Normothermic ex vivo perfusion of the limb allograft depletes donor leukocytes prior to transplantation.

Introduction: The donor immune compartment plays a central role in graft rejection of the vascularised composite allograft (VCA) by contributing to ‘direct presentation’. Using our limb ex vivo normothermic machine perfusion (EVNP) protocol designed for prolonged allograft preservation, this study aimed to assess whether donor leukocytes responsible for allograft rejection are mobilised from the donor compartment. Methods: Five genetically different pig forelimbs underwent perfusion via the brachial and radial collateral artery for 6 h after 2 h of cold storage. Oxygenated haemodilute leucocyte-deplete blood was recirculated at normothermia using an extracorporeal perfusion system. Tissue perfusion was evaluated clinically and biochemically via blood perfusate. The temporal kinetics of donor leucocyte extravasation, cytokine secretion and cell-free DNA was characterised in the circulating perfusate. Results: Flow cytometry revealed increasing populations of viable leukocytes over time, reaching 49 billion leukocytes by 6 h. T (3.0 × 10 ⁹ cells) and B cells (3.1 × 10 ⁸ cells) lymphocytes, monocytes (2.7 × 10 ⁹ cells), granulocytes (8.1 × 10 ⁹ cells), NK (6.3 × 10 ⁸) and γδ (8.1 × 10 ⁸) cells were all identified. Regulatory T cells comprised a minor population (1.6 × 10 ⁷ cells). There was a cumulative increase in pro-inflammatory cytokines suggesting that the donor limb has the capacity to elicit significant inflammatory responses that could contribute to leucocyte activation and diapedesis. Conclusion: EVNP not only acts as a preservation tool, but could also be utilized to immunodeplete the VCA allograft prior to transplantation. This has clinical implications to mitigate acute rejection and prevent graft dysfunction and supports the future application of machine perfusion in graft preservation and immune modulation.