sensitization and agglutination l.
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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

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antigen antibody reaction
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
Agglutination Reactions
  • 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.

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 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


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

factors affecting red cell sensitization15
Factors affecting Red Cell Sensitization

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
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



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
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 like (Rh) 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

300 Ao

250-300 Ao

150 Ao