Laboratory methods of Blood Grouping techniques.p

MLS 412
BLOOD GROUPING TECHNIQUES
AKINBO D.B. Lecture Series

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.

• 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 antisera to avoid clotting.

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.

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.

• 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.

Laboratory methods of Blood Grouping techniques.p

• 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.

Laboratory methods of Blood Grouping techniques.p

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