The identification of HLA class I alloantibodies is important for organ transplantation and platelet transfusion in alloimmunized patients. Because microcytotoxicity testing against frozen trays of lymphocyte panels is rapid and efficient for determining specificities of unknown antibodies, a simple method was devised to increase test sensitivity to weak antibodies. Standard anti-human globulin (AHG)-facilitated microcytotoxicity was modified by the insertion of a double addition-of-serum (DAS) step, and reagent and patient's sera were evaluated by both methods. DAS modification increased antibody titers and, more significantly, made the identification of weak specificities easier because of the twofold to threefold increase in reactivity rates (29-42% for AHG vs. 75-82% for DAS) of panel cells that were expected to be positive, while low (approx. 1%) "extra" reaction rates were maintained for cells that were expected to be negative. DAS was relatively unaffected by variations in serum volumes or target cell preparation, and its use did not significantly increase test time or costs. In a program of platelet donor selection driven by donor antibody rather than donor-recipient antigen matching, DAS greatly facilitated platelet transfusion support for alloimmunized patients.