RED CELL AGGLUTINATION

An Antigen-Antibody Reaction
 In-vitro testing for the detection of antigens
or antibodies may be accomplished by
commonly used techniques such as: (1)
hemagglutination; (2) Precipitation; (3)
agglutination inhibition; (4) hemolysis.

 Other techniques that are used to quantify

antigens or antibodies uses: (1) isotopes; (2)
enzymes; (3) Fluorescent labels.
 HEMAGGLUTINATION

◦ AGGLUTINATION

 Is the antibody-mediated clumping of particles that

express antigen on their surface.

 It is the endpoint for most tests involving red cells and

blood group antibodies.

 2 stages:
 Sensitization
 Lattice Formation
 Sensitization
◦ The attachment of antibody to antigen on the red
cell membrane.

◦ Factors that affect Sensitization:

 Chemical Bonding
 For sensitization to occur, a non-covalent bond must form
between antigen and antibody. These bonds are generally
weak and are active only over a very short distance.
 Hydrogen bonds
 Electrostatic bonds or Ionic bonds
 van del Waals forces
 Equilibrium Constant of the Antibody (Ko)
 The Ko reflects the degree to which antibody and antigen
associate and bind to one another and the speed of
reaction.
 The higher the Ko value the better the association or fit.

 Temperature
 Most blood group antibodies react within restricted
temperature ranges.
 2 categories:
 Cold reacting antibodies ( ABO, Lewis, Ii, P, MN)
 Warm reacting antibodies ( Rh, Kell, duffy, Kidd, Ss)

 pH
 Changes in pH can affect electrostatic bonds
 For most routine testing, a pH around 7.0 should be used.
 Incubation Time
 The time needed to reach equilibrium differs for different
blood group antibodies.
 Most clinically significant antibodies are detected at 30 to
60 minutes of incubation at 37˚C.

 Ionic Strength
 Lowering the ionic strength of the reacting medium
enhances electrostatic attractions between antigens and
antibodies.

 Antigen-Antibody Proportions
 Antigen excess reduces the number of antibody molecules
bound per red cell. Limiting their ability to aggluninate.
 Antibody excess is therefore more desirable in most
routine testings.
 Lattice Formation
◦ Results from the formation of bridges between the
sensitized red cells.
 Factors that affect Lattice Formation
◦ Size of Immunoglobulin
 IgG: monomer, takes two to activate complement.
 IgM: Pentamer, takes one to activate complement.

◦ Number of Binding Sites

 IgG: two binding sites
 IgM: ten binding sites

◦ Location and Density of Antigen Sites

 A,B,M,N antigens are on the outer edges of the red cell
glycoproteins and have higher densities allowing IgG
antibodies to crosslink.
◦ Centrifugation
 Brings Antigens and Antibodies close to proximity

◦ Zeta Potential
 Red cells suspended in saline have a net negative
charge at their surface and therefore repel one
another.

◦ Waters of Hydration
 Acts as insulating bubble around RBC.
 Water molecules are tightly bound to the hrdrophilic
macromolecules on the red cell surface.
 READING AND INTERPRETING
HEMAGGLUTINATION REACTIONS

 4+ 1+
 ONE SOLID AGGLUTINATE
SMALL AGGLUTINATES
TURBID BACKGROUND

 3+
 SEVERAL LARGE AGGLUTINATES
HEMOLYSIS

 2+ 0
 MEDIUM-SIZE AGGLUTINATES, NO
AGGLUTINATION
 CLEAR BACKGROUND