Use of N -acetylcysteine during liver procurement: A prospective randomized controlled study

ORIGINAL ARTICLE

Use of N-Acetylcysteine During LiverProcurement: A Prospective RandomizedControlled StudyFrancesco D’Amico,1 Alessandro Vitale,1 Donatella Piovan,2 Alessandra Bertacco,1 Rafael RamirezMorales,3 Anna Chiara Frigo,4 Domenico Bassi,1 Pasquale Bonsignore,1 Enrico Gringeri,1 MicheleValmasoni,1 Greta Garbo,1 Enrico Lodo,1 Francesco Enrico D’Amico,1 Michele Scopelliti,1 AmedeoCarraro,1 Martina Gambato,5 Alberto Brolese,1 Giacomo Zanus,1 Daniele Neri,1 and Umberto Cillo1

1Department of Hepatobiliary Surgery and Liver Transplantation, University Hospital of Padua, Padua, Italy;2Medical Pharmacology, University Hospital of Padua, Padua, Italy; 3Veneto Institute of OncologyIOV–IRCCS, Padua, Italy; 4Department of Biostatistics and Epidemiology, University Hospital of Padua,Padua, Italy; and 5Division of Gastroenterology, University Hospital of Padua, Padua, Italy

Antioxidant agents have the potential to reduce ischemia/reperfusion damage to organs for liver transplantation (LT). In thisprospective, randomized study, we tested the impact of an infusion of N-acetylcysteine (NAC) during liver procurement onpost-LT outcomes. Between December 2006 and July 2009, 140 grafts were transplanted into adult candidates with chronicliver disease who were listed for first LT, and according to a sequential, closed-envelope, single-blinded procedure, thesepatients were randomly assigned in a 1/1 ratio to an NAC protocol (69 patients) or to the standard protocol without NAC [71patients (the control group)]. The NAC protocol included a systemic NAC infusion (30 mg/kg) 1 hour before the beginning ofliver procurement and a locoregional NAC infusion (300 mg through the portal vein) just before cross-clamping. The primaryendpoint was graft survival. The graft survival rates at 3 and 12 months were 93% and 90%, respectively, in the NAC group and82% and 70%, respectively, in the control group (P ¼ 0.02). An adjusted Cox analysis showed a significant NAC effect on graftsurvival at both 3 months [hazard ratio ¼ 1.65, 95% confidence interval (CI) ¼ 1.01-2.93, P ¼ 0.04] and 12 months (hazard ra-tio ¼ 1.73, 95% CI ¼ 1.14-2.76, P " 0.01). The incidence of postoperative complications was lower in the NAC group (23%)versus the control group (51%, P < 0.01). In the subgroup of 61 patients (44%) receiving suboptimal grafts (donor risk index >1.8), the incidence of primary dysfunction of the liver was lower (P ¼ 0.09) for the NAC group (15%) versus the control group(32%). In conclusion, the NAC harvesting protocol significantly improves graft survival. The effect of NAC on early graft functionand survival seems higher when suboptimal grafts are used. Liver Transpl 19:135-144, 2013. VC 2012 AASLD.

Received April 3, 2012; accepted July 7, 2012.

Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase; CI, confidence interval; DRI, donor risk index;GSH, glutathione; ICU, intensive care unit; INR, international normalized ratio; IRI, ischemia/reperfusion injury; LT, livertransplantation; MELD, Model for End-Stage Liver Disease; NAC, N-acetylcysteine; PDF, primary dysfunction; PNF, primarynonfunction; POD, postoperative day.

Francesco D’Amico contributed to the study concept and design, the acquisition and analysis of data, and the supervision of thestudy and manuscript. Alessandro Vitale contributed to the analysis and interpretation of data, the drafting of the manuscript,and the statistical analysis. Anna Chiara Frigo contributed to the statistical analysis. Umberto Cillo contributed to the analysisand interpretation of data and the supervision of the study. Alessandra Bertacco contributed to the acquisition and analysis ofdata and the critical revision of the manuscript. Donatella Piovan, Domenico Bassi, Rafael Ramirez Morales, Pasquale Bonsignore,Enrico Gringeri, Michele Valmasoni, Greta Garbo, Enrico Lodo, Francesco Enrico D’Amico, Michele Scopelliti, Amedeo Carraro,Martina Gambato, Alberto Brolese, Giacomo Zanus, and Daniele Neri contributed to the critical revision of the manuscript forimportant intellectual content and the acquisition of data.

Francesco D’Amico had full access to all the data and takes full responsibility for the veracity of the data and the statistical analysis.

The present study is registered with ClinicalTrials.gov, identifier NCT01394497.

Address reprint requests to Francesco D’Amico, M.D., Ph.D., F.E.B.S., Department of Hepatobiliary Surgery and Liver Transplantation, University Hospitalof Padua, Via Giustiniani 2, 35128 Padua, Italy. Telephone:þ39 049 821-8624; FAX:þ39 049 821-1816; E-mail: [email protected]

DOI 10.1002/lt.23527View this article online at wileyonlinelibrary.com.LIVER TRANSPLANTATION.DOI 10.1002/lt. Published on behalf of the American Association for the Study of Liver Diseases

LIVER TRANSPLANTATION 19:135-144, 2013

VC 2012 American Association for the Study of Liver Diseases.

See Editorial on Page 118

Liver transplantation (LT) is the treatment of choicefor end-stage liver disease. Ischemia/reperfusioninjury (IRI) during the conventional cold storage andpreservation of donated livers is a key determinant ofearly graft function after LT.1

In recent years, suboptimal livers have beenincreasingly used because of organ shortages.2

Unfortunately, the use of marginal livers may havenegative effects on initial graft function,3 with pro-longed IRI being the main source of graftdysfunction.1

The pathophysiology of IRI is mainly due to the oxida-tive stress and cytotoxicity arising from the imbalancebetween oxidants and antioxidants.4 Glutathione (GSH)is the main endogenous antioxidant, and the liver is themain organ producing GSH under normal conditions(normothermia and normoperfusion/oxygenation).Because of the donor’s stay in the intensive care unit(ICU) and the period of ischemia, potential intrahepaticGSH depletion can lead to the liver being less able toovercome reperfusion oxidative stress. Hepatic GSHsynthesis is a cysteine/methionine-dependent processstrictly linked to the dietary introduction of its precur-sors. N-Acetylcysteine (NAC) is the acetylated precursorof both the amino acid L-cysteine and reduced GSH. Ex-perimental animal models have shown a potential posi-tive impact of NAC or GSH infusions in decreasing IRIduring cold liver preservation.4,5

NAC is commonly used in clinical practice as a mu-colytic agent for chronic respiratory illness, as anantidote for hepatotoxicity due to acetaminophenoverdose, and as prophylaxis against renal injury dueto radiocontrast agents.6 NAC has a very good safetyprofile, and there is no documented evidence of dose-dependent side effects.

The aim of this prospective, randomized study wasto test the impact of systemic and locoregional infu-sions of NAC during liver procurement on post-LToutcomes.

PATIENTS AND METHODS

Trial Design and Participants

This was a prospective, single-blinded, randomizedphase 2 study of 2 parallel groups designed to evalu-ate the clinical benefits of NAC infusions during liverprocurement versus a standard procedure.

All subjects who met the inclusion criteria wererandomized in a 1:1 ratio to receive an organ perfusedor not perfused with NAC during the harvestingprocedure.

This study included all consecutive subjects withchronic liver disease who underwent LT for the first timewith deceased donor livers at our institution betweenDecember 2006 and July 2009. The following subjectswere excluded: (1) recipients with acute liver disease, (2)pediatric patients or adult patients receiving livers from

pediatric donors, (3) patients undergoing multiorgantransplantation, (4) patients undergoing re-LT, and (5)patients undergoing living donor LT.

Data were collected during the organ procurementphase, surgical interventions, and follow-up medicalvisits performed at our facilities.

This study was reviewed and approved by our insti-tutional ethics committee, which considered consentand authorization for research necessary only for therecipients.

Interventions

In this study, the selection of patients for LT and theassignment of donor organs strictly adhered to ourclinical practice and were thus based on a transpar-ent and well-defined allocation protocol using theModel for End-Stage Liver Disease (MELD) score tostage the severity of liver cirrhosis.7 This allocationscheme was established at our center 6 months beforethe initiation of this study.

All the recipients were at our institution (ie, thiswas a single-center study). Organ procurement, onthe contrary, was performed throughout the Italianregion of the center and sometimes also extraregion-ally. However, the surgical recovery team alwaysbelonged to our institution.

In Italy, organ procurement is performed exclusivelywith heart-beating donors.

Consecutive deceased donor livers with eligiblepotential recipients were randomly assigned to eitherthe standard procurement procedure or the NACprotocol.

In addition to the standard procedure, the NAC pro-tocol included a 15-minute systemic NAC infusion (30mg/kg for a maximum dose of 3000 mg) diluted intoa 5% glucose solution (500 mL) 1 hour before the ini-tiation of liver harvesting and a locoregional infusion(150 mg/kg of estimated liver weight for a maximumdose 300 mg) into the portal vein just before cross-clamping. The protocol infusion doses were estab-lished according to the following criteria: (1) the sys-temic NAC dose (30 mg/kg) corresponded to the main-tenance dose used for the treatment of acute hepaticfailure, and (2) the locoregional NAC dose (150 mg/kgof liver weight) corresponded to the loading dose usedfor the treatment of acute liver failure.

As previously described,8 the standard procurementprocedure at our center was based on a modified dou-ble perfusion technique (aortic and portal cooling withtourniquet clamping of the splenomesenteric veininflow). Single aortic perfusion was used only withsplit or multivisceral harvesting procedures. Donorlivers were gravity-perfused in situ via the aorta andportal vein with Celsior solution at 4$C. Approximately60% of the solution volume (30 mL/kg via the portalvein and 60 mL/kg via the aorta in the double perfu-sion procedure and 90 mL/kg via the aorta with thesingle aortic perfusion technique) was infused rapidly(10-15 minutes) after aortic cross-clamping. Perfusionwas then slowed for the remaining 40% of the solution

136 D’AMICO ET AL. LIVER TRANSPLANTATION, February 2013

until the harvesting was completed (20-40 minutes).After hepatectomy, donor livers were further perfusedat the back table with Celsior (700 mL via the portalvein and 300 mL via the hepatic artery) and thenstored in conventional bags containing the same solu-tion at 4$C until transplantation.

LT was always performed with the preservation ofthe retrohepatic vena cava (the piggyback technique)and without the use of a biopump. Transplant patientswere given dual- or triple-drug immunosuppressivetherapy, which included cyclosporine or tacrolimuscombined with corticosteroids with or without myco-phenolate mofetil. Liver biopsy was performed aftertransplantation only when it was clinically indicated.

Outcomes and Definitions

The primary endpoint of this study was graft survival.The secondary endpoints were as follows: (1) patient

survival; (2) the incidence of primary dysfunction(PDF); (3) differences in transaminase, internationalnormalized ratio (INR), and bilirubin values on post-operative days (PODs) 2, 7, and 15; (4) lengths of hos-pital stays, blood-derived transfusions, and days oftherapy with vasoactive amines; (5) postoperativecomplications (surgical and medical complications,including infections); and (6) acute rejection. We con-sidered a complication to be any clinical adverse eventgraded as 3 or higher according to Common Terminol-ogy Criteria for Adverse Events version 4.0.

The quality of donor livers was described with thedonor risk index (DRI).3

Graft PDF was defined according to Pokorny et al.9

as the sum of primary nonfunction (PNF) and initialpoor function. PNF was defined as non–life-sustainingfunction of the graft leading to death or retransplanta-tion within 7 days. Initial poor function was definedas an aspartate aminotransferase (AST) level > 2500IU/L and clotting factor support for more than 2 daysduring the first 5 days after the operation.

In this study, we sought maximal adherence to ournormal clinical practice. Consequently, we did not per-form protocol-specific biopsy at the time of LT or at 3months, but we performed liver biopsy only when itwas clinically indicated (to discriminate between acuterejection, hepatitis, and other kinds of liver damage).

Sample Size

The sample size was calculated on the basis of the fol-lowing assumptions:

• A type I error rate of 5%.• A power of 90%.• A 1:1 distribution ratio of enrolled patients in the2 groups.

• A 1-sided test.• An accrual period of 30 months.• A minimum follow-up period of 12 months for liv-ing enrolled patients.

To reach these goals, we decided to recruit 176patients (88 per group).

An interim analysis was planned for 15 months af-ter the initiation of enrollment.10 No a priori criteriawere set for an early end of the study.

Because of the monocentric and spontaneous na-ture of this study and the well-known positive safetyprofile of NAC, the inclusion of an external data safetymonitoring board in the study design was not deemednecessary or required.

Randomization

between December 2006 and July 2009, all potentialgrafts for recipients considered eligible for this studywere randomly assigned to 1 of 2 arms: (1) deceaseddonors received systemic and locoregional infusions ofNAC, or (2) the standard harvesting procedure (with-out NAC infusions) was performed. The designation oforgans to either group was performed according tocomputer-generated block randomization (n ¼ 10 perblock). Random ranking per block was determinedwith the RAND function of Microsoft Excel, and it wasused in the sequential attribution of treatments. Ran-domization was performed by the principal investiga-tor immediately after he received the notification callfrom our organ-sharing organization for a potentialliver from a deceased donor (this included organs thatwere deemed unsuitable for transplantation after anaccurate evaluation), and this was communicated tothe surgical team at the moment of organ retrieval.Patients were blinded to the type of preservation treat-ment that the organ had received, and at no pointduring the study were they unblinded.

Between December 2006 and July 2009, 214 poten-tial grafts were included in the procedure (107 in thestudy group and 107 in the control group). In 74cases (38 in the study group and 36 in the controlgroup), the livers were subsequently consideredunsuitable for transplantation because of high-gradesteatosis (steatosis > 50% after biopsy; 10 cases),liver cirrhosis (13 cases), cancer in the donor (9cases), liver fibrosis (13 cases), sepsis in the donor (4cases), hepatitis C virus positivity (4 cases), hepatictrauma (2 cases), liver ischemic injury (6 cases), andother causes (13 cases).

The remaining 140 livers were transplanted into therecipients enrolled in this study.

Statistical Analysis

All demographic and baseline variables weredescribed as follows: categorical data were describedas frequencies and percentages, and continuous datawere described as medians and ranges. Missing datawere excluded from the final analysis.

For subgroup comparisons, quantitative variableswere compared with the Student t test or the Wilcoxonrank-sum test, and categorical variables were com-pared with the v2 test or Fisher’s exact test asappropriate.

Enrolled patients were followed until April 2011when the final data analysis was performed.

LIVER TRANSPLANTATION, Vol. 19, No. 2, 2013 D’AMICO ET AL. 137

The follow-up and survival periods were expressedas medians and ranges. Survival curves were calcu-lated according to the Kaplan-Meier method and werecompared with a log-rank analysis. The Cox propor-tional hazards model was used to calculate univariateand multivariate hazard ratios and 95% confidenceintervals (CIs). In the multivariate model, the effect ofNAC on graft and patient survival at 3 and 12 monthswas adjusted for the following covariates: recipientage, recipient MELD score at LT, period of LT, andDRI. The study was dichotomized into nonoverlapping15-month periods.

We used targeted subgroup analyses to obtain amore accurate interpretation of the results. For quan-titative variables, the cutoff was the median value.

Statistical significance was set at P < 0.05. Calcula-tions were performed with the JMP package (SASInstitute, Inc., 1989-2003).

RESULTS

Participant Flow, Recruitment,and Baseline Data

The enrollment period started in December 2006 andended in July 2009 when the established enrollmentperiod of 30 months was reached.

As shown in Fig. 1, 184 consecutive patients wereassessed for eligibility; 44 were excluded according tothe inclusion/exclusion criteria. One hundred fortypatients were enrolled and randomized: 69 patients inthe study group and 71 patients in the control group. Allenrolled patients attained the primary endpoint (therewere no losses or exclusions after randomization).

A bar graph of patient accrual by 6-month intervalsis presented in Fig. 2. The established sample size (88patients per arm) was not met because of slower thanexpected accrual. Moreover, the encouraging results

Figure 1. Consolidated Standards of Reporting Trials (CONSORT) 2010 flow diagram.


of the interim analysis10 prompted us to not prolongthe established enrollment period, although no a pri-ori criteria for ending the study early were originallyplanned.

Tables 1 and 2 describe the baseline characteristicsof the donors and recipients. As expected, there wereno significant differences between the groups.

The characteristics of the liver grafts showed a rele-vant prevalence of donor factors with a potentiallynegative impact on post-LT outcomes (Table 1). Inparticular, we observed a high median DRI value inboth groups. A DRI value of 1.8 (corresponding to themedian value in the control group) was used for sub-group analyses.

As for recipient characteristics, the prevalence ofpatients with hepatocellular carcinoma in both groupshas to be underlined (Table 2).

Primary Endpoint

The median follow-up was 33.4 months (range ¼ 0.1-52.8 months).Figure 2. Bar graph of patient accrual by 6-month intervals.

TABLE 1. Donor Characteristics of the NAC and No-NAC Groups

Variable No NAC (n ¼ 71) NAC (n ¼ 69) P Value

Age (years)* 56 (18-86) 54 (19-79) 0.71Partial grafts [n (%)] 11 (15) 5 (7) 0.18Body mass index (kg/m2)* 24.7 (15.6-33.2) 24.7 (18.4-40.0) 0.64Hemodynamic instability [n (%)] 31 (44) 24 (35) 0.37Ultrasound steatosis [n (%)] 19 (27) 21 (30) 0.42Sodium (mmol/L)* 149 (122-167) 147 (134-188) 0.98AST (U/L)* 36 (7-1700) 36 (4-293) 0.64ALT (U/L)* 23 (4-933) 31 (4-227) 0.92Bilirubin (mg/dL) 0.8 (0.2-3.9) 0.6 (0.2-7.2) 0.08Time from start of surgery (NAC infusion) tocross-clamping (minutes)*

180 (100-385) 195 (70-350) 0.21

DRI* 1.8 (0.9-3.0) 1.7 (0.9-2.4) 0.15

*The data are presented as medians and ranges.

TABLE 2. Recipient Characteristics and Technical Aspects of the NAC and No-NAC Groups


Age (years)* 58 (30-68) 55 (27-68) 0.17Sex: female [n (%)] 20 (28) 14 (20) 0.33Main indication for LT [n (%)] 0.87Hepatitis C virus 13 (18) 19 (28)Hepatitis B virus 7 (10) 3 (4)Alcohol 4 (6) 6 (9)Hepatocellular carcinoma 36 (51) 34 (49)Other 11 (15) 7 (10)

Child-Pugh-Turcotte score* 8 (6-14) 9 (6-14) 0.32MELD score* 17 (7-35) 16 (7-37) 0.85Cold ischemia time (minutes)* 500 (253-620) 489 (140-605) 0.12Warm ischemia times (minutes)* 30 (15-70) 31 (15-85) 0.32Total LT time (minutes)* 460 (310-855) 420 (300-930) 0.21Cell saver (mL)* 1000 (0-9000) 755 (224-6875) 0.24Rapid infusion system (mL)* 9669 (850-63,033) 10,132 (1200-31,115) 0.31Red blood cells (mL)* 1500 (0-7800) 1350 (0-5700) 0.91Plasma (mL)* 1600 (0-5000) 2000 (0-12,000) 0.13



We recorded 38 graft failures (27%): 13 (34%) in thestudy group and 25 (66%) in the control group (P ¼0.03). Six cases of graft failure did not result inpatient death because of successful liverretransplantation.

Nineteen graft failures (14%) occurred within thefirst 3 months after LT; the main causes were PDF (8cases or 42%), sepsis complicating initial PDF (5cases or 26%), sepsis with regular initial liver function(3 cases or 16%), heart failure (2 cases or 11%), andportal/arterial thromboses (1 case or 5%).

A substantial proportion of graft failures (6.4%) alsooccurred 3 to 12 months after LT. Seven of these fail-ures (78%) occurred in the control group: 3 due tohepatitis C virus recidivism, 2 due to delayed graftfunction/sepsis, 1 due to a de novo tumor, and 1 dueto intra-abdominal bleeding after liver biopsy. We hadonly 2 failures in the same period in the study group

(P < 0.05): 1 due to hepatitis C virus recidivism and 1due to hepatocellular carcinoma recurrence.

The graft survival rates at 3, 12, and 24 monthswere 93%, 90%, and 86%, respectively, in the studygroup and 82%, 70%, and 67%, respectively, in thecontrol group (P ¼ 0.02; Fig. 3). The difference in graftsurvival at 3 months was 11% (95% CI ¼ 2%-20%, P¼ 0.04), and although it was significant, the differencewas lower than that assumed in the sample sizedetermination. The calculated difference in graft sur-vival at 12 months was 20% (95% CI ¼ 9%-31%, P <0.01), and the sample size determined the power ofFisher’s exact test in estimating this difference to be91%.

To better understand the effects of NAC on graftsurvival at different time points, we used 2 separateCox regressions at 3 and 12 months and consideredboth unadjusted and adjusted models (Table 3). Thisanalysis confirmed an increasing impact of NAC ongraft survival from 3 to 12 months that was not influ-enced by other recipient or donor variables.

Secondary Endpoints

During the study period, we registered 32 patientdeaths: 10 (31%) in the study group and 22 (69%) inthe control group (P ¼ 0.02). The patient survivalrates at 3, 12, and 24 months were 99%, 94%, and90%, respectively, in the study group and 86%, 75%,and 72%, respectively, in the control group (P ¼ 0.01;Fig. 4).

Similarly to the graft survival analysis, the effect ofNAC on patient survival was independent of other re-cipient or donor covariates (Table 3).

We recorded 14 PDF events in the study group(20%) and 16 PDF events (23%) in the control group.The difference did not reach statistical significance(Table 4).

Among the other secondary endpoints, we found thefollowing significant or marginally significant differen-ces (Table 4): (1) the POD 15 AST levels were 31 U/L(range ¼ 15-150 U/L) in the study group and 38 U/L(range ¼ 13-326 U/L) in the control group (P ¼ 0.02),(2) the median hospital stay was 17 days (range ¼ 7-

Figure 3. Kaplan-Meier curves showing graft survival in the 2groups. The log-rank test showed a statistically significantdifference between the 2 curves (P ¼ 0.0223).

TABLE 3. Cox Proportional Hazards Models Showing the Unadjusted and Adjusted Impacts of NAC on

Graft and Patient Survival

Survival Endpoint

Hazard Ratio (95% CI)/P Value

3 Months 12 Months

Graft survivalAdjusted 0.33 (0.10-0.91)/0.04 0.30 (0.12-0.70)/<0.01Unadjusted 0.35 (0.11-0.91)/0.03 0.31 (0.12-0.70)/<0.01

Patient survivalAdjusted 0.09 (0.00-0.46)/<0.01 0.18 (0.05-0.51)/<0.01Unadjusted 0.09 (0.00-0.45)/<0.01 0.20 (0.06-0.52)/<0.01

NOTE: In the multivariate model, the effects of NAC on graft and patient survival at 3 and 12 months were adjusted for thefollowing covariates: recipient age, recipient MELD score at LT, period of LT, and DRI.


95 days) in the study group and 19 days (range ¼ 8-155 days) in the control group (P ¼ 0.07), (3) the num-ber of days of therapy with vasoactive amines duringthe ICU stay was lower in the study group [0 days(range ¼ 0-18 days)] versus the control group [1 day(range ¼ 0-55 days), P ¼ 0.08], (4) the overall inci-dence of postoperative complications was 23% in thestudy group and 51% in the control group (P < 0.01),and (5) the incidence of acute rejection episodes wassignificantly higher in the study group [20% (14

patients)] versus the control group [3% (2 patients), P< 0.01]. In all, the diagnosis of acute rejection washistologically proven in 16 cases. Biopsy was per-formed in 8 other cases (all in the control group). Thediagnoses were sepsis (6 cases) and ischemia/reper-fusion damage (2 cases).

The numbers of septic events in the 2 groups werenot significantly different (and thus did not explainthe difference in the complication rates).

Subgroup Analysis

We evaluated the impact of suboptimal liver grafts(DRI > 1.8) on the incidence of PDF and early graftsurvival. Sixty-one patients (44%) received a subopti-mal liver: 27 patients (39%) in the study group and34 patients (48%) in the control group (P > 0.05).

The incidence of PDF events was clearly lower in thestudy group (15%) versus the control group (32%); thedifference reached marginal statistical significance (P¼ 0.09).

Conversely, the incidence of PDF events was similarin the 2 groups when only optimal grafts were consid-ered (DRI " 1.8).

When the subgroup analysis was applied to theearly graft survival endpoint, we found that the differ-ences between the 2 groups were concentrated exclu-sively in patients receiving suboptimal grafts. In par-ticular, we had 7 early graft failures (21%) in thecontrol group and only 1 early graft failure (4%) in thestudy group when donors with DRIs > 1.8 were used(P ¼ 0.05). When the specific causes of failure wereinvestigated, we found that the difference was mainlydue to the 5 cases of early graft failure due to sepsisfollowing PDF (all in the control group). We found that

Figure 4. Kaplan-Meier curves showing patient survival in the2 groups. The log-rank test showed a statistically significantdifference between the 2 curves (P ¼ 0.01).

TABLE 4. Secondary Endpoints


PDF [n (%)] 16 (23) 14 (20) 0.84PNF [n (%)] 4 (6) 3 (4) 0.45POD 2 AST (U/L)* 705 (47-11,933) 571 (65-10,160) 0.85POD 2 ALT (U/L)* 670 (34-8632) 538 (35-9600) 0.89POD 2 bilirubin (mg/dL)* 2.9 (0.6-46.7) 2.6 (0.4-19.8) 0.49POD 2 INR* 1.5 (1.0-6.4) 1.4 (1.1-3.6) 0.52POD 7 AST (U/L)* 48 (12-2987) 48 (17-1087) 0.36POD 7 ALT (U/L)* 101 (9-1580) 105 (21-1160) 0.70POD 7 bilirubin (mg/dL)* 5.0 (0.7-37.4) 5.1 (0.7-20.1) 0.33POD 7 INR* 1.2 (1.0-2.1) 1.2 (1.0-1.8) 0.80POD 15 AST (U/L)* 38 (13-326) 31 (15-150) 0.02POD 15 ALT (U/L)* 68 (10-938) 62 (12-720) 0.45POD 15 bilirubin (mg/dL)* 2.7 (0.9-26.2) 2.4 (0.9-23.0) 0.78POD 15 INR* 1.1 (1.0-1.8) 1.1 (0.9-1.9) 0.71Hospital stay (days)* 19 (8-155) 17 (7-95) 0.07Plasma in ICU (mL)* 1650 (0-15,000) 2250 (0-16,100) 0.40Vasoactive amines (days in ICU)* 1 (0-55) 0 (0-18) 0.08Postoperative complications [n (%)] 36 (51) 16 (23) <0.01Acute rejection [n (%)] 2 (3) 14 (20) <0.01



the main factor influencing septic graft failure afterLT was a previous PDF event.

DISCUSSION

To the best of our knowledge, no randomized clinicaltrials have evaluated the use of antioxidant agentsinfused during liver procurement and their impact onpost-LT outcomes.

The main result of this study is that our NAC pro-curement protocol was able to significantly improvegraft survival after LT with respect to the conventionaltechnique (Fig. 2).

This result was confirmed both in the very earlypost-LT phase (3-month graft survival) and in the in-termediate follow-up period (12-month graft survival).However, when this was analyzed in detail, the maindifference between the 2 groups in specific causes ofgraft failure was related to the occurrence of sepsiscomplicating a PDF event. In particular, 5 cases ofgraft failure were clustered in the subgroup ofpatients receiving suboptimal grafts (DRI > 1.8). Thesubgroup analysis of the 61 patients receiving subop-timal livers also revealed that there was a significantupward trend for the incidence of PDF events in thecontrol group versus the study group. On a purelyspeculative basis, one could interpret the observedpositive effect of NAC on graft survival as the result ofa protective effect of this antioxidant agent againstIRI, especially when suboptimal livers are used. Thiseffect may also be a likely explanation for other signif-icant differences between the 2 groups with respect tosecondary endpoints such as patient survival (Fig. 3),lengths of hospital stays, and postoperativecomplications.

Three to 12 months after LT, the control group hada higher incidence of graft loss due to delayed graftfunction or early and aggressive hepatitis C virusrecidivism, and it is well known that both pathophys-iological events may be influenced by the extent ofIRI.1,2,11 These results suggest a potential impact ofthe NAC protocol in the intermediate term as well.

When we analyzed the prognostic impact of all vari-ables included in the DRI formula, only donor agehad a significant impact (data not shown). When weseparately considered the effect of donor age on thestudy group versus the control group, this variablemaintained a significant impact on graft survival onlyin the latter group. This finding supports a potentialprotective effect of NAC on post-LT outcomes, particu-larly when older donors are used.

Although liver biopsy is important for directly esti-mating ischemia/reperfusion damage at the momentof LT, GSH reserves, and the quality of graft recovery3 months after LT, we deliberately chose not to per-form protocol-specific biopsy because we felt that ad-herence to our daily clinical practice was essential forlimiting any potential bias in the selection and care ofpatients.

Moreover, it is possible for data on acute rejectionto be biased by the biopsy rate. However, in our clini-

cal practice, we have adopted an extremely aggressiveapproach to biopsy, which is immediately performedwhenever liver functional tests show any alterations.This mitigates the potential bias related to the ab-sence of protocol-specific biopsy samples.

It is very difficult to understand the higher inci-dence of acute rejection in the study group. Recentreports11 suggest a potential interaction between IRIand the recipient’s immune system, which could bethe interpretative key to understanding the linkbetween NAC and acute rejection.

In terms of donor and recipient characteristics(Tables 1 and 2), our population was similar to onereported in a recent Italian multicenter prospectivestudy.12 In particular, we had a donor populationcharacterized by a higher median DRI versus a USpopulation.13,14 It must be stressed that the medianDRI of our 2 groups was also higher than the medianDRI of the Italian Liver Match study cohort.12 Thepoor results for the controls could be due to this highproportion of extended criteria donors (Table 1). Thegraft survival rates 3 and 12 months after LT were infact 82% and 70%, respectively, in the control group.The survival figures were very similar to thosedescribed in a US population with a DRI of approxi-mately 1.8.3

As for recipient characteristics, we compared theresults of our study to those for a historical cohortfrom our center (January 2003 to December 2006).We selected an additional group of 223 consecutivepatients who fulfilled the study’s inclusion criteria butwere not present in the original study. In 2006, ourcenter introduced a MELD-based organ allocation sys-tem.7 The overall graft survival rates for this retro-spective cohort of patients were 88% and 85% at 3and 12 months, respectively. These rates were inter-mediate between those of the study and controlgroups enrolled in our randomized clinical trial. Theonly relevant difference between these 2 populationswas the median MELD score at LT, which changedsignificantly in non–hepatocellular carcinoma patientsfrom 15 (range ¼ 6-41) in the retrospective cohort to22 (range ¼ 9-37) in this study (data not shown). Thistrend is evident for all Italian centers with respect tohistorical cohorts.12 It must be emphasized that theMELD score variable had a significant prognostic rolein our Cox multivariate analyses at both 3 and 12months.

MELD-based allocation alone may not explain ourpoor control results. However, clinical evidence fromour center favors the hypothesis that the particularinteraction between high MELD values and high-DRIgrafts leads to a deterioration in post-LT survival.This phenomenon seems to have been confirmed byseveral Italian12 and European centers.15

Taken together, these considerations of donor andrecipient factors strengthen our conclusions regardingthe excellent results obtained for the study group andthus the positive effects of the NAC protocol.

Although we did not reach the predetermined sam-ple size and survival difference at the 3-month graft


survival endpoint, the graft survival gain of 20% at 12months was a very strong result, and Fisher’s exacttest showed statistical significance with a power of91%. The final sample size was sufficient to determinea significant positive impact of the NAC protocol on 1-year graft survival.

Previous studies have thus far failed to show a rele-vant clinical impact of NAC on post-LT outcomes.16,17

The originality of our protocol was the systemic NACinfusion 30 minutes before the initiation of the har-vesting procedure. This methodological aspect wasobserved in only 1 previous study.18 Incidentally, thatparticular work is to date the only other study show-ing a positive impact of NAC on post-LT outcomes. Allother studies known to us have administered NAC ei-ther in later phases of harvesting or during the recipi-ent’s operation. We think that an early systemic infu-sion of NAC is essential for the sought-after effectbecause it gives time for the donor’s liver to replenishits metabolic reserve of GSH.6 This may be particu-larly important when we are using extended criteriagrafts, whose GSH reserves may be depleted.

NAC has a very good safety profile, and there is nodocumented evidence of dose-dependent side effects.6

In our study, we did not record any drug-relatedadverse events. Moreover, a preliminary investigationof the outcomes of other organs harvested fromdonors into which NAC was infused did not show anysignificant adverse events that could be attributed tothe use of NAC (data not shown). We are now perform-ing further data analyses to determine whether theNAC protocol has beneficial effects on organs otherthan the liver.

The good safety performance of the NAC protocol,the very low costs of this antioxidant agent, and itsstrong effect on graft survival make the adoption ofthe NAC protocol highly cost-effective. For this reason,an ancillary study is now beginning at our institutionwith the aim of producing a detailed cost-effectivenessanalysis of the NAC protocol.

Several limitations of this study must be noted. Itwas a monocentric experience; thus, our resultsshould be considered with caution until larger, betterdesigned, and externally validated studies becomeavailable. Although randomization was used, sometrends toward differences in the characteristics ofdonors of the control and study groups could beobserved (partial graft use, bilirubin levels, and DRIvalues). We cannot exclude the idea that these trendshad an impact on the study results. This was not adouble-blinded study with a risk of bias in either theselection of patients or their care. In this study, theselection of patients for LT was based on a transpar-ent and well-defined allocation protocol essentiallybased on the MELD score.7 In addition to allocation,with the randomization of the harvesting procedureand the study inclusion criteria, we have sought tolimit the impact of any potential selection bias.

As for the care of the patients, although caregiverswere not explicitly blinded for the purposes of thisstudy, patients in both arms were treated according

to the center’s standard of care. The extremely stand-ardized nature of the harvesting and transplantationprocedures8 and the specific lifesaving feature of LTwith its ethical and clinical implications are such thatthe non–double-blinded design had limited influenceon the physicians’ care of graft recipients. In ourassessment, unblinded caregivers had marginal ornegligible effects on the results of this study.

The main problem of this study is that the signifi-cant difference in terms of graft survival (the primaryendpoint) is not well justified by significant differencesbetween conventional post-LT variables such astransaminase and lactate levels and PDF (Table 4).This fact may be due to at least 2 causes. First, it iswell known that conventional variables are not accu-rate predictors of early graft function and IRI. Thus,the absence of a correlation between NAC administra-tion and these conventional variables does notexclude a correlation between NAC and IRI. Second,the main methodological problem of this study is thatwe did not perform protocol biopsy to study IRI on ahistological and molecular basis. A prospective studyfocused on this particular matter is currently underelaboration by our institution.

Finally, the impact of the NAC protocol on mid- tolong-term outcomes (eg, hepatitis C virus recurrence)should be analyzed in greater detail.14

The question of the potential impact of this studyon everyday clinical practice remains. Our study sug-gests that NAC could be an effective tool for reducingthe risk of PDF and graft failure after LT, especiallywhen suboptimal livers are used. The NAC procure-ment protocol could be selectively used when a poten-tially marginal donor organ is to be allocated.

NAC could be a complementary tool used in addi-tion to other approaches for optimizing the post-LToutcomes of candidates receiving suboptimal grafts ina multimodal strategy of sorts. Our group has previ-ously shown the importance of the donor harvestingtechnique.7 The adoption of low cold ischemia timesand routine biopsying are also advisable, especially inthose cases involving potentially at-risk donors.19 Theuse of machine perfusion methods for graft preserva-tion during the procurement procedure may alsoimprove the post-LT outcomes of patients receivingsuboptimal livers.20 Finally, an optimal donor-recipi-ent match represents a crucial issue for overcomingthe risk of post-LT graft failure.21

In conclusion, we propose that organ harvestingwith the NAC protocol is one of the possible tools forimproving LT results. This harvesting procedureshould be used in the context of a multimodal strat-egy aimed at improving the outcomes of LT, increasingthe use of suboptimal livers, and extending andstandardizing the use of such relevant resources.

ACKNOWLEDGMENTThe authors thank Charles Miller, Myron Schwartz,Sukru Emre, and Gabriel Gondolesi for the interestand support shown to Francesco D’Amico during his


stay as a research fellow at Mount Sinai Hospital ofNew York and to all the 2000 and 2004-2005 trans-plant fellows.

This article is dedicated to the memory of AugustoD’Amico.

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Use of N -acetylcysteine during liver procurement: A prospective randomized controlled study

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