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Original Clinical Science—General

Ex Vivo Normothermic Perfusion of Human Upper Limbs

Rezaei, Majid DDS, MSc1; Ordenana, Carlos MD1; Figueroa, Brian A. MD1; Said, Sayf A. MD, MPH1; Fahradyan, Vahe MD1; Dalla Pozza, Edoardo MD1; Orfahli, Lynn M. BA1; Annunziata, Michael J. BS1; Rohde, Elizabeth MA1; Madajka, Maria PhD1; Papay, Francis MD1; Rampazzo, Antonio MD, PhD1; Bassiri Gharb, Bahar MD, PhD1

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doi: 10.1097/TP.0000000000004045
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Improvements in the field of immunosuppression and advances in solid organ transplantation opened the doors for vascularized composite allotransplantation (VCA).1,2 Technical success has been achieved in hand, face, abdominal wall, uterus, and penile transplantation, with over 210 vascularized composite allografts performed worldwide.3 From a logistical perspective, however, the field of VCA remains unchanged. Since the first vascularized composite transplant in the United States, a laryngeal transplant performed at Cleveland Clinic (Cleveland, OH) in 1998, allograft procurement and transplantation have been center-specific and coordinated at a local level.4 Seventy percent of the 28 vascularized composite allografts, performed between 2014 and 2017, at 14 transplant centers, were procured approximately 100 miles away from the recipient hospitals.4 This limited radius for allograft procurement is dictated by tissue susceptibility to ischemia. Composite vascularized allografts consist of multiple tissue types, of which muscle is the least tolerant to ischemia and reperfusion because of its high metabolic activity.5 Irreversible muscle injury can occur in 4–6 h of warm ischemia with the best functional results achieved when the organ is reperfused in <2 h.2,6 The current clinical practice of VCA preservation is based on static cold storage (SCS). The 2 main principles are the reduction of metabolic activity (by cooling) and prevention of cellular swelling (by preservation solution). However, during hypothermia, anaerobic metabolism continues, resulting eventually in depletion of energy stores,7 cell swelling, and lysis.8 Accumulation of metabolic products triggers the production of toxic molecules after reperfusion, promoting downstream pathways of ischemia-reperfusion injury.7-9

Moreover, during SCS, the quality of the limb cannot be assessed or enhanced before revascularization; the injury sustained during the ischemic period cannot be reversed, and preservation times are limited.10

Ex vivo normothermic perfusion (EVNP) is an emerging preservation technology that provides organs with oxygenation and nutrition to maintain physiological metabolism,11 avoiding the deleterious effects of both hypoxia and cooling.12 Longer preservation and the ability to assess the viability and functionality of the preserved organ or to reverse the injury before transplantation are other advantages of EVNP.2 The efficacy of EVNP has been proven preclinically for solid organs, and clinical trials for liver, heart, lung, and kidney are ongoing.12

The aim of this study was to investigate the outcomes of extended EVNP of human upper extremities compared with SCS.


This study was approved by Cleveland Clinic Institutional Review Board (IRB# 15-1429) and conducted with the support and assistance of Lifebanc, Northeast Ohio’s organ and tissue recovery organization.

Bilateral upper limbs were procured from adult patients following declaration of brain death and after consent for research procurement was obtained from the donors’ families by the Lifebanc staff. Exclusion criteria for this study were unresolved sepsis, hepatitis B virus+, hepatitis C virus+, HIV/AIDS, and evidence of prior trauma or surgery of the upper extremity.

Limb Procurement

A circumferential incision was made 15 cm above the medial epicondyle to preserve the integrity of all forearm compartments. Cephalic vein, brachial artery, and vein, as well as median, ulnar, and radial nerves, were isolated 5 cm proximal to the incision. Arm muscles were divided, and a humerus osteotomy was performed. After the upper limb was completely isolated, on the vascular pedicle, the brachial artery and concomitant veins were divided.

Limbs were weighed immediately after procurement. The brachial artery was cannulated and flushed with preservation solution (University of Wisconsin preservation solution Cold Storage Solution, Belzer, Columbia, SC) until venous outflow was clear. Limbs were weighed again, placed in a polyurethane bag, and transported to the laboratory. One limb was connected to ex vivo normothermic limb perfusion (EVNLP) system, and the contralateral limb was preserved on ice slurry at 4 °C, as cold storage control.

The EVNLP system12 is shown in Figure 1. EVNLP was terminated at 48 h or earlier if any of the following signs were present: maximum arterial pressure 115 mm Hg and above (indicating increasing resistance within the limb), 20% decrease of tissue oxygen saturation compared with baseline, and fullness of muscle compartments (confirmed by compartment pressure >30 mm Hg).13

Schematic illustration of the perfusion system that consists of a roller pump, oxygenator, gas regulator/mixer, heat exchanger, and arterial line filter. Venous drainage from the limb occurs freely and is collected in a perfusate reservoir. The limb rests on a support tray and is covered by a heated dome set at 37 °C.


The perfusate consisted of 1200 mL of packed red blood cells, 900 mL fresh frozen plasma, and 350 mL of albumin 25% solution. Blood products were type-matched and obtained from the Cleveland Clinic Blood Bank. Heparin (5000 U), vancomycin (250 mg), and cefazolin (250 mg) were added to the perfusate. Methylprednisolone (500 mg) was added for endothelial protection and minimization of edema. Dextrose and insulin were added as needed to prevent an extracellular potassium shift and to guarantee enough energetic substrates to the musculature.

Oxygenation was achieved using a diffusion membrane oxygenator (Quadrox-I, Maquet, Getinge, Sweden) with humidified 100% oxygen. Perfusate flow rates were gradually increased during the first hour of perfusion until a mean arterial perfusate pressure of 90 mm Hg was achieved. Vascular resistance was calculated through the hydrostatic variation of the Ohms equation R = (mean arterial pressure − venous pressure)/flow with the venous pressure set to 0 because the circuit had an open venous return.

Perfusate exchanges (500 mL) were performed every 3 h starting at time point 6 to maintain electrolyte concentrations within physiological ranges and minimize metabolite accumulation.

EVNLP Monitoring

Tissue oxygen saturation was measured by near-infrared spectroscopy (ViOptix, Fremont, CA). Venous and arterial perfusate gases were sampled hourly (Abaxis i-STAT 1, Union City, CA). Arterial and venous oxygen content was calculated using the following formula: oxygen content = (1.36 × hemoglobin × O2Sat/100) + (0.0031 × PO2). Oxygen consumption was measured as O2 consumption = flow × (arterial O2 content – venous O2 content). Oxygen uptake rate was calculated as (([O2]in – [O2]out)/100) × flow rate/limb weight, where [O2]in is the oxygen content in mLO2/mL of arterial perfusate and [O2]out is the oxygen content in mLO2/mL of venous perfusate.14 Complete blood count, creatine kinase (CK), and albumin levels were measured every 6 h (Siemens ADVIA 120 Hematology System, Munich, Germany). Surface temperature of the limb was assessed hourly by infrared thermography (Fluke TiS Thermal Imager, Everett, WA). Muscle and nerve functionality were hourly assessed by electrical stimulation of motor nerves with a modified transepidermal nerve stimulation unit (TENS Ultima 20, Largo, FL) in continuous waveform mode at a pulse rate of 3 Hz and a pulse width of 250 µs (default settings). Contractility was graded using the Medical Research Council grading system on a 0–5 scale, where 0 represented no contraction and 5 represented full contraction against gravity.

Biopsies were collected from biceps brachii muscle at procurement (time point 0) and at 6, 12, 18, 24, 36, and 48 h from beginning of EVNLP. At the endpoint, biopsies were taken also from forearm flexor and extensor muscles. Specimens were stored in 10% formalin and 70% ethanol and stained with hematoxylin and eosin. The slides were scanned by a high-resolution slide scanner (Aperio AT Turbo, Leica Biosystems, Buffalo Grove, IL) and evaluated for muscle fiber damage, as described by McCormack et al,15 by an investigator blinded to the preservation group.

At perfusion endpoint, compartment pressures were measured in the forearm flexor compartment (Stryker intracompartmental pressure monitor, Munich, Germany). Indocyanine green angiography was used to investigate uniformity of the peripheral perfusion (Stryker SPY Elite System, Munich, Germany).

Metabolomic Analysis

Muscle samples were obtained at 24 h and stored at –80ºC for metabolomic analysis. Samples were processed for untargeted metabolomics by adding 1 mL of chilled 70% methanol, 20% water, and 10% chloroform containing internal standards to 35 mg of muscle followed by tissue homogenization. Ten microliters of each homogenate were used for protein concentration measurements. Each sample was then subjected to liquid chromatography–mass spectrometry analysis. The data yielded from liquid chromatography–mass spectrometry analysis were used for principal component analysis, putative identification assignment, and pathway enrichment analysis via the Kyoto Encyclopedia of Genes and Genomes (KEGG). Databases used for MS identification include KEGG, the Human Metabolome Database, LipidMaps, BioCyc, Reactome, and ChEBI databases, and mzCloud MS/MS database using Compound Discoverer version 2.1 (Thermo Scientific, Fremont, CA).

Outcome Measures and Statistical Analyses

Outcome measures for this study were EVNLP duration, muscle contractile response to electrical stimulation, limb weight, compartment pressure, and muscle injury scores. To detect significant difference in myocytes injury scores between EVNLP and SCS groups, a sample size of 10 limbs per study arm was estimated on the basis of prior EVNLP experiments using porcine limbs,16 with a standardized effect size of 1.3 at a 2-sided α of 0.05 and 80% power.

Variables are reported as mean and SD (x ± SD) or median and interquartile range (M [IQR]). To compare muscle contractions, compartment pressures, and muscle injury scores between EVNLP and SCS groups, the Mann–Whitney U test, Kruskal–Wallis test, and Student t test were applied, respectively. To calculate the rate of change for a variable concentration in relation to time, a linear regression model was conducted with the variable in question set as the dependent variable and the time point as the independent variable. The rate of change was reported as β for each hour and the P value. Spearman’s rank correlation coefficient test was used to assess the correlation between discrete variables, and the results were presented as ρ and P value. To investigate the influence of predictor variables (sodium, potassium, calcium, pH, and lactate) on muscle contraction, a multinomial logistic regression was modeled. In metabolomic analysis, the ion presence threshold was set at 0.7 in each study group. Data were then log-transformed and analyzed for statistical significance via Mann–Whitney U test (P < 0.05). Ions present in just a subset of samples were analyzed as categorical variables for presence status via Fisher exact test. Data analysis was performed using SPSS 25.0 for Mac (IBM Corporation, New York, NY) and STATA for Windows (StataCorp. 2019. Stata Statistical Software: Release 16. College Station, TX: StataCorp LLC). A P value <0.05 was considered significant.


The initial warm ischemia was 59.6 ± 20.9 min for the EVNLP group and 64.9 ± 21.1 min for the SCS group. The average duration of EVNLP was 41.6 ± 9.4 h.


The average flow was 0.41 ± 0.06 L/min. Vascular resistance was high at the beginning of EVNLP (254.3 ± 43.7 mm Hg × min/L), decreased to a nadir level of 142.25 ± 19.52 mm Hg × min/L at a median time point of 9.5 (8.8) h of perfusion, and remained relatively stable with a slow upward trend reaching an average of 187.3 ± 26.9 mm Hg × min/L at the end of the perfusion (Table S1, SDC,; Figure 2).

Changes in electrolytes and gases over time for ex vivo normothermic perfused limbs. Asterisks denote perfusate exchanges starting on time point 6 and every 3 h consequently. See the text for a detailed explanation. HL, human limb.


PaO2 averaged 509.5 ± 91.4 mm Hg. The average baseline tissue oxygen saturation was 90 ± 9%. The average tissue oxygen saturation during EVNLP was 87.4 ± 11.4%. Oxygen uptake rate averaged 5.7 ± 2.8 mL/min/g. Venous PCO2 (PvCO2) averaged 15.7 ± 30.2 mm Hg. Average perfusate pH was 7.465 ± 0.206 (Table S1, SDC,; Figure 2).


Sodium concentration increased 0.545 mmol/L/h (95% confidence interval, 0.51-0.58; P < 0.001). Potassium concentration decreased from 6.0 ± 1.7 mmol/L to a nadir of 3.7 ± 1.5 mmol/L at 6 (2) h. Thereafter, the potassium concentration increased 0.092 mmol/L/h (95% confidence interval, 0.084-0.101; P < 0.001) reaching an average level of 7.6 ± 0.9 mmol/L toward perfusion endpoint. Sodium and potassium increments were minimally responsive to perfusate exchanges. Perfusate analysis did not show evidence of hemolysis. Calcium concentration remained stable throughout the perfusion (0.4 ± 0.1 mmol/L) (Table S1, SDC,; Figure 2).


Lactic acid increased to 20 mmol/L (I-Stat detection limit) at a median time point of 15 (6) h. Glucose decreased from 433.6 ± 116.9 to 200.4 ± 78.6 mg/dL at the end of the perfusion. CK concentration increased from 956 (3781) U/L within the first hour to 49 020 (48 585) U/L at endpoint. Initial myoglobin concentration in the perfusate was 5370 (9170) ng/mL incrementing to 34 730 (83 081) ng/mL at the end of the perfusion (Table S2, SDC,; Figure 2).

Limb Parameters

Muscle temperature improved from 29.5 ± 4.7 °C reaching an average of 35.1 ± 1.7 °C. Infrared thermography confirmed gradual improvement of peripheral perfusion (Figure 3). Baseline weight (before flushing with preservation solution) was 2889 ± 642 g for the perfused limbs and 2881 ± 501 g in the SCS group. Following flushing, no significant difference in limb weight in neither group was noted (EVNLP: 2889 ± 642 g [before flushing] versus 2934 ± 556 g [after flushing]; P = 0.84; SCS: 2881 ± 501 g [before flushing] versus 2883 ± 515 g [after flushing]; P = 0.99). The weight change at the end of the perfusion was 0.4% ± 12.2% of the initial weight. Limb weight remained unchanged at the end of the experiments in the SCS control limbs. The difference in weight change between groups at the end of EVNLP was not statistically significant (P = 0.21). The average flexor compartment pressure at the end of the perfusion was 21.7 ± 15.5 mm Hg (P = 0.003). Limb 3 developed an expanding hematoma at time point 6; as a result, the limb became increasingly hard to perfuse. A flexor compartment fasciotomy was performed and the EVNLP proceeded for 39 h afterward. In the control group, the average compartment pressure at the end of the experiments was 1.8 ± 0.4 mm Hg (P = 0.003) (Table S2, SDC,

Procured HLs were often cold, edematous, and with ecchymosis close to level of vascular accesses. Infrared thermography showed hypothermia at the beginning of the perfusion. Limb surface temperature improved during the perfusion and remained within the physiological range until the end. ICG angiography at the end of perfusion showing well-perfused fingertips in most of the limbs, as indicated by the uniform dye distribution. HL, human limb; ICG, indocyanine green.

Indocyanine green angiography confirmed the presence of peripheral perfusion until the termination of EVNLP (Figure 3). (See Video S1, SDC,, which shows the preservation of peripheral circulation at the end of EVNLP.)

Changes in muscle contractility are shown in Figure 4. Contractions improved from 0 (1.25) at the beginning of the perfusion to 5 (1) at 5 (4.5) h (see Video S2, SDC,, which shows the improvement of muscle contractility during the initial hours of EVNLP). Maximum contractility (5/5) was maintained for a median duration of 7 (9.25) h. Muscle contraction was observed until a median of 30.5 (15.8) h, whereas SCS limbs had no muscle contraction during the entire experiment (P < 0.001). There was a negative correlation between the contractions and the concentration of potassium (ρ = –0.65, P < 0.001).

Muscle contractility map. HLs undergoing ex vivo normothermic perfusion exhibited the most forceful muscle contractions for 5 (4) h and were sustained for 30.5 (16) h. HL, human limb.

At EVNLP endpoint, 28.9 ± 11.5% of muscle fibers had evidence of injury with broken borders, inconsistent texture and color throughout the myocytes, and presence of holes. In the SCS control group, 90.2 ± 11.8% of myocytes displayed severe damage at the end of the experiment (P < 0.001) (Table S2, SDC,; Figure 5). In the EVNLP group, myocyte injury scores did not significantly differ among time points 0, 12, 24, 36, and 48 h (P = 0.46). In the control group, myocyte injury score was significantly higher at endpoint compared with time point 0 (P = 0.009).

Representative H&E–stained muscle at the beginning of perfusion (A), after 12 h (B), after 24 h (C), and at the end of perfusion (D). (E and F) Representation of the control group at the beginning and after 48 h of cold storage, respectively. At experiment endpoint, SCS control limb myocytes showed heterogeneous staining with fragmented muscle fibers. EVNLP, ex vivo normothermic limb perfusion; H&E, hematoxylin and eosin; SCS, static cold storage.

The effects of perfusate sodium, potassium, calcium, pH, and lactate change on the likelihood of muscle contraction are summarized in Table 1. Lactate (P = 0.27) and sodium (P = 0.05) concentrations had no effect on muscle contractions against gravity. The probability of limbs contracting at M5 increased as potassium decreased (P < 0.001). Calcium and alkaline pH shift also demonstrated a highly significant (P < 0.001) positive direction of change with muscle contractility against gravity.

TABLE 1. - Results of multinomial logistic regression. Effect of predictor variables on muscle contraction
M1 contraction M2 contraction M3 contraction M4 contraction M5 contraction
Sodium –0.029(0.041) –0.111*(0.053) 0.062(0.047) 0.106(0.047) 0.129(0.043)
Potassium –0.537*(0.220) –0.953***(0.270) –1.455***(0.282) –1.511***(0.272) –1.513***(0.266)
Calcium 14.483***(3.862) 24.770***(4.943) 25.468***(4.708) 26.164***(4.669) 27.615***(4.709)
pH –0.902(2.490) 3.350(2.858) 6.175***(2.664) 9.132***(2.886) 8.119***(2.884)
Lactate 0.144(0.133) 0.478(0.176) 0.284(0.133) 0.237(0.156) 0.158(0.130)
M0 contraction used as the baseline outcome. Coefficients with the respective standard errors are within parentheses.
*P < 0.05.
**P < 0.01.
***P < 0.001.
M0, equals no contraction visible or palpable; M1, trace contraction; M2, movement with gravity eliminated, M3, movement against gravity; M4, movement, against gravity and resistance; M5, normal power.

Human Limb Metabolomic Profiling

A total of 13 694 spectral features were identified. Taurine and tryptophan were among the most significantly perturbed ions at 24 h. Pathway enrichment analysis using the BioCyc and KEGG databases showed that metabolites belonging to neuroactive ligand–receptor interaction (P = 0.031) and amino acid metabolism (P = 0.036), including tyrosine (P = 0.015) and tryptophan metabolism (P = 0.002), were the most perturbed at 24 h. Taurine abundance decreased (P = 0.002) and tryptophan abundance increased (P = 0.002) at 24 h.


We were able to maintain EVNLP for an average of 41.6 ± 9.3 h. At the time of procurement, most (8/10) upper extremities were edematous and cold (Figure 3). The limb condition improved during the first hours of perfusion with normalization of electrolytes, muscle, and surface temperature, and most importantly, restoration of muscle contraction that was maintained for 30.5 (15.8) h. Vascular resistance decreased and remained stable. Infrared thermography confirmed an improvement in peripheral perfusion with increase in temperature of the fingertips.

The first report of EVNLP was published by Delorme et al in 1964.17,18 Amputated human upper and lower limbs were perfused at physiological temperature and pressure with oxygenated whole blood and dextran for 4.5 h, with preservation of muscle contraction during perfusion and loss of contractility upon cooling the limb. Fifty-two years later, Werner et al preserved 5 human upper extremities in subnormothermic conditions (30–33 °C) with a perfusate flow of 310 mL/min for 24 h. Their results demonstrated ongoing oxygen consumption, stable vascular resistance, and intact response to nerve stimulation, all indicators of limb viability after 24 h of ex vivo perfusion.19 Haug et al20 perfused 3 human limbs for 24 h with oxygenated Steen solution at an average 30.4 mL/min flow and a perfusate pressure of 30 mm Hg at 10 °C. They reported 4.3% weight increase, median potassium levels of 5.7 mmol/L, and lactic acid of 2.8 mmol/L. Muscle contraction and oxygen uptake were not assessed.

Brockmann et al21 reported improved posttransplant survival (27%–86%, P = 0.026), in livers undergoing EVNP for 20 h compared with cold storage. Koerner et al22 showed that EVNP, in heart transplantation, contributes to better outcomes after transplantation with regard to recipient survival, incidence of primary graft dysfunction, and incidence of acute rejection. Our findings and these reports highlight the potential of EVNP in eliciting organ repair and suggest a role in organ reconditioning by reversing energy depletion and the immediate effects of warm ischemia.11

Currently, there are no objective criteria that define transplantability of the donor arms. Limb number 3 appeared cold, edematous, and displayed multiple ecchymosis at the antecubital fossa and evidence of multiple arterial punctures on distal forearm at the time of procurement. The limb developed compartment syndrome within the first 6 h of perfusion. A decision was made to perform fasciotomies and the EVNLP continued for 39 h afterward.

We had previously established muscle contractility as an indicator of maintenance of critical limb physiology.12 The best contractile strength was observed between 3 and 18 h. It seems that the initial poor condition of the limb, secondary to compromised hemodynamics of the donor patient and ischemia, can be reversed by EVNLP. Although 80% of the limbs achieved maximal contractility within the first 6 h of perfusion, limb 6 never regained a high level of contractility. The ability of the limb to contract indicates structural and functional integrity of multiple tissues such as nerves, the neuromuscular endplate, and contraction of at least 80% of all myocytes within the contracting group of muscles.19 In the clinical setting, lack of contraction may raise suspicion about transplantability of a perfused limb. However, muscle contractility is influenced by multiple variables during EVNLP, such as perfusate potassium, calcium, and pH, and unless all these parameters are in physiological range, we no longer use contractility as a primary indicator of limb viability during EVNLP. Contractility is inversely correlated with perfusate potassium levels. This correlation of potassium level and muscle paralysis is rarely seen in clinical practice because cardiac manifestations usually precede muscle weakness. Patients with hyperkalemic periodic paralysis may become symptomatic at a plasma concentration around 5.5 mEq/L.23 During EVNLP, muscle contractions declined and stopped at a potassium concentration of 6.2 ± 0.6 and 7.6 ± 0.9 mmol/L, respectively. Increasing perfusate potassium during ongoing machine perfusion may be the result of water loss by evaporation, lack of intrinsic regulation of the concentration (absence of kidney), spillage of intracellular potassium ions from the cut end of the limb, and secondary to myocyte destruction.

EVNLP limbs gained an average 0.4 ± 12.2% (P = 0.21) of the initial weight. Petrasek et al24 demonstrated that weight gain in ischemic muscle was related to the severity of muscle injury, finding 4% weight gain in the least injured muscles and a 32% weight gain in the most severely damaged muscles.

Average perfusate pH was kept within the physiological range. After an initial period of alkalosis, the perfusate became slightly acidotic as time progressed, accompanied by rising lactate levels. Lactate levels are a result of anaerobic muscle, skin, and red blood cell metabolism. In physiological conditions, lactate removal takes place via its oxidation to pyruvate in liver, kidneys, and to a lesser extent heart. Pyruvate may be either oxidized to carbon dioxide producing energy or transformed into glucose. In the absence of metabolizing organs, lactate levels are expected to raise during EVNLP as reported by others.19,25-29 Despite adequate oxygenation, the tissues change to anaerobic metabolism at some point during EVNLP, resulting in metabolic acidosis and increased lactate.30 Mitochondrial dysfunction over time can also contribute to increased lactate production. Finally, water loss by evaporation over time, despite perfusate addition, contributes to increase of electrolytes and metabolites concentration. Slater et al31 perfused porcine flaps with either University of Wisconsin preservation solution, HTK, or autologous blood (normothermic machine perfusion). In the blood-perfused grafts, metabolic byproducts, especially lactate, rose continuously. Ozer et al26,27 used heparinized autologous blood in their perfusion system and found a steady increase in lactate level until the end of perfusion at 24 h. The significance of lactate levels, as a marker of cellular stress and tissue hypoxia, during EVNP is yet to be completely understood. In an EVNP model of perfused porcine hearts, a significant correlation was found between myocardial injury scores and lactate levels.32 In a clinical trial of ex vivo normothermic heart perfusion, lactate was used as the primary determinant of transplantation potential.33 Increases in lactate have been observed during the ex situ lung perfusion, and good outcomes after transplantation were achieved despite elevated lactate concentrations.34 Our results did not show a direct influence of lactate levels on muscle contractility.

Myoglobin and CK were measured as markers of muscle injury. However, we did observe a linear increment of both markers over time (from time point zero, differently from other parameters) that did not correlate with the other markers of injury. We hypothesize that both myoglobin and CK are continuously released in the perfusate from the cut end of the arm interfering with detection of the smaller quantities released within the limb.

Metabolomic analysis showed a decrease in taurine abundance at 24 h of EVNLP as compared to the control. Taurine is required for normal respiratory chain function.35 Without supplementation within the perfusate, taurine will be used and depleted during perfusion. Cellular taurine depletion reduces respiratory function and elevates mitochondrial superoxide generation, which damages mitochondria and increases oxidative stress.36 Taurine metabolism could signal damage to myocytes and release of intracellular proteins containing the amino acid. It has been shown that taurine exhibits antioxidant effects and can decrease tissue oxidation caused by reactive oxygen species (ROS), decrease inflammatory activity with no known side effects,35,36 and reduce ischemia-reperfusion–induced compartment syndrome in experimental settings.37 Taurine supplementation could prevent mitochondrial damage and subsequent oxidative stress and improve perfusion outcome.

Tryptophan presence and catabolism during EVNLP could potentially signify skeletal muscle injury. Tryptophan is considered both a glucogenic and ketogenic amino acid and can only be metabolized in the liver.38 Because the EVNLP system lacks a liver and hepatic enzyme, the presence of this amino acid could simply mean that the muscle tissue itself is not able to metabolize tryptophan.

The results of the current study need to be examined in light of some of its limitations. There was no randomization of the limbs. The selection of limbs without indwelling arterial and venous lines for EVNLP could have led to selection bias. Additionally, the formation of ROS following reperfusion was not directly quantified. Our study design included the use of 100% oxygen, which could have enhanced membrane peroxidation and a further increase in the formation of ROS. Furthermore, our model did not include a formal analysis of reperfusion injury. This was determined by difficulty in procuring whole blood to simulate reperfusion from the donors. We were able to collect whole blood only from 3 donors because of donor hemodynamic instability. Warm reperfusion in the presence of mediators of inflammation might have provided additional insight into the quality of the preservation and the viability of limb allografts.

EVNLP termination criteria have been modified in our porcine studies; weight increases of >5% have been implemented as a criterion for termination of perfusion before irreversible injury occurs. Porcine EVNLP data showed an increase in the vascular resistance after a weight increase of >5%, indicating fluid accumulation and initial ischemic injury. These results should be validated, with outcomes of perfused extremities following transplantation.


EVNLP is a promising technology that can overcome the limitations of cold preservation, not only extending preservation times but also enabling assessment of limb quality and allowing reconditioning of the limbs before transplantation. EVNLP may impact access and long-term outcomes of limb transplantation.


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