Ex-vivo flush of the limb allograft reduces inflammatory burden prior to transplantation

Background: Passenger leucocytes and inflammatory debris transferred from the donor limb to the recipient can induce allorecognition, which activates the host immune response. This is the first study to evaluate whether the transfer of this inflammatory burden can be reduced via post-preservation flush prior to revascularisation, and whether this is influenced by ischaemia. Methods: Bilateral forelimbs from the same pig were procured and infused with preservation flush and stored on ice. Each limb from the same pig underwent a post-preservation intravascular flush with isotonic solution at either 2 or 6 h. Venous effluent underwent flow cytometry to phenotype leucocyte populations, with additional quantification of cytokines and cell-free DNA. Results: We identified large populations of viable leucocytes in the flush effluent (8.65 × 10⁸ ± 3.10 × 10⁸ cells at 2 h and 1.02 × 10⁹ ± 2.63 × 10⁸ at 6 h). This comprised T cells, B cells, NK cells and monocytes. Post-preservation flush yielded significant concentrations of pro-inflammatory cytokines including IL-6, IL-18, GM-CSF, IL-1β IL1α and CXCL-8 and mitochondrial DNA. The regulatory cytokine, IL-10 was undetectable. Conclusions: This study supports the finding that a post-preservation flush removes leucocytes and inflammatory components that are responsible for direct presentation. This study also gives an indication of how ischaemia impacts on the inflammatory burden transferred to the recipient upon reperfusion.