BLOOD GROUPING-TUBE METHOD
Principle
In the ABO Rh test (blood group typing) tube method, both serum and red blood
cells are utilized to identify the ABO and RhD blood group. Serum, the liquid
portion of blood remaining after clotting, contains antibodies that react with
specific antigens on red blood cells. Red blood cells possess antigens that are
either present or absent, determining the individual’s blood group. The principle
of ABO Rh test (blood group typing) involves two distinct processes: forward
grouping and reverse grouping.
Forward grouping, also known as cell grouping, directly identifies the antigens
present on an individual’s red blood cells. It utilizes antisera, which contain
antibodies specifically directed against A and B antigens. These antisera are added
to a suspension of the patient’s red blood cells. If the patient’s red blood cells
possess the corresponding antigen (A or B), they will agglutinate or clump
together due to the antigen-antibody reaction.
Reverse Grouping
Reverse grouping, also known as serum grouping, identifies the antibodies
present in an individual’s serum, which are indicative of the antigens they lack on
their red blood cells. It utilizes red blood cells of known A, B and O blood groups.
The patient’s serum is added to each tube, and agglutination of the corresponding
red blood cells indicates the presence of antibodies against that antigen (anti-A or
anti-B).
Materials
Polypropylene/glass tubes 10 mm x 75 mm
Centrifuge
Refrigerated centrifuge
Pasteur pipettes
Commercially prepared anti-A, -B, -A,B and -D sera
Commercially prepared known A, B and O cells
Patient’s EDTA whole blood
Normal saline
Procedure
Preparation of 2 – 5% red cell suspension and plasma
1. Centrifuge patient’s whole blood at 1000 – 2000 x g for 10 minutes using
refrigerated centrifuge.
2. Label two tubes with the patient/donor’s name and ID, designating one for red cell
suspension and the other for plasma.
3. Use a pipette to carefully extract the serum/plasma, avoiding touching the red
blood cells layer.
4. Transfer the extracted serum/plasma into the appropriately labeled plasma tube.
5. Carefully insert the tip of the pipette into the center of the red cell layer and
aspirate a small quantity of red cells.
6. Dispense one drop the aspirated red cells into the appropriately labeled tube for
red cell suspension.
7. Add saline solution to the tube containing the red cells until it is approximately
three-quarters full (approximately 0.5 – 1 mL of saline).
8. Thoroughly resuspend the red cells.
Forward Grouping (Cell Grouping):
1. Label tubes with donor/patient and test identification.
2. Prepare cell suspension for cells being tested (Refer SP 015)
3. Place two drops of anti-A, anti-B and anti-AB reagent in the
appropriately labelled tubes.
4. Add to each tube one drop of a 2 – 5% cell suspension (in normal saline,
serum or plasma) of the red cells to be tested.
5. Mix the contents of the tubes gently and incubate at room temperature
for 15 minutes.
6. Centrifuge at 1000 rpm for 1 minute.
Reverse Grouping (Serum Grouping):
1. Label tubes with donor-patient and test identification.
2. Add 2 drops of test serum in all tubes in the corresponding column.
3. Prepare cells for testing of A, B and O groups by pooling 3 samples of
each group.
4. Add 1 drop of 2% pooled A red cell suspension in tube labelled A
5. Add 1 drop of 2% pooled B red cell suspension in tube labelled B
6. Add 1 drop of 2% pooled O red cell suspension in tube labelled O
7. Mix the contents of the tubes gently and incubate the test for minimum
15 minutes at room temperature.
8. Centrifuge all tubes at 1000 rpm for 1 minute.
9. Gently resuspend the red cell button & examine for agglutination.
Assessment of red cell agglutination test grading
Agglutination
Symbol Description
score
4+ /
Complete 12 Macroscopically visible cell button with a clear supernatant.
(C)
Macroscopically visible large clumps of cell button with
3+ 10
a clear supernatant.
Macroscopically visible small clumps of cell button with
2+ 8
a clear supernatant.
Just macroscopically visible fine granular clumps
1+ 5
of the cell button and the supernatant is turbid and reddish.
or weak Only microscopically visible fine granules
3
(w) of the cell button and the supernatant is turbid.
0 0 No agglutination seen. The supernatant is clear and reddish in color.
MF (mixed
MF A mixture of agglutinated and unagglutinated red cells seen.
field)
Complete hemolysis of the patient sample.
H
The supernatant is grossly red with no evidence of red cells.
In forward grouping, four distinct tubes are prepared:
1. Tube 1 contains anti-A serum and patient’s red blood cells.
2. Tube 2 contains anti-B serum and patient’s red blood cells.
3. Tube 3 contains anti-A,B serum and patient’s red blood cells.
4. Tube 4 contains anti-D serum and patient’s red blood cells.
Based on the agglutination patterns observed in these tubes, the ABO blood group
is determined:
1. Agglutination in tubes 1 and 3 (anti-A serum) indicates type A blood.
2. Agglutination in tubes 2 and 3 (anti-B serum) indicates type B blood.
3. Agglutination in tubes 1, 2 and 3 indicates type AB blood.
4. Absence of agglutination in tubes 1, 2 and 3 indicates type O blood.
5. Agglutination in tube 4 indicates RhD positive blood.
6. Absence of agglutination in tube 4 indicates RhD negative blood.
In reverse grouping
Based on the agglutination patterns observed in the tubes, the RhD blood group is
determined:
1. Agglutination in tube 1 indicates the presence of anti-A antibodies,
corresponding to type B or type O blood.
2. Agglutination in tube 2 indicates the presence of anti-B antibodies,
corresponding to type A or type O blood.
3. Absence of agglutination in both tubes indicates the absence of anti-A and
anti-B antibodies, corresponding to type AB blood.
4. Agglutination in both tubes indicates the presence of anti-A and anti-B
antibodies, corresponding to type O blood.
5. Agglutination in tube 3 could indicate presence of alloantibodies.
