BACHELOR OF BIOMEDICAL SCIENCES

BASIC TRANSFUSION SCIENCE AND BLOOD BANKING

BMC 125

CHAPTER 6: BLOOD GROUPING SEROLOGY

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 Learning Outcome
At the end of the lecture, students should able
to
• Describe different methods of ABO& Rh
grouping
• Explain requirements of ABO & Rh grouping
• Differentiate different types of blood groups

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 Different methods
• Slide or Tile method
• Tube method
• Microplate method
• ID-Micro typing system.
• Automated and semi automated methods.

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 Slide/ Tile method
• Used only in emergency
• Mainly in outdoor camps
• It is insensitive and not reliable for weak
antigen
• It having drying effect.
• Tile method should be confirmed by using
tube or other methods.

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 Meterials
• Glass slide/White tile.
• Reagent antibody/test serum.
• Blood
• Applicator sticks.

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 Procedure
• Preparation of 10% cell suspension
Mix 10 drops packed cells with 90drops
saline(1drop packed cell with 9 drop saline)
1) Take tile /slide and marked as A,B,AB, &O
2) Place a drop of antiserum ‘A’ in the square marked
as A, antiserum ‘B’ in the square marked as B , ‘AB’
and O add respective squares.
3) Add one drop of cell suspension to each square
make sure that the dropper does not touch the
antisera on either square.

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4) Mix the cells with the antisera using separate
applicator sticks.
5) Gently rock the slide back and forth and
observe for the appearance of agglutination .
Doubtful cases may be confirmed by using
microscope.

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 Observation
Anti A Anti B Anti AB AntiD Group
+ - + - A Neg
- + + - B Neg
+ + + - AB Neg
+ - + + A Pos
- + + + B Pos
- - - + O Pos
- - - - O Neg
+ + + + AB Pos

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 Tube method
• More reliable as it capable of detecting a
weaker antigen –antibody reaction.
• This method is very sensitive and avoid drying
effect.
• Negative and positive controls can also be
done simultaneously using known A cell, B
cells and O cells.

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 Procedure
• Preparation of 5% cell suspension .
1) Take 3-5 drops blood in a test tube
2) Add Saline and mix well, then centrifuge at
3000 rpm for 1 mts
3) Decant the supernatant and repeat the
procedure at least 3 times.
4) Take 1 drop of final supernatant and 19
drops of saline.(5% suspension).

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• Tube method 2 types
1) Immediate spin method
2) Saline room temperature method
Immediate spin method
Materials and method
1. Test tube
2. Test serum
3. Test red cells.

Procedure
1)Set up a Four rows of test tube and labeled them as A,B,AB, & O.
2)Add 2 drops of antiserum i.e Anti A, Anti B ,Anti AB and Anti D in
pre labeled tubes respectively.
3) Add one drops of 3-5% cell suspension to each tube.
4) Centrifuge at 1500 rpm for 1-2 mts
5) Look for agglutination against well lighted back ground
6) Record the list

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 Reverse grouping / Serum grouping

• Confirmation of blood group is done by using this

method.
• Patient serum is mixed with a washed cell suspension
of known A-cell, B-cell, and O cell.
• Always done in tube method
• Commonly used in blood bank

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 Preparation of known A,B,&O cell
suspension
• Collect the blood from known individuals of A, B, O
blood in different tubes and mark respectively.
• Prepare 3-5% suspension of each cell type.
• Take 3-5 drops of blood in different tube and mark
respectively.
• Add Normal saline , mix well and centrifuge 3000rpm
for 1mts
• Decant the supernatant and repeat the procedure 3
times .

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• Prepare each cells 3-5% solution
Take one drop of cell button and add 19 drops
of saline mix well (Cherry red colour ) then
marked A,B and O .

• Then check by using the anti serum A &B.

Store in refrigerator

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 Materials and Method

• Test tube
• Known A cell ,B cell, and O cell suspension.
• Patient serum.
• Centrifuge.

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 Procedure

• Arrange three small tubes in a rack and label them as ‘A’ ‘B’ &
‘O’
• Place 2 drops of serum to be tested in each tube.
• Add 1 drop of freshly prepared saline suspension of known
group A cell to tube marked ‘A’, B cell to tube marked ‘B’, and O
cells to tube marked ‘O’
• Mix well and Centrifuge at 1500 rpm for 1-2 mts
• Gently rotate the tube to dislodge cell sediment and examine
for agglutination.
• Avoid hard shaking because it breaking agglutination

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Reaction Patterns for ABO Groups

Blood Group Agglutination Agglutination

with A cells with B cells
A - +

B + -

AB - -

O + +

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Grading of Agglutination:
• Negative (0) ------ No clumps or aggregates
• Weak (+/-) ------ Tiny clumps or aggregates barely
visible macroscopically or to the naked eye
• 1+ Few small aggregates
visible macroscopically
• 2+ Medium-sized aggregates
• 3+ Several large aggregates
• 4+ One solid aggregate

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 Microplate method
• It is used for testing large number of blood
samples
• Microplates are polysterene plates ,having 96
small wells
• Each of which can hold 200-300 micro ltrs of
reagent.
• The wells are either ‘V’ type, Flat bottom, or U
type (U type commonly used)
• It is disposable ,some times it reuse.
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 Materials and method
• Microplate
• Reagent or test serum.
• Reagent or test red cell.
Method
-Add one drop of antiserum in to appropriate
wells of the plate .
- Add one drop of 2-5% saline suspension of red
cell to each of the anti sera in to the wells .

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Mix the contents by gently shaking the plate.
- Incubate the plate at room temperature for
20-30 mts
- Look for any haemolysis or agglutination

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 Microtyping system (Gel method)
Principle:
• Microtubes in the forms cards, filled with buffered
dextran gel are used which may be neutral or
impregnated with antisera.
• In negative reaction red cells are pass through gel
upon centrifugation where as in positive reaction the
agglutinated red cells are trapped on the top of the
gel or suspended within it.

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BIOMEDICAL SCIENCE, FACULTY OF MEDICINE
 Antiserum reagent
• Anti ‘A’ - Blue colour – Methylene blue
• Anti ‘B’ - Yellow colour- Acriflavin
• Anti ‘AB’ – Colourless
• Anti ‘O’ - Colour less

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 REFERENCES
• Denise M. Harmening PhD MLS(ASCP)
CLS(NCA),2012.Modern Blood Banking &
Transfusion Practices 6th Edition.by F.A.Davis
company united sate of America.
• Eva D.Quinley Ms MT
(ASCP)SBB.2010.immunohaematology Principles and
practice,3rd ed( Lippincott Williams and Wikins)
• Jeffrey Mc Cullough.2011.Trnasfusion Medicine 3 rd
ed,wiley Blackwell publication ,United states.
• Harvey G.Klein,David J Anstee 2013,Mollisons blood
transfusion in clinical medicine,12 th ed,Wiley-Blackwell.
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Thank you

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