Coombs test (antiglobulin test) is used to detect the presence of ‘incomplete’ Rh antibodies i.e. IgG antibodies capable of sensitizing RBCs but incapable of causing agglutination of RBCs (hemagglutination). The antiglobulin (Coombs) test was introduced by Coombs and colleagues in 1945.
Anti-Rh antibodies are of IgG type, but they normally do not agglutinate Rh-positive RBCs (RBCs containing Rh antigen) so anti-Rh antibodies are also called incomplete antibodies as opposed to ‘complete’ IgM antibodies, which do agglutinate red cells.
Complete vs. Incomplete Antibodies
Red blood cells contain various antigens on their surface. RBC containing antigens A, B, and AB on its surface are designated as blood groups A, B, and AB respectively. Blood group ‘O’ does not contain both antigen ‘A’ and antigen ‘B’ on its surface. These blood group antigens are carbohydrate molecules. Our immune system usually makes IgM class of antibodies against carbohydrate antigen (as it gets no sufficient signals for class switching). These IgM antibodies (which are pentameric in nature) can bind specific antigens present on the surface of RBCs and can agglutinate RBC. So antibodies against ABO blood group antigens are called complete antibodies.
RBC also contains another antigen on its surface, which is called the ‘Rh’ antigen. Rh antigens are protein in nature. When a person lacking Rh antigen (e.g. having a negative blood group) is exposed to Rh antigen (during a blood transfusion or mother during delivery of children having a positive blood group), his/her immune system makes antibodies against Rh antigen. As this Rh antigen is a protein, its exposure generates IgG (class switching occurs).
IgG antibodies thus formed bind specifically to the Rh antigens present on the surface of RBCs but they become unable to agglutinate RBCs (there is no cross-linking or clumping) perhaps due to the presence of insufficient antigenic determinants (epitope) on the RBCs to permit the antibody to overcome normal electrostatic repulsion that exists among RBCs and/or because of the inherent property of IgG (monomeric form). So, the antibodies formed against Rh antigens are called incomplete antibodies.
Negative charge on the surface of RBCs 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.
The standard antiglobulin reagent contains antibodies against all four classes of IgG and components of complement (usually C3 and C4).
Direct Coombs Test
The direct Coombs test, also known as the direct antiglobulin test (DAT) uses antibodies directed against human proteins (primarily immunoglobulin G [IgG] and complement [C3]) to detect whether these proteins are attached to the surface of RBCs. This involves the addition of Coombs serum directly to a patient’s washed RBCs.
The occurrence of agglutination means that the patient’s RBCs have been sensitized in vivo by the antibody. Direct Coombs testing is vital for diagnosing autoimmune hemolytic anemias such as;
- hemolytic transfusion reaction,
- hemolytic disease of the fetus and newborn (HDFN) and
- autoimmune hemolytic anemia (AIHA) etc.
Indirect Coombs test
The indirect Coombs test, also known as indirect antiglobulin test (IAT) detects antibodies against human RBCs in the patient’s serum. This involves incubating a patient’s serum with RBCs of a known type and adding Coombs serum. If in vitro sensitization occurs, agglutination will result, which indicates that antibodies are present against the known blood type.
The indirect Coombs test is used in crossmatching before blood transfusions and in prenatal testing of pregnant women.
An indirect antiglobulin crossmatch is performed to assure compatibility of red cell units for transfusion in certain patients by incubating the recipient’s plasma or serum and donor red cells.
- A. If ‘nonagglutinating’ or ‘incomplete’ antibodies against red blood cell antigens are present in patient’s serum, they will coat the RBCs of the donor but won’t be able to clump them together. When the coombs reagent (anti-human globulin) reagent is added the antihuman antibodies will bind to the Fc portion of non-agglutinating antibodies (IgG) attached to RBCs causing agglutination of the red cells.
- B: If incomplete antibodies are absent in the patient’s serum: anti-human antibodies won’t be able to attach to RBCs so will be washed away. There will be no agglutination of RBCs.