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

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

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 * Antisera are products of “Cypress-diagnostic”. They are murine monoclonal IgM antibodies.

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.

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

(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 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 touching antisera.

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.

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.

Blood typeSerum Reaction Anti-AAnti-BAnt-ABAnti-D AB B A O Enter your results in the following table

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

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

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