Described in 1945 by coombs ,mourant and race, Coombs test, also known as the antiglobulin test, is a laboratory procedure used to detect the presence of antibodies on the surface of red blood cells (RBCs).
There are two main types of Coombs tests: the Direct Coombs Test (Direct Antiglobulin Test or DAT) and the Indirect Coombs Test (Indirect Antiglobulin Test or IAT)
The Direct Coombs Test (Direct Antiglobulin Test or DAT)
PRINCIPLE
In certain medical conditions, such as autoimmune hemolytic anemia or hemolytic disease of the newborn, the immune system may generate antibodies (IgG) that can adhere to a patient’s RBCs, or complement proteins (C3b or C3d) may become attached to the RBCs. These antibodies or complement proteins render the RBCs “sensitized.“
Sensitized RBCs do not spontaneously clump together when mixed or centrifuged because the antibodies or complement proteins by themselves lack the ability to induce RBC aggregation.
To promote agglutination of sensitized RBCs, an additional antibody (antihuman globulin or Coombs reagent) is introduced to the system. This added antibody interacts with the Fc region of the IgG antibodies on the RBC surface or the RBCs coated with complement.
The antihuman globulin or Coombs reagent acts as a bridge, connecting the antibodies on the RBC surface to the antibodies in the added reagent. This connection causes the sensitized RBCs to come together and form clumps or agglutination.
The DAT is used to detect antibodies that are attached to RBCs. This can be done on a blood sample from a patient or on a sample of donor blood before a transfusion.
PROCEDURE
Direct Antiglobulin Test (DAT): The DAT is used to detect antibodies or complement proteins that have already bound to a patient’s red blood cells. This is commonly used to diagnose autoimmune hemolytic anemia.
Procedure:
Collect a blood sample from the patient in an EDTA anticoagulated tube.
preparation of 5% RBC suspension in Isotonic saline
Centrifuge the blood sample to separate the plasma and packed red blood cells.
Prepare 5% RBC suspension
To make a 5% cell suspension add 50 microlitre of the packed RBC’s to test tube and add normal saline till tube is filled 2/3rd.
Wash the Cells with Normal Saline:
Add a Drop of Cell Suspension to a Small Tube using a clean pipette, add one drop of the prepared 5% red blood cell suspension to a small tube ,then add normal saline till tube is filled 2/3rd .Centrifuge the tube for a short duration (e.g., 1-2 minutes) at a low speed (e.g., 1500 RPM) to separate the red blood cells from the saline. The saline can be decanted after centrifugation.
Repeat this cell washing 2 times more. cell washing is done to remove any unbound antibodies that can potentially interfer with results
After the final wash and decanting, add two drops of anti-human serum (Coombs reagent) to the washed red blood cells in the small tube.
Mix the contents of the tube well to ensure proper contact between the anti-human serum and the red blood cells .Incubate the mixture at 37°C for 30 minutes to allow for antibody binding, if present.
After incubation now centrifuge the tube for one minute at 1500 RPM to facilitate the reaction between the anti-human serum and any antibodies that may be bound to the red blood cells.
Examine the red blood cells under a microscope for agglutination (clumping). The presence of agglutination is a positive result, indicating the presence of antibodies on the surface of the red blood cells.
Method of DCT by gel tube method:
- Preparation of cell suspension (5%)
- Add 50 micro litre of packed RBC cells of test
sample in 1 ml normal saline solution and
mix uniformly - Open the wells of cassette
- Add 10 microlitre of 3.5% RBC suspension
- Centrifuge the cassette for 5 minutes
- Read the result
