Brain-dead humans as preclinical reference models for xenotransfusion: bridging nonhuman primates and clinical applications through in vitro evaluation

Background Xenotransfusion using genetically engineered (GE) pig red blood cells (RBCs) offers a promising solution to blood shortages, particularly in emergency settings. Although nonhuman primates (NHPs) have been widely used in preclinical studies, their translational relevance is limited by species-specific immune responses and logistical challenges. This study aimed to evaluate whether brain-dead humans could serve as a human translational reference model by characterizing and comparing their hematologic, biochemical, and immunologic profiles with those of patients with acute blood loss (ABL). The goal was to generate baseline data to inform the design and interpretation of future in vivo studies involving the transfusion of GE pig RBCs. Materials and methods Comprehensive clinical and immunological analyses were performed on donation after brain death (DBD) subjects (n=179) and patients with ABL requiring transfusion (n=104). The parameters included hematological indices, electrolytes, coagulation factors, inflammatory biomarkers, and arterial blood gases. Immune assays were conducted on sera from DBD subjects (n=31) and patients with ABL (n=102) to examine IgM/IgG binding and complement-dependent cytotoxicity (CDC) against triple-knockout (TKO) pig RBCs lacking Gal, Neu5Gc, and Sda antigens. Results Across most measured parameters, overlapping ranges in hematologic and biochemical indices were observed between DBD subjects and patients with ABL. Anti-TKO IgM/IgG binding and CDC were not detectably different between the two groups under the conditions tested. However, differences were observed in several other immune parameters, including hemagglutination, cytokine profiles, total immunoglobulin levels, and complement components. Conclusion Brain-dead humans may represent an ethically feasible human translational reference model for xenotransfusion research. While DBD subjects do not fully reproduce the physiologic and inflammatory milieu of patients with ABL, they provide useful baseline human data for assessing selected early xenoreactive responses to GE pig RBCs and may help bridge the translational gap between NHP studies and future clinical application.