1. Lab Work 4SDV1- Physiology IIBlood Grouping Dr Than Kyaw 26 March 2012

2. Lab 4 – Blood Grouping Aim: To determine the blood group of a given blood sample Principle • Although there are many blood types in man, only blood groups (A, B, AB and O) are used in practice including rhesus factor • Rhesus factor (Rh) is important for fetal hemolysis during pregnancy • Refer to lecture notes

3. Principle • Basis for this test - agglutinogen agglutinin reaction. • Agglutinogen (antigen) - present on the RBC membrane • Agglutinins (antibody) – present in the serum • Mixing of RBC containing agglutinogens with serum • containing specific agglutinin causes agglutination of RBCs due to antigen-antibody reaction

4. Requirements • Glass slides • Needles/lancets • Blood sample • Antisera* – A, B, and AB; anti-D (for Rh factor) • 0.9% normal saline (or 3.8% Na citrate solution) • Test tubes • Filter paper • tooth pricks • Spirit, cotton • Microscope *Antiseraare products of “Cypress-diagnostic”. They are murine monoclonalIgM antibodies.

5. Procedure (a) Direct mixing of blood and antisera • Prepare 4 clean slides and label each slides. • -- anti-A, anti-B, anti-AB and anti-D, respectively. • - Swab the finger tip with spirit and prick with sterile needle or • a lancet. • Place one drop of blood on each slides. • Place a drop of specific antisera on the slides marked anti-A • anti-B, anti-AB and anti-D, respectively. • Be careful not to touch the dropper with the blood. • Stir the blood-antisera mixtures with separate tooth pricks • Gently rock the slides back and forth. • Observe for the presence of agglutination after 1 minute.

6. Slide for antigen A Slide for antigen B A B Blood drop Blood drop Slide for antigen D Slide for antigen AB AB D Preparation of slides for Blood grouping.

7. (b) Dilution method • As in method (a), prepare and label 4 clean slides • -- anti-A, anti-B, anti-AB and anti-D, respectively • After pricking a finger, transfer the drops of blood from • the finger into the test tube containing 3 - 5 ml of 0.9% normal saline. • It is done by blocking the mouth of the test tube with the • pricked finger and inverting the test tube. • Place a drop of specific antisera on the slides marked • anti-A, anti-B, anti-C and anti-D, respectively. • Place one or two drops of diluted blood on each slide with • the help of a dropper without touchingantisera.

8. Procedure (continued) • Stir the blood-antisera mixtures with separate tooth pricks • Gently rock the slides back and forth • Observe whether there is agglutination in the mixtures • within 2 to 3 minutes • - Confirm the agglutination by observing under the low power • of microscope • Record the antisera with which agglutination has occurred.

9. Time to use blood sample • Use blood samples as soon as possible. • If testing the blood samples is delayed, store at 2°-8°C. • If the blood is used heparin or oxalate as anticoagulants, test within 2 days. • If sodium citrate or EDTA -- test within 14 days.

10. Enter your results in the following table

11. Precautions • Use always clean and dry slides • Do not let the blood drop clot; do as fast as possible. • Do not intermix the antisera • Do not use same stick for mixing different antisera mixture • Pseudoagglutination in the form of rouleaux may mislead the result • For dubious results examine the specimen under low power of the microscope

12. Purposes of blood grouping • To avoid transfusion reactions • Organ transplantation • Medicolegal conditions (parent dispute, criminal identification • To prevent development erythroblastosisfetalis (Rh test) • Research

13. What you have to do • All 8 groups must do direct agglutination test. • One student must volunteer for taking blood sample. • Dilution methodwill be demonstrated. Special care! • Take care not to waste reagents, etc. • Properly clean every pieces of litters and staffs you have used.