Direct Coomb Test (DCT): Introduction, Principle, Test Requirements, Test Procedure, Result and Interpretation, and Clinical Significance

Introduction of Direct Coomb Test (DCT)

Direct Coomb Test (DCT) is a type of Coomb test also called the anti-globulin test. It is of two types-Direct Coomb’s test (DCT) and the Indirect Coomb’s test (ICT). DCT uses to detect sensitized red blood cells (RBCs) while ICT detects the presence of ‘incomplete’ Rh antibodies i.e. IgG antibodies. IgG antibodies are capable of sensitizing RBCs but incapable of causing agglutination of RBCs (hemagglutination). Coomb’s test was introduced by Cambridge immunologists Robin Coomb and his et. al. (Arthur Mourant and Rob Race) in 1945.

Coomb Test Reagent
Fig. Coomb Test Reagent

Principle of Direct Coomb Test (DCT)

Direct anti-human globulin test is used to detect the sensitized red blood cells in particular Immunoglobulin gamma (IgG) and Immunoglobulin meu (IgM) that is present in patient red blood cells.

Requirements for DCT

Following equipment and reagents are needed to proceed direct Coomb’s test.

  1. Refrigerator
  2. Tabletop centrifuge
  3. View box/Microscope
  4. Water bath
  5. Anti-human globulin (AHG)
  6. Normal saline
  7. Glass test tubes (10 × 75  mm)
  8. Test tube rack
  9. Pasteur pipette or micro pipette
  10. Marker
  11. Container for waste disposal
  12. Tissue paper
  13. Specimen-Blood-Particularly EDTA  is preferred but oxalate, or clotted,  citrated whole blood may be used ( the specimen needs to be a fasting sample).

The procedure of the Direct Coomb Test

  1. Take 3 test tubes and label them as T, N, and P for the test sample, negative control, and positive control respectively.
  2. Add 2/2 drops of washed patient cells, washed O red cells, and sensitized red blood cells in T, N, and P test tubes respectively.
  3. Add 1/1 drop of anti-human globulin to all test tubes.
  4. Incubate at room temperature for 5 minutes.
  5. Now, mix it and centrifuge at 1000 rpm for 1 minute.
  6. Gently re-suspend the red cell bottom and examine for agglutination or clumping.
  7. Observe macroscopically.
  8. If it is doubtful in macroscopic observation, proceed microscopically.
Direct Coomb Test (DCT) Observation
Fig. Direct Coomb Test (DCT) Observation

Result and Interpretation of Direct Coomb Test (DCT)

  • Negative control: No agglutination
  • Positive control: Agglutination
  • Test: Agglutination or no agglutination
  • Presence of agglutination or clumping: Direct Coomb Test (DCT) Positive
  • Absence of agglutination or clumping: Direct Coomb Test (DCT) Negative

 Direct Coomb Test (DCT) Negative

 Direct Coomb Test (DCT) Negative
Fig.  Direct Coomb Test – Negative

Direct Coomb Test (DCT) Positive

Direct Coomb Test (DCT) Positive
Fig. Direct Coomb Test – Positive

Clinical Significance of DCT

This test is performed to detect sensitized RBCs or anti-D antibodies or other antibodies attached to the red blood cell surface within the bloodstream. This occurs in the following  conditions:

  1. Erythroblastosis fetalis or hemolytic disease of the newborn (HDN): When there is an Rh-positive baby in the womb of a sensitized  Rh-negative woman, the antibodies produced in the mother’s serum cross the placenta, and after entering the baby’s bloodstream, these antibodies will attach to the bloodstream, these antibodies will attach to the baby’s Rh-positive red blood cells. These coated ( or sensitized) cells are clumped and removed from the circulations causing hemolytic anemia. When the baby is born, the baby’s blood (cord blood collected from the umbilical cord) is collected and tested for the Direct Coomb test.
  2. Transfusion reaction
  3. Drug-induced red cell  sensitization
  4. Autoimmune hemolytic anemia

Keynotes on DCT

  1. Anti-Rh antibodies are of IgG type that normally do not agglutinate Rh-positive RBCs so anti-Rh antibodies are also known as incomplete antibodies as resistant to ‘complete’ IgM antibodies, which do agglutinate red cells.
  2. Anti-human globulin (AHG) is also called Coomb’s reagent
  3. The sensitivity of the test can be increased by incubation at room temperature for 5 to 10 minutes and by re-centrifugation.
  4. In a simple setup, DCT is performed in test tubes while in the advanced laboratory, it is commonly done using micro-array and gel technology.
  5. Coomb’s reagent contains antibodies against all four classes of IgG and components of complement (usually C3 and C4).

Further Reading

  • Technical Manual of the American Association of Blood Banks-13th Edition, 1999
  • Introduction to Transfusion Medicine –Zarin Bharucha & D.D. Chouhan,1st Edition, 1990

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