Haemagglutination Test – Principle, Procedure, Result, Material

• George Hirst, an American virologist, created the hemagglutination assay or haemagglutination assay (HA) and the hemagglutination inhibition test (HI or HAI) in 1941–1942 as procedures for determining the relative concentration of viruses, bacteria, or antibodies.
• Hemagglutination is a process that causes red blood cells, such as the influenza virus, to clump together in the presence of some enveloped viruses.
• Hemagglutinin, a glycoprotein on the viral surface, interacts with red blood cells, causing red blood cell clustering and the creation of a lattice.
• In the absence of an enveloped virus, red blood cells precipitate to form a red dot at the bottom of a container.
• In the presence of a virus, however, red blood cell clusters are scattered and do not form a red dot. This is the fundamental principle underlying hemagglutination assays.

Haemagglutination Test Principle

• HA and HI use the process of hemagglutination, in which sialic acid receptors on the surface of red blood cells (RBCs) attach to the hemagglutinin glycoprotein found on the surface of influenza virus (and several other viruses) to form a network of interconnected RBCs and virus particles.
• The agglutinated lattice suspends the RBCs, resulting in a diffuse, scarlet solution.
• When the relative virus concentration is too low, the RBCs are not limited by the lattice and sink to the bottom of the well.
• In the presence of staphylococci, vibrios, and other bacterial species, hemagglutination is found, similarly to how viruses cause erythrocyte agglutination.
• RBCs employed in HA and HI assays often come from chickens, turkeys, horses, guinea pigs, or humans, depending on the selectivity of the targeted virus or bacterium and the RBC’s associated surface receptors.

Materials Required

1. Adjustable multi-channel micropipette (50-300 µl) 
2. Adjustable single-channel micropipette (50-200µl)
3. Adjustable single-channel micropipette (5-50µl) 
4. Disposable tips 
5. Reagent troughs 6. Waste discarders

Reagents and test kits

• Micro plates (U bottom)
• Sample diluents 
• Control cells (uncoated) 
• Test cells (sensitized with T.pallidum antigen) 
• Kit controls

Procedure of Haemagglutination Test

Rapid haemagglutination test

1. Bring kit components to room temperature (15-30°C) approximately 30 minutes before beginning the test procedure. Gently combine the liquid reagents. Determine the total number of testable samples and the number of plates necessary for the assay.
2. If necessary, provide a unique plate ID in the upper-middle portion of the plate.
3. Label the plate with the last three numbers of the donor’s ID on the lower left side of the well to indicate the placements. Include one negative and one positive controls every sample batch.
4. Arrange the samples in a sampling rack according to the plate map.
5. Use the first additional well based on the quantity of samples, and add 190l of sample diluent.
6. Then, using a new pipette tip for each addition, combine 10l of specimen with 190l of sample diluent in the same position in the extra well/plate.
7. Then, place 25ml of the diluted sample in each of the control and test wells.
8. After sampling, place the sample in the same/different sampling rack position.
9. Add 25l to the negative and positive controls, respectively.
10. Gently combine the control and test cell bottles to create a homogenous suspension, then add 1 drop (75 l) of the suspension to each well, including the positive and negative control wells.
11. Mix the contents of the well by slowly turning the plate in a circular motion. Maintain the plate on a level, stable surface. Examine the results after one hour of incubation at room temperature.

Interpretation of the test result

• Each control well should have a button-like structure. If any control well lacks button formation, the test must be repeated to rule out a technical problem. (Agglutination of both the control and test cells shows the presence of anti-cell antibody; in this case, the test is invalid and should be redone after absorption of the test serum. To accomplish this, dilute 1/4 of the test serum with control cells and allow to stand at room temperature. After centrifuging the sample for 5 minutes at 1000 rpm. Dilute the supernatant by one-fifth with the diluent. Test this dilution directly, without further dilution, using suspensions of test and control cells.
• If a test well contains a compact button, the result is termed nonreactive.
• Specimens are regarded to be reactive for T. pallidum if they have a typical ring pattern or net of cells, whereas the control well displays a compact button formation.
• TPHA should repeat all positive test results.
• Identify the sample, isolate it, and retest it with RPR if it also tests positive after a second test.

Agglutination has frequently been used to assess whether or not a patient has a bacterial infection. For instance, if typhoid fever is suspected, the patient’s serum is combined with a culture of Salmonella typhi. If an agglutination reaction develops, as seen by the germs clumping together, the patient had or has a S. typhi infection. Since specific antibodies might remain in a patient’s blood for years after the patient has recovered from an illness, a positive reaction does not necessarily indicate that the patient is currently infected. To assess whether a patient is now afflicted with typhoid fever, the quantity or titer of the antibody will be measured at the commencement of disease and two weeks later.

Micro haemagglutination test in a V-bottom microwell plate

This approach is useful for determining the presence or absence of haemagglutinin in the allantoic fluid of a large number of embryonated eggs. A solution of 1 percent red blood cells is utilised. In V-bottom plates, the cells settle faster and the contrast between positive and negative results is greater than in U-bottom plates.

Materials

• Inoculated eggs, chilled for at least 2 hours, preferably overnight
• Negative and positive control samples
• V-bottom microwell plate and lid
• Micropipette and tips to measure 50 µL
• 1 percent suspension of red blood cells
• 70 percent alcohol solution
• Cotton wool
• Forceps and/or small scissors
• Absolute alcohol
• Discard tray
• Microwell plate recording sheet. 

Method

1. On a recording sheet, record the details of the samples being analysed. The wells will be filled with samples and controls as indicated on this sheet.
2. Using a micropipette, withdraw 50 mL of allantoic fluid from each egg and place it in a microwell plate well. Use a distinct tip for every sample.
3. Include positive and negative control samples of allantoic fluid on one of the plates.
4. Inject 50 mL of PBS into each of two wells. These wells will serve as controls for autoagglutination of red blood cells.
5. Add 25 mL of red blood cells at 1% to each well.
6. Tap the sides of the plate gently to combine. Put a lid on the platter.
7. Allow the plate to rest at room temperature for 45 minutes.
8. Observe and document the outcomes.

Results

• Different settling patterns exist for single and agglutinated red blood cells. Individual cells roll down the sides of the V-shaped well and settle as sharp buttons. Agglutinated cells do not form a button by rolling down the sides of the well. They settle instead as a diffuse film.
• Negative HA outcome equals a sharp button
• Positive HA result = a diffuse film
• Control of red blood cells equals a sharp button
• Notate the HA results on the microwell sheet.

Titration to establish haemagglutinin (HA) titre of a suspension of virus

The haemagglutination test is utilised to determine the concentration of Newcastle disease virus in a suspension. This is accomplished by doing two-fold serial dilutions of the viral suspension in a microwell plate, followed by testing to establish an endpoint. This result is then used to determine the concentration of haemagglutinin in the suspension and is expressed as a HA titre.

Materials

• 96 well V-bottom microwell plate and cover
• 25 mL single and multi-channel micropipettes and tips
• PBS
• 1 percent chicken red blood cells
• Sample to be titrated
• Reagent troughs
• Microwell plate recording sheet.

Procedure

1. Record on recording sheets the method of sample distribution on microwell plates.
2. Distribute 25 L of PBS per well in the microwell plate.
3. Place 25 L of test samples in the first well of each row and column of the first row. If necessary, samples can be tested in duplicate or triple.
4. Use a multichannel pipette to perform two-fold successive dilutions until Column 11 across the plate. Instructions for carrying out twofold serial dilutions can be found in Appendix 4.
5. Pour 25 L of PBS per well.
6. Add 25 L of red blood cells at 1 percent to each well, including Column 12. This column contains control wells containing simply PBS and red blood cells.
7. Tap the sides of the plate gently to combine. Put a lid on the platter.
8. Allow the plate to rest at room temperature for 45 minutes.
9. Read and record each well’s results. All control wells should be negative for HA.
10. HA-negative: A pinpoint of red blood cells at the base of the V-shaped well.
11. HA-positive: A hazy layer of red blood cells, with either no button or a very small button of red blood cells at the bottom of the V-bottom well.
12. Determine the conclusion. This is the final well that will demonstrate complete haemagglutination and it contains one haemagglutinating unit.

Calculation of the HA titre of the test sample

The HA titre is equal to the reciprocal of the dilution that yields one unit of HA.

Figure displays an example of HA titration.

• A 1 in 64 (1/64) dilution contains 1 HA unit.
• Therefore, the HA concentration of the test sample is equal to the reciprocal of 1/64 = 64 = 26
• The titre of the Newcastle disease virus suspension can be stated as either 64 or 26 HA units per 25 mL.

Applications of Hemagglutination Assay

• Against any disease or infectious agent, it can be utilised to detect the body’s humoral immune response.
• It can be used to classify blood cells into their respective categories.Infections with viruses like paramyxovirus, influenza, etc., can be identified and measured with this tool.
• Diverse hemagglutination-based quick diagnostic test kits are developed. An RDT kit that can detect HbsAg in the event of a Hepatitis B infection is one example.
• As an added bonus, it may pick up on a wide range of bacterial infections including syphilis.

Advantages of Hemagglutination Assay

• Work is straightforward and needs little instruction.
• It’s cheaper than a lot of other diagnostic procedures.
• The tools and supplies needed are typically readily available.
• Rapid result analysis (as in the case of RDT).

Limitations of Hemagglutination Assay

• The concentration of RBCs and the length of time they are incubated can cause errors that can invalidate the results.
• It’s important to get specific with the many reaction components. Their generality could lead to a false conclusion.
• Values in the quantitative realm can only be determined and interpreted by those with the proper training.
• Due to the lack of digital data, the many observers’ interpretations of the results may contain inaccuracies or variations.

References

• S. Miyaishi, F. Moriya, (2005) Encyclopedia of Analytical Science (Second Edition)
  Parija S.C., (2009), Textbook of Microbiology and Immunology, 2nd edition, Elsevier, a division of Reed Elsevier India Private Limited, pg. 108
• Sukhadeo B. Barbuddhe and Deepak B. Rawool, Methods in Microbiology, 2020
• Townsend, A., Rijal, P., Xiao, J. et al. A haemagglutination test for rapid detection of antibodies to SARS-CoV-2. Nat Commun 12, 1951 (2021). https://doi.org/10.1038/s41467-021-22045-y
• Thangavelu, C. P., & Koshi, G. (1980). Micro-indirect hemagglutination test for detection of antibodies to the Ibc protein of group B Streptococcus. Journal of clinical microbiology, 12(1), 1–6. https://doi.org/10.1128/jcm.12.1.1-6.1980
• https://nios.ac.in/media/documents/dmlt/Microbiology/Lesson-60.pdf
• https://www.fao.org/3/ac802e/ac802e0d.htm
• https://www.news-medical.net/health/An-Overview-of-Hemagglutination.aspx
• https://www.tandfonline.com/doi/pdf/10.1080/03079457708418208
• http://neradslab.res.in/document/SOP/NDV_HA_HI.pdf