Student name: ______________________
Date: ___________________________
Practical session 2
Learning objectives
To perform and interpret ABO and Rh (D) forward and reverse blood grouping
techniques correctly.
To perform a weak D(Du) testing.
Introduction
ABO BLOOD GROUPING METHOD
Forward grouping:
Forward grouping refers to the testing of the RBCs to determine the presence of A or B
antigen on the surface. Other common terms used to describe forward grouping include
front typing, cell typing, cell grouping, and forward typing. Forward grouping is
accomplished by testing a sample of RBCs with known anti-A and anti-B. Anti-A, B may be
used for typing all group O donors.
Reverse Grouping:
Reverse grouping refers to the testing of a serum to determine the presence or absence of
anti-A or anti-B. Other common names for this testing include serum grouping, confirmation
grouping, and back typing.
Reverse grouping is routinely accomplished by reacting the serum to be tested with a
suspension of known A cells and known B cells.
A. Red cell washing
Purpose:
o To remove unwanted substances like proteins, antibodies.
o To remove damaged red cells and the released hemoglobin.
Reagents and Equipment:
Normal saline
Pasteur pipette
Centrifuge
Clean glass tube
Test tube racks
Dropper bottles
Procedure:
a. Take a blood sample into a test tube(about 0.5 ml)
b. Centrifuge the sample for 3min at 2500rpm.
c. Remove the plasma/serum with the help of a Pasteur pipette.
d. Add normal saline to the test tube by means of a dropper bottle up to ¾ of the
tube.
e. Centrifuge at 3500 rpm for 3 minutes.
f. Discard the supernatant.
g. Repeat the procedure from d 3 times.
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Haematology IV Practicals
B. Detection of Rh Antigens
Tube testing
a. Label two test tubes. One tube as ‘D’ and the other as ‘control’.
b. Wash the red blood cells to be tested in normal saline.
c. Prepare a 5% suspension of the washed cells in normal saline.
d. Add one drop of anti-D to the tube labeled as ‘D’.
e. Add one drop of Rh control to the tube labeled as ‘control’.
f. Centrifuge for 15 seconds at 1000rpm.
g. Gently resuspend the cell button, watching for agglutination as resuspension
occurs.
Interpretation:
Agglutination in the tube indicates Rh positive cells.
A negative result is absence of agglutination
C. Weak D (Du) testing
On all negative Rhesus (D) groupings the following additional procedures should be
employed to test for the Du subgroup.
1. perform steps a-g as described in Rh(D) blood grouping technique(Tube testing)
2. If the reaction is less than 2+, incubate the test and the control at 37°C for 15 minutes.
3. After incubation wash the cells in each tube with normal saline.
4. Fill the tube with saline, mixing well. Be sure to completely resuspend the cell button in
the saline.
5. Centrifuge for 3 min at 2500rpm.
6. Decant the saline completely.
7. Repeat steps 4-6 for 3 washes.
8. Add 2 drops of AHG (Coombs Reagent), centrifuge for 15 seconds at 1000rpm and
resuspend gently.
9. Resuspend gently, and observe for agglutination.
10. Record the results.
Activity
1. Perform and interpret ABO forward blood grouping techniques correctly.
2. Perform and interpret ABO reverse blood grouping techniques correctly.
3. Carry out a Du test for the sample provided.
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Haematology IV Practicals
Student name:_______________________
Date:________________________________
Practical session 2
1. Record results of ABO and reverse groupings
2. Record the result of Du test
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Haematology IV Practicals
