Harnessing whole human liver ex situ normothermic perfusion for preclinical AAV vector evaluation.

Developing clinically predictive model systems for evaluating gene transfer and gene editing technologies has become increasingly important in the era of personalized medicine. Liver-directed gene therapies present a unique challenge due to the complexity of the human liver. In this work, we describe the application of whole human liver explants in an ex situ normothermic perfusion system to evaluate a set of fourteen natural and bioengineered adeno-associated viral (AAV) vectors directly in human liver, in the presence and absence of neutralizing human sera. Under non-neutralizing conditions, the recently developed AAV variants, AAV-SYD12 and AAV-LK03, emerged as the most functional variants in terms of cellular uptake and transgene expression. However, when assessed in the presence of human plasma containing anti-AAV neutralizing antibodies (NAbs), vectors of human origin, specifically those derived from AAV2/AAV3b, were extensively neutralized, whereas AAV8- derived variants performed efficiently. This study demonstrates the potential of using normothermic liver perfusion as a model for early-stage testing of liver-focused gene therapies. The results offer preliminary insights that could help inform the development of more effective translational strategies.

Incorporating a hemodialysis filter into a commercial normothermic perfusion system to facilitate long-term preservation of human split-livers.

BACKGROUND: Normothermic machine perfusion (NMP) allows for the assessment and resuscitation of ex-vivo human livers prior to transplantation. Commercially available NMP systems are closed circuits that accumulate metabolic waste and cytokines over time, potentially limiting organ preservation times. Dialysis has been proposed as a method to remove waste and excess fluid from such systems. This study aimed to demonstrate the utility of integrating dialysis into a commercially available system by quantifying solute removal. METHODS: A dialysis filter was attached in parallel to a commercially available liver perfusion system. Three livers declined for transplantation were split before undergoing long-term NMP with blood using the modified system. During perfusion, dialysate flow rates were set in the range of 100-600 mL/h for short periods of time. At each flow rate, perfusate and spent dialysate samples were collected and analyzed for solute clearance. RESULTS: The addition of dialysis to a commercial NMP system removed water-soluble waste and helped regulate electrolyte concentrations. Interleukin-6 was successfully removed from the perfusate. Solute clearance was proportional to dialysate flow rate. A guide for our perfusion setup was created for the appropriate selection of dialysis flow rates and duration based on real-time perfusate composition. CONCLUSIONS: Dialysis circuits can efficiently remove waste and regulate perfusate composition, and can be easily incorporated to improve the performance of commercially available systems. Quantification of the effect of dialysis on perfusate composition enables refined dialysis control to optimize electrolyte profiles and avoid the over- or under-correction of key solutes.

Microbial Contamination During Long-term Ex Vivo Normothermic Machine Perfusion of Human Livers.

BACKGROUND: Normothermic machine perfusion permits the ex vivo preservation of human livers before transplantation. Long-term perfusion for days-to-weeks provides the opportunity for enhanced pretransplant assessment and potential regeneration of organs. However, this risks microbial contamination and infection of the recipient if the organ is transplanted. An understanding of perfusate microbial contamination is required to inform infection control procedures and antimicrobial prophylaxis for this technology. METHODS: We modified a liver perfusion machine for long-term use by adding long-term oxygenators and a dialysis filter. Human livers that were not suitable for transplantation were perfused using a red-cell-based perfusate under aseptic and normothermic conditions (36 °C) with a goal of 14 d. Cephazolin was added to the perfusate for antimicrobial prophylaxis. Perfusate and bile were sampled every 72 h for microbial culture. RESULTS: Eighteen partial human livers (9 left lateral segment grafts and 9 extended right grafts) were perfused using our perfusion system. The median survival was 7.2 d. All organs surviving longer than 7 d (9/18) had negative perfusate cultures at 24 and 48 h. Half of the grafts (9/18) became culture-positive by the end of perfusion. Microbial contaminants included Gram-negative ( Pseudomonas species, Proteus mirabilis, Stenotrophomonas maltophilia ) and Gram-positive bacteria ( Staphylococcus epidermidis , Enterococcus faecalis , and Bacillus species) as well as yeast ( Candida albicans ). CONCLUSIONS: Microbial contamination of perfusate is common during long-term perfusion of human livers with both exogenous and endogenous sources. Enhanced infection control practices and review of targeted antimicrobial prophylaxis are likely to be necessary for translation into the clinical arena.

Indocyanine green: A novel marker for assessment of graft quality during ex situ normothermic machine perfusion of human livers.

BACKGROUND: Ex situ machine perfusion facilitates the assessment of livers prior to transplantation. However, currently available markers of liver function poorly predict long-term graft function. Indocyanine green (ICG) is a liver-specific dye which, although common in vivo, has never been comprehensively evaluated for the assessment of graft quality during ex situ machine perfusion. This study aimed to assess the utility of ICG in the ex situ setting. METHODS: Using a customized long-term perfusion system, human livers that were not suitable for transplantation were perfused using a red cell-based perfusate. ICG was delivered into the perfusate on days 0, 1, and 4 to assess ICG clearance (spectrophotometric absorbance at 805 nm) and ICG fluorescence (near-infrared camera). RESULTS: Sixteen partial livers were perfused for a median duration of 172 h (7.2 days). On day 0, the median ICG perfusate disappearance rate (PDR) was 7.5%/min and the median ICG retention at 15 min was 9.9%. Grafts that survived ≥7 days had a significantly higher median ICG PDR on day 0 (14.5%/min vs. 6.5%/min, p = 0.005) but not on days 1 or 4. ICG perfusion demonstrated that long-surviving grafts had a significantly lower median red-value (89.8 vs. 118.6, p = 0.011) and a significantly lower median blue-value (12.9 vs. 22.6, p = 0.045) than short-surviving grafts. CONCLUSION: ICG is a novel marker for the assessment of liver function during ex situ normothermic machine perfusion. ICG PDR and quantitative ICG perfusion can distinguish between long- and short-surviving grafts and demonstrate the utility of ICG in the assessment of graft quality prior to transplant.

Histological Assessment of the Bile Duct before Liver Transplantation: Does the Bile Duct Injury Score Predict Biliary Strictures?

INTRODUCTION: Histological injury to the biliary tree during organ preservation leads to biliary strictures after liver transplantation. The Bile Duct Injury (BDI) score was developed to assess histological injury and identify the grafts most likely to develop biliary strictures. The BDI score evaluates the bile duct mural stroma, peribiliary vascular plexus (PVP) and deep peribiliary glands (DPGs), which were correlated with post-transplant biliary strictures. However, the BDI score has not been externally validated. The aim of this study was to verify whether the BDI score could predict biliary strictures at our transplant centre. METHODS: Brain-dead donor liver grafts transplanted at a single institution from March 2015 to June 2016 were included in this analysis. Bile duct biopsies were collected immediately before transplantation and assessed for bile duct injury by two blinded pathologists. The primary outcome was the development of clinically significant biliary strictures within 24 months post-transplant. RESULTS: Fifty-seven grafts were included in the study which included 16 biliary strictures (28%). Using the BDI score, mural stromal, PVP and DPG injury did not correlate with biliary strictures including Non-Anastomotic Strictures. Severe inflammation (>50 leucocytes per HPF) was the only histological feature inversely correlated with the primary outcome (absent in the biliary stricture group vs. 41% in the no-stricture group, p = 0.001). CONCLUSIONS: The current study highlights limitations of the histological assessment of bile duct injury. Although all grafts had bile duct injury, only inflammation was associated with biliary strictures. The BDI score was unable to predict post-transplant biliary strictures in our patient population.

An unsupervised learning approach to identify immunoglobulin utilization patterns using electronic health records.

BACKGROUND: Managing Canada's immunoglobulin (Ig) product resource allocation is challenging due to increasing demand, high expenditure, and global shortages. Detection of groups with high utilization rates can help with resource planning for Ig products. This study aims to uncover utilization subgroups among the Ig recipients using electronic health records (EHRs). METHODS: The study included all Ig recipients (intravenous or subcutaneous) in Calgary from 2014 to 2020, and their EHR data, including blood inventory, recipient demographics, and laboratory test results, were analyzed. Patient clusters were derived based on patient characteristics and laboratory test data using K-means clustering. Clusters were interpreted using descriptive analyses and visualization techniques. RESULTS: Among 4112 recipients, six clusters were identified. Clusters 1 and 2 comprised 408 (9.9%) and 1272 (30.9%) patients, respectively, contributing to 62.2% and 27.1% of total Ig utilization. Cluster 3 included 1253 (30.5%) patients, with 86.4% of infusions administered in an inpatient setting. Cluster 4, comprising 1034 (25.1%) patients, had a median age of 4 years, while clusters 2-6 were adults with median ages of 46-60. Cluster 5 had 62 (1.5%) patients, with 77.3% infusions occurring in emergency departments. Cluster 6 contained 83 (2.0%) patients receiving subcutaneous Ig treatments. CONCLUSION: The results identified data-driven segmentations of patients with high Ig utilization rates and patients with high risk for short-term inpatient use. Our report is the first on EHR data-driven clustering of Ig utilization patterns. The findings hold the potential to inform demand forecasting and resource allocation decisions during shortages of Ig products.

Long-term ex situ normothermic perfusion of human split livers for more than 1 week.

Current machine perfusion technology permits livers to be preserved ex situ for short periods to assess viability prior to transplant. Long-term normothermic perfusion of livers is an emerging field with tremendous potential for the assessment, recovery, and modification of organs. In this study, we aimed to develop a long-term model of ex situ perfusion including a surgical split and simultaneous perfusion of both partial organs. Human livers declined for transplantation were perfused using a red blood cell-based perfusate under normothermic conditions (36 °C) and then split and simultaneously perfused on separate machines. Ten human livers were split, resulting in 20 partial livers. The median ex situ viability was 125 h, and the median ex situ survival was 165 h. Long-term survival was demonstrated by lactate clearance, bile production, Factor-V production, and storage of adenosine triphosphate. Here, we report the long-term ex situ perfusion of human livers and demonstrate the ability to split and perfuse these organs using a standardised protocol.

Ex Vivo Preservation of Ovine Periosteum Using a Perfusion Bioreactor System.

Periosteum is a highly vascularized membrane lining the surface of bones. It plays essential roles in bone repair following injury and reconstruction following invasive surgeries. To broaden the use of periosteum, including for augmenting in vitro bone engineering and/or in vivo bone repair, we have developed an ex vivo perfusion bioreactor system to maintain the cellular viability and metabolism of surgically resected periosteal flaps. Each specimen was placed in a 3D printed bioreactor connected to a peristaltic pump designed for the optimal flow rates of tissue perfusate. Nutrients and oxygen were perfused via the periosteal arteries to mimic physiological conditions. Biochemical assays and histological staining indicate component cell viability after perfusion for almost 4 weeks. Our work provides the proof-of-concept of ex vivo periosteum perfusion for long-term tissue preservation, paving the way for innovative bone engineering approaches that use autotransplanted periosteum to enhance in vivo bone repair.

Liver splitting during normothermic machine perfusion: a novel method to combine the advantages of both in-situ and ex-vivo techniques.

BACKGROUND: Split liver transplantation permits the transplant of two recipients using a single donor liver. Liver splitting can be performed using the ex-vivo technique (more convenient), or the in-situ technique (shorter cold ischaemic time). We aimed to develop a technique for liver splitting during normothermic machine perfusion which combines the advantages of both techniques and permits graft assessment prior to transplant. METHODS: Human livers declined for transplantation were perfused at 36 °C using a modified-commercial perfusion machine. We developed a six-step method to split whole livers into left lateral segment grafts and extended right grafts. Both partial livers were then perfused on separate machines for individual assessment. RESULTS: Using our technique, 10 whole livers were successfully split during normothermic perfusion resulting in 20 partial grafts. Apart from a single graft which failed due to a technical error, all grafts survived for 24-h after splitting. Survival was demonstrated by lactate clearance, bile production and synthesis of coagulation factors. CONCLUSIONS: Liver splitting during normothermic machine perfusion has the potential to revolutionise split liver transplantation. We describe a novel technique that reliably achieves two grafts from a single donor liver. This raises the possibility of semi-elective transplantation, and sophisticated graft assessment prior to implant.

Does an Additional Bile Duct Flush With Low-viscosity Preservation Solution Reduce Bile Duct Injury? A Single-blinded Randomized Clinical Trial.

Biliary complications are a common cause of morbidity after liver transplantation and associated with bile duct injury. To reduce injury, a bile duct flush is performed with high-viscosity preservation solution. It has been suggested that an earlier additional bile duct flush with low-viscosity preservation solution may reduce bile duct injury and biliary complications. This study aimed to investigate whether an earlier additional bile duct flush would reduce bile duct injury or biliary complications. Methods: A randomized trial was conducted using 64 liver grafts from brain dead donors. The control group received a bile duct flush with University of Wisconsin (UW) solution after donor hepatectomy. The intervention group received a bile duct flush using low-viscosity Marshall solution immediately after the onset of cold ischemia and a bile duct flush with University of Wisconsin solution after donor hepatectomy. The primary outcomes were the degree of histological bile duct injury, assessed using the bile duct injury score, and biliary complications within 24 mo of transplant. Results: Bile duct injury scores were not different between the 2 groups. Similar rates of biliary complications occurred in the intervention group (31% [n = 9]) and controls (23% [n = 8]) (P = 0.573). No difference between groups was observed for anastomotic strictures (24% versus 20%, P = 0.766) or nonanastomotic strictures (7% versus 6%, P = 1.00). Conclusions: This is the first randomized trial to investigate an additional bile duct flush using low-viscosity preservation solution during organ procurement. The findings from this study suggest that performing an earlier additional bile duct flush with Marshall solution does not prevent biliary complications and bile duct injury.

Long-term normothermic perfusion of human livers for longer than 12 days.

In this case report, we preserved human livers for up to 13 days under normothermic conditions using a modified commercial perfusion system. Two whole livers were split into two left lateral segment grafts and two extended right grafts without interruption to blood flow and then perfused on separate machines. Not only does this provide the basis for a meaningful study of liver function in the long term, but this could also facilitate the development of a model of ex situ liver regeneration.

Is it safe to expand the indications for split liver transplantation in adults? A single-center analysis of 155 in-situ splits.

INTRODUCTION: Split liver transplantation (SLT) enables two recipients to be transplanted using a single donor liver; typically, an adult and a child. Despite equivalent long-term outcomes to whole grafts in selected adults, the use of these grafts in high-risk adult recipients with high model for end-stage liver disease (MELD) scores (≥30), a poor pre-transplant clinical status (ICU or hospital-bound), acute liver failure or retransplantation remains controversial. METHODS: We retrospectively analyzed all deceased donor adult liver transplants performed between July 2002 and November 2019 at a single high-volume center and performed a propensity score-matched analysis. A subgroup analysis was performed to assess utility of these grafts for high-risk recipients. RESULTS: A total of 1090 adult liver transplants were performed, including 155 SLT (14%). Graft survival at 1-, 3- and 5-years were comparable between recipients of split and whole liver grafts (82%, 79% and 74% vs. 86%, 81% and 77%, respectively, log rank P = .537), as was patient survival at 1-, 3- and 5-years. Recipients of split grafts were more likely to have biliary complications and hepatic artery thrombosis, but equivalent long-term survival. Recipients with high MELD scores or a poor pre-transplant clinical status had similar patient and graft survival and complication profiles irrespective of whether they received split or whole grafts. CONCLUSIONS: SLT is an important method for addressing donor shortages and provides comparable long-term outcomes in adult recipients despite an increase in short-term complications. SLT use in high-risk recipients should be considered to allow for sickest-first allocation policies.

Current and Potential Applications for Indocyanine Green in Liver Transplantation.

Indocyanine green (ICG) is a fluorescent dye taken up and almost exclusively cleared by the liver. Measurement of its clearance and visualization of its fluorescence make it suitable for a number of potential applications in liver transplantation including assessment of liver function and real-time assessment of arterial, venous, and biliary structures. ICG clearance can be used to assess donor graft quality before procurement and graft metabolic function before transplant using normothermic ex vivo machine perfusion. ICG clearance in the post-liver transplantation period is able to predict recipient outcomes with correlations to early allograft dysfunction and postoperative complications. After absorbing light in the near-infrared spectrum, ICG also emits fluorescence at 835 nm. This allows the assessment of vascular patency after reconstruction and patterns of liver perfusion in real time. ICG perfusion patterns after revascularization are also associated with posttransplant graft function and survival. ICG fluorescence cholangiography is routine in a number of centers and acts as an aid to identifying the optimal point of bile duct division during living donor liver transplantation to optimize safety for both donor and recipient. In summary, ICG is a versatile tool and has a number of useful applications in the liver transplantation journey including assessment of liver function, perfusion assessment, and cholangiography. Further research and clinical trials are required to validate and standardize its routine use in liver transplantation.

Biliary complications in donation after circulatory death liver transplantation: the Australian National Liver Transplantation Unit's experience.

BACKGROUND: Biliary complications are the most common complications of donation after circulatory death (DCD) liver transplantation and the international experience with DCD transplants suggests increased rates of biliary complications compared to donation after brain death transplants. Therefore, it is important to understand factors that are associated with the development of biliary complications within the Australian DCD context in order to inform future practice. The aim of this study is to determine the incidence of biliary complications after DCD liver transplantation at the Australian National Liver Transplantation Unit and identify factors associated with this outcome. METHODS: A retrospective analysis of all adult DCD liver transplants at the Australian National Liver Transplantation Unit from 2007 to 2015 was undertaken. The primary outcome measure was the incidence of biliary complications and was censored on 31 December 2016. Recipients were then stratified into groups based on the development of biliary complications and risk factor analysis was performed. RESULTS: Biliary complications occurred in 35% of DCD transplants, including seven anastomotic strictures and 10 non-anastomotic strictures. Higher donor risk index scores (P = 0.03), post-transplant portal vein complications (P = 0.042) and peak gamma-glutamyl transferase levels within 7 days post-transplant (P = 0.047) were associated with biliary complications. CONCLUSION: Findings from this study demonstrate that biliary complications remain common in DCD liver recipients. Recipients who developed a biliary complication tended to have higher donor risk index, elevated peak gamma-glutamyl transferase levels within 7 days post-transplant or a portal vein complication. The presence of any of these factors should prompt close monitoring for post-transplant biliary complications.

Colonic lymphangioma presenting with intermittent pain and intussusception.

BACKGROUND: Cystic lymphangiomas are rare benign tumours and their actual incidence in the colon is unclear. Within the large bowel, these lesions are often submucosal and are incidental findings on colonoscopy. CASE REPORT: A 43-year-old man with colonic lymphangioma presented with a 7-month history of abdominal pain and altered bowel habits. Computed tomography showed a cystic mass lesion at the distal descending colon. Colonoscopy revealed a smooth, polypoid mass, initially thought as an inverting diverticulum. He underwent laparoscopic high anterior resection. The pathological analysis revealed the descending colon mass to be a lymphangioma with no evidence of diverticulosis. CONCLUSION: There is increasing incidence in lymphangioma due to the increasing accessibility to colonoscopy, particularly for asymptomatic or subacute disease. Although colonoscopic excision, sclerotherapy and use of steroids or fibrin glue have been advocated as possible modes of treatment, surgical excision is still considered to be the treatment of choice.