Liver transplantation has emerged as an effective and recognized therapeutic option for patients with progressive or life-threatening hepatic disease. The first successful human liver transplants were performed in 1963, and over the succeeding years, the procedure has seen improvements in organ preservation, surgical technique, and immunosuppression. These refinements have translated into improved patient survival and have led to a marked surge in the number of operations performed. Currently over 3,000 livers are transplanted annually in the United States, with regular reports of one-year survival rates exceeding 80% and three-year survival rates greater than 70%.^9,98,109,117

The surgical pathologist plays several roles in the transplant process, beginning, of course, with the diagnosis and evaluation of the recipient's primary disease. With the expanding indications for liver transplantation, these disorders fill an ever-growing list, encompassing chronic end-stage disease produced by many causes, acute hepatic failure of various etiology, various inborn errors of metabolism, and, to a lesser extent, malignant neoplasms.

The donor liver may be biopsied at the time of transplantation to document any preexisting changes. Most of these specimens are normal or show only minor portal fibrosis or ductular proliferation. No features in the donor liver have been consistently associated with poor graft function, although in some studies, severe macrovesicular steatosis correlates with a high risk of primary graft failure.^13,47,103

After transplantation, the liver biopsy becomes one of the primary means of assessing the status of the allograft. Specimens are commonly obtained to evaluate graft dysfunction evinced by clinical or laboratory abnormalities, but because these are imperfect indicators, protocol biopsies are also performed at specified posttransplant intervals to detect early or subclinical problems. The grafted liver is subject, not only to any disease affecting nontransplanted livers, but to a multitude of additional conditions: preservation injury, allograft rejection, various secondary complications, and recurrent disease. The graft can be concurrently afflicted by more than one of these processes, further compounding the diagnostic challenge for both clinician and pathologist.^15,18,42

These various insults to the allograft yield several major histologic patterns (Table 13-1). Given the limited repertoire of hepatic responses, few of these patterns are entirely diagnostic in the absence of clinical correlation, and they furthermore provide a only an immediate glimpse of the liver's state at a particular time. An appreciation of the clinical situation combined with close communication with the attending clinicians is therefore critical, just as in all liver biopsy interpretation. Additional diagnostic clues are provided by the timing of the biopsy, since certain processes are more apt to occur during particular posttransplant periods.

Preservation injury is a loose generic term used to designate the nonimmunologic graft damage that results from the perioperative procedures involved in the harvesting, transportation, and reperfusion of the donor liver. Typically this injury appears early, within the first two posttransplant weeks, and spontaneously regresses over several weeks, although the recovery is prolonged for several months in some cases. The milder forms are associated with good graft function and few sequelae, but with more severe injury, graft function can be compromised and graft failure may result.^18,102

The associated histologic features include a variable combination of hepatocyte ballooning, canalicular cholestasis, and hepatocyte necrosis. Additional changes are often noted in baseline specimens taken at the time of transplantation (time-zero biopsies). For example, focal clusters of neutrophils and scattered acidophilic bodies are commonly found; a familial alteration in any surgical biopsy specimen, this appearance is sometimes referred to as surgical hepatitis (Figure 13-1). Microvesicular fatty change of mild degree is another common but innocuous feature in these specimens.

In hepatocyte ballooning, the liver cells are enlarged and display cytoplasmic rarefaction (Figure 13-2). These changes primarily affect the centrilobular region, but in severe cases, they can extend to involve the entire lobule. Parenchymal mitoses, focal hepatocyte necroses, and acidophilic bodies are occasional accompaniments, and canalicular cholestasis is often present. Hepatocyte ballooning can be associated with marked elevations of serum aminotransferases, but it does not generally carry ominous prognostic implications unless complicated by confluent necrosis.^34,73,88 Conditions other than preservation injury may, of course, be responsible for hepatocyte ballooning and these should be considered when the change is first encountered after the second posttransplant week; among the possibilities are acute and chronic rejection, technical vascular complications and ischemia, and occasional examples of recurrent viral hepatitis.

Canalicular cholestasis is a regular but nonspecific finding in allograft liver biopsies and is associated a wide array of possible causes. These include acute and chronic rejection, biliary obstruction, sepsis and other infections, and drug-induced injury.^43,112 As a manifestation of preservation injury, however, cholestasis develops in the absence of a clearly defined etiology and has thus been referred to as functional cholestasis.^112,115 It is characterized by prominent canalicular bile plugs, varying degrees of attendant hepatocyte injury, and, in more prominent examples, bile ductular proliferation with associated portal inflammation (Figure 13-3). Ductular and ductal cholestasis are occasionally recognized, but in this setting, they do not have the same diagnostic significance they do in nongrafted livers, where they suggest the presence of sepsis and biliary obstruction, respectively.

As preservation injury increases in severity, hepatocyte necrosis becomes more conspicuous, and zones of confluent hepatocyte destruction are seen (Figure 13-4). These zones are typically located in the centrilobular region, but they sometimes exhibit a periportal or subcapsular distribution and may lead to hepatic infarction.⁹⁰ Hepatocyte ballooning and canalicular cholestasis are typically present, and a portal tract reaction with prominent proliferation of bile ductules and inflammatory infiltration often develops, yielding an appearance that mimics biliary obstruction. Confluent hepatocyte necrosis need not result in problems with graft survival, although that is frequently the outcome with severe diffuse involvement; centrilobular scarring may remain as persistent evidence of the necrosis (Figure 13-5).³⁴ Outside of the setting of preservation injury, confluent necrosis can be noted with vascular complications, drug-induced injury, and, as an indicator of poor prognosis, acute and chronic allograft rejection.^61,95

Hepatic allograft rejection designates the injury to the engrafted liver caused by the immunologic response of the host. As advances in organ preservation, surgical technique, and immunosuppression have improved patient survival, rejection has become a principal problem following liver transplantation. It currently represents the major cause of graft dysfunction and one of the leading factors in graft failure.^50,85

The immunologic reactions potentially contributing to hepatic allograft rejection are multiple and varied. The primary process appears to involve the activation of recipient T-lymphocytes by allogenic antigens expressed on the graft. This stimulation can, in turn, trigger a variety of effector mechanisms, both cell-mediated and immunoglobulin-mediated, that act to destroy the graft cells. In addition, the elaboration of cytokines during the reaction can induced enhanced or aberrant expression of major histocompatibility antigens, possibly amplifying the immune attack.^21,106 Whatever the exact mechanisms involve, the main targets of the immunologic reaction are bile duct epithelium and vascular endothelium; damage to these structure constitute the chief pathologic features of rejection. Two major forms of liver allograft rejection are conventionally delineated -- acute cellular rejection and chronic rejection -- although rare cases of humoral-related hyperacute rejection are also described.^24,43,94

Acute and chronic rejection can be defined on the basis of clinical features, morphologic abnormalities, or a combination of both. Each approach has its strengths, but because of the nonspecificity of the clinical manifestations, the histologic characteristics better serve as the foundation for classification. From a clinical perspective, for example, acute rejection implies an early onset and reversibility with immunosuppressive therapy, whereas chronic rejection suggests a later inception and lack of responsiveness to treatment. These implications, although generally the case, are not always accurate. Some cases of acute rejection arise late in the posttransplant course or fail to respond to therapy. Similarly, chronic rejection sometimes presents within the first few weeks after transplantation or regresses either spontaneously or following treatment.¹¹⁰

Because of these inappropriate chronological and clinical connotations, the acute and chronic terminology has been criticized.^1,43,57 Alternative designations have been offered, but none has gained complete acceptance.⁵⁸ Therefore the traditional terms entrenched in the literature are adopted for this discussion, but they are used strictly in a pathologic sense to designate particular histologic patterns irrespective of the clinical setting.

Hyperacute rejection denotes the rapid, antibody-mediated form of graft failure caused by preformed circulating antibodies directed against donor-specific antigens. These antibodies react with the graft vasculature and cause endothelial damage with complement activation, vasospasm, and thrombosis, ultimately resulting in necrosis of the transplanted organ. Although well recognized with renal and cardiac allografts, hyperacute rejection is a rare complication of liver transplantation. It is described in only a few reports and does not predictably develop even when major blood-group incompatibilities or positive cytotoxic crossmatches are present.^6,39,97 The reasons for the liver's relative resistance to hyperacute rejection are not clear, but the potentially injurious antibodies may be bound by Kupffer cells or by soluble histocompatibility antigens released into the circulation.²¹

Clinical evidence of progressive graft failure typically appears within the first postoperative week, although it may be preceded by several hours or days of stable graft function. In some instances, however, graft dysfunction begins immediately after transplantation, just as with hyperacute rejection of other organs. The liver function tests are markedly elevated, arteriograms may demonstrate diffuse luminal narrowing indicative of vascular spasm, and eventually hepatic insufficiency requiring retransplantation evolves.¹⁹

The early histologic alterations include sinusoidal accumulations of neutrophils, fibrin deposits, and sludged erythrocytes together with centrilobular hepatocyte swelling, canalicular cholestasis, and small foci of hepatocyte necrosis and parenchymal hemorrhage. The portal tracts exhibit a variable neutrophil infiltrate and bile ductular proliferation; at times, small hepatic arteries demonstrate a necrotizing or neutrophilic arteritis. With progression, widespread zones of hemorrhagic infarction develop and dominate the histologic picture. These zones are variably accompanied by neutrophil exudation, vascular thrombosis and intimal proliferation, and, in some cases, evidence of superimposed acute rejection.^6,19,23,39 By immunohistochemical techniques, deposits of immunoglobulins and complement components can be demonstrated within arterial walls, particularly of larger vessels; patchy venular or sinusoidal involvement is sometimes seen.

The differential diagnosis of hyperacute rejection includes, in the initial phases, preservation injury and, in the later stages, other causes of extensive graft necrosis such as ischemia or sepsis. Distinguishing among these conditions on histologic grounds can be a difficult task. Arteritis and portal neutrophil infiltrates are suggestive features, but they are not invariably present. Confirmatory evidence of hyperacute rejection is provided by identifying vascular immune deposits, although this cannot be documented in all cases and must be carefully differentiated from nonspecific leakage of proteins into damaged vessel walls.^19,23 Because of the problems in establishing the diagnosis, some examples of primary graft failure of unknown cause may, in fact, be due to unrecognized hyperacute rejection.^44,94

This form of rejection is characterized by the accumulation of a cellular inflammatory infiltrate and evidence of damage to the immunologic targets of the liver, the bile ducts and venular endothelium. Although this is a morphologic definition, clinical correlation is nonetheless necessary for a final diagnosis. In particular, it is important in helping to exclude those conditions that share one or another of the histologic features of acute rejection, including viral hepatitis, biliary obstruction, and drug-induced injury.

Clinical features. Acute cellular rejection is a common complication of hepatic transplantation, affecting over 50% of recipients in various reported series. Generally it develops within the first three weeks following transplantation with the median onset ranging between 7 to 10 days, but it can occur at any time during the posttransplant course.^20,50 Late presentations are generally related to decreased immunosuppression, resulting from either inadequate absorption of drugs or poor patient compliance.⁶⁹

There are no specific clinical or laboratory features of acute cellular rejection. Some patients complain of malaise or anorexia, and fever, graft tenderness, or a change in the color or quantity of bile is occasionally observed. The serum aminotransferase, alkaline phosphatase, and bilirubin are frequently abnormal, but the values vary greatly and the biochemical profiles are not distinctive.^10,41 Indeed, in some patients undergoing protocol biopsy, acute rejection is found despite normal liver function tests. This emphasizes the need for histologic examination in confirming the diagnosis of rejection. Although several noninvasive diagnostic tests have been examined as a substitute for liver biopsy, none has been demonstrated to be completely reliable.⁷⁷

Treatment of acute cellular rejection consists of a short-term course of augmented immunosuppression. High-dose intravenous corticosteroids are usually employed, followed in patients who do not respond, by antilymphocyte preparations or other immunosuppressive agents. This therapeutic strategy is effective in about 90% of patients; the remaining 10% tend to progress to chronic rejection, although a few individuals require early retransplantation because of severe uncontrollable acute rejection.^2,110 In occasional instances mild acute rejection has been reported to resolve spontaneously.^37,114

Pathologic features.^{20,28,46,83,88,96,114} The histologic hallmarks of acute cellular rejection center on three main features: portal tract inflammation, bile duct injury, and endothelialitis (Figure 13-6). None of these features is entirely specific for rejection, and controversy persists over their relative roles as diagnostic criteria.^49,92,113 Nonetheless in the proper context, a combination of changes enables a reasonably firm and reproducible diagnosis to be rendered.^16,101

The portal infiltrate, usually of moderate to marked density, is distinguished by a mixed population of inflammatory cells (Figure 13-7). Small lymphocytes typically predominate, but other mononuclear cells including large activated lymphocytes or immunoblasts, macrophages, and plasma cells are also seen, together with varying numbers of neutrophils and eosinophils. In some cases, eosinophils are especially numerous and may prompt consideration of drug-induced injury.⁹² The infiltrate typically expands the portal tract, but it may be focally and unevenly distributed. On occasion, the inflammatory cells extend into the adjacent parenchyma, sometimes accompanied by periportal hepatocyte necrosis or, in extreme cases, by portal-portal bridging necrosis (Figure 13-8).¹⁵

As expected, immunophenotypic analysis demonstrates that the cells of the portal infiltrate consist largely of T lymphocytes, with contributions by both CD4- and CD8-positive subtypes. Although such investigations are not generally useful in the routine clinical setting, some evidence suggests that a predominance of CD4-positive cells correlates with a better therapeutic response.^68,80

Bile duct injury is a requisite feature of acute cellular rejection and assumes a spectrum of histologic alterations (Figure 13-9). In milder cases, epithelial cells of the interlobular ducts exhibit cytoplasmic vacuolation or eosinophilia and enlarged nuclei with variable anisonucleosis, nuclear crowding, prominent nucleoli, and occasional mitotic figures. More striking abnormalities include intraepithelial infiltration by lymphocytes or neutrophils, focal necrosis of epithelial cells, and, in severe cases, destruction and loss of ducts.^49,107

The ductal changes can be focal, and their identification can therefore require careful examination of several levels in the paraffin block. At times, the injured ducts are obscured by particularly dense portal infiltrates, and special stains (trichrome or diastase-PAS) or cytokeratin immunostains can aid in their identification. Bile ductular proliferation can also be seen, although it is not typically a prominent feature. Only interlobular bile ducts should be evaluated for evidence of rejection-associated duct injury, however, since proliferated ductules -- which can be seen as a nonspecific feature in several posttransplant settings -- regularly display reactive epithelial alterations.

Endothelialitis (or endotheliitis) refers to the inflammatory involvement of venular endothelium by mononuclear cells, primarily lymphocytes.⁶⁰ In mild examples, lymphocytes accumulate along the luminal surface of the vein and are attached to enlarged, damaged endothelial cells, sometimes by means of a thin cytoplasmic bridge. With advancing degrees of involvement, the lymphocytes infiltrate into the subendothelial zone, detaching the endothelial cells from their underlying connective tissue and, at times, resulting in destruction of the endothelium (Figure 13-10). Both portal veins and central veins can be affected, but central vein involvement is typically easier to identify because the confounding portal inflammatory infiltrate is not present.

Endothelialitis is often considered to be the most specific feature of acute cellular rejection, but its prevalence in various series has ranged approximately from 0% to 100%.^{46,49,88,95,96} This wide variation derives partially from differences in diagnostic criteria, but it also reflects the focal distribution of the lesions and the usual modest grades of involvement. The endothelialitis is of mild degree in over 80% of cases, and it is, moreover, a transitory lesion that regresses rapidly once antirejection therapy is begun.^37,60,95 Adding to the diagnostic problem is the finding of mild endothelialitis in conditions other than acute rejection, including acute and chronic hepatitis and confluent centrilobular necrosis. Therefore, the histologic context must be taken into consideration when interpreting this lesion. In other cases, occasional lymphocytes are found attached to undamaged endothelial cells; although the diagnostic implications of this finding are not clear, it should be distinguished from endothelialitis.

A variety of additionally features are inconsistently present in acute cellular rejection. Canalicular cholestasis and centrilobular hepatocyte swelling are commonly noted, but in many instances, their presence probably reflects coexisting preservation injury. Sparse lobular inflammation by mononuclear cells is occasionally seen, perhaps representing a form of sinusoidal endothelialitis, and scattered focal hepatocyte necrosis or acidophilic bodies may be present.⁶⁰

Less common but more ominous features seen in severe acute rejection include arteritis and confluent hepatocyte necrosis. The arteritis is characterized by lymphocytic or neutrophilic infiltration or, less often, mural necrosis; the large hilar vessels tend to be involved more frequently than are the small hepatic artery branches usually seen in needle biopsy specimens.^18,95 Confluent necrosis in acute rejection has usually been attributed to ischemia resulting from arteritis, central vein endothelialitis, or generalized blood flow reduction, although primary immunologically-mediated hepatocyte damage has also been conjectured.^43,61 The necrosis is typically centrilobular in location and often develops in a background of hepatocyte ballooning; extreme cases are complicated by the development of bridging or multilobular necrosis with parenchymal hemorrhage.^34,95 Although small foci of confluent necrosis can be an innocuous finding, the lesion is a cause for concern since its persistence is a marker of poor prognosis and high risk of chronic rejection.^24,61,95

Several systems for the histologic grading of acute cellular rejection have been proposed (Table 13-2).^24,49,95,114 Although useful in expressing the histologic range of acute rejection, these schemes have not been rigorously tested for their ability to predict therapeutic responsiveness or progression to chronic rejection, and none has been uniformly accepted. Nevertheless, acute rejection that is complicated by loss of bile ducts, confluent centrilobular necrosis, or arteritis, or develops early after transplantation (by day five) has been associated with a lesser response rate to initial treatment and a greater risk of subsequent chronic rejection.^7,88,95,110

Follow-up biopsy specimens are often obtained to assess the efficacy of therapy. With appropriate therapy, the inflammatory infiltrate and active duct injury generally resolve within seven to ten days, although the biliary epithelium and endothelium can show lingering reactive alterations for several weeks (Figure 13-11). In some cases of treated rejection, the specimens show persistent portal neutrophil infiltration, bile ductular proliferation, cholestasis, and mild portal fibrosis, resulting in an appearance that can be mistaken for biliary obstruction.⁹⁶ Persistent or worsening rejection is a signal for continued or increased immunosuppression.

Differential diagnosis. Although the diagnosis of acute cellular rejection can usually be established with reasonable confidence, biopsy interpretation nonetheless poses several problems. The diagnostic lesions can be focal, particularly in mild cases, and sampling error thus becomes a consideration with smaller specimens. In some cases, the diagnostic abnormalities are inconspicuous or additional atypical features of uncertain significance are present.

The various causes of graft dysfunction all enter into the differential diagnosis of acute rejection, but recurrent or de novo viral hepatitis is prominent source of difficulty. With viral hepatitis, the degree of lobular inflammation and parenchymal injury generally exceeds that seen in rejection, and the portal infiltrate tends to be restricted to mononuclear cells. There are exceptions to these generalizations, however, and the distinction can then become quite difficult on histologic grounds alone. In some cases, both rejection and hepatitis are concurrently present, although this may become apparent only with ancillary information and appropriate follow-up evaluations.^17,32

Another difficulty arises in distinguishing between nonspecific portal infiltration and mild acute rejection. The nonspecific infiltrates are typically composed primarily of mononuclear cells and lack the polymorphous composition and ductal-centered nature characteristic of rejection-associated inflammation (Figure 13-12). Furthermore, more distinctive features of rejection such as endothelialitis are absent. In other cases, however, minor evidence of bile duct damage is seen, and the picture becomes more equivocal, hinting at rejection but falling short of the diagnosis. An indeterminate designation of "consistent with rejection" is then appropriate; in some of these cases, overt rejection evolves, but in others, the alterations regress without complication.

Chronic allograft rejection is defined in histologic terms by destruction and loss of the interlobular bile ducts, often in conjunction with distinctive obliterative alterations of the hepatic arteries. This condition has been less well delineated than acute rejection, however, and conflicting diagnostic criteria have been applied in different reports. As a result, many synonyms have been employed for chronic rejection, including vanishing bile duct syndrome, ductopenic rejection, and vascular rejection.⁵⁸ These various names nonetheless emphasize the major defining histologic features, and they further indicate that chronic rejection represents one of the several disorders associated with bile duct paucity (see Chapter 5).

The pathogenesis of chronic rejection is poorly understood, and whether the processes involved are the same as those implicated in acute rejection is not known.¹⁰⁶ Two mechanisms have been proposed to account for the loss of bile ducts -- direct immunocytotoxicity and ischemic injury -- and perhaps both play a role under certain circumstances. A direct immunologic attack on ductal epithelium is suggested by ultrastructural and immunohistochemical studies showing lymphocytes in close apposition with bile duct remnants.^31,68 Possibly contributing to this process are mismatches in major histocompatibility antigens or positive lymphocytotoxicity tests, but this remains controversial.^4,27 Similarly, cytomegalovirus infection has been incriminated as a critical cofactor in the development of chronic rejection, although other evidence suggests that it is instead an innocent bystander.^3,75,118

Since the blood supply of the bile ducts derives solely from the hepatic arteries, biliary damage could also arise from ischemia produced by the obliterative arteriopathy of chronic rejection.^55,111 This mechanism is supported by the concordance between bile duct loss and the presence of arterial lesions and the coexistence, in many cases, of centrilobular ischemic alterations.⁷⁶ Despite this, however, other examples of chronic rejection fail to demonstrate arteriopathic changes or lobular evidence of ischemia, and therefore bile duct destruction cannot be completely ascribed to this mechanism.³³ The pathogenesis of the arteriopathy is similarly uncertain, although immunologically-mediated endothelial damage is the likely initiating factor.⁵⁵

Clinical Features. Chronic rejection develops in approximately 8% to 10% of liver allograft recipients and is thus much less common than acute cellular rejection.^33,111 In recent studies, however, the prevalence appears to be declining, presumably because of improvements in immunosuppression and patient management.^50,82,105

The usual onset varies between six weeks and six months following transplantation, although some cases develop as early as the second postoperative week, and rare examples arise insidiously later in the course.^1,62,110 Typically chronic rejection is preceded by one or more episodes of acute rejection that fail to respond to immunosuppressive therapy but instead progress with gradually increasing serum bilirubin and alkaline phosphatase levels. In most instances, this process leads to deteriorating hepatic function, irreversible graft failure, and the eventual need for retransplantation; this scenario has been termed the vanishing bile duct syndrome.⁶² Unfortunately, the risk of recurrent chronic rejection in the retransplanted grafts is high, ranging in the few reported series from 25% to 90%.^1,105

Occasional cases are described, however, with a more favorable natural history. After a period of clinical and biochemical deterioration, the condition slowly and spontaneously improves. The laboratory values return toward normal, and the histologic abnormalities partially or completely resolve over many months to years. Although these spontaneously resolving cases demonstrated no distinguishing features in some series, other studies describe an association with preserved hepatic synthetic function and an absence of ischemic features (centrilobular necrosis or fibrosis) on histologic examination.^33,45,74

Pathologic Features. Histologically, chronic rejection is distinguished by two principal abnormalities: disappearance of bile ducts and obliterative arteriopathy.^33,38,83,107 Although these features can be described in a straightforward manner, their evaluation can be difficult in any particular biopsy specimen. Consequently, the diagnosis of chronic rejection tends to be less reliable than that of acute rejection.¹⁶ Assessment of sequential changes in repeated biopsy specimens is often useful in establishing the diagnosis and evading interpretative problems.

The loss of bile ducts -- also referred to as ductopenia -- is a progressive process that evolves over several weeks to months. Interlobular ducts of all sizes are lost, typically in a patchy fashion, although the smallest ducts with diameters of less than 35 μm are especially affected (Figure 13-13). Since bile ducts are normally accompanied by comparably-sized hepatic artery branches, the decrease in numbers is recognized by finding arteries or portal tracts that lack a corresponding duct (see Chapter 5). In quantitative terms, the absence of more than 50% of the bile ducts is considered diagnostically important, and in well-established cases of chronic rejection, over 75% may be missing.^33,62,76