Donor Animal Characteristics and Elapsed Time
Donor animal characteristics and the elapsed time at each stage of the experiment are shown in Table 1. There were no significant differences between groups in terms of donor animal weight, baseline PaO2/FiO2, or elapsed time from ET tube clamp to asystole, with a mean weight for all animals of 30 ± 0.7 kg, baseline PaO2/FiO2 of 494 ± 22 mm Hg, and time from clamp to asystole of 22.2 ± 1.7 minutes. Since groups were randomized based on duration of WIT, there were significant differences in time from ET tube clamp to cold flush, time from ET tube clamp to initiation of EVLP, and total elapsed time from ET tube clamp to completion of EVLP.
Physiologic Changes During Hypoxia
The ABG samples were obtained every 1 to 2 minutes during hypoxia. Changes over time from ET tube clamp to asystole for each parameter are shown in Figure 1. Changes in MAP and heart rate are shown in Figure 2. The normal range is indicated with horizontal shading to identify the time at which the parameter became abnormal. The mean time of death was 22.2 minutes (vertical line, Figures 1 and 2), which displays how abnormal each parameter was on average upon initiation of the prescribed warm ischemia period. Within 3 minutes of inducing hypoxia, pH, PaCO2, HCO3, and base excess were all abnormal. After 6 minutes, MAP and lactic acid levels were abnormal, and within 8 minutes, PO2 values fell below the normal range. Heart rate fell below the normal range at 14 minutes.
Oxygenation Capacity and PA Pressures During EVLP
Mean PO2/FiO2 at each hour during EVLP is shown in Figure 3A. Final PO2/FiO2 after 4 hours of EVLP was not significantly different between groups (60 WIT: 518.3 ± 33.7 vs 90 WIT: 477.2 ± 31.7 vs 120 WIT: 427 ± 36.2 mm Hg, P = 0.28), with all 3 meeting the oxygenation threshold for transplant suitability (final PO2/FiO2 > 400 mm Hg).17 Oxygenation capacity increased during EVLP for all groups, with no significant difference between groups in percent change of PO2/FiO2 (Figure 3B). EVLP significantly improved the oxygenation capacity of 60 WIT (P = 0.01) and 90 WIT (P = 0.01) lungs, but did not for 120 WIT lungs (P = 0.6) (Figure 3C). The PA PCO2 levels were within the target range of 35 to 45 mm Hg for all animals, with no difference between groups (60 WIT: 41.1 ± 0.9 vs 90 WIT: 40.7 ± 0.5 vs 120 WIT: 42.1 ± 0.5 mm Hg, P = 0.15).
Mean PA pressures during EVLP were different between groups at hours 1 (P = 0.02) and 2 (P = 0.04), but not at the completion of EVLP (60 WIT: 18.7 ± 1.7 vs 90 WIT: 18.7 ± 0.9 vs 120 WIT: 22 ± 1.3 mm Hg, P = 0.12) (Figure 3D). Pulmonary artery pressure increased during EVLP for all 3 groups. The percent change remained below the exclusion criteria of 15% for groups 60 WIT (14 ± 7.7%) and 120 WIT (7.7% ± 6.4%), but not for group 90 WIT (30.8 ± 5.0%, P = 0.04) (Figure 3E). Final PA pressures were significantly higher compared to starting PA pressures for group 90 WIT (P = 0.001) (Figure 3F). LA pressures were maintained within the target range for all groups (60 WIT: 2.3 ± 0.5 vs 90 WIT: 2.5 ± 0.3 vs 120 WIT: 2.8 ± 0.2 mm Hg, P = 0.5).
Airway Pressures During EVLP
There were no significant differences in plateau or peak airway pressures between groups at each hour of EVLP (Figure 4A and D). The percent change in airway pressures was also not different between groups, with all groups having mean changes in plateau and peak airway pressures that fell below the exclusion criteria of 15% (Figure 4B and E). Final airway pressures were not significantly different from starting airway pressures in all 3 groups (Figure 4C and F).
Compliance During EVLP
Static and dynamic compliance at each hour of EVLP were not significantly different between groups (Figure 5A and D). Percent change in static compliance (60 WIT, −11.4% ± 5.4% vs 90 WIT, 2.6% ± 11.3% vs 120 WIT, 25.2% ± 23.2%; P = 0.43) and dynamic compliance (60 WIT, −10.7% ± 8.3% vs 90 WIT, 4.0% ± 12.2% vs 120 WIT, 15.6% ± 16.3%; P = 0.53) were not significantly different between groups, with all groups having mean changes within the acceptable limit for transplant suitability (<15% deterioration) (Figures 5B and E). Ex vivo lung perfusion did not significantly change static and dynamic compliance values for any group (Figures 5C and F).
Pulmonary Edema After EVLP
Lung wet-to-dry weight ratios were calculated to assess pulmonary edema. There were no significant differences between groups in wet-to-dry weight ratios (60 WIT, 6.3 ± 0.5 vs 90 WIT, 7.1 ± 0.7 vs 120 WIT, 7.4 ± 0.3, P = 0.36).
Histologic Assessment After EVLP
Composite lung injury severity scores were not significantly different between groups (60 WIT, 2.1 ± 0.5 vs 90 WIT, 2.6 ± 0.7 vs 120 WIT, 3.8 ± 0.3, P = 0.14) (Figure 6); however, lung injury severity scores did rise somewhat as WIT increased.
Using a porcine DCD model, the present study sought to evaluate the effect of warm ischemia after circulatory arrest on ex vivo lung function. After confirming asystole, donor lungs were exposed to 60, 90, or 120 minutes of warm ischemia followed by 4 hours of EVLP. Transplant suitability was determined at the completion of EVLP using the Toronto exclusion criteria (PO2/FiO2 < 400 mm Hg, >15% increase in PA pressure, and >15% deterioration of airway pressures and compliance).17,20 All 3 groups (60 WIT, 90 WIT, and 120 WIT) met the criteria for oxygenation, with no significant difference between groups in final PO2/FiO2. Pulmonary artery pressure increases were acceptable for transplantation in groups 60 WIT and 120 WIT, but not in group 90 WIT (30.8% ± 5.0%). All 3 groups met the transplant criteria for acceptable changes in airway pressures and compliance. There were also no significant differences between groups in edema accumulation or histologic lung injury severity scores. These results suggest that DCD lungs with WIT up to 120 minutes still meet transplant criteria after 4 hours of EVLP.
In recent years, many institutions both in the United States and internationally have expanded their procurement protocols to accept Maastricht category III “controlled” DCD lungs.5,21 These patients undergo planned withdrawal of treatment, often in the operating room, with transplant teams nearby who begin organ procurement shortly after confirmation of death.8 Outcomes after lung transplantation with “controlled” DCD lungs have been comparable to outcomes with heart-beating donors.22,23 Although the addition of EVLP has not been shown to improve survival after transplantation with DCD lungs, there is potential that EVLP may allow for inclusion of DCD lungs with longer WITs that would otherwise be rejected.24,25 “Uncontrolled” Maastricht category I and II donors have yet to be incorporated into routine transplant protocols due to associated logistic, physiologic, and ethical challenges, including timely notification of the procurement team, sufficient communication with family members, and proper management of the donor.26 One center in Spain was able to navigate the challenges associated with patients who die outside of the hospital and have reported their experience.27-29
For the present study, we sought to better understand the associated physiologic challenges after hypoxic cardiac arrest using our established porcine model of lung procurement. Although this model does not reflect all the various pathways that can lead to circulatory arrest, it does parallel donors who suffer respiratory arrest, which includes prolonged hypoxia from pulmonary disease and acute respiratory emergencies from airway obstructions and drowning. Published data in a porcine lung transplant model show that premortem hypoxia is associated with significant deterioration in graft function.30
The present study monitored all animals with frequent ABGs and continuous cardiac monitoring during the hypoxic period to better characterize the physiologic changes associated with this model. Certain parameters (pH, PaCO2, HCO3, and base excess) became abnormal within a few minutes of hypoxia while others took longer (MAP, lactic acid levels, PO2, and heart rate). We believe these data are valuable in helping to understand the quality of lungs that can be expected after certain types of death. Lungs exposed to warm ischemia after hypoxic cardiac arrest will likely function differently compared with lungs after sudden cardiac arrest.
After exposure to increasing amounts of warm ischemia, lungs from all 3 groups met all transplant criteria after 4 hours of EVLP, except the 90 WIT group on 1 parameter (PA pressure increase >15%). Additionally, when oxygenation capacity, airway pressures, and compliance were compared between groups, there were no significant differences after 4 hours of EVLP. This finding highlights the value of EVLP assessment of lungs before determining transplant suitability, as it appears that increasing WIT alone (up to 120 minutes) does not predict lung function after EVLP. Additional studies are necessary to determine a defined upper limit of acceptable WIT. It likely will be prudent to assess all potential donor lungs (within a large window of warm ischemic exposure) via EVLP to determine transplant suitability.
In the present study, EVLP provided a useful platform for assessing graft quality but did not confer much in terms of reconditioning. Ex vivo lung perfusion significantly improved oxygenation capacity for groups 60 WIT and 90 WIT, but not for group 120 WIT. Pulmonary artery pressures were significantly worse at the completion of EVLP for group 90 WIT, which was unexplainable but could be related to air trapping, vasoconstriction due to decreased organ temperature, or persistent clot burden. Although airway pressures and compliance did not differ significantly different between groups, group 120 WIT appeared to benefit the most with static compliance improvements of 25.2% ± 23.2% and dynamic compliance improvements of 15.6% ± 16.3%. Despite the lack of a statistical difference in improvement between hours 1 and 4 of EVLP for most parameters, all parameters at the completion of EVLP except for PA pressures in group 90 WIT were within acceptable limits for transplantation. Additionally, supporting data such as pulmonary edema and histologic lung injury severity scores were not different between groups. Although the definitive test will be lung function after transplantation, it appears that increasing WIT alone does not worsen lung function after 4 hours of EVLP.
Although Maastricht category I and II lungs comprise a potential solution to the supply-demand mismatch, there are significant hurdles that must be overcome before they can become an acceptable, routine option.31 Heparin was not administered to animals in the present study, as any premortem interventions will likely not be possible in an “uncontrolled” donor scenario. Clot within the pulmonary vasculature was present in most donor lungs procured after 90 and 120 minutes of warm ischemia, but was able to be flushed out with Perfadex. Published data on nonheparinized category III lungs is encouraging, but WITs are shorter under those circumstances.22,32 Other uncertainties include the cause of death (eg, respiratory arrest, cardiac arrest, exsanguination) and any resuscitation efforts that are attempted (eg, rescue breathing, chest compressions). This variability in patient presentation makes EVLP a near necessity to fully assess graft function and potentially rehabilitate damaged organs. Our laboratory and others have evaluated various treatment options during EVLP that may help recondition injured lungs.12,33-35 As these therapies are developed, the likelihood increases that we may one day have protocols that allow for routine, successful transplantation of Maastricht category I and II lungs.
The present study does have limitations. First, although this study was designed to set a baseline for acceptable warm ischemic injury by testing ex vivo lung function, the findings are limited because the organs were not subsequently transplanted into recipient animals and reperfused. The effect of ischemia-reperfusion injury after transplant on graft function will be an important next step of investigation. Second, there is inherent variability present in large animal studies. Our study was limited to n = 3 for the 60 WIT group and n = 6 for the 90 and 120 WIT groups, and it is possible that the use of higher numbers of animals per group could have revealed small but significant differences between groups in some parameters (eg, histologic lung injury scores). Based on previous work from our laboratory using 60 minutes of warm ischemia after hypoxic cardiac arrest, the number of animals randomized to group 60 WIT was limited to 3.10 Third, our study was also limited by the hypoxia method used to induce cardiac arrest; however, it provided consistent, reproducible injury. Finally, our findings are limited by not having a group that failed to meet transplant criteria after EVLP, which would have helped define the upper limit of acceptable WIT. Based on increasing variability in the data for the 120 WIT group, especially PO2/FiO2 and PA pressures (Figure 3) and airway pressures (Figure 4), we speculate that 120 minutes of WIT may be approaching the upper limit of acceptability.
In conclusion, for DCD donors to become a routinely utilized source of lungs for transplantation, understanding the effect of increasing WIT after different types of death is necessary. The present study shows that DCD lungs exposed to WIT up to 120 minutes after hypoxic cardiac arrest still meet transplant criteria after 4 hours of normothermic EVLP. As the field of lung transplantation continues to evolve to meet the ever-growing demand for organs, Maastricht category I and II lungs DCD lungs may eventually become acceptable for transplantation in conjunction with routine assessment by EVLP.
The authors wish to thank Tony Herring, Cynthia Dodson, and Sheila Hammond for their dedication to this project, as well as the University of Virginia Histology Core for their efficient processing of tissue samples.
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© 2018 The Authors. Published by Wolters Kluwer Health, Inc.
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