CLLS 3311

Advanced Clinical
Immunohematology
Antigen and Antibody
Reactions

Demonstration of red cell antigenantibody reactions is key to

immunohematology.
AABB Technical Manual

 Red cell antigen-antibody reactions

that we study and perform include
Patient ABO grouping, Rh typing,
Antibody screens and identifications,
Crossmatching, etc.
An awareness of how antigens and
antibodies react is necessary to
accurately perform the required
testing including: quality control,
trouble shooting, maintenance, etc.

Immune Response
Definition: Physiological mechanism to fight
disease or clear foreign substances.
Primary immune response: First exposure
of a foreign agent. IgM is the predominant
antibody produced that attaches to and fights
the foreign agent. In our case a foreign red
blood cell antigen.
Secondary immune response: Subsequent
exposure of the same foreign agent. Rapid
response in which IgG is the predominant
antibody produced.

Antigen/Antibody Reactions
1. Antigen:

1. Substance capable of eliciting an

immune response when introduced
into a immunocompetent individual
to whom it is foreign.

2. Antigenicity: 2. Ability of an antigen to react with

the products of an immune response
3. Epitopes:

3. Antigenic determinants - structural

site where the antibody combines
with the antigen.

Characteristics of Antigens
1. Antigenicity of an
Antigen is affected
by its:
2. Foreign:
3. Size:
4. Most common
forms of Blood
Group Antigens:

1. Size, shape, rigidity, location,

number of determinants
(epitopes), dose, route of
entry, and genetic factors.
2. Normallydont forget
autoimmune processes
3. >10,000 daltons
Glycoproteins: HLA
Glycolipids: ABH, Le, Ii
and P antigens (Ag)
Proteins: Rh, M, N antigens

Characteristics of
Immunoglobulins (Antibodies)
RBC Immune Vs. Non-RBC Immune
RBC Immune Antibody: antibody that results
from exposure to foreign red cell antigen either
by transfusion or pregnancy (anti-D, etc.)
Non-RBC Immune Antibody: antibody that is
present without any evidence of exposure to
foreign red cell antigen (anti-A, anti-N, etc.)

Clinically Significant Antibody: An antibody

that decreases the red cell survival. Able to
destroy red blood cells in vivo. (anti-A,B,
Anti-Jka, etc.)

Characteristics of IgG Class Antibodies

1. Structure

1. Monomer

2. Phase of Reactivity 2. Warm: reacts best at 37oC

3. Placenta
4. Complement (C)
Activation
5. Clinical
Significance

3. Can cross the placental

barrier
4. Poor to good C activators.
Two IgG required to activate
C to completion.
5. Usually clinically significant
RBC immune Antibody

Characteristics of IgM Class Antibodies

1. Structure

1. Pentamer

2. Phase of reactivity

2. Cold: reacts best at 4-10oC

3. Placenta

3. Cannot cross the placental

barrier
4. GOOD (most) complement
activators - one IgM can
activate C to completion
5. Usually NOT clinically
significant, Non RBC immune.

4. Complement
Activation
5. Clinical
Significance

 We just stated that IgM antibodies

are usually NOT clinically significant.
There is a notable exception to this
statement! What is it?
ABO antibodies are VERY CLINICALLY
SIGNIFICANT. Most transfusion fatalities
are the result of an ABO incompatibility.
Yet anti-M, anti-N, etc. are usually NOT
clinically significant IgM antibodies.

IgG Antibody

IgM Antibody

(Monomer Structure)

(Pentamer Structure)

Images provided by textbook: Figure 14.3, page 97, Kuby

Immunology, 4th Edition, W.H. Freeman and Company

1. Cold reacting

IgM
Antibodys

2. Good complement
activator

3. Pentamer
4. ABO, Ii, Lewis, MN, & P
1. Warm Reacting

IgG
Antibodys

2. Poor complement
activators
3.

Monomer

4. Rh, Kell, Kidd, Duffy, and Ss

Separation of Red Blood Cells

In vivo red blood cells are built in such a way as to
keep their distance from each other. It is a good
thing. Theories include:
Waters of Hydration: Water bound by RBC
membrane glycoproteins helps maintain the distance
between rbcs. (Water envelope)
Electrostatic charges: Electron cloud surrounds
RBC with a net negative charge called the zeta
potential.
To observe Ag/Ab reactions in vitro these forces
need to be overcome to enable Abs to attach to their
corresponding Ag.

In vitro (in the test tube)

indicators of Ag/Ab Rxns
1. Antibody coating RBC without
Agglutination

Sensitization: Attachment of antibody to antigen

that requires antihuman globulin to detect.

2. RBC Hemolysis

Immune mediated lysis of red blood cells.

3. Agglutination

Antibody mediated clumping of red blood cells

that express corresponding antigens on their
surface.

Antibody Coating RBC Membrane

without Agglutination
Attachment of antibody to corresponding
antigen on RBC membrane, ONLY.
This reaction is NOT SEEN at the Immediate
Spin (IS), Room Temperature (RT), or 37 oC
(LISS) phases.
This reaction requires the use of an Antihuman
Globulin reagent in the Direct (DAT) or
Indirect Antiglobulin Tests (IAT) to observe.

Hemolysis
When antibody (IgG or IgM) has
activated Complement to completion.
The complement (C) cascade is the
mechanism that actually destroys the red
blood cell.
Antibody (immunoglobulin) by itself
cannot tear a hole in the RBC
membrane. It is the C that does that.
Seen when supernatant is CLEAR and RED.
Not seen at the AHG phase! Why??

Agglutination Reactions
Two Stage Process:
Stage 1

Sensitization: attachment of Antibody to

Antigen on the RBC membrane. TM

Stage 2

Lattice formation: formation of bridges

between the sensitized red cells to form the
lattice that constitutes agglutination. TM

This represents what occurs during stage

one of agglutination: Sensitization

Stage 1

Antibody molecules attach to their corresponding

Antigenic site (epitope) on the red blood cell
membrane. There is no visible clumping.

This represents what occurs during stage

2 of agglutination: Lattice Formation

Stage 2

Antibody molecules crosslink RBCs forming a lattice

that results in visible clumping or agglutination.

Stage 1 of Agglutination:

Sensitization

Attachment of antibody to antigen

Attachment between Antigen and Antibody
is dependent on spatial complementarity
(Lock and Key concept) and on weak nonspecific intermolecular forces including:
Electrostatic forces, Hydrogen Bonds,
Hydrophobic forces, and van der Waals forces.
(Does NOT involve covalent bonding.)

Weak Non-specific
Intermolecular Forces
Electrostatic forces
Attraction of oppositely charged or ionized molecules
Force of attraction is inversely proportional to square
of the distance between charges: closer you get the
stronger the attachment of Ag to Ab.

Hydrogen bonds
Weak, reversible hydrogen bridges between
hydrophilic groups which are stronger at lower
temperatures.

Weak Non-specific
Intermolecular Forces
Hydrophobic forces
Water hating groups (valine, leucine) come into
close contact and exclude water between them,
resulting in lower free energy system disrupting
the water envelope.
Disruption of water envelope is the result of
hydrophobic forces: requires close proximity

Weak Non-specific
Intermolecular Forces
van der Waals bonds
Temporary disruption of electrons in one
molecule effectively forms a dipole which
causes the formation of a dipole in another
molecule, the two dipoles then exhibit an
attraction for each other: Requires close
proximity.

LAW OF MASS ACTION

Due to weakness of these forces Ag/Ab
complexes obey the Law of Mass Action
which is:

Antigen and antibody complexing is

REVERSIBLE.
Equilibrium is the point at which the
number of bonds being formed equals
the number of bonds being broken.

Factors Affecting
Stage 1 of Agglutination
Temperature

Cold (4-10oC): Exothermic reactions occur when

H+ bonds are made with carbohydrates at LOW
temperatures.
ABO, P1, Lewis antibodies bond better at low
temperatures due to carbohydrate nature of antigens

Warm (37oC): Entropy driven reactions associated

with hydrophobic bonding to proteins.
Rh antibodies bond better at 37oC due to protein nature
of Rh antigens.

Factors Affecting
Stage 1 of Agglutination
pH
Changes in pH can affect electrostatic bonds.
Optimal range is physiologic pH
Some Abs like lowered pH particularly anti-M

Incubation Time
The time needed to reach equilibrium
Saline systems: 30-60 minutes at 37oC
Enhancement media can reduce incubation time
significantly: LISS is 10-15 minutes

Factors Affecting
Stage 1 of Agglutination
Ionic Strength
In normal saline, Na+ and Cl- ions cluster around
and partially neutralize opposite charges on Ag
and Ab molecules, which hinders the association
of Ab with Ag.
If you lower the ionic strength of the test system
then you increase rate of Ag-Ab association.
Using LISS (low ionic strength solution)
decreases incubation time.

Stage 2 of Agglutination:
Lattice Formation
Lattice formation: formation of bridges
between the sensitized red cells to form the
lattice that results in agglutination (clumping)
of red blood cells.

Concentration of Ag and Ab

also affects agglutination reactions, both

the first and second stages.

Factors Affecting
Stage 2 of Agglutination
Size of the Immunoglobulin
IgG: Monomer, takes two to activate C, etc.
IgM: Pentamer, takes one to activate C, etc.

Number of binding sites

IgG: Two binding sites (anti-D, anti-Jk a, etc.)
IgM: Ten binding sites (anti-A,B, anti-I, etc.)

Factors Affecting
Stage 2 of Agglutination
Location and Number of Antigenic
Determinants
A, B, M and N antigens: 600,000 to >1,000,000
antigen sites per RBC
Kidd: approximately 10-20,000 Ag sites/RBC

Centrifugation
Brings Abs and Ags into close proximity
Undercentrifugation: may result in false negative
Overcentrifugation: may result in false positive

Factors Affecting
Stage 2 of Agglutination
Zeta Potential
Net negative charge surrounding RBC
Now considered a minor player

Waters of Hydration
Acts as an insulating bubble around RBC
Water molecules tightly bound to
hydrophilic macromolecules on the red cell
surface.

Reading and Interpreting

Ag/Ab Reactions
Agglutination
Grade reaction strength: Neg to 4+
Harmening Color Plate 2.

Hemolysis
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.

Reading and Interpreting

Ag/Ab Reactions
Rouleaux
Increased proteins can cause RBCs to
clump and stack - false positive

Mixed Field Agglutination

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.

Potentiators
We have not covered the topic of
potentiators. This is found on pages 60-62
in Harmening and is Objective #10.
We also did not cover Solid Phase and Gel
testing techniques. This is found on page
62-63 in Harmening and is Objective #6.