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The antiglobulin test was first introduced into clinical medicine in 1945 by R.R. Coombs who showed that it could be used to detect non-agglutinating red cell antibodies (indirect antiglobulin test, IAT) or sensitized red cells (direct-antiglobulin test, DAT).  Most non-agglutinating (incomplete) antibodies are IgG, although some antibodies are IgM.  It appears that these antibodies do not spontaneously cause agglutination.  This is due to a strong electronegative charge on the red cell surface that prevents the cells from coming into close proximity, a requirement for an antibody to cause agglutination.  The antiglobulin reagent is able to bridge these negative forces.  Current antiglobulin reagent (COOMBS reagent) preparations contain a “cocktail” of monoclonal antibodies directed against human IgG and C3.  The latter is more effective than an anti-IgM antibody for detecting IgM antibodies because a single IgM molecule will bind numerous complement molecules to the red cell surface and IgM antibodies tend to spontaneously dissociate from the red cell membrane.

DIRECT ANTIGLOBULIN TEST (DAT)

The DAT is used to detect IgG or C3 bound to the surface of the red cell.  In patients with hemolysis, the DAT is useful in determining whether there is an immune etiology.  Non-immune causes of hemolysis, such as DIC, thrombotic thrombocytopenic purpura, mechanical hemolysis, such as those due to artificial valves or burns, hemoglobinopathies (sickle cell, thalassemia), red cell enzyme deficiencies (G6PDP, pyruvate kinase), and red cell membrane defects (hereditary spherocytosis, PNH) will have a negative DAT.  Immune causes of hemolysis, including autoimmune hemolytic anemias, drug induced hemolysis, and delayed or acute hemolytic transfusion reactions, are characterized by a positive DAT.  Positive DATs without hemolysis occur in patients with SLE, CLL, other autoimmune diseases and some infectious diseases, such as infectious mononucleosis.  A small proportion of normal individuals will also have a positive DAT without evidence of decreased red cell survival.  Thus, a positive DAT, by itself, does not mean that the patient has an immune hemolytic anemia. 

The DAT is a five to ten minute procedure performed by incubating patient red cells with the antiglobulin reagent.  A positive DAT due to IgG is seen most frequently in patients with warm autoantibodies.  Approximately half of these patients also have C3 on the red cell membrane.  IgG coated red cells may also be seen in patients who have received an incompatible transfusion.  Thus, the DAT is routinely performed as part of all transfusion reaction investigations.  IgG bound to the red cell surface can be eluted and its specificity determined.  Eluted auto- antibodies usually bind to all red cells (panagglutinin) but occasionally have specificity within the Rh system.  Eluted alloantibodies can usually be given a distinct specificity.  Red cells sensitized with IgG may be destroyed by extravascular hemolysis.  The primary site of removal is the spleen via Fc receptors on phagocytic cells.  Factors that determine whether hemolysis will occur include: antibody titer, number of IgG molecules on the red cell, number of antigen sites on the red cell, IgG subclass, and splenic function.   

A positive DAT due to complement (C3) alone is seen in patients with cold autoantibodies, paroxysmal cold hemoglobinuria, and in some drug induced hemolytic anemias.  The offending antibodies are typically of the IgM isotype and efficiently bind complement.  IgM antibodies are not directly detected by the DAT, but are detected indirectly by the presence of C3 on the red cell surface.  Cold autoimmune hemolytic anemias may be associated with intravascular hemolysis due to complement mediated lysis.  Extravascular removal of C3 coated cells can occur via complement receptors on phagocytes in the liver. 

Rarely, patients with immune hemolysis may have a negative DAT.  Many of these patients have IgG, IgM, or IgA antibodies detected on the red cell with more sensitive techniques

DIRECT ANTIGLOBULIN TEST (DAT)

The IAT is used to detect red cell antibodies in patient serum.  In clinical practice, this is referred to as the “antibody screen” and is part of the type, screen, and crossmatch procedure.  Approximately five percent of patients have a positive IAT due to IgG antibodies, IgM antibodies, or both.  Most clinically significant alloantibodies are IgG antibodies that react best at 37°C and are formed as a result of previous exposure via transfusion or pregnancy.  Examples include antibodies to Rh, Kell, Kidd, and Duffy red cell antigens.  IgM antibodies are usually not clinically significant (except for ABO antibodies) but are a source of invitro serologic difficulty that may delay transfusion.  Examples include antibodies to the Lewis, I, P, M, and N red cell antigens.  IgM antibodies react best at cold temperatures (4°C) and are usually naturally occurring in that they do not require a sensitizing event.  A positive IAT in the absence of a positive DAT does not indicate an autoantibody and is not consistent with a diagnosis of autoimmune hemolytic anemia.   

The IAT is performed by incubating patient serum with reagent screening red cells (antibody screen) or with red cells from a unit of blood intended for transfusion (crossmatch).  The IAT takes approximately 20 minutes to perform.  If the antibody screen or crossmatch is positive, additional testing is required to determine the specificity of the antibody. 

If transfusion is necessary, patients with clinically significant red cell alloantibodies should receive antigen negative red cells.  Compatible blood may be difficult to find if antigen negative blood is rare or if multiple antibodies are present.  Consultation with the transfusion service is helpful in developing a transfusion strategy in these cases.

SUMMARY

The IAT and DAT are used to detect red cell antibodies in the serum and on the red cell, respectively.  The DAT is used to determine whether patients with hemolysis have an immune etiology.  The IAT is used to identify clinically significant red cell alloantibodies that are important in choosing compatible blood products.

For questions regarding Coomb's Testing, please contact Darrell J. Triulzi, M.D. at: (412) 209-7304.