How to perform Direct and Indirect coombs test

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How to perform Direct and Indirect coombs test mltgeeks

The antiglobulin test (Coombs test) was introduced in 1945 by Coombs, Mourant and Race. The test has been developed to detect antibodies that can bind the antigen on the surface of erythrocytes but can not agglutinate them( incomplete antibodies).

What is the Principle of Coombs test

The test is based on the principle that anti- human globulin( AHG) antibodies are combined with red cells coated with human immunoglobulin or complement( in vivo or in vitro). The AHG acts as a bridge and causes an agglutination of the erythrocytes.

Two types of antiglobulin tests exist : Direct Antiglobulin Test (DAT) and Indirect Antiglobulin Test (IAT)

Direct Antiglobulin Test (DAT)

To determine if the RBCs of a patient are coated with immunoglobulin, complement or both, AHG with reactivity to human immunoglobulin and/or compliment is added to the RBCs of the patient. If cross- link and subsequent agglutination occurs, the direct Coomb test is positive.

Materials required for Direct antiglobulin test

  • Test tubes
  • AHG reagent
  • Positive control cells (IgG coated)
  • Centrifuge and microscope

Type of specimen: EDTA blood

Direct antiglobulin test procedure

  1. Make 5 percent of the patient’s blood cell suspension by washing with normal saline three times.
  2. After the last wash, decant the supernatant completely.
  3. In the test tube, take 1 drop of cell suspension of the patient.
  4. Add two drops of A.H.G reagent to the cell suspension of the patient in the test tube.
  5. Mix well and centrifuge the mixture for 1 minute at 1500 rpm
  6. Shake the tube gently and examine it with naked eye and under a microscope to see the agglutination.
  7. If the result of the test is negative, add a drop of control cells.
  8. Mix well and centrifuge the mixture for 1 minute at 1500 rpm and search for agglutination. If  there is no agglutination, the result is invalid.
Also read  Various methods used for hemoglobin measurement in the laboratory


Presence of agglutination means a positive DAT. This indicates the presence of human immunoglobulin or a complement bound to erythrocytes.

The lack of agglutination means a negative DAT

What are the uses of Direct Antiglobulin Test

  1. In order to detect hemolytic transfusion reactions when incompatible blood is transfused.Donor cells are coated with the antibodies of the recipient and DAT is positive.
  2. To detect Newborn hemolytic disease: detects the presence of maternal antibodies attached to fetal RBCs.
  3. To detect autoantibodies against RBC, such as warm autoimmune hemolytic anemia( AHA).
  4. To detect Drug- induced antibodies.


Reagent RBCs( Coombs control cells) are incubated in the presence of patient ’s serum. With addition of AHG reagent. agglutinated IgG- coated erythrocytes or red cells agglutinate

Materials required for Indirect antiglobulin test

  • O positive red blood cells (Coombs control cells)
  • Anti D serum
  • Normal saline
  • Test tubes
  • AHG reagent
  • Positive control cells (IgG coated)
  • Centrifuge and microscope

Type of specimen : Patient’s Serum

How to prepare “O” positive red cells

  1. A pool of “O” positive blood group cells from three different donors is taken.
  2. These cells are washed  with saline at least 3 times and a 5 percent  saline suspension of the washed cells is made.

Indirect antiglobulin test procedure

  1. Take three test tubes that are labeled test, positive control and negative control.
  2. To the tube labelled Test (T) add 2 drops of test serum and  1 drop of coombs control cells
  3. To the tube labelled Positive Control (P) add 1 drop of Anti D serum and 1 drop of coombs control cells
  4. To the test tube labelled Negative Control (N) add 1 drop of normal saline and 1 drop of coombs control cells.
  5. Wash all the cells in the tubes separately at least three times with normal saline to remove any traces of attached serum.
  6. Now add 2 drops of anti human globulin to each test tube
  7. Incubate for 5-10 min. at room temperature
  8. Centrifuge at 1500 RPM for 1 minutes
  9. Resuspend the cells and observe all the tubes for agglutination with naked eye and microscopically
Also read  Hemolytic Disease of the Newborn (Causes and investigation)

Observation and Result

TestPositive Control Negative ControlResult
AgglutinationAgglutinationNo AgglutinationPositive
No Agglutination Agglutination No Agglutination Negative

If positive control shows no agglutination or negative control shows agglutination, the test has not been correctly performed and must be repeated.

Result interpretation

  • A positive test means the serum of the patient contains anti- D antibodies.
  • A negative test means that patients with serum do not contain anti- D antibodies

How to increase the Sensitivity of IAT

  • By optimizing the temperature at 37ˆC
  • Increasing the ratio of serum to red cells
  • Addition of LISS (low ionic strength saline) albumin and enzymes

What are the uses of Indirect Antiglobulin Test

  • Cross matching To detect the presence of RBC donor- based antibodies.
  • It is used for the Detection of the presence of Anti D antibodies in a Rh negative woman married to an Rh positive man.
  • Also used for the typing of erythrocyte antigen

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About the Author: Arthur Westmann

DEFFE ARTHUR (AMOEBAMANN) is the founder and author of MLTGEEKS and MLTEXPO.He’s from Cameroon and is currently a Final year State Medical Laboratory Technician (MLT MA). Beyond lab works, he’s a passionate internet user with a keen interest in web design and blogging. Furthermore He likes traveling, hanging around with friends and social networking to do in his spare time.

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