Viability and Functional Outcomes of Limb Allografts Following Sub-normothermic Plasma and Red Blood Cell Based Perfusion

OBJECTIVE: Vascularized composite allotranplantation (VCA) is an evolving area to reconstruct composite tissue defects. One of the challenges in VCA is the narrow window of tolerable ischemia time. Current standard of care, static cold storage, is only effective for short periods and may result in severe tissue damage and acute rejection when prolonged. Ex-situ perfusion has the potential to overcome these challenges by extending allowable ischemia time. However, optimum perfusate composition, preservation temperature, and hemodynamic parameters of perfusion are still debated. Over the past decade, the benefits of addition of fresh plasma in hemorrhagic shock resuscitation and attenuation of reperfusion injury have been recognized. Whether these observations are due to high or low molecular weight (HMW and LMW, respectively) plasma proteins require further investigations. This study investigates the effects of addition of fresh plasma, separated based on molecular weight of proteins, on the viability of perfused limb allografts. METHODS: All limb transplantations were performed orthotopically between male Lewis rats. In group 1, amputated limbs were transplanted immediately. In group 2, amputated limbs were preserved at 4°C. In other groups, amputated limbs were perfused for 6 hours at 20° to 22°C using one of the following perfusates (in less than 90 minutes): RBC only (G3), RBC + whole plasma (G4), RBC + LMW plasma (G5), RBC + HMW plasma (G6). 3 months post-transplantation, surviving recipients had end-point assessment using electromyography to measure compound muscle action potentials (CMAP) and muscle force generation. RESULTS: Recipients in the group perfused with RBC and HMW plasma proteins had severe reperfusion injury and did not survive beyond 3 days. Among surviving recipients, the addition of whole plasma did not appear to improve amplitude and latency compared to immediately transplanted limbs. Groups perfused with RBC only and RBC with LMW plasma demonstrated excellent amplitude and latencies on gastrocnemius and extensor digitrum longus (EDL) muscles with no significant difference compared to immediately transplanted limbs (p>0.05). The EDL muscle in these groups also generated as much tetanic and twitch forces compared to immediate transplantation group (p>0.05). CONCLUSION: Ex-situ perfusion at 20 to 22C for 6 hours using RBC and LMW plasma appears to protect both fast and slow twitch muscles and improve axonal regeneration and myelin sheath thickness comparable with immediately transplanted group. CORRESPONDING AUTHOR: Stephen WP Kemp, PhD. 1150 W Medical Center Drive, MSRB II, A570. P: 734-764-8750, F: 734-615-32-92. Email: [email protected].