VCA (Videos Available)

Thursday July 05, 2018 from 09:45 to 11:00

Room: N-112

622.7 Optimization of an ex-vivo limb perfusion protocol for vascularized composite allograft transplantation (Video Available)

Kavit Amin, United Kingdom

Clinical Trainee in Plastic Surgery/PhD in Regenerative Medicine
Manchester Collaborative Centre for Inflammation Research (MCCIR), Division of Infection, Immunity and Respiratory Research, School of Biology, Medicine and Health. Manchester Academic Health Science Centre
University of Manchester, Manchester, M13 9NT. The Transplant Centre, Manchester University Hospitals NHS Foundation Trust, M23 9LT

Abstract

Optimization of an Ex-Vivo Limb Perfusion Protocol for Vascularized Composite Allograft Transplantation

Kavit Amin1,2, John P Stone1,2, Rebecca J Edge1,2, Jacob Parkes1,2, Jak Kerr2, Leena Joseph3, Jason Wong4, James E Fildes1,2.

1The Transplant Centre, Manchester University Hospitals NHS Foundation Trust, Manchester, United Kingdom; 2Manchester Collaborative Centre for Inflammation Research (MCCIR), University of Manchester, Manchester, United Kingdom; 3Department of Pathology, University Hospital South Manchester NHS Foundation Trust, Manchester, United Kingdom; 4Plastic Surgery Research Blonde McIndoe Laboratories, University of Manchester, Manchester, United Kingdom

Introduction: The current protocol for vascularized composite allograft (VCA) preservation involves cold static storage on ice. This severely limits storage times of the graft and is associated with several deleterious processes that augment ischaemia reperfusion injury and acute rejection. As a result there is a major drive to develop ex-vivo perfusion (EVP) protocols that can improve preservation. Critical parameters for successful EVP include temperature of the circulating perfusate and mean arterial pressure that have yet to be defined in the literature for composite tissues.
Methods: A total of 30 pig forelimbs were used in this study. Each experiment consisted of n=5 bilateral limbs to enable paired analysis. Limbs were cold stored for 2 hours and perfused for six hours via the axillary and radial artery at normothermia (NMP, 38°C), subnormothermia (SNMP, 28°C) or hypothermia (HMP, 10°C). Perfusion was evaluated and accurate assessment of temperature was performed in real-time. Cytokines/chemokines and circulating cell free mitochondrial (mtDNA) and genomic DNA (gDNA) were quantified as markers of inflammation and cell death. Pre and post-perfusion biopsy samples (skin, muscle and vessel) were interrogated by a study blinded histopathologist. 
Results: Tissue viability was maintained in all limbs via visual criteria. Limbs perfused via NMP demonstrated higher flow rates of 80.25±7.83ml/kg/min, followed by SNMP 71.66±6.32ml/kg/min and HMP 22.70±2.39ml/kg/min.Lactic acidosis was present in all limbs, which was physiologically buffered by bicarbonate. Perfusate pH, base excess and electrolyte levels remained stable until the end of perfusion. Glucose consumption was significantly higher in the NMP group compared with SNMP and HMP, indicating higher metabolic activity.
At the end of perfusion, the concentration of cytokines (figure 1 a & b) and cell free DNA (figure 2) were significantly higher in the NMP group compared with SNMP and HMP. Histologically HMP demonstrated superior preservation of skin, muscles and vessel compared with SNMP and NMP. NMP had greater neutrophilic margination and exudation into the interstitium with more marked inflammation.

Conclusion: Composite tissues can be optimally preserved via HMP using a blood-based perfusate.  This is supported by the reduction in markers of cellular injury, inflammation and better tissue integrity. This is the first limb preservation protocol that systematically provides valuable insight into how composite tissues respond to temperature during machine perfusion. 

British Society for Surgery of the Hand (BSSH). Royal College of Surgeons (RCS). EPSRC & MRCS PhD in Regenerative Medicine.



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