antiserum. These must be immediately stored in the refrigerator after use. All the three bottles of the kit must be used carefully with a caution not to mix the lid or dropper of any with the other. Procedure for the experiment This procedure is called the RAPID SLIDE TEST 1. Sterilize the finger tip with 90% alcohol by wiping the tip of finger with alcohol soaked in cotton. 2. Prick the finger with sterilized needle. 3. Take 3 drops of blood separately on a clean side as shown in the figure. 4. Quickly, take 1 drop each of anti A serum, anti B serum and anti Rh with the help of separate droppers and put one by one on the 3 blood drops. 5. Immediately mix drop of blood and antiserum with help of a needle. Observe/look for agglutination or clumping after 3-4 minutes under the microscope. 4
5 LIFE SCIENCES (LAB MANUAL) BLOOD GROUPING Sources of error and precautions Care is required at each stage of the experiment and also during handling of chemicals and solutions. Finger and needle should be properly sterilized. Slide should be clean. Finger should not be pressed to squeeze out to get the blood. Mix the antigen antiserum immediately. Separate needle should be used for mixing antigen and antiserum. Observations Observations of the tests performed on the slide are shown in the small table. Agglutination of RBC by a reagent indicates +ve test. Absence of agglutination indicates ve test. 5
6 LIFE SCIENCES (LAB MANUAL) Results Blood group A has antigen A on the red blood cells and has antibodies b in their plasma and thus shows agglutination with antiserum A Blood group B has antigen B on the red blood cells and has antibodies a in their plasma and thus shows agglutination with antiserum B Blood group AB has both antigen A and B on the red blood cells but has no antibodies in their plasma and thus shows agglutination with both anti serum A and antiserum B. Blood group O has no antigen on the red blood cells but has antibodies to both A and B in their plasma and thus shows no agglutination Rh+ve blood group will have Rh factor on their red blood cells and hence will show agglutination with antiserum D 6
7 LIFE SCIENCES (LAB MANUAL) BLOOD GROUPING Discussion / Interpretation of the expected results: 7
8 LIFE SCIENCES (LAB MANUAL) Questions 1. What are blood groups? 3. What is the basis of blood grouping and why is it called ABO system? 4. What is meant by Universal Donors and Universal Recipients? 5. In blood, where are the agglutinins and agglutinogens present? Applications of the procedure In blood transfusion. In pregnancy (Rh incompatibility). Investigating cases of paternity disputes. Medico legal value. Blood group antigen help in cell recognition. References 1. Human Physiology by Moffett, Moffett and Schauf. 2. Biology for Today, Volume 2 by Ernest G. Neal & Keith R.C. Neal 3. Immunology by Ivan Roitt, J. Brostoff and David Male. 8
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