Background: Non‐ABO alloantibodies are frequently implicated in hemolytic transfusion reactions and are a leading cause of transfusion‐related mortality. Detection of clinically significant non‐ABO alloantibodies is reliant on an antibody screen, which is prone to clerical, technical, and reagent error. Data on the frequency of false‐negative antibody screens due to the occurrence of these errors are scarce, and the true incidence of false‐negative antibody screens in everyday practice is unknown.STUDY DESIGN AND METHODS: Monitoring for false‐negative antibody screens is routinely performed in our institution. All cases of false‐negative antibody screens involving clinically significant antibodies were identified through review of the blood bank quality assurance records from 2004 to 2007. The clinical impact was recorded in each case.RESULTS: Twenty‐one cases of false‐negative antibody screens due to clinically significant antibodies were detected. Sources of error included testing error (12 cases), reagent red blood cell (RBC) failure (one case), and reagent limitations (one case). The cause of error was inconclusive in seven cases. Nine patients were found to have received antigen‐incompatible blood as a consequence of these errors, resulting in a single nonfatal hemolytic transfusion reaction.CONCLUSIONS: The identification and investigation of false‐negative antibody screens is a valuable quality assurance measure which can serve to monitor staff performance, identify cases of reagent RBC failure, and identify patients who have received antigen‐incompatible blood at risk for hemolytic transfusion reactions.