M. Zaharna Blood Bank Lab. 2009 Sensitization and Agglutination.

M. Zaharna Blood Bank Lab. 2009

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.


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 1: Sensitization


This represents what occurs during stage 2 of agglutination:

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

Stage 2: Lattice Formation


• 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



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



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



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



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


• 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

• Most naturally occurring cold Abs are of no clinical significance– Compatibility testing is done at 37oC

4oC 37oC



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



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



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



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



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

IgMAg

300 Ao

IgG IgG

150 Ao250-300 Ao


Summary

Sens. Agg.

Removal of RBCs sialic acid by enzymes

Introduction of bipolar albumin ___

Increasing ionic strength of the medium

decreasing ionic strength of the medium

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M. Zaharna Blood Bank Lab. 2009 Sensitization and Agglutination.

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