Hemagglutination

Hemagglutination Reaction

1. Hemagglutination

2. 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 non agglutinating antibodies. The test can also detect complement attached to red blood cells. The Coombs’ test is often employed when a new-born has  jaundice , yellowing of the skin caused by high blood concentrations of bilirubin, a product of the breakdown of haemoglobin 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  haemolytic transfusion reactions ,  autoimmune haemolytic anaemia , 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).

3. 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 neighbouring 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  haemolytic disease of the new-born .

4. 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.

5. A serial dilution  viral agglutination assay is used to measure the  titre  or estimate the amount of virus produced in cell culture or for vaccine production. A viral titre 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 titre. 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