Machine Perfusion or Cold Storage in Deceased-Donor Kidney Transplantation

Two different forms of organ preservation — static cold storage and hypothermic machine perfusion — are used clinically for renal allografts obtained from deceased donors. In static cold storage, the kidney is flushed, cooled with one of several cold preservation solutions, and transported on ice. In hypothermic machine perfusion, after an initial washout of blood, the kidney is connected to a perfusion device, and a solution is pumped continuously through the renal vasculature at temperatures between 1 and 10°C.1 The typical deceased kidney donor today is older and has been exposed to more concomitant disease than donors were several decades ago; these factors may have a detrimental effect on allograft quality.2,3 In addition, the use of organs received from donors after cardiocirculatory death is increasing in most countries.4 Such allografts are known to have significantly higher rates of delayed graft function.4,5 Evidence suggests that organs that do not function immediately after transplantation have an increased risk of acute rejection, and allograft survival may be inferior.6,7 In addition, delayed graft function increases the costs of kidney transplantation.8,9 Retrospective studies have suggested that machine perfusion could result in a better short-term outcome, with lower rates of delayed graft function after transplantation of kidneys from all types of deceased donors.9-11 Therefore, interest in machine perfusion is increasing. Our international randomized, controlled trial compared machine perfusion with cold-storage preservation in deceased-donor kidney transplantation with a primary end point of delayed graft function.
Methods
Study Design

This investigator-driven, international randomized, controlled study included the Netherlands, Belgium, and the federal state of North Rhine–Westphalia in Germany. All consecutive deceased-donor kidney pairs identified in these regions that met the initial inclusion criteria were eligible for randomization by Eurotransplant, the international organ-exchange organization of Austria, Belgium, Croatia, Germany, Luxemburg, the Netherlands, and Slovenia (Croatia became a member after the present study was completed). Since we aimed to include the whole spectrum of deceased donors, no previous selection of donor types to be included was made. Thus, the study reflects the effect of machine perfusion as compared with cold storage in everyday practice within an international organ-exchange organization. From each donor, one kidney was randomly assigned to machine perfusion and the contralateral organ to cold storage. The organ could be transplanted into any recipient within the Eurotransplant region.12 Approval for the study was obtained from the ethics review boards in each trial region and from the Eurotransplant Ethical Advisory Committee and Kidney Advisory Committee. Since the random assignment of kidneys to a preservation method was limited to organs isolated before transplantation, no informed consent from recipients was required for this intervention.

An independent scientific steering committee composed of clinicians and scientists from each trial region was solely responsible for the design, conduct, data analysis, and manuscript preparation for this study.
Inclusion and Exclusion Criteria

Organ donors had to be 16 years of age or older. Only kidney pairs from deceased donors were included in the study, either from donation after brain death or donation after cardiocirculatory death. The category for donors without a heartbeat had to be Maastricht category III (awaiting cardiocirculatory death after withdrawal of treatment) or IV (cardiocirculatory death in a brain-dead donor).13 Kidney pairs were included only if both organs were actually transplanted into two different recipients. If one kidney was transplanted into the same recipient together with another organ, this kidney pair was excluded. The only exclusion criterion for recipients was the death of the patient in the first week after transplantation, since a follow-up of at least 1 week was required to determine the primary end point.
Randomization

A randomization scheme based on permuted blocks within regions was used with separate randomization lists for each trial region. A detailed description of the randomization process is available in the Supplementary Appendix, available with the full text of this article at NEJM.org. Surgical teams were allowed to switch preservation methods only if the kidney assigned to machine perfusion had an aortic patch that was too small or if it had too many renal arteries for a reliable connection to the machine-perfusion device; this switch in preservation methods changed the initial randomization.
Logistics

In each trial region, a team of trained perfusionists was on hand 24 hours per day, 7 days per week to respond when a donor became available. The perfusionists transported the machine-perfusion device to the donor hospital and assisted donor surgeons with connecting one kidney to the machine. No changes were made to the existing Eurotransplant rules for organ allocation or to transportation protocols. Kidneys that underwent machine perfusion as well as those that were preserved with cold storage were transported to their respective recipient center without any monitoring.
Hypothermic Machine Perfusion

LifePort Kidney Transporter machines (Organ Recovery Systems) were used for perfusion, delivering a pulsatile flow of University of Wisconsin machine preservation solution (Kidney Preservation Solution-1)14 at 1 to 8°C, with no changes in perfusion settings throughout the preservation period. The systolic perfusion pressure was fixed at 30 mm Hg, and the kidneys underwent machine perfusion from organ procurement until transplantation. To prevent bias in clinical decisions about transplanting or discarding an organ, intravascular resistance and flow readings were never revealed to the transplantation team.
Cold Storage

No changes were made to the standard cold-storage protocols. After an initial vascular washout, kidneys were submerged in the preservation solution and stored on melting ice, according to the established Eurotransplant routine.
Data Collection

Follow-up data were provided by each participating transplantation center through a secure online database hosted by Eurotransplant. A random sample of 10% of all patients was audited externally; no relevant irregularities were found.
Study End Points

The primary end point was delayed graft function, defined as the requirement for dialysis during the first week after transplantation. The secondary end points were the duration of delayed graft function, primary nonfunction (permanent lack of function of the allograft from the time of transplantation), the area under the curve of the daily serum creatinine level at days 1 to 14, the creatinine clearance at day 14, biopsy-proven acute rejection, toxicity of the calcineurin inhibitor, the length of the recipient's hospital stay, and survival of the graft and patient up to 1 year after transplantation. Data on graft survival were censored at the time of death in patients who died with a functioning allograft. In addition to the primary end point, which was defined in terms of the requirement for dialysis after transplantation, we also examined delayed graft function as a secondary end point. This secondary end point, functional delayed graft function, was defined in terms of the absence of a decrease in the serum creatinine level of at least 10% per day for at least 3 consecutive days in the first week after transplantation, not including patients in whom acute rejection, toxicity of the calcineurin inhibitor, or both developed within the first week.15 All end points described above were prespecified in the study protocol, except primary nonfunction, which was added post hoc.
Statistical Analysis

This study was powered to detect a reduction in delayed graft function of at least 10%, based on a presumed incidence of 35% among recipients of kidneys that had been preserved by means of cold storage. With a statistical power of 0.8 and a one-sided type I error of 0.05, the minimum required sample size was 300 kidney pairs; this is equivalent to the required sample size for a logistic-regression analysis with a two-sided type I error of 0.05 and similar power.16 The primary analysis of the primary end point — delayed graft function — consisted of a logistic-regression model, which examined whether machine perfusion as compared with cold-storage preservation, in the context of other relevant factors, influenced the risk of delayed graft function.7,17 Covariates for this model (see the Supplementary Appendix) were prespecified in the study protocol and were based on relevant literature.18,19 The final model was determined by entering all covariates together in the analysis, with a built-in normal gamma frailty term for the donor to account for the paired study design.20 For end-point variables, univariate differences between the groups were assessed with the use of McNemar's test or the Wilcoxon signed-rank test. For demographic variables, differences were assessed with the use of Fisher's exact test or the Mann–Whitney test. The Kaplan–Meier method was used to analyze graft and patient survival. Differences between survival curves were determined with the use of log-rank tests. A Cox proportional-hazards model was applied to examine which variables significantly influenced the risk of graft failure.21 To construct this model, an approach similar to the logistic-regression model for delayed graft function was followed.

We performed prespecified subgroup analyses to determine the treatment effect on the primary end point according to donation after cardiocirculatory death versus donation after brain death and according to expanded-criteria donation versus standard-criteria donation.22 Expanded-criteria donation was defined as a donor age of 60 years or more or a donor age between 50 and 60 years, with at least two of the following additional donor characteristics: history of hypertension, death due to a cerebrovascular cause, and a serum creatinine level of more than 132 μmol per liter (1.5 mg per deciliter) before removal of the kidney.23

All reported P values are two-sided and not adjusted for multiple testing. A P value of 0.05 or less was considered to indicate statistical significance. Analyses were conducted with the use of the SPSS, SAS, and R software packages and were based on all organ pairs that met the inclusion criteria.

No interim analyses of study end points were carried out. At regular intervals, confidential safety analyses were performed by the trial safety board, which compared the reported rates of adverse events between the two trial groups. The sponsor was not involved in the conduct of the study, the analysis or storage of the data, or the preparation of the manuscript. The scientific steering committee vouches for the accuracy and completeness of the data and analyses.
Results

From November 1, 2005, through October 31, 2006, there were 654 potential deceased kidney donors 16 years of age or older in the three trial regions. Figure 1Figure 1Enrollment, Assignment of Kidney Pairs to Machine Perfusion or Cold Storage, Follow-up, and Assessment. shows a flow diagram of the 336 kidney pairs (672 recipients) included in our analysis. In 25 donors (4.6%), preservation methods were switched because of the aberrant vascular anatomy of the kidney assigned to machine perfusion. Vascular anomalies were not observed to have a significant effect on delayed graft function. Aberrant vascular anatomy did not significantly increase the risk of graft failure, and the addition of this factor to the Cox model had no effect on the hazard ratio for graft failure associated with machine perfusion versus cold storage (see the Supplementary Appendix).

The 20 “other reasons for exclusion” of the kidney pairs (Figure 1) were as follows: 12 adverse events that occurred during the donor procedure, 5 cases in which the donor had one kidney, 2 cases in which the consent for kidney donation was withdrawn just before procurement, and 1 procedure involving a donor after cardiocirculatory death that was planned as a Maastricht category III donation but was changed to a Maastricht category II donation (cardiocirculatory death after unsuccessful resuscitation).
Study Patients

Table 1Table 1Characteristics of Donors, Recipients, and Transplants and Univariate Differences between the Groups. summarizes the characteristics of the study groups. All kidneys donated after cardiocirculatory death were in Maastricht category III, as defined earlier. There were no significant differences between the two groups with regard to relevant baseline characteristics.
Delayed Graft Function

Delayed graft function occurred in 70 recipients in the machine-perfusion group (20.8%) as compared with 89 patients in the cold-storage group (26.5%). Table 2Table 2Multivariate Analysis of the Risk of Delayed Graft Function and Graft Failure. shows the results of analysis using the logistic-regression model. As compared with cold storage, machine perfusion significantly reduced the risk of delayed graft function (adjusted odds ratio, 0.57; P=0.01).
Subgroup Analysis

In September 2006, when enrollment of donors in the study was nearly complete, the scientific steering committee expected that an insufficient number of donors would be enrolled at trial completion to conduct a meaningful subgroup analysis for donation after cardiocirculatory death. At the suggestion of the steering committee and with the permission of all centers, the inclusion of additional donors after cardiocirculatory death was extended by an amendment to the protocol, until a total of 82 donors were enrolled on August 17, 2007 (see the Supplementary Appendix for details). Solely for the subgroup analysis involving donation after brain death versus donation after cardiocirculatory death, these inclusions were added to the main group of patients to provide more statistical power. Figure 2Figure 2Forest Plot of the Treatment Effect in Prespecified Subgroup Analyses. shows a forest plot of the treatment effect in the prespecified subgroup analyses. In the main data set, we found no significant difference in the magnitude of the treatment effect on delayed graft function after standard-criteria donation versus expanded-criteria donation (P=0.75) and after donation after brain death versus donation after cardiocirculatory death (P=0.42). In the extended data set, the effect of the preservation method on delayed graft function did not differ significantly between patients who received kidneys from donors after brain death versus patients who received kidneys from donors after cardiocirculatory death (P=0.26).
Secondary End Points

Functional delayed graft function occurred in 77 recipients in the machine-perfusion group and in 101 recipients in the cold-storage group (22.9% vs. 30.1%, P=0.03). The incidence of primary nonfunction in the cold-storage group (4.8% vs. 2.1%, P=0.08) was more than two times higher than in the machine-perfusion group, but this difference did not reach statistical significance. If delayed graft function developed, its duration was 3 days shorter after machine perfusion as compared with cold storage (10 days vs. 13 days, P=0.04). There were no significant differences between the study groups in creatinine clearance at 14 days after transplantation, length of hospital stay of recipients, the incidence of toxicity of the calcineurin inhibitor, and acute rejection rate in the first 14 days after transplantation. Daily serum creatinine values in the first 2 weeks after transplantation were significantly lower in recipients in the machine-perfusion group than in recipients in the cold-storage group (median area under the curve, 1456 [range, 385 to 5782] vs. 1787 [range, 288 to 6500]; P=0.01) (see Fig. S2 in the Supplementary Appendix).
Patient and Graft Survival

In the cold-storage group, one patient died within 1 week after transplantation because of cardiac arrhythmia and was therefore excluded from the study along with the recipient of the contralateral kidney. At 1 year after transplantation, patient survival was 97% in both groups. Between 7 days and 1 year after transplantation, 11 patients in the machine-perfusion group died and 9 patients in the cold-storage group died (Table 3Table 3Adverse Events and Deaths Reported in the First Year after Transplantation.). One-year graft survival (Figure 3Figure 3Graft Survival after Transplantation.) in the machine-perfusion group was significantly higher than in the cold-storage group (94% vs. 90%, P=0.04). Cox regression analysis (Table 2) showed that machine perfusion significantly reduced the risk of graft failure in the first year after transplantation, with a hazard ratio of 0.52 (P=0.03). A post hoc analysis in which delayed graft function was added as a time-dependent covariate to the Cox model indicated that recipients with delayed graft function had a significantly increased risk of graft failure (hazard ratio, 1.69; P<0.001); p="0.08)">

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