Coombs Test (Antiglobulin Test), including both the Direct and Indirect versions


🧪 1. Objective
The objective of the Coombs test was to detect antibodies (or complement proteins) bound to red blood cells (Direct Coombs) or free antibodies in a patient’s serum that could react with red cells (Indirect Coombs). It was used to diagnose immune-mediated hemolytic anemia, hemolytic disease of the fetus and newborn (HDFN), and for crossmatching in transfusions.

🧪 2. Principle
The test relied on the use of Coombs reagent (anti-human globulin, AHG), which binds to human IgG and/or complement (C3d) attached to red cells.

  • In the Direct Coombs test (DAT), it detected in vivo sensitization of RBCs.

  • In the Indirect Coombs test (IAT), it detected antibodies in the serum capable of binding to RBC antigens in vitro.

Agglutination after addition of AHG indicated a positive result.

🧪 3. Materials

  • Patient’s red blood cells (for DAT) or serum (for IAT)

  • Screening or panel red cells (for IAT)

  • Anti-human globulin reagent (polyspecific or monospecific: anti-IgG, anti-C3d)

  • Test tubes or gel card system

  • Normal saline, centrifuge, incubator (37°C)

🧪 4. Procedure

A. Direct Coombs Test (DAT)

  1. Patient’s red cells were washed 3–4 times with saline.

  2. 1 drop of washed RBCs was mixed with 2 drops of Coombs reagent.

  3. The tube was centrifuged for 20 seconds.

  4. The cell button was gently resuspended and checked for agglutination.

B. Indirect Coombs Test (IAT)

  1. Patient’s serum was mixed with reagent red cells and incubated at 37°C for 15–30 minutes.

  2. The cells were washed to remove unbound antibodies.

  3. Coombs reagent was added.

  4. The mixture was centrifuged and checked for agglutination.

🧪 5. Result (Example)

Test TypeObservationInterpretation
DAT (Polyspecific)Positive (+2)IgG or C3d on patient RBCs → Immune hemolysis
IATNegativeNo unexpected antibodies detected
Control CellsAgglutinatedValidates a true negative IAT

🧾 Interpretation: A positive DAT indicated autoimmune hemolytic anemia or alloimmunization. A negative IAT suggested absence of clinically significant antibodies.

🧪 6. Uses

  • Diagnose autoimmune hemolytic anemia

  • Investigate transfusion reactions

  • Screen for alloantibodies in pregnancy

  • Crossmatch blood for transfusion

  • Detect HDFN (hemolytic disease of the fetus and newborn)

🧪 7. Conclusion
The Coombs test was an essential immunohematology tool for detecting RBC-bound or circulating antibodies. It provided critical information in transfusion safety and immune-related hemolytic conditions.

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