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Department of Cardiology, Aarhus University Hospital, Aarhus, DenmarkDepartment for Clinical Medicine—Comparative Medicine Lab, Aarhus University, Aarhus, DenmarkDepartment for Cardiothoracic Surgery, Aarhus University Hospital, Aarhus, Denmark
Heart transplantation in donation after circulatory death (DCD) relies on warm perfusion using either in situ normothermic regional perfusion (NRP) or ex situ normothermic machine perfusion. In this study, we explore an alternative: oxygenated hypothermic machine perfusion (HMP) using a novel clinically applicable perfusion system, which is compared to NRP with static cold storage (SCS).
In a porcine model, a DCD setting was simulated, followed by either (1) NRP and SCS (2) NRP and HMP with the XVIVO Heart preservation system or (3) direct procurement (DPP) and HMP. After preservation, heart transplantation (HTX) was performed. After weaning from cardiopulmonary bypass (CPB), biventricular function was assessed by admittance and Swan-Ganz catheters.
Only transplanted hearts in the HMP groups showed significantly increased biventricular contractility (end-systole elastance) 2 hour post-CPB (left ventricle absolute change: NRP HMP: +1.8 ± 0.56, p = 0.047, DPP HMP: +1.5 ± 0.43, p = 0.045 and NRP SCS: +0.97 ± 0.47 mmHg/ml, p = 0.21; right ventricle absolute change: NRP HMP: +0.50 ± 0.12, p = 0.025, DPP HMP: +0.82 ± 0.23, p = 0.039 and NRP SCS: +0.28 ± 0.26, p = 0.52) while receiving significantly less dobutamine to maintain a cardiac output >4l/min compared to SCS. Diastolic function was preserved in all groups. Post-HTX, both HMP groups showed significantly less increments in plasma troponin T compared to SCS.
In DCD HTX, increased biventricular contractility post-HTX was only observed in hearts preserved with HMP. In addition, the need for inotropic support and signs of myocardial damage were lower in the HMP groups. DCD HTX can be successfully performed using DPP followed by preservation with HMP in a preclinical setting.
Two clinically used strategies exist to achieve functional recovery following the warm ischemic period. The first strategy is in situ restoration by normothermic regional perfusion (NRP) using central extracorporeal membrane oxygenation for fast donor organ reperfusion.
A novel approach in DCD HTX to achieve metabolic restoration is ex situ hypothermic oxygenated machine perfusion (HMP). HMP has already proven successful in DCD kidney and DCD liver transplantations as this method restores metabolic performance of organ cells.
have demonstrated the importance of both perfusion and oxygenation during HMP. It has been suggested that rapid succinate oxidation during normothermic reperfusion play an important role in ischemia reperfusion injuries by reverse electron transfer, while hypothermia and low-flow oxygenation may attenuate reverse electron transfer upon succinate oxydation.
In this study, we investigated the effect of oxygenated HMP with the XVIVO Heart preservation system after either NRP or DPP on DCD heart function following orthotopic transplantation. For control, we used a clinically applied method of NRP and static cold storage.
Female Danish Landrace pigs (76-85 kg) were used for 24 orthotopic DCD cardiac transplantations. The study was conducted as a prospective intervention study and included the following groups: (1) NRP followed by SCS to serve as a clinically practiced control group, (2) NRP followed by HMP, and (3) DPP followed by HMP. The experimental timeline is shown in Figure S1. The study was approved by the Danish Animal Experimentation Inspectorate (#2018-15-0201-01603).
Donor anesthesia, monitoring and instrumentation have previously been described
Both donor and recipient received 1.5 g Cefuroxime and 1,000 mg methyl-prednisolon at start of the procedure. All donor animals underwent same surgical procedure as described earlier with central cannulation for NRP.
After instrumentation, baseline measurements including hemodynamic, pressure-volume and blood gas measurements were recorded; and blood and tissue from LV were sampled. Following baseline, circulatory death was instituted by discontinuation of mechanical ventilation, resulting in asphyxiation and circulatory arrest (CA), defined as central venous pressure = mean arterial pressure (MAP). Functional warm ischemic time (FWIT) was defined as the time from systolic blood pressure <50 mmHg to onset of NRP or cardioplegic flush in the DPP group. We used 10 min CA to obtain a solid reference in the NRP SCS group. In the NRP HMP and DPP HMP groups, we used 15 min CA to simulate clinical practice.
At the end of the warm ischemic period, the supraaortic vessels and infrarenal aorta were clamped to prevent cerebral blood flow before 60 min NRP was commenced with flow rates previously described.
Vasoinotropic support was achieved with norepinephrine (0.0-0.9 μg/kg/min) to maintain MAP >60 mmHg during NRP, and dobutamine (2.5 μg/kg/min) was started 15 min prior to weaning. Arterial and venous blood gasses were analyzed at 5, 15, 30, and 55 min during NRP, and repeated at 15 and 30 min post-NRP. Hemodynamic function was assessed 30 min post-NRP with bi-ventricular pressure-volume measurements. The heart was subsequently procured by cross-clamping the aorta and flushed with 1-1.5 liter, 4°C, cardioplegia; Histidine-tryptophan-ketogluterate (HTK) solution in the SCS group and XVIVO Heart Solution for the HMP groups. In the SCS group, the donor heart was placed in an organ bag containing 500 ml HTK and stored at 4°C for 120 min.
DPP and HMP
After the warm ischemic period, the heart was decompressed by the two-stage venous cannula in the right atrium before the aorta was cross-clamped and the heart was flushed. The donor heart in the HMP groups was excised, weighed, and cannulated for preservation; additionally, a coronary sampling tube was placed through the hemi-azygos vein which was subsequently ligated.
During HMP with the XVIVO heart box, hearts were perfused with 2.5 liter oxygenated XVIVO Heart Solution supplemented with 500 ml leukocyte filtered and isolated erythrocytes with a constant aortic root pressure of 20 mmHg for 180 min.
Median hematocrit was (IQR) 8.2% (6.4-8.6). During HMP, coronary flow was measured and arterial (from the oxygenator) and venous (from the coronary sinus) samples were collected at 5, 90, and 180 min after onset of preservation. Hearts were weighed and biopsied after preservation. Calculations for oxygen consumption are described in the supplementary section. The HMP protocol was the same in the NRP HMP and DPP HMP groups.
Anaesthesia of the recipient animal was maintained with propofol (3.5 mg/kg/hour) and fentanyl (15 μg/kg/hour) supplemented with inhaled (1%) sevoflurane for lung protection.
Animal preparation, monitorization and thoracic exposure were performed as in the donor protocol. Systemic anticoagulation with 40.000 IU heparin and ascending aortic and bi-caval cannulation were used to institute normothermic CPB. Nitroprusside (0.5-2.0 μg/kg/min) and CPB flow rates were adjusted to maintain MAP 50-70 mmHg. We performed orthotropic HTX using bi-caval technique and topical cooling (to the authors knowledge first to report this technique in a porcine model
). In the SCS group, 250 ml antegrade HTK cardioplegia was administered after the first three anastomoses, while no additional cardioplegia was administered to the HMP hearts during implantation. An LV-vent was placed in the apex, and de-airing maneuvers were performed before removal of the aortic cross-clamp and reperfusion. Internal defibrillation (30 J) was used to treat ventricular arrhythmias. Dobutamine (1.5-2.5 μg/kg/min) and a bolus of 3.5 mg Milrinone and norepinephrine (0.05-1.0 μg/kg/min) were used for inotropic and vasoconstrictive support, respectively. Following implantation, hearts were reperfused unloaded for 60 min. Subsequently, weaning from CPB was initiated by gradually decreasing flow and volume-loading the hearts. Weaning was considered successful when the animal maintained MAP >60 mmHg for over 30 min. The recipient was observed for 120 min after successfully weaning. At 60 and 120 min postweaning, hemodynamic measurements were performed. In addition, arterial and venous blood gasses were evaluated every 30 min. At 120 min post-CPB, a biopsy was withdrawn from the apex of the LV, and the experiment was terminated.
Assessment of cardiac function
Continuous cardiac output (CO) and pressure measurements were obtained from the PA catheter (7.5Fr, CCOmbo, Edwards lifescience, USA). The pressure-volume admittance catheters (EMKA Technologies, Paris, France) were calibrated according to manufactures specifications (Transonic Science, London, Canada). Contractility of the LV and RV were assessed using the end-systolic elastance (Ees), preload recruitable stroke work (PRSW), dP/dtmax and LV ejection fraction (EF). Diastolic function was assessed using the active relaxation constant τ, dP/dtmin and End-diastolic elastance (Eed).
Arterial and mixed venous samples were analyzed (ABL90 Flex Plus; Radiometer Medical, Copenhagen, Denmark). Creatinine kinase-muscle/brain (CK-MB) and Tronopin T (TnT) were measured by chemiluminescence (Advia Centaur XPT; Siemens Healthcare Diagnostics, E Walpole, MA) and (Cobas e601, Roche Diagnostics International AG, Rotkreuz, Switzerland)
Data were checked for normality by qq-plots and histograms and model validation was controlled by residual plots. Normally distributed variables are presented as mean ± SD. Non-normally distributed data are presented as median (interquartile range). Graphical data is presented as individual data points and medians. Differences between groups and time were compared by mixed-effects model analysis to allow for analysis of parameters containing missing data. The model was used to compare outcome between intervention groups with intervention-group and time as fixed effects and subjects as random effects. Time was modeled as a categorical variable. Only donor baseline and post-TX time points were included in the statistical analysis. Measurements post-NRP and HMP are displayed for reference. Nonrepeated data was compared using one-way ANOVA or Kruskal-Wallis test where appropriate. Tukey's test was used for adjustment for multiple comparison in the mixed effects model and ANOVA, while Dunn's correction was used as adjustment in the Kruskal-Wallis test. p-values <0.05 were considered statistically significant. GraphPad Prism 9.4 (GraphPad Software, CA, USA) was used for analyses.
A total of 24 transplantations were performed; hereof 16 were successful with 6/9 in the NRP SCS group, 5/7 in the NRP HMP group, and 5/8 in the DPP HMP group. See detailed description and CONSORT diagram in the supplementary section (S2). All hearts in the NRP groups were successfully reanimated, preserved, and went on to transplantation. All hearts in the HMP groups showed similar perfusion and oxygen extraction trends during HMP (Figure 1, S3) Two donor animals in the DPP HMP group had a FWIT of more than 25 min and developed ischemic contraction before procurement and could not be weaned posttransplantation. These hearts would not have been procured in a clinical setting. Two hearts from the NRP SCS group developed PGD despite good function post-NRP, hence no hearts with good function prior to HMP developed PGD after HMP and TX. Only animals that completed the full follow up were included in the analyses.
Due to the protocol differences for the control NRP SCS group and the HMP groups, the FWIT was significantly longer in the HMP groups (Table 1). However, the time from withdrawal from life support to onset of circulatory arrest was similar in the three groups. Total out of body time was significantly shorter in the NRP SCS group. Conversely, ischemic time was significantly longer in the NRP SCS due to the non-oxygenated storage method.
Table 1Important times during donation and transplantation.
All weaned hearts displayed good hemodynamic function post TX after weaning from CPB with MAP>60 mmHg, central venous pressure<12 mmHg, and cardiac output >4 liter/min with similar doses of norepinephrine in all 3 groups; of note, cardiac output was significantly decreased from baseline in all groups (Figure 2). Systemic vascular resistance was stable throughout the experiment. All groups showed increased mPAP and PVR after weaning from CPB post-TX as has been described as a complication of CPB.
Supplementary Table 1 shows exploratory correlation of post-NRP function and HMP perfusion parameters to LV Ees 1hour post-CPB. There was no correlation between ischemia time and LV function. Post-NRP LV Ees and TnT showed tendencies to correlate with post HTX LV function. End-HMP O2-extraction was strongly correlated to post-HTX LV function, most pronounced in the NRP HMP group.
HMP versus SCS
Both HMP groups were significantly faster to wean from CPB and received significantly less dobutamine post-TX compared to the SCS group (Table 1, Figure 3). The HMP hearts did not display macroscopic signs of edema formation during HMP. The NRP HMP hearts nonsignificantly reduced weight by −15 grams 95%CI (−35 to 5) and the DPP HMP nonsignificantly increased by 6 grams 95%CI (−14 to 26) after HMP. After HMP, LV, and RV Ees were significantly increased at 60 and 120 min post-CPB compared to donor baseline, whereas no significant increase was found in the NRP SCS group (Figure 4); at 60 min post-CPB the NRP HMP group showed significantly higher Ees than the SCS group. No significant changes over time or between groups were observed in PRSW. Only the HMP groups showed clear improvement of RV dP/dtmax at 60 and 120 min post-CPB, changes that were significantly more pronounced compared to the SCS group. No significant between-group differences were found in any of the diastolic parameters for either RV or LV (Figure 5). However, compared to baseline, active LV relaxation (τ) was only improved in the NRP HMP group. Diastolic function remained at baseline levels in the SCS group. TnT was significantly elevated in the NRP SCS group at 1 hour post-CPB compared to HMP groups with similar tendencies at the other time points post-TX (Figure 6).
NRP versus DPP
Post weaning from NRP, hearts showed preserved systolic contractile function NRP seen as biventricular improvement of dP/dtmax, Ees, and PRSW, thus showing a preserved contractile reserve under stimulation with inotropes (Figure 4). Diastolic function was similarly preserved as τ and dP/dtmin decreased from baseline indicating improved active relaxation and Eed remained at baseline levels post-DCD and NRP (Figure 5). Post-TX biventricular contractile function was improved in all groups compared to baseline, and there was no significant between group differences in contractile measures, Ees, PRSW, dP/dtmax; DPP HMP was on similar levels as the NRP groups. The DPP HMP group even showed larger improvements in contractile biventricular function than NRP SCS measured in LV and RV Ees at 120 min post-CPB. DPP HMP LV dP/dtmax did not increase post-TX compared to baseline and showed no between-group differences, but was significantly increased from baseline in the NRP groups. Overall LV function was marginally better in the NRP HMP group compared to the other groups. Post-TX biventricular diastolic function was preserved seen as Eed, with no between-group differences. Active LV relaxation was significantly improved from baseline in the NRP HMP group, whereas it was preserved at baseline levels in the NRP SCS and DPP HMP group. Arterial lactate was increased from baseline in the NRP groups, though only significant in the SCS group, whereas DPP lactate levels remained near baseline levels post-TX (Figure 6).
We investigated the feasibility of a novel technique for DCD heart preservation in the form of oxygenated HMP using the XVIVO Preservation technology. We used a porcine DCD-model and compared hemodynamic outcomes to a clinically relevant NRP SCS protocol. We used the HMP (1) in conjunction with NRP and (2) as sole preservation after DCD with DPP. We found that porcine DCD hearts procured with DPP and HMP performed on par with DCD hearts reanimated with NRP.
We introduced HMP for donor heart preservation and clinical DCD HTX that has so far relied on warm perfusion using either in situ NRP or ex situ NMP or a combination hereof.
Wyss et al demonstrated the importance of oxygenation during HMP in a DCD DPP rat model for restoration of cellular ATP at the end of oxygenated HMP compared to nonoxygenated HMP, and they found improved contractile function during warm perfusion.
The key objective of this study is whether HMP can restore DCD hearts safely for subsequent transplantation. In both kidney and liver transplantation, restoration of vital cells after DCD donation is achieved with cold oxygenated perfusion,
The current study is the first DCD heart study to explore HMP using the XVIVO heart box, including both DPP and NRP. We found that DCD hearts preserved with DPP HMP had preserved contractile function after 180 min preservation and subsequent transplantation. These results may be encouraging enough to warrant a pilot clinical trial of DPP HMP.
There are important limitations of our study. Despite the small number of animals, we found relevant and consistent data that proved good cardiac function in the early post-TX phase in all groups. We used young (approximately 120 days old) healthy female pigs for donor and recipient. These animals are younger and healthier than the typical organ donor and recipient, thus the hearts may tolerate ischemia differently than the human counterpart. However, it is known from the literature that porcine hearts tolerate ischemia more poorly than human hearts.
Hence, we believe that the findings of this study are valid for human translation. By protocol, we used a shorter CA time (10 min) for the control NRP SCS group than for the NRP HMP and DPP HMP groups (15 min). The herewith-introduced bias of less warm ischemic damage was based on pilot experiments where longer ischemic damage led to nonfunction. Even with the shorter CA time the post-Tx performance was inferior to HMP with a higher requirement of inotropic support; hence, we found no positive bias and deem this group as a good control. The control group using NRP and SCS represent a clinically applicable method for DCD heart preservation and transplantation. Today, the most widely used method is direct procurement with subsequent NMP using the Transmedics organ care system. We, however, did not have access to this system to make a comparison.
We demonstrated that DCD hearts can be directly procured and preserved using the novel ex situ HMP technique. Both HMP groups showed improved contractile function compared to hearts preserved with SCS. HMP can serve as a valuable alternative to warm ex situ perfusion with an additional safety feature of the hearts being stored cold if the machine perfusion were to arrest and, at the same time, avoiding the ethical issues of NRP. Further testing is needed to determine viability of the heart while being preserved and explore if extended periods of HMP for DCD hearts is possible.
All authors agree with the content of the manuscript and have made substantial contributions to the work; NM, ME and HE conceived and planned the experiments and NM, ME, IE, MH and FD carried out the experiments. NM, ME, HE interpreted the results and LI, PR and contributed to the surgical protocol and provided constructive feedback. NM wrote the first draft of the manuscript. All authors provided critical feedback and helped shape the research, analysis, and manuscript.
Niels Moeslund received a scholarship from Aarhus University. Novo Nordisk, Independent Research foundation Denmark and the A.P. Moller Foundation also generously supported the study.
XVIVO® provided the heart box, disposables and heart preservation solution and partial funding for the running costs for the experiments.
Michiel Erasmus holds a patent with XVIVO perfusion and received a research grant from XVIVO for 2020-2021. No other author declares any conflicts of interest.
Kira Sonnichsen Graahede and Trine Louise Bang Østergaard for invaluable assistance with the handling of animals and preparation of surgical facilities. Sif Bay, Lasse Tiroke, and Agnete Madsen for all the hours and help with pig handling and hard work in the basement. Ceren Ünal, Karsten Lund Soeberg, Peter Johansen, Oddvar Nils Klungreseth and Debbie Richards for resuscitating the pigs with excellent handling of the heart-lung machine. Casper C Elkjaer provided great assistance with biochemical analysis.