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Blood Bank

Antiglobulin Sera

Antiglobulin Sera, more commonly known as Anti-Human Globulin (AHG) or sometimes historically called Coombs’ Serum, is one of the most crucial reagents in the blood bank. Honestly, without AHG, we’d miss a huge number of clinically significant antibodies! It’s the key that unlocks the detection of many IgG antibodies and complement proteins bound to red blood cells

Think of it like this: some antibodies (especially IgG) are like tiny little hands that can grab onto antigens on red blood cells, but they’re often too small or don’t bind in a way that makes the cells clump together (agglutinate) directly. They coat the cells invisibly! AHG is like a bigger molecule that comes along and says, “Hey, I see you little IgG hand stuck there!” and grabs onto it, and also grabs onto another one on a nearby cell, effectively linking the cells together so we can see the clumping

The Fundamental Purpose: Detecting Invisible Sensitization

The main job of AHG is to detect red blood cells that have been sensitized (coated) with:

  • IgG antibodies: Many clinically significant antibodies that cause transfusion reactions or HDFN are IgG. They bind at body temperature (37°C) but often don’t cause direct agglutination
  • Complement components: Sometimes, antibodies (IgG or IgM) bind and then activate the complement cascade. Fragments of complement proteins (like C3b, C3d) can remain stuck to the red cell surface

AHG allows us to visualize this otherwise invisible coating by causing agglutination

How is AHG Made? (The Basics)

Historically, AHG was made by injecting human serum proteins (like IgG or complement) into animals (like rabbits). The animals would produce antibodies against these human proteins. These animal antibodies were then harvested and purified to create the reagent. This produces polyclonal AHG (a mix of different antibodies recognizing different parts of the target human protein)

Modern methods also include monoclonal antibody technology using hybridomas. This creates highly specific antibodies that recognize a single epitope (part) of the target human protein (IgG or C3d). Many current reagents are blends of monoclonal antibodies, or sometimes a mix of monoclonal and polyclonal

Types of AHG Reagents: Know Your Tools!

This is super important because using the right type tells you different things:

  • Polyspecific AHG
    • Contains: BOTH Anti-IgG AND Anti-C3d (and often other anti-complement components like anti-C3b)
    • Use: Often used as the initial reagent in the Direct Antiglobulin Test (DAT). A positive result tells you something (IgG or complement or both) is coating the patient’s red cells in vivo. Also sometimes used in routine antibody screening (IAT), though many labs now prefer Anti-IgG for screening
    • Think of it as: The broad searchlight. It tells you if anything is there
  • Monospecific AHG
    • Contains: Antibodies against ONLY ONE specific component
    • Types & Use
      • Anti-IgG: Contains only antibodies against the Fc portion of human IgG
        • Crucial for: The Indirect Antiglobulin Test (IAT) – used in antibody screening, antibody identification, Weak D testing, antigen typing, and the AHG crossmatch to detect IgG antibodies bound in vitro
        • Also used in: The DAT after a positive polyspecific result, to determine specifically if IgG is coating the cells
      • Anti-C3d: Contains only antibodies against the C3d fragment of complement (C3d is stable and remains on the cell surface after complement activation)
        • Use: Primarily in the DAT after a positive polyspecific result, to determine specifically if complement is coating the cells (common in Cold Agglutinin Disease, some HTRs, some drug-induced reactions). Rarely used in routine IAT
      • (Less common: Anti-C3b, Anti-C4, etc. - used in specific reference lab investigations)
    • Think of it as: The specific spotlights. They tell you exactly what kind of coating you found (IgG? Complement?)

Applications: Where We Rely on AHG

AHG is the cornerstone reagent for:

  • Indirect Antiglobulin Test (IAT): Detecting in vitro sensitization (antibody in plasma binding to reagent cells in the test tube/system)
    • Antibody Screening
    • Antibody Identification
    • Weak D Testing
    • Antigen Typing (for some antigens requiring IAT)
    • AHG Crossmatch
  • Direct Antiglobulin Test (DAT): Detecting in vivo sensitization (cells coated with antibody/complement inside the patient’s body)
    • Transfusion Reaction Workups
    • HDFN Investigations
    • Autoimmune Hemolytic Anemia (AIHA) Workups
    • Drug-Induced Hemolytic Anemia (DIHA) Workups

Mechanism of Action: The Bridge

Whether it’s Anti-IgG binding to the Fc portion of IgG molecules coating two adjacent red cells, or Anti-C3d binding to C3d molecules coating adjacent cells, the AHG molecule acts as a bridge. This bridging overcomes the natural repulsive forces between red cells, allowing the formation of a stable lattice structure that we see as agglutination

Quality Control & Critical Considerations

  • Washing is KING!: For both IAT and DAT, inadequate washing of red cells before adding AHG is the #1 cause of false-negative results. Why? Any unbound human globulins (IgG) left in the surrounding fluid will neutralize the AHG reagent before it can bind to the globulins on the cells
  • Check Cells (Coombs Control Cells): These are essential! They are IgG-coated red cells that are added to all negative AHG tests (IAT and sometimes DAT depending on policy/method). They MUST agglutinate. If they don’t, it means something was wrong (AHG wasn’t added, was inactive, or washing was poor), and the negative test result is INVALID
  • Proper Reaction Reading: Need correct technique (gentle resuspension) and lighting
  • Reagent Storage & Handling: Follow manufacturer instructions precisely

Mastering the use and interpretation of AHG reagents is fundamental to safe and effective blood banking. It allows us to detect those hidden dangers – the antibodies and complement proteins that can lead to red cell destruction! Keep practicing those DATs and IATs!

Key Terms

  • Antiglobulin Sera (Anti-Human Globulin / AHG): Reagents containing antibodies directed against human immunoglobulins (primarily IgG) and/or complement components (primarily C3d). Used to detect red blood cells sensitized with these proteins
  • Coombs’ Test: The historical name for antiglobulin tests (Direct and Indirect), named after the inventors (Coombs, Mourant, and Race)
  • Sensitization: The coating of red blood cells with antibodies or complement components, either in vivo (inside the body) or in vitro (in the test tube)
  • Polyspecific AHG: AHG reagent containing both anti-IgG and anti-C3d activity
  • Monospecific AHG: AHG reagent containing activity against only one specific component, typically either Anti-IgG or Anti-C3d
  • Anti-IgG: Monospecific AHG reagent that detects IgG antibodies bound to red blood cells
  • Anti-C3d: Monospecific AHG reagent that detects the C3d component of complement bound to red blood cells
  • Indirect Antiglobulin Test (IAT): Test procedure used to detect in vitro sensitization of red blood cells (used for antibody screening, ID, crossmatching, Weak D). Requires incubation of plasma/serum with cells, washing, and addition of AHG
  • Direct Antiglobulin Test (DAT): Test procedure used to detect in vivo sensitization of red blood cells (used for HTR, HDFN, AIHA investigations). Involves washing patient red cells and directly adding AHG
  • Agglutination: Visible clumping of red blood cells, indicating a positive reaction in antiglobulin tests
  • Neutralization (in AHG testing): Inactivation of the AHG reagent by unbound human globulins remaining after inadequate washing, leading to false-negative results
  • Check Cells (Coombs Control Cells): IgG-coated red blood cells added to all negative AHG tests to validate the test system’s integrity (confirming active AHG was present and washing was adequate)