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Unlabelled: The aim of this study was to determine the frequencies and specificities of "enzyme-only" detected red blood cell (RBC) alloantibodies in the routine antibody screening and antibody identification in patients hospitalized in Austria. Routine blood samples of 2420 patients were investigated. The antibody screening was performed with a 3-cell panel in the low-ionic strength saline- (LISS-) indirect antiglobulin test (IAT) and with an enzyme-pretreated (papain) 3-cell panel fully automated on the ORTHO AutoVue Innova System. The antibody identification was carried out manually with an 11-cell panel in the LISS-IAT and with an enzyme-pretreated (papain) 11-cell panel. In total 4.05% (n = 98) of all patients (n = 2420) had a positive RBC antibody screening result. Of them 25.51% (25/98) showed "enzyme-only" detected specific or nonspecific RBC alloantibodies. Rhesus and Lewis system antibodies were found the only specificities of "enzyme-only" RBC alloantibodies: all in all 4.8% (4/98) were detected with anti-E, 3.06% (3/98) with anti-Le(a), 3.06% (3/98) with anti-D after anti-D prophylaxis and 1.02% (1/98) with anti-e. In total, 14.29% (14/98) showed a nonspecific RBC alloantibody result with the enzyme test. The results of the present study demonstrate that a high number of unwanted positive reactions with the enzyme technique overshadows the detection of "enzyme-only" RBC alloantibodies. (, Trial Registration: K-37-13).