CHAPTER 16 The Liver in Organ Transplantation
Introduction
The pathologist is often asked to examine liver biopsies obtained to evaluate liver dysfunction in transplant patients, including recipients of liver, renal and bone marrow grafts. For liver transplantation, liver biopsy remains the diagnostic ‘gold standard’ when jaundice and allograft dysfunction develop, since biochemical tests do not adequately discriminate between rejection and other conditions that may develop in the allograft.1 Moreover, even when serum liver function tests are normal, histological abnormalities (including rejection lesions) may be present.2,3 At the time of harvesting or engraftment, the donor liver may also require assessment, sometimes by frozen section, for lesions that may determine whether or not the graft can be used, and that can affect the postoperative course and appearance of subsequent post-transplantation biopsies. This chapter reviews the histopathological features of liver transplant rejection and other conditions affecting the allograft. The concluding sections discuss liver disease in recipients of renal and bone marrow transplants.
Liver transplantation
Assessment of the donor liver
The source of the donor allograft is an important consideration when evaluating liver biopsies obtained prior to transplantation, after graft revascularisation or at later times. Biopsies from potential living donors in apparent good health may disclose conditions such as steatohepatitis or primary biliary cirrhosis, which disqualifies their candidacy.4 Living donor left or right lobe grafts, if small in relation to the recipient’s size, may show cholestasis or congestion associated with ascites, prolonged coagulation parameters and impaired metabolic function immediately after transplantation (small-for-size syndrome5) due to problems in venous drainage6 and portal hyperperfusion.5 Postoperative portal tract biliary obstructive changes may also be present due to ischaemia or mechanical obstruction of the large bile ducts associated with this type of procedure.7 Cadaveric livers are subject to preservation (ischaemia/reperfusion) injury8 after harvesting and transport to the site of surgery, which in early baseline biopsies is evident as variable degrees of perivenular necrosis, liver-cell ballooning and/or apoptosis. The increased risk of postoperative biliary strictures and bile leaks with ‘donation after cardiac death’ grafts9 should be kept in mind if cholestasis and features of biliary obstruction are seen in a post-transplantation biopsy. If an allograft is used from a donor with known chronic hepatitis B or C (‘extended donor criteria’), a baseline biopsy warrants careful attention to the grade and stage of the chronic hepatitis (see Ch. 9) for later comparison with subsequent biopsies. Allograft biopsies obtained soon after transplantation sometimes disclose an unsuspected condition in the donor such as α1-antitrypsin deficiency, iron overload or amyloidosis.10
Frozen section of potential donor livers may be requested to exclude pre-existing disease. Pathologists providing frozen section coverage should be aware of three common reasons that frozen sections are requested: (1) to determine whether steatosis is present, and its degree; (2) to exclude changes of chronic hepatitis if the donor is known to be positive for antibodies to hepatitis B core antigen but negative for surface antigen (i.e. possible occult hepatitis B), or is positive for hepatitis C virus; and (3) to evaluate a mass found in the donor liver. Concern about a substantially fatty liver is based on the increased incidence of primary graft dysfunction or non-function when this is present.11 The degree of macrovesicular (large droplet) fatty change should be categorised according to the percentage of parenchymal involvement as either absent, mild (<30%), moderate (30–60%) or marked (>60%).12,13 Transplant surgeons have considered the last category unsuitable for use because of the high risk of primary dysfunction or non-function associated with severe steatosis.11,13 Microvesicular (small droplet) fat is held not to be a contraindication, however,14 but should also be graded and merits discussion with the transplant team if substantial. Diffuse portal mononuclear inflammatory cell infiltrates in donors with markers of hepatitis B or C viral infection support the presence of chronic hepatitis. The significance of this finding needs to be considered by the transplant team. With regard to mass lesions in the donor liver, demonstration of a malignant or metastatic tumour is an obvious contraindication to its use. However, the features of benign lesions such as focal nodular hyperplasia (see Ch. 11) are important to recognise since they are often encountered in this setting.
The liver allograft biopsy: general considerations
Needle liver biopsies are obtained as part of a liver transplantation protocol or because of clinical deterioration.15 Discussion with the clinical team and careful review of pertinent radiographic, biochemical and microbiological findings are critical to biopsy interpretation and institution of appropriate therapy. Serial biopsies may be necessary to resolve difficult diagnostic problems.
There are many causes of allograft injury in addition to rejection (Table 16.1) and these should be considered in the context of the time elapsed since transplantation16 (Fig. 16.1). For several weeks following transplantation, functional cholestasis may be present and must, if possible, be distinguished from the cholestasis of acute rejection, bile-duct obstruction, hepatitis, drug toxicity and sepsis. Bile is present within hepatocytes and canaliculi. This impairment of bile flow can be explained by exposure of the donor liver to cold ischaemia and reperfusion injury (‘preservation injury’) with resultant damage to liver-cell organelles.17 Liver-cell death due to preservation injury actually shows features of both necrosis and apoptosis (‘necrapoptosis’).18 Early postoperative cholestasis may also be due to a ‘small-for-size’ graft.19 Cholestasis may be accompanied by hepatocellular ballooning in perivenular regions (Fig. 16.2) or in a diffuse distribution.20,21 In the absence of frank perivenular necrosis, ballooning does not confer an unfavourable prognosis.20 Hypoperfusion liver damage in the perioperative period may result in necrosis in periportal or perivenular regions and sometimes an irregular subcapsular band of infarction.22 If the donor liver is fatty, rupture of hepatocytes affected by preservation injury may rarely cause sinusoidal engorgement by lipid vacuoles (lipopeliosis)23 (Fig. 16.3).
Table 16.1 Pathological considerations in the transplant liver
In evaluating post-transplant biopsies, special attention should be paid to the portal tracts, the major sites of rejection lesions. The type of cellular infiltrate, the bile ducts, portal-vein branches and hepatic arterioles are examined to distinguish rejection from other conditions with portal tract pathology, particularly bile-duct obstruction, recurrent viral hepatitis, drug toxicity and immunosuppression-related lymphoproliferative disease (see Differential diagnosis in transplant biopsies). The perivenular region also requires inspection for possible preservation injury, cholestasis or inflammation, and for necroinflammation which may accompany portal tract lesions in more severe cases of acute rejection.24 The lobular parenchyma shows few alterations in rejection apart from cholestasis and the occasional apoptotic bodies and scattered liver-cell mitoses which develop as the allograft equilibrates to the appropriate size for the recipient. As a result, in cases where confusion arises in the interpretation of portal changes, it is important to carefully evaluate the lobular parenchyma for evidence of intercurrent diseases such as viral or drug hepatitis. The pathologist should always bear in mind that a given biopsy may show superimposed features attributable to several different post-transplantation complications.
Graft rejection
The histopathological lesions of liver allograft rejection have been well characterised25–29 and are classified as humoral rejection, acute (cellular) rejection and chronic (ductopenic) rejection, as recommended by an international working party which met in 1994.24 Acute and chronic rejection are the most common forms seen in clinical practice.
Humoral rejection
Humoral rejection (antibody-mediated or hyperacute rejection) is rare after liver transplantation, developing in patients with pre-formed antibodies or subsequently formed antibodies to a donor liver that is incompatible with ABO blood groups. Microvascular damage evolves over the first few hours after transplantation, consisting of sinusoidal infiltrates of neutrophilic leucocytes, fibrin and red blood cells associated with focal haemorrhages. This progresses to portal and periportal oedema with coagulative and haemorrhagic necrosis over the next few days30 (Fig. 16.4). Immunofluorescent studies show linear deposits of IgG or IgM, complement fractions C1q, C3 and C4 and fibrinogen in arterial walls.24,31 The graft may remain stable in some patients for the first few days, however, possibly because of Kupffer-cell protection against the effects of circulating antibodies.32 Graft failure within 2–4 weeks is associated with a progressive and marked rise in serum aminotransferase activity. The liver appears mottled and cyanotic at gross examination. Recipients of ABO-unmatched livers may also develop graft-versus-host haemolysis, associated with erythrophagocytosis and Kupffer-cell siderosis.33
Acute (cellular) rejection
Acute (cellular) rejection, the most common form of rejection, is a cell-mediated immune injury directed at bile-duct epithelium and the endothelium of portal-vein branches and terminal hepatic venules. This usually occurs within the first month to 6 weeks after transplantation,34 but may be seen later if immunosuppression is lowered or discontinued. The characteristic histological triad of cellular rejection includes portal inflammation, bile-duct damage and endotheliitis (endothelialitis). Endotheliitis is not present in all cases. The portal inflammatory lesion is typically heterogeneous, with lymphocytes predominating among plasma cells, neutrophils and, occasionally, large lymphoid cells, some in mitosis (Fig. 16.5). Eosinophils are often abundant (Fig. 16.6), which is a very helpful diagnostic sign that acute rejection is present.35,36
Bile ducts are surrounded and infiltrated by immune cells and damage to their epithelium takes the form of variation in nuclear size, vacuolation of cytoplasm, regions of cell stratification or cell loss, and irregularity of duct outlines (Figs 16.5–16.7). Endotheliitis comprises attachment of lymphoid cells to the endothelium of portal-vein branches or terminal hepatic venules, variable degrees of endothelial damage, subendothelial inflammation (Figs 16.5, 16.6) and lifting-off of endothelial cells from the underlying vein wall (Fig. 16.8). Sinusoidal endotheliitis is occasionally also present. Mild focal endotheliitis is sometimes found in association with hypoperfusion damage in baseline biopsies, but extensive endotheliitis in the postoperative period is very characteristic of rejection.25 Necrosis of perivenular hepatocytes, accompanied by endotheliitis of terminal hepatic venules and expansive portal inflammatory lesions involving the periportal parenchyma, is indicative of severe acute rejection.37 Central perivenulitis (central venulitis) – characterised by endotheliitis of terminal venules, dropout and apoptosis of nearby hepatocytes (sometimes with focal sinusoidal congestion and dilatation) – often presages later episodes of acute rejection and chronic ductopenic rejection.38–40 Central perivenulitis is a common expression of rejection in paediatric allografts.41 It is sometimes the chief manifestation of rejection, with few or no portal tract changes, as isolated central perivenulitis42 in both paediatric and adult allografts, many months or longer after transplantation (see Late liver allograft dysfunction, below).
Descriptive and semi-quantitative grading of acute rejection can effectively be accomplished using the scoring system presented in the Banff international consensus document37 (Table 16.2). Using the semi-quantitative approach of assigning a numerical score to each component of the acute rejection triad, a total Rejection Activity Index (RAI) can be conveyed in the biopsy report. Alternatively, a simpler global assessment of the biopsy as showing indeterminate, mild, moderate or severe changes of acute rejection can be used (Table 16.3). The choice of grading system, as with grading and staging for chronic hepatitis, should be made after discussion with clinicians.
Table 16.2 Banff grading scheme for acute rejection*
| Category | Criteria | Score |
|---|---|---|
| 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 | 2 | |
| Marked expansion of most or all of the triads by a mixed infiltrate containing numerous 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 nucleus–cytoplasm 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 vacuolisation of the epithelium | 2 | |
| As above for 2, with most or all of the ducts showing degenerative changes or focal luminal disruption | 3 | |
| Venous endothelial inflammation | Subendothelial lymphocytic infiltration involving some, but not the majority, of the portal and/or hepatic venules | 1 |
| Subendothelial infiltration involving most or all of the portal and/or hepatic venules | 2 | |
| As above for 2, with moderate or severe perivenular inflammation that extends into the perivenular parenchyma and is associated with perivenular hepatocyte necrosis | 3 |
Note: total score = sum of components. Criteria that can be used to score liver allograft biopsies with acute rejection are as defined in the World Gastroenterology Consensus Document.
* The Rejection Activity Index (RAI) is the sum of the scores for each of the three components of acute rejection. RAI ≥ 4 (mild), RAI ≥ 6 (moderate or severe).
Source: Reprinted with permission from International Panel.37
Table 16.3 Descriptive terminology for acute rejection
| Global assessment* | Criteria |
|---|---|
| Indeterminate | Portal inflammatory infiltrate that fails to meet the criteria for the diagnosis of acute rejection (see text) |
| Mild | Rejection infiltrate in a minority of the triads that is generally mild and confined within the portal spaces |
| Moderate | Rejection infiltrate, expanding most or all of the triads |
| 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 |
Note: global assessment of rejection grade is made on a review of the biopsy and after the diagnosis of rejection has been established.
* Verbal description of mild, moderate or severe acute rejection could also be labelled as grades I, II and III, respectively.
Source: Reprinted with permission from International Panel.37
Chronic (ductopenic) rejection
Chronic (ductopenic) rejection (‘vanishing bile-duct syndrome’) is defined as obliterative vasculopathy and loss of bile ducts occurring 60 days or longer after transplantation.24,43 The incidence of chronic rejection in liver transplant patients has declined to less than 5% in some series44–46as immunosuppression regimens have improved. In most cases, vasculopathy and ductopenia occur together, but in a minority they can be present independently.47
The diagnosis of chronic rejection can be problematic even for experienced hepatic pathologists,48,49 particularly in the early stages.24 Atrophy, nuclear pleomorphism and pyknosis of small ducts (bile-duct ‘dystrophy’) often precede frank ductopenia.50 The presence of ductopenia is established when a formal count of small bile ducts and hepatic artery branches within portal tracts demonstrates loss of bile ducts from over 50% of portal tracts (Fig. 16.9). Progressive bile-duct loss results from a destructive cholangitis, which in most cases stems from bouts of acute rejection that are not controlled by immunosuppression. Cytokeratin immunostaining may help identify remnants of bile-duct epithelium.28 Portal tract hepatic arterioles may also be lost.43 Over time, portal inflammation becomes sparse and bile ducts disappear from the majority of portal tracts, usually without a ductular reaction43 (Fig. 16.9). Episodes of acute rejection with increased inflammation and endotheliitis may develop superimposed on changes of chronic rejection. The pathology report in chronic rejection should therefore include consideration of the following points:43 (1) whether acute rejection is present; (2) the degree of bile-duct loss in portal tracts; (3) the presence of perivenular necrosis or fibrosis; and (4) the degree of hepatic arteriole loss in relation to the total number of portal tracts.
The presence of obliterative vasculopathy (rejection arteriopathy) may be more difficult to demonstrate on needle biopsies, since the characteristic subintimal accumulations of foamy histiocytes and myointimal cells predominantly affect the large-calibre arteries of the liver hilum47,51 (Fig. 16.10). However, foam-cell lesions can sometimes be demonstrated in medium-sized portal arterioles present in biopsies and occasionally in portal veins and sinusoids (Fig. 16.11). The presence of arteriopathy in most cases must be inferred when perivenular ischaemic necrosis and fibrosis are seen in liver biopsies obtained in the appropriate time frame of chronic rejection. Demonstration of perivenular necrosis in repeated biopsies indicates a poor prognosis.52 Mismatch of recipient and donor histocompatibility antigens, activation of the complement membrane attack complex and persistent cytomegalovirus infection in the allograft have been invoked in the pathogenesis of bile-duct loss and arteriopathy.28,53–57
Figure 16.10 Rejection arteriopathy.
(Case kindly provided by Dr Jurgen Ludwig, Rochester, MN, USA.)
Chronic rejection usually leads to irreversible graft failure, although some patients may recover.28,58 The late stage characteristically shows marked cholestasis and bile-duct loss, portal and periportal fibrosis, perivenular fibrosis and variable numbers of bridging fibrous septa linking portal tracts or central veins to portal tracts. Cirrhosis develops after liver transplantation in only a minority of patients and is typically due to recurrent or acquired viral hepatitis, rather than chronic rejection59 (see Recurrent disease).
Other causes of graft dysfunction
Infection
Cytomegalovirus (CMV) is a common pathogen in liver allografts, most cases of CMV hepatitis occurring 4–8 weeks after transplant.60 Typical intranuclear and cytoplasmic CMV inclusions (see Ch. 15) can be found in hepatocytes, bile-duct epithelium (see Fig. 15.5) and endothelial cells. CMV infection should be suspected when small microabscess-like foci of necrosis with an infiltrate of neutrophils are present (Fig. 16.12). Smaller collections of parenchymal neutrophils (‘mini-microabscesses’) are occasionally seen in patients without CMV infection, apparently without adverse effects on the graft.61 CMV infection may also lead to formation of epithelioid granulomas. Immunohistochemical staining for CMV antigens is a sensitive method of demonstrating occult infection.62
Epstein–Barr virus infection should be considered if portal tracts and sinusoids contain a preponderance of atypical lymphocytes and immunoblasts.63–65 The possibility that as yet unidentified hepatitis viruses may cause post-transplantation liver dysfunction has been considered.66
Infection by Gram-negative bacilli may produce hepatocellular and canalicular cholestasis or, with sepsis, the more unusual picture of inspissated bile in periportal bile ductules (‘bile ductular cholestasis’) (see Ch. 15 and Fig. 15.11). Cholestasis due to infection and/or sepsis must be distinguished from that seen in bile-duct obstruction and rejection. Assessment of portal tract changes as well as results of microbiological studies are important in making these distinctions. Culture and special stains of liver biopsy specimens that show microabscesses or granulomas are the best means of documenting bacterial, fungal or other infections.67
Thrombosis
Thrombosis of the hepatic artery68,69 or portal vein70 (the latter particularly in children) may develop within the first few weeks or months of transplantation, leading to infarction of the liver (see Ch. 12). Needle biopsy specimens may not be representative owing to the irregular distribution of infarcted liver parenchyma. Thrombosis, stricture or foam-cell arteriopathy of perihilar arteries may cause necrosis, stricture or cholangiectases of perihilar bile ducts due to impaired duct perfusion.71 Liver biopsy in such cases may show features of biliary obstruction.71
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