Elderly Liver Grafts are More Susceptible to Ischemia‐Reperfusion After Prolonged Cold Ischemia than Grafts from Young Donors—An Ex Vivo Liver Machine Perfusion Analysis in Sprague Dawley Rats

In an experimental murine liver clamping model, we aimed to investigate the efficacy of real-time confocal microscopy (RCM) in assessing viability of steatotic livers in comparison to standard assessment tools, including histopathological evaluation. C57Bl/6 mice were subjected to a methionine-choline-deficient diet causing nonalcoholic fatty liver disease or to Lieber DeCarli diet causing ethanol-induced liver injury. Untreated animals served as controls. Liver biopsies were analyzed following challenge with 45 min of warm ischemia time and either 4 h of reperfusion or 24 h of cold storage. Organ quality assessment was performed at defined time points by RCM, histological staining, measurement of serum alanine aminotransferase activity, and expression analyses of proinflammatory cytokines. Additionally, survival analysis was performed. Cold as well as warm ischemia time resulted in a significant decrease in cell viability when compared with naive livers as well as nonischemic-challenged steatotic livers (P < 0.05) as assessed by RCM. Furthermore, RCM revealed the actual cellular damage at early time points, while established methods including H&E-staining and serum transaminase profile failed. In a translational attempt, we demonstrate that RCM is a suitable diagnostic tool to obtain information about functional damage of the liver apart from standard approaches.

Kidney transplantation holds much promise as a treatment of choice for patients with end-stage kidney disease. The impact of cold ischemia time (CIT) on acute renal transplant rejection (ARTR) remains to be fully studied in a large cohort of renal transplant patients. From the Organ Procurement and Transplantation Network database, we analyzed 63 798 deceased donor renal transplants performed between 2000 and 2010. We assessed the association between CIT and ARTR. We also evaluated the association between recipient age and ARTR. Six thousand eight hundred two (11%) patients were clinically diagnosed with ARTR. Longer CIT was associated with an increased risk of ARTR. After multivariable adjustment, compared with recipients with CIT < 12 hours, the relative risk of ARTR was 1.13 (95% confidence interval, 1.04-1.23) in recipients with CIT ≥ 24 hours. The association of CIT and ARTR was more pronounced in patients undergoing retransplantation: compared with recipients with CIT less than 12 hours, the relative risk of ARTR was 1.66 (95% confidence interval, 1.01-2.73) in recipients with CIT of 24 hours or longer. Additionally, older age was associated with a decreased risk of ARTR. Compared with recipients aged 18 to 29 years, the relative risk of ARTR was 0.50 (95% confidence interval, 0.45-0.57) in recipients 60 years or older. Longer CIT was also associated with increased risk of death-censored graft loss. Compared with recipients with CIT less than 12 hours, the hazard ratio of death-censored graft loss was 1.22 (95% confidence interval, 1.14-1.30) in recipients with CIT of 24 hours or longer. Prolonged CIT is associated with an increased risk of ARTR and death-censored graft loss. Older age was associated with a lower risk of ARTR.

The aim of our study was to examine, in a recent cohort of kidney transplant recipients who have received modern immunosuppressive therapy, the respective role of cold ischemia time (CIT) and delayed graft function (DGF) on acute rejection (AR) rates and long-term graft survival. We retrospectively reviewed the charts of 611 renal transplantations between 1996 and 2005. Most patients received a calcineurin inhibitor as maintenance therapy, either cyclosporine (43%) or tacrolimus (52%) and 76% of the patients received an antilymphocyte induction therapy. Study endpoints were DGF, first-year AR, and long-term graft survival. Uni- and multivariate analyses were performed to determine factors that may have influenced the study outcomes. DGF was observed in 16.2% of patients. Both older donor age and longer CIT were significant risk factors for DGF. DGF rates were similar whether patients received a calcineurin inhibitor before transplantation or not. AR occurred in 16.5% of grafts during the first year. Independent predictors of AR by multivariate analysis were duration of dialysis, CIT, current panel-reactive lymphocytotoxic antibody more than 5%, and the number of human leukocyte antigen-A, B, and DR mismatches. Each hour of cold ischemia increases the risk of rejection by 4%. With respect to death-censored graft survival, three pretransplant parameters emerged as independent predictors of graft loss: younger recipient age, peak panel-reactive lymphocytotoxic antibody more than 5% and longer CIT. The detrimental effect of CIT on graft survival was entirely because of its propensity to trigger AR. When AR was added to the multivariate Cox model, CIT was no longer significant whereas first-year AR became the most important predictor of graft loss (Hazards ratio, 4.6). Shortening CIT will help to decrease not only DGF rates but also AR incidence and hence graft loss. Patients with prolonged CIT should receive adequate immunosuppression, possibly with antilymphocyte preparations, to prevent AR occurrence.

The purpose of this study was to analyze the combined effect of cold ischemia time (CIT) and donation after cardiac death (DCD, with requisite warm ischemia time, WIT) on kidney transplant (KT) outcomes. Single center retrospective review of DCD KT recipients stratified by CIT. From 6/08 to 10/20, we performed 446 DCD KTs (115 CIT ≤20, 205 CIT 20-30, 88 CIT 30-40, 38 CIT ≥40h). Mean WITs (26/25/27/23min) and KDPI values (59%/55%/55%/59%) were similar while mean CITs (16.4/23.6/33.4/42.5h) and pump times (10.3/13.6/16.1/20.4h) differed across groups (P<.05). With a mean 6-year follow-up, patient survival (84%/84%/74%/84%) was similar. Kidney graft survival (GS) (72%/72%/56%/58%) and death censored GS (DCGS) (82%/80%/63%/67%) rates decreased whereas rates of primary nonfunction (PNF, .9%/2.4%/9.1%/7.9%) and delayed graft function (DGF) (36%/48%/50%/69%) increased with longer CIT (≥30h, P<.05). Meaningful years free of dialysis, which we refer to as Allograft Life Years, were achieved in all cohorts (4.5/4.3/3.9/4.3 years per patient transplanted). DCD donor kidneys with prolonged CIT (≥30h) are associated with increased rates of DGF and PNF, along with decreased GS and DCGS. Despite this, Allograft Life Years were gained even with longer CITs, demonstrating the utility of using these allografts.

We would like to highlight to the readership a case that demonstrates utility of normothermic machine perfusion (NMP), to facilitate a complex liver retransplant, pushing the boundaries in liver transplantation (LT) overcoming unpredictable clinical circumstances. Over the last 5 years, there is a growing body of evidence supporting the safety and efficacy of NMP in LT.1 The question now is not “can NMP be used,” but how best should it be used and in which circumstances? We present a case of a 34-year-old man listed for his third liver transplant for late hepatic artery thrombosis, transplanted aged 4 years for biliary atresia, and aged 26 years for graft cirrhosis with unsuccessful hepatic vein stenting causing caval narrowing. His previous operations were complicated by significant intraabdominal adhesions, extensive portal hypertension, cocoon abdomen, enterocutaneous fistula, and multiple relaparotomies. This surgical complexity meant an extended explant time was anticipated, which would have caused a prolonged cold ischemia time (CIT) for any graft. A fast-tracked liver graft from a donor after brain stem death was accepted (44-y-old woman, bilateral intracranial hemorrhage, body mass index 17 kg/m2, alanine aminotranferase 60 IU/L) that had been declined by other centers due to positive screening for the human T-lymphotropic virus (HTLV) 1 antibody. Considering deterioration of the patients health, this graft was accepted, because he had lower limb edema progressing up to the thoracic wall, and a computed tomography scan demonstrated thrombosis of the inferior vena cava (IVC) and right hepatic vein, without extension to the right atrium on echocardiogram and a negative thrombophilia screen. His United Kingdom End Stage Liver Disease (UKELD) and Model for End Stage Liver Disease (MELD) were 63 and 17, respectively. With the recognized high false-positive rate of HTLV screening assays,2 we utilized NMP after 07:34 hours of CIT, allowing additional time to repeat tests and clear doubts of HTLV status (Figure 1A).FIGURE 1.: A, Liver perfusion circuit; (B) transesophageal echocardiogram identifying right atrial thrombus; (C) extracted right atrial thrombus; (D) schematic of heart bypass circuit utilized. A, donor liver on NMP with the OrganOx Metra (OrganOx Ltd, Oxford, United Kingdom): note the color coding of the cannulae: red—hepatic arterial cannula, yellow—portal venous cannula, and blue—inferior vena cava (IVC) return cannula; (B) dimensions of right atrial thrombus on echocardiogram marked by crosses and dotted lines; (C) extracted right atrial thrombus from with fleshy appearance denoting recent propagation. Histology of this extracted thrombus was rich with platelets, and there are plentiful neutrophils and relatively scanty fungal spores. Microbiology of this thrombus grew Candida albicans, and the patient was treated with an extended course of antimicrobials; and (D) superior vena cava (SVC) and left common femoral vein (LCFV) drainage with the return into the main pulmonary artery (PA). The SVC was snared before opening the right atrium (RA) as the cannula in the LCFV was short (32 cm); there was no need to snare the IVC.The graft fulfilled the NMP Birmingham Viability Criteria for transplantation,3 and the patient was anesthetized for transplantation. Transesophageal echocardiogram had a new finding of a propagated IVC thrombus into the right atrium (Figure 1B), with significant risk of acute dislodgment and embolization within hours to days. Proceeding with the transplant held a risk of on-table death but represented the only chance of survival. With the graft on NMP, the cardiothoracic team were consulted, proposing open extraction of the right atrial thrombus (Figure 1C) on a beating heart utilizing a novel right heart bypass support circuit (Figure 1D). LT was followed with the bypass support, with a total of 23 hours of NMP and a combined preservation time of 30.5 hours. A successful transplant operation was performed (extubated on D5, transferred to the ward on D26, and discharged home on D40). The patient has an excellent quality of life without any significant complications at 12 months follow-up. We believe this case demonstrates how NMP is a vital tool to facilitate complex LT, where it is anticipated there will be prolonged CIT, the importance, in this case, being to mitigate the further increased CIT due to evolving unpredictable clinical events.

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

Normothermic machine perfusion may prevent regulated cell death following renal ischemia-reperfusion injury.

Impact of Cold Ischemia Time on Initial Graft Function and Survival Rates in Renal Transplants From Deceased Donors Performed in Andalusia

Donor, technical, and recipient risk factors cumulatively impact survival and health-related quality of life after liver transplantation. Retrospective study. Tertiary care center. A total of 483 adults undergoing primary orthotopic liver transplantation between January 1, 1991, and July 31, 2003. Graft and patient survival, Karnofsky functional performance scores, Medical Outcomes Study Short Form 36 Health Survey scores, and Psychosocial Adjustment to Illness Scale scores as influenced by potential risk factors including donor age, weight, warm ischemia time, cold ischemia time (CIT), sex, United Network for Organ Sharing (UNOS) status (1 or 2A vs 2B or 3), recipient age and disease, bilirubin level, and creatinine level. Five-year graft survival was 72% for recipients of donors younger than 60 years and 35% for recipients of donors 60 years and older (P<.001). A CIT of 12 hours or more was associated with shorter 5-year graft survival (71% vs 58%; P = .004). Five-year graft survival for UNOS status 2B or 3 was 71% vs 60% for status 1 or 2A (P = .02). A comparable pattern was seen for patient survival in relation to donor age (P = .003), CIT (P = .005), and urgency status (P = .03). Urgent UNOS status, advanced donor age, and prolonged CIT were independently associated with shorter graft and patient survival (P<.05). Functional performance and health-related quality of life were not affected by donor, recipient, or technical characteristics. Combining advanced donor age, urgent status, and prolonged CIT adversely affects graft and patient survival, and the cumulative effects of these risk factors can be modeled to predict posttransplant survival.

Ongoing shortage of donor organs still represents a relevant problem in transplantation medicine. Use of extended criteria donor organs, living donation, and ex vivo splitting have been the major strategies to extend the donor pool for liver transplantation within the past years.1 Recently, the revival of ex vivo machine perfusion has opened up new opportunities to fight organ shortage.2 Especially extended criteria donor organs have been shown to substantially profit from hypothermic oxygenated perfusion (HOPE) prior to transplant.3,4 Combining several of the aforementioned approaches thus might generate synergistic effects and further increase the rate of organs deemed suitable for transplantation. By this means, Mabrut et al5 demonstrated the general feasibility of performing the ex vivo splitting procedure during normothermic machine perfusion. Thorne et al6 showed that extended right lobes (ERLs) generated from ex vivo splitting, usually exposed to prolonged cold ischemic time (CIT), indeed benefit from HOPE. HOPE was recently successfully applied even in pediatric liver transplantation.7 We have now applied HOPE for pretransplant conditioning of a pediatric ERL to avoid loss of the potential graft due to prolonged CIT or posttransplant cholangiopathy. In brief, a 9-y-old female child suffering from cirrhosis due to familiar intrahepatic cholestasis type 2 in reduced condition (hepatic osteopathy and recurrent decompensation amongst others) was offered an ERL following ex situ splitting of a 6-y-old male donor organ. The donor was diagnosed brain dead following traumatic brain injury. Regarding the course of the laboratory findings and the organ quality and anatomy, the donor liver (803 g) was deemed suitable for an ex vivo splitting procedure. Following an uncomplicated split, the ERL of 508 g was accepted for our patient in an extended allocation following rejection of the ERL by the primary center due to size mismatch (ERL accepted for an adult recipient). Because of the overall demanding logistics, the estimated CIT eventually was expected to be >16 h. In order to preserve the organ for transplantation, we thus decided ad hoc to perform HOPE of the ERL to counter the expected increased ischemia reperfusion injury following the prolonged CIT. HOPE via the portal vein was performed using oxygenated University of Wisconsin perfusion solution (VitaSmart, Bridge to Life, UK) with maximum pressure and flow set at 3 mm Hg and 175 mL/min, respectively (Figure 1). HOPE was maintained for 200 min followed by flush of the ERL at the end of perfusion with ice-cold custodiol solution. The transplant procedure itself was uneventful with reconstruction of the right liver artery of the donor to an aberrant right liver artery of the recipient by end-to-end anastomosis. Reperfusion of the graft likewise was uneventful without any signs of postreperfusion syndrome despite 16 h 23 min of CIT. Primary closure of the abdomen was achieved with immediate uptake of liver function regarding regular lactate clearance and courses of transaminases, international normalized ratio, and bilirubin (Figure 1). The child was extubated the same day and discharged from intensive care unit on the next day (total length of hospital stay: 21 d). Apart from a biopsy-proven, mild acute rejection episode in week 5, well responsible to steroid treatment, the postoperative course was uneventful with regular graft function at 3 mo after transplantation and absence of any signs of posttransplant cholangiopathy.FIGURE 1.: Hypothermic oxygenated perfusion of a pediatric extended right lobe: Following ex situ splitting of the liver and cold static storage of 13 h 3 min, the extended right lobe was treated for 200 min by hypothermic oxygenated perfusion (HOPE) via the portal vein prior to implant (A). Transplantation was uneventful with rapid homogenous reperfusion and no signs of postreperfusion syndrome, and the liver graft showed regular lactate clearance (B) and uptake of liver function in the course (C). ALT, alanine transaminase; γ-GT, gamma-glutamyl transferase; INR, international normalized ratio.To our knowledge, this is the first report demonstrating the effective use of HOPE for transplantation of a pediatric ERL. Based on this single-case experience, pressure-controlled perfusion of the pediatric organ seems safe. Potential benefits of HOPE in terms of reducing ischemia reperfusion injury might also apply to pediatric organs.

Delayed Graft Function: State of the Art, November 10–11, 2000. Summit Meeting, Scottsdale, Arizona, USA

As the field of kidney transplantation searches for new ways to expand utilization of deceased donor kidneys while maintaining adequate outcomes, learning from past experience and deciding on the most promising avenues to expand practice continue. In the article by Helantera et al. ([1][1]), data

Synergistic effect of cold and warm ischemia time on postoperative graft function and outcome in human liver transplantation

How can we define expanded criteria for liver donors?

The use of donation after circulatory death (DCD) liver allografts has been constrained by limitations in the duration of donor warm ischemia time (DWIT), donor agonal time (DAT), and cold ischemia time (CIT). The purpose of this study is to assess the impact of longer DWIT, DAT, and CIT on graft survival and other outcomes in DCD liver transplants. The Scientific Registry of Transplant Recipients was queried for adult liver transplants from DCD donors between 2009 and 2015. Donor, recipient, and center variables were included in the analysis. During the study period, 2107 patients underwent liver transplant with DCD allografts. In most patients, DWIT and DAT were <30 minutes. DWIT was <30 minutes in 1804 donors, between 30 and 40 minutes in 248, and >40 minutes in 37. There was no difference in graft survival, duration of posttransplant hospital length of stay, and readmission rate between DCD liver transplants from donors with DWIT <30 minutes and DWIT between 30 and 40 minutes. Similar outcomes were noted for DAT. In the multivariate analysis, DAT and DWIT were not associated with graft loss. The predictors associated with graft loss were donor age, donor sharing, CIT, recipient admission to the intensive care unit, recipient ventilator dependence, Model for End-Stage Liver Disease score, and low-volume transplant centers. Any CIT cutoff >4 hours was associated with increased risk for graft loss. Longer CIT was also associated with a longer posttransplant hospital stay, higher rate of primary nonfunction, and hyperbilirubinemia. In conclusion, slightly longer DAT and DWIT (up to 40 minutes) were not associated with graft loss, longer posttransplant hospitalization, or hospital readmissions, whereas longer CIT was associated with worse outcomes after DCD liver transplants.