DAT

Direct Antiglobulin Test is often called the DAT or sometimes the Direct Coombs test. This is a fundamental test in the blood bank, and understanding it helps us figure out if something is going wrong with red blood cells inside the patient’s body

Think of it this way: the Antibody Screen and Crossmatch (using the Indirect Antiglobulin Test - IAT) look for antibodies floating around in the plasma that could attach to red cells. The DAT, however, asks a different question: “Are the patient’s red blood cells already coated with antibodies or complement proteins right now, while circulating in their bloodstream?”

The Core Purpose: Detecting In Vivo Sensitization

The primary goal of the DAT is to detect red blood cells (RBCs) that have been sensitized in vivo

• In Vivo: Means “within the living body” – the coating happened inside the patient’s circulation
• Sensitized: Means the RBCs are coated with immunoglobulins (primarily IgG) and/or complement components (often C3d is what we detect)

Why Would We Perform a DAT? (Clinical Indications)

We usually run a DAT when we suspect something is causing red cell destruction or interaction within the patient. Key scenarios include:

• Transfusion Reaction Workup: Essential test if a patient shows signs/symptoms of a hemolytic transfusion reaction (HTR) after receiving blood. A positive DAT on the post-reaction sample (compared to the pre-reaction sample) suggests the transfused cells are being coated by recipient antibodies
• Hemolytic Disease of the Fetus and Newborn (HDFN): Testing the newborn’s cord blood or peripheral blood cells. A positive DAT suggests maternal IgG antibodies have crossed the placenta and coated the baby’s RBCs (if the baby has the corresponding antigen)
• Autoimmune Hemolytic Anemia (AIHA) Investigation: Patients may present with unexplained anemia, jaundice, or signs of hemolysis. A positive DAT is a hallmark finding in AIHA, indicating the patient is making antibodies against their own RBCs (Warm AIHA, Cold Agglutinin Disease, Mixed-Type AIHA)
• Drug-Induced Hemolytic Anemia (DIHA): Certain medications can cause antibodies to form that lead to RBC sensitization and destruction. A DAT is part of the investigation
• Unexplained Anemia/Hemolysis: Sometimes ordered to investigate anemia when the cause isn’t clear

How the DAT is Performed (The Basic Principle)

It’s actually quite straightforward conceptually:

1. Sample We need the patient’s Red Blood Cells. These are typically collected in an EDTA (lavender top) tube. EDTA chelates calcium, preventing in vitro (in the tube) activation and binding of complement, which could otherwise cause false-positive results for complement
2. WASH, WASH, WASH! This is arguably the MOST CRITICAL STEP. The patient’s RBCs must be washed thoroughly (usually 3-4 times) with saline
   • Why?: To remove all traces of unbound plasma proteins. If unbound immunoglobulins (IgG) are left behind in the plasma surrounding the cells, they will neutralize the AHG reagent added next, leading to a FALSE-NEGATIVE result. We only want the AHG to react with antibodies/complement stuck to the cells
3. Add Anti-Human Globulin (AHG) Reagent After washing, AHG reagent (Coombs’ serum) is added to the patient’s RBC button
   • Polyspecific AHG: Often used first. Contains both anti-IgG and anti-C3d (and sometimes other anti-complement components). A positive result tells you something is coating the cells, but not specifically what
   • Monospecific AHG: If the polyspecific DAT is positive, we follow up with monospecific reagents:
     • Anti-IgG: Contains only antibodies against human IgG
     • Anti-C3d: Contains only antibodies against the C3d component of complement
4. Centrifuge and Read The tube (or gel card/microplate) is centrifuged gently to facilitate agglutination
   • Mechanism: If the patient’s RBCs were coated in vivo with IgG or C3d, the corresponding antibodies in the AHG reagent will bind to these molecules on adjacent RBCs, forming bridges and causing visible agglutination
5. Interpretation
   • Agglutination = Positive DAT: Indicates in vivo sensitization. The pattern with monospecific reagents helps determine what is coating the cells (IgG, C3d, or both)
   • No Agglutination = Negative DAT: Indicates no detectable coating with IgG or C3d. (Note: Check cells are generally not added to a DAT, as a positive reaction is the endpoint we’re looking for. Controls usually involve ensuring the cells don’t spontaneously agglutinate in saline)

Interpreting DAT Results and Clinical Significance

The DAT result provides a piece of the puzzle, but MUST be interpreted in the context of the patient’s clinical picture, history, and other lab findings

• Positive Polyspecific DAT: Requires reflex testing with monospecific Anti-IgG and Anti-C3d
• Monospecific Results
  • Positive IgG, Negative C3d: Common in Warm AIHA, HDFN, some DIHA, delayed HTRs
  • Negative IgG, Positive C3d: Classic for Cold Agglutinin Disease (CAD), Paroxysmal Cold Hemoglobinuria (PCH), some complement-mediated HTRs, some DIHA
  • Positive IgG, Positive C3d: Seen in Warm AIHA (especially severe cases or those associated with conditions like lupus), some DIHA, some HTRs
• Strength of Reaction: A 4+ reaction is generally more concerning for active hemolysis than a weak positive (w+) reaction, but any confirmed positive DAT in the right clinical setting is significant
• A Positive DAT Doesn’t Always Mean Hemolysis: Some individuals, particularly the elderly or hospitalized patients, may have a positive DAT without clinical evidence of increased red cell destruction. The reason for the positive DAT still needs consideration
• Impact on Other Testing: A positive DAT (especially with IgG) can interfere with subsequent Rh typing (particularly Weak D testing) because the cells are already coated with antibody, potentially causing false positives. Special techniques might be needed if accurate typing is required

Key Takeaways

• The DAT detects IgG and/or C3d coating RBCs in vivo (inside the patient)
• It’s crucial for investigating HTRs, HDFN, AIHA, and DIHA
• Washing: the patient’s RBCs thoroughly is essential to prevent false negatives
• Testing usually starts with Polyspecific AHG, followed by Monospecific Anti-IgG and Anti-C3d if positive
• Results must be correlated with the clinical context. A positive DAT indicates sensitization, but not necessarily active hemolysis on its own

Key Terms

• Direct Antiglobulin Test (DAT) (Direct Coombs Test): A laboratory test used to detect immunoglobulin (IgG) and/or complement components (C3d) that have bound to red blood cells in vivo (within the patient’s bloodstream)
• In Vivo Sensitization: The coating of red blood cells with antibodies (primarily IgG) or complement proteins while circulating within the body
• Anti-Human Globulin (AHG) Reagent (Coombs’ Serum): Reagent containing antibodies directed against human IgG and/or complement components (like C3d). Used in both DAT and IAT to detect sensitization by bridging antibody/complement-coated cells, causing agglutination
• Polyspecific AHG: AHG reagent containing both anti-IgG and anti-C3d antibodies (and potentially other anti-complement antibodies). Used for initial DAT screening
• Monospecific AHG: AHG reagent containing antibodies against only one type of molecule, typically either anti-IgG or anti-C3d. Used to determine the specific nature of the coating when the polyspecific DAT is positive
• Washing (in DAT/IAT): The critical process of repeatedly suspending red blood cells in saline and centrifuging to remove unbound plasma/serum proteins, preventing neutralization of the AHG reagent and ensuring test accuracy
• Agglutination: The visible clumping of red blood cells, which is the endpoint reaction indicating a positive result in the DAT (and many other blood bank tests)
• Hemolytic Transfusion Reaction (HTR): An adverse reaction to transfusion where transfused red blood cells are destroyed, often due to an antibody-antigen reaction. A DAT is a key test in the workup
• Hemolytic Disease of the Fetus and Newborn (HDFN): A condition where maternal IgG antibodies cross the placenta and destroy fetal/newborn red blood cells possessing the corresponding antigen. A positive DAT on the infant’s cells is characteristic
• Autoimmune Hemolytic Anemia (AIHA): A group of disorders where the immune system mistakenly produces autoantibodies that attack the individual’s own red blood cells, often resulting in a positive DAT and hemolysis
• Drug-Induced Hemolytic Anemia (DIHA): Hemolysis resulting from an immune response triggered by a medication, often involving red blood cell sensitization detectable by a DAT
• EDTA (Ethylenediaminetetraacetic acid): An anticoagulant commonly used for collecting blood samples for DAT testing because it chelates calcium, preventing the in vitro activation of the complement cascade which could interfere with the detection of in vivo complement binding