BACKGROUND: The clinical significance of natural and treatment-emergent antibodies specific for amustaline/glutathione pathogen-reduced red blood cells (PRRBCs) is not known. STUDY DESIGN AND METHODS: A Phase 3, randomized clinical trial of PRRBCs (ReCePI) compared PRRBCs with conventional RBCs in cardiac or thoracic-aorta surgery. Subjects transfused during and for 7 days after surgery were screened for PRRBC-specific antibodies at baseline, 28 and 75 days post-surgery. Subjects with treatment-emergent antibodies were assessed for evidence of hemolysis. Cryopreserved subject RBC samples were assayed by flow cytometry for circulating PRRBCs using an acridine-specific (2S197-2M1) monoclonal antibody, and for human IgG-coated RBCs. RBC-surface acridine density was quantitated using a commercial calibrated phycoerythrin (PE)-bead panel. RESULTS: Five of 159 (3.1%) PRRBC and zero of 162 conventional RBC recipients developed treatment-emergent PRRBC-specific IgG, low titer antibodies detected 26-80 days post-surgery after exposure to 1-3 PRRBC units, without clinical evidence of hemolysis. DAT and eluate were weak (w+) positive and PRRBC-specific in one subject. A monocyte monolayer assay (MMA) was non-reactive in the three subjects with an interpretable result. Flow cytometry demonstrated circulating PRRBCs in all five subjects expressing surface acridine concentrations at the limit of detection (approximately 150-301 PE molecules/RBC) compared with freshly transfused PRRBCs (approximately 7500 PE molecules/RBC). In some samples, loss of surface acridine expression could not be distinguished from clearance of the PRRBCs. DISCUSSION: Treatment-emergent PRRBC-specific antibodies with the characteristics of nonclinically significant antibodies were detected in five subjects transfused with PRRBCs. Flow cytometry demonstrated persistent circulating PRRBCs with minimal surface acridine expression. (www. CLINICALTRIALS: gov Identifier NCT03459287).