General aspects of liver transplantation 881
Historical overview and survival following liver transplantation 881
Indications for liver transplantation 881
Complications of liver transplantation 882
Pathological assessments in liver transplantation 882
Examination of native hepatectomy specimens 883
Pathological changes in post-reperfusion biopsies of donor liver and preservation/reperfusion injury 884
Pre-existing donor lesions 884
Preservation/reperfusion injury 886
Reduced-size grafts and small-for-size syndrome 888
Liver allograft rejection 888
Definition and classification of rejection 888
Antibody-mediated rejection 888
Acute cellular rejection 892
Chronic rejection 897
Relationship between acute and chronic rejection 902
Immunopathogenesis of acute and chronic rejection 902
Response to rejection-induced liver injury 904
Graft tolerance 904
General aspects 905
Opportunistic viral infections 905
Bacterial infections 909
Fungal and parasitic infections 910
Vascular problems 910
Biliary complications 911
Disease recurrence 913
General aspects 913
Recurrent viral infection 913
Recurrent autoimmune disease 923
Recurrent fatty liver disease 925
Hepatic neoplasms 925
Other recurrent diseases 927
De novo disease 928
Acquired viral hepatitis 928
De novo autoimmune hepatitis (plasma cell hepatitis, plasma cell-rich rejection) 928
Nonalcoholic fatty liver disease 929
De novo neoplasia 930
Other de novo diseases 930
Other histological findings in post-transplant biopsies 930
Unexplained (‘idiopathic’) chronic hepatitis (idiopathic post-transplant hepatitis) 930
Other causes of unexplained graft fibrosis 932
Hepatic structural abnormalities 933
Drug toxicity in liver allograft recipients 933
Liver disease after haematopoietic cell (bone marrow and peripheral stem cell) transplantation 934
General aspects 934
Graft-versus-host disease 934
Hepatitis B and C infections 935
Iron overload 936
General aspects of liver transplantation
Historical overview and survival following liver transplantation
The first successful human liver transplant was performed in the United States in 1967, with the first European case the following year. The two pioneering surgeons, Thomas Starzl and Roy Calne, were awarded the Lasker-DeBakey Clinical Research Award in 2012 for developing this intervention that now saves more than 20,000 lives each year. Results were poor during the first decade of clinical liver transplantation, with fewer than 30% of patients surviving longer than 1 year. Transplant activity during this period was confined to a small number of centres. Subsequent improvements in preservation of donor organs, surgical technique and immunosuppressive drug therapy have greatly improved the outcome following liver transplantation. Current survival figures exceed 80%, 70%, 65% and 50% at 1, 5, 10 and 20 years, respectively. The improved outcome for liver allograft recipients during the last 20 years is largely related to a reduction in complications during the first 6–12 months after transplant. As a consequence, there has been a steady increase in transplant activity worldwide, with almost 24,000 transplants performed in 2012.
After an exponential increase in the number of transplant operations during the 1980s and early 1990s, the rate of increase subsequently slowed down, principally due to a lack of donor organs. This has led to extending the use of cadaveric organs, including splitting livers for use in two recipients (usually one adult, one paediatric) and using donor livers previously considered unsuitable, also referred to as ‘marginal’ or ‘extended-criteria donor’ (ECD) grafts. Use of living-related liver transplantation (LRLT) has also increased, mainly in regions where there are problems with obtaining cadaveric organs (e.g. Asia, Middle East), although LRLT also accounts for a small proportion of transplants (<5%) in Europe and North America. The use of alternative transplant strategies is associated with increased complications compared with standard liver transplant operations. Examples include a higher rate of ‘initial poor function’ or ‘primary nonfunction’ in recipients of marginal grafts (e.g. livers with increased steatosis or prolonged cold ischaemia) and an increased risk of biliary and vascular complications in reduced-size and marginal grafts.
Indications for liver transplantation
Liver transplantation (LT) is now well-established as a treatment for many otherwise incurable liver diseases. The indications for LT can be divided into three main groups: end-stage chronic liver disease, acute liver failure and hepatic neoplasms ( Table 14.1 ).
|Indication||Cases (%)||Of these cases (%)|
|Chronic liver disease||67|
|Viral + Alcohol||3|
|Primary cholestatic diseases||9|
|Acute liver failure *||7|
The most common indication for LT is end-stage chronic liver disease, which accounts for approximately 70% of all transplant operations. Within this large group, the relative proportions of specific disease types vary from centre to centre. In countries such as the United Kingdom, primary biliary cirrhosis (cholangitis) has a high prevalence and has been among the most frequent indications for LT but is now waning. In many other centres worldwide, chronic hepatitis C virus (HCV) infection is now the most common indication for LT, with alcoholic liver disease (ALD) and malignancy, mainly hepatocellular carcinoma (HCC), also being frequent indications. LT for nonalcoholic fatty liver disease (NAFLD) has increased almost 10-fold in the last 10 years and now accounts for up to 20% or more of liver transplants in some U.S. centres. Just as end-stage hepatitis C overtook hepatitis B as an indication when antiviral therapies for the latter became available, the development of more effective anti-HCV drugs is likely to see the proportion of transplants performed for NAFLD increase further. In the paediatric population the most common indication for LT continues to be extrahepatic biliary atresia, with cirrhosis related to metabolic diseases also a frequent indication in this group.
Approximately 10% of liver transplant operations are for acute or subacute hepatic failure. The two commonly identified causes within this group are viral agents (mainly hepatitis A and B) and drugs (mostly paracetamol/acetaminophen). A small number of cases may represent an acute presentation of autoimmune hepatitis. However, in some patients undergoing LT for acute liver failure, no obvious cause can be identified; these cases may be labelled clinically as ‘seronegative hepatitis’. Survival rates in these patients, who are often extremely ill at transplantation, have steadily improved in the past 20 years but remain lower than those observed in patients with LT for cirrhosis. Recipients >50 years old receiving grafts from donors >60 years have a high mortality rate (>50%) during the first year.
Liver transplantation has also been used in the treatment of primary hepatic neoplasms, particularly HCC. Early results for HCC were poor because of problems with disease recurrence. However, careful preoperative selection to exclude patients with a high risk of recurrence (mainly based on tumour size and number) has resulted in patients transplanted with HCC and cirrhosis having survival rates similar to those transplanted for cirrhosis alone.
In addition to metabolic diseases associated with liver damage (e.g. α1-antitrypsin deficiency, haemochromatosis, tyrosinaemia, Wilson disease), LT has also been done to correct metabolic defects in which the liver itself shows minimal or no signs of damage (e.g. urea cycle disorders, protein C deficiency, familial hypercholesterolaemia, type 1 hyperoxaluria, Crigler–Najjar syndrome, familial amyloid polyneuropathy).
Approximately 5–10% of patients who undergo LT require retransplantation for graft failure related to complications of LT ( Table 14.2 ). About 1% undergo two or more retransplant operations. Compared with primary LT, the overall graft and patient survival tends to diminish with successive retransplant operations. Among 5596 retransplant operations reported to the European Liver Transplant Registry from 1988 to 2009, the five main indications were vascular complications (27%), primary nonfunction (25%), rejection (19%), recurrent disease (11%) and biliary complications (10%). The overall rate of retransplantation has declined, mainly because of a reduction in the frequency of rejection and surgical complications.
|Indication||Frequency||Timing after transplantation|
|Primary nonfunction||Up to 5–10%||First few days|
|Graft ischaemia/infarction||Up to 5%||First month|
|Massive haemorrhagic necrosis||Now very rare (≪1%)||1–3 weeks|
|Biliary complications (ischaemic cholangiopathy)||Uncommon (<1%)||1–6 months|
|Chronic rejection||Incidence declining (now <2%)||Typically during first 12 months. Late cases (>12 months) becoming more common and may have different histological features|
|Hepatitis C virus||Increasingly frequent; most common indication for late retransplantation in recent years. Likely to become less frequent with advent of new antiviral drugs.||From 2–3 years|
|Hepatitis B virus||Now very rare (due to prophylactic treatment)||From 2–3 years|
|PBC||Very rare (<1%)||Late (>10 years)|
|Autoimmune hepatitis||Rare||>1 year|
|NASH||Very rare||From 2–3 years|
|Other late complications|
|De novo autoimmune hepatitis||Very rare||>1 year|
|‘Idiopathic’ chronic hepatitis||Very rare||>5 years|
Complications of liver transplantation
The main complications of LT include the following:
Problems with the preservation and reperfusion of the donor organ (preservation/reperfusion injury)
Technical/surgical complications involving vascular and/or biliary structures
Complications of immunosuppressive therapy (e.g. opportunistic infections, post-transplant lymphoproliferative diseases, other solid malignancies, drug toxicity)
Recurrence of the original disease for which transplantation was performed
Acquired liver disease (e.g. ‘ de novo autoimmune hepatitis’ or fatty liver disease)
Many of the complications just listed result in morphological changes within the liver allograft itself, as discussed in this chapter. Complications also frequently involve other organs. Important examples include cardiovascular disease and chronic renal failure, both of which occur as complications of long-term immunosuppression.
Pathological assessments in liver transplantation
Histopathological assessments have an important role at all stages in the management of patients undergoing LT. The starting point is an examination of the native (host) liver removed at transplantation. The protocol used for obtaining post-transplant biopsies varies from centre to centre. In many centres a biopsy of the donor liver is done immediately after reperfusion. This ‘time zero’ biopsy is used as a baseline assessment to detect pre-existing disease in the donor liver and to identify changes related to organ preservation and reperfusion. Until the mid-1990s, protocol biopsies were frequently taken on or around day 7 post-transplantation. This was done because the end of the first week was recognized as the time when morphological changes of acute cellular rejection were generally first manifest. However, the discovery that histological features of rejection are often present in patients with stable graft function, and that such cases do not require additional immunosuppression, has led to protocol day 7 biopsies being discontinued in most centres. In some centres, protocol biopsies are also obtained in long-term survivors as part of an annual review. These specimens frequently show histological abnormalities, even in people who are clinically well with normal or near-normal liver biochemistry. However, uncertainty about the clinical significance and therapeutic implications of such findings has led most transplant units to discontinue the practice of obtaining protocol biopsies.
For some conditions where liver biopsy is taken to investigate graft dysfunction, histology can be regarded as the gold standard for diagnosis. The best example is liver allograft rejection, for which no other reliable diagnostic marker currently exists. For other conditions (e.g. hepatitis C infection), a likely cause of graft dysfunction may have been identified using other methods, but liver biopsy provides important additional information regarding morphological changes in the liver (e.g. severity of necroinflammatory activity and fibrosis) and may point to the presence of other, coexisting causes of graft dysfunction. In some patients, liver biopsy may provide the first clue to a biliary or vascular problem, which is subsequently confirmed radiologically. Fine-needle aspiration (FNA) cytology has also been used as an adjunct to liver biopsy in the postoperative assessment of liver allograft recipients but is not used routinely in most major transplant centres. Table 14.3 summarizes the main pathological changes that may be seen at different times after LT.
|‘Time-zero’ (post-reperfusion)||Pre-existing donor disease||Macrovesicular steatosis|
|Preservation/reperfusion injury||Changes generally mild at this stage|
|First month||Rejection||Hyperacute (very rare)|
|Opportunistic infection||CMV hepatitis (other organisms rarely seen in liver biopsy specimens)|
|Recurrent disease||Hepatitis B and C|
|>12 months||Recurrent disease||Hepatitis C (common)|
|PBC, autoimmune hepatitis, PSC (less common)|
|Others, e.g. alcohol, hepatitis B (uncommon)|
|‘Idiopathic’ chronic hepatitis|
|Rejection||Acute and chronic rejection both rare. May have different histological features to cases presenting earlier|
Examination of native hepatectomy specimens
The explant should be sampled according to a protocol, which includes sections from both lobes, subcapsular and deep parenchyma, left and right hilar regions perpendicular to the major hilar structures and any focal lesions or abnormalities. In many cases, examination of hepatectomy specimens obtained at LT confirms previous histological diagnoses. An important exception relates to focal hepatocellular lesions in cirrhotic livers, the great majority of which are diagnosed radiologically before transplantation without the use of liver biopsy. For occasional cases of acute or chronic liver disease, particularly when LT is done without prior liver biopsy, examination of the hepatectomy specimen also leads to a change in diagnosis. Examples where a pretransplant diagnosis is subsequently revised include patients presenting with severe portal hypertension attributed to cirrhosis found to have intrahepatic noncirrhotic portal hypertension and cases of acute liver failure from neoplastic hepatic infiltration mistakenly diagnosed as severe acute hepatitis. The presence of α1-antitrypsin (AAT) globules or unusually prominent hepatocellular siderosis in cirrhotic livers may reveal previously unsuspected metabolic disorders, which have probably acted as cofactors in the pathogenesis of chronic liver disease.
The opportunity to examine entire livers in a well-preserved state has provided a better understanding of the distribution of diseases within the liver as a whole. For many chronic liver diseases, particularly those associated with bile duct loss, the severity of fibrosis may be extremely variable, and it is possible to see areas of advanced cirrhosis alongside areas where normal architecture is still clearly retained. Examples include primary biliary cirrhosis/cholangitis (PBC), primary sclerosing cholangitis (PSC), biliary atresia and liver disease related to cystic fibrosis ( Fig. 14.1 ). These observations have raised concerns regarding the accuracy of traditional histological staging systems for PBC and PSC in needle biopsy specimens. More recently described staging systems which incorporate other features related to disease progression, such as ductopenia and copper-associated protein deposition, may offer advantages in this respect (see Chapter 9 ).
Another example of patchy disease distribution occurs in cases of fulminant hepatic failure associated with submassive hepatic necrosis. Large areas of panacinar necrosis may be seen alongside areas of nodular regeneration in which there is often pronounced cholestasis. A liver biopsy taken from an area of panacinar necrosis may overestimate the severity of disease present in the liver as a whole, whereas a biopsy taken from a cholestatic regeneration nodule may provide no clues to either the nature or the severity of liver injury present. For some liver diseases the removal of the whole liver allows the study of larger biliary or vascular structures, which would not normally be sampled in needle biopsy specimens. Examples include PSC, in which a spectrum of bile duct lesions affecting ducts of all sizes can be seen, and Budd–Chiari syndrome, in which lesions can be seen in hepatic vein branches of varying sizes.
Transplantation for HCC requires confirmation of the pretransplant radiological diagnosis and staging. Studies correlating findings in hepatectomy specimens with pretransplant radiological diagnoses have shown that up to 44% of imaging studies may overdiagnose or underdiagnose the extent of HCC. A recent study of 4500 hepatectomy specimens obtained from patients with suspected HCC found that radiological assessments under- and overestimated the final pathological stage in approximately equal proportion of cases (22.7% and 21.5%, respectively). In addition to confirming the diagnosis of HCC, histological examination can also be used to assess the efficacy of pretransplant treatment (e.g. extent of necrosis in nodules treated by radiofrequency ablation) and to identify additional prognostic features such as histological grade and microscopic vascular invasion that are predictive for tumour recurrence (discussed later). A variable proportion of cirrhotic livers contain previously unsuspected HCC. The incidence is highest in children with tyrosinaemia and adults with chronic HCV infection, with an overall frequency of 12–30% in three series published 10–15 years ago. The frequency of incidental HCC has declined since then, presumably because of improved imaging techniques. Most incidental HCCs are small lesions with little impact on recurrence-free survival. However, a small proportion of cases may have adverse prognostic features such as vascular invasion, and some cases of recurrent ‘incidental’ HCC have been documented. Foci of HCC have been identified in approximately 35% of patients undergoing LT for multiple hepatocellular adenomas (adenomatosis); some of these have also recurred after transplantation. The possibility of clinically undetected malignancy should also be considered in patients undergoing LT for PSC. In early studies, up to 10% of PSC livers contained previously undiagnosed cholangiocarcinoma. These tumours were usually hilar in location, often incompletely excised, and were thus associated with a high risk of recurrence. The incidence of cholangiocarcinoma (CC) incidentally discovered at LT appears to be declining. This may reflect a tendency to perform transplantation earlier, before neoplastic transformation has occurred, and the use of more sensitive imaging methods to exclude patients with PSC-associated CC who otherwise might have been transplanted. Nevertheless, in two more recent studies, 3–5% of patients undergoing LT for PSC still had previously unsuspected biliary neoplasms in their explanted livers.
Pathological changes in post-reperfusion biopsies of donor liver and preservation/reperfusion injury
Pre-existing donor lesions
With the widespread use of liver transplantation as therapy for end-stage liver disease, organ shortage has become an increasing problem in many countries, forcing the use of more marginal donor grafts. Extended-criteria donor (ECD) grafts are defined as those with poorer graft function or the potential to transmit disease to the recipient. Examples of the former include donor age >65 years, intensive care unit (ICU) stay (ventilated) >7 days, body mass index (BMI) >30 kg/m 2 , graft steatosis >40%, serum sodium >165 mmol/L, increased donor transaminases and bilirubin >3 mg/dL. Donor disease transmission can occur with the use of livers from hepatitis B (HBV) core antibody-positive or hepatitis C (HCV)-positive patients, donors with other infections or those with history of malignancy or metabolic disease such as familial amyloid polyneuropathy. More recently, use of donation after cardiac death (DCD) has increased as a further source of grafts, and although there is a reported increase of complications such as primary nonfunction and ischaemic cholangiopathy, centres with significant experience are reporting good results. Furthermore, novel techniques for graft preservation, such as normothermic and subnormothermic machine perfusion, have been shown to improve the quality and viability of marginal grafts that might otherwise be discarded. To optimize the use of more marginal grafts in the face of growing demand, several scores have been developed to quantify the risk of graft failure, including the donor risk index and the balance of risk (BAR) score.
No clear protocols exist to guide the use of donor liver biopsies before transplantation. The main indications for a pretransplant frozen section are to determine the nature of unexplained focal liver lesions identified at organ retrieval and to assess the severity of steatosis in patients with suspected fatty liver. Steatosis may be suspected because of rounded edges of the lower liver border or loss of fine surface ‘scratches’, even if parenchymal pallor is not evident. A lower threshold for frozen section is probably advisable in ECD livers, particularly if there are multiple ECD factors, as well as for DCD grafts or if the recipient is a high-risk patient.
Both micro- and macrovesicular steatosis are common findings in donor liver biopsies; analysis of the combined U.S. and European databases report a prevalence of 20% for microvesicular steatosis and 23% for macrovesicular steatosis, and others find both even more frequently. They often coexist. Macrovesicular steatosis is defined as one or more cytoplasmic fat vacuoles that are larger than the hepatocyte nucleus and that typically displace it peripherally. The vacuoles in microvesicular steatosis are smaller than the nucleus, and the nucleus remains central. Steatosis is graded as mild (5–30%), moderate (30–60%) or severe (>60%), according to the estimated percentage of liver parenchyma affected. It should be noted that the small fat droplets, which are referred to as ‘microvesicular’ in the context of assessing donor liver biopsies, are probably best regarded as a small-droplet variant of macrovesicular steatosis ; the term ‘mediovesicular steatosis’ has been proposed to describe such cases. ‘True’ microvesicular steatosis, as seen in conditions associated with defects in heptocellular β-mitochondrial oxidation, rarely occurs in this setting. Macrovesicular steatosis is the more important finding in pretransplant biopsies; implicated donor factors include age, increased BMI and alcohol consumption, although in other cases, no definite cause can be found.
Because macroscopic appearances may not be reliable in assessing the severity of steatosis, a frozen section of the donor liver is often obtained in cases where fatty change is suspected clinically. Fatty change is usually distributed fairly uniformly within the liver, and one or two needle biopsies are sufficient to grade the severity of steatosis, two being more useful if there is clinical suspicion. Although frozen-section assessments are useful and reproducible in providing an estimate of overall amount of fat, they lack sensitivity in the ‘boundary zones’ between the different grades of steatosis severity. The use of specific fat stains such as Oil Red O generally increases the amount of fat visualized, but these methods are prone to considerable technical variation. There is often a discrepancy between the severity of steatosis determined on frozen section and that seen in subsequent paraffin histology, but this discrepancy appears to have little clinical impact. Because of problems with the subjective assessment of steatosis, the use of image analysis has been suggested as providing more objective and reproducible findings, but the utility of this alternative approach in clinical practice requires further study.
Donor grafts with up to 30% macrovesicular steatosis can be safely used and represent 97% of all steatotic grafts transplanted in the United States. Moderate steatosis is often associated with graft dysfunction in the early post-transplant period, usually manifest as elevation of serum transaminases, sometimes also with prolongation of prothrombin times, and may result in reduced graft survival; however, these grafts can be used in appropriate clinical circumstances. The majority of fat is cleared by macrophages over 1–3 weeks following transplantation.
Clinically, most transplant surgeons will not use donor livers with severe fatty change. This is not universal practice, however, and some report similar long-term outcomes for severely steatotic grafts if carefully managed. These grafts have an increased risk of graft nonfunction in the immediate post-transplant period (‘primary nonfunction’), resulting in death or retransplantation within the first week. The pathogenesis is multifactorial. Hepatocytes distended with large fat droplets can obstruct the sinusoidal microvasculature, resulting in problems with perfusion of the donor liver at retrieval and subsequent reperfusion in the recipient. Steatotic hepatocytes are significantly more sensitive to oxidative injury and death, causing the release of free lipid from hepatocytes, further compromising sinusoidal blood flow and enhancing lipid peroxidation in sinusoidal endothelial cells (SECs). Other consequences of fatty change on graft function include an increased susceptibility of SECs to cold and warm ischaemic damage, depletion in glycogen content and mitochondrial abnormalities in hepatocytes, a shift to necrosis as the main mechanism for cell death after reperfusion (compared with apoptosis as the predominant pathway in nonfatty livers) and an impaired regenerative capacity after cell loss caused by preservation/reperfusion injury. Complement also plays a role in the development of ischaemia/reperfusion injury in steatotic livers. Histological studies of failed liver allografts with severe fatty change have shown large extracellular fatty aggregates associated with areas of hepatocyte necrosis, haemorrhage and disruption of the sinusoidal framework. The release of fat droplets from necrotic hepatocytes may be associated with the formation of cystic lesions resembling peliosis hepatis (‘lipopeliosis’) ( Fig. 14.2 ). However, electron microscopic analysis has shown that the free lipid is extrasinusoidal, with only more severe cases resulting in rupture into the sinusoidal space.
Microvesicular (small-droplet) steatosis is also seen frequently in donor liver biopsies. As noted earlier, ‘true’ microvesicular steatosis with innumerable tiny steatotic vacuoles filling most or all of the hepatocytes is rare and was seen in only 1% of donors in a recent series. Small fat droplets can be difficult to detect in conventional haematoxylin and eosin (H&E)-stained sections, and the incidence and severity of microvesicular steatosis increase if special stains for fat (e.g. Sudan) are used on frozen sections, but in practice this is not generally done. Although microvesicular steatosis is well recognized as a rare cause of liver failure in the nontransplanted liver (e.g. Reye syndrome), its presence in donor livers, even to a marked degree, is not generally associated with poor graft function. A recent study suggested that small-droplet steatosis does not exacerbate the effects of macrovesicular steatosis.
Donor biopsy in living-related liver transplantation
Because of cadaveric donor shortages, particularly for paediatric livers and in some regions where deceased organ donation is negligible, a number of liver transplant units are utilizing living-related liver allografts for paediatric and adult transplantation. Donor safety is of paramount importance, and careful selection of donors is thus required to minimize the risks involved with liver resection. Although donors are generally in good health, the diagnostic pretransplant workup aims to detect occult hepatic or extrahepatic disease. Most centres do not routinely biopsy all potential donors, but guidelines on when to biopsy are lacking. In studies where biopsies have been obtained from potential living donors (some routinely, others after progressing beyond initial screening process), <50% are normal. The most common abnormality is macrovesicular steatosis, generally a manifestation of NAFLD and usually associated with an increased BMI. Steatosis is present in 20–60% of patients and is typically mild in severity. About 15–30% have either moderate or severe steatosis, and 1–15% have steatohepatitis. As with cadaveric liver grafts, mild degrees of steatosis are tolerated, but donors are excluded if macrovesicular steatosis is >25% or 33%, because of the risk to both donor and recipient of compromised regeneration. Steatohepatitis is also regarded as a contraindication to donation. A number of other histological findings have been found in a smaller proportion of donor biopsies. Portal/periportal and lobular inflammatory changes, in some cases producing a chronic hepatitis-like appearance, have been observed in up to 25% of donors but are usually less common. These are typically mild in severity and have no obvious cause. Unexplained portal fibrosis of variable degree may also be seen. Other rare lesions described include occult primary liver disease (PBC, PSC, AAT deficiency), nodular regenerative hyperplasia, sinusoidal dilation, portal eosinophilia, granulomas of uncertain cause, schistosomiasis and siderosis.
Other donor factors
Older donors are increasingly being considered for use. Particularly over 60 years of age, these have an increased risk of graft loss that is most evident after transplantation for hepatitis C, but there is also an increased risk of primary nonfunction, hepatic artery thrombosis, preservation/reperfusion injury and biliary complications. Avoidance of long cold ischaemic time (<7 hr) and performance of protocol liver biopsy have been shown to improve outcomes. Conventional microscopy shows no difference between young and older liver, but there is reduced hepatocyte volume, thickening and defenestration of the SECs and an increased susceptibility to preservation/reperfusion injury.
A number of other factors may cause damage to the donor liver before its removal for transplantation, without necessarily resulting in abnormalities that are recognizable morphologically. These include episodes of hypotension (leading to ischaemic graft injury), poor nutritional state (resulting in depletion of hepatic glycogen stores) and endotoxaemia, possibly related to mucosal injury of the gut. Moderate or severe hepatocellular siderosis, presumably related to genetic haemochromatosis, has been reported as an incidental finding in a small number of donor liver biopsies. Donor HFE mutations have been demonstrated in a few cases. In most cases, siderosis diminishes after LT, but in some patients, iron stores are slow to mobilize, and occasionally, severe siderosis has persisted for years after LT, suggesting that the liver itself may be abnormal. AAT deficiency can be transmitted with a liver graft, and this becomes problematic if there is concurrent hepatitis, which accelerates globule deposition even in the MZ phenotype.
In the period from organ harvesting (or after circulatory collapse in DCD) until after implantation, a series of intragraft events cause a variable degree of damage, known as preservation/reperfusion injury (PRI) or ‘ischaemia/reperfusion injury’. Two main phases of preservation injury are recognized, targeting different cell types. Cold ischaemia, which typically lasts for several hours, principally targets SECs. Warm ischaemia, occurring during implantation, lobar division in living donors and before organ removal in DCD, has its major effect on the hepatocytes because of glycogen depletion and metabolic stress. Most of the graft injury, however, occurs after reperfusion of the liver.
Key components of reperfusion injury are the death of hepatocytes and SECs, Kupffer cell activation and formation of reactive oxygen species (ROS), followed by accelerating immune activation and further parenchymal injury. PRI may also be aggravated by donor factors such as macrovesicular steatosis, as discussed earlier. Ischaemic injury to hepatocytes and SECs results in adenosine triphosphate (ATP) depletion and death, which may result from apoptosis, necrosis or an intermediate form of cell death in which there appears to be regulated necrosis, referred to as ‘necroptosis’. Reperfusion injury subsequently induces an inflammatory cascade that is amplified by the innate and adaptive immune systems. Formation of ROS is an important early step after reoxygenation. The initial source is from Kupffer cells, which are activated within 15 min of reperfusion, with subsequent amplification from neutrophils as they are recruited. Kupffer cells also promote a proinflammatory state through the production of various cytokines and factors involved with Toll-like receptor 4 (TLR4) signalling, which bind ligands from injured tissues such as extracellular matrix (ECM) components and heat shock proteins, and exogenous lipopolysaccharide (LPS) from gut bacteria translocated by mucosal oedema related to portal vein clamping. Microvascular injury is amplified by endothelial injury with platelet aggregation and by activation of the complement system and procoagulant factors, resulting in a hypercoagulable state. Deposition of the complement fragment C4d can be demonstrated immunohistochemically in areas of hepatocyte necrosis. Extrusion of dead hepatocytes into sinusoidal spaces and loss of the ECM may also further impair sinusoidal blood flow. As leukocytes are recruited to the liver, there is upregulation of the expression of cytokines such as tumour necrosis factor (TNF) α, interferon-γ and platelet-activating factor (PAF), upregulation of cell adhesion molecules (CAMs) and chemokines involved in mediating the adhesion and transmigration of neutrophils, and induction of co-stimulatory molecules on antigen-presenting cells (APCs), both professional and nonprofessional. T lymphocytes, particularly CD4+ T cells, are recruited by similar mechanisms and have been implicated in the pathogenesis of PRI.
Histologically, the changes vary depending on the severity and time elapsed since the injury. ‘Time zero’ post-reperfusion biopsies frequently have morphological abnormalities. SECs affected by cold ischaemia show swelling, and ultrastructurally there is vacuolation of the cytoplasm, enlargement of fenestrae and blebs in sinusoidal lumen, with detachment and sloughing into the sinusoid in more severe injury. Other changes are generally mild, tending to be most marked in the centrilobular regions, and include hepatocyte ballooning, necrosis or apoptosis of single hepatocytes, neutrophil polymorph aggregates in sinusoids or around hepatocytes and cholestasis. Neutrophil polymorphs are typically seen around damaged hepatocytes and sometimes have an intracytoplasmic location. Larger areas of confluent necrosis are uncommon but may be the harbinger of evolving severe injury. Similar changes may be seen, generally to a lesser degree, in occasional biopsies obtained from donor livers before reperfusion. Given that changes seen in time zero biopsies reflect a relatively short period of liver injury, it is not surprising that changes seen at this stage are relatively mild. In many cases, however, these appear to represent the earliest stages of more severe damage that can be observed in the first few days or weeks after LT ( Fig. 14.3 ).
A recent study found that time zero biopsy grading could be performed reproducibly, after 45–60 min of reperfusion. Mild PRI was characterised by occasional hepatocyte detachment and single or rare clustered neutrophils in the sinusoids. Moderate injury showed clustered neutrophils and some hepatocyte necrosis or apoptosis, and severe injury had zonal (usually centrilobular) hepatocyte necrosis, neutrophilic infiltrates in these areas and clustered neutrophils in more distant sinusoids. The presence of severe PRI was predictive of adverse outcomes in the early post-transplant period, including an increased risk of primary nonfunction and death within the first 90 days. Hepatocyte ballooning is a common finding in the early post-transplant period. It tends to be most marked in centrilobular areas but in severe cases can be seen throughout the lobule. In most cases this lesion can be attributed to the effects of PRI. An association with high serum transaminase levels during the first 48 h after LT supports this. If ballooning persists beyond the first 2 weeks after transplant, other possible causes should be considered, particularly when associated with centrilobular necrosis.
Cholestasis is also a common finding in early post-transplant biopsies. The cholestatic changes are most often centrilobular, but in severe cases of PRI, cholangiolar cholestasis, similar to that seen in sepsis, may also be present. Cholestasis is not specific for PRI, and the many other possible causes include rejection, small-for-size syndrome, viral infection, sepsis, biliary obstruction and drug toxicity. A syndrome of ‘pure’ cholestasis, sometimes also referred to as ‘functional’ cholestasis, has been reported to occur in the absence of any obvious cause. Some of these cases probably represent a delayed manifestation of PRI, and cholestasis gradually resolves, sometimes over several weeks. Although fatty change is mainly considered to be a pre-existing donor lesion, some evidence suggests that graft ischaemia and reperfusion injury may lead to the development of steatosis in the early post-transplant period and may be microvesicular in type.
Primary graft dysfunction (PDF), initial poor function (IPF; also known as ‘early allograft dysfunction’) and primary nonfunction (PNF) are related terms used to describe grafts functioning poorly in the immediate post-transplant period. A number of donor and recipient factors have been implicated, but damage related to PRI is likely to be a major factor in most cases. These terms should not be used to describe patients with other peri- or postoperative factors, such as vascular occlusion or antibody-mediated rejection, which can also result in graft dysfunction or failure in the immediate post-transplant period. Clinical features include hyperbilirubinaemia, marked elevation of transaminases (>2000 U/L), prolonged international normalized ratio (INR) with haemostatic problems, hypoglycaemia, hyperkalaemia, metabolic acidosis and renal failure. The diagnosis is usually made in the first 24–48 h following transplantation. There are problems with establishing precise diagnostic criteria for these three syndromes, accounting for the wide variation in their reported frequency. IPF can be regarded as a less severe form with potentially reversible graft dysfunction, whereas PNF is a more severe form with graft failure incompatible with its survival. PDF has been suggested to describe all grafts that function poorly in the immediate post-transplant period (IPF and PNF). The reported incidence of PNF ranges from 2% to 23%, but recent analysis indicates <5% of transplants. Histological studies of grafts obtained at retransplantation have shown areas of coagulative hepatocyte necrosis, either centrilobular or panlobular in distribution, suggesting an ischaemic mechanism.
It has been suggested that time zero biopsies may be of prognostic value in predicting subsequent poor graft function when certain changes (apart from significant steatosis as already discussed) are present. However, schemas to grade these lesions and the relationships with graft outcomes have varied. Caution is needed when subcapsular wedge biopsies are taken as a baseline assessment because these may contain areas of zonal necrosis, possibly reflecting the susceptibility of the subcapsular region to ischaemic damage or creasing of the liver from retraction, with no apparent bearing on subsequent graft function. Recent studies suggest that the use of alternative methods such as metabolomics and genomic profiling to analyse pre- and postimplantation donor liver biopsies may also be helpful in predicting early allograft dysfunction.
In addition to direct liver injury, PRI can be involved in the pathogenesis of other post-transplant complications as well. Induction of an inflammatory state predisposes to the subsequent development of graft rejection. Prolonged cold ischaemia and PRI play a role in the pathogenesis of ischaemic bile duct injury and other complications occurring later in liver allografts.
Reduced-size grafts and small-for-size syndrome
Transplantation of a single liver lobe, either left or right, is performed in LRLT and as a means of overcoming size mismatch in paediatric patients. A cadaveric graft can be split to provide two grafts, often to a paediatric and an adult recipient. Although there is no reduction in graft or patient survival, the reduced size and extra surgical handling of split organs impose extra risks, particularly for anastomotic stenosis, with increased biliary complications, hepatic artery thrombosis and venous outflow obstruction. Reduced or variable perfusion of segments I and IV by the right hepatic artery can also be associated with segmental ischaemia in these regions. Reduced-size grafts aim to provide a graft with at least 30–40% of the expected liver volume or a graft/recipient weight ratio of >0.8%. When an insufficient liver volume is transplanted, the major complication is portal hyperperfusion or small-for-size syndrome (SFSS). Characterized by early graft dysfunction or nonfunction in a partial liver allograft with no other identifiable cause, SFSS often manifests as hyperbilirubinaemia, coagulopathy and ascites in the first postoperative week. The underlying pathogenesis of SFSS is uncertain. The most likely explanation is portal venous hyperperfusion leading to portal hypertension, which in turn results in venous endothelial damage and reflex arterial vasospasm due to the hepatic artery buffer response. Other mechanisms may also play a role, including p21-dependent inhibition of hepatocyte regeneration.
Histological changes in these grafts can be subdivided into early and late phases. The initial damage is characterized by endothelial cell denudation and swelling, resulting in haemorrhage into portal tracts, which may extend into the parenchyma in severe cases. Subsequent arterial vasospasm results in lumenal occlusion and myocyte vacuolation, parenchymal infarcts and ischaemic bile duct injury. These changes can be seen in failed allografts, but in peripheral needle biopsies the characteristic (but not specific) histological triad is centrilobular microvesicular steatosis, canalicular cholestasis and a periportal ductular reaction with mild periportal neutrophilia. Cholangiolar cholestasis may be seen. Portal venous endothelial changes are seen infrequently in biopsies, but cellular loss and vacuolation have been reported. In allografts surviving the initial insult, late complications relate to either arterial insufficiency or portal-arterial flow mismatch. The former leads to biliary strictures and biliary sludge, whereas the latter may be associated with nodular regenerative hyperplasia of variable degree and portal vein obliteration.
Liver allograft rejection
Definition and classification of rejection
Rejection can be defined as an immunological response to foreign antigens in the donor organ that has the potential to result in graft damage. Varying degrees of immune activation occur in all allograft recipients, although these are modified by immunosuppressive drugs. In the context of LT, an important distinction needs to be made between morphological changes seen in the absence of any significant clinical or biochemical abnormalities (‘biological’ or ‘subclinical’ rejection) and those accompanied by clinical signs of graft dysfunction (‘clinical’ rejection).
Three main patterns of rejection are recognized in solid-organ allografts based on the time course: ‘hyperacute rejection’, occurring immediately from preformed antibodies; ‘acute rejection’, developing quickly over a few days; and ‘chronic rejection’, evolving more gradually over weeks or longer. From a pathophysiological perspective, this subdivision has been modified as our understanding of the role of antibodies in rejection has increased, and rejection can be classified as antibody-mediated rejection (AMR), acute cellular rejection (ACR) and chronic rejection (CR). More recently, the Banff Working Group has suggested that the term ‘T cell-mediated rejection’ (TCMR) may be preferable to (acute) cellular rejection, and that TCMR can have different histological appearances depending on whether it presents early or late after LT. At present, however, the terms ACR and CR are still widely used and understood and therefore are used in this chapter.
Although initially the liver allograft was believed to be resistant to AMR because of the apparent absence of hyperacute rejection after transplantation of crossmatch-positive and ABO-mismatched grafts, it was subsequently shown that the presence of donor-specific antibodies (DSAs) could have an adverse impact on graft and patient survival through vascular complications. There has been renewed interest in this area, but currently some uncertainty remains about the exact role that antibodies play in liver allograft dysfunction. Certainly, the presence of DSAs may not be associated with any allograft dysfunction, and the nature of any injury depends on factors such as the immunoglobulin subclass, DSA quantity and ability to fix complement. When it occurs, injury may range from severe, early haemorrhagic and necrotic graft damage or acute steroid-resistant rejection to slower, less understood injury such as direct endothelial antibody binding with endothelial cell activation, intimal proliferation and fibrosis.
The tempo of injury in AMR varies, and other solid-organ allografts show hyperacute rejection, acute AMR or chronic AMR forms. In the liver allograft these patterns are less distinct, and when acute AMR occurs, it is often in association with T cell-mediated acute cellular rejection. Moreover, the exact nature of chronic AMR is still unclear and remains under investigation.
Definition and related terms
Primary AMR is caused by preformed antidonor antibodies, with the greatest and most predictable risk being with ABO-incompatible grafts. Severe cases present with very early graft failure within days ( hyperacute rejection). The liver appears to have a relative resistance to antibody-mediated injury, and often, less severe changes occur in the setting of preformed blood group or anti-HLA antibodies, presenting with early graft dysfunction in the first month ( acute AMR). DSAs directed against HLA class II antigens appear to be more strongly associated with the development of acute AMR, particularly if they have a mean fluorescence intensity (MFI) >10,000. However, the sensitivity for detecting DSAs varies from one laboratory to another, and further standardization is required. DSAs can also develop de novo after LT and have been implicated in the pathogenesis of acute and chronic rejection. De novo DSAs directed at HLA class II antigens, especially HLA-DQ, appear to be particularly important in the pathogenesis of chronic AMR.
Hyperacute rejection with rapid haemorrhagic necrosis of the allograft is distinctly rare in the liver. Further, in contemporary series it is no longer seen even in ABO-incompatible grafts with appropriate management, including rituximab administration and plasma exchange. A recent study reporting outcomes in 235 adults receiving ABO-incompatible grafts showed 3-year graft and patient survival rates of about 90%, comparable to that seen in recipients of ABO-compatible grafts. Acute AMR was the more common expression of ABO incompatibility in the 1980s, before the advent of effective immunosuppressive regimens, and caused graft failure within 30 days in about half the recipients. Currently, acute AMR is rare, probably occurring in about 5% of the 10–20% of allograft recipients with high titres of DSA and in approximately 7% of ABO-incompatible grafts, which amounts to ≤1% of liver transplants in total. However, this incidence increases to ≥10% when considering idiopathic early allograft failure before 3 months.
Chronic AMR is still an evolving concept in liver allograft pathology, so its significance remains uncertain. However, several studies in children, for whom recurrent liver disease is not a confounder, have shown the presence of late DSAs in about half of recipients and a significant association with increased graft inflammation and fibrosis in many of these. A lower frequency was found in a recent study of adult patients (20% had DSAs, with mean fluorescence intensity >5000), and this was also associated with increased graft fibrosis and graft failure after controlling for hepatitis C.
Clinical features and diagnostic criteria
AMR is of variable severity. Hyperacute AMR, now exceptionally rare due to the use of pretreatment for ABO-incompatible grafts, presents with severe graft dysfunction within the first week of transplantation. In contrast to renal allografts, where signs of hyperacute rejection are visible within a few minutes of reperfusion, changes in liver allografts may take several hours or even days to become manifest. An initial period of stable graft function is followed by a rapid rise in serum transaminases, coagulopathy and signs of acute liver failure. Decreased platelet count and total serum complement activity also occur from consumption and are indirect signs of humoral-mediated injury.
Acute AMR is more common and is characterized by unexplained graft dysfunction in the first few weeks, accompanied by falling platelet count and complement activity. Hyperbilirubinaemia is typically present, and transaminase levels may also be disproportionately high compared with the anticipated severity of PRI in the early post-transplant period. Acute AMR may manifest as severe steroid-resistant rejection, and correlation with DSA results is useful in these patients. Bortezomib, a proteasome inhibitor effective in depleting plasma cells, has been used in a small number of cases. With the increased use of effective anti-B lymphocyte pretreatment, AMR is more often milder and delayed and may be associated with late complications, including biliary strictures. The following four recommended diagnostic criteria for acute AMR include a combination of clinical, histological and laboratory findings :
Histopathological pattern of injury consistent with acute AMR
Positive serum DSAs
Presence of diffuse microvascular C4d deposition (C4d score = 3; see later)
Exclusion of other insults that might cause a similar pattern of graft injury
The clinical and biochemical features of chronic AMR are not well characterized. Many of the histological changes occurring in late biopsies that are potentially related to chronic AMR (e.g. unexplained graft inflammation and/or fibrosis) have been observed in protocol biopsies obtained from patients who appear to be clinically well and have normal liver biochemistry. The Banff Working Group recently proposed the following four features as suggestive of chronic AMR :
Histopathological pattern of injury consistent with chronic AMR (both a and b required)
At least mild portal inflammation with interface hepatitis and/or perivenular inflammation with necroinflammatory activity
At least moderate fibrosis (periportal/sinusoidal/perivenular)
Recent circulating DSAs
At least focal microvascular C4d deposition (C4d score ≥2)
Exclusion of other insults that might cause a similar pattern of graft injury
Hyperacute AMR is largely confined to historical studies when ABO-incompatible grafts were transplanted without pretreatment. Histopathological findings vary according to the severity of the humoral reaction and the time when tissue samples are obtained. In clinical LT, severe coagulopathy precluded liver biopsy in most patients. In patients for whom liver biopsy was feasible, a typical sequence of events has been observed. The earliest changes in biopsies obtained 2–6 h after implantation include the deposition of fibrin, platelets, neutrophils and red blood cells in small vessels and hepatic sinusoids. As a consequence of endothelial injury, there is widespread neutrophilic exudation, congestion and coagulative hepatocyte necrosis, which can be seen in biopsies obtained 1–2 days after implantation. In severe cases this results in a characteristic picture of massive haemorrhagic necrosis throughout the liver ( Fig. 14.4 ). Lack of lymphocyte infiltration, or other typical features of cellular rejection, is another characteristic feature.
Immunohistological studies have shown deposition of immunoglobulins (IgG and IgM), complement (C1q, C3, C4) and fibrinogen in vascular and sinusoidal endothelium. These deposits tend to be most marked during the earlier stages of hyperacute AMR, between 2 h and 2 days after graft insertion, and rapidly diminish thereafter. Examination of failed allografts reveals more focal deposits of IgM and C1q, mostly confined to arteries. One case of possible hyperacute rejection was associated with strong C4d staining of the endothelium of portal vessels, particularly hepatic arteries, with focal staining of central venules and minimal staining of sinusoids.
Acute antibody-mediated rejection
The presence of preformed antidonor antibodies has been associated with a higher incidence and greater severity of acute cellular rejection. Importantly, there may also be atypical histological features that indicate the presence of AMR, such as portal/periportal oedema, portal haemorrhage, neutrophil-rich inflammatory infiltration in portal tracts and prominent ductular reaction, producing changes resembling those seen in biliary obstruction ( Fig. 14.5 A ). Careful inspection also shows more diagnostic changes in dilated portal microvessels, including capillaries of the peribiliary plexus and the portal venules that extend from the portal vein for a short distance into the lobule (inlet venules). Endothelial cells in these vessels show hypertrophy and cytoplasmic eosinophilia indicating activation, which is accompanied by microvasculitis. This differs from the endothelial injury seen in acute cellular rejection, because the inflammatory cells are within the lumen rather than beneath the endothelium, and the infiltrating cells are macrophages/monocytes, neutrophils and eosinophils rather than activated lymphocytes. Injury to tiny vessels forming the vasa vasorum of the biliary tract most likely underlies the later development of biliary strictures. There are usually changes of acute cellular rejection coexisting, sometimes progressing to chronic (ductopenic) rejection if not controlled. Changes described in the liver parenchyma include centrilobular hepatocellular swelling and canalicular cholestasis and sinusoidal neutrophil infiltrates.
Immunostaining for the complement component C4d, which is deposited at sites of classical complement pathway activation, can be done on routinely processed tissues and is used as a marker of AMR in the renal allograft and more recently in liver. However, interpretation of C4d staining in liver is more difficult than in other organs because of nonspecific reactivity and differing staining protocols. Deposition of C4d in liver biopsies from both ABO-incompatible and ABO-compatible grafts with features suggestive of AMR has been described ( Fig. 14.5 B ). The C4d deposits persist for at least several days but become more patchy. Positive staining is typically present in a strong linear or finely granular pattern, which may be present on both the lumenal and the basal surfaces of endothelial cells. Several different patterns have been described, the significance of which is still incompletely understood. Reviewing these in the context of their own extensive experience, Demetris et al. have suggested that typically, deposition should be both strong and diffuse (present in >50% of tracts), most frequently staining the endothelium in the proximal microvasculature (i.e. portal veins, portal capillaries, hepatic arteries, periportal sinusoids). The Banff Working Group recently proposed a similar approach for scoring C4d staining on a scale of 0–3; a C4d score of 3, which is needed to make a definitive diagnosis of AMR in a recipient of an ABO-compatible graft, requires the presence of C4d staining in >50% of portal microvascular endothelia (portal veins, capillaries, often extending into portal inlet venules or periportal sinusoids) involving >50% of portal tracts. Care is needed interpreting arteries because of nonspecific staining of the elastic lamina. Portal stromal staining has also been described in ABO-incompatible grafts, possibly reflecting more severe microvascular injury and release of immune complexes from the vessels. Staining of central vein endothelium and sinusoids is less frequently seen. Sinusoidal C4d expression may be the most reliable marker of AMR when immunofluorescence staining is done on frozen sections, although this has not been confirmed in a more recent study. Diffuse cytoplasmic C4d staining is seen in hepatocytes undergoing necrosis, regardless of the underlying cause, and therefore is not useful in the diagnosis of AMR. As discussed earlier, C4d immunoreactivity cannot be interpreted in isolation, since it can be seen in native livers with a variety of disorders, as well as in allografts with a variety of insults, including PRI, otherwise typical acute cellular rejection, chronic rejection, vascular thrombosis, biliary obstruction, recurrent viral hepatitis (hepatitis B and C), recurrent autoimmune disease (autoimmune hepatitis [AIH] and PBC) and de novo AIH.
Chronic antibody-mediated rejection
Chronic AMR has not been fully characterized in the liver allograft, but reports are emerging suggesting that the presence of high-titre DSA, either preformed or de novo, is associated with late graft inflammation and fibrosis, particularly in the paediatric population, as discussed later. Biopsies from liver recipients with DSA have shown significantly increased interface hepatitis and lobular hepatitis, including plasma cell inflammation, with increased HLA class II expression demonstrated in the inflamed areas. Fibrosis in putative chronic AMR is characterized by a paucicellular ‘densification’ of portal collagen that has been termed ‘portal collagenization’; this may be associated with obliteration of portal veins (portal venopathy). Subsinusoidal fibrosis is also increased in some studies and is often centrilobular in location. Progression to fibrous septa and cirrhosis is described in more severe cases. A recently proposed scoring system for chronic AMR incorporates histological features related to interface hepatitis, lobular inflammation, portal tract collagenization, portal venopathy and sinusoidal fibrosis—a high chronic AMR score in combination with DSA positivity identified individuals at risk for graft loss. Positive immunostaining for C4d in portal microvessels is also helpful in supporting a diagnosis of chronic AMR, but this tends to be less extensive than that seen in acute AMR.
Other antibody-mediated lesions in the liver allograft
Recent interest in antibody-mediated graft reactions has implicated these in a range of allograft injuries, although at this stage definite causality has not been proved in many cases ( Table 14.4 ). Antidonor antibodies have been implicated in the pathogenesis of early centrilobular changes of hepatocyte ballooning and cholestasis, resembling changes seen in a severe form of PRI. The presence of DSA has also been shown to correlate with some cases of early, otherwise unexplained allograft loss, as discussed earlier. Chronic injury to the liver microvasculature may contribute to late biliary and lobular lesions and may be involved in the pathogenesis of the bile duct loss and arteriopathy which characterize chronic rejection, ischaemic bile duct injury and other vascular complications. Humoral immunity may also play a role in poorly characterized but potentially progressive allograft inflammation and fibrosis long term, but the role of histological assessments in management of these processes currently remains uncertain.
In isolation, the histological features previously described are not specific for AMR, and the clinical, histological and laboratory findings must be considered together. Massive haemorrhagic graft necrosis now occurs infrequently, and a clinical diagnosis of hyperacute rejection requires exclusion of other causes of graft failure occurring in the early postoperative period, in particular those associated with the syndrome of primary nonfunction. In contrast to PNF, most cases of hyperacute rejection follow an initial period of graft function. Acute graft failure in the early post-transplant period associated with the histological picture of massive haemorrhagic necrosis (MHN) has also been described in the absence of any demonstrable humoral mechanism. Nonhumoral factors implicated in this setting include ischaemia related to hepatic arterial kinking, gram-negative sepsis, opportunistic viral infections (e.g. herpes simplex, herpes zoster, adenovirus, enterovirus), recurrent infection with togavirus-like particles and a single-organ Shwartzman reaction. Although idiopathic MHN is rarely seen now, occasional cases with a similar pattern of graft injury are still being reported.
Acute AMR can mimic changes seen in bile duct obstruction, with a ductular reaction, portal oedema and a neutrophil-rich inflammatory infiltrate. The presence of interstitial haemorrhage, portal oedema, microvasculitis and interstitial rather than periductal neutrophilia should prompt C4d staining and testing for DSA; in this context, portal endothelial or stromal C4d staining favours a diagnosis of AMR. There is an increasing recognition that acute AMR interacts with T cell-mediated acute rejection, and overlapping histological features may thus occur. Features which favour AMR as the main diagnosis include portal vein endothelial cell hypertrophy, portal eosinophilia and eosinophilic venulitis, whereas lymphocytic portal inflammation and lymphocytic venulitis are less prominent in AMR. Similarly, acute cellular rejection with atypical features (e.g. prominent macrophage, neutrophil and eosinophil infiltrates) or ACR unresponsive to steroids should suggest that AMR is contributing to the graft injury.
In comparison with other organs such as the kidney, the liver is unusually resistant to AMR, as evidenced by the ability to carry out transplantation successfully in the face of positive antidonor crossmatching, including ABO incompatibility. Postulated reasons for the reduced susceptibility of the liver to AMR include (1) the presence of a dual blood supply, which may protect the organ from ischaemic damage; (2) a large sinusoidal vascular bed, which has more limited endothelial HLA expression compared with other solid organs and can dilute antibody binding across a larger endothelial surface; (3) release of soluble class I major histocompatibility complex (MHC) antigens into the circulation, which can bind to preformed antidonor antibodies; (4) the capacity for Kupffer cells and SECs to scavenge immune complexes and platelets and (5) the high regenerative capacity of the liver. Further evidence for the liver having a privileged immunological status in the context of organ transplantation has come from studies showing that transplantation of liver allografts into sensitized recipients is able to protect kidneys transplanted into the same individuals from developing hyperacute rejection.
The antibodies mediating AMR are heterogeneous. Isoagglutinins directed against blood group antigens are potent at inducing injury. The early and severe vascular injury that occurred in some patients has been modified with the increased use of novel therapeutic strategies, including anti-B lymphocyte therapies such as rituximab and plasmapheresis with or without splenectomy, so that most patients affected by AMR present later and less dramatically. In ABO-compatible grafts, DSAs are generally directed against HLA, usually class II, with variability in titre, complement-binding ability and immunoglobulin subclass that impacts their pathogenicity. The DSAs are now detected by flow cytometry and solid-phase immunoassays with increased sensitivity. Preformed DSAs disappear from the circulation in the majority of patients within the first week but can persist, especially when present at high titre. De novo DSAs, most frequently anti-HLA class II antibodies, develop at high titre in just under 10% of patients later after transplantation, usually in the context of reduced immunosuppression. Acute injury occurs through complement activation, causing coagulation and recruitment of innate immune cells including macrophages, neutrophils and eosinophils. Simultaneous development of acute cellular rejection is common. In other organs, complement-independent direct antibody binding to endothelial cells may have a role in chronic allograft injury, and a similar mechanism, possibly including direct binding of DSAs to activate hepatic stellate cells, may occur in liver to stimulate gradual fibrosis in the liver allograft.
Acute cellular rejection
Definition and related terms
Acute cellular rejection (ACR) can be defined as T cell-mediated damage to the liver allograft characterized by cellular infiltrates, principally present in portal areas and associated with damage to bile ducts and vascular structures. Inflammatory changes are also usually seen in the liver parenchyma, mainly around terminal hepatic venules. Most cases occur in the early postoperative period and are responsive to immunosuppression. ACR has also been called ‘acute rejection’ or ‘cellular rejection’ in the past, but the term ACR allows unambiguous distinction from acute AMR. Other terms include nonductopenic rejection, rejection without duct loss, early rejection and reversible rejection. As mentioned earlier, ‘T cell-mediated rejection’ (TCMR) has recently been suggested as the most appropriate term to use.
Incidence and risk factors
ACR is the most common form of liver allograft rejection. The incidence varies according to whether ACR is defined on the basis of clinically significant rejection (i.e. rejection accompanied by graft dysfunction requiring additional immunosuppression) or whether it is defined simply on the basis of histological abnormalities. In early studies, histological features compatible with a diagnosis of ACR have been observed in up to 80% of protocol biopsies obtained about the end of the first week after LT and it was recognized even then that not all these patients required treatment. A similar frequency of histological rejection (77%) was observed in a more recent study where protocol biopsies were obtained at the end of the first week. The incidence of clinically significant rejection is lower and appears to be declining, probably related to improvements in immunosuppressive therapy. In 2002 a systematic review showed biopsy-proven ACR with graft dysfunction in 35% of patients, but more recently the incidence under current immunosuppressive regimens has been 11–25% within the first year. A higher incidence of ACR has been noted in patients undergoing LT for autoimmune liver diseases and in those transplanted for hepatitis C, although the latter may reflect different approaches to the use of immunosuppression and the assessment of post-transplant biopsies in HCV-positive patients. Conversely, a lower incidence of ACR has been documented in patients undergoing transplantation for alcoholic liver disease and chronic HBV infection and in recipients of HLA-zero-mismatched grafts. A putative lower risk after LRLT remains controversial. Late ACR, occurring >3–6 months after LT, also occurs in 7–19% of recipients.
The majority of ACR episodes occur within the first month of transplantation. Clinical manifestations of acute rejection include pyrexia, graft enlargement and tenderness and reduced bile flow. Biochemical abnormalities typically have a predominantly cholestatic pattern. A sudden rise in serum transaminases may be a manifestation of parenchymal-based rejection changes. Peripheral blood leukocytosis and eosinophilia are also usually present. Clinical and biochemical abnormalities are nonspecific, and the diagnosis therefore requires histological confirmation. Late ACR cases often have atypical features, as discussed next.
Liver biopsy specimens show various combinations of a diagnostic portal-based triad, described by Snover et al. and subsequently confirmed in other studies of post-transplant liver biopsies (reviewed by the Banff Working Group). In recent years, there has been increased interest in a spectrum of changes involving terminal hepatic venules and the surrounding liver parenchyma.
Portal tract lesions in acute cellular rejection
The three components of the diagnostic triad are portal inflammation, bile duct damage and venular endothelial inflammation (also known as endothelitis, endotheliitis or endothelialitis). At least two of these three features are required for a diagnosis of acute rejection. Because the inflammatory lesions occurring in acute rejection can vary considerably in intensity in different parts of a single biopsy specimen, it is recommended that sections be obtained from a series of levels and that a minimum of five portal tracts be available for examination.
Portal inflammation begins as a lymphocytic infiltrate. By the time that rejection presents clinically, there is typically a mixed infiltrate of cells, including lymphocytes (mostly T cells), large activated ‘blast’ cells, macrophages, neutrophils and eosinophils ( Fig. 14.6 A ). All these cell types are also involved in mediating damage to bile ducts and endothelial cells. The presence of large numbers of eosinophils may be helpful in identifying a more severe form of rejection less likely to respond to additional immunosuppressive therapy. Plasma cells occur in some biopsies and increase with severity of rejection. The presence of prominent interface hepatitis with spillover of inflammatory cells into the lobule is also a feature of more severe cellular rejection. Mast cells may be present in varying numbers. Portal tract granulomas are rarely seen in acute rejection.
The initial damage to bile ducts is probably mediated by lymphocytes, but by the time rejection is clinically evident, there is usually a mixed infiltrate, in some cases including a prominent component of neutrophils ( Fig. 14.6 B ). Bile ducts are typically cuffed and focally infiltrated by inflammatory cells and may show degenerative changes in the form of cytoplasmic vacuolation, pyknosis and focal disruption of the basement membrane. In cases where portal inflammation is particularly intense, bile ducts can be effaced by inflammatory cells and are difficult to identify in routinely stained sections. Immunostaining for bile duct keratins is useful in demonstrating that bile ducts are still present in this situation. In some cases the presence of large numbers of neutrophils, including lumenal aggregates of pus cells, may mimic changes seen in ascending infective cholangitis. Large numbers of neutrophils have also been identified in samples of bile obtained from patients with ACR.
Venular inflammatory changes are seen in portal and hepatic vein branches. In early or mild cases there is focal lymphoid attachment to the lumenal surface of endothelial cells. In more advanced or severe cases there is subendothelial infiltration, associated with lifting and sometimes disruption of endothelial cells ( Fig. 14.6 C ). Cells associated with endothelial damage are mostly lymphocytes. However, a mixed infiltrate resembling that seen in bile ducts may also be present. In most cases, endothelial inflammation only affects a small segment of the vessel. Involvement of the entire circumference of the venule is generally confined to cases with severe rejection. Venular endothelial inflammation has generally been regarded as the most specific feature of liver allograft rejection, but it is not invariably present, particularly in cases occurring beyond the early post-transplant period. Furthermore, venular endothelial inflammation can be seen in many other conditions in which there is inflammatory infiltration of portal tracts or the liver parenchyma, including viral hepatitis, PBC, AIH and lymphoproliferative diseases.
Arterial lesions including endothelial inflammation and fibrinoid necrosis have been reported but are rarely seen in needle biopsy specimens ( Fig. 14.6 D ). When present, these have been regarded as a sign of severe damage, possibly reflecting concomitant AMR, with an increased likelihood of progression to chronic rejection. Angiographic studies demonstrating attenuation of large and medium-sized arteries suggest that these vessels may also be affected in acute rejection.
Bile ductular reaction is often seen in biopsies showing features of ACR and may in part be a response to other portal tract changes, especially bile duct damage. Other possible causes of bile ductular reaction in the early post-transplant period include delayed effect of PRI, acute AMR and SFSS.
Parenchymal changes in acute cellular rejection including central perivenulitis
Lobular inflammatory lesions comprise a spectrum of changes, principally involving hepatic venules and the surrounding liver parenchyma for which the term ‘central perivenulitis’ (CP) is now most widely used. In some cases there may be a more diffuse lobular hepatitis or a predominantly sinusoidal pattern of lymphocytic infiltration. Other terms used to describe these changes include central venulitis, centrilobular necrosis, centrilobular necroinflammation, centrilobular alterations, centrilobular changes and ‘hepatitic phase’ of rejection. Other parenchymal changes frequently seen in association with acute rejection include cholestasis, hepatocyte ballooning, fatty change and focal apoptotic (acidophil) body formation. These lesions tend to be most marked in perivenular regions and in some cases may be causally related to ACR. Particularly in the early post-transplant period, however, much of the parenchymal damage is more likely to be related to PRI than rejection.
The histological features and significance of CP depend on when it is seen (early or late) and whether portal changes of ACR are also present. At one end of the spectrum, usually seen in early post-transplant biopsies, central vein endotheliitis is a prominent feature ( Fig. 14.7 A ). Portal tract changes of ACR are typically also present, usually at least moderate in severity. The diagnosis and grading of rejection in these cases are relatively straightforward. In other cases, usually occurring significantly later after transplant, centrilobular necroinflammatory lesions are present with minimal or no central vein inflammation ( Fig. 14.7 B ) and sometimes also with minimal or mild portal inflammatory changes, also referred to as ‘isolated central perivenulitis’(ICP). A diagnosis of rejection in such cases is less easily established, and other causes of centrilobular damage also need to be considered ( Table 14.5 ) (see later discussion). In two studies, features of ICP were observed in 22% of children biopsied >3 months after LT and 28% of adults undergoing protocol biopsy >3 years after transplant. The inflammatory infiltrate is mainly mononuclear with lymphocytes predominating, sometimes with conspicuous plasma cells. Foci of mild congestion and pigmented macrophages may also be seen. Perivenular necroinflammatory lesions may also be associated with the gradual development of parenchymal fibrosis in zone 3, with linkage in more severe cases.
|Vascular occlusion (hepatic artery, portal vein, hepatic vein)|
|Viral hepatitis (recurrent or acquired)||Hepatitis B|
|Autoimmune hepatitis (recurrent or acquired)|
|Other||‘Idiopathic’ chronic hepatitis|
The prognostic significance of centrilobular inflammation and dropout depends on the histological context. Evidence suggests that the presence of perivenular necroinflammatory lesions with portal features of ACR in the first few months after LT indicates a more severe form of acute rejection, which is less likely to respond to immunosuppression and is more likely to progress to chronic rejection. In many patients the centrilobular changes appear to be present at an early stage, before bile duct loss is evident; recognition of this process and initation of appropriate immunosuppressive therapy may prevent progression to irreversible changes of chronic rejection. On the other hand, when present as ICP, the prognosis is generally favourable. Most patients, if treated at all, respond to bolstered immunosuppression, but the lesion may persist, recur or fluctuate. If there is no response to increased baseline immunosuppression or a single steroid bolus, the inflammation may behave more like de novo AIH and may respond to the reintroduction of steroids or mycophenolate mofetil for a time rather than more aggressive depleting therapies such as OKT3. When untreated, there may be progression to centrilobular fibrosis. A grading scheme for the severity of CP proposed by the Banff Working Group has been shown to correlate with adverse outcomes in one study of ICP.
In some patients, inflammation of hepatic venular endothelium may be associated with the development of veno-occlusive lesions and more severe congestive changes resembling venous outflow obstruction ( Fig. 14.8 ). Many of these patients have ACR with particularly severe endothelial inflammation. However, the lesion can also develop more insidiously, without overt portal changes of ACR, although it still appears to be responsive to optimization of immunosuppression.
Late acute cellular rejection
Several studies have suggested that late ACR may have different histological features from early ACR. These include a predominantly mononuclear portal inflammatory infiltrate (contrasting with the mixed population of cells seen in early biopsies), less inflammation of bile ducts and portal venules and more prominent interface hepatitis. The overall appearances may thus come to resemble those seen in chronic viral or AIH. As discussed above, centrilobular necroinflammatory lesions falling within the spectrum of central perivenulitis, either with or without portal inflammation, are a common feature of late ACR. Cases with predominantly lobular changes often present with raised transaminase levels, contrasting with the cholestatic profile that is more typically seen in early portal-based acute rejection, or the LFTs may be normal. However, the presence of cholestasis may identify patients with a more severe form of late ACR, which is less likely to steroid–responsive. Late ACR is associated in many cases with inadequate immunosuppression and is associated with an increased risk of developing a number of adverse outcomes including further episodes of ACR and progression to chronic rejection. Those with prominent interface hepatitis and/or CP can progress to de novo AIH or develop gradual centrilobular fibrosis. As discussed later, there are many similarities among late ACR, de novo AIH and idiopathic post-transplant hepatitis, suggesting that these three conditions may be regarded as part of an overlapping spectrum of immune-mediated damage in the liver allograft. As discussed earlier, it has been suggested more recently that some cases of otherwise unexplained, late graft inflammation may be related to chronic AMR.
Grading of acute cellular rejection
The Banff Schema devised by an international panel of liver transplant pathologists, physicians, surgeons and scientists has been widely used for grading the severity of ACR. A slightly modified version of the original grading scheme, which incorporates perivenular necroinflammatory changes as well as portal-based inflammatory lesions, appears in the latest consensus document produced by the Banff Working Group. The schema incorporates two components: a global assessment of the overall rejection grade ( Table 14.6 ) and scoring of the three main features of liver allograft rejection semiquantitatively on a scale of 0 (absent) to 3 (severe), to produce an overall rejection activity index (RAI) ( Table 14.7 ). Studies have shown that the Banff Schema is simple to use, reproducible and clinically useful in making decisions regarding therapy and may also be helpful prognostically. In one study of 575 ACR episodes, the presence of moderate or severe rejection correlated with higher transaminase levels, development of perivenular fibrosis and increased risk for developing chronic rejection. However, another study in a similar-sized cohort receiving tacrolimus immunosuppression did not find any significant association of RAI with either steroid responsiveness or progression to chronic rejection.
|Global assessment *||Criteria|
|Indeterminate||Portal and/or perivenular inflammatory infiltrate that fails to meet the criteria for the diagnosis of mild acute rejection|
|Mild||Rejection-type infiltrate in a minority of portal tracts or perivenular areas that is generally mild, and is confined within the portal spaces (for portal-based rejection) and is without confluent necrosis/dropout (for cases with isolated perivenular infiltrates)|
|Moderate||Rejection-type infiltrate, expanding most or all of portal tracts and/or perivenular areas with confluent necrosis/dropout limited to a minority of perivenular areas|
|Severe||As above for moderate, with spillover into periportal areas and moderate to severe perivenular inflammation that extends into the hepatic parenchyma and is associated with perivenular hepatocyte necrosis involving a majority of perivenular areas|
|Portal inflammation||Mostly lymphocytic inflammation involving, but not noticeably expanding, a minority of the triads.||1|
|Expansion of most or all of the triads, by a mixed infiltrate containing lymphocytes with occasional blasts, neutrophils and eosinophils. If eosinophils are conspicuous and accompanied by oedema and prominent microvascular endothelial cell hypertrophy, acute antibody-mediated rejection should be considered.||2|
|Marked expansion of most or all of the triads by a mixed infiltrate containing blasts and eosinophils with inflammatory spillover into the periportal parenchyma.||3|
|Bile duct inflammation/damage||A minority of the ducts are cuffed and infiltrated by inflammatory cells and show only mild reactive changes such as increased nuclear/cytoplasmic ratio of the epithelial cells.||1|
|Most or all of the ducts infiltrated by inflammatory cells. More than an occasional duct shows degenerative changes such as nuclear pleomorphism, disordered polarity and cytoplasmic vacuolization of the epithelium.||2|
|As above for score 2, with most or all of the ducts showing degenerative changes or focal lumenal disruption.||3|
|Venous endothelial inflammation||Subendothelial lymphocytic infiltration involving some, but not a majority, of the portal and/or hepatic venules.||1|
|Subendothelial infiltration involving most or all of the portal and/or hepatic venules with or without confluent necrosis involving a minority of perivenular regions.||2|
|As above for score 2, with moderate or severe perivenular inflammation that extends into the perivenular parenchyma and is associated with perivenular hepatocyte necrosis involving a majority of perivenular regions.||3|
Problems with applying the Banff Schema may be encountered when biopsies are taken beyond the early post-transplant period, partly because the histological features at this stage can be different, but also because there are other potential causes for cellular infiltration in the liver allograft. This particularly applies to biopsies obtained from HCV-positive individuals (discussed later) and those with unexplained chronic hepatitis. Importantly, when any uncertainty exists regarding the overall diagnosis of rejection, grading should not be done in these patients.
The diagnosis of ACR rarely poses problems during the first month after transplant, because other causes of graft inflammation are infrequently seen during this period. Greater problems exist beyond the early post-transplant period, when other causes of graft inflammation become more common. Particularly important in this respect is recurrent hepatitis C infection, discussed later. Other diseases associated with a predominantly portal-based inflammatory infiltrate include AIH (recurrent or de novo ) and Epstein–Barr virus-associated post-transplant lymphoproliferative disease. In addition to the combination of changes seen in the typical diagnostic triad, a useful feature pointing to a diagnosis of ACR is the presence of a mixed population of inflammatory cells, which is rarely seen to a marked degree in the other allograft conditions associated with portal inflammation.
Another problem with the assessment of late acute rejection is that the features may mimic chronic hepatitis. When centrilobular necroinflammatory changes predominate, usually beyond the early post-transplant period, the diagnosis of ACR is straightforward if the triad of diagnostic portal changes is also present. Many cases can still be attributed to rejection in the absence of typical portal tract changes, although other causes of centrilobular injury should first be excluded (see Table 14.5 ), as discussed later. The presence of persistent centrilobular necrosis should raise the suspicion of progression to chronic rejection and warrants careful examination of the bile ducts.
In cases where bile ductular reaction appears unduly prominent, the possibility of biliary tract pathology should be considered, particularly if there is also portal oedema and an infiltrate disproportionately rich in neutrophil polymorphs. Large bile duct obstruction should be relatively easy to exclude radiologically. However, in some cases, subtle biliary features may represent the early stages of ischaemic bile duct damage, which may escape radiological recognition. Biliary features may also be a manifestation of acute AMR. If suggestive portal microvasculitis with macrophages, eosinophils and neutrophils rather than lymphocytes is present, it should prompt C4d staining and assessment of DSAs.
Response to treatment
Mild (often subclinical) ACR often resolves spontaneously without the need for additional immunosuppression. The development of mild rejection in the early post-transplant period may have a beneficial effect in inducing long-term graft tolerance. A small number of patients with more severe forms of ACR, including clinical and biochemical signs of graft dysfunction, have resolved spontaneously without additional immunosuppression. The concept of ‘self-limiting rejection’ is well recognized in animal models of LT and may also be relevant to human LT.
In the majority of patients in whom histological features of ACR are accompanied by clinical/biochemical signs of graft dysfunction, the administration of additional immunosuppression results in resolution, with no adverse impact on long-term graft function. In early studies of follow-up biopsies obtained after treatment for acute rejection, these showed bile duct atypia and features resembling those seen in large bile duct obstruction. Repeat biopsies are no longer obtained if there is adequate biochemical response to additional immunosuppression. In cases where features of cellular rejection persist despite treatment with corticosteroids (steroid-resistant rejection), treatment with other drugs (e.g. OKT3 or mycophenolate mofetil) may result in resolution of rejection. A small number of patients are unresponsive to all forms of immunosuppression (‘intractable rejection’), and many of these either have or will develop features of chronic rejection. A role for acute AMR in some cases of severe rejection unresponsive to conventional immunosuppression is now recognized.
Definition and related terms
Chronic rejection (CR) can be defined as immune-mediated damage to the liver allograft that is associated with potentially irreversible injury to the bile ducts, arteries and veins. It is characterized histologically by two main features: dystrophic epithelial changes and the loss of small bile ducts and an obliterative vasculopathy affecting large and medium-sized arteries. CR occurs later than acute rejection; many cases evolve from ACR incompletely or unresponsive to immunosuppression. Because bile duct loss is generally considered to be the most important diagnostic feature in needle biopsy specimens, the term ‘ductopenic rejection’ has been most widely used as an alternative to chronic rejection. Other terms that have been used but fail to recognize the full scope of the changes seen include late rejection, irreversible rejection, vanishing bile duct syndrome, rejection with bile duct loss and vascular rejection.
Incidence and risk factors
CR is considerably less common than acute rejection. The incidence in series reporting patients transplanted before 1991 ranged from 2% to 20%, a wide variation that may partly reflect different criteria used to define CR. The incidence of CR is declining, presumably because of more effective immunosuppression, and now results in graft failure in <2% of cases.
Risk factors identified for CR can be divided into two main categories, as follows:
Donor/recipient factors include transplantation for autoimmune liver disease, male-to-female sex mismatching of donor to recipient, non-European recipient ethnic origin, young recipient age, old donor age and presence of high-titre DSAs of IgG3 subclass. A lower rate of CR has been observed in recipients of living-related grafts than in those with cadaveric donor organs.
Post-transplant factors include the severity and number of episodes of acute rejection, late presentation of acute rejection (>1 month post-transplant), cyclosporine use (versus tacrolimus), cytomegalovirus infection, HBV and HCV infection and interferon therapy for viral hepatitis. Those undergoing retransplantation for CR have an increased risk of developing CR in subsequent grafts.
CR often occurs as a consequence of repeated episodes of ACR that are unresponsive to immunosuppression. Many cases occurred in the first year with a peak incidence at 2–6 months after LT in studies from the 1980s and 1990s. In some cases there was a more acute presentation, with rapid progression to graft failure within a few weeks of transplantation (‘acute vanishing bile duct syndrome’). Classic cases of CR presenting with graft failure during the first year after transplant are now less common, reflecting improvements in immunosuppression. Instead, more cases are diagnosed later, often in the context of poor compliance or reduced immunosuppression for infection. These patients may have different clinical features, including a more insidious presentation and more gradual graft damage. Histological features may also be different and, in some cases, are further modified by interaction with other graft complications, such as recurrent HCV infection, making liver biopsy assessment difficult.
Clinically, CR is characterized by progressive jaundice accompanied by cholestatic liver biochemistry. The transition from acute to chronic rejection may be associated with an elevation in aspartate transaminase (AST) levels, most likely related to the presence of central perivenulitis. In common with acute cellular rejection, the clinical and biochemical manifestations of CR are nonspecific, and the diagnosis therefore requires histological confirmation.
Portal tract changes
Two main diagnostic features have been described for CR: (1) dystrophic or ‘dysplastic-like’ biliary epithelial changes or loss of bile ducts from >50% of portal tracts and (2) foam cell arteriopathy. Characteristic but less specific changes are also present in the liver parenchyma. Some cases of late CR may have different histological features, including chronic hepatitis-like changes.
During the early stages of CR, bile ducts show inflammatory infiltration, indistinguishable from that seen during potentially reversible acute cellular rejection. When there has been an incomplete biochemical response to additional immunosuppression, the overall degree of inflammation in portal tracts may be reduced. However, bile ducts show continued lymphocytic infiltration associated with nuclear pleomorphism, disordered polarity and focal attenuation and/or disruption of biliary epithelium. The cytoplasm is often eosinophilic, and with the irregular nuclei, this produces a ‘dysplastic-like’ or atrophic appearance ( Fig. 14.9 A ). These changes are associated with features of ‘replicative senescence’ (e.g. nuclear p21 expression) and are widely regarded as an early sign of impending bile duct loss. When these appearances are seen without loss of the majority of bile ducts, there is a greater likelihood of reversal. Interestingly, a recent study suggested that the expression on biliary epithelial cells of the senescence-associated factor p21 and a marker of mesenchymal differentiation (S100A4) may be occurring as a relatively early stress-related response in liver allograft rejection, which is potentially reversible if the hostile environment is modulated.
As the disease progresses to a later stage, there is loss of bile ducts, typically associated with a diminishing cellular infiltrate that eventually produces a characteristic ‘burnt-out’ appearance in end-stage livers ( Fig. 14.9 B ). Bile duct loss principally affects the small interlobular bile ducts and is thus readily diagnosed in needle biopsy specimens. In normal liver allografts, at least 70–80% of portal tracts should contain a bile duct of equivalent diameter to the hepatic artery branch. Bile duct loss should be seen in >50% of portal tracts to make a firm diagnosis of late CR. However, there are problems in counting bile ducts accurately, particularly in small needle biopsy specimens. Loss of both the artery and the bile duct from a portal tract may also impede counting. An adequate sample, usually 16G passes with at least 11 portal tracts and 20–30 mm combined length and ideally containing 20 or more portal tracts, and/or the demonstration of ductopenia in several biopsies may be required before a confident diagnosis of bile duct loss can be made. In hepatectomy specimens obtained at retransplantation, there may be loss of epithelium from medium-sized (septal) and large (hilar) ducts. The latter sometimes show a distinctive pattern of lumenal obliteration by fibrous tissue and inflammatory cells ( Fig. 14.9 C ).
A notable feature is the absence of bile ductular reaction or periportal fibrous expansion in the majority of cases, the main exception being an associated distal biliary stricture. This is in contrast to other diseases associated with loss of bile ducts, in which these secondary changes are almost always present. Studies have attributed the lack of ductular reaction in CR to an increase in apoptosis or a reduction in proliferative activity within the ductular compartment. A close relationship has been observed between ductular reaction and periportal neovessel formation in a number of liver diseases, and a lack of these two reparative responses has been implicated in the pathogenesis of irreversible bile duct loss in CR. Staining for biliary keratins such as K7 helps to confirm the absence of bile ducts and lack of a ductular reaction in CR and often also shows prominent positive staining of periportal cells with an intermediate hepatobiliary phenotype ( Fig. 14.9 D ).
The characteristic vascular lesions of CR are seen in large and medium-sized arteries and are typically manifest as intimal aggregates of lipid-laden foamy macrophages ( Fig. 14.10 A ), although other layers of the arterial wall can also be affected. These occlusive arterial foam cell lesions may produce abnormalities that can be detected angiographically. In some cases with a more acute presentation, there is a prominent infiltrate of inflammatory cells ( Fig. 14.10 B ), mainly T lymphocytes, suggesting an overlap with ACR. Conversely, in cases with a more prolonged course, there are increasing numbers of myofibroblasts associated with varying degrees of intimal fibrosis as well as fragmentation of the internal elastic lamina. However, advanced fibromuscular intimal thickening of the type classically seen in end-stage CR affecting renal or cardiac allografts is only rarely found in liver allograft rejection ( Fig. 14.10 C ). The macrophages and mesenchymal cells in arterial lesions are of recipient origin. Because these arterial lesions rarely affect small vessels of the size sampled in needle biopsy specimens, the definitive diagnosis of chronic vascular rejection is usually only made when the whole liver is available for examination ( Fig. 14.10 D ). However, smaller portal tracts may show a reduced number of small arterial branches and other microvascular channels. These changes can occur during the early stages of CR, before bile duct loss is present.
Inflammatory and/or foam cell lesions are also seen in portal and hepatic venules in some cases of CR, particularly those associated with a more acute presentation, again suggesting areas of overlap with ACR. Inflammatory lesions in hepatic venules may also result in fibrous lumenal obliteration, producing changes similar to those seen in hepatic veno-occlusive disease. Similar changes can also occur in small portal vein branches ( Fig. 14.11 ). A combination of fibroinflammatory occlusive lesions involving portal and hepatic veins appears to be important in the pathogenesis of the parenchymal fibrosis in CR, in some cases resulting in a cirrhosis-like appearance, with a venocentric pattern. A similar mechanism has been postulated for the development of fibrosis and cirrhosis in nontransplanted liver.
In the majority of CR cases, bile duct loss and occlusive arteriopathy are both present. Morphometric studies have demonstrated a parallelism between the severity of these two components of CR and have suggested that ischaemia may be a factor contributing to bile duct loss in the liver allograft. However, there are well-documented cases with a purely ductopenic or a purely vascular form of CR. In a combined series of 72 cases from three centres, 51 (71%) had both lesions, 10 (14%) had ductopenia alone and 11 (15%) had a purely vascular form of CR.
Perivenular bilirubinostasis is a prominent finding in CR and in most cases is presumably related to bile duct loss. Cholestasis can also be seen with the purely vascular form of CR, suggesting that ischaemia may also be a factor in some cases. Sinusoidal foam cells are frequently seen and are probably also a response to cholestasis.
Perivenular necrosis is also a common finding in CR ( Fig. 14.12 A ) and typically occurs as a sequela to the necroinflammatory lesions, which are seen during the preceding phase of acute cellular rejection. Humoral mechanisms may also be involved; the deposition of C4d has been described in portal and central venules as well as perivenular sinusoids. In cases with incomplete biochemical response to additional immunosuppression, the cellular infiltration in perivenular regions often subsides, but hepatocellular dropout persists. Necrosis typically has a lytic pattern and is accompanied by reticulin collapse and immature collagen fibre deposition. In some cases there may be more extensive necrosis with bridging and nodule formation ( Fig. 14.12 B ). Even in cases where zonal necrosis has been detected in serial biopsies over several months, the lesions frequently retain an appearance suggesting acute damage, without the formation of mature collagen or elastic fibres. This suggests a dynamic equilibrium between hepatocyte loss and regeneration in perivenular regions. However, in some cases there is development of more mature fibrous lesions, which may ultimately progress to cirrhosis-like changes. A range of rejection-related ischaemic mechanisms has also been implicated in the pathogenesis of parenchymal necrosis and fibrosis; these include occlusive lesions in large and medium-sized arteries, loss of small arterial branches and occlusive lesions involving portal and hepatic veins. Although centrilobular necrosis has been regarded as a ‘surrogate marker’ of rejection-related arteriopathy, a definite association between these two processes has not been convincingly demonstrated.
Late chronic rejection
As noted earlier, ‘classic’ cases of CR progressing to graft failure during the first 12 months after LT are now uncommon. In addition to a lower overall prevalence, more cases now occur later, often with a more insidious presentation and an indolent course. Cases presenting later may have slowly progressive bile duct loss occurring over several years. Ductopenia related to previous rejection has also been seen in late protocol biopsies from patients who are clinically well with good graft function ; whether this represents a low-grade subclinical form of CR is uncertain. In contrast to CR presenting during the first year, cases occurring later may also be associated with ductular reaction and periportal fibrosis, which occasionally progresses to a biliary pattern of cirrhosis.
By a process of exclusion, it has been suggested that many cases of otherwise unexplained chronic hepatitis presenting >12 months after transplant may represent a hepatitic form of CR, which can result in the development of progressive fibrosis, in some cases progressing to cirrhosis. Findings suggesting that chronic AMR may also be playing a role, particularly with the presence of high rates of DSA and C4d positivity in some studies, need further clarification. The relationship of ‘idiopathic’ post-transplant hepatitis to late rejection is discussed later.
Staging of chronic rejection
The Banff Schema was updated by the Banff Working Group in 2000 to incorporate features related to the staging of CR. A slightly modified version of the 2000 staging scheme, which recognizes different patterns of fibrosis that may be related to CR, appears in the latest consensus document produced by the group ( Table 14.8 ). In staging changes related to CR, a distinction is made between changes occurring during the early phase, when the disease is potentially still reversible, from those indicating advanced or irreversible disease, for which retransplantation is generally the only therapeutic option. In the liver biopsy diagnosis of early CR, particular emphasis is placed on the recognition of biliary epithelial atypia in the majority of bile ducts, which can be seen before bile duct loss has occurred, and zone 3 necroinflammatory lesions, which also occur at an early stage in the disease process. Application of Banff staging schema has been shown to improve greatly the sensitivity of needle biopsies for diagnosing CR at an early stage. More importantly, the use of potent immunosuppressive agents at an early stage in the disease process may prevent the development of irreversible graft damage. Late CR is recognized if at least two of the advanced features listed in Table 14.8 are present. Although bile duct loss has been the most widely used feature indicating progression to late (irreversible) CR, there are problems in counting bile ducts accurately in needle biopsy specimens, particularly concerning sampling variation. Examination of hepatectomy specimens with end-stage disease sometimes reveals marked variation in the degree of bile duct loss from one part of the liver to another. Perivenular fibrosis with bridging may also be indicative of irreversible graft damage, although this is not always a reliable criterion. As discussed earlier, areas of overlap exist between acute and chronic rejection, and an individual biopsy may show features of ongoing inflammatory activity, which can be graded, as well as signs of progressive liver injury (e.g. bile duct loss), which can be staged.
|Structure||Early CR *||Late CR *|
|Small bile ducts (<60 µm)||Senescence changes involving a majority of ducts||Degenerative changes in residual bile ducts|
|Bile duct loss in <50% of portal tracts||Bile duct loss in ≥50% of portal tracts|
|Portal tract hepatic arterioles||Occasional loss involving <25% of portal tracts||Loss involving >25% of portal tracts|
|Terminal hepatic venules and zone 3 hepatocytes||Lytic zone 3 necrosis and inflammation||Variable inflammation|
|Mild perivenular fibrosis.||Moderate-severe (bridging) fibrosis (see note below)|
|Intimal/lumenal inflammation||Focal obliteration|
|Large perihilar hepatic artery branches||Intimal inflammation, focal foam cell deposition without lumenal compromise||Lumenal narrowing by subintimal foam cells |
|Large perihilar bile ducts||Inflammation damage and focal foam cell deposition||Mural fibrosis|
|Other||So-called transition hepatitis with spotty necrosis of hepatocytes||Sinusoidal foam cell accumulation: marked cholestasis|
Some of the other features listed in Table 14.8 (e.g. lesions affecting large arteries and bile ducts) can only be diagnosed reliably in hepatectomy specimens. For these lesions, the distinction between ‘early’ and ‘late’ CR is based on theoretical concepts, which cannot be applied to needle biopsy assessment. Other features (e.g. cholestasis, sinusoidal foam cells) are nonspecific findings and can be seen in a variety of other conditions.
A number of diseases in the liver allograft may be associated with portal inflammatory infiltration, bile duct damage and in some cases bile duct loss. These include recurrent viral hepatitis (in particular hepatitis C), recurrent biliary disease (PBC, PSC) and ischaemic cholangiopathy which, as discussed later, has histological features closely resembling those seen in PSC.
The relationship between recurrent HCV infection and liver allograft rejection is considered in more detail later. Cases with combined features of hepatitis C and CR pose particular problems for diagnosis and management. Although CR is similar to PBC and PSC (and ischaemic cholangiopathy) in that all three are vanishing bile duct diseases, there are important histological differences. The granulomatous bile duct lesions in PBC and the sclerosing duct lesions in PSC are different from the inflammatory and ‘dysplastic-like’ bile duct lesions seen during the early stages of CR. Perhaps the most useful feature distinguishing typical cases of CR from other diseases with bile duct loss is the absence of secondary biliary features such as ductular reaction, periportal fibrosis and deposition of copper-associated protein. However, as discussed earlier, these secondary biliary features may be seen in rare cases of late CR developing several years after LT. Bilirubinostasis, as seen in CR, is rarely prominent in other biliary tract diseases unless there is advanced fibrosis/cirrhosis, severe ductopenia or, in the case of PSC, a major bile duct stricture associated with features of large duct obstruction. The finding of severe unexplained cholestasis occurring beyond the early post-transplant period unaccompanied by portal tract changes suggesting biliary obstruction should therefore prompt a careful search for subtle bile duct lesions that might suggest a diagnosis of early CR.
The differential diagnosis of the centrilobular lesions which occur in chronic (and acute) rejection is more difficult. Although many cases are rejection related, a number of other factors, including vascular problems, viral hepatitis, AIH (recurrent or acquired) and drug toxicity may also be implicated (see Table 14.5 ).
Among the vascular causes of centrilobular necrosis (CLN), preservation/reperfusion injury is unlikely to result in lesions persisting beyond the early postoperative period. Even in biopsy specimens obtained the first week after transplant, CLN is most likely to be rejection related, particularly if accompanied by a lymphocytic inflammatory infiltrate. Rejection-associated changes resulting in hepatic veno-occlusive lesions and surrounding congestion and necrosis may be indistinguishable from other causes of venous outflow obstruction. In many cases the presence of rejection-related inflammatory changes, particularly with marked endothelial inflammation, either with or preceding histological features suggesting venous outflow obstruction, is helpful in showing rejection as the likely cause.
Centrilobular inflammation and CLN may be seen in association with recurrent or, more rarely, acquired viral or autoimmune hepatitis. Knowledge of the original indication for LT and post-transplant viral and immunology studies are helpful in the diagnosis of recurrent viral or autoimmune hepatitis. Features favouring a diagnosis of rejection include prominent hepatic venular endothelial inflammation together with bilirubinostasis and/or ballooning in surrounding viable hepatocytes. The presence of conspicuous bile duct injury and bile duct loss and portal venular endothelial inflammation are also features that favour a diagnosis of rejection. Conversely, the presence of a plasma cell-rich infiltrate, portal interface hepatitis and serological detection of autoantibodies indicates a diagnosis of AIH.
CLN and hepatic veno-occlusive lesions can also be seen as a toxic injury related to use of the immunosuppressive drug azathioprine and as a manifestation of ‘idiopathic’ post-transplant chronic hepatitis.
Response to treatment and outcome
The treatment of CR depends on the stage at which the disease is diagnosed. Patients with early CR, in whom bile duct atypia is present with minimal or no duct loss, may respond to a range of immunosuppressive agents, including tacrolimus, OKT3, mycophenolate mofetil and rapamycin (sirolimus). In contrast, patients with advanced CR, typically associated with severe biochemical cholestasis and advanced ductopenia, are generally unresponsive to immunosuppression and often require retransplantation. However, the exact point of irreversibility cannot be defined histologically. A small number of patients with apparently advanced CR (including bile duct loss in >50% of portal tracts) have recovered spontaneously or with the use of additional immunosuppression. Interestingly, some of these patients have had follow-up liver biopsies showing a persistent paucity of bile ducts without other histological features of CR. Ductopenia has also been noted as an incidental finding in protocol biopsies taken at annual review from patients who are clinically well with no previous biopsies suggesting CR. Other cases may have gradual bile duct loss over 5–10 years without progressing to graft failure. These observations suggest that some patients may have permanent duct loss as a result of rejection, but that sufficient ducts remain to allow the graft to function well. Because histological features in needle biopsy specimens are not reliable in identifying irreversible graft damage caused by CR, clinical features and biochemical markers are also used to determine the point at which retransplantation is required.
Relationship between acute and chronic rejection
The subdivision of liver allograft rejection into acute and chronic forms is based on three main diagnostic features: time of onset, behaviour and histological changes ( Table 14.9 ). Although this approach is useful clinically, areas of overlap exist for each of these three features. Acute and chronic rejection are thus best regarded as different ends of a spectrum of immune-mediated damage occurring within the liver allograft. Histologically, a broad spectrum of changes exists between the cellular infiltrates that characterize early acute cellular rejection (ACR) and the ductopenia and obliterative vasculopathy, which are seen in end-stage chronic rejection (CR). Most cases of CR are preceded by episodes of ACR. Many biopsies obtained during the evolution of CR have features of ongoing cellular rejection, and these features may also be seen in hepatectomy specimens obtained at retransplantation.
|Time of onset||Early||Late|
|Response to treatment||Generally good||Poor|
|Histological features||Cellular infiltrates||Bile duct loss |
Immunopathogenesis of acute and chronic rejection
Targets for immune damage in liver allograft
Allograft rejection is a vigorous immunological reaction to foreign antigens of another member of the same species and is a complex interplay between the innate and adaptive arms of the immune system. This reaction is primarily to foreign, polymorphic major histocompatibility complex (MHC) antigens that are present on all nucleated cells. In uncontrolled ACR, mediated mainly by T cells, the vigour of the response results from the high frequency of antigraft lymphocytes, which are 100–1000 times more numerous than in usual immune responses. All the nucleated cells of the liver are potential targets for immune-mediated damage in acute and chronic rejection. Lesions involving bile ducts, vascular endothelial cells and hepatocytes (mainly in centrilobular zones) are well recognized, and Kupffer cells and other sinusoidal cells may also be destroyed in liver allograft rejection, to be replaced by cells of recipient phenotype.
The MHC complexes have a central peptide groove that holds endogenous or exogenous peptide fragments for presentation to the T-cell receptor (TCR). In the alloresponse the polymorphic MHC proteins are recognized as foreign. Additionally, because of varying shape and charge of the peptide groove and side pockets, the polymorphic MHC molecules bind different peptide fragments, even from the same protein, and these can also be recognized as ‘foreign’. Thus, alloreactive T cells may recognize the allogeneic MHC, the allogeneic peptide or in most cases, both. The diversity of MHC molecules and the large array of different peptides that they can bind in their grooves likely explain the vigour of the alloreaction. The alloreactive T cells in acute rejection are a combination of T cells specifically reactive against the donor MHC, as well as significant numbers of pathogen-specific T cells that cross-react with the foreign MHC.
Stages in allograft rejection
The stages in allograft rejection include allorecognition, T-cell activation and the alloresponse. Initially, allorecognition, or the afferent arm, involves the presentation of graft alloantigens and the recognition of these antigens by recipient T lymphocytes. Following T-cell activation and cytokine release, the alloresponse, or efferent arm, involves recruitment and activation of effector cells that mediate damage to target structures within the allograft.
Afferent arm of immune response: allorecognition
The initial phase of the allograft response involves the presentation of graft alloantigens and the recognition of these antigens by recipient T cells. Central to this process is the antigen-specific interaction between the MHC molecules expressed on the surface of antigen-presenting cells (APCs) and the TCR expressed on the surface of recipient T lymphocytes ( Fig. 14.13 ). Interactions involving CD4+ T cells and MHC class II-expressing ‘professional’ APCs are primarily involved in the initial stages of graft recognition. The principle APCs in normal liver are dendritic cells (DCs) located in portal areas and, to a lesser extent, in centrilobular regions of the liver parenchyma.
Three main pathways of antigen presentation are described: direct, indirect and semidirect. In the direct pathway, the main pathway involved in the early post-transplant period, donor DCs transferred as passenger leukocytes in the liver at transplantation migrate to regional lymph nodes and spleen, maturing during transit, where they present donor MHC antigens and activate alloreactive T lymphocytes. The DC mobilization occurs as a nonspecific reaction to surgical and ischaemic injury, stimulated by the release of cytokines such as interleukin-1 (IL-1), IL-6 and tumour necrosis factor α (TNFα).
Later, the donor APC population is depleted and replaced by host APCs. These process and present other donor alloantigens shed from the graft, but this time in the context of host MHC molecules. This indirect pathway is slower and less vigorous than the direct pathway, but still appears to be important in sustaining an ongoing persistent immune response, particularly a humoral response. The semidirect pathway has been characterized more recently, following the recognition that host APCs could acquire whole allogeneic MHC-peptide complexes through exosomes released by graft cells and could express them on their surface. The semidirect pathway can stimulate T-cell responses and potentially links the direct and indirect pathways.
The interaction between T cells and graft cells involves other important signalling pathways, including adhesion molecules, which enable T cells to bind to graft cells, and co-stimulatory molecules, which are required for naive T-cell activation ( Fig. 14.13 ). These other factors are not antigen specific. Two important co-stimulatory molecule-ligand pairs are CD28/B7 and CD40/CD154. In the absence of appropriate co-stimulatory signals, T cells do not become fully activated and either become unresponsive to further antigenic stimuli (anergic) or undergo apoptosis. Co-inhibitory pathways involving interactions between programmed death 1 (PD-1) receptor, which is expressed on graft-infiltrating T cells in acute rejection, and its ligand PD-L1, expressed on hepatocytes, cholangiocytes and sinusoidal cells, may also be important in regulating immune responses in the liver allograft.
Cells in the normal liver have a limited expression of MHC antigens, adhesion molecules and other molecules involved in the alloimmune response. After LT, there is cytokine-mediated upregulation of many of these molecules on bile ducts, hepatocytes and endothelial cells. The functional significance of these changes is uncertain, but they may be involved in both enhancing antigen presentation and promoting T-cell activation and effector mechanisms.
T-cell activation and cytokine release
The binding of the TCR to MHC antigens triggers a cascade of intracellular signalling events, leading to the activation and proliferation of responding T cells and the subsequent migration of these cells to the liver allograft, where they are recruited due to the expression of adhesion molecules and chemokines. There they secrete cytokines that promote effector functions not only of T lymphocytes themselves but also of other effector cells, including macrophages, natural killer (NK) cells and other inflammatory cells.
Several lineages of activated CD4+ T cells (T-helper or Th cells) are recognized, which develop differentially depending on the inflammatory microenvironment during activation. These populations, including Th1, Th2, Th17, Th9 and Th22 cells, emerge in variable proportions and have differing cytokine signatures. For example, Th1 responses are characterized by IL-2 and interferon-γ (IFN-γ) production, leading to CD8+ cytotoxic T lymphocyte (CTL, Tc) and macrophage activation, whereas Th2 cytokines preferentially induce humoral responses and eosinophil infiltration through secretion of cytokines that include IL-4 and IL-5. Although the Th1 response is dominant in acute rejection, Th2 cells also contribute. Currently, there is interest in the role of Th17 cells in allograft rejection, with data suggesting a possible role in the liver and other organs. This lineage also appears to be related to regulatory T cells, with apparent plasticity between these two responses.
Effector mechanisms in liver allograft rejection
The main effector mechanisms in liver allograft rejection are summarized in Table 14.10 . Antigen-specific damage to graft cells is mediated principally by T lymphocytes, which are predominant in graft infiltrates. A mixed population of CD4+ and CD8+ cells has been identified in varying proportions. CD8+ T cells appear to be particularly involved with mediating damage to the principal targets of liver allograft injury—bile ducts and vascular endothelium —and are also the predominant cell found in parenchymal infiltrates, particularly in perivenular regions in cases of chronic rejection. CD8+ Tc cells, activated by a cell cluster comprising an APC and a CD4+ Th cell, interact directly with MHC class 1 molecules expressed on graft cells, resulting in death of graft cells by apoptosis. Two main pathways for T cell-mediated apoptosis have been identified. The first involves the synthesis and secretion of cytolytic granules that contain perforin, granzymes and granulysin. The second pathway involves Fas–Fas ligand (Fas-L) interaction. Fas-L, upregulated on the surface of CTLs, binds trimeric Fas expressed on the surface of target cells which include hepatocytes, bile ducts and endothelial cells (sinusoidal and vascular), causing target cell apoptosis. However, hepatocytes are relatively resistant to Fas-mediated apoptosis, and members of the TNF superfamily (CD40, TNFR1, TNFR2 and TRAIL) are more important in cellular injury. Interactions involving CD40 on target cells and CD40 ligand, expressed on effector cells, have also been implicated in amplifying Fas-mediated apoptosis of hepatocytes and biliary epithelial cells in rejecting liver allografts. CD40-L-positive macrophages appear to play an important role in this process and may also be involved in other aspects of immune activation in the liver allograft.
Some cytokines released by activated T cells (e.g. IFN-γ, TNFα) may result in damage to adjacent structures in a non-antigen-specific manner. In addition, T cell-derived cytokines result in the recruitment and activation of a number of other inflammatory cells, including neutrophils, eosinophils, macrophages and NK cells. All these cells can augment T cell-mediated damage to target structures in a non-antigen-specific manner. The role of NK cells in mediating graft damage remains unclear, and these may also be involved in the induction of graft tolerance. Mast cells have been implicated in the pathogenesis of bile duct injury in chronic rejection and are also increased in acute rejection, as well as other graft complications such as recurrent HCV infection.
As discussed earlier, antibody-mediated mechanisms have been implicated in very early graft dysfunction, accelerated and steroid-resistant acute rejection in the early post-transplant period and also late allograft dysfunction. The possible role of vascular lesions in mediating damage to bile ducts, whose blood supply is entirely derived from the hepatic arterial system, and also perivenular hepatocytes has been discussed earlier, but the mechanisms linking antigraft antibodies to late fibrosis and inflammation in the liver remain poorly understood. Vascular lesions that have been implicated in mediating bile duct loss in chronic rejection include occlusive lesions in large and medium-sized arteries, loss of terminal hepatic arterioles and destruction of portal tract microvasculature. Morphometric studies have shown that the severity of arterial lesions correlates with the degree of bile duct loss, and that the loss of portal tract vessels precedes bile duct loss in liver allograft rejection.
Response to rejection-induced liver injury
The liver has a considerable capacity for regeneration. Increased hepatocyte proliferation occurs within the early post-transplant period in response to preservation/reperfusion injury. In cases of chronic rejection, increased proliferative activity can be seen in surviving hepatocytes surrounding perivenular zones of hepatocyte necrosis ( Fig. 14.14 ). Proliferation of biliary epithelial cells can also be seen in liver allograft rejection, but this may fail in chronic rejection due to cyclosporine-dependent transforming growth factor β (TGFβ) expression or vascular loss. The final outcome of rejection in the liver allograft thus depends on the balance between factors leading to cell death and the capacity for a compensatory proliferative response.
Bone marrow-derived progenitor cells may be recruited to the liver and have the potential to differentiate into the various cell types present in the liver. This process may represent an alternative repair pathway after liver allograft damage, particularly in cases where the regenerative capacity of the liver allograft is impaired (e.g. severe recurrent HCV infection), and has also been implicated in the development of tolerance. However, with the exception of Kupffer cells and, at a seemingly lower rate, endothelial cells, which are extensively replaced by cells of recipient origin, the majority of other cells in the liver allograft retain a donor phenotype.
The liver is at the forefront in the quest to perform solid-organ allografting without the need for long-term immunosuppression. As well as the cost of immunosuppression, there is a high cost to the patient in terms of infectious and neoplastic complications, diabetes, renal dysfunction and lipid disorders. The appeal of liver allografts in exploring immunosuppression withdrawal is several-fold. In porcine and rodent models, spontaneous tolerance following MHC-mismatched liver transplantation is well described. LT in these models is also associated with donor-specific protection of other solid-organ grafts. In clinical LT, the liver is an immune-privileged organ with a lower rate of acute and chronic rejection and reversibility of early chronic rejection; this protective advantage is conferred to other solid organs co-transplanted from the same donor. More intriguingly, approximately 20% of liver allograft recipients have the potential to be weaned from immunosuppression completely after a period of time. These observations are likely related to the liver having a normal immunoregulatory function, whereby various components of allogeneic immune responses are modulated to produce a tolerogenic microenvironment favouring the development of graft tolerance.
Several types of tolerance are recognized. True transplantation tolerance is an absence of donor-reactive cells in vivo and in vitro and occurs in only some tolerance models. Operational tolerance is an absence of acute and chronic rejection and indefinite graft survival without immunosuppression in an immunocompetent recipient. This is the usual form in patients tolerant of their allografts and in many spontaneously-tolerant rodent models, where T-cell alloresponsiveness can still be detected in vitro despite the stable graft function. Prope tolerance refers to a state of near-tolerance in patients who require only minor and subtherapeutic levels of immunosuppression to maintain normal graft function, without complete withdrawal of immunosuppression.
Tolerance is complex, multifactorial and remains incompletely understood. Central tolerance is characterized by the thymic deletion of alloreactive as well as self-reactive precursor T lymphocytes during TCR rearrangement and relies on the presence of donor antigens at that site. Thymic induction of regulatory or ‘suppressor’ T cells (Tregs), which suppress immune responses, is also described. Conversely, peripheral tolerance occurs outside the thymus and is typified by a combination of post-thymic T-cell deletion, unresponsiveness (anergy) and Treg differentiation. These are likely the dominant processes in operational tolerance of liver. A large number of tolerogenic protocols and tolerance-breaking manoeuvres have been described, but with respect to liver tolerance, several key features have emerged. Tolerance is a process that evolves, with induction and maintenance phases that theoretically have differing dominant mechanisms. Successful tolerogenesis requires a careful balance of bidirectional immune responses directed against both the graft and the host. In the induction phase, deletion of alloreactive T cells is likely to be an important mechanism limiting the alloresponse, and some form of lymphocyte depletion appears to be necessary to allow tolerance to develop. Factors that could have a role in lymphocyte deletion include the characteristic nature of liver-derived APCs and apoptosis of graft-infiltrating T cells on contacting the various parenchymal and nonparenchymal liver cells.
In the later maintenance phase of tolerance, the favoured mechanism is the emergence of donor-specific cells with a regulatory phenotype (mainly Tregs). The repertoire of regulatory cells continues to increase, and regulatory networks include CD4+ and CD8+ T cells, double-negative T cells, γδ T cells, regulatory B cells, tolerogenic DCs, NKT cells, myeloid-derived suppressor cells and mesenchymal stromal cells. The liver may preferentially facilitate Treg differentiation. It has been postulated that chronic exposure to gut-derived endotoxin delivered by the portal vein promotes expression of anti-inflammatory cytokines such as IL-10 and TGFβ by Kupffer cells and sinusoidal endothelial cells. This milieu, as well as direct lipopolysaccharide tolerance, may condition liver DCs to promote more tolerogenic responses. Other nonparenchymal cells (e.g. hepatic stellate cells) may also play a role.
Diagnosis and monitoring of graft tolerance
It has long been recognized that a small number of liver allograft recipients are able to cease immunosuppression after transplantation. As theoretical studies of tolerance move to clinical practice, pathology is likely to have an expanded role in monitoring patients before, during and after weaning from immunosuppression. However, although many patients worldwide are currently free of immunosuppression, the long-term benefits of immunosuppression withdrawal are as yet unproved, and this remains an experimental procedure. Since much is still unknown in the practical application of operational tolerance, it has been recommended that protocol biopsy should be performed at baseline and at defined time points after weaning, even in apparently stable patients, although this is not done in all centres. Weaning has generally been carried out over months, beginning more than 2–3 years after LT, in highly select patients with stable graft function and no evidence of significant pathology apart from recurrent hepatitis C.
With regard to factors associated with successful immunosuppression weaning, time since transplantation appears to be particularly important. A recent European study of operational tolerance in liver recipients found that weaning was possible in 12.5% of patients transplanted for <5.7 years, but this increased to 79.2% in patients transplanted for ≥10.6 years. Younger children and older adults appear to be more favourably disposed to immunosuppression withdrawal, although probably for differing reasons. Other factors reported to be associated with a more favourable outcome include the absence of early rejection and the presence of low levels of baseline immunosuppression before weaning. Factors associated with a reduced likelihood of successful weaning include initial transplantation for autoimmune disease, presence of C4d staining in baseline biopsies and presence of DSAs. However, a recent study of children with follow-up 5 years after withdrawal of immunosuppression suggested that DSAs (persistent or de novo ) are frequently present in operationally tolerant individuals without impacting graft histology.
Biopsies taken before weaning allow a baseline assessment of inflammation (portal/interface and centrilobular), fibrosis or other features that might indicate subclinical rejection. The presence of more than a mild degree of any of these changes may preclude proceeding with immunosuppression withdrawal. Conversely, features observed in preweaning biopsies found to be predictive of tolerance include absence of portal inflammation, absence of lobular CD3+ and CD8+ T cells, higher stage of fibrosis in HCV-positive patients and presence of portal FoxP3+ T cells. Studies on peripheral blood and liver samples have identified a range of putative biomarkers associated with a ‘tolerogenic profile’ which are under continued evaluation with prospective analyses and validation cohorts.
Postweaning biopsies have been obtained to investigate episodes of graft dysfunction and on a protocol basis. Rejection is the most common cause of graft dysfunction after immunosuppression withdrawal and is the most common reason for failure to achieve graft tolerance. Most cases present during the period of immunosuppression weaning or within the first few months thereafter. Liver biochemistry is not reliable in distinguishing rejection from other possible causes of graft dysfunction, and this remains an important indication for liver biopsy. Most cases have features resembling typical acute cellular rejection (ACR), are relatively mild in severity and respond well to reinitiation of baseline immunosuppression. A smaller number of cases with more severe ACR or features suggestive of early chronic rejection (CR) may require more aggressive immunosuppressive therapy. Rare cases have progressed to CR leading to graft failure. Some cases of rejection occurring after immunosuppression withdrawal lack the typical histological features seen in ACR and instead have features resembling the hepatitic picture more typically seen in late acute rejection. Comparison with baseline preweaning biopsies may be particularly helpful in this setting. Later protocol biopsies in an LRLT cohort have shown a significant increase of portal fibrosis in some operationally tolerant patients compared with those on immunosuppression. It was initially postulated that the higher numbers of Tregs seen in these patients, thought to be involved in mediating tolerance, may also be associated with profibrogenic cytokine profile. However, the observation that portal fibrosis reversed with the reintroduction of immunosuppression suggests that some of these apparently tolerant patients may have had a mild, subclinical form of rejection.
A more recent study showed that increased numbers of portal T lymphocytes, with enrichment of Tregs, were present in biopsies obtained 1–3 years after weaning from operationally tolerant patients with no signs of rejection and no evidence of fibrosis. Minor degrees of bile duct atypia or ductopenia have also been observed in postweaning biopsies, suggesting the possibility of low-grade CR. Histological features thought to be of concern in postweaning biopsies have been identified by the Banff Working Group and include the following :
Increased portal or lobular inflammation compared with baseline preweaning biopsy, especially if accompanied by lymphocytic bile duct inflammation or interface or venous endothelitis
New-onset central perivenulitis that is increased compared with baseline
Bile duct changes, including dystrophic/senescent changes or ductopenia, if other causes such as bile duct obstruction or stenosis are reasonably excluded
Fibrosis that is increased over baseline and is not explained by another cause
Arterial changes, such as foam cell or obliterative arteriopathy
Further investigation in this area is required. As experience grows, pathologists will have an important role in clinical decision making through biopsy diagnosis, documenting new patterns of injury and providing insights into the microanatomical processes occurring during operational tolerance.
Infections are very common in liver allograft recipients, particularly in the first 2 postoperative months. Overall, 60–80% of patients will have at least one episode of infection, of which bacteria account for 50–60% of cases, viruses for 20–40%, fungi for 5–15% and other organisms <10%. The prevalence of infection as a cause of death has declined from >50% before 1980 to <10% more recently. Common examples include wound infections complicating major abdominal surgery, persistent or recurrent viral infection in the recipient (discussed later), infections superimposed on other transplant complications (e.g. bile duct infection in ischaemic bile duct necrosis) and a broad spectrum of opportunistic infections arising from immunosuppression. Most infections occurring in liver allograft recipients are diagnosed by nonhistological methods and do not involve the graft itself. The following discussion focuses on histological aspects of infection within the liver allograft.
Opportunistic viral infections
The liver is involved variably as part of a systemic infection. Most episodes occur during the first few months of transplantation, when levels of immunosuppression are highest. Primary infection developing in patients with no previous exposure (usually children) tends to be more severe than reactivation of latent infection, which occurs as a consequence of immunosuppression. The most common example in post-transplant biopsies is cytomegalovirus. Epstein–Barr virus is mainly of interest because of its involvement in the pathogenesis of post-transplant lymphoproliferative diseases, which can sometimes involve the liver allograft. Other opportunistic viruses such as adenovirus, herpes simplex and varicella-zoster usually involve the liver as part of an overwhelming infection and are thus mainly seen in autopsy material.
Cytomegalovirus (CMV) was a common cause of graft infection in the 1980s and early 1990s, when it occurred in 30–50% of patients, was symptomatic in up to 25% of cases and was associated with significant morbidity and mortality. The clinical frequency and mortality have considerably declined due to the use of prophylactic and pre-emptive antiviral therapy, and symptomatic disease now occurs in only 2–10% of patients. Although the peak time of occurrence in untreated patients is between 4 and 12 weeks after transplantation, the use of antiviral prophylaxis with ganciclovir or valganciclovir for the first 3 months after transplantation has seen an increasing prevalence of delayed-onset infection, which may present up to 1 year or more after discontinuation of prophylaxis. Although most of these late infections are associated with mild disease, occasional studies still implicate CMV infection as an important cause of death at 1 year.
The most important risk factor for the development of CMV disease is the combination of a seropositive donor and a CMV-seronegative recipient (D + R − ). Late disease still occurs in this group, even with antiviral prophylaxis, with a prevalence of 19–27% at 1 year. Other risk factors include transplantation for acute fulminant hepatitis, amount and type of immunosuppressive therapy (particularly antilymphocyte antibodies, mycophenolate or high-dose steroids), previous episodes of acute rejection, hepatitis C infection, infection with human herpesviruses 6 and 7, female gender and polymorphisms associated with innate immunity.
CMV infection is common and is distinguished from CMV disease. Infection indicates the presence of virus in blood, fluids or tissues regardless of symptoms, but the presence of clinical symptoms or signs is termed ‘CMV disease’ and is generally associated with higher levels of virus. CMV disease manifests as CMV syndrome (fever and marrow suppression) in up to two-thirds of affected patients, with the remainder having tissue-invasive CMV. In solid-organ transplantation the allograft is particularly prone to CMV infection, so the liver has been the most common site of organ involvement in liver allograft recipients. Other manifestations of CMV disease include gastrointestinal (GI) disease, pneumonia, neurological complications and chorioretinitis. As delayed-onset infection becomes more common, the incidence of infection outside the liver has increased, particularly in the GI tract.
The characteristic and most common histological finding in CMV hepatitis is the presence of scattered neutrophil polymorph aggregates (microabscesses) within the liver parenchyma ( Fig. 14.15 A ). Parenchymal microgranulomas can also be seen. There is occasionally a picture of spotty lobular inflammation and lobular disarray, which is indistinguishable from other forms of viral hepatitis. Other reported features include mild portal inflammation—sometimes associated with mild inflammation of bile duct and portal venules, suggesting mild cellular rejection—Kupffer cell enlargement and hepatocellular changes, including anisocytosis, anisokaryosis, nuclear hyperchromatism and increased numbers of mitoses. Viral inclusions are most often seen within the infected hepatocytes, usually in the vicinity of inflammatory lesions, and less often in biliary epithelial cells and endothelial cells of sinusoids and vessels. Examination of serial sections and deeper levels is useful because inclusions can be rare. In one study of serial post-transplant liver biopsies, CMV infection was first detected within sinusoidal cells, several days before typical histological features of CMV hepatitis became apparent. Increasingly, hepatitis with suggestive inflammatory lesions but no obvious viral inclusions is seen. Immunohistochemical (IHC) staining for CMV antigens is useful in confirming the presence of the virus ( Fig. 14.15 B ). In situ hybridization (ISH) techniques have also been used but are not widely available and do not appear to be more sensitive than IHC staining.
Although parenchymal neutrophil microabscesses are highly suggestive of CMV hepatitis, these lesions may also occur in cases when no CMV infection can be detected. Suggested associations include other infections (bacterial, viral or fungal), graft ischaemia and biliary obstruction/cholangitis. In some cases, no other obvious disease can be found. One group found that neutrophil aggregates in CMV-negative cases were smaller and more numerous than those occurring in association with CMV infection, prompting the term ‘mini microabscesses’. Conversely, a subsequent study found that greater numbers of microabscesses (>9) indicated CMV infection.
CMV infection has been implicated as an indirect factor contributing to an increased risk of other allograft complications, such as acute and chronic rejection, hepatic artery thrombosis and biliary complications. The relationship between CMV infection and rejection is complex and remains poorly understood. Whereas the use of high-dose immunosuppression to treat episodes of rejection is a risk factor for the emergence of opportunistic infection, CMV can infect bile ducts and vascular endothelial cells (VECs), which are targets for immune-mediated damage in allograft rejection. Infection also stimulates immune responses such as inflammatory cell recruitment and the release of cytokines, which may augment rejection. However, CMV-infected sinusoidal endothelial cells (SECs) can also recruit Tregs and may also affect T-cell differentiation, since recent evidence suggests that primary CMV infection in seronegative recipients (D + R − ) leads to the emergence of hyporesponsive T cells and reduced late acute rejection. Some of the other complications may also relate to the capacity of the virus to infect VECs and biliary epithelium. For example, CMV infection of VECs is associated with a rapid procoagulant response, which may predispose to thrombosis. whereas CMV infection of biliary epithelium has been implicated in causing cholangitis, which may lead to the development of nonanastomotic biliary strictures.
Epstein–Barr virus and post-transplant lymphoproliferative disorder
Epstein–Barr virus (EBV) is an important pathogen and is associated with a broad spectrum disorders, from asymptomatic viraemia to a range of lymphoproliferative diseases, usually of B-cell origin, in immunocompromised individuals. Most adults (up to 95%) have been exposed to EBV, with latent viral infection usually controlled by NK and T cells. Impaired immune responses to primary and reactivation EBV infection with immunosuppression can result in post-transplant lymphoproliferative disorder (PTLD). At greatest risk are EBV-naive allograft recipients, which includes 50% of children, who may be unable to control the primary infection adequately and may develop subsequent B-lymphocyte lymphoproliferation. Monitoring of patients at high risk of primary infection by longitudinal EBV viral load measurement using quantitative polymerase chain reaction (PCR) shows seroconversion of 60% by 3 months. Detection of increased viral load does not necessarily indicate clinically significant disease, but a sustained viral detection for ≥6 months identifies a group at increased risk of PTLD. Other risk factors for PTLD include high-dose immunosuppression. Patients receiving grafts after 2000 fare better because of improved EBV monitoring. Other suggested risk factors, such as older age, transplantation for cholestatic liver disease, hepatitis C and CMV infection, are more controversial.
PTLD develops in 2–5% of liver transplant recipients overall, with a higher incidence of 5–10% in children without antiviral prophylaxis. PTLD usually develops within the first year in children, but later in adults. Up to one-third of patients present with allograft dysfunction and infiltration of the graft, usually in early-presenting cases; in others, lymphadenopathy, tonsillar enlargement or infiltration of other organs is seen. Most PTLD cases occurring in liver allograft recipients are derived from recipient lymphoid cells, although some cases of donor or mixed donor and recipient origin have been described. The last group was EBV negative and showed multiple clonal paraproteins. It is increasingly recognized that EBV may not be causative in up to a half or more of contemporary cases. A recent study using comparative genomic hybridization analysis showed that post-transplant cases of EBV-negative diffuse large B-cell lymphoma (DLBCL) had a genomic profile that was different to that seen in EBV-positive DLBCL and more closely resembled the profile seen in DLBCL occurring in immunocompetent individuals, suggesting that different molecular pathways are involved in the pathogenesis of EBV-positive and EBV-negative PTLD.
Clinically, typical signs of EBV infection, in particular fever, jaundice and lymphadenopathy, are only variably seen at presentation. There is a wide spectrum of disease, from mild hepatitis to polyclonal proliferations and monomorphic PTLD, as well as EBV-associated smooth muscle tumours. This spectrum is continuous, and clinicians should remember that there may be evolution in a single patient from benign to aggressive disease, as well as heterogeneity between multiple lesions.
Histologically, EBV hepatitis is characterized by portal mononuclear cell infiltrates with lobular disarray, swollen and apoptotic hepatocytes and accumulation of lymphoid cells in the sinusoids. Lymphoid cells can also be seen beneath the endothelium of portal and central vein branches. In some cases, sinusoidal mononuclear cells predominate with a mononucleosis-like pattern. The lymphoid cells in EBV infection are predominantly B cells, with plasma cells and immunoblasts present in the infiltrate. B cells can be highlighted by CD20 or CD79a immunostaining. Eosinophils and neutrophils are rarely seen. These features help to distinguish EBV hepatitis from the major differential diagnosis of acute rejection, which usually has a T-cell predominant infiltrate, few plasma cells or immunoblasts and conspicuous eosinophils. Additionally, although subtle duct injury can be seen in EBV hepatitis, it is only minor compared with the degree of inflammation, whereas in rejection, duct injury increases in proportion with the inflammatory infiltrate. Patients with mixed features of EBV hepatitis and rejection pose problems for diagnosis and management.
PTLDs are most often B-cell proliferations, with <15% showing T/NK cell differentiation, the latter less often EBV associated. The World Health Organization (WHO) classification of neoplastic lymphoid diseases recognizes four main categories of PTLD :
Early lesions: plasmacytic hyperplasia, infectious mononucleosis-like PTLD
Polymorphic PTLD: polyclonal (rare) or monoclonal
Monomorphic PTLD: B-cell and T/NK-cell types
Classic Hodgkin lymphoma-type PTLD
Early lesions are more typically seen in lymph node and tonsillar biopsies. In most cases, hepatic infiltration is characterized by a diffuse and heavy, predominantly portal infiltrate of lymphoid and plasma cells. The polymorphic PTLD is considered to be distinctive and only seen in immunocompromised individuals. There is a heavy portal infiltrate of mixed cells showing the full range of B-lymphocyte maturation; this effaces the portal architecture. Necrosis and conspicuous mitotic activity may be present, and some more monomorphous areas can occur. The pattern has a lymphomatous appearance, and the cells may be monoclonal and light chain-restricted despite the mixed pattern of the infiltrate. Histological workup should include routine histology, immunophenotyping, including assessment of light-chain restriction, and EBV-encoded nuclear RNAs (EBER) ISH. If only paraffin-embedded tissue is available, IHC assessment of light-chain restriction will generally fail unless plasmacytic differentiation is present. Genetic studies to determine clonality are a useful adjunct.
Monomorphic PTLD has essentially the same features as lymphoma occurring in immunocompetent individuals, with most of the infiltrating cells having an atypical appearance, prominent nucleoli, a high mitotic rate and areas of necrosis. Most are of B-cell type with morphology of DLBCL, Burkitt lymphoma or rarely, plasma cell myeloma. A small number of cases of EBV-associated T-cell lymphoma have also been documented in liver allograft recipients, and these may appear as peripheral T-cell lymphoma–not otherwise specified (NOS) or hepatosplenic T-cell lymphoma. In some patients, particularly those in whom PTLD is donor derived, the infiltrate is confined to the liver itself. These patients may show formation of localized masses, particularly in the porta hepatis, which can present with features of biliary obstruction.
Distinction from other causes of portal inflammation, particularly rejection, may be difficult, and in some cases, EBV-related PTLD may coexist with rejection or precipitate it when immunosuppression is reduced. As discussed, features favouring an EBV-related lymphoid infiltration are the presence of an infiltrate with many plasmacytoid cells, immunoblasts and frequent mitoses and sparse eosinophils ( Fig. 14.16 A ). IHC demonstration of B-lymphocyte predominance with CD20 or CD79a is also helpful. Immunohistochemistry or ISH can be used to demonstrate light- or heavy-chain restriction if plasmacytic differentiation is present, but immunoglobulin gene rearrangement analysis is more sensitive for demonstrating clonal proliferation of B cells in PTLD. IHC staining for virus-associated proteins (e.g. latent membrane protein 1) or EBER-ISH can also be used to detect the virus in tissue sections ( Fig. 14.16 B ); ISH is the more sensitive. A small number of EBV-positive cells may be found among portal inflammatory cells in other conditions, including acute and chronic rejection, and in inflammatory diseases occurring in a nontransplanted liver. Lymphoid infiltration should therefore only be ascribed to EBV infection if a substantial proportion of the cells can be shown to contain the virus.
Guidelines for management of PTLD have been published. During the early stages of polyclonal B-cell proliferation, the disease is frequently reversible with a reduction in immunosuppression. Treatment with antiviral agents may also be of benefit at this stage, although most of the EBV in transformed B cells is latent and not accessible to antivirals. Progression to a monoclonal and monomorphic B-cell proliferation indicates more aggressive disease, which is less likely to be responsive to changes in immunosuppression and requires other therapeutic approaches, including combination chemotherapy, anti-B-cell monoclonal antibodies and, in some specialized centres, adoptive immunotherapy with in vitro expanded virus-specific T cells. Recent data showing a 90% complete or partial response rate to sequential anti-B-cell antibodies followed by chemotherapy suggest improving outcomes.
Other rare opportunistic viruses
Adenovirus is a common pathogen that can cause significant hepatitis in immunocompromised patients. A recent review found that 48% of reported cases were associated with LT, usually in the first 6 months after grafting. It is more often seen in paediatric liver transplant recipients, but cases have also been described in adults. In the early stages there may be parenchymal inflammatory lesions resembling those seen in CMV hepatitis, with more severe cases showing small or larger areas of confluent parenchymal necrosis that may have a prominent haemorrhagic picture. The characteristic nuclear inclusions have an irregular contour, producing a ‘smudge cell’ effect ( Fig. 14.17 A ). Immunohistochemistry using an anti-adenovirus group antibody is useful to confirm the diagnosis ( Fig. 14.17 B ). Adenovirus infection can also involve the biliary tree with necrotizing cholangitis and loss of interlobular bile ducts.
Herpes simplex virus (HSV) infection is a rare cause of allograft hepatitis. It is characterized by foci of hepatocyte necrosis containing neutrophils and macrophages ( Fig. 14.18 A ). These may develop into more extensive areas of confluent necrosis associated with high mortality. Similar changes have also been observed with varicella-zoster virus (VZV) infection. Dense nuclear inclusions are seen in viable cells at the edge of necrotic areas ( Fig. 14.18 B ), only rarely detected in liver biopsy specimens. IHC staining for HSV antigens is helpful to confirm the diagnosis ( Fig. 14.18 C ).
Human herpesviruses 6 and 7 (HHV6 and HHV7) are relatively recently recognized members of the Betaherpesvirinae subfamily and are closely related to CMV. Infection with these viruses is common after organ transplantation, but clinical disease develops in only 1% of patients. HHV6 infection, present as a latent infection of lymphocytes and monocytes in 95% of humans, occurs in up to 80% of liver allograft recipients, usually between 2 and 8 weeks after transplant. Most cases represent reactivation and result in subclinical disease or a mild febrile illness. Hepatic involvement may be associated with a mild degree of graft dysfunction, a mild/moderate portal lymphocytic inflammation (without damage to bile ducts or portal vessels) and foci of parenchymal inflammation, including neutrophil aggregates similar to those seen in CMV hepatitis. The HHV6 antigens can also be demonstrated on IHC staining in portal lymphocytes. Rarely reported features include syncytial giant cell hepatitis, giant cell transformation of biliary epithelium (after cardiac transplantation) and periportal confluent necrosis/apoptosis causing a targetoid appearance around portal tracts. Although direct effects of HHV6 infection on the liver allograft appear fairly mild, the virus is an immunomodulator and has been implicated as potentiating other graft complications, including CMV disease, invasive fungal infection, possibly more aggressive recurrent HCV infection, rejection and an increased risk for graft loss in patients with unexplained graft hepatitis. HHV6-induced upregulation of adhesion molecules and lymphocyte activation may have a role in the pathogenesis of liver allograft rejection.
HHV7 infection has been implicated in the pathogenesis of CMV infection but does not appear to have any direct effects on the liver allograft itself.
HHV8 varies in frequency geographically and can cause viraemia in a minority of patients. Rare cases of Kaposi sarcoma associated with HHV8 infection have been reported in liver graft recipients. One case of donor-transmitted dengue viral infection developed 6 days after living-donor transplantation. The liver graft biopsy showed cholestasis.
Although bacterial infections are common in liver allograft recipients, it is uncommon to see direct morphological evidence of bacterial infection in liver biopsy specimens. Gram-negative sepsis is associated with a characteristic picture of severe cholestasis with ductular reaction and bile plugging at the periphery of portal tracts, and this needs to be considered in the differential diagnosis of intrahepatic cholestasis in the early postoperative period. Ascending cholangitis should be suspected if signs of biliary obstruction are combined with pus cells in the lumina of interlobular bile ducts. Lumenal pus cells are also seen in acute cellular rejection (ACR; see earlier), but the presence of other portal inflammatory changes helps to distinguish this from ascending infection. Ischaemic necrosis of larger bile ducts (see later) is frequently associated with bile sludging, and superadded bacterial infection is frequently seen in these areas.
Fungal and parasitic infections
The most common opportunistic fungal infections observed in liver allograft recipients are Aspergillus and Candida . Although the liver may be involved in systemic infection with these organisms, it is extremely rare to observe fungi in post-transplant needle biopsies. Superadded fungal infection is typically seen in association with necrotic bile ducts in hepatectomy specimens removed for ischaemic bile duct necrosis, and it may also be found in primary explants with biliary stasis. Rarely, fungal infection of the hepatic artery may result in thrombosis or pseudoaneurysm formation and rupture.
Infection of the graft with Schistosoma mansoni ova is rarely described in endemic regions, usually transferred from living donors. Because of the parasitic life cycle, only the donor needs to be treated.
Liver transplantation involves three sets of vascular anastomoses: hepatic artery, portal vein and vena cava. All three may be associated with technical complications, such as anastomotic stricture or kinking, or thrombosis resulting in vascular occlusion. Vascular complications occur in 7–15% of patients overall, with the higher rate in recipients of living-donor liver transplants. The effects of vascular impairment are mostly similar to those occurring in the native liver, although some important differences exist. Early thrombotic occlusion of the hepatic artery or portal vein within the first month often causes severe ischaemic injury because of a lack of collateral development. Later, stenosis or thrombosis is usually better tolerated and may be clinically silent. The transplanted liver is also more vulnerable to hepatic arterial compromise because of the greater sensitivity of the biliary tree to arterial ischaemia. Depending on timing and severity, a range of biliary lesions can be seen, from severe biliary necrosis to bile leaks, abscesses, sepsis or nonanastomotic biliary strictures.
Hepatic artery complications
Hepatic artery thrombosis is the most common vascular complication, occurring in 2.5–11% of liver transplants. Although a high incidence of up to 29% was described in paediatric recipients when whole-liver grafts were used and the arterial diameter was smaller, subsequent experience using reduced-size grafts with larger arterial branches for anastomosis shows a lower frequency, approaching that of adults. Predisposing factors include small-calibre arteries, abnormal arterial anatomy, a small-for-size or a segmental graft or previous transplantation and nontechnical factors such as CMV infection, ABO incompatibility, a thrombotic tendency, increased donor age, transplantation for porphyria and previous episodes of rejection.
Nonocclusive arterial insufficiency also occurs and has several causes. Hepatic artery stenosis is caused by anastomotic narrowing in approximately 5% of patients and, although occasionally seen early, usually occurs >3 months after LT. Biliary complications develop in about one-third of patients, and the diagnosis and management are usually radiological. Splenic arterial steal syndrome is an under-recognized early cause of graft ischaemia, occurring in up to 6–7% of allografts. Initial descriptions suggested that it was caused by siphoning of hepatic to splenic arterial flow because of hypersplenism, but more recently it is proposed to occur because of portal hyperperfusion causing reflex hepatic arterial hypotension and reversed diastolic flow. Although less often seen now, ischaemia is a cause of graft failure in the early postoperative period, even without overt lesions in the hepatic or portal vessels. In early studies, up to 50% of failed allografts removed within the first month after LT had morphological evidence of ischaemic damage. Many cases have a multifactorial aetiology. In some cases, thrombi can be detected in hepatic arteries and/or portal vein branches, although in many instances there is no demonstrable vascular occlusion. Improvements in patient selection, graft preservation and surgical technique have probably all contributed to a reduced incidence of nonocclusive infarction in the early post-transplant period.
Because histological changes are often patchy and variable in pattern, radiological imaging including Doppler ultrasound is important in the clinical diagnosis of arterial insufficiency. In the early post-LT period, graft ischaemia is characterized by irregular ‘geographical’ areas of infarction with surrounding haemorrhagic borders ( Fig. 14.19 A ). Histology shows coagulative necrosis of the liver parenchyma with variable infiltration by neutrophil polymorphs ( Fig. 14.19 B ). There is relative sparing of portal tracts, and in less severe cases, necrosis may be confined to peripheral acinar regions. Post-transplant biopsies may be useful in establishing that ischaemic damage is present but are not reliable in determining the severity because there is considerable sampling variation. Small peripheral infarcts are frequently seen in liver allografts that are otherwise functioning well and can cause concern if they are inadvertently biopsied. Conversely, even livers showing extensive infarction frequently contain large areas in which hepatocytes are well preserved. Changes seen in graft ischaemia occurring in the immediate postoperative period merge with those observed in cases of severe preservation/reperfusion injury, ‘primary nonfunction’ and also unexplained massive haemorrhagic graft necrosis. A recent study suggested that a combination of increased hepatocellular mitosis and apoptosis without significant lobular inflammation may be an indication of hepatic arterial compromise in the absence of overt ischaemic lesions.
An important complication of later hepatic artery thrombosis is ischaemic bile duct necrosis, typically affecting large intrahepatic bile ducts. The areas of necrosis are associated with ulceration, bile staining of surrounding parenchyma and colonization by bacterial or fungal organisms; abscesses may supervene. Later, arterial occlusion is often more insidious and can be associated with the development of nonanastomotic strictures and dilation of the ducts, and biliary sepsis can be a major problem. In patients undergoing retransplantation for ischaemic biliary complications, there is usually little damage to the liver parenchyma.
Hepatic artery aneurysm and pseudoaneurysm are complications related to infection at the arterial anastomosis. Intrahepatic aneurysms are iatrogenic, more rarely seen and arise secondary to biopsy or stent placement. Rupture with high mortality can occur.
Portal vein thrombosis (PVT) and stenosis are rare, occurring more frequently after adult living-related liver transplantation (LRLT) and also in the paediatric population. Early thrombosis is a cause of severe graft dysfunction and graft failure, whereas later thrombosis is associated with portal hypertension and ascites. Histological changes seen in biopsies from children with late PVT include portal fibrosis, centrilobular fibrosis, hepatocyte ballooning and cholestasis. Occasional cases have been associated with cirrhosis. Early management is with thrombolysis, or later with angioplasty of stenotic lesions.
Occlusion of the hepatic vein or inferior vena cava caused by anastomotic stricture, kinking or thrombosis is also rare, with an overall frequency of 1% in a recent study. Occlusion of the vena cava may result in the Budd–Chiari syndrome (BCS), mainly seen in the context of recurrent disease. An acute BCS has also been described as a rare complication of using the ‘piggyback’ technique for hepatic venous anastomosis. In some cases of ‘piggyback syndrome’, hepatic venous outflow obstruction appears to be aggravated by an upright posture and may not be detected radiologically when the patient is lying flat; liver biopsy plays an important diagnostic role in such cases ( Fig. 14.19 C ). The histological diagnosis of venous outflow obstruction in the liver allograft is problematic, because congestive changes indistinguishable from those occurring in BCS can be also seen as a result of rejection or drug-related hepatic veno-occlusive lesions.
Other vascular lesions
The three main forms of rejection may all be associated with graft ischaemia. Changes are most marked in severe acute antibody-mediated rejection, which produces a picture resembling nonthrombotic infarction of the liver. It has also been suggested that sinusoidal fibrosis in centrilobular regions, sometimes progressing to a veno-occlusive disease pattern, may be a manifestation of chronic antibody-mediated injury. Alterations in blood flow have also been noted in acute cellular rejection, although it is not clear if these changes produce lesions that are detected histologically. In chronic rejection, subtle fibrous occlusive lesions involving hepatic arteries, portal veins and hepatic veins have been implicated in the pathogenesis of bile duct loss, perivenular hepatocyte necrosis and parenchymal fibrosis. Early immunological injury of endothelial cells followed by fibrosis and vascular thrombosis is the likely pathogenesis.
Focal nodular hyperplasia (FNH) has recently been described as a late complication of LT in four patients, three of whom had conditions associated with altered hepatic vascular perfusion, which is thought to be important in the pathogenesis of FNH occurring in the native liver.
Nodular regenerative hyperplasia (NRH) is a form of parenchymal nodularity without significant fibrosis that results from vascular injury. NRH is a common finding in late post-transplant biopsies and is discussed in a later section.
Biliary complications are a common cause of graft dysfunction. These may present as leaks, strictures, casts, sludge or stones and have an overall prevalence ranging from 10% to 25%. The risk is higher, sometimes double or greater, with increasing donor complexity related to LRLT and split-liver transplantation, as well as the more frequent use of extended-criteria donors. Despite improvements in their management, biliary complications remain an important cause of post-transplant morbidity and are associated with a reduction in graft survival.
The aetiology of biliary complications is multifactorial and includes surgical, vascular and immunological causes. Relying on the hepatic artery alone for their afferent supply via the peribiliary vascular plexus, the bile ducts are more vulnerable to ischaemic injury than other areas of the liver. Since the blood flow in the larger ducts is from peripheral to lumenal, the lining biliary epithelium is particularly prone to damage; in fact, almost all grafts develop extensive loss of lining epithelium immediately after grafting, with variable injury in the surrounding stroma and peribiliary glands of the large bile ducts. However, clinically significant ischaemic injury is much less frequent due to regeneration of the biliary lining, which appears to rely on progenitor cells in the deep peribiliary glands; ischaemic damage to these or to the small arterioles of the vascular plexus is associated with a high rate of biliary complications.
Biliary complications can be subdivided into two main types. Anastomotic complications develop at the site of the surgical anastomosis of the donor bile duct and are mainly caused by technical problems. Nonanastomotic complications involve large bile ducts within the liver allograft itself and are mostly related to vascular and immunological events. However, the distinction between these two processes is not always clear-cut, and vascular and immunological factors have also been implicated in the pathogenesis of anastomotic biliary complications.
Historically, complications related to the biliary anastomosis were a major cause of morbidity and mortality after LT, and biliary reconstruction was considered the ‘technical Achilles heel’ of LT. Refinements in surgical technique, including the widespread use of duct-to-duct anastomosis, have significantly reduced the rate of biliary complications. A higher incidence in children presumably reflects the small size of the bile ducts and their vascular supply.
Anastomotic complications present either as bile leaks, usually occurring in the early postoperative period, or as bile duct strictures, which present somewhat later, usually during the first 6 months.
Histological features of biliary obstruction are similar to those seen in a nontransplanted liver. Acute biliary obstruction is characterized by portal changes of oedema, bile ductular reaction and neutrophilic infiltration accompanied by variable cholestasis. Varying numbers of eosinophils may also be present in portal areas in cases of biliary obstruction. In some cases, biliary obstruction in the liver allograft can remain asymptomatic for a considerable period. In such patients, biopsies may show signs of chronic cholestasis, including deposition of copper associated protein and progressive periportal fibrosis, resulting eventually in biliary cirrhosis. Liver biopsy is useful in determining the severity of biliary fibrosis in these patients, in addition to identifying nonbiliary causes for graft dysfunction. Rare cases of late biliary obstruction have been attributed to the development of traumatic neuroma at the liver hilum.
Nonanastomotic biliary complications have become increasingly recognized as important after LT, occurring in 5–20% of recipients. There may be an identifiable ischaemic basis, such as hepatic artery thrombosis, low flow due to hepatic artery stenosis or arterial steal syndrome of the coeliac axis. If there is no clear ischaemic cause, the term ‘ischaemic-type biliary lesion’ (ITBL) has been used, with a wide range of risk factors identified. Ischaemic mechanisms include preservation/reperfusion injury associated with prolonged cold ischaemia of >10–13 h or warm ischaemia, vascular sludging in the small capillaries caused by viscous UW preservation solution, use of marginal donors, particularly non-heart-beating (DCD) donors, and small-for-size grafts, where excess portal flow causes reflex hepatic arterial spasm. Predominantly immunological causes of ITBL have also been postulated, including the use of ABO-incompatible grafts, ABO-compatible antibody-mediated rejection, transplantation for primary sclerosing cholangitis (PSC) or autoimmune hepatitis, previous CMV infection, the obliterative vasculopathy of chronic rejection, gender-mismatched grafts or CC chemokine receptor 5 (CCR5) delta-32 mutation, which impairs the recruitment of Tregs. Toxicity due to bile acids may play a role in potentiating injury.
Nonanastomotic bilary stricturing is not a uniform disorder, and two patterns have been suggested. Most cases appear early in the first year afer LT, presenting as strictures of large ducts at or below the main duct bifurcation and correlating with ischaemic mechanisms such as long cold or warm ischaemic times. A second group is more peripheral, occurs later (>1 year post-transplant) and also affects smaller ducts. These late cases correlate with immunological factors. There is some overlap, particularly in patients transplanted before routine immunsuppressive regimens were used for ABO-incompatible grafts, in whom disease presented early and was often extensive.
Ischaemic biliary complications present as either strictures or dilations involving one or more large intrahepatic bile ducts. The radiological appearances can resemble those seen in PSC. Superadded infection frequently leads to the formation of one or more intrahepatic abscesses, for which retransplantation is frequently required. In some patients presenting later, sludging of cholesterol, calcium bilirubinate and mucus may occur as a result of ischaemic biliary dysfunction, leading to the formation of debris, biliary casts and stones, which exacerbate the obstruction. The term ‘biliary cast syndrome’ has been used to describe cases with prominent bile sludging and cast formation.
Liver biopsies obtained from patients with ischaemic biliary complications typically show features of biliary obstruction. However, the changes are frequently patchy in distribution, and only minor histological abnormalities may be present in biopsies of patients who have gross radiological abnormalities in the hilum. Interlobular bile ducts may show cytological atypia with an atrophic appearance resembling changes seen during the early stages of chronic rejection. Perivenular hepatocyte dropout, ballooning and cholestasis, also features of chronic rejection, have been documented in cases of ischaemic cholangiopathy.
Examination of failed allografts at retransplantation typically shows irregular collections of bile-stained material centred on necrotic, ulcerated and inflamed large- and medium-sized bile ducts. These often contain biliary sludge or bile casts ( Fig. 14.20 ). There is frequently superadded bacterial or fungal infection. Bile extravasation may extend for some distance into surrounding tissue, accompanied by necrosis and inflammation. In some cases, there are also fibro-obliterative lesions involving medium-sized ducts, disappearance of small interlobular ducts and secondary changes of ductular reaction and biliary fibrosis resembling changes seen inPSC.
Several of the features of biliary obstruction can also be seen in acute cellular rejection. Changes favouring a diagnosis of acute rejection rather than simple biliary obstruction include a prominent mononuclear portal infiltrate, conspicuous bile duct damage and the presence of venular endothelial inflammation.
Cases of biliary obstruction with atrophic/dystrophic changes in ducts or with perivenular dropout can appear similar to chronic rejection. However, CR is characterized by lesions predominantly affecting small bile ducts, occurring in the absence of secondary biliary features such as bile ductular reaction or stellate periportal fibrosis. By contrast, large ducts are predominantly affected in ischaemic cholangitis, with changes in smaller portal areas occurring as a secondary event.
The prominent ductular reaction in cholestatic hepatitis B or C is associated with a prominent periportal ductular reaction, lobular disarray, pallor of hepatocytes and relatively less proliferation of the ducts in portal areas. The degree of ductular reaction is disproportionate to other features of duct obstruction usually seen, and accumulation of copper-associated protein is not a feature. Very high viral loads support a diagnosis of viral hepatitis.
Recurrent PSC and ischaemic cholangitis have very similar appearances radiologically and histologically. The difficulty in distinguishing them is further compounded by similar risk factors identified for both diseases, as well as transplantation for PSC being a risk factor for late ITBS. Although imperfect, it has been suggested that recurrent PSC should be defined by strict criteria, including confirmed PSC before LT, diagnosis >90 days after LT, characteristic strictures on cholangiography and/or fibro-obliterative lesions or fibrous cholangitis histologically and absence of hepatic artery thrombosis/stenosis, chronic rejection, anastomotic strictures alone, nonanastomotic strictures before day 90 or ABO incompatibility.
With improved long-term survival following transplantation, problems related to disease recurrence are becoming increasingly important. Most of the common diseases for which LT is performed in adults can recur. The incidence and clinical consequences of disease recurrence vary considerably ( Table 14.11 ). For some conditions (e.g. hepatitis C), recurrence is common and early and has an important impact on graft function, in some cases resulting in graft failure or death. Other diseases (e.g. PBC) recur in a mild, often subclinical form, with no obvious impact on graft or patient survival. Recurrent disease is less problematic in children, the great majority of whom are transplanted for conditions that do not recur, such as biliary atresia and metabolic diseases.
|Hepatitis B||<10%||Higher frequency in 1980s–early 1990s (15–85%). Incidence and clinical impact now greatly reduced by use of antiviral therapy before and after transplant.|
|Hepatitis C||>90%||Recurrent infection universal. Most cases associated with graft damage. Progressive disease common: 20–50% cirrhotic by 5–10 years. Likely to become much less frequent with the advent of new antiviral therapies.|
|Primary biliary cholangitis (PBC)||20–50%||Most cases have mild/asymptomatic disease, frequently diagnosed on protocol biopsies. Rare cases (<1%) have progressed to cirrhosis or graft failure.|
|Primary sclerosing cholangitis (PSC)||20–30%||More frequently clinically symptomatic than recurrent PBC. Approximately 10% progress to graft failure. Histological and radiological features may be difficult to distinguish from ischaemic cholangiopathy.|
|Autoimmune hepatitis||20–30%||Most cases occur as a result of suboptimal immunosuppression and respond to immunosuppressive therapy. Diagnosis based on a combination of biochemical, immunological and histological findings.|
|Alcoholic liver disease (ALD)||10–30%||Return to drinking is common, but serious graft complications are relatively uncommon. Fatty change is the most common finding. Occasional cases progress to steatohepatitis or fibrosis.|
|Nonalcoholic fatty liver disease (NAFLD)||20–40%||Risk factors for NAFLD often persist after transplantation and may be exacerbated by immunosuppressive drugs and other transplant-related factors; 10–40% progress to steatohepatitis, and up to 12% become cirrhotic.|
|Hepatic neoplasms||Variable||Early studies reported 30–50% recurrence rates for hepatocellular carcinoma. Incidence now reduced (<10–20%), due to better pretransplant selection criteria.|
|High recurrence rates (up to 90%) reported in early studies of cholangiocarcinoma, due to hilar location and incomplete resection. Improved survival now achieved in some centres using better selection criteria.|
The histological diagnosis of recurrent disease is influenced by two important transplant-related factors. First, there may be similarities or interactions with other complications of LT. Probably the most challenging example of two conditions with overlapping histological features and complex pathogenic interactions involves hepatitis C and acute cellular rejection. Other examples with overlapping morphological features include recurrent biliary disease (PBC or PSC) and chronic rejection; recurrent PSC and ischaemic cholangiopathy; and recurrent autoimmune hepatitis (AIH) and graft hepatitis related to alloimmune mechanisms (e.g. late cellular rejection, de novo AIH). The second issue relates to the effects of immunosuppressive therapy. Diseases thought to be immune mediated (e.g. AIH, PBC) are likely to be prevented from recurring or may progress more slowly as a result of immunosuppression. Conversely, viral infections behave in a more aggressive manner in immunocompromised individuals and may display atypical features not seen in immunocompetent persons.
Recurrent viral infection
Incidence and risk factors
During the 1980s and early 1990s, recurrence of HBV infection was a common and important complication of LT. In a large, combined European series of 372 patients the overall actuarial risk of recurrent infection 3 years after LT was 50%. This was associated with a marked reduction in overall graft survival that was <50% at 5 years. The most important risk factor for recurrent infection was the presence of active viral replication at transplantation. The highest incidence of recurrent HBV infection (>80%) thus occurred in cirrhotic patients who were HBV-DNA positive. Conversely, fulminant hepatitis B infection was associated with a much lower risk of reinfection (<20%) because a massive immune response often resulted in the elimination of viral antigens. Co-infection with hepatitis delta virus (HDV) was likewise associated with a lower risk of recurrent HBV infection, due to the inhibitory effect of HDV on HBV replication.
The use of antiviral therapy before and after transplant has resulted in a marked reduction in both incidence and severity of recurrent HBV infection in recent years. Pretransplant treatment with lamivudine and more recently, entecavir, adefovir or tenofovir, is mainly directed at reducing viral DNA levels at transplant. Post-transplant prophylaxis with anti-HBs immunoglobulin (HBIg) and/or a nucleos(t)ide inhibitor is then used to prevent recurrence. The risk of developing recurrent HBV infection is now significantly less than 10% and in some centres is approaching zero. High pretransplant HBV-DNA levels and concomitant hepatocellular carcinoma (HCC) continue to be recognized as risk factors for recurrent HBV infection. Survival in patients with HBV-related cirrhosis is now comparable with that seen in other cirrhotic patients undergoing LT. Viral mutations are the basis for HBV reinfection in the setting of prophylactic antiviral therapy and can be detected before serum HBV-DNA levels rise or biochemical signs of liver injury are manifest. Even in patients who develop recurrent HBV infection, a good outcome can be achieved with the use of other nucleos(t)ide inhibitors. Small amounts of HBV-DNA can be detected in the serum or liver of patients with no histological or serological evidence of recurrent HBV infection, providing a potential source for recurrent disease if antiviral therapy is discontinued.
Histopathological features and natural history
Histopathological findings in HBV infection of the liver allograft can be divided into two main categories. Typical features of HBV infection, as seen in the nontransplanted liver, occur in the majority of cases of recurrence. More rarely, atypical patterns of liver damage occur, presumably related to other factors present in liver allograft recipients.
Typical features of recurrent HBV infection
Three main phases of reinfection have been identified. In the initial incubation phase (first 3 months post-transplant), changes directly attributable to HBV are rarely present, although a slight disarray of hepatocytes is sometimes seen. Positive IHC staining for HBV-associated antigens is usually the first sign of recurrent infection, typically in the form of focal cytoplasmic or nuclear staining for core antigen ( Fig. 14.21 ). During the second phase of early reinfection (usually 1–6 months post-transplant), biopsies typically show features of lobular hepatitis, which is usually mild. Varying degrees of portal inflammation may also be present. Portal and periportal inflammatory changes generally become more prominent as the disease progresses to the third stage of established ( chronic ) infection (>6 months post-transplant) and resemble those seen in chronic hepatitis B involving the nontransplanted liver. In early studies the severity and speed of progression were considerably greater in the liver allograft than in the native liver. In a combined series of 42 patients reinfected with HBV prior to 1991, 41 developed acute hepatitis, 20 progressed to chronic hepatitis and eight had become cirrhotic within a period ranging from 7 to 70 months.
Atypical features of recurrent HBV infection
Three main atypical patterns of reinfection have been identified: hepatocyte ballooning, fatty change and a distinctive cholestatic syndrome (‘fibrosing cholestatic hepatitis’). These frequently occur in combination and appear to be part of a spectrum of HBV infection occurring in immunocompromised individuals. The use of antiviral strategies, which reduce viral levels even in cases where recurrent infection occurs, has prevented these atypical manifestations of recurrent HBV infection from occurring.
Hepatocyte ballooning typically occurs without significant accompanying inflammation and is associated with diffuse, often panacinar, cytoplasmic and nuclear immunostaining for HBcAg ( Fig. 14.21 B and C ). A similar pattern of immunostaining has also been described for HBeAg. These observations suggest that massive viral replication can result in direct cytopathic damage to hepatocytes. Hepatocyte ballooning may be accompanied by varying degrees of fatty change. In cases where steatosis is particularly prominent, the term ‘steatoviral hepatitis’ has been used.
The term ‘fibrosing cholestatic hepatitis’ (FCH) was used to describe a distinctive pattern of graft damage, which typically presented during the first few months after LT, when levels of immunosuppression are highest. FCH is characterized histologically by periportal fibrosis extending as thin perisinusoidal strands for varying distances into the liver lobule, accompanied by flat ductular structures without an identifiable lumen ( Fig. 14.21 D–F ). Liver parenchyma shows prominent hepatocyte ballooning, cholestasis and widespread immunoreactivity for HBcAg (nuclear and cytoplasmic) and to a variable degree for HBsAg. Inflammatory changes are generally mild or absent. Prior to the availability of preventive measures and specific antiviral therapy, this type of injury usually progressed rapidly to liver failure, but it is no longer seen in current practice.
HBV-associated nucleic acids have been identified in peripheral blood mononuclear cells and several extrahepatic sites, and these are presumed to be the source of reinfection in the liver allograft. Two main pathways have been implicated in causing graft damage following reinfection with HBV. First, immune-mediated mechanisms, as seen in the nontransplanted liver, mainly involve HLA class I-restricted viral recognition by HBV-specific CD8 T cells and are probably responsible for producing the more typical inflammatory changes seen in recurrent HBV infection. Second, direct cytopathic damage related to uncontrolled viral replication occurring in the setting of immunosuppression is the more likely mechanism for producing atypical patterns of recurrent HBV infection. The immunosuppressive agents used in LT may have a direct effect in promoting HBV replication. Corticosteroids can activate a steroid-responsive promoter in the HBV genome, and azathioprine may also directly promote viral replication. HLA-matching is not done routinely in LT, and host-viral interactions involving recognition of class I MHC molecules by cytotoxic T lymphocytes are thus likely to be impaired in most cases. HLA class I-independent HBcAg-specific T-cell responses have been demonstrated in liver allograft recipients with recurrent HBV infection.
The development of mutations in the HBV genome is an important mechanism enabling the virus to escape from immune recognition or antiviral therapy. Examples include the precore and pre-S mutants, which are associated with more severe disease in the liver allograft ; mutations in the HBs protein, which have been detected in patients developing HBV reinfection despite hepatitis B immune globulin (HBIG) prophylaxis ; and mutations in the YMDD locus of the HBV polymerase gene that occur in cases where ‘viral escape’ occurs with lamivudine therapy. Other mutations have been described as a mechanism for viral escape in patients treated with other nucleos(t)ide inhibitors. In FCH the massive viral burden in hepatocytes is likely to interfere with basic cell function, including the capacity for replication, acting as a stimulus for progenitor cell activation. This could account for the extensive ductular reaction observed in this situation.
HBV infection in other immunocompromised individuals
More severe HBV infection has also been described in other immunocompromised individuals, including patients undergoing haemopoietic cell transplantation (discussed later) and recipients of renal and cardiac allografts. Manifestations of more severe disease in these cases include more rapid progression to cirrhosis and the development of FCH. The advent of effective antiviral therapies has improved the outlook for HBV-positive individuals undergoing renal transplantation.
Incidence and risk factors
The recent emergence of direct-acting antiviral agents (DAAs) as safe, effective and well-tolerated therapy for chronic HCV infection is likely to transform LT for this indication in the coming years, similar to the evolution of grafting for HBV over the past two decades. Early promising results in experimental studies suggest that the use of DAAs will be transferred to routine transplant practice in coming years and can be expected to reduce the significant burden of recurrent disease. Tolerance and efficacy of therapy are greater than with interferon-based regimens. Use of DAAs is likely to impact HCV infection in the liver allograft in a number of ways. Pretransplant viral eradication will reduce the need for LT and should also reduce the risk of graft reinfection in patients undergoing transplantation. The role of DAAs in treating patients who are viraemic after transplant is evolving. Early (pre-emptive) therapy could theoretically prevent problems with graft reinfection. However, in many centres the use of DAAs is currently restricted to patients who have advanced disease or are at high risk of aggressive HCV recurrence (e.g. early fibrosis, cholestatic HCV), and liver biopsy continues to play a role in the assessment of such cases.
For patients who are viraemic at transplantation, reinfection is universal. Graft reinfection probably begins during reperfusion, and viral replication commences within a few hours thereafter. Viral RNA levels typically decrease in the immediate postoperative period, but rapidly return to pretransplant levels, often within a few days, and subsequently increase further, typically reaching up to 100 times pretransplant levels 2–3 months after transplant. About 50% of patients remain asymptomatic during the first 12 months after transplant, but the majority (70–95%) will eventually develop histological features of graft hepatitis. Risk factors predisposing to more severe liver disease are defined either as progression to fibrosis/cirrhosis in post-transplant biopsies or development of graft failure ( Table 14.12 ). The most important risk factors relate to the use of immunosuppression, either as baseline therapy or to treat episodes of acute rejection. The increased viral replication that occurs in the setting of immunosuppression predisposes to more severe HCV-related graft damage, which may be aggravated when the immune system is reconstituted after tapering of immunosuppression, particularly if this is done rapidly. Levels of viraemia are also important and appear to correlate both with the onset of clinical episodes of acute graft hepatitis and with subsequent progression to chronic liver disease or graft failure. Older donor age has emerged as an important risk factor. Development of features related to the metabolic syndrome is more common in patients undergoing LT for HCV cirrhosis than in those transplanted for other chronic liver disease, and it is increasingly recognized as a risk factor for more severe HCV-associated disease in the liver allograft. Patients co-infected with HCV and human immunodeficiency virus (HIV) have been shown to have an increased risk of more rapid fibrosis progression after orthoptic LT compared with monoinfected patients.
|Viral factors||HCV genotype (1b or 4)|
|HCV-RNA levels (pre- and post-transplant)|
|Lack of viral quasispecies diversity|
|African American receiving race-mismatched graft|
|Gender (conflicting data)|
|Co-infection with HIV|
|Transforming growth factor β1 polymorphism|
|Combination of gene polymorphisms|
|Hepatic iron concentration|
|Co-infection with herpesviruses (CMV, HHV6)|
|Histological findings in early post-transplant biopsies||Fatty change (in first month and at 1 year)|
|Progenitor cell expansion/ductular reaction|
|Hepatocyte cell cycle arrest or replicative senescence|
|Hepatic stellate cell activation|
The natural history of recurrent infection in individual patients appears to be determined relatively early, and histological features seen in biopsies taken during the first 12 months after LT may also be predictive of more aggressive disease behaviour. Examples include the presence or severity of necroinflammatory activity at first presentation with HCV hepatitis during the first 6 months and in protocol biopsies obtained at 4–12 months ; presence or severity of macrovesicular steatosis in biopsies obtained during the first month or at 1 year ; amount of hepatocyte apoptosis ; presence of prominent hepatocyte ballooning and/or cholestasis ; early periportal sinusoidal fibrosis ; extent of hepatic stellate cell (HSC) activation demonstrated by immunostaining for α-smooth muscle actin (αSMA) in biopsies obtained 3–6 months post-transplant or by immunostaining for glial fibrillary acidic protein (GFAP), a putative marker of early HSC activation ; increased hepatocyte replicative arrest as detected by either hepatocellular expression of the cell cycle entry protein minichromosome maintenance protein 2 (mcm-2), p21 or senescence-associated β-galactosidase ; degree of progenitor cell expansion and/or ductular reaction ; fibrosis stage at 1-year protocol biopsy (Ishak stage ≥2) ; and extent of collagen-proportionate area by image analysis at 1 year or when advanced fibrosis is present. The rate of initial fibrosis progression also predicts progression to cirrhosis and hepatic decompensation.
Histopathological features and natural history
Typical features of HCV infection, resembling those seen in the nontransplanted liver, occur in the majority of cases. More rarely, there are atypical patterns of liver damage, presumably related to other factors present in liver allograft recipients.
The role of liver biopsy in the diagnosis and management of recurrent HCV infection is changing. Distinction between recurrent HCV and other graft complications such as rejection cannot be made on the basis of clinical or biochemical changes alone, and liver biopsy is thus important in establishing a diagnosis of HCV infection in cases where the cause of graft dysfunction is uncertain. Liver biopsies have also been used to assess disease severity, particularly fibrosis. Histological abnormalities are frequently observed in protocol biopsies from HCV-positive patients who are clinically well with good graft function, and the changes seen in these biopsies have implications for prognosis and treatment. For example, the presence and severity of fibrosis at 1 year are predictive for subsequent progression to cirrhosis and graft failure. Patients who develop early fibrosis are thus more likely to benefit from treatment with antiviral therapy. As with HCV infection in the native liver, the use of noninvasive markers of fibrosis, especially transient elastography (TE), has substantially reduced the frequency of liver biopsy in liver allograft recipients. A number of studies have shown that noninvasive methods are reliable both for detecting significant or advanced fibrosis and for predicting disease progression. As a result of these observations, the use of protocol biopsies in HCV-infected liver allograft recipients has been discontinued in most centres. Liver biopsies continue to be obtained from patients undergoing assessment for antiviral therapy with novel DAAs, particularly in the context of clinical trials. The utility of liver biopsy in this setting includes establishing a diagnosis of HCV infection (and excluding other causes of graft dysfunction such as rejection) as well as assessing disease severity.
Typical features of recurrent HCV infection
Three main phases can be identified. During the initial stage of graft reinfection (0–2 months post-transplant), when viral RNA levels are already high, the assessment of HCV-related changes is difficult due to the frequent presence of other causes of graft damage, such as preservation/reperfusion injury and acute rejection. HCV-related inflammation is rarely seen at this stage. Instead, there is typically a picture of lobular disarray associated with hepatocyte ballooning, acidophil bodies and sometimes increased mitotic activity. Fatty change may also be seen at this stage. Severe steatosis may be the initial histological manifestation of recurrent hepatitis in cases of HCV genotype 3. The second stage of established graft infection (2–4 months post-transplant) is characterized by more typical features of acute hepatitis. Inflammation is generally mild, predominantly lobular in location and is associated with varying degrees of lobular disarray, hepatocellular ballooning, acidophil bodies and Kupffer cell enlargement ( Fig. 14.22 A ). There may also be evidence of increased hepatocyte turnover, with variation in hepatocyte size, scattered mitotic figures and an increased Ki-67 labelling index ( Fig. 14.22 B ). Sinusoidal lymphocytosis is sometimes a feature. Varying degrees of portal inflammation may also be seen at this stage. HCV hepatitis occasionally occurs within the first 2 months, with cases documenting inflammatory changes attributed to recurrent HCV infection as early as 9 days after transplant. The third stage of progressive liver damage (>6 months post-transplant) is characterized by histological features of chronic hepatitis resembling those seen in chronic HCV infection of the nontransplanted liver ( Fig. 14.22 C ). There is a predominantly mononuclear portal inflammatory infiltrate, typically associated with lymphoid aggregates and focal lymphocytic infiltration of bile ducts. Variable interface hepatitis is seen. There is also usually spotty lobular inflammation, fatty change (usually macrovesicular) and focal acidophil body formation. Approximately 50% of patients have histological evidence of chronic hepatitis at 1 year after transplant, this figure increasing with longer follow-up.
There is increasing evidence that HCV infection behaves more aggressively in the liver allograft than in the nontransplanted liver. Histologically, more severe degrees of necroinflammatory activity have been noted, including areas of confluent and bridging necrosis, which are very rarely, if at all, seen in nonimmunocompromised individuals. In such cases, the possibility of an additional cause for centrilobular necroinflammation (‘central perivenulitis’) should be considered, the main ones being acute rejection and de novo AIH. However, in some cases these changes can be attributed to HCV infection alone ( Fig. 14.22 D ). When the diagnosis is uncertain histologically, information relating to recent changes in immunosuppression and/or antiviral therapy and HCV-RNA levels usually help in deciding whether changes seen are related to viral infection or an allo/autoimmune response. The severity of inflammation correlates with fibrosis stage at biopsy and may be important in the pathogenesis of the more rapid progression of fibrosis seen with HCV infection in the liver allograft compared with the native liver. Fibrosis is frequently present in biopsies obtained 1 year after LT, and a few patients are already cirrhotic by this time. Approximately 20–40% are cirrhotic 5 years after transplant, and 50% by 7–10 years. Connective tissue stains are required for accurate assessment of fibrosis ; morphometry may also be helpful in assessing the severity of fibrosis. There have been occasional cases of de novo HCC occurring as a complication of recurrent HCV infection with cirrhosis. Most occurred within 7 years of transplantation, again supporting an accelerated disease progression in the liver allograft, although one has been described >10 years after interferon treatment and viral clearance in a noncirrhotic graft. Recurrent HCV infection is now recognized as a major cause of late graft failure and in some centres has become the leading indication for retransplantation in patients surviving >12 months post-transplant.
Atypical features of recurrent HCV infection
A number of atypical histological manifestations of recurrent HCV infection have been recognized. These are probably related to HCV infection occurring in a setting of immunosuppression and resemble those seen in cases of atypical recurrent HBV infection.
Cholestatic hepatitis C (‘fibrosing cholestatic hepatitis’)
A small proportion of cases develop a severe cholestatic syndrome that resembles fibrosing cholestatic hepatitis (FCH) of recurrent HBV infection. This typically occurs in the early post-transplant period (1–6 months) and is associated with high serum and intrahepatic HCV-RNA levels and an impaired HCV immune response, suggesting that it may represent a form of severe hepatocellular injury related to direct cytopathic effects of HCV. The reported prevalence varies from 2% to 13%, possibly reflecting variation in the criteria used to make a diagnosis of cholestatic HCV. A higher frequency of up to 19% has been reported in HCV/HIV co-infected individuals. Early histological features include severe bilirubinostasis and centrilobular hepatocyte ballooning, which may be accompanied by lobular disarray. Lobular inflammation is typically mild or absent. Foci of delicate perisinusoidal fibrosis may also be detected at this stage ( Fig. 14.22 G ). Subsequently, there is portal tract expansion with prominent marginal zones of ductular reaction, mimicking changes seen in biliary obstruction ( Fig. 14.22 E and F ). Varying degrees of portal inflammation, usually mild, may also be present. More extensive fibrosis, sometimes with a bridging pattern, is also evident at this stage. Other causes of cholestasis (e.g. biliary obstruction, drug toxicity) should be excluded before attributing cholestasis to HCV infection alone. Histological features favouring a diagnosis of cholestatic HCV over biliary obstruction include hepatocyte ballooning and lobular disarray, periportal sinusoidal fibrosis ( Fig. 14.22 G ) and a prominent K7-positive ductular reaction without K7-positive interemediate cells in the liver parenchyma ( Fig. 14.22 F ).
In some biopsies a definitive diagnosis of FCH cannot be made despite the presence of some cholestatic features. Biochemical cholestasis also appears to be predictive of disease progression in hepatitis C, without necessarily being associated with typical histological features of FCH. These cases may represent the mild end of a disease spectrum where hepatocyte swelling and the ductular reaction are relatively subtle, without satisfying the consensus diagnostic criteria for FCH, which include very high viral load and cholestatic liver enzymes more than five times normal. A recent study of recurrent HCV assessed the presence of each of the four key histological features of FCH (ductular reaction, cholestasis, hepatocyte ballooning, periportal sinusoidal fibrosis) and found that the presence of an increasing number of these features correlated with the rate of disease progression, with three or four features correlating with a diagnosis of full-blown FCH.
Until recently, patients with typical cholestatic HCV infection progressed rapidly to graft failure or death within a few months of diagnosis, and therapies were generally ineffective. The overall mortality in cases reported from 1990 to 1999 was 50%. Examination of failed allografts obtained at retransplantation has shown varying histological changes, including persistent severe cholestasis and hepatocyte ballooning, multiacinar necrosis, bridging fibrosis and cirrhosis. Recent studies suggest that the use of new DAA regimens may be effective in treatment, even in severe cases.
HCV with ‘autoimmune features’ (‘plasma cell hepatitis’)
Several studies have identified features resembling autoimmune hepatitis (AIH) in patients transplanted for HCV infection. These have occurred in two main settings. Antiviral therapy in the form of pegylated interferon (IFN) and ribavirin has been associated with the development of immune-mediated dysfunction in the graft, principally with AIH-like features that more recently have been termed ‘plasma cell hepatitis’ (PCH). Cases with PCH and concurrent acute cellular rejection (ACR) and with pure rejection are also described in IFN-treated HCV-positive patients, suggesting an overlapping spectrum of immune-mediated graft injury in this setting. The prevalence has varied from 3% to 20%, and the risk is increased if plasmacytic infiltrates are present before commencing antiviral treatment. Most patients are HCV-RNA negative at diagnosis and respond to treatment with increased immunosuppression, suggesting that this may be an alloimmune response triggered by IFN-induced stimulation of the host immune system. Other studies have identified histological features suggestive of AIH, unrelated to antiviral therapy, in approximately 15% of patients with recurrent HCV infection. Although some are associated with autoantibodies and increased serum IgG levels, others appear to be associated with suboptimal immunosuppression, suggesting that this may be a form of rejection. One study showed that the presence of a plasma cell-rich infiltrate in the explanted liver was a risk factor for the subsequent development of PCH, suggesting that some patients have an immune system that predisposes them to develop PCH. Histological features include plasma cell-rich portal and lobular inflammatory infiltrates (thus the term ‘plasma cell hepatitis’) associated with interface hepatitis, resembling changes seen in AIH in the native liver and de novo AIH in the liver allograft. Up to 90% of cases are associated with centrilobular necroinflammatory features of central perivenulitis. Patients with autoimmune features have a worse prognosis than those with ‘typical’ recurrent HCV but usually respond to treatment with immnuosuppression.
HCV with granulomas
A few studies have identified lobular and portal granulomas as a possible manifestation of recurrent HCV infection. One study suggested that the development of granulomas may be related to interferon therapy. The presence of granulomas has no obvious implications for HCV disease progression. An unusual pattern of recurrent HCV infection with granulomatous bile duct destruction, closely resembling changes seen in PBC, was noted in a single biopsy obtained 18 months after transplant from a woman with recurrent hepatitis C. Granulomatous bile duct lesions have subsequently been reported in association with HCV infection in the nontransplanted liver.
Hepatitis C and rejection
There is a complex relationship between HCV and rejection. On the one hand, the immunosuppressive therapy used to treat episodes of ACR predisposes to more severe HCV infection. On the other hand, a higher incidence of acute and chronic rejection has been identified in HCV-positive patients compared with those transplanted for other diseases. This association may reflect different approaches to immunosuppression in HCV-positive individuals, shared pathways of inflammatory damage or the effects of antiviral therapy used to treat HCV infection. After IFN treatment, up to 25% of patients have episodes of acute rejection and up to 17% develop features of chronic rejection, in a small proportion of cases leading to graft failure. IFN therapy is thought to predispose to rejection by a number of mechanisms, including nonspecific stimulation of the recipient’s immune system, release of both viral and alloantigens from lysed cells and accelerated hepatocellular metabolism of immunosuppressive drugs occurring as a result of viral clearance from hepatocytes. However, it has been suggested that the higher reported frequency of rejection in HCV-positive patients may partly reflect confounding factors such as a tendency to biopsy these patients more frequently and difficulties in distinguishing recurrent HCV from acute rejection. Although patients treated with direct-acting antiviral agents (DAAs) might theoretically be at risk of developing rejection, for similar reasons to those postulated for IFN-induced rejection, episodes of rejection are rarely seen in patients treated with nucleotide analogues; the overall frequency of rejection was only 1% (3/262) in two recent studies.
The distinction between recurrent HCV infection and ACR continues to be a common problem in the assessment of post-transplant biopsies, because the two conditions have overlapping histological features, including predominantly portal-based inflammation, bile duct inflammation, portal venous endothelial inflammation and portal tract eosinophils. Table 14.13 summarizes features that are helpful in distinguishing recurrent HCV hepatitis from ACR. Although no absolutely specific features exist for either condition, in most cases a careful assessment of the pattern of inflammation and extent of damage allows identification of the main cause of graft damage. Changes such as bile duct and endothelial inflammation or reactive changes in the biliary epithelium should be present in the majority (>50%) of structures to diagnose rejection. The timing of events is helpful; most ACR episodes occur in the first month after transplant, and HCV-related inflammatory changes, particularly in portal tracts, are unlikely to be present at this time. A recent study showed that the combination of venous endothelial inflammation and time <6 weeks post-transplant excluded HCV as a cause of graft dysfunction. Conversely, ACR is rare >12 months post-transplant, when portal inflammatory changes related to recurrent HCV infection are likely to be present. Serum HCV-RNA levels tend to be higher in HCV patients with recurrent disease than in those with pure rejection, but there is considerable overlap between the two groups. In a small proportion of biopsies, distinction between hepatitis C and ACR may be difficult or impossible. It is likely that the changes present in many such cases reflect a combination of HCV- and rejection-related graft damage. In the majority of cases where a dual pathology is suspected, rejection-related changes are at worst ‘mild’ in severity; additional immunosuppression is not indicated in these patients, and recurrent HCV is best regarded as the primary diagnosis. Increased immunosuppression should only be considered as a treatment option if features of ACR are at least ‘moderate’ in severity, or if there are features suggestive of progression to chronic rejection.
|Hepatitis C infection||Acute cellular rejection|
|Portal and periportal changes|
|Portal inflammation||Mononuclear cells (lymphoid aggregates)||Mixed infiltrate (lymphocytes, macrophages, blast cells, neutrophils, eosinophils)|
|Bile duct inflammation||Mild (lymphocytes)||Prominent (mixed infiltrate)|
|Bile duct loss||None||Variable (in cases progressing to chronic rejection)|
|Venous endothelial inflammation||None/mild||Yes|
|Parenchymal inflammation||Generally mild||Variable|
|Associated features||Lobular disarray||Hepatic vein endothelial inflammation|
|Cholestasis||Rare (except FCH-like cases)||Common|
|Fatty change||Yes (macrovesicular)||No|
|Acidophil bodies||Common||Less numerous|