Haemagglutination Test

Theoretical Background/Context

• Hemagglutination is used for the diagnosis of some enveloped viruses such as influenza viruses. 
• This method relies on the specific feature of some enveloped viruses that can adsorb to red blood cells (RBCs). 
• Specifically, hemagglutinin5 (HA), an envelope glycoprotein of some enveloped viruses, imparts this property. 
• In the absence of virus particles, RBCs precipitate by gravity to the bottom of the well, giving rise to a distinct red-colored dot in a conical shaped well. 
• In the presence of virus particles, RBCs clump together as a result of interaction between HA proteins of virus particles and RBC, leading to a lattice formation. In this case, as RBCs are dispersed as a clump, a red dot is not formed. 
• In a given sample, a red dot will appear beyond a certain dilution fold. To carry out a hemagglutination assay, a twofold serial dilution of virus-containing samples is dispensed into individual wells of a 96-well microtiter plate. 

Then, aliquots of RBC are added to each well. The highest dilution at which clumping is observed is regarded as the HA titer of the sample. The virus titer in a sample can be estimated by multiplying the dilution fold.

Principle Work of Haemagglutination Test

• In the Haemagglutination virus simulation lab, hemagglutination is a classical method for viral diagnosis and it is still used for the diagnosis of influenza virus today. 
• One outstanding advantage of this method is that it does not require any equipment. Moreover, it is a robust and rapid diagnostic tool, but the sensitivity is somewhat limited.
• Haemagglutination assay applies the process of hemagglutination, in which sialic acid receptors on the surface of red blood cells (RBCs) bind to the hemagglutinin glycoprotein found on the surface of influenza virus (and several other viruses) and create a network, or lattice structure, of interconnected RBCs and virus particles. 
• The agglutinated lattice maintains the RBCs in a suspended distribution, typically viewed as a diffuse reddish solution. 
• The formation of the lattice depends on the concentrations of the virus and RBCs, and when the relative virus concentration is too low, the RBCs are not constrained by the lattice and settle to the bottom of the well.