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The current gold standard of VCA preservation is static cold storage (SCS), which limits ischemia time to 12 hours for large extremity segments. The current literature provides supportive evidence for the introduction of machine preservation of extremities as an alternative to SCS with potential for translation into clinical practice. The goal of this review is to provide a comprehensive qualitative analysis of published literature on machine perfusion of limbs. We present elements of circuit design, use of animal models, perfusate solutions, temperature and oxygenation during perfusion, strategies to prevent muscle damage, methods to assess viability, and the future of ex-vivo limb perfusion. The future of machine preservation research focuses on the application of ex-vivo perfusion in scenarios where limb reconditioning or testing for viability prior to transplantation or replantation are warranted.


Delorme et al. 1 conducted the first known ex-vivo extremity perfusion of human lower limbs in 1964. Six lower extremities were perfused using autologous blood. Normal force, duration and of muscle contraction with direct electrical were reported. However, the concept of machine perfusion gained little traction in the 1960s 2 due to the relative simplicity, low cost, and efficacy of limb preservation by cooling. In the last decade, refueling of interest for machine preservation in solid organs, as a mean to increase the number of organs for transplantation, has also inspired research in ex-vivo preservation of limbs.

In 1998, the first hand transplant took place in Lyon, France in a patients with a prior traumatic mid-forearm amputation 3. Later that same year, a 37-year-old male underwent a left hand transplantation in Louisville, Kentucky 4. To date, more than 150 hand transplants have been performed worldwide 5. Machine perfusion literature has shown evidence that ex-vivo perfusion is a suitable method for reducing ischemia time and preserving limbs for an extended period of time by provision of oxygen and nutrients prior to replantation or transplantation 6-11.

As evidence of machine perfusion’s advantages over SCS continues to accumulate, research efforts have focused on translatable animal models, protocol optimization, and system refinement. Nearly 80% of English literature on ex-vivo machine perfusion of extremities has been published in the last 10 years, while 41% has been published in the last 3 years; this review encompasses all 34 published original articles (Tab. I).


Skeletal muscle is the most metabolically active tissue in the limb and thus most susceptible to ischemic injury. Clinical reports of hand transplantation indicate that shorter ischemia results in superior graft function. Herzberg et al. indicted increased ischemia time (9 h) for modest functional recovery following bilateral hand transplant 12. Landin et al. reported perioperative ischemic injury (3 h of cold and 3.5 h of warm ischemia) resulting in fibrotic contracture of forearm muscles following transplantation.13 However, Piza-Katzer et al., in a bilateral hand transplant with shorter ischemia time (2.5-2.8 h), found improved fine motor function and recovery of sensation 14. Ischemic injury is also documented as a risk factor for acute and chronic rejection after limb transplantation due to activation of immune responses 15. In experimental models, ischemia time between 1-3 hours have been shown to result in sufficient musculocutaneous reperfusion injury to increase the risk of acute rejection 16.


Optimal perfusate temperature in ex-vivo perfusion has been a topic of controversy in recent years. While multiple studies have shown the superiority of machine perfusion over SCS, there is no consensus on the optimal perfusate temperature. Hence, a wide range of temperatures (4°C to 39 °C) have been tested during machine perfusion of extremities. Karangwa et al 17. standardized nomenclature for perfusate temperature: hypothermic (0-12 °C) 18-24, midthermic (13-24C°) 21-27, subnormothermic (25-34 °C) 7 15,17,24-32, and normothermic (35-38 °C)18,30,36-41.

Hypothermic perfusion (0-12 °C)

Kruit et al. 7 successfully replanted six porcine limbs after 18 h of hypothermic perfusion with University of Wisconsin solution, achieving a 3-fold elongation of the current maximum SCS time. Although they found increased muscle damage in perfused limbs compared to the contralateral SCS controls, muscle contractility was preserved. Perfused limbs in their study gained 18.6% weight and SCS 11.6%, but this difference was not statistically significant. Additionally, Amin et al. 18 compared hypothermic (10°C), subthermic (28°C), and normothermic (38°C) perfusion and found that porcine forelimbs could be optimally preserved via hypothermic perfusion, supported by the reduction in markers of cellular injury, inflammation, and better tissue integrity. Gok et al. compared the outcomes of limb transplantation after SCS versus hypothermic perfusion with HTK and concluded that hypothermic perfusion preserved limb viability according to amino acid metabolism and energy stores, but failed to restore muscle force 20,42. In a study by Steward et al., the normothermic perfused limb group maintained a near physiologic pH, while the hypothermic group developed acidosis and increased perfusate potassium, which remained throughout perfusion despite the addition of THAM, which was also associated with hyperkalemia. Muller et al. 40 also found that levels of all studied immune response markers were lower in hypothermic perfusion than normothermic.

Midthermic perfusion (13-24 °C)

As a compromise between hypothermic and normothermic perfusion temperatures, midthermic perfusion has been investigated by a few authors 21-27. Burlage et al. 25 perfused rat hindlimbs with three different acellular perfusates (bovine serum albumin (BSA), BSA ± polyethylene glycol (PEG), or HBOC-201 (Hemopure, HbO2, Therapeutics LLC)) at 21ºC for 6 hours. They reported that energy charge ratios were higher in perfused limbs than SCS and that HBOC-21 (Hemopure, HbO2, Therapeutics LLC) perfusions resulted in significantly less edema. Following perfusion, limbs were also transplanted successfully.

Subnormothermic perfusion (25-34 °C)

Temperatures near normothermia are used in an attempt to decrease the amount of peripheral vasoconstriction and shunting 45. Gok et al. 46 perfused rat hindlimbs for 6 hours using STEEN (XVIVO Perfusion, Göteborg, Sweden) solution with swine erythrocytes while maintaining the temperature between 30-35ºC. They found that gastrocnemius energy stores were maintained at the end of perfusion from metabolomic analysis and that the muscles remained viable, as metabolic activity was maintained. There was no difference in the levels of total high-energy phosphates compared to contralateral cold storage controls. On muscle H&E histology they did not find any qualitative evidence of ischemic necrosis or myocyte degeneration 46. Werner et al. perfused human limbs with RBCs for 24 hours between 30-33ºC with no control group. Muscle fiber cross-sectional area and force of contraction did not change significantly, while histological sections of the flexor carpi radialis also revealed no differences in fascicular architecture or shape throughout perfusion (0, 12, 24 h) 8.

Normothermic perfusion (35-38 °C)

Fahradyan et al. compared subnormothermic (33.0 ± 2.6°C) and normothermic (35.4 ± 1.7°C) conditions. Their study demonstrated that ex-vivo normothermic limb perfusion preserves amputated limbs in a near-physiologic state with maintained contractility of the skeletal muscle in response to electrical stimulation, for at least 24 hours 31. Rezaei et al. perfused 10 human upper limbs for 41.6 ± 9.4 h demonstrated the presence of active metabolism in and metabolic derangement toward the end of 24-hour perfusion, correlating with mitochondrial structure, swelling, and elongation. They concluded that EVNLP extends preservation time and enables assessment of human limb quality and allows reconditioning 36,38.


Oxygenation is closely related to perfusion temperature as lower temperatures mean lower metabolic rates and therefore lower energy and oxygen demands. Hypothermic perfusion relies on the decreased baseline metabolism and oxygen demand to oxygenate the perfusate through passive diffusion from the air contained within the reservoir 48,49. Therefore, typical hypothermic perfusion devices do not need to have an oxygenator. However, to support a physiologic metabolic rate, oxygen delivery is necessary, and an oxygenator is typically used in normothermic perfusion. Most normothermic and subnormothermic studies used a membrane oxygenator with 95-100% O218,24,26,31,32,34,36,37,39,40,50, while only one group used a Paragonix machine with 95% O2 (Paragonix Technologies, Inc; Cambridge, MA, USA) 18. Duraes et al. 9 kept the partial pressure of oxygen within physiologic range using a combination of 100% O2, 7% CO2, and 93% N2. Werner et al. 8 had a partial pressure of 300 mmHg by using 40-60% O2 with 5-10% N2/CO2, however.


A large variety of solutions have been experimented in ex-vivo limb perfusion including commercially available preservatives, and cellular-based solutions with modifications and/or additives 50. Red blood cell (RBC)-based perfusate are thought to have superior oxygen-carrying capabilities because of the presence of hemoglobin and possibly reduced edema formation due to the closest resemblance to whole blood 52. However, acellular solutions like University of Wisconsin’s 24,27,53, Perfadex 11,25, modified Perfadex 22, histidine-tryptophan-ketoglutarate 20, or STEEN (XVIVO Perfusion, Göteborg, Sweden) 21,23,34,46 have also been used in hypo, mid, sub, and normothermic conditions.

Given the limitations of using human RBC in organ perfusion, including limited availability, need for cross match, mechanical hemolysis, activation of pro-inflammatory proteins, transmission of infectious diseases, and patient refusal to accept human blood products, Said et al. investigated the feasibility of HBOC-201 (Hemopure, HbO2, Therapeutics LLC), ultra-purified bovine hemoglobin polymerized with glutaraldehyde. Under normothermic conditions, HBOC-201 outcomes were similar to RBCs with preservation of muscle contractility and mitochondrial structure 41. These findings were recently validated by Figueroa et al. 35, who confirmed that the outcomes of normothermic perfusion using HBOC-201 (Hemopure, HbO2, Therapeutics LLC) are similar to RBC-based perfusion.

Kruit et al. performed ex-vivo perfusion of six porcine forelimbs, with University of Wisconsin (UW®, Northbrook, USA) solution at 8-10 ºC, for 18 hours. All limbs were then replanted. They reported superior muscle contraction, however there was evidence of ischemia-reperfusion injury on histology 12 hours after replantation. They also reported 19% weight increase prior to replantation, which is compatible with the reported histology evidencing muscle injury.

Kuckelhaus et al. 11 used oxygenated STEEN (XVIVO Perfusion, Göteborg, Sweden) to perfuse five porcine limbs for 12 h each, (10-12 ºC) after which they found a mean of 44% weight gain. They found less evidence of hypoxic cells on histology of perfused limb muscle samples compared to SCS controls. After 24 hours of perfusion with a modified STEEN (XVIVO Perfusion, Göteborg, Sweden) solution (8 ºC), Krezdorn et al. 23 found that porcine limb weight increased 42%, though aside from edema, histology showed preserved muscle architecture. The perfused limbs were then replanted and followed for one week. The SCS control limbs in this study were preserved for 4 hours and showed evidence of freezing damage with minimally disrupted muscle fibers.

Perfusate additives

Common perfusate additives include antibiotics; steroids, to prevent inflammation and maximize cell membrane permeability; anticoagulants, most commonly heparin; pH buffers, such as bicarbonate; glucose; and insulin 7,22,49. The addition of the and correct dosing of each antibiotic have been derived from clinical indications; their relative efficacies have not yet been investigated. Under normothermic conditions with presence of cefazolin only in the perfusate, we have confirmed growth of pseudomonas during porcine EVLP. Subsequent experiments with the addition of ceftazidime to the perfusate have solved this issue (unpublished data). Valdivia et al. 49 studied rat hindlimb preservation, and following perfusion, added streptomycin and amphotericin B to tissue cultures, but did not report on any bacterial or fungal culture results from during the perfusion.

During subnormothermic perfusion, authors investigated additional interventions, such as transgenic expression of HLA-E/hCD46 32, perfusion in combination with lentiviral vectors 34, and addition of a C1 esterase inhibitor (C1 INH) 29. Puga Yung et al. 32 perfused six porcine limbs with transgenic expression of HLA-E/hCD46 using human blood for 12 hours and found that this partially protected porcine limbs from human NK-mediated xenorejection responses compared to the wild-type porcine limbs. Similarly, Valdivia et al. 50 studied rejection through genetic modification with the aim to decrease expression of genes involved in ischemia-reperfusion injury, inflammation, and rejection (e.g., cytokines, but by adding lentiviral vectors to the Steen (XVIVO Perfusion, Göteborg, Sweden) perfusate of rat hindlimbs for 4 hours. Abdelhafez et al. 28 found that limbs pretreated with C1-INH before perfusion had a significant reduction of complement, bradykinin, fibrin, IgM, and IgG depositions, decreased edema formation, and IRI compared to those without after 12 h of ex-vivo perfusion. During 24-hour normothermic perfusion of rat hindlimbs, Araki et al. 29 added artificial oxygen-carrying hemoglobin vesicles, which resulted in maintenance of aerobic respiration in the gastrocnemius muscle and successful replantation and functional gait at 3 month follow-up.

Gene expression, genetic markers, and metabolomics

Kruit et al. 7,50 have reported that ex-vivo perfused limbs and flaps had similar genetic expression for tumor necrosis factor receptor, regulators of G-protein coupled signaling, hypoxia inducible factor, caspase, and other genes, to their respective cold storage controls, though with small sample sizes. They concluded that machine perfusion preserves limbs in an acceptable state longer than SCS (> 12 hours). However, studies by this group were limited by hypothermic perfusion temperatures and the inevitable downregulation of gene transcription that aligns with intentionally slowed metabolism. There has yet to be a study on genetic expression in normothermic limb perfusion.

Metabolomic analysis entails the identification and quantification of metabolites within a biological sample 52, which allows for pathway analysis downstream of gene expression. Rohde et al. demonstrated preservation of active metabolic activity in normothermic perfusion of 7 human limbs 37. By 24 hours, there was evidence of deranged metabolism (e.g., tryptophan catabolism, decreased taurine levels) 36. Dysregulated metabolism at later time points correlated with evidence of altered mitochondrial structure and swelling.


A common finding among ex-vivo limb perfusion studies is an improved outcome after at least 6 hours of perfusion when compared to cold storage. Current evidence does not allow conclusions to be drawn about the superiority of one method over another, nor resolve the controversy of presented outcomes across the literature. We can, however, recommend the minimum parameters that should be presented as additional data allow more rigorous analysis of outcomes. In future studies of ex-vivo limb perfusion, there should be analysis of basic parameters that are included in most studies to date, such as temperature, hemodynamics, electrolytes, weight, and compared to a contralateral cold storage control. These parameters should be supported by biopsy data (e.g., H&E histology or electron microscopy) and other downstream effects, such as gene expression, metabolomics, protein expression (immunohistochemistry), and muscle contractility. Analyses of these data and correlation with limb outcomes after replantation or transplantation will allow determination of evidence-based viability criteria that can be monitored in real-time, as well as the ability to determine the optimal protocol for limb perfusion. Direct comparisons of slightly different protocols with one factor changed, like temperature or oxygenation, and all others held constant, could provide enough evidence to help us achieve this determination and standardize EVLP protocols in the future.

Conflict of interest statement

The authors declare no conflict of interest.


This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Authors’ contributions

AM, MA, DDB: contributed to study design, data collection, interpretation, preparation of the manuscript, and critical revision of the manuscript; VK, AR: contributed to study design and critical revision of the manuscript; BBG conceived the idea, contributed to study design, interpretation, and critical revision of the manuscript.

Ethical consideration

Not applicable.

Figures and tables

Author year institution Species Control group(s) Ex-vivo perfusion group(s) Perfusate temperature Perfusion duration (hours) Perfusate composition Perfusate oxygenation Perfusion pressure (mmHg), flow rate (mL/min) Perfusate additives Outcome measures
Burlage et al., 2022 Brigham and Women’s Hospital 25 Rat Hindlimb 1. SCS for 6 hours (n = 10) Transplantation controls: Hindlimbs preserved for 6 h (n = 4), 24 h (n = 5) and fresh controls (n = 5) Perfusion groups: Perfusion with BSA (n = 4) Perfusion with BSA+PEG (n = 4) Perfusion with HBOC-201 (n = 4) Midthermic (21°C) 6h BSA, PEG, HBOC-201 95% O , 5% CO 30-40mmHg, NR Insulin, heparin, dexamethasone, hydrocortisone, antibiotics MAP, flow, pH, O saturation electrolytes, PaO , PvO , Glucose, lactate, O consumption, energy change
Transplantation group: After 6 h of perfusion with HBOC-201(n = 13)
Figueroa et al., 2022 Cleveland Clinic 35 Pig Forelimb 1. SCS (n = 12) Perfusion groups: Perfusion with HBOC-201 (n = 6) Perfusion with RBC (n = 6) Normothermic (38°C) 22.5±1.71h HBOC-201 RBC NR HBOC: 78.50 ± 10.75 mmHg, 325.00 ± 25.00 mL/min RBC: 85.70 ± 19.90 mmHg, 444.73 ± 50.60 ml/min Insulin, heparin, vancomycin, albumin, calcium gluconate, methylprednisolone MAP, flow, pH, O saturation electrolytes, PaO , PvO , weight, temperature muscle contractility, compartment pressure, Glucose, lactate, O consumption, CK, myoglobin, metHb, albumin, IR thermography, ICG angiography, muscle histology
Transplantation group: NR
Rezaei et al., 2022 Cleveland Clinic 36 Human Arm 1. SCS (n = 10) Perfusion (n = 10) Normothermic (38°C) 41.6 ± 9.4h pRBC 100% O 90mmHg, 0.41± 0.06 L/min Albumin, heparin, vancomycin, cefazolin, methylprednisolone, dextrose, insulin MAP, flow, electrolytes, pH, O saturation, weight, temperature, compartment pressure Glucose, lactate, O2 consumption, CK, metabolomics: taurine, tryptophan; IR thermography, ICG angiography, muscle contractility, H&E histology
Transplantation group: NR
Valdivia et al., 2022 Institute of Transfusion Medicine and Transplant Engineering 33 Rat Hindlimb 1. SCS for 4h (n = 15) Perfusion groups: Perfusion with lentiviral vectors (n = NR) Perfusion without lentiviral vectors (n = NR) Subnormothermic (33°C) 4h STEEN solution and Sterofundin ISO (1:1 ratio) 95% O , 5% CO 90-120mmHg, 5–9ml/min NaHCO2, cefazolin. After rewarming: protamine sulfate. Transduced limbs: lentiviral vectors NanoLuc / NeonGreen. Pressure, flow, temperature, PaO Perfusate cytokine quantification, myoglobin, lactate and LDH activity, artery, muscle, and skin biopsies for bioluminescence detection, thigh skin for culture and isolation of keratinocytes, fibroblasts, and microvascular endothelial cells
Transplantation group: NR
Amin et al., 2021 University of Manchester 34 Pig Forelimb No control groups Perfusion after 2h of SCS (n = 5) Normothermic (38°C) 6h Bovine pRBC 95% O , 5% CO 70mmHg, 356 ± 131.5 ml/min Albumin, heparin, meropenem, glucose, methylprednisolone, NaHCO2 Perfusate samples were collected at baseline (prior to limb attachment) and from the venous outflow at 3 and 6 h of EVNP Perfusate flow cytometry, cytokine assay, and quantification of circulating cell-free genomic (g) DNA and mitochondrial (mt) DNA via real-time qPCR
Transplantation group: NR
Kruit et al., 2021 Raboud University 7 Pig Forelimb 1. SCS for 4h with RPL (n = 6) Perfusion with RPL (n = 6) Midthermic (16°C) 18h UW solution 95% O , 5% CO NR, 16±1.7ml/min Methylprednisolone Flow, pressure, electrolytes, pH, O saturation, PaO , PvO , weight, temperature, doppler signal Glucose, lactate, ICG angiography, muscle contractility
Replantation group: After 18 h of perfusion
Rohde et al., 2021 Cleveland Clinic 37 Human Arm 1. SCS (n = 7) Perfusion (n = 7) Normothermic (38°C) 41.6 ± 9.4h pRBC 100% O 90mmHg, 0.41± 0.06 L/min Albumin, heparin, vancomycin, cefazolin, methylprednisolone, dextrose, insulin MAP, flow
Transplantation group: NR Metabolomics
Amin, et al., 2020 University of Manchester 18 Pig Forelimb Experiment 1: No control Experiment 1: NMP at 70mmHg (n = 5, other n = 5 for validation) 2.SNMPat 70mmHg (n = 5) SNMP at 50mmHg (n = 5) HMP at 30mmHg (n = 5) Hypothermic (10°C) Subnormothermic (28°C) Normothermic (38°C) 6h pRBC 95% O , 5% CO NR, 102.3 ± 34.8 ml/kg/min Bovine serum Albumin, ringers’ solution, insulin, Nutriflex, heparin, meropenem, glucose, methylprednisolone, NaHCO2 MAP, flow, electrolytes, weight, temperature, pH, HCO , Ca, glucose, hematocrit, Muscle and skin histology, blood gas analysis
Experiment 2: SCS for 8 h + perfusion for 4 h Transplantation group: NR
Experiment 2: 1. NMP at 70mmHg (n = 5)
Transplantation group: NR
Duraes et al., 2017 Cleveland Clinic 9 Pig Forelimb 1. SCS for 12h (n = 5) Perfusion (n = 5) Normothermic (39°C) 12h Heparinized autologous blood 100% O Gradually increased during the first hour to reach a physiologic arterial pressure THAM, albumin, glucose, vancomycin, methylprednisolone, insulin, CO , nitrogen O saturation, surface temperature, muscle temperature, compartment pressure, electrolytes, CBC PaO , PaCO , pH, lactate, glucose, CK, myoglobin, weight, CK, myoglobin, ICG angiography, Muscle, skin, and nerve histology, muscle and nerve functionality (electrical stimulation)
Kueckelhaus et al., 2017 Brigham and Women’s Hospital 11 Pig Forelimb 1. SCS for 4h with RPL (n = 4) 1.Perfusion (n = 3) Hypothermic (10°C) 12h Perfadex NR 30 mmHg, adjusted to maintain BP Dextrose, insulin, methylprednisolone Blood gas analysis Muscle histology and electron microscopy for quantification of IRI and hypoxia markers, lactate, myoglobin
Replantation group: After 12 h of perfusion (n = 3)
Puga Yung et al., 2017 University Hospitals and Medical Faculty of Geneva 32 Pig Forelimb 1. Perfusion of wild-type limbs (n = 6) Perfusion of HLA-E/human CD46 double-transgenic limbs (n = 6) Subnormothermic (32°C) 12h Heparinized whole human blood NR NR, NR Heparin WBC count, PBMC count, muscle histology and immunofluorescence analysis of NK cell infiltration and percentage, NK cytotoxicity through immunoassay
Transplantation group: NR
Werner et al., 2017 University of Michigan 8 Human Arm No control groups Perfusion (n = 5) Subnormothermic (30-33°C) 24h pRBC 40–60% O , 5-10% CO , 30-55% N 93 ± 2 mmHg, 310 ± 20 ml/min Albumin, NaHCO2, THAM, calcium chloride, heparin, dextrose, insulin, methylprednisolone, antibiotics MAP, flow, temperature pH, PaO , PaCO , O saturation, NaHCO2, Na, K, Hemoglobin concentration, skin temperature at palm. lactate, myoglobin, muscle histology, fiber contractility, muscle function, and the maximum isometric contractile force of permeabilized single muscle fiber, neuromuscular electrical stimulation
Transplantation group: NR
Kueckelhaus et al., 2016 Brigham and Women’s Hospital 24 Pig Hindlimb 1. SCS for 12h (n = 5) Perfusion (n = 5) Hypothermic (10°C) 12h Perfadex NR 30 mmHg, adjusted to maintain BP THAM, dextrose, insulin, glucose, methylprednisolone pH, PaCO , base excess, K, Ca, glucose uptake muscle histology and IHC
Ng et al., 2017 Brigham and Women’s Hospital 53 Pig Forelimb No control groups Perfusion (n = 2) Subnormothermic (NR) 3h William’s E medium NR NR, 270-320 ml/min Dexamethasone, insulin, heparin MAP, flow ATP, lactate clearance analysis, perfusate biochemical analysis
Ozer et al., 2016 University of Michigan 31 Pig Forelimb 1. SCS for 6h with RPL (n = 4) Perfusion (n = 4) Subnormothermic (27-32°C) 24h Autologous blood 95% O , 5% CO 60-80 mmHg, NR Glucose, insulin MAP, flow, weight, temperature, O delivery and consumption, acid-base status, electrolytes, glucose, muscle histology and fiber contractility, lactate, blood gas analysis, functional electro stimulation
Replantation group: After 24 h of perfusion (n = 4)
Araki et al., 2015 University of Tokyo 29 Rat Hindlimb 1. SCS for 6h with RPL (n = 4) Perfusion groups: Perfusion with ETK (n = 4) Perfusion with ETK/HbV (n = 4) Subnormothermic (23-27°C) 6h ETK NR NR, 1 NR PaO , PaCO
Replantation groups: After 6 h of perfusion with ETK (n = 4) After 6 h of perfusion with ETK/HbV (n = 4) HbV Muscle histology, lactate, walking track analysis, walking appearance
Ozer et al., 2015 University of Michigan 10 Pig Forelimb 1. SCS for 6h with RPL (n = 3) Perfusion with RPL (n = 4) Subnormothermic (27-32°C) 12h Autologous blood 95% O , 5% CO 60-80 mHg, 10-120ml/min Glucose, insulin Pressure, flow, weight, PaO , PaCO , Electrolytes, pH, glucose, blood gas analysis, lactate, single muscle fiber contractility, neuromuscular electrical stimulation
Replantation group: After 12 h of perfusion (n = 4)
Müller et al., 2013 Bern University Hospital 40 Pig Forelimb Normothermic (36°C) Perfusion groups: 6 h ischemia + 12 h perfusion (n = 7) 2.12 h ischemia + 5 h perfusion (n = 6) Subnormothermic (32°C) 12h Autologous blood 21% O NR, 100-150 ml/min Methylprednisolone, glucose, insulin Muscle, nerve and blood vessel histology, immunofluorescence, serum cytokine and complement immunoassay, blood gas analysis, neuromuscular electrical stimulation
Replantation groups: 12 h perfusion + replantation (n = 11) 6h ischemia + 12h perfusion+ replantation (n = 8)
Constantinescu et al., 2011 Bern University Hospital 43 Pig Forelimb 1. SCS for 12h (n = 8) Perfusion (n = 8) Subnormothermic (32°C) 12h Autologous blood 21% O 33 mmHg, 100-150 ml/min Insulin, glucose Pressure, O saturation, compartment pressure, temperature blood gas analysis, Electrostimulation, nerve stimulation, histology and immunofluorescence, skin and muscle color, capillary refill
Tsuchida et al., 2003 Kumamoto University 47 Rat Hindlimb 1. WS (n = 6) Perfusion groups: 1.Perfusion (n = 6) No perfusion (n = 6) Subnormothermic (25°C) 5h UW NR 100 mmHg, 1-2 ml/min NR Muscle ATP analysis, serum CPK analysis, reperfusion blood flow, and vascular endothelial exocrine function
Replantation groups: After perfusion for 5 h (n = 6) After WS for 5h (n = 6)
Tsuchida et al., 2001 Kumamoto University 48 Rat Hindlimb WS for 4h (n = 4) WS for 5h (n = 4) Perfusion groups: Perfusion with EC at 40 mmHg (n = 4) Perfusion with EC at 100 mmHg (n = 4) Perfusion with UW solution at 40 mmHg (n = 4) Perfusion with UW solution at 100 mmHg (n = 4) Subnormothermic (25°C) 4h EC NR 40/100 mmHg, NR NR ATP
Transplantation group: NR 5h UW
Gordon et al., 1992 California Pacific Medical Center 26 Dog Hindlimb 1. Storage at 13°C for 12h (n = 6) Perfusion (n = 6) Midthermic (13°C) 12h UW solution NR 70mmHg, NR NR Muscle histology and magnetic resonance spectroscopy
Transplantation group: NR
Domingo et al., 1991 Centre de Cirurgia Experimental de la Mutua Sabadellenca 19 Dog Hindlimb NR Perfusion groups: Perfusion (n = 9) Hypothermic (NR) 24h Preserved blood NR > 100 mmHg, 500 ml/min Ringer’s lactate solution, Rheomacrodex, mannitol, NaHCO2, heparin, prednisolone, piperacillin, nitroglycerin Biochemical analysis, muscle histology
Replantation group: Immediate replantation (n = 6) Perfusion for 24 h + replantation(n = 6)
Usui et al., 1985 Sapporo Medical College 27 Dog Hindlimb Storage at room temperature (n = 15) Storage in iced water (n = 10) Perfusion + Replantation groups: Intermittent perfusion with FC at room temperature + RPL (n = 9) Intermittent perfusion with FC in iced water + RPL (n = 6) Continuous perfusion with FC at room temperature+ RPL (n = 6) Continuous perfusion with FC in iced water + RPL (n = 5) Continuous perfusion with HA in iced water + RPL (n = 5) Midthermic (room temperature) 6h FC NR 50 mmHg, NR NR Weight, K, pH
HA Lactate, CK, GOT-m, LDH
Delorme et al., 1964 Massachusetts General Hospital 1 Human Leg No control groups Perfusion with human blood (n = 6) NR (“cooled” and “rewarmed”) NR (“several hours”) Human blood Kay-cross disc oxygenator NR none Muscle temperature, pH, PaCO , PaO , contractility, electrical studies on muscles and isolated nerves
Table I. Summary of studies involving machine preservation of extremities. There was no species limitation, and all studies published on machine perfusion of extremities are included; the earliest was published in 1964 and most recent in 2022.


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Abigail Meyers - Department of Plastic Surgery, Cleveland Clinic, Cleveland, USA

Daniela Duarte-Bateman - Department of Plastic Surgery, Cleveland Clinic, Cleveland, USA

Mazen Almalak - Department of Plastic Surgery, Cleveland Clinic, Cleveland, USA

Varun Kopparthy - Department of Plastic Surgery, Cleveland Clinic, Cleveland, USA

Antonio Rampazzo - Department of Plastic Surgery, Cleveland Clinic, Cleveland, USA

Bahar Bassiri Gharb - Department of Plastic Surgery, Cleveland Clinic, Cleveland, USA

How to Cite
Meyers, A., Duarte-Bateman, D., Almalak, M., Kopparthy, V., Rampazzo, A. and Bassiri Gharb, B. 2023. EX-VIVO LIMB PERFUSION. European Journal of Transplantation. 2, (Mar. 2023), 143–154. DOI:
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