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Discuss about the Virology Hemagglutination Assay.



The hemagglutination  (HA) assay is a simple and quantitative technique of culturing influenza virus based on their ability to attach to molecules present on the red blood cells. In case of research related to the avian influenza virus, the tool is used for screening of the cell culture extracted from the emryonating chicken eggs with type A influenza (1).  The surface of the Influenza A virus consist of hemagglutinin (HA) and neuraminidase protein (NA) (2). The hemagglutinin protein present on the Influenza A virus has the potential to bind with N-acetylneuraminic acid, which contains the proteins, found on the avian and mammalian erythrocytes. In case of high concentration of the Influenza virus, combination of the protein leads to an agglutination reaction. This causes both the erythrocytes to link together and consequently form a diffused lattice. Agglutination may be caused by other viruses too, such as paramyxovirus, adenovirus and bacteria with hemaggutinating property (1). Therefore, HA cannot be regarded as an identification assay, hemagglutination inhibition assay is to necessary to identify the type of virus present in mammalian cells.


From the analysis of the results obtained from HA assay, it can be said that it is an easy and sensitive technique that gives direct results within 30 minutes. Expected outcome was possible because every steps in the experiment was done accurately performed. However, slight difference in reading was seen due to the temperature of the laboratory, equipments used, technical expertise of the experimenter and the quality of chicken erythrocytes obtained (3). The HA assay may not be efficient in detecting viable virus, however live and inactivated viruses are readily detected from this technique. The HA assay also has the potential to detect non-infectious virus particles and inactivated or degraded viral particles.

During the experiment, it was found that erythrocytes did not settle in control wells even after 20-30 minutes. This might have occulted due to poor quality of hemolysed erthyrocyte suspension or incorrect pH of PBS or contamination of the well with viral antigen. This could have been avoided by performing all the steps in a Class II biological safety cabinet to prevent contamination. Secondly, aerosol resistant tips and use of asecptic technique while taking the aliquot from the virus containing tube might reduce the problem during the experiment.

Other safe and accurate technique is that HA assay plate should be read only when the erythrocytes present in the control well have settled down well. However, plates should not be kept for longer time before reading and reading should be taken as far as possible. The neuraminidase protein found on the surface of the Influenza virus can break virus bond cells and facilitate breaking apart the lattice formed by the virus and erythrocytes. In some viral strains, there might be very high neuraminidase activity, which might hamper proper haemagglutination process (4).


The report summarized the efficacy of HA assay in detecting type of virus in culture cells. The subtype of virus is identified on the basis of agglutination reaction when erythrocytes combine to form a lattice. As other virus and bacteria might also have hemagglutinating properties, the type of virus is confirmed by hemagglutination inhibition assay. The discussion related to experiment revealed that following accurate and aseptic technique is important to avoid error in reading and misinterpretation of the study results.



  1. Killian ML. Hemagglutination assay for the avian influenza virus. Avian Influenza Virus. 2008:47-52.
  2. Influenza Type A Viruses | Avian Influenza (Flu) [Internet]. 2017 [cited 3 May 2017]. Available from:
  3. Truelove S, Zhu H, Lessler J, Riley S, Read JM, Wang S, Kwok KO, Guan Y, Jiang CQ, Cummings DA. A comparison of hemagglutination inhibition and neutralization assays for characterizing immunity to seasonal influenza A. Influenza and other respiratory viruses. 2016 Nov 1;10(6):518-24.
  4. Monto AS, Petrie JG, Cross RT, Johnson E, Liu M, Zhong W, Levine M, Katz JM, Ohmit SE. Antibody to influenza virus neuraminidase: an independent correlate of protection. Journal of Infectious Diseases. 2015 Apr 8:jiv195.

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