Coomb’s test also termed as Antiglobulin Test are of two types:
- Direct Coombs test
- Indirect Coombs test
Direct Coombs test
Antibodies when bind to erythrocytes, do not always result in agglutination. Those antibodies that bind to erythrocytes (RBC) and do not cause agglutination of RBCs are referred to as incomplete antibodies. To detect presence of non-agglutinating antibodies on red blood cells, a second antibody directed against Ig/Ab coating the red cells is added. This anti-immunoglobulin can now cross link red blood cells and result in agglutination. Thus it is called Direct Coomb’s test.
In direct comb’s test (anti-globulin test), anti-human globulin reagent is used to detect antibodies adsorbed to red blood cells in vivo. Direct Antiglobuloin Test (DAT) detect antibodies bound to RBC in vivo. It is also used to is used to detect if complement system factors have bound to RBC surface antigens in vivo.
It may help to explain why one is not feeling great or have symptoms that suggest trouble related to blood. For e.g. antibodies destroying own red blood cells because of diseases like lupus and leukemia, Infections such as mononucleosis, medicines including penicillin etc.
This test is done on the newborn’s blood sample, usually in setting of a newborn with jaundice. The two most commonly recognized forms of antibody-mediated hemolysis in newborns are Rh incompatibility and ABO incompatibility.
- Prepare 5% cell saline suspension of cells (Cells washed 3-4 times with normal saline) to be tested.
- Label 3 tubes as Test, Positive Control and Negative Control.
- Take 2 drops of 5% saline cell suspension to be tested in tube labeled as T (Test).
- In the test tube labeled as PC (Positive control), 1 drop of anti D sera and 1 drop of Rh +ve pooled cells.
- Take 1 drop of normal saline and one drop of Rh +ve pooled cells in test tube labeled as NC (Negative control).
- Add 2 drops of Anti human globulin to each of the tubes.
- Mix well and centrifuge for 1 minute at 1500 rpm.
- Re-suspend cells by gentle agitation and examine macroscopically and microscopically for agglutination.
Note Point: If no agglutination is not seen on control tube, test is invalid. For direct antiglobulin test, blood drawn into EDTA is preferred (to prevent fixation of complement in vitro), but oxalated, citrated, or clotted whole blood may be used.
- Presence of agglutination means a positive test. This indicates presence of human immune globulin or complement bound to RBCs.
- Absence of agglutination means a negative test.
Positive test is a visual indication that antibodies or complement proteins are bound to surface of RBCs and may be causing destruction of those cells.
Direct Coomb’s test is used to detect:
- To detect hemolytic transfusion reactions when incompatible blood is transfused. (donor cells get coated with recipient’s antibodies and the DAT is positive).
- Antibodies induced by drugs.
- Presence of autoantibodies against RBC as in case of warm autoimmune hemolytic anemia (AHA).
- To detect hemolytic disease of Newborn (detects presence of maternal antibodies attached to fetal RBCs).
Download Link: Direct Coomb’s test.pdf