Sensitization and
Agglutination
M. Zaharna Blood Bank Lab. 2009
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
M. Zaharna Blood Bank Lab. 2009
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.
M. Zaharna Blood Bank Lab. 2009
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
M. Zaharna Blood Bank Lab. 2009
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.
M. Zaharna Blood Bank Lab. 2009
• 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
M. Zaharna Blood Bank Lab. 2009
M. Zaharna Blood Bank Lab. 2009
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
M. Zaharna Blood Bank Lab. 2009
• 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
M. Zaharna Blood Bank Lab. 2009
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
M. Zaharna Blood Bank Lab. 2009
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
M. Zaharna Blood Bank Lab. 2009
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
M. Zaharna Blood Bank Lab. 2009
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
M. Zaharna Blood Bank Lab. 2009
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)
M. Zaharna Blood Bank Lab. 2009
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 37oC
M. Zaharna Blood Bank Lab. 2009
• 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
M. Zaharna Blood Bank Lab. 2009
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
M. Zaharna Blood Bank Lab. 2009
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
M. Zaharna Blood Bank Lab. 2009
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
M. Zaharna Blood Bank Lab. 2009
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
M. Zaharna Blood Bank Lab. 2009
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
M. Zaharna Blood Bank Lab. 2009
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
M. Zaharna Blood Bank Lab. 2009
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
M. Zaharna Blood Bank Lab. 2009
___
Agg.