Discovery

• Karl Landsteiner
– Drew blood from himself
and colleagues
– Performed the first forward
and reverse blood grouping
• John Bernstein
– Theory for the inheritance
• Chromosome 9q34.2
– One position is occupied by
A, B or O gene (which
codes for
glycosyltransferase)
– O is amorph
– A & B are codominant
• Individual inherits one
ABO gene from each
parent
• Simple Mendelian
genetics
Summary
 Antibodies
• Potent and naturally
occurring antibodies
against antigens not
found on their RBCs
• Detectable at 3-6 months
of age
• Ab’s in cord blood is of
maternal origin
• Peaks at 5-10 years and
declines later in life
• Found universally in
 Antibodies
• Strong agglutination
• Rapid intravascular
hemolysis
• IgG is predominant in O
and in AB persons
sensitized by prior
pregnancy
• IgM predominates in
other instances
• Reacts efficiently at 20-
 Antigens
• Presence or absence of two
antigens (A & B) defines
the four blood types
• Determined genetically by
the inheritance of a gene or
genes that code for the
production of
GLYCOSYLTRANSFERASES
• Gene interaction of ABO,
Hh & Se
• Develop as early as 37th
day of fetal life but fully
 Antigens
• Type 2 precursor
oligosaccharide
substance or
paragloboside (Beta 1-4
linkage)
• Page 112 of Harmening
…
• Bombay phenotype
• Soluble antigens
• Subgroups
Soluble Antigens
Antigens
Summary
 QUIZ
1. ISBT number
2. Chromosome number
3. Pattern of inheritance
4. Time of appearance of ABO
antigens
5. Time of appearance of ABO
antibodies
6. Glycosyl transferase for B
antigen
7. Lectin for O antigen
8. Immunodominant sugar for
group O
9. Proponent of ABO grouping
 Basic Principle
ABO TYPING:
- Forward
- Backward

• Problems and their

causes
• Discrepancies
• Approaches to resolving
discrepancies
 Reminders
1. Should be done at room
temperature (2O-24oC) or
lower; testing in hot
environment weakens the
reaction.
2. Must include both cell and
serum testing as each serves
as a check on the other.
3. Antisera used in the ABO
grouping must be used as per
the manufacturer’s
instructions and checked for
contamination.
4. Adequate controls should be
put with each batch of tests
 Reminders
5. Tubes, slides, tiles, microplates
or gel cards should be clean,
dry and labelled properly. One
should not rely on the colour of
the dye to identify the
antiserum.
6. Serum should be added before
adding cells and each tube
should be examined after
serum has been added to
ensure that none has been
missed.
7. Results should be recorded
immediately after observation.
8. Concave mirror (agglutination
viewer) or microscope may be
 ABO grouping sera
- Commercially prepared
- Polyclonal (human source) or
monoclonal (tissue culture)
antisera are available
- Monoclonal antisera give more
specific reactions and are very
sensitive for weaker reactions.
- The potency of any antisera
deteriorates rapidly if kept for
too long at ambient temperature,
best to keep at 4°C or as
directed by the manufacturer
when not in use. Frozen antisera
must be completely thawed
before use and no refreezing
should be done.
Blood sample for ABO
grouping
1. Properly labelled samples of
non-hemolyzed, unclotted
blood collected in screw-
capped plain test tubes are
most suitable
2. May be stored at 4°C but
preferably tested within 48
hours.
3. Separated serum should be
placed in a pre-labelled tube
for serum grouping and other
serological tests.
4. Cells from the test sample
 Preparation of Red
Cells for ABO Testing
• Reagent red cells are
fresh and well-selected

‘A’ cells

Pool known fresh A group

cells from at least 3
donors (why?). Wash
three times with normal
saline, make a 2-5%
suspension in saline for
use.
 Preparation of Red
Cells for ABO Testing
• Reagent red cells are
fresh and well-selected

‘0’ cells

Pool known fresh O group

cells from 1-2 donors.
Wash three times with
saline and make a 2%
suspension in saline for
 Preparation of Red
Cells for ABO Testing
• Reagent red cells are
fresh and well-selected

‘B’ cells

Pool known fresh B group

cells from 1-2 donors.
Wash three times with
normal saline, make a 2-
5% suspension in saline
for use.
 Preparation of Red
Cells for ABO Testing
• Cells are washed in isotonic saline to
remove all traces of plasma or serum.
They must then be accurately diluted
to give a standard suspension in
saline by either using a graduated
tube (Wintrobe’s tube) or by counting
drops to make a 2-5% cell
suspension.
When prepared, the cells should be
checked against anti-A and anti-B.
The cells should be kept at 4°C in the
refrigerator when not in use.
If low-ionic strength saline solution
(LISS) is being used in the laboratory
as an enhancing medium, then the
last wash should be in LISS and the
cells are thereafter suspended in LISS
 Preparation of Red
Cells for ABO Testing
• Patient and donor cells
The test cells also should
be washed at least once
with saline and suspended
to make a 2-5% suspension
in saline. When dealing
with large number of
samples, adequate
precautions must be taken
to avoid mislabelling of
tubes and the cells must be
dispensed in already
labelled tube for washing.
 ABO Blood group
system
ABO TYPING (Problems):
1. Abnormally weak
agglutination
2. Mixed field agglutination
3. Rouleaux or RBC
aggregation
4. Clumping
5. RBC’s trapped as
bystanders
6. No agglutination
7. Agglutination in all cell
 ABO Blood group
system
ABO TYPING (Causes):
1. Incorrect, contaminated,
expired reagents
2. Failure to add a
component
3. Failure to follow
manufacturer’s
instructions
4. Clerical error
5. Wrong specimen
6. Improper reading and
interpretation
This cassette consists of six wells. Each well
consists of a gel (or small glass beads).
To the first three wells is pre added  the
typing antisera ( anti-A, Anti-B and Anti-
A,B) as illustrated.  A different cassette is
used for the reverse group. This consists
of just the gel. (To this is added the
patients serum and cells of a known ABO
group i.e. A, B and O cells).
 Advantages of
Automation
1. Easy to rapidly train scientists
2. Easy to multi-skill scientists to
perform blood grouping who
don't have previous experience.
3. Standardized methods. These
results are not prone to
individual scientist subjectivity
to interpret positive and
negative reactions.
4. Cassettes are stable. This
means that doubtful are
problem reactions can be stored
for review by a supervisor.
5. Suited to high volume
workflows such as typing donor
units at Red Cross.
 Disadvantages of
Automation
1. Very expensive!
2. Not random access. That
means it is best suited to
processing batches of
specimens. This is a problem
if an urgent group is
required.
3. There is a possibility that the
scientist may rely too much
on the instrument to interpret
the group and not consider
the interpretation them
selves.
4. Less need for trained
scientists!
 Definition
• When forward and
backward typing does not
match
 Definition
1. Record of patient’s blood
type does not match
current test results
2. Weak agglutination in the
cell and/or serum typing
that does not match
strong(er) agglutination
patterns in the reverse or
cell typing
3. Suspicious or abnormal
looking agglutination in the
discrepant (cell or serum)
typing
4. All positive or all negative
 Types
1. Type I – unexpected
agglutination in forward
typing
2. Type II – lack of expected
agglutination in the
forward typing
3. Type III – unexpected lack
of agglutination in the
reverse typing
4. Type IV – lack of expected
agglutination in the
reverse typing
 Causes of TYPE I
1. Spontaneous agglutination due
to antibody coated patients
RBC’s (eg cold reactive
agglutinins)
2. Transplant chimerism
3. RBC polyagglutination
4. Genetic or “acquired” anomalies
(chimerism, acquired antigens)
5. Patient has antibody to a
component in the reagent
6. Abnormally high levels of serum
proteins or macromolecules
 Causes of TYPE II

1. Disease conditions (leukemia,

Hodgkin’s)
2. Weak Antigens (premature,
newborn)
3. ABO subgroup or some other
genetic anomaly (reduced #
of Ag sites, qualitative
differences in transferases)
4. Recent blood transfusion
5. Abnormally high
concentrations of A and/or B
substance in patient’s serum
which may neutralize ABO
typing reagent
 Causes of TYPE III
1. Contaminants in
patient’s serum
2. Abnormal serum protein
levels or elevated
fibrinogen levels (eg
MM, Waldenstrom’s
macroalbuinemia)
3. Unexpected
alloantibody in the
patient’s serum eg:
subgroup, cold reactive
 Causes of TYPE IV
1. Recent transfusion with
compatible but not
group specific plasma
(weakening of ABO
reactions)
2. Hypogammaglobulinemi
a (eg
immunosuppressive
drugs, congenital,
leukemia,
immunodeficiency
 Causes of TYPE IV
4. Bone marrow or
progenitor cell
transplants
5. Unusual inheritance
involving blood forming
tissue e.g. Inherited
chimerism
 Resolving
Discrepancies
GENERAL
1. Consult manufacturer’s
instructions and repeat
test procedure
2. Obtain a new patient
sample
3. Inspect sample
collection date and
determine its age
4. Determine or confirm
patient’s age
5. Obtain patient’s
 Resolving
Discrepancies
6. Obtain patient’s
transfusion history and
current status
7. Obtain patient’s RBC’s
with NSS
8. Determine if
polyagglutination is
present
9. Investigate the sample
for possibility of A or B
 Resolving
Discrepancies
10. Identify interference due to
cold antibody
11. Identify and characterize
ABO typing problems due to
polyagglutination
T & Tk – transient, due to
bact’l enz act
Cad – inherited, enhanced
Sda
Tn – somatic mutation in
hematopoietic clone cells,
may be persistent
 Resolving
Discrepancies
SPECIFIC
A-like Ag- treat with
proteolytic enzymes
Spontaneous Agglutination
due to Ab coated RBCs –
elute w/ warm saline,
treat w/ DTT
Acquired B Ag – treat w/
acetic anhydride, retype
using monoclonal rgts &
anti-serum, test
 Resolving
Discrepancies
Weak reactions – use
extended incubation;
proteolytic enzymes,
adsorption and elution
of anti-A and/or anti-B
from patient’s RBCs; use
secretions
 CLINICAL
SIGNIFICANCE
• HTR
• HDN