The Hemagglutination


Related Posts:


A) Diagram of direct Coomb’s test. 1.  Blood sample from a patient with hemolytic anemia with antibodies attached to red blood cells. 2. Antibodies coat the patient’s red blood cells. 3. A few drops of Coomb’s reagent (containing anti-human antibodies) is mixed in with the patient’s blood sample. 4. Agglutination reaction (clumping) is visible after cross-linkage of antibodies. B) Diagram of indirect Coomb’s test. 1. Patient's serum containing antibodies is drawn. 2. Donor blood is added. 3. Patient's antibodies bind to donor's red blood cells. 4. A few drops of Coombs’ reagent are mixed with the sample. 5. Agglutination reaction is observed.
Source: OpenStax Microbiology

OpenStax Microbiology

Agglutination of red blood cells is called hemagglutination. One common assay that uses hemagglutination is the direct Coombs’ test, also called the direct antihuman globulin test (DAT), which generally looks for nonagglutinating antibodies. The test can also detect complement attached to red blood cells.

The Coombs’ test is often employed when a newborn has jaundice, yellowing of the skin caused by high blood concentrations of bilirubin, a product of the breakdown of hemoglobin in the blood. The Coombs’ test is used to determine whether the child’s red blood cells have been bound by the mother’s antibodies. These antibodies would activate complement, leading to red blood cell lysis and the subsequent jaundice. Other conditions that can cause positive direct Coombs’ tests include hemolytic transfusion reactions, autoimmune hemolytic anemia, infectious mononucleosis (caused by Epstein-Barr virus), syphilis, and Mycoplasma pneumonia. A positive direct Coombs’ test may also be seen in some cancers and as an allergic reaction to some drugs (e.g., penicillin).

The antibodies bound to red blood cells in these conditions are most often IgG, and because of the orientation of the antigen-binding sites on IgG and the comparatively large size of a red blood cell, it is unlikely that any visible agglutination will occur. However, the presence of IgG bound to red blood cells can be detected by adding Coombs’ reagent, an antiserum containing antihuman IgG antibodies (that may be combined with anti-complement). The Coombs’ reagent links the IgG attached to neighboring red blood cells and thus promotes agglutination.

There is also an indirect Coombs’ test known as the indirect antiglobulin test (IAT). This screens an individual for antibodies against red blood cell antigens (other than the A and B antigens) that are unbound in a patient’s serum. IAT can be used to screen pregnant women for antibodies that may cause hemolytic disease of the newborn. It can also be used prior to giving blood transfusions.

Antibodies that bind to red blood cells are not the only cause of hemagglutination. Some viruses also bind to red blood cells, and this binding can cause agglutination when the viruses cross-link the red blood cells. For example, influenza viruses have two different types of viral spikes called neuraminidase (N) and hemagglutinin (H), the latter named for its ability to agglutinate red blood cells. Thus, we can use red blood cells to detect the presence of influenza virus by direct hemagglutination assays (HA), in which the virus causes visible agglutination of red blood cells. The mumps and rubella viruses can also be detected using HA.

Most frequently, a serial dilution viral agglutination assay is used to measure the titer or estimate the amount of virus produced in cell culture or for vaccine production. A viral titer can be determined using a direct HA by making a serial dilution of the sample containing the virus, starting with a high concentration of sample that is then diluted in a series of wells. The highest dilution producing visible agglutination is the titer. The assay is carried out in a microtiter plate with V- or round-bottomed wells. In the presence of agglutinating viruses, the red blood cells and virus clump together and produce a diffuse mat over the bottom of the well. In the absence of virus, the red blood cells roll or sediment to the bottom of the well and form a dense pellet, which is why flat-bottomed wells cannot be used.

A photo of nonagglutination shows a red stripe. A diagram shows that without virus the cells from a compact pellet at the bottom of the well. A photo of agglutination shows a red spot and pink surrounding area. A diagram shows that the red blood cells and viruses form a clump.
A viral suspension is mixed with a standardized amount of red blood cells. No agglutination of red blood cells is visible when the virus is absent, and the cells form a compact pellet at the bottom of the well. In the presence of virus, a diffuse pink precipitate forms in the well. (credit bottom: modification of work by American Society for Microbiology)

A modification of the HA assay can be used to determine the titer of antiviral antibodies. The presence of these antibodies in a patient’s serum or in a lab-produced antiserum will neutralize the virus and block it from agglutinating the red cells, making this a viral hemagglutination inhibition assay (HIA). In this assay, patient serum is mixed with a standardized amount of virus. After a short incubation, a standardized amount of red blood cells is added and hemagglutination is observed. The titer of the patient’s serum is the highest dilution that blocks agglutination.

A drawing of a well plate. The label at the top reads: Dilution. The rows are labeled: 1:1, 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128. The columns are labeled: Sample A, titer = 128 (this spans columns 1 and 2). Sample B, no neutralizing antibody (spans columns 3 and 4). Sample C, titer = 64 spans columns 5 and 6.). Columns 1 and 2 have red dots in all rows but the bottom one. Columns 3 and 4 have no red dots. Columns 5 and 6 have red dots in all rows but the bottom two.
In this HIA, serum containing antibodies to influenzavirus underwent serial two-fold dilutions in a microtiter plate. Red blood cells were then added to the wells. Agglutination only occurred in those wells where the antibodies were too dilute to neutralize the virus. The highest concentration at which agglutination occurs is the titer of the antibodies in the patient’s serum. In the case of this test, Sample A shows a titer of 128, and Sample C shows a titer of 64. (credit: modification of work by Evan Burkala)

Source:

Parker, N., Schneegurt, M., Thi Tu, A.-H., Forster, B. M., & Lister, P. (n.d.). Microbiology. Houston, Texas: OpenStax. Access for free at: https://openstax.org/details/books/microbiology