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Feasibility Of Extremity Transplantation Following Ex Vivo Normothermic Limb Perfusion (EVNLP)
Carlos X. Ordenana, MD, Majid Rezaei, DDS MS, Vahe Fahradyan, MD, Sayf Said, MD, Lynn Orfahli, BM, Brian Figueroa, MD, Edoardo Dalla Pozza, MD, Maria Madajka, PhD, Francis Papay, MD, Antonio Rampazzo, MD, PhD, Bahar Bassiri, MD, PhD.
Cleveland Clinic, Cleveland, OH, USA.

PURPOSE:
Ischemia-reperfusion injury remains a major limiting factor for limb replantation and transplantation. Ex-vivo normothermic limb perfusion has been proven to preserve viability and function of amputated limbs longer than cold storage. Our aim was to investigate the feasibility of limb transplantation following EVNLP and possible vascular, inflammatory and infectious complications in a mid-humerus porcine forelimb transplant model.
METHODS:
Eight Yucatan miniature pigs, 4 donors and 4 recipients, were used. Right forelimbs of donor animals were amputated at mid-humeral level, to maintain integrity of forearm fascial compartments, and preserved with EVNLP until replantation. The right forelimb of the recipient animal was amputated and a central venous line was placed in the contralateral neck. Perfusate electrolytes, gases, O2 saturation, muscle contractility, surface temperature (Infrared thermography) and peripheral perfusion (ICG agiography) were assessed during EVNLP. The humerus was fixed with a single 3.5mm LC-DCP plate; microsurgical anastomosis of the brachial artery, cephalic vein, and repair of radial, ulnar, and median nerves were performed under an operative microscope. Tendons of the biceps and triceps were repaired with pulvertaft weave technique. The first animal did not receive systemic immunosuppression. Systemic immunosuppression for the remaining 3 pigs included induction with antithymoglobulin, followed by daily cyclosporine (CSA), mycophenolate mofetil and methylprednisolone. CSA trough levels were measured daily. Limbs were monitored clinically and histologically for signs of rejection. Bone healing was confirmed with CT scan at euthanasia. The endpoint of the study was 90 days.
RESULTS:
Warm ischemia time during limb procurement was 20.69 minutes and 2.20.25 hours during limb transplantation. EVNLP lasted an average of 4.30.52 hours. Total time to revascularize was 6.80.5 hours. PO2, pH and Lactate where 55772mmHg, 7.50.1, 5.60.9mmol/L respectively. Muscle contractions were 4/5 during EVNLP. All forelimbs were successfully transplanted with no vascular failure. CSA trough levels were on average 678450[BGB1] [F2] , 369445, and 336468ng/mL. Animals 2, 3 and 4 developed septic thrombophlebitis of the central line, which was replaced on POD 14, 51, and 28. Animals 3 and 4 lost IV access on POD 54 and 64 respectively and were transitioned to PO medications. The first transplanted limb (animal 1) showed evidence of acute rejection on POD 4 and the animal was euthanized on POD 6. In the remaining animals, the incisions healed and initial edema resolved by day 14.The third animal developed angioinvasive aspergillosis on POD 20 with two areas of full thickness skin necrosis of trunk. Voriconazol was started and the lesions were resected on POD51. The animal lost the central line on POD 55, showed evidence of rejection on POD 57 and was euthanized on POD 60. Animals 2, 3 and 4 showed bone healing and consolidation with the presence of bony callus on CT scans. At endpoint animal 2 and 4 had reached complete weight bearing on the transplanted limb, at the hoof and wrist respectively.
CONCLUSION:
Extremity transplantation can be successfully performed following EVNLP. EVNLP does not increase the risk of vascular or early infectious complications. The mid-humerus porcine transplantation model is feasible and reproducible.


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