Agglutination Test

Agglutination Test, Agglutination Assay, Agglutination, Flocculation Test, Flocculation, Hemagglutination, Agglutinin, Latex Fixation Test, Latex Agglutination, Passive Agglutination, Indirect Agglutination, Direct Agglutination, Active Agglutination, Agglutinogen

Definitions

Agglutinin
1. Substance that induces particles (e.g. Bacteria, viruses, cells) to stick together, forming a clump or mass
2. Agglutinin examples include antibodies, lectins, and viral Proteins
Agglutinogen
1. Antigens to which Agglutinins (e.g. antibodies) bind, resulting in Agglutination
Agglutination
1. Clumping together of material suspended in solution in response to exposure to specific agents
2. Allows for Antigen-Antibody complexes to become visible in solution
Flocculation
1. Similar to Agglutination, but the aggregated complexes are smaller, finer and of a cloudy appearance
Hemagglutination
1. Red Blood Cell aggregation by Agglutinins
Direct Agglutination (Active Agglutination)
1. Antigen-Antibody complexes readily agglutinate without aid of other agents
2. Original Agglutination technique used by Lancefield et. al. in the 1920s to identify Beta-hemolytic Streptococcus strains
Indirect Agglutination (Passive Agglutination, Latex Agglutination)
1. Agglutination of particles is made evident by attaching Antigen to inert substances (typically latex beads)
Latex Agglutination (Latex Fixation)
1. Passive Agglutination test in which specific Antigen is attached to latex particles
2. Particles clump (or aggregate) when exposed to Antibody specific for the embedded Antigen

Types
Hemagglutination Examples

ABO Typing
1. Combine patient's Red Blood Cells with known anti-A serum and observe for Agglutination
2. Combine patient's Red Blood Cells with known anti-B serum and observe for Agglutination
Direct Coombs Test
1. Detects presence of antibodies that have already bound to Red Blood Cells (e.g. Hemolytic Anemia)
2. Antiglobulin (Antibody to Antibody) is added to a patient's blood sample
3. Agglutination occurs if the patient's Red Blood Cells are coated with Antibody against the Red Blood Cells
Indirect Coombs Test
1. Detects presence of antibodies that may bind to specific Red Blood Cells when exposed (e.g. pre-transfusion, Rh factor)
2. Donor Red Blood Cells are added to patient's serum
3. Antiglobulin (Antibody to Antibody) is added to the combined sample
4. Agglutination occurs if the patient has Antibody to the donor's Red Blood Cells
Cold Agglutinins
1. Autoantibodies to Red Blood Cells that are activated by cold
2. Cold Agglutinins are present in low levels in normal patients
  1. Cold Agglutinins may increase with Mycoplasma pneumoniae, Lymphoma and other conditions
  2. Cold Agglutinins may also be increased in a rare Autoimmune Hemolytic Anemia
Heterophil Antibody (Mononucleosis)
1. Antibodies react to multiple Antigens
2. Heterophil Antibody agglutinates sheep RBCs, and in the case of Monospot Test, horse RBCs

Types
Direct Agglutination Examples

VDRL (Syphilis Screening, requires confirmation)
1. Based on finding that Treponema organisms release cardiolipin Antigen
2. Colorless Alcohol based solution of beef cardiolipin, Cholesterol, and lecithin is added to patient's serum
3. Fluid is examined on glass slide for microflocculation
4. Microflocculation suggests possible Syphilis, but has high False Positive Rate and requires confirmation
RPR (Syphilis Screening, requires confirmation)
1. Modified VDRL test using charcoal bound cardiolipin, easier to detect without microscope
2. RPR test is performed on a detection card and can be read with the naked eye
Widal Test (Typhoid Fever, requires confirmation)
1. Salmonella typhi Antigen is exposed to patient's serum
2. Agglutination suggests possible Typhoid Fever, but has a high False Positive Rate and requires confirmation

Types
Latex Agglutination Examples

Rheumatoid Factor
1. Rheumatoid Factor is an Antibody to the Fc portion of IgG found in Rheumatoid Arthritis
2. Latex Agglutination test in which IgG-coated latex beads are exposed to a patients serum
3. Rheumatoid Factor IgM in a patient's serum will agglutinate the suspended beads
Human Chorionic Gonadotropin (Urine bHCG or qualitative Pregnancy Test)
1. Patient urine is mixed with anti-HCG antibodies
2. HCG-coated latex particles added to the patient's urine
  1. Agglutination occurs if the patient is NOT pregnant (xHCG binds HCG-coated latex)
  2. Agglutination does NOT occur if patient is pregnant (xHCG binds HCG in urine)

Resources

Agglutination Assay (Lumen Microbiology)
1. https://courses.lumenlearning.com/microbiology/chapter/agglutination-assays/

References

Goldberg (2014) Clinical Physiology, Medmaster, Miami, p. 83-5