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Immunohematology
Transfusion Medicine
Blood Bank
History 101 of Blood Bank
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1492: first attempt at using blood for
therapeutic use.
1665: First animal to animal-Richard
Lower
1667: Jean Baptiste Denys, first successful
IV transfusion of blood from animal to
human
1818: James Blundell First to transfuse
human to human.
1901: Karl Landsteiner: Discovered ABO
blood grouping
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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 foctalis (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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Antigen and Antibody in blood banking
Antibody production is result of blood
group antigen (foreign-transfusion or
pregnancy), or can be naturally occurring.
Blood group antigens are integral part of
the RBC membrane
B Cells produce antibody molecules that
are specific for a target antigen (part of
the surface of RBC)
Antigenic determinants: on RBC-elicit the
production of different antibodies
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Complement System: set or group of serum
proteins that generates membrane attack that
causes cell destruction. (Hemolysis)
Classical vs Alternative pathway
Antibody-Antigen complex activates
Complement.
Complement activation
Anaphylatoxins
Vasoactive amines
Chemotactic
Opsonins
Receptors
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Antigens-have different levels of
immogenicity-cause immune response.
Factors effecting immunogenecity
Chemical composition and complexity
Degree of foreignness
Size
Dosage
Route of administration
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Red Blood Cell Antigens
23 blood group systems with more than
200 RBC antigens
RBC antigens are determined by genetic
inheritance pattern
Antigen immunogenicity based on
stimulation production upon exposure
Types of antigen RBC, HLA, Platelet
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Antibodies-protein (immunoglobulin-Ig)
5 Classification:
IgG- Clinically significant in BB
IgM- Can be Clinically significant in BB
IgA
IgE
IgD
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IgM antibodies:
Produced initially in response to foreign
antigen
Large pentamer structure
Contains 10 potential antigenic sites
In BB – reaction in saline procedures
5-10 % of Ig class
Short half-life 5-6 days
Activates Complement with great
efficiency.
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IgG antibodies
Monomer
Accounts for 80% of Ig
Found in extravascular fluid
2 antigen binding sites
In BB- reaction less apparent in saline
procedures, need additive to show reaction.
Half-life 23 days
Cross placenta
Activates complement
Subclasses
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Immune response (Primary vs. Secondary)
Immune response influenced by:
Age
Route of exposure
Genetic make-up
Health
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Antigen-Antibody interaction:
Formation of antigen –antibody complex
causes an immune reaction.
Amount is determined by:
Goodness to fit
Size
Shape
Charge
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Antigen-Antibody reaction in Vivo vs. Vitro
Primary concern in Blood Bank-Vivo
Want to reduce the potential of exposure
to foreign antigens which would result in
antibody production.
Identify antigens present on the RBC
Identify any antibodies produced – found
in the serum/plasma
Identify antigen-antibody complex by:
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Hemolysis (Complement reaction
associated with complex)
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Agglutination (antigen/antibody
complex), like clotting of the blood
Don’t want agglutination2 stages of agglutination:
1. Sensitization- antibody binds to an
antigen. Is influenced by amount of
antigen and antibody present.
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Sensitization enhanced by:
Serum to cell ratio
Temperature
Incubation
pH
Ionic strength
Lattice Formation stage (cell interaction,
see visible agglutination, linkage
between antigen and antibody)
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Factors that influence Lattice formation
1. Zeta potential
2. Serum to cell ratio (zone of equivalence)
3. Prozone effect
4. Centrifugation
Grading agglutination (lattice formation)
0(neg), 1+, 2+, 3+, 4+
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Indicators of antibody-antigen complex
• Hemolysis
• Agglutination
Hemolysis is an indication of antigenantibody complex that results in cell
destruction. Identified usually in the
Antiglobulin Test.
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Antiglobulin Test (Anti-humanglobulin or
AHG)
Discovered by Coombs, Mourant and Race
in 1945- found that RBC can become
sensitized without visible agglutination.
Reagent base test
Identifies IgG antibodies and Complement
proteins
Polyspecific and monospecific AHG
reagent.
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AHG reagent will react with bound and/or free
IgG and Complement proteins.
Must get rid of the free to I.D. the bound
forms that can cause reaction.
WASH cells (RBC for testing-cell suspension)
If cells wash properly and add AHG reagent
and agglutination forms this is a positive
reaction (NOT GOOD)
2 Types of AHG test:
Direct antiglobulin test (DAT)
Indirect Antiglobulin test (IAT or antibody
screen)
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Direct antiglobulin Test:
Detects antibodies bound to RBC in vivo
Results in clinical event or illness
(+) DAT indicates an immune response;
patients cells have attached IgG and/or
Complement)
EDTA is sample choice for DAT
Indirect Antiglobulin Test (IAT)
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Detects in vitro sensitization of RBC
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2 Step process:
1. Incubation at 37°C serum with donor
cells
False positive and False negative reaction for
various reasons.
Positive IAT indicates a specific reaction
between antigen and antibody in serum
of patient.
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Potentiator Reagent: enhances antibody and
antigen complex
A.K.A. as enhancement media
Enhances antibody uptake
Promotes agglutination
4 Types potentiators
Low ionic strength solution (LISS)
Bovine serum albumin
Polyethylene glycol (PEG)
Proteolytic enzyme (3 Types)
Papain
Ficin
Bromelin
Chapter 2: Blood Bank Reagents
Basic blood banking reagents depends on the
source of antigens and source of antibody in
testing. (What are we looking for?)
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Detect an antigen present or absent on RBC
(donor/patient cells)
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Detect antibody present or absent in serum
(donor/patient serum)
Have to have a known source of antigen to detect
antibody or known antibody to detect antigen.
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Source of Antigens:
Commercially prepared RBC suspension
with known antigens to detect unknown
antibodies.
In BB- usually patient RBC antigen type is
unknown and we test the cells to identify
the type of antigen present on the RBC by
testing with a known antibody
I.D. blood type (forward typing)
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Source of Antibody
Anti-sera commercially prepared or can be
patient serum or plasma in BB.
Commercially prepared anti-sera contain
known RBC antibodies to identify unknown
RBC antigens.
Reverse Typing
Routine Testing procedure:
1. ABO/Rh typing
2. Antibody screen (IAT)
3. Antibody Identification
4. Cross match
Only do antibody identification if IAT is
positive. If IAT is negative-indicates
there are no unknown antibodies present
in patients sample.
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Important to understand reagent so that
you understand what you are looking for
or identifying
Antigen
Antibody
ABO/Rh
Pt. RBC
Commercial
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Anti-A, Anti-B, Anti-D
Antibody Screen Screen cells
Antibody I.D. Panel Cells
Cross-match Donor cells
Pt. Serum
Pt. Serum
Pt. Serum
Reagents – 4 basic groups
1. Reagent RBC- Known RBC antigens
2. Antisera- Known RBC antibodies
3. Antiglobulin (AHG)
4. Potentiators
Reagents are regulated by the FDA:
minimum standards for reagents used for
testing.
1. Specificity
2. Potency
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Reagent Quality Control
Technical procedures to determine
analytical testing phase work properly.
Specific Q.C. requirements
Checks:
Reagents
Equipment
Reagents are:
1. Monoclonal (single clone of cellsspecificity)
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Advantage vs disadvantage
2. Polyclonal (human source: mixture of
cells-contains multiple antibodies)
Assist in identifying antigen present on
patients RBC that may cause and
reaction in vivo.
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Anti-Sera for ABO typing (forward typing):
• Anti-A and Anti-B determines the presence of
A,B or no antigens on RBC.
• Test performed using Donor/Patient RBC with
known Anti-sera
• (+) agglutination = antigen present
• (=) agglutination = antigen absent
• Identifies the 4 major blood groups
A: posses A antigens
B: posses B antigens
AB: posses both A and B antigens
O: lack antigens (has no antigens)
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Anti-sera for Rh typing:
There are multiple antigens in the Rh
system (c,C,D,E,e), most prevalent and
most important to identify is D.
Identify the presence (+) or absence (=)
of the D antigen on patient RBC.
Use anti-D reagent combined with testing
RBC.
Agglutination: D present, D (+)
No agglutination: D absent, D(=)
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Antiglobulin Reagent: (AHG)
Detects IgG antibodies and Complement
protein that have attached to RBC.
2 Types
Polyspecific
Monospecific
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Check Cells (Coomb’s Control Cells)
Required control system by AABB
standards
Control system:
RBC commercially prepared with IgG
antibodies
Identified true negative reactions-false
negative
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Reagent RBC
Reagent A1- RBC with known antigen (A)
mixed with serum to identify or confirm
ABO typing
Reagent B- RBC with known antigen (B)
mixed with serum to identify or confirm
ABO typing
Testing phase known as Reverse typing or
Back typing.
Patient posses the antibody directed
against the antigen of their ABO group.
 Example: Group A individual: A antigens
on RBC; Lack B antigen; produce B
antibodies (in serum or plasma). Serum
agglutinates with B cell reagent.
 Group B individual: B antigens on RBC;
Lack A antigen; produce A antibodies (in
serum or plasma).
Serum agglutinates with A cell reagent
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Screen Cells-Group of known reagents that
contain known antibodies.
Looks for antibodies with specificity to RBC
antigens in patient and donor sample (naturally
occurring or from exposure)
Commercially available: Group O donor source
AABB standards states antibodies test performed
on recipient specimen required
Blood group antigens expressed on or in screen
cells: D, C, E, c, e, M, N, S, s, P1, Lewis,
Lutheran, Kell, Duffy and Jka, Jkb
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Panel Cells (10 + reagent based testing
system)
Group of test to determine the specificity
of RBC antibody that was Identified in
antibody screen.
Antigenic profile is important.
Lectins which are useful in identifying
certain antibodies through panel cells.
Lectins: pg 50
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Testing procedure uses tube method and
slide method.
New method include:
Gel technology
Microplate
Solid phase – serological methodautomative
Chapter 3: Genetics
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Blood type is determined by genetic inherited
patterns.
Phenotype: observable trait
Genotype: actual genetic make-up
Predict genotype, if you know phenotype and
can predict phenotype, if you know genotype.
Blood type is determined by the antigen present
on the RBC.
Punnet Square
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Genes: unit of inheritance on a
chromosome. They are located on specific
areas of the cells called genetic loci.
Alleles: Form or different forms of a gene
of a given loci
Ex: A, B and O alleles on the ABO gene
locus.
Polymorphic: having two or more alleles at
a given locus. (Rh system)
Inheritance Pattern:
1. Co-dominant (equal expression of a gene
on an individual)
2. Recessive or dominant ( only one alleles
is expressed on the cell)
3. Amorphic expression: gene present, but
does not express detectable product.
Mandelian Principle: Independent
segregation of traits
4. Mutations
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Homozygous vs Heterozygous inheritance
Homozygous: 2 alleles for a given trait
are the same-genotype are identical genes
Heterozygous: 2 alleles are inherited are
different-genotype are different.
Agglutination reactions will be effected by
inheritance pattern.
Homozygous pattern- stronger reactions
Heterozygous pattern- weaker reactions
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Inheritance pattern with Cis and Trans
position. (related to the Rh system and
how it expresses itself)
Cis : gene expression is from the same
chromosome
Trans: gene expression is from different or
opposite Chromosome.
Can help determine agglutination reaction.
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Silent Genes- “Amorph”, present and
cause problems, but do not produce
detectable antigen.
Result in unusual phenotype
Example is a “Null” type individual- blood
type is not apparent or predictable- see
with the Rh system.
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Paternity Testing
Direct exclusion: child has a trait present
that neither parent posses.
Indirect exclusion: child lacks a gene that
should be inherited from the parent in
question.
Inclusion: when the child has the
predictable traits that are expected form
the parent in question.