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Significance of Reactions at Different Phases of Testing

Antibodies have optimum temperatures for reactivity. Reaction readings can be made at different phases: after immediate spin, after incubation at 37° C, and after the addition of antihuman globulin (AHG) and centrifugation.

Reactivity in a certain phase will help to determine whether the antibody is cold reacting (IgM) or warm reacting (IgG). It will also help to distinguish between antibodies that are clinically significant and not significant.

Clinically significant antibodies that are capable of causing acute and delayed hemolytic transfusion reactions (HTR) or hemolytic disease of the newborn (HDN) are usually IgG and react best in the AHG phase.

Readings can be done at all three phases, if a tube method is used. If a gel method or solid phase is used, readings are done only at AHG.

  • Immediate spin: Antibodies reacting in this phase tend to be cold reactive. They are usually IgM class and not clinically significant (with the exception of the A and B antibodies).
  • 37°C: Antibodies that react in this phase include strong IgM or IgG antibodies. After incubation, the tubes are examined for the presence of hemolysis. If complement was bound during incubation then hemolysis could be seen. NOTE: This reaction would only occur in serum samples. If EDTA plasma samples are used for testing, the complement cascade has been halted. Magnesium and calcium ions are not available for complement to be activated.
  • AHG: Antibodies reacting in this phase are considered clinically significant. They are usually warm reactive and IgG.
Antibodies in the Duffy, Kidd, and Kell blood group systems (anti-Fya, anti-Jka, and anti-K) react most commonly at the AHG phase of testing. Antibodies in the Lewis, MNSs and P blood group systems (anti-Lea, anti-M, and anti-P1) are commonly IgM antibodies and react optimally at room temperature and below. Antibodies in the Rh system (anti-E, anti-D, and anti-c) generally react at both 37° C and AHG.