Coombs Test - Introduction, History, Purpose, Principle
Introduction to Coombs Test
The Coombs Test is a form of antiglobulin test used to demonstrate the presence of an antibody (immunoglobulin) or complement that binds to red blood cell (RBC) membranes. It uses the anti-human globulin to form visible agglutination – indicating in vivo sensitization.
It is also known as Direct Antiglobulin Test (DAT) or Direct Anti-human Globulin Test.
The Coombs test is important in the assessment of both immune and drug-induced hemolytic anemias, HDFN, and the evaluation of blood transfusion reactions.
History of Coombs Test
The Coombs Test was first developed in 1908 by Morescho. Decades later, in 1945, Robin Coombs introduced this reaction to demonstrate RBC agglutination in the presence of what was believed to be an “incomplete” or “blocking” antibody.
Purpose of Coombs Test
It is one of the most widely used assays in the laboratory. The purpose of the Coombs test is as follows:
Diagnosis of autoimmune-mediated hemolytic anemia (destruction of red blood cells).
Investigation of blood transfusion reaction.
Diagnosis of hemolytic diseases in newborns - Hemolytic disease of fetus and newborn (HDFN)
Principle of Coombs Test
The Coombs test detects incomplete anti-Rh antibodies that do not agglutinate Rh+ erythrocytes in saline. It is produced by injecting human globulin into animals - which produces polyclonal antibodies with specificity to human immunoglobulins and/or human complement system factors.
When RBC coated with IgG antibodies or complement is centrifuged, it does not agglutinate and is said to be sensitized with IgG or complement. For agglutination to occur, additional antibodies must be added. This anti-human globulin uses 2 Fab sites present in the reagent antibody or the C3b or C3d component of complement to bind to the Fc portion of the antibody which has been coating target RBCs.
The use of anti-human globulin formed in the RBC coating antibody causes a “bridge” which results in the formation of visible RBC agglutination.
When plasma was tested for the presence of Rh antibody by using Rh antigen–positive RBCs, the reaction did not result in agglutination, although the RBCs appeared to be sensitized by the respective Rh antibody. In addition, RBCs could no longer bind with their respective “complete” or agglutinating antibodies.
However, the Coombs test has limited sensitivity as it may give false-positive and false-negative results.
Types of Coombs test
Coombs test can be direct or indirect.
Direct Coombs Test (Direct Antiglobulin Test- DAT)
Indirect Coombs Test (Indirect Antiglobulin Test- DAT)
Direct Coombs Test (Direct Antiglobulin Test- DAT)
Direct Coombs Test (Direct Antiglobulin Test- DAT) is used for the detection of antibodies (IgG or C3) bound to the RBC surface.
In the direct Coombs method, in vivo sensitization of red blood cells (RBCs) with incomplete antibodies takes place. It uses antiserum against human immunoglobulin to agglutinate a patient’s RBC and detects cell-bound antibodies.
This test is done on newborn blood samples, mostly on infants with jaundice for diagnosis of Rh incompatibility and ABO incompatibility.
Indirect Coombs Test (Indirect Antiglobulin Test- DAT)
The purpose of the Indirect Coombs Test (Indirect Antiglobulin Test- DAT), is to determine the presence of free-flowing antibodies against certain red blood cells and is mostly done in cases of blood transfusion reaction.
In the indirect Coombs method, RBCs are sensitized with incomplete antibodies in vivo. Antiserum is added to human immunoglobulin while sample serum is mixed with normal RBCs. If antibodies are present in the serum, agglutination occurs.
Also referred to as an antibody screen, this test is done on the mother's blood. It identifies minor antigens that could cause problems in the newborns or the mother if a blood transfusion is necessary.
Approximately 5% of patients have positive IAT due to immunoglobulin G or immunoglobulin M.