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Blood Grouping

This document discusses various methods for blood grouping and determining blood types, including slide, tube, and microplate methods. The slide method allows for preliminary blood typing but is less sensitive than the tube method, which is considered the gold standard. The tube method involves forward and reverse typing, where antisera are mixed with a patient's red blood cells to determine antigens (forward typing) and with their serum to detect antibodies (reverse typing). Grading scales are used to classify the strength of agglutination reactions, which indicate blood type. Proper controls, washing of red blood cells, antisera to antigen ratios, and interpretation of results are essential to accurate blood grouping.

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211 views47 pages

Blood Grouping

This document discusses various methods for blood grouping and determining blood types, including slide, tube, and microplate methods. The slide method allows for preliminary blood typing but is less sensitive than the tube method, which is considered the gold standard. The tube method involves forward and reverse typing, where antisera are mixed with a patient's red blood cells to determine antigens (forward typing) and with their serum to detect antibodies (reverse typing). Grading scales are used to classify the strength of agglutination reactions, which indicate blood type. Proper controls, washing of red blood cells, antisera to antigen ratios, and interpretation of results are essential to accurate blood grouping.

Uploaded by

gacruz1010
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© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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BLOOD GROUPING

SLIDE/TUBE METHODS

BEA NICOLE C. REYES

https://stdominiccollege.edu.ph
INTRODUCTION
ABO blood group antigens present on red blood cells and
IgM antibodies present in the serum
BLOOD GROUPING TESTS
• Slide test for determination of ABO group of RBCs
• Tube test for determination of ABO group of RBCs and
serum
SLIDE GROUPING
ADVANTAGES:
• Preliminary typing tests
• Use during camps
DISADVANTAGES:
• Not routine test
• Less sensitive
• Drying of reaction giving to false positive results
ANTISERA
SLIDE GROUPING

• Test should be done at room temperature or lower


• Tubes, slides should be dry and labeled properly
• Antisera should always be added before adding cells
• Results should be recorded immediately after observation
• Hemolysis is interpreted as positive result
BLOOD SAMPLE
FIRST ADD ANTISERA TO SLIDE
SAMPLES ADDED TO SLIDES
OBSERVE FOR AGGLUTINATION
(SAMPLE 1)
SAMPLE 2
SLIDE TEST FOR DETERMINATION OF
ABO GROUP OF RBCs
Tube Test for Determination of
ABO group of RCBs
Forward and Reverse Typing
1. ABO typing is the first thing to be done
before transfusion.
2. A person must receive ABO matched blood
because ABO incompatibilities are the
major cause of fatal transfusion reactions
3. To guard against these incompatibilities
typing is done in two steps:
1- Forward typing
2- Reverse typing
1 – Forward Typing
1. ABO typing is the first thing to be
done before transfusion.
2. A person must receive ABO
matched blood because ABO
incompatibilities are the major
cause of fatal transfusion reactions
3. To guard against these
incompatibilities typing is done in
two steps:
1- Forward typing
2- Reverse typing
Reverse Typing - 2
Back or reverse type with A and B cells
Grading System for Reactions
Ratio of Serum to RBCs
1. The ratio of serum to red cells may dramatically affect the
sensitivity of agglutination tests.
2. Consistent preparation of either 2 to 5% red cell suspension is
critical to any agglutination test.
WASHED 3% CELL SUSPENSION
Principle
1. 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.
2. 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.
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%
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.
Test Tube Method
Recommended Method (Gold Standard)

• Allows longer incubation of antigen and antibody mixture without drying


• Tubes can be centrifuged to enhance reaction
• Can detect weaker antigen / antibody

Two Steps in ABO grouping


Cell Grouping (Forward Grouping)

• Tests the patients red cells with known Anti-A & Anti- B to determine the antigen
expressed
Serum Grouping (reverse grouping)

• Test the patients serum with known A & B cells to determine the presence

of antibody.
Cell Grouping
(Forward Grouping)
• Prepare 2-5% suspension of test sample in normal
saline
• Set three tubes , label them as A,B, D
• Add two drops of anti A , anti-B, anti D in three
different tubes
• Add one drop of 2-5% cell suspension (Ratio of 2:1)
• Mix contents well and centrifuge at 1500 rpm for 1
minute
• Observe for hemolysis
• Gently disperse cell button and check for
agglutination
• Confirm negative results under microscope
TUBE
METHOD
SAMPLE ADDED
MIX TUBE CONTENTS
CENTRIFUGE LOADING
CENTRIFUGE AT 1500 RPM
RH VIEWING BOX
GRADING AGGLUTINATION
Serum Grouping
(Reverse Grouping)
• Prepare 2-5% suspension of pooled cells A,B,O

• Label three tubes A cells, B cells and O cells

• Place two drops of serum in each tube

• Add one drop of cell suspension ( A cell to A tube, B cell to B tube and one drop

of O cell to O tube

• Centrifuge tubes at 1500 rpm for 1 minute

• Gently disperse for agglutination

• Negative results check by microscope


GRADING OF AGGLUTINATION
Other Methods for Blood Grouping
Gel Cards
• The cards containing specific typing reagents (monoclonal antibodies
to the various red cell antigens).

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 Test
• 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 microplates.
• Add reagent and patient sample( red cells/ serum)
• Incubation,
• Centrifugation
• Red cell resuspension,
• Reading of results
• Interpretation of results
PROCEDURE
Reagents
Anti-A antibodies
Anti-B antibodies
Anti-AB antibodies (optional)
Anti-D
Group A & B RBCs
RBCs Testing (Forward)
• Place one drop of anti-A serum in a clean labeled test tube, anti-B
serum in a second clean labeled test tube & anti-D in a third one
• Add to each tube one drop of a 2-5 % suspension of RBCs to be tested
• 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.
Serum Testing (Reverse)
• Label 2 clean test tubes (A, B )
• Add 2-3 drops of serum to each tube
• Add one drop of (A) reagent RBCs to the tube labeled A
• Add one drop of (B) reagent RBCs to the tube labeled B
• Mix the contents of the tubes gently & then centrifuge for 15-30 seconds
at 900-1000 x g
• 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 Results
Thank You
For Your Attention

https://stdominiccollege.edu.ph

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