Clinical Features

Reliable early signs of significant preservation/reperfusion injury after complete revascularization include poor bile production and persistent elevation of the serum lactate level. This condition is usually associated with marked (>2500 IU/mL) elevations of the serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels during the first few days after transplantation,⁹ followed by a rapid normalization of the ALT/AST rato during the first week and a prolonged cholestatic phase characterized by persistent elevation of total bilirubin and GTP values. Grafts that recover from early significant injury have gradual resolution of abnormal liver injury test results, but they are also at risk for ischemic cholangiopathy, which causes biliary sludge syndrome.^67,68

Pathologic Features

Postreperfusion needle biopsies obtained within several hours of complete revascularization can reliably gauge the extent of preservation/reperfusion injury.^9,69 The mild damage that occurs in many liver allografts includes microvesicular steatosis, which is usually attributable to warm ischemia, hepatocellular cytoaggregation (i.e., detachment of individual hepatocytes from each other and accumulation of the cytoplasm so that the cell assumes a rounded appearance), and hepatocellular swelling.^9,50 Severe injury is characterized by zonal or confluent coagulative necrosis, particularly if it is periportal or bridging, and severe neutrophilic inflammation.^9,69 The pathologist should not overinterpret surgical hepatitis, which is characterized by perivenular sinusoidal neutrophilia without necrosis, or manipulation injury, which manifests as necrosis and neutrophilia in the immediate subcapsular parenchyma in wedge biopsies, as a preservation/reperfusion injury.

Repair responses usually begin 1 to 2 days after injury and are directly proportional to the severity of the insult. Mild injury is usually followed by hepatocellular mitosis, thickening of the plates, and nuclear enlargement. Persistent injury manifests as mild centrilobular hepatocellular swelling, and hepatocanalicular cholestasis often coexists and persists for several weeks (Fig. 52.3). Severe preservation/reperfusion injury is usually accompanied by marked centrilobular hepatocellular swelling and hepatocanalicular and cholangiolar cholestasis.^9,50 These features often persist for 1 or 2 months (Fig. 52.4). Cholangiolar proliferation is usually triggered by periportal and confluent bridging necrosis with collapse of the reticulin framework.^9,50 Coexistent sepsis, which is common during this period, can contribute to the pattern of injury. If the graft recovers, normal architecture can be restored, but patients are at risk for ischemic cholangiopathy.

Preservation/reperfusion injury of donor livers with more than 20% macrovesicular steatosis causes death of some fat-containing hepatocytes, which then release large lipid droplets into the sinusoids. These droplets coalesce into even larger globules, which trigger local fibrin deposition, neutrophilia, red blood cell congestion, and local obstruction of sinusoidal blood flow (Fig. 52.5).⁷ If the liver recovers, the large fat globules become surrounded by macrophages and eventually resolve over several weeks. Because normal hepatocytes require only 4 to 6 hours to undergo the entire apoptotic cycle, recognition of apoptotic hepatocytes or coagulative necrosis in a biopsy obtained more than several days after transplantation should arouse suspicion of another, usually ischemic, insult.⁷⁰

Differential Diagnosis

Biliary obstruction, pancreatitis, sepsis, AMR, and cholestatic hepatitis can produce histopathologic changes that resemble preservation/reperfusion injury. Detailed donor information, including age and organ type (e.g., ECD, DCD), cold and warm ischemic times, operative difficulties, recipient’s clinical profile, blood culture, blood crossmatch, and results for DSAs and C4d staining help to determine the likely source of injury.⁵³ Preservation/reperfusion injury and operative technical difficulties most often are the causes of liver injury early after transplantation.

Examination of the true bile ducts (not cholangioles) contained within the original portal tract stroma and cholangioles at the interface zone provide useful clues for distinguishing preservation/reperfusion injury from biliary tract obstruction or stricturing. The latter usually cause at least some degree of periductal lamellar edema or produce stellate septal bile duct lumens with neutrophils in the lumen or infiltrating between biliary epithelial cells. Preservation/reperfusion injury does not usually show true bile duct changes unless it is associated with or leads to ischemic cholangiopathy. Instead, neutrophils surround the interface zone cholangioles. Centrilobular hepatocanalicular, cholangiolar cholestasis and intralobular neutrophil clusters are common to both disorders. The clinical context, history, and laboratory results are often needed to distinguish sepsis from preservation/reperfusion injury.

ACR superimposed on preservation injury is recognized by the typical rejection-type inflammatory infiltrate in the portal and perivenular regions. The infiltrate consists of blastic and smaller lymphocytes and especially eosinophils, which are an excellent early marker of an emerging rejection reaction, as are findings of lymphocytic cholangitis and lymphocytic central perivenulitis. Distinguishing preservation injury from AMR is discussed later (see Antibody-Mediated Rejection).

Cholestatic hepatitis can be difficult to distinguish from preservation injury and cholestatic hepatitis without knowledge of the clinical history. Cholestatic hepatitis has been reported only for patients infected with HBV or HCV, and it is distinctly unusual during the first 3 to 4 weeks after transplantation. Cholestatic hepatitis usually worsens with time, unless the patient is treated with decreased immunosuppression or antiviral therapy, whereas the trend is gradual improvement for patients with a preservation/reperfusion injury.

Clinical Features

Dahm and colleagues defined PHP/SFSS as at least two of the following complications occurring on 3 consecutive days in the first several weeks after transplantation: elevated bilirubin (>100 µmol/L), international normalized ratio (INR) higher than 2, and grade 3 or 4 encephalopathy after exclusion of technical, immunologic, or infectious complications.⁸⁴ Surgeons are usually aware of the potential for PHP/SFSS, but the diagnosis is difficult to establish with certainty. Clinical manifestations are not specific and may be caused by other insults (e.g., portal vein thrombosis).

It is often difficult to determine whether PHP/SFSS contributes to complications that may otherwise be deemed technical, especially because the latter are encountered with increased frequency in cases of reduced-size allografts. For example, a strong arterial buffer response can lead to significant arterial vasospasm and predispose to thrombosis, especially if blood vessels are a smaller caliber than normal.⁶¹ This event may be misinterpreted as a technical complication.

Pathologic Features

Initial events in PHP/SFSS have been observed reliably in experimental animal models^73,81,85,86 and occasionally in postreperfusion or early posttransplantation allograft biopsies obtained within the first several days in humans. Denudation of portal vein and periportal sinusoidal endothelium occurs as early as 5 minutes after transplantation.^{61,73,81,85,86} In severe cases, microvascular rupture at the portal vein–sinusoidal junction can result in hemorrhage into the portal and periportal connective tissue and dissect into hepatic parenchyma.^87,88

If the allograft survives the initial crisis, reparative changes occur, including endothelial cell hypertrophy and subendothelial edema accompanied by growth of myofibroblasts and endothelial cells into the subendothelial space. Eventually, this process leads to fibrointimal hyperplasia or intimal thickening and luminal obliteration or recanalization of thrombi (Fig. 52.6). Venous findings typical of PHP/SFSS are uncommon in peripheral core-needle biopsies. The pathologist instead often observes a constellation of nonspecific findings that may include centrilobular hepatocanalicular cholestasis, centrilobular hepatocyte steatosis or hepatocyte atrophy with sinusoidal dilation, centrilobular hepatocyte necrosis, and a low-grade ductular reaction (Fig. 52.7).

Review of the operative and radiographic reports and clinical history and a discussion with the surgeon help to determine whether pathologic changes in peripheral needle biopsy can be attributed to PHP/SFSS. Hilar sections of allografts that fail because of PHP/SFSS frequently show changes of traumatic injury to large portal vein endothelium, focal fibrointimal hyperplasia of vein branches, evidence of arterial vasospasm, and, in some cases, ischemic cholangitis, particularly if the hepatic artery has thrombosed.⁶¹

If the graft recovers, portal hypertension and ascites resolve during a period of several weeks. This is accompanied by restoration of normal architecture, except that some grafts eventually develop significant nodular regenerative hyperplasia as a result of portal venopathy from the initial injury or from suboptimal perfusion with associated collapse.⁶¹

Differential Diagnosis

Suboptimal arterial flow resulting from arterial thrombosis or stricturing, sepsis, hypotension, biliary tract obstruction or stricturing, or ischemic cholangitis can cause pathologic changes similar to those of PHP/SFSS. Although preservation injury is a differential diagnostic consideration, living donor grafts are usually not affected significantly.

Because portal hyperperfusion can lead to diminished arterial flow and thrombosis, it is not surprising that more than one complication may occur. Biliary tract obstruction or stricturing alone is usually not accompanied by portal tract connective tissue hemorrhage, centrilobular hepatocyte ischemic changes, or significant nodular regenerative hyperplasia. When these changes are found, PHP/SFSS should be considered.

Arterial vasospasm is usually detected microscopically only in failed allografts and only when severe. The vessels most commonly affected are medium-sized perihilar arteries.

Clinical Features

Most early hepatic artery thromboses manifest clinically with fever, leukocytosis, septic shock, and severe elevations of liver injury laboratory test results.^89,90 Late hepatic artery thrombosis may be asymptomatic, or it may maniest insidiously along with cholangitis, relapsing fever, sepsis (related to hepatic infarcts), abscesses, and ischemic cholangiopathy with subsequent impaired bile flow.^89,90

Ultrasonography is often used as a screening tool to inspect hepatic arterial blood flow, but angiography is the most reliable method of establishing a diagnosis. Urgent revascularization is often attempted to salvage the graft, but even if successful, the patient can develop ischemic cholangiopathy.

Pathologic Features

Hepatic artery thrombosis can be diagnosed on peripheral core-needle biopsies. However, this approach is often not reliable, because needle biopsies sample subcapsular parenchyma, which is only sometimes affected.^50,93 The structures more susceptible to ischemic injury are the perihilar tissue and large bile ducts, which are not routinely sampled.

Peripheral core-needle biopsies show frank centrilobular coagulative necrosis, marked centrilobular hepatocyte swelling, and biliary tract complications such as cholangiolar proliferation, acute cholangiolitis, and obstruction or stricturing. The peripheral biopsy may be unremarkable because vascular collaterals render the thrombosis inconsequential or the necrosis of bile ducts and biliary sludging have not developed in the peripheral liver parenchyma. Occasionally, hepatic artery thrombosis can lead to spotty acidophilic necrosis of hepatocytes, called ischemic hepatitis, and can mimic acute viral hepatitis. Chronic suboptimal arterial flow can cause centrilobular hepatocellular atrophy and sinusoidal widening.

Examination of failed allografts with hepatic artery thrombosis (Fig. 52.8) often reveals necrosis of the hilar or perihilar bile ducts (Fig. 52.9) and leakage of bile into the surrounding connective tissue. Biliary sludge, biliary abscesses seeded with fungi and bacteria, infarction of hepatic hilar lymph nodes, and patchy parenchymal infarction may also be seen.

Differential Diagnosis

Because hepatic artery thrombosis or arterial narrowing can mimic almost every type of liver allograft syndrome, uncommon manifestations can easily be misdiagnosed. Ischemic necrosis, which is often centrilobular, most reliably indicates hepatic artery thrombosis, but suboptimal flow can also result in centrilobular hepatocyte swelling and biliary tract obstruction or cholangitis. Chronic suboptimal arterial flow can cause biliary epithelial cell senescence that resembles chronic rejection. Ischemic hepatitis can appear similar to the lobular phase of acute or recurrent viral hepatitis (e.g., HCV infection). However, Liu and colleagues suggest that a combination of apoptosis and mitosis (i.e., low ratio of apoptosis to mitosis) without inflammation is more common in ischemic hepatitis than in recurrent hepatitis C, in which apoptosis predominates.⁹⁴ The relationship between arterial thrombosis and biliary tract complications is common, and examination of hepatic arterial patency should be performed when biliary tract complications are encountered.

Clinical Features

Portal vein thrombosis in a noncirrhotic allograft can cause widespread necrosis (Fig. 52.10, A). It manifests with bleeding varices, fulminant hepatic failure, and portal hypertension with massive ascites and edema.⁹⁵ If the thrombus is partial, small infarcts can develop, or the liver can become seeded by intestinal bacteria, which may cause relapsing fever. Suboptimal flow can result in periportal and midzonal hepatocyte atrophy, necrosis, and diffuse macrovesicular steatosis (see Fig. 52.10, B).⁹⁷ The clinical presentation of portal vein thrombosis in a cirrhotic allograft is the same as that in a native liver with cirrhosis: variceal hemorrhage, splenomegaly, and ascites.

Pathologic Features

Findings depend on the severity of portal vein flow compromise, time after transplantation, and the condition or structure of the allograft. Complete portal vein obstruction early after transplantation in a noncirrhotic allograft often causes massive coagulative necrosis. Suboptimal portal vein flow because of strictures, kinks, or persistent collateral circulation can cause periportal or midzonal hepatocyte atrophy, coagulative necrosis (see Fig. 52.5), unexplained zonal or panlobular steatosis, and nodular regenerative hyperplasia. Suboptimal portal vein blood flow, which is usually associated with persistent venous collaterals that have failed to close or have reopened after transplantation, can also manifest with hepatic steatosis. Bacterial or fungal infection of a partial portal vein thrombus can result in miliary abscesses of the liver.

Differential Diagnosis

Suboptimal portal vein blood flow can be difficult to distinguish from suboptimal hepatic venous drainage. Linear zones of ischemic necrosis or hepatocellular atrophy favor the former, whereas red blood cell congestion in central veins and centrilobular sinusoids and obliterative central venopathy suggest suboptimal hepatic venous drainage. Ultrasonography and angiography often are needed to specify the cause of the vascular abnormality. Cases of suboptimal portal vein flow that manifest with intrahepatic steatosis need to be distinguished from recurrent or de novo steatosis and steatohepatitis.

Clinical Features

The clinical presentation depends on the severity of outflow tract compromise. Severe stenosis or thrombosis leads to Budd-Chiari syndrome, with hepatic enlargement, tenderness, ascites, and edema. Less severe stenosis may cause only pathologic manifestations or an increase in the portal vein–vena cava pressure gradient. In less severe cases, it is often difficult to determine whether surgical intervention is required.

Pathologic Features

Congestion and hemorrhage involving the hepatic venules and surrounding perivenular sinusoids are the most reliable pathologic findings in patients with suboptimal hepatic venous drainage (Fig. 52.11). Bland centrilobular hepatocyte necrosis and dropout are usually identified. Chronic changes include nodular regenerative hyperplasia, perivenular fibrosis, and central vein occlusion with venocentric cirrhosis, particularly if outflow obstruction is severe or of long duration. Chronic changes can also be accompanied by a prominent ductular reaction at the interface zone. Ductular reaction can also occur in perivenular zones, which can make it difficult to recognize architectural landmarks and to exclude biliary obstruction.

Differential Diagnosis

If any of the previously described perivenular changes are not accompanied by noticeable inflammation, a mechanical or outflow obstruction should be suspected. Studies of long-surviving allografts, especially in children, show that acute or chronic AMR may contribute to noninflamed perivenular subsinusoidal fibrosis.^98–100 Recognition of an immune-mediated cause may be aided by the finding of increased perivenular and central vein endothelial C4d and CD20 cell staining and by detection of serum DSAs of relatively high titers (>10,000 median fluorescence intensity [MFI]).^98–100 Many affected children had previous episodes of ACR. If acute or chronic centrilobular vascular changes are accompanied by significant lymphocytic, histiocytic, or lymphoplasmacytic inflammation, an immunologically mediated cause of injury such as acute or chronic rejection, plasma cell–rich or autoimmune hepatitis (AIH), or an adverse drug reaction should be suspected.

Perivenular inflammation can be transient in some cases of immune-mediated centrilobular injury. It can manifest at a later stage, when it is indistinguishable from mechanical venous outflow obstruction.¹⁰¹

Clinical Features

Liver injury tests that show selective elevation of γ-glutamyltransferase (GGT) and alkaline phosphatase levels are the usual methods of detecting early or minor biliary tract complications such as strictures and stones. Clinical signs and symptoms are uncommon unless the patient is jaundiced or develops acute cholangitis. Biliary tract problems are also often first suspected on routine biopsy evaluation, but cholangiography (e.g., magnetic resonance cholangiopancreatography [MRCP], duct-to-duct endoscopic retrograde cholangiopancreatography [ERCP], percutaneous transhepatic cholangiography [PTC]) is needed to confirm the diagnosis and localize the defects.^108,112,115

More serious biliary tract complications, such as obstruction, cholangitic abscesses, and ascending cholangitis, usually manifest with fever, jaundice, right upper quadrant pain, and intermittent bacteremia. During the first several months after transplantation, T-tube stents provide access for cholangiograms, which are routinely performed before clamping at 1 week postoperatively and again at the time of T-tube removal.¹⁰²

Pathologic Features

Allograft biliary tract complications are pathologically identical to those seen in native livers. Portal, periportal, periductal, and pericholangiolar edema; predominantly neutrophilic portal inflammation; intraepithelial and intraluminal neutrophils in true bile ducts; an interface ductular reaction of some severity; centrilobular hepatocanalicular cholestasis; and small clusters of neutrophils throughout the lobules are typical findings of stricturing or obstructive cholangiopathy (see Fig. 52.12). With time, chronic biliary tract strictures are often associated with mixed or predominantly chronic portal inflammation, biliary epithelial cell senescence changes, and low-grade ductopenia involving small bile ducts. More than 1 year after transplantation, in addition to the classic features described previously, biliary strictures are a relatively common cause of portal eosinophilia.

Biliary-vascular fistulas are characterized by red blood cells in the lumen of bile ducts or by bile concretions in blood vessels. Occasionally, an inordinate elevation of the serum bilirubin level is associated with a fistula. In contrast, periductal hemorrhage surrounding small interlobular bile ducts is an inconsequential finding in asymptomatic patients when a biopsy is obtained within 1 or 2 days after transhepatic cholangiography.¹¹⁶

Differential Diagnosis

The differential diagnosis is heavily influenced by the time after transplantation and the patient’s original disease. Within the first several weeks after transplantation, biliary obstruction or cholangitis can be difficult to distinguish from preservation/reperfusion injury and ACR, particularly if the patient was treated with increased immunosuppression before the biopsy was obtained.

Features that favor biliary tract obstruction or stricturing to ACR include neutrophilic-predominant portal inflammation, periductal edema, retention of the normal nucleus-to-cytoplasm ratio in biliary epithelial cells, and an absence of perivenular mononuclear inflammation. Acute rejection is favored when the mixed portal inflammation is composed of blastic and small lymphocytes, plasma cells, and eosinophils. Lymphocytic cholangitis, an increased nucleus-to-cytoplasm ratio in biliary epithelial cells, and perivenular inflammation also are seen. Portal eosinophilia in early acute rejection can be quite striking, especially in patients treated with steroid-sparing immunosuppressive regimens.¹¹⁷

Late-onset chronic biliary tract complications occasionally manifest with predominantly mononuclear portal inflammation, biliary epithelial cell senescence changes, and low-grade ductopenia.¹¹⁸ Late-onset chronic biliary strictures can also cause portal eosinophilia and can mimic acute and chronic rejection, viral hepatitis, and recurrent autoimmune disorders.

It can be difficult to differentiate chronic rejection caused by biliary strictures from other causes, especially recurrent PSC, in peripheral core-needle biopsies.¹¹⁹ The at-risk populations are similar. Both conditions can cause intrahepatic cholestasis, biliary epithelial cell senescence changes, and small bile duct loss. Careful examination of the clinical history, evaluation of serial biopsies, and analysis of the pathology are needed to distinguish between biliary strictures and chronic rejection.¹¹⁹

In analyzing needle biopsies, features that favor biliary strictures or recurrent primary sclerosis cholangitis instead of chronic rejection include a history of biliary tract complications, PSC as the original disease, periductal lamellar edema involving true bile ducts, stellate portal expansion, portal neutrophilia, a ductular reaction affecting at least some portal tracts, and deposition of copper or copper-associated protein in periportal hepatocytes. Features that favor acute or chronic rejection include a previous history of rejection, inadequate immunosuppression, lymphoplasmacytic portal inflammation, small portal tracts, absence of a ductular reaction, active central perivenulitis, and perivenular fibrosis.

Additional findings in failed allografts can be used to distinguish biliary strictures or recurrent PSC from chronic rejection.¹¹⁹ Chronically rejected livers are usually of normal or slightly increased size and weight, whereas those with obstructive cholangiopathy are usually significantly enlarged. In hilar lymph nodes, obstructive cholangiopathy usually causes bile-pigmented sinus histiocytosis, whereas chronically rejected nodes are usually atrophic and fibrotic. Perihilar arteries are normal or show mild focal eccentric fibrointimal hyperplasia. Foam cell arteriopathy and significant concentric fibrointimal hyperplasia are typical of chronic rejection. In obstructive cholangiopathy, extrahepatic or large intrahepatic bile ducts often show focal ulceration, periductal lymphoplasmacytic inflammation, and fibrosis. In chronic rejection, large duct inflammation and ulceration are unusual. When the infiltrate shows plasma cell–rich infiltrates, the possibility of recurrent or de novo immunoglobulin G4 (IgG4)–associated sclerosing disease should be considered.¹²⁰

Cholangitis favors a biliary tract complication, whereas cholangiolitis and lobular disarray favor chronic hepatitis. A cholestatic liver laboratory test profile favors obstructive cholangiopathy unless the patient has the cholestatic variant of viral hepatitis. Review of HBV or HCV nucleic acid levels in blood helps to distinguish between the two, because cholestatic viral hepatitis can be associated with very high levels of viral replication (e.g., 20 to 30 million IU/mL of HCV RNA). Deposits of copper or copper-associated protein in periportal hepatocytes indicate obstructive cholangiopathy because they are not seen in chronic rejection alone or in cholestatic hepatitis.

Angiography and cholangiography (e.g., ERCP) are useful in making the distinction between obstructive cholangiopathy and chronic rejection. Pruning of peripheral arterial and biliary trees and poor peripheral filling are seen in chronic rejection. In cases of obstructive cholangiopathy, some intrahepatic duct dilation is observed, but arterial changes are not seen or are insignificant.

Clinicopathologic correlation can help to distinguish late-onset biliary tract complications from late-onset acute rejection. Adequate immunosuppressive drug levels, with preferential elevation of the GGT and alkaline phosphatase levels,¹²¹ favors obstructive cholangiopathy because late-onset acute rejection is unusual in this circumstance. It is not possible to reliably distinguish recurrent PSC from other causes of biliary tract obstruction or stricturing on the basis of needle biopsy evaluation alone.

Pathophysiology

The consequences of anti-donor antibodies depend on the subclass, titer, and timing of the antibody response and on the density and distribution of target antigens in the liver.^107,136,137 Antibodies can cause various degrees of damage and can even be protective in some circum­stances.^138–140 Complement-fixing, high-titer, preformed antibodies directed at antigens expressed on endothelial cells, such as isoagglutinins and lymphocytotoxic (anti-HLA class I and II) antibodies, are the most dangerous.¹⁴¹ MHC class I antigens and ABO antigens are moderately to strongly expressed on the vasculature in normal livers. The former can be upregulated when the graft is inflamed for other reasons. MHC class II antigens are not expressed or are expressed at low levels in the hepatic microvasculature in the normal liver. However, they are upregulated significantly after transplantation.^142–144

ABO incompatibility places recipients at risk for AMR, especially when isoagglutinins are present in high titers.^123,129 Isoagglutinins also cause more severe damage than lymphocytotoxic antibodies; the latter do not reliably cause clinically relevant allograft dysfunction unless present at relatively high titer (>1 : 32) and do not routinely influence short-term survival.^{53,54,145,146}

Resistance of liver allografts to AMR from anti-HLA antibodies has been attributed to (1) secretion of soluble MHC class I antigens that bind to and neutralize anti-class I HLA¹⁴⁷; (2) Kupffer cell phagocytosis of immune complexes and activated platelet aggregates; (3) a dual afferent hepatic blood supply; (4) unique hepatic sinusoidal microvasculature (which is devoid of a conventional basement membrane)¹⁰⁷; and (5) a homologous source of complement in xenografts.¹⁴⁸

Positive lymphocytotoxic crossmatches (mostly anti-HLA antibodies) are encountered in 8% to 12% of recipients.^{53,54,145,146} Most recipients are female. Only 5% to 20% of recipients with a positive crossmatch have titers high enough to cause clinically significant liver injury.^53,123 IgG usually causes more damage than immunoglobulin M (IgM) lymphocytotoxic antibodies.^107,149

Because the patient population at risk for severe AMR because of anti-HLA antibodies is small, AMR is often overlooked as a cause of liver dysfunction or failure. This occurs because the incidence of AMR in ABO-compatible allografts early after transplantation is relatively low (approximately 5% among patients harboring anti-HLA antibodies¹²⁴); many programs do not routinely conduct pretransplantation crossmatching or DSA determinations; and even if crossmatches are performed, the titer and specificity are not determined, and posttransplantation monitoring is not routinely carried out. The long-term consequences of intermittent or persistent DSAs require further study.^53,150

Antibodies cause damage by binding to allograft endothelium and fixing complement. This triggers the complement cascade and causes endothelial damage, deposition of platelet-fibrin thrombi, and initiation of the clotting and fibrinolytic cascades. In severe, rapidly evolving cases, this leads to microvascular thrombosis, arterial vasospasm, and coagulopathy that act in concert to impair blood flow and cause hemorrhagic necrosis of the liver.^138,139 Less precipitous injury can occur during the transition from acute to chronic rejection when destruction of peribiliary capillary plexuses contributes to ductopenic or chronic rejection.^{141,151–153}

Clinical Presentation

Hyperacute AMR, the most severe form, has largely been eliminated by avoiding ABO-incompatible transplants without pretreatment.^128,129 When it does occur, hyperacute rejection begins immediately after reperfusion. Even in severe cases, allograft dysfunction evolves during a period of hours to days, as with hyperacute rejection in other organs.¹⁰⁷ Initial signs of dysfunction usually develop in the operating room after complete revascularization. It can be recognized by uneven reperfusion, swelling, a dusky appearance, and cessation of bile flow. These signs are often accompanied by coagulopathy, which manifests as difficulty in achieving hemostasis and an inordinate need for platelets and other blood components.⁴⁶ Precipitous allograft failure is unusual.

A more common manifestation of AMR is a persistent elevation of serum bilirubin and ALT/AST levels (depending on whether the antibodies are isoagglutinins or anti-HLA) during the first several days to weeks after transplantation. This is often accompanied by persistence of the positive crossmatch, the presence of DSAs, unexplained and often refractory thrombocytopenia, and low serum complement activity levels.^{47,123,124,149} In severe cases, hepatic angiograms, obtained to exclude an arterial thrombosis, usually show segmental narrowing (indicative of immunologically mediated arterial vasospasm) and diffuse luminal narrowing with poor peripheral blood flow.¹⁰⁷

Pathologic Features

Pathologic manifestations of AMR depend on the timing of the biopsy; the subclass, titer, and specificity of the anti-donor antibodies; and whether ACR coexists.¹⁰⁷ Isoagglutinins usually cause more red blood cell congestion and necrosis than anti-HLA or lymphocytotoxic antibodies (Fig. 52.13).

High-titer isoagglutinins (>1 : 64) often cause prominent red blood cell and focal neutrophil sludging in the sinusoids and platelet-fibrin thrombi in periportal sinusoids, portal veins, and central veins.^46,154,155 Acidophilic hepatocyte necrosis develops within 2 to 6 hours after reperfusion in untreated recipients. This is often quickly followed by portal or periportal edema, necrosis, focal hemorrhage, C4d deposits in portal stroma,¹⁵⁴ and diffuse endothelial C4d deposits in portal veins, capillaries, and sinusoids.^53,154

In severe untreated cases, confluent coagulative necrosis, prominent sinusoidal and venous congestion, and edema and hemorrhage into the portal tracts appear within 1 to 5 days.^46,53,154 Portal veins often show fibrin deposition. If sampled, medium-size arteries show endothelial cell hypertrophy and evidence of arterial vasospasm, such as mural myocyte vacuolization, wrinkling of the elastic lamina, and thickening of the wall with narrowing of the lumen. Neutrophilic or necrotizing arteritis is only occasionally observed. Portal neutrophilia, cholangiolar proliferation, and small areas of confluent hepatic necrosis begin to appear at 2 to 3 days after transplantation. If untreated, progressive hemorrhagic infarction occurs in ABO-incompatible organs during the next 1 to 2 weeks.

ABO-incompatible allografts that fail because of untreated AMR are often grossly enlarged, cyanotic, and mottled and show areas of geographic necrosis. Capsular ruptures, hepatic artery, or portal vein thrombosis can develop in extreme cases. Microscopically, changes in the hilum or perihilar region can be particularly helpful in recognizing that AMR caused or contributed to allograft failure. These changes include congestion and leukocyte margination in the peribiliary vascular plexus, partially organized thrombi in arterial branches, focal mural necrosis of large septal bile ducts, and inflammatory and necrotizing arteritis. Late sequelae of AMR include biliary sludge and stricturing caused by obstructive cholangiopathy, obliterative arteriopathy, loss of small bile ducts, and chronic rejection.^{46,128,130,156,157}

Reperfusion biopsies from patients with high-titer (>1 : 32) lymphocytotoxic antibodies show platelet aggregates in the portal and terminal hepatic veins more often than crossmatch-negative controls.^9,123 Spotty acidophilic necrosis of hepatocytes and centrilobular hepatocellular swelling, accompanied by cholangiolar proliferation and hepatocanalicular cholestasis, occurs during the first week after transplantation in patients with high-titer antibodies. Inflammatory and necrotizing arteritis is rare, but it is almost diagnostic when detected. Coexistent ACR with portal eosinophilia is common.¹²⁴ The pathologic changes closely resemble those of preservation/reperfusion injury. Clinicopathologic correlation with exclusion of other causes of dysfunction and staining for C4d and other immune deposits are needed to confirm the diagnosis.^47,48,53,153

An AMR diagnosis in ABO-compatible liver allografts should be based on a biopsy showing changes consistent with an AMR-related pattern of injury, clinicopathologic exclusion of causes of a similar pattern of injury, serologic evidence of DSAs, and evidence of strong and diffuse antibody or complement (C4d) deposition within the injured allograft (Fig. 52.14).^{46,48,53,123,158} The latter is defined by strong portal vein, capillary, and usually periportal sinusoidal endothelial staining involving most portal tracts.¹²⁴ Compared with other allografts, a diagnosis of isolated liver allograft AMR because of anti-HLA antibodies is difficult to establish with certainty.

Difficulties can be encountered in establishing a correct diagnosis when liver allografts are large and able to absorb high antibody loads (especially anti–class I HLA antibodies)¹⁴⁷ and are therefore resistant to AMR-related damage.^{9,107,123,136,159} In some cases, the immune deposits are ephemeral^46,123 or can be associated with other insults.¹³⁴ Clinicopathologic similarities exist between AMR and preservation injury, sepsis, and biliary or vascular complications.^46,123

Intrahepatic immune deposits are ephemeral in AMR, even when evaluated by immunofluorescence in frozen tissue sections.^{46–48,123,158} In severe cases, selective deposits of IgG, IgM, C3, or C4 are usually detectable in the sinusoids and in perihilar arteries, portal veins, and the peribiliary plexus.^46,47,123 Thereafter, the distribution of immune deposits becomes patchy, and they may be difficult to distinguish from background staining unless antibody titers are high. If AMR is suspected, saving frozen tissue for immunofluorescence testing is important.

Recognition that C4d persists for several days, can be detected in FFPE tissues and correlates with circulating DSAs makes an AMR diagnosis easier to establish in kidney allografts.^160,161 Interpretation and utility of C4d staining in liver allografts is less clearcut.^{154,158,162–169}

Bellamy⁴⁸ and Hubscher¹⁵⁸ reviewed the literature on C4d immunohistochemistry in liver allografts. Normal liver and allograft biopsies are usually negative for C4d staining. Portal vein, arterial, and capillary C4d and sinusoidal endothelial C4d have been detected in crossmatch-positive recipients more often than in crossmatch-negative controls¹⁶⁷ and in patients with isolated AMR.^124,134,169 Endothelial cell C4d staining is most specific for AMR, but portal vein C4d stromal staining has also been described in cases of ABO-incompatible AMR,¹⁵⁴ ACR,¹⁶⁷ and chronic rejection.^153,170 Kozlowski and coworkers¹³⁴ suggest that C4d staining of FFPE tissue sections is nonspecific and that linear sinusoidal C4d deposits in frozen tissue specimens are needed to establish AMR. We found that C4d staining in FFPE tissue is reliable and that endothelial deposits are most specific. However, clinicopathologic correlation is needed to establish a diagnosis of AMR with certainty. In contrast, portal and perivenular stromal staining is commonly observed and difficult to interpret. In nonliver allografts, capillary microvascular staining is used mainly to determine whether antibodies contribute to allograft injury.

As in other allografts, C4d deposits are often accompanied by ACR.^{53,153,154,162–169} In some studies, the burden of C4d deposits is directly proportional to the Banff grade.^{154,162–169} Necrotic and steatotic hepatocytes can show nonspecific C4d staining.

Portal vein and capillary C4d deposits can be detected in other causes of allograft dysfunction, including biliary obstruction,¹⁶² recurrent HBV infection,¹⁶³ recurrent HCV infection,¹⁶⁵ and de novo autoimmune hepatitis.¹⁷¹ C4d staining has also been described in portal vein and capillary, sinusoidal, central vein, and arterial endothelial cells; in lymphoid nodules; and in periductal and portal stromal cells in native pediatric livers with hepatitis B, hepatitis C, autoimmune hepatitis, and overlap syndromes that include autoimmune hepatitis and PSC.¹⁷² Endothelial C4d deposits in non–rejection-related allograft disorders are less widespread than in severe AMR or ACR. Similar to kidney and heart allografts, liver C4d deposits have been associated with macrophage and plasma cell infiltrates.¹⁶⁴

The possibility that chronic or low-grade, persistent AMR may contribute to indolent but progressive fibrosis has received increased attention, particularly for pediatric recipients.^98–100 Evidence supporting this association includes increased prevalence and titer of DSAs associated with tissue C4d deposits and suboptimal immunosuppression.^98–100

Differential Diagnosis

Distinguishing AMR from preservation/reperfusion injury can be difficult. The presensitization state, persistence of circulating antibodies, diffuse C4d staining,⁵³ and posttransplantation clinical and laboratory profile can provide discriminating information. AMR should be suspected in female liver allograft recipients with high-titer DSAs or lymphocytotoxic antibodies who have received a liver with a short cold ischemic time but who show persistent DSAs after transplantation and develop graft dysfunction, refractory and otherwise unexplained thrombocytopenia, and circulating low complement levels in the first several weeks after transplantation.^{47,53,123,149}

Alloantibody-mediated AMR should be favored instead of preservation/reperfusion injury when any of the following histopathologic findings are detected: (1) Neutrophils, macrophages, eosinophils, and to a lesser extent, lymphocytes may be marginating on the luminal aspect and beneath the portal and central veins; this finding may be combined with diffuse endothelial cell reactivity. (2) Occasional blastic lymphocytes and eosinophils and other features of ACR may be seen in the portal or perivenular areas. (3) Diffuse C4d staining may be detected in the microvasculature.⁵³ Diffuse staining is defined as positive staining in most portal veins and capillaries, with extension into the periportal sinusoids. Portal or periportal edema, necrosis, and hemorrhage should raise the possibility of ABO incompatibility.

Precipitous allograft failure from severe AMR is rare, except in ABO-incompatible livers. When it occurs, it is difficult to distinguish from hemorrhagic liver necrosis caused by hypotension, poor perfusion, sepsis, or vascular thrombosis. Unless unequivocal evidence of AMR is detected, such as inflammatory or necrotizing arteritis or diffuse immunoglobulin and complement (C4d) deposition combined with serologic evidence of anti-donor antibodies, the cause of allograft failure can be extremely difficult to determine with certainty. More commonly, changes diagnostic of AMR are not identified. The diagnosis of AMR requires thorough clinicopathologic correlation.

Clinical Features

ACR usually manifests clinically between 5 and 30 days after transplantation, depending on the immunosuppressive regimen. ACR affects approximately 30% of liver transplant recipients.¹⁷⁵ Earlier manifestations can be seen among presensitized patients or patients who receive less than optimal baseline immunosuppression. Later manifestations can occur in recipients who have been treated with lymphocyte-depleting antibodies or recipients with inadequate maintenance immunosuppression. Clinical findings are often absent early in the course of disease, when tissue damage is mild. Later in the course or in cases of severe acute rejection, fever and allograft enlargement, cyanosis, and tenderness may occur.

Early after transplantation, when biliary T-tubes are in place, decreased bile flow and thin, pale bile are commonly seen and do not necessarily denote acute rejection. Likewise, ascites may develop because of increased intrahepatic hydrostatic pressure that results in liver swelling and increased lymph production.¹⁸⁰ These clinical findings alone are not sufficient to implicate acute rejection.

Biochemical evidence of liver dysfunction because of acute rejection usually includes nonselective elevation of some or all of the standard liver injury test values. Leukocytosis and eosinophilia are frequently identified.¹⁸⁰ The levels of a variety of proteins (e.g., interleukins, neopterin, amyloid A protein) are increased in the peripheral blood, but none of the proteins is routinely assayed to monitor recipients. Instead, ACR is usually suspected on clinical grounds and then confirmed by a core-needle biopsy.

Risk factors for the development of early acute rejection depend on the type of immunosuppressive regimen. This condition can affect any recipient subgroup, including young recipients, those with advanced-age donors, healthy recipients (e.g., normal serum creatinine level, low Child-Pugh classification), donor-recipient HLA-DR mismatches, patients with immune dysregulated syndromes (e.g., PSC, autoimmune hepatitis, PBC), those with long cold ischemic times, and those with an HLA-C genotype.^175,181 Late-onset acute rejection more than 1 year after transplantation may have a different pathologic appearance. It is more often associated with inadequate immunosuppression, is more difficult to control, and more frequently leads to allograft injury and failure.^177,181,182

Pathologic Features and Grading

ACR is characterized by predominantly mononuclear but mixed portal inflammation containing blastic or activated lymphocytes, neutrophils, and eosinophils; by subendothelial inflammation of portal and terminal hepatic venules; and by bile duct inflammation and damage.^180,183 Minimal diagnostic criteria needed to establish a diagnosis of acute rejection include at least two of these pathologic findings. The diagnosis is strengthened if more than 50% of the ducts or terminal hepatic veins are damaged or unequivocal endotheliitis of portal or terminal hepatic vein branches are identified. Pathologic evidence of severe injury, which is used for grading, includes perivenular inflammation, centrilobular necrosis, arteritis, and inflammatory features such as central-to-central, bridging inflammation and necrosis.^176,180

Acute rejection-type infiltrates are encountered mostly in portal tracts but occasionally surround central veins. Endotheliitis (i.e., endothelialitis) refers to lymphocytes located underneath the portal and central vein endothelium. This characteristic feature of acute rejection may be seen in other types of allograft dysfunction.¹⁸⁴ The lymphocytes are predominantly T cells, including CD8-positive cells that often surround and invade damaged bile ducts.^185,186 B cells usually comprise a minor fraction of the infiltrate. Macrophages and other leukocytes are present and can predominate in severe acute rejection.^185–187 Immunophenotypic analysis is not prognostically or clinically useful for establishing a diagnosis of acute rejection, except when attempting to distinguish acute rejection (T-cell predominant) from a posttransplantation lymphoproliferative disorder (B-cell predominant).

Lymphocytic cholangitis involving small bile ducts (<30 µm in diameter) is an important feature of ACR. Lymphocytes are located inside ductal basement membranes in association with biliary epithelial cell injury. The morphologic features of acute injury include paranuclear vacuolization, apoptotic bodies, increased nucleus-to-cytoplasm ratio, increased mitoses, and prominent nucleoli. Cytoplasmic eosinophilia and multinucleation usually signal senescence-related changes and chronic injury.¹¹⁸ Breaks in the basement membrane indicate severe bile duct damage. Portal or peribiliary granulomas are not a feature of acute or chronic rejection. When encountered, a non–rejection-related cause of duct injury, such as recurrent PBC, a coexistent mycobacterial or fungal infection, or sarcoidosis, should be suspected.

Inflammatory, necrotizing arteritis is a defining feature of severe acute rejection, but it is rarely detected in needle biopsies because the arteries that are most commonly affected are located in the hilum of the liver. Recognition of arteritis in peripheral needle biopsies is a poorly reproducible finding.¹⁸⁸ Arteritis is not usually included in grading schemes. When present, this feature indicates a severe rejection episode. The portal tract–parenchyma interface zone is usually unremarkable in cases of early mild and moderate ACR. In severe cases, portal inflammatory cells involve the periportal parenchyma.¹⁷⁸

Rejection-type infiltrates can be seen in the perivenular sinusoids and extracellular matrix surrounding terminal hepatic venules (i.e., central perivenulitis).¹⁷⁸ The infiltrates are identified in as many as 30% cases of acute rejection. Perivenulitis occurs later (>100 days) after transplantation. Severe ACR is diagnosed only when typical portal changes of acute rejection are accompanied by perivenular inflammation and zonal centrilobular congestion, hemorrhage, and hepatocyte necrosis and dropout.

The Banff grading scheme for acute and chronic rejection was constructed on data generated by recognized experts in liver transplant pathology, hepatology, and surgery from many of the major hepatic transplantation centers in North America, Europe, and Asia (Tables 52.3 and 52.4).¹⁸³ It is widely used¹⁸⁹ easy to apply, and reproducible.¹⁹⁰ It is scientifically correct and has prognostic significance.^176,191

The Banff system uses descriptive grades of indeterminate, mild (Fig. 52.15), moderate (Fig. 52.16), and severe (Fig. 52.17 and Table 52.3). It also uses a semiquantitative rejection activity index (RAI) (Table 52.4),¹⁸³ which is a remnant of the European grading system¹⁹² and equivalent to the hepatitis activity index.¹⁹³ RAI semiquantitatively scores the prevalence and severity of three separate pathologic features, each on a scale of 0 to 3: portal inflammation, bile duct damage, and subendothelial inflammation. The individual components are then summed to determine a total RAI score.

There is direct correlation between the total RAI score and rejection grade and correlation with increased risk of persistent or recurrent acute rejection, chronic rejection, and graft failure.¹⁷⁶ The ranges of RAI scores are indeterminate (1 to 2), mild (3 to 4), moderate (5 to 6), and severe (>6). The maximum possible total RAI score is 9, but biopsies rarely achieve this score.¹⁷⁶ Instead, most episodes are mild, have a total RAI score of less than 6, and respond to increased immunosuppression. These cases do not lead to significant fibrosis, bile duct loss, or arteriopathy as determined in subsequent or follow-up biopsies.¹⁷⁶ Graft failure from ACR is distinctly unusual.

Additional immunosuppression causes some pathologic changes more difficult to detect due to resolution of some characteristic findings, such as subendothelial infiltration of veins, can occur within 24 hours. Immunosuppressive treatment before biopsy can lead to centrilobular hepatocyte swelling and hepatocanalicular cholestasis, causing further confusion. Typically, 7 to 10 days are required for rejection-related changes to resolve after therapy.

Late acute rejection (>100 days) is characterized by features found in early ACR. However, slightly atypical features can develop,¹⁷⁸ such as fewer blastic lymphocytes, more necroinflammatory interface activity, less venous subendothelial inflammation, a higher incidence of perivenular inflammation, and slightly more lobular activity. The features more closely resemble those of chronic hepatitis.¹⁷⁸

Late ACR can manifest with prominent perivenular inflammation, hepatocyte dropout, and minimal or no portal tract changes (i.e., isolated central perivenulitis) (Fig. 52.18).^194–196 These cases may evolve into typical chronic rejection with ductopenia and perivenular fibrosis. Subendothelial inflammation of the portal or central veins is not a required finding. Perivenular fibrosis and a Budd-Chiari syndrome or a veno-occlusive disease–like clinical syndrome can develop as a consequence of the severe perivenular injury.^101,197 Evidence suggests that chronic AMR may contribute to perivenular sinusoidal fibrosis.^98–100 The Banff Working Group proposal is described in Table 52.5.

Minimal and mild cases may resolve spontaneously.¹⁹⁶ Severe perivenular changes probably warrant more aggressive treatment, but no prospective studies have been performed.

Differential Diagnosis

The differential diagnosis of ACR depends on the time since transplantation. ACR should be distinguished from preservation injury and obstructive cholangiopathy or cholangitis during the first several months after transplantation.

Distinguishing acute rejection from recurrent hepatitis B or C and autoimmune hepatitis can be difficult. Hepatitis and rejection show predominantly mononuclear portal inflammation, bile duct damage, and acidophilic necrosis of hepatocytes. The severity and prevalence of bile duct damage, interface activity, lobular changes, and perivenular inflammation and hepatocyte dropout should be assessed.¹⁹⁸ Features that favor acute rejection include inflammatory bile duct damage and perivenular inflammation involving most ducts and central veins, respectively, and low-grade or absent lobular and interface necroinflammatory activity. Conversely, recurrent or new-onset viral hepatitis or autoimmune hepatitis is favored when interface and lobular necroinflammatory activity predominate over bile duct and perivenular changes.

Allograft autoimmune hepatitis¹⁹⁹ resembles viral hepatitis in most aspects, except that autoimmune hepatitis more often shows conspicuous plasma cells as a component of the infiltrate and often shows more prominent interface activity. Some allograft recipients with autoimmune hepatitis have a prominent plasma cell–rich central perivenulitis that involves most central veins (similar to autoimmune hepatitis affecting native livers). Severe interface activity, plasma cell predominance in the inflammatory infiltrate, and relatively mild bile duct damage favor allograft autoimmune hepatitis over acute rejection.

Pathophysiology

Immunologic mechanisms of injury that contribute to allograft damage during acute rejection also contribute to the development of chronic rejection,^{176,200,203,206–209} including an antibody component.^{125,141,151,205} Bile duct loss in chronic rejection has been attributed to a combination of direct immunologic and indirect ischemic damage resulting from obliterative arteriopathy, small artery or arteriolar loss, and destruction of the peribiliary capillary plexus,^152,210 with a possible contribution from alloantibodies.^123,125,151 Cumulative damage triggers biliary epithelial cell senescence.¹¹⁸ Several studies have shown that the early phase of chronic rejection is potentially reversible,^{118,208,211,212} provided that ductules and surrounding microvasculature are preserved.²¹³

Clinical Features

Chronic rejection usually occurs in patients with a history of acute rejection in whom progressive cholestasis and an increase in canalicular enzymes unresponsive to antirejection treatment develops.¹⁸⁰ Two clinical settings are typical: culmination of multiple or unresolved episodes of ACR and AMR and an indolent manifestation without preceding clinically recognizable episodes of acute rejection. The first scenario is most common, especially in the first year after transplantation. The latter presentation is relatively uncommon and likely reflects inadequate monitoring. Late-onset chronic rejection (>1 year after transplantation) typically occurs in inadequately immunosuppressed patients as a result of noncompliance or because of infectious, neoplastic, or toxic complications of chronic immunosuppression.²⁰³

Standard liver function tests usually show a cholestatic or biliary pattern of injury, with preferential elevation of GGT and alkaline phosphatase levels.^121,180,214 Persistent elevation of ALT and total bilirubin levels usually marks the transition from acute to chronic rejection and can presage allograft failure.^194,203,208 Clinical symptoms resemble those of acute rejection until dysfunction becomes significant enough to cause jaundice. Biliary sludging, biliary strictures, hepatic infarcts, and loss of hepatic synthetic function are other late findings that often occur immediately before allograft failure.¹⁸⁰ Hepatic angiograms show pruning of the intrahepatic arteries with poor peripheral filling and segmental narrowing, reflecting the obliterative arteriopathy of chronic rejection.^180,215

Pathologic Features and Staging

Chronic rejection primarily affects portal tracts and the perivenular parenchyma (Fig. 52.19). It is divided into early (Fig. 52.20) and late (Fig. 52.21) stages according to the Banff scheme (Table 52.6).²⁰⁰ Early-stage chronic rejection is characterized by mild portal inflammation, lymphocytic cholangitis, biliary epithelial cell senescence changes involving most small bile ducts, and some degree of small bile duct loss (usually involving less than 50% of portal tracts). Compared with acute rejection, chronic rejection usually shows less severe inflammation and fewer eosinophils. The inflammatory infiltrate is composed primarily of lymphocytes, plasma cells, and mast cells.²¹⁶

Recognition of biliary epithelial cell senescence is critical to the diagnosis of early chronic rejection.¹¹⁸ Features include nuclear enlargement, hyperchromasia, and uneven spacing resembling cytologic dysplasia; syncytia formation; eosinophilic transformation of the cytoplasm; and bile ducts only partially lined by biliary epithelial cells (Figs. 52.19 and 52.20). Immunohistochemical markers of senescence and downregulated cell cycle progression, such as CDKN2A (p16) and CDKN1A (p21), become upregulated in cells under severe stress.^118,217 Downregulation of epithelial junctional proteins combined with upregulation of mesenchymal proteins may occur in response to injury.²¹⁸

Late-stage chronic rejection is characterized by bile duct loss involving more than 50% of portal tracts. Arteriolar loss can also be seen.

Crawford and colleagues²¹⁹ defined a portal tract as “a focus within the parenchyma containing connective tissue (by Masson trichrome stain) and at least two luminal structures, each with a continuous connective tissue circumference.” In normal liver sampled by percutaneous liver biopsy, bile ducts and hepatic artery branches are seen in 93% ± 6% and 91% ± 7% of portal tracts, respectively.²¹⁹ These percentages may be lower in larger tissue samples.¹⁵² With two standard deviations from the normal as a cutoff, bile duct loss is diagnosed when less than 80% of portal tracts contain bile ducts. Arterial loss is diagnosed when less than 77% of portal tracts contain hepatic artery branches.

A similar approach uses the concept of bile duct–artery parallelism. Ductopenia is defined by at least one unpaired artery in more than 10% of all portal tracts or two unpaired arteries in different portal tracts.²²⁰ Unpaired arteries were defined as arteries without an accompanying bile duct within a distance of 10 hepatic artery diameters from the edge of the artery.

Late chronic rejection can cause bile duct and arterial loss,^152,210 which can present difficulties when trying to apply these diagnostic algorithms. Portal tract recognition should be based primarily on the parenchymal location of the putative structure; cholestasis in chronic rejection is centrilobular.

A ductular reaction at the interface zone is unusual in chronic rejection, unless the liver is in a recovery phase.^208,211,212 Staining with cytokeratins 19 and 7 can help to document bile duct loss in chronic rejection and detect ductular metaplasia of periportal hepatocytes during recovery.⁴⁹ When a ductular reaction is easily noticeable by routine light microscopy, it signals the regrowth of bile ducts^208,211,212 or a downstream biliary stricture. The latter can be documented by identifying periportal hepatocyte copper deposits, which are not seen in chronic rejection.

Early chronic rejection in the terminal hepatic venules and surrounding perivenular parenchyma is characterized by subendothelial and perivenular mononuclear inflammation (see Fig. 52.19). The perivenular infiltrate usually consists of lymphocytes, pigment-laden macrophages, and plasma cells,^200,209 often accompanied by hepatocyte dropout and mild perivenular fibrosis.²⁰⁰ Spotty acidophilic hepatocytes elsewhere in the parenchyma can be seen during evolution from early to late chronic rejection.²²¹

Perivenular changes in late chronic rejection are characterized by severe (bridging) perivenular fibrosis, at least focal central-to-central or central-to-portal bridging, and sometimes obliteration of terminal hepatic venules (Fig. 52.21).²⁰⁰ Well-developed cirrhosis arising from chronic rejection is unusual, until the very late stages, when venous obliteration leads to parenchymal extinction and venocentric cirrhosis.²⁰⁶ True regenerative nodules are uncommon, presumably because coexistent obliterative arteriopathy blunts any regenerative response.²²² Perivenular hepatocyte ballooning and dropout, centrilobular hepatocanalicular cholestasis, nodular regenerative hyperplasia, and intrasinusoidal foam cell clusters are other common findings in late chronic rejection.

The final diagnosis of chronic rejection should be based on a combination of clinical, radiologic, laboratory, and pathologic findings. In a biopsy specimen, minimal diagnostic criteria for chronic rejection are senescent changes affecting most bile ducts with or without bile duct loss, convincing foam cell obliterative arteriopathy, or bile duct loss affecting more than 50% of portal tracts.²⁰⁰

A diagnosis of chronic rejection is much easier to establish in explanted failed allograft specimens because diagnostic findings can be seen in first-, second-, and third-order branches of the hepatic artery (Fig. 52.22). Obliterative arteriopathy is usually found in at least some of the perihilar arteries, except in cases characterized by bile duct loss and perivenular fibrosis alone. Accumulation of foamy macrophages usually occurs initially in the intima, which triggers proliferation of intimal and migration of medial donor-derived myofibroblasts. This causes intimal thickening, luminal narrowing, and medial thinning as arteries attempt to dilate and compensate for reduced arterial flow. Eventually, compensatory mechanisms fail, and the entire arterial wall may be replaced by foam cells. The artery may undergo thrombosis, causing necrosis of large bile ducts and ischemic cholangiopathy.

Foamy macrophages may be seen around bile ducts and veins and within the connective tissue. Large perihilar bile ducts can show sloughing of the epithelium, papillary hyperplasia, mural fibrosis, and acute and chronic inflammation.

Staging of chronic rejection assumes that the diagnosis has already been established.²⁰⁰ The term early chronic rejection implies that a significant potential for recovery exists if the source of immunologic injury can be controlled or reversed. The term late chronic rejection implies that the potential for recovery is limited and that retransplantation should be considered. It has not been established that all patients proceed sequentially from early to late stage chronic rejection. Some patients persist in the early stage for months or years, whereas severe fibrosis and late changes rapidly develop within the first year after transplantation or within weeks or months after the initial onset of chronic rejection. Some cases show predominantly or exclusively bile duct loss or arteriopathy alone. Both features usually occur together.²⁰⁰

One important practical implication of chronic rejection staging is that biopsy findings do not define the point of no return. Instead, the biopsy provides information about the likelihood of reversal, which should be correlated with other clinical and laboratory parameters, such as persistently elevated serum levels of bilirubin (>20 mg/dL), progressive decline in synthetic function, superimposed hepatic artery thrombosis, and bile duct necrosis or biliary sludging.

Differential Diagnosis

In percutaneous needle biopsy specimens, a diagnosis of chronic rejection diagnosis is primarily based on damage and loss of small bile ducts and perivenular fibrosis. Arteries with pathognomonic changes are rarely seen in biopsy specimens.²⁰⁰ Duct injury and ductopenia can also occur because of non–rejection-related complications, such as obstructive cholangiopathy, hepatic artery stricturing or thrombosis, cholangitic adverse drug reactions, and CMV infection. Perivenular fibrosis can also be caused by suboptimal hepatic venous drainage and other causes of perivenular injury. A diagnosis of chronic rejection based on biliary epithelial senescence or loss or on perivenular fibrosis alone should first exclude other non–rejection-related causes of ductal injury and perivenular fibrosis.

Chronic rejection is very difficult to distinguish from biliary tract obstruction or stricturing and recurrent PSC.¹¹⁹ Features that favor obstructive cholangiopathy are bile duct loss in some portal tracts, accompanied by a ductular reaction in others; neutrophil clusters within the lobules; bile infarcts; deposition of copper or copper-associated protein in periportal hepatocytes; and hepatocanalicular cholestasis out of proportion to the prevalence of ductopenia (<50%). Features that favor chronic rejection are bile duct changes combined with central perivenulitis or fibrosis, an absence of changes typical of recurrent PSC, and a history of persistent acute rejection or suboptimal immunosuppression. Cholangiography or angiography may be required to distinguish chronic rejection from biliary obstruction. Studies usually show pruning and poor peripheral filling in patients with chronic rejection.

Isolated ductopenia involving less than 50% of portal tracts can be seen without significant elevations of liver function tests. Whether these uncommon cases represent an early phase of chronic rejection remains uncertain. Isolated perivenular fibrosis can be caused by mechanical outflow obstruction, adverse drug reactions, chronic and persistent DSAs,^98–100 and nonrejection causes of hepatic outflow obstruction.

The safest approach to diagnosing chronic rejection is to review prior biopsies and correlate the pathologic findings with the clinical course. Particular attention should be paid to a history of severe or unresolved acute rejection preceding the development of histologic findings interpreted as chronic rejection.

Clinical Features

Depending on the extent of viral dissemination, any organ system can be involved. The most common signs and symptoms of active CMV infection are gastrointestinal. They include fever, diarrhea, ulcers, leukopenia, and low-grade hepatitis; the latter has modestly elevated liver function test results.^224,225 Respiratory insufficiency and retinitis are signs of severe disseminated disease. CMV can cause a syndrome that mimics EBV-associated posttransplantation lymphoproliferative disorder, with lymphadenopathy, fever, and atypical lymphocytosis.²²⁶

Pathologic Features

CMV hepatitis is characterized by spotty lobular necrosis, Kupffer cell hypertrophy, mild lobular disarray, and patchy lobular inflammation. Under current management schemes, infected hepatocytes rarely contain diagnostic nuclear or cytoplasmic inclusions. Instead, inclusions are usually limited to patients who are overimmunosuppressed and not adequately monitored or treated. Any cell type can be infected. Diagnostic features include large eosinophilic intranuclear inclusions surrounded by a clear halo and accompanied by small basophilic or amphophilic cytoplasmic inclusions. In severe cases, which are largely of historical significance, numerous cells containing CMV inclusions can be found throughout the liver. CMV infection alone does not cause submassive or massive necrosis.²²⁶

Infected cells are often surrounded by neutrophils, microabscesses, or clusters of macrophages and lymphocytes, called microgranulomas. CMV hepatitis can be associated with mild lymphoplasmacytic portal inflammation with bile duct infiltration and damage that can resemble acute or early chronic rejection (Fig. 52.23). Bile duct loss and chronic rejection have been associated with persistent CMV infection in allografts.^224,225

Viral inclusions are rare. CMV hepatitis often manifests with parenchymal alterations but without inclusions. Instead, fragmented nuclear CMV inclusions occur but are often difficult to recognize without immunoperoxidase staining or in situ hybridization. Rapidly dividing tissues such as young granulation tissue, proliferating cholangioles, the edges of infarcts, abscesses, or other intraparenchymal defects are fertile soil for CMV growth.⁵⁰ Immunoperoxidase stain for an epitope of the CMV matrix phosphoprotein 65 (pp65) may be more sensitive, but it requires frozen tissue. It is used less frequently than the monoclonal antibody against CMV p52, an early nuclear protein that works better in FFPE specimens.²²⁴

Differential Diagnosis

CMV hepatitis can be difficult to distinguish from the early phase of HBV or HCV recurrence and EBV hepatitis. CMV can also be difficult to distinguish from HSV hepatitis because both disorders can cause multinucleation and intranuclear eosinophilic inclusions surrounded by halos. CMV inclusion–containing cells can show small basophilic or amphophilic cytoplasmic inclusions, which helps to distinguish them from HSV-infected cells. Circumscribed foci of coagulative necrosis characteristic of HSV is not a feature of CMV hepatitis.

In CMV cases without inclusions, subtle clues that enable distinction from early acute HBV or HCV hepatitis include less lobular disarray and hepatocyte swelling. Microabscesses and microgranulomas are not usually seen in the early lobular phase of HBV or HCV infection. A definitive diagnosis of CMV hepatitis requires characteristic inclusions, which are rare, or demonstration of viral antigens or nucleic acids with immunostaining or in situ hybridization.

CMV hepatitis can resemble EBV hepatitis because both may show mild lymphoplasmacytic portal and lobular inflammation and contain blastic and atypical lymphocytes. EBV hepatitis usually causes more cytologic atypia of the lymphocytes, whereas CMV hepatitis causes more intralobular foci of inflammation (e.g., microgranulomas, microabscesses). Deeper sections, staining for EBV and CMV viral antigens, and in situ hybridization for EBV nucleic acid are usually required to make the distinction.

CMV hepatitis most commonly develops in patients who have recently completed an augmented immunosuppressive regimen for rejection. In some cases, it is difficult to determine whether the liver injury is attributable to residual CMV hepatitis, relapse of acute rejection, or development of chronic rejection. O’Grady and colleagues showed an association between CMV infection and chronic rejection,^227,228 further complicating the issue. Others have not found this association.²²⁹ Priority may be given to the finding of CMV inclusions, antigens, or nucleic acid, followed by anti-CMV therapy and follow-up biopsy after 1 to 2 weeks, if liver function abnormalities persist.

Pathologic Features

Recognition and prompt reporting of HSV and VZV hepatitis is essential because effective pharmacologic therapy is available. Without treatment, the infection can be rapidly fatal. Localized and diffuse patterns of HSV hepatitis have been described.²³⁰ Both pathologic patterns cause circumscribed areas of coagulative-type necrosis (Fig. 52.24).^226,230 The center of the necrotic zones is typically occupied by ghosts of hepatocytes intermixed with neutrophils and nuclear debris. Viable hepatocytes rim the periphery and usually contain HSV or VZV hepatitis inclusions. Infected cells are usually slightly enlarged and contain ground-glass nuclei or characteristic Cowdry type A eosinophilic inclusions. Multinucleate smudgy cells are occasionally seen. However, diagnostic inclusions of HSV or VZV hepatitis may be absent on H&E-stained slides.

Immunoperoxidase stains for HSV antigens are confirmatory. In our experience, antibody preparations used to detect HSV 1 and 2 can show cross-reactivity with each other and with VZV hepatitis, making it difficult to distinguish these viruses. In our experience, antibodies used to detect the VZV hepatitis precursor and mature glycoprotein are more discriminating. If a histopathologic diagnosis of HSV or VZV hepatitis is considered on the basis of H&E staining, the physicians should be immediately notified.²³² This will prompt effective antiviral therapy that can be discontinued if the diagnosis is not confirmed on immunostaining.

Clinical Features

Clinical manifestations include hepatitis; gastroenteritis; B, T, and natural killer (NK) cell posttransplantation lymphoproliferative diseases; Hodgkin disease; smooth muscle stromal tumors; and a variety of cancers (e.g., nasopharyngeal, gastric).^240–244 EBV-related syndromes often resemble infectious mononucleosis, manifesting with fever, lymphadenitis, pharyngitis, and jaundice. Atypical signs and symptoms include jaw pain, arthralgia, joint space effusions, diarrhea, encephalitis, pneumonitis, mediastinal lymphadenopathy, and ascites. Liver allograft hepatitis or posttransplantation lymphoproliferative disease (PTLD) involvement usually produces mildly elevated ALT and AST values, and atypical lymphocytes are see in the circulation. Pancytopenia is occasionally found.^245–247

Risk factors for the development of serious EBV-associated disease include primary infection, high-dose immunosuppression, underlying Langerhans cell histiocytosis, and coexistent CMV disease. The risk of developing PTLD late after transplantation is not influenced by the type of immunosuppressive agents but is increased by the duration and intensity of immunosuppression.^240,241,248

Peripheral blood monitoring for EBV nucleic acid is used to monitor viral replication.²⁴⁰ When levels are elevated, the patient is examined for lymphadenopathy and other signs of EBV-related disease, and a preemptive reduction in immunosuppression is usually made before more serious EBV-related disease develops.²⁴⁹ The treatment for significant EBV-related disease depends on the clinicopathologic presentation and clonality of the proliferating lymphocytes. Polyclonal infectious mononucleosis-like syndromes usually respond to a reduction of immunosuppression, whereas monomorphic PTLD resembling clonal lymphomas often requires more aggressive immunotherapy or chemotherapy, or both.^{240–242,244}

Pathologic Features

EBV causes morphologic changes ranging from acute hepatitis characterized by a mild nonspecific portal and sinusoidal lymphocytosis to PTLDs (Fig. 52.26) that resemble diffuse large B-cell lymphomas.^{245–247,250} Even patients with enhanced EBV replication and viremia have increased intragraft T and B cells,²⁵¹ with the latter occasionally containing EBV-encoded small RNA (EBER) sequences. Even in patients without overt EBV disease, EBER-positive cells may be admixed with other inflammatory cells associated with other causes of allograft dysfunction.²⁵⁰ The contention that EBV causes chronic hepatitis or recurrent acute relapses is controversial.²⁵²

EBV hepatitis manifests as mild portal lymphoplasmacytic inflammation combined with sinusoidal lymphocytosis composed of small or mildly atypical lymphocytes. Lymphocytes lining up in the sinusoids should suggest an EBV-related disorder, as in native livers. Other lobular changes include focal hepatocellular swelling, mild acidophilic necrosis of hepatocytes, mild lobular disarray, and regenerative activity.

In the liver, PTLDs show cytologically atypical lym­phoplasmacytic portal infiltrates. In early or polymorphic lesions, atypical cells are usually mixed with small and blastic lymphocytes, plasmacytoid lymphocytes, and plasma cells. In monomorphic lesions, atypical cells predominate and manifest as maplike enlargement of portal tracts due to sheets of atypical immunoblastic cells that obscure the normal portal anatomic landmarks. Atypical cells can be seen in the sinusoids. Some cases have significant hepatic necrosis. Hodgkin lymphoma–like PTLDs with classic Reed-Sternberg cells can occur, and they may be associated with bile duct loss. Subendothelial localization of lymphocytes in portal and central veins can mimic acute rejection.

Outside of the liver, PTLDs usually occur in the lymph nodes and intestines and are categorized according to the World Health Organization (WHO) classification scheme²⁵³ as early lesions (i.e., reactive plasmacytic hyperplasia and infectious mononucleosis-like), polymorphic (i.e., hyperplasia and lymphoma), monomorphic (i.e., T- and B-cell subtypes), and Hodgkin and Hodgkin-like lesions.

Diagnosis of any EBV-related disorder is confirmed by in situ hybridization for EBV RNA (i.e., EBER sequence). Routine workup should include immunohistochemical stains or in situ hybridization for κ and λ light chains and CD20 to determine possible responsiveness to anti-CD20 antibodies.²⁴² If fresh tissue is available, a portion may be submitted for flow cytometry and molecular analyses, which enable a more detailed phenotypic characterization and study of immunoglobulin gene rearrangement, respectively.

Differential Diagnosis

EBV hepatitis and PTLD are difficult to distinguish from severe ACR. Features that favor acute rejection include pleomorphic, rejection-type portal or perivenular inflammatory infiltrates, conspicuous eosinophils, and severe bile duct damage that is proportional to the severity of inflammation. Features favoring EBV include a relatively monomorphic portal infiltrate, consisting primarily of activated and immunoblastic mononuclear cells. Many of the cells show plasmacytic differentiation, and some show atypical cytologic features. Patchy and mild inflammatory bile duct damage is less severe than would be expected based on the severity of the portal inflammatory infiltrate. Eosinophils are usually less conspicuous.

EBV hepatitis can be difficult to distinguish from nonspecific reactive hepatitis and acute HBV, HCV, or CMV hepatitis. HCV and EBV hepatitis can show sinusoidal lymphocytosis, but EBV-related disorders usually contain atypical cells. In contrast, lymphocytes associated with HCV hepatitis are usually small, round, and inactive appearing, and they form nodular aggregates in the portal tracts.^{245–247,250} Clinical suspicion and increased peripheral blood EBV levels also point to the correct diagnosis.

In most cases, the final diagnosis of any type of EBV-related disorder depends on in situ hybridization for EBV RNA (i.e., EBER probe). However, results should be interpreted with caution,²⁵⁴ because rare EBER-positive cells are found in lymphoid tissues from the general population. These cells are found with slightly increased frequency in allograft recipients, the significance of which is controversial.²⁵⁴ However, clustering of EBER-positive cells or the presence of EBER-positive cells in tissues that show other pathologic features of EBV-associated disease indicates enhanced EBV replication. These patients are at increased risk for EBV-related disease.

Pathologic Features

Adenovirus hepatitis is uncommon, and nuclear inclusions are difficult to recognize with certainty. Typical cases show poxlike granulomas, consisting of macrophages with or without neutrophils that are spread randomly throughout the liver parenchyma (Fig. 52.27). In other cases, the granulomas surround small, maplike areas of necrosis.^255–257 Viral inclusions are usually found in nuclei of viable hepatocytes near the edge of necrotic zones or granulomas. Infected cells have smudgy appearance with crowding of chromatin near the nuclear membrane, which makes the nucleus appear like a baked muffin. Immunohistochemical staining is usually required to confirm the diagnosis. Given that there are 51 serotypes, it is prudent to use antibodies reactive with the hexon protein common to all serotypes (e.g., MAB805, blend of clones 20/11 and 2/6, Chemicon International, Temecula, Calif.).²⁵⁸