1. Sensitization and Agglutination

2. 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

4. 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.

5. 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.

7. 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.

8. 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

9. 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.

11. 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

13. 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

14. 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

15. Factors affecting the Zeta Potential

16. 1- Using Enzyme

17. 2. Introduction of bipolar Albumin
Albumin dissipates some of the +ve charges around cells, reducing zeta potential
Albumin

19. 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

20. Increase the ionic strength of the medium

21. 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

22. Decreasing the ionic strength of medium by using low ionic strength saline (LISS)

23. 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

24. Factors affecting Red Cell Sensitization
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)

25. 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

26. 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

27. 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

29. Factors Influencing RBCs Agglutination
Agglutination occurs when RBCs are close enough allowing the Ab to bridge adjacent cells

30. 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)

31. 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)

32. 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

33. 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

34. 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 Ao
300 Ao
150 Ao
IgM Ag
IgG
IgG

35. 7- Antibody Characteristics

36. 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