of antibodies on erythrocytes and is usually positive in AIHA A positive DAT prompts additional testing by the blood bank to further characterize the antibody Acute hemolysis is most commonly associated with a warm (37°C)-reactive IgG antibody with or without complement (C3) This type of hemolysis is usually extravascular, occurring in the spleen IgM-mediated cold agglutinin disease is less common in children than in adults, but can occur following mycoplasma (anti-I) or infectious mononucleosis (antii) These antibodies bind to RBCs in the cold and characteristically cause intravascular hemolysis as complement is bound and activated at warmer temperatures Cold-reactive IgG antibodies (Donath–Landsteiner test) cause paroxysmal cold hemoglobinuria (PCH), which in children frequently follows a viral infection A negative DAT does not definitively exclude an immune-mediated process Rarely, IgA or warm–reactive-IgM antibodies may be present but not detected by the Coombs reagent Additionally, in rare cases, the causative warm-IgG antibodies are below the level of detection When there is a high degree of suspicion for an immune-mediated process despite a negative DAT, specialized assays are required for antibody detection