Previously known as the direct and IATs, these assays play a major role in transfusion medicine as well as in the diagnosis of immune hemolytic anemias (see p. 377), because they detect antibodies either bound to RBCs (the DAT), or in serum (the indirect antiglobulin test, IAT). In patients that have not been transfused within the preceding 3 months, a positive DAT almost always reveals autoimmune antibodies.
The IAT is used to demonstrate in vitro reactions between RBCs and antibodies that sensitize red cells that express the corresponding antigen. The patients serum or plasma is incubated with red cells, which are then washed to remove unbound globulins. Agglutination that develops when the antiglobulin reagent is added indicates a reaction between serum antibodies (usually the result of immunization from previous transfusions) and red cells.
The antiglobulin reagent consists in most cases of rabbit antibodies directed against human IgG. Other reagents used in the DAT assay are anticomplement (anti-C3dg) or a mixture of anti-IgG and anti-C3dg. If the DAT is positive following recent transfusions, the antibodies can be eluted from RBCs and the eluted antibodies must be identified.
Use
The DAT is used whenever hemolysis of red cells is suspected as being caused by autoantibodies. The assay determines if red cells have been coated in vivo with immunoglobulins, complement, or both.
The utility of the IAT in blood banking stems from its great sensitivity in detecting various IgG antibodies in the recipients serum prior to transfusions. It is part of the antibody screening test. It is used to detect the presence of alloantibodies directed against non-ABO blood group antigens.
In cases of severe autoimmune hemolytic anemia, both the DAT and the IAT may be positive because the excess antibodies elute from the red cell membranes and spill out into serum.
Interpretation
Both the DAT and the IAT are reported and interpreted as either positive or negative.
The DAT is positive whenever the patients red cells are coated with autoantibodies that developed against the patient's own red cells. It is also positive when alloantibodies in a recipient's circulation react with antigens on recently transfused red cells, as well as alloantibodies in maternal circulation, which cross the placenta and coat fetal red cells. Antibodies directed against certain drugs may also bind to red cell membranes and result in a positive test.
The IAT is positive in the presence of serum alloantibodies in patients previously transfused and immunized against non-self " ôred cell antigens.
Limitations
Finding of a positive DAT indicates the presence of red cell autoantibodies, alloantibodies following transfusions, or of coating of red cells with excess immunoglobulins. It requires additional workup to elucidate the etiology of the immunoglobulins by performing tests for antibody specificity: cold agglutinins (see p. 377 under hemolytic anemias), Donath-Landsteiner antibody (see p. 379), and also serum protein electrophoresis or immunofixation when a plasmacytic disease (see p. 432) is suspected. The administration of certain drugs (α-methyldopa, IV penicillin, or procainamide) and recent transfusions must also be excluded.
A negative DAT does not rule out hemolysis but only hemolysis of autoimmune etiology. For instance, DAT is negative in some cases of drug-induced hemolytic anemias, hemoglobinopathies, hereditary spherocytosis, and other hereditary hemolytic anemias.
A positive IAT requires further investigation to identify more precisely the offending antigen(s).