Hemagglutination Assay – Its Principle, Procedure and Interpretation


In 1941-1942, an American virologist named George Hirst developed the hemagglutination assay (HA) and the hemagglutination inhibition test (HI or HAI), procedures for detecting the relative concentration of viruses, bacteria, or antibodies.

In the presence of some enveloped viruses, hemagglutination causes red blood cells to clump together.

Hemagglutinin, a glycoprotein found on the viral surface, interacts with red blood cells, causing them to clump and form a lattice. Red blood cells precipitate to form a red dot at the bottom of a container in the absence of an enveloped virus. However, in the presence of a virus, red blood cell clusters are dispersed and do not form a red dot. This is the basic concept of hemagglutination tests.

What is hemagglutination?

The hemagglutination assay and hemagglutination inhibition assay use the hemagglutination process, which involves the adhesion of sialic acid receptors on the surface of red blood cells (RBCs) with the hemagglutinin glycoprotein present on the surface of certain viruses, including the influenza virus and others. This type of interaction results in the creation of a network or lattice structure in which RBCs and virus particles are linked together.

RBCs stay suspended in a dotted bright red solution as a result of this lattice development. The creation of the lattice depends on virus and RBC concentrations. When the virus concentration is low, the lattice is unable to enclose RBCs, resulting in them to settle at the well’s bottom. This hemagglutination phenomenon is not confined to viruses, but can also be observed in the presence of some bacterial species such as staphylococci, vibrio, and others. These bacteria use a similar mechanism to cause erythrocyte agglutination.

RBCs from chickens, turkeys, horses, guinea pigs, or humans are used to perform HA and HI experiments, depending on the selectivity necessary for the specific virus or bacterium being targeted and associated surface receptors on the RBCs.

For many years, the hemagglutination assay and hemagglutination inhibition assay have been vital tools in virology and bacteriology. They enable researchers to determine the concentration of viruses, bacteria, or antibodies in a sample, which provides important information about their presence and activity. These assays are still frequently utilized in a variety of scientific and medical sectors, aiding in the understanding of infectious illnesses and the development of diagnostic tests and vaccinations.

Hemagglutination assay Principle

The basic idea of the hemagglutination test depends upon the interaction of antigens and antibodies on the surface of Red Blood Cells. When these antigens come into contact with their corresponding complementary antibodies, they bind together, causing agglutination or clumping of the RBCs. This agglutination process is clearly visible and can be used to distinguish between a positive and negative test result.

The presence or absence of agglutination in this test offers important information regarding the interaction of antigens and antibodies. When the specific antigen and its complementary antibody are present in the sample, they will interact, resulting in evident RBC clumping. This clumping can easily be seen with the naked eye or under a microscope. Agglutination will not occur if the antigen and its associated antibody are not present or do not interact. The absence of clumping in these situations shows a negative test result.

Figure 1: the figure represents the principle of HA test (Takahara et al. 2019)

Requirements of hemagglutination assay

RBC Suspension

In the assay, RBC suspension acts as a carrier particle. It is coated with either antigens or antibodies that are specific to the antigens to be detected. The RBCs that are used determined by the assay and the desired target. RBCs from human beings, sheep, chicks, and other animals can all be used.

Negative and positive control samples

These are required in hemagglutination assays to validate the accuracy and reliability of the results. Positive control samples include a known concentration of the target material to validate the assay’s sensitivity and specificity, whereas negative control samples do not contain the target substance and serve as a baseline for comparison.

A serum or blood sample

They carry the antibodies, antigens, or viruses. This sample is used in the test to determine whether or not the target substance is present.

Microtitre Plates

In hemagglutination assays, microtitre plates, such as 96-well V-bottomed plates, are often used.

Phosphate Buffered Saline (PBS)

It is a common diluent used in hemagglutination experiments. It creates an ideal environment for the reaction of the RBCs with the target substance. PBS aids in the maintenance of the pH and ionic balance required for effective hemagglutination.

Kits for Rapid Hemagglutination Assay (RDT)

Rapid hemagglutination assay kits may be used in some cases. These kits often comprise slides, reagents, and control samples that have been specifically designed for quick and easy testing.

Hemagglutination assay Procedure

96 well plate method

The hemagglutination Assay is a useful tool for determining the presence or absence of hemagglutinin in allantoic fluid derived from embryonated eggs. While it is a more time-consuming approach than the Rapid Diagnosis Test (RDT), it has its own set of advantages and applications.

Red blood cells in a 1% solution are dissolved in the Micro-hemagglutination Assay. This provides for a standardized cell suspension for testing. In this test, V-bottom plates are recommended because the cells settle more quickly and efficiently in the V-shaped wells. During the analysis, the settling pattern of the red blood cells is an important feature to examine.

The Micro-hemagglutination Assay has a major advantage over U-bottom plates in that the difference between positive and negative results is more evident. This improves the precision and accuracy of result interpretation. While this procedure takes more time and attention than the Rapid Diagnosis Test, it gives important information about the presence or absence of hemagglutinin in the allantoic fluid.

The Micro-hemagglutination Assay is especially beneficial in research settings where a more detailed hemagglutinin analysis is required. Using this procedure, scientists can learn more about the hemagglutinating properties of samples and gain a better understanding of the nature of the tested agents.

Requirements of 96 wells plate HA test

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

Procedure of hemagglutination test in a V-bottom microwell plate

  • Start the procedure by completing a recording sheet with relevant details about the samples to be analyzed. This sheet will aid in the tracking and recording of assay results.
  • Take a sample of approximately 50 µl with a micropipette and dispense it into a specified well of the microwell plate. To avoid contamination between samples, it is critical to use a new pipette tip for each one.
  • On one of the plates, place negative and positive controls. These controls are critical for comparing and validating test results.
  • To each well, add 50 µl of Phosphate Buffered Saline (PBS). These wells will act as red blood cell auto-agglutination controls.
  • Pour 25 µl of 1% red blood cells into each well. In the experiment, red blood cells will serve as the carrier particles.
  • Gently tap the plate’s sides to ensure that the contents of each well are properly mixed. This will make it easier for the samples to engage with the red blood cells.
  • To get ideal conditions for the assay, cover the plate with a plate cover.
  • Allow the plate to stand for around 40 minutes to allow the reaction to take place.
  • Observe and thoroughly record the data during the incubation period. In each well, take note of any obvious agglutination or absence of agglutination.

Results interpretation of hemagglutination test in a V-bottom microwell plate

Negative HA result

Red blood cells settle down the edges of the V-shaped well, generating a sharp button at the bottom, resulting in a negative HA result. This shows that the red blood cells have not agglutinated or clumped, and the result can be regarded as negative for the specific antigen or antibody being tested.

Positive HA Result

When agglutination occurs, a positive result is observed. In this situation, the red blood cells do not form a distinct button as they settle down the well’s edges. They instead form a diffuse film, indicating the presence of agglutinated cells. This indicates the presence of the antigen or antibody under investigation.

Control of Red Blood Cells

A control using only red blood cells is provided as part of the test. Individual red blood cells should settle down the sides of the control wells, generating a sharp button identical to the negative result of HA. This ensures that the red blood cells are working properly and serves as a baseline against which the test results may be compared.

Figure 2: the figure represents the HA titer of two samples, agglutination represents the positive result and dot or bead represents negative results (Sharma et al.)

Noting the HA Results: It is critical to record the micro-hemagglutination test results on the microwell sheet or a designated recording sheet. For each sample tested, the HA outcomes, whether negative (sharp button) or positive (diffuse film), should be carefully noted.

Rapid Hemagglutination Assay

The Rapid Hemagglutination Assay, commonly known as the Rapid Diagnosis Test (RDT), was developed to check the presence of a hemagglutinating component in a sample quickly. It is so named because it can produce results in under a minute. When performing this test on numerous samples, it is sufficient to test the negative and positive control samples only once.

It is essential to evaluate the settling pattern of the red blood cell suspension during the hemagglutination test. This is accomplished by combining the diluent and allowing the red blood cells to settle. Here’s how to investigate the settling pattern:

  • Fill the designated area with diluent.
  • Gently mix the red blood cells with the diluent to ensure adequate mixing.
  • Leave the red blood cells to settle for some time.
  • Examine the settled cell pattern. The cells should settle in a typical pattern in the Rapid Hemagglutination Assay, with no evidence of auto-agglutination or clumping. A negative result should include a distinct button of cells and an even suspension.

The Rapid Hemagglutination Assay is useful because it produces quick results, allowing for the rapid detection of the presence of a hemagglutinating substance. Potential difficulties such as auto-agglutination can be recognized by analyzing the settling pattern of the red blood cells, ensuring the assay’s accuracy.

Procedure of Rapid Hemagglutination Assay

  • Before beginning the test, ensure that all kit components are at room temperature (15-30°C). Allow them to adjust for around 30 minutes. Mix the liquid reagents gently.
  • Calculate the total number of samples to be tested as well as the number of plates needed for the assay.
  • To differentiate them, establish a unique plate ID in the upper-middle area of each plate if necessary.
  • Label the plate with the last three numbers of the donor’s ID on the lower left side of each well to identify the well. Remember to have one negative control and one positive control in each sample batch.
  • To make sure proper placement, arrange the samples in a sampling rack according to the plate map.
  • Add 190 µl of sample diluent to the first additional well based on the number of samples.
  • In the same spot in the extra well or plate, combine 10 µl of specimen with 190 µl of sample diluent using a fresh pipette tip for each addition.
  • Fill each control and test well with 25 µl of the diluted sample.
  • Place the sample in the same or a different position in the sampling rack after sampling.
  • Fill the negative and positive control wells with 25 µl of the control cell suspension, respectively.
  • To ensure a homogenous solution, gently mix the control and test cell bottles. Then, add one drop of the suspension (about 75 µl) to each well, including the positive and negative control wells.
  • By slowly rotating the plate in a circular motion, mix the contents of each well. Maintain a level and stable surface for the plate.
  • Incubate the plate for one hour at room temperature.
  • Examine the data after the incubation time. Examine each well for the presence or absence of agglutination. Agglutination refers to a positive result, whereas the absence of agglutination means a negative result.

Results of Rapid Hemagglutination Assay

Control Well

Each control well should have a different button-like structure which means accurate functioning of the test. If any control well fails to create buttons, this indicates a technical issue, and the test should be repeated to ensure correct results.

Agglutination of Control and Test Cells

Agglutination of both the control and test cells indicates the presence of anti-cell antibodies. In this situation, the test happens to be invalid, and the serum should be absorbed. Dilute 1/4 of the test serum with control cells and set aside at room temperature. After 5 minutes of centrifugation at 1000 rpm, dilute the supernatant by one-fifth with the diluent. Use suspensions of test and control cells to test this diluted material directly, without further dilution.

Nonreactive Result

If a test well gives a compact button without agglutination, the result is classified as nonreactive, indicating the absence of the substance being tested for.

Examples of HA results

T. pallidum Reactive Result

A reactive result for T. pallidum detection is indicated by the presence of a typical ring pattern or net of cells in the test well, whereas the control well exhibits a compact button formation.

Positive Results Confirmation

Positive TPHA (Treponema pallidum hemagglutination assay) test results should be repeated to validate the results.

Additional Testing

If a sample is consistently positive, it is critical to identify and isolate the sample. Retesting using additional assays, such as RPR (Rapid Plasma Reagin), can assist in determining the existence of infection or antibodies.

Applications of HA test

  • The hemagglutination assay is useful in determining the body’s humoral immune response against an infection or infectious agents. It can provide information on the presence and quantity of certain antibodies in a patient’s serum by evaluating the agglutination reaction between red blood cells and antibodies. This is essential for detecting and monitoring immunological responses.
  • Hemagglutination principles have been used to create quick diagnostic test kits. These kits use hemagglutination-based responses to quickly detect particular diseases or antigens. For example, hemagglutination is a critical component in the detection process of an RDT kit designed to detect Hepatitis B surface antigen (HbsAg).
  • The hemagglutination assay is used to detect and quantify viral infections. It is especially useful for viruses with hemagglutination characteristics, such as paramyxovirus and influenza. The assay can aid in the diagnosis and monitoring of viral infections by evaluating the agglutination reaction between the virus and red blood cells.
  • HA is also used to detect certain bacterial infections. For example, it is utilized in the diagnosis of syphilis, where the assay aids in the detection of particular antibodies against the microorganisms that cause illness.
  • The hemagglutination assay is useful in distinguishing blood cell types or groupings. It identifies specific antigens on red blood cells, allowing for accurate blood typing and matching in blood transfusion procedures and organ transplants.

Advantages of HA test

  • Hemagglutination test equipment, such as microtitre plates, pipettes, and centrifuges, are commonly used in laboratories. Because these equipments are neither specific for one test nor unique to the assay, these are cheaper and require fewer investments.
  • The hemagglutination assay allows for rapid interpretation of data in specific forms, such as the Rapid Diagnostic Test (RDT). RDTs are intended to produce quick and easily readable results in a brief period of time. This particular feature is very useful in point-of-care settings or cases requiring quick decision-making for further diagnosis.
  • Because the hemagglutination assay is relatively easy to conduct, it is suitable for a wide range of laboratories and healthcare settings. Typically, the technique includes mixing the samples, red blood cells, and particular reagents, followed by evaluating the agglutination reaction. Its simple protocol makes implementation easier and lowers the possibility of procedural errors.
  • The hemagglutination assay is frequently less expensive than other diagnostic tests. The chemicals and equipment needed for the assay are often inexpensive and widely available, making it a viable option for less resource settings or when budget limitations are a factor.

Disadvantages of HA test

  • Without the assistance of computerized data analysis, the hemagglutination assay typically relies on manual result interpretation. This reliance on visual perception might lead to variation and subjective interpretation. Different observers’ analyses may differ slightly, resulting in conflicts or inaccuracies in result interpretation. The problem can be overcome by making interpretation standards and providing frequent training to workers.
  • The hemagglutination assay is sensitive to experimental factors such as incubation time and RBC concentration. The variations from the recommended conditions can result in inaccurate results. To produce reliable and reproducible results, constant and accurate incubation periods and suitable RBC concentrations must be provided.
  • The determination of quantitative values and interpretation of results in the hemagglutination assay may necessitate the use of experienced and qualified persons. Titrations and dilutions are frequently used in the assay, and while interpreting results, it may demand a thorough comprehension of the assay principles and applicable standards. To achieve precise and trustworthy quantitative analysis, skilled professionals are required.
  • The specificity of the factors involved, such as antibodies, antigens, and reagents, is critical to the success of the hemagglutination experiment. Non-specific or cross-reactive responses might produce a false-positive or false-negative interpretation of the result. To reduce the possibility of incorrect interpretations, it is critical to use well-characterized and specified components.


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  • 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
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Mubashir Iqbal
Mubashir Iqbal

Mubashir Iqbal is a highly dedicated and motivated Microbiologist with an MPhil in Microbiology from the University of Veterinary and Animal Sciences. Currently, he is researching the efficacy of commercially available SARS Cov-2 vaccines to neutralize the omicron variant in Pakistan. He holds a Bachelor's degree in Microbiology and has experience in chemical and microbiological analysis of water samples, managing SOPs and documents according to standard ISO 17025. Additionally, he has worked as an internee in BSL 3, Institute of Microbiology, UVAS, where he gained experience in RNA extraction, sample processing, and microscopy.

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