Direct Coomb Test (DCT): Introduction, Principle, Test Requirement

Introduction of Direct Coomb Test (DCT)

Table of Contents

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.

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.

Refrigerator
Tabletop centrifuge
View box/Microscope
Water bath
Anti-human globulin (AHG)
Normal saline
Glass test tubes (10 × 75 mm)
Test tube rack
Pasteur pipette or micro pipette
Marker
Container for waste disposal
Tissue paper
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

Take 3 test tubes and label them as T, N, and P for the test sample, negative control, and positive control respectively.
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.
Add 1/1 drop of anti-human globulin to all test tubes.
Incubate at room temperature for 5 minutes.
Now, mix it and centrifuge at 1000 rpm for 1 minute.
Gently re-suspend the red cell bottom and examine for agglutination or clumping.
Observe macroscopically.
If it is doubtful in macroscopic observation, proceed microscopically.

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) 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:

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.
Transfusion reaction
Drug-induced red cell sensitization
Autoimmune hemolytic anemia

Keynotes on DCT

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.
Anti-human globulin (AHG) is also called Coomb’s reagent
The sensitivity of the test can be increased by incubation at room temperature for 5 to 10 minutes and by re-centrifugation.
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.
Coomb’s reagent contains antibodies against all four classes of IgG and components of complement (usually C3 and C4).

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