ABO Blood Grouping
• ABO blood group-A, B, AB, or O— is based on the
presence or absence of the A and B antigens on
the red blood cells of an individual.
• The A blood type has only the A antigen while the
B blood type has only the B antigen.
• AB blood types have both the A and B antigens,
and the O blood types have neither A nor B
antigen.
• As at the age of six months, an individual naturally
would have developed antibodies against the
antigens his red blood cells lack.
ABO Blood Grouping
• However, the distribution of each of the four ABO
blood types varies between racial groups while O
remains the most common and AB is the least
common.
• The ABO grouping is the first test done on blood
when it is tested for transfusion.
• The ABO grouping test is based on the principle of
haemagglutination reaction.
• Haemagglutination of red cells occur during the
reaction of red cell antigens (agglutinogens) with
their corresponding antibody.
ABO Blood Grouping
• A positive agglutination is represented by
the lattices of red cells as seen on the slide
or by button formation in the test tube
• While a negative reaction is represented by
uniform distribution of red cells on slide and
lack of button formation in test tube.
ABO Blood Grouping
Techniques
• Two basic methods to observe the
haemagglutination reactions in ABO blood grouping;
(i) slide method and (ii) test tube method.
• The former is easier to perform and the latter is
more sensitive.
• Numerous laboratories in the developing countries
perform the tube test only in situations where the
result of the slide test is doubtful.
• The tube method is however recommended for
reliable results following internationally approved
guidelines.
ABO Blood Grouping
Techniques
• Slide Method: The requirements include Glass
slides, Pastuer pipettes, Applicator sticks and
centrifuge.
• The reagents are:
- Anti-A sera (blue color): Human polyclonal or murine
monoclonal.
- Anti-B sera (yellow color): Human polyclonal or
murine monoclonal.
- Normal saline: 0.9 g/dl sodium chloride in distilled
water.
Slide Method for ABO Blood
Grouping
• Specimen: Clotted blood is generally used, the
clotted blood is centrifuged at 1500rpm for few
minutes to separate serum.
• The red cells are then separated from the clot using
a Pastuer pipette and suspended in saline.
• Anti-coagulated blood with proper anticoagulant like
EDTA can also be used.
• The specimen should be stored at 2-8C if there is
any delay in examination.
• Blood obtained by finger puncture may be tested
directly by the slide method and mixed quickly with
Slide Method for ABO Blood
Grouping
Procedure:
1. Prepare a 10% suspension of red blood cells in
normal saline;
(i)Mix 0.05 ml (5 drops) of sedimented red cells with
2 ml of normal saline,
(ii)Centrifuge at 1,500 rpm for 1 to 2 min and discard
supernatant,
(iii)Add 2 ml of normal saline to the sedimented red
cells, mix well to gives a 10% suspension of red cells.
Slide Method for ABO Blood
Grouping
Procedure:
2. Place 1 drop of anti-A sera on one-half of a glass
slide.
3. Place 1 drop of anti-B sera on the other-half slide.
4. Add a drop of the red cell suspension to each half
of the slide using a clean Pastuer pipette.
5. Mix each cell-serum mixture well using separate
applicator sticks.
6. Tilt the slide back and forth and observe for
agglutination.
Slide Method for ABO Blood
Grouping
• Results Interpretation:
- Tests that show no agglutination within two minutes are
considered negative.
- Do not interpret peripheral drying or fibrin stands as
agglutination.
• Observation:
- If any agglutination occurs it is visible with naked eyes as
dark reddish lattices of different sizes.
- If agglutination is minimal it can be confirmed by examining
it under microscope.
Slide method result
ABO Blood Grouping
Techniques
• Tube Method: The requirements include test tubes (10
x 75 mm or 12 x 75 mm) and microscope.
• Procedure:
1. A 5% red blood cell suspension in normal saline is
prepared as follows:
(i) Mix 0.05 ml (5 drops) of sedimented red cells with 2 ml
of normal saline,
(ii) centrifuge at 1,500 rpm for 1 to 2 minutes and discard
supernatant,
(iii) Add 4 ml of normal saline to the sedimented red cells,
mix well to get a 5% suspension of red cells.
Tile Method for ABO Blood
Grouping
• Procedure:
2. To a small test tube, add one drop of anti-A sera.
3. To a second test tube, add one drop of anti-B sera.
4. Add one drop of 5% red cell suspension to each of
the two test tubes using a Pastuer pipette
5. Mix well and centrifuge both the tubes at 1,500
rpm for one min. or incubate at room temperature
for one min.
Tile Method for ABO Blood
Grouping
• Procedure:
6. Examine for agglutination and red cell sediments called a
button will be seen at the bottom of the tube if the tube is
centrifuged.
- Gently tap the button from the tube by a spring action of right
index finger and dislodge the cell button.
- If red cells form one or more lattices with clear supernatant
fluid, the agglutination is present.
- If red cells re-suspend easily, without any visible lattice formed,
agglutination is absent.
7. In case of any doubt, take a drop of the suspension on a slide
and observe under the 10X objective for agglutination.
ABO Forward and Reverse
Grouping
• There are two ways in determining the ABO blood
group;
• Forward grouping or front typing or cell
typing procedure is when the individual’s red
cells are tested with a known anti – A and anti- B
sera.
• Reverse grouping or back typing or serum
typing procedure when the individual's serum is
tested with known group A cells and group B cells.
Rh Blood Typing
• Also called Rh blood grouping is next important to ABO
blood grouping and detects only the presence of Rh
antigen (or D antigen) out of all Rh factors on the red
cells.
• Its principle is based upon haemagglutination where the
red cells with Rh antigen (D antigen) will form a lattice
with anti-D antiserum at room temperature (in the
presence of protein).
• The technique is similar to ABO blood grouping and
hence, Rh typing is done along with ABO blood grouping.
• The Rh typing can also be done by two methods which
are the Slide method and Tube method.
Rh Blood Typing
Techniques
Slide Method:
•The requirements are same as that of ABO
slide method.
•The reagents include the Anti-D sera (human
polyclonal or human monoclonal), and normal
saline.
•The specimen is same as that of ABO
method.
Slide method for Rh
Blood Typing
Procedure:
1. Place one drop of anti-D serum on a pre-warmed glass
slide.
2. Add one drop of 10% suspension of red blood cells (in case
of anaemic patients, use one drop of sedimented red cells)
using a Pastuer pipette.
3. With an applicator stick, mix cell-serum mixture well.
4. Tilt the slide back and forth and observe for agglutination.
5. Tests that show no agglutination within three to five
minutes are considered negative.
Rh Blood Typing
Techniques
Tube Method:
•The requirements are same as that of ABO
tube method.
•The reagents include Anti-D sera (human
polyclonal or human monoclonal) and normal
saline.
•The specimen is same as that of ABO method.
Tube method for Rh
Blood Typing
Procedure:
1. Prepare a 5% suspension of red blood cells in normal
saline.
2. Add one drop of anti-D serum to a test tube .
3. Add one drop of cell suspension using a Pasture pipette.
4. Mix well and centrifuge tubes at 1,500 rpm for one
minute or incubate at room temperature for two minutes.
5. Examine the agglutination reaction in each tube by
dislodging the button gently and if necessary, use a
magnifying hand lens.
Tube method for Rh
Blood Typing
6. Interpretation: Agglutination will be recognized by
the formation of small lattices in a clear liquid.
- As the bottom of the test tube is tapped, the lattices
whirl up and then settle down and this will be marked
as positive reaction with the cells identified as Rh-
positive.
- If the red cells re-suspend homogeneously with no
visible lattices, it should be marked as negative
reaction and the cells are identified as Rh- negative.
7. All the Rh negative cells must be tested for Du.
