It’s Monday 10 April 2017. It’s been a busy day at work.
I bet you’re wondering why am I wanting to mention something so outdated in a world of enzyme immunoassays, robotics and automation?
Well I got my start in pathology because of hæmagglutination inhibition or HAI. In 1982 when I was in grade twelve I did a biology project at a local medical testing laboratory. The pathologist in charge was very encouraging. The original project involved looking for antibodies to rubella virus after immunising mice with the rubella vaccine. The assay I used to measure the antibodies was an hæmagglutination inhibition assay. Part II of the study involved comparing HAI with an immunofluorescence assay and an enzyme immunoassay.
So, what is hæmagglutination inhibition? It’s probably easier to first describe hæmagglutination assays (HA).
Hæmagglutination occurs when erythrocytes (that is, red blood corpuscles) bind with proteins on viruses and form a lattice or matrix of interconnected erythrocytes and viruses. When agglutinated, the erythrocytes and viruses stay suspended and give an appearance of a red solution in the bottom of the test tube. If there is an excess of erythrocytes and not enough virus the erythrocytes settle and gravitate to the bottom of the test tube or well in a microtitre plate to form a button of erythrocytes.
The erythrocytes used for HA and HI assays usually come from chicks, turkeys, horses, guinea pigs and humans.
I was used to bleeding 3-day old chicks for their red blood cells (here’s a quiz question, why do humans have red blood corpuscles and chicks have red blood cells?) for the rubella HAI. I’d anaesthetise the chicks in a chamber with chloroform and then decapitate them with a scalpel blade. I’d hold the chicks upside-down over a bowl with a citrate solution. I’d kill 50 chicks every Monday afternoon. I did that for years until we moved to an immunoassay.
The basic principle of the HA is to determine how much virus you may have in a sample. Serial dilutions are made and a standard preparation of red blood cells are added. The test tubes or microtitre plates are incubated and then read. The tubes with high concentrations of virus look like they contain a reddish solution while the tubes with low virus concentrations have a button of red blood cells in the bottom.
In the HAI, we’re looking to determine the antibody titre. Serum samples are pipetted in serial dilution and then a standardised preparation of virus is added. The tubes or microtitre plates are incubated and in that time the wells or tubes containing antibody will bind to virus and render it incapable of hæmagglutination. Red blood cells are then added and incubated. In wells with high titres of antibody, there is no virus available for hæmagglutination so the excess red blood cells settle and form a button. Low titre antibody titre wells or no antibody titre wells have virus present and when the red blood cells are added, hæmagglutination occurs. The highest titre demonstrating no hæmagglutination is the antibody titre.
For example, if you were testing for an antibody to a viral illness you may start with a neat and 1 in 10 dilution of serum. Serial doubling dilution would result in dilutions 1 in 20, 1 in 40, 1 in 80, 1 in 160 and so on. Higher titres correspond with higher concentrations of antibody.
Hæmagglutination and hæmagglutination inhibition assays are not commonly performed anymore. We now mainly go to enzyme immunoassays and preferably direct detection of pathogens by nucleic acid amplification techniques rather than the witchcraft of serology.
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