Period Of Static Cold Storage Research Articles

O242: Prolonged subnormothermic acellular machine perfusion of human kidneys

Abstract Introduction Prolonged periods of ex vivo perfusion are required to effectively deliver regenerative therapies directly to a kidney before transplantation. This provides time to start repair processes and reduce the likelihood of early graft dysfunction. The aim of this study was to assess the capability of extending the preservation interval using subnormothermic acelllular machine perfusion (SNAP). Methods After a period of static cold storage, 12 human kidneys declined for transplantation were perfused with an oxygenated human serum albumin-based solution at 32°C for 6, 12 or 24h (n=4 per group). After preservation all kidneys were reperfused with a red blood cell-based solution at 37°C for 4h for assessment. Results Functional parameters remained stable during SNAP in all kidneys. However, the perfusate flow rate was significantly lower in the 24h group (24h 166±45, 12h 266±45, 6h 214±36 mL/min/100g; P=0.008). After reperfusion, levels of urinary neutrophil gelatinase-associated lipocalin (uNGAL), insulin-like growth factor binding protein 7 (IGFBP7) and liver-type fatty acid-binding protein (L-FABP) were similar between groups (P>0.05). Levels of oxygen consumption were significantly lower in 24h kidneys compared to 12h (24h 1.45±0.86, 12h 4.3±1.78 mL/min/100g; P=0.0294). Histological evaluation showed a preserved renal morphology in all kidneys, except for the 24h group which showed a mild increase in tubular and endothelial damage. Conclusion This study demonstrated that human kidneys can be successfully preserved with SNAP for up to 12h. There was some evidence of additional ischaemic injury in the 24h SNAP kidneys which warrants further investigation.

Metabolic perturbation studies using a Nash Equilibrium model of liver machine perfusion: modeling oxidative stress and effect of glutathione supplementation

The current clinical standard of Static Cold Storage (SCS) which involves preservation on ice (about +4°C) in a hypoxic state limits storage to a few hours for metabolically active tissues such as the liver and the heart. This period of hypoxia during can generate superoxide and other free radicals from purine metabolism, a well-established component of ischemia/reperfusion injury (IRI). Machine perfusion is at the cutting edge of organ preservation, which provides a functional, oxygenated preservation modality that can avoid/attenuate IRI. In clinical application, perfusion usually follows a period of SCS. This presentation of oxygen following hypoxia can lead to superoxide and hydrogen peroxide generation, but machine perfusion also allows manipulation of the temperature profiles and supply of antioxidant treatments, which could be used to minimize such issues. However, metabolomic data is difficult to gather, and there are currently no mathematical models present to allow rational design of experiments or guide clinical practice. In this article, the effects of a gradual warming temperature policy and glutathione supplementation to minimize oxidative stress are studied. An optimal gradual warming temperature policy for mid-thermic machine perfusion of a liver metabolic model is determined using a combination of Nash Equilibrium and Monte Carlo optimization. Using this optimal gradual warming temperature policy, minimum GSH requirements to maintain hydrogen peroxide concentrations in the normal region are calculated using a different Monte Carlo optimization methodology. In addition, the dynamic behavior of key metabolites and cofactors are determined. Results show that the minimum GSH requirement increases and that the ratio of GSH/GSSG decreases with increasing hydrogen peroxide concentration. In addition, at high concentrations of hydrogen peroxide it is shown that cytochrome C undergoes dysfunction leading to a decrease in useful oxygen consumption and ATP synthesis from the electron transport chain and an overall reduction in the energy charge for the liver cells.

Letter to the Editor: Can ex-situ normothermic perfusion improve graft survival among donation after circulatory death liver allografts?

To the editor, We read with great interest the manuscript by Ibeabuchi et al recently published in Liver Transplantation.1 The authors found that ex-situ normothermic liver perfusion (ENLP) improved graft survival compared to static cold storage among donation after circulatory death (DCD) liver allografts by identifying the United Network of Organ Sharing database. This was consistent with the results of a randomized trial, which confirmed that ENLP was associated with a 50% lower level of graft injury, with these benefits being greatest in the DCD subgroup.2 Besides, considering that ENLP DCDs were performed at 20 (19.8%) of the 101 centers, the authors conducted a subanalysis restricted to the 20 centers to eliminate potential baseline differences in graft outcomes related to center experience, and the results were largely unchanged. Despite this, the results should still be interpreted with caution. The number of ENLP DCDs performed among these 20 centers ranged from 1 to 10 (median: 2; interquartile range: 1–4.5). In only a few cases in each center, it was not clear in what circumstances ENLP was selected, which inevitably led to significant limitations. All the time, the time window for evaluating the feasibility of donor livers is relatively short, and the time for surgery is severely limited. These all lead to the urgency of liver transplantation and sometimes to the fact that part of the liver available for transplantation has to be wasted. The advantages of ENLP are to create a more physiological environment in vitro and maintain the normal physiological function of donor livers, which can provide opportunities for precise evaluation and intervention treatment of donor livers. ENLP intervention allows longer total preservation times, achieves higher organ use rates, and may indeed be a promising option for organ preservation in vitro.3 However, the ENLP process is relatively complex and requires advanced equipment and technological capabilities. Once problems occur in the operation process, it is likely to cause further damage to the liver. Meanwhile, ENLP does not appear to be suitable for split liver transplantation because it may increase the warm ischemia time through additional rewarming steps. Last, whether ENLP is required for the full duration of livers’ preservation or can equally well be applied after a short period of static cold storage when the livers reach the transplant center.

Inulin Clearance During Ex vivo Normothermic Machine Perfusion as a Marker of Renal Function.

Normothermic machine perfusion (NMP) offers a unique opportunity to objectively assess donor organ quality. This study describes the evaluation of inulin clearance as a potential marker for the ex vivo function of porcine kidneys during NMP. The function assessment was performed in both kidneys from slaughterhouse pigs (n = 20) and kidneys from pigs in a laboratory setting (n = 28). The kidneys were exposed to different warm ischemia times (WIT). After a period of static cold storage, the kidneys underwent a 4-hour NMP with autologous whole blood. Inulin clearance, hemodynamic parameters, and urine output were measured. Based on the inulin excretion behavior laboratory pig kidneys were assigned to three classes (functional, limited functional, and nonfunctional), slaughterhouse pig kidneys to two classes (limited functional and nonfunctional), respectively. Contrary to the marginal kidneys of the slaughterhouse pigs, the functional variation of kidneys of the laboratory pigs was associated with the WIT. A correlation between functional kidneys and a WIT less than 25 min was shown. Because none of the slaughterhouse pig kidneys could be assigned to the functional class, only the laboratory pig kidneys were used for examinations with functional markers. Renal blood flow and urine output during NMP correlated significantly ( p < 0.01) with ex vivo kidney function. This study demonstrated that inulin is a marker of high quality for the evaluation of suggested kidney function after NMP with whole blood. Furthermore, surrogate markers measured during NMP can be used to describe and predict the physiologic behavior of kidneys before transplantation.

Hypothermic machine perfusion can safely prolong cold ischemia time in deceased donor kidney transplantation. A retrospective analysis on postoperative morbidity and graft function.

In deceased donor kidney transplantation (KT), a prolonged cold ischemia time (CIT) is a negative prognostic factor for KT outcome, and the efficacy of hypothermic machine perfusion (HMP) in prolonging CIT without any additional hazard is highly debated. We conducted a retrospective study on a cohort of 154 single graft deceased donor KTs, in which a delayed HMP, after a preliminary period of static cold storage (SCS), was used to prolong CIT for logistic reasons. Primary outcomes were postoperative complications as well as 1year graft survival and function. 73 cases (47.4%) were managed with HMP and planned KT, while 81 (52.6%) with SCS and urgent KT. The median CIT in HMP group and SCS group was 29hour:57minutes [27-31hour:45minutes] and 11hour:25minutes [9-14hour:30minutes], respectively (P<.001). The period of SCS in the HMP group was significantly shorter than in the SCS group (10 vs. 11hour:25minutes, P=.02) as well as the prevalence of expanded criteria donors was significantly higher (43.8% vs. 18.5%, P<.01). After propensity score matching for these two baseline characteristics, the HMP and SCS groups showed comparable outcomes in terms of delayed graft function, vascular, and urologic complications, infections, and episodes of graft rejection. At 1 year follow-up, serum creatinine levels were comparable between the groups. Therefore, the use of HMP to prolong the CIT and convert KT into a planned procedure seemed to have an adequate safety profile, with outcomes comparable to KT managed as an urgent procedure and a CIT nearly three time shorter.

Normothermic Machine Preservation of Marginal Livers in Australasia - Initial Brisbane Experience Using a “Back-to-Base” Approach

Introduction: The assessment of marginal liver suitability using empirical criteria remains imprecise. Ex-vivo normothermic machine perfusion (NMP) allows for a period of graft assessment under near-physiological conditions, which might improve the selection of such livers for transplantation. We describe the first 11 clinical applications of NMP in Australasia using a “back-to-base” approach. Methods: Eleven marginal livers were accepted for preservation and assessment on the OrganOx metra device following a period of static cold storage (post-SCS-NMP). Average donor risk index was 1.53 (1.08-1.93), with five grafts donated after brain death (DBD) and six donated after circulatory death (DCD). All DCD grafts were outside of established local criteria. Average cold ischaemic time and NMP time was 5.2 (4.2-6.5) hours and 12.0 (9.1-17.6) hours respectively. Recipient MELD score was 17 (11-20), including one combined liver-kidney transplant and one re-transplant. Results: All livers met pre-established viability criteria and were successfully transplanted. Five (45%) recipients developed early allograft dysfunction based solely on peak AST >2000 U/L (average 1860 (629-8910) U/L, Figure 1). Two patients (18%) required revisional surgery for biliary anastomotic complications following initial Roux-en-Y hepaticojejunostomy. No clinically significant biliary problems were observed in the nine patients receiving duct-to-duct anastomosis. All cases have satisfactory graft function to date. Conclusions: Post-SCS NMP was successfully implemented in 11 cases, enabling the safe utilisation of grafts deemed non-transplantable using static cold storage preservation alone. The technique was user friendly, improved transplant logistics and surgeon confidence, and increased local transplant activity by 10%.

Effects of Delayed Hypothermic Machine Perfusion on Kidney Grafts with a Preliminary Period of Static Cold Storage and a Total Cold Ischemia Time of Over 24 Hours.

BackgroundHypothermic machine perfusion (HMP) appears to exert a reconditioning effect on the ischemic damage of kidney grafts. However, some concerns still remain about its real effectiveness when it is delayed after a preliminary period of static cold storage (SCS) or with prolonged overall cold ischemia time (CIT).Material/MethodsThe effect of HMP on hemodynamic, metabolic, histological and ultrastructural features of grafts was investigated in 21 single-kidney grafts treated with a delayed HMP after SCS and with a total CIT of over 24 h.ResultsThe mean CIT, SCS, and HMP times were 29 h, 12 h, and 18 h, respectively. Longer SCS was associated with higher vascular resistance and lower arterial flow. In the pre- vs. post-HMP comparison, a significant decrease in arterial resistances and increase of flow were recorded. The hemodynamic improvement was independent of HMP duration. The perfused grafts retained some metabolic activity, with a statistically significant decrease of pH, pO2, and glucose levels, and increase of lactates in the perfusion liquid, by the end of HMP. Longer SCS was associated with higher pH and greater pO2 decrease during HMP. Light microscopy and transmission electronic microscopy revealed no significant variations in nuclear, cytoplasmic, or ultrastructural damage. SCS, HMP, and CIT were not identified as risk factor for delayed graft function or rejection.ConclusionsA delayed and extended HMP can recover the graft hemodynamic function, maintain some metabolic activity, and stabilize the accumulated ischemic damage due to a preliminary SCS.

Cell-free microRNAs as early predictors of graft viability during ex vivo normothermic machine perfusion of human donor livers.

BackgroundCell‐free microRNAs (miRs) have emerged as early and sensitive biomarkers for tissue injury and function. This study aimed to investigate whether the release of hepatocyte‐derived microRNAs (HDmiRs) and cholangiocyte‐derived miRs (CDmiRs) correlates with hepato‐cholangiocellular injury and function during oxygenated, normothermic machine perfusion (NMP) of human liver grafts.MethodsDonor livers (n = 12), declined for transplantation, were subjected to oxygenated NMP (6 hours) after a period of static cold storage (median 544 minutes (IQR 421‐674)). Perfusate and bile samples were analyzed by qRT‐PCR for HDmiR‐122 and CDmiR‐222. Spearman correlations were performed between miR levels and currently available indicators and classic markers.ResultsBoth HDmiR‐122 and CDmiR‐222 levels in perfusate at 30 minutes of NMP strongly correlated with hepatocyte injury (peak perfusate AST) and cholangiocyte injury (peak biliary LDH). In bile, only CDmiR‐222 correlated with these injury markers. For hepato‐cholangiocellular function, both miRs in perfusate correlated with total bilirubin, while HDmiR‐122 (in perfusate) and CDmiR‐222 (in bile) correlated with bicarbonate secretion. Both the relative ratio of HDmiR‐122/CDmiR‐222 and AST in perfusate at 30 minutes significantly correlated with cumulative bile production, but only the relative ratio was predictive of histopathological injury after 6 hours NMP.ConclusionEarly levels of HDmiR‐122 and CDmiR‐222, in perfusate and/or bile, are predictive of excretory functions and hepato‐cholangiocellular injury after 6 hours NMP. These miRs may represent new biomarkers for graft viability and function during machine perfusion.

Viability testing and transplantation of marginal livers (VITTAL) using normothermic machine perfusion: study protocol for an open-label, non-randomised, prospective, single-arm trial

IntroductionThe use of marginal or extended criteria donor livers is increasing. These organs carry a greater risk of initial dysfunction and early failure, as well as inferior long-term outcomes. As...

The use of ex-vivo normothermic perfusion for the resuscitation and assessment of human kidneys discarded because of inadequate in situ perfusion.

BackgroundMany kidneys are rejected for transplantation due to inadequate in situ perfusion during organ retrieval because of the risk of additional ischaemic injury and microvasculature thrombosis. This study describes the use of ex vivo normothermic perfusion (EVNP) for the resuscitation and assessment of human kidneys that were discarded after inadequate in situ perfusion.MethodsTwenty-two human kidneys were retrieved but then deemed unsuitable for transplantation, primarily due to inadequate in situ perfusion. After a period of static cold storage, kidneys were perfused for 60 min with an oxygenated red cell based solution at 36 °C.ResultsNineteen out of 22 kidneys (86 %) were from DCD donors. During EVNP, kidneys were assessed and scored based on their macroscopic appearance, measures of renal blood flow and urine production. Kidneys were scored from 1 indicating the least injury to 5, indicating the worst. Twelve kidneys had an EVNP score of 1–2, 7 scored 3–4 and 3 kidneys scored 5. The EVNP score 5 kidneys had a low level of tubular function compared to the score 1–4 kidneys. Their perfusion parameters did not improve during EVNP and they were considered non-transplantable. There was no association between the histological evaluation and EVNP parameters.ConclusionEVNP restores function ex vivo and enables an assessment of kidneys that have been declined for transplantation due to inadequate in situ perfusion.