Storage Differentially Impacts Immunization to Red Cell Antigens
Authors: Maier Cheryl L , Jajosky Ryan , Verkerke Hans , Patel Seema R , Allen Jerry William , Fuller Megan , Stowell Sean R 
Published: November 2021

Abstract
Introduction: The impact of red blood cell (RBC) storage on alloimmunization rates after transfusion remains controversial, with clinical experience demonstrating conflicting results. Indeed, some reports suggest increased rates of alloimmunization associated with longer storage of RBC units, while others suggest no association between alloimmunization rates and length of storage. We hypothesized that this discrepancy may reflect the differential impact of storage on alloimmunization related to each unique antigen and the immune response elicited. Here we define the effect of red cell storage on two distinct antigens, including the Hel-Ova-Duffy (HOD) and KEL antigens, using a preclinical murine model of RBC storage and transfusion.
Methods: RBCs singly expressing HOD (HOD RBC) or KEL (KEL RBC) antigens, in addition to those dually expressing both HOD and KEL (HOD x KEL RBC) antigens, were collected from respective transgenic donor mice into CPDA. Units of HOD, KEL or HOD x KEL RBC were generated and used immediately (fresh RBC), or stored for 8 (D8) or 14 (D14) days at 4C before transfusion into wildtype recipients. Recipient cytokine profiles were assessed 2 hours post-transfusion using a multiplex inflammation panel. Donor RBC survival at 24 hours post-transfusion was assessed by flow cytometry. Serum was collected from recipients on days 5 and 14 post-transfusion and analyzed for IgM or IgG development by flow-cytometric crossmatch using HOD or KEL RBC targets.
Results: Post-transfusion RBC survival was decreased with increased storage, but was not significantly different between donor cell types (HOD, KEL or HOD x KEL). Recipient cytokine profiles demonstrated a distinct inflammatory profile associated with storage. Development of IgM antibodies against HOD in mice transfused fresh versus D8 or D14 HOD or HOD x KEL RBCs were not significantly different; however, significantly increased (p&lt;0.05) IgG antibodies were detected in recipients transfused with D8 and D14 HOD or HOD x KEL RBCs as compared to those transfused fresh HOD or HOD x KEL RBCs. In contrast, and while the development of anti-KEL IgM antibodies was not significantly different in mice transfused fresh versus D8 or D14 KEL or HOD x KEL RBCs, the IgG response to KEL was abrogated with longer storage, with recipients generating significantly less (p&lt;0.05) anti-KEL IgG following transfusion with stored KEL or HOD x KEL RBCs as compared to fresh HOD or HOD x KEL RBCs.
Conclusions: Although the impact of RBC storage on various measures of transfusion efficacy have been determined, the impact of storage on alloimmunization remains incompletely understood. Using a murine model of transfusion, we demonstrate that storage differentially impacts immunization to red cell antigens, increasing the response to certain antigens, like HOD, while decreasing the response to others, like KEL. Although it is unclear whether these findings apply to other RBC antigens and what relevance this holds for human patients, our study provides insight into the impact of storage on red cell alloimmunization and may enlighten future storage protocols.

Disclosures
Stowell: Argenx: Speakers Bureau; Grifols: Speakers Bureau; Alexion: Consultancy.