University of Groningen

Transplantation of Suboptimal Donor Livers: Exploring the Boundariesvan Leeuwen, Otto

DOI:10.33612/diss.132816502

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TransplantationofSuboptimalDonorLivers:ExploringtheBoundaries

OttoBoudewijnvanLeeuwen

Fortheprintingofthisthesis,financialsupportofthefollowinginstitutionsandcompaniesisgratefullyacknowledged:UniversityofGroningenResearchInstituteGUIDENederlandseTransplantatieVerenigingOrganAssistBridgetoLife

©Copyright2020O.B.vanLeeuwen,Groningen.Allrightsreserved.Nopartofthisthesismaybereproduced,storedinaretrievalsystem,ortransmittedinanyformorbyanymeans,withoutpriorpermissionoftheauthor.Layoutandcover:EvelienJagtman–evelienjagtman.comPrinting:Ridderprint–www.ridderprint.nlISBN:978-94-034-2539-9(Book)ISBN:978-94-034-2538-2(Epub)

Transplantation of Suboptimal Donor Livers: Exploring the

Boundaries

Proefschrift

ter verkrijging van de graad van doctor aan de Rijksuniversiteit Groningen

op gezag van de rector magnificus prof. dr. C. Wijmenga

en volgens besluit van het College voor Promoties.

De openbare verdediging zal plaatsvinden op

woensdag 04 november 2020 om 14.30 uur

door

OttoBoudewijnvanLeeuwen

geboren op 15 november 1995 te Apeldoorn

Promotores

Prof.dr.R.J.Porte

Prof.dr.J.A.Lisman

Copromotor

Dr.V.E.deMeijer

Beoordelingscommissie

Prof.dr.H.G.D.Leuvenink

Prof.dr.D.Monbaliu

Prof.dr.J.K.G.Wietasch

Paranimfen

Drs.C.A.T.vanLeeuwen

Dhr.W.Wierbos

Tableofcontents

Chapter1 Generalintroductionandoutlineofthisthesis 11

PartI:Transplantationofsuboptimaldonorlivers:determiningtheboundariesChapter2 Biliarycomplicationsfollowinglivertransplantation 19 In:ClavienPA,TrotterJF,editors.MedicalandSurgicalCareofLiverTransplantationPatients.Wiley-Blackwell;2020Chapter3: Donorhepatectomytimeinfluencesischemia-reperfusion 45 injuryofthebiliarytreeindonationaftercirculatorydeath livertransplantation Surgery.2020(inpress)

Chapter4: Donorbloodcompositionisariskfactorforbiliaryinjury 65indonationaftercirculatorydeathlivertransplantationSubmittedforpublication

Chapter5: Selectedlivergraftsfromdonationaftercirculatory 85deathcanbesafelyusedforretransplantationTransplInt.2020(inpress)

PartII:Transplantationofsuboptimaldonorlivers:expandingtheboundaries

Chapter6: Hypothermicoxygenatedmachineperfusionreduces 105bileductreperfusioninjuryaftertransplantationofdonationaftercirculatorydeathliversLiverTranspl.2018;24:655-664

Chapter7: Extendedhypothermicoxygenatedmachineperfusion 125enablesexsitupreservationofporcineandhuman|liversforupto24hoursJHEPRep.2020(inpress)

Chapter8: Transplantationofhigh-riskdonorliversafterexsitu 149resuscitationandassessmentusingcombinedhypo-andnormothermicmachineperfusion:aprospectiveclinicaltrial.AnnSurg.2019;270:906-914

Chapter9: Exsitumachineperfusionofhumandonorliversviathe 171surgicallyreopenedumbilicalvein:aproofofconcept.Transplantation.2019;103:2130-2135.

Chapter10: Summary,DiscussionandFuturePerspectives 185

AppendixI: CasereportrelatedtoChapter6:

Thefirstreportofsuccessfultransplantationof 199apediatricdonorlivergraftafterhypothermicmachineperfusionPediatrTransplant.2019;23:e13362

AppendixII: CommunicationrelatedtoChapter8:

Viabilitycriteriaforfunctionalassessmentofdonor 211liversduringnormothermicmachinepreservationLiverTranspl.2018;24:1333-1335

Nederlandsesamenvatting 217

Listofpublications 225

Dankwoord 229

Curriculumvitae 236

1Generalintroductionandoutlineofthisthesis

Livertransplantation:pastandpresentLiver transplantation is currently the standard life-saving treatment for patientswith

end-stageliverdiseaseaswellasforselectedpatientswithhepatobiliarymalignancies

or certainmetabolicdiseases.The first liver transplantwasperformedbydr.Thomas

StarzlandhisteaminDenver,Colorado,in1963.1Unfortunately,asaresultofmassive

bleeding,thethree-years-oldrecipientdiedintra-operatively.Problemspersistedduring

the following five transplants, with patient survival no more than 23 days. In 1967,

Starzlperformedthefirstsuccessfullivertransplantwithpatientsurvivalexceedingone

year.2 Twelve years later, after Calne’s development of cyclosporine and Starzl’s

introduction of tacrolimus, liver transplantation steadily progressed towards a

standardizedtreatmentwithacceptableoutcomes.3,4Nowadays,1yeargraftandpatient

survivalratesofover90%areseenglobally.

Thedemandfordonor livers,however,severelyexceedsthenumberofsuitabledonor

livers available. Until the late 1990’s liver grafts were mainly donated after

determinationofbraindeathinthedonor(DBDdonation). Inanattempttowidenthe

potentialdonorpool,donationaftercirculatorydeath(DCD)wasre-introduced.5Inthe

earlyphasesofthisdevelopment,excellentresultswithDCDlivertransplantationwere

achieved.However,byextendingtheacceptancecriteriaforDCDlivers,theprevalence

of biliary complications increased. Post-transplant cholangiopathy (PTC) became the

mostfrequentcomplicationfollowingDCDlivertransplantation.6Thelargestcomponent

ofPTCsare thenon-anastomotic strictures (NAS)of thebileduct in thepresenceofa

patenthepaticartery.IncidencesofNAShavebeenreportedinupto30%ofDCDliver

recipients.6

Organpreservation

In the early 1980’s, Belzer’s group developed the University of Wisconsin (UW)

preservation solution, which became a standardized fluid used to preserve organs.

Duringorganprocurement,theabdominalcompartmentisflushedwithseverallitersof

ice-coldUWsolution,afterwhichtheliverisstoredinabagcontainingUWsolutionina

box with ice. This static preservation technique, unfortunately, does not sufficiently

prevent NAS. As a result, dynamic preservation techniques of donor livers by using

machine perfusion is increasingly studied in an attempt to improve outcomes after

(suboptimal) liver transplantation.7-10Ex situmachine perfusion can be performed at

different temperatures and time points. For example, hypothermicmachine perfusion

(4-12°C) has the potential to reduce ischemia-reperfusion injury, whereas

normothermic machine perfusion (37°C) allows viability testing and potential graft

treatment.9

Outlineofthisthesis

This thesis focuses on transplantation of suboptimal donor livers, especially livers

donated after circulatory death. Part 1 contains observational studieswhich aimed to

identifytheboundariesintransplantationofsuboptimaldonorlivers,andtorecognize

risk factorsthatdeterminetheseboundaries.Part2containsobservational,preclinical

and clinical studies with a common goal to investigate safe and successful

transplantationofsuboptimallivers.

Part I: Transplantation of suboptimal donor livers: determining the boundaries

Biliary complications, including NAS, remain the Achilles heel in DCD liver

transplantation.6 About half of the patients that develop NAS undergo re-

transplantation,withanother25%passingawaybeforeasuitabledonorliverbecomes

available.6 Several studies have focused on predicting graft survival after DCD liver

transplantation, but the prediction of NAS still remains highly difficult. Known risk

factorssuchashighdonorage,prolongeddonorwarmischemiatimesandcoldischemic

preservationperiodsarenotpresentinasignificantproportionofNAScases.Chapter2

provides a review of literature on biliary complications after liver transplantation.

Although ischemia and subsequent reperfusion injury is a main risk factor for the

development of NAS, other mechanisms, such as immune-mediated injury, bile salt

toxicity, and insufficient regeneration of the epithelium also play a role in the

pathogenesis, and are discussed in this review. Chapter 3 describes a study on the

influenceofthetimebetweenstartofcoldflushingandtheendofliverretrievalonthe

development of biliary complications after DCD liver transplantation. During liver

retrieval, donor livers maintain a temperature of around 15-20°C and therefore

continuestosufferlukewarmischemia.12Therefore,itishypothesizedthattheduration

of this period is a substantial determinant of the development of NAS. Chapter 4

contains a study on the involvement of DCD donor blood composition on the

development of biliary complications. In addition to peribiliary gland injury,

arteriolonecrosis inthebileductwall isconsideredtobeoneoftheprimeriskfactors

for thedevelopmentofNASafter transplantation.13Weconsideredthatgraft flush-out

upon procurement, and thereby the severity of the arteriolonecrosis, would be

influencedby the cellular compositionof donorblood. Inchapter5, it is investigated

whetherDCDliverscanbesafelyusedforpatientsrequiringare-transplantation.

Part II: Transplantation of suboptimal donor livers: expanding the boundaries

Overthelastyears,dynamicpreservationofdonorliversbyusingmachineperfusionis

slowlymaking itsway into clinic.The first randomized controlled trialshave recently

finished with patient accrual, however, study protocols are highly different over the

globeandtheresultsofaforementionedtrialsremainawaited.8Substantialbenefitsof

end-ischemic hypothermic machine perfusion have been reported, however, viability

testing(especiallyofthebiliarytree)remainsnotpossibleusingthistechnique.9-11End-

ischemic normothermicmachine perfusion has the benefit that it does allow viability

testing,butitexposestheorgantoanextrahitofischemia-reperfusioninjuryanddoes

notmitigate reperfusion injury.8 In the studydescribed inchapter6,we investigated

the effect of dual hypothermic oxygenated machine perfusion (DHOPE) on the

development of biliary injury during DCD liver transplantation. Biopsies were taken

upon arrival and after reperfusion in DHOPE livers and control livers, to assess the

potentialbenefitofDHOPE.Inchapter7,wedescribeastudyonthesafetyofprolonged

DHOPE, and compared liver function after 2, 6 and 24 hours of DHOPE. Prolonged

DHOPE can simplify logistics and may potentially facilitate day-time liver

transplantation. In chapter 8, we report the results from a single-arm prospective

intervention trial. In this study, end-ischemic dynamic preservation using sequential

hypo- and normothermicmachine perfusionwas applied in an attempt to resuscitate

andassessthefunctionofpreviouslydiscardedhumanlivers,andtosubsequentlyallow

safe transplantation. Chapter 9 contains a preclinical study to potentially simplify

ischemia-free liver transplantation.14 Rather than portal perfusion via an end-to-side

anastomosedautologousiliacveingraft,weinvestigatedthefeasibilityofportalvenous

machineperfusionviathesurgicallyreopenedumbilicalvein.

Locatedafterchapter10aretwoappendices.AppendixIisacasereportusingthe

techniquedescribedinChapter6.AppendixIIcontainsaneditorialinwhichviability

criteriaforfunctionalassessmentofdonorliversarediscussed.

References

1. Starzl TE, Marchioro TL, Vonkaulla KN, Hermann G, Brittain RS, Waddell WR.Homotransplantationoftheliverinhumans.SurgGynecolObstet.1963;117:659–676.

2. Starzl TE, Groth CG, Brettschneider L, Penn I, Fulginiti VA, Moon JB, et al.Orthotopichomotransplantationofthehumanliver.AnnSurg.1968;168(3):392–415.

3. CalneRY,RollesK,WhiteDJ,ThiruS,EvansDB,McMasterP,etal.CyclosporinAinitiallyastheonlyimmunosuppressantin34recipientsofcadavericorgans:32kidneys,2pancreases,and2livers.Lancet.1979;2(8151):1033–1036.

4. StarzlT,FungJ,VenkatarammanR,TodoS,DemetrisA,JainA.FK506forliver,kidneyandpancreastransplantation.TheLancet.1989;334(8670):1000-1004.

5. Monbaliu D, Pirenne J, Talbot D. Liver transplantation using Donation afterCardiacDeathdonors.JHepatol.2012;56(2):474-485.

6. de Vries Y, von Meijenfeldt F, Porte R. Post-transplant cholangiopathy:Classification,pathogenesis,andpreventivestrategies.BiochimBiophysActaMolBasisDis.2018;1864(4):1507-1515.

7. JamiesonNV,SundbergR,LindellS,etal.Preservationofthecanineliverfor24-48hours using simple cold storage with UW solution. Transplantation1988;46(4):517-522

8. NasrallaD,CoussiosCC,MergentalH,etal.Arandomizedtrialofnormothermicpreservationinlivertransplantation.Nature2018;557:50-56.

9. Schegel A,Muller X, KalisvaartM, et al. Outcomes of DCD liver transplantationusing organs treated by hypothermicmachine perfusion before implantation. JHepatol2019;70:50-57.

10. DeMeijerVE,FujiyoshiM,PorteRJ.Exsitumachineperfusionstrategiesinlivertransplantation.JHepatol.2019;70:203-205.

11. VanRijnR,KarimianN,MattonAPM,etal.Dualhypothermicoxygenatedmachineperfusion in liver transplants donated after circulatory death. Br J Surg2017;104:907-917.

12. VillaR, Fondevila C, Erill I, et al. Real-timedirectmeasurement of human liverallograft temperature from recovery to transplantation. Transplantation.2006;81(3):483-486.

13. OpdenDriesS,WesterkampAC,KarimianN,etal.Injurytoperibiliaryglandsandvascular plexus before liver transplantation predicts formation of non-anastomoticbiliarystrictures.JHepatol2014;60:1172-1179.

14. HeX,GuoZ,ZhaoQetal.Thefirstcaseofischemia-freeorgantransplantationinhumans:Aproofofconcept.AmJTransplant2017;18(3):737-744.

PartI:Transplantationofsuboptimaldonorlivers:determiningtheboundaries

Biliarycomplicationsfollowinglivertransplantation

OttoB.vanLeeuwen,IrisE.M.deJong,RobertJ.PorteIn:ClavienPA,TrotterJF,editors.MedicalandSurgicalCareofLiverTransplantationPatients.Wiley-Blackwell;2020

2

Introduction

Biliary complications are a major cause of morbidity and graft failure after liver

transplantation.Althoughadvancesinthesurgicaltechniqueoflivertransplantation

have led to a better overall outcome and fewer surgical complications, biliary

complications still occur in10–40%of recipientsandareassociatedwithmortality

rates of 8–15%.1-3 The high biliary complication rate in liver transplantation can

partlybeexplainedbytheincreasingdiversityoflivergraftsusedfortransplantation

inrecentyears.Theshortageofgraftsavailablehasledtotheincreaseduseoflivers

that have been donated after cessation of blood flow in the donor, or so called

donationaftercirculatorydeath(DCD)donors.TheorgansofDCDdonorssufferedan

extraperiodofwarmischemiacomparedtodonationafterbraindeath(DBD)livers,

andarethereforemoresusceptibletodevelopbiliarycomplications.TheuseofDCD

organs andother extended-criteriadonor livers is inevitable in an attempt to scale

down the worldwide shortage of organs. In order to expand the pool of potential

donors, split-liver transplantation and living donors have also evolved as surgical

alternatives and numbers have increased in recent years, providing particularly

youngchildrenwithanopportunitytoreceiveagraft in time.Therateofsplit-and

livingdonortransplantationshowslargevariationsamongcountries. InsomeAsian

countries, the percentage of living-donor liver transplantation rate reaches almost

100%, whereas in the US and most European countries the percentage is around

10%.Diversityinthequalityandtypeoftransplantedorgans,variationsinrecipient

risk factors, and variations in the applied surgical technique lead to a diversity in

biliary complications that may occur after liver transplantation. The three most

common types of biliary complications can are: non-anastomotic strictures (NAS),

anastomotic strictures and biliary leakage.4 These and other less frequent biliary

complicationsaresummarizedinbox32.1andwillbediscussedinthischapter.First,

aspects of organ procurement that are relevant for the prevention of biliary

complicationswillbecovered.Hereaftersurgicalaspectsofbileductreconstruction

willbediscussed,followedbyadiscussionofdiagnosticandimagingmethodsanda

descriptionofthepathogenesis,clinicalpresentation,andmanagementofthevarious

typesofbiliarycomplicationsafterlivertransplantation.

Table1.

Classificationofbiliarycomplicationsafterlivertransplantation4

1. Biliaryleakage

1A.Frombiliaryanastomosis

1B.Fromhepaticbiopsyorparenchymalinjury

1C.Fromgallbladderfossaorcysticductstump

1D.Afterremovalofbiliarydrain

2. Anastomoticstenosisof:

2A.Choledocho-choledochostomy

2B.Hepatico-jejunostomy

3. Post-transplantcholangiopathy

3A. Non-anastomotic biliary strictures (of extrahepatic and large intrahepatic

ducts)

3B.Intraductalbiliarycasts

3C.Bileductnecrosiswithintrahepaticleakageandbilomaformation

4. Biliaryabnormalitiesduetohepaticarterystenosisofthrombosis

5. Biliarystricturesduetorecurrentdisease(i.e.primarysclerosingcholangitis)

2.Surgicaltechniqueinrelationtobiliarycomplications

2.1Organprocurementandpreservation

Efforts tominimize the risk of biliary complications after liver transplantation should

start with proper surgical and preservation techniques during the donor procedure.

Aspectsof liverprocurementandpreservationthathavebeendemonstratedtoreduce

the risk of biliary complications include: 1) efforts tominimize ischemic injury of the

bileducts,2)preservationof thevasculatureof theextrahepaticbileductbyavoiding

dissectiontooclosetothebileduct,3)thoroughrinsingofthebileductlumentoremove

toxicbile,4)adequatearterialperfusionoftheliverwithpreservationfluidtopreserve

theperibiliarycapillaryplexusand5)rapidprocurementafterinitiationofcoldflushing

inthedonor.

Theseaspectsarerelevantasbiliaryepithelialcells(cholangiocytes)areverysensitive

to ischemia/reperfusion injury. In addition to primary preservation-related ischemic

injury,ischemicdamageoftheperibiliaryplexuswillresultinsecondaryischemicinjury

ofthebiliaryepithelium.Thestrongrelationshipbetweenischemiaandbileductinjury

is illustrated by studies demonstrating an association between both cold and warm

ischemia time and thedevelopment ofNAS.As long as the cold ischemia time is kept

below 10 h, the incidence of NAS is not increased, however more prolonged cold

ischemiaisclearlyassociatedwithahigherriskofthesestrictures.Warmischemiatime

has also been identified as a risk factor in several studies. The relevance of warm

ischemia isalso illustratedby thehigh incidenceofNASafter transplantationof livers

fromDCDdonors,which suffer an inevitable period ofwarm ischemia prior to organ

procurement.5,6

During organ procurement, surgeons should avoid “stripping” of the extrahepatic bile

duct, which will damage its microvascularization. The extrahepatic bile duct should

alwaysremainsurroundedbyanadequateamountof tissuetoensuresufficientblood

supply.

Preservation injury results in increased arterial resistance andmay cause circulatory

disturbancesinsmallcapillaries,suchasthebiliaryplexus.Sincethebloodsupplytothe

biliary tract is solely dependent on arterial inflow, disturbances in the blood flow

through the peribiliary plexusmay result in insufficient oxygenation and subsequent

damageofthebiliaryepithelium.

Gentle retrograde flushing of the bile ducts with preservation fluid is considered an

important method to remove bile from the bile duct lumen. Bile contains bile salts,

whicharecytotoxicduetotheirdetergentproperties.Severalstudieshaveshownthat

bile salts may contribute to toxic damage of the biliary epithelium both during liver

preservationandafterlivertransplantation.7,8Atthismoment,thereisnoconsensuson

whichflushingsolutionismostadequateforsuccessfulbileductpreservation.

University of Wisconsin (UW) solution and Histidine-tryptophan-ketoglutarate (HTK)

have been recognized as the gold standard preservation solutions. Although some

studies have suggested that highly viscous preservation solutions such as the UW

solutionmay result in an incomplete flush-out of the small donor peribiliary arterial

plexus,resultinginahigherincidenceofNAS,9,10thiscouldnotalwaysbeconfirmedin

other studies.11 Therefore, it remains debatable whether low viscosity preservation

fluids, such as HTK, are associated with a lower incidence of biliary complications.

Adequatelypoweredrandomized,controlledtrialswithlong-termfollowupareneeded

to determine whether the type of preservation fluid has an impact on biliary

complicationsafterlivertransplantation.

Onemethodtoovercomeinadequateflush-outandpreservationoftheperibiliaryplexus

is theapplicationofhighpressurearterial infusionofpreservation fluideither in vivo

duringprocurementorimmediatelyafterwardsduringtheback-tableprocedure.Some

retrospective studies have shown that additional flushing of the peribiliary plexus by

controlledarterialback-tablepressureperfusionmayresultinaconsiderablereduction

in the incidence ofNAS.12However, a prospective, randomized controlled trial on the

efficacyof additional arterial ex situback-tableperfusiondemonstrated that thisdoes

not prevent NAS after transplantation.13 Better flush-out and preservation of the

peribiliarycapillaryplexusmayalsobeachievedbymachinepreservation.Severalsmall

studies have shown that end-ischemic hypothermic oxygenated machine perfusion is

safelyapplicable in livertransplantation,andtheresults lookpromising.14-18Asof this

moment,norandomizedcontrolledtrialshavebeenfinishedyetontheoutcomesafter

theuseofhypothermicmachineperfusioninlivertransplantation.

Recently,theperiodbetweenthestartofcoldflushofthedonororgansandtheendof

liverretrievalhasbeenshowntoinfluencegraftsurvival.19Duringorganprocurement,

thetemperatureoftheabdominalorgansdoesnotdropbelow15-20°C.whichdoesnot

protecttheliverandbileductsagainstwarmischemicinjury.Therefore,afterinitiation

ofinsitucoldflushing,adonorlivershouldbeexcisedasrapidlyaspossibleandplaced

in a bowl with preservation fluid with sterile ice, where it will finally reach a

temperature<4°C.20Hereafter, the livershouldbestoredassoonaspossible insterile

bagsandaboxwithice.

2.2Biliaryreconstruction

Thetwomaintypesofbiliaryreconstructionusedinlivertransplantationtodayare:1)

choledocho-choledochostomy,alsocalledtheduct-to-ductanastomosis(usingeitheran

end-to-endanastomosisora side-to-sideanastomosis), and2)ahepatico-jejunostomy

usingaRoux-Y jejunal loop.Theuseofonetypeofreconstructioninsteadoftheother

largelydependsontheanatomicalsituationoftherecipient’sextrahepaticbileductsand

sometimesthesurgicalpreference.

In case of a duct-to-duct choledocho-choledochostomy, an anastomosis is created

between donor and recipient choledochal ducts (common bile duct). An end-to-end

anastomosisisgenerallyeasiertoperformthanaside-to-sideanastomosis,theformeris

therefore usedmore frequently. In a prospective, randomized trial comparing end-to-

end anastomosis with side-to-side anastomosis, no major differences in outcome

between the two techniqueswere found.21 An end-to-end reconstruction restores the

physiologic anatomical situation and does not carry the risk of bile sludge or cast

formationascanoccurinthedeadendsofaside-to-sideanastomosis.

In case of a Roux-Y hepatico-jejunostomy, an end-to-side anastomosis is constructed

betweenthedonorhepaticductandaRoux-Yjejunalloopcreatedintherecipient.Roux-

Yhepatico-jejunostomyismainlyusedinpatientswhosenativeextrahepaticbileductis

notsuitableforanastomosiswiththebileductofthedonorliver.Themainindications

for using a Roux-Y loop for biliary reconstruction are primary sclerosing cholangitis

with involvement of the extrahepatic bile duct, biliary atresia, significant size

discrepancy between the donor and recipient choledochal duct, and in some cases,

retransplantation.22,23Althoughahepatico-jejunostomymaybeasafealternativewhen

duct-to-duct anastomosis is not feasible, the disadvantage is that it creates an open

connectionbetween the intrahepaticbileductsof thegraftand thebowel lumen.This

may result in reflux of small bowel content into the bile ducts and subsequently

ascending bacterialmigration and (recurrent) cholangitis. An additional advantage of

using a choledocho-choledochostomy is easier access for diagnostics and therapy

comparedwithaRoux-Yhepatico-jejunostomy.Itis,therefore,generallyagreedthatthe

preferred method of biliary reconstruction in liver transplantation should be a

choledocho-choledochostomywheneverpossible.

Fewcentershaveadvocatedandreportedontheuseofadirectconnectionbetweenthe

donorbileductandtherecipientduodenum(so-calledcholedocho-duodenostomy)asa

safealternativetoahepatico-jejunostomy.24

2.3Useofabiliarydrain

Whenreconstructingthebiliarysysteminalivertransplantrecipient,thiscanbedone

eitherwithorwithouttheinsertionofabiliarydrain.AbiliarydraincanbeeitheraT-

tubeorastraight(opentip)catheter.AT-tube isa flexibletubethat is inserted inthe

choledochal duct in the proximity of the end-to-end anastomosis in case of a

choledocho-choledochostomy. This tube allows the bile to drain in two directions:

towardstheduodenumandoutwardof thebody.Alternatively,astraightcathetercan

beused,withtheadvantageofalowerriskofbileleakageuponremovalofthedrainas

itresultsinasmallerholeinthebileductafterextraction.

Choledocho-choledochostomy reconstructions over T-tubes have been the subject of

controversyformanyyears,butithasneverthelessremainedcommonpracticeinsome

transplantcenters.Yet,withincreasingsurgicalexperience,manycentershavebegunto

abandontheroutineuseofbiliarydrainsintheirlivertransplantrecipients.25,26

Thebenefitsofusingabiliarydrainincludedirectvisualevaluationofthequalityofbile

produced by the recently implanted graft and easy access to the biliary tree for

radiologicimaging.Especiallyinlivergraftsthathaveahigherriskofdevelopingbiliary

complications (e.g. livers fromDCDdonors) this could be an advantage. Some studies

have suggested that placement of a T-tube may reduce the incidence of anastomotic

strictures.27 In addition a T-tubemay result in adequate decompression of the biliary

treeandareductionoftheintraductalpressure,whichmaysubsequentlycontributetoa

lowerrateofintrahepaticbiliarystrictureandleakage.

ThemaindrawbackofusingT-tubesistheirassociationwithanincreasedrateofbiliary

complications,especiallybileleakageatthesiteofthedraininsertionafteritsremoval

occurring in5–15%ofpatients.21 Inaddition, theuseofaT-tube increases theriskof

ascendingcholangitisandperitonitis,duetoanopenconnectionofthecholedochalduct

withtheexterior.Inonesystematicreviewandmeta-analysisofstudiesfocusingonthe

useofbiliarydrainsinlivertransplantationitwasconcludedthatbiliarydrainssuchas

T-tubes should be abandoned.25 Although this meta-analysis showed lower rates of

anastomoticandNAS inpatientswithaT-tube, the incidenceof interventionswasnot

diminished in comparison topatientswithoutaT-tube.PatientswithoutaT-tubehad

fewer episodes of cholangitis and fewer episodes of peritonitis. Yet, patients with or

without a T-tube had equivalent outcomes with respect to anastomotic bile leaks or

fistulas, the need for biliary interventions, incidence of hepatic artery thrombosis,

retransplantationrate,andmortalityduetobiliarycomplications.Twoothersystematic

reviewsandmeta-analysesshowthattheuseofaT-tubemightreducetheincidenceof

biliarystrictures,but that there isnohardevidence towardsstandardizeduse in liver

transplantation.26,27

The use of alternative devices, such as internal stents, have been reported by some

centers, but these stents have been associated with increased rates of serious

complications,includingobstruction,migration,anderosionwithhemobilia.28

The use of biliary drains such as a T-tube in liver transplant recipients, therefore,

remainscontroversial.ProbablytheonlyremainingargumenttouseaT-tubeistoallow

accurate monitoring and easy access to the biliary tree in liver grafts that carry an

increasedriskofbiliarycomplications,liversfromDCDdonorsforexample.

3.Diagnosticmodalities

Inmostcases,thesuspicionofabiliarycomplicationwillariseafteranincreaseinliver

enzymes is noted. There is no specific pattern to reliably distinguish a biliary

complication from other causes of graft dysfunction, although an increase in serum

bilirubin,alkalinephosphataseand/orgamma-glutamyltransferasehasbeensuggested

to bemost specific. Alternatively, patients can presentwith upper abdominal pain or

bacterialcholangitis.Inmanyinstancesofliverenzymedisturbances,aliverbiopsywill

beperformedaftergrossbiliarycongestionandbileductdilatationhavebeenexcluded

byultrasonography.Thepresenceof specificpathologic features suchas centrilobular

cholestasis andportal changes including edema,predominantlyneutrophil polymorph

infiltration,ductularproliferationandcholangiolitismaybeindicativeofthepresenceof

a biliary complication.28 These findings, however, are not very specific and can be

absent. In addition, biopsy findings are not informative with regard to the type and

severityofbiliaryabnormalities.

Thediagnosticwork-upofan increase in liverenzymeswillalwaysdependonclinical

context such as primary disease, time after transplantation, local experience, and

informationonthebiliaryanatomy.AgeneralalgorithmisprovidedinFigure1.

Figure1:Schematicpresentationoftheclinicaldecisionsanddiagnosticstepsin

thework-upofalivertransplantrecipientwithasuspectedbiliarycomplication.

3.1Transabdominalultrasonography

Transabdominal ultrasonography is a useful primary diagnostic tool when a biliary

complicationissuspected.Allograftvascularizationcanbeassessed(especiallypatency

of the hepatic artery), fluid collections can be identified, liver parenchyma can be

studied, and dilatation of bile ducts can be identified. It should be noted that the

transplanted liver behaves differently from a normal liver, in that the biliary system

doesnot dilate as easily in thepresenceof a biliary obstruction as innormal livers.29

This leads to a limited sensitivity of approximately 60% of transabdominal

ultrasonographytodetectbiliarystrictures.29,30Thepredictivevalueoftransabdominal

ultrasonographytodetectNASisratherlow.Therefore,evenanormalultrasonography

of the livergraft inapatientwithclinicalorbiochemicalevidenceofbiliarypathology

warrantsfurtherinvestigation.

3.2Magneticresonancecholangiographyandcomputedtomography

Magnetic resonance cholangiography (MRC) is an established diagnostic tool for the

detection of biliary abnormalities. It has the strong advantage of providing excellent

anatomic information without being invasive. MRC is useful in the detection of both

leakages and strictures. The use of an additional magnetic resonance imaging or

magneticresonanceangiographyscanningprotocolcanalsoprovideinformationabout

the liverparenchymaandvasculature.The reported sensitivity and specificityofMRC

forthedetectionofbiliarycomplicationsiswellover90%.31Afterultrasonography,MRC

isthepreferreddiagnostictoolwhenabiliarycomplicationissuspected.However,one

study showed that MRC is indeed a reliable tool to detect or exclude biliary

complications, but that its reliability to grade severity of these strictures is low.32

Recently, also computed tomography (CT) scanninghasbeensuggested tobeofvalue

for the detection of post-transplant biliary complications – it has a higher spatial

resolution compared toMRC.However, the experiencewith CT cholangiography after

liver transplantation is very limited: 1) it can only be performed using a contrast

medium, 2) it is associated with significant radiation, and 3) it is less reliable in the

presence of biliary obstruction or high serum bilirubin levels. The use of CT

cholangiography to detect a biliary complication should still be considered

experimental.

3.3Directcholangiography

Direct cholangiography, either percutaneously or through endoscopic retrograde

cholangiography (ERC), has been the gold standard for the detection of biliary

abnormalitiesforalongtime.Ithastheinherentadvantageofbiliaryaccesstofacilitate

therapeuticmeasures.Sincetheuseofabiliarydrain(e.g.T-tube)isnolongerroutine

practiceinmosttransplantcenters,ERCisafrequentlyusedmethodtodetectandtreat

biliarycomplications.However,overthelastyearsthelessinvasiveMRCisincreasingly

used when compared to ERC. There is no data to suggest that ERC after liver

transplantationisassociatedwithmorecomplicationsthantheuseofERCinthegeneral

population. Considering the safety, diagnostic yield, and therapeutic potential of ERC,

this should be considered the preferred invasive method. In the presence of altered

biliary anatomy, such as a Roux-Y hepatico-jejunostomy, ERC is more difficult to

perform. In these cases, percutaneous transhepatic cholangiography (PTC) or PTC

drainage isagoodalternativemethod toobtainadequate imagingof thebileducts. In

several series successful ERC in the presence of a Roux-Y reconstruction has been

reportedusingeitheranormalduodenoscopeordouble-balloonendoscopes.33,34PTCis

most easily obtained in the presence of dilated bile ducts. In experienced hands,

however,thiscanbeasafeprocedurealsowithundilatedbileducts.35Itnotonlyallows

adequate imaging of the bile ducts, but also provides access for therapeutic

interventionssuchasballoondilatation(asdiscussedbelow).

3.4Hepatobiliaryscintigraphy

Hepatobiliary scintigraphy can be used as a diagnostic tool to detect post-transplant

biliary obstruction and leakage. It has a sensitivity of approximately 60% for these

indications.36 Themain advantage is its non-invasive nature; itsmain disadvantage is

lowresolutionandlackofdirectvisualizationofthebiliaryanatomy.Thesensitivityof

hepatobiliary scintigraphy to detect NAS is not known. With the increasing use and

availability of MRC, scintigraphy is today rarely anymore used to detect biliary

strictures.Itcouldstillbeofvalueinthosepatientsinwhomanobstructionatthelevel

oftheRoux-Yjejunal loopissuspectedorwhenMRCisnotpossible(i.e.presenceofa

pacemaker).

3.5Otherdiagnostictools

Endoscopic ultrasonography is an emerging tool for the detection of hepatobiliary

diseases. It has excellent diagnostic properties for the distal bile duct. Endoscopic

intraductal ultrasonography can be used for the characterization of intraductal

abnormalities.Useofthesetechniquesinlivertransplantrecipientsisstillanecdotal.A

potentially more valuable tool is direct cholangioscopy. With this technique, a small

endoscope (cholangioscope) can be advanced through a normal duodenoscope to

directly visualize the bile ducts. This can provide information about the biliary

epitheliumandthepresenceofstones,sludgeandstrictures.Itcanalsobeatherapeutic

tooltoadvanceguidewiresortoremovebileductstones.Thenumberofindicationsfor

thesehighlyspecializedtechniques,however,isstilllimited.

4.Pathogenesis,clinicalpresentation,andmanagement

A broad variety of biliary complications can occur after liver transplantation and the

pathophysiologyisoftenmultifactorial.Itspresentationmaybeaspecificandphysicians

canidentifybiliarycomplicationsbyoneormoreofthefollowingsymptoms:abdominal

pain, cholangitis, elevated liver enzymes and jaundice if the bile duct becomes

obstructed. In general, critical mechanisms in the development of post-transplant

cholangiopathyincludeischemia-reperfusioninjury,alteredandthereforetoxicbilesalt

composition,insufficientprotectionbytheHCO3-umbrella,aninsufficientregeneration

ofthebiliaryepitheliumbycholangiocytesandperibiliaryglands,anddifferentimmune-

mediatedinjuries.Eachofthesemechanismscanconcomitantlycontributetobileduct

damage during and after liver transplantation and result in subclinical and clinical

manifestations. Accordingly, various biliary complications overlap and share common

pathogeneses. For example, hepatic artery thrombosis (HAT) results in tremendous

ischemicaldamage, lossof cholangiocytes, andbileductwallnecrosis.NAS, casts, and

eventually,intrahepaticbileductleakagecandevelop.Inthiscase,lossoftheepithelial

barrier leads to infiltrationof toxicbile into thebileductwall and this in turn causes

morebileductdamage,necrosis,andintrahepaticbilomaformation.Castsdevelopfrom

the cumulating epithelial cells that are sloughedoff from thebile ductwall.However,

NASarenotalwaysprecededbyHATandintraductalcastsandsludgecanbedetected

withoutsignsofNAS.Thisexplainstheheterogeneityofbiliarystricturesandtherefore

we propose the term post-transplant cholangiopathy to describe the spectrum of

pathologies of the larger bile ducts in the absence of hepatic artery thrombosis or

stenosis without signs of recurrent diseases (i.e. primary sclerosing cholangitis). A

completeclassificationofbiliarycomplicationsaftertransplantationisdepictedinBox

32.1,ofthese,themostcommontypesarebiliaryleakageandbileductstrictures.

4.1Biliaryleakage

Pathogenesisandclinicalpresentation

Bileleakageafterlivertransplantationisreportedin1–25%ofrecipients.Theincidence

ofbileleakageisthehighestaftertransplantationofasplitliveroragraftfromaliving

donorduetothehepaticresectionsurface.29,37Bile leakagecaneitherbesymptomatic

or asymptomatic, and may be discovered coincidentally on a postoperative

cholangiogram. Symptomatic patients may present with abdominal pain, localized or

generalized peritonitis, fever, and sometimes elevated serum liver enzymes and/or

bilirubin.

Biliary leakage can occur at various sites and intervals after transplantation. The

majorityofpostoperativeleaksoccuratthesiteofanastomosisortheT-tubeinsertion

site,butalsotheresectionsurfaceofthegraftincaseofaliving-donororasplit-donor

transplantation is a common site for leakage. Bile leakage early after liver

transplantationmostlikelyoriginatesfromtheanastomosisortheT-tubeinsertionsite.

Anastomotic leaks aremainly related to errors in surgical technique and/or ischemic

necrosisattheendofthebileduct.Insufficientbloodsupplyortractionofthestitches

causesischemia,whichcanresultinbileleakage.Ahepaticarterythrombosiscanleadto

massivebiliarynecrosisresultingindehiscenceofthebiliaryanastomosis.Bileleakage

attheT-tubeinsertionsitecanoccurimmediatelyaftertransplantationorafterremoval

of theT-tubedue toan insufficiently formed fistulaaround the tractof thebiledrain.

Occasionally, bile leakage occurs after percutaneous liver biopsy or iatrogenic duct

damage.

Management

The management of bile leaks depends on the type of biliary anastomosis, clinical

presentation,severity,andlocalizationofthebileleak.Themajorityofbileleaksaredue

toleakageatthesiteofthebiliaryanastomosis.Ifaleakpresentsshortlyaftersurgery,

ultrasonographyshouldbemadetoconfirmarterialperfusionofthegraft.

A small anastomotic bile leak can sometimes be managed conservatively, especially

when thepatient isasymptomatic.Earlyanastomotic leakagecanbestbe treatedbya

relaparotomyandasurgicalrevisionofthebiliaryanastomosis.Symptomaticorinfected

bile collections should be treatedwith a radiologically placed percutaneous drain. An

anastomotic bile leak without disruption of the anastomosis can be successfully

managed primarily nonsurgically. Stenting of the bile duct, nasobiliary drainage,

sphincterotomyandacombinationofthesehaveallbeenusedwithasuccessrateof85–

100%. Since sphincterotomy may lead to specific complications (bleeding and

perforation),itshouldnotberoutinelyperformed.Theoptimaltimingofstentremoval

afterresolutionofsymptomsisstillunclear,but8weekshasbeenprovensuccessful.38

Inthepresenceofahepatico-jejunostomy,ERCPcanbeattempted,butisfrequentlynot

successful.Alternatively,aPTCdraincanbeplaced,eveninthepresenceofnon-dilated

bileducts.35

In the rare case of a complete disruption of the anastomosis, prompt surgery with

conversion to a hepatico-jejunostomy is most appropriate. In selected cases a repeat

choledochocholedochostomycanbeconsidered.Inthecaseofdiffusebiliousperitonitis

withhemodynamicinstabilityorsepsis,directlaparotomyshouldalwaysbeconsidered.

Leakageafterremovalofabiledraincanbemanagedsuccessfullyinone-thirdofcases

by conservative measures, including intravenous fluids, antibiotics, analgesics, and

observation.39 In the absence of improvement, ERCP with stent placement should be

performed. A laparotomy is indicatedwhen clinical signs of biliary peritonitis persist

despiteadequatedrainageofthebiliarysystem.

4.2Anastomoticstenosis

Pathogenesisandclinicalpresentation

Isolated strictures at the site of the bile duct anastomosis, so-called anastomotic

strictures, are reported in 4–9% of patients after liver transplantation. In general,

anastomoticstricturesdonotremainsubclinicalandaredetectedaftertheoccurrence

of cholestatic laboratory liver function tests, jaundice, or cholangitis.40 Anastomotic

strictures are thought to resultmainly from surgical technique and/or local ischemia,

leading to fibrotic scarring of the anastomosis. Surgical factors include inadequate

mucosa-to-mucosa adaptationat the anastomosis anddamageofmicrovascularization

duetodissectiontooclosetothebileduct.41Tominimizetheriskoflocalischemiaatthe

distal end of the donor choledochal duct, the bile duct should therefore remain

surrounded by an adequate amount of tissue. Generalized hepatic ischemia due to

hepaticartery thrombosiscanalsoresult inanastomoticstricturing.Otherrisk factors

for the development of anastomotic structures are anastomotic bile leakage after

transplantationandasexmismatchbetweendonorandrecipient.41,42

Liver transplantation using a split graft or a liver derived from a living donor is

associatedwithahigherriskofdevelopingananastomoticbileductstricture,becauseof

the frequent discrepancy between the diameter of the hepatic duct of the graft and

choledochalductintherecipient.Inaddition,vascularizationofthehepaticductcanbe

compromisedwhenapartialgraftisderivedfromalivingdonororsplitliver.Theseand

other surgical aspects of living-donor and split-liver transplantation are discussed in

moredetailinchapters23and24.

Management

The most frequently used therapeutic approach to an anastomotic stricture is

endoscopic balloon dilatation and stenting of the stenosis. This treatment has been

widelystudiedandisbothsafeandeffective.Technicalsuccessisobtainedin90–100%,

and long-termresolutionof thestricture in70–100%ofcases.43Althoughdisputedby

some,mostcentersobtainthebestresultswithaprotocolofprogressivestentingevery

8–12 weeks with increasing numbers and diameters of stents until resolution of the

stenosisisobtained.44Insomecases,thestenosisrecursdespiteeffectiveinitialtherapy.

Somecentershaveusedacoveredexpandablemetal stent to treata refractorybiliary

stenosisaftertransplantation.This,however,isnotroutinepractice.Presentationofan

anastomotic stricture more than 6 months after transplantation and previous bile

leakageatthesiteoftheanastomosisareriskfactorsfordifficult-to-managestrictures.40

When an anastomotic stenosis does not respond to repeated dilatation and stenting,

surgical revision or conversion to a Roux-en-Y hepatico-jejunostomy anastomosis is a

good alternative with excellent long-term success.40 Incidentally, narrowing at the

anastomosis can be detected while it remains unclear whether this is a clinically

relevantstricture.Insuchcases,ashorttrialofstentingcanbeofvalue.45

In the presence of a hepatico-jejunostomy, where the anastomosis is not easily

accessiblebyendoscopy,percutaneoustranshepatictreatmentbyballoondilatationand

temporarystentingisusuallysuccessful.Thisapproachcanalsobeusedaftersplit-liver

orliving-donorlivertransplantation,althoughresultsarenotasgood,possiblybecause

compromised microvascularization and local ischemia are more frequently the

underlyingcause.43,46

4.3Post-transplantcholangiopathy

The term post-transplant cholangiopathy coversmultifocal biliary abnormalities after

liver transplantation that include NAS, intraductal sludge and casts, and bile duct

necrosiswithintrahepaticleakageandbilomaformation.4Thesebileductabnormalities

represent different aspects of the post-transplant cholangiopathywith necrosis of the

bileductwallandsubsequentleakageofbileintotheliverparenchymabeingthemost

severesideofthespectrum.Othertermsusedinliteraturethatattempttodescribepost-

transplant biliary abnormalties are ischemic-type biliary lesions (ITBL) and ischemic

cholangiopathy. Yet the term post-transplant cholangiopathy is preferred since the

pathogenesisisbelievedtobemultifactorialandcannotalwaysbeidentified.

4.3.1Non-anastomoticstrictures

Pathogenesisandclinicalpresentation

NAS are strictures at any location in the donor bile duct other than the anastomosis.

Biliarystricturesmaybeconfinedtothehepaticbifurcation,butmayalsopresentasa

morediffusetypeincludingnarrowingofthemoreperipheralbileductsintheliver.This

typeofbileductstricturesisregardedasthemosttroublesomebiliarycomplicationas

thestricturesareoftenresistanttotherapyandoneofthemostfrequentindicationsfor

retransplantatie.43,47Asstatedbefore,NAScanbeaccompaniedbyintraductalsludgeor

cast formation. The clinical presentation of patients with NAS is often not specific;

symptomsmay include fever due to cholangitis, abdominal complaints, and increased

cholestaticliverfunctiontests,eitherwithorwithoutclinicaljaundice.

The reported incidence of NAS after liver transplantation varies between different

studies, ranging from1–20%,5,22,23which can partly be explained by variations in the

definitionofNASusedindifferentstudies.AbouthalfofallNASoccurwithin1yearafter

transplantation, and the remainder can be detected up to several years after

transplantation.43,47 In liversobtained fromDCDdonors, the incidenceofNAS isabout

10%higherandtheymayoccurearlierthaninliversobtainedfromDBDdonors.41,45

Knowledge about the pathogenesis of NAS is slowly emerging from clinical and

experimentalstudies.Severalriskfactorsforthistypeofbiliarycomplicationhavebeen

identified, strongly suggesting a multifactorial origin. In general, the mechanisms

underlying NAS can be grouped into three categories: 1) preservation or ischemia

related damage to the bile duct wall without sufficient regeneration of the biliary

epithelium 2) cytotoxic injury induced by hydrophobic bile salts, and 3) immune-

mediatedinjury.

These pathological mechanisms contribute, whether simultaneously or not, to

disastrous damage of the biliary epithelium. Generally, in case of epithelial loss,

cholangiocytesproliferateinanattempttorepopulatethedecayedepithelium.However,

ifthedamageisdetrimentaltoalmostallcholangiocytes,thismechanismalonecannot

restoretheintegrityofthebileduct.Asasecondrepairmechanism,stemcellssituated

in the peribiliary glands are activated to proliferate and differentiate and thereby

restoretheepitheliallining.48Thesestemcellsareresistanttoischemiaandresideinthe

bileductwallgroupedtogetherinsmallislets,theperibiliaryglands.49Inprogressionto

post-transplant cholangiopathy, also this resource of new cholangiocytes falls short,

whichmakestheuncoveredandunprotectedbileductwallsusceptibleforintrusionof

toxicbilesaltsandinfections.DamagetothePVPduetoischemiaandhistologicalinjury

totheperibiliaryglandshavebeenassociatedwiththedevelopmentofNAS.50Thelack

of adequate supply of oxygen and nutrients in this case could explain the poor

regeneration by the peribiliary glands. However, further studies are required to

understandwhy this secondmechanism tends to fail in course of biliary strictures or

otherbiliarycomplications.

Inone large clinical study inwhichpatientswere groupedbasedon the time interval

between transplantation and the occurrence of NAS, it was suggested that ischemia-

mediatedmechanisms aremainly responsible for the development of NASwithin the

first year after transplantation, whereas immune-mediated mechanisms play a more

importantroleinthepathogenesisofstricturesoccurringbeyondthefirstyear.9

The high incidence of post-transplant cholangiopathy after DCD liver transplantation

andtheradiologicsimilaritiesbetweenthebileductabnormalitiesofNASandbileduct

abnormalities seen in the presence of hepatic artery thrombosis strongly suggest an

ischemicfactorintheoriginofthesestrictures.Therelevanceofadequatebloodsupply

and the impact of ischemia on the bile ducts have been discussed in more detail in

paragraph2.1(Organprocurementandpreservation).

Anotherrelevantfactorinthepathogenesisofpost-transplantcholangiopathyistoxicity

caused by hydrophobic bile salts. Hydrophobic bile salts have potent detergent

propertiestowardscellularmembranesofhepatocytesandbiliaryepithelialcells.Under

physiological circumstances the toxic effects of bile salts are prevented by complex

formationwithphospholipidsandcholesterol(mixedmicelle).However,earlyafterliver

transplantation,thebalanceinbiliaryexcretionofthesethreecomponentsisdisturbed,

leading to the formation of more toxic bile.7 Evidence for a pivotal role of bile salt-

mediated toxicity in the pathogenesis of bile duct injury and subsequent bile duct

stricturing has gradually emerged during the last decade. Both experimental animal

studiesandclinical studieshavedemonstrated thatbiliarybile salt toxicityearlyafter

transplantationisassociatedwiththedevelopmentofmicroscopicaswellmacroscopic

bileduct injury.7Bilesalt toxicityactssynergisticallywith ischemia-mediatedinjuryof

thebiliaryepitheliumwithoutsufficientregeneration.8

Inthisview,nontoxichydrophilicbilesalts(e.g.ursodeoxycholicacid)mayhavepositive

effectsontheincidenceofpost-transplantcholangiopathy.Inarandomizedclinicaltrial,

administrationofursodeoxycholicacidearlyafterDCDtransplantationdidnotdecrease

theincidenceofNAS.Interestingly,however,biliarysludgeandcastswheresignificantly

diminished within the first year postoperative.51 More (large) studies are needed to

confirm a positive effect of administration of nontoxic bile salts to liver transplant

recipientsonpost-transplantcholangiopathy.

Several studies have provided evidence for an immunologic component in the

pathogenesisofNAS.NAShavebeenassociatedwithvariousimmunologicallymediated

processes,suchasABO-incompatiblelivertransplantation,pre-existingdiseaseswitha

presumed autoimmune component (such as primary sclerosing cholangitis and

autoimmunehepatitis), cytomegalovirus infection, chronic rejection,and finallywitha

geneticpolymorphisminoneoftheCCchemokinereceptors.21

Management

Incontrasttoanastomoticstrictures,NASaremuchmoreheterogeneousinlocalization

andseverity.General recommendationsregardingmanagementarehard tomake,and

good-quality prospective studies are rare. In the case of diffuse and severe biliary

strictureswithprogressivejaundiceandbacterialcholangitisorbiliaryfibrosis,usually

re-transplantationisthemostfavorableoption.Inmostpatients,thestricturesaremore

localizedandcirrhosishasnotyetdeveloped.Manycasesareamenable toendoscopic

therapy. In endoscopic therapy, repeated endoscopies with balloon dilatation and

multiple stents are used. With this approach, success rates are 50–75%.41 As in

anastomoticstrictures,PTCcanbeusedwhenendoscopicaccessisnotfeasible.Incase

ofNASthatareconfinedtotheextrahepaticbileducts,surgicalresectionofthediseased

part and construction of a hepatico-jejunostomy should be considered. In case of

recurrent cholangitis, maintenance antibiotics may result in long-term relief of

symptoms.Althoughwidelyused,thereisnoclinicalevidencethatsupportstheuseof

ursodeoxycholicacid.

Whilemost typesofbiliary complicationscanusuallybemanagedsuccessfully (either

surgically or by endoscopic techniques) or run a self-limiting course, NAS remain the

most challenging typeofbiliary complication, as theyare frequently therapy resistant

and frequently associated with long-term sequelae. Up to 50% of patients with NAS

eitherdieorrequireretransplantation.Mortalityratesdiffermarkedlyamongstudies.23

4.3.2Biliarycastsandsludge

BiliarycastsandsludgearefrequentlyfoundincompanyofNASandcanbeconsidered

asasequelofbileductstricturesalthoughtheycouldalsoappear independentlyor in

combination with other pathologies. Casts and sludge present as filling defects on

cholangiography. Sludge is a viscous collection of mucus, calcium bilirubinate, and

cholesterol. When left untreated, biliary casts can develop. Casts consist of retained

lithogenicmaterialmorphologicallyconfinedtobileductdimensions.Biliarysludgeand

caststendtooccurwithinthefirstyearaftertransplantation.

Multiple factors may contribute to sludge and/or cast formation, including ischemia,

infection,andpreservationinjury.5Theoretically,anythingthatincreasestheviscosityof

bile or reduces bile flow can predispose to casts or sludge. It is likely that ischemia

contributestotheformationofcastsorsludgeboththroughstasisofbile(asaresultof

strictures)andthroughitsdirectinjurytothebiliaryepithelium,resultingintherelease

ofcelldebrisintothebileductlumenaswellasincreasingtheepithelialsusceptibilityto

precipitationof lithogenicmaterials.Otherpathogenic factors thatareassociatedwith

casts or sludge arebiliary cholesterol content, bacterial infection in relation to stents,

thepresenceofahepatico-jejunostomy,fungalinfectionsandtheuseofcyclosporine.23

Regardlessofthecauseofcastsorsludgeaftertransplantation,anincidenceof5.7%was

reportedinthelargeststudysofar,including1650transplantedlivers.52

Mostpatientswithbiliarycastsand/orsludgepresentwithcholangitisandonlyasmall

percentagepresentwithabdominalpain.Despitetherelativeinfrequency,studieshave

shown an increased rate ofmorbidity andmortality as a result of biliary sludge and

casts,whichhavecausedrecurrentcholangitis,repeatedneedforsurgery,graftloss,and

death.53

Intraductal casts and sludge of the biliary tree can almost universally be managed

successfullybyendoscopic removal.However, the long-termsuccessof this treatment

willdependontheunderlyingcause. If the formationofcastsorsludge iscausedbya

localobstructionsuchasabiliarydrainorananastomoticstricturethatcanbetreated

successfully,removaloftheobstructionmaybecurative.However,whenbiliarycastsor

sludge are a symptom of ischemic bile duct injury, the severity of the latter will

determinethelong-termsuccessofcastremovalandwilldeterminethefateofthegraft.

4.4Biliaryabnormalitiesduetohepaticarterystenosisorthrombosis

HATis themostcommonandseriousvascularcomplicationwithareported incidence

that varies between 2% to 11%.54,55 More detailed information regarding vascular

complications can be found in chapter 31, however, we will cover shortly the main

impactonthepost-transplantbileductinthischapter.Toobtainasufficientamountof

oxygenandnutrients,thebiliarytreereliesonbloodsupplyfromthehepaticarteryand

the arterial branches of the gastroduodenal artery. These arteries continue in a fine

vascular network encircling the bile duct, called the peribiliary vascular plexus. After

transplantation,blood supply to thebileductsdependsentirelyon thehepatic artery.

Accordingly,incaseofHAT,thebileductexperienceswidespreadischemia,whichmay

resultinnecrosisandeventuallybileleakage.HATcanbedividedin2categories:early

HATandlateHAT,withthetimeframeusedinliteraturevaryingbetween2weeksand

100daysafter transplantation.56,57Whereas lateHATmayhavea relativemildcourse

due to the formation of vascular collaterals, earlyHAT is associatedwithwidespread

biliary ischemia and subsequent necrosis and bile leakage. Nevertheless, biliary

complicationsmayresult lateafterthediagnosisofHATandsuccessfulrestorationsof

arterialflowtotheliver.

4.5Biliarystricturesduetorecurrentdisease

Recurrent primary sclerosing cholangitis (PSC) may be another cause of biliary

strictures occurring late (>6–12 months) after transplantation. A large retrospective

multicenteranalysisevaluatingtheincidenceofbiliarystricturesafterOLTinacohortof

PSC patients reported an incidence of 36.1%.58 This is approximately 3.5-fold higher

thaninnon-PSCpatients.Moredetailedinformationregardingrecurrentdiseasesofthe

bileductcanbefoundinchapter36.

4.6Bacterialcholangitis

Bacterial cholangitis is not uncommon in immunosuppressed liver transplant patients

andcanresultinalifethreateningillness.Ingeneral,theriskofcholangitisisincreased

in patients in whom a T-tube is used, in patients who underwent a hepatico-

jejunostomy,andinpatientscomplicatedbyanastomoticorNAS.Alloftheseconditions

mayfacilitateascendingmigrationofbacteriaintothebiliarytree.Whenabiliarydrain

ispresent,positivebacterialculturesfromthebilemaysupportthediagnosis,although

it should be noted that colonization of bile is not infrequent. In other patients the

diagnosischolangitisisrarelysupportedbypositivebileculturesandusuallymadeafter

exclusion of other causes of fever. Bacterial cholangitis after liver transplantation

usuallypresentswithhighfeverwithorwithoutchills incombinationwithcholestatic

liver function test.Management of acute cholangitis after transplantation is similar to

thatrecommendedtonontransplantpatientsandshouldincludeappropriateantibiotic

therapyaftertheexclusionofananatomicalcause(e.g.anastomoticstrictures).

5.Summary

Biliary complications are a frequent cause of morbidity after liver transplantation.

Advancesinsurgicaltechniquesandpreservationmethodsduringthelastdecadeshave

ledtobetterresults,butbiliarycomplicationsstilloccurin10–40%oftherecipientsand

are associatedwithmortality rates of 8–15%. Partial liver grafts (e.g. split livers and

livers from living donors) as well as livers from extended-criteria donors (e.g. DCD

donors)areassociatedwitharelativelyhighriskofbiliarycomplications.Ofallbiliary

complications, bile duct strictures and bile leakage are most common after liver

transplantation.Whilebile leakageandanastomoticbileductstricturescanusuallybe

managedsuccessfullywithout long-termsequelae,NASarethemost troublesometype

of biliary complications. NAS are often multifocal and can be difficult to treat by

endoscopic techniques. When associated with recurrent cholangitis, jaundice or even

secondary biliary fibrosis, retransplantation may be the only treatment option left.

Future studies should focusonbetterdefining themechanismunderlyingNASandon

the development of effective preventive measures. In this respect, development and

potential implementation in liver transplant protocols of machine preservation is of

greatrelevance.

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Donorhepatectomytimeinfluencesischemia-reperfusioninjuryofthebiliarytreeindonationaftercirculatorydeathlivertransplantation

OttoB.vanLeeuwen,MarjoleinvanReeven,DannyvanderHelm,JanN.M.Ijzermans,VincentE.deMeijer,AadP.vandenBerg,SarwaDarwishMurad,BartvanHoek,IanP.Alwayn,RobertJ.Porte,WojciechG.PolakSurgery.2020(inpress)

3

ABSTRACT

Background: Donor hepatectomy time is associated with graft survival after liver

transplantation.Theaimofthisstudywastoidentifytheimpactofdonorhepatectomy

timeonbiliaryinjuryduringdonationaftercirculatorydeathlivertransplantation(DCD-

LT)

Methods:First,bileductbiopsiesof livers included in (pre)clinicalmachineperfusion

researchwereanalyzed.Secondly,ofthesamelivers,bilesampleswerecollectedduring

normothermic machine perfusion (NMP). Lastly, a nationwide retrospective cohort

studywasperformedincluding273adultpatientsundergoingDCD-LTbetween01-01-

2002 and01-01-2017. Primary endpointwasdevelopment of non-anastomotic biliary

strictures (NAS) within two years of DCD-LT. Cox Proportional-Hazards regression

analyseswereusedtoassesstheinfluenceofhepatectomytimeondevelopmentofNAS.

Results: Livers with severe histological bile duct injury had a higher median

hepatectomy time (p=0.03). During NMP, livers with hepatectomy time >50min had

lower biliary bicarbonate and bile pH levels. In the nationwide retrospective study,

donorhepatectomytimewasanindependentriskfactorforNASafterDCD-LT(HR1.18

pertenminutesincrease,95%CI1.06-1.30,p-value=0.002).

Conclusions:Donorhepatectomytimenegativelyinfluenceshistologicalbileductinjury

prior to NMP and bile composition during NMP. Additionally, hepatectomy time is a

significantindependentriskfactorforthedevelopmentofNASafterDCD-LT.

INTRODUCTION

The imbalance between the number of patients on the waiting list for liver

transplantation(LT)andthenumberofavailablegraftsfromdonationafterbraindeath

(DBD)donorshasresultedinanincreaseduseofliversfromdonationaftercirculatory

death (DCD) donors. In 2018, 38%of all deceased donor LT in theNetherlandswere

performedwithaDCDgraft1.

LT fromDCD donors can lead to inferior outcomes compared to LTwith DBD grafts,

especially with respect to graft survival 2-5, which is related to a higher chance of

developing early allograft dysfunction and post-transplant cholangiopathy 6-10. Among

post-transplant cholangiopathies, non-anastomotic strictures (NAS), also known as

ischemic type biliary lesions (ITBL) or ischemic cholangiopathy (IC), is the most

hazardoustype,withastrongnegativeimpactongraftsurvival11-13.

AnimportantdeterminantofoutcomeafterLTisischemiareperfusioninjury(IRI).IRI

occursinbothDBDandDCD-LT.However,DCDgraftssufferfromanadditionalperiod

ofwarmischemiainthedonorbetweenwithdrawaloflifesupportandinitiationofcold

flush out, the so called donor warm ischemia time (dWIT). Several studies have

indicatedthatthelengthofthedWITisacriticalriskfactorfornegativeoutcomeafter

DCD-LT2,14,15.

Unfortunately, thestartof in situ cold flushoutandcoolingdoesnot lead toadequate

protectionagainst ischemic injury,becausethecoretemperatureof the livergenerally

does not drop below 15-20oC during surgery16. At this temperature, organs are still

metabolically active, resulting in rapid depletion of adenosine tri-phosphate and

accumulationofmetabolitesduringanaerobicmetabolism.Livercoretemperaturefirst

reachesarelativelysafetemperature(<4oC)whenorgansarestoredinabagwithcold

preservationsolutioninaboxwithice.Therefore,itishypothesizedthat,apartfromthe

dWIT, the duration of the hepatectomy time provides an additional risk factor for

ischemicinjuryandcouldthereforeimpactoutcomeafterLT.

A recent study published by Jochmans et al. based on data from the Eurotransplant

Registry supported this hypothesis17. In this study, donor hepatectomy time was an

independent risk factor for patient mortality and graft loss. Moreover, DCD grafts

appeared to bemore susceptible to donor hepatectomy time than DBD grafts. More

recently, Farid et al. assessed the influence of the donor hepatectomy time on the

outcomes of DCD-LT in the United Kingdom, concluding that a hepatectomy time of

more than60minuteswas associatedwith ahigher riskof primarynon function and

graftfailure18.Neitherstudy,however,didassesstheeffectofdonorhepatectomytime

on the development of post-transplant cholangiopathy after DCD-LT, neither did they

evaluatewhetherhepatectomytimewasdifferentamongprocurementteams.

Several studies have shown a strong relation between bile duct injury prior to

implantation and the development of NAS after transplantation19, 20. If donor

hepatectomytimeinfluencestheratesofNAS,thiswouldbedisplayedintheseverityof

biliaryinjurypriortoimplantation.Additionally,bilecompositionduringnormothermic

machineperfusion(NMP)oflivergraftscanbestudiedtoassessbileductinjury21,22.

TheaimofthisstudywastoassesstheimpactofdonorhepatectomytimeinDCDdonors

onthedevelopmentofbiliaryinjuryduringDCD-LT.First,bileductbiopsiestakenupon

arrival in one of the three recipient centers are analyzed. Secondly, bile composition

duringNMP isstudied.Last, the influenceofhepatectomytimeon thedevelopmentof

NASafterDCD-LTisstudiedinanationwideretrospectivedatabasestudy.

METHODS

Donationprocedureandorganprocurement

Untilrecently,alldonorprocedures/procurementsintheNetherlandswereperformed

byoneoffiveregionalprocurementteams,eachcoveringacertainregionofthecountry.

Each procurement team consists of a surgeon, surgical assistant, anesthesiologist and

two operation room assistants. In the Netherlands, withdrawal of life support in a

patienteligibleforDCDorgandonationgenerallytakesplaceattheintensivecareunit.

Pre-mortemcannulationofthepatientisnotperformed,andsystemicheparinizationis

prohibited by Dutch law. When circulatory arrest has been determined, there is a

mandatory five minutes ‘no-touch’ period. After this ‘no-touch’ period, the donor is

transportedtotheoperatingtheatre.Asuper-rapidsterno-laparotomywithpressurized,

aortic-only perfusion is used as standard perfusion technique. Cold perfusion is

currentlyexecutedwithBelzerUniversityofWisconsincoldstoragesolution(Bridgeto

Life, London,UK) (UW).Whether the liver is retrieved separately or en-blocwith the

pancreas is based on the preferences of the surgeon. On the back table, the liver is

flushed via the portal veinwith at least 500ml cold preservation solution until clear

perfusate is established. The CBD and intrahepatic biliary tree are flushed with low

pressure UW 23. As there are no clear Dutch guidelines on the sequence of organ

procurement, the lungs in a DCD donor are usually procured before the abdominal

organs. The implantation is usually executedwith a caval sideclampand veno-venous

anastomosis, end-to-end arterial and portal anastomosis and duct-to-duct biliary

anastomosis.Thestandardreperfusiontechniqueusedisinitialportalveinreperfusion.

Studydesign

Thisstudyconsistsofthreeparts.First,bileductbiopsiesandbilecompositionofDCD

liverswereanalyzedforapotentialinfluenceofhepatectomytime(partA-B).Hereafter,

to validate the findings, a nationwide retrospective database analysis was performed

(partC).

PartA:Histologicalanalysisofbileducts

Of all DCD livers that underwent preclinical and clinical normothermic machine

perfusionintheUniversityMedicalCenterGroningenbetween01-01-2013and01-01-

2019,bileductbiopsiesprior tomachineperfusionwere collected.Theonly criterion

required for inclusion was that the donor hepatectomy time was available. Biopsies

were taken from the distal common bile duct beforemachine perfusion, fixed in 4%

formalin,andsubsequentlyembedded inparaffin.Slicesof4µmwerecutandstained

withHematoxylin&Eosin,andsubsequentlyexaminedusinglightmicroscopy.Thebile

ductinjury(BDI)scorewasdeterminedinablindedfashionbytworesearchers,usinga

clinicallyrelevanthistologicalgradingsystem20, 22.TheBDIconsistedofthecombined

scores for deep peribiliary gland injury, peribiliary vascular plexus injury and stroma

necrosis. The used cut-off value between low and high BDI was 4.75, as described

previously22.ComparisonsbetweengroupswereperformedwiththeChi-Squaretest,or

Fisher’sExact testwhere appropriate.ROC (ReceiverOperatingCharacteristic) curves

wereusedtoidentifymostappropriatecut-offvalues.

PartB:Normothermicmachineperfusion

AllpreclinicalandclinicalNMPprocedureswereperformedwiththeLiverAssistdevice

(Organ Assist, Groningen, the Netherlands). Protocols and outcomes are reported

elsewhere22,24,25.Tomonitorbiliarytreeviability,bilewascollectedfroma8Frbiliary

drain in the common bile duct. During NMP, bile samples were collected every 30

minutes undermineral oil to determine biliary pH, bicarbonate and glucose, as these

parameters are biomarkers of bile duct viability 22. Bile composition was compared

betweenthegroupsatdifferenttimepointsusingtheMann-Whitneytest.

PartC:Retrospectivenationwidestudy

Inthisnationwide,retrospectivecohortstudy,alladultLTperformedwithaDCDgraft

in the Netherlands between 01-01-2002 and 01-01-2017 were included. Exclusion

criteria were multi organ DCD transplants, DCD retransplantations, transplantations

involvingmachineperfusionandprocurementofDCDgraftsbya foreignprocurement

team. Additionally, cases with missing information on hepatectomy times or donor

agonalphasewereexcluded.Donorcharacteristicsandinformationontheprocurement

procedure and the regional procurement team were obtained via the Donor Data

Application of Eurotransplant. Data of recipients and transplantation outcomes were

obtained fromthedatabasesof theparticipatingcenters, completedwithmissingdata

fromtheelectronicalpatients’medicalrecords.

Donorhepatectomytimewasdefinedastheperiodbetweenthestartofcoldflushinthe

donorandthestorageoftheliverinabowlwithcoldpreservationfluidandmeltingice

on the back-table. The dWIT was calculated as the time between withdrawal of life

support and cold flush in the donor. Since in the normal situation the donor

hepatectomytime ispartof thecold ischemia time, thedefinitionof thecold ischemia

timehasbeenaltered tominimize the chanceof confounding; cold ischemia timewas

definedastheperiodbetweentheendofthedonorhepatectomyandtheremovalofthe

liverfromicepriortoimplantation.Finally,recipientwarmischemiatimewasdefined

as the time between removal of the liver from ice until either portal or arterial

reperfusion,whichevercamefirst.

TheendpointoftheretrospectivestudywasthedevelopmentofNASwithintwoyears

aftertransplantation.NASwasdefinedasdonorbileductstricturesatanylocationbut

theanastomosis,inabsenceofhepaticarterythrombosis.Tomeettheendpoint,patients

wererequiredtohaveclinicalsymptomsofcholestasis(e.g.jaundice,itch,elevatedtotal

bilirubin)withsubsequentimagingdemonstratingbileductstrictures.IfNASdeveloped

afterthisperiod, itwasconsideredtoberelatedtorecipientfactorsratherthandonor

factors. Univariable and multivariable Cox Proportional-Hazards regression models

wereusedtoevaluateindependentriskfactorsforNAS.Inbothmodels,subjectsthatdid

not develop NAS within two years were censored at two years post transplantation.

Patients who died or underwent retransplantation within the first two years after

transplantation were censored at their date of death or date of retransplantation,

respectively. Variables were included in the multivariable, backward stepwise, Cox

modelifunivariableCoxregressionyieldedap-value<0.20.Thethresholdof0.20was

chosentodecreasetheriskofoverfittingofthemodel.Thereportedhazardratios(HR)

for donor hepatectomy time refer to an increase of ten-minutes in hepatectomy time.

For the cold ischemia time and recipient warm ischemia time, the HR represent an

increaseofonehourandoneminute,respectively.

Inallthreeprojectsincorporatedinthisstudy,continuousvariableswerepresentedas

median with both total range and interquartile range (IQR), whereas categorical

variableswerepresentedasnumber[percentage]Alltestshadatwo-sideddesignwith

ap-valuebelow0.05consideredassignificant.TheanalyseswereperformedusingSPSS

version 24 (IBM, Chicago, Illinois). This study was approved by the Medical Ethical

CommitteeoftheUniversityMedicalCenterGroningen(METC.2017/504).

RESULTS

PartA:Histologicalanalysisofthebileduct

Of 40 consecutive normothermic machine perfusion procedures between 2014 and

2019,39bileductbiopsieswerecollected.Afterexclusionofbiopsieswithanunknown

donorhepatectomytime,27biopsieswere included in theanalysis.Liverswithahigh

bile duct injury score had a significant longer median donor hepatectomy time

compared to graftswith lowbile duct injury score (56 versus44 minutes, p = 0.03).

(Figure1A).ROC-curveanalysisshowedadonorhepatectomytimeof50.0minutesas

themost suitablecut-offpoint.Of liverswithhepatectomy time≤50.0minutes,17.0%

displayed high bile duct injury, versus 64% in livers with a hepatectomy time >50.0

minutes(p=0.01)(Figure1B).

Figure1:Hepatectomy time influencesbiliary injuryprior to transplantation. A:

Livers grafts with a high bile duct injury score had a longermedian hepatectomy time

comparedto liverswith lowbileduct injuryscore(p=0.027).Datapresentedasmedian

(IQR).B:Liversgraftswithadonorhepatectomytime>50minuteshavemoreseverebile

ductinjurycomparedtoliverswithahepatectomytime≤50minutes(p=0.016).*depicts

asignificant(p<0.050)difference.Abbreviations:BDI,bileductinjuryscore;min;minutes.

PartB:Normothermicmachineperfusion

Ofthe27 livers, liverswithahepatectomytime≤50.0minuteshadmorealkaloticbile

duringthefirstfourhoursofNMP.Subsequentlybiliarybicarbonatelevelswerehigher

in livers with a hepatectomy time below 50 minutes. (Figure 2A-B). Glucose

reabsorption,displayedbytheglucoseratiobetweenbileandperfusate,didnotseemto

besignificantlyinfluencedbyhepatectomytime(Figure2C).

Figure 2: Hepatectomy time influences bile composition during NMP. A:

Hepatectomy time influenced biliary bicarbonate levels during NMP. B: bile pH was

significantlylowerinthegroupwithhepatectomytime≤50minutes.C:hepatectomytime

didnotseemtosignificantlyinfluencebiliaryglucosereabsorption.*depictsasignificant

(p<0.050)difference.Abbreviations:BDI,bileductinjuryscore;min;minutes.

PartC:Retrospectivestudy

Atotalof376DCD-LTwereperformedintheNetherlandsbetween01-01-2002and01-

01-2017.Hundred-and-threecasesmetoneormoreoftheexclusioncriteria,resulting

ina totalof273 included in thisstudy(Figure3).Themedian followupperiodof the

complete cohort was 4.36 (IQR 2.81-7.08, range 0-16.8 years) years. Baseline

characteristicsarepresentedinTable1.Mediandonorhepatectomytimefortheentire

cohort was 63.0 (IQR 52.5-80.5, range 23-140) minutes. Lung procurement led to a

significantly longer donor hepatectomy time of 69 (IQR 59-80) minutes, when

compared toahepatectomy timeof61 (IQR49-81)minutes indonors inwhich lungs

werenotprocured (p=0.02).TheoutcomesafterDCD-LT in the complete cohort are

shown in Table 1. Actuarial one-, three-, and five-year graft survival rateswere 75%,

64%and60%,and87%,79%,and74%forpatientsurvivalrespectively.

Figure3:Flowchartofincludedsubjectsintheretrospectivedatabasestudy.

Table1:Donorandrecipientdemographics

Characteristic Result(n=273)Donor Age(years) 47.0(35.5-54.0)

Range12-74 Gender Male 155[56.8%] Female 118[43.2%] Bodymassindex(kg/m2) 24.0(22.0-26.0)

Range13-34 CVAascauseofdeath No 151[55.3%] Yes 122[44.7%] ALTpeak(U/L) 43.0(23.0-87.0)

Range6-7385 LastγGT(U/L) 34.0(20.0-65.5)

Range4-747Procurement Donorwarmischemiatime(min)* 32.0(26.0-38.0)

Range15-80 Donorhepatectomytime(min)† 63.0(52.5-80.5)

Range23-140 Lungprocurement 84.0[30.8%]Recipient Age(years) 57.0(49.0-63.0)

Range22-70 Gender Male 197[72.2%] Female 76[27.8%] Bodymassindex(kg/m2) 25.7(23.4-29.1)

Range17-46 HCCasindicationfortransplantation No 175[64.1%] Yes 98[35.9%] LaboratoryMELDscore 14.6(10.0-21.0)

Range6-44Transplantation Coldischemiatime(min)‡ 359(302-431)

Range131-743 Recipientwarmischemiatime(min)§ 34.0(26.0-42.0)

Range17-144Outcomes ASTpeak(u/L) 2115(1165-4252)

Range129-20280 ALTpeak(u/L) 1620(771-2857)

Range162-10944

Intensivecareunitstay(days) 2.0(1.0-5.0)Range0-185

Totalhospitalstay(days) 18.0(13.0-27.0)Range0-235

Primarynon-function|| 8[2.90%] Non-anastomoticstrictures 70[25.6%] Withintwoyearspost-transplantation 66[24.2%] Hepaticarterythrombosis 14[5.10%] Actuarialgraftsurvival 1year 75% 3year 64% 5year 60% Actuarialpatientsurvival 1year 87% 3year 79% 5year 74%

Valuesarepresentedasmedian(interquartilerange)ornumber[%].*Thetimebetween

withdrawal of life support and cold flush in the donor. †Theperiod of timebetween the

startofcoldflushinthedonorandthestorageoftheliveroniceontheback-table.‡The

timebetweentheendofthedonorhepatectomyandtheremovaloftheliverfromiceprior

to implantation. §The time between removal of the liver from ice until either portal or

arterialreperfusion.||Patientdeathorretransplantationwithin7daysoftransplantation

without clear cause. Abbreviations: ALT, alanine aminotransferase; , AST; aspartate

aminotransferase, CVA , Cerebrovascular accident; γGT, gamma-glutamyl transferase;

HCC,hepatocellularcarcinoma;MELD,modelforend-stageliverdisease.

Sixty-sixpatients(24.2%)werediagnosedwithNASwithintwoyearsofLT.Duringthe

complete followup,25patientshaveundergonearetransplantationasaresultof this

complication. Baseline characteristics, stratified by recipient development of NAS are

provided in supplementary table 1. In a univariable Cox Proportional-Hazards

regression model, donor hepatectomy time was an independent risk factor for the

development ofNAS (HR1.14, 95%CI 1.03-1.26, p-value0.02).After adjusting for all

covariateswith a p-value below 0.2 in univariable analyses, donor hepatectomy time

remainedanindependentriskfactorfordevelopingNASwithinthefirsttwoyearspost-

LT (adjusted HR 1.18, 95% CI 1.06-1.30, p-value 0.002, Table 2 and 3). Besides

hepatectomy time, donor age and cold ischemia timewere significant risk factors for

NAS.

Table2:UnivariableCoxProportional-HazardsregressionmodelfordevelopingNAS

Hazardratio 95%CI p-valueDonor Age(years) 1.03 1.01-1.05 0.004 Gender Male REF Female 1.14 0.70-1.86 0.59 Bodymassindex(kg/m2) 0.99 0.92-1.08 0.88 CVAascauseofdeath No REF Yes 1.83 1.13-2.98 0.02 ALTpeak(U/L) 1.00 1.00-1.00 0.09 LastγGT(U/L) 1.00 1.00-1.00 0.41Procurement Donorwarmischemiatime(minutes)* 1.03 1.01-1.05 0.01 Donorhepatectomytime(10-minutes)† 1.14 1.03-1.26 0.02 Lungprocurement 0.60 0.34-1.06 0.08Recipient Age(years) 0.994 0.97-1.02 0.60 Gender Male REF Female 1.22 0.72-2.07 0.45 Bodymassindex(kg/m2) 1.02 0.97-1.07 0.55 HCCasindicationfortransplantation No REF Yes 0.77 0.46-1.30 0.33 LaboratoryMELDscore 0.98 0.95-1.02 0.35Transplantation Coldischemiatime(hours)‡ 1.17 1.04-1.33 0.01 Recipientwarmischemiatime(minutes)§ 1.02 1.00-1.04 0.17

Valuesarepresentedasmedian(interquartilerange)ornumber[%].*Thetimebetween

withdrawal of life support and cold flush in the donor. †Theperiod of timebetween the

startofcoldflushinthedonorandthestorageoftheliveroniceontheback-table.‡The

timebetweentheendofthedonorhepatectomyandtheremovaloftheliverfromiceprior

to implantation. §The time between removal of the liver from ice until either portal or

arterial reperfusion.Abbreviations:ALT,alanineaminotransferase;CVA, cerebrovascular

accident;γGT,gamma-glutamyltransferase;HCC,hepatocellularcarcinoma;MELD,model

for end-stage liver disease; PBC, primary biliary cirrhosis; PSC, primary sclerosing

cholangitis.

Table3:MultivariableCoxProportional-HazardsregressionmodelforNAS

Hazardratio 95%CI p-valueDonor Age(years) 1.03 1.01-1.05 0.01 CVAascauseofdeath - - 0.29 No Yes ALTPeak 1.00 1.00-1.00 0.05Procurement Donorwarmischemiatime* - - 0.50 Donorhepatectomytime† 1.18 1.06-1.30 0.002 Lungprocurement 0.47 0.26-0.84 0.01Transplantation Coldischemiatime‡ 1.22 1.08-1.38 0.001 Recipientwarmischemiatime§ - - 0.47

Multivariablemodelwasconductedviabackwardstepwiseapproach.Adash(-)indicates

thatvariablewasremovedfromthemodel.*Thetimebetweenwithdrawaloflifesupport

andcoldflushinthedonor.†Theperiodoftimebetweenthestartofcoldflushinthedonor

and the storage of the liver on ice on the back-table. ‡The time between the end of the

donorhepatectomyandtheremovaloftheliverfromicepriortoimplantation.§Thetime

between removal of the liver from ice until either portal or arterial reperfusion.

Abbreviations:ALT,alanineaminotransferase;γGT,gamma-glutamyltransferase.

DISCUSSION

This is thefirststudythatdemonstratesthe impactofdonorhepatectomytimeonthe

developmentofbiliary injuryduringandafterDCDlivertransplantation.Hepatectomy

time influences the severity of histological bile duct injury prior to transplantation.

Moreover,prolongedhepatectomytimesnegativelyinfluencedbilecompositionduring

NMP.Additionally,theretrospectivestudyindicatesthateveryten-minutesincreasein

donorhepatectomytimeleadstoan18percentincreasein,theriskofdevelopingNAS.

OpdenDriesetal.haveshownthatbileducthistologyishighlypredictiveofNASafter

livertransplantation20.Inthecurrentstudy,itisobservedthatprolongedhepatectomy

timesleadstoanincreasedBDIscore,depictingincreasedratesofdeepperibiliarygland

injury, peribiliary vascular plexus injury and stroma necrosis. The results from this

histologystudydemonstratethattheimpactofhepatectomytimeisalreadyvisibleprior

to graft reperfusion. In addition to histology, NMP can be used to assess biliary

function21,22. Similar results were observed during NMP, livers with prolonged

hepatectomytimeproducedbileofinferiorquality.

The results of the current study are roughly in line with those reported in the

EurotransplantregistrystudybyJochmansetal.andtheUKbasedstudyfromFaridand

colleagues: a prolonged donor hepatectomy time impairs the outcome of DCD-LT.

However,neitherofthestudieswereabletoassesstheinfluenceofdonorhepatectomy

time on the development of biliary complications. Surprisingly, the median donor

hepatectomytimeintheDutchcohortinthecurrentstudywassubstantiallylongerthan

thatoftheDCD-LTsubgroupinthestudyofJochmansetal.(63versus50minutes)17.As

within the Eurotransplant region, only theNetherlands, Belgium andAustria perform

DCD organ procurements, this implies that the donor hepatectomy time in the

Netherlandsissubstantiallylongercomparedtotheothertwocountries.Moreover,the

medianhepatectomytimeinourcohortwasalsoconsiderablylongerthanintheUnited

KingdomasreportedbyFaridetal.(63versus35minutes)18.Asaresultofthisfinding,

the Dutch Committee on Independent Procurement Teams implemented several

strategiestolowerthehepatectomytime,suchasraisingawarenessontheimpactofthe

donor hepatectomy time and endorsing knowledge and skill exchange between the

teams.Since2018,thishasresultedinasubstantialdecreaseofthedonorhepatectomy

time in theNetherlands (mean of 42minuteswith standarddeviation of 12minutes)

without an increase in liver injuries, highlighting the importance of training in organ

procurementandregularevaluation26.

ThegraftsurvivalratesreportedbyFaridetal.aresubstantiallyhigherthanthoseinour

cohort(1,3,and5yeargraftsurvivalrateof86.5%,80.9%and77.7%intheUKversus

75%,64%and60%intheDutchcohort,Table1).Sincethepatientsurvivalrateshave

notbeenreportedbyFaridetal.,itisnotpossibletoevaluatewhetherthehigherrateof

graft loss in the Netherlands is the result of more patient deaths or of more

retransplantations. However, it could possibly be explained by the difference in

hepatectomy time between the two cohorts. Nevertheless, it would be valuable to

thoroughlyinvestigatethissubstantialdifferenceingraftsurvivalrates.

Surprisingly, procurement of the lungs seemed to have a protective effect on the

development of NAS, despite the fact that lung procurement leads to a prolonged

hepatectomytime.Thisfindingisprobablytheresultofthestrictacceptancecriteriafor

DCDlungdonationhandledbythoracicsurgeonsandlungphysicians.Only lungsfrom

optimal DCD donors are accepted, otherwise the lungs are not procured. Lung

procurementisinthatcaseaproxyforamoreoptimaldonor.

Jochmansetal.statedthatportalperfusion,nexttostandardinsituaorticcoldflush,can

accelerate liver cooling and might prevent the detrimental effect of prolonged

hepatectomytime17.Inarecentpublishedstudy,Hameedetal.concludedthatinhigh-

risk donors DBD donors, dual perfusion is superior 27. Furthermore, Ghinolfi et al.

concluded recently that dual perfusion has a protective effect on the development of

ITBL after LT with grafts from octogenarian donors 28. However, a randomized

controlled trial comparing aortic flush only and combined aortic and portal flush in

DBD-LT, showed no difference in the incidence of post-transplant cholangiopathy 29.

SinceDCDgraftscouldalsobeconsideredashigh-riskgrafts, itwouldbe justifiableto

evaluate the effect of dual perfusion versus aortic only perfusion in the DCD-LT

population. Another method to potentially minimize the detrimental effect of both

donor warm ischemia time and hepatectomy time on the outcomes after liver

transplantation is the use of normothermic regional perfusion (NRP). A recently

publishedstudybyHessheimeretal.showedthatwiththeuseofNRPtheratesofbiliary

complicationsandgraftlosscouldbereducedsubstantiallywhencomparedtoasuper-

rapidrecovery30.

Recently,Kalisvaartetal.showedtheimportanceoftheagonalphaseoftheDCDdonor

andits influenceontheoutcomesaftertransplantation,consideringanarterialoxygen

saturation level below 80% as starting point for the functional donorwarm ischemia

time31.Unfortunately,inourcohort,dataonbloodpressuresandsaturationduringthe

agonal phasewere unavailable or improperly recorded. Therefore,wewere forced to

use another definition of the donor warm ischemia time. Since the agonal phase has

proven to be of importance, we chose to use the period between withdrawal of life

supportandtheinitiationofcoldflushingasthedWITinthisstudyratherthanthetime

betweencardiacarrestandcoldperfusion.Additionally,asshownbefore,thisstudyalso

underlinestheimportanceofashortcoldischemiatimeforDCDgrafts2232.Everyhour

ofcoldischemiawasassociatedwitha22%increasedriskofNAS.Finally,donorageis

onceagainshowntobeanimportantriskfactorforbiliarycomplications.

An importantstrengthof this study is the fact thathistologicalanalysesarecombined

withastudyofbilecompositionduringNMPandanationwideretrospectivedatabase

study. Another strength is that donor hepatectomy time is incorporated as an

continuousvariableintothemultivariablemodelratherthanasadichotomousvariable

setaroundacertaincut-offofdonorhepatectomytime.Thislatterwouldhaveledtoa

lossofvaluable information.Anotherstrongaspectof thisstudy is the follow-upofall

patients with detailed information on the development of biliary complications. One

limitationofthedatabasestudyistheretrospectivedesignandrelativelysmallcohorts.

In addition, aspartCwasused tovalidate the findings inpartAandB, these cohorts

consist of differentpatients.Moreover, in a substantial numberof cases, hepatectomy

timeand/ordonorwarm ischemia timewasmissing, leading to ahighexclusion rate.

Sincewe could not guarantee these variables to bemissing at random, imputation of

thesevariableswasnotdesirable.Wedonotsuspectthatourresultswereconfounded

bythus,howeverbiascannotentirelybeexcluded.

Inconclusion,donorhepatectomy timestrongly influencesbiliary injuryduring

and after DCD-LT. The donor hepatectomy time should be kept as short as possible,

especiallyinthepresenceofotherriskfactorssuchasanolderdonororprolongedcold

ischemiatime.

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Donorbloodcompositionisariskfactorforbiliaryinjuryduringdonationaftercirculatorydeathlivertransplantation

OttoB.vanLeeuwen,MarjoleinvanReeven,DannyvanderHelm,JanN.M.Ijzermans,IanP.J.Alwayn,BartvanHoek,WojciechG.Polak,TonLisman,VincentE.deMeijer,RobertJ.Porte.Submittedforpublication

4

ABSTRACT

Post-transplant cholangiopathy following ischemia-reperfusion injury (IRI) inhibits

widespreadapplicationofdonationafter circulatorydeath liver transplantation (DCD-

LT). Blood composition of the donor could influence graft flush-out and IRI. We

hypothesized that platelets, leukocytes and hematocrit of the DCD donor influence

biliaryinjuryduringDCD-LT.

Theassociationofhematocrit,plateletandleukocytecountswithbileducthistologyand

bile composition during (pre)clinical normothermic machine perfusion (NMP) was

assessed.Secondly,inanationwideretrospectivestudy,allDCD-LTbetween2010-2017

were included to assess the role of blood composition in the development of non-

anastomoticbiliarystrictures(NAS).

High donor platelet counts (OR 2.553, 95%CI 1.082-6.021, p=0.029) and low donor

leukocyte counts (OR 0.734 95%CI 0.581-0.927, p=0.009) were associated with

increased bile duct injury of 40NMP livers, and donor platelet count associatedwith

biliary bicarbonate and pH. In a multivariate Cox Proportional-Hazards regression

analysis including 235 DCD-LT, donor platelet count (HR 1.047 95%CI 1.007-1.089,

p=0.022) and hematocrit (HR 1.044 95%CI 1.005-1.083, p=0.025) were significant

independentriskfactorsforthedevelopmentofNAS.

Donor platelets and leukocytes are associatedwith biliary injury prior to and during

NMP.Additionally,plateletcountandhematocritaresignificantindependentriskfactors

forthedevelopmentofNASafterDCD-LT.

INTRODUCTION

Liver transplantation is the preferred treatment for patients with end-stage liver

disease.Theimbalancebetweenorganavailabilityanddemandhasledtotheincreased

use of donation after circulatory death (DCD) livers. The main limitation of the

widespread use of DCD livers is the frequent development of post-transplant

cholangiopathyfollowingtransplantation.Post-transplantcholangiopathyisaspectrum

of bile duct abnormalities, including non-anastomotic biliary strictures (NAS),

intraductalcastsand/orintrahepaticbilomaformation.1NASisobservedinupto30%

ofpatientsthatreceiveaDCDliver,whereasafterdonationafterbraindeath(DBD)liver

transplantation,thisvariesbetween3and13%.2-4

Injuryofperibiliaryglands(PBG)anddamageto theperibiliaryvascularplexus(PVP)

havebeen identifiedasrisk factors for thedevelopmentofNAS.Peribiliaryglandsare

essential in the regeneration of biliary epithelium after severe bile duct injury, as

encounteredduringtransplantation.5Recently,weobservedinthedatafromthestudy

byOpdenDries et al. that all biopsies gradedwith>50%deepPBGdamage alsohad

severe(>50%)damagetothePVP.Therefore,wehypothesizedthatPBGandPVPinjury

developparallelduringthedonorwarmischemicperiodandsubsequentcoldischemia

time prior to DCD liver transplantation. Accordingly, we hypothesized that if the

pathophysiology of PVP injury could be clarified, subsequent potential solutions that

decreasePVPinjurywouldalsopositivelyaffectPBGdamage.

Plateletsareoneofthemainthree(cellular)componentsofblood,andcontainseveral

prohemostatic and proinflammatory mediators.6 Several studies have shown an

influenceofplateletsonischemia-reperfusioninjuryinischemichearts,lungs,pancreata

and livers.7-14 In the early 1990’s Cywes et al. have shown that the level of platelet

adherence in donor livers grafts can predict the outcome of transplantation.15 An

increased level of platelet adherence was associated with inferior post-transplant

outcomes, and up to one-third of donor livers demonstratedmoderate to high-grade

plateletadhesion,suggestingsevereendothelialcelldamage.15Additionally,Cywesetal

haveshownthatadditionofplateletsduringperfusionofratliversaggravatedischemia-

reperfusion injury.14 At the beginning of this century, Sindram et al. have shown that

plateletsinducesinusoidalendothelialcellapoptosisofthecoldischemicratliver.16This

studyshowedthatpharmacologicalpreventionofplateletadhesionreducedsinusoidal

epithelial cell apoptosis. We hypothesize that donor platelets may influence biliary

injury by adhesion to endothelial cells in the PVP during thewarm hypoxic/ischemic

periodthatDCDgraftsencounter,subsequentlyleadingtoincreasedPVPinjury.

Inadditiontoplatelets,wehypothesizedthatalsoredbloodcellscouldinfluencebiliary

injury.Underhypoxicconditions,erythrocytesgeneratereactiveoxygenspecies(ROS).17

ROSarekeyinthedevelopmentofischemia-reperfusioninjury.18Moreover,inhypoxia,

erythrocytesinducepro-inflammatoryendothelialactivation,potentiallycausinginjury

tothePVP.19The lastof thethreecellularcomponentsofblood, leukocytes,couldalso

impact endothelial function, as in hypoxic conditions leukocytes are associated with

endothelialdamage.20

We aimed to study the potential association of platelet and leukocyte counts and

hematocritonbiliary injury inachronologicalmethod.First,bileductbiopsiesofDCD

liversprospectivelytakenpriortonormothermicmachineperfusion(NMP)wereused

to asses biliary injury at baseline. Secondly, bile produced during normothermic

machineperfusion (NMP)of thesameDCD liverswasstudied.Lastly,weperformeda

nationwideretrospectivestudy toassess the influenceofdonorplateletand leukocyte

countsandhematocritonthedevelopmentofNASafterDCD-LT.

METHODS

Studydesign

The first part of this study is a prospective cohort study including all DCD livers that

underwent preclinical and clinical NMP in the University Medical Center Groningen

(2013-2019).Extrahepaticbileductbiopsiespriortomachineperfusionwerecollected,

fixed in4% formalin,andsubsequentlyembedded inparaffin.Bileduct slicesof4µm

werecutandstainedwithhematoxylin&eosin,andsubsequentlyexaminedusinglight

microscopy.Thebileductinjury(BDI)scorewasdeterminedinablindedfashionbytwo

independentresearchers,usingaclinicallyrelevanthistologicalgradingsystem.21,22The

BDIscorewasbasedonascaleof0to7andacut-offscoreof4.75wasusedtodefine

low and high BDI, as described previously.21 Both preclinical and clinical NMP

procedureswereperformedwith theLiverAssistdevice(OrganAssist,Groningen, the

Netherlands),withprotocolsandoutcomesreportedelsewhere.21,23,24Bilewascollected

from a 8Fr biliary drain in the common bile duct. During NMP, bile samples were

collectedevery30minutesundermineraloil todeterminebiliarypH,bicarbonateand

glucoseasdescribedpreviously.21,23,24

Subsequently, in a national retrospective cohort study all DCD liver transplantations

between 01-01-2010 and 01-01-2017 were included. Exclusion criteria were

retransplantation, pediatric liver transplantation (<18 years) and the use of

hypothermicmachineperfusion.Dataondonorandorganprocurementcharacteristics

wereobtained throughtheEurotransplantDonordatadatabase.Dataof recipientsand

transplantation outcomes were obtained from the databases of participating centers,

completedwithmissingdatafromtheelectronicalpatients’medicalrecords.Thisstudy

was approved by the Medical Ethical Committee of the University Medical Center

Groningen(METC.2017/504).

Definitions

Donor hematocrit, leukocyte and platelet countwere defined as the last known value

prior towithdrawal of life-support.Non-anastomotic strictures of the bile ductswere

definedasbiliarystricturesatanyplaceinthebiliarytreeexceptfortheanastomosisin

theabsenceofhepaticarterythrombosis.ToclassifyforNAS,patientswererequiredto

haveclinicalsymptomsofcholestasiswithsubsequentimagingdemonstratingbileduct

strictures within two years after DCD liver transplantation. The total donor warm

ischemiatimewasdefinedastheperiodfromcardiacarrestuptotheinitiationofcold

flushinginthedonor.Theagonalphaseandtheasystolephasecomprisedoftheperiod

betweenwithdrawaloflife-supportandcirculatoryarrest,andcirculatoryarresttocold

perfusion,respectively.Coldischemiatimewasdefinedasthetimebetweenthestartof

coldflushandthemomentthattheliveristakenoficeforimplantationintherecipient.

Recipientwarmischemiatimewasthetimebetweenremovaloftheliverfromiceuntil

eitherportalorarterialreperfusion,whichevercamefirst.

Donationaftercirculatorydeathprocedure

In theNetherlands,withdrawalof lifesupport isgenerallyperformedon the intensive

care unit. Cannulation prior to circulatory arrest is not performed, and systemic

heparinizationisprohibitedbyDutchlaw.Afteramandatory5-minutesnotouchperiod

followingcirculatoryarrest,thedonoristransportedtotheoperatingtheatre.Asuper-

rapid sterno-laparotomy followed by aortic-only perfusion with heparinized Belzer

University of Wisconsin (UW) cold storage solution (Bridge to Life, London, UK) is

performed. The common bile duct and intrahepatic biliary tree are flushed with low

pressureUWsolution.Afterhepatectomy,theliverisflushedwithat least500mlcold

UWpreservationsolutionthroughtheportalveinuntilcleareffluentisobserved.

Statisticalanalysis

Continuous variables were presented as median and interquartile range (IQR), and

difference between groups were tested using Mann-Whitney or Kruskal-Wallis test.

Categoricalvariableswerepresentedasnumber[percentage]anddifferencesbetween

groupsweretestedusingPearson’schi-squaretestorFisher’sexacttest.

In the histological analysis, logistic regression models were used to evaluate

independentriskfactorsforahighbileductinjuryscore.Variableswereincludedinthe

multivariatelogisticregressionanalysisifunivariateanalysesyieldedap-value<0.20.

ROCcurveswereformedtoidentifythemostsuitablecut-offvalues.

Intheretrospectivecohortstudy,univariateandmultivariateCoxProportional-Hazards

regression models were used to evaluate independent risk factors for NAS. Subjects

without the event were censored at two years post transplantation. Variables were

included in a multivariate Cox Proportional-Hazards regression analysis if univariate

analyses yielded a p-value < 0.20. The reported Hazard ratios (HR) for donor

hematocrit, leukocyte and platelet count refer to an increase of 1%, 1x109/L and

50x109/L, respectively. All tests had a two-sided design with a p-value below 0.05

considered as significant. All analyses were performed using SPSS version 24 (IBM,

Chicago,Illinois).

RESULTS

HistologicalanalysisofbileductbiopsiesfromDCDlivers

Of40 consecutivepreclinical and clinicalNMPprocedures, 39bileductbiopsieswere

obtained upon arrival in our center. In a multivariate logistic regression analysis

includingdonorageanddonorcauseofdeath,donorplateletcount (OR2.553,95%CI

1.082-6.021,p-value=0.032)andcoldischemiatime(OR2.251,95%CI1.192-4.251,p=

0.012) were identified as independent risk factors for high BDI, whereas donor

leukocytecount(OR0.728,95%CI0.572-0.926,p=0.010)wasassociatedwithlowBDI

(Table 1). Cut-off values of 181 x 109/L for platelets and 12.5 x 109/L for leukocytes

wereidentifiedusingROCcurves.Liversfromdonorswithplateletsbelow181x109/L

andleukocytesabove12.5x109/LrarelyhadahighBDI,whereasthecombinationofan

increaseddonorplateletcountwithlowleukocytesoftenresultedinahighBDI.(Figure

1).

Figure1:Impactofdonorleukocyteandplateletcountonbileductinjuryscoreof

donationaftercirculatorydeathlivers.Thecombinationofaplateletcountbelow181

x109/Landleukocytecount>12.5x109/Lappearedtobeprotectiveforbileduct injury.

GreencirclesindicateliverswithalowBDI,redtrianglesindicateliverswithahighBDI.

Table1:Univariateandmultivariatelogisticregressionanalysisforahighbile

ductinjuryscore.

Univariateanalysis MultivariateanalysisVariable OR 95%CI P-value OR 95%CI P-

valueDonor GenderMale(ref)Female

ref0.437

ref0.113-1.681

0.549ref0.228

Age(years) 1.041 0.980-1.105 0.191 NS BMI 1.065 0.956-1.186 0.253 CauseofdeathCVA(ref)TraumaAnoxia

ref0.0600.056

ref0.006-0.5740.005-0.669

ref0.0150.023

NS

Hematocrit(%) 1.009 0.913-1.116 0.857 Leukocytecount(per1x109/L)

0.924 0.823-1.036 0.176 0.734 0.581-0.927

0.009

Plateletcount(per50x109/L)

1.283 0.932-1.766 0.099 2.553 1.082-6.021

0.029

WarmischemiatimeAgonalphase(min)Aystolictime(min)

1.0090.9971.086

0.979-1.0400.963-1.0310.932-1.266

0.5650.8430.292

Coldischemiatime(hours)

1.006 1.000-1.011 0.043 1.675 1.093-2.566

0.013

Abbreviations:BMI;bodymassindex,CVA;cerebralvascularattack,OR;oddsratio.NSindicatesvariablewasremovedfromtheforwardLRmodel.

Bilecomposition

Duringthe40NMPprocedures, the liversofdonorswithaplateletcountbelow181x

109/Lshowedhigherbicarbonatelevelsinbile,andahigherbilepHcomparedtobileof

liverswithaplateletcountabove181x109/L.(Figure2A-B).Leukocytecountdidnot

significantlyassociatewithbicarbonatesecretion.During the first60minutesofNMP,

liverswithaleukocytecount>12.5x109/LhadalowerbilepH.

Figure2:CompositionofbileduringNMP.Platelet count significantly influences

biliarybicarbonatelevelsandbilepH.*indicatesasignificantdifferencebetweenthe

twogroupsatthattimepoint(p<0.050).

Retrospectivecohortstudy:baselinecharacteristics

Atotalof252DCD liver transplantationswereperformed in theNetherlandsbetween

01-01-2010 and01-01-2017.After applicationof the exclusion criteria, the remaining

235caseswereincludedinthisstudy.BaselinecharacteristicsarepresentedinTable2.

Actuarialone-andtwo-yeargraftsurvivalrateswere81%and75%,and90%and89%

forpatientsurvivalrespectively.

Table2:Baselinecharacteristicsofdonor,procurementandrecipient,and

outcomesaftertransplantation.

Variable Median(IQR)N=235

Donor Age(yrs) 48(35-54)GenderMaleFemale

140(59%)95(41%)

BodyMassIndex 24(22-26)Causeofdeath:TraumaAnoxiaCVAOther

57(24%)69(29%)90(38%)19(8%)

ALTpeak(u/L) 48(24-101)GGTpeak(u/L) 34(20-68)Hematocrit(%) 34(30-38)Plateletcount(x109/L) 182(145-244)Leukocytecount(x109/L) 12.5(9.4-17.6)Procurement Donorwarmischemiatime-Agonalphase(min)-Asystolictime(min)

15(11-20)15(13-18)

Recipient Age(yrs) 55(47-63)BMI 26(24-29)GenderMaleFemale

165(70%)70(30%)

LabMELDscore 15(10-21)Transplantation Coldischemiatime(min) 423(366-494)Secondwarmischemiatime(min)# 33(26-42)OutcomesafterDCD-LT Actuarialgraftsurvival 1year 81%2year 75%Actuarialpatientsurvival 1year 90%2year 89%Primarynon-function 3(1%)Non-anastomoticstrictures<2yearsofLT 61(26%)Hepaticarterythrombosis 15(6%)Intensivecareunitstay(days) 2(1-4)Totalhospitalstay(days) 16(13-26)ASTpeak(u/L) 2019(1105-4287)ALTpeak(u/L) 1502(719-2767)

Abbreviations:ALT;alanineaminotransferase,AST,aspartaneaminotransferase,CVA;

cerebralvascularattack,GGT;gammaglutamyltransferase,LT;livertransplantation,

MELD;modelend-stageliverdisease#thetimeframefromremovaloftheliverfromice

untilgraftreperfusion.

Retrospectivecohortstudy:non-anastomoticbiliarystrictures

Sixty-one (26%) patients developed NAS within two years of transplantation. The

resultsofunivariateanalysisareshowninTable3.InthemultivariateCoxProportional-

Hazardregressionmodels,donorhematocrit(HR1.044,95%CI1.005-1.083,p=0.025)

anddonorplateletcount(HR1.047,95%CI1.007-1.089,p=0.022)wereidentifiedas

significantindependentriskfactorsforthedevelopmentofNAS(Table4).Additionally,

donor age and cold ischemia time were significant independent risk factors for NAS.

DonorleukocytecountdidnotseemtoinfluenceratesofNASintheunivariateanalysis

(Table3).Inthedonorpopulationover40years,plateletsandhematocritwerestrong

predictors ofNASafterDCD liver transplantation (HR1.122, 95%CI1.013-1.241, p=

0.027 and HR 1.050, 95% CI 1.009-1.093, p = 0.017, respectively) whereas in the

population below40 years, no influencewas observed of platelets and hematocrit on

NAS(HR1.058,95%CI0.881-1.270p=0.574andHR0.988,95%CI0.911-1.071,p=

0.764,respectively).

Table3:UnivariateCoxRegressionanalysisforthedevelopmentofnon-anastomoticbiliarystrictureswithintwoyearsoftransplantation.

Variable HR 95%CI P-valueDonor Gender

Male(ref)Female

0.957

0.574-1.593

0.864

Age(years) 1.030 1.008-1.051 0.007 BMI 0.994 0.913-1.082 0.891 Causeofdeath

CVA(ref)TraumaAnoxiaOther

0.6640.4511.398

0.344-1.2830.225-0.9070.660-2.963

0.2230.0260.382

ALTpeak(u/L) 0.998 0.996-1.001 0.186 LastGGT(u/L) 1.000 0.998-1.003 0.866 Hematocrit(%) 1.051 1.013-1.090 0.024 Leukocytecount(x109/L) 1.020 0.980-1.062 0.340

Plateletcount(per50x109/L) 1.051 1.013-1.090 0.008Transplantation Warmischemiatime

Agonalphase(min)Aystolictime(min)

1.0171.004

0.961-1.0760.987-1.022

0.5620.650

Coldischemiatime(hours) 1.274 1.122-1.447 <0.001Recipient Age(years) 0.994 0.971-1.017 0.585 BMI 0.993 0.939-1.051 0.820 Lab-MELD 0.980 0.946-1.016 0.269 Warmischemiatime(min) 1.028 1.010-1.047 0.003

Abbreviations:ALT;alanineaminotransferase,BMI;bodymassindex,CVA;cerebral

vascularattack,GGT;gammaglutamyltransferase,MELD;modelend-stageliverdisease,

Table4:MultivariateCoxRegressionanalysisforthedevelopmentofnon-anastomoticbiliarystrictureswithintwoyearsoftransplantation.

Variable HR 95%CI P-valueDonor Age(years) 1.031 1.009-1.054 0.006 Causeofdeath

CVA(ref)TraumaAnoxiaOther

NS NS NS

ALTpeak(u/L) NS NS NS Hematocrit(%) 1.044 1.005-1.083 0.025 Plateletcount(per50x109/L) 1.047 1.007-1.089 0.022 Coldischemiatime(hours) 1.252 1.098-1.427 0.001Recipient Warmischemiatime(min) 1.031 1.010-1.053 0.004

NSindicatesvariablewasremovedfromtheforwardLRmodel.Abbreviations:ALT;

alanineaminotransferase,CVA;cerebrovascularattack.

DISCUSSION

Inthispaperwepresentdatasuggestingthatdonorplateletsinfluencebiliaryinjuryin

theprocessofDCD liver transplantation. In addition toplatelets, also leukocyte count

was inversely associated with biliary injury prior to graft reperfusion. Platelet count

and, additionally, hematocrit were significant independent risk factors for the

developmentofNASwithintwoyearsofDCDlivertransplantation.

Following theevidence thatbiliarycomplicationsarepredictedby theseverityofPBG

and PVP injury, several studies have been performed on the role of PBGs in liver

transplantation.25-28 As NAS is hypothesized to be a result of reduced regenerative

capacity from the PBGs after liver transplantation, regenerative medicine offers a

potential treatment for NAS.25 However, the pathophysiology of PVP injury in the

developmentofNAShashardlybeenstudied,whereasintegrityofthePVPismostlikely

requiredtoperformanyPBGregenerativetreatment.

To our knowledge, the involvement of platelets in ischemia-reperfusion injury of the

biliarytreehasnotbeenreportedbefore.AsCywesetal.andSindrametal.haveshown

before,plateletsplayanimportantroleinischemia-reperfusioninjuryoflivergrafts.14-16

Cywes et al have shown that activation of platelets increase the extent of reperfusion

injury,whereasSindrametalobservedthatplateletsinducesinusoidalendothelialcell

apoptosis upon reperfusion. Interestingly, the current study suggests that platelets

alreadyinfluencebiliaryinjurypriortograftreperfusion.Onepotentialexplanationfor

this is that during the hypoxic/ischemic period that DCD grafts encounter, severe

endothelialdysfunctionoccurs.

Vascular endothelial dysfunction is a characteristic of human ageing, displayed by

reduced nitric oxide bioavailability, shifts from predominantly vasodilator to

vasoconstrictor prostanoid production, and lastly an increased endothelin-1mediated

vasoconstriction.29-31 Especially this last characteristic offers a potential

pathophysiologicalexplanationforthe involvementofplatelets inbiliary injury.Under

hypoxicconditions,plateletsincreasinglyadheretoendothelialcells.32Inpatientswith

endothelial dysfunction, platelet-endothelium interaction leads to enhanced release of

endothelin-1, initiating vasoconstriction.31 Vasoconstriction during the agonal and

asystolicphasewould lead to insufficient flushingof thePVP.Tostudy this,we tested

whetherthepotentialimpactofplateletsonNASwasdifferentamongtheyoungerand

the older donor population, as endothelial function decreases over age. We did not

observe a significant influence of platelet count and hematocrit in DCD liver

transplantationwithdonoragebelow40years. Indonorsabove40years, therewasa

strong influence of platelets and hematocrit on NAS. Using a rat model. Tyagi and

colleagueshavedescribed thatacutesimulatedhypoxiaresults inadeceasedbleeding

propensity and increased platelet activity.31 In the same paper, inhibition of platelet

calpain activity demonstrated reversal of hypoxia-induced platelet hyperreactivity 31.

These findings offer a potential for a therapeutic intervention, however, in many

countriesadministrationofmedicationthatisnotintendedforstabilizingthedonorbut

focussesonpost-transplantoutcomes,isforbiddenbylaw.Inafewcountries,systemic

heparinizationpriortowithdrawaloflife-supportisallowed.Gaoetal.haveshownthat

heparin potentiates platelet activity33, and Hessheimer et al showed that heparin

administrationtoporcineliverdonorsimprovesoutcomeinDCDlivertransplantation.34 Itwouldprovidevaluable information toassess the influenceofplateletsonbiliary

injury,priortoreperfusion,aswellinthecountrieswereheparinizationisallowed.

Additionally, we observed that the donor leukocyte count was associated with the

development of biliary injury prior to NMP. Large amounts of research have been

performedontherecipient’sinnateimmuneresponsefollowingtransplantation,which

constitutes a large proportion of ischemia-reperfusion injury.35,36 Interestingly, now

donorleukocytesseemtoinfluencetheischemicinjuryofthebiliarytreeintheabsence

of recipient leukocytes, and high leukocyte counts seems to be protective for biliary

injury.However,thebeneficialeffectondonorleukocytesdisappeareduponreperfusion

ontheperfusiondeviceaswellas intherecipient.Unfortunately,donorreports inthe

Eurotransplant region only state the leukocyte count, and further information of

leukocytedifferentiationisnotavailable.ArecentpaperbyRichardsetal.demonstrated

that in older adults the peripheral vasodilation during systemic hypoxia is severely

impairedcomparedtoyoungercontrols.37Impairedvasodilationfollowingthehypoxia

andischemiaoftheagonalandasystolicperiodcouldleadto lessthoroughflushingof

themicrocirculationofalivergraft.Therefore,toexplaintheprotectiveeffectofahigh

leukocyte count on biliary injury prior to reperfusion, we hypothesize that a high

leukocyte count could contribute to local vasodilation, and thereby more efficient

flushingofthePVP.Themechanismofimpairedvasodilationinolderpeoplecouldalso

beanexplanationfortheobservationthattheinfluenceofhematocritandplateletswas

only observed in the donor population > 40 years. An interesting way to test the

hypothesis that impairedhypoxicvasodilationplaysa role in thedevelopmentofPVP

injury, would be to add a strong vasodilator to the flushing solution. Potentially, this

couldalsoleadtoareductionintheinfluenceofplateletsandhematocritonNAS,asthat

wasmainlyobservedinolderdonors.

Additionally,weobservedthatdonorhematocritwasassociatedwiththedevelopment

ofNASafterDCD-LT.Morariuetal.haveshownthatUniversityofWisconsin(UW)Cold

Storagesolutionpromotesseverehyperaggregationofredbloodcells(RBC’s)compared

toRBC innormalblood.38Ashematocrit increases, there isprogressiveaggregationof

RBC’s.39Themaximumobservedsizeoftheseaggregatesis4.332µm2,comparedto137

µm2 in normal blood samples without UW.38 The presence of these aggregates could

result in inferiormicrocirculation,andthereby leadingto injuryof thePVP.There isa

large size difference between these RBC aggregates and leukocytes, which have a

diameter averaging between 10 µm and 20µm.40 Therefore we hypothesize that

leukocyteswouldnotleadtomicrovascularobstruction,whereastheseRBCaggregates

could. However, it remains unclearwhy donor hematocrit influenced the rate of NAS

after transplantation,but that itdidnot seemto influencehistologicalbileduct injury

andbileproductionduringNMP.Anotherhypothesisfortheinvolvementofhematocrit

inbiliaryinjuryisthatduringhypoxicconditionserythrocytesexcretereactiveoxygen

species (ROS). ROS cause severe injury following graft reperfusion in transplantation,

whichwould explain that the impact of hematocrit on biliary injury is only observed

after transplantation. Consequently, a higher hematocrit would thereby lead to an

increasedROSexcretion.

An interesting aspect of this study is that we suggested new factors involved in the

pathophysiologyofpost-transplantcholangiopathy.Wedobelievethatastrongpointof

this work is that histology, bile analysis and a retrospective database analysis are

combinedtothoroughlyinvestigatetheinfluenceofdonorcellularbloodcompositionon

biliaryinjuryduringDCDlivertransplantation.Anotherstrongaspectistheprospective

collection of biopsies and bile for other protocols. We are interested whether the

involvementofplatelets,leukocytesandhematocritonischemia-reperfusioninjurycan

alsobefoundintransplantationofotherorgans.Thisstudyprovidesarationaleforthe

usageofnormothermicregionalperfusion(NRP),asNRPallowsforrapidrestorationof

(microcirculatory)bloodflowfollowingthedonorwarmischemiatime.A limitationof

this study is the retrospective character of the database study, and its limited size.

Moreover,thenumbersinthehistologyandbilecompositionstudyarerelativelysmall.

In conclusion, this study shows that the cellular blood composition of the donor

influencesbiliaryinjuryduringDCDlivertransplantation.Inahistologicalanalysisprior

toreperfusion,donorleukocyteandplateletcountwereassociatedwithbileductinjury.

Plateletcount>181x109/Lalsohadanegativeimpactonbiliaryepithelialcellfunction

markersduringNMP,suchasbiliarybicarbonatesecretionandbilepH. Inaccordance

withthis,donorhematocritandplateletcountweresignificantindependentriskfactors

forthedevelopmentofNASafterDCDlivertransplantation.

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Selectedlivergraftsfromdonationaftercirculatorydeathcanbesafelyusedforretransplantation

MarjoleinvanReeven,OttoB.vanLeeuwen,SarwaDarwishMurad,AadP.vandenBerg,BartvanHoek,IanP.J.Alwayn,WojciechG.Polak,RobertJ.Porte.Transpl.Int.2020(inpress)

5

ABSTRACTBackground and aims: Due to the growing number of liver transplantations (LTs),

there is an increasingnumberof patients requiring retransplantation (reLT).There is

littledataontheuseofgraftsfromextendedcriteriadonors(ECD),especiallydonation

after circulatory death (DCD), for reLT. We aimed to assess the outcome of patients

undergoingreLTusingaDCDgraftintheNetherlandsbetween2001and2017.

Materialandmethods:EachDCD-reLTwasmatchedwithtwoDBD-reLTcases,based

onnumberofsuccessivereLT,BAR-score,intervalbetweenreLTandpriorLTandyear

ofreLT.Primaryoutcomeswerepatientandgraftsurvival,calculatedviaKaplanMeier

estimator.NineteenDCD-reLTwerecomparedwith38matchedDBD-reLTs.

Results:DonorsintheDCD-reLTgroupweresignificantlyyounger(38versus54years,

p<0.001)andhadalowerBMI(22versus25kg/m2,p=0.003).Comparisonofrecipient

demographics and ischemia times yielded no significant differences. Patient and graft

survivalrateswerecomparablebetweenthetwogroups.Furthermore, theoccurrence

ofnon-anastomoticstrictureswithinthefirstyearafterreLTdidnotdiffersignificantly

betweenthetwogroups(26.3%versus10.5%,p=0.143).

Conclusions: reLTwithDCD grafts does not result in inferior outcomes compared to

DBD grafts in selected patients. Therefore, DCD liver grafts should not routinely be

declinedforpatientsawaitingreLT.

INTRODUCTION

Liver transplantation(LT) is thebest treatmentoption forpatientssuffering fromend

stageliverdisease.Toexpandthedonorpoolintimesofincreasingdemand,theuseof

graftsfromextendedcriteriadonors(ECD)hasincreasedsubstantially,ofwhichgrafts

fromdonationaftercirculatorydeath(DCD)isamainparameter.1In2018,aDCDgraft

wasusedin38%and9%ofalldeceaseddonorLTintheNetherlandsandUnitedStates

of America, respectively.2,3 In the United Kingdom, 26% of deceased donor LT were

executedwithDCDgrafts(between01.04.2017and31.03.2018).4

LivertransplantationwithDCDgrafts(DCD-LT)isconsideredtobeinferiorcomparedto

DBD-LT,due to the increased riskof complications suchasearlyallograftdysfunction

(EAD) and biliary complications.5-8 Among biliary complications, non-anastomotic

strictures (NAS) are the most feared complications as they often require multiple

interventions for biliary drainage, are largely irreversible and are known to have a

negative impact on recipient andgraft survival.9The incidenceofNAS, alsoknownas

ischemic cholangiopathy (IC) or Ischemic Type Biliary Lesions (ITBL), after DCD-LT

variesbetween3and39%.6

Since the use of grafts frommarginal donors has increased, it is assumed that more

recipients will suffer from a suboptimal graft. Furthermore, due to improvements in

surgical techniques, post-operative care and immunosuppressive regimes, the short-

term survival after LT has improved significantly,10 resulting in a larger population

surviving long enough to develop late graft failure. A retransplantation of the liver

(reLT)iscurrentlytheonlydefinitivetreatmentforallograftfailure.However,itiswell

known that reLT is associated with inferior patient and graft survival compared to

primaryLT.11,12

Despite DCD liver grafts being widely accepted, transplant physicians and surgeons

tend to avoid the use of DCD grafts for reLT. However, since in some countries the

availability ofDBD grafts has decreased,13 thewaiting time for an optimal, preferably

DBDlivertobecomeavailable forareLTcandidatecouldbetoo longwithsubsequent

riskofdeteriorationofthepatient’scondition,makinghimorherineligibleforreLT.

ThereisverylittlereportedontheuseofDCDgraftsforpatientsrequiringareLT.Only

onestudyhasassessedtheoutcomesoftenpatientsundergoingreLTusingDCDgrafts.14

TheauthorsconcludedthattheuseofDCDgraftshouldbeavoidediftherecipienthasa

moderate to highModel for End-Stage Liver Disease (MELD) score. Unfortunately, no

comparison was made with reLT using DBD grafts. Since DCD-LT is common in the

Netherlands,andreLTisnotanofficialcontra-indicationfortheuseofaDCDliver,we

aimedtocomparetheoutcomesofreLTwithDCDgraftsintheNetherlandswiththatof

matchedDBDcases.

MATERIALS&METHODS

Inthismulticenterretrospectivestudy,allpatientswhounderwentare-LTusingaDCD

liver graft (DCD-reLT) in one of the three liver transplant centers in the Netherlands

from the beginning of the DCD-LT program in 2001 until January 1st 2017, were

included. Pediatric LT (<18 years), multi-organ transplantation and grafts preserved

with machine perfusion were excluded. A matched control group of DBD

retransplantations (DBD-reLT) was formed. For each DCD-reLT, two DBD-reLT were

selectedbasedonthefollowingmatchingcriteria(inorderof importance):Transplant

center, Balance of Risk score (BAR),15 number of consecutive reLT, year of reLT and

intervalbetweenpriorLTandreLT.Thislattermatchingcriterionwaschosensincean

earlyreLT(withinthreemonths), isontheonehandtechnically lesschallenging than

latereLT(easierhepatectomywithlessadhesions),butontheotherhandisperformed

inpatientswhomaybesickerpre-reLTthanpatientsundergoinga latereLT.16,17Data

on donor and organ procurement characteristics were obtained through the

Eurotransplant Donordata database. Recipient data and data on follow-up were

collected from prospective maintained databases and patients’ electronic medical

records.ThestudyhasbeenapprovedbytheInstitutionalReviewBoardoftheErasmus

MCUniversityMedicalCenterRotterdam(MEC-2019-0316).

In all DCD organ procurements in the Netherlands, withdrawal of life support takes

placeattheICUorregularward.Aftercirculatoryarrestamandatorynotouchperiodof

fiveminutesiscarriedoutafterwhichthedonoristransportedtotheoperatingtheatre.

AsdescribedintheNationalProtocolPost-mortemDonorOrganProcurement,asuper-

rapidretrievaltechniqueisusedinDCDdonorstominimizethedonorwarmischemia

time.Aftercannulationofaortaandinferiorvenacava,coldperfusionwithUniversityof

Wisconsin (UW) solution is started.18 Since pre-mortem administration of heparin is

prohibitedby law,heparin isaddedtotheperfusionsolution.Thestandardmethodof

implantation is with a piggyback caval vein anastomosis, an end-to-end arterial and

portalanastomosis,andaduct-to-ductbiliaryanastomosis.

Thetotaldonorwarmischemiatime(dWIT)wasdefinedastimebetweenwithdrawalof

life supporting treatmentandstartof coldperfusion.Thedefinitionofasystolicdonor

warm ischemia timewas the timebetween circulatory arrest and cold perfusion. The

cold ischemia time (CIT) was defined as the period between the start of the cold

perfusion in the donor and the removal of the liver from ice during the recipient

procedure.Thedefinitionofrecipientwarmischemiatime(rWIT)usedinthisstudyis

theintervalbetweenremovaloftheliverfromiceandgraftreperfusion(i.e.inthevast

majorityportalreperfusion).

The primary outcomemeasures of this studywere patient and graft survival. Patient

survivalwasdefinedastimebetweenreLTanddeath,withorwithoutfunctioninggraft.

Graft survival was calculated as time between the reLT and patient death (with or

withoutfunctioninggraft)orasuccessiveretransplantation.Secondaryoutcomeswere

incidenceofbileleakageandanastomoticstricturesaswellasthedevelopmentofNAS

withinthe firstyearaftertransplantation.NASwasdefinedasanystrictureof thebile

duct except those localized near the biliary anastomosis and in absence of an hepatic

arterythrombosis.

Continuousdatawerepresentedasmedianandinterquartilerange(IQR)andcompared

with the Mann-Whitney U test. Categorical variables were presented as number and

percentages and comparedwith the Pearson chi-square test or the Fisher’s exact test

where appropriate. Survival analyses was conducted using the Kaplan Meier method

and comparisonsweremadewith the log-rank test. All testwere two-sidedwith a p-

valuebelow0.05consideredassignificant.Thestatisticalanalyseswasperformedusing

SPSSversion24(IBM,Chicago,Illinois,UnitedStatesofAmerica).

RESULTS

Between the start of the DCD-LT program in 2001 and January 1st 2017, 19 patients

underwent a retransplantation with a DCD liver. These caseswerematchedwith 38

DBD-reLTcases.Donorand recipientdemographicsaregiven inTable1.Therewasa

male predominance in both donor groups (DCD-reLT: 52.6%, DBD-reLT: 57.9%).

Compared to DBD-reLT donors, DCD-reLT donors were significantly younger (38.0

versus53.5years,p<0.001)andhadalowerBMI(22.0versus24.5kg/m2,p=0.003).

Furthermore,DCDdonorshadmoreoftendiedfromtraumawhereasDBDdonorsdied

more from cerebrovascular accidents. In DCD-reLT themedian asystolic donorwarm

ischemiatimewas15minutes(11-18minutes)whereasthetotaldWITwas28minutes

(22–32minutes).

Themajorityof the recipientswasmale,with amedianageof51.0 years (IQR,48.0 -

58.0years)intheDCD-reLTgroupand49.5years(IQR,42.5–59.3years)intheDBD-

reLT group (p = 0.72). The most common indication for reLT was (ischemic)

cholangiopathy(42%intheDCD-reLTgroup,53%intheDBD-reLTgroup),followedby

primarynon-functionandrecurrenceof theprimarydisease.Themajorityof thereLT

were of the late type, being performed at least threemonths after the prior LT. The

medianlaboratoryMELDscoreattimeofreLTwassubstantiallylowerintheDCD-reLT

groupcomparedtotheDBD-reLTgroup(18versus26),howeverthisdifferencewasnot

statisticallysignificant(p=0.417).

Table1:Donorandrecipientdemographics

TotalgroupN=57

DCD-reLTN=19

DBD-reLTN=38

P-value

Donor Male 32(56.1) 10(52.6) 22(57.9) 0.71Age(years) 50.0

(26.5-57.0)38.0(20.0-45.0)

53.5(39.0-59.0)

<0.001

BMI(kg/m2) 24.0(22.0-26.0)

22.0(19.0-24.0)

24.5(22.0-28.0)

0.003

CauseofdeathTraumaCVAAnoxiaOther

36(63.2%)11(19.3%)9(15.8%)1(1.8%)

7(36.8%)6(31.6%)5(26.3%)1(5.3%)

29(76.3%)5(13.2%)4(10.5)0

0.03

Lastγ-GT(U/L) 29.0(16.5-42.0)

30.0(18.0-36.0)

27.5(14.8-43.3)

0.93

LastALT(U/L) 26.0(14.0-45.0)

23.0(15.0-46.0)

27.5(14.0-44.8)

0.83

AsystolicdWIT(minutes)† n/a 15.0(11.0-18.0)

n/a n/a

TotaldWIT(minutes)‡ n/a 28.0(22.3-31.8)

n/a n/a

Recipient Male 33(57.9) 11(57.9) 22(57.9) 0.999Age(years) 51.0

(44.0-59.0)51.0(48.0-58.0)

49.5(42.5-59.3)

0.72

BMI(kg/m2) 22.8(20.7-26.5)

22.5(21.3-26.5)

23.3(19.7-26.8)

0.87

LaboratoryMELDscore 22.5(10.0-28.3)

17.5(10.3-24.0)

25.5(10.0-29.0)

0.42

IndicationforreLTPNFVascular(e.g.HAT/PVT)Biliary(e.g.ITBL)RecurrentprimarydiseaseOther

12(21.1)6(10.5)28(49.1)7(12.3)4(7.0)

3(15.8)3(15.8)8(42.1)3(15.8)2(10.5)

9(23.7)3(7.9)20(52.6)4(10.5)2(5.3)

0.70

NumberofreLT123

47(82.5)8(14.0)2(3.5)

16(84.2)2(10.5)1(5.3)

31(81.6)6(15.8)1(5.3)

0.78

TypeofreLTEarlyreLT(<3months)LatereLT(>3months)

18(31.6)39(68.4)

4(21.1)15(78.9)

14(36.8)24(63.2)

0.23

TimebetweenreLTandpriorLT(days)

617(55-3549)

775(239-3903)

326(12-3158)

0.41

GrafttypeofpriorLTDBDgraftDCDgraft

42(73.7)15(26.3)

13(68.4)6(31.6)

29(76.3)9(23.7)

0.52

Dataareshownasmedian(IQR)andfrequency(proportion).†:AsystolicdWITisdefined

asthetimebetweencirculatoryarrestandstartofcoldperfusion.‡:TotaldWITisdefined

astimebetweenwithdrawaloflifesupportingtreatmentandcoldperfusion.ALT:Alanine

transaminase,BMI:BodyMassIndex,CVA:Cerebrovascularaccident,DBD:Donationafter

BrainDeath,DCD:DonationafterCirculatoryDeath,dWIT:donorWarmIschemiaTime,γ-

GT:Gamma-glutamyltransferase,LT:livertransplantation,MELD:ModelforEndstage

LiverDisease,reLT:liverretransplantation

Table2 showsoperativedataaswell asdataon thepost-operativeoutcomes.Neither

the cold ischemia time (CIT) nor the recipient warm ischemia time (rWIT) differed

significantlybetweenthetwogroups.However,atrendtowardssignificancewasfound

in perioperative blood loss (4319 milliliters in the DCD-reLT group versus 2500

millilitersintheDBD-reLTgroup,p=0.07).Patientsweredischargedfromthehospital

afteramedianof25daysintheDCD-reLTgroupand20daysintheDBD-reLTgroup(p

=0.37).

Table2:Surgicalandpost-operativedemographics

TotalgroupN=57

DCD-reLTN=19

DBD-reLTN=38

P-value

Operation

rWIT(minutes) 39.0(34.0-46.0)

43.0(35.0-48.0)

37.5(32.5-43.0)

0.18

CIT(minutes) 452(395-525)

440(374-517)

454(400-528)

0.59

Bloodloss(ml)† 2800(2150-7000)

4319(2675-8125)

2500(1500-5875)

0.07

BARscore 12.0(7.3-17.0)

11.0(8.0-18.0)

12.0(7.0-17.0)

0.86

Post-operativeoutcomes

ICUstay(days) 3.0(2.0-9.0)

2.0(2.0-5.0)

3.5(2.0-9.3)

0.37

Hospitalstay(days) 23.0(13.5-34.5)

25.0(14.0-34.0)

19.5(13.0-40.3)

0.37

PeakALTwithin1stweek 1217(613-2499)

1238(582-2522)

1159(634-2461)

0.82

Bileleak 6(10.5) 2(10.5) 4(10.5) 0.999

Anastomoticstrictures 13(22.8) 4(21.1) 9(23.7) 0.999

Non-anastomoticstrictures‡

9(15.8) 5(26.3) 4(10.5) 0.143

Death 14(24.6) 5(26.3) 9(23.7) 0.999

Retransplantation 4(7.0) 1(5.3) 3(7.9) 0.999

Dataareshownasmedian(IQR)andfrequency(proportion).†:Proportionofmissingdata

forthisvariableis19.3%.‡:incidencewithinfirstyearafterreLT.ALT:Alanine

transaminase,BAR:Balanceofrisk,CIT:ColdIschemiaTime,DBD:DonationafterBrain

Death,DCD:DonationafterCirculatoryDeath,ICU:IntensiveCareUnit,LT:liver

transplantation,MELD:ModelforEndstageLiverDisease,reLT:liverretransplantation,

rWIT:recipientWarmIschemiaTime

Survivalrates

Themedianfollow-upofthetotalcohortwas5.35years(IQR,1.82-9.26years).The30

days, 1-year, 5-year and 10-year recipient survival in the DCD-reLT group was 95%,

79%,79%and79% respectively, compared to 87%,84%,78%and78% in theDBD-

reLT group (p =0.915, Figure 1). The most common cause of death after reLT was

infection(Table3).

Figure 1: KaplanMeier curve of patient survival after DCD-reLT andDBD-reLT.

Patient survival isdefinedasdeath (withorwithout functioninggraft). DBD-reLT: liver

retransplantation with graft from donation after brain death. DCD-reLT: liver

retransplantationwithgraftfromdonationaftercirculatorydeath

Patient survival

0 2 4 6 8 100

20

40

60

80

100DCD-reLTDBD-reLT

Log Rank: p=0.915

Number entering interval 0 2 4 6 8 10

DCD-reLT 19 12 11 8 7 5

DBD-reLT 38 30 23 17 11 6

Years

Cum

ulat

ive

surv

ival

Table3:Causesofdeath

Patient Grafttype IntervalbetweenreLTanddeath(days)

Causeofdeath

1. DCD-reLT 1 Myocardialinfarctioninsepticpatient

2. DBD-reLT 9 Multipleorganfailureinsepticpatient

3. DBD-reLT 12 Progressiveliverfailure,probablyasresultofhypoperfusion

4. DBD-reLT 13 Multipleorganfailureinpatientwithimmunethrombocytopenicpurpura(transmittedfromdonor)andportalveinthrombosis

5. DBD-reLT 13 Neurologicalinfaustprognosisafterintra-operativeexsanguination

6. DBD-reLT 14 Multipleorganfailureinsepticpatient

7. DCD-reLT 129 Multipleorganfailureinsepticpatient

8. DCD-reLT 129 Recurrentgiantcellhepatitis

9. DBD-reLT 174 Criticalillnessneuropathy

10. DCD-reLT 205 PseudomonasinfectioninpatientwithrecurrenthepatitisCinfection

11. DBD-reLT 1333 Recurrentperitonitisduetomultidrugresistantbacteria.Patientwishednofurthertreatment

12. DBD-reLT 1405 Pulmonaryhypertensionandmesenterialthrombosis.Patientwishednofurthertreatment

13. DBD-reLT 3994 Bacterialperitonitisincirrhoticliver.Patientwishednofurthertreatment

14. DCD-reLT 4941 Recurrentlivercirrhosis,decompensatedaftersurgeryforhipfracture

The30days,1-year,5-yearand10-yeargraftsurvivalwas95%,79%,79%and79%for

theDCD-reLTgroupand84%,79%,70%and70%intheDBD-reLTgroup(p=0.603)

(Figure 2). Four patients needed a subsequent retransplantation: three because of

biliary complications (one in theDCD-reLT group and two in theDBD-reLT group, of

whichonecasewasaccompaniedbyrecurrenceofPSC)andonepatientintheDBD-reLT

groupduetoanearlyhepaticarterythrombosis.

Figure 2: Kaplan Meier curve of graft survival after DCD-reLT and DBD-reLT.

Graft survival is defined as death (with or without functioning graft) or consecutive

retransplantation.DBD-reLT:liverretransplantationwithgraftfromdonationafterbrain

death.DCD-reLT:liverretransplantationwithgraftfromdonationaftercirculatorydeath

Biliarycomplications

In total, 10.5%of the recipients in both groups had a bile leakage. Furthermore, four

recipients in the DCD-reLT group (21.1%) and nine in the DBD-reLT group (23.7%)

developedananastomoticstricture(p=0.999).Theproportionofrecipientsdeveloping

NAS within the first year after reLT was substantially higher in the DCD-reLT group

(26.3 % versus 10.5%), however this difference was not statistically significant (p =

0.143).ThemediantimeintervalbetweenreLTanddiagnosisofNASwas204days(IQR

118–343days).

Graft survival

0 2 4 6 8 100

20

40

60

80

100

Log Rank: p=0.603

DCD-reLTDBD-reLT

Number entering interval 0 2 4 6 8 10

DCD-reLT 19 12 11 8 7 5

DBD-reLT 38 28 21 15 10 5

Years

Cum

ulat

ive

surv

ival

DISCUSSION

TherelativeshortageofavailablelivergraftshasledtoamorewidespreaduseofDCD

grafts.However,theoutcomesafterreLTwithaDCDgrafthaverarelybeenreportedin

literature.This is the firststudytoanalyzetheoutcomesafterDCD-reLTandcompare

thesewithoutcomesaftermatchedDBD-reLT.OurresultssuggestthatreLTwithaDCD

graft in selected recipients does not result in inferior outcome when compared to

matchedDBD-reLTs.

OursurvivalratesafterDCD-reLTaresubstantiallyhigherthanpresentedinaprevious

study on DCD-reLT performed by Perry et al. in 2011.14 This could be due to the

substantiallowerMELDscoreinourpopulation(medianof17.5versusamedianof27.0

reportedbyPerryetal.).Unfortunately,itisunclearwhetherinthestudybyPerryetal.

theMELDscoreincluded(non)standardexceptionpoints.Sinceourmedianlaboratory

MELDscoreisthatmuchlower,weareunabletorefuteorendorsetheconclusionfrom

Perry et al. that the use of DCD grafts should be avoided in high MELD recipients

awaiting reLT. However, a recent published study by Taylor et al. concluded that

acceptingaDCDgrafthasasurvivaladvantageoverwaitingforaDBDliver,especiallyin

recipients with a high MELD score.19 As this study only included first-transplant

recipients,itisdoubtfulwhethertheconclusionsmadebyTaylorandcolleaguescanbe

extrapolatedtothefieldof liverretransplantation.Basedonourresults, it is indicated

that at least in recipientswith a low-moderateMELD score the use of a DCD graft is

justifiableforreLT.

Thesignificantdifference indonoragebetweentheDCD-reLTandDBD-reLTgroup in

this study is attributable to the strict upper age limit for DCD donors that was

maintained in the Netherlands until recently and to the individual choice of the

transplant surgeon or physician to accept only the best DCD livers (i.e. DCD grafts

withoutotherextendedcriteriaandrelevantcomorbidities) for the indicationofreLT.

This latter isprobablythecase inmosttransplantprogramsperformingDCD-reLT.To

assesswhethertheexcellentsurvivalratesintheDCD-reLTgroupcouldbeattributedto

the relatively lowdonor age in this group, donor agewas tested in anunivariate Cox

Proportional-Hazardsregressionmodelforbothrecipientandgraftsurvival,yieldingno

significantresult(datanotshown).Thisisinlinewithliteraturethatagealonedoesnot

impair survival outcomes after DCD-LT.20,21 Since none of the matching variables

differedsignificantlybetweenthetwogroups,itishighlyunlikelythatthedifferencein

donorageistheresultofincorrectmatching.

The difference in donor cause of death and donor BMI between the two groups are

probably theresultof the lowerage in theDCD-reLTgroup;youngerdonorsdiemore

often from trauma, whereas older donors die predominantly from cerebrovascular

accidents,andyoungerdonorstendtohavealowerBMI.22-24However,thelowerdonor

BMIintheDCD-reLTgroupcouldalsobeduetoselectionbytransplantphysiciansand

surgeons. Since there seems to be some association between BMI and degree of

steatosis,aknownriskfactorforpooroutcomeafterLT,25,26thelowerBMIintheDCD-

reLTgroupcouldtosomeextenthaveinfluencedtheoutcomeinthisgroup.

WhencomparedwithDBDgrafts,livertransplantationwithDCDgraftsaregenerallyat

higher risk of developing biliary complications post-transplantation, especiallyNAS.A

similartrend,albeitnon-significantcanbeseeninthecurrentstudy.Itisexpectedthat

withanincreasedsamplesize,thisdifferencewouldhavebecomestatisticallysignificant

between the DCD-reLT and DBD-reLT group. Surprisingly, the incidence of NAS after

especiallyDBD-reLTinthecurrentstudyishigherthanexpected.Therecouldbeseveral

explanationsforthis.First,thehighNASincidenceintheDBD-cohortcouldbetheresult

of thematching.Furthermore,untilrecentlythedonorhepatectomytime(i.e. thetime

betweenthestartofcoldperfusioninthedonorandtheliverbeingstoredonice)was

relatively long in theNetherlands.Researchhas shown that aprolongedhepatectomy

timeisariskfactorforthedevelopmentofNAS.27,28Finally,thehighincidencesofNAS

inthisreLTcohortcouldalsoimplythatareLT,independentofgrafttype,hasahigher

riskofdevelopingpost-operativeNAS.Unfortunately,literatureonthistopicislacking.

Lastly, it should be noted that of the nine patients who developed NAS, only three

requiredanewtransplant,indicatingthatNASinthisgroupofpatientswasrathermild

andcouldbetreatedconservatively.

With the renewed interest in theuseofDCDgrafts,webelieve that the resultsof our

study are very relevant for further practice in these centers. With careful selection,

recipientandgraftsurvivalafterDCD-reLTappearsimilartothesurvivalinDBD-reLT.

Therefore, grafts for reLTshouldnotbe rejectedbasedon theDCDstatusalonebuta

carefulassessmentofadditionaldonor factors isneededforacase-by-casedecisionto

use these grafts. Furthermore,making use of DCD donors for reLTmay facilitate the

currentethicaldebateregardingreLT.Thatis,iftransplantsurgeonsandphysicianswill

accept DCD grafts for retransplantation, more DBD grafts will remain available for

recipientsonthewaitinglistawaitingtheirfirsttransplant.Atthesametime,expansion

ofthedonorpoolwithDCDdonorswillresultinmoreexpeditedreLTforthoseinneed.

Finally, with the emerging technologies in the field of machine perfusion, it can be

anticipatedthat thequalityofDCDgraftscanbe improved,resulting inamongothera

decreasedincidenceofischemiccholangiopathy.29-31

OnestrengthofthisstudyisthecomparisonofoutcomeafterDCD-reLTwithamatched

controlgroupofDBD-reLTcases.Thishasmadeapropercomparisonofthetwogroups

possible, fromwhich itcanbeconcludedthatsurvivalafterDCD-reLTisundercertain

circumstances similar to that after DBD-reLT. This study also has several limitations.

Firstly,wehadtodefinedonorwarmischemiatimeastimebetweenwithdrawaloflife

supportandcoldperfusion.Wewereunabletocalculatethemoreimportantfunctional

warmischemiatimeinthedonorsincedataonhemodynamicstatusduringtheagonal

phase is lacking or improperly recorded. Furthermore, the study had a retrospective

design,whichispronetobiasandconfounding.Finally,thesamplesizeofthisstudyis

relatively small,whichmade detailed statistical analysis such asmultivariate analysis

impossible.

In conclusion, liver retransplantationwith a DCD graft can yield similar outcomes as

liver retransplantation with donation after brain death. Therefore, DCD status itself

should not preclude the use of such donors in patients awaiting retransplantation.

However, careful selection of the offered DCD livers probably remains mandatory.

Largerstudiesareneededareneededtoconfirmourresults.

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21. Schlegel A, Scalera I, Perera M, Kalisvaart M, Mergental H, Mirza DF, et al.Impactofdonorageindonationaftercirculatorydeathlivertransplantation:Isthecutoff"60"stillofrelevance?LiverTranspl.2018;24(3):352-62.

22. DariusT,MonbaliuD,JochmansI,MeurisseN,DesschansB,CoosemansW,etal.Septuagenarianandoctogenariandonorsprovideexcellentlivergraftsfortransplantation.TransplantProc.2012;44(9):2861-7.

23. SinghalAK,ShengX,DrakosSG,Stehlik J. Impactofdonorcauseofdeathontransplant outcomes: UNOS registry analysis. Transplant Proc.2009;41(9):3539-44.

24. Rabelo AV, Bastante MD, Raya AM, Méndez CSM, Ramirez ARG, Suarez YF.Liver Transplantation Results by Donor Age. Transplantation Proceedings.2016;48(9):2994-6.

25. GabaRC,KnuttinenMG,BrodskyTR,PalestrantS,OmeneBO,OwensCA,etal.Hepaticsteatosis:correlationsofbodymassindex,CTfatmeasurements,andliverdensitywithbiopsyresults.DiagnIntervRadiol.2012;18(3):282-7.

26. WuC,LuC,XuC.Short-termandlong-termoutcomesoflivertransplantationusing moderately and severely steatotic donor livers: A systematic review.Medicine(Baltimore).2018;97(35):e12026.

27. VanReevenM,VanLeeuwenOB,FujiyoshiM,DeKleineRHJ,DeBoerMT,DeJonge J, et al. Donor hepatectomy time in donation after circulatory deathdonorsisanindependentriskfactorfordevelopmentofbiliarystricturesandearlygraftlossaftertransplantation.ILTSAnnualCongres.Lisbon;2018.

28. Gilbo N, Fieuws S, Meurisse M, Pirenne J, Jochmans I, Monbaliu D. Donorhepatectomytimeandimplantationtimeincreasetheriskofnon-anastomoticbiliarystricturesandallograftdysfunctionafter livertransplantation. ESOT.Copenhagen;2019.

29. DutkowskiP,PolakWG,MuiesanP,SchlegelA,VerhoevenCJ,ScaleraI,etal.First Comparison of Hypothermic Oxygenated PErfusion Versus Static ColdStorage of Human Donation After Cardiac Death Liver Transplants: AnInternational-matched Case Analysis. Ann Surg. 2015;262(5):764-70;discussion70-1.

30. van Rijn R, KarimianN,Matton APM, Burlage LC,WesterkampAC, van denBerg AP, et al. Dual hypothermic oxygenated machine perfusion in livertransplantsdonatedaftercirculatorydeath.BrJSurg.2017;104(7):907-17.

31. Boteon YL, Boteon AP, Attard J,Wallace L, Bhogal RH, Afford SC. Impact ofmachine perfusion of the liver on post-transplant biliary complications: Asystematicreview.WorldJTransplant.2018;8(6):220-31.

PartII:Transplantationofsuboptimaldonorlivers:expandingtheboundaries

Hypothermicoxygenatedmachineperfusionreducesbileductreperfusioninjuryaftertransplantationofdonationaftercirculatorydeathlivers

RiannevanRijn,OttoB.vanLeeuwen,AlixP.M.Matton,LauraC.Burlage,MariusC.vandenHeuvel,RubenH.J.deKleine,MariekeT.deBoer,AnnetteS.H.Gouw,RobertJ.Porte.LiverTranspl.2018May;24(5):655-664

6

INTRODUCTION

Theworldwideshortageofdonorliversfortransplantationhasledtoeffortstoincrease

thenumberofavailablegrafts.IncountriessuchastheNetherlands,SpainandtheUK,

thishasledtoamorefrequentuseofdonationaftercirculatorydeath(DCD)liversfor

transplantation.1,2Unfortunately,DCDlivergraftshavea3-foldhigherriskofdeveloping

non-anastomotic biliary strictures (NAS) after transplantation, compared to donation

afterbraindeath(DBD)livergrafts.ThereportedincidenceofNASrangesbetween16-

31% in DCD versus 3-13% in DBD liver grafts.3-5 This type of biliary complication is

regarded as amajor complication after DCD liver transplantation as it often requires

multiple endoscopic interventions and leads to re-transplantation in 16% of patients

anddeathin6%.6,7

Although the etiology of NAS is not fully understood, the duration of cold andwarm

ischemiaduringtransplantationhasbeenrecognizedasamajorriskfactorforNAS.8-10

Ischemic conditions lead to a complex cascade of events resulting in ischemia-

reperfusion(IR)injury.11Threeindependentclinicalstudiesrecentlydemonstratedthat

themajorityofdonor livershavehistologicalevidenceofextensivebiliary IR injuryat

the time of transplantation.12-14 Especially the degree of biliary epithelial loss, mural

necrosis,andinjuryofthedeepperibiliaryglands(PBG)andperibiliaryvascularplexus

(PVP)at thetimeof transplantationhasbeenassociatedwiththedevelopmentofNAS

after transplantation.12-14 The PBG contain biliary stem/progenitor cells that are

involvedintheregenerationandrepairofthebileductepitheliumaftersevereinjury.15-

17 Therefore, it has been hypothesized that reduced regenerative capacity of the bile

ductsduetodamageofthePBGplaysanimportantroleinthedevelopmentofNASafter

transplantation.14,18

Ashortperiodofhypothermicmachineperfusion(HMP)afterconventionalstaticcold

storage(SCS)hasbeenshowntoreduceIRinjuryofdonorlivers.19,20End-ischemicHMP

results inareductionofhepatocyteapoptosisandnecrosis,mitochondrialandnuclear

injury, endothelial injury, Kupffer cell activation, and the subsequent host immune

response.19,23Furthermore,animalstudieshavesuggestedthatHMPreducesIRinjuryof

thebileducts,asindicatedbyanimprovedbiliaryepithelialcellfunction,reducedbiliary

injury markers, and less histological bile duct wall necrosis, epithelial cell loss, and

arteriolonecrosisofthePVP,comparedtoSCSalone.24-26Thefirstclinicalseriesofend-

ischemic HMP have demonstrated that this method is safe and may lead to a lower

incidenceofbiliarycomplicationsaftertransplantation.27-31

Despite the recognizedbeneficial effect ofHMPon IR injury and a suggested reduced

riskofNASaftertransplantation,therearenostudiesthathaveexaminedtheeffectof

HMP on IR injury of human bile ducts. In the present study, we aimed to determine

whetherdualoxygenatedHMP(DHOPE)reducesreperfusioninjuryofthebileductsin

DCDlivertransplantation,byperformingasystematichistologicalcomparisonofdonor

bileductsbeforeandaftergraftreperfusion.

MATERIALS&METHODS

Studypopulation

Arecentlyperformedphase1studyinourcenterincludedtenconsecutivepatientswho

underwentDCD liver transplantationbetweenAprilandNovember2014.31Thedonor

livers were preserved with DHOPE for two hours after conventional SCS. Informed

consent for DHOPE was obtained. The study protocol conformed to the ethical

guidelinesofthe1975DeclarationofHelsinkiandwasapprovedbytheMedicalEthics

CommitteeoftheUMCG(approvalnumberMETc2014.100).

AcontrolgroupconsistedofpatientswhounderwentDCDlivertransplantation inour

centerbetweenFebruary2012andSeptember2015.Allpatientswithavailablebileduct

biopsieswereincludedinthecontrolgroup.Nopatientswereexcludedfromthecontrol

group.

Studyprotocol

AllDCDliverswereprocuredbyoneoftheregionalmulti-organrecoveryteamsusinga

rapidprocurementprotocolwithaorticcoldflush-outandsubsequentSCS.Uponarrival

in our center, the liver was inspected and prepared for perfusion by placement of

cannulas in theportal vein andaorta.The cannulaswere connected to thedisposable

tubing set of the Liver Assist device (Organ Assist, Groningen, The Netherlands). The

liverswereperfusedforatleasttwohourswith4LofUWMachinePerfusionSolution

(Bridge-to-Life Ltd, Norfolk, UK) at a temperature of 10-12 °C. Two rotary pumps

enabled pressure-controlled perfusion with pulsatile mean arterial pressure of 25

mmHg and a continuous portal pressure of 5 mmHg. The perfusion solution was

oxygenatedbytwohollowfibermembraneoxygenatorswith100%FiO2at500ml/min

peroxygenator.

Bileductbiopsies

Bileductbiopsieswereobtainedfromthecommonbileduct,asdescribedpreviously.14

The biopsies were at the end of SCS (baseline) and after graft reperfusion in the

recipient, short before constructing the bile duct anastomosis. Baseline biopsieswere

collectedafterSCSduringthebacktableprocedure,whichwasfollowedbyDHOPE.All

sampleswerefixedin4%formalin,subsequentlyembeddedinparaffinand4µmthick

slideswere stainedwithhematoxylin&eosin (H&E).The slideswere examinedusing

lightmicroscopyandscannedwithaHamamatsudevice.

Histologicalgradingofbiliaryinjury

Biliary injury was graded according to an established semi-quantitative histological

gradingsystemdescribedbyHansenetal.andmodifiedtoOpdenDriesetal.13-14(Table

1). Biopsies were scored separately by two investigators without knowledge of the

clinical data (RvR and OBvL) under supervision of two dedicated liver pathologists

(ASHGandMvdH).Incaseofdiscordantresults,slideswereexaminedbyathird

Table1:Histologicalgradingofbileductinjury

Item HistologicalCharacteristic

Grading

1. Biliary epitheliumloss

Absenceofepithelialcellsliningthebileductlumen

0:noloss1:≤50%ofthebileductwithabsentepitheliallining2:>50%

2.Muralstromanecrosis

Necrosisofthebileductwall

0:nonecrosis1:≤25%ofthewallnecrotic2:>25%and≤503:>50and≤754:>75%

3.Peribiliaryvascularplexusdamage

Damagetothevesselssuchassubendothelialedema

0:novascularlesions1:≤50%ofthevesselsdamaged2:>50%

4.Arteriolonecrosis Lossofendothelialnucleiofarteriolesandmediadegeneration

0:noarteriolonecrosis1:≤50%ofthearteriesnecrotic2:>50%

5.Thrombosis Presenceofmicro-thrombi

0:nomicrothrombi1:microthrombipresent

6.Intramuralbleeding

Presenceoferythrocytesinthebileductwall

0:nobleeding1:≤50%ofthebileductwall2:>50%

7.PeriluminalPBGloss

AbsenceofepithelialcellsinthePBGclosetothelumen

0:noloss1:≤50%loss2:>50%loss

8.DeepPBGloss AbsenceofepitheliuminthePBGlocateddeepinthebileductwall

0:noloss1:≤50%loss2:>50%loss

Abbreviations:PBG;peribiliaryglands.

Statisticalanalyses

Continuousvariableswerepresentedasmedian(interquartilerange)ormean(standard

deviation) when appropriate and were compared between groups with the 2-tailed

Mann-Whitney test.Categoricalvariableswerepresentedasnumber (percentage)and

compared between groupswith the Pearson chi-square test or the Fisher’s exact test

whereappropriate.Pvalues< .05weredefinedassignificant.Statisticalanalyseswere

performedusingSPSSversion22.0forWindows.

RESULTS

Donorandrecipientcharacteristics

BetweenAprilandNovember2014, tenpatientswere included in theDHOPEphase1

trial. Detailed clinical results have been reported previously.31 The control group

consistedof twentypatientswhopreviouslyunderwentaDCDliver transplantation in

our center and in whom bile duct biopsies were obtained during the transplant

procedurebetween2012and2014.Baselineclinicalcharacteristicsaresummarizedin

Table2.TheEurotransplantdonor risk index (ET-DRI)was similar in theDHOPEand

the control group. Thedonors in theDHOPE grouphad a higher latest serumalanine

aminotransferase(ALT)andpeakserumALTconcentration,comparedtothedonorsin

thecontrolgroup:latestALT72U/L(39-125U/L)vs.29U/L(19-46U/L),respectively

(P= .008);peakALT121U/L(42-271U/L)vs.33U/L(20-46U/L), respectively (P<

.001). The cold ischemia time was shorter in the DHOPE group, but the total

preservation time in theDHOPEgroupwas longer than in thecontrolgroup:521min

(469-592min)vs.430min(407-485min),respectively(P=.002).

Table2:Donorandrecipientcharacteristics

Characteristic DHOPEgroup

(n=10)

Controlgroup

(n=20)

Pvalue

Donorcharacteristics

Eurotransplantdonorriskindex* 2.30(1.81-2.53) 2.22(1.67-2.54) .98

Age(years) 53(47-57) 49(34-55) .18

BMI(kg/m2) 23.0(24.1-19.9) 23.6(22.0-26.0) .25

LatestALT(U/L) 72(39-125) 29(19-46) .008

PeakALT(U/L) 121(42-271) 33(20-46) .004

LatestGGT(U/L) 50(19-102) 39(17-70) .75

Preservationcharacteristics

Preservationfluid(UWvs.HTK) 10(100%) 18(90%) .54

Asystoletime(min)# 15(13-17) 15(12-19) .95

Donorwarmischemiatime(min)^ 26(23-42) 33(29-41) .35

Coldischemiatime(min)** 358(314-398) 426(402-485) .002

Totalpreservationtime(min)## 521(469-592) 430(407-485) .002

Anastomosistime(min)^^ 34(30-49) 33(31-43) .88

Recipientcharacteristics

Age(years) 57(54-62) 55(47-63) .50

Sex(male) 6(60%) 9(45%) >.99

MELDscore*** 16(15-22) 20(13-24) .56

Underlyingdisease: .08

Alcoholiccirrhosis 3(30%) 3(15%)

NASH 5(50%) 3(15%)

Primarysclerosingcholangitis 1(10%) 4(20%)

Primarybiliarycirrhosis 0 0

Autoimmunehepatitis 0 3(15%)

HepatitisBorC 1(10%) 1(5%)

Hepatocellularcarcinoma 0 0

Cryptogenic 0 3(15%)

Other 0 4(20%)

*Eurotransplantdonorriskindexisavalidatedtooltoassesstheriskoflivergraftfailure

(37).#Asystoletimewasdefinedastimebetweencirculatoryarrestandinsituaorticcold

flush.^Donorwarmischaemiatimewasdefinedasthetimeintervalbetweenwithdrawal

ofdonor life supportand initiationof in situaortic cold flush. ** Cold ischemia timewas

defined as the interval between start aortic cold flush and end of static cold storage

excluding thedurationofDHOPE. ##Totalpreservation timewasdefinedas the interval

between start aortic cold flush in the donor and portal reperfusion in the recipient. ^^

Anastomosis time was defined as the interval between donor liver out of ice and

revascularization. ***MELDscorewasdefinedas thehighestof laboratoryderivedMELD

score or the (non) standard exception MELD score. Abbreviations: ALT, alanine

aminotransferase; DCD, donation after circulatory death; GGT, gamma-glutamyl

transferase; DHOPE, dual hypothermic oxygenated machine perfusion; HTK, histidine-

tryptophan-ketoglutarate;MELD,model for end stage liver disease;NASH, non-alcoholic

steatohepatitis;UW,UniversityofWisconson.

Histologicalevidenceofbileductinjury

Theresultsbileductinjuryscoringsbasedonthesemi-quantitativehistologicalgrading

systemaresummarizedinTable3.Therewerenodiscordantresultsbetweenthetwo

investigators.Asexpected,atbaselinetherewerenosignificantdifferencesbetweenthe

twogroupsforanyitemofthehistologicalgradingsystem(Fig.1).Inthecontrolgroup,

the histological bile duct damage after reperfusionwasmore severe than at baseline.

Especially the degree of mural stroma necrosis and the degree of deep PBG injury

increasedafterreperfusion(P=.002andP=.02,respectively).Incontrasttothecontrol

group, there was no increase in the severity of histological biliary injury after

reperfusionintheDHOPEgroup(Table3).

Whencomparingtheseverityofpost-reperfusionbileductinjurybetweenthetwostudy

groups,liversintheDHOPEgroupdisplayedsignificantlylessinjuryandlossofcellsin

theperiluminal(P=.04)andthedeepPBG(P=.04),comparedtocontrols(Fig.1-3).No

differenceswereobservedintheseverityofarteriolonecrosis,stromanecrosis,orinjury

ofthePVPbetweenthetwogroupsafterreperfusion.

Fig.1.DegreeofinjuryofthebileductsofDCDliverstreatedwithDHOPEversus

controls after static cold storage and after reperfusion in the recipient. (A) The

degreeofmural stromanecrosis increasedafterreperfusioncompared tobaseline in the

controlgroup(P<.001),butnotintheDHOPEgroup.(B)Nodifferenceswereobservedfor

the degree of injury of the peribiliary vascular plexus. (C) The periluminal PBG of livers

treatedwithDHOPEdemonstratedlessinjuryafterreperfusionthaninthecontrolgroup

(P = .04). Additionally, the injury of the deep PBG in the control group increased after

reperfusion compared to baseline (P= .02). (D) ThedeepPBG in the livers treatedwith

DHOPEdemonstrated lessdamageafter reperfusion than in the controlgroup (P= .04).

Asterisks indicatedaPvalue< .05.Abbreviations:DCD,donationaftercirculatorydeath;

DHOPE, dual hypothermic oxygenated machine perfusion; PBG, peribiliary glands; PVP,

peribiliaryvascularplexus

Fig.2.RepresentativehistologicexamplesofperiluminalPBGinthecommonbile

duct. (A) Bile duct at baseline of a DCD liver in the DHOPE group. (B) Bile duct after

reperfusionofaDCDliverintheDHOPEgroup.(C)BileductatbaselineofaDCDliverin

thecontrolgroup.(D)BileductafterreperfusionofaDCDliverinthecontrolgroup.The

insertrepresentsahighermagnificationoftheperiluminalPBG(400x).Bileductsoflivers

preserved with DHOPE displayed significantly less epithelial cell loss of the periluminal

PBG, compared to control livers. Original magnification was 200x. Arrows indicate

periluminalPBG.Asterisks indicate lumenof thebile duct. Abbreviations:DCD, donation

after circulatory death; DHOPE, dual hypothermic oxygenated machine perfusion; PBG,

peribiliaryglands.

Fig.3.RepresentativehistologicexamplesofdeepPBGinthecommonbileductof

DCD livergrafts.(A)BileductatbaselineofaDCD liver in theDHOPEgroup.(B)Bile

ductafterreperfusionofaDCDliverintheDHOPEgroup.(C)BileductatbaselineofaDCD

liver in the control group. (D) Bile duct after reperfusion of a DCD liver in the control

group. The insert represents a higher magnification of the deep PBG (400x). Arrows

indicate deep PBG. Asterisks indicate the lumen of the bile duct. Bile ducts of livers

preserved with DHOPE displayed significantly less epithelial cell loss of the deep PBG,

comparedtocontrollivers.Originalmagnificationwas200x.Abbreviations:DCD,donation

after circulatory death; DHOPE, dual hypothermic oxygenated machine perfusion; PBG,

peribiliaryglands.

Table3:Comparisonofhistologicalbileductinjury

Bileductwall

component

DHOPEgroup Controlgroup

Baseline

After

reperfusion

P

value

Baseline After

reperfusion

P

value

Biliaryepitheliumloss >.99 -

Grade0 - - - -

Grade1 10% 14% - -

Grade2 90% 86% 100% 100%

Muralstromanecrosis .25 .002

Grade0 90% 57% 50% 9%

Grade1 - - 43% 9%

Grade2 10% 29% - 36%

Grade3 - 14% 7% 46%

Peribiliaryvascular

plexusdamage

.38 .72

Grade0 50% 57% 43% 36%

Grade1 50% 29% 43% 36%

Grade2 - 14% 14% 28%

Arteriolonecrosis >.99 .57

Grade0 90% 100% 93% 82%

Grade1 10% - - -

Grade2 - - 7% 18%

Thrombosis >.99 >.99

Grade0 90% 100% 93% 91%

Grade1 10% - 7% 9%

Intramuralbleeding - .11

Grade0 100% 100% 100% 73%

Grade1 - - - 18%

Grade2 - - - 9%

PeriluminalPBGloss .24 .23

Grade0 - 14% - -

Abbreviations:DHOPE,dualhypothermicoxygenatedmachineperfusion;PBG,peribiliary

glands;SCS,staticcoldstorage.

Clinicaloutcomes

Theoverall clinical outcomeafter transplantationof thepatients in theDHOPEgroup

hasbeendescribedindetailpreviously.31Completeone-yearfollowupwasavailablein

allpatients.TheincidenceofNASwas10%intheDHOPEgroupand35%inthecontrol

group (P = .15). One recipient of a DHOPE preserved liver developed local NAS in

segment2and3of the liver,whichwassuccessfully treatedwithendoscopicstenting.

None of the patients in the DHOPE groups required re-transplantation for NAS. In

contrast,4(20%)inthecontrolgrouprequiredre-transplantationinforNAS.

DISCUSSION

In the current study, we aimed to determine whether end-ischemic oxygenated

hypothermicmachineperfusion(DHOPE)ofhumanDCDlivergraftsreducesIRinjuryof

the bile ducts after transplantation. The results of this study clearly demonstrated a

reduction inbiliary IR injuryofDHOPE-preservedDCD livers, compared toDCD livers

that did not undergo DHOPE. These findings provide important new insight in the

protective mechanism of end-ischemic DHOPE and are in line with the clinically

observedreductionintheincidenceofNASafterDCDlivertransplantationwhenDHOPE

isapplied.27-31

Grade1 30% 29% 21% -

Grade2 70% 57% 79% 100%

DeepPBGloss .64 .02

Grade0 40% 43% 36% -

Grade1 40% 43% 64% 73%

Grade2 20% 14% - 27%

Inaccordancewithpreviousreports,12-14allDCDliversincludedinthisstudydisplayed

histologicalsignsofsubstantialbileductinjuryattheendofSCS.Asexpected,thedegree

ofbileductinjuryafterSCS(baseline)wasnotdifferentbetweenliversthatunderwent

DHOPEornot.IncontrolliversthatwerenottreatedwithDHOPE,thedegreeofbiliary

injury worsened after graft reperfusion in the recipient, a phenomenon that was not

observed inDHOPEpreserved livers.Especially, theseverityofmuralstromanecrosis

andinjuryofthedeepperibiliaryglandsincreasedafterreperfusionofthecontrollivers,

but not of theDHOPEpreserved livers.When comparing post-reperfusion biopsies of

bileductsofDHOPE-preserved liversandcontrols, significantly lesshistological injury

ofboththeperiluminalanddeepPDGwasfoundintheDHOPEgroup.

In a large clinical study including 128 liver transplant recipients we have previously

shownthattheseverityofbileductinjuryattheendofSCSisastrongriskfactorforthe

development of NAS after transplantation.14 Upon reperfusion of a liver graft in the

recipient, the degree of biliary injury increases further and this may result in

irreversible damage of essential components of the bile duct wall, such as themural

stroma, peribiliary vasculature, and PBG.12,14 Obviously, end-ischemic DHOPE cannot

haveaprotectiveeffectonbiliaryinjurythatisalreadypresentafterSCS.However,the

currentstudyindicatesthatDHOPEdoespreventfurtherworseningofbileductinjury

aftergraftreperfusion.

It is well known that the majority of tissue damage due to IR injury occurs in the

reperfusionperiodafterrestorationofbloodflowtothegraft.11Someofthekeyfactors

in IR injury are the depletion of cellular energy content (especially intracellular

adenosinetriphosphate(ATP))andtheformationofreactiveoxygenspecies(ROS)due

tomitochondrial dysfunction.11,32WhileROS formation results in oxidative damage of

cellularstructures,suchascellmembranesandnuclearDNA,depletionandinsufficient

restorationofATPresultsincelldeathduetoinsufficientmetabolicfunction.Although

the conditions for IR injury are generated during cold ischemic preservation, the

sequelaeofeventsthatleadtothefullblownIRinjuryisnotactivateduntilafterwarm

reperfusion.11Experimentalandclinicalstudieshavedemonstratedthatoneofthekey

protective mechanisms of hypothermic oxygenated machine perfusion is a full

restoration of cellular ATP content and “resuscitation” ofmitochondria, resulting in a

significantreductionofROSproductionaftersubsequentwarmgraftreperfusioninthe

recipient.21,22,33,34 In addition, and downstream of this, oxygenated HMP results in a

reductionofKupffercellactivationandlesssecondaryactivationoftheinnateimmune

system.21,22,33,34 Altogether, these protective effects have been shown to result in a

reductionofhepatocellularIRinjury.OurdataonthereductionofbiliaryIRinjuryarein

linewiththesepreviousstudiesanddemonstratethatnotonlyhepatocellularIRinjury,

butalsoIRinjuryofthebileductsisattenuatedbyoxygenatedHMP.

TheDHOPE-preservedliversinthepresentstudydemonstratedsignificantlylesssevere

injuryofthedeepandperiluminalPBGafterreperfusion,comparedtothecontrollivers

that did not undergo DHOPE. This histological finding is clinically relevant since the

PBGshavebeenidentifiedasa localnicheofbiliaryprogenitorcells thatcontributeto

theregenerationofbiliaryepitheliumafterinjury.15-17,35SevereinjuryofthedeepPBGat

the time of transplantation is a significant risk factor for the development of NAS.14

Therefore, the protective effect of DHOPE on PBG may lead to better preserved

regenerative biliary capacity followed by a reduction of the incidence of NAS. This is

supportedbythe(non-significant)lowincidenceofNASobservedaftertransplantation

of DCD livers preserved with HMP in the first clinical series.27-31 However, formal

evidence that oxygenated HMP reduces the incidence of NAS should come from an

adequatelypoweredrandomized,controlled trial.Suchamulticenter trialhasrecently

been initiated in transplant centers in the Netherlands, Belgium and United Kingdom

(ClinicalTrials.govNCT02584283).

Bile duct biopsies in this studywere obtained from the extrahepatic bile ducts,while

NASmainlyoccursintheintrahepaticbileducts.However,previousstudieshaveshown

thatthedegreeofinjuryoftheextrahepaticbileductcorrelateswellwiththedegreeof

injury of the intrahepatic bile ducts36 and histological assessment of the extrahepatic

bileductcanpredictthedevelopmentofNAS.14

IncontrasttoapreviousstudyonDHOPEusingaporcineDCDlivermodel,wedidnot

findareduction in thedegreeof injuryof thePVPandarteriolonecrosis in thehuman

liverbileductsafterDHOPE.24Infact,inthecurrentstudywedidnotfindasignificant

increase inthedegreeofPVPinjuryandarteriolonecrosisafterreperfusion,compared

to baseline in both groups. In the current study the post-reperfusion bile duct biopsy

was taken1-2hours after graft reperfusion and this time intervalmayhavebeen too

shortforhistologicallydetectablevascularinjurytodevelop.

Limitations of this study are the sample size and the use of historical controls. The

controlsusedinthishistologicalstudywereslightlydifferentfromtheonesusedinour

previous report on clinical outcome after transplantation of DHOPE-preserved DCD

livers,34becausethecontrolgroupconsistedofallavailableparaffinembeddedbileduct

biopsies fromDCDlivergraftswithoutapplicationofexclusioncriteria.Althoughmost

baselinecharacteristicssuchasET-DRIweresimilarbetweenthetwostudygroups,the

donors in the DHOPE group had significantly higher serum ALT levels, compared to

donors in the control group.AsALT is amarker forhepatocellular injury, theDHOPE

groupconsistedof liverswithslightlymorepre-existing injurythanthecontrolgroup.

Therefore,theobservedbeneficialeffectsofDHOPEmighthavebeenmorepronounced

if the ALT levels would have been equivalent between the groups. As expected, the

preservationmethod DHOPE affected the length of preservation periods in the study

groups resulting ina longer totalpreservation timeandshorter cold ischemia time in

theDHOPEcomparedtothecontrolgroup.Intheinterventiongroupthedonorliveris

machine perfusedwhile the recipient surgery is performed. In the control group, the

donorliverremainsintheiceboxduringthisperiod.Theshortercoldischemiatimein

theDHOPE groupmay have caused an advantage,while the longer total preservation

timemayhavecausedadisadvantage.However,themediandifferenceincoldischemia

timewasonly30minutesandbaseline levelsofbileduct injuryweresimilarbetween

thegroups.

Inconclusion,thisstudydemonstratesthatDHOPEattenuatesIRinjuryofthebileducts

after transplantation ofDCD liver grafts. In particular, DHOPE contributed to a better

preservation of the PBG and, as such, may preserve the regenerative capacity of the

donorbileductsleadingtoareducedriskofbiliarycomplicationsaftertransplantation.

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3. denDulkAC,SebibKorkmazK,deRooijBJ,SuttonME,BraatAE,IndersonA,et al. High peak alanine aminotransferase determines extra risk fornonanastomotic biliary strictures after liver transplantation with donationaftercirculatorydeath.TransplInt2015;28:492-501.

4. DubbeldJ,HoekstraH,FaridW,RingersJ,PorteRJ,MetselaarHJ,etal.Similarliver transplantation survival with selected cardiac death donors and braindeathdonors.BrJSurg2010;97:744-753.

5. O'NeillS,RoebuckA,KhooE,WigmoreSJ,HarrisonEM.Ameta-analysisandmeta-regressionofoutcomesincludingbiliarycomplicationsindonationaftercardiacdeathlivertransplantation.TransplInt2014;27:1159-1174.

6. Blok JJ, DetryO, PutterH,Rogiers X, PorteRJ, vanHoekB, et al. Long-termresults of liver transplantation from donation after circulatory death. LiverTranspl2016;22:1107-1114.

7. VerdonkRC,BuisCI,PorteRJ,vanderJagtEJ,LimburgAJ,vandenBergAP,etal. Anastomotic biliary strictures after liver transplantation: causes andconsequences.LiverTranspl2006;12:726-735.

8. Detry O, Donckier V, Lucidi V, Ysebaert D, Chapelle T, Lerut J, et al. Livertransplantation from donation after cardiac death donors: initial Belgianexperience2003-2007.TransplInt2010;23:611-618.

9. Gilbo N, Jochmans I, Sainz M, Pirenne J, Meurisse N, Monbaliu D. ReducingNon-AnastomoticBiliaryStricturesinDonationAfterCirculatoryDeathLiverTransplantation:ColdIschemiaTimeMatters!AnnSurg2016;[Epubaheadofprint].

10. TanerCB,BulataoIG,PerryDK,SibuleskyL,WillinghamDL,KramerDJ,etal.Asystoletocross-clampperiodpredictsdevelopmentofbiliarycomplicationsinlivertransplantationusingdonationaftercardiacdeathdonors.TransplInt2012;25:838-846.

11. vanGolenRF, vanGulik TM,HegerM. The sterile immune response duringhepaticischemia/reperfusion.CytokineGrowthFactorRev2012;23:69-84.

12. BrunnerSM, JungerH,RuemmeleP, SchnitzbauerAA,DoeneckeA,KirchnerGI,etal.Bileductdamageaftercoldstorageofdeceaseddonorliverspredictsbiliary complications after liver transplantation. J Hepatol 2013;58:1133-1139.

13. HansenT,HollemannD,PittonMB,HeiseM,Hoppe-LotichiusM,SchuchmannM,etal.Histologicalexaminationandevaluationofdonorbileductsreceivedduring orthotopic liver transplantation--a morphological clue to ischemic-typebiliarylesion?VirchowsArch2012;461:41-48.

14. op den Dries S, Westerkamp AC, Karimian N, Gouw AS, Bruinsma BG,Markmann JF, et al. Injury to peribiliary glands and vascular plexus beforelivertransplantationpredictsformationofnon-anastomoticbiliarystrictures.JHepatol2014;60:1172-1179.

15. CarpinoG,CardinaleV,OnoriP,FranchittoA,BerlocoPB,RossiM,etal.Biliarytreestem/progenitorcellsinglandsofextrahepaticandintrahepticbileducts:ananatomicalinsitustudyyieldingevidenceofmaturationallineages.JAnat2012;220:186-199.

16. DipaolaF,ShivakumarP,PfisterJ,WaltersS,SablaG,BezerraJA.Identificationof intramural epithelial networks linked to peribiliary glands that expressprogenitor cell markers and proliferate after injury in mice. Hepatology2013;58:1486-1496.

17. NakanumaY,HosoM,SanzenT,SasakiM.Microstructureanddevelopmentofthe normal and pathologic biliary tract in humans, including blood supply.MicroscResTech1997;38:552-570.

18. KarimianN,OpdenDriesS,PorteRJ.Theoriginofbiliarystricturesafterlivertransplantation: is it the amount of epithelial injury or insufficientregenerationthatcounts?JHepatol2013;58:1065-1067.

19. Dutkowski P, Graf R, Clavien PA. Rescue of the cold preserved rat liver byhypothermic oxygenated machine perfusion. Am J Transplant 2006;6:903-912.

20. Guarrera JV,Estevez J,Boykin J,BoyceR,Rashid J,SunS,etal.Hypothermicmachineperfusionof liver grafts for transplantation: technicaldevelopmentinhumandiscardandminiatureswinemodels.TransplantProc2005;37:323-325.

21. SchlegelA,KronP,GrafR,DutkowskiP,ClavienPA.Warmvs.coldperfusiontechniquestorescuerodentlivergrafts.JHepatol2014;61:1267-1275.

22. Schlegel A, Rougemont O, Graf R, Clavien PA, Dutkowski P. Protectivemechanisms of end-ischemic cold machine perfusion in DCD liver grafts. JHepatol2013;58:278-286.

23. WesterkampAC,KarimianN,MattonAP,MahboubP,vanRijnR,Wiersema-Buist J, et al. Oxygenated Hypothermic Machine Perfusion After Static ColdStorage ImprovesHepatobiliaryFunctionofExtendedCriteriaDonorLivers.Transplantation2016;100:825-835.

24. OpdenDriesS,SuttonME,KarimianN,deBoerMT,Wiersema-BuistJ,GouwAS, et al. Hypothermic oxygenated machine perfusion preventsarteriolonecrosis of the peribiliary plexus in pig livers donated aftercirculatorydeath.PLoSOne2014;9:e88521.

25. Schlegel A, Graf R, Clavien PA, Dutkowski P. Hypothermic oxygenatedperfusion(HOPE)protectsfrombiliaryinjuryinarodentmodelofDCDlivertransplantation.JHepatol2013;59:984-991.

26. WesterkampAC,MahboubP,Meyer SL,HottenrottM,Ottens PJ,Wiersema-Buist J, et al. End-ischemic machine perfusion reduces bile duct injury indonationaftercirculatorydeathratdonorliversindependentofthemachineperfusiontemperature.LiverTranspl2015;21:1300-1311.

27. DutkowskiP,PolakWG,MuiesanP,SchlegelA,VerhoevenCJ,ScaleraI,etal.First Comparison of Hypothermic Oxygenated PErfusion Versus Static ColdStorage of Human Donation After Cardiac Death Liver Transplants: AnInternational-matchedCaseAnalysis.AnnSurg2015;262:764-771.

28. DutkowskiP,SchlegelA,deOliveiraM,MullhauptB,NeffF,ClavienPA.HOPEfor human liver grafts obtained from donors after cardiac death. J Hepatol2014;60:765-772.

29. Guarrera JV, Henry SD, Samstein B, Odeh-Ramadan R, Kinkhabwala M,Goldstein MJ, et al. Hypothermic machine preservation in human livertransplantation:thefirstclinicalseries.AmJTransplant2010;10:372-381.

30. Guarrera JV, Henry SD, Samstein B, Reznik E, Musat C, Lukose TI, et al.Hypothermic machine preservation facilitates successful transplantation of"orphan"extendedcriteriadonorlivers.AmJTransplant2015;15:161-169.

31. van Rijn R, KarimianN,Matton APM, Burlage LC,WesterkampAC, van denBerg AP, et al. Dual hypothermic oxygenated machine perfusion in livertransplantsdonatedaftercirculatorydeath.BrJSurg2017;104:907-917.

32. Schlegel A, Kron P, Dutkowski P. Hypothermic Oxygenated Liver Perfusion:BasicMechanisms and Clinical Application. Curr Transplant Rep 2015;2:52-62.

33. HenrySD,NachberE,Tulipan J, Stone J,BaeC,ReznikL, et al.Hypothermicmachine preservation reduces molecular markers of ischemia/reperfusioninjuryinhumanlivertransplantation.AmJTransplant2012;12:2477-2486.

34. SchlegelA,KronP,GrafR,ClavienPA,DutkowskiP.HypothermicOxygenatedPerfusion(HOPE)downregulatestheimmuneresponseinaratmodeloflivertransplantation.AnnSurg2014;260:931-938.

35. Irie T, Asahina K, Shimizu-Saito K, Teramoto K, Arii S, Teraoka H. Hepaticprogenitorcellsinthemouseextrahepaticbileductafterabileductligation.StemCellsDev2007;16:979-987.

36. KarimianN,WeederPD,BomfatiF,GouwAS,PorteRJ.Preservationinjuryofthedistalextrahepaticbileductofdonorliversisrepresentativeforinjuryoftheintrahepaticbileducts.JHepatol2015;63:284-287.

37. Braat AE, Blok JJ, Putter H, Adam R, Burroughs AK, Rahmel AO, et al. TheEurotransplant donor risk index in liver transplantation: ET-DRI. Am JTransplant2012;12:2789-2796.

Extendedhypothermicoxygenatedmachineperfusionenablesexsitupreservationofporcineliversforupto24hours

IsabelM.A.Brüggenwirth,OttoB.vanLeeuwen,YvonnedeVries,SilkeB.Bodewes,JelleAdelmeijer,JannekeWiersema-Buist,TonLisman,PauloN.Martins,VincentE.deMeijer,RobertJ.Porte.JHEPRep.2020(inpress)

7

ABSTRACT

Background:End-ischemichypothermicoxygenatedmachineperfusion(HOPE)of the

donor liver for 1-2h mitigates ischemia-reperfusion injury during subsequent liver

transplantation. Extended preservation time can be desired to facilitate difficult

recipienthepatectomyortooptimizelogistics.Wethereforeinvestigatedwhetherend-

ischemicdualHOPE(DHOPE)couldextendpreservationtimeupto24husingaporcine

liverreperfusionmodel.

Methods: Porcine liverswith 30minuteswarm ischemiawere subjected to 2h static

cold storage (SCS), followed by 2h, 6h, or 24h DHOPE (n=6 per group). Subsequent

normothermic reperfusion was performed for 4h using autologous blood. Two livers

preservedby24hSCS servedas additional controls.Aproof of principle confirmation

was carried out in two discarded human livers subjected to extended DHOPE.

Hepatocellular and cholangiocyte injury and function were assessed. Oxidative stress

levelsandhistologywerecomparedbetweengroups.

Results: Perfusion flows remained stableduringDHOPE, regardlessofduration.After

normothermic reperfusion, 24h perfused DHOPE livers had similar lactate clearance,

bloodpH,glucose,andalanineaminotransferaselevels,andbiliarypH,bicarbonate,and

LDHlevels,whencomparedto2hand6hperfusedlivers.Levelsofmalondialdehydeand

high-mobilitygroupbox1 inserumand liverparenchymaweresimilar forallgroups.

Histologicalanalysisofbileductsandliverparenchymarevealednodifferencesbetween

the groups. ExtendedDHOPE in discardedhuman livers preservedhepatocellular and

cholangiocyte functionandhistologyafterreperfusion. Incontrast, liverspreservedby

24hSCSwerenon-functioning.

Conclusion:Extendedend-ischemicDHOPEenabledsuccessfulpreservationofporcine

and discarded human donor livers for up to 24h. Extended DHOPE enables safe

extension of preservation time, which may facilitate logistics of allocation and

transplantation,andfurtherexpandthedonorpool.

INTRODUCTION

Preservation using ex situ machine perfusion has gained considerable interest to

facilitate enhanced utilization of liver grafts for transplantation, albeit with several

technical variations.1 While normothermic machine perfusion (NMP) enables

hepatobiliary viability assessment and therapeutic interventions prior to

transplantation, hypothermic oxygenatedmachine perfusion (HOPE) reconditions the

graftbyinducingahypometabolicstatewhilstrestoringmitochondrialfunctionthrough

delivery of oxygen.1 End-ischemic HOPE is a relatively simple approach. Livers are

preserved by static cold storage (SCS) after procurement and are then subjected to

machineperfusionuponarrivalattherecipientcenter.DynamicpreservationbyHOPE

for1-2hoursissufficienttomitigateorgandamageandrestoremitochondrialfunction

andcellularenergystores,resultinginpostoperativelyreducedgraftfailure.2

Previously, we have shown that a short period (2 hours) of dual HOPE (DHOPE)

improvedhepatobiliaryfunctionanddecreasedinjurytothelivergraftandbiliarytree

in discarded human livers.3,4 In a prospective cohort study, 2 hours of end-ischemic

DHOPEwas shown to be feasible and safe in resuscitating donation after circulatory

death (DCD) liver grafts with 100% one-year graft and patient survival after

transplantation.5 Another important finding was that DHOPE attenuates injury of the

biliarytreeaftertransplantationofDCDlivergraftscomparedtonon-DHOPEpreserved

livers.6 In 2015, our center initiated a large international multicenter randomized

controlledtrialcomparingDHOPEversusSCSinDCDlivertransplantationofwhichthe

firstresultsareexpectedmid2020.7

In addition to graft reconditioning, viability assessment, and potential therapeutic

interventions,machine perfusionmay also have the potential to prolong preservation

time. Traditionally, graft preservation using static cold storage (SCS) may keep good

qualityliversviablefortransplantationforseveralhours.Machineperfusion,however,

may extend preservation times, particularly in extended criteria donor (ECD) organs

which are more vulnerable to cold ischemia.8–10 Extended preservation by HOPE, for

example, could facilitate logistics for allocation and transplantation. As a proof-of-

concept,ahumandiscardedliverhasbeenpreservedfor86hoursusingexsituNMP11,

and an initially declined human liver has successfully been transplanted after

preservationfor26hours,ofwhich17.5hoursSCSand8.5hourswithNMP.12However,

little data are available regarding extended graft preservation by using (dual) HOPE,

and,currently,maximumreportedpreservationtimeusingHOPEis8hours.10,13,14

We investigated the effects of extended preservation by applying end-ischemic dual

HOPE (DHOPE) in a porcine liver ischemia reperfusion injury model. Our endpoints

included graft viability after warm reperfusion and various markers of injury during

machine perfusion, and after warm reperfusion. In addition, a proof of principle

confirmationofend-ischemicDHOPEpreservationwascarriedoutindiscardedhuman

livers.

MATERIALS&METHODS

Porcinedonationaftercirculatorydeathliverprocurement

Livers from 5-month-old white female landrace pigs were retrieved after circulatory

death. Pigs were sacrificed by a standardized procedure of electrocution followed by

exsanguination.2Lofautologousbloodwascollected ina containerwith25,000 IUof

heparin(heparinLEO5000IU/ml,LEOPharmaceuticalProducts,Denmark).Bloodwas

then stored in bags supplemented with the anticoagulant citrate-phosphate-dextrose

(Sanquin, Amsterdam, the Netherlands) and cold stored (4°C) until subsequent use.

Within30minutesaftercirculatorydeath, liverswereflushedbygravityviatheportal

vein with 1L of cold (4°C) NaCl 0.9% (Baxter BV, Utrecht, the Netherlands)

supplementedwith25,000IUofheparin,followedby2LofcoldUniversityofWisconsin

(UW)solution(BridgetoLife,Ltd,London,UnitedKingdom).Afterportalflush,theaorta

wascannulated,andsidebrancheswereclippedfollowedbycoldarterialflushoutwith

UWsolutionusingasyringe.Thecysticductwasligated,andthecommonbileductwas

cannulatedwithabilecannula(8Fr,OrganAssist,Groningen,theNetherlands).Livers

werestaticcoldstored(4°C)inUWsolutionfor2hours.Theporcineliversusedinthe

present study were retrieved from a slaughterhouse (Kroon, Groningen, the

Netherlands)wherehumanecircumstancesareappliedaccordingtonationallegislation.

According to the Dutch law, no institutional approval is needed when using a

slaughterhousemodel.

Dualhypothermicoxygenatedmachineperfusion(DHOPE)

AfterSCS,graftswererandomlyassignedtoeither2hDHOPE(DHOPE-2),6h(DHOPE-

6), or 24h DHOPE (DHOPE-24) (n=6 per group). Machine perfusion was performed

usingtheLiverAssistdevice(OrganAssist,Groningen,theNetherlands).15Liverswere

perfusedwith 2L Belzer UWmachine perfusion solution (Bridge to Life, Ltd, London,

UnitedKingdom),oxygenatedwith100%O2.Temperaturewasmaintainedbetween8–

10°C. Pressures were limited to 25 mmHg in the hepatic artery and 3 mmHg in the

portalvein.Perfusatesampleswerecollectedfromthearterialinflowcannulaattheend

of preservation. Liver tissue sampleswere obtained after procurement, at the end of

DHOPEpreservation,andattheendofwarmreperfusion.

Exsitunormothermicoxygenatedwholebloodreperfusion

After extended DHOPE preservation for 2h, 6h, or 24h, liver grafts were flushed by

gravitywith1Lofcold(4°C)salinesolutionand1Lofsalineatroomtemperature.Livers

were then transferred to a second Liver Assist device and reperfused at 37°C with

autologouswhole blood.Whole bloodwas used during this phase to simulate clinical

transplantation. Sodium bicarbonate (B. Braun Medical, Melsungen, Germany) was

addedinthefirsthourafterreperfusiontoadjusttoaphysiologicalpH.Thebloodwas

oxygenatedwithacarbogenmixtureof95%O2and5%CO2at1L/min.Theportalvein

was perfused continuously at a pressure of 11 mmHg, and the hepatic artery was

perfusedwithapulsatileflowatameanpressureof70mmHg.Perfusatesampleswere

taken before reperfusion, and at 5, 60, 120, 180, and 240minutes after reperfusion.

Arterial blood gas samples were taken before reperfusion, and every 30 minutes

thereafter. Partial oxygenpressure,Hb, pH, glucose, and lactateweremeasuredusing

thei-STATclinicalanalyzer(AbbotPointofCareInc.,Princeton,NJ).Bileproductionwas

measuredgravimetricallythroughoutreperfusion.Everyhour,bilewascollectedunder

mineral oil, as described previously,16 and biliary pH, bicarbonate and lactate

dehydrogenase (LDH) levels were measured. As controls, two porcine livers were

preserved with static cold storage (4°C) in UW-solution for 24 hours, followed by 4

hoursofnormothermicreperfusionwithwholebloodasdescribedabove.

Outcomes

Hepatocellularinjuryafterwarmreperfusionwasassessedbyalanineaminotransferase

(ALT) levels, using a standardized laboratorymethod. Nuclear subcellular injurywas

measured by release of highmobility group box-1 protein (HMGB-1) using a specific

ELISA (IBL International GmbH, Fujioka, Japan). Cell-freeDNA (cfDNA)was used as a

marker of cell damage and necrosis and measured as described previously.17 Tumor

necrosis factor alpha (TNF�) and interleukin6 (IL-6) levelsweremeasuredbyELISA

(Biotechne, Abingdon, UK). Lipid peroxidation, a marker of oxidative stress, was

quantified by measurement of malondialdehyde (MDA) in the perfusate and liver

biopsies.15MDA levels in liver parenchymawere corrected for the amount of protein

(Bradfordassay,Bio-Rad,Hercules,USA).Inaddition,8-hydroxy-2-deoxyguanosine(8-

OHdG)wasmeasured(FineTest,Wuhan,China)asaproductofoxidativedamageofDNA

by reactive oxygen and nitrogen species. Soluble thrombomodulin (sTM) was

determined using a commercially available enzyme-linked immunosorbent assay

(ELISA)(Biotechne,Abingdon,UK).18Hepaticcontentofadenosinetriphosphate(ATP)

was used as an indicator of energy status of grafts before and after DHOPE. For this

purpose, liverbiopsieswereimmediatelyfrozeninliquidnitrogenandlaterprocessed

forATPmeasurement,asdescribedpreviously.19

Histologicalanalysis

Liver biopsies were fixed in formalin and embedded in paraffin. We performed

hematoxylin-eosin(H&E)stainingtoassessnecrosisandVonWillebrandFactor(VWF)

staining to assess endothelial activation. Quantification of necrosis and VWF-positive

endothelial cells was determined in 3 random visual fields (20x). Biopsies of the

extrahepatic bile ducts were taken after procurement and at the end of reperfusion.

After H&E staining, biliary injury was assessed using a modified scoring system as

describedbyOpdenDries et al.20 Scoringwas conducted in ablinded fashionby two

independentinvestigators.

Humanprotocol

Human application of extended DHOPE was performed as a preclinical study using

discarded human liver grafts. Family of the donors gave consent for research. Donor

liverswerestaticcoldstoredoniceafterprocurementandtransferredtoourhospital.

Tomimicclinicalpracticeatourcenterascloseaspossible,graftsweresubjectedtoour

clinically used DHOPE-COR-NMP protocol.21 Extended 20-hour end-ischemic DHOPE

preservationwas applied using UWmachine perfusion solution. The timeframe of 20

insteadof24hourswaschosenbecauseoflogisticalreasons.AfterDHOPEpreservation,

UWwaschangedtoaperfusionsolutioncontainingahemoglobin-basedoxygencarrier,

as described previously.22 In accordance with our clinical protocol, discarded human

livers subsequently underwent 1 hour of controlled oxygenated rewarming (10°C to

37°C) followed by normothermic (37°C) reperfusion and liver viability testing for an

additional4hours.21

Statistics

All values are expressed asmeans ± standard error of themean for n=6 animals per

group.DifferencesbetweenthegroupsweretestedusingtheKruskal-Wallistestunless

statedotherwise.A two-sidedp-value less than0.05was considered tobe significant.

AnalyseswereperformedusingIBMSPSSsoftwareversion25forwindows.Forfurther

detailsregardingmaterialsandmethodsused,pleaserefertotheCTATtable.

RESULTS

Ischemiatimes

There were no significant differences in warm and cold ischemia times between the

groups.Meanwarmischemiatimewas24±2minutesforDHOPE-2,22±2minutesfor

DHOPE-6,and22±1minutesforDHOPE-24livers(p=0.847).Meancoldischemiatime

was129±11minutesforDHOPE-2livers,122±5minutesforDHOPE-6livers,and122

±5minutesforDHOPE-24livers(p=0.890).

FlowsduringDHOPE

The perfusate temperature was maintained at 10�C throughout all perfusions. In

addition, portal venous and hepatic arterial flows remained stable in all livers

throughoutthepreservationperiod.MeanarterialflowattheendofDHOPEwas104.8±

29.2ml/min in theDHOPE-2 group, 104.3 ± 35.2ml/min in theDHOPE-6 group, and

149.3±41.7ml/minintheDHOPE-24group(p=0.676).Meanportalflowattheendof

DHOPEwas 311.7 ± 49.6ml/min in theDHOPE-2 group, 282.0 ± 72.2ml/min in the

DHOPE-6group,and193.3±16.3ml/minintheDHOPE-24group(p=0.219).

LivergraftandbileductviabilityafterextendedDHOPE

Warmreperfusionwasusedtoassesslivergraftviabilityafterextendedpreservationby

24-hourDHOPE.Portal(Fig.1A)aswellasarterial(Fig.1B)flowssteadilyincreasedin

all liver grafts upon reperfusion. There were no significant differences between the

groups. In all groups, all livers cleared lactate (Fig. 1C). Perfusate pH was adjusted

withinthefirsthourafterreperfusionbyaddingbicarbonate.Therewerenosignificant

differencesintheamountofaddedbicarbonatebetweenthegroups.Afterthefirsthour,

nofurthercorrectionswereneeded,andpHremainedstableduringallliverperfusions

(Fig. 1D). Starting one hour after reperfusion, in all livers perfusate glucose levels

graduallydecreased(Fig.1E).

All livers produced sufficient quantities of bile,without differences in cumulative bile

production (Fig. 2A). In addition, bile duct viability was assessed during 4 hours

normothermicreperfusion.Inallgroups,biliarypHandbicarbonateincreasedduring4

hourswarmreperfusion.LevelsofbiliarypH,bicarbonateandLDHweresimilar inall

groups at the end of reperfusion (Fig. 2B-D). In this porcinemodel of DCD livers,we

observed extensive loss of biliary epithelial lining immediately after procurement (30

minofwarmischemia).Thiswasconsistentwithpreviousfindings.20Attheendofwarm

reperfusion, bile ducts of all livers displayed signs of bile duct injury. Histological

comparisonofbileductsrevealedsimilarinjuryattheendofreperfusioninallgroups

(Fig.2E,Fig.S1).

Figure 1. Perfusion flows and markers of liver function during 4 hours

normothermicreperfusionaftereither2,6,or24hoursDHOPE.(A)Portalvenous

flow,(B)hepaticarteryflow,(C)lactateconcentration,(D)perfusatepH,(E)glucoselevels.

All liversshowgoodliverfunctionduringreperfusionafterpreservationbyeither2,6,or

24hoursDHOPE.Shownherearethemeanandstandarderrorofthemeanforn=6livers

pergroup.Therewerenosignificantdifferencesbetweenthegroupsinallmarkersshown

(Kruskal-Wallistest).DHOPE=dualhypothermicoxygenatedmachineperfusion.

Figure 2. Biliary function during 4 hours normothermic reperfusion. (A)

Cumulativebile productionduring reperfusion, (B)biliarypH, (C) biliarybicarbonateat

the end of reperfusion, (D) biliary LDH at the end of reperfusion. All livers show good

biliary functionafterpreservationby either2, 6, or24hoursDHOPEand therewereno

significantdifferencesbetweenthegroups inallparametersshown(Kruskal-Wallis test).

(E)Histologyof extrahepaticbileducts, *=bileduct lumen,arrow=absenceof luminal

epithelium.Shownhereare themeanand standarderrorof themean forn=6 liversper

group. DHOPE = dual hypothermic oxygenated machine perfusion; LDH = lactate

dehydrogenase.

Next,weinvestigatedthedegreeofhepatocellularinjuryafterDHOPEpreservationfor

2,6and24hours.Weanalyzedseveralmarkersforcellularinjuryattheendof4hours

reperfusion.LevelsofALT(Fig.3A;p=0.687),HMGB-1(Fig.3B;p=0.690),orcfDNA(Fig.

3C; p=0.229) in the perfusate were not elevated by extended DHOPE preservation.

Histological analysis of liver parenchyma revealed that DHOPE preservation up to 24

hours did not result in more necrosis after reperfusion compared to shorter

preservationtimes(Fig.3DandE).

Figure 3. Markers for cellular injury at the end of normothermic reperfusion.

Perfusatelevelsof(A)ALT(p=0.687),(B)HMGB-1(p=0.690),(C)andcfDNA(p=0.229).

There were no statistically significant differences between the groups at the end of

reperfusion (Kruskal-Wallis test). (D) percentage of necrosis on histological analysis of

liverparenchymaafterH&Estaining, (E)H&Estainingof liverparenchyma.Shownhere

are the mean and standard error of the mean for n=6 livers per group. ALT = alanine

aminotransferase;cfDNA=cell-freeDNA;DHOPE=dualhypothermicoxygenatedmachine

perfusion; H&E = hematoxylin and eosin; HMGB-1 = high-mobility group 1; SCS = static

coldstorage.

Since livers were perfused for up to 24 hours with an oxygenated solution, we

investigatedmarkersforoxidativestressattheendofDHOPEpreservationandafter4

hours warm reperfusion. Extended preservation was not associated with increased

levelsofMDAintheperfusateattheendofDHOPE(Fig.4A;p=0.312),nor inbloodat

theendofwarmreperfusion(Fig.4B;p=0.308).ExtendedDHOPEpreservationwasalso

not associated with increased levels of MDA in liver biopsies at the end of DHOPE

(p=0.125), nor at the end of reperfusion (p=0.604) (Fig. 4C). Perfusate 8-OHdG

measuredattheendofwarmreperfusionwassimilarinallgroups(p=0.172)(Fig.4D).

Figure 4. Markers related to oxidative stress at the end of DHOPE and

normothermicreperfusion. (A)MDA inperfusateat the endofDHOPE (p=0.312), (B)

MDAinliverparenchymaattheendofDHOPE(p=0.125)andreperfusion(p=0.604)),(C)

MDA in perfusate at the end of normothermic reperfusion (p=0.308), (D) 8-OHdg in the

perfusate at the end of normothermic reperfusion (p=0.172). Therewere no statistically

significantdifferencesbetweenthegroupsonallparameters(Kruskal-Wallistest).24-hour

DHOPE preservation does not induce oxidative injury compared to shorter preservation

times. Shown here are the mean and standard error of the mean for n=6 animals per

group. 8-OHdG = 8-hydroxydeoxyguanosine; DHOPE = dual hypothermic oxygenated

machineperfusion;MDA=malondialdehyde.

Extended DHOPE preservation was not associated with increased release of

inflammatory cytokines TNF-a and IL-6. Mean TNF-a was 334.4 ± 55.8 pg/ml for

DHOPE2, 252.3 ± 57.8 pg/ml for DHOPE6, and 347.3 ± 123.2 pg/ml for DHOPE24

preserved livers, respectively (p=0.692) (Fig.5A).Mean IL-6was13.9±5.5pg/ml for

DHOPE2,23.9±8.7pg/ml forDHOPE6,and19.7±8.2pg/ml forDHOPE24preserved

livers,respectively(p=0.578)(Fig.5B).Finally,weinvestigatedthedegreeofendothelial

injury after prolonged DHOPE preservation. Levels of soluble thrombomodulin (sTM)

werenotaffectedbyprolongedDHOPE(p=0.985)(Fig.5C). IntensityofVWFstaining

didnotrevealmoreactivationofendothelialcellsonhistologicalanalysis(Fig.5Dand

E).

Figure5.Endothelialcellactivationandlevelsofsolublethrombomodulinduring

reperfusion.(A)TNF-ainperfusate(p=0.692),(B)IL-6inperfusate(p=0.578),(C)soluble

thrombomodulin in perfusate (p=0.985) (D) VWF staining, (E) percentage VWF positive

endothelial cells per high-power field. There were no statistically significant differences

between the groups at the end of reperfusion (Kruskal-Wallis test). Preservation by 24-

hour DHOPE did not induce more injury to endothelial cells compared to shorter

preservation times. DHOPE = dual hypothermic oxygenated machine perfusion; HPF =

high-power field; IL-6= interleukin6;TNF-a= tumornecrosis factoralpha; SCS= static

coldstorage;sTM=solublethrombomodulin;VWF=VonWillenbrandFactor.

24-hourSCSpreservedlivers

Asacontrolgroup,twoporcineliverswerepreservedbySCSfor24hours.Subsequent

warmreperfusionwasperformed for4hours to investigateviability.Macroscopically,

both livers preserved by 24 hours SCS depict large, patchy dark and greyish areas.

During 4 hours warm reperfusion of these grafts, perfusion flow rates gradually

decreased, bile production was absent, pH was decreased to <7.0, and lactate in the

perfusatereached>8mmol/Lat theendofreperfusion.Fig.6AandBdepictaporcine

liver after 24-hours DHOPE preservation, and subsequent 4 hours normothermic

reperfusion with macroscopically normal appearance. In contrast, Fig. 6C shows

normothermic reperfusion after 24-hour preservation by SCS, demonstrating severe

hemorrhagic injury. Because of the severe degree of injury after 24-hours SCS

preservation(i.e.,non-functioninggrafts),a further increase innumberof livers in the

24-hourSCSgroupwasdeemedfutile.

Figure 6. Representative pictures. (A) porcine liver at the end of 24-hour DHOPE

preservation, (B)porcine liverduringreperfusionafter24-hourDHOPEpreservation, (C)

porcineliverduringreperfusionafter24-hourSCSpreservation.Clearly,liverspreservedby

24-hour SCS depict large patchy dark and greyish areas, suggestive of a failing graft.

DHOPE=dualhypothermicoxygenatedmachineperfusion;SCS=staticcoldstorage

HumanpreclinicalexperienceinextendedDHOPE

Finally,we investigatedwhether the observations in our porcinemodel could also be

applied to clinical liver transplantation. Therefore,weperfused twodiscardedhuman

donorliversusingaprotocolcurrentlyusedatourcenter(Fig.7A).21Thefirstliverwas

from a 71-year old DCD donor, with a BMI of 29, history of smoking, macroscopic

hepaticsteatosisof40%,121minutesbetweenwithdrawaloflifesupportanddeath,a

warmischemiatimeof16minutes,and668minutesofSCS.Thesecondgraftwasfroma

59-yearolddonationafterbraindeathdonor,withaBMIof31,ahistoryofsmokingand

hypertension, and a SCS time of 466 minutes. This liver was discarded by another

transplant center because of a macroscopic appearance of poor perfusion, and

subsequently rejected for transplantation nationwide. These human liver grafts were

preserved by traditional SCS during transportation from the donor center and then

subjected to extended DHOPE for 20 hours. After 1 hour of gradual controlled

oxygenatedrewarmingfollowedbyanadditional4hoursofnormothermicreperfusion,

bothgraftsmettheviabilitycriteriathatweuseinourpractice(Fig.7B).Fig.7Bshows

data from our prospective clinical trial, inwhich the greenmargins depict livers that

weredeemedviableafter2.5hoursnormothermic reperfusion.23Thepurpleandblue

lines represent both human livers included in this study. After 2.5 hours reperfusion,

bothliversmettheviabilitycriteria,basedonlactateclearance,thedeltaofbiliaryand

arterial pH, the biliary/arterial glucose ratio, and the delta of biliary and arterial

bicarbonate. Hepatic ATP content was increased 3–5 fold during 20 hours DHOPE

preservation(Fig.7C).PerfusatelevelsofHMGB-1,TNF-a,IL-6,andcfDNAremainedlow

in both livers during the entire period ofDHOPE (Fig. 7C). At the end of reperfusion,

histologyof theextrahepaticbileducts, the intrahepaticbileductsaswell as the liver

parenchyma revealed remarkable preservationwith onlyminor cholangiocellular and

hepatocellularinjury(Fig.7D).

Figure7.24-hourDHOPEpreservationofdiscardedhumanlivergrafts.(A)pictures

of a human liver at the end of DHOPE preservation (left) and during normothermic

reperfusion (right). (B) Markers for graft viability. The green margins depict historical

data of clinically perfused livers thatwere deemed viable after 2.5 hours normothermic

perfusionandthusacceptedfortransplantation.Theredmarginsdepicthistoricaldataof

livers that were deemed non-viable after 2.5 hours reperfusion, which were secondarily

discardedfortransplantation.Thepurpleandbluelinesrepresentthetwoliversincluded

inthisstudy.Bothliversfollowthegreenmargins,indicativeofaviablegraft.Shownare

lactate clearance, the delta of biliary and arterial pH, the biliary/arterial glucose ratio,

and the delta of biliary and arterial bicarbonate. After 2.5 hours, both livers meet the

viability criteria. (C) Hepatic ATP content at the end of DHOPE, and perfusate levels of

HMGB-1, TNF-a, IL-6, and cfDNA during DHOPE preservation. (D) H&E staining of

extrahepatic bile ducts (left), intrahepatic bile ducts (middle), and liver parenchyma

(right),*=bileductlumen.

DISCUSSION

In this studywe have shown that in a porcine DCDmodelwith 30minutes ofwarm

ischemia,liverspreservedbyDHOPEremainedviableforatleast24hours,whereas24-

hourpreservationbySCS resulted innon-viable grafts.24,25DHOPE forup to24hours

appearstobefeasibletoextendexsitulivergraftpreservationtimes.

The results described in this paper provide evidence that DHOPE may substantially

extendliverpreservationtimes.Thesefindingsareimportantforseveralreasons.First,

extendedDHOPEcouldfacilitatelogisticsoforganallocation.Itmaybeusefultoextend

preservation times in cases of suboptimal livers that are more difficult to allocate.

Second,thistechniquecanbefavoredwhenliversneedtobetransportedacrossregions

oflargecountries(e.g.UnitedStates),orbetweencountries(e.g.Eurotransplant).Atthe

same time, it may reduce transportation costs when livers can be shipped with

commercial flights instead of chartered jets. Lastly, storage time can be prolonged in

cases of logistical issues at the recipient center (lack of operating rooms or medical

teams),ortoscheduletransplantationsurgerythenextdayinsteadofduringthenight,

sincethelatterhasbeenassociatedwithagreaterriskofmorbidityandmortality.26

Up to now, it has been shown that short-term (1–2 hours) (D)HOPE prior to

transplantation is an effective approach to mitigate ischemia-reperfusion injury by

slowingdownmitochondrialrespiration.2ThepresentstudyshowsthatDHOPEcanalso

be used to extend preservation times. Only a few studies investigated extended cold

perfusion of donor livers.8,27 Belzer and colleagues were the first who managed to

preserveandtransplantgoodqualitycanineliversafter72-hourhypothermicmachine

perfusion (HMP).8 More than a decade later, Xu et al. published a study on 24-hour

preservationbyHMPinaratreperfusionmodel,butwithoutverygoodresults.27Atthe

endof30minutesreperfusion,LDHwashigherintheHMPgroupcomparedtotheSCS

group, ICG clearance was similar between the groups, and hepatic hyaluronic acid

uptake showed severely impaired hepatic sinusoidal endothelial cells after 24-hour

HMP. Incontrast toourstudy,Xuandcolleaguesdidnotuseanoxygenatedperfusion

solution. It has been shown that complete absence of oxygen during HMP triggers

hepatocyte cell death via mitochondria, and fails to prevent reperfusion injury.2 The

liver is a metabolically high-demanding organ, even under hypothermic conditions.

Therefore, active oxygenation is needed, especially in grafts with more preservation

injury,suchasDCDlivers.2,28,29

A potential disadvantage of HMP is the risk of undesired endothelial injury (shear

stress)intheliversinusoidscausedbyhighervascularresistancesinthecold.30Inour

experiments,we have observed stable perfusion flows during the entire preservation

periodof24hours.Thisisincontrasttowhatotherauthorshavedescribedforextended

HMP.27,31–33 Ifmachine settings are adjustedatportalpressures≤3mmHgandarterial

pressures≤25mmHg,shearstresscanbeavoided.2,34Inthisstudy,usinglowperfusion

pressures,wedidnotobserveactivationofendothelialcells,indicatedbyVWFstaining

aswellaslowlevelsofsTM.sTMisreleasedfromthevascularendotheliumwhenliver

sinusoidsareinjuredby,forexample,graftpreservation.35LevelsofsTMhavealsobeen

correlatedwithelevated liverenzymesand increasedadherenceof leukocytes in liver

tissue.18

Research groupsworld-wide arepushing theboundaries of organpreservation times.

Morerecently,thefeasibilityofextendedpreservationbyNMPwasdemonstrated.11,36,37

The group from Cleveland even shows 86-hour liver perfusion by NMP in a non-

transplantationmodel.11Normothermicconditionsallowtestingoforganviability,butit

alsobearsa riskof severe injuryas theorgan ismuchmoremetabolically active.38 In

addition, normothermic perfusion requires more intensive labor, since the liver

produces waist products and the composition of the perfusate needs continuous

monitoringandadjusting.Hypothermicperfusioncanbeadvantageoussincetheorgan

is in a hypometabolic state with less production of waste products. In addition,

comparedtoNMP,itpreventsgraftlossiftheperfusionsystemfails(thegraftwouldstill

be preserved in SCS). This study shows that temperature can bemaintained at 10°C

without interventions, it does not require an oxygen carrier (e.g. blood), and no

adjustments of the perfusate needed to be done, making it a relatively easy and

substantiallycheaperapproach,whencomparedtoNMP.

Clearlytheseresultsmustbeassessed inthecontextofwhat isanexperimentalstudy

usingporcinelivers.Wedidnotconfirmourfindingsinatransplantationmodel,butan

ex-situ whole blood reperfusion model was used to test liver viability, as done

previously by others.15,39–41 In addition, it was recently shown that 2.5 hours of

normothermicreperfusionissufficienttoassesshepatobiliaryviability.21Itisgenerally

accepted that the pig liver is a very rigorous model of organ preservation, with

maximum successful SCS preservation times that are substantially shorter than those

regularlyachievedinclinicalpractice.36Incombinationwithourpreliminaryresultson

thetwodiscardedhumanlivergrafts,thesearegroundsforoptimismthatcomparable

resultsmaybeachievedintheclinicalsetting.Althoughtheresultsofthecurrentstudy

are encouraging, experimental work with subsequent transplantation to validate this

approach remains necessary. Until then, extended DHOPE should only be performed

withinaresearchsetting.

Inconclusion,thisisthefirststudytoshowsuccessfulextendedpreservationbyDHOPE

ofDCDporcinelivers.Ifconfirmedtotheclinicalsetting,extendedDHOPEcouldbeused

globallytofacilitatetransplantationlogisticsandexpandthedonorpool.

ACKNOWLEDGEMENTS

WearegratefultoArjanvanderPlaats,MartinKuizenga,MarkSlotemaker,andEmma

Offringa (Organ Assist, Groningen, The Netherlands) for their technical support and

assistanceduringtheperfusionexperiments.Wewouldalsoliketothanktheemployees

ofSlagerKroon,Groningen,fortheirwellwillingcooperation.

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8. OpdenDriesS,SuttonME,KarimianN,etal.Hypothermicoxygenatedmachineperfusion prevents arteriolonecrosis of the peribiliary plexus in pig liversdonated after circulatory death. PLoS One. 2014;9(2):e88521.doi:10.1371/journal.pone.0088521

9. MattonAPM,deVriesY,BurlageLC,etal.BiliaryBicarbonate,pHandGlucoseAreSuitable Biomarkers of Biliary Viability During Ex Situ NormothermicMachinePerfusion of Human Donor Livers. Transplantation. November 2018:1.doi:10.1097/TP.0000000000002500

10. vonMeijenfeldtFA,BurlageLC,BosS,AdelmeijerJ,PorteRJ,LismanT.Elevatedplasma levels of cell-free DNA during orthotopic liver transplantation areassociated with activation of coagulation. Liver Transplant. August 2018.doi:10.1002/lt.25329

11. Sido BP, Datsis K, Mehrabi A, et al. Soluble Thrombomodulin—a Marker OfReperfusion Injury After Orthotopic Liver Transplantation. Transplantation.1995;60(5):462-466.

12. SuttonME,opdenDriesS,KarimianN,etal.Criteria forviabilityassessmentofdiscardedhumandonor liversduringexvivonormothermicmachineperfusion.PLoSOne.2014;9(11):e110642.doi:10.1371/journal.pone.0110642

13. opdenDriesS,WesterkampAC,KarimianN,etal.Injurytoperibiliaryglandsandvascular plexus before liver transplantation predicts formation of non-anastomotic biliary strictures. J Hepatol. 2014;60(6):1172-1179.doi:10.1016/j.jhep.2014.02.010

14. Vries Y, Matton APM, Nijsten MWN, et al. Pretransplant sequential hypo- andnormothermicmachineperfusionof suboptimal liversdonatedaftercirculatorydeath using a hemoglobin-based oxygen carrier perfusion solution. Am JTransplant.2019;19(4):1202-1211.doi:10.1111/ajt.15228

15. vanLeeuwenOB,deVriesY,FujiyoshiM,etal.Transplantationofhigh-riskdonorlivers after ex situ resuscitation and assessment using combined hypo- andnormothermicmachineperfusion:Aprospectivetrial.AnnSurg.2019.

16. van Leeuwen OB, de Vries Y, Fujiyoshi M, et al. Transplantation of High-riskDonorLiversAfterExSituResuscitationandAssessmentUsingCombinedHypo-and Normothermic Machine Perfusion. Ann Surg. August 2019:1.doi:10.1097/SLA.0000000000003540

17. MannerM,ShultW,SenningerN,MachensG,OttoG.Evaluationofpreservationdamage after porcine liver transplantation by assessment of hepaticmicrocirculation.Transplantation.1990;50(6):940-943.

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19. LonzeBE,ParsikiaA,FeyssaEL,etal.OperativeStartTimesandComplicationsAfter Liver Transplantation. Am J Transplant. 2010;10(8):1842-1849.doi:10.1111/j.1600-6143.2010.03177.x

20. XuH,LeeCY,ClemensMG,ZhangJX.Pronlongedhypothermicmachineperfusionpreserveshepatocellularfunctionbutpotentiatesendothelialcelldysfunctioninratlivers.Transplantation.2004;77(11):1676-1682.

21. LüerB,KoettingM,EfferzP,MinorT.Roleofoxygenduringhypothermicmachineperfusion preservation of the liver. Transpl Int. 2010;23(9):944-950.doi:10.1111/j.1432-2277.2010.01067.x

22. Westerkamp AC, Mahboub P, Meyer SL, et al. End-ischemicmachine perfusionreduces bile duct injury in donation after circulatory death rat donor liversindependent of the machine perfusion temperature. Liver Transpl.2015;21(10):1300-1311.doi:10.1002/lt.24200

23. Schlegel A, Dutkowski P. Role of hypothermic machine perfusion in livertransplantation.TransplInt.2015;28(6):677-689.doi:10.1111/tri.12354

24. JainS,XuH,DuncanH,etal.Ex-vivostudyofflowdynamicsandendothelialcellstructureduringextendedhypothermicmachineperfusionpreservationoflivers.Cryobiology.2004;48(3):322-332.doi:10.1016/j.cryobiol.2004.01.010

25. Minor T, Manekeller S, Sioutis M, Dombrowski F. Endoplasmic and VascularSurface Activation During Organ Preservation: Refining Upon the Benefits ofMachinePerfusion.Am JTransplant.2006;6(6):1355-1366.doi:10.1111/j.1600-6143.2006.01338.x

26. van der Plaats A,MaathuisMHJ, ’t Hart NA, et al. The Groningen HypothermicLiverPerfusionPump:FunctionalEvaluationofaNewMachinePerfusionSystem.AnnBiomedEng.2006;34(12):1924-1934.doi:10.1007/s10439-006-9207-4

27. Debbaut C, Monbaliu D, Casteleyn C, et al. From Vascular Corrosion Cast toElectrical Analog Model for the Study of Human Liver Hemodynamics andPerfusion. IEEE Trans Biomed Eng. 2011;58(1):25-35.doi:10.1109/TBME.2010.2065229

28. VerhoevenCJ,FaridWRR,deJongeJ,MetselaarHJ,KazemierG,vanderLaanLJW.Biomarkerstoassessgraftqualityduringconventionalandmachinepreservationin liver transplantation. J Hepatol. 2014;61(3):672-684.doi:10.1016/J.JHEP.2014.04.031

29. VogelT,BrockmannJG,PigottD,etal.Successfultransplantationofporcinelivergrafts following 48-hour normothermic preservation. Gruttadauria S, ed. PLoSOne.2017;12(11):e0188494.doi:10.1371/journal.pone.0188494

30. Vogel T, Brockmann JG, Quaglia A, et al. The 24-hour normothermic machineperfusionofdiscardedhumanlivergrafts.LiverTransplant.2017;23(2):207-220.doi:10.1002/lt.24672

31. Schlegel A, Muller X, Dutkowski P. Hypothermic Machine Preservation of theLiver: State of the Art. Curr Transplant reports. 2018;5(1):93-102.doi:10.1007/s40472-018-0183-z

32. Liu Q, Nassar A, Farias K, et al. Comparing Normothermic Machine PerfusionPreservation With Different Perfusates on Porcine Livers From Donors AfterCirculatoryDeath.AmJTransplant.2016;16(3):794-807.doi:10.1111/ajt.13546

33. vonHornC,HannaertP,HauetT,etal.Coldflushafterdynamicliverpreservationprotects against ischemic changes upon reperfusion - an experimental study.TransplInt.October2018.doi:10.1111/tri.13354

34. Minor T, Efferz P, Fox M, Wohlschlaeger J, Lüer B. Controlled oxygenatedrewarmingofcoldstoredlivergraftsbythermallygraduatedmachineperfusionprior to reperfusion. Am J Transplant. 2013;13(6):1450-1460.doi:10.1111/ajt.12235

SupplementaryFig.1.Degreeofinjuryoftheextrahepaticbileductsofliverstreatedwith2,6,or24hoursDHOPEpriortoSCSandafter4hoursnormothermicreperfusion.Thedegreeof(A)biliaryepithelialloss(p=1.000),(B)muralstromanecrosis(p=0.276),(C)arteriolonecrosis(p=0.159),(D)injurytothePBGs(p=1.000),and(E)intramuralbleeding(p=0.570).(F)Presenceofthrombosis(p=0.105).Therewerenostatisticallysignificantdifferencesbetweenthegroupsattheendofreperfusion(Chi-squaretest).Shownherearepercentagesforn=6animalspergroup.

Transplantationofhigh-riskdonorliversafterexsituresuscitationandassessmentusingcombinedhypo-andnormothermicmachineperfusion:aprospectiveclinicaltrial.

OttoB.vanLeeuwen,YvonnedeVries,MasatoFujiyoshi,MaartenW.N.Nijsten,RinseUbbink,GertJanPelgrim,MaureenJ.M.Werner,KoenM.E.M.Reyntjens,AadP.vandenBerg,MariekeT.deBoer,RubenH.J.deKleine,TonLisman,VincentE.deMeijer,RobertJ.Porte.AnnSurg.2019Nov;270(5):906-914

8

ABSTRACT

Objective:Toevaluatesequentialhypothermicandnormothermicmachineperfusionas

atooltoresuscitateandassessviabilityofinitiallydeclineddonorliverstoenablesafe

transplantation.

SummaryBackgroundData:Machineperfusionisincreasinglyusedtoresuscitateand

test the function of donor livers. While (dual) hypothermic oxygenated perfusion

([D]HOPE) resuscitates livers after cold storage, normothermic machine perfusion

(NMP)enablesassessmentofhepatobiliaryfunction.

Methods:Inaprospectiveclinicaltrial,nationwidedeclinedliversweresubjectedtoex

situ NMP (viability assessment phase), preceded by one-hour DHOPE (resuscitation

phase)andonehourofcontrolledoxygenatedrewarming(COR),usingaperfusionfluid

containing an hemoglobin-based oxygen carrier. During the first 2.5h of NMP,

hepatobiliary viability was assessed, using predefined criteria: perfusate lactate

<1.7mmol/L,pH7.35-7.45,bileproduction>10mLandbilepH>7.45.Liversmeetingall

criteria were accepted for transplantation. Primary endpoint was 3-months graft

survival.

Results:SixteenliversunderwentDHOPE-COR-NMP.All liverswerefromdonorsafter

circulatory death, with median age of 63 (range 42-82) years and median

Eurotransplant donor risk index of 2.82. During NMP, all livers cleared lactate and

producedsufficientbilevolume,but in5 liversbilepHremained<7.45.The11(69%)

livers thatmet all viability criteriawere successfully transplanted,with100%patient

and graft survival at 3 and6months. Introduction ofDHOPE-COR-NMP increased the

numberofdeceaseddonorlivertransplantsby20%.

Conclusions: SequentialDHOPE-COR-NMPenabled resuscitation and safe selection of

initiallydeclinedhigh-riskdonorlivers,therebyincreasingthenumberoftransplantable

liversby20%.

INTRODUCTION

Althoughlivertransplantationisalife-savingtreatmentforpatientswithend-stageliver

disease,mortalityon thewaiting list ishighdue to shortageof suitabledonororgans.

Therefore,‘extended-criteriadonor’(ECD)organsofsuboptimalqualityareincreasingly

usedfortransplantation.However,ahighnumberofECDliversisstilldeclinedbecause

theriskofprimarynon-functionorotherseverecomplicationsisconsideredtoohigh.1

Particularly livers fromdonorsaftercirculatorydeath(DCD)arepronetoseveregraft

injury and early failure due to prolonged donor circulatory arrest and hypotension

(warm ischemia).2 The biliary tract is especially vulnerable to ischemic injury,

illustrated by a 3-fold higher incidence of ischemic cholangiopathy after DCD liver

transplantation,compared to transplantationof livers fromdonationafterbraindeath

(DBD).3-5

Ex situ oxygenated machine perfusion is increasingly applied to improve graft

preservation and reduce the risk of graft failure, and various protocols are currently

tested in clinical trials.6-8 Single or dual hypothermic oxygenated machine perfusion

([D]HOPE)reducesischemia-reperfusioninjurybyresuscitatingmitochondria,resulting

inrestorationofcellularenergystoresanddecreasedreleaseofreactiveoxygenspecies

duringtransplantation.9Controlledoxygenatedrewarming(COR)aimstominimizethe

potential deleterious transition between cold preservation and warm reperfusion.10

Normothermic machine perfusion (NMP) reduces ischemic injury and allows ex situ

assessmentofhepatobiliaryfunctionbecausetheorganismetabolicallyactiveat37oC.11

However, when applied after a period of static cold storage, NMP may not provide

optimalprotectionagainstischemia-reperfusioninjury.12-14

Intwopreclinicalstudiesusingdiscardedhumanlivers,ashortperiodof(D)HOPEprior

to NMP resulted in better hepatobiliary function and less injury, compared to NMP

only.12,13AftersequentialDHOPEandNMP,hepaticadenosinetriphosphatecontentand

levels of biliary bicarbonate and bilirubin were higher compared to livers that

underwentNMPonly.12

We, therefore, hypothesized that a combination of DHOPE and NMPwouldmaximize

utilization of the machine perfusion potential when applied after a period of cold

preservation.Totestthishypothesis,weinitiatedaprospectiveclinicaltrialofcombined

DHOPE, COR, and NMP with the aim to resuscitate and test the viability of initially

nationwidedeclinedliverstoallowsafetransplantation.

METHODS

Trialdesign

In this prospective clinical trial, all livers that were nationwide declined for regular

transplantationbetweenAugust2017andOctober2018wereincluded(Figure1).The

study protocol (www.trialregister.nl; NTR5972) was approved by themedical ethical

reviewcommitteeofourcenter(METc2016.281).Allrecipientsgavewritten informed

consent.

Objective of this study was to safely increase utilization of donor livers by ex situ

resuscitation and viability assessment using a protocol combining DHOPE, COR, and

NMP. Primary endpointwas graft survival after 3months. Secondary endpointswere

graftandpatientsurvivalat6months,theincidenceofprimarynon-functionandpost-

transplantcholangiopathy(definedasaspectrumofbileductabnormalities, including

non-anastomotic biliary strictures, intraductal casts and/or intrahepatic biloma

formation),15andbiochemicalserummarkersofgraftfunctionandischemia-reperfusion

injuryatpostoperativeday1-7,andafter1and3months. Inaddition,graftutilization

ratewasdetermined.

Procurementofdonorlivers

Allliverswereprocuredinastandardizedmannerbyadedicatedprocurementteam.16

AlthoughbothDBDandDCDliverswereeligibleforinclusioninthetrial,coincidentally

all included liverswere fromDCDdonors.DCDorganprocurement in theNetherlands

includesamandatory5min‘notouch’periodafterdeclarationofcirculatorydetermined

deathandheparinizationofthedonorisnotpermitted.Afterrapidinsitucoldflushout

andprocurement,liverswerepreservedandtransportedusingstaticcoldpreservation

inUniversityofWisconsin(UW)coldstoragesolution(BridgetoLife,London,UK).Time

betweenstartofcold flushingandeitherthebeginningofmachineperfusion(DHOPE-

COR-NMP) or graft reperfusion in the comparator cohorts, was defined as static cold

ischemiatime.

Perfusionsolution

Toallowbothhypothermicandnormothermicmachineperfusionwithout theneed to

interrupt perfusion to replace the perfusion fluid, an acellular perfusion solution

containing a hemoglobin-based oxygen carrier, HBOC-201, (Hemopure, HbO2

TherapeuticsLLC,Souderton,USA)wasusedforalltemperaturephases.17-19Inaddition

to HBOC-201, the perfusion solution contained gelofusine, albumin, metronidazole,

glutathione, cefazolin, heparin, insulin and sodium bicarbonate (Supplementary Table

1). After the first five perfusions, clinical grade taurocholate came available and was

addedtotheperfusionfluid(50mgatstartofNMPfollowedbycontinuous infusionof

7.7mg/h).Taurocholatewasproducedaccordingtogoodmanufacturingpracticebyour

hospitalpharmacy.

Machineperfusionprocedure

Machine perfusion procedures were performed with the Liver Assist device (Organ

Assist, Groningen, the Netherlands) (Figure 2A). Prior to machine perfusion, a portal

veinflushwasperformedwith2Lofcoldsaline.Allliversunderwent1hourofDHOPE

(8-12ºC)withportalveinandhepaticarterypressuresof5and25mmHg,respectively.

DuringDHOPE,1L/minof100%O2wasadministered,resultinginaPaO2>80kPa.After

1hofDHOPE,temperaturewasstepwiseincreased(1°Cper2minutes),until37°C(COR

phase).Additionally,theportalveinandhepaticarterypressureswereslowlyincreased

to11and70mmHg,respectively.Afterperfusion fluid temperaturereached37ºC, the

NMP phase started to allow viability assessment. During NMP an air/oxygenmixture

wasusedforventilation,aimingatahepaticvenousoxygensaturationof55-75%.

Every 30 minutes, arterial perfusate samples were collected and analyzed with an

ABL90Flex blood gas analyzer (Radiometer, Brønhøj, Denmark). Additionally, every

hour venous outflow samples were collected to determine SvO2 and PvO2. Bile was

collected froma 8Fr biliary drain and volumewasdetermined gravimetrically. At 30-

minute intervals samplesofbilewerecollectedundermineraloil todeterminebiliary

pH,bicarbonate,andglucose.20,21

Viabilityassessment

During the first 2.5h of NMP, hepatobiliary function was tested using the following

criteria:lactate<1.7mmol/L,perfusatepH7.35-7.45,bileproduction>10ml,andbiliary

pH >7.45. When a liver met all viability criteria, the liver was accepted for

transplantation. In non-viable livers, NMPwas discontinued and the liver secondarily

discarded. Viable donor livers remained on NMP until recipient hepatectomy was

completedandimplantationcouldbeinitiated.AfterNMP,liverswereflushedoutwith

2Lof coldUWto remove themachineperfusionsolution.After implantation, the liver

was flushed with 500mL recipient blood via the caval anastomosis following portal

venousreperfusion.

Histologicalanalysis

Biopsiesweretakenfromthedistalcommonbileductbeforemachineperfusion,fixedin

4% formalin, and subsequently embedded in paraffin. Slices of 4 µm were cut and

stainedwithhematoxylin&eosinforexaminationusinglightmicroscopy.Thebileduct

injury (BDI) score was determined in-duplo in a blinded fashion, using a clinically

relevanthistologicalgradingsystem.20,22

Comparatorcohorts

ContemporarycomparatorcohortsofallconsecutiveadultDCDorDBDlivertransplants

performed in our center between January 2017 and October 2018 were formed.

Recipientsofmulti-organtransplantswereexcluded.

Statisticalanalysis

Continuous variables were presented as median (interquartile range; IQR) and

compared using the Mann-Whitney test. Categorical variables were presented as

number (percentage) and compared using Chi-square or Fisher’s exact test where

appropriate. Graft survival was determined with the Kaplan-Meier method, and

differences were determined with the log-rank test. Two-sided P-values <0.05 were

considered statistically significant. Analyses were performed using SPSS, version 23

(IBM,Armonk,NewYork,USA).

RESULTS

Inclusions

Between August 2017 and October 2018, 42 livers were offered for inclusion in the

DHOPE-COR-NMP trial afternationwidedecline for regular transplantation (Figure1).

Ofthese,16liversunderwentmachineperfusion.Donorcharacteristicsaresummarized

inTable1.AllliverswerefromDCDdonorswithamedianageof63(range42-82)years

andamedianEurotransplantdonorriskindex(ET-DRI)of2.82.23

Figure1:FlowchartoftheliversofferedforinclusionintheDHOPE-COR-NMPtrial.

A total of 42 nationwide declined high-risk livers was offered. Twenty-six livers did not

undergo machine perfusion as a result of logistic reasons, too long agonal phase or

macroscopic findingsduringorganprocurement. In theNetherlands,organprocurement

teamswithdraw ifnocirculatoryarrestoccurswithin twohoursofventilator switch-off.

Sixteen livers underwent DHOPE-COR-NMP. Abbreviations: COR; controlled oxygenated

rewarming,DHOPE;dualhypothermicoxygenatedperfusion,NMP;normothermicmachine

perfusion.

Viabilitytesting

DuringNMP,11of16(69%)perfusedlivers(depictedasgreenlinesinFigures2-3)met

all viability criteria (Figure 2B-D). The five livers secondarily declined for

transplantation(depictedasredlinesinFigures2-3)allhadabilepH<7.45at2.5hof

NMP(Figure2D).Allliversclearedlactateandproduced>10mLofbile.Nodifferencesin

portal vein and hepatic artery flows were observed between transplanted and non-

transplanted livers (Table 1). There were no major differences in baseline

characteristicsof transplantedandnon-transplanted livers, apart froma longerdonor

hepatectomytimeandstaticcoldischemiatimeinthelattergroup.

Table 1. Donor and perfusion characteristics of transplanted and non-

transplantedlivers.

Variable Transplanted(n=11)

Non-transplanted(n=5)

p-value

Donorcharacteristics Age(years) 63(52-72) 63(52-71) 0.961Bodymassindex(kg/m2) 25(21-26) 28(24-32) 0.052Gender*MaleFemale

8(73%)3(27%)

4(80%)1(20%)

0.635

Causeofdeath*TraumaCerebrovascularattackAnoxiaOther

2(18%)5(46%)3(27%)1(9%)

2(40%)1(20%)1(20%)1(20%)

0.648

Time from withdrawal of lifesupport to circulatory arrest(min)

14(11-18) 14(8-21) 0.851

Time from circulatory arrest tocoldperfusion(min)

16(14-16) 16(14-20) 0.743

Total donor warm ischemia time(min)

32(25-33) 30(18-37) 0.913

Lastsodium(mmol/L) 143(141-153) 140(133-148) 0.177LastAST(u/L) 60(32-95) 79(53-158) 0.441LastALT(u/L) 62(36-127) 81(33-178) 0.743LastGGT(u/L) 64(30-167) 55(24-543) 0.743LastALP(u/L) 63(52-78) 105(76-346) 0.013Hepatectomytime(min) 44(28-54) 70(44-93) 0.040Staticcoldischemiatime(min) 270(241-294) 326(286-480) 0.018ET-DRI# 2.81(2.60-2.90) 2.82(2.47-3.26) 0.743Perfusion characteristicsat150minutesofNMP

Portalveinflow(ml/min) 1790(1550-2070) 1750(1665-1750) 0.955Hepaticarteryflow(ml/min) 478(420-653) 515(323-871) 0.462PerfusatepH 7.42(7.40-7.44) 7.38(7.34-7.39) 0.115

Perfusatelactate 0.7(0.4-1.1) 1.7(1.3-1.9) 0.068Perfusateglucose 24.0(19.3-29.5) 27.0(24.5-29.0) 0.955BilepH 7.57(7.521-7.61) 7.39(7.32-7.40) 0.002Bilebicarbonate 27.6(24.9-29.6) 18.3(16.9-19.7) <0.001Bileglucose 13.5(6.6-17.0) 20.4(20.2-21.7) 0.055BilepH–perfusatepH^ 0.15(0.10-0.19) 0.01(-0.05-0.05) 0.005Bile bicarbonate – perfusatebicarbonate^

8.6(6.7-11.7) 2.0(1.3-3.1) 0.013

Bileglucose–perfusateglucose^ -9.8(-13.8--8.1) -4.3(-8.0--2.0) 0.052Glucoseratiobileandperfusateº 0.50(0.30-0.63) 0.82(0.76-0.90) 0.013

Continuousdataarepresentedasmedian(IQR),categoricaldataasnumber(percentage).

*Shownasnumber(percentage).#Avalidatedtooltoassesstheriskoflivergraftfailure.23

^Thedifferencebetween thebileand theperfusate. ºTheglucose level in theperfusate

divided by the glucose level in the bile. Abbreviations: AFP; alkaline phosphatase, ALT;

alanine aminotransferase, AST; aspartate aminotransferase, DBD; donation after brain

death, DCD; donation after circulatory death, ET-DRI; Eurotransplant Donor Risk Index,

GGT;gammaglutamyltransferase.

Figure 2: Viability testing during DHOPE-COR-NMP. The green lines indicate the

transplantedlivers,theredlinesrepresentthenon-transplantedliver.Thelightgreenline

indicates the transplanted liver that developed post-transplant cholangiopathy. In livers

thatdidnotmeetalviabilitycriteriaduringthefirst2.5hofNMP,NMPwasdiscontinued

and liversweresecondarilydiscarded. Inviable livers thatmetallviabilitycriteria,NMP

wascontinueduntilrecipienthepatectomywascompleted.A,Liverfroma82-yearoldDCD

donor with a functional warm ischemia time of 33 minutes. This liver met the viablity

criteria and was successfully transplanted. Portal vein and supratruncal aorta were

cannulated,anddrainswereplacedinthecommonbileductandthesuprahepaticinferior

vena cava. B, Lactate decreased during NMP. C, Perfusate pH normalized during NMP

except forone liver.D,BilepH increasedduringNMP,but remainedbelow7.45 in the5

non-transplanted livers. E, Bile glucose decreased during NMP. F, Bile bicarbonate

increasedduringNMP,butremained lowinthe5non-transplanted livers.Abbreviations:

COR;controlledoxygenatedrewarming,DHOPE;dualhypothermicoxygenatedperfusion,

min;minutes,NMP;normothermicmachineperfusion.

ClinicaloutcomesDHOPE-COR-NMPversuscomparatorcohorts

Themedian follow up of the patients that received a DHOPE-COR-NMP liver was 12

months(range8-22months).UsingtheDHOPE-COR-NMPprotocolforresuscitationand

viability testing of initially declined livers,we achieved actuarial 3-, 6-, and12-month

graftsurvivalratesof100%.Patientsurvivalwas100%atthesetimepoints.Outcomes

werecomparedtocontemporarycomparatorcohortsofregularDBDorDCDliversthat

were transplanted in our center outside the DHOPE-COR-NMP protocol (Table 2).

Notably,donorsofregularDCDliversweresignificantlyyounger(52vs.63years),and

had a significantly lower ET-DRI (2.34 vs. 2.81) (Table 2). Actuarial graft and patient

survivalratesintheDBDcomparatorcohortwere100%at3,6and12months.Actuarial

graftsurvivalratesintheDCDcomparatorcohortwere83%,83%and80%at3,6and

12months, respectively, and patient survival was 92%, 92%, and 88% at these time

points.GraftandpatientsurvivalwerenotsignificantlydifferentbetweentheDHOPE-

COR-NMP cohort and the two comparator cohorts. During execution of the trial, 56

regular deceased donor liver transplantations were performed, indicating a 20%

increaseduetothemachineperfusionprotocol.

Table2.Donor,recipientandtransplantationcharacteristicsandpost-transplant

outcomesofthetransplantedDHOPE-COR-NMPliversversusthecomparator

cohorts.

ComparatorcohortVariable DHOPE-COR-NMP

(n=11)DBD(n=36)

DCD(n=24)

p-valueDHOPE-COR-NMPversusDBD

p-valueDHOPE-COR-NMPversusDCD

Donor Typeofdonor*DBDDCD

0(0%)11(100%)

36(100%)0(0%)

0(0%)24(100%)

--

--

Age(years) 63(52-72) 61(47-66) 52(48-56) 0.227 0.005Gender(male)* 8(73%) 20(56%) 15(63%) 0.485 0.682Bodymassindex(kg/m2) 25(21-26) 26(24-28) 27(22-29) 0.057 0.092Causeofdeath*TraumaCVAAnoxiaOther

2(18%)5(46%)3(27%)1(9%)

7(19%)21(58%)7(19%)1(3%)

6(25%)8(33%)5(21%)5(21%)

0.116 0.452

Timefromwithdrawaloflifesupporttocirculatoryarrest(min)

14(11-18) - 12(8-15) - 0.046

Timefromcirculatoryarresttocoldperfusion(min)

16(14-16) - 16(14-20) - 0.445

Totalwarmischemiatime(min)

32(25-33) - 28(23-33) - 0.274

Lastsodium(mmol/L) 140(133-148) 146(144-149)

143(140-146)

0.479 0.540

LastGGT(u/L) 64(30-167) 44(21-76) 41(18-63) 0.268 0.186LastALT(u/L) 62(36-127) 40(22-69) 33(18-55) 0.123 0.025LastAST(u/L) 60(32-95) 49(28-79) 49(19-78) 0.364 0.211LastALP(u/L) 63(52-78) 66(49-85) 61(50-97) 0.904 0.923ICUstay(days) 3(1-5) 2(1-2) 3(1-5) 0.090 0.923Hepatectomytime(min) 44(28-54) 46(35-55) 48(37-84) 0.296 0.114ET-DRI# 2.81(2.60-2.90) 1.75(1.48-

1.90) 2.34(2.14-

2.49)<0.001 <0.001

Recipient Age(years) 61(55-66) 52(40-

60) 56(48-61) 0.017 0.198

Gender(male)* 7(64%) 22(61%)

13(54%) 1.000 0.721

Bodymassindex(kg/m2) 28(25-32) 25(22-28)

27(23-30) 0.150 0.494

LabMELD-score° 14(13-15) 15(10-22)

18(11-24) 0.405 0.268

ICUstaypriortotransplant

1(9%) 4(11%) 1(4%) 1.000 0.536

Retransplantation*Transplantindication*

1. NASH/NAFLD2. Post-alcoholic

cirrhosis3. Biliarydiseases≠4. Other

0(0%) 9(25%) 3(12%) 0.092 0.5364(37%)3(27%)3(27%)1(9%)

3(8%)5(14%)11(31%)17(47%)

6(25%)4(17%)6(25%)8(33%)

0.070 0.632

Transplantation Staticcoldischemiatime(min)

270(241-294) 407(352-471) 446(379-492)

<0.001 <0.001

Totalpreservationtime(min)

868(805-924) 407(352-471) 446(379-492)

<0.001 <0.001

Anastomosistime(min) 38(32-44) 42(37-51) 42(34-52) 0.124 0.352Estimatedbloodloss(ml) 4000(2400-7700) 3450(1960-

6250) 3475(1987-4886)

0.382 0.409

RBCtransfusion(units) 5(3-8) 3(1-8) 5(0-10) 0.183 0.586FFPtransfusion(units) 0(0-0) 0(0-3) 0(0-4) 0.703 0.445Implantation(piggyback)*8(73%) 32(89%) 21(88%) 0.330 0.352Post-operativeresults Actuarialgraftsurvival3-months

100%

100%

83.3%

1.000

0.159

6-months 100% 100% 83.3% 1.000 0.15912-months 100% 100% 80% 1.000 0.207PeakALT(u/L) 683(282-757) 1195(600-

2285) 1598(652-3191)

0.009 0.001

PeakAST(u/L) 751(483-1757) 1646(627-2494)

2406(1102-4573)

0.165 0.004

Bilirubinday7(umol/L) 16(8-27) 21(14-62) 16(12-37) 0.143 0.510INRday7 1.0(1.0-1.0) 1.0(1.0-1.1) 1.1(1.0-1.1) 0.123 0.076Post-transplantcholangiopathy*

1(9%) 3(8.3%) 4(18%) 1.000 0.643

Anastomoticstricture* 3(27%) 11(31%) 4(18%) 1.000 0.656Primarynon-function* 0(0%) 0(0%) 2(8.3%) 1.000 1.000Hepaticarterythrombosis*

0(0%) 0(0%) 0(0%) - -

Acuterejection* 1(9%) 3(8.3%) 1(4%) 1.000 1.000

Continuousdataarepresentedasmedian(IQR),categoricaldataasnumber(percentage).

* Shown as number (percentage). # A validated tool for assess the risk of liver graft

failure23. ° Defined as the last laboratory-derived Model for End-stage Liver Disease

(MELD) score. ≠ Including primary biliary cirrhosis, primary sclerosing cholangitis.

Abbreviations:AFP;alkalinephosphatase,ALT;alanineaminotransferase,AST;aspartate

aminotransferase,CVA,cerebrovascularaccidentCPR;cardiopulmonaryresuscitation,ET-

DRI; Eurotransplant Donor Risk Index, FFP; fresh frozen plasma, GGT; gamma

glutamyltransferase,ICU;intensivecareunit,INR;internationalnormalizedratio,NAFLD;

non-alcoholicfattyliverdisease,NASH;non-alcoholicsteatohepatitis,RBC;redbloodcell.

Developmentofpost-transplantcholangiopathy

OnerecipientofaDHOPE-COR-NMP liverdevelopedcholangiopathyat4monthsafter

transplantation, characterized by recurrent jaundice, pruritus and cholangitis. Biliary

anastomosis in this patientwas constructed using a Roux-en-Y hepatico-jejunostomy.

Magnetic resonance cholangiography revealed biliary irregularities with intraductal

casts (Figure3A). Thepatient underwent surgical removal of thebiliary casts (Figure

3B)withshorteningoftheextrahepaticbileductandre-anastomosisofthejejunalloop,

afterwhichbileflowwasrestored.Intraoperativecholangioscopydemonstratedinjury

oftheintrahepaticbileducts(Figure3C).

Duringpretransplantmachineperfusionof this liver,perfusate lactatewas<1mmol/L,

perfusatepH7.46,andbilepHwas7.45after2.5hofNMP(lightgreenlineinFigures2-

3).Althoughthislivermetallviabilitycriteria,inretrospectwehavelearnedfromthis

casethatthedifferencebetweenbileandperfusatepH,bicarbonateandglucosemaybe

morepredictiveofbileductviabilitythantheabsolutebiliaryvalues.(Figure3E-H).

Baselinebileductbiopsyof this liver showedsignsof severe injury to theextramural

peribiliaryglands,necroticvesselsanddiffusestromanecrosis(BDIscore6;Figure3D).

AnalysisofallbaselinebileductbiopsiesrevealedasignificantlyhigherBDIintheliver

whichdevelopedcholangiopathyand theothernon-transplanted livers (medianBDI5

[4-6]),whencomparedtothetransplantedliverswithoutsymptomsofpost-transplant

cholangiopathy(medianBDI3[2-4];p=0.031).

Figure3.Developmentofpost-transplant cholangiopathyafterDHOPE-COR-NMP

anddiscriminatingpowerofbilecomposition.

A, Magnetic resonance cholangiography of the patient that developed post-transplant

cholangiographyrevealedbiliaryirregularitieswithintraductalcastformation.B,Alarge

intraductal cast was subsequently removed. C, Intraoperative cholangioscopy

demonstratedinjuryoftheintrahepaticbileducts.D,Thebaselinebileductbiopsyofthe

liver that developed post-transplant cholangiopathy revealed severe injury to the

extramuralperibiliaryglands,necroticvesselsanddiffusestromanecrosis.Magnification:

10x.E-H,thedifferencebetweenbileandperfusatepH,bicarbonateandglucoseappeared

to bemore indicative of biliary damage than the absolute value of pH, bicarbonate and

glucoseinthebile.G,theglucosevalueinthebiledividedbytheglucoseintheperfusate

showed a clear difference in glucose reabsorption by the biliary epitheliumbetween the

transplanted and non-transplanted livers. Abbreviations: COR; controlled oxygenated

rewarming, DHOPE; dual hypothermic oxygenated perfusion, min; minutes, NMP;

normothermicmachineperfusion.

DISCUSSION

Thisprospective clinical trial suggests that sequentialDHOPE,CORandNMP is a safe

andefficaciousinterventiontoallowsuccessfultransplantationofinitiallydeclinedhigh-

risk donor livers. After transplantation of DHOPE-COR-NMP livers, graft and patient

survival rates were at least similar when compared to contemporary comparator

cohortsofregularDCDorDBDlivergrafts.

To the best of our knowledge, two studies have been published on transplantation of

discardedhumandonorlivers.InthefirstseriesbytheBirminghamgroup,sixdiscarded

humandonorliversunderwentviabilitytestingbyusingend-ischemicNMP,ofwhich5

liversweresubsequentlytranplanted.24Theaveragedonorageofthesefiveliverswas

45 years, and all grafts functioned well after transplantation. In this study, viability

criteria duringNMP included lactate clearance, perfusate pH >7.30, stable portal vein

andhepaticarteryflows,andahomogeneousgraftperfusion.Noviabilitytestingofthe

biliary tree was performed. In the second study conducted by the Cambridge group,

twelve discarded human donor livers underwent end-ischemic NMP, followed by

transplantation.25Inthisstudy,thecompositionofbilewasmeasured,butnotusedfor

viability testing. Of the twelve livers, one developed primary non-function and three

livers(27%)developedpost-transplantcholangiopathy.Anassociationbetweenlowbile

pHduringNMPanddevelopmentofpost-transplantcholangiopathywasfoundandthe

authors suggested bile pH as a potential viability criterion. In a follow-up paper, the

same authors described a correlation between bile pH, glucose and bicarbonate,with

biliary injury.26 Previous clinical series ofDCD liver transplantation indicated a lower

incidenceofpost-transplant cholangiopathy if liversunderwent (D)HOPE.9,27Although

these results still have to confirmed by ongoing randomized controlled trials,28 we

decidedtoperformDHOPEprior toNMPtoattenuatethebiliary ischemia-reperfusion

injury and to include biliary viability testing, to allow more safe transplantation

comparedtotheseearlierexperiences.

Inanotherrecentlycompletedstudyontransplantationofinitiallydiscardedlivers,the

VITTAL-studybytheBirminghamgroup,onlyhepatocellularfunctionwastestedduring

NMP, while viability testing of the biliary treewas not performed.29 One of themain

strengthsinthecurrentstudyisthatbothhepatocellularandcholangiocellularfunction

were assessed, based on previously determined biomarkers of bile duct viability.20 In

ourstudy,all16liversmetthehepatocellularviabilitycriteriausedintheVITTAL-study,

but only 11 met our criteria for cholangiocellular function (bile pH >7.45). In the

VITTAL-study,45%ofpatientsdevelopedbileductirregularitiesonmagneticresonance

cholangiographyand18%requiredretransplantationforbiliarystrictures.30,31Although

this may provide a strong rationale for additional biliary viability testing, definitive

proofwouldrequirearandomizedcontrolledtrial.

Withamedianfollowupof12months(range8-22months),wehaveobservedonlyone

case of post-transplant cholangiopathy after DHOPE-COR-NMP of high-risk DCD liver

graftswithamedianageof63(range42-82)years.Healthybiliaryepitheliummodifies

bile composition by secreting bicarbonate, leading to a protective alkalotic biliary

environment.20,32,33 Additionally, biliary epithelium reabsorbs glucose, decreasing

glucoselevelsinbile.20,34-37DuringtheNMP-phaseoftheoneliverthatdevelopedpost-

transplant cholangiopathy, the bile pH, bicarbonate, and glucosewere similar to their

levelsintheperfusate,suggestingimpairedbiliaryepithelialfunction.Sincethiscase,we

considerthedifferenceofpH,bicarbonate,andglucosebetweenperfusateandbileasa

markerofbiliaryviabilityinsteadofabsolutevaluesonly.

While end-ischemicDHOPE reduces reperfusion injury inDCD liver transplantation38,

post-ischemic biliary injury already present before machine perfusion cannot be

repaired.We therefore presume thatwith our currentDHOPE-COR-NMPprotocol,we

have safely pushed the boundaries of end-ischemic machine perfusion for high-risk

donorlivers,butthistechniquedoesnotenableactiverepairofpre-existingseverebile

ductinjury.Therefore,alternativestrategiessuchasnormothermicregionalperfusionof

DCDdonorsmayprovideadditionalbenefitandhelp toavoidbileduct injuryprior to

staticcoldpreservation.39,40

A limitationof thisstudy is the lackofrandomizationbetweenmachineperfusionand

traditionalstaticcoldpreservationonly.Inourcountry,post-transplantcholangiopathy

hasbeenobservedinupto30%ofDCDliverrecipientsandthereforeDCDliverswitha

donor age >60 years are usually declined for regular transplantation.3-5 Ethical

constraints refrained us from accepting these and other high-risk livers for

transplantationwithoutaddinganyinterventionandwiththesoleintentiontoserveas

a control group in this trial. We, therefore, used comparator cohorts of all adult

recipientsofregularDCDorDBDlivergraftsduringthestudyperiod.Patientandgraft

survival were not different between these groups. Another possible limitation is that

livers declined for transplantation based on a low bile pH during NMP, were not

transplantedandthereforehavenofollowup.Althoughwehavenocertaintythatthese

livers would have developed post-transplant cholangiopathy, previous (pre)clinical

studiesindicatedasubstantiallyincreasedrisk.20,22,26

In conclusion, this prospective clinical trial demonstrates the safety and feasibility of

transplantation of high-risk ECD liver grafts using sequential DHOPE, COR, and NMP.

Pretransplantresuscitationandviabilityassessmentoftheseinitiallydeclinedliversled

toa20%increaseinnumberofdeceaseddonorlivertransplantsinourcenter.

ACKNOWLEGDEMENTS

HBOC-201(Hemopure)waskindlyprovidedbyZafirisZafirelis(HBO2TherapeuticsLLC,

Souderton,USA).WearegratefultotheDutchtransplantcoordinatorsfortheirhelpin

identifying potential donors, whose livers could be eligible for the DHOPE-COR-NMP

trial.TheperfusiondeviceandsolutionusedinthisstudyarenotFDAapproved.

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25. Watson CJE, Kosmoliaptsis V, Randle LV, et al. Normothermic perfusion in theassessmentandpreservationofdeclinedliversbeforetransplantation:hyperoxiaand vasoplegia – important lessons from the first 12 cases. Transplantation.2017;101:1084-1098.

26. WatsonCJE,KosmoliaptsisV,PleyC,etal.Observationsontheexsituperfusionofliverfortransplantation.AmJTransplant.2018;18;2005-2020.

27. Dutkowski P, Polak WG, Muiesan P, et al. First Comparison of HypothermicOxygenated PErfusion Versus Static Cold Storage of Human Donation AfterCardiac Death Liver Transplants: An International-matched Case Analysis. AnnSurg.2015;262:764-770.

28. VanRijnR,vandenBergAP,Erdmann JI, etal.Studyprotocol foramulticenterrandomized controlled trial to compare the efficacy of end-ischemic dualhypothermicoxygenatedmachineperfusionwithstaticcoldstorageinpreventingnon-anastomotic biliary strictures after transplantation of liver grafts donatedafter circulatory death: DHOPE-DCD trial. BMC Gastroentrol. 2019;19:40. DOI:10.1186/s12876-019-0956-6.

29. Laing RW, Mergental H, Yap C, et al. Viability testing and transplantation ofmarginallivers(VITTAL)usingnormothermicmachineperfusion:studyprotocolfor an open-label, non-randomised, prospective, single-arm trial. BMJ Open.2017;7:e017733.

30. Laing RW, Boteon YL, Kirkham A, et al. Transplantation of discarded liversfollowing viability testing with normothermic machine perfusion: the VITTAL(VIability Testing and Transplantation of mArginal Livers) trial outcomes.

[Abstract]. InternationalLiverTransplantationSociety,25thAnnualInternationalCongres2019(https://2019.ilts.org).

31. NeilD,HubscherS,LaingR,etal.Viabilitytestingandtransplantationofmarginaldonor livers VITTAL trial outcomes: bile duct injury assessment during thenormothermic machine perfusion of discarded livers. [Abstract]. InternationalLiver Transplantation Society, 25th Annual International Congres 2019(https://2019.ilts.org)..

32. SutorDJ,WilkieLI.Diurnal variations in thepHofpathological gallbladderbile.Gut.1976;17:971-974.

33. BoyerJL.Bileformationandsecretion.ComprPhysiol.2013;3:1035-1078.34. LazaridisKN,PhamL,VromanB,deGroenPC,LaRussoNF.Kineticandmolecular

identification of sodium-dependent glucose transporter in normal ratcholangiocytes.AmJPhysiol.1997;272:1168-1174.

35. MasyukAI,MasyukTV,TietzPS,SplinterPL,LaRussoNF.Intrahepaticbileductstransport water in response to absorbed glucose. Am J Physiol Cell Physiol.2002;283:785-791.

36. ScheinCJ,ZumoffB,KreamJ,CassoutoJ,HellmanL.Ablood-bileglucosebarrierinman.Gastroenterology.1968;54:1094-1097.

37. GuzelianP,Boyer JL.Glucosereabsorption frombile.Evidence forabiliohepaticcirculation.JClinInvest.1974;53:526-535.

38. vanRijnR,vanLeeuwenOB,MattonAPM,etal.Hypothermicoxygenatedmachineperfusion reduces bile duct reperfusion injury after transplantation of donationaftercirculatorydeathlivers.LiverTranspl.2018;24:655-664.

39. WatsonCJE,Hunt F,Messer S, et al. In situnormothermicperfusionof livers incontrolledcirculatorydeathdonationmayprevent ischemic cholangiopathyandimprove graft survival. Am J Transplant. 2018 Dec 27; [Epub ahead of print].Accessedon02/11/2019DOI:10.1111/ajt.15241

40. HessheimerAJ,CollE,TorresF,etal.Normothermicregionalperfusionvs.super-rapidrecoveryincontrolleddonationaftercirculatorydeathlivertransplantation.JHepatol.2019;70:658-665.

Exsitumachineperfusionofhumandonorliversviathesurgicallyreopenedumbilicalvein:aproofofconcept.

OttoB.vanLeeuwen,MasatoFujiyoshi,RinseUbbink,MaureenJ.M.Werner,IsabelM.A.Brüggenwirth,RobertJ.Porte,VincentE.deMeijer.Transplantation.2019Oct;103(10):2130-2135.

9

ABSTRACT

Background: Machine perfusion of donor livers is typically performed via the portal

veinmainstem.Instead,cannulationofareopenedumbilicalveincouldallowmachine

perfusion during organ procurement and subsequent implantation in the recipient

withoutinterruptionoftheportalvenouscirculation.Weaimedtoassessthefeasibility

ofportalvenousmachineperfusionviatheumbilicalvein.

Methods:Duringbacktableinspectionoffivehumanliversdeclinedfortransplantation,

the umbilical veinwas surgically reopened, dilated and cannulated. Hypothermic and

normothermic oxygenated machine perfusion (NMP) were performed using the

umbilicalveinforportalinflow.Threeliverswereperfusedwithhypothermicmachine

perfusion,one full livergraftunderwentNMP for fourhours, andone left lateral split

procedurewasperformedundercontinuousNMPwithportalperfusionviatheumbilical

vein.

Results: In all livers access to the portal venous system via the umbilical vein was

successfully achieved, with good portal flows and macroscopically homogeneous

perfusion.ThefulllivergraftthatunderwentNMPviatheumbilicalveinforfourhours

showedgoodlactateclearance,normalizedpHandachievedgoodbileproductionwith

pH>7.55.DuringthesplitprocedureundercontinuousNMPviatheumbilicalvein,the

left lateral segment and extended right lobe remained equally perfused, as

demonstratedbyDoppler-ultrasound.

Conclusion:Machineperfusionwithportalperfusionviatheumbilicalveinis feasible.

Portalvenousflowsweresimilartothoseobtainedaftercannulationoftheportalvein

main stem. This technique enables continuous oxygenated perfusion of liver grafts

duringprocurement,splittingandimplantation.

INTRODUCTION

Livertransplantationisthetreatmentofchoiceforpatientswithend-stageliverdisease.

However, donor organ shortage remains a limiting factor in the wider application of

liver transplantation.Thisdiscrepancybetweenorganavailabilityanddemandhas led

totheincreaseduseofso-called‘extendedcriteria’donor(ECD)grafts.TheuseofECD

livers is associated with increased rates of ischemia-reperfusion injury-related

complications, such as primary graft non-function, early allograft dysfunction, and

biliary complications.1 This has led to a large number of livers being declined for

transplantation. Ex situ oxygenatedmachine perfusion is increasingly applied to ECD

liverstoreducetheriskofgraftfailure,andhasmadethetransitiontoclinicaltrials.2

Recently,Heetal.reportedthefirstcaseofischemia-freelivertransplantation(IFLT)in

humans.3 Their innovative technique of continuous normothermic machine perfusion

(NMP) during liver procurement, preservation and transplantation is a major

breakthrough, because for the first time a liverwas successfully transplantedwithout

any ischemia or reperfusion. The authors used a donor iliac vein graft that was

anastomosedend-to-sidetothedonorportalveinmainstemtoenablecannulationand

perfusionof theportalvenoussystemwithout interruptingthebloodcirculation,even

duringporto-portalanastomosisintherecipient.3However,thisadditionalend-to-side

anastomosis may predispose to thrombosis, potentially leading to portal venous

complications.

Our group recentlypostulated theuseof the surgically reopenedumbilical vein as an

alternativeroute to theportalvenoussystemtoovercome theneed toanastomosean

iliacveingraft to theportalveinmainstem.4Access to theportalvenoussystemviaa

surgically reopenedumbilical veinhaspreviouslybeenused for successful retrograde

tumorthrombectomyoftheportalveinmainstem.5Therefore,wehypothesizedthata

surgically reopened umbilical vein would provide good access to the portal venous

system formachineperfusion (Figure1A-B).Weherepresentour first experiencesof

human livermachine perfusionwith portal perfusion via the umbilical vein in a pre-

clinicalsetting.

METHODS

BetweenJanuaryandMay2018, fivehumanECDliversdeclinedfortransplantation in

theEurotransplant regionwere offered to our center formachineperfusion research.

This studywas approved by themedical ethical committee of the UniversityMedical

CenterGroningenandtheDutchTransplantSociety,thecompetentauthorityfororgan

donationintheNetherlands.Donorrelativesprovidedconsentforresearchpriortothe

donationprocedure.Foralllivers,donationaftercirculatorydeathcombinedwithdonor

ageexceeding60yearswasthereasonforgraftdiscard.Allorganswereprocuredusing

asuper-rapidprocurementtechniquedescribedpreviously.6

Organinspectionandpreparationformachineperfusion

Allliverswerepreparedfordualmachineperfusionusingtheumbilicalveinforportal

perfusion and the supratruncal aorta for arterial perfusion. Opening of the umbilical

veinwasperformedbycarefulexplorationoftheroundligamentoftheliver(Figure1C).

Assoonastheumbilicalveinwasidentifiedanddissected,aFogartyballooncatheter(8

Fr,EdwardsLifeSciences,Nyon,Switzerland)was introduced todilate theobliterated

vessel(Figure1D)toallowadequateopeningof theentrance intothe left intrahepatic

portal veinbranch (Figure1E). Inall five livers, a connectionbetween theportalvein

and umbilical vein could be obtained easily (Figure 1F).Hereafter, the portal cannula

from the Liver Assist device (Organ Assist, Groningen, the Netherlands)was inserted

intotheumbilicalveinwithitstipclosetothelumenoftheleftportalveinbranchand

wassecured(Figure1G).Theproximalstumpoftheportalveinmainstemwasclosed

withanon-traumaticvascular clamp (Figure1H).The first three liverswereperfused

hypothermically (10-12 ºC) with University of Wisconsin (UW) Machine Perfusion

solution(BridgetoLife,London,UK).Livers#4and#5wereperfusednormothermically

(37ºC) with our clinically used NMP solution containing a hemoglobin-based oxygen

carrier.6Thehypothermicandnormothermicoxygenatedmachineperfusionprocedures

were performed using the Liver Assist, a pressure-controlled perfusion device, as

describedpreviously.6,7

Figure 1. A: Schematic drawing of human portal venous anatomy with usual machine

perfusion via the portal vein main stem. B: Schematic drawing of machine perfusion

through the umbilical veinwith alternative flowpattern. C: Theumbilical ligament of a

humandonorliverpriortodissectionoftheumbilicalvein.D:IntroductionoftheFogarty

balloon catheter to pneumatically dilate the obliterated umbilical vein. E. Adequately

dilatedinsertionoftheumbilicalveinintotheleftintrahepaticportalveinbranch(denoted

by the *). F: Connection between umbilical vein and portal vein main stem ensured

(demonstratedbyaprobe).G:Cannulationof theumbilicalveinwitha24Frcannula.H:

Normothermicmachineperfusionvia theumbilicalveinand thesupratruncalaorta.The

proximal stump of the portal veinmain stemwas closedwith a non-traumatic vascular

clamp. Abbreviations: FBC: Fogarty balloon catheter, PV: portal vein, SA: supratruncal

aorta,UL:umbilicalligament,UV:umbilicalvein.

RESULTS

Hypothermicmachineperfusionviatheumbilicalvein

In the first liver, a smaller cannula (12 Fr) than usual (24 Fr)was introduced in the

umbilical vein. However, due to the increased resistance of the smaller cannula,

umbilicalveinflowswerelower(80-100mL/minatapresetpressureof5mmHg)than

previouslydescribedduringhypothermicmachineperfusion(i.e.,200-300mL/minat5

mmHg).5Inlivers#2and#3,theumbilicalveinwascannulatedwiththeusualcannula

forportalveincannulation(24Fr).Inthesecases,umbilicalveinflowratesweresimilar

tovaluesobtainedduringnormalhypothermicperfusionviatheportalveinmainstem.

(i.e.,>200mL/minatapressureof5mmHg).6Pulsatilearterialflowswerecomparable

(75-100mL/min at a preset pressure of 25mmHg) to flows as described previously

duringdualhypothermicmachineperfusion(i.e.,50-100mL/minat25mmHG).6

Normothermicmachineperfusionviatheumbilicalvein

The fourth liver was prepared for NMP through the umbilical vein and supratruncal

aorta (Figure2A).DuringNMP, this full livergraft cleared lactate (Figure2B)and the

perfusatepHreachednormalvalueswithintwohours(Figure2C).Dopplerultrasound

analysisconfirmedaccuratepositionofthetipoftheperfusioncatheterjustpriortothe

lumenoftheleftportalveinbranch(Figure2D),demonstratedadequatebloodflowin

both the left and the right portal venous branches (Figure 2E), as well as in all the

hepatic veins (Figure 2F). To analyze the bile production from the left and right liver

lobeseparately,boththe leftandrighthepaticbileductwerecannulatedandbilewas

collectedfromeachliverlobe.Theamountofbileproducedwas>10ml.Afteronehour

ofNMP,thepHofthebilefrombothliverlobeswas>7.55,andaftertwohoursofNMP

>7.65.

Figure2.A:Normothermicmachineperfusionviatheumbilicalvein.B:Lactateclearance

of the full liver graft during normothermic machine perfusion. C: Normalization of

perfusate pH of the full liver graft during normothermicmachine perfusion. No sodium

bicarbonate was administered. D: Ultrasound image with doppler acknowledging the

position of the 24Fr perfusion cannula (denoted by the *) in the umbilical vein. E:

Ultrasound image with doppler demonstrating the patency of peripheral portal vein

branches in the right liver lobe.F: Ultrasound imagewith doppler visualizing adequate

venousoutflowfromtherighthepaticvein.Abbreviations:LPV:leftportalvein,PV:portal

vein,RHV:righthepaticvein,SA:supratruncalaorta,S7PV:segment7portalveinbranch,

UV:umbilicalvein.

Experimentalexsituleftlateralsegmentsplitprocedure

ThefifthliverwaspreparedforNMPthroughtheumbilicalveinandsupratruncalaorta

toassessthefeasibilityofaleftlateralsegment(LLS)splitprocedureundercontinuous

NMPwith subsequent continuedmachine perfusion of the LLS graft. The portal vein

cannula (24 Fr) was introduced in the umbilical vein and the arterial cannula in the

supratruncalaortaafterwhichNMPwasinitiated(Figure3A).Theorganwasperfused

at 37 ºC during transection of the liver parenchyma using the CUSA device (Excel+,

Integra LifeSciences, Tullamore, Ireland), followedby sharp division of the hilar plate

(Figure3B,C). Finally, under continuousoxygenatedNMP the right hepatic artery and

rightportalveinweredividedandthelefthepaticveinwasdissectedfromtheinferior

vena cava. The extended right lobe was removed from the reservoir while the LLS

remainedundercontinuousNMP(Figure3D).Theextendedrightlobewasimmediately

flushedandpreservedinabowlwithUWsolutionandsterileice.Inthehourfollowing

the splittingprocedure, theLLSgraft cleared lactateand thepHof theperfusion fluid

maintained within normal values (between 7.35-7.45). The umbilical venous and

pulsatile arterial flows of the LLS remained >400 mL/min and 150-200 mL/min,

respectively.

Figure3.A:Experimentalsettingforthesplittingofliver5.Theportalveinwasclamped

and the whole liver graft was perfused normothermically via the umbilical vein. B:

Experimental setting for the splittingof liver5.TheLLSwasperfusedwithNMPvia the

umbilicalveinandsupratruncalaortaafterthesplitting.TheERLwasremovedfromthe

perfusion device andpreserved on ice. C: Left lateral split procedure during continuous

normothermic machine perfusion. D: Normothermic machine perfusion of left lateral

segmentaftersplittingundernormothermicmachineperfusionviatheumbilicalveinand

supratruncal aorta. E: Conceptual clinical ex situ splitting procedure: until portal vein

dissection, continuous oxygenated machine perfusion via the portal vein main stem is

performed. The umbilical vein is cannulated but clamped to prevent blackflow. F:

Conceptual ex situ split procedure: after portal vein dissection, hypothermic machine

perfusion via both the portal vein main stem and umbilical vein allows continuous

oxygenation of both partial liver grafts during and after splitting. Abbreviations: ERL:

extended right lobe, LHV: lefthepatic vein, LLS: left lateral segment,PV:portal vein, SA:

supratruncal,aortaUV:umbilicalvein.

Proposed ex situ split procedure using continuous oxygenated hypothermic

machineperfusion.

Theabovementionedconceptcouldalsobeappliedbyusingsingle(portalvenousonly)

ordual(portalvenousandarterial)hypothermicmachineperfusiontoallowcontinuous

oxygenatedLLS andextended right lobe split, or even full-left and full-right lobe split

procedure.Todoso,portalveinmainstemandumbilicalveinshouldbothbecannulated

with a portal vein cannula (24 Fr). For dual perfusion, the arterial cannula should be

introduced in the supratruncal aorta. For the parenchymal transection phase

hypothermicoxygenatedmachineperfusioncanbeinitiatedviathecannulaintheportal

veinmainstem,while thecannula in theumbilicalveinshouldbeclamped toprevent

backflow(Figure3E).Afterdivisionofthehilarplate,theleftportalveincanbedivided

andclamped.Portalvenousperfusionviatheumbilicalveincanbeinitiatedtoperfuse

theLLSor full-left lobe,while continuousperfusionof theextendedright lobeor full-

rightlobeismaintainedviatheportalveinmainstem(Figure3F).Afterdivisionofthe

left hepatic artery, perfusion via the supratruncal aorta allows dual hypothermic

oxygenatedmachineperfusionoftheextendedrightlobeorfull-rightlobe,whereasthe

LLSorfull-leftlobecanbeperfusedviatheumbilicalveinonly.

DISCUSSION

In this proof of concept study, we show for the first time that machine perfusion of

humandonorliversviathesurgicallyreopenedumbilicalveininsteadoftheportalvein

main stem is technically feasible. This modification may simplify the previously

described technique of IFLT as it may allow procurement, preservation as well as

implantation with a standard end-to-end porto-portal anastomosis in the recipient

under continuousoxygenatedportal venousmachineperfusionvia theumbilical vein,

withouttheneedtoanastomoseaniliacveingrafttotheportalveinmainstem.Wealso

demonstrate the technical feasibility of a left lateral segment split procedure under

continuousNMPviathesurgicallyreopenedumbilicalveinaswellasthesupratruncal

aorta.

The first three liverswere perfused hypothermically, duringwhich it is currently not

possible to perform viability testing. We have assessed the flow rates through the

umbilical vein and compared this with the usual portal vein flows observed in our

clinical hypothermic machine perfusions in humans.6 We noticed that when using a

standard24Frcannula forumbilicalveinperfusion, flowswerecomparable to typical

hypothermicperfusionviatheportalveinmainstem(200-300ml/min).Meticulouscare

should be taken to avoid twisting of the umbilical vein cannula becausewe observed

thatthisimmediatelyimpairedportalvenousflow.

The fourth liver perfusion experiment demonstrated that our clinically used viability

criteria for donor livers during NMP2 (after 150 minutes of NMP: perfusate lactate

<1.7mmol/L, perfusate pH between 7.35-7.45, bile pH >7.45, Dutch Trial Register,

#NTR5972)werereachedbyusingportalvenousperfusionviatheumbilicalvein.

Inthefifthliver,wehaveperformedaLLSsplitprocedureundercontinuousNMPusing

portalvenousperfusionviathesurgicallyreopenedumbilicalvein.Aftercompletionof

thesplitprocedure,NMPoftheleftlateralsegmentwascontinued.Inthefollowinghour,

lactate was cleared and the pH remained within normal values. We propose that by

introducingacannulaintheportalveinmainstemaswellasacannulainthesurgically

reopened umbilical vein,machine perfusion of the LLS or full-left lobe aswell as the

extendedrightorfull-rightlobecanbemaintainedevenaftercompleteseparationofthe

twoliverparts.

Weenvisionthatsplittingundercontinuousoxygenatedhypothermicmachineperfusion

could be beneficial, for example, for logistical reasons to allow sequential liver

transplantation in the same centre (conceptualized in Figure 3E,F). Also, hypothermic

oxygenatedmachineperfusion is lessprone to technical failureand isassociatedwith

lowerflowrateswhenusingapressure-controlledmachineperfusiondevice.Wehave,

however,notexperiencedtroublewithflowobstructionduringtheNMPsplitprocedure.

Whenusingcontinuousoxygenatedhypothermicmachineperfusion, the transfer from

onemachinetoanotherortostaticcoldstoragepriortoimplantationcanbeperformed

without additional warm ischemic injury. At normothermic temperatures, we do not

consideritbeneficialtoperfuseboththeextendedrightorfull-rightlobeandtheLLSor

full left lobe simultaneously. For NMP, arterial perfusion is required and arterial

cannulationofeithertheleftortherighthepaticarterymayriskadditionalinjurytothe

vascularendothelium.Therefore,weproposethatcontinuoushypothermicoxygenated

machineperfusionduringsplittingwillbemostbeneficialinclinicalpractice.

In this proof of concept study,we have demonstrated for the first time thatmachine

perfusion through the surgically reopenedumbilical vein indiscardedhuman livers is

technically feasible, thereby achieving adequate portal venous flows. Future studies

should investigate theadditional clinicalutilityofmachineperfusionvia theumbilical

vein,whichshouldincludehistopathologicalanalysisoftheparenchymaandbiliarytree.

REFERENCES

1. Durand F, Renz JF, Alkofer B, et al. Report of the Paris consensus meeting onexpandedcriteriadonors in livertransplantation.LiverTranspl.2008;14:1694–1707.

2. DeMeijerVE,FujiyoshiM,PorteRJ.Exsitumachineperfusionstrategiesinlivertransplantation.JHepatol.2018;doi:10.1016/j.jhep.2018.09.019

3. HeX,GuoZ,ZhaoQ,etal.Thefirstcaseofischemia-freeorgantransplantationinhumans:Aproofofconcept.AmJTransplant.2018;18(3):737-744.

4. vanLeeuwenOB,UbbinkR,deMeijerVE,PorteRJ.Thefirstcaseofischemia-freeorgan transplantation in humans: A proof of concept. Am J Transplant.2018;18(8):2091.

5. Soyama A, Eguchi S, Takatsuki M, et al. Tumor thrombectomy via a surgicallyreopenedumbilicalveincombinedwithrighthemi-hepatectomyinapatientwithhepatocellularcarcinoma.DigSurg.2011;28(3):222-225.

6. vanRijnR,KarimianN,MattonA, et al.Dualhypothermicoxygenatedmachineperfusion in liver transplants donated after circulatory death. Br J Surgery.2017;104(7):907-917.

7. MattonAPM,BurlageLC, vanRijnR, et al.Normothermicmachineperfusionofdonorliverswithouttheneedforhumanbloodproducts.LiverTransplantation.2018;24(4):528-538.

Summary,DiscussionandFuturePerspectives

10

SUMMARYPART1

The first part of this thesis contains a review and three observational studies on

identifyingtheboundariesoftransplantationofsuboptimaldonorlivers.

Afterageneralintroductioninchapter1,areviewwaspresentedinchapter2focusing

on biliary complications after liver transplantation. Bile leaks and bile duct strictures

(anastomoticornon-anastomotic)are themostcommontypesofbiliarycomplication.

Of these, non-anastomotic strictures (NAS) are themost troublesome and difficult to

treat.Patientsreceivingadonationaftercirculatorydeath(DCD)livergraft,livingdonor

graft, or a split liver graft are at increased risk of developing biliary complications.

Although ischemia and subsequent reperfusion injury of the bile ducts is amain risk

factorforthedevelopmentofNAS,othermechanisms,suchasimmune-mediatedinjury,

bilesalt toxicity,and insufficientregenerationof theepitheliumalsoplayarole in the

pathogenesis.

Inchapter3theresultsofanationwideretrospectivestudyontheassociationbetween

donor hepatectomy time and the development of biliary injury during DCD liver

transplantationarepresented.Donorhepatectomytimewasdefinedastheperiodfrom

theinitiationofcoldflushinguntiltheliverwasremovedfromtheabdominalcavityand

placedintoabowlwithice-coldpreservationsolution.Theimpactofdonorhepatectomy

timeonbiliary injurywas studied ina chronological fashion.First, itwasobserved in

humanbileductbiopsiesthatdonorhepatectomytimenegativelyinfluencedthedegree

ofbileductinjurypriortotransplantation.Secondly,liverswithprolongedhepatectomy

time produced bile of inferior quality during normothermic machine perfusion,

displayedbylowerbiliarybicarbonatesecretionandthereforealowerbilepH.Lastly,in

anationwideretrospectivedatabasestudy,donorhepatectomytimewasidentifiedasan

independent significant risk factor for the development of NAS after DCD liver

transplantation. Inconclusion:donorhepatectomytime influences thedevelopmentof

biliary injury in DCD liver transplantation. The results from this study suggest that

donor hepatectomy time should be kept as short as possible to reduce rates of post-

transplantcholangiopathy.

Chapter4containsfindingsthat implicatetheroleofdonorbloodcompositiononthe

development of NAS after DCD liver transplantation. We hypothesized that donor

hematocrit, thrombocyte and leukocyte counts could influence the rates of NAS after

transplantation.Forexample,hematocritcouldaffecttheviscosityoftheblood,platelets

can play a role in endothelial activation, and similar effects can be attributed to

leukocytes, which also can initiate local vasodilation, and all of these factors can

influencegraft flush-out,andthereby influencetheseverityofbiliary injury.First, ina

histological analysis of 40 bile duct biopsies of human donor livers, increased donor

platelet counts and reduced leukocyte counts were associated with a high bile duct

injury score (Odds Ratio 2.553, 95%CI 1.082-6.021, p=0.029 and Odds Ratio 0.734

95%CI 0.581-0.927, p=0.009, respectively). Secondly, during normothermic machine

perfusion(NMP)oftheabovementionedlivers,graftsofdonorswithincreasedplatelet

countandreducedleukocytecountproducedbilewithlowerbiliarybicarbonateandpH

levels, indicating more severe biliary injury. Lastly, in a nationwide retrospective

database analysis, donor hematocrit and platelet count were identified as significant

independent risk factors for the development of NAS after DCD liver transplantation

(HazardRatio1.04795%CI1.007-1.089,p=0.022andHazardRatio1.04495%CI1.005-

1.083,p=0.025).Altogether, thesedatasuggest thatbloodcompositionof thedonor is

associatedwiththedevelopmentofbiliaryinjuryduringDCD-LT.

In the studydescribed inChapter5,weaimed to assess the safetyof theuseofDCD

livers forpatients that requirea re-transplantationof the liver. In theWesternworld,

DBD livers are the golden standard when it comes to graft selection for re-

transplantation.However,over the last years, the three liver transplant centers in the

Netherlands have performed several re-transplantations with a DCD liver. In this

observational study, we compared the outcomes after DCD re-transplantation with a

matched cohort of DBD re-transplantations. Interestingly, graft and patient survival

weresimilaramongbothgroups.RatesofNASintheDCDcohortwerehigh(38%),but

therequirementforretransplantationfortheseNAScaseswaslow.Thisstudysuggests

thatforselectedre-transplantationcandidates,usingaDCDgraftfromayoung,healthy

donormayleadtonon-inferioroutcomes.DCDliversshouldthereforenotberoutinely

declinedforre-transplantationcandidates.

SUMMARYPARTII:

The second part of this thesis contains studies aiming to expand the boundaries of

transplantationofsuboptimaldonorlivers.

In Chapter 6, a histological analysis was performed of bile duct biopsies of 10 DCD

liversthatunderwentend-ischemicdualhypothermicmachineperfusion(DHOPE)prior

to graft implantation, compared with a historical cohort of 20 DCD livers without

machineperfusionofwhichbileductbiopsiesweretaken.Uponbaseline,prior toany

intervention, no difference was observed between the DHOPE group and the control

group.Aftergraftreperfusion,liversthathadunderwentDHOPEdisplayedlessstroma

necrosis and less periluminal and deep peribiliary gland injury after reperfusion,

comparedtothecontrolgroupofliversthatwerecontinuouslypreservedonice.From

this study, it was concluded that DHOPE attenuates the histological sequelae of

ischemia-reperfusioninjuryofthebiliarytree.

In Chapter 7, the results of a preclinical study on extended hypothermic dynamic

preservation are presented. We investigated the safety of prolonged DHOPE by

comparing liver graft function after 2, 6 and 24 hours of DHOPE. Following dynamic

preservationof2,6or24hoursusingDHOPE,reperfusionwithautologousbloodwas

performed and liver graft function was tested. Interestingly, no differences in lactate

clearance, pH stabilization, bile production and bile composition were observed

between the groups. All analyzed markers of endothelial distress and/or injury

displayednodifferencesbetweenthe2,6and24hoursofDHOPEgroups.Additionally,

as proof of concept two human livers declined for transplantation underwent

hypothermicdynamicpreservationusingDHOPEfor20hourspriortorewarmingand

reperfusion. These human liver grafts also showed excellent hepatobiliary viability

duringNMP.Fromthisstudy, itwasconcludedthatitseemssafetoextendDHOPEfor

up to24hours.Afterconfirmation ina transplantation-setting, thisstrategymayhave

substantial clinical impact because it may simplify the difficult logistics around

transplantsandcaninitiateday-timelivertransplantsurgery.

Inchapter8,wedescribetheresultsfromaprospectiveclinicaltrial,theDHOPE-COR-

NMPtrial.Inthisstudy,allnationwidedeclinedliverswereofferedforinclusioninthe

protocol.Mainly,theofferedgraftswereDCDliverswithadonorageexceeding60years,

or DCD grafts with high percentage of steatosis. Livers first underwent one hour of

DHOPE, to resuscitate themitochondriaandprevent subsequent ischemia-reperfusion

injury. After 60 minutes of controlled oxygenated rewarming (COR) to 37°C,

normothermicmachine perfusion (NMP)was continued.Within the first 2.5 hours of

NMP, livershadtomeet the followingcriteria:perfusate lactate<1.8mmol/L,pH7.35-

7.45,bileproduction>10mLandbiliarypH>7.45.Withinthetrialperiod,sixteenlivers

underwent DHOPE-COR-NMP. All livers showed adequate liver parenchymal function

andclearedlactateandproducedbile.However,onlyelevenofsixteenliversreacheda

bilepH>7.45andwere thereforesuitable for safe transplantation.One-yeargraftand

patientsurvival following theseeleven transplantswere100%.With theDHOPE-COR-

NMP protocol, 69% of livers that were previously discarded were successfully

transplantedwitha100%graftsurvivalat12months,resultinginanover20%increase

in the number of post-mortal liver transplantations in the University Medical Center

Groningen.

Theaimofthestudyinchapter9wastofindamethodtosimplifyischemia-freeliver

transplantationasperformedby the transplant team in theFirstAffiliatedHospitalof

SunYat-senUniversity, Guangzhou, China. InGuangzhou, a small interposition vein is

placed end-to-side on the portal vein, to allow cannulation and continuous perfusion

duringprocurement,preservationandimplantation.Wehypothesizedthatcannulation

ofthesurgicallyreopenedumbilicalveinwouldallowcontinuousperfusionaswell.To

test this,weperfusedfivedonor liversvia theumbilicalveinandmonitoredperfusion

closely.Thefirstthreeliverswereperfusedhypothermically,andadequateflowswere

observed with low pressures, similar to regular (D)HOPE. The next two livers were

perfusednormothermically.Thefirstclearedlactate,normalizedpHandproducedhigh

qualitybile.Thiswasalsoobservedinthesecondliver,whichwas‘splitonthepump’.In

this chapter,we have also described amethod for splitting liver grafts ‘on the pump’

withthefeasibilityofsubsequentischemia-freeimplantationoftheleftlateralsegment

graft.

Discussionandfutureperspectives

Through the studies in this thesis we have attempted to elucidate the boundaries of

transplantation of suboptimal donor livers. However, large parts of the grey area

between transplantable and non-transplantable livers remain unexplored. Future

studiescanhelprecognizethepathophysiologybehindcomplicationsthatrepresentthe

actual boundaries. Once themechanisms of these complications are fully understood,

onecanattempttopushtheboundariesbyapplyingnoveltherapies.Inthispartofthe

thesis,theresultsofthestudiesdescribedwillbediscussed,andfutureperspectivesfor

studiesonsuboptimallivertransplantationareconsidered.

In an attempt to introduce a chronological order to this thesis, chapter 2 provided

backgroundandrationaleforchapter3-5.BiliarycomplicationsformtheAchilles’heel

ofDCDlivertransplantation.Overthelastyears,thepathophysiologyofpost-transplant

cholangiopathyhasbeenextensivelystudied.1OpdenDriesetal showed in2014 that

biliarycomplicationsarelargelypredictedbytheseverityofhistologicalinjuryobserved

upon arrival of the graft in the transplant center.2 Two specific aspects of the biliary

injury have a very strong predictive value for NAS: damage to the peribiliary glands

(PBG’s)anddamagetotheperibiliaryvascularplexus(PVP)locatedinthebileductwall.

Interestingly, in the study by op den Dries, a large increase in biliary damage was

observeddirectly after graft reperfusion in the recipient, demonstrating the impactof

reperfusion injury following ischemia.2 This offers the potential for a therapeutic

intervention in the period between arrival of the organ and graft reperfusion, the so-

called‘end-ischemicperiod’,andthisformedtherationaleforinitiatingtheclinicaltrial

of which chapter 7 was a satellite study.3 Possible end-ischemic interventions can

attenuatereperfusioninjury,andtherebyreduceratesofNASaftertransplantation.

However,intheabsenceofregenerativetherapy,goingbeyondattenuatingreperfusion

injuryisnotyetpossible.Futurestudiesaimingatrepairingthedamagethatisalready

presentuponarrivalofthegraft,areeagerlyawaited.Asactiverepairisnotyetfeasible,

someofthechaptersinthisthesisarefocusedonidentifyingmechanismsthatinfluence

theseverityofbiliaryinjuryalreadybeforearrivalinthetransplantcenter.Thisincludes

chapter3,wherethedonorhepatectomytimewasidentifiedasacrucialperiodinthe

development of biliary injury. If this period is kept short, biliary injury isminimized.

However, over the last decades, training and regular feedback for organprocurement

teams has apparently been insufficient in the Netherlands. In the same period in the

United Kingdom, regulations required a donor hepatectomy time to be below 45

minutes.4 In theNetherlands, themedianhepatectomy timeover the last fifteenyears

has been 63 minutes, with cases exceeding two hours. This can partly explain the

extremelyhigh ratesofNAS in theNetherlands thathavebeenreportedpreviously.5-7

Futureperspectivesinthepreventionofhepatectomytimeinducedbiliaryinjurymainly

comprises education of donor surgeons and introduction of normothermic regional

perfusion (NRP).With NRP, impressive results have been observed in terms of post-

transplant cholangiopathy rates and graft survival.8,9 NRP is a method in which a

perfusiondevice(e.g.extracorporealmembraneoxygenationdevice)isusedtoperfuse

the abdominal (and potentially also the thoracic) cavity via large cannulas after

circulatoryarrestofthedonor.Thismethodquicklyleadstorestorationofintracellular

adenosinetriphosphatelevelsandhypotheticallyconvertsaDCDprocedureintoamore

controlledprocedure.8,9Sofar,norandomizedcontrolledtrialshavebeenperformedor

initiated to our knowledge, but NRP appears to have the potential to reduce rates of

biliarycomplicationsafterDCDlivertransplantation.

Another study on the mechanisms influencing biliary injury prior to arrival in the

recipient transplant center is described in chapter 4, which provides a significant

amountoffuelforfutureperspectives.Thereweretworationalesforthisstudy.First,it

still remains uncertain whether the two aspects of biliary injury that predict post-

transplant cholangiopathy, namely peribiliary gland and peribiliary vascular plexus

injury,bothsimplyoccursimultaneouslyasaresultofdonorwarmischemiaintheDCD

donor,or that theyoccursequential.So far,nostudieshavebeenperformed involving

bileductbiopsiestakenuponorganprocurement,butitwouldbehighlyinterestingto

see if both PBG and PVP injury are already present prior to static cold preservation

(SCS),orwhethertheydevelopsimultaneouslyduringSCS.Itcanbehypothesizedthatif

PVP injurywould develop prior to PBG injury, perhaps PBG injury can be prevented

wheninterventionsagainstPVPinjuryareinitiated.

Secondly, it is still uncertainwhether the additionalwarm ischemia that DCD donors

encounterincomparisontoDBDdonorsissolelyresponsiblefortheincreaseinbiliary

injury following DCD liver transplantation. Other mechanisms that can lead to PVP

injury are present in a DCD donor in the process of dying. Unfortunately, not much

research has been performed on how the vasculature responds to an approaching

circulatory arrest and to the first minutes after cessation of blood flow. Severe

peripheral vasoconstriction would for example lead to less effective flush-out and

therefore potentially aggravate peripheral PVP injury. The circulatory arrest and

subsequent blood stasis is also pro-thrombotic, and can cause flush-out problems by

microthrombiformation.

However,interestingly,therehasbeenquitealotofresearchperformedonthevascular

response to hypoxia,which is largely dependent on endothelial cell function.10-13DCD

donorsexperienceasignificantperiodofhypoxiapriortocirculatoryarrest.Endothelial

cellfunctionisdependentonmanyaspects,includingageandatheroscleroticdisease.It

has been described that young healthy people respond to hypoxia with peripheral

vasodilation, but that this response completely disappears as people age.10 This can

explainourobservationthatbiliary injuryuponarrival in therecipientcenter ismore

extremeinelderlydonors.Overthelastyears,afteridentifyingdonorageasariskfactor

forNAS, ithasbeenhypothesizedthatafter transplantation,oldergraftshavereduced

regenerativecapacityfromtheperibiliaryglands.However,theobservationthatbiliary

injury is already more severe upon arrival in the recipient center prior to graft

reperfusion, partially contradicts this hypothesis, and suggests involvement of other

mechanismsearlierinthetransplantprocess.

Inchapterfour,especiallythedonorplateletcountappearedtobestronglycorrelatedto

thedevelopmentofbiliaryinjury.Plateletsareknowntoinduceendothelialdysfunction

uponhypoxiaandwethereforehypothesizedinchapter5thatincreasedplateletcount

leads to more vasoconstriction and less efficient arterial flush-out, and thereby

influences biliary injury.14Minor et al observed early in this century that a pre-flush

withstreptokinasepriortoregularice-coldflushingimprovedgraftviability.15Asthisis

an easy intervention that can improve preservation ofmicrovasculature, the effect of

thisdonorpre-flushonbiliaryinjuryshouldbestudied.However,hopefully,theneedto

studysuperior flushing techniquesdisappearswhenNRPbecomesstandardof care in

DCDliverdonors.

Wehypothesized that the associationbetweenblood composition andpost-transplant

outcomes isparticularlynotablewith respect tobiliary complications.Livergraftsare

flushedthroughtheportalveinuponprocurement,andthereforeadequatecoolingcan

be achieved for the liverparenchymaevenwhen thearterial flushdidnot adequately

rinsethemicrocirculation.However,asthebileductislargelydependentonthehepatic

artery forblood supply, the importanceofhigh-quality arterial flushing is essential in

thepreventionofbiliaryinjury.

Thestudy inchapter5 removeda smallboundary thatmanycentersheldon to: that

DCD livers by definition should not be accepted for patients requiring a re-

transplantation. However, we observed no difference in graft survival between DCD

liversusedforre-transplantationandamatchedcohortofDBDlivers.Weenvisionthat

whenDHOPEwillbeusedregularly,DCDliverscanbeeasilyacceptedforstablepatients

listedforretransplantation.Asinchapter8itisdescribedthatthereisnodifferencein

livergraftfunctionafter2,6and24hoursofDHOPE,therecipienthepatectomyincase

ofaretransplantationcanbeperformedwithouttimepressurewhentheorganisplaced

ontheperfusiondevice.

TheeffectsofDHOPEhavebeenstudiedinseveralchaptersinthisthesis.Inchapter6,

itwasobservedthatDHOPEreducesreperfusioninjuryofthebiliarytree.Moreover,in

appendix I we described the first case of liver transplantation after hypothermic

machine perfusion of a pediatric liver graft, with excellent outcome. Subsequently in

chapter 7, DHOPE allowed safe preservation of liver grafts for up to 24 hours. This

studyprovides rationale for initiatingaday-time liver transplantationprogram.Every

donor liver that is expected to arrive after, for example, 20:00, can be placed on the

perfusiondeviceuntilthenextmorning.Comparisonwiththeinterventiongroupofthe

DHOPE randomized controlled trial (short periods of DHOPE) can be made to

adequatelyassessthesafetyofprolongedDHOPE.Thebenefitofday-timesurgeryisthat

anoptimalteamcanbearrangedandthatthetransplantteamiswell-rested.

With the DHOPE-COR-NMP protocol, in chapter 8 all livers showed excellent

hepatocellular viability. We are convinced that DHOPE is a safe technique and that

centers should first introduce cold perfusion into the clinic, prior to initiating a NMP

program. Starting a NMP programwithout proper experience in the field ofmachine

perfusion isrisky.16 Incontrast tootherstudieswithNMPthatattempttoassessgraft

functionofdiscardedliverstoallowtransplantation,DHOPE-COR-NMPissofartheonly

protocolleadingto100%ofliversmeetinghepatocellularviabilitycriteria.17-19Whether

thisistheresultoftheDHOPEprecedingtheNMP,shouldbestudiedinthefuture.Large

trialswillhavetodemonstratethedirecteffectofDHOPEonNASandgraftsurvival,and

as two large trials in hypothermic machine perfusion have currently closed their

inclusions,theresultsareeagerlyawaited.20,21InappendixII,viabilitycriteriathatcan

beusedtoassessgraftfunctionduringNMParediscussed.

The drawback of DCD liver transplantation remains the biliary injury. Even in the

DHOPE-COR-NMP trial, 31% of the livers were considered ‘non-viable’ based on bile

composition, which reflects bile duct viability. Including the one liver that developed

NAS, thatwouldwith the current experience be declined for transplantation, actually

38% of the livers had too severe biliary injury prior to DHOPE-COR-NMP. Therefore,

even with end-ischemic (D)HOPE for regular DCD livers, such as in the DHOPE-DCD

randomized controlled trial, the percentage of NAS will not be zero, as there will be

always be livers that already arrive with too severe biliary damage. Short donor

hepatectomyandcold ischemia timescanminimize thedegreeofbiliary injurybefore

implantation. In conclusion; DHOPE, and DHOPE-COR-NMP, have stretched the

boundariesoftransplantationofsuboptimaldonorlivers.Topushtheboundarieseven

further,severalinterventionsinthedonorhospitalshouldbestudied,suchasNRP.

LasttodiscussisaveryhopefuldevelopmentinGuangzhou,China,whereatransplant

team succeeded in performing the first series of ischemia-free liver transplantation

(IFLT)worldwide.22With this technique, biliary injurywill hopefullyno longerplay a

major role in liver transplantation. However, it will take some time before

implementation in theWestern world can begin, as a result of the complexity of the

procedureandthelogistics.InGuangzhou,alldonorsweretransferredtotherecipient

hospital,which isnotallowedinmostWesterncountries. InaDCDsetting,completely

ischemia-free transplantation is not possible, as a result of the donorwarm ischemia

time.However,withtheuseofNRPfurtherischemiawillbeavoidedandtheriskofNAS

aftertransplantationwillbelow.OnewaytosimplifyIFLTisdescribedinchapter9.

The studies described in this thesis aimed to allow safe transplantation of high-risk,

suboptimaldonor livers.However, still large amountsofdonor livers aredeclined for

transplantation.Aslongasasignificantnumberofpatientsdieonthewaitinglistforan

organ,transplantphysiciansshouldstudysafetransplantationofthesehigh-riskdonor

organs. As described in an editorial in the New England Journal of Medicine: “risk

aversion has contributed to a shift in the research portfolios of many transplant

programs,awayfromdiscoveryresearchandtowardhealthservicesscienceandclinical

outcomes studies”.23 The study aiming to allow safe transplantation of previously

declineddonorlivers(chapter8),demonstratesthatamutualrisktakenbypatientand

physiciancanleadtoalargestepforwardinthereductionofwaitinglistmortality.Many

future studies are required to decrease waiting list mortality and reduce rates of

complicationsafter transplantation.Toachieve thisgoal,physiciansshouldnotrefrain

fromrisks,but theyshouldenter theboldpath tomedicaladvances togetherwith the

patient.

REFERENCES

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2. OpdenDriesS,WesterkampAC,KarimianN,etal.Injurytoperibiliaryglandsandvascular plexus before liver transplantation predicts formation of non-anastomoticbiliarystrictures.JHepatol2014;60:1172-1179.

3. VanRijnR,KarimianN,MattonAPM,etal.Dualhypothermicoxygenatedmachineperfusioninlivertransplantsdonatedaftercirculatorydeath.BrJSurg;104:907-917.

4. FaridSG,AttiaMS,VijayanandD,etal.ImpactofDonorHepatectomyTimeduringOrgan Procurement in Donation after Circulatory Death Liver Transplantation:TheUnitedKingdomExperience.Transplantation.2018.

5. den Dulk AC, Sebib Korkmaz K, de Rooij BJ, et al. High peak alanineaminotransferase determines extra risk for nonanastomotic biliary stricturesafter liver transplantation with donation after circulatory death. Transplantinternational2015;28:492-501.

6. DubbeldJ,HoekstraH,FaridW,etal.Similarlivertransplantationsurvivalwithselected cardiac death donors and brain death donors. Br J Surg 2010;97:744-753.

7. O'Neill S, Roebuck A, Khoo E, et al. A meta-analysis and meta-regression ofoutcomes including biliary complications in donation after cardiac death livertransplantation.TransplInt2014;27:1159-1174.

8. WatsonCJE,HuntF,MesserS,etal. Insitunormothermicperfusionof livers incontrolledcirculatorydeathdonationmaypreventischemiccholangiopathyandimprovegraftsurvival.AmJTransplant.2019Jun;19(6):1745-1758.

9. HessheimerAJ,CollE,TorresF,etal.Normothermicregionalperfusionvs.super-rapid recovery in controlled donation after circulatory death livertransplantation.JHepatol.2019Apr;70(4):658-665.

10. Richards JC, Crecelius AR, Larson DG, Luckasen GJ, Dinenno FA. Impairedperipheralvasodilationduringgradedsystemichypoxia inhealthyolderadults:role of the sympathoadrenal system. Am J Physiol Heart Circ Physiol.2017;312(4):H832-H841.

11. TaddeiS,VirdisA,GhiadoniL,etal.Age-relatedreductionofNOavailabilityandoxidativestressinhumans.Hypertension.2001;38(2):274-279.

12. TaddeiS,VirdisA,MatteiP,etal.Agingandendothelialfunctioninnormotensivesubjectsandpatientswithessentialhypertension.Circulation.1995;91(7):1981-1987.

13. Westby CM, Weil BR, Greiner JJ, Stauffer BL, DeSouza CA. Endothelin-1vasoconstriction and the age-related decline in endothelium-dependentvasodilatationinmen.ClinSci(Lond).2011;120(11):485-491.

14. Tyagi T, Ahmad S, Gupta N, et al. Altered expression of platelet proteins andcalpain activity mediate hypoxia-induced prothrombotic phenotype. Blood.2014;123(8):1250-1260.

15. Yamauchi J, Richter S, Vollmar B, Menger M, Minor T. Warm preflush withstreptokinase improvesmicrovascularprocurementand tissue integrity in livergraft retrieval from non-heart-beating donors. Transplantation.2000;69(9):1780-1784.

16. Bral M, Gala-Lopez B, Bigam D et al. Preliminary Single-Center CanadianExperienceofHumanNormothermicExVivoLiverPerfusion:ResultsofaClinicalTrial.AmJTransplant.2016;17(4):1071-1080.

17. VanLeeuwenOB,deVriesY,FujiyoshiM,etal.Transplantationofhigh-riskdonorlivers after ex situ resuscitation and assessment using combined hypo- andnormothermic machine perfusion: A prospective clinical trial. Ann Surg. 2019.DOI10.1097/SLA.0000000000003540

18. NeilD,HubscherS,LaingR,etal.Viabilitytestingandtransplantationofmarginaldonor livers VITTAL trial outcomes: bile duct injury assessment during thenormothermic machine perfusion of discarded livers. [Abstract]. InternationalLiver Transplantation Society, 25th Annual International Congres 2019(https://2019.ilts.org)

19. Watson CJE, Kosmoliaptsis V, Randle LV, et al. Normothermic perfusion in theassessmentandpreservationofdeclinedliversbeforetransplantation:hyperoxiaand vasoplegia – important lessons from the first 12 cases. Transplantation.2017;101:1084-1098.

20. van Rijn R, van den Berg A, Erdmann J et al. Study protocol for amulticenterrandomized controlled trial to compare the efficacy of end-ischemic dualhypothermic oxygenated machine perfusion with static cold storage inpreventingnon-anastomoticbiliarystricturesaftertransplantationoflivergraftsdonated after circulatory death: DHOPE-DCD trial.BMC Gastroenterol.2019;19(1).doi:10.1186/s12876-019-0956-6

21. Jochmans I, Akhtar MZ, Nasralla D, et al. Past, present, and future of dynamickidney and liver preser- vation and resuscitation. Am J Transplant2016;16:2545–2555.

22. HeX,GuoZ,ZhaoQetal.Thefirstcaseofischemia-freeorgantransplantationinhumans:Aproofofconcept.AmJTransplant2017;18(3):737-744.

23. MontgomeryR.Gettingcomfortablewithrisk.NEJM.2019Oct24;381(17):1606-1607

AppendixI: FirstReportofSuccessfulTransplantationofaPediatricDonorLiverGraftafterHypothermicMachinePerfusion

MaureenJ.M.Werner,OttoB.vanLeeuwen,IrisE.M.deJong,FrankA.J.A.Bodewes,MasatoFujiyoshi,OlafC.Luhker,RenéScheenstra,YvonnedeVries,RubenH.J.deKleine,RobertJ.Porte

PediatrTransplant.2019May;23(3):e13362

ABSTRACT

Oneof themain limiting factors inpediatric liver transplantation isdonoravailability.

Foradults,donationaftercirculatorydeath (DCD) livergraftsare increasinglyused to

expand the donor pool. To improve outcome after DCD liver transplantation, ex situ

machine perfusion is used as an alternative organ preservation strategy, with the

supplementalvalueofprovidingoxygentothegraftduringpreservation.Weherereport

the first successful transplantation of a pediatric DCD liver graft after hypothermic

oxygenatedmachineperfusion.

Thefullsizelivergraftwasderivedfroma13-year-old,femaleDCDdonorandwasend-

ischemicpre-treatedwithdualhypothermicoxygenatedmachineperfusion.Arterialand

portalpressuresweresetat18and4mmHg,slightly lowerthanprotocolizedsettings

foradultlivers.During2hoursofmachineperfusion,portalandarterialflowsincreased

from100to210ml/minand30to63ml/minrespectively.Thepre-treatedlivergraft

was implanted in a 16-year-old girl with progressive familial intrahepatic cholestasis

type 2. Postoperative aspartate aminotransferase, alanine aminotransferase and

prothrombintimenormalizedwithinaweek.Therecipientquicklyrecoveredandwas

discharged from the hospital after 18 days. One year after transplantation she is in

excellentconditionwithacompletelynormalliverfunctionandhistology.

This case is the first reportof successful transplantationofapediatricDCD livergraft

afterhypothermicoxygenatedmachineperfusionandillustratesthepotentialroleofex

situ machine perfusion in expanding the donor pool and improving outcome after

pediatriclivertransplantation.

INTRODUCTION

Orthotopiclivertransplantationistheonlyeffectivetherapyinpatientswithend-stage

liverdisease.Still,donoravailabilityisthemainlimitingfactorinlivertransplantation,

especially inpediatricpatients,whoneedaperfect size-appropriate graft.1To expand

thenumberofsuitablelivergraftsforpediatricrecipients,severaltechnicalvariantsare

practiced, including splittingofdeceasedadultdonor livergrafts and theuseof living

donors. Despite this, waiting list mortality rate is up to 20%.2 Moreover, during the

period on the waiting list children are at great risk of growth and developmental

retardation.

Foradults,donationaftercirculatorydeath (DCD) livergraftsare increasinglyused to

expand the donor pool. Good results with transplantation of DCD liver grafts are

reported, but a major concern remains the high rate of biliary complications. For

children,theuseofDCDgraftsisstillcontroversialandtheavailabledataarelimitedto

smallseries.3,4

To improve outcome of DCD liver transplantation, ex situ hypothermic machine

perfusion(HMP)is increasinglyusedasanalternativestrategyfororganpreservation,

withthesupplementalvalueofprovidingoxygentothegraftduringpreservation.5The

initial experience in adults has demonstrated that end-ischemic HMP provides better

preservationofDCD liver grafts.HMPameliorates ischemia-reperfusion injury inDCD

livergraftsbyrestoringmitochondrialfunctionbeforeimplantationanditoffersbetter

preservationofthebileductsandtheirvasculature.6,7Thisisanimportantstepforward

inreducingbiliarycomplicationsafterDCDlivertransplantation.

Sofar,machineperfusionhasonlybeenreportedinadulttoadultlivertransplantation.

Wehere report the first successful transplantationof apediatricDCD liver graft after

oxygenatedHMP.

CASEPRESENTATION

Thelivergraftwasderivedfroma13-year-old,femaleDCDdonor(65kg,167cm),who

was resuscitated after an out of hospital cardiac arrest. She was admitted to the

intensivecareunitfor7days.Lastserumalanineaminotransferasebeforeprocurement

was65U/L,lastserumsodiumwas162mmol/L.Theagonalphasebetweenwithdrawal

fromlifesupportuntilcirculatoryarrestwas19minutes.Afteramandatory5minutes

‘notouchperiod’,rapidcannulationoftheaortawasperformedandtheliverwasinsitu

perfused with ice-cold Belzer University of Wisconsin (UW) cold storage solution

(supplementedwithheparin).Thetotalperiodfromwithdrawaloflifesupporttoinsitu

coldperfusionendured34minutes.Thebileductsweregentlyflushedinaretrograde

fashionwithUWpreservation solution. Subsequently, the liverwaspacked static cold

storageandtransportedtoourcenter.

In our center the liver graftwas inspected and appeared to be of good quality. Liver

weight was 1509 gram. Because the liver was derived from a DCD donor and to

minimizefurtherischemicinjuryasmuchaspossible,itwasdecidedtopreparetheliver

graft for oxygenated HMP during recipient hepatectomy. A conventional back table

procedureofthegraftwasperformedafterwhichtheportalveinandsupratruncalaorta

were cannulated for machine perfusion. Subsequently, the liver underwent pressure-

controlled dual hypothermic oxygenated machine perfusion using the Liver Assist

(OrganAssist,Groningen,TheNetherlands).Theperfusion fluid consistedof4000mL

Belzer UW machine perfusion solution, supplemented with 3420 mg glutathione.

Perfusionfluidwasoxygenatedwith1L/min100%O2toobtainaPaO2of>70mmHg

andtemperaturewaskeptat10°C,accordingtoourHMPprotocol.6Arterialandportal

pressures were set at 18 and 4mmHg respectively, which is slightly lower than our

protocolizedsettingsforadultlivers(25and5mmHgrespectively).DuringHMPportal

flow increased adequately from 100 to 210 ml/min and arterial flow from 30 to 63

ml/min, whereas pressure and temperature remained stable (Figure 1). Perfusate

glucose level increased in the first 30minutes of HMP from 8.8 to 12.5mmol/L, and

remained stable thereafter. The perfusate lactate level decreased from 2.4 to 1.7

mmol/L.After2hoursofHMPthe liverwasdisconnectedfromtheperfusionmachine

andtransplanted.

Figure1.Portalandarterialflowratesduringtwohoursofdualhypothermic

oxygenatedmachineperfusionofan13-year-oldDCDlivergraft.

Theperfusionmachinewaspressure-controlledwithportalpressuresetat4mmHgand

arterialpressuresetat18mmHg.DCD,donationaftercirculatoryarrest.

Theselectedrecipientwasa16-year-oldgirl(42kg,156cm),whowasdiagnosedinthe

neonatal phase with progressive familial intrahepatic cholestasis type 2. To prevent

progressive damage of the hepatocytes by retention and accumulation of bile salts, a

partialexternalbiliarydiversionprocedurewasperformedwhenshewas4yearsold.8

Despite this, at the ageof14years shewas listed for liver transplantationbecauseof

deterioration of cholestasis with icterus and itching, and bile stoma bleedings. The

recipientandherparentsgaveconsenttoreceiveaHMP-preservedDCDliver.

TheHMP-pretreated full size liver graftwas implantedusing thepiggyback technique

withend-to-endportalandarterialanastomoses.Perioperativebloodlosswas1800ml

and the recipient received one red blood cell transfusion (280 ml) intraoperatively.

Totalcoldpreservationtimeofthedonorlivergraftwas512minutes,consistingof384

minutes of cold ischemic storage and 128 minutes of oxygenated HMP. Subsequent

warm ischemia timewas 33minutes. Immediately after transplantation the recipient

was extubated and admitted to the pediatric intensive care unit where vasopressive

support could be reduced to zero and intravenous heparin was administered as is

routine practice after pediatric transplantation in our center. Postoperative aspartate

aminotransferase, alanine aminotransferase and prothrombin time rapidly decreased

andnormalizedwithinaweek(Figure2a).Alkalinephosphataseandgamma-glutamyl

transferase normalized within a month and remained stable afterwards. Immediate

postoperative lactatewas3.5mmol/Land levels steadilydecreased thereafter,with a

small second peak on postoperative day 4 when an intra-abdominal bleeding was

diagnosedwhichrequiredsurgicalintervention(Figure2b).Surgicalinspectionshowed

diffuse oozing with a potential bleeding focus at the inferior vena cava which was

clippedandadditionallyahematomawasevacuated.Afterthis,therecipienthadaquick

and further uneventful recovery until she was discharged from the hospital on

postoperative day 18. One year later, the recipient is in excellent condition with a

completely normal liver function with a serum alanine aminotransferase of 16 U/L,

bilirubinof7µmol/Landanormalliverhistologyonroutineliverbiopsy.Therewereno

clinical or histological signs of biliary complications, additional imaging was not

performed.

Figure2.PanelA:SerumAST,ALT,ALPandGGTlevelsina16-year-oldrecipientafter

successfultransplantationofahypothermicoxygenatedmachineperfusedpediatricDCD

livergraft.PanelB:Serumbilirubinandlactateina16-year-oldrecipientaftersuccessful

transplantationofahypothermicoxygenatedmachineperfusedpediatricDCDlivergraft.

AST,aspartateaminotransferase;ALT,alanineaminotransferase;ALP,alkaline

phosphatase;GGT,gamma-glutamyltransferase;DCD,donationaftercirculatoryarrest.

DISCUSSION

This case reportdescribes the first successful transplantationof apediatricDCD liver

graftafterexsituoxygenatedHMP.Thereareonlyafewdescriptionsofpediatric liver

transplantationswith grafts fromDCDdonors in the current literature.3,4,9Hong et al.

reported a matched case-control study of 7 DCD liver transplantations in pediatric

patientswithexcellentlong-termoutcomes.3Abiliaryanastomoticstrictureoccurredin

onlyoneof the recipientsand the incidenceofbiliary complicationswasnotdifferent

betweenDCDanddonationafterbraindeath liver transplantations.AlsoGozinnietal.

havesuggestedthatlivergraftsfromyoungDCDdonorswithshortischemiatimescan

besafelyusedinpediatrictransplantation.9Moreover,vanRijnetal.demonstratedthat

transplantationofpediatricDCDlivergraftsresultsingoodlong-termoutcomes,when

the donorwarm ischemia time is kept under 30minutes.4 Patient- and graft survival

rates were comparable to those of pediatric donation after brain death liver grafts.

Moreover, the incidence of non-anastomotic biliary strictures after transplantation of

pediatric DCD livers was remarkably low. These studies support the use of pediatric

DCDlivergraftsfortransplantation.

AmajordrawbackofDCDlivergraftsisthedevastatingeffectofwarmwithsubsequent

coldischemia,leadingtodepletionofintracellularenergysources,suchasadenosine5'-

triphosphate, combined with other metabolic disturbances. This results into cellular

injuryanddysfunctiondue to reperfusion injuryduring transplantation.10,11 Ischemia-

reperfusioninjuryisamajorcauseofprimarynonfunction,earlyallograftdysfunction

andbiliarycomplicationsaftertransplantation.12

Inadultlivertransplantation,ithasbeendemonstratedthatashortperiod(1-2hours)

of oxygenated HMP after traditional static cold storage restores the hepatic energy

status in liver grafts, reduces ischemia-reperfusion injury, and improves early graft

survival.5,6,13Basedontheseexperiences,wedecidedtoapplyend-ischemicHMPtothe

pediatric DCD liver graft offered to our recipient. Compared to adult liver grafts,

pediatricliversaresmallerandpotentiallymoresusceptibletointravascularpressure-

induced damage. This is important because one of the potential risks of HMP is

endothelial injury due to shear stress. Shear stress occurs in case of high perfusion

pressures, especially at low temperatures when endothelial cell membranes are

susceptible to injury.14 Perfusion induced endothelial cell injury can be prevented by

using low perfusion pressures and a pressure-controlled perfusion system.13,15

Therefore, we used a pressure-controlledmachine perfusion devicewith arterial and

portalperfusionpressureslowerthanvaluesgenerallyusedforadultlivergrafts.16

In the reported casewedemonstratedHMPof a13-year-oldDCD liver graft,which is

relative large.TodetermineoptimalportalandarterialpressuresforHMPinpediatric

liver grafts, more experiences and research are required. In our opinion, perfusion

pressuresinpediatriclivergrafts,shouldbeloweredbasedondonorage,toadjustfor

the lower physiological pressure in the liver graft. For HMP in adult liver grafts,

protocolized portal and arterial perfusion pressures are set at 3-5 and 25 mmHg

respectively.6,13 Normally, an adult liver graft is used to a physiologicalmean arterial

blood pressure of 90 mmHg in the donor, whereas pediatric liver grafts are used to

lower systemic blood pressures. Perhaps we should lower perfusion pressures for

pediatriclivergraftsbasedondonormeanarterialbloodpressuresaccordingtodonor

age. For example, a 5-year old pediatric liver graft, is used to a mean arterial blood

pressureof65mmHg,whichisabout30%lowerwhencomparedto90mmHginadults.

Therefore it seems reasonable to reduce the portal and arterial pressure with 30%,

leading to a portal and arterial perfusion pressure of 3-4 and 18mmHgduringHMP,

respectively.

Inthecomingyears,furtheradvancesinorganpreservation,suchasmachineperfusion

may provide a solution to the problem of donor organ scarcity for pediatric patients.

Machine perfusion of DCD donor grafts might reduce part of the risks of DCD liver

transplantation.With this casewedemonstrated thatHMPof apediatric liver graft is

feasibleandcanbeperformedsafelywithadjustedperfusionpressures.

Apart from providing a better preservation method, machine perfusion can also

facilitate pediatric liver transplantation by enabling a split procedure of a liver graft

under continuous oxygenated perfusion. The concept of splitting a liver graft during

machine perfusion was recently shown by Stephenson et al.17 These investigators

successfullyperformeda splitprocedureof anadult liver graft resulting in a segment

2/3 and an extended right lobe graft. In addition, with the upcoming technique of

normothermicmachine perfusion, which enables ex situ functional assessment of the

liver graft prior to implantation, it might be useful to estimate the suitability of a

suboptimal liver graft for a pediatric recipient.18 Altogether, this may increase the

numberoflivergraftsthataresuitableforpediatriclivertransplantation.

In conclusion,we present the first successful transplantation of a pediatric DCD liver

graftafterexsituhypothermicoxygenatedmachineperfusion.Thiscase illustrates the

potentialroleofexsitumachineperfusiontechnologyinexpandingthedonorpooland

improvingoutcomeafterpediatriclivertransplantation.

REFERENCES

1. Wertheim JA, Petrowsky H, Saab S, Kupiec-Weglinski JW, Busuttil RW. Majorchallenges limiting liver transplantation in the United States. Am J Transplant.2011;11(9):1773-1784.

2. vanderDoefHPJ,vanRheenenPF,vanRosmalenM,RogiersX,VerkadeHJ, forpediatric liver transplantation centers of Eurotransplant.Wait-list mortality ofyoungpatientswithbiliaryatresia:Competingriskanalysisofaeurotransplantregistry-basedcohort.LiverTranspl.2018;24(6):810-819.

3. Hong JC,VenickR,YersizH, etal. Liver transplantation in childrenusingorgandonationafter circulatorydeath:A case-control outcomesanalysisof a20-yearexperienceinasinglecenter.JAMASurg.2014;149(1):77-82.

4. vanRijnR,HooglandPER,LehnerF,vanHeurnELW,PorteRJ.Long-termresultsafter transplantation of pediatric liver grafts from donation after circulatorydeathdonors.PLoSOne.2017;12(4):e0175097.

5. Dutkowski P, Polak WG, Muiesan P, et al. First comparison of hypothermicoxygenatedperfusionversusstaticcoldstorageofhumandonationaftercardiacdeath liver transplants: An international-matched case analysis. Ann Surg.2015;262(5):764-70

6. vanRijnR,KarimianN,MattonAPM,etal.Dualhypothermicoxygenatedmachineperfusion in liver transplants donated after circulatory death. Br J Surg.2017;104(7):907-917.

7. van Rijn R, van Leeuwen OB, Matton APM, et al. Hypothermic oxygenatedmachineperfusion reducesbileduct reperfusion injury after transplantationofdonationaftercirculatorydeathlivers.LiverTranspl.2018;24(5):655-664.

8. YangH,PorteRJ,VerkadeHJ,DeLangenZJ,Hulscher JB.Partialexternalbiliarydiversion in children with progressive familial intrahepatic cholestasis andalagilledisease.JPediatrGastroenterolNutr.2009;49(2):216-221.

9. GozziniS,PereraMT,MayerDA,etal.Livertransplantationinchildrenusingnon-heart-beatingdonors(NHBD).PediatrTransplant.2010;14(4):554-557.

10. de Rougemont O, Breitenstein S, Leskosek B, et al. One hour hypothermicoxygenated perfusion (HOPE) protects nonviable liver allografts donated aftercardiacdeath.AnnSurg.2009;250(5):674-683.

11. XuH,BerendsenT,KimK, et al. Excorporeal normothermicmachineperfusionresuscitates pig DCD livers with extended warm ischemia. J Surg Res.2012;173(2):e83-8.

12. DubbeldJ,HoekstraH,FaridW,etal.Similarlivertransplantationsurvivalwithselectedcardiacdeathdonorsandbraindeathdonors.BrJSurg.2010;97(5):744-753.

13. Schlegel A, de Rougemont O, Graf R, Clavien PA, Dutkowski P. Protectivemechanisms of end-ischemic cold machine perfusion in DCD liver grafts. JHepatol.2013;58(2):278-286.

14. Fondevila C, Hessheimer AJ, Maathuis MH, et al. Hypothermic oxygenatedmachineperfusion inporcinedonationafter circulatorydeterminationofdeathlivertransplant.Transplantation.2012;94(1):22-29.

15. Schlegel A, Dutkowski P. Role of hypothermic machine perfusion in livertransplantation.TransplInt.2015;28(6):677-689.

16. Shore PM, Huang R, Roy L, et al. Potential for liver and kidney donation aftercirculatorydeathininfantsandchildren.Pediatrics.2011;128(3):e631-8.

17. Stephenson BTF, Bonney GK, Laing RW, et al. Proof of concept: Liver splittingduringnormothermicmachineperfusion.JSurgCaseRep.2018;2018(3):rjx218.

18. Ravikumar R, JassemW,Mergental H, et al. Liver transplantation after ex vivonormothermicmachinepreservation:Aphase1(first-in-man)clinicaltrial.AmJTransplant.2016;16(6):1779-1787.

AppendixII: Viabilitycriteriaforfunctionalassessmentofdonorliversduringnormothermicmachinepreservation

OttoB.vanLeeuwen,VincentE.deMeijer,RobertJ.Porte

LiverTranspl.2018Oct;24(10):1333-1335

Persistingdonororganshortagehasledtoanincreaseduseofsuboptimaldonorlivers

for transplantation. Transplantation of these extended criteria donor (ECD) livers,

however, is associated with high rates of complications, including early allograft

dysfunction(EAD)andprimarynon-function(PNF).Asaresult,alargenumberofECD

liversarediscarded.ThedecisiontoacceptordeclineanECDliverfortransplantationis

largely based on empiric and rather subjective criteria available at the time of donor

organoffer,butitmaynotreflecttheactualconditionofanorganafterseveralhoursof

staticcoldstorage(SCS).Exsitumachineperfusionisincreasinglybeingstudied

as a method to resuscitate and functionally assess donor organs shortly before

transplantation. When performed at 37°C, ex situ normothermic machine perfusion

(NMP) enables a metabolic assessment of high-risk liver grafts. However, there is

currentlynoconsensusonthecriteriathatcanbeusedduringNMPtoselectlivergrafts

thatcanbetransplantedsafely,despiteinitialdeclinebasedonanestimatedtoohighof

ariskforEAD/PNF.

In this issue of Liver Transplantation, Mergental et al. report an interesting study in

which they aimed to identify criteria that can be used during NMP to predict PNF of

high-risk donor livers.1 A total of 12 discarded human donor livers underwent NMP

using a perfusion solution based on packed red blood cells for up to 6 hours after a

period of SCS. Perfusate blood gas profiles, bile production, and vascular flow

characteristicswereexaminedtoidentifyparametersthatareassociatedwithpoorliver

functionandahighdegreeofinjuryonhistologyofliverbiopsies.Initially,theauthors

identified lactate clearance as a marker for graft viability. The authors observed 2

distinctgroups:agroupof6organswithasharpdecreaseinlactatelevels,whereasthe

other6showed“fluctuationsandrises”ofthelactatelevelsintheperfusionsolution.

After this observation, further analyses were performed by comparing these lactate-

clearingandnonlactate-clearinggroups.Second,theauthorsnotedlesshepatocellular

injuryonhistology,higherhepaticadenosinetriphosphatelevels,morestableperfusate

pH, and higher bile production in the lactate-clearing group compared with the

nonlactate-clearinggroup.Thesefindingssupportedtheiruseoflactateasanindicator

ofhepatocellular functionandcorroboratespreviousresults fromothergroups.2,3Bile

production,however,wasobservedinonly4ofthe6liversinthelactate-clearinggroup

versus 1 out of 6 livers in the nonlactate-clearing group. The authors conclude that

measurable bile production can be used as a marker of good liver function, but the

absence of bile production does not necessarily imply poor function. Similar

observationshavebeenmadebytransplantgroupsfromCambridgeandCleveland.2,3

Inourexperience,wehavenoted that very loworabsentbile flowcan sometimesbe

explained by a technical issue. For example, the tip of a biliary catheter can be stuck

againstthebileductwallobstructingitslumenandprecludingbileflow.Inaddition,we

havenotedthatbileflowthroughabiliarydrainisdependentonthesizeofthedrain.If

thediameterofthedrainistoolarge,bileflowmaynotcommenceeasily,especiallyas

the drain often makes an uprising loop at the rim of the reservoir (Fig. 1B,C in the

currentarticle).Theuseofasmallfeedingtubecatheter(8-Fr)withanopentipandside

holesmayminimize the risk of these technical issues inmeasuringbile production.A

small-sized catheter provides a capillary force, which facilitates bile flow. These

potential issuesshouldbeconsideredwhenliversseemtoproduceminimalornobile

despitebiochemicalevidenceofgood functional recovery, suchasadecreasing lactate

level.

Collection of bile produced during NMP is important because it may aid in the

assessmentofbileductviabilityduringNMP.4Thisisatopicnotcoveredinthecurrent

studybyMergentaletal.,butitisclinicallyveryrelevant,especiallyintheassessmentof

high-risk livers fromdonationafter circulatorydeath (DCD).DCD livergraftsnotonly

havean increasedriskofhepatocellulardysfunctionthatmay leadtoEADorPNF,but

theyarealsopronetocholangiopathyduetocholangiocellularinjury.5Experimentaland

preclinical studies have indicated that biliary pH, bicarbonate, and lactate

dehydrogenase during NMP may reflect biliary viability.4 Cholangiocytes (biliary

epithelial cells) actively modify bile composition by secretion of bicarbonate and

resorption of glucose, resulting in an alkalotic pH and low glucose level in bile at the

level of the common bile duct. Observations made by the Cambridge group during

clinical applicationofNMP recently supported thepotential value of biliarypH in the

assessment of biliary viability (and thus the risk of posttransplant cholangiopathy)

during NMP.2 However, more research will be needed to establish the diagnostic

accuracyofbilecompositioninassessingbiliaryviability.Identificationandvalidationof

biomarkers and criteriaof bileduct viabilityduringNMPwouldbe an important step

forwardinthewideruseofDCDlivergrafts.

Interestingly,Mergentaletal.notedan increase in injuryof the intrahepaticsmallbile

ductsduringNMPinallbut1oftheliversinthenonlactate-clearinggroupandin2of

the6inthelactate-clearinggroup.Altogether,approximately50%oftheliverssuffered

an increase in biliary injury duringNMP.Unfortunately, histological data on the large

intrahepatic and or extrahepatic bile ductswere not available. Typically, these larger

bile ducts are at risk of ischemia/reperfusion injury, resulting in posttransplant

cholangiopathy.5 Although NMPmaymitigate ischemia/reperfusion injury due to the

absenceofleukocytesandplateletsintheperfusionfluid,injuryduetotheformationof

radicaloxygenspecies(ROS)maystilloccur.Infact,thefirstclinicalstudiesofliverNMP

have not shown a reduction in the incidence of biliary complications after

transplantation compared with SCS.2,6 To improve results, preceding NMP by

oxygenatedhypothermicmachineperfusion(HMP)maybemoreeffectiveasthelatter

has been shown to resuscitatemitochondria, reduce postreperfusion ROS production

andinflammation,andmitigatereperfusioninjuryofthebileducts.7Inaccordancewith

this,2preclinicalstudiesusingdiscardedhumandonorlivershaverecentlyshownthat

a short period of HMP prior to NMP improves metabolic recovery and attenuates

oxidative stress and tissue inflammation.8,9 Therefore, it may be important to

sequentially apply HMP and NMP, especially when NMP is used to assess the

transplantabilityofhigh-riskDCDlivers,which inadditionto theriskofEADandPNF

haveahighriskofdevelopingbiliarycomplicationsaftertransplantation.Liverviability

testingremainsahighlydifficultresearchfieldasthetranslationtoclinicalapplication

mustbeperformedwithexceptionalcare.On thebasisof theprincipleofprimumnon

nocere, patient safety has to be the main concern. This concern has forced clinical

researchgroupstoremainonthesafesidewhenitcomestotransplantationofinitially

declined livers after a positive viability judgment by NMP. On the other hand, a too

prudentialapplicationishamperingtheidentificationofclinicallyrelevantcutoffvalues

ofviabilitycriteria.Withincreasingexperience,however,itcanbeexpectedthatcenters

will startpushing theboundariesbystretching theacceptancecriteria.As longas this

process is carefully monitored and outcome data are published in a standardized

fashion,10 we will collectively learn from this process and be able to effectively and

safelyusethepotentialbenefitsofexsitumachineperfusioninincreasingthenumberof

suitableorgansfortransplantation.

REFERENCES

1. Mergental H, Stephenson B, Laing RW, Kirkham A, Neil D, Wallace L et al.Developmentofclinicalcriteriaforfunctionalassessmenttopredictprimarynon-function of high-risk livers using normothermic machine perfusion. LiverTranspl.2018

2. WatsonCJE,KosmoliaptsisV,PleyC,RandleL,FearC,CrickKetal.Observationson the ex situ perfusion of livers for transplantation. Am J Transplant.2018;18:2005-2020

3. LiuQ,NassarA,BucciniL,IuppaG,SolimanB,PezzatiD,etal.Lipidmetabolismand functionalassessmentofdiscardedhuman liverswithsteatosisundergoing24hoursnormothermicmachineperfusion.LiverTranspl.2018Feb;24:233-245

4. WeederPD,vanRijnR,PorteRJ.Machineperfusioninlivertransplantationasatool to prevent non-anastomotic biliary strictures: Rationale, current evidenceandfuturedirections.JHepatol.2015;63:265-275

5. de Vries Y, von Meijenfeldt FA, Porte RJ. Post-transplant cholangiopathy:Classification, pathogenesis, and preventive strategies. Biochim Biophys Acta.2018;1864:1507-1515

6. NasrallaD,CoussiosCC,MergentalH,AkhtarMZ,ButlerAJ,CeresaCDLetal.Arandomizedtrialofnormothermicpreservation in livertransplantation.Nature.2018;557:50-56.

7. SchlegelA,MullerX,DutkowskiP.Hypothermicliverperfusion.CurrOpinOrganTransplant.2017;22:563-570

8. Westerkamp AC, Karimian N,Matton APM,Mahboub P, van Rijn R,Wiersema-BuistJ,etal.Oxygenatedhypothermicmachineperfusionafterstaticcoldstorageimproves hepatobiliary function of extended criteria donor livers.Transplantation2016;100:825-835

9. Boteon YL, Laing RW, Schlegel A,Wallace L, Smith A, Attard J et al. Combinedhypothermicandnormothermicmachineperfusionimprovesfunctionalrecoveryofextendedcriteriadonorlivers.LiverTranspl.2018.DOI:10.1002/lt.25315

10. KarangwaSA,DutkowskiP,FontesP,FriendPJ,GuarreraJV,MarkmannJFetal.Machine perfusion of donor livers for transplantation: a proposal forstandardized nomenclature and reporting guidelines. Am J Transplant2016;16:2932-2942.

Nederlandsesamenvatting

Eenlevertransplantatieistegenwoordigdestandaardbehandelingvoorpatiënteninhet

eindstadiumvaneenleverziekte.Deeerstelevertransplantatiewerduitgevoerddoordr.

Thomas Starzl endiens team inDenver, Colorado, in 1963.Helaas stierf de driejarige

ontvanger peroperatief als gevolg vanmassaal bloedverlies. Hetzelfde probleem deed

zich voor gedurende de volgende vijf transplantaties, met een patiëntoverleving van

maximaal 23 dagen. In 1967 slaagde Starzl erin om de eerste succesvolle

levertransplantatie uit te voeren met een patiëntoverleving van meer dan een jaar.

Twaalf jaar later, na de ontwikkeling van cyclosporine door Calne en tacrolimusdoor

Starzl,ontwikkeldedelevertransplantatiezichtoteengestandaardiseerdebehandeling

metacceptabeleuitkomsten.Opditmomentwordtwereldwijdeen1-jaartransplantaat-

enpatiëntoverlevingvanbovende90%gezien.

Helaasblijfthet tekortaandonororganen immeraanwezigenoverstijgtdevraagnaar

donorlevershetaanbod.Toteindjaren’90werdendemeestedonorleversuitgenomen

uithersendodedonoren.Omhetdonoraanboduittebreidenwerdenvanafdatmoment

ook organen uitgenomen uit patiënten die waren overleden na het stoppen van de

bloedsomloop (donation after circulatory death [DCD] donoren). In het begin werden

goede resultaten behaald bij levertransplantatie met DCD organen. Echter, toen na

enkele jaren de criteria voor DCD lever acceptatie verbreed werden, nam het aantal

postoperatieve complicaties sterk toe. Vooral galwegcomplicaties werden frequent

geziennaDCD-levertransplantatie,meteenincidentievanbijna30%.

In de vroege jaren ’80 ontwikkelde de groep van Folkert Belzer de University of

Wisconsinpreservatievloeistof(UW),dievoorvele jarendegoudenstandaardwerdin

abdominale orgaantransplantatie. Het abdominale compartiment van de donor wordt

tijdens de donoroperatie via de aorta geflushed met meerdere liters ijskoude UW.

HiernawordendeorganenzosnelmogelijkineenzakmetUWopijsineenorgaandoos

gelegd.Dezetechniekvankoudestatischepreservatieishelaasnietvoldoendegebleken

om complicaties naDCD-levertransplantatie te voorkomen. De afgelopen jaren is veel

onderzoek gedaan naar machineperfusie van donorlevers, in een poging om de

overleving na DCD-levertransplantatie te vergroten. Enerzijds wordt koude

(hypotherme, 10-12 graden Celsius) machineperfusie gebruikt om de energieniveaus

van de lever weer te verhogen voorafgaand aan transplantatie, waardoor ischemie-

reperfusieschadeverminderdkanworden.Anderzijdsbiedtwarme(normotherme,37

gradenCelsius)perfusiedemogelijkheidomdelever-engalwegfunctievoorafgaandaan

transplantatietechecken.

Ditproefschrift focustopde transplantatievansuboptimale levers;vooralDCD levers.

Het eerste deel bevat met name observationele studies die gericht zijn op het

identificerenvandegrenzenvansuboptimalelevertransplantatie,enophetherkennen

van risicofactoren die deze grenzen bepalen. Deel twee bevat observationele,

preklinische en klinische studies met het gezamenlijke doel om veilig en succesvol

transplanterenvansuboptimaleleversmogelijktemaken.

Hierondervolgteensamenvattingvanheteerstedeelvanhetproefschrift.

Hoofdstuk1dientalsalgemeneinleidingenbeschrijftderationalevoorendeopzetvan

ditproefschrift.

Hoofdstuk 2 bevat een review van de literatuur over galwegcomplicaties na

transplantatie. Het ontstaan van galwegcomplicaties wordt besproken evenals

verschillende risicofactoren hiervoor. Gallekkage en galwegstricturen zijn de meest

voorkomende galwegcomplicaties. Van de galwegcomplicaties zijn met name de niet-

anastomotische stricturen (NAS) van de intrahepatische galweg het meest

problematisch.PatiëntendieeenDCD-lever,split-lever,of levende-donor-leverkrijgen,

hebben een verhoogd risico op galwegcomplicaties. Een groot deel van het risico op

galwegcomplicatieswordt bepaald door ischemie-reperfusie schade,maar ook andere

mechanismen spelen hier een rol, zoals bijvoorbeeld immuun-gemedieerde schade,

galzout-toxiciteitenonvoldoenderegeneratievanhetepitheel.

In hoofdstuk 3 worden de resultaten van een nationale retrospectieve studie

beschreven naar de associatie tussen donor-hepatectomietijd en het ontwikkelen van

galwegschade tijdens DCD levertransplantatie. Donor-hepatectomietijd werd

gedefinieerdalsdeperiodetussendestartvanhetkoudeflushenvandedonor,tothet

verwijderenvanhetorgaanuitdebuikholte.Deinvloedvandonor-hepatectomietijdop

galwegschadewerdbestudeerdineenchronologischevolgorde.Allereerstwerdgezien

bijgalwegbioptengenomenvóórmachineperfusiedatdehoeveelheidschadebeïnvloed

werd door de donor-hepatectomietijd. Ten tweede werd tijdens normotherme

machineperfusiegeziendatleversdieeenlangehepatectomietijdhaddengehad,galvan

minder goede kwaliteit produceerden. Vervolgens is met behulp van een nationale

retrospectieve studie aangetoond dat donor-hepatectomietijd een risicofactor is voor

het ontstaan van galwegcomplicaties. Concluderend: donor-hepatectomietijd moet zo

kortmogelijkgehoudenwordenomhetrisicoopgalwegcomplicatiesteverkleinen.

Hoofdstuk4bevateenstudienaardeassociatietussendesamenstellingvanhetbloed

vandeDCD-donorenhetontstaanvangalwegschadevanhetdonororgaan.Wevormden

dehypothesedathethematocrietendehoeveelhedenwittebloedcellenenbloedplaatjes

vandedonorhetrisicoopNASkunnenbeïnvloeden.Deredenvoordezehypothesewas

dathethematocrietdeviscositeitbepaalt,bloedplaatjesendotheelactivatieveroorzaken

bijhypoxie,enleukocytenvasodilatatiebeïnvloeden,enaldezefactorenkunnenleiden

tot eenminder succesvol flushen van het donororgaan. Allereerstwerd gezien in een

histologischeanalysevan40galwegbioptenvanhumanedonorleversdatbloedplaatjes

en leukocyten de ernst van galwegschade beïnvloeden voorafgaand aan

machineperfusie (OddsRatio2.553, 95%CI1.082-6.021, p=0.029 enOddsRatio0.734

95%CI 0.581-0.927, p=0.009, respectievelijk). Vervolgens, tijdens normotherme

machineperfusie, werd deze associatie wederom geobserveerd. In een landelijke

retrospectieve database studie werd aansluitend gezien dat donor hematocriet en

bloedplaatjeshetrisicoopNASbeïnvloeden.Debloedsamenstellingvandedonor lijkt,

concluderend, sterk geassocieerd met het ontstaan van galwegschade tijdens DCD-

levertransplantatie.

Inhoofdstuk 5 worden de resultaten beschreven van een studie die als doel had te

onderzoeken of DCD-levers veilig gebruikt kunnen worden in patiënten die een

retransplantatie van de lever moeten ondergaan. Tot nu toe worden voor

retransplantatiesindeWestersewereldvrijwelaltijdDBD-leversgebruikt.Inderecente

jaren zijn door de Nederlandse centra af en toe DCD-retransplantaties uitgevoerd. In

deze observationale studie hebben we de uitkomsten na DCD-retransplantatie

vergeleken met de uitkomsten van een gematched cohort DBD-retransplantaties. De

uitkomsten na DCD-retransplantatie waren vergelijkbaar met het cohort van DBD-

retransplantaties. De resultaten van deze studie suggereren dat DCD-levers niet

standaard afgewezen worden voor patiënten in relatief goede conditie die een

retransplantatiemoetenondergaan.

Inhettweededeelvanhetproefschriftzijnhoofdstukkentevindendieeropgerichtzijn

degrenzenvantransplantatievansuboptimaledonorleversteverleggen.

Inhoofdstuk6wordteenhistologischeanalysebeschrevenvandegalwegbioptenvan

10 DCD-levers die koude machineperfusie (DHOPE) ondergingen alvorens

transplantatie in het UMCG. De ontwikkeling van galwegschade van deze gepompte

levers werd vergeleken met de schade bij 20 controlegroep DCD-levers. Bij baseline

hadden beide groepen evenveel galwegschade. Na transplantatie bleken de levers die

DHOPE hadden ondergaan minder galwegschade te hebben ten opzichte van de

controlegroeplevers. Bijdecontrolegroepleverswerdeenverergeringvandeschade

gezien na reperfusie. Deze verergering van schade was afwezig bij de DHOPE levers.

Conclusie:DHOPEvermindertreperfusieschadevandegalwegen.

Hoofdstuk 7 bevat de resultaten van een preklinische studie naar het verlengen van

DHOPE om donorlevers langer te kunnen bewaren. We hebben de leverfunctie

onderzochtna2,6 en24uurDHOPEbij zowel varkens- alshumane leversdoordeze

met autoloog bloed te reperfunderen. Opvallend genoeg bleek er geen verschil in

leverfunctiewaarneembaar.Allegeanalyseerdemarkersvanendotheelschadelietenook

geenonderscheidzientussendegroepen.Tweehumaneleverswerden20uurgepompt

metbehulpvanDHOPE,voorafgaandaanopwarmenenreperfusie.Dezeleversvoldeden

vervolgens aandeklinischenormthermemachineperfusie criteria voor transplantatie.

Of>12uurDHOPEindepraktijkevenveiligblijktals2uurDHOPE,zalmoetblijkenuit

eenprospectieveklinischestudie.

In hoofdstuk 8 hebben we de resultaten van een prospectief klinisch onderzoek

beschreven,deDHOPE-COR-NMPtrial.Indezestudiewerdenallenationaalafgekeurde

donorleversaangebodenvoorinclusie.IndepraktijkbetroffenditmetnameDCDlevers

vandonoren>60jaaroud,en/ofzeersteatotischelevers.Donorleversondergingeneerst

eenuurDHOPE,omdemitochondriaterevitaliserenendeischemie-reperfusieschade

teverminderen.Vervolgenswerderopgewarmdnaar37gradenCelsius(normotherme

machineperfusie[NMP]).Binnen2.5uurvanNMPmoestdedonorleveraandevolgende

criteria voldoen om getransplanteerd te worden: perfusaat lactaat <1.8mmol/L, pH

7.35-7.45,galproductie>10mLengalpH>7.45.Binnendestudieperiodeondergingen

zestien levers DHOPE-COR-NMP. Alle afgekeurde levers lieten een adequate

parenchymfunctiezien.Echter,elfvandezestienlevershaddenookeengalpH>7.45en

wordendustransplantabelgeacht.MethetDHOPE-COR-NMPprotocolwerd69%vande

levers die afgekeurd waren tóch getransplanteerd, met een 100%

transplantaatoverlevingna1jaar.Ditleiddetoteen>20%toenamevanhetaantalpost-

mortalelevertransplantatiesinhetUMCG.

Hoofdstuk 9 bevat een techniek die is ontwikkeld naar aanleiding van een recente

nieuwetransplantatiemethodeuitGuangzhou,China.InhetFirstAffiliatedHospitalvan

Sun Yat-sen University zijn ze erin geslaagd om als eerste ter wereld ischemie-vrije

orgaantransplantatieuittevoeren.Bijdezetechniekwerdeenkleininterpositievatend-

to-sideopdevenaportaegeanastomoseerd,waardoorcannulatieencontinueperfusie

tijdens uitname, preservatie en implantatie mogelijk was. In Groningen ontstond het

ideeom,inplaatsvanhetinterpositievatopdevenaportae,devenaumbilicalisvande

donorleverhiervoor tegebruiken.Dezebleekeenvoudig teopenenen tedilateren, en

machineperfusiedoordevenaumbilicalisbleekvervolgensnietinferieuraanreguliere

perfusie via de vena portae hoofdstam. Tevens wordt in dit hoofdstuk een nieuwe

techniekvoor‘splittenaandepomp’beschreven,metbehulpvanmachineperfusieviade

venaumbilicalis.

CONCLUSIE:

Destudiesdieinditproefschriftwordenbeschrevenhebbenhetdoelomtransplantatie

van suboptimale, hoog-risico donorlevers op veilige wijze mogelijk te maken. Helaas

wordt inNederlandnogsteeds tegende30%vanbeschikbaredonorleversafgewezen

voor transplantatie. Zo lang als ernog veel patiëntenoverlijdenopdewachtlijst voor

een donororgaan, zouden transplantatieprofessionals nieuwe technieken moeten

onderzoeken om deze afgewezen donorlevers toch te kunnen transplanteren. Zoals

recent beschreven in een editorial in de New England Journal of Medicine, “het

vermijdenvan risico’sheeft geleid tot eenverschuivingvanonderzoeksportfolio’svan

veel transplantatiecentra: ver weg van pionierswerk en juist in de richting van

leefstijlgeneeskundeenlangetermijnobservationelestudies”.Destudiewaaringetracht

is afgekeurde donorlevers toch te transplanteren met behulp van machine perfusie

(hoofdstuk8)toontaandateengezamenlijkerisicodatwordtgenomendoorpatiënten

artsleidttoteengrotestapvoorwaartsinhetminimaliserenvanwachtlijststerfte.Veel

toekomstige studies zijn nodig om wachtlijststerfte te verbannen uit

orgaantransplantatie en om de uitkomsten na transplantatie te verbeteren. Om dit te

bereiken, zullen artsen risico’s niet moeten vermijden, maar het dappere pad naar

medischeinnovatiesamenmetdepatiëntbetreden.

Listofpublications

• deJongIEM,vanLeeuwenOB,LismanT,GouwASH,PorteRJ.Repopulatingthe

biliarytreefromtheperibiliaryglands.BiochimBiophysActaMolBasisDis.2018

Apr;1864:1524-1531.

• vanRijnR,vanLeeuwenOB,MattonAPM,BurlageLCWiersema-BuistJ,vanden

HeuvelMetal.Hypothermicoxygenatedmachineperfusionreducesbileduct

reperfusioninjuryaftertransplantationofdonationaftercirculatorydeathlivers.

LiverTranspl.2018May;24(5):655-664.

• vanLeeuwenOB,UbbinkR,deMeijerVE,PorteRJ.TheFirstCaseofIschemia-

freeOrganTransplantationinHumans:aProofofConcept.AmJTransplant.2018

Aug;18(8):2091.

• van Leeuwen OB, de Meijer VE, Porte RJ. Viability criteria for functional

assessment of donor livers during normothermic machine perfusion. Liver

Transpl.2018Oct;24(10):1333-1335.

• deVriesY,vanLeeuwenOB,MattonAPM,FujiyoshiM,deMeijerVE,PorteRJ.Ex

situnormothermicmachineperfusionofdonorliversusingahemoglobinbased

oxygen carrier: a viable alternative to red blood cells. Transpl Int. 2018

Nov;31(11):1281-1282.

• deVriesY,MattonAPM,NijstenMWN,WernerMJM,vandenBergAP, deBoer

MT,Buis CI, FujiyoshiM, deKleineRHJ,vanLeeuwenOB, et al. Pretransplant

Sequential Hypo- and Normothermic Machine Perfusion of Suboptimal Livers

Donated after Circulatory Death Using a Hemoglobin-based Oxygen Carrier

PerfusionSolution.AmJTransplant.2019Apr;19(4):1202-1211.

• deVriesY,BerendsenTA,FujiyoshiM,vandenBergAP,BlokzijlH,deBoerMT,

van der Heide F, de Kleine RHJ,van LeeuwenOB,Matton APM,WernerMJM,

LismanT,deMeijerVE,PorteRJ.Transplantationofhigh-riskdonorliversafter

resuscitationandviabilityassessmentusingacombinedprotocolofoxygenated

hypothermic, rewarming and normothermicmachine perfusion: study protocol

foraprospective,single-armstudy(DHOPE-COR-NMPtrial).BMJOpen.2019Aug

15;9(8):e028596.

• WernerMJM,vanLeeuwenOB,de Jong IEM,BodewesFAJA,FujiyoshiM,etal.

First report of successful transplantation of a pediatric donor liver graft after

hypothermicmachineperfusion.PediatrTransplant.2019May;23(3):e13362.

• van Leeuwen OB, FujiyoshiM, Ubbink R,WernerMJM, Brüggenwirth IMA, de

Meijer VE, Porte RJ. Ex situ machine perfusion of human donor livers via the

surgically reopened umbilical vein: a proof of concept. Transplantation. 2019

Oct;103(10):2130-2135.

• vanLeeuwenOB,deVriesY,FujiyoshiM,NijstenMWN,UbbinkR,PelgrimGJ,et

al. Transplantation of high-risk donor livers after ex situ resuscitation and

assessment using combined hypo- and normothermic machine perfusion: a

prospectiveclinicaltrial.AnnSurg.2019Nov;270(5):906-914.

• HeX,ChenG,ZhuZ,ZhangZ,YuanX,HanM,ZhaoQ,ZhengY,TangY,HuangS,

WangL,vanLeeuwenOB,WangX,ChenC,MoL,JiaoX,LiX,WangC,HuangJ,

CuiJ,GuoZ.TheFirstCaseofIschemia-FreeKidneyTransplantationinHumans.

FrontMed(Lausanne).2019Dec11;6:276.

• van LeeuwenOB, de Jong IEM, Porte RJ. Biliary Complications following liver

transplantation. In:ClavienPA,Trotter JF, editors.Medical andSurgicalCareof

LiverTransplantationPatients.Wiley-Blackwell;2020

• vanLeeuwenOB,vanReevenM,vanderHelmD,IJzermansJNM,deMeijerVE,

vandenBergAP,DarwishMuradS,vanHoekB,AlwaynIPJ,PorteRJ,PolakWG.

Donor hepatectomy time influences ischemia-reperfusion injury of the biliary

tree indonation after circulatorydeath liver transplantation. Surgery. 2020 (in

press).

• vanReevenM,vanLeeuwenOB,DarwishMuradS,vandenBergAP,vanHoekB,

Alwayn IPJ, Polak WG, Porte RJ. Selected livers of donation after circulatory

deathcanbesafelyusedforretransplantation.TransplInt.2020(inpress).

• Brüggenwirth IMA, van Leeuwen OB, de Vries Y, Bodewes SB, Adelmeijer J,

Wiersema-Buist J, Lisman T, Martins PN, de Meijer VE, Porte RJ. Extended

hypothermic oxygenated machine perfusion enables ex situ preservation of

porcineliversforupto24hours.JHepRep.2020(inpress).

• vanReevenM,GilboN,MonbaliuD,vanLeeuwenOB,PorteRJ,YsebaertD,van

Hoek B, Alwayn IPJ,MeurisseN, Detry O, Coubeau L, Cicarelli O, Berrevoets F,

RogiersX,IJzermansJNM,PolakWG.Liversdonatedaftereuthanasia:ajustifiable

optiontoexpandthedonorpool.Submittedforpublication.

• vanLeeuwenOB,vanReevenM,vanderHelmH,IjzermansJNM,AlwaynIPJ,van

HoekB,PolakWG,LismanT,deMeijerVE,PorteRJ.Donorbloodcompositionisa

risk factor for biliary injury during donation after circulatory death liver

transplantation.Submittedforpublication.

• deVriesY,BrüggenwirthIMA,vonMeijenfeldtFA,vanLeeuwenOB,BurlageLC,

de Jong IEM, Gouw ASH, Lisman T, Porte RJ. Dual versus single oxygenated

hypothermic machine perfusion of porcine donation after circulatory death

livers: impact on hepatobiliary and endothelial cell injury. Submitted for

publication.

Dankwoord

Toen ik in 2014 het geluk had in het studenten leverteam te worden aangenomen,

merkte ik meteen hoe warm en stimulerend het onderzoeksklimaat binnen de

onderzoeksgroeprondomdeleverperfusiewas.Teamworkheeftaltijdvooropgestaan;

gezamenlijkwerdenprojectenvlotenmeestalsuccesvol toteeneindegebracht.Het is

een groot voorrecht om de afgelopen jaren in deze omgeving te hebben mogen

functioneren waarin ik veel heb kunnen opsteken van de chirurgen, hepatologen en

bovenaldeanderepromovendi.Ditproefschriftvoeltvoormijnietalsheteindevaneen

tijdperk,maaralseeneerstetraptrede.

Professordr.R.J.Porte,besteRobert,watbenikontzettenddankbaarvoorallekansen

envrijheiddiejemijdeafgelopenjarengebodenhebt.Ikhebheelveelrespectvoorhoe

je dag in dag uit het hoogst haalbare nastreeft en nooit akkoord zal gaan met een

binnenbocht.Indecommunicatiezijnweallebeigraag‘kortenbondig’.Jeafsprakenmet

anderepromovendi liepenvaakuit,maar ik stondmeestalbinnendehelft vande tijd

weerbuiten.HoopmaardathiergeldtzoalsMilanKunderaooit treffendschreef:“You

can't measure the mutual affection of two human beings by the number of words they

exchange”…Wehebbenveelmeegemaaktafgelopenjaren:vanmoedelozemomentenbij

afgekeurde levers na urenlange NMP’s tot het aanschouwen van ischemie-vrije

levertransplantatie inGuangzhou.Er zijnnogveel ideeënoveren ikhoopdat ik jede

komendejarenkanblijvenbijstaanbijdeuitvoeringvanhiervan.Ikhadmegeenbetere

promotorkunnenwensen.

Professordr.T.Lisman,Ton,ondanksdatiknaarjouwwetenschappelijkemaatstaven

hoogstwaarschijnlijknietdeidealepromovenduswas,hebjemijmetveelenthousiasme

gesteund. Je bent een kundig voorbeeld van denken in oplossingen in plaats van

problemen. Je scherpe analyses hebben mij regelmatig uit de klinische/praktische

tunnelvisie gehaald. Ik hoop dat we gezamenlijk de komende jaren het project met

bloedsamenstelling/stolling/endotheelactivatie en de linkmet NAS verder uit kunnen

werken.Dankvoorjesupport!

Dr.V.E.deMeijer,Vincent,watbenikonderdeindrukvanwatjeindeafgelopenjaren

in het UMCG al bereikt hebt met je enorme ambitie! Met inmiddels je geaccepteerde

Veni-aanvraagopzakweetikzekerdatjeheelveelzalbereikenkomendejaren.Ikvond

hetergfijndatwevanuitonzewerkplekkenelkaarkondenzienendatikdaardoorveel

bijjelangskonhoppenvooroverleg.Dathielddesnelheidinonzeprojectenhoog.Los

vanjewetenschappelijkeexpertisebenikeveneensdankbaarvoorhetfeitdatikmetjou

enRobert tweehele goede chirurgen als promotores heb gehad.Hierdoor heb ik niet

alleen opwetenschappelijk gebied,maar ook op klinisch vlak ontzettend veel kunnen

leren.

Beoordelingscommissie: Prof. H.G.D. Leuvenink, prof. D. Monbaliu, prof. J.K.G.

Wietasch, heel veel dank voor jullie interesse in het proefschrift en de inhoudelijke

feedback.

OLT1&OLT2’s:prof.dr.RobertPorte,dr.MariekedeBoer,drs.RubendeKleine,

dr.CarlijnBuis,dr.VincentdeMeijer,prof.dr.JoostKlaase,dr.MaartenNijkamp,

dr.FrederikHoogwater,ikbenontzettenddankbaardatjulliemijals18/19/20-jarige

leverstudentbijheelveelOLT’senelectieveHPB-operatieshebben latenassisterenen

meekijken.Datheeftbijmijvooreenhoopmotivatiegezorgdvoorditpromotietrajecten

hetheefthetmijduidelijkgemaaktwaar inhet ziekenhuismijnpassie ligt.Bovenal is

door jullie ondersteuning de machineperfusie in de afgelopen jaren volledig

geïntegreerdinhetUMCG!

Dr.M.Fujiyoshi,dearMasato, Iamthankful for thegreatyearswehad inGroningen.

Youwere always first to discuss ideas on transplantation andmachine perfusion and

youalwaysprovidedmewithsupportonmyprojects.WespentalotoftimeintheOPR

(and Burgerking) together and without your everlasting help the machine perfusion

researchintheUMCGwouldhavedevelopedmuchslower.Ihopetobeabletocometo

Japanagaininthecomingyears!

Kantoorgenoten: drs. M. Meerdink, drs. C.R. Hoepel, drs. S.J.S Ruiter, drs. A.N.

Tournicourt,BesteMark,Carlos,SimeonenAn,ikbenjulliedankbaardatjulliemijals

‘benjamin’ op de kamer accepteerden. Het heeft voor mij continu als motivatie

gefungeerdomjullieklinischeverhalentehoren.Ikhoopdatiklaternogeenkeeronder

julliebegeleidingmagwerkenindekliniek!

Chirurgisch Onderzoeks Laboratorium: Jacco Zwaagstra, Petra Ottens, Jelle

Adelmeijer, Janneke Wiersema-Buist. Ik weet niet hoe vaak ik Jacco heb horen

benadrukken: “Otto, diewerkt hier eigenlijk nooit”, Desondanks liggen van een groot

deel van mijn projecten de wortels in het COL. Ik ben jullie dankbaar voor de

behulpzaamheid en prettige werksfeer die ik altijd heb ervaren. Susanne Veldhuis,

Suus,heelveeldankvoorjehulpbijhetvarkensexperimentmetFienenmij.Wewaren

metname inhetbeginergonbeholpenennietaltijdevenwetenschappelijk,maarmet

jouwhulphebbenweereenvolwassenexperimentvankunnenmaken!

Kroon Vlees Groningen: Gert, Jan, Luitzen, en de vele anderen die ervoor gezorgd

hebbendatwe inmiddelsalredelijkwat jarensuccesvolvarkensexperimentenkunnen

uitvoeren:heelveeldankvoorjulliehulpengastvrijheid!

Drs.M.vanReeven,besteMarjolein,watwehebbentocheenprettigesamenwerking

gehad ondanks de obstakels die we zijn tegengekomen. Het meest voorkomende

probleemkwamechtervanmijnkant:dehumane-envarkensperfusiesdieweer inde

weglagen.Ikbenjedankbaarvoorjegeduldéndrivewaarmeejeonzeprojectentoteen

goedeindehebtwetentebrengen.Tevensveeldankaandr.W.G.Polak,prof.dr.J.N.M.

IJzermans,prof.dr.I.P.J.Alwayn,prof.dr.B.vanHoekendr.D.vanderHelmvoor

demooienationalesamenwerking!

(Oud) transplantatiecoordinatoren UMCG, beste TC’ers, heel veel dank voor jullie

hulpmethetidentificerenvanpotentiëledonorenvoorNMP!Dehoogtepuntenvanonze

samenwerking vonden echter plaats in de late uurtjes: in de Poelestraat of op de

congressen!

OrganAssist;WilfreddenHartog,ArjanvanderPlaats,TonMulderij,veeldankvoor

jullie support en het toestaan van onze varkensexperimenten waarmee we voor een

hoop gedoe hebben gezorgd aan de Bornholmstraat.. Ik kijk terug op een prettige

samenwerking.EmmaOffringa,Mark Slotemaker, Ernst Buiter, Martin Kuizenga:

jullie waren simpelweg geweldig!! De congressen werden met jullie altijd een groot

feest,maarookdevarkensexperimenten.Erwasaltijdeengoedesfeerenwaarweook

waren,julliegavenaltijdeengevoelvan‘thuiskomen’.

Studenten van leverteam, waaronder Silke Bodewes, dank voor jullie inzet bij het

organiserenenuitvoerenvansample-afnamesenhetverwerkenrondomde(perfusie)

levertransplantaties.

Orgaanperfusionisten, drs. Rinse Ubbink, dr. Gert Jan Pelgrim, drs. Maureen

Werner, en nu sinds kort ookdrs.MartijnHaring, Veerle Lantinga en Leonie van

Leeuwen;watbeniktrotsopderesultatendiewedeafgelopenjarenbehaaldhebben!

Het heeft de nodige flexibiliteit gekost, maar jarenlang stondenwe klaar voor iedere

perfusie.Dankvoorjullieteamworkenenthousiasme!

Leveronderzoekers:dr.AlixMatton,dr.LauraBurlage,drs.YvonnedeVries,drs.

ShaniceKarangwa,IrisdeJong,ikrealiseermewateenontzettendeluxehetisomhet

goed te kunnenvindenmet je collega’s.Alle perfusies,maarmetnamede congressen

(van Bootcongressen tot Oslo/Seattle/Toronto) werden geweldige ervaringen door

jullie!

Dr.R.vanRijn,Rianne,als18/19-jarigejongenwerdikdoorRobertaanjegekoppeld

enmocht ik jewatgaanhelpenmet jeonderzoeken.Heb(totopdedagvanvandaag)

ontzettend genoten van je indrukwekkende puurheid. Ik ben dankbaar voor je

vertrouwenendekansendiejemehebtgebodenenikgaervanuitdatikindetoekomst

nogveelvanjegaleren.

FienvonMeijenfeldt,Fien,wathebbenweeenmooivarkensexperimentneergezetmet

de retrograde flush en DNAse therapie. Lange dagen, maar altijd met goede sfeer

ondanksdesoms licht tragischeresultaten!Bedanktvoorde fijnesamenwerkingen je

verfrissendeblikopzakendievoormijvoorheenkraakhelderleken.

Drs. M.J.M. Werner, Maureen, mede onderzoeker en orgaanperfusionist, wat ben ik

onderdeindrukgeweestvanjenietaflatendeopgewektheid.Zaljecontinuesupportop

m’npersoonlijkedieptepuntopdezeiknatteHighway5vanVancouvernaarSeattleook

nooit vergeten, nog steeds dankbaar voor... De vele nachten diewe gezamenlijk in de

OPRhebbendoorgebrachtwerdendoorjoueengrootfeest!Ondanksdatwesomsmet

een lege energietank tóch weer aan een NMP moesten beginnen, sleepte je ons er

allemaal doorheen met je optimisme! Denk dat ook menig donorlever door jouw

aanstekelijkeenthousiasmetochnétaandeviabilitycriteriagingvoldoen…

IsabelBrüggenwirth,Isabel,wathebbenweveelmeegemaaktdeafgelopenjaren.Van

eindeloos veel hardlopen, inclusief marathon, tot ontzettend veel humane- en

varkensperfusies. Denk nog steeds dat we het leverperfusierecord in handen hebben

metdecombinatie‘verlengdeDHOPEvarkensexperiment+succesvollehumaneDHOPE-

COR-NMPprocedure’,waarinwevandinsdag05:00 totwoensdag18:00non-stop (37

uur) stonden tevlammen. Jebentaltijdm’neersteaanspreekpuntgeweestbijnieuwe

(en regelmatig slechte) ideeën en hersenspinsels en ik hoop dat dat nog lang zomag

blijven!

Willem Wierbos, Willem, waar menig vriendschap zou lijden onder de vele

leverperfusiesvandeafgelopen jaren,hebbenwijdaarnooit lastvangehad.Dankbaar

voor je support en trots op je ontwikkeling als zanger/acteur/poëet naast je

geneeskundestudie.Vereerddatjem’nparanimfwiltzijn!

Dr.R.B.vanLeeuwen,drs.M.A.vanLeeuwen-Buinink,mr.dr.B.J.vanLeeuwenen

drs.C.A.T.vanLeeuwen;Roeland,Marie-Anne,BarendenClaar,hetheeftdeafgelopen

jarenheelwatgekost,maarwatwashethetwaard!Hebvanjongsafaangoedvanjullie

allenkunnenafkijkenhoeveelwerkerverzetmoetwordenomietstebereikenenben

dankbaarvoordevoorbeeldfunctiesdie jullievoormijgeblekenzijn.Debezoekenaan

Apeldoornwarennietaltijdevenlangdurigdeafgelopenjarendoordefrequentelevers,

maaromBarendsingezettetraditievoorttezettenwas iksteedsopreisenaltijdthuis.

Claar;ikbentrotsopjerolalsparanimf,enbinnenkortstaanwebij jouwverdediging;

ikkijkernaaruit.Heeldankbaarvoorjulliesupport!

Curriculumvitae

OttovanLeeuwenwasbornon15thNovember1995inApeldoorn,theNetherlands.He

receivedhis secondaryeducationat the ‘GymnasiumApeldoorn’ inApeldoorn.Forhis

final research project at secondary school, Otto spent one month studying surgical

woundcare inMwananyamalaHospital,DarEsSalaam,Tanzania.Aftergraduation,he

movedtoTampa,USA.AttheUniversityofTampa,hemajoredineconomics.However,

mostofhisclassesfocussedonliteratureandacademicwriting.AfteroneyearinTampa,

he was admitted to study Medicine at the University of Groningen. During the three

years of his bachelor’s programme, Otto worked in the student liver transplantation

teamand thePrometheusnierteamat theUniversityMedical CenterGroningen.From

thefirstyearofhisbachelor’sprogramme,Ottoenjoyedworkingforprof.dr.R.J.Porte

attheDepartmentofSurgeryandstartedassistingwithresearchprojects.Hewrotehis

bachelor thesis “Increasing thequalityof suboptimaldonor livers for transplantation:a

bridgebetweenclinicandresearch”andmasterthesis“Riskfactorsassociatedwithbiliary

complications and early graft loss following donation after circulatory death liver

transplantation”underthesupervisionofprof.dr.R.J.Portebeforeapplying foraMD-

PhDposition.HewasacceptedintotheMD-PhDtrajectoryinJune2018.FromMarchto

April2018,OttoworkedinGuangzhou,China,topractisehumanandporcineischemia-

free liver transplantation. Otto is currently finishing his Master’s degree inMedicine,

hopingtopursuehisdreamofbecominga(transplant)surgeon.