Immunohematology and Blood Banking Techniques Class Notes

IMMUNOHEMATOLOGY
&
BLOOD BANKING TECHNIQUES
Dr MANOJ K PATRO, MD [PATH]; MKCG MC BRAHMAPUR
INTRODUCTION:
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IMMUNO
HEMATO
IMMUNOHEMATOLOGY
LOGY
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IMMUNOLOGY:
Study of the immune system

and the immune response.
Study of antigen-antibody
reactions in vivo.
SEROLOGY:
Study of antigen-antibody
reactions in vitro.
HEMATOLOGY:
Study
of
the
cellular
components of the blood.
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IMMUNITY:
Protection against infection or

disease caused by foreign
particles.
DEFINITIONS:
Three functions:
Defense
Homeostasis
Surveillance
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The Immune System
The Immune System
Markers of Self
Markers of Non-Self
Markers of Self:
Major Histocompatibility Complex
Organs of the Immune System
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Components:
Lymphocytes: T-cells, B-cells & NK cells.

Phagocytic cells: N, E, B, Monocytes,
Macrophages, Dendritic cell, etc.
Chemical mediators:
Complement system.
Chemokines.
Cells:
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Cells of the Immune System
B Cells
Antibody
Immunoglobulins
Antibody Genes
T Cells
Cytokines
Killer Cells: Cytotoxic Ts and NKs
Phagocytes and Their Relatives
Phagocytes in the Body
Complement
Immunity: Active and Passive
IMMUNOHEMATOLOGY:
DETECTION,
IDENTIFICATION, and/or
QUANTITATION of antibodies involved primarily with

red cells [although white cells and platelets may also
be involved].
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DEFINITIONS:
Basically this branch of science related with red cell

antigens and their corresponding antibodies.
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BLOOD BANK:
Work primarily with patient's

blood.
Primary
areas
of
responsibility:
Blood typing.
Antibody
detection
and
identification.
Compatibility
testing
(crossmatching).
Blood component therapy
(hemotherapy).
Transfusion
reaction
workups.
Autoimmune
hemolytic
anemia workups.
Hemolytic disease of the
newborn (HDN) workups.
Determining
Rh
immune
globulin eligibility.
Works
primarily
with
donor blood.
Major
areas
of
responsibility:
Donor recruitment.
Donor screening.
Blood collection.
Testing
(typing,
infectious
disease
screening).
Blood
component
preparation.
Component preservation.
May provide reference
lab services.
May store rare donor
blood.
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TRANSFUSION SERVICE:
IMMUNOHEMATOLOGY FACILITIES:
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HISTORICAL OVERVIEW
26
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1665: Richard Lower, English Physiologist 1st animal-toanimal [dog to dog] blood transfusion.
1667: Jean Bapiste Denys Unsuccessful transfusion of

animal-to-human [sheep to human] blood transfusion.
1667 1818: Transfusions prohibited.
1818: James Blundell of England 1st successful human-tohuman blood transfusion. This species specific transfusion
had 50% success rate, the rest resulted in death.
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1616: Sir William Harvey Described circulation of blood.
1900: Karl Landsteiner Discovered the ABO blood

groups.
27
Described the ABO
Blood Groups.
Karl Landsteiner:
28
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Discovered
that
the
incompatibility
of
many
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Karl Landsteiners discovery:
now known as Antigens.
Postulated two things:
Each species has unique species specific factors on

red dells.
Even in each species has some common and some

uncommon factors to each other.
Introduced
the
Immunological
era
of
blood
transfusion was due to certain factors on red cells
transfusion began the era of scientific based
transfusion
therapy
foundation
IMMUNOHEMATOLOGY as a science.
of
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system.
1939,40: Levine, Stetson, Landsteiner and Weiner

discovers Rh system and its role in erythroblastosis
foetalis (HDN).
1946-Kell system discovered by Coombs, Mourant and

Race.
1950-51: Duffy, Kidd, Lutheran system discovered.
Landsteiner and Alexanders lead to the discovery of

>800 Blood group systems.
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1927: Landsteiner and Levine discovered M,N and P
30
ANTIGENS:
31
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ANTIGEN is a substance that either
combines with an antibody or
is processed and binds to a T lymphocyte to
stimulate an IMMUNE RESPONSE.
IMMUNOGEN - An antigen that stimulates an immune

response.
HAPTENS - small chemical substances that must be
bound to a larger molecule to provide sufficient

molecular
weight
for
stimulation
of
antibody
production.
Proteins
Complex
Lipopolysaccharides.
carbohydrates
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PROPERTY
Foreignness
Size
Chemical
composition
Complexity
DESCRIPTION
Non-self more likely to stimulate
antibody production
>10,000 M.W.
Proteinbest immune response.
Complex carbohydratesecond best
immune response.
Lipids, Nucleic acid weak immune
response.
More complex molecules produce
better immune response.
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Properties of Molecules that Contribute to

Immunogenicity
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Some antigens protrude from the cell surface, while
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Antigen location:
Physical location impacts antibody stimulation as well
as the physical ability of the antigen to react with an

antibody once it is produced.
Red Blood Cell Antigens:
Red blood cell antigens and corresponding antibodies

provide the foundation for blood bank testing.
More than 20 blood group systems that contain
others are an integral part of the membrane.
greater than 200 red blood cell antigens.
ABO and Rh antigens are matched between donor and

recipient.
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ANTIBODIES
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Produced in response to stimulation with an antigen.
Specific for the stimulating antigen and will react with

that antigen.
IMMUNOGLOBULIN 5 classes, IgG, IgM, IgA, IgD &

IgE.
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Protein.
36
Primary immunological components are antigens and

antibodies.
Cardinal rule for antigens and antibodies [blood

bank]: Antigens on RBC surfaces & Antibodies in
serum / plasma.
Primary & Secondary Immune responses:
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Immunological principles:
37
Each antibody reacts with the antigen that stimulated its

production.
Bonding:
Noncovalent bonds.
Physical fit:
The fit of the antigen and antibody depend on compatible

shapes that allow the antigen and antibody to physically
touch - a lock and key mechanism.
Concentration of
antigen
and
antibody:
Antigens and antibodies must be present in optimal

concentrations; excess antibodies will result in a situation
known as prozone phenomenon.
Temperature:
Optimal temperature of reactivity for a specific antibody will

expedite the combination of antigen and antibody.
Time:
Incubation time must be that which is optimal for the specific

antibody. General guidelines are a range of 1560 minutes for
optimal antigen-antibody attachment.
pH:
A pH range of 7.27.4 is maintained for most antigenantibody reactions.
Surface charge:
A net negative charge known as zeta potential surrounds the

red cells. The reduction of this charge influences the ability
of antigen and antibody to combine.
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Specificity:
Antigen-Antibody Reactions: Factors influencing:
38
LOCK & KEY
AGGLUTINATION
CONCENTRATION
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2 methods:
Agglutination.
Hemolysis.
Precipitation.
Agglutination involves a particulate antigen or an antigen that
is attached to a particle (such as a red blood cell).
Agglutination occurs in when:

1.
An antibody molecule attaches to a single antigen on a

single cell with one antigen-binding site.
2.
The free arm of the immunoglobulin molecule attaches

to an antigen on a second red cell. This creates a
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Visualization of Ag-Ab reaction in BB:
crosslink.
3.
Multiple cross links create a lattice.
4.
The lattice is visualized as agglutination.
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Grading of Agglutination:
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42
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BLOOD GROUP GENETICS
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Chromosomes & Genes:
44
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45
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Genetics: Study of Inheritance.
Inheritance of transmissible characteristics or

traits: such as blood group antigens found on red
blood cells.
Each parent
information.
contributes
1/2
of
The
genetic
information
is
on chromosomes composed of DNA
the
genetic
contained
Humans
have
23
pairs
of
chromosomes
a.
22 matched (autosomal) chromosomes and
b. 1pair of sex chromosomes.
System
ABO
Rh
Common Genes
A, B, O
D, C, E, c, e
Located on
Chromosome
9
1
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Basic Principles:
47
Genes
are
the
units
of
inheritance
within
the
chromosomes.
Alleles: At each loci, different forms of the genes. E.g.

ABO Blood Group System - A1, A2, B, and O as common
alleles.
Genotype: Genetic composition for a particular trait.
Homozygous: When the two inherited alleles are
the same. E.g. OO / AA / BB.
Heterozygous: when the two inherited alleles are

different. E.g. AO / AB / BO.
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Basic Principles:
48
Phenotype: The expression of a genotype.
Dominant: The dominant gene will express itself if

present
both
in
homozygous
as
well
as
heterozygous state. E.g. Rh (D).
Co-dominant: Both the alleles express. E.g. A & B.
Recessive: They express in homozygous state only.
Amorph: No gene product. e.g. O.
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Basic Principles:
49
Dosage: In some blood group systems, persons

homozygous for an allele have MORE antigen on their
red cells than persons heterozygous for an allele.
Variation in antigen expression due to the number of

alleles present is called DOSAGE.
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Basic Principles:
50
BLOOD GROUP SYSTEMS
51
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In 1901, Karl Landsteiner used his blood and the

blood of his colleagues:
Mixed the serum of some individuals with cells of

others.
Discovered three groups A, B & O.
His colleagues discovered the 4th group AB.
LANDSTEINERS LAW / RULE:
ABO antigens on red cells and the reciprocal

agglutinating antibodies in the serum of the same
individual (e.g. A antigens on red blood cells and
anti-B in the serum).
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HISTORICAL PERSPECTIVE:
52
ABO & H SYSTEM ANTIGENS:
53
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Present on RBC surface.
Also
on
lymphocytes,
thrombocytes,
endothelial cells, and epithelial cells.
Detectable at 5 to 6 weeks of gestation.
Newborns - weaker antigens.
Fully developed by two to four years of age.
organs,
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ABO ANTIGENS:
54
One factor contributing to the difference in ABO

antigen strength between newborns and adults is the
number of branched oligosaccharides.
Adults
greater
numbers
of
branched
chains
compared to newborns - more linear chains.
The branched chains permit attachment of more

molecules to determine antigen specificity.
Phenotype
Number of Ag sites
A1 adult
8,10,000 to 1,170,000
A1 cord
2,50,000 to 3,70,000
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ABO ANTIGENS:
55
Simple
Mendelian
fashion
from
an
individuals
parents.
Each individual possesses a pair of genes.
FOUR genes H, A, B & O.
Hh Chromosome 19 HH / Hh / hh.
hh Bombay phenotype Oh.
A, B & O - Chromosome 9.
A and B genes produce a detectable products while
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INHERITANCE:
the O gene is an amorph.
The expression of the A and B genes is codominant.
56
Gene Combination
Phenotype
AO
AA
BO
BB
AB
AB
OO
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INHERITANCE:
57
GENE
TRANSFERASE
-L-fucosyltransferase
-3-N-acetyl-D-galactosaminyl
Transferase
-3-D-acetyl-D-galactosyl
Transferase
No Transferase.
Basic common core structure - an oligosaccharide chain attached

to either a protein or a lipid molecule present on cell surface.
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GENE PRODUCTS & BIOCHEMICAL COMPOSITION

OF BLOOD GROUP SUBSTANCES:
58
The L-fucose is the immunodominant sugar for the H
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GENE PRODUCTS & BIOCHEMICAL COMPOSITION

OF BLOOD GROUP SUBSTANCES:
antigen.
The H antigen serves as a precursor for A and B

antigens.
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SECRETOR STATUS
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Nonsecretors:
Saliva, Sweat, Tears, Semen, Serum & Amniotic fluid.
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Secretors:
Ability to secrete ABH antigens in

body secretions.
Chromosome 19: Se / se [amorph].
Gene product L- fucosyltransferase.
A & B transferases are found in the
secretions of A / B persons regardless
of their secretor status.
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SUBGROUPS OF A & B
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2 major subgroups:
~ 80% A1
~ 20% A2.
2 major subgroups of AB:

~ 80% A1B
~ 20% A2B.
Group A1
Group A2
Qualitative differences:
Reaction with Anti-A in Forward

Grouping
4+
4+
Number of Antigen Sites-Adults
10,00,000
2,50,000
Number of Antigen SitesNewborn
3,00,000
1,40,000
Quantitative differences:
Reaction with Anti-A1
Positive
Negative
Anti-A1 in serum
Absent
? Present
-3-N-acetyl-D-galactosaminyl
Transferase Activity
Normal
Diminished
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Subgroups of A:
63
branched chain oligosaccharides than A2.
2 mutations Pro156Leu / Single nucleotide deletion

1060 reduced enzyme activity of A2.
Routine antisera NO DIFFERENCE in reaction.
The LECTIN Dolichos biflorus is used to obtain an

extract with anti-A1 specificity.
A2 individuals can develop antibodies to the A1

antigen.
Additional A subgroups: Aintr, A3, Ax, Am, Aend, Ael,and

Abantu Fewer antigenic sites on their surface.
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A1 more antigens on the cell surface more
64
Subgroups of B are very rare and encountered less

frequently than subgroups of A.
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Subgroups of B:
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ABO ANTIBODIES:
66
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Antibodies directed against ABO antigens are the

most important antibodies in transfusion medicine.
It is the only example of a blood group where each

individual
produces
antibodies
to
present on the red cells.
Newborns have NO ABO ANTIBODIES.
antigens
not
67
indicates BLOOD GROUP OF THE MOTHER.
The child will begin antibody production and have a

detectable titre at 3 6 months of age peaks at 5
10 years of age.
Originally thought to be NATURAL ANTIBODIES

formed with no antigenic stimulus.
Proposed mechanism some naturally occurring

substances resemble A & B antigens and stimulate
production of complementary antibodies.
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REVERSE GROUPING of Cord blood / Newborn serum
68
Age related:
Immunodeficient
individuals:
Immunosuppresse
d patients:
Newborns and young infants

Elderly individuals
Congenital conditions
Congenital hypogammaglobulinemia
Congenital agammaglobulinemia
Immunosuppressive therapy
Chronic lymphocytic leukemia
Bone marrow transplant
Multiple myeloma
Acquired hypogammaglobulinemia
Acquired agammaglobulinemia
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Conditions with decreased levels of ABO antibodies:
69
ABO antibodies ISOAGGLUTININS Saline agglutinins
with optimal reactivity at 40C.
Mostly IgM.
IgG & IgA.
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Immunoglobulin class:
70
Group O do not have A / B antigens.
Produce anti-A, anti-B and anti-AB.
anti-AB cross-reactive antibody reacts with a

common molecular structure in both antigens.
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Anti AB:
71
As per Landsteiners Law, group B and O individuals
produce anti-A.
This
anti-A
can
be
separated
procedures - anti-A and anti-A1.
by
absorption
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Anti-A1:
72
ABO HDFN:
Rh HDFN:
Affects 1st pregnancy.

MC: O mother with A
baby.
Sensitization occurs in
1st pregnancy and
affects
subsequent
positive pregnancies.
Rh negative mother
Rh positive baby.
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Cause both
HDFN Hemolytic disease of fetus and new born &
HTR Hemolytic transfusion reaction.
HDFN: usually presents itself with a maternal IgG
antibody to an antigen on the surface of the babys
red cells.
Clinical significance of ABO antibodies:
73
Occurs when a recipient is transfused with red cells

that
are
an
ABO
group
incompatible
with
the
antibodies in his or her serum.
Because of the complement-binding ability of the ABO

antibodies, this is always a life-threatening situation.
As
the
recipient
antibodies
react
with
the
incompatible red cells, complement is activated and
in vivo hemolysis, agglutination, and red blood cell

destruction occurs.
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Hemolytic transfusion reaction:
74
RH BLOOD GROUP SYSTEM
75
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One of the most polymorphic and antigenic blood group
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INTRODUCTION:
2nd only to ABO in importance in:
Blood transfusion &
A primary cause of HDFN.
The principal antigen is D and the terms Rh positive or

Rh negative refers to presence / absence of this antigen.
Other 4principal antigens are C, c, E and e.
50 other rare antigens detected.
Rh negative phenotype common in Caucasians 15 17%.
95% Indian population: Rh Positive.
systems.
76
Chromosome 1.
2 genes: RhD & RhCE.
The proteins encoded by these 2 differ by 32 to 35
AAs. That is why RhD is so antigenic in Rh negative

persons.
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GENES:
77
Discovered in the 1940s.
3 systems:
Fisher & Race: 3 closely linked genes D at one

locus, C / c at the 2nd locus & E / e at the 3rd locus.
Modified Weiner terminology: Supposes a single

gene.
ISBT terminology: Assigns each antigen a number

D: Rh1, C: Rh2, E:Rh3, c:Rh4 & e:Rh5.
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NOMENCLATURE:
78
Very antigenic D+ for presence / D- for absence.
Variants: due to a point mutation causing single AA

differences.
1.
2.
3.
Weak D [formerly Du, obsolete now]: 1 to 57

types: Type 1 to 3 90% cases.
Del: Not detected by routine testing but requires
adsorption-elution studies / molecular RHD
genotyping.
Partial D: Due to hybrid genes portion of RHD is

replaced by corresponding portion of RHCE gene.
The RBCs type D+ve but make anti-D following
transfusion / pregnancy.
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D ANTIGEN:
79
No Rh system antigens.
2 pathways:
Rh-negative person (lacking RHD) who also has an

inactive RHCE gene, referred to as an Rhnull amorph.
More often, inheritance of inactive RHAG gene,

referred to as an Rhnull regulator. RhAG protein is
required for expression and trafcking of RhCE and
RhD to the RBC membrane.
The serum of the people who form these antibodies

agglutinates cells from all people except another
Rhnull.
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Rh null:
80
Principally RBC stimulated.

Most are of the IgG class, usually the IgG1 or IgG3 subclass.
Can cross placenta.
May occur in mixtures with a minor component of IgM.

The antibodies usually appear between 6 weeks and 6
months after exposure to the Rh antigen.
IgG Rh system antibodies react best at 370C and are
enhanced when enzyme-treated RBCs are tested.
D is the most immunogenic of the common Rh antigens,
followed in decreasing order of immunogenicity by c, E, C,
and e.
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Rh ANTIBODIES:
81
30% to 85% of D-ve persons who will make anti-D following

exposure to D+ve RBCs Responders.
LABORATORY DETERMINATION OF THE

ABO SYSTEM
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83
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RBC
Glucose
Precursor
Substance
(stays the
same)
Galactose
N-acetylglucosamine
Galactose
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RBC PRECURSOR STRUCTURE
84
RBC
Glucose
H antigen
Galactose
N-acetylglucosamine
Galactose
Fucose
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FORMATION OF H Ag
85
RBC
Glucose
Galactose
N-acetylglucosamine
Galactose
Fucose
N-acetylgalactosamine
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FORMATION OF A Ag
86
RBC
Glucose
Galactose
N-acetylglucosamine
Galactose
Fucose
Galactose
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FORMATION OF B Ag
87
88
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ANTISERUM
89
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solution containing known antibody, which is used

to know the presence or absence of antigen on cells
and to phenotype ones blood group.
Antiserum is named on the basis of the antibody it

contains:
Antisera
Antibody present
Anti-A
anti-A
Anti-B
anti-B
Anti-AB
anti-A & anti-B
Anti-D
anti-D
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An antiserum is a purified, diluted and standardized
90
MONOCLONAL ANTIBODIES
Animal inoculation.
Serum from an individual who has been sensitized to

the
antigen
through
transfusion,
pregnancy
injection.
Serum from known blood group persons.
or
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Sources:
91
Antiserum must meet certain criterias to be acceptable for
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Criterias:
Qualities of a good antisera:
Specific: does not cross react, and only reacts with its
own corresponding antigen,
Avid: the ability to agglutinate red cells quickly and
strongly and,
Stable: maintains it specificity and avidity till the expiry

date.
It should also be clear [as turbidity may indicate bacterial

contamination] and free of precipitate and particles.
use.
It should be labelled and stored properly.
92
The
observable
combination
of
reactions
a
red
resulting
cell
antigen
from
with
the
its
corresponding antibody are agglutination and/ or
haemolysis.
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Manifestation of Ag-Ab reaction:
93
Is the clumping of particles with antigens on their surface,
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Agglutination:
bridges between the antigenic determinants.
Hemagglutination.
Agglutinogen antigen.
Agglutinin antibody.
Two stages:
1.
Sensitization: Abs
attach
to
the
Ags
on
RBC
Sensitized RBC.
2.
such as erythrocytes by antibody molecules that form
Agglutination: Ab binding to Ag on >1 RBC Lattice

formation.
94
Ab Hemolysin.
Complement mediated lysis of the RBC membrane

with release of Hb to stain the plasma.
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Hemolysis:
95
Zeta potential keeps RBCs 25 nm apart.
IgG Ab max span 14 nm so can only bind to Ag

and sensitize them [can not cause agglutination] in
saline media.
IgM Ab larger and pentameric can bridge a wider

gap cause agglutination.
2.
pH: Optimum pH 7.0.
3.
Temperature:
Optimum
temperature
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Ab size:
1.
Right condition for the RBCs to agglutinate / hemolysis:
varies
depending upon Ab type: IgG 370C, IgM 4 220C.
96
Low ionic strength increases agglutination.
LISS 0.2% NaCl in 7% glucose is used.

Number of Ag sites:
5.
Seen that IgG Abs of Rh system fail to agglutinate

RBCs suspended in saline where as IgG Abs of ABO
system can agglutinate because number of ABO
sites are 100 times more in D sites.
6.
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Ionic strength:
4.
Centrifugation: at high speed attempts to overcome

the problem of distance in sensitized cells by
physically forcing the cells together.
97
Enzyme treatment:
Treatment with weak proteolytic enzymes [Trypsin /

Papain] removes surface sialic acid residue on RBC
lowers zeta potential promotes agglutination.
Has a disadvantage destroys some blood group Ags.
Colloidal suspension: [Bovine albumin]
8.
Can reduce the zeta potential helps IgG Abs of Rh

system to agglutinate.
Ratio of Ag & Ab:
9.
Excess Ab Prozone phenomenon Use serial dilutions
7.
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of the antisera.
Avoid heavy RBC suspension as it may mask the presence

of a weak Ab.
98
ABO GROUPING TECHNIQUES
99
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METHODS:
100
Anti-A antibodies.
Anti-B antibodies.
Anti-AB antibodies (optional).
Group A & B RBCs.
Slides, or Test tubes.
Wooden applicator.
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REAGENTS:
101
Verify
that
patient
information
on
the
sample
matches information on the worksheet.
Centrifuge the sample and remove the serum to a

labelled tube.
Prepare a washed 2 - 5% RBC suspension.
Use Patient cell suspension in Forward typing.
Use
Patient
serum
for
confirmation
Reverse
grouping or backtyping.
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IMPORTANT THINGS TO FOLLOW:
At the End, Discard all materials in the isolation

trash containers.
102
1. Perform all tests according to the manufacturers
direction.
2. Always label tubes and slides fully and cleanly.
3. Do not perform tests at temperature higher than
room temperature.
4. REAGENT ANTISERA SHOULD BE TESTED DAILY

WITH ERYTHROCYTES OF KNOWN ANTIGENICITY.
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ABO GROUPING: RULES FOR PRACTICAL WORK:
This eliminates the need to run individual controls

each time the reagents are used.
103
5. Do not rely on colored dyes to identify reagent

antisera.
6. Always add serum before adding cells.

7. Observe for agglutination against a welllighted
background, and record results immediately after
observation.
8. Use an optical aid to examine reactions that appear
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ABO GROUPING: RULES FOR PRACTICAL WORK:
to the naked eye to be negative.

104
Important to the accuracy of testing in the blood

bank.
Can be prepared directly from anticoagulated blood

or from packed red cell (after separating the serum
or plasma).
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ABO GROUPING: PREPARATION OF RBC SUSPENSION:
105
Place 1 to 2ml of anticoagulated blood in a test tube.

Fill the tube with saline and centrifuge the tube.
Aspirate or decant the supernatant saline.
Repeat (steps 2 and 3) until the supernatant saline is
clear.
Pipette 10 ml of saline in to another clean test tube.
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Procedure: (as an example preparation of 2% RBC

suspension of 10 ml volume):
ABO GROUPING: PREPARATION OF RBC SUSPENSION:
Add 0.2 ml of the packed cell button to the 10 ml of

saline.
Cover the tube until time of use. Immediately before
use, mix the suspension by inverting the tube several
times until the cells are in suspension.
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DIRECT ABO GROUPING:
107
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The direct blood grouping also called
Cell grouping / Forward grouping
employs known anti sera to identify the antigen

present or their absence on an individuals RBC.
19-09-2015
DIRECT ABO GROUPING: PRINCIPLE:
It can be performed by the
Slide or Test tube method.
108
Make rings on the slide and label one ring as anti- A

and the other ring as anti-B.
First add corresponding antisera to the rings.
Add 10% cell suspension to both rings.
Mix using separate applicator sticks.
Observe the reaction within 2 minutes by rotating
19-09-2015
DIRECT ABO GROUPING: SLIDE METHOD:
the slide back and forth.

109
Interpret the results.
Strong
agglutination
of
19-09-2015
any ABO grouping reagent

constitutes
positive
result.
A smooth suspension of
RBCs
at
minutes
the
is
end
a
of
negative
result.
Samples that give weak or
RBCs in the presence of
doubtful reactions should

be retested by Tube test
ABO grouping.
110
Take two tubes, label one tube anti- A and the second
anti- B.
First add corresponding antisera to the tubes.
Put one drop of the 2-5% cell suspension to both tubes.
Mix the antiserum and cells by gently tapping the base of
each tube with the finger or by gently shaking.
Leave the tubes at RT for 5 minutes.
Centrifuge at low speed (2200-2800 rpm) for 30 seconds.
Read the results by tapping gently the base of each tube
19-09-2015
DIRECT ABO GROUPING: TEST TUBE METHOD:
looking for agglutination or haemolysis against a well

lighted white background.
111
19-09-2015
- Label Test tubes
- Add 2 drops of Anti sera A, B ,

and D
- Prepare 2-5% cell suspension
112
- 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
19-09-2015
- Add one drop of 2-5% Patient

Red Blood Cell suspension.
113
Interpretation
Anti-A
Anti-B
Cell Ag
ABO Group
No Ag
A, B
AB
19-09-2015
Reaction of cells
tested with
TUBE METHOD READING OF RESULTS:
114
INDIRECT ABO GROUPING:
115
19-09-2015
The indirect blood grouping also called
Serum grouping / Reverse grouping

employs RBCs possessing known antigen to identify
the type of antibodies present or absent in the serum
of an individual.
It can be performed by the Test tube method.
Slide reverse grouping is not reliable.
19-09-2015
INDIRECT ABO GROUPING: PRINCIPLE:
116
Take two tubes, label A- Cells and B cells.
Put one drop of the serum to be tested each tube.
Add one drop of 2-5% A cells to the tube labeled A cells
and one drop of 2-5% B cells to the tube labeled B cells.
Mix the contents of the tubes.
Leave the tubes at RT for 5 minutes.
Centrifuge at low speed (2200-2800 rpm) for 30 seconds.
Read the results by tapping gently the base of each tube
19-09-2015
INDIRECT ABO GROUPING: TEST TUBE METHOD:
looking for agglutination or haemolysis against a well
lighted white background.
117
agglutination in serum tests constitutes positive test

results.
A smooth suspension of RBCs after resuspension of

an RBCs button is a negative result.
19-09-2015
Agglutination in any tube of RBCs test or hemolysis or
118
SERUM TESTED WITH
BLOOD GROUP
A CELL
B CELL
Negative
Positive
Positive
Negative
Negative
Negative
AB
Positive
Positive
19-09-2015
INDIRECT ABO GROUPING: INTERPRETATION:
119
Anti-A
Reaction of serum tested

against
Interpretation
Anti-B A cells B Cells O cells ABO Group
AB
Reaction of cells
tested with
19-09-2015
INTERPRETATION OF BOTH:
120
OTHER METHODS OF BLOOD GROUPING:
121
19-09-2015
Gel Cards containing Anti-A, Anti-B, and Anti-A1B are used
19-09-2015
GEL CARDS:
absence of the A and/or B antigens.
The results of red blood cell grouping should be confirmed

by reverse (serum) grouping, i.e. testing the individuals
serum with known A1 and B red blood cells.
In the Gel Test, the specific antibody (Anti-A, Anti-B, or

Anti-D) is incorporated into the gel. This gel has been pre-
filled into the microtubes of the plastic card. As the red

blood cells pass through the gel, they come in contact with
to test patient or donor red blood cells for the presence or
the antibody. Red blood cells with the specific antigen will
agglutinate
when
combined
with
the
corresponding
antibody in the gel during the centrifugation step.
122
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.
A positive reaction in the Control microtube indicates a

false positive reaction, thus invalidates the tests.
19-09-2015
A positive reaction is recorded when red cells are
GEL CARDS: INTERPRETATION OF RESULTS
Drying, discoloration, bubbles, crystals, other artefacts,

opened or damaged seals may indicate product alteration.
123
19-09-2015
PROCEDURE:
124
Microplate techniques can be used to test for antigens on
19-09-2015
MICROPLATE TECHNIQUE:
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 microplate.
Add reagent and patient sample( red cells/ serum)
Incubation,
Centrifugation
Red cell resuspension,
Reading of results.
Interpretation of results.
red cells and for antibodies in serum.
125
ABO GROUPING DISCREPANCIES
126
19-09-2015
Most errors are technical in nature & can be

resolve by careful repeating the test procedure.
Common errors are:

1.
Contaminated reagents.
2.
Dirty glass ware.
3.
Over / Under centrifugation.
4.
Incorrect serum:cell ratio.
5.
Incorrect incubation temperature.
6.
Failure to add test specimen / reagents.
19-09-2015
ABO GROUPING DISCREPANCIES: TECHNICAL ERRORS
127
If after careful repeat the same agglutination

pattern is obtained than the causes can be:
1.
Missing / Weak reacting Abs.
2.
Missing / Weak Abs.
3.
Additional Ab.
4.
Plasma abnormalities.
19-09-2015
ABO GROUPING DISCREPANCIES: NON-TECHNICAL

ERRORS
128
Age:
Infants before producing own Abs or who possess passively

acquired maternal Abs.
Elderly persons whose Ab levels have declined.

Hypogammaglobulinemia:
2.
Lymphoma.
Leukemia.
Immunodeficiency disorders / Use of immnosuppressive drugs.
Following BM transplantation.
RESOLUTION:
enhancing
reaction
in
reverse
grouping
by
incubating test serum with RBCs at RT for 15 mins / at 40C or 160C

for 15 mins.
1.
19-09-2015
ABO GROUPING DISCREPANCIES: MISSING / WEAK Abs.
129
1.
Subgroups of A / B Ags. [RESOLUTION: Subgroup the sample.]
2.
Diseases
Leukemia:
ABO
Ags
greatly
depressed.
[RESOLUTION: Investigate the diagnosis.]

3.
Blood group specific substances: Ovarian cysts / carcinomas.
[RESOLUTION: Wash the cells in saline.]

4.
Acquired B Ag: Effect of bacterial enzymes & adsorption of

bacterial polysaccharide on to the group A / O RBCs B
specificity weak B Ag reaction in the forward grouping.

[RESOLUTION: Acidify the anti-B reagent to pH 6 rules out
acquired B.]
5.
Additives to sera. [RESOLUTION: Wash the cells in saline.]
6.
Mixtures of blood: recent BT / BM

[RESOLUTION: Investigate.]
transplant recipient.
like
19-09-2015
ABO GROUPING DISCREPANCIES: MISSING / WEAK Ags.
130
1.
AutoAb.: Cold autoAb - spontaneous agglutination of the A & B
19-09-2015
ABO GROUPING DISCREPANCIES: ADDITIONAL Ab.
coated
with
sufficient
Ab
to
promote
spontaneous
agglutination.
[RESOLUTION: Wash RBCs in warm 370C to establish cold Abs. Treat
cells with Chloroquine diphosphate to eliminate bound warm Abs]
1.
Anti A1: A2 & A2B individuals may produce naturally occurring

anti-A1 which cause discrepant ABO typing.
[RESOLUTION: Investigate the diagnosis.]

1.
Irregular Ab: Irregular antibodies in some other blood group

system may be present that react with antigens on the A or B
cells in reverse grouping. Warm AIHA patients may have RBCs
cells used in reverse grouping.

[RESOLUTION: Use A & B cells that are negative for corresponding
Ag.]
131
1.
Increased globulin.
2.
Abnormal proteins.
3.
Whartons jelly.
All these cause increased rolueaux formation that can be

mistaken as agglutination.
19-09-2015
ABO GROUPING DISCREPANCIES: PLASMA

ABNORMALITIES
[RESOLUTION: Wash with NS to remove proteins.]

132
LABORATORY DETERMINATION OF THE

RH SYSTEM
133
19-09-2015
No Indirect / Reverse grouping.
No naturally occurring Rh antibodies are not found

in the serum of persons lacking the corresponding
Rh antigens.
In performing Rh grouping:
The number of drops,
Time &
Speed of centrifugation shall be determined by

manufacturers directions.
19-09-2015
Direct Slide / Tube testing method.
134
Place a drop of anti- D on a labelled slide.
Place a drop of Rh control (albumin or other control

medium) or another labelled slide.
Add two drops of 40-50% suspension of cells to each slides.
Mix the mixtures on each slide using separate applicator

sticks, spreading the mixture evenly over most of the slide.
Interpretation or results:
Agglutination of red cells- Rh positive.
No red cell agglutination- Rh negative.
A smooth suspension of cell must be observed in the
control.
Note: Check negative reactions microscopically.
19-09-2015
Rh TYPING: SLIDE TEST METHOD
135
Make a 2-5% red cell suspension.
Mark D on a test tube and add two drops of anti-D.
Place a drop of Rh control (albumin or other control
medium) on another labelled tube.
Add one drop of a 2-5% cell suspension to each tube.
Mix well and centrifuge at 2200-2800 rpm for 60 seconds.
Gently resuspend the cell button and look for agglutination

and grade the results (a reaction of any grade is interpreted
as Rh positive) a smooth suspension of cells must be
19-09-2015
Rh TYPING: TUBE TEST METHOD
observed in the control.
Collect a weakly positive and negative sample to perform

the Du test.
136
Use the initial Rh D typing tube and control in procedure
19-09-2015
Rh TYPING: Du TYPING USING IAT
samples and the control at 370C for 30 minutes.
Wash cells in both test and control tube 3-4 times with
normal saline.
Add one drop of the poly specific antihuman globulin

(Coombs) to each tube and mix well.
Centrifuge at 2200-2800 rpm for 10 second.
Gently
suspend
the
cell
button
and
observe
for
agglutination.
Interpretation: the positive result is agglutination in the
above and incubate the Rh - negative or weakly reactive
tube containing antiD and the control is negative. A
negative result is absence of agglutination in both the test

& control.
137
THE ANTI-GLOBULIN TEST
138
19-09-2015
It is a sensitive technique in the detection of
Incomplete antibodies,
Antibodies
agglutinate
that
RBCs
can
sensitize
suspended
but
in
which
saline
at
fail
19-09-2015
Introduced in to clinical medicine by Coombs in 1945.
to
room
temperature, mainly IgG.
Complements.
PRINCIPLE:
Anti-IgG / Anti-C3 in antiglobulin serum agglutinates the
incomplete Abs / Sensitizing Abs on neighboring RBCs

/ Complements by cross-linking them.
139
140
19-09-2015
It is made by injecting rabbits, sheep or goat with

purified human IgG or C3.
The reagent may be polyspecific or monospecific.
Polyspecific Anti-human Globulin: contains a blend

of Anti-IgG & Anti-C3b, -C3d and sometimes C4
Monospecific reagents:
Anti-C3b,-C3d alone
contains Anti-IgG alone or
19-09-2015
AHG Reagent:
141
Positive Control:
Sensitized O Rh (D) positive cells.
Negative Control:
Sensitized 0 Rh (D) negative cells.
Unsensitized 0 Rh (D) positive cells.
19-09-2015
Preparation of coombs control check cells (CCC):
142
Take 0.5 mL of 5-6 times washed and packed 0 Rh (D)

+ve red cells in a test tube.
Add 2-3 drops of IgG anti-D (select a dilution titre

[1:4] of anti-D which coats the red cells but does not
agglutinate them at 37C).
Mix and incubate at 37C for 30 minutes. If there is

agglutination, repeat the procedure using more
diluted anti-D.
Wash 3-4 times and make 5% suspension in saline for
19-09-2015
Preparation of Positive Coombs control check

cells (CCC):
use.
Perform a DAT which should give a 2+ reaction. If no
agglutination occurs, repeat using less diluted anti-D.
143
0 Rh(D) negative sensitized red cells are also prepared

by treating 0 Rh(D) negative cells in the same manner.
The
preparation
should
antiglobulin test (DAT).
give
negative
direct
19-09-2015
Preparation of Negative Coombs control check

cells (CCC):
144
1.
Direct Antiglobulin Test [Direct Coombs Test]

DAT.
2.
Indirect Antiglobulin Test [Indirect Coombs Test]

IAT.
19-09-2015
TYPES:
145
It is used to demonstrate whether RBCs have been

sensitized (coated) with antibody or complement in vivo,
as in case of
HDN,
Autoimmune haemolytic anemia,
Drug induced haemolytic anemia, and
Transfusion reactions.
Principle:
Patients erythrocytes are washed to remove free plasma
19-09-2015
DAT:
proteins and directly mixed with AHG, and if incomplete

antibodies are present, agglutination occurs.
146
147
19-09-2015
red cells with IgG and/or complement components

C3b and C3d in vivo.
In
vivo
coating
of
red
cells
with
IgG
and/or
complement may occur in any immune mechanism is

attacking the patient's own RBC's.
This
mechanism
could
be
autoimmunity,
alloimmunity or a drug-induced immune mediated

mechanism.
19-09-2015
The direct antiglobulin test (DAT) detects sensitized
DAT:
148
Requirements:
Test tubes: (10x75 mm)
Pasteur pipettes
Incubator
Centrifuge
Reagent: Anti-human globulin serum.
Specimen: Blood drawn into EDTA is preferred but
19-09-2015
DAT: Requirements
oxalated, or clotted, citrated whole blood may be used
(specimen need not be fasting sample).
149
Prepare a 5 % suspension in isotonic saline of the red
19-09-2015
DAT: Procedure
With clean Pasture pipette add one drop of the prepared
cell suspension to a small tube.
Wash three times with normal saline to remove all the

traces of serum.
Decant completely after the last washing.
Add two drops of Antihuman globulin.
Mix well and incubate at 370C for 30 mins.
Centrifuge for one minute at 1500 RPM.
Resuspend the cells by gentle agitation and examine
macroscopically and microscopically for agglutination.
blood cells to be tested.
150
151
19-09-2015
antibodies or other antibodies in patients serum in

case of the following:
a.
To check whether an Rh-negative women (married

to Rh-positive husband) has developed Anti Rh
antibodies.
b.
Rh incompatible blood transfusions.
2.
To detect Du Ag.
3.
In cross-matching to detect Abs that might reduce

the survival of transfused cells.
19-09-2015
This test is performed to detect presence of Rh
1.
IAT:
152
The serum containing antibodies is incubated with

erythrocytes containing antigens that adsorb the
incomplete antibodies. After washing to dilute the
excess antibody in the serum, the addition anti
globulin
serum
produces
agglutination
in
the
19-09-2015
IAT: Principle
presence of incomplete antibodies.

153
154
19-09-2015
Requirements:
Test tubes: (10x75 mm)
Pasteur pipettes
Incubator
Centrifuge
Specimen: Serum (need not be fasting)
Reagents:
Antihuman globulin
IgG Anti-D serum
Coombs control cells: Make a pooled O Rho (D) positive
cells from at least three different O positive blood
19-09-2015
IAT: Procedure
samples. Wash these cells three times in normal saline
(these cells should be completely free from serum with no

free antibodies).
155
1.
Label three test tubes as T (test serum) PC

(Positive control) and NC (negative control).
2.
In the tube labelled as T, add two drops of Test

serum.
3.
In the tube PC add two drops of 1:4 diluted IgG

Anti-D.
4.
In the tube NC add two drops of NS.
19-09-2015
IAT: Procedure
156
5.
Add two drops of 5 % saline suspension of the
19-09-2015
IAT: Procedure
6.
Incubate all the three tubes for one hour at 37C.
7.
Wash the cells three times in normal saline to

remove excess serum with no free antibodies, (in the
case of inadequate washings of the red cells,
negative results may be obtained).

8.
Add two drops of Coombs serum (anti human

globulin) to each tube. Keep for 5 minutes and then
pooled O Rh (D) positive cells in each tube.
centrifuge at 1,500 RPM for one minute.

9.
Resuspend the cells and examine macroscopically as

well as microscopically.
157
158
19-09-2015
Agglutination.
Conclusion
Correct test.
PC
No agglutination. Incorrect test, repeat.
No agglutination. Correct test.

NC
Agglutination.
Incorrect test, repeat.
Agglutination.
Positive Patient serum contains Ab.
Test
Observation
19-09-2015
IAT: Interpretation
No agglutination. Negative.
159
Temperature:
Optimal temperature: 370C.
Incubation at higher / lower temperature may give false

positive results.
Serum Cell ratio: Increasing the ratio of serum to cells
increases the antibody coating. Commonly used ratio in

saline suspension is 2:1 but in LISS suspending cells, use
equal volume of serum and 2% cell suspension.
Incubation time:
Saline, Albumin or enzyme technique : 45-60 minutes.
LISS suspended cells - Routine 15 minutes.
Suspension medium: The sensitivity of IAT can be

increased with addition of 22% bovine albumin, enzyme or
by using LISS suspended cells.
19-09-2015
Factors affecting sensitivity of IAT
160
1.
Inadequate cell washing.
2.
Delay in adding antiglobulin reagent
after the
washing step.
3.
Presence of small fibrin clots among the cells.
4.
Inactive, or forgotten antiglobulin reagent .
19-09-2015
False Negative Antiglobulin test results:
161
1.
Using improper sample (clotted cells instead of

EDTA for Direct Antiglobulin Test, DAT).
2.
Spontaneous agglutination (cells heavily coated with

IgM).
3.
Non-specific agglutination ("sticky cells").
19-09-2015
False Positive Antiglobulin test results:
162
CROSS-MATCHING
163
19-09-2015
To select donors blood that will not cause any

adverse reaction like hemolysis or agglutination in
the recipient.
2.
Also to help the patient to receive maximum benefit

from transfusion of red cells, which will survive
maximum in his circulation.
However,
cross
match
will
not
prevent
immunization of the patient, and will not guarantee

normal survival of transfused erythrocytes or detect
all unexpected antibodies in a patients serum.
19-09-2015
1.
Purpose:
164
1.
Major.
2.
Minor.
19-09-2015
Types of Cross Match:
165
RECIPIENTS / PATIENTS SERUM with DONORs RBCs.
It is much more critical for assuring safe transfusion

than the minor compatibility test.
It is called major because the antibody with the
recipients serum is most likely to destroy the donors

red cells and that is why it is called major cross
match.
19-09-2015
Major Cross Match:
166
DONORs SERUM with PATIENTs RBCs.
It is usually thought that any antibody in the donors

serum will be diluted by the large volume of the
recipients blood, so it causes relatively less problem
and so called minor cross match.
19-09-2015
Minor Cross Match:
167
When whole blood is to be transfused, the blood

selected for 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.

Group of
Patient
Choice of Blood
1st
2nd
3rd
4th
---
---
---
---
---
---
---
AB
AB
A*
19-09-2015
Selection of blood for Cross Match:
168
Group A is the second choice of blood because anti-B in Gp
A blood is likely to be weaker than anti-A in Gp B blood.
1.
4 PHASES:
1.
Saline.
2.
Protein.
3.
AHG.
4.
Enzyme.
Saline tube technique at RT: provides the optimum
temperature and medium for the detection of IgM
antibodies
of
ABO
system
and
other
potent
cold
agglutinins.
2.
19-09-2015
Procedure of Cross Match:
Saline 370C: is the optimum for the detection of warm

agglutinin, of which are saline reactive IgG antibodies of
the Rh/ Hr system.
169
3.
AHG: is highly efficient for the detection of most kinds of
19-09-2015
4.
Enzyme technique- is a very sensitive one for the

detection of some low affinity Rh antibodies, which are
not detected by other methods including the antiglobulin
technique.
Procedure:
1.
Put 3 drops of patients serum in to a test tube.
2.
Put one drop of donors 3% red cells suspension.
3.
Mix and centrifuge at 3400 rpm for 15 seconds.
4.
Examine for agglutination or haemolysis, if compatible
incomplete antibodies.
proceed with the next phase.

5.
Mix the contents of the tube and incubate at 370C for 20

30 min.
170
potentiators such as a drop of 22% albumin may be
added at this
6.
phase to increase the sensitivity of the test.
Centrifuge at 3400 rpm for 15 seconds and examine for
agglutination or hemolysis. If there is no hemolysis or

agglutination proceed with the next phase.
7.
Wash the contents of the tube 3-4 times with normal

saline.
8.
After the last wash, decant all saline and add two drops of
AHG reagent and mix.

9.
Centrifuge at 3400 rpm for 15 seconds.
10.
Gently
re
suspend
the
cells
button
and
examine
macroscopically and microscopically for agglutination or

hemolysis.
Note:
19-09-2015
171
Enzyme cross match can be performed by using different
19-09-2015
Bromelin / Ficin / Papain / Trypsin.
Two methods are available to carry out enzyme cross

match:
One stage &
Two stage methods.
The one-stage technique involves enzyme, patients serum

and donors red cell incubated together.
enzymes:
The two-stage technique involves red cells pre-treated with

enzyme and then tested with the patients serum.
172

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IMMUNOHEMATOLOGY

Dr MANOJ K PATRO, MD [PATH]; MKCG MC BRAHMAPUR

Dr MANOJ K PATRO, MD [PATH]; MKCG MC BRAHMAPUR

Study of the immune system

Protection against infection or

Dr MANOJ K PATRO, MD [PATH]; MKCG MC BRAHMAPUR

The Immune System

The Immune System

Organs of the Immune System

Lymphocytes: T-cells, B-cells & NK cells.

Dr MANOJ K PATRO, MD [PATH]; MKCG MC BRAHMAPUR

Cells of the Immune System

Killer Cells: Cytotoxic Ts and NKs

Phagocytes and Their Relatives

Phagocytes in the Body

Immunity: Active and Passive

QUANTITATION of antibodies involved primarily with

Basically this branch of science related with red cell

Work primarily with patient's

Dr MANOJ K PATRO, MD [PATH]; MKCG MC BRAHMAPUR

Dr MANOJ K PATRO, MD [PATH]; MKCG MC BRAHMAPUR

1667: Jean Bapiste Denys Unsuccessful transfusion of

1667 1818: Transfusions prohibited.

1616: Sir William Harvey Described circulation of blood.

Dr MANOJ K PATRO, MD [PATH]; MKCG MC BRAHMAPUR

1900: Karl Landsteiner Discovered the ABO blood

Described the ABO

Karl Landsteiners discovery:

now known as Antigens.

Postulated two things:

Each species has unique species specific factors on

Even in each species has some common and some

Dr MANOJ K PATRO, MD [PATH]; MKCG MC BRAHMAPUR

transfusion was due to certain factors on red cells

transfusion began the era of scientific based

1939,40: Levine, Stetson, Landsteiner and Weiner

1946-Kell system discovered by Coombs, Mourant and

1950-51: Duffy, Kidd, Lutheran system discovered.

Landsteiner and Alexanders lead to the discovery of

1927: Landsteiner and Levine discovered M,N and P

Dr MANOJ K PATRO, MD [PATH]; MKCG MC BRAHMAPUR

Dr MANOJ K PATRO, MD [PATH]; MKCG MC BRAHMAPUR

ANTIGEN is a substance that either

combines with an antibody or

is processed and binds to a T lymphocyte to

stimulate an IMMUNE RESPONSE.

IMMUNOGEN - An antigen that stimulates an immune

HAPTENS - small chemical substances that must be

bound to a larger molecule to provide sufficient

Dr MANOJ K PATRO, MD [PATH]; MKCG MC BRAHMAPUR

Properties of Molecules that Contribute to

Some antigens protrude from the cell surface, while

Physical location impacts antibody stimulation as well

as the physical ability of the antigen to react with an

Red Blood Cell Antigens:

Red blood cell antigens and corresponding antibodies

More than 20 blood group systems that contain

Dr MANOJ K PATRO, MD [PATH]; MKCG MC BRAHMAPUR

others are an integral part of the membrane.

greater than 200 red blood cell antigens.

ABO and Rh antigens are matched between donor and

Dr MANOJ K PATRO, MD [PATH]; MKCG MC BRAHMAPUR

Produced in response to stimulation with an antigen.

Specific for the stimulating antigen and will react with