IMMUNOHAEMATOLOGY

WHAT IS AN ANTIGEN?
An antigen (Ag) is a substance or any molecule that
induces an immune response and is cable of binding to
the subsequently produced antibodies.

An antigen is often foreign or toxic to the body for

example bacteria or virus.
 WHAT IS THE IMMUNE SYSTEM?
Immune system is the biological structures and
processes within an organism that protects against
diseases by identifying and killing pathogen and tumor
cells.

It detects a variety of antigens and distinguish them

from the organism’s own healthy cells and tissues in
order to function properly.
 WHAT IS AN ANTIBODY?
An antibody (Ab), also known
as Immunoglobulin (Ig) , is a
“Y” shaped protein produced by
plasma cells that is used by the
immune system to identify and
neutralize antigens.

Each antibody recognizes a

specific antigen unique to its
target.
FUNCTIONS OF ANTIBODIES
BLOOD GROUP ANTIBODIES
DIFFERENCE BETWEEN IgG and IgM
IgG
1. Small molecule (150 KD)
2. Incomplete Ab
3. Clinical relevant
4. Can cross the placenta
5. Weak Agglutinins need
AHG
6. Reactive at warm
temperature (37oC)
7. React to RBC in protein or
potentiating medium
8. Weakly active the
complement
IgM
1. Big molecule (900 kD)
2. Complete Ab
3. Clinical less relevant (except
anti-A and anti-B)
4. Cannot cross placenta
5. Strong agglutinins (3-4+
reaction)
6. Reactive at cold temperature
4oC to room temp
7. React to cell suspended in saline
8. Fix/activate complement
 CLINICAL IMPORTANCE OF
BLOOD GROUP ANTIBODIES
 Hemolytic Transfusion Reaction (HTR)

Hemolytic Disease of the Fetus and

Newborn (HDFN)

Autoimmune Hemolytic Anemia

HEMOLYTIC DISEASE OF THE
NEWBORN
 HEMOLYTIC ANEMIAS
 Hereditary Spherocytosis  Thrombotic
Glucose-6- Phosphate Thrombocytopenic
dehydrogenase Purpura (TTP)
Deficiency Thrombocytopenia and
Most common enzyme
microgiopathic
defect in RBC hemolytic anemia, fever,
X-linked renal insufficiency,
Brisk hemolysis when neurologic symptoms
patient exposed to oxidative Schistocytes on smear
stress from drug, infections
or toxins
 HEMOLYTIC ANEMIAS
Autoimmune Hemolytic
 Hemolytic Uremic
Anemia
Syndrome
a. Warm antibody mediated
 Thrombocytopenia,
• IgG binds to RBC surface
microgiopathic hemolytic
• Most common
anemia, renal insufficiency
• Diagnosed by positive
coomb’s test
 Infections: Malaria,
• Can be cause by drugs
Babesiosis and sepsis
b. Cold Agglutinin Disease
 Trauma:
IgM antibodies bind to RBC
some snake, insect bites surface
ANTIBODY SCREENING
 AUTO-LOGOUS CONTROL
Auto-logous control is considered as part of the antibody
screening. It can be performed in parallel with antibody
screen and involves testing the patient’s serum against
the patient’s RBC.

A positive auto-logous control is an abnormal finding

and usually means that the patient has a positive DAT.
A PANEL RESULTS USING GEL CARD

AC

AT.19.001
Panel Red Cells
Group O red blood cells obtained from donors
 Panel

Red Cells
An autocontrol (AC) should also be run with ALL
panels

Autocontrol
Patient RBCs +
Patient serum
You have agglutination…now what?
CC


2+ 0 0

0 0 0

0 0 0

2+ 0 0

0 0 0
0 0 0 

2+ 0 0 

0 0 0

2+ 0 0
0 0 0 

0 0 0 

0 0 0

??
 GUIDELINES
Again, it’s important to look at:
 Autocontrol
 Negative - alloantibody
 Positive – autoantibody

 Phases
 IS – cold (IgM)
 37° - cold (some have higher thermal range) or warm reacting
 AHG – warm (IgG)…significant!!

 Reaction strength
 1 consistent strength – one antibody
 Different strengths – multiple antibodies or dosage
 GUIDELINES
Matching the pattern

 Single antibodies usually shows a pattern that matches

one of the antigens (see previous panel example)

 Multiple antibodies are more difficult to match because

they often show mixed reaction strengths
INTERPRETING ANTIBODY PANELS
There are a few basic steps to follow when
interpreting panels:
1. “Ruling out” means crossing out antigens
that did not react
2. Circle the antigens that are not crossed out
3. Consider antibody’s (from the circled)
usual reactivity
4. Look for a matching pattern
 ALWAYS REMEMBER:
An antibody will only react with cells
that have the corresponding antigen;
antibodies will not react with cells that
do not have the antigen
HERE’S AN EXAMPLE:
 1. RULING OUT

2+ 0 0

0 0 0

0 0 0

2+ 0 0
0 0 0 

0 0 0 

2+ 0 0 

0 0 0

2+ 0 0
0 0 0 

0 0 0 

0 0 0

Cross out antigens that show NO REACTION in any phase; do

NOT cross out heterozygous antigens that show dosage.
ABOUT REACTION STRENGTHS……
Strength of reaction may be due to “dosage”
If panel cells are homozygous, a strong reaction may
be seen
If panel cells are heterozygous, reaction may be weak
or even non-reactive
Panel cells that are heterozygous for an antigen
should not be crossed out because antibody may be
too weak to react
Blood groups which show dosage:
Kidd, Duffy, Rh(no D) and MNSs
2. CIRCLE ANTIGENS NOT CROSSED OUT

2+ 0 0

0 0 0

0 0 0

2+ 0 0
0 0 0 

0 0 0 

2+ 0 0 

0 0 0

2+ 0 0
0 0 0 

0 0 0 

0 0 0
3. CONSIDER ANTIBODY’S USUAL REACTIVITY


2+ 0 0

0 0 0

0 0 0

2+ 0 0
0 0 0 

0 0 0 

2+ 0 0 

0 0 0

2+ 0 0
0 0 0 

0 0 0 

0 0 0
Lea is normally a Cold-Reacting antibody (IgM), so it makes sense that
we see the reaction in the IS phase of testing; The E antigen will
usually react at warmer temperatures
4. LOOK FOR A MATCHING PATTERN
E doesn’t match and it’s
a warmer rxn Ab


2+ 0 0

0 0 0

0 0 0

2+ 0 0
0 0 0 

0 0 0 

2+ 0 0 

0 0 0

2+ 0 0
0 0 0 

0 0 0 

0 0 0

…Yes, there is a matching pattern!

Interpretation:
anti-
Lea
 RULE OF THREE
The rule of three must be met to confirm the presence
of the antibody
A p-value ≤ 0.05 must be observed
This gives a 95% confidence interval
How is it demonstrated?
Patient serum MUST be:
 Positive with 3 panel cells with the antigen, +ve reaction.
 Negative with 3 cells without the antigen and should not be

reacting.
 OUR PREVIOUS EXAMPLE
FULFILLS THE “RULE OF THREE”

2+ 0 0

0 0 0
3 Positive 
0 0 0
cells 
2+ 0 0
0 0 0 

0 0 0 

2+ 0 0 

0 0 0
3 Negative 
2+ 0 0
cells 0 0 0 

0 0 0 

0 0 0
Panel Cells 1, 4, and 7 are positive for the antigen and gave a reaction at IS
Panel Cells 8, 10, and 11 are negative for the antigen and did not give a reaction at IS
 WHAT IF THE “RULE OF THREE” IS NOT
FULFILLED?
If there are not enough cells in the
panel to fulfill the rule, then additional
cells from another panel could be used

Most good labs carry different lot

numbers of panel cells
FURTHER TESTING MAY INCLUDE:
Phenotyping- helps to confirm identification of alloantibody
by demonstrating lack of antigen

Adsorption- removing antibodies from sample by incubation

with antigen- positive RBCs

Elution- removal of RN}BC-bound antibodies (heat, cold,

chemical)

Proteolytic enzymes- may change Ag expression/Ab binding

(ficin and papain)
ALLOANTIBODY vs AUTOANTIBODY
ALLOANTIBODY- antibody directed against antigens
that the individual doesn’t possess:
May results from: Transfusion, Pregnancy and
injection of immunogenic materials (needle sharing)

AUTOANTIBODY- antibody directed against one’s

own antigens
 REFERENCES:
http://
ib.bioninja.com.au/higher-level/topic-11-animal-physi
ology/111-antibody-production-and/antibodies.html
https://en.wikipedia.org/wiki/Blood_type

http://slideplayer.com/slide/7249985/
THE END
ANY QUESTION??