Immunohematology and Blood Banking Techniques PDF

IMMUNOHEMATOLOGY
&
BLOOD BANKING TECHNIQUES
INTRODUCTION:
Dr MANOJ K PATRO, MD [PATH]; MKCG MC BRAHMAPUR 19-09-2015

IMMUNO
HEMATO
IMMUNOHEMATOLOGY
LOGY

DEFINITIONS:
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• Protection against infection or
IMMUNITY: disease caused by foreign
particles.
Dr MANOJ K PATRO, MD [PATH]; MKCG MC BRAHMAPUR

• Study of the immune system
and the immune response.
IMMUNOLOGY:
• Study of antigen-antibody
reactions in vivo.
• Study of antigen-antibody
SEROLOGY:
reactions in vitro.
• Study of the cellular

HEMATOLOGY: 4
components of the blood.
The Immune System
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 Three functions:
 Defense
 Homeostasis
 Surveillance
5
The Immune System
Markers of Self
Markers of Non-Self
Markers of Self:
Major Histocompatibility Complex
Organs of the Immune System
Components:
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Cells:

• Lymphocytes: T-cells, B-cells & NK cells.
• Phagocytic cells: N, E, B, Monocytes,
Macrophages, Dendritic cell, etc.
Chemical mediators:
• Complement system.
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• Chemokines.
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
DEFINITIONS:
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IMMUNOHEMATOLOGY:

 DETECTION,
 IDENTIFICATION, and/or
 QUANTITATION of antibodies involved primarily with

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

antigens and their corresponding antibodies. 24
IMMUNOHEMATOLOGY FACILITIES:
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TRANSFUSION SERVICE: BLOOD BANK:
• Work primarily with patient's • Works primarily with

blood. donor blood.
• Primary areas of • Major areas of
responsibility: responsibility:
• Blood typing. • Donor recruitment.
• Antibody detection and
identification.
• Donor screening.
• Compatibility testing • Blood collection.
(crossmatching). • Testing (typing,
• Blood component therapy infectious disease
(hemotherapy). screening).
• Transfusion reaction • Blood component
workups. preparation.
• Autoimmune hemolytic • Component preservation.
anemia workups.
• Hemolytic disease of the
• May provide reference
newborn (HDN) workups. lab services. 25
• Determining Rh immune • May store rare donor
globulin eligibility. blood.
HISTORICAL OVERVIEW
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 1616: Sir William Harvey – Described circulation of blood.
 1665: Richard Lower, English Physiologist – 1st animal-to-

animal [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-to-

human blood transfusion. This species specific transfusion
had 50% success rate, the rest resulted in death.
 1900: Karl Landsteiner – Discovered the ABO blood

groups. 27
Blood Groups.
Described the ABO
Karl Landsteiner:
28

Karl Landsteiner’s discovery:
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 Discovered that the incompatibility of many
transfusion was due to certain factors on red cells

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 – began the era of scientific based
transfusion therapy – foundation of 29
IMMUNOHEMATOLOGY as a science.
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 1927: Landsteiner and Levine discovered M,N and P
system.

 1939,40: Levine, Stetson, Landsteiner and Weiner
discovers Rh system and it‟s 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. 30
ANTIGENS:
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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.
32
 Proteins – Complex carbohydrates –
Lipopolysaccharides.
Properties of Molecules that Contribute to
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Immunogenicity
PROPERTY DESCRIPTION

Foreignness Non-self more likely to stimulate
antibody production
Size >10,000 M.W.
Chemical Protein—best immune response.
composition Complex carbohydrate—second best
immune response.
Lipids, Nucleic acid —weak immune
response.
Complexity More complex molecules produce 33
better immune response.

Antigen location:
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 Some antigens protrude from the cell surface, while
others are an integral part of the membrane.

 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
greater than 200 red blood cell antigens.
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 ABO and Rh antigens are matched between donor and
recipient.
ANTIBODIES
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 Protein.
 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.
36
Immunological principles:
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 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:
37
Antigen-Antibody Reactions: Factors influencing:
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•Each antibody reacts with the antigen that stimulated its
Specificity:
production.
Bonding: •Noncovalent bonds.

•The fit of the antigen and antibody depend on compatible
Physical fit: shapes that allow the antigen and antibody to physically
touch - a lock and key mechanism.
Concentration of •Antigens and antibodies must be present in optimal

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

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

Time: antibody. General guidelines are a range of 15–60 minutes for
optimal antigen-antibody attachment.
•A pH range of 7.2–7.4 is maintained for most antigen-

pH:
antibody reactions.
38
•A net negative charge known as zeta potential surrounds the
Surface charge: red cells. The reduction of this charge influences the ability
of antigen and antibody to combine.
LOCK & KEY
CONCENTRATION
AGGLUTINATION
39

Visualization of Ag-Ab reaction in BB:
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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
crosslink.
3. Multiple cross links create a lattice. 40
4. The lattice is visualized as agglutination.
Grading of Agglutination:
41

42

BLOOD GROUP GENETICS
43

Chromosomes & Genes:
44

45

46

Basic Principles:
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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 contributes 1/2 of the genetic
information.
 The genetic information is contained
on chromosomes composed of DNA
 Humans have 23 pairs of chromosomes
a. 22 matched (autosomal) chromosomes and
b. 1pair of sex chromosomes.
Located on
System Common Genes
Chromosome
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ABO A, B, O 9
Rh D, C, E, c, e 1
Basic Principles:
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 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. 48
Basic Principles:
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 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.
49
Basic Principles:
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 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.
50
BLOOD GROUP SYSTEMS
51

HISTORICAL PERSPECTIVE:
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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.
LANDSTEINER’S 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 52
anti-B in the serum).
ABO & H SYSTEM ANTIGENS:
53

ABO ANTIGENS:
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 Present on RBC surface.

 Also on lymphocytes, thrombocytes, organs,
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.
54
ABO ANTIGENS:
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 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

55
A1 cord 2,50,000 to 3,70,000
INHERITANCE:
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 Simple Mendelian fashion from an individual‟s
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

the O gene is an amorph.
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 The expression of the A and B genes is codominant.
INHERITANCE:
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Gene Combination Phenotype

AO A
AA A
BO B
BB B
AB AB
OO O
57
GENE PRODUCTS & BIOCHEMICAL COMPOSITION
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OF BLOOD GROUP SUBSTANCES:
GENE TRANSFERASE

H α-L-fucosyltransferase
α-3-N-acetyl-D-galactosaminyl
A
Transferase
α-3-D-acetyl-D-galactosyl
B
Transferase
O No Transferase.
58
 Basic common core structure - an oligosaccharide chain attached
to either a protein or a lipid molecule present on cell surface.
GENE PRODUCTS & BIOCHEMICAL COMPOSITION
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OF BLOOD GROUP SUBSTANCES:

 The L-fucose is the immunodominant sugar for the H
antigen.
59
 The H antigen serves as a precursor for A and B
antigens.
SECRETOR STATUS
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• Ability to secrete ABH antigens in
body secretions.
• Chromosome 19: Se / se [amorph].

Secretors: • Gene product – L- fucosyltransferase.
• A & B transferases are found in the
secretions of A / B persons regardless
of their secretor status.
Nonsecretors:
Saliva, Sweat, Tears, Semen, Serum & Amniotic fluid.

61
SUBGROUPS OF A & B
62

Subgroups of A:
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 2 major subgroups:  2 major subgroups of AB:
 ~ 80% A1  ~ 80% A1B
 ~ 20% A2.  ~ 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 Sites-
3,00,000 1,40,000
Newborn
Quantitative differences:
Reaction with Anti-A1 Positive Negative
Anti-A1 in serum Absent ? Present 63
α-3-N-acetyl-D-galactosaminyl
Normal Diminished
Transferase Activity
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 A1 – more antigens on the cell surface – more
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 64
Abantu – Fewer antigenic sites on their surface.

Subgroups of B:
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 Subgroups of B are very rare and encountered less
frequently than subgroups of A.
65
ABO ANTIBODIES:
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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 antigens not
present on the red cells.
 Newborns have NO ABO ANTIBODIES.

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 REVERSE GROUPING of Cord blood / Newborn serum
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.
68
Conditions with decreased levels of ABO antibodies:
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• Newborns and young infants
Age related:

• Elderly individuals
• Congenital conditions
Immunodeficient
• Congenital hypogammaglobulinemia
individuals:
• Congenital agammaglobulinemia
• Immunosuppressive therapy
• Chronic lymphocytic leukemia
Immunosuppresse • Bone marrow transplant
d patients: • Multiple myeloma 69
• Acquired hypogammaglobulinemia
• Acquired agammaglobulinemia
Immunoglobulin class:
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 ABO antibodies – ISOAGGLUTININS – Saline agglutinins
with optimal reactivity at 40C.
 Mostly IgM.
 IgG & IgA.
70
Anti AB:
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 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.
71
Anti-A1:
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 As per Landsteiner‟s Law, group B and O individuals
produce anti-A.
 This anti-A can be separated by absorption

procedures - anti-A and anti-A1.
72
Clinical significance of ABO antibodies:
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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 baby‟s

red cells.
ABO HDFN: Rh HDFN:
• Affects 1st pregnancy. • Sensitization occurs in

• MC: O mother with A 1st pregnancy and
baby. affects subsequent
positive pregnancies.
• Rh negative mother 73
Rh positive baby.
Hemolytic transfusion reaction:
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 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.
74
RH BLOOD GROUP SYSTEM
75

INTRODUCTION:
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 One of the most polymorphic and antigenic blood group
systems.

 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%. 76

 95% Indian population: Rh Positive.
GENES:
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 Chromosome 1.
 2 genes: RhD & RhCE.
 The proteins encoded by these 2 differ by 32 to 35

AA‟s. That is why RhD is so antigenic in Rh negative
persons.
77
NOMENCLATURE:
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 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.
78
D ANTIGEN:
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 Very antigenic – D+ for presence / D- for absence.
 Variants: due to a point mutation causing single AA

differences.
1. Weak D [formerly Du, obsolete now]: 1 to 57
types: Type 1 to 3 – 90% cases.
2. Del: Not detected by routine testing but requires

adsorption-elution studies / molecular RHD
genotyping.
3. Partial D: Due to hybrid genes – portion of RHD is

replaced by corresponding portion of RHCE gene.
The RBC‟s type D+ve but make anti-D following 79
transfusion / pregnancy.
Rh null:
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 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 trafﬁcking of RhCE and
RhD to the RBC membrane.
 The serum of the people who form these antibodies

agglutinates cells from all people except another 80
Rhnull.
Rh ANTIBODIES:
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 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.
81
 30% to 85% of D-ve persons who will make anti-D following
exposure to D+ve RBCs – Responders.
ABO SYSTEM
LABORATORY DETERMINATION OF THE
82

83

RBC PRECURSOR STRUCTURE
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RBC
Glucose
Galactose
Precursor
Substance
(stays the N-acetylglucosamine
same)
Galactose
84
FORMATION OF H Ag
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RBC
Glucose
H antigen Galactose
N-acetylglucosamine
Galactose
85
Fucose
FORMATION OF A Ag
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RBC
Glucose
Galactose
N-acetylglucosamine
Galactose
86
N-acetylgalactosamine
Fucose
FORMATION OF B Ag
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RBC
Glucose
Galactose
N-acetylglucosamine
Galactose
87
Galactose
Fucose
88

ANTISERUM
89

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 An antiserum is a purified, diluted and standardized
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 90
Anti-D anti-D
Sources:
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 MONOCLONAL ANTIBODIES

 Animal inoculation.
 Serum from an individual who has been sensitized to
the antigen through transfusion, pregnancy or
injection.
91
 Serum from known blood group persons.
Criterias:
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 Antiserum must meet certain criterias to be acceptable for
use.

 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. 92
 It should be labelled and stored properly.

Manifestation of Ag-Ab reaction:
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 The observable reactions resulting from the
combination of a red cell antigen with its
corresponding antibody are agglutination and/ or
haemolysis.
93
Agglutination:
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 Is the clumping of particles with antigens on their surface,
such as erythrocytes by antibody molecules that form

bridges between the antigenic determinants.
 Hemagglutination.
 Agglutinogen – antigen.
 Agglutinin – antibody.
 Two stages:
1. Sensitization: Abs attach to the Ags on RBC –

Sensitized RBC.
2. Agglutination: Ab binding to Ag on >1 RBC – Lattice

formation. 94
Hemolysis:
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 Ab – Hemolysin.
 Complement mediated lysis of the RBC membrane
with release of Hb to stain the plasma.
95
Right condition for the RBCs to agglutinate / hemolysis:
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1. Ab size:
 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 varies

depending upon Ab type: IgG – 370C, IgM – 4 – 220C. 96
19-09-2015
4. Ionic strength:
 Low ionic strength increases agglutination.

 LISS – 0.2% NaCl in 7% glucose is used.
5. Number of Ag sites:
 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. Centrifugation: at high speed attempts to overcome

the problem of distance in sensitized cells by 97
physically forcing the cells together.

19-09-2015
7. 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.
8. Colloidal suspension: [Bovine albumin]
 Can reduce the zeta potential – helps IgG Abs of Rh
system to agglutinate.
9. Ratio of Ag & Ab:
 Excess Ab – Prozone phenomenon – Use serial dilutions
of the antisera.
98
 Avoid heavy RBC suspension as it may mask the presence
of a weak Ab.
ABO GROUPING TECHNIQUES
99

METHODS:

100
REAGENTS:
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 Anti-A antibodies.
 Anti-B antibodies.

 Anti-AB antibodies (optional).
 Group A & B RBCs.
 Slides, or Test tubes.
 Wooden applicator.
101
IMPORTANT THINGS TO FOLLOW:
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 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.
 At the End, Discard all materials in the isolation

trash containers. 102
ABO GROUPING: RULES FOR PRACTICAL WORK:
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1. Perform all tests according to the manufacturer’s

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.
This eliminates the need to run individual controls
each time the reagents are used. 103
ABO GROUPING: RULES FOR PRACTICAL WORK:
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5. Do not rely on colored dyes to identify reagent
antisera.
6. Always add serum before adding cells.
7. Observe for agglutination against a well–lighted

background, and record results immediately after
observation.
8. Use an optical aid to examine reactions that appear

to the naked eye to be negative.
104
ABO GROUPING: PREPARATION OF RBC SUSPENSION:
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 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).
105
ABO GROUPING: PREPARATION OF RBC SUSPENSION:
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Procedure: (as an example preparation of 2% RBC
suspension of 10 ml volume):

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.
Add 0.2 ml of the packed cell button to the 10 ml of

saline.
106
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.
DIRECT ABO GROUPING:

107
DIRECT ABO GROUPING: PRINCIPLE:
19-09-2015
 The direct blood grouping also called

Cell grouping / Forward grouping
employs known anti sera to identify the antigen
present or their absence on an individual‟s RBC.
 It can be performed by the

108
 Slide or Test tube method.
DIRECT ABO GROUPING: SLIDE METHOD:
19-09-2015
 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
the slide back and forth.

109
 Interpret the results.
19-09-2015
 Strong agglutination of
RBCs in the presence of

any ABO grouping reagent
constitutes a positive
result.
 A smooth suspension of
RBCs at the end of 2
minutes is a negative
result.
 Samples that give weak or
doubtful reactions should
be retested by Tube test 110
ABO grouping.
DIRECT ABO GROUPING: TEST TUBE METHOD:
19-09-2015
 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

looking for agglutination or haemolysis against a well
lighted white background. 111

19-09-2015
- Prepare 2-5% cell suspension

- Label Test tubes
- Add 2 drops of Anti sera A, B ,

and D
112
19-09-2015
- Add one drop of 2-5% Patient
Red Blood Cell suspension.

- 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
113
TUBE METHOD READING OF RESULTS:
19-09-2015
Reaction of cells Interpretation
tested with

Anti-A Anti-B Cell Ag ABO Group
- - No Ag O
+ - A A
- + B B
+ + A, B AB
114
INDIRECT ABO GROUPING:

115
INDIRECT ABO GROUPING: PRINCIPLE:
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. 116

INDIRECT ABO GROUPING: TEST TUBE METHOD:
19-09-2015
 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

looking for agglutination or haemolysis against a well
lighted white background.
117
19-09-2015
 Agglutination in any tube of RBCs test or hemolysis or
agglutination in serum tests constitutes positive test

results.
 A smooth suspension of RBCs after resuspension of

an RBCs button is a negative result.
118
INDIRECT ABO GROUPING: INTERPRETATION:
19-09-2015
SERUM TESTED WITH

BLOOD GROUP
A CELL B CELL
Negative Positive A
Positive Negative B
Negative Negative AB
Positive Positive O
119
INTERPRETATION OF BOTH:
19-09-2015
Reaction of cells Reaction of serum tested Interpretation
tested with against

Anti-A Anti-B A cells B Cells O cells ABO Group
- - + + - O
+ - - + - A
- + + - - B
+ + - - - AB
120
OTHER METHODS OF BLOOD GROUPING:

121
GEL CARDS:
19-09-2015
 Gel Cards containing Anti-A, Anti-B, and Anti-A1B are used
to test patient or donor red blood cells for the presence or

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 individual‟s
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
the antibody. Red blood cells with the specific antigen will
agglutinate when combined with the corresponding 122
antibody in the gel during the centrifugation step.
GEL CARDS: INTERPRETATION OF RESULTS
19-09-2015
 A positive reaction is recorded when red cells are
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.
 Drying, discoloration, bubbles, crystals, other artefacts,

123
opened or damaged seals may indicate product alteration.
PROCEDURE:

124
MICROPLATE TECHNIQUE:
19-09-2015
 Microplate techniques can be used to test for antigens on
red cells and for antibodies in serum.

 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. 125

ABO GROUPING DISCREPANCIES

126
ABO GROUPING DISCREPANCIES: TECHNICAL ERRORS
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. 127

ABO GROUPING DISCREPANCIES: NON-TECHNICAL
19-09-2015
ERRORS

 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. 128

ABO GROUPING DISCREPANCIES: MISSING / WEAK Abs.
19-09-2015
1. Age:
 Infants before producing own Abs or who possess passively
acquired maternal Abs.

 Elderly persons whose Ab levels have declined.
2. Hypogammaglobulinemia:
 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 129
for 15 mins.
ABO GROUPING DISCREPANCIES: MISSING / WEAK Ags.
19-09-2015
1. Subgroups of A / B Ags. [RESOLUTION: Subgroup the sample.]
2. Diseases like 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.]

130
6. Mixtures of blood: recent BT / BM transplant recipient.
[RESOLUTION: Investigate.]
ABO GROUPING DISCREPANCIES: ADDITIONAL Ab.
19-09-2015
1. AutoAb.: Cold autoAb - spontaneous agglutination of the A & B
cells in reverse grouping. Warm AIHA patients may have – RBCs
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 used in reverse grouping.
131
[RESOLUTION: Use A & B cells that are negative for corresponding
Ag.]
ABO GROUPING DISCREPANCIES: PLASMA
19-09-2015
ABNORMALITIES

1. Increased γ globulin.
2. Abnormal proteins.
3. Wharton’s jelly.
All these cause increased rolueaux formation that can be
mistaken as agglutination.
[RESOLUTION: Wash with NS to remove proteins.]
132
RH SYSTEM
LABORATORY DETERMINATION OF THE

133
19-09-2015
 Direct Slide / Tube testing method.
 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. 134
Rh TYPING: SLIDE TEST METHOD
19-09-2015
 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. 135
 Note: Check negative reactions microscopically.

Rh TYPING: TUBE TEST METHOD
19-09-2015
 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
observed in the control.
 Collect a weakly positive and negative sample to perform 136
the Du test.
Rh TYPING: Du TYPING USING IAT
19-09-2015
 Use the initial Rh D typing tube and control in procedure
above and incubate the Rh - negative or weakly reactive
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
tube containing anti–D and the control is negative. A
negative result is absence of agglutination in both the test 137
& control.
THE ANTI-GLOBULIN TEST

138
19-09-2015
 Introduced in to clinical medicine by Coomb’s in 1945.
 It is a sensitive technique in the detection of

 Incomplete antibodies,
 Antibodies that can sensitize but which fail to

agglutinate RBCs suspended in saline at 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
AHG Reagent:
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: contains Anti-IgG alone or
Anti-C3b,-C3d alone 141

Preparation of coombs control check cells (CCC):
19-09-2015
 Positive Control:

 Sensitized O Rh (D) positive cells.
 Negative Control:
 Sensitized 0 Rh (D) negative cells.
 Unsensitized 0 Rh (D) positive cells.
142
Preparation of Positive Coombs control check
19-09-2015
cells (CCC):
 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 37°C).
 Mix and incubate at 37°C 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
use.
143
 Perform a DAT which should give a 2+ reaction. If no
agglutination occurs, repeat using less diluted anti-D.
Preparation of Negative Coombs control check
19-09-2015
cells (CCC):

 0 Rh(D) negative sensitized red cells are also prepared
by treating 0 Rh(D) negative cells in the same manner.
The preparation should give a negative direct
antiglobulin test (DAT).
144
TYPES:
19-09-2015
1. Direct Antiglobulin Test [Direct Coomb‟s Test] –
DAT.
2. Indirect Antiglobulin Test [Indirect Coomb‟s Test] –

IAT.
145
DAT:
19-09-2015
 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

proteins and directly mixed with AHG, and if incomplete
antibodies are present, agglutination occurs. 146
147
DAT:
19-09-2015
 The direct antiglobulin test (DAT) detects sensitized
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

148
mechanism.
DAT: Requirements
19-09-2015
 Requirements:
 Test tubes: (10x75 mm)

 Pasteur pipettes
 Incubator
 Centrifuge
 Reagent: Anti-human globulin serum.
 Specimen: Blood drawn into EDTA is preferred but

oxalated, or clotted, citrated whole blood may be used
149
(specimen need not be fasting sample).
DAT: Procedure
19-09-2015
 Prepare a 5 % suspension in isotonic saline of the red
blood cells to be tested.

 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.

150
 Resuspend the cells by gentle agitation and examine
macroscopically and microscopically for agglutination.
151
IAT:
19-09-2015
1. This test is performed to detect presence of Rh
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. 152
IAT: Principle
19-09-2015
 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
presence of incomplete antibodies.

153
154
IAT: Procedure
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
samples. Wash these cells three times in normal saline
(these cells should be completely free from serum with no 155
free antibodies).
IAT: Procedure
19-09-2015
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. 156

IAT: Procedure
19-09-2015
5. Add two drops of 5 % saline suspension of the
pooled „O‟ Rh (D) positive cells in each tube.

6. Incubate all the three tubes for one hour at 37°C.
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 Coomb‟s serum (anti human
globulin) to each tube. Keep for 5 minutes and then
centrifuge at 1,500 RPM for one minute.
9. Resuspend the cells and examine macroscopically as 157
well as microscopically.
158
IAT: Interpretation
19-09-2015
Observation Conclusion

Agglutination. Correct test.
PC
No agglutination. Incorrect test, repeat.
No agglutination. Correct test.

NC
Agglutination. Incorrect test, repeat.
Agglutination. Positive – Patient serum contains Ab.

Test
No agglutination. Negative.
159
Factors affecting sensitivity of IAT
19-09-2015
 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
160
increased with addition of 22% bovine albumin, enzyme or
by using LISS suspended cells.
False Negative Antiglobulin test results:
19-09-2015
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 .
161
False Positive Antiglobulin test results:
19-09-2015
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").
162
CROSS-MATCHING

163
Purpose:
19-09-2015
1. To select donor‟s 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, a cross match will not prevent

immunization of the patient, and will not guarantee
normal survival of transfused erythrocytes or detect
164
all unexpected antibodies in a patient‟s serum.
2.
1.
Major.
Minor.
Types of Cross Match:

165
Major Cross Match:
19-09-2015
 RECIPIENT‟S / PATIENT‟S SERUM with DONOR‟s RBC‟s.
 It is much more critical for assuring safe transfusion

than the minor compatibility test.
 It is called major because the antibody with the

recipient‟s serum is most likely to destroy the donor‟s
red cells and that is why it is called major cross
match.
166
Minor Cross Match:
19-09-2015
 DONOR‟s SERUM with PATIENT‟s RBC‟s.
 It is usually thought that any antibody in the donor‟s

serum will be diluted by the large volume of the
recipient‟s blood, so it causes relatively less problem
and so called minor cross match.
167
Selection of blood for Cross Match:
19-09-2015
 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 Choice of Blood
Patient 1st 2nd 3rd 4th
A A O --- ---
B B O --- ---
O O --- --- ---
AB AB A* B O
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.
Procedure of Cross Match:
19-09-2015
 4 PHASES:
1. Saline.

2. Protein.
3. AHG.
4. Enzyme.
1. 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. Saline 370C: is the optimum for the detection of warm

agglutinin, of which are saline reactive IgG antibodies of 169
the Rh/ Hr system.

19-09-2015
3. AHG: is highly efficient for the detection of most kinds of
incomplete antibodies.
Enzyme technique- is a very sensitive one for the

4.
detection of some low affinity Rh antibodies, which are

not detected by other methods including the antiglobulin
technique.
 Procedure:
1. Put 3 drops of patient‟s serum in to a test tube.
2. Put one drop of donor‟s 3% red cells suspension.
3. Mix and centrifuge at 3400 rpm for 15 seconds.
4. Examine for agglutination or haemolysis, if compatible
proceed with the next phase.
170
5. Mix the contents of the tube and incubate at 370C for 20 –
30 min.
19-09-2015
Note: potentiators such as a drop of 22% albumin may be
added at this phase to increase the sensitivity of the test.

6. 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 171
hemolysis.
19-09-2015
 Enzyme cross match can be performed by using different
enzymes:

 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, patient‟s serum

and donor‟s red cell incubated together.
 The two-stage technique involves red cells pre-treated with

enzyme and then tested with the patient‟s serum. 172

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IMMUNOHEMATOLOGY

Dr MANOJ K PATRO, MD [PATH]; MKCG MC BRAHMAPUR 19-09-2015

Dr MANOJ K PATRO, MD [PATH]; MKCG MC BRAHMAPUR 19-09-2015

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

• Study of the cellular

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

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

 QUANTITATION of antibodies involved primarily with

 Basically this branch of science related with red cell

• Work primarily with patient's • Works primarily with

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

Dr MANOJ K PATRO, MD [PATH]; MKCG MC BRAHMAPUR 19-09-2015

 1665: Richard Lower, English Physiologist – 1st animal-to-

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

 1667: Jean Bapiste Denys – Unsuccessful transfusion of

 1667 – 1818: Transfusions prohibited.

 1818: James Blundell of England – 1st successful human-to-

 1900: Karl Landsteiner – Discovered the ABO blood

Dr MANOJ K PATRO, MD [PATH]; MKCG MC BRAHMAPUR 19-09-2015

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

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

 1946-Kell system discovered by Coombs, Mourant and

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

 Landsteiner and Alexanders lead to the discovery of

Dr MANOJ K PATRO, MD [PATH]; MKCG MC BRAHMAPUR 19-09-2015

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

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

Complexity More complex molecules produce 33

better immune response.

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

Dr MANOJ K PATRO, MD [PATH]; MKCG MC BRAHMAPUR 19-09-2015

 Produced in response to stimulation with an antigen.

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

 IMMUNOGLOBULIN – 5 classes, IgG, IgM, IgA, IgD &

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

Bonding: •Noncovalent bonds.

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

Concentration of •Antigens and antibodies must be present in optimal

•Optimal temperature of reactivity for a specific antibody will

•Incubation time must be that which is optimal for the specific

•A pH range of 7.2–7.4 is maintained for most antigen-

Dr MANOJ K PATRO, MD [PATH]; MKCG MC BRAHMAPUR 19-09-2015

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

Dr MANOJ K PATRO, MD [PATH]; MKCG MC BRAHMAPUR 19-09-2015

Dr MANOJ K PATRO, MD [PATH]; MKCG MC BRAHMAPUR 19-09-2015

Dr MANOJ K PATRO, MD [PATH]; MKCG MC BRAHMAPUR 19-09-2015

Dr MANOJ K PATRO, MD [PATH]; MKCG MC BRAHMAPUR 19-09-2015

Dr MANOJ K PATRO, MD [PATH]; MKCG MC BRAHMAPUR 19-09-2015

Dr MANOJ K PATRO, MD [PATH]; MKCG MC BRAHMAPUR 19-09-2015

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

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

 Genotype: Genetic composition for a particular trait.

 Homozygous: When the two inherited alleles are

 Heterozygous: when the two inherited alleles are

 Dominant: The dominant gene will express itself if

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

 Co-dominant: Both the alleles express. E.g. A & B.

 Recessive: They express in homozygous state only.

 Amorph: No gene product. e.g. O.

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

 Variation in antigen expression due to the number of

Dr MANOJ K PATRO, MD [PATH]; MKCG MC BRAHMAPUR 19-09-2015

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