Sensitization and

Agglutination

Antigen-Antibody reaction
Red cell Ag-Ab reaction can detected by a
number of techniques
Most frequently used
Hemolysis
Occurs if the entire complement sequence is
activated following Ag-Ab interaction
Frequently stops at C3 no lysis

Agglutination
Used as indicator of Ag-Ab reaction

Agglutination Reactions
Two Stage Process:
Stage 1 Sensitization:
attachment of Antibody to Antigen on the RBC
membrane.

Stage 2 Lattice formation (agglutination):

formation of bridges between the sensitized red
cells to form the lattice that constitutes
agglutination.

Stage 1: Sensitization
This represents what occurs during stage one of agglutination.

Antibody molecules attach to their corresponding

antigenic site (epitope) on the red blood cell membrane.
There is no visible clumping.
Red cells must be close enough for the Fab portion of
Ab to bind and make bridges between cells

Stage 2: Lattice Formation

This represents what occurs during stage 2 of agglutination:

Antibody molecules crosslink RBCs forming a lattice
that results in visible clumping or agglutination.

 Sensitization by IgG does not result in

agglutination
IgG is too small to span the distance
between two red cells
IgM can easily cause agglutination
For agglutination to occur, the repulsive
forces keeping red cells apart must be
overcome

The Zeta Potential

The electric repulsion between cells
This explains why cells do not agglutinate
Red cells have negative charge due to
sialic acid molecules
When red cells are in solution containing
free ions:
Cations are attracted to the vely charged red
cells
This forms a repelling cloud around the cell

 The Zeta Potential can

be varied by altering
the charge on red
cells
This can affect both
sensitization and
agglutination
Reducing the cloud
density allow Abs to
approach the cells,
sensitize and then
agglutinate them

Factors affecting the Zeta Potential

1. Removal of sialic acids by enzymes
2. Introduction of bipolar Albumin
Albumin dissipates some of the +ve charges
around cells, reducing zeta potential

Albumin

Factors affecting the Zeta Potential

3. Increase the ionic strength of the medium
Increasing conc. of cations in medium cause

Increase in the density of ions around the red cell

which cause

Size of cloud of cations is decreased

Zeta potential decreases
Red cell approach each other easily
Agglutination is facilitated

Factors affecting the Zeta Potential

4. Decreasing the ionic strength of medium by
using low ionic strength saline (LISS)
Decreasing conc. of cations in medium
Leads to decrease in density of ions around
red cells
This increases sensitization
But decreases agglutination

Factors affecting Red Cell Sensitization

1- Ratio of Ab to Ag
Sensitization occurs easily when at higher
conc. of Ab
This can be done by increasing conc. of
serum containing the Ab to conc. of cells

Factors affecting Red Cell Sensitization

2- The pH of reaction
mixture
At a pH below the pI, Abs
have +ve charges
This makes it easier for the
Ab to bind to the vely
charged red cells
Optional pH for sensitization
is 6.5 to 7.5 (Ab +vely
charged)

pH 8

pH 7

Factors affecting Red Cell Sensitization

3- Temperature
Ag- Ab reactions are exothermic
Therefore, Abs bind to a greater degree at
lower temperature
But at lower temperatures, rate of reaction is
reduced
To speed up reaction, tests are done at 37 oC

 Temperature can also affect Ag accessibility on

red cells
Some IgM Abs bind best at 4oC (cold Abs)

Temperature can make conformational changes

in the Ag
More Ag sites are exposed as the temperature is
lowered allowing increased binding of Ab
37oC

4oC

Most naturally occurring cold Abs are of no

clinical significance
Compatibility testing is done at 37oC

Factors affecting Red Cell Sensitization

4- Ionic strength of the medium
When RBCs are suspended in LISS the cloud
of ions around the cell is less dense than in
isotonic saline
Reduced conc. of cations surrounding RBCs
allow +vely charged Abs easier to access Ag
sites
Rate of sensitization increases

Factors Influencing RBCs Agglutination

Agglutination occurs when RBCs are close
enough allowing the Ab to bridge adjacent
cells
1- Ionic strength of Medium
Rate of sensitization increases in LISS
Agglutination impaired because of increase in
zeta potential

Factors Influencing RBCs Agglutination

2- Presence of Albumin in medium
3- Enzyme treatment of red cells

Remove vely charged sialic acid

& therefore reduce the zeta potential
This make cells come closer & agglutinate
But, certain Ags can be destroyed by enzymes (M,
N, S, Fya, Fyb)

4- Temperature

Factors Influencing RBCs Agglutination

5- Antigen Density
The greater the number of Ags on red cell, the
greater the sensitization
Binding of +vely charged Abs to red cells lower the
zeta potential
And therefore enhances agglutination

Increased Ag density also increases chance

of bridging

Factors Influencing RBCs Agglutination

6- Ag Clustering and Mobility
Clustering facilitates agglutination by
increasing likelihood of Ab binding at that site
Cluster of some Ags can occur after enzyme
treatment of cells

Clustering of Ags

Factors Influencing RBCs Agglutination

7- Antibody Characteristics
Ability of Ab to agglutinate cells depend on the Ig
class
IgM has a wider span than IgG, and therefore more
effective agglutination
IgG can be chemically modified to increase its span
250-300 Ao

300 Ao

150 Ao

A
g

IgG

IgG

IgM

Summary
Sens.
Removal of RBCs sialic acid by
enzymes
Introduction of bipolar albumin
Increasing ionic strength of the
medium
decreasing ionic strength of the
medium

___

Agg.