- Email: [email protected]
Liver Preservation with SCOT 15 Solution Decreases Posttransplantation Cholestasis Compared with University of Wisconsin Solution: A Retrospective Study
Liver Preservation with SCOT 15 Solution Decreases Posttransplantation Cholestasis Compared with University of Wisconsin Solution: A Retrospective Study E. Savier, B. Granger, F. Charlotte, N. Cormillot, J.M. Siksik, J.C. Vaillant, and L. Hannoun ABSTRACT Background. SCOT 15 is a new solution to preserve abdominal organs for transplantation. Its principal characteristic is the use of polyethylene glycol. Herein We report our experience using SCOT 15 compared with the reference University of Wisconsin (UW) solution for hepatic transplantation. Methods. We compared 2 groups: SCOT 15 (n ⫽ 33; 2009 –2010) versus UW (n ⫽ 34; 2008 –2010), which were paired for cold and warm ischemic times, donor ages, and graft weights. Endpoints were biologic tests in the first 2 months after the operation. A linear mixed model was used to evaluate longitudinal changes and influences of each solution. Results. No primary failure was observed. At postoperative day 0, transaminase values were higher in the SCOT 15 than in the UW group: aspartate transaminase: 2,435 ⫾ 399 vs 589 ⫾ 83 IU/L (P ⬍ .01); alanine transaminase: ALT: 1,207 ⫾ 191 vs 484 ⫾ 64 IU/L (P ⬍ .05), then returned to low levels in both groups. From day 0 to 8, coagulation factors reached normal values; there was no difference between the 2 groups. Total bilirubin decreased similarly in the 2 groups. However, from the second postoperative week (W1) to W8, the SCOT 15 group showed a slow decrease in the mean values of gammaglutamyltranspeptidase (gGT) from 233 ⫾ 125 to 130 ⫾ 161 IU/L, which were significantly lower than those in the UW group, where the gGT remained around 300 IU/L (P ⬍ .01). The End-Stage Liver Disease, Child-Pugh, or United Network for Organ Sharing scores, primary liver diseases, hepatitic C virus status, arterial or biliary complications, and male/ female ratio, which was different in the 2 groups, did not statistically influence these results. Conclusions. The main effect of cold storage of human liver using SCOT 15 compared with UW solution was to decrease cholestasis following transplantation. epatocellular insufficiency, end-stage chronic liver disease, and several cancers can be treated by hepatic transplantation. This intervention requires a graft whose functionality depends both on the donor and on the preservation conditions. In the case of a brain-dead donor, parameters such as the medical history, underlying hepatopathy, and hemodynamic conditions cannot be modified at the time of harvest. There is only one choice, to accept or refuse the organ, which is constrained by the limited number of donors. This shortage leads to the use of grafts with an increased postoperative risk for complications or primary nonfunction. Expanded donor criteria have been established to limit these risks.1 In such situations, graft preservation must be of the highest quality possible by
H
reducing the cold ischemia time, controlling the hypothermia, and using optimal preservation solutions.2,3 Since the 1990s, the reference solution for hepatic transplantation has From the Service de Chirurgie Digestive et Hépato-BilioPancréatique - Transplantation Hépatique, Groupe Hospitalier Pitié-Salpêtrière (E.S., J.M.S. J.C.V., L.H.), and Modélisation en Recherche Clinique (B.G.), Université Pierre et Marie Curie; and Unité de Biostatistiques et Informatique Médicale (B.G.), Service d’Anatomie Pathologique (F.C.), and Fédération de Transplantation (N.C.), Groupe Hospitalier Pitié-Salpêtrière, Paris, France. Address reprint requests to Eric Savier, MD, Service de Chirurgie Digestive et Transplantation Hépatique, Groupe Hospitalier Pitié-Salpêtrière, 47-83 Boulevard de l’Hôpital, 75651 Paris Cedex 13, France. E-mail: [email protected]
0041-1345/11/$–see front matter doi:10.1016/j.transproceed.2011.09.054
© 2011 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
3402
Transplantation Proceedings, 43, 3402–3407 (2011)
LIVER PRESERVATION WITH SCOT 15
been Belzer solution (Viaspan), which was developed at the University of Wisconsin and commonly called UW.4 The intracellular composition of UW includes oxygen radical scavengers and colloid hydroxyethylstarch (HES) to maintain oncotic pressure.5 Nowadays, new preservation concepts include machine perfusion, protective molecules supplements, and genetic manipulations.3,6,7 Extracellular solutions have been used for preservation and for a fast rinse of the graft just before reperfusion.8 New solutions (Custodiol, Celsior, IGL-1) of extracellular composition are emerging, although clinical trials have so far failed to demonstrate any major differences from the criteria standard, UW.9-12 Polyethylene glycol (PEG) a macromolecule used in some solutions to ensure oncotic pressure (IGL-1: PEG 35 kD, 1 g/L), is used as PEG 20 kD, 15 g/L in a newly developed solution, SCOT 15.13 An interesting property of the PEG, which is polymerized to 20 kD molecular weight, is an associated decrease in allograft rejection in several models.14-18 The main hypothesis to explain these results is that PEG may block the immunologic synapse,19 consequently decreasing the initial immune response. This effect could be important, because a main consequence of reperfusion after cold ischemia is the induction of a temporary but major inflammatory chain reaction that stimulates the innate immunological response.20 It is now clearly established that these pathophysiologic effects have long-term consequences.16,21,22 Therefore, preserving an organ with SCOT 15 could be a step forward in liver transplantation. The goal of the present study was to report our results with this new solution versus those with UW. METHODS Definitions D0 was the day of the transplantation, W0 (D0 to D7) the week after, and M0 (D0 to D30) the month after the operation. Consequently, M1 was the second month after grafting.
Surgical Technique Every human liver transplantation was performed as follows. After cooling with SCOT 15 or UW infused through the aorta and the inferior mesenteric vein, the liver was harvested from the donor according to Starzl’s technique,23 and subsequently prepared with infusion of cold SCOT 15 (1,500 mL) or UW (1,000 mL) by the portal vein. In the recipient, we performed a total hepatectomy including the retrohepatic vena cava. Just before portal reperfusion, the graft was rinsed with cold solution, either 500 –1,000 mL human albumin (4%) following UW cold storage or 500 mL NaCl (0.9%) solution following SCOT 15 cold storage, seeking to decrease the potassium concentration in the effluent. An arterial anastomosis was then performed under magnification. The biliary anastomosis was performed with or without biliary drainage (silicon T-tube or polyurethane transcystic tube), according to the biliary anatomy and personal experience of the surgeon. Biliary drainage was removed after M2 in all cases. Posttransplantation immunosuppressive therapy included cyclosporine or tacrolimus, steroid, and mycophenolate mofetil for 3 months before switching to a 2-drug regimen, if possible. Liver biopsy was performed
3403 according to blood liver test results and in cases of suspected acute rejection episodes.
Selection of the Patients and Exclusion Criteria Exclusion criteria were split grafts, live donor grafts, non– heartbeating donors, domino liver transplantations, pediatric recipients, combined heart and liver recipients, grafts procured by other surgical teams, and grafts stored in SCOT 30 (PEG 20 kD, 30 g/L). The transplanted patients whose livers had been preserved in SCOT 15 were compared with a retrospective group of recently transplanted patients with median dates of transplantation January 15, 2009, for the UW group and February 11, 2010, for the SCOT 15 group. Among 35 transplantations using SCOT 15 between July 2009 and July 2010, 2 were excluded: one owing to vena caval thrombosis, leading to a surgical deobstruction on D3 with the patient alive at the time of writing, and the other owing to a split graft. Control group patients were selected among the transplantations with preservation in UW performed between January 2008 and September 2010. They were paired for cold ischemia time, warm ischemia time, namely, arterial revascularization minus portal reperfusion time, graft weight, and donor age (Table 1). Among 40 selected patients, 6 were excluded for the following reasons: renoportal anastomosis in the absence of portal trunk (n ⫽ 1); grafts divided with a pediatric team (n ⫽ 3); combined heart transplant (n ⫽ 1), and death at D0 from sepsis (n ⫽ 1). One patient’s data including heart failure were removed from D5 until retransplantation. Thus, the SCOT 15 group included 33 liver transplantations in 33 patients and the UW group 34 transplantations in 33 patients.
Measured Parameters Biologic parameters were assayed by hospital departments. When several values were available for 1 period of time (day or week), we included the maximum value for aspartate transaminase (AST) and alanine transaminase (ALT) and the average value for gammaglutamyltranspeptidase (gGT), bilirubin, factor V, prothrombin time, and platelet count. Liver biopsies, obtained with a 14-gauge Menghini needle or with a wedge surgical biopsy, were fixed, prepared and stained using standard techniques. All hepatic biopsies were evaluated by the same pathologist (F.C.) in blinded fashion; rejections were graded according to Banff rejection activity index.
Statistical Analyses Baseline characteristics of patients were compared with the Fisher exact test for categoric variables or the Mann-Whitney Wilcoxon test for continuous variables. Linear mixed models, with fixed subject random intercepts, were used to evaluate longitudinal changes (time effect) in each parameter. Using all available time points, this approach yielded greater statistical power than considering only the first and last points. Linear mixed models took into account the solution (the parameter levels over time were different between solutions), interactions between time and solution (the kinetics of evolution of parameter was different between solutions) with gender as covariates. The same linear mixed model was used to evaluate the effects of the solution on each parameter. Data were analyzed using the R version 2.11.0 and SigmaPlot version 9.01 of SigmaStat version 3.11 (Systat Software).
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SAVIER, GRANGER, CHARLOTTE ET AL Table 1. Statistical Comparisons Between SCOT 15 and University of Wisconsin (UW) Groups
Cold ischemia time (min) Warm ischemia time (min)* Donor age (y) Graft weight (g) MELD score Child-Pugh score UNOS score 1: Intensive care unit 2: In-hospital stay 3: Permanent care 4: Waiting at home Main indication for liver transplantation Cirrhosis or chronic hepatocellular insufficiency† Acute liver failure Hepatocellular carcinoma Metabolic Anti-HCV antibody Negative Positive PCR HCV Negative Positive Gender ratio (M/F) Biliary anastomosis with biliary tube‡ Yes No Gamma-glutamyl transpeptidase (gGT) Week 0 Week 1 Week 2 Week 3 Week 4 Week 5 Week 6 Week 7 Week 8
SCOT 15 (n ⫽ 33)
UW (n ⫽ 34)
P Value
458 ⫾ 14 50 ⫾ 3 50 ⫾ 3 1,533 ⫾ 67 15.7 ⫾ 1.7 9 ⫾ 0.5
472 ⫾ 11 53 ⫾ 3 53 ⫾ 2 1,440 ⫾ 54 18.4 ⫾ 1.9 9.2 ⫾ 0.5
.459 .199 .390 .311 .269 .721 .769
13 3 2 15
10 2 2 20
17 3 13 0
21 2 10 1
20 13 (39%)
24 10 (29%)
23 10 (30%) 29/4
27 7 (20%) 21/13
18 (55%) 15
16 (47%) 18
125 ⫾ 68 233 ⫾ 125 182 ⫾ 125 153 ⫾ 100 146 ⫾ 132 161 ⫾ 170 128 ⫾ 166 129 ⫾ 156 130 ⫾ 161
125 ⫾ 69 324 ⫾ 165 261 ⫾ 243 282 ⫾ 277 237 ⫾ 256 305 ⫾ 305 244 ⫾ 275 332 ⫾ 357 310 ⫾ 388
.635
.114
1
.023 .628
Time: .0002 Gender: .358 Solution: ⬍.01 Time* solution: .291
Abbreviations: HCV, hepatitis C virus; MELD, Model for End-Stage Liver Disease; PCR, polymerase chain reaction; UNOS, United Network for Organ Sharing. *Time between portal and arterial liver reperfusion. †Cirrhosis from any cause and chronic sclerosing cholangitis at end stage. ‡T-Tube or transcystic tube inserted at the time of the transplantation. Baseline characteristics of patients were compared with the Fisher exact test or the Mann-Whitney Wilcoxon test depending on the type of data. Consecutive periods of postoperative time were compared between the 2 groups from week 0 to 8 for gGT. A linear mixed model was used to evaluate the longitudinal change and the influence of solution on each parameter as described in the Methods. Results are presented as mean ⫾ SD for quantitative data and frequency for qualitative data. P ⬍ .05 for statistical significance.
RESULTS Biologic Results
At D0, transaminase release in the SCOT 15 group showed a median value of 1,792 UI/L (range 346 –7,791) for AST, and 750 UI/L (range 205– 4,870) for ALT. This peak of transaminase disappeared after 48 hours for AST (Fig 1A) and after 4 days for ALT (Fig 1B). Interestingly, from W1 to W8, AST and ALT decreased more quickly among the SCOT 15 group than the UW group (data not shown), with a slight increase between D8 and D12 for ALT only in the UW group (P ⫽ NS; Fig 1B). The evolution of prothrombin time was identical in the 2 groups (Fig 2A). Factor V increased from 40% at D0 to 80% at D5, remaining ⬎80% beyond D8 in both groups (data not shown). The average bilirubin decreased from D7–D14 to D21–D28 to a value
below twice the normal result among the SCOT 15 group, whereas it normalized above normal in the UW group, who showed a similar evolution. (Fig 2B). The platelet count increased between D0 and D21 from 60,000/mm3 to 250,000/mm3, without any difference between the 2 groups (Fig 2C). The evolution of gGT was significantly different over the first 2 postoperative months (Fig 3). In the SCOT 15 group, the average gGT was maximal at W1, gradually decreasing to 2–3 times normal at W7 (Table 1). Among the UW group, in contrast, the gGT reached high levels that rose from W1 to W7 (Table 1). Histologic Results
A hepatic biopsy was performed when there were blood test anomalies or the clinical context required histologic data to
LIVER PRESERVATION WITH SCOT 15
3405
patient died 0.8 month later from multiple organ failure. The second patient, transplanted for alcoholic cirrhosis with a major reperfusion syndrome, displayed an arterial thrombosis at D27. She was retransplanted but died from sepsis and multiple organ failure 16 days later. We did not include the second transplantation because the preservation solution was IGL-1. One patient in each group experienced a main bile duct stricture at M1; both were successfully
Fig 1. Release of transaminases after liver transplantation with SCOT 15 or University of Wisconsin (UW) solutions. Serum aspertate transaminase (AST) (A) and alanine transaminase (ALT) (B) were measured by the Pitié-Salpêtrière hospital laboratories. Values shown are the maximal of every available value measured during each time period (day). A significant difference was observed at day 0 between the SCOT 15 group and the UW group. Mann- Whitney rank sum test (n ⫽ 33 and 34: *P ⬍ .05; **P ⬍ .01. Results are shown as mean ⫾ SEM.
adapt the immunosuppressive therapy. Between W0 and W12, 37 biopsies were performed: 11 in the SCOT 15 (33%) and 26 in the UW group (76%). In the SCOT 15 group we identified 3 acute rejection episodes involving 3 patients: total Banff score 6 at D7, Banff 4 at D16, and Banff 2 at D32. Among the UW group, we identified 7 acute rejection episodes involving 6 patients: Banff 3 at D10, Banff 4 at D11, Banff 3 at D14 and D55, Banff 2 at D37, Banff 4 at D14, and Banff 3 at D56. Preservation injury was observed in 1 SCOT 15 and 6 UW group cases. Postoperative Follow-Up
No primary liver failure was noticed in either group. Mortality at 3 months was 2 patients in the UW and 0 in the SCOT 15 group. The first patient had a pericardial fluid collection, leading to heart failure with considerable repercussion on the liver graft, as demonstrated by explantation of a necrotic graft at D28. Despite retransplantation, the
Fig 2. Prothrombin time, total bilirubin and platelets count evolution following liver transplantation with SCOT 15 or University of Wisconsin (UW) solutions.Prothrombin time (A), total bilirubin (B), and platelet count (C) were measured by the PitiéSalpêtrière hospital laboratories. Values shown are the means of every available value measured during each time period (day). A linear mixed model was used to evaluate the longitudinal change, and the influence of solution on each parameter as described in Methods. No difference was observed between the SCOT 15 group and the UW group (n ⫽ 33 and 34). Results are shown as mean ⫾ SEM.
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SAVIER, GRANGER, CHARLOTTE ET AL
Postoperative Cytolysis
Fig 3. Release of gGT following liver transplantation with SCOT 15 or University of Wisconsin (UW) solutions. Serum activity of gamma-glutamyltranspeptidase (gGT) was measured by the Pitié-Salpêtrière hospital laboratories. Values shown are the means of every available value measured during each time period. Week 0 includes postoperative days 0 –7. A linear mixed model was used to evaluate the longitudinal change and the influence of solution on each parameter as described in the Methods. A significant difference was observed, as detailed in Table 1, between the SCOT 15 group and the UW group (n ⫽ 33 and 34). Results are shown as mean ⫾ SEM.
treated with endoscopic retrograde stenting. The median follow-up of the grafts was 9.6 months (range 3.2–16) among the SCOT 15 group and 17.1 months (range 0.8 – 34.3) among the UW group. DISCUSSION
This report on the use of SCOT 15 solution in clinical liver transplantation used a retrospective comparison with UW. Our study was homogeneous regarding to surgical procedures, assays, and postoperative management. Therefore, missing data were negligible for observations with statistical significance. The quality of graft preservation with SCOT 15 was assessed by the postoperative evolution of prothrombin time, factor V, platelets, and bilirubin. Results and evolution of these parameters postoperatively were identical between the SCOT 15 and the UW group (Fig 2). However, 2 parameters were significantly different: postoperative cytolysis (Fig 1) and gGT values from W0 to W8 (Fig 3; Table 1). Among parameters that could have influenced our results was the gender ratio, which was significantly different between the 2 groups. We hesitate to attribute a major impact the observed differences in transaminase release to gender. However, the normal value of gGT is higher for men (55 IU/L) than for women (36 IU/L). Therefore, the increased male/female ratio would tend to increase the observed values. Consequently, this male/female partition would tend to increase gGT in the SCOT 15 group. However, according to our statistical analysis, gender did not influence the results.
Although immediate release of transaminase was higher following cold storage in SCOT 15 than UW solution, we failed to observe any consequence on graft functionality. The large range of results, in the SCOT group, reflected by the large SEM (Fig 1), suggested that other parameters could have influenced our results. We gave special attention to the possibility that some distal vascular territory of the graft might not have been correctly rinsed by SCOT 15 solution. Supporting this assumption, SCOT solution was initially developed with PEG at 30 g/L (SCOT 30). At that time, many areas visible at the surface of the grafts were badly rinsed after cooling (personal experience, not included in this study). Therefore, clinical use of SCOT 30 was as continued to be replaced by SCOT 15, which displays a lower viscosity. In fact, macromolecules such as HES impaire red blood cell deformability.24-26 We assume that PEG interacts with blood cells, which could affect the microcirculation, impairing the quality of the preservation in some vascular territories, thereby leading to cell necrosis with immediate release of transaminase after reperfusion. However, such areas might be too small to have consequences for graft functionality.
Postoperative Decrease of the Anicteric Cholestasis
A decreased gGT release in the SCOT 15 compared with the UW group was observed from W1 to W8. The reasons for gGT release after liver transplantation include bile tree hypoxia,27,28 bile duct obstruction,29 preservation injury to the bile ducts, recurrent viral infection,30 regeneration phenomena,31 and immunologic reactions.32 The assumption of an arterial complication can be excluded because we observed only 1 arterial thrombosis within 1 month after transplantation in the UW group. Obstruction of the main bile duct due to an anastomotic stricture was observed once in each group (3%). Inversely, external drainage of the bile duct artificially decreases pressure in the bile duct and thereby gGT release. However, the rate of biliary drainage during liver transplantation was similar in both groups; we failed to observe any statistical effect of this parameter (Table 1). We thus considered that a technical biliary complication could not explain the observed statistical difference. Liver regeneration can increase gGT, which would be lower in the SCOT 15 than the UW group, which contradicts the findings of postoperative release of ALT (Fig 1). Rinsing the graft with saline solution in the SCOT 15 group could have affected the reperfusion injury and the bile tree injury following reperfusion. In fact, albumin, used in the UW group, has been demonstrated to protect against this injury.33 But histologic results and gGT levels (Fig 3) did not support increased preservation injury among the SCOT 15 group rinsed with saline solution. Therefore, although we could not exclude better protection against IRI of the bile tree in the cases involving SCOT 15 versus those
LIVER PRESERVATION WITH SCOT 15
involving UW, the role of the rinse of the solution was not a consistent hypothesis. Hepatitis C virus (HCV) infection, as well as immunologic reactions may increase gGT release. In this study, the presence of antibody against HCV, or HCV replication demonstrated by polymerase chain reaction, was similar between the 2 groups, exerting no significant influence on the results (Table 1). Therefore, the role of HCV to explain the differences observed in the gGT could be excluded. ALT is known to best reflect hepatocyte cytolysis. Although the increased ALT during W1 was not significant, the phenomen was observed only in the UW group. As already reported, PEG 20 kDa can block the immunologic synapse (immunocamouflage phenomenon), decreasing early recognition of surface antigens expressed after ischemia-reperfusion21 and explaining the decreased incidence of rejection episodes observed in kidney,16,22 heart,14 small bowel,15 and pancreatic islet transplantations.17,18 Taken together, after excluding other hypotheses, these results were concordant with the hypothesis that the presence of PEG 20 kDa in SCOT 15 solution decreased alloantigen recognition after liver reperfusion, which would decrease the immunologiccytoxic reactions in the bile ducts. Experimental and clinical studies are warranted to consolidate this assumption. ACKNOWLEDGEMENTS The authors gratefully thank Pr T. Hauet, Pr R. Thuillier, J. Beachner, and Wm. F. Beachner for reviewing this manuscript.
REFERENCES 1. Durand F, Renz JF, Alkofer B, et al: Report of the Paris consensus meeting on expanded criteria donors in liver transplantation. Liver Transpl 14:1694, 2008 2. Belzer FO, Southard JH: Principles of solid organ preservation by cold storage. Transplantation 45:673, 1988 3. Fuller BJ, Lee CY: Hypothermic perfusion preservation: the future of organ preservation revisited? Cryobiology 54:129, 2007 4. Belzer FO, Kalayoglu M, d’Alessandro AM, et al: Organ preservation: experience with University of Wisconsin solution and plans for the future. Clin Transplant 4:73, 1990 5. Neveux N, de Bandt JP, Charrueau C, et al: Deletion of hydroxyethylstarch from University of Wisconsin solution induces cell shrinkage and proteolysis during and after cold starage of rat liver. Hepatology 25:678, 1997 6. Guarrera JV, Karim NA: Liver preservation: is there anything new yet? Curr Opin Organ Transplant 13:148, 2008 7. Feng XN, Xu X, Zheng SS: Current status and perspective of liver preservation solutions. Hepatobiliary Pancreat Dis Int 5:490, 2006 8. Ben AH, Steghens JP, Margonari J, et al: High-Na⫹ low-K⫹ UW cold storage solution reduces reperfusion injuries of the rat liver graft. Transpl Int 11:223, 1998 9. Rayya F, Harms J, Martin AP, et al: Comparison of histidinetryptophan-ketoglutarate solution and University of Wisconsin solution in adult liver transplantation. Transplant Proc 40:891, 2008 10. Lopez-Andujar R, Deusa S, Montalva E, et al: Comparative prospective study of two liver graft preservation solutions: University of Wisconsin and Celsior. Liver Transpl 15:1709, 2009 11. Garcia-Gil FA, Arenas J, Guemes A, et al: Preservation of the liver graft with Celsior solution. Transplant Proc 38:2385, 2006
3407 12. Duca WJ, da Silva RF, Arroyo PC Jr, et al: Liver transplantation using University of Wisconsin or Celsior preserving solutions in the portal vein and Euro-Collins in the aorta. Transplant Proc 42:429, 2010 13. Hauet T, Eugene M: A new approach in organ preservation: potential role of new polymers. Kidney Int 74:998, 2008 14. Collins GM, Wicomb WN, Levin BS, et al: Heart preservation solution containing polyethyleneglycol: an immunosuppressive effect? Lancet 338:890, 1991 15. Itasaka H, Burns W, Wicomb WN, et al: Modification of rejection by polyethylene glycol in small bowel transplantation. Transplantation 57:645, 1994 16. Faure JP, Petit I, Zhang K, et al: Protective roles of polyethylene glycol and trimetazidine against cold ischemia and reperfusion injuries of pig kidney graft. Am J Transplant 4:495, 2004 17. Giraud S, Claire B, Eugene M, et al: A new preservation solution increases islet yield and reduces graft immunogenicity in pancreatic islet transplantation. Transplantation 83:1397, 2007 18. Lee DY, Park SJ, Lee S, et al: Highly poly(ethylene) glycolylated islets improve long-term islet allograft survival without immunosuppressive medication. Tissue Eng 13:2133, 2007 19. Grakoui A, Bromley SK, Sumen C, et al: The immunological synapse: a molecular machine controlling T cell activation. Science 285:221, 1999 20. Gallucci S, Matzinger P: Danger signals: SOS to the immune system. Curr Opin Immunol 13:114, 2001 21. Hauet T, Goujon JM, Baumert H, et al: Polyethylene glycol reduces the inflammatory injury due to cold ischemia/reperfusion in autotransplanted pig kidneys. Kidney Int 62:654, 2002 22. Thuillier R, Renard C, Rogel-Gaillard C, et al: Effect of polyethylene glycol-based preservation solutions on graft injury in experimental kidney transplantation. Br J Surg 98:368, 2011 23. Starzl TE, Miller C, Broznick B, et al. An improved technique for multiple organ harvesting. Surg Gynecol Obstet 165:343, 1987 24. Chmiel B, Cierpka L. Organ preservation solutions impair deformability of erythrocytes in vitro. Transplant Proc 35:2163, 2003 25. van der Plaats A, ‘t Hart NA, Morariu AM et al. Effect of University of Wisconsin organ-preservation solution on haemorheology. Transpl Int 17:227, 2004 26. Panzera P, Rotelli MT, Salerno AM, et al: Solutions for organ perfusion and storage: haemorheologic aspects. Transplant Proc 37:2456, 2005 27. Li S, Stratta RJ, Langnas AN, et al: Diffuse biliary tract injury after orthotopic liver transplantation. Am J Surg 164:536, 1992 28. Pirenne J, Monbaliu D, Aerts R, et al: Biliary strictures after liver transplantation: risk factors and prevention by donor treatment with epoprostenol. Transplant Proc 41:3399, 2009 29. Shastri YM, Hoepffner NM, Akoglu B, et al: Liver biochemistry profile, significance and endoscopic management of biliary tract complications post orthotopic liver transplantation. World J Gastroenterol 13:2819, 2007 30. Ueda Y, Takada Y, Marusawa H, et al: Clinical features of biochemical cholestasis in patients with recurrent hepatitis C after living-donor liver transplantation. J Viral Hepat 17:481, 2010 31. Eisenbach C, Encke J, Merle U, et al: An early increase in gamma glutamyltranspeptidase and low aspartate aminotransferase peak values are associated with superior outcomes after orthotopic liver transplantation. Transplant Proc 41:1727, 2009 32. Chiu KW, Chen YS, de Villa VH, et al: Characterization of liver enzymes on living related liver transplantation patients with acute rejection. Hepatogastroenterology 52:1825, 2005 33. Adam R, Astarcioglu I, Castaing D, et al: Ringer’s lactate vs serum albumin as a flush solution for UW preserved liver grafts: results of a prospective randomized study. Transplant Proc 23: 2374, 1991
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reducing the cold ischemia time, controlling the hypothermia, and using optimal preservation solutions.2,3 Since the 1990s, the reference solution for hepatic transplantation has From the Service de Chirurgie Digestive et Hépato-BilioPancréatique - Transplantation Hépatique, Groupe Hospitalier Pitié-Salpêtrière (E.S., J.M.S. J.C.V., L.H.), and Modélisation en Recherche Clinique (B.G.), Université Pierre et Marie Curie; and Unité de Biostatistiques et Informatique Médicale (B.G.), Service d’Anatomie Pathologique (F.C.), and Fédération de Transplantation (N.C.), Groupe Hospitalier Pitié-Salpêtrière, Paris, France. Address reprint requests to Eric Savier, MD, Service de Chirurgie Digestive et Transplantation Hépatique, Groupe Hospitalier Pitié-Salpêtrière, 47-83 Boulevard de l’Hôpital, 75651 Paris Cedex 13, France. E-mail: [email protected]
0041-1345/11/$–see front matter doi:10.1016/j.transproceed.2011.09.054
© 2011 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
3402
Transplantation Proceedings, 43, 3402–3407 (2011)
LIVER PRESERVATION WITH SCOT 15
been Belzer solution (Viaspan), which was developed at the University of Wisconsin and commonly called UW.4 The intracellular composition of UW includes oxygen radical scavengers and colloid hydroxyethylstarch (HES) to maintain oncotic pressure.5 Nowadays, new preservation concepts include machine perfusion, protective molecules supplements, and genetic manipulations.3,6,7 Extracellular solutions have been used for preservation and for a fast rinse of the graft just before reperfusion.8 New solutions (Custodiol, Celsior, IGL-1) of extracellular composition are emerging, although clinical trials have so far failed to demonstrate any major differences from the criteria standard, UW.9-12 Polyethylene glycol (PEG) a macromolecule used in some solutions to ensure oncotic pressure (IGL-1: PEG 35 kD, 1 g/L), is used as PEG 20 kD, 15 g/L in a newly developed solution, SCOT 15.13 An interesting property of the PEG, which is polymerized to 20 kD molecular weight, is an associated decrease in allograft rejection in several models.14-18 The main hypothesis to explain these results is that PEG may block the immunologic synapse,19 consequently decreasing the initial immune response. This effect could be important, because a main consequence of reperfusion after cold ischemia is the induction of a temporary but major inflammatory chain reaction that stimulates the innate immunological response.20 It is now clearly established that these pathophysiologic effects have long-term consequences.16,21,22 Therefore, preserving an organ with SCOT 15 could be a step forward in liver transplantation. The goal of the present study was to report our results with this new solution versus those with UW. METHODS Definitions D0 was the day of the transplantation, W0 (D0 to D7) the week after, and M0 (D0 to D30) the month after the operation. Consequently, M1 was the second month after grafting.
Surgical Technique Every human liver transplantation was performed as follows. After cooling with SCOT 15 or UW infused through the aorta and the inferior mesenteric vein, the liver was harvested from the donor according to Starzl’s technique,23 and subsequently prepared with infusion of cold SCOT 15 (1,500 mL) or UW (1,000 mL) by the portal vein. In the recipient, we performed a total hepatectomy including the retrohepatic vena cava. Just before portal reperfusion, the graft was rinsed with cold solution, either 500 –1,000 mL human albumin (4%) following UW cold storage or 500 mL NaCl (0.9%) solution following SCOT 15 cold storage, seeking to decrease the potassium concentration in the effluent. An arterial anastomosis was then performed under magnification. The biliary anastomosis was performed with or without biliary drainage (silicon T-tube or polyurethane transcystic tube), according to the biliary anatomy and personal experience of the surgeon. Biliary drainage was removed after M2 in all cases. Posttransplantation immunosuppressive therapy included cyclosporine or tacrolimus, steroid, and mycophenolate mofetil for 3 months before switching to a 2-drug regimen, if possible. Liver biopsy was performed
3403 according to blood liver test results and in cases of suspected acute rejection episodes.
Selection of the Patients and Exclusion Criteria Exclusion criteria were split grafts, live donor grafts, non– heartbeating donors, domino liver transplantations, pediatric recipients, combined heart and liver recipients, grafts procured by other surgical teams, and grafts stored in SCOT 30 (PEG 20 kD, 30 g/L). The transplanted patients whose livers had been preserved in SCOT 15 were compared with a retrospective group of recently transplanted patients with median dates of transplantation January 15, 2009, for the UW group and February 11, 2010, for the SCOT 15 group. Among 35 transplantations using SCOT 15 between July 2009 and July 2010, 2 were excluded: one owing to vena caval thrombosis, leading to a surgical deobstruction on D3 with the patient alive at the time of writing, and the other owing to a split graft. Control group patients were selected among the transplantations with preservation in UW performed between January 2008 and September 2010. They were paired for cold ischemia time, warm ischemia time, namely, arterial revascularization minus portal reperfusion time, graft weight, and donor age (Table 1). Among 40 selected patients, 6 were excluded for the following reasons: renoportal anastomosis in the absence of portal trunk (n ⫽ 1); grafts divided with a pediatric team (n ⫽ 3); combined heart transplant (n ⫽ 1), and death at D0 from sepsis (n ⫽ 1). One patient’s data including heart failure were removed from D5 until retransplantation. Thus, the SCOT 15 group included 33 liver transplantations in 33 patients and the UW group 34 transplantations in 33 patients.
Measured Parameters Biologic parameters were assayed by hospital departments. When several values were available for 1 period of time (day or week), we included the maximum value for aspartate transaminase (AST) and alanine transaminase (ALT) and the average value for gammaglutamyltranspeptidase (gGT), bilirubin, factor V, prothrombin time, and platelet count. Liver biopsies, obtained with a 14-gauge Menghini needle or with a wedge surgical biopsy, were fixed, prepared and stained using standard techniques. All hepatic biopsies were evaluated by the same pathologist (F.C.) in blinded fashion; rejections were graded according to Banff rejection activity index.
Statistical Analyses Baseline characteristics of patients were compared with the Fisher exact test for categoric variables or the Mann-Whitney Wilcoxon test for continuous variables. Linear mixed models, with fixed subject random intercepts, were used to evaluate longitudinal changes (time effect) in each parameter. Using all available time points, this approach yielded greater statistical power than considering only the first and last points. Linear mixed models took into account the solution (the parameter levels over time were different between solutions), interactions between time and solution (the kinetics of evolution of parameter was different between solutions) with gender as covariates. The same linear mixed model was used to evaluate the effects of the solution on each parameter. Data were analyzed using the R version 2.11.0 and SigmaPlot version 9.01 of SigmaStat version 3.11 (Systat Software).
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SAVIER, GRANGER, CHARLOTTE ET AL Table 1. Statistical Comparisons Between SCOT 15 and University of Wisconsin (UW) Groups
Cold ischemia time (min) Warm ischemia time (min)* Donor age (y) Graft weight (g) MELD score Child-Pugh score UNOS score 1: Intensive care unit 2: In-hospital stay 3: Permanent care 4: Waiting at home Main indication for liver transplantation Cirrhosis or chronic hepatocellular insufficiency† Acute liver failure Hepatocellular carcinoma Metabolic Anti-HCV antibody Negative Positive PCR HCV Negative Positive Gender ratio (M/F) Biliary anastomosis with biliary tube‡ Yes No Gamma-glutamyl transpeptidase (gGT) Week 0 Week 1 Week 2 Week 3 Week 4 Week 5 Week 6 Week 7 Week 8
SCOT 15 (n ⫽ 33)
UW (n ⫽ 34)
P Value
458 ⫾ 14 50 ⫾ 3 50 ⫾ 3 1,533 ⫾ 67 15.7 ⫾ 1.7 9 ⫾ 0.5
472 ⫾ 11 53 ⫾ 3 53 ⫾ 2 1,440 ⫾ 54 18.4 ⫾ 1.9 9.2 ⫾ 0.5
.459 .199 .390 .311 .269 .721 .769
13 3 2 15
10 2 2 20
17 3 13 0
21 2 10 1
20 13 (39%)
24 10 (29%)
23 10 (30%) 29/4
27 7 (20%) 21/13
18 (55%) 15
16 (47%) 18
125 ⫾ 68 233 ⫾ 125 182 ⫾ 125 153 ⫾ 100 146 ⫾ 132 161 ⫾ 170 128 ⫾ 166 129 ⫾ 156 130 ⫾ 161
125 ⫾ 69 324 ⫾ 165 261 ⫾ 243 282 ⫾ 277 237 ⫾ 256 305 ⫾ 305 244 ⫾ 275 332 ⫾ 357 310 ⫾ 388
.635
.114
1
.023 .628
Time: .0002 Gender: .358 Solution: ⬍.01 Time* solution: .291
Abbreviations: HCV, hepatitis C virus; MELD, Model for End-Stage Liver Disease; PCR, polymerase chain reaction; UNOS, United Network for Organ Sharing. *Time between portal and arterial liver reperfusion. †Cirrhosis from any cause and chronic sclerosing cholangitis at end stage. ‡T-Tube or transcystic tube inserted at the time of the transplantation. Baseline characteristics of patients were compared with the Fisher exact test or the Mann-Whitney Wilcoxon test depending on the type of data. Consecutive periods of postoperative time were compared between the 2 groups from week 0 to 8 for gGT. A linear mixed model was used to evaluate the longitudinal change and the influence of solution on each parameter as described in the Methods. Results are presented as mean ⫾ SD for quantitative data and frequency for qualitative data. P ⬍ .05 for statistical significance.
RESULTS Biologic Results
At D0, transaminase release in the SCOT 15 group showed a median value of 1,792 UI/L (range 346 –7,791) for AST, and 750 UI/L (range 205– 4,870) for ALT. This peak of transaminase disappeared after 48 hours for AST (Fig 1A) and after 4 days for ALT (Fig 1B). Interestingly, from W1 to W8, AST and ALT decreased more quickly among the SCOT 15 group than the UW group (data not shown), with a slight increase between D8 and D12 for ALT only in the UW group (P ⫽ NS; Fig 1B). The evolution of prothrombin time was identical in the 2 groups (Fig 2A). Factor V increased from 40% at D0 to 80% at D5, remaining ⬎80% beyond D8 in both groups (data not shown). The average bilirubin decreased from D7–D14 to D21–D28 to a value
below twice the normal result among the SCOT 15 group, whereas it normalized above normal in the UW group, who showed a similar evolution. (Fig 2B). The platelet count increased between D0 and D21 from 60,000/mm3 to 250,000/mm3, without any difference between the 2 groups (Fig 2C). The evolution of gGT was significantly different over the first 2 postoperative months (Fig 3). In the SCOT 15 group, the average gGT was maximal at W1, gradually decreasing to 2–3 times normal at W7 (Table 1). Among the UW group, in contrast, the gGT reached high levels that rose from W1 to W7 (Table 1). Histologic Results
A hepatic biopsy was performed when there were blood test anomalies or the clinical context required histologic data to
LIVER PRESERVATION WITH SCOT 15
3405
patient died 0.8 month later from multiple organ failure. The second patient, transplanted for alcoholic cirrhosis with a major reperfusion syndrome, displayed an arterial thrombosis at D27. She was retransplanted but died from sepsis and multiple organ failure 16 days later. We did not include the second transplantation because the preservation solution was IGL-1. One patient in each group experienced a main bile duct stricture at M1; both were successfully
Fig 1. Release of transaminases after liver transplantation with SCOT 15 or University of Wisconsin (UW) solutions. Serum aspertate transaminase (AST) (A) and alanine transaminase (ALT) (B) were measured by the Pitié-Salpêtrière hospital laboratories. Values shown are the maximal of every available value measured during each time period (day). A significant difference was observed at day 0 between the SCOT 15 group and the UW group. Mann- Whitney rank sum test (n ⫽ 33 and 34: *P ⬍ .05; **P ⬍ .01. Results are shown as mean ⫾ SEM.
adapt the immunosuppressive therapy. Between W0 and W12, 37 biopsies were performed: 11 in the SCOT 15 (33%) and 26 in the UW group (76%). In the SCOT 15 group we identified 3 acute rejection episodes involving 3 patients: total Banff score 6 at D7, Banff 4 at D16, and Banff 2 at D32. Among the UW group, we identified 7 acute rejection episodes involving 6 patients: Banff 3 at D10, Banff 4 at D11, Banff 3 at D14 and D55, Banff 2 at D37, Banff 4 at D14, and Banff 3 at D56. Preservation injury was observed in 1 SCOT 15 and 6 UW group cases. Postoperative Follow-Up
No primary liver failure was noticed in either group. Mortality at 3 months was 2 patients in the UW and 0 in the SCOT 15 group. The first patient had a pericardial fluid collection, leading to heart failure with considerable repercussion on the liver graft, as demonstrated by explantation of a necrotic graft at D28. Despite retransplantation, the
Fig 2. Prothrombin time, total bilirubin and platelets count evolution following liver transplantation with SCOT 15 or University of Wisconsin (UW) solutions.Prothrombin time (A), total bilirubin (B), and platelet count (C) were measured by the PitiéSalpêtrière hospital laboratories. Values shown are the means of every available value measured during each time period (day). A linear mixed model was used to evaluate the longitudinal change, and the influence of solution on each parameter as described in Methods. No difference was observed between the SCOT 15 group and the UW group (n ⫽ 33 and 34). Results are shown as mean ⫾ SEM.
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SAVIER, GRANGER, CHARLOTTE ET AL
Postoperative Cytolysis
Fig 3. Release of gGT following liver transplantation with SCOT 15 or University of Wisconsin (UW) solutions. Serum activity of gamma-glutamyltranspeptidase (gGT) was measured by the Pitié-Salpêtrière hospital laboratories. Values shown are the means of every available value measured during each time period. Week 0 includes postoperative days 0 –7. A linear mixed model was used to evaluate the longitudinal change and the influence of solution on each parameter as described in the Methods. A significant difference was observed, as detailed in Table 1, between the SCOT 15 group and the UW group (n ⫽ 33 and 34). Results are shown as mean ⫾ SEM.
treated with endoscopic retrograde stenting. The median follow-up of the grafts was 9.6 months (range 3.2–16) among the SCOT 15 group and 17.1 months (range 0.8 – 34.3) among the UW group. DISCUSSION
This report on the use of SCOT 15 solution in clinical liver transplantation used a retrospective comparison with UW. Our study was homogeneous regarding to surgical procedures, assays, and postoperative management. Therefore, missing data were negligible for observations with statistical significance. The quality of graft preservation with SCOT 15 was assessed by the postoperative evolution of prothrombin time, factor V, platelets, and bilirubin. Results and evolution of these parameters postoperatively were identical between the SCOT 15 and the UW group (Fig 2). However, 2 parameters were significantly different: postoperative cytolysis (Fig 1) and gGT values from W0 to W8 (Fig 3; Table 1). Among parameters that could have influenced our results was the gender ratio, which was significantly different between the 2 groups. We hesitate to attribute a major impact the observed differences in transaminase release to gender. However, the normal value of gGT is higher for men (55 IU/L) than for women (36 IU/L). Therefore, the increased male/female ratio would tend to increase the observed values. Consequently, this male/female partition would tend to increase gGT in the SCOT 15 group. However, according to our statistical analysis, gender did not influence the results.
Although immediate release of transaminase was higher following cold storage in SCOT 15 than UW solution, we failed to observe any consequence on graft functionality. The large range of results, in the SCOT group, reflected by the large SEM (Fig 1), suggested that other parameters could have influenced our results. We gave special attention to the possibility that some distal vascular territory of the graft might not have been correctly rinsed by SCOT 15 solution. Supporting this assumption, SCOT solution was initially developed with PEG at 30 g/L (SCOT 30). At that time, many areas visible at the surface of the grafts were badly rinsed after cooling (personal experience, not included in this study). Therefore, clinical use of SCOT 30 was as continued to be replaced by SCOT 15, which displays a lower viscosity. In fact, macromolecules such as HES impaire red blood cell deformability.24-26 We assume that PEG interacts with blood cells, which could affect the microcirculation, impairing the quality of the preservation in some vascular territories, thereby leading to cell necrosis with immediate release of transaminase after reperfusion. However, such areas might be too small to have consequences for graft functionality.
Postoperative Decrease of the Anicteric Cholestasis
A decreased gGT release in the SCOT 15 compared with the UW group was observed from W1 to W8. The reasons for gGT release after liver transplantation include bile tree hypoxia,27,28 bile duct obstruction,29 preservation injury to the bile ducts, recurrent viral infection,30 regeneration phenomena,31 and immunologic reactions.32 The assumption of an arterial complication can be excluded because we observed only 1 arterial thrombosis within 1 month after transplantation in the UW group. Obstruction of the main bile duct due to an anastomotic stricture was observed once in each group (3%). Inversely, external drainage of the bile duct artificially decreases pressure in the bile duct and thereby gGT release. However, the rate of biliary drainage during liver transplantation was similar in both groups; we failed to observe any statistical effect of this parameter (Table 1). We thus considered that a technical biliary complication could not explain the observed statistical difference. Liver regeneration can increase gGT, which would be lower in the SCOT 15 than the UW group, which contradicts the findings of postoperative release of ALT (Fig 1). Rinsing the graft with saline solution in the SCOT 15 group could have affected the reperfusion injury and the bile tree injury following reperfusion. In fact, albumin, used in the UW group, has been demonstrated to protect against this injury.33 But histologic results and gGT levels (Fig 3) did not support increased preservation injury among the SCOT 15 group rinsed with saline solution. Therefore, although we could not exclude better protection against IRI of the bile tree in the cases involving SCOT 15 versus those
LIVER PRESERVATION WITH SCOT 15
involving UW, the role of the rinse of the solution was not a consistent hypothesis. Hepatitis C virus (HCV) infection, as well as immunologic reactions may increase gGT release. In this study, the presence of antibody against HCV, or HCV replication demonstrated by polymerase chain reaction, was similar between the 2 groups, exerting no significant influence on the results (Table 1). Therefore, the role of HCV to explain the differences observed in the gGT could be excluded. ALT is known to best reflect hepatocyte cytolysis. Although the increased ALT during W1 was not significant, the phenomen was observed only in the UW group. As already reported, PEG 20 kDa can block the immunologic synapse (immunocamouflage phenomenon), decreasing early recognition of surface antigens expressed after ischemia-reperfusion21 and explaining the decreased incidence of rejection episodes observed in kidney,16,22 heart,14 small bowel,15 and pancreatic islet transplantations.17,18 Taken together, after excluding other hypotheses, these results were concordant with the hypothesis that the presence of PEG 20 kDa in SCOT 15 solution decreased alloantigen recognition after liver reperfusion, which would decrease the immunologiccytoxic reactions in the bile ducts. Experimental and clinical studies are warranted to consolidate this assumption. ACKNOWLEDGEMENTS The authors gratefully thank Pr T. Hauet, Pr R. Thuillier, J. Beachner, and Wm. F. Beachner for reviewing this manuscript.
REFERENCES 1. Durand F, Renz JF, Alkofer B, et al: Report of the Paris consensus meeting on expanded criteria donors in liver transplantation. Liver Transpl 14:1694, 2008 2. Belzer FO, Southard JH: Principles of solid organ preservation by cold storage. Transplantation 45:673, 1988 3. Fuller BJ, Lee CY: Hypothermic perfusion preservation: the future of organ preservation revisited? Cryobiology 54:129, 2007 4. Belzer FO, Kalayoglu M, d’Alessandro AM, et al: Organ preservation: experience with University of Wisconsin solution and plans for the future. Clin Transplant 4:73, 1990 5. Neveux N, de Bandt JP, Charrueau C, et al: Deletion of hydroxyethylstarch from University of Wisconsin solution induces cell shrinkage and proteolysis during and after cold starage of rat liver. Hepatology 25:678, 1997 6. Guarrera JV, Karim NA: Liver preservation: is there anything new yet? Curr Opin Organ Transplant 13:148, 2008 7. Feng XN, Xu X, Zheng SS: Current status and perspective of liver preservation solutions. Hepatobiliary Pancreat Dis Int 5:490, 2006 8. Ben AH, Steghens JP, Margonari J, et al: High-Na⫹ low-K⫹ UW cold storage solution reduces reperfusion injuries of the rat liver graft. Transpl Int 11:223, 1998 9. Rayya F, Harms J, Martin AP, et al: Comparison of histidinetryptophan-ketoglutarate solution and University of Wisconsin solution in adult liver transplantation. Transplant Proc 40:891, 2008 10. Lopez-Andujar R, Deusa S, Montalva E, et al: Comparative prospective study of two liver graft preservation solutions: University of Wisconsin and Celsior. Liver Transpl 15:1709, 2009 11. Garcia-Gil FA, Arenas J, Guemes A, et al: Preservation of the liver graft with Celsior solution. Transplant Proc 38:2385, 2006
3407 12. Duca WJ, da Silva RF, Arroyo PC Jr, et al: Liver transplantation using University of Wisconsin or Celsior preserving solutions in the portal vein and Euro-Collins in the aorta. Transplant Proc 42:429, 2010 13. Hauet T, Eugene M: A new approach in organ preservation: potential role of new polymers. Kidney Int 74:998, 2008 14. Collins GM, Wicomb WN, Levin BS, et al: Heart preservation solution containing polyethyleneglycol: an immunosuppressive effect? Lancet 338:890, 1991 15. Itasaka H, Burns W, Wicomb WN, et al: Modification of rejection by polyethylene glycol in small bowel transplantation. Transplantation 57:645, 1994 16. Faure JP, Petit I, Zhang K, et al: Protective roles of polyethylene glycol and trimetazidine against cold ischemia and reperfusion injuries of pig kidney graft. Am J Transplant 4:495, 2004 17. Giraud S, Claire B, Eugene M, et al: A new preservation solution increases islet yield and reduces graft immunogenicity in pancreatic islet transplantation. Transplantation 83:1397, 2007 18. Lee DY, Park SJ, Lee S, et al: Highly poly(ethylene) glycolylated islets improve long-term islet allograft survival without immunosuppressive medication. Tissue Eng 13:2133, 2007 19. Grakoui A, Bromley SK, Sumen C, et al: The immunological synapse: a molecular machine controlling T cell activation. Science 285:221, 1999 20. Gallucci S, Matzinger P: Danger signals: SOS to the immune system. Curr Opin Immunol 13:114, 2001 21. Hauet T, Goujon JM, Baumert H, et al: Polyethylene glycol reduces the inflammatory injury due to cold ischemia/reperfusion in autotransplanted pig kidneys. Kidney Int 62:654, 2002 22. Thuillier R, Renard C, Rogel-Gaillard C, et al: Effect of polyethylene glycol-based preservation solutions on graft injury in experimental kidney transplantation. Br J Surg 98:368, 2011 23. Starzl TE, Miller C, Broznick B, et al. An improved technique for multiple organ harvesting. Surg Gynecol Obstet 165:343, 1987 24. Chmiel B, Cierpka L. Organ preservation solutions impair deformability of erythrocytes in vitro. Transplant Proc 35:2163, 2003 25. van der Plaats A, ‘t Hart NA, Morariu AM et al. Effect of University of Wisconsin organ-preservation solution on haemorheology. Transpl Int 17:227, 2004 26. Panzera P, Rotelli MT, Salerno AM, et al: Solutions for organ perfusion and storage: haemorheologic aspects. Transplant Proc 37:2456, 2005 27. Li S, Stratta RJ, Langnas AN, et al: Diffuse biliary tract injury after orthotopic liver transplantation. Am J Surg 164:536, 1992 28. Pirenne J, Monbaliu D, Aerts R, et al: Biliary strictures after liver transplantation: risk factors and prevention by donor treatment with epoprostenol. Transplant Proc 41:3399, 2009 29. Shastri YM, Hoepffner NM, Akoglu B, et al: Liver biochemistry profile, significance and endoscopic management of biliary tract complications post orthotopic liver transplantation. World J Gastroenterol 13:2819, 2007 30. Ueda Y, Takada Y, Marusawa H, et al: Clinical features of biochemical cholestasis in patients with recurrent hepatitis C after living-donor liver transplantation. J Viral Hepat 17:481, 2010 31. Eisenbach C, Encke J, Merle U, et al: An early increase in gamma glutamyltranspeptidase and low aspartate aminotransferase peak values are associated with superior outcomes after orthotopic liver transplantation. Transplant Proc 41:1727, 2009 32. Chiu KW, Chen YS, de Villa VH, et al: Characterization of liver enzymes on living related liver transplantation patients with acute rejection. Hepatogastroenterology 52:1825, 2005 33. Adam R, Astarcioglu I, Castaing D, et al: Ringer’s lactate vs serum albumin as a flush solution for UW preserved liver grafts: results of a prospective randomized study. Transplant Proc 23: 2374, 1991