Sensitization and Agglutination - PowerPoint PPT Presentation

Sensitization and Agglutination

• 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

• Two Stage Process:
• Stage 1 Sensitization:
• attachment of Antibody to Antigen on the RBC membrane.
• Stage 2 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: agglutination

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

• This can affect both sensitization and agglutination
• Reducing the cloud density allow Abs to approach the cells, sensitize and then agglutinate them

Albumin

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

• 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

• Decreasing conc. of cations in medium
• Leads to decrease in density of ions around red cells
• This increases sensitization
• But decreases agglutination

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

• 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

• Ab -Ag 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

• 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

37oC

4oC

• 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

• 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

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

• 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

• Clustering facilitates agglutination by increasing likelihood of Ab binding at that site
• Cluster of some Ags like (Rh) can occur after enzyme treatment of cells

Clustering of Ags

IgM

Ag

IgG

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

300 Ao

250-300 Ao

150 Ao

IgG