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Outcomes Of Ex-vivo Normothermic Limb Perfusion (EVNLP) With A Hemoglobin-based Oxygen Carrier (HBOC-201)
Lynn M. Orfahli, BM1, Majid Rezaei, DDS, MSc1, Carlos X. Ordenana, MD1, Brian A. Figueroa, MD1, Sayf Said, MD1, Michael J. Annunziata, BS1, Henri Brunengraber, MD, PhD2, Srinivasan Dasarathy, MD1, Francis A. Papay, MD1, Antonio Rampazzo, MD, PhD1, Bahar Bassiri Gharb, MD, PhD1.
1Cleveland Clinic Foundation, Cleveland, OH, USA, 2Case Western Reserve University School of Medicine, Cleveland, OH, USA.

PURPOSE:
Ex-vivo normothermic limb perfusion (EVNLP) has been proven to preserve limb viability and function for a longer duration than static cold storage (SCS). Perfusates containing red blood cells (RBCs) as oxygen carriers improve outcomes when compared to acellular perfusates. However, the use of blood products is hampered by limited availability, need for cross-matching, and potential transmission of blood-borne infections. Hemoglobin-Based Oxygen Carrier-201 (HBOC-201), a polymerized, highly purified bovine hemoglobin, has a 3-year shelf life at room temperature, low immunogenicity, and an oxygen-carrying capacity similar to that of RBCs. The aim of this study was to compare outcomes of EVNLP with HBOC-201 to EVNLP with an RBC-based perfusate.
METHODS:
Eighteen porcine forelimbs were procured from Yorkshire pigs following euthanasia. Twelve limbs underwent EVNLP until mean perfusate pressure reached 115 mmHg. Six were perfused with an HBOC-201-based perfusate, six with an RBC-based perfusate, and six were stored at 4C (SCS control). Hemodynamics (flow and pressure), perfusate electrolytes, arterial and venous gases, O2 uptake rates (OUR), methemoglobin, glucose, and lactate were measured. Limb temperature, weight, compartment pressure, contractility, and peripheral perfusion, assessed by indocyanine green (ICG) angiography and infrared thermography, were recorded. Skin and muscle sections were stained with hematoxylin and eosin for histological analysis.
RESULTS: EVNLP duration in HBOC-201- and RBC-perfused limbs was 22.51.7 and 28.27.3 hours, respectively (p=0.04). Throughout perfusion, HBOC-201 and RBC forelimbs exhibited comparable results regarding vascular flow (325.025.0 vs. 444.750.6 mL/min; p=0.39), vascular resistance (214.869.8 vs. 190.958.3 mmHg*min/mL; p=0.18), OUR (2.01.45 vs. 1.30.92 mLO2/min*g of tissue; p=0.80), and weight change (23.13.0% vs. 13.222.7%; p=0.07). Methemoglobin levels in HBOC-201 limbs increased, reaching an average of 47.912.1% at endpoint. In both groups, sodium and potassium concentrations increased steadily throughout EVNLP; minimal correction of potassium was achieved with partial perfusate exchanges. Overall pH was alkalotic in both (7.530.25 vs. 7.500.23, p=0.82). RBC limbs maintained maximal muscle contractility longer than HBOC-201 limbs; however, this did not reach statistical significance (p=0.57). Flexor (28.322.0 vs. 27.510.6 mmHg; p=1) and extensor (31.522.9 vs. 28.814.5 mmHg; p=0.82) compartment pressures at the end of perfusion were borderline physiologic in both groups. Creatine kinase and myoglobin concentrations were 42% and 22% higher at endpoint in the HBOC-201 limbs, but statistical significance was not reached (p=0.49, p=0.07, respectively). Lactate concentrations at endpoint were significantly higher in HBOC-201 limbs (20.22.8 vs. 14.63.9 mmol/L; p=0.02). The preservation of peripheral tissue perfusion was confirmed by infrared thermography and ICG angiography in both groups.
CONCLUSION: The results of this study suggest a trend towards better EVNLP outcomes with an RBC-based perfusate; however, for shorter durations of EVNLP, it may still be feasible to use HBOC-201 as an alternative oxygen carrier without significantly compromising limb quality. The authors believe that modifiable factors, including metabolism, may be responsible for the differences seen in the outcomes of HBOC-201-perfused limbs. Optimization of these factors could allow for the achievement of extended EVNLP with HBOC-201, overcoming the significant logistical constraints inherent with the use of traditional blood products.


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