Indirect Coombs Test (Indirect Antiglobulin Test- IAT) - Principle, Procedure, Result, Interpretation
Indirect Coombs Test (Indirect Antiglobulin Test- IAT)
The purpose of the Indirect Coombs Test (Indirect Antiglobulin Test- IAT), 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 antenatal antibody screen, this test is done on the mother's blood. It identifies minor antigens that could cause problems in the newborns (hemolytic disease of the newborn) or the mother if a blood transfusion is necessary.
Since it detects very low concentrations of antibodies present in a patient's plasma/serum, the Indirect Coombs test is often done pre-blood transfusion. In addition to crossmatching, other applications of the Indirect Coombs Test (Indirect Antiglobulin Test- DAT) include antibody identification, RBC phenotyping, and titration studies.
Approximately 5% of patients have positive IAT due to immunoglobulin G or immunoglobulin M.
Anti-human reagents in Coombs Test
For the Coombs Test, there are three types of reagents that may be used:
monospecific reagents to detect bound immunoglobulin G (IgG)
monospecific reagents to detect bound complement (C3)
polyspecific reagents that can simultaneously detect IgG and/or C3
For the step of screening RBCs, the most commonly used reagents are polyspecific reagents that can simultaneously detect IgG and/or C3. If the result is positive, the RBC sample is then subsequently tested with monospecific reagents, either bound immunoglobulin G or bound complement. This finalizes the identification of a substance that is bound to the RBC surface.
Procedure of Direct Coombs Test (Direct Antiglobulin Test- DAT)
Historically, the Indirect Coombs Test (Indirect Antiglobulin Test- DAT) is done by tube method. Meanwhile, automated methods such as solid phase or antiglobulin gel (microcolumn method) are now being commonly used.
The procedure for the Indirect Coombs Test (Indirect Antiglobulin Test- DAT) is as follows:
Take three clean test tubes and label them T (test serum), PC (Positive control), and NC (negative control).
Place two drops of test serum in tube T (test serum).
Take one drop of anti-D serum in tube PC (Positive control).
Put one drop of normal saline in tube NC (negative control).
In each tube, add one drop of 5 % saline suspension of the pooled ‘O’ Rho (D) positive cells.
Incubate for one hour at 37ºC.
Wash the blood sample with saline three times to remove excess, unbound IgG or complement present in the serum.
* such unbound IgG or complement may preferentially bind and hence inhibit the reactivity of the added reagent.
Add two drops of anti-human globulin (Coombs serum) to each tube.
* This step should be followed immediately as delay in a test or inadequate RBC washing can cause the dissociation of antibodies from the RBC sample which can result in false-negative reports.
* If complement reagent is used instead of polyspecific reagent, a second incubation can be done to allow enhancement of weak anti-complement reactions.
Leave the tubes for 5 minutes.
Centrifuge the tubes at 1500 RPM for 1 minute.
Gently agitate the tube to resuspend.
Examine the test tubes for visible agglutination – both microscopically and microscopically.
The agglutination is graded from 0 to 4+, with 4+ representing a solid button of agglutination.
Result, Interpretation of Coombs Test
Negative result
Absence of agglutination, both microscopically and macroscopically.
Positive result
The presence of agglutination is interpreted as a positive result.
This means antibodies are bound to RBCs of the test blood sample i.e. immune system is attacking the patient’s RBCs. This condition may be autoimmunity, alloimmunity, or a drug-induced immune-mediated mechanism.