Hemagglutination

Many  viruses (including influenza virus)  have  the  ability  to agglutinate red  blood cells (RBCs),  a  phenomenon  known as  hemagglutination, by  linking cells  together such that they  form  a mesh or network. In order for the reaction to occur, the virus needs to be present in sufficient concentration to form cross-bridges between RBCs, causing the agglutination. Below this concentration, nonagglutinated RBCs remain separate and as such will sink in a solution to form a pellet. The hemagglutination assay is performed by mixing RBCs with diluted virus in a multi-well plate with hemispherical (round-bottomed) wells. Nonagglutinated RBCs pellet to sit as a tight  red  button at the base  of the  well, while agglutinated RBCs form  a lattice-work structure which coats the sides  of the  well, remaining as a diffuse red  coloration throughout the  well. The assay determines the number of hemagglutinating particles in a given suspension of virus, i.e. it is a particle count assay, not a measure of infectivity. Nevertheless it is one of the most routinely used indirect methods for the determination of virus titer.  The assay uses an end-point titration method; serial two-fold dilutions of virus are mixed with an equal volume of RBCs and the wells are observed for agglutination. The end point of the titration is the last dilution showing complete agglutination that by definition is said to contain one HA unit.  HA titer of a virus suspension is therefore defined as the reciprocal (inverse) of the highest dilution that causes complete agglutination

Figure:  Schematic of hemagglutination (a) and  typical results  for a hemagglutination assay(b). Serial doubling dilutions of virus show the  agglutination end  point  at 1:512.

and is expressed as the  number of HA units per unit  volume. The example upon which a calculation of the HA titer can be made is shown in Figure. The end point in this figure is the well containing virus at a dilution of 1⁄512. If 0.2 ml virus dilution was added per well the HA titer would be 512 HA units per 0.2 ml or 2560 HA units ml^-1.