Blood

Grouping
Lecture 7
Type of Blood Grouping :
 forward grouping direct grouping Use Patient cell
suspension (Ag ).
 reverse grouping indirect grouping . Use patient serum
for confirmation (Ab).
Methods for determination of
ABO group of RBCs
Slide method
Tube method
Gel card method
Microplate
method
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.
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
).
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 .
Results
:Negative: No agglutination of red cells, cells are floating homogenously.
Positive: little clumps of red cells are seen floating in a clear liquid.
Anti A Anti B Anti D Blood group
 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.
Tube Methods - Forward
Typing - Prepare 2-5% cell suspension

- Label Test tubes

- Add 2 drops of Anti sera A, B ,

and D
Results
:Positive: red cell form one or more clumps with clear supernatant.
Negative: red cell re-suspended easily, without any visible clumps.
Indirect blood
grouping
(reverse
grouping)
Specimen: Serum

Materials:
Pasteur pipettes.
12x75 mm plastic test tubes.
Rack.
Grouping cells (A cells, B
cells and O cells).
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
Results:
Positive: red cell form one or more clumps with clear supernatant.
Negative: red cell re-suspended easily, without any visible clumps.
Sample Anti-A Anti-B Anti-D A cell B cell O cell B.G
No
 Other methods for blood grouping
Gel Cards
Gel C ards 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
c a rd. 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.
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.
 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.
 Microplate Technique

Microplate techniques ca n 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