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Modification of red cell antigenic characteristics via covalent modification with branched poly (ethylene glycol)

Background: Red Blood Cell (RBC) transfusions are associated with the risk of rejection and sensitization for the recipient. Covalent modification of human RBC using poly (ethylene glycol)) (PEG) is currently being investigated as a method to modify the antigenic characteristics of RBC in order to develop an antigenically silent RBC transfusion product. The focus of this study is to determine whether a branched PEG, 4PEGss [MW 10,000] can concurrently camouflage and stabilize the RBC membrane, giving the red cell higher osmotic resistance. Study Design and Methods: Human blood was collected from consenting donors into EDTA tubes. R B C were separated, counted, washed and incubated with 4PEGss stock solution for up to 120 minutes at room temperature (RT). Agglutination assays and flow cytometry were used to determine polymer-dependent antigen masking while scanning electron microscopy (SEM), erythrocyte sedimentation rate (ESR) assays, osmotic fragility assays, hemolysis measurements and generation of hemoglobin oxidation species were used to measure the impact of 4PEGss treatment on RBC function. ' Results: Agglutination assays and flow cytometry showed that 4PEGss treatment significantly camouflaged RhD and A and B antigens in a dose-dependent manner. However, S E M and ESR assays indicated 4PEGss treatment may cause cell aggregate formation. Osmotic fragility assays and hemolysis measurements showed a 4PEGss-mediated increase of hemolysis. Conclusions: At all concentrations investigated, 4PEGss modification yielded antigenically camouflaged (both A B O and RhD) yet less stable RBC compared to non-treated controls.