Observations on the ex situ perfusion of livers for transplantation

Normothermic ex situ liver perfusion might allow viability assessment of livers before transplantation. Perfusion characteristics were studied in 47 liver perfusions, of which 22 resulted in transplants. Hepatocellular damage was reflected in the perfusate transaminase concentrations, which correlated with posttransplant peak transaminase levels. Lactate clearance occurred within 3 hours in 46 of 47 perfusions, and glucose rose initially during perfusion in 44. Three livers required higher levels of bicarbonate support to maintain physiological pH, including one developing primary nonfunction. Bile production did not correlate with viability or cholangiopathy, but bile pH, measured in 16 of the 22 transplanted livers, identified three livers that developed cholangiopathy (peak pH < 7.4) from those that did not (pH > 7.5). In the 11 research livers where it could be studied, bile pH > 7.5 discriminated between the 6 livers exhibiting >50% circumferential stromal necrosis of septal bile ducts and 4 without necrosis; one liver with 25‐50% necrosis had a maximum pH 7.46. Liver viability during normothermic perfusion can be assessed using a combination of transaminase release, glucose metabolism, lactate clearance, and maintenance of acid‐base balance. Evaluation of bile pH may offer a valuable insight into bile duct integrity and risk of posttransplant ischemic cholangiopathy.     

Pretransplant sequential hypo‐ and normothermic machine perfusion of suboptimal livers donated after circulatory death using a hemoglobin‐based oxygen carrier perfusion solution

Ex situ dual hypothermic oxygenated machine perfusion (DHOPE) and normothermic machine perfusion (NMP) of donor livers may have a complementary effect when applied sequentially. While DHOPE resuscitates the mitochondria and increases hepatic adenosine triphosphate (ATP) content, NMP enables hepatobiliary viability assessment prior to transplantation. In contrast to DHOPE, NMP requires a perfusion solution with an oxygen carrier, for which red blood cells (RBC) have been used in most series. RBC, however, have limitations and cannot be used cold. We, therefore, established a protocol of sequential DHOPE, controlled oxygenated rewarming (COR), and NMP using a new hemoglobin‐based oxygen carrier (HBOC)‐based perfusion fluid (DHOPE‐COR‐NMP trial, NTR5972). Seven livers from donation after circulatory death (DCD) donors, which were initially declined for transplantation nationwide, underwent DHOPE‐COR‐NMP. Livers were considered transplantable if perfusate pH and lactate normalized, bile production was ≥10 mL and biliary pH > 7.45 within 150 minutes of NMP. Based on these criteria five livers were transplanted. The primary endpoint, 3‐month graft survival, was a 100%. In conclusion, sequential DHOPE‐COR‐NMP using an HBOC‐based perfusion fluid offers a novel method of liver machine perfusion for combined resuscitation and viability testing of suboptimal livers prior to transplantation.     

Successful lung transplantation with graft recovered after thoracoabdominal normothermic perfusion from donor after circulatory death

Lung transplantation with lungs procured from donors after circulatory death (DCD) has been established as an alternative technique to traditional donation after brain death (DBD) with comparable outcomes. Recently, in situ thoracoabdominal normothermic regional perfusion (TA-NRP) has emerged as a novel technique employed in the procurement of cardiac allografts after circulatory death. TA-NRP, in contrast to ex situ machine perfusion, has the advantage of allowing in situ assessment of donor organs prior to final acceptance. However, there are some concerns that this technique may adversely impact the quality of lung allografts. Here, we present a case of a successful bilateral sequential lung transplantation in a patient with postinflammatory pulmonary fibrosis due to acute respiratory distress syndrome (ARDS), with lungs procured after normothermic in situ lung perfusion. Apart from the lungs, heart, liver, and kidneys were also successfully transplanted from this donor.     

Impact of machine perfusion after long static cold storage on delayed graft function incidence and duration and time to hospital discharge

Delayed graft function (DGF) is very high in our center (70%‐80%), and we usually receive a kidney for transplant after more than 22 hours of static cold ischemia time (CIT). Also, there is an inadequate care of the donors, contributing to a high rate of DGF. We decided to test whether machine perfusion (MP) after a CIT improved the outcome of our transplant patients. We analyzed the incidence of DGF, its duration, and the length of hospital stay (LOS) in patients who received a kidney preserved with MP after a CIT (hybrid perfusion—HP). We included 54 deceased donors kidneys preserved with HP transplanted from Feb/13 to Jul/14, and compared them to 101 kidney transplants preserved by static cold storage (CS) from Nov/08 to May/12. The median pumping time was 11 hours. DGF incidence was 61.1% vs 79.2% (P = .02), median DGF duration was 5 vs 11 days (P < .001), and median LOS was 13 vs 18 days (P < .011), for the HP compared to CS group. The observed reduction of DGF with machine perfusion did not occur in donors over 50 years old. In the multivariate analysis, risk factors for DGF, adjusted for CIT, were donor age (OR, 1.04; P = .005) and the absence of use of MP (OR, 1.54; P = .051). In conclusion, the use of HP contributed to faster recovery of renal function and to a shorter length of hospital stay.     

Use of ex vivo normothermic machine perfusion after normothermic regional perfusion to salvage a poorly perfused DCD kidney

Normothermic regional perfusion (NRP) and normothermic machine perfusion (NMP) have both been used in the procurement and conditioning of abdominal organs from donation after circulatory death donors with reported improved outcomes for the recipients. Here, we describe an unusual case of a kidney that underwent NMP after NRP. After 2 hours of abdominal NRP, the intra‐abdominal organs were cold flushed in situ. The liver and right kidney were well flushed, but the left kidney was poorly flushed. Further attempts to clear the left kidney by flushing on the backtable were unsuccessful, and the kidney was thought to be unsuitable for transplant. The left kidney then underwent a 1‐hour period of NMP using a red cell–based perfusate. During NMP, the kidney met previously described quality assurance criteria for transplant with good global perfusion and adequate renal blood flow and urine production. The kidney was transplanted into a suitable recipient who had slow early graft function but did not require dialysis posttransplant. The recipient was discharged 6 days posttransplant, and the serum creatinine level was 160 μmol/L (1.8 mg/dL) at 2 months posttransplant.     

Opposite acute potassium and sodium shifts during transplantation of hypothermic machine perfused donor livers

Liver transplantation is frequently associated with hyperkalemia, especially after graft reperfusion. Dual hypothermic oxygenated machine perfusion (DHOPE) reduces ischemia/reperfusion injury and improves graft function, compared to conventional static cold storage (SCS). We examined the effect of DHOPE on ex situ and in vivo shifts of potassium and sodium. Potassium and sodium shifts were derived from balance measurements in a preclinical study of livers that underwent DHOPE (n = 6) or SCS alone (n = 9), followed by ex situ normothermic reperfusion. Similar measurements were performed in a clinical study of DHOPE‐preserved livers (n = 10) and control livers that were transplanted after SCS only (n = 9). During DHOPE, preclinical and clinical livers released a mean of 17 ± 2 and 34 ± 6 mmol potassium and took up 25 ± 9 and 24 ± 14 mmol sodium, respectively. After subsequent normothermic reperfusion, DHOPE‐preserved livers took up a mean of 19 ± 3 mmol potassium, while controls released 8 ± 5 mmol potassium. During liver transplantation, blood potassium levels decreased upon reperfusion of DHOPE‐preserved livers while levels increased after reperfusion of SCS‐preserved liver, delta potassium levels were ‐0.77 ± 0.20 vs. +0.64 ± 0.37 mmol/L, respectively (P = .002). While hyperkalemia is generally anticipated during transplantation of SCS‐preserved livers, reperfusion of hypothermic machine perfused livers can lead to decreased blood potassium or even hypokalemia in the recipient.     

Organ‐specific metabolic profiles of the liver and kidney during brain death and afterwards during normothermic machine perfusion of the kidney

We investigated metabolic changes during brain death (BD) using hyperpolarized magnetic resonance (MR) spectroscopy and ex vivo graft glucose metabolism during normothermic isolated perfused kidney (IPK) machine perfusion. BD was induced in mechanically ventilated rats by inflation of an epidurally placed catheter; sham‐operated rats served as controls. Hyperpolarized [1‐¹³C]pyruvate MR spectroscopy was performed to quantify pyruvate metabolism in the liver and kidneys at 3 time points during BD, preceded by injecting hyperpolarized[1‐¹³C]pyruvate. Following BD, glucose oxidation was measured using tritium‐labeled glucose (d ‐6‐3H‐glucose) during IPK reperfusion. Quantitative polymerase chain reaction and biochemistry were performed on tissue/plasma. Immediately following BD induction, lactate increased in both organs (liver: eµ_d0.21, 95% confidence interval [CI] [?0.27, ?0.15]; kidney: eµ_d0.26, 95% CI [?0.40, ?0.12]. After 4 hours of BD, alanine production decreased in the kidney (eµ_d0.14, 95% CI [0.03, 0.25], P < .05). Hepatic lactate and alanine profiles were significantly different throughout the experiment between groups (P < .01). During IPK perfusion, renal glucose oxidation was reduced following BD vs sham animals (eµ_d0.012, 95% CI [0.004, 0.03], P < .001). No differences in enzyme activities were found. Renal gene expression of lactate‐transporter MCT4 increased following BD (P < .01). In conclusion, metabolic processes during BD can be visualized in vivo using hyperpolarized magnetic resonance imaging and with glucose oxidation during ex vivo renal machine perfusion. These techniques can detect differences in the metabolic profiles of the liver and kidney following BD.     

Normothermic kidney perfusion: An overview of protocols and strategies

Normothermic machine perfusion (NMP) technologies are emerging as an important adjunct in organ preservation and transplantation. NMP can enable the reduction or avoidance of cold ischemia and allows for pretransplant measurement of function and metabolic status to assess the suitability of the organ for transplantation. The key requirement of NMP is to provide an environment that is protective to the organ, ensures optimal oxygen delivery and supports metabolic function. Red blood cell-based solutions, artificial hemoglobin solutions, and acellular solutions have all been utilized in NMP. However, there is no clear consensus on perfusion protocols. A period of NMP after hypothermic preservation is the most commonly used strategy. As an alternative, several groups have developed and tested the feasibility of more prolonged periods of NMP. There are only a few reports of the application of NMP in clinical kidney transplantation and each uses different approach and conditions. This review details the rationale for NMP protocols considering duration of NMP and different perfusate compositions in experimental and clinical models. We also include a discussion on the mechanistic action of NMP, comparison of subnormothermic and hypothermic conditions, the different logistical approaches and future requirements.     

常温机械灌注修复边缘性供肝的临床疗效：单中心报道

目的探讨常温机械灌注（normothermic machine perfusion, NMP）修复边缘性供肝的安全性和有效性。 方法2018年9月至2019年9月,使用NMP进行6例边缘性供肝体外评估和修复,供肝来自4例心死亡和2例高胆红素血症患者。记录灌注过程的灌注参数、灌注液血气分析及生化检验指标,结合供肝外观等评估边缘性供肝是否适合移植。术后随访至少3个月,记录移植后7 d内肝功能指标、生化指标、并发症发生情况等。 结果NMP期间灌注参数稳定,肝动脉灌注流量为110~334 ml/min,门静脉灌注流量为540~1 180 ml/min。灌注液pH、PO₂、PCO₂在灌注0.5 h后基本恢复正常,乳酸水平迅速下降;肝酶水平无明显升高,胆汁pH>7.5。所有供肝灌注均匀,质地柔软,均被用于移植。受者肝移植后恢复情况良好,无一例发生原发性移植肝无功能或缺血性胆道病变,至末次随访所有患者及移植物均存活。 结论本科室在临床肝移植中应用NMP技术的初步经验提示,NMP用于边缘性供肝的保存是安全、有效的。     

Human liver stem cell-derived extracellular vesicles reduce injury in a model of normothermic machine perfusion of rat livers previously exposed to a prolonged warm ischemia

Livers from donors after circulatory death (DCD) are a promising option to increase the donor pool, but their use is associated with higher complication rate and inferior graft survival. Normothermic machine perfusion (NMP) keeps the graft at 37°C, providing nutrients and oxygen supply. Human liver stem cell-derived extracellular vesicles (HLSC-EVs) are able to reduce liver injury and promote regeneration. We investigated the efficacy of a reconditioning strategy with HLSC-EVs in an experimental model of NMP. Following total hepatectomy, rat livers were divided into 4 groups: (i) healthy livers, (ii) warm ischemic livers (60 min of warm ischemia), (iii) warm ischemic livers treated with 5 × 10⁸ HLSC-EVs/g-liver, and (iv) warm ischemic livers treated with a 25 × 10⁸ HLSC-EVs/g-liver. NMP lasted 6 h and HLSC-EVs (Unicyte AG, Germany) were administered within the first 15 min. Compared to controls, HLSC-EV treatment significantly reduced transaminases release. Moreover, HLSC-EVs enhanced liver metabolism by promoting phosphate utilization and pH self-regulation. As compared to controls, the higher dose of HLSC-EV was associated with significantly higher bile production and lower intrahepatic resistance. Histologically, this group showed reduced necrosis and enhanced proliferation. In conclusion, HLSC-EV treatment during NMP was feasible and effective in reducing injury in a DCD model with prolonged warm ischemia.     

Predictor parameters of liver viability during porcine normothermic ex situ liver perfusion in a model of liver transplantation with marginal grafts

Normothermic ex situ liver perfusion (NEsLP) offers the opportunity to assess biomarkers of graft function and injury. We investigated NEsLP parameters (biomarkers and markers) for the assessment of liver viability in a porcine transplantation model. Grafts from heart‐beating donors (HBD), and from donors with 30 minutes (donation after cardiac death [DCD]30′), 70 minutes (DCD70′), and 120 minutes (DCD120′) of warm ischemia were studied. The HBD, DCD30′, and DCD70′‐groups had 100% survival. In contrast, 70% developed primary nonfunction (PNF) and died in the DCD120′‐group. Hepatocellular function during NEsLP showed low lactate (≤1.1 mmol/L) in all the groups except the DCD120′‐group (>2 mmol/L) at 4 hours of perfusion (P = .04). The fold‐urea increase was significantly lower in the DCD120′‐group (≤0.4) compared to the other groups (≥0.65) (P = .01). As for cholangiocyte function, bile/perfusate glucose ratio was significantly lower (<0.6) in all the groups except the DCD120′‐group (≥0.9) after 3 hours of perfusion (<0.01). Bile/perfusate Na+ ratio was significantly higher (≥1.2) after 3 hours of perfusion in all the groups except for the DCD120′‐group (≤1) (P < .01). Three hours after transplantation, the DCD120′‐group had a significantly higher international normalized ratio (>5) compared to the rest of the groups (≤1.9) (P = .02). Rocuronium levels were higher at all the time‐points in the animals that developed PNF during NEsLP and after transplantation. This study demonstrates that biomarkers and markers of hepatocellular and cholangiocyte function during NEsLP correlate with the degree of ischemic injury and posttransplant function.     

The future of marginal kidney repair in the context of normothermic machine perfusion

Normothermic machine perfusion (NMP) is a technique that utilizes extracorporeal membrane oxygenation to recondition and repair kidneys at near body temperature prior to transplantation. The application of this new technology has been fueled by a significant increase in the use of the kidneys that were donated after cardiac death, which are more susceptible to ischemic injury. Preliminary results indicate that NMP itself may be able to repair marginal organs prior to transplantation. In addition, NMP serves as a platform for delivery of therapeutics. The isolated setting of NMP obviates problems of targeting a particular therapy to an intended organ and has the potential to reduce the harmful effects of systemic drug delivery. There are a number of emerging therapies that have shown promise in this platform. Nutrients, therapeutic gases, mesenchymal stromal cells, gene therapies, and nanoparticles, a newly explored modality, have been successfully delivered during NMP. These technologies may be effective at blocking multiple mechanisms of ischemia‐ reperfusion injury (IRI) and improving renal transplant outcomes. This review addresses the mechanisms of renal IRI, examines the potential for NMP as a platform for pretransplant allograft modulation, and discusses the introduction of various therapies in this setting.     

The first case of ischemia‐free organ transplantation in humans: A proof of concept

Ischemia and reperfusion injury (IRI) is an inevitable event in conventional organ transplant procedure and is associated with significant mortality and morbidity post‐transplantation. We hypothesize that IRI is avoidable if the blood supply for the organ is not stopped, thus resulting in optimal transplant outcomes. Here we described the first case of a novel procedure called ischemia‐free organ transplantation (IFOT) for patients with end‐stage liver disease. The liver graft with severe macrovesicular steatosis was donated from a 25‐year‐old man. The recipient was a 51‐year‐old man with decompensated liver cirrhosis and hepatocellular carcinoma. The graft was procured, preserved, and implanted under continuous normothermic machine perfusion. The recipient did not suffer post‐reperfusion syndrome or vasoplegia after revascularization of the allograft. The liver function test and histological study revealed minimal hepatocyte, biliary epithelium and vascular endothelium injury during preservation and post‐transplantation. The inflammatory cytokine levels were much lower in IFOT than those in conventional procedure. Key pathways involved in IRI were not activated after allograft revascularization. No rejection, or vascular or biliary complications occurred. The patient was discharged on day 18 post‐transplantation. This marks the first case of IFOT in humans, offering opportunities to optimize transplant outcomes and maximize donor organ utilization.     

Twenty‐four–hour normothermic perfusion of discarded human kidneys with urine recirculation

Transportable normothermic kidney perfusion for 24 hours or longer could enable viability assessment of marginal grafts, increased organ use, and improved transplant logistics. Eleven clinically declined kidneys were perfused normothermically, with 6 being from donors after brain death (median cold ischemia time 33 ± 36.9 hours) and 5 being from donors after circulatory death (36.2 ± 38.3 hours). Three kidneys were perfused using Ringer’s lactate to replace excreted urine volume, and 8 kidneys were perfused using urine recirculation to maintain perfusate volume without fluid replenishment. In all cases, normothermic perfusion either maintained or slightly improved the histopathologically assessed tubular condition, and there was effective urine production in kidneys from both donors after brain death and donors after circulatory death (2367 ± 1798 mL vs 744.4 ± 198.4 mL, respectively; P = .44). Biomarkers, neutrophil gelatinase–associated lipocalin, and kidney injury molecule‐1 were successfully detected and quantified in the perfusate. All kidneys with urine recirculation were readily perfused for 24 hours (n = 8) and exhibited physiological perfusate sodium levels (140.7 ± 1.2 mmol/L), while kidneys without urine recirculation (n = 3) achieved a reduced normothermic perfusion time of 7.7 ± 1.5 hours and significantly higher perfusate sodium levels (159.6 ± 4.63 mmol/:, P < .01). Normothermic machine perfusion of human kidneys for 24 hours appears to be feasible, and urine recirculation was found to facilitate the maintenance of perfusate volume and homeostasis.     

The effect of pulsatile pump perfusion on hepatitis C transmission in kidney transplantation: A prospective pilot study

With increasing utilization of hepatitis C (HCV) viremic donor organs, there may be a role for kidney pump perfusion to reduce viral load and prevent HCV transmission. We performed a prospective pilot study of HCV viremic donors; one kidney from each donor pair was pumped with perfusate exchanges and viral load testing at least every 4 hours. Donor, recipient, and transplant characteristics were obtained with clinical outcomes. Linear regression was performed to quantify the association between pump time and perfusate viral load. Six HCV viremic donors for six pairs of aviremic recipients were included. Perfusate of the pumped kidneys showed detectable virus throughout the pump cycles. Although perfusate viral levels decreased with increasing pump times, this was not statistically significant (β = −.48, P = .36). All recipients had detectable HCV RNA postoperatively. The pumped cohort had an insignificantly reduced mean viral load compared to pumped recipients (1352 ± 2006 vs 26 170 ± 61 211, P = .09). Time to initiation of direct-acting antiviral was 32 ± 12 vs 26 ± 7 days (P = .17) and to undetectable levels was 66 ± 27 vs 55 ± 22 days (P = .82) for the pumped and unpumped cohorts, respectively. Pulsatile perfusion alone does not appear adequate to decrease HCV transmission. Future studies will need to explore additional ex vivo interventions to pumping.     

Synthetic hemoglobin‐based oxygen carriers are an acceptable alternative for packed red blood cells in normothermic kidney perfusion

Normothermic machine perfusion presents a novel platform for pretransplant assessment and reconditioning of kidney grafts. Maintaining the metabolic activity of a preserved graft at physiologic levels requires an adequate oxygen supply, typically delivered by crystalloid solutions supplemented with red blood cells. In this study, we explored the feasibility of using a synthetic hemoglobin‐based oxygen carrier (HBOC) in human kidney normothermic perfusion. Fourteen discarded human kidneys were perfused for 6 hours at a mean temperature of 37°C using a pressure‐controlled system. Kidneys were perfused with a perfusion solution supplemented with either HBOC (n = 7) or packed red blood cells (PRBC) (n = 7) to increase oxygen‐carrying capacity. Renal artery resistance, oxygen extraction, metabolic activity, energy stores, and histological features were evaluated. Throughout perfusion, kidneys from both groups exhibited comparable behavior regarding vascular flow (P = .66), oxygen consumption (P = .88), and reconstitution of tissue adenosine triphosphate (P = .057). Lactic acid levels were significantly higher in kidneys perfused with PRBC (P = .007). Histological findings were comparable between groups, and there was no evidence of histological damage caused by the HBOC. This feasibility experiment demonstrates that a HBOC solution can offer a logistically more convenient off‐the‐shelf alternative to PRBC in normothermic machine perfusion of human kidneys.     

Evaluation of outcomes in renal transplantation with hypothermic machine perfusion for the preservation of kidneys from expanded criteria donors

In 2012, an expert working group from the French Transplant Health Authority recommended the use of hypothermic machine perfusion (HMP) to improve kidney preservation and transplant outcomes from expanded criteria donors, deceased after brain death. This study compares HMP and cold storage (CS) effects on delayed graft function (DGF) and transplant outcomes. We identified 4,316 kidney transplants from expanded criteria donors (2011‐2014) in France through the French Transplant Registry. DGF occurrence was analyzed with a logistic regression, excluding preemptive transplants. One‐year graft failure was analyzed with a Cox regression. A subpopulation of 66 paired kidneys was identified: one preserved by HMP and the other by CS from the same donor. Kidneys preserved by HMP (801) vs CS (3515) were associated with more frequent recipient comorbidities and older donors and recipients. HMP had a protective effect against DGF (24% in HMP group and 38% in CS group, OR = 0.49 [0.40‐0.60]). Results were similar in the paired kidneys (OR = 0.23 [0.04‐0.57]). HMP use decreased risk for 1‐year graft failure (HR = 0.77 [0.60‐0.99]). Initial hospital stays were shorter in the HMP group (P < 0.001). Our results confirm the reduction in DGF occurrence among expanded criteria donors kidneys preserved by HMP.     

Brief O2 uploading during continuous hypothermic machine perfusion is simple yet effective oxygenation method to improve initial kidney function in a porcine autotransplant model

With oxygenation proposed as a resuscitative measure during hypothermic models of preservation, the aim of this study was to evaluate the optimal start time of oxygenation during continuous hypothermic machine perfusion (HMP). In this porcine ischemia‐reperfusion autotransplant model, the left kidney of a ±40 kg pig was exposed to 30 minutes of warm ischemia prior to 22 hours of HMP and autotransplantation. Kidneys were randomized to receive 2 hours of oxygenation during HMP either at the start (n = 6), or end of the perfusion (n = 5) and outcomes were compared to standard, nonoxygenated HMP (n = 6) and continuous oxygenated HMP (n = 8). The brief initial and continuous oxygenated HMP groups were associated with superior graft recovery compared to either standard, nonoxygenated HMP or kidneys oxygenated at the end of HMP. This correlated with significant metabolic differences in perfusate (eg, lactate, succinate, flavin mononucleotide) and tissues (eg, succinate, adenosine triphosphate, hypoxia‐inducible factor‐1α, nuclear factor erythroid 2‐related factor 2) suggesting superior mitochondrial preservation with initial oxygenation. Brief initial O₂ uploading during HMP at procurement site might be an easy and effective preservation strategy to maintain aerobic metabolism, protect mitochondria, and achieve an improved early renal graft function compared with standard HMP or oxygen supply shortly at the end of HMP preservation.