Ex vivo normothermic preservation of amputated limbs with a hemoglobin-based oxygen carrier perfusate

Figueroa, Brian; Said, Sayf; Rezaei, Majid; Ordenana, Carlos; Dubé, Gregory P.; Papay, Francis; Brunengraber, Henri; Dasarathy, Srinivasan; Rampazzo, Antonio; Gharb, Bahar Bassiri

doi:10.1097/ta.0000000000003395

Cited by 9 publications

(13 citation statements)

References 59 publications

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“…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.…”

Section: Methodsmentioning

confidence: 99%

Ex Vivo Normothermic Perfusion of Human Upper Limbs

et al. 2022

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Original Clinical Science-General Background. Ischemia-reperfusion injury remains a primary concern in upper extremity transplantation. Ex vivo normothermic perfusion (EVNP) enables near-physiological organ preservation, avoiding the deleterious effects of hypoxia and cooling. We investigated the effectiveness of human limb EVNP compared with static cold storage (SCS). Methods. Twenty human upper extremities were procured. Ten were perfused at 38 °C with an oxygenated red blood cell-based solution, and contralateral limbs served as SCS control (4 °C). EVNP was terminated with systolic arterial pressure ≥115 mm Hg, compartment fullness, or a 20% decline in oxygen saturation. Weight, contractility, compartment pressure, tissue oxygen saturation, and uptake rates were assessed. Perfusate fluid dynamics, gases, electrolytes, and metabolites were measured. Myocyte injury scores and liquid chromatography-mass spectrometry analysis were performed. Results. EVNP duration was 41.6 ± 9.4 h. Vascular resistance averaged 173.0 ± 29.4 mm Hg × min/L. Weight change and compartment pressures were 0.4 ± 12.2% (P = 0.21) and 21.7 ± 15.58 mm Hg (P = 0.003), respectively. Arterial and venous carbon dioxide partial pressure, oxygen saturation, and pH were 509.5 ± 91.4 mm Hg, 15.7 ± 30.2 mm Hg, 87.4 ± 11.4%, and 7.3 ± 0.2, respectively. Oxygen uptake rates averaged 5.7 ± 2.8 mL/min/g. Lactate reached 20 mmol/L after 15 (interquartile range = 6) h. Limb contractility was preserved for 30.5 (interquartile range = 15.8) h (P < 0.001) and negatively correlated with perfusate potassium (ρ = -0.7, P < 0.001). Endpoint myocyte injury scores were 28.9 ± 11.5% (EVNP) and 90.2 ± 11.8% (SCS) (P < 0.001). A significant increase in taurine (P = 0.002) and decrease in tryptophan (P = 0.002) were detected. Infrared thermography and indocyanine green angiography confirmed the presence of peripheral perfusion. Conclusions. EVNP can overcome the limitations of cold preservation by extending preservation times, enabling limb quality assessment, and allowing limb reconditioning before transplantation.

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Section: Methodsmentioning

confidence: 99%

Ex Vivo Normothermic Perfusion of Human Upper Limbs

et al. 2022

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“…EVNLP was terminated at 24 h or when maximal arterial pressure was ≥115 mm Hg, indicating increasing resistance within the limb, fullness of muscle compartments (confirmed by measurement of a compartment pressure > 30 mm Hg), or 20% reduction of tissue oxygen saturation compared with baseline. 11 , 13 , 19 , 20 …”

Section: Methodsmentioning

confidence: 99%

“…Although extended preservation of the extremities can be achieved with EVNLP, 13 the machine perfusion systems lack metabolic, endocrine, neurologic, and excretory homeostatic functions. Gradual loss of tissue viability occurs, resulting in increased vascular resistance and compartment pressure that necessitates perfusion termination.…”

Section: Introductionmentioning

confidence: 99%

“… 18 There is no consensus on the threshold of parameters beyond which the limb is no longer considered suitable for transplantation or replantation. 13 , 18 , 19 , 20 , 21 Real‐time, accurate monitoring of key indicators is vital for the assessment of limb condition during EVNLP. 18 There is no consensus on the threshold of parameters beyond which the limb is no longer considered suitable for transplantation or replantation.…”

Section: Introductionmentioning

confidence: 99%

“… 18 There is no consensus on the threshold of parameters beyond which the limb is no longer considered suitable for transplantation or replantation. 13 , 18 , 19 , 20 , 21 Weight gain can be measured as a proxy for limb edema, which is a key component in the pathophysiology of compartment syndrome due to increased volume within a confined fascial space 10 , 14 , 19 , 22 , 23 , 24 Limb weight increases greater than 40% have been reported within 24 h of ex vivo limb perfusion. 25 We hypothesized that limb weight gain could be used as an early marker of skeletal muscle injury and viability during EVNLP.…”

Section: Introductionmentioning

confidence: 99%

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Weight gain is an early indicator of injury in ex vivo normothermic limb perfusion (EVNLP)

et al. 2022

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Purpose There are no established criteria for discontinuing ex vivo normothermic limb perfusion (EVNLP) before irreversible damage occurs. This study evaluates weight gain as an indicator of injury during EVNLP. Methods Sixteen Yorkshire pig forelimbs were procured and preserved using EVNLP with a hemoglobin‐based oxygen carrier (HBOC‐201) or static cold storage. EVNLP continued until termination criteria were met: arterial pressure ≥ 115 mm Hg, compartment pressure > 30 mm Hg, or 20% reduction of oxygen saturation. Limb weight, contractility, hemodynamics, perfusate electrolytes, metabolites and gases were recorded. Muscles were biopsied 6‐h, and muscle injury scores (MIS) calculated. Forearm compartment pressures and indocyanine green (ICG) angiography were recorded at endpoint. Outcomes were compared at 2%, 5%, 10%, and 20% limb weight gain. Results EVNLP lasted 20 ± 3 h. Weight gain was observed after 13 ± 5 h (2%), 15 ± 6 h (5%), 16 ± 6 h (10%), and 19 ± 4 h (20%). Weight correlated positively with MIS (ρ = 0.92, p < 0.0001), potassium (ρ = −1.00, p < 0.0001), pressure (ρ = 0.78, p < 0.0001), and negatively with contractility (ρ = −0.96, p = 0.011). At 5% weight gain, MIS (p < 0.0001), potassium (p = 0.03), and lactate (p < 0.0001) were significantly higher than baseline. Median muscle contractility was 5 [3–5] at 2% weight gain, 4 [1–5] at 5%, 3 [0–4] and 2 [0–2] at 10% and 20%, respectively. At 20% weight gain, contractility was significantly lower than baseline (p = 0.003). Percent weight gain correlated negatively with endpoint ICG hoof fluorescence (r = −0.712, p = 0.047). Conclusions Weight gain correlated with microscopic muscle injury and was the earliest evidence of limb dysfunction. Weight gain may serve as a criterion for discontinuation of EVNLP.

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Orthotopic forelimb transplantation in a Yucatan minipig model: Anatomic and in vivo study

Figueroa,

Ordenana,

Rezaei

et al. 2024

Microsurgery

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IntroductionAbove elbow transplants represent 19% of the upper extremity transplants. Previous large‐animal models have been too distal or heterotopic, did not use immunosuppression and had short survival. We hypothesize that an orthotopic forelimb transplant model, under standard immunosuppression, is feasible and can be used to address questions on peri‐transplant ischemia reperfusion injury, and post‐transplantation vascular, immunologic, infectious, and functional outcomes.Materials and methodsFour forelimbs were used for anatomical studies. Four mock transplants were performed to establish technique/level of muscle/tendon repairs. Four donor and four recipient female Yucatan minipigs were utilized for in‐vivo transplants (endpoint 90‐days). Forelimbs were amputated at the midarm and preserved through ex vivo normothermic perfusion (EVNP) utilizing an RBC‐based perfusate. Hourly perfusate fluid‐dynamics, gases, electrolytes were recorded. Contractility during EVNLP was graded hourly using the Medical Research Council scale. EVNP termination criteria included systolic arterial pressure ≥115 mmHg, compartment pressure ≥30 mmHg (at EVNP endpoint), oxygen saturation reduction of 20%, and weight change ≥2%. Indocyanine green (ICG) angiography was performed after revascularization. Limb rejection was evaluated clinically (rash, edema, temperature), and histologically (BANFF classification) collecting per cause and protocol biopsies (POD 1, 7, 30, 60 and endpoint). Systemic infections were assessed by blood culture and tissue histology. CT scan was used to confirm bone bridging at endpoint.ResultsAnimals 2, 4 reached endpoint with grade 0‐I rejection. Limbs 1, 3 presented grade III rejection on days 6, 61. CsA troughs averaged 461 ± 189 ng/mL. EVNLP averaged 4.3 ± 0.52 h. Perfusate lactate, PO2, and pH were 5.6 ± 0.9 mmol/L, 557 ± 72 mmHg and 7.5 ± 0.1, respectively. Muscle contractions were 4 [1] during EVNLP. Transplants 2, 3, 4 showed bone bridging on CT.ConclusionWe present preliminary evidence supporting the feasibility of an orthotopic, mid‐humeral forelimb allotransplantation model under standard immunosuppression regimen. Further research should validate the immunological, infectious, and functional outcomes of this model.

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Ex vivo normothermic preservation of amputated limbs with a hemoglobin-based oxygen carrier perfusate

Cited by 9 publications

References 59 publications

Ex Vivo Normothermic Perfusion of Human Upper Limbs

Ex Vivo Normothermic Perfusion of Human Upper Limbs

Weight gain is an early indicator of injury in ex vivo normothermic limb perfusion (EVNLP)

Orthotopic forelimb transplantation in a Yucatan minipig model: Anatomic and in vivo study

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