Antihuman Information and Courses from MediaLab, Inc.

The auto control and all panel cells are showing reactivity at the AHG phase which indicates a possible autoantibody (warm).IS = Immediate Spin; AHG = Antihuman Globulin Phase; CC = Check Cells; AC = Auto Control; ND= Not done

Reaction PhaseLook at the phase in which reactions are occuring and see if the results match the reaction patterns on your panel. Reactions occurring only at immediate spin phase could indicate a possible IgM antibody, cold agglutinin, or rouleaux. Reactions occurring in the AHG phase could indicate a possible IgG antibody. Reactions occurring in both reaction phases could indicate a combination of both IgM and IgG antibodies or a strong IgM antibody that carries through to AHG phase. If a tube method is used, reactions are usually read at immediate spin and AHG phase. If a gel method is used, reaction readings are done only at AHG phase. PatternComparing the pattern of reactivity of the sample reactions with that of the panel cells will help to determine which antibody or antibodies are present. If the reactions match, or closely match, a specific panel cell, that could be the antibody.The strength of the reaction does not correlate with the clinical significance of the antibody present. It does correlate to the amount of antibody that is available to react. If reactions are strong at immediate spin (IS) phase and get weaker at antihuman globulin (AHG) phase, it is possible that a strong IgM antibody is present that is carrying through to the AHG phase (e.g., 4+ at IS and 1+ at AHG).The presence of multiple antibodies should be considered if reactions vary in strength or there are two separate reaction patterns in the IS and AHG phases.

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 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°: 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.

A 49-year-old male with pneumonia was treated with penicillin. He became jaundiced with yellow sclera. Observe the photograph of his peripheral blood smear. Anisocytosis was observed with pale-centered microcytes and polychromatophilic macrocytes. Since penicillin is a classic offender for autoimmune hemolytic disease, the clinician asked for an antihuman globulin (AHG) test, also known as the Coombs test. A positive AHG reaction occurs when the antibody stimulated by penicillin becomes attached to red blood cells. Hemolysis follows, leaving the patient with jaundice and a peripheral blood smear, as demonstrated in the photograph.