Normothermic Preservation Research Articles

Sneaking Islets Into the Liver During Ex-vivo Normothermic Preservation - A Cunning Strategy to Avoid Ischemia and IBMIR.

Sneaking Islets Into the Liver During Ex-vivo Normothermic Preservation - A Cunning Strategy to Avoid Ischemia and IBMIR.

A-154 Non-standard body fluid validation of d-dimer analysis of organox liver perfusate to support clinical evaluations of donor livers prior to transplantation

Abstract Background Non-standard body fluid analysis is considered a challenge to laboratories since very few have been approved by the FDA for routine testing. As medical procedures evolve, the laboratory must balance the requirement to uphold regulatory standards while also supporting procedures that improve patient care. The LUMC Liver Transplant Team recently implemented the use of the FDA-approved OrganOx Metra normothermic machine perfusion instrument for maintaining donor livers under physiologic conditions prior to transplantation. The team mitigates risk for biliary complications associated with fibrin microthrombi by perfusing a combination of alteplase rTPA with fresh frozen plasma and monitoring the prevalence of fibrin through the release of D-dimers over the course of ex situ normothermic preservation. Liver perfusate is not an FDA-approved specimen type for the analysis of D-dimer and requires laboratory verification as a compatible specimen for analysis. Methods The D-dimer HS 500 assay was performed on an ACL TOP 750 analyzer. Carry-over studies evaluated activated partial thromboplastin time (aPTT) of a normal control (citrated plasma) specimen initially, and repeatedly in-between sets of D-dimer measurements of heparinized perfusate specimen. Recovery studies were performed by mixing liver perfusate with citrated plasma spiked with D-dimer in known ratios, allowing for the calculation of an expected value of D-dimer. Results Carry-over studies measuring aPTTs of normal plasma specimens provided %CVs of 1 and 3%. Recovery of D-dimer in seven sets of independent add-mixture series (three mixed specimen per set) was on average 105% with a range from 95 to 113% recovery. Conclusions Concern for cross-contamination of perfusate specimen containing heparin anticoagulant on analyzers used to evaluate routine coagulation testing was mitigated by the results of the carry-over study, which provided %CV within the manufacturer limit for intraday precision. Recovery studies evaluating matrix effect for the analysis of D-dimer on liver perfusate generated under the LUMC liver transplant protocol indicated that the matrix of the perfusate did not impact D-dimer analysis, as compared to the FDA-approved specimen type.

The impact of normothermic and hypothermic preservation methods on kidney lipidome-comparative study using chemical biopsy with microextraction probes.

Normothermic ex vivo kidney perfusion (NEVKP) is designed to replicate physiological conditions to improve graft outcomes. A comparison of the impact of hypothermic and normothermic preservation techniques on graft quality was performed by lipidomic profiling using solid-phase microextraction (SPME) chemical biopsy as a minimally invasive sampling approach. Direct kidney sampling was conducted using SPME probes coated with a mixed-mode extraction phase in a porcine autotransplantation model of the renal donor after cardiac death, comparing three preservation methods: static cold storage (SCS), NEVKP, and hypothermic machine perfusion (HMP). The lipidomic analysis was done using ultra-high-performance liquid chromatography coupled with a Q-Exactive Focus Orbitrap mass spectrometer. Chemometric analysis showed that the NEVLP group was separated from SCS and HMP groups. Further in-depth analyses indicated significantly (p < 0.05, VIP > 1) higher levels of acylcarnitines, phosphocholines, ether-linked and longer-chain phosphoethanolamines, triacylglycerols and most lysophosphocholines and lysophosphoethanolamines in the hypothermic preservation group. The results showed that the preservation temperature has a more significant impact on the lipidomic profile of the kidney than the preservation method's mechanical characteristics. Higher levels of lipids detected in the hypothermic preservation group may be related to ischemia-reperfusion injury, mitochondrial dysfunction, pro-inflammatory effect, and oxidative stress. Obtained results suggest the NEVKP method's beneficial effect on graft function and confirm that SPME chemical biopsy enables low-invasive and repeated sampling of the same tissue, allowing tracking alterations in the graft throughout the entire transplantation procedure.

Ex vivo normothermic preservation of a kidney graft from uncontrolled donation after circulatory death over 73 hours.

The transplant community is focused on prolonging the ex vivo preservation time of kidney grafts to allow for long-distance kidney graft transportation, assess the viability of marginal grafts, and optimize a platform for the translation of innovative therapeutics to clinical practice, especially those focused on cell and vector delivery to organ conditioning and reprogramming. We describe the first case of feasible preservation of a kidney from a donor after uncontrolled circulatory death over a 73-h period using normothermic perfusion and analyze hemodynamic, biochemical, histological, and transcriptomic parameters for inflammation and kidney injury. The mean pressure and flow values were 71.24 ± 9.62mmHg and 99.65 ± 18.54mL/min, respectively. The temperature range was 36.7°C-37.2°C. The renal resistance index was 0.75 ± 0.15mmHg/mL/min. The mean pH was 7.29 ± 0.15. The lactate concentration peak increased until 213mg/dL at 6h, reaching normal values after 34h of perfusion (8.92mg/dL). The total urine output at the end of perfusion was 1.185mL. Histological analysis revealed no significant increase in acute tubular necrosis (ATN) severity as perfusion progressed. The expression of KIM-1, VEGF, and TGFβ decreased after 6-18h of perfusion until 60h in which the expression of these genes increased again together with the expression of β-catenin, Ki67, and TIMP1. We show that normothermic perfusion can maintain a kidney graft viable ex vivo for 3days, thus allowing a rapid translation of pre-clinical therapeutics to clinical practice.

Bile proteome reveals biliary regeneration during normothermic preservation of human donor livers

Normothermic machine perfusion (NMP) after static cold storage is increasingly used for preservation and assessment of human donor livers prior to transplantation. Biliary viability assessment during NMP reduces the risk of post-transplant biliary complications. However, understanding of molecular changes in the biliary system during NMP remains incomplete. We performed an in-depth, unbiased proteomics analysis of bile collected during sequential hypothermic machine perfusion, rewarming and NMP of 55 human donor livers. Longitudinal analysis during NMP reveals proteins reflective of cellular damage at early stages, followed by upregulation of secretory and immune response processes. Livers with bile chemistry acceptable for transplantation reveal protein patterns implicated in regenerative processes, including cellular proliferation, compared to livers with inadequate bile chemistry. These findings are reinforced by detection of regenerative gene transcripts in liver tissue before machine perfusion. Our comprehensive bile proteomics and liver transcriptomics data sets provide the potential to further evaluate molecular mechanisms during NMP and refine viability assessment criteria.

Ischemia-Induced Metabolic Patterns Associate With Kidney Function During Normothermic Kidney Perfusion, a Preclinical Study.

To investigate if ischemia alters donor kidney metabolism and whether these changes associate with organ function. An unmet need in kidney transplantation is the ability to predict post-transplant organ function before transplantation. Key to such viability testing is a profound understanding of the organ's complex biochemistry and how ischemia, inevitable during the transplantation process, influences this. First, metabolic changes in glucose, lactate and 20 amino acids induced by no, 1h of warm, or 22h of cold ischemia were investigated during 4h perfusion of pig kidneys with autologous whole blood (n=6/group), simulating the ischemia-reperfusion phase of transplantation. Next, we confirmed similar metabolic changes during normothermic preservation of pig (n=3/group; n=4 for cold ischemia) and discarded human kidneys (n=6) perfused with a red-blood cell based perfusate. At 2h of perfusion with autologous whole blood, abundances of 17/20 amino acids were significantly different between groups, reflecting the type of ischemia. Amino acid changes at 15 min and 2h of perfusion correlated with future kidney function during perfusion. Similar metabolic patterns were observed during perfusion preservation of pig and discarded human donor kidneys, suggesting an opportunity to assess kidney viability before transplantation. Perfusate metabolite changes during normothermic kidney perfusion represent a unique non-invasive opportunity to assess graft viability. These findings now need validation in transplant studies.

123. Comparison of Ex Vivo Normothermic Preservation of Vascularized Composite Allografts in a Porcine Model: 12 vs 24 Hour Perfusion?

123. Comparison of Ex Vivo Normothermic Preservation of Vascularized Composite Allografts in a Porcine Model: 12 vs 24 Hour Perfusion?

PC33. Comparison of Ex Vivo Normothermic Preservation of Vascularized Composite Allografts in A Porcine Model: 12 vs 24 Hour Perfusion

PURPOSE: Ex vivo normothermic perfusion (EVNP) extends the ischemia time for soft tissue flaps. We hypothesized that extended EVNP of flaps for 24-hour was feasible and outcomes were equivalent to 12-hour preservation. METHODS: Eighteen superior epigastric artery flaps were procured from Yorkshire pigs. Flaps were preserved by EVNP for 12 (PFP12; n=5) and 24 hours (PFP24; n=4). Contralateral flaps were preserved at 4°C as static cold storage controls (SCS;(n=9). Outcome measures, including perfusate dynamics, flap temperature, gases, metabolites, electrolytes, and flap weight, were monitored and evaluated using Mann-Whitney U test. Indocyanine Green (ICG) angiography was utilized to analyze skin perfusion before division of the pedicle and at perfusion endpoint. RESULTS: Warm ischemia time for PFP12 and PFP24 was 15.7±5.2min and 13.50±7.05min (p=0.42), respectively. Flap temperatures were 29.0±1.9°C (PFP12) and 28.15± 2.06 (PFP24)(p= 0.84). Average arterial pressures were 45±12 mmHg (PFP12) and 35.75±4.11 mmHg (PFP24)(p=0.19). Arterial PaO2 was 453±77mmHg(PFP12) and 580±35.52(PFP24)(p=0.56). Perfusate pH was not significantly different between groups (7.30±0.04 and 7.39±0.06;p=0.41). Lactate was 5.1±0.8 mmol/L(PFP12) and 7.37±0.52(PFP24)(p=0.99). Creatine kinase increased (TP0.5 864±468 U/L vs. TP12 6340±1764 U/L (PFP12)(p=0.001); TP0.5 1593±5.41 U/L vs. TP24 5467±2966 U/L (PFP24)(p=0.001). Potassium and sodium were not significantly different (PFP12)3.9±0.22mEq/L; (PFP24)3.75±0.31(p=0.13) and (PFP12)159±3mEq/L;(PFP24)155±0.82(p=0.71) between groups. Flap weight did not change between groups (TP0 0.222±0.041kg vs. TP12 0.223±0.044kg(p=0.48);[PFP24]TP0.5 0.18±0.06 vs. TP24 0.18±0.06; p=0.99). ICG angiography fluorescence scores were significantly higher in the PFP24 ([PFP12]21.23±13.26 vs. (PFP24)34.95±2.90; p=0.03). CONCLUSION: EVNP can preserve soft tissue flaps for 24 hours. Outcome at 24 hours were not significantly different from 12 hours.

State-of-the-Art and Future Directions in Organ Regeneration with Mesenchymal Stem Cells and Derived Products during Dynamic Liver Preservation.

Transplantation is currently the treatment of choice for end-stage liver diseases but is burdened by the shortage of donor organs. Livers from so-called extended-criteria donors represent a valid option to overcome organ shortage, but they are at risk for severe post-operative complications, especially when preserved with conventional static cold storage. Machine perfusion technology reduces ischemia-reperfusion injury and allows viability assessment of these organs, limiting their discard rate and improving short- and long-term outcomes after transplantation. Moreover, by keeping the graft metabolically active, the normothermic preservation technique guarantees a unique platform to administer regenerative therapies ex vivo. With their anti-inflammatory and immunomodulatory properties, mesenchymal stem cells are among the most promising sources of therapies for acute and chronic liver failure, but their routine clinical application is limited by several biosafety concerns. It is emerging that dynamic preservation and stem cell therapy may supplement each other if combined, as machine perfusion can be used to deliver stem cells to highly injured grafts, avoiding potential systemic side effects. The aim of this narrative review is to provide a comprehensive overview on liver preservation techniques and mesenchymal stem cell-based therapies, focusing on their application in liver graft reconditioning.

313.5: Meeting Packed Red Blood Cell Requirement for the Transmedics Organ Care System (OCS) Liver Transplantation Trial (PROTECT Trial) At Henry Ford Hospital

Introduction: In January 2016, HFHS Transplant Institute began the PROSPECT trial, an interventional trial studying normothermic preservation of the donated liver before transplant using the Liver Organ Care System (OCSTM). The Henry Ford Hospital Transfusion Service (TS) provided the required packed red blood cells (PRBC). Background: The TransMedics (OCSTM) liver trial was an international, randomized trial studying the intervention of a portable liver system to preserve donor livers for transplant. The system utilizes an oxygenated blood-based perfusate mixed with nutrient solution in normothermic perfusion for organ preservation. The TS provided PRBC needed for the study, final PRBC used dictated by the condition and size of the retrieved liver. TS study goal was to secure proper transfusion needs for liver perfusion while also adhering to blood inventory management. Methods: Organ procurement candidates for the PROTECT trial were selected based on pre-determined conditions and consented according to that protocol. Once the patient was consented and the liver transplantation was scheduled, the PROTECT trial team notified the TS,and 5 units of group O Positive, leukoreduced PRBC less than 10 days old were selected, and placed inside a temperature-controlled packaging container along with a remote temperature monitoring device. The container was then transported with the procurement team to the harvesting location. Once the insulated container was returned to the TS, the temperature-monitoring device information was uploaded to verify that the returned PRBC have been stored within their proper temperature limits. Results: The TS support for the PROTECT trial at HFHS occurred from January 2016 until September 2021. The table below shows the activity related to this trial. - Issued Utilized Returned Wasted PRBC units 240 132 104 4 In summary, 29/48 study events used 4 units; 3/48 study events used 5 units; on 15/48 events the organ harvesting was cancelled; all units returned had an acceptable temperature range and returned to the available inventory. For one study event, 4 PRBC were spiked in preparation for use in the perfusate prior to the event being cancelled. These PRBC were discarded and deemed wasted. Conclusions: During the PROTECT trial no PRBCs were wasted due to inappropriate storage conditions. The joint use of a temperature-controlled container and remote temperature monitoring device allowed for the appropriate return of unused PRBCs to the TS blood inventory, thus avoiding any wastage. Only 3/48 events used 5 units, the\us issuing 4 PRBC to meet the OCS™ support may suffice, further decreasing the risk for wastage of any PRBC units. After receiving FDA in September 2021, alternatives to maintain the required PRBC temperature should be addressed to minimize wastage at this time of limited blood availability for the support of the TransMedics Organ Care System (OCSTM) for liver organ perfusion.

Normothermic liver machine perfusion as a dynamic platform for regenerative purposes: What does the future have in store for us?

Liver transplantation has become an immense success; nevertheless, far more recipients are registered on waiting lists than there are available donor livers for transplantation. High-risk, extended criteria donor livers are increasingly used to reduce the discrepancy between organ demand and supply. Especially for high-risk livers, dynamic preservation using machine perfusion can decrease post-transplantation complications and may increase donor liver utilisation by improving graft quality and enabling viability testing before transplantation. To further increase the availability of donor livers suitable for transplantation, new strategies are required that make it possible to use organs that are initially too damaged to be transplanted. With the current progress in experimental liver transplantation research, (long-term) normothermic machine perfusion may be used in the future as a dynamic platform for regenerative medicine approaches, enabling repair and regeneration of injured donor livers. Currently explored therapeutics such as defatting cocktails, RNA interference, senolytics, and stem cell therapy may assist in the repair and/or regeneration of injured livers before transplantation. This review will provide a forecast of the future utility of normothermic machine perfusion in decreasing the imbalance between donor liver demand and supply by enabling the repair and regeneration of damaged donor livers.

337.2: Comparison of Hypothermic Static and Normothermic Ex-situ Donor Heart Preservation in Heterotopic Heart Transplantation With the Murine Model

Introduction and Background: Hypothermic static and Normothermic ex-situ preservation methods are the most widely used preservation technique worldwide. While cold static storage is associated with subsequent ischemic injury, Normothermic perfusion preservation has the potential to elongate preservation time with less ischemia. The current study compares the hypothermic and normothermic preservation methods in terms of graft performance, morphologic changes, and acute immune response in an experimental model. Materials and Method: Twenty-five rats underwent heterotopic abdominal heart transplantation after two hours of donor heart hypothermic storage (HT group; n=10) or normothermic ex-situ preservation (NT group; n=15). Blood samples were obtained just before and after 4-hours of implantation to analyze surface markers of immune cells (CD4, CD8, CD161, CD45R). ECG and Echocardiography were performed before harvesting and after implantation. Hearts were extracted after four hours of implantation for Hematoxylin-Eosin (HE) and TUNEL-staining, and recipient rats were euthanized. Results: 19 (76%) rats successfully survived after implantation (HT group: 100%, NT group: 60%). The mean ischemic time of the donor heart was 162.7±8.34 minutes in the HT group and 45±3.7 minutes in the NT group. The NT group showed no significant change in the heart rate before and after implantation but significantly decreased in the first hour of implantation in the HT group (259.5±24.3rpm) and normalized at 3 hours (338.3±34.1rpm). Ejection fraction and fractional shortening significantly decreased after implantation in both groups but were less significant in the NT group (P=0.001). Granulocyte was less significantly increased in the NT group compared to the HT group after the experiment (p=0.037). Although the gross structure was well preserved in both groups in HE-staining, the number of TUNEL positive cells was significantly higher in the HT group. Conclusions: Our findings suggest that normothermic ex-situ preservation is associated with well-preserved donor hearts but similar recipient immune response in comparison with hypothermic static preservation.

Transplantation of a human liver following 3 days of ex situ normothermic preservation.

Current organ preservation methods provide a narrow window (usually <12 hours) to assess, transport and implant donor grafts for human transplantation. Here we report the transplantation of a human liver discarded by all centers, which could be preserved for several days using ex situ normothermic machine perfusion. The transplanted liver exhibited normal function, with minimal reperfusion injury and the need for only a minimal immunosuppressive regimen. The patient rapidly recovered a normal quality of life without any signs of liver damage, such as rejection or injury to the bile ducts, according to a 1-year follow up. This inaugural clinical success opens new horizons in clinical research and promises an extended time window of up to 10 days for assessment of viability of donor organs as well as converting an urgent and highly demanding surgery into an elective procedure.

Molecular and proteomic signatures associated with preservation related graft injury: insight from human liver normothermic achine perfusion (NMP)

Fungai Dengu1; Sadr Shaheed1; Letizia Lo Faro1; Adam Thorne1; Honglei Huang1; Peter Friend1,Rutger Ploeg1.&#x0D; 1. Oxford Organ Perfusion Lab, Nuffield Department of Surgical Sciences and Oxford BiomedicalResearch Centre, University of Oxford, Oxford, UK&#x0D; BackgroundContinuous liver NMP is a novel technology associated with safe extension of organpreservation time, increased organ utilisation and reduced early graft injury1. Increasingly, it isutilised as a ‘back to base’ application with cold storage for organ transport and NMP initiatedat the implanting centre prior to transplantation2. We aimed to evaluate the impact ofadditional cold ischaemia time (CIT) on the proteomic and molecular signature of NMP livers.&#x0D; MethodsLiver tissue samples (N= 57) from a prospective clinical trial of ‘back to base’ NMP wereanalysed. Collection occurred at the end of cold storage (LT1), end of NMP/total preservation(LT2) and after organ reperfusion (LT3). Unbiased, label-free-quantitative (LFQ) proteomicanalysis was conducted using liquid chromatography with tandem mass spectrometry andtrapped ion mobility spectrometry (TIMS) to time-of-flight (TOF) mass analysis (LC-MS/MS TIMSTOF).Differential expression and Gene Ontology/Pathway analysis were performed.&#x0D; ResultsLT2 samples with prolonged CIT (&gt;6hr) prior to NMP had significant differential expression ofproteins associated with liver-specific oxidative stress, cellular haemostasis and removal ofdamaged or misfolded proteins (e.g. CYP3A5, PSMB1). LT3 samples, similarly, had reducedproteins involved in autophagy and cell-cycle regulation (e.g. STBD1, CD2AP, GADD45GIP1,) andincreased expression of proteins involved in neutrophil chemotaxis, adhesion and aggregation(e.g. S100A9).&#x0D; DiscussionThe molecular signature of grafts at LT2 and LT3 varies depending on the length of CIT prior toNMP. Further exploration of the molecular signatures associated with preservation related graftinjury is required to determine how best to apply this novel technology clinically.&#x0D; &#x0D;

Molecular and proteomic signatures associated with preservation related graft injury: insight from human liver normothermic machine perfusion (NMP)

Fungai Dengu1; Sadr Shaheed1; Letizia Lo Faro1; Adam Thorne1; Honglei Huang1; Peter Friend1,Rutger Ploeg1.&#x0D; 1. Oxford Organ Perfusion Lab, Nuffield Department of Surgical Sciences and Oxford BiomedicalResearch Centre, University of Oxford, Oxford, UK&#x0D; &#x0D; &#x0D; BackgroundContinuous liver NMP is a novel technology associated with safe extension of organ preservation time, increased organ utilisation and reduced early graft injury1. Increasingly, it is utilised as a ‘back to base’ application with cold storage for organ transport and NMP initiated at the implanting centre prior to transplantation2. We aimed to evaluate the impact of additional cold ischaemia time (CIT) on the proteomic and molecular signature of NMP livers.&#x0D; Methods Liver tissue samples (N= 57) from a prospective clinical trial of ‘back to base’ NMP were analysed. Collection occurred at the end of cold storage (LT1), end of NMP/total preservation (LT2) and after organ reperfusion (LT3). Unbiased, label-free-quantitative (LFQ) proteomic analysis was conducted using liquid chromatography with tandem mass spectrometry and trapped ion mobility spectrometry (TIMS) to time-of-flight (TOF) mass analysis (LC-MS/MS TIMS-TOF). Differential expression and Gene Ontology/Pathway analysis were performed.&#x0D; Results LT2 samples with prolonged CIT (&gt;6hr) prior to NMP had significant differential expression of proteins associated with liver-specific oxidative stress, cellular haemostasis and removal of damaged or misfolded proteins (e.g. CYP3A5, PSMB1). LT3 samples, similarly, had reduced proteins involved in autophagy and cell-cycle regulation (e.g. STBD1, CD2AP, GADD45GIP1,) and increased expression of proteins involved in neutrophil chemotaxis, adhesion and aggregation (e.g. S100A9).&#x0D; Discussion The molecular signature of grafts at LT2 and LT3 varies depending on the length of CIT prior to NMP. Further exploration of the molecular signatures associated with preservation related graft injury is required to determine how best to apply this novel technology clinically.

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

Ex vivo normothermic limb perfusion (EVNLP) preserves amputated limbs under near-physiologic conditions. Perfusates containing red blood cells (RBCs) have shown to improve outcomes during ex vivo normothermic organ perfusion, when compared with acellular perfusates. To avoid limitations associated with the use of blood-based products, we evaluated the feasibility of EVNLP using a polymerized hemoglobin-based oxygen carrier-201 (HBOC-201). Twenty-four porcine forelimbs were procured from Yorkshire pigs. Six forelimbs underwent EVNLP with an HBOC-201-based perfusate, six with an RBC-based perfusate, and 12 served as static cold storage (SCS) controls. Ex vivo normothermic limb perfusion was terminated in the presence of systolic arterial pressure of 115 mm Hg or greater, fullness of compartments, or drop of tissue oxygen saturation by 20%. Limb contractility, weight change, compartment pressure, tissue oxygen saturation, oxygen uptake rates (OURs) were assessed. Perfusate fluid-dynamics, gases, electrolytes, metabolites, methemoglobin, creatine kinase, and myoglobin concentration were measured. Uniformity of skin perfusion was assessed with indocyanine green angiography and infrared thermography. Warm ischemia time before EVNLP was 35.50 ± 8.62 minutes (HBOC-201), 30.17 ± 8.03 minutes (RBC) and 37.82 ± 10.45 (SCS) (p = 0.09). Ex vivo normothermic limb perfusion duration was 22.5 ± 1.7 hours (HBOC-201) and 28.2 ± 7.3 hours (RBC) (p = 0.04). Vascular flow (325 ± 25 mL·min-1 vs. 444.7 ± 50.6 mL·min-1; p = 0.39), OUR (2.0 ± 1.45 mL O2·min-1·g-1 vs. 1.3 ± 0.92 mL O2·min-1·g-1 of tissue; p = 0.80), lactate (14.66 ± 4.26 mmol·L-1 vs. 13.11 ± 6.68 mmol·L-1; p = 0.32), perfusate pH (7.53 ± 0.25 HBOC-201; 7.50 ± 0.23 RBC; p = 0.82), flexor (28.3 ± 22.0 vs. 27.5 ± 10.6; p = 0.99), and extensor (31.5 ± 22.9 vs. 28.8 ± 14.5; p = 0.82) compartment pressures, and weight changes (23.1 ± 3.0% vs. 13.2 ± 22.7; p = 0.07) were not significantly different between HBOC-201 and RBC groups, respectively. In HBOC-201 perfused limbs, methemoglobin levels increased, reaching 47.8 ± 12.1% at endpoint. Methemoglobin saturation did not affect OUR (ρ = -0.15, r2 = 0.022; p = 0.45). A significantly greater number of necrotic myocytes was found in the SCS group at endpoint (SCS, 127 ± 17 cells; HBOC-201, 72 ± 30 cells; RBC-based, 56 ± 40 cells; vs. p = 0.003). HBOC-201- and RBC-based perfusates similarly support isolated limb physiology, metabolism, and function.

2: Ex-vivo Normothermic Preservation Of Amputated Limbs With A Hemoglobin-based Oxygen Carrier (HBOC-201) Perfusate

Background:Ex-vivo normothermic perfusion (EVNP) has been used as an alternative to static cold storage (SCS) to improve allograft quality in solid organ and vascularized composite allotransplantation (VCA). Perfusates containing red blood cells (RBCs) have shown to improve outcomes during ex vivo normothermic organ perfusion when compared to acellular perfusates. However, the use of blood products is challenging due to limited availability, the need for cross-matching, and potential blood-borne infection transmission. To avoid limitations associated with the use of blood-based products, we evaluated the feasibility of EVNLP utilizing a polymerized Hemoglobin-Based Oxygen Carrier-201 (HBOC-201).Methods:Twenty-four porcine forelimbs were procured from Yorkshire pigs following euthanasia. Six forelimbs underwent EVNLP with an HBOC-201 based perfusate, six with an RBC-based perfusate, and twelve served as static cold storage (SCS) controls. EVNLP termination criteria included systolic arterial pressure ≥115 mmHg, fullness of compartments, or tissue oxygen saturation drop by 20%. Limb contractility, weight change, compartment pressure, tissue oxygen saturation, oxygen uptake rates (OUR) were assessed. Perfusate fluid-dynamics, gases, electrolytes, metabolites, methemoglobin (MetHb), creatine kinase (CK), and myoglobin concentration were measured. Limb viability was assessed with indocyanine green (ICG) angiography, infrared thermography (IRT), and muscle histology.Results:Warm ischemia time before EVNLP was 35.50±8.62 min in HBOC-201 perfused limbs and 30.17±8.03 min in RBC-perfused limbs (p=0.07). EVNLP duration in HBOC-201 and RBC-perfused limbs was 22.5±1.7 and 28.2±7.3 hours, respectively (p=0.04). Vascular flow (325±25 vs. 444.7±50.6 ml/min; p=0.39), OUR (2.0±1.45 vs. 1.3±0.92 mlO2/min*g of tissue; p=0.80), lactate (14.66±4.26 vs. 13.11±6.68 mmol/L; p=0.32), and perfusate pH (7.53±0.25 HBOC-201; 7.50±0.23 RBC; p=0.82) were not significantly different between treatment groups. Additionally, flexor (28.3±22.0 vs. 27.5±10.6; p=0.99) and extensor (31.5±22.9 vs. 28.8±14.5; p=0.82) compartment pressures, contractility (3±2 vs. 4±1 p=0.57), and percent weight change (23.1±3.0% vs. 13.2±22.7%; p=0.07) were not significantly different between HBOC-201 and RBC groups. In HBOC-201 perfused limbs, MetHb levels increased, reaching 47.8±12.1% at endpoint. Methemoglobin saturation did not affect OUR (ρ = -0.15, r2 = 0.022; p=0.45). Distal tissue preservation was confirmed by IRT and ICG angiography in both EVNLP groups. Hypoxic cell clusters were identified in the SCS control group at endpoint and were absent in both treatment arms.Conclusion:HBOC-201- and RBC-based perfusates similarly support isolated limb physiology, metabolism, and function. Optimization of modifiable factors, including HBOC-201 oxidation, may extend EVNLP durations employing HBOC-201 and overcome logistical constraints of the utilization of traditional blood products.

O-39: Comparison of the Effects of Normothermic Machine Perfusion and Cold Storage Preservation on Porcine Intestinal Allograft Regenerative Potential and Viability

O-39: Comparison of the Effects of Normothermic Machine Perfusion and Cold Storage Preservation on Porcine Intestinal Allograft Regenerative Potential and Viability

Complications during multiorgan retrieval and pancreas preservation

In pancreas transplantation, complications can arise at each step of the process, from the initial selection of donors and recipients through the surgical technique itself and the post-operative period, when lifelong immunosuppression is required. In the early steps, careful retrieval and preservation of the pancreas are crucial for the viability of the organ and ultimate success of the transplant. The pancreas is a low-flow gland, making it highly sensitive to transplantation conditions and presenting risk of pancreatitis due to periods of ischemia. The two groups of donors - after brain death (DBD) or after cardiac arrest (DCD) - require different strategies of retrieval and preservation to avoid or reduce the risk of complications developing during and after the transplantation. For DBD donor transplantation, multiorgan retrieval and cold preservation is the conventional technique. Asystole donor (DCD) transplantation, in contrast, can benefit from the newest technologies, such as hypothermic and especially normothermic preservation machines (referred to as NECMO), to optimize organ preservation. The latter has led to an increase in the pool of donors by facilitating recuperation of organs for transplantation that would have been discarded otherwise.

Development of a human heart-sized perfusion system for metabolic imaging studies using hyperpolarized [1-13 C]pyruvate MRI.

Increasing worldwide demand for cardiac transplantation has spurred new developments to increase the donor pool. Normothermic preservation of heart grafts for transplantation is an emerging strategy to improve the utilization of marginal grafts. Hyperpolarized MR using metabolic tracers such as [1-13 C]pyruvate, provide a novel means of investigating metabolic status without the use of ionizing radiation. We demonstrate the use of this methodology to examine ex vivo perfused porcine heart grafts. Hearts from three 40-kg Danish domestic pigs were harvested and subsequently perfused in Langendorff mode under normothermic conditions, using an MR-compatible perfusion system adapted to the heart. Proton MRI and hyperpolarized [1-13 C]pyruvate were used to investigate and quantify the functional and metabolic status of the grafts. Hearts were perfused with whole blood for 120 min, using a dynamic contrast-enhanced perfusion experiment to verify successful myocardial perfusion. Hyperpolarized [1-13 C]pyruvate MRI was used to assess the metabolic state of the myocardium. Functional assessment was performed using CINE imaging and ventricular pressure data. High lactate and modest alanine levels were observed in the hyperpolarized experiment. The functional assessment produced reduced functional parameters. This suggests an altered functional and metabolic profile compared with corresponding in vivo values. We investigated the metabolic and functional status of machine-perfused porcine hearts. Utilizing hyperpolarized methodology to acquire detailed myocardial metabolic information-in combination with already established MR methods for cardiac investigation-provides a powerful tool to aid the progress of donor heart preservation.