Agglutination Reactions

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The page below is a sample from the LabCE course Introduction to the ABO Blood Group System. Access the complete course and earn ASCLS P.A.C.E.-approved continuing education credits by subscribing online.

Learn more about Introduction to the ABO Blood Group System (online CE course)
Agglutination Reactions

Antibodies of the ABO system cause agglutination of saline-suspended red cells at 4ºC to 20ºC. Heating to 37ºC weakens the reaction. "Naturally" occurring ABO antibodies may not be strong enough to agglutinate cells without centrifugation. Thus, testing serum for the presence of anti-A or anti-B has classically been performed using the tube system in which serum and cells added to a test tube are centrifuged and then evaluated for agglutination. A slide test has also been performed for forward reactions. Although tube tests are still widely used especially in educational settings, newer systems utilizing other technology such as gel agglutination are becoming more prevalent.
The image on this page illustrates agglutination reactions observed with the tube system, from 4+ in the topmost image to 0 in the lowest image. ABO reactions should be strong. Weak or missing reactions occur but must be resolved before blood products can be released.
  • 4+ agglutination: Red blood cell button is a solid agglutinate; clear background
  • 3+ agglutination: Red blood cell button breaks into several large agglutinates; clear background
  • 2+ agglutination: Red blood cell button breaks into many medium-sized agglutinates; clear background; no free red blood cells
  • 1+ agglutination: Red blood cell button breaks into many small clumps barely visible macroscopically; background is turbid; many free red blood cells
  • 0: No agglutinated red blood cells present; red cells are observed flowing off the red blood cell button during the process of grading
Other reactions that may occur are mixed field reactions, in which mixtures of agglutinated and unagglutinated red blood cells are present, and hemolysis, in which red cells are hemolyzed by the antibody. Both of these patterns are considered positive reactions.

4+ to 0 agglutination reaction