Indirect Coombs Test Procedure

The Indirect Coombs Test Procedure looks for antibodies that are floating around in the blood. These antibodies might act against certain red blood cells. This test is most often done to determine if you may be having a reaction to a blood transfusion.

Also Known asIndirect Coombs TestIndirect Antiglobulin TestIATIndirect Anti-human Globulin Test
Test PurposeIndirect Coombs Test looks for antibodies that are floating around in the blood
Test PreparationsNo Need any Preparations
Test ComponentsIndirect Coombs Test
SpecimenIndirect Coombs: 2 ML (1 ML Min.) Serum From 1 SST. 
Stability RoomIndirect Coombs : 2 hours
Stability RefrigeratedIndirect Coombs : 48 hours
Stability FrozenIndirect Coombs : N/A
MethodErythrocyte Magnetised Technology
Download Report Download Report
Indirect Coombs Test Procedure
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Principle of Coombs’ Test

Red blood cells coated with complement or IgG antibodies do not directly agglutinate when centrifuged. These cells are said to be IgG or complement sensitized. For agglutination to occur, an additional antibody must be added to the system that reacts with the Fc portion of the IgG antibody, or with the C3b or C3d component of complement. This will form a “bridge” between the antibodies or complement that coats the red blood cells, causing agglutination.

Sample :

2 ML (1 ML Min.) Serum From 1 SST.

Sample Preprations:

No need any Preprations

Indirect Coombs Test Procedure:

  • Label three test tubes as T (test serum), PC (positive control), and NC (negative control).
  • In the tube labeled T (Test), take 2 drops of test serum.
  • In the test tube labeled PC (Positive Control), take 1 drop of anti D serum.
  • In the test tube labeled NC (Negative Control), take 1 drop of normal saline.
  • Add one drop of 5% saline to the pooled ‘O’ Rho (D) positive cells in each tube.
  • Incubate all three tubes for one hour at 37°C.
  • Wash cells three times in normal saline to remove excess serum lacking free antibodies (inadequate red blood cell washes may lead to negative results).
  • Add two drops of Coombs’ serum (anti-human serum) to each tube.
  • Hold for 5 minutes and then centrifuge at 1500 RPM for one minute.
  • Resuspend cells and examine both macroscopically and microscopically.

Results:

  • Negative: No cell agglutination (no clumping). This means that you do not have antibodies against red blood cells.
  • Positive: Clumping (agglutination) of blood cells. It means you have antibodies on your red blood cells.

Indirect Coombs Test Negative Result Mean:

No clumping of cells (no agglutination). This means you have no antibodies to red blood cells.

Indirect Coombs Test Positive Result Mean:

Clumping (agglutination) of the blood cells during a direct Coombs test means that you have antibodies on the red blood cells and that you may have a condition that causes the destruction of red blood cells by your immune system (hemolysis). This may be due to

  • Hemolytic anemia,
  • Chronic lymphocytic leukemia or similar disorder,
  • Erythroblastosis fetalis (hemolytic disease of the newborn),
  • Infectious mononucleosis,
  • Mycoplasmal infection,
  • Syphilis,
  • Systemic lupus erythematosus and
  • Transfusion reaction, such as one due to improperly matched units of blood.
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