HEM 2133

Immunohaematology I
Lesson 3: ABO Typing Procedure

ABO Tube Grouping

A more sensitive and reliable method
Is used in blood banks and in clinical
laboratories
Requires dilution of the blood with saline to
make a 2% to 5% suspension of cells

 ABO tube grouping consists of:

Direct or forward grouping, which identifies
the antigens on the cells

Reverse or confirmatory grouping, which
identifies the blood group antibodies in the
serum

Performing Forward Grouping

Forward or direct grouping identifies the
antigens present on red blood cells by reacting
a suspensions of cells with commercial anti-A
and anti-B sera and observing for
agglutination after centrifugation
If a centrifuge is not available, the reactions
can be observed after allowing the tubes to sit
undisturbed at room temperature for 15 to 30
minutes

 A 2% to 5% red blood cell suspension is made by

adding 18 to 19 volumes of saline to one volume
of the patients blood
Two tubes labeled A and B are set up: one drop of
anti-A serum is placed in the A tube and one drop
of anti-B serum is placed in the B tube
One drop of the patients 2% to 5% cell
suspension is added to each tube, and the
contents are mixed
The tubes are centrifuged for 30 seconds to
enhance the reaction

Interpretation of Forward Grouping

The tubes are tapped gently to loosen the
cells from the bottom of the tube and the cells
are observed for agglutination
Clumping of the cells is a positive reaction
indicating the antigen present on the cells
corresponds to the antibody placed in the test
tubes
Reactions should be graded using a plus
system

 neg (no visible clumps or agglutinates)

w + (a few small, very fine aggregates barely
visible to the naked eye)
1 + (button breaks into numerous tiny clumps.
Background becomes cloudy)
2+ (button breaks into many medium-sized
clumps. Background is clear)
3+ (button breaks into a few large clumps.
Background is clear)
4+ (button is one or two large clumps after
being dislodged. Background is clear)

Reaction of Antisera with Red Cells

The reaction between the antiserum and red
cells can occur in various ways:
1. Agglutination

If the red cells and the antiserum contain
corresponding antigen and antibody, they
will react to bring about agglutination of the
red cells

If either antigen or antibody is absent, there
will be no agglutination

2. Hemolysis

Sometimes, the antigen-antibody reaction
may result in hemolysis due to activation of
the complement system

Complement, if present in the antiserum, can
bind to the antigen-antibody complex, and
lyse the red cells

Complement can be easily inactivated by
heating at 56C for 30 minutes

3. Rouleaux formation

A high concentration of globulin in patients
serum can hold the red cells together to
appear like a stack of coins

This is rouleaux formation and can be
mistaken for agglutination

Performing Reverse Grouping

Reverse grouping identifies the antibodies
present in a patients serum or plasma by
reacting the plasma with a 2% to 5%
suspension of group A cells and a commercial
2% to 5% group B cells and observing for
agglutination
Two drops of the patients plasma are added
to each of three tubes marked a, b and control

 One drop of the group A cell suspension is

added to tube a, one drop of group B cell
suspension is added to tube b, and one drop
of a 2% to 5% suspension of patient cells is
added to the control tube
The contents of the tubes are mixed, and the
tubes are centrifuged for 30 seconds

Interpretation of Reverse Grouping

The tubes are tapped gently, and the cells are
observed for agglutination and the reactions
graded
A positive test, agglutination, indicates that the
antibody present in the patients plasma
corresponds to the antigen on cells added to the
tube
The control tube should always be negative for
agglutination since it contains only the patients
plasma and cells
Reverse grouping results should confirm the
results of forward grouping

ABO Forward Grouping Results

Blood Group Reactions of cells with
Anti-A

Anti-B

AB

ABO Reverse Grouping Results

Blood
Group

Reactions of plasma with

A cells

B cells

O cells

AB

Forward and Reverse Grouping

Gel Typing
Automated and semi-automated systems are
available for blood grouping and
crossmatching
Solid-phase and gel or column typing methods
can be automated allowing some walk-away
testing

Gel typing
Sensitive and specific
The procedure can be standardized, verified
and validated
Testing is performed in a card prefilled with
gels mixed with the appropriate reagent
A dilution of patient cells is pipetted onto the
gel column and the card is incubated
The card is centrifuged and read

 Agglutinated cells will not travel through the

gel but remain at the top of the column, a
positive reaction
Non-agglutinated cells travel through the gel
to the bottom of the column, a negative
reaction
Since some gel typing reactions are stable for
several hours, tests can be retained and
reread if necessary

Advantages of Gel Typing

Minimal handling of reagents and specimens
increases biosafety
Eliminates variables due to work technique
Clear, stable, well-defined endpoints
objective and reproducible interpretation of
test results
Reduce needs to repeat test

Anomalous Results in ABO Blood

Grouping
A situation that exist when the results
between forward and reverse grouping is of
no match (odd)
May lead to false negative or false positive
reactions

Technical Problems - mostly

Dirty tubes or glassware

Contamination or inactivation of reagent
Failure to add reagent or serum
Failure to follow manufacturers direction
Warming during centrifugation
Uncallibrated centrifuge
Over/undercentrifuge

Improper cell to serum concentration

Cell suspension too light or too heavy
Failure to identify hemolysis as positive
Careless readings
Clerical errors (incorrect recording of results
or interpretation/readings)
Failure to incubate tests at 20-25C or below
Improper technique

Missing or Weak Reaction Antibodies

Reason depressed Ab production or cannot
produce the ABO antibodies
Cause false negative
1. Age newborn infants (not producing own
Abs yet) or elderly (declined Ab)
2. Disease patients with leukaemia,
lymphomas, using immunosuppressive drugs,
immunodeficiency disease, bone marrow
transplant

3. Chimerism possess dual population of red

cells (mixed cell population)
How to resolve?

Eliminate all technical errors

Incubate patients serum with red cells at
room temperature for 15 minutes

Chimerism establish whether the patient has
transfusion or transplant bone marrow, fetal
maternal bleeding

Missing or Weak Reaction Antigens

Unusual genotype subgroups of A and B
Disease leukaemia, Hodgkins disease
Acquired B-like activity resulting from the
action of gram-negative organisms/ intestinal
obstruction/ colon or rectum cancer/ other
lower intestinal tract disorders
Colouring dyes
Antibodies in the reagent

Plasma Abnormalities
1. Increased gamma globulin disease e.g.
multiple myeloma, Hodgkins lymphoma
rouleaux formation
2. Abnormal proteins causing rouleaux
formation
3. Whartons jelly found only when cord blood
is used
Rouleaux - stacks of red blood cells (RBCs)
which form because of the unique discoid
shape of the cells

Rouleaux