Indirect coombs test or indirect antiglobulin test is a type of coombs test,it an important test for detection of circulating antibodies in the plasma, which do not agglutinate cell suspended in the saline.
It depends on the combination of in vitro of an antibody with its specific antigen.
Indirect coombs test principle
In the indirect coombs test consist of two steps. In first step, “O-positive” group cells are exposed to a serum which are 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
Indirect coombs test is a two step procedure,in the 1st step, red blood cells are incuabted with suspected serum allow antibodies to coat on red blood cell.
In second step, Red blood cells are tested (direct coomb method) , whether thet are sensitized or not.
Here is the porocedure of Indirect coombs test .
- Place two drops of serum in the test tube.
- One drop of 3% O-positive red cell suspension in the serum test tube.
- Mix well, incubate at room temperature for 45 to 50 mints.
- After incubation, wash them three times with noraml saline.
- After washing, add two to three drops of coombs reagent. Mix well.
- Mix and cetrifuge for 10 seconds.
- Look for agglutination, confirm by microscopy.
- If result is negative, add one drop of check cells to check the validity of coomb reagents.
What is the function of LESS in indirect coomb test?
LESS or low ionic strength solution reduce the incubation from 30 mints to 10 mints. when an equal volume of LESS is add in the serum.
Indirect coombs test purpose
Indirect coomb test indicate the presence of antibodies in the suspected serum. If known antiserum is used test will indicate the presence of coresponding antigen..
Indirect coombs test negative means
If Indirect coombs test negative it means, patient serum is free form agglutinating antibodies.
Indirect coombs test positive means
If Indirect coombs test is positive, it means patient serum has agglutinating antibodies. It will sensitized the red blood cells and result in agglutination.
What is the Difference between direct and indirect coombs test?
Coomb direct test 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 indirect coomb test?
Antiglobulin reagent or Coomb reagent should be checked with known SENSITIZED red blood cells. If it detect known red blood cell, it means coomb reagent is in good condetion.
But if it did not detect the known sensitized red blood cells, it mean our reagent is an expiry.
How to prepare coomb check cells
Take one meli letter serum from D-negative patient, who has been already sensitized by D-positive fetal.
The titer of anti-D antibodies should be 1/16.
Mix the serum containing anti-D IgG with washed o-positve. Incubate at 37 C for thirty minutes. Wash the coated red blood cell and make 3% suspension with saline. These cells used to check the validity of coomb test.