Antiglobulin Coombs Test

Nevin Hughes-Jones, Molecular Immunopathology Unit, Medical Research Council, Blood Transfusion Centre, Cambridge, UK

The anti-globulin test was developed to detect human blood group immunoglobulin G (IgG) antibodies by agglutination. The negative charge on the surface of red cells prevents them from coming closer than within 20 nm of each other, thus IgG antibodies are unable to cross-link red cells as the Fab arms, even when fully extended, are unable to span this distance. On the other hand, the Fc portion of cell-bound antibody projects outwards sufficiently far to allow an anti-IgG antibody to cross-link between IgG molecules on one cell and those bound to neighboring red cells, thus bringing about agglutination (Figure 1). The development of the test occurred following the discovery of the Rh blood group system in the early 1940s. It was soon realized that there were two types of antibody involved in this system -those that would directly agglutinate red cells in saline (retrospectively identified as IgM antibodies), and the so-called 'incomplete' or 'blocking' antibodies (now identified as IgG) which would not directly agglutinate but would block the action of the direct agglutinators.

R.R.A. Coombs, A.E. Mourant and R.R. Race, working in Cambridge in the early 1940s, were investigating the various specificities of the antibodies that appeared to be associated with the Rh system. As reported in their publication of 1945, the seminal idea was that red cells sensitized with nonagglutinating anti-Rh antibody 'had absorbed antibody globulin at some points on their surface and that an

Figure 1 Cross-linking of cell bound IgG.

anti-human globulin serum might be expected to react with this in some observable way'. The principle of the test was soon established. In a later publication, they indicated that the test could probably be used to detect any nonagglutinating antibody present in serum (the indirect Coombs' test) by initially absorbing the antibody on to red cells; they also showed that it could also be used to detect antibody on the surface of fetal red cells in hemolytic disease of the newborn (direct Coombs' test).

It is interesting to note that the use of an antiglobulin serum in this way was first described by the Italian physiologist, Moreschi, in 1908. Moreschi showed that if rabbit red cells were reacted with a goat anti-rabbit red cell antibody at too low a concentration to bring about agglutination, they could subsequently be agglutinated by the addition of a goat serum-specific antibody. At that time, human nonagglutinating antibodies were unknown and hence the significance of the reagent was not appreciated and its description was forgotten.

The test is widely used in two areas in clinical immunohematology. First, it is of major importance in blood transfusion in cross-matching for determining compatibility between donor and recipient. Donor red cells are added to the recipient's serum and any nonagglutinating antibody which binds to the cells can be detected by the addition of the antiglobulin reagent. Second, it is used in the diagnosis of acquired hemolytic anemias for demonstrating the presence of antibodies bound to red cells in vivo.

Antiglobulin reagents can be made specific for IgM, IgA and the four subclasses of IgG. The reagents used in cross-matching also contain antibodies specific for the complement component C3, because some antibodies are best detected by testing for the bound complement which is deposited as the result of the antigen-antibody reaction.

See also: Antibodies, detection of; Blood transfusion reactions; Gammaglobulin; Hemolytic disease of the newborn; Maternal antibodies; Rh antigens; Transfusion.

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