Whether a blood or a blood stain belongs to group A, B, AB or O, can be known directly by testing for antigen or agglutinogen, present on the surface of the blood cells if the cell structure is intact, or indirectly by testing for the antibody or agglutinin present in the serum or the antigen present in the stain when the cell structure is lost.


For group specific,antigen or agglutinogen —

1. When the red cells are intact then direct agglutination test with the help of known antisera can be undertaken either by precipitin tube method or by tile method. Tube method is preferred to the other one.

(a) In TUBE METHOD precipitin tubes of size 5 cm long and 5.5 mm inner diameter with round bottom are used.

The red cells to be tested are washed with normal saline and suspended in normal saline. One drop (0.02 – 0.03 ml.) of cell suspension is added to equal volumes of each of anti-A, anti-B and O group serum in separate tubes. These are left in room temperature for 1 to 2 hours. When the reaction is strongly positive then naked eye clumping due to agglutination is well appreciated. When naked eye clumping is not clearly visible, one drop of the mixture is taken out from each tube on separate glass slides with the help of separate pasteur pipettes, and examined under microscope for detection of microscopic clumps of small number of cells.

(b) In TILE METHOD, one drop of the cell suspension and one drop of each antisera are mixed separately in different wells of the tile, shaken by hand and examined for clumping by naked eye and under microscope. Here much time cannot be allowed for the reaction to occur as the samples get dry quickly.

Interpretation of ABO grouping test for unknown cells
Sample cells or test cells (unknown) Serum (Known) Findings Inference (group of the tested blood
anti-A clumping
no clumping
Group A
Group B or O
anti-B clumping
no clumping
Group B
Group A or O
antisera of group O blood clumping
no clumping
Group A, B or AB
Group O

2. When blood group is to be determined from unknown serum then known cell suspensions of A, B, and O group are taken and the test is carried on as above.

Interpretation of ABO grouping test for unknown serum
Sample serum or test serum Group cells (known) Findings Inference (group of the tested blood)
A clumping
no clumping
sera of B or O group blood
sera of A or group AB blood
B clumping
no clumping
sera of A or O group blood
sera of B or group AB blood
O no clumping sera of O, A, B or AB group blood

Whenever necessary, the subgroup A1 or A2 can be determined for both unknown cells or serum by using anti A1 sera or latex of Dolichos biflorus plant and cells of-A1 or A2 respectively.

3. Absorption inhibition technique of determination of blood group may be used when the cells’ structure is damaged and they cannot be subjected to agglutination test. Even if the cells’ structure is lost, the antigenic group factor or the agglutinogen is present in a blood stain.

The test : To the stain extract, known antisera, anti-A, anti-B and anti-H (sera of blood group O) are added in different dilutions (dilution is increased by double dilution method). The mixtures are left for two hours at a temperature of 4°C.

In the second phase, 2% suspension of known cells of group A, B, and O are added separately in different tubes with mixtures of stain extract and antisera of different dilutions.

Result: With the use of anti-A sera, A-group cell factor or A-agglutinogen will be reacted upon and anti-A sera will be neutralized. Thus, if the blood stain contains A or AB group factor then, there will be no clumping of the used known A cells. This is inhibition test. Similar interpretations are made with use of anti-B sera and B cells and anti-H sera and AB group cells. But to give reasonable emphasis on the inhibition test (when there is no clumping), dilution of antisera showing inhibition, should not be very high because the diluted weak antisera themselves may be incapable to agglutinate the cell antigen.

Interpretation of results of absorption inhibition test
Stain extract Known antisera of different dilutions Indicating cells Reaction Interpretation
anti-A A cell clumping B or O group stain
anti-A A cell no clumping A or AB group stain
Unknown anti-A B cell
anti-A O cell
anti-B A cell
anti-B O cell
group anti-B B cell clumping A or O group stain
anti-B B cell no clumping B or AB group stain
anti-H A cell clumping B or AB group stain
anti-H A cell no clumping A group stain
factor anti-H B cell clumping A or AB group stain
anti-H B cell no clumping B group stain
anti-H O cell

4. Mixed agglutination test:

The stained area of the cloth is cut into pieces of 2-3 mm. length and the fibres are separated from each other either as such or after being softened by treating with a little normal saline solution. To each fibre, in separate siliconized glass tubes, anti-A, anti-B, and anti-H sera are added in neat strength and in 1 in 10 dilution separately. The tubes are incubated at 37°C for 2-3 hours. The fibres are then washed with saline for 3-4 times. In each tube are then added known indicator red cell suspensions. The tubes are then placed in rotator for 30 minutes. The fibres are then taken out, placed on a siliconized glass slide and examined under microscope. Clumps of red cells will be seen adhered along the fibre with a few clumps being scattered away from the fibre, if the test is positive. Positive test means that the blood group of the indicator cells which have clumped along the length of the fibre is the group of the blood stain on the cloth. For example, if the cloth was stained with A-group blood then, A-group factor present in the stained fibre will absorb anti-A sera which will stick to the fibre even after washing and will subsequently attract and clump A-group cells.

5. Absorption elution technique :

As in case of mixed agglutination test, the cut pieces of fibres are first treated with antisera, washed and then the fibres are transferred in the wells of a tile and known cell suspensions are then added. The tile is then placed inside a moist chamber at 55°C for 5-6 minutes. Then the slide is taken out of the chamber and rotated swiftly and the fibre is pushed to one side of the tile. The tile is then left in a rotator for 15-20 minutes at room temperature. The content of the wells of the tile are then examined by naked eye and if necessary under a microscope. Clumps will be noticed if the test is positive. As in case of mixed agglutination test, in case of a positive result, the group of the known indicator cells is the group of the bloodstain on the cloth.

Absorption elution test and mixed agglutination test are the direct tests to know the group of the blood stain, as they give the group of the stain when the tests are positive. The disadvantage of these tests are that with weak or diluted antisera the result of the test may be wrongly concluded.

Mixed agglutination technique has another advantage. This test is helpful to know the group factor of a person from body tissue like squamous cells of mouth and vagina or cells of the hair bulb. In these cases, the procedure being same as with stained fibres, the known indicator R.B.C.s get adhered and clump around the bigger squamous cells tested for knowing the group factor.