IMMUNOHEMA

• Antihuman Globulin Reagent (AHG)


  Detects IgG or complement fractions that have attached to the RBC
• Coombs, Mourant and Race


  1945
• Goal: Detection of IgG or complement fractions bound to patient's RBCs in vivo


  1. Patient's RBC coated in vivo with IgG or complement fractions

  2. Anti-human globulin (AHG) Agglutination (positive reaction)

  3. Patient's RBC not coated with IgG or complement fractions

  4. No agglutination (negative reaction)
• Polyclonal Antihuman Globulin


  Antibody collected and purified from multiple rabbits
• Monospecific Polyclonal


  Contains only one antibody specificity: either anti-IgG or antibody to specific components of complement, such as C3b or C3d
• Polyspecific AHG


  Contains antibody to human IgG and to the C3d component of complement
• Monoclonal


  Derived from one clone of plasma cells and recognize a single epitope
• Direct AHG


  Washed (3x's) Patient Red Cells (sensitized in Vivo) + AHG Reagent
• FDA Licensed Antihuman Globulin Reagents

  • Rabbit polyclonal
  • Rabbit/murine monoclonal blend
  • Anti-IgG (Rabbit polyclonal)
  • Anti-IgG (Murine monoclonal IgM antibody)
  • Anticomplement Anti-C3d
• Direct Antiglobulin Test (DAT)


  Detects sensitization of RBC with IgG or complement components
• Clinical Application In Vivo Sensitization

  • HDFN
  • HTR
  • AIHA
• DAT Panel: Patterns of Reactivity in Autoimmune Hemolytic Anemia


  • Anti-IgG + Anti-C3d
  • Anti-IgG + Anti-C3d + (WAIHA)
  • Anti-IgG + Anti-C3d (CAS; PCH, WAIHA)
  • Mixed-type AIHA (cold and warm)
• DAT detects a level of 100 to 500 IgG molecules per RBC and 400 to 1,100 molecules of C3d per RBC
• In Vivo Phenomena Associated With a Positive DAT

  • Recipient alloantibody and donor antigen
  • Donor antibody and recipient antigen
  • Type I (hapten-dependent Ab) Drug Induced
  • Type II (autoantibody) Drug Induced
  • Type III (drug-dependent Ab) Drug Induced
  • WAIHA (IgG and/or C3)
  • CAS (C3)
  • PCH (IgG)
  • Maternal alloantibody crosses placenta (IgG)
  • Absorbed proteins; administration of equine preparations of antilymphocyte globulin (ALG) and antithymocyte globulin (ATG)
  • Administration of high-dose IV gamma globulin and hypergammaglobulinemia
• Indirect AHG


  Detects sensitization of RBC with IgG or complement components
• Indirect AHG Procedure


  1. Incubate anti-sera

  2. Wash 3x

  3. Add AHG reagent

  4. Incubate reagent red cells

  5. Wash 3x

  6. Add AHG reagent
• Tasks and Purposes of the Indirect Antiglobulin Test

  • Incubate RBCs with antisera
  • Perform a minimum of three saline washes
  • Add antiglobulin reagent
  • Centrifuge
  • Examine for agglutination
  • Grade agglutination reactions
  • Add antibody-coated RBCs to negative reactions
• Applications of the Indirect Antiglobulin Test

  • Antibody detection
  • Compatibility testing
  • Antibody screening
  • Antibody identification
  • Antibody titration
  • Rh antibody titer
  • RBC phenotype
  • RBC antigen detection(ex: weak D, K, Fy)
• IAT detects a level of 100 and 200 IgG or C3 molecules per red cell
• Factors Affecting AHG Test

  • Cell:serum ratio
  • Reaction medium
  • Temperature
  • Incubation time
  • Centrifugation
  • Washing
• Sources of Error in Antihuman Globulin Testing

  • False-Positive Results
  • False-Negative Results
• Comparison of AHG Methodologies

  • Saline-tube testing
  • LISS-tube testing
  • PEG-tube testing
  • Gel
  • Solid phase