MLS311: IHTM (LECTURE) FUNDAMENTALS OF IMMUNOLOGY

FUNDAMENTALS OF IMMUNOLOGY
ANTIGEN & ANTIBODY REACTIONS
• Demonstration of red cell antigen-antibody reactions is key to
immunohematology. -AABB Technical Manual
RED CELL ANTIGENS
• Most common forms of blood group antigens
• Glycoproteins: HLA
• Glycolipids: ABH, Lewis, Ii and P blood group antigens
• Proteins: Rh, M, N blood group antigens
RELATIVE IMMUNOGENICITY OF DIFFERENT BLOOD
GROUP ANTIGENS
Blood Group Blood Group Immunogenicity IGA
Antigen System (%)* • About 30% of anti-A and anti-B antibodies are of the IgA class
D (Rh0) Rh 50 • Anti-IgA antibodies can cause severe anaphylaxis if IgA are
K Kell 5
transfused in plasma products to patients who are deficient in
c (hr’) Rh 2.05
IgA.
E (rh’’) Rh 1.69
k Kell 1.50
o Individuals who are IgA deficient must be given washed
e (hr’’) Eh 0.56 components.
Fy3 Duffy 0.23 • IgA can increase the effect of IgG induced RBC hemolysis.
C (rh’) Rh 0.11 IGE
Jka Kidd 0.07 • The FC portion of the IgE molecule attaches to basophils
S MNSs 0.04 and mast cells and facilitates histamine release when an
Jkb Kidd 0.03 allergen binds to the Fab portion of the molecule and cross-
s MNSs 0.03 links with a second molecule on the cell surface
• Percentage of transfusion recipients lacking the blood group antigen • Presence of IgE antibodies may cause urticaria
(in the first column) who are likely to be sensitized to a single
transfusion of red cells containing that antigen.
IMMUNOGLOBULINS (ANTIBODIES)
RBC Immune Vs. Non-RBC Immune
• RBC Immune Antibody
o found in the serum of individuals who were previously
exposed to RBC antigens by transfusion, injection, or
pregnancy IGD
o Rh, Kell, Duffy, Kidd, and Ss blood group systems • Bound to the membrane of B cells
o Usually IgG • Least significant in blood banking
• Non-RBC Immune Antibody/Naturally-occurring • We just stated that IgM antibodies are usually NOT clinically
o found in the serum of individuals who have never been significant. There is a notable exception to this statement!
previously exposed to RBC antigens by transfusion, What is it?
injection, or pregnancy. • ABO antibodies are VERY CLINICALLY SIGNIFICANT.
o ABH, Hh, Ii, Lewis, MN, and P blood group systems). Most transfusion fatalities are the result of an ABO
o Usually IgM incompatibility.
o Probably produced in response to substances in the
environment that resemble RBC antigens such as pollen
grains and bacteria membranes
• Clinically Significant Antibody:
o An antibody that decreases the red cell survival.
o Able to destroy red blood cells in vivo
o anti-A,B, Anti-Jka , etc.
o Reactive at 37°C
• Alloantibody: Antibody produced in response to foreign
antigens
• Autoantibody: Antibody produced in response to self-
antigen. May be warm or cold reacting.
CHARACTERISTICS OF IGG CLASS ANTIBODIES
Structure Monomer IGM ANTIBODY’S
Phase of Reactivity Warm: reacts best at 37oC 1. Cold Reacting
Placenta Can cross the placental barrier except IgG2
2. Good complement activator
Complement Poor to good C’ activators. Two IgG required to
(C’) Activation activate C’ to completion except IgG4
3. Pentamer
Clinical Usually clinically significant RBC immune 4. ABO, Ii, Lewis, MN, P, Lua
Significance Antibody IGG ANTIBODY’S
CHARACTERISTICS OF IGM CLASS ANTIBODIES 1. Warm reacting
Pentamer: can be dissociated by sulfhydryl reducing 2. Poor complement activators
reagents such as β-2 mercaptoethanol (2-ME) or 3. Monomer
Structure dithiothreitol (DTT), 2-aminoethylisothiouronium 4. Rh, Kell, Kidd, Duffy, Lub, SsU
bromide (AET)
Phase of Reactivity Cold: reacts best at 4-10oC
Placenta Cannot cross the placental barrier
Complement GOOD (most) complement activators - one IgM
(C’) Activation can activate C’ to completion
Clinical “Usually” NOT clinically significant, Non RBC
Significance immune.

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MLS311: IHTM (LECTURE) FUNDAMENTALS OF IMMUNOLOGY

HEMOLYSIS
• When antibody (IgG or IgM) has activated complement to
completion, i.e. MAC(C5b-9) is formed.
SEPARATION OF RED BLOOD CELLS
• Antibody (immunoglobulin) by itself cannot tear a hole in the
• In vivo red blood cells are built in such a way as to keep their
RBC membrane. It is the C’ that does that.
distance from each other. It is a good thing. Theories include:
• Seen when supernatant is CLEAR and RED.
o Waters of Hydration: Water bound by RBC membrane
• Not seen at the AHG phase! Why??
glycoproteins helps maintain the distance between
COMPLEMENT
rbc’s. (Water envelope)
o Electrostatic charges: Electron cloud surrounds RBC • C1 molecules bind with two adjacent Ig Fc regions. A
with a net negative charge called the zeta potential. pentameric IgM molecule provides two Fc regions side by
side, thereby binding complement.
• To observe Ag/Ab reactions in vitro these forces need to be
overcome to enable Ab’s to attach to their corresponding Ag • A monomeric IgG molecule binds C1q less efficiently, and
IN VITRO (IN THE TEST TUBE) INDICATORS OF AG/AB two IgG molecules are needed in close proximity to bind
RXN’S complement.
1. Sensitization • Antibodies against the Rh antigens usually do not bind
o Antibody coating RBC without agglutination complement due to the low level of Rh antigens on RBC
o Attachment of antibody to antigen surfaces
2. RBC Hemolysis
o Complement mediated lysis of red blood cells.
3. Agglutination
o Antibody mediated clumping of red blood cells that
express corresponding antigens on their surface.
SENSITIZATION
• Attachment of antibody to corresponding antigen on RBC
membrane, ONLY.
• This reaction is NOT SEEN at the Immediate Spin (IS), Room
Temperature (RT), or 37oC (LISS) phases.
• This reaction requires the use of an Antihuman Globulin
reagent in the Direct (DAT) or Indirect Antiglobulin Tests
(IAT) to observe.
DIRECT COOMB’S TEST

AGGLUTINATION REACTIONS
Two Stage Process:
• Stage 1
o Sensitization: attachment of Antibody to Antigen on the
RBC membrane.
• Stage 2
o Lattice formation: formation of bridges between the
sensitized red cells to form the lattice that constitutes
agglutination.

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MLS311: IHTM (LECTURE) FUNDAMENTALS OF IMMUNOLOGY

BINDING FORCES
1. Hydrophobic bonds: major bond
2. Hydrogen bonds
o formation of hydrogen bridges between appropriate
atoms (O-H-O, N-H-N)
3. Van der Waal’s Force / London Dispersion force
o nonspecific
o Linkages occur between electrically neutral molecules
when they are so close to each other
4. Electrostatic attraction/force
o Attraction between a (-) with a (+) pole

STAGE 1: SENSITIZATION
• Antibody molecules attach to their corresponding Antigenic
site (epitope) on the red blood cell membrane. There is no
visible clumping.
• Attachment of antibody to antigen
• Attachment between Antigen and Antibody is dependent on
spatial complementarity (Lock and Key concept) and on
weak non-specific intermolecular forces including:
o Electrostatic forces
o Hydrogen Bonds
o Hydrophobic forces
o van der Waals forces
o Does NOT involve covalent bonding

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MLS311: IHTM (LECTURE) FUNDAMENTALS OF IMMUNOLOGY
LAW OF MASS ACTION • Centrifugation
• Due to weakness of these forces Ag/Ab complexes obey the o Brings Ab’s and Ag’s into close proximity
Law of Mass Action which is: o Undercentrifugation: may result in false negative
o Antigen and antibody complexing is REVERSIBLE. o Overcentrifugation: may result in false positive
• Equilibrium is the point at which the number of bonds being • Zeta Potential
formed equals the number of bonds being broken. o Net negative charge surrounding RBC
FACTORS AFFECTING STAGE 1 OF AGGLUTINATION • Waters of Hydration
• Temperature o Acts as an insulating bubble around RBC
o Cold (4-10oC) o Water molecules tightly bound to hydrophilic
▪ IgM antibodies like ABO, P1 , Lewis antibodies macromolecules on the red cell surface.
bond better at low temperatures due to ZETA POTENTIAL ON RBC
carbohydrate nature of antigens
o Warm (37oC)
▪ IgG antibodies Rh antibodies bond better at 37oC
due to protein nature of Rh antigens.
• pH
o Changes in pH can affect electrostatic bonds.
o Optimal reaction phase is 6.5-7.5
o Some Ab’s like lowered pH particularly anti-M and
anti-Pr (pH 6.5)
• Incubation Time
o The time needed to reach equilibrium phase
o Saline systems: 60-120 minutes at 37oC
o Use of enhancement media like BSA and LISS can
reduce incubation time significantly: EFFECT OF CENTRIFUGATION
▪ High Protein System (i.e. BSA): 30-60 minutes
at 37oC
▪ LISS: 10-15 minutes
• Ionic Strength
o In normal saline, Na+ and Clions cluster around and
partially neutralize opposite charges on Ag and Ab
molecules, which hinders the association of Ab with
Ag.
o If you lower the ionic strength of the test system then
you increase rate of Ag-Ab association.
o Using LISS (low ionic strength solution) 0.2% NaCl EFFECT OF ENHANCEMENT MEDIA AND POTENTIATORS
decreases incubation time. • Protein Media
STAGE 2: LATTICE FORMATION • LISS
• Lattice formation: formation of bridges between the • Macromolecule additives
sensitized red cells to form the lattice that results in o PEG
agglutination (clumping) of red blood cells. • Polycations
• Heidelberg Phenomenon Illustrate the effect of antigen and o Polybrene
antibody mass on lattice formation o Protamine
• Enzymes
o Ficin
o Papain
o Trypsin
o Bromelin
• Albumin
• AHG

FACTORS AFFECTING STAGE 2 OF AGGLUTINATION

• Size of the Immunoglobulin
o IgG: Monomer, requires AHG to cause lattice formation
o IgM: Pentamer, does not require AHG to cause lattice
formation
• Number of binding sites
o IgG: Two binding sites (anti-D, anti-Jka , etc.)
o IgM: Ten binding sites (anti-A, anti-I, etc.

• Location and Number of Antigenic Determinants

o A, B, M and N antigens: 600,000 to >1,000,000 antigen
sites per RBC
o Kidd: approximately 10-20,000 Ag sites/RBC

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MLS311: IHTM (LECTURE) FUNDAMENTALS OF IMMUNOLOGY
MICROSCOPIC VIEW

READING AND INTERPRETING AG/AB REACTIONS

• Agglutination
o Grade reaction strength: Neg to 4+
• Hemolysis
o ALWAYS observe the supernatant of the test tube after
centrifugation. If the supernatant is clear and red
Hemolysis is indicated. THIS IS A POSITIVE
REACTION! And indicates a Nasty antibody.
• Rouleaux
o Increased proteins can cause RBCs to clump and
stack (false positive)
• Mixed Field Agglutination
o Presence of two cell populations: such as Group O
cells in a Group A patient. Anti-A will only agglutinate A
cells resulting in a mixture of clumping cells and free cells

MIXED-FIELD AGGLUTINATION

BLOOD SAMPLES REQUIRED FOR TESTING

• Different tests in the blood bank require different samples.
• Serum preferred for complement studies.
• EDTA chelates divalent ions (Ca++ and Mg++) and therefore
prevents complement activation. Sodium heparin on the other
hand, prevents cleavage of C4
• For DAT and elution studies, plasma is preferred since fibrin
clot cause false positive reactions
• Years of testing with both samples have shown that for most
tests, plasma is comparable to serum. Currently, however,
plasma is used routinely in place of seru

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