Blood Grouping

E MTENAN H AS SAN

BY: DR. EMTENAN HASAN

Type of Blood Grouping :
 forward grouping direct grouping Use Patient cell suspension
(Ag ).
 reverse grouping indirect grouping . Use patient serum for
confirmation (Ab).

BY: DR. EMTENAN HASAN

Methods for determination of ABO
group of RBCs
Slide method
Tube method
Gel card method
Microplate method

BY: DR. EMTENAN HASAN

Direct blood groping (forward grouping)
Principle:
A suspension of red cells of the specimen is reacted with known
reagent anti-sera (anti-A anti-B and anti-D), agglutination indicates the
presence of corresponding antigen on the red cells.

BY: DR. EMTENAN HASAN

Slide Method
Specimen:
5 % Red cell suspension .
Materials:
Normal saline .
Pasteur pipettes.
Glass slides
Wooden stick
Commercial anti-sera (Anti-A, anti-B and anti D ).

BY: DR. EMTENAN HASAN

Procedure (Slide method):
Take 3 slides and label them as A, B, D two halves of the same slide can
also be used, label it in the same way.
Place one drop of anti-A on the slide marked A , one drop of anti-B on
the slide marked B and anti-D on the slide marked D.
Add one drop of the cell suspension to each slide.
Mix the antisera and cells with wooden stick .
Rotate the slide back and forth to complete the mixing
Examine for agglutination against white backgroud within two minutes .

BY: DR. EMTENAN HASAN

Results:
Positive: little clumps of red cells are seen floating in a clear liquid.
Negative: No agglutination of red cells, cells are floating homogenously.

BY: DR. EMTENAN HASAN

Anti A Anti B Anti D Blood group

BY: DR. EMTENAN HASAN

 Procedure (Tube method):
Arrange 3 small test tubes (75X10mm) and label them as A ,B and D.
Add one drop of anti-A in the test tube marked 'A', one drop of anti-
B in the test tube marked 'B' and one drop of anti-D in the test tube
marked 'D'.
Add one drop of the cell suspension to each tube and mix gently.
Centrifuge immediately at low speed (1000 rpm) for one minute.
Examine for agglutination: if the tube is centrifuged, red cell
sediment will be seen at the bottom of the tube, gently tap the
bottom of the tube by spring action of your right index finger and
dislodge the cell bottom. Watch the behavior of the red cell button
against a white background or magnifying lens.

BY: DR. EMTENAN HASAN

Tube Methods - Forward Typing
- Prepare 2-5% cell suspension

- Label Test tubes

- Add 2 drops of Anti sera A, B ,

and D

BY: DR. EMTENAN HASAN

BY: DR. EMTENAN HASAN
Results:
Positive: red cell form one or more clumps with clear supernatant.
Negative: red cell re-suspended easily, without any visible clumps.

BY: DR. EMTENAN HASAN

BY: DR. EMTENAN HASAN
Indirect blood
grouping (reverse
grouping)

BY: DR. EMTENAN HASAN

Specimen: Serum

Materials:
Pasteur pipettes.
12x75 mm plastic test tubes.
Rack.
Grouping cells (A cells, B cells and O cells).

BY: DR. EMTENAN HASAN

Procedure:
Label three (12x75 mm) test tubes as A, B and O cells.
Add 2 drops of serum under test to each of the tubes.
Add 1 drop of cell suspensions (reagent known cells) from A, B and O
blood groups into the respective tubes.
Read the agglutination immediately (by holding the tube on a
magnifying concave mirror or against a lighted background at eye level)
after low centrifugation1000 rpm for 1 minute or leave for 30 min to
one hour

BY: DR. EMTENAN HASAN

Results:
Positive: red cell form one or more clumps with clear supernatant.
Negative: red cell re-suspended easily, without any visible clumps.

BY: DR. EMTENAN HASAN

Sample Anti-A Anti-B Anti-D A cell B cell O cell B.G
No

BY: DR. EMTENAN HASAN

 Other methods for blood grouping
Gel Cards
Gel Cards containing Anti-A, Anti-B, and Anti-A,B are
used to test patient or donor red blood cells for the
presence or absence of the A and/or B antigens.
The results of red blood cell grouping should be
confirmed by reverse (serum) grouping, i.e. testing the
individual’s serum with known A1 and B red blood cells.
In the Gel Test™, the specific antibody (Anti-A, Anti-B, or
Anti-D) is incorporated into the gel. This gel has been pre-
filled into the microtubes of the plastic card. As the red
blood cells pass through the gel, they come in contact
with the antibody. Red blood cells with the specific
antigen will agglutinate when combined with the
corresponding antibody in the gel during the
centrifugation step.
BY: DR. EMTENAN HASAN
ABO/D + Reverse group cards

Procedure:
1. Suspend 50 µL WB or 25 µL RBCs in 0.5 ml diluent.
2. Identify the card with patient's name.
3. To microtubes l, 2, 3 & 4 add l0 µL of suspension. To microtube 5
add 50 µL Al cells + 50 µL plasma. To microtube 6 add 50 µL B cells
+ 50 µL plasma.
4. Centrifuge for l0 minutes and read.

BY: DR. EMTENAN HASAN

 Interpretation of Results
◦ A positive reaction is recorded when red cells are retained in
or above the gel column after centrifugation
◦ A negative reaction is recorded when a distinct button of
cells sediment to the bottom of the column after
centrifugation.

BY: DR. EMTENAN HASAN

 Microplate Technique

Microplate techniques can be used to test for antigens

on red cells and for antibodies in serum.
A microplate can be considered as a matrix of 96
“short” test tubes; the principles that apply to
hemagglutination in tube tests also apply to tests in
microplate.
◦ Add reagent and patient sample( red cells/ serum)
◦ Incubation,
◦ Centrifugation
◦ Red cell resuspension,
◦ Reading of results
◦ Interpretation of results

BY: DR. EMTENAN HASAN