Jonathan C. Yeung

Technique for Prolonged Normothermic Ex Vivo Lung Perfusion

BACKGROUND The inhibition of cellular metabolism induced by hypothermia obviates the possibility of substantial reparative processes occurring during organ preservation. The aim of this study was to develop a technique of extended (12-hour) ex vivo lung perfusion (EVLP) at normothermia for assessment and protective maintenance of the donor lung. METHODS Six double-lung blocks from 35-kg pigs and 5 single human lungs were subjected to 12 hours of normothermic EVLP using acellular Steen Solution. In the animal studies, the left lung was transplanted into recipients at the end of EVLP and reperfused for 4 hours to evaluate the impact of prolonged EVLP on post-transplant lung function. A protective mode of mechanical ventilation with controlled perfusion flows and pressures in the pulmonary vasculature were employed during EVLP. Lung oxygenation capacity (DeltaPo(2)), pulmonary vascular resistance and airway pressures were evaluated in the system. Red blood cells were added to the perfusate to a hematocrit of 20% at the end of human lung EVLP to study lung functional assessment with and without cells. RESULTS Lung function was stable during 12 hours of EVLP. This stability during prolonged normothermic EVLP translated into excellent post-transplant lung function (Pao(2)/Fio(2): 527 +/- 22 mm Hg), low edema formation (wet/dry ratio: 5.24 +/- 0.38) and preserved lung histology after transplantation. The acellular perfusion assessment of lung function accurately correlated with post-transplant graft function. CONCLUSIONS Twelve hours of EVLP at physiologic temperatures using an acellular perfusate is achievable and maintains the donor lungs without inflicting significant added injury. This system can be used to assess, maintain and treat injured donor lungs.

Functional Repair of Human Donor Lungs by IL-10 Gene Therapy

Treatment of damaged donor lungs with the cytokine interleukin-10 improves their function, allowing previously unacceptable organs to be used for transplantation. Living Well After Lung Replacement Bumper stickers that counsel motorists to “just breathe” abound—easier said than done when it comes to patients with serious lung disorders. Lung transplantations are on the rise, from 203 in 1990 to more than 1200 in 2008 in the United States. Earlier this year, transplant surgeons at Johns Hopkins presented evidence that more is better—hospitals that perform 20 or more lung transplants per year have the best patient survival rates. However, successful surgeries require healthy donor lungs, a resource that remains in short supply. Now, Keshavjee and colleagues describe a gene therapy treatment protocol to repair lungs after removal from the donor and before transplantation into patients. Candidates for lung transplantation are patients suffering from end-stage lung diseases, such as emphysema, cystic fibrosis, pulmonary fibrosis, and pulmonary arterial hypertension. Organ donors are people who have undergone brain death, a process that is as violent as it sounds: Brain death is accompanied by the spewing of inflammation-inducing molecules called cytokines that damage more than 80% of donated lungs. These injured organs are highly inflamed, and their alveoli—the gas exchange machinery in lungs—are disrupted and only mildly functional. To avoid primary graft dysfunction—lung damage that occurs within the 72 hours after transplantation—transplant surgeons usually reject such injured organs. A method is needed to heal these fixer-upper organs so that they can be used to give patients a new lease on life. Using IL-10, an anti-inflammatory cytokine, Keshavjee’s team devised a treatment to quell inflammation in the injured donor lungs and refurbish the alveoli. Although the standard technique for the handling of organs is to keep them on ice in a sealed bag, this IL-10 gene therapy approach must be performed at body temperature so that the lung’s cellular machinery can express the gene efficiently. The researchers then carried out prolonged ex vivo lung perfusion (EVLP) and kept the lungs breathing outside the body in conditions that mimic physiological ones. Pig lungs that were subjected to IL-10 gene therapy and EVLP for 12 hours displayed reduced inflammation and enhanced function when transplanted into donor pigs, relative to control organs. The same treatment was applied to human lungs deemed unsuitable for transplantation, and these organs, relative to controls, displayed the presence of anti-inflammatory cytokines, repair of alveoli, and improved function, determined by measuring gas exchange and pulmonary vascular resistance. This procedure can yield a larger number of usable lungs and thus more successful transplantations so that patients can “just breathe.” More than 80% of potential donor lungs are injured during brain death of the donor and from complications experienced in the intensive care unit, and therefore cannot be used for transplantation. These lungs show inflammation and disruption of the alveolar-capillary barrier, leading to poor gas exchange. Although the number of patients in need of lung transplantation is increasing, the number of donors is static. We investigated the potential to use gene therapy with an adenoviral vector encoding human interleukin-10 (AdhIL-10) to repair injured donor lungs ex vivo before transplantation. IL-10 is an anti-inflammatory cytokine that mainly exerts its suppressive functions by the inactivation of antigen-presenting cells with consequent inhibition of proinflammatory cytokine secretion. In pigs, AdhIL-10–treated lungs exhibited attenuated inflammation and improved function after transplantation. Lungs from 10 human multiorgan donors that had suffered brain death were determined to be clinically unsuitable for transplantation. They were then maintained for 12 hours at body temperature in an ex vivo lung perfusion system with or without intra-airway delivery of AdhIL-10 gene therapy. AdhIL-10–treated lungs showed significant improvement in function (arterial oxygen pressure and pulmonary vascular resistance) when compared to controls, a favorable shift from proinflammatory to anti-inflammatory cytokine expression, and recovery of alveolar–blood barrier integrity. Thus, treatment of injured human donor lungs with the cytokine IL-10 can improve lung function, potentially rendering injured lungs suitable for transplantation into patients.

Normothermic Ex Vivo Perfusion Prevents Lung Injury Compared to Extended Cold Preservation for Transplantation

Treatment of injured donor lungs ex vivo to accelerate organ recovery and ameliorate reperfusion injury could have a major impact in lung transplantation. We have recently demonstrated a feasible technique for prolonged (12 h) normothermic ex vivo lung perfusion (EVLP). This study was performed to examine the impact of prolonged EVLP on ischemic injury. Pig donor lungs were cold preserved in Perfadex® for 12 h and subsequently divided into two groups: cold static preservation (CSP) or EVLP at 37°C with Steen™ solution for a further 12 h (total 24 h preservation). Lungs were then transplanted and reperfused for 4 h. EVLP preservation resulted in significantly better lung oxygenation (PaO2 531 ± 43 vs. 244 ± 49 mmHg, p < 0.01) and lower edema formation rates after transplantation. Alveolar epithelial cell tight junction integrity, evaluated by zona occludens‐1 protein staining, was disrupted in the cell membranes after prolonged CSP but not after EVLP. The maintenance of integrity of barrier function during EVLP translates into significant attenuation of reperfusion injury and improved graft performance after transplantation. Integrity of functional metabolic pathways during normothermic perfusion was confirmed by effective gene transfer and GFP protein synthesis by lung alveolar cells. In conclusion, EVLP prevents ongoing injury associated with prolonged ischemia and accelerates lung recovery.

Experience with the first 50 ex vivo lung perfusions in clinical transplantation.

OBJECTIVE Normothermic ex vivo lung perfusion is a novel method to evaluate and improve the function of injured donor lungs. We reviewed our experience with 50 consecutive transplants after ex vivo lung perfusion. METHODS A retrospective study using prospectively collected data was performed. High-risk brain death donor lungs (defined as Pao(2)/Fio(2) <300 mm Hg or lungs with radiographic or clinical findings of pulmonary edema) and lungs from cardiac death donors were subjected to 4 to 6 hours of ex vivo lung perfusion. Lungs that achieved stable airway and vascular pressures and Pao(2)/Fio(2) greater than 400 mm Hg during ex vivo lung perfusion were transplanted. The primary end point was the incidence of primary graft dysfunction grade 3 at 72 hours after transplantation. End points were compared with lung transplants not treated with ex vivo lung perfusion (controls). RESULTS A total of 317 lung transplants were performed during the study period (39 months). Fifty-eight ex vivo lung perfusion procedures were performed, resulting in 50 transplants (86% use). Of these, 22 were from cardiac death donors and 28 were from brain death donors. The mean donor Pao(2)/Fio(2) was 334 mm Hg in the ex vivo lung perfusion group and 452 mm Hg in the control group (P = .0001). The incidence of primary graft dysfunction grade 3 at 72 hours was 2% in the ex vivo lung perfusion group and 8.5% in the control group (P = .14). One patient (2%) in the ex vivo lung perfusion group and 7 patients (2.7%) in the control group required extracorporeal lung support for primary graft dysfunction (P = 1.00). The median time to extubation, intensive care unit stay, and hospital length of stay were 2, 4, and 20 days, respectively, in the ex vivo lung perfusion group and 2, 4, and 23 days, respectively, in the control group (P > .05). Thirty-day mortality (4% in the ex vivo lung perfusion group and 3.5% in the control group, P = 1.00) and 1-year survival (87% in the ex vivo lung perfusion group and 86% in the control group, P = 1.00) were similar in both groups. CONCLUSIONS Transplantation of high-risk donor lungs after 4 to 6 hours of ex vivo lung perfusion is safe, and outcomes are similar to those of conventional transplants. Ex vivo lung perfusion improved our center use of donor lungs, accounting for 20% of our current lung transplant activity.

Normothermic Acellular Ex Vivo Liver Perfusion Reduces Liver and Bile Duct Injury of Pig Livers Retrieved After Cardiac Death

We compared cold static with acellular normothermic ex vivo liver perfusion (NEVLP) as a novel preservation technique in a pig model of DCD liver injury. DCD livers (60 min warm ischemia) were cold stored for 4 h, or treated with 4 h cold storage plus 8 h NEVLP. First, the livers were reperfused with diluted blood as a model of transplantation. Liver injury was determined by ALT, oxygen extraction, histology, bile content analysis and hepatic artery (HA) angiography. Second, AST levels and bile production were assessed after DCD liver transplantation. Cold stored versus NEVLP grafts had higher ALT levels (350 ± 125 vs. 55 ± 35 U/L; p < 0.0001), decreased oxygen extraction (250 ± 65 mmHg vs. 410 ± 58 mmHg, p < 0.01) and increased hepatocyte necrosis (45% vs. 10%, p = 0.01). Levels of bilirubin, phospholipids and bile salts were fivefold decreased, while LDH was sixfold higher in cold stored versus NEVLP grafts. HA perfusion was decreased (twofold), and bile duct necrosis was increased (100% vs. 5%, p < 0.0001) in cold stored versus NEVLP livers. Following transplantation, mean serum AST level was higher in the cold stored versus NEVLP group (1809 ± 205 U/L vs. 524 ± 187 U/L, p < 0.05), with similar bile production (2.5 ± 1.2 cc/h vs. 2.8 ± 1.4 cc/h; p = 0.2). NEVLP improved HA perfusion and decreased markers of liver duct injury in DCD grafts.

Initial experience with lung donation after cardiocirculatory death in Canada.

BACKGROUND Organ donation after cardiac death (DCD) has the potential to alleviate some of the shortage of suitable lungs for transplantation. Only limited data describe outcomes after DCD lung transplantation. This study describes the early and intermediate outcomes after DCD lung transplantation in Canada. METHODS Data were collected from donors and recipients involved in DCD lung transplantations between June 2006 and December 2008. Described are the lung DCD protocol, donor characteristics, and the occurrence of post-transplant events including primary graft dysfunction (PGD), bronchial complications, acute rejection (AR), bronchiolitis obliterans syndrome (BOS), and survival. RESULTS Successful multiorgan controlled DCD increased from 4 donors in 2006 to 26 in 2008. Utilization rates of lungs among DCD donors were 0% in 2006, 11% in 2007, and 27% in 2008. The lung transplant team evaluated 13 DCD donors on site, and lungs from 9 donors were ultimately used for 10 recipients. The 30-day mortality was 0%. Severe PGD requiring extracorporeal membrane oxygenation occurred in 1 patient. Median intensive care unit stay was 3.5 days (range, 2-21 days). Hospital stay was 25 days (range, 9-47 days). AR occurred in 2 patients. No early BOS has developed. Nine (90%) patients are alive at a median of 270 days (range, 47-798 days) with good performance status and lung function. One patient died of sepsis 17 months after transplantation. CONCLUSION DCD has steadily increased in Canada since 2006. The use of controlled DCD lungs for transplantation is associated with very acceptable early and intermediate clinical outcomes.

Physiologic assessment of the ex vivo donor lung for transplantation

BACKGROUND The evaluation of donor lungs by normothermic ex vivo acellular perfusion has improved the safety of organ utilization. However, this strategy requires a critical re-evaluation of the parameters used to assess lungs during ex vivo perfusion compared with those traditionally used to evaluate the donor lung in vivo. Using a porcine model, we studied the physiology of acellular lung perfusion with the aim of improving the accuracy of clinical ex vivo evaluation. METHODS Porcine lungs after 10 hours of brain death and 24 hours of cold ischemia and uninjured control lungs were perfused for 12 hours and then transplanted. PaO2, compliance, airway pressure and pulmonary vascular resistance were measured. Ventilation with 100% nitrogen and addition of red blood cells to the perfusate were used to clarify the physiologic disparities between in vivo blood perfusion and ex vivo acellular perfusion. RESULTS During 12 hours of ex vivo perfusion, injured lungs developed edema with decreased compliance and increased airway pressure, but ex vivo PO2 remained stable. After transplantation, injured lungs demonstrated high vascular resistance and poor PaO2. A reduced effect of shunt on ex vivo lung perfusion PO2 was found to be attributable to the linearization of the relationship between oxygen content and PO2, which occurs with acellular perfusate. CONCLUSIONS Ex vivo PO2 may not be the first indication of lung injury and, taken alone, may be misleading in assessing the ex vivo lung. Thus, evaluation of other physiologic parameters takes on greater importance.

Ex Vivo Adenoviral Vector Gene Delivery Results in Decreased Vector-associated Inflammation Pre- and Post–lung Transplantation in the Pig

Acellular normothermic ex vivo lung perfusion (EVLP) is a novel method of donor lung preservation for transplantation. As cellular metabolism is preserved during perfusion, it represents a potential platform for effective gene transduction in donor lungs. We hypothesized that vector-associated inflammation would be reduced during ex vivo delivery due to isolation from the host immune system response. We compared ex vivo with in vivo intratracheal delivery of an E1-, E3-deleted adenoviral vector encoding either green fluorescent protein (GFP) or interleukin-10 (IL-10) to porcine lungs. Twelve hours after delivery, the lung was transplanted and the post-transplant function assessed. We identified significant transgene expression by 12 hours in both in vivo and ex vivo delivered groups. Lung function remained excellent in all ex vivo groups after viral vector delivery; however, as expected, lung function decreased in the in vivo delivered adenovirus vector encoding GFP (AdGFP) group with corresponding increases in IL-1β levels. Transplanted lung function was excellent in the ex vivo transduced lungs and inferior lung function was seen in the in vivo group after transplantation. In summary, ex vivo delivery of adenoviral gene therapy to the donor lung is superior to in vivo delivery in that it leads to less vector-associated inflammation and provides superior post-transplant lung function.

Update on donor assessment, resuscitation, and acceptance criteria, including novel techniques--non-heart-beating donor lung retrieval and ex vivo donor lung perfusion.

The shortage of adequate organ donors remains a great challenge in clinical lung transplantation. With increasing experience in the medical management and surgical technique of lung transplantation, gradual expansion of the criteria for lung donor selection has occurred with beneficial effects on the donor pool. Interest in donation after cardiac death also is increasing as the gap increases between donors and the needs of listed patients. Successful use of these new sources of lungs depends on the accurate assessment and prediction of transplanted lung function. Promising techniques for lung assessment and diagnostics include investigating key genes associated with graft failure or good graft performance using molecular approaches, and ex vivo evaluation. Further studies are needed to answer remaining questions about the best technique and solution to reperfuse human lungs for several hours without edema formation. As the predictive ability to discern good from injured donor lungs improves, strategies to repair donor lungs become increasingly important. Prolonged normothermic EVLP seems to be a platform on which many reparative strategies can be realized. With these new methods for assessing and resuscitating lungs accurately, it is hoped that inroads will be made toward providing every listed patient a chance for successful lung transplantation.

Lung Transplantation With Donation After Circulatory Determination of Death Donors and the Impact of Ex Vivo Lung Perfusion

The growing demand for suitable lungs for transplantation drives the quest for alternative strategies to expand the donor pool. The aim of this study is to evaluate the outcomes of lung transplantation (LTx) with donation after circulatory determination of death (DCDD) and the impact of selective ex vivo lung perfusion (EVLP). From 2007 to 2013, 673 LTx were performed, with 62 (9.2%) of them using DCDDs (seven bridged cases). Cases bridged with mechanical ventilation/extracorporeal life support were excluded. From 55 DCDDs, 28 (51%) underwent EVLP. Outcomes for LTx using DCDDs and donation after neurological determination of death (DNDD) donors were similar, with 1 and 5‐year survivals of 85% and 54% versus 86% and 62%, respectively (p = 0.43). Although comparison of survival curves between DCDD + EVLP versus DCDD‐no EVLP showed no significant difference, DCDD + EVLP cases presented shorter hospital stay (median 18 vs. 23 days, p = 0.047) and a trend toward shorter length of mechanical ventilation (2 vs. 3 days, p = 0.059). DCDDs represent a valuable source of lungs for transplantation, providing similar results to DNDDs. EVLP seems an important technique in the armamentarium to safely increase lung utilization from DCDDs; however, further studies are necessary to better define the role of EVLP in this context.