Mechanism of the Indirect Coombs Test
The indirect Coombs (antiglobulin) test detects circulating antibodies in serum that can bind to red blood cells by incubating patient serum with donor red blood cells, then using anti-human immunoglobulin reagent to detect antibody binding. 1
Technical Methodology
The indirect antiglobulin test operates through a two-stage process:
Stage 1 (Sensitization): Patient serum containing potential antibodies is incubated with reagent red blood cells of known antigen phenotype, allowing any circulating antibodies to bind to corresponding antigens on the RBC surface 1, 2
Stage 2 (Detection): After washing away unbound antibodies, anti-human immunoglobulin (Coombs reagent) is added, which binds to any IgG antibodies or complement components attached to the red blood cells, causing visible agglutination 1
What the Test Detects
The indirect test specifically identifies:
Free-floating antibodies in serum rather than antibodies already bound to the patient's own red blood cells (which would be detected by the direct Coombs test) 2, 3
IgG antibodies are the primary target, though the test can also detect IgA, IgM, and complement components depending on the reagent used 1, 4
Alloantibodies from prior transfusions or pregnancy, as well as autoantibodies that may cause hemolysis under certain conditions 5, 3
Clinical Applications
The indirect antiglobulin test serves three main purposes:
Prenatal screening: Performed on maternal serum to detect antibodies that could cause hemolytic disease of the newborn, with serial monitoring of antibody titers recommended when positive 5, 3
Pre-transfusion compatibility testing: Used as part of the crossmatch procedure to ensure donor blood lacks antigens corresponding to recipient antibodies 6
Antibody identification: When positive, further testing determines the specific antibody present (anti-D, anti-Kell, anti-E, etc.) to guide clinical management 3, 4
Critical Distinction from Direct Coombs Test
The key mechanistic difference is the antibody location:
Indirect test: Detects unbound antibodies circulating in serum/plasma that have the potential to bind RBCs 2, 3
Direct test: Detects antibodies or complement already coating the patient's own red blood cells in vivo 2, 4
Important Clinical Caveat
A positive indirect Coombs test does not automatically indicate active hemolysis or require treatment—it simply confirms the presence of circulating antibodies that may persist for months to years after sensitization 5, 7. Clinical correlation with hemolysis markers (LDH, haptoglobin, indirect bilirubin, reticulocyte count) is essential before initiating therapy 7, 8.