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IMMUNOHEMATOLOGY
CHAPTER FOUR
INTRODUCTION TO IMMUNOHEMATOLOGY
 Refersto immunologic reactions
involving blood components

 It
deals with the concepts and clinical
techniques related to modern
transfusion therapy

 Is
the analytical process of finding
antigens on the cells of blood by
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principle of antibody binding to antigen.
INTRODUCTION TO IMMUNOHEMATOLOG……..

 It is an application of the principles of immunology

to the study of
o red cell antigens and
o their corresponding antibodies in blood for
resolving the problems of blood transfusions.

 ANTIGEN:Is a substance that stimulates immune

system to produce antibody against it. It may be
part of the host cell
 ANTIBODY: is a protein also known as

immunoglobulin, that help to remove foreign 3

invaders or antigens.
HISTORICAL BACKGROUND

 The era of blood transfusion began

when William Harvey described the
circulation of blood in 1616.

 In 1665, Richard Lower,

successfully performed the first
animal-to-animal blood transfusion 4
HISTORICAL BACKGROUND…

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 In1667, jean Bapiste Denys transfused, blood
from the carotid artery of a lamb into the vein

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of a young man, which at first seemed
successful using animal blood, but they were
unsuccessful.

 Later,
it was found that it is impossible to
successfully transfuse the blood of one species
of animal into another species.
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 HISTORICAL BACKGROUND…

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 Transfusions were prohibited from 1667 to 1818
Due to the disastrous consequences resulting.
 In 1818, James Blundell of England

successfully transfused human blood to women

suffering from hemorrhage at childbirth.

 Suchspecies-specific transfusions seemed to

work sometimes but mostly the result was
death. 6
 HISTORICAL BACKGROUND…
Karl Landsteiner

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o discovered the ABO blood groups in

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1900,
o introduced the immunological era of
blood transfusion
 It became clear that the incompatibility of
many transfusion was caused by the
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presence of certain factors on red blood
HISTORICAL BACKGROUND…
 From the discovery of scientists, two main postulates

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were drawn:

1. Each species of animal or human have certain

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factors on the red cells that are unique to that
species, and

2. Each species have some common and some

uncommon factors to each other
 This landmark event initiated the era of science
based transfusion therapy and was the
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foundation of immunohematology as a science
BLOOD GROUP GENETICS

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 Concerned with the way in which the

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different blood groups are
inherited
 Genes code for different inherited physical
characteristics, including blood groups

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BLOOD GROUP GENETICS…
Genotype versus phenotype

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 Phenotype
o Physical expression of inherited traits,

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o Determined by reacting red cells with known
antibody
 Genotype
o Actual genes inherited from each parent
o Family studies are required to determine the actual
genotype
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TABLE 1: THE ABO PHENOTYPES AND
THEIR CORRESPONDING GENOTYPES

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Phenotype Genotype

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A AA, AO

B BB,BO

AB AB

O OO
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HOMOZYGOSITY &
HETROZYGOSITY

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Homozygous

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o The Genotype is made up of identical
genes, such as AA, BB, or OO

Heterozygous
o The Genotype is made up of different
alleles from each parent, such as AO, AB,
or BO
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 BLOOD CELL ANTIGENS

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Red blood cell antigens
 A unique set of red blood cell Ag is determined through
genetic inheritance

 These antigens project from the surface of the RBC in

three dimensional configurations
o As a result, they are accessible to Antibody molecules
for agglutination reaction

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BLOOD GROUP ANTIBODIES & THEIR
STIMULATION

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Blood group antibodies are classified into:

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o Natural and
o Immune antibodies
Natural / Non Red Cell Immune Antibodies
o Are RBC Abs in the serum of an individual that
are not provoked by previous RBC
sensitization
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 IMMUNE ANTIBODIES
 Produced due to previous antigenic stimulation either
by transfusion or pregnancy

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 Mainly IgG type
 Optimally react at 370C: warm agglutinins
 Causes more serious transfusion reactions than the
naturally occurring ones
 Can cross the placental barrier
Antigen - Antibody Interactions
 This union complies with the principles of a chemical
reaction that has reached equilibrium
 When Ag and Ab combines, an immune complex is 15

produced
 THE ANTI-SERUM
 It is a highly purified solution of antibody

 Used to determine a person’s blood type,

 A substance that must be available to show what
antigens are present on the red cell

 The substance used for this purpose is referred to as

anti serum
 Named on the basis of the antibody it contains
For Example: 16

o Solution of Anti-B antibodies is called anti –B antiserum

IN VITRO DETECTION OF ANTIGEN
AND ANTIBODY REACTION

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 The presence of In vitro antigen and

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antibody interaction can be detected
by:
 Hemolysis

 Precipitation

 Agglutination (Most commonly used)

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 THE ABO BLOOD GROUP SYSTEM
General Characteristics of the ABO Antigens
ABO antigens are widely distributed and located on

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 red blood cells
 lymphocytes

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 platelets
 tissue cells
 bone marrow, and
 organs such as the kidneys
 Soluble forms of the ABO antigens can be
o synthesized and secreted by tissues & cells
o found in association with cellular membranes and
o Found in all body fluids except CSF
o Glycoproteins 18
ABO SYSTEM ANTIBODIES
 Present in individuals with no known exposure to

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blood or blood products
 Are not detected in the serum of new born until 3 to
6 months of age

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 There are 2 types of ABO antibodies: Anti-A & Anti-B.
 General Characteristics of Human Anti- A and

Anti – B
 Anti- A and anti- B are found:
o In group B, group A & group O individuals
o are primarily IgM with small amounts of IgG
o In group O individuals they are primarily
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composed of IgG class

 ABO GROUPING
 ABO grouping is based on the presence or
absence of blood group antigens(A & B
antigens) on the red blood cells.
 Group A individuals have A antigens on their

red cells.
 Group B individuals have B antigens on their

red cells.
 Group AB individuals have both A and B

antigens on their red cells.

 Group O individuals have neither A nor B

antigens on their red cells. 20

 ABO GROUPING…

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Principle
When an antigen is mixed with its

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corresponding antibody under the right
conditions it causes agglutination or
haemolysis of the red cells.
Clinical significance of ABO grouping
 For safe blood transfusion
 Prevention of Hemolytic Disease of the 21

Fetus and Newborn (HDFN)

 ABO GROUPING…
ABO phenotyping has two components:

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1. Forward grouping
2. Reverse grouping

Forward grouping
 Red cell antigens of a person are identified by
mixing a person’s blood with typing serum (Anti-
A and Anti-B) and looking for agglutination.
 This is called forward or direct grouping.

 If a person’s red blood cells agglutinate (clump)

when mixed with a typing antiserum(i.e anti A),
they have that antigen(A antigen) on their red 22
blood cells.
 ABO GROUPING…

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Anti-A Serum Anti-B Serum Blood group
No
Agglutination
agglutination A

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(+)
(-)
No
Agglutination
agglutination B
(+)
(-)
Agglutination Agglutination
AB
(+) (+)
No No
agglutination agglutination(- O
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(-) )
ABO GROUPING…

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Pictures of ABO Blood Grouping/Typing

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 ABO GROUPING…

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Reverse grouping
 Humans have naturally occurring ABO
antibodies in their plasma or serum

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against blood antigens they do NOT
have.

 Forexample: Group A individuals have

Anti-B antibodies; Group B individuals
have Anti-A antibodies.

 Detectionof these antibodies is called

reverse or indirect grouping. 25
 ABO GROUPING…

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 Reverseor indirect blood grouping&
presence of Anti-A & Anti-B antibodies:

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Blood Anti-A Anti-B
Group Abs Abs
A No Yes
B Yes No
AB No No
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O Yes Yes
ILLUSTRATION OF THE FORWARD AND
REVERSE GROUPING REACTION PATTERNS
OF THE ABO GROUPS

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 ABO GROUPING…

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 Group O packed red blood cells are
sometimes referred to as the “Universal
Donor”.

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 In an emergency, it can be given to a person
with any blood type because the red cells do
not have antigens against which the patient’s
plasma antibodies might react.

 The blood is “packed”, so that it does not

have plasma antibodies.
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 ABO GROUPING…

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 GroupAB is sometimes referred to as the
“Universal Recipient”.

 In
an emergency, individuals with AB blood
group can receive any blood type because
they do not have any plasma antibodies to
react against A or B antigens.

 However, it is always best to give the

patient group or type specific blood. 29
 THE RH BLOOD GROUP SYSTEM
Historical Background of Rh Blood Grouping
 In 1940 Landsteiner & Wiener reported the discovery

of a human blood factor, which they called Rhesus

factor.
 They immunized guinea pigs and rabbits with blood

from the Macacus rhesus monkey, and the antiserum

obtained agglutinated not only the red cells of the
rhesus monkey but also 85% of human blood.
 They realized that this serum which they called anti-

Rh was about detecting an unknown human blood

group antigen, which was independent of all other
blood groups discovered before that time.
 They used it to type those donors whose red cells

were agglutinated by the new anti-Rh antibody as Rh

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positive, and those whose red cells were not
 HISTORICAL BACKGROUND…..
 This discovery followed the detection of an
antibody by Levine & Stetson in 1939.
 This antibody occurred in the serum of a woman

who delivered a stillborn fetus.

 The woman suffered a hemolytic reaction to her

husband’s ABO compatible blood transfused

shortly after delivery.
 The antibody was found to agglutinate

approximately 80% of randomly selected ABO

compatible donor’s and latter was shown to be
anti-Rh in specificity.
 Levine and Stetson also postulated that the

antibody had arisen as the result of 31

immunization of the mother by a fetal antigen
which had been inherited from the father.
 RH FACTOR

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 Rh is another antigen that individuals may
have on their red blood cells.

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 Another name for Rh factor is D antigen.
 If a person has the Rh factor (D antigen) on
their red blood cells, they are called Rh
positive.

 If a person does NOT have the Rh factor (D

antigen) on their red blood cells, they are
called Rh negative.

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 RH FACTOR

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 When a person is blood grouped or
typed, Rh is always determined, in
addition to ABO grouping.

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 Rh (D) grouping is also performed
using anti-serum (anti-D), which is
mixed with patient’s blood.

 Ifthere is agglutination, the person is

called Rh positive; if there is NO
agglutination, the person is called Rh
negative. 33
COMPATIBILITY (PRETRANSFUSION) TESTING
 Purpose: To select blood components that

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will not cause harm to the recipient and
will have acceptable survival when
transfused.

 When performed properly, pretransfusion

tests will confirm ABO compatibility
between the donor and the recipient, as
well, it will detect most clinically
significant unexpected antibodies.

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PRE-TRANSFUSION TESTING

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1.Donor & recipient basic testing
A. Donor Basic Testing

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 ABO Grouping: Forward and Reverse
 Rh typing: Complete with Weak D
 Blood borne disease testing: HIV, HCV, &
HBV, syphilis
B.Recipient Basic Testing:
 ABO Grouping: Forward and Reverse
 Rh typing

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PRE-TRANSFUSION
TESTING….CONTINUED
2. Cross-matching
 Is a test to determine the compatibility b/n
recipient’s blood and donor’s blood
 A procedure will be performed before
transfusion
o To select donor’s blood that will not cause any
adverse reaction, (hemolysis /agglutination)
 The procedure helps the patient to receive
maximum benefit from the transfusion of red cells

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 Cross-matching Cont…

 Main purpose is to prevent a transfusion

reaction
 Transfusion reaction may resulted due to:
o Unexpected antibodies in the patient’s
/donor’s serum
o ABO incompatibilities and
o Technical or labeling errors (clerical errors)

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Cross-matching Cont…

Transfusion reaction –
 Is the term used to describe the clinical effects
caused by the destruction of:-
o RBCs of recipient by the donor’s antibodies
or
o RBCs of donor by the recipient antibodies
during an incompatible transfusion

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 2 Types of Cross-match

Major and Minor Cross-Match

Major Cross-Match:
 Involves mixing recipient’s serum with the
donor’s red cells
 Is much more critical for assuring safe transfusion
than the minor compatibility test
 Called major b/c the Abs in the recipient’s serum are
most likely to destroy the donor’s RBC

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 Types Cont….

Minor Cross-Match:
 Involves mixing the donor’s serum with recipient’s
red cells
 Called minor because
o Any Ab in the donor’s serum will be diluted by the
large volume of the recipient’s blood
o The destructed RBCs of the patient may be
compensated by the transfused RBC of the donors

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SELECTION OF BLOOD FOR CROSS
MATCH

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 Generally,when whole blood is to be
transfused, the blood selected for

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cross- match should be of the same
ABO and Rh (D) group as that of the
recipient.

 However, Rh positive recipients may

receive either Rh positive or Rh
negative blood.
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HEMOLYTIC TRANSFUSION REACTIONS

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 Immediate intravascular hemolytic Transfusion reactions

 Delayed hemolytic transfusion reactions

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Intravascular Hemolytic TX RXS
 Usually due to ABO incompatibility
 IgM complement-binding antibodies
 Ab to Rh can cause IHTR
 Lysis of transfused RBCs is usual scenario
 Rarely, lysis of recipient RBC due to Tx of plasma
containing antibodies (anti-A)
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