blood grouping.ppt
- 1. Immunohematology To perform blood grouping of given sample
- 2. Immuno-hematology is an application of principle of immunology to the study of red cell antigen and their corresponding antibody in blood. These principles are employed in blood banking and transfusion medicine. There are certain lipoproteins and or glycoproteins on the surface of red blood cell which act as antigen. The antigen character of red cell is inherited. On the basis of these antigen a number of blood group system are known. So far nearly 300 blood group system have been discovered of which most important are ABO system and Rh system. Example of other system are P system, MNS system, Lutheran system, Kell system, duffy system, Lewis system etc.
- 3. Use of blood grouping 1.Blood transfusion. 2.Diagnosis and treatment of hemolytic disease of newborn. 3.Dispute over paternity of person. 4.Medico legal use.
- 4. ABO system Landsteiner discovered the ABO blood group system.This system depend on the presence or absence of A and B antigen on the cell membrane of the RBC.According to ABO system all human being can be divided into 4 major blood group i.e. A, B, AB, O group.He also found that if a particular antigen were present on RBC corresponding antibodies would always be present in serum. It is necessary that ABO blood group should match between donor and recipient for blood transfusion. In case of mismatch serious transfusion reaction may occur which could even lead to the death of the recipient.
- 5. Blood group Antigen on RBC surface Antibody in serum % of population in india A A Anti B 27% B B Anti A 31% AB A and B No antibody 8% O No antigen Anti A and B 34%
- 6. ABO Blood Grouping • ABO blood group-A, B, AB, or O— is based on the presence or absence of the A and B antigens on the red blood cells of an individual. • The A blood type has only the A antigen while the B blood type has only the B antigen. • AB blood types have both the A and B antigens, and the O blood types have neither A nor B antigen. • As at the age of six months, an individual naturally would have developed antibodies against the antigens his red blood cells lack.
- 7. ABO Blood Grouping • However, the distribution of each of the four ABO blood types varies between racial groups while O remains the most common and AB is the least common. • The ABO grouping is the first test done on blood when it is tested for transfusion. • The ABO grouping test is based on the principle of haemagglutination reaction. • Haemagglutination of red cells occur during the reaction of red cell antigens (agglutinogens) with their corresponding antibody.
- 8. ABO Blood Grouping • A positive agglutination is represented by the lattices of red cells as seen on the slide or by button formation in the test tube • While a negative reaction is represented by uniform distribution of red cells on slide and lack of button formation in test tube.
- 9. ABO Blood Grouping Techniques • Two basic methods to observe the haemagglutination reactions in ABO blood grouping; (i) slide method and (ii) test tube method. • The former is easier to perform and the latter is more sensitive. • Numerous laboratories in the developing countries perform the tube test only in situations where the result of the slide test is doubtful. • The tube method is however recommended for reliable results following internationally approved guidelines.
- 10. ABO Blood Grouping Techniques • Slide Method: The requirements include Glass slides, Pastuer pipettes, Applicator sticks and centrifuge. The reagents are: - Anti-A sera (blue color): Human polyclonal or murine monoclonal. -Anti-B sera (yellow color): Human polyclonal or murine monoclonal. -Sodium azide is added to prevent the growth of bacteria. -Antisera are kept stored at 4-6*c to preserve their potency. - Normal saline: 0.9 g/dl sodium chloride in distilled water.
- 11. Slide Method for ABO Blood Grouping • Specimen: Clotted blood is generally used, the clotted blood is centrifuged at 1500rpm for few minutes to separate serum. • The red cells are then separated from the clot using a Pastuer pipette and suspended in saline. • Anti-coagulated blood with proper anticoagulant like EDTA can also be used. • The specimen should be stored at 2-8*c if there is any delay in examination. • Blood obtained by finger puncture may be tested directly by the slide method and mixed quickly with antisera to avoid clotting.
- 12. Slide Method for ABO Blood Grouping Procedure: 1. Prepare a 10% suspension of red blood cells in normal saline; (i) Mix 0.05 ml (5 drops) of sedimented red cells with 2 ml of normal saline, (ii) Centrifuge at 1,500 rpm for 1 to 2 min and discard supernatant, (iii) Add 2 ml of normal saline to the sedimented red cells, mix well to gives a 10% suspension of red cells.
- 13. Slide Method for ABO Blood Grouping Procedure: 2. Place 1 drop of anti-A sera on one-half of a glass slide. 3. Place 1 drop of anti-B sera on the other-half slide. 4. Add a drop of the red cell suspension to each half of the slide using a clean Pastuer pipette. 5. Mix each cell-serum mixture well using separate applicator sticks. 6. Tilt the slide back and forth and observe for agglutination.
- 14. Slide Method for ABO Blood Grouping • Results Interpretation: - Tests that show no agglutination within two minutes are considered negative. - Do not interpret peripheral drying or fibrin stands as agglutination. • Observation: - If any agglutination occurs it is visible with naked eyes as dark reddish lattices of different sizes. - If agglutination is minimal it can be confirmed by examining it under microscope.
- 17. ABO Blood Grouping Techniques • Tube Method: The requirements include test tubes (10 x 75 mm or 12 x 75 mm) and microscope. • Procedure: 1. A 5% red blood cell suspension in normal saline is prepared as follows: (i) Mix 0.05 ml (5 drops) of sedimented red cells with 2 ml of normal saline, (ii) centrifuge at 1,500 rpm for 1 to 2 minutes and discard supernatant, (iii) Add 4 ml of normal saline to the sedimented red cells, mix well to get a 5% suspension of red cells.
- 18. Tube Method for ABO Blood Grouping • Procedure: To a small test tube, add one drop of anti-A sera. To a second test tube, add one drop of anti-B sera. Add one drop of 5% red cell suspension to each of the two test tubes using a Pastuer pipette Mix well and centrifuge both the tubes at 1,500 rpm for one min. or incubate at room temperature for one min.
- 19. Tube Method for ABO Blood Grouping Examine for agglutination and red cell sediments called a button will be seen at the bottom of the tube if the tube is centrifuged. - Gently tap the button from the tube by a spring action of right index finger and dislodge the cell button. - If red cells form one or more lattices with clear supernatant fluid, the agglutination is present. - If red cells re-suspend easily, without any visible lattice formed, agglutination is absent. In case of any doubt, take a drop of the suspension on a slide and observe under the 10X objective for agglutination.
- 20. ABO Forward and Reverse Grouping • There are two ways in determining the ABO blood group; • Forward grouping(cell grouping)procedure is when the individual’s red cells are tested with a known anti – A and anti- B sera. • Reverse grouping(serum grouping)procedure when the individual's serum is tested with known group A cells and group B cells.
- 21. Rh Blood Typing • Also called Rh blood grouping is next important to ABO blood grouping and detects only the presence of Rh antigen (or D antigen) out of all Rh factors on the red cells. • Its principle is based upon haemagglutination where the red cells with Rh antigen (D antigen) will form a lattice with anti-D antiserum at room temperature (in the presence of protein). • The technique is similar to ABO blood grouping and hence, Rh typing is done along with ABO blood grouping. • The Rh typing can also be done by two methods which are the Slide method and Tube method.
- 22. Rh Blood Typing Techniques Slide Method: • The requirements are same as that of ABO slide method. • The reagents include the Anti-D sera which is colorless solution (human polyclonal or human monoclonal), and normal saline. • The specimen is same as that of ABO method.
- 23. Slide method for Rh Blood Typing Procedure: 1. Place one drop of anti-D serum on a pre-warmed glass slide. 2. Add one drop of 10% suspension of red blood cells (in case of anaemic patients, use one drop of sedimented red cells) using a Pastuer pipette. 3. With an applicator stick, mix cell-serum mixture well. 4. Tilt the slide back and forth and observe for agglutination. 5. Tests that show no agglutination within three to five minutes are considered negative.
- 24. Rh Blood Typing Techniques Tube Method: • The requirements are same as that of ABO tube method. • The reagents include Anti-D sera (human polyclonal or human monoclonal) and normal saline. • The specimen is same as that of ABO method.
- 25. Tube method for Rh Blood Typing Procedure: 1. Prepare a 5% suspension of red blood cells in normal saline. 2. Add one drop of anti-D serum to a test tube . 3. Add one drop of cell suspension using a Pasture pipette. 4. Mix well and centrifuge tubes at 1,500 rpm for one minute or incubate at room temperature for two minutes. 5. Examine the agglutination reaction in each tube by dislodging the button gently and if necessary, use a magnifying hand lens.
- 26. Tube method for Rh Blood Typing 6. Interpretation: Agglutination will be recognized by the formation of small lattices in a clear liquid. - As the bottom of the test tube is tapped, the lattices whirl up and then settle down and this will be marked as positive reaction with the cells identified as Rh- positive. - If the red cells re-suspend homogeneously with no visible lattices, it should be marked as negative reaction and the cells are identified as Rh- negative.
- 27. Thank You