Normothermic Ex-vivo Perfusion Research Articles

Reanimating Pancreatic Grafts Subjected to Prolonged Cold Ischemic Injury Using Normothermic Ex Vivo Perfusion.

Pancreas transplant volumes are limited because of poor utilization of "extended criteria grafts." Prolonged cold ischemia is a risk factor associated with poor allograft survival. We aimed to establish the feasibility of transplantation using grafts subjected to prolonged cold ischemia and determine whether these grafts could be optimized using normothermic ex vivo perfusion (NEVP) in a porcine model. The study population consisted of 35 to 40 kg male Yorkshire pigs in an allotransplantation model with a 3-d survival plan for recipients. Control grafts were subjected to cold storage (CS) in a University of Wisconsin solution for 21 to 24 h (n = 6), whereas the test group received an additional 3 h NEVP after CS of 21 h (n = 5). The 3-d survival was 60% in the NEVP arm versus 0% in the control arm (P = 0.008; log rank). Graft parenchyma was 60% to 70% preserved in the NEVP arm at necropsy on gross appearance. In addition, the islet function was well preserved, and both the pancreas (including the islets) and the duodenal morphology were maintained histologically. The intravenous glucose tolerance test on the day of euthanasia was in the normoglycemic range for 80% of cases in the NEVP arm. Optimization of pancreas grafts exposed to extended CS with NEVP seems promising at rescuing and reanimating these grafts for transplantation, resulting in significantly improved survival in a porcine pancreas transplant model.

O197: Quality Assessment of Human Kidneys by Ex-vivo Normothermic Machine Perfusion prior to Transplantation

Abstract Introduction Ex-vivo normothermic machine perfusion (NMP) is a technique of preservation that can be used to assess the quality and suitability of a kidney for transplantation. This may prevent the unnecessary discard and increase the number of transplanted kidneys. Methods A total of 22 kidneys declined for transplant by all UK transplant centres, were assessed over a three-year period across three centres in the UK. Prior to NMP, most kidneys were declined due to poor quality cold flushing at retrieval. All kidneys were assessed during 1h of NMP. A quality assessment score (QAS) based on the appearance, blood flow and urine output was applied to each kidney. Scores ranged from 1 (best) to 5 (worst). Results Sixteen kidneys (72%) had a QAS of 1 to 3 and were transplanted into matched recipients. One patient died with a non-functioning kidney 12 days post-transplant due to a cardiac arrest. Six recipients (37.5%) had delayed graft function. The mean serum creatinine levels and estimated glomerular filtration rate at 3 months was 154±74µmol/L and 45±15mL/min/1.73m2, respectively. Graft and patient survival at 12 months were 94%. Of the 6 non-transplanted kidneys, 3 were deemed unsuitable after assessment during NMP, 1 was not transplanted due to logistical reasons and a prolonged cold ischaemic time, and the remaining 2 due to chronic degenerative changes assessed by histology scores. Conclusion This clinical research study demonstrated the ability to use NMP technology to successfully increase the number of transplants by assessing kidneys that would otherwise be discarded.

O132: Impaired oxygen unloading from stored blood results in diffusion-limited oxygen release at tissues: evidence from twinned-circuit human kidney perfusion

Abstract Background The storage lesion is a multifactorial degradation effect seen in units of banked blood, and results in an increase in the time-constant of oxygen-unloading from red cells (tau). We have previously shown the relevance of this effect in observational data from a recent clinical trial of prolonged normothermic kidney perfusion as a preservation technique ahead of transplantation. We sought direct confirmatory evidence for diffusion-limited oxygen release by stored red cells (RBCs) during normothermic kidney perfusion in a precisely controlled experimental setting. Methods A novel twinned-circuit kidney perfusion system using common, recirculated dialysis was developed, achieving physical separation of RBCs of two conditions whilst allowing non-cellular components of the perfusate to equilibrate throughout. Using human kidneys unsuitable for transplantation, perfusion was alternated between stored and rejuvenated RBCs (treated with PIPA solution) originating from the same donation, to manipulate oxygen-unloading whilst holding all non-RBC parameters constant, without interrupting blood flow. Results Tau improved with PIPA treatment (1.6 to 1.1s). Rejuvenated oxygen-unloading kinetics reversibly and significantly improved the kidney’s oxygen diffusion capacity and increased cortical oxygen tension by 60%. This supported observations from our clinical trial, where increased tau was strongly associated with restricted renal oxygen consumption. Conclusions We have confirmed that oxygen delivery to tissues can become diffusion-limited during normothermic perfusion with stored blood. This effect is reversible by treatment with PIPA solution. This has important implications for ex-vivo normothermic organ perfusion, and potentially for other scenarios such as major hemorrhage, elective surgery with high blood product utilisation, and paediatric transfusion.

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.

(152) Metabolomic Profiling During Ex-Vivo Normothermic Perfusion Prior to Heart Transplantation Defines Patterns of Substrate Utilization and Correlates with Markers of Allograft Injury

(152) Metabolomic Profiling During Ex-Vivo Normothermic Perfusion Prior to Heart Transplantation Defines Patterns of Substrate Utilization and Correlates with Markers of Allograft Injury

Ex-Vivo Preservation of Heart Allografts—An Overview of the Current State

As heart transplantation continues to be the gold-standard therapy for end-stage heart failure, the supply-demand imbalance of available organs worsens. Until recently, there have been no advances in increasing the donor pool, as prolonged cold ischemic time excludes the use of certain donors. The TransMedics Organ Care System (OCS) allows for ex-vivo normothermic perfusion, which allows for a reduction of cold ischemic time and allows for long-distance procurements. Furthermore, the OCS allows for real-time monitoring and assessment of allograft quality, which can be crucial for extended-criteria donors or donation after cardiac death (DCD) donors. Conversely, the XVIVO device allows for hypothermic perfusion to preserve allografts. Despite their limitations, these devices have the potential to alleviate the supply-demand imbalance in donor availability.

Heart Transplantation in High-Risk Recipients Employing Donor Marginal Grafts Preserved With Ex-Vivo Perfusion.

Extending selection criteria to face donor organ shortage in heart transplantation (HTx) may increase the risk of mortality. Ex-vivo normothermic perfusion (EVP) limits ischemic time allowing assessment of graft function. We investigated the outcome of HTx in 80 high-risk recipients transplanted with marginal donor and EVP-preserved grafts, from 2016 to 2021. The recipients median age was 57years (range, 13-75), with chronic renal failure in 61%, impaired liver function in 11% and previous cardiac surgery in 90%; 80% were mechanically supported. Median RADIAL score was 3. Mean graft ischemic time was 118 ± 25min, "out-of-body" time 420 ± 66min and median cardiopulmonary bypass (CPB) time 228min (126-416). In-hospital mortality was 11% and ≥moderate primary graft dysfunction 16%. At univariable analysis, CPB time and high central venous pressure were risk factors for mortality. Actuarial survival at 1 and 3years was 83% ± 4%, and 72% ± 7%, with a median follow-up of 16months (range 2-43). Recipient and donor ages, pre-HTx extracorporeal life support and intra-aortic balloon pump were risk factors for late mortality. In conclusion, the use of EVP allows extension of the graft pool by recruitment of marginal donors to successfully perform HTx even in high-risk recipients.

Pharmacological agents for defatting livers by normothermic machine perfusion.

Ex-vivo normothermic machine perfusion (NMP) preserves the liver metabolism at 37°C and has rapidly developed as a promising approach for assessing the viability and improving the performance of organs from expanded criteria donors, including fatty liver grafts. NMP is an effective method for defatting fatty livers when combined with pharmaceutical therapies. Pharmacological agents have been shown to facilitate liver defatting by NMP. This systematic review summarizes available pharmacological therapies for liver defatting, with a particular emphasis on defatting agents that can be employed clinically as defatting components during liver NMP as an ex vivo translational paradigm. NMP provides an opportunity for organ treatment and can be used as a defatting platform in the future with defatting agents. Nagrath's cocktail is the most commonly used defatting cocktail in NMP; however, its carcinogenic components may limit its clinical application. Thus, the combination of a defatting cocktail with a new clinically applicable component, for example, a polyphenolic natural compound, may be a novel pharmacological option.

Modelling acute antibody-mediated rejection of human kidney transplants using ex-vivo warm machine perfusion.

Transplant rejection is a major cause of graft loss and morbidity. Currently, no human models of antibody-mediated rejection (AMR) exist, limiting mechanistic investigation and organ-specific targeted therapy. Here, using 12 human kidneys and ex-vivo normothermic machine perfusion, we demonstrate phenotypes of AMR after addition of antibodies against either human HLA class I or blood group antigens (A, B), thus modelling clinical AMR that can follow HLA incompatible (HLAi) or blood group incompatible (ABOi) transplantation. Discarded human kidneys with wide ranging demographics and cold ischaemia times (11-54h) were perfused with red blood cells and fresh frozen plasma (FFP) as a source of complement/coagulation factors. For the HLAi model, 600μg of W6/32 anti-class 1 HLA antibody was added to the circuit (time '0'). For the ABOi model, high titre FFP of the relevant blood group antibody was added. Renal blood flow index (RBFi, mL/min/100 g), C3 desArg, prothrombin fragments 1+2 and histology were determined. Our endpoints included haemodynamic changes, thrombosis, and biopsy proven complement deposition. Compared to control kidneys perfused without anti-donor antibodies, both models demonstrated haemodynamic collapse after antibody perfusion with only the HLAi model showing glomerular C4d deposition. We show that a clinically relevant human kidney model of AMR is feasible, and anticipate that these models, with refinements, could provide a basis to test different strategies to prevent AMR. The Rosetrees and Stonygate Trust, The Royal College of Surgeons of England Fellowship Grant, NIHR Biomedical Research Centre/KCL Early Career Grant, Kidney Research U.K.

Breaking the limits of experimental pancreas transplantation: Working toward the clinical ideal graft

Pancreas transplantation is, at present, the only curative treatment for type-1 diabetes that maintains normoglycemia thus avoiding complications arising from poor glycemic control. Despite its great benefits, the number of pancreas transplants has decreased significantly since its inception in the late 1960s, largely due to demographic changes and the consequent suboptimal quality of donors. The selection criteria for pancreas donors mainly depend on morphological variables such as fatty infiltration, fibrosis, or edema, as well as both functional (amylase and lipase) and clinical variables of the donor. However, the final criterion in the decision-making process is the somewhat subjective assessment of a trained surgeon. That being said, the recent incorporation of graft perfusion machines into clinical practice seems to be changing the work dynamics of the donor organ retrieval team, facilitating decision-making based on objective morphological and functional criteria. Normothermic perfusion using perfusate with supplemental oxygen replicates near physiological parameters thus being a promising strategy for organ preservation. Nevertheless, optimum perfusion parameters are difficult to establish in pancreas transplantation given its complex vascular anatomy combined with an intrinsically low blood flow. The objective of this work is to analyze the results published in the recent literature relating to the considerations of ex-vivo normothermic graft perfusion machines and their usefulness in the field of pancreas transplantation.

334.10: Development and Evaluation of a Cell Therapy for Ex Vivo Porcine Kidney Graft Conditioning Using Exosomes Derived From Porcine Urine Progenitor Cells

Introduction: Kidneys from extended criteria donors are particularly sensitive to ischemia/reperfusion injury. Among strategies to improve graft conditions, our study aims to test a cell therapy based on exosomes derived from porcine Urine Progenitor Cells (pUPCs) combined with Hypothermic Machine Perfusion (HMP) followed by Ex Vivo Normothermic Perfusion (EVNP). Materials and Methods: pUPCs were isolated and characterized by flow cytometry, immunofluorescence and western blotting. pUPC-derived exosomes were extracted by ultracentrifugation and characterized by western blotting, electronic microscopy and Nanosight Tracking Analysis. Porcine kidneys obtained from slaughterhouse were submitted to 32 min of warm ischemia then preserved by HMP for 24 hours at 4°C. Afterwards, kidney early function recovery was assessed using EVNP for 5 hours at 37°C. For cell therapy, pUPC-derived exosomes were injected via the porcine kidney artery. Three experimental groups were planned (n=6/group): Group 1: HMP/vehicle + EVNP/vehicle, Group 2: HMP/ pUPC-derived exosomes + EVNP/vehicle, Group 3: HMP/ pUPC-derived exosomes + EVNP-/ pUPC-derived exosomes. Analyses include functional parameters, histology, biomarkers, protein/cytokine and gene expression analyses in tissue samples and HMP and EVNP perfusates; as well as perfusate effect on in vitro porcine aortic cell cultures. Results: Firstly, pUPCs were successfully isolated and amplified from porcine urine samples. Isolated pUPCs expressed stem cell markers such as SSEA4 embryonic stem cell marker, CD90 mesenchymal stem cell marker and are negative for CD45 hematopoietic stem cell marker and CD31 endothelial marker. Cells expressed kidney channel water markers such as AQP1 and AQP2 and can gain epithelial phenotype when differentiated into tubular cells. pUPC-derived exosomes expressed Alix and TSG101 exosomal markers and were of expected size (40-100nm of diameter) and delimited with a double membrane. Impact of exosome treatment during HMP and EVNP protocol is currently assessed. Preliminary results seem to show that pUPC-exosome injection seems feasible and does not induce acute kidney dysfunction. The potential beneficial effect will be assessed soon after the inclusion of the 18 porcine kidneys (13 performed for now). Conclusion: This study is showing the feasibility to use porcine urine stem cells derived exosomes for renal graft conditioning, and will assess the impact of this therapy on kidney graft quality and early function. Keywords: Kidney, Exosomes, Hypothermic machine perfusion, Ex vivo Normothermie perfusion, Stem cells, Cell therapy.INSERM UMR-1313 IRMETIST, Poitiers, F-86021, France. Université de Poitiers, Faculté de Médecine et de Pharmacie, Poitiers, F-86073, France.

P11.14: The Use of Ex Vivo Normothermic Perfusion to ‘Pause’ Cold Ischaemic Time to Allow for Third Recipient to Be Selected And Undergo Kidney Transplant

Introduction: On arrival to the hospital on receipt of a transplant offer, recipients can be found to be unfit precluding transplantation. In this instance, the graft is offered back to NHS Blood and Transplant. Grafts often, however, remain locally to reduce cold ischaemic time inherent in further relocation. When another suitable recipient is not found, and cold ischaemic time (CIT) increases to undesirable levels, grafts can unfortunately be deemed unusable. Case Presentation: Herein we describe a case in which ex vivo normothermic perfusion (EVNP) facilitated the admittance of a third potential recipient for a 66yo DCDkidney. The first two allocated recipients who were deemed unfit: the first recipient was found to have an infected lower limb ulcer; the second patient was found to have raised inflammatory markers in the context of an aorto-bifemoral graft. At the time a third recipient (55yo, pre-dialysis) was selected the CIT on the graft was 19 hours. The patient was admitted to the ward and EVNP was used to ‘pause’/limit CIT in order for the patient to be prepared, assessed and consented for transplantation. Outcome: EVNP assessment score = 1 (one hour perfusion duration), with excellent perfusion demonstrated and good urine output (>100ml); total CIT was 23 hours at in situ reperfusion. The patient was successfully transplanted and the graft achieved primary function with a creatinine of 166µmol at time of discharge. At 5 months the creatinine is 151µmol and eGFR 32mls/min/m2. Discussion: Without EVNP this graft would have likely been discarded. Cold ischaemic time was effectively paused by the perfusion technology allowing the graft to be assessed and utilised, and ultimately prevented graft discard. EVNP offers a technique to improve organ utilisation.

313.3: Domino Kidney Transplant Following Nephrectomy for Renal Artery Stenosis With Arterial Reconstruction and Viability Assessment Using Ex Vivo Normothermic Perfusion: A Case Series

Introduction: Ex vivo normothermic perfusion (EVNP) is increasingly recognised as a viability tool to increase organ utilisation. We report use of EVNP to assess graft perfusion of potential domino transplants following therapeutic nephrectomy and backbench arterial reconstruction in four cases of refractory hypertension secondary to renal artery stenosis (RAS) unsuitable for endovascular treatment. Case Detail: Patient A and Patient B had isolated unilateral RAS presumed secondary to fibromuscular dysplasia. Pre-operative imaging and functional assessment revealed a split function of the affected kidneys to be 38% and 43%, for Patient A and Patient B, respectively. Patient C and Patient D had a wider distribution of vascular occlusive disease. Patient C had an occluded left renal artery with an atrophic left kidney and no evidence of function on isotope imaging. Following unsuccessful angioplasty and stenting, Patient D had developed in stent occlusion; subsequent imaging demonstrated hypo-perfused right kidney with 6% estimated split function. Outcome: Following nephrectomy, all kidneys were prepared on the backbench for EVNP. For Patient A and Patient B, a common stem was created using spatulation of the renal artery and reconstruction with collateral vessels (plus saphenous vein patch in Patient B). Both grafts perfused well with excellent global perfusion and urine output (EVNP assessment score=1). Beyond the stent stenosis, the renal artery from Patient C was short but allowed cannulation following dilatation. Patient D required separate cannulation (to renal artery and main collateral) with 14G cannula. Patient C and Patient D demonstrated high resistance and poorer perfusion (EVNP assessment score=4). The kidneys from Patient A and Patient B were successfully transplanted into two dialysis-dependent patients who achieved primary function and eGFR of 58 and 62ml/min/1.73m2, respectively. Discussion: The demonstration of adequate arterial reconstruction plus excellent graft perfusion whilst on EVNP, alongside favourable pre-operative functional imaging, provided confidence to transplant two marginal domino grafts.

315.6: Assessment of Biliary Regeneration During Long Term (>5 Days) Ex-vivo Normothermic Machine Perfusion

315.6: Assessment of Biliary Regeneration During Long Term (>5 Days) Ex-vivo Normothermic Machine Perfusion

P11.02: Application of Ex-vivo Normothermic Machine Perfusion To Assess Renal Allograft Viability Following Renal Vein Thrombosis

Background: Ex-vivo Normothermic Machine perfusion (NMP) has been shown to improve transplantation outcomes and assess the viability of discarded grafts. We report an application of NMP in assessing renal allograft viability in a case of renal vein thrombosis (RVT) following deceased renal transplantation in the immediate postoperative period. Method: A 44-year-old female patient of unknown cause chronic kidney disease, on maintenance haemodialysis since 2018, underwent a deceased donor renal transplant from a young adult male donor after negative virtual crossmatch with CIT of 7 hrs 8 min. She had a history of fistula failure on multiple occasions and one abortion; her pro-coagulant workup was negative. She was on peritoneal dialysis for the past year, and she had multiple anti-HLA antibody specificities in class I and II loci (highest MFI 9709). She received Induction ATG, tacrolimus, mycophenolate, and steroids. The surgical procedure required reconstruction of the right renal vein. Allograft function was immediate and good with nadir creatinine 1.0mg/ml on postoperative day 6. On day 7, she had rapid onset anuria. Ultrasonogram showed clots in the graft pelvis with flow reversal, and CT angiogram showed RVT. On surgical re-exploration, the graft was congested, and the renal vein had an occlusive thrombus. After explantation and thrombectomy, the graft was placed on the Organ AssistTM using a red cell-based perfusate. It was perfused for six hours to check for graft viability on which there was the restoration of blood flow as evidenced by improving vascular resistance (peak 3mmHg/ml/min dropping to 1.3mmHg/ml/min) and blood flow (20ml/min rising to 220ml/min) and stabilization of lactate levels with a good macroscopic appearance. However, there was persistent haemorrhagic urine output despite an adequate and steady increase in renal blood flow. Result: Despite demonstrating viability on NMP, the allograft could not be re-implanted due to haemorrhagic urine output on NMP, given the potential for bleeding and the hemodynamic instability in the recipient. The recipient was discharged on day 18 on dialysis. The allograft nephrectomy specimen showed isolated vascular acute T cell-mediated rejection- C4d negative with superimposed venous thrombosis. Conclusion: EVNMP can help decision making in complex situations where graft viability assessment is not readily apparent. Received Funding from PGIMER Chandigarh.

247.6: Preliminary Results of Ex-vivo Normothermic Machine Perfusion in Deceased Donor Renal Allograft Transplantation – First Clinical Experience From Asia

247.6: Preliminary Results of Ex-vivo Normothermic Machine Perfusion in Deceased Donor Renal Allograft Transplantation – First Clinical Experience From Asia

Normothermic Ex Vivo Pancreas Perfusion for the Preservation of Pancreas Allografts before Transplantation.

Pancreas transplantation (PTx) is a curative treatment for people who live with the burden of a diagnosis of diabetes mellitus (DM). However, due to organ shortages and increasing numbers of patients being listed for PTx, new strategies are needed to increase the number of available grafts for transplantation. Static cold storage (SCS) is considered the gold standard for standard criteria organs. However, standard criteria donors (SCD) are becoming scarce and new strategies that can increase the rate of organ acceptance from extended criteria donors (ECD) are urgently needed. Normothermic ex vivo perfusion (NEVP) is one of the strategies that has become increasingly popular over the past couple of decades. This preservation method has already been used successfully in other organs (liver, kidneys, and lungs) but has been minimally explored in pancreas transplantation. The few papers that describe the method for pancreas show little success, edema being one of the major issues. The following manuscript describes the successful NEVP method and setup developed by our group to perfuse swine pancreas.

O004 Blood group antigen removal of a human kidney using ex-vivo normothermic machine perfusion technology

Abstract Introduction A novel strategy to overcome the constraints of ABO compatibility in solid organ transplantation is the enzymatic removal of blood group antigens using an alpha-galactosidase enzyme from Bacteroides fragilis during ex-vivo normothermic machine perfusion (NMP). Methods Human kidneys rejected for transplantation and offered for research were used in this study which was approved by the National Research Ethics committee and Research and Development office (NRES: 15/NE/0408). Blood group antigen removal using the alpha-galactosidase enzyme GH110B from B. fragilis was first investigated on formalin-fixed paraffin-embedded (FFPE) cortex sections from human kidneys of blood group B (n=5). Immunofluorescence co-staining of the blood group B antigens (isolectin GSIB4) and vascular endothelium (CD-31) was completed. A human kidney of blood group B was then treated with 0.0025mg/ml of alpha-galactosidase during 6hrs of NMP using an acellular perfusate solution. Serial biopsies taken throughout perfusion were stained and analysed for antigen removal. Results Alpha-galactosidase treatment of FFPE sections at all concentrations examined (0.0013mg/ml 0.5mg/ml) caused significant reduction of the Pearson correlation coefficient for B antigen and CD-31 co-localisation compared to the untreated control (p<0.001). Following NMP of the treated B kidney, the Pearson correlation coefficient dropped from 0.182±0.042 (pre-treatment level) to a minimum of 0.017±0.102 after 6hrs (p<0.0001), indicating a near-complete loss of the blood group antigens. Conclusion This is the first example of the enzymatic removal of type B blood group antigens from a human kidney during NMP. This strategy may have the potential to increase the pool of ABO compatible donor kidneys for transplantation. Take-home message We have shown that an alpha-galactosidase enzyme added during ex-vivo normothermic machine perfusion can be used to enzymatically remove the blood group B antigens from human kidneys, providing the first example of whole organ conversion in humans. This strategy has the potential to massively increase the pool of ABO compatible donor kidneys for transplantation.

Perfusate Composition and Duration of Ex-Vivo Normothermic Perfusion in Kidney Transplantation: A Systematic Review.

Ex-vivo normothermic perfusion (EVNP) is an emerging strategy in kidney preservation that enables resuscitation and viability assessment under pseudo-physiological conditions prior to transplantation. The optimal perfusate composition and duration, however, remain undefined. A systematic literature search (Embase; Medline; Scopus; and BIOSIS Previews) was conducted. We identified 1,811 unique articles dating from January 1956 to July 2021, from which 24 studies were deemed eligible for qualitative analysis. The perfusate commonly used in clinical practice consisted of leukocyte-depleted, packed red blood cells suspended in Ringer's lactate solution with Mannitol, dexamethasone, heparin, sodium bicarbonate and a specific nutrient solution supplemented with insulin, glucose, multivitamins and vasodilators. There is increasing support in preclinical studies for non-blood cell-based perfusates, including Steen solution, synthetic haem-based oxygen carriers and acellular perfusates with supraphysiological carbogen mixtures that support adequate oxygenation whilst also enabling gradual rewarming. Extended durations of perfusion (up to 24h) were also feasible in animal models. Direct comparison between studies was not possible due to study heterogeneity. Current evidence demonstrates safety with the aforementioned widely used protocol, however, extracellular base solutions with adequate oxygenation, supplemented with nutrient and metabolic substrates, show promise by providing a suitable environment for prolonged preservation and resuscitation. Systematic Review Registration: https://www.crd.york.ac.uk/prospero/display_record.php?ID=CRD42021231381, identifier PROSPERO 2021 CRD42021231381.

Artificial oxygen carriers in organ preservation: Dose dependency in a rat model of ex-vivo normothermic kidney perfusion.

Organ preservation through ex-vivo normothermic perfusion (EVNP) with albumin-derived perfluorocarbon-based artificial oxygen carriers (A-AOCs) consisting of albumin-derived perfluorodecalin-filled nanocapsules prior to transplantation would be a promising approach to avoid hypoxic tissue injury during organ storage. The kidneys of 16 rats underwent EVNP for 2h with plasma-like solution (5% bovine serum albumin, Ringer-Saline, inulin) with or without A-AOCs in different volume fractions (0%, 2%, 4%, or 8%). Cell death was determined using TdT-mediated dUTP-biotin nick end labeling (TUNEL). Aspartate transaminase (AST) activity in both perfusate and urine as well as the glomerular filtration rate (GFR) were determined. The hypoxia inducible factors 1α and 2α (HIF-1α und -2α) were quantified in tissue homogenates. GFR was substantially decreased in the presence of 0%, 2%, and 8% A-AOC but not of 4%. In accordance, hypoxia-mediated cell death, as indicated by both AST activity and TUNEL-positive cells, was significantly decreased in the 4% group compared to the control group. The stabilization of HIF-1α and 2α decreased with 4% and 8% but not with 2% A-AOCs. The dosage of 4% A-AOCs in EVNP was most effective in maintaining the physiological renal function.