The Coomb’s Test

The Coomb’s Test

PREPARED BY MR. ABHIJIT DAS

Antihuman Globulin (AHG)  (or Coombs Reagent)

• It is an antibody made in animals (usually rabbits) against human immunoglobulins (IgG) or complement proteins (C3d).
• AHG binds to human antibodies or complement that are already coated on red blood cells (RBCs) but not visible to the naked eye.

 

Antiglobulin Test (Coombs Test)

The Antiglobulin Test detects antibodies or complement proteins attached to red blood cells (RBCs), which do not cause visible agglutination on their own.

Principle (Both DAT & IAT)

• When RBCs coated with IgG antibodies or complement.
• The Antihuman Globulin (AHG or Coombs reagent) binds to these antibodies or complement.
• This causes visible agglutination, confirming presence of antibody or complement.

 

Direct Antiglobulin Test (DAT)

Purpose:

• Detects IgG or complement bound to RBCs inside the body (in vivo).

Procedure:

1. Collect patient’s blood sample.
2. Wash RBCs 3–4 times to remove free antibodies.
3. Add Coombs reagent (AHG) to washed RBCs.
4. Centrifuge and observe for agglutination.

Interpretation:

• Agglutination = Positive DAT → Antibodies/complement coated RBCs (seen in Hemolytic disease of newborn, autoimmune hemolytic anemia, transfusion reactions).
• No agglutination = Negative DAT.

 

Indirect Antiglobulin Test (IAT)

Purpose:

• Detects free antibodies in the serum before they attach to RBCs.
• Used in:
• Crossmatching
• Antibody screening

Procedure:

1. Mix patient’s serum with test RBCs (known antigen).
2. Incubate at 37°C to allow antibody binding.
3. Wash to remove unbound antibodies.
4. Add Coombs reagent (AHG).
5. Centrifuge and observe for agglutination.

Interpretation:

• Agglutination = Positive IAT → Antibody present in serum.
• No agglutination = Negative IAT.