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Incubate at Room Temperature or In the Refrigerator

Naturally occurring ABO antibodies are IgM by nature.
IgM antibodies react better at colder temperatures.
When encountering Group I ABO typing discrepancies, it can be effective to incubate patient plasma and reagent red cells at room temperature or 4°C to enhance the potential of reactivity.
Room Temperature Incubation
  1. Incubate patient plasma and reagent red cells at room temperature for 15 to 30 minutes.
  2. Centrifuge sample and examine for macroscopic agglutination.
  3. If unresolved, proceed to incubation at 4°C.
4°C Incubation
  1. Incubate patient plasma and reagent red cells at 4°C for 15 minutes.
  2. To ensure that no underlying cold reacting antibody interferes with the reaction, an auto control and O cell control should be included.
    1. Auto control is used to screen for a cold reacting autoantibody
    2. O cell control is used to screen for a cold reacting alloantibody.
  3. After a 15 minutes incubation, centrifuge and read for macroscopic agglutination.