Indirect coombs test procedure

Indirect Coombs test or indirect antiglobulin test is a type of coombs test, it is an important test for the detection of circulating antibodies in the plasma, which do not agglutinate cells suspended in the saline.

It depends on the combination of in vitro of an antibody with its specific antigen.

Indirect Coombs test principle

The indirect Coombs test consists of two steps. In the first step, “O-positive” group cells are exposed to a serum that is suspected of containing antibodies.

After washing to see whether red blood cells are sensitized or not.

indirect coombs test sample collection

Indirect coombs test procedure

The indirect Coombs test is a two-step procedure, in the 1st step, red blood cells are incubated with suspected serum allowing antibodies to coat on red blood cells.

In the second step, Red blood cells are tested (direct coomb method) , whether they are sensitized or not.

Here is the procedure of the Indirect coombs test.

1. Place two drops of serum in the test tube.
2. One drop of 3% O-positive red cell suspension in the serum test tube.
3. Mix well, and incubate at room temperature for 45 to 50 minutes.
4. After incubation, wash them three times with normal saline.
5. After washing, add two to three drops of coombs reagent. Mix well.
6. Mix and centrifuge for 10 seconds.
7. Look for agglutination, confirm by microscopy.
   1. If the result is negative, add one drop of check cells to check the validity of coomb reagents.

What is the function of LESS in the indirect coomb test?

LESS or low ionic strength solution reduces the incubation from 30 minutes to 10 minutes. when an equal volume of LESS is added to the serum.

Indirect Coombs test purpose

The indirect coomb test indicates the presence of antibodies in the suspected serum. If known antiserum is used test will indicate the presence of the corresponding antigen.

Indirect Coombs test negative means

If Indirect coombs test negative it means, the patient’s serum is free from agglutinating antibodies.

Indirect Coombs test positive means

If the Indirect coombs test is positive, it means the patient’s serum has agglutinating antibodies. It will sensitize the red blood cells and result in agglutination.

What is the Difference between direct and indirect coombs tests?

Coomb direct test is used to detect the SENSITIZED red blood cells. While In the indirect coomb test, floating antibodies in the serum are assessed which sensitized the red blood cells.

what is the quality control in an indirect coomb test?

Antiglobulin reagent or Coomb reagent should be checked with known SENSITIZED red blood cells. If it detects known red blood cells, it means the coomb reagent is in good condition.

But if it did not detect the known sensitized red blood cells, it means our reagent is expiry.

How to prepare coombs check cells

Take one-mile letter serum from a D-negative patient, who has been already sensitized by a D-positive fetus.

The titer of anti-D antibodies should be 1/16.

Mix the serum containing anti-D IgG with washed o-positive. Incubate at 37 C for thirty minutes. Wash the coated red blood cell and make a 3% suspension with saline. These cells are used to check the validity of the coomb test.