Uniiversity of Pisa
P
DIPARTIMEN
NTO DI PAT
TOLOGIA CHIRURGICA, MEDIICA,
MOL
LECOLARE
E E DELL’A
AREA CRITICA
Dotto
orato di riceerca in Scien
nze dei Trap
pianti
Teesi di dottorrato
TITOLO
Molecularr-size excclusion chromato
c
ographyy of
gamma
a-glutam
myltransfferase fractions:
a tool fo
or investtigating pathoph
p
hysiologyy
of liveer diseasse and liv
ver transplant
Settore scienttifico discipllinare: Med
d05
Candidate
n Abdelazizz Morsi Ahm
med Elawad
di
Dr. Hassan
Tutor
Prof. Aldoo Paolicchi
Doctorate C
Coordinator
Prof. Fran
nco Filippon
ni
Finall Exam yearr 2013
Academ
mic year: 20
008-2009
Index
INDEX
ABSTRACT.......................................................................................................... v
1. INTRODUCTION .............................................................................................. 1
1.1 General .......................................................................................................... 1
1.1.1 The possible policies for organ allocation .................................................... 3
1.1.2 Strategies and techniques to expand the donor organ pool ........................ 8
1.1.3 Sources of organ. ........................................................................................ 8
1.1.4 Types of OLT ............................................................................................... 8
1.2 Selection of recipients and organ allocation ............................................. 9
1.2.1 The US model for liver allocation ................................................................. 9
1.2.2 The European model for liver allocation ...................................................... 10
1.3 Scoring systems ........................................................................................... 11
1.3.1 The Child-Turcotte-Pugh (CTP) score ......................................................... 11
1.3.2 CTP limitations ............................................................................................ 12
1.3.3 MELD score: “sickest first policy” ................................................................ 13
1.3.4 MELD calculation: ....................................................................................... 14
1.3.5 Upgrading of MELD score for Hepatocellular Carcinoma (HCC) ................. 15
1.3.6 MELD limitations ......................................................................................... 15
1.3.7 Classification of Candidates for Liver Transplants According to Old UNOS 18
1.3.8 Other Scoring Systems................................................................................ 18
1.4 Indications and Contraindications for Liver Transplantation................... 22
1.4.1 Indication of liver transplantation in adults ................................................... 23
1.4.2 Indications in children .................................................................................. 24
1.4.3 Variant syndromes requiring liver transplantation ........................................ 24
1.4.4 Transplantation For Acute Liver Failure (ALF)............................................. 24
1.4.5 Criteria for liver transplantation in acute liver failure (ALF) .......................... 26
1.4.6 Transplantation For Alcoholic Liver Disease (ALD) ..................................... 27
1.4.7 Transplantation for Chronic Liver Disease................................................... 27
1.4.8 Transplantation For Hepatic Malignancy ..................................................... 29
1.4.9 Transplantation For Metabolic Liver Disease .............................................. 32
1.4.10 Transplantation For Vascular Disorders .................................................... 32
1.4.11 Other indications ....................................................................................... 33
i
Index
1.4.12 Contraindications to Liver Transplantation ................................................. 33
1.4.13 Retransplantation ....................................................................................... 35
1.4.14 Delisting Criteria ......................................................................................... 36
1.4.15 Living Donor Liver Transplantation............................................................. 36
1.4.16 Contraindications for LDLT ........................................................................ 38
1.5 Patient Evaluation ......................................................................................... 38
1.5.1. Evaluation Aim And Purpose ...................................................................... 38
1.5.2 Timing of referral for liver transplantation evaluation.................................... 40
1.5.3 The Process of Liver Transplant Evaluation ................................................. 41
1.5.4 Evaluation of potential donors for living donor liver transplantation ............. 45
1.5.5 Listing for transplantation and organ allocation ............................................ 45
1.5.6 Medical issues to be considered during evaluation ...................................... 45
1.5.7 Specific Consideration For Liver Transplantation ......................................... 49
1.6 Management While Waiting for Transplantation ........................................ 58
1.6.1 Lab values Recertification schedule of Meld data ........................................ 59
1.6.2 Disease-Specific Considerations ................................................................. 59
1.7 The Donor ...................................................................................................... 73
1.7.1 Brain Death ................................................................................................. 73
1.7.2 Strategies and techniques to expand the donor organ pool include ............. 76
1.7.3 Donor age .................................................................................................... 80
1.7.4 Influence Of Hepatic steatosis ..................................................................... 84
1.7.5 Non–heart-beating donor (NHBD) livers ...................................................... 90
1.7.6 Orthotopic Liver Transplantation with partial allografts................................. 97
1.7.7 Other risk factors ......................................................................................... 101
1.7.8 Donor-transmitted diseases ......................................................................... 104
1.8 Living donor liver transplantation ............................................................... 108
1.9 Immunology ................................................................................................... 116
1.9.1 Hyperacute rejection .................................................................................... 121
1.9.2 Acute rejection ............................................................................................. 121
1.9.3 Chronic rejection .......................................................................................... 123
1.9.4 Immunosuppressive therapy general considerations ................................... 124
1.10 Post-liver transplantation complications .................................................. 128
1.10.1 The main complications in the immediate postoperative period ................. 132
1.10.2 Long-term complications ............................................................................ 151
ii
Index
1.10.3 Graft Monitoring and Post Transplant Pathology ....................................... 161
1.10.4 Early New Onset Diseases/Injuries in the Liver Allograft ........................... 165
1.10.5 Later new-onset disease/injuries in the liver allograft ................................ 199
1.11 Cell Therapy ................................................................................................ 211
1.11.1 Hepatocytes .............................................................................................. 212
1.11.2 Embryonic stem cells................................................................................. 215
1.11.3 Mesenchymal stromal cells ....................................................................... 217
1.11.4 Amnion epithelial (AE) cell transplantation ................................................ 219
1.11.5 Induced Pluripotent Cells ( iPSC) .............................................................. 221
1.12 Biomarkers of liver fibrosis ....................................................................... 223
1.13 γ-Glutamyltransferase................................................................................ 231
1.13.1 Generalities and tissue distribution ............................................................ 231
1.13.2 Physiological functions of γ-glutamyltransferase ....................................... 233
1.13.3 Serum GGT: origin and chemical and physical characteristics.................. 240
1.13.4 Predictive value of serum GGT in hepatobiliary diseases ......................... 242
1.13.5 Serum γ-glutamyltransferase: cardio - vascular diseases ......................... 244
1.13.6 Fractional GGT analysis ............................................................................ 250
1.13.7 GGT fractions in the Framingham Heart Study ......................................... 251
2. AIM ................................................................................................................... 255
3. MATERIALS AND METHODS ......................................................................... 257
3.1 Patient selection ............................................................................................. 257
3.2 Laboratory analysis ........................................................................................ 258
3.3 Total and fractional GGT determination ......................................................... 258
3.4 Statistical analysis .......................................................................................... 260
3.5 Patient selection for immunohistochemical analysis ....................................... 261
3.6 Tissue Microarray Technique (TMA) .............................................................. 261
3.7 Immunohistochemical analysis ....................................................................... 261
3.8 Collection of samples of primary human bile and analysis of the fractions ..... 262
3.9 Half-life activity of bile GGT ............................................................................ 262
3.10 Activation of papain ...................................................................................... 262
3.11 Treatment with papain and bile deoxycholic acid ......................................... 263
iii
Index
4. RESULTS AND DISCUSSION ......................................................................... 265
4.1 Accuracy of bGGT fraction for the diagnosis of NAFLD .................................. 265
4.2 Cirrhotic patient evaluation – pretransplant ..................................................... 271
4.3 Characterization of GGT fractions in primary human bile................................ 289
4.4 Fractional GGT evaluation after liver transplant .............................................. 297
5. CONCLUSION .................................................................................................. 315
6. REFERENCES ................................................................................................. 321
iv
Abstract
ABSTRACT
The aim of this study is test the diagnostic power of GGT fraction for hepatic diseases in
comparison with that of total GGT and its usefulness in the setting of liver
transplantation. Cirrhosis and chronic liver failure, and HCC are leading causes of
morbidity and mortality worldwide. The diagnosis of cirrhosis and the determination of
the etiology remain complex, in fact, no serologic test or radiological study can
accurately diagnose cirrhosis. Besides, assays in most standard liver panels do not
reflect the function of the liver correctly. With appropriately selected patients, liver
transplantation is a definitive curative therapy for long-term survival and good quality of
life for patients with end stage liver disease facing death. Accurate diagnosis and
prognosis are essential for patients management pre and post-transplant, and for
patients prioritization for organ allocation for liver transplantation. This requires the
choice of biomarkers that provide adequate diagnostic information, at the minimum cost
to more accurately select candidates for liver transplantation, to monitor post-transplant
outcome and provide an optimal treatment regimen.
Serum gamma-glutamyltransferase (GGT) activity is a sensitive marker of liver
dysfunction, but its specificity is modest, in fact, its value increases in all liver
dysfunctions. GGT has been already included in diagnostic algorithm (i.e.: the Fatty
Liver Index, FLI), its specificity was high if considered with other markers, but low if
considered alone. The currently used laboratory GGT assays do not allow
discriminating among the different causes of GGT increase, thus reducing the clinical
value and specificity of this otherwise sensitive disease biomarker. A new method
based on molecular-size exclusion chromatography, followed by a GGT-specific postcolumn reaction, allowed to identify and quantify, in healthy subjects, 4 plasma GGT
fractions with high sensitivity, specificity and reproducibility. These fractions, named bigGGT (b-GGT), medium-GGT (m-GGT), small-GGT (s-GGT), and free-GGT (f-GGT)
showed different molecular weight (MW), i.e. 2000, 1000, 250 and 70 kDa, respectively.
It has been previously shown that in healthy subjects f-GGT is the most abundant
fraction, while b-GGT showed the highest degree of correlation with established
cardiovascular risk factors. Interestingly b-GGT has been found in atherosclerotic
plaques together with products deriving from the pro-oxidant reactions catalysed by the
enzyme, The liver is one of the main organs that generates free radicals, one of the
mechanisms of hepatocyte injury in response to diverse insults occurring in different
pathological conditions. For example, oxidative stress has been demonstrated to be
v
Abstract
implicated as a cause of hepatic fibrosis. Besides, liver damage is characterized by
increased iron storage which elicits a free-radical mediated peroxidation.
In the period between February 2008 and April 2011, 264 patients during evaluation for
liver transplant [215 men; median (25th – 75th percentile); age 54.5 (50-60 years)] were
enrolled at the Department of Surgery, Liver Transplantation Unit of the University
Hospital of Pisa. At the visit, attendees underwent anamnestic-physical examination and
blood sampling for the laboratory assessment of liver function. In this cohort: 39 patients
were diagnosed with metabolic cirrhosis (MC), 96 with viral cirrhosis (VC) 129 with viral
cirrhosis and hepatocellular carcinoma (HCC). As control 200 blood donors were
selected and studied for the determination of fractional GGT reference values. Blood
samples were also collected from 14 LT recipients preoperatively before native liver
hepatectomy (T0), and for 10 consecutive days post-transplant. Bile samples were
collected intra-operatively during duct anastomosis (T0) and 10 days following the
surgical procedure of transplantation through Kehr-tube. Standard assay of all blood
tests were simultaneously performed according to the standard clinical laboratory
procedures by automated analysers at the Clinical Laboratories of the University
Hospital of Pisa.
Analysis of total and fractional GGT was performed using an FPLC (fast protein liquid
chromatography) system. Separation of fractional GGT was obtained by gel filtration
chromatography and the enzymatic activity was quantified by post-column injection of
the fluorescent substrate for GGT. The area under chromatogram peak is proportional
to fractional GGT activity. Total area and fractional GGT area was calculated by a
MatLab program. Localization of GGT protein in liver biopsies was performed by
automated indirect immunohistochemical analysis, using a polyclonal antibody directed
against the C-terminal 20 amino acids of GGT heavy chain. Histological sections were
analysed using the image software MetaAnalisys.
Different GGT fraction patterns were observed in cirrhotic patients and within the three
sub cohorts (VC, MC, HC). s-GGT showed a broader and double profile not seen in
controls, defined as s1-GGT and s2-GGT. The b/s ratio was lower in patients than
controls. The diagnostic value of the b/s ratio was independent of the absolute values of
total GGT and from the aetiology of the cirrhosis and the presence of liver cancer.
Variations of the GGT fractions reflect different aspects of the liver cirrhosis: b-GGT
vi
Abstract
behaves as a positive index of liver function, and reflects the progression of portal
hypertension and splenomegaly; s2-GGT fraction reflects hepatocellular damage.
GGT activity in human bile is higher than that found in plasma, showing only two peaks
corresponding to plasma b-GGT and f-GGT fractions, while m- and s-GGT fractions
were not detectable. Regarding the nature and characteristics of biliary complex
corresponding to the plasma b-GGT, the part of b-GGT fraction insensitive to the direct
action of papain can be released into the bile associated with membrane vesicles such
as exosomes. Immunolocalization of GGT in patients and control biopsy demonstrated
different abundance and tissue distribution all over the section and quantification of
GGT in liver tissue suggest that there is not a direct relationship between tissue and
circulating GGT enzyme levels.
The post-operative course of the selected 14 patients was uneventful and there were no
events of acute rejection. Soon after transplantation (24h), a sharp decline in total
plasma GGT is observed and reflected on all fractions, in particular b-GGT. In 5-6 days
after there has been a gradual increase in total plasma GGT. Plasma f-GGT fraction
shows minor alterations, while other fractions have a similar trend as total GGT. In bile
sample T0 GGT is present mainly as b-GGT and in less extent as f-GGT. The first 24 h
post-transplant bile b-GGT activity is decreased followed by a sudden increase in its
activity with a peak observed in the fourth day while bile f-GGT fraction shows minimal
changes. An increase of bile GGT activity and an apparent peak of f-GGT preceded by
an abrupt drop in b-GGT activity a day before is observed at days 6 and 10 in two
patients: and a reversal of the proportions between the bile b-and f-GGT fractions in
favour of b-GGT fraction has been observed in one of these patient at day 10 (T10) and
in another patient on days 7 (T7) and 8 (T8). All fractions behave as positive index of
cholestasis and liver function. Interestingly all fractions showed a positive correlation
with direct bilirubin apart from s1-GGT, which showed a strict negative correlation.
Unexpectedly, all fractions were negative associated with LDH, and b-GGT and m-GGT
showed a negative correlation also with transaminases AST and ALT. Thus plasma
GGT fractions, in particular b-GGT and m-GGT, were primarily related to ischemic-type
biliary lesions following liver transplantation.
In conclusion the main findings of this study are:
vii
Abstract
1) patients with NAFLD and CHC display different GGT fraction patterns, despite similar
total GGT activity values.
2) Collected data showed that the b/s ratio, independently of the absolute values of total
GGT and its fractions, displays a high sensitivity and specificity for liver cirrhosis, and
the values of the b/s ratio were lower than controls independently of the cause of the
cirrhosis (viral or cryptogenetic) or the presence of associated liver cancer. This
suggests that the b/s ratio is a specific biomarker of architectural and functional damage
of the liver.
3) the elution profile of bile GGT activity showed the presence of only two forms
corresponding to plasma fractions b-GGT and f-GGT, respectively. Similar to that found
in plasma GGT fractions, the biliary f-GGT fraction consists of soluble protein and bGGT fraction of exosomes. But, unlike plasma b-GGT, biliary b-GGT fraction is in part
sensitive ti papain action; likely, the portion of biliary b-GGT sensitive to the proteolytic
action might be consistuted of bile acids micelles.
4) Plasma b-GGT and m-GGT levels, in the first 10 days after liver transplant, were
primarily related to ischemic-type biliary lesions following liver transplantation
The precise nature of GGT fractions has not yet been established, and at present it is
not possible to speculate on the possible reasons conducting to different GGT fraction
patterns in NAFLD and CHC and cirrhosis. Data collected suggest that GGT fraction
pattern specificity might depend on its ability to reflect the different extents of
inflammatory, structural and functional derangement in liver disease.
Further study on the nature and biological significance of plasma GGT fractions in
health and disease might allow to improve the use of this sensitive but otherwise poorly
specific biomarker in the numerous contexts in which it is employed, including
multimarker algorithms comprising plasma GGT for the assessment of liver steatosis
and fibrosis. Extensive investigation on the diagnostic value of GGT fractions might
provide a novel diagnostic tool for liver diseases; understanding the nature, properties,
and pathophysiological variations of GGT fraction pattern might allow a better
understanding of the pathogenesis of the diseases associated with increased GGT.
viii
Introduction
1. INTRODUCTION
1.1 General
Liver transplantation is not a palliative but a definitive, curative therapy for a wide range
of diseases. The aim is not only to prolong survival but also to improve the quality of life
of recipients. The procedure has undergone major improvements. Continuous advances
in
surgical
techniques,
improvement
of
intra-operative
management,
better
management of complications, immunosuppressions, and better organ preservation
together with better selection of candidates for transplantation and allocation of donor
organs according to more objective criteria have led to a great success to the
procedure. It is now a routine, safe, standardized procedure performed in many
transplant centers with a substantially improved graft and patient survival and accepted
morbidity rates. Currently, survival rates of over 90-95% and 70% at one year and five
years post-transplantation, respectively are expected (Roberts MS, et al. 2004; Lucey
MR, et al. 1997; Belle SH, et al. 1997; Demetris r AJ, et al. 2009), three-year patient and
graft survival rates in liver transplant recipients are currently 79% and 74%, respectively
(Freeman RB, et al. 2008) and 1-year graft survival rates now exceed 80% (Waki K
2008) with good quality of life. This great success has resulted in one hand first,
broadening of the indications to include previously contraindicated conditions, second,
provided innovations to the field of complex hepatobiliary surgery, laparoscopic liver
procedures, trauma surgery, surgical intensive care, and surgical education. In the other
hand, it is challenged by many obstacles that need to be surpassed and problems to be
resolved. The most important being the shortage of donors in face of great number of
patients awaiting for transplantation and prolonged waiting list time, the need of timely
availability of suitable livers, and the need for an expanded number of useable donor
organs make from liver allocation a true challenge. In addition, the need for improved
therapies to treat recurrent hepatitis C after transplantation, and the need for improved
detection, and risk stratification to combat hepatocellular carcinoma. Liver grafts for
transplantation can be obtained either from deceased donors (DDs) or living donors
(LDs). Living donor liver transplantation (LDLT) was introduced to overcome the
increasing demand for donor organs and to tight the widening gap between the
resource (deceased donor) and demand (recipient) and is the main procedure in
countries where there was virtually no deceased donor programme due to particular
reasons. In deceased donor liver transplantation (DDLT) programme, prioritization of
1 Introduction
patients for organ allocation is crucial. Living donor liver transplantation (LDLT)
programe is different where the prospective donor is usually a close relation. In both
situations, a measure such as a scoring system is important in prognosticating the
outcome following transplantation. There has to be equilibrium between the patient’s
medical reserves to endure or withstand the complex major surgical procedure of liver
transplantation and its probable outcome. A patient is considered too healthy to undergo
LT if the expected survival is greater without LT. Therefore, criteria are needed in order
to select patients who can most benefit from transplantation. Prioritization for liver
transplantation (LT) has evolved over the past 20 years (Adam R, et al. 2009). The
objective of the allocation system is to minimize the total number of deaths to the patient
population. Allocation policies must serve the patients most in need and achieve the
best post-transplant results (Patrizia Burra, et al. 2006). In the context of deceased
donor livers; medical urgency, utility and transplant benefit are the three frequently
discussed organ allocation schemes (Merion RM, et al. 2005, Schaubel DE, et al.
2009). In the urgency policy (sickest first), patients with worse outcomes on the waiting
list are given higher priority for transplantation. DDLT organ allocation was initially
based on whether the patient is at home, in hospital or in an intensive care unit, and the
time length on the waiting list (United Network for Organ Sharing-UNOS status), then
based on their United Network of Organ Sharing (UNOS) status (2A, 2B and 3) based
on their Child Turcotte Pugh (CTP) classification system and its variations to stratify
patients with chronic liver disease to predict the mortality and morbidity. Since 2002, the
Organ Procurement and Transplantation Network, along with the United Network of
Organ Sharing (UNOS), developed a new system based on the model for end-stage
liver disease (MELD for adults and PELD for paediatric recipients) adopting the sickest
first policy for organ allocation (Freeman RB, et al. 2008, Durand F, 2008) to prioritize
patients on the waiting list. These are mathematical regression models which objectively
assess the need for liver transplantation and more accurately predict the short-term
mortality while on the transplantation waiting list (Merion RM, et al. 2005, Kamath PS, et
al. 2001, Longheval G, et al. 2003). In the Eurotransplant countries, the Child-Pugh
Turcotte score was replaced by the MELD score in December 2006. In UK the UK
organ allocation defines donor pools based on patient-specific characteristics, but organ
allocation to individual patients remains at the center’s discretion. United Kingdom
model for End-stage Liver Disease (UKELD) score has been adopted for many years
2 Introduction
and published (Neuberger J, et al. 2008). MELD and UKELD scores poorly predict
outcomes after liver transplantation due to the absence of donor factors.
1.1.1 The Possible Policies For Organ Allocation
a) Medical urgency models
1. Child- Turcotte- Pugh score
2. MELD score
3. Modifications of MELD score
4. UKMELD
b) Utility-based score
1. Donor risk index(DRI)
2. D-MELD
3. Model based on ELTR
c) Transplant Benefit models
Child-Turcotte-Pugh (CTP) Scoring System to Assess Severity of Liver Disease
Points
1
2
3
Encephalopathy grade
None
1 and 2
3 and 4
Ascites
Absent
Slight
Moderate
Bilirubin(mg/dl)
1-2
2-3
>3
For primary biliary cirrhosis Bil.(mg/dl)
1-4
4-10
>10
Albumin (g/dl)
3.5
2.8-3.5
>2.8
Prothrombine Time ( seconds prolonged)
1-4
4-6
>6
Or, INR
>1.7
1.7-2.3
>2.3
According to grading of Trey, Burns, and Saunders.21 Trey C, Burns DG, Saunders SJ.
Treatment of hepatic coma by exchangeblood transfusion. New Engl J Med 1966;
274:473-481.
MELD score according to the UNOS database:
MELD score (UNOS current version) =
9.57 × ln(creatinine) (mg/dl) + 3.78 × ln(Tot.Bil.) (mg/dl) + 11.20 × ln(INR) + 6.43.
- Any value < 1 is considered equal to 1
- If the patient has been dialyzed twice within the last 7 day => serum creatinine = 4.0 mg/dL
- Creatinine >4 was automatically calculated as 4
- Patients with a diagnosis of HCC will be assigned a MELD score based on how advanced the cancer is
United Kingdom Model for End-stage Liver Disease (UKELD).
UKELD = 5 x{1.5 x ln(INR) + 0.3 x ln(Creat) + 0.6 x ln(Br) x13 x ln (Na) + 70}.
Where
INR = international normalized ratio
Creat = serum creatinine (lmol/l)
Br = serum bilirubin (lmol/l)
Na = serum sodium (mmol/l)
3 Introduction
Donors meeting specific criteria offering the liver for splitting is obligatory, the left lateral
segment going to a child at one of three national paediatric centers and the remaining
right liver to the retrieving center.
Splitting criteria.
Donor livers should be split if not required for super urgent transplantation or
multivisceral grafting and the following criteria are met:
1. Donor age <40
2. Weight >50 kg
3. ICU stay less than 5 days
The decision to split is based solely on these criteria and if a segmental graft is
required for a child in any paediatric center the splitting process should be initiated
independent of any decision on allocation of the right liver to an adult patient.
The utility-based systems are based on post-transplant outcome taking into account
donor and recipient characteristics. The transplant benefit models rank patients
according to the net survival benefit that would derive from transplantation. These
models would be based on the maximization of the lifetime gained through liver
transplantation. Regarding survival benefit, there are several methods to characterize
the survival benefit associated with liver transplantation. One method calculates the
covariate-adjusted ratio of post- to pre-transplant mortality rates, and is the direct output
of a standard Cox regression model. Using such a model and with a maximum of 1 year
of post-transplant follow-up, transplant recipients with a MELD score ≥17 derived
significant survival benefit, including patients at the maximum MELD score of 40
(Merion RM, et al. 2005). In contrast, patients at low MELD scores had lower mortality
risk on the waiting list and hence did not derive a survival benefit from liver
transplantation. No current model has all the best characteristics. The lab MELD score
is a numerical scale using the three laboratory parameters and ranging from 6 (less ill)
to 40 (severely ill). In a large study (Merion RM, et al. 2005) investigating the survival
benefit of LT candidates, those transplanted with a MELD score <15 had a significantly
higher mortality risk as compared to those remaining on the waiting list, while
candidates with a MELD score of 18 or higher had a significant transplant benefit.
However, the MELD score does not accurately predict mortality in approximately 1520% of patients. Therefore, MELD-based allocation allows exceptions for patients
whose score may not reflect the severity of their liver disease. These exceptions include
hepatocellular carcinoma (HCC), non-metastatic hepatoblastoma, adult polycystic liver
4 Introduction
degeneration, primary hyperoxaluria type 1, small for size syndrome, cystic fibrosis,
familial
amyloid
polyneuropathy,
hepatopulmonary
syndrome,
portopulmonary
hypertension, urea cycle disorders, hereditary hemorrhagic telangiectasia (OslerWeber-Rendu disease), hemangioendothelioma of the liver, biliary sepsis, primary
sclerosing cholangitis (PSC) and cholangiocarcinoma. Patients with standard
exceptions will be assigned a higher MELD score (match MELD) than patient’s
laboratory test results (lab MELD), consequently, resulting in an increasing number of
patients transplanted for HCC and other exceptions over time (Massie 2011). MELD has
proved to be accurate as a predictor of waiting list mortality, but has shown to be less
accurate to predict post-transplant outcome. For instance, MELD allocation resulted in
decreased waiting list mortality; whereas post-transplant morbidity has increased due to
transplantation of a higher proportion of sicker recipients with MELD scores >30
(Dutkowski 2011). Moreover, since the introduction of MELD, the quality of donor
organs has been impaired and the threshold for organ allocation has increased from a
match MELD of 25 to 34 (Schlitt 2011). A potential modification of the MELD allocation
system currently under investigation is to allocate organs by not only taking into account
pretransplant mortality but also donor-related factors for estimation of the donor risk
index (DRI) (Feng 2006) and post-transplant mortality. Furthermore, standardization of
laboratory assays and variants of MELD including incorporation of parameters such as
sodium or cholinesterase have been proposed to overcome the limitations of the current
scoring system (Choi 2009; Weissmüller 2008). Additional parameters also include
serum ferritin (SF) (Walker NM, et al. 2010). Na, Fe are easily determined and available
in routine clinical chemistry laboratories. They can also indicate patient morbidity, which
may influence prognosis and outcome following LT. This has been described for
impaired renal function (as a part of MELD parameters), serum sodium (Londono MC,
et al. 2006), as well as for elevated SF and prognosis in hemodialysis patients, (Hasuike
Y, et al. 2010; Jenq CC, et al. 2009; Kalantar-Zadeh K, et al. 2001), hematological
diseases (Lim ZY, et al. 2010; Mahindra A, et al. 2009), and iron overload prior to
LT(Tung BY, et al. 1999). Several studies have analyzed data to define prognostic
models associated with outcome following LT, which include only pre-LT recipient
factors (age, serum creatinine, cholinesterase; SALT [survival after LT] score)
(Weismuller TJ, et al. 2008), or recipient, donor, and surgery-related data (survival
outcomes following LT [SOFT] score) (Rana A, et al. 2008). SALT reached a c-statistic
of 0.79 (MELD ¼ 0.57; 6-month post-LT survival) in an LT cohort with a mean MELD of
5 Introduction
14.5. This score identified a high-risk group and a low-risk group with a specificity of
87.3% and a sensitivity of 68.75% (Weismuller TJ, et al. 2008). SOFT, developed in a
large cohort with a mean MELD of 20.6, showed superior outcome prediction than
MELD (c-statistic for SOFT ¼ 0.7; for MELD ¼ 0.63; 3-month post-LT survival) with the
main variables being previous LT and pre-LT life support (Rana A, et al. 2008). In 2010,
SF was reported as a prognostic parameter in patients on the waiting list (Walker NM, et
al. 2010). Well-designed prospective studies and simulation models are necessary to
establish the optimal allocation system in liver transplantation, as no current model has
all the best characteristics.
Preallocation score to predict survival outcomes following liver transplantation
(P-SOFT)
Risk factor
Age >60
BMI>35
One previous transplant
Two previous transplants
Previous abdominal surgery
Albumin < 2.0 g/dL
Dialysis prior to transplantation
Intensive care unit pretransplant
Admitted to hospital pretransplant
MELD score >30
Life support pretransplant
Encephalopathy
Portal vein thrombosis
Ascites pretransplant
points
4
2
9
14
2
2
3
6
3
4
9
2
5
3
Score to predict survival outcomes following liver transplantation (SOFT)
risk
points
P-SOFT score
Total
Portal bleed 48 h pretransplant
6
Donor age 10–20 years
-2
Donor age > 60 years
3
Donor cause of death from cerebral vascular accident
2
Donor creatinine > 1.5 mg/dL
2
National allocation
2
Cold ischemia time 0–6 h
-3
Low risk 0-5 points, low moderate 6-15 points, high moderate 16-35 points, high 36-40
points, futile >40 points.
6 Introduction
SALT score
was calculated as ‘SALT = 0.04 age (years) + 0.003 CREA (lmol/l) ) 0.349*CHE (kU/l)’
‘SALT = 0.04*age (years) + 0.003*CREA (µmol/l) − 0.349*CHE (kU/l).
Calculation: Donor risk index
Donor risk index = exp[(0.154 if 40≤ age <50) + (0.274 if 50≤ age <60) + (0.424 if 60≤
age <70) + (0.501 if 70 ≤ age) + (0.079 if COD = anoxia) + (0.145 if COD = CVA) +
(0.184 if COD = other) + (0.176 if race = African American) + (0.126 if race = other) +
(0.411 if DCD)+(0.422 if partial/split)+(0.066 ((170–height)/10))+(0.105 if regional
share)+(0.244 if national share)+(0.010×cold time)].
In order to expand the donor pool, strategies and techniques have been adopted as well
as legislative measures, mass media campaigns, and optimization of available organ
allocation. Extended criteria donors ECD or marginal donors are accepted to overcome
the organ shortage. The hypothesis supporting EDC utilization is that the benefit of
earlier access to transplantation afforded by an EDC allograft outweighs the combined
risk associated with the specific allograft and the risk of additional waiting for LTX. The
definition of ECD are somewhat center-based but in general term they are defined as
those with greater risk of initial poor function IPF or graft failure, and the presence of a
disease within the donor that may be transmitted to the recipient and therefore
associated with an increase risk for recipient morbidity and mortality (Busuttil RW, et al.
2003). Currently, some marginal donors are being routinely used: elderly donors,
steatotic grafts, non-heart beating donors (livers from donation after cardiac death
DCD), hepatitis C virus-positive (HCV+) or hepatitis B core antibody-positive. Although
these organs may not be optimal, they represent an alternative to decrease waiting list
mortality. Other alternatives include living-donor liver transplantation, reuse of grafts as
domino transplantation, ex situ and in situ (Rogiers X, et al. 1996) split liver
transplantation, reduced-size liver transplantation. Other potential alternatives to liver
transplantation including bioartificial liver for acute liver failure patients awaiting for
transplantation, cell-based therapies (Ctx) using cell sources from humans or animals
are under investigations in an attempt to decrease waiting list mortality due to scarcity of
donors. Ctx has shown a great deal of promise, and the progress made over the past
several decades of preclinical and clinical studies provides a growing amount of
rationale for its use to treat a variety of liver disorders. The most promising cells types
are hepatocytes, embryonic stem cells (ESC), mesenchymal stromal cells (MSC),
7 Introduction
amnion epithelial (AE) cells, and induced pluripotent stem cells (iPSC). Each cell type
has its own associated risks and benefits. Improvement of cell engraftment remains the
single biggest challenge to overcome. New methods to modulate the immune reaction
and relieve changes in vascular pressures after cell transplant are currently being
investigated to enhance engraftment and improve patient outcome. Preconditioning
protocols of the recipient liver, such as hepatic irradiation, portal vein embolization, and
surgical resection, may also help to improve engraftment by giving donor cells selected
growth advantage (Soltys et al. 2010; Puppi et al. 2011). Future work is required to
enhance utility of this novel branch of regenerative medicine. Xenotransplantation is
investigated as well but no clinical relevant system of xenotransplantation exist.
1.1.2 Strategies And Techniques To Expand The Donor Organ Pool
Use of donor livers with extended criteria
Use of steatotic donor organs
HCV-positive donor organs for HCV-positive recipients
Use of high-risk CDC donor organs
Donation after cardiac death
Split liver transplantation
Living donor liver transplantation
Domino liver transplantation
1.1.3 Sources of Organ
Excluding xeno-transplantation, The majority of livers are procured from cadaveric
donors they can be: Brain-dead donors, Non-heart-beating donors, and living donors.
1.1.4 Types of OLT
Conventional LTx,
Living-donor LTx,
Reuse of grafts as domino transplantation,
Ex situ as well as in situ split LTx,
Reduced-size LTx.
Most liver transplants are performed using a whole liver from a deceased donor. Types
of liver transplantation include Orthotopic liver transplantation: where donor liver is
placed in the orthotopic position, Split liver transplantation donor organs can be divided
and the separate parts transplanted into two recipients (Keeffe EB. 2001). A portion of
the left lobe of an adult donor organ can be transplanted into a child and the remaining
8 Introduction
portion used to transplant the liver into an adult. (Otte JB, et al. 1998; Malago M, et al.
2002; Gridelli B, et al. 2003; Renz JF, et al. 2003). In living donor transplantation where
only a portion of the donor liver is removed for transplantation; a portion of the left lobe,
is a well-established procedure for children (Otte JB, et al. 1998; Malago M, et al. 2002)
while for adults, the donor right lobe is transplanted but donor safety remains an
ongoing concern. (Trotter JF, et al. 2002; Surman OS, et al. 2002). Under ideal
circumstances, a deceased donor organ also can be split and transplanted into two
adult recipients (Renz JF, et al. 2004). Perioperative complications are higher but longterm patient survival are comparable with that of deceased orthotopic liver
transplantation (Renz JF, et al. 2004; Settmacher U, et al. 2004) Liver transplantation is
a complex, time-consuming operation that requires vascular reconstruction of the
hepatic artery, the portal vein, and the hepatic venous drainage to the inferior vena
cava. Biliary reconstruction usually is accomplished using an end-to-end anastomosis of
the proximal donor bile duct to the distal recipient duct; however, in recipients with
diseased ducts, the donor duct is usually anastomosed to the jejunum using a Roux-enY loop.
A number of complications can be anticipated after liver transplantation,
including perioperative and surgical complications, immunologic and infectious
disorders, and a variety of medical complications.
1.2 Selection of recipients and organ allocation.
Selection of recipients and organ allocation vary in different countries.
1.2.1 The US model for liver allocation
The model of organ allocation in the USA was set to be patient-based due to
heterogeneity among more than 118 centers, in the size of the waiting list and organ
availability, as well as large distances. For more than 20 years the Organ Procurement
and Transplantation Network of the United States (OPTN) suggested to use Child–
Turcotte–Pugh (CTP) score (Brown Jr RS, et al. 2002) ABO blood type and time on the
waiting list the concept of “first come, first served” in managing the waiting list to
establish the priority of organ allocation. In 2000 the Model for End Stage Liver Disease
(MELD) was developed. Further studies established that model for end-stage liver
disease (MELD) scoring system was superior to the CTP score in predicting the 3month survival of cirrhotic patients a waiting for liver transplantation. (Wiesner RH, et al.
2003). Since 2002 MELD was implemented in the USA, and subsequently many other
9 Introduction
countries, modifying the liver allocation system and started to use the MELD score to list
candidates for liver transplantation (Kamath PS, et al. 2001). The corresponding scoring
system in children is called PELD.
1.2.2 The European model for liver allocation
There are no uniform rules or systems for organ allocation in Europe or within European
Union. The organ exchange organizations for different geographical areas include
Eurotransplant (ET; Germany, The Netherlands, Belgium, Luxembourg, Austria,
Slovenia, and Croatia), United Kingdom Transplant, Organizacion Nacional de
Transplantes in Spain, Scandiatransplant (Sweden, Finland, Norway, Denmark, and
Iceland), North Italian transplant, and Etablissement francais des Greffes in France. The
organs are allocated and transplanted within each organization, and in collaboration
among these organizations. The organ allocation within Eurotransplant is patient-based
as in the USA but, in Spain, Scandiatransplant, and UK, is center-directed. Italy has no
single unique allocation policy but MELD is currently used in many Transplant Centres.
Greece and Scandinavian countries have no formal agreement on listing criteria and
allocation is left to the discretion of clinicians (Neuberger J, et al. 2008). Recently, the
United Kingdom and France started to manage patients waiting for LT with a new
scoring system. The British model Liver allocation in the UK was initially based on each
center being allocated a portion of the nation’s donor pool reflecting its previous
transplant activity and its nationally contracted (and funded) activity with the National
Specialist Commissioning Advisory Group (NSCAG). Units were required to target
recipients with an expected post transplant survival of more than 50% at 5 years
(Neuberger J, et al. 1999). Predictably, waiting times and waiting list mortality varied
widely. Therefore agreed national minimal listing criteria (Neuberger J, et al. 2008) were
introduced with a minimum disease severity based on the United Kingdom Model for
End-stage Liver Disease (UKELD) Additionally for donors meeting specific criteria
offering the liver for splitting is obligatory, the left lateral segment going to a child at one
of three national paediatric centers and the remaining right liver to the retrieving center.
Thus UK organ allocation defines donor pools based on patient-specific characteristics,
but organ allocation to individual patients remains at the center’s discretion In UK they
use UKMELD, in France, L’ “Agence de la Biomedicine” utilizes an allocation system
(liver score) based on specific variables for each liver disease, which has not yet been
validated (Jacquelinet C, et al. 2008). In ET, allocation is directed by different national
10 Introduction
laws. The Eurotransplant Liver Allocation System (ELAS) from 2000 to the end of 2006,
the selection of potential recipients was based on the medical urgency, donor weight,
ABO blood group, waiting time, and donor region. The selected potential recipients were
ranked using a scoring system, and the higher the scores the priority to receive the
organ. Some urgency categories within ET were given based on the respective medical
urgencies and not scoring system. Due to increasing waiting list mortality under ELAS
and because of the positive experience with the implementation of MELD/PELD scores
in 2002 in the USA, the board of ET decided to acquire the MELD score system for
listing and prioritizing the potential recipients as of December 16th 2006 (Eurotransplant
International Foundation 2008). In Italy, where there is no formal priority score for
patients in the waiting list (Burra P, et al. 2000) precedence for LT was assigned
conventionally according to UNOS statuses in view of the fact that the specifications for
MELD usage were established by UNOS Policy 3.6, released on February 2002
(United Network for Organ Sharing. Allocation of Livers Proposed Amended UNOS
Policy 3.6. 2002. Available at http://www.unos.org). In Italy, in the second half of 2002,
the MELD model came into use because of a growing number of Italian transplantation
centers side by side with UNOS statuses only. For this reason, the MELD score was not
included in the parameters meanwhile adopted by the Italian Ministry of Health (IMH) to
evaluate retrospectively the quality of the national liver transplantation activity referred
to
the
previous
two-year
period
(Liver
Transplant
Activities.
Available
at:
http://www.ministerosalute.it/trapianti [accessed February], 2003).
1.3 Scoring Systems
1.3.1 The Child-Turcotte-Pugh (CTP) Score
The Child and Turcotte classification (1964) and the Pugh’s modification (1973) (ChildTurcotte-Pugh [CTP] score) (Pugh R, et al. 1973) were originally deviced for the
assessment of the severity of liver disease in predicting the outcome of patients with
cirrhosis in whom surgical therapy for portal hypertension was planned. It was then
extended for endoscopic treatment of varices or transjugular intrahepatic portosystemic
shunt therapy (TIPS), for prognosis in general, and more recently to stratify patients on
the waiting list for LT. (Christensen E, et al. 2004; Cholongitas E, et al. 2005). CTP
provides accurate prognostic information of various cirrhosis-related complications
(Merkel C, et al. 2000; Shetty Ket, et al. 1997) and is very usefull as a prognostic tool to
assess the mortality risk of patients with end-stage liver disease (Huo TI, et al. 2004).
11 Introduction
Until 2002, the CTP score and the time on the waiting list, although never formally
validated, was used to stratify the risk of death of patients awaiting LT in most Liver
Transplant Centres worldwide (Rudow DL, et al. 2008).
1.3.2 CTP Limitations
The use of CTP, particularly for prioritizing potential liver transplant recipients, has
several limitations and drawbacks (Rudow DL, et al. 2008; Durand F, et al. 2005). The
variables, ascites and encephalopathy, are subjective and assessed by physical
examination alone; and when other methods are used (ultrasonography, psychometric
testing, EEG), a different degree of severity is diagnosed. Ascites and encephalopathy
are influenced by therapy such as diuretics, albumin, and lactulose. Measurement of
prothrombin time in different laboratories is variable and depends on the sensitivity of
the thromboplastin reagent used (Robert A, et al. 1996). Serum bilirubin of 3 or 13
mg/dL or prothrombin time increased by 6 or 16 seconds will not alter CTP score
(Kamath PS, et al. 2001). In addition, the “ceiling” and “floor” effect in terms of the limits
set to the laboratory parameters of bilirubin, albumin, and prothrombin time in the
grades A, B, and C and changes of serum bilirubin concentrations with therapy (e.g.,
with ursodeoxycholic acid) do not allow assessment using a continuous scale of
severity. The absence of an assessment of renal function, which is a well-established
prognostic marker in cirrhosis (Durand F, et al. 2005) is another limitation of the CTP
score. The Child-Turcotte-Pugh (CTP) places the patient in a class A (good, 4% 3month mortality), B (intermediate, 14% 3-month mortality), or C (poor, 51% 3-month
mortality). For patients on the waiting list for LT, CTP score is within a narrow range of
7-15 (Child B or C), and some patients may have identical CTP score; in such case, the
waiting time on the waiting list is then taken as a tie-breaker, which is unreliable
(Freeman RB Jr, et al. 2000). Up to 1996, allocation of organ for deceased donor liver
transplant (DDLT), was based on CTP score, time on the waiting list and whether the
patient is at home, in hospital or intensive care unit (ICU). However, the minimal criteria
for registering in the waiting list and for admission in an ICU, are not well defined and
hence these parameters – longest on the waiting list or in an ICU, are not useful.
Furthermore, these parameters do not accurately identify, the sickest patient on the
waiting list for LT (Freeman RB Jr, et al. 2000).
12 Introduction
1.3.3 MELD SCORE: “sickest first policy”
In 1999, the controversial state of the United Network for Organ Sharing waiting list for
liver transplantation and the resulting pressure to formulate a more objective and
temporally discriminatory assessment tool led to the development of the Model for EndStage Liver Disease (MELD) score. This tool was developed by physicians at the Mayo
Clinic and was validated for predicting survival in 3 months in cirrhotic patients, initially
for 231 patients undergoing Transjugular Intrahepatic Portosystemic Shunt (TIPS)
(Malinchoc M, et al. 2000), a short-term bridge therapy to liver transplantation and later
for those on the waiting list for LT (United Network for Organ Sharing (UNOS: Feb. 27,
2002) (Kamath PS, et al. 2001). MELD is calculated from a validated predictive equation
based on the patient’s serum bilirubin (mg/dL), serum creatinine (mg/dL), International
Normalised Ratio (INR) for prothrombin time and also included the aetiology of liver
disease: (zero for cholestatic or alcoholic, one score for other aetiology) (Kamath PS, et
al. 2001, Wiesner RH, et al. 2001). In 2000, the aetiology of liver failure was dropped
from the MELD score because it proved prognostically insignificant (Pagliaro L, et al.
2002), but the coefficient of this variable 6,4 remained in the formula. Many clinical
studies have compared CTP and MELD in various populations either undergoing TIPS,
orthotopic liver transplantation, or no surgery at all (del Olmo JA, et al. 2003; Jakab F, et
al. 1993; Malinchoc M, et al. 2000) These studies have shown MELD to be at least
comparable and perhaps slightly better at predicting short-term mortality. The
differences between CTP and MELD are that MELD includes renal functions. Liver and
renal function are strictly dependent on each other in advanced cirrhosis, and the
severity of the liver disease correlates directly with the severity of the renal disease and
renal function influence as well the course of liver disease (Huo TI, et al. 2004; Kamath
S, et al. 2007). MELD score utilizes only laboratory values, making it a continuous
score, more objective, and possessing a wide range of scores which is more accurate in
discriminating among patients in similar clinical conditions (Kamath S, et al. 2007)
making the listing process more precise and without biases related to subjective or
personal opinions. MELD is superior to CTP in predicting short and mid-term survival
among cirrhotic patients (Botta F, et al. 2003), and has been shown to predict the 3month survival more accurately than CTP for both UNOS status 2A (e.g. CTP score ≥10
plus cirrhosis-related complications such as active variceal haemorrhage, hepato-renal
syndrome,
refractory
ascites/hepatic
hydrothorax,
or
stage
3
or
4
hepatic
encephalopathy) and status 2B (e.g. CTP score ≥10, or score ≥7 plus complications)
13 Introduction
(Wiesner RH, et al. 2003). Nevertheless, a recent review showed that of 11 studies,
only four (4512 patients) demonstrated a statistical superiority of the MELD in
comparison with the CTP system, whereas seven studies (8020 patients) showed no
statistical difference. However, no studies reported the MELD to be statistically inferior
to the CTP system (Wiesner RH, et al. 2003). Since February 2002, most Liver
Transplant Centres in the USA have adopted the MELD score to allocate livers to the
sickest recipients rather than to those who had been on the waiting list for a longer time
(Rudow DL, et al. 2008). The MELD score is currently used in many countries. To
classify patients awaiting LT according to the severity of their liver disease, with the
exception of fulminant MELD seems to be more reliable in predicting survival in patients
with higher scores (Huo TI, et al. 2005) and data analysis has shown that MELD is
effective in reducing waiting list mortality (Wiesner RH, et al. 2001; Freeman RB, et al.
2004) without changing patient and graft survival. MELD score, almost always gave a cstatistic for 3-month survival > 0.80 in all groups of patients with cirrhosis, without any
significant improvement by adding complications such as ascites, encephalopathy,
variceal bleeding, and spontaneous bacterial peritonitis. c- statistic >0.80 implies
excellent diagnostic accuracy, but still means that there will not be an accurate
prediction in approximately 20% of occasions (Kamath PS, et al. 2001)
1.3.4 MELD Calculation
MELD is calculated from:
3.8x ln(bilirubin mg/dL) + 11.2x ln(INR) + 9.6x ln(creatinine mg/dL) + 6.4
(creatinine value is assumed 4 for patients on dialysis if dialyzed within last week twice;
values<1 are considered 1)
MELD
MORTALITY % in next three month
40 or more
30-39
20-29
10-19
<9
71.3%
52.6%
19.6%
6%
1.9%
If the MELD score is >25, 19-24, 11-18, ≤ 10, it is recalculated every 7 days, 1, 3, 12
months respectively. As shown in the table above, in cirrhotic liver patients MELD score
is more accurate than CTP score, (MELD score >40: 71% mortality, <10: 2%), in
predicting mortality in next 3 months (Wiesner RH et al 2001).
14 Introduction
1.3.5 Upgrading of MELD score for Hepatocellular Carcinoma (HCC)
Most USA and European LT Centres currently use additional points for HCC patients
that are set according to tumour size. This policy has significantly reduced the number
of drop-outs among HCC patients awaiting LT and today more than the 25% of donated
livers are used for these candidates (Ioannou G, et al. 2008). Patients with HCC who
fulfill the Milan criteria (1 nodule <= 5 cm in diameter, or <=3 nodules and <= 3 cm in
diameter) are indicated for liver transplantation (Mazzaferro V, et al. 1996) Patients with
HCC are likely to develop intrahepatic and/ or extrahepatic complications and hence
were allotted additional fixed points (approved by regional review board: RRB) as
follows: (Wiesner RH, 2001, Sharma P, et al. 2004) patients with a single lesion <2 cm:
20 points, patients with a single lesion 2-5 cm or ≤ 3 lesions which are not greater than
3 cm: 24 points, and for every 3 months on the waiting list : 10% additional; now these
additional fixed points have been reduced to 22 points for stage 2 (T2), and no priority
score to stage 1 score (Wiesner RH, et al. 2004). Following the upgrading of MELD
score for HCC patients, the number of DDLT performed for HCC have increased and
their waiting list period significantly reduced (2.3 to 0.7 years) (Sharma P, et al. 2004).
Eighty-seven per cent of HCC patients received LT within 3 months of wait listing,
indicating excessive priority for HCC especially for small HCC which have a low risk of
progression to advanced disease or complications, for the first year (Yao FY, et al.
2002). Furthermore following LT, 5 year survival is 60% (Europe), 45% (US) and 10
year survival is 47% (Europe) (Pelletier SJ, et al. 2009; Dutkowski P, et al. 2010).
1.3.6 MELD Limitations
MELD score is a good predictor of survival prior to LT but in 15% of patients MELD
score does not accurately predict survival (Kamath S, et al. 2007). Limitations of MELD
have been emphasized by a few authors (Neuberger J, et al. 2004, Freeman RB et al
2005). The laboratory test values included in the equation are subject to inter laboratory
variability. Regarding serum creatinine; the use of different laboratory methodology
(O’Leary modified Jaffe, compensated kinetic Jaffe, enzymatic and standard kinetic
Jaffe) for determination of serum creatinine resulted in marked variations in
measurements and investigators found that there is poor agreement among different
assays for creatinine (Cholongitas E. 2007). The raised serum creatinine (as a late
event) is a known predictor of poor prognosis in liver cirrhosis (Ruf AE, et al. 2005) but
15 Introduction
serum creatinine values may be lowered due to reduced muscle mass, selection of one
creatinine value amongst the few fluctuating values in a decompensated cirrhotic on
diuretic therapy, and the arbitrarily selected value of 4 for dialysis patients. Serum
creatinine is also influenced by age and gender as well as ethnicity, which may lead to
discrimination against women, white, or malnourished patient. Female patients with liver
disease have lower glomerular filtration rates than males for the same creatinine levels
in cohorts with abnormal liver function tests, as well as candidates for liver
transplantation (Cholongitas E, et al 2007). Correcting the creatinine in females for the
same glomerular filtration rate in males showed that the current MELD scoring may
generate significantly lower MELD scores in females despite a similar renal function,
and thus a lower priority for liver transplantation compared with males. Various methods
for creatinine measurement have introduced to overcome this interference but there is
little concordance between different assays and no accepted consensus on the best
method (Cholongitas E, et al. 2005). Regarding, total serum bilirubin: MELD score
includes total bilirubin, which is a sum of direct (hepatic) and indirect (non-hepatic)
bilirubin. In cirrhosis, increased indirect bilirubin may result from glucose-6 phosphate
deficiency (G6PD), thalessaemia trait, spur cell anaemia, ribavirin, anti-retroviral drugs.
Whether inclusion of direct bilirubin instead of total bilirubin for measuring the MELD
score, improves its accuracy or not, is still not clear that the direct fraction could be a
more accurate predictor of survival than the total value (Kamath S, et al. 2007). In
addition, the accuracy of INR in representing the coagulative status of the patient has
been questioned, considering that coagulopathy in cirrhosis affects different sites of the
coagulation pathway (Kamath S, et al. 2007) and that it is designed to standardize the
anticoagulate effect of warfarin and may not reflect the severity of the disease
(Cholongitas E, et al. 2005). The most common severe cirrhosis complications, such as
hepatic encephalopathy, oesophageal variceal bleeding, and spontaneous bacterial
peritonitis, are not scored by MELD, and patients high mortality is not properly rated by
MELD (Huo TI, et al. 2005). Other clinical conditions in which MELD cannot adequately
predict short-term survival and hence to manage patients on the waiting list include:
polycystic liver disease, Budd–Chiari syndrome, malnutrition, hepato-pulmonary
syndrome, hereditary haemorrhagic telangectasia, cystic fibrosis, recurrent biliary
sepsis, and unusual tumours (Freeman Jr RB. 2008). It has been reported also that
MELD may not be reliable in predicting survival of HIV infected patients, indeed to give
additional points to these candidates can be appropriate (Samuel D, et al. 2008). MELD
16 Introduction
accuracy in predicting survival also seems to be lower for patients awaiting retransplantation. In general, re-transplantation has a worse outcome than first transplant,
mainly because of surgical technical difficulties (Zhu ZJ, et al. 2007; Onaca N, et al.
2006). Moreover, the MELD score failed to predict patient or graft survival in living donor
liver transplant recipients (Hayashi PH, et al. 2003) and it did not correlate with the
severity of the disease of patients affected by malignancy or metabolic disorders (Llado
L, et al. 2002) or with the degree of encephalopathy and ascites (Yoo HY, et al. 2003).
Additionally, studies carried out in patients undergoing TIPS, the original source of the
MELD score, found that MELD model and CTP can be used with equal accuracy for
prognosis (Angermayr B, et al 2003; Schepke M, et al. 2003) and that mortality was
unpredictable in patients with refractory ascites by using pretransplant variables
(Thuluvath PJ, et al. 2003). The use of MELD for allocation is a ‘justice’ and not a ‘utility’
score, as it does not consider outcome after liver transplantation (LT), and donor factors
are not considered (Adam R, et al. 2000). As a result, both pre-LT MELD and change in
MELD in the course of the disease (Northup PG, et al 2004) do not correlate with postLT survival, with only a c-statistic of 0.58 in the UK (Jacob M, et al. 2004). C-statistic for
3-month survival on the waiting list is as low as 0.75 (Heuman D, et al. 2003). Use of
MELD outside the USA, has also given poor predictive accuracy in individual patients
and poor generalisability (Llado L, et al. 2002).
17 Introduction
1.3.7 Classification of Candidates for Liver Transplants According to Old UNOS
System United network for organ-sharing (UNOS) liver status classification.
Status 1
Fulminant liver failure with life expectancy <7 days
(i) Fulminant hepatic failure as traditionally defined
(ii) Primary graft nonfunction <7 days of transplantation
(iii) Hepatic artery thrombosis <7 days of transplantation
(iv) Acute decompensated Wilson’s disease
Status 2a
Hospitalized in ICU for chronic liver failure with life expectancy <7 days, with a Child-Pugh score of ≥10
and one of the following:
(i) unresponsive active variceal hemorrhage
(ii) hepatorenal syndrome
(iii) refractory ascites/hepatic hydrothorax,
(iv) Stage 3 or 4 hepatic encephalopathy
Status 2B
Requiring continuous medical care, with a Child-Pugh score of ≥10, or a Child-Pugh score ≥7 and one
of the following:
(i) unresponsive active variceal hemorrhage
(ii) hepatorenal syndrome
(iii) spontaneous bacterial peritonitis
(iv) refractory ascites/hepatic hydrothorax,
or presence of hepatocellular carcinoma
Status 3
Requiring continuous medical care, with a Child-Pugh score of ≥7, but not meeting criteria for Status 2B
Status 7 Temporary inactive
The MELD score is calculated. If the MELD score is ≥30 the patient’s UNOS listing status is 2a, if it is
24–29, it is 2b, and if it is less than 24, it is 3. A Status 1 patient is given priority following which those
with a MELD/PELD score ≥15 and later those having a score of ≤14
From http://www.unos.org/ initially implemented in July 1997 later modified in January
1998 and August 1998.
1.3.8 Other Scoring Systems
The model for end-stage liver disease (MELD) scoring System does not include any
parameter correlated with complications of cirrhosis in its formula. Liver cirrhosis alter
vascular haemodynamics resulting in dilutional hyponatraemia associated with
refractory ascites, hepato-renal syndrome and increased mortality (Porcel A, et al. 2002;
Arroyo V, et al. 2003; Sersté T, et al. 2008; Fernández-Esparrach G, et al. 2001; Borroni
G, et al. 2000). Dilutional hyponatremia (free water retention) results from a higher rate
of renal retention of water despite increased total body sodium due to antidiuretic
hormone mediated reduction in free water clearance (Schrier RW, et al. 1988; Gines P,
et al. 1998). Free water retention positively correlate with the severity of portal
hypertension (Freeman RB, et al. 2004) and serum sodium (SNa) level may inversely
reflect the severity of portal hypertension. Hyponatremia in this setting has been
correlated with increased mortality (Llach J, et al. 1988; Biggins SW, et al. 2006),
18 Introduction
consequently patients with low MELD scores who have persistent ascites and low SNa
are at a disadvantage and at a higher risk of mortality than that predicted by the MELD
score alone (Srikureja W, et al. 2005) Some studies have indicated that serum sodium
is an independent predictor of mortality in patients with cirrhosis (Wang YW, et al. 2007;
Selcuk H, et al. 2007) and the incorporation of Na into the MELD may enhance its
prognostic accuracy (Biggins SW, et al. 2005; Ruf AE, et al. 2005). Consequently new
scoring systems have been proposed with new mathematical equations based on both
MELD and Na, known as the MELD with the incorporation of serum sodium (MELD-Na)
(Biggins SW, et al. 2006) the integrated MELD (iMELD) score (Luca A, et al. 2007) and
the MELD to sodium (MESO) index (Huo TI, et al. 2007). Serum sodium <126 mEq/L in
cirrhotic patients listed for LT is an independent predictor of 3- and 6-month mortality
(Biggins SW, et al. 2005). Persistent ascites (including hydrothorax) and low serum
sodium are independent predictors of 6-month survival, especially in patients with
MELD below 21, and concurrent ascites and serum sodium <135 mEq/L is more
predictive of survival than MELD score alone (Heuman DM, et al. 2005). It was reported
that hyponatraemia (≤130mEq/L) was an excellent predictor of outcome in cirrhotic
patients awaiting LT (Ruf AE, et al. 2005). For values between 120 and 135mEq/L for
each unit decrease in serum sodium concentration there is an increased mortality risk
increase by 12% (Londo˜no MC, et al. 2007) addition of serum sodium to the MELD
score in this study did not seem to significantly improve MELD prognostic accuracy.
Considering these preliminary results, Biggins et al. (Biggins SW, et al. 2007) and
subsequently Kim et al. (Kim SY, et al. 2007) proposed a new MELD-based score,
called MELD-Na, obtained through the integration of serum sodium and traditional
MELD parameters using the following formula:
[MELD-Na =MELD + 1.59 (135−Na)].
Compared to the traditional MELD score, the MELD-Na score showed a more accurate
6-month survival for cirrhotic patients awaiting LT.
Kim WR, et al. (2008) reported another equation:
[MELDNa =MELD-Na−(0.025)×MELD×(140−Na) + 140],
suggesting that MELD-Na may provide better short-term mortality prediction for
candidates awaiting LT. In this study, the majority of the patients showed serum sodium
concentrations above 135 mmol/L; for those patients, the MELD-Na score was
essentially equal to the MELD score. Moreover, for candidates with MELD scores above
19 Introduction
30, the effect of hyponatraemia was quite small. However, for patients with moderate
MELD scores, the effect may be considerable. Luca et al proposed a score called
iMELD, calculated considering age, serum sodium and MELD. According to the
reported data, the iMELD was better than MELD in predicting three, 6- and 12-month
survival of enrolled patients (Luca A, et al. 2007).
United Kingdom Model for end-stage liver disease. The UK Liver Transplant Units
(Barber KM, et al. 2007) described the UKELD (United Kingdom Model for end-stage
liver disease) calculated using serum bilirubin, INR, creatinine and serum sodium.
Delta MELD. Delta MELD (D-MELD) is the difference between the MELD score
calculated at two separate time points. The rate of increase in D-MELD is calculated by
dividing D- MELD in the interval in months between the first and the second
determination. In an attempt to measure the dynamic change in residual liver function
over time. Patients with a D-MELD greater than 5 points showed a higher waiting list
mortality risk than those for whom the MELD score increased more gradually.
MELD-XI. In certain clinical situations e.g. patients with Budd–Chiari syndrome, or other
thrombophilic syndromes managed by using anticoagulants e.g. warfarin and/or low
molecular weight heparins (Horton JD, et al. 2008) which may interfere with vitamin Kdependent gamma carboxylation of clotting factors resulting in an increasing INR in the
MELD modifying the final MELD score (Suttie JW.1987). A new score has been
proposed omitting INR and the equation is as follow: [MELDXI = 5.11 Ln(B) + 11.76
Ln(Cr) + 9.44]. (Heuman, et al. 2007).
MELD modified by gender. Female cirrhotic patients usually have a lower glomerular
filtration rate than male patients with comparable creatinine values (Cholongitas E, et al
2007). In this setting another new MELD score has been obtained by correcting
creatinine according to gender (MELD modified by gender) to provide an equal priority
to female patients on the waiting list as male patients (Huo, et al. 2007). Its predictive
efficacy is more reliable for mid-term survival (9 and 12 months).
MESO Index. Another score called the MESO index has been proposed. MESO index
had a higher significant predictive value than the traditional MELD score.
20 Introduction
MESO index = (MELD Score/SNa mEq/L) x 10.
Updated MELD. Another modification of MELD score called updated MELD, assigning
a lower weight to creatinine and INR and a higher weight to bilirubin. This score was
reported to be a better survival predictor than traditional MELD at all time points.
(Sharma, et al. 2008).
Artificial neural network (ANN). ANN application permits correlating simultaneously
many different variables which can provide a more complete analysis of prognostic
factors than traditional statistical techniques (Cross S, et al. 1995) The variables
included in the ANN were: aspartate aminotransferase (IU/L), total serum bilirubin
(mg/dL), gamma-glutamyl transpeptidase (GGT) (IU/L), alkaline phosphatase (IU/L)
serum creatinine (mg/dL), serum albumin value (g/dL), INR value, platelet count
(×103/mm3), white cell count (×103/mm3) and haemoglobin concentration (g/dL). The
ANN analysis was shown to be superior to MELD in predicting the 3-month survival in
patients awaiting LT (Cucchetti A, et al 2007). Specific software is required for
calculation. Some other scoring models have been developed to predict the survival of
patients with cirrhosis not eligible for LT such as Modified CTP Score (Huo et al 2006),
MESO Index.
Modified CTP Score (Huo, et al. 2006). An additional class is introduced (class D)
giving an additional point to patients with serum albumin <2.3 g/dL, bilirubin >8 mg/dL or
prothrombin time prolongation >11 to overcome the ceiling effect of the original CTP
system.
Modified Child–Turcotte–Pugh (modified CTP) scoring system
Score
1
2
3
Ascites
None
Not controlled
Severe
Encephalopathy Grade
None
Grade I-II
Grade III-IV
Bilirubin (mg/dL)
<2
2-3
3.1-8
>8
Albumin g/dL
>3.5
3.5-2.8
2.3-2.7
<2.3
Prothrombine Time Prolongation (sec)
<4
4-6
6-11
>11
Scores 5–6 = Class A
Scores 7–9 =Class B 10–15
4
Scores 16-18 = Class C
Scores 16–18 =Class D
21 Introduction
Scoring
system
Score Formula
MELD-Na=
MELD-Na−(0.025)×MELD×(140−Na)+140
iMELD =
MELD+ (age×0.3)−(0.7×Na) + 100
UKELD=
=[(5.395×ln(INR))+(1.485×ln(creatinine))+(3.13×ln(bilirubin))−(81.565×ln(Na))] + 435
Delta MELD
= MELD2 −MELD1
MELD-XI =
11.2×ln(INR)+9.57×ln[(186×(Age)−0.203/female GFR(1/1.154)]+3.78×ln[bilirubin]+6.43
ANN =
Specific software required for calculation
Updated
MELD =
1.266×ln(1+creatinine)+0.939×ln(1+bilirubin)+1.658×ln(1+INR)
ANN = Specific software required for calculation
Notes: MELD: Mayo clinic model for end stage liver disease; Na: serum sodium; INR:
international normalized ratio; GFR: glomerular filtration rate; Bilirubin and Creatinine
are expressed in mg/dL.
1.4 Indications For Liver Transplantation:
The list of indications for liver transplantation includes all the causes of end stage liver
disease, which are irreversible and curable by the procedure. In 1997 the American
Society of Transplant Physicians and the American Association for the Study of the
Liver Disease put forward the minimal listing criteria for patients with end stage liver
disease. To qualify for the listing, the patient’s expected survival should be ≤90% within
1 year without transplantation. Liver transplantation should lead to prolonged survival
and an improved quality of life (M. R. Lucey, et al. 1997). Hepatitis C virus (HCV) or
alcohol-induced liver disease account for the most common disease indications in
adults with liver cirrhosis (http://www.eltr.org). Other indications include cholestatic liver
disorders (primary biliary cirrhosis, primary sclerosing cholangitis) (PBC, PSC), hepatitis
B virus (HBV) infection, autoimmune hepatitis (AIH), inherited metabolic diseases
(Wilson’s
Disease,
hemochromatosis,
α-1-antitrypsin
deficiency),
nonalcoholic
steatohepatitis, HCC, and acute or acute - on chronic hepatic failure. In children, biliary
atresia and metabolic liver diseases are the most common indications.
22 Introduction
Indications for liver transplantation (LT). Primary diseases leading to LT in Europe
1988 - 2010 (Data kindly provided from European Liver Transplant Registry,
http://www.eltr.org).
1.4.1 Indication Of Liver Transplantation In Adults (Mieth M, 2006; Fink SA and
Brown RS Jr 2005)
Benign disorders
Chronic liver diseases
Liver cirrhosis (62%)
Liver cirrhosis caused by HBV, HCV, HDV
Liver cirrhosis caused by autoimmune hepatitis
Ethyl toxic liver cirrhosis
Cryptogenic liver cirrhosis
Cholestatic liver disorders (10%)
Primary sclerosing cholangitis (PSC)
Primary biliary cirrhosis (PBC)
Secondary sclerosing cholangitis
Familial cholestatic syndrome
Atresia of the biliary tract
Metabolic/genetic disorders (20%)
α-1 antitrypsin deficiency
Wilson disease
Hereditary hemochromatosis
Glycogen storage diseases
Galactosemia
Tyrosinemia
β-Thalasemia
Mucoviscidosis
Familial amyloidotic polyneuropathy
Other disorders
Congenital cystic liver
Echinoccocosis of the liver
Chronic Budd-Chiari Syndrome
Adult polycystic liver disease
Nodular regenerative hyperplasia
Caroli’s disease
Severe graft-versus-host disease
Veno-occlusive disease
Benign disorders
Acute liver diseases (6%)
Fulminant hepatic failure
Pregnancy-associated liver failure
Severe liver trauma
Postoperative liver failure following resection/LTx
Malignant disorders
Primary malignant diseases of the liver (2%)
Primary hepatocellular carcinoma (HCC)/on
cirrhosis
Fibrolamellar hepatocellular carcinoma
Liver metastases of endocrine tumors
Hepatoblastoma
Epithelioid hemangio endothelioma
23 Introduction
1.4.2 Indications In Children (Mieth M,2006, Strasberg SM 1994)
Benign disorders
Cholestatic liver diseases
Extrahepatic bile duct atresia (62%)
Bile duct hypoplasia
Alagille syndrome (arteriohepatic dysplasia; <1%)
Familial cholestatic syndrome (7%)
Primary sclerosing cholangitis (3%)
Secondary biliary cirrhosis (1%)
Metabolic/genetic disorders
α-1 antitrypsin deficiency (8%)
Wilson disease (<1%)
Crigler–Najjar syndrome (<1%)
Mucoviscidosis
Tyrosinemia type I (2%)
Gylcogenosis type III, IV (<1%)
Urea cycle defects (<1%)
Organic acidopathy
Neonatal hemochromatosis
Congenital hepatic fibrosis (<1%)
Cystic fibrosis (1%)
Post hepatic cirrhosis
Autoimmune hepatitis (2%)
Post-viral hepatitis (HBV, HCV)
Idiopathic neonatal hepatitis (2%)
Acute liver failure
Fulminant hepatitis (5%)
Autoimmune hepatitis
Toxic liver injury
Idiopathic liver failure
Malignant disorders
Hepatoblastoma (1%)
Hepatocellular carcinoma
1.4.3 Variant Conditions and Syndromes Requiring Liver Transplantation
- Intractable ascites: Diuretic resistant, Nonresponsive to TIPS or, TIPS contraindicated;
- Hepatopulmonary Syndrome: Shunt fraction >8%, pulmonary vascular dilatation;
- Chronic hepatic encephalopathy
- Persistent and intractable pruritus
1.4.4 Transplantation For Acute Liver Failure (ALF)
Acute liver failure (often used synonymously with fulminant liver failure) is defined as
acute hepatic deterioration without antecedent chronic liver disease, which has
progressed from the onset of jaundice to the development of hepatic encephalopathy in
less than 8 weeks (Benhamou JP. 1991). Refinements in the definition of acute liver
failure include a distinction between fulminant (<2 weeks) and subfulminant hepatic
failure (>2 weeks), a difference that reflects the greater predominance of brain edema
and intracranial hypertension in patients with the shorter interval. Both drug-induced
hepatic failure and an indeterminate etiology seem to be more commonly associated
with a longer interval. Fulminant hepatic failure (ALF and subfulminant hepatic failure),
which is characterized by encephalopathy, jaundice, and coagulopathy. It accounts for
5-6% of all patients undergoing liver transplantation (US Scientific Registry (UNOS)
2006). Acetaminophen toxicity is the leading cause of ALF in western countries,
followed by hepatitis A, E, B and seronegative hepatitis as other common aetiological
factors. The major cause of subfulminant hepatic failure is idiosyncratic drug induced
liver injury (W. M. Lee, 1993). Patients who meet the King’s College Criteria for urgent
transplantation need to undergo transplantation, as soon as possible without any delay.
There is a 100% percent mortality if these selected patients do not undergo
24 Introduction
transplantation and this is either due to liver failure per se or because of sepsis and
multiorgan failure (W. M. Lee, 1993). Patients with subacute failure have a poor
outcome with almost universal mortality if not transplanted; these patients might require
transjugular liver biopsy to establish the presence of massive or submassive liver cell
necrosis. Timely referral is important in these patients because in the absence of
transplantation death may occur from sepsis and cerebral oedema. There are several
scoring systems for listing a patient for urgent liver transplantation: King’s College
criteria, UK Blood and Transplant criteria, Clichy criteria (acute viral hepatitis), and
Wilson’s prognostic index/revised Wilson’s prognostic index (Wilson’s disease with
fulminant hepatitis) (J. G. O’Grady, et al. 1989; G. C. MacQuillan, et al. 2005; H. Nazer,
et al. 1986; A. Dhawan, R. M. et al. 2005).
UK blood and transplant criteria for registration as a super-urgent transplant.
Paracetamol poisoning
Category 1: pH < 7.25 more than 24 hours after overdose and fluid resuscitation
Category 2: Coexisting prothrombin time >100 s or INR > 6.5 and serum creatinine
>300 µmol/L or anuria, and grade 3-4 encephalopathy
Category 3: Serum Lactate >24 hours after overdose > 3.5 mmol/L on admission or
>3 mmol/L after fluid resuscitation
Category 4: Two of the three criteria from category 2 with clinical evidence of
deterioration (e.g. increased ICP, Fi02 > 50%, increasing inotrope requirement) in the
absence of clinical sepsis
Seronegative hepatitis, hepatitis A, B, or an idiosyncratic drug reaction
Category 5: Prothrombin time >100 s or INR > 6.5, and any grade of encephalopathy
Category 6: Any grade of encephalopathy, and any three from the following:
unfavourable aetiology (idiosyncratic drug reaction, seronegative hepatitis), age > 40
years jaundice encephalopathy interval >7 days, serum bilirubin >300 µmol/L,
prothrombin time >50s or INR > 3.5
Category 7: Acute presentation of Wilson’s disease, or Budd-Chiari syndrome. A
combination of coagulopathy, and any grade of encephalopathy.
Category 8: Hepatic artery thrombosis on days 0 to 21 days after liver transplantation
Category 9: Early graft dysfunction on days 0 to 7 after liver transplantation with at
least 2 of the following: AST > 10,000 IU/L, INR > 3.0, serum lactate > 3 mmol/L,
absence of bile production.
Category 10: Any patient who has been a liver donor who develops severe liver failure
within 4 weeks of the donor operation.
25 Introduction
1.4.5 Criteria For Liver Transplantation In Acute Liver Failure (ALF).
King’s College Criteria
Acetaminophen-induced ALF
Nonacetaminophen ALF
(1) Arterial pH < 7.3 irrespective of grade of
encephalopathy
1) INR > 6.5 (PT > 100 sec) irrespective of grade of
encephalopathy
OR any 3 of the following:
(a) PT > 100 sec
(b) Serum creatinine >3.4mg/dL
(c) Stage 3 or 4 encephalopathy
OR any 3 of the following:
(a) INR > 3.5 (PT > 50 sec)
(b) Age < 10 or >40 years
(c) Serum bilirubin >18 mg/dL
(d) Jaundice to encephalopathy interval >7 days
(e) Non-A, non-B hepatitis, idiosyncratic drug reaction.
Prognostic index in fulminant Wilsons hepatitis (WPI) ( H. Nazer, R et al 1986)
Score
0
1
2
3
4
Serum bilirubin
(reference range 3–20 mmol/L)
<100
100-150
151-200
201-300
>300
Serum aspartate transaminase
(reference range 7–40 IU/L)
<100
100-150
151-200
201-300
>300
Prothrombin time prolongation
(seconds)
<4
4-8
9-12
13-20
>20
Patients with a WPI score ≥7 need urgent liver transplantation
Revised Wilson prognostic index (RWPI) (A. Dhawan, et al 2005)
Score
Bilirubin
(µmol/L)
INR
AST
(IU/L)
WCC
9
(10 /L)
Albumin
(g/L)
0
0-100
0–1.29
0-100
0–6.7
>45
1
101-150
1.3–1.6
101-150
6.8–8.3
34–44
2
151-200
1.7–1.9
151-300
8.4–10.3
25–33
3
201-300
2.0–2.4
301-400
10.4–15.3
21–24
4
>300
>2.5
>400
>15.4
<20
Patients with a RWPI ≥11 needed urgent liver transplantation
Clichy criteria (Hospital Paul-Brousse, Villejuif (J. Bernuau, D. et al 1991)
Hepatic encephalopathy, and factor V level: <20% in patients <30 years of age, or
<30% in patients’ ≥30 years of age.
26 Introduction
1.4.6 Transplantation For Alcoholic Liver Disease (ALD)
A period of abstinence is mandatory in patients with ALD to ensure that they do not
relapse and also to give a trial of an alcohol-free period during which the liver function
might recover. The period is not uniform but currently a 6-month rule of abstinence is
applied in US and European liver transplant programmes (J. E. Everhart and T. P.
Beresford, 1997). Patients enlisted are those abstinent who have had an evaluation with
a psychiatrist provided that CTP score is ≥7, or having portal hypertensive bleed, or an
episode of spontaneous bacterial peritonitis (J. Neuberger, et al 2002). These patients
may have a concurrent infection with hepatitis B or C virus, which needs further
evaluation. They are also more prone to develop hepatocellular carcinoma. Acute
alcoholic hepatitis (AAH) is a contra-indication for liver transplantation as the required
period of abstinence is lacking, and there is very little and mixed experience of liver
transplantation in this situation. The severity of AAH is assessed using the Maddrey
discriminant function (DF) score, which predicts the risk of early death. Patients with a
DF score of ≥32 imply poor outcome with one month mortality ranging between 35% to
45% , they are put on medical therapy (Bonet H, et al. 1993; Shakil AO, et al. 1997).
There have been recent reports from France where transplantation is being proposed
for patients with AAH; however, it is still not accepted as an indication elsewhere (H.
Castel, et al. 2009).
Maddrey discriminant function (DF) score:
(4.6 x (PT test - control))+ S.Bilirubin in mg/dl.
1.4.7 Transplantation for Chronic Liver Disease.
In general patients who have a projected 1-year mortality of 10% without liver
transplantation are enlisted.
Viral Hepatitis.
Hepatitis C-related chronic liver disease, it is essential to define the pretransplant
viral load and genotype; to predict post transplant prognosis. Patients with
decompensated HCV-related chronic liver disease do not tolerate interferon therapy,
and disease reccurence is significantly in those with high viral loads. According to the
International Liver Transplantation Society (ILTS) guidelines patients with a child’s score
of 8–11 may be considered for antiviral treatment while they are listed for
27 Introduction
transplantation (Wiesner RH, et al. 2003). Post-transplantation serological recurrence is
universal in patients who have viraemia at the time of transplantation. Many factors
adversely
affecting
patient
survival
including
the
pre-transplant
viral
load,
cytomegalovirus status, advanced recipient age, hyperbilirubinaemia, a raised INR, and
advanced donor age (Charlton M, et al. 2004). Retransplantation in patients with
recurrent HCV infection and cirrhosis is controversial in the setting of DDLT. The
efficacy of antiviral therapy in the presence of a recurrence is questionable. Patients
with early (within one year) aggressive recurrence and graft failure have a poor outcome
following retransplantation.
Hepatitis B virus-related chronic liver disease was previously associated with a high
prevalence of recurrent infection in the graft. However, the availability of hepatitis B
immunoglobulin (HBIG) and oral nucleoside or nucleotide therapy reinfection of the graft
and recurrent hepatitis B disease is now rarely encountered. The duration of HBIG
therapy and oral antiviral therapy is still controversial; a few programmes give HBIG for
one year while others are using it life- long (S. A. Fink and R. S. Brown, 2006).
Cholestatic Liver Disease. The severity of cholestatic liver diseases such as primary
biliary cirrhosis (PBC) and primary sclerosing cholangitis (PSC) is taken into
consideration apart from using the child’s score (≥7) and the Mayo models for PSC and
PBC with a risk score predicting > than 10% mortality at one year without
transplantation (Lucey MR, et al. 1997).
Prognostic models in primary sclerosing cholangitis
Mayo model
0.535 loge serum bilirubin (mg/dl) + 0.486 histological stage + 0.041 age (years) +
0.705 if splenomegaly present
28 Introduction
Prognostic models in primary biliary cirrhosis
European model
2.51x loge serum bilirubin (µmol/l) + ((age exp (age [yrs] – 20)/10) + 0.88 if cirrhosis
present – 0.05 serum albumin (g/l) + 0.68 if central cholestasis present
+ 0.52 if not treated with azathioprine
Mayo model
0.871 loge serum bilirubin (mg/dl) – 2.53 loge albumin (g/dl) + 0.039 age (years) +
2.38 loge prothrombin time (seconds) + 0.859 if peripheral oedema present
Christensen model
2.53 log serum bilirubin (µmol/l) – 1.53 + 1.39 if ascites present – 0.085 serum
albumin (g/l) – 34.3 + 0.4 age (years) – 55 + 0.065 if gastrointestinal bleeding present
Unos Scale
(I) Full-time employment
(II) Part-time employment
(III) Housebound
(IV) Hospitalised
(V) Intensive care unit
(VI) Intentsive care unit, life support
Quality of life issues like recurrent cholangitis requiring repeated drainage procedures
(endoscopic or percutaneous), intractable itching, xanthomatous neuropathy, and
severe metabolic bone disease are some of the other indications for transplantation. In
paediatric patients, biliary atresia and sclerosing cholangitis are the commonest
cholestatic disorders requiring transplantation, with biliary atresia being the foremost
cause (60–70%) in those undergoing liver transplantation (UNOS Policy 3 Appendix 3B,
2011). Liver transplantation is required in most patients with biliary atresia irrespective
of a previous Kasai’s procedure. Other cholestatic disorders, which can lead to cirrhosis
and decompensation requiring transplantation are the Alagille syndrome and Byler’s
disease.
1.4.8 Transplantation For Hepatic Malignancy.
Cirrhosis is associated with a 2 to 8% annual incidence of hepatocellular carcinoma (M.
Sherman, 2005). Transplantation has become the mainstay of treatment for HCC in the
early stages; it offers the advantage of being curative with minimal risk of recurrence.
There have been several criteria for listing these patients for transplantation. These
criteria have been modified over a period of time so as to include as many patients who
would benefit from transplantation and who would have a 5-year survival of >50%. The
Milan criterion defines early stage HCC as those with a single lesion < 5 cm, or no more
than 3 lesions, with none > than 3 cm, in the absence of vascular invasion and
29 Introduction
metastases (Mazzaferro V, et al 1996). However, using the University of California, San
Francisco, (UCSF) criteria (a single lesion ≤ 6.5 cm or 3 or fewer lesions with the largest
being ≤4.5 cm and a total tumour burden of 8 cm or less), patients had a similar
outcome following transplantation compared to those within the Milan criteria (Yao FY,
et al 2001). The MELD score in patients with HCC might be low, and this might prevent
these patients from being given priority or even being listed in spite of the fact that their
disease is fatal if left untreated. Therefore, these patients are prioritized by giving a
score of 22 to T2 lesions. While waiting for transplantation, they usually undergo either
transarterial chemoembolisation or radiofrequency ablation as a bridge to more
definitive therapy. Other primary malignancies of the liver, which are indications for
transplantation are epitheloid haemangioendothelioma and hepatoblastoma. Metastatic
lesions of the liver have a poor prognosis; hence, they do not form an indication for
transplantation; however, neuroendocrine tumors after the removal of the primary may
have a good outcome following the procedure.
HCC staging system:
Several staging systems have been proposed for HCC. All systems include an
assessment of tumor stage and most also include liver function. An exception is the
tumor necrosis metastasis (TNM) system, which measures fibrosis rather than liver
function. Only a few staging systems capture variables related to health status such as
Eastern Cooperative Oncology Group (ECOG) performance or Karnofsky score. In fact,
only the Barcelona Clinic Liver Cancer (BCLC) system includes ECOG performance
score; the French Groupe d'Etude de Traitement du Carcinome Hepatocellular system
includes Karnofsky score, and the Chinese University Prognostic Index (CUPI) system
includes the presence of symptoms. In the West, the staging systems that are most
widely applied are the BCLC system and, less so, the TNM system. The Japanese
Integrated Staging System score and the CUPI system are widely used in Asia.
30 Introduction
Eu-U.S. system
Tumor Staging
Liver Function
Endorsement
GETCH/French
PVT;
AFP < 35 or > 35ug/L
Bilirubin,
alkaline phosphatase
CLIP
Nodules number, tumor
> or <50% area of the
liver and PVT,
AFP<400 or = or
>400ng/mL
CTP
AHPBA
BCLC
Nodules number,
Tumor size, and PVT
CTP
AASLD. EASL
TNM
Nodules number,
Tumor size, PVT, and
presence of metastasis
No
AJCC
ASIA System
Tumor staging
Liver function
JIS
TNM
CTP
Okuda/Tokyo
Tumor > or <50% of crosssectional area of the liver
Ascites, albumin, bilirubin
CUPI
TNM; AFP < 500 or > 500ng/L
Bilirubin, ascites, alkaline
phosphatase.
BCLC, Barcelona Clinic Liver Cancer; CLIP, Cancer of Liver Italian Program; CTP,
Child-Turcotte-Pugh; CUPI, Chinese University Prognostic Index; GETCH, Groupe
d'Etude de Traitement du Carcinome Hepatocellular; HCC, hepatocellular carcinoma;
JIS, Japanese Integrated Staging System; TNM, tumor node metastasis.
Tumor-Node-Metastasis (TNM) Staging Classification
TX, NX, MX Not assessed
TO, NO, MO Not found
T1 1 nodule <or=1.9 cm
T2 One nodule 2.0–5.0 cm; 2 or 3 nodules, all < 3.0 cm
T3 One nodule >5.0 cm; 2 or 3 nodules, at least 1 >3.0 cm
T4a 4 or more nodules, any size
T4b T2, T3, or T4a plus gross intrahepatic portal or hepatic vein involvement as
indicated by CT, MRI, or ultrasound
N1 Regional (portal hepatis) nodes, involved
M1 Metastatic disease, including extrahepatic portal or hepatic vein involvement
Stage I T1
Stage II T2
Stage III T3
Stage IVA1 T4a
Stage IVA2 T4b
Stage IVB Any N1, any M1
31 Introduction
Extended criteria for liver transplantation for hepatocellular carcinoma
Institution
Living or
Deceased
Diagnosis
Size and Number
Tumor Marker
UCSF, US
(Yao 2007)
Deceased
Preoperative
imaging
Solitary tumor ≤ 6.5 cm, or 3
or fewer
nodules with the largest lesion
≤4.5 cm
and total tumor diameter ≤ 8 cm
Alberta, Canada
(Toso 2009)
Deceased
Preoperative
imaging
Total tumor volume ≤ 115 cm
Kyoto, Japan
(Takada 2007)
Living
Preoperative
imaging
All tumor ≤ 5 cm and
number ≤10
And DCP
≤ 400 mAU/mL
Kyushu, Japan
(Soejima 2007)
Living
Preoperative
imaging
All tumors <5 cm
Or DCP
< 300 mAU/mL
Asan, Korea
(SungGyu 2010)
Living
Living
Pathology
All tumors ≤5 cm and
number ≤6 ,
no gross vascular invasion
Multicenter, Japan
(Todo 2007)
Living
Preoperative
imaging
Solitary tumor ≤ 5 cm, or 3
or fewer nodules
with the largest lesion ≤3 cm
3
And AFP
≤ 400 ng/mL
And AFP
≤400 ng/mL
and DCP
≤ 100 mAU/mL
AFP, alpha-fetoprotein; DCP, des-gamma-carboxy prothrombin; UCSF, University of
California San Francisco.
1.4.9 Transplantation For Metabolic Liver Disease
Metabolic liver diseases, which cause decompensation and irreversible damage, are
indications
for
transplantation.
These
include
Wilson’s
disease,
hereditary
haemochromatosis, and α1-antitrypsin disease. Metabolic liver diseases affect other
organ systems; hence, pre-transplant evaluation includes assessment of the concerned
system to rule out systemic disease, which would otherwise preclude transplantation.
Other metabolic disorders can also affect extrahepatic organs while the synthetic liver
functions are intact such as in case of Type-1 hyperoxaluria or familial homozygous
hypercholesterolaemia, these are indications for transplantation as the concerned
metabolic disorder gets corrected. In childhood, the metabolic disorders, which form an
indication for transplantation, are the urea cycle defects, Criggler-Najjar syndrome,
tyrosinaemia, and cystic fibrosis.
1.4.10 Transplantation For Vascular Disorders
The Budd-Chiari syndrome is characterized by obstruction to the hepatic venous outflow
either at the level of the hepatic veins and/or the inferior vena cava. It is associated with
32 Introduction
myeloproliferative disorders (50%), malignancy (10%), hypercoagulable states (15%),
webs in the inferior vena cava IVC, and paroxysmal nocturnal haemoglobinuria (5%).
No cause is found in about 20% of patients. Indications for transplantation in these
patients are established cirrhosis and acute decompensation. These patients generally
require lifelong anticoagulation after the transplant procedure (Klein AS, 2006).
1.4.11 Other Indications
- Complicated polycystic liver disease (combined with or without kidney disease) with
haemorrhage, infection, pain, massive cystic enlargement, portal hypertension, biliary
obstruction, and rarely malignant transformation. These patients might have well
preserved synthetic functions.
- Auto immune hepatitis (AIH) either alone or as an overlap syndrome with PSC/PBC.
- Nonalcoholic steatohepatitis.
1.4.12 Contraindications to Liver Transplantation
Absolute contraindications
Severe cardiopulmonary disease
Extrahepatic malignancy (oncologic criteria for cure not met)
Active alcohol/substance abuse
Acute alcoholic hepatitis
Active infection/uncontrolled sepsis
Lack of psychosocial support/inability to comply with medical treatment
Brain death
Relative contraindications
Advanced age
Acquired immune deficiency syndrome
Cholangiocarcinoma
Diffuse portal vein thrombosis
Severe Cardiopulmonary Disease.
Severe pulmonary hypertension
Symptomatic coronary artery disease unless ischemia is resolved.
Severe ventricular dysfunction
Advanced cardiomyopathy
Severe valvular heart disease: aortic stenosis having poor ventricular function are
absolute contraindications for transplantation
Patients with severe pulmonary hypertension or hypoxaemia resulting from the
hepatopulmonary syndrome (HPS) are at high risk. A mean pulmonary arterial pressure
(PAP) of ≥ 50mmHg is an absolute contraindication for transplantation as the
postoperative mortality reach 100%. Those with (PAP) between 35–50mmHg have a
50% mortality after transplantation. Patients with mild pulmonary hypertension with a
33 Introduction
mean PAP of <35mmHg are suitable for transplantation (M. J. Krowka, et al 2000). The
mortality in patients with HPS increases to about 30% in the presence of arterial
hypoxaemia (<50mmHg PaO2) (M. J. Krowka, et al 1997). Oxygen-dependent chronic
obstructive pulmonary disease (COPD) and advanced pulmonary fibrosis are
contraindications for transplantation. In the other hand reactive airway disease, hepatic
hydrothorax, muscle wasting and infection, are only relative contraindications as these
conditions are reversible. Symptomatic coronary artery disease (CAD), severe
ventricular dysfunction, advanced cardiomyopathy, severe valvular heart disease, and
aortic stenosis having poor ventricular function are absolute contraindications for
transplantation. Patients could be listed for transplantation if myocardial ischaemia is
resolved following bypass surgery or revascularization and angioplasty.
Active Alcohol and Substance Abuse. Active alcohol intake or substance abuse or
poly drug abuse (opiates, sedatives, and cannabinoids), active tobacco abuse are
absolute contraindication for transplantation in all programmes. A pre-transplant period
of abstinence is a must for listing in most transplant programmes, although the period of
abstinence is not well defined, 6 months is generally required (Limand JK, et al. 2004).
This period of abstinence allows the liver to recover and provides an opportunity for
psychosocial assessment and preparation to minimize the chance of recidivism
following transplantation. About 20– 26% of patients resume heavy alcohol intake within
4.5 years of transplantation; which adversely affects the graft survival (DiMartini A, et al.
2006). There is insufficient data on the outcome of transplantation in these patients as
there is no period of abstinence (Bonet H, et al. 1993; Shakil AO, et al. 1997).
Age.
Patients over the age of 60–65 have been shown to have lower survival rates at 1 year
and 5 years than those who are younger (Keswani RN, et al 2004). However, many
centres now accept 70 years as the cut off limit for transplantation and have shown
good results with this policy (Lipshutz GS, et al 2007). Extensive evaluation is required
to rule out the absolute contraindications like severe cardiopulmonary disease and
malignancy.
34 Introduction
Obesity.
Morbidly obese patients (BMI>40) have an increased 5-year mortality after
transplantation due to the associated cardiovascular morbidity (Nair S, et al. 2002).
Recipients who have a BMI > 35 kg/m2 require an individualized approach according to
the policy of the centre.
Infection
(I) HIV Infection
HIV used to be an absolute contraindication for transplantation due to the fear of
progression of disease with immunosuppression. Now with the availability of highly
effective antiretroviral drugs, transplantation is now being offered selectively in
collaboration with experts in the management of HIV infections. The absolute
contraindication to transplantation in these patients includes uncontrolled HIV disease
with multi drug resistance, leukoencephalopathy, advanced malnutrition, life support
requirement, and opportunistic infections (S. A. Fink and R. S. Brown, 2009)
(II) Other Infections.
Pneumonia, sepsis, bacteraemia, osteomyelitis, and fungal infection are all absolute
contraidications and should be treated adequately before transplantation.
(III) Portal vein thrombosis. Is no longer a contraindication except in the presence of
diffuse thrombosis (Manzanet G, et al. 2001).
(IV) Patients with extrahepatic malignancy .
At least 5-year tumour-free interval is a must before transplantation can be considered
(A. Ahmed and E. B. Keeffe. 2007). Cholangiocarcinoma used to be an indication for
liver transplantation, is now a relative contraindication due to the poor outcome
especially in those with advanced disease.
1.4.13 Retransplantation
The rate of retransplantation has been gradually declining, in particular, the rate of
retransplantation for HCV. The most common indications for retransplantation include
primary nonfunction, hepatic artery thrombosis (HAT), acute and chronic rejection, and
recurrent disease. Use of DCD allografts is associated with a 13% retransplantation
rate, which has remained stable over the last decade. Outcome after retransplantation
is generally worse than after initial OLT but is slowly improving. Patients requiring
retransplantation are at increased risk for infection, HAT, and acute renal failure.
35 Introduction
Recurrent HCV is associated with worse outcomes following retransplantation overall,
and most centers avoid retransplantation for this indication. Factors portending a poor
prognosis include preoperative mechanical ventilation, HCV infection, elevated
creatinine and bilirubin, and prolonged donor cold ischemia. Retransplantation for
recurrent HCV infection, autoimmune hepatitis, alcoholic liver disease, nonalcoholic
steatohepatitis, and hepatocellular carcinoma are some of the controversial areas
though not contraindications in themselves. This is because the survival of both patient
and graft is suboptimal in the long term. Previous abdominal surgery increases the
length of operation, blood loss, and complications related to the transplantation
procedure.
1.4.14 Delisting Criteria
There is no guideline for delisting candidates except in patients with HCC who develop
metastatic disease and fall out of the listing criteria. However, while in waiting list, if the
liver disease progresses to such an extent that the survival benefit from transplantation
(50% 5 year survival) no longer holds, which generally occurs if the MELD score is >40,
then it is probably better to delist the patient. Patients who resume alcohol intake or
substance abuse should be delisted. Temporary deactivation is done for patients who
have clinical deterioration in the form of mechanical ventilation, haemodialysis, and
fungal or resistant bacterial infection.
1.4.15 Living Donor Liver Transplantation
Living donor liver transplant candidate recipients’ indications
Pre-MELD
Hepatocellular carcinoma (stages T1 and T2)
Fulminant hepatic failure
Patients not likely to receive cadaveric organ, with life expectancy less than 6 months
Post-MELD
Hepatocellular carcinoma (exceeding T2 criteria)
Complications of cirrhosis, low MELD score
Gastrointestinal bleeding
Hepatic encephalopathy
Intractable pruritus
Recurrent cholangitis
Fulminant hepatic failure
36 Introduction
The indications for liver transplantation and listing criteria are generally the same
(child’s score ≥7, MELD >10) as in DDLT. Patients with cholestatic liver disease who
have
lower
MELD
scores,
and
those
with
recurrent
cholangitis,
recurrent
encephalopathy, and severe itching, who might not get listed in a DDLT program may
gain access to LDLT. These patients can benefit from partial liver grafts as they have
otherwise stable liver disease. Studies have revealed that the average MELD score in a
patient having LDLT is less than the score of a DDLT recipient (14.8 versus 23.5) (R. B.
Freeman 2004). The risk of transplantation is increased compared with its benefit if the
MELD score is <14 or more than (R. B. Freeman, 2004). The advantages of LDLT are
that almost all transplants are planned and elective (except for those with ALF), the
recipient’s functional status can be optimized before surgery, and the graft cold
ischaemia time is reduced. Waiting period reduction is an another advantage of LDLT
over DDLT particularly for HCC patient with low MELD score, and patients fulfilling the
Milan or UCSF criteria, depending upon the programme, get transplanted earlier in an
LDLT setting before metastases occur and the outcome is equally good. Other patients
who have low MELD scores and would benefit from LDLT are those with symptomatic
benign liver lesions (haemangioma, haemagioendothelioma, and polycystic liver
disease), metabolic disorders (familial amyloidosis, hyperoxaluria, tyrosinaemia,
glycogen storage disease), or complicated cholestatic liver disease. These patients
otherwise would have to wait for a longer period to get a deceased donor graft.
Improvement in donor and patient selection, and in surgical technique in both the donor
and recipient surgery in LDLT has resulted in improvement in 1- year graft and patient
survival to 81% and 89%; reduction of the vascular and biliary complications (hepatic
artery thrombosis <5%, biliary complications 5–20%) (Tan HP, et al. 2005; Gondolesi,
GE, et al. 2004; Marcos A, et al. 2003; Soin AS, et al 2010). It is very important to
ensure donor safety in an LDLT program, and so far the reported donor mortality is
<0.2– 0.5%, morbidity is between 10–15%, and donor biliary complication is <5% (Tan
HP, et al. 2005; Gondolesi GE, et al. 2004; Marcos A, et al. 2003; Soin AS, et al. 2010;
Lo CM, et al. 2004).
37 Introduction
1.4.16 Contraindications for LDLT
Absolute Contraindications
 Donor having macrosteatosis (>20%) on liver biopsy are rejected.
 Remnant liver volume less than 25% especially when right lobe graft is big. It is never an issue when
the left lateral segment is the proposed graft and is rarely an issue if the left lobe graft is taken.
 Unrelated donor: In India, The Human Organ Transplantation Act, does not allow unrelated donation;
to prevent donation under any kind of coercion and to avoid any organ trade. However unrelated
donation is acceptable in other countries like Hong Kong, Korea, China, Japan, and so forth.
 Age: Living donor should be between 18 and 55 years of age. Lower limit is the age at which legal
consent can be given.
Relative Contraindications.
 Body mass index (BMI)>30 of the donor is generally associated with macrosteatosis, such donors
must reduce their weight, and liver biopsy is needed to rule out >20% steatosis. If there are other
potential donors in the family, they are rejected as liver donors.
 Liver attenuation index of <5 on plain CT scan is suggestive of steatosis; hence, such donors are
either rejected or in the absence of other donors need to reduce weight and have a biopsy to rule out
>20% macrosteatosis.
 Donors are rarely rejected on anatomical grounds. Double artery, double portal vein, or more than 2
hepatic veins can be easily tackled during implantation, and these no longer preclude donation.
However, certain anatomical anomalies, for example, a Type E portal vein in the donor where there
are multiple right-sided segmental portal vein tributaries draining into the left portal vein is a
contraindication for LDLT (Nakamura T, et al. 2002) .
 All types of biliary anatomy in the donor (as classified by Huang) is acceptable (Huang TL, et al.
1996). Very rarely if there are multiple ducts in the donor (more than 3 bile ducts) to be anastomosed,
then the donor is rejected (Soin AS, et al. 2010).
1.5 PATIENT EVALUATION
1.5.1. Evaluation Aim And Purpose
Liver transplantation is a major procedure. Complications can be anticipated after liver
transplantation, including perioperative and surgical complications, immunologic and
infectious disorders, and a variety of medical complications. The purpose of the LT
evaluation is to ensure that the candidate is suitable for transplantation. Evaluation must
first, ensure that liver transplantation will offer the patient the best chance for long-term
survival and that no other options would serve the patient better, then to define
comorbid medical or psychosocial conditions that reduce the benefit of transplantation
or would preclude successful recovery from the procedure and to manage such relative
contraindications to allow transplantation procedure, and management of the patient
while awaiting transplant. Finally, to determine the urgency of proceeding with
transplantation, the natural history of the patient’s disease must be carefully compared
with the anticipated survival after liver transplantation. The clinical tools most widely
used to determine prognosis in patients with chronic liver diseases include, the ChildTurcotte-Pugh (CTP) classification, the prognostic model for end-stage liver disease
(MELD), and disease-specific indices for primary biliary cirrhosis (Dickson ER, et al.
1989; Murtaugh PA, et al 1994) and sclerosing cholangitis (Dickson ER, et al. 1992).
38 Introduction
The CTP score is useful as a rapid means of assessing the relative risk of mortality
among groups of patients with cirrhosis. Although never formally validated as a
prognostic tool, CTP score has been widely adopted for risk-stratifying patients before
transplantation because of its simplicity and ease of use (Conn HO, et al. 1981). The
CTP score is as effective as quantitative liver function tests in determining short-term
prognosis among groups of patients awaiting liver transplantation (Conn HO, et al.
1991). More than one third of patients with CTP scores of 10 or more (class C) who are
waiting for transplantation can be expected to die within 1 year (Shetty K, et al. 1997;
Oellerich M, et al. 1991). In contrast, patients with CTP scores of 7 to 9 (class B) have
an 80% chance of surviving 5 years, and those with CTP scores of 5 to 6 (class A) have
a 90% chance of surviving more than 5 years without transplantation (Shetty K et al
1997, Propst A, et al 1995, Lucey MR, 1995). The MELD was originally developed to
assess short term prognosis in patients undergoing transjugular intrahepatic
portosystemic shunts (TIPS). Using the MELD model, patients are assigned a score in a
continuous scale from 6 to 40, which equates to estimate 3-month survival rates from
90% to 7%, respectively (Malinchoc M, et al. 2000). Subsequent studies of this model
demonstrated its usefulness as an effective tool for determining the prognosis of groups
of patients with chronic liver disease (Kamath PS, et al. 2001). A modification of this
model not including the diagnosis of the disease is now used to prioritize patients for
donor allocation has been shown useful both in predicting short-term survival in groups
of patients on the waiting list for liver transplantation as well as the risk of postoperative
mortality (Wiesner R, et al. 2003; Freeman RB, et al. 2004). A similar model has been
developed for pediatric end stage liver disease (PELD). The variables included in this
model are: age younger than 1 year, serum albumin level, serum bilirubin, INR and
growth failure (<2 SD below the age-based mean). (Wiesner RH, et al 2001, McDiarmid
SV et al 2002). The higher the PELD score, the lower the likelihood of 3-month survival
without transplantation. This model has been useful in predicting deaths of pediatric
patients waiting for transplantation (Freeman RB, et al. 2001). The development of
ascites, variceal bleeding, hepatic encephalopathy, spontaneous bacterial peritonitis, or
hepatorenal syndrome also have a significant impact on the prognosis of patients with
cirrhosis. The 5-year survival rate of individuals in whom any of these complications
develop is only 20% to 50% of patients with compensated cirrhosis (Fattovich G, et al.
1997; Gines P, et al. 1987). The most ominous complications are spontaneous bacterial
peritonitis and rapid-onset (type I) hepatorenal syndrome. One year survival can be
39 Introduction
expected in less than 50% of those in whom spontaneous bacterial peritonitis develops,
whereas the median survival among patients with type I hepatorenal syndrome is less
than 2 weeks (Andreu M, et al. 1993; Gines A, et al. 1993). Patients with a MELD score
of 15 or more and a CTP score of 7 or more can be expected to achieve improved
survival with liver transplantation (Lucey MR, et al. 1997; Wiesner R, et al. 2003;
Freeman RB, et al. 2004).
1.5.2 Timing of Referral for Liver Transplantation Evaluation
Generally patients should be considered for liver transplantation if they have evidence
of fulminant hepatic failure, a life-threatening systemic complication of liver disease, or a
liver-based metabolic defect causing systemic disease. The most common indication for
LT is cirrhosis. The decision about when to refer a patient for liver transplant evaluation
is based upon how advanced the cirrhosis is and the occurrence or the presence of
complications i.e. 1) When chronic liver disease decompensate: ascites, spontaneous
bacterial peritonitis, variceal hemorrhage, protein calorie malnutrition, portosystemic
encephalopathy, severe metabolic bone disease (hepatic osteodystrophy), persistent
and
severe
coagulopathy,
uncontrolled
biliary
sepsis,
certain
hepatobiliary
malignancies. 2) When biochemical tests of liver function worsen and the patient has a
Child- Turcotte-Pugh score of >7: bilirubin>3mg/dL, serum albumin<3,0 g/dL,
prothrombine time> 3second over control or INR >1,8. 3) Patients with a Model of Endstage Liver Disease score (MELD) of greater than 10. 4) Patients with HCC fufiling
Milan criteria (who have three or fewer lesions less than three cm or one lesion up to
five cm) are potential liver transplant candidates and may receive priority for liver
transplantation. These patients should be immediately referred for evaluation. Children
with liver disease should be referred for liver transplant evaluation when they fall off
their growth curve, or when their liver decompensates. Early referral is essential,
allowing for pre-transplant problems to be addressed and resolved while the liver
disease is relatively well-compensated. There is no advantage, however, of early
referral in terms of waiting time for transplantation. The complications of cirrhosis are
managed effectively, but the natural history of the disease could change and therefore
transplantation is considered. Referral for evaluation does not mean neither immediate
transplantation nor immediate insertion in waiting list. Referral to a liver transplant
center is followed by a detailed medical evaluation to ensure that transplantation is
technically feasible, medically appropriate, and in the best interest of both the patient
40 Introduction
and society. The patient should be carefully assessed to determine the need for the
operation, to confirm that all other effective treatments have been attempted, and finally,
the patient’s likelihood of being an appropriate candidate for transplantation i.e.
Patient's fitness for surgery psychosocial preparedness, potential for recovery, no preexisting medical conditions unrelated to the liver disease that would make
transplantation unnecessarily risky. Patients approved for transplantation are enlisted
on a waiting list.
Referral of patient for transplantation:
1. Patients with cirrhosis should be referred for transplantation when they develop
evidence of hepatic dysfunction (CTP > 7 and MELD > 10) or when they experience
their first major complication (ascites, variceal bleeding, or hepatic encephalopathy.)
Referral before the patient’s anticipated mortality (estimated one year mortality<90%)
exceeds that of the estimated postoperative survival is important.
2. Children with chronic liver disease should be referred when they deviate from
normal growth curves or develop evidence of hepatic dysfunction or portal
hypertension.
3. Patients with type I hepatorenal syndrome should have an expedited referral for
liver transplantation.
4. Every option for disease-specific treatment should be considered in patients with
chronic liver disease. Only when there is no effective alternative therapy or when
treatment has been shown to be ineffective should liver transplantation be considered.
In critically ill patients in whom the outcome of medical therapy is uncertain, it is
appropriate to simultaneously begin specific treatment for the disease and to initiate
evaluation for potential liver transplantation.
1.5.3 The Process of Liver Transplant Evaluation
1) Referral To transplant center.
2) Medical evaluation.
3) Transplant surgery evaluation.
4) Anesthesia evaluation.
5) Psychiatry or psychology or psychosocial assessment.
6) Other consultants: gynecologist, urologist, neurologist, dentist, ENT physician.
7) Nutritional support assess nutritional status and patient education.
The process is standardized, uniform and involves a multidisciplinary team approach
usually carried as an outpatient LT and designed to be completed in few days. The
transplant team includes transplant hepatologists, transplant surgeons, anaesthesist,
transplant nurse coordinators, social workers, and transplant psychiatrists/psychologists
41 Introduction
with expertise in substance abuse issues. The use of other consultants varies according
to the individual transplant center and the patient needs.
Medical evaluation include:
Hepatology assessment: To confirm diagnosis and optimize management, which
means to confirm the necessity of LT and to implement a plan for the management of
the complications of cirrhosis, to evaluate disease-specific issues that may potentially
impact on outcome after LT, and to assess other comorbid conditions and possible
contraindications to LT.
Laboratory testing baseline laboratory testing, hepatic synthetic function, electrolytes,
renal function, viral serologies and microbiologic screening, markers of other causes of
liver disease, autoantibodies, tumor markers, ABO-Rh blood typing; inulin clearance or
24-hour urine for creatinine clearance; urinalysis and urine drug screen, thyroid function
tests prostate-specific antigen level (males), Pap smear (female).
Cardiac evaluation is done prior to LT to exclude coronary heart disease, valvular
heart disease, and cardiac failure due to other etiologies. Special attention should be
given to the patient with alcoholic liver disease and hemochromatosis as they are at
increased
risk
for
cardiomyopathies.
Electrocardiography
and
2-dimensional
echocardiography, stress testing and with special emphasis if risk factors are present
and/or age 40 years or older.
Hepatic imaging Ultrasonography with Doppler to document portal vein patency, triplephase computed tomography or gadolinium magnetic resonance imaging for tumor
screening
Abdominal MRI or CT scan
Chest radiograph
Arterial blood gas analysis, and pulmonary function testing are routinely done in
most transplant centers. It is of major importance to identify and distinguish both of the
uncommon but clinically significant pulmonary syndromes; hepatopulmonary syndrome
and portopulmonary hypertension among patients with cirrhosis.
Upper and lower endoscopy.
Mammogram (females).
Transplant surgery evaluation to assess technical issues and risks of procedure
Extensive portal and mesenteric venous thrombosis, previous abdominal surgery near
the hepatic hilum, and severe obesity can make the surgery difficult, if not impossible.
42 Introduction
The most common surgical contraindication to liver transplantation is absence of a
viable splanchnic venous inflow system, from portal vein thrombosis in adults or
cavernous transformation of the portal vein in children. Patients with occlusion or
hypoplasia of the splanchnic blood supply require careful anatomical evaluation before
transplantation because of the increased risk of perioperative mortality and graft loss.
Computed tomographic and magnetic resonance angiography can provide accurate
preoperative assessment of both hepatic arterial anomalies and the integrity of portal
inflow to the liver (Smith PA, et al. 1998; Eubank WB, et al. 2002). Such studies also are
valuable in assessing both the donor and recipient vasculature before living-related
transplantation. (Erbay N, et al. 2003). Thrombosis of the main portal vein can be
successfully bypassed; however, if the entire portal venous system is occluded or
atrophied, attempts at transplantation are associated with a high risk of graft loss and
perioperative mortality (Yerdel MA, et al. 2000; Manzanet G, et al. 2001; Sobhonslidsuk
A, et al. 2001).
Anesthesia evaluation to identify high operative risk, eg, portopulmonary hypertension,
hypertrophic obstructive cardiomyopathy, previous anesthesia complications.
Psychiatry or psychology or psychosocial assessment is an integral part of pre-LT
evaluation process to address the issues related to substance abuse, risk of relapse,
compliance, and adequacy of social support, prior history of substance abuse,
psychiatric illness, or adjustment difficulties and the possible impact of transplantation
on patient’s personal and social system Individuals should meet reasonable
expectations of compliance before placement on a donor waiting list and every effort
should be made to provide expert counseling and treatment of disorders that may
adversely
affect
postoperative
compliance.
The
most
frequently
encountered
contraindication to transplantation is continued destructive behavior resulting from drug
and alcohol addiction. Any form of addictive behavior also should be addressed and be
well controlled before patients are accepted for transplantation. Significant psychiatric
disorders must be under excellent medical control with assurance that the patient can
be compliant after transplantation. In addition, patients must have adequate support
from family or friends. Prisoners and children with mental retardation pose significant
logistical and ethical challenges. Patients receiving methadone maintenance who are
otherwise good candidates for transplantation should not be denied consideration for
the operation Three small studies have indicated that post transplantation outcome and
compliance among patients on methadone maintenance is comparable with that of
43 Introduction
other transplant recipients, although more pain medications and higher doses of
methadone may be required during the perioperative period. (Kanchana TP, et al. 2002;
Liu LU, et al. 2003; Weinrieb RM, 2004).
Other consultants: gynecologist, urologist, neurologist, dentist, ENT physician.
Nutritional support assess nutritional status and patient education.
Evaluation of potential recipients before liver transplantation
General evaluation:
Age and body mass index
Blood group for listing purposes HLA typing
History survey and physical examination
History of alcohol consumption and substance abuse
Infection: syphilis, cytomegalovirus, Epstein–Barr virus and herpes
simplex virus
Abdominal imaging (Doppler ultrasound, CT, MR angiography and
MR cholangiopancreatography to determine vascular and biliary
anatomy, calculate liver volume and assess steatosis
Screening for colon, breast, cervical and prostate cancer
Cardiopulmonary status evaluation: chest roentgenography,
electrocardiogram, 2-D echocardiogram
Thallium stress test and coronary angiography for high-risk patients
Pulmonary function tests
Studies for patients with
the following conditions:
Hepatitis B: HBV–DNA, HBeAg, anti-HBe Ab, anti-delta Ab
Hepatitis C: HCV-RNA, HCV genotype
Autoimmune hepatitis: immunoglobulin G, antinuclear antibody,
antismooth muscle antibody, liver–kidney microsomal antibody
a1-Antitrypsin deficiency: a1-antitrypsin level and phenotype
Wilson disease: ceruloplasmin, 24-h urine copper, hepatic copper
Hemochromatosis: iron saturation, ferritin, HFE gene test
HCC: bone scan, chest radiography
Hepatopulmonary syndrome: arterial blood gas, transthoracic
contrast echocardiography, arterial oxygen response to 100%
oxygen, quantification of intrapulmonary shunting using
macroaggregated albumin scan
Primary sclerosing cholangitis: colonoscopy (to exclude ulcerative colitis)
and ERCP (to exclude cholangiocarcinoma)
To detect underlying
contraindicated
conditions:
Arterial blood gas to screen for the presence of severe portopulmonary
hypertension
Serum a-fetoprotein, CA-199, liver ultrasound, CT, and/or MRI: to
exclude HCC, cholangiocarcinoma
Doppler ultrasound to exclude portal vein thrombosis
Bone densitometry to check the presence of severe osteoporosis
Neuropsychological testing: optional
Infection: HIV
CT, computed tomography; delta Ab, antibody to hepatitis D virus;
ERCP, (Endoscopic retrograde cholangiopancreatography); HBeAg,
hepatitis Be
antigen; HBV–DNA, hepatitis B viral DNA; HCC, hepatocellular
carcinoma; HCV, hepatitis C virus; HFE, hemochromatosis; HLA, human
leukocyte antigen; MR, magnetic resonance; MRI, magnetic resonance
44 Introduction
1.5.4 Evaluation of potential donors for living donor liver transplantation
Age, relation to recipient, body mass index, medical history and blood group.
History survey and physical examination
History of alcohol consumption and substance abuse
Contraindicated surgical history: previous major abdominal surgery
Contraindicated major medical conditions: diabetes, severe or uncontrolled
hypertension, hepatic, cardiac, renal or pulmonary disease
Laboratory survey:
* Complete blood count with differential count
* Coagulation profiles
* Liver and kidney biochemistries, triglycerides, cholesterol
* Fasting blood glucose level
* Ferritin, transferring saturation, a-1-antitrypsin, ceruloplasmin, antinuclear antibody.
Virus infection: hepatitis B and C, HIV, cytomegalovirus, Epstein–Barr virus and
herpes simplex virus
Cardiopulmonary survey: arterial blood gas, electrocardiography, chest
radiography, pulmonary function test, echocardiography and Doppler ultrasound
Liver volume and vascular, biliary anatomy estimation: abdominal ultrasound, CT or
MRI
Presence of steatosis: liver images and/or liver biopsy
Optional: celiac angiography, ERCP, stress electrocardiography
Psychological and ethical evaluation
CT, computed tomography; ERCP, endoscopic retrograde cholangiopancreatography;
MRI, magnetic resonance imaging.
1.5.5 Listing for transplantation and organ allocation
Based on the pre-LT evaluation testing, a multidisciplinary selection committee makes a
decision regarding the candidacy for LT for the candidates. The results of the liver
transplant evaluation are reviewed in detail by a selection committee composed of
transplant surgeons, hepatologists, anesthesiologists, psychiatrists or psychologists,
transplant coordinators, or others who might offer insight on a particular case as an
oncologist, a cardiologist. Approval by the committee leads to listing of the patient on
the donor organ waiting list.
1.5.6 Medical issues to be considered during evaluation:
Age.
There is no age limitation to successful liver transplantation (Starzl TE, et al. 1987;
Zetterman RK, et al. 1998). Older patients have diminished long-term survival after
transplantation compared with younger individuals, because of an increased risk of
death from malignancies (Collins BH, et al. 2000; Herrero JI, et al. 2003).
45 Introduction
Coronary Artery Disease.
Perioperative mortality after liver transplantation is high in patients with coronary artery
disease (Plotkin JS, et al. 1996). Chronic smokers, patients over the age of 50, and
those with a clinical or family history of heart disease or diabetes should undergo
evaluation for coronary artery disease. Dobutamine stress echocardiography is an
effective screening test, (Plotkin JS, et al. 1998; Donovan CL, et al. 1996; Williams K, et
al. 2000) positive test results should be confirmed with cardiac catheterization to
delineate further the extent of the coronary disease. (Plotkin JS, et al. 1996, Plotkin JS,
et al 1998).
The Hepatopulmonary Syndrome (HPS).
Patients with cirrhosis and severe hepatopulmonary syndrome (chronic liver disease,
arterial deoxygenation, and widespread intrapulmonary vasodilation) have an extremely
poor prognosis without transplantation. The median survival of patients with cirrhosis
and severe HPS is less than 12 months (Schenk P, et al. 2003). The condition is
reversible after liver transplantation, therefore patient should be referred without any
delay for evaluation for urgent transplantation (Krowka MJ, et al. 1997; Santamaria F, et
al. 2002). Preoperative evaluation of suspected patients include arterial blood pO2
determination, transthoracic contrast echocardiography, arterial oxygen response to
100% oxygen administration, and quantification of intrapulmonary shunting using a
macroaggregated albumin (MAA) scan (Arguedas MR, et al. 2003). Patients with severe
hypoxia have increased perioperative mortality (Arguedas MR, et al. 2003; Collisson
EA, et al. 2002). Preoperative PaO2 of 50 mmHg or less alone or in combination with a
MAA shunt fraction of 20% or more are the strongest predictors of postoperative
mortality (Arguedas MR, et al. 2003). Patients with clinical evidence of HPS and PaO2
of less than 60 mmHg on room air with no underlying lung disease can receive
enhanced prioritization for organ allocation to allow them a reasonable possibility of
receiving a deceased donor organ within 3 months.
Portopulmonary Hypertension.
Portopulmonary hypertension is seen in 2% to 4% of patients with cirrhosis (Hadengue
A, et al. 1991; Colle IO, et al. 2003). All patients undergoing evaluation for potential liver
transplantation should undergo screening for pulmonary hypertension. Doppler
echocardiography is a sensitive method of detecting the presence of pulmonary
hypertension (Colle IO, et al. 2003; Kim WR, et al. 2006; Genesca J et al. 2001; Cotton
CL, et al. 2002). The positive predictive value of the test is low and should be confirmed
46 Introduction
with right heart catheterization. Patients with severe pulmonary hypertension should be
considered for liver transplantation only if the condition can be effectively controlled with
medical therapy. A number of studies have found that mild pulmonary hypertension
(systolic pulmonary artery pressure, 30-44 mmHg) and moderate pulmonary
hypertension (systolic pulmonary artery pressure, 45-59 mmHg) are not associated with
an increased risk of liver transplantation (Ramsay MA, et al. 1997; Krowka MJ, et al.
2000; Starkel P, et al. 2002). Severe pulmonary hypertension is associated with high
perioperative mortality and, if not successfully treated, represent a contraindication to
liver transplantation (Ramsay MA, et al. 1997; Krowka MJ, et al. 2000). Patients with
severe pulmonary hypertension who have been successfully treated with medical
therapy have undergone transplantation safely and pulmonary hypertension gradually
resolves within 4 to 6 months after transplantation (Plotkin JS, et al. 1998; Ramsay MA,
et al. 1999; Mair P, et al. 2001).
Obesity.
Morbid obesity (BMI >40 kg/m2) should be considered a contraindication to liver
transplantation. Obesity has an adverse impact on both immediate and long-term
survival. Obesity was more common in women and in patients with cryptogenic
cirrhosis. Morbid obesity was associated with decreased 30-day, 1-year, and 2-year
postoperative survival. Five-year survival was reduced both in patients with morbid and
severe obesity (BMI >35 kg/m2) (Nair S, 2002a et al. 2002).
Cigarette Smoking.
All patients considered for liver transplantation should abstain from smoking. Recent
studies have demonstrated the deleterious effects of smoking on outcomes after
transplantation. The risk of hepatic artery thrombosis appears to be significantly
increased among chronic smokers (Pungpapong S, et al. 2002). This effect disappears
in chronic smokers who discontinue nicotine use 2 years before transplantation
(Pungpapong S, et al. 2002). Long-term postoperative survival of smokers also is
decreased because of an increase in cardiac mortality and death from malignancies
(Herrero JI, et al. 2003). It has been confirmed that active smoking is independently
associated with a higher risk of malignancy after liver transplantation (Herrero JI, et al.
2011; Vallejo GH, et al. 2005). In contrast to alcohol, the level of concern about this
deadly addiction remains low: only 1 series is available (Dew MA, et al. 2008; van der
Heide F, et al. 2009). Before transplantation, 17% of patients are active smokers (the
rate is 52% for patients with alcoholic liver disease), and only one-third of these patients
47 Introduction
succeed in quitting. In addition, smoking is an independent and dose related factor
contributing to hepatocellular carcinoma (Braillon A, et al. 2010).
Renal Failure.
Pre-transplantation evaluation of renal function is important. A number of studies have
identified elevated serum creatinine (one of the major variables in the MELD model) as
an independent risk factor for the development of renal failure and decreased survival
after liver transplantation (Lafayette RA, et al. 1997; Bilbao I, et al. 1998; Nair S2002b,
et al. 2002). As a result, an increasing number of patients with renal insufficiency are
being selected for liver transplantation. The presence of renal insufficiency is an
important predictor of postoperative renal failure and mortality after liver transplantation.
Hepatorenal syndrome (type 1) which is a rapidly progressive, life threatening condition,
is usually reversed by transplantation, therefore, patients with such condition should be
referred for evaluation without delay. Acute renal failure from the hepatorenal syndrome
usually improves dramatically after liver transplantation and does not appear to have an
impact on post-transplant survival (Seu P, et al. 1991; Gonwa TA, et al. 1991). In
contrast, patients with preexisting chronic renal disease have diminished survival and
an increased risk of requiring dialysis after transplantation (Lafayette RA, et al. 1997).
However, it is quite difficult to distinguish these two conditions in patients with severe
liver disease (Davis CL, et al. 2002). Selected patients with chronic renal and liver
disease should be considered for combined liver–kidney transplantation (Grewal HP, et
al 2000; Rogers J, et al 2001).
Extrahepatic Malignancies.
Close consultation between a patient’s oncologist and transplantation physicians should
occur before evaluation for liver transplantation in patients with extrahepatic
malignancies. Patients with a history of extrahepatic malignancy are at high risk for
recurrent
disease
because
of
the
immunosuppression
required
after
liver
transplantation. The natural history and chance of recurrence varies with different
tumors.
Osteoporosis.
All patients with chronic liver disease should be screened for osteoporosis during
evaluation for liver transplantation. It is a common complication of cirrhosis (Sokhi RP,
et al. 2004) particularly in postmenopausal women, patients with cholestatic disorders
such as PBC and PSC, and patients who have received prolonged corticosteroid
therapy. (Menon KV, et al. 2001). Osteoporosis is also common in patients with chronic
48 Introduction
hepatitis C and alcoholic cirrhosis (Floreani A, et al. 2001; Carey EJ, et al. 2003). It is of
particular concern in patients being considered for liver transplantation because of the
loss of bone density and the risk for pathological fractures that can occur in the
perioperative period (Trautwein C, et al 2000). Patients with significant bone loss, efforts
to improve bone density and to prevent pathological fractures should be pursued both
before and after transplantation
Patients With HIV Infection.
Liver transplantation in patients with HIV infection requires a well-coordinated,
multidisciplinary team with expertise both in transplantation and HIV management
(Roland ME, et al. 2003). With the widespread use of highly active antiretroviral therapy
(HAART), both the natural history of HIV infection and the outcome after transplantation
have improved dramatically. An increasing number of patients with HIV infection are
being referred for liver transplantation. Recent results suggest that short-term survival
after transplantation in patients with HIV infection that is well controlled with HAART is
comparable with that seen in HIV-negative recipients (Neff GW, et al. 2003; Stock PG,
et al 2003). Most patients have undetectable HIV RNA after the operation; however, a
number of serious interactions have been reported between antiretroviral drugs and the
immunosuppressive agents used after liver transplantation (Schvarcz R, et al. 2000;
Sheikh AM, et al. 1999)
1.5.7 Specific Consideration For Liver Transplantation
Chronic Noncholestatic Liver Disorders.
Accounts for more than 60% of all transplants performed annually and include endstage liver disease secondary to chronic viral hepatitis, autoimmune hepatitis, and
alcoholic cirrhosis. Postoperative survival for this group of patients is slightly less than
for transplantation recipients with cholestatic liver disorders (1 year, 86%; 3 years, 77%)
(Roberts MS, et al. 2004).
Chronic Hepatitis C.
10-year survival rate of patients with well-compensated cirrhosis is more than 80%, 5year survival is less than 50% after development of complications (Fattovich G, et al
1997). Patients with cirrhosis secondary to chronic hepatitis C also have a 2% to 8%
annual risk of developing HCC (Befeler AS, et al. 2002). Patients with clinically
decompensated cirrhosis from chronic hepatitis C infection should be referred for
consideration of liver transplantation. Antiviral therapy should be considered in patients
49 Introduction
who have been accepted as candidates for liver transplantation, with close monitoring
for adverse effects.
Chronic Hepatitis B.
An estimated 350 million persons worldwide are infected with HBV. HBV carriers,
particularly those who acquire the disease at birth or in early childhood, are at risk for
the development of cirrhosis and HCC. HBV carriers with compensated cirrhosis have
an 84% 5-year survival rate and a 68% 10-year survival rate. Patients with
decompensated cirrhosis have a 5-year survival rate of only 14% (Lok AS and
McMahon BJ. 2001).
Autoimmune hepatitis.
Some patients who achieved biochemical and histological remission of disease develop
intractable portal hypertension and slowly progressive liver failure despite medical
therapy. Liver transplantation is the only effective treatment for patients with severe
autoimmune hepatitis who fail to respond to immunosuppressive therapy or who
develop advanced decompensated disease despite treatment.
Alcoholic Cirrhosis.
Abstinence from alcohol is required to avoid the risk of unnecessary surgery as many
patients with advanced alcoholic liver disease who completely abstain can recover to
the degree that transplantation is not required (Veldt BJ, et al. 2002). There is no
effective means of predicting which patients will have such a dramatic response.
Transplantation programs require 6 months of abstinence and careful evaluation by
professional counselors to directly address the addiction to alcohol before
transplantation (Everhart JE, et al. 1997). Patients who have CTP scores of 11 or more
(Child C disease), despite at least 6 months of abstinence, have improved survival with
transplantation compared with the natural history of disease predicted from prognostic
models (Poynard T, et al. 1999).
Cholestatic Liver Disorders.
Liver transplantation is the only effective treatment for adults with end-stage liver
disease secondary to PBC and PSC. Biliary atresia is the most common indication for
liver transplantation in children, accounting for 60% to 70% of all procedures performed.
Survival after transplantation for either adults or children with cholestatic disorders is
excellent, with 1-year postoperative survival of more than 90% and 3-year survival
approximating 85% (Roberts MS, et al. 2004).
50 Introduction
Primary Biliary Cirrhosis.
The disease most commonly affects women in the fourth to seventh decades of life.
Liver transplantation is the only effective treatment for liver failure secondary to primary
biliary cirrhosis and may be indicated in selected patients for uncontrolled pruritus with
good liver functions after exploring every possible medical treatment. After liver
transplantation, 70% of patients with PBC survive at least 10 years after the operation
(Roberts MS, et al. 2004; Pasha TM, et al. 1997; Liermann Garcia RF, et al. 2001;
Tinmouth J, et al. 2002). Numerous studies using disease-specific prognostic models
have documented improved survival after transplantation compared with estimated
survival without surgery (Pasha TM, et al. 1997; Liermann Garcia RF, et al. 2001). The
survival benefit of transplantation is evident as soon as 3 months after surgery, and 2year survival of transplanted patients is more than twice that predicted for those treated
conservatively (Pasha TM, et al. 1997). PBC recurrence after transplantation has been
well documented, it has not had a major impact on long-term postoperative survival
(Liermann Garcia RF, et al. 2001).
Primary Sclerosing Cholangitis.
The disease typically occurs in young men, 70% to 75% of whom have inflammatory
bowel disease. Most patients with symptomatic disease develop liver failure within 10 to
12 years (Harnois DM, et al. 1997). No specific medical treatment has been shown to
improve survival in patients with PSC (Harnois DM, et al. 1997; Lee YM, et al. 2002).
Liver transplantation is the only effective treatment for decompensated cirrhosis
secondary to primary sclerosing cholangitis. Because of the high incidence of colon
cancer, regularly scheduled colonoscopies should be performed both before and after
transplantation in all patients who have inflammatory bowel disease. Patients with PSC
and cholangiocarcinoma should be excluded from transplantation. Most studies have
reported transplantation outcomes for PSC patients that equal or surpass those
reported for PBC, with 3-year survival rates of more than 90%. (Roberts MS, et al. 2004;
Abu-Elmagd KM, et al. 1993; Narumi S, et al. 1995; Ricci P et al. 1997; Goss JA, et al.
1997; Graziadei IW, 1999a, et al. 1999). Another report demonstrated higher
retransplantation rates and lower long term survival among patients with PSC
(Maheshwari A, et al. 2004). Nevertheless, survival of patients with PSC after liver
transplantation has been shown to be superior to that predicted for patients treated
conservatively (Dickson ER, et al. 1992; Farrant JM, et al. 1991; Farges O, et al. 1995).
51 Introduction
Recurrent disease is common after transplantation, this has not had a significant impact
on long-term postoperative survival. (Goss JA, et al. 1997; Graziadei IW, et al. 1999b ).
Childhood Cholestatic Diseases.
Chronic cholestasis in children can result from a variety of conditions, including biliary
atresia, alpha 1-antitrypsin deficiency, cystic fibrosis, various types of intrahepatic
cholestasis, and PSC. Liver transplantation is indicated in appropriately selected
children with biliary atresia if portoenterostomy is unsuccessful, or if intractable portal
hypertension or liver failure develops despite successful portoenterostomy. Liver
transplantation should be considered for its ability to significantly prolong survival and
improve quality of life by reducing pruritus in syndromic and nonsyndromic forms of
intrahepatic cholestasis in children. Children with Alagille syndrome should have
preoperative assessment for congenital heart disease, which is common in this
condition. In evaluating patients with cystic fibrosis for liver transplantation, careful
assessment of lung disease should be performed.
Metabolic Diseases.
A variety of metabolic diseases can result in progressive liver injury and cirrhosis. The
most common metabolic diseases in adults are alpha1-antitrypsin deficiency, Wilson
disease, hereditary hemochromatosis, and nonalcoholic steatohepatitis (NASH).
Common metabolic disorders that can cause liver failure in children include alpha1antitrypsin deficiency, Wilson disease, tyrosinemia, glycogen storage diseases, and
neonatal hemochromatosis. The outcome of transplantation in adults is excellent (1year survival, 88%; 3-year survival, 84%) and is better in children (1-year survival, 94%;
5-year survival, 92%) (Roberts MS, et al. 2004; Kayler LK, et al. 2003). Liver
transplantation is the only effective treatment for decompensated cirrhosis secondary to
alpha1-antitrypsin deficiency, careful assessment for lung disease should be performed
before transplantation in such patients. Urgent liver transplantation is the only effective
option for patients with fulminant hepatic failure resulting from Wilson disease but is not
recommended as primary treatment for neurological Wilson disease because the liver
disease is stabilized by medical therapy in most of these individuals, and outcomes with
liver transplantation are not always beneficial. Liver transplantation should be
considered for selected patients with decompensated cirrhosis secondary to
nonalcoholic steatohepatitis (NASH). The posttransplantation care of these patients
should include metabolic monitoring. Liver transplantation should be considered for
selected patients with decompensated cryptogenic cirrhosis. These patients should be
52 Introduction
screened for metabolic dysregulation because of the possibility of underlying
nonalcoholic steatohepatitis. All patients with newly diagnosed cirrhosis should be
screened for hemochromatosis using serologic tests, with genetic testing in equivocal
cases. Survival of transplanted patients with hereditary hemochromatosis is lower than
in those transplanted for other causes of liver disease. Due to the increased risk of
cardiac complications, a pretransplantation cardiac evaluation is essential. Efforts
should be made to phlebotomize these patients before transplantation. Liver
transplantation is the only effective treatment for infants with severe neonatal
hemochromatosis. Urgent evaluation at a transplant center is recommended. Children
with tyrosinemia who develop hepatocellular carcinoma (HCC) and meet the criteria for
liver transplantation for HCC, should be high-priority candidates. Children with
tyrosinemia and glycogen storage diseases unresponsive to medical management
should be considered for transplantation. Consideration of extrahepatic complications of
the underlying disease must be carefully considered in potential transplant candidates
as these children can have a variety of renal, cardiac, or neurological abnormalities that
may compromise the likelihood of survival with good quality of life after liver
transplantation, and must therefore be considered during the evaluation for the
operation (Matern D, et al. 1999). Selected patients with metabolic diseases may
require liver transplantation, not for liver failure but to prevent severe extrahepatic
manifestations of the disease. Children and adults with inborn errors in metabolism for
which liver transplantation is performed to correct the enzyme deficiency and halt
progression of extra-hepatic organ damage have normally functioning livers. Hyperoxaluria and amyloidosis are the most common conditions in adults, whereas the most
frequent conditions in children are the urea cycle defects and defects in branched-chain
amino acid metabolism. Patients with hereditary amyloidosis, in which the mutant
amyloid precursor protein is produced by the liver, may benefit from liver transplantation
(Suhr OB, et al. 2002). The variants for which liver transplantation has been most
successful include mutations of the transthyretin, apolipoprotein A-1, and fibrinogen Aa
amyloid precursors. The ideal timing of transplantation seems to be within the first year
of symptoms and before the development of severe cardiac, renal, gastrointestinal, or
neurological involvement (Adams D, et al. 2000). Patients with type 1 primary
hyperoxaluria also may benefit from liver or combined liver and renal transplantation.
Other metabolic conditions that result in significant extrahepatic morbidity include urea
cycle defects (ornithine transcarbamylase deficiency, citrullinemia, carbamyl phosphate
53 Introduction
synthetase deficiency, argininosuccinic aciduria, and arginase deficiency) and disorders
of branched-chain amino acids (maple syrup urine disease, methylmalonic acidemia,
propionic acidemia, and isovaleric acidemia). In patients having aggressive disease not
satisfactorily managed with standard dietary and pharmacological interventions, liver
transplantation has been effective (Kayler LK, et al. 2002; Yorifuji T, et al. 2000). High
rate of neurological complications after transplantation has been observed in children
with some of these conditions, particularly the branched-chain amino acid disorders
(van’t Hoff W, et al. 1999; Chakrapani A, et al. 2002). In considering these patients for
liver transplantation, prompt evaluation of the reversibility of the enzyme deficiency with
whole or partial organ liver transplantation especially if parent-to-child living-donor
transplantation is being considered, because these are usually autosomal recessive
disorders in which parents have reduction of enzyme activity. The major reason for liver
transplantation is to prevent the progression of neurological injury, the potential for
functional health after transplantation must be estimated, based on the child’s health at
the time of evaluation. Liver transplantation is indicated in children with metabolic
diseases that cause progressive extra-hepatic injury resulting in significant morbidity
and mortality that are not responsive to disease-specific medications or dietary
modification and for which liver transplantation would result in the reversal of the
enzyme deficiency and metabolic derangement. Living related transplantation should be
considered only if the enzyme activity of the donor would satisfactorily reverse the
deficiency of the recipient. The degree of neurological injury before transplantation
should be considered when selecting patients for liver transplantation. Based on the
PELD and MELD scoring systems, these patients would never have a score that would
select them of a deceased donor organ. However, the need is urgent. Consequently,
these patients can be given priority for deceased donor organs.
Hepatic Malignancies.
Timely liver transplantation often is the most effective treatment for many patients with
primary hepatic malignancies. The exception is cholangiocarcinoma, which usually
recurs rapidly after transplantation.
Hepatocellular Carcinoma (HCC).
HCC causes approximately 1 million deaths worldwide each year. Patients with chronic
hepatitis B, chronic hepatitis C, and hemochromatosis are at particularly high risk for
HCC. In addition, almost all untreated children with tyrosinemia surviving to early
childhood develop HCC. The prognosis depends both on the stage of the tumor and the
54 Introduction
degree of liver function impairment (Llovet JM 1999a et al 1999). Liver transplantation
should be viewed as the treatment of choice for selected patients with hepatocellular
carcinoma who are not candidates for surgical resection and in whom malignancy is
confined to the liver. Optimal results following transplantation are achieved in patients
meeting Milan criteria (with a single lesion 2 cm or larger and less than 5 cm, or no
more than three lesions, the largest of which is less than 3 cm, and no radiographic
evidence of extrahepatic disease). Patients who meet these criteria should receive a
donor organ within 6 months of listing for transplantation. Primary hepatic resection has
long been considered the treatment of choice for HCC, 5-year tumor-free survival rates
are less than 50% (Cha CH, et al 2003). Furthermore, most patients referred for
resection are rejected because the tumor is unresectable or because of inadequate
hepatic reserve (Bruix J, et al 2000). Even in patients with well-compensated cirrhosis,
perioperative mortality after surgical resection is extremely high in those patients with
portal hypertension or high serum bilirubin values (Llovet JM, et al 1999).
Radiofrequency ablation and percutaneous alcohol injection are effective in tumors
smaller than 3 cm but are far less successful for larger tumors (Castells A, et al 1993,
Curley SA, 2000). In selected patients with otherwise untreatable tumors but relatively
well-preserved liver function, chemoembolization has been shown to improve survival;
however, these patients have much lower survival rates than those who are candidates
for surgical or ablative therapy. (Llovet JM, et al 2002, Lo CM, et al 2002, Llovet JM, et
al 2003)
Hepatoblastoma.
Hepatoblastoma is the most common primary hepatic malignancy in children. It is
usually locally invasive and has a better long-term prognosis than HCC. Liver
transplantation should be considered for children in whom the tumor is unresectable
and confined to the liver. Chemotherapy prior to transplantation results in an excellent
prognosis and long-term tumor free survival. (Srinivasan P, et al 2002). The PELD
scoring system does not reflect the need for transplantation of these children as they do
not have an underlying liver disease. Transplantation center may submit a request for
enhanced prioritization for deceased donor organs.
Fibrolamellar Hepatocellular Carcinoma and Hemangioendothelioma.
Fibrolamellar variant of HCC and epithelioid hemangioendothelioma have better
prognoses than HCC (El Serag HB, et al 2004, Makhlouf HR, et al 1999).
Transplantation is uncommon in most cases due to the absence of an underlying liver
55 Introduction
disease. Large tumors are not among the contraindications for liver transplantation.
(Ben-Haim M, et al 1999) and transplantation should be considered in unresectable
tumors provided that there is no evidence of extrahepatic disease.
Cholangiocarcinoma.
The outcome of liver transplantation for cholangiocarcinoma has been very poor.
Transplantation should be limited to well established centers with well designed clinical
trials approval by a local institutional review board and informed consent of potential
recipients.
Fulminant Hepatic Failure.
Fulminant hepatic failure (FHF) is defined as the development of hepatic
encephalopathy and profound coagulopathy within 8 weeks of the onset of symptoms in
patients without preexisting liver disease. Patients with fulminant hepatic failure should
be referred to a transplant center as quickly as possible for critical care management.
Patients predicted to have little chance of spontaneous recovery should undergo
transplantation as soon as possible. Patients with FHF can develop cerebral edema,
multiorgan failure, or cardiovascular collapse within days to weeks after clinical
presentation (Ellis A, et al 1996, McCormick PA, et al 2003). Consequently, any delay in
obtaining a donor organ can have fatal consequences. To address this urgency, a
special category (status1) was created to allow these patients to receive first preference
for any deceased donor organ. There is no specific therapy for FHF (Lee WM, et al
1996). However, if given appropriate critical care support, many patients spontaneously
recover. In these instances, recovery typically is complete, with no evidence of residual
liver injury. The prognosis for spontaneous recovery depends on the patient’s age, the
underlying etiology of disease, and the degree of encephalopathy. (Lee WM, et al 2003,
O’Grady JG, et al 1988, O’Grady J, et al 1989). Other important prognostic factors
include acidosis, prolongation of prothrombin time values, and elevated APACHE II
scores.(Bailey B, et al 2003). Survival after liver transplantation for FHF has improved
dramatically over the past few years. (Bismuth H, et al 1997, McCashland TM, et al
1996, Farmer DG, et al 2003).
Miscellaneous Conditions.
Other less frequent indications for liver transplantation include liver failure secondary to
hepatic vein occlusion (Budd-Chiari syndrome), selected metastatic neuroendocrine
tumors, and polycystic disease.
56 Introduction
Budd-Chiari Syndrome.
The selection of patients for liver transplantation for Budd-Chiari syndrome must be
individualized, considering alternative therapeutic options. Survival after transplantation
depends on the severity of disease at the time of transplantation, the extent of the
thromboses, and the underlying cause of the condition. The best results have been
achieved in patients who have thrombosis limited to the hepatic veins, in whom the
underlying cause of the syndrome can be corrected by liver replacement (Ringe B, et al
1995).
Metastatic Neuroendocrine Tumors.
Metastases from neuroendocrine tumors often are slow growing and can be confined to
the liver for long periods. A variety of options are available for managing these patients,
including systemic somatostatin or radioactive metaiodobenzylguanidine therapy,
surgical excision, radiofrequency ablation, chemoembolization, and liver transplantation
(Sutcliffe R et al 2004, Sarmiento JM, et al 2003). Liver transplantation for metastatic
neuroendocrine tumors should be confined to highly selected patients who are not
candidates for surgical resection in whom symptoms have persisted despite optimal
medical therapy or tumors causing life-threatening hormonal symptoms (Olausson M, et
al 2002).
Polycystic Liver Disease.
Liver failure is uncommon in patients with polycystic disease. Some patients are so
debilitated as a result of abdominal pain, anorexia, or fatigue in such case liver
transplantation is considered. Dramatic improvement in symptoms and quality of life are
typical after transplantation (Jeyarajah DR, et al 1998, Chui AK, et al 2000, Koyama I, et
al 2002). Importantly, these patients seem to be unusually susceptible to infection after
the procedure.
Retransplantation.
Retransplant operations account for approximately 10% of all liver transplants.
Indications for retransplantation are primary graft nonfunction, hepatic artery
thrombosis, allograft rejection, and recurrent disease. Retransplantation should be
considered before patients develop severe hepatic and renal failure. The outcome of
retransplantation is lower than for primary transplantation with 1-,3-, and 5-year survival
rates
approximately
20%
lower
than
for
primary
transplantation
(http://www.optn.org/latestdata/rptstrat.asp). Retransplantation for liver failure from
recurrent hepatitis C has been associated with particularly poor survival (Yoo HY, et al
57 Introduction
2003). The prognosis depends on the urgency of retransplantation, serum bilirubin and
creatinine levels, CTP score of 10 or more, and MELD score of more than 25 all are
associated with a poor prognosis after retransplantation (Azoulay D, et al 2002, Biggins
SW, et al 2002, Sieders E, et al 2001, Yao FY, et al 2004). Retransplantation should be
avoided in subgroups of patients with little chance of success.
Hospice Care.
Patients will be considered to be in the terminal stage of liver disease (life expectancy of
six months or less) and eligible for hospice care, if they meet the following criteria (1
and 2 must be present; factors from 3 will lend supporting documentation):
1. The patient should show both a and b:
a. Prothrombin time prolonged more than 5 seconds over control, or International
Normalized Ratio (INR)> 1.5
b. Serum albumin <2.5 gm/d1
2. End stage liver disease is present and the patient shows at least one of the following:
a. ascites, refractory to treatment or patient non-compliant
b. spontaneous bacterial peritonitis
c. hepatorenal syndrome (elevated creatinine and BUN with oliguria
(<400ml/day) and urine sodium concentration <10 mEq/l)
d. hepatic encephalopathy, refractory to treatment, or patient non-complaint
e. recurrent variceal bleeding, despite intensive therapy
3. Documentation of the following factors will support eligibility for hospice care:
a. progressive malnutrition
b. muscle wasting with reduced strength and endurance
c. continued active alcoholism (> 80 gm ethanol/day)
d. hepatocellular carcinoma
e. HBsAg (Hepatitis B) positivity
f. hepatitis C refractory to interferon treatment
1.6 Management While Waiting for Transplantation
All enlisted patients should be managed by transplant hepatologist at regular interval
depending on MELD score. The aim is to avoid unnecessary complications of cirrhosis,
to optimize management of complications, to screen for changes in the medical
condition that might change the priority for transplantation, and to ensure that the
patient is still fit for transplantation and in the best possible condition when a donor
58 Introduction
organ becomes available. A MELD score of 10 warrants testing every 6 months to 1
year, while patients with MELD scores between 11 and 18, every 3 months, monthly for
MELD scores of 19–24 and for patients with MELD scores =or>25 tests are performed
weekly.
1.6.1 Lab values Recertification schedule of MELD data
Score
Recertification
Recertification
≥25
every 7 days
≤48 hours old
24-19
every 30 days
≤ 7 days old
18-11
every 90 days
≤14 days old
≤10
every year
≤30 days old
In every visit regular reporting of the MELD score along with a full battery of liver tests,
creatinine, electrolytes, complete blood count, and prothrombin time. Assessment of
clinical or subclinical portosystemic encephalopathy, ascites, and edema. Periodic
ultrasound and computed tomography (CT) / magnetic resonance imaging of the liver
for the development of HCC. The patients at risk for HCC include those with cirrhosis,
but especially those associated with hepatitis B and C, and certain metabolic disorders
like hemochromatosis and alpha-1-antitrypsin deficiency. (El-Serag HB, et al 2002).
Patients developing problems may remain temporarily inactive while remaining on the
list (termed status 7) or permanently delisted. Possible problems are the development of
extrahepatic malignancy or other comorbid conditions, pregnancy or the patient may
die. Sometimes patient condition improve, become stable and transplantation is no
more needed. When an organ becomes available, patients are admitted to the hospital
for a brief evaluation to ensure that they are still appropriate candidates.
1.6.2 Disease-Specific Considerations
Hepatitis C
Up to 20% of HCV-infected patients progress to cirrhosis after 20 years of infection
(Freeman AJ, et al 2001). Among patients with HCV-induced cirrhosis, 4% per year
decompensate and 1%–4% per year develop HCC. (Serfaty L, et al 1998, Fattovich G,
et al 1997, El-Serag HB, et al 2004). Hepatic failure represents the clinical trigger point
for referring these patients for transplantation as five year survival is only about 50%
(Fattovich G, et al 1997, El-Serag HB, et al 2004). Viral clearance in patients with
59 Introduction
advanced fibrosis nearly eliminates the subsequent risk of liver failure and markedly
reduces the chance of developing HCC (Bruno S, et al 2007). Treatment before
transplantation in patients who tolerate therapy is imperical because nearly all patients
with chronic hepatitis C who undergo liver transplantation with detectable virus will
reinfect the graft (Garcia-Retortillo M, et al 2002). Antiviral treatment of chronic hepatitis
C is effective in eradicating infection in approximately 50% of treated patients
(Sangiovanni A, et al 2006, Manns MP et al 2001). The number of studies investigating
the tolerability and efficacy of antiviral therapy in HCV patients before LT is limited
(Crippin JS, et al 2002, acobellis A, et al 2007, Everson GT et al 2005, Triantos C wt al
2005). Noncirrhotic patients have a 40%–80% sustained virologic response (SVR) to
pegylated interferon and ribavirin, depending on their genotype (El-Serag HB, et al
2004, Sangiovanni A, et al 2006). SVR occurs in only 13% of patients with genotype 1
and 50% of patients with genotype 2 and 3 (Everson GT, et al 2005). In compensated
patients with bridging fibrosis or cirrhosis, the SVR rate is about 10% lower than in
patients without significant fibrosis, but SVR eliminates the risk of further progression
and liver failure however the risk of HCC remains (Garcia-Retortillo M, et al 2002).
Patients who have decompensated cirrhosis and already evaluated and enlisted for
transplantation can still be treated with dose escalation with careful monitoring for doselimiting cytopenia and should clearly understand the risks of recurrent disease.
Eradication of the virus (SVR) before transplantation improves the long-term outcome
post-transplantation (Everson GT, et al 2005). Recently protease and polymerase
inhibitors therapy permitting SVR in many patients and hence decreasing the need for
transplantation. The clinical course for patients with recurrent HCV after liver
transplantation is variable, in general patients progress more quickly to cirrhosis. Longterm survival of patients who undergo transplantation with HCV is inferior to that of
patients who undergo transplantation for other indications (Thuluvath PJ, et al 2007).
Rapid recurrence and graft loss due to fibrosing cholestatic hepatitis occur in 1%–10%
of patients, and most of these individuals die within 1 year (Gane E. et al 2003). In
those without fibrosing cholestatic hepatitis, 20%–40% develop cirrhosis in 5 years.
(Wiesner RH, et al 2003) and decompensation occurs in 40% of these cirrhotic patients
within 1 year, and 50% of them die in nearly one year (Wiesner RH, et al 2003,
Berenguer M, et al 2000). Patients can be treated for HCV after liver transplantation, but
SVR rates are low (10%–30%) because most have previously been treated
unsuccessfully and cytopenias prevent achieving optimal doses in most cases.
60 Introduction
Hepatitis B.
Chronic hepatitis B accounts for about 5% of liver transplants. It is advisable to treat
such patients while they await transplantation to reduce their viral load and risk of
disease recurrence posttransplantation (antigenemia and liver disease). In all viremic
(>300 copies/mL) patients awaiting liver transplantation for HBV-related liver damage,
efficient antiviral therapy is required. Suppression of HBV DNA may lead to clinical
stabilization resulting in removal from the waiting list or in postponing liver transplant.
Monitoring for HBV DNA at least every three months is recommended. A major concern
of long-term lamivudine (LAM) therapy is the emergence of mutations in the YMDD
motif of the DNA polymerase (Cornberg M, et al 2008, Tillmann HL. 2007, Trotter JF.
2003, Beckebaum S (a), et al 2008, Ono SK, et al 2001, Mutimer D, et al 2000). LAM
has been proposed to be downgraded from first-line to second-line therapy (Seehofer D,
et al 2001). In patients with end stage liver disease, a more potent nucleos(t)ide
analogue (entecavir [ETV] or tenofovir [TDF]) is preferred because development of
resistance to LAM could result in clinical decompensation. In patients with LAM
resistance, recent results have shown that adefovir dipivoxil (ADV) and LAM
combination therapy is superior to ADV monotherapy (Tan J, et al 2007, Vassiliadis T,
et al 2007). The introduction of high-dose intravenous hepatitis B immunoglobulin
reduced the chance of recurrence and allowed transplantation to be performed with
excellent long-term survival (Lampertico P, et al 2007). Nucleos(t)ide analogues
decrease the viral load before transplantation and nearly eliminate the chance of
chronic hepatitis B posttransplantation, particularly when used in combination with
hepatitis B immunoglobulin(Samuel D, et al 1991). Most centers use hepatitis B
immunoglobulin indefinitely with a nucleoside analogue.
Alcohol.
Alcoholic liver disease alone accounts for 10%–12% of liver transplants and also
contributes to more rapid progression of other causes of liver disease to cirrhosis and
hepatic failure, particularly hepatitis C (Markowitz P, et al 1998). All patients must stop
drinking all alcohol at least 6 months before they can be listed for a liver transplant.
Liver function can improve significantly in some patients to a point where transplantation
is no longer required patients who undergo transplantation for alcohol induced cirrhosis
have a 19%–33% risk of recidivism after liver transplantation (Corrao G, et al 1998,
Osorio RW, et al 1994). Appropriately selected patients with alcohol-induced cirrhosis
have excellent survival posttransplantation. In patients who experience a relapse, the
61 Introduction
pattern of drinking posttransplantation is variable, but even patients who return to
occasional drinking rarely experience graft loss (DiMartini A, et al 2006). A minority of
patients returns to abusive drinking, and this can result in graft loss and decreased
survival (Pfitzmann R, et al 2007).
Cryptogenic Cirrhosis and Nonalcoholic Steatohepatitis.
Cryptogenic cirrhosis accounts for about 9%–10% of liver transplants. Cryptogenic
cirrhosis represents a diverse group of patients with liver disease resulting from a
previous hepatic insult such as drug injury, alcohol use, resolved viral infection,
autoimmune disease, or more commonly, nonalcoholic steatohepatitis (Caldwell SH et
al 1999, Poonawala A et al 2000) is the predominant cause of cryptogenic cirrhosis.
Due high prevalence of obesity, insulin resistance or diabetes, hyperlipidemia, and other
manifestations of the metabolic syndrome among these patients. It is often difficult to
attribute cryptogenic cirrhosis to nonalcoholic steatohepatitis with certainty because fat
may decrease or completely disappear as cirrhosis ensues (Adams LA, et al. 2005).
The reasons for this are not clear but might relate to weight loss due to hepatic
decompensation, the catabolic state associated with cirrhosis, or metabolic changes in
hepatocytes related to a decrease in portal blood flow. The risk of hepatic
decompensation among patients with nonalcoholic steatohepatitis–induced cirrhosis is
as high as 40%–60% within 5–7 years in some highly selected groups (Adams LA, et al.
2005; Teli MR, et al. 1995) these patients should be considered for transplantation early
with particular evaluation because they have an increased risk of death from
cardiovascular and cancer related diseases (Kim WR, et al. 1996). Nonalcoholic
steatohepatitis recurrence is uncommon after liver transplantation, and graft loss is rare.
Cholestatic Liver Diseases.
Primary biliary cirrhosis, primary sclerosing cholangitis (PSC), and secondary biliary
cirrhosis are the most common causes of chronic cholestatic liver disease in adults.
Primary biliary cirrhosis accounts for about 5% of liver transplants, and is gradually
decreasing (Kim WR, et al. 2007; Angulo P, et al. 2002). The course of the disease is
reasonably well predicted by a mathematical model using common laboratory tests,
permitting the determination of time for evaluation for transplantation (Murtaugh PA, et
al 1994). Liver transplantation should be considered when the predicted 2-year survival
begins to decline, and this usually coincides with the onset of hyperbilirubinemia or
other manifestations of hepatic decompensation. Ursodeoxycholic acid delays
62 Introduction
progression of the disease (Poupon RE, et al. 1991) but it is ineffective once fibrosis
develops.
A simple method for calculating the prognosis (Kim WR, et al 1998)
Age: 38 years 0 points, 38–62 years 1 point, ¼ / . 63 years 2 points
Bilirubin (mg/dL):.1 0 points; 1–1.6 1 point, 1.7–6.4 2 points, >6.4 3 points.
Albumin (g/dL): .4.1 0 points, 2.8–4.0 1 point, <2.8 2 points.
Prothrombin time: Normal 0 points, prolonged 1 point.
Oedema: Absent 0 points, present 1 point.
A score of 6 correlates with the minimal listing criteria for transplantation.
PSC accounts for about 5% of all transplants. Although some patients with PSC
undergo transplantation for hepatic decompensation, it is more common for them to
develop dominant strictures with jaundice or recurrent biliary sepsis. Many become
dependent on endoscopic or percutaneous biliary drains. Because these patients may
not accumulate many MELD points, some regional review boards grant exceptions and
extra points when patients experience recurrent biliary sepsis or tube dependence. All
patients with PSC should receive a choledochojejunostomy at the time of liver
transplantation. The incidence of recurrent PSC posttransplantation is debated but may
occur in 15%–20% of patients (Jeyarajah DR, et al. 1998; Graziadei IW, et al. 1999).
There is a strong association between PSC and inflammatory bowel disease (LaRusso
NF, et al. 2006). All patients with PSC should undergo a colonoscopy with biopsies
regardless of age or symptoms to determine if they have inflammatory bowel disease.
Indications for liver transplantation in PSC patients
Jaundice which cannot be alleviated endoscopically (dilatation and/orstenting)
Jaundice which cannot be treated medically (e.g. steroids in ‘overlap’syndromes
Severe, recurrent cholangitis
Cirrhosis with reduced liver function and/or symptoms of this
HCC is demonstrated or CC is suspected
As PSC patients run a high risk of developing hepatobiliary malignancies, this should
always be considered when evaluating a PSC patient for Ltx. Patients with PSC and
HCC should be handled according to general guidelines. Liver resection for a HCC is
rarely considered in a PSC patient with advanced liver disease as previous
hepatobiliary surgery is a poor prognostic factor in PSC patients receiving a liver
allograft (Brandsæter B, et al. 2003). The problems of identifying PSC patients with a
63 Introduction
particular risk of developing Cholangiocarcinoma (CC) are far more complicated. No
adequate biochemical tumour markers exist. The tumour marker CA-19-9 in
combination with carcinoembryonic antigen (CEA) was claimed to be of value in
identifying PSC patients with CC (Ramage JK, et al 1995) but CA-19-9 has a low
sensitivity and a low specificity in diagnosing CC, (Fisher A, et al. 1995). The finding of
(CC) has traditionally been considered as a contraindication to Ltx. The experience with
(CC) in PSC patients undergoing Ltx, is limited. And exact guidelines for management
do not exist. Data from two studies indicate, however, that the results of Ltx in highly
selected PSC patients with (CC) are satisfactory if the patients are offered
chemotherapy and local radiation therapy prior to Ltx (De Vreede J, et al 2000; Sudan
D, et al. 2002 ). The diagnosis of (CC) can be extremely difficult and PSC patients are
often evaluated for Ltx with a present suspicion of (CC). They are accepted for Ltx while
the final decision to perform a transplantation is taken during the laparotomy when a
thorough examination of the abdominal cavity for possible extrahepatic spread of (CC)
is done. Patients where a (CC) is found per- or postoperatively and in whom no signs of
extrahepatic growth is detected, seem to have a fair prognosis (Brandsæter B, et al.
2004) which should justify transplantation. If extrahepatic growth of (CC) is found during
laparotomy the transplantation should be stopped and the patient offered palliative
therapy. The value of routine brush cytology of the larger biliary ducts for early
diagnosis of CC has not been estimated. In some cases, however, patients with PSC
have been found to have severe dysplasia in cytological smears obtained by brush
cytology during endoscopic retrograde cholangiography (ERC). These patients should
be considered as running a very high risk for (CC) development and should be
evaluated for Ltx (Boberg KM et al 2003) No consensus exists as to what imaging
modality should be preferred in order to detect a possible (CC). Positron emission
tomography (PET-scanning) has been claimed to be of value in diagnosing (CC)
(Keiding S, et al. 1998). Patients with PSC and inflammatory bowel disease are at
increased risk for colon cancer compared with patients with inflammatory bowel disease
(IBD) alone. When assessing a PSC patient for Ltx, a colonoscopy should be
performed. It is important both to diagnose and to characterise the activity of a possible
coexisting (IBD). Patients with severe ulcerative colitis (UC) or mucosal dysplasia
should be considered for colectomy though particular attention should be paid to the risk
of possible severe and life-threatening decompensation of the liver function following
colectomy (Post AB, et al. 1994). Patients previously treated surgically for colorectal
64 Introduction
cancer should be very thoroughly assessed for both recurrent malignancy and for
hepatobiliary cancer (Brandsæter B, et al. 2004). Secondary biliary cirrhosis is most
often caused by a previous surgical procedure. However, pathologic obstruction of the
biliary system by stones, malignancy, cysts, or parasites must be considered.
Unfortunately, timely biliary decompression often fails to prevent progression of liver
injury in these patients, who then may come to liver transplantation.
Malignancy.
The incidence of HCC is rising, the majority of cases is attributable to HCV. LT is
considered an optimal strategy that addresses both the underlying disease Liver
transplantation is restricted to patients who have limited tumor burden as defined by the
Milan criteria: a single tumor <or=5 cm or up to 3 tumors with none exceeding 3 cm and
no evidence of macrovascular invasion (portal vein thrombosis) or metastasis
(Mazzafero V, et al 1996). MELD priority is given to patients with stage T2 HCC who
meet the Milan criteria these patients reach transplantation without tumor progression
(Sharma P, et al. 2004) and recurrence is extremely low with a 5-year survival rate of
71%–75% (Jonas S, et al. 2001). Many centers have proposed expanding tumor size
limits with a modest extension of criteria (Duffy JP, et al. 2007; Onaca N, et al. 2007).
Downsizing tumors by radiofrequency ablation, chemoembolization, bland embolizition,
percutaneous alcohol ablation, radiotherapy, or other means it is not known whether
downsizing sufficiently changes the biology of the tumor and therefore the likelihood of
recurrence (Schwartz M, 2004) long waiting times for transplantation, with disease
progression may lead to Waiting-list drop-out or death while on the waiting list. Waitinglist drop-out rates may be reduced by the application of bridging therapies such as
transarterial chemoembolization or radiofrequency ablation (Roayaie, 2007). Under
MELD allocation, patients meeting the Milan criteria qualify for exceptional HCC waiting
list consideration by being allotted extra points which used to be as follow: patients with
a single lesion <2 cm: 20 points : patients with a single lesion 2-5 cm or ≤ 3 lesions
which are not greater than 3 cm : 24 points and for every 3 months on the waiting list :
10% additional . Now only T2 patients are awarded 22 extra points. Following the
upgrading of MELD score for HCC patients, the number of DDLT performed for HCC
have increased and their waiting list period significantly reduced (2.3 to 0.7 years)
(Freeman RB, et al. 2004) .The assesent should include CT scan or MRI of the
abdomen and chest and a bone scan. Tri-monthly routine follow-up examinations (MRI
65 Introduction
or CT scan) of wait-listed HCC patients for early detection of disease progression are
required. Accurate discrimination of HCC patients with good and poor prognosis by
appropriate criteria (genomic or molecular strategies) is highly warranted and still in the
exploratory phases (Marsh 2003, Finkelstein 2003). Patients may be registered at a
MELD score equivalent to a 15% probability of pre-transplant death within 3 months.
Patients will receive additional MELD points equivalent to a 10% increase in pretransplant mortality to be assigned every 3 months until these patients receive a
transplant or become delisted due to HCC progression. In patients with alcohol-related
liver disease and HCC, a multidisciplinary approach and thorough work-up of both the
alcoholic
and
oncologic
problem
is
mandatory
(sotiropoulos
2008a).
Cholangiocarcinoma is an aggressive malignancy with a poor prognosis. Most cases
are associated with PSC (Kahn SA, et al. 2002). Results following transplantation for
cholangiocarcinoma have been disappointing due to early recurrence in half of patients
and 5-year survival of only 23% (Meyer C, et al. 2000). The Mayo Clinic found that
highly selected patients with cholangiocarcinoma who are treated with an aggressive
protocol of radiotherapy and chemotherapy pretransplantation can achieve excellent
survival posttransplantation (Hassoun Z, et al. 2002).
Groups in Whom HCC Screening and Surveillance Is Recommended.
Hepatitis B carriers (HBsAg positive)
Asian males >40 y
Asian females >50 y
All cirrhotic hepatitis B carriers
Family history of HCC
Africans over age 20 y
Nonhepatitis B cirrhosis
Hepatitis C
Alcoholic cirrhosis
Genetic hemochromatosis
Primary biliary cirrhosis
Possibly: alpha1-antitrypsin deficiency, nonalcoholic steatohepatitis,
autoimmune hepatitis
66 Introduction
University of California, San Francisco [UCSF] criteria:
solitary tumor < or = 6.5 cm, or three or fewer nodules with the largest lesion < or = 4.5
cm and total tumor diameter < or = 8 cm, without gross vascular invasion
German guidelines for organ transplantation diagnosis of HCC is confirmed if the
following criteria are given: (Bundesärztekammer 2008):
(1) liver biopsy-proven or (2) AFP >400 ng/ml and hypervascular liver lesion
detectable in one imaging technique (magnetic resonance imaging (MRI), spiral
computed tomography (CT), angiography) or (3) hypervascular liver lesion detectable
in 2 different imaging techniques.
Summary of Therapeutic Modalities for HCC and Their Outcomes
Treatment
Survival
Special issues
Surgical resection
1 y: 97%
3 y: 84%
5 y: 26%–57%
Choice of therapy for patients without cirrhosis (low
morbidity)
5%–15% of HCC patients eligible
Right hepatectomy has higher risk than left
hepatectomy
Pre/postresection adjunct therapy not recommended
Transplantation (LT)
1 y: 91%
2 y: 75%
5 y (MILAN): >70%
5 y (extended): 50%
Curative treatment for chronic disease and HCC
MELD exception points for HCC
Effective corresponding to UNOS criteria (1 tumor
=/<5 cm; up
to 3 tumors <3 cm
Live donor LT considered for HCC progression
outside MILAN criteria
UCSF criteria not implemented in current MELD
exception allocation policy
Radiofrequency
ablation (RFA)
1 y: 90%
3 y: 74%
5 y: 40%–50%
Effect is more predictable in all tumor sizes than
following PEI
Superior to PEI in larger tumors; equivalent in small
tumors
Requires fewer treatment sessions
Percutaneous
ethanol injection
(PEI)
1 y: 85%
3 y: 50%
5 y: 40%–50%
Early HCC patients not suitable to resection or OLT
or RFA not
available or contraindicated
Highly effective for small HCC (_2 cm)
Low rate of AEs
Transarterial
chemoembolization
(TACE)
1 y: 82%
2 y: 63%
Nonsurgical patients with large/multifocal HCC w/o
vascular
invasion or extrahepatic spread
AE, adverse events; OLT, orthotopic liver transplantation.
67 Introduction
Fulminant Liver Failure
Fulminant hepatic failure accounts for less than 5% of liver transplants. Acetaminophen
hepatotoxicity is the most common cause approximately65% of patients survive
acetaminophen overdose without transplantation when appropriate treatment is
provided early (Larson AM, et al. 2005). Idiosyncratic reactions to other medications are
less common but have a worse prognosis. Other causes of fulminant hepatic failure
include acute hepatitis A or B, Wilson’s disease, autoimmune hepatitis, herbal
supplement toxicity, cardiogenic liver failure, Budd–Chiari syndrome, pregnancy related
complications, diffuse liver metastases, and unknown causes (Khan SA, 2006b et al.
2006). Transplant evaluation should be initiated immediately. Several scoring systems
have also been developed to predict outcome, including King’s College criteria, Clichy
criteria, and the MELD score. King’s College criteria utilize different parameters
depending on the etiology of liverfailure (acetaminophen vs nonacetaminophen
(O’Grady JG, et al. 1989). The Clichy criteria utilize the grade of hepatic
encephalopathy and serum factor V activity, with transplantation recommended for
patients with grade 3 or 4 encephalopathy or factor V levels less than 20% of normal
(Bemuan J, et al. 1986). Recently, a MELD score of more than 35 was also found to be
an excellent predictor of mortality in adults with non–acetaminophen-induced fulminant
hepatic failure (Katoonizadeh A, et al. 2007). The King's College Criteria identify two
groups of patients that have a poor prognosis with acetaminophen induced liver failure:
Arterial pH < 7.3 (taken by sampling of blood from an artery). All three of an (INR) of
greater than 6.5, serum creatinine of greater than 300 micromoles per litre and the
presence of encephalopathy (of grade III or IV). In patients with non-acetaminophen
acute liver failure, the following criteria were identified as being associated with a poor
prognosis. INR greater than 6.5; or, three of the following five criteria: patient age of less
than 11 or greater than 40; Serum creatinine of greater than 300 micromoles per litre;
Time from onset of jaundice to the development of coma of greater than seven days;
INR greater than 3.5; or, Drug toxicity, regardless of whether it was the cause of the
acute liver failure. Clichy’s criteria indicate a poor prognosis when hepatic
encephalopathy grade3 or 4 is associated with factor V. concentrations <20% for
patients aged <30 yr or <30% for those older than 30 yr (Bernuau J, et al. 1986.
Bernuau J, et al. 1991). Other parameters that have been reported to predict outcome
include rising alpha-fetoprotein, blood lactate, and phosphorus levels, but these have
not replaced current criteria (Schiødt FV, et al. 2006; Bernal W, et al. 2002a; Bernal W,
68 Introduction
2003b). In practice, both these standardized criteria and good clinical judgment are
required Overall survival after liver transplantation is 40%–90%, depending on the
etiology (O’Grady JG, 2005). Early referral to a liver transplantation center is essential
because (1) it is difficult to predict which patients will recover spontaneously; (2)
deterioration can occur suddenly; (3) the chance of receiving a liver transplant increases
with early placement on the waiting list; and (4) once brainstem herniation has occurred,
patients are not salvageable by any means, including liver transplantation.
Hepatic support.
Because of a severe shortage of human donors, many patients with acute liver failure
die while waiting for a suitable donor organ. Patients should be referred to centers that
cannot only perform liver transplantation, but can support such patients until a donor
liver becomes available. In addition to standard medical support, several strategies are
being developed for temporary hepatic support, including artificial liver support devices,
bioartificial livers, hepatocyte transplantation, and extracorporeal liver perfusion.
Artificial liver support systems.
Although some studies have suggested that charcoal hemoperfusion systems have a
survival advantage for certain causes of fulminant hepatic failure, most patients do not
seem to benefit (O’Grady JG, et al. 1988). Other artificial liver support systems have
included dialysis-like systems coupled with absorbent; a technology, where dialysis fluid
containing charcoal and a cation exchange resin is used to bind toxic substances in the
blood. A pilot study showed that this system was well tolerated and could produce
biochemical improvements in acute liver failure, but did not retard the progression to
terminal brain swelling. In the molecular absorbents recirculating system, the dialysis
system contains albumin impregnated in the polysulphone membrane and has a
dialysate enriched with albumin to facilitate removal of toxic metabolites (Stange J, et al.
1998). In the microsphere-based detoxification system, plasma is recirculated at high
flow rates with the entire flow exposed to particle-size absorbents that provide a large
surface area for absorption (Weber C, et al. 1994).
Bioartificial liver support systems.
Another approach consists of a bioartificial liver, wherein plasma, obtained by a
centrifugal plasma separator, is perfused through microcarrier-bound porcine
hepatocytes (Rozga J, et al. 1994). In the extracorporeal liver assist device, blood is
perfused through hollow fiber membranes surrounded by cell of a human tumor
hepatocyte line (Sussman NL, et al. 1994).
69 Introduction
Hepatocyte transplantation.
More recently, hepatocyte transplantation has been successfully used to treat certain
metabolic disorders, even when associated with acute liver failure (Fox IJ, et al. 1993).
The number of cryopreserved hepatocytes required to achieve success, however, may
limit the use of this approach.
Extracorporeal liver perfusion.
Extracorporeal liver perfusion overcomes the following problems associated with the
previous approaches: inability to support all the hepatic functions, and inability to
provide sufficient hepatic support to overcome the derangement from fulminant hepatic
failure. In extracorporeal liver perfusion, a centrifugal pump and tissue oxygenator
pumps blood derived from the femoral vein through an extracorporeal circuit, containing
human or porcine livers, and then returns the blood to the patient through the jugular or
axillary vein. This approach has successfully provided biochemical and neurologic
improvement in patients, and can successfully provide a bridge to liver transplantation
(Chari RS, 1994). The limiting factor with porcine livers is vascular rejection, which
occurs 2 to 4 hours after initiating perfusion, because of preformed human antibodies to
porcine endothelium. Because of a severe donor organ shortage, strategies have been
developed
to
overcome
the
early
rejection
associated
with
pig-to-primate
xenotransplantation (Harland RC, et al. 1995) development of pigs that are transgenic
for human complement regulatory proteins. In this situation, complement is not activated
in pig endothelium and early rejection may be avoided. Transplantation of kidneys from
transgenic pigs to nonhuman primates extends graft survival from hours to weeks when
compared with kidneys from nontransgenic pigs.
Metabolic Diseases.
Metabolic diseases are responsible for less than 5% of liver transplants in adults.
Metabolic disorders that may present with chronic liver disease in adulthood and require
transplantation include genetic hemochromatosis, alpha 1-antitrypsin deficiency,
Wilson’s disease. Genetic or hereditary hemochromatosis accounts for less than 1% of
liver transplants. The outcome after transplantation is poor, with 1- and 5-year survival
rates of 64% and 34%, respectively (Kilpe V, et al. 1993; Kowdley KV, et al. 2005), due
to sepsis from uncommon pathogens and cardiac complications (Brandhagen DJ, 2000;
Brandhagen D, 2001). Patients with genetic hemochromatosis survival shows some
improvements due to better disease understanding, selection of transplant candidates,
and adequate phlebotomy before transplantation, alpha1-Antitrypsin defficiency with the
70 Introduction
PiZZ phenotype 10% develop severe liver disease with portal hypertension and
cirrhosis (Sverger T, et al. 1995). Heterozygous states may also be a factor that
predisposes to more rapid progression of other forms of liver disease (Bowlus CL, et al.
2005). Alpha1-Antitrypsin deficiency accounts for 1.6% of adult liver transplants. One
and 5-year survival rates are near that observed for other indications (Vennarecci G, et
al. 1996). The recipient assumes the alpha 1-antitrypsin phenotype of the organ donor
and produces normal alpha 1-antitrypsin levels within weeks of transplantation.
(Filipponi F, et al. 1994). Whether this phenotype recovery has any impact on
pulmonary function is still not known. Wilson’s disease is transmitted as an autosomal
recessive defect that is associated with abnormal copper transport mechanisms that
result in intrahepatic copper accumulation leading to progressive liver injury (Zhang K,
et al. 2007). Increased copper deposition also occurs in other organs, including the
brain, causing an array of neuropsychiatric manifestations, and the cornea, resulting in
Kayser–Fleischer rings. Hepatic manifestations of Wilson’s disease include chronic
hepatitis, cirrhosis, or fulminant hepatic failure. (Zhang K, et al. 2007). The diagnosis is
often difficult because the disease can have such a varied presentation and laboratory
results can be misleading. The hallmarks of the diagnosis are low serum ceruloplasmin
level, a copper level in urine greater than 100 micro g/24 h, the presence of Kayser–
Fleischer rings detected by slit lamp examination, and an elevated hepatic copper level
>250 micro g/g dry wt (Ala A, et al. 2004). Prompt diagnosis allows for the initiation of
chelation therapy that may prevent further tissue injury (Schilsky ML, et al. 2001). Liver
transplantation offers a cure of the metabolic defect. Long-term survival after
transplantation is excellent, although survival is lower in patients with neurologic
manifestations of their disease (Medici V, et al. 2005). Thus, liver transplantation in
patients with isolated or advanced neurologic deficits remains controversial (Geissler I,
et al 2003; Marin C, et al. 2007). Although uncommon, patients with primary oxaluria,
familial amyloidosis, Crigler–Najar syndrome, and urea cycle defects can benefit from
liver transplantation. Many of these cases present during childhood.
Pediatric Indications.
The major reasons for liver transplantation in children are biliary atresia, cholestatic liver
disease, metabolic disorders, malignancies including hepatoblastoma, and fulminant
hepatic failure. Biliary atresia is the most common indication for liver transplantation in
children and accounts for 41% of cases, of which about 25% occur in infants younger
than 12 months (McDiarmid SV, et al. 2004), most patients undergo a Kasai
71 Introduction
portoenterostomy, to restore bile flow to prevent or delay the need for liver
transplantation for many years. Survival after liver transplantation for patients with biliary
atresia is over 80% (Fouquet V, et al. 2005). Cholestatic disorders, including Alagille
syndrome and sclerosing cholangitis, are the second most common indications for
transplantation in children (McDiarmid SV, et al. 2004). The most common metabolic
indication for transplantation is alpha 1-antitrypsin deficiency (Tiao G, et al. 2005).
Cholestasis during infancy is the most common presentation, which resolves
spontaneously by 6 months of age but some infants develop bile duct paucity with
prolonged jaundice and cirrhosis (Ibarguen E, et al. 1990). Less common metabolic
indications include urea cycle enzyme deficiencies, glycogen storage disease, Wilson’s
disease, tyrosemia, and primary hyperoxaluria. Children with cystic fibrosis develop
thick biliary secretions that plug the bile ducts, causing biliary obstruction, stasis
choledocholithiasis, and occasionally sclerosing cholangitis. Secondary biliary cirrhosis
leads to chronic cholestasis, loss of liver function, and portal hypertension. Infants with
cystic fibrosis undergoing liver transplantation are a challenge because they often have
advanced nutritional deficiencies due to exocrine pancreatic deficiency and/or shortbowel syndrome. Some have abnormal pulmonary function tests and are often
colonized with Aspergillus (Molmenti E, et al. 2003). Children primary hepatic
malignancies in which liver transplantation Hepatoblastoma treated with surgical
resection and adjuvant chemotherapy before and after transplantation has achieved the
best long-term survival rates. (Tiao G, et al. 2005) HCC is less common in children.
Liver transplantation in children is challenged by a limited donor pool of appropriate
size. Children can receive either a size-matched organ from a deceased or living donor
or an organ from an adult that has been reduced in size or split. This improvement in
organ allocation did not affect transplant outcomes
Retransplantation.
Early graft loss due to primary nonfunction of the graft or hepatic artery thrombosis
accounts for 70% of graft loss during the first year and need early retransplantation. (US
Scientific Registry (UNOS). 2007; Burton J, et al. 2006; Testa G, et al. 2000). Late graft
loss due to disease recurrence or chronic rejection, late retransplantation is
controversial. Primary liver diseases at risk for recurrence include hepatitis B and C,
autoimmune hepatitis, primary biliary cirrhosis, and PSC. Recurrent hepatitis C is the
most common. Late retransplantation is controversial, particularly for patients with
recurrent hepatitis C. Survival following retransplantation is inferior to that for primary
72 Introduction
grafts, with 3-year survival of only 40%–50%. (Testa G, et al. 2000; McCashland T, et
al. 2007; Markmann JF, et al. 1997).
1.7 THE DONOR
Donor characteristics significantly impact liver transplantation outcomes. With the
increasing success of liver transplantation, maturation of the hepatitis C virus (HCV)
epidemic, and broadening of the indications for liver transplantation to include
previously contraindicated diagnoses, the demand for liver grafts has far outpaced the
supply and the donor pool will never be sufficient for the demand. Liver transplant
community has tried to expand the criteria that define what grafts are acceptable for
transplantation while recognizing that these broader criteria also often confer additional
risks to recipients in comparison with a reference donor.
1.7.1 Brain Death (BD)
Is defined as the irreversible loss of brain and brain stem function, usually caused by
major hemorrhage, hypoxia, or metabolic dysregulation (Wijdicks EFM, et al. 2010).
Brain death (BD) and its consequences are related to impaired graft quality and
accelerated immunogenicity resulting in compromised patient and graft survival. (BD)
diagnosis is based on a comprehensive neurologic assessment with the absence of
brain stem reflexes and apnea under standardized conditions (blood alcohol content
below 0.08%, core temperature N36°C, systolic blood pressure N100 mm Hg, and
exclusion
of
central
nervous
system
depressant
drugs).
Other
tests:
electroencephalography, transcranial Duplex-ultrasound, or cerebral angiography, can
be performed to confirm the diagnosis. Brain death consequences include
hemodynamic, hormonal, metabolic, and inflammatory changes, affecting the quality
and immune activation of potential donor organs. Hemodynamic changes initiate by an
immediate short period of hypertension and bradycardia, followed by a prompt release
of catecholamines and hyperdynamic cardiovascular responses. This “catecholamine
storm” and facilitated increased cardiac output based on an elevated contractility and
heart rate, despite increased systemic and pulmonary vascular resistance. Increased
cardiac indices and enhanced tissue oxygenation have been registered in brain dead
patients (Belzberg H, et al. 2007). Later, after BD, a decline of serum catecholamine
levels and peripheral vascular resistance is observed, which finally results into a
cardiovascular collapse compounded by hypovolemia (Chen EP, et al. 1996; Novitzky
73 Introduction
D, et al. 1984). As a result of deteriorating hemodynamics, a compromised perfusion,
particularly of abdominal organs, takes place (Herijgers P, et al. 1996). Consequently, a
shift from aerobic to anaerobic metabolism and acidosis is evident which is detected
clinically by elevated serum levels of lactate and free fatty acids and promoted by
decreasing insulin secretion and hyperglycemia (Herijgers P, et al 1996, Novitzky D,
Cooper DK, Morrell D, et al 1988). Thus, initial hallmarks of BD include hemodynamic
instability with compromised organ perfusion and reduced oxygenation. Hormonal
changes due to Brain death are due to the cessation of the hypothalamicpituitary axis
effects systemic hormone regulation. Vasopressin secretion decreases to undetectable
levels and overt diabetes insipidus becoming evident, aggravating the decline of
hemodynamic stability with increasing hypovolemia furthermore (Herijgers P, et al.
1996; Chen JM, et al. 1999). At the same time, levels of thyroid and
adrenocorticotrophic hormones decline, contributing to an accelerated acidosis and an
augmented hemodynamic instability, which demands an increased need for inotropic
support (Herijgers P, et al. 1996; Novitzky D, et al. 1988; Smith M. 2004; Cooper DK, et
al. 2009). Decreased cortisol levels, at the same time, promote an inflammatory and
immunologic activation. Inflammatory events after BD is a result of the activation and
interaction of vascular endothelium, complement, and the coagulation system as well as
components of the innate and adaptive immune response. Small animals studies, has
shown that BD causes a systemic release of proinflammatory cytokines, including
interleukin (IL) 1, IL-6, tumor necrosis factor α, and IFNγ. HLA molecules become upregulated and increase the immunogenicity of donor organs in which lymphocytes,
macrophages, and neutrophils are accumulating. BD can be aggravated by hypotension
but are also evident under conditions of normotension (Van der Hoeven JA, et al. 1999;
Bittner HB, et al. 1996). Circulating macrophage-associated activation factors and other
serum components may play an important role in communicating systemic inflammatory
events (Miñambres E, et al. 2003; Takada M, et al.1998). Moreover, inflammatory
activation triggered by BD results inaccelerated rejection rates, and brain dead donor
organs provoked a distinct immune response reflected by cellular infiltrates,
inflammatory cytokines, chemokines, and adhesion molecules expressed on both
leukocytes and endothelial cells (Wilhelm MJ, et al. 2000; Pratschke J, et al. 2000). Of
note, consequences of hypotension and BD may overlap, as hypotension per se has
been shown to increase IL-6 serum levels and CD11b expression in small animal
models (Okamoto S, et al. 2000). Finally, BD induces apoptosis in solid organs, a
74 Introduction
mechanism that may link the inflammatory response to an accelerated immunogenicity
(Birks EJ, et al. 2000; Van Der Hoeven JA, et al. 2003; Koudstaal LG, et al. 2008).
Endothelial activation may represent an initial step in the inflammatory cascade after BD
(Van der Hoeven JA, 1999; Kusaka M, et al. 2000; Wilhelm MJ, et al. 2000). The
relevance of a time-dependent up-regulation of E- and P-selectin has been shown in rat
and canine models (Morariu AM, et al. 2008; Schuurs TA, Morariu AM, 2006; Szabo G,
et al. 2002) endothelial activation may also be related to cardiac dysfunction as shown
by a decreasing coronary flow in hearts of brain dead dogs Szabo G, et al. 2002). In
experimental models, an increased VCAM-1 synthesis has been associated with a more
severe intimal hyperplasia in the allogeneic setting, suggesting strong relation between
alloimmunity and consequences of BD (Zweers N, et al. 2004). Neuronal damage is a
result of the acute release of proinflammatory S-100 proteins, myeloid-related proteins 8
and 14, and proinflammatory cytokines (IL-6, IL-8, and MCP-1), which trigger an
endothelial activation (Kusaka M, 2007; Viemann D, et al. 2005). Ligated to
parenchymal cell receptors, elevated IL-6 serum levels communicate a rapid activation
of several signal transduction pathways through the phosphorylation of MAP kinases.
Downstream, p53, and NFκB pathways are being activated and lead to the expression
of a proinflammatory gene profile, which, in turn, triggers cellular graft infiltration (van
der Hoeven JA, et al. 2000; Bouma HR, et al. 2009). The coagulation system are getting
activated after BD. The important role of endothelial activation is evident as reflected by
treatment success of targeting selectin ligands (Takada M, et al. 1998). Complement
activation participates in the pathogenesis of organ injury, and increased C3a serum
levels have been reported in a mouse brain dead model. Extensive complement
deposition was also found in hearts shortly after the induction of BD. Complement
deposition explains the presence of myocardial damage and higher cardiac troponin I
serum levels but and has another role for the expression of P-selectin, VCAM-1, and
ICAM-1 after BD. The critical role of complement is supported by a reduced endothelial
activation in C3 knockout mice (Atkinson C, et al. 2009).
Clinical Relevance In Liver Transplantation
More pronounced inflammatory changes are observed in livers originating from BD
donors. CD3+ lymphocytes and macrophages/monocytes infiltrate livers from brain
dead donors in high numbers, whereas a more pronounced ICAM-1 expression was
observed. Clinical events after BD included more frequent acute rejection episodes and
75 Introduction
increased lymphocytic infiltrates (Jassem W, et al. 2003). Brain death results in an
increased serum levels of inflammatory cytokines and increased transaminases with
more frequent acute rejection episodes and higher rates of primary nonfunction after
liver transplantation (Kuecuek O, et al. 2005; Weiss S, et al. 2007). Recently, DCD
donor organs have also been used more frequently in liver transplantation. Although the
increased sensitivity of the liver to prolonged warm ischemia is recognized (Waki K,
2006), clinical studies with adjusted DCD populations (controlled Maastricht III criteria
donors) demonstrated comparable graft and patient survival of DCD and BD livers (Lee
KW, et al. 2006; Dubbeld J, et al. 2010). Biliary complications are significantly more
frequent in DCD livers underlining the high vulnerability of the biliary system to
hemodynamic instability and ischemic injury (Dubbeld J, et al. 2010).
1.7.2 Strategies And Techniques To Expand The Donor Organ Pool
Use of donor livers with extended criteria, use of steatotic donor organs, use of HCVpositive donor organs for HCV-positive recipients, use of high-risk CDC donor organs,
donation
after
cardiac
death,
Split
liver
transplantation,
Living
donor
liver
transplantation, Domino liver transplantation.
Extended Donor Criteria
The hypothesis supporting EDC utilization is that the benefit of earlier access to
transplantation afforded by an EDC allograft outweighs the combined risk associated
with the specific allograft and the risk of additional waiting for LTX. Extended-donor
criteria liver allografts do not meet traditional criteria for ideal or reference donor for
transplantation i.e. age below 40 years, trauma as the cause of death, hemodynamically
stable, no steatosis, and no transmittable disease (Feng S, et al. 2006; Durand F, et al.
2008).
Definition for ECD
By the suggestion of Chung et al includes age > 65 years, macrovesicularsteatosis >
40%, serum sodium > 155 mmol/L, positive serological data, extrahepatic carcinoma,
DCD, and split-graft liver transplantation (Chung et al. 2010). In a large retrospective
cohort study of 1153 OLT (Cameron et al. 2006), the ECD included donor age over 55
years, donor hospital stay >5 days, CIT >10 hours and warm ischemia time (WIT) > 40
minutes. In this report, the addition of several factors showed progressive impacts on
graft failure and 1-year mortality rates; there was an important significance when three
76 Introduction
or more factors were present. This study also showed that advanced age and urgent
recipient status worsened the outcome for each donor risk score.
Marginal Donors
Marginal donors were considered by Pokorny et al as follows (Pokorny et al. 2005):
older than 60 years, a prolonged intensive care unit (ICU) stay > 4 days with ventilatory
support, a prolonged CIT >10 hours, a high vasopressor support (high-dose dopamine
or any other vasoactive amines), a donor peak serum sodium > 155 mEq/L, a donor
serum creatinine >1.2 mg/100 mL and BMI > 30. When patients had more than three
cumulative marginal donor criteria, the rate of PGNF was 36% (Pokorny et al., 2005). In
a study from the Spanish registry of 5150 OLT, other donor factors were significantly
related to graft survival: donor age >55 years, body mass index >25, stroke as a cause
of death, use of inotropes, intensive care unit stay >6 days, bicarbonate level >18
mEq/L, increased liver enzymes, and history of treated hypertension>3 years. (Cuende
N, et al. 2005).
Criteria for ideal or reference donor for transplantation
- age below 40 years,
- trauma as the cause of death,
- hemodynamically stable,
- no steatosis, and no transmittable disease
The use of donor who do not meet all these criteria has become common practice
(Durand F, et al. 2008; Attia M, et al. 2008). These organs offer immediate expansion of
the donor pool, but in the other hand, transplantation of such liver allografts increases
potential short- and long-term risk to the recipient manifested as impaired allograft
function or donor-transmitted disease. In a LDLT risks are short- as well as long-term
risks (Brown RS Jr, et al. 2003; Trotter JF, et al. 2002). Early complications in the
recipient from surgical technique or insufficient hepatic mass increase the risk for
impaired allograft function. Recipient risk is compounded by short- and long-term risks
incurred by the donor who undergoes major hepatic resection (Renz JF, et al. 2000).
Current outcomes include a LDLT within the sphere of EDC (Renz JF, et al. 2005; Renz
J, et al. 2005). Guidelines defining this category of donor, level of acceptable risk,
principles of consent, and post-transplantation surveillance have not been defined. EDC
recipients are typically selected by the transplant center rather than allocated according
to wait list priority. No data exist on utilizing EDC allografts in an allocation scheme that
77 Introduction
mirrors the allocation of optimal hepatic allografts. However, Literature to date reflects
this operational paradigm with significant increases in access to transplantation, wait-list
mortality, and survival results that approach those observed utilizing optimal allografts
(Mor E, et al. 1992; Mirza D, et al. 1994; Renz JF, et al. 2005; Hertl M, et al. 1997;
Lopez-Navidad A, et al. 2003; Tisone G, et al. 2004; Rocha MB, et al. 2004; Montalti R,
et al, 2004; Cameron A, et al. 2005; Gruttadauria S, et al. 2005).
High risk donor characteristics associated with graft dysfunction
Donor characteristics associated with an increased risk of delayed graft function or
primary nonfunction include: age older than 60 years, (Ploeg RJ, et al. 1993; Busuttil
RW, et al. 2003; Tisone G, et al. 2004; Gruttadauria S, et al. 2005; Detre KM, et al.
1995; Strasberg SM, 1994; Feng S, et al. 2006; Busquets J, et al. 2001; Alexander JW,
et al 1991, Adam R, et al1993, De Carlis L, et al 1999, Marino IR, et al 1995),
hypernatremia exceeding 155 meq/L, (Gruttadauria S et al 2005, Totsuka E,et al 2004)
macrovesicular steatosis exceeding 40%, ( Ploeg RJ,et al1993, Busuttil RW et al
2003, Gruttadauria S et al 2005 Strasberg SM, 1994, Todo S, et al 1989, Adam R et al.
1991; Selzner M, et al. 2001; Verran D, et al. 2003; Soejima Y, et al. 2003; Briceno J, et
al. 2005), cold ischemia time exceeding 12 hours, (Ploeg RJ, et al. 1993; Busuttil RW,
et al. 2003; Gruttadauria S, et al. 2005; Briceno J, et al. 1997; Totsuka E, et al. 2004),
partial-liver allografts (SLT, RLT aLDLT) (Ploeg RJ, et al. 1993; Feng S, et al. 2006;
Marino IR, et al. 1995) and DCD. (Busuttil RW, et al; Feng S, et al. 2006; Foley DP, et
al 2005; Abt PL et al 2004).
Donor risk index.
In an attempt to develop a quantitative donor risk index; (DRI), Feng et al analyzed data
from deceased donors reported to the Scientific Registry of Transplant Recipients. They
identified seven donor or technical factors that were significantly associated with graft
failure. Donor age >70 years was the strongest risk factor with a relative risk (RR) of
graft failure of 1.65 compared to donors younger than 40 years; however, every age
group over 40 showed a significant increase in RR, DCD, and split grafts were also
strongly associated with graft failure Other risk factors were: African- American donors,
reduced donor height, cerebrovascular accident as the cause of brain death were more
modestly associated with graft failure. Each additional hour of CIT beyond 8 hours was
associated with a 1% increased in the risk of graft loss. A graft from another transplant
78 Introduction
area was also a risk factor for graft loss. A formula to calculate the DRI was developed
whereas a DRI of 1 or less was associated with a 87.6% 1-year survival, it was 76.9%
for a DRI of 1.6 to 1.8 and 71.4% for a DRI> 2. Grafts with an increased donor risk
index have been preferentially transplanted into older candidates (>50 years of age)
with moderate disease severity (nonstatus 1 with lower model for end-stage liver
disease (MELD) scores (MELD score 10–14)) and without hepatitis C. This quantitative
assessment of the risk of donor liver graft failure using a DRI is an essential first
element in the development of an allocation system that takes both recipient and donor
variables into consideration. However, several donor-related variables that are not
included in this DRI may be relevant as well, including length of stay in the ICU and
degree of steatosis (Durand F et al. 2008; Briceno J et al. 2005). A formula to calculate
the DRI was developed.
Calculation: Donor risk index
Donor risk index = exp[(0.154 if 40≤ age <50) + (0.274 if 50≤ age <60) + (0.424 if 60≤
age <70) + (0.501 if 70 ≤ age) + (0.079 if COD = anoxia) + (0.145 if COD = CVA) +
(0.184 if COD = other) + (0.176 if race = African American) + (0.126 if race = other) +
(0.411 if DCD)+(0.422 if partial/split)+(0.066 ((170–height)/10))+(0.105 if regional
share)+(0.244 if national share)+(0.010×cold time)].
Donor factors defining ECD
Risk of Impaired Graft Function
Risk of Disease Transmission
Donor age (>60 years)
Donor obesity
Steatotic livers (>40% macro)
Donation after cardiac death
Hypernatremia (serum Na _ 155 mEq/L)
Hypotension and inotropic support
Prolonged intensive care stay
Long ischemia times (CIT >12 hours)
Partial liver grafts (split/live donor)
Positive hepatitis B and C serologies
Unexplained cause of death
Known donor malignancy
“High-risk” lifestyle
Active bacterial/viral infections
Elderly donors
Using US data from the SRTR (Scientific Registry of Transplant Recipients), (Schaubel
DE et al. 2008) calculated the survival benefits of liver transplantation as a function of
candidate disease severity as expressed by the MELD score and donor quality as
expressed by the DRI. All recipients with MELD 20 or more had a significant survival
benefit from transplantation, regardless of DRI. These authors concluded that pairing of
high-DRI livers with low-MELD candidates fails to maximize survival benefit and may
79 Introduction
deny life saving organs to high-MELD candidates who are at high risk of death without
transplantation.
1.7.3 Donor Age
Physiologic and anatomic characteristics associated with older liver allografts
Recognition of unique physiologic and anatomic characteristics associated with older
liver allografts is essential for successful utilization of such grafts. Physiological and
morphological studies suggest that the liver seems to age fairly well and may be more
immune to senescence because of its large functional reserve, regenerative capacity,
and dual blood supply, which exceeds its metabolic needs. (Popper H et al. 1996).
Routine liver function tests do not show age-associated changes as evidenced from
clinical practice where hepatic functions are maintained in advanced age , and a liver
from a donor aged 86 years, have been transplanted successfully (Schmucker, 1998).
Macroscopically the liver is undergoing brown atrophy with old age; became smaller in
size, with darker texture and may have developed fibrous thickening of the capsule
(Jimenez RC et al. 1999), increased steatosis, and arterial atherosclerosis. (Busuttil
RW, et al. 2003; Tsukamoto I et al. 1993). In a study, it has been shown that in the
elderly, the liver may not be morphologically smaller, but the hepatocyte volume
decreases, i.e., it has fewer larger hepatocytes histologically (Wakabayashi H, et al
2002), this problem may result in a physiologically mismatched graft despite its size
being appropriate. In autopsy studies, aging was found to be associated with a 24%
reduction in liver weight in males and a 18% reduction in females. This trend has been
confirmed with different techniques, and in general, the reduction of liver size is noted to
be in the order of 25–35% (Le Couteur and McLean, 1998) . Old age is associated with
a reduction in hepatic blood flow of about 35–40% due to a diminished splanchnic blood
flow which reduced input of blood into the portal vein. This has been documented using
a variety of technical methods including dye dilution and indicator clearance, indicator
distribution and Doppler ultrasound (Wynne et al. 1989; Woodhouse and Wynne. 1992;
Zoli et al.1999). Bile flow and bile salt formation are reduced by about 50% reflecting, at
least in part, impairment of energy dependent and microtubule-dependent transport
processes (Le Couteur and McLean, 1998). In animal models, the aging liver has been
shown to accumulate mitochondrial deletions, thus leading to an inability to cope with
the production of reactive oxygen species (ROS) that enhances apoptosis and
subsequent fibrosis. (Gadaleta MN, et al. 1998) The cytochrome P450 content of liver
80 Introduction
specimens has been reported to decline from the age of 40 to 69 years by 16% and
further decline by 32% after age 70 (Zeeh J, et al 2002). Ultrastructural changes in the
aging liver include pseudocapillarization of the sinusoidal endothelium, defenestration
with reduced porosity, thickening of the endothelium, infrequent development of basal
lamina, and only minor collagen deposits in the space of Disse. These changes may
restrict the availability of oxygen and other substances (Briceno J, et al 2002)
Furthermore, preperfusion biopsies of livers from donors >60 years show higher rates of
moderate to severe microvesicular steatosis compared with those from <60 year-old
donors, as well as higher values of bilirubin and prothrombin time (Jimenez Romero C,
et al 1999) Older livers allografts have a lower tolerance for preservation. (Busuttil RW
et al 2003) and are more susceptible to endothelial cell injury from cold ischemia, with
impaired adenosine triphosphate (ATP) synthesis post reperfusion and result in
decreased regenerative capacity and synthetic function (Gordon Burroughs & Busuttil,
2009, Washburn WK,et al 1996. Emre S, et al 1996) which occurs early in older
allografts; consequently will increase the risk of inflammation, thrombosis, and T cell–
mediated rejection (Busuttil RW et al 2003, Tsukamoto I, et al 1993). The more
prevalent steatosis found in the eldery (Karatzas T et al 1997, Adam R et al 1993)
potentiate cold preservation injury (Adam R et al. 1995) and show decreased adenosine
triphosphate (ATP) synthesis after reperfusion
which in turn will impend synthetic
function and regenerative capacity of the liver (Busuttil RW et al 2003; Tsukamoto I, et
al 1993; Kimura F, et al 1996). This may be potentiated by the more prevalent steatosis
found in livers of the, decreased ATP synthesis after reperfusion, decrease regenerative
capacity (Karatzas T et al. 1997; Adam R et al. 1993) which and potentiate cold
preservation injury (Adam R et al. 1995) impaired synthetic function, and delayed
function with a notable cholestatic pattern after implantation (Nardo B, et al 2004; Yersiz
H, et al 1995) however, over than 75% of the recipients have regained normal liver
function (Yersiz H, et al. 1995). Furthermore, by maintaining cold ischemia time (CIT) to
8 hours or less, long-term graft function was shown to be equivalent in donors greater
and less than 50 years of age. (Yersiz H, et al 1995). Allografts from older donors
demonstrate increased delayed graft function and prolonged cholestasis consistent with
significant ischemia- reperfusion injury (Renz JF, et al. 2005). Therefore, older donors
need to be carefully selected, and each organ requires an assessment based on other
risk factors, especially steatosis and CIT.
81 Introduction
Influence Of Donor Age
Studies using the large databases of either SRTR/UNOS or ELTR clearly identified
donor age as an important risk factor for poor outcome after liver transplantation
(Burroughs AK et al 2006; Ioannou GN. et al 2006; Feng S, et al 2006). Donor age 65
years and older represents the largest expanding component of the current donor pool.
Donor age >50 years was thought to be associated with poor graft outcomes but studies
(Mor E, et al; Karatzas T et al 1997; Briceno J, et al; Wall WJ et al,1990; Grande L, et al
1998; Oh CK et al. 2000), have shown that aged donors (>50 years) without additional
risk factors have similar outcomes to younger donors and age itself should not be a
contraindication to liver donation. Donor age over 60 years significantly increased 3month patient mortality in the analysis of the European Liver Transplant Registry
(Burroughs AK, et al 2006) and donor age of more than 70 years, was found to be
associated with lower patient and graft survival (Busquets J, et al 2001). However, in
ideal recipients group for donors >70 years described as: patients >45 years old with
body mass index <35; non-HCV+; non-United Network for Organ Sharing (UNOS)
status 1 registration; first transplants with a cold ischemia time <8 hours.(Segev DL et al
2007). In these ideal recipients, OLT using donors >70 years was followed by similar
graft and patient 3-year survivals as when compared with ideal donors <40 years: 75%
and 81% versus 77% and 81%, respectively.
Donor age Liver volume and age (Wynne et al., 1989; Zeeh and Platt, 1990; Iber et
al., 1994; Le Couteur and McLean, 1998)
Technique
n
Age range
(years)
Decline
relative (%)
Decline
absolute(ml)
Year of
publication
Autopsy
1582
20-80
24% male
18% female
Ultrasound
26
25-80
24%
Ultrasound
50
50-80
32%
Ultrasound
65
24-91
37%
1474-934
1989
Ultrasound
32
55-70
20 %
1446-1157
1994
1933
1303-990
1978
1988
Post-transplant complications and survival
Regarding post-transplant complications and survival, old livers have been reported to
be more susceptible to rejection episodes, biliary complications (Busquets J, et al
2001), increased risk of vascular complications due to arteriosclerosis of the hepatic
82 Introduction
artery (Grazi GL et al 2001), and greater risk of transmission of occult tumours (Detry O,
et al 1997, Healey PJ, et al 1998). Liver biopsies has identified donor age as a major
independent determinant of the long-term histological prognosis of liver grafts (Rifai K,
et al 2004). A review of the liver transplantation database of the National Institute of
Diabetes and Digestive and Kidney Diseases (NIDDK) has declared the donor age >59
years as one of the three independent clinical characteristics most associated with
resource utilization (Showstack J, et al 1999). On the other side, others have observed
no significant increase in the incidence of acute rejection, nonischemic biliary stenosis,
FK-506 and cyclosporine toxicity, renal failure necessitating dialysis, level of
immunosuppressive drugs Renz JF, et al 2005 and intensive care unit or total hospital
stay (Neipp M, et al 2004). Regarding increased vascular complications, although celiac
axis atherosclerosis may be evdent (DeBakey ME, et al 1985) the hepatic arterial tree is
generally spared even in the elderly (Wall WJ, et al 1990) Persistently elevated gammaglutamyl-transpeptidase (GGT), alkaline phosphatase, and total bilirubin have been
reported to almost resume to normal values at 12 months after transplantation (Neipp
M, et al 2004). Reports on patient and graft survival following transplantation of older
donor livers are also highly variable, while some observed significantly lower short term
graft survivals with older livers, others have found significant differences only in longer
terms, or no differences at all (Figueras J et al 1996; Renz JF, et al 2005; Busquets J, et
al 2001; Neipp M et al 2004; Moore DE et al 2005; Markmann JF et al 2001). Regarding
the utilization of elderly donors in the setting of hepatitis C virus (HCV), convincing data
from single centers as well as the UNOS Transplant Registry demonstrate earlier HCV
recurrence and diminished survival of HCV recipients of donor allografts older than 60
years. (Cameron A et al 2005; Baccarani U et al 2004; Berenguer M et al 2002;
Condron SL, et al. 2005; Jain A, et al. 2005; Mutimer DJ, et al. 2006, Velidedeoglu E, et
al 2004; Gonzalez FX et al 1994). In recipients with HCV who receive organs from older
donors fibrosis is more rapid and cirrhosis is more common. (Wali M et al. 2002). May
be due to the rapid decrease of hepatocyte lifespan in viral hepatitis due to hepatocyte
telomere shortening (Aikata H et al 2000; Zeeh J, et al 2002). Moreover, grafts from
older HCV positive donors have been reported to cause significantly more advanced
fibrosis compared to HCV positive grafts from younger donors (Khapra AP et al 2006).
Various studies have also shown more severe HCV recurrence and reduced graft and
patient survivals after transplantation from elderly donors (Machicao, et al 2004,
Mutimer DJ et al 2006). Five-year graft survival less than 50% was described with
83 Introduction
donors over 70 years among HCV+ patients (Briceno J, et al 2000; Gastaca M et al
2007; Alonso O et al 2005). If HCV positive recipients receive grafts from older donors,
antiviral therapy should be used early after liver transplantation (Berenguer M, et al
2002). Some investigators recommend matching the graft to the recipient (i.e. marginal
grafts for low-risk patients as opposed to replacement in high-risk recipients e.g.,
fulminant hepatic failure (Busuttil RW, et al. 2003). Donor age has been a wellrecognized factor affecting PGNF. In the study by Feng et al, donor age over 40 years
was associated significantly with the relative risk of graft failure (Feng et al. 2006). In the
multivariate analysis by Lake et al, donor age over 40 was related with a 1.67 increased
risk of graft failure in HCV-infected recipients and with 2.21 increased risk of graft failure
when donor age was more than 60 years (Lake et al. 2005). In the SRTR analysis by
Johnson et al, donor age more than 40 years was an independent factor for the
prediction of PGNF (Johnson et al. 2007). Inversely, in other studies, donor age was not
confirmed to increase the incidence of PGNF (Busquets et al. 2001; Grande et al.,1998;
Washburn et al.,1996). However, these studies were based on a much smaller scale.
1.7.4 Influence Of Hepatic steatosis
The utilization of steatotic livers can significantly contribute to the expansion of the
donor pool. Hepatic steatosis is more common in donors of advanced age, as well as in
those with a history of obesity, dyslipidemia, metabolic disorders, or diabetes. In
deceased organ donors, liver steatosis has been documented in up to 30% (Angele MK
et al 2008; Marsman WA, et al 1996; Verran D et al 2003) and between 15% and 50%
of livers from cadaveric brain dead adult and child donors for liver transplantation exhibit
significant fatty liver disease, as defined by more than 30% of hepatocytes showing
steatosis. (Todo S, et al 1989; Strasberg SM, et al 1994; Trevisani F et al 1996; Selzner
and Clavien, 2001; Alwayn and Porte, 2007), moreover, 5–6% of cadaveric livers are
discarded due to steatosis.(Koneru B, et al 2002). However, steatosis, especially
macrosteatosis, is an established risk factor for primary nonfunction and IPGF (Selzner
&Clavien, 2001). Microsteatosis, which often appears as a mixed form with
macrosteatosis, has a smaller effect on ischemia-reperfusion injury than macrostatosis
(McCormack L, et al 2007), and livers with predominantly microvesicular steatosis show
less injury and allograft survival rates are similar to those in nonsteatotic grafts
(Fishbein et al. 1997). While mild macrosteatosis (<30%) is acceptable for use in
transplantation if cold ischemia time is short, the allocation of livers with moderate
84 Introduction
macrosteatosis (30–60%) remains challenging (Durand F et al. 2008) due to the
significantly increased risk of primary graft failure. The decreased allograft survival rate
after using fatty livers is seen in the early posttransplant period (Verran et al. 2003). The
most recent and largest study on post-transplant outcome of donor liver steatosis
originates from the USA and refers to 5051 liver transplanted patients (Spitzer AL, et al.
2010). In this registry, the presence of more than 30% of macrosteatosis was found to
be an independent risk factor associated with lower one year graft survival (relative risk
1.71). Importantly, when cold ischemia extended beyond 11 h, also lower degrees of
macrosteatosis (20%, 25%, and 30%) were associated with an increased risk of graft
loss (relative risk 1.51). The data additionally suggested that donor livers with >30%
macrosteatosis (MaS) may be successfully used, if other donor risk factors are
eliminated (e.g. donor age <40y, cold ischemia <5 h, no donation after cardiac death)
(Spitzer AL et al 2010). Interestingly, an Italian group demonstrated that transplanting
livers with moderate to severe MaS is an independent risk factor for the development of
biliary complications after LT (Baccarani U, et al. 2009). Steatosis diminishes the tissue
perfusion with compromised microcirculation in human liver grafts. A significant
reduction in hepatic microcirculation has been demonstrated in human steatotic donor
livers as assessed by laser Doppler flowmetry (Seifalian AM, et al. 1998). An
experimental study (Seifalian AM, et al. 2001) in an animal model of steatosis showed
that moderate steatosis caused a significant reduction in portal and total hepatic blood
flow, and impairment of hepatic microcirculation. These have suggested that the poor
initial function of fatty livers after transplantation is associated with an impairment of the
microcirculation (Seifalian AM, et al 1998; Seifalian AM, et al 2001; Teramoto K, et al
1993) and decreased ATP production (Selzner M, Clavien PA. 2001). By using highresolution in vivo microscopy, the most remarkable features of fatty liver graft in terms of
microcirculatory disturbance were characterized by narrow and irregular sinusoids,
vascular congestion, and blood cell adhesion to the sinusoidal walls (Teramoto K, et al
1993). This injury was accompanied by a significant reduction in the functional
sinusoidal density and mitochondrial membrane potential as assessed by Rh123associated fluorescence in the steatotic liver (Sun CK, et al 2001).The number of
adhesions of blood cells to the sinusoidal wall increased as the cold preservation time
increased, and sinusoidal blood flow decreased as preservation time increased, the
latter being correlated with survival in both normal and fatty liver grafts (Teramoto K, et
al 1993). Kupffer cells also have been implicated in an important role in graft injury that
85 Introduction
is correlated to the impairment of hepatic microcirculation in transplanted fatty livers, as
evidence shows that the inactivation of Kupffer cells by gadolinium chloride has
minimized graft damage by improving hepatic microcirculation and diminishing lipid
peroxidation (Zhong Z et al 1996). The phagocytic activity of Kupffer cells in preserved
fatty livers was greater than that in normal livers. Together with impaired hepatic
microcirculation, these features may cause liver cell death and contribute to primary
graft nonfunction after the transplantation of a fatty liver. (Teramoto K, et al. 1993).
Macrovesicular steatosis may impaire mitochondrial oxidation of fatty acids, increase
synthesis and delivery of fatty acids to hepatocytes, reduce the removal of hepatocyte
triglycerides, and microcirculatory disruption with narrowing of the hepatic sinusoids by
enlarged, fat-laden hepatocytes (Reddy & Rao, 2006). Further more, macrovesicular
steatosis may increase the oxidative injury of endothelial cell and hepatocyte and the
vulnerability to secondary insults, including the cytokine surge associated with brain
death as well as cold ischemic injury (Gordon Burroughs & Busuttil, 2009). In addition,
the occurrence of IPGF or PGNF may be due to the release of free lipids from fatty
hepatocytes, probably as a consequence of cold ischemia. Free lipids result in the
production of reactive oxygen species after reperfusion initiating a cascade, which
finally leads to sinusoidal endothelial cell damage (Selzner & Clavien, 2001; KupiecWeglinski & Busuttil, 2005). Severe fatty livers are more susceptible to warm and cold
ischemia reperfusion injury than normal ones (Kukan & Haddad, 2001). The type of
damage is not through the pathway of cellular apoptosis, but necrosis (Selzner &
Clavien, 2000). Overexpression of the mitochondrial uncoupling protein 2 in fatty livers
may contribute to decreased cellular ATP levels (Serviddio et al. 2008), which reduces
the capacity for hepatic regeneration (Selzner, et al. 2000). There are some other
factors which may play a role in fatty livers after reperfusion, like the down-regulation of
peroxisome proliferator-activated receptor-α, an important regulator of the hepatic
inflammatory response to ischemia reperfusion, and overexpression of adiponectin, a
fat cell-secreted hormone with antidiabetic and antiinflammatory activities (MassipSalcedo et al., 2008). Ureña et al considered that hepatic allografts with moderate
macrovesicular steatosis (30% – 60%) can be used selectively in critical situations; mild
macrovesicular steatosis (< 30%) is relatively safe, and severe cases (> 60%) enhance
the rate of PGNF (Ureña et al., 1998; D'Alessandro et al. 1991). In the study by Verran
et al, 6% of patients receiving grafts with severe macrovesicular fat required
retransplantation within 3 months versus 1.4% of those receiving mildly steatotic grafts
86 Introduction
(Verran et al., 2003). In another multivariate analysis by Salizzoni et al, cumulative
adverse factors on the incidence of PGNF included donor age, recipient HCV viremia,
and prolonged CIT by use of grafts with over 15% macrovesicular steatosis (Salizzoni et
al., 2003) Recipients receiving a fatty liver, show a dramatic decrease in fatty infiltration
shortly after (Marsman WA et al 1996 , McCormack L, et al 2007, Li J, et al 2009). The
mechanism of this phenomenon remains elusive. Donor age (>50 years) and prolonged
cold ischemia time (>12 h) are independent factors that negatively affect this reversal of
steatosis. ( Li J, et al 2009). The presence of moderate to severe MaS before LT did not
affect long-term organ survival. (Angele MK, et al 2008) Importantly, LT recipients are
particularly at risk for de novo development of NAFLD as they cumulate several risk
factors ; cyclosporine has been associated with a high incidence of hypertension and
hyperlipemia, and tacrolimus or sirolimus may cause a variety of adverse effects,
including diabetes mellitus. (Johnston O, et al 2008, Pagadala M, et al 2009) Moreover,
major changes in nutritional status, especially those with history of alcoholic disease,
which may contribute to some metabolic dysfunctions (Duvnjak M, et al 2009). The
grafts itself may contribute to the pathogenesis of NAFLD, as its own personal history
and genetic predisposition may influence its response to the new and different
environment provided by the recipient. Interestingly, steatosis in the liver graft has been
identified as a negative prognostic factor for HCV recurrence ( Yilmaz N et al 2009
Briceno J et al 2007, Briceno J, et al 2009). However, given the fact that steatosis
disappears early after LT, there is no obvious mechanism by which steatosis in the liver
graft synergizes HCV recurrence after LT. In contrast with previous data, a
study
suggested that steatotic grafts do not exacerbate the progression of fibrosis nor
negatively affect long-term survival in HCV recipients (Burra P, et al 2009). Donor age
limitation and exclusion of moderately to severely steatotic livers were proposed to
minimize the severity of HCV recurrence (Berenguer M. et al 2007).The literature is
divided on the effect of donor graft steatosis as a facilitator or stimulator of fibrosis on
patients with post-LT HCV recurrence. (Yilmaz N et al 2009 , Feng S. et al 2009, Burra
P, et al 2009). To optimize results when using fatty liver grafts other risk factors must be
minimized. Donor age below 40 years and a cold storage beyond 5 h were shown to be
protective in combination with up to 30% of graft MaS. In addition, the general condition
of the recipient is likewise the single most important factor (MELD <25). Some
promising approaches preventing activation of the inflammatory cascade are under
investigations in a number of experimental and clinical protocols, such as attenuation of
87 Introduction
cytokine activation (mitogen activated protein kinase, MAPK), blockade of endothelin
receptors, modulation of the heme oxygenase system, or inhibition of mitochondrial
dysfunction (Theruvath TP et al 2008, ] Mittler J, et al 2008) The use of machine based
liver perfusion systems may also offers benefits and perhaps a way to test the function
of the organ prior to implantation. The new preservation concepts include in situ warm
oxygenated perfusion before harvest (normothermic concept)(Fondevila C, et al 2007
)or hypothermic machine perfusion after organ procurement and transport to the
transplantation center (hypothermic concept)( de Rougemont O, et al, ] Dutkowski P,
2008, Dutkowski P et al 2006a, Dutkowski P et al 2006b , Bessems M, et al 2007) While
the perfusion system may enable to determine the viability potential of the graft, wide
application of perfusion system in marginal graft such as severe steatotic livers will need
long-term data after LT.Manipulation of the chemical composition of hepatic lipids may
evolve as a useful strategy to expand the donor pool and improve the outcome after LT.
A very promising option to prevent post-transplant complications appears to be the use
of a pretreatment with omega- 3 FAs. This approach is only feasible in living donation
since it requires oral administration of omega-3 FAs before organ procurement.
However, machine liver perfusion of any liver graft with omega-3 FAs before
implantation may emerge as an easily applicable method to reverse an abnormal
omega-3:omega-6 fatty acids ratio and decrease reperfusion injury.
The quantitative evaluation of steatosis
The quantitative evaluation of steatosis is based on the percentage of hepatocytes
containing cytoplasmic fat inclusions. In the clinical setting, steatosis is usually reported
as mild (less than 30%,), moderate,( 30% -60%)
or severe ( more than 60%) of
hepatocytes contain fat vacuoles within the cytoplasm (Nocito A et al 2006, McCormack
L. et al 2005,
Selzner M, et al 2001). In addition, fatty infiltration is divided
quantitatively into two categories, macro and microsteatosis. Macrosteatosis (MaS) is
characterized by a single, bulky fat vacuole in hepatocytes, displacing the nucleus to the
edge of the cell. This type Is most commonly associated with obesity, diabetes,
hyperlipidemia, and alcohol abuse. The underlying pathogenesis is related to an
excessive triglyceride accumulation in the liver, mainly due to an increased uptake of
fatty acids released from adipose tissue and/or an augmented de novo synthesis
(Nocito A, et al. 2006; McCormack L. et al 2005, Selzner M, et al 2001). Additionally, a
defective hepatic export, caused by reduced lipoprotein synthesis or impaired b88 Introduction
oxidation of fatty acids, further increases hepatic triglyceride content (Donnelly KL, et
al. 2005). In microsteatosis (MiS), the cytoplasm of the hepatocytes contains tiny lipid
vesicles without nuclear dislocation. MiS is usually encountered in mitochondrial
disruption following acute viral, toxin- or drug-induced injury, sepsis, and in some
metabolic disorders (Silva MA. 2009). Importantly, other histo-pathological features
should be carefully assessed in the presence of steatosis including inflammation,
fibrosis, and ballooning degeneration (Silva MA. 2009 , El-Badry AM, et al. 2009). MaS
and MiS often present simultaneously at different degrees in the liver.
Assessment of fatty liver graft
An initial evaluation, by inspection and palpation of the graft, is done during
procurement , criteria such as color and texture of the graft depend on the experience of
the explanting surgeon, and thus remain subjective. A study analyzing explanted, but
not transplanted livers, confirmed that neither preoperative evaluation by ultrasound nor
macroscopic evaluation during harvesting were reliable in steatosis evaluation. ( Rey
JW, et al 2009). Imaging modalities like CT or MRI may help in a more objective
assessment of hepatic fat, but such information is rarely available before procurement. (
Nickkholgh A et al 2007) There is a general agreement that the gold standard to assess
hepatic steatosis is liver biopsy (Silva MA. 2009 , El-Badry AM et al 2009). However, it
is not routinely performed in many centers. A European survey showed that liver biopsy
at the time of procurement for LT is rarely performed (McCormack L et al 2007) .Only
23% of liver transplant recipients in the United Network for Organ Sharing (UNOS) had
a liver donor biopsy recorded, 50%of the transplant surgeons in the UK never integrate
a liver biopsy into their decision making process (Imber CJ, et al 2002) .However,
several transplant programs consider a liver biopsy mandatory before discarding a
potential liver( Rey JW, et al McCormack L et al 2007) 2009 Spitzer AL, et al 2010). As
another strategy, 38% of liver transplant surgeons in the UK and 47% in the US proceed
with the histological examination of the graft, when steatosis is suspected at inspection
at the time of procurement. (Imber CJ, et al 2002a). The interobserver variability in
interpretation for both quantitative and qualitative assessments of the histologic features
of liver steatosis. (El-Badry AM et al 2009), staining techniques can affect detection and
grading of steatosis. Sample size errors that lead to misleading interpretation may be
related to focal steatosis, hypersteatosis, or hepatic fatty sparing . (El-Badry AM et al
2009). Two biopsy cores from the right and left liver were regarded to best predict
89 Introduction
overall liver histological characteristics (Frankel WL et al 2008). A recent study
confirmed that H&E-stained frozen biopsy overestimates MiS but underestimates MaS,
when compared with permanent sections using more specific staining modality . (Lo IJ,
et al 2008) Alternative methods to detect steatosis with higher sensitivity are Sudan-III,
toluidine blue, and oil red O staining ( McCormack L. et al 2005 Silva MA. 2009, ElBadry AM et al 2009 Imber CJ, et al 2002, Imber CJ, et al 2002b) but are rarely used in
the decision to accept or not a potential graft. In order to minimize inter-observer
variability, computerized programs have been developed to more objectively quantitate
hepatic steatosis by determining the area occupied by lipid droplets in a given field of a
liver section. (El-Badry AM et al 2009) although these computer methods determine the
total amount of fat and not the size of the fat droplet (i.e., microvesicular vs.
macrovesicular steatosis). Measurement of the omega-6 and omega-3 FAs and
prostanoid levels in liver biopsy samples, may help prediction of the magnitude of
reperfusion injury . (Silva MA. 2009)
1.7.5 Non–heart-beating donor (NHBD) livers
They are a potential means of expanding the donor pool. NHBDs are classified into 4
Maastricht categories: (Kootstra G, et al 1995a). Category 1 dead on arrival, Category 2
unsuccessful resuscitation, Category 3 awaiting cardiac arrest (usually after planned
withdrawal of support), Category 4 cardiac arrest while brain dead. Categories 1 and 2
are termed “uncontrolled” (UCNHBD), Category 3 is termed “controlled” (CNHBD) as
there is enough time to obtain family consent and mobilize the retrieval team prior to
withdrawal of support, consequently, warm ischemia time can be reduced. In
(UCNHBD), there is no enough time to organize the process of organ donation, the
process of retrieval is only initiated after the declaration of death, and consequently
these organs necessarily suffer a prolonged period of warm ischemia. Several ethical
issues are involved in retrieval of organ from NHBDs regarding care of the donor and
recipient declaration of death and the duration of the mandatory no-touch period( hands
off period) after cardiac arrest before organ retrieval. The hands-off period is to ensure
that there is no auto-resuscitation after cardiopulmonary arrest. Current data suggests
that this does not occur after 2 minutes. In contrast to HBDs, where death is defined by
neurological criteria, NHBDs death is declared only after cardiac arrest which mean that
NHBD is alive, until cardiac arrest takes place. The time between cardiac arrest and the
start of the organ retrieval process varies in different institutions. Intervals ranging from
90 Introduction
no waiting,. Olson L, et al 1996 , to 2 minutes,. (Casavilla A, et al 1995)
5 minutes,(
Reich DJ, et al 2000 ) or 10 minutes (Kootstra G 1995b) have been reported for
intervention following the declaration of death. The first international workshop in
Maastricht, the Netherlands, held in 1995, recommended that a 10-minute period after
cardiopulmonary arrest should be allowed before intervention by the transplant
team.(Kootstra G 1995b). In the United States, the Institute of Medicine guidelines
recommend a 5-minute hands-off period after cardiopulmonary arrest before organ
retrieval. Clinical and moral requirements are governing NHBD deceased donor organ
procurement policy. (Youngner SJ, et al 1993, Obermann K, et al 1995) to ensure that
organs are taken from dead donors , and that nothing is done to a donor prior to death
that is not in his interest and that an Informed Consent is Obtained Prior to Retrieval.
Regarding the intervention, practices are different in different countries. In the United
Kingdom administration of drugs (including heparin), prolongation of ventilation, or the
insertion of cannulas are prohibited. In the United States, the Institute of Medicine
recommends that the withdrawal of support and provision of palliative care should be
the same for both donors and non-donors of organs. Life-sustaining mechanical
ventilation of CNHBDs is allowed until provision can be made for retrieval of organs.
With the consent of the family cannulas may be placed, drugs including heparin and
phentolamine may be administered. Consent is usually handled in the same way as for
HBDs. However in the case of a UCNHBD, it is likely that the next of kin will not be
available at the moment of death. In this situation, practice varies in different countries
and in different institutions in the same country. Most countries, however, practice a
system of “opting in,” and consent must be sought from the family of the donor.
Because any delay will cause irreparable damage to the organs most centers that
retrieve organs from UCNHBDs, is to allow cannulation of the femoral vessels and in
situ cooling of abdominal organs prior to obtaining consent, but to delay retrieval
surgery until consent has been obtained. As stated above, the fundamental problem
with NHBD organs is prolonged warm ischemia. The first international workshop in
Maastricht, the Netherlands, held in 1995, recommended that warm ischemia should be
counted from the moment of cardiac arrest until the start of hypothermic flush out.
(Kootstra G. 1995b). A uniform definition of warm ischemia is lacking in the published
literature for liver transplantation. It has been defined as time between withdrawal of
support and cold flushing of the organs, (D’Alessandro AM, et al 2000) time between
hypotension (blood pressure <35 mm Hg) or low oxygen saturation (<25%) and flushing
91 Introduction
of the organs, (Fukumori T et al 2003) or time from extubation to aortic cross clamp.
(Abt P et al 2003). As prolonged WIT is common with uncontrolled DCD, standardized
criteria in donor selection have not been established and limited data concerning its use
have been reported. The allowable ischemic times for these liver grafts are short: 30 to
45 minutes for warm ischemia and 8 hours for cold ischemia. (Foley D et al 2005,
Bernat J, et al 2006). Algorithms have been developed by both the University of
Wisconsin and the United Network for Organ Sharing (UNOS) to predict the length of
time to patient expiration after withdrawal of support to help determine which patients
have the highest likelihood of becoming donors. (Lewis J et al 2003, United Network for
Organ Sharing) In these controlled situations, expansion of the donor pool can be
achieved by using DCD grafts with an incremental decrement in expected graft and
patient survival compared with the typical cadaveric liver graft . Foley D et al 2005. Abt
P, et al 2004, Manzarbeitia C, et al 2004, Mateo R, et al 2006). D'Alessandro et al
reported that the average WIT was 16.4 minutes in 19 cases of OLT using a DCD and
the rate of PGNF was 10.5% (D'Alessandro et al., 2000). Gomez et al also reported that
with 5-15 minutes average WIT, IPGF occurred in 6 of 8 cases and PGNF in the other
two cases (Gomez et al., 1997). In a matched-pair analysis, PGNF was occurred 5.1%
in livers of DCD versus 0% in those of DBD (Pine et al., 2009). In another retrospective
study, PGNF was presented 3.7% in livers of DCD versus 1.4% in those of DBD
(Grewal et al, 2009). In the study of 141 patients by de Vera et al, the incidence of
PGNF was 12% in livers of DCD versus 3% in livers of DBD. WIT over 20 minutes was
associated with poorer DCD outcomes (de Vera et al., 2009). In the study by Chen et al,
the average WIT was significantly longer in the IPGF group than in the non-IPGF (Chen
et al., 2007). Changes after warm ischemia were seen in liver biopsies before OLT.
Furthermore, WIT was 7 minutes in only one case who suffered from PGNF. From
logistic regression analysis, the possibility of IPGF was enhanced significantly when
WIT exceeded 3 minutes. These results suggested that extension of WIT is a direct risk
factor in bringing on IPGF. Recently, an analysis of OPTN/UNOS data demonstrates
donor age > 60 years, WIT > 30 minutes, CIT > 10 hours, retransplantation, and
recipient cardiopulmonary support pre-OLT to be the most important predictors of
significantly PGNF and patient survival after transplantation of a DCD graft (Mateo et
al., 2006). Similarily, the University of California, Los Angeles (UCLA) reported with
controlled DCD that PGNF occurred only in 2.6% of the recipients with about 30
minutes of mean WIT (Gordon Burroughs & Busuttil, 2009). Donor selection in some
92 Introduction
centers use only young donors. Abt P, et al 2003 while in others centers donors from
the age of 11 to 69 years have been used. It has been reported that organs from older
donors (>55 years) can be safely used for transplantation. (Fukumori T et al 2003).
Reich DJ, et al 2000)However, an analysis of the United Network of Organ Sharing
database33 (Abt PL et al 2004) revealed that the use of organs (12 transplants) from
donors greater than 60 years of age was associated with a very high PNF (25%). In
respect to organ retrieval process, the goal is to minimize warm ischemia.( Olson L, et
al 1999) and most units do not retrieve the livers if cardiac arrest does not occur within 1
hour of withdrawal of support. (D’Alessandro AM, et al 2000, Reich DJ, et al 2000)This
is to avoid retrieving organs that have been subjected to prolonged hypoxia and
hypotension prior to cardiac arrest. Strategies designed to recirculate oxygenated blood
following cardiac arrest have been utilized prior to and during organ retrieval from
UCNHBDs. Closed external cardiac massage has been used manually or mechanically
(Thumper, Michigan Instruments, Grand Rapids, MI) with simultaneous mechanical
ventilation (Nicholson ML, et al 2000) Compression of both the chest and abdomen is
performed to improve the cardiac output. Donors have also been placed on
cardiopulmonary bypass (CPB) either at normothermic ( Valero R, et al 2000) or
hypothermic (Alvarez J, et al 2000) temperatures to recirculate oxygenated blood.
These techniques are designed to bring about repletion of ATP before cold
preservation. The length and time that cells and organs can remain without oxygenated
perfusion is limited. Early studies indicated that the hepatic graft would not recover
when ischemia times were longer than one hour at room temperature. (Belzner FO,
Southard JH 1988).
Organ preservation and transplantation is associated with ischemia reperfusion
injury. Cold preservation at 4°C slows metabolism and provides a milieu to limit the
effect of ischemia. Metabolism is slowed 1.5- to 2-fold for every 10°C drop in
temperature, but considerable metabolic activity still occurs at 1°C. (Clavien PA, et al
1992) Adenosine triphosphate (ATP) is depleted and lack of oxygen converts aerobic
metabolism to anaerobic metabolism, leading to accumulation of lactate and
hypoxanthine, lowering pH and intracellular acidosis is developed. ATP is required to
maintain the integrity of sodium / potassium pumps that maintain electrolyte balance
throughout all cellular compartments. (Bronk SF, et al 1991) . Depletion of ATP leads to
pump failure, creating a loss of electrolyte gradients and membrane integrity, causing
93 Introduction
cellular edema. (Carini R, et al 1999). Membrane dysfunction allows calcium to enter
cells uninhibited, and the intracellular acidic environment uncouples calcium from
cellular proteins. (Gasbarrini A, et al 1992). Calcium activates phospholipases,
proteases, and nucleases, initiating the enzymatic cascades of inflammation and the
degradation pathways of cell death. Phospholipase A2 activation contributes to
impairment of the electron transport chain, ATPases, and adenine nucleotide
translocase activities, and can aggravate cellular edema by altering the cytoskeleton
through protease activation. ( Schroeder RA et al 1999). Adenosine, produced by
complete dephosphorylation of ATP, is broken down to hypoxanthine, which is normally
a substrate for xanthine dehydrogenase. However, under ischemic conditions, xanthine
dehydrogenase is converted into xanthine oxidase, which upon reperfusion converts
hypoxanthine to xanthine and urate, accompanied by release of free radicals. These
cause lipid peroxidation, a potent cause of graft dysfunction.(Goode HF, et al 1994).
Concomitantly, there is activation of Kupffer cells with release of reactive oxygen
species, nitric oxide, and proinflammatory cytokines. There is also expression of
adhesion molecules, which leads to recruitment and trapping of leukocytes, contributing
to progression of injury. The production of tumor necrosis factor alpha also affects
distant organs (this manifests as myocardial dysfunction and pulmonary edema
following liver reperfusion). (Lentsch AB, et al 2000) Simultaneous complement
activation contributes to cellular injury and further leukocyte recruitment into the graft.
(Lehmann TG, et al 1998, Scoazec JY et al1997), Cold preservation solution has been
designed to abrogate these effects and has contributed to the success of liver
transplantation.( Kalayoglu M, et al 1988). In NHBD organs, the effects of cold ischemia
are superimposed on the injury sustained during warm ischemia. Warm ischemia rapidly
causes depletion of ATP in organs, which are then subjected to a period of cold
ischemia leading to further injury. Cold ischemia leads to initial injury to sinusoidal
endothelial cells whereas warm ischemia mainly injures the hepatocytes.( Ikeda T et al
1992). NHBD organs have the benefit of not having been exposed to the cytokinemediated effects of brain death. The deleterious effects of brain death, resulting in
upregulation of inflammatory markers pre- and posttransplantation, have been identified
in animal models and in clinical transplantation. (Van Der Hoeven JA, et al 2000, Van
der Hoeven JA, et al 2001 , Compagnon P, et al 2002, Jassem W, et al 2003) Careful
donor organ selection, rapid cooling of organs after cardiac arrest, and minimization of
the duration of cold ischemia are essential to reduce ischemia reperfusion injury and
94 Introduction
subsequently improve results. A major problem with liver transplantation from NHBDs is
the risk of PNF. There are no reliable tests of pretransplant viability.( Vilca Melendez H,
et al 2000) and up to 40% of retrieved organs have been discarded due to doubts
about viability .The liver is assessed by the quality of perfusion and texture at retrieval.(
Casavilla A et al1995, Reich DJ,et al 2000) Organs showing patchy and nonhomogeneous perfusion are usually discarded. Additionally, biopsies proven organs
showing significant degree of steatosis or hepatocellular degeneration are discarded as
well.(Reich DJ, et al 2000).The use of UCNHBD livers is associated with a very high risk
of PNF. Reich DJ, et al. 2000 reported no PNF, with graft and patient survival of 100%
at 18 months but the early cholestasis and rejection were twice that of HBD. In contrast
to the UCNHBD livers, CNHBD livers are being increasingly used, with acceptable
results. Although , some have reported patient and graft survival of only 50% at 1 year,
and no PNF (Casavilla A, et al. 1995). Another report from the University of Wisconsin
reported an increased incidence of PNF in NHBD compared to HBD,(D’Alessandro AM,
et al 2000). Abt et al.( Abt P et al 2003) reported that patients with CNHBD livers have
similar long-term patient and graft survival compared to HBD, together with a higher
incidence of ischemic-type biliary strictures in case of CNHBD. The rate of major biliary
complications in patients who receive DCD grafts is about 33%, compared with 10% in
patients who receive standard grafts (Foley D, et al 2005, Abt P, et al 2003) To date,
there has been a lack of standardization with regard to many aspects of DCD, such as
precise definitions of terminology, technique, use of vasodilatory drugs, antioxidants,
preservation solutions, and the use of anticoagulation.(Alkofer B et al 2006) In an effort
to standardize procurement protocols and refine reporting of data, updated practice
guidelines for organ procurement have been published by UNOS, the Institute of
Medicine and the Society of Critical Care Medicine, (Reich DJ, et al 2010) and in 2009
the American Society of Transplant Surgeons issued recommendations on controlled
DCD based on evidence and expert opinion ( Reich DJ et al 2009). These American
Society of Transplant Surgeons guidelines spread on all aspects of controlled DCD
organ procurement including such issues as donor criteria, consent, withdrawal of
support, operative technique, biliary concerns, ischemia times, and recipient
considerations ( Reich DJ et al 2009). With ischemic cholangiopathy being the Achilles
heel of DCD OLT, various authors have proposed recommendations on maneuvers to
prevent biliary problems. These include performing an expeditious in situ biliary flush,
(Reich DJ et al 2006 Reich DJ et al 2009) considering arterial revascularization before
95 Introduction
or simultaneously with portal revascularization, (Abt P et al 2003 ,Reich DJ et al 2009)
use of a T-tube for easy access to the ducts postoperatively for stricture dilation and
sludge removal to prevent bile casts,( Reich DJ, et al. 2006 Reich DJ et al 2009) and
using the bile acid ursodeoxycholic acid posttransplantation. Other suggestions to
counter the specter of postoperative ischemic cholangiopathy have included using
thrombolytic agents and anticoagulants and replacing the more viscous University of
Wisconsin solution with histidine tryptophan ketoglutarate preservation solution. (Fung
JJ, et al 2007). It has been suggested that the use of low viscosity solutions for initial
flushing of the liver results in better perfusion of the microcirculation36 Tojimbara T et al
1997) and hence hyperosmolar citrate or a combination of Ringer’s lactate and
UWsolution have been used for initial flushing of the aorta.( Olson L, et al 1999)
Several transplant groups, including the group at the University of Michigan, use
postmortem extracorporeal membrane oxygenation to facilitate restoration of the flow of
warm oxygenated blood to the intra-abdominal organs during the interval between death
and organ procurement.(Magliocca JF et al 2005) Exciting new research endeavors in
organ preservation are in development, such as using ex vivo machine perfusion of the
liver. (Guarrera JV, et al 2010, Schreinemachers M et al 2007, de Rougemont O, et al
2009 , Imber CJ, et al 2002). A technique by Hong and colleagues proposed the novel
concept of regulated hepatic reperfusion to modulate ischemia and reperfusion injury
during organ revascularization (Hong JC,
et al 2009)These and other innovative
strategies potentially applicable to DCD are in early development and not yet ready for
transfer from bench to bedside.( Reich DJ, et al 2010) In order to diminish the ischemia
reperfusion injury, heparin with phentolamine are admisterated to the donor and
prostaglandin E1, vitamin E, and N-acetylcysteine to the recipient.3 D’Alessandro AM,
et al 2000. Transplantation of an ECD organ into a recipient who has a high (MELD)
score may contribute to worsened graft and patient outcomes. Therefore, many
transplantation centers have devised ECD recipient lists of specific patients who have
low MELD scores and an urgent need for transplantation.These patients, in consultation
with the transplantation team, may agree to accept a graft from an ECD donor that
probably could not be used in patients who have higher MELD scores (Busuttil R, et al
2003 )
96 Introduction
1.7.6 Orthotopic Liver Transplantation with partial allografts
Partial allograft from cadaver or living donors has become a viable option for some
selected patients with cirrhosis and end stage liver disease(Yersiz H, et al 2006).
Partial-liver allografts are associated with impaired allograft function and increased
recipient morbidity. Complications result from anatomic variations as well as from both
donor and recipient physiology. Liver volume must be sufficient to meet the metabolic
demands of the recipient, graft positioning is of great importance to optimize vascular
flow and biliary drainage. Complications include parenchyma bile leak, hepatic arterial
thrombosis, obstruction of hepatic venous flow, infection from remnant necrotic tissue,
and poor graft function secondary to insufficient hepatic volume.
Reduced-liver transplantation (RLT) is the surgical reduction of the size of a whole
adult cadaver allograft to fit for a child (Bismuth H, et al 1984 ). Studies demonstrated
satisfactory outcomes in children (Hemptinne B et al 1987, . Burdelski M, et al1988,
Ringe B, et al 1990). Although improved outcomes were achieved, the shortcomings of
RLT, namely the discarding of a right hemiliver (Broelsch CE, et al 1988) and the
increased competition between adult and pediatric candidates for the same donor
pool,(Zitelli BJ, et al 1987, Emond JC et al 1989, Otte JB, et al 1990) made the
procedure impractical. Currently, RLT is rarely performed in adults.
Split livers are part of the cadaveric donor pool it is now a routine operation and
considered to increase the supply of liver grafts by providing 2 transplants from a single
allograft serving a child who received the left-lateral lobe and an adult, who received
the extended right lobe. (Azoulay D, et al 1996, Rogiers X, et al 1996). Alternatively, to
split the liver into 2 hemigrafts and use the left side for a small adult or a teenager and
the right for a medium-sized adult patient. After introduction of in situ SLT, from 1988 to
2008 a total of 4103 split cadaveric and 3079 living donor transplantations were carried
out in Europe (European Liver Transplant Registry). Split liver transplantation (SLT) will
increase the available grafts for the pediatric patients; consequently, increasing waiting
list mortality in this group. All splitting procedures are based on the segmental anatomy
of the liver described by Couinaud ( Couinaud C: 1957).The 8 described segments are
based on the vertical division of the liver by the 3 hepatic veins in portal sectors and the
horizontal segmenting by the portal pedicles. Functionally, the main portal fissure
divides the liver along the line of Cantlie in the right (segments V-VIII) and left hemiliver
97 Introduction
(segments I-IV), which are supplied by the right and left hepatic artery and portal vein,
respectively. Regarding the contribution of the liver volume, the right hemiliver generally
provides two thirds and the left lobe one third of the total liver volume, which typically
represents at least 2% to 2.7% of the body weight. (Henderson JM, et al 1981).The split
procedure is a graft injury by itself with different consequences therefore, the
requirements must be stricter than for whole organ grafts.as well as considerations for
left lateral and full split procedure. The general technical preconditions to achieve 2
functional split liver grafts are the preservation of the venous drainage of all segments
and bile duct vascularization, as well as adequate portal and arterial perfusion.
Anatomic variations that will limit any of these factors may interfere with the split
procedure. In case of left lateral split procedure, no criteria about the ideal split-donor
are uniformly defined. The transplant centers have their own policies, but in general
there is an agreement about basic factors (Wilms C et al 2006, Broering DC, et al 2002
a, Ghobrial RM, et al 2000, Emond JC, et al 2002) regarding liver quality such as donor
age less than 40 to 50 years because the liver’s regeneration capacity is compromised
by aging (Schlitt HJ et al 2002), and greater than 10 to 14 years; hemodynamically
stable condition; intensive care unit (ICU) stay less than 5 days; normal liver enzymes;
and aspartate aminotransaminase (AST), alanine aminotransferase (ALT), and gammaglutamyltransferase (GGT) levels less than double of the normal value , serum sodium
less than 160 mEq/L fatty degeneration of the liver less than 30%; and no history of liver
disease. Older donors with acceptable liver function are also suitable. Donor variables
include as well donor liver anatomy (specifically variations of the hepatic artery, portal
vein, and biliary anatomy precluding splitting), Regarding the left lateral split procedure,
there are almost no anatomic variants to give a contraindication. If an aberrant left
artery from the left gastric artery or an aberrant right artery from the superior mesenteric
artery are found , this could support the split liver transplantation, provided that the
variation was recognized and respected by the harvesting surgeon. Regarding the
biliary anatomy there are only very rare variations prohibiting the left lateral split (eg, the
main bile duct bifurcation is located behind the left portal vein). (Broering DC, et al
2002b) Another contraindication would be the missing of a main portal vein bifurcation
with the absence of a left portal vein, (Couinaud C et al 1991 ) which is described with
an incidence of 0.9% (Broering DC, et al 2002b, Couinaud C. 1989, Couinaud C et al
1991). The macroscopic appearance of the donor liver, the consistency of the liver,
deciding the fat content by finger printing test, and, if necessary, obtaining a liver frozen
98 Introduction
section biopsy. Transection of the common bile duct and observing the quality and
production of bile during the procedure provides valuable information to the
procurement team with regard to the quality of the liver, the body weight and degree of
illness of the potential recipient. The liver can be split on the back table (ex situ) or in
the donor hospital before the donor cross-clamp (in situ ) splitting technique.
Advantages of in situ splitting include decreased total ischemia time and increased
possibility of inter-center sharing, additionally, in the in situ split technique, the hilar
dissection and parenchymal transection are performed during the warm dissection of
the procurement operation. The cut surface can be carefully inspected for bleeding and
bile leaks as well as evaluation of the viability of segment IV , and better control of
bleeding or bile leaks from inspection of the cut surface upon reperfusion on the
recipient. It is also important to ensure that the donor receives adequate nutrition before
procurement procedure because liver glycogen stores are depleted within 8–12 hours of
fasting. The use N-acetyl-cysteine 150 mg/kg IV as an oxygen free radical scavenger 1–
3 hours before procurement is also considered. It is ideal for the liver procurement team
to start procedure at least 1–2 hours ahead of other procurement teams. The splitting
procedure can be done by Cavitron ultrasonic aspirator or other devices for splitting;
either finger fracture or clamp crush techniques suffice to perform liver partition. If liver
is split through the main fissure to create right and left lobe splitting, performing
intraoperative cholangiography is mandatory; as some anatomic variations of the biliary
anatomy such as crossing bile ducts may preclude the splitting procedure. For a right to
left split, intraoperative ultrasonography enable the delineation of the major segment 5
and 8 veins draining into the middle hepatic vein.
Liver allograft /size
The calculation of allograft/recipient size matching is of great importance. In infants and
newborns, large graft size is a problem. In this regard, measuring the allograft length
(both horizontal and anterior posterior dimension) and comparing that with the
estimated abdominal domain of the recipient. If graft recipient weight ratio is equal or
less than 4, abdominal closure can be achieved without compression of the allograft
and its vascular supplies. In adults, small graft size is always the primary concern. The
minimal graft/ recipient weight ratio of 0.8 suffices to cope with the metabolic needs of
the recipient without developing small for-size syndrome. For small-for-size syndrome, it
is important to understand the concept of “functional graft size.” Compromised venous
99 Introduction
drainage of the graft, severity of the portal hypertension of the recipient, technical
complications such as bile leak, and infectious complications immediately after
transplant facilitate the development of small-for-size syndrome even if the
graft/recipient weight ratio is >0.8 (Hill MJ, et al 2009). Another important concern is the
possible bile duct problems, including necrosis of the bile duct which may be caused by
extensive dissection around the bile ducts injuring the biliovascular sheet surrounding
the bile ducts causing bile leak in the recipient and damaging the vascular supply of the
bile duct resulting in stricture formation in the recipient. In adult recipients, venous
drainage of the hemiliver grafts is extremely important because graft size is
compromised. Any problem with outflow obstruction may result in graft loss and
possible mortality. In right lateral grafts, it is also imperative to drain prominent segment
V and VIII veins as well as any short hepatic veins >0.5 cm in diameter before
reperfusion in the recipient. The right/left lobe splitting for 2 adults is more complex.
Although series were encouraging (Azoulay D, et al 2001, . Humar A et al 2001) , one
discouraging study was published by an Italian group (Giacomoni A,et al 2008). Even if
an optimal donor is selected, SLT is hampered by logistical constraints requiring short
CIT and recipient limitations.(Durand F, et al 2008) In general, right allografts have
yielded better results than left allografts.(Humar A, et al 2001, Sommacale D, et al
2000). A match pair analysis of patients after whole versus SLT using an extended right
liver lobe donor found no difference in either short- or long-term morbidity or mortality.
(Wilms C, et al 2006) Left grafts remain a technically challenging procedure with a high
risk of PNF owing to insufficient parenchymal volume and complex biliary and vascular
anastomoses. (Azoulay D, et al 2001)
Although data emerging from certain high-
volume centers in both the United States (Ghobrial RM, et al 2000) and multicenter
study from Italy reports the results from more than 300 split grafts with a median followup of 22 months(Cardillo M, et al 2006) . Three-year patient and graft survival were
similar in recipients of a left-lateral segment split graft and in whole-graft recipients and
split donation decreased the dropout rate on the waiting list in this study from 27% to
16%. Multivariate comparison again underscores the importance of both donor and
recipient selection as, for example, 3-year survival in healthy, nonurgent recipients
approached 90% but fell significantly, to 65%, among urgent split-graft recipients.
(Ghobrial RM, et al 2000) This notion is supported by Feng’s analysis of thelarge SRTR
database where SLT or partial grafts were associated with 52% higher risk of graft
failure.( Feng S, et al 2006)
100 Introduction
1.7.7 Other risk factors
The role of other risk factors such as obesity, elevated liver function tests, hypotension,
vasopressor use, nutrition, and length of stay in the intensive care unit is less clear and
were not found to confer increased risk of graft failure in the most recent study (Feng S,
et al. 2006). Certainly, donors at the extremes of these characteristics should be used
cautiously. Donors with a prolonged ICU stay are at increased risk of infection. A
multivariate analysis of the results of microbiologic cultures obtained before and at
harvesting from 610 consecutive liver donors has shown an ICU stay of >3 days to be
the only significant donor characteristic to predict donor infection . (Cerutti E, et al 2006)
Most of the larger series investigating the bacteraemic donors suggest that livers
procured from bacteraemic donors are likely to function well and pose little if any
increased risk to the recipient, provided that the recipient is treated with antibacterial
agents active against the donor bacterial isolate .( Little D et al 1997, Lumbreras C, et al
2001, Freeman RB et al 1999, Zibari G et al 2000) However, there is no controlled trial
indicating the optimal duration of antibacterial treatment for recipients of organs from
bacteraemic donors. Five to 7 days of appropriate therapy seems to be the most
frequently cited regime.( Angelis M, et al 2003). Some studies have identified donor
gender (female gender) as a risk factor for worse post-OLT outcome(Ioannou GN. Et al
2006) whereas others have failed to confirm this(Feng S, et al 2006)- Donor race
consistently seems to have worse recipient outcome(Feng S, et al 2006, Ioannou GN.
Et al 2006).With regard to the donor two parameters reflecting donor size, only height
(less height) but not weight has been shown to be independently associated with graft
failure and recipient outcome (Feng S, et al 2006)
Ischemia time
One of the major reasons for graft dysfunction is ischemic injury to the graft. Cold
preservation increases anaerobic metabolism and cellular acidosis. Metabolic activity is
reduced with mitochondrial energy uncoupling. Energy stores are depleted with an
accumulation of hypoxanthine, a substrate for the generation of toxic, reactive oxygen
species during reperfusion.(Busuttil RW,et al 2003). Prolonged cold ischemia time is an
independent risk factor for the development of preservation injury and delayed graft
function. Reperfusion following prolonged cold ischemia in human and animal models is
associated with inflammatory changes within the allograft that include sinusoidal cell
damage, complement activation, small vessel hypercoagulability, and increased
101 Introduction
circulating levels of interleukin 6 (IL-6) and IL-8. (Busuttil RW,et al 2003 Schmidt A,et al
2004 Shen XD,et al 2005). Prolonged CIT increases the incidence of short-term
complications as well as the incidence of long-term biliary complications.(Scotte M, et al
1994, Figueras J et al 1996) More than 14 h of cold ischemia has been consistently
associated with an increased preservation damage associated with a prolonged
postoperative course, biliary strictures and decreased graft survival (Briceno J, et al
2002, Piratvisuth T, et al 1995, Ploeg RJ,et al 1993) Accordingly, the risk of graft loss
increases by 1% for each additional hour of cold ischemia (Feng S, et al 2006) The
precise threshold for significant cold preservation injury varies with the individual
allograft. In allografts from otherwise healthy donors who are not older than 60 years,
the threshold for reduced allograft function secondary to prolonged cold ischemia lies
between 14 and 16 hours. A report of 315 LTX procedures from a European multicenter
study group, found that cold ischemia time greater than 16 hours was associated within
creased PNF and reduced long-term graft survival.(Porte RJ et al 1998). Others report
that cold ischemia times exceeding 14 to 16 hours are associated with a roughly twofold
increase in complications related to allograft function. (Ploeg RJ, et al 1993, Briceno J,
et al 2002 Hoofnagle JH et al 1996, Piratvisuth T et al 1995). Hepatic allografts from
older donors (age>60 years) are much more sensitive to preservation injury and
demonstrate optimal function when cold ischemia is under 8 hours. (Yersiz H, et al
1995)Several authors have implicated a synergistic effect of prolonged cold and warm
ischemia time on postoperative graft outcome.(Piratvisuth T, et al 1995, Totsukali E, et
al 2004) Cold and warm ischemia times have been identified as independent risk factors
for mortality; they should be kept as short as possible to mitigate unfavourable donor
characteristics. An ELTR analysis of 34664 primary adult liver transplants has identified
a total ischemia time >13 hours to be associated with significantly increased mortality at
3- and 12-months post-transplantation (Burroughs AK, et al 2006) As a previous ELTR
analysis had identified a cut-off of 12 hours (Adam R, et al 2000 ) the current
recommendation is to try to keep total ischemia time below 12 hours although the
precise threshold for improved outcome is unclear [4]. Programs using significant
numbers of EDC livers should consider using the piggyback technique for all cases to
minimize warm ischemia time (Mieth M et al 2006, Tector AJ et al 2006, Tzakis A, Todo
S, et al 1989).
102 Introduction
Hypotension and Inotropic Support
Previous United Network for Organ Sharing (UNOS) data have shown that donor
organs subjected to prolonged hypotension have no significant increase in
posttransplantation graft loss. However, graft loss was increased in liver transplant
recipients when donors received norepinephrine.(Opelz G, et al. 1994). In other studies,
dopamine dose of 10 micro g/kg/min( Markmann JF, et al. 2001) or 6 microg/kg/min
(Mimeault R, et al. 1989) had a significant effect on early graft function.
Donor Hypernatremia
Hypernatremia is a frequent clinical finding within the donor population that has a
negative impact upon hepatic allografts. (Gonzalez FX et al 1994)Hypernatremia may
result from aggressive treatment of cerebral edema, decreased antidiuretic hormone
secretion secondary to cerebral ischemia, or inadequate donor fluid management.
Allograft function is impaired because hepatocytes increase their intracellular osmolality
to minimize cellular damage associated with the extracellular hypertonic state. This
occurs through the influx of sodium and cytoplasmic osmoles, principally amino acids,
methylamines, and polyols. During normalization of hypernatremia, intracellular water
may rapidly accumulate,resulting in cell swelling and injury. (Busuttil RW, et al 2003
Gonzalez FX et al 1994)Donor hypernatremia is an independent predictor of post-LTX
graft dysfunction. It has been reported that there is a direct correlation between donor
serum sodium concentration and peak serum aminotransferase following LTX.71
(Avolio AW, et al 1991)another study reported that donor hypernatremia is the strongest
predictor of early graft dysfunction.(Gonzalez FX et al 1994). It has been reported that
donor plasma sodium exceeding 155 mmol/L at procurement
wasindependently
associated with an increased rate of retransplantation and decreased actuarial graft
survival( Figueras J,et al 1996) and that donor serum sodium equal or more than 170
mEq/dL had independent prognostic value in predicting graft survival after primary
LTX.(Markmann JF, et al 2001)One-year graft survival for donor serum sodium<170
mEq/dL was 75% versus 61% for donor serum sodium 170 mEq/dL or more.(Markmann
JF, et al 2001) Hypernatremia is a clear indication to slow down the donation process as
correction of serum hypernatremia prior to recovery abrogates its negative effect upon
organ function. In circumstances where hypernatremia cannot be corrected prior to
organ recovery, precool perfusion with 1 L of 5% dextrose in water for donor serum
103 Introduction
sodium levels above 160 meq/dL can be done.(Busuttil RW,et al2003, Renz J et al
2005)
1.7.8 Donor-Transmitted Diseases
The routine screening of potential donors for specific pathogens, includes HIV, HBV,
HCV,
syphilis,
cytomegalovirus,
tuberculosis,
and
Epstein–Barr
virus.(Organ
Procurement and Transplantation Network. Policy 2) Donors with an increased risk of
disease transmission include those with positive serologic data (HCV, hepatitis B virus
[HBV], human T cell lymphotrophic virus [HTLV I/II], carcinoma outside the liver, and
Centers for Disease Control [CDC] high-risk behavior). Guidelines on utilization of these
EDC allografts and posttransplantation screening for donor-disease transmission have
not been established.
Bacterial infections in the donor do not represent by themselves a risk factor for liver
graft failure. The risk of transmitting a bacterial infection in the case of bacteremia in the
donor is low. Early fever and positive cultures in the recipient as well as the presence of
yeast justify empiric therapy. (Freeman RB, et al 1999, Cerutti E, et al 2006, Lumbreras
C, et al 2001. Donors with documented bacterial meningitis do not preclude
transplantation ( Lopez-Navidad A, et al 1997). With regard to WNV infection, current
recommendations include excluding potential donors with meningoencephalitic
symptoms of undetermined etiology who live in regions of WNV activity, screening with
nucleic acid testing (NAT) as close to the time of procurement as possible, and being
suspicious when transplant recipients have postoperative fever and/or neurologic
symptoms not otherwise explained. Serologic testing of the donor and all recipients from
that donor should be performed (as well as lumbar puncture as indicated). At this time
there is no specific treatment for WNV.(Teperman L. 2010). Recipients of an allograft
with an increased risk of donor-transmitted disease should receive additional screening
after LTX. CDC high-risk allograft recipients should be tested at an appropriate time for
HBV, HCV, HTLV, and human immunodeficiency virus (HIV). HTLV I/II (+) allografts are
usable for select recipients. Donor serology positive for HTLV I/II should be confirmed
by Western blot analysis. Recipients of an allograft from a donor with a history of
extrahepatic carcinoma do not receive specific screening unless clinically indicated.
104 Introduction
Hepatitis
C
Transplantation
because
of
hepatitis
C
(HCV)
cirrhosis
has
increased.(Charlton M. et al 2001) HCV(+) grafts, constitute approximately 2% to 5% of
potential organ pool (Candinas D, et al 1994,Pereira BJ, et al 1995) and may be
implanted in HCV+recipients, as shown by several studies.(Everhart JE et al 1999,
Testa G, et al 1998, Velidedeoglu E, et al 2002) and under exceptional circumstances,
could be used in HCV-recipients. Utilization of HCV (+) allografts among HCV (+)
recipients who are active viral replicators of genotype 1 or 4 should be encouraged and,
these situations should not be considered EDC.(Renz JF et al 2005) . A review of
UNOS registry regarding the clinical outcome of a large series of HCV+ recipients of
HCV+ liver allografts showed that donor hepatitis C status does not impact on graft or
patient survival after liver transplantation for HCV+ recipients. Their survival was
equivalent, if not better, compared with a control group of HCV+ recipients of HCV(-)
livers. (Marroquin CE et al 2001) Data from other large centres have yielded similar
results. (Vargas HE, et al 1999, A 10-year experience of liver transplantation for
hepatitis C: analysis of factors determining outcome in over 500 patients (Ann Surg
2001; 234: 384 ). The time to recurrence and the course of HCV disease as well as
vector of means of alanine aminotransferase and total bilirubin parallel that in patients
who received non infected organs in a matched-pair analysis over a 3-year followup(Saab S et al 2003). It has been reported that HCV recurrence rate was similar in 2
patients groups who underwent transplantation due to HCV one group received HCV+
donor graft and the other HCV- donor graft (42% versus 55%;respectively) Patient and
graft survival at 4 years posttransplantation was 84% and 72% in the HCV+ donor
grafts, compared with 79% and 76% in the HCV- donor grafts. (Testa G et al
1998)Similar rates of HCV recurrence, patient survival, and graft survival have been
reported by other centers using HCV+ liver grafts for patients requiring transplantation
for HCV cirrhosis. (Velidedeoglu E, et al 2002. Vargas HE, et al 1999). Data from the
UNOS Scientific Registry showed that patient survival at 2 years was higher in 96
recipients of HCV+ than in 2,827 recipients of HCV- grafts (90% versus 77%;
P=.01).(Marroquin CE, et al 2001). In the largest series from a single institution, the
outcomes of 59 patients who underwent transplantation for HCV receiving HCV_grafts
were similar to those of 419 patients who did not receive a HCV+ graft. (Ghobrial RM, et
al 2001) although protocol biopsies were not performed and no data regarding fibrosis
scores and virological parameters was available. However, recipients of HCV+ grafts
105 Introduction
from older donors have higher rates of death and graft failure, and develop more
extensive fibrosis than HCV- graft recipients from older donors. ( Khapra AP et al 2006)
Hepatitis B Liver transplantation is a highly efficient mechanism for transmission of
HBV from an HBV-core antibody donor (Wachs ME et al 1995, . Prieto M, et al 2001)
The transmission rate of HBV infection to HBV-negative recipients through this route
has been reported to be 17–94% without prophylaxis (Grob P et al 2000,. Jilg W et al
1995, Dodson SF et al 1997, Anselmo DM et al 2002, Douglas DD et al 1997, Uemoto
S, et al 1998, Castells L, et al 1999, . Boyacioglu S et al2001 Two to 15% of liver donors
are anti-HBc positive. The proportion of positive anti-HBc livers in donors >60 years
may rise to 25% . (Prieto M, et al 2001), Use of hepatitis B core antibody-positive
donors in orthotopic liver transplantation. Arch Surg 2002; 137: 572). The use of
hepatitis B immune globulin (HBIG), with or without lamivudine, is now used to prevent
recurrence of HBV in the recipient as well as transmission from donor to recipient in
cases of donor anti-HBc positivity.( Mieth M, et al 2006) The 5-year patient and graft
survival rate in recipients of anti-HBc positive livers who received dual HBV prophylaxis
with HBIG and lamivudine has been reported to be significantly higher than for patients
who received single prophylaxis or no prophylaxis . (Saab S, et al 2003) Anti-HBc
positive donor livers must be directed selectively first to HBsAg positive recipients as
they will require life-long HBIG anyway. Secondly, these livers should be directed to
anti-HBs positive patients as they do not seem to require HBIG. It is not clear whether
or not to treat anti-HBs negative, anti-HBc positive patients with HBIG. Finally, HBVnegative recipients should only receive these livers in case of critical conditions. Lifelong
HBIG
is
mandatory
(Donataccio
D,
2006).
Given
the
very
costly
immunoprophylaxis therapy, there are recommendations for the use of such donors in
order to obtain the most justified economic approach. In this context, serology is an
insufficient tool to guide the therapy, and determination of donor HBV-DNA status is
mandatory at the time of transplantation to allow safe and efficacious use of anti-HBc
positive livers. Combined HBIG and lamivudine prophylactic therapy is thus
recommended when, at least, donor or recipient is HBV-DNA positive. Lamivudine
therapy alone is recommended when donor and recipient are both HBV-DNA negative.
If the recipient is HBsAg negative but anti-HBs positive, no prophylaxis is
recommended. When HBV-DNA is not available, lamivudine is administered when the
recipient is HBsAg and anti-HBs negative . Nery JR et al 2003 However, if no virological
106 Introduction
testing is available, long-term immunoprophylaxis is necessary to avoid de novo
infection (Nery JR et al 2003). Liver transplantation with grafts from donors positive for
both anti-HBc and HCV has been followed by similar graft and patient survivals as
transplants with anti-HBc+ or HCV+grafts. Saab et al 2003 described 22 patients
transplanted with both anti-HBc +/HCV+livers; their graft survivals at 1, 3, and 5 years
were 86%, 77%, and 69% with patient survivals of 91%, 81%, and 74%,
respectively.
Transmission of Malignancy
The risk of malignancy increases with donor age; transplanting organs from elderly
donors may increase the risk of transmitting defined and undefined malignancies.
(Durand F et al 2008).The incidence of cancer in donors is approximately 3%, and the
risk of transmitting malignancy by organ transplantation is roughly 0.01%. (Kauffman
HM, et al 2002, Kauffman HM, et al 2000, Buell JF et al 2006 ).A review on this subject
using UNOS data showed a total of 21 donor-related malignancies among 108,062
transplant recipients over 8 years, giving an incidence of tumor transmission to be
0.02%. Morath C, et al 2005 . In general, nonmelanoma skin cancer, low-grade
neurologic tumors as the risk of tumor transmission from donors with a primary CNS
malignancy is small, and in situ carcinoma seem to be a safe source of solid organs for
transplantation (Morath C, et al 2005). Certain tumor types, such as glioblastoma and
medulloblastoma, carry a higher risk of transmission and should be avoided unless the
recipient status warrants the extra risk. (Kauffman HM, et al 2002). Donors who have
had previous craniotomies and ventricular peritoneal shunts may have a greater risk of
extracranial metastasis. A history of melanoma, choriocarcinoma, lymphoma, or
carcinoma of the breast, lung, and
colon seem to possess a high rate of cancer
transmission, even after long apparent cancer-free survival (Morath C, et al 2005).
Tumors that may possess the potential of unpredictable recurrence include breast,
colon, lung, melanoma, and renal cell carcinoma (Myron Kauffman H, et al 2002). Any
metastatic malignancy in the donor should exclude donation. Recipients of donors with
malignancies
should
have
their
immunosuppression
modulated
because
overimmunosuppression reduces immune surveillance that can accelerate tumor
growth. The potential benefit from the mammalian target or rapamycin inhibitors, which
have both immunosuppressive and antiangiogenic properties, requires further
investigation. (Vignot S et al 2005)
107 Introduction
1.8 Living donor liver transplantation
Overview:
LDLT was introduced in 1989 with a successful series of pediatric patients (Broelsch
1991). Adult-to-adult LDLT (ALDLT) was first performed in Asian countries where
cadaveric organ donation is rarely practiced due to particular reasons. (Sugawara 1999,
Kawasaki 1998). LDLT is considered as an alternative option to DDLT. LDLT peaked in
the US in 2001 (Qiu 2005) but the numbers declined by 30% over the following years
(Vagefi 2011). A decline over time was also observed in Europe. The evaluation of
donors is a cost-effective but time-consuming process. Clinical examinations, imaging
studies, special examinations, biochemical parameters, and psychosocial evaluation
prior to donation varies from center to center.(Valentin-Gamazo 2004). Due to the
increasing number of potential candidates and more strict selection criteria, rejection of
potential donors has been reported in about 69-86% of cases (Valentin- Gamazo 2004,
Pascher 2002). Since LDLT is performed under elective circumstances, the advantages
of LDLT include timing of the operation when medically indicated especially in patients
with hepatocellular carcinoma (HCC) thus reducing waiting time mortality and lower the
dropout rate, studies using hypothetical decision analytical models have demonstrated
theoretical survival benefits for LDLT over DDLT.(Cheng SJ, et al 2001, Sarasin FP, et
al 2001 ) However, it is unclear whether the shorter waiting time and lower dropout rate
really provide survival benefits for patients with HCC in clinical settings. The short cold
ischemia time (CIT) is an advantage as long (CIT) is a well known risk factor for acute
cellular rejection (ACR) and graft loss in DDLT( Shaked A, et al 2009, Reese PP, et al
2008) Additionly, preoperative interventions can be planned for both the donor and
recipient, including nutritional treatment. In the surgical procedures in the recipient, bile
duct reconstruction has proven to be the most challenging part of the procedure with
biliary complications ranging from 15% to 60% (Sugawara 2005). Regarding donor
outcome, morbidity rates are variable in the literature (Patel 2007, Beavers 2002).
Possible complications include wound infection, pulmonary problems, vascular
thrombosis with biliary leaks, strictures, and incisional hernia. Biliary complications are
the most common postoperative complication in LDLT and occur in up to 7% of donors
(Perkins 2008, Sugawara 2005). Liver regeneration can be documented with imaging
studies and confirmed by normalization of bilirubin, liver enzymes, and synthesis
parameters.
108 Introduction
Living donor liver transplantation (LDLT) as an option to expand the donor pool.
LDLT is considered an option to expand the scarce donor pool ( Strong R, et al 1990,
Broelsch C,et al 1991). Every patient eligible for cadaveric liver transplantation is also a
candidate for LDLT. LDLT recipients were mostly children, but LDLT represent another
option for adult recipients. (Trotter JF, et al 2002) and adult-to-adult LDLT should be
considered EDC as outcomes do not meet expectations of optimal cadaver donors.
(Olthoff KM, et al2005) The incidence of technical complications is higher among aLDLT
recipients, and outcomes are inferior, when applied to patients in urgent medical need
of LTX. The Vancouver Forum stated that the expected graft and patient survival of
LDLT should be approximately the same as for a recipient of a deceased donor
transplant with the same disease (Barr ML, et al2006) Patients with severely
decompensated liver disease do not tolerate LDLT (Marcos A, et al2000). In patients
with chronic liver disease and severe decompensation (MELD > 30) the long-term
mortality following LDLT (57%) was significantly higher compared to deceased donor
transplant (18% historical control) (Testa G et al 2002, Kam I 2002). Therefore, it has
been recommended not to offer LDLT if MELD score >25 (Tan HP,et al 2005).
Candidates for LDLT are those patiens who have life-threatening complications and a
relatively low MELD score including patients with cholestatic liver disease and severe
pruritus or patients with ascites and/or encephalopathy, but still well-preserved liver
function, and those who have hepatocellular carcinoma (Trotter J,2002b) in which
progression of disease may lead to death or clinical deterioration while waiting for a
deceased-donor graft. Selected recipients have lower MELD scores at LTX than
patients undergoing deceased-donor liver transplantation (DDLT) (mean MELD score,
15.6 versus 22)(Olthoff K, et al 2005) and the most frequent complication leading to
aLDLT recipient mortality is allograft dysfunction. Additional risk is also transferred to
the donor. (Trotter J,et al 2002a, Kam I. 2002, Trotter J,et al 2002b, Neuhaus P. 2005).
Cold ischemic time is shorter in LDLT than in DDLT. Prolonged CIT is closely related to
the occurrence of various complications, including ACR and graft loss after
DDLT.(Shaked A, et al 2009, Reese PP, et al 2008) The risk of ACR for LDLT with a
CIT of 50 min is similar to that for DDLT with a CIT of 380 min, although longer CIT was
associated with increased risk of rejection in both types of transplantation. This finding
suggests that living donor allografts are much more susceptible to prolonged cold
ischemia than deceased donor allografts. Immunological molecules activated in the
immediately early regenerative process shown in the living donor allograft may
109 Introduction
unfavorably affect the occurrence of ACR. The 1- and 3-year patient survival rates after
adult LDLT are 89.1% and 80.3%, respectively, similar to the DDLT 1- and 3-year
patient survival rates of 85.7% and 77.7%, respectively. Graft survival rates at 1 and 3
years were similar also, 79.3% and 70.1%, respectively, for LDLT and 80.7% and
71.1%, respectively, for DDL (The Organ Procurement and Transplant Network.).
Complications are similar in both LDLT and DDLT recipients but biliary complications
are more frequent in LDLT compared to DDLT recipients, occurring in 24% – 67% ( Liu
CL et al 2005). Possible risk factors associated with an increased risk of postoperative
biliary complications included multiple ductal openings (Gondolesi GE, et al 2004), high
preoperative MELD (MELD>35) (Liu CL et al 2004), older donor age and previous
history of a bile leak (Shah SA, et al 2007). Whereas initially more Roux-en-Y (R–Y)
anastomoses were performed, today most authors would agree that duct to duct
anastomosis is safe and has the advantage of providing access for future endoscopic
therapy in cases of leak or stricture. This increased complication rate also leads to
higher hospitalization rates in LDLT recipients (Merion R, et al 2007). In the first year
the increased number of hospitalizations was due to biliary and nonbiliary
complications, whereas after the first year, these were mostly because of biliary
complications. In pediatric LDLT excellent results have been achieved, some centers
reported survival rates even for the youngest children below 2 years exceeding the
outcome after transplantation with deceased donor organs.( Roberts JP, et al 2004) In
large series of adult and pediatric LDLT, the 1-year survival rates are reported to be
between 73% and 98%(Adam R, et al 2003, Roberts MS, et al 2004, Kim JS, et al
2005, Thuluvath PJ, et al 2004, Maluf DG, et al 2005, Liu CL, et al 2006, Bourdeaux
C, et al 2007, Emiroglu R, et al 2007) .Liver cirrhosis due to chronic hepatitis C is the
most common indication for liver transplantation in most countries. Therefore, the
outcome of this patient group after LDLT was of special interest. Outcomes for LDLT
recipients infected with hepatitis C have been a point of some controversy.
Initial
reports suggested that HCV recurrence in LDLT recipients occurred early and in more
severe, whereas these differences were no longer consistently observed in more recent
studies(Foster R, et al 2007, Sugawara Y, et al 2005). Some centers have found no
difference in patient and graft survival in this group of patients, but other centers have
reported opposite findings(Gaglio P, et al 2003 , Pan S, et al 2004, Fahmy A, et al
2004, Vleirberghe H, et al 2004 ,Gordon F, et al 2004, Bozorgzadeh A, et al 2004,
Russo M, et al 2004, Thuluvath P, et al 2004, Shiffman M, et al 2004, Maluf D, et al
110 Introduction
2005, Humar A, et al 2005 , Rodriguez-Luna H, et al 2003, Guo L, et al 2006, Schiano
T, et al 2005)
LDLT for HCC
After the adoption of the Milan criteria (MC)( Mazzaferro V, et al. 1996) favorable
survival outcomes could be obtained after LT for HCC with survival rates comparable to
those in patients receiving transplants for nonmalignant diseases. Problems such as a
high dropout rate from the waiting list due to tumor progression (15% at 6 months, 25%
at 12 months)(Maddala YK, et al 2004) are still remaining. Since the living donor graft is
a dedicated gift directed exclusively to the recipient; LDLT can thus shorten the waiting
time and lower the dropout rate. Studies using hypothetical decision analytical models
have demonstrated theoretical survival benefits for LDLT over DDLT.(Cheng SJ, et al
2001, Sarasin FP, et al 2001) The role of LDLT and the intention-to-treat survival
benefits over DDLT among HCC patients were demonstrated(Lo CM, et al 2004 ) and
reported outcomes for a cohort of 51 patients with unresectable HCC who were
accepted on lists for both LDLT and DDLT in a single center. Median waiting time was
significantly shorter for LDLT than for DDLT (24 days vs 344 days, P <0.005), with a
dropout rate of up to 70%. Intention-to-treat survival rates of HCC patients with
voluntary live donors were significantly higher than those of patients without voluntary
live donors (4-year survival, 66% vs 31%, P = 0.029). In contrast, the multicenter Adultto-Adult Living Donor Liver Transplantation Retrospective Cohort Study (A2ALL)
reported that LDLT recipients displayed a significantly higher rate of HCC recurrence at
3 years than DDLT recipients (29% vs 0%, P = 0.002), although LDLT recipients had
shorter waiting times than DDLT recipients (mean 160 vs 469 days, P< 0.0001)( Fisher
RA, et al 2007). LDLT is a less radical oncological procedure due to the surgical
techniques—such as greater manipulation of the native liver, which leads to tumor
embolization through the hepatic veins—and a need to keep vascular margins closer to
the liver. Moreover, promotion of tumor growth and invasiveness by factors upregulated
during the natural course of liver regeneration in a partial liver graft may influence the
high rate of tumor recurrence after LDLT.(Ninomiya M, et al 2003). A retrospective study
from a single institute found no significant differences in long-term overall and
recurrence-free survival rates among HCC patients who received LDLT and DDLT when
analogous selection criteria were used for candidates: about two-thirds of patients met
the MC at transplantation( Di Sandro S, et al 2009). The effect of the shorter waiting
111 Introduction
time for LDLT on patient outcomes is thus unclear. Intention-to-treat analysis in largescale prospective randomized controlled studies or a detailed meta-analysis is
necessary to clarify the advantages of a shorter waiting time for LDLT over that for
DDLT. The advantage of LDLT involves the more liberal criteria compared with those for
DDLT. Interestingly, the evolution of expanded criteria for LDLT for HCC contrasts
strongly with that for DDLT. LDLT centers, mainly in Asian countries, have been
narrowing the selection criteria, while DDLT centers, mainly in Western countries, have
been expanding the selection criteria. indications for LDLT for HCC are decided based
on the balance between risks to the live donor and benefits to the recipient. As a result,
many Asian transplantation centers have adopted expanded criteria beyond standard
criteria such as the MC and UCSF criteria from the beginning of LDLT for HCC. Among
these, the Kyoto group started an LDLT program in February 1999 for patients with
HCC meeting extended criteria that include any size or number of tumors provided that
no distant metastases or gross vascular involvement are identified on preoperative
imaging.(Kaihara S, et al 2003); survival rates were similar for patients who met the
MC and those who did not.(Takada Y, et al 2007).
Donor selection
Appropriate donor evaluation is crucial in LDLT(Trotter JF, et al 2002).The aim of the
evaluation process is to ensure a safe outcome for the donor, to exclude donors with an
increased risk for morbidity and mortality, and to ensure that a suitable graft for the
recipient can be obtained. Acceptance rate of donor candidates dropped significantly in
USA and higher donor rejection rates are reported from Europe (86%) (ValentinGamazo C, et al 2004). Donor BMI, age and relatedness were donor-specific factors
influencing the acceptance rate The living donor should be a healthy volunteer and
should present normal liver function, no medical comorbidities, and no history of major
abdominal surgery The body mass index must be below 30 kg/m2 to prevent
thromboembolic events. (Kucher N, et al 2005) Any other procoagulatory disorders and
risk factors for thrombosis must be excluded to prevent perioperative development of
pulmonary embolism as one of the most feared complications in living donation.
(Durand F, et al 2002). The donor must be with a comprehensible close relationship
with the recipient, not limited to blood-born relatives in most countries. The donor age is
usually between 18 and 55 years of age, have an identical or compatible blood type to
the recipient, although there are reports about successful ABO-incompatible donation in
112 Introduction
children, and adults using different immunosuppressive regimens leading to survival
rates of 60% to 80% (Troisi R, et al 2006, Kozaki K et al 2005, Egawa H, et al 2004,
Egawa H et al 2008). Extensive evaluations are performed with assessments of the
medical risk, assessment of the remnant liver, suitability of the potential graft for the
recipient, such evaluation includes a thorough history, physical examination,
psychosocial assessment,(Broering DC, et al. 2003) laboratory testing, and crosssectional hepatic imaging to evaluate the liver for parenchymal abnormalities, steatosis,
biliary and vascular anatomy, and volumetric assessment of the graft and residual liver.
In fact, only about one third of the potential donors are suitable, (Middleton PF, et al
2006) thus, the invasive and cost-effective steps are performed in late stages of
evaluation. Data from the A2ALL cohort have shed light on the donor evaluation
process where an overall acceptance rate of 40% was achieved among more than 1000
potential donor candidates. (Trotter JF et al 2007) The decision regarding which kind of
graft should be used depends on the demand of the recipient matched with the
individual situation in the donor. To fulfill the recipient’s metabolic demands, a liver
volume of at least 0.8% to 1% of the body weight (graft-to recipient- weight ratio,
GRWR) should be targeted. Living donation for children up to 25 kg will require
resection of the left lateral liver lobe (segments II + III) in the donor. Children and small
adults between 30 and 60 kg need the implantation of a full left lobe (segments I-IV),
representing approximately 40% of liver volume of the donor. Donation for adult
recipients weighing more than 60 kg mainly necessitates harvesting a right liver lobe
(segments V-VIII). The examination of the liver quality and especially remnant liver
volume is of high importance to the donor’s outcome. Particularly in adult-to-adult LDLT,
where right liver lobe donation is planned, the donor must be safeguarded from
postoperative liver failure due to insufficient remnant volume. The critical threshold of
remnant liver volume has been assumed to be 30% of the standard liver volume to
provide a safety margin, since the lowest limit was reported to be 27%.(Fan ST, et al
2000) Presence of relevant steatosis must be checked as well, as it would lower the
functional liver mass. To exclude fatty degeneration of the liver, liver biopsy in cases of
right lobe donation is demanded during evaluation. Examination of the vascular
anatomy is also crucial to identify potential contraindications. Such as, the absence of
the main portal vein bifurcation or significant biliary or arterial malformations, explaining
the need for MRI or CT angiography and cholangiography. Again, right lobe donation
will require the closest investigations, particularly regarding the arterial blood supply of
113 Introduction
segment IV, which can arise from the right side as well as from the left side and also
regarding the various biliary anatomy of the right hemiliver. In situations in which the
remnant liver volume is too small for the donor or the graft size is not sufficient for the
recipient or both is transplantation of 2 smaller grafts from 2 donors can result in a
sufficient combined graft volume. (Lee SG, et al 2001, Broering DC, 2007)
Donor Morbidity and Mortality
The risks to the healthy live donor represent the greatest disadvantage for LDLT. The
healthy live donor undergoes major surgery for no direct, physical benefit. Even with the
best evaluation process complications and even death cannot be completely avoided.
The Japanese Liver Transplantation Society collected data from 3565 live donors and
reported that 299 donors (8.4%) suffered complications related to live donation, with
one donor death.(Hashikura Y, et al 2009) . A worldwide systematic review reported that
donor morbidity ranged from 0% to 100%, with a median of 16.1%.( Middleton PF et al
2006).and mortality is higher for adult to adult (0.24–0.4%)compared to adult to child
donation (0.09–0.2%). This is explained by the fact that adult to adult donation mostly
encompasses a right lobe and adult to children mostly a left lobe donation. Left lobe
donation has been associated with a lower mortality (0.05–0.21%), compared to right
lobe (0.23–0.5%) (Middleton PF,et al 2006).Regarding mortality; from the literature 18
early and 5 late donor deaths; of those, 3 early and 2 late deaths were reported after left
lateral donation; 12 early and 1 late donor deaths occurred after harvesting of the right
lobe and the rest were not specified (Nadalin S, et al 2007) a number of 12,000 living
donations worldwide, the mortality rate after left lateral lobe donation reaches
approximately 0.1%(Otte JB. 2003, Barr ML, et al 2006) whereas it is 0.4% to 0.5% (,
Renz JF, et al 2005 ) after right lobe donation. This range may be due to the lack of
standardized definitions for complications as well as the different reporting policies. A
standardized classification system of reporting morbidity, which could be adopted to the
Clavien system (Clavien PA, et al 1994). Donation of the right hemiliver is associated
with a higher risk for complications than donation of the left lateral liver lobe (Nanashima
A, et al, Umeshita K, et al 2003) The typical reported complications include biliary
leakages and strictures, wound infections, pleural effusion, small bowel obstruction,
pneumonia, and incisional hernia. Biliary complications and infections were the most
commonly reported donor morbidities, with median frequencies of 6.2% and 5.8%,
respectively. Based on the estimate of 14 000 LDLTs performed worldwide, the donor
114 Introduction
death rate is 0.1–0.3%. (Ringe B et al 2008) The A2ALL group investigated the rate and
severity of complications in 393 living donors.48 Eighty-two donors (21%) experienced
one complication, and 66 (21%) had two or more. In particular, 103 donors (26%)
experienced potentially lifethreatening complications and 11 (3%) experienced severe
or lifethreatening complications. Furthermore, the A2ALL retrospective cohort study
recently demonstrated that the LDLT benefit was magnified, with a mortality hazard ratio
of 0.35 (95% confidence interval, 0.23–0.53, P < 0.001), as centers gained greater
experience. (Berg CL, et al 2007).
The Clavien-Dindo Classification of Surgical Complications
Full Scale
Contracted Form
Grades
Definition
Grades
Definition
Grade I:
Any deviation from the normal postoperative
course without the need for pharmacological
treatment or surgical, endoscopic and radiological
interventions.
Allowed therapeutic regimens are: drugs as
antiemetics, antipyretics, analgetics, diuretics and
electrolytes and physiotherapy. This grade also
includes wound infections opened at the bedside.
Grade I:
Same as for Full Scale
Grade II:
Requiring pharmacological treatment with drugs
other than such allowed for grade I complications.
Blood transfusions and total parenteral nutrition
are also included.
Grade II:
Same as for Full Scale
Requiring surgical, endoscopic or radiological
intervention
Grade III-a: intervention not under general anesthesia
Grade III-b: intervention under general anesthesia
Grade III:
Grades IIIa & IIIb
Grade III:
Grade IV:
Life-threatening complication (including CNS Grade IV: Grades IVa & IVb
complications)‡ requiring IC/ICU-management
Grade IV-a: single organ dysfunction (including dialysis)
Grade IV-b: multi organ dysfunction
Grade V:
Death of a patient
Suffix 'd':
If the patients suffers from a complication at the
time of discharge, the suffix “d” (for ‘disability’) is
added to the respective grade of complication. This
label indicates the need for a follow-up to fully
evaluate the complication.
Grade V:
Same as for Full Scale
‡brain hemorrhage, ischemic stroke, subarrachnoidal bleeding,but excluding transient
ischemic attacks (TIA);IC: Intermediate care; ICU: Intensive care unit. Dindo D.,
Demartines N., Clavien P.A.; Ann Surg. 2004; 244: 931-937
115 Introduction
1.9 IMMUNOLOGY
Liver allografts were considered to be immunologically privileged, evidenced by the
absence of hyperacute rejection even in case of a positive T cell cross-match, the low
incidence of graft loss due to chronic rejection, and the potential for hepatocyte
regeneration after tissue injury. Importantly, in clinical transplantation, some liver
transplant recipients who cease taking immunosuppressive drugs maintain allograft
function. However, acute liver allograft rejection occurs in approximately 50% to 75% of
liver transplant recipients, such condition can be reversed by immunosuppressive
regimen to treat cellular rejection. In the other hand immunosuppressive drugs also
produce significant toxic effects that increase patient morbidity and mortality (Lechler R I
et al., 2005; Sayegh M H et al., 2004). The current immunosuppressive regimens do not
prevent the development of chronic rejection. Most studies have also shown that a
variety of autoimmune diseases with unknown aetiologies target the liver, including
primary biliary cirrhosis, primary sclerosing cholangitis, autoimmune hepatitis, and
biliary atresia (Duclos-Vallee J C et al., 2009; Schramm C et al., 2010; Guichelaar MM
et al., 2003). After liver transplantation antibody-mediated hyperacute vasculitic
rejection, can take place in individuals with preformed antibodies against the donor's
MHC class I–encoded antigens, Acute allograft rejection is the best-characterised graftspecific form of immune rejection and is initiated by the large number of recipient T cells
that recognise donor alloantigens (Stefanova I et al., 2003). Therefore, the
transplantation of MHC histoincompatible tissues elicits a strong, cytopathic, T cell
dependent immune response to donor tissues. Graft alloantigens are processed by
antigen presenting cells (APCs). Graft MHC molecules are internalised by donor and
recipient APCs, following intracellular processing, MHC peptide fragments are
presented to the recipient’s T cells (Watschinger B, 1995; Afzali B et al. 2008). Clinically
, this is manifested by a sudden deterioration in allograft function; liver biopsy shows
infiltration by host T cells , mononuclear leucocytes and damaged graft. CD4 and CD8 T
cells both participate in acute rejection. The rejection response is mediated primarily by
CD4 T cells which are activated by direct and indirect pathways, (Watschinger B.,
1995). Many activated CD8 T cells infiltrate the transplant tissue at the time of rejection,
along with other mononuclear leucocytes (Strom TB et al., 1975). The cells of the innate
immune system participate as well in rejection process; natural killer (NK) cells, are
present in allografts during rejection. NK cells can recognise alloantigens because they
constitutively express inhibitory receptors that are specific for self-MHC class I antigens;
116 Introduction
in addition, cytokines secreted by activated CD4 or CD8 T cells can promote the
activation of NK cells, which can initiate and aggravate the rejection response (Dollinger
MM et al., 1998). The humoral immune response is also important in the mediation of
allograft rejection although relatively uncommon in liver transplantation. The production
of anti-donor MHC antibodies is associated with acute and chronic graft damage,
usually in the form of graft vasculopathy. These antibodies can damage the graft by
activating complement and mononuclear cells with Fc receptors that recognise the
heavy chain of antibodies. Thus, Fc receptor–expressing leucocytes can be activated by
antibody coated donor cells. Anti-donor antibodies can also directly inhibit signalling
cascades within endothelial cells (Li F et al., 2009). Humoral-mediated rejection of
allografts is often observed following kidney, heart and lung transplantation, but liver
allografts appear to recover in relation to the development of humoral-mediated
rejection. Most transplant organs manifest insidious and inexorable dysfunction as time
passes. Although this process was formerly called ‘chronic rejection’, it is not clear that
donor-specific immune rejection is the sole or even the primary cause in many
conditions (Seetharam A et al., 2010). Fibrosis and atrophy are often present in the
absence of infiltration by T cells and other mononuclear leucocytes. Potential additional
causes for chronic allograft failure include viral infection, recurrence of the original
disease and drug toxicity.
Pathways of alloantigen presentation.
A) Direct pathway, recipient T cells recognise intact allogeneic MHC molecules on the
surface of donor APCs. The direct pathway is responsible for the large proportion of T
cells that have reactivity against alloantigens due to the cross-reactivity of the T cell
receptor (TCR) with self and foreign MHC molecules.
B) Indirect pathway, recipient APCs trafficking through the allograft phagocytose
allogeneic material are shed by donor cells (mostly peptides derived from allogeneic
MHC molecules) and presented to the T cells on recipient MHC molecules
Following T cell activation and proliferation, homeostasis of the adaptive immune
system is restored by cell death via “neglect” of most antigen-specific T cells. A small
number of T cells, however, survive and become long-lasting memory cells Memory T
cells can be divided into central memory and effector memory subsets, based on their
circulation pattern and functional responsiveness. Central memory T cells are
responsible for recall antigen responses, and effector memory T cells survey peripheral
117 Introduction
tissues and immediately respond to invading pathogens (Sallusto F et al., 2004). In the
setting of organ transplantation, upon re-exposure to donor antigens donor-reactive
memory T cells are more sensitive to antigens, acting more rapidly, produce effector
cytokines, and survive longer than naïve T cells and directly or indirectly produce
cytolytic effects on the transplanted tissue (Ku C C et al., 2000; Sallusto F et al., 2000;
Garcia S et al., 1999 & Barber DL et., 1999). As a consequence of continuous exposure
to foreign antigens, memory T cells accumulate with time and represent approximately
50% of the total T cell pool in adults. Recipients who have not received a transplanted
graft can still generate donor-reactive T cells, which can appear through immunisation
by direct exposure to alloantigens via pregnancy or blood transfusion (Bingaman A W
et. 2002). Furthermore, donor-reactive memory T cells can be generated in the absence
of alloantigen exposure through heterologous immunity. Some memory T cells are
therefore primed by an antigenic pathogen-derived peptides and cross-react with
allogeneic peptides presented by the self or the donor MHC molecules. Alloreactive
naïve T cells can acquire a memory phenotype and generate a substantial pool of
donor-reactive memory T cells after transplantation, even when a recipient is under
immunosuppressive therapy. Furthermore, the use of antibodies that deplete host T
cells can amplify this phenomenon by inducing homeostatic T cell proliferation in
response to lymphopenia (Wu Z et al., 2004). Because of their capacity to rapidly
generate effector immune responses upon rechallenge, memory T cells appear to be
particularly efficient at mediating allograft rejection (Zheng X X et al., 1999 & Schenk A
D et al., 2008). In addition, memory T cells are less sensitive than naïve T cells to many
immunosuppressive strategies. Compared with conventional T cells, memory T cells are
less sensitive to T cell-depleting antibodies and therapeutics that block the CD28 and
CD154 costimulatory signallers which inhibit the mammalian target of rapamycin (Pearl
J P et al., 2005; Vu M D et al., 2006; Adams A B et al., 2003 & Araki K et al., 2009). The
effects of memory T cells on the allograft response have been well delineated in animal
models of allograft tolerance, wherein the generation of memory T cells by presensitisation, heterologous immunity or homeostatic proliferation prevents the graftprotecting effects of most tolerising therapeutic strategies (Koyama I et al., 2007 &
Valujskikh A et al., 2002). In contrast to human recipients, animals live in the protected
environments of transplantation laboratories and do not usually contain substantial
numbers of memory T cells. This is one of the reasons that may explain the difficulties
of translating into the clinic the results of protocols capable of creating allograft
118 Introduction
tolerance in rodent models. But the results cannot be applied in clinical conditions.
Conventional immunosuppressive drugs showed little effects on activated memory
lymphocytes, and memory T cells also exert harmful effects and pose a significant
barrier to inducing tolerance to allografts (Chalasani G et al., 2002; Zhai Y et al., 2002 &
Adams AB et al., 2003) in clinical transplantation. T cells must receive two distinct but
coordinated signals in order to achieve optimal activation. The first signal is provided by
the TCR engagement with recognition of peptide/ MHC I or II on APCs, and the second
signal is achieved by the interaction of costimulatory molecules on the T cells and their
ligands on APCs. The importance of costimulation was found through experimental
models in which its inhibition was achieved by some means, Signal 1 in the absence of
signal 2 – as likely occurs in the liver – leads to a state of T cell non responsiveness (or
anergy) in which T cells can recognise cognate antigens through the TCR, but fail to
mount a functional response upon reencounters with the antigen. So, there have been
significant efforts to inhibit or block co-stimulatory pathways as a means of achieving
allograft tolerance. There are two co-stimulatory pathways that are important in the
generation of a complete T cell response are CD28/B7 and CD40/CD154 in the costimulatory field. The role of CD28 has perhaps been that most intensively investigated
in the co-stimulatory field. CD28 represents the prototypical T cell co-stimulatory
molecule. In humans, CD28 is expressed on 90% of CD4 T cells and 50% of CD8 T
cells; moreover, ligands for CD28, B7-1 (CD80) and B7-2 (CD86) are found on a variety
of APCs including DCs, B cells and macrophages. The expression of CD86 is greater
than for CD80 on APCs, although CD80 expression is enhanced during APC activation.
The expression of CD80 and CD86 has been examined by immunohistochemistry or
real-time polymerase chain reaction in livers following transplantation (Kwekkeboom J
et al., 2003). CD80 was expressed only sporadically on normal liver but was present on
at least 25% of the Kupffer cells in 45% of the transplanted livers. CD86 was found on
the majority of Kupffer cells in all transplanted liver tissue and in normal liver tissue.
Ligation of CD28 by either CD86 or CD80 increases cytokine synthesis and enhance
proliferation by various intracellular signalling. Immunohistochemical analysis of CD86
expression in biopsies of liver recipients demonstrated an association with the
increased expression of CD86 in the graft during severe acute cellular rejection (Bartlett
A S et al., 2003). CTLA4 (CD152) is a CD28-related protein that binds to CD86 and
CD80. Whereas CD28 delivers a positive co-stimulatory signal to T cells, CD152
delivers a negative signal that attenuates T cell function. CD152 expression is
119 Introduction
enhanced after T cell activation, and it has a higher affinity for CD86 or CD80 than does
CD28; it has been proposed that the physiologic function of CD152 is to downregulate T
cell responses. Therefore, specific activation of CD152 could potentially yield
immunoinhibitory function and achieve allograft tolerance, but this ideal approach has
been reached by the lack of suitable reagents. The CD40/CD154 co-stimulatory
pathway is a second important co-stimulatory pathway that is critical in the immune
response of allotransplantation. CD40 is mainly expressed on APCs (including DCs, B
cells, and macrophages) but it can also be expressed on nonimmune cells (including
endothelial cells, mast cells, platelets and epithelial cells). However, CD154 is mainly
expressed on CD4 T cells following activation, and to a lesser extent on NK cells, B
cells, and CD8 T cells. CD154 combines with CD40, which is critical for the activation of
DCs, B cells, and macrophages. In DCs, CD40 upregulates interleukin12 (IL-12)
production, and in macrophages it results in the production of various proinflammatory
cytokines. CD154 was also detected on Kupffer cells and on sinusoidal macrophages in
livers during chronic rejection, but not in stable liver allografts or normal liver (Gaweco A
S et al., 1999). The most widely-used measure to block CD28-B7 interactions has been
CTLA immunoglobulin (Ig). In the orthotopic rat liver transplantation model, repeated
administration of CTLA-Ig – beginning with CTLA-Ig in combination with donor
splenocytes – leads to extended graft survival of >100 days, whereas the delayed
administration of CTLA4-Ig alone or donor splenocytes alone did not (Neumann U P, et
al., 2002). In recent years, many studies have shown that B7 cross-linking on APCs by
CTLA4-Ig induces indoleamine 2, 3-dioxygenase (IDO), which itself inhibits local T cell
activation (Mellor A L et al., 2003; Li W et al., 2009). Gene therapy approaches to
deliver CTLA4-Ig to liver allografts have been successfully used in some animal
experiments. Adenoviral-mediated gene delivery of CTLA4-Ig through ex vivo perfusion
of cold preserved livers resulted in indefinite survival of rat liver allografts and in the
generation of donor-specific unresponsiveness (Olthoff K et al., 1998). An interesting
report suggests that CD154/CD40 interaction plays a role in promoting dendritic cellmaturation in the absence of CD4+CD25+ regulatory lymphocytes, whilst these cells
promote the maintenance of immaturity (Serra P et al., 2003; Misra N et al., 2004).
Allograft rejection mainly involves host-versus-graft reaction in liver transplantation,
which is the rejection of the transplant by the recipient's body. The graft rejection has
been divided into three groups: hyperacute rejection, acute rejection and chronic
rejection
120 Introduction
Type of rejection
Time taken
Cause
Hyperacute
Minutes-hours
Pre-existing anti-donor antibodies and complement activation
Acute
Days - weeks
Primary activation of T cells
Chronic
Months – years
Causes unclear:
antibodies, slow cellular reactions,
immune complexes, recurrence of disease.
1.9.1 Hyperacute rejection
Hyperacute rejection often occurs within minutes to hours after the host blood vessels
are to graft vessels. The rejection is mediated by pre-existing antibodies specific to the
graft antigens (including ABO blood type antigens, VEC antigens and HLA antigens).
Furthermore, these different antigens can activate the complement of the host and lead
to damage to the endothelial cell. Studies have reported that the process is often
accompanied with platelets activation and results in thrombosis and vascular occlusion
(Fiane A E et al., 1999). In addition, the massive recruitment of neutrophils occurs,
followed by rapid inflammation after transplantation. The pathological changes of
hyperacute rejection are thrombotic occlusion of the graft vasculature ischemia,
denaturation and necrosis This rejection is relatively rare in liver transplantation.
1.9.2 Acute rejection
Acute rejection occurs within days and up to three months after transplantation (80-90%
of cases occur within one month). The rejection occurs due to donor HLA interaction
with the host T cells, creating a cascade of immune responses initiated by that interface.
After a solid organ transplant, there is an immunological milieu of activity. The
mechanisms of the process involve abundant immune factors, such as humoral and/or
cellular mechanisms. Antibodies can injure the graft by activating complement and
mononuclear cells with Fc receptors that recognise alloantigens on the endothelial cell,
resulting in vasculitis. Cytotoxic T cells (CD8+) will recognise alloantigens on an antigen
presenting cell (APC) by direct presentation on the donor tissue and endothelial cells,
which promotes the apoptosis of transplanted tissue. It has been shown that CD8+ cells
alone are sufficient for the mediation of acute allograft rejection, but with the help of
CD4+ cytokines secretion – such as IL-2 – clonal expansion and the expression of
cytotoxic attack molecules will be upregulated (Kreisel D et al., 2002). The Fas/Fas
ligand (FasL) pathway is another death inducing pathway which is utilised by CD8+
cells. Whereas FasL is specifically induced upon CD8+ cells’ activation, Fas is
121 Introduction
ubiquitously expressed on lymphoid and non-lymphoid tissue, including the liver. The
Fas/FasL pathway is thought to play an important role in a variety of hepatic
pathologies, and there is evidence that this pathway is also active during liver allograft
rejection (Tannapel A et al., 1999; Ogura Y et al., 2001). Delayed hypersensitivity also
has an important role in acute rejection, being initiated by alloantigen primed CD4+
cells specific to the donor class II (Carrodeguas L et., 1999). CD4+ cells release IFN-γ
by re-exposure to specific alloantigens, a proinflammatory cytokine that can cause the
activation of macrophages and the subsequent release of a variety of inflammatory
mediators. These inflammatory mediators can augment the cellular anti-graft response
or else can cause direct tissue damage. The pathological features of acute rejection are
acute vasculitis and parenchymal cell necrosis, along with the infiltration of lymphocytes
and macrophages.
Late acute rejection Late ACRs are those occurring after 6 months. Predisposing
factors and causes include poor compliance to medication, biliary complications, and
obligatory reduction in immunosuppressive regimen due to infections (such as
tuberculosis, CMV, recurrent HCV) or PTLD. However, in many patients, no identifiable
reasons are apparent. (D’Antiga L, et al 2002, Mor E, et al 1992 , Cakaloglu Y,et al
1995) The pattern and nature of inflammatory infiltrates differ from early ACR, in having
more hepatitic features (interface activity, central perivenulitis and lobular inflammation),
tendency towards monotypic/less mixed portal infiltrates, and less prominent duct injury.
(Demetris AJ, et al ,Pappo O, et al 1995) these atypical features are more likely to be
observed in the older grafts in terms of time elapsed from transplant. Central
perivenulitis (CPV). can be mild, moderate or severe, and occurs with or without
perivenular hepatocellular necrosis. (Demetris AJ. , et al 2006) Allograft CPV can be
isolated or associated with hepatitic lobular inflammation, and/or portal based features
of ACR, including duct injury.( Hassoun Z, et al 2004, Krasinskas AM.,et al 2008,
Sundaram SS, et al 2006)
here the diagnosisis is established and the treatment
approach follows the established anti-rejection protocols. Isolated CPV or in association
with portal changes other than those of ACR, the aetiology of CPV become debatable
and the feature is difficult to characterize as it can present with normal or only minimally
elevated liver enzymes. (Krasinskas AM, et al 2008).
122 Introduction
1.9.3 Chronic rejection
Chronic rejection is less well-defined than either hyperacute or acute rejection,
developing months or years after acute rejection reactions have subsided. Chronic
rejection is an indolent but progressive form of allograft injury that is usually irreversible.
It is the most significant obstacle to morbidity-free long-term survival. By five years after
transplantation, it affects as many as 30-50% of heart, lung, pancreas and kidney
allograft recipients, but only 4-8% of patients who undergo liver replacement (Demetris,
A J et al., 1997). Liver allografts differ from other solid organs in that chronic rejection is
potentially reversible. This feature has been mainly attributed to its unique
immunobiological privilege and the regenerative capacity of the process. Livers with
chronic rejection have a decreased number of bile ducts on biopsy. This is referred to
as "vanishing bile duct syndrome" (Demetris A et al., 2000). Chronic rejection is
characterised by vasculopathy, fibrosis and a progressive loss of organ function.
Chronic rejection may be mediated by a low-grade, persistent, delayed hypersensitivity
response in which activated macrophages secrete mesenchymal cell-growth factors. Of
potential importance are the persistent viral infections which induce cellular immune
responses which in turn may synergise with donor-specific alloreactive T cells within the
allograft. Chronic rejection may also reflect chronic ischemia secondary to the injury of
blood vessels by antibody or cell-mediated mechanisms. Vascular occlusion may also
occur as a result of smooth muscle cell proliferation in the intimae of arterial walls.
Severe or very late-stage chronic rejection results in the loss bile ducts small branches
of the hepatic artery. The majority of liver transplant centres regard blood group
compatibility as the primarily immunological selection criterion. Recently, many
transplantation centres have used ABO-incompatible liver grafts, and the outcomes
have been shown to be similar to that of blood-type-matched transplantations in some
centres. However, infection is the major cause of morbidity and mortality after ABOincompatible liver transplantation (Tanabe M et al., 2010). The transplantation of
compatible but not identical livers is common practice, especially for recipients with the
less common blood groups. Interestingly, the results of ABO identical grafts were
slightly better than the ABO compatible but non-identical grafts (Gugenheim J et al.,
1990). An occasional complication with compatible, non-identical grafts is the
occurrence of allograft rejection, due to the immunocompetent passenger lymphocytes
within the transplanted liver producing antibodies against the recipient erythrocytes.
Some surgeons ignore HLA-matching in patient selection for donor shortages.
123 Introduction
Retrospective data has not shown any clear survival advantages associated with good
HLA-matching (Navarro V et al., 2006).
1.9.4 Immunosuppressive therapy general considerations
The immunosuppressive therapy used in liver transplantation includes corticosteroids,
calcineurin inhibitor (CNI),antimetabolites, inhibitors of TOR, and monoclonal and
polyclonal antibodies which have different patterns of action (Beaudreuil S et al., 2007).
Corticosteroids form complexes with cytosolic receptors, leading to their translocation to
the nucleus where they bind to glucocorticoid-response elements in the promoter
regions of cytokine genes, thereby blocking T cell-mediated cytokine expression. It is a
mainstay of treatment during the early days after transplantation, but are often
accompanied by many side-effects within a few years. Calcineurin inhibitor is the first
routinely employed immunosuppressive agent, including cyclosporine A (CyA) and
tacrolimus (FK-506). CyA selectively inhibits T lymphocyte proliferation by forming a
complex with cyclophilin. This complex can inhibit the calcium and calmodulindependent phosphatase calcineurin. Calcineurin is a key enzyme involved in controlling
the transcription of IL-2 and other cytokines (Friman S et al., 1996). Therefore, impairing
IL-2 transduction has a profound effect on the immune process of rejection by inhibiting
calcineurin. However, the CyA metabolism is complex in liver transplant patients.
Because it is metabolised primarily in the intestine and the liver, it increases the burden
on the liver .Fk506 is similar to CyA in action and side effects, and more potent. It binds
to the FK-binding protein 12.forming a complex inhibiting calcineurin, which regulates
the transcription of the genes encoding IL-2, IL-3, IL-4, IL-8, as well as various
chemotactic factors (Komolmit P et al., 1999). Antimetabolites were not initially used in
liver transplantation. Mycophenolate mofetil (MMF) has been shown to inhibit T and B
cell proliferation, to reduce the rate of acute rejection in renal transplantation. They can
be used together with an antibody against the IL2 receptor, to delay the introduction of
CINs. this led to the use of these drugs in liver transplantation. Combination therapy
with tacrolimus and MMF may significantly reduce the incidence of acute liver allograft
rejection, allow a significant reduction in tacrolimus dosage, and decrease the incidence
of nephrotoxicity (Eckhoff D E et al., 1998). In addition, the side-effects of MMF were
relatively few. Inhibitors of TOR mainly include Rapamicine and Everolimus.
Rapamicine is a macrocyclic triene antibiotic that is structurally similar to tacrolimus. It
forms a complex with the FK506-binding protein but it does not inhibit calcineurin. The
124 Introduction
complex blocks the cytokine response to T cell and B cell activation, preventing cell
cycle progression and proliferation. Its principal side-effects are leukopenia,
thrombocytopenia, high serum cholesterol and triglyceride levels, anaemia, lymphocele,
wound dehiscence and mouth ulcers (Levitsky J., 2011). The biggest advantage of
Rapamicine is associated with the lack of any significant nephrotoxicity (Vivarelli M et
al., 2010). Compared with Rapamicine, Everolimus has greater bioavailability and a
shorter half-life. The antibodies used in transplantation may be monoclonal or
polyclonal. At present, monoclonal antibodies primarily include IL-2R antibodies and
anti-CD52 antibodies. Two humanised IL-2R antibodies have been put on the market:
basiliximab and daclizumab, which inhibit T cell proliferation by the competitive
antagonism of IL-2- induced T cell proliferation, and they are accompanied with very few
side-effects. OK3 is also currently the most widely-used monoclonal antibody, which
binds to part of the T cell receptor (CD3) complex. The major impact of OK3 has been in
the reversal of steroid resistant, acute rejection (Cosimi A B et al., 1981). Polyclonal
antibodies are IgG fractions from animals inoculated with human lymphocytes,
thymocytes or cultured lymphoblast. Polyclonal antibodies have more profound and
long-lasting biological depleting effects than other antibodies (Rebellato L M). However,
polyclonal antibodies often induce the oversuppression of the immune system,
increasing the risk of infectious diseases, lymphoproliferative syndrome and tumours.
Tregs are a promising substance for the achievement of transplant tolerance.T
regulatory cells (Tregs), a subset of CD4+CD25+Foxp3+ lymphocytes, have the
functional ability to suppress alloimmune responses both in vitro and in vivo. Increasing
evidence from animal transplant research shows that Tregs can play a key role in
promoting immunological unresponsiveness to allograft transplants (Pilat N et al., 2010;
Webster KE et al., 2009). Regulatory T cells are the key cell-types in the induction of
immune tolerance, and so the modulation of such cells may provide new strategies in
creating transplant tolerance. However, there are several challenges to translating
Tregs into the clinic. Tregs only account for about 5-10% of the total CD4+ T cells in the
periphery, the limitation of cell number restricted the clinical application. There are a
number of studies demonstrating the functional instability of Tregs in vivo, which can
become IL-17 producing T effector cells in the presence of IL-6 (Yang XO et al., 2008).
Furthermore, T effector cells activated under inflammatory conditions are highly
resistant to Tregs-mediated suppression (Korn T et al., 2007).
125 Introduction
Immunosuppression regimen:
The optimal immunosuppression (IS) regimen remains the holy grail of organ
transplantation until tolerogenic interventions succeed is achieved, i.e. the level of drug
therapy which leads to graft acceptance with least suppression of systemic immunity.
This approach is further complicated by a lack of standardization in (IS)between
transplant programs and the management of chronic and, to a lesser extent, acute
cellular rejection (ACR)(A. J. Demetris, 2006). Current protocols use a combination of
drugs with different modes of action and toxicities directed at specific sites of the T-cell
activation cascade, thus allowing lower doses of each drug (V. K. Sharma, B et al
1994). Induction therapy refers to the practice of administering potent antibody therapy
in the perioperative period (when the risk of allograft rejection is greatest) and delaying
the introduction of maintenance therapy such as calcineurin inhibitors (CNI’s) which
have been the backbone of most immunosuppressive regimens in LT. Due to the wellknown adverse effects of long-term CNI use, alternative strategies such as CNI
minimization or even complete avoidance have been attempted (T. A. Gonwa, et al
2001, A. O. Ojo, et al 2003, A. B. Jain, et al 1998) .The standard immunosuppressive
regimen is a triple therapy containing either cyclosporine A (Neoral®, Sandimun®,
Novartis), methylprednisolon (Urbason®), and mycophenolate mofetil (MMF; CellCept®,
Roche)/mycophenolic acid (MPA; Myfortic®, Novartis) or tacrolimus (FK506, Prograf®,
Astellas), methylprednisolon, and MMF. CNIs such as cyclosporine A (CsA) and
tacrolimus are the most widely used in most liver transplant centers They inhibit the
calcineurin-calmodulin complex and therefore IL-2 production. adverse effects of CsA
are severe nephrotoxicity. The incidence of chronic renal dysfunction has reached to
70% of patients (Afonso RC, et al 2008; Ziolkowski J, et al.2003). End stage renal
disease has been described to occur in 18% of patients during a follow-up of 13 years
after LT(Gonwa TA, et al.2001)(3), dyslipidemia, and hypertension. In LT patients with
CNI-induced nephrotoxicity, a complete replacement of CNI with conversion to MMF
has shown conflicting results with respect to occurrence of rejection ranging between
0% and 60% (Créput C, et al. 2007; Moreno JM, et al. 2003; Moreno Planas JM, et al.
2004; Stewart SF, et al. 2001; Schlitt HJ, et al. 2001). MMF inhibits inosine
monophosphate dehydrogenase, an important enzyme in the de novo pathway of purine
synthesis. Results from previous studies with immunosuppressive regimens including
MMF and minimal CNI treatment suggest a significant improvement in renal function in
this patient group (Cicinnati VR (a), et al.2007; Beckebaum S (a),et al. 2004; Raimondo
126 Introduction
ML, et al. 2003; Cantarovich M, et al 2003; Garcia CE, et al. 2003). Beside potential
nephrotoxicity, CNI therapy is associated with cardiovascular complications, tremor,
headache, electrolyte abnormalities, hyperuricemia, hepatotoxicity, and gastrointestinal
symptoms, gingival hyperplasia and hirsutism. Cardiovascular side-effects due to CNI
and steroids include hyperlipidemia, arterial hypertension, and diabetes (Beckebaum
2004b). Treatment of hyperlipidemia with reductase inhibitors (statins) is safe and well
tolerated. Corticosteroids are a fixed part of initial and maintenance treatment.They
pocess a dose-dependent side effects including osteoporosis, diabetes, Cushing
syndrome, hypertension, and hyperlipidemia, and promotion of viral replication (HBV,
HCV), development of cataracts. Tapering and discontinuation of the therapy are
recommended during 6 months posttransplant. Steroid avoidance has been studied,
reports are encouraging and findings are promising with steroid-free protocols including
basiliximab induction therapy (Filipponi F, et al. 2004; Neuhaus P, et al 2002)
Tacrolimus therapy, show lower incidence of hypertension and hyperlipidemia, but has
the same nephrotoxicity and higher incidence of diabetes and neurotoxicity.
Neurotoxicity, including, tremor, paresthesia, muscle weakness, and seizures. Recent
reports showed a shift toward using more tacrolimus than CsA (Haddad EM, et al.
2006; Kaufman DB, et al. 2004) .The new prolonged-release tacrolimus (Advagraf®),
the dosage is reduced to one daily for better compliance (Wente MN,et al 2006).
Several studies including a large multicenter trial have shown that the use of MMF in
combination with tacrolimus or CsA has reduced the incidence of acute cellular rejection
in LTx and allowed the steroid withdrawal and CNI reduction (Papatheodoridis GV, et
al. 1999; Wiesner R, et al. 2001). MMF adverse effects include bone marrow
suppression, gastrointestinal symptoms, and increase incidence of lymphoproliferative
diseases, and opportunistic infections. The new potent immunosuppressive agents,
which belong to the inhibitor of the mammalian target of rapamycin (mTOR inhibitors)
include sirolimus (rapamycin; Rapamune®, Wieth) and everolimus (Certican®,
Novartis). Because of the additional and unique antiproliferative characteristics of
sirolimus, it has drawn the attention in the transplantation field especially in patients
undergoing LTx for hepatocellular carcinoma a satisfactory outcome and potential
survival benefit was reported in HCC patients with SRL-based immunosuppression
(Kneteman NM, et al 2004; Zimmerman MA, et al. 2008; Toso C, et al. 2007). Sirolimus
(SRL) is a macrolide isolated from streptomyces hygroscopius. It binds to a highly
conserved cellular protein, FKBP12, and to the rapamycin/FKBP12 complex targets,
127 Introduction
and it inactivates mTOR, which is considered a master switch for cell cycle progression
(Luan FL, et al. 2003). Reported side-effects of SRL include increased incidence of
wound infection and dehiscence due to its antifibrotic effect resulting in impaired wound
healing (Watson CJ, et al. 1999), HAT, hyperlipidemia, thrombocytopenia, leucopenia,
and anemia. Structurally, it resembles tacrolimus and binds the same receptor (FK
binding protein 12) but has different therapeutic and side effect profile because of a
different signaling pathway. The most important adverse effects of sirolimus include
impaired wound healing, hyperlipidemia, non-infectious pneumonitis, and prothrombotic
effect on hepatic artery (Scherer MN, et al. 2007; Mehrabi A, et al.2006). Individual
studies have demonstrated a benefit of SRL/MMF combination therapy (Kniepeiss D, et
al. 2003; Maheshwari A, et al. 2006) A second mTOR inhibitor, EVL, may exhibit
improved bioavailability as compared to SRL. The half-life of SRL and EVL is 62h and
approximately 30h, respectively, in stable kidney recipients treated with CSA. The
pharmacokinetics of EVL are less influenced by CSA. A study demonstrated that
everolimus in combination with oral CSA had an acceptable safety and tolerability
profile (Levy G, et al. 2006). However, the side effects were more frequent in the ERL
as compared to the ERL-free control group. Of note, there was no difference in the
incidence of thrombocytopenia or leukopenia between the groups. In some centers the
regimen includes induction agents added to the standard immunosuppressive agents
aiming for preventing or reducing the incidence of early rejection .The induction therapy
consists
of
antibodies
genetically
(basiliximab,
(alemtuzumab),
or
modified
non-depleting
daclizumab),
depleting
an
polyclonal
anti
monoclonal
CD52
antibodies
anti-CD25-receptor
monoclonal
antibodies
(thymoglobulin®,
ATG
Fresenius®,lymphoglobulin®). OKT3, the murine-depleting monoclonal anti-CD3
antibody, is currently used only in the setting of steroid-resistant rejection. Because of
severe side effects, such as pulmonary edema, fever, and gastrointestinal toxicity, it is
not used in most centers. The usage of depleting antibodies has the higher risk of
developing posttransplantation lymphoproliferative disorders (Mehrabi A, et al. 2007;
Scherer MN, et al. 2007).
1.10 Post-liver transplantation complications
OVERVIEW
Liver transplantation is accompanied by significant morbidity and mortality.(Lucey MR,
et al 1997, Belle SH, et al 1997; Devlin J, et al 1999, Hepp J, et al 2004) despite of the
128 Introduction
currently, high survival rates of over 90-95% and 70% at one year and five years posttransplantation, respectively (Roberts MS, et al 2004, Lucey MR, et al 1997, Belle SH,
et al. 1997), three-year patient and graft survival rates in liver transplant recipients are
currently 79% and 74%, respectively(RB Freeman et al 2008) and 1-year graft survival
rates exceed 80%.(K Waki. 2008). Transplanted patients survive longer, consequently,
posttransplant complications and causes of mortality has changed and new problems
have been encountered as patients survive longer. During long-term follow-up new
problems
affecting
transplant
recipients
have
been
identified
e.g.
metabolic
complications affecting both quality of life and long-term survival. The expanded use of
non-ideal donors to increase the transplant rate transplant (Delmonico et al., 2005)
increased the incidence of graft dysfunction in addition to the inevitable graft injury
during all the procedures Patient and allograft survival during the first several weeks
after transplantation are dependent primarily on parenchymal function, long term
allograft viability is determined primarily by biliary wound healing and adequate bile
drainage (Demetris AJ et al 2006, Busuttil RW, Tanaka K. 2003). In general
posttransplant complications can be divided into early and late complications with
considerable variation in the timing of all posttransplant complications.The main concern
in the first period is related to the immediate post-surgical survival together with
prevention of acute rejection. Recurrent disease is the commonest recognized cause of
late graft dysfunction (Hubscher SG. 2006), acute and chronic rejection are uncommon
at this time and may have different histological features to those seen in the early posttransplant period. (The period of 3–6 months represents an intermediate time, when
early and late changes overlap. Most of the main complications that occur during the
early posttransplant period can also be seen in late post-transplant biopsies (>1 year
post-LT) (Hubscher SG, Portmann BC. 2007) in different frequency. The liver interacts
with all body systems and many physiological changes take place in the recipient of a
liver graft During and in the immediate postoperative period, the liver is exposed to
different factors leading to different outcome Factors affecting the postoperative
outcome of each patient varies, some are related the patient’s(recipient) preoperative
state including hypotension, hypoxia, ischaemia and hepatotoxic drugs, others related
to the quality of the donor and the graft such as hepatic steatosis, use of vasoactive
drugs, hemodynamic changes, as well as technical problems related to the surgical
procedure such as intra- or postoperative hemorrhage, vascular complications, and
biliary complications. (Keeffe EB. 2001, Murray KF, 2005), and immune responses.
129 Introduction
Early and accurate diagnosis of such complications, as well as prompt appropriate
interventions are essential for optimal patient and graft outcome. Establishing the
correct diagnosis is essential as the differential diagnosis is not simple due to the
similarities of clinical manifestations and laboratory results abnormalities of most liver
transplant complications After the patient is wheeled from the operating theater to the
intensive care ward, several important problems may arise, for which the laboratory may
be central to the diagnosis and management. Assessment of the liver transplant
recipient requires the choice of tests that provide adequate diagnostic information, at
the minimum cost. Many tests have been proposed as being potentially useful in
assessing the posttransplant recipient but most of the routine tests that are used in
assessing the liver transplant recipient are individually nonspecific; they identify the
presence of a problem, but not the problem itself. However, the use of tests in
combination increases their diagnostic efficacy.
Donor, graft, and recipient factors that have been shown to adversely affect
posttransplant patient survival rates. ICU, intensive care unit; UNOS, United
Network for Organ Sharing.
Donor Factors
Recipient Factors
Graft Factors
Advaced age
Severity of illness:
Prolonged ICU stay
Prolonged hospital stay
History of hypertrension
History of diabetes
Low serum bicarbonate.
Advanced age
Severity of illness i.e. Urgent
UNOS status.
Donation after cardiac death
Prolonged cold ischemia time
Macrovesicular steatosis:
Moderate-Severe.
Liver function tests available at all times
Function assessed
Analyte
Synthetic
Albumin; Ammonia; PT
Multicompartmental
Bilirubin (total)
Biliary
ALP; GGT
Hepatocyte integrity
Aspartate transaminase; Alanine transaminasi
Dynamic real-time test
Monoethylglycine xylidide (this test is performed sparingly)
130 Introduction
Early Complications After Liver Transplantation
Type
Complication
Pulmonary
Cardiovascular
Renal
Metabolic
Infectious
TRALI/ARDS, pneumonia, pleural effusions, persistent shunting
CHF, hypotension, myocardial ischemia, arrhythmias
ATN, hepatorenal syndrome, CNI nephrotoxicity
Hyperglycemia/diabetes mellitus, electrolyte abnormalities
Persistent encephalopathy, seizures, posterior leukoencephalopathy, central
pontine myelinolysis
Wound infections, seromas, intraabdominal abscess, sepsis
Abdominal
Surgical bleeding or bleeding as a result of coagulopathy
Vascular
Hepatic artery or portal vein stenosis/thrombosis, IVC obstruction
Biliary
Biliary stricture or leaks
Rejection
Cellular rejection
Immunosuppressant toxicity
Seizures, kidney failure
Neurologic
ARDS, acute respiratory distress syndrome; ATN, acute tubular necrosis; CHF,
congestive heart failure; CNI, calcineurin inhibitor; IVC, inferior vena cava; TRALI,
transfusion-related acute lung injury
Hepatic
Postsurgical
Primary nonfunction
Early graft dysfunction
Vascular
Hepatic artery thrombosis
Portal vein thrombosis
Hepatic vein thrombosis
Biliary
Bile leak
Biliary stricture
Immunologic
Acute cellular rejection
Hepatic
Immunologic
Chronic rejection
Disease recurrence
Hepatocellular carcinoma
Viral hepatitis
Autoimmune disease
Alcoholic liver disease
Nonalcoholic fatty liver disease
EARLY COMPLICATIONS:
Nonhepatic
Bacterial infection
Fungal infection
Cytomegalovirus infection
LATE COMPLICATIONS:
Nonhepatic
Metabolic/cardiovascular
Hypertension
Dyslipidemia
Diabetes mellitus
Obesity
Renal failure
Atherosclerotic cardiovascular disease
Neoplastic
Cutaneous malignancy
Posttransplant lymphoproliferative disease
Solid organ malignancy
Infectious CMV late onset
131 Introduction
Post OLT complications can be categorized according the time of occurence, and
the type of complications (The period of 3–6 months represents an intermediate time,
when early and late changes overlap).
TIME
TYPES OF COMPLICATIONS
0-1 Month
Infections:
–Bacterial (related to the procedure) wound infection, pneumonia, biliary sepsis, col.
Difficile infection, catheter related
–Viral: HSV stomatitis, HCV, Hepatitis B, (if without prophylaxis)
–Fungal: Candida, Aspergillus
–Parasites: Strongyloides
Allograft dysfunction:
–PNF in first two weeks
–Acute cellular rejection
–Small-for-size Syndrome
Biliary tract:
–Bile leaks
–Anastomosis disruption
–Hepatic duct stricture/hepatic artery thrombosis
•Disease recurrence:unusual
1-6 Months
Infections:
–Viral: HHV6, Adenovirus, RSV, Viral reactivation (CMV, EBV,VZV,HCV,HBV),
–Bacterial: Listeria, Nocardia, TB,
–Fungal: Pneumocystis, Aspergillus, Cryptococcus, Hystoplasma, Coccidioides,
–Parasites: Toxoplasma, Strongyloides, Leishmania, -Trypanosoma
Allograft dysfunction:
–Recurrent HCV
–Rejection
–Hepatic artery thrombosis
Biliary tract:
–Biliary stricture
–Leak associated with T-tube removal
Disease recurrence:
–HCV,
–PBC,
–PSC (if after > 90 days),
–Alcohol (rarely)
>6 months
Infections:
–Community acquired infections (UTI, pneumonia)
–VZV, CMV, influenza, papillomavirus, PTLD
•Allograft dysfunction:
–Chronic Rejection
–Lymphoproliferative Syndrome (PTLD)
–Underlying Disease
•Biliary tract: < 4% per year
•Disease recurrence: HCV, PBC, PSC, alcoholism within two years
1.10.1 The main complications in the immediate postoperative period
The main Immediate complications are related to the function of the graft (dysfunction
and rejection), the surgical technique, infections: bacterial, fungal, and viral (Mazariegos
GV et al 1999), and systemic problems (pulmonary, renal, or neurological).
132 Introduction
Complications during follow up period.
Immediate complications:
Medical complications:
Hemodynamic complications
Respiratory changes
Renal dysfunction
Neurological complications
Technical complications
Postoperative hemorrhage
Vascular complications
Biliary tract complications
Liver graft dysfunction
Initial poor function
Acute cellular rejection
Recurrent viral hepatitis
Infections
Bacterial
Viral
Fungal
Long-term complications
Chronic rejection
Renal failure
Arterial hypertension
Diabetes mellitus
Dyslipidemia
Obesity
Bone complications
Neurological complications
Malignancy
Allograft dysfunction and surgical complications occurring in the immediate
postoperative period.
Allograft dysfunction
Primary non function
Initial poor function
Acute cellular rejection
Recurrent viral hepatitis
Drug hepatotoxicity
Surgical complications
Postoperative hemorrhage
Vascular complications
Hepatic artery thrombosis
Portal vein thrombosis
Hepatic venous obstruction
Other
Biliary tract complications
Bile leak or fistula
Biliary stricture
133 Introduction
Technical complications The prevalence of technical complications is on average
26% and consists of haemorrhage, vascular complications, and biliary complications.
Technical complications after liver transplantation: type and onset
Complication
Type (onset)
Abdominal bleeding
Anastomoses (immediate)
Site of implantation (immediate)
Vascular complications
Hepatic artery thrombosis (early)
Hepatic artery stenosis (late)
Portal vein thrombosis (early)
Portal vein stenosis (immediate)
Suprahepatic/infrahepatic vena caval obstruction
(immediate)
Biliary complications
Biliary leakage (early)
Biliary strictures (late)
Stenosis of papilla vateri (early)
Non-specific surgical complications
Infections (early/late)
Small bowel obstruction (early/late)
Injury of intra-abdominal organs (immediate)
Previous operations (immediate)
Postoperative Hemorrhage in the immediate postoperative period
Causes for early postoperative haemorrhage
Patient with serious preaoperative coagulopathy and thrombocytopenia.
Initial poor function, primary non-function.
Liver surface injury.
Anastomotic leak.
Heparin induced.
Associated with interventions, including liver biopsies.
Mycotic aneurysm, rare event.
Hepatic artery stenosis and thrombosis
Postoperative haemorrhage of different causes necessitating surgical intervention
occurs in approximately 10–15% of liver-transplanted patients. (Gordon RD, van Thiel
DH & Starzl TE. 1993). The most important risk factor of early postoperative
haemorrhage is the preexisting severe coagulopathy and thrombopenia. Patients with
poor initial graft function are also subjects of such complication due to insufficient
synthesis of coagulation factors (immediate poor synthetic function). Fibrinolysis is
activated on full reperfusion as a result of the ischemia/reperfusion injury leading to
significant hemorrhage during surgery. Postoperative bleeding most frequently result
134 Introduction
from the donor procedure from lacerations of the right hepatic lobe during harvesting or
can results during the recipient hepatectomy. Other sources for postoperative
haemorrhage include the gall bladder bed, cystic artery, and non-detected small veins
draining into the vena cava are further. During recipient hepatectomy, adrenal gland
injury is an important cause of repeated postoperative haemorrhage. Postoperative
haemorrhage from different vascular anastomoses is another source, it is usually
diagnosed within the first 48 hours post-transplantation in such condition liver function
remain unaffected. The diagnosis is predominantly clinical. The patient can present with
hemodynamic instability; hypotension, tachycardia, abdominal distension, hemorrhagic
abdominal drainages (percutaneous drains can fail if the haemorrhage occurs more
than 2 days after transplantation even when the drains are still properly placed),
decrease in mixed central venous oxygen saturation, and deterioration of kidney
function and serial determination of the hematocrit/hemoglobin. Ultrasound of the
abdomen shows sub- or perihepatic haematoma and intraperitoneal fluid. Using the
angio-CT scan, intrahepatic haematoma can be sensitively differentiated from the less
dangerous extrahepatic haematoma as well as from other processes like liver
abscesses or areas of malperfusion. Management is conservative, however, reoperation is needed in 10-15% of cases, and the cause of the hemorrhage is found in
only 50% of these. (Motschman TL, et al 1989, De Boer MT et al 2005) Guidelines for
reoperation include the use of more than 4–6 units of packed red blood cells within 24
hours and haemodynamic instability of the patient. In all patients, with postoperative
haemorrhage, coagulation studies should be closely performed and coagulation factors
including antithrombin III and factor XIII substituted accordingly).
135 Introduction
Vascular Complications: are either arterial or venous complications
Time of occurence
Leading symptoms
Treatment
Early
Fulminant increase in LFTs
Acute liver failure Hemodynamic
instability
Urgent acute thrombectomy or
urgent retransplantation
Late
Biliary complications Strictures,
intrahepatic abscesses
Cholangitis and sepsis
Management of biliary
complications using ERC, PTC
Rt-PA lysis therapy Elective
retransplantation
Hepatic artery stenosis
Slight increase in LFTs Mild or
late biliary complications
Reoperation with resection of
the anastomosis and end-to-end
reconstruction
Early
Acute liver failure, fulminant
increase in LFTs, hemodynamic
instability Ascites, variceal
bleeding
Urgent thrombectomy
Urgent retransplantation
Late
Slight increase in LFTs Portal
hypertension,
ascites, variceal bleeding
Endoscopic treatment Rt-PA
lysis therapy
Elective retransplantation
Slight increase in LFTs Portal
hypertension, ascites
Resection and end-to-end
reconstruction
Hepatic artery thrombosis:
Portal vein thrombosis:
Portal vein stenosis
ERC, endoscopic retrograde cholangiopancreatography; LFT, liver function tests; PTC,
percutaneous transhepatic cholangiopancreatography; Rt-PA, recombinant tissue-type
plasminogen activator.
Arterial complications
Hepatic Artery Thrombosis (HAT) a native non-transplanted liver might function very
well without arterial blood supply, which is not true for a transplanted liver. The liver
allograft is devoid of a collateral arterial circulation making it more susceptible to
ischemic injury especially early in the post transplantation period. The majority of
hepatic artery thromboses occur during the early postoperative period, although they
can also occur several months after transplantation. Advanced surgical techniques have
decreased its incidence, but still remains the most frequent cause of vascular
complications after liver transplantation. (Starzl TE, Demetris AJ 1990). Children are
more susceptible for the development of hepatic artery thrombosis because of the small
diameter of arterial vessels. The incidence of hepatic artery thrombosis ranges between
2.5 and 10% in adults and 15–20% in children (Tzakis AG, et al 1993, Sanchez-Bueno
F, et al. 1994; Valente J, et al 1996.) The need for vascular extension grafts increases
136 Introduction
the risk for the development of arterial thrombosis by up to 70% in children. This is
especially the case if the extension graft is placed to the infrarenal aorta. The use of the
supracoeliac aorta has reduced the incidence of hepatic artery thrombosis to 12.5% in
children and is not related to an increased risk for the development of arterial
thrombosis compared with primary anastomosis (2.5–10%) in adults. In general, early
occurrence of hepatic artery stenosis and thrombosis are more dangerous in terms of
graft and patient survival. The presentation of HAT varies from dramatic elevations of
aminotransferases indicating fulminant acute allograft ischemia and/or infarction. HAT
may be clinically silent and can be discovered incidently during radiographic
assessment of the allograft, particularly if it occurs late after. Undetected non
reconstructed
right
hepatic
artery
during
the
donor
procedure
and
during
transplantation, will result in complete necrosis of the right hepatic lobe requiring urgent
retransplantation.
Predisposing factors for hepatic artery thrombosis
Multiple recipient arteries (28%).
Inadequate inflow secondary to proximal stenosis Coeliac trunk stenosis.
Lienalis steel syndrome.
Violation of the intima of the donor hepatic artery, haematoma.
Multiple donor arteries requiring reconstruction.
Infrarenal interposition graft (not to be done).
Children, split, and living related liver transplantation due to smaller vessel diameter.
Use of bypass grafts, arterial intima trauma or injury, and anomalous anatomy
necessitating complex back table arterial reconstruction procedures.
HAT diagnosis and management
Depend on the time of occurrence and clinical consequences. Diagnosis of HAT is
suggested when duplex ultrasound cannot identify flow in the hepatic artery, and should
be confirmed by visceral angiography. It occurs due to multiple causes including poor
arterial flow, increased sinusoidal resistance, preservation injury, stenosis of the
anastomosis and a state of hypercoagulability. Hepatic artery thrombosis (HAT) can be
broadly divided into early (within the first 1 to 2 months after OLT) and late (> 6 months
post transplantation) differening in etiologies, clinical presentation and patient
manifestations, and treatments. When occurring at an early stage, it typically leads to
137 Introduction
ischemia/necrosis of the graft; but when it occurs at a later time point, it results in biliary
complications which take the form of intrahepatic biliomas and biliary stenosis with
preserved graft functions.
Early HAT occurs within the first 1 to 2 months after OLT and has a mean incidence of
2.9% in adult OLT patients, and of 8% to 10% in the pediatric population (Bekker et al,
2009; Farmer et al, 2007). Early HAT results in graft loss and death in 53% and 33%,
respectively (Bekker et al, 2009). Technical, donor, and recipient factors contribute to an
increased risk of HAT (Bekker et al, 2009; Del Gaudio et al, 2005; Duffy et al, 2009;
Jurim et al, 1995; Soin et al, 1996; Vivarelli et al, 2004). Donor factors include smallcaliber vessels, aberrant anatomy that requires complex arterial reconstruction, use of
aortic conduits, cytomegalovirus (CMV) seropositivity, and donor-recipient mismatch.
Recipient factors include variable recipient anatomy. Early HAT may be asymptomatic,
or often presents with bile leak, cholangitis, or sepsis because hepatic artery is the sole
blood supply to the donor bile duct. It frequently results in massive hepatocellular injury
and the patiens represent with increased transaminase levels and impaired hepatic
synthetic function. Duplex ultrasound (US) is diagnostic in adults, although visceral
angiography remains the gold standard. Emergency exploration with attempted
thrombectomy and revascularization may salvage the graft. Some reports suggest that
endovascular
procedures—intraarterial
thrombolysis,
percutaneous
transluminal
angioplasty, and endoluminal stenting—can be successful in hepatic arterial
revascularization (Singhal et al. 2010); however, a majority of patients eventually
require retransplantation (Bekker, et al. 2009, Duffy et al, 2009). Because of the severity
of this complication, some authors advocate the routine use of postoperative duplex US.
Late HAT Patients may be asymptomatic with no clinical consequences (Gunsar et al,
2003). Other patients may present with fever secondary to perihepatic abscess or biliary
leak, biliary strictures, or cholangitis. Factors affecting late HAT occurence are active
tobacco abuse; coagulation abnormalities such as factor V Leiden; cerebrovascular
accident as donor cause of death; donor age death > 50 years; recipient CMV positivity;
and use of donor iliac interposition graft (Del Gaudio et al, 2005; Gunsar et al, 2003;
Pascual et al, 1997; Pungpapong et al, 2002; Stewart et al, 2009; Vivarelli et al, 2004).
Treatment may be attempted with endoscopic or percutaneous biliary decompression,
stenting, or even systemic anticoagulation. Retransplantation is less required.
Antiplatelet therapy used post peratively may reduce the rate of late HAT in high-risk
patients (Vivarelli et al, 2007)
138 Introduction
Hepatic Artery Stenosis
Hepatic artery stenosis (HAS) incidence is of 4% to 11% (da Silva et al, 2008). The use
of DCD allografts is main a risk factor (Pine et al. 2009). Initially, the patients present a
mild increase in aminotransferase levels with or without associated graft dysfunction or
biliary complications. Doppler US demonstrating an increased resistance in hepatic
arterial flow confirmed by arteriography is the diagnostic gold standard. Therapeutic
options include angioplasty with or without stenting. Restenosis can occur in up to one
third of patients within 1 year after stenting (Ueno et al, 2006).
Portal vein thrombosis.
Is a less frequent complication than HAT and adversely affects the overall survival after
OLT (Duffy et al, 2009). The prevalence is of 2-3%, and the incidence less than 2% in
adult recipients and 10% in pediatric recipients (Duffy et al, 2009; Lerut et al,
1987; Millis et al, 1996). PVT). Pretransplantation factors including prexisting portal
thrombosis, splenectomy, and previous portal hypertension surgical intervention, low
portal flow, veins with small-diameter (<5mm), donor-recipient vessel size mismatch,
and the use of vascular grafts for reconstruction are the main risk factors for PVT
occurrence. (Cheng et al, 2004). PVT is typically symptomatic, and patients can present
early with acute hepatic failure, similar to HAT, or later portal hypertension
complications e.g. ascites, splenomegaly, and variceal/gastrointestinal hemorrhage
(Duffy et al, 2009). Diagnosis is made using duplex US or contrast-enhanced CT portal
venography. Management of such patient depend on the severity and the time of the
presentation. In patients presenting with fulminant hepatic failure, exploration and portal
revascularization are performed. Some particular patients with combined PVT and HAT
require retransplantation. Portocaval shunt to augment flow through the reconstructed
portal vein has been described (Bakthavatsalam et al, 2001), as has the use of
transjugular intrahepatic portocaval shunt (TIPS) in conjunction with thrombolytics
(Ciccarelli et al, 2001). Systemic anticoagulant may be sufficient in patients with
preserved graft function (Duffy et al, 2009). In case of preserved graft function
symptoms of portal hypertension can be managed medically with standard therapies for
ascites in combination with variceal banding or sclerotherapy; however, the graft
salvage rate is less than 50% (Duffy et al, 2009).
139 Introduction
Portal Vein Stenosis
Portal vein stenosis, is frequently diagnosed on routine screening US in asymptomatic
patients. The most common area of stenosis is the extrahepatic portal venous
anastomotic site. Narrowing of the main portal vein diameter by more than 50%,
presence of a poststenotic jet, or lack of visualized flow on Doppler imaging are
diagnostic. Management includes percutaneous transhepatic balloon angioplasty and
stenting (Woo et al, 2007),or surgical resection followed by direct anastomosis
with/without a venous graft. (Pastacaldi S, et al, Vivarelli et al 2004)Biliary complications Biliary complications occur early and late in the post-transplant
course. They are considered the Achilles’ heel of liver transplantation, particularly in the
setting of live donor liver transplantation. The clinical picture is variable and depends on
the time of development, lead time to diagnosis, and the presence of a T-tube. Some
criteria are indicatives of
biliary problems, these include the lack of bile formation
through the drainage, the formation of a bilioma radiologically confirmed, increase of
cholestatic enzymes (GGT, ALP), and discrete leukocytosis. Treatment is initially by
interventional radiology and/or endoscopy, surgical intervention in up to 10-20% is
required for a definitive resolution. At the time of transplantation, reconstruction of the
biliary tract occurs in the form of a duct-to-duct anastomosis or choledochojejunal
anastomosis. Mucosal and/mural damage may occur in the process and lead to biliary
tract complications, such as bile leaks, and anastomotic or intrahepatic strictures. The
process of biliary wound healing takes place and may or may not be ineffectual. This
can affect the small extrahepatic biliary tree and/or the large extrahepatic biliary tree. In
the extrahepatic large bile ducts, biliary healing may lead to scarring and stricture
formation. In the small extrahepatic bile ducts, impaired proliferation of the bile duct
epithelium or exuberant responses can contribute to liver injury.
Biliary sludge syndrome
Cold ischaemic-preservation injury depletes energy stores in microvascular endothelial
cells and bile duct epithelium. As a result, metalloproteinases are activated. Biliary
epithelium
and
endothelium
are
detached
from
underlying
matrix.
In
the
microvasculature, detachment of endothelium predisposes to thrombosis after
reperfusion. (Kukan M, Haddad PS. 2001, Teoh NC, Farrell GC. 2003) Leucocytes
become activated by tissue damage and release effector molecules, causing more
140 Introduction
tissue damage and further promote thrombogenesis. (Kukan M, Haddad PS. 2001,
Teoh NC, Farrell GC. 2003) Several factors, including increased sensitivity of bile duct
cells to reperfusion injury, poor functional recovery after ATP depletion, invasion of
polymorphonuclear leucocytes into bile ducts, and hydrophobic bile salts, appear to
contribute to preservation-related injury of bile ducts. (Kukan M, Haddad PS. 2001,
Teoh NC, Farrell GC. 2003, Noack K, et al 1993, Carrasco L, et al 1996) Damaged
biliary epithelial cells are sloughed into the bile, the underlying stroma become exposed
to bile which form a nidus for crystallisation of biliary sludge ( Demetris AJ, et al 2006)
Injury of bile ducts is associated with hyperbilirubinaemia and underlies the long lasting
phase of reperfusion graft injury. (Kukan M, Haddad PS. 2001, Teoh NC, Farrell GC.
2003). Morphological changes take place in the extrahepatic large bile ducts and
intrahepatic large bile ducts, as well as in the small intrahepatic ducts. Biopsies usually
sample the peripheral liver, therefore small intrahepatic ducts are that what is
encountered in biopsies. There is prominent ductular reaction consisting of biliary cells
and periductal myofibroblasts due the increased pressure in the biliary tree distal to the
point of luminal obliteration. The proliferating ductules and myofibroblasts form a wedge
of tissue that arises from the portal tract and distorts the liver architecture. Large bile
ducts are usually seen at the time of re-transplantation in the excised failed graft. There
is biliary sludge, mucosal ulcers and inflamed granulation tissue and myofibroblast
proliferation in the wall of extrahepatic bile ducts and large intrahepatic bile ducts
(Demetris AJ, et al 2006) As a result of exposure of the underlying stroma; inflammation
and activation of myofibroblasts take place which lead to wound contraction and
fibrosis, and strictures in large-calibre ducts. Complete fibrous obliteration of the bile
duct lumens by concentric rings of fibrous tissue occurs.
Biliary fistula Occurs initially in the first or the third month. In the first month it is related
to anastomotic dehiscence secondary to technical errors or biliary tract ischaemia. In
the third month it is related to the T-tube withdrawal. Management of biliary
complications
depends on the patient’s condition and the postoperative time.
Conservatively; opening the T-tube and appropriate antibiotic coverage can be used.
Other cases
require endoscopic papillotomy and/or percutaneous drainage of the
bilioma . Failure of the above or in case of peritonitis, open surgery is considered.
141 Introduction
Biliary obstruction
Anastomotic stenosis, intrahepatic stenosis and coledolithiasis result in this type of
complication. The clinical picture is variable ranging from cholestatic enzymes elevation
in an asymptomatic patient to a septic shock due to bacterial cholangitis (Moser MA, et
al 2001).
MEDICAL COMPLICATIONS
When the transplant evolves favorably, the patient is awake, hemodynamically stable,
with spontaneous respiration, preserved renal function, and with progressively
improving liver activity. When complications develop, the stay in the intensive care unit
is
prolonged
and
mortality
increases.
The
global
mortality
in
this
early
posttransplantation period is approximately 5-10%. The most frequent medical
complications that can be expected during this early post-transplant period are
hemodynamic alterations, and respiratory, renal and neurological complications.
Hemodynamic complications are frequent during the early post-transplant period. The
most commonly encountered is arterial hypertension,caused by the effect of
immunosuppressive drugs, the presence of intense pain, or due to hypervolemia
secondary to excessive hydrous replacement.It is managed by calcium inhibitors and/or
diuretics.
Electrolytic alterations of sodium, potassium, calcium, and magnesium, due to hepatic
reperfusion and to the transplant , can cause cardiac arrhythmia most frequently
bradycardia and to lesser extent supraventricular arrhythmias (particularly atrial
fibrillation) and need to be quickly managed if they persist, additional factors such as
acidosis, renal or liver failure, must be excluded. In patients with a history of ischemic,
hypertensive or valvular disease once transplantation has been performed and despite
a careful pre-transplant cardiologic evaluation, the cardiopathy may destabilize
(Mazariegos GV, et al 1999).
Respiratory changes Any abdominal surgery causes reduced ventilation capacity,
together with the reduction in diaphragm motility and/or the presence of ascitis. Pleural
leakage, predominantly on the right, is the most frequent complication with a prevalence
reported to be as high as 100% in some patient group. Atelectasias, pneumo- or
142 Introduction
hemothorax are less frequent. Prior hypoproteinemia, fluid replacement in large
amounts during surgery and the development of renal insufficiency are important
determinant factors as they can result in interstitial edema and acute pulmonary edema.
Primary graft failure, hemorrhage, respiratory infection, respiratory distress syndrome or
emboligenic problems secondary to surgery may complicate removal of the mechanical
ventilation (Snowden CP, et al 2000).
Renal dysfunctions Changes in renal function during this period: are affected by many
factors such as the prior existence of renal dysfunction, peri-operative hemorrhage,
vascular clamping with hypotension, the use of nephrotoxic drugs, sepsis, a state of
shock, and graft dysfunction. Renal dysfunction is defined by a creatinine level above 23 mg/dL and/or an increase in the basal seric creatinine greater than 50%. The clinical
manifestations are oliguria, diuresis of less than 0.5 mL/kg/h, electrolytic changes,
ascitis, edema and acid/base disorders with increases in the levels of creatinine
between the second and fourth days postoperatively. A state of euvolemia has to be
maintained
with
adequate
renal
perfusion
pressures,
colloid-based
hydrous
replacement should be aggressive. Early dialysis must be considered at all times if
necessary (Bilbao I, et 1998). Liver transplant recipients have a high risk of developing
posttransplant chronic renal failure, with an incidence of 18% at 5 years (Ojo et al,
2003). The average functional decline in renal function is a 38% decrease in glomerular
filtration rate, and it is correlated with time elapsed since transplantation (decline by 36
2
mL/min/1.73m ) (Bucuvalas et al, 2006; Karie-Guigues et al, 2009). The use of
calcineurin inhibitors (CNIs) is associated with increased risk of renal failure in patients
after OLT, although this decline may be partially attenuated with concomitant
administration of mycophenolate mofetil and calcineurin dose reduction (Karie-Guigues
et al, 2009). Other risk factors for the onset of post-OLT renal dysfunction include age
and gender of the recipient, history of HCV infection, DM, and pretransplantation renal
insufficiency, CAD, and primary nonfunction (Ojo et al, 2003; Pawarode et al, 2003).
Mortality following OLT is four times greater in recipients who develop posttransplantation renal failure (Ojo et al, 2003).
Neurological complications. Neurologic complications occur with greater frequency in
recipients of liver allografts than in other solid-organ recipients (Senzolo et al, 2009).
The aggregate prevalence of neurologic sequelae is 25%, although such have occurred
143 Introduction
in more than 60% of patients in some series (Amodio et al, 2007; Bronster et al, 2000;
Emiroglu et al, 2006; Ghaus et al, 2001; Lewis & Howdle, 2003; Saner et al, 2006).
Posttransplantation encephalopathy is the most common neurologic complication,
followed by seizures (Bronster et al, 2000; Lewis & Howdle, 2003; Saner et al, 2006;
Senzolo et al, 2009). Encephalopathy can be caused by anoxia, sepsis, medications
(especially CNIs), primary graft nonfunction, renal failure, rejection, and central pontine
myelinolysis (CPM) (Erol et al, 2007). Infection, stroke, and CPM are also common
causes of seizures (Senzolo et al, 2009). Other neurologic complications include
posterior leukoencephalopathy, cerebellar syndrome, focal deficits, headache, tremor,
sleep disorders, and peripheral neuropathy. Risk factors for neurologic complications
include an operative time longer than 10 hours, high CTP score, and a history of hepatic
encephalopathy
(Dhar
et
al,
2008).
Older
age
and
higher
MELD
scores
pretransplantation are associated with increased risk of tacrolimus-related neurotoxicity
(DiMartini et al, 2008). In cases of CNI-related neurotoxicity, patients can frequently be
switched to a different drug within the same class with successful resolution of
symptoms (Emiroglu et al, 2006; Erol et al, 2007). Most central nervous system (CNS)
complications (80%) occur within 1 month after OLT but may be seen up to several
years after transplantation (Bronster et al, 2000). The incidence of neurologic complications in LDLT recipients (17%) approximates that of cadaveric allograft recipients, and
outcomes from these complications appear to be similar (Saner et al, 2010).
LIVER GRAFT DYSFUNCTION
Normal postoperative course, is manifested by progressive decrease of transaminases,
increase of factor V, prothrombin and platelets, control of acidosis, normalization of
ammonium, good biliary production, and absence of encephalopathy. Dysfunction of the
graft may occur in the immediate postoperative period (early dysfunction) or late during
the follow-up of the patient typically related to the recurrence of the original disease
(viral hepatitis, primary biliary disease, sclerosing cholangitis, alcohol or autoimmune
liver disease) or chronic rejection. Graft dysfunction can be a result of various factors
including status of donor, quality of hepatic graft, organ harvesting, ischemiareperfusion injury, SFSS, primary liver disease, status of liver function of recipients and
operative techniques, etc. Although ECD from older donors or steatotic grafts provide a
solution for the shortage of allografts, donor factors could still predispose recipients to
IPGF and/or PGNF. With extended application of living donor liver transplantation
144 Introduction
(LDLT), SFSS has become the main problem. The occurrence of SFSS depends on a
number of recipient, graft and technical factors. Clinically, primary dysfunction is defined
as IPGF or PGNF. Primary graft non-function (PGNF) is the most severe type of graft
damage after OLT, followed by initial poor graft function (IPGF) (Chui et al., 2000).
Incidence is variable ranges from 2% and 23%. Ploeg et al reported the rates of IPGF
and PGNF for 22% and 6%, respectively (Ploeg et al., 1993). Ardite et al's study
showed the rates of IPGF and PGNF for 19% and 0% (Ardite, et al.1999) in contrast to
29.5% and 0.93%, respectively in Chui et al's investigation (Chui, et al. 2000). In the
study by Chen et al, the rates of IPGF and PGNF were 36.25% and 1.3%, respectively
(Chen et al., 2007). In The Scientific Registry for Transplant Recipients (SRTR) analysis
enrolling 10545 deceased donors, adult first transplants, 613 (5.8%) cases of PNGF
occurred (Johnson et al., 2007). In another single-center analysis of donors after
cardiac death (DCD), PGNF occurred in 6.4% of brain dead donors (DBD) vs. 11.8% of
DCD (Abt et al., 2004) IPGF and PGNF are influenced and affected by many factors,
such as status of donor, quality of hepatic graft, long-term warm ischemia, cold
ischemia, primary liver disease, status of liver function of recipients and operative
techniques (Brokelman et al., 1999). For evaluation of the donor hepatic allograft with
regard to pre-existing diseases, in particular macrovesicular steatosis and posttransplant evaluation of hepatic graft function, liver biopsy is the most challenging and
valuable clinical practice. Recently, some progress has been made in the prevention
and treatment of early hepatic graft dysfunction.
Causes of early dysfunction of the graft
Early graft dysfunction can be due to problems of the graft itself (primary
dysfunction/malfunction, nonspecific cholestatic syndrome, rejection), complications of
the surgical technique (biliary and/or vascular (arterial, portal thrombosis, poor drainage
of the suprahepatic veins) or other causes such as drug-related liver toxicity (e.g.,
cyclosporine) or infections (CMV, bacterial). The problem in many of these cases is the
differential diagnosis, since although from a clinical and biological point of view, they
share many manifestations, the therapeutic approach is completely different.
Factors affecting occurence IPGF and PGNF status of donor (age, gender, obesity,
weight, height, BMI, elevated liver functions, hypotension/increased administration of
vasopressor and hypernatremia, cause of donor death), quality of hepatic graft (e.g.
145 Introduction
graft steatosis), organ harvesting, ischemia-reperfusion injury, SFSS, recipient primary
liver disease, status of liver function of recipients, operative techniques.
Primary graft failure The concept of primary graft dysfunction is not clear. Clinically,
primary dysfunction is defined as IPGF or PGNF. Primary graft non-function (PGNF) is
the most severe type of graft damage after OLT, followed by initial poor graft function
(IPGF) (Chui et al., 2000). Emergency hepatic retransplantation is necessary because
of the extreme high mortality of PGNF The difference between IPGF and PGNF
depends on the degree of dysfunction, the time length after liver transplantation and the
need for urgent retransplantation. The exact cause of this severe complication is
unknown. Predisposing factors include advanced age, hemodynamic instability,
suboptimal donors, cold ischemia time, reperfusion damage, release of intestinal
endotoxins, drug-related liver toxicity. Histopathology findings are those of ischemic
hepatic necrosis. Prostaglandins can be used within the first hours of the procedure, in
an attempt to improve microcirculation of the liver. However, if regression of the clinical
situation is not observed after 24-48 hours, retransplantation must be considered as
soon as possible to avoid the development of multi-organ failure although the mortality
associated with retransplantation is very high. (Deschenes M, et al 1998 , Demetris AJ.
2001)
PGNFDefinition
Generally, PGNF is defined as the clinical situation in which there is poor liver function
to maintain the individual’s life leading to death of the patient or retransplantation during
the first seven postoperative days. It is one of the most serious situations in the early
post-transplant setting, its incidence is estimated at 5-10% after reperfusion, and is
manifested by hepatic cytolysis and rapidly immediate rising transaminases, absence of
bile production, severe liver-related coagulation deficit, hypoglycemia, high lactate
levels, and hepatic hemodynamic instability (Uemura et al., 2007). According to the
United Network for Organ Sharing (UNOS), PGNF is defined as irreversible graft
function requiring emergency liver replacement during the first 10 days after liver
transplantation. It is characterized by an AST ≥ 5000 UI/L, an international normalized
ratio of prothrombin (INR) ≥ 3.0, prothrombin time < 60% despite administration of
plasma and coagulopathy Factor V < 20%, and lactic acidosis (pH ≤ 7.3 and/or lactate
concentration ≥ 2× normal) that cannot be corrected, hepatic encephalopathy the
146 Introduction
patient does not wake up and cannot be extubated, and elevated ammonium values
and acidosis. Silberhumer et al (Silberhumer et al., 2007) proposed four grades of initial
graft function over the first postoperative 5 days as:
Proposed grades of initial graft function over the first postoperative 5 days
(1) good function, AST maximal 1000 UI/L and spontaneous prothrombin time > 50%;
(2) fair function, AST 1000-2500 UI/L, clotting factor support < 2 days;
(3) IPGF, AST > 2500 UI/L, clotting factor support > 2 days; and (4) PGNF,
retransplantation required within 7 days.
Initially Poor Graft Function (IPGF) is a severe clinical complication after OLT, with
elevation of serum aminotransferase. Some patients may further develop PGNF (Mor et
al., 1992), which is manifested by hepatocellular necrosis, rapidly rising transaminases,
absence of bile production, severe liver-related coagulation deficit, high lactate levels,
systemic hemodynamic instability and acute renal failure (Pokorny et al., 2000). In
partial liver transplantation, the biochemical profile of small for-size syndrome (SFSS)
includes cholestasis with elevated conjugated bilirubin, mild to moderate elevation of
transaminases, and prolonged prothrombin time. Approximately 50% of recipients with
SFSS will die of sepsis within 4 – 6 weeks after septicemia (Heaton, 2003). The
diagnostic standard for IPGF has not been set yet, and there are different opinions
among some reported definitions The criteria of Ploeg et al (Ploeg et al., 1993) and
Gonzalez et al (Gonzalez et al., 1994) are adopted by some earlier studies and recently
Nanashima et al's criteria (Nanashima et al., 2002) has been introduced.
Criteria according to Gonzalez et al. for classifying graft function (after OLT)
Parameter
Serum ALAT (U/L)
< 1000
1000 – 2500
>2500
Bile output (ml/24 hr.)
>100
40-100
<100
Prothrombin activity (%)
>60
>60 while receiving FFP
Assigned value
1
2
3
1
2
3
1
2
Severe graft dysfunction: the sum was
7-9; moderate graft function if the sum
was 5 or 6; good early graft function if
the sum was 3 or 4.
147 Introduction
Evaluation of IPGF is determined by a high level of alanine aminotransferase (ALT)
and/or aspartate aminotransferase (AST). IPGF directly influences the survival in the
hepatic graft. Ultimately, some grafts recover completely while others need to be
retransplanted (Pokorny et al., 2000). Child-Pugh classification had no influence on the
occurrence of IPGF (Chen et al., 2007 The MELD score, had no significant impact on
initial graft function (Silberhumer et al.,2007). In the SRTR analysis by Johnson et al,
MELD score as a compound of risk factors was not included in models to predict PGNF
(Johnson et al., 2007) Elderly recipient and urgent recipient status with the use of ECD
graft were associated with an increased risk of death by 50% (Cameron et al., 2006).
Although the recipient's age and clinical status before OLT were related to IPGF
(Moreno Sanz et al., 1999), it was not confirmed in a recent study (Chen et al., 2007). In
addition, the incidence of PGNF was more in male recipients receiving grafts from
female
donors
than
in
patients
with
male
donors
(Marino
et
al.
1995).
Hyperbilirubinemia before OLT was associated with PGNF (Avolio et al., 1999). In the
STRT anslysis it was showed that mechanical ventilation, use of inotropes,
hemodialysis, initial status 1 and use of a shared transplant were risk factors for PGNF
by univariate analysis on recipients (Johnson et al., 2007). In the multivariate model,
only recipient serum creatinine, bilirubin, on life support and status 1 at transplant were
significant risk factors for PGNF. For pediatric recipients, diagnosis of tumor, dialysis
prior to transplant, recipient body weight ≤ 6 kg increased the risk of graft failure (Lee et
al., 2008). Regarding the morbidity and mortality of patients undergoing OLT at the
extremes of the body mass index (BMI). An extremely BMI is defined as a BMI < 18.5
kg/m2 or a BMI > 40 kg/m2. In a study of the UNOS database, which reviewed 73,538
adult liver transplants, there was no significant impact of underweight or very severely
obese on the occurrence of PGNF though these patients experienced significantly
higher rates of morbidity and mortality compared with recipients with intermediate BMI
range (Dick et al.,2009).
Rejection
Rejection can be divided into hyperacute, acute, and chronic. Hyperacute responses
occur within minutes to hours, are antibody and complement mediated, and are
generally irreversible. Acute rejection is cell mediated, although its prevalence is
declining, around 20–40% of patients still have one or more episodes requiring
treatment with additional immunosuppression (Shaked A,et al 2009) The majority of
148 Introduction
episodes
occur during the first few weeks(days to months) of transplantation and
typically present with predominantly portal-based inflammation, which is associated with
inflammation of bile ducts and portal venules (Hubscher SG, et al 2007) and can be
reversed using a variety of currently available drugs. Chronic rejection generally occurs
over a span of months, can be unresponsive to current therapy, and continues to be a
source of graft loss. (Deschenes M, et al 1998 , Demetris AJ. 2001). During episodes of
acute rejection, patients may be asymptomatic, or may describe general malaise or
discomfort in the upper quadrant. Liver transplant recipients patient with rising serum
transaminase levels, particularly if this is accompanied by sub-therapeutic blood levels
of immunosuppressive agents diagnosis of rejection must be considered, in such case
liver biopsy is mandatory to confirm the diagnosis. The treatment is based on increases
in baseline immunosuppressive doses, switching to a more potent agent (for instance,
from cyclosporine to tacrolimus) introduction of an additional agent (i.e. mycophenolate
mofetil) and pulse boluses of intravenous corticosteroids. Repeated episodes of acute
rejection may indicate the need for introduction of a second line immunosuppressive
agent.(Lovell MO et al 2004, Varotti G, et al 2005).
Infections occur in 50% or more of transplanted patients infection and are responsible
of more than half of the deaths in liver transplant recipients. The source of the infecting
organism can be: a) the donor organ and transfused blood products (especially viral
infections, such as cytomegalovirus, Epstein-Barr virus, hepatitis-B and hepatitis-C
virus)
b) the reactivation of previous infection
c) invasion by exogenous micro-organisms or by endogenous flora.
Predisposing factors include repeated surgical intervention (Gayowski T, et al 1998)
breakage of the muco-cutaneous defense barriers, excessive exposure to pathogenic
micro-organisms due to prolonged hospitalization, patient’s poor condition prior to
transplantation (presence of cytopenias, other illnesses, malnutrition, etc.) as well as by
the immunosuppression used to avoid rejection. The infecting organism and type of
infection is closely related to the time post-transplantation. During the first month,
infections are typically of nosocomial origin. Surgical technique-related infection is
located in the abdomen, liver and biliary tract, and includes superficial and deep
infection of the surgical bed (surgical wound, intra-hepatic and extra-hepatic abscess,
peritonitis and cholangitis). Thus, intra-hepatic abscess is associated with the existence
149 Introduction
of hepatic ischaemia zones secondary to thrombosis or stenosis of the hepatic artery.
Extra-hepatic abscess is produced by infection of perisurgical bloody collections or
infection of biliomas secondary to biliary fistula. Cholangitis is a consequence of
stenosis or obstruction (due to microlithiasis or lithiasis) of the biliary tract. In case of
prolonged hospitalization infection is nosocomial and includes pneumonia, bacteremia
and urinary infection. Pneumonia is related to prolonged intubation and to re-intubation;
urinary infection, to bladder catheterization, and bacteremia, to intra-vascular
catheterization. In the intermediate period, from the second to the sixth months, the
higher immunosuppression period, bacterial infections (opportunistic bacteria) are less
common than viral infections (especially cytomegalovirus, recurrence of HCV, EpsteinBarr and adeno-viruses). Viral infections are followed in decreasing order of frequency,
by fungi (Pneumocystis carinii, Candida, Aspergyllus, Cryptococcus), bacteria
(Mycobacteria, Nocardia and Listeria) and parasites.24-26 Regardless of the cause of
the liver disease, cytomegalovirus (CMV) is the most frequently isolated micro-organism
liver transplantation. The graft from a seropositive donor implanted in a seronegative
recipient, polytransfusion and the use of anti-lymphocyte antibodies are considered risk
factors for this complication. In the absence of prophylaxis, between 23% and 85% of
patients will present cytomegalic infection, but only 10-40% develop the disease.
Infection by cytomegalovirus is associated with increased post-transplantation mortality
and loss of the graft. After the sixth month, with the transplanted organ functioning
normally and minimum immunosuppressive doses, the frequency of bacterial infections
is reduced to figures similar to those of the general population and the causes are
pathogenic bacteria of the community. Infections in this period affect mainly the
respiratory tract and are caused principally by Pneumococcus and Haemophilus
influenzae.( refer)Infection in the liver transplant patient is diagnosed in the same way
as in the non-transplanted population. Prompt work-up is needed to reach a diagnosis in
absence of focalized fever or in bacterimia, due to the wide differential diagnosis and
because of the use of immunosuppressive medication. For initial assessment, urgent
chest x-ray (to discard pneumonia), Doppler abdominal ultrasound and CT scan of the
abdomen (intra-abdominal collections) are indicated. Other explorations, such as
cholangiography (through the Kehr or trans-hepatic tube), endoscopic cholangiography,
or cholangio-MRI are indicated to discard the existence respectively, of fistulae or
stenoses of the biliary tract. All intra-abdominal collections must be aspirated in order to
confirm infection and identify the microorganism. Methods for early detection of viral
150 Introduction
infection, in the case of cytomegalovirus, are periodic determination of CMV
antigenemia in peripheral blood leukocytes and PCR techniques to detect the blood
viral genome. When a bacterial etiology is probable, or the patient’s situation
deteriorates, empirical treatment is recommended with prior blood sample cultured for
microbiological diagnosis. The choice of empirical treatment should be based on the
type of infection, and the antibiotic sensitivity of the causative micro-organisms (Losada
I, et al 2002). When choosing an antibiotic, it is important to be aware of drug-drug
interactions between any antimicrobials and immunosuppressive drugs. Drug interaction
occurs with antimicrobial agents that use the P450-3A hepatic cytochrome system, the
main metabolic route of cyclosporine and tacrolimus. Antimicrobial agents that inhibit
this system increase serum concentrations of immunosuppressive drugs, nephrotoxicity
and neurotoxicity. In contrast, antimicrobials that induce P450 cytochrome, increase the
metabolism of cyclosporine/ tacrolimus, decrease their serum concentrations, and
increase the risk of acute rejection. The prophylaxis of bacterial infection includes the
following strategies:
a) selective intestinal decontamination
b) administration of systemic antibiotics peri-operatively
c) antibiotic prophylaxis before invasive explorations of the biliary tract.
d) personnel hand hygiene
e) strict asepsis in all invasive procedures. (Arnow PA. et al 2001, Paterson DL, et al
2003).
Another form of prevention, mainly targeted to avoiding the development of clinically
manifest CMV disease, is the treatment of infection in the pre-symptomatic stage.
Universal prophylaxis is useful mainly in high-risk patients (donor+/recipient- CMV, high
transfusion requirements, rejection episodes, treatment with steroids, acute renal and
liver failure, etc.) and can be done effectively and safely with oral drugs (e.g., oral
ganciclovir 3 g/day or oral valganciclovir 900 mg/day for 100 days). Anticipated
treatment is also an effective and probably most cost-effective strategy. (Seehofer D, et
al 2002, Singh N, et al 2005)
1.10.2 Long-term complications
Long
term
complications,
are
mainly
the
consequences
of
the
prolonged
immunosuppressive therapy, and include: diabetes mellitus, systemic arterial
hypertension, de novo neoplasia, and
organ toxicities, especially nephrotoxicity.
151 Introduction
(Munoz SJ, et al 2000), and chronic renal failure, dyslipidemia, obesity, bone or
neurological complications and the development of de novo tumors (Reuben A. 2001)
One of the main problems affecting long-term survival and graft loss and not considered
a transplantation-derived complication is the recurrence of the original disease because
in most cases, the transplant procedure does not eliminate the underlying disease that
was the cause of the failure of the native live. (Wiesner R et al 2003).
Long-term complications
• Chronic rejection
• Renal failure
• Arterial hypertension
• Diabetes mellitus
• Dyslipidemia
• Obesity
• Bone complications
• Neurological complications
• Malignancy
Chronic rejection
Chronic rejection is usually not evident until at least 6 months after transplant. The
pathogenesis is still unclear. Clinical and biochemical cholestasis is the predominant
form of presentation. Liver biopsy is required for diagnosis and confirmation of chronic
rejection. Histologically there is evidence of loss of small bile ducts and obliterative
angiopathy. In the early stages, the changes may mimic acute rejection, with a dense
portal tract infiltration and bile duct endothelitis. The presence of foamy macrophage
infiltration of arterial branches supports the diagnosis. (Backman L,
et al 1993,
Demetris AJ. 2000) . The treatment is based on the same principles than acute
rejection. Response to immunosuppressive treatment is poor and uncommon especially
when bilirubin is greater than 10 mg/ dl and liver retransplantation represent the only
consideration.
Renal failure
Post-transplantation chronic renal failure is closely related to the use of calcineurin
inhibitors (CNI) (cyclosporine and tacrolimus). The prevalence is variable, and depends
152 Introduction
on the criterion used to define renal failure and to the method used to assess renal
function. Serum creatinine measurement may underestimate the presence of renal
failure. Significant renal failure is defined by a serum creatinine level above 2.3 mg/dl or
a glomerular filtrate rate below 50 ml/min. Chronic nephrotoxicity due to calcineurin
inhibitors is characterized by vascular damage (arteriopathy), tubular atrophy and
interstitial fibrosis. Risk factors of renal failure in the first post-transplantation year
are
1. Advanced age of the recipient.
2.Renal support and Dialysis during surgery or in the immediate postoperative stage.
3. Post-transplantation infection due to cytomegalovirus.
4.Retransplantation.
The treatment of chronic renal dysfunction related to CNI’s is not well established; other
causes have to be discarded. In patients with mild renal dysfunction, the reduction of
the CNI dose may be sufficient to normalize the renal function. Some other patients
need the addition of/or increase of another immunosuppressive agent without renal
toxicity (such as azathioprine, mycophenolate mofetil, sirolimus). Another strategy,
particularly in patients with severe damage, is the progressive withdrawal of the CNI
drug and its replacement by a nonnephrotoxic immunosuppressive drug.35,36 Gonwa
TA a. 2001a, Gonwa TA,b et al 2001b).
Cardiovascular complications. Advances in medical therapy and surgical techniques
have led to improvement of patient and graft survival rates following orthotopic liver
transplantation (OLT), with 1- and 5-year patient survival rates of 84 and 67%
respectively in those patients who, otherwise, have little or no hope of survival (
Futagawa Y et al 2006), and acceptance of patients with higher risk profile in large
centers
.The
prevalence
of
medical
complications
such
as
hypertension,
hyperlipidaemia, weight gain and diabetes has been rising along with increased life
expectancy after liver transplantation( Bianchi G, et al 2008). The presence of
hypertension, hyperlipidaemia, diabetes, and obesity post-transplant leads to an
increased risk of atherosclerotic heart disease, with liver transplant recipients having a
higher risk of cardiovascular death and ischemic events as compared with an age- and
sex matched population without liver transplantation.( Bianchi G, et al 2008, Johnston
SD et al 2002). Liver transplantation stresses the cardiovascular system,(Al-Hamoudi
WK et al 2010; Myers RP et al 2006) and limited cardiac reserve preoperatively may be
153 Introduction
associated with poor outcomes postoperatively.( Johnston SD et al 2002) .
Cardiovascular complications ; CAD, peripheral vascular disease particularly stroke,
heart failure (which may be due
to be due to cirrhotic cardiomyopathy syndrome
Alqahtani SA et al 2008) and arrhythmia occur in 25% to 70% of patients after OLT
(Therapondos G, et al 2004). Evidence of pulmonary edema is observed in 22% to 56%
of transplant recipients during the first postoperative month (Therapondos G 2004)
Despite the careful cardiovascular assessment in the evaluation process It has been
estimated that as many as 27% of patients considered for LT may have underlying
coronary artery disease (CAD) (Carey WD 1995) and CV complications are frequent in
these patients (Plotkin JS et al 1996, Dec GW et al 1995, Diedrich DA, et al 2008) The
low systemic vascular resistance and increased cardiac output commonly seen in
patients with cirrhosis normalize perioperatively or soon after transplantation
(Therapondos G et al 2004, Alqahtani SA et al 2008) and may lead to additional stress if
the cardiac microcirculation is impaired. Additionally significant morbidity and mortality
from cardiovascular complications in transplant recipients are reported without any
known predisposing risk factor. This elevated cardiovascular event rate ranges from 9%
at 5 years post-transplant (Mazuelos F et al2003) to 25% at 10 years post-transplant
(Ciccarelli O et al 2005). Cardiovascular disease causes 21% of deaths among liver
transplant patients with functioning grafts surviving more than 3 years (Pruthi J et al
2001) and remains a common cause of death in OLT patients (Kashyap R et al 2001).
Moreover, mortality associated with cardiac causes accounts for up to 7% of deaths in
the early- to medium-term posttransplant period (Therapondos G 2004). Post-OLT
cardiovascular complications result from a combination of factors that include preexisting disease before transplantation and chronic exposure to immunosuppressive
agents following OLT (Gonwa A et 2001). Most liver transplant recipients have
advanced cirrhosis, cardiac abnormalities in patients with cirrhosis are usually
subclinical before transplantation due marked peripheral vasodilatation of end-stage
cirrhosis hiding potentially latent heart failure so they may respond poorly to the stress
of transplantation. These patients with subclinical or asymptomatic heart disease create
the greatest challenge in pretransplant risk stratification. A simple and reliable marker
for identifying such patients is not yet available until now. Several previous studies have
examined predictive factors for cardiovascular events in OLT recipients. A study found
that cardiovascular complications occurred in more than 70% of liver transplant
recipients and showed that preexisting cardiac disease and older age at transplantation
154 Introduction
were the only independent predictors of a major complication (Dec GW et al 1995) while
another study focused on the development of CAD events in the perioperative period
(defined as up to 30 days after transplantation) found that a history of stroke, CAD,
postoperative sepsis, and increased interventricular septal thickness were markers of
poor perioperative cardiac outcomes (Safadi A, et al 2009) a retrospective study (Fouad
TR et al 2009) showed that the independent predictors of cardiovascular complications
were : intraoperative cardiovascular event, a preoperative history of cardiac disease or
hypertension,
and
the
integrated
Model
for
End-Stage
Liver.
Preoperative
cardiovascular tests and imaging may provide some predictive ability for postoperative
cardiovascular events. Single-photon emission computed tomography (SPECT)
scanning and using coronary angiography as a gold standard test they found that the
sensitivity of SPECT imaging was only 37%, and the specificity was 63%. The positive
predictive value was only 22%, and the negative predictive value was 77%. It is,
therefore, clear that SPECT scanning is unreliable as a predictive test for OLT patients (
Davidson CJ et al 2002). It has been reported that 31% of patients with cirrhosis and no
prior history of CVD had an elevated troponin I level and this elevation was linked to a
decrease in the stroke volume index and left ventricular mass, and this implied an
element of subclinical left ventricular myocardial damage as a potential etiology
(Pateron D et al 1999). Troponin I and T isoforms are elaborated by cardiomyocytes in
response to conditions that injure or severely stress the heart, such as ischemia,
ventricular dilatation or failure, cardiomyopathy, and inflammation.( Omland T 2010). A
retrospective study (Coss E et al 2011) showed that an abnormal pretransplant troponin
I level (unlike troponin T isoform, the I isoform is not dependent on glomerular filtration
for elimination and is thus unaffected by renal dysfunction)>0.07 ng/ mL) was among of
only four factors predicting postoperative cardiovascular complications up to 8 years
after transplantation. The other 3 factors were a previous cardiovascular history,
smoking, and pretransplant diabetes. Pretransplant troponin I levels increase correlated
with markers of cardiac dysfunction/disease, such as left ventricular wall thickness and
a low ejection fraction. In the same study neither troponin T nor other serum markers of
inflammation such as C-reactive protein showed any predictive value. Other studies
have demonstrated that MELD scores and renal dysfunction are associated with cardiac
complications after OLT. (Fouad TR, et al 2009, Ghobrial RM et al 2002, Brandao A et
al 2009) The ejection fraction and the cardiac index frequently decrease after
transplantation, and it has been suggested that the cardiac index can decrease as much
155 Introduction
as 35% within 6 to 13 months of transplantation (although not commonly) (Park SC, et
al 1985 , Piscaglia F, et al 1999 ) so a lower ejection fraction before transplantation may
be reflecting relative cardiac dysfunction. The cardiac calcium score, also called the
coronary artery calcium (CAC) score, uses computerized tomography to measure the
buildup of calcium in the arterial wall plaque. CAC scoring has been most thoroughly
evaluated in asymptomatic patients with an intermediate risk of major adverse
cardiovascular events as predicted by the Framingham risk score. In this population, the
annual risk of CAD death or myocardial infarction is 0.4% with a CAC score of 0 to
99,1.3% with a CAC score of 100 to 399, and 2.4% with a CAC score higher than 399
(Greenland P, et al 2007). The relative risk of all coronary disease events is 1.9 with a
CAC score of 1 to 99, 10.2 with a CAC score of 100 to 399, and 26.2 with a CAC score
higher than 399 (Arad Y,et al 2005). Recently, a study demonstrated a strong
relationship between coronary artery calcium and a number of known cardiovascular
risk factors in the setting of liver transplantation (McAvoy NC et al 2008).
Risk factors for cardiovascular complications
Cardiovascular complication
Incidence (%)
Risk factors
Hypertension
31–82
Cyclosporine, tacrolimus, glucocorticoids
Hyperlipidaemia
29–50
Sirolimus, glucocorticoids, cyclosporine, tacrolimus
Diabetes mellitus
10–64
Glucocorticoids, tacrolimus, cyclosporine,
hepatitis C, cytomegalovirus, male gender
Obesity
22–64
glucocorticoids, greater recipient BMI, greater
donor BMI, being married, absence of acute
cellular rejection, family history of: diabetes,
arteriosclerotic heart disease and hypertension.
Arterial hypertension (AHT) is the most common and frequent complication in liver
transplant recipients. The prevalence of de novo arterial hypertension is as high as 77%
posttransplant (DA Neal et al.2004). Its prevalence varies between 50-70% in the first
post-transplantation months but decreases thereafter probably due to the reduction of
the immunosuppressive doses. Hypertension mechanisms include reversal of pretransplant portal hypertension–mediated systemic vasodilation (Moller, JH Henriksen. Heart,
87 (2002), pp. 9–15), immunosuppressive medications e.g. Calcineurin inhibitors,
particularly CYA, have been clearly demonstrated to cause arterial hypertension via
stimulation of renin release, up-regulation of angiotensin II receptors (PV Avdonin et
156 Introduction
al.1999) increased thromboxane release, and impairment of prostacyclin and nitric
oxide-mediated vasodilation (GK Oriji, HR Keiser. 1998). They have also been shown to
cause sympathetic activation, all of these result in systemic vasoconstriction, resulting in
hypertension. AHT is less frequent and late in those immunosuppression protocols that
are based on tacrolimus than in those based on cyclosporine. Corticosteroids have also
been reported to contribute to hypertension via renin-angiotensin system activation,
increased sympathetic responsiveness, reduction of vasodepressor systems, and direct
activation of vascular smooth muscle glucocorticoid receptors. Steroids withdrawal is
associated with improved blood pressure. Management include in addition to lifestyle
modification e.g. weight loss, dietary sodium restriction, smoking cessation, regular
exercise, and avoidance of excessive alcohol intake, pharmacologic management of
hypertension. The drugs of first choice are those that induce vasodilatation as Calcium
channel
blockers
due
to
their
proposed
ability
to
counteract
CNI-induced
vasoconstriction (DA Neal et al.2004) (Gonwa TA.2001a, Gonwa TA, 2001b, Rimola A,
et al 2004, Rabkin JM, et al 2002). The use of angiotensin-converting enzyme (ACE)
inhibitors for treatment of hypertension may be ineffective in the first transplant year, but
possibly efficacious afterward. Because circulating renin levels are low during the first
year after liver transplantation, then steadily increase after the first posttransplant year.
β-Blockers have also been used with success in liver transplant patients. ACE inhibitors
must be used with particular caution, however, due to the prevalence of renal failure
posttransplant. Modification of immunosuppressive regimens has also been shown to
improve hypertension in liver transplant patients. Strategies involving replacement of
CYA with TAC, as well as elimination of CNIs altogether, with or without substitution
with MMF, have been shown to improve hypertension. Corticosteroid withdrawal late
after transplant improves hypertension.
Diabetes mellitus prior to transplantation is a frequent finding in liver transplant
recipients, particularly those with alcoholic cirrhosis or cirrhosis secondary to chronic
infection by the hepatitis C virus. (Navasa M, et al 1996, Correia MITD, et al 2003) Post
transplantation a variable percentage of patients, 4-20% will develop diabetes mellitus
following transplantation (de novo DM). It is very frequent in the initial posttransplantation period due to the use of high CNI and steroid doses. The use of longterm steroids predisposes a state of insulin resistance. In addition, cyclosporine and
tacrolimus can cause altered insulin synthesis and secretion. The prevalence depends
157 Introduction
on the time elapsed since transplantation and on the immunosuppressive drugs. Risk
factors are
1.recipient advanced age,
2.family history of diabetes
3.obesity
4.number of rejection episodes.
Dyslipidemia
Patients
with
cholestatic
disease,
frequently
present
hypercholesterolemia tied to bile secretion alteration, cirrhotic patients as well have
synthesis-reduction related hypocholesterolemia. In the post-transplantation sertting 1766% develop serum lipids changes that can require dietary and/or pharmacological
treatment. The etiology of post-transplantation hyperlipidemia involves many factors,
such as the diet, genetic predisposition, de novo DM, posttransplantation kidney
dysfunction, and immunosuppressive especially steroids play a significant role in
hyperlipidemia onset which is mediated by increased hepatic secretion and conversion
of VLDL to LDL. CNI is also involved in the development of hypercholesterolemia and
hypertriglyceridemia. Sirolimus major side effect is the development of hyperlipemia.
Treatment targets patients of persistent dyslipidemia, particularly if they have concurrent
cardiovascular risk factors. Appropriate diet, weight reduction, strict control of DM and
arterial hypertension along with smoking or drinking cessation are initial measures.
Secondarily, HMG-CoA reductase inhibitor drugs such as pravastatin can be used as
second line alternatives.
Obesity is a very frequent complication in transplanted patients with a prevalence that
ranges between 15 and 40% one year after transplantation, the period when the
greatest weight gain is seen. Factors involved in this complication, include : pretransplantation obesity, post-transplantation sedentary life style, and greater food intake
following transplantation. Drugs also play a significant role; the frequency of obesity
seems to be higher with cyclosporine than with tacrolimus. Withdrawal of steroids within
the first 6 months can be useful in these patients. The treatment of obesity is focused to
its prevention since the treatment of morbid obesity is frustrating and has few effective
results. The initial steps must include ongoing dietary advice and progressive
introduction of physical exercise. (Navasa M, et al 1996 ,Correia MITD,et al 2003)
158 Introduction
Bone complications Osteopenia is a frequent finding in patients with cholestatic
disease and those with advanced chronic liver disease. Screening with bone
densitometry should therefore begin prior to LT A further increase in bone turnover has
been described after LT and may be associated with resolution of cholestasis,
increased parathormone secretion and/or CNI administration (Moreira Kulak 2010).
Metabolic bone disease is therefore a common cause of morbidity after LT. There is no
specific therapies for posttransplant osteoporosis other than for nontransplanted
patients. General interventions to reduce fracture risk include adequate intake of
calcium and vitamin D. Liver transplant recipients present atraumatic bone fractures; the
prevalence reach 40% globally and rises to 65% in patients transplanted due to
cholestatic disease and in retransplant patients. The most frequent locations are the
vertebrae and the ribs. Multifactors have been involved importantly : hormonal changes
associated with the pathogenesis of the liver disease, such as, hypogonadism,
secondary hyperparathyroidism and adverse lifestyle factors vitamin D deficiency,
prolonged immobilization, and immunosuppressive treatment, particularly steroids.
Immunosuppression affects bone density through its influence on the cytokines that
intervene in bone metabolism. In addition, some of the drugs directly suppress
osteoblast function, inhibit intestinal absorption of calcium, and stimulate its secretion
through the kidneys. Calcium, vitamin D, calcitonine and biphosphonates have been
used to avoid post-transplantation osteoporosis, but no consensus has been reached
yet as to the best approach. (Bjoro K, et al 2003, Hay JE, et al 2005) Bisphosphonates
are currently the most effective agents for treatment of posttransplant osteoporosis
(Ebeling 2007, Moreira Kulak 2010). A metaanalysis and systematic review of
randomised controlled trials demonstrated that bisphosphonate therapy within the first
12 months after LT is associated with reduced accelerated bone loss and improved
bone mineral density at the lumbar spine (Kasturi 2010).
Neurological complications A large proportion of liver transplant recipients develop
some degree of neurotoxicity secondary to CNI. The prevalence seems to be slightly
higher with tacrolimus than with cyclosporine. Tremor, the most frequent symptom,
usually responds to calcineurin inhibitors dose reduction. Headache, paraesthesia or
insomnia are other complaints that can actually become very disabling. Chronic
headache may improve with reduction of the CNI doses; if no other cause is identified,
159 Introduction
beta blockers, tricyclic anti-depressants and calcium antagonists may be useful. (Lewis
M, et al 2003).
Malignancy
5-15% of patients who receive a solid organ transplant develop a de novo tumor, with a
prevalence of cancer doubling that seen in the normal population. After transplantation
development of De novo malignancy is a well-known complication.
Factors associated with such complication include:
- The duration and intensity of immunosuppression.
- The type of transplant
- Initial disease.
The natural history of malignant tumors in the transplant patient tends to be different
from that of the normal population; they appear at an earlier age, tend to be in a more
advanced stage when diagnosed, and their evolution is more aggressive, causing high
mortality directly related to the tumor. Malignant tumors can appear at any time after
transplantation, Kaposi’s sarcoma followed by lymphoproliferative disorders are the
earliest that usually develop. The later ones are skin tumors and carcinomas of the
vulva and perineum. The highest risks in the transplant setting are nonmelanoma skin
cancers, mainly as squamous cell carcinoma and basal cell carcinoma which range
from 6-70% of the tumors observed followed by PTLD (4.3-30%)(Yao 2006; Vallejo
2005). Squamous cell carcinoma and basal cell carcinoma are increased by factors of
~100 and 10, respectively, in organ transplant recipients as compared to the
immunocompetent population (Ulrich 2008) An annual routine dermatological follow-up
exam, limitation of sun exposure and sun protective measures including sunscreens are
highly recommended for transplant patients. A trend has been recently reported toward
an increased incidence of advanced colon polyps and colon carcinoma in
immunosuppressed patients after LT. Posttransplant colon cancer surveillance should
be performed more frequently than in the non-transplant setting (Rudraraju 2008).
Recent studies reported a significantly higher incidence of aerodigestive cancer
including lung cancer among patients who underwent LT for alcohol-related liver
disease (Vallejo 2005; Jimenez 2005) The immunosuppressant sirolimus (SRL) exerts
antiangiogenic activities resulting decrease in production of vascular endothelial growth
factor (VEGF) and marked inhibitory response of vascular endothelial cells to
stimulation by VEGF(Guba, et al, 2002.). SRL increase the expression of E-cadherin
160 Introduction
suggests its ability to block regional tumor growth and for inhibiting metastatic
progression. Patients transplanted for HCC and those with de novo malignancies after
LT should be given special consideration for SRL-based immunosuppressive regimens.
Important risk factors for tumor development include alcohol, HCV ( Berenguer M, et al
1998 , Benlloch S, et al 2004), and higher frequency of oropharyngeal cancer has been
described in transplanted patients for alcoholic cirrhosis, as well as increased
presentation of lymphoproliferative syndromes in those transplanted for HCV-cirrhosis.
1.10.3 GRAFT MONITORING AND POST TRANSPLANT PATHOLOGY
Follow-up of transplanted liver involves monitoring for evidence of graft injury. A wide
spectrum of pathological findings and processes in the liver allograft can be seen
including post transplant related and non related findings. Post-transplant pathology
occurs early or late in the posttransplant period. It may reveal presence of rejection,
recurrence of the initial liver disease, and sometimes other new processes that may
occur during post transplant period. In addition, two or more clinicopathological
processes may occur in the same patient. Liver enzymes changes and liver
abnormalities of liver synthetic function (bilirubin, albumin and coagulation parameters)
denote impairment or even failure of normalization or as a result of post-transplant
injuries. Liver enzyme abnormalities and other clinical parameters are not always clearcut in differentiating between diverse conditions potentially affecting the allograft in
most cases liver allograft biopsies are performed in response to changes in liver
enzyme levels, abnormality in one or more liver function parameters, imaging
abnormalities or functional abnormalities, to follow-up an earlier biopsy, or as part of a
protocol that requires time-specific biopsies. Early graft dysfunction refers to changes
occurring within the first 3 months of transplantation, while late changes refer to those
occurring after 6 months (D’Antiga L et al 2002, Junge G, et al 2005).
161 Introduction
Biomarker
ALT
ALT1
ALT2
AST
Total
bilirubin
ALP
GGT
Bile
acids
SDH
GLDH
localization
Activity
Cytoplasm
mitochondria
periportal
Cytoplasm
mitochondria
NH-group reductive
transfer from aa
Cytoplasm
mitochondria
Cytoplasm
mitochondria
Cell
membrane
Cell
membrane
Cytoplasm
mitochondria
Mitochondria
l matrix
Tissue
localization
Broad
Damage
Detection
Comments
Necrosis
Colorimetri
c
Gold standard
NH-group reductive
transfer from aa
Liver restricted
Necrosis
Immunoenymatic
NH-group reductive
transfer from aa
NH-group reductive
transfer from aa
Hemoglobin
degradation
Phosphatase
Skeletal muscle,
heart
Broad
Necrosis
Indirect (serum),
direct (hepatic)
Broad
Gamma-glutamyl
transfer
Cholesterol
metabolism
Sorbitol redox,
fructose, NADH
Amino acid
oxidation urea
production
Kidney > liver,
pancreas
Bile duct
Cholestasi
s, biliary
Cholestasi
s, biliary
Cholestasi
s, biliary
Functional
Liver, kidney
Necrosis
Liver specific >
kidney
Necrosis
Immunoenymatic
Colorimetri
c
Colorimetri
c
Colorimetri
c
Colorimetri
c
Colorimetri
c MS–MS
Colorimetri
c
Colorimetri
c
Possible new
standard,
isozyme
Extrahepatic,
isozyme
Current standard
Necrosis
PNP
Cytoplasm of
endothelial
cells,
kupffer cells,
hepatocytes
Key enzyme in
purine salvage
pathway
Liver > muscle >
heart
Necrosis
Colorimetri
c
MDH
Cytoplasm
mitochondria
periportal
Constituent enzyme
of citric acid cycle
Liver > heart >
muscle > brain
Necrosis
Colorimetri
c
PONI
Cytoplasm,
microsomal,
nuclear, ER
Protects HDL and
LDL from lipid
peroxidation;
cholesterol
metabolism;
detoxifies
organophosphates
Necrosis
Colorimetri
c
Serum
protein F
akka
HPD
GST
Primarily
cytoplasm
Tyrosine catabolism
Liver
specific>kidney
Necrosis
EIA,
pending
Cytoplasm,
centrolobular
cells
Cytoplasm
Phase II detox
enzyme
Liver specific
Necrosis.
prodromal
EIA,
Arginine catabolism
Liver
Necrosis
EIA,
pending
Arginase
I
Conventional
biliary
Conventional
biliary
Conventional
biliary
Total
Enzymatic
instability
Centrilobular
enzyme;
literature cites ↑
with ANIT, CCl4
in rat
Released into
hepatic sinusoids
with necrosis;
literature cites ↑
with
galactosamine,
endotoxin in rats
Periportal
enzyme,
literature cites ↑
with APAP, TAA
in rat
HDL-esterase ↓
with
phenobaribital in
rats and ↓ in
human with
chronic liver
disease, gene
polymorphism
Elevations in
human
hepatocellular
damage
↑ with TAA, ANIT,
BrB; in rats, gene
polymorphism
Literature cites ↑
with TAA in rat
Alanine aminotransferase activity (ALT), alanine aminotransferase isoform 1 (ALT1),
alanine aminotransferase isoform 2 (ALT2), aspartate aminotransferase activity (AST),
alkaline phosphatase activity (ALP), gamma-glutamyl transferase activity (GGT); for
enzymatic hepatotoxicity assays: sorbitol dehydrogenase activity (SDH), glutamate
dehydrogenase activity (GLDH), malate dehydrogenase activity (MDH), purine
nucleoside phosphorlyase activity (PNP), paraoxonase-1 activity (PON1); and for ELISA
(EIA)hepatotoxicity assays: hydroxyphenylpyruvate dioxygenase (HPD) and
glutathione-S-transferase (GST). Amino acid (aa) is indicated in biological activity
162 Introduction
EARLY INDICATIONS OF LIVER ALLOGRAFT BIOPSY
Worsening or failure of liver function or enzymes to normalize post-transplant
(primary or secondary non-function) as a result of:
1.Technical problems (anastomotic: duct or vascular; nonanastomotic vascular factors.
eg, HAT
2. Immunological (cellular rejection, ABO incompatibility/ antibody-mediated rejection)
3. Donor factors (‘‘marginal’’ grafts including fatty liver, long warm and/or cold ischaemic
period; small-for-size syndrome in live donor grafts)
4. Extreme preservation/reperfusion injury
Rise in liver enzymes after initial fall as a result of
Immunological factors (rejection)
Infection (new or reactivated)
Delayed manifestation of anastomotic problems
Adverse drug reaction
Recurrence of primary disease
Donor factors
Post-transplant lymphoproliferative disease
Less than expected normalisation of liver enzymes following a treated event
Wrong initial clinical and/or pathological diagnosis
Correct initial diagnosis, but no response to treatment
Correct initial diagnosis, but missed or unmasked other pathology
Adverse reaction to medication
Patient non-compliance
Follow-up to a prior biopsy:
Compare response to prior intervention, progression and QA prior biopsy
Other factors dependent on indication for follow-up biopsy
Abnormalities of post-transplant imaging
Poor flow (ischaemic parenchymal injury, vascular thrombi, bile duct necrosis/ischaemic
cholangitis, outflow obstruction, sinusoidal obstruction)
Collections (haematoma, abscess, infarct, neoplasm
163 Introduction
Protocol biopsy (time defined):
Compare with prior biopsies if available
Document any pathology or absence of any
Fibrosis staging
LATE INDICATIONS OF LIVER ALLOGRAFT BIOPSY
New-onset abnormality in liver function/rise in liver enzymes from baseline:
– Recurrent disease
– Infection (new or reactivated)
– Immunological (cellular rejection, ductopenic rejection)
– De novo post-transplant neoplasm (post-transplant lymphoproliferative disease,
other)
– Recurrent neoplasm (usually hepatocellular carcinoma)
– Adverse drug reaction
– Newly acquired liver disease (eg. de novo hepatitis or any other form of liver
disease seen in native livers)
– Late anastomotical complications (eg, biliary stricture)
– Vasculopathies (chronic rejection related, sinusoidal obstruction syndrome,
cirrhosis)
– Metastatic neoplasm
– Liver involvement by another systemic disease
Less than expected liver enzymes normalisation following a treated event:
– Wrong initial clinical and/or pathological diagnosis
– Correct initial diagnosis, but no response to treatment
– Correct initial diagnosis, but missed or unmasked other pathology
– Adverse reaction to medication
– Patient non-compliance
Follow-up to a prior biopsy
Imaging abnormalities:
– Neoplasm (primary, recurrent, metastatic)
– Non-neoplastic mass lesions
Protocol (time defined):
– Compare with prior biopsies if available
– Document any pathology or important negatives
– Fibrosis staging
Early indications f
164 Introduction
Focal lesions in the liver as important differential diagnosis in frozen section
Simple cysts vs. Echinococcal cyst
Congenital cystic disease
Cystic mesenchymal hamartoma
Hemangioma (capillary vs cavernous)
Hemangioendothelioma
Biliary hamartoma
Lymphangiomatosis
Angiomyolipoma
Focal fatty change
Heterotopia
prolonged ischemia (cold/warm)
Focal nodular hyperplasia
Liver Cell Adenoma
Nodular, regenerative Hyperplasia
Peliosis hepatis
Biliary adenoma
HCC in normal liver
Sarcomas
1.10.4 EARLY NEW ONSET DISEASES/INJURIES IN THE IN LIVER ALLOGRAFT
Preservation and reperfusion injury
Cellular structural and biochemichal changes and damage take place in the hypoxic
organ and are accentuated following the restoration of blood flow and oxygen delivery.
In liver transplantation setting the damage is sustained during cold preservation of the
liver following explantation from the donor, and during subsequent warm reperfusion at
implantation into the recipient. In non–heart beating donor, or in systemic low flow
states and hypoxia occurring perioperatively either to donor or recipient an additional
warm ischemic damage takes place until hepatic cold perfusion initiate. The main
factors affecting the degree of IR injury are: the duration and nature of ischemia applied
to the liver, as well as liver parenchymal condition. The longest and continuous is the
duration ,the more are the damage and the dysfunction . Other factors include age ; the
more aged the livers the more is the damage and sex (in animal models, males are
less tolerant of ischemic insults than are females; however there is insufficient evidence
for a firm clinical recommendation on the role of sex in liver IR injury). Direct IR to the
liver does not affect only the liver but also distant organ; lungs, heart, kidneys, and
blood vessels and have all been shown to sustain remote dysfunction secondary to
direct liver IR. In liver transplantation IR injury is responsible for many post operative
complications either directly to the hepatobilary system i.e. increasing incidence of
primary graft non-function, primary graft dysfunction, and intrahepatic biliary strictures
or indirectly through its effects on distant organs. Liver IR injury is an interaction of
cellular participants involving( Kupffer cells, CD4+ lymphocytes, neutrophils, sinusoidal
endothelial cells and heptocytes) ,and humoral molecular contributors : cytokines ,
chemokines, and complement proteins of the inflammatory
response in various
pathways ending by cellular death via a combination of apoptosis and necrosis. Hepatic
165 Introduction
ischemia reperfusion injury is an important factor related to IPGF (Mueller et al.,1997).
During
the
course
of
clinical
liver
transplantation,
warm
ischemia,
cold
ischemia,rewarming ischemia, and reperfusion occur sequentially in the allograft.
Severe ischemia reperfusion injury leads to immediate graft non-function and triggers
irreversible ischemic biliary lesions. Preservation injury occuring at the time of organ
harvesting results in tissue damage causing graft dysfunction immediately after
transplantation. Most biopsy specimens were essentially normal before transplantation
except for focal mild spotty acidophilic necrosis, a slight increase in sinusoidal
inflammatory cells and mild hepatocellular swelling. Reperfusion biopsies can predict
IPGF or PGNF during the first few weeks post operation. Many factors contribute to
such type of injury. These include donor and recipient hypotension, warm ischaemia,
cold ischaemia during organ preservation, and reperfusion injury.
Cold ischaemia occurs during storage of the liver in preservation fluid and ice bath
immersion. The hepatic microenvironment involved in the pathogenesis of preservation
injury includes lymphocytes, hepatocytes, bile duct epithelium, sinusoidal cells, Kupffer
cells, neutrophilic leukocytes and platelets. Sinusoidal endothelial cells are the first
affected then hepatic parenchymal cells (Clavien, 1998), because of activation of
Kupffer cells and neutrophilic leukocytes, and the release of inflammatory mediators,
which leads to impairment of hepatic allografts (Jaeschke, 2006). During the phase of
cold ischemia, loss of mitochondrial respiration and ATP depletion occur consequently
though hypothermia reduces the metabolic rate and prolongs the time that anoxic cells
can retain essential metabolic functions (Selzner et al., 2003). Energy-dependent
metabolic pathways and transport processes deteriorate and proteinases and
metalloproteinases are activated. Ultrastructural analysis revealed that the sinusoidal
microvasculature was more sensitive to organ procurement and cold preservation than
the endothelium of larger vessels or hepatocytes (Kakizoe et al., 1990). These changes
at the end of cold ischemia before transplantation included endothelial cell vacuolization
and a partial or complete detachment of individual cells, resulting in denudation with
loss of the space of Disse. The sinusoids contained cellular debris, presumably
fragments of hepatocytes, detached endothelial cells and occasional inflammatory cells.
The hepatocellular changes detected were relatively mild and included cytoplasmic fat
vacuolization, a decrease in the mitochondrial matrix, formation of hepatocellular
cytoplasmic blebs protruding into the sinusoids and occasional loss of hepatocyte
microvilli on the sinusoidal surface.These changes lead to the sinusoidal endothelial
166 Introduction
cells to be lifted away from the underlying matrix together with loss of sinusoidal
microvascular integrity and function(D’Antiga L et al 2002, Kakizoe S, et al 1990, Bilzer
M, et al 2000) and the degree of endothelial damage has been correlated with functional
impairment of the liver following reperfusion. After reperfusion, increased sinusoidal
cellular debris, focal sinusoidal endothelial cell denudation and occasional active
appearing Kupffer cells that contained cytoplasmic vacuoles and electron-dense
material are observed. Inflammatory cells were often clustered in areas of
microarchitectural distortion and sinusoidal lining cell denudation. They were also seen
near Kupffer cells and directly adherent to hepatocytes or amidst cellular debris.
Hepatocyte alterations were relatively mild. The changes included an increase in lipid
vacuolization, detachment of cytoplasmic blebs and, in some areas, formation of
electrondense material in the cytoplasm. The mitochondria in some cases showed mild
swelling, and the rough endoplasmic reticulum showed focal mild fusiform dilatation
when compared with samples taken before transplantation.It has been reported that the
rate of IPGF was significantly higher when CIT was more than 720 minutes (Piratvisuth
et al., 1995)and that AST level increased significantly after OLT if cold ischemia time
was above 600 minutes (Janny et al., 1997). The possibility of hepatic allograft loss was
significantly enhanced when cold ischemia time was over 720 minutes (Adam et al.,
1992). It has been suggested that CIT over 8 hours was significantly related to the
occurence of PGNF (de Vera et al., 2009). In the study by Chen et al, the cold
preservation time in all cases was within 1000 minutes, averaging 622 minutes in the
IPGF group and 515 minutes in the non-IPGF group (Chen et al., 2007). A significant
difference was shown by univariate analysis and not by multi-regression analysis. The
results above suggested that extension of CIT is a potential risk factor for IPGF and
PGNF. Cold ischaemia damages sinusoidal endothelial cells with lifting of the sinusoidal
endothelial cells away from the underlying matrix together with loss of sinusoidal
microvascular integrity and function. (D’Antiga L et al 2002, Kakizoe S, et al 1990,
Bilzer M, et al 2000) Severe cold preservation reperfusion damage is one of the major
reasons for PGNF. Histologically, it is characterized by massive necrosis, which
becomes evident within the first 48 hours after transplantation (Chazouillères et al.,
1993). The integrity of the sinusoidal lining cells could not be evaluated reliably with
immersion fixed, paraffin-embedded and hematoxylin and eosin-stained slides of biopsy
specimens
before
transplantation.
By
contrast
to
biopsy
specimens
before
transplantation, various pathological findings after reperfusion are demonstrated. In
167 Introduction
severe cold ischemia reperfusion injury under the examination of light microscopy
(Kakizoe et al., 1990), larger areas of necrosis appeared, which were classified as focal
or zonal with periportal or bridging necrosis, and severe neutrophilic exudation. The
focal or zonal necrosis was either centrilobular, periportal, or both in its distribution.
Warm ischaemia occurs when the organ is maintained at body temperature but is
inadequately perfused with blood and in livers harvested from cardiac death donors. It
preferentially damages hepatocytes. However, if the duration is less than 120 min in
duration, it poses almost no problem.(Takada Y et al 1998, Kootstra G, et al 2002) The
process of warm ischemia reperfusion injury is two phases process which involves
activation of immune pathways and is dominated by hepatocellular injury. The early
phase (less than 2 hours after reperfusion) is marked by activation of immune cells
(CD4+T cells and Kupffer cells) and production of oxidant stress; the later injury (6 to 48
hours after reperfusion) is characterized by neutrophil-mediated inflammation and
hepatocellular injury (Klune & Tsung, 2010). In addition, warm ischemia also damages
endothelial cells (Selzner et al., 2003; Teoh & Farrell, 2003). Prolonged WIT is common
with uncontrolled DCD, standardized criteria in donor selection have not been
established and limited data concerning its use have been reported. It has been
reported that the average WIT was 16.4 minutes in 19 cases of OLT using a DCD and
the rate of PGNF was 10.5% (D'Alessandro et al., 2000) and that with 5-15 minutes
average WIT, IPGF occurred in 6 of 8 cases and PGNF in the other two cases (Gomez
et al., 1997). In a matched-pair analysis, PGNF was occurred 5.1% in livers of DCD
versus 0% in those of DBD (Pine et al., 2009). In another retrospective study, PGNF
was presented 3.7% in livers of DCD versus 1.4% in those of DBD (Grewal et al, 2009).
WIT over 20 minutes was associated with poorer DCD outcomes (de Vera et al., 2009).
Changes after warm ischemia were seen in liver biopsies before OLT. More injury
occurs in the reperfusion phase after restoration of blood flow where a cascade of
processes is triggered leading to an imbalance of vasoconstrictors over vasodilators
which contributes to microcirculatory failure which in turn is reflected on the biliary tree
and the hepatocytic parenchyma. Bile duct cells are directly susceptible to preservation
and reperfusion injury.
The biliary sludge syndrome is a caused by the pathophysiological mechanisms
relevant to preservation/reperfusion injury and wound healing in the biliary tree. (
Demetris AJ a2006 . Demetris AJ,b2006) . Reperfusion injury begin after
revascularization, the degree of inflammation increases and parallels the degree of
168 Introduction
necrosis. There is mild hepatocellular injury, such as microvesicular steatosis, roundingup of hepatocyte cytoplasm with detachment from adjacent hepatocytes, and mild
hydropic hepatocellular swelling. (Kakizoe S et al 1991, Demetris AJ, et al 1987), focal
hepatocellular cytoaggregation were detected. In more severe injury, there is zonal or
confluent coagulative necrosis, sometimes with periportal or bridging necrosis, and
severe neutrophilic exudation. The subcapsular parenchyma show a more severe
pathological process than the deeper parenchyma. If hepatocellular necrosis was
mainly in zone 3, centrilobular hepatocyte dropout is seen. Repair responses in such
cases take place and the adjacent viable zone 2 hepatocytes proliferate to restore the
liver parenchyma, and mitoses are seen as well as thickening of the cell plates and
nuclear enlargement, mild zone 3 hepatocellular swelling and canalicular cholestasis
may be present. If periportal necrosis and bridging necrosis are present, the
parenchymal collapse triggers ductular reaction that can link adjacent portal tracts and
distort the architecture. Portal inflammation and ductular reaction at the portal/periportal
interface are usually absent in mild injury. In more severe injury, where hepatocellular
necrosis was mainly in zone 3, centrilobular hepatocyte dropout can be seen. The
adjacent viable zone 2 hepatocytes proliferate to restore the liver parenchyma, and
mitoses are seen. If periportal necrosis and bridging necrosis are present, the
parenchymal collapse triggers ductular reaction (Kakizoe S et al 1991, Demetris AJ, et
al 1987) that can link adjacent portal tracts and distort the architecture. More severe
injury is usually accompanied by centrilobular hepatocellular swelling, canalicular and
cholangiolar cholestasis. (Kakizoe S et al. 1991, Demetris AJ, et al 1987) The
pathological differential diagnosis includes sepsis, biliary obstruction, antibody-mediated
rejection and cholestatic hepatitis. Correlation is needed in such cases with the clinical
history (donor age, donation after cardiac death liver, details of cold and warm
ischaemic times, operative note and microbiological studies).
Distinguishing between preservation injury and obstruction/cholangitis requires
careful examination of the bile ducts located within the portal tract connective tissue and
comparing them with the ductules located at the interface zone. In obstruction or
cholangitis, there is concentric periductal lamellar oedema, accompanied by neutrophils
within the lumen or infiltrating between biliary epithelial cells. These bile duct changes
are not seen in preservation injury. There is acute pericholangiolitis in preservation
injury. Both disorders can show marked zone 3 hepatocanalicular and/or cholangiolar
cholestasis and intralobular neutrophil clusters (Demetris r AJ c2009)
169 Introduction
Lipopeliosis is seen in approximately 5% of transplants. (Ferrell L et al 1992, Cha I et
al 1994) It is a lesion that occurs in the early post-transplant period directly related
steatotic donor liver due to hepatocyte necrosis occurring in a steatotic graft after
transplantation due to ischaemia or preservation injury. The fat globules are then
released from the injured hepatocytes and are sequestered in the sinusoids which
appear full of fat and/or the space of Disse. The clinical outcome of lipopeliosis is
variable and depends on the severity of the underlying hepatocellular necrosis (Cha I et
al 1994). Lipopeliosis may be associated with prolonged post-transplant cholestasis
(Cha I et al 1994. Lipopeliosis by itself is reversible and not toxic to the liver but is
indicative of a more severe form of preservation injury (Ferrell L et al 1992, Cha I et al
1994).
Small-for-size graft syndrome
Patients with cirrhosis coming to liver transplantation have markedly increased portal
blood flow (Marcos A, et al 2000). The arterial buffer response regulates a balanced
portal vein and hepatic artery inflow (Marcos A, et al 2000, Lautt WW.1985, Smyrniotis
V, et al 2002). There is reciprocal regulation between portal vein and hepatic arterial
inflow. Increased portal venous flow diminishes hepatic artery flow, whereas decreased
portal flow increases hepatic artery flow. A constant release of the vasodilator
adenosine among the hepatic arterioles and portal venules maintains balanced inflows.
Increased portal flow decreases local adenosine concentrations resulting in hepatic
artery branch constriction and a reduction in arterial flow. This is observed in PHP/SFSS
liver allografts. Conversely, decreased portal flow results in decreased adenosine and
hepatic artery vasodilatation. (Marcos A, et al 2000, Lautt WW.1985) In early SFSS
there is increase portal blood flow, arterial vasospasm and poor arterial flow. The portal
hyperperfusion (PHP) or small-for-size graft syndrome (SFSS) occurs primarily in living
donor or reduced-size liver allografts. The principal pathogenesis of SFSS is the
unbalance between the accelerated liver regeneration and the increased demand of
liver function, leading to severe graft dysfunction with prolonged hyperbilirubinemia and
increased ascites (Ikegami et al., 2008). SFSS is caused by multiple factors including
graft quality, recipient conditions and technique problems. SFSS is seen most frequently
when the graft volume/standard liver volume ratio (GV/SLV) is less than 30% or partial
liver grafts with graft weight/recipient weight ratios (GW/RW) less than 0.8% (Kiuchi T,et
al. 1999; Lo CM, et al.; Ludwig J, et al. 1992; Nishizaki T, et al. 2001). Another
important factor leading to SFSS is portal venous hypertension (Shimamura et al.,
170 Introduction
2001) that is when a transplanted liver is not large enough to accommodate the
markedly increased portal vein blood flow. However PHP/SFSS also occurs following
transplantation of whole cadaveric livers and partial allografts that are greater than 0.8%
body weight. It has been reported that GW/RW did not appear to be the only
determinant of outcome after partial liver transplantation and the occurrence of SFSS
was influenced not only by the graft size but also by other factors such as the degree of
portal hypertension as well (Hill et al., 2009). For adult patient receiving right lobe graft,
low intraoperative body temperature, graft size of < 35% of the estimated standard graft
weight, and middle hepatic vein occlusion were significantly independent factors in
determining hospital mortality (Fan et al., 2003). Other factors may impact the
occurrence of SFSS (Emond et al., 1996; Yoshizumi et al., 2008) donor-related factors
including advanced donor age and steatotic graft and recipient-related factors including
higher MELD scores, septic complications, rejection and biliary complications. Donor
age over 50 years is associated with reduced regenerative capacity, increased
susceptibility to prolonged cold ischemia, increased rates of IPGF/PGNF and prolonged
cholestasis. Fatty infiltration of 30% or more in grafts in splitting or auxiliary liver
transplantation may increase incidence of SFSS (Heaton & Rela, 2001).
Histological findings due to portal hyperperfusion include portal vein and periportal
sinusoidal endothelial denudation and focal haemorrhage into the portal tract connective
tissue; when severe, it dissects into the periportal hepatic parenchyma, and results in
functional dearterialisation, ischaemic cholangitis and parenchymal infarcts. Late
sequelae are small portal vein branch thrombosis with occasional luminal obliteration or
recanalisation, nodular regenerative hyperplasia and biliary strictures.24 (Demetris AJ
d2006 et al 2006) SFSS changes are present in the peripheral and central liver. Since
core biopsies sample the peripheral liver parenchyma, it follows that not all features of
SFSS will be captured in a core biopsy. In peripheral core needle biopsies, affected
grafts most commonly show the following triad: centrilobular hepatocanalicular
cholestasis, centrilobular hepatocyte microvesicular steatosis, and a ductular reaction at
the interface zone, venous findings are uncommon in peripheral core needle biopsies
(Demetris AJ d2006 et al 2006). Changes in zone 3 and ductular reaction are not
specific for SFSS the differential diagnosis include suboptimal arterial flow because of
hepatic artery thrombosis or bile duct stricturing not related to the SFSS, and systemic
causes such as sepsis with or without systemic hypotension.
171 Introduction
Hepatic artery thrombosis
The liver allograft is devoid of a collateral arterial circulation making it more susceptible
to ischemic injury especially early in the post transplantation period. Although advanced
surgical techniques have decreased its incidence, it remains the most frequent cause of
vascular complications after liver transplantation. (Starzl TE, Demetris AJ 1990). The
hilum and large bile ducts are the most commonly affected due to their predominant or
exclusive dependence on arterial supply. These structures are not routinely sampled in
the liver biopsy. Peripheral core needle biopsies thus may show variable changes not
always reliable for establishing a diagnosis of hepatic artery thrombosis. (Demetris AJ,
et al. 1987) Findings can range from completely normal to marked centrilobular
hepatocyte swelling (later centrilobular hepatocellular atrophy and sinusoidal widening),
ductular reaction, with or without bile plugs, and acute cholangiolitis or frank coagulative
necrosis. In some cases, spotty acidophilic necrosis of hepatocytes, so-called ischaemic
hepatitis, can mimic acute viral hepatitis, while duct ischaemia often leads to biliary tract
injury and structuring (Demetris r AJ, et al 2009).
REJECTION
Liver allografts are prone to immunologically mediated rejection, but the roles played by
the major histocompatibility complex (MHC) antigens are not as well defined (Steinhoff
G 1990). Many programmes do not perform donor-recipient human leucocyte antigen
comparison or cross-matches prior to transplant. Types of rejection in liver allografts
include:
1. Acute cellular (cell-mediated) rejection occuring in the early post-transplant period
2. Late occurring cellular rejection. Those occurring 6 months post-transplant or later,
and have been estimated to occur in 6–10% of adult patients, and are more likely to
show ‘‘atypical’’ histopathological features, be resistant to treatment, require rescue
therapy, or progress to ductopenic rejection. (Junge G, et al 2005, Florman S,et al 2004,
Anand AC, et al 1995, Dousset B, et al 1998 , Mor E, et al 1992).
3. Chronic rejection
4. Antibody-mediated rejection. Antibody-mediated rejection can be hyperacute (very
rare outside of ABO mismatch) or it may occur days to weeks post-transplant.
172 Introduction
Rejection Activity Index from Banff Schema for Acute Hepatic Graft Rejection
Category
Criteria
Score
Portal
inflammation
Mostly lymphatic inflammation involving a minority of the triads
Expansion of most triads by a mixed infiltrate containing lymphocytes,
neutrophils, and eosinophils
Marked expansion of most or all triads by a mixed infiltrate containing
numerous blasts, with spillover into periportal parenchyma
Minority of ducts infiltrated by inflammatory cells, with only mild reactive
changes in epithelial cells
Most or all ducts infiltrated by inflammatory cells, with occasional
degenerative duct changes, such as nuclear pleomorphism, disorder polarity,
and vacuolization
As above, with most or all ducts showing degenerative changes
Subendothelial lymphocytic infiltration of some portal or hepatic venules
Subendothelial infiltration involving most or all portal or hepatic venules
As above, with perivenular inflammation extending into surrounding
parenchyma and associated hepatocyte necrosis
1
2
Bile duct
damage
Venous
endothelial
inflammation
3
1
2
3
1
2
3
This index has a range from 0 to 9, classified as follows: 0 to 3, minimal acute
rejection; 4 to 6, mild acute rejection; 7 to 9, moderate to severe acute rejection.
Modified from the : Banff consensus 1997 schema for grading liver allograft
rejection: an international consensus document. Hepatology 25:658-663.
Acute cellular rejection
Acute cell-mediated rejection (ACR) remains the commonest cause of early graft
dysfunction, with incidence ranging from 24–80%, with a mean of 49.8%. The reported
incidence often includes ACR diagnosed clinically, with or without confirmatory biopsies.
The Banff 1997 document defining criteria for scoring ACR on a scale of 0–9 is widely
used and the total score of all the rejection features present in a given biopsy is known
as the Rejection Activity Index (RAI). Factors determining the incidence of ACR include
site of transplant, type of immunosuppression, perioperative factors (ischaemia,
infections), type of post-transplant surveillance, donor characteristics and other
factors.29–34 (Desai M, et al 2009 , Fisher LR, et al 1995 , Florman S, et al 2004 ,
Shaked A, et al 2009 , Tippner C, et al 2001 ,Yilmaz F, et al 2006 , Anand AC, et al
1995 ) The definition of ACR is not based on time of occurrence from transplant, but
rather on characteristic morphological changes
173 Introduction
Relative diagnostic performance of some proposed markers of acute rejection.
Analyte
Blood specimen:
Bilirubin
GGT
ALP
ALT/ASP
a-GST
TNF
g-INF
IL-1
IL-2
IL-5
IL-6
IL-2R
ICAM-1
b2-M
Neopterin
Bile specimen:
b2-M
Neopterin
IL-2R
IL-6
Histology
Liver biopsy
Sensitivity
Specificity
Convenience, cost,
and availability
+
++
+
+/+/++
++
++
++
++
++
++
++
+
+
+/+/+/+/+/+/+/+/+/+/+/+/+/+
++
++
++
++
+/+/+/+/+/+/+/+/+/+/-
++
++
++
++
+
+
+
+
-
++
+
+/-
Histopathological features of ACR
ACR is an immunologically mediated injury targeting bile duct epithelium and vascular
endothelial cells. (Adams DH, et al 1989, Ludwig J, et al 1989, Scholz M, et al 1997).
Therefore inflammatory infiltrates of cellular rejection are found around these targets,
namely portal tracts and in perivenular areas of zone 3. The lobular regions between the
portal tract and zone 3 venules show no significant involvement by the immune effector
cells; which helps differentiating typical ACR from hepatitis. In ACR, since the portal
vein endothelium and bile ducts are targets, the infiltrates, except in the more severe
forms, tend to cluster around these targets with little to no spillover to the lobule through
the interface hepatocytes. When the terminal hepatic venules are involved, the infiltrates
are seen under the endothelial cells (endotheliitis, phlebitis), but in the more severe
forms of rejection these infiltrates involve the perivenular parenchyma, with or without
hepatocellular necrosis. The 1997 Banff scoring system for liver allografts is the most
widely used scoring system for liver allograft injuries, including ACR, (Banff schema for
grading liver allograft rejection). The three histological parameters underlying ACR
(portal inflammation, bile duct injury, and portal and/or terminal hepatic venule
endothelial injury) are each scored on a scale of 0–3 to give a total RAI on a scale of 0–
9. Portal infiltrates in ACR are usually always mixed, and may include activated
174 Introduction
lymphocytes (including blast forms), eosinophils and neutrophils. These infiltrates range
from mild to severe depending on the density, and can involve few to all sampled portal
tracts. The density and extent of portal tract involvement assign a score for this aspect
of ACR. Bile duct injury in cellular rejection is characterised by the presence of
inflammatory cells within duct epithelial cells associated with evidence of epithelial
injury, such as high N:C ratio, variation in nuclear size, cytoplasmic vacuolisation and
disruption of lumen. Necrosis of ducts can be present in the most severe injuries.
Endothelial injury to the portal and/or terminal hepatic vein endothelium comprises
inflammatory infiltrates beneath the endothelium, referred to as endotheliitis or phlebitis
this ranges from only lifting of the endothelium to ‘‘embolisation’’ into the vascular lumen
and/or nuclear atypia. In the more severe forms, it is accompanied by perivenular
extension (usually around terminal hepatic venules) of inflammation into the lobule
producing necrosis of surrounding hepatocytes. Phlebitis can be seen in non-rejection
processes including recurrent chronic hepatitis C virus (HCV). Histological features of
ACR can also be seen at the hepatic hilum resulting in inflammation of hilar nerve twigs
and/or large hepatic arterial intimal inflammation with endothelial injury. Since ACR is an
inflammatory process, evaluation of liver allografts for cellular rejection not only requires
the recognition of diagnostic histopathological features but also involves the exclusion of
non-rejection independent pathological processes especially other inflammatory
processes, due to viral infections, de novo non-viral and non-infective hepatitis, and
lymphoproliferative diseases. The infective agents that pose the most problems are
recurrent and less commonly de novo viral hepatitis B or C. Other viral infections
(Epstein–Barr virus (EBV), cytomegalovirus (CMV) and others) should always be
considered in this context of immunosuppression. Drug induced hepatitis and de novo
autoimmune hepatitis are also diagnoses of exclusion. The distinction between ACR
and HCV represents the most commonly encountered diagnostic dilemma.
175 Introduction
Comparison of histological changes occurring in hepatitis C infection and acute
cellular rejection of the liver allograft.
Hepatitis C
Rejection
Portal inflammation
Mainly mononuclear cells
(lymphoid aggregates)
Interface hepatitis
Variable (generally mild)
Mixed infiltrate
(lymphocytes, macrophages,
blast cells neutrophils,
eosinophils)
Usually mild
Bile duct inflammation
None/mild (lymphocytes)
Variable, may be prominent
(mixed infiltrate)
Bile duct loss
None
Venous endothelial inflammation
None/mild
Variable (in cases progressing to
chronic rejection)
Variable, may be prominent
Fibrosis
Yes
No (except in cases with chronic
hepatitic features)
Lobular inflammation
Severity
Pattern
Distribution
Associated features
Generally mild
Spotty
Random
Lobular disarray
Variable
Confluent
Perivenular
Hepatic vein endothelitis
Cholestasis
Rare (except FCH-like cases)
Common
Rare (except FCH-like cases)
Yes (macrovesicular)
No
Acidophil bodies
Common
Less numerous
Rate and timing of liver enzyme
changes
Usually smouldering,
rarely steep except in
FCH(fibrosing
cholestatic epatiti)
Recent change from baseline
and may be associated with
Suboptimum immunosuppression
Predominant enzyme pattern
ALT/AST
ALT/AST, ALP or mixed
Late rejection in general is related to suboptimal immunosuppression (Anand AC et al
1995, Florman S et al 2004) but this is not always the case (Uemura T, et al 2008) or
withdrawal of immunosuppressive medication, erratic absorption of immunosuppressive
agents, or poor compliance, which occurs particularly in the teenage population. (Anand
AC, et al 1995) Histological features of late acute rejection are different from those of
early acute rejection (Pappo O, et al 1995; Kemnitz J, et al. 1989; Cakaloglu Y, et al.
1995) these include a predominantly mononuclear portal inflammatory cell infiltrate in
contrast with the mixed population of cells more typically seen in early AR, less severe
inflammation of bile ducts and portal venules, interface hepatitis and lobular hepatitis
are more prominent and sometimes associated with periportal fibrosis (Hubscher
SG.a2009; Hubscher S. b2009). Presense prominent centrilobular inflammatory
changes often associated with foci of centrilobular or bridging necrosis (Krasinskas AM,
et al 2001; Demetris AJ, et al 2006; Sundaram SS, et al 2006; Gouw AS, et al 2002;
176 Introduction
Neil DA, et al 2002 ,Hassoun Z, et al 2004 , Riva S, et al 2006 , Demirhan B, et al
2008, Hubscher SG. 2008). All these changes are collectively known as ‘‘central
perivenulitis’’ (CP) (Demetris AJ, et al 2006, Hubscher SG. 2008) and can occur in the
absence of significant portal inflammation (‘‘isolated central perivenulitis’’) Krasinskas
AM a2001, et al 2001; Demetris AJ, et al 2006; Hubscher SG. 2008; Abraham SC, et al
2008; Krasinskas AM b2008). Two recent studies found that isolated CP was present in
22% of children biopsied >3 months post-LT (Abraham SC, et al 2008) and 28% of
adults undergoing protocol biopsy >3 years post-LT (Krasinskas AM b2008). Hepatic
venous endothelitis, which is typically seen in association with CP in early acute
rejection, is rarely seen in late rejection. Late rejection with features of CP often
presents with raised transaminase levels, contrasting with the cholestatic liver
biochemistry that is more typically seen in early portal-based AR (Neil DA, et al 2002;
Hassoun Z, et al 2004; Junge G, et al 2005). It is less responsive to
immunosuppression and is associated with an increased frequency of adverse
outcomes including further episodes of acute rejection (Demirhan B, et al 2008; Lovell
MO, et al 2004) , progression to chronic rejection (Gouw AS, et al 2002 , Neil DA, et al
2002 Krasinskas AM b2008 , Lovell MO, et al 2004 , D’Antiga L et al 2002, Sebagh M et
al 2002 Miloh T, et al 2009), and the development of de novo autoimmune hepatitis
(Krasinskas AM b2008, D’Antiga L et al 2002). Grading of late rejection with features of
CP is often difficult according to the conventional Banff criteria, which require the
presence of typical portal tract changes of AR (International P. Banff schema for grading
liver allograft rejection: an international consensus document). However, a system
proposed by the Banff Working Group for grading the severity of CP (Demetris AJ, et al
2006) appears to have some value in predicting adverse outcome (Krasinskas AM
b2008).
Chronic rejection CR
Most CR is diagnosed several months post-transplant, it occurs in less than 3% of liver
transplant patients at 5 years with a mean of 25.1 months. Rarely, accelerated chronic
rejection can occur within few weeks posttransplant, and this is usually seen in highly
sensitised or suboptimally immunosuppressed patients. The incidence does not
increase with increasing post transplant time. Factors favoring CR occurrence are
determined pre transplant and early in post transplant periods. These factors include
repeated ACR, CMV infection, high donor age, long cold ischaemic period, and
177 Introduction
inadequate/suboptimum immunosuppression. (Pappo O, et al 1995, Demetris AJ 2006;
Quiroga J, et al 1991; Blakolmer K, et al 2000; Wiesner RH, et al 1999). Chronic
rejection may appear indolently and might only become apparent as liver test injury
abnomalities (gGT, AP, bilirubin, transaminases). The diagnosis needs to be confirmed
by histopathologic examination. The most widely recognized manifestation of chronic
rejection is obliterative arteriopathy CR is an immunological injury targeting the vascular
endothelium of the hepatic artery and peribiliary plexus, as well as the bile duct
epithelium. The resulting ductal injury is characterised by epithelial senescence, loss of
small bile ducts (ductopenia). Hepatic artery shows intimal thickening with accumulation
of foamy macrophages. The 2000 Banff document describes the criteria for grading,
recognition and scoring of chronic rejection (CR) in liver allografts. (Demetris A, et al
2000).
Banff Schema for Chronic Hepatic Rejection (Demetris A, et al, 2000)
Structure
Early Chronic Rejection
Late Chronic Rejection
Small bile
ducts
(<60 µm)
Degenerative changes involving most
ducts: increased nuclear-to-cytoplasmic
ratio, nuclear hyperchromasia, uneven
nuclear spacing, ducts partially lined with
epithelium
Bile duct loss in <50% of portal tracts
Degenerative changes in remaining bile
ducts
Bile duct loss in >50% of portal tracts
Terminal
hepatic
venules
Intimal/luminal inflammation
Lytic zone 3 necrosis and inflammation
Mild perivenular fibrosis
Focal obliteration
Variable inflammation
Severe (bridging) fibrosis
Portal tract
hepatic
arterioles
Occasional loss involving <25% of portal
tracts
Loss involving >25% of portal tracts
Large
perihilar
hepatic
artery
branches
Intimal inflammation, focal foam-cell
deposition
Luminal narrowing by subintimal foam cells
and fibrointimal proliferation
Large
perihilar bile
ducts
Inflammation damage, focal foam-cell
deposition
Mural fibrosis
Other
“Transition” hepatitis with spotty necrosis of
hepatocytes
Sinusoidal foam cell accumulation, marked
cholestasis
Clinical history could include prior (multiple) or ongoing cellular rejection episodes, low
serum levels of immunosuppression, and rising alkaline phosphatase; bilirubin elevation
is usually late. A minimum of at least seven fully sampled portal tracts should be
reviewed in needle biopsy. Histological features of CR include senescence or atrophy
178 Introduction
affecting the majority of interlobular bile ducts with less than 50% ductopenia in early
CR, or duct loss in more than 50% in late ACR. Senescence or atrophy of bile ducts is
characterised by epithelial disruption with irregular spacing, cytoplasmic eosinophilia,
high nucleocytoplasmic ratio, nuclear hyperchromasia and luminal narrowing.
Expansion of portal tracts is absent or minimal, and ductular proliferation or copper
retention is not seen; inflammation is minimal, some other features include zone 3
perivenular fibrosis, hepatocellular cholestasis with or without accentuation in zone 3,
sinusoidal foam cells, and foam cell arteriopathy (if hilar structures are included).
Ductular reaction is absent except in the recovery phase following increase in or switch
of immunosuppressive agent (Demetris AJ 2006.) The use of cytokeratin 7 immunostain
(or other biliary epithelial markers) should be used in graft sample from patients with
unexplained elevation of cholestatic liver enzymes because early duct senescence
and/or loss can be subtle on routine stains. It is important to note that CR is an evolving
process and several biopsies may be needed before a definite histological diagnosis is
feasible. Switching the baseline immunosuppression from CSA to TAC and initiating
myco phenolate mofetil (MMF) rescue therapy represent a treatment option in these
patients, anti-interleukin (IL)-2 receptor antibodies (daclizumab and basiliximab) for
steroid-resistant rejection revealed a poor histologic response.
Recurrent primary sclerosing cholangitis (PSC) is difficult to distinguish from CR.
Other causes of ductopenia in the liver allograft include vanishing duct syndrome should
always be considered, excluded, especially those caused from drugs such as antibiotics
like SeptraH54 and ACE-inhibitors, to which many transplant patients are invariably
exposed
179 Introduction
Comparison of the features of recurrent primary sclerosing cholangitis and
chronic rejection
Recurrent primary sclerosing cholangitis
Chronic rejection
Clinical features
Original disease PSC, years after
transplantation, selective rise of ALP/ GGT
Typically within 1st year posttransplant, inadequate
immunosuppression,
unresolved ACR, or after multiple
episodes of ACR
Cholangiogram
Mural irregularity, diverticulum-like
outpouchings, beading and ‘‘pruning’’ of bile
ducts
‘‘Pruning’’ of peripheral bile ducts
Uneven, portal expansion by mixed infiltrate,
periductal lamellar oedema, and
pericholangitis; focal biliary epithelial
degenerative changes
No significant expansion, biliary
epithelial
degenerative changes in most portal
tracts, duct loss
Interface
changes
Oedema, cholatestasis, ductular reaction,
copper deposits in periportal hepatocytes
Usually not significant
CPV
Variable
Often present
Cholestasis
Slow progression in time, biliary type
Usually present
Liver biopsy:
Portal changes
Fibrosis
Perivenular with or without bridging
septa, if present.
Antibody-mediated rejectionThe liver is relatively not absolutely protected from
hyperacute AMR because of the role of the Kupffer cells in getting rid of deleterious
antibodies, immense vascular reserve due to the presence of dual circulation. Antibodymediated rejection (AMR), although uncommon, occurs in liver allografts, either rapidly
in the immediate perioperative period (immediately post-reperfusion; hyperacute), and is
usually but not only due to ABO incompatibility (ABO isoagglutinins), or it occurs later in
less aggressive form during the first few days or within the first week post-transplant
(Terminology for hepatic allograft rejection. International Working Party 1995, DellaGuardia B, et al 2008, Demetris AJ, et al 1988, Haga H, et al 2004). The clinical picture
includes hypotension, rapidly evolving coagulopathy, progressive hyperbilirubinaemia,
renal failure and refractory thrombocytopenia; accelerating hepatic failure process.
Imaging studies can reveal portal vein thrombosis and parenchymal necrosis.
Serological demonstration of donor–recipient incompatibility at the ABO, MHC or other
levels is present, as well as relevant donor-specific antibodies. The pathogenesis stems
from transplantation into a sensitised host with preformed antidonor antibodies. The
titre, class and specificity of these preformed antibodies determine the degree of injury,
180 Introduction
and the higher titres are more likely to result in more severe injury (Furuya T, et al 1992,
Demetris AJ, et al 1992). Pathological gross examination reveals liver enlargement up
to twice the preengraftment weight (Demetris AJ, et al 1988) and congested/motteled
capsule and cut surfaces. Patchy foci of haemorrhage and necrosis can be grossly
recognised. Microscopically the liver shows haemorrhagic and coagulative areas of
necrosis and, in the less affected areas, features mimicking preservation/reperfusion
injuries (zone 3 hepatocellular ‘‘loosening’’ and cholestasis) can be identified. Despite
the short duration post-grafting ischaemic necrosis of bile ducts of all calibres is evident
together with loss of small bile ducts. Small, intermediate and sometimes large vessels
show evidence of thromboses and vasculitis with neutrophilic exudation and fibrin
deposits in and around vascular walls. Tissue demonstration of antibody activity by the
presence of C4d, C1q or immunoglobulins (almost always IgG, IgM) in vascular and
sinusoidal walls can be seen, but is hardly necessary. During the first week post
transplant less aggressive form characterized by slow normalisation of and/or
increasing liver enzymes levels can occur. The diagnosis is clinical and serological with
the demonstration of donor–recipient mismatch and the presence of donor-specific
antibody. The histopathological examination shows features that overlap with
preservation/reperfusion injury including zone-3 accentuated cholestasis, portal
expansion with oedema and ductular proliferation in this setting obstruction/strictures
must be excluded. C4d deposition is seen in the walls of portal capillaries and veins and
hepatic venule; sinusoidal C4d can be seen, but its specificity is yet to be determined
(Demetris AJ, et al 1988, Demetris AJ et 1992). These findings are more pronounced in
patients with preformed circulating donor antibodies (Demetris AJ et 1992). If perihilar
tissue is included in the biopsy, arterial injury and thrombi are present, as evidenced by
endothelial hypertrophy and myocyte necrosis, vacuolization and thickening, while
necrosis of large bile ducts and congestion of peribiliary plexus complete the picture.
(Demetris AJ et 1992, Nakamura K et al 1993)
DISEASE RECURRENCE
The major cause of graft dysfunction and loss after 6 months is disease
recurrence (Desai M, et al 2009)
Disease recurrence does occur in patients transplanted for viral hepatitis, tumor
disease, autoimmune or cholestatic or alcohol-related liver diseases. With universal
recurrence of HCV in all replicative patients, hepatitis C continues to pose one of the
181 Introduction
greatest challenges for preventing disease progression in the allograft. However, liver
transplantation is a curative therapy of many liver diseases without recurrence, such as
a1-antitrypsin deficiency disease, Wilson disease and cystic fibrosis, and most
metabolic liver diseases, do not recur after transplantation. Post-transplant outcome for
patients with haemochromatosis is not yet certain, but there seems to be little impact up
to 5 years after transplantation. Most of the main complications that occur during the
early posttransplant period can also be seen in late post-transplant biopsies (>1 year
post-LT) (Hubscher SG, et al 2007).
182 Introduction
Summary of the main diseases (excluding neoplasms) that recur post liver
transplantation
Recurrent
disease
Frequency
%
Histology >12m post-transplant
Comment
Hepatitis B
<10
Chronic hepatitis (typically mild).
Fibrosis now rarely more than mild in
severity
Incidence and clinical impact now
greatly reduced by use of anti-viral
therapy pre- and post-transplant.
Hepatitis C
>90
transplantation in many centres.
Most cases result in graft damage,
severity variable. Most frequent
cause of late graft failure.
PBC
20 -50
Chronic hepatitis, typically
resembling changes seen in the
native liver. More severe cases may
be associated with prominent
interface hepatitis ± confluent or
bridging necrosis. Some cases have
“autoimmune features” Progressive
disease common - 20-50% cirrhotic
by 5-10 years.
Lymphocytic or granulomatous
cholangitis. Portal mononuclear
inflammation-typically focal. May
precede development of typical bile
duct lesions by several years.
Progressive disease associated with
ductopenia and features of chronic
cholestasis. Progression to cirrhosis
rare.
PSC
20-30
Fibrous cholangitis (rarely seen in
liver biopsies). Diagnosis more often
based on findings of chronic
cholestasis, ductopenia, ductular
reaction and a “biliary pattern” of
fibrosis. Approximately 25% have
bridging fibrosis or cirrhosis by 5
years post-transplant.
More frequently clinically
symptomatic than recurrent PBC.
Approximately 10% progress to graft
failure. Histological and radiological
features difficult to distinguish from
ischaemic cholangiopathy. Diagnosis
therefore requires exclusion of other
causes of biliary tract disease.
AIH
20-30
Portal tract plasma cell-rich
inflammatory infiltrate associated
with interface hepatitis. Lobular
inflammation frequently present severe cases include foci of
confluent/bridging necrosis. Lobular
inflammatory changes may resemble
“central perivenulitis” and can occur
as the first manifestation of recurrent
disease.
Most cases occur as a result of
suboptimal immunosuppression and
respond to immunosuppressive
therapy. Diagnosis based on a
combination of biochemical,
serological, and histological findings.
ALD
10-30
Fatty change common (>60% of
cases) Steatohepatitis and fibrosis
less common. Progression to
cirrhosis rare.
Recurrent alcohol consumption
common, but serious graft
complications are rare.
NAFLD
20-40
Fatty change common (60-100% of
cases). 10-40% progress to
steatohepatitis and up to 12%
become cirrhotic.
Risk factors for NAFLD often persist
and may be exacerbated by
immunosuppressive drugs and other
transplant-related factors. Many
people with recurrent NAFLD have
normal LFTs.
Most cases have mild/asymptomatic
disease, frequently diagnosed on
protocol biopsies. Rare cases (<1%)
progress to graft failure
183 Introduction
Disease recurrence after liver transplantation is highly influenced by the aetiology of the
primary liver disease (Desai M, et al 2009, Abouljoud MS,et al 2001, Kotlyar DS, et al
2006, Yusoff IF, et al 2002) and the presence of a different set of human leucocyte
antigen (HLA) molecules in the allograft which may alter the recognition and response
of the body toward viral particles. In addition, the immunosuppressive environment,
which facilitates viral replication and can underlie aggressive behaviour when the
disease recurs (Demetris r AJ et al 2009). Recurrent HCV, HBV are encountered the
most; primary biliary cirrhosis (PBC), PSC and AIH may all recur after liver
transplantation and diagnostic criteria for recurrence may differ from the ones used for
similar native liver disease (Faust TW. 2001, Gautam M, et al 2006, Mottershead M,et al
2008, Schreuder TC, et al 2009; Tamura S, et al 2008). Recurrence of alcoholic liver
disease, non-alcoholic fatty liver disease and non-alcoholic steatohepatitis have also
been reported. Liver transplantation for primary hepatic malignancies (hepatocellular
carcinoma or cholangiocarcinoma) is based on stage of disease, and the likelihood of
recurrence is dependent on several risk factors, such as microscopic vascular invasion,
multiple tumours and tumour burden, for hepatocellular carcinoma. (Schreibman IR, et
al 2006, Heimbach JK. a2008, Heimbach JK.b 2008, Rosen CB, et al 2008, Said A,
Lucey MR. 2008, Tamura S, et al 2001, Kato T, et al 2001, Zimmerman MA, et al 2008)
Cholangiocarcinoma has a poor prognosis after liver transplantation and its recurrence
influenced largely by disease stage (Heimbach JK. a2008, Heimbach JK.b 2008,
Rosen CB, et al 2008).
Recurrent autoimmune
hepatitis
Recurrent primary sclerosing
cholangitis
Recurrent primary biliary
cirrhosis
Liver transplant for autoimmune
hepatitis
Liver transplant for primary
sclerosing cholangitis
Liver transplant for primary biliary
cirrhosis
Autoantibodies in significant titre
(>1:40)
Multiple nonanastomotic biliary
strictures
Persistence of antimitochondrial
antibodies
Sustained rise in serum
aminotransferase activity (more
than two times normal)
Exclusion of other causes (ie,
rejection, infection, ischaemia)
Elevated immunoglobulins
Elevated serum immunoglobulins
Diagnostic or compatible liver
histology
Corticosteroid dependency
Exclusion of other causes of graft
dysfunction (eg, HCV infection,
rejection
184 Diagnostic or compatible liver
histology
Diagnostic or compatible liver
histology
Exclusion of other causes of graft
damage
Introduction
Recurrent HBV infection
The course and sources of post-transplant HBV infection are well known (OliveraMartinez MA, et al 2007) Sources of
reinfection include patient’s circulation and
extrahepatic replicating sites(Olivera-Martinez MA, et al 2007). Currently, combination
therapy by oral antiviral agents (eg, lamivudine, adefovir) and hepatitis B
immunoglobulin in the pretransplant and posttransplant setting achieves nearly 100% of
protection against adverse outcomes from graft reinfection (Demetris AJ, et al 2005,
Eisenbach C, et al 2006, Marzano A, et al 2007). The course similar to that which
occurs in non-transplanted patients with HBV infection with more severe activity and
progression. It runs in three phases; an incubation period (approximately 3 months posttransplant), followed by acute infection (up to 6 months) and chronic infection (more
than 6 months). Inadequate antiviral treatment or the development of resistant viral
strains may result in progression from acute to chronic hepatitis and cirrhosis.
Resolution of disease can sometimes occur after a bout of acute hepatitis. (Longerich T,
Schirmacher P. 2006) Coinfection with HCV or HDV reduces HBV viral replication
resulting in attenuation of HBV activity and progression (Rosen CB, et al 2008)
Fulminant recurrent HBV and HDV have been reported in patients with HDV infection
and active HBV replication (Thung SN.2006). The main clinical feature of acute
hepatitis B in the allograft is mild elevation of liver enzymes; nausea, vomiting, jaundice
and signs of hepatic failure characterize its severe form.
An atypical pattern of recurrent HBV infection, known as fibrosing cholestatic
hepatitis (FCH), can occur within the first month post transplant and may rapidly lead to
graft failure. Patients with chronic HBV infection may be asymptomatic or may complain
of non-specific gastrointestinal symptoms. (Thung SN.2006). Liver damage in allograft
recipients is secondary to virally directed immunological injury. Viral antigens are
recognized by memory T helper cells leading to expansion and activation of antigenspecific TH1-type CD4+ lymphocytes. (Marinos G, et al 2000) resulting in macrophage
activation and pro-inflammatory cytokine production. Interferon (IFN) gamma and
tumour necrosis factor (TNF) alpha cause damage by recruiting and activating nonspecific inflammatory cells, upregulating TNF-receptor expression, exerting a direct
cytotoxic effect on hepatocytes expressing HBsAg and inducing local mediators of
tissue
injury
such
as
nitric
oxide.
It
has
been
suggested
that
despite
immunosuppression, HLA-class-I-independent immune mechanisms have a significant
pathogenic role in liver damage associated with HBV recurrence after liver
185 Introduction
transplantation. (Marinos G, et al 2000) Fibrosing cholestatic hepatitis, which usually
affects over immunosuppressed patients, is associated with massive viral replication,
and liver injury may be attributable to a direct HBV-mediated cytopathic effect.
The acute phase
This corresponds to the onset of necroinflammatory activity, Kupffer cell hypertrophy,
lobular disarray and portal inflammation (Abouljoud MS, et al 2001, Phillips MJ, et al
1992, Thung SN.2006) Scattered nuclear and cytoplasmic staining for hepatitis B core
antigen (HBcAg) can be demonstrated by immunohistochemistry 2–5 weeks after
transplantation in biopsies reinfected by HBV. Subsequently, surface antigen is
expressed, but ground glass cells are not easily found during this acute phase (Thung
SN.2006). Clinically, these changes are associated with graft dysfunction. Individual
hepatocytes undergoing eosinophilic or ballooning degeneration (spotty necrosis) can
be found sporadically in the lobules. Patients with low immunosuppression may develop
bridging or submassive necrosis, which is a reflection of more robust immune response
against the virus.
Recurrent chronic HBV infection
Has a more aggressive course which rapidly progresses to fibrosis due to enhanced
viral replication and attenuated host response. (Thung SN.2006). Liver biopsies show
lymphoplasmacytic portal inflammation and fibrosis, interface hepatitis, ductular
reaction,
lobular
disarray,
Kupffer
cell
hypertrophy
and
variable
lobular
necroinflammatory activity. There may be ground glass cells or sanded-appearing nuclei
corresponding to HBV surface antigen or core antigen expression, respectively. These
can be demonstrated by immunostaining for HBsAg and HBcAg. A complete absence of
stainable HBcAg should raise the possibility of other causes of graft dysfunction,
including coinfection with HCV or HDV.
Fibrosing cholestatic hepatitis (FCH) is an atypical pattern of liver injury associated
with HBV infection, and it can also affect renal transplant recipients (Thung SN.2006).
Histologically, FCH is characterised by diffuse hepatocellular swelling, cholestasis,
prominent ductular reaction and perisinusoidal fibrosis, but lack of significant
inflammatory infiltrate. The hepatocytes are swollen, often showing cytoplasmic and
nuclear immunoreactivity for HBcAg as indicative of viral replication (Thung SN.2006).
186 Introduction
Some other atypical forms of recurrent HBV infection associated with heavy viral load
have been described as fibrosing cytolytic hepatitis, fibroviral hepatitis B and steatoviral
hepatitis B (Phillips MJ, et al 1992,Thung SN.2006). In HDV/HBV coinfection, hepatitis
D
antigen
can
be
identified
in
the
nuclei
of
infected
hepatocytes
by
immunohistochemistry (Thung SN.2006). Recurrent HDV infection can also be
confirmed by HDV RNA detection by molecular studies or the presence of anti-HDV IgM
(Thung SN, 2006). HDV hepatitis associated with non-replicative HBV infection can
result in hepatitic lesions similar to fibrosing cholestatic, fibrosing cytolytic or steatoviral
hepatitis, but without HBcAg expression. In contrast, in the presence of active HBV
replication, combined HBV/HDV hepatitis in allografts is histologically similar to that in
non-allograft livers (Rosen CB, et al 2008). The differential diagnosis of recurrent HBV
infection includes HCV and non-hepatotropic viruses, drug-induced liver injury and
immune-mediated hepatitis. There may be some overlapping features between
recurrent HBV infection and acute or chronic rejection. Preferential lobular involvement
and serological data are helpful in distinguishing HBV infection from rejection. (Demetris
AJ, et al 1986).
Recurrent HCV infection
HCV infection is the most common indication for OLT. Viral recurrence is universal and
graft injury occurs routinely and is among the leading causes of graft loss and the need
for retransplantation (Brown RS. 2007, Dixon LR, Crawford JM. 2007, Khettry U, et al
2007, Petrovic LM. 2006, Watt KD, et al 2006, Asanza CG, et al 1997, Berenguer M.
2003, Berenguer M.2005, McCaughan GW, et al 2004, Belli LS, et al 1996, Charlton M.
2001, Gane E. 2003, Gane EJ. 2008 ,Gawrieh S, et al, Lake JR. 2003, McCaughan
GW, et al 2003, Ramirez S, et al 2008, Rodriguez-Luna H, Douglas DD. 2004 Rosen
HR. 2002, Russo MW, et al 2005, Zekry A, et al 2002, Ziarkiewicz-Wroblewska B, et al
2008). Reinfection occurs during allograft reperfusion, and pretransplant viral titres are
reached in about 72 h (Brown RS. 2007). Histological recurrence with hepatitis due to
HCV occurs in up to 90% of individuals by 5 years after transplant (Brown RS. 2007,
Dixon LR, Crawford JM. 2007, Ziarkiewicz-Wroblewska B, et al 2008). Progression of
HCV infection is variable. The clinical presentation of allograft recipients with recurrent
HCV hepatitis is similar to that of non-allograft patients with primary infection. Liver
enzymes increase in parallel with histological evidence of hepatitis, usually within 3–6
weeks after transplantation. Severe recurrent HCV can cause fibrosing cholestatic
187 Introduction
hepatitis (FCH). This is usually associated with overimmunosuppression and is
clinically manifested by fatigue, jaundice and a marked increase of serum bilirubin,
alkaline phosphatase (ALP) and gamma glutamyl transpeptidase (GGT). The presence
of markedly elevated HCV RNA levels is important to establish a correct diagnosis.
Cirrhosis will develop in 5–20% of patients because of recurrent HCV hepatitis. (Khettry
U, et al 2007, Petrovic LM. 2006) Several factors related to the virus (ie, genotype 1b,
viral genomic heterogeneity), the host, the environment and the donor are implicated in
the outcome (Berenguer M. 2003, Berenguer M.2005, Belli LS, et al 1996, Gane EJ
2008). The immune status is the most influencing disease severity: the more intense the
immunosuppression the worse are the outcomes (Brown RS. 2007. Watt KD, et al
2006). Donor age, donor steatosis, length of cold ischaemic, host immunogenetic
background (ie, HLA matching), and timing of recurrence and early histological findings
are other predictors of HCV infection after liver transplantation. (Brown RS. 2007
Berenguer M. 2005.) Severe recurrent HCV infection could be reduced by rapid tapering
doses of steroids and steroid-free immunosuppression, with or without induction
antibodies (Brown RS. 2007, Berenguer M. 2003, Berenguer M, et al 2006, Tisone G, et
al 2006). Pretransplant viral eradication by antiviral therapy prevents disease
progression and improves survival, whereas post-transplant treatment before or after
histological recurrence has shown variable outcomes(Brown RS. 2007 Berenguer M.
2005 Gane EJ.2008.). The presence of coexistent CMV infection after transplant and a
history of acute allograft rejection are also associated with increased severity of HCV
recurrence. (Demetris r AJ, CJ, Minervini MI, et al. 2009). Obesity and alcohol
influences are to those in nontransplanted patients. The initial biochemical and
histological hepatitis usually occurs between 1 and 3 months after transplant (Brown
RS. 2007). The influence of HCV infection on allograft histology is highly variable. The
liver injury can vary from absent or mild disease despite high viral burden to cirrhosis in
the allograft. Some patients who achieve viral response under therapy still have
progression of liver fibrosis (Cicinnati 2007b) HCV RNA concentrations in the medium
term and long term after LT do not correlate with the severity of inflammation in the liver.
Thus, regular histological evaluation of posttransplant chronic hepatitis C in 1-year (or
maximum 2-year) intervals is recommendable to determine the grade of inflammation
and fibrosis stage. In particular, the biopsy result is important for therapy decision, to
exclude signs of rejection prior to antiviral therapy and to determine the efficacy of
antiviral therapy. It has been reported that patient and graft survival in HCV-infected
188 Introduction
transplant recipients is worse compared to those with other indications) (Berenguer
2007; Forman 2002; Testa 2000) After the diagnosis of cirrhosis, the decompensation
risk appears to be accelerated 17% and 42% at 6 and 12 months, respectively
(Berenguer 2000) and patient survival is significantly decreased (66% and 30% at 1 and
5 years, respectively (Saab 2005).
Immunosupressant impact on HCV reccurence
The relationship between immunosuppressive agents and clinical expression of HCV
recurrence has been studied. TAC and CSA do not seem to be significantly different
(Berenguer 2006a; Lake 2003; Martin 2004; Hilgard 2006) with respect to their impact
on the course of hepatitis C recurrence. CSA has a strong suppressive effect on HCV
replication using the HCV replicon cell culture system (Watashi 2003) and is associated
with a higher sustained viral response in interferon-treated HCV recipients (Cescon
2009; Firpi 2009). Both CNI can increase TGF-b gene transcription and thus contribute
to the development of chronic and progressive disease. However, the accelerated
fibrosis observed in LT patients with hepatitis C recurrence does not seem to be related
to a greater amount of activated hepatic stellate cells and TGFb-1 expression in the
grafts of these patients as compared to non-LT patients with chronic hepatitis C. In LT
patients, the amount of activated hepatic stellate cells and TGFb-1 expression
correlated with the fibrosis stage and progression without any apparent influence of the
type of CNI administered (Cisneros 2007). Slowly tapering off corticosteroids over time
may prevent progression to severe forms of recurrent disease (Brillanti 2002; Berenguer
2002; McCaughan 2003). Induction with MMF is reported to be associated with more
severe recurrence of HCC (Berenguer 2003). Other investigators have found that MMF
has no impact on patient survival, rejection, or rate of HCV recurrence in HCV-infected
transplant recipients based on biochemical changes and histological findings (Jain
2002). A study showed significantly better patient survival and graft survival for HCVinfected patients treated with MMF, TAC, and steroids than for patients treated only with
TAC and steroids (Wiesner 2005). Another study has shown a positive effect of MMF in
combination with CNI taper for 24 months on fibrosis progression, graft inflammation,
and alaninaminotransferase levels (Bahra 2005). MMF may prevent fibrosis progression
through an antiproliferative effect on myofibroblast-like cells as well as to the inhibition
of adhesion molecules involved in the migration of immune cells towards the allograft,
reduced nitric oxide production and subsequent suppressed allograft injury. Results
189 Introduction
from a randomized controlled, multicenter study revealed that IL-2 induction therapy
was associated with a significantly lower mortality and rate of allograft loss 6 and 12
months after LT (Calmus 2002).
Role of non-invasive biomarkers
Studies evaluating the predictability of fibrosis using FibroScan in the LT setting are
scanty. The diagnostic value of single laboratory tests, combinations of routinely
available laboratory values with or without clinical parameters, direct biochemical
markers of hepatic extracellular matrix turnover, and more complex assays based on a
statistical approach has been assessed in immunocompetent patients. The diagnostic
use of many of these non-invasive tests, remains to be determined in LT patients. To
date, there is no model available for transplant recipients to be used irrespective of the
indication for LT
The acute phase of HCV recurrent infection
Is characterized by a peak of HCV replication and induction of hepatocyte apoptosis
and proliferation, CD8/NKT cellular infiltrate in the graft, and specific anti-HCV CD4
response (Spengler U, Nattermann J.2007). Persistent HCV infection in the allograft
most often evolves to chronic hepatitis (6–12 months). This phase is characterised by:
(1) enhanced inflammatory response and upregulation of IFN gamma-inducible genes.
(2) induction of antiviral IFN alpha-inducible genes that are not associated with reduced
viral replication.
(3) HCV-driven enhanced proliferation, apoptosis and fibrosis response in the allograft
(McCaughan GW, Zekry A.2004)
Infiltrating inflammatory cells often lack a specific HCV-directed antigen response
(Demetris r AJ, CJ, Minervini MI, et al. 2009). Less than 10% of patients may develop
severe liver injury (ie, fibrosing cholestatic hepatitis C). In this particular case, a reduced
immune response with undetectable HCV-specific CD4 response and stable
quasispecies takes place as a result of immunosuppression (McCaughan GW, Zekry
A.2004). These allografts typically show a non-specific TH2 cytokine response, with
high levels of interleukin (IL) 10 and/or IL4. Together, these events are believed to allow
rapid HCV replication resulting in extremely high viral burdens (HCV RNA levels in
serum >30 million IU/ml), and cytopathic allograft injury (Demetris r AJ, CJ, Minervini
MI, et al. 2009, Thung SN. 2006). Early hepatic stellate activation has been shown to
190 Introduction
occur in patients at greater risk to develop progressive fibrosis and more aggressive
recurrent HCV infection in the allograft (Gawrieh S, et al 2005, Russo MW,et al 2005,
Demetris AJ, Lunz JG 3rd. 2005).
Histological findings
The pathological features of HCV in liver allografts are similar to those of primary
infection in non-allograft livers. Histological recurrence may be evident within 4–6 weeks
after transplant, or sometimes as early as 10–14 days.
Liver biopsies performed during the acute phase of recurrent HCV infection
Lobular disarray,
Kupffer cell hypertrophy,
Hepatocyte apoptosis,
Mild sinusoidal lymphocytosis and
Mild mononuclear portal inflammation.
Periportal and mid-zonal large droplet steatosis is often seen.
Mild bile duct injury may be present in the form of intraepithelial lymphocytes and
scattered biliary epithelial reactive changes. Such mild duct injury needs to be
interpreted with caution so as not to overcall ACR in this setting.
The chronic phase of HCV recurrent infection
Disease progresses into a chronic phase, usually beginning at 6 –12 months after
transplant, the portal inflammation increases often with lymphoid aggregates, interface
hepatitis of variable severity, lobular disarray, and mild necroinflammatory activity.
Inflammatory bile injury is mild and focal and may be absent. Bile duct loss is not a
feature of recurrent HCV infection. Perivenular (zone 3) inflammation can be present
involving a minority of hepatic veins (Demetris AJ, et al 2006).
Fibrosing cholestatic HCV
This is an aggressive variant of recurrent HCV that occurs in occasional patients with
rapid deterioration. This is characterized histologically by: Extensive dense portal
fibrosis with immature pericellular/sinusoidal fibrous bands; Extensive hepatocyte
swelling and degeneration; Ductular reaction; Marked canalicular and cellular
bilirubinostasis; and Moderate mononuclear inflammation.
FCH due to HCV needs to be distinguished from large duct obstruction and hepatic
artery thrombosis (ischaemic cholangitis). Bile duct obstruction is identified by portal
191 Introduction
oedema and ductular reaction with or without acute cholangitis (Hubscher SG PB.
2007). It has been shown that a small subset of patients with recurrent HCV infection
present clinical and morphological features that overlap with AIH (ie, post-liver
transplant AIH like hepatitis) (Khettry U, et al 2007). In these particular cases, the
allograft biopsies show a prominent portal, periportal and lobular plasma-cell-rich
infiltrate, and perivenular (zone 3) necrosis. Some of these patients also have positive
autoimmune serology with increased serum globulins, presence of anti-nuclear antibody
and antismooth muscle antibody. Its recognition is clinically important because of
increased fibrosis progression; however, this may be difficult to separate from de novo
AIH and atypical acute rejection.
Differential diagnosis
Distinction between ACR and recurrent HCV infection is very important these two
conditions may also coexist in the liver allograft. Distinguishing recurrent HCV from
other viral hepatitides, de novo AIH, drug-induced hepatitis, PBC and PSC is based
primarily on a combination of clinical, biochemical, serological and histopathological
findings. AIH usually shows more prominent plasma cell inflammation and less steatosis
compared with recurrent HCV infection. Recurrent HCV (ie, non-FCH) may coexist with
a biliary problem or another cause of cholestasis (eg, adverse drug reaction or sepsis).
The histological features may be very similar to FCH. It is very important to rule out an
infectious process, or history of new medications. Imaging of the biliary tree may be
helpful.
192 Introduction
Laboratory features of different forms of acute hepatitides
Type
AST/ALT
ALP
Bilirubin
PT
Serology
Viral
8-50xURL
<3 URL
5-15 mg/dL
<15 sec
positive
HAV
IgM anti-HAV
HBV
HBsAG, IgM
anti HBc
HCV
HCV RNA,
+/- anti-HCV
Alcoholic
<8xURL
Toxic
Other
5-15mg/dL
<15 sec
neg
AST>ALT
>50xURL
>3xURL
in25%
normal
<5 mg/dL
>15 sec
neg
Ischemic
>50xURL
normal
<5 mg/dL
>15 sec
neg
Toxin usually
detectable, acute
renal failure
common
Acute renal failure
common
Drug
induced
8-50xURL
>3xURL
in50%
5-15mg/dL
neg
Eosinophilia, skin
rash common
Autoimmune
8-50xURL
<3 xURL
5-15mg/dl
Positive ANA
or ASMA
Low albumin, high
globulins
Wilson
8-50xURL
Low normal
or decreased
5-15 mg/dL
neg
Hemolytic anemia,
renal failure
common, low
ceruloplasmin
oftenly absent
Recurrent autoimmune hepatitis
Recurrent autoimmune hepatitis diagnostic criteria
Liver transplant for autoimmune hepatitis
Autoantibodies in significant titre (1>40)
Sustained rise in serum aminotransferase activity ( more than two times normal)
Elevated serum immunoglobulins
Diagnostic or compatible liver histology
Corticosteroid dependency
Exclusion of other causes of graft dysfunction (eg, HCV infection, rejection)
Autoimmune hepatitis is a relatively uncommon indication of liver transplant. Outcomes
are good with 1-year and 5-year patient survival rates of about 87% and 80–90%,
respectively. Graft survival rates at 1 year and 5 years are 84% and 74–76%,
respectively (Olivera-Martinez MA, Gallegos-Orozco JF2007, Ayata G, et al 2000,
Gonzalez-Koch A, et al 2001, Hubscher SG. 2001, Ratziu V, et al 1999, Reich DJ, et al
2000). The reported recurrence rate for AIH in most studies is in the range of 17–42% at
5 years (Faust TW.2001, Gautam M, et al 2006, Schreuder TC, Hubscher SG,
Neuberger J. 2009, Said A, Lucey MR. 2008). Recurrent AIH occurs at variable time
193 Introduction
periods after transplantation, slowly progressing Ayata G. et al 2000) and responds well
to increases in immunosuppression or addition of corticosteroids (Faust TW.2001). The
pathogenesis of AIH is unknown. Autoimmune response is influenced by the genetic
susceptibility to present self or cross-reacting antigens, the sensibility to aetiological
triggers (ie, viruses or toxins), and the composition cytokine environment (Said A, Lucey
MR. 2008, Gonzalez-Koch A, et al 2001). Aberrant exposure of HLA-II antigens and
enhanced presentation of normal constituents on hepatocytes with subsequent
activation and proliferation of cytotoxic T lymphocytes may take place. Hepatocellular
damage seems to be secondary to proinflammatory cytokines released by sensitised T
cells (Faust TW. 2001, Vergani D, Mieli-Vergani G. 2004). There are no consistent risk
factors for recurrence, but recurrent AIH has been shown to be more common in
transplant recipients who were HLA-DR3 positive or HLA-DR4 positive in one
study.(Schreuder TC, et al 2009). Furthermore, a greater incidence of recurrent is
disease associated with the presence of type I autoimmune disease, and severe
inflammation
in
the
native
liver
before
transplantation
and
suboptimal
immunosuppression (Ayata G, et al 2000, Hubscher SG 2001). Diagnostic criteria for
recurrent AIH are similar to those used in the non-transplanted liver (Alvarez F, et al
1999) but the difficulty to apply them in the allograft liver is due to biochemical and
histological overlap with ACR, the immunosuppressive environment, and the possibility
of alloimmune disease directed against allograft antigens (Hubscher SG. 2001).
Because of the lack of reliable disease markers liver biopsy remains the main diagnostic
tool for identifying recurrent AIH in the allograft.
Histological findings. The histological changes attributed to recurrent AIH are not
specific and need to be distinguished from other causes of chronic hepatitis, ACR,
chronic rejection, adverse drug reactions and recurrent PBC and PSC. Early changes
include lobular hepatitis with hepatocyte ‘‘rossetting’’ (Schreuder TC, Hubscher SG,
Neuberger J. 2009, Hubscher SG. 2001, Demetris AJ, et al 2006) The chronic phase is
usually marked by portal infiltrate composed of lymphocytes and plasma cells with
prominent interface activity. Lobular necroinflammatory activity is variable, and confluent
and bridging necrosis may be present. Perivenular (zone 3) inflammation can be
present in amajority of hepatic venules similar to rejection.
Bile duct inflammatory
damage involves a minority of ducts (Hubscher SG. 2001, Demetris AJ, et al 2006).
194 Introduction
Recurrent PBC
Recurrent primary biliary cirrhosis diagnostic criteria
Liver transplant for primary biliary cirrhosis
Persistence of antimitochondrial antibodies
Elevated immunoglobulins
Diagnostic or compatible liver histology
Exclusion of other causes of graft damage
PBC is considered to be a disease of disordered immune regulation characterised by
progressive loss of interlobular and septal bile ducts leading to cholestasis and
cirrhosis. Antimitochondrial antibodies are present in 95% of patients. Liver
transplantation is indicated for advanced PBC, with excellent overall patient and graft
outcomes. The 5-year survival rate after deceased donor liver transplantation is
approximately 80%. Recurrent PBC after transplantation is controversial, but has now
become accepted. Recurrent PBC is seen in 17% of patients at a mean of 36 months,
and 30% at 10 years. The reported median time recurrence is between 3.7 and 5 years.
(Faust TW. 2001 Gautam M, et al 2006, Mottershead M, Neuberger J. 2008, Schreuder
TC, Hubscher SG, Neuberger J.2009, Tamura S, et al 2008, Dmitrewski J, et al 1996,
Hashimoto E, et al 2001, Liermann Garcia RF, et al 2001, Neuberger J. 2003). The role
of ursodeoxycholic acid in the treatment and prevention of recurrent PBC is
controversial (Mottershead M, Neuberger J. 2008). Disease recurrence is more
common after living-related liver transplantation and after corticosteroid withdrawal
(Hashimoto E, et al 2001). Other factors such as donor and recipient age, cold and
warm ischaemia time, and type of immunosuppression used, may influence disease
recurrence.
Elevated
serum
immunoglobulins
and
persisting
antimitochondrial
antibodies are not sufficient for the diagnosis of disease recurrence. A diagnosis of
recurrent PBC is made by characteristic histology, which may be present in the allograft,
even in the absence of biochemical abnormalities (Mottershead M, Neuberger J. 2008).
Other causes of graft damage should be ruled out.
Histological findings
Changes of recurrent PBC are similar to those present in native livers. Liver biopsies
with recurrent PBC may show one or more of the following features: Variable portal
inflammation with mononuclear (or mixed) infiltrate, Lymphoid aggregates with germinal
centres, Lymphocytic cholangitis with biliary epithelial eosinophilia, and periductal
195 Introduction
epithelioid non-necrotising granulomatous reaction (Neuberger J.2003, Hubscher SG
PB. 2007).
The diagnostic lesions (ie, epithelioid granulomas and florid duct lesions) are often
focal, and therefore may not be present in needle biopsies in the early stages). As
disease progresses, there is development of lymphoplasmacytic interface activity
resembling AIH, and biliary interface activity with cholate stasis. Additional features
include ductular reaction, portal and periportal fibrosis, small bile duct loss and
periportal oedema (halo sign). The parenchyma may show spotty necrosis or even
scattered foci of lytic necrosis, and deposition of copper and copper-associatedproteins
at the portal/parenchymal interface provides supportive evidence of recurrent PBC if
other causes of biliary tract disease have been excluded. Bile duct injury or loss due to
recurrent PBC needs to be distinguished from ACR, chronic rejection, adverse drug
reaction, CMV and HCV infection, recurrent PSC, ischaemic cholangitis, recurrent or de
novo AIH, and graft versus host disease. Usually, the clinical scenario, serological
investigations and imaging results are very important to make the diagnosis clear. A
diagnosis of recurrent PBC can be definitive when granulomatous bile duct destruction
and/or florid bile duct lesions are present in the proper clinical context. In the absence of
these features, the presence of a prominent but focal lymphocytic cholangitis,
accompanied by portal-based lymphoid aggregates with germinal centres and bile
ductular reaction, are highly suggestive, although not diagnostic, of recurrent
PBC.(Hubscher SG PB. 2007). Sometimes the time frame of rise of ALP is a clue: a
sudden rise in ALP is unlikely to be due to recurrent PBC.
Recurrent primary sclerosing cholangitis
Liver transplant for primary sclerosing cholangitis
Multiple nonanastomotic biliary strictures
Exclusion of other causes (ie, rejection, infection, ischaemia)
Diagnostic or compatible liver histology
PSC is a progressive cholestatic disease of unknown aetiology that usually involves
both the extrahepatic biliary tree and the intrahepatic biliary tree, and has a close
association with inflammatory bowel disease. The hallmark clinical lesion of PSC is an
abnormal cholangiogram. Endoscopic retrograde cholangiopancreatography and
magnetic resonance cholangiopancreatography typically show irregular strictures,
beading, diverticular outpouching, and pruning of bile ducts. Liver transplantation is
196 Introduction
indicated for patients with end-stage disease. The long-term outcome after
transplantation is very good, with survival rates of 86% at 5 years, and 70% at 10 years.
(Abouljoud MS, et al 2001). A higher incidence of acute and chronic and steroidresistant rejection in PSC patients has been reported, especially in the presence of
coexistent inflammatory bowel disease (Demetris AJ. 2006, Abouljoud MS, et al 2001).
Recurrence of PSC after transplantation ranges from 9% to 47% (Abouljoud MS, et al
2001, Kotlyar DS, et al 2006, Schreibman IR, Schiff ER. 2006, Faust TW. 2001,
Schreuder TC, et al 2009, Goss JA, et al 1997, Graziadei IW, et al a1997, Graziadei IW,
et al b1999, Gordon F. 2006, Gow PJ, Chapman RW. 2000, Graziadei IW. 2002,
Oldakowska-Jedynak U, et al 2006). Risk factors for disease recurrence include donor–
recipient gender mismatch, male gender, and intact colon at the time of transplantation.
(Vera A, et al 2002). The presence of cholangiocarcinoma is considered an absolute
contraindication to transplantation at most centres due to the high risk of recurrence in
the graft (Gordon F. 2006). The presence of hilar cholangiocarcinoma before
transplantation significantly decreases survival after transplantation. There is yet no
effective treatment to delay the presentation or progression of recurrent PSC in the
allograft (Gordon F. 2006). Selective elevation of ALP and GGT due to PSC recurrence
usually manifests 1 year after transplantation. It is very difficult to separate recurrent
PSC from other causes of biliary strictures (eg, choledochojejunal anastomotic stricture,
hepatic artery thrombosis, preservation injury, chronic ductopenic rejection, ABO blood
group incompatibility, viral/bacterial biliary tract infection, SFSS in living donors, nonheartbeating donors) (Gordon F. 2006). Non-anastomotic intrahepatic strictures that
develop within 90 days after transplantation are not considered recurrent disease. The
diagnosis of recurrent PSC requires cholangiographic and histological evaluation.
Histological findings
The histological features of recurrent PSC are identical to those seen in the native livers
with PSC. Early changes in the peripheral liver include mild non-specific acute and
chronic ‘‘pericholangitis’’ and mild ductular reaction. (Demetris AJ, et al 2006, Gautam
M, et al 2006). As disease progresses, there is periductal lamellar oedema with
increased ductules and mixed portal inflammation with eosinophils and neutrophils,
periportal oedema, ductular reaction and scattered small duct loss (Hubscher SG PB.
2007). Later stages are featured by biliary cirrhosis, cholestasis, marked copper
deposition, and Mallory bodies in paraseptal hepatocytes. Periductal concentric fibrosis
197 Introduction
and duct loss involve small and medium-sized bile ducts. These so-called ‘‘fibroobliterative duct lesions’’ can also be seen in patients with ischaemic cholangitis
(hepatic artery thrombosis) and other post-transplant causes of secondary sclerosing
cholangitis. The large intrahepatic and extrahepatic bile ducts may show ulceration,
biliary sludge and marked periductal lymphoplasmacytic infiltrate (Demetris AJ. 2006).
The distinction between recurrent PSC and chronic rejection may be challenging, as
both cause a cholestatic pattern of liver enzyme elevation and duct loss .The clinical
history, evaluation of serial biopsies and histopathological findings are useful to
separate these two conditions (Hubscher SG PB. 2007).
Recurrent alcoholic liver disease, non-alcoholic fatty liver disease and nonalcoholic steatohepatitis
Alcoholic liver disease represents a leading cause indication for liver transplantation
with short-term survival rates comparable to those for patients who undergo liver
transplantation for other conditions.( Lim JK, Keeffe EB. 2004, Lucey MR, 1997, Tang
H, Boulton R, et al. 1998). The rate of alcohol relapse is considered low, and
resumption of alcohol seems to begin within the first year after transplantation (Mackie
J,et al. 2001, Burra P, Lucey MR.2005). Fatty liver and steatohepatitis are the main
histological features of alcohol relapse. (Burra P, Lucey MR.2005). More severe
recidivism can lead to frank alcoholic hepatitis with Mallory’s hyaline, foamy
degeneration of hepatocytes and perivenular fibrosis. Accurate data on the percentage
of liver transplants performed for non-alcoholic steatohepatitis (NASH)-related cirrhosis
are not available, in part because many cases identified as cryptogenic cirrhosis may in
fact represent ‘‘burnt out’’ NASH. (Burke A, Lucey MR .2004). Steatosis has been
reported to occur within 6–12 months and cirrhosis within 2 years of transplantation in
patients undergoing liver transplantation for NASH. (Burke A, Lucey MR.2004, Contos
MJ, et al 2001). Recurrent NASH seems to occur at later times than fatty liver alone,
with increasing incidence over time during follow-up.
Recurrent metabolic diseases In disorders such as type 1 tyrosinaemia, a1-antitrypsin
deficiency, Wilson disease, neonatal haemochromatosis, and glycogen storage disease
types 1, 3 and 4, the liver is replaced by a genetically normal one that is not susceptible
to recurrent disease (Jaffe R. 1998). The risk of recurrence is higher in patients with
198 Introduction
metabolic defects involving extrahepatic sites, and the effects on the liver are largely
secondary in those that are at highest risk of recurrence (eg, Niemann–Pick disease,
Gaucher’s disease, cystinosis and erythropoietic protoporphyria) (Jaffe R. 1998).
Inborn Errors of Metabolism that Have Been Treated by Liver Transplantation
Liver Affected
Other Organs Also Affected
α1-Antitrypsin deficiency
Primary hyperoxaluria
Wilson disease
Crigler-Najjar syndrome
Protoporphyria
Primary hypercholesterolemia
Tyrosinosis
Niemann-Pick disease
Tyrosinemia
Sea-blue histiocyte disease (may recurs after transplantation)
Galactosemia
Hemophilia A and B
Glycogen storage disease types I, IV
Protein C deficiency; Protein S deficiency
Byler disease
Hemochromatosis
Cystic fibrosis (may recur)
Gaucher disease (may recur)
Urea cycle enzyme deficiencies
1.10.5 LATER NEW-ONSET DISEASES/INJURIES IN THE LIVER ALLOGRAFT
Biliary complications occur early and late in the post-transplant course. At the time of
transplantation, reconstruction of the biliary tract is either a duct-to-duct anastomosis or
choledochojejunal anastomosis. Mucosal and/mural damage may occur in the process
and lead to biliary tract complications, such as bile leaks, and anastomotic or
intrahepatic strictures (Sanchez-Urdazpal L, et al. 1993, Sanchez-Urdazpal L, Gores
GJ, et al. 1992). The process of biliary wound healing occurs and may or may not be
ineffectual. This can affect the small and/or large extrahepatic biliary tree. In the
extrahepatic large bile ducts, biliary healing may lead to scarring and stricture formation.
In the small extrahepatic bile ducts, impaired proliferation of the bile duct epithelium or
exuberant responses can contribute to liver injury (Demetris AJ, 2006). Radiological
tests such as MRI and/or allograft biopsies may be performed in the course of
investigation of biliary complication post-transplant.
Biliary sludge syndrome
Cold ischaemic-preservation injury depletes energy stores in microvascular endothelial
cells and bile duct epithelium. As a result, metalloproteinases are activated. Biliary
199 Introduction
epithelium
and
endothelium
are
detached
from
underlying
matrix.
In
the
microvasculature, detachment of endothelium predisposes to thrombosis after
reperfusion. (Kukan M, Haddad PS. 2001, Teoh NC, Farrell GC. 2003) Leucocytes
become activated by tissue damage and release effector molecules, causing more
tissue damage and further promote thrombogenesis. (Kukan M, Haddad PS. 2001,
Teoh NC, Farrell GC. 2003) Several factors, including increased sensitivity of bile duct
cells to reperfusion injury, poor functional recovery after ATP depletion, invasion of
polymorphonuclear leucocytes into bile ducts, and hydrophobic bile salts, appear to
contribute to preservation-related injury of bile ducts. (Kukan M, Haddad PS. 2001,
Teoh NC, Farrell GC. 2003, Noack K, et al 1993, Carrasco L, et al 1996) Damaged
biliary epithelial cells are sloughed into the bile, the underlying stroma become exposed
to bile, which form a nidus for crystallisation of biliary sludge (Demetris AJ, et al 2006).
Injury of bile ducts is associated with hyperbilirubinaemia and underlies the long lasting
phase of reperfusion graft injury. (Kukan M, Haddad PS. 2001, Teoh NC, Farrell GC.
2003) Morphological changes take place in the extrahepatic large bile ducts and
intrahepatic large bile ducts, as well as in the small intrahepatic ducts. Biopsies usually
sample the peripheral liver, therefore small intrahepatic ducts are that what is
encountered in biopsies. There is prominent ductular reaction consisting of biliary cells
and periductal myofibroblasts due the increased pressure in the biliary tree distal to the
point of luminal obliteration. The proliferating ductules and myofibroblasts form a wedge
of tissue that arises from the portal tract and distorts the liver architecture. Large bile
ducts are usually seen at the time of re-transplantation in the excised failed graft. There
is biliary sludge, mucosal ulcers and inflamed granulation tissue and myofibroblast
proliferation in the wall of extrahepatic bile ducts and large intrahepatic bile ducts
(Demetris AJ, et al 2006) As a result of exposure of the underlying stroma; inflammation
and activation of myofibroblasts take place which lead to wound contraction and
fibrosis, and strictures in large-calibre ducts. Complete fibrous obliteration of the bile
duct lumens by concentric rings of fibrous tissue occurs.
Bile duct strictures
Blood is supplied to intrahepatic bile ducts and extrahepatic bile ducts exclusively
through hepatic arteries (HAs) (Takasaki S, Hano H. 2001). Over 50% of the blood
conveyed by HAs is primarily destined to the bile ducts (Ekataksin W, ZZ, Wake K, et
al.1997). Intrahepatic arteries course in close proximity to the bile ducts. They drain into
200 Introduction
the peribiliary plexus, which is a rich microvascular network surrounding bile ducts
(Ekataksin W, ZZ, Wake K, et al.1997). Blood supplying the bile ducts drains into the
sinusoids via the portal system. Ischaemia-induced bile duct lesions have been
collectively labelled as ischaemic cholangitis (Batts KP. 1998). The biliary epithelium is
susceptible to injury when arterial blood flow is compromised. Ischaemic cholangitis
manifests as segmental strictures with resultant mechanical impairment of bile flow and,
occasionally, secondary infection of the biliary system. Biliary strictures in liver
transplant recipients are related to a combination of large HA occlusion related to
surgical reconstruction or sepsis, and/or damage to small-sized arteries and peribiliary
plexus—due to preservation, reperfusion, rejection, ABO incompatibility or CMV
infection. (Deltenre P, Valla DC. 2006).
Biliary strictures may be anastomotic or nonanastomotic.
Anastomotic biliary strictures
They result from technical surgical problems or local ischaemia. Most anastomotic
strictures appear within the first several months after transplantation, but they may also
develop, less frequently, many years after transplantation. The incidence has been
reported to be 10% for deceased donor liver transplantation, and up to 30%, for living
donor liver transplantation.( Verdonk RC, et al. 2006, Greif F, et al. 1994, Mosca S, et al
2000,
Chahin NJ, et al. 2001, et al 2004, Yazumi S, Chiba T. 2005, Pascher A,
Neuhaus P. 2005)
Nonanastomotic strictures
Occur later after transplantation, generally progressive, resistant to treatment, and
adversely impact graft and patient survival. Intrahepatic biliary strictures result primarily
from hepatic artery thrombosis (Deltenre P, Valla DC. 2006) but in the absence of
hepatic artery occlusion, they are related to chronic ductopenic rejection, ABO
incompatibility, ischaemia–reperfusion injury, or recurrence of primary disease, such as
PSC or AIH. (Sanchez-Urdazpal L, et al 1992, Noack K, et al. 1993, Pascher A,
Neuhaus P. 2005, Colonna J, et al. 1992, Li S, et al 1992, Guichelaar MM, et al.2003).
The biopsy features of duct obstruction in allografts are the same as those encountered
in native livers. Most complications show predominantly neutrophilic portal inflammation,
periductal oedema, and intraepithelial and intraluminal neutrophils within true portal bile
ducts. Mild ductular proliferation, centrilobular hepatocanalicular cholestasis and small
201 Introduction
clusters of neutrophils throughout the lobules are also commonly seen. (Demetris A, et
al 2009). Chronic biliary tract strictures may be associated with chronic portal
inflammation and biliary epithelial cell senescence.
The differential diagnosis of early duct obstruction (within 6 months post OLT) is
preservation injury with biliary sludge syndrome and ACR. Late obstruction, i.e. after 6
months post transplantation, biliary obstruction/stricture mimics a broader spectrum of
processes that include: Acute and chronic rejection, Viral hepatitis and Recurrent
autoimmune disorders.
Chronic intermittent biliary obstruction or cholangitis, as in patients with the biliary
sludge syndrome, can be associated with a mixed, or a predominantly mononuclear,
portal infiltrate and biliary epithelial cell senescence changes. Portal fibrosis with mild
duct proliferation, mild portal neutrophilic or eosinophilic inflammation, and mild
centrilobular cholestasis are features that suggest obstructive cholangiopathy.
202 Introduction
Differential diagnosis of biliary stricture
Histological feature
Biliary stricture
Preservation injury
Acute rejection
Portal inflammation
Predominantly
neutrophilic
Mild non-specific
inflammation
Lymphocytes, plasma
cells, and eosinophils
(which may predominate
when patients are treated
with corticosteroidsparing
immunosuppressive
regimens)
Bile duct epithelium
Relatively normal
nucleus-tocytoplasm
ratio
+/-
Reactive changes;
increased nucleus-tocytoplasm ratio
Perivenular
mononuclear
inflammation
Absent
Absent
Present
Ductular reaction
Usually present
May be prominent if
biliary sludge
syndrome present
Usually absent
Periductal oedema
Usually present
Neutrophils and bile
ducts
Intraepithelial and
intraluminal neutrophils
may be present in
interlobular ducts
Neutrophilic
pericholangitis (if
severe
No; duct injury by
lymphocytes seen
infiltrating biliary
epithelium
Periportal architectural
collapse
Absent
May be present in
severe injury
Usually absent
Parenchyma
Centrilobular
cholestasis in
hepatocytes and
canaliculi; small clusters
of neutrophils in lobules
may be seen
Zonal confluent
necrosis early.
Hepatocellular,
swelling, rounding up,
centrilobular
cholestasis in
hepatocytes and
canaliculi
Absent
INFECTIOUS COMPLICATIONS The scope of infections in liver allograft patients is
broad, and include viral including EBV, CMV, newly acquired hepatitis B or hepatitis C
infection and non-viral agents that are either new or re-activated (mostly opportunistic)
infections.
Post-transplant EBV infection
The incidence of EBV infection post-transplant is variable and occurs in 63–80% of
patients who are seronegative at the time of transplantation. Reactivation infections
occur in 20–22% of patients exposed to the virus before transplantation (Lamy ME, et al
1990, Smets F, et al 2000). Many patients are asymptomatic. In a paediatric series, only
203 Introduction
15% of patients with primary post-transplant EBV hepatitis were symptomatic (Smets F,
et al 2000).
Typical EBV hepatitis pattern shows mild portal and sinusoidal mononuclear
infiltrates. These infiltrates are composed of small and mildly atypical lymphocytes
arranged in a single file pattern within the sinusoids. In situ hybridisation for EBVencoded RNA (EBER) is confirmatory.
Lobular changes include focal hepatocellular swelling, acidophilic necrosis of
hepatocytes and mild lobular disarray. Granulomas may occur (Ishak K. 1983).
Pattern of nonspecific reactive hepatitis. The liver sinusoids show mild
lymphocytosis. Portal mononuclear inflammatory infiltrates are of variable severity.
Portal tracts with sparse infiltrates coexist with dense portal lymphocytic infiltrates
elsewhere in the same biopsy. Characteristic constellation of features consists of mixed
mononuclear portal and sinusoidal infiltrates containing atypical large non-cleaved
mononuclear cells and immunoblasts, associated hepatitic lobular activity, and relatively
mild duct damage disproportionate to the severity of the infiltrate (Randhawa PS, et al
1990). Serological confirmation can be obtained. The differential diagnosis of a
mononuclear infiltrate in the post-transplant setting is cellular rejection, which may
coexist especially in biopsies taken as follow-up after treatment for EBV hepatitis that
includes reduction of immunosuppression. EBV-related bile duct damage and phlebitis
may occur (Chang MY, Campbell WG Jr.1975) .The presence of occasional EBVinfected cells was thought to reflect an increased circulating viral burden in these
patients.171. (Randhawa P, et al.2001). Positive in situ hybridisation for EBER in liver
biopsies is only seen in the context of a high viral load in patients with PTLD or those at
high risk for developing this complication. (Randhawa PS et al.1992).
Post-transplant CMV infection
In liver transplant recipients, the overall incidence of CMV disease has reached 29%
and 2–17%. (Paya CV, RR.2003, Lautenschlager I, et al 2006 Seehofer D et al 2005) in
CMV hepatitis as a significant complication of CMV infection after liver transplantation.
Transplantation of an organ from a CMV-serology-positive donor to a serologynegative
patient (D+/R2) carries the highest risk and may be as high as 44–65% if no prophylaxis
is given. (Gane E, et al 1997, Paya CV, et al 1989). CMV infection of the liver transplant
204 Introduction
is characterised by graft dysfunction. The diagnosis should be based on liver biopsy.
(Colina F, et al 1995, Washington K.2005) .The histological features of CMV hepatitis
are variable. In high-risk patients (ie, heavily immunosuppressed or naive recipients),
CMV cytopathic effects may occur in any cell type of the liver. The characteristic CMV
inclusions are large eosinophilic and intranuclear, surrounded by a clear halo.
Occasional small basophilic or amphophilic cytoplasmic inclusions may be present
(Demetris A et al 1996). CMV hepatitis is characterised by spotty lobular necrosis, and
mild lobular disarray. There is mononuclear, or mixed, portal inflammation, focal bile
duct damage and aggregates of macrophages (microgranulomas) scattered throughout
the parenchyma. Hepatocytes containing CMV inclusions may be associated with small
clusters of neutrophils (microabscesses). CMV hepatitis can sometimes be difficult to
distinguish from early recurrent HCV or HBV. CMV hepatitis may also posses
overlapping features with EBV hepatitis: mild lymphoplasmacytic portal and lobular
inflammation. If present, microabscesses and microgranulomas are helpful, as they are
not generally associated with HBV or HCV infection, but immunoperoxidase staining for
viral antigens (CMV, HSV and HBV) or in situ hybridisation for EBV should be
performed, as either is usually diagnostic in these difficult cases. The demonstration of
CMV inclusions or its antigens by immunostaining takes precedence, resulting in
reduction in immunosuppression and initiation of ganciclovir therapy. Correlation of time
lines of enzyme improvement or worsening with immunosuppression lowering and/or
initiation of ganciclovir treatment can be helpful in resolving the differential diagnosis.
DE NOVO HEPATITIS
Idiopathic chronic hepatitis Unexplained chronic hepatitis
Idiopathic chronic hepatitis incidence in the adult allograft recipient is variable ranging
from 10% and 50% in different series. (Sebagh M, et al, Berenguer M, et al 2001,
Rosenthal P, et al 1997, Slapak GI, et al 1997, Heneghan MA, et al 2003, Burra P, et
al 2001). The features are largely similar to those seen in chronic hepatitis in the nontransplant setting. (Scheuer PJ 1991) but bile duct injury or vascular lesions
characteristic of acute or chronic rejection are minimal or absent (Evans HM et al 2006).
ICH is characterised by a predominantly portal mononuclear inflammatory infiltrate
associated with interface hepatitis. Lobular inflammation is of variable degrees, and
hepatocyte necrosis or apoptosis is frequently present. Factors affecting long-term
outcome or late biopsy findings are: the use of extended-criteria organs with influence
205 Introduction
from donor factors and the early postoperative course, severity of early acute rejection,
undetected low-grade rejection, variation in terminology and histological threshold for
diagnosis of ICH (‘‘portal and lobular mononuclear inflammation’’ at one centre may be
diagnosed as ICH, whereas a similar infiltrate may be termed as ‘‘nonspecific
inflammation’’ in another centre). The differential diagnosis includes cellular rejection,
infections and de novo post OLT AIH, recurrent or newly acquired HCV or HBV, viral
hepatitides include EBV, and hepatitis E virus which has been hypothesised that it may
be a cause of chronic hepatitis in liver transplant recipients. Unidentified hepatotropic
viruses have been suggested as a possible cause of chronic hepatitis, (Shaikh OS et al
2007, Kamar N et al 2008). The histological features of ICH on routine stains are
nonspecific and thus, often, the aetiology cannot be determined on histological grounds
alone. Correlation of histological findings with clinical events and time lines of enzyme
elevation, if any, is required (eg, recent reduction in immunosuppression, recent illness
interfering with absorption of medications, introduction of new drugs that may cause
drug-induced liver injury or affect levels of immunosuppressants). Autoimmune and viral
(including hepatotropic and non-hepatotropic viruses, eg, EBV) serology and
immunoglobulin levels, particularly IgG, are an integral part of evaluation to complement
histology, immunostains for other viruses, such as herpes simplex and CMV, may be
performed. In situ hybridisation for EBV performed on the liver biopsy should also be
considered. ICH has a benign rather than a significantly adverse clinical outcome,
(Burra Pet al 2001, Sebagh M et al 2003, Berenguer M, et al 2001, Rosenthal P et al
1997, Slapak GI et al 1997, Heneghan MA et al 2003) although there are a few studies
that show evidence to the contrary as in a retrospective study by the Birmingham group
reported mild inflammatory activity and mild to moderate fibrosis in the initial biopsies of
12 of 30 recipients with ICH. Approximately 41% of these 12 recipients subsequently
developed marked graft dysfunction, new or progressive fibrosis was noted in
approximately 50%, and three patients developed cirrhosis (Syn WK et al 2007).
De novo AIH
In native liver, AIH does not have a pathognomonic feature, and its laboratory,
serological and histological manifestations are found in acute and chronic liver disease
of diverse causes. In 1993, the International Autoimmune Hepatitis Group proposed
diagnostic criteria, which were revised in 1999. Due to the complexity of these criteria
206 Introduction
and their insufficient validation, the International Autoimmune Hepatitis Group devised a
simplified scoring system for wider applicability in routine clinical practice.
Autoimmune
Hepatitis:
Revised
Scoring
System
(1999)
(International Autoimmune Hepatitis Group, J. Hepatology 31: 929-938, 1999)
Feature
-5
-4
-3
-2
Sex
ALP/ALT or
ALP/AST (note 1)
>3
0
+1
+2
Male
Female
1.5-3.0
<1.5
Serum globulins
or IgG above
normal
<1x normal
1-1.5x
normal
1.5-2x
normal
ANA, SMA, or
LKM1 (note 2)
<1:40
1:40
1:80
AMA
Positive
Hepatitis viral
markers (note 3)
Negative
Positive
Drug history
(note 4)
Yes
No
Average alcohol
intake
Histology
> 60 g/day
Absence of
all of the
following:
interface
hepatitis,
lymphoplasmacyti
c infiltrate,
and liver
cell
rosettes
Biliary
changes
(note 5) or
other
defined
changes
(note 6) (-3
each)
<25 g/day
Predomina
ntly
lymphoplasmacyti
c infiltrate,
liver cell
rosettes (1
each)
207 Introduction
Simplified Score for the Diagnosis of Autoimmune Hepatitis (AIH) (Hennes et al.,
2008)
Clinical feature
ANA or SMA
≥1:40
≥1:80 or LKM1 ≥1:40 or SLA-positive
Serum IgG
>upper limit of normal
>1.1 times upper limit of normal
Histologic findings
Compatible with AIH
Typical of AIH
Hepatitis viral markers
Negative
Aggregate score without treatment
Definite AIH
Probable AIH
Points
+1
+2
+1
+2
+1
+2
+2
≥7
≥6
Histologically
Mononuclear infiltrates that are plasma-cell predominant and interface activity are
reasonably good markers of AIH in native livers (Czaja AJ, Carpenter HA (a) 1997,
Czaja AJ, Carpenter HA(b) 1993). De novo AIH in the liver allograft is diagnosed by a
plasma-cell rich infiltrate showing significant necroinflammatory interface and
perivenular activity. In the posttransplant setting, a new diagnosis of AIH is complicated
by the need to distinguish this entity from recurrent HCV and rejection. HCV infection
appears to induce a genetic susceptibility to autoimmune processes, including in the
liver (Dai YD, et al 2005, Vanderlugt CL, Miller SD.2002, Demetris AJ, Sebagh M. 2008)
and HCV infection by itself in the non-transplant setting can be associated with multiple
immunemediated extrahepatic manifestations, and chronic HCV liver disease can be
associated with AIH-like features in native liver (Czaja AJ, Carpenter HA (a) 1997,
Kessel A, Toubi E 2007). It has been reported the presence of higher serum levels of
gammaglobulin and immunoglobulin G, higher frequency of cirrhosis, a higher mean
Knodell score, a higher frequency of HLA-DR3, and a high titre of smooth muscle
antibodies associated with the AIH-like pattern of HCV-induced liver injury in the general
population (Czaja AJ, Carpenter HA (a) 1997). There is evidence in the literature to
suggest that patients transplanted for HCV liver disease, like non-transplant patients
with HCV, can develop AIH-like features in the graft associated with recurrent HCV or,
after successful HCV therapy, with HCV RNA clearance (Khettry U et al 2007, Berardi S
208 Introduction
et al 2007, Fiel MI et al 2008). The features of post-transplant AIH are similar to those
considered in the native liver and include: portal inflammation with numerous plasma
cells, prominent interface hepatitis, and lobular inflammation (plasma cell rich) with zone
3 necrosis. Perivenular inflammation, and necrosis involving a majority of central veins,
are not typical features of recurrent HCV in allografts; when they are found, an
alternative
explanation
that
includes
an
accompanying
immune-mediated
injury/cellularrejection should always be considered and further investigated clinically.
DRUG-INDUCED LIVER INJURY
Drug-induced liver injury can mimic many patterns of transplant-related and nontransplant-related liver pathology. Some drugs are commonly used in the posttransplant setting: immunosuppressants and sulfamethoxazole– trimethoprim (Septra).
Ciclosporin (CyA) and tacrolimus (KF506) hepatotoxicity were reported in liver allograft
recipients in the early 1990s. However, newer insights indicate that many of the features
thought to represent CyA and/or FK506 toxicities, such as perivenular necrosis/fibrosis,
bile duct epithelial changes and sinusoidal foam cells, are in fact due to chronic
rejection. (Wisecarver JL, 1992, Kassianides C, et al 1990, Fisher A, et al 1995) In
practical terms, these calcineurin inhibitors are more likely to present with renal toxicity
leading to dose adjustments before hepatic injury becomes clinically apparent, and may
explain why they are almost never reported in clinical practice. Sirolimus and
azathioprine probably cause hepatotoxicity. In cases of liver allografts biopsied for
elevated liver enzymes, including acute hepatic injury in patients on sirolimus (alkaline
phosphatase and transaminases) or preferentially alkaline phosphatase elevation in the
case of azathioprine, in the absence of other contending explanations and with
nonspecific histopathological findings (such as centrilobular cholestasis, focal feathery
degeneration of hepatocytes, peliosis and sinusoidal dilatation), drug toxicity should be
considered (Hebert MF, TS, Carithers RL. 2003, Neff GW, et al 2004, Niemczyk M, et al
2005, Degott C, et al 1978) Importantly azathioprine has the tendency to cause
endothelial injury, sometimes resulting in veno-occlusive disease (VOD) (Holtmann M,
et al 2003, Haboubi NY, et al 1988)
Sulfamethoxazole–trimethoprim (Bactrim, Septra) is unfortunately a known cause of
cholestatic liver injury, which may be prolonged to 1–2 years after discontinuation, and
sometimes presenting with pruritus. (Mohi-ud-din R, Lewis JH. 2004) Liver biopsy in
Septra toxicity is primarily cholestatic, sometimes causing ‘‘vanishing ducts’’ but minimal
209 Introduction
hepatocellular necrosis or inflammation. (Kowdley KV, et al 1992). Most times the
cholestatic nature of injury is apparent, but sometimes cytokeratin 7 or other bile duct
epithelial markers should be obtained once this possibility becomes relevant in the
individual case.
VASCULAR ABNORMALITIES
Nodular regenerative hyperplasia
Nodular regenerative hyperplasia NRH has been reported to develop up to 20 years
after Stem Cell and other solid organ transplantation (Snover DC et al 1989). It is
recognised in liver allografts especially in the context of SFSS. NRH is presumed to be
due to chronic lowgrade vascular injury, which in the post-transplant setting may be
partly due to abnormal activation of the immune system. A localised imbalance of portal
vein and hepatic arterial inflow leads to periportal hepatocyte proliferation and NRH
changes that may be regional or focal rather than diffuse. Regional hypoperfusion of
small portal vein branches late after transplantation because of thrombosis and luminal
obliteration can locally increase hepatic arterial flow. (Kondo F. 2001). The diagnosis of
NRH can be difficult in small biopsy; therefore a reticulin stain is a recommended tool
when NRH is suspected; this stain helps to outline the typically subtle nodularity of liver
parenchyma, with compressed reticulin fibres forming the boundaries of each nodule.
Veno-occlusive disease (sinusoidal obstruction syndrome)
VOD is a recognised complication of bone marrow transplantation (Bearman SI. 1995)
and kidney transplantation in the context of azathioprine immunosuppression.
(Eisenhauer T, et al 1984, Katzka DA, et al 1986). The term VOD of the liver refers to a
form of liver injury characterised clinically by the development of hepatomegaly, ascites
and jaundice. VOD occurs between 1 and 20 weeks post-transplant may be in
association with ACR. The histological features are marked sinusoidal fibrosis, necrosis
of zone 3 hepatocytes, and narrowing and eventual fibrosis of small hepatic venules. It
has been suggested that the primary site of the toxic injury is sinusoidal endothelial
cells, resulting in series of biological processes that lead to circulatory compromise of
centrilobular hepatocytes, fibrosis and obstruction of liver blood flow, primarily at the
sinusoidal level, making ‘‘sinusoidal obstruction syndrome’’ a more appropriate term
(Wang X, et al 2000, DeLeve LD, 2002).
210 Introduction
1.11 Cell Therapy
Cell-based therapies are emerging as an alternative to whole-organ transplantation in
the setting of liver transplantation. The procedure is less expensive, less invasive and
incomparable with the major surgery of transplantation and serious post operative and
life-long immunosuppression therapy complications. Other advantages include retain of
the native liver and the overcome of procedural timing. HTx would not require obtaining
livers that could be used for OLT, which would only further stress an already stressed
system. HTx would be using liver tissue that would otherwise be discarded. In addition,
multiple patients could be treated with hepatocytes from a single tissue donor, and
potentially, in cases of metabolic disease, a patient’s autologous hepatocytes could be
collected, genetically manipulated to correct the deficiency, and infused back into the
patient. Cells for transplant can be banked and cryopreserved for almost instant
availability. Stable patients, such as those with a metabolic disease could be given an
infusion of cells as an outpatient procedure. Indications are numerous and include acute
liver failure as a rescue to provide functional liver support until native liver regeneration
occurs, the long term supplementation of liver function in patients with chronic liver
disease to serve as a “bridge therapy” until OLT, and the treatment of inborn errors of
metabolism as it may help in avoiding OLT by replacing the missing enzyme function in
such metabolic conditions (Strom & Ellis, 2011).
Approaches that might be applied to cell transplantation are substantial. The source and
type of liver cells to be used as well as the sites for implantation must be defined.
Techniques of cells implantation, survival, function and proliferation of the implanted
cells need to be optimized. The most promising cells types are hepatocytes, embryonic
stem cells (ESC), mesenchymal stromal cells (MSC), amnion epithelial (AE) cells, and
induced pluripotent stem cells (iPSC). Each cell type has its own associated risks and
benefits.
211 Introduction
Summary of clinical HTx to treat chronic liver failure, acute liver failure, and
inherited metabolic diseases
Liver Disease
Outcome
References
α1-antitrypsin (A1AT)
No clinical benefit likely due to thepresence of
fibrosis
Reversal of disease
Strom 1997a
Stromet 1999
Fisher 2000
Soriano 2002
Fisher & Strom 2006
Ott 2006
Stephenne 2006
Acute liver failure
Argininosuccinate lyase
deficiency
Complete correction
Biliary atresia
Partial correction - slow and continuous decrease in
bilirubin levels
Khan 2008
Chronic liver failure
Bridge to OLT
Citrullinemia
Partial correction – decreased citrulline and
circulating ammonia at 6 months post- cell infusion
Bilir 2000
Strom 1997b
Fisher & Strom 2006
Strom 1999
Meyburg 2009a
Crigler-Najjar type 1
Partial correction - slow and continuous decrease in
bilirubin levels; evidence of long term correction by
hepatocyte graft (one patient was followed for > 1.5
years)
Fox 1998
Dhawan 2004
Ambrosino 2005
Familial cholesterolemia
Partial correction – cholesterol decrease and
transgenic expression >4 months
Grossman 1991
Glycogen storage
disease type 1a & 1b
Partial correction – patients could maintain blood
glucose between meals as well as higher and
sustained glucose levels at meals
Muraca 2002
Lee 2007
Infantile refsum disease
Partial correction – improved fatty acid metabolism,
reduced pipecolic acid and bile salt levels, improved
strength and weight gain
Sokal 2003
Inherited Factor VII
deficiency
Partial correction – reduced FVII requirement 80%
Dhawan 2004
Ornithine
transcarbamylase
deficiency (OTC)
Partial correction – ammonia and glutamine levels
were normalized following transplant. Most required
OLT at a later date
Progressive familiar
intrahepatic cholestasis
No clinical benefit likely due to the presence of
fibrosis
Strom 1997a
Horslen 2003
Mitry 2004
Stephenne 2005
Puppi 2008
Meyburget 2009a,b
Hughes 2005
1.11.1 Hepatocytes
The major source of hepatocytes for HTx are livers that were unsuitable for OLT.
The adult human liver consists of approximately 250 billion hepatocytes constituting
about 65-80% of the cell population of the liver and representing the basic metabolic cell
of the liver. The liver is largely quiescent with only 1:1000 hepatocytes in mitosis at any
212 Introduction
given time. Hepatocytes are regarded as unipotent stem cells as they are capable of
rapid proliferation as well as complete and functional regeneration of the liver following
injury. Hepatocyte transplantation (HTx) can be regarded as a potential therapy for a
number of liver diseases. Approximately 3.5-7.5% of liver mass can safely be
transplanted in one transplant event (Fox et al., 1998), whereby the transplant may be
divided in up to 6 separate infusions over a number of hours. Studies with various
animal models have showed the efficacy of hepatocyte transplantation to support liver
function, improve survival in acute liver failure and correct metabolic liver disease such
as glucuronosyltransferase-deficiency, phenylketonuria (PKU) ( Mali H & Gupta, 2001;
Groth et al., 1977 Harding & Gibson 2010; Gupta & Chowdhury, 2002; Hamman et al.,
2005; Skvorak et al., 2009a; 2009b).
Clinically, therapeutic benefit has been seen in the management of disorders of the urea
cycle (citrullinemia, OTC, argininosuccinase lyase deficiency), familial cholesterolemia,
Crigler-Najjar, biliary atresia, infantile refsum disease, Factor VII, and Glycogen storage
disease type 1a & 1b. HTx is more often done as therapy for inborn errors of hepatic
metabolism in which a specific absent protein can be measured from transplanted
unmodified donor hepatocytes expressing the gene. Hepatocyte infusions to correct
inborn errors of metabolism is used when a specific metabolic deficiency, with wellstudied animal modelling, can be measured. Then, after infusion of donor liver cells
natively expressing the required gene, objective measures of required hepatocyte mass,
engraftment percent, and survival advantage can be obtained. To avoid the need for
immunosuppression or the risk of rejection, transplantation of genetically modified
autologous hepatocytes may also be an option, such as in a clinical study to treat
familial cholesterolemia (Grossman et al., 1991). In this study, retrovirus was used to
transduce and correct a patient’s deficient hepatocytes, which were then infused back
into the patient to yield a partial correction of the disease. HTx to treat progressive
familiar intrahepatic cholestasis and A1AT were also attempted, but without clinical
benefit due to native liver fibrosis (Strom et al., 1997a; Strom et al., 1999; Hughes et al.,
2005). Regarding long-term engraftment, transplanted hepatocytes must gain a
selection advantage over the recipient’s cells which is triggered by native liver injury or
damage promoting rapid proliferation of healthy hepatocytes which would provide
transplanted hepatocytes a selected growth advantage over native cells in patients with
acute or chronic liver failure. In the other hand, in most cases of metabolic liver
213 Introduction
diseases the native liver is not injured therefore, transplanted cells would likely not
receive selection advantage over the recipient’s cells and hence higher numbers of
transplanted hepatocytes as well as repeated transplantations are needed for better cell
engraftment, and successful treatment of metabolic liver diseases. Cells infusion into
the liver via the portal vein is the preferred method of transplant in cases where liver
architecture is intact (i.e., metabolic diseases, or in the case of acute liver failure).
Animal studies demonstrated that hepatocytes infused via the portal vein disperse with
the portal blood flow and finally translocate to the hepatic sinusoids in the periportal
region of the liver lobules (Sokal et al., 2003).
Single cells succeed in traversing the endothelial barrier and integrate into the
parenchyma. After re-establishing intercellular contacts with neighbouring host cells,
transplanted hepatocytes may start to proliferate when sufficient space is made for the
infused cells forming clusters repopulating the recipient liver. Treatment of cirrhotic
livers by CTx requires extra consideration regarding transplant site, cell number, and
overall safety of the procedure. Cell therapy of end-stage liver disease in cirrhotic livers
is problematic due to hepatic architecture derangement and, decreased functional
hepatocytes, portal hypertension in addition to the presence of intrahepatic portal
venous shunts preventing the exchange between hepatocytes and blood plasma, and
cell infusions via portal vein may cause prolonged portal hypertension and embolization
in the lung (Strom et al., 1999) due to portal shunting (Gupta et al., 1993). Therefore,
transplantation into the spleen is preferable (Strom et al., 1997b; Fisher & Strom, 2006).
Direct intrasplenic injection produced better engraftment than that obtained by splenic
artery infusion with less serious complications (Nagata et al.; 2003). Alcoholic cirrhotic
patients showed only transient clinical improvement after treatment by splenic HTx
(Strom et al., 1999; Sterling & Fisher, 2001).
Bioartificial liver (BAL) can be used to support metabolic function and regeneration
(Koenig et al., 2005; Carpentier et al., 2009). In general, possible complications of CTx
include the use of immunosuppressors, embolisation of the pulmonary vascular system,
sepsis, or hemodynamic instability, and increase in portal pressures as blood flow is
restricted by plugs of donor hepatocytes (Gupta et al., 1999). However, if transplanted
cells are in the range of 5% of the total liver mass, this increased portal pressure usually
resolves within minutes or hours. HTx would be using liver tissue that would otherwise
be discarded but there are still many problems associated with the use of hepatocytes.
214 Introduction
The current major limitation is the availability fresh human liver tissue and human
hepatocytes because primary hepatocytes proliferate very poorly in vitro they lose their
hepatic potential, and display very limited survival (Tanaka et al., 2006, Nahmias et al.,
2007). The numbers and/or quality of hepatocytes isolated from non-transplantable
livers will not allow a widespread application of HTx. Successful cryopreservation is
needed for establishment of cell banks, which would allow cryopreserved hepatocytes
to be available for emergency use in acute and chronic liver diseases, or for planned or
repeated use in patients with liver-based metabolic disorders. Another major limitation is
the consistently poor quality of cells after cryopreservation. Hepatocytes are very
sensitive to freezing damage, and three distinct modes of cell death have been
identified: cell rupture by the formation of ice crystals, necrosis, and apoptosis (Baust,
2002). Loss of membrane integrity, and thus leakage of important enzymes and
cofactors which affect liver function, low attachment efficiency, and a loss in viability of
50% or greater is typical.
Stem cell derived hepatocyte-like cells should demonstrate characteristic hepatic gene
expression and function, express appropriate transport proteins and transcription
factors, metabolize ammonia and billirubin, produce albumin and/or bile acids, and no
longer express genes characteristic of ESC or other cell types. Therapeutically useful
hepatocyte-like cells must be safe (i.e. nontumorigenic), contribute to liver function in
vivo, and importantly, must express hepatic genes at a level comparable to mature
hepatocytes. Currently there are no definitive reports of any stem cell-derived
hepatocytes with these ideal characteristics.
1.11.2 Embryonic stem cells
(Clinical ESC therapy for liver disease is not currently realistic. ESCs also carry
religious, political, and ethical concerns, and there is legislation restricting or banning
their use in certain countries.)
Embryonic stem cells (ESC) are derived from totipotent cells of the inner cell mass of
the blastocyst, (Thomson et al., 1998). They are pluripotent and express many specific
gene factors. Common markers include stage specific embryonic antigens (SSEA) 3 &
4, and the tumor rejection antigens (TRA) 1-60 & 1-81 (Thomson et al., 1998), OCT-4,
SOX-2, and Nanog, as well as high expression of telomerase reverse transcriptase
(TERT) (Thomson et al., 1998; Chambers et al., 2003), which is important in a cell’s
replicative lifespan (Vaziri & Benchimol, 1998). High levels of telomerase activity are
also found in 80-90% of human tumor samples (Chen & Chen, 2011). ESCs will readily
215 Introduction
become tumorigenic in vivo when injected into severe combined immunodeficient
(SCID) mice forming either teratomas, or teratocarcinomas (Ben-David & Benvenisty,
2011). In vitro, ESCs display aneuplody, a characteristic of cancer cells (Spits et al.,
2008). Very low human leukocyte antigen (HLA) class I antigens are expressed by
ESCs sufficient to induce acute rejection through the action of cytotoxic T-cells and
affect treatment tolerance (Robertson et al., 2007; Drukker et al., 2006) suggesting the
need of immunosuppression if patients recieved stem cell-derived CTx. HLA class II
antigens and co-stimulatory factors (Drukker et al., 2006) are almost undetected.
Sustaining pluripotency in vitro requires continued expression of Nanog and OCT-4
(Chambers et al., 2003). The expression of these factors are maintained through coculture with a feeder cell layer, most commonly mouse embryonic fibroblasts (MEFs),
and the addition of basic fibroblast growth factor (bFGF) for human ESC. ESCs will
spontaneously differentiate simply by removing factors and/or allowing the formation of
spheroid clumps known as embryoid bodies (EB) in culture. In a developing embryo,
signals from the cardiac mesoderm and septum transversum mesenchyme specify
endoderm to accept a hepatic fate. It was eventually determined that FGFs and bone
morphogenic proteins (BMPs) can mimic the appropriate signals and thus induce
endoderm towards a hepatic fate (Jung et al., 1999). Targets of BMPs and FGFs, FoxA
genes and the GATA and hepatocyte nuclear factor (HNF) transcription factors, defined
additional molecules tested in differentiation studies to produce hepatocyte-like cells
from ESC. There are many published protocols to differentiate ESC into various cell
types from all three germ layers (Trounson, 2006; Zaret & Grompe, 2008; SotoGutierrez et al., 2008; Sancho-Bru et al., 2009). Hepatocyte-like cells that express
alpha-fetoprotein (AFP), albumin, cytochrome P450 (CYP450), cytokeratin (CK) 18, and
display epithelial-like morphology have all been extensively described. However,
expression of these few factors does not guarantee the differentiated stem cell is a
“hepatocyte”; hepatocyte-like stem cells may express a few hepatic genes, but they
could also be negative for many others important to hepatic function (Soto-Gutierrez et
al., 2008). In addition, some of these hepatic markers are not limited to hepatocyte
expression, such as CYP450. There have been many articles describing ESC derived
hepatocyte-like cells transplanted into liver-damaged mice (Banas et al. 2007) but few
have determined the cells significantly contribute to improved liver function and
regeneration. Induction rates remain low regardless of the method used, and general
hepatic function of the cells, even once transplanted, were very limited when compared
216 Introduction
to mature hepatocytes (Sharma et al., 2008). However, a successful report described
ESCs demonstrating liver function able to overcome liver damage in mice (Heo et al.,
2006). Clinical ESC therapy for liver disease is not currently realistic, but at present
there are four ongoing ESC clinical trials targeting other organs (Trounson et al., 2011).
Two trials are in Phase I and are targeting spinal cord injuries or spinal muscular
atrophy, while the remaining two are in Phase I/II and are targeting Macular
Degeneration. All trials involve ESCs that were first differentiated in culture prior to
transplantation.
1.11.3 Mesenchymal stromal cells
Mesenchymal stromal cells (MSC) originate from mesoderm. They are multipotent nonhematopoietic adult stem cells that have been isolated from a variety of tissues: bone
marrow, adipose tissue, Wharton’s jelly, umbilical cord blood, and different
compartments of the placenta (Parolini et al., 2008). MSCs show in vitro differentiation
potential into adipogenic, chondrogenic, and osteogenic cell lineage that are highly
proliferative fibroblast-like cells displaying plastic adherence in culture and express
specific surface markers (i.e. positive for CD105/CD90/CD73, and negative for
CD34/CD45/CD11b, or CD14/CD19, or CD79 alpha/HLA-DR1) (Dominici et al. 2006).
Importantly, MSCs are TERT-negative (Zimmerman et al. 2003). MSCs tumorigenicity is
lower than that of ESC, and assist tumor growth by transformation and suppression of
the antitumor immune response (Ren et al. 2009). MSCs display reduced
immunogenicity, but demonstrate a powerful immunomodulatory response in vivo
(Hematti, 2008; Bifari et al., 2010). MSCs interfere with antigen-presenting cells and
suppress B-cell differentiation causing inhibition of Natural Killer (NK) cells and cytotoxic
T cells. They express various anti-inflammatory cytokines and chemokines (Banas et
al.; 2008) and inhibit local and systemic proinflammatory responses through inhibition of
TNF-alpha and interleukin (IL)-1 and hence prevent tissue damage (Lin et al., 2011). In
addition, MSCs express low levels of HLA class I antigens and lymphocyte functionassociating antigen (LFA)-3, and do not express HLA class II antigens or co-stimulatory
molecules which could function to upregulate HLA class II antigens in vivo (Bifari et al.,
2010). MSCs significantly lower the incidence of graft-versus-host disease, autoimmune
diseases, and can induce tolerance upon transplantation (Le Blanc et al, 2004; 2005;
2007). MSC therapy may also increase the vulnerability to viral infections such as
herpes (Sundin et al., 2006). MSCs are able to differentiate into various cell types by
217 Introduction
stimulation with specific growth factors. A recent review describes a number of protocols
for differentiating along a hepatic lineage (Puglisi et al., 2011). MSCs have been
differentiated into hepatocyte-like cells (Schwartz et al., 2002) performing hepatic
functions such as albumin production, urea synthesis, glycogen storage, and lowdensity lipoprotein uptake (Jiang et al., 2002). MSCs stimulated with hepatocyte growth
factor (HGF), epidermal growth factor (EGF), and FGF are able to differentiate into
hepatocyte-like cells (Lange et al., 2005) able to engraft in the liver upon transplantation
improving hepatic function and regeneration (Kuo et al., 2008). Bone marrow MSCs
significantly increased the number of hepatic stellate cells and myofibroblasts,
contributing to the fibrotic cascade (Russo et al., 2006). Whether MSCs are able to
become hepatic cells through differentiation or by cell fusion, this is not yet clear,
homing mechanism by which intravenously injected MSCs can preferentially recruit to
the injured liver is not well understood (Sakaida et al., 2004). Inflammation might
provide regulatory factors in the targeted migration of MSCs towards injured sites (Kuo
et al., 2008). MSCs have been used to treat acute graft-versus-host disease and
osteogenesis imperfecta in children (Le Blanc et al., 2005). MSCs also secrete several
factors suppressing hepatocyte apoptosis, inflammatory responses, and liver fibrosis, in
the other hand stimulating hepatocyte proliferation and function e.g. HGF which helps in
liver regeneration (Lin et al., 2011; Zhou et al., 2009). Preclinical and clinical studies
have suggested that MSC transplantation can moderately restore liver function and
enhance survival rates in fulminant hepatic failure and end-stage liver disease (Yagi et
al., 2009; Kuo et al., 2008; Banas et al., 2009). There is little evidence to date that
verifies whether MSCs are able to form mature hepatocytes, either in culture or once
transplanted. However, growing evidence does suggest that MSCs may improve
cirrhotic liver function once infused into patients. For example, bone marrow MSCs
transplantation reduced liver fibrosis, and improved liver function and survival in mice
(Sakaida et al., 2004) and rats (Abdel Aziz et al., 2007). This provided rationale for the
use of autologous bone marrow MCSs for cell therapy to treat cirrhosis, which spurred
several clinical trials investigating cell safety and feasibility (Kharaziha et al., 2009;
Mohamadnejad et al., 2007; Salama et al., 2010). At present, there are 123 ongoing
clinical trials involving MSCs investigating a variety of applications including bone,
cartilage, and heart repair, immune rejection and autoimmune diseases, as well as
treatment for cancer, gastrointestinal, and neurodegenerative diseases (Trounson et al.,
2011). ESCs both differentiated and undifferentiated, possess the ability of unlimited
218 Introduction
self-renewal, which is also critical because self-renewal, genetic instability, and
tumorigenicity are all characteristics shared by ESCs and cancer cells (Stutchfield et al.,
2010). In addition, ESCs also carry religious, political, and ethical concerns, and there is
legislation restricting or banning their use in certain countries. MSCs have fewer ethical
concerns; they exhibit a lower risk of spontaneous tumors. However, MSCs have been
shown to contribute to tumor growth in vivo and increased risk of viral infections.
Therefore, high-risk patients may not be feasible candidates for MSC transplantation.
Stem cells are abundantly proliferative and it is currently not possible to provide the
required number of cells for transplantation to treat liver disorders. Furthermore,
differentiated cells display minimal hepatocyte function both in vitro and in vivo,
engraftment as well is very low to contribute for tissue regeneration making
questionable the usefulness of stem cell-derived hepatocyte-like cells in managing liver
disease.
1.11.4 Amnion epithelial (AE) cell transplantation (Many characteristics identify
AE cells as similar to ESC, but not identical)
AE cells meet many important criteria for clinically relevant cells AE cells. They maintain
a stable karyotype and are nontumorigenic in both an undifferentiated and differentiated
state, they are nonimmunogenic, and express anti-inflammatory factors and have been
used
in
clinical
studies
for
more
than
sixty
years
(since
1947)
without
immunosuppression and without evidence of acute rejection. Undifferentiated AE cells
are proposed to become hepatocyte-like once engrafted in the liver parenchyma and
have contributed to liver function in animal models of disease. AE cells are clearly the
safest alternative to hepatocytes from the stem cells, but their effectiveness to correct
liver disease is currently unknown. Isolation is relatively easy, an average of 100 million
cells can be isolated from a single term placenta, and AE is able to proliferate robustly
in culture; about 100 million AE cells could be expanded to 10-60 billion cells within six
passages. (Miki, et al. 2005). It is unknown whether differentiation prior to
transplantation will be necessary; one should assume that differentiation into the
required cell type would be the most clinically efficient and effective method of
treatment. Unlike hepatocytes, AE cell viability and morphology are also very stable
when cryopreserved long term at -80°C. Current umbilical cord blood stem cell
guidelines could be used as a template to set up similar procurement and banking
procedures for placental-derived stem cells (Serrano-Delgado et al., 2009).
219 Introduction
Amnion
epithelial
cells
have
stem
cell-like
pluripotent
characteristics,
low
immunogenicity, and anti-inflammatory properties. AE cells in culture express stem cell
surface markers (e.g. SSEA-3 & 4, TRA 1-60 & 1-81) as well as molecular markers of
stem cells (e.g. OCT-4, Nanog, SOX-2, FGF-4, and Rex-1), and unlike ESC, do not
require feeder cell layers to maintain OCT-4 and Nanog expression (Miki et al., 2005;
Miki & Strom, 2006).
Interestingly, AE cells do not express the stem cell marker TERT (Miki et al., 2005). AE
cells consistently display a normal karyotype and are nontumorigenic when transplanted
into SCID mice (Miki & Strom, 2006; Marongiu et al., 2011). In addition, AE cells are
derived from neonatal tissue without environmental and age-acquired DNA damage
(Miki, 2011). Amnion does not express HLA class II antigens and only expresses class I
antigens at low levels. AE cells were also found to secrete anti-inflammatory and
immunosuppressive factors, which inhibited inflammation and reduced the proliferation
of T- and B-cells in vitro (Li et al., 2005). Volunteers transplanted with AE cells did not
experience any immunological reaction, and to date no tumors have ever formed as a
result (Akle et al., 1981; Yeager et al, 1985; Scaggiante et al, 1987; Sakurgawa et al.,
1992). Important for the treatment of liver diseases by CTx, AE cells demonstrate
hepatic gene expression and functions at a level of mature hepatocytes following
implantation into the livers of SCID mice, which suggest they differentiate into
hepatocyte-like cells once engrafted in the liver parenchyma (Miki & Strom, 2006;
Marongiu et al., 2011). Undifferentiated AE cells were able to functionally engraft into
the livers of immunocompromised mouse models of liver damage resulting in a
reduction of hepatic fibrosis, inflammation, and hepatocyte apoptosis (Manuelpillai et al.,
2010; Marongiu et al., 2011).
AE cells have also been used in clinics to correct lysosomal storage diseases with no
adverse effects (Yeager et al, 1985; Scaggiante et al, 1987; Sakurgawa et al., 1992).
AE cells could partially rescue a mouse model of intermediate MSUD (Skvorak et al.,
2010), AE cell transplantation partially corrected iMSUD mice similarly to the partial
correction previously obtained with hepatocyte transplantation (Skvorak et al, 2009a;
2009b) and >70% of animals survived to day of life 100 (Skvorak et al., 2010).
Immunosuppression was not used and there was no evidence of rejection. Recently,
studies have shown lung protection following human AE cell transplantation in a SCID
220 Introduction
mouse model of bleomycin-induced lung injury (Moodley et al., 2010; Murphy et al.,
2010). Studies have also shown the efficacy of AE cells on corneal resurfacing in
horses (Plummer, 2009), rabbits (Wan et al., 2011), and human patients (Nubile et al.,
2011), in which amniotic membranes were transplanted as a graft over the injury site.
These studies were done without immunosuppression and without evidence of acute
rejection. Differentiated AE have also been used to treat a rat model of Parkinson’s
disease (Kakishita et al, 2000; 2003).
1.11.5 Induced Pluripotent Cells ( iPSC)
In the blastocyst stage, a cell becomes specified by controlling the expression of certain
genes through specific signals and not by changing its DNA sequence. The first attempt
to manipulate a cell’s developmental potential was known as somatic cell nuclear
transfer, which led to the birth of live lambs (Wilmut et al., 1997). Pluripotent ESC-like
cells from murine somatic cells were derived through forced expression of the
reprogramming factors OCT-3/4, SOX-2, c-MYC, and KLF-4 by lentiviral induction
(Takahashi & Yamanaka, 2006). Similar to ESC, these “induced pluripotent stem cells”
(iPSC) were similar to ESCs in morphology, growth properties, expression of ESC
marker genes. They display an unstable karyotype, tumorigenicity and teratoma
formation in SCID mice. Human iPSC from adult skin fibroblasts were generated using
the same four factors as mice (Takahashi et al., 2007), and also by forced expression of
a new set of four factors: OCT-4, SOX-2, Nanog, and Lin-28 (Yu et al., 2007). Recently,
iPSCs have been derived from a variety of human tissues, such as umbilical cord matrix
(Cai et al., 2010), fetal and juvenile tissues (Park et al., 2008a; Li et al., 2010; Aasen et
al., 2008), placental tissue (Nagata et al., 2009; Cai et al., 2010; Zhao et al., 2010), and
primary human hepatocytes. Pluripotent iPSC should express the stem cell markers
SSEA3, SSEA-4, TRA-1-60, TRA-1-81, OCT-4, alkaline phosphatase, TERT, SOX-2
and Nanog, and form teratomas in vivo. It has also been reported that a specific
expression profile of stem cell markers corresponds to either a completely or partially
reprogrammed cell (Chan et al., 2009).
It is now evident that iPSC retain epigenetic memory of their cells of origin for both
mouse (Kim et al., 2010; Polo et al., 2010) and human (Hu et al., 2010). Cell source to
generate iPSC for the treatment of liver disease would be hepatocytes; hepatic-like cells
differentiated from hepatocyte-derived iPSC would most closely resemble their primary
cell counterparts. Generation of iPSC from a variety of inherited diseases has been
221 Introduction
described, such as Huntington’s, Duchene’s and Beckers’s muscular dystrophy,
diabetes mellitus type 1, Down’s syndrome, and Parkinson’s (Park et al. 2008b). iPSC
have also been generated from inherited metabolic disease patients with A1AT, CriglerNajjar, tyrosinemia type 1, familial hypercholesterolemia, and glycogen storage disease
type 1a (Rashid et al., 2010), which were then differentiated into hepatocytes to model
the disease in vitro. More recently, iPSC-derived hepatocyte-like cells generated from
the dermal fibroblasts of a Wilson’s disease patient was shown to mimic the disease
phenotype in vitro (Zhang et al., 2011). Importantly, iPSCs of metabolic disease could
be genetically corrected in culture, differentiated into hepatocytes possessing the ability
to make normal protein, and potentially infused back into patients to cure their disease.
The use of autologous cells would also reduce the risk of immune issues and rejection
theoretically avoiding the need for immunosuppression. In addition, iPSC-derived
hepatocyte-like cells modeling Wilson’s disease were corrected in vitro using either
lentiviral gene therapy or treatment of the drug curcumin (Zhang et al., 2011). There are
several reports of differentiation of iPSC along a hepatic lineage using ESC protocols
(Si-Tayeb et al., 2010; Song et al., 2009; Liu et al., 2011). However, in most reported
cases iPSC-derived hepatocytes displayed very low hepatic function and gene
expression in vitro when compared to primary hepatocytes. Unlike ESC, patient specific
iPSC could be generated, corrected, and infused back into a patient without the need
for immunosuppression. iPSC could theoretically provide an unlimited pluripotent source
of cells that could be banked and differentiated into hepatocytes for transplant when
needed. It has ben estimated that an iPSC bank with only 30 stem cell lines could
match the HLA haplotypes in >80% of the Japanese population (Nakatsuji et al. 2008).
Importantly, there are no religious, ethical, or political controversies associated with the
use of iPSCs. The major concern with iPSC use is the high risk of their tumorigenicity
more than ESC as iPSC exhibit genetic instability, express TERT, and can produce
teratomas in vivo (Ben-David & Benvenisty, 2011). Furthermore, the most reliable,
reproducible, and efficient method to currently generate iPSC is through an integrating
viral vector, which could induce cancers. Additionally, cell differentiation protocols and
methods to enhance engraftment have not yet been optimized, making uncertain their
long-term survival in vivo. Further research is needed to generate iPSC that are safe,
effective, and therapeutically useful before these cells can be used for clinical cell
therapies.
222 Introduction
1.12 BIOMARKERS OF LIVER FIBROSIS
Biomarkers of liver fibrosis
Liver transplantation is a, curative therapy for a wide range of chronic liver diseases.
The natural history of CLD is variable and long-term evolution differs from patient to
another. Accurate prognosis of CLD represents a main stay for prioritization of patient
for organ allocation for liver transplantation (appropriate selection of candidates and
timing of LT (Forman LM, et al. 2001).
Criteria are needed in order to select patients who can profit (benefit) more from LT,
reduce mortality, and improve outcome in LT recipients especially in the era of scarce
(limited) donor pool in face increasing demands. Among these criteria is the
identification of non-invasive biomarkers which are able to assess, quantify, monitor,
reflect the dynamic bidirectional process of liver fibrosis (fibrogenesis/fibrolysis); the
main characteristic of CLD which progresses at variable rates depending on the cause
of liver disease, environmental factors, and host factors (Bircher J, et al. 1999; Schiff
ER, et al 2003) in the other hand has the potential for regression, reversal, and
resolution. Recent data suggest that cirrhosis regression or even reversal is possible.
(Desmet VJ, et al. 2004; Wanless IR, et al. 2000) These markers must be sensitive to
the changes in fibrosis because of the treatment or due to the natural history of disease
progression, and precisely define the stage fibrosis, which is the main determinant of
prognosis and management as they both depend on the quantity and the progression of
LF. The most widely used scores to judge CLD prognosis and hence used in liver
allocation are The Pugh modification of Child-Turcotte classification (CTP score) and
The Model for End-Stage Liver Disease (MELD) score beside their potential
modifications (under investigations) e.g. Modified CTP, Meso index (both are for
cirrhotic patients not listed for transplantation), MELD-Na, iMELD, Delta MELD, MELDXI, MELD modified by gender, Updated MELD, and Arificial neural network(ANN) .
These scores have some limitations and none accurately assess (estimate) the amount
of architectural disorganization (disorder, distortion) as a result of progressive liver
tissue fibrogenesis and extensive intrahepatic vascular remodeling.
Serum Markers of Fibrosis
Serum markers of hepatic fibrosis are either indirect including simple routine blood tests
reflecting the alterations in hepatic function or direct markers, which reflect serum ECM
turnover. They are used either individually or in combination. They are suitable for the
223 Introduction
cross-sectional diagnosis of fibrosis stage but cannot determinate the rate of fibrosis
progression or regression and could not differentiate stages of fibrosis accurately
(Parkes J, et al. 2006) and have limited accuracy for the diagnosis of significant
fibrosis(Lackner C, et al 2005)). Scores combining direct and indirect markers using
mathematical formulae have been used (formulated). The FibroTest is the most widely
used (Imbert-Bismut F, et al. 2001). Several other scores have been proposed mainly in
patients with chronic hepatitis C, including the Forns index (Forns X, et al. 2002) the
Aspartate to Platelet Ratio Index (APRI) (Wai CT, et al 2003), the FibroSpect II (Patel K,
et al 2004), the MP3 (Leroy V, et al 2004), the Glycocirrhotest (Callewaert N, et al.
2004) the European Liver Fibrosis score (ELF) (Rosenberg WM, et al. 2004) the
Fibrosis Probability Index (FPI) (Sud A, Hui JM, et al. 2004), the Lok index (Lok AS, et
al. 2005), the Gotebörg University Cirrhosis Index (GUCI) (Islam S, et al 2005), the
Hepascore (Adams LA, et al. 2005), the Fibrometer (Cales P, et al 2005), the ViraHep-C
model (Fontana RJ, et al 2006), the Fibroindex (Koda M,et al. 2007), the FIB-4 (ValletPichard A, et al. 2007) and lastly the Halt-C model (Fontana RJ, et al 2007). Other
scores have been proposed specifically in patients with hepatitis B (Hui AY, et al 2005;
Zeng MD, et al. 2005) or NAFLD (Angulo P, et al. 2007; Harrison SA, et al 2008). None
is liver-specific and they may be influenced by changes in their clearance and excretion.
Increased levels of hyaluronate occur in the post-prandial state (Fraser JR, et al.2005)
or in aged patients with chronic inflammatory processes such as rheumatoid arthritis.
Also, the reproducibility of measurement of some parameters included in ‘indirect’
serum markers, such as AST levels or platelet count, is questionable (Piton A, et al.
1998).
Indirect Biomarkers
No true serum marker that would act as a surrogate marker of hepatic fibrosis has been
validated to date. Indirect markers of liver fibrosis reflect abnormal hepatic structure or
function but not ECM dynamics. They have been used either individually or in
combination to apply a score to the patient. The use of the alanine aminotransferase
(ALT) or aspartate aminotransferase (AST) level alone to diagnose the stage of fibrosis
is not clinically useful (Assy N et al. 2000; Anderson FH, et al. 2000). AST/ALT ratio is
confounded by the use of alcohol (Oberti F, et al 1997). The ratio of >1 suggests the
diagnosis of cirrhosis. (Park GJ, et al. 2000; Schalm SW. 1997). AST to Platelet Ratio
Index (APRI test) worsening and progression of fibrosis results in portal hypertension,
224 Introduction
reduction in thrombopoetin production, increase platelets sequestration by the spleen
(Kawasaki T, et al 1999; Aster RH. 1966), and decrease AST clearance (Kamimoto Y,
et al. 1985) Therefore, potentially useful in detection of significant fibrosis or cirrhosis.
Studies were concerned mainly in patients infected with hepatitis C virus (HCV) or
HCV/human immunodeficiency virus (HIV) coinfection (Wai CT, et al. 2003; Castera L,
et al. 2005; Schiavon LL, et al. 2007) and alcoholic liver disease (Lieber CS, et al. 2006)
and useful for excluding significant fibrosis in HCV (Shaheen AA, 2007). PGA index has
been validated in a variety o CLD especially in ALD its accuracy for detecting cirrhosis
ranges from 66% to 72% (Oberti F, et al 1997; Poynard T 1991). Fibrotest a group of
biochemical markers where the results are formulated to determine 3 different
categories of fibrosis: mild (METAVIR F0–F1), significant fibrosis (METAVIR F2–F4),
and indeterminate. The sensitivity and specificity for the detection of significant fibrosis,
by their definition, were 75% and 85% respectively. The inter-laboratory reproducibility
of such score enables its clinical use practice (Imbert-Bismut F, et al. 2004; Cales P, et
al 2008). The interpretation need a carefull analysis to avoid false positive or falsenegative results e.g. The existence of haemolysis or Gilbert syndrome can lead to falsepositive results (by a decrease haptoglobin or an increase in bilirubin, respectively)
(Poynard T, et al. 2004). ActiTest is a modification of the FibroTest and reflects both
hepatic fibrosis and necroinflammatory activity, thus valuable for the detection of
advanced fibrosis associated with severe histologic inflammation. (Halfon P, et al. 2002)
The Forns index based on 4 routine clinical variables: age, platelet count, cholesterol
levels, and GGT (Forns X, et al. 2002). It is useful at excluding patients with minimal or
no fibrosis but not for the identification of advanced fibrosis. Criticisms of the Forns
index are about dyslipidemia, the effects of medications and the variability of platelet
measurements. The FibroIndex (platelets count and GGT) accuracy is currently being
determined (Koda M, et al 2007). The Hepascore combines clinical and laboratory
variables: age, gender, bilirubin, GGT, hyaluronic acid (HA), and alpha2-macroglobulin
to create a score. The FIB-4 combines biochemical variables (platelet count, AST, and
ALT) with age. It had reasonably good accuracy for predicting advanced fibrosis in
patients with chronic HCV (Vallet-Pichard A, et al. 2007; Sterling RK, et al 2006). The
NAFLD fibrosis score was described and examined in patients with NAFLD (Angulo P,
et al. 2007)
225 Introduction
Direct biomarkers
None of the currently available direct biomarkers completely fulfills the criteria for an
ideal biomarker as none is liver specific and most are affected by metabolism,
clearance, or excretion. HA is a glycosaminoglycan synthesized by HSC (hep. Stellate
cells) and cleared by both liver and kidney. It is a component of the ECM (McGary CT,
et al 1989) used individually or incorporated in the SHASTA index, Hepascore, and
FibroSpect scores. High levels of HA denote increased fibrogenesis (Guechot J, et al.
1996). In chronic HCV patients HA levels correlate with the degree of hepatic fibrosis
(Guechot J, et al. 1996; McHutchison JG, et al. 2000; Murawaki Y, et al 2001) while in
alcoholic liver disease, correlate with both the degree of fibrosis and the severity of
inflammation (Pares A, et al 1996). Increased HA levels occur in the postprandial state
(Idobe Y, et al. 1998) or in advanced age associated with chronic inflammatory
processes such as rheumatoid arthritis. PIIINP is the most widely studied marker of
hepatic
fibrosis.
High
levels
are
found
in
acute
hepatitis
correlating
with
aminotransferase levels. Serum levels of PIIINP reflect the degree of fibrosis in
alcoholic liver disease, viral hepatitis, and primary biliary cirrhosis (Teare JP, et al.
1993; Montalto G, et al. 1996; Trinchet JC, et al. 1991) but in chronic HCV, do not
correlate with the degree of fibrosis but with the scores for necrosis (Giannini E, et al.
2001; Gabrielli GB, et al 1997). It is a useful test for the evaluation of liver injury in
patient treated with hepatotoxic drugs such as methotrexate (Maurice PD, et al. 2005;
Chalmers RJ, et al 2005). Type I and Type IV Collagens Serum messenger RNA and
protein levels of type I collagen are increased in liver fibrosis and correlate with fibrosis
score but not necroinflammatory score(Giannini E, et al 2001).Type IV collagen has
been immunolocalized to the periportal interstitium and large fibrotic bands in alcoholic
liver disease(Hahn E, et al 1980). Serum type IV collagen is increased in
hemochromatosis patients with advanced fibrosis compared with normal controls
(George DK, et al. 1999). In patients with alcoholic liver disease, there is a significant
correlation between type IV collagen levels and fibrotic stage, particularly periportal
fibrosis (Ueno T, et al. 1992). Laminin Serum levels of laminin and the laminin P1
fragment are elevated in patients with chronic liver disease due to alcohol and viral
hepatitis (Walsh KM, et al 2000). Laminin is superior to PIIINP but inferior to type IV
collagen in predicting fibrosis in chronic viral hepatitis. (Misaki M, et al 1990). Matrix
Metalloproteinases (MMPs) and Tissue Inhibitors of Metalloproteinases (TIMPs) are
group of proteins acting locally, having multiple activities including activation of growth
226 Introduction
factors, affecting cell proliferation, and inhibition of apoptosis. They are involved in the
control of matrix degradation by their complex interaction (Benyon RC, et al. 2001;
Arthur MJ. 2000). MMPs are expressed in hepatic injury suggesting that degradation of
normal ECM may contribute to hepatic fibrosis(Fabris P, et al. 1999). Studies examining
the correlation of MMP-2 in chronic HCV have yielded conflicting results (Kasahara A, et
al. 1997; Walsh KM, et al. 1999; Boeker KH, et al 2002).
Little is known about the role of MMP-3 (stromelysin) in liver injury. MMP-9 (gelatinaseB) plasma levels have been shown to be increased in patients with hepatocellular
carcinoma but not those with chronic hepatitis or cirrhosis (Hayasaka A, et al. 1996).
MMP-13 is decreased in activated stellate cell cultures, but TIMP-1 and TIMP-2 are
increased. This corresponds to findings in cirrhotic liver explant tissue showing an
increase in TIMP-1 and TIMP-2 in patients with sclerosing cholangitis, primary biliary
cirrhosis, autoimmune hepatitis, and biliary atresia (Benyon RC, et al. 1996; Iredale JP,
et al 1995). The study by Boeker et al found that TIMP-1 levels had a sensitivity of
100% for the prediction of cirrhosis, with a specificity of between 56% and 75%(Boeker
KH, et al. 2002). A recent study showed reasonable sensitivity and specificity in
predicting fibrosis in HIV/HCV coinfected patients (Larrousse M, et al. 2007). YKL-40 or
Chondrex is a novel marker of hepatic fibrosis. Immunohistochemical staining has
demonstrated positivity for YKL-40 in areas of hepatic fibrosis and fibrogenesis
(Johansen JS, et al. 2000) more accurate than HA in measuring hepatic fibrosis
because of schistosomiasis (Zheng M, et al. 2005). In alcoholic liver disease it could
function as a marker of clinical outcomes (Nojgaard C, et al. 2003). A number of
cytokines mediating hepatic fibrogenesis have been studied as potential markers of
fibrosis. In patients with chronic HCV, there was a correlation between TGF-B and
severity of fibrosis (Nelson DR, et al. 1997) and the rate of fibrosis progression (Kanzler
S, et al 2001). Tumor necrosis factor (TNF)-alpha has been associated with liver injury
in patients with alcoholic liver disease(McClain CJ, et al. 1989). Platelet derived growth
factor is up regulated following liver injury (Ikura Y, 1997), and levels may correlate with
the degree of liver injury (Shiraishi T, et al 1994).
Combinations of Indirect and Direct Biomarkers
SHASTA Index
The SHASTA index consists of measurements of serum HA, AST, and albumin and was
developed in a cohort of 95 patients with HCV/HIV infection. It was capable of
227 Introduction
classifying mild fibrosis and advanced fibrosis and had similar accuracy to FibroTest
and performed significantly better than APRI (Kelleher TB, et al. 2005)
FibroSpect involves 3 parameters: HA, TIMP-1, and alpha2-macroglobulin. In clinical
practice, the assay is used for prediction and the presence of mild and advanced
fibrosis but performs less well for intermediate stages (Patel K, et al 2004) in excluding
advanced fibrosis( Zaman A, et al. 2007; Christensen C, et al. 2006).
The European Liver Fibrosis (ELF) group reported an assay in a multicenter cohort of
1021 patients with chronic HCV, NAFLD, and alcoholic liver disease. An algorithm using
age, HA, PIIINP, and TIMP-1 was developed. The algorithm accurately predicted the
presence of fibrosis with a sensitivity of 90% and the absence of fibrosis with a negative
predictive value of 92% (Rosenberg WM, et al 2004).
Proteomics and Glycomics GlycoCirrhoTest is a novel DNA sequencer-based serum
glycomics test which could be both cost-effective and rapidly determine a signature
profile for fibrosis of n-glycans (Callewaert N, et al. 2004). Combining GlycoCirrhoTest
with the FibroTest gave a sensitivity of 79% and specificity of 86% in distinguishing
cirrhosis from noncirrhotic disease.
Liver biopsy
Histological staging of fibrosis assesses the amount of fibrosis and architectural
disorganization. It is based on semi-quantitative scoring systems including the
histological activity index (Knodell RG, et al 1981) the Ishak’s score (Ishak K, et al
1995), and the METAVIR scoring system (Bedossa P, et al. 1996) or viral hepatitis as
well as Brunt (Brunt EM, et al. 1999) and Kleiner scores (Kleiner DE, et al. 2005) for
Non Alcoholic Fatty Liver Disease (NAFLD). The two main endpoints are the presence
of significant fibrosis which is an indication for antiviral treatment in chronic hepatitis B
and C and the presence of cirrhosis which is an indication for specific monitoring of
complications in relation to portal hypertension and to the increased risk of developing
hepatocellular carcinoma (European Association For The Study Of The Liver. 2009;
Ghany MG, et al. 2009).
Simultaneous evaluation of necro-inflammation (portal tract inflammation, interface
hepatitis, lobular inflammation) identify whether fibrosis is the result of an old event
stabilized, regressing, or progressing towards advanced stages. Liver biopsy also
detects associated lesions such as steatosis, steato-hepatitis, and iron overload, which
help in patient management and prognosis (Bedossa P, et al. 2007) Liver biopsy is an
228 Introduction
invasive procedure with higher risk specially with increasing passes and performance of
a biopsy in patients with sepsis or the need for correction of coagulopathy (Perrault J, et
al. 1978; Terjung B, et al. 2003). Hospital admission or prolonged hospital stay is
required in 1% to 5% of patients, and mortality has been is between 1 in 1000 patients
and 1 in 10,000 patients (Froehlich F, et al. 1993; Thampanitchawong P, et al 1999).
The procedure is associated with transient pain, anxiety and discomfort in around 30%
of cases (Castera L, et al. 1999; Castera L, et al. 2001) and rare but potentially lifethreatening complications (haemorrhage in 0.3% of cases and mortality in 0.01%)
(Piccinino F, et al 1986). The accuracy of liver biopsy to assess fibrosis is subject to
sampling errors and intra- and inter-observer variability that may lead to over- or understaging. Most studies have shown excellent inter- and intraobserver reproducibility for
the staging of fibrosis but inconsistent reproducibility of hepatic inflammatory
activity(Oberti F, et al. 1997; O’Brien MJ, et al 2000) The size of the biopsy specimen
varies in length and diameter and represents 1/50,000 of the total mass of the liver
therefore prone to substantial sampling error, only 65% of 15-mm biopsies and 75% of
25-mm biopsies were correctly staged (Bedossa P, et al 2003). Also a difference of at
least one fibrosis stage between the right and left lobes has been reported in around
30% of cases (Regev A, et al 2002). Cirrhosis may be missed on a single blind liver
biopsy in 10–30% of cases (Maharaj B, et al. 1986). Apart from the size of the liver
biopsy, the type of needle used to perform the biopsy can also affect the diagnostic
accuracy (Colombo M, et al. 1988; Vargas-Tank L, et al. 1985). In clinical practice, liver
biopsy should always be performed after weighting risks of the procedure with potential
benefits in terms of patient management.
Transient Elastography and its LIMITATIONS
TE given its likely prognostic value in cirrhosis, offer a mean for rapid discrimination of
different steps of progression within the stage of compensated cirrhosis. This will greatly
help allocating cirrhotic patients in different categories of risk and guide the need for
further evaluation. Liver stiffness measurements can be difficult in obese patients or
with narrow intercostal space and impossible in patients with ascites. (Sandrin L, et al
2003) TE reproducibility has been shown to be excellent for inter observer and intraobserver agreement (Fraquelli M, et al 2007; Boursier J, et al 2008) with intra class
correlation coefficient (ICC) of 0,98 (Boursier J, et al 2008; 4,5). However, interobserver agreement was significantly reduced in patients with lower degrees of hepatic
229 Introduction
fibrosis, with hepatic steatosis, and with increased body mass index (Fraquelli M et al.
2007) as well as for liver stiffness values <9 kPa (Boursier J, et al 2008). Liver stiffness
measurements were not interpretable in nearly one in five cases (failure to obtain any
measurement
in
4%
and
unreliable
results
not
meeting
manufacturer’s
recommendations in 17%) (Castera L, et al. 2010). Because the liver is a wrapped in a
distensible but non-elastic envelope (Glisson’s capsula), any space-occupying lesion,
as in cases of oedema and inflammation, cholestasis and congestion, may interfere with
liver stiffness measurement (LSM), independently of fibrosis. The extent of necroinflammatory influence TE measurements in patients with viral hepatitis with a steady
increase of liver stiffness values in parallel with the degree of histological activity (Arena
U, et al 2008; Fraquelli M, et al 2007; Chan HL, et al. 2009).The risk of overestimating
liver stiffness values has been reported in case of ALT flares in patients with acute viral
hepatitis or chronic hepatitis B(Coco B, et al 2007; Arena U, et al. 2008) as well as in
cases of extrahepatic cholestasis (Millonig G, et al. 2008) or congestive heart failure
(Millonig G, et al. 2010). Also TE measurements need to be standardized, since in
patients with cirrhosis its values increased by over 25% simply after a light meal, as
compared with fasting patients (Berzigotti A, et al. 2010)
230 Introduction
1.13 γ-Glutamyltransferase
1.13.1 Generalities and tissue distribution.
γ-Glutamyltransferase (GGT, EC 2.3.2.2) is a dimeric glycoprotein consisting of a heavy
(HSU 55-62 KDa) and a light subunit (LSU 20-30 KDa) linked by non covalent bonds. In
the N-terminal portion of the heavy subunit there is a hydrophobic domain which allows
the enzyme to be anchored to cell plasma membrane; in particular, both subunits are
exposed in the extracellular environment (Finidori et al., 1984). GGT catalytic site is
localized in the light subunit, thus the enzyme acts on extracellular substrates (Tate and
Meister, 1977; Ikeda et al., 1995).
GGT has a central role in glutathione (γ-glutamyl-cysteinyl-glycine, GSH) metabolism
and in the γ-glutamyl cycle, which includes synthesis and degradation of GSH (Meister,
1995). In this tripeptide glutamic acid and cysteine are linked by a particular peptide
bond in which the carboxylic group on γ-carbon of glutamic acid binds the amino group
on α-carbon of cysteine (Figure 1). The γ-glutamyl bond makes GSH resistant to
peptidase, but not to GGT which is able to hydrolyze it or to transfer the glutamic acid to
an acceptor (amino acid or dipeptide). GSH is the most abundant substrate for GGT,
but it is not the only one, actually all γ-glutamyl-compound are substrate for the enzyme,
e.g.:
GSH
conjugates
of
xenobiotics,
leukotrien
C4
(Whitfield,
2001),
S-
nitrosoglutathione (Hogg et al., 1997)
Figure 1. Structure of the tripeptide Glutathione (γ-glutamyl-cysteinyl-glycine).
The arrow indicates the γ-glutamyl bond.
GGT is widely distributed, being found in bacteria (Sakai et al., 1996), plants (Martin
and Slovin, 2000), as well as in all members of the animal kingdom. The distribution of
immunoreactive GGT in normal human tissues was studied by Hanigan and Frierson
(1996). They showed GGT is present in the plasma membrane of virtually all cells, but it
is principally localized in epithelial tissues with secretory or absorptive functions. The
231 Introduction
highest activity is present in the kidney, where GGT is localized to the luminal surface of
the proximal tubule cells, while it is virtually absent in distal tubules and glomeruli. In
liver, GGT is concentrated in biliary epithelial cells and bile canaliculi, and hence it is
secreted in the bile. In pancreas the major GGT activity is in the acinar cells. A strong
immunoreactivity is observed also in endothelial cells lining the capillaries in the brain
(e.g. choroid plexus), ciliary body and spinal cord. GGT positive are also cells of sweat
and submandibular glands, galactophorous duct, bronchial epithelium, epididymis,
seminal vesicle and prostate.
On the other hand a number of cell types show significant GGT activity but do not
conform to the generalization that is particularly associated with membrane transport.
GGT is expressed in almost all blood cell. In granulocytic and lymphocytic cell lineages
is a surface marker reflecting differentiation in normal and neoplastic cells (Novogrodsky
et al., 1976; Khalaf and Hayhoe, 1987; Grisk et al., 1993; Sener and Yardimci, 2005);
while in platelets GGT is present in the secretory granules (Bolodeoku et al., 1997).
Furthermore, GGT is normally found in serum as result of organ release, thus level
reflect the quantitative modification of its production and release in the blood (Huseby
and Ingebretsen, 1993). It has been supposed that the liver is the main source of
plasma GGT but the mechanism of its release in the circulation is not yet known as well
as GGT carriers were not identified. GGT activity in the blood has long been known to
be associated with several complexes of different molecular weights, densities and
charges, but their structures and clinical significance have not yet been characterized.
Serum GGT values reach abnormally high levels in different types of hepatic tissue
injuries regardless of the etiology (Whitfield, 2001). As such, serum GGT activity has
long been the parameter of choice in many conditions e.g. alcohol abuse monitoring
and its association with morbidity was initially reported in this perspective. Studies
conducted over the past 10 years have shown that serum GGT values within the
reference range (men >28 U/L; women >19 U/L) have been positively associated with
the risk of cardiovascular events, hypertension, type II diabetes, and metabolic
syndrome, independently of liver disease and alcohol consumption (Emdin et al., 2005;
Ruttmann et al., 2005; Paolicchi et al., 2006b).
The currently used laboratory GGT assays do not allow the discrimination of the
different causes of increased plasma GGT level, consequently reducing the clinical
value and specificity of this otherwise sensitive disease biomarker.
232 Introduction
1.13.2 Physiological functions of γ-glutamyltransferase.
Antioxidant role: glutathione synthesis.
GSH has multiple important biological functions (Pompella et al., 2003), and these
include conjugation of electrophiles, thiol-disulfide exchange reactions in the
maintenance of normal cellular redox status, and antioxidant functions as scavenger or
as
co-factor
of
antioxidant
enzymes
(i.e.
GSH-peroxidase,
GSH-dependent
dehydroascorbate reductases). Consequently, maintenance of the reduced intracellular
GSH pool is important for the general welfare of cells, especially in situation of severe
oxidative stress.
Intracellular GSH level depends on the equilibrium existing between its consumption
and synthesis, the latter is regulated by the availability of the three precursor amino
acids (Glu, Cys, Gly). GGT catalyzes the first step in the degradation of extracellular
GSH, thus making possible the uptake of the three separate amino acids by cells
(Figure 2). In this process, GGT transfers Glu from GSH to an amino acid or a dipeptide
and releases Cys-Gly. The main acceptors for Glu are: cistine (Cis), glutamate, alanylglycine, serylglycine, glycyl-glycine (GlyGly) (Meister, 1988). γ-Glutamyl-amino acids
and γ-glutamyl-dipeptides thus formed are carried inside cells where are transformed by
γ-glutamyl cyclotransferase into the respective amino acids and 5-oxoprolinase, the
latter is converted into Glu which can be used for GSH synthesis. Cys-Gly is further
hydrolyzed by membrane dipetidases (e.g aminopeptidase N and membrane bound
dipeptidase, Kozak and Tate, 1982) into Cys and Gly which are uptake separately by
cells to be used for GSH synthesis (Griffith et al., 1979: Tate and Meister, 1981). Two
are the main dipeptidases possibly involved in Cys-Gly hydrolysis: Aminopeptidase N
(ApN, CD13) and Mebrane Bound Dipeptidase (MBP) (Kozak and Tate, 1982). ApN is
specific for reduced Cys-Gly (Km 2.5 Mm); MBP activity, instead, is inhibited by reduced
Cys-Gly, but not by cystinyl-bis-glycine which is an excellent substrate of MBdP (Km 0.6
mM). Thus, MBP appears to play a far greater role in GSH metabolism. Tissue
expression pattern of MBD and GGT has been studied in mouse (Habib et al., 1996):
both are expressed at high levels in the kidney and small intestine, but in other tissues
these enzymes showed an independent expression pattern. E.g. in the lung, MBD is
expressed at high levels, whereas GGT is almost undetectable; the reverse is true in
the seminal vesicles and fetal liver. Thus, although both enzymes may function in
concert to metabolize glutathione in kidney and small intestine, in other tissues they
233 Introduction
appear to act independently, suggesting that they have independent roles in other
biological processes.
Figure 2 γ-Glutamyl cicle (Lieberman et al., 1995)
Gluthatione and cysteine supply.
Another important functions of GSH is to store Cys because the latter is extremely
unstable extracellularly and rapidly auto-oxidizes to cystine, in a process producing
potentially toxic oxygen free radicals (Lu, 1999). GGT allows the efficient utilization of
GSH as Cys storage releasing Cys-Gly from GSH. Besides, cystine is a preferred
acceptor for transpeptidation reaction by GGT (Meister, 1988), thus γ-glutamyl-cystine
represents another way to transport Cys inside cells (Figure 3). Cys is the limiting
substrate for GSH synthesis, but it is especially important for protein synthesis, being an
essential aminoacids (Lu, 1999; Zhang et al., 2005).
Figure 3. GGT and thiol supply.
(1) hydrolysis of Cys-Gly is hydrolyzed by membrane dipeptidases;
(2) formation of γ-Glu-Cis. (Pompella et al., 2006)
234 Introduction
γ-Glutamyltransferase and prooxidant reactions.
Stark et al. (1993) first proposed that GGT-dependent catabolism of GSH can drive
prooxidant reactions in particular in presence of ferric iron. GGT was thus shown to
stimulate lipid peroxidation (LPO) in several systems involving GSH as substrate, Fe(III)
complexes as redox catalysts, Gly-Gly as transpeptidation acceptor and having as
targets: linoleic acid (Stark et al., 1993), isolated LDL (Paolicchi et al., 1999) or cells
expressing GGT (e.g. hepatic pre-neoplastic lesions or isolated hepatocytes and HepG2
cells; Pompella et al., 1996; Paolicchi et al. 1997).
The mechanism of GGT-dependent LPO is based on Cys-Gly, the sulfydryl group of
which is predominantly dissociated in the anion form at pH 7.4. Cys-Gly thiolate anion,
turning into thiyl radical, can redox-couple with Fe(III) thus triggering production of
reactive oxygen species (ROS), superoxide anion and hydrogen peroxide(H2O2) in first
place (Figure 4). ROS and thiyl radicals are then responsible of LPO (Zalit et al., 1996).
GSH itself can reduce iron (Paolicchi et al., 1999), but it has been showed the reaction
rates increased significantly when GGT and Gly-Gly where included, while no reduction
was observed in absence of GSH. On the other hand, Cys-Gly was found to reduce
ADP-chelated Fe(III) more effectively than did GSH, forming Fe(II) to the same extent
as observed with GSH in presence of GGT (Paolicchi et al., 1999). The higher reactivity
of Cys-Gly is due to a lower pKa of its thiol group in comparison with GSH (6.4 vs. 8.6
respectively. Stark et al., 1989) but also to the enzymatic removal of γ-gluatmate. GSHdependent iron reduction is limited by the chelating properties of the α-carboxyl group of
γ-glutamate which affects redox interactions of the cysteine thiol with Fe(III) (Spear and
Aust, 1994). In fact GSH dimethyl ester (GSH-DME), where Glu is in conjunction with
Gly through a methyl group, is as effective as Cys-Gly in reducing ADP-Fe(III) (Paolicchi
et al., 1999), even if the thiol groups of GSH and GSH-DME share very similar pKa
(Spear and Aust, 1994). Enzymatic action of GGT removes exactly γ-glutamate and its
chelating effect, thus enhancing the redox activity of the remaining thiol group.
235 Introduction
Figure 4. GGT-dependent prooxidant reactions.
LPO, lipoperoxidation
GGT-dependent prooxidant reactions were observed also in presence of physiological
sources of iron, i.e. transferrin and ferritin, that means these reactions can take place in
vivo and, more, that reducing power of Cys-Gly, originated by GGT, is sufficient to effect
the reductive release of redox-active iron from its storage protein (Stark et al., 1993;
Drozdz et al., 1998; Corti et al., 2004). Thus GSH catabolism by GGT could represent a
mechanism to increase locally the availability of free iron (Paolicchi et al., 2002a). The
role of copper and ceruloplasmin was also investigated (Glass and Stark, 1997), both
participate in GGT-dependent prooxidant reactions enhancing the transfer of electrons
from thiol to iron. Release of free iron from its storage sites can occur also in
inflammatory events, in fact superoxide and hydrogen peroxide, produced by phagocytic
cells, cause iron to be released from ferritin (Halliwell and Gutteridge, 1999).
γ-Glutamyltransferase and prooxidant reactions:
Effects on low density lipoproteins (LDL).
Redox cycling of iron is a recognized factor in initiation of lipid peroxidation (Minotti,
1993). Accordingly, GSH/GGT-dependent iron reduction was repeatedly shown to result
in the promotion of lipid peroxidation in several distinct experimental models;
subsequent studies were thus dedicated to verify the possibility that GGT-mediated
production of Cys-Gly, during GSH catabolism, might serve as a mechanism to promote
iron reduction and hence LDL peroxidation (Paolicchi et al., 1999). Experiments showed
that in systems including ADP-Fe(III) complexes GSH itself could reduce some iron, but
the reaction rate increased significantly when GGT was included to remove the γ-
236 Introduction
glutamate residue. This effect was observed over a broad range of GGT activities (0200 mU/ml) and GSH concentrations (0-2 mM; Figure 5).
Figure 5. GSH and GGT dependence of LDL oxidation.
Incubations (1ml final volume) contained LDL (0.1 mg prot/ml), Gly-Gly (20 mM) and
ADP-Fe(III) (150 µM ADP-15 µM FeCl3) in PBS pH 7.4, 37°C.
(A) The reactions were started by adding GSH (2mM), plus increasing amounts of
GGT. (B) GGT was held constant (50 mU/ml) and the reactions were started by
adding increasing concentrations of GSH. (Paolicchi et al., 1999)
Importantly additional experiments indicated that GSH-dependent LDL oxidation could
be efficiently promoted both by GGT in presence of transferrin as iron source and by
cells expressing significant levels of GGT activity at their surface such as HepG2
hepatoblastoma or U937 monoblastoid cells (Paolicchi et al., 1999). Cells must be
considered as source of physiological GGT but also of GSH, in fact, the latter is
normally released by cells (Meister, 1995). These last two findings are a first indication
that LDL oxidation mediated by GSH/GGT might occur in physiological conditions.
S-Nitrosoglutathione metabolism.
S-Nitrosoglutathione (GSNO) is a metabolite of GSH and nitric oxide (Hart, 1985).
GSNO has been shown to have several pharmacological activities; these include the
inhibition of platelet aggregation (De Belder et al., 1994) and the activation of soluble
guanylyl cyclase. In addition, GSNO has a protective effect in cardiac reperfusion injury
and during the exposure of cells to oxidants (Konorev et al., 1995), it has also a dual
role in apoptosis, being both protective and toxic depending on concentration and cell
type (Sandau and Brüne, 1996; Dimmeler, 1998). GSNO is considered a carrier and a
donor of NO, but very little is known about the decomposition and metabolism of GSNO.
GSNO belongs to the class of γ-glutamyl compound, so the possibility that GSNO is a
substrate for GGT has been investigated. Hogg et al. (1997) showed in vitro that GGT,
in presence of Gly-Gly as transpetidation acceptor, catalysed the conversion of GSNO
237 Introduction
to S-nitroso-cysteinyl-glycine (CG-SNO) with a Km of 28 µM, while the Km for GSH was
5-10 µM (Singh et al., 1995). CG-SNO in presence of transition metal ions
spontaneously dissociated into Cys-Gly and NO. Zeng et al. (2001) investigated the
mechanism of GSNO decomposition using bovine aorta endothelial cells (BAEC). They
concluded that the mechanism of GSNO decomposition by endothelial cells involved the
reduction of extracellular disulfides to form thiols, which then reduced GSNO. The
authors, thus, suggested that GSNO was degraded by a cellular modification of the
extracellular environment rather than metabolized by a specific protein on the cell
surface. If the case, GGT could still participate in this mechanism triggering prooxidant
reactions in the extracellular environment and thus modulating the redox status of the
latter (Paolicchi et al., 2002a)
Detoxifying role: mercapturic acid formation.
GGT participates to detoxification processes having a role in the synthesis of
mercapturic acids which derive from GSH conjugates (Figure 6). Being a nucleophile,
GSH can directly reacts with electrophile compound, otherwise GSH conjugates are
also formed by the cytosolic enzyme GSH S-transferase. Anyway, GSH-adducts are
actively secreted from the cell where enter the pathway of mercapturic acid formation,
after the removal of Glu from GSH by GGT activity. The metabolism of GSH conjugates
to mercapturic acids begins either in the biliary tree, intestine, or kidney and they are
eliminated in bile and urine (Hinchman et al., 1998; Kearns and Hall, 1998).
Fig 6 GGT in the synthesis of
mercapturic acids. The
electrophile agent X is
conjugated to GSH, then is
secreted by a specific carrier
(GS-X pump).
Outside cell, GSH-adducts are
transformed into a Cys-adduct
(Cys-X) by the action of GGT
and membrane bound
dipeptidase (DPase).
Conjugates Cys-X are
transformed into mercapturic
acid and then excreted.
(Kearns and Hall, 1998)
238 Introduction
Leukotrien metabolism.
Leukotrienes (LT) belong to a class of arachidonic acid-derived lipid inflammatory
mediators produced by lipoxygenase pathways. They include the cysteinyl LTs LTC4,
LTD4, and LTE4, representing biologically active constituents of the long-known “slowreacting substance of anaphylaxis” and the dihydroxyeicosatetraenoate LTB4.
Leukotriene C4 is conjugated to a GSH molecule and it is converted into leukotriene D4
by GGT activity, The cleavage of Gly from LTD4 yields LTE4 (Lewis et al., 1990).
Leukotrienes, C4 and D4 included, bind to specific receptors on smooth muscle cells
causing prolonged bronchoconstriction (Anderson et al., 1982; Bernstrom and
Hammarstrom, 1982).
Cysteinyl LTs metabolism has been recently investigated in three patients with GGT
deficiency (Mayatepek et al., 2004). Patients displayed an abnormal profile of LTs in
urine and in plasma with increased concentrations of LTC4 and absence of LTD4 as
well as LTE4, whereas LTB4 synthesis was not affected. GGT deficiency thus can be
regarded as an inborn error of cysteinyl LT synthesis. LTC4 synthesis deficiency has
been found to be associated with a fatal developmental syndrome, including severe
muscular hypotonia, psychomotor retardation, failure to thrive, and microcephaly
(Mayatepek et al., 1998). Variable neurological disorders are associated with GGT
deficiency too; therefore Mayatepek et al. (2004) proposed that LT metabolic defect,
either excessive LTC4 or more likely lack of LTD4 and LTE4, may contribute to some or
even all of the observed symptoms.
γ-glutamyltransferase and cytokine-like function.
Recently a new and provocative function has been proposed for GGT, Niida et al.
(2004), in fact, reported that the addition of GGT protein to mouse bone marrow culture
effectively induced formation of osteoclasts. The same result was obtained after
inhibition of the enzymatic activity by acivicin, but not in presence of an antibody which
recognized GGT without affecting the enzymatic activity. Furthermore, it was shown that
both native and inactive GGT stimulated the expression of the receptor activator of NFkB ligand (RANKL) mRNA and protein from bone marrow stromal cells. Thus GGT
seems to posses a cytokine-like biological function independently of its enzymatic
activity. A subsequent study showed that urinary excretion of GGT changes in parallel
with established biochemical markers of bone resorption (i.e. deoxypyridinoline and type
239 Introduction
I collagen N-telopeptide) both in animal models and human subjects (Asaba et al.,
2006).
Structural studies designed to identify disulfide bridges in mammalian GGT predicted
disulfide formations between Cys49 and Cys73 and between Cys191 and Cys195, in
particular Cys191 and 195 are arranged in a CX3C motif (Kinlough et al., 2005). CX3C
disulfide motif is associated with several biological meaning:
1) it is a signature motif for one class of chemokines (Bazan et al., 1997), the only
known member of which is fractalkine/neuroactin (Stievano et al., 2004).
2) it is an essential coordinate for copper binding in several yeast proteins (Balatri et
al., 2003), and in GGT CX3C motif is probably in a solvent-accessible site (Kinlough
et al., 2005).
3) it is essential for the functioning of the vaccinia virus protein A2.5L, a thiol
oxidoreductase that controls disulfides formation in viral membrane proteins
(Senkevich et al., 2002).
1.13.3 Serum GGT: origin and chemical and physical characteristics
Since 1961, the determination of serum GGT is used as a diagnostic marker of hepatic
dysfunction (Szczeklik et al., 1961), including cancer and excessive alcohol
consumption (Whitfield et al., 2001). The measurement of serum GGT assay with the
substrate γ-glutamyl-p-nitroanilide is a simple laboratory tests, considered the most
sensitive marker in the diagnosis of cholestasis and hepatic steatosis, also induced by
alcohol or drugs. The elevation of serum GGT can be detected even in patients with
chronic liver disease due to HCV infection and represents an unfavorable prognostic
index of response to treatment with interferon (Baptized et al., 1992; Paolicchi et al.,
2005). Despite the sensitivity of the test, the diagnostic use of GGT serum is limited by
the low specificity..
In the genome exists a family of genes for GGT, but only the gene GGT1 is transcribed
and translated into the complete and catalytically active protein (Courtay et al., 1994).
GGT has no enzymatic isoforms in the sense of protein structure, in contrast,
glycosylation is tissue specific and contribute to the heterogeneity of molecular weight
and charge observed in the proteins extracted from various tissues. Serum GGT seems
to be of hepatic origin as suggested by studies conducted by Huseby and collaborators
(1981), from which it appears that the GGT present in the serum had the same
240 Introduction
characteristics as the one extracted from the liver regarding the molecular weight (MW),
the content of sialic acid and the degree of glycosylation. These parameters were
distinct from those of GGT recovered in the urine, kidney and pancreas (Huseby et al.,
1981).
Subsequent studies have demonstrated the presence of two different fractions of GGT
in serum, a hydrophilic one and a hydrophobic one, differing in charge, MW and density.
The hydrophilic form, apparently identical to that obtained after the proteolytic treatment
used for the purification of GGT from tissue, was present in small quantities and
seemed to circulate in the blood as free enzyme (Huseby et al., 1982a; Huseby et al.,
1982b; Huseby et al., 1982c). Regarding the hydrophobic fraction, it has been proposed
that it was constituted by GGT transported by serum lipoproteins VLDL, LDL, HDL and
chylomicrons. It was assumed that the association of GGT with lipoproteins was
mediated by the N-terminal peptide responsible for the normal insertion of GGT into the
plasma membrane. In fact, the enzyme associated with LDL consisted of a whole heavy
chain, this indicates that the amphipathic enzyme form was involved (Paolicchi et al.,
2003a).
The association of the enzyme GGT with lipoproteins has been studied by Huseby and
colleagues (1982) in the serum of patients with hepatobiliary diseases. It was observed
that 60-80% of the total GGT activity was associated with lipoproteins. The latter could
be separated into two major fractions by gel filtration chromatography or electrophoresis
in agarose gel. A fraction was characterized by a high molecular weight (MW> 600 kDa)
and β-mobility and had been found in the sera of most patients with cholestasis. The
other fraction containing GGT eluted in the range of MW from 250 to 450 kDa and
migrated with α1α2 mobility. Such MW were compatible with complexes between
lipoproteins and this association appeared to be confirmed by the presence of GGT
activity in the fractions of plasma lipoproteins separated by ultracentrifugation for
density gradient. Most of the activity of the first fraction (MW> 600 kDa) seemed
associated with VLDL lipoprotein - LDL, while GGT present in the second fraction (MW:
250 – 450 kDa) seemed to be associated with HDL lipoproteins as observed in 70% of
serum of patients not jaundiced. Finally both fractions were heterogeneous with respect
to size, charge and density (Huseby et al., 1982a).
In a study conducted successively by Wenham and collaborators (1984), serum GGT
was separated, by molecular exclusion chromatography, into three fractions with
241 Introduction
relative MW of 1000, 250-500 and about 120 kDa. In this study, attention was focused
mainly on the fraction of GGT with intermediate MW (250-500 kDa) isolated from
patients with liver disease. Even in this study it was shown that this fraction consisted of
a complex of GGT and high density lipoproteins (HDL). In addition the physical
properties of this fraction, both the charge that the MW, could be altered by incubation
of the serum with the bile (Wenham et al., 1984).
1.13.4 Predictive value of serum GGT in hepatobiliary diseases
The problem of the physical nature of the isoforms (fractions) of serum GGT cannot be
separated from that of its diagnostic and predictive value, although the GGT is
performed as routine examination until the 60s (Szceklik et al., 1961) only recently the
complexity of his involvement with the human disease has been highlighted.
Traditionally the elevation of serum GGT is associated with hepatobiliary disease and
alcohol abuse; recent studies have revealed an extraordinary complexity: while high
GGT values are diagnostic for liver disease, GGT values within the upper reference
range have predictive value in respect of cardiovascular disease associated with
atherosclerosis (Emdin et al., 2006). These concepts are evident in an Austrian cohort
study of 283,438 subjects aimed to investigate the relation of serum GGT levels to longterm mortality (Kazemi-Shirazi et al., 2007). Patients were stratified, according to GGT
and gender, in: 1-normally low (<9 U/L for women and <14 U/L for men), 2-normally
high (9 to 17 U/L and 14 to 27 U/L), 3-moderately high (18 to 26 U/L and 28 to 41 U/L),
4-high (27 to 35 U/L and 42 to 55 U/L) and 5-very high (>35 U/L and and > 55); it was
observed that '"hazard ratio" (HRs) for death from hepatobiliary disease increased
progressively from 1 in subgroup 1, to 1.4, 3.2, and 4.9 in the 4th group and jumped to
15.1 in the 5th group; the mortality risk for hepatoma was 1 in subgroup 1, 5.2 in the 4th
and 18.05 in the 5th (Figure 7A). On the contrary, the risk of death from cardiovascular
diseases increased only within the normal range, but without any increase between the
4th and the 5th group, thus demonstrating that GGT values higher than the upper
reference limit (5th group) are associated to death from liver disease, non-neoplastic and
neoplastic, but not at higher risk of death from cardiovascular disease (Figure 7B;
Kazemi-Shirazi et al., 2007).
242 Introduction
A
Hazard ratios (95% CI)
Rischio relativo 95% IC
20-­‐
15-­‐
10-­‐
5-­‐
0-­‐
1
2
3
4
5
Mortalità epatobiliare
Hepatobiliary
mortality
1
2
3
4
5
Mortalità per
Hepatocarcinoma
epatocarcinoma
mortality
Rischio
(95%
Hazardrelativo
ratios (95% CI) IC)
B
1 2 3 4 5
All
Tutte
1 2 3 4 5 1 2 3 4 5 1 2 3 4 5
Non Cause
non
neoplastic
neoplastiche
Neoplastic
Neoplasie
Vascular
Cause
vascolari
Cause di mortalità
MORTALITY CAUSES
1 2 3 4 5 1 2 3 4 5
Ischemic
Cardiopatia
heart
ischemica
disease
cerebro-­‐
Malattie
vascular
cerebrodisease
vascolari
Figure 7. Serum GGT levels and mortality risk for A) hepatic diseases or B) other
causes. Serum GGT quintiles: (1) < 9 U/L women, < 14 U/L men; (2) 9-17 U/L e 14-27
U/L; (3) 18-26 U/L e 28-41 U/L; (4) 27-35 U/L e 42-55 U/L; (5) >35 U/L e > 56 U/L. the
lowest category served as reference (Kazemi-Shirazi et al.,2007).
243 Introduction
1.13.5 Serum γ-glutamyltransferase: a prognostic marker in cardio - vascular
diseases.
Conigrave et al. (1993) were the first to appreciate that GGT can have a predictive
value irrespective of hepatic disease or alcohol consumption. Such a role of GGT as an
independent predictor of mortality from all causes has been confirmed by several
subsequent studies (Brenner et al., 1997; Arndt et al., 1998; Karlson et al., 2000). With
respect to cardiovascular diseases, it was precociously observed that serum GGT levels
can be correlated with increased risk of myocardial infarction (Betro et al., 1973; Hood
et al., 1990; Wannamethee et al., 1995). More recently, the association of the risk of
stroke with serum GGT was also observed (Jousilahti et al., 2000; Bots et al., 2002).
These associations are partly explained by the known correlations of GGT with
recognized factors in the pathogenesis of cardiovascular disorders, such as changes in
blood lipid (Van Barnevel et al., 1989; Nilssen et al., 1990; Jousilahti et al., 2000), body
mass index (Nilssen et al., 1990; Daeppen et al., 1990), hypertension (Miura et al.,
1994; Yamada et al., 1995), glucose intolerance (Umeki et al., 1989), insulin resistance
(Rantala et al., 2000) and type 2 diabetes (Perry et al., 1998; Lee et al., 2003). GGT
levels also correlate positively with novel risk factors such as C-reactive protein,
fibrinogen and F2-isoprostanes (Lee et al., 2003). It is worth emphasizing that most of
the studies mentioned above refer to serum GGT values within the laboratory reference
range, which would otherwise raise no specific health concern.
Of particular interest was the study by Wannamethee et al. (1995), where the attention
was focused on ischemic heart disease (IHD). In a large prospective study (7613
middle-aged men, with a 11-years follow-up), GGT levels in the normal range were
strongly associated with all-cause mortality, and the association was largely due to a
significant increase in deaths from ischemic heart disease in the top quintile of GGT
distribution. Serum GGT was positively correlated with preexisting ischemic heart
disease, diabetes mellitus, antihypertensive medication, systolic and diastolic blood
pressure, total and HDL cholesterol, heart rate and blood glucose, while a negative
association was observed with physical activity and lung function. After adjustment for
these variables, elevated GGT (highest quintile, ≥24 U/l, vs. the rest) was still
associated with a significant increase in mortality from all causes and from IHD. The
increased risk of IHD mortality was more marked for patients with evidence of ischemic
heart disease at screening, particularly those with previous myocardial infarction
(Wannamethee et al., 1995), these findings clearly pointed to a connection of GGT with
underlying atherosclerotic coronary artery disease.
244 Introduction
The supposed connection of GGT with atherosclerotic disease has received a detailed
assessment in a prospective study of Emdin et al. (2001). The study included a six-year
follow-up of 469 patients with ischemic syndrome and angiographically documented
coronary artery disease (CAD). After correction for other cardiovascular disease risk
factors (age, smoking, serum cholesterol, left ventricular ejection fraction, body mass
index, diabetes mellitus), or confounding factors (serum alanine aminotransferase, selfreported alcohol consumption), the prognostic value of serum GGT activity for cardiac
death and non-fatal infarction was confirmed. In particular, the significance of serum
GGT was more evident in a subset of patients prone to plaque complications, i.e.
characterized by the association of diffuse atherosclerosis (“multivessel disease”) and a
history of previous myocardial infarction (approx. 36% of the whole population). The risk
was increasing using two different GGT cut-off values (25 or 40 U/l, both considered
within the normal range), and the event excess was concentrated within the first three
years (Figure 8A). The prognostic significance of serum GGT was thus correlated with
the diffusion of coronary artery disease. More interestingly, the significance of serum
GGT appeared to depend on the instability of plaque as well, as indicated by the fact
that the prognostic value of GGT disappeared after revascularization by angioplasty
(Figure 8B). This procedure is considered as a means of plaque stabilization (Amoroso
et al., 2001). Thus, the unfavourable prognosis signalled by elevated serum GGT
seems to apply specifically to patients with vulnerable plaques, suggesting that
connections of some kind must exist between GGT and the processes involved in
plaque instability.
245 Introduction
Figure 8. Event-free survival according to serum GGT activity.
(A) 6-years survival of 168 patients with CAD, history of previous myocardial infarction
and multiple vessel disease (119 with serum GGT < 40 U/L; 49 > 40 U/L). (B) 6-Years
survival in a subset of the same population having undergone revascularization by
angioplasty. Vertical lines represent confidence intervals.
(Emdin et al., 2001; Pompella et al., 2004).
A recent prospective study by Ruttmann et al. (2005), carried out in as many as 163,944
Austrian adult subjects, has now rather conclusively shown that serum GGT levels are
an independent prognostic factors for fatal events of chronic forms of CAD, congestive
heart failure and ischemic or hemorrhagic stroke. The finding was true for both-sexes,
with a clear dose-response relationship and a stronger significance as far as younger
subjects were concerned. Ruttmann and colleagues established the prognostic value of
GGT for cardiovascular events at serum levels that lie within normal values. The
receiver operating characteristics analysis suggested GGT cut-off values of 15.5 U/L for
men and 10.5 U/L for women, corresponding to 27.6 U/L and 18.7 U/L, respectively, for
measurements made at 37°C.
In the last year (2006) other three prospective studies, carried out in smaller groups of
subjects, confirmed the association between GGT and cardiovascular disease related to
atherosclerosis:
1. Meisinger et al., MONICA/KORA study group, Germany.
Conclusion - Serum GGT is a strong predictor of acute coronary events in
apparently healthy men (1,878 subjects) from general population, independent of
other risk factors for cardiovascular disease.”
2. Lee D.H. et al., Finland
246 Introduction
Conclusion- This study suggested an independent mechanism linking serum
GGT to coronary heart disease (CHD) among general population (28,838 subjects).
… stronger associations were observed among subjects aged < 60 and among
alcohol drinkers. Especially, measurement of serum GGT among type-2 diabetics
may be helpful to predict the future risk of CHD.”
3. Lee D.S. et al., the Framingham Heart Study
Conclusion – An increase in serum GGT predicts onset of metabolic syndrome,
incident CVD, and death suggesting that GGT is a marker of metabolic and
cardiovascular risk.”
Alcohol consumption is a common confounding factor in studies involving serum GGT.
The possible connections among the three factors – alcohol consumption, serum GGT
and cardiovascular risk – were analyzed by Jousilahti et al. (2002). In a random sample
of 3666 men aged 25-74, prevalent coronary heart disease (CHD) was correlated with
serum GGT, carbohydrate-deficient transferrin (CDT, the other recognized marker of
alcohol consumption), and self-reported alcohol consumption. CDT levels were
inversely and GGT levels positively correlated with CHD risk. Besides, in a composite
risk assessment, men with elevated GGT levels (> 80 U/L) and normal CDT (≤ 20 U/L)
had nearly 8-fold adjusted risk of CHD, as compared to subjects with normal GGT and
elevated CDT levels. Self-reported alcohol consumption had an inverse association with
CHD risk, which disappeared after adjustment for the other risk factors. As CDT is to be
taken as a more reliable marker of alcohol consumption than GGT itself (Wuyts and
Delanghe, 2003), these data seem to indicate that alcohol consumption can have a
protective effect on cardiovascular prognosis, as previously reported (Muntwyler et al.,
1998), and that the prognostic association of serum GGT must be independent of
alcohol-related liver injury.
Altogether, to date the reported studies have confirmed in multivariable analyses the
value of serum GGT in predicting the clinical evolution of cardiac and cerebrovascular
diseases towards life-threatening events, such as myocardial infarction, stroke and
cardiac death, irrespective of the occurrence of hepatic disease, alcohol consumption
and other traditional risk factors.
Interestingly, a recent report points to a prognostic value of elevated serum GGT also in
stent restenosis (Ulus et al., 2006). The correlation of stent restenosis with serum levels
of GGT was valuated in a cohort of 120 patients, free of hepatobiliary diseases, with a
history of coronary stent implantation, 60 with restenosis and 60 without. Multivariate
247 Introduction
analysis revealed that serum GGT as well as with C-reactive protein and alkaline
phosphatase levels were independent predictors of stent restenosis. It is worth that the
prognostic value of GGT in predicting stent restenosis was particularly clear in patients
with serum GGT activity > 40 U/L.
γ-glutamyltransferase inside plaques: possible connections with serum GGT.
Epidemiological studies mentioned in the previous paragraph suggest a relationship of
circulating GGT with the evolution of atherosclerosis, but it is not known how the
phenomena are connected. A clue was independently provided by studies showing the
presence of GGT inside atherosclerotic lesions. Histochemical studies, in fact, revealed
intense GGT activity in the intimal layers of human atherosclerotic lesions, where it
apparently locates in CD68+ macrophage-derived foam cells (Paolicchi et al., 1999;
Paolicchi
et
al.,
2004).
GGT-positive
cells
were
found
to
co-localize
with
immunoreactive oxidized LDL (Figure 9; Emdin et al., 2002), and catalytically active
GGT could also be detected in microthrombi adhering to the surface of atheromas
(Figure 10; Dominici et al., 2003a).
Figure 9. Colocalization of
GGT activity with oxidized
LDL.
Human cerebral artery
plaque, serial sections.
A) Histochemical demonstration of GGT activity.
B) Indirect immunofluorescence for oxidized LDL
(Emdin et al., 2002).
Figure 10. Enzymatically
active GGT is in thrombi
adhering to a human
atherosclerotic plaque.
(A) Haematoxylin/eosin;
(B) Histochemical demonstration of GGT activity.
(Dominici et al., 2003a)
248 Introduction
The origin of GGT activity detectable in atherosclerotic lesions is however unclear. A
possibility is that the expression of GGT is for some reason up-regulated in the
macrophages present in the intimal space, in fact, monocytes/macrophages normally
express low levels of GGT activity (Bauvois et al., 1995). As an alternative, the GGT
activity found in atherosclerotic plaques might originate from circulating GGT, with a
mechanism similar to the extravasation of LDL lipoprotein into the intimal space.
In recent study (Franzini et al., 2009) the chemico-physical characteristic of plaque GGT
have been compared with those of plasma GGT. Collected data suggest that plaque
GGT may derive both from insudation of specific GGT-rich high molecular weight
complexes, as well as from endogenous synthesis by cellular elements of likely
inflammatory nature (i.e. macrophages). Once accumulated in the plaque environment,
GGT retains its enzymatic activity, as shown by previous histochemical studies and can
promote pro-oxidant effects. In fact, significant levels of protein-bound cysteinyl-glycine
have been detected in plaque material (Franzini et al., 2009); such a biochemical
marker in fact documents that GGT-mediated redox reactions have taken place (Corti et
al., 2005). Redox events can play relevant roles in several processes favouring the
evolution of atherosclerotic plaques towards instabilization and rupture, which could at
least partly explain the reported association of increased serum GGT with unfavourable
prognosis of cardiovascular diseases. In fact, the hypothesis implies that the role of
GGT activity in plaque destabilization is mediated by the known ability of GGT to affect
redox equilibria and antioxidant status of cells and tissues (Figure 11).
A
B
Fig 11 GGT activity within plaques
(A) Histochemical demonstration of GGT activity within a frozen section of coronary
atheroma from endoarteriectomy in vivo.
(B) GGT metabolism of GSH within the plaque. The hydrolysis of GSH originates CysGly, which is a powerful reductant of Fe(III), able to simultaneously generate Fe(II)
and a free thiyl radical. Subsequent reactions lead to the formation of ROS which
might contribute to LDL oxidation and likely to other processes, such as
metalloproteinase activation, cell proliferation, and apoptosis. (Emdin et al., 2005)
249 Introduction
1.13.6 Fractional GGT analysis
Numerous hepatic diseases are associated with abnormal GGT values, which can be
also detected in kidney disease (Ryu S. e 2007) or alcohol abuse as well (Whitfield et
al, 2001). Furthermore, it is now established that variations within the normal range
have clinical significance in the field of cardiovascular and metabolic diseases.
However, the determination of serum GGT can neither distinguish between these
groups of patients, nor identify people who show high GGT levels in the absence of liver
disease or alcohol abuse. For this reason in recent years, several techniques capable of
highlighting different fractions of GGT, each associated with a different profile of risk of
disease, have been made available (Nemesanszky et al., 1985). Unfortunately, such
techniques were burdened by a lack of sensitivity and an equally poor reproducibility, so
they never entered clinical practice.
Recently, at the Department of Experimental Pathology, a technique with high sensitivity
capable of detecting different fractions of GGT in serum or plasma has been developed
(Franzini et al., 2008). This technique has a detection limit of 0.5 mU/mL, lower than
that of the routine technique, and it is based on a molecular exclusion chromatographic
followed by post-column injection of a fluorescent reagent. In contrast to the previous
techniques, which allowed the assessment of GGT fractions only in subjects with very
high values of the enzyme, the new procedure allows the study of GGT in all individuals,
including those with GGT levels within the reference range.
By using this method, in a previous thesis, the distribution of GGT fractions in 200
healthy subjects, blood donors (100 men and 100 women; Figure 8) was characterized.
In all examined subjects four fractions were identified namely big-GGT GGT (b-GGT,
2000 kDa), medium-GGT (m-GGT, 940 kDa), small-GGT (GGT-s, 140 kDa) and free GGT (f-GGT, 70 kDa). The molecular weights of the fractions b-GGT, m-and s-GGT
GGT are compatible with those of complexes with lipoproteins VLDL, LDL, HDL,
respectively, while that of f-GGT is indicative of a free enzyme form..
250 Introduction
Figure 8 Elution profile of fractional GGTactivity corresponding to the 25th (dashed line),
50th (solid line) and 75th (dotted line) percentile of total GGT in healthy males (A, n=100)
and healthy females (B, n=100). Fractional GGT analysis was performed on plasma–
EDTA samples by high performance gel-filtration chromatography, GGT activity was
specifically detected by an on-line post-column reaction with the fluorescent substrate,
γGlu-7-amido-4-methylcoumarin.
1.13.7 GGT fractions in the Framingham Heart Study: correlates and reference
limits.
The reference values of GGT fractions, as well as their clinical correlates have been
assessed in a large reference sample of healthy subjects from the Offspring Cohort of
the Framingham Heart Study (Franzini et al., 2013).
The analysis of Framingham Offspring cohort showed that the correlates of plasma
activity vary for each GGT fraction: the b-GGT fraction is mostly associated with serum
triglyceride levels in both sexes, while m- and s-GGT are mostly correlated with either
alcohol consumption and HDL or LDL cholesterol level. Prominent correlates of f-GGT
in both sexes were PAI-1 level and triglyceride level and alcohol consumption in men,
and blood glucose in women.
The reference values for each of the four GGT fractions has been established in a
subgroup of healthy subjects (n= 432). Fractional GGT analysis showed significant
differences in activity of all fractions between men and women.
Analysis of the clinical correlates in the whole community sample confirmed the results
of bivariate correlations. Plasma total GGT activity is positively associated with already
described factors (Whitfield, 2001) such as alcohol consumption, triglycerides, LDL
cholesterol, blood pressure, body mass index, waist circumference, serum glucose,
fibrinogen and CRP, and negatively with HDL cholesterol, physical activity. The
multivariable analysis showed that alcohol consumption, triglycerides, HDL and LDL
251 Introduction
cholesterol and CRP were the only independent correlates, together with PAI-1 hereby
described for the first time.
As expected, bivariate correlations and multivariable linear regression analyses
conducted separately for each GGT fraction showed that biological and clinical
correlations described for total GGT actually depended on the diverse association of the
above mentioned factors with specific fractions.
Alcohol consumption showed a prominent association with the m- and s-GGT fractions:
in fact, we have previously reported that fractional GGT profile of alcohol addicts is
characterized by a greatest increase in m- and s-GGT levels vs. other fractions
(Franzini et al., 2012b). We have also previously observed the elevation of s-GGT
fraction in patients with chronic hepatitis C, this suggesting the s-GGT as a marker of
hepatocellular damage (Franzini et al., 2012a). The negative correlation found between
s-GGT and plasma fibrinogen in the Framingham cohort might correspond to underlying
liver dysfunction.
Markers of metabolic syndrome (BMI, DBP, glucose, triglycerides) showed the highest
positive correlation with the b- and f-GGT fractions. These results confirm and support
the recent finding that b-GGT fraction holds the best specificity and sensitivity for the
diagnosis of NAFLD (Franzini et al., 2012a). b- and f-GGT showed a negative
correlation with physical activity as it had been previously showed for total GGT activity.
It is well known that regular exercise improves many cardiovascular and metabolic risks
factors, including the intrahepatic triglyceride content (Magkos, 2010).
The existence of a correlation between PAI-1 and total GGT levels has been previously
reported only in small selected cohorts of hypertriglyceridemic and insulin-resistant
patients (Bastard et al., 1996); in the present investigation we observed that plasma
PAI-1 is among the strongest independent predictors of especially b-GGT and f-GGT.
Enhanced expression of both PAI-1 and GGT has been shown in a variety of liver injury
models, including bile duct ligation and alcohol- induced liver injury [35-36], affecting
hepatic protein synthesis. Furthermore, plasma PAI-1 levels were strongly correlated
with the cluster of variables defining the metabolic syndrome (i.e.: insulin-resistance,
obesity, glucose and lipid metabolic imbalance; Henry et al., 1998).
Comparison of total GGT values between the subsets of healthy subjects and of
subjects affected by CVD, metabolic syndrome, diabetes, or characterized by heavy
alcohol consumption confirmed that total plasma GGT activity is a sensitive but
nonspecific marker. On the other hand, each subset was characterized by a specific
252 Introduction
fractional GGT pattern, better described by the b/s ratio. Based to our correlation
analysis, heavy alcohol intake was characterized by the highest values of s-GGT and
the lowest b/s ratio, while individuals with metabolic syndrome and diabetes had the
highest values of both b-GGT and b/s ratio. As a perspective, the estimation of b/s ratio
could improve the interpretation of total GGT elevation, as already observed in small
selected cohorts of patients affected by liver steatosis or chronic viral hepatitis C, where
an increase has been associated with a metabolic liver dysfunction or a decrease with
hepatocellular damage (Franzini et al., 2012a).
In conclusion, the present study indicates that known cardiovascular and metabolic risk
markers are important correlates of GGT fractions, in particular of b-GGT. The study of
GGT fractions could permit a better understanding of the pathogenesis of diseases
associated with GGT increase, thus allowing a better clinical use of the GGT test.
Prospective studies are needed to establish the risk for metabolic disease and
cardiovascular events associated with each GGT fractions.
Fractional GGT reference limits (U/L).
Men (n = 194)
2.5
th
50
Women (n = 238)
th
97.5
th
2.5
th
50
th
97.5
Total GGT
11.4
23.1
108.8
8.8
16.8
87.3
b-GGT
0.8
3.1
18.1
0.8
2.5
30.1
m-GGT
0.01
0.48
4.19
0.01
0.29
3.51
s-GGT
2.0
6.3
71.6
1.4
4.1
46.4
f-GGT
6.9
11.7
24.2
5.6
9.4
20.9
b/s ratio
0.16
0.46
1.25
0.18
0.59
1.61
th
Data are from empirical quantile estimation.
253 Introduction
254 Aim
2. AIM
Serum gamma-glutamyltransferase (GGT) activity is a sensitive marker of liver
dysfunction, but its specificity is modest, as its value increases with common conditions
causing liver dysfunction, such as steatosis and viral hepatitis. GGT value has been
already included in diagnostic algorithm (i.e.: the Fatty Liver Index, FLI) to test the
presence of steatosis and its specificity was high if considered with other markers, but
low if considered alone.
A new method based on molecular-size exclusion chromatography, followed by a GGTspecific post-column reaction, allowed to identify and quantify, in healthy subjects, 4
plasma GGT fractions with high sensitivity, specificity and reproducibility. These
fractions, named big-GGT (b-GGT), medium-GGT (m-GGT), small-GGT (s-GGT), and
free-GGT (f-GGT) showed different molecular weight (MW), i.e. 2000, 1000, 250 and 70
kDa, respectively. It has been previously shown that in healthy subjects f-GGT is the
most abundant fraction, while b-GGT showed the highest degree of correlation with
established cardiovascular risk factors, such as level of serum triglycerides, LDLcholesterol, C-reactive protein, diastolic blood pressure. Interestingly b-GGT has been
found in atherosclerotic plaques together with products deriving from the pro-oxidant
reactions catalysed by the enzyme, suggesting that only b-GGT, rather than total serum
GGT itself, is responsible of the association between serum GGT and cardiovascular
disease in population and clinical studies.
The present study was aimed to test the diagnostic power of GGT fraction for hepatic
diseases in comparison with that of total GGT.
The study has been divided into four phases:
1) ACCURACY
DISEASE:
OF
GGT
FRACTIONS FOR THE DIAGNOSIS OF NON-ALCOHOLIC FATTY LIVER
The first study on diagnostic specificity of GGT fractions has been
conducted on patients with non-alcoholic fatty liver diseases (NAFLD) or chronic
viral hepatitis C (CHC): NAFLD has been chosen as model of metabolic disease,
while CHC as model of hepatocellulare damage. Results of this part of the
project have been published, the article has been reported in the Results
section.
2) CIRRHOTIC
PATIENT EVALUATION
–
PRETRANSPLANT:
in the second part of the PhD
project, fractional GGT pattern has been investigated in the end-stage liver
255
Material and Methods
disease, to establish if the four GGT fractions are still present during liver
cirrhosis, whether each fraction has a different correlation with the different
clinical aspects of the progression of liver disease, and to verify if architectural
changes of hepatic tissue might affect GGT fraction release.
3) CHARACTERIZATION
OF GGT FRACTIONS IN PRIMARY HUMAN BILE:
plasma GGT is
supposed to origin from liver, but the mechanism of its release is not known yet;
bile is rich in GGT activity, which might contribute to plasma activity, thus, to
deepen this topic, GGT fractions present in human bile have been characterized
and compared with the plasmatic one.
4) FRACTIONAL GGT EVALUATION AFTER LIVER TRANSPLANT: in the last part of the PhD
project, the trend of the total GGT and its fractions, in plasma and bile samples,
have been evaluated in 14 patients undergoing liver transplantation, with the aim
to correlate GGT fraction release in both fluid with the ischemic-reperfusion
injury and the recovery of hepatic function.
256
Material and Methods
3 MATERIALS AND METHODS
3.1 Patient selection
In the period between February 2008 and April 2011, 264 patients with liver cirrhosis
who were undergoing liver transplant evaluation [215 men; median (25th – 75th
percentile); age 54.5 (50-60 years)] were enrolled at the Department of Surgery,
Liver Transplantation Unit of the University Hospital of Pisa. At the visit, attendees
underwent anamnestic-physical examination and blood sampling for the laboratory
assessment of liver function. Blood samples were obtained following an overnight
fasting. Cirrhosis had been diagnosed on the basis of history, physical examination,
liver function tests panel, targeted serologic studies, and abdominal ultrasonography
with Doppler consistent with cirrhosis and potential complications, CT and CT portal
phase imaging MRI, MRA. Liver biopsy was performed in those cases of uncertain
diagnosis or undetermined aetiology. In this cohort: 39 patients were diagnosed with
metabolic cirrhosis (MC), defined as the absence of known cause for biopsy proven
cirrhosis after careful serological, virological and histological workup; 96 with viral
cirrhosis (VC) defined as positive for HBV (HbsAg, Anti-HBs, Anti-HBc, HBV DNA),
and/or for HDV (IgG and IgM anti-HDV), and/or for HCV (anti-HCV antibodies, a
positive result for HCV RNA in serum by nested reverse transcription-PCR); 129 with
viral cirrhosis and hepatocellular carcinoma (HCC). In the HCC group, the diagnosis
was established in the presence of histological proof of HCC when a focal lesion was
>2 cm in diameter, assessed by early arterial hypervascularization, using two
contrast-enhanced methods (Triple phase helical CT-scan, Triple phase Dynamic
contrast, enhanced MRI, arteriography), or when there was an association between
serum AFP level of >400 ng/mL plus early arterial hypervascularization, assessed by
one contrast enhanced method (21). Patients enlisted for transplantation are those
fulfilling Milan criteria (22). Two hundred subjects were selected out of 256 blood
donors already characterized and studied for the determination of fractional GGT
reference values (16), in order to obtain a cohort of healthy subjects to be compared
with cirrhotic patients.
The Institutional Ethics Committee approved the study and all subjects gave informed
consent.
257
Material and Methods
3.2 Laboratory analysis
Standard assay of all blood tests were simultaneously performed according to the
standard clinical laboratory procedures by automated analysers at the Clinical
Laboratories of the University Hospital of Pisa. Estimated glomerular filtration rate
(eGFR) was estimated by the Cockcroft-Gault formula [eGFR ml/min = [(140età)*peso Kg /(creatinina mg/dl * 72)]*0.85 if woman)]; LDL cholesterol was
calculated using the Friedewald formula [LDL-C = total cholesterol – HDL cholesterol
– (triglycerides/5)].
The Model for End-Stage Liver Disease (MELD) score has been calculated according
to UNOS modified formula:
MELD score (UNOS version) =
9.57 × ln(creatinine) (mg/dl) + 3.78 × ln(Tot.Bil.) (mg/dl) + 11.20 × ln(INR) + 6.43.
- Any value < 1 is considered equal to 1
- If the patient has been dialyzed twice within the last 7 day => serum creatinine =
4.0 mg/dL
- Creatinine >4 was automatically calculated as 4
- Patients with a diagnosis of HCC will be assigned a MELD score based on how
advanced the cancer is
3.3 Total and fractional GGT determination
Analysis of total and fractional GGT was performed, as previously described (15,16)
(pat. pend. WO2009/001290-A3, The University of Pisa), on plasma-EDTA
(ethylenediamine-tetra-acetic acid) samples using an FPLC (fast protein liquid
chromatography) system (AKTA purifier, GE Healthcare Europe, Milan, Italy)
equipped with a gel filtration column (Superose 6 HR 10/300 GL, GE Healthcare
Europe) and fluorescence detector (Jasco FP-2020, Jasco Europe, Lecco, Italy).
Separation of fractional GGT was obtained by gel filtration chromatography and the
enzymatic activity was quantified by post-column injection of the fluorescent
substrate
for
GGT,
gamma-glutamyl-7-amido-4-methylcoumarin
(gGluAMC).
Enzymatic reaction, in the presence of gGluAMC 0.030 mmol/L and glycylglycine 4.5
mmol/L, proceeded for 4.5 min in a reaction coil (PFA, 2.6 mL) kept at the 37 °C in a
water bath (Figure 1). The fluorescence detector operating at excitation/emission
wavelengths of 380/440 nm detected the AMC signal; the intensity of the
258
Material and
a Methodss
fluorrescence signal
s
was expressed
d in arbitra
ary fluoresc
cence unitss (f.u.), and the area
a
unde
er curve is proportion
nal to GGT
T activity.
Tota
al area and
d fractionall GGT area
a was calc
culated by a MatLab program (Version 7
Math
hWorks, In
nc.) to reso
olve overlap
pping peak
ks; the curv
ve fitting w
was conduc
cted with a
nonlinear leasst-squares minimizattion algorithm using
g four expponentially
y modified
Gaussian (EMG) curves.. The reacttion was calibrated analysing
a
pplasma sam
mples with
know
wn total GG
GT activity
y (standard
ds). The slope of the calibrationn curve wa
as used to
o
convvert total and fraction
nal GGT arrea to U/L.
A 4.5 mmol/L
L stock solution of gGluAMC
C was prepared in ethanol 30% w/w
w
05 N NaOH
H and storred at –20°°C. This so
olution wass daily dilutted 25-fold
conttaining 0.00
into 0.25 M Tris-HCl bufffer pH 8.5 ((25°C).
S
represen tation of the instrumentation for GGT
T fractionss
Fiigure 1 Schematic
sepa
aration and
d quantifica
ation.
259
9
Material and
d Methods
The corre
espondencce between
n elution vvolume and
d molecula
ar mass w
was determ
mined
using puriified proteins with known moleccular mass
s (Figure 2).
2
MW
W (Da)
LogMW
Elution
n volume (m
ml)
•
Carbonic anh
hydrase
29000 4.46
•
Alb
bumin
66000 4.82
22.56
•
Am
mylase
200000
5.30
20.95
•
Apo
oferritin
443000
5.65
19.78
•
Thrryoglobulin
n
•
Blu
ue dextran 2000000
669
9000
6.30
5.83
24.4
44
17.7
7
11.77
Figura 2: Molecu
ular weight calibration
n curve.
stical analy
ysis
3.4 Statis
Statistical analysis was
w condu
ucted by S
Student’s t-test or 1-w
way ANOV
VA followe
ed by
Bonferron
ni’s multiple
e comparis
son test. T
Total, b-, mm and s-GGT, as weell as b-GG
GT/sGGT ratio
o triglycerid
de and BM
MI values w
were ln-transformed to reduce the distribution
260
Material and Methods
skewness. f-GGT fraction data were not ln-transformed because they were normally
distributed (16).
Bivariate linear correlations between biological variables and fractional GGT activity
were presented as Spearman correlation coefficient.
Receiver Operating Characteristics curve analysis (ROC) was performed to establish
the diagnostic power of total and fractional GGT and of the b-GGT/s-GGT ratio
values. ROC analysis and the comparison between ROC curves were performed with
MedCalc 11.5 analysis software.
3.5 Patient selection for immunohistochemical analysis.
Indirect immunohistochemistry analysis was performed to localize GGT protein within
the hepatic tissue samples obtained from explanted livers. Depending on the
etiology, patients were set into 4 equal groups (cirrhosis with HCC, viral cirrhosis,
metabolic cirrhosis and biliary cirrhosis). Each group consists of ten patients, five
having the highest total plasma GGT levels and five having the lowest ones. The aim
is to check if different plasma GGT levels correspond with different tissue
expressions of GGT. Hepatic tissues selected for analysis were derived from the
largest tumor noduIe in cases of HCC while for all other cases of cirrhosis from the
right hepatic lobe B4. The tissue portions for Tissue Microarray were obtained from
the corresponding blocks containing paraffin-embedded tissue preserved in the
department of Anatomical Pathology of the University Hospital of Pisa.
3.6 Tissue Microarray Technique (TMA)
Tissue microarrays (TMAs) consist of paraffin blocks in which up to 1000 separate
tissue cores are assembled in array fashion making possible multiplex histological
analysis. The instrument consists of two hollow needles of 1.5 mm diameter each,
the first needle is used to make holes in the virgin paraffin block in which tissue cores
will be thereafter inserted by the second needle. As control, five spots of histological
sections from a single block containing healthy hepatic tissue were included. 20
sections from each block of TMA were collected for histological staining.
3.7 Immunohistochemical analysis
Localization of GGT protein GGT was performed by automated indirect
immunohistochemical analysis, using a polyclonal antibody directed against the C261
Material and Methods
terminal 20 amino acids of GGT heavy chain (Hanigan and Frierson, 1996). Prior to
TMA analysis, the appropriate concentrations of the primary antibody were
determined, dilutions 1:2000 was found to be optimal for antiserum, and 1:50 for IgG
purified from the antiserum, incubated for 44 min. at room temperature. The antigenantibody reaction was detected using a secondary antibody conjugated with
peroxidase and 3,3'-diaminobenzidine as a substrate. At the end of the
immunohistochemistry, TMA was observed by optical microscope connected to a
digital camera for images acquisition. The software MetaAnalisys ^ B made possible
the quantification of GGT protein found within the entire histologic section and
specifically in bile canaliculi and the endothelium of vessels.
3.8 Collection of samples of primary human bile and analysis of the fractions.
Bile samples were collected daily from hospitalized patients post operatively through
the Kehr’s T - tube inserted in the common bile duct during liver allograft
implantation. Patients were hospitalized by the “Unione Operativa di Chirurgia
Epatica e del Trapianto di Fegato, del Dipartimento di Oncologia, dei Trapianti e delle
Nuove Tecnologie, dell’Azienda Ospedaliera Universitaria Pisana”.
Primary human bile samples were obtained from patients undergoing liver
transplantation at the Department of Oncology, Transplants and New Technologies in
Medicine, University of Pisa and unpretentious. Bile samples were collected daily
from the drain placed at the level of the common bile duct.
GGT fractions present in bile were analyzed using the same method and the same
reaction conditions described for the plasma. In order to decrease the viscosity of the
samples and the concentration of bile pigments that have non-specific interactions
with the matrix of the chromatographic column, the sample is diluted 10 times with
running buffer, 20 μl of diluted sample were injected. To obtain the activity in the
original sample, values calculated from the chromatogram were multiplied by 10.
3.9 Half-life activity of bile GGT
To evaluate the best storage conditions, a sample of hepatic bile was divided into two
aliquots of 200μl one of these was placed at -20 ºC and the other at 4 ºC and after 24
h, we analyzed the elution profile of the fractions of GGT by gel filtration.
262
Material and Methods
To investigate how long it was possible to keep the bile at 4 º C on another sample of
human bile, with total activity of GGT in 45.44 U/L was divided into 10 aliquots of 200
μl each, stored at 4 ºC. The profile of the fractions of GGT was analyzed every day
for a week, then after 15, 24 and 30 days.
3.10 Activation of papain
In order to activate papain enzymatic activity, pre-incubation period of 3 min. at room
temperature with cysteine is required. For this purpose, 28μl of enzyme suspension
(corresponding to 0.8 mg of papain) were mixed with 22 μl of cysteine acidic solution
(Cys-HCl, 57 mM, final concentration in the mixture: 25 mM).
3.11 Treatment with papain and bile deoxycholic acid
Human bile sample with total GGT activity of 127.5 U/L were treated with papain and
deoxycholic acid.
Bile samples were treated using 1 mg of papain to about 20 mg of protein. First,
papain treatment was performed by mixing 100 μl of bile with 25 μl of activated
papain (containing 0.8 mg of papain) for an incubation period of 24h at room
temperature in vivacious agitation. Second, bile samples treatment with detergents
and papain was performed by adding 10 μl of sodium-desoxycholate (DOC, 10% w/v)
to 100 μl of each sample then incubated for 1h, at room temperature, in vivacious
stirring. 25 μl of activated papain were then added and samples were left in vivacious
stirring for 24h at room temperature. The same bile sample was used for each test
and for each treated sample the respective control, consisting of 100 uL of bile and
Dulbecco's PBS buffer in place of papain and deoxycholic acid was prepared.
263
Material and Methods
264
Results and discussion
4. RESULTS AND DISCUSSION
265 Results and discussion
266 Results and discussion
267 Results and discussion
268 Results and discussion
269 Results and discussion
270 Results and discussion
4.2 CIRRHOTIC PATIENT EVALUATION – PRETRANSPLANT.
Patients’ characteristics
Clinical and biochemical baseline characteristic of the study population and of 200
healthy controls, used as a reference population, are presented in Table 1. As
expected, cirrhotic patients showed higher serum aspartate aminotransferase (AST),
alanine aminotransferase (ALT), alkaline phosphatase (ALP), bilirubin, lactate
dehydrogenase (LDH), international normalized ratio (INR), triglycerides and lower
albumin, cholesterol, platelet counts as compared to healthy controls. Total plasma
GGT, as well as all its fractions displayed higher values in the cirrhotic patients; s-GGT
showed the largest increase, and was the most represented fraction in the cirrhotic
patients. The b/s ratio was significantly lower in patients with cirrhosis as compared to
healthy controls. In both groups total plasma GGT, as well as all fractions and the b/s
ratio, showed a right-skewed distribution (P < 0.0001). Out of 264 cirrhotic patients, 113
(9 females) showed total GGT values [median (25th – 75th percentile): 35.6 (25.8 – 45.7)
U/L; Table 2] within the reference range (16) [males: total GGT < 60.5 U/L, females: <
30.9].
Receiving Operative Curve analysis of the GGT fractions
When comparing healthy subjects with patients with cirrhosis, ROC analysis showed
that, among all GGT fractions, s-GGT had the highest specificity and sensitivity for liver
cirrhosis (AUC, 95% C.I.: 0.924, 0.897-0.947), but the ratio b/s showed an AUC (0.951,
0.927-0.969) significantly higher than s-GGT (P=0.046) and total GGT itself (0.900,
0.869-0.925; P=0.010) (Table 3, Figure 1A). Unexpectedly, the diagnostic value of the
b/s ratio was independent of the absolute values of total GGT: in the subset of 113
patients with total GGT values within the reference range the AUC of the b/s ratio
(0.940, 0.900-0.968) was substantially the same than in the whole population, while that
of all other fractions dropped to much lower (b-, m-, and s-GGT) or insignificant values
(f-GGT); (Table 3, Figure 1B).
Fractional GGT activity (U/L) in cirrhotic patients
In order to understand the reasons of the different behaviour of the GGT fractions in
liver cirrhosis, the three sub-cohorts of patients with VC, MC, or HC were compared.
Figure 2 shows the calculated elution profiles of each of the three subgroups,
271 Results and discussion
corresponding to the 25th, 50th and 75th percentile of the distribution, and the actual
chromatographic profile of a representative individual patient of each group. In all cases,
as compared with healthy controls, the s-GGT elution profile showed a significantly
broader profile, that was found to correspond to the presence of two Gaussian
components, that were mathematically defined and named s1-GGT and s2-GGT, The
double profile of s-GGT was not seen in healthy controls (not shown). Differences
emerged among groups as concerns the values of total GGT as well as of the individual
fractions (Table 4): in the VC group, b-GGT values and the b/s ratio values were lower
than in the metabolic cirrhosis group, while HC showed higher values of total, b-, m-,
and s1-GGT as compared to the VC group, but not to the MC group.
Noticeably, in all the three groups (Table 4) the values of b/s ratio were below the value
of controls (see Table 1), thus making this value independent not only from the absolute
values of total GGT, but also from the aetiology of the cirrhosis and the presence of liver
cancer.
Correlations of total and fractional GGT with biological variables of cirrhotic
patients
To see whether the variations of the GGT fractions may reflect different aspects of the
liver cirrhosis, we performed a Spearman correlation analysis of the GGT fractions with
the main laboratory biomarkers used to describe the progression of liver disease. While
triglycerides and total cholesterol displayed a positive correlation with all the GGT
fractions, for other biological variables a specific pattern of associations emerged (Table
5); in particular, b-GGT showed a positive association with albumin and fibrinogen, and
negative association with INR, thus behaving as a positive index of liver function, but no
or negative correlation with AST, and ALT, ALP, bilirubin and LDH, the biomarkers of
liver cell damage and of cholestasis. In fact b-GGT showed a negative correlation with
the MELD score. In line with this, only the b-GGT showed a positive association with the
platelet count, which reflects the progression of portal hypertension and hypertrophy of
the spleen. As opposed to b-GGT, the s2-GGT fraction displayed a statistically
significant positive correlation with AST, ALT, LDH, ALP, and bilirubin, but negative
association with serum albumin and no association with INR and fibrinogen, thus
apparently reflecting only hepatocellular damage, this observation is supported by the
positive correlation with the MELD score. Noticeably, the two fractions s1-GGT and s2GGT showed a quite independent behaviour (e.g. Albumin, INR, and fibrinogen, showed
272 Results and discussion
a significant correlation only with one of the s-GGT fractions), and sometimes opposite
behaviour (bilirubin and LDH showed opposite correlations with s1 and s2-GGT); mGGT and f-GGT displayed also differential association with biological variables, the fGGT mostly corresponding to s2-GGT.
Immunolocalization of GGT in liver biopsies
Immunohistochemical assay, which has the advantage of localizing GGT within tissues,
was performed on liver sections from explanted native livers of patients who underwent
orthotopic liver transplantation. The analysis was possible in the following cases:
- 10 cases diagnosed as hepatocellular carcinoma (HCC) divided equally into two
groups according to the total serum GGT level of each case as follow: 5 cases with total
serum GGT level >230 U/L each, and 5 cases each with total GGT serum level ranging
between 18 and 30 U/L
- 8 cases diagnosed as metabolic cirrhosis, divided into two equal groups; the first
group consists of 4 cases, with total GGT serum level >90 U/L each, while the second
group of 4 cases with total serum GGT level ranging between 29 and 44 U/L
- 10 cases diagnosed as viral cirrhosis, divided equally into two groups as follow: 5 with
total serum GGT> 100 U/L each, and the 5 other cases each with total serum GGT level
between 19 and 34 U/L
- 3 cases of biliary cirrhosis among which 1 case with total serum GGT of 180 U/L, and
3 other cases with total serum GGT ranging between 22 and 50 U/L.
Tables 6 shows the sequential order by which the liver sections were mounted on the
two Tissue Microarray (TMA) made; in reference to a system of Cartesian axes relevant
to the recognition of each individual case. Figure 3 shows the TMA-2 after
immunohistochemical staining.
As a control, the immunolocalization of GGT was evaluated on 5 sections obtained from
a liver biopsy of a healthy individual subjected to liver biopsy having total serum GGT of
15 U/L. In these control histological sections (Figure 4), GGT is found in 6-11% of the
total tissue area and predominantly localized in the bile canaliculi and bile ducts, but
absent at cytosolic level. Immunohistochemical analysis of liver biopsies of patients with
biliary cirrhosis revealed an abundant distribution of GGT all over the entire tissue
section in an area representing approximately 36% of total tissue area. The enzyme is
273 Results and discussion
uniformly distributed and in abundance along the hepatocytes membrane delimiting bile
canaliculi, in bile ducts or within them, in blood vessels endothelium, and in abundance
in the cytosol (Figure 5A).
Similarly, on the liver section of patients with metabolic cirrhosis, the enzyme is
localized in the bile canaliculi membrane and in the cytosol (Figure 5B), and cover
about 30-38% of the total area of the section.
Immunolocalization of GGT on histological sections of viral liver cirrhosis: the area
covered by the enzyme is variable ranging between 19% and 33%. The enzyme is
localized at the bile canaliculi membrane, the endothelium of blood vessels, and
modestly found in the cytosol (Figure 5C).
In HCC liver biopsy, GGT is found in abundance and distributed all over the entire
surface of the histological section covering area ranging between 20 and 28% of the
total tissue area. Importantly, this come in agreement with the previously reported data
that the distribution and concentration of GGT show many variations from that found in
normal tissues, and confirms that in tumor tissues an over expression of the gene
GGT1, and an increase in GGT synthesis takes place (Hanigan, 1999). GGT is
predominantly confined to the membranes; the interface of the hepatocytes membranes
forming bile canaliculi, and partly to blood vessels endothelium of (Figure 5D).
The immunohistochemistry is able to localize GGT activity within the tissues, but in nonquantitave manner. Therefore, quantification has been made by the mean of an
automated data acquisition system able to calculate the percentage of the area of the
section occupied by the protein of interest making the determination of the total protein
GGT amount present in the section possible. In addition, through a procedure of
calculation associated with the manual system of data acquisition, called "counts to the
touch" (Figure 6), the amount of GGT present at the bile canaliculi and vessels
endothelium was evaluated. This has permitted to achieve a comparative assessment
of expression levels in the two given districts, expressed with the ratio of the signal
associated to the bile canaliculi and the endothelium.
Table 7 shows the data for each section on which the quantification was performed.
The data obtained from both immunolocalization and quantification of GGT in liver
tissue suggest that there is not a direct relationship between tissue and circulating GGT
enzyme levels (Figure 7). The increased tissue expression of GGT is associated with
274 Results and discussion
the presence of the protein cytosol, on the membranes of bile canaliculi and on the
endothelium of hepatic sinusoids. This particular location could be a result of increased
expression of the enzyme with a consequent increase in the amount of GGT at the level
of the endoplasmic reticulum and Golgi apparatus.
275 Results and discussion
Table 1. Baseline characteristics of healthy subjects (n=200) and patients affected by
end-stage liver cirrhosis (n=264).
Males, n (%)
Age, years
2
BMI*, kg/m
SBP, mmHg
DBP, mmHg
Heart rate, bpm
Glucose, mg/dL
Creatinine, mg/dL
eGFR, ml/min
Total cholesterol, mg/dL
HDL cholesterol, mg/dL
LDL cholesterol, mg/dL
Triglycerides*, mg/dL
AST, U/L
ALT, U/L
Total bilirubin, mg/dL
ALP, U/L
LDH, U/L
Albumin, g/dL
Hemoglobin, g/dL
9
Leukocytes, x10 L
9
Platelets, 10 /L
INR
Fibrinogen, mg/dl
total GGT*, U/L
b-GGT*, U/L
m-GGT*, U/L
s-GGT*, U/L
f-GGT, U/L
b/s ratio*
Healthy Subjects
Cirrhosis
100 (50%)
41.5
35.0 - 50.0
24.3
22.2 - 26.6
120.0
110.0 - 120.0
80.0
70.0 - 80.0
70.0
64.0 - 75.5
93.0
86.0 - 100.0
0.9
0.8 - 1.0
104.5
91.5 - 119.2
187.5
160.8 - 209.3
51.0
43.0 - 62.0
116.5
97.0 - 135.0
73.0
50.0 - 106.5
17.0
14.0 - 21.0
17.0
12.0 - 24.5
0.7
0.6 - 0.9
n.a.
n.a.
n.a.
14.7
13.6 - 15.8
6.2
5.4 - 7.6
249.0
212.0 - 283.0
n.a.
n.a.
18.8
13.8 - 28.7
1.6
0.9 - 3.0
0.6
0.4 - 1.1
5.6
3.2 - 10.2
10.4
8.6 - 13.4
0.28
0.20 - 0.40
215 (81%)
54.5
50.0 - 60.0
24.8
23.1 - 26.8
120.0 110.0 - 130.0
70.0
70.0 - 80.0
68.0
60.0 - 75.0
101.0 93.0 - 116.0
0.8
0.7 - 0.9
107.5 89.5 - 130.7
139.5 118.8 - 166.0
49.0
37.0 - 61.0
75.0
56.8 - 96.0
83.5
69.5 - 112.0
70.0
43.8 - 106.3
50.0
33.0 - 81.0
1.6
0.9 - 2.9
129.5 94.8 - 182.0
198.5 166.0 - 244.8
3.9
3.6 - 4.3
12.9
11.9 - 14.5
4.3
3.6 - 5.7
75.5
58.0 - 111.5
1.4
1.2 - 1.5
217.0 183.8 - 270.3
61.1
40.3 - 113.5
2.6
1.4 - 5.3
3.1
1.5 - 6.7
36.6
21.0 - 81.4
16.2
13.6 - 20.4
0.06
0.04 - 0.10
P
< 0.0001
n.s.
n.s.
0.0003
n.s.
< 0.0001
n.s.
n.s.
< 0.0001
0.0306
< 0.0001
0.0008
< 0.0001
< 0.0001
< 0.0001
< 0.0001
< 0.0001
< 0.0001
< 0.0001
< 0.0001
< 0.0001
< 0.0001
< 0.0001
< 0.0001
Data are reported as median, 25th – 75th percentile. ALP: Alkaline phosphatase; ALT:
alanine aminotransferase; AST: aspartate aminotransferase; BMI: body mass index;
DBP: diastolic blood pressure; eGFR: estimated glomerular filtration rate; INR:
international normalized ratio; GGT: gamma-glutamyltransferase; LDH: lactate
dehydrogenase; SBP: systolic blood pressure. eGFR has been estimated by the
Cockcroft-Gault formula; LDL cholesterol has been estimated by the Friedewald
formula. ALP, LDH, albumin, INR and fibrinogen were not assessed in healthy subjects
(blood donors). *Student’s t test performed on ln-transformed data. n.a.: not available.
n.s.: not significant.
276 Results and discussion
Table 2. Total and fractional GGT activity (U/L) in healthy subjects (n=109; 9 women)
and patients affected by hepatic cirrhosis and total GGT values within the reference
range (n=113; 9 women).
Healthy Subjects
Hepatic cirrhosis
P
Males/Females
100/9
104/9
Age, years
44.0 38.0 – 50.0
54.0 49.0-60.0
<0.0001
Total GGT*
25.4 19.0 – 37.0
35.6 25.8 – 45.7
<0.0001
b-GGT*
2.5
1.5 – 4.9
1.2
0.7 – 2.0
<0.0001
m-GGT*
1.0
0.6 – 1.6
1.2
0.8 – 1.9
0.0124
s-GGT*
9.2
5.1 – 14.2
18.1 11.8 – 26.6
<0.0001
f-GGT
12.9 10.2 – 15.8
13.2 10.5 – 15.7
n.s.
b/s*
0.29 0.20 – 0.40
0.06 0.04 –0.10
<0.0001
Data are reported as median, 25th – 75th percentile. Total GGT upper reference limit:
60.5 U/L men; 30.9 U/L women. *Student’s t test performed on ln-transformed data.
n.s.: not significant.
277 Results and discussion
Table 3. Diagnostic power of total, fractional GGT and of the b-GGT/s-GGT ratio in
differentiating patients affected by hepatic cirrhosis from healthy subjects (HS).
HS (n=200) vs.
Cirrhosis (n=264)
P value
vs. b/s
ratio
Tot.GGT
within ref. range:
HS (n=109) vs.
Cirrhosis (n=113)
P value
vs. b/s
ratio
Total GGT
0.900
0.869 – 0.925
0.010
0.661
0.595 – 0.723
< 0.0001
b-GGT
0.616
0.570 – 0.661
< 0.0001
0.771
0.711 – 0.825
< 0.0001
m-GGT
0.868
0.833 – 0.897
< 0.0001
0.593
0.525 – 0.658
< 0.0001
s-GGT
0.924
0.897 – 0.947
0.0460
0.751
0.688 – 0.806
< 0.0001
f-GGT
0.805
0.766 – 0.840
0.0001
0.519
0.451 – 0.586
< 0.0001
b/s ratio
0.951
0.927 – 0.969
0.940
0.900 – 0.968
Data are presented as ROC-AUC (95% CI). ROC-AUC: area under receiver operating
characteristic curve.
278 Results and discussion
Figure 1. ROC analysis of total and fractional GGT in differentiating patients affected by
hepatic cirrhosis from healthy subjects: A) whole population; B) total GGT within
reference range. b/s: black solid line; b-GGT: dotted line; s-GGT: dashed line; total
GGT: gray solid line.
279 Results and discussion
Figure 2. Calculated elution profile (A-C) of fractional GGT activity corresponding to the
25th (dotted line), 50th (solid line) and 75th (dashed line) percentile and a representative
chromatogram (D-F) of patients affected by viral hepatic cirrhosis (A, D) or metabolic
hepatic cirrhosis (B, E) or hepatic cirrhosis and hepatocellular carcinoma (C, F).
Fractional GGT analysis was performed on plasma-EDTA samples by high performance
gel-filtration chromatography, GGT activity was specifically detected by an on-line postcolumn reaction with a fluorescent substrate.
280 Results and discussion
Table 4. Total and fractional GGT activity (U/L) in patients affected by viral hepatic
cirrhosis (VC), metabolic hepatic cirrhosis (MC) or hepatic cirrhosis and hepatocellular
carcinoma (HC).
VC (n=96)
MC (n=39)
HC (n=129)
P
VC vs.
MC
P
P
VC vs. MC vs.
HC
HC
Tot GGT*
53.0
34.4-91.2
54.8
41.7-142.9
68.7 42.6-128.0
n.s.
< 0.01
n.s.
b-GGT*
1.8
0.9-3.6
3.8
1.5-5.5
2.8 1.6-5.7
< 0.05
< 0.01
n.s.
m-GGT*
2.9
1.0-4.7
2.5
1.4-9.7
3.4 1.5-7.6
n.s.
< 0.05
n.s.
s1-GGT*
24.2
13.9-58.9
27.5
14.6-77.8
40.1 18.6-88.2
n.s.
< 0.05
n.s.
s2-GGT*
4.7
3.0-7.1
4.8
3.0-9.9
5.2 2.8-7.5
n.s.
n.s.
n.s.
f-GGT
15.2
11.6-18.3
18.0
13.3-22.6
16.7 13.6-20.6
n.s.
n.s.
n.s.
b/s*
0.05
0.04-0.08
0.09
0.06-0.13
0.06 0.04-0.11
< 0.01
n.s.
n.s.
Data are reported as median, 25th – 75th percentile. HCC: hepatocarcinoma. Statistical
analysis: 1-way ANOVA followed by Bonferroni’s multiple comparison test; *statistical
analysis performed on ln-transformed data. n.s.: not significant.
281 Results and discussion
Table 5. Linear correlation analysis between biological variables and fractional GGT
activity.
Variables
Tot.GGT
b-GGT
m-GGT
s1-GGT
s2-GGT
f-GGT
MELD score
-0.216†
-0.283‡
n.s.
-0.300‡
0.166§
n.s.
Total cholesterol, mg/dL
0.456‡
0.508‡
0.480‡
0.473‡
0.166§
0.277‡
Triglycerides, mg/dL
0.337‡
0.351‡
0.420‡
0.301‡
0.214†
0.289‡
Platelets, 109/L
n.s.
0.180§
n.s.
n.s.
n.s.
n.s.
Total Bilirubin, mg/dL
-0.181
‡
-0.268
‡
n.s.
0.175
§
n.s.
0.155*
0.415
§
0.359‡
-0.264
ALP, U/L
0.232
†
n.s.
0.315
AST, U/L
0.157*
n.s.
0.199§
n.s.
0.413‡
0.283‡
ALT, U/L
0.276‡
n.s.
0.255‡
0.249‡
0.409‡
0.320‡
LDH, U/L
n.s.
-0.181§
n.s.
-0.148*
0.200‡
0.183§
Albumin, g/dL
n.s.
0.260‡
n.s.
n.s.
-0.240†
n.s.
n.s.
n.s.
n.s.
0.163§
INR
Fibrinogen, mg/dL
-0.269
0.264
‡
‡
-0.361
0.438
‡
‡
‡
‡
-0.172
0.228
§
†
-0.327
0.286
‡
‡
Data are reported as Spearman correlation coefficients. ALP: alkaline phosphatase;
ALT: alanine aminotransferase; AST: aspartate aminotransferase; INR: international
normalized ratio; LDH: lactate dehydrogenase. Statistical significance levels: *P < 0.05;
§
P < 0.01; †P < 0.001; ‡P < 0.0001. n.s.: not significant.
282 Results and discussion
Table 6. Immunolocalization of GGT protein in liver biopsies obtained from explanted
native livers of cirrhotich patients who underwent orthotopic liver transplantation. The
table reflects the order of the spots on the slide of the tissue microarray TMA-1 and
TMA-2. For each case the values of plasma total and fractional GGT (U/L) are show.
TMA-1
D1
HCV Cirr/HCC
D2
HCV Cirr/HCC
D3
Alcohol Cirr/HCC
D4
Alcohol Cirr/HCC
D5
Alcohol Cirr/HCC
Tot GGT 435.69
b-GGT 13.53
m-GGT 25.46
s1-GGT 360.49
s2-GGT 0.16
f-GGT 29.86
Tot GGT 349.10
b-GGT 28.69
m-GGT 32.76
s1-GGT 244.36
s2-GGT 15.01
f-GGT 24.29
Tot GGT 18.07
b-GGT 0.64
m-GGT 0.63
s1-GGT 1.35
s2-GGT 1.89
f-GGT 13.49
Tot GGT 18.07
b-GGT 0.64
m-GGT 0.63
s1-GGT 1.35
s2-GGT 1.89
f-GGT 13.49
.
Tot GGT 18.07
b-GGT 0.64
m-GGT 0.63
s1-GGT 1.35
s2-GGT 1.89
f-GGT 13.49
C1
Biliary Cirr
C2
Biliary Cirr
C3
Biliary Cirr
C4
Biliary Cirr
Tot GGT 184.39
b-GGT 6.98
m-GGT 19.29
s1-GGT 119.92
s2-GGT 18.80
f-GGT 18.03
Tot GGT 38.61
b-GGT 1.27
m-GGT 1.38
s1-GGT 23.79
s2-GGT 3.00
f-GGT 8.91
Tot GGT 50.38
b-GGT1.25
m-GGT 3.26
s1-GGT 15.04
s2-GGT 5.71
f-GGT 24.80
Tot GGT 22.97
b-GGT 3.49
m-GGT 0.93
s1-GGT 10.47
s2-GGT 0.82
f-GGT 6.87
B1
Alcohol Cirr
B2
Alcohol Cirr
B3
Alcohol Cirr
B4
Metabolic Cirr
Tot GGT 263.26
b-GGT 5.33
m-GGT 10.32
s1-GGT 189.58
s2-GGT 16.42
f-GGT 41.67
Tot GGT 425.95
b-GGT26.87
m-GGT 42.75
s1-GGT 272.72
s2-GGT52.23
f-GGT 30.67
Tot GGT 29.53
b-GGT 0.37
m-GGT 1.01
s1-GGT 7.33
s2-GGT 3.47
f-GGT 17.18
Tot GGT 41.11
b-GGT 1.78
m-GGT 1.82
s1-GGT 13.26
s2-GGT 5.44
f-GGT 18.54
A1
HCV Cirr
A2
HCV Cirr
A3
HCV Cirr
A4
HCV Cirr
A5
HCV Cirr
Tot GGT 19.03
b-GGT 0.36
m-GGT0.47
s1-GGT 7.32
s2-GGT 1.75
f-GGT 9.11
Tot GGT 173.06
b-GGT 8.80
m-GGT 10.34
s1-GGT 127.12
s2-GGT 9.25
f-GGT 16.15
Tot GGT 157.81
b-GGT 6.89
m-GGT 9.05
s1-GGT 99.24
s2-GGT 8.44
f-GGT 34.05
Tot GGT 22.14
b-GGT 0.35
m-GGT 0.53
s1-GGT 6.42
s2-GGT 2.76
f-GGT 12.07
Tot GGT 22.14
b-GGT0.35
m-GGT 0.53
s1-GGT 6.42
s2-GGT 2.76
f-GGT 12.07
A4: B6 right lobule; A5: B2 right lobule; D3: A9, III segment nodule; D4: A12, III segment nodule; D5: A10,
III segment nodule:
Cirr: cirrhosis
283 Results and discussion
TMA-­‐2 D1
Control
D2
Control
D3
Control
D4
Control
D5
Control
Tot GGT 15
Tot GGT 15
Tot GGT 15
Tot GGT 15
Tot GGT 15
C1
Metabolic Cirr
C2
Metabolic Cirr
C3
Metabolic Cirr
C4
Metabolic Cirr
Tot GGT 44.06
b-GGT 3.18
m-GGT 1.29
s1-GGT 26.20
s2-GGT 2.95
f-GGT 10.07
Tot GGT 44.45
b-GGT 2.44
m-GGT 1.98
s1-GGT 22.75
s2-GGT 3.49
f-GGT 13.30
Tot GGT 161.44
b-GGT 18.17
m-GGT 10.74
s1-GGT 108.27
s2-GGT 5.78
f-GGT 15.59
Tot GGT 90.73
b-GGT 4.07
m-GGT 6.76
s1-GGT 55.99
s2-GGT 10.94
f-GGT 13.00
B1
HCV Cirr
B2
HCV Cirr
B3
HCV Cirr
B4
HCV Cirr
B5
HBV Cirr
Tot GGT 31.12
b-GGT 0.76
m-GGT 0.90
s1-GGT 12.22
s2-GGT 2.98
f-GGT 14.27
Tot GGT 34.65
b-GGT 0.80
m-GGT 0.87
s1-GGT 19.40
s2-GGT 3.00
f-GGT 10.49
Tot GGT 103.86
b-GGT 3.14
m-GGT 6.00
s1-GGT 66.67
s2-GGT 8.78
f-GGT 18.64
Tot GGT 103.86
b-GGT 3.14
m-GGT 6.00
s1-GGT 66.67
s2-GGT 8.78
f-GGT 18.64
Tot GGT 109.42
b-GGT 3.64
m-GGT 5.19
s1-GGT 79.48
s2-GGT 6.02
f-GGT 13.93
A1
HBV Cirr/HCC
A2
HBV Cirr/HCC
A3
HCV Cirr/HCC
A4
HCV Cirr/HCC
Tot GGT 24.29
b-GGT 2.71
m-GGT 0.88
s1-GGT 11.72
s2-GGT 0.03
f-GGT 8.78
Tot GGT 27.48
b-GGT 1.61
m-GGT 3.30
s1-GGT 2.94
s2-GGT 4.99
f-GGT 14.53
Tot GGT 343.30
b-GGT 31.23
m-GGT 37.40
s1-GGT 223.39
s2-GGT 15.63
f-GGT 30.59
Tot GGT 231.31
b-GGT 9.24
m-GGT 18.47
s1-GGT 159.32
s2-GGT 17.88
f-GGT 25.62
D1-D5: B4, right lobe; B3: A4 nodule; B4: small cell dysplastic nodule.
Cirr: cirrhosis.
284 Results and discussion
Figure 3. Reconstruction of the TMA-2. The images of the spots were obtained after
immunohistochemical staining for GGT
285 Results and discussion
Figure 4. Immunolocalization of the protein GGT on a liver biopsy of control
Figure 5. Immunolocalization of protein GGT in liver biopsies obtained from a patient
suffering from: A) Biliary Cirrhosis (total plasma GGT 50 U/L, GGT area / total area
section: 36.9%; TMA1-section C3), B) Cirrhosis metabolic (plasma GGT total 90.7 U/L,
GGT area / total area section: 36.4%; TAM2 section C4), C) viral cirrhosis (total plasma
GGT 109 U/L, GGT area / total area section: 34% section; TMA2-B5 ) D) cirrhosis with
HCC (total plasma GGT 27.5 U/L, GGT area / total area section: 18.7%; TMA2-section
A2 section).
286 Results and discussion
Figure 6. Immunolocalization of GGT on a section of liver tissue (nodule HCC).
Assessment of the distribution and endothelial canalicular using the manual procedure
of "counts touch" associated with the computer system metaanalysis^B. The areas in
blue indicating the presence of GGT at the level of bile canaliculi, the areas in red brown
correspond to the endothelium of blood vessels.
Metabolic Viral HCC Biliary 45 Tissue GGT (%) 40 35 30 25 20 15 10 5 0 0 50 100 150 200 250 300 350 400 Plasma total GGT (U/L) Figure 7. Comparison between plasma total GGT activity and the enzyme expressed in
liver tissue.
287 Results and discussion
Table 7. Quantification of the protein GGT in of liver biopsies through the computerized
system metaanalysis.
Mean ± SD
Total GGT (U/L)
15
GGTarea/tot area(%)
10.8 ± 0.3
Control
Control
Control
Control
Control
11.32
12.15
14.74
8.60
6.97
Metab.Cirr
Metab.Cirr
Metab.Cirr
Metab.Cirr
Total GGT (U/L)
85.2 ± 55.4
44.06
44.45
90.73
161.44
GGTarea/tot area(%)
31.1 ± 9.3
18.17
30.72
36.42
38.98
GGTcan/GGTendot
1.2 ± 0.5
1.18
1.76
0.58
1.26
Biliary canaliculi (n)
5295.8 ±1560.1
5575
5575
3147
6886
Endothelial Vessel (n)
3990.3 ±
17797.8
1774
3326
5411
5450
HCV Cirr
HCV Cirr
HCV Cirr*
HCV Cirr **
HBV Cirr
Total GGT (U/L)
76.6 ± 40.0
31.12
34.65
103.9
103.8
109.42
GGTarea/tot area(%)
22.5 ± 8.7
21.48
19.91
26.42
10.47
33.98
GGTcan/GGTendot
1.0 ± 0.4
0.46
0.80
1.27
1.04
1.56
Biliary canaliculi (n)
1727.9 ± 1036.0
1676
1088
1519
868
3488
Endothelial Vessel (n)
1858.2 ± 1109.9
3624
1353
1253
833
2228
HBV
Cirr/HCC
HCV
Cirr/HCC
HCV
Cirr/HCC
HCV
Cirr/HCC
Total GGT (U/L)
156.6 ± 157.7
24.29
27.48
231.31
343.30
GGTarea/tot area(%)
21.2 ± 5.2
16.2
18.67
21.46
28.32
GGTcan/GGTendot
1.3 ± 0.3
1.2
1.29
1.77
0.98
Biliary canaliculi (n)
1949.0 ± 1268.2
540
1763
3622
1871
Endothelial Vessel (n)
1437.3 ± 721.1
450
1357
2039
1903
Biliary Cirr
Biliary Cirr
Biliary Cirr
Total GGT (U/L)
85.9 ± 86.4
22.97
50.38
184.39
GGTarea/tot area(%)
21.2 ± 5.2
12.47
36.89
14.35
GGTcan/GGTendot
1.3 ± 0.3
1.25
2.02
0.66
Biliary canaliculi (n)
1949.0 ± 1268.2
1220
7122
1667
Endothelial Vessel (n)
1437.3 ± 721.1
969
3510
2502
Control: B4 right lobe
Can.: canlicular; Cirr.: cirrhosis; Endot.: endothelial.
*A4 nodule; **Small cell dysplastic nodule
288 Results and discussion
4.3 CHARACTERIZATION OF GGT FRACTIONS IN PRIMARY HUMAN BILE
Fractions of GGT in human bile liver
In the same laboratory where the experiments were conducted, characterization of the
sensitivity of plasma GGT fractions to deoxycholic acid (DOC, 1% final concentration; a
secondary bile acid known to possess detergent properties), as well as to papain were
performed. Papain is a cysteine protease acting on GGT protein resulting in the release
of a soluble enzymatically active form, free from 30 amino acids of the N-terminal of the
GGT heavy chain. Results of this study demostrated that two fractions namely m-GGT
and s-GGT- consist of micelles of bile acids and GGT, while b-GGT fraction would be
made up of membrane microvesicles and f-GGT from soluble protein [Fornaciari et al.,
2012 ]. Relying upon the fact that the liver is the primary source of circulating GGT; I
decided to characterize the elution profile of GGT fractions in human bile. Bile samples
were collected from hospitalized patients post operatively through the Kehr’s T - tube
inserted in the common bile duct during liver allograft implantation. Patients were
hospitalized by the “Unione Operativa di Chirurgia Epatica e del Trapianto di Fegato, del
Dipartimento di Oncologia, dei Trapianti e delle Nuove Tecnologie, dell’Azienda
Ospedaliera Universitaria Pisana”. These samples were analyzed by gel filtration.
Unlike plasma, bile elution profile revealed the presence of only two peaks
corresponding to plasma b-GGT and f-GGT fractions (Figure 8A and 8B). The same
sample was also analyzed in the absence of GGT fluorescent substrate in order to
assess the existence of other complexes, if any in the bile samples that might emit
fluorescence at the same wavelengths used. The results confirmed that the observed
peaks were due to the presence of GGT (Figure 8A) in accordance and confirming
what had been previously demonstrated by Orlowski [1963], that GGT activity in human
bile is higher than that found in plasma (median: 112.5 U/L, 25th-75th percentile: 73.9183.1 U/L, min-max from 55.8 to 218.8 U/L, n= 7). In contrast to data obtained from the
DOC treated plasma, s-GGT fraction of the enzyme is absent in bile samples and those
found were mainly (90%) in a form corresponding to the molecular weight of plasma bGGT fraction and to a lesser extent in form of f-GGT; this result confirms as well what
had already been observed by Wenham [1978] that in the bile there are two forms of
GGT; one of a high molecular weight (b-GGT) and the other corresponding to the free
enzyme (f-GGT) obtained by treatment with papain and detergents.
289 Results and discussion
GGT half-life in the bile
Bile preservation and enzyme stability were studied on order to know how samples can
be conserved and for how long. First, bile sample (GGT 45.44 U/L) was equally divided
into two aliquots of 200µl each, one has been placed at - 20 º C and the other at 4 º C,
and both were analyzed after 24 h to evaluate the GGT fractions profile. The
comparison of the elution profiles (Figure 9) reveals a great reduction of GGT activity
bile stored at -20 ºC in contrast to that stored at 4 ºC due to its precipitation with the
pellets formed in the tube. Therefore, bile is better preserved and stored at 4 C. Second,
the same sample of bile kept at 4 ºC was subdivided into 200µl aliquots and kept at 4 º
C. GGT fractions profile was analyzed daily for seven consecutive days, then on days
15 (T15), 24(T24) and 30 (T30). From the results (Figure 10; Table 8) it is obvious that
total GGT activity remains constant for the first seven days, then decreased from 42.92
U/L on day 8 (T8) to 30.95 U/L on (T15) till (T24), followed by further decrease from
32.46 U/L (T24) to 23.9 U/L (T30). It can therefore be concluded that b-and f-GGT
fractions follow the same trend of the total GGT, bile can be stored at 4 ºC for 7 days,
and total GGT value of total as well as its fractions remains constant for about a week.
Treatment with papain and deoxycholic acid
To understand the nature and characteristics of biliary complex corresponding to the
plasma b-GGT fraction, a sample of bile (GGT = 127.5 U/L) was treated with papain
and with Doc. The treatment with papain alone has been done to verify the eventual
presence of the membrane insertion peptide present in b-GGT fraction protein in bile.
Papain hydrolyses the N-terminal of the heavy chain responsible of the enzyme
anchorage to the membrane resulting in the release of an enzymatically active protein,
free from 30 amino acids of N-terminal in the solution [Tate and Meister, 1985; Tate et
al., 1988; Thioudellet et al., 1994]. The comparison of the elution profile of GGT
fractions between the papain treated and untreated bile (Figure 11A) demonstrates a
significant reduction of b-GGT peak in papain treated sample the peak b-GGT while
maintaining the same elution volume. In contrast, f-GGT peak increases greatly.
Calculation of the activity (Figure 11B) shows 50% reduction of b-GGT activity following
papain treatment and a higher than expected f-GGT activity (= f-GGT activity + 50%
activity b-GGT), this could be due to the partial inhibition of GGT enzymatic activity by
bile b-GGT components or papain may have favored the formation of complexes that
emit fluorescence at the wavelengths used eluting in correspondence to the f-GGT.
290 Results and discussion
Sample was then treated for 1 h at 37 º C with the DOC (w/v 1%, final concentration)
and then for 24h with papain. The elution profile of bile treated sample in comparison to
the untreated one (Figure 12A and B) shows the complete disappearance of b-GGT
and higher f-GGT fraction activity (Figure 12B). In conclusion, GGT present in bile bGGT fraction retains its membrane insertion peptide and is partially directly sensitive to
the action of papain, and complete sensitivity takes place only after DOC treatment.
Accordingly, we can assume that the part of b-GGT fraction insensitive to the direct
action of papain can be released into the bile associated with membrane vesicles such
as exosomes.
It has been reported that bile contains a high molecular weight GGT probably consisting
of GGT associated with membrane fragments [Wenham et al., 1978], De Broe [1975] as
well had shown that some bile enzymes are released associated with membrane
vesicles. Recent studies have reported that bile contains vesicles of similar density and
size to the exosomes, probably released from hepatocytes, which interact with the
primary cilia of cholangiocytes regulating intracellular mechanisms and proliferation
[Masyuk et al., 2010].
A part of this GGT could be extracted from exosomes or directly from bile canaliculi cell
membrane membrane from bile acids present in the bile and included in detergent
micelles that tend to aggregate with each other allowing the formation of
submicroscopic particles, called micelles, capable to host different lipid molecules,
otherwise insoluble in water, such as lecithin and cholesterol, in this way constituting a
high molecular weight GGT which could correspond to that part of b-GGT directly
sensitive to the action of papain.
291 Results and discussion
900
Fluorescence (mV)
810
A
720
630
540
450
360
270
180
90
0
10
12
14
16
18
20
22
24
26
22
24
26
Elution volume (ml)
30
27
B
Fluorescence (mV)
24
21
18
15
12
9
6
3
0
10
12
14
16
18
20
Elution volume (ml)
Figure 8. A) Elution profile of GGT fractions (dashed line) and autofluorescence
(continuous line) of primary human bile sample; B) elution profile of a plasma sample
from the same patient from which bile sample was taken.
292 Results and discussion
900
810
Fluorescence (mV)
720
630
540
450
360
270
180
90
0
10
12
14
16
18
20
22
24
26
Elution volume (ml)
Figure 9. Elution profile of GGT fractions of primary human bile sample (GGT = 44.45
U/L) stored 24h at 4 º C (dashed line) and -20 º C (continuous line).
293 Results and discussion
GGT activity (U/L)
120
b-­‐G GT
100
f-­‐G GT
80
total G GT
60
40
20
0
0
10
20
Days
30
40
Figure 10. Trend of GGT fractions (b-and f-GGT GGT) GGT and total sample of
primary human bile (GGT = 44.45U/L) over time from day 1 (T1) to day 30 (T30)
Table 8. Comparison between the activity of GGT fractions (b-and f-GGT GGT) and
total GGT of primary human bile sample (GGT = 44.45 U/L) from day 1 (T1) to day 30
(T30). 294 Day
b-GGT (U/L)
f-GGT (U/L)
GGT tot (U/L)
T1
32.39
13.05
45.44
T2
27.24
9.65
36.89
T4
29.17
8.85
38.02
T5
30.87
10.21
41.08
T6
32.67
10.39
43.06
T7
32.76
9.3
42.06
T8
29.34
13.58
42.92
T15
21.6
9.35
30.95
T24
24.32
8.14
32.46
T30
14.1
9.8
23.9
Results and discussion
350
A
315
Fluorescence (mV)
280
245
210
175
140
105
70
35
0
10
12
14
16
18
20
22
24
26
Elution volume (ml)
B
b-GGT
(U/L)
f-GGT
(U/L)
GGT tot
(U/L)
Bile
(control)
106.16
21.32
127.48
Bile
papain
62.14
109.08
171.22
Figure 11. A) Elution profile GGT fractions of a bile sample (GGT = 127.5 U/L) before
(continuous line) and after (dashed line) the treatment with papain 24h in ambient
temperature. B) Comparison between the activities of GGT fractions (b- and f-GGT) and
total GGT of bile sample before and after treatment with papain.
295 Results and discussion
600
540
A
Fluorescence (mV)
480
420
360
300
240
180
120
60
0
10
12
14
16
18
20
22
24
26
Elution volume (ml)
B
b-GGT
(U/L)
f-GGT
(U/L)
GGT tot
(U/L)
Bile
(control)
91.73
9.37
101.10
Bile
Doc 1% – papain
19.57
197.98
217.56
Figure 12 A) Elution profile of GGT fractions of a sample of bile (GGT = 127.5 U/L)
before (continuous line) and after (dashed line) treatment with 1% DOC 1h at 37 º C
and papain for 24h in room temperature. B) Comparison between the activities of the
GGT fractions (b and f-GGT GGT) and total GGT of bile sample before and after
treatment with DOC 1% 1h at 37 º C and papain for 24h at room temperature.
296 Results and discussion
4.4 FRACTIONAL GGT EVALUATION AFTER LIVER TRANSPLANT
Plasma and bile fractional GGT activity
The trend of the total GGT and its fractions was evaluated in 14 patients undergoing
liver transplantation. Blood samples were collected preoperatively before native liver
hepatectomy (T0), then for 10 consecutive days post-transplant. Bile samples were
collected intraoperatively during duct anastomosis (T0) and 10 days following the
surgical procedure of transplantation through Kehr-tube.
The postoperative course of these patients was uneventful and there were no events of
acute rejection. In the first postoperative day (T1) all patients had increased levels of
cytolysis indices (AST: 892 ± 826 U/L, ALT: 715 ± 748 U/L, LDH: 339 ± 351 U/L, Figure
13) and a reduction in liver synthetic function tests (total protein: 4.7 ± 0.5 mg/dL,
albumin: 2.1 ± 0.3 mg/dL; Fibrinogen: 193 ± 101 mg/dL, INR: 1.9 ± 0.7, Figure 13).
During the following 10 days these indices showed a tendency toward normalization
with reduction of cytolysis indices of and an increase in those of synthetic functions
(T10, AST: 46 ± 21 U/L, ALT: 115 ± 88 U/L, LDH: 238 ± 81 U/L; Total protein: 5.9 ± 0.8
mg/dL, albumin: 3.0 ± 0.5 mg/dL; Fibrinogen: 405 ± 113 mg/dL, INR: 1.1 ± 0.1, Figure
13).
On the contrary, indices of cholestasis, showed an inverse trend (Figure 13), which are
slightly altered 24h after transplantation (T1, Total Bilirubin: 2.3 ± 1.2 mg/dL Direct
bilirubin: 1.7 ± 1.0 mg/dL, ALP: 87 ± 50 U/L, GGT tot: 48 ± 18 U/L), and during the
following days show a gradual but steady increase (T10, Total Bilirubin: 5.7 ± 5.3 mg/dL
Direct bilirubin: 5.1 ± 5.1 mg/dL, ALP: 189 ± 83 U/L, GGT total: 204 ± 103 U/L).
Figures 14 show the trends of the average mean values of plasma and bile GGT
fractions in 10 days after liver transplantation. Figure 15 shows the trends for each
patient.
Soon after transplantation (24h), a sharp decline in total plasma GGT is observed (T0:
96.6 ± 91.6 U/L vs. T1: 57.7 ± 38.9 U/L; Table 8), which is reflected on all fractions, in
particular b-GGT (T0: 15.5 ± 32.6 U/L vs. T1: 5.2 ± 3.9 U/L; Table 8). In 5-6 days after
there has been a gradual increase in total plasma GGT (T6: 197.5 ± 139.3 U/L),
followed in some cases by a peak of activity in the tenth day (T10: 220.3 ± 106.3 U/L).
Plasma f-GGT fraction shows minor alterations (T1: 12.8 ± 5.3 U/L vs. T6: 20.6 ± 7.9
297 Results and discussion
U/L), while other fractions have a similar trend as total GGT, with b-GGT (T1: 5.2 ± 3.9
U/L vs. T6: 92.7 ± 63.2 U/L) and m-GGT (T1: 3.3 ± 3.6 U/L vs. T6: 20.7 ± 21.5 U/L)
showing the greatest increase in activity (Table 8, Figure 14)
In this phase of the study it was possible to examine samples of hepatic bile collected
intraoperatively during duct anastomosis, even in these sample GGT is present mainly
as b-GGT (T0: 217.9 ± 223.9 U/L; Table 9) and in less extent as f-GGT (T0: 30.4 ±
37.19 U/L; Table 9), as already described in samples of hepatic bile obtained posttransplantation (see Results, section-2).
The first posttransplant 24 h is associated with decreased bile b-GGT activity followed
immediately by a sudden increase in its activity with a peak observed in the fourth day
(T4: 1678 ± 2034 U/L), in some cases, bile b-GGT activity tends to return to baseline in
the following days, in other cases, a second peak is observed in the tenth day (T10:
2947 ± 6760 U/L). On the contrary, bile f-GGT fraction shows minimal changes (T1:
36.38 ± 43.79 U/L vs. T4: 47.80 ± 40.67 U/L) (Table 9, Figure 14).
The apparent peak of f-GGT activity observed at days 6 and 10 (Figure 14) is found in
two patients: 8-MR, 5-FG, respectively (Figure 15) in these days there is an increase of
bile GGT activity as well (8-MR T6: b-GGT= 5246 U/L, f-GGT= 2111 U/L; 8-MR T10: bGGT= 10 U/L, f-GGT= 1401 U/L, 5-FG T10: b-GGT= 16,742 U/L, f-GGT= 1349 U/L).
Another interesting event is the reversal of the proportions between the bile b-and fGGT franctions in favor of b-GGT fraction. This phenomenon has been observed in the
patient 8-MR day 10 (T10) and in the patient 14-RH on days 7 (T7) and 8 (T8) (Figure
15, Table 10), in each case in the previous day, an abrupt drop in b –GGT activity is
observed, which becomes quantitatively less than f-GGT and increased again in the
following days, and f-GGT follows the opposite trend. Clinically, there is no evidence of
significant clinical change in these two patients (Table 10), both were on Tacrolimus
therapy and had a post-operative course within the norm.
Further studies, including a more detailed characterization of bile composition will be
needed to clarify this point.
Correlations between plasma indexes of liver function and fractional GGT activity.
To see whether the variations of the GGT fractions may reflect the ischemic-reperfusion
injury and/or the recovery of liver functionality during the first 10 days after liver
298 Results and discussion
transplant, we performed a Spearman correlation analysis between plasma GGT
fractions and the main laboratory biomarkers used to monitor hepatobiliary damage and
liver function (Table 11). All fractions showed a positive correlation with ALP, total
protein, albumin and fibrinogen, thus behaving as positive index of cholestasis and liver
function. Interestingly all fractions showed a positive correlation with direct bilirubin
apart from s1-GGT, which showed a strict negative correlation. Unexpectedly, all
fractions were negative associated with LDH, and b-GGT and m-GGT showed a
negative correlation also with transaminases AST and ALT.
Thus plasma GGT fractions, in particular b-GGT and m-GGT, were primarily related to
ischemic-type biliary lesions following liver transplantation [Cutrin et al., 1996; Cursio e
Gugenheim, 2012]
299 Results and discussion
LDH
ALT
800
800
600
600
400
400
200
200
LDH (U/L)
1000
0
0
1
2
3
4
5
6
7
8
9 10
ALT(U/L)
1000
0
Days
Fibrinogen
Albumine
5
4
400
3
2
200
1
0
0
1
2
3
4
5
6
7
8
9 10
Albumin (mg/dL)
Fibrinogen (mg/dL)
600
0
Days
ALP
Direct bilirubin
10
ALP (U/L)
8
200
6
4
100
2
0
0
1
2
3
4
5
6
7
8
9 10
0
Dir. Bilirubin (mg/dL)
300
Days
Figure 13. Trend of plasma indexes of hepatocellular damage, liver function and
cholestasis in the first 10 days after liver transplant
300 Results and discussion
GGT activity (U/L)
125
Plasma
b-GGT
m-GGT
s1-GGT
s2-GGT
f-GGT
100
75
50
25
0
0
1
2
3
4
5
6
7
8
9 10
Days
GGT activity (U/L)
3000
2500
Bile
b-GGT
f-GGT
2000
1500
1000
500
0
0
1
2
3
4
5
6
7
8
9 10
Days
Figure 14. Trend of plasma and bile fractional GGT activities in the first 10 days after
liver transplant.
301 Results and discussion
Table 8. Mean values and standard deviation (SD) for plasma total and fractional GGT
activities in the first 10 days after liver transplant.
GGT tot
b-GGT
m-GGT
s1-GGT
s2-GGT
f-GGT
N
12
11
10
13
14
12
11
9
11
7
8
Days
0
1
2
3
4
5
6
7
8
9
10
Mean
96.6
57.7
61.7
89.1
135.2
162.8
197.5
179.8
184.4
207.5
220.3
SD
91.6
38.9
68.0
74.8
114.9
106.3
139.3
137.2
159.1
158.6
106.3
Mean
15.0
5.2
6.1
25.0
49.4
68.9
92.7
83.0
82.8
109.9
117.8
SD
32.6
3.9
7.1
35.4
59.4
60.0
63.2
67.9
69.8
95.8
74.9
Mean
5.1
3.3
3.6
8.0
13.8
15.1
20.7
20.2
19.0
20.3
19.8
SD
5.2
3.6
4.7
11.2
18.2
15.1
21.5
24.1
20.9
22.1
11.4
Mean
40.0
24.9
21.4
21.8
29.5
34.8
26.8
23.5
25.5
19.6
31.0
SD
35.7
23.4
31.5
26.1
37.7
46.9
19.0
20.3
18.5
17.2
28.2
Mean
19.2
11.5
16.1
17.8
22.2
21.2
24.7
21.9
21.6
23.2
24.1
SD
27.5
8.2
19.3
11.3
17.3
18.3
21.8
20.9
22.3
16.8
15.7
Mean
17.4
12.8
14.0
15.4
18.1
19.2
20.6
21.6
21.9
21.7
22.8
SD
8.1
5.3
6.7
5.2
7.9
7.3
7.9
7.6
14.6
12.5
9.6
Table 9. Mean values and standard deviation (SD) for bile total and fractional GGT
activities in the first 10 days after liver transplant.
GGT tot
b-GGT
f-GGT
302 N
9
8
9
11
14
11
9
9
8
4
6
Days
0
1
2
3
4
5
6
7
8
9
10
Mean
382.3
184.0
423.5
1338
1976
953.7
1446
654.2
603.4
655.6
5026
SD
355.9
111.3
450.0
1171
2209
716.9
2302
791.1
811.3
307.2
11040
Mean
217.9
115.8
304.9
1136
1678
751.7
1070
482.9
448.9
381.8
2947
SD
223.9
70.24
329.7
1097
2034
586.6
1633
682.8
710.4
221.7
6760
Mean
30.04
36.38
21.38
47.45
47.80
24.88
253.3
33.55
35.04
39.18
464.9
SD
37.19
43.79
11.34
31.93
40.67
19.36
697.0
54.19
59.07
44.87
705.3
Results and discussion
Figure 15. Total and fractional GGT activities in plasma and hepatic bile in 14 patients,
before, during and after liver transplant. AP: blood sample obtained from portal vein
during anhepatic phase; AS: systemic blood sample obtained during anhepatic phase;
RS: systemic blood sample obtained after reperfusion.
Patient #1-­‐AV
Bile
b-GGT
f-GGT
GGT activity U/L
3000
2000
1000
0
0
AP
AS
RS
1
2
3
4
5
6
7
8
9
10
days
Plasma - GGT tot
GGT activity U/L
300
200
100
0
0
AP
AS
RS
1
2
3
4
5
6
7
8
9
10
6
7
8
9
10
days
bGGT U/L
mGGT U/L
s1GGT U/L
s2GGT U/L
fGGT U/L
GGT activity U/L
150
Plasma - Fractional GGT
100
50
0
0
AP
AS
RS
1
2
3
4
5
days
303 GGT activity U/L
GGT activity U/L
GGT activity U/L
0
50
100
150
0
50
100
150
200
250
0
1000
2000
3000
4000
0
0
0
AS
AS
AP
AS
bGGT U/L
mGGT U/L
s1GGT U/L
s2GGT U/L
fGGT U/L
AP
AP
b-GGT
f-GGT
RS
RS
RS
1
days
3
4
5
2
days
3
4
Plasma - GGT tot
2
5
1
2
days
3
4
5
Plasma - Fractional GGT
1
Bile
6
6
6
7
7
7
8
8
8
9
9
9
GGT activity U/L
GGT activity U/L
304 GGT activity U/L
Patient #2-­‐BP
0
20
40
60
80
0
50
100
150
0
100
200
300
400
500
0 AP AS RS 1
0 AP AS RS 1
0 AP AS RS 1
b-GGT
f-GGT
2
2
2
3
3
3
7
8
days
9 10 11 12 13 14 15 16 17 18
7
8
days
4 5a 5b 6
7
8
days
9 10 11 12 13 14 15 16 17 18
bGGT U/L
mGGT U/L
s1GGT U/L
s2GGT U/L
fGGT U/L
9 10 11 12 13 14 15 16 17 18
Plasma - Fractional GGT
4 5a 5b 6
Plasma - GGT tot
4 5a 5b 6
Bile
Patient #3-­‐DNC
Results and discussion
GGT activity U/L
GGT activity U/L
GGT activity U/L
0
50
100
150
0
50
100
150
200
250
0
500
1000
1500
2000
2500
0
0
0
RS
AS RS
AS
AP AS RS
bGGT U/L
mGGT U/L
s1GGT U/L
s2GGT U/L
fGGT U/L
AP
AP
b-GGT
f-GGT
1
1
1
2
2
2
6
days
5
7
4
6
days
5
7
Plasma - GGT tot
4
8
8
3
4
6
days
5
7
8
Plasma - Fractional GGT
3
3
Bile
9
9
9
10
10
10
11
11
11
12
12
12
13
13
13
14
14
14
GGT activity U/L
GGT activity U/L
GGT activity U/L
Patient #4-­‐DE
0
100
200
300
250
300
350
400
450
500
0
5000
10000
15000
20000
0
0
0
AS
RS
AP AS RS
bGGT U/L
mGGT U/L
s1GGT U/L
s2GGT U/L
fGGT U/L
AP AS RS
AP
b-GGT
f-GGT
1
1
1
2
2
2
6
days
5
7
8
4
6
days
5
7
Plasma - GGT tot
4
8
9
3
4
6
days
5
7
8
Plasma - Fractional GGT
3
3
Bile
Patient #5-­‐FG
9
9
10
10
10
11
11
11
12
12
12
13
13
13
14
14
14
Results and discussion
305 GGT activity U/L
GGT activity U/L
GGT activity U/L
2
days
1
2
RS
1
2
Plasma - Fractional GGT
RS
3
3
3
4
4
4
5
5
5
days
0
100
150
200
250
0
100
200
300
400
0
AS
1
days
Plasma - GGT tot
RS
1000
2000
3000
4000
0
AP
AS
AS
b-GGT
f-GGT
50
0
AP
AP
bGGT U/L
mGGT U/L
s1GGT U/L
s2GGT U/L
fGGT U/L
0
0
Bile
10
20
30
40
50
0
50
100
150
0
500
1000
1500
2000
GGT activity U/L
GGT activity U/L
306 GGT activity U/L
Patient #6-­‐LA
0
0
0
AS
AS
AP
AS
bGGT U/L
mGGT U/L
s1GGT U/L
s2GGT U/L
fGGT U/L
AP
AP
b-GGT
f-GGT
RS
RS
RS
1
2
2
4
days
5
6
3
days
4
5
Plasma - GGT tot
3
2
3
days
4
5
6
Plasma - Fractional GGT
1
1
Bile
Patient #7-­‐LF
6
7
7
8
7
8
9
8
9
10
9
10
11
10
11
11
Results and discussion
GGT activity U/L
GGT activity U/L
GGT activity U/L
0
50
100
150
0
100
200
300
0
2000
4000
6000
0
0
0
AS
AS
RS
RS
AP
AS
RS
bGGT U/L
mGGT U/L
s1GGT U/L
s2GGT U/L
fGGT U/L
AP
AP
f-GGT
b-GGT
1
1
1
2
2
2
5
6
Days
7
4
6
days
5
7
8
8
3
4
6
Days
5
7
8
Plasma - Fractional GGT
3
4
Plasma - GGT tot
3
Bile
9
9
9
10
10
10
11
11
11
12
12
12
13
13
13
14
14
14
GGT activity U/L
GGT activity U/L
GGT activity U/L
Patient #8-­‐MR
0
50
100
150
0
50
100
150
200
0
200
400
600
800
1000
0
0
0
AP AS RS
bGGT U/L
mGGT U/L
s1GGT U/L
s2GGT U/L
fGGT U/L
AP AS RS
AP AS RS
1
1
1
2
2
2
5
6
days
7
8
4
5
days
6
7
8
Plasma - GGT tot
4
9
9
3
4
5
days
6
7
8
9
Plasma - Fractional GGT
3
3
Bile
Patient #9-­‐PA
10
10
10
11
11
11
12
12
12
13
13
13
14
14
14
15
b-GGT
f-GGT
15
15
Results and discussion
307 GGT activity U/L
GGT activity U/L
GGT activity U/L
0
50
100
150
0
50
100
150
200
250
0
2000
4000
6000
8000
0
0
0
AS
AS
AP
AS
bGGT U/L
mGGT U/L
s1GGT U/L
s2GGT U/L
fGGT U/L
AP
AP
RS
RS
RS
3
days
4
5
2
3
days
4
5
Plasma - GGT tot
2
1
2
3
days
4
5
Plasma - Fractional GGT
1
1
Bile
6
6
6
7
7
7
8
8
8
9
9
9
f-GGT
b-GGT
10
10
10
GGT activity U/L
GGT activity U/L
308 GGT activity U/L
Patient #10-­‐PN
0
50
100
150
200
0
100
200
300
0
100
200
300
400
0
0
0
AS
AS
AP
AS
bGGT U/L
mGGT U/L
s1GGT U/L
s2GGT U/L
fGGT U/L
AP
AP
b-GGT
f-GGT
RS
RS
RS
3
days
4
5
2
3
days
4
5
Plasma - GGT tot
2
6
1
2
3
days
4
5
Plasma - Fractional GGT
1
1
Bile
Patient #11-­‐PA
6
6
7
7
7
8
8
8
9
9
9
10
10
10
Results and discussion
GGT activity U/L
GGT activity U/L
GGT activity U/L
0
20
40
60
80
0
50
100
150
0
100
200
300
400
Bile
Plasma - Fractional GGT
days
0 APASRS 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27
bGGT U/L
mGGT U/L
s1GGT U/L
s2GGT U/L
fGGT U/L
days
0 APASRS 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27
Plasma - GGT tot
days
0 APASRS 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27
b-GGT
f-GGT
Patient #13-­‐TM
Results and discussion
309 GGT activity U/L
GGT activity U/L
GGT activity U/L
AS
AS
RS
days
3
4
2
days
3
4
Plasma - GGT tot
2
5
5
1
2
3
4
5
Plasma - Fractional GGT
1
1
6
6
6
7
7
7
8
8
8
9
9
9
40
60
80
100
0
50
100
150
0
Days
0
AS
RS
RS
2000
4000
6000
0
AP
bGGT U/L
mGGT U/L
s1GGT U/L
s2GGT U/L
fGGT U/L
AP
AP
f-GGT
b-GGT
20
0
0
0
Bile
50
100
150
200
250
0
200
400
600
800
0
100
200
300
400
500
GGT activity U/L
GGT activity U/L
310 GGT activity U/L
Patient #14-­‐RH, before rejection
0
0
0
AS
AS
AP
AS
bGGT U/L
mGGT U/L
s1GGT U/L
s2GGT U/L
fGGT U/L
AP
AP
RS
RS
RS
2
days
3
4
2
days
3
4
1
2
days
3
4
Plasma - Fractional GGT
1
Plasma - GGT tot
1
Bile
Patient #12-­‐PA
5
5
5
6
6
6
7
7
7
8
8
8
b-GGT
f-GGT
Results and discussion
GGT activity U/L
GGT activity U/L
GGT activity U/L
0
50
100
150
200
150
200
250
300
0
100
200
300
400
1
bGGT U/L
mGGT U/L
s1GGT U/L
s2GGT U/L
fGGT U/L
1
1
2
2
2
Days
3
Plasma - Fractional GGT
days
3
Plasma - GGT tot
days
3
Bile
4
4
4
Patient #14-­‐RH, after rejection
5
5
5
f-GGT
b-GGT
Results and discussion
311 Results and discussion
Table 10. Bile fractional GGT values and plasma index of hepatocellular damage of the
patients 8-MR and 14-RH.
8-MR
Days of hospitalisation
8
9
10
11
12
b-GGT, U/L
2149
342
10
208
959
f-GGT, U/L
58
105
1401
65
195
37.1
3.3
0.007
3.2
4.9
Direct Bilirubin, mg/dL
4.2
3.7
2.4
2.0
1.5
AST, U/L
57
55
45
47
41
ALT, U/L
578
437
308
252
203
GGT tot, U/L
140
254
263
216
153
ALP, U/L
104
180
190
205
200
LDH, U/L
174
166
141
144
138
b-GGT/f-GGT
Plasma:
14-HR
Days of hospitalisation
5
6
7
8
9
b-GGT, U/L
417
104
65
70
f-GGT, U/L
50
74
170
173
b-GGT/f-GGT
8.3
1.4
0.4
0.4
Direct Bilirubin, mg/dL
6.1
8.8
11.9
14.2
16.1
AST, U/L
99
88
64
57
58
ALT, U/L
172
166
143
115
104
GGT tot, U/L
336
544
503
603
440
ALP, U/L
257
413
453
437
504
LDH, U/L
202
207
219
216
214
Plasma:
ALP: alkaline phosphatase; ALT: alanine aminotransferase; AST: aspartate
aminotransferase; LDH: lactate dehydrogenase.
312 Results and discussion
Table 11. Correlation analysis between plasma indexes of liver function and plasma
fractional GGT activity (U/L).
GGT tot
b-GGT
m-GGT
s1-GGT
s2-GGT
f-GGT
0.343†
0.364†
0.365‡
n.s.
n.s.
0.462‡
Tot. Bilirubin, mg/dL
n.s.
n.s.
0.281§
-0.543‡
n.s.
0.275§
Direct Bilirubin, mg/dL
n.s.
0.200*
0.343†
-0.543‡
n.s.
0.272§
n.s.
n.s.
n.s.
n.s.
ns
n.s.
ALP, U/L
0.647‡
0.642‡
0.699‡
0.213*
0.379‡
0.487‡
AST, U/L
-0.312†
-0.487‡
-0.493‡
n.s.
n.s.
-0.221*
ALT, U/L
-0.286§
-0.300§
-0.293§
n.s.
n.s.
n.s.
LDH, U/L
-0.502‡
-0.486‡
-0.504‡
-0.256§
-0.411‡
-0.330†
Total Protein, mg/dL
0.494‡
0.382‡
0.361†
0.531‡
0.201*
0.458‡
Albumin, mg/dL
0.442‡
0.368‡
0.304§
0.423‡
0.216*
0.338†
n.s.
-0.324§
-0.213*
n.s.
n.s.
n.s.
0.236*
0.339†
0.369†
0.226*
0.224*
n.s.
Platelets, 109/L
Indirect Bilirubin,
mg/dL
INR
Fibrinogen, mg/dL
Data are reported as Spearman correlation coefficients (r) and refer to 10 days followup of the 14 patients. ALP: alkaline phosphatase; ALT: alanine aminotransferase; AST:
aspartate
aminotransferase;
INR:
international
normalized
§
ratio;
LDH:
lactate
†
dehydrogenase. Statistical significance levels: *P < 0.05; P < 0.01; P < 0.001; ‡P <
0.0001. n.s.: not significant.
313 Results and discussion
314 Conclusions
5. CONCLUSIONS
The main findings of this study are:
1) Patients with NAFLD and CHC display different GGT fraction patterns, despite similar
total GGT activity values.
Among all the GGT fractions, b-GGT provided the best specificity and sensitivity for the
diagnosis of NAFLD, with an accuracy equivalent or even greater to those provided by
available diagnostic algorithms such as SteatoTest (ROC-AUC 0.79, SE 0.03) (Poynard
et al., 2005) or Fatty Liver Index (ROC-AUC 0.85, 95%CI 0.81-0.88) (Bedogni et al.,
2006). Interestingly, these algorithms include total serum GGT activity and it is likely
that replacing GGT with b-GGT would increase their sensitivity and specificity.
In subjects with CHC, b-GGT did not show any significant change, while s-GGT showed
a prominent increase, thus suggesting that the ratio between these fractions was a key
aspect of the disease-associated GGT fraction pattern, and indeed the b/s ration
showed the highest specificity for distinguishing between CHC and NAFLD, as well as
for the diagnosis of CHC as compared to total GGT and all individual fractions. All other
ratios between the remaining fractions were also tested, but their ROC-AUCs were
significantly lower (not shown).
The precise nature of GGT fractions has not yet been established, and at present it is
not possible to speculate on the possible reasons conducting to different GGT fraction
patterns in NAFLD and CHC. The fact that in NAFLD the increase of serum GGT occurs
through a proportional increase of b-GGT and s-GGT, while in viral hepatitis occurs
through a prominent increase of s-GGT suggests that GGT fraction pattern specificity
might depend on its ability to reflect the different extents of inflammatory, structural and
functional derangement in liver disease.
These findings for the first time open the perspective of a positive diagnosis of NAFLD
that might be helpful as a screening test or to perform large population studies on the
prevalence of NAFLD and related diseases.
The use of GGT fractional analysis might also contributes to the understanding of the
complex epidemiological association between serum GGT, NAFLD, and cardiovascular
disease. In fact, during the last decade, large population studies have shown that serum
GGT (independently from alcohol consumption) is one of the strongest predictors of
metabolic syndrome, diabetes as well as final events (myocardial infarction and stroke)
315 Conclusions
associated with the atherosclerotic disease, thus opening new perspectives as concerns
the association between liver damage, metabolism and atherosclerosis. It has been
showed that GGT activity, in the presence of trace iron and glutathione, its physiological
substrate, catalyses the production of free radicals and reactive oxygen species, thus
promoting LDL oxidation in vitro, and it has been found that human atherosclerotic
plaques contain GGT activity in correspondence of the oxidized LDL and CD68+ foam
cells. The GGT extracted from human carotid plaques was found to correspond in part
to the human serum b-GGT fraction. The fact that b-GGT, which has a potential
atherogenic role, was found in this study to increase specifically in NAFLD, a condition
strictly associated with metabolic syndrome, iperinsulinemia, and dyslipidemia, suggests
that NAFLD act as cardiovascular risk factor by promoting b-GGT accumulation and
GGT-related oxidative events within the plaque. Thus, results of this study might help to
define a new pathway connecting dysmetabolism with cardiovascular disease.
On the basis of the present results, further studies on larger population will show if GGT
fraction analysis might also be used to identify subjects with NAFLD progressing
towards parenchymal inflammation and fibrosis, which might experience a progressive
decrease of the b/s ratio.
2) Collected data showed that the b/s ratio, independently of the absolute values of total
GGT and its fractions, displays a high sensitivity and specificity for liver cirrhosis. The
area under the ROC curve was substantially the same (exceeding 94%) in the whole
cohort and in the subset of patients with total GGT values within the reference range,
and the values of the b/s ratio were lower than controls independently of the cause of
the cirrhosis (viral or cryptogenetic) or the presence of associated liver cancer. This
suggests that the b/s ratio is a specific biomarker of architectural and functional damage
of the liver, and that the causes of decreased b/s ratio are not the same causing the
increase of total GGT in liver disease. In fact, we found decreased values of b/s ratio in
chronic HCV hepatitis, but not in NAFLD; noticeably, the values of b/s ratio found in
cirrhotic patients in this study (0.06, 0.04-0.1) are lower than those found in patients with
CHC (0.10, 0.07-015), suggesting that a progressive decrease of the b/s ratio
accompanies the evolution of chronic liver disease towards liver cirrhosis and failure,
316 Conclusions
and pointing to the b/s ratio as a potential progression biomarker of chronic liver
disease.
As obvious, the shift in the b/s ratio depends on the different association of the
individual GGT fraction with different aspects of the progression of liver disease that are
still to be investigated in detail. In this cohort of patients, a simple linear correlation
analysis revealed that b-GGT is associated with biomarkers of liver function and
structure (positive association with serum albumin, fibrinogen, platelet counts, negative
association with INR), while s-GGT, and in particular its component s2-GGT behaves as
a rather selective biomarker of cell damage and cholestasis (positive association with
ASL, ALT, LDH, ALP, bilirubin, negative association with serum albumin).
Histochemical analysis showed that cirrhotic patients had higher tissue GGT activity
than control, independently from circulating GGT levels. This suggests that the
alteration of the hepatic architecture, as consquences of cirrhosis, might influence GGT
fraction release.
3) Based on the assumption that both plasma m-GGT and s- GGT fractions are formed
by micelles of bile acids and GGT, the elution profile of GGT fractions in human bile was
analyzed to be compared with those of plasma, with the aim to determine the
contribution of the liver to plasma GGT enzyme. Unexpectedly, the elution profile of bile
GGT activity showed the presence of only two forms corresponding to plasma fractions
b-GGT and f-GGT, respectively. This may be due to the low concentration of bile acids
in hepatic bile which is about 10 times lower than the amount of DOC (%) used to
observe the conversion of plasma b-GGT into s-GGT. For this reason it will be
necessary also analyze the elution profile of GGT activity in the gallbladder bile, whose
solutes concentration are 5-10 times higher than that of hepatic bile.
Similar to that found in plasma GGT fractions, the biliary GGT fraction consists of
soluble protein (f-GGT) and exosomes (b-GGT). But, unlike plasma b-GGT, biliary bGGT fraction is in part sensitive to papain action; likely, the portion of biliary b-GGT
sensitive to the proteolytic action might be consistuted of bile acids micelles. In fact,
about 80% of biliary GGT activity can be recovered in the fraction with a density of
1.123 g / mL, which is also the most rich in proteins (55% of total) and bile acid (35%),
317 Conclusions
but with the lower content of phospholipids (17%) and even free of cholesterol (Accatino
et al., 2005).
In the bile, following treatment with papain, in either the absence or presence of DOC,
the formation of a peak of f-GGT activity greater than expected was observed, a
phenomenon that has not been observed in plasma. Probably, the molecular context in
which GGT is incorporated in biliary b-GGT might affect the kinetic of the enzyme
exerting an inhibitory effect, for example by influencing the accessibility of the substrate
to the active site of the enzyme. Therefore, the protein in f-GGT appears to have a
faster kinetic. In the light of these results it is clear that the problem of the physical
nature of the molecular forms of plasma GGT is fundamental in order to understand the
role of GGT in the pathogenesis of diseases associated with its increase and that
cannot be separated from the its diagnostic and predictive value.
4) Post-transplant changing of plasma GGT fractions, in particular b-GGT and m-GGT,
were primarily related to ischemic-type biliary lesions following liver transplantation.
Collected data about transaminases and cholestatic indexes trends observed in
transplanted patients depends on ischemic-reperfusion injury, to which biliary system is
more sensible than hepatic parenchyma. Hepatic cytolysis and cholestasis consistently
occur in patients who have undergone orthotopic liver transplantation, a typical
ischemia-reperfusion syndrome. Liver transplant dependent cytolysis is an early but, in
general, transient event. Unless episodes of tissue rejection occur, plasma
transaminases return to the normal range, or close to it, within one week. While
intrahepatic cholestasis following liver transplantation is common, one of the main
causes of intrahepatic cholestasis after LT is cold and warm ischemic-reperfusion injury.
Under normal conditions, an intact cytoskeleton is required for bile canalicular
contraction, which is based on a pericanalicular web of contractile proteins, actin
microfilaments, and cytokeratin intermediate filaments acting as a pump to facilitate bile
flow into the intrahepatic canalicular system. The bile canaliculus is one of the liver
structures that is early damaged by ischemic-reperfusion injury. This oxidative stressdependent structural damage contributes to perturbate the bile acid transport during
ischemia. The resulting loss of microvilli and the canalicular atony, decrease the bile
flow and lead to cholestasis.
318 Conclusions
Further study on the nature and biological significance of plasma GGT fractions in
health and disease might allow to improve the use of this sensitive but otherwise poorly
specific biomarker in the numerous contexts in which it is employed, including
multimarker algorithms comprising plasma GGT for the assessment of liver steatosis
and fibrosis. Extensive investigation on the diagnostic value of GGT fractions might
provide a novel diagnostic tool for liver diseases; understanding the nature, properties,
and pathophysiological variations of GGT fraction pattern might allow a better
understanding of the pathogenesis of the diseases associated with increased GGT.
319 Conclusions
320 References
6. REFERENCES
2001 Annual Report of the U.S. Scientific Registry of Transplant Recipients and the Organ Procurement
and
Transplantation
Network:
Transplant
Data
1989-1998.
Available
at
http://
www.optn.org/data/annualReport.asp. Accessed April 10, 2003.
Aasen T, Raya A, Barrero MJ, et al. Efficient and rapid generation of induced pluripotent stem cells from
human keratinocytes. Nat Biotechnol 2008; 26:1276-1284.
Abdel Aziz MT, Atta HM, Mahfouz S, et al. Therapeutic potential of bone marrowderived mesenchymal
stem cells on experimental liver fibrosis. Clin Biochem. 2007; 40, (12):893-899.
Abouljoud MS, Escobar F, Douzdjian V, et al. Recurrent disease after liver transplantation. Transplant
Proc 2001; 33:2716–9.
Abraham SC, Freese DK, Ishitani MB, et al. Significance of central perivenulitis in pediatric liver
transplantation. Am J Surg Pathol 2008; 32:1479–1488.
Abt P, Crawford M, Desai N, et al. Liver transplantation from controlled non-heartbeating donors: an
increased incidence of biliary complications. Transplant. 2003; 75:1659–63.
Abt P, Desai N, Crawford M, et al. Survival following liver transplantation from non-heart-beating donors.
Ann Surg. 2004; 239:87–92
Abu-Elmagd KM, Malinchoc M, Dickson ER, et al. Efficacy of hepatic transplantation in patients with
primary sclerosing cholangitis. Surg Gynecol Obstet 1993; 177:335-344.
Accatino L, Pizarro M, Solís N, et al. Association of canalicular membrane enzymes with bile acid
micelles and lipid aggregates in human and rat bile. Biochim Biophys Acta 1995;1243(1):33-42.
Adam R, Astarcioglu I, Azoulay D, et al. Liver transplantation from elderly donors. Transplant Proc 1993;
25:1556-1557.
Adam R, Bismuth H, Diamond T, et al. Effect of extended cold ischaemia with UW solution on graft
function after liver transplantation. Lancet 1992; 340 (8832):1373-1376.
Adam R, Cailliez V, Majno P et al. Normalised intrinsic mortality risk in liver transplantation: European
Liver Transplant Registry study. Lancet 2000; 356:621–627.
Adam R, Hoti E. Liver transplantation: the current situation. Semin Liver Dis 2009; 29:3-18.
Adam R, Reynes M, Johann M, et al. The outcome of steatotic grafts in liver transplantation. Transplant
Proc 1991; 23:1538–1540.
Adam R, Sanchez C, Astarcioglu I, Deleterious effect of extended cold ischemia time on the
posttransplant outcome of aged livers. Transplant Proc. 1995; 27:1181-1183.
Adams AB, et al. Adams AB, Williams MA, Jones TR, et al. Heterologous immunity provides a potent
barrier to transplantation tolerance. J Clin Invest 2003; 111:1887–1895.
Adams D, Samuel D, Goulon-Goeau C, et al. The course and prognostic factors of familial amyloid
polyneuropathy after liver transplantation. Brain 2000; 123(7):1 495-1504.
Adams DH, Wang L, Hubscher SG, et al. Hepatic endothelial cells. Targets in liver allograft rejection?
Transplantation 1989; 47:479–82.
Adams LA, Bulsara M, Rossi E, et al. Hepascore: an accurate validated predictor of liver fibrosis in
chronic hepatitis C infection. Clin Chem 2005; 51(10):1867–73.
Adams LA, Lymp JF, St Sauver J, et al. The natural history of nonalcoholic fatty liver disease: a
population-based cohort study. Gastroenterology 2005; 129:113–121.
Afonso RC, Hidalgo R, Zurstrassen MP, et al. Impact of renal failure on liver transplantation survival.
Transplant Proc 2008; 40(3):808-10.
Afzali B, Lombardi G, Lechler RI. Pathways of major histocompatibility complex allorecognition. Curr Opin
Organ Transplant 2008; 13:438–444.
Ahmed A and Keeffe EB, Current Indications and Contraindications for Liver Transplantation. Clinics in
Liver Disease 2007; 11(2):227–247.
321
References
Aikata H, Takaishi H, Kawakami Y et al. Telomere reduction in human liver tissues with age and chronic
inflammation. Exp Cell Res 2000; 256:578–582.
Akle CA, Adinolfi M, Welsh KI, et al. Extended-donor criteria liver allografts. Semin Liver Dis 2006;
26:221–33.
Akle CA, Adinolfi M, Welsh KI, et al. Immunogenicity of human amniotic epithelial cells after
transplantation into volunteers. Lancet 1981; 2 (8254):1003-1005.
Ala A, Schilsky M. Wilson’s disease: pathophysiology, diagnosis, treatment and screening. Clin Liver Dis
2004; 8(4):778–805, viii. Review.
Alexander JW, Vaughn WK. The use of ‘‘marginal’’ donors for organ transplantation: the influence of
donor age on outcome. Transplantation 1991; 51:135–141.
Al-Hamoudi WK, Alqahtani S, Tandon P, et al. Hemodynamics in the immediate post-transplantation
period in alcoholic and viral cirrhosis. World J Gastroenterol 2010; 16:608-612.
Alonso O, Loinaz C, Moreno E, et al. Advanced donor age increases the risk of severe recurrent hepatitis
C after liver transplantation. Transpl Int 2005; 18:902.
Alqahtani SA, Fouad TR, Lee SS. Cirrhotic cardiomyopathy. Semin Liver Dis 2008; 28:59-69.
Alvarez F, Berg PA, Bianchi FB, et al. International Autoimmune Hepatitis Group Report: review of criteria
for diagnosis of autoimmune hepatitis. J Hepatol 1999; 31:929–38.
Alvarez J, del Barrio R, Arias J, et al. Non-heart-beating donors from the streets: an increasing donor pool
source. Transplantation 2000; 70:314–317.
Alwayn IP and Porte RJ . How to make steatotic livers suitable for transplantation. Liver Transpl 2007;
13:480–482.
Amodio P, Biancardi A, Montagnese S, et al. Neurological complications after orthotopic liver
transplantation. Dig Liver Dis 2007; 39:740-747.
Anand AC, Hubscher SG, Gunson BK, et al Timing, significance, and prognosis of late acute liver
allograft rejection. Transplantation 1995; 60:1098–103.
Anderson FH, Zeng L, Rock NR, et al. An assessment of the clinical utility of serum ALT and AST in
chronic hepatitis C. Hepatol Res 2000; 18:63–71.
Anderson ME, Allison RD, Meister A. Interconversion of leukotrienes catalyzed by purified gammaglutamyl transpeptidase: concomitant formation of leukotriene D4 and gamma-glutamyl amino acids. Proc
Natl Acad Sci USA 1982;79:1088-91.
Andreu M, Sola R, Sitges SA, et al. Risk factors for spontaneous bacterial peritonitis in cirrhotic patients
with ascites. Gastroenterology 1993; 104:1133-1138.
Angele MK, Rentsch M, Hartl WH, et al. Effect of graft steatosis on liver function and organ survival after
liver transplantation. Am J Surg 2008; 195:214–220.
Angelis M, Cooper JT, Freeman RB. Impact of donor infections on outcome of orthotopic liver
transplantation. Liver Transpl 2003; 9:451–462.
Angermayr B, Cejna M, Karnel F, et al. Child-Pugh versus MELD score in predicting survival in patients
undergoing transjugular intrahepatic portosystemic shunt. Gut 2003; 52:879.
Angulo P, Hui JM, Marchesini G, et al. The NAFLD fibrosis score: a noninvasive system that identifies
liver fibrosis in patients with NAFLD. Hepatology 2007; 45(4):846–54.
Angulo P, Lindor KD. Primary biliary cirrhosis. In: Feldman M, Friedman L, Sleisenger MH, eds.
Gastrointestinal and liver disease. 7th ed. Philadelphia: Saunders 2002:1474–1485.
Anselmo DM, Ghobrial RM, Jung LC et al. New era of liver transplantation for hepatitis B: a 17-year
single-center experience. Ann Surg 2002; 235:611–619.
Arad Y, Goodman KJ, Roth M, et al. Coronary calcification, coronary disease risk factors, Creactive
protein, and atherosclerotic cardiovascular disease events: the St. Francis Heart Study. J Am Coll Cardiol
2005; 46:158-165.
Araki K, Turner AP, Shaffer VO, et al. mTOR regulates memory CD8 T cell differentiation. Nature
2009;460:108–112.
322
References
Ardite E, Ramos C, Rimola A, et al. Hepatocellular oxidative stress and initial graft injury in human liver
transplantation. J Hepatol 1999; 31:921-927.
Arena U, Vizzutti F, Abraldes JG, et al. Acute viral hepatitis increases liver stiffness values measured by
transient elastography. Hepatology 2008; 47:380–384.
Arena U, Vizzutti F, Abraldes JG, et al. Reliability of transient elastography for the diagnosis of advanced
fibrosis in chronic hepatitis C. Gut 2008; 57:1288–1293.
Arguedas MR, Abrams GA, Krowka MJ, et al. Prospective evaluation of outcomes and predictors of
mortality in patients with hepatopulmonary syndrome undergoing liver transplantation. Hepatology 2003;
37:192-197.
Arnow PA. Antibiotic prophylaxis: the role of selective bowel decontamination. Curr Opin Organ
Transplant 2001; 6:301-4.
Arroyo V, Colmenero J. Ascites and hepatorenal syndrome in cirrhosis: pathophysiological basis of
therapy and current management. J Hepatol 2003; 38(1):S69–89.
Arthur MJ. Fibrogenesis II. Metalloproteinases and their inhibitors in liver fibrosis. Am J Physiol
Gastrointest Liver Physiol 2000; 279:G245–G249.
Asanza CG, Garcia-Monzon C, Clemente G, et al. Immunohistochemical evidence of
immunopathogenetic mechanisms in chronic hepatitis C recurrence after liver transplantation. Hepatology
1997; 26:755–63.
Assy N, Minuk GY. Serum aspartate but not alanine aminotransferase levels help to predict the
histological features of chronic hepatitis C viral infections in adults. Am J Gastroenterol 2000; 95:1545–
1550.
Aster RH. Pooling of platelets in the spleen: role in the pathogenesis of “hypersplenic” thrombocytopenia.
J Clin Invest 1966; 45:645–657.
Atkinson C, Varela JC, Tomlinson S. Complement-dependent inflammation and injury in a murine model
of brain dead donor hearts. Circ Res 2009; 105:1094-101.
Attia M, Silva MA, Mirza DF. The marginal liver donor – an update. Transpl Int 2008; 21:713.
Avolio AW, Agnes S, Magalini SC, et al. Importance of liver blood chemistry (AST, serum sodium) in
predicting liver transplant outcome. Transplant Proc 1991; 23:2451–2452.
Ayata G, Gordon FD, Lewis WD, et al. Liver transplantation for autoimmune hepatitis: a long-term
pathologic study. Hepatology 2000; 32:185–92.
Azoulay D, Astarcioglu I, Bismuth H, et al. Split-liver transplantation. The Paul Brousse policy. Ann Surg
1996; 224:737.
Azoulay D, Castaing D, Adam R, et al. Split-liver transplantation for two adult recipients: feasibility and
long-term outcomes. Ann Surg 2001; 233:565.
Azoulay D, Linhares MM, Huguet E, et al. Decision for retransplantation of the liver: an experience- and
costbased analysis. Ann Surg 2002; 236:713-721.
Baccarani U, Adani GL, Toniutto P, et al. Liver transplantation from old donors into HCV and non-HCV
recipients. Transplant Proc 2004; 36:527–528.
Baccarani U, Isola M, Adani GL, et al. Steatosis of the hepatic graft as a risk factor for post-transplant
biliary complications. Clin Transplant 2010; 23(2):239.
Backman L, Gibbs J, Levy M, et al. Causes of late graft loss after liver transplantation. Transplantation
1993; 55:1078-82.
Bailey B, Amre DK, Gaudreault P. Fulminant hepatic failure secondary to acetaminophen poisoning: a
systematic review and meta-analysis of prognostic criteria determining the need for liver transplantation.
Crit Care Med 2003; 31:299-305.
Bakthavatsalam R, Marsh CL, Perkins JD, et al. Rescue of acute portal vein thrombosis after liver
transplantation using a cavoportal shunt at re-transplantation. Am J Transplant 2001; 1:284-287.
Banas A, Teratani T, Yamamoto Y, et al. In vivo therapeutic potential of human adipose tissue
mesenchymal stem cells (AT-MSCs) after transplantation into mice with liver injury. Stem Cells 2008;
26:2705-2712.
323
References
Banas A, Teratani T, Yamamoto Y, et al. Rapid hepatic fate specification of adipose-derived stem cells
and their therapeutic potential for liver failure. J Gastroenterol Hepatol 2009; 24(1):70-77.
Banas A, Teratani T, Yamamoto Y, et al. Stem cell plasticity: learning from hepatogenic differentiation
strategies. Developmental Dyn 2007; 236:3228-3241.
Banff schema for grading liver allograft rejection: an international consensus document. Hepatology 1997;
25:658–63.
Barber DL, Wherry EJ, Ahmed R. Cutting edge: rapid in vivo killing by memory CD8 T cells. J Immunol
2003; 171:27–31.
Barber KM, S. Pioli, J. E. Blackwell, D. Collett, J. M. Neuberger, and A. E. Gimson, “Development of a UK
score for patients with end-stage liver disease,” Hepatology, vol. 46, article 510A, 2007.
Barr ML, Belghiti J, Villamil FG, et al. A report of the Vancouver Forum on the care of the live organ
donor: lung, liver, pancreas, and intestine data and medical guidelines. Transplantation 2006; 81:1373–
1385.
Bartlett AS, McCall JL, Ameratunga R, Yeong, et al. Analysis of intragraft gene and protein expression of
the co-stimulatory molecules, CD80, CD86 and CD154, in orthotopic liver transplant recipients. Am J
Transplant 2003; 3:1363-1368.
Bartlett AS, McCall, Ameratunga R, et al. Costimulatory blockade prevents early rejection, promotes
lymphocyte apoptosis and inhibits the upregulation of inter-graft interleukin 6 in an orthotopic liver
transplant model in the rat. Liver Transpl 2002; 8: 458-468.
Batts KP. Ischemic cholangitis. Mayo Clin Proc 1998; 73:380–5.
Baust JM. Molecular mechanisms of cellular demise associated with cryopreservation failure. Cell
Preservation Technol 2002; 1:17–31.
Bearman SI. The syndrome of hepatic veno-occlusive disease after marrow transplantation. Blood 1995;
85:3005–20.
Beaudreuil S, Samuel D, Rouas-Freiss N, et al. New aspect of immunosuppressive treatment in liver
transplantation. How could you induce tolerance in liver transplantation? Transpl Immunol. 2007; 17(2):
98-107.
Beavers KL, Sandler RS, Shrestha R. Beavers KL, et al. Donor morbidity associated with right lobectomy
for living donor liver transplantation to adult recipients. Liver Transpl 2002; 8:110-7.
Beckebaum S, Cicinnati V, Brokalaki E, et al. CNI-sparing regimens within the liver transplant setting:
experiences of a single center. Clin Transpl 2004; 215-20.
Beckebaum S, Cicinnati VR, Broelsch CE. Future directions in immunosuppression. Transplant Proc
2004; 36(2):574S-6S
Beckebaum S, Sotiropoulos G, Gerken G, et al. Hepatitis B and liver transplantation: 2008 update. Rev
Med Virol 2008; 19(1):7-29. Review.
Bedossa P, Dargère D, Paradis V. Sampling variability of liver fibrosis in chronic hepatitis C. Hepatology
2003; 38:1449–57.
Bedossa P, Moucari R, Chelbi E, et al. Evidence for a role of nonalcoholic steatohepatitis in hepatitis C: a
prospective study. Hepatology 2007; 46(2):380–7.
Bedossa P, Poynard T. An algorithm for the grading of activity in chronic hepatitis C. The METAVIR
cooperative study group. Hepatology 1996; 24:289–93.
Befeler AS, Di Bisceglie AM. Hepatocellular carcinoma: diagnosis and treatment. Gastroenterology 2002;
122:1609-1619.
Bekker J, loem S, de Jong KPl. Early hepatic artery thrombosis after liver transplantation: a systematic
review of the incidence, outcome and risk factors. Am J Transplant 2009; 9:746-757.
Bellamy CO, DiMartini AM, Ruppert K, et al. Liver transplantation for alcoholic cirrhosis: long term followup and impact of disease recurrence. Transplant 2001; 72:619–626.
Belle SH, Porayko MK, Hoofnagle JH, et al. Changes in quality of life after liver transplantation among
adults. National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) Liver Transplantation
Database (LTD). Liver Transpl Surg 1997; 3:93-104.
324
References
Belli LS, Silini E, Alberti A, et al. Hepatitis C virus genotypes, hepatitis, and hepatitis C virus recurrence
after liver transplantation. Liver Transpl Surg 1996; 2:200–5.
Belzberg H, Shoemaker WC, Wo CC, et al. Hemodynamic and oxygen transport patterns after head
trauma and brain death: implications for management of the organ donor. J Trauma 2007; 63:1032-42.
Belzner FO, Southard JH: Principles of solid-organ preservation by cold storage. Transplant1988; 45:673.
Bemuan J, Guodau A, Poynard T, et al. Multivariate analysis of prognostic factors in fulminant hepatitis B.
Hepatology 1986; 6:648–651.
Ben-David, U. & Benvenisty, N. The tumorigenicity of human embryonic and induced pluripotent stem
cells. Nature Rev. Cancer 2011; 11: 268-277.
Ben-Haim M, Roayaie S, Ye MQ, Thung SN, et al. Hepatic epithelioid hemangioendothelioma: resection
or transplantation, which and when? Liver Transpl Surg 1999; 5:526-531.
Benhamou JP. Fulminant and sub-fulminant hepatic failure: definition and causes. In:Williams R, Hughes
RD, editors. Acute liver failure: improved understanding and better therapy. London: Mitre Press; 1991;
6–10.
Benlloch S, Berenguer M, Prieto M, et al. De novo internal neoplasms after liver transplantation:
increased risk and aggressive behavior in recent years? Am J Transplant 2004; 4(4):596-604.
Benyon RC, Arthur MJ. Extracellular matrix degradation and the role of hepatic stellate cells. Semin Liver
Dis 2001; 21:373–384.
Benyon RC, Iredale JP, Goddard S, et al. Expression of tissue inhibitor of metalloproteinases 1 and 2 is
increased in fibrotic human liver. Gastroenterology 1996; 110:821–831.
Berardi S, Lodato F, Gramenzi A, et al. High incidence of allograft dysfunction in liver transplanted
patients treated with pegylated-interferon a-2b and ribavirin for hepatitis C recurrence: possible de novo
autoimmune hepatitis? Gut 2007;56:237–42.
Berenguer M, Aguilera V, Prieto M, et al. Significant improvement in the outcome of HCV-infected
transplant recipients by avoiding rapid steroid tapering and potent induction immunosuppression. J
Hepatol 2006; 44:717–22.
Berenguer M, Prieto M, Bustamante M, et al. Incidence of de novo neoplasms after liver transplantation.
Med Clin Barc 1998; 111:481-4.
Berenguer M, Prieto M, Rayón JM, et al. Natural history of clinically compensated hepatitis C virus-related
graft cirrhosis after liver transplantation. Hepatology 2000; 32:852–858.
Berenguer M, Prieto M, San Juan F, et al: Contribution of donor age to the recent decrease in patient
survival among HCV-infected liver transplant recipients. Hepatology 2002; 36:202-210.
Berenguer M, Rayon JM, Prieto M, et al. Are posttransplantation protocol liver biopsies useful in the long
term? Liver Transpl 2001; 7:790–6.
Berenguer M. Host and donor risk factors before and after liver transplantation that impact HCV
recurrence. Liver Transpl 2003; 9:S44–7.
Berenguer M. Risk of extended criteria donors in hepatitis C virus-positive recipients. Liver Transpl 2008;
14 (2):S45–50.
Berenguer M. What determines the natural history of recurrent hepatitis C after liver transplantation? J
Hepatol 2005; 42:448–56.
Berg CL, Gillespie BW, Merion RM, et al. Improvement in survival associated with adult-to-adult living
donor liver transplantation. Gastroenterology 2007; 133:1806–13.
Bernal W, Donaldson N, Wyncoll D, et al. Blood lactate as an early predictor of outcome in paracetamolinduced acute liver failure: a cohort study. Lancet 2002; 359:558–563.
Bernal W, Wendon J. More on serum phosphate and prognosis of acute liver failure. Hepatology 2003;
38:533–534.
Bernat J, D’Alessandro A, Port T, et al. Report of a national conference on donation after cardiac death.
Am J Transplant 2006; 6:281–91.
325
References
Bernuau J, Benhamou JP. Fulminant and subfulminant liver failure. In: McIntyre N, Benhamou JP, Bircher
J, Rizzeto M, Rodes J, editors. Oxford Textbook of Clinical Hepatology. Oxford: Oxford Medical
Publications, 1991:923-942.
Bernuau J, D. Samuel, F. Durand et al., “Criteria for emergency liver transplantation in patients with acute
viral hepatitis and factor V (FV) below 50% of normal: a prospective study (abstract),” Hepatology, vol. 14,
article 49A, 1991.
Bernuau J, Goudeau A, Poynard T, et al. Multivariate analysis of prognostic factors in fulminant hepatitis
B. Hepatology 1986; 6:648- 651.
Berzigotti A, de Gottardi A, Delgado MG, et al. Liver stiffness increases after a meal in patients with
cirrhosis but its change does not correlate with the changes in HVPG (abstract). Hepatology 2010;
52:1074A.
Bessems M, Doorschodt BM, Kolkert JK, et al. Preservation of steatotic livers: a comparison between
cold storage and machine perfusion preservation. Liver Transpl 2007; 13:497–504.
Bianchi G, Marchesini G, Marzocchi R, et al. Metabolic syndrome in liver transplantation: relation to
etiology and immunosuppression. Liver Transpl 2008; 14:1648–54.
Bifari F, Pacelli, L. & Krampera, M. Immunological properties of embryonic and adult stem cells. World J.
Stem Cells 2010; 2 (3):50-60.
Biggins SW, Beldecos A, Rabkin JM, et al. Retransplantation for hepatic allograft failure: prognostic
modeling and ethical considerations. Liver Transpl 2002; 8:313-322.
Biggins SW, Kim WR, Terrault NA, et al. Evidence-based incorporation of serum sodium concentration
into MELD. Gastroenterology 2006; 130:1652–60.
Biggins SW, Rodriguez HJ, Bacchetti P, et al. Serum sodium predicts mortality in patients listed for liver
transplantation. Hepatology 2005; 41:32–39.
Bilbao I, et al. Risk factors for acute renal failure requiring dialysis after liver transplantation. Clin
Transplant 1998; 12:123-129.
Bilzer M, Gerbes AL. Preservation injury of the liver: mechanisms and novel therapeutic strategies. J
Hepatol 2000; 32:508–15.
Bingaman AW, Farber DL. Memory T cells in transplantation: generation, function, and potential role in
rejection. Am J Transplant 2004; 4:846–852.
Bioulac-Sage P, Balabaud C, Ferrell L. Lipopeliosis revisited: should we keep the term? Am J Surg Pathol
2002; 26:134–5.
Bircher J, Benhamou JP, McIntyre N, Rizzetto M, Rodes J, eds. Oxford textbook of clinical hepatology,
2nd edn. Oxford: Oxford University Press, 1999.
Birks EJ Yacoub MH, Burton PS, et al. Activation of apoptotic and inflammatory pathways in dysfunctional
donor hearts. Transplantation 2000; 70:1498-506.
Bismuth H, Houssin D. Reduced-sized orthotopic liver graft in hepatic transplantation in children. Surgery
1984; 95:367–370.
Bismuth H, Samuel D, Castaing D, et al. Liver transplantation in Europe for patients with acute liver
failure. Semin Liver Dis 1997; 16:415-425.
Bittner HB, Chen EP, Craig D, et al. Preload-recruitable stroke work relationships and diastolic
dysfunction in the brain-dead organ donor. Circulation 1996; 94:II320-5.
Bjoro K, Brandsaeter B, Wiencke K, et al. Secondary osteoporosis in liver transplant recipients: a
longitudinal study in patients with and without cholestatic liver disease. Scand J Gastroenterol
2003;38:320-7.
Blakolmer K, Jain A, Ruppert K, et al. Chronic liver allograft rejection in a population treated primarily with
tacrolimus as baseline immunosuppression: longterm follow-up and evaluation of features for
histopathological staging. Transplantation 2000; 69:2330–6.
Boberg KM, Jebsen P, Clausen OP, et al. Cholangiocarcinoma in situ in primary sclerosing cholangitis
diagnosis by brush cytologi and treatment by liver transplantation. J Hepatol 2003; 39:453.
326
References
Boeker KH, Haberkorn CI, Michels D, et al. Diagnostic potential of circulating TIMP-1 and MMP-2 as
markers of liver fibrosis in patients with chronic hepatitis C. Clin Chim Acta 2002;316:71– 81.
Bonet H, R. Manez, D. Kramer et al., “Liver transplantation for alcoholic liver disease: survival of patients
transplanted with alcoholic hepatitis plus cirrhosis as compared with those with cirrhosis alone,”
Alcoholism, vol. 17, no. 5, pp. 1102–1106,1993.
Borroni G, Maggi A, Sangiovanni A, et al. Clinical relevance of hyponatraemia for the hospital outcome of
cirrhotic patients. Dig Liver Dis 2000; 32:605–10.
Bots M, Salonen J, Elwood P et al. Gamma-Glutamyltransferase and risk of stroke: the EUROSTROKE
project Epidemiol Community Health. 2002 February; 56(Suppl 1): i25–i29.
Botta F, Giannini E, Romagnoli P, et al. MELD scoring system is useful for predicting prognosis in
patients with liver cirrhosis and is correlated with residual liver function: a European study. Gut 2003;
52:134–9.
Bouma HR, Ploeg RJ, Schuurs TA, et al. Signal transduction pathways involved in brain death–induced
renal injury. Am J Transplant 2009; 9:989-97.
Boursier J, Konate A, Guilluy M, et al. Learning curve and interobserver reproducibility evaluation of liver
stiffness measurement by transient elastography. Eur J Gastroenterol Hepatol 2008; 20:693–701.
Bowlus CL, Willner I, Zern MA, et al. Factors associated with advanced liver disease in adults with alpha
1-antitrypsin deficiency. Clin Gastroenterol Hepatol 2005; 3:390–396.
Boyacioglu S, Arslan H, Demirhan B, et al. Is there risk of transmitting hepatitis B virus in accepting
hepatitis B core antibody-positive donors for living related liver transplantation? Transplant Proc 2001;
33:2802–2083.
Bozorgzadeh A, Jain A, Ryan C, et al. Impact of hepatitis C viral infection in primary cadaveric liver
allograft versus primary living donor allograft in 100 consecutive liver transplant recipients receiving
tacrolimus. Transplantation 2004; 77:1066–70.
Braillon A. Liver transplantation: from guidelines to randomized controlled trials. Ann Surg. 2010;
252(4):705-6.
Braillon A. Screening for hepatocellular carcinoma: from lack of evidence to common sense. Hepatology
2010; 52:1863-1864.
Brandao A, Fuchs SC, Gleisner AL, et al. MELD and other predictors of survival after liver transplantation.
Clin Transplant 2009; 23:220-227.
Brandhagen D. Liver transplantation for hereditary hemochromatosis. Liver Transpl 2001; 7:663–672.
Brandhagen DJ, Alvarez W, Therneau TM, et al. Iron overload in cirrhosis-HFE genotypes and outcome
after liver transplantation. Hepatology 2000; 31:456–460.
Brandsæter B, Friman S, Broome U, et al. Outcome following liver transplantation for primary sclerosing
cholangitis in the Nordic countries. Scand J Gastroenterol 2003; 38:776–1783.
Brandsæter B, Isoniemi H, Broome´ U, et al. Liver transplantation for primary sclerosing cholangitis;
predictors and consequences of hepatobiliary malignancy. J Hepatol 2004; 40(5):815-22.
Briceno J, Ciria R, Pleguezuelo M, et al. Contribution of marginal donors to liver transplantation for
hepatitis C virus infection. Transplant Proc 2007; 39:2297–2299.
Briceno J, Ciria R, Pleguezuelo M, et al. Impact of donor graft steatosis on overall outcome and viral
recurrence after liver transplantation for hepatitis C virus cirrhosis. Liver Transpl 2009; 15:37–48.
Briceno J, Lopez-Cillero P, Rufian S, et al. Impact of marginal quality donors on the outcome of liver
transplantation. Transplant Proc 1997; 29:477-480.
Briceno J, Marchal T, Padillo J, et al. Influence of marginal donors on liver preservation injury.
Transplantation 2002; 74:522–526.
Briceno J, Padillo J, Rufia´n S, et al. Assignment of steatotic livers by the Mayo model for end-stage liver
disease. Transpl Int 2005; 18:577–583.
Briceno J, Solorzano G, Pera C. A proposal for scoring marginal liver grafts. Transpl Int 2000; 13 [Suppl
1]:S249–S252
327
References
Broelsch C, Whitington PF, Emond JC, et al. Liver transplantation in children from living related donors.
Surgical techniques and results. Ann Surg 1991; 214:428–437.
Broelsch CE, Emond JC, Thistlethwaite JR, et al. Liver transplantation with reduced-size donor organs.
Transplantation 1988; 45:519–524
Broering DC, Schulte AM Esch J, et al. Consequences of anatomy for the split liver surgeon. In: Rogiers
X, Bismuth H, Busuttil RW, Broering DC, Azoulay D, editors. Split Liver Transplantation. Darmstadt:
Springer; 2002:46-62.
Broering DC, Sterneck M, Rogiers X. Living donor liver transplantation. J Hepatol 2003; 38 (1):S119-35.
Broering DC, Topp S, Schaefer U, et al. Split liver transplantation and risk to the adult recipient: analysis
using matched pairs. J Am Coll Surg 2002; 195:648-57.
Broering DC, Walter J, Rogiers X. The first two cases of living donor liver transplantation using dual grafts
in Europe. Liver Transpl 2007; 13:149-53.
Bronk SF, Gores GJ. Efflux of protons from acidic vesicles contributes to cytosolic acidification of
hepatocytes during ATP depletion. Hepatology 1991; 14(4 Pt 1): 626–633.
Bronster DJ, Emre S, Boccagni P, et al: Central nervous system complications in liver transplant
recipients: incidence, timing, and long-term follow-up. Clin Transplant 2000;14: 1-7.
Brown Jr RS, et al. Model for end-stage liver disease and Child–Turcotte–Pugh score as predictors of
pretransplantation disease severity, posttransplantation outcome, and resource utilization in United
Network for Organ Sharing status 2A patients. Liver Transpl 2002; 8: 278–84.
Brown RS Jr, Russo MW, Lai M, et al. A survey of liver transplantation from living adult donors in the
United States. N Engl J Med 2003; 348:818–825.
Brown RS. Hepatitis C and liver transplantation. Nature 2005; 436(7053):973–8.
Bruix J, Sherman M, Llovet JM, et al. Clinical management of hepatocellular carcinoma. Conclusions of
the Barcelona-2000 EASL conference. European Association for the Study of the Liver. J Hepatol 2001;
35:421-430
Bruno S, Stroffolini T, Colombo M, et al. Sustained virological response to interferon-alpha is associated
with improved outcome in HCV-related cirrhosis: a retrospective study. Hepatology 2007; 45:5 79–587.
Brunt EM, Janney CG, Di Bisceglie AM, et al. Nonalcoholic steatohepatitis: a proposal for grading and
staging the histological lesions. Am J Gastroenterol 1999; 94(9):2467–74.
Bucuvalas JC, Campbell KM, Cole CR, et al: Outcomes after liver transplantation: keep the end in mind. J
Pediatr Gastroenterol Nutr 2006; 43(1):S41-48.
Buell JF, Alloway RR, Steve Woodle E. How can donors with a previous malignancy be evaluated? J
Hepatol 2006;45:503–7.
Burdelski M, Ringe B, Rodeck B, et al. [Indications and results of liver transplantation in childhood].
Monatsschr Kinderheilkd 1988; 136:317–322.
Burke A, Lucey MR. Non-alcoholic fatty liver disease, non-alcoholic steatohepatitis and orthotopic liver
transplantation. Am J Transplant 2004; 4:686–93.
Burra P, Loreno M, Russo FP, et al. Donor livers with steatosis are safe to use in hepatitis C viruspositive recipients. Liver Transpl 2009; 15:619–628.
Burra P, Lucey MR. Liver transplantation in alcoholic patients. Transpl Int 2005; 18: 491–8.
Burra P, Mioni D, Cecchetto A, et al. Histological features after liver transplantation in alcoholic cirrhotics.
J Hepatol 2001; 34:716–22.
Burra P, Porte RJ. Should donors and recipients be matched in liver transplantation? Forum on Liver
Transplantation / Journal of Hepatology 2006; 45: 483–513.
Burra P, Smedile A, Angelico M, et al. Liver transplantation in Italy: current status. Study Group on Liver
Transplantation of the Italian Association for the Study of the Liver (A.I.S.F.). Dig Liver Dis 2000; 32:249.
Burroughs AK, Sabin CA, Rolles K, et al. European Liver Transplant Association. 3-month and 12-month
mortality after first liver transplant in adults in Europe: predictive models for outcome. Lancet 2006;
367:225–232.
328
References
Burton J, Rosen H. Diagnosis and management of allograft failure. Clin Liver Dis 2006; 10:407–435.
Busquets J, Xiol X, Figueras J et al. The impact of donor age on liver transplantation: influence of donor
age on early liver function and on subsequent patient and graft survival. Transplantation 2001; 71:1765–
1771.
Busuttil R, Tanaka K. The utility of marginal donors in liver transplantation. Liver Transpl 2003; 9:651–63.
Cai J, Li W, Su H, et al. Generation of human induced pluripotent stem cells from umbilical cord matrix
and amniotic membrane mesenchymal cells. J Biol Chem 2010; 285:11227-11234.
Cakaloglu Y, Devlin J, O’Grady J, et al. Importance of concomitant viral infection during late acute liver
allograft rejection. Transplantation 1995;59: 40–5.
Caldwell SH, Oelsner DH, Iezzoni JC, et al. Cryptogenic cirrhosis: clinical characterization and risk factors
for underlying disease. Hepatology 1999; 29:664–669.
Cales P, Oberti F, Michalak S, et al. A novel panel of blood markers to assess the degree of liver fibrosis.
Hepatology 2005; 42(6):1373–81.
Cales P, Veillon P, Konate A, et al. Reproducibility of blood tests of liver fibrosis in clinical practice. Clin
Biochem 2008; 41(1–2):10–8.
Callewaert N, Van Vlierberghe H, Van Hecke A, et al. Noninvasive diagnosis of liver cirrhosis using DNA
sequencer-based total serum protein glycomics. Nat Med 2004; 10:429-434.
Calne RY, Sells RA, Pena JR, et al. Induction of immunological tolerance by porcine liver allografts.
Nature 1969; 223:472-476.
Cameron A, Busuttil RW. AASLD/ILTS transplant course: is there an extended donor suitable for
everyone? Liver Transpl 2005; 11(2):S2–S5.
Cameron AM, Ghobrial RM, Yersiz H, et al. Optimal utilization of donor grafts with extended criteria: a
single-center experience in over 1000 liver transplants. Annals of Surgery 2006; 243(6):748-753.
Candinas D, Joller-Jemelka HI, Schlumpf R, et al. RNA prevalence in a Western European organ donor
pool and virus transmission by organ transplantation. J Med Microbiol 1994; 41:220–223.
Cantarovich M, Tzimas GN, Barkun J, et al. Efficacy of mycophenolate mofetil combined with very lowdose cyclosporine microemulsion in long-term liver-transplant patients with renal dysfunction.
Transplantation 2003; 76:98-102.
Cardillo M, De Fazio N, Pedotti P, et al. Split and whole liver transplantation outcomes: a comparative
cohort study. Liver Transpl 2006; 12:402–10.
Carey EJ, Balan V, Kremers WK, et al. Osteopenia and osteoporosis in patients with end-stage liver
disease caused by hepatitis C and alcoholic liver disease: not just a cholestatic problem. Liver Transpl
2003; 9:1166-1173.
Carey WD, Dumot JA, Pimentel RR, et al. The prevalence of coronary artery disease in liver transplant
candidates over age 50. Transplantation 1995; 59:859-864.
Carini R, Autelli R, Bellomo G, et al. Alterations of cell volume regulation in the development of
hepatocyte necrosis. Exp Cell Res 1999; 248:280–293.
Carpentier B, Gautier A. & Legallais C. Artificial and bioartificial liver devices: present and future. Gut
2009; 58:1690-1702.
Carrasco L, Sanchez-Bueno F, Sola J, et al. Effects of cold ischemia time on the graft after orthotopic
liver transplantation. A bile cytological study. Transplantation 1996; 61:393–6.
Carrodeguas L, Orosz CG, Waldman WJ, et al. Transvivo analysis of human delayed-type
hypersensitivity reactivity. Hum Immunol 1999; 60:640-651.
Casavilla A, Ramirez C, Shapiro R, et al. Experience with liver and kidney allografts from non-heartbeating donors. Transplantation 1995; 59:197–203.
Castel H, C. Moreno, T. Antonini, J. Duclos-Vallee, J. Dumortier, and V. Leroy, “Early transplantation
improves survival of non-responders to steroids in severe alcoholic hepatitis: a challenge to the 6 month
rule of abstinence,” Hepatology, vol. 4, pp. 307A–308A, 2009.
329
References
Castells A, Bruix J, Bru C, et al. Treatment of small hepatocellular carcinoma in cirrhotic patients: a cohort
study comparing surgical resection and percutaneous ethanol injection. Hepatology 1993; 18:1121-1126.
Castells L, Vargas V, Rodriguez-Frias F et al. Transmission of hepatitis B virus by transplantation of livers
from donors positive for antibody to hepatitis B core antigen. Transplant Proc 1999; 31: 2464–2465.
Castera L, Foucher J, Bernard PH, et al. Pitfalls of liver stiffness measurement: a 5-year prospective
study of 13,369 examinations. Hepatology 2010; 51:828–835.
Castera L, Negre I, Samii K et al. Pain experienced during percutaneous liver biopsy. Hepatology 1999;
30(6):1529–30.
Castera L, Negre I, Samii K, et al. Patient-administered nitrous oxide/oxygen inhalation provides safe and
effective analgesia for percutaneous liver biopsy: a randomizedplacebo-controlled trial. Am J
Gastroenterol 2001; 96(5):1553–7.
Castera L, Vergniol J, Foucher J, et al. Prospective comparison of transient elastography, Fibrotest,
APRI, and liver biopsy for the assessment of fibrosis in chronic hepatitis C. Gastroenterology 2005;
128:343–350.
Cerutti E, Stratta C, Romagnoli R, et al. Bacterial- and fungal-positive cultures in organ donors: clinical
impact in liver transplantation. Liver Transpl 2006; 12:1253–1259.
Cha CH, Ruo L, Fong Y, et al. Resection of hepatocellular carcinoma in patients otherwise eligible for
transplantation. Ann Surg 2003; 238:315-321.
Cha I, Bass N, Ferrell LD. Lipopeliosis. An immunohistochemical and clinicopathologic study of five
cases. Am J Surg Pathol 1994; 18:789–95.
Chahin NJ, De Carlis L, Slim AO, et al. Long-term efficacy of endoscopic stenting in patients with stricture
of the biliary anastomosis after orthotopic liver transplantation. Transplant Proc 2001; 33:2738–40.
Chakrapani A, Sivakumar P, McKiernan PJ, et al. Metabolic stroke in methylmalonic acidemia five years
after liver transplantation. J Pediatr 2002; 140:261-263.
Chalasani G, Dai Z, Konieczny BT, et al. Recall and propagation of allospecific memory T cells
independent of secondary lymphoid organs. Proc Natl Acad Sci USA 2002; 99:6175–6180.
Chalmers RJ, Kirby B, Smith A, et al. Replacement of routine liver biopsy by procollagen III aminopeptide
for monitoring patients with psoriasis receiving long-term methotrexate: a multicentre audit and health
economic analysis. Br J Dermatol 2005; 152:444–450.
Chambers I, Colby D, Robertson M, et al. Functional expression cloning of Nanog, a pluripotency
sustaining factor in embryonic stem cells. Cell 2003; 113:643-655.
Chan E.M, Ratanasirintrawoot S, Park I.H, et al. Live cell imaging distinguishes bona fide human iPSC
from partially reprogrammed cells. Nat Biotechnol 2009; 27:1033-1037.
Chan HL, Wong GL, Choi PC, et al. Alanine aminotransferase-based algorithms of liver stiffness
measurement by transient elastography (Fibroscan) for liver fibrosis in chronic hepatitis B. J Viral Hepat
2009; 16:36–44.
Chapman MJ, Goldstein S, Lagrange D, et al. A density gradient ultracentrifugal procedure for the
isolation of the major lipoprotein classes from human serum. J Lipid Res. 1981;22:339-58.
Chari RS, Collins BH, Magee JC. Brief report: treatment of hepatic failure with ex vivo pig-liver perfusion
followed by liver transplantation. N Engl J Med 1994; 331:234–7.
Charlton M, K. Ruppert, S. H. Belle et al., “Long-term results and modeling to predict outcomes in
recipients with HCV infection: results of the NIDDK liver transplantation database,” Liver Transplantation,
vol. 10, no. 9, pp. 1120–1130, 2004.
Charlton M. Hepatitis C infection in liver transplantation. Am J Transplant 2001; 1:197-203.
Chazouillères O, Calmus Y, Vaubourdolle M, et al. Preservation-induced liver injury. Clinical aspects:
mechanisms and therapeutic approaches. Journal of Hepatology 1993; 18(1):123-134.
Chen C.H. & Chen, R.J. Prevalence of telomerase activity in human cancer. J. Formos. Med. Association
2011; 110(5):175-189.
Chen EP, Bittner HB, Kendall SW, et al. Hormonal and hemodynamic changes in a validated animal
model of brain death. Crit Care Med 1996; 24:1352-9.
330
References
Chen H, et al. Multi-factor analysis of initial poor graft function after orthotopic liver transplantation.
Hepatobiliary & Pancreatic Diseases International 2007; 6(2):141-146.
Chen JM, Cullinane S, Spanier TB, et al. Vasopressin deficiency and pressor hypersensitivity in
hemodynamically unstable organ donors. Circulation 1999; 100(19):II244-6.
Cheng SJ, Pratt DS, Freeman RB, et al. Living-donor versus cadaveric liver transplantation for nonresectable small hepatocellular carcinoma and compensated cirrhosis: a decision analysis.
Transplantation 2001; 72:861–8.
Cheng YF, Chen CL, Jawan B, et al. Multislice computed tomography angiography in pediatric liver
transplantation. Transplantation 2003; 76:353-357.
Cheng YF, Peng CH, Shen BY, et al: Risk factors for intraoperative portal vein thrombosis in pediatric
living donor liver transplantation. Clin Transplant 2004; 18:390-394.
Choi PC, Kim HJ, Choi WH, et al. Model for end-stage liver disease, model for end-stage liver diseasesodium and Child-Turcotte-Pugh scores over time for the prediction of complications of liver cirrhosis.
Liver Int 2009;29:221-6.
Cholongitas E, Marelli L, Kerry A, et al. Different methods of creatinine measurement significantly affect
MELD scores. Liver Transpl 2007; 13:523-9.
Cholongitas E, Marelli L, Kerry A, et al. Different methods of creatinine measurement significantly affect
MELD scores. Hepatology 2005; 42:204A.
Cholongitas E, Marelli L, Kerry A, et al. Female liver transplant recipients with the same GFR as male
recipients have lower MELD scores- a systematic bias. Am J Transpl 2007; 7:685–92.
Cholongitas E, Papatheodoridis GV, Vangeli M, et al. Systematic review: The model for end-stage liver
disease—should it replace Child–Pugh’s classification for assessing prognosis in cirrhosis? Aliment
Pharmacol Ther 2005; 22:1079–89.
Christensen C, Bruden D, Livingston S, et al. Diagnostic accuracy of a fibrosis serum panel (FIBROSpect
II) compared with Knodell and Ishak liver biopsy scores in chronic hepatitis C patients. J Viral Hepat
2006; 13:652– 658.
Christensen E. Prognostic models including the Child-Pugh, MELD and Mayo risk scores—Where are we
and where should we go? J Hepatol 2004; 41:344-350.
Chui AK, Koorey D, Pathania OP,et al. Polycystic disease: a rare indication for combined liver and kidney
transplantation. Hong Kong Med J 2000;6:116-118.
Chui AK, Shi LW, Rao AR, et al. Primary graft dysfunction after liver transplantation. Transplant Proc
2000; 32:2219-2220.
Chung, HY, Chan SC, Lo CM. & Fan ST. Strategies for widening liver donor pool. Asian Journal of
Surgery 2010; 33(2):63-69.
Ciccarelli O, Goffette P, Laterre PF, et al. Transjugular intrahepatic portosystemic shunt approach and
local thrombolysis for treatment of early posttransplant portal vein thrombosis. Transplantation 2001;
72:159-161.
Ciccarelli O, Kaczmarek B, Roggen F, et al. Long-term medical complications and quality of life in adult
recipients surviving 10 years or more after liver transplantation. Acta Gastroenterol Belg 2005; 68: 323–
30.
Cicinnati VR, Yu Z, Klein CG, et al. Clinical trial: switch to combined mycophenolate mofetil and minimal
dose calcineurin inhibitor in stable liver transplant patients-assessment of renal and allograft function,
cardiovascular risk factors and immune monitoring. Aliment Pharmacol Ther 2007; 26(9):1195-208.
Clavien PA, Camargo CA Jr, Croxford R, Langer B, Levy GA, Greig PD. Definition and classification of
negative outcomes in solid organ transplantation. Application in liver transplantation. Ann Surg 1994;
220:109-20.
Clavien PA, Harvey PR, Strasberg SM. Preservation and reperfusion injuries in liver allografts. An
overview and synthesis of current studies. Transplantation 1992; 53:957–978.
Clavien, PA. (1998) Sinusoidal endothelial cell injury during hepatic preservation and reperfusion.
Hepatology, Vol.28, No.2, (August 1998), pp. 281-285, ISSN: 1665-2681.
331
References
Coco B, Oliveri F, Maina AM, et al. Transient elastography: a new surrogate marker of liver fibrosis
influenced by major changes of transaminases. J Viral Hepat 2007; 14:360–369.
Cocucci E, Racchetti G, Meldolesi J. Shedding microvesicles: artefacts no more. Trends Cell Biol
2009;19(2):43-51.
Colina F, Juca NT, Moreno E, et al. Histological diagnosis of cytomegalovirus hepatitis in liver allografts. J
Clin Pathol 1995; 48:351–7.
Colle IO, Moreau R, Godinho E, et al. Diagnosis of portopulmonary hypertension in candidates for liver
transplantation: a prospective study. Hepatology 2003; 37:401-409.
Collins BH, Pirsch JD, Becker YT, et al. Long-term results of liver transplantation in older patients 60
years of age and older. Transplantation 2000; 70:780- 783.
Collisson EA, Nourmand H, Fraiman MH, et al. Retrospective analysis of the results of liver
transplantation for adults with severe hepatopulmonary syndrome. Liver Transpl 2002; 8:925-931.
Colombo M, Del Ninno E, de Franchis R, et al. Ultrasoundassisted percutaneous liver biopsy: superiority
of the Tru-Cut over the Menghini needle for diagnosis of cirrhosis. Gastroenterology 1988; 95:487–489.
Colonna JO 2nd, Shaked A, Gomes AS, et al. Biliary strictures complicating liver transplantation.
Incidence, pathogenesis, management, and outcome. Ann Surg 1992; 216:344–50.
Compagnon P, Wang H, Lindell SL, et al. Brain death does not affect hepatic allograft function and
survival after orthotopic transplantation in a canine model. Transplantation 2002; 73:1218–1227.
Condron SL, Heneghan MA, Patel K, et al. Effect of donor age on survival of liver transplantation
recipients with hepatitis C infection. Transplantation 2005; 80:145–148.
Conn HO Burdelski M, Lautz H-U, et al. Predictors of one-year pretransplant survival in patients with
cirrhosis. Hepatology 1991; 14:1029-1034.
Conn HO. A peek at the Child-Turcotte classification. Hepatology 1981; 1:673-676.
Contos MJ, Cales W, Sterling RK, et al. Development of nonalcoholic fatty liver disease after orthotopic
liver transplantation for cryptogenic cirrhosis. Liver Transpl 2001; 7:363–73.
Cooper DK, Novitzky D, Wicomb WN, et al. A review of studies relating to thyroid hormone therapy in
brain-dead organ donors. Front Biosci 2009; 14:3750-70.
Cornberg M, Protzer U, Dollinger MM, et al. The German guideline for the management of hepatitis B
virus infection: short version. J Viral Hepatitis 2008; 15:1-20.
Corrao G, Arico S. Independent and combined action of hepatitis C virus infection and alcohol
consumption on the risk of symptomatic liver cirrhosis. Hepatology 1998; 27:914–919.
Correia MITD, Rego LO, Lima AS. Post-liver transplant obesity and diabetes. Curr Opin Clin Nutr Metab
Care 2003; 4: 457-60.
Cosimi AB, Burton RC, Colvin RB, et al. Treatment of acute renal allograft rejection with OKT3
monoclonal antibody. Transplantation. 1981; 32(6):535-9.
Coss E, Watt KDS, Pedersen R, et al. Predictors of cardiovascular events after liver transplantation: a
role for pretransplant serum troponin levels. Liver Transpl 2011; 17:23-31.
Cotton CL, Gandhi S, Vaitkus PT, et al. Role of echocardiography in detecting portopulmonary
hypertension in liver transplant candidates. Liver Transpl 2002; 8:1051-1054.
Couinaud C, Houssin D. Analysis of the anatomical difficulties of bipartition. Paris: Couinaud; 1991.
Couinaud C. Surgical Anatomy of the Liver Revisited. Paris: Couinaud; 1989.
Couinaud C: Le Foie: Etudes Anatomiques et Chirurgicales. Paris: Masson; 1957
Créput C, Blandin F, Deroure B, et al. Long-term effects of calcineurin inhibitor conversion to
mycophenolate mofetil on renal function after liver transplantation. Liver Transpl 2007;13(7):1004-10.
Crippin JS, McCashland T, Terrault N, et al. A pilot study of the tolerability and efficacy of antiviral therapy
in hepatitis C virus-infected patients awaiting liver transplantation. Liver Transpl 2002; 8:350-5.
Cross S, Harrison RF, Kennedy RL. Introduction to neural networks. Lancet 1995; 346:1075–9.
332
References
Cucchetti A, Vivarelli M, Heaton ND, et al. Artificial neural network is superior to MELD in predicting
mortality of patients with end-stage liver disease. Gut 2007; 56:253–8.
Cuende N, Miranda B, Canon JF, et al: Donor characteristics associated with liver graft survival.
Transplantation 2005; 79:1445.
Curley SA, Izzo F, Ellis LM, et al. Radiofrequency ablation of hepatocellular cancer in 110 patients with
cirrhosis. Ann Surg 2000; 232:381-391.
Curry MP, “Hepatitis B and hepatitis C viruses in liver transplantation,” Transplantation, vol. 78, no. 7, pp.
955–963, 2004.
Cursio R, Gugenheim J. Ischemia-Reperfusion Injury and Ischemic-Type Biliary Lesions following Liver
Transplantation. J Transplant. 2012;2012:164329.
Cutrin JC, Cantino D, Biasi F, Chiarpotto E, Salizzoni M, Andorno E, Massano G, Lanfranco G, Rizzetto
M, Boveris A, Poli G. Reperfusion damage to the bile canaliculi in transplanted human liver. Hepatology.
1996;24:1053-7.
Czaja AJ, Carpenter HA. Histological findings in chronic hepatitis C with autoimmune features.
Hepatology 1997; 26:459–66.
Czaja AJ, Carpenter HA. Sensitivity, specificity, and predictability of biopsy interpretations in chronic
hepatitis. Gastroenterology 1993; 105:1824–32.
D’Antiga L et al 2002,. Preservation injury of the liver: mechanisms and novel therapeutic strategies. J
Hepatol 2000;32:508–15.
D’Antiga L, Dhawan A, Portmann B, et al. Late cellular rejection in paediatric liver transplantation:
aetiology and outcome. Transplantation 2002; 73:80–4.
da Silva RF, Raphe R, Felício HC, et al: Prevalence, treatment, and outcomes of the hepatic artery
stenosis after liver transplantation. Transplant Proc 2008; 40:805-807.
Dai YD, Carayanniotis G, Sercarz E. Antigen processing by autoreactive B cells promotes determinant
spreading. Cell Mol Immunol 2005; 2:169–75.
D'Alessandro AM, Hoffmann RM, Knechtle SJ, et al. Liver transplantation from controlled non-heartbeating donors. Surgery 2000; 128 (4): 579-588.
Das S.K., Dhanya L., Vasudevan D.M.
Scand.J.Clin.Lab.Invest. 2008; 68, 81–92.
Biomarkers
of
alcoholism:
an
updated
review.
Davidson CJ, Gheorghiade M, Flaherty JD, et al. Predictive value of stress myocardial perfusion imaging
in liver transplant candidates. Am J Cardiol 2002; 89:359-360.
Davis CL, Gonwa TA, Wilkinson AH. Pathophysiology of renal disease associated with liver disorders:
implications for liver transplantation. Part I. Liver Transpl 2002; 8:91-109.
De Boer MT, Molenaar IQ, Hendriks HG, et al. Minimizing blood loss in liver transplantation: progress
through research and evolution of techniques. Dig Surg 2005; 22: 265-75.
De Carlis L, Colella G, Sansalone CV, et al. Marginal donors in liver transplantation: the role of donor
age. Transplant Proc 1999; 31:397–400.
de Hemptinne B, Salizzoni M, Yandza TC, et al. Indication, technique, and results of liver graft volume
reduction before orthotopic transplantation in children. Transplant Proc 1987; 19:3549–3551.
de Rougemont O, Breitenstein S, Leskosek B, et al. One hour hypothermic oxygenated perfusion (HOPE)
protects nonviable liver allografts donated after cardiac death. Ann Surg 2009; 250:674–83.
de Rougemont O, Dutkowski P, Clavien PA. Biological modulation of liver ischemia-reperfusion injury.
Curr Opin Organ Transplant; 15:183–189.
de Vera ME, Lopez-Solis R, Dvorchik I, et al. Liver transplantation using donation after cardiac death
donors: long-term follow-up from a single center. American Journal of Transplantation 2009; 9(4): 773781.
De Vreede J, Steers JL, Burch PA, et al. Prolonged disease-free survival after orthotopic liver
transplantation plus adjuvant chemoirradiation for CC. Liver Transpl 2000; 6:309–316.
333
References
DeBakey ME, Lawrie GM, Glaeser DH. Patterns of atherosclerosis and their surgical significance. Ann
Surg 1985; 201:115–131.
Dec GW, Kondo N, Farrell ML, et al. Cardiovascular complications following liver transplantation. Clin
Transplant 1995; 9:463-471.
Degott C, Rueff B, Kreis H, et al. Peliosis hepatis in recipients of renal transplants. Gut 1978; 19:748–53.
Del Gaudio M, Grazi GL, Ercolani G, et al. Outcome of hepatic artery reconstruction in liver
transplantation with an iliac arterial interposition graft. Clin Transplant 2005; 19:399-405.
del Olmo JA, Flor-Lorente B, Flor-Civera B, et al. Risk factors for nonhepatic surgery in patients with
cirrhosis. World J Surg 2003; 27:647– 52.
DeLeve LD, Shulman HM, McDonald GB. Toxic injury to hepatic sinusoids: sinusoidal obstruction
syndrome (veno-occlusive disease). Semin Liver Dis 2002; 22:27–42.
Della-Guardia B, Almeida MD, Meira-Filho SP, et al. Antibody-mediated rejection: hyperacute rejection
reality in liver transplantation? A case report. Transplant Proc 2008; 40:870–1.
DeLong ER, DeLong DM, Clarke-Pearson DL. Comparing the areas under two or more correlated
receiver operating characteristic curves: a nonparametric approach. Biometrics 1988; 44: 837-45.
Deltenre P, Valla DC. Ischemic cholangiopathy. J Hepatol 2006; 44:806–17.
Demetris A, CJ, Minervini M, et al Transplantation pathology of the liver. In: In: Odze RD, Goldblum JR,
eds. Surgical pathology of the GI tract, liver, biliary tract and pancreas. Philadelphia: Saunders Elsevier,
2009:1177–80.
Demetris A, et al, Update of the International Banff Schema for Liver Allograft Rejection: working
recommendations for the histopathologic staging and reporting of chronic rejection: an international panel.
Hepatology 2000;31:792-799.
Demetris A, TA, Delaney CP, et al. Pathology of liver transplantation In: Busuttil R, KG, eds.
Transplantation of the liver. Philadelphia: WB Saunders, 1996:681–723.
Demetris AJ, a2006 , Fontes P, Lunz JG 3rd, et al. Wound healing in the biliary tree of liver allografts. Cell
Transplant 2006;15(Suppl 1):S57–65.
Demetris AJ, Adeyi O, Bellamy CO, et al. Liver biopsy interpretation for causes of late liver allograft
dysfunction. Hepatology 2006; 44:489–501.
Demetris AJ, b2006, Lunz JG 3rd, Specht S, et al. Biliary wound healing, ductular reactions, and IL6/gp130 signaling in the development of liver disease. World J Gastroenterol 2006;12:3512–22.
Demetris AJ, Jaffe R, Sheahan DG, et al. Recurrent hepatitis B in liver allograft recipients. Differentiation
between viral hepatitis B and rejection. Am J Pathol 1986;125:161–72.
Demetris AJ, Jaffe R, Starzl TE. A review of adult and pediatric post-transplant liver pathology. Pathol
Annu 1987; 22:347–86.
Demetris AJ, Jaffe R, Tzakis A, et al. Antibody-mediated rejection of human orthotopic liver allografts. A
study of liver transplantation across ABO blood group barriers. Am J Pathol 1988; 132:489–502.
Demetris AJ, Kelly DM, Eghtesad B, et al. Pathophysiologic observations and histopathologic recognition
of the portal hyperperfusion or small-for-size syndrome. Am J Surg Pathol 2006; 30:986–93
Demetris AJ, Lunz JG 3rd. Early HCV-associated stellate cell activation in aggressive recurrent HCV:
what can liver allografts teach about HCV pathogenesis? Liver Transpl 2005; 11:1172–6.
Demetris AJ, Murase N, Lee R.G, et al. Chronic rejection. A general overview of histopathology and
pathophysiology with emphasis on liver, heart and intestinal allografts. Ann Transplant 1997; 2(2):27-44.
Demetris AJ, Murase N, Nakamura K, et al. Immunopathology of antibodies as effectors of orthotopic liver
allograft rejection. Semin Liver Dis 1992; 12:51–9.
Demetris AJ, Sebagh M. Plasma cell hepatitis in liver allografts: Variant of rejection or autoimmune
hepatitis? Liver Transpl 2008; 14:750–5.
Demetris AJ. Central venulitis in liver allografts: considerations of differential diagnosis. Hepatology 2001;
33:1329-30.
334
References
Demetris AJ. Distinguishing between recurrent primary sclerosing cholangitis and chronic rejection. Liver
Transpl 2006; 12(11 Suppl 2): S68–72.
Demetris AJ. Spectrum of chronic hepatic allograft rejection and arteriopathy and the controversy of
centrilobular necrosis. Liver Transpl 2000; 6: 102-3.
Demetris r AJ, CJ, Minervini MI, et al. Transplantation pathology of the liver. In: Odze RD, Goldblum JR,
eds. Surgical pathology of the GI tract, liver, biliary tract, and pancreas. 2nd edn. Philadelphia:
Saunders/Elsevier, 2009:1169–229.
Demirhan B, Bilezikci B, Haberal AN, et al. Hepatic parenchymal changes and histologic eosinophilia as
predictors of subsequent acute liver allograft rejection. Liver Transpl 2008; 14:214–219.
Desai M, Neuberger J. Chronic liver allograft dysfunction. Transplant Proc 2009;41:773–6.
Deschenes M, Belle SH, Krom RA,et al. Early allograft dysfunction after liver transplantation: a definition
and predictors of outcome. National Institute of Diabetes and Digestive and Kidney Diseases Liver
Transplantation Database. Transplantation 1998; 15(66): 302-10.
Desmet VJ, Roskams T. Cirrhosis reversal: a duel between dogma and myth. J Hepatol 2004; 40: 860–
67.
Detre KM, Lombardero M, Belle S, et al. Influence of donor age on graft survival after liver
transplantation: United Network for Organ Sharing registry. Liver Transpl Surg 1995; 1:311–31932.
Detry O, Bonnet P, Honore P, et al. What is the risk of transferral of an undetected neoplasm during
organ transplantation? Transplant Proc 1997; 29: 2410–2411.
Devlin J, O’Grady J. Indications for referral and assessment in adult liver transplantation: a clinical
guideline. British Society of Gastroenterology. Gut 1999; 45 Suppl 6: VI1-VI22.
Dew MA, DiMartini AF, Steel J, et al. Meta-analysis of risk for relapse to substance use after
transplantation of the liver or other solid organs. Liver Transpl 2008; 14:159-172.
Dhar R, Young GB, Marotta P: Perioperative neurological complications after liver transplantation are
best predicted by pre-transplant hepatic encephalopathy. Neurocrit Care 2008; 8:253-258.
Dhawan AR, Taylor M, Cheeseman P, et al. Wilson’s disease in children: 37-year experience and revised
King’s for liver transplantation. Liver Transplantation 2005; 11(4): 441– 448.
Di Sandro S, Slim AO, Giacomoni A et al. Living donor liver transplantation for hepatocellular carcinoma:
long-term results compared with deceased donor liver transplantation. Transplant. Proc. 2009; 41: 1283–
5.
Dickson ER, Grambsch PM, Fleming TR, et al. Prognosis in primary biliary cirrhosis: model for decision
making. Hepatology 1989; 10:1-7.
Dickson ER, Murtaugh PA, Wiesner RH, et al. Primary sclerosing cholangitis: refinement and validation of
survival models. Gastroenterology 1992; 103:1893-1901.
Diedrich DA, Findlay JY, Harrison BA, et al. Influence of coronary artery disease on outcomes after liver
transplantation. Transplant Proc 2008; 40:3554-3557.
DiMartini A, Fontes P, Dew MA, et al: Age, Model for End-stage Liver Disease score, and organ
functioning predict posttransplant tacrolimus neurotoxicity. Liver Transpl 2008; 14:815-822.
DiMartini A, N. Day, M. A. Dew et al. Alcohol consumption patterns and predictors of use following liver
transplantation for alcoholic liver disease. Liver Transplantation 2006; 12(5):813–820.
Dixon LR, Crawford JM. Early histologic changes in fibrosing cholestatic hepatitis C. Liver Transpl 2007;
13:219–26.
Dmitrewski J, Hubscher SG, Mayer AD, et al. Recurrence of primary biliary cirrhosis in the liver allograft:
the effect of immunosuppression. J Hepatol 1996; 24:253–7.
Dodson SF, Issa S, Araya V et al. Infectivity of hepatic allografts with antibodies to hepatitis B virus.
Transplantation 1997; 64: 1582–1584
Dollinger MM, Howie SE, Plevris JN, Graham AM, Hayes PC, Harrison DJ. Intrahepatic proliferation of
'naïve' and 'memory' T cells during liver allograft rejection: primary immune response within the allograft.
FASEB J. 1998;12(11):939-47.
335
References
Dominici M, Le Blanc K, Mueller I, et al. Minimal criteria for defining multipotent mesenchymal stromal
cells. The International Society for Cellular Therapy Position Statement. Cytotherapy 2006; 8(4): 315-317.
Donataccio D, Roggen F, De Reyck C, Verbaandert C, Bodeus M, Lerut J. Use of anti-HBc positive
allografts in adult liver transplantation: toward a safer way to expand the donor pool. Transpl Int 2006;
19: 38–43.
Donnelly KL, Smith CI, Schwarzenberg SJ,et al. Sources of fatty acids stored in liver and secreted via
lipoproteins in patients with nonalcoholic fatty liver disease. J Clin Invest 2005; 115: 1343–1351.
Donovan CL, Marcovitz PA, Punch JD, et al. Two-dimensional and dobutamine stress echocardiography
in the preoperative assessment of patients with end-stage liver disease prior to orthotopic liver
transplantation. Transplantation 1996; 61:1180-1188.
Douglas DD, Rakela J, Wright TL, et al. The clinical course of transplantation-associated de novo
hepatitis B infection in the liver transplant recipient. Liver Transpl Surg 1997; 3: 105–111
Dousset B, Conti F, Cherruau B, et al. Is acute rejection deleterious to long-term liver allograft function? J
Hepatol 1998; 29:660–8.
Dresske B, Lin X, Huang DS, Zhou X, et al. Spontaneous tolerance: experience with the rat liver
transplant model. Hum Immunol. 2002; 63(10):853-61.
Drukker, M.; Katchman, H.; Katz, G.; et al. Human embryonic stem cells and their differentiated
derivatives are less susceptible to immune rejection than adult cells. Stem Cells 2006; 24:221–229.
Dubbeld J, Hoekstra H, Farid W, et al. Similar liver transplantation survival with selected cardiac death
donors and brain death donors. Br J Surg 2010; 97:744-53.
Duclos-Vallee JC, Sebagh M. Recurrence of autoimmune disease, primary sclerosing cholangitis, primary
biliary cirrhosis, and autoimmune hepatitis after liver transplantation. Liver Transpl. 2009;15 Suppl 2:S2534.
Duffy JP, Hong JC, Farmer DG, et al. Vascular complications of orthotopic liver transplantation:
experience in more than 4,200 patients. J Am Coll Surg 2009; 208:896-903.
Duffy JP, Vardanian A, Benjamin E, et al. Liver transplantation criteria for hepatocellular carcinoma
should be expanded: a 22- year experience with 467 patients at UCLA. Ann Surg 2007; 246: 502–509.
Durand F, Ettorre GM, Douard R, et al. Donor safety in living related liver transplantation: underestimation
of the risks for deep vein thrombosis and pulmonary embolism [see comment]. Liver Transpl 2002; 8:11820.
Durand F, Renz JF, Alkofer B et al. Report of the Paris consensus meeting on expanded criteria donors in
liver transplantation. Liver Transpl 2008; 14:1694–1707.
Durand F, Valla D. Assessment of prognosis of cirrhosis. Semin Liver Dis 2008; 28:110-122.
Durand F, Valla D. Assessment of the prognosis of cirrhosis: Child-Pugh versus MELD. J Hepatol 2005;
42(Suppl): S100-S107.
Dutkowski P, 2006aFurrer K, Tian Y, Graf R, Clavien PA. Novel short-term hypothermic oxygenated
perfusion (HOPE) system prevents injury in rat liver graft from non-heart beating donor. Ann Surg
2006;244:968–976, Discussion 976–967.
Dutkowski P, 2006bGraf R, Clavien PA. Rescue of the cold preserved rat liver by hypothermic
oxygenated machine perfusion. Am J Transplant 2006;6: 903–912.
Dutkowski P, de Rougemont O, Clavien PA. Machine perfusion for ‘marginal’ liver grafts. Am J Transplant
2008; 8:917–924.
Dutkowski P, De Rougemont O, Müllhaupt B, et al. Current and future trends in liver transplantation in
Europe. Gastroenterology 2010; 138: 802-9.
Dutkowski P, Oberkofler CE, Béchir M, et al. The model for end-stage liver disease allocation system for
liver transplantation saves lives, but increases morbidity and cost: a prospective outcome analysis. Liver
Transpl 2011; 17: 674-84.
Duvnjak M, Tomasic V, Gomercic M, et al. Therapy of nonalcoholic fatty liver disease: current status. J
Physiol Pharmacol 2009; 60 (Suppl 7):57–66.
336
References
Eason JD, Loss GE, Blazek J, et al. Steroid-free liver transplantation using rabbit anti-thymocyte globulin
induction: results of a prospective randomized trial. Liver Transplant 2001; 7(8):693–7.
Eckhoff DE, McGuire BM, Frenette LR, et al. Tacrolimus (FK506) and mycophenolate mofetil combination
therapy versus tacrolimus in adult liver transplantation. Transplantation. 1998; 27;65(2):180-7.
Egawa H, Oike F, Buhler L, Shapiro AM, et al. Impact of recipient age on outcome of ABO-incompatible
living-donor liver transplantation. Transplantation 2004; 77:403-11.
Egawa H, Teramukai S, Haga H,et al. Present status of ABO-incompatible living donor liver
transplantation in Japan. Hepatology 2008; 47:11-3.
Eisenbach C, Sauer P, Mehrabi A, et al. Prevention of hepatitis B virus recurrence,after liver
transplantation. Clin Transplant 2006; 20(Suppl 17):111–6.
Eisenhauer T, Hartmann H, Rumpf KW, et al. Favourable outcome of hepatic venoocclusive disease in a
renal transplant patient receiving azathioprine, treated by portacaval shunt. Report of a case and review
of the literature. Digestion 1984; 30:185–90.
Ekataksin W, ZZ, Wake K, et al. The hepatic microcirculatory subunits: an over three-century-long search
for the missing link between an exocrine unit and an endocrine unit in mammalian liver nodules. In: Motta
PM, ed. Recent advances in microscopy of cells, tissues and organs. Rome: University of Rome La
Sapienza Press, 1997:375–80.
El Serag HB, Davila JA. Is fibrolamellar carcinoma different from hepatocellular carcinoma? A US
population-based study. HEPATOLOGY 2004; 39:798-803.
El-Badry AM, Breitenstein S, Jochum W, et al. Assessment of hepatic steatosis by expert pathologists:
the end of a gold standard. Ann Surg 2009; 250:691–697.
El-Badry AM, Moritz W, Contaldo C, et al. Prevention of reperfusion injury and microcirculatory failure in
macrosteatotic mouse liver by omega-3 fatty acids. Hepatology 2007; 45(4):855-63.
Ellis A, Wendon J. Circulatory, respiratory, cerebral, and renal derangements in acute liver failure:
pathophysiology and management. Semin Liver Dis 1996;16:379-388.
El-Serag HB, Marrero JA, Rudolph L, Reddy KR. Diagnosis and treatment of hepatocellular
carcinoma.Gastroenterology 2008; 134: 1752-1763.
El-Serag HB. Hepatocellular carcinoma and hepatitis C in the United States. Hepatology 2002; 36:S74–
S83.
El-Serag HB. Hepatocellular carcinoma: recent trends in the United States. Gastroenterology 2004;
127(Suppl 1):S27–S34.
Emdin M, Passino C, Donato, et al. Serum gamma-glutamyltransferase as a risk factor of ischemic stroke
might be independent of alcohol consumption. Stroke 2002; 33: 1163-4.
Emdin M, Passino C, Michelassi C, et al. Prognostic value of serum gamma-glutamyl transferase activity
after myocardial infarction. Eur Heart J 2001;22:1802-1807.
Emiroglu R, et al: Tacrolimus-related neurologic and renal complications in liver transplantation: a singlecenter experience. Transplant Proc 2006; 38:619-621.
Emond JC, Freeman RB Jr, Renz JF, et al. Optimizing the use of donated cadaver livers: analysis and
policy development to increase the application of split-liver transplantation. Liver Transpl 2002; 8:863-72.
Emond JC, Renz JF, Ferrell LD et al. Functional analysis of grafts from living donors. Implications for the
treatment of older recipients. Annals of Surgery 1996; 224(4):544-552.
Emond JC, Whitington PF, Thistlethwaite JR, et al. Reduced-size orthotopic liver transplantation: use in
the management of children with chronic liver disease. Hepatology 1989; 10:867–872.
Emre S, Schwartz ME, Altaca G et al. Safe use of hepatic allografts from donors older than 70 years.
Transplantation 1996; 62: 62–65.
Erbay N, Raptopoulos V, Pomfret EA, et al. Living donor liver transplantation in adults: vascular variants
important in surgical planning for donors and recipients. AJR Am J Roentgenol 2003; 181:109-114.
Erol I, Alehan F, Ozcay F, et al: Neurological complications of liver transplantation in pediatric patients: a
single center experience. Pediatr Transplant 2007; 11:152-159.
337
References
Eubank WB, Wherry KL, Maki JH, et al. Preoperative evaluation of patients awaiting liver transplantation:
comparison of multiphasic contrast-enhanced 3D magnetic resonance to helical computed tomography
examinations. J Magn Reson Imaging 2002; 16:565-575.
European Association For The Study Of The Liver. EASL clinical practice guidelines: management of
chronic hepatitis B. J Hepatol 2009;50(2):227–42.
European Liver Transplant Registry. Available: www.eltr.org
Eurotransplant International Foundation (2008). http://www.eurotransplant.nl/
Evans HM, Kelly DA, McKiernan PJ, et al. Progressive histological damage in liver allografts following
pediatric liver transplantation. Hepatology 2006; 43:1109–17.
Everhart JE and T. P. Beresford, “Liver transplantation for alcoholic liver disease: a survey of
transplantation programs in the United States,” Liver Transplantation and Surgery, vol. 3, no. 3, pp. 220–
226, 1997
Everhart JE, Beresford TP. Liver transplantation for alcoholic liver disease:a survey of transplantation
programs in the United States. Liver Transpl Surg 1997; 3:220-226.
Everhart JE, Wei Y, Eng H, Charlton MR, et al. Recurrent and new hepatitis C virus infection after liver
transplantation. Hepatology 1999; 29:1220-1226.
Everson GT, Trotter J, Forman L, et al. Treatment of advanced hepatitis C with a low accelerating dosage
regimen of antiviral therapy Hepatology 2005;42:255–262.
Fabris P, Marranconi F, Bozzola L, et al. Fibrogenesis serum markers in patients with chronic hepatitis C
treated with _-IFN.J Gastroenterol 1999; 34:345–350.
Fahmy A, O’Mahony A, Kaul H, et al. Living donor liver transplantation (LDLT) is safe and effective for
hepatitis C recipients [abstract]. Am J Transplant 2004; 4(s8):355.
Fan ST, Lo CM, Liu CL, et al. Determinants of hospital mortality of adult recipients of right lobe live donor
liver transplantation. Annals of Surgery 2003; 238(6):864-869.
Fan ST, Lo CM, Liu CL, et al. Safety of donors in live donor liver transplantation using right lobe grafts.
Arch Surg 2000; 135:336-40.
Farges O, Malassagne B, Sebagh M, et al. Primary sclerosing cholangitis: liver transplantation or biliary
surgery. Surgery 1995; 117:146-155.
Farges O, Morris PJ, Dallman MJ. Spontaneous acceptance of rat liver allografts is associated with an
early downregulation of intragraft interleukin-4 messenger RNA expression. Hepatology 1995; 21(3):76775.
Farmer DG, Anselmo DM, Ghobrial RM, Yersiz H, McDiarmid SV, Cao C, et al. Liver transplantation for
fulminant hepatic failure: experience with more than 200 patients over a 17-year period. Ann Surg 2003;
237:666-675.
Farmer et al, 2007. Farmer DG, et al: Predictors of outcomes after pediatric liver transplantation: an
analysis of more than 800 cases performed at a single institution. J Am Coll Surg 2007; 204:904914.discussion 914-906
Farrant JM, Hayllar KM, Wilkinson ML, et al. Natural history and prognostic variables in primary
sclerosing cholangitis. Gastroenterology 1991; 100:1710-1717.
Fattovich G, Giustina G, Degos F, et al. Morbidity and mortality in compensated cirrhosis type C: a
retrospective follow-up study of 384 patients. Gastroenterology 1997; 112:463–472.
Faust TW. Recurrent primary biliary cirrhosis, primary sclerosing cholangitis, and autoimmune hepatitis
after transplantation. Liver Transpl 2001; 7(11 Suppl 1):S99–108.
Feng S, Goodrich NP, Bragg-Gresham JL, et al. Characteristics associated with liver graft failure: the
concept of a donor risk index. Am J Transplant 2006; 6:783–90.
Feng S. Increased donor risk: who should bear the burden? Liver Transpl 2009; 15:570–573.
Fernández-Esparrach G, Sánchez-Fueyo A, Ginès P, et al. A prognostic model for predicting survival in
cirrhosis with ascites. J Hepatol 2001; 34:46–57.
338
References
Ferrell L, Bass N, Roberts J, et al. Lipopeliosis: fat induced sinusoidal dilatation in transplanted liver
mimicking peliosis hepatis. J Clin Pathol 1992; 45:1109–10.
Fiane AE, Videm V, Johansen HT, et al. C1-inhibitor attenuates hyperacute rejection and inhibits
complement, leukocyte and platelet activation in an ex vivo pig-to-human perfusion model.
Immunopharmacology 1999;42(1-3):231-43.
Fiel MI, Agarwal K, Stanca C, et al. Posttransplant plasma cell hepatitis (de novo autoimmune hepatitis) is
a variant of rejection and may lead to a negative outcome in patients with hepatitis C virus. Liver Transpl
2008; 14:861–71.
Fierabracci V, Franzini M, Baggiani A, et al. Developmental variations of plasma gammaglutamyltransferase fractions in humans and in laboratory mammalians. Biomarkers. 2012; 17: 43-7.
Figueras J, Busquets J, Grande L, et al. The deleterious effect of donor high plasma sodium and
extended preservation in liver transplantation: a multivariate analysis. Transplantation 1996;61:410–413.
Filipponi F, Callea F, Salizzoni M, et al. Double-blind comparison of hepatitis C histological recurrence
rate in HCV+ liver transplant recipients given basiliximab + steroids or basiliximab + placebo, in addition
to cyclosporine and azathioprine. Transplantation 2004; 78:1488-95.
Filipponi F, Soubrane O, Labrousse F, et al. Liver transplantation for end-stage liver disease associated
with alpha-1-antitrypsin deficiency in children: pretransplant natural history, timing and results of
transplantation. J Hepatol 1994; 20:72–78.
Finidori J, Laperche Y, Haguenauer-Tsapis R, et al. In vitro biosynthesis and membrane insertion of
gamma-glutamyl transpeptidase. J Biol Chem 1984;259:4687-90.
Fink SA and R. S. Brown, “Current indications, contraindications, delisting criteria, and timing for liver
transplantation,” in Textbook on Liver Transplantation, R. W. Busuttil and G. B. Klintmalm, Eds., chapter
7, pp. 95–113, Elsevier, New York, NY, USA, 2nd edition, 2009.
Fink SA, Brown RS Jr (2005) Current indications, contraindications, delisting criteria, and timing for liver
transplantation. In: Busuttil RW, Klintmalm GB (eds) Transplantation of the liver.Elsevier Saunders,
Philadelphia, pp 95–114
Finkelstein SD, Marsh W, Demetris AJ, et al. Microdissection-based allelo-typing discriminates de novo
tumor from intrahepatic spread in hepatocellular carcinoma. Hepatology 2003; 37:871-9.
First” liver transplantation in cirrhotic patients requiring anticoagulant therapy. Liver Transpl 2007; 13:30–
7.
Fisher A, Mor E, Hytiroglou P, et al. FK506 hepatotoxicity in liver allograft recipients. Transplantation
1995; 59:1631–2.
Fisher A, Theise ND, Min A, Mor E, Emre S, Pearl A, et al. CA 19-9 does not predict cholangiocarcinoma
in patients with primary sclerosing cholangitis undergoing liver transplantation. Liver Transpl Surg 1995;
1:94–98.
Fisher LR, Henley KS, Lucey MR. Acute cellular rejection after liver transplantation: variability, morbidity,
and mortality. Liver Transpl Surg 1995; 1:10–5.
Fisher RA, Kulik LM, Freise CE et al. A2ALL Study Group. Hepatocellular carcinoma recurrence and
death following living and deceased donor liver transplantation. Am. J. Transplant. 2007; 7: 1601–8.
Fisher, R.A. & Strom, S.C. Human hepatocyte transplantation: worldwide results. Transplantation 2006;
82:441-449.
Floreani A, Mega A, Tizian L, et al. Bone metabolism and gonad function in male patients undergoing
liver transplantation: a two-year longitudinal study. Osteoporos Int 2001; 12:749- 754.
Florman S, Schiano T, Kim L, et al. The incidence and significance of late acute cellular rejection (>1000
days) after liver transplantation. Clin Transplant 2004; 18:152–155.
Foley DP, Fernandez LA, Leverson G, et al. Donation after cardiac death: the University of Wisconsin
experience with liver transplantation. Ann Surg 2005; 242:724–731.
Fondevila C, Hessheimer AJ, Ruiz A, Calatayud D, Ferrer J, Charco R, et al. Liver transplant using
donors after unexpected cardiac death: novel preservation protocol and acceptance criteria. Am J
Transplant 2007; 7:1849–1855.
339
References
Fontana RJ, Goodman ZD, Dienstag JL, Bonkovsky HL, Naishadham D, Sterling RK, et al. Relationship
of serum fibrosis markers with liver fibrosis stage and collagen content in patients with advanced chronic
hepatitis C. Hepatology 2008; 47 (3):789–98.
Fontana RJ, Kleiner DE, Bilonick R, Terrault N, Afdhal N, Belle SH, et al. Modeling hepatic fibrosis in
African American and Caucasian American patients with chronic hepatitis C virus infection. Hepatology
2006; 44(4):925–35.
Forman LM, Lucey MR. Predicting the prognosis of chronic liver disease: an evolution from Child to
MELD. Hepatology 2001; 33:473-5.
Forns X, Ampurdanes S, Llovet JM, et al. Identification of chronic hepatitis C patients without hepatic
fibrosis by a simple predictive model. Hepatology 2002; 36(4 Pt 1):986–92.
Foster R, Zimmerman M, Trotter JF. Expanding donor options marginal, living, and split donors. Clin Liver
Dis 2007; 11:417–429.
Fouad TR, Abdel-Razek WM, Burak KW, et al. Prediction of cardiac complications after liver
transplantation.Transplantation 2009; 87:763-770.
Fouquet V, Alves A, Branchereau S, et al. Long-term outcome of pediatric liver transplantation for biliary
atresia: a 10-year follow- up in a single center. Liver Transpl 2005; 11:152–160.
Fox IJ, Langnas AN, Fristoe LW, et al. Successful application of extracorporeal liver perfusion: a
technology whose time has come. Am J Gastroenterol 1993; 88:1876–81.
Fox, I.J.; Chowdhury, J.R.; Kaufman, S.S.; et al. Treatment of the Crigler-Najjar syndrome type 1 with
hepatocyte transplantation. New England J. Medicine 1998; 228:1422-1426.
Frankel WL, Tranovich JG, Salter L, Bumgardner G,et al. The optimal number of donor biopsy sites to
evaluate liver histology for transplantation. Liver Transpl 2002; 8:1044–1050.
Franzini M, Bramanti E, Ottaviano V, et al. A high performance gel filtration chromatography method for
gamma-glutamyltransferase fraction analysis. Anal Biochem. 2008; 374:1-6.
Franzini M, Bramanti E, Ottaviano V, et al. A high performance gel filtration chromatography method for
gamma-glutamyltransferase fraction analysis. Anal Biochem 2008;374:1-6.
Franzini M, Corti A, Fornaciari I, et al. Cultured human cells release soluble gamma-glutamyltransferase
complexes corresponding to the plasma b-GGT. Biomarkers. 2009;14:486-92.
Franzini M, Corti A, Martinelli B, et al. Gamma-Glutamyltransferase activity in human atherosclerotic
plaques-biochemical similarities with the circulating enzyme. Atherosclerosis 2009;202(1): 119-27.
Franzini M, Fornaciari I, Fierabracci V, et al. Accuracy of b-GGT fraction for the diagnosis of non-alcoholic
fatty liver disease. Liver Int. 2012; 32: 629-34.
Franzini M, Fornaciari I, Vico T, et al. High-sensitivity gamma-glutamyltransferase fraction pattern in
alcohol
addicts
and
abstainers.
Drug
Alcohol
Depend.
2012
(in
press:
http://dx.doi.org/10.1016/j.drugalcdep.2012.06.004)
Franzini M, Ottaviano V, Fierabracci V et al. Fractions of plasma gamma-glutamyltransferase in healthy
individuals: reference values. Clin Chim Acta. 2008; 395: 188-9.
Franzini M, Paolicchi A, Fornaciari I, et al. Cardiovascular risk factors and gamma-glutamyltransferase
fractions in healthy individuals. Clin Chem Lab Med. 2010; 48: 713-7.
Fraquelli M, Rigamonti C, Casazza G, Conte D, Donato MF, Ronchi G, et al. Reproducibility of transient
elastography in the evaluation of liver fibrosis in patients with chronic liver disease. Gut 2007;56:968–973.
Fraser A, Harris R, Sattar N, et al. Alanine aminotransferases, and incident diabetes: the British Women’s
Heart and Health Study and meta-analisys. Diabetes Care 2009; 32:741-50.
Fraser A, Harris R, Sattar N, et al. Gamma-glutamyltransferase is associated with incident vascular
events independently of alcohol intake: analysis of the British Women's Heart and Health Study and
Meta-Analysis. Arterioscler Thromb Vasc Biol. 2007;27:2729-35.
Fraser JR, Gibson PR. Mechanisms by which food intake elevates circulating levels of hyaluronan in
humans. J Intern Med 2005;258(5):460–6.
Freeman AJ, Dore GJ, Law MG, et al. Estimating progression to cirrhosis in chronic hepatitis C virus
infection. Hepatology 2001; 34:809–816.
340
References
Freeman Jr RB. Model for end-stage liver disease (MELD) for liver allocation: a 5-year score card.
Hepatology 2008;47:1052–7.
Freeman RB et al. Liver and intestine transplantation in the United States, 1997-2006 Am J Transplant, 8
(2008), pp. 958–976
Freeman RB Jr, Edwards EB. Liver transplant waiting time does not correlate with waiting list mortality:
implications for liver allocation policy. Liver Transpl 2000;6:543-52.
Freeman RB, “Overview of the MELD/PELD system of liver allocation indications for liver transplantation
in the MELD era: evidence-based patient selection,” Liver Transplantation, vol. 10, no. 10, pp. S2–S3,
2004.
Freeman RB, Giatras I, Falagas ME et al. Outcome of transplantation of organs procured from bacteremic
donors. Transplantation 1999; 68: 1107–1111
Freeman RB, Jamieson N, Schaubel DE, et al. Who should get a liver graft? J Hepatol 2009;50:664-673.
Freeman RB, Rohrer RJ, Katz E, Lewis WD, Jenkins R, Cosimi AB, et al. Preliminary results of a liver
allocation plan using a continuous medical severity score that de-emphasizes waiting time. Liver Transpl
2001;7: 173-178.
Freeman RB, Wiesner RH, Edwards E, Harper A, Merion R, Wolfe R. Results of the first year of the new
liver allocation plan. Liver Transpl 2004; 10: 7-15
Freeman RB, Wiesner RH, Edwards E, Harper A, Merion R, Wolfe R. United Network for Organ Sharing
Organ Procurement and Transplantation Network Liver and Transplantation Committee. Results of the
first year of the new liver allocation plan. Liver Transpl 2004; 10: 7–15
Freeman RB, Wiesner RH, Roberts JP, McDiarmid S, Dykstra DM, Merion RM. Improving liver allocation:
MELD and PELD. Am J Transplant 2004;4(Suppl):114–31.
Freeman RB. MELD: the holy grail of organ allocation? J Hepatol 2005;42:16-20.
Friman S, Backman L. A new microemulsion formulation of cyclosporine: pharmacokinetic and clinical
features. Clin Pharmacokinet 1996;30 (3):181–93.
Froehlich F, Lamy O, Fried M, et al. Practice and complications of liver biopsy. Results of a nationwide
survey in Switzerland. Dig Dis Sci 1993;38:1480–1484.
Fukumori T, Kato T, Levi D, Olson L, Nishida S, Ganz S, et al. Use of older controlled non-heart-beating
donors for liver transplantation. Transplantation 2003;75:1171–1174.
Fung JJ, Eghtesad B, Patel-Tom K. Using livers from donation after cardiac death donors—a proposal to
protect the true Achilles heel. Liver Transpl 2007;13:1633–6.
Furuya T, Murase N, Nakamura K, et al. Preformed lymphocytotoxic antibodies: the effects of class, titer
and specificity on liver vs. heart allografts. Hepatology 1992;16:1415–22.
Futagawa Y, Terasaki PI, Waki K, Cai J, Gjertson DW. No improvement in long-term liver transplant graft
survival in the last decade: an analysis of the UNOS data. Am J Transplant 2006; 6: 1398–406.
Gabrielli GB, Capra F, Casaril M, et al. Serum laminin and type III procollagen in chronic hepatitis C.
Diagnostic value in the assessment of disease activity and fibrosis. Clin Chim Acta 1997;265:21–31.
Gadaleta MN, Cormio A, Pesce V, Lezza AM, Cantatore P. Aging and mitochondria. Biochimie 1998; 80:
863–970
Gaglio P, Malireddy S, Levitt B, et al. Increased risk of cholestatic hepatitis C in recipients of grafts from
living versus cadaveric liver donors. Liver Transpl 2003;9:1028–35.
Gane E, Saliba F, Valdecasas GJ, et al. Randomised trial of efficacy and safety of oral ganciclovir in the
prevention of cytomegalovirus disease in liver-transplant recipients. The Oral Ganciclovir International
Transplantation Study Group [corrected]. Lancet 1997;350(9093):1729–33.
Gane E. The natural history and outcome of liver transplantation in hepatitis C virus-infected recipients.
Liver Transpl 2003; 9(Suppl 3):S28–S34.
Gane EJ. The natural history of recurrent hepatitis C and what influences this. Liver Transpl
2008;14(Suppl 2):S36–44.
341
References
Garcia CE, Ribeiro HB, Garcia RL, et al. Mycophenolate mofetil in stable liver transplant patients with
calcineurin inhibitor-induced renal impairment: single-center experience. Transplant Proc 2003;35:1131-2.
Garcia S, DiSanto J, Stockinger B (1999) Following the development of a CD4 T cell response in vivo:
from activation to memory formation. Immunity 11:163–171.
Garcia-Retortillo M, Forns X, Feliu A, et al. Hepatitis C virus kinetics during and immediately after liver
transplantation. Hepatology 2002;35:680–687
Gasbarrini A, Borle AB, Farghali H, Bender C, Francavilla A, Van Thiel D. Effect of anoxia on intracellular
ATP, Na+i,Ca2+i, Mg2+i, and cytotoxicity in rat hepatocytes. J Biol Chem 1992;267:6654–6663.
Gastaca M, Fernandez JR, Valdivieso A, et al: Influence of donor age in HCV recurrence after liver
transplantation: very old donors really do worse. Am J Transplant 7(suppl 2):876A, 2007
Gautam M, Cheruvattath R, Balan V. Recurrence of autoimmune liver disease after liver transplantation:
a systematic review. Liver Transpl 2006;12:1813–24.
Gaweco AS, Wiesner RH, Yong S, Krom R, Porayko M, Chejfec G, et al. CD40L (CD154) expression in
human liver allografts during chronic ductopenic rejection. Liver Transpl Surg 1999;5:1-7.
Gawrieh S, Papouchado BG, Burgart LJ, et al. Early hepatic stellate cell activation predicts severe
hepatitis C recurrence after liver transplantation. Liver Transpl 2005;11:1207–13.
Geissler I, Heinemann K, Rohm S, et al. Liver transplantation for hepatic and neurological Wilson’s
disease. Transplant Proc 2003;35:1445–1446.
Genesca J, Gonzalez A, Evangelista A, Mora A, Margarit C, et al. Role of Doppler echocardiography in
the assessment of portopulmonary hypertension in liver transplantation candidates. Transplantation
2001;71:572-574.
George DK, Ramm GA, Walker NI, et al. Elevated serum type IV collagen: a sensitive indicator of the
presence of cirrhosis in haemochromatosis. J Hepatol 1999;31:47–52.
Ghany MG, Strader DB, Thomas DL, Seeff LB. Diagnosis, management, and treatment of hepatitis C: an
update. Hepatology 2009;49(4):1335–74.
Ghobrial RM, Gornbein J, Steadman R, Danino N, Markmann JF, Holt C, et al. Pretransplant model to
predict posttransplant survival in liver transplant patients. Ann Surg 2002;236:315-322.
Ghobrial RM, Steadman R, Gornbein J, Lassman C, Holt CD,Chen P, et al. A 10-year experience of liver
transplantation for hepatitis C: Analysis of factors determining outcome in over 500 patients. Ann Surg
2001;234:384-393; discussion 393- 394.
Ghobrial RM, Yersiz H, Farmer DG, Amersi F, Goss J, Chen P, et al. Predictors of survival after In vivo
split liver transplantation: analysis of 110 consecutive patients. Ann Surg 2000;232:312-23.
Giacomoni A, Lauterio A, Donadon M, et al: Should we still offer split-liver transplantation for two adult
recipients? A retrospective study of our experience. Liver Transpl 14:999, 2008
Giannini E, Caglieris S, Ceppa P, et al. Serum pro-collagen III peptide levels are related to lobular
necrosis in untreated patients with chronic hepatitis C. Eur J Gastroenterol Hepatol 2001;13:137–141.
Gines A, Escorsell A, Gines P, Salo J, Jimenez W, Inglada L, et al. Incidence, predictive factors, and
prognosis of the hepatorenal syndrome in cirrhosis with ascites. Gastroenterology 1993;105:229-236.
Gines P, Berl T, Bernardi M, Bichet DG, Hamon G, Jimenez W, et al. Hyponatremia in cirrhosis: from
pathogenesis to treatment. Hepatology 1998;28:851–864.
Gines P, Quintero E, Arroyo V, Teres J, Bruguera M, Rimola A, et al. Compensated cirrhosis: natural
history and prognosis factors. HEPATOLOGY 1987;7:122-128.
GK Oriji, HR Keiser. Role of nitric oxide in cyclosporin A-induced hypertension. Hypertension, 32 (1998),
pp. 849–855
Gómez, M.; Garcia-Buitrón, JM.; Fernandez-Garcia, A.; Vilela, D.; Fernández-Selles, C.; Corbal, R.;
Fraguela, J.; Suárez, F.; Otero, A.; Alvarez, J. & Mánez, R. (1997) Liver transplantation with organs from
non-heart-beating donors. Transplantation Proceedings, Vol.29, No.8, (December 1997), pp. 3478-3479,
ISSN: 0041-1345
Gondolesi GE, Varotti G, Florman SS, Munoz L, Fishbein TM,Emre SH, et al. Biliary complications in 96
consecutive right lobe living donor transplant recipients. Transplantation 2004;77:1842–1848.
342
References
Gonwa A. Hypertension and renal dysfunction in longterm liver transplant recipients. Liver Transpl 2001;
7: S22–26.
Gonwa TA, Mai ML, Melton LB, Hays SR, et al. End-stage renal disease (ESRD) after orthotopic liver
transplantation (OLTX) using calcineurin-based immunotherapy: risk of development and treatment.
Transplantation 2001;72(12):1934-9.,
Gonwa TA, Morris CA, Goldstein RM, Husberg BS, Klintmalm GB. Long-term survival and renal function
following liver transplantation in patients with and without hepatorenal syndrome: experience in 300
patients.Transplantation 1991;51:428-430.
Gonzalez FX, Rimola A, Grande L, et al. Predictive factors of early postoperative graft function in human
liver transplantation. Hepatology 1994;20:565–573
Gonzalez-Koch A, Czaja AJ, Carpenter HA, et al. Recurrent autoimmune hepatitis after orthotopic liver
transplantation. Liver Transpl 2001;7:302–10.
Goode HF, Webster NR, Howdle PD, Leek JP, Lodge JP, Sadek SA, Walker BE. Reperfusion injury,
antioxidants and hemodynamics during orthotopic liver transplantation. Hepatology 1994;19:354–359.
Gordon Burroughs, S. & Busuttil, RW. (2009) Optimal utilization of extended hepatic grafts. Surgery
Today, Vol.39, No.9, (September 2009), pp. 746-751, ISSN: 0941-1291
Gordon F, Pomfret E, Pomposelli J, et al. The severity of recurrent hepatitis C (HCV) in living donor adult
liver transplant (LDALT) recipients is the same as cadaver (CAD) recipients [abstract]. Am J Transplant
2004;4(S8):400.
Gordon F. Recurrent primary sclerosing cholangitis: clinical diagnosis and long-term management issues.
Liver Transpl 2006;12(11 Suppl 2):S73–5.
Gordon RD, Iwatsuki S, Esquivel CO, Tzakis A, Todo S, Starzl TE. Liver transplantation across ABO
blood groups. Surgery. 1986 Aug;100(2):342-8.
Gordon RD, van Thiel DH & Starzl TE, Liver transplantation. In Schiff L & Schiff E (eds.) Diseases of the
Liver, vol. 2. Philadelphia: JP Lippincott, 1993, pp. 1210–1235.
Goss JA, Shackleton CR, Farmer DG, et al. Orthotopic liver transplantation for primary sclerosing
cholangitis. A 12-year single center experience. Ann Surg 1997;225:472–81; discussion 481–3.
Gouw AS, van den Heuvel MC, van den Berg AP, Slooff MJ, de Jong KP, Poppema S. The significance
of parenchymal changes of acute cellular rejection in predicting chronic liver graft rejection.
Transplantation 2002;73:243–247.
Gow PJ, Chapman RW. Liver transplantation for primary sclerosing cholangitis. Liver 2000;20:97–103.
Gowans JL. The fate of parental strain small lymphocytes in F1 hybrid rats. Ann N Y Acad Sci. 1962 Oct
24;99:432-55.
Grande L, Matus D, Rimola A, Manyalic M, Cabrer C, Garcia- Valdecasas JC, Visa J. Expanded liver
donor age over 60 years for hepatic transplantation. Clin Transpl 1998;297-301.
Grazi GL, Cescon M, Ravaioli M et al. A revised consideration on the use of very aged donors for liver
transplantation. Am J Transplant 2001; 1: 61–68
Graziadei IW, Wiesner RH, Batts KP, et al. Recurrence of primary sclerosing cholangitis following liver
transplantation. Hepatology 1999;29:1050–6.
Graziadei IW, Wiesner RH, Marotta PJ, et al. Long-term results of patients undergoing liver
transplantation for primary sclerosing cholangitis. Hepatology 1999;30:1121–7.
Graziadei IW. Recurrence of primary sclerosing cholangitis after liver transplantation. Liver Transpl
2002;8:575–81.
Graziadei IW1999a, Wiesner RH, Marotta PJ, Porayko MK, Hay JE, CharltonMR, et al. Long-term results
of patients undergoing liver transplantation for primary sclerosing cholangitis. HEPATOLOGY
1999;30:1121-1127.
Greenland P, Bonow RO, Brundage BH, Budoff MJ, Eisenberg MJ, Grundy SM, et al. ACCF/AHA 2007
clinical expert consensus document on coronary artery calcium scoring by computed tomography in
global cardiovascular risk assessment and in evaluation of patients with chest pain: a report of the
American College of Cardiology Foundation Clinical Expert Consensus Task Force (ACCF/AHA Writing
343
References
Committee to Update the2000 Expert Consensus Document on Electron Beam Computed Tomography).
Circulation 2007;115:402-426.
Greif F, Bronsther OL, Van Thiel DH, et al. The incidence, timing, and management of biliary tract
complications after orthotopic liver transplantation. Ann Surg 1994;219:40–5.
Grewal HP, Brady L, Cronin DC, Loss GE, Siegel CT, Oswald K, et al. Combined liver and kidney
transplantation in children. Transplantation 2000;70:100-105.
Grewal, HP.; Willingham, DL.; Nguyen, J.; Hewitt, WR.; Taner, BC.; Cornell, D.; Rosser, BG.; Keaveny,
AP.; Aranda-Michel, J.; Satyanarayana, R.; Harnois, D.; Dickson, RC.; Kramer, DJ. & Hughes, CB. (2009)
Liver transplantation using controlled donation after cardiac death donors: an analysis of a large singlecenter experience. Liver Transplantation, Vol.15, No.9, (September 2009), pp. 1028-1035, ISSN: 15276473
Gridelli B, Spada M, Petz W, Bertani A, Lucianetti A, Colledan M, et al. Split-liver transplantation
eliminates the need for living-donor liver transplantation in children with end-stage cholestatic liver
disease. Transplantation 2003;75:1197-1203.
Grob P, Jilg W, Bornhak H et al. Serological pattern ‘anti-HBc alone’: report on a workshop. J Med Virol
2000; 62: 450–455
Grossi E, Colombo R, Cavuto S, et al. The REALAB project: a new method for the formulation of
reference intervals based on current data. Clin Chem. 2005;51:1232-40.
Grossman, M.; Raper, S.E. & Wilson, J.M. (1991) Towards liver-directed gene therapy: retrovirusmediated gene transfer into human hepatocytes. Somat. Cell Mol. Gen. Vol. 17, No. 6, pp. 601-607
Groth, C.G.; Arborgh, B.; Björkén, C.; et al. (1977) Correction of hyperbilirubinemia in the
glucuronyltransferase-deficient rat by intraportal hepatocyte transplantation. Transplant Proc. Vol. 9, No.
1, pp. 313-316group.
Gruttadauria S, Cintorino D,Mandala L, et al. Acceptance of marginal liver donors increases the volume of
liver transplant: early results of a single-center experience. Transplant Proc 2005;37:2567–2568
Guarrera JV, Henry SD, Samstein B, et al. Hypothermic machine preservation in human liver
transplantation: the first clinical series. Am J Transplant 2010;10: 372–81.
Guechot J, Laudat A, Loria A, et al. Diagnostic accuracy of hyaluronan and type III procollagen aminoterminal peptide serum assays as markers of liver fibrosis in chronic viral hepatitis C evaluated by ROC
curve analysis. Clin Chem 1996;42:558–563.
Gugenheim J, Samuel D, Reynes M, Bismuth H. Liver transplantation across ABO blood group barriers.
Lancet. 1990 Sep 1;336(8714):519-23.
Guichelaar MM, Benson JT, Malinchoc M, et al. Risk factors for and clinical course of non-anastomotic
biliary strictures after liver transplantation. Am J Transplant 2003;3:885–90.
Guichelaar MM, Benson JT, Malinchoc M, Krom RA, Wiesner RH, Charlton MR. Risk factors for and
clinical course of non-anastomotic biliary strictures after liver transplantation. Am J Transplant. 2003
Jul;3(7):885-90.
Gunsar et al, 2003. Gunsar F, et al: Late hepatic artery thrombosis after orthotopic liver transplantation.
Liver Transpl 2003; 9:605-611.
Guo L, Orrego M, Rodriguez-Luna H, et al. Living donor liver transplantation for hepatitis C-related
cirrhosis: no difference in histological recurrence when compared to deceased donor liver transplantation
recipients. Liver Transpl 2006;12:560–5.
Gupta, S. & Chowdhury, J.R. (2002) Therapeutic potential of hepatocyte transplantation. Semin Cell Dev
Biol. Vol. 13, No. 6, pp.439-46
Gupta, S.; Rajvanshi, P.; Sokhi, R.; et al. (1999) Entry and integration of transplanted hepatocytes in rat
liver plates occur by disruption of hepatic sinusoidal endothelium. Heptatology. Vol. 29, pp. 509-519
Gupta, S.; Yerneni, P.R.; Vemura, R.P.; et al. (1993) Studies on the safety of intrasplenic hepatocyte
transplantation: relevance to ex vivo gene therapy and liver repopulation in acute hepatic failure. Human
Gene Ther. Vol. 4, pp. 249-257
344
References
Habib GM, Barrios R, Shi ZZ, Lieberman MW. Four distinct membrane-bound dipeptidase RNAs are
differentially expressed and show discordant regulation with gamma-glutamyl transpeptidase. J Biol
Chem. 1996; 271: 16273-80.
Haboubi NY, Ali HH, Whitwell HL, et al. Role of endothelial cell injury in the spectrum of azathioprineinduced liver disease after renal transplant: light microscopy and ultrastructural observations. Am J
Gastroenterol 1988;83:256–61.
Haddad EM, McAlister VC, Renouf E, Malthaner R, Kjaer MS, Gluud LL (2006) Cyclosporin versus
tacrolimus for liver transplanted patients. Cochrane Database Syst Rev, vol. 4, article no. CD005161
Hadengue A, Benahyoun M, Lebrec D, Benhamou JP. Pulmonary hypertension complicating portal
hypertension: prevalence and relation to splanchnic hemodynamics. Gastroenterology 1991;100:520-528.
Haga H, Egawa H, Shirase T, et al. Periportal edema and necrosis as diagnostic histological features of
early humoral rejection in ABO-incompatible liver transplantation. Liver Transpl 2004;10:16–27.
Hahn E, Wick G, Pencev D, et al. Distribution of basement membrane proteins in normal and fibrotic
human liver: collagen type IV, laminin, and fibronectin. Gut 1980;21:63–71.
Halfon P, Imbert-Bismut F, Messous D, et al. A prospective assessment of the inter-laboratory variability
of biochemical markers of fibrosis (FibroTest) and activity (ActiTest) in patients with chronic liver disease.
Comp Hepatol 2002;1:3.
Hamman, K., Clark, H., Montini, E.; et al. (2005) Low therapeutic threshold for hepatocyte replacement in
murine phenylketonuria. Molecular Therapy. Vol. 12, No. 2, pp. 337- 344
Hanigan MH, Frierson HF Jr. Immunohistochemical detection of gammaglutamyl transpeptidase in normal
human tissue. J Histochem Cytochem 1996;44:1101-8.
Hanzal-Bayer MF, Hancock JF. Lipid rafts and membrane traffic. FEBS Lett 2007;581:2098-104.
Harding, C.O. & Gibson, K.M. (2010) Therapeutic liver repopulation for phenylketonuria. J. Inherited.
Metabolic Dis. Vol. 33, No. 6, pp. 681-7
Harland RC, Platt JL. Prospects for xenotransplantation of the liver. J Hepatol 1996;25: 248–58.
Harnois DM, Lindor KD. Primary sclerosing cholangitis: evolving concepts in diagnosis and treatment. Dig
Dis Sci 1997;15:23-41.
Harrison SA, Oliver D, Arnold HL, Gogia S, Neuschwander-Tetri BA. Development and validation of a
simple NAFLD clinical scoring system for identifying patients without advanced disease. Gut
2008;57(10):1441–7.
Hashikura Y, Ichida T, Umeshita K et al. Donor complications associated with living donor liver
transplantation in Japan. Transplantation 2009; 88: 110–14.
Hashimoto E, Shimada M, Noguchi S, et al. Disease recurrence after living liver transplantation for
primary biliary cirrhosis: a clinical and histological follow-up study. Liver Transpl 2001;7:588–95.
Hassoun Z, Gores G, Rosen C. Preliminary experience with liver transplantation in selected patients with
unresectable hilar cholangiocarcinoma. Surg Oncol Clin North Am 2002;11:909–921
Hassoun Z, Shah V, Lohse CM, et al. Centrilobular necrosis after orthotopic liver transplantation:
association with acute cellular rejection and impact on outcome. Liver Transpl 2004;10:480–7.
Hasuike Y, Nonoguchi H, Tokuyama M, Ohue M, Nagai T, Yahiro M, et al. Serum ferritin predicts
prognosis in hemodialysis patients: the Nishinomiya study. Clin Exp Nephrol 2010;14:349–355.
Hay JE, Guichelaar MM. Evaluation and management of osteoporosis in liver disease. Clin Liver Dis
2005; 9: 747-66.
Hayasaka A, Suzuki N, Fujimoto N, et al. Elevated plasma levels of matrix metalloproteinase-9 (92-kd
type IV collagenase/gelatinase B) in hepatocellular carcinoma. Hepatology 1996;24: 1058–1062.
Hayashi PH, Forman L, Steinberg T, et al. Model for endstage liver disease score does not predict patient
or graft survival in living donor liver transplant recipients. Liver Transpl 2003; 9: 737.
Healey PJ, Davis CL. Transmission of tumours by transplantation. Lancet 1998; 352: 2–3
Heaton, N. & Rela, M. (2001) Auxiliary liver transplantation. In: Transplantation of the liver, 3rd Ed.
Philadelphia : Lippincott Williams & Wilkins; 2001; 121–130.
345
References
Hebert MF, TS, Carithers RL. Immunomodulating agents and the transplant situation. In: Kaplowitz N,
Deleve L, eds. Drug-induced liver disease. New York: Marcel Dekker, 2003:633–51.
Heimbach JK a2008. Successful liver transplantation for hilar cholangiocarcinoma. Curr Opin
Gastroenterol 2008;24:384–8.
Heimbach JK b2008. Liver transplantation for hepatocellular carcinoma. Cancer J 2008;14:95–9.
Hematti, P. (2008) Role of mesenchymal stromal cells in solid organ transplantation. Transplant Rev
(Orlando). Vol. 22, No. 4, pp. 262-73
Henderson JM, Heymsfield SB, Horowitz J, Kutner MH. Measurement of liver and spleen volume by
computed tomography. Assessment of reproducibility and changes found following a selective distal
splenorenal shunt. Radiology 1981; 141:525-7.
Heneghan MA, Zolfino T, Muiesan P, et al. An evaluation of long-term outcomes after liver transplantation
for cryptogenic cirrhosis. Liver Transpl 2003;9:921–8.
Hennes EM,Zeniya M,Czaja AJ,Parés A,Dalekos GN,Krawitt EL,Bittencourt PL,Porta G,Boberg KM Hofer
H,Bianchi FB,Shibata M,Schramm C,Eisenmann de Torres B,Galle PR,McFarlane I,Dienes HP,Lohse
AW;International Autoimmune Hepatitis Group. Simplified criteria for the diagnosis of autoimmune
hepatitis. Hepatology 2008;48(1):169-179.
Heo, J.; Factor, V.M.; Uren, T.; et al. (2006) Hepatic precursors derived from murine embryonic stem cells
contribute to regeneration of injured liver. Hepatology. Vol. 44, pp. 1478-1486
Hepp J, Innocenti FA. Liver transplantation in Latin America: current status. Transplant Proc 2004; 36:
1667-8.
Herijgers P, Leunens V, Tjandra-Maga TB, et al. Changes in organ perfusion after brain death in the rat
and its relation to circulating catecholamines. Transplantation 1996;62:330-5.
Herrero JI, Lucena JF, Quiroga J, Sangro B, Pardo F, Rotellar F, et al. Liver transplant recipients older
than 60 years have lower survival and higher incidence of malignancy. Am J Transplant 2003;3:14071412.
Herrero JI, Pardo F, D’Avola D, Alegre F, Rotellar F, In˜arrairaegui M, et al. Risk factors of lung, head and
neck, esophageal, and kidney and urinary tract carcinomas after liver transplantation: the effect of
smoking withdrawal. Liver Transpl 2011;17:402- 408.
Hertl M, Malago M, Rogiers X, Burdelski M, Broelsch CE. Surgical approaches for expanded organ usage
in liver transplantation. Transplant Proc 1997;29:3683–3686
Heuman D, Mihas A. Utility of the MELD score for assessing 3- month survival in patients with liver
cirrhosis: one more positive answer. Gastroenterology 2003;125:992–993.
Heuman DM, Abou-Assi SG, Habib A, et al. Persistent ascites and low serum sodium identify patients
with cirrhosis and low MELD scores who are at high risk for early death. Hepatology 2004;40:802–10.
Heuman DM,Mihas AA, Habib A, et al. MELD-XI: A rational approach to “sickest first” liver transplantation
in cirrhotic patients requiring anticoagulant therapy. Liver Transpl 2007, 13: 30-37
Hill MJ, Hughes M, Jie T, et al: Graft weight/recipient weight ratio: how well does it predict outcome after
partial liver transplants? Liver Transpl 15:1056, 2009
Hill, MJ.; Hughes, M.; Jie, T.; Cohen, M.; Lake, J.; Payne, WD. & Humar, A. (2009) Graft weight/recipient
weight ratio: how well does it predict outcome after partial liver transplants? Liver Transplantation, Vol.15,
No.9, (September 2009), pp. 1056-1062,
Holtmann M, Schreiner O, Kohler H, et al. Veno-occlusive disease (VOD) in Crohn’s disease (CD) treated
with azathioprine. Dig Dis Sci 2003;48:1503–5.
Hong JC, Koroleff D, Xia V, et al. Regulated hepatic reperfusion attenuates postreperfusion syndrome
and improves survival after prolonged warm ischemia in a swine model (abstract). Am J Transplant
2009;9:S204.
Hoofnagle JH, Lombardero M, Zetterman RK, et al. Donor age and outcome of liver transplantation.
Hepatology 1996; 24:89–96
Horton JD, San Miguel FL, Ortiz JA. Budd–Chiari syndrome: illustrated review of current management.
Liver Int 2008;28:455–66.
346
References
Hu, Q.; Friedrich, A.M.; Johnson, L.V.; et al. (2010) Memory in induced pluripotent stem cells:
reprogrammed human retinal pigmented epithelial cells show tendency for spontaneous re-differentiation.
Stem Cells. Vol. 28, pp. 1981-1991
Huang TL, Y. F. Cheng, C. L. Chen, T. Y. Chen, and T. Y. Lee, “Variants of the bile ducts: clinical
application in the potential donor of living-related hepatic transplantation,”Transplantation Proceedings,
vol. 28, no. 3, pp. 1669–1670, 1996.
Hubscher S. b2009 What does the long-term liver allograft look like for the pediatric recipient? Liver
Transpl 2009;15:S19–S24.
Hubscher SG a2009. Transplantation pathology. Semin Liver Dis 2009;29:74–90.
Hubscher SG PB. Transplantation Pathology. In: Burt AD, PB, Ferrell LD, eds. MacSween’s pathology of
the liver. 5th edn. London: Churchill Livingstone, 2007:815–79.
Hubscher SG. Central perivenulitis: a common and potentially important finding in late posttransplant liver
biopsies. Liver Transpl 2008;14: 596–600.
Hubscher SG. Recurrent and de-novo disease in the liver allograft. Curr Opin Organ Transpl
2006;11:283–288.
Hubscher SG. Recurrent autoimmune hepatitis after liver transplantation: diagnostic criteria, risk factors,
and outcome. Liver Transpl 2001;7:285–91.
Hughes, R.D.; Mitry, R.R. & Dhawan, A. (2005) Hepatocyte transplantation for metabolic liver disease:
UK experience. J R Soc Med. Vol. 98, pp. 341–345
Hui AY, Chan HL, Wong VW, Liew CT, Chim AM, Chan FK, et al. Identification of chronic hepatitis B
patients without significant liver fibrosis by a simple noninvasive predictive model. Am J Gastroenterol
2005;100(3):616–23.
Humar A, Horn K, Kalls A, et al. Living donor and split-liver transplants in hepatitis C recipients: does liver
regeneration in crease the risk for recurrence? Am J Transplant 2005; 5:399–405.
Humar A, Ramcharan T, Sielaff TD, et al. Split liver transplantation for two adult recipients: an initial
experience. Am J Transplant 2001;1:366–72.
Huo SC, Huo TI, Lin HC, et al. Is the corrected-creatinine model for end-stage liver disease a feasible
strategy to adjust gender difference in organ allocation for liver transplantation? Transplantation
2007;84:1406–12.
Huo TI, Lin HC, Lee FY, et al. Occurrence of cirrhosis-related complications is a time dependent
prognostic predictor independent of baseline model for end stage liver disease score. Liver Int
2006;26:55–61.
Huo TI, Lin HC,Wu JC, et al. Proposal of amodified Child–Turcotte–Pugh scoring system and comparison
with the model for end-stage liver disease for outcome prediction in patients with cirrhosis. Liver Transpl
2006;12:65–71
Huo TI, Wang YW, Yang YY, Lin HC, Lee PC, Hou MC, Lee FY, Lee SD. Model for end-stage liver
disease score to serum sodium ratio index as a prognostic predictor and its correlation with portal
pressure in patients with liver cirrhosis. Liver Int 2007; 27: 498-506 transplantation for end-stage liver
disease. Dig Dis Sci 2007; 52: 3217-3223
Huo TI, Wu JC, Lin HC, et al. Different model for end-stage liver disease score block distributions may
have a variable ability for outcome prediction in cirrhotic patients. Transplantation 2005; 80: 1414–8.
Huo TI,Wu JC, Huang YH, et al. Acute renal failure after transarterial chemoembolization for
hepatocellular carcinoma: a retrospective study of the incidence, risk factors, clinical course and longterm outcome. Aliment Pharmacol Ther 2004; 19: 999–1007.
Huo TI,Wu JC, Lee SD. MELD in liver transplantation: the da Vinci code for the Holy Grail? J Hepatol
2005;42:477–8.
Huseby NE. Hydrophilic form of γ-glutamyltransferase: proteolitic formation in liver homogenates and its
estimation in serum. Clin Chim Acta 1982;124:113-21.
Huseby NE. Multiple form of γ-glutamyltransferases: Biochemical characterization. Adv Biochem
Pharmacol 1982;3:47-54.
347
References
Huseby NE. Multiple forms of γ-glutamyltransferase. Association of the enzyme with lipoproteins. Clin
Chim Acta 1982;124:130-12.
Iacobellis A, Siciliano M, Perri F, et al. Peginterferon alfa-2b and ribavirin in patients with hepatitis C virus
and decompensated cirrhosis: a controlled study. J Hepatol 2007;46(2):185-8.
Ibarguen E. Liver disease in alpha 1-antitrypsin deficiency and prognostic indicators. J Pediatr
Gastroenterol Nutr 1990;117:864– 870.
Iber, F.L., Murphy, P.A., Connor, E.S., 1994. Age-related changes in the gastrointestinal system. Drugs
and Aging 5, 34–48.
Idobe Y, Murawaki Y, Ikuta Y, et al. Post-prandial serum hyaluronan concentration in patients with chronic
liver disease. Intern Med 1998;37:568–575.
Ikeda T, Yanaga K, Kishikawa K, Kakizoe S, Shimada M, Sugimachi K. Ischemic injury in liver
transplantation: difference in injury sites between warm and cold ischemia in rats. Hepatology
1992;16:454–461.
Ikegami, T.; Shimada, M.; Imura, S.; Arakawa, Y.; Nii, A.; Morine, Y. & Kanemura, H. (2008) Current
concept of small-for-size grafts in living donor liver transplantation. Surgery Today, Vol.38, No.11,
(October 2008), pp. 971-982, ISSN: 0941-1291
Ikura Y, Morimoto H, Ogami M, et al. Expression of plateletderived growth factor and its receptor in livers
of patients with chronic liver disease. J Gastroenterol 1997;32:496 –501.
Imber CJ 2002 b, St Peter SD, Handa A, Friend PJ. Hepatic steatosis and its relationship to
transplantation. Liver Transpl 2002;8:415–423.
Imber CJ 2002a, St Peter SD, Lopez I, Guiver L, Friend PJ. Current practice regarding the use of fatty
livers: a trans-Atlantic survey. Liver Transpl 2002;8: 545–549
Imber CJ, St Peter SD, Lopez de Cenarruzabeitia I, et al. Advantages of normothermic perfusion over
cold storage in liver preservation. Transplantation 2002;73: 701–9.
Imbert-Bismut F, Messous D, Thibaut V, Myers RB, Piton A, Thabut D, et al. Intra-laboratory analytical
variability of biochemical markers of fibrosis (Fibrotest) and activity (Actitest) and reference ranges in
healthy blood donors. Clin Chem Lab Med 2004;42(3):323–33.
Imbert-Bismut F, Ratziu V, Pieroni L, Charlotte F, Benhamou Y, Poynard T. Biochemical markers of liver
fibrosis in patients with hepatitis C virus infection: a prospective study. Lancet 2001;357(9262):1069–75.
International P. Banff schema for grading liver allograft rejection: an international consensus document.
Hepatology 1997;25:658–663.
Ioannou G, Perkins JD, Carhiters RL. Liver transplantation for hepatocellular carcinoma: impact of the
MELD allocation system and predictors of survival. Gastroenterology 2008;134:1342–51.
Ioannou GN. Development and validation of a model predicting graft survival after liver transplantation.
Liver Transpl 2006;12:1594–1606.
Iredale JP, Goddard S, Murphy G, et al. Tissue inhibitor of metalloproteinase-I and interstitial collagenase
expression in autoimmune chronic active hepatitis and activated human hepatic lipocytes. Clin Sci (Lond)
1995;89:75–81
Ishak K, Baptista A, Bianchi L, Callea F, De Groote J, Gudat F, et al. Histological grading and staging of
chronic hepatitis. J Hepatol 1995;22(6):696–9.
Ishak K. Granulomas of liver. In: Ioachim HL, ed. Pathology of granulomas. New York: Raven, 1983:308–
9.
Islam S, Antonsson L, Westin J, Lagging M. Cirrhosis in hepatitis C virus-infected patients can be
excluded using an index of standard biochemical serum markers. Scand J Gastroenterol 2005;40(7):867–
72.
Jacob M, Copley LP, Lewsey JD, Gimson A, Toogood GJ, Rela M, et al. Pretransplant MELD score and
post liver transplantation survival in the UK and Ireland. Liver Transpl 2004;10:903–907.
Jacquelinet C, Audry B, Pessione F, Antoine C, Loty B, Calmus Y. Rules for allocation of livers for
transplantation. Presse Med 2008;37:1782–6.
348
References
Jaeschke, H. (2006) Mechanisms of Liver Injury. II. Mechanisms of neutrophil-induced liver cell injury
during hepatic ischemia-reperfusion and other acute inflammatory conditions. American Journal of
Physiology-Gastrointestinal and Liver Physiology, Vol.290, No.6, (June 2006), pp. G1083-1088, ISSN:
1522-1547
Jaffe R. Liver transplant pathology in pediatric metabolic disorders. Pediatr Dev Pathol 1998;1:102–17.
Jain A B, L. D. Yee, M. A. Nalesnik, et al., “Comparative incidence of de novo nonlymphoid malignancies
after liver transplantation under tacrolimus using surveillance epidemiologic end result data,”
Transplantation, vol. 66, no. 9, pp. 1193–1200, 1998
Jain A, Orloff M, Abt P, et al. Transplantation of liver grafts from older donors: impact on recipients with
hepatitis C virus infection. Transplant Proc 2005;37:3162–3164
Jakab F, Rath Z, Sugar I, et al. Complications following major abdominal surgery in cirrhotic patients.
Hepatogastroenterology 1993;40:176 –9.
Janny, S.; Sauvanet, A.; Farges, O.; LeMée, J.; Maillochaud, JH.; Marty, J. & Belghiti, J. (1997) Outcome
of liver grafts with more than 10 hours of cold ischemia. Transplantation Proceedings, Vol.29, No.5,
(August 1997), pp. 2346-2347, ISSN: 0041-1345
Jassem W, Koo DD, Cerundolo L, et al. Leukocyte infiltration and inflammatory antigen expression in
cadaveric and living-donor livers before transplant. Transplantation 2003 Jun 27;75:2001-7.
Jassem W, Koo DD, Muiesan P, Cerundolo L, Rela M, Fuggle SV, Heaton ND. Non-heart-beating versus
cadaveric and livingdonor livers: differences in inflammatory markers before transplantation.
Transplantation 2003;75:1386–1390.
Jenq CC, Hsu CW, Huang WH, Chen KH, Lin JL, Lin-Tan DT. Serum ferritin levels predict all-cause and
infection-cause 1-year mortality in diabetic patients on maintenance hemodialysis. Am J Med Sci
2009;337:188–194.
Jeyarajah DR, Gonwa TA, Testa G, Abbasoglu O, Goldstein R, Husberg BS, et al. Liver and kidney
transplantation for polycystic disease. Transplantation 1998;66:529-532.
Jeyarajah DR, Netto GJ, Lee SP, et al. Recurrent primary sclerosing cholangitis after orthotopic liver
transplantation: is chronic rejection part of the disease process? Transplantation 1998;66:1300–1306.
Jiang, Y.; Jahagirdar, B.N.; Reinhardt, R.L.; et al. (2002) Pluripotency of mesenchymal stem cells derived
from adult marrow. Nature. Vol. 41, No. 6893, pp. 41-9.
Jilg W, Sieger E, Zachoval R, Schatzl H. Individuals with antibodies against hepatitis B core antigen as
the only serological marker for hepatitis B infection: high percentage of carriers of hepatitis B and C virus.
J Hepatol 1995; 23: 14–20
Jimenez RC, Moreno GE, Colina RF, Palma CF, Loinaz SC, Rodriguez GF, et al. Use of octogenarian
livers safely expands the donor pool. Transplantation 1999;68:572-575.
Johansen JS, Christoffersen P, Moller S, et al. Serum YKL-40 is increased in patients with hepatic
fibrosis. J Hepatol 2000;32: 911–920.
Johnson, SR.; Alexopoulos, S.; Curry, M. & Hanto, DW. (2007) Primary nonfunction (PNF) in the MELD
Era: An SRTR database analysis. American Journal of Transplantation,
Johnston O, Rose CL, Webster AC, Gill JS. Sirolimus is associated with newonset diabetes in kidney
transplant recipients. J Am Soc Nephrol 2008;19: 1411–1418.
Johnston SD, Morris JK, Cramb R, Gunson BK, Neuberger J. Cardiovascular morbidity and mortality after
orthotopic liver transplantation. Transplantation 2002; 73: 901–6.
Jonas S, Bechstein WO, Steinmüller T, et al. Vascular invasion and histopathologic grading determine
outcome after liver transplantation for hepatocellular carcinoma in cirrhosis. Hepatology 2001;33:1080–
1086.
Jung, J.; Zheng, M.; Goldfarb, M. & Zaret, K.S. (1999) Initiation of mammalian liver development from
endoderm by fibroblast growth factors. Science. Vol. 284, pp.1998-2003
Junge G, Tullius SG, Klitzing V, et al. The influence of late acute rejection episodes on long-term graft
outcome after liver transplantation. Transplant Proc 2005;37:1716–7.
349
References
Jurim et al, 1995. Jurim O, et al: Reduced-size grafts—the solution for hepatic artery thrombosis after
pediatric liver transplantation?. J Pediatr Surg 1995; 30:53-55.
Kahn SA 2002a, Davidson BR, Goldin R, et al. Guidelines for the diagnosis and treatment of
cholangiocarcinoma: consensus document. Gut 2002;51:1–9.
Kaihara S, Kiuchi T, Ueda M et al. Living-donor liver transplantation for hepatocellular carcinoma.
Transplantation 2003; 75: S37–40.
Kakishita, K.; Elwan, M.A.; Nakano, N.; Itakura, T. & Sakuragawa, N. (2000) Human amnionic epithelial
cells produce dopamine and survive after implantation into the struatum of a rat model of Parkinson’s
disease: a potential source of donor for transplantation therapy. Experimental Neurology. Vol. 165, No. 1,
pp. 27-34
Kakishita, K.; Nakao, N.; Sakuragawa, N. & Itakura, T. (2003) Implantation of human amniotic epithelial
cells prevents the degeneration of nigral dopamine neurons in rats with 6 hydroxydopamine lesions. Brain
Research. Vol. 980, Vol. 1, pp. 48-56
Kakizoe S, Yanaga K, Starzl TE, et al. Evaluation of protocol before transplantation and after reperfusion
biopsies from human orthotopic liver allografts: considerations of preservation and early immunological
injury. Hepatology 1990;11:932–41
Kalantar-Zadeh K, Don BR, Rodriguez RA, Humphreys MH. Serum ferritin is a marker of morbidity and
mortality in hemodialysis patients. Am J Kidney Dis 2001;37:564–572.
Kalayoglu M, Sollinger HW, Stratta RJ, D’Alessandro AM, Hoffmann RM, Pirsch JD, Belzer FO. Extended
preservation of the liver for clinical transplantation. Lancet 1988;1(8586):617–619.
Kam I. Adult-adult right hepatic lobe living donor liver transplantation for status 2a patients: too little, too
late. Liver Transpl 2002;8:347–349
Kamar N, Selves J, Mansuy JM, et al. Hepatitis E virus and chronic hepatitis in organ-transplant
recipients. N Engl J Med 2008;358:811–7.
Kamath PS, Wiesner RH, Malinchoc M, Kremers W, Therneau TM, Kosberg CL, et al. A model to predict
survival in patients with end-stage liver disease. HEPATOLOGY 2001;33:464-470.
Kamath S, Kim WR. The model for end-stage liver disease (MELD). Hepatology 2007; 45: 797–805.
Kamimoto Y, Horiuchi S, Tanase S, et al. Plasma clearance of intravenously injected aspartate
aminotransferase isozymes: evidence for preferential uptake by sinusoidal liver cells. Hepatology
1985;5:367–375.
Kanchana TP, Kaul V, Manzarbeitia C, Reich DJ, Hails KC, Munoz SJ, et al. Liver transplantation for
patients on methadone maintenance. Liver Transpl 2002;8:778-782.
Kanzler S, Baumann M, Schirmacher P, et al. Prediction of progressive liver fibrosis in hepatitis C
infection by serum and tissue levels of transforming growth factor-B1. J Viral Hepat 2001;8:430 – 437.
Karatzas T, Olson L, Ciancio G, Burke GW, Spires G, Cravero L, et al. Expanded liver donor age over 60
years for hepatic transplantation. Transplant Proc 1997;29:2830-2831.
Karie-Guigues S, et al: Long-term renal function in liver transplant recipients and impact of
immunosuppressive regimens (calcineurin inhibitors alone or in combination with mycophenolate mofetil):
the TRY study. Liver Transpl 2009; 15:1083-1091
Kasahara A, Hayashi N, Mochizuki K, et al. Circulating matrix metalloproteinase-2 and tissue inhibitor of
metalloproteinase-1 as serum markers of fibrosis in patients with chronic hepatitis C. Relationship to
interferon response. J Hepatol 1997;26: 574–583.
Kashyap R, Jain A, Reyes J, et al. Causes of death after liver transplantation in 4000 consecutive
patients: 2 to 19 year follow-up. Transplant Proc 2001; 33: 1482–3.
Kassianides C, Nussenblatt R, Palestine AG, et al. Liver injury from cyclosporine A. Dig Dis Sci
1990;35:693–7.
Katoonizadeh A, Decaestecker J, Wilmer A, et al. MELD score to predict outcome in adult patients with
non-acetaminopheninduced acute liver failure. Liver Int 2007;27:329–334.
Katzka DA, Saul SH, Jorkasky D, et al. Azathioprine and hepatic venocclusive disease in renal transplant
patients. Gastroenterology 1986;90:446–54.
350
References
Kauffman HM, McBride MA, Cherikh WS, et al. Transplant tumor registry: donors with central nervous
system tumors. Transplantation 27 2002;73:579–82.
Kauffman HM, McBride MA, Delmonico FL. First report of the United Network for Organ Sharing
Transplant Tumor Registry: donors with a history of cancer. Transplantation 2000;70:1747–51.
Kaufman DB, Shapiro R, Lucey MR, Cherikh WS, R TB, Dyke DB (2004) Immunosuppression: practice
and trends. Am J Transplant 4(Suppl 9):38–53
Kawasaki S, Makuuchi M, Matsunami H, et al. Living related liver transplantation in adults. Ann Surg
1998;227:269-74.
Kawasaki T, Takeshita A, Souda K, et al. Serum thrombopoietin levels in patients with chronic hepatitis
and liver cirrhosis. Am J Gastroenterol 1999;94:1918–1922.
Kayler LK, Merion RM, Lee S, Sung RS, Punch JD, Rudich SM, et al. Long-term survival after liver
transplantation in children with metabolic disorders. Pediatr Transplant 2002;6:295-300.
Kayler LK, Rasmussen CS, Dykstra DM, Punch JD, Rudich SM, Magee JC, et al. Liver transplantation in
children with metabolic disorders in the United States. Am J Transplant 2003;3:334-339.
Keeffe EB. Liver transplantation: current status and novel approaches to liver replacement.
Gastroenterology 2001; 120: 749-762.
Keiding S, Hansen SB, Rasmussen HH, Gee A, Kruse A, Roelsgaard K, et al. Detection of
cholangiocarcinoma in primary sclerosing cholangitis by positron emission tomography. Hepatology
1998;28: 700–706.
Kelleher TB, Mehta SH, Bhaskar R, et al. Prediction of hepatic fibrosis in HIV/HCV co-infected patients
using serum fibrosis markers: the SHASTA index. J Hepatol 2005;43:78–84.
Kemnitz J, Gubernatis G, Bunzendahl H, Ringe B, Pichlmayr R, Georgii A. Criteria for the
histopathological classification of liver allograft rejection and their clinical relevance. Transplant Proc
1989;21:2208–2210.
Kessel A, Toubi E. Chronic HCV-related autoimmunity: a consequence of viral persistence and
lymphotropism. Curr Med Chem 2007;14:547–54.
Keswani RN, A. Ahmed, and E. B. Keeffe, “Older age and liver transplantation: a review,” Liver
Transplantation, vol. 10, no. 8, pp. 957–967, 2004.
Khan SA 2006b, Shah N, Williams R, et al. Acute liver failure: a review. Clin Liver Dis 2006;10:239–258.
Khapra AP, Agarwal K, Fiel MI et al. Impact of donor age on survival and fibrosis progression in patients
with hepatitis C undergoing liver transplantation using HCV+ allografts. Liver Transpl 2006; 12: 1496–
1503
Kharaziha, P.; Hellström, P.M.; Noorinayer, B.; et al. (2009) Improvement of liver function in liver cirrhosis
patients after autologous mesenchymal stem cell injection: a phase III clinical trial. Eur J Gastroenterol
Hepatol. Vol. 21, No. 10, pp. 1199-1205
Khettry U, Huang WY, Simpson MA, et al. Patterns of recurrent hepatitis C after liver transplantation in a
recent cohort of patients. Hum Pathol 2007;38:443–52.
Kilpe V, Krakauer H, Wren RE. An analysis of liver transplant experience from 37 transplant centers as
reported to Medicare. Transplantation 1993;56:554–561.
Kim SY, Yim HJ, Lee J, et al. Comparison of CTP, MELD, and MELD-Na scores for predicting short term
mortality in patients with liver cirrhosis. Korean J Gastroenterol 2007;50:92–100.
Kim WR, Benson JT, Hindman A, et al. Decline in the need for liver transplantation for end stage liver
disease secondary to hepatitis B in the US (abstr). Hepatology 2007;46(Suppl 1):238A
Kim WR, Biggins SW, Kremers WK, et al. Hyponatremia and mortality among patients on the livertransplant waiting list. N Engl J Med 2008;359: 1018–26.
Kim WR, Krowka MJ, Plevak DJ, Lee J, Rettke SR, Frantz RP, et al. Accuracy of Doppler
echocardiography in the assessment of pulmonary hypertension in liver transplant candidates. Liver
Transpl 2000;6:453- 458.
Kim WR, Poterucha JJ, Porayko MK, et al. Recurrence of nonalcoholic steatohepatitis following liver
transplantation. Transplantation 1996;62:1802–1805.
351
References
Kim WR, Wiesner RH, Therneau TM, Poterucha JJ, Porayko MK, Evans RW, et al. Optimal timing of liver
transplantation for primary biliary cirrhosis. Hepatology 1998;28:33–38.
Kim, K.; Doi, A.; Wen, B.; et al. (2010). Epigenetic memory in induced pluripotent stem cells. Nature. Vol.
467, pp. 285-292.
Kimura F, Miyazaki M, Suwa T, et al. Reduction of hepatic acute phase response after partial
hepatectomy in elderly patients. Res Exp Med (Berl) 1996;196:281–290
Kiuchi T, Kasahara M, Uryuhara K, et al. Impact of graft size mismatching on graft prognosis in liver
transplantation from living donors. Transplantation 1999;67:321–7.
Klein AS, “Management of Budd-Chiari syndrome,” Liver Transplantation, vol. 12, no. 11, pp. S23–S28,
2006.
Kleiner DE, Brunt EM, Van Natta M, Behling C, Contos MJ, Cummings OW, et al. Design and validation
of a histological scoring system for nonalcoholic fatty liver disease. Hepatology 2005;41(6):1313–21
Klune, JR. & Tsung, A. (2010) Molecular biology of liver ischemia/reperfusion injury: established
mechanisms and recent advancements. Surgery Clinics of North America, Vol.90, No.4, (August 2010),
pp. 665-677, ISSN: 1042-3699
Kneteman NM, Oberholzer J, Al Saghier M, et al. Sirolimus-based immunosuppression for liver
transplantation in the presence of extended criteria for hepatocellular carcinoma. Liver Transpl.
2004;10(10):1301- 11.
Kniepeiss D, Iberer F, Grasser B, Schaffellner S, Tscheliessnigg KH. Sirolimus and mycophenolate
mofetil after liver transplantation. Transpl Int 2003;16(7):504-9.
Knodell RG, Ishak KG, Black WC, Chen TS, Craig R, Kaplowitz N, et al. Formulation and application of a
numerical scoring system for assessing histological activity in asymptomatic chronic active hepatitis.
Hepatology 1981;1(5):431–5,
Koda M, Matunaga Y, Kawakami M, et al. FibroIndex, a practical index for predicting significant fibrosis in
patients with chronic hepatitis C. Hepatology 2007;45:297–306.
Koenig, S.; Stoesser, C.; Krause, P.; et al. (2005) Liver repopulation after hepatocellular transplantation:
integration and interaction of transplanted hepatocytes in the host. Cell Transplant. Vol. 14, pp. 31–40
Komolmit P, Davies MH. Tacrolimus in liver transplantation. Expert Opin Investig Drugs 1999;8(8):1239–
54.
Kondo F. Benign nodular hepatocellular lesions caused by abnormal hepatic circulation: etiological
analysis and introduction of a new concept. J Gastroenterol Hepatol 2001;16:1319–28.
Koneru B, Dikdan G. (2002). Hepatic steatosis and liver transplantation current clinical and experimental
perspectives. Transplantation 73:325–330.
Kootstra G, Kievit J, Nederstigt A. Organ donors: heartbeating and nonheartbeating. World J Surg
2002;26:181–4.
Kootstra G,1995a Daemen JH, Oomen AP. Categories of non-heartbeating donors. Transplant Proc
1995;27:2893–2894.
Kootstra G.1995b Statement on non-heart-beating donor programs. Transplant Proc 1995;27:29653.
D’Alessandro AM, Hoffmann RM, Knechtle SJ, Odorico JS, Becker YT, Musat A, et al. Liver
transplantation from controlled non-heart-beating donors. Surgery 2000;128:579–588.
Korn T, Reddy J, Gao W, Bettelli E, Awasthi A, Petersen TR, Bäckström BT, Sobel RA, Wucherpfennig
KW, Strom TB, Oukka M, Kuchroo VK. Myelin-specific regulatory T cells accumulate in the CNS but fail to
control autoimmune inflammation. Nat Med. 2007 Apr;13(4):423-31.
Kotlyar DS, Campbell MS, Reddy KR. Recurrence of diseases following orthotopic liver transplantation.
Am J Gastroenterol 2006;101:1370–8.
Koudstaal LG, 't Hart NA, Ottens PJ, et al. Brain death induces inflammation in the donor intestine.
Transplantation 2008;86: 148-54.
Kowdley KV, Brandhagen DJ, Gish RG, et al. Survival after liver transplantation in patients with hepatic
iron overload: the national hemochromatosis transplant registry. Gastroenterology 2005;129:494–503.
352
References
Kowdley KV, Keeffe EB, Fawaz KA. Prolonged cholestasis due to trimethoprim sulfamethoxazole.
Gastroenterology 1992;102:2148–50.
Koyama I, Fuchinoue S, Urashima Y, Kato Y, Tsuji K, Kawase T, et al. Living related liver transplantation
for polycystic liver disease. Transpl Int 2002;15:578-580.
Koyama I, Nadazdin O, Boskovic S, et al. Depletion of CD8 memory T cells for induction of tolerance of a
previously transplanted kidney allograft. Am J Transplant 2007;7:1055–1061.
Kozak EM, Tate SS. Glutathione-degrading enzymes of microvillus membranes. J Biol Chem. 1982; 257:
6322-7.
Kozaki K, Egawa H, Kasahara M, Oike F, Yoshizawa A, Fukatsu A, et al. Therapeutic strategy and the
role of apheresis therapy for ABO incompatible living donor liver transplantation. Ther Apher Dial
2005;9:285-91.
Krasinskas AM a2001, Ruchelli ED, Rand EB, Chittams JL, Furth EE. Central venulitis in pediatric liver
allografts. Hepatology 2001;33:114
Krasinskas AM b2008, Demetris AJ, Poterucha JJ, Abraham SC. The prevalence and natural history of
untreated isolated central perivenulitis in adult allograft livers. Liver Transpl 2008;14:625–632.
Krasinskas AM, Demetris AJ, Poterucha JJ, et al. The prevalence and natural history of untreated
isolated central perivenulitis in adult allograft livers. Liver Transpl 2008;14:625–32.
Kreisel D et al. Non-hematopoietic allograft cells directly activate CD8+ T cells and trigger acute rejection:
an alternative mechanism of allorecognition. Nat Med 2002; 8(3): pp 233-9.
Krowka MJ, D. J. Plevak, J. Y. Findlay, C. B. Rosen, R. H. Wiesner, and R. A. F. Krom, “Pulmonary
hemodynamics and perioperative cardiopulmonary-related mortality in patients with portopulmonary
hypertension undergoing liver transplantation,” Liver Transplantation, vol. 6, no. 4, pp. 443–450, 2000.
Krowka MJ, M. K. Porayko, D. J. Plevak et al., “Hepatopulmonary syndrome with progressive hypoxemia
as an indication for liver transplantation: case reports and literature review,” Mayo Clinic Proceedings, vol.
72, no. 1, pp. 44–53,1997.
Krowka MJ, Plevak DJ, Findlay JY, Rosen CB, Wiesner RH, Krom RA. Pulmonary hemodynamics and
perioperative cardiopulmonary-related mortality in patients with portopulmonary hypertension undergoing
liver transplantation. Liver Transpl 2000;6:443-450.
Ku CC, Murakami M, Sakamoto A, Kappler J, Marrack P (2000) Control of homeostasis of CD8+ memory
T cells by opposing cytokines. Science 288:675–678.
Kucher N, Tapson VF, Goldhaber SZ. Risk factors associated with symptomatic pulmonary embolism in a
large cohort of deep vein thrombosis patients. Thromb Haemost 2005;93:494-8.
Kuecuek O, Mantouvalou L, Klemz R, et al. Significant reduction of proinflammatory cytokines by
treatment of the brain-dead donor. Transplant Proc 2005;37:387-8.
Kukan M, Haddad PS. Role of hepatocytes and bile duct cells in preservationreperfusion injury of liver
grafts. Liver Transpl 2001;7:381–400.
Kuo, T.K.; Hung, S.P.; Chuang, C.H.; et al. (2008) Stem cell therapy for liver disease: parameters
governing the success of using bone marrow mesenchymal stem cells. Gastroenterology. Vol. 134, No. 7,
pp. 2111-2121
Kupiec-Weglinski, JW. & Busuttil, RW. (2005) Ischemia and reperfusion injury in liver transplantation.
Transplantation Proceedings, Vol.37, No.4, (May 2005), pp. 1653- 1656, ISSN: 0041-1345
Kusaka M, Kuroyanagi Y, Kowa H. Genomewide expression profiles of rat model renal isografts from
brain dead donors. Transplantation 2007;83:62-70.
Kusaka M, Pratschke J, Wilhelm M, et al. Activation of inflammatory mediators in rat renal isografts by
donor brain death. Transplantation 2000;69:405-10
Kwekkeboom J, Kuijpers MA, Bruyneel B, Mancham S, De Baar-Heesakkers E, Ijzermans JN, et al.
Expression of CD80 on Kupffer cells is enhanced in cadaveric liver transplants. Clin Exp Immunol
2003;132:345-351.
Lackner C, Struber G, Liegl B, Leibl S, Ofner P, Bankuti C, et al. Comparison and validation of simple
noninvasive tests for prediction of fibrosis in chronic hepatitis C. Hepatology 2005;41(6):1376–82.
353
References
Lafayette RA, Pare G, Schmid CH, King AJ, Rohrer RJ, Nasraway SA.Pretransplant renal dysfunction
predicts poorer outcome in liver transplantation. Clin Nephrol 1997;48:159-164.
Lake JR. The role of immunosuppression in recurrence of hepatitis C. Liver Transpl 2003;9:S63–6.
Lake, JR.; Shorr, JS.; Steffen, BJ.; Chu, AH.; Gordon, RD. & Wiesner, RH. (2005) Differential effects of
donor age in liver transplant recipients infected with hepatitis B.; hepatitis C and without viral hepatitis.
American Journal of Transplantation, Vol.5, No.3, (March 2005), pp. 549-557, ISSN: 1600-6143
Lampertico P, Vigano M, Manenti E, Iavarone M, Sablon E, Colombo M. Low resistance to adefovir
combined with lamivudine: a 3-year study of 145 lamivudine-resistant hepatitis B patients.
Gastroenterology 2007;133:1445-51.
Lamy ME, Favart AM, Cornu C, et al. Epstein–Barr virus infection in 59 orthotopic liver transplant
patients. Med Microbiol Immunol 1990;179:137–44.
Lange, C.; Bassler, P.; Lioznov, M.V.; et al. (2005) Liver-specific gene expression in mesenchymal stem
cells is induced by liver cells. World J Gastroenterol. Vol. 11, No. 29, pp. 4497-504
Larrousse M, Laguno M, Segarra M, et al. Noninvasive diagnosis of hepatic fibrosis in HIV/HCVcoinfected patients. J Acquir Immune Defic Syndr 2007;46:304 –311.
Larson AM, Polson J, Fontana RJ, et al. Acetaminophen-induced acute liver failure: results of a United
States multicenter, prospective study. Hepatology 2005;42:1364–1372.
LaRusso NF, Shneider BJ, Black D, et al. Primary sclerosing cholangitis: summary of a workshop.
Hepatology 2006;44:746–764.
Lautenschlager I, Halme L, Hockerstedt K, et al. Cytomegalovirus infection of the liver transplant:
virological, histological, immunological, and clinical observations. Transpl Infect Dis 2006;8:21–30.
Lautt WW. Mechanism and role of intrinsic regulation of hepatic arterial blood flow: hepatic arterial buffer
response. Am J Physiol 1985;249:G549–56.
Le Blanc, K. & Ringden, O. (2007) Immunomodulationby mesenchymal stem cells and clinical
experience. J Intern Med. Vol. 262, pp.509–525.
Le Blanc, K.; Gotherstrom, C.; Ringden, O., et al. (2005) Fetal Mesenchymal Stem-Cell Engraftment in
Bone after In Utero Transplantation in a Patient with Severe Osteogenesis Imperfecta. Transplantation.
Vol. 79, pp. 1607-1614
Le Blanc, K.; Rasmusson, I.; Sundburg, B.; et al. (2004) Treatment of severe acute graftversus-host
disease with third party haploidentical mesenchymal stem cells. Lancet. Vol. 363, pp. 1439-1441
Le Couteur, D.G., McLean, A.J., 1998. The aging liver. Drug clearance and an oxygen diffusion barrier
hypothesis. Clin. Pharmacokinet. 34, 359–373.
Lechler RI, Sykes M, Thomson AW, Turka LA. Organ transplantation: how much of the promise has been
realized? Nat Med 2005; 11:605–613.
Lee DH, Ha MH, Kim KY, et al. Gamma-glutamyltransferase: an effect modifier in the association
between age and hypertension in a 4-year follow-up study. J Hum Hypertens. 2004; 18: 803–807.
Lee DH, Jacobs DR Jr, Gross M, et al. Gamma-glutamyltransferase is a predictor of incident diabetes and
hypertension: the Coronary Artery Risk Development in Young Adults (CARDIA) Study. Clin Chem
2003;49:1358-1366.
Lee DH, Jacobs DR Jr, Gross M, Steffes M. Serum gamma-glutamyltransferase was differently
associated with microalbuminuria by status of hypertension or diabetes: the Coronary Artery Risk
Development in Young Adults (CARDIA) Study. Clin Chem. 2005; 51: 1185–1191.
Lee DS, Evans JC, Robins SJ, et al. Gamma glutamyl transferase and metabolic syndrome,
cardiovascular disease, and mortality risk: the Framingham Heart Study. Arterioscler Thromb Vasc Biol
2007;27:127-33.
Lee KW, Simpkins C, Montgomery R, et al. Factors affecting graft survival after liver transplantation from
donation after cardiac donors. Transplantation 2006;82:1683-8.
Lee SG, Hwang S, Park KM, Kim KH, Ahn CS, Lee YJ, et al. Seventeen adult-to-adult living donor liver
transplantations using dual grafts. Transplant Proc 2001;33:3461-3.
354
References
Lee WM, “Acute liver failure,” The New England Journal of Medicine, vol. 329, no. 25, pp. 1862–1872,
1993.
Lee WM. Acute liver failure in the United States. Semin Liver Dis 2003; 23:217-226.
Lee WM. Management of acute liver failure. Semin Liver Dis 1996;16: 369-378.
Lee YM, Kaplan MM. Management of primary sclerosing cholangitis. Am J Gastroenterol 2002;97:528534.
Lehmann TG, Koeppel TA, Kirschfink M, Gebhard MM, Herfarth C, Otto G, Post S. Complement inhibition
by soluble complement receptor type 1 improves microcirculation after rat liver transplantation.
Transplantation 1998;66:717–722.
Lentsch AB, Kato A, Yoshidome H, McMasters KM, Edwards MJ. Inflammatory mechanisms and
therapeutic strategies for warm hepatic ischemia / reperfusion injury. Hepatology 2000; 32:169–173.
Leroy V, Monier F, Bottari S, Trocme C, Sturm N, Hilleret MN, et al. Circulating matrix metalloproteinases
1, 2, 9 and their inhibitors TIMP-1 and TIMP-2 as serum markers of liver fibrosis in patients with chronic
hepatitis C: comparison with PIIINP and hyaluronic acid. Am J Gastroenterol 2004;99(2):271–9.
Lerut et al, 1987. Lerut J, et al: Complications of venous reconstruction in human orthotopic liver
transplantation. Ann Surg 1987; 205:404-414.
Levitsky J. Operational tolerance: past lessons and future prospects. Liver Transpl. 2011 Mar;17(3):22232.
Levy G, Schmidli H, Punch J, et al. Safety, tolerability, and efficacy of everolimus in de novo liver
transplant recipients: 12- and 36-month results. Liver Transpl 2006;12(11):1640-8.
Lewis J, Peltier J, Nelson H, et al. Development of the University of Wisconsin donation after cardiac
death evaluation tool. Prog Transplant 2003;13:265–73.
Lewis M, Howdle P. Neurologic complications of liver transplantation in adults. Neurology 2003; 61: 11741178.
Li F, Atz ME, Reed EF. Human leukocyte antigen antibodies in chronic transplant vasculopathymechanisms and pathways. Curr Opin Immunol 2009;21:557–562.
Li J, Liu B, Yan LN, Zuo YX, Li B, Zeng Y, et al. Reversal of graft steatosis after liver transplantation:
prospective study. Transplant Proc 2009;41: 3560–3563.
Li J, Sherman-Baust CA, Tsai-Turton M, et al. Claudin-containing exosomes in the peripheral circulation
of women with ovarian cancer. BMC Cancer 2009;9:244.
Li S, Stratta RJ, Langnas AN, et al. Diffuse biliary tract injury after orthotopic liver transplantation. Am J
Surg 1992;164:536–40.
Li W, Li B, Fan W, Geng L, Li X, Li L, Huang Z, Li S. CTLA4Ig gene transfer alleviates abortion in mice by
expanding CD4+CD25+ regulatory T cells and inducing indoleamine 2,3-dioxygenase. J Reprod Immunol.
2009 Jun;80(1-2):1-11.
Li, H.; Niederkorn, J.Y.; Neelam, S.; et al. (2005) Immunosuppressive factors secreted by human amniotic
epithelial cells. Invest. Ophtalmol. Visual Science. Vol. 46, No. 3, pp. 900-907
Li, Y.; Zhao, H.; Lan, F.; et al. (2010). Generation of human-induced pluripotent stem cells from gut
mesentery-derived cells by ectopic expression of OCT4/SOX2/NANOG. Cell Reprogram. Vol. 12, pp.
237-247.
Lieber CS, Weiss DG, Morgan TR, et al. Aspartate aminotransferase to platelet ratio index in patients with
alcoholic liver fibrosis. Am J Gastroenterol 2006;101:1500–1508.
Liermann Garcia RF, Evangelista Garcia C, McMaster P, et al. Transplantation for primary biliary
cirrhosis: retrospective analysis of 400 patients in a single center. Hepatology 2001;33:22–7.
Lim JK, Keeffe EB. Liver transplantation for alcoholic liver disease: current concepts and length of
sobriety. Liver Transpl 2004;10(10 Suppl 2):S31–8.
Lim ZY, Fiaccadori V, Gandhi S, Hayden J, Kenyon M, Ireland R, et al. Impact of pre-transplant serum
ferritin on outcomes of patients with myelodysplastic syndromes or secondary acute myeloid leukaemia
receiving reduced intensity conditioning allogeneic haematopoietic stem cell transplantation. Leuk Res
2010;34:723–727.
355
References
Limand JK, E. B. Keeffe, “Liver transplantation for alcoholic liver disease: current concepts and length of
sobriety,” Liver Transplantation, vol. 10, no. 10, pp. S31–S38, 2004.
Lin, H.; Xu, R.;Zhang, Z.; et al. (2011) Implications of the immunoregulatory functions of mesenchymal
stem cells in the treatment of human liver diseases. Cell Mol Immunol. Vol. 8, No. 1, pp. 19-22.
Lipshutz G. S, J. Hiatt, R. M. Ghobrial et al., “Outcome of liver transplantation in septuagenarians: a
single-center experience,” Archives of Surgery, vol. 142, no. 8, pp. 775–781, 2007.
Little D, Farell J, Cunningham P, Hickey DP. Donor sepsis is not a contraindication to cadaveric organ
donation. Q J Med 1997; 90: 641–642
Liu CL, Lo CM, Chan SC, Fan ST. Safety of duct-to-duct biliary reconstruction in right-lobe live-donor liver
transplantation without biliary drainage. Transplantation 2004;77:726–732.
Liu CL, Lo CM, Fan ST. What is the best technique for right hemiliver living donor liver transplantation?
With or without the middle hepatic vein? Duct-to-duct biliary anastomosis or Rouxen- Y
hepaticojejunostomy? J Hepatol 2005;43:17–22.
Liu LU, Schiano TD, Lau N, O’Rourke M, Min AD, Sigal SH, et al. Survival and risk of recidivism in
methadone-dependent patients undergoing liver transplantation. Am J Transplant 2003;3:1273-1277.
Liu LU, Schiano TD, Min AD, Kim-Schluger L, Schwartz ME, Emre S, Fishbein TM, Bodenheimer HC Jr,
Miller CM. Syngeneic living-donor liver transplantation without the use of immunosuppression.
Gastroenterology. 2002 Oct;123(4):1341-5.
Liu, H.; Kim, Y.; Sharkis, S.; et al. (2011). In vivo liver regeneration potential of human induced pluripotent
stem cells from diverse origins. Sci Transl Med. Vol. 3, pp. 82ra39.
Liver Transplant Activities. Available at http://www.ministerosalute.it/trapianti [accessed February, 2003]
Llach J, Gines P, Arroyo V, Rimola A, Tito L, Badalamenti S, et al. Prognostic value of arterial pressure,
endogenous vasoactive systems, and renal function in cirrhotic patients admitted to the hospital for the
treatment of ascites. Gastroenterology 1988;94:482–487.
Llado L, Figueras J, Memba R, Xiol X, Baliellas C, Vazquez S, et al. Is MELD really the definitive score
for liver allocation? Liver Transpl 2002;8:795–798.
Llovet JM 1999a, Bru C, Bruix J. Prognosis of hepatocellular carcinoma: the BCLC staging classification.
Semin Liver Dis 1999;19:329-338.
Llovet JM, Bruix J. Systematic review of randomized trials for unresectable hepatocellular carcinoma:
chemoembolization improves survival. HEPATOLOGY 2003;37:429-442.
Llovet JM, Fuster J, Bruix J. Intention-to-treat analysis of surgical treatment for early hepatocellular
carcinoma: resection versus transplantation.HEPATOLOGY 1999;30:1434-1440.
Llovet JM, Real MI, Montana X, Planas R, Coll S, Aponte J, et al. Arterial embolisation or
chemoembolisation versus symptomatic treatment in patients with unresectable hepatocellular
carcinoma: a randomised controlled trial. Lancet 2002;359:1734-1739.
Lo CM, et al., “Lessons learned from one hundred right lobe living donor liver transplants,” Annals of
Surgery 2004; 240(1):151–158.
Lo CM, Fan ST, Chan JK, et al. Minimum graft volume for successful adult-to-adult living donor liver
transplantation for fulminant hepatic failure. Transplantation 1996;62:696–8.
Lo CM, Fan ST, Liu CL, Chan SC, Wong J. The role and limitation of living donor liver transplantation for
hepatocellular carcinoma. Liver Transpl. 2004; 10: 440–7.
Lo CM, Ngan H, Tso WK, Liu CL, Lam CM, Poon RT, et al. Randomized controlled trial of transarterial
lipiodol chemoembolization for unresectable hepatocellular carcinoma. HEPATOLOGY 2002;35:11641171.
Lo IJ, Lefkowitch JH, Feirt N, Alkofer B, Kin C, Samstein B, et al. Utility of liver allograft biopsy obtained at
procurement. Liver Transpl 2008;14: 639–646.
Logozzi M, De Milito A, Lugini L, et al. High Levels of Exosomes Expressing CD63 and Caveolin-1 in
Plasma of Melanoma Patients. PLoS One 2009;4:5219.
356
References
Lok AS, Ghany MG, Goodman ZD,Wright EC, Everson GT, Sterling RK, et al. Predicting cirrhosis in
patients with hepatitis C based on standard laboratory tests: results of the HALT-C cohort. Hepatology
2005;42(2):282–92.
Lok AS, McMahon BJ. Chronic hepatitis B. HEPATOLOGY 2001;34: 225-1241
Londo˜no MC, Cárdenas A, Guevara M, et al.MELDscore and serum sodium in the prediction of survival
of patients with cirrhosis awaiting liver transplantation. Gut 2007;56:1283–90.
Londono MC, Guevara M, Rimola A, Navasa M, Taura P, Mas A, et al. Hyponatremia impairs early
posttransplantation outcome in patients with cirrhosis undergoing liver transplantation. Gastroenterology
2006;130:1135–1143.
Longerich T, Schirmacher P. General aspects and pitfalls in liver transplant pathology. Clin Transplant
2006;20(Suppl 17):60–8.
Longheval G, Vereerstraeten P, Thiry P, et al., “Predictive models of short- and long-term survival in
patients with nonbiliary cirrhosis,” Liver Transplantation, 2003; 9 (3): 260–267
Lopez-Navidad A, Caballero F. Extended criteria for organ acceptance: strategies for achieving organ
safety and increasing organ pool. Clin Transplant 2003;17:308–324
Lopez-Navidad A, Domingo P, Caballero F, et al. Successful transplantation of organs retrieved from
donors with bacterial meningitis. Transplantation 1997;64: 365–8.
Losada I, Cuervas-Mons V, Millan I, Damaso D. Early infection in liver transplant recipients: incidence,
severity, risk factors and antibiotic sensitivity of bacterial isolates. Enferm Infecc Microbiol Clin 2002; 20:
422-30.
Lovell MO, Speeg KV, Halff GA, Molina DK, Sharkey FE. Acute hepatic allograft rejection: a comparison
of patients with and without centrilobular alterations during first rejection episode. Liver Transpl
2004;10:369–373.
Luan FL, Ding R, Sharma VK, Chon WJ, Lagman M, Suthanthiran M. Rapamycin is an effective inhibitor
of human renal cancer metastasis. Kidney Int 2003;63:917-26.
Luca A, Angermayr B, Bertolini G, Koenig F, Vizzini G, Ploner M, Peck-Radosavljevic M, Gridelli B, Bosch
J. An integrated MELD model including serum sodium and age improves the prediction of early mortality
in patients with cirrhosis. Liver Transpl 2007; 13: 1174-1180
Lucey MR, Brown KA, Everson GT, Fung JJ, Gish R, Keeffe EB, et al. Minimal criteria for placement of
adults on the liver transplant waiting list: a report of a national conference organized by the American
Society of Transplant Physicians and the American Association for the Study of Liver Diseases. Liver
Transpl Surg 1997;3:628-637.
Lucey MR, Carr K, Beresford TP, et al. Alcohol use after liver transplantation in alcoholics: a clinical
cohort follow-up study. Hepatology 1997;25:1223–7.
Ludwig J, Batts KP, MacCarty RL. Ischemic cholangitis in hepatic allografts. Mayo Clin Proc
1992;67:519–26.
Ludwig J, Batts KP, Ploch M, et al. Endotheliitis in hepatic allografts. Mayo Clin Proc 1989;64:545–54.
Lumbreras C, Sanz F, Gonzalez A et al. Clinical significance of donor-unrecognized bacteremia in the
outcome of solid-organ transplant recipients. Clin Infect Dis 2001; 33: 722–726
Machicao, VI, Bonatti H, Krishna M, et al: Donor age affects fibrosis progression and graft survival after
liver transplantation for hepatitis C. Transplantation 77:84, 2004
Mackie J, Groves K, Hoyle A, et al. Orthotopic liver transplantation for alcoholic liver disease: a
retrospective analysis of survival, recidivism, and risk factors predisposing to recidivism. Liver Transpl
2001;7:418–27.
MacQuillan G. C, M. S. Seyam, P. Nightingale, J. M. Neuberger, and N. Murphy, “Blood lactate but not
serum phosphate levels can predict patient outcome in fulminant hepatic failure,” Liver Transplantation,
vol. 11, no. 9, pp. 1073–1079, 2005.
Maddala YK, Stadheim L, Andrews JC et al. Drop-out rates of patients with hepatocellular cancer listed
for liver transplantation: outcome with chemoembolization. Liver Transpl. 2004; 10: 449–55.
357
References
Maddrey, WC., Schiff, ER. & Sorrell, MF. eds, Lipincott Williams and Wilkins, pp. 121,ISBN-10
0781720397, ISBN-13 978-0781720397, Philadelphia
Magliocca JF, Magee JC, Rowe SA, et al. Extracorporeal support for organ donation after cardiac death
effectively expands the donor pool. J Trauma 2005;58:1095–101.
Maharaj B, Maharaj RJ, Leary WP, Cooppan RM, Naran AD, Pirie D, et al. Sampling variability and its
influence on the diagnostic yield of percutaneous needle biopsy of the liver. Lancet 1986;1(8480):523–5.
Maheshwari A, Torbenson MS, Thuluvath PJ. Sirolimus monotherapy versus sirolimus in combination
with steroids and/or MMF for immunosuppression after liver transplantation. Dig Dis Sci.
2006;51(10):1677-8
Maheshwari A, Yoo HY, Thuluvath PJ. Long-term outcome of liver transplantation in patients with PSC: a
comparative analysis with PBC. Am J Gastroenterol 2004;99:538-542.
Mahindra A, Sobecks R, Rybicki L, Pohlman B, Dean R, Andresen S, et al. Elevated pretransplant serum
ferritin is associated with inferior survival following nonmyeloablative allogeneic transplantation. Bone
Marrow Transplant 2009;44:767–768.
Mair P, Kaehler CH, Pomaroli A, Schwarz B, Vogel W, Margreiter R. Orthotopic liver transplantation in a
patient with severe portopulmonary hypertension. Acta Anaesthesiol Scand 2001;45:513-518.
Makhlouf HR, Ishak KG, Goodman ZD. Epithelioid hemangioendothelioma of the liver: a clinicopathologic
study of 137 cases. Cancer 1999; 85:562-582.
Malago M, Hertl M, Testa G, Rogiers X, Broelsch CE. Split-liver transplantation: future use of scarce
donor organs. World J Surg 2002;26:275- 282.
Mali, H. & Gupta, S. (2001) Hepatocyte transplantation: new horizons and challenges. J. Hepatobiliary
Pancreat. Surg. Vol. 8, pp. 40-50
Malinchoc M, Kamath PS, Gordon FD, Peine CJ, Rank J, ter Borg PC. A model to predict poor survival in
patients undergoing transjugular intrahepatic portosystemic shunts. HEPATOLOGY 2000;31:864-871.
Maluf D, Stravitz R, Cotterell A, et al. Adult living donor versus deceased donor liver transplantation: a 6year single center experience. Am J Transplant 2005;5:149–56.
Manns MP, McHutchison JG, Gordon SC, et al. Peginterferonalfa2b plus ribavirin for initial treatment of
chronic hepatitis C: a randomized trial. Lancet 2001;358:958–965.
Manuelpillai, U.; Tchongue, J.; Lourensz, D.; et al. (2010) Transplantation of human amnion epithelial
cells reduces hepatic fibrosis in immunocompetant CCl4-treated mice. Cell Transplant. Vol. 19, No. 9, pp.
1157-1168
Manzanet G, F. Sanju´an, P. Orbis et al., “Liver transplantation in patients with portal vein thrombosis,”
Liver Transplantation, vol. 7, no. 2, pp. 125–131, 2001.
Manzarbeitia C, Ortiz J, Jeon H, et al. Long-term outcome of controlled, non-heart-beating donor liver
transplantation. Transplantation 2004;78:211–5.
Marcos A, Fisher RA, Ham JM, Olzinski AT, Shiffman ML, Sanyal AJ, et al. Selection and outcome of
living donors for adult to adult right lobe transplantation. Transplantation 2000;69:2410–2415.
Marcos A, M. Killackey, M. S. Orloff, L. Mieles, A. Bozorgzadeh, and H. P. Tan, “Hepatic arterial
reconstruction in 95 adult right lobe living donor liver transplants: evolution of anastomotic technique,”
Liver Transplantation, vol. 9, no. 6, pp. 570–574, 2003.
Marcos A, Olzinski AT, Ham JM, et al. The interrelationship between portal and arterial blood flow after
adult to adult living donor liver transplantation. Transplantation 2000;70:1697–703.
Marin C, Robles R, Parrilla G, et al. Liver transplantation in Wilson’s disease: are its indications
established? Transplant Proc 2007;39:2300–2301.
Marino IR, Doyle HR, Aldrighetti L, et al. Effect of donor age and sex on the outcome of liver
transplantation. Hepatology 1995;22:1754–1762
Marinos G, Rossol S, Carucci P, et al. Immunopathogenesis of hepatitis B virus recurrence after liver
transplantation. Transplantation 2000;69:559–68.
358
References
Markmann JF, Markmann JW, Markmann DA et al. Preoperative factors associated with outcome and
their impact on resource use in 1148 consecutive primary liver transplants. Transplantation 2001; 27; 72:
1113–1122
Markmann JF, Markowitz JS, Yersiz H, et al. Long-term survival after retransplantation of the liver. Ann
Surg 1997;226:408– 420.
Markowitz P, Martin JA, Conrad J, et al. Prophylaxis against hepatitis B recurrence following liver
transplantation using combination of lamivudine plus hepatitis B immunoglobulin. Hepatology
1998;28:585–589.
Markus BH, Duquesnoy RJ, Gordon RD, Fung JJ, Vanek M, Klintmalm G, Bryan C, Van Thiel D,
StarzlTE. Histocompatibility and liver transplant outcome. Does HLA exert a dualistic effect?
Transplantation. 1988 Sep;46(3):372-7.
Marongiu, F.; Gramignoli, R.; Dorko, K.; et al. (2011) Hepatic differentiation of amnion epithelial cells.
Hepatology. Vol. 53, No. 5, pp. 1719-29
Marroquin CE, Marino G, Kuo PC et al. Transplantation of hepatitis C-positive livers in hepatitis C-positive
patients is equivalent to transplanting hepatitis C-negative livers. Liver Transpl 2001; 7: 762–768
Marsh JW, Finkelstein SD, Demetris AJ, et al. Genotyping of hepatocellular carcinoma in liver transplant
recipients adds predictive power for determining recurrence-free survival. Liver Transpl 2003:9:664-71.
Marsman WA, Wiesner RH, Rodriguez L, Batts KP, Porayko MK, Hay JE, et al. Use of fatty donor liver is
associated with diminished early patient and graft survival. Transplantation 1996;62:1246–1251.
Marzano A, Angelucci E, Andreone P, et al. Prophylaxis and treatment of hepatitis B in
immunocompromised patients. Dig Liver Dis 2007;39:397–408.
Massie AB, Caffo B, Gentry SE, Hall EC. MELD Exceptions and Rates of Waiting List Outcomes. Am J
Transplant, 2011; 11: 2362-71
Massip-Salcedo, M.; Zaouali, MA.; Padrissa-Altés, S.; Casillas-Ramirez, A.; Rodés, J.; Roselló-Catafau,
J. & Peralta, C. (2008) Activation of peroxisome proliferatoractivated receptor-alpha inhibits the injurious
effects of adiponectin in rat steatotic liver undergoing ischemia-reperfusion. Hepatology, Vol.47, No.2,
(February 2008), pp. 461-472, ISSN: 0168-8278
Masyuk AI, Huang BQ, Ward CJ, et al. Biliary exosomes influence cholangiocyte regulatory mechanisms
and proliferation through interaction with primary cilia. Am J Physiol Gastrointest Liver Physiol
2010;299(4):G990-9.
Mateo R, Cho Y, Singh G, et al. Risk factors for graft survival after liver transplantation from donation
after cardiac death donors: an analysis of OPTN/UNOS data. Am J Transplant 2006;6:791–6.
Matern D, Starzl TE, Arnaout W, Barnard J, Bynon JS, Dhawan A, et al. Liver transplantation for glycogen
storage disease types I, III, and IV. Eur J Pediatr 1999;158(Suppl 2):S43–S48.
Maurice PD, Maddox AJ, Green CA, et al. Monitoring patients on methotrexate: hepatic fibrosis not seen
in patients with normal serum assays of aminoterminal peptide of type III procollagen. Br J Dermatol
2005;152:451–458.
Mazariegos GV, Molmenti EP, Kramer DJ. Early complications after orthotopic liver transplantation. Surg
Clin North Am 1999; 79: 109-29.
Mazuelos F, Abril J, Zaragoza C, et al. Cardiovascular morbidity and obesity in adult liver transplant
recipients. Transplant Proc 2003; 35: 1909–10.
Mazzafero V, Regalia E, Doci R, et al. Liver transplantation for the treatment of small hepatocellular
carcinomas in patients with cirrhosis. N Engl J Med 1996;334:693–699.
McAvoy NC, Kochar N, McKillop G, Newby DE, Hayes PC. Prevalence of coronary artery calcification in
patients undergoing assessment for orthotopic liver transplantation. Liver transpl 2008; 12:1725-31
McCashland T, Watt K, Lyden E, et al. Retransplantation for hepatitis C: results of a U.S. multicenter
retransplant study. Liver Transpl 2007;9:1246–1253.
McCashland TM, Shaw BW Jr., Tape E. The American experience with transplantation for acute liver
failure. Semin Liver Dis 1996;16:427-433.
359
References
McCaughan GW, Zekry A. Impact of immunosuppression on immunopathogenesis of liver damage in
hepatitis C virus-infected recipients following liver transplantation. Liver Transpl 2003;9:S21–7.
McCaughan GW, Zekry A. Mechanisms of HCV reinfection and allograft damage after liver
transplantation. J Hepatol 2004;40:368–74.
McClain CJ, Cohen DA. Increased tumor necrosis factor production by monocytes in alcoholic hepatitis.
Hepatology 1989;9: 349 –351.
McCormack L, Clavien PA. Understanding the meaning of fat in the liver. Liver Transpl 2005;11:137–139.
McCormack L, Petrowsky H, Jochum W, Mullhaupt B, Weber M, Clavien PA. Use of severely steatotic
grafts in liver transplantation: a matched case-control study. Ann Surg 2007; 246: 940–946; Discussion
946–948.
McCormick PA, Treanor D, McCormack G, Farrell M. Early death from paracetamol (acetaminophen)
induced fulminant hepatic failure without cerebral oedema. J Hepatol 2003;39:547-551
McDiarmid SV, Anand R, Lindblad AS. Development of a pediatric end-stage liver disease score to
predict poor outcome in children awaiting liver transplantation. Transplantation 2002;74:173-181.
McDiarmid SV, Anand R, Lindblad AS. SPLIT Research Group. Studies of Pediatric Liver
Transplantation: 2002 update. An overview of demographics, indications, timing, and immunosuppressive
practices in pediatric liver transplantation in the United States and Canada. Pediatr Transplant
2004;8:284–294.
McGary CT, Raja RH, Weigel PH. Endocytosis of hyaluronic acid by rat liver endothelial cells. Evidence
for receptor recycling.Biochem J 1989;257:875–884.
McHutchison JG, Blatt LM, de Medina M, et al. Measurement of serum hyaluronic acid in patients with
chronic hepatitis C and its relationship to liver histology. Consensus Interferon Study Group. J
Gastroenterol Hepatol 2000;15:945–951.
Medici V, Mirante VG, Fassati LR, et al. Liver transplantation for Wilson’s disease: the burden of
neurological and psychiatric disorders. Liver Transpl 2005;11:1056–1063.
Mehrabi A, Fonouni H, Kashfi A, Schmied BM, Morath C, Sadeghi M, Schemmer P, Encke J, Sauer P,
Zeier M, Weitz J, Buchler MW, Schmidt J (2006) The role and value of sirolimus administration in kidney
and liver transplantation. Clin Transplant 20(Suppl 17):30–43
Mehrabi A, Mood Zh A, Sadeghi M, Schmied BM, Muller SA, Welsch T, Kuttymuratov G, Wente MN,
Weitz J, Zeier M, Morath C, Riediger C, Schemmer P, Encke J, Buchler MW, Schmidt J (2007)
Thymoglobulin and ischemia reperfusion injury in kidney and liver transplantation. Nephrol Dial
Transplant 22(Suppl 8): viii54–viii60
Meister A. Glutathione metabolism and its selctive modification. J Biol Chem. 1988;263:17205-8.
Mellor AL, Baban B, Chandler P, Marshall B, Jhaver K, Hansen A, Koni PA, Iwashima M, Munn DH.
Cutting edge: induced indoleamine 2,3 dioxygenase expression in dendritic cell subsets suppresses T cell
clonal expansion. J Immunol. 2003 Aug 15;171(4):1652-5.
Menon KV, Angulo P, Weston S, Dickson ER, Lindor KD. Bone disease in primary biliary cirrhosis:
independent indicators and rate of progression. J Hepatol 2001;35:316-323.
Merion R, Shearon TH, Berg CL, Everhart JE, Abecasis R, Shaked A, et al. Hospitalisations rates before
and after adult-toadult living donor or deceased donor liver transplantation. Hepatology 2007;46:234A.
Merion RM, Schaubel DE, Dykstra DM, Freeman RB, Port FK, Wolfe RA. The survival benefit of liver
transplantation. Am J Transpl 2005; 5: 307–313
Merkel C, Bolognesi M, Sacerdoti D, et al. The hemodynamic response to medical treatment of portal
hypertension as a predictor of clinical effectiveness in the primary prophylaxis of variceal bleeding in
cirrhosis. Hepatology 2000;32:930–4
Meyer C, Penn I, James L. Liver transplantation for cholangiocarcinoma: results in 207 patients.
Transplantation 2000;69:1633– 1637.
Middleton PF, Duffield M, Lynch SV, Padbury RT, House T, Stanton P, et al. Living donor liver
transplantation—adult donor outcomes: a systematic review. Liver Transplantation 2006;12:24-30.
360
References
Mieth M, Schemmer P, Encke J et al. Heidelberger Manual
Universita¨tsklinikum Heidelberg, 2nd Edition 2006; ISBN 3-9808751-1-3
der
Lebertransplantation.
Miki, T. & Strom, S.C. (2006) Amnion-derived pluripotent/multipotent stem cells. Stem Cell Reviews. Vol.
2, pp.133-142
Miki, T. (2011) Amnion-derived stem cells: in quest of clinical applications. Stem Cell Research &
Therapy. Vol. 2, No. 25.
Miki, T.; Lehmann, T.; Cai, H.; Stoltz, D. & Strom, S.C. (2005) Stem cell characteristics of amnion
epithelial cells. Stem Cells. Vol. 23, pp. 1549-1559
Millis et al, 1996. Millis JM, et al: Portal vein thrombosis and stenosis in pediatric liver transplantation.
Transplantation 1996; 62:748-754
Millonig G, Friedrich S, Adolf S, Fonouni H, Golriz M, Mehrabi A, et al. Liver stiffness is directly influenced
by central venous pressure. J Hepatol 2010;52:206–210.
Millonig G, Reimann FM, Friedrich S, Fonouni H, Mehrabi A, Buchler MW, et al. Extrahepatic cholestasis
increases liver stiffness (FibroScan) irrespective of fibrosis. Hepatology 2008;48:1718–1723.
Miloh T, Magid MS, Iyer K, Kerkar N, Morotti RA. Chronic rejection preceded by central perivenulitis,
rapidly ensuing after liver transplantation in a pediatric patient. Semin Liver Dis 2009;29:134–138.
Mimeault R, Grant D, Ghent C, Duff J, Wall W. Analysis of donor and recipient variables and early graft
function after orthotopic liver transplantation. Transplant Proc 1989;21: 3355
Miñambres E, Cemborain A, Sánchez-Velasco P, et al. Correlation between transcranial interleukin-6
gradient and outcome in patients with acute brain injury. Crit Care Med 2003;31:933-8.
Mirza D. Policies in Europe on ‘‘marginal quality’’ donor livers. Lancet 1994;344:1480–1483
Misaki M, Shima T, Yano Y, et al. Basement membrane-related and type III procollagen-related antigens
in serum of patients with chronic viral liver disease. Clin Chem 1990;36:522–524.
Misra N, Bayry J, Lacroix-Desmazes S, Kazatchkine MD, Kaveri SV. Cutting edge: human CD4+CD25+ T
cells restrain the maturation and antigen-presenting function of dendritic cells. J Immunol. 2004 Apr
15;172(8):4676-80. PubMed PMID: 15067041.
Mitchell A, John PR, Mayer DA, Mirza DF, Buckels JA, de Ville dG. Improved technique of portal vein
reconstruction in pediatric liver transplant recipients with portal vein hypoplasia. Transplantation 2002;73:
1244-1247.
Mittler J, Pascher A, Neuhaus P, Pratschke J. The utility of extended criteria donor organs in severely ill
liver transplant recipients. Transplantation 2008;86:895–896.
Mohamadnejad, M.; Namiri, M.; Bagheri, M.; et al. (2007) Phase 1 human trial of autologous bone
marrow-hematopoietic stem cell transplantation in patients with decompensated cirrhosis. World J
Gastroenterol. Vol. 13, No. 24, pp. 3359-3563
Mohi-ud-din R, Lewis JH. Drug- and chemical-induced cholestasis. Clin Liver Dis 2004;8:95–132, vii.
Moller, JH Henriksen Cirrhotic cardiomyopathy: a pathophysiological review of circulatory dysfunction in
liver disease Heart, 87 (2002), pp. 9–15
Molmenti E, Squires R, Nagata D, et al. Liver transplantation for cholestasis associated with cystic fibrosis
in the pediatric population. Pediatr Transplant 2003;7:93–97.
Montalti R, Nardo B, Bertelli R, et al. Donor pool expansion in liver transplantation. Transplant Proc 2004;
36:520–522
Montalto G, Soresi M, Aragona F, et al. Procollagen III and laminin in chronic viral hepatopathies. Presse
Med 1996;25: 59–62.
Moodley, Y.; Ilancheran, S.; Samuel, C.; et al. (2010) Human amnion epithelial cell transplantation
abrogates lung fibrosis and augments repair. American Journal of Respiratory and Critical Care Medicine.
Vol. 182, No. 5, (September 2010), pp.643-651
Moore DE, Feurer ID, Speroff T et al. mpact of donor, technical, and recipient risk factors on survival and
quality of life after liver transplantation. Arch Surg 2005; 140: 273–277
361
References
Mor E, Gonwa TA, Husberg BS, et al. Late-onset acute rejection in orthotopic liver transplantation—
associated risk factors and outcome. Transplantation 1992;54:821–4.
Mor E, Klintmalm GB, Gonwa TA, Solomon H, Holman MJ, Gibbs JF, et al. The use of marginal donors
for liver transplantation. A retrospective study of 365 liver donors. Transplantation1992;53:383-386.
Morariu AM, Schuurs TA, Leuvenink HG, et al. Early events in kidney donation: progression of endothelial
activation, oxidative stress and tubular injury after brain death. Am J Transplant 2008;8: 933-41.
Morath C, Schwenger V, Schmidt J, et al. Transmission of malignancy with solid organ transplants (Abs.).
Transplantation 2005;80:S164.
Moreno JM, Rubio E, Pons F, et al. Usefulness of mycophenolate mofetil in patients with chronic renal
insufficiency after liver transplantation. Transplant Proc 2003;35:715-7.
Moreno Planas JM, Cuervas-Mons Martinez V, Rubio Gonzalez E, et al. Mycophenolate mofetil can be
used as monotherapy late after liver transplantation. Am J Transplant 2004;4:1650-5.
Mosca S, Militerno G, Guardascione MA, et al. Late biliary tract complications after orthotopic liver
transplantation: diagnostic and therapeutic role of endoscopic retrograde cholangiopancreatography. J
Gastroenterol Hepatol 2000;15:654–60.
Moser MA, Wall WJ. Management of biliary problems after liver transplantation. Liver Transpl 2001;
7(Suppl 1): S46-52.
Motschman TL, Taswell HF, Brecher ME, Rakela J, Grambsch PM, Larson-Keller J, et al. Intraoperative
blood loss and patient and graft survival in orthotopic liver transplantation: their relationship to clinical and
laboratory data. Mayo Clin Proc 1989; 64: 346-55.
Mottershead M, Neuberger J. Transplantation in autoimmune liver diseases. World J Gastroenterol
2008;14:3388–95.
Munoz SJ, Rothstein KD, Reich D, Manzarbeitia C. Long-term care of the liver transplant recipient. Clin
Liver Dis 2000; 4: 691-710.
Murawaki Y, Ikuta Y, Okamoto K, et al. Diagnostic value of serum markers of connective tissue turnover
for predicting histological staging and grading in patients with chronic hepatitis C. J Gastroenterol
2001;36:399–406.
Murphy, S.; Lim, R.; Dickinson, H.; et al. (2010). Amnion epithelial cells prevent bleomycin induced
induced lung injury and preserve lung function. Cell Transplant. November 2010,
Murray KF, Carithers RL Jr. AASLD practice guidelines: Evaluation of the patient for liver transplantation.
Hepatology 2005; 41: 1407-32.
Murtaugh PA, Dickson ER, Van Dam GM, et al. Primary biliary cirrhosis: prediction of short-term survival
based on repeated patient visits. Hepatology 1994;20:126–134.
Mutimer D, Dusheiko G, Barrett C, et al. Lamivudine without HBIg for prevention of graft reinfection by
hepatitis B: long-term follow-up. Transplantation 2000;70:809-15.
Mutimer DJ, Gunson B, Chen J, et al: Impact of donor age and year of transplantation on graft and patient
survival following liver transplantation for hepatitis C virus. Transplantation 81:7-14, 2006
Myers RP, Lee SS. Cirrhotic cardiomyopathy and liver transplantation. Liver Transpl 2000;6 (4 Suppl
1):S44-S52.
Myron Kauffman H, McBride MA, Cherikh WS, Spain PC, Marks WH, Roza AM. Transplant tumor
registry: Donor related malignancies. Transplantation 2002;74:358-362.
Nadalin S, Malago M, Radtke A, Erim Y, Saner F, Valentin-Gamazo C, et al. Current trends in live liver
donation. Transpl Int 2007;20:312-30.
Nagata, S.; Toyoda, M.; Yamaguchi, S.; et al. (2009). Efficient reprogramming of human and mouse
primary extra-embryonic cells to pluripotent stem cells. Genes Cells. Vol. 14, pp. 1395-1404.
Nahmias, Y.; Berthiaume, F. & Yarmush, M.L. (2007) Integration of technologies for hepatic tissue
engineering. Adv. Biochem. Eng. Biotechnol. Vol. 103, pp. 309-329
Nair S 2002a, Verma S, Thuluvath PJ. Obesity and its effect on survival in patients undergoing orthotopic
liver transplantation in the United States. HEPATOLOGY 2002;35:105-109.
362
References
Nair S 2002b, Verma S, Thuluvath PJ. Pretransplant renal function predicts survival in patients
undergoing orthotopic liver transplantation. HEPATOLOGY 2002;35:1179-1185.
Nakamura K, Murase N, Becich MJ, et al. Liver allograft rejection in sensitized recipients. Observations in
a clinically relevant small animal model. Am J Pathol 1993;142:1383–91
Nakamura T, K. Tanaka, T. Kiuchi et al., “Anatomical variations and surgical strategies in right lobe living
donor liver transplantation: lessons from 120 cases,” Transplantation, vol. 73, no. 12, pp. 1896–1903,
2002.
Nakatsuji, N.; Nakajima, F. & Tokunaga, K. (2008) HLA-haplotype banking and iPS cells. Nature
Biotechnology. Vol. 26, pp. 739-740
Nakazawa Y, Neil D, Pappo O, Parizhskaya M, Randhawa P, Rasoul-Rockenschaub S, Reinholt F,
Reynes M, Robert M, Tsamandas A, Wanless I, Wiesner R, Wernerson A, Wrba F, Wyatt J, Yamabe H.
Update of the International Banff Schema for Liver Allograft Rejection: working recommendations for the
histopathologic staging and reporting of chronic rejection. An International Panel. Hepatology. 2000
Mar;31(3):792-9
Nanashima A, Pillay P, Verran DJ, Painter D, Nakasuji M, Crawford M, et al. Analysis of initial poor graft
function after orthotopic liver transplantation: experience of an australian single liver transplantation
center. Transplant Proc 2002;34:1231-1235
Nanashima A, Yamaguchi H, Shibasaki S, Ide N, Morino S, Sumida Y, et al. Comparative analysis of
postoperative morbidity according to type and extent of hepatectomy. Hepatogastroenterology
2005;52:844-8.
Nardo B, Masetti M, Urbani L et al. Liver transplantation from donors aged 80 years and over: pushing the
limit. Am J Transplant 2004; 4: 1139–1147
Narumi S, Roberts JP, Emond JC, Lake J, Ascher NL. Liver transplantation for sclerosing cholangitis.
HEPATOLOGY 1995;22:451-457.
Navarro V, Herrine S, Katopes C, Colombe B, Spain CV. The effect of HLA class I (A and B) and class II
(DR) compatibility on liver transplantation outcomes: an analysis of the OPTN database. Liver Transpl.
2006 Apr;12(4):652-8.
Navasa M, Bustamante J, Marroni C, Gonzalez E, Andreu H, Esmatjes E, Garcia-Valdecasas JC, Grande
L, Cirera I, Rimola ARodes J. Diabetes mellitus after liver transplantation: prevalence and predictive
factors. J Hepatol 1996; 25(1): 64-71.
Nazer H, R. J. Ede, A. P. Mowat, and R. Williams, “Wilson’s disease: clinical presentation and use of
prognostic index,” Gut, vol. 27, no. 11, pp. 1377–1381, 1986.
Neal, David A. J.; Brown, Morris J.; Wilkinson, Ian B.; Byrne, Christopher D.; Alexander, Graeme J. M.
Hemodynamic effects of amlodipine, bisoprolol, and lisinopril in hypertensive patients after liver
transplantation. Transplantation, 77 (2004), pp. 748–766 - Issue 5
Neff GW, Bonham A, Tzakis AG, Ragni M, Jayaweera D, Schiff ER, et al. Orthotopic liver transplantation
in patients with human immunodeficiency virus and end-stage liver disease. Liver Transpl 2003;9:239247.
Neff GW, Ruiz P, Madariaga JR, et al. Sirolimus-associated hepatotoxicity in liver transplantation. Ann
Pharmacother 2004;38:1593–6.
Neil DA, Hubscher SG. Histologic and biochemical changes during the evolution of chronic rejection of
liver allografts. Hepatology 2002;35: 639–651.
Neipp M, Bektas H, Lueck R et al. Liver transplantation using organs from donors older than 60 years.
Transpl Int 2004; 17: 416–423
Nelson DR, Gonzalez-Peralta RP, Qian K, et al. Transforming growth factor-B1 in chronic hepatitis C. J
Viral Hepat 1997;4: 29 –35.
Nery JR, Nery-Avila C, Reddy KR et al. Use of liver grafts from donors positive for antihepatitis B-core
antibody in the era of prophylaxis with hepatitis-B immunoglobulin and lamivudine. Transplantation 2003;
75: 1179–1186
363
References
Neuberger J, Gimson A, Davies M, Akyol M, O’Grady J,Burroughs A, et al. Liver Advisory Group; UK
blood and transplant. Selection of patients for liver transplantation and allocation of donated livers in the
UK. Gut 2008;57:252–257.
Neuberger J, James O. Guidelines for selection of patients for liver transplantation in the era of donororgan shortage. Lancet 1999;354:1636–1639.
Neuberger J, K. H. Schulz, C. Day et al., “Transplantation for alcoholic liver disease,” Journal of
Hepatology, vol. 36, no. 1, pp. 130–137, 2002.
Neuberger J. Allocation of donor livers--is MELD enough? Liver Transpl 2004;10:908-10.
Neuberger J. Incidence, timing, and risk factors for acute and chronic rejection. Liver Transpl Surg. 1999
Jul;5(4 Suppl 1):S30-6.
Neuberger J. Recurrent primary biliary cirrhosis. Liver Transpl 2003;9:539–46
Neuhaus P, Clavien PA, Kittur D, et al. Improved treatment response with basiliximab immunoprophylaxis
after liver transplantation: results from a double-blind randomized placebo-controlled trial. Liver Transpl
2002;8:132-42.
Neuhaus P. Live donor/split liver grafts for adult recipients: when should we use them? Liver Transpl
2005;11:S6–9.
Neumann UP, Fischer U, Schmitz V, Lang M, Langrehr JM, Neuhaus P. Long-term graft acceptance after
rat liver allograft transplantation induced by application of CTLA4-Ig and donor specific spleen cell
administration. Transplant Proc 2002;34:1400-1401.
Nichols TC, Guthridge JM, Karp DR, et al. Gamma-glutamyl transpeptidase, an ecto-enzyme regulator of
intracellular redox potential, is a component of TM4 signal transduction complexes. Eur J Immunol
1998;28:4123-4129.
Nicholson ML, Metcalfe MS, White SA, Waller JR, Doughman TM, Horsburgh T, Feehally J, et al. A
comparison of the results of renal transplantation from non-heart-beating, conventional cadaveric, and
living donors. Kidney Int 2000;58:2585–2591
Nickkholgh A, Weitz J, Encke J, Sauer P, Mehrabi A, Buchler MW, et al.Utilization of extended donor
criteria in liver transplantation: a comprehensive review of the literature. Nephrol Dial Transplant 2007;22
(Suppl 8):viii 29–viii 36.
Niemczyk M, Wyzgal J, Perkowska A, et al. Sirolimus-associated hepatotoxicity in the kidney graft
recipient. Transpl Int 2005;18:1302–3.
Ninomiya M, Harada N, Shiotani S et al. Hepatocyte growth factor and transforming growth factor beta1
contribute to regeneration of small-for-size liver graft immediately after transplantation. Transpl. Int. 2003;
16: 814–19.
Nishizaki T, Ikegami T, Hiroshige S, et al. Small graft for living donor liver transplantation. Ann Surg
2001;233:575–80.
Noack K, Bronk SF, Kato A, et al. The greater vulnerability of bile duct cells to reoxygenation injury than
to anoxia. Implications for the pathogenesis of biliary strictures after liver transplantation. Transplantation
1993;56:495–500.
Nocito A, El-Badry AM, Clavien PA. When is steatosis too much for transplantation? J Hepatol
2006;45:494–499.
Nojgaard C, Johansen JS, Christensen E, et al. Serum levels of YKL-40 and PIIINP as prognostic
markers in patients with alcoholic liver disease. J Hepatol 2003;39:179 –186.
Northup PG, Berg CL. Preoperative delta-MELD score does not independently predict mortality after liver
transplantation. Am J Transpl 2004;4:1643–1649.
Novitzky D, Cooper DK, Morrell D, et al. Change from aerobic to anaerobic metabolism after brain death,
and reversal following triiodothyronine therapy. Transplantation 1988;45:32-6.
Novitzky D, Wicomb WN, Cooper DKC, et al. Electrocardiographic, hemodynamic and endocrine changes
occurring during experimental brain death in the chacma baboon. J Heart Transplant 1984;4:63-9.
364
References
Nubile, M.; Dua, H.S.; Lanzini, M.; et al. (2011) In vivo analysis of stromal integration of multilayer
amniotic membrane transplantation in corneal ulcers. American Journal of Ophthalmology. Vol. 151, No.
5, pp.809-822.e1.
O’Brien MJ, Keating NM, Elderiny S, et al. An assessment of digital image analysis to measure fibrosis in
liver biopsy specimens of patients with chronic hepatitis C. Am J Clin Pathol 2000;114:712–718.
O’Grady JG a, Alexander GJ, Hayllar KM, et al. Early indicators of prognosis in fulminant hepatic failure.
Gastroenterology 1989; 97:439–445.
O’Grady JG b. Acute liver failure. Postgrad Med J 2005;81:148–154.
O’Grady JG, Gimson AES, O’Brien CJ, Pucknell A, Hughes RD, Williams R. Controlled trials of charcoal
hemoperfusion and prognostic factors in fulminant hepatic failure. Gastroenterology 1988;94:1186-1192.
Obermann K, Nagel E, Pichlmayr R. Ethical considerations in procuring organs from non-heart-beating
donors after sudden cardiac death. Transplant Proc 1995;27:2924–2925.
Oberti F, Valsesia E, Pilette C, et al. Noninvasive diagnosis of hepatic fibrosis or cirrhosis.
Gastroenterology 1997;113: 1609–1616.
Oellerich M, Burdelski M, Lautz H-U, Binder L, Pichlmayr R. Predictors of one-year pretransplant survival
in patients with cirrhosis.HEPATOLOGY 1991;14:1029-1034.
Ogura Y, Martinez OM, Villanueva JC, Tait JF, Strauss HW, Higgins JPT, et al. Apoptosis and allograft
rejection in the absence of CD8 T cells. Transplantation 2001;71:1827- 1834.
Oh CK, Sanfey HA, Pelletier SJ, Sawyer RG, McCullough CS, Pruett TL. Implication of advanced donor
age on the outcome of liver transplantation. Clin Transplant 2000;14:386-390.
Ojo AO, Held PJ, Port FK, Wolfe RA, Leichtman AB, Young AW, et al. Chronic renal failure after
transplantation of a non-renal organ. N Engl J Med 2003;349(10):931–40.
Okamoto S, Corso CO, Leiderer R, et al. Role of hypotension in braindeath associated impairment of liver
microcirculation and viability. Transpl Int 2000;13:428-35.
Olausson M, Friman S, Cahlin C, Nilsson O, Jansson S, Wangberg B, et al. Indications and results of liver
transplantation in patients with neuroendocrine tumors. World J Surg 2002;26:998-1004
Oldakowska-Jedynak U, Nowak M, Mucha K, et al. Recurrence of primary sclerosing cholangitis in
patients after liver transplantation. Transplant Proc 2006;38:240–3.
Olivera-Martinez MA, Gallegos-Orozco JF. Recurrent viral liver disease (hepatitis B and C) after liver
transplantation. Arch Med Res 2007;38:691–701.
Olson L, Castro VL, Ciancio G, Burke G, Nery J, Cravero LB, et al. Twelve years’ experience with nonheart-beating cadaveric donors. J Transpl Coord 1996;6:196–199.
Olson L, Davi R, Barnhart J, Burke G, Ciancio G, Miller J, Tzakis A. Non-heart-beating cadaver donor
hepatectomy ‘the operative procedure’. Clin Transplant 1999;13(1 Pt 2):98–103.
Olthoff K, Judge TA, Gelman AE, Xhen XD, Hancock WW, Turka LA, Shaked A. Adenovirus-mediated
gene transfer into cold-preserved liver allografts: survival pattern and unresponsiveness following
transduction with CTLA4Ig. Nat Med 1998;4: 194-200.
Olthoff KM, Merion RM, Ghobrial RM, et al. Outcomes of 385 adult-to-adult living donor liver transplant
recipients: a report from the A2ALL consortium. Ann Surg 2005; 242:314–323
Omland T. New features of troponin testing in different clinical settings. J Intern Med 2010;268:207-217
Onaca N, Davis GL, Goldstein RM, et al. Expanded criteria for liver transplantation in patients with
hepatocellular carcinoma: a report from the International Registry of Hepatic Tumors in Liver
Transplantation. Liver Transpl 2007;13:391–399.
Onaca N, Levy MF, Ueno T, et al. An outcome comparison between primary liver transplantation and
retransplantation based on the pretransplant MELD score. Transpl Int 2006;19:282–7.
Ono SK, Kato N, Shiratori Y, et al. The polymerase L528M mutation cooperates with nucleotide bindingsite mutations, increasing hepatitis B virus replication and drug resistance. J Clin Invest 2001;107:449-55.
Opelz G, Wujciak T. The influence of HLA compatibility on graft survival after heart transplantation. The
Collaborative Transplant Study. N Engl J Med 1994;330:816-819.
365
References
Organ Procurement and Transplantation Network. Policy 2: minimum procurement standards for an organ
procurement organization (OPO). Rockville (MD): US Department of Health and Human Services; 2009.
Osorio RW, Ascher NL, Avery M, et al. Predicting recidivism after orthotopic liver transplantation for
alcoholic liver disease. Hepatology 1994;20:105–110.
Otte JB, de Ville de Goyet J, Sokal E, et al. Size reduction of the donor liver is a safe way to alleviate the
shortage of size matched organs in pediatric liver transplantation. Ann Surg 1990;211:146–157
Otte JB, Ville-de-Goyet J, Reding R, Van-Obbergh L, Veyckemans F, Carlier MA, et al. Pediatric liver
transplantation: from the full-size liver graft to reduced, split, and living related liver transplantation.
Pediatr Surg Int 1998;13:308-318.
Otte JB. Donor complications and outcomes in live-liver transplantation. Transplantation 2003;75:1625-6.
Pagadala M, Dasarathy S, Eghtesad B, McCullough AJ. Posttransplant metabolic syndrome: an epidemic
waiting to happen. Liver Transpl 2009;15:1662–1670.
Pagliaro L. MELD: the end of Child-Pugh classification? J Hepatol 2002;36:141–2.
Pan S, Nissen N, Steven C, et al. Comparisons of rejection and hepatitis C recurrence in live donor and
deceased donor liver transplantation [abstract]. Am J Transplant 2004;4(S8):295.
Paolicchi A, Dominici S, Pieri L, Maellaro E, Pompella A. Glutathione catabolism as a signaling
mechanism. Biochem Pharmacol. 2002; 64: 1027-35.
Paolicchi A, Dominici S, Pieri L, Maellaro E, Pompella A. Glutathione catabolism as a signaling
mechanism. Biochem Pharmacol. 2002a; 64: 1027-35.
Paolicchi A, Emdin M, Ghliozeni E, Ciancia E, Passino C, Popoff G, Pompella A. Human atherosclerotic
plaques contain gamma-glutamyl transpeptidase enzyme activity. Circulation. 2004; 109: 1440.
Paolicchi A, Emdin M, Passino C, Lorenzini E, Titta F, Marchi S, Malvaldi G, Pompella A. Betalipoprotein- and LDL-associated serum gamma-glutamyltransferase in patients with coronary
atherosclerosis. Atherosclerosis. 2006a; 186: 80-5.
Paolicchi A, Franzini M, Emdin M, Passino C, Pompella A. The potential roles of gammaglutamyltransferase activity in the progression of atherosclerosis and cardiovascular diseases. Vasc Dis
Prev. 2006b; 3: 205-9.
Paolicchi A, Lorenzini E, Perego P, Supino R, Zunino F, Comporti M, Pompella A. Extra-cellular thiol
metabolism in clones of human metastatic melanoma with different gamma-glutamyl transpeptidase
expression: implications for cell response to platinum-based drugs. Int J Cancer. 2002b; 97: 740-5.
Paolicchi A, Minotti G, Tonarelli P, Tongiani R, De Cesare D, Mezzetti A, Dominici S, Comporti M,
Pompella A. Gamma-glutamyl transpeptidase-dependent iron reduction and LDL oxidation--a potential
mechanism in atherosclerosis. J Investig Med. 1999; 47: 151-60.
Paolicchi A, Pompella A, Tonarelli P, Gadducci A, Genazzani AR, Zunino F, Pratesi G, Tongiani R.
Gamma-glutamyltranspeptidase activity in human ovarian carcinoma. Anticancer Res. 1996; 16: 3053-8.
Paolicchi A, Sotiropuolou M, Perego P, Daubeuf S, Visvikis A, Lorenzini E, Franzini M, Romiti N, Chieli E,
Leone R, Apostoli P, Colangelo D, Zunino F, Pompella A. gamma-Glutamyl transpeptidase catalyses the
extracellular detoxification of cisplatin in a human cell line derived from the proximal convoluted tubule of
the kidney. Eur J Cancer 2003; 39: 996-1003.
Paolicchi A, Tongiani R, Tonarelli P, Comporti M, Pompella A. gamma-Glutamyl transpeptidasedependent lipid peroxidation in isolated hepatocytes and HepG2 hepatoma cells. Free Rad Biol Med.
1997; 22: 853-60.
Papatheodoridis GV, O’Beirne J, Mistry P, Davidson B, Rolles K,Burroughs AK (1999) Mycophenolate
mofetil monotherapy in stable liver transplant patients with cyclosporine-induced renal impairment: a
preliminary report. Transplantation 68(1):155–157
Pappo O, Ramos H, Starzl TE, et al. Structural integrity and identification of causes of liver allograft
dysfunction occurring more than 5 years after transplantation. Am J Surg Pathol 1995;19:192–206
Pares A, Deulofeu R, Gimenez A, et al. Serum hyaluronate reflects hepatic fibrogenesis in alcoholic liver
disease and is useful as a marker of fibrosis. Hepatology 1996;24:1399–1403.
366
References
Park GJ, Lin BP, Ngu MC, et al. Aspartate aminotransferase: alanine aminotransferase ratio in chronic
hepatitis C infection: is it a useful predictor of cirrhosis? J Gastroenterol Hepatol 2000;15:386–390.
Park SC, Beerman LB, Gartner JC, Zitelli BJ, Malatack JJ, Fricker FJ, et al. Echocardiographic findings
before and after liver transplantation. Am J Cardiol 1985;55:1373-1378.
Park, I.H.; Arora, N.; Huo, H; et al. (2008b). Disease-specific induced pluripotent stem cells. Cell. Vol.
134, pp. 877-886.
Park, I.H.; Zhao, R.; West, J.A.; et al. (2008a). Reprogramming of human somatic cells to to pluripotency
with defined factors. Nature. Vol. 451, pp. 141-146.
Parkes J, Guha IN, Roderick P, et al. Performance of serum marker panels for liver fibrosis in chronic
hepatitis C. J Hepatol 2006;44:462–474.
Parolini, O.; Alviano, F.; Bagnara, G.P.; et al. (2008) Concise review: Isolation and characterization of
cells from human term placenta: Outcome of the first International Workshop on Placenta Derived Cells.
Stem Cells. Vol. 26, pp. 300-311
Pascher A, Neuhaus P. Bile duct complications after liver transplantation. Transpl Int 2005;18:627–42.
Pascher A, Sauer IM, Walter M, et al. Donor evaluation, donor risks, donor outcome, and donor quality of
life in adult-to-adult living donor liver transplantation. Liver Transpl 2002;8:829-37.
Pascual et al, 1997. Pascual M, et al: Anticardiolipin antibodies and hepatic artery thrombosis after liver
transplantation. Transplantation 1997; 64:1361-1364.
Pasha TM, Dickson ER. Survival algorithms and outcome analysis in primary biliary cirrhosis. Semin Liver
Dis 1997;17:147-158.
Pastacaldi S, Teixeira R, Montalto P, Rolles K, Burroughs AK. Hepatic artery thrombosis after orthotopic
liver transplantation: a review of nonsurgical causes. Liver Transpl 2001; 7: 75-81.
Patel K, Gordon SC, Jacobson I, et al. Evaluation of a panel of non-invasive serum markers to
differentiate mild from moderate- to-advanced liver fibrosis in chronic hepatitis C patients. J Hepatol
2004;41:935–942.
Patel S, Orlaoff M, Tsoulfas G, et al. Living donor liver transplantation in the United States: identifying
donors at risk for perioperative complications. Am J Transplant 2007;7:2344-9.
Pateron D, Beyne P, Laperche T, Logeard D, Lefilliatre P, Sogni P, et al. Elevated circulating cardiac
troponin I in patients with cirrhosis. Hepatology 1999;29:640-643
Paterson DL, Rihs JD, Squier C, Gayowski T, Sagnimeni A, Singh N. Lack of efficacy of mupirocin in the
prevention of infections with Staphylococcus aureus in liver transplant recipients and candidates.
Transplantation 2003; 75: 194-8.
Pavel V Avdonin, Florence Cottet-Maire, Galina V Afanasjeva, Svetlana A Loktionova, Philippe Lhote and
Urs T Ruegg. Cyclosporine A up-regulates angiotensin II receptors and calcium responses in human
vascular smooth muscle cells. Kidney Int, 55 (1999), pp. 2407–2414
Pawarode A, Fine DM, Thuluvath PJ: Independent risk factors and natural history of renal dysfunction in
liver transplant recipients. Liver Transpl 2003; 9:741-747.
Paya CV, Hermans PE, Wiesner RH, et al. Cytomegalovirus hepatitis in liver transplantation: prospective
analysis of 93 consecutive orthotopic liver transplantations. J Infect Dis 1989;160:752–8.
Paya CV, RR. Cytomegalovirus infection after solid organ transplantation. In: Bowden RA, Ljungman P,
Paya CV, eds. Transplant infections. Philadelphia: Lippincott, Williams and Wilkins, 2003:298–325.
Pearl JP, Parris J, Hale DA, et al. Immunocompetent T cells with a memory-like phenotype are the
dominant cell type following antibody-mediated T cell depletion. Am J Transplant 2005;5: 465–474.
Pelletier SJ, Fu S, Thyagarajan V, et al. An intention-to-treat analysis of liver transplantation for
hepatocellular carcinoma using organ procurement transplant network data. Liver Transpl 2009; 15: 85968.
Pereira BJ, Wright TL, Schmid CH, Levey AS. A controlled study of hepatitis C transmission by organ
transplantation. The New England Organ Bank Hepatitis C Study Group. Lancet 1995; 345: 484–487
367
References
Perkins JD. Biliary tract complications: The most common postoperative complication in living liver
donors. Liver Transpl 2008:14:1372-7.
Perrault J, McGill DB, Ott BJ, et al. Liver biopsy: complications in 1000 inpatients and outpatients.
Gastroenterology 1978;74: 103–106.
Petrovic LM. Early recurrence of hepatitis C virus infection after liver transplantation. Liver Transpl
2006;12(11 Suppl 2):S32–7.
Pfitzmann R, Schwenzer J, Rayes N, et al. Long-term survival and predictors of relapse after orthotopic
liver transplantation for alcoholic liver disease. Liver Transpl 2007;13:197–205.
Phillips MJ, Cameron R, Flowers MA, et al. Post-transplant recurrent hepatitis B viral liver disease. Viralburden, steatoviral, and fibroviral hepatitis B. Am J Pathol 1992;140:1295–308.
Piccinino F, Sagnelli E, Pasquale G, Giusti G. Complications following percutaneous liver biopsy. A
multicentre retrospective study on 68,276 biopsies. J Hepatol 1986;2(2):165–73.
Pilat N, Baranyi U, Klaus C, Jaeckel E, Mpofu N, Wrba F, Golshayan D, Muehlbacher F,Wekerle T. Tregtherapy allows mixed chimerism and transplantation tolerance without cytoreductive conditioning.
Transplant Proc. 2010 Apr;10(4):751-62.
Pine et al, 2009. Pine JK, et al: Liver transplantation following donation after cardiac death: an analysis
using matched pairs. Liver Transpl 2009; 15:1072-1082.
Piratvisuth T, Tredger JM, Hayllar KA, Williams R. Contribution of true cold and rewarming ischemia times
to factors determining outcome after orthotopic liver transplantation. Liver Transpl Surg 1995;1:296
Piscaglia F, Zironi G, Gaiani S, Mazziotti A, Cavallari A, Gramantieri L, et al. Systemic and splanchnic
hemodynamic changes after liver transplantation for cirrhosis: a long-term prospective study. Hepatology
1999;30:58-64.
Piton A, Poynard T, Imbert-Bismut F, Khalil L, Delattre J, Pelissier E, et al. Factors associated with serum
alanine transaminase activity in healthy subjects: consequences for the definition of normal values, for
selection of blood donors, and for patients with chronic hepatitis C. MULTIVIRC Group. Hepatology
1998;27(5):1213.
Ploeg RJ, D’Alessandro AM, Hoffmann RM, et al. Impact of donor factors and preservation on function
and survival after liver transplantation. Transplant Proc 1993;25:3031–3033
Ploeg RJ, D’Alessandro AM, Knechtle SJ, et al. Risk factor for primary dysfunction after liver
transplantation: a multivariate analysis. Transplantation 1993;55:807–813
Plotkin JS, Benitez RM, Kuo PC, Njoku MJ, Ridge LA, Lim JW, et al. Dobutamine stress
echocardiography for preoperative cardiac risk stratification in patients undergoing orthotopic liver
transplantation. Liver Transpl Surg 1998;4:253-257.
Plotkin JS, Kuo PC, Rubin LJ, Gaine S, Howell CD, Laurin J, et al. Successful use of chronic
epoprostenol as a bridge to liver transplantation in severe portopulmonary hypertension. Transplantation
1998;65:457- 459.
Plotkin JS, Scott VL, Pinna A, Dobsch BP, De Wolf AM, Kang Y. Morbidity and mortality in patients with
coronary artery disease undergoing orthotopic liver transplantation. Liver Transpl Surg 1996;2:426- 430
Plummer, C.E.; Ollivier, F.; Kallberg, M.; et al. (2009) The use of amniotic membrane transplantation for
ocular surface reconstruction: a review and series of 58 equine clinical cases (2002-2008). Vet
Ophtamology. Suppl. 1, pp.17-24
Pokorny, H.; Gruenberger, T.; Soliman, T.; Rockenschaub, S.; Langle, F. & Steininger, R. (2000) Organ
survival after primary dysfunction of liver grafts in clinical orthotopic liver transplantation. Transplant
International, Vol.13, No.Suppl 1, (2000), pp. S154- 157, ISSN: 1432-2277
Pokorny, H.; Langer, F.; Herkner, H.; Schernberger, R.; Plöchl, W.; Soliman, T.; Steininger, R. &
Muehlbacher, F. (2005) Influence of cumulative number of marginal donor criteria on primary organ
dysfunction in liver recipients. Clinical Transplantation, Vol.19, No.4, (August 2005), pp. 532-536, ISSN:
0902-0063
Polo, J.M.; Liu, S.; Figueroa, M.E.; et al. (2010). Cell type of origin influences the molecular and functional
properties of mouse induced pluripotent stem cells. Nat Biotechnol. Vol. 28, pp.848-855.
368
References
Pompella A, De Tata V, Paolicchi A, Zunino F. Expression of gamma-glutamyltransferase in cancer cells
and its significance in drug resistance. Biochem Pharmacol. 2006; 71: 231-8.
Pompella A, Emdin M, Passino C, Paolicchi A. The significance of serum gamma-glutamyltransferase in
cardiovascular diseases. Clin Chem Lab Med. 2004; 42: 1085-91.
Pompella A, Paolicchi A, Dominici S, Comporti M, Tongiani R. Selective colocalization of lipid
peroxidation and protein thiol loss in chemically induced hepatic preneoplastic lesions: the role of gammaglutamyltranspeptidase activity. Histochem Cell Biol. 1996; 106: 275-82.
Pompella A, Paolicchi A, Emdin M, Mikhailidis DP. Platelet activation, gamma-glutamyltransferase and
stent restenosis. Atherosclerosis 2007 (in press).
Pompella A, Visvikis A, Paolicchi A, De Tata V, Casini AF. The changing faces of glutathione, a cellular
protagonist. Biochem Pharmacol. 2003; 66: 1499-503.
Pompili M, Addolorato G, Pignataro G, et al. Evaluation of the albumin-gamma-glutamyltransferase
isoenzyme as a diagnostic marker of hepatocellular carcinoma-complicating liver cirrhosis. J
Gastroenterol Hepatol 2003;18(3):288-95.
Poonawala A, Nair SP, Thuluvath PJ. Prevalence of obesity and diabetes in patients with cryptogenic
cirrhosis: a case-control study. Hepatology 2000;32:689–692.
Popper H. Aging and the liver. Prog Liver Dis 1986;8:659-683.
Porcel A, Diaz F, Rendon P, Macìas M, Martin-Herrera L, Giron-Gonzalez JA. Dilutional hyponatremia in
patients with cirrhosis and ascites. Arch Intern Med 2002;162:323–8.
Porte RJ, Ploeg RJ, Hansen B, et al. Long-term graft survival after liver transplantation in the UW era: late
effects of cold ischemia and primary dysfunction. European Multicentre Study Group. Transpl Int
1998;11:S164–S167
Post AB, Bozdech JM, Lavery I, Barnes DS. Colectomy in patients with inflammatory bowel disease and
primary sclerosing cholangitis. Dis Colon Rectum 1994;37:175–178.
Poupon RE, Balkau B, Eschwege E, et al. A multicenter, controlled trial of ursodiol for the treatment of
primary biliary cirrhosis. N Engl J Med 1991;324:1548–1554
Poynard T, Aubert A, Bedossa P, et al. A simple biological index for detection of alcoholic liver disease in
drinkers. Gastroenterology 1991;100:1397–1402.
Poynard T, Munteanu M, Imbert-Bismut F, Charlotte F, Thabut D, Le Calvez S, et al. Prospective analysis
of discordant results between biochemical markers and biopsy in patients with chronic hepatitis C. Clin
Chem 2004;10:10.
Poynard T, Naveau S, Doffoel M, Boudjema K, Vanlemmens C, Mantion G, et al. Evaluation of efficacy of
liver transplantation in alcoholic cirrhosis using matched and simulated controls: 5-year survival.
Multicentre group. J Hepatol 1999; 30: 1130–1137.
Pratschke J, Wilhelm MJ, Kusaka M, et al. A model of gradual onset brain death for transplant-associated
studies in rats. Transplantation 2000;69:427-30.
Prieto M, Gomez MD, Berenguer M et al. De novo hepatitis B after liver transplantation from hepatitis B
core antibodypositive donors in an area with high prevalence of anti-HBc positivity in the donor
population. Liver Transpl 2001; 7: 51–58
Propst A, Propst T, Sangeri G, Ofner D, Judmaier G, Vogel W. Prognosis and life expectancy in chronic
liver disease. Dig Dis Sci 1995;40:1805- 1815.
Pruthi J, Medkiff KA, Esrason KT, et al. Analysis of causes of death in liver transplant recipients who
survived more than 3 years. Liver Transpl 2001; 7: 811–15.
Pugh R, Murray-lyon I, Dawson J. Transcection of the oesophagus for bleeding oesophageal varices. Br J
Surg 1973;60:646-649.
Puglisis, M.A.; Saulnier, N.; Piscaglia, A.C.; et al. (2011) Adipose tissue-derived mesenchymal stem cells
and hepatic differentiation: old concepts and future perspectives. Eur. Rev. Med. Pharmacol. Sci. Vol. 15,
No. 4, pp. 355-64
Pungpapong et al, 2002. Pungpapong S, et al: Cigarette smoking is associated with an increased
incidence of vascular complications after liver transplantation. Liver Transpl 2002; 8:582-587.
369
References
Puppi J, et al. Improving the techniques for human hepatocyte transplantation: report from a consensus
meeting in London. Cell Transplant. 2011, April 1, ePub ahead of print, PMID: 21457616
Qiu J, Ozawa M, Terasaki PI. Liver transplantation in the United States. Clin Transpl 2005:17-28
Quiroga J, Colina I, Demetris AJ, et al. Cause and timing of first allograft failure in orthotopic liver
transplantation: a study of 177 consecutive patients. Hepatology 1991;14:1054–62.
Raimondo ML, Dagher L, Papatheodoridis GV, et al. Long-term mycophenolate mofetil monotherapy in
combination with calcineurin inhibitors for chronic renal dysfunction after liver transplantation.
Transplantation 2003;75:186-90.
Ramage JK, Donaghy A, Farrant JM, Iorns R, Williams R. Serum tumor markers for the diagnosis of
cholangiocarcinoma in primary sclerosing cholangitis. Gastroenterology 1995;108:865–869.
Ramirez S, Perez-Del-Pulgar S, Forns X. Virology and pathogenesis of hepatitis C virus recurrence. Liver
Transpl 2008;14(Suppl 2):S27–35.
Ramsay MA, Simpson BR, Nguyen AT, Ramsay KJ, East C, Klintmalm GB. Severe pulmonary
hypertension in liver transplant candidates. Liver Transpl Surg 1997;3:494-500.
Ramsay MA, Spikes C, East CA, Lynch K, Hein HA, Ramsay KJ, et al. The perioperative management of
portopulmonary hypertension with nitric oxide and epoprostenol. Anesthesiology 1999;90:299-301.
Rana A, Hardy MA, Halazun KJ, Woodland DC, Ratner LE, Samstein B, et al. Survival outcomes
following liver transplantation (SOFT) score: a novel method to predict patient survival following liver
transplantation. Am J Transplant 2008;8:2537–2546.
Randhawa P, Blakolmer K, Kashyap R, et al. Allograft liver biopsy in patients with Epstein–Barr virusassociated posttransplant lymphoproliferative disease. Am J Surg Pathol 2001;25:324–30.
Randhawa PS, Jaffe R, Demetris AJ, et al. Expression of Epstein–Barr virusencoded small RNA (by the
EBER-1 gene) in liver specimens from transplant recipients with post-transplantation lymphoproliferative
disease. N Engl J Med 1992;327:1710–4.
Randhawa PS, Markin RS, Starzl TE, et al. Epstein–Barr virus-associated syndromes in
immunosuppressed liver transplant recipients. Clinical profile and recognition on routine allograft biopsy.
Am J Surg Pathol 1990;14:538–47.
Rantala AO, Lilja M, Kauma H, et al. Gamma-glutamyl transpeptidase and the metabolic syndrome. J
Intern Med. 2000; 248: 230–238.
Rashid, S.T.; Corbineau, S.; Hannan, N.; et al. (2010). Modeling inherited metabolic disorders of the liver
using human induced pluripotent stem cells. J Clin Invest. Vol. 120, pp. 3127-3136.
Ratziu V, Samuel D, Sebagh M, et al. Long-term follow-up after liver transplantation for autoimmune
hepatitis: evidence of recurrence of primary disease. J Hepatol 1999;30:131–41.
Rebellato LM, Gross U, Verbanac KM, Thomas JM. A comprehensive definition of the major antibody
specificities in poyclonal rabbit anti-thymocyte globulin. Transplantation 1994;57(5):685–94.
Reddy, JK. & Rao, MS. (2006) Lipid metabolism and liver inflammation. II. Fatty liver disease and fatty
acid oxidation. American Journal of Physiology-Gastrointestinal and Liver Physiology, Vol.290, No.5,
(May 2006), pp. G852-858, ISSN: 1522-1547
Reese PP, Sonawane SB, Thomasson A, Yeh H, Markmann JF. Donor age and cold ischemia interact to
produce inferior 90-day liver allograft survival. Transplantation 2008; 85: 1737–44.
Regev A, Berho M, Jeffers LJ, Milikowski C, Molina EG, Pyrsopoulos NT, et al. Sampling error and
intraobserver variation in liver biopsy in patients with chronic HCV infection. Am J Gastroenterol
2002;97(10):2614–8..
Reich DJ, Fiel I, Guarrera JV, et al. Liver transplantation for autoimmune hepatitis. Hepatology
2000;32:693–700.
Reich DJ, Hong JC. Current status of donation after cardiac death liver transplantation. Curr Opin Organ
Transplant 2010;15:316–21.
Reich DJ, Mulligan DC, Abt PL, et al. ASTS recommended practice guidelines for controlled donation
after cardiac death organ procurement and transplantation. Am J Transplant 2009;9:2004–11.
370
References
Reich DJ, Munoz SJ, Rothstein KD, Nathan HM, Edwards JM, Hasz RD, Manzarbeitia CY. Controlled
non-heart-beating donor liver transplantation: a successful single center experience, with topic update.
Transplantation 2000;70:1159–1166.
Reich DJ, Munoz SJ, Rothstein KD, Nathan HM, Edwards JM, Hasz RD, Manzarbeitia CY. Controlled
non-heart-beating donor liver transplantation: a successful single center experience, with topic update.
Transplantation 2000;70:1159–1166.
Reich DJ. Nonheartbeating donor organ procurement. In: Humar A, Payne WD, Matas AJ, editors. Atlas
of organ transplantation. London: Springer; 2006. p. 23–33.
Ren, G.; Su, J.; Zhang, L.; et al. (2009) Species variation in the mechanisms of mesenchymal stem cellmediated immunosuppression. Stem Cells. Vol. 27, No. 8, pp. 1954-62.
Renz J. Outcomes of living donor liver transplantation. In: Busuttil RW, ed. Transplantation of the Liver.
Philadelphia: Elsevier; 2005:713–724
Renz J. The donor operation. In: Busuttil RW, ed. Transplantation of the Liver. Philadelphia: Elsevier;
2005: 545–559
Renz JF, Emond JC, Yersiz H, Ascher NL, Busuttil RW. Split-liver transplantation in the United States:
outcomes of a national survey. Ann Surg 2004;239:172-181
Renz JF, Kin C, Kinkhabwala M, et al. Utilization of extended donor criteria liver allografts maximizes
donor use and patient access to liver transplantation. Ann Surg 2005; 242:556–565
Renz JF, Kin CJ, Saggi BH, Emond JC. Outcomes of living donor liver transplantation. In: Busuttil RW,
editor. Transplantation of the Liver. Philadelphia: Elsevier; 2005:713-24
Renz JF, Roberts JP. Long-term complications of living donor liver transplantation. Liver Transpl
2000;6(suppl 2): S73–S76
Reuben A. Long-term management of the liver transplant patient: diabetes, hyperlipidemia, and obesity.
Liver Transpl 2001; 7(Suppl 1): S13-21.
Rey JW, Wirges U, Dienes HP, Fries JW. Hepatic steatosis in organ donors: disparity between surgery
and histology? Transplant Proc 2009;41: 2557–2560.
Ricci P, Therneau TM, Malinchoc M, Benson JT, Petz JL, Klintmalm GB, et al. A prognostic model for the
outcome of liver transplantation in patients with cholestatic liver disease. HEPATOLOGY 1997;25:672677.
Rifai K, Sebagh M, Karam V et al. Donor age influences 10-year liver graft histology independently of
hepatitis C virus infection. J Hepatol 2004; 41: 446–453
Ringe B, Burdelski M, Rodeck B, Pichlmayr R. Experience with partial liver transplantation in Hannover.
Clin Transpl 1990:135–144
Ringe B, Lang H, Oldhafer K-J, Gebel M, Flemming P, Georgii A, et al. Which is the best surgery for
Budd-Chiari syndrome: venous decompression or liver transplantation? A single-center experience with
50 patients. HEPATOLOGY 1995;21:1337-1344.
Ringe B, Strong RW. The dilemma of living liver donor death: to report or not to report? Transplantation
2008; 85: 790–3.
Riva S, Sonzogni A, Bravi M, Bertani A, Alessio MG, Candusso M, et al. Late graft dysfunction and
autoantibodies after liver transplantation in children: preliminary results of an Italian experience. Liver
Transpl 2006;12: 573–577.
Roayie K, Feng S. Allocation policy for hepatocellular carcinoma in the MELD era: room for
improvement? Liver Transpl 2007;13:S36-43.
Robert A, Chazouillères O. Prothrombin time in liver failure: time, ratio, activity percentage, or
international normalized ratio? Hepatology 1996;24:1392-4.
Roberts MS, Angus DC, Bryce CL, Valenta Z, Weissfeld L. Survival after liver transplantation in the
United States: a disease specific analysis of the UNOS database. Liver Transpl 2004; 10: 886-897.
Robertson, N.J.; Brook, F.A.; Gardner, R.L.; et al. (2007) Embryonic stem cell-derived tissues are
immunogenic but their inherent immune privilege promotes the induction of tolerance. Proc Natl Acad Sci
USA. Vol. 104, pp. 20920–20925
371
References
Rocha MB, Boin IF, Escanhoela CA, Leonardi LS. Can the use of marginal liver donors change recipient
survival rate? Transplant Proc 2004;36:914–915
Rodriguez-Luna H, Douglas DD. Natural history of hepatitis C following liver transplantation. Curr Opin
Infect Dis 2004;17:363–71.
Rodriguez-Luna H, Vargas H, De Petris G, et al. HepatitisC virus recurrence in living donor liver
transplantation vs. cadaveric liver transplantation [abstract]. Am J Transplant 2003; 3(S5):160.
Rogers J, Bueno J, Shapiro R, Scantlebury V, Mazariegos G, Fung J, et al. Results of simultaneous and
sequential pediatric liver and kidney transplantation.Transplantation 2001;72:1666-1670.
Rogiers X, Malago M, Gawad K, Jauch KW, Olausson M, Knoefel WT, Gundlach M, Bassas A,Fischer L,
Sterneck M, Burdelski M, Broelsch CE In situ splitting of cadaveric livers. The ultimate expansion of a
limited donor pool. Ann Surg 1996; 224(3): 331-339.
Roland ME, Stock PG. Review of solid-organ transplantation in HIV infected patients. Transplantation
2003;75:425-429.
Rosalki SB, Rau D. Serum-glutamyl transpeptidase activity in alcoholism. Clin Chim Acta 1972; 39: 41-7.
Rosen CB, Heimbach JK, Gores GJ. Surgery for cholangiocarcinoma: the role of liver transplantation.
HPB (Oxford) 2008;10:186–9
Rosen HR. Hepatitis C in the liver transplant recipient: current understanding and treatment. Microbes
Infect 2002;4:1253–8.
Rosenberg WM, Voelker M, Thiel R, Becka M, Burt A, Schuppan D, et al. Serum markers detect the
presence of liver fibrosis: a cohort study. Gastroenterology 2004;127(6):1704–13.
Rosenthal P, Emond JC, Heyman MB, et al. Pathological changes in yearly protocol liver biopsy
specimens from healthy pediatric liver recipients. Liver Transpl Surg 1997;3:559–62.
Rozga J, Podesta L, LePage E, et al. A bioartificial liver to treat severe acute liver failure. Ann Surg
1994;219:538–46.
Rudow DL, Goldstein MJ. Critical care management of the liver transplant recipient. Crit Care Nurs Q
2008;31:232–43
Ruf AE, Kremers WK, Chavez LL, Descalzi VI, Podesta LG, Villamil FG. Addition of serum sodium into
the MELD score predicts waiting list mortality better than MELD alone. Liver Transpl 2005; 11: 336-343
Russo M, Galanko J, Beavers K, et al. Patient and graft survival in hepatitis C recipients after adult living
donor liver transplantation in the United States. Liver Transpl 2004;10: 340–6.
Russo MW, Firpi RJ, Nelson DR, et al. Early hepatic stellate cell activation is associated with advanced
fibrosis after liver transplantation in recipients with hepatitis C. Liver Transpl 2005;11:1235–41.
Russo, F.P.; Alison, M.R.; Bigger B.W.; et al. (2006) The bone marrow functionally contributes to liver
fibrosis. Gastroenterology. Vol. 130, No. 6, pp. 1807-1821
Ruttmann E, Brant LJ, Concin H, et al. Gamma- Glutamyltransferase as a risk factor for cardiovascular
disease mortality. An investigation in a cohort of 163,944 austrian adults. Circulation 2005;112:21302137.
Saab S, Chang AJ, Comulada S, et al: Outcomes of hepatitis C- and hepatitis B core antibody-positive
grafts in orthotopic liver transplantation. Liver Transpl 9:1053, 2003
Saab S, Ghobrial RM, Ibrahim AB et al. positive grafts may be used in orthotopic liver transplantation: a
matched analysis. Am J Transplant 2003; 3: 1167–1172
Safadi A, Homsi M, Maskoun W, Lane KA, Singh I, Sawada SG, Mahenthiran J. Perioperative risk
predictors of cardiac outcomes in patients undergoing liver transplantation surgery. Circulation
2009;120:1189-1194.
Said A, Lucey MR. Liver transplantation: an update 2008. Curr Opin Gastroenterol 2008;243):339–45.
Sakaida, I.; Terai, S.; Yamamoto, N.; et al. (2004) Transplantation of bone marrow cells reduces CCl4induced liver fibrosis in mice. Hepatology. Vol. 40, No. 6, pp. 1304-1311
Sakuragawa, N.; Yoshikawa, H. & Sasaki, M. (1992) Amniotic tissue transplantation: clinical and
biochemical evaluations for some lysosomal storage diseases. Brain Development. Vol. 14, pp. 7-11
372
References
Salama, H.; Zekri, A.N., Bahnassy, A.A.; et al. (2010) Autologous CD34+ and CD133+ stem cells
transplantation in patients with end stage liver disease. World J. Gastroenterology. Vol. 16, No. 42, pp.
5297-5305
Salizzoni, M.; Franchello, A.; Zamboni, F.; Ricchiuti, A.; Cocchis, D.; Fop, F.; Brunati, A. & Cerutti, E.
(2003) Marginal grafts: finding the correct treatment for fatty livers.Transplant International, Vol.16, No.7,
(July 2003), pp. 486-493, ISSN: 1432-2277
Sallusto F, Geginat J, Lanzavecchia A. Central memory and effector memory T cell subsets: function,
generation, and maintenance. Annu Rev Immunol 2004;22:745–763.
Sallusto F, Langenkamp A, Geginat J, et al. Functional subsets of memory T cells identified by CCR7
expression. Curr Top Microbiol Immunol 2000;251:167–171.
Samuel D, Bismuth A, Mathieu D, et al. Passive immunoprophylaxis after liver transplantation in HBsAgpositive patients. Lancet 1991;337:813–815.
Samuel D, Weber R, Stock P, Duclos-Vallée JC, Terrault N. Are HIV-infected patients candidates for liver
transplantation? J Hepatol 2008;48:697–707.
Sanchez-Urdazpal L, Gores GJ, Ward EM, et al. Diagnostic features and clinical outcome of ischemictype biliary complications after liver transplantation. Hepatology 1993;17:605–9.
Sanchez-Urdazpal L, Gores GJ, Ward EM, et al. Ischemic-type biliary complications after orthotopic liver
transplantation. Hepatology 1992;16:49–53.
Sancho-Bru, P.; Najimi, M.; Caruso, M.; et al. (2009) Stem and progenitor cells for liver repopulation: can
we standardize the process from bench to bedside? Gut. Vol. 58,
Sandrin L, Fourquet B, Hasquenoph JM, Yon S, Fournier C, Mal F, et al.Transient elastography: a new
noninvasive method for assessment of hepatic fibrosis. Ultrasound Med Biol 2003;29:1705–1713.
Saner F, et al: Neurological complications after cadaveric and living donor liver transplantation. J Neurol
2006; 253:612-617.
Saner FH, et al: Neurologic complications in adult living donor liver transplant patients: an underestimated
factor?. J Neurol 2010; 257:253-258.
Sangiovanni A, Prati GM, Fasani P, et al. The natural history of compensated cirrhosis due to hepatitis C
virus: a 17-year cohort study of 214 patients. Hepatology 2006;43:1303–1310.
Santamaria F, Sarnelli P, Celentano L, Farina V, Vegnente A, Mansi A, et al. Noninvasive investigation of
hepatopulmonary syndrome in children and adolescents with chronic cholestasis. Pediatr Pulmonol
2002;33:374- 379.
Sarasin FP, Majno PE, Llovet JM, Bruix J, Mentha G, Hadengue A. Living donor liver transplantation for
early hepatocellular carcinoma: a life-expectancy and cost-effectiveness perspective. Hepatology 2001;
33: 1073–9.
Sarmiento JM, Que FG. Hepatic surgery for metastases from neuroendocrine tumors. Surg Oncol Clin N
Am 2003;12:231-242.
Sayegh MH, Carpenter CB. Transplantation 50 years later: progress, challenges, and promises. N Engl J
Med 2004; 351:2761–2766.
Scaggiante, B.; Pineschi, A.; Sustersich, M.; et al. (1987) Successful therapy of Niemann-Pick disease by
implantation of human amniotic membrane. Transplantation. Vol. 44, pp. 59-61
Schalm SW. The diagnosis of cirrhosis: clinical relevance and methodology. J Hepatol 1997;27:1118–
1119.
Schaubel DE, Guidinger MK, Biggins SW, Kalbfleisch JD, Pomfret EA, Sharma P, et al. Survival benefitbased deceased donor liver allocation. Am J Transpl, 2009; 9 (part 2): 970-981
Schaubel DE, Sima CS, Goodrich NP, Feng S, Merion RM. The survival benefit of deceased donor liver
transplantation as afunction of candidate disease severity and donor quality. Am J Transpl 2008;8:419–
425.
Schenk AD, Nozaki T, Rabant M, et al. Donor-reactive CD8 memory T cells infiltrate cardiac allografts
within 24-h post-transplant in naïve recipients. Am J Transplant 2008;8:1652–1661.
373
References
Schenk P, Schoniger-Hekele M, Fuhrmann V, Madl C, Silberhumer G, Muller C. Prognostic significance
of the hepatopulmonary syndrome in patients with cirrhosis. Gastroenterology 2003;125:1042-1052.
Schepke M, Roth F, Fimmers R, et al. Comparison of MELD, Child-Pugh, and Emory model for the
prediction of survival in patients undergoing transjugular intrahepatic portosystemic shunting. Am J
Gastroenterol 2003; 98: 1167.
Scherer MN, Banas B, Mantouvalou K, Schnitzbauer A, Obed A, Kramer BK, Schlitt HJ (2007) Current
concepts and perspectives of immunosuppression in organ transplantation. Langenbecks Arch Surg
392(5):511–523
Scheuer PJ. Classification of chronic viral hepatitis: a need for reassessment. J Hepatol 1991;13:372–4.
Schiano T, Gutierrez J, Walewski J, et al. Accelerated hepatitis C virus kinetics but similar survival rates
in recipients of liver grafts from living versus deceased donors. Hepatology 2005;42:1420–8.
Schiavon LL, Schiavon JL, Filho RJ, et al. Simple blood tests as noninvasive markers of liver fibrosis in
hemodialysis patients with chronic hepatitis C virus infection. Hepatology 2007;46: 307–314.
Schiff ER, Sorrell MF, Maddrey EC, eds. Schiff ’s diseases of the liver, 9th edn. Philadelphia, PA:
Lippincott, Williams & Wilkins, 2003.
Schilsky ML. Treatment of Wilson’s disease: what are the relative roles of penicillamine, trientine, and
zinc supplementation? Curr Gastroenterol Rep 2001;3:54–59.
Schiødt FV, Ostapowicz G, Murray N, et al. Alpha-fetoprotein and prognosis in acute liver failure. Liver
Transpl 2006;12:1776– 1781.
Schlitt HJ, Barkmann A, Boker KH, et al. Replacement of calcineurin inhibitors with mycophenolate
mofetil in liver- transplant patients with renal dysfunction: a randomised controlled study. Lancet
2001;357:587-91.
Schlitt HJ, Loss M, Scherer M, et al. Current developments in liver transplantation in Germany: MELDbased organ allocation and incentives for transplant centres. Z Gastroenterol 2011;49: 30-8. PMID
21225535.
Schlitt HJ: Which liver is splitable? In Rogiers X, Bismuth H, Busuttil RW, et al, eds: Split-liver
transplantation—theoretical and practical aspects. Darmstadt, Germany: Steinkopff Verlag; 2002, p 63
Schmidt A, Tomasdottir H, Bengtsson A. Influence of cold ischemia time on complement activation,
neopterin, and cytokine release in liver transplantation. Transplant Proc 2004;36:2796–2798
Schmucker, D.L., 1998. Aging and the liver: an update. J. Gerontol. 53A, B315–B320.
Scholz M, Auth MK, Markus BH. The immunological role of biliary epithelial cells in human liver transplant
rejection. Transpl Immunol 1997;5:142–51.
Schramm C, Bubenheim M, Adam R, Karam V, Buckels J, O'Grady JG, Jamieson N, Pollard S, Neuhaus
P, Manns MM, Porte R, Castaing D, Paul A, Traynor O, Garden J, Friman S, Ericzon BG, Fischer L, Vitko
S, Krawczyk M, Metselaar HJ, Foss A, Kilic M, Rolles K, Burra P, Rogiers X, Lohse AW; European Liver
Intestine Transplant Association. Primary liver transplantation for autoimmune hepatitis: a comparative
analysis of the European Liver Transplant Registry. Liver Transpl. 2010 Apr; 16(4):461-9.
Schreibman IR, Schiff ER. Prevention and treatment of recurrent Hepatitis B after liver transplantation:
the current role of nucleoside and nucleotide analogues. Ann Clin Microbiol Antimicrob 2006;5:8.
Schreinemachers M, Doorschodt BM, van Gulik TM, et al. Machine perfusion preservation of the liver: a
worthwhile clinical activity? Curr Opin Organ Transplant 2007;12:224–30.
Schreuder TC, Hubscher SG, Neuberger J. Autoimmune liver diseases and recurrence after orthotopic
liver transplantation: what have we learned so far? Transpl Int 2009;22:144–52.
Schrier RW, Arroyo V, Bernardi M, Epstein M, Henriksen JH, Rodes J. Peripheral arterial vasodilation
hypothesis: a proposal for the initiation of renal sodium and water retention in cirrhosis. Hepatology
1988;8:1151–1157.
Schroeder RA, Kuo PC. Local consequences of reperfusion following transplantation. In: Grace PA,
Mathie RT, eds. Ischemiareperfusion injury. London: Blackwell Science, 1999:113–122.
Schuurs TA, Morariu AM, Ottens PJ, 't Hart NA, Popma SH, Leuvenink HG, Ploeg RJ. Time-dependent
changes in donor brain death related processes. Am J Transplant 2006;6:2903-11.
374
References
Schvarcz R, Rudbeck G, Soderdahl G, Stahle L. Interaction between nelfinavir and tacrolimus after
orthoptic liver transplantation in a patient coinfected with HIV and hepatitis C virus (HCV). Transplantation
2000;69:2194-2195.
Schwartz M. Liver transplantation for hepatocellular carcinoma. Gastroenterology 2004;127:S268–276
Schwartz, R.E.; Reyes, M.; Koodie, L.; et al. (2002) Multipotent adult progenitor cells from bone marrow
differentiate into functional hepatocyte-like cells. J Clin Invest. Vol, 109, No. 10, pp. 1291-302
Scoazec JY, Borghi-Scoazec G, Durand F, Bernuau J, Pham BN, Belghiti J, et al. Complement activation
after ischemia-reperfusion in human liver allografts: incidence and pathophysiological relevance.
Gastroenterology 1997;112:908–918.
Scotte M, Dousset B, Calmus Y, et al. The influence of cold ischemia time on biliary complications
following liver transplantation. J Hepatol 1994;21:340–346
Seaberg EC, Belle SH, Beringer KC, et al. Liver transplantation in the United States from 1987- 1998:
updated results from the Pitt-UNOS Liver Transplant Registry. In: Cecka JM, Terasaki PI, eds. Clinical
Transplants 1998. Los Angeles: UCLA Tissue Typing Laboratory 1999:17-37
Sebagh M, Blakolmer K, Falissard B, Roche B, Emile JF, Bismuth H, et al. Accuracy of bile duct changes
for the diagnosis of chronic liver allograft rejection: reliability of the 1999 Banff schema. Hepatology
2002;35: 117–125.
Sebagh M, Rifai K, Feray C, et al. All liver recipients benefit from the protocol 10- year liver biopsies.
Hepatology 2003;37:1293–301.
Seehofer D, Rayes N, Neumann UP, et al. Changing impact of cytomegalovirus in liver transplantation—a
single centre experience of more than 1000 transplantations without ganciclovir prophylaxis. Transpl Int
2005;18:941–8.
Seehofer D, Rayes N, Steinmuller T, et al. Occurrence and clinical outcome of lamivudine-resistant
hepatitis B infection after liver transplantation. Liver Transpl 2001;7:976-82.
Seehofer D, Rayes N, Tullius SG, Schmidt CA, Neumann UP, Radke C, Settmacher U, Muller AR,
Steinmuller T, Neuhaus P. CMV hepatitis after liver transplantation: incidence, clinical course, and longterm follow-up. Liver Transpl 2002; 8: 1138-46.
Seetharam A, Tiriveedhi V, Mohanakumar T. Alloimmunity and autoimmunity in chronic rejection. Curr
Opin Organ Transplant 2010;15:531–536.
Segev DL, Maley WR, Simpkins CE, et al: Minimizing risk associated with elderly liver donors by
matching to preferred recipients. Hepatology 46:1907, 2007
Seifalian AM, Chidambaram V, Rolles K, Davidson BR. (1998). In vivo demonstration of impaired
microcirculation in steatotic human liver grafts. Liver Transpl Surg 4:71–77.
Seifalian AM, El Desoky A, Davidson BR. (2001). Hepatic indocyanine green uptake and excretion in a
rabbit model of steatosis. Eur Surg Res 33:193–201.
Selcuk H, Uruc I, Temel MA, Ocal S, Huddam B, Korkmaz M, Unal H, Kanbey M, Savas N, Gur G, Yilmaz
U, Haberal M. Factors prognostic of survival in patients awaiting liver transplantation for end-stage liver
disease. Dig Dis Sci 2007; 52:3217-3223
Selzner and Clavien, 2001; Selzner M, Clavien PA. 2001. Fatty liver in liver transplantation and surgery.
Semin Liver Dis 21:105–113.
Selzner N, Rudiger H, Graf R, et al. Protective strategies against ischemic injury of the liver.
Gastroenterology 2003;125:917–36.
Selzner N, Selzner M, Jochum W, Amann-Vesti B, Graf R, Clavien PA. Mouse livers with macrosteatosis
are more susceptible to normothermic ischemic injury than those with microsteatosis. J Hepatol 2005.
Selzner, M. & Clavien, PA. (2000) Failure of regeneration of the steatotic rat liver: disruption at two
different levels in the regeneration pathway. Hepatology, Vol.31, No.1, (January 2000), pp. 35-42, ISSN:
0168-8278
Selzner, M.; Rudiger, HA.; Sindram, D.; Madden, J. & Clavien, PA. (2000) Mechanisms of ischemic injury
are different in the steatotic and normal rat liver. Hepatology, Vol.32, No.6, (December 2000), pp. 12801288, ISSN: 0168-8278
375
References
Selzner, N.; Rudiger, H.; Graf, R. & Clavien, PA. (2003) Protective strategies against ischemicinjury of the
liver. Gastroenterology, Vol.125, No.3, (September 2003), pp. 917-936, ISSN: 0016-5085
Senzolo M, Ferronato C, Burra P: Neurologic complications after solid organ transplantation. Transpl Int
2009; 22:269-278.Transpl Int 2009; 22:269-278.
Serfaty L, Aumaitre H, Chazouilleres O, et al. Determinants of outcome of compensated hepatitis C virusrelated cirrhosis. Hepatology 1998;27:1435–1440.
Serra P, Amrani A, Yamanouchi J, Han B, Thiessen S, Utsugi T, Verdaguer J, Santamaria P. CD40
ligation releases immature dendritic cells from the control of regulatory CD4+CD25+ T cells. Immunity.
2003 Dec;19(6):877-89.
Serrano-Delgado, V.M.; Novello-Garza, B. &Valdez-Martinez, E. (2009) Ethical issues relating to the
banking of umbilical cord blood in Mexico. BMC Medical Ethics. Vol. 10, pp. 12
Sersté T, Lebrec D, Valla D, Moreau R. Incidence and characteristics of type 2 hepatorenal syndrome in
patients with cirrhosis and refractory ascites. Acta Gastroenterol Belg 2008;71:9–14.
Serviddio, G.; Bellanti, F.; Tamborra, R.; Rollo, T.; Capitanio, N.; Romano, AD.; Sastre, J.; Vendemiale,
G. & Altomare, E. (2008) Uncoupling protein-2 (UCP2) induces mitochondrial proton leak and increases
susceptibility of non-alcoholic steatohepatitis (NASH) liver to ischaemia-reperfusion injury. Gut, Vol.57,
No.7, (July 2008), pp. 957-965, ISSN: 1468-3288
Settmacher U, Theruvath T, Pascher A, Neuhaus P. Living-donor liver transplantation—European
experiences. Nephrol Dial Transplant 2004; 19(Suppl 4):iv16-iv21.
Seu P, Wilkinson AH, Shaked A, Busuttil RW. The hepatorenal syndrome in liver transplant recipients.
Am Surg 1991;57:806-809.
Shah SA, Grant DR, McGilvray ID, Greig PD, Selzner M, Lilly LB, et al. Biliary strictures in 130
consecutive right lobe living donor liver transplant recipients: results of aWestern center. Am J Transplant
2007;7:161–167.
Shaheen AA, Myers RP. Diagnostic accuracy of the aspartate aminotransferase-to-platelet ratio index for
the prediction of hepatitis C-related fibrosis: a systematic review. Hepatology 2007;46:912–921.
Shaikh OS, Demetris AJ. Idiopathic posttransplantation hepatitis? Liver Transpl 2007;13:943–6.
Shaked A, Ghobrial RM, Merion RM, Shearon TH, Emond JC, Fair JH, et al. Incidence and severity of
acute cellular rejection in recipients undergoing adult living donor or deceased donor liver transplantation.
Am J Transplant 2009;9:301–308.
Shakil AO, A. Pinna, J. Demetris, R. G. Lee, J. J. Fung, and J. Rakela, “Survival and quality of life after
liver transplantation for acute alcoholic hepatitis,” Liver Transplantation and Surgery, vol. 3, no. 3, pp.
240–244, 1997.
Sharma P, Balan V, Hernandez JL, et al. Liver transplantation for hepatocellular carcinoma: the MELD
impact. Liver Transpl 2004;10: 36-41.
Sharma P, Schaubel DE, Sima CS, Merion RM, Lok AS. Re-weighting the model for end-stage liver
disease score components. Gastroenterology 2008;135:1575–81.
Sharma V K, B. Li, A. Khanna, P. K. Sehajpal, and M. Suthanthiran, “Which way for drug-mediated
immunosuppression?” Current Opinion in Immunology, vol. 6, no. 5, pp. 784–790, 1994.
Sharma, A.D.; Cantz, T.; Vogel, A.; et al. (2008) Murine embryonic stem cell-derived hepatocyte
progenitor cells engraft in recipient livers with limited capacity of liver tissue formation. Cell Transplant.
Vol. 17, No. 3, pp. 313-323
Shaw LM, London JW, Petersen LE. Isolation of gamma-glutamyltransferase from human liver, and
comparison with the enzyme from human kidney. Clin Chem 1978;24:905-15.
Sheikh AM, Wolf DC, Lebovics E, Goldberg R, Horowitz HW. Concomitant human immunodeficiency
virus protease inhibitor therapy markedly reduces tacrolimus metabolism and increases blood levels.
Transplantation 1999;68:307-309.
Shen XD, Gao F, Ke B, et al. Inflammatory responses in a new mouse model of prolonged hepatic cold
ischemia followed by arterialized orthotopic liver transplantation. Liver Transpl 2005;11:1273–1281
376
References
Sherman M, “Hepatocellular carcinoma: epidemiology, risk factors, and screening,” Seminars in Liver
Disease, vol. 25, no. 2, pp. 143–154, 2005.
Shetty K, Rybicki L, Carey WD. The Child–Pugh classification as a prognostic indicator for survival in
primary sclerosing cholangitis. Hepatology 1997;25:1049–53.
Shiffman M, Stravitz R, Contos M, et al. Histologic recurrence of chronic hepatitis C virus in patients after
living donor and deceased donor liver transplantation. Liver Transpl 2004;10: 1248–55.
Shimamura T et al. Excessive portal venous inflow as a cause of allograft dysfunction in smallfor-size
living donor liver transplantation. Transplant Proc. 2001; 33 (1- 2): 1331.
Shimamura, T.; Taniguchi, M.; Jin, MB.; Suzuki, T.; Matsushita, M.; Furukawa, H. & Todo, S. Shiraishi T,
Morimoto S, Koh E, et al. Increased release of platelet-derived growth factor from platelets in chronic liver
disease. Eur J Clin Chem Clin Biochem 1994;32:5–9.
Showstack J, Katz PP, Lake JR et al. Resource utilization in liver transplantation: effects of patient
characteristics and clinical practice. NIDDK Liver Transplantation Database Group. JAMA 1999; 281:
1381–1386
Sieders E, Peeters PM, TenVergert EM, de Jong KP, Porte RJ, Zwaveling JH, et al. Retransplantation of
the liver in children. Transplantation 2001;71:90-95.
Silberhumer, GR.; Pokorny, H.; Hetz, H.; Herkner, H.; Rasoul-Rockenschaub, S.; Soliman, T.; Wekerle,
T.; Berlakovich, GA.; Steininger, R. & Muehlbacher, F. (2007) Combination of extended donor criteria and
changes in the Model for End-Stage Liver Disease score predict patient survival and primary dysfunction
in liver transplantation: a retrospective analysis. Transplantation, Vol.83, No.5, (May 2007), pp. 588-592,
ISSN:1534-0608
Silva MA. Putting objectivity into assessment of steatosis. Transplantation 2009;88:620–621
Simpson RJ, Lim JW, Moritz RL, et al. Exosomes: proteomic insights and diagnostic potential. Expert Rev
Proteomics 2009;6(3):267-83.
Singh N, Wannstedt C, Keyes L, Wagener MM, Cacciarelli TV. Who among cytomegalovirus-seropositive
liver transplant recipients is at risk for cytomegalovirus infection? Transpl 2005; 11: 700-704.
Singh N. The current management of infectious diseases in the liver transplant recipient. Clin Liver Dis
2000; 4: 657-73.
Singhal et al, 2010. Singhal A, et al: Endovascular treatment of hepatic artery thrombosis following liver
transplantation. Transpl Int 2010; 23:245-256
Si-Tayeb, K.; Noto, F.K.; Nagaoka, M.; et al. (2010). Highly efficient generation of human hepatocyte-like
cells from induced pluripotent stem cells. Hepatology. Vol. 51, pp. 297-305.
Skvorak, K.J.; Dorko, K.; Hansel, M.C.; et al. (2010) Human amnion epithelial (hAE) stem cell transplant
significantly improves disease phenotype and survival in the Intermediate Maple Syrup Urine Disease
(iMSUD) mouse model. Hepatology. Vol. 52, No. 4 Supplimental, pp. 413A, abstract #181, Proceedings
of AASLD Annual Meeting, Boston, MA, Oct. 29-Nov. 2, 2010
Skvorak, K.J.; Hager, E.J.; Arning, E.; et al. (2009b) Hepatocyte transplantation (HTx) corrects selected
neurometabolic abnormalities in murine intermediate maple syrup urine disease (iMSUD). Biochim.
Biophys. Acta. Vol. 1792, No. 10, pp. 1004-10
Skvorak, K.J.; Paul, H.S.; Dorko, K.; et al. (2009a) Hepatocyte transplantation improves phenotype and
extends survival in a murine model of intermediate maple syrup urine disease. Molecular Therapy. Vol.
17, No. 7, pp. 1266-1273
Slapak GI, Saxena R, Portmann B, et al. Graft and systemic disease in long-term survivors of liver
transplantation. Hepatology 1997;25:195–202.
Smets F, Bodeus M, Goubau P, et al. Characteristics of Epstein–Barr virus primary infection in pediatric
liver transplant recipients. J Hepatol 2000;32:100–4.
Smith M. Physiologic changes during brain stem death—lessons for management of the organ donor. J
Heart Lung Transpl 2004;23:217-22.
Smith PA Klein AS, Heath DG, Chavin K, Fishman EK. Dual-phase spiral CT angiography with volumetric
3D rendering for preoperative liver transplant evaluation: preliminary observations. J Comput Assist
Tomogr 1998;22:868-874.
377
References
Smyrniotis V, Kostopanagiotou G, Kondi A, et al. Hemodynamic interaction between portal vein and
hepatic artery flow in small-for-size split liver transplantation. Transpl Int 2002;15:355–60.
Snover DC, Weisdorf S, Bloomer J, et al. Nodular regenerative hyperplasia of the liver following bone
marrow transplantation. Hepatology 1989;9:443–8.
Snowden CP, Hughes T, Rose J, Roberts DR. Pulmonary edema in patients after liver transplantation.
Liver Transpl 2000; 6: 466-70.
Sobhonslidsuk A, Reddy KR. Portal vein thrombosis: a concise review. Am J Gastroenterol 2002;97:535541.
Soejima Y, Shimada M, Suehiro T, et al. Use of steatotic graft in living-donor liver transplant.
Transplantation 2003;76: 344–348
Soejima Y, Taketomi A, Yoshizumi T et al. Extended indication for living donor liver transplantation in
patients with hepatocellular carcinoma. Transplantation 2007; 83: 893–9.
Soin AS, V. Kumaran, A. N. Rastogi et al., “Evolution of a reliable biliary reconstructive technique in 400
consecutive living donor liver transplants,” Journal of the American College of Surgeons, vol. 211, no. 1,
pp. 24–32, 2010.
Soin et al, 1996. Soin AS, et al: Donor arterial variations in liver transplantation: management and
outcome of 527 consecutive grafts. Br J Surg 1996; 83:637-641.
Sokal, E.M.; Smets, F.; Bourgois, A.; et al. (2003) Hepatocyte transplantation in a 4-year-old girl with
peroxisomal biogenesis disease: technique, safety, and metabolic followup. Transplantation. Vol. 76, pp.
735–738
Sokhi RP, Anantharaju A, Kondaveeti R, Creech SD, Islam KK, Van Thiel DH. Bone mineral density
among cirrhotic patients awaiting liver transplantation. Liver Transpl 2004;10:648-653.
Soltys, K.; Soto-Gutierrez, A.; Nagaya, M.; et al. (2010) Barriers to the successful treatment of liver
disease by hepatocyte transplantation. Hepatology. Vol. 53, pp. 769-774
Sommacale D, Farges O, Ettorre GM, et al. In situ split liver transplantation for two adult recipients.
Transplantation 2000;69:1005–7.
Song, Z.; Cai, J.; Liu, Y.; et al. (2009). Efficient generation of hepatocyte-like cells from human induced
pluripotent stem cells. Cell Res. Vol. 19, pp. 1233-1242.
Sotiropoulos CG (a), Beckebaum S, Lang H, et al. Single-center experience on liver transplantation for
hepatocellular carcinoma arising in alcoholic cirrhosis: results and ethical issues. Eur Surg Res
2008;40:7-13.
Soto-Gutierrez, A.; Basma, H.; Navarro-Alvarez, N.; et al. (2008) Differentiating stem cells into liver.
Biotech. Genetic Enginering Rev. Vol. 25, pp. 149-164
Spengler U, Nattermann J. Immunopathogenesis in hepatitis C virus cirrhosis. Clin Sci (Lond)
2007;112:141–55.
Spitz, C.; Mateizel, I.; Geens, M.; et al. (2008) Recurrent chromosomal abnormalities in human embryonic
stem cells. Nature Biotech. Vol. 26, pp. 1361-1363
Spitzer AL, Lao OB, Dick AA, Bakthavatsalam R, Halldorson JB, Yeh MM, et al. The biopsied donor liver:
incorporating macrosteatosis into high-risk donor assessment. Liver Transpl 2010;16:874–884..
Srikureja W, Kyulo NL, Runyon BA, Hu KQ. MELD score is a better prognostic model than Child-TurcottePugh score or Discriminant Function score in patients with alcoholic hepatitis. J Hepatol 2005; 42: 700706
Srinivasan P, McCall J, Pritchard J, Dhawan A, Baker A, Vergani GM, et al. Orthotopic liver
transplantation for unresectable hepatoblastoma. Transplantation 2002;74:652-655.
Stange J, Ramlow W, Mitzner S, et al. Dialysis against a recycled albumin solution enables the removal
of albumin-bound toxins. Artif Organs 1993;17:809–13.
Starkel P, Vera A, Gunson B, Mutimer D. Outcome of liver transplantation for patients with pulmonary
hypertension. Liver Transpl 2002;8: 382-38
Starzl TE, Demetris AJ. Liver transplantation: a 31-year perspective. Part I. Curr Probl Surg 1990;27:49–
116.
378
References
Starzl TE, Fung J, Tzakis A, Todo S, Demetris AJ, Marino IR, Doyle H, Zeevi A, Warty V, Michaels M,
Kusne S, Rudert W. A, Trucco M. Baboon-to-human liver transplantation. Lancet. 1993 Jan
9;341(8837):65-71.
Starzl TE, Todo S, Gordon RD, Makowka L, Tzakis A, Iwatsuki S, et al. Liver transplantation in older
patients. New Engl J Med 1987;316:484- 485.
Stefanova I, Dorfman JR, Tsukamoto M, et al. On the role of self-recognition in T cell responses to foreign
antigen. Immunol Rev 2003;191:97–106.
Steinhoff G. Major histocompatibility complex antigens in human liver transplants. J Hepatol 1990;11:9–
15.
Sterling RK, Lissen E, Clumeck N, et al. Development of a simple noninvasive index to predict significant
fibrosis in patients with HIV/HCV coinfection. Hepatology 2006;43:1317–1325.
Sterling, R.K. & Fisher, R.A. (2001) Liver Transplantation: Living Donor, Hepatocyte, and
Xenotransplantation. In: Current Future Treatment Therapies for Liver Disease. Clinics in Liver Disease,
Gish, R. (ed.) Philadelphia: WB Saunders.
Stewart et al, 2009. Stewart ZA, et al: Increased risk of graft loss from hepatic artery thrombosis after liver
transplantation with older donors. Liver Transpl 2009; 15:1688-1695.
Stewart SF, Hudson M, Talbot D, Manas D, Day CP. Mycophenolate mofetil monotherapy in liver
transplantation. Lancet 2001;357:609-10.
Stock PG, Roland ME, Carlson L, Freise CE, Roberts JP, Hirose R, et al. Kidney and liver transplantation
in human immunodeficiency virus-infected patients: a pilot safety and efficacy study. Transplantation
2003; 76:370-375.
Stranges S, Trevisan M, Dorn JM, et al. Body fat distribution, liver enzymes, and risk of hypertension:
evidence from the Western New York Study. Hypertension. 2005; 46: 1186–1193.Epub 2005 Oct 3.
Strasberg SM, Howard TK, Molmenti EP, Hertl M. Selecting the donor liver: risk factor for poor function
after orthotopic liver transplantation. Hepatology 1994;20:829–
Strom TB, Tilney NL, Carpenter CB, et al. Identity and cytotoxic capacity of cells infiltrating renal
allografts. N Engl J Med 1975; 292:1257–1263.
Strom, S.C. & Ellis, E.C.S. (2011) Cell therapy of liver disease: From hepatocytes to stem cells, In:
Principles of Regenerative Medicine, 2nd Edition, Atala, A.; Lanza, R.; Thomson, R.A. & Nerem, R.
(eds.), Elsevier, pp. 305-326, ISBN 9780123814227
Strom, S.C.; Choudhury, J.R. & Fox, I.J. (1999) Hepatocyte transplantation for the treatment of human
disease. Semin Liver Dis. Vol. 19, pp. 39-48
Strom, S.C.; et al. (1997a) Transplantation of human hepatocytes. Transplant Proc. Vol. 29, pp. 21032106
Strom, S.C.; Fisher, R.A.; Thompson, M.T.; et al. (1997b) Hepatocyte transplantation as a bridge to
orthotopic liver transplantation in terminal liver failure. Transplantation. Vol. 63, pp, 559–569
Strong R, Lynch SV, Ong TH, Matsunami H, Koido Y, Balderson GA. Successful liver transplantation from
a livingdonor to her son. N Engl J Med 1990;322:1505–1507.
Stutchfield, B.M.; Forbes, S.J. & Wigmore, S.J. (2010) Prospects for stem cell transplantation in the
treatment of hepatic disease. Liver Transplantation. Vol. 16, pp. 827-836
Sud A, Hui JM, Farrell GC, Bandara P, Kench JG, Fung C, et al. Improved prediction of fibrosis in chronic
hepatitis C using measures of insulin resistance in a probability index. Hepatology 2004;39(5):1239–47.
Sudan D, DeRoover A, Chinnakotla S, Fox I, Byers S, McCashland T, et al. Radiochemotherapy and
transplantation allow long-term survival for non-resectable hilar carcinoma. Am J Transplant 2002; 2:774–
779
Sugawara Y, Makuuchi M. Living donor liver transplantation: present status and recent advances. Br Med
Bull 2005;75-76:15-28.
Sugawara Y, Makuuchi M. Should living donor liver transplantation be offered to patients with hepatitis C
virus cirrhosis? J Hepatol 2005;42:472–475.
379
References
Sugawara Y, Makuuchi M. Technical advances in living-related liver transplantation. J Hepatobiliary
Pancreat Surg 1999;6:245-53.
Suhr OB, Ericzon BG, Friman S. Long-term follow-up of survival of liver transplant recipients with familial
amyloid polyneuropathy (Portuguese type). Liver Transpl 2002;8:787-794
Sun CK, Zhang XY, Zimmermann A, Davis G, Wheatley AM. (2001). Effect of ischemia-reperfusion injury
on the microcirculation of the steatotic liver of the Zucker rat. Transplantation 72:1625–1631
Sundaram SS, Melin-Aldana H, Neighbors K, et al. Histologic characteristics of late cellular rejection,
significance of centrilobular injury, and long-term outcome in pediatric liver transplant recipients. Liver
Transpl 2006;12:58–64.
Sundin, M.; Orvell, C.; Rasmusson, I.; et al. (2006) Mesenchymal stem cells are susceptible to human
herpes viruses, but viral DNA cannot be detected in the healthy seropositive individual. Bone Marrow
Transplant. Vol. 37, No. 11, pp. 1051-9
SungGyu Lee, ChulSoo Ahn, TaeYong Ha, DeokBog Moon, KunMoo Choi Æ GiWon Song, DongHwan
Chung Æ GilChun Park, YoungDong Yu, NamKyu Choi . KwanWoo Kim Æ KiHun Kim Æ Shin Hwang
Liver transplantation for hepatocellular carcinoma: Korean experience J Hepatobiliary Pancreat Sci
(2010) 17:539–547 DOI 10.1007/s00534-009-0167-6
Surman OS. The ethics of partial-liver donation. New Engl J Med 2002; 346:1038.
Sussman NL, Gislason GT, Conlin CA, et al. The Hepatix extracorporeal liver assist device: initial clinical
experience. Artif Organs 1994;18:390–6.
Sutcliffe R, Maguire D, Ramage J, Rela M, Heaton N. Management of neuroendocrine liver metastases.
Am J Surg 2004;187:39-46.
Suttie JW. The biochemical basis of warfarin therapy. Adv Exp Med Biol 1987;214:3–16.
Sverger T, Erikson S. The liver in adolescents with alpha 1-anti-trypsin deficiency. Hepatology
1995;22:514–517.
Syn WK, Nightingale P, Gunson B, et al. Natural history of unexplained chronic hepatitis after liver
transplantation. Liver Transpl 2007;13:984–9.
Szabo G, Buhmann V, Bahrle S, et al. Brain death impairs coronary endothelial function. Transplantation
2002;73:1846-8.
Takada M, Nadeau KC, Hancock WW, et al. Effects of explosive brain death on cytokine activation of
peripheral organs in the rat. Transplantation 1998;65(12):1533-42.
Takada Y, Ito T, Ueda M et al. Living donor liver transplantation for patients with HCC exceeding the
Milan criteria: a proposal of expanded criteria. Dig. Dis. 2007; 25: 299–302.
Takada Y, Taniguchi H, Fukunaga K, et al. Prolonged hepatic warm ischemia in nonheart- beating
donors: protective effects of FK506 and a platelet activating factor antagonist in porcine liver
transplantation. Surgery 1998;123:692–8.
Takahashi, K. & Yamanaka, S. (2006). Induction of pluripotent stem cells from mouse embryonic and
adult fibroblast cultures by defined factors. Cell. Vol. 126, pp. 663- 676
Takahashi, K.; Tanabe, K.; Ohnuki, M.; et al. (2007). Induction of pluripotent stem cells from adult human
fibroblasts by defined factors. Cell. Vol. 131, pp. 861-872.
Takasaki S, Hano H. Three-dimensional observations of the human hepatic artery (arterial system in the
liver). J Hepatol 2001;34:455–66.
Tamura S, Kato T, Berho M, et al. Impact of histological grade of hepatocellular carcinoma on the
outcome of liver transplantation. Arch Surg 2001;136:25–30; discussion 31.
Tamura S, Sugawara Y, Kaneko J, et al. Recurrence of cholestatic liver disease after living donor liver
transplantation. World J Gastroenterol 2008;14:5105–9.
Tan HP, K. Patel-Tom, and A. Marcos, “Adult living donor liver transplantation: who is the ideal donor and
recipient?” Journal of Hepatology, vol. 43, no. 1, pp. 13–17, 2005
Tan J, Lok AS. Antiviral therapy for pre- and post-liver transplantation patients with hepatitis B. Liver
Transpl 2007;13:323-6
380
References
Tanabe M, Kawachi S, Obara H, Shinoda M, Hibi T, Kitagawa Y, Wakabayashi G, Shimazu M, Kitajima
M. Current progress in ABO-incompatible liver transplantation. Eur J Clin Invest. 2010 Oct;40(10):943-9.
Tanaka, K.; Soto-Gutierrez, A.; Navarro-Alvarez, N.; et al. (2006) Functional hepatocyte culture and its
application to cell therapies. Cell Transplant. Vol. 15, pp. 855-864
Tang H, Boulton R, Gunson B, et al. Patterns of alcohol consumption after liver transplantation. Gut
1998;43:140–5.
Tannapel A, Kohlhaw K, Ebelt J, Hauss J, Liebert U, Berr F, Wittekind C. Apoptosis and the expression of
Fas and Fas ligand (FasL) antigen in rejection and re-infection in liver allograft specimens.
Transplantation 1999;15:1079-1083.
Teare JP, Sherman D, Greenfield SM, et al. Comparison of serum procollagen III peptide concentrations
and PGA index for assessment of hepatic fibrosis. Lancet 1993;342:895–898.
Tector AJ, Mangus RS, Chestovich P et al. Use of extended criteria livers decreases wait time for liver
transplantation without adversely impacting posttransplant survival. Ann Surg 2006; 244: 439–450
Teli MR, James OF, Burt AD, et al. The natural history of nonalcoholic fatty liver: a follow-up study.
Hepatology 1995;22:1714– 1719.
Teoh NC, Farrell GC. Hepatic ischemia reperfusion injury: pathogenic mechanisms and basis for
hepatoprotection. J Gastroenterol Hepatol 2003;18:891–902.
Teperman L. Donor-transmitted diseases. Liver Transplantation 2010;16:S40–4.
Teramoto K, Bowers JL, Kruskal JB, Clouse ME. (1993). Hepatic microcirculatory changes after
reperfusion in fatty and normal liver transplantation in the rat. Transplantation 56:1076–1082.
Terjung B, Lemnitzer I, Dumoulin FL, et al. Bleeding complications after percutaneous liver biopsy. An
analysis of risk factors. Digestion 2003;67:138–145.
Terminology for hepatic allograft rejection. International Working Party. Hepatology 1995;22:648–54.
Testa G, Crippin J, Netto G, et al. Liver transplantation for hepatitis C: Recurrence and disease
progression in 300 patients. Liver Transpl 2000;6:553–561.
Testa G, Goldstein RM, Netto G, Abbasoglu O, Brooks BK, Levy MF, et al. Long-term outcome of patients
transplanted with livers from hepatitis C-positive donors. Transplantation 1998;65:925-929.
Testa G, Malago M, Nadalin S, Hertl M, Lang H, Frilling A, et al. Right-liver living donor transplantation for
decompensated end-stage liver disease. Liver Transpl 2002;8:340–346.
Thampanitchawong P, Piratvisuth T. Liver biopsy: complications and risk factors. World J Gastroenterol
1999;5:301–304.
The Organ Procurement and Transplant Network. National data reports, liver Kaplan-Meier patient
survival rates for transplants performed: 1995–2002. Available at www.optn.org.
Therapondos G, Flapan AD, Plevris JN, Hayes PC. Cardiac morbidity and mortality related to orthotopic
liver transplantation. Liver Transpl 2004;10:1441-1453.
Theruvath TP, Zhong Z, Pediaditakis P, Ramshesh VK, Currin RT, Tikunov A, et al. Minocycline and Nmethyl-4-isoleucine cyclosporin (NIM811) mitigate storage/reperfusion injury after rat liver transplantation
through suppression of the mitochondrial permeability transition. Hepatology 2008;47: 236–246.
Thethy S, Thomson B, Pleass H, et al. Management of biliary tract complications after orthotopic liver
transplantation. Clin Transplant 2004;18:647–53.
Thomson, J.A.; Itskovitz-Eldor, J.; Shapiro, S.S.; et al. (1998) Embryonic stem cell lines derived from
human blastocysts. Science. Vol. 282, pp. 1145-1147
Thuluvath P, Yoo H. Graft and patient survival after adult live donor liver transplantation compared to a
matched cohort who received a deceased donor transplantation. Liver Transpl 2004;10(10):1263–8.
Thuluvath PJ, Bal JS, Mitchell S, Lund G, Venbrux A. TIPS for management of refractory ascites:
response and survival are both unpredictable. Dig Dis Sci 2003; 48: 542.
Thuluvath PJ, Krok KL, Segev DL, et al. Trends in post-liver transplant survival in patients with hepatitis C
between 1991 and 2001 in the United States. Liver Transpl 2007;13:719–724.
Thung SN. Histologic findings in recurrent HBV. Liver Transpl 2006;12(11 Suppl 2):S50–3
381
References
Tiao G, Bobey N, Allen S, et al. The current management of hepatoblastoma: a combination of
chemotherapy, conventional resection, and liver transplantation. J Pediatr 2005;146:204–211.
Tillmann HL. Antiviral therapy and resistance with hepatitis B virus infection. World J Gastroenterol
2007;13:125- 40.
Tinmouth J, Tomlinson G, Heathcote EJ, Lilly L. Benefit of transplantation in primary biliary cirrhosis
between 1985- 1997. Transplantation 2002;73:224-227.
Tippner C, Nashan B, Hoshino K, et al. Clinical and subclinical acute rejection early after liver
transplantation: contributing factors and relevance for the long-term course. Transplantation
2001;72:1122–8.
Tisone G, Manzia TM, Zazza S, et al. Marginal donors in liver transplantation. Transplant Proc
2004;36:525–526
Tisone G, Orlando G, Cardillo A, et al. Complete weaning off immunosuppression in HCV liver transplant
recipients is feasible and favourably impacts on the progression of disease recurrence. J Hepatol
2006;44:702–9.
Todo S, Demetris AJ, Makowka L, et al. Primary nonfunction of hepatic allografts with preexisting fatty
infiltration. Transplantation 1989;47:903–905
Todo S, Furukawa H, Tada M, Japanese Liver Transplantation Study Group. Extending indication: role of
living donor liver transplantation for hepatocellular carcinoma. Liver Transpl. 2007; 13: S48–54.
Tojimbara T, Wicomb WN, Garcia-Kennedy R, Burns W, Hayashi M, Collins G, Esquivel CO. Liver
transplantation from non-heart beating donors in rats: influence of viscosity and temperature of initial
flushing solutions on graft function. Liver Transpl Surg 1997;3:39–45.
Toso C, Asthana S, Bigam DL, Shapiro AM, Kneteman NM. Reassessing selection criteria prior to liver
transplantation for hepatocellular carcinoma utilizing the Scientific Registry of Transplant Recipients
database. Hepatology 2009; 49: 832–8.
Toso C, Meeberg GA, Bigam DL, et al. De novo sirolimus-based immunosuppression after liver
transplantation for hepatocellular carcinoma: long-term outcomes and side effects. Transplantation.
2007;83(9):1162-8.
Totsuka E, Fung U, Hakamada K, et al. Analysis of clinical variables of donors and recipients with respect
to short-term graft outcome in human liver transplantation. Transplant Proc 2004;36:2215–2218
Totsukali E, Fung JJ, Ishizawa Y, et al. Synergistic effect of cold and warm ischemia time on
postoperative graft outcome in human liver transplantation. Hepatogastroenterology 2004; 51:1413–1416
Trautwein C, Possienke M, Schlitt HJ, Boker KH, Horn R, Raab R, et al. Bone density and metabolism in
patients with viral hepatitis and cholestatic liver diseases before and after liver transplantation. Am J
Gastroenterol 2000;95:2343-2351
Trevisani F, Colantonit A, Caraceni P, Van Thiel DH. 1996. The use of donor fatty liver for transplantation:
a challenge or quagmire? J Hepatol 22:114–121.
Triantos C, Samonakis D, Stigliano R, Thalheimer U, Patch D, Burroughs A. Liver transplantation and
hepatitis C virus: systematic review of antiviral therapy. Transplantation 2005;79:261-8
Trinchet JC, Hartmann DJ, Pateron D, et al. Serum type I collagen and N-terminal peptide of type III
procollagen in chronic hepatitis. Relationship to liver histology and conventional liver tests. J Hepatol
1991;12:139–144.
Troisi R, Noens L, Montalti R, Ricciardi S, Philippe J, Praet M, et al. ABO mismatch adult living donor liver
transplantation using antigen-specific immunoadsorption and quadruple immunosuppression without
splenectomy. Liver Transpl 2006;12:1412-7.
Trotter J,2002b Campsen J, Bak T, et al. Outcomes of donor evaluations for adult-to-adult right hepatic
lobe living donor liver transplantation. Am J Transplant 2006;6:1882–9.
Trotter J2002a, Wachs M, Everson G, et al. Adult-to-adult transplantation of the right hepatic lobe from a
living donor. N Engl J Med 2002;346:1074–82.
Trotter JF, Wisniewski KA, Terrault NA, et al. Outcomes of donor evaluation in adult-to-adult living donor
liver transplantation. Hepatology 2007;46:1476–84.
382
References
Trotter JF. Sirolimus in liver transplantation. Transplant Proc 2003;35(3 Suppl): 193S-200S.
Trounson, A. (2006) The production and directed differentiation of human embryonic stem cells. Endocr
Rev. Vol. 2, No. 2, pp. 208-19
Trounson, A.; Thakar, R.G.; Lomax, G. & Gibbons, D. (2011) Clinical trials for stem cell therapies. BMC
Medicine. Vol 9, pp. 52-58
Tsukamoto I, Nakata R, Kojo S. Effect of ageing on rat liver regeneration after partial hepatectomy.
Biochem Mol Biol Int 1993;30:773-778.
Tung BY, Farrell FJ, McCashland TM, Gish RG, Bacon BR, Keeffe EB, et al. Long-term follow-up after
liver transplantation in patients with hepatic iron overload. Liver Transpl Surg 1999;5:369–374.
Tzakis A, Todo S, Starzl TE. Orthotopic liver transplantation with preservation of the inferior vena cava.
Ann Surg 1989; 210: 649–452
Uemoto S, Inomata Y, Sannomiya A et al. Posttransplant hepatitis B infection in liver transplantation with
hepatitis B core antibody-positive donors. Transplant Proc 1998; 30: 134–135
Uemura T, Ikegami T, Sanchez EQ, Jennings LW, Narasimhan G, McKenna GJ, et al. Late acute
rejection after liver transplantation impacts patient survival. Clin Transplant 2008;22:316–323..
Uemura, T.; Randall, HB.; Sanchez, EQ.; Ikegami, T.; Narasimhan, G.; McKenna, GJ.; Chinnakotla, S.;
Levy, MF.; Goldstein, RM. & Klintmalm, GB. (2007) Liver retransplantation for primary nonfunction:
analysis of a 20-year single-center experience. Liver Transplantation, Vol.13, No.2, (February 2007), pp.
227-233, ISSN: 1527-6473
Ueno T et al, 2006. Ueno T, et al: Clinical outcomes from hepatic artery stenting in liver transplantation.
Liver Transpl 2006; 12:422-427.
Ueno T, Inuzuka S, Torimura T, et al. Significance of serum type-IV collagen levels in various liver
diseases. Measurement with a one-step sandwich enzyme immunoassay using monoclonal antibodies
with specificity for pepsin-solubilized type-IV collagen. Scand J Gastroenterol 1992;27:513–520.
Umeshita K, Fujiwara K, Kiyosawa K, Makuuchi M, Satomi S, Sugimachi K, et al. Operative morbidity of
living liver donors in Japan. Lancet 2003;362:687-90.
United Network for Organ Sharing. Allocation of Livers. Proposed Amended UNOS Policy 3.6. 2002.
Available at http://www.unos.org [accessed on February, 2003].
United Network for Organ Sharing. Donor management; the critical pathway for the organ donor; critical
pathway for donation after cardiac death. Available at: www.unos.org. Accessed August 22, 2006
UNOS Policy 3 Appendix 3B, 2011,
http://optn.transplant.hrsa.gov/PoliciesandBylaws2/policies/pdfs/policy 15.pdf
Ureña MA.; Ruiz-Delgado, FC.; González, EM.; Segurola, CL.; Romero, CJ.; García, IG.; González-Pinto,
I. & Gómez Sanz, R. (1998) Assessing risk of the use of livers with macro and microsteatosis in a liver
transplant program. Transplantation Proceedings, Vol.30, No.7, (November 1998), pp. 3288-3291, ISSN:
0041-1345
US Scientific Registry (UNOS). Richmond (VA): United Network for Organ Sharing, 2006,
http://www.unos.org/.
Use of hepatitis B core antibody-positive donors in orthotopic liver transplantation. Arch Surg 2002; 137:
572
Vagefi PA, Ascher NL, Freise CE, Dodge JL, Roberts JP. The use of living donor liver transplantation
varies with the availability of deceased donor liver transplantation. Liver Transpl 2011; doi:
10.1002/lt.22455.
Valentin-Gamazo C, Malago M, Karliova M, Lutz JT, Frilling A, Nadalin S, et al. Experience after the
evaluation of 700 potential donors for living donor liver transplantation in a single center. Liver Transpl
2004;10:1087–1096.
Valero R, Cabrer C, Oppenheimer F, Trias E, Sanchez-Ibanez J, De Cabo FM, et al. Normothermic
recirculation reduces primary graft dysfunction of kidneys obtained from non-heartbeating donors. Transpl
Int 2000;13:303–310.
383
References
Vallejo GH, Romero CJ, de Vicente JC. Incidence and risk factors for cancer after liver transplantation.
Crit Rev Oncol Hematol 2005;56:87-99.
Vallet-Pichard A, Mallet V, Nalpas B, Verkarre V, Nalpas A, Dhalluin-Venier V, et al. FIB-4: an
inexpensive and accurate marker of fibrosis in HCV infection. comparison with liver biopsy and fibrotest.
Hepatology 2007;46(1):32–6.
Valujskikh A, Pantenburg B, Heeger PS. Primed allospecific T cells prevent the effects of
costimulatoryblockade on prolonged cardiac allograft survival in mice. Am J Transplant 2002;2:501–509.
van der Heide F, Dijkstra G, Porte RJ, Kleibeuker JH, Haagsma EB. Smoking behavior in liver transplant
recipients. Liver Transpl 2009;15:648-655.
Van der Hoeven JA, Lindell S, van Schilfgaarde R, Molema G, Ter Horst GJ, Southard JH, Ploeg RJ.
Donor brain death reduces survival after transplantation in rat livers preserved for 20 hr. Transplantation
2001;72:1632–1636.
Van Der Hoeven JA, Moshage H, Schuurs T, et al. Brain death induces apoptosis in donor liver of the rat.
Transplantation 2003;76:1150-4.
Van der Hoeven JA, Ploeg RJ, Postema F, et al. Induction of organ dysfunction and up-regulation of
inflammatory markers in the liver and kidneys of hypotensive brain dead rats: a model to study marginal
organ donors. Transplantation 1999;68:1884-90.
Van Der Hoeven JA, Ter Horst GJ, Molema G, de Vos P, Girbes AR, Postema F, et al. Effects of brain
death and hemodynamic status on function and immunologic activation of the potential donor liver in the
rat. Ann Surg 2000;232:804–813.
van der Hoeven JA, Ter Horst GJ, Molema G, et al. Effects of brain death and hemodynamic status on
function and immunologic activation of the potential donor liver in the rat. Ann Surg 2000;232:804-13.
van’t Hoff W, McKiernan PJ, Surtees RA, Leonard JV. Liver transplantation for methylmalonic acidaemia.
Eur J Pediatr 1999;158(Suppl 2): S70–S74.
Vanderlugt CL, Miller SD. Epitope spreading in immune-mediated diseases: implications for
immunotherapy. Nat Rev Immunol 2002;2:85–95.
Vargas HE, Laskus T, Wang LF, Lee R, Radkowski M, Dodson F, et al. Outcome of liver transplantation
in hepatitis C virus– infected patients who received hepatitis C virus–infected grafts. Gastroenterology
1999;117:149-153.
Vargas-Tank L, Martinez V, Jiron MI, et al. Tru-cut and Menghini needles: different yield in the histological
diagnosis of liver disease. Liver 1985;5:178–181.
Vassiliadis T, Giouleme O, Koumerkeridis G, et al. Adefovir dipivoxil plus lamivudine combination
treatment is superior to adefovir dipivoxil monotherapy in lamivudine-resistant hepatitis B e antigennegative chronic hepatitis B patients. Hepatology 2007;46:662A.
Vaziri, H. & Benchimol, S. (1998) Reconstitution of telomerase activity in normal human cells leads to
elongation of telomeres and extended replicative life span. Current Biol. Vol. 8, No. 5, pp. 279-282
Veldt BJ, Laine F, Guillygomarc’h A, Lauvin L, Boudjema K, Messner M, Brissot P, Deugnier Y, Moirand
R. Indication of liver transplantation in severe alcoholic liver cirrhosis: quantitative evaluation and optimal
timing. J Hepatol 2002;36:93-98.
Velidedeoglu E, Desai NM, Campos L, Olthoff KM, Shaked A, Nunes F, et al. The outcome of liver grafts
procured from hepatitis C-positive donors. Transplantation 2002;73:582-587.
Velidedeoglu E, Mange KC, Frank A, et al. Factors differentially correlated with the outcome of liver
transplantation in HCV + and HCV- recipients. Transplantation 2004;77:1834–1842
Vennarecci G, Gunson BK, Ismail T, et al. Transplantation for end stage liver disease related to alpha 1
antitrypsin. Transplantation 1996;61:1488–1495.
Vera A, Moledina S, Gunson B, et al. Risk factors for recurrence of primary sclerosing cholangitis of liver
allograft. Lancet 2002;360(9349):1943–4.
Verdonk RC, Buis CI, Porte RJ, et al. Anastomotic biliary strictures after liver transplantation: causes and
consequences. Liver Transpl 2006;12:726–35.
Vergani D, Mieli-Vergani G. Mechanisms of autoimmune hepatitis. Pediatr Transplant 2004;8:589–93.
384
References
Verran D, Kusyk T, Painter D, Fisher J, Koorey D, Strasser S, et al. Clinical experience gained from the
use of 120 steatotic donor livers for orthotopic liver transplantation. Liver Transpl 2003;9:500–505.
Viemann D, Strey A, Janning A, et al. Myeloid-related proteins 8 and 14 induce a specific inflammatory
response in human microvascular endothelial cells. Blood 2005;105:2955-62.
Vignot S, Faivre S, Aguirre D, et al. mTOR-targeted therapy of cancer with rapamycin derivatives. Ann
Oncol 2005;16:525–37.
Vilca Melendez H, Rela M, Murphy G, Heaton N. Assessment of graft function before liver transplantation:
quest for the lost ark? Transplantation 2000;70:560–565
Vivarelli et al, 2004. Vivarelli M, et al: Ischemic arterial complications after liver transplantation in the
adult: multivariate analysis of risk factors. Arch Surg 2004; 139:1069-1074.
Vivarelli et al, 2007. Vivarelli M, et al: Can antiplatelet prophylaxis reduce the incidence of hepatic artery
thrombosis after liver transplantation?. Liver Transpl 2007; 13:651-654
Vivarelli M, Dazzi A, Cucchetti A, Gasbarrini A, Zanello M, Di Gioia P, Bianchi G, Tamè MR, Gaudio MD,
Ravaioli M, Cescon M, Grazi GL, Pinna AD. Sirolimus in liver transplant recipients: a large single-center
experience. Transplant Proc. 2010 Sep;42(7):2579-84.
Vleirberghe H, Troisi R, Colle I, et al. Hepatitis C infection-related liver disease: patterns of recurrence
and outcome in cadaveric and living donor liver transplantation in adults. Transplantation 2004;77:210–4.
Vu MD, Clarkson MR, Yagita H, et al. Critical, but conditional, role of OX40 in memory T cell-mediated
rejection. J Immunol 2006;176:1394–1401.
Wachs ME, Amend WJ, Ascher NL, et al. The risk of transmission of hepatitis B from HBsAg(-),
HBcAb(+),HBIgM(-) organ donors. Transplantation 1995;59:230–234
Wai CT, Greenson JK, Fontana RJ, et al. A simple noninvasive index can predict both significant fibrosis
and cirrhosis in patients with chronic hepatitis C. Hepatology 2003;38:518–526.
Wak Ki. UNOS.Liver Registry: ten year survivals Clin Transpl (2006), pp. 29–39
Wakabayashi H, Nishiyama Y, Ushiyama T, Maeba T, Maeta H. Evaluation of the effect of age on
functioning hepatocyte mass and liver blood flow using liver scintigraphy in preoperative estimations for
surgical patients: comparison with CT volumetry. J Surg Res 2002; 106: 246–253
Wali M, Harrison RF, Gow PJ, Mutimer D. Advancing donor liver age and rapid fibrosis progression
following transplantation for hepatitis C. Gut 2002; 51: 248–252
Walker NM, Stuart KA, Ryan RJ, Desai S, Saab S, Nicol JA, et al. Serum ferritin concentration predicts
mortality in patients awaiting liver transplantation. Hepatology 2010;51:1683–1691.
Wall WJ, Mimeault R, Grant DR, Bloch M. The use of older donor livers for hepatic transplantation.
Transplantation 1990; 49:377-381.
Walsh KM, Fletcher A, MacSween RN, et al. Basement membrane peptides as markers of liver disease in
chronic hepatitis C. J Hepatol 2000;32:325–330.
Walsh KM, Timms P, Campbell S, et al. Plasma levels of matrixmetalloproteinase-2 (MMP-2) and tissue
inhibitors of metalloproteinases- 1 and -2 (TIMP-1 and TIMP-2) as noninvasive markers of liver disease in
chronic hepatitis C: comparison using ROC analysis. Dig Dis Sci 1999;44:624–630.
Wan, P.; Wang, X.; Ma, P.; et al. (2011) Cell delivery with fixed amniotic membrane reconstructs corneal
epithelium in rabbits with limbal stem cell deficiency. Investigative Ophthalmology & Visual Science.
Vol.52, No.2, pp.724-30.
Wang X, Kanel GC, DeLeve LD. Support of sinusoidal endothelial cell glutathione prevents hepatic venoocclusive disease in the rat. Hepatology 2000;31:428–34.
Wang YW, Huo TI, Yang YY, Hou MC, Lee PC, Lin HC, Lee FY, Chi CW, Lee SD. Correlation and
comparison of the model for end-stage liver disease, portal pressure, and serum sodium for outcome
prediction in patients with liver cirrhosis. J Clin Gastroentrol 2007; 41:706-712
Wanless IR, Nakashima E, Sherman M. Regression of human cirrhosis. Morphologic features and the
genesis of incomplete septal cirrhosis. Arch Pathol Lab Med 2000; 124: 1599–607.
Wannamethee SG, Shaper AG, Lennon L, et al. Hepatic enzymes, the metabolic syndrome, and the risk
of type 2 diabetes in older men. Diabetes Care. 2005; 28: 2913–2918.
385
References
Washburn WK, Johnson LB, Lewis WD, Jenkins RL. Graft function and outcome of older (> or = 60 years)
donor livers. Transplantation 1996; 61: 1062–1066
Washington K. Update on post-liver transplantation infections, malignancies, and surgical complications.
Adv Anat Pathol 2005;12:221–6.
Watschinger B. How T cells recognize alloantigen: evidence for two pathways of allorecognition. Nephrol
Dial Transplant. 1995;10(9):1556-8.
Watson CJ, Friend PJ, Jamieson NV, et al. Sirolimus: a potent new immunosuppressant for liver
transplantation. Transplantation 1999;67:505-9.
Watt KD, Burak K, Deschenes M, et al. Recurrent hepatitis C post-transplantation: where are we now and
where do we go from here? A report from the Canadian transplant hepatology workshop. Can J
Gastroenterol 2006;20:725–34.
Weber C, Rajnoch C, Loth F, et al. The microspheres based detoxification system (MDS): a new
extracorporeal blood purification procedure based on recirculated microspherical absorbent particles. Int J
Artif Organs 1994;17:595–602.
Webster KE, Walters S, Kohler RE, Mrkvan T, Boyman O, Surh CD, Grey ST, Sprent J. In vivo expansion
of T reg cells with IL-2-mAb complexes: induction of resistance to EAE and long-term acceptance of islet
allografts without immunosuppression. J Exp Med. 2009 Apr 13;206(4):751-60.
Weinrieb RM, Barnett R, Lynch KG, DePiano M, Atanda A, Olthoff KM. A matched comparison study of
medical and psychiatric complications and anesthesia and analgesia requirements in methadonemaintained liver transplant recipients. Liver Transpl 2004;10:97-106.
Weismuller TJ, Prokein J, Becker T, Barg-Hock H, Klempnauer J, Manns MP, et al. Prediction of survival
after liver transplantation by pre-transplant parameters. Scand J Gastroenterol 2008;43:736–746.
Weiss S, Kotsch K, Francuski M, et al. Brain death activates donor organs and is associated with a worse
I/R injury after liver transplantation. Am J Transpl 2007;7:1584-93.
Wenham PR, Horn DB, Smith AF. In vitro studies upon the release of gamma-glutamyltransferase from
human liver. Clin Chim Acta 1986;160:223-33.
Wente MN, Sauer P, Mehrabi A, Weitz J, Buchler MW, Schmidt J, Schemmer P (2006) Review of the
clinical experience with a modified release form of tacrolimus [FK506E (MR4)] in transplantation. Clin
Transplant 20(Suppl 17):80–84
Whitfield JB. Gamma glutamyl transferase. Crit Rev Clin Lab Sci 2001;38:263-355.
Wiesner R, Edwards E, Freeman R, Harper A, Kim R, Kamath P, et al. Model for end-stage liver disease
(MELD) and allocation of donor livers. Gastroenterology 2003;124:91-96.
Wiesner R, Rabkin J, Klintmalm G, McDiarmid S, Langnas A, Punch J, McMaster P, Kalayoglu M, Levy
G, Freeman R, Bismuth H, Neuhaus P, Mamelok R, Wang W (2001) A randomized double-blind
comparative study of mycophenolate mofetil and azathioprine in combination with cyclosporine and
corticosteroids in primary liver transplant recipients. Liver Transpl 7(5):442–450
Wiesner R, Rakela J, Ishitani M, Mulligan D, Spivey J, Steers J, et al. Recent advances in liver
transplantation. Mayo Clinic Proceedings 2003; 78: 197-210.
Wiesner RH, Batts KP, Krom RA. Evolving concepts in the diagnosis, pathogenesis, and treatment of
chronic hepatic allograft rejection. Liver Transpl Surg 1999;5:388–400.
Wiesner RH, Freeman RB, Mulligan DC. Liver transplantation for hepatocellular cancer; the impact of the
MELD allocation policy. Gastroenterology 2004; 127(suppl. 1): S261-7
Wiesner RH, McDiarmid SV, Kamath PS, Edwards EB, Malinchoc M, Kremers WK, et al. MELD and
PELD: application of survival models to liver allocation. Liver Transpl 2001;7:567-580.
Wiesner RH, Sorrell M, Villamil F, et al. Report of the first international liver transplantation society expert
panel consensus conference on liver transplantation and hepatitis C. Liver Transpl 2003;11(Suppl 3):S1–
S9.
Wijdicks EFM, Varelas PN, Gronseth GS, et al. Evidence-based guideline update: determining brain
death in adults: report of the quality standards subcommittee of the American Academy of Neurology.
Neurology 2010;74:1911-8.
386
References
Wilhelm MJ, Pratschke J, Beato F, et al. Activation of the heart by donor brain death accelerates acute
rejection after transplantation.Circulation 2000;102:2426-33.
Williams K, Lewis JF, Davis G, Geiser EA. Dobutamine stress echocardiography in patients undergoing
liver transplantation evaluation. Transplantation 2000;69:2354-2356.
Wilms C, Walter J, Kaptein M, et al. Long-term outcome of split liver transplantation using right extended
grafts in adulthood: a matched pair analysis. Ann Surg 2006; 244:865–72.
Wilmut, I.; Schnieke, A.E.; McWhir, J.; et al. (1997). Viable offspring derived from fetal and adult
mammalian cells. Nature. Vol. 385, pp. 810-813.
Wisconsin experience with liver transplantation. Ann Surg 2005;242:724–31.
Wisecarver JL, Earl RA, Haven MC, et al. Histologic changes in liver allograft biopsies associated with
elevated whole blood and tissue cyclosporine concentrations. Mod Pathol 1992;5:611–6.
Woo et al, 2007. Woo DH, et al: Management of portal venous complications after liver transplantation.
Tech Vasc Interv Radiol 2007; 10:233-239.
Woodhouse, K., Wynne, H.A., 1992. Age-related changes in hepatic function. Implications for drug
therapy. Drugs and Aging 2, 243–255.
Wu Z, Bensinger SJ, Zhang J, et al. Homeostatic proliferation is a barrier to transplantation tolerance. Nat
Med 2004;10: 87–92.
Wynne, H., Cope, L., Mutch, E., Rawlins, M., Woodhouse, K., James, O.F.W., 1989. The effect of age
upon liver volume and apparent liver blood flow in healthy man. Hepatology 9, 297–301.
Yagi, H.; Parekkadan, B.; Suganuma, K.; et al. (2009) Long-term superior performance of a stem
cell/hepatocyte device for the treatment of acute liver failure. Tissue Eng Part A. Vol. 1, No. 11, pp. 337788
Yang XO, Nurieva R, Martinez GJ, Kang HS, Chung Y, Pappu BP, Shah B, Chang SH, Schluns KS,
Watowich SS, Feng XH, Jetten AM, Dong C. Molecular antagonism and plasticity of regulatory and
inflammatory T cell programs. Immunity. 2008 Jul 18;29(1):44-56.
Yang YJ, Chen DZ, Li LX, Kou JT, Lang R, Jin ZK, Han DD, He Q. Sirolimus-based immunosuppressive
therapy in liver transplant recipient with tacrolimus-related chronic renal insufficiency. 2008
Jun;40(5):1541-4.
Yao F. Y, Ferrell L, Bass NM, et al. Liver transplantation for hepatocellular carcinoma: expansion of the
tumor size limits does not adversely impact survival. Hepatology 2001; 33(6):1394–1403.
Yao FY, Bass NM, Nikolai B, et al. Liver transplantation for hepatocellular carcinoma: analysis of survival
according to the intention-to-treat principle and dropout from the waiting list. Liver Transpl 2002; 8: 87383.
Yao FY, Saab S, Bass NM, Hirose R, Ly D, Terrault N, et al. Prediction of survival after liver
retransplantation for late graft failure based on preoperative prognostic scores. HEPATOLOGY 2004;
Yao FY, Xiao L, Bass NM, Kerlan R, Ascher NL, Roberts JP. Liver transplantation for hepatocellular
carcinoma: validation of the UCSF-expanded criteria based on preoperative imaging. Am. J. Transplant.
2007; 7: 2587–96.
Yazumi S, Chiba T. Biliary complications after a right-lobe living donor liver transplantation. J
Gastroenterol 2005;40:861–5.
Yeager, A.M.; Singer, H.S.; Buck, J.R.; et al. (1985) A therapeutic trial of amniotic epithelial cell
implantation in patients with lysosomal storage diseases. Am. J. Medical Genetics. Vol. 22, pp. 347-355
Yerdel MA, Gunson B, Mirza D, Karayalcin K, Olliff S, Buckels J, et al. Portal vein thrombosis in adults
undergoing liver transplantation: risk factors, screening, management, and outcome. Transplantation
2000;69: 1873-1881.
Yersiz H, Cameron AM, Carmody I, et al. Split liver transplantation. Transplant Proc 2006; 38:602–3.
Yersiz H, Shaked A, Olthoff K, et al. Correlation betweendonor age and the pattern of liver graft recovery
after liver transplantation. Transplantation 1995;60:790–794
Yilmaz F, Aydin U, Nart D, et al. The incidence and management of acute and chronic rejection after
living donor liver transplantation. Transplant Proc 2006;38:1435–7.
387
References
Yilmaz N, Shiffman ML. Impact of the donor liver with steatosis in patients with hepatitis C virus: not so
Fast. Liver Transpl 2009;15:4–6.
Yoo HY, Edwin D, Thuluvath PJ. Relationship of the model for end-stage liver disease (MELD) scale to
hepatic encephalopathy, as defined by electroencephalography and neuropsychometric testing, and
ascites. Am J Gastroenterol 2003; 98: 1395.
Yoo HY, Maheshwari A, Thuluvath PJ. Retransplantation of liver: primary graft nonfunction and hepatitis
C virus are associated with worse outcome. Liver Transpl 2003;9:897-904.
Yorifuji T, Muroi J, Uematsu A, Nakahata T, Egawa H, Tanaka K. Living-related liver transplantation for
neonatal-onset propionic acidemia. J Pediatr 2000;137:572-574.
Yoshizawa A, Takada Y, Fujimoto Y, Koshiba T, Haga H, Nabeshima S, Uemoto S. Liver transplantation
from an identical twin without immunosuppression, with early recurrence of hepatitis C. Am J Transplant.
2006 Nov;6(11):2812-6.
Yoshizumi, T.; Taketomi, A.; Soejima, Y.; Uchiyama, H.; Ikegami, T.; Harada, N.; Kayashima, H.;
Yamashita, Y.; Shimada, M. & Maehara, Y. (2008) Impact of donor age and recipient status on left-lobe
graft for living donor adult liver transplantation. Transplant International, Vol.21, No.1, (January 2008), pp.
81-88,
Youngner SJ, Arnold RM. Ethical, psychosocial, and public policy implications of procuring organs from
non-heart-beating cadaver donors. JAMA 1993;269:2769–2774.
Yu, J.; Vodyanik, M.A.; Smuga-Otto, K.; et al. (2007). Induced pluripotent stem cell lines derived from
human somatic cells. Science. Vol. 318, pp. 1917-1920.
Yusoff IF, House AK, De Boer WB, et al. Disease recurrence after liver transplantation in Western
Australia. J Gastroenterol Hepatol 2002;17:203–7.
Zaman A, Rosen HR, Ingram K, et al. Assessment of FIBROSpect II to detect hepatic fibrosis in chronic
hepatitis C patients. Am J Med 2007;120:280.
Zaret, K.S. & Grompe, M. (2008) Generation and regeneration of cells of the liver and pancreas. Science.
Vol. 322, No. 5907, pp. 1490-1494
Zeeh J, Platt D. The aging liver: structural and functional changes and their consequences for drug
treatment in old age. Gerontology 2002; 48: 121–127.
Zeeh, J., Platt, D., 1990. Alternsvera¨nderungen der Leber: Konsequenzen fu¨ r die Arzneimitteltherapie.
Fortschr. Med. 108, 651–653 (in German).
Zekry A, Bishop GA, Bowen DG, et al. Intrahepatic cytokine profiles associated with posttransplantation
hepatitis C virus-related liver injury. Liver Transpl 2002;8:292–301.
Zeng MD, Lu LG, Mao YM, Qiu DK, Li JQ, Wan MB, et al. Prediction of significant fibrosis in HBeAgpositive patients with chronic hepatitis B by a noninvasive model. Hepatology 2005;42(6):1437–45.
Zetterman RK, Belle SH, Hoofnagle JH, Lawlor S, Wei Y, Everhart J, et al. Age and liver transplantation:
a report of the Liver Transplantation Database. Transplantation 1998;66:500-506.
Zhai Y, Meng L, Gao F, Busuttil RW, Kupiec-Weglinski JW (2002) Allograft rejection by primed/memory
CD8+ T cells is CD154 blockade resistant: therapeutic implications for sensitized transplant recipients. J
Immunol 169:4667–4673.
Zhang K, Tung B, Kowdley K. Liver transplantation for metabolic liver diseases. Clin Liver Dis
2007;11:265–281.
Zhang, S.; Chen, S.; Li, W.; et al. (2011) Rescue of ATP7B function in hepatocyte-like cells from Wilson’s
disease induced pluripotent stem cells using gene therapy or the chaperon drug curcumin. Human Mol.
Genetics. Vol. 20, No. 16, pp. 3176-3187
Zhao, H.X.; Li, Y.; Jin, H.F.; et al. (2010). Rapid and efficient reprogramming of human amnion-derived
cells into pluripotency by three factors OCT4/SOX2/NANOG. Differentiation. Vol. 80, pp. 123-129.
Zheng M, Cai WM, Zhao JK, et al. Determination of serum levels of YKL-40 and hyaluronic acid in
patients with hepatic fibrosis due to schistosomiasis japonica and appraisal of their clinical value. Acta
Trop 2005;96:148 –152.
388
References
Zheng XX, Markees TG, Hancock WW, et al. CTLA4 signals are required to optimally induce allograft
tolerance with combined donor-specific transfusion and anti-CD154 monoclonal antibody treatment. J
Immunol 1999;162:4983–4990.
Zhong Z, Connor H, Mason RP, Qu W, Stachlewitz RF, Gao W, Lemasters JJ, Thurman RG. (1996).
Destruction of Kupffer cells increases survival and reduces graft injury after transplantation of fatty livers
from ethanol-treated rats. Liver Transpl Surg 2:383–387.
Zhou, P.; Hohm, S.; Olusanya, Y.; et al. (2009) Human progenitor cells with high aldehyde
dehydrogenase activity efficiently engraft into damaged liver in a novel model. Hepatology. Vol. 49, No. 6,
pp. 1992-2000
Zhu ZJ, Rao W, Zheng H, et al. Analysis of survival rate and risk factors of liver retransplantation.
ZhonghuaWai Ke Za Zhi 2007;45:1012–4.
Ziarkiewicz-Wroblewska B, Wroblewski T, Wasiutynski A. Morphological features and differential
diagnosis of hepatitis C recurrence after liver transplantation- literature review and results of single
transplantation center. Ann Transplant 2008;13:12–20.
Zibari G, Lipka J, Zizzi A, Abreo K, Jacobbi L, McDonald J. The use of contaminated donor organs in
transplantation. Clin Transpl 2000; 14: 397–400
Zimmerman MA, Ghobrial RM, Tong MJ, et al. Recurrence of hepatocellular carcinoma following liver
transplantation: a review of preoperative and postoperative prognostic indicators. Arch Surg
2008;143:182–8; discussion 188.
Zimmerman MA, Trotter JF, Wachs M, et al. Sirolimus-based immunosuppression following liver
transplantation for hepatocellular carcinoma. Liver Transpl 2008;14(5):633-8.
Zimmermann, S.; Voss, M.; Kaiser, S.; et al. (2003) Lack of telomerase activity in human mesenchymal
stem cells. Leukemia. Vol. 17, pp. 1146-1149
Ziolkowski J, Paczek L, Senatorski G, et al. Renal function after liver transplantation: calcineurin inhibitor
nephrotoxicity. Transplant Proc 2003;35(6):2307-9.
Zitelli BJ, Gartner JC, Malatack JJ, et al. Pediatric liver transplantation: patient evaluation and selection,
infectious complications, and life-style after transplantation. Transplant Proc 1987;19:3309–3316
Zoli, M., Magalotti, D., Bianchi, G., Gueli, C., Orlandini, C., Grimaldi, M., Marchesini, G., 1999. Total and
functional hepatic blood flow decrease in parallel with aging. Age and Ageing 28, 29–33.
Zweers N, Petersen AH, van der Hoeven JA, et al. Donor brain death aggravates chronic rejection after
lung transplantation in rats. Transplantation 2004;78:1251-8.
389
References
390
Acknowledgements
Acknowledgements
I would like to thank every person who cooperated with me during the realization of my
PhD project, in particular:
- the medical and nursing staff of the “UO di Chirurgia Epatica e del Trapianto di
Fegato” for the collaboration in the collection of biological samples;
- Prof. Daniela Campani, of the “SOD Anatomia Patologica Sperimentale” for being
available for the histological analysis;
- dr. Maria Franzini, of Scuola Superiore Sant'anna, for having made available the
instrumentation for fractional GGT analysis;
- dr. Vanna Fierabracci, who followed me during all the project helping me in the
management and analysis of data
- Mrs Anna Maida, unique help for everything concerning administrative aspects.
391