Coombs Test (Direct and Indirect Antiglobul) test
1. Objective
Direct Coombs Test (DCT):
The objective was to detect antibodies or complement proteins already bound to the patient's red blood cells, indicating in vivo sensitization.
Indirect Coombs Test (ICT):
The objective was to detect free antibodies present in the patient’s serum that could bind to red blood cells, usually used for blood compatibility testing.
2. Principle
DCT:
The test was based on adding Coombs reagent (antihuman globulin) to washed red blood cells. If antibodies or complement were already bound to the cells, the reagent caused agglutination.
ICT:
The test worked by mixing the patient’s serum with donor red cells, incubating to allow antibody binding, then washing and adding Coombs reagent to detect bound antibodies via agglutination.
3. Materials
- Patient's blood sample (for serum and RBCs)
- Coombs reagent (Antihuman globulin)
- Red blood cells (from patient for DCT; from donor for ICT)
- Test tubes
- Centrifuge
- Normal saline
- Microscope
- Pipettes
- Glass slides and coverslips
4. Procedure (Microscopic)
Direct Coombs Test (DCT)
1. Patient’s RBCs were washed 3 times with normal saline.
2. A 2–5% RBC suspension was prepared.
3. One drop of Coombs reagent was added to one drop of RBC suspension.
4. The tube was incubated at 37°C for 15–30 minutes.
5. It was centrifuged at 1000 rpm for 1 minute.
6. The tube was gently shaken, and a drop was placed on a slide and examined microscopically for agglutination.
Indirect Coombs Test (ICT)
1. Patient’s serum was mixed with donor RBCs and incubated at 37°C for 30–60 minutes.
2. The cells were washed 3 times with saline to remove unbound antibodies.
3. One drop of Coombs reagent was added to the washed cells.
4. After centrifugation, a drop of the suspension was placed on a slide and observed under the microscope for agglutination.
5. Result
Positive Test (DCT or ICT):
Agglutination observed under the microscope indicated the presence of antibodies.
Negative Test:
No agglutination meant antibodies or complement were absent.
6. Uses
Direct Coombs Test:
Diagnosis of autoimmune hemolytic anemia
Hemolytic disease of the newborn (HDN)
Hemolytic transfusion reactions
Drug-induced hemolysis
Indirect Coombs Test:
Pre-transfusion compatibility testing (crossmatch)
Antibody screening in pregnancy (Rh incompatibility)
Detecting alloantibodies in recipients/donors
7. Consultation
Test results were interpreted with clinical history and consultation with a hematologist or physician. A positive DCT suggested ongoing immune-mediated hemolysis, while a positive ICT indicated alloimmunization risk, especially in transfusion or pregnancy scenarios. Further immunohematological workup was often advised.
[C] MediLab BD
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