Ex-vivo Perfusion Research Articles

Clinical use of controlled oxygenated rewarming of kidney grafts prior to transplantation by ex vivo machine perfusion. A pilot study.

Sudden restoration of normothermic conditions upon reperfusion of cold-stored grafts has been suggested to entail a massive energy demand not yet met by the cells that still suffer from hypothermic torpor. An adapted and gentle rise of graft temperature by ex-vivo machine perfusion has, therefore, been proposed. This should now be tested in the clinical setting. In a first clinical series, six ECD-kidneys were subjected to controlled oxygenated rewarming (COR) during short term pre-implantation machine perfusion. Matched kidneys that were conventionally kept on ice served as controls. Early allograft function after transplantation was significantly improved by COR. On post-operative day 7, clearance of creatinine was more than twofold higher after COR and fractional excretion of sodium in the normal range, while significantly elevated in control kidneys. Good correlations were seen between ulterior graft function and real-time parameters obtained during pre-transplant machine perfusion (Lactate: r2 =.9; TIMP2: r2 =.74). Conventional denominators of graft viability like kidney donor risk index KDRI were far less predictive (r2 =.26). It is concluded that COR can be safely applied to renal grafts and appears to be a valuable tool to predict and improve early renal function after transplantation.

Placental transport of parabens studied using an ex-vivo human perfusion model.

Parabens are a group of chemicals widely used as preservatives in daily consumer products such as cosmetics, food items, pharmaceuticals and household commodities. They have been broadly detected in human samples indicating a general human exposure, and concerns arose from their potential endocrine disrupting effect. Especially the exposure to parabens during pregnancy is concerning, as the time of fetal development is a particularly vulnerable period. The aim of this study was to investigate the transport and metabolism of four commonly used parabens: methyl-, ethyl-, propyl- and butylparaben (MeP, EtP, PrP and BuP) and the metabolite para-hydroxybenzoic acid (PHBA) across the human placenta. An ex-vivo human placental perfusion model was used. The test compounds were added in the maternal compartment (with initial concentrations of 1mM or 0.1mM). Placental transport was evaluated by fetal-maternal concentration ratios (FM-ratio), transport index (TI) and indicative permeability (IP). Information about parabens kinetics was taken from 10 perfusions and PHBA from 7 perfusions. Paraben metabolism was not detected. The placental transport of MeP, EtP, PrP, BuP and PHBA revealed a transfer from maternal to fetal circulations with FM120 of 0.86±0.27 (MeP), 0.98±0.28 (EtP), 1.00±0.28 (PrP), 1.12±0.59 (BuP) and 0.82±0.37 (PHBA). The test substances accumulated in the perfused tissue in some degree. The average kinetic parameters FM-ratio, TI and IP were not different between chemicals. The present study shows that the placenta barrier is permeable to all four parabens and the metabolite, which implies potential fetal exposure.

Inhaled sphingosine has no adverse side effects in isolated ventilated and perfused pig lungs

Ex-vivo lung perfusion (EVLP) systems like XVIVO are more and more common in the setting of lung transplantation, since marginal donor-lungs can easily be subjected to a performance test or be treated with corticosteroids or antibiotics in high dose regimes. Donor lungs are frequently positive in bronchoalveolar lavage (BAL) bacterial cultures (46–89%) which leads to a donor-to-recipient transmission and after a higher risk of lung infection with reduced posttransplant outcome. We have previously shown that sphingosine very efficiently kills a variety of pathogens, including Pseudomonas aeruginosa, Staphylococcus aureus and epidermidis, Escherichia coli or Haemophilus influenzae. Thus, sphingosine could be a new treatment option with broadspectrum antiinfective potential, which may improve outcome after lung transplantation when administered prior to lung re-implantation. Here, we tested whether sphingosine has any adverse effects in the respiratory tract when applied into isolated ventilated and perfused lungs. A 4-h EVLP run using minipig lungs was performed. Functional parameters as well as perfusate measurements where obtained. Biopsies were obtained 30 min and 150 min after inhalation of sphingosine. Tissue samples were fixed in paraformaldehyde, embedded in paraffin and sectioned. Hemalaun, TUNEL as well as stainings with Cy3-coupled anti-sphingosine or anti-ceramide antibodies were implemented. We demonstrate that tube-inhalation of sphingosine into ex-vivo perfused and ventilated minipig lungs results in increased levels of sphingosine in the luminal membrane of bronchi and the trachea without morphological side effects up to very high doses of sphingosine. Sphingosine also did not affect functional lung performance. In summary, the inhalation of sphingosine results in an increase of sphingosine concentrations in the luminal plasma membrane of tracheal and bronchial epithelial cells. The inhalation has no local side effects in ex-vivo perfused and ventilated minipig lungs.

Graft utilization after normothermic regional perfusion in controlled donation after circulatory death-a single-center perspective from France.

Normothermic regional perfusion (NRP) in controlled donation after circulatory death (cDCD) is a promising procurement strategy. However, a detailed analysis of graft utilization rates is lacking. This retrospective study included all cDCD donors proposed to a single center for NRP procurement of at least one abdominal organ from 2015 to 2020. Utilization rates were defined as the proportion of transplanted grafts from proposed donors in which withdrawal of life sustaining therapies (WLST) was initiated. In total, 125 cDCD donors underwent WLST with transplantation of at least one graft from 109 (87%) donors. In a total of 14 (11%) procedures NRP failure led to graft discard. Utilization rates for kidney and liver grafts were 83% and 59%, respectively. In 44% of the discarded livers, the reason was poor graft quality based on functional donor warm ischemia >45min, macroscopic aspect, high-transaminases release, or pathological biopsy. In this study, abdominal NRP in cDCD lead to transplantation of at least one graft in the majority of cases. While the utilization rate for kidneys was high, nearly half of the liver grafts were discarded. Cannulation training, novel graft viability markers, and ex-vivo liver graft perfusion may allow to increase graft utilization.

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.

Reconditioning of circulatory death hearts by ex-vivo machine perfusion with a novel HTK-N preservation solution

Warm ischemia followed by blood reperfusion is associated with reduced myocardial contractility. Circulatory death (CD) hearts are maintained by machine perfusion (MP) with blood. However, the impact of MP with histidine-tryptophane-ketoglutarate (HTK) or novel HTK-N solution on reconditioning of CD-heart contractility is unknown. In a porcine model, native hearts were directly harvested (control), or CD was induced before harvesting, followed by left ventricular (LV) contractile assessment. In MP-groups, CD-hearts were maintained for 4h by MP with blood (CD-B), cold oxygenated HTK (CD-HTK) or HTK-N (CD-HTK-N) before contractile evaluation (all groups n=8). We performed immunohistochemistry of LV myocardial samples. We profiled myocardial expression of 84 oxidative stress-related genes and correlated the findings with myocardial contractility via a machine learning algorithm. HTK-N improved end-systolic pressure (ESP=172±10 vs 132±5mmHg, p=0.02) and maximal slope of pressure increment (dp/dtmax=2161±214vs 1240±167mmHg/s, p=0.005) compared to CD, whereas CD-B failed to improve contractility. Dp/dtmax (2161±214 vs 1177±156, p=0.08)and maximal rate of pressure decrement (dp/dtmin=-1501±228 vs -637±79, p=0.005) were also superior in CD-HTK-N compared to CD-B. In CD-HTK-N, myocardial 4-hydroxynonenal (marker for oxidative stress; p<0.001), nitrotyrosine (marker for nitrosative stress; p=0.004), poly(adenosine diphosphate-ribose)polymerase (marker for necrosis; p=0.028) immunoreactivity and cell swelling (p=0.008) were decreased compared to CD-B. Strong correlation of gene expression with ESP was identified for oxidative stress defense genes in CD-HTK-N. During harvesting procedure, MP with HTK-N reconditions CD-heart systolic and diastolic function by reducing oxidative and nitrosative stress and preventing cardiomyocytes from cell swelling and necrosis.

Lung transplantation using allografts with more than 8 hours of ischemic time: A single-institution experience

Six hours was historically regarded as the limit of acceptable ischemic time for lung allografts. However, broader sharing of donor lungs often necessitates use of allografts with ischemic time >6 hours. We characterized the association between ischemic time ≥8 hours and outcomes after lung transplantation using a contemporary cohort from a high-volume institution. Patients who underwent primary isolated bilateral lung transplantation between 1/2016 and 5/2020 were included. Patients bridged to transplant with extracorporeal membrane oxygenation or mechanical ventilation, and ex-vivo perfusion cases were excluded. Recipients were stratified by total allograft ischemic time <8 hours (standard) vs ≥8 hours (long). Perioperative outcomes and post-transplant survival were compared between groups. Of 358 patients, 95 (26.5%) received long ischemic time (≥8 hours) lungs. Long ischemic time recipients were more likely to be male and have donation after circulatory death donors than standard ischemic time recipients. On unadjusted analysis, long and standard ischemic time recipients had similar survival, and similar rates of grade 3 primary graft dysfunction at 72 hours, extracorporeal membrane oxygenation post-transplant, acute rejection within 30 days, reintubation, and post-transplant length of stay. After adjustment, long and standard ischemic time recipients had comparable risks of mortality or graft failure. In a modern cohort, use of lung allografts with "long" ischemic time ≥8 hours were associated with acceptable perioperative outcomes and post-transplant survival. Further investigation is required to better understand how broader use impacts post-lung transplant outcomes and the implications for smarter sharing under an evolving national allocation policy.

Hyperkalemia: Major but still understudied complication among heart transplant recipients.

Hyperkalemia is a recognized and potentially life-threatening complication of heart transplantation. In the complex biosystem created by transplantation, recipients are susceptible to multiple mechanisms for hyperkalemia which are discussed in detail in this manuscript. Hyperkalemia in heart transplantation could occur pre-transplant, during the transplant period, or post-transplant. Pre-transplant causes of hyperkalemia include hypothermia, donor heart preservation solutions, conventional cardioplegia, normokalemic cardioplegia, continuous warm reperfusion technique, and ex-vivo heart perfusion. Intra-transplant causes of hyperkalemia include anesthetic medications used during the procedure, heparinization, blood transfusions, and a low output state. Finally, post-transplant causes of hyperkalemia include hemostasis and drug-induced hyperkalemia. Hyperkalemia has been studied in kidney and liver transplant recipients, but there is limited data on the incidence, causes, management, and prevention in heart transplant recipients. Hyperkalemia is associated with an increased risk of hospital mortality and readmission in these patients. This review describes the current literature pertaining to the causes, pathophysiology, and treatment of hyperkalemia in patients undergoing heart transplantation and focuses primarily on post-heart transplantation.

Thrombolytic Therapy During ex-vivo Normothermic Machine Perfusion of Human Livers Reduces Peribiliary Vascular Plexus Injury.

Background: A major limitation in expanding the use of donation after circulatory death (DCD) livers in transplantation is the increased risk of graft failure secondary to ischemic cholangiopathy. Warm ischemia causes thrombosis and injury to the peribiliary vascular plexus (PVP), which is supplied by branches of the hepatic artery, causing higher rates of biliary complications in DCD allografts.Aims/Objectives: We aimed to recondition discarded DCD livers with tissue plasminogen activator (tPA) while on normothermic machine perfusion (NMP) to improve PVP blood flow and reduce biliary injury.Methods: Five discarded DCD human livers underwent 12 h of NMP. Plasminogen was circulated in the base perfusate prior to initiation of perfusion and 1 mg/kg of tPA was administered through the hepatic artery at T = 0.5 h. Two livers were split prior to perfusion (S1, S2), with tPA administered in one lobe, while the other served as a control. The remaining three whole livers (W1-W3) were compared to seven DCD control liver perfusions (C1-C7) with similar hepatocellular and biliary viability criteria. D-dimer levels were measured at T = 1 h to verify efficacy of tPA. Lactate, total bile production, bile pH, and difference in biliary injury scores before and after perfusion were compared between tPA and non-tPA groups using unpaired, Mann-Whitney tests.Results: Average weight-adjusted D-dimer levels were higher in tPA livers in the split and whole-liver model, verifying drug function. There were no differences in perfusion hepatic artery resistance, portal vein resistance, and arterial lactate between tPA livers and non-tPA livers in both the split and whole-liver model. However, when comparing biliary injury between hepatocellular and biliary non-viable whole livers, tPA livers had significantly lower PVP injury scores (0.67 vs. 2.0) and mural stroma (MS) injury scores (1.3 vs. 2.7).Conclusion: This study demonstrates that administration of tPA into DCD livers during NMP can reduce PVP and MS injury. Further studies are necessary to assess the effect of tPA administration on long term biliary complications.

Donation after circulatory death and liver transplantation: a cohort study.

Donations after circulatory death (DCD) are still challenging in Italy because of prolonged ischemia time (tWIT) due to the law and logistical issues. This cohort study was primarily aimed at assessing the association between successful transplantation and DCD types in the North Italy Transplant program. Adjusted risk ratios (RR) and 95% confidence intervals (CIs) for type III versus type II DCD were estimated using a Poisson regression model with a robust error variance. All consecutive DCD between 2008 and 2020 were included. Among 142 DCD, 102 were eligible for liver donation, and 96 were proposed: 68/69 (99%) and 28/33 (85%) type III and II DCD, respectively. Sixty-nine livers were recovered, 51/68 (75%) from type III and 18/28 (64%) from type II DCD, respectively (RR: 1.18; 95% CI: 0.87-1.60). After ex-vivo perfusion, 50/68 (74%) and 14/28 (50%) livers from type III and type II DCD were transplanted (RR: 1.49; 95% CI: 1.01-2.19). The estimate decreased after further controlling for tWIT (RR: 1.11; 95% CI: 0.55-2.24). Five patients (7.8%) experienced a PNF, 3/50 and 2/14 from type III and type II DCD, respectively. Type III DCD livers were more likely to be transplanted than type II. Warm ischemia time might explain this difference.

Syncytiotrophoblast Extracellular Vesicles From Late-Onset Preeclampsia Placentae Suppress Pro-Inflammatory Immune Response in THP-1 Macrophages

Syncytiotrophoblast derived Extracellular Vesicles (STBEV) from normal pregnancy (NP) have previously been shown to interact with circulating monocytes and B cells and induce pro-inflammatory cytokine release. Early-onset preeclampsia (EOPE) is associated with an exacerbated inflammatory response, yet there is little data regarding late-onset PE (LOPE) and immune function. Here, using a macrophage/monocyte cell line THP-1, we investigated the inflammatory potential of STBEV, comprising medium/large-STBEV (>200nm) and small-STBEV (<200nm), isolated from LOPE (n=6) and normal (NP) (n=6) placentae via dual-lobe ex-vivo placental perfusion and differential centrifugation. THP-1 cells bound and internalised STBEV isolated from NP and LOPE placentae, as revealed by flow cytometry, confocal microscopy, and ELISA. STBEV-treated THP-1 cells were examined for cytokine gene expression by RT-qPCR and the cell culture media examined for secreted cytokines/chemokines. As expected, NP medium/large-STBEV significantly upregulated the transcriptional expression of TNF-α, IL-10, IL-6, IL-12, IL-8 and TGF-β compared to PE medium/large-STBEV. However, there was no significant difference in the small STBEV population between the two groups, although in general, NP small STBEVs slightly upregulated the same cytokines. In contrast, LOPE STBEV (medium and large) did not induce pro-inflammatory responses by differentiated THP-1 macrophages. This decreased effect of LOPE STBEV was echoed in cytokine/chemokine release. Our results appear to suggest that STBEV from LOPE placentae do not have a major immune-modulatory effect on macrophages. In contrast, NP STBEV caused THP-1 cells to release pro-inflammatory cytokines. Thus, syncytiotrophoblast extracellular vesicles from LOPE dampen immune functions of THP-1 macrophages, suggesting an alternative mechanism leading to the pro-inflammatory environment observed in LOPE.

Prolonged ex-vivo normothermic kidney perfusion: The impact of perfusate composition.

Normothermic machine perfusion (NMP) of donor kidneys provides the opportunity for improved graft preservation and objective pre-transplant ex-vivo organ assessment. Currently, a multitude of perfusion solutions exist for renal NMP. This study aimed to evaluate four different perfusion solutions side-by-side and determine the influence of different perfusate compositions on measured renal perfusion parameters. Porcine kidneys and blood were obtained from a slaughterhouse. Kidneys underwent NMP at 37°C for 7 hours, with 4 different perfusion solutions (n = 5 per group). Group 1 consisted of red blood cells (RBCs) and a perfusion solution based on Williams’ Medium E. Group 2 consisted of RBCs, albumin and a balanced electrolyte composition. Group 3 contained RBCs and a medium based on a British clinical NMP solution. Group 4 contained RBCs and a medium used in 24-hour perfusion experiments. NMP flow patterns for solutions 1 and 2 were similar, solutions 3 and 4 showed lower but more stable flow rates. Thiobarbituric acid reactive substances were significantly higher in solution 1 and 4 compared to the other groups. Levels of injury marker N-acetyl-β-D glucosaminidase were significantly lower in solution 2 in comparison with solution 3 and 4. This study illustrates that the perfusate composition during NMP significantly impacts the measured perfusion and injury parameters and thus affects the interpretation of potential viability markers. Further research is required to investigate the individual influences of principal perfusate components to determine the most optimal conditions during NMP and eventually develop universal organ assessment criteria.

Abstract No. 171 Validating an ex-vivo bovine kidney pulsatile perfusion model with micro-CT evaluation of distal angioembolization

Development of a reliable and affordable ex-vivo bovine kidney perfusion model has great potential for further advancing the field of interventional radiology (IR) and minimizing in-vivo animal research. We aim to validate our model for ex-vivo bovine kidney pulsatile perfusion by utilizing microscopic computed tomography (mCT) evaluation of distal angioembolization. Bovine kidneys were obtained directly from a local slaughterhouse. Immediately, the renal artery was flushed with heparinized saline. Organs were then transported on ice to the perfusion lab. Renal artery was cannulated to 3/8” tubing, and renal vein was cannulated with an inflated 16-Fr Foley catheter for collection of venous output. A roller pump was used for pulsatile arterial perfusion. Arterial tubing was set up with an in-line blood pressure (BP) monitor and a one-way valve for intraprocedural BP control. A 5 Fr sheath was placed in the arterial tubing to serve as vascular access. The kidney was mounted over a reservoir of heparinized saline perfusate. The pump would receive perfusate from this reservoir. Kidneys were perfused for 10 minutes prior to beginning the experiment. Two integrated-IR residents were primary operators with attending intraprocedural supervision in a DSA lab. A series of 4 ex-vivo bovine kidney perfusion protocols were successfully performed. Segmental embolization was performed using a 2.8 Fr microcatheter supported by a 5 Fr guiding catheter. Embolization was to contrast stasis. The following variables were collected: kidney pre- and post-perfusion weight, intraprocedural BP, embolic material volume delivered, DSA images, and distal embolization assessed by mCT vascular distribution. Average pre- and post-perfusion weights were found to have a statistically significant increase (P< .05). Intraprocedural BP averaged 140+25mmHg systolic and 61+14mmHg diastolic. 57 minutes were spent perfusing the organ. All post embolic DSAs revealed successful embolization. In a pulsatile perfused ex vivo bovine kidney, microbeads demonstrated stability, fair homogeneity, and microembolization below 40 um vessels without significant venous distribution. Our ex-vivo kidney pulsatile perfusion model is reliable, replicable, and provides a platform where microembolization experiments can be performed. This perfusion model has applications in translational and educational research.

A Closed-Circuit Ex Vivo Perfusion System for Pediatric Solid Organ Transplantation

<h3>Purpose</h3> Donation after cardiac death and ex-vivo organ perfusion are increasingly used strategies to mitigate donor organ shortage in cardiac transplantation. These concepts have mainly been established in adults, while neglecting the specific needs for application in children. To address this gap, we have developed an adjustable closed-circuit ex-vivo perfusion system that reduces priming volume, accommodates a wide range of organ sizes, and allows longer support times. <h3>Methods</h3> Silicone-infused thermoplastic polyurethane was used to construct a flexible biocompatible structure to closely envelop the heart. The cone-shaped bottom of the envelope serves as a drainage port to recirculate coronary sinus blood to a pump and oxygenator. Additional ports allow for aortic perfusion, left ventricular venting, deairing, pacing, ECG, and other monitoring. T-slot aluminum extrusion frames were constructed to suspend the device. A microprocessor with custom-built software was incorporated into the design to monitor and store key parameters, such as ECG, temperature, pressure in various compartments, and flow. <h3>Results</h3> Feasibility of ex-vivo perfusion was tested in a mock-loop setup. The design allowed use of commercially available and clinically approved oxygenators (Quadrox-i, Getinge, Sweden) and pumps (Rotaflow, Getinge, Sweden). Envelope design eliminated blood-air contact, while maintaining flow rates up to and above expected resting coronary blood flow values in adult hearts of 300ml/min. Flow consistency could be shown for extended runs of >3h. Perfusion of an isolated pig heart was performed to prove technical validity of the model. <h3>Conclusion</h3> We have developed an initial prototype for a novel ex-vivo organ perfusion capable of accommodating hearts of variable sizes from neonates to adults, ideally suited for the pediatric population. The system further aims at a reduction of blood trauma and foreign surface contact to increase support times without increasing inflammatory response and organ damage.

Characterization of Extracellular Vesicles from Ex Vivo Perfused Human Lungs and Their Relationship with Allograft Injury

<h3>Purpose</h3> Primary graft dysfunction (PGD) is an important cause of morbidity and mortality following lung transplantation and results in part from injuries sustained in the donor. Extracellular vesicles (EVs), membrane-bound sacs released by cells, are increasingly being recognized as key modulators of inflammation. EVs have been implicated in the anti-donor response to the lung allograft and may play a role in the pathogenesis of PGD. Perfusate from ex-vivo lung perfusion (EVLP) allows interrogation of donor EVs which may serve as predictive biomarkers for lung injury. In this study, we aim to identify and characterize EVs derived from EVLP perfusates of marginal donor lungs. <h3>Methods</h3> Hourly perfusate samples of lungs undergoing EVLP were collected, precleared and ultracentrifuged at 100,000x<i>g</i> for 90mins to isolate small EVs. The presence of EVs was validated by Nanoparticle tracking analysis (NTA), Western blot (WB), and flow cytometry (FACS). Next, EV protein arrays were used to investigate differences in protein content between lungs accepted for transplant (n=20), and injured lungs declined for transplant (n=20). <h3>Results</h3> Our analyses show the presence of EV-sized particles (≤300nm) positive for canonical EV markers. CD9 and CD81 were identified by both WB(Fig1) and FACS, while CD63 was only detectable by FACS(Fig2). Using EV arrays, CD63 and CD31, an endothelial adhesion marker, were found to be elevated in EVs from damaged lungs declined for transplantation (p=0.0183; p=0.0167). There was no difference observed in levels of EV-associated CD9(Fig3). <h3>Conclusion</h3> Our findings show that EVLP perfusates contain donor EVs. CD63+ and CD31+ EVs may reflect damage in lungs declined for transplantation. Next, we aim to characterize and compare the RNA cargo of EVs acquired from the same cohort of donor samples, to determine whether differences can be observed at the transcript level. Together, these findings will provide an insight to the potential role of EVs as biomarkers for lung injury.

Rupture of the Ascending Aorta in a Donor Heart during Ex-Vivo Transport: A Case Report

<h3>Purpose</h3> Problems encountered during transport of an ex-vivo donor heart perfusion device can have drastic consequences on graft function. We describe a case of aortic rupture occurring during ex-vivo transport for donation after cardiac death (DCD) heart transplantation. <h3>Methods</h3> Cardiac procurement in a DCD donor was performed in the standard fashion. Upon connection to the Transmedics Organ Care System, bleeding was noted to be originating from the posterior aortic wall, likely secondary to injury caused by the cardioplegia needle, inserted into the flaccid aorta of relatively small diameter. The site was successfully repaired and transport begun. The aorta was noted to be fragile during repair. Organ function parameters remained in the acceptable ranges throughout the transfer. <h3>Results</h3> As the transport vehicle approached the recipient center, an abrupt movement of the vehicle over a speed hump was followed by sudden drop in the perfusion pressure of the donor heart. Catastrophic bleeding was noted from a defect in the ascending aorta. Bleeding was controlled with digital pressure resulting in return of satisfactory perfusion pressures. The donor heart had minimal perfusion pressure for a total of five minutes. The donor heart was transported on the OCS emergently to the operating room where cardioplegia was delivered and the organ topically cooled. A defect adjacent to the original repair site was noted (image). As this was 2cm above the sinotubular junction, this area was trimmed and the heart was implanted in the standard fashion. Subsequent graft function was good, and the recipient was separated from cardiopulmonary bypass with routine levels of support. The recipient made an uneventful post-operative recovery. <h3>Conclusion</h3> This case illustrates an injury to the posterior wall of the aorta with subsequent major bleeding occurring during ex-vivo transport. Procurement teams should be familiar with the process of emergent conversion to conventional cold static preservation during organ transport.

Reconditioning of Donation after Cardiac Death Hearts by Ex-Vivo Machine Perfusion with a Novel HTK-N Preservation Solution

Purpose Hearts donated from cardiac death donors (DCD) can be successfully maintained by ex-vivo machine perfusion (EVMP) with blood. Donor heart function will be deteriorated depending on the warm ischemia time (WIT). However, the reconditioning capability of machine perfusion with a novel crystalloid preservation solution histidine-tryptophane-ketoglutarate-N (HTK-N) has not been investigated. Methods In a pig model of DCD, cardiac death was induced by termination of ventilation, followed by harvesting of donor hearts. After a total WIT of 30 min, hearts underwent either 4 h of EVMP with warm donor blood (DCD-B) in a beating state or cold EVMP with oxygenated HTK-N (DCD-HTK-N), both followed by reperfusion in Langendorff fashion, or were directly undertaken reperfusion (DCD) (N=8/group). During reperfusion, hemodynamic function was measured by a left ventricular (LV) balloon catheter. Oxidative and nitrosative stress as well as necrosis pathways and morphology of LV-tissue samples were analyzed by 4-hydroxy-2-nonenal (HNE), nitrotyrosine (NT), poly(adenosine diphosphate-ribose)polymerase (PARP) and hematoxylin-eosin (HE) staining. Results After 60 min of reperfusion, DCD-HTK-N hearts showed significantly improved end-systolic pressure (ESP = 172±10 mmHg, p=0.02) and rate of pressure increasement (dp/dtmax = 2161±214 mmHg/s, p=0.005) compared to immediately reperfused DCD-hearts (ESP = 132±5 mmHg, dp/dtmax = 1240±167 mmHg/s) at 20 mL of balloon volume. dp/dtmax and rate of pressure decreasement (dp/dtmin = -1501±228, p=0.005) were also significantly improved after EVMP with HTK-N compared to blood perfusion (dp/dtmax = 1177±156, dp/dtmin = - 637±79). After HTK-N perfusion, oxidative and nitrosative stress as well as necrosis were significantly decreased compared to direct reperfusion (HNE: p=0.005, NT: p=0.002, PARP: p=0.019) or blood perfusion (HNE: p Conclusion EVMP with HTK-N is able to recondition systolic function of DCD-hearts and leads to improved diastolic donor heart function compared to EVMP with blood by reducing oxidative stress, nitrosative stress as well as preventing cardiomyocytes from morphologic change.

Real World Experience with Transmedics Organ Care System in Cardiac Transplantation with Donor Organs Associated with Marginal Risk Factors

<h3>Purpose</h3> Donor organ acceptance is limited by preservation method using cold storage. We sought to evaluate the efficacy of normothermic TransMedics OCS Heart to preserve BD donor organ associated with pre-donation criteria that may increase risk of primary graft dysfunction (expanded criteria). <h3>Methods</h3> All cardiac transplantations between 2017-19 were evaluated in this single-centre, observational analysis. Expanded criteria was defined as: (I) cold ischaemic time between 4-8 hours, (ii) donor age >55 year-old with normal coronary angiography, (iii) reported circulatory down-time > 20mins, (iv) LVEF <50% where impairment is thought to be reversible, and (v) LV hypertrophy (wall thickness 12-16mm). Final transcoronary lactate level < 5 mmol/L at the end of the ex-vivo perfusion period was required for final organ acceptance. Clinical outcomes of interest include: total cold ischaemic time, index hospitalisation length of stay, and all-cause mortality at 90-day. <h3>Results</h3> Between July 2017 and December 2019, the OCS Heart Retrieval team was dispatched on 9 occasions, with 8 cases of ex-vivo organ retrievals. Of the 8 cases, 6 were successfully transplanted. With a total of 43 heart transplants were undertaken, availability of OCS Heart effectively allowed 14% increase in successful transplantation (6/43). Donor organ retrieval using TransMedics OCS Heart was associated with significant reduction in the total cold ischaemic time compared to cold storage (192.4mins vs 80.33mins, p=0.0005). Despite the expanded criteria, donor hearts that were successfully preserved were associated with comparable clinical outcomes: index hospitalization length of stay (P=0.97) and excellent post-transplant survival (Figure). <h3>Conclusion</h3> Normothermic donor heart preservation, with TransMedics OCS, allows comparatively safe transplantation with expanded criteria BD donor organ compared to conventional cold storage method with conservation donor characteristics.

Lung Transplantation Using Allografts with Extreme Ischemic Time: A Single-Institution Experience

<h3>Purpose</h3> Six hours has historically been regarded as the limit of acceptable ischemic time (IT) for lung allografts. However, smarter sharing of donor lungs often necessitates use of allografts with IT >6 hours. We characterized the association between IT ≥8 hours and outcomes after lung transplantation (LTx) using a contemporary cohort from a high-volume institution. We hypothesized that longer ITs do not predict greater mortality or graft failure. <h3>Methods</h3> Patients who underwent primary isolated bilateral orthotopic LTx between 1/2016-5/2020 were included. Patients bridged to LTx with ECMO or mechanical ventilation, and ex-vivo perfusion cases were excluded. Recipients were stratified by allograft IT <8h vs ≥8h. Survival to a composite outcome of death or graft failure and perioperative outcomes were compared between IT strata. Unadjusted survival was estimated using the Kaplan-Meier method; adjusted survival was modeled using multivariable Cox regression. <h3>Results</h3> Of 358 patients, 95 (26.5%) received IT ≥8h lungs. IT ≥8h recipients were more likely to be male (69.5% vs 54.4%, p=0.01) and have DCD donors (20.0% vs 5.3%, p<0.01) than IT <8h recipients. On unadjusted analysis, IT ≥8h and <8h recipients had similar survival (<b>Figure</b>) and similar rates of PGD3 at 72 hours (7.4% vs 6.8%, p=0.9), ECMO post-transplant (16.8% vs 10.6%, p=0.1), acute rejection within 30 days (15.8% vs 16.3%, p=0.9), reintubation during the transplant hospitalization (26.3% vs 20.9%, p=0.3), and similar lengths of stay (median 25 vs 21 days, p=0.2). After adjustment, IT ≥8h and <8h recipients had comparable risks of mortality or graft failure (HR 0.83, 95% CI 0.46-1.51). <h3>Conclusion</h3> In a modern cohort, use of lung allografts with IT ≥8h was associated with similar perioperative outcomes and post-transplant survival. Further investigation is required to better understand how broader use impacts post-LTx outcomes and the implications for smarter sharing under an evolving national allocation policy.

Monitoring of perfusion quality and prediction of donor heart function during ex-vivo machine perfusion by myocardial microcirculation versus surrogate parameters

Currently, lactate (Lac) is used to evaluate machine perfusion (MP) of hearts, donated after circulatory death (DCD). We hypothesize that monitoring of myocardial microcirculation (mLDP) by Laser-Doppler-Perfusion is superior to Lac to evaluate perfusion and predict contractility. In a pig model, DCD-hearts were perfused 4 hours followed by reperfusion and left ventricular contractility measurement. Lac and mLDP were measured every 30 min in successfully (N = 9) and unsuccessfully (N = 7) maintained hearts. Successfully maintained hearts showed decreasing Lac (5.6 to 2.8mmol/L) and slightly downregulated (92%) mLDP. In unsuccessfully maintained hearts Lac first decreased (5.1 to 3.8mmol/L) followed by increase and mLDP dropped to 39%. In a single-variable regression only mLDP showed a significant r² for systolic (0.514, p = 0.045) and diastolic (0.501, p = 0.049) parameters. The combination of mLDP and Lac (r2 = 0.876, p = 0.005) showed best results. mLDP seems to be superior to Lac to show perfusion disorders and predict DCD-heart contractility.