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SOP

Direct Antiglobulin Test (DAT / Direct Coombs)

SOP for detecting in vivo sensitization of RBCs with IgG and/or C3d.

Last verified 1 month ago 1 min read Blood Bank / Transfusion Medicine Immunohematology #blood bank #dat #direct coombs #hemolytic anemia #immunohematology
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Document Control
Version
1.0
Effective
Aug 28, 2026
Reviewed
Aug 29, 2026
Department
Blood Bank / Transfusion Medicine
Principle

AHG reagent bridges the gap between IgG or C3d molecules coating the patient's RBCs, causing visible agglutination. Washing removes free serum globulins that could neutralize the AHG reagent.

Standard Operating Procedure: Direct Antiglobulin Test (DAT)

1. Purpose: Detect antibodies or complement bound to patient RBCs in vivo (e.g., HDFN, autoimmune hemolytic anemia, drug-induced hemolysis).

2. Specimen: EDTA whole blood (preferred to prevent in vitro complement activation).

3. Reagents: Polyspecific AHG (Anti-IgG + Anti-C3d), IgG monospecific AHG, C3d monospecific AHG, Coombs control cells (check cells).

4. Procedure:

  • Wash patient RBCs 3-4 times with saline to remove unbound globulins.
  • Prepare 3-5% washed RBC suspension.
  • Add 1 drop suspension to tubes labeled Anti-IgG, Anti-C3d, and Control (saline).
  • Add 1 drop corresponding reagent. Mix and centrifuge.
  • Read for agglutination.
  • Add check cells to all negative tubes; centrifuge and read to confirm AHG activity.

5. Interpretation: Agglutination = Positive DAT. No agglutination + check cell agglutination = Negative DAT.

Frequently Asked Questions

They confirm that the AHG reagent was active and not neutralized by inadequate washing, preventing false-negative results.