Long-term biochemical stability of fresh-frozen plasma from Asian elephants (Elephas maximus) stored at −20°C: Implications for emergency transfusion protocols for elephant endotheliotropic herpesvirus hemorrhagic disease

Background and Aim: Elephant endotheliotropic herpesvirus hemorrhagic disease (EEHV-HD) is a leading cause of fatal hemorrhagic illness in juvenile Asian elephants (Elephas maximus), often requiring urgent plasma transfusion. However, the biochemical stability of fresh-frozen plasma (FFP) during long-term storage has not been systematically evaluated in this species. This study assessed the stability of key plasma proteins, fibrinogen, clotting factor VIII, immunoglobulin G (IgG), and albumin, in FFP stored at −20°C for 4, 8, and 12 months, and compared them with fresh plasma to determine suitability for emergency clinical use. Materials and Methods: Plasma samples were collected from 20 healthy elephants and processed into fresh and frozen aliquots. Fibrinogen concentrations were quantified using the Clauss assay, factor VIII activity via a one-stage clotting assay, and IgG and albumin concentrations using colorimetric methods. A repeated-measures generalized linear model evaluated the effects of storage duration on protein stability, with post hoc Tukey adjustments. Results: Fibrinogen concentrations remained stable during storage, with no significant differences at 8 or 12 months compared with fresh plasma. Factor VIII activity declined progressively, with a significant 16% reduction after 12 months (p < 0.001), though values remained within clinically acceptable ranges. Conversely, IgG and albumin concentrations increased significantly during frozen storage, with 37% and 21% higher values, respectively, at 12 months, likely reflecting cryoconcentration. Neither sex nor other covariates significantly influenced protein stability. Conclusion: This study provides the first evidence that elephant FFP stored at −20°C retains acceptable biochemical stability for up to 12 months. Although factor VIII activity decreases over time, fibrinogen remains stable, and immunoproteins increase, supporting the clinical utility of stored plasma in EEHV-HD emergencies. These findings provide foundational guidance for establishing elephant plasma banking protocols, improving readiness for rapid intervention, and advancing One Health–aligned conservation strategies for endangered megafauna. Keywords: Asian elephant, coagulation factors, EEHV hemorrhagic disease, factor VIII activity, fibrinogen stability, fresh-frozen plasma, plasma banking protocols, wildlife transfusion medicine.