Antigen-Antibody reaction • Red cell Ag-Ab reaction can be 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 • In vitro, lysis of RBC’s rarely occurs. • Agglutination • Used as indicator of Ag-Ab reaction
Sensitization • Sensitization is whereby the Ab binds to Ag on the surface of the cell. • Sensitization does not always result in visible agglutination. • IgG is too small to span the distance between two red cells. • So IgG does not always result in visible agglutination.
Agglutination • Agglutination occurs when bound Ab links adacent red cells forming clumps. • Red cells must be close enough for the Fab portion of Ab to bind and make bridges between cells. • IgM can easily induce agglutination.
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.
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
2. Introduction of bipolar Albumin • Albumin dissipates some of the +ve charges around cells, reducing zeta potential Albumin
Factors affecting the 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
Factors affecting the Zeta Potential • 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
Decreasing the ionic strength of medium by using low ionic strength saline (LISS)
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 isoelectric point, Abs have +ve charges This makes it easier for the Ab to bind to the –vely charged red cells Optimal pH for sensitization is 6.5 to 7.5 (Ab +vely charged) Factors affecting Red Cell Sensitization
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
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 37oC 4oC
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
Factors Influencing RBCs Agglutination 1- Ionic strength of Medium • Rate of sensitization increases in LISS BUT: • Agglutination is impaired because of increase in zeta potential • (Rule: reduced zeta potential facilitate agglutination)
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 (previously discussed)
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
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
IgM Ag IgG 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 300 Ao 250-300 Ao 150 Ao IgG