The Coombs test, also called the antiglobulin test, is used to detect antibodies that may attach to red blood cells and cause them to be destroyed prematurely.
It was discovered in 1945 by Coombs, Mourant, and Race.
The test uses Coombs reagent, which is an antihuman globulin.
Coombs reagent is produced by injecting human globulin into animals, prompting them to generate polyclonal antibodies that specifically target human immunoglobulins and components of the human complement system.
Principle of Coombs test
Red blood cells coated with complement or IgG antibodies do not directly agglutinate when centrifuged.
Such red blood cells are considered sensitized with IgG or complement.
For agglutination to take place, an additional antibody must be introduced that reacts with the Fc portion of the IgG antibody or with the C3b or C3d component of the complement system.
This additional antibody creates a “bridge” between the antibodies or complement bound to the red blood cells, leading to agglutination.
Types of Coombs test
Direct Coombs Test (Direct Antiglobulin Test- DAT)
The direct Coombs test, also called the Direct Antiglobulin Test (DAT), is performed to detect antibodies (IgG or C3) that are attached to the surface of red blood cells.
Various diseases and certain drugs can lead to the presence of such antibodies.
These antibodies may destroy red blood cells, resulting in anemia.
The test is often carried out on a newborn’s blood sample, particularly in cases of newborn jaundice.
The two most common forms of antibody-mediated hemolysis in newborns are Rh incompatibility and ABO incompatibility.
Procedure of Direct Coombs Test
Prepare a 5% suspension of the red blood cells to be tested in isotonic saline.
Using a clean pipette, add one drop of the prepared cell suspension into a small test tube.
Wash the cells three times with normal saline to eliminate all traces of serum.
Completely decant the saline after the final wash.
Add two drops of anti-human serum to the tube.
Mix thoroughly and centrifuge for one minute at 1500 RPM.
Gently resuspend the cells by agitation and check for agglutination both macroscopically and microscopically.
Indirect Coombs Test (Indirect Antiglobulin Test- IAT)
The indirect Coombs test, also known as the Indirect Antiglobulin Test (IAT), is used to detect free-floating antibodies against specific red blood cells.
It is most commonly performed to assess the risk of a reaction to a blood transfusion.
This test is conducted on a mother’s blood sample as part of routine prenatal laboratory work and is often called the “antibody screen.”
The antibody screen identifies a wide range of minor antigens that could cause complications in newborns or lead to problems for the mother if a blood transfusion becomes necessary.
Around 5% of patients have a positive IAT result, which may be due to the presence of IgG antibodies, IgM antibodies, or both.
Procedure of Indirect Coombs Test
Label three test tubes as T (Test serum), PC (Positive control), and NC (Negative control).
In the tube labeled T (Test), add 2 drops of test serum.
In the tube labeled PC (Positive control), add 1 drop of anti-D serum.
In the tube labeled NC (Negative control), add 1 drop of normal saline.
To each tube, add 1 drop of a 5% saline suspension of pooled ‘O’ Rho (D) positive red blood cells.
Incubate all three tubes for 1 hour at 37°C.
Wash the cells three times with normal saline to remove excess serum and free antibodies; inadequate washing may result in false-negative results.
Add 2 drops of Coombs serum (anti-human serum) to each tube.
Allow to stand for 5 minutes, then centrifuge at 1,500 RPM for 1 minute.
Resuspend the cells and examine for agglutination both macroscopically and microscopically.
Result Interpretation of Coombs Test
Negative Result:
No clumping of cells (no agglutination) is observed.
Indicates absence of antibodies against red blood cells.
Positive Result:
Clumping (agglutination) of blood cells during a direct Coombs test indicates the presence of antibodies on red blood cells.
Suggests a condition where the immune system is destroying red blood cells (hemolysis).
Possible causes include:
Hemolytic anemia
Chronic lymphocytic leukemia or related disorders
Erythroblastosis fetalis (hemolytic disease of the newborn)
Infectious mononucleosis
Mycoplasmal infection
Syphilis
Systemic lupus erythematosus
Transfusion reaction, such as one caused by improperly matched blood units
