Agglutination assays are powerful tools for detecting specific antigens or antibodies in samples. They rely on the clumping of cells or particles when antibodies bind to matching antigens. These tests are widely used in blood typing, disease diagnosis, and bacterial identification.

Blood typing through agglutination is crucial for safe transfusions. It determines ABO and Rh blood types by mixing red blood cells with specific antibodies. Cross-matching ensures compatibility between donor and recipient blood, preventing potentially life-threatening transfusion reactions.

Agglutination Assays

Direct vs indirect agglutination

• Direct agglutination involves antibodies binding directly to antigens on the surface of cells or particles (bacteria, red blood cells) used to detect the presence of specific antigens in a sample
  • Blood typing determines ABO and Rh blood groups by mixing red blood cells with antibodies specific to A, B, and D antigens
  • Bacterial identification groups Streptococcus species based on specific cell wall antigens
• Indirect agglutination involves antibodies binding to soluble antigens, which then cross-link particles or cells (latex beads, red blood cells) used to detect the presence of specific antibodies in a sample
  • Widal test diagnoses typhoid fever by detecting antibodies against Salmonella typhi antigens
  • Rheumatoid factor test detects autoantibodies in rheumatoid arthritis that agglutinate IgG-coated particles
• Antibody titer can be determined by serial dilutions of the sample to find the highest dilution that still produces visible agglutination

Applications of hemagglutination

• Hemagglutination involves the agglutination of red blood cells (RBCs) in the presence of specific antibodies or antigens
• Viral hemagglutination occurs when viruses (influenza) have surface proteins that bind to receptors on RBCs causing agglutination
  • Hemagglutination inhibition test detects antibodies against these viruses in a patient's serum that inhibit agglutination
• Bacterial hemagglutination occurs when bacteria (Mycoplasma pneumoniae) produce adhesins that bind to RBCs causing agglutination
  • Indirect hemagglutination test detects antibodies against these bacteria in a patient's serum that agglutinate bacteria-coated RBCs

Blood Typing and Transfusion

Blood typing through agglutination

• ABO blood typing determines blood type based on the presence or absence of A and B antigens on RBCs and anti-A and anti-B antibodies in the serum
  • Agglutination occurs when the corresponding antigen and antibody are mixed
  • Four blood types: A (A antigen), B (B antigen), AB (both A and B antigens), O (neither antigen)
• Rh blood typing determines Rh status based on the presence or absence of the Rh factor (D antigen) on RBCs
  • Agglutination occurs when Rh-positive RBCs are mixed with anti-D antibodies
  • Rh-positive individuals have the D antigen, Rh-negative individuals lack the D antigen

Cross-matching for blood transfusions

• Cross-matching ensures compatibility between donor and recipient blood to prevent transfusion reactions
• Steps in cross-matching:
  1. ABO and Rh typing of both donor and recipient blood
  2. Screening recipient serum for the presence of unexpected antibodies (antibody screen)
  3. Mixing recipient serum with donor RBCs to check for agglutination (cross-match)
     • Agglutination indicates incompatible donor blood that cannot be used
     • No agglutination indicates compatible donor blood that can be safely transfused
• Agglutination in cross-matching indicates the presence of antibodies in the recipient's serum that can react with antigens on the donor's RBCs
  • Transfusing incompatible blood can lead to severe transfusion reactions (hemolytic reactions, acute kidney injury)
• Cross-reactivity can occur when antibodies recognize similar epitopes on different antigens, potentially leading to false-positive results

Additional Considerations in Agglutination Assays

• Prozone effect: A phenomenon where excess antibodies in a sample can interfere with agglutination, leading to false-negative results in high-concentration samples
• Cold agglutinins: Antibodies that cause agglutination at low temperatures, which can affect blood typing and transfusion procedures
• Complement fixation: A modification of the agglutination assay where complement proteins are used to enhance the sensitivity of the test, particularly useful in detecting certain bacterial and viral infections