Practical
Blood Bank
ABO Grouping
Introduction
 ABO blood group antigens present on red blood
cells (Natural Antigens)
 IgM antibodies present in the serum
RBC Precursor Structure
RBC
Glucose
Precursor
Substance
(stays the
same)
Galactose
N-acetylglucosamine
Galactose
Formation of the H antigen
RBC
Glucose
Galactose
H antigen
N-acetylglucosamine
Galactose
Fucose
Formation of the A antigen
RBC
Glucose
Galactose
N-acetylglucosamine
Galactose
Fucose
N-acetylgalactosamine
Formation of the B antigen
RBC
Glucose
Galactose
N-acetylglucosamine
Galactose
Fucose
Galactose
Methods for determination of ABO group of RBCs
Reagents
◦
◦
◦
◦
◦
Anti-A antibodies
Anti-B antibodies
Anti-AB antibodies (optional)
Anti-D
Group A & B RBCs
◦ Slides, or Test tubes.
◦ Wooden applicator.
◦ …..
Procedure

Verify that patient information on the sample
matches information on the worksheet.

Centrifuge the sample and remove the serum
to a labeled tube.

Add 2-3 drops of the patient’s cells to the WC
tube, and prepare a washed 3% suspension.

Use Patient cell suspension in forward typing.

Use patient serum for confirmation ( reverse
grouping or backtyping).

At the End, Discard all materials in the isolation
trash containers.
Blood Grouping:
Forward and reverse typing



ABO typing is the first thing to be done
before transfusion
A person must receive ABO matched
blood because ABO incompatibilities
are the major cause of fatal transfusion
reactions
To catch these incompatibilities, typing
is done in two steps:
 Forward typing
 Reverse typing
Forward typing
Front or forward type using monoclonal
anti-A and anti-B (commercial) , Anti-D
 Patient blood is mixed with serum that
contains antibodies against type A
blood, and type B blood.
 Determination of the blood type is
based on the whether or not the blood
agglutinate in the presence of these
sera

Reverse Typing
Back or reverse type with A and B cells
• Commercially available A and B cells
are used.
• Patient serum added to the known cells.
• Used as confirmatory for the forward
method.
Grading System for Reactions
Washed 3% Cell Suspension



Used in forward typing , tube methods.
The ratio of serum to red cells may
dramatically affect the sensitivity of
agglutination tests.
Consistent preparation of either 2 to 5%
red cell suspension is critical to any
agglutination test.
Washed 3% Cell Suspension
Principle

Washing cells to be tested removes
serum or plasma which may contain
 proteins that interfere with testing, causing
non-specific agglutination or rouleaux
formation.
 Washing also removes fibrinogen, which
may cause small clots.

The ratio of serum to cells markedly
affects the sensitivity of agglutination
tests. Preparation of a 2-5% cell
suspension provides cells in an optimum
concentration to detect weak
antibodies.
Washed 3% Cell Suspension
Procedure
1. Label a 12 x 75 mm tube
2. Transfer 2-4 drops of blood from the sample to
the labeled tube
3. Forcibly squirt saline from the wash bottle into
the tube until it is about 3/4 full
4. Centrifuge 45-60 seconds at high speed (3400
rpm)
5. Decant supernatant and shake to resuspend
completely
6. If gross hemolysis is present, repeat steps 3 to 5
until supernatant is reasonably clear
7. After the final wash, shake the tube to
completely resuspend the cells and add
saline to a final concentration of
approximately 3%
Washed 3% Cell Suspension
Notes and Precautions

To prevent contamination, do not touch
tubes with the tip of the saline bottle.

Resuspend the cell button thoroughly
between washes before adding more saline
to ensure complete washing.

Do not attempt to mix a tube full of saline.

Do not mix cells by using your gloved finger
as a stopper.

To prevent cells from spraying out during
centrifugation, fill tubes no more than 3/4 full.

To ensure good resuspension of cells, add
the saline in a forceful stream.
Slide Method – Forward Typing

Principle:
When red cells are mixed with various
reagent antiseras (soluble antibody),
agglutination will occur on the slides
containing cells positive for (possessing
the antigen) the corresponding antigen.
No agglutination will occur when the red
cells do not contain the corresponding
antigen.
Procedure:
1. On the section of slide labeled anti-A place
one drop of antibody A.
2. On the section of slide labeled anti-B place
one drop of antibody B.
3. On the section of slide labeled anti-AB place
one drop of antibody AB.
4. On new slide labeled anti-D place one drop
of antibody D.
5. Place one drop of cells in each antibody
containing circle.
6. Carefully mix each solution with a separate
applicator stick.
7. Tilt slowly for one minute, then observe for the
agglutination.
Interpretation of the results:



Strong agglutination of
RBCs in the presence
of any ABO grouping
reagent constitutes a
positive result.
A smooth suspension
of RBCs at the end of 2
minutes is a negative
result.
Samples that give
weak or doubtful
reactions should be
retested by Tube test
ABO grouping
Tube Methods - Forward Typing
- Prepare 2-5% cell suspension
- Label Test tubes
- Add 2 drops of Anti sera A, B ,
and D
- Add one drop of 2-5% Patient
Red Blood Cell suspension.
- Mix the contents of the tubes
gently and centrifuge for 15-30
seconds at approx. 900-1000 x g
- Gently resuspend the RBCs
buttons and examine for
agglutination
If the Rh test is negative, add a
second drop of anti-D and
incubate 15 minutes at 37oC,
then centrifuge and read again.
Read and record agglutination reaction
Reaction of cells tested
with
Interpretation
Anti-A
Anti-B
Cell Ag
ABO Group
-
-
No Ag
O
+
-
A
A
-
+
B
B
+
+
A, B
AB
Serum testing (Reverse)
1. Label 2 clean test tubes (A, B )
2. Add 2-3 drops of serum to each tube
3. Add one drop of (A) reagent RBCs to the tube
labeled A
4. Add one drop of (B) reagent RBCs to the tube
labeled B
5. Mix the contents of the tubes gently & then
centrifuge for 15-30 seconds at 900-1000 x g
6. Examine the tubes for evidence of hemolysis.
Gently resuspend the RBCs buttons and
examine them for agglutination
Interpretation of results

Agglutination in any tube of RBCs test or hemolysis or
agglutination in serum tests constitutes positive test
results
• A smooth suspension of RBCs after resuspension of
an RBCs button is a negative result
Interpretation of Both
(Forward and Reversed Typing)
Reaction of cells
tested with
Reaction of serum tested Interpretation
against
A
Anti-A Anti-B
cells
B
Cells
O
cells
ABO
Group
-
-
+
+
-
O
+
-
-
+
-
A
-
+
+
-
-
B
+
+
-
-
-
AB
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, AntiB, 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.

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.
A positive reaction in the MTS Control microtube indicates
a false positive reaction may have occurred in the
corresponding blood grouping microtube, thus invalidating
the blood grouping tests.
Drying, discoloration, bubbles, crystals, other artifacts,
opened or damaged seals may indicate product
alteration
A buffered gel suspension is contained in two (2)
microtubes of the A/B/D Monoclonal and Reverse
Grouping Card™.
Sodium Azide (0.1% final concentration) is added as a
preservative.
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.
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.

◦
◦
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◦
◦
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Add reagent and patient sample( red cells/ serum)
Incubation,
Centrifugation
Red cell resuspension,
Reading of results
Interpretation of results