The direct antiglobulin test (DAT) is used to detect in vivo sensitization of erythrocytes by γglobulins or complement fractions. It is first performed using a polyspecific Coombs serum. DAT positivity occurs with the presence of at least 500 immunoglobulin molecules attached to the red blood cells, and can be traced back to one of the following factors:
• Auto antibodies directed to the patient’s own erythrocyte antigens
• Allo antibodies present in a recently transfused patient that react with antigens present on the donor’s red cells
• Passively acquired antibodies by administration of plasma or immunoglobulins or, in the case of maternal antibodies, that have crossed the placenta and react with fetal red blood cells (neonatal hemolytic disease)
• Antibodies directed to drugs that bind to the erythrocyte membrane (such as penicillin)
• Nonspecific binding (mediated by drugs usually cephalosporins) of immunoglobulins to the erythrocyte membrane
• Presence of complement fractions on the surface of red blood cells with a mechanism mediated by drug-induced immune complexes
• Autoimmune diseases
Figure 1 shows two markedly positive samples for the direct antiglobulin test. The sample above is reactive with polyvalent Coombs serum (AHG.CD), with monospecific serum for IgG (IgG.IG) and C3d (C3d.IG); therefore, on the surface of these red cells there are IgG and complement fractions. The sample below is reactive with polyvalent Coombs serum and with monospecific serum for IgG but not for C3d; therefore, on the surface of these red cells there are IgG but not complement fractions.
Fig1. Direct antiglobulin test. Patient shows a marked positivity (++++) with the polyspecific Coombs serum. The positivity is confirmed for IgG (++++) and for C3b (+++−), the auto-control is negative. The patient therefore presents both IgG and complement on the surface of the red cells. The second patient shows marked positivity (++++) with polyspecific Coombs serum. The positivity is confirmed for IgG (++++) but both the C3b and the auto-control are negative. The patient therefore presents exclusively IgG on the surface of the red cells
In case of positive TCD with polyspecific Coombs serum, the antibody adhered to the red blood cells is then identified by TCD with monospecific sera and using elution methods. The direct antiglobulin test with monospecific sera is used to detect in vivo sensitization of erythrocytes by di γ-globulins or complement fractions. Secondly, monospecific Coombs sera (anti-IgG, anti-IgA, anti-IgM, anti-C3, anti-C3b, anti C4) can be tested.
The elution methods are used to remove the antibodies adhered to the red blood cells, and to be able to proceed with their study using the eluate. Several methods of antibody elution based on physical methods are available: hot elution, particularly effective for IgM; by freezing and thawing, usable in cases of ABO related HNFN; or chemical methods: for example, elution with organic solvents or with acid glycine which is widely indicated in the elution of IgG antibodies. Once the antibodies have been eluted, it is also possible to try to identify their specificity.
