Antiglobulin/Coombs Test

 

 Introduction

·         IgG/IgM antibodies occasionally coat red blood cells (RBCs) without causing agglutination due to their monomeric structure.

·         The small size of antibody molecules prevents cross-linked bridges formation.

·         Coombs, Mourant, and Race developed a test in 1945 to detect these non-agglutinating, coating antibodies.

 

 Principle

·         RBCs coated with incomplete antibody (IgG) or C3 complement do not cause agglutination.

·         Coombs reagent contains antibodies against human IgG/IgM/complement.

·         Anti-Human Globulin (AHG) reacts with human globulin molecules, either bound to RBCs or free in serum.

 

 FDA Licensed Antihuman Globulin Reagents

- Polyspecific

  1. Rabbit polyclonal: Contains anti-IgG and anti-C3d.

  2. Rabbit/murine monoclonal blend.

- Monospecific

  1. Anti-IgG (Rabbit polyclonal): Contains anti-IgG with no anticomplement activity.

  2. Anti-IgG (Gamma-done AHG): Murine monoclonal IgM antibody.

  3. Anticomplement Anti-C3d: Contains a murine monoclonal antibody to C3d.

 

 Coombs/Antiglobulin Test

- Direct (Coombs) Antiglobulin Test (DAT)

·         Detects in vivo sensitization of RBCs with IgG or complement components.

- Indirect (Coombs) Antiglobulin Test (IAT)

·         Determines in vitro sensitization of RBCs.

·         Used in compatibility testing and antibody screening.

 

 In Vivo Phenomena Associated With Positive DAT

·         Conditions like Hemolytic disease of the fetus and newborn (HDFN), Hemolytic transfusion reaction (HTR), Autoimmune and drug-induced hemolytic anemia (AIHA) can result in RBCs coated with antibodies or complement.

 

 In Vitro Application

·         The IAT is used for antibody detection, compatibility testing, antibody screening, antibody identification, antibody titration, and RBC phenotype determination.

 

 Factors Affecting the Antiglobulin Test

·         Various factors such as serum-to-cell ratio, temperature, reaction medium, incubation time, washing of RBCs, and centrifugation affect the test results.

 

 Sources of Error in AHG Testing

·         Errors leading to false-positive or false-negative results include specimen issues, procedural errors, and environmental factors.

 

 Comparison of AHG Methodologies

·         Different testing methodologies have advantages and disadvantages based on factors like sensitivity, cost, and procedural complexity.

 

References:

1.      Nayak, R., & Rai, S. (Eds.). (Undated). Essentials in Hematology and Clinical Pathology.

2.      Harmening, D. (Undated). Modern Blood Banking & Transfusion Practices, Seventh Edition.