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Practical Blood Bank
Lab 1 ABO Grouping
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ABO blood group antigens present on red blood cells (Natural Antigens)
IgM antibodies present in the serum
IntroductionIntroduction
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RBC Precursor StructureRBC Precursor Structure
Glucose
Galactose
N-acetylglucosamine
Galactose
Precursor Substance (stays the
same)
RBC
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Formation of the H Formation of the H antigenantigen
Glucose
Galactose
N-acetylglucosamine
Galactose
H antigen
RBC
Fucose
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Formation of the A antigenFormation of the A antigen
Glucose
Galactose
N-acetylglucosamine
Galactose
RBC
FucoseN-acetylgalactosamine
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Formation of the B antigenFormation of the B antigen
Glucose
Galactose
N-acetylglucosamine
Galactose
RBC
FucoseGalactose
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MethodsMethods for determination of ABO group for determination of ABO group of RBCs of RBCs
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ReagentsReagents◦ Anti-A antibodies◦ Anti-B antibodies◦ Anti-AB antibodies (optional)◦ Anti-D◦ Group A & B RBCs
◦ Slides, or Test tubes.◦ Wooden applicator.◦ …..
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ProcedureProcedure 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 typingforward typing.
Use patient serum for confirmation ( reverse grouping or backtyping).
At the End, Discard all materials in the isolation trash containers.
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Blood Grouping: Blood Grouping: Forward and reverse typingForward 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
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Forward typingForward 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 blood agglutinate in the presence of these sera
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Reverse TypingBack or reverse type with A and B cellsBack 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.
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Grading System for Grading System for ReactionsReactions
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Washed 3% Cell SuspensionWashed 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.
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Principle Principle Washing cells to be tested removes removes
serum or plasmaserum or plasma which may contain proteinsproteins that interfere with testing,
causing non-specific agglutination or rouleaux formation.
Washing also removes fibrinogenfibrinogen, which may cause small clots.
The ratio of serum to cells markedly affects the sensitivity of agglutination sensitivity of agglutination tests. Preparation of a 2-5% cell suspension provides cells in an optimum concentration to detect weak antibodies.
Wash
ed 3
% C
ell
Wash
ed 3
% C
ell
Susp
ensi
on
Susp
ensi
on
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ProcedureProcedure1. Label a 12 x 75 mm tube 2. Transfer 2-4 drops of blood from the sample
to the labeled tube3. Forcibly squirt saline from the wash bottle
into the tube until it is about 3/4 full4. 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 clear7. After the final wash, shake the tube to
completely resuspend the cells and add saline to a final concentration of approximately 3%
Wash
ed 3
% C
ell
Wash
ed 3
% C
ell
Susp
ensi
on
Susp
ensi
on
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Notes and Precautions Notes and Precautions To prevent contamination, do not touch do not touch
tubes with the tip of the saline bottlesaline bottle.
Resuspend the cell button 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 gloved finger as a stopper.
To prevent cells from spraying out during centrifugation, fill tubes no more than 3/4 3/4 fullfull.
To ensure good resuspension of cells, add the saline in a forceful streamforceful stream.
Wash
ed 3
% C
ell
Wash
ed 3
% C
ell
Susp
ensi
on
Susp
ensi
on
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Slide Method – Forward Slide Method – Forward TypingTypingPrinciple:
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.
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Procedure: 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.
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Interpretation of the results: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
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Tube Methods - Forward Typing Tube Methods - Forward Typing
- Prepare 2-5% cell suspension
- Label Test tubes
- Add 2 drops of Anti sera A, B , and D
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- 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 .
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Read and record agglutination reaction
Reaction of cells tested with
Interpretation
Anti-AAnti-BCell AgABO Group
--No AgO
+-AA
-+BB
++A, BAB
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Serum testing (Reverse)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
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Interpretation of resultsInterpretation 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
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Interpretation of Both Interpretation of Both (Forward and Reversed Typing)(Forward and Reversed Typing)
Reaction of cells tested with
Reaction of serum tested against
Interpretation
Anti-AAnti-BA
cellsB
CellsO
cellsABO
Group
--++-O
+--+-A
-++--B
++---AB
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Other methods for blood Other methods for blood groupinggroupingGel 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.
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Interpretation of ResultsInterpretation of Results◦ A positive reactionA positive reaction is recorded when red cells are
retained in or above the gel column after centrifugation◦ A negative reaction 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 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.
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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.
ABO/D + Reverse group cards
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Microplate TechniqueMicroplate 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
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Thank
You