IMHM321 WEEK 4 LABORATORY - ABO BLOOD GROUPING.pdf

UNIT OBJECTIVES: To achieve this unit, a learner must: 1. Perform the ABO blood grouping procedure accurately and precisely. 2. Determine and interpret the results of the test correctly. 3. Understand the clinical significance of blood grouping. ABO BLOOD GROUP SYSTEM  Most important of all bl...

UNIT OBJECTIVES: To achieve this unit, a learner must: 1. Perform the ABO blood grouping procedure accurately and precisely. 2. Determine and interpret the results of the test correctly. 3. Understand the clinical significance of blood grouping. ABO BLOOD GROUP SYSTEM  Most important of all blood groups in transfusion practice  It is the only blood group system in which individuals have antibodies in their serum to antigens that are absent from their RBCs. This occurs without any exposure to RBCs through transfusion or pregnancy (naturally occurring)  EX. BLOOD GROUP/TYPE A  ANTI-B  Due to the presence of these antibodies, transfusion of an incompatible ABO type may result in immediate lysis of donor RBCs. This produces a very severe, if not fatal, transfusion reaction in the patient  Testing to detect ABO incompatibility between a donor and potential transfusion recipient is the foundation on which all other pre-transfusion testing is based. THE LANDSTEINER LAW  The antigen is present on the RBC surface and determines the blood group/type  The corresponding antibody is NEVER FOUND in the individual’s serum  The OPPOSITE naturally occurring antibody is always present in the individual’s serum Blood Antigens on the Naturally occurring Percentage in the groups RBC surface antibody in serum American population A A Anti-B 40% B B Anti-A 10% AB A and B None 5% O H Anti-A and Anti-B 45% ABO BLOOD GROUPING/TYPING SLIDE METHOD Materials: Applicator sticks Disposable blood lancet or pricker Glass Marker or labeling material Typing Sera A (anti-A) Typing Sera B (anti-B) PROCEDURE: 1. Place 1 drop of anti-A and 1 drop of Anti-B reagent separately on a labeled slide. 2. Add 1 drop of 20% test red cell suspension or a drop of whole blood from a capillary puncture to each drop of the typing antiserum (the suspension may be prepared by adding 20 parts of red cells to 80 part of normal saline). 3. Mix the cells and reagent using a clean applicator stick. Spread each mixture evenly on the slide over an area of 10-15 mm diameter. PROCEDURE: 4. Tilt the slide for 2 minutes at room temperature (22°C – 24°C) and observe for agglutination. Do not read results after 2 minutes. 5. Read and Record the result. 6. Report as “+” for agglutination and “0” for no agglutination. 7. Dispose all biohazardous waste in a puncture-proof waste container. ABO BLOOD GROUPING/TYPING TUBE METHOD ABO FORWARD GROUPING/TYPING ABO REVERSE GROPING/TYPING  for ABO blood group system only  ABO FORWARD GROUPING/TYPING  Also known as cell, direct typing  Uses known sources of commercial anti-sera to detect antigens on an patient’s RBC  Specimen: patient’s Red cells  Reagent: anti-sera (Anti-A and Anti-B)  ABO REVERSE GROUPING/TYPING  Also known as serum, indirect, back typing  Detects ABO antibodies in the patient’s serum by using known reagent RBC’s (A and B cells)  Only unique to the ABO blood group system; checks results of forward typing Specimen: patient’s serum/plasma Reagent: Known Red cells ABO FORWARD GROUPING/TYPING Materials: Test tube Centrifuge Glass marker or an alternative labeling device Typing sera A (anti-A antisera) Typing sera B (anti-B antisera) Group “O” type serum (anti-A,B) PROCEDURE: 1. Prepare three clean test tubes and label as follows: anti-A, anti-B, and anti-A,B NOTE: Labeling should be done with care since clerical errors are the most frequent errors in the blood bank. 2. Place two drops of the appropriate reagent (anti-A, Anti-B and O serum). NOTE: Use a free falling drop. Do not touch the dropper to the side of the tube. Always add antisera before cells. Always check for the clarity and expiration of the antisera. 3. Add one drop of 2% to 5% suspension of red blood cells to be tested to each tube. NOTE: Use a free falling drop. Do not touch the dropper to the side of the tube. PROCEDURE: 4. Mix the reagent and RBC suspension and centrifuge at 3400 rpm for 15 seconds. NOTE: Time may vary with each centrifuge. Check the calibration information for each individual centrifuge. Never open the lid of the centrifuge before the spinning motion stops. 5. Gently resuspend the RBC button and then observe for agglutination or hemolysis macroscopically. 6. Observe suspected weakly reacting results by viewing the mixture under low power objective of the compound microscope. 7. Grade reactions and record the results. 8. Dispose all the biohazardous waste in the puncture –proof container. ABO REVERSE GROUPING/TYPING Materials: Test tube Droppers Centrifuge Glass marker or an alternative labeling device Normal Saline Solution (NSS) A, B, AB and O red blood cell suspensions PROCEDURE: 1. Prepare 2-5% suspensions of A1 red cells, B red cells, AB cells and O red cells in saline 2. Label 4 tubes: A, B, O, AB 3. Place 2 drops of serum into each of three tubes identified as specimen and labeled “A”, “B” , “O” and “AB”. 4. Add 1 drop of the suspension of A cells to tube “A”, 1 drop of the suspension of B cells to the tube“B”, 1 drop of suspension of O cells to tube “O” and 1 drop of suspension of AB cells to tube “AB”. 5. Mix by shaking gently and centrifuge for 15 seconds at 3400 rpm. 6. Gently dislodge the cell button and examine for hemolysis or agglutination. 7. Grade each reaction and record the results. 8. Dispose all the biohazardous waste in the puncture-proof container. 4+ ONE SOLID AGGLUTINATE 3+ SEVERAL LARGE AGGLUTINATE; CLEAR BACKGROUND 2+ MEDIUM SIZED AGGLUTINATES; CLEAR BACKGROUND 1+ SMALL AGGLUTINATES TURBID BACKGROUND W+ TINY AGGLUTINATES TURBID BACKGROUND 0: NO AGGLUTINATION OR NO HEMOLYSIS CHARACTERISTIC OF ABO ANTIGENS They can be demonstrated as early as second month of fetal life The A antigen appears to be weak at birth but at the age of one year, agglutinogens reach final strength They persist throughout life analtered. However, abnormal antigens may be found as acquired characteristics in leukemia, and cancer (abnormal secretion of ABH substance) They may be found in saliva, pancreatic secretions, gastric secretions of people who are secretors They may be found in bacteria and other species CHARACTERISTIC OF ABO ANTIBODIES  Not normally present at birth. If present at birth, they originated from the mother through placental leakage during delivery  They develop 3-6 months after birth  Predominantly IgM and react at room temperature or colder  Occur in two forms: Naturally occurring antibodies Immune antibodies produced during incompatible transfusion or incompatible pregnancies  They are present in some animals and plants as lectins. Lectins are plants or seed extracts diluted to agglutinate specific human blood group antigens  Dolichos biflorus: agglutinates A1 or A1B cells (anti-A1lectin)  Bandeiraea simplicifolia: agglutinates B cells (anti-B lectin)  Ulex europaeus: agglutinates O cells (H specificity) and other ABO blood groups depending on the amount of H antigen (anti-H lectin)  Present in low titer or even absent in cases of acquired and congenital hypogammaglobulinemia and agammaglobuleniemia