Pathology of liver diseases 9781118895030, 1118895037

Table of contents :
fmatter
ch1_2
ch2
ch3_2
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ch6_2
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ch8
ch9
ch10_2
ch11
ch12
ch13
ch14
ch15_3
index

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Pathology of Liver Diseases

Pathology of Liver Diseases Gary C. Kanel, M.D. Clinical Professor of Pathology Keck Medical Center of USC Department of Pathology and Laboratory Medicine Los Angeles, CA, USA

This edition first published 2017 © 2017 John Wiley & Sons Ltd. All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, except as permitted by law. Advice on how to obtain permission to reuse material from this title is available at http://www.wiley.com/go/permissions. The right of Gary C. Kanel to be identified as the author of this work has been asserted in accordance with law. Registered Offices John Wiley & Sons, Inc., 111 River Street, Hoboken, NJ 07030, USA John Wiley & Sons Ltd., The Atrium, Southern Gate, Chichester, West Sussex, PO19 8SQ, UK Editorial Office 9600 Garsington Road, Oxford, OX4 2DQ, UK For details of our global editorial offices, customer services, and more information about Wiley products visit us at www.wiley.com. Wiley also publishes its books in a variety of electronic formats and by print-on-demand. Some content that appears in standard print versions of this book may not be available in other formats. Limit of Liability/Disclaimer of Warranty The contents of this work are intended to further general scientific research, understanding, and discussion only and are not intended and should not be relied upon as recommending or promoting scientific method, diagnosis, or treatment by physicians for any particular patient. The publisher and the authors make no representations or warranties with respect to the accuracy and completeness of the contents of this work and specifically disclaim all warranties, including without limitation any implied warranties of fitness for a particular purpose. In view of ongoing research, equipment modifications, changes in governmental regulations, and the constant flow of information relating to the use of medicines, equipment, and devices, the reader is urged to review and evaluate the information provided in the package insert or instructions for each medicine, equipment, or device for, among other things, any changes in the instructions or indication of usage and for added warnings and precautions. Readers should consult with a specialist where appropriate. The fact that an organization or website is referred to in this work as a citation and/or potential source of further information does not mean that the author or the publisher endorses the information the organization or website may provide or recommendations it may make. Further, readers should be aware that websites listed in this work may have changed or disappeared between when this works was written and when it is read. No warranty may be created or extended by any promotional statements for this work. Neither the publisher nor the author shall be liable for any damages arising herefrom. Library of Congress Cataloging-in-Publication Data Names: Kanel, Gary C., author. Title: Pathology of liver diseases / Gary C. Kanel. Description: Hoboken, NJ : John Wiley & Sons, Inc., 2017. | Includes bibliographical references and index. Identifiers: LCCN 2017001716 (print) | LCCN 2017002257 (ebook) | ISBN 9781118895030 (cloth) | ISBN 9781118895023 (pdf ) | ISBN 9781118895009 (epub) Subjects: | MESH: Liver Diseases—pathology Classification: LCC RC845 (print) | LCC RC845 (ebook) | NLM WI 700 | DDC 616.3/62—dc23 LC record available at https://lccn.loc.gov/2017001716 Cover images: Courtesy of the author. Set in 10/12pt Warnock Pro by Aptara Inc., New Delhi, India 10 9 8 7 6 5 4 3 2 1

v

Contents Preface vi About the companion website viii Chapter 1

Normal Liver

Chapter 2

Viral Hepatitis

Chapter 3

Fatty Liver Diseases

Chapter 4

Diseases of the Biliary Tract

Chapter 5

Non-Viral Infectious Diseases

Chapter 6

Granulomatous Hepatitis

Chapter 7

Autoimmune Hepatitis

Chapter 8

Vascular Disorders

Chapter 9

Genetic and Metabolic Hepatic Diseases

Chapter 10

Developmental Hepatobiliary Disorders and Cystic Diseases

Chapter 11

Drug- and Toxin-Induced Liver Diseases 215

Chapter 12

Liver Transplantation

Chapter 13

Hepatic Tumors, Benign

Chapter 14

Hepatic Tumors, Malignant

Chapter 15

Miscellaneous Hepatic Disorders

Index

354

1 24 50 72 95

123

131

143

231 266 289 337

162 198

vi

Preface With all of the advancements in hepatology, including invasive and non-invasive imaging, laboratory tests targeting specific diseases, molecular biology, and genomics, one might think that liver biopsy material in patients with suspected acute or chronic liver diseases or hepatic tumors may not be as necessary in arriving at a specific diagnosis as it was in the past; however, what still persists is an inconsistency oftentimes in the way patients clinically present with laboratory tests and what in fact the biopsy shows. This therefore leads to the continued importance of liver biopsy interpretation. The settings where liver biopsies are especially crucial tools in patient care are numerous, are discussed in more detail in Chapter 1, and include assessing patients who present with clinical signs of acute or chronic liver disease but with normal liver tests, patients with abnormal liver tests inconsistent with the suspected diagnosis (e.g., clinical diagnosis of acute viral hepatitis with an unexpectedly high alkaline phosphatase value), and patients in acute liver failure and those with transaminitis of no known cause. Biopsies are often warranted in the evaluation of various space-occupying lesions when imaging, cultures or fine needle aspirates are not conclusive. Biopsies are often critical in specifically evaluating liver transplant patients where the clinical presentation and liver test abnormalities can hint at any of a whole range of possibilities including acute or chronic rejection, biliary strictures, infections, or recurrent disease, all of which necessitate different treatment approaches. Lastly biopsies

also remain the gold standard in the staging and grading of chronic liver diseases to determine the appropriate individual therapies. For all of these reasons, useful reference material, as presented in this book Pathology of Liver Diseases, is most helpful to the pathologist and the patients’ attending physicians in arriving at the diagnosis or best possibilities that fit each individual patient. Pathology of Liver Diseases uses both book and online material in the presentation of the whole range of liver diseases seen in both the community hospitals as well as academic medical centers. The book in fifteen chapters emphasizes not only the pathology seen in biopsy and surgically resected or transplanted and autopsy material, but also the most pertinent clinical and laboratory findings including epidemiology and the various etiologic and pathophysiologic concepts, imaging when appropriate, and the differential diagnostic possibilities with references. Furthermore, what significantly adds to the overall usefulness of the book is the online material. An online Library Images that contains over 850 images of the various liver diseases is offered that significantly adds to the over 540 images already in the book. Additional online Tables to those already in the book are added so that the reader can have a more detailed reference to the grading and staging systems of fatty liver diseases, viral and autoimmune hepatitis, and liver transplant rejection. In addition 140 Case Examples from patients seen at the Keck Medical Center of USC as well as from consult

Preface vii

case material are presented in PowerPoint format that demonstrate the various ways many of these disease entities present clinically and the pathology seen, including both classic and common examples such as alcoholic hepatitis as well as cases infrequently seen such as progressive familiar intrahepatic cholestasis. Finally, courtesy of Dr. Rick Selby, Professor of Surgery and Chief of Hepatobiliary, Pancreatic Surgery and Abdominal Organ Transplantation at the Keck Medical Center of USC, the PowerPoint presentation Liver Transplantation – Surgical Procedure is also included with photographs from

the operating table of the step-by-step process in liver transplantation. The amount of information targeting the numerous fields of medicine has exponentially increased over recent years. The addition of this book and online material will not only be most valuable in enabling the viewer to have access to the wealth of material in diagnostics but also in gaining a better overall understanding of these hepatic and hepatobiliary disorders. Gary C. Kanel, Los Angeles, 2017

viii

About the companion website This book is accompanied by a companion website: www.wiley.com/go/kanel/liverpathology

The website includes the following to supplement each chapter: ● ●

●

A complete Reference List 140 Case Examples, which include over 420 images that demonstrate the various ways many of these disease entities clinically present. A Library that contains over 860 images of the various liver diseases, which adds to over 540 images that are in the book itself.

There are also: ●

●

Additional Tables that address in detail the grading and staging of various liver diseases such as viral hepatitis and fatty liver diseases. A PowerPoint presentation entitled “Liver Transplantation – Surgical Procedure”, which includes photographs from the operating table of the step-by-step process in liver transplantation.

To access the website, you will need to enter the password, which is the first word of the first paragraph in Chapter 15.

   1

1 Normal Liver The liver is a unique organ that has numerous structural and physiological functions. It is most important when discussing liver pathology that one understands first the normal liver histology before one can best understand the basic pathophysiologic concepts of the numerous liver diseases. The pathologist plays a fundamental role in assessing the various morphologic features seen in liver tissue, whether by fine needle aspirates, needle or wedge biopsies, partial hepatectomies, liver explants, or autopsy material. The pathologist also has not only routine but also numerous special histochemical and immunohistologic stains as well. Yet correlating the histologic findings with the most pertinent clinical and laboratory data enables the pathologist to better arrive at a diagnosis and the most pertinent differential possibilities. This introductory chapter addresses all aspects of the normal liver, reviewing the embryologic development, gross and microscopic features, the pertinent intracytoplasmic components and how their function varies with their location within the hepatic lobule, and the importance of stem cell function within the liver. Additionally the various useful stains and laboratory values will also be presented, as well as a brief outline of how best to organize pathologic readings and signouts of liver biopsy specimens.

Embryology The hepatic primordium anlage initially appears at the end of the third week of gestation and is

first seen as a hollow midline outgrowth stalk (hepatic diverticulum) of the endodermal epithelium at the distal aspect of the foregut. By the fourth week, the diverticulum enlarges from proliferation of the endodermal cell strands (hepatoblasts) and projects cranially into the mesoderm of the septum transversum, eventually giving rise to the liver hepatic parenchyma and intrahepatic ducts. The cephalic end ultimately develops into the right and left hepatic lobes, while the stalk between the diverticulum and foregut narrows and forms the extrahepatic biliary system and gallbladder. Solid cords are initially formed by proliferating endodermal cells. These eventually anastomose to form vesicles and cribriform tubules with centrally located lumenal structures (biliary canaliculi). The cords eventually merge and develop small channels and capillaries that subdivide the cords to eventually form the hepatic sinusoids. The individual hepatoblasts are progenitor cells that develop into mature hepatocytes, with those immediately adjacent to the portal mesenchyme becoming the ductal plates. The rapid growth rate of the hepatic cords enables the development of sheets of cells (muralium multiplex) that persist until birth, after which the cell sheets narrow to two cells (muralium duplex) and eventually evolve within the first year of life into a one cell thick trabecular cord (muralium simplex). The perisinusoidal cells and Kupffer cells appear by three months gestation. The mesoderm from the septum transversum initially surrounds the liver and is directly in

Pathology of Liver Diseases, First Edition. Gary C. Kanel. © 2017 John Wiley & Sons, Ltd. Published 2017 by John Wiley & Sons, Ltd. Companion website: www.wiley.com/go/kanel/liverpathology

2   1 Normal Liver

contact with the lesser curvature of the stomach, duodenum, and ventral body wall. The mesoderm eventually forms the lesser omentum, the falciform, coronary, and triangular ligaments, with a portion developing into the liver (Glisson) capsule. The mesoderm on the liver surface is also in continuity with the peritoneum, and the portion that makes contact with the future diaphragm remains uncovered (bare area). The developing hepatic artery and vagus nerve branches follow the mesoderm along and adjacent to the portal vein. The mesoderm is the main focus in the development of hematopoiesis, which begins at about 6 weeks and becomes most active during the fifth month of gestation. This process regresses with increase in bone marrow activity. The erythroid precursors are most prominent during fetal development within the hepatic sinusoids while the myeloid and megakaryocytic precursors reside mostly within the portal structures (Figure 1.1). This hematopoiesis is responsible for the enlarged size of the liver (up to 10% body weight by the tenth week of gestation, with the right and left lobes taking up an equal volume), but this size significantly regresses at birth (5% of body weight) at which time only rare small clusters of normoblasts can be seen. By 4 weeks of age hematopoietic activity has usually ceased.

Figure 1.1  Embryonic development. A developing bile ductule is seen at the border of the portal tract and parenchyma. The portal tract and sinusoids contain hematopoietic precursors (extramedullary hematopoiesis).

Additionally with time the left lobe diminishes in size, and the caudate and quadrate lobes develop as subdivisions of the right lobe. The vascular network, originally derived from the development of the vitelline and umbilical veins, occurs at the same time as proliferation of the hepatoblasts, with the sinusoids forming from anastomosis of the hepatic cords and vessels. By the fifth week of gestation most of the major vessels are present and include the right and left umbilical veins, the transverse portal sinus, and the ductus venosus, which shunts blood from the umbilical vein into the inferior vena cava. The portal vein initially develops from the vitelline vein and then subdivides into the right and left branches. The hepatic and portal vein branches divide the parenchyma into the individual lobules and acini. At birth, a sphincter mechanism closes the ductus venosus, resulting in cessation of blood flow through the umbilical vein, with the liver now receiving blood from the left branch of the portal vein. The biliary apparatus develops from membranous infoldings between the junctional complexes located between individual hepatoblasts and initially appears as intercellular spaces with no distinct wall. The biliary canaliculi are first seen at 6 weeks of gestation, with synthesis of bile occurring by the ninth week and secretion of bile by the twelfth week. The ductal plate, which is initially two layers thick, is formed from the periportal hepatoblasts. A lumen develops by the third month (see Figure 1.1) with eventual formation of double-layered tubular (ductular) structures. The true interlobular bile ducts occur immediately after birth from remodeling of these ductular elements. This biliary network receives its blood supply from a complex of arterioles and capillaries formed from the peribiliary plexus. The extrahepatic biliary tree develops from the stalk of the original hepatic outgrowth. Individual cell functions become apparent at different stages of the embryologic development. α-Fetoprotein, found in high amounts at birth, initially is seen within the hepatocytes by one month gestation and continues throughout fetal development, with high serum levels at

Gross Anatomy   3

birth. Fatty change (steatosis), glycogen and glycogen synthesis become most apparent by two to three months gestation, with the glycogen eventually diminishing due to rapid glycogenolysis. Hemosiderin is usually seen early on but gradually decreases, with some often occurring in the periportal hepatocytes at birth.

Gross Anatomy The adult liver weighs from 1200 to 1800  g, dependent on the overall body size, takes up the majority of the right upper abdominal cavity beneath the rib cage, and extends from the right lateral aspect of the abdomen 15–20 cm transversely toward the xiphoid process. Although the weight of the adult liver constitutes about 1.8–3.1% of the total body weight, at birth the Right posterolateral

Right anterolateral

liver is larger compared with adjacent thoracic and abdominal viscera and constitutes about 5–6% of the body weight. Anatomically, the liver has four lobes: right, left, caudate, and quadrate. The right lobe accounts for one-half to two-thirds of the total liver volume and is divided from the left lobe by the falciform ligament on gross inspection; however, functionally the right and left lobes are of about equal size and are divided by a line extending from the inferior vena cava superiorly to the middle of the gallbladder fossa inferiorly. A total of eight functional segments are present, each having its own vascular supply and biliary drainage: the right posterolateral (VI and VII), right anterolateral (V and VIII), left anterior (IV), left posterior (II and III), and the caudate lobe (I), the latter being a watershed area of both the right and left lobes blood supply (Figure 1.2). Left anterior

Left posterior

Middle hepatic vein Right hepatic vein Left hepatic vein

II

VII

VIII

I IV III Umbilical vein (remnant)

VI V

Inferior vena cava Hepatic artery Portal vein Hepatic duct

Figure 1.2  Schematic anatomical vascular arrangements of the liver. The liver is divided into eight functional anatomical segments, each having its own vascular supply and biliary drainage: the right posterolateral (VI and VII), right anterolateral (V and VIII), left anterior (IV), left posterior (II and III), and the caudate lobe (I). Source: Wanless IR. Physioanatomic considerations. In: Schiff’s Diseases of the Liver, 11th edn. Oxford: Wiley Blackwell, 2012. Reproduced with permission of John Wiley & Sons.

4   1 Normal Liver

The portal vein, which is the main route of vascular drainage of the gastrointestinal tract, is formed by merger of the superior mesenteric and splenic veins, with additional blood supply from the coronary and cystic veins. The portal vein divides at the porta hepatis into the right and left main branches. The right branch divides early into anterior and posterior segments, while the left branch divides into the pars transversus, which extends to the left in the porta hepatis, and the pars umbilicus, which descends into the umbilical fossa. The caudate lobe veins arise from both the right and left main portal vein branches. The hepatic vein is composed of three major tributaries: right, middle, and left. The middle and left hepatic veins often converge to form a single outflow vessel before draining into the inferior vena cava, while the right hepatic vein opens through a separate ostium. The caudate lobe drains directly into the inferior vena cava. The hepatic artery is a branch of the celiac artery and ascends along the hepatoduodenal ligament, eventually dividing into the right and left main branches. The right hepatic artery, usually located behind the common hepatic duct after giving rise to the cystic artery, eventually divides into the anterior and posterior branches. The left hepatic artery passes obliquely upward and to the left in the porta hepatis, eventually dividing into the medial and lateral branches. The quadrate lobe is fed by a branch of the middle hepatic artery, while the caudate lobe is fed by both right and left hepatic artery branches. The biliary system originally arises from the bile canaliculi within the hepatic lobule and is first seen on gross inspection in the larger interlobular branches. The biliary drainage of the right lobe is derived from anterior and posterior segmental branches that merge to form the right hepatic duct, while the lateral and medial segmental branches merge to form the left hepatic duct that drains the left lobe. The caudate lobe is drained from three duct branches directly into the right and left hepatic ducts. The smaller interlobular bile ducts do not have

a wall, but the larger septal branches have a thin wall of collagen fibers. The intra- and extrahepatic bile ducts are directly fed by the hepatic artery and its anastomosing branches, which parallel the ducts as they progress through the various hepatic divisions. The lymphatic channels are divided into deep and superficial branches. The deep branches parallel the portal and hepatic vein branches, while the superficial branches arise from Glisson capsule and drain through the adjacent falciform ligament, diaphragm, esophagus, and hilar lymph nodes. The nerve supply parallels the main hepatic artery and portal vein and is divided into parasympathetic and sympathetic fibers. The nerve supply enters the hepatic hilum through both anterior and posterior routes, feeds the arteries and bile ducts through sympathetic innervation, and branches through the main portal tracts, with smaller unmyelinated branches feeding the periportal hepatocytes. Many of the nerve fibers terminate on endothelial cells lining the smallest arterioles and along Kupffer cells, stellate (fat-storing) cells, and hepatocytes.

Histology Basic Architecture Arrangement

The basic microanatomical structure of the liver can best be appreciated on a three-dimensional drawing of the portal tracts, parenchyma, and vascular blood flow (Figure 1.3). The portal tract – terminal hepatic (central) venule – portal tract arrangement of the hepatic lobule is evenly distributed throughout all of the hepatic lobules. Although the architecture may at times be difficult to assess when acute hepatic injury occurs, on recovery the arrangements return to normal; however, architectural distortion seen in advanced chronic liver disease and cirrhosis is for the most part irreversible, although regression of fibrosis has been documented in patients with chronic viral hepatitis with severe fibrosis who respond to anti-viral therapy.

Histology   5 Arterial capillary emptying into paraportal sinusoid

Arterial capillary emptying into paraportal sinusoid Perisinusoidal space of Disse

Portal Limiting vein plate

Periportal connective tissue

Central (hepatic) Lymph vessel veins

Sinusoids

Central (hepatic) veins

Perisinusoidal space of Disse Sublobular vein

Central (hepatic) veins

Central (hepatic) veins Intralobular cholangiole

Arterial capillary emptying into intralobular sinusoid

Inlet venules

Bile canaliculi on the surface of liver plates (not frequent) Portal Bile Hepatic vein duct artery Portal tract

Limiting plate

Cholangioles in portal canals

Figure 1.3  Structure of the normal liver. Source: Sherlock S and Dooley J. Diseases of the Liver and Biliary System, 11th edn, Blackwell Science, 2002. Reproduced with permission of John Wiley & Sons.

Portal Tracts

The portal tracts are composed of five individual components (Figure 1.4). The interlobular bile ducts number from one to two per portal tract, although in infants the ducts appear early on to be slightly less frequent. The ducts are usually seen immediately adjacent to the hepatic arterioles, which are responsible for their blood supply. The hepatic arterioles are usually present singly. The portal venules are a single vascular structure. It is important to note that transverse cuts of the portal tracts in biopsies can errantly appear as if two or more ducts and vessels are present; however, a true increase in portal venules and lymphatics are characteristic of portal hypertension and are usually seen in conjunction with severe portal fibrosis or cirrhosis. The fibroconnective tissue is composed of mature collagen and supports the major portal

tract components. It often varies in degree, depending on the distance of the portal tracts from the hepatic hilum, and can mistakenly be

Figure 1.4  Portal tract. This normal portal structure exhibits a single interlobular bile duct, portal venule, and small hepatic arteriole.

6   1 Normal Liver

described as fibrotic when the biopsy specimen is taken toward the hepatic hilum. The cellular inflammatory components within the portal tracts consist usually of scattered lymphocytes. Although in the normal liver these portal inflammatory cells are usually absent, it is not infrequent that a few scattered lymphocytes may appear within these portal structures in an otherwise normal liver. Parenchyma

The hepatic lobules comprise about 80% of the total hepatic volume and are mainly composed of liver cell cords one cell thick made up of polyhedral hepatocytes. The adjacent sinusoids are lined by both endothelial and Kupffer cells, with the perisinusoidal space located between the endothelial cells and hepatocytes. Stellate cells and collagen fibers also occur along the perisinusoidal space, but on routine H&E staining of normal liver tissue these structures are usually inconspicuous. The sinusoids drain from the portal venule and hepatic arterioles into the terminal hepatic (central) venules (Figure 1.5). Hepatocyte

The hepatocyte comprises about two-thirds of the total number of cells within the liver

Figure 1.5  Parenchyma. The hepatic cords are one cell thick lined by flattened Kupffer and endothelial cells. The sinusoids are open with the circulating blood draining into a terminal hepatic (central) venule.

and about four-fifths of the total liver volume. Hepatocytes measure 25–40  μm in diameter, the size dependent on the age of the patient and the zonal location. The cells are polyhedral, are arranged in cords one cell thick, and have three dimensional boundaries (1) the sinusoidal (basolateral) surface area lined by both microvilli that extend into the perisinusoidal spaces and plasma membranes that exhibit various membrane pits and infoldings for both secretory and absorptive functions, (2) the lateral (intercellular) membranes lying between adjacent hepatocytes that form gap junctions, intermediate and tight junctions, all related in various degrees to transport of metabolites, cell membrane resilience, and membrane permeability to macromolecules, and (3) the canalicular membranes lined by microvilli and containing various contractile microfilaments allowing transport of bile secretions. Nucleus

The liver cell nucleus is round to oval, contains clumped chromatin and small nucleoli, and measures approximately 10 μm in diameter. The nuclear membrane is composed of two envelopes separated by a narrow zone, these membranes having numerous apertures or pores that provide communication between the cytoplasm and the nucleus. The outer membrane is often lined by ribosomes, which are in direct communication with adjacent rough endoplasmic reticulum (ER) fibers. The intranuclear chromatin fibers are composed of heterochromatin (round to irregular dense metabolically inactive granules) and euchromatin which actively transcribes RNA. From one to six nucleoli are often present. The hepatocyte usually contains one nucleus, although bilobed forms may also be seen predominantly in the perivenular zone (zone 3 of Rappaport) and are more frequently present in the elderly patient population. Various nuclear inclusions may be present and include glycogen (most frequent in diabetic patients), lipid, and cytoplasmic invaginations (pseudo-inclusions, nuclear membrane irregularities).

Histology   7

Cytoplasm

●●

The cytoplasm constitutes about 90% of the volume of the hepatocyte and contains numerous functionally important organelles (Figure 1.6). The superstructure of the cell is maintained by the cytoskeleton of the hepatocyte and includes three major subdivisions: microfilaments, microtubules, and intermediate filaments that are responsible for the overall three-dimensional framework of the hepatocytes as well as organization of the various intracellular functions. Additionally the structure and function of the cells vary depending on their zonal location (Tables 1.1 and 1.2). The cytoplasm contains the following important components: ●●

Mitochondria are one of the most prominent intracellular organelles that average up to 2200 per hepatocyte, are oval to oblong and measure 0.4–3.5  μm in diameter. They are divided into outer and inner membranes separated by a gap, with folds (cristae) projecting into the body of the mitochondria, increasing the total mitochondrial membrane area. They have numerous critical functions that include oxidative phosphorylation and fatty acid oxidation and contain components essential for the urea and citric acid cycles.

●●

●●

ER is composed of a convoluted network of cisternae, saccules, tubules, and vesicles that are distributed throughout the liver cell cytoplasm. The ER is divided into two components: the rough ER (seen predominantly surrounding the nucleus and biliary channels) and smooth ER (forming a meshwork of small tubules that are devoid of ribosomes and often communicate with the Golgi apparatus). The ER is where various important functions such as protein synthesis and fatty acid metabolism occur. The cytochrome p450 oxidative system is also located in the ER and plays a large part in drug metabolism and toxin degradation. Golgi apparatus complexes are composed of highly polarized, parallel, flattened, dilated saccules or vesicles, are about 1 μm in diameter, and may number from 40 to 60 per liver cell. Vesicles arranged at the periphery of the sacs detach and transfer secretory material such as lipoproteins into the sinusoids or biliary canaliculi. Golgi function also includes bile secretion, incorporation of carbohydrates into proteins, and membrane synthesis and repair. Lysosomes appear as electron-dense, pleomorphic, single membrane-bound vesicles

rer

Figure 1.6  Liver cell cytoplasm. Using electron microscopy, the cytoplasm is composed of rough endoplasmic reticulum (rer), mitochondria (m), glycogen (gly), lipid droplets (L), peroxisomes (p), and secondary lysosomes (*). Source: Phillips. The Liver: An Atlas and Text of Ultrastructural Pathology. Raven Press, 1987. Reproduced with permission from Wolters Kluwer Health.

L

m

m

gly

m

p

L m

8   1 Normal Liver

Table 1.1  Liver cell structural zonal variations Zone 3 (perivenular)

Zone 1 (periportal)

Mitochondria round, less numerous and smaller, fewer inner membranes Peroxisomes prominent Lysosomes numerous Bile canaliculi with fewer microvilli, smaller in diameter Surface area of smooth endoplasmic reticulum larger Sinusoids form parallel vessels that open into terminal hepatic venules, are wider (30 μm), fewer in number Endothelial cells more numerous but smaller, increase in number of fenestrations and porosity Larger nuclear volumes Increase in number of microbodies Slight increase in stellate (Ito) cellsa Predominant collagen types I, III, VI

Mitochondria oval and oblong, larger diameter, larger volume and number, greater cristae area Rough endoplasmic reticulum more abundant Bile canaliculi with numerous microvilli, larger in diameter Kupffer cells more numerous Sinusoids form interconnecting polygonal network, are smaller (6 μm), more tortuous, more numerous in number Abundant Golgi-rich volume Endothelial cells larger, endothelial fenestrations larger but less numerous Numerous large granular lymphocytes (pit cells) Predominant collagen types IV, V

a

Zone distribution varies considerably depending on the nutritional state. Source: Adapted from Kanel GC. Anatomy, microscopic structure, and cell types of the liver. In: Yamada T (ed.) Textbook of Gastroenterology, 4th edn. London: Wolters Kluwer–Lippincott Williams & Wilkins, 2003.

Table 1.2  Liver cell functional zonal variations

a

Zone 3 (perivenular)

Zone 1 (periportal)

Glycolysis Glycogen synthesis from glucose Lipogenesis Removal of ammonia from blood by glutamine Detoxification, biotransformation of the majority of drugs and toxins (p450 enzymes)a Ketogenesis Bile acid synthesis Bile salt-independent fraction of bile formation, bile acid uptake (sodium independent) Glucuronidation Mixed function oxidase Increase in Kupffer cell phagocytic activity

Gluconeogenesis Glycogen synthesis from lactate β-Oxidation of fatty acids Amino acid catabolism Urea synthesis Cholesterol synthesis Bile acid secretion Bile salt-dependent fraction of bile formation; bile acid uptake (sodium dependent)

Certain drugs and toxins (e.g., allyl formate, phosphorus) are metabolized and may cause liver cell injury in zone 1 due to different pathophysiological mechanisms. Source: Adapted from Kanel GC. Anatomy, microscopic structure, and cell types of the liver. In: Yamada T (ed.) Textbook of Gastroenterology, 4th edn. London: Wolters Kluwer–Lippincott Williams & Wilkins, 2003.

Histology   9

●●

containing and storing enzymes such as acid phosphatase, esterases, proteases, and lipases. They are most frequently identified adjacent to the canalicular membrane and are divided into primary and secondary lysosomes. The primary lysosomes digest intracellular degradation products (“auto-phagocytosis”), forming secondary lysosomes that secrete these vacuoles into the biliary system. Various pigments such as lipochrome, hemosiderin, and copper also may accumulate within lysosomes with time and form residual bodies. Peroxisomes (microbodies) are single membrane intracellular organelles that vary from 0.2 to 1.3 μm, are round to oval, exhibit a finely granular homogeneous matrix, and contain various oxidases and catalases. Their primarily function involves the oxidation and degradation of numerous substrates with formation of hydrogen peroxides. They are also involved in oxidation of long-chain fatty acids.

There are various other intracellular components seen by light microscopy that contribute to the function and appearance of the hepatocyte. ●●

●●

●●

●●

Lipochrome is a finely to coarsely granular brown pigment that is derived from an increase in lysosomal activity and intracellular condensation of various cellular remnants. Bile is a clumped green to green-yellow globular pigment that is positive on the Hall stain for bilirubin. Usually the presence of intracellular bile is also accompanied by intracanalicular bile. Hemosiderin is a coarsely granular and golden brown pigment that is highlighted by the Perl iron stain and represents red blood cell degradation remnants. Intracellular lipids appear as clear distinct rounded vacuoles and are usually composed of neutral triglycerides. The size of the fat droplets varies, with the extremely small variants (microvesicular or foamy) often difficult to see on routine light microscopy and may require thin 1-micron sections on routine

●●

●●

H&E stain or fat stains (Oil Red O) on frozen section material. The small lipid droplets can also be demonstrated by immunoperoxidase staining using perilipin and adipophilin, two proteins that play a role in lipid metabolism and appear at the rim of the lipid droplets. Intracellular glycogen is distributed throughout the cytoplasm but is more easily seen by periodic acid–Schiff (PAS) stain on frozen section material. Nuclear glycogen, when abundant, gives the nucleus a clear appearance and is more often present in a number of liver diseases including non-alcoholic steatohepatitis and Wilson disease.

Sinusoidal Lining Cells Kupffer cells

Kupffer cells are sinusoidal lining cells that function as tissue macrophages. Although originally derived from the circulation, they eventually rest along the sinusoidal borders but maintain the ability to divide and migrate along the sinusoidal spaces, especially into regions of liver cell damage where it is not uncommon to find hyperplastic and hypertrophic Kupffer cell clusters and aggregates. Kupffer cells have oval to elongated nuclei, abundant pyramidal stellate cytoplasm, and measure up to 9 μm in length. They overlie but do not form junctional complexes with the smaller endothelial cells but may be seen in gaps between adjacent endothelial cells, with cytoplasmic processes extending through endothelial fenestrations. Kupffer cells contain various lysosomes, with their primary functions related to (1) phagocytosis and eventual clearance of particulate material, (2)  clearance of bacteria, endotoxins, and degenerating cellular components, (3) synthesis and catabolism of lipids, (4) clearance of senescent erythrocytes, (5) sequestration of antigens, and (6) clearance of immune complexes. Endothelial cells

Endothelial cells are flattened elongated sinusoidal cells that range from 50 to 80  nm and

10   1 Normal Liver

contain numerous cytoplasmic projections and clustered fenestrae or gaps varying from 0.1 to 0.2 μm. These fenestrae function as a filtration barrier. These cells have slightly different functions from more typical endothelial cells seen in other organ systems, as they do not bind lectin or factor VIII-related antigen, and they normally express little CD31 or CD34, although activation and expression of these latter markers is common in endothelial cells lining trabecular cords in chronic liver diseases and in many benign and most malignant hepatocellular neoplasms. Although endothelial cells synthesize various substances such as prostaglandins and cytokines, their main function involves filtering of various macromolecules from the sinusoidal blood by receptor-mediated endocytosis, enabling substances such as glycoproteins and polysaccharides direct contact with the hepatocyte but excluding and protecting the liver cell from numerous larger cellular components. Stellate (perisinusoidal, fat-storing, Ito) cells

Stellate cells are located within the perisinusoidal liver cell recesses along the space of Disse, range from 2 to 10 μm, and contain small star-shaped nuclei without prominent nucleoli. The cytoplasm often contains variably sized lipid droplets having a high concentration of vitamin A (retinoyl palmitate) that can easily be demonstrated on frozen sections by intensely green rapidly fading fluorescence when excited at a wavelength of 328  nm. Besides being the major source of vitamin A storage, the cells synthesize extracellular matrix by way of cytokine activation and resultant transformation to myofibroblasts in response to liver injury, with enhancement of protein and collagen synthesis. These cells also produce hepatocyte growth factor and play a role in the expression of the vascular contour of sinusoids. Pit cells (liver-associated lymphocytes)

The pit cells are non-parenchymal T cells distributed within the sinusoidal lumen in loose contact with the endothelial and Kupffer cells, although they can occasionally be seen within portal tracts. These cells function as natural

lymphocyte-activated killer (NK) cells and contain multivesicular body-related dense granules and rod-cored vesicles. These cells are often seen in direct contact with the endothelium in response to various immunologic mechanisms, produce cytokines, and can be targeted in viral hepatitis, acute post-transplant cellular rejection, and various primary and metastatic neoplastic processes where they are felt to play a role in the host immune reaction. Stroma (Extracellular Matrix)

The stroma overall supports the basic hepatic architectural arrangement, produces intercellular cohesion and communication, and effects cellular differentiation. The capsule of Glisson, composed of dense hypocellular collagen, surrounds the liver and extends at the hilum into the hepatic parenchyma, forming the tensile structure of the portal tracts. Extension within the sinusoids into the space of Disse as reticulin fibers maintains the intralobular framework. Five basic types of collagen are seen, with types I and III representing more than 95% of the total collagen. Type I represents mature collagen fibers and is present predominantly within the portal tracts but also around the terminal hepatic venules, sublobular veins and hepatic veins, while type III represents new collagen fibers which, along with the type IV collagen, comprise the sinusoidal reticulin framework. Type IV collagen is also present in the basal lamina (membrane) around small vascular structures and ducts, and represents about 1% of the total hepatic collagen. The non-collagenous proteins are numerous matrix glycoproteins and include (1) laminin, the major glycoprotein component within the basement membranes responsible in part for cell adhesion and formation of capillaries within the sinusoids, (2) fibronectin, synthesized by perisinusoidal cells responsible for collagen adhesion, and (3) elastin, which stabilizes blood vessel walls. Collagen deposition is often triggered by activation of stellate cells, the best example being alcoholic hepatitis where perivenular sinusoidal fibrosis is most prominent.

Histology   11

Biliary Network

The main function of the biliary tract is to transport bile synthesized in the hepatocyte into the gastrointestinal tract by way of the intra- and extrahepatic biliary network. The transport proteins synthesized by biliary epithelium aid in both the secretion of bicarbonate-rich fluid and the reabsorption of various fluids and solutes that generally enhance bile flow. The biliary tract can be divided into its various structural components: ●●

●●

Biliary canaliculi are located along the intercellular spaces between hepatocytes, range in diameter from 0.5 to 1 μm, and are lined by microvilli (Figures 1.7 and 1.8). The canaliculi have numerous anastomotic connections and may undergo contractions secondary to actin, myosin, and tropomyosin, enabling and enhancing forward bile flow. Terminal bile ductules, periportal cholangioles, and canals of Hering are formed from

●●

●●

Figure 1.7  Scanning electron micrograph of the canalicular biliary system. Source: Sherlock S and Dooley J. Diseases of the Liver and Biliary System, 11th edn. Blackwell Science, 2002. Reproduced with permission of John Wiley & Sons.

canaliculi that enter into the portal structures, are derived from hepatocytes located at the periportal limiting plate, and provide communication with the interlobular bile ducts. These ductules develop a basement membrane and have both liver cell and duct ultrastructural and histochemical features. Although in the normal liver these ductules are usually inconspicuous on routine light microscopy, their proliferation (duct transformation, duct metaplasia) is most apparent in instances of (1) liver cell damage, and (2) biliary tract obstruction and other cholestatic processes due to the accumulation of bile acids which trigger bile duct reduplication. Interlobular bile ducts are larger ducts that range from 15 to 20  μm, are located within the smaller portal structures, and are lined by a single layer of cuboidal cells with discrete round nuclei, usually inconspicuous nucleoli, and scanty eosinophilic cytoplasm. The luminal surface contains numerous pinocytotic vacuoles, with complex interdigitations present in adjoining duct epithelium. A basement membrane is apparent and easily demonstrated on PAS stain. Although the smaller ducts have no apparent wall, the larger interlobular ducts which measure up to 100 μm in diameter develop a small periductal fibrous sheath. The main blood supply is the smaller branches of the hepatic artery and the peribiliary plexus, which run in parallel with the duct structures. These ducts express class  I major histocompatibility antigens, with cytokine-mediated class II expression in instances of liver allograft rejection and certain chronic biliary tract diseases that attack ducts, such as primary biliary cirrhosis. Interlobar and septal ducts measure more than 100 μm in diameter, have a fibrous wall, and are lined by a single layer of cuboidal to columnar epithelium, with nuclei located towards the basement membrane. Some degree of periductal fibrous tissue is common but should not be confused with the distinct periductal concentric fibrosis seen in long-term bile duct obstruction and primary sclerosing cholangitis.

12   1 Normal Liver

lys

ves

m G

mv bc

ect m cm G

ves p

m

Figure 1.8  Biliary pole. Using electron microscopy, the bile canaliculi (bc) can be seen to the left of the field, surrounded by microvilli (mv) and pericanalicular ectoplasm (ect). Golgi complexes (G), vesicles (ves), mitochondria (m), peroxisomes (p), and lysosomes (lys) can also be seen in the adjacent liver cell cytoplasm. cm, liver cell membrane. Source: Phillips. The Liver: An Atlas and Text of Ultrastructural Pathology. Raven Press, 1987. Reproduced with permission from Wolters Kluwer Health.

●●

Segmental ducts are formed from the interlobar and septal ducts and measure up to 800  μm in diameter. These ducts eventually form the major hilar ducts that measure up to 1.5 mm in diameter. The hilar ducts ultimately branch into the main right and left hepatic ducts. The hilar ducts are lined by columnar mucus-secreting epithelium, have a distinct fibromuscular wall, and are associated with both intramural and extramural seromucinous peribiliary glands that communicate with the bile duct lumen.

Vascular and Lymphatic Networks

The major blood vessels that supply the liver are the portal vein and hepatic artery, the former supplying approximately two-thirds of the total blood flow. The portal vein develops into interlobar, segmental, interlobular veins and pre-terminal branches, with the terminal portal

venules measuring about 20–30 μm in diameter that are present in the smaller portal tracts. The hepatic artery branches accompany the portal vein and divide within the smaller portal tracts into two segments: the periportal plexus, which branches around the portal vein and drains into the sinusoids, and the peribiliary plexus, which provides blood supply to the accompanying interlobular bile ducts by way of small capillaries that are layered around the ducts. Various connections are seen between the small arterioles and the sinusoids that are most prominent in the periportal zone (zone 1 of Rappaport). There are a number of organizational approaches in assessing the structure as well as function of the liver lobule. On the basis of vascular injection studies, Rappaport et al. described the concept of the liver acinus, with a stratified order of simple acini, complex acini, and acinar agglomerates. The simple acinus (Figure 1.9) is the smallest functional parenchymal unit and

Histology   13

PS PS

ACIN

R LIVE

atory periph rcul er oci r y c i M

THV

Figure 1.9  Hepatic acinus. The simple liver acinus demonstrates the three hepatic zones and their relationship to the microcirculatory blood supply. PS, portal structures; THV, terminal hepatic venule. Source: Sherlock S and Dooley J. Diseases of the Liver and Biliary System, 11th edn. Blackwell Science, 2002. Reproduced with permission of John Wiley & Sons.

US

3

2

1

centers around the pre-terminal portal venule, hepatic arteriole, and terminal bile ductule. The acinus is divided into three zones (zones of Rappaport): periportal (zone 1) which includes the limiting plate, midzone (zone 2), and perivenular (zone 3) with the terminal hepatic venule at its outer lateral margin. Biliary drainage runs parallel to the vascular sinusoidal circulation. The complex acinus is derived from three adjacent simple acini and is fed by a pre-terminal portal venule and arterial branches. The acinar agglomerate is composed of about four complex acini and is fed by a portal venous branch measuring 300–1200 μm in diameter. The space of Disse lies between the hepatocyte and the endothelial cells, measures 0.2–1.0 μm in width, and forms a space that is not appreciated on routine light microscopy. Numerous microvilli can be seen projecting from the liver cell membranes into the space of Disse. The discontinuity of the adjacent endothelial cells allows plasma to have access to the liver cell

1

2

3′

1′

2′

PS

membranes. The space of Disse contains reticulin fibers, and stellate or perisinusoidal cells (Ito cells) also protrude into this space. The sinusoids eventually drain into the terminal hepatic venules, which have no fibrous wall. These vessels then drain into the terminal hepatic and sublobular intercalated veins, and then exit the liver from the three main hepatic vein branches into the inferior vena cava. Hepatic lymph is mostly derived from the space of Disse, whereas a minority comes from capillary leakage from the peribiliary plexus. Its main function is to drain excess proteinaceous fluid from the interstitial hepatic spaces. The hepatic lymph drainage within the space of Disse travels into the smallest lymphatic vessels within the portal tracts by way of endothelial massaging by circulating erythrocytes and leukocytes within the sinusoids. Small lymphatic branches can be seen along the hepatic venous outflow vessels. A lymphatic plexus is also present within Glisson capsule and communicates

14   1 Normal Liver

with the intrahepatic lymphatics through anastomotic channels. Most lymphatics leave the liver at the porta hepatis, although lymphatic drainage is prominent through the capsule of Glisson in instances when the hepatic venous drainage is impaired (e.g., acute and chronic hepatic venous outflow obstruction, cirrhosis). Neural Network

The nerve fibers are composed of both parasympathetic and sympathetic branches and release neurotransmitters from intrasinusoidal fibers that contribute to modulation of liver cell function including regulation of carbohydrate metabolism and sinusoidal blood flow. Small nerve segments can be seen within the larger portal tracts, but smaller unmyelinated fibers can be discerned by way of electron microscopy and immunohistochemical studies within the space of Disse.

Structural and Functional Components The hepatocytes within the various parenchymal zones have many different specialized structural and physiological functions (see Tables 1.1 and 1.2). This functional heterogeneity applies not only to liver cells but also to the sinusoidal and perisinusoidal spaces, Kupffer and endothelial cells, the extracellular matrix, and bile duct epithelial cells. These functions are manifestations of (1) nutrient and hormonal gradients delivered to the various zones, (2) sinusoidal vascular perfusion and oxygen concentration gradients, (3) availability of innumerable substrates and co-factors, and (4) expression of various enzyme activities through gene expression and local (zonal) genetic variations.

from blastocysts and first give rise to somatic stem cells followed by multipotent tissue-specific stem cells. Oval cells are a heterogeneous population of cells that under certain circumstances are activated and proliferate. These cells express various histologic markers of both hepatocytes (α-fetoprotein, albumin) and bile duct epithelium (cytokeratins 7 and 19) and are also known as facultative stem cells. Oval cells also express various isozymes of aldolase, pyruvate kinase, lactic dehydrogenase, and glucose-6-phosphatase, the latter a typical hepatocyte marker. Oval cells also express various markers of hematopoietic stem cells (e.g., Thy-1, CD45, Sca-1), although it is not felt that oval cells have a bone marrow origin. These cells are seen along the canals of Hering, behave like progenitor and stem cells, and have the ability to replicate and differentiate into hepatocytes under certain conditions. In fact, these cells appear as prominent bile ductular proliferation that is seen after significant confluent hepatic necrosis, with these ductular cells having the capacity to develop into mature bile ducts and hepatocytes (Figure 1.10). This is seen also in liver cell regeneration after partial hepatectomy and in partial livers after living-related transplantation; however, in chronic hepatitis with cirrhosis, the replicative ability of these cells is quite diminished.

Progenitor and Stem Cells Progenitor and stem cells play a significant role in liver cell development and regeneration. The pluripotential embryonic stem cells are derived

Figure 1.10  Stem cells. Prominent bile ductular proliferation is seen in this example of severe hepatitis with confluent necrosis, the ductules derived from facultative stem cells.

Evaluating a Liver Biopsy Specimen   15

A very small number of hematopoietic stem cells are also present in fetal livers and may remain in adult livers as well. They are induced to proliferate by similar conditions that cause oval cell proliferation, and have been shown to have the ability to mobilize and migrate into the liver with differentiation into hepatocytes and duct epithelial cells. These bone marrow cells can generate into hepatocytes in transplanted livers as well, although the frequency is quite low with such cells not always detectable. Although experimentally it has been shown by some that oval cells in the liver can be generated from hematopoietic stem cells, the numbers are extremely small, with some studies also showing no evidence of oval cells generated from hematopoietic stem cells.

Evaluating a Liver Biopsy Specimen Indications for Liver Biopsy

The liver biopsy is a useful tool in evaluating patients with known or suspected liver diseases. Often the clinical history and liver tests, and when appropriate special studies, provide the clinician with enough information to make a diagnosis and treatment choice without necessitating a liver biopsy; however, certain scenarios can occur that make a biopsy necessary (Table 1.3).

Table 1.3  Indications for liver biopsy Clinical signs and symptoms of acute or chronic liver disease associated with normal liver tests Clinical signs and symptoms of acute or chronic liver diseases associated with abnormal liver tests inconsistent with the suspected clinical diagnosis Staging and grading of known chronic liver diseases Evaluation of space-occupying lesions Evaluation of liver transplant specimens Acute liver failure of no known cause Abnormal liver tests of no known cause

Clinical Signs and Symptoms of Acute or Chronic Liver Disease Associated with Normal Liver Tests

If a patient has signs of chronic liver disease such as esophageal varices, ascites, and/or splenomegaly, but the liver tests including albumin are normal, a biopsy may be necessary to see if chronic liver disease with cirrhosis is indeed present, as at times patients with advanced liver disease may in fact have normal liver tests; however, if the liver biopsy is normal or shows non-specific changes, then other non-hepatic causes of ascites and portal hypertension (e.g., portal vein thrombosis, cardiac failure with “cardiac” ascites, peritonitis) may be present and necessitate appropriate workup. Additionally, in certain conditions such as non-cirrhotic portal fibrosis and nodular regenerative hyperplasia, signs of portal hypertension may be clinically present with associated normal liver tests, with a biopsy again aiding in the correct diagnoses of these lesions as well. Clinical Signs and Symptoms of Acute or Chronic Liver Diseases Associated with Abnormal Liver Tests Inconsistent with the Suspected Clinical Diagnoses

Another indicator for biopsy are patients with a known liver disease where the liver test results do not fit, hence bringing up the possibility of either an incorrect clinical diagnosis or two coexisting liver diseases. For example, patients with known alcoholic liver disease after binge drinking who a week later present with jaundice, hepatomegaly, and an abdominal bruit, all features most suggestive of alcoholic hepatitis, but have only minimally abnormal aspartate transaminase (AST) and alanine aminotransferase (ALT) values but a markedly elevated alkaline phosphatase activity, would be biopsied to rule out various causes of those liver test abnormalities including bile duct obstruction. Staging and Grading of Known Chronic Liver Disease

Patients with chronic viral hepatitis periodically have liver biopsies for staging and grading of the disease even when the liver tests are only mildly abnormal, as even in those cases the

16   1 Normal Liver

degree of transaminase elevations may not parallel the degree of necroinflammatory change and fibrosis seen on biopsy. The histologic features would then aid the clinician as to whether therapy is indicated. Additionally, even if the liver tests border on normal, a biopsy showing a bridging fibrosis alone may warrant therapy to prevent a severe fibrosis and eventual cirrhosis developing. Staging and grading systems are also used in patients with non-alcoholic fatty liver diseases and autoimmune hepatitis. Evaluation of Space-Occupying Lesions

In patients with a hepatic lesion seen on imaging, a biopsy is frequently warranted for diagnosis. Even when the clinical indicators are most suggestive of a benign process such as a hepatocellular adenoma (e.g., in a young woman on oral contraceptive therapy with normal liver tests and a single solid liver mass on imaging), a biopsy is often necessary to rule other causes of a benign process such as focal nodular hyperplasia or cavernous hemangioma that require different treatment and follow-up. In addition a tissue diagnosis of hepatocellular carcinoma or other primary or metastatic malignant lesions is also oftentimes indicated before initiation of therapy. Evaluation of Liver Transplant Specimens

It is not uncommon for various patients postliver transplant to have identical abnormal liver tests yet have completely different histologic findings on biopsy, leading to different therapies. For example, acute cellular rejection, bile duct obstruction, bile duct ischemia, and harvesting injury can all be associated with hyperbilirubinemia with high alkaline phosphatase values and modest elevations of the aminotransferases, all indicators of the value of post-transplant liver biopsies. Acute Liver Failure of No Known Cause

About 10–20% of patients with severe liver cell necrosis and liver failure have no known clinical cause. Transjugular liver biopsies are

often then performed to assess for possible histologic changes (e.g., abundant plasma cells in unsuspected autoimmune disease, necrosis without inflammation in ischemic injury or due to certain medications such as acetaminophen) that would lead to a correct diagnosis. Abnormal Liver Tests of No Known Cause

Although liver test values can often correlate with the clinical aspects of certain liver diseases, such as a high alkaline phosphatase with hyperbilirubinemia in known bile duct obstruction, at other times patients may present with abnormal liver tests for no known cause, especially when associated with fever or other systemic findings such as rash or lymphadenopathy. The biopsy can often then point to a specific cause or group of possibilities, such as multiple granulomas on biopsy (suggestive of an infectious process and other causes) or numerous portal eosinophils (suggestive of a possible drug-induced hepatitis). Organizational Approach in Liver Biopsy Evaluation

Individual morphologic features are often not diagnostic of specific liver diseases; however, assessing the whole complex of portal tract and parenchymal changes oftentimes may lead to likely diagnoses with differential possibilities. Even in instances where the clinical diagnosis is known, all aspects of the morphology should be assessed to prevent missing an unexpected or additional diagnosis. The basic architectural arrangement should be first evaluated as to whether it is intact, with regularly arranged portal tracts and terminal hepatic (central) venules, or whether there is portal fibrosis with bridging, incomplete, or complete fibrous septa (cirrhosis with regenerative nodule formation). Each portal tract is then evaluated for a number of different parameters. The degree of portal fibrosis (if any) along with the degree of portal inflammation and the type of inflammatory

Evaluating a Liver Biopsy Specimen   17

cells are assessed. Additionally the presence or absence and the degree of periportal interface inflammatory activity are evaluated. The bile ducts are reviewed as to whether they are normal in number and appearance or whether there is duct proliferation, duct dilatation (ectasia), periductal fibrosis, and inflammatory cells targeted towards the ducts (cholangitis). Duct cytologic atypia and the absence of ducts (ductopenia) are also important indicators of a number of different disorders (e.g., chronic allograft rejection). The portal vessels (arterioles, portal venules) should also be assessed as to whether there is thrombosis or occlusion, inflammatory cells targeted to the arteries (arteritis) or venules (pylephlebitis) and whether the venules are increased in number (a manifestation of portal hypertension). The parenchyma is assessed as to the degree and type of inflammatory infiltrates and the degree of lobular necrosis, with any zonal accentuation noted. The individual hepatocytes are evaluated for the presence or absence of Mallory–Denk bodies, steatosis (degree and type [macrovesicular, microvesicular]), cholestasis, granuloma formation, pigments (bile, lipochrome, hemosiderin), and inclusions (nuclear [e.g., cytomegalovirus], cytoplasmic [e.g., “ground glass cells” in chronic hepatitis B virus infection]). The sinusoids are evaluated as to whether there is dilatation and congestion, acute hemorrhage, sinusoidal fibrosis (zonal, patchy, diffuse), and red blood cell extravasation into hepatic trabeculae. The Kupffer cells are noted as to whether they are hyperplasic or hypertrophic and whether cytoplasmic material (e.g., red blood cells) or microorganisms (e.g., Histoplasma) are present. Certainly the present of tumor cells (benign and malignant, primary and metastatic) and other space-occupying lesions (e.g., abscesses, cysts) are crucial features as well. Routine and Special Histologic Stains in Liver Biopsy Specimens

Table 1.4 lists the routine and special histochemical stains used in liver biopsy evaluation.

Each laboratory sets up a basic “panel” that includes routine H&E, trichrome and iron stains, but the addition of PAS after diastase and reticulin stains are also used in some centers as well. Examples of these special stains are shown in Figures 1.11, 1.12, 1.13, 1.14, 1.15, and 1.16. It is best to include at least two or three H&E stained slides for each biopsy, with these slides not consecutive in the block. This can avoid missing an important feature that is eventually seen on deeper special stains that may preclude accurate evaluation, especially in instances of a granuloma or neoplasm. The table also lists additional stains that are available to best evaluate other possible diagnoses. Of note is that if a clinical diagnosis is suspected that could require special stains for diagnosis (e.g., Grocott’s methenamine silver, acid–fast stains for non-viral infection, immunohistochemical markers for neoplasms), then it may be appropriate to order unstained slides ahead of time for possible later use, as ordering them afterwards often means loss of tissue from the initial shavings when the paraffin block is re-set in the microtome. Routine Laboratory Tests Aiding in Liver Biopsy Evaluation

The routine and special laboratory tests performed in evaluating patients with known or suspected hepatic disease are listed in Table 1.5. The most common tests include the transaminases (AST, ALT), total bilirubin and direct, alkaline phosphatase, albumin, and globulin values (“liver panel”). Aminotransferases

The AST or SGOT (glutamic oxaloacetic transaminase) is a pyridoxal phosphatedependent transaminase enzyme that catalyzes the reversible transfer of an α-amino group between aspartate and glutamate, and is found in multiple organ systems besides the liver that include heart, skeletal muscle, kidneys, brain, and red blood cells. Two isoenzymes are present: a cytosolic isoenzyme derived mainly from red blood cells and myocardial fibers, and

18   1 Normal Liver

Table 1.4  Routine and special stains for liver biopsy interpretation Specific stain

Features

Clinical correlation

Routine histology panel for liver biopsies Hematoxylin and eosin Masson trichrome

Periodic acid–Schiff after diastase digestion (DiPAS)

Perl’s iron, Prussian blue

Routine assessment of liver histology Collagen (dark blue)

Chronic liver diseases

Lobular confluent necrosis (light blue)

Severe (confluent, submassive, massive) hepatic necrosis (fulminant hepatitis)

Lysosomal activity in portal macrophages, Kupffer cells

Areas of liver cell necrosis, dropout, and phagocytosis (both mild non-specific changes and acute/chronic hepatitis)

α1-Antitrypsin inclusions in periportal hepatocytes

α1-Antitrypsin deficiency (heterozygous and homozygous)

Lipochrome pigment

Older patient population (perivenular hepatocytes)

Lipochrome-like pigment

Dubin–Johnson syndrome, Gilbert syndrome

Hemosiderin

Hepatocellular iron: hemosiderosis, idiopathic hemochromatosis Kupffer cell iron: hemolytic anemias, multiple blood transfusions

Additional useful special stains Periodic acid–Schiff (PAS)

Glycogen Neutral polysaccarides Bile duct basement membranes

Steatosis of hepatocytes with microvesicular fat droplets highlighted (fatty liver diseases) Storage cells (Gaucher, Niemann–Pick diseases)

von Gieson

Elastic tissue fibers (medium and large vessels)

Fibrointimal thickening, vascular occlusion (non-cirrhotic portal fibrosis, allograft vascular rejection)

Reticulin

Assessment of the basic hepatic cord-sinusoid reticulin (type 3 collagen) framework

●●

Fibrosis

Chronic liver diseases

Confluent necrosis (condensed fibers)

Severe (confluent, submassive, massive) hepatic necrosis (fulminant hepatitis)

Fouchet, Hall

Bile

Cholestatic liver diseases

Sirius red

Direct staining of mature (type 1) collage fibers

Fibrosis in chronic liver diseases

Congo red

Amyloid

Amyloidosis confirmed with positive applegreen birefringence under polarized light

Gram

Gram-positive and Gram-negative cocci and bacilli

Bacterial sepsis, abscesses

Grocott’s methenamine silver (GMS)

Fungal micro-organisms, parasites

Infection by Aspergillus, Candida, Cryptococcus, Pneumocystis, and others

Evaluation of tumors: Normal cord thickness: 1–2 cells ●● Thickened cords >2–3 cells thick,   decrease to absent staining (hepatocellular carcinoma)

(continued)

Evaluating a Liver Biopsy Specimen   19

Table 1.4  (continued) Specific stain

Features

Clinical correlation

Ziehl–Neelson acid–fast (AFB)

Mycobacterium

Tuberculosis (M. tuberculosis) MAI infection (M. avium-intracellulare complex)

Warthin–Starry reaction

Spirochetes

Syphilis (Treponema)

Phosphotungstic acid hematoxylin (PTAH)

Fibrin (sinusoids, vessels)

Toxemia of pregnancy HELLP syndrome Q-fever Humoral allograft rejection

Rubeanic acid, rhodanine

Copper

Wilson disease

Orcein (Shikata), Victoria blue

Copper-binding protein (granular staining)

Indian childhood cirrhosis Certain chronic biliary tracts disorders (e.g., primary biliary cirrhosis)

Orcein (Shikata), Victoria blue

Hepatitis B surface antigen (diffuse cytoplasmic staining)

Chronic viral hepatitis type B

Oil Red O, Sudan Black

Neutral fats (frozen sections)

Fatty liver diseases

Useful immunohistochemical stainsa HBsAg

Cytoplasmic staining of hepatocytes

Chronic viral hepatitis B

HBcAg

Nuclear, sometimes cytoplasmic staining of hepatocytes

Chronic viral hepatitis B

HCV Ag

Cytoplasmic staining of hepatocytes

Chronic viral hepatitis C

Delta antigen

Nuclear staining of hepatocytes

Acute and chronic delta hepatitis

Ubiquitin

Mallory–Denk bodies

Active alcoholic and non-alcoholic fatty liver diseases Chronic biliary tract disorders

α1-Antitrypsin

Periportal intracytoplasmic inclusions α1-Antitrypsin deficiency

Cytokeratins 7, 19

Bile duct and ductular epithelium

Liver diseases associated with interlobular bile duct loss (e.g., primary biliary cirrhosis, chronic allograft rejection) with absent cytokeratin staining

Cytomegalovirus

Nuclear staining of hepatocytes

Cytomegalovirus infection, active and latent

Adenovirus

Nuclear “smudge” staining of hepatocytes

Adenovirus infection

Herpesvirus

Nuclear, sometimes cytoplasmic staining of hepatocytes

Herpesvirus infection

Epstein–Barr encoded RNA (EBER) probe

Intrahepatic latent Epstein–Barr virus

Lymphoma Post-transplant lymphoproliferative disorder (PTLD)

Polymerase chain reactions (PCR)

Viruses, infectious agents, gene mutations

Chronic HBV, HCV hepatitis Hemochromatosis (HFE genes)

Gene micro-array analysis

Gene mutations

Hemochromatosis (HFE genes)

Molecular techniques

a

Excluding tumor markers (see Chapters 13 and 14).

20   1 Normal Liver

Figure 1.11  Trichrome stain. Mature type I collagen representing mature fibrous bands in this cirrhotic liver is present.

Figure 1.12  Trichrome stain. Type III sinusoidal collagen fibers in an alcoholic fibrotic liver are demonstrated.

Figure 1.13  PAS stain after diastase digestion (DiPAS). Increase in lysosomal activity in areas of liver cell necrosis is seen.

Figure 1.14  PAS stain after diastase digestion (DiPAS). α1-Antitrypsin inclusions in periportal hepatocytes are highlighted with this stain.

Figure 1.15  Perl’s iron stain. Hemosiderin pigment stains intensely blue in the hepatocytes in this example of hereditary hemochromatosis.

Figure 1.16  Reticulin stain. The reticulin fibers line the hepatocyte trabecular cords that are one cell thick.

Evaluating a Liver Biopsy Specimen   21

Table 1.5  Routine and special laboratory tests for liver biopsy evaluation Laboratory test

Cause of abnormal values

Examples

AST (aspartate transaminase)

⇑ In hepatocellular damage

Various acute and chronic hepatitic reactions

ALT (alanine aminotransferase)

⇑ In hepatocellular damage

Various acute and chronic hepatitic reactions

Alkaline phosphatase

⇑ In cholestatic injury, bile duct damage, infiltrative processes

Biliary tract diseases including duct damage in transplant rejection Granulomatous hepatitis Primary and metastatic tumors

Total bilirubin, direct

⇑ In cholestatic injury, bile duct damage, functional hepatocellular damage

Biliary tract obstruction Hepatitis, acute and chronic with increased activity Advanced liver disease with liver failure Dubin–Johnson syndrome, Gilbert syndrome

Albumin

Values parallel liver cell synthetic function

Cirrhosis (decreased values)

Globulin

Values manifestations of liver injury

Cirrhosis, autoimmune hepatitis (increased values)

INR (prothrombin activity)

⇑ In liver dysfunction

Severe hepatitis, cirrhosis

γ-Glutamyl transferases (GGTP)

⇑ In bile duct damage

Biliary tract disorders

5′-Nucleotidase (5′NT)

⇑In bile duct damage

Biliary tract disorders

Lactic dehydrogenase (LDH)

⇑ In hepatocellular damage

Drug-induced injury Liver cell ischemia

α-Fetoprotein (AFP)

⇑ In liver cell neoplasms ⇑ In liver cell regeneration

Hepatocellular carcinoma Fulminant hepatitis with hepatocellular regeneration

Iron/iron-binding capacity

⇑ With increased hepatic absorption ⇑ In hemolysis

Hemochromatosis Hematologic disorders (hemolysis, ineffective erythropoiesis)

α1-Antitrypsin

⇑ In inflammatory diseases ⇓In inherited disease

Infections (increased values) α1-Antitrypsin deficiency (decreased values)

Ceruloplasmin

⇓In copper storage disorders, copper deficiency

Wilson disease Aceruloplasminemia Menke disease

Hepatic tissue iron quantitation

⇑ In primary and secondary iron deposition

Hemochromatosis

Hepatic tissue copper quantitation

⇑ In hepatic copper

Wilson disease Indian childhood cirrhosis

Routine liver tests

Additional useful tests

Viral and autoimmune hepatitis serologies See Table 2.2 Viral hepatitis – serologic markers See Table 7.2 Autoimmune-associated liver diseases – serologies

22   1 Normal Liver

a mitochondrial isoenzyme present mainly in the liver. The ALT or SGPT (glutamic pyruvic transaminase) catalyzes the transfer of an amino group from l-alanine to α-ketoglutarate and requires the coenzyme pyridoxal phosphate. The enzyme is found in the cytosol of the hepatocytes. It is found in low concentrations in many other sources but is most frequently present and more specifically associated with liver damage. The AST and ALT values are elevated to various degrees in almost all liver diseases at one point or another. Although the degree of enzyme elevations can give hints suggestive of certain liver diseases while making other less likely (e.g., marked elevations >1000  U/L in acute viral hepatitis, values in 100–200 range in chronic viral hepatitis), there is considerable overlap, as in chronic viral hepatis with reactivation associated with high aminotransferases. Of importance, the aminotransferases are not manifestations of true liver function but liver status. For instance, although the aminotransferases can be in the thousands in fulminant hepatitis, these values in the same disease can then decrease and even approach near normal associated with a rise in bilirubin values and INR due to the fact that there are few viable liver cells remaining to release the enzymes. The AST : ALT ratio can often be very useful. In alcoholic liver disease, especially alcoholic hepatitis, the AST  :  ALT ratio is characteristically 2–3/1, with the AST ranging from 100 to 300 while the ALT is only slightly elevated or can even be normal. Conversely, in non-alcoholic fatty liver disease this ratio does not occur. In acute or chronic viral and autoimmune hepatitis, the AST and ALT are approximately the same, with the ALT often slightly higher, although in the cirrhotic stage the AST is usually higher than the ALT. In direct and indirect hepatotoxic (non-hypersensitivity-induced) injury from certain drugs (e.g., acetaminophen) and toxins and in ischemic liver injury the AST can be markedly elevated with the ALT only modestly increased.

Bilirubin

Bilirubin, a heme degradation molecule excreted from the liver via the biliary system, is water insoluble and requires glucuronidation by the enzyme bilirubin uridine diphosphate (UDP)-glucuronyltransferase (bilirubin-UGT) into the water-soluble bilirubin mono- and di-glucuronide forms for secretion into the biliary canaliculi. It is divided into both the unconjugated (indirect) and the conjugated (direct, a combination of the mono- and di-glucuronide) forms in laboratory testing. The bilirubin values are elevated in a wide variety of liver diseases that hamper bile secretion. In various causes of severe hepatitis with or without liver failure (e.g., acute and fulminant viral or drug-induced hepatitis), elevated bilirubin values are the rule. Extrahepatic or large intrahepatic bile duct obstruction or stricture are also common causes. Elevated bilirubin values can also occur as a response to intracytoplasmic pathologic processes such as hereditary hyperbilirubinemias (e.g., Dubin–Johnson syndrome) and developmental disorders (e.g., paucity of duct syndrome). Alkaline Phosphatase

The alkaline phosphatase enzymes are zinc metalloenzymes present in most tissues and are localized in the liver within the microvilli of the bile canaliculi. It is a hydrolase enzyme responsible for removing the phosphate groups from many molecules including nucleotides, proteins, and alkaloids (dephosphorylation). It is also present within bone, with elevated levels occurring during pregnancy due to its placental origin as well. The alkaline phosphatase, like the serum bilirubin, is often elevated in acute and chronic biliary tract obstruction. It also is elevated in immune-mediated processes that attack interlobular ducts themselves (e.g., primary biliary cirrhosis, autoimmune cholangitis, post-transplant acute cellular rejection) while the bilirubin may be normal. The alkaline phosphatase is also increased in infiltrative processes such as granulomatous hepatitis, amyloidosis, or rapidly growing primary or metastatic tumors.

Selected Reading   23

Serum Proteins Albumin and Globulin

Albumin is one of the most abundant proteins in the circulation and has many functions including transport of fatty acids, various metabolites, and drugs. It is synthesized in the hepatocyte as a pre-proalbumin, with the N-terminal peptide removed before the nascent protein is released from the ER as proalbumin. It is then glycosylated and released as the albumin protein. Although there are a number of non-hepatic causes for low values (hypothyroidism, nephrotic syndrome, malnutrition), its value also parallels the number of viable hepatocytes in the liver, hence low values often correlate with advanced liver disease and cirrhosis. The serum globulin includes the α1 and α2, β, and γ (IgG, IgM, IgA) globulins. The values are elevated in numerous non-hepatic conditions (arthritis, infections, multiple myeloma) but are also useful in correlating with various liver diseases, as the value is usually elevated in advanced liver disease and cirrhosis and may be markedly elevated in autoimmune hepatitis. Special Laboratory Tests

A wide range of additional testing is also listed in Table 1.5 that includes laboratory values targeted to specific liver diseases (e.g., low serum ceruloplasmin values in Wilson disease). The critical viral and autoimmune hepatitis serologic markers are discussed in Chapters 2 (Viral hepatitis) and 7 (Autoimmune hepatitis) and listed in the appropriate tables in each chapter.

Selected Reading Crawford JM, Burt AD. Anatomy, pathophysiology and basic mechanisms of disease. In: Burt A, Portmann B, Ferrell L (eds). MacSween’s Pathology of the Liver, 6th edn. Elsevier: Edinburgh, 2012:1–77. Eleazar JA, Memeo L, Jhang JS, et al. Progenitor cell expansion: an important source of hepatocyte regeneration in chronic hepatitis. J Hepatol 2004;41:983–91. Friedman SL. The cellular basis of hepatic fibrosis. N Engl J Med 1993;328:1828. Gaudio E, Carpino G, Cardinale V, et al. New insights into liver stem cells. Dig Liver Dis 2009;41:455–62. Jungermann K, Kietzmann T. Zonation of parenchymal and nonparenchymal metabolism in liver. Annu Rev Nutr 1996;16:179. Kanel GC, Korula J. General aspects of the liver and liver diseases. In: Atlas of Liver Pathology, 3rd edn. Oxford: Elsevier, 2011:3–15. Lamers WH, Hilberts A, Furt E, et al. Hepatic enzymic zonation: a reevaluation of the concept of the liver acinus. Hepatology 1989;10:72. Lefkowitch JH. Special stains in diagnostic liver pathology. Semin Diagn Pathol 2006;23:190. Phillips MJ, Poucell S, Patterson J, et al. The Liver: An Atlas and Text of Ultrastructural Pathology. New York: Raven Press, 1987:1. Teutsch HF. The modular microarchitecture of human liver. Hepatology 2005;42:317. Turner R, Lozoya O, Wang Y, et al. Human hepatic stem cell and maturational liver lineage biology. Hepatology 2011;53:1035. Wanless IR. Physioanatomic considerations. In: Schiff ER, Sorrell MF, Maddrey WC (eds) Schiff ’s Diseases of the Liver, 9th edn. Philadelphia: Lippincott Williams & Wilkins, 2003:17.

Additional material for this chapter can be found online at: www.wiley.com/go/kanel/liverpathology This includes a full list of References, Case Examples, and Library Images to supplement this chapter.

24   

2 Viral Hepatitis Viral hepatitis is an acute and chronic necroinflammatory process of the liver that can be subdivided into infection by either one of the hepatotropic viruses (viral infection mostly localized to the liver) or by a system virus affecting the liver but other organ systems as well. This chapter discusses the epidemiology, virology, and clinical and biological behavior of these viruses with the characteristic pathologic changes seen in liver biopsy material.

Viral Hepatitis Secondary to Infection by the Hepatotropic Viruses General Characteristics and Clinical Presentations

Table 2.1 lists the six hepatotropic viruses – hepatitis A, B, δ, C, E, and G virus (HAV, HBV, δ [delta], HCV, HEV, and GVH-C) – and their epidemiology and clinical course, with the most pertinent serologic markers summarized in Table 2.2. Although each of the viruses has various individual characteristic, the general basic features of acute and chronic hepatitis are discussed below. In acute hepatitis, patients in general may present with malaise, flu-like symptoms, anorexia, low-grade fever, arthralgias, and myalgias, with jaundice and dark urine present in approximately 20% of cases oftentimes paralleling the severity of the disease or the specific virus involved. In many instances, especially associated with HCV virus, patients may be asymptomatic or have vague non-specific complaints.

Hepatomegaly may at times be present, and gastrointestinal symptoms (e.g., diarrhea) occur less frequently in approximately 25% of cases of acute HAV infection. The length of the prodromal period and the likelihood of clinical symptoms vary with each of the specific viruses. Extrahepatic manifestations may also occur that include serum sickness-like syndromes (rash, angioneurotic edema, urticaria), polyarteritis nodosa (HBV infection), cryoglobulinemia, and renal disease (mild proteinuria, abnormal urinary sediments, rarely glomerulonephritis). Liver tests show elevations of aspartate transaminase (AST) and alanine aminotransferase (ALT) usually greater than 500 IU/L and oftentimes over a thousand, with the ALT more specific for liver cell injury. The alkaline phosphatase value is usually normal or only slightly elevated (two times normal). When hyperbilirubinemia and jaundice occur the bilirubin value ranges from 5 to 20  mg/dL, depending on the degree of infection and necroinflammatory changes. Importantly the prothrombin time (INR) and serum albumin levels are normal in uncomplicated cases. Recovery is the rule in the vast majority (∼98%) of patients, with liver tests returning to normal usually within a few weeks after clinical presentation, although a prolonged course lasting a few months can occur with acute HAV infection. In a very small percentage of patients (1–2%), however, a fulminant course can occur with any of the hepatotropic viruses, but in particular with acute HEV infection in pregnant women and in patients with coexisting acute HBV and

Pathology of Liver Diseases, First Edition. Gary C. Kanel. © 2017 John Wiley & Sons, Ltd. Published 2017 by John Wiley & Sons, Ltd. Companion website: www.wiley.com/go/kanel/liverpathology

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Fecal–oral

Resolution with fulminant course 1.1 g/dL) with the total protein in the ascitic fluid often >3.0 g/dL, in contrast to values in cirrhosis where the gradient is ≤1.1 g/dL with low ascitic fluid protein. Diagnosis is made by various imaging techniques including Doppler ultrasound, venography, and MRI. Pathology

In the acute phase of the disease the liver is usually enlarged and shows a “nutmeg” appearance on cut section. Microscopically sinusoidal congestion, dilatation, and acute hemorrhage occur in the perivenular zones (Figure 8.1), with extension into the midzones in the more severe cases. The hepatocytes early on show a coagulative ischemic necrosis. In addition, extravasation of red blood cells into the space of Disse, replacing the damaged hepatocytes that have become atrophic and dropped out, is present, with the sinusoids remaining open (red blood cell – trabecular lesion) (Figure 8.2). This is caused by perivenular ischemia of hepatocytes associated with increase in sinusoidal pressure from outflow obstruction, with the establishment of an extrasinusoidal circulatory plexus for these

Figure 8.1  Budd–Chiari syndrome, acute. Perivenular and midzonal sinusoidal dilatation, congestion, and acute hemorrhage are present.

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Figure 8.2  Budd–Chiari syndrome, acute. Perivenular sinusoidal dilatation is seen. Red blood cells are present not in the sinusoids but instead in the hepatic cords, replacing damage hepatocytes that have dropped out due to ischemia (“red blood cell – trabecular lesion”).

extravasated red blood cells. It is speculated that the sinusoidal blood flows co-axially with the terminal venules in an attempt to bypass the hepatic vein occlusion. These outflow features often vary from one lobule to another, ranging from severe hemorrhage to only mild sinusoidal dilatation in the same biopsy specimen. In addition, biopsies taken from the caudate lobe may show little change due to its separate vascular outflow. The viable hepatocytes in the midand periportal zones may exhibit mild steatosis, but little necroinflammatory change is seen. Cholestasis may occur in the severe cases but is unusual. The portal tracts are initially normal in size with normal bile ducts and little portal inflammation. With time chronic changes can occur. The liver is normal in size but may be slightly small, is firm but does not show distinct regenerative nodules. Microscopically intraluminal fibrosis of the terminal hepatic venules develops with accompanying perivenular and pericellular sinusoidal fibrosis (Figure 8.3). This tends to merge with adjacent lobules, with eventual perivenular bridging fibrosis that leads to small nodules of liver cells with normal centrally located portal tracts (reversed lobulation or cardiac cirrhosis). Secondary thrombosis of the portal vein and its major tributaries can also occur, and indis-

Figure 8.3  Budd–Chiari syndrome, chronic. Prominent perivenular and intrasinusoidal fibrosis is seen due to long-term vascular outflow impairment.

crete septa between portal tracts and terminal hepatic venules can with time develop (venoportal cirrhosis). In long-term cases, portal to perivenular bridging fibrosis develops eventually leading to a cirrhosis of a micronodular type. The sublobular and hepatic veins often exhibit variable degrees of thickening of the wall with organizing thrombosis and recanalization that can occur in early and late stage disease (Figures 8.4 and 8.5). In addition inflammation of the sublobular and hepatic veins can develop with endothelial damage and resultant partial or even complete thrombosis of these inflamed outflow vessels (phlebitis). Sometimes a thin fibrous valve-like membrane can occur in the main hepatic veins as they enter the inferior vena cava, with resultant thrombosis and vascular outflow impairment. Right-Sided (Congestive) Heart Failure Clinical Presentation

Also termed congestive hepatopathy, patients present with signs and symptoms of congestive heart failure which is most often due to severe vascular and valvular disease, cardiomyopathy, and congenital heart diseases. Patients may be asymptomatic with only mildly abnormal liver tests, or a dull aching right upper quadrant pain may occur. Pulsatile hepatomegaly and increase

Hepatic Veins   147

Figures 8.4 and 8.5  Budd–Chiari syndrome, chronic. The sublobular vein shows intraluminal acute hemorrhage with early fibrin and new collagen deposition and the formation of small capillaries (Figure 8.4). The trichrome stain shows prominent intraluminal fibrosis (Figure 8.5).

in jugular venous distension with hepatojugular reflux may also be present on physical examination. Ascites is infrequent unless constrictive pericarditis is also present. In long-standing heart failure, manifestations of chronic liver disease with esophageal varices and ascites may occur, although variceal bleeding is uncommon. The liver tests are usually normal or only minimally increased; however, in severe heart failure, ischemia to the liver cells may occur secondary to hypoxia, with elevations of the liver tests, in particular the aspartate transaminase (AST), that can sometimes reach values of 1000  IU/L or higher. Notably, the alanine aminotransferase (ALT) can be elevated as well but not nearly to the extent of the AST values. Elevated bilirubin values are uncommon but may occur associated with severe ischemia. In patients with ascites, SAAG is usually elevated (>1.1 g/dL), although if cirrhosis has developed the gradient may decrease due to portal hypertension. Pathogenesis

The disease mechanisms overall relate to liver cell ischemia. In congestion there is impediment in blood flow, with stasis of red cells within the perivenular (zone 3) sinusoids. This directly impairs oxygen supply to the hepatocytes, resulting in liver cell ischemia with atrophy and

eventual loss of these hepatocytes, with resultant perivenular fibrosis over months to years. Ischemia may also be due to hypotension from cardiac insufficiency, similarly resulting in liver cell atrophy of the perivenular hepatocytes which are the last hepatocytes to receive blood and hence oxygen from the hepatic artery blood supply. Eventually hepatocellular cell loss occurs. Pathology

The liver on gross examination shows a characteristic “nutmeg” pattern on cross sections. Perivenular (pericentral) sinusoidal dilatation and congestion without an accompanying inflammatory infiltrate is seen on microscopic examination (Figure 8.6). The trabecular cords show atrophic hepatocytes having scanty cytoplasm due to the increase in sinusoidal pressure, although remarkably these liver cells are still quite functional. Eosinophilic round discrete hyaline droplets measuring up to 4  μ in diameter can sometimes be seen free within the perivenular sinusoids as well as within the cytoplasm of the perivenular hepatocytes. The portal tracts show minimal changes and can be entirely normal, with the interlobular bile ducts unremarkable. In severe cases of heart failure, perivenular acute hemorrhage and coagulative ischemic necrosis of hepatocytes may occur,

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Figure 8.6  Right-sided heart failure, acute. Perivenular sinusoidal dilatation and congestion are seen. The hepatic cords show small somewhat atrophic liver cells due to increased sinusoidal pressure from the vascular impairment.

infrequently associated with cholestasis. With time, perivenular sinusoidal fibrosis can occur (Figure 8.7) with perivenular–perivenular bridging and the formation of a “cardiac” cirrhosis, with the nodules containing uninvolved centrally located portal tracts (reversed lobulation) (Figure 8.8). As the lesion progresses there may be eventual portal–perivenular and portal–portal bridging fibrosis as well, resulting in a micronodular cirrhosis over many years.

Figure 8.7  Right-sided heart failure, acute on chronic (trichrome stain). Sinusoidal collagen with time is laid down in the subendothelial regions and the space of Disse.

Figure 8.8  Right-sided heart failure, “cardiac” cirrhosis (trichrome stain). Bridging fibrosis between the perivenular areas may occur with time forming a nodule; however, the portal tracts early on are spared and can be seen in the center of these nodules (“reverse lobulation”). As the disease further progresses, bridging fibrosis occurs between the perivenular and portal regions forming a micronodular cirrhosis.

Additionally in long-term right-sided heart failure, persistent increased venous pressure may cause phlebosclerosis of the medium and large hepatic vein branches. Differential Diagnoses of Hepatic Venous Outflow Impairment and Hepatic Vein Thrombosis

Cardiac failure, whether primary (e.g., vascular, valvular diseases) or secondary (e.g., constrictive pericarditis, pulmonary disorders) is a leading cause of hepatic venous outflow impairment and most often occurs with a patent hepatic vein. The various causes of hepatic vein thrombosis with hepatic venous outflow impairment are listed in Table 8.1. Hypercoagulable states, outflow impairment due to space-occupying lesions (both neoplastic and non-neoplastic), and advanced liver disease (cirrhosis) of almost any origin are some of the more common differentials. Extensive workup including various imaging studies, in particular venography, Doppler imaging with real-time evaluation of the hepatic and portal veins and inferior vena cava, and gadolinium-enhanced MRI, strongly contribute to determining the cause. Liver biopsy

Portal Veins   149

is oftentimes an important factor and is complimentary to the clinical assessment and imaging. Biopsies can be most useful in distinguishing the Budd–Chiari syndrome from sinusoidal obstruction syndrome (SOS), to determine the grade and stage of the acute and chronic disease, and to determine whether cirrhosis is present and a possible differential cause of the cirrhosis.

Portal Veins Portal Vein Thrombosis

Thrombosis of the portal vein and its major intrahepatic branches is caused by a number of factors (Table 8.2) and can clinically present in

both acute and chronic phases. In about onethird of cases a specific localized cause such as inflammatory processes can be seen while most cases are associated with systemic diseases such as myeloproliferative disorders. In the acute phase of the disease abdominal or lower back pain and fever may occur, with the severity of the disease correlating with the extent of portal and oftentimes mesenteric vein involvement. Gastrointestinal symptoms such as ileus and colicky pain, with diarrhea and low-grade fever, sometimes occur. When there is propagation of the thrombus, ischemia of the bowel with infarction is a serious complication. In chronic disease with thrombosis and recanalization of the vessel, small tortuous collateral

Table 8.2  Portal vein thrombosis – etiology factors Vascular thrombosis (systemic diseases) Hypercoagulative states ●● Acquired prothrombin disorders –– Myeloproliferative diseases (polycythemia vera, essential thrombocythemia, agnogenic myeloid metaplasia) –– Paroxysmal nocturnal hemoglobinuria –– Hyperhomocysteinaemia ●● Inherited prothrombin disorders –– Protein S deficiency –– Protein C deficiency –– Antithrombin III deficiency –– Prothrombin G20210A mutation –– Factor V Leiden deficiency –– Methylene-tetrahydrofolate reductase (MTHFR) mutation ●● Oral contraceptives ●● Pregnancy, puerperium Vascular stasis ●● Cirrhosis of any cause ●● Localized large hepatic lesions –– Abscesses –– Benign and malignant tumors Localized vascular invasion ●● Hepatocellular carcinoma ●● Cholangiocarcinoma ●● Metastatic neoplasms

Vascular inflammation and injury Appendicitis ●● Pancreatitis ●● Pylephlebitis ●● Diverticulitis ●● Cholecystitis ●● Inflammatory bowel disease ●● Surgical manipulation, status post liver transplantation, portacaval shunt, splenectomy ●● Status post chemoembolization ●● Abdominal trauma, surgery Small portal vein and venular occlusion, obliteration ●● Cirrhosis ●● Schistosomiasis ●● Non-cirrhotic portal fibrosis ●● Congenital hepatic fibrosis ●● Primary biliary cirrhosis ●● Systemic lupus erythematosus ●● Umbilical vein catheterization ●● Portal vein compression by lymph nodes (e.g., tuberculosis, lymphoma) ●●

Source: Adapted from Seijo S and DeLeve LD. Vascular diseases of the liver. In: Podolsky DK et al. (ed). Yamada’s Textbook of Gastroenterology, 6th edn, Wiley Blackwell, 2016. Reproduced with permission of John Wiley & Sons; and adapted from Jain D and West AB. Histologic Diagnosis. In: DeLeve LD and Garcia-Tsao G (eds). Vascular Liver Disease. New York: Springer, 2011, pp103–123.

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veins develop (“cavernous transformation”). Esophageal varices, splenomegaly, ascites, and portal hypertensive gastropathy can occur due to portal hypertension. Of note is that although cirrhosis in and of itself can cause portal vein thrombosis, it is not common and is seen in only about 5% of cirrhotic livers; however, in the presence of hepatocellular carcinoma, a not uncommon complication of cirrhosis, the incidence rapidly rises to almost one-third of cases in the larger lesions. Pathology

Gross examination of the extrahepatic portal veins may show total or eccentric partial occlusion by thrombosis with various degrees of organization. Histology may demonstrate an organizing thrombus (Figure 8.9) with fibrin, red blood cells, and inflammatory cells which may be neutrophils when infection also occurs. Variable new small vessel formation with fibrosis develops with resultant portal vein thickening. Calcifications can occur. Tumor thrombi most often involve the major intrahepatic portal vein branches with extension into the main proximal portal vein. The small portal vein branches and venules may show a number of changes. Dilatation

Figure 8.10  Portal venular thrombus. The portal venule shows fibrin and leukocytes within the lumen (pylephlebitis). Adjacent portal edema with acute and chronic inflammatory infiltrates is also present.

with small increase in portal venous radicals can occur, but diminishment of the vessels with obliteration of the small venules can also develop. Inflammation to variable degrees may be seen with inflammatory cells attached to the endothelium and oftentimes infiltrating into the larger portal vein walls, sometimes resulting in thrombosis with vascular occlusion (pylephlebitis) (Figure 8.10). Decreased portal vein perfusion can occur particularly in cirrhotic livers, with resultant atrophy of the hepatic parenchyma in the affected hepatic lobe (“hepatic extinction”). This feature tends to more commonly involve the left portal vein segment and can result in left lobe atrophy. Differential Diagnoses

Figure 8.9  Portal vein thrombosis. This low power cross-section image of the extrahepatic portal vein shows partial intraluminal occlusion with acute hemorrhage and early thrombus formation. Extrahepatic portal vein thrombosis is also seen in non-cirrhotic portal fibrosis (“non-cirrhotic portal hypertension”).

The various causes of portal vein thrombosis are listed in Table 8.2. Imaging of the hepatic and portal veins with portal pressure measurements and clinical correlation are oftentimes most important in determining the specific cause, as the liver biopsy findings are often quite subtle with the differential possibilities often numerous. One of the causes of small portal vein occlusion, non-cirrhotic portal fibrosis, is discussed in more detail in the following section.

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Non-Cirrhotic Portal Fibrosis (“Non-Cirrhotic Portal Hypertension”)

Non-cirrhotic portal fibrosis is a liver disease associated with portal hypertension in the absence of cirrhosis. It is uncommon in the United States but occurs not infrequently in India and Japan, and is believed to account for up to one in four of all cases of portal hypertension in India. Associated autoimmune diseases such as systemic lupus erythematosus, progressive systemic sclerosis, thyroiditis, and mixed connective diseases have been associated with non-cirrhotic portal fibrosis in Japan, with up to a quarter of the patients being ANA positive. It occurs about twice more often in men and usually presents insidiously in young to middle-aged patients with marked splenomegaly, esophageal varices, and sometimes ascites. Jaundice and hepatic encephalopathy are uncommon. Laboratory tests often show anemia, leukopenia, and thrombocytopenia due to the marked splenomegaly and splanchnic pooling of blood. Liver tests are usually near normal. Imaging and vascular pressure measurements show evidence of a pre-sinusoidal portal hypertension. Portal venous pressure is increased while wedged hepatic venous pressure is normal or minimally elevated. Ultrasound demonstrates dilated portal and splenic veins. The hepatic arterial blood flow is normal to slightly increased, and angiography shows distortion and sudden diminution of the second- and third-order portal venous radicles along with aberrant vessels. Portal hypertension develops as a manifestation of initial thrombosis of the portal vein with recanalization, intimal thickening and eventual sclerosis with resultant increase in the portal vein pressures (hepatoportal sclerosis).

Figure 8.11  Non-cirrhotic portal fibrosis. The portal venule shows prominent ectasia. In addition an increase in small portal venous branches is also seen.

light microscopy of tissue sections by the Verhoeff ’s elastic tissue stain. Organizing thrombus with recanalization can be seen in the portal vein (see Figure 8.9). Microscopically the portal tracts are normal in size or show a stellate or arachnoid periportal fibrosis without significant intrasinusoidal collagen deposition. Little if any portal inflammation is seen and the bile ducts are normal; however, dilated portal veins and venules and increased small portal venous radicals are present (Figures 8.11 and 8.12). Many smaller portal tracts show a decrease to absence

Pathology

The liver on gross examination is usually unremarkable, although there is some resistence on cut section. The medium and larger intrahepatic segments of the portal vein and the extrahepatic portal vein usually show variable irregular intimal thickening which can be highlighted on

Figure 8.12  Non-cirrhotic portal fibrosis. The portal tract shows numerous small portal venous radicals. A mild portal lymphocytic infiltrate is also present.

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of the portal venular segments. Abnormal proliferating intralobular sinusoidal and vascular channels sometimes occur (Figure 8.13), and the sinusoids may show mild dilatation. The hepatocytes show variable hydropic change, but lobular inflammation is minimal to absent. Although most cases show only little portal fibrosis, portal bridging fibrosis can infrequently occur without the development of a well-established cirrhosis. Differential diagnoses

A number of liver diseases can be associated with clinical signs and symptoms of portal hypertension in the absence of cirrhosis (Table 8.3). Many of the listed diseases are not difficult to differentiate from non-cirrhotic

Figure 8.13  Non-cirrhotic portal fibrosis. The parenchyma shows numerous irregularly spaced outflow vessels (terminal hepatic venular branches).

Table 8.3  Causes of non-cirrhotic portal hypertension Vascular disorders

Arteriovenous fistulas Budd–Chiari syndrome Extrahepatic portal vein thrombosis Non-cirrhotic portal fibrosis Sinusoidal obstruction syndrome Sinusoidal infiltrates ●● Leukemia and myeloproliferative disorders, extensive extramedullary hematopoiesis in myelofibrosis of bone marrow, plasma cell dyscrasias, Gaucher disease, amyloidosis, fibrin in toxemia, primary and metastatic tumors, sinusoidal fibrosis in alcoholic and non-alcoholic fatty liver diseases Veno-occlusive changes Peliosis hepatis

Infectious disorders

Leishmaniasis Schistosomiasis Bacterial sepsis with pylephlebitis

Developmental and inherited disorders

Congenital hepatic fibrosis Fibropolycystic disease Gaucher disease Hereditary hemorrhagic telangiectasia

Drug- and toxininduced injury

Ethanol (acute and chronic alcoholic liver disease) Arsenicals Copper sulphate (vineyard sprayers) Vinyl chloride monomers Hypervitaminosis A 6-Mercaptopurine Azathioprine

Other disorders

Constrictive pericarditis Severe restrictive cardiomyopathy Nodular regenerative hyperplasia Partial nodular transformation Primary biliary cirrhosis Sarcoidosis Hemodialysis

Source: Adapted from Kanel GC and Korula J. Atlas of Liver Pathology, 3rd edn. Elsevier, 2011.

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portal fibrosis in the appropriate clinical setting (e.g., known schistosomiasis, Gaucher disease), although sometimes with limited clinical information and only small biopsy material the diagnoses can be quite problematic.

Hepatic Artery and Systemic Circulation Hepatic Artery Hypoperfusion and Hypoxia, Vasculitis, Hepatic Artery Thrombosis

Hepatic ischemia (“shock liver”) is most often a consequence of poor cardiac output and reduction in systemic blood flow secondary to severe atherosclerotic cardiovascular disease and heart failure but can occur from a number of different causes (Table 8.4). Because the hepatic artery blood flow directly relates to cardiac output, poor perfusion with resultant hepatic ischemia often occurs in severe hypotensive states regardless of its etiology. The hepatocytes in the perivenular zone are most susceptible to low oxygen tension and hence are most affected by the hypoxemic states. In most instances the liver can maintain proper function through the portal vein blood flow and arterial collaterals; however, in severe arterial impairment, especially in the post-transplant state where collaterals for the new implanted livers have not developed, ischemia may occur. Although liver tests are usually normal, in some cases, depending on the severity and duration of the hypotension, the transaminases with the AST predominating are usually mildly to moderately elevated, with hyperbilirubinemia sometimes occurring. The aminotransferases may infrequently exceed 1000  IU/L and clinically mimic an acute hepatitis. Serum lactate dehydrogenase is also characteristically elevated in hypoxemia and is useful in distinguishing viral hepatitis from ischemic necrosis as well. The abnormal liver tests often rapidly improve and return to normal within a few days after resolution of the hypotensive episode, in contrast to acute viral hepatitis where the resolution of

Table 8.4  Causes of hepatic arterial blood flow impairment Severe atherosclerosis cardiovascular disease with cardiac failure ●● Cardiac arrhythmias ●● Myocardial infarction Small hepatic artery and arteriolar branch occlusion ●● Disseminated intravascular coagulation ●● Diabetic arteriopathy Aortic and hepatic artery aneurysms Cardiomyopathies Cor pulmonale Pulmonary embolism Pericarditis Severe dehydration Severe acute intra-thoracic, intra-abdominal hemorrhage Pericardial tamponade Sepsis (septic shock) Hepatic artery thrombosis from septic emboli Amyloidosis (cardiac, small hepatic arteriolar involvement) Antiphospholipid syndrome Hypercoagulable states Vasculitis ●● Rheumatoid arthritis ●● Polyarteritis nodosa ●● Kawasaki disease ●● Secondary syphilis ●● Churg–Strauss syndrome Post-transplant disorders ●● Thrombosis at anastomotic site ●● Humoral (hyperimmune) rejection ●● Chronic vascular rejection (obliterative vasculopathy) Trauma with hepatic artery injury Heat stroke, hyperpyrexia Eclampsia

abnormal liver tests is more gradual. Patients may clinically present with signs of bile duct obstruction, as ischemia to the larger ducts (extrahepatic, hilar, and large intrahepatic bile ducts) results in injury and secondary stricture. Secondary (acquired) sclerosing cholangitis is a complication. Hepatomegaly may at times occur, and ascites may also be present in instances of right-sided (congestive) heart failure. Pathology

The hepatic artery and its smaller segments show variable pathologic changes, dependent

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on the etiology. In cardiac failure due to the variant causes, the small vessels show little change with the medium and larger arteries usually showing atheromatous features. In vasculitis usually the small vessels may be spared with the medium and larger arteries showing inflammatory and necrotizing fibrinoid necrosis with fibrin thrombi, the inflammatory infiltrates within the vessel wall consisting of lymphocytes and neutrophils, with increased eosinophils at times. A granulomatous inflammatory response can also occur within the wall (e.g., polyarteritis nodosa). Secondary thrombosis of the adjacent hepatic venular segments can also be present. The very small vessels can also show vasculitic changes with thrombosis in post-transplant humoral rejection and severe acute cellular rejection. Small vessel thickening can also be seen in diabetic arteriopathy and in amyloidosis, although luminal occlusion usually does not occur and hepatocellular ischemia is uncommon unless larger vessel pathology is also clinically apparent. The large bile duct damage due to hepatic artery thrombosis in ischemic cholangiopathy consists of irregular eosinophilia of duct epithelium which can lead to epithelial necrosis and loss, with extravasation of bile into the damaged ischemic vessel wall of the larger ducts (ischemia-induced cholangitis) (Figure 8.14). The smaller ducts can also be affected with eventual bile duct loss in some instances (e.g., post-transplant hepatic artery thrombosis with bile duct ischemia), with adjacent bile ductular reaction sometimes present in the smaller affected portal tracts. The major changes that affect the hepatic parenchyma are usually the same regardless of the cause. Although mildly fluctuating hypotensive episodes alone often show little pathologic hepatic changes, perivenular (zone 3) coagulative confluent ischemic necrosis of hepatocytes can occur in more severe instances of poor perfusion (Figure 8.15), and in the early stages there is no accompanying inflammation. Within a few days liver cell

Figure 8.14  Bile duct ischemia due to decrease in hepatic artery blood flow. The hilar bile duct shows extensive ischemic necrosis involving the entire vessel, with extravasation of bile into the necrotic vascular wall, secondary to hepatic artery thrombosis.

dropout occurs with a corresponding histiocytic reaction and perivenular collapse of the reticulin framework. Sometimes neutrophils can predominant although that is not the rule. In severe cases of ischemia the necrosis can extend to the midzones. Panlobular necrosis with hepatic infarction is uncommon but can occur in total hepatic artery occlusion in the post-transplant setting or status post inadvertent hepatic artery ligation in surgery for traumatic injury. Infarction can also be intended in embolism therapy for treatment of primary and metastatic tumors. The adjacent viable

Figure 8.15  Lobular ischemia due to decrease in hepatic artery blood flow. The perivenular and midzonal hepatocytes show early coagulative confluent ischemic necrosis.

Hepatic Artery and Systemic Circulation   155

hepatocytes show variable hydropic changes with little inflammation. Cholestasis can occur in some cases. Liver cell regeneration of the viable hepatocytes occurs with cessation of the hypotensive events. Usually the degree of ischemic changes may show variability from one lobule to another. This is due to heterogeneous arterial perfusion of the hepatic lobules, which is especially obvious in cirrhotic livers where severe ischemic necrosis can be seen after esophageal or gastric variceal bleeding in one regenerative nodule with the adjacent nodules spared. Of interest is that in instances of severe leftsided heart failure in the absence of hypotension, the perivenular hepatocytes still undergo ischemic necrosis and dropout; however, the sinusoids stay open, with red blood cells entering the hepatic cords via the space of Disse (“red blood cell – trabecular lesion”) without sinusoidal congestion and hemorrhage (Figure 8.16). This type of red blood cell lesion can also occur in acute hepatic venous outflow obstruction (acute Budd–Chiari syndrome) (Figure 8.2), although usually there is some degree of accompanying sinusoidal congestion and acute hemorrhage as well.

Figure 8.16  Left-sided heart failure in the absence of hypotension. Perivenular sinusoidal dilatation with extravasation of red blood cells into Disse space, partially replacing damaged hepatocytes, is seen.

Heat Stroke and Hyperpyrexia

Heat stroke is a severe illness with a mortality of up to 60%, patients having elevated body temperatures of over 40°C (hyperpyrexia) with neurologic abnormalities and multiorgan system failure. It can be caused by extreme overexertion and by environmental exposure to very hot and humid climates. The mechanism relates to the increase in metabolic processes causing hypoxemia, direct cytotoxic injury from extreme heat, and activation of various mediators causing endothelial activation and release of various cytokines. The hypoxic events then secondarily involve the liver. In fact up to 12% of patients with hyperpyrexia can develop liver failure. Patients initially may present with sweating, muscle cramps and dizziness, and high-grade fevers, and can go on to severe hypotension and coma with a high mortality. Liver aminotransferases, especially the AST, are markedly elevated (over 1000 IU/L) with hyperbilirubinemia in the more severe cases. Severe hypophosphatemia and elevated lactate dehydrogenase (LDH) commonly occur. Liver biopsies show striking perivenular and midzonal confluent coagulative (ischemic) necrosis (Figure 8.17) that can oftentimes be panlobular. Some degree of microvesicular steatosis can sometimes be seen in the viable hepatocytes,

Figure 8.17  Hyperpyrexia (heat stroke). Extensive confluent coagulative necrosis of hepatocytes is seen due to ischemia.

156   8 Vascular Disorders

these vessels remain open in about 45% of these patients. The main primary abnormality originates within the sinusoids, renaming this disorder the Sinusoidal Obstruction Syndrome (SOS). Epidemiology and pathophysiology

Figure 8.18  Hyperpyrexia (heat stroke). The portal tract shows a mild lymphocytic infiltrate. In this example the coagulative necrosis has extended to involve the periportal hepatocytes as well (panlobular necrosis).

as can cholestasis at times. Although in the initial stages of the disease no lobular inflammation is seen, early on a neutrophilic infiltrate with phagocytosis of the damaged hepatocytes sets in. The portal tracts show a mild to moderate lymphocytic infiltrate (Figure 8.18). Although the interlobular bile ducts are usually normal, bile ductular proliferation is usually present, sometimes associated with a neutrophilic infiltrate amongst the ductules. An acute cholangitis can also infrequently occur. Differential Diagnoses of Hypoxic Lesions due to Impaired Hepatic Artery Perfusion

Table 8.4 lists the various causes of impaired hepatic arterial blood flow resulting in ischemic liver cell injury. The clinical history is usually sufficient at arriving at the exact cause, although coexisting processes such as severe atherosclerosis and sepsis are certainly not uncommon.

Hepatic Sinusoids Sinusoidal Obstruction Syndrome (Venoocclusive Disease)

Although the disorder veno-occlusive disease was initially believed to be a process involving the occlusion of terminal hepatic venules, in fact

There are in general two causes of SOS (1) induction by the use of pyrrolizidine alkaloids (ingestion of “bush tea” prepared from boiling leaves of Crotalaria fulva and Senecio in the West Indies), and (2) the use of various chemotherapeutic conditioning regimens (e.g., actinomycin D, azathioprine, busulfan, cyclophosphamide) in bone marrow transplantation, the latter a much more common cause today. It can also occur in graft-versus-host disease in bone marrow transplant recipients, in renal allograft patients, rarely in liver transplant recipients, and in association with certain toxins (e.g., vinyl chloride, arsenicals). The incidence of SOS varies with the type and intensity of the conditioning regimen used in bone marrow transplant recipients, and can range from almost zero to up to 50%. The frequency has declined over the years due to better recognition and adaptation of the treatment doses, knowledge of the medications that are most associated with SOS (in particular cyclophosphamide), and better therapeutic drug monitoring. The pathogenesis of SOS involves first direct injury to the sinusoidal endothelial lining cells in the perivenular zone, with gaps forming within and between these cells allowing red blood cells to enter the space of Disse, dissecting off the sinusoidal lining cells and causing their sloughing. These extravasated red blood cells then hinder sinusoidal blood flow, with fibrin deposition within the terminal hepatic venular lumen, hypoxemia of the perivenular hepatocytes, and finally stellate cell activation with perivenular and intraluminal obliterative fibrosis. Clinical Presentation

Patients present 10–20 days after the start of therapy with right upper quadrant pain and tenderness, renal sodium retention, and increase in weight. Jaundice can then occur up

Hepatic Sinusoids   157

to 1 week thereafter, with renal dysfunction, ascites, and thrombocytopenia additional features. Transaminitis occurs 2–4 weeks after the start of treatment and is often due to hepatic ischemia from impediment of the sinusoidal vascular blood flow. Chronic disease occurring months to years later may have manifestations of portal hypertension with esophageal varices. Pathology

In the acute phase of SOS, the terminal hepatic venules 1.9

Source: Bacon BR, et al. Hemochromatosis and Iron ­Storage Disorders. In: Schiff ER, Maddrey WC, Sorrell MF (eds). Schiff ’s Diseases of the Liver, 11th edn, 2012, Wiley Blackwell. Reproduced with permission of John Wiley & Sons.

174    9  Genetic and Metabolic Hepatic Diseases

Figures 9.17 and 9.18  Hereditary hemochromatosis. The hepatocytes contain abundant golden-brown coarsely granular pigment that represent hemosiderin (Figure 9.17), confirmed on the Perl’s iron stain (Figure 9.18).

also stage the extent of the disease progression. The hallmark feature in established cases of the homozygous C282Y mutation is abundant golden brown coarsely granular pigment (hemosiderin) (Figure 9.17), confirmed on positive Prussian blue or Perl’s iron stain (Figure 9.18). Usually the pigment is concentrated in the pericanalicular region of the liver cells. The pigment can involve virtually all of the hepatocytes, and is also seen in bile duct and ductular epithelium (Figure 9.19) and to variable degrees in endothelial and connective tissue cells. Although the Kupffer cells are usually spared, in instances of lobular inflammation with liver cell necrosis the pigment can then be

Figure 9.19  Hereditary hemochromatosis. The interlobular bile ducts and ductules also contain the hemosiderin pigment.

deposited within the Kupffer cells involved in phagocytosis of the damaged hepatocytes. Mild focal necroinflammatory change can occur within the lobules but is usually quite mild. Variable but usually mild macrovesicular steatosis and occasional glycogenated nuclei of liver cells are also often seen, the latter more prominent in the diabetic patients as a result of iron overload in the pancreas. In the rare neonatal variant where the infants are often stillborn or premature, cholestasis, severe confluent necrosis, and syncytial giant cell changes of the hepatocytes can be seen, oftentimes associated with severe fibrosis or even cirrhosis. In the early-stage disease the amount of hemosiderin may be only mild to moderate and the portal fibrosis may be minimal to absent. There is a tendency even in early-stage disease for iron to accumulate within bile duct epithelium. The iron is usually concentrated first in the periportal hepatocytes; however, as the disease progresses and the iron deposition becomes more prominent, portal fibrosis with eventual bridging fibrosis and a predominantly micronodular cirrhosis occurs (Figures 9.20 and 9.21). At this stage the iron is more prominent in the periseptal hepatocytes; however, in both the pre-cirrhotic and cirrhotic stages the iron also may diffusely involve virtually all of the hepatocytes. In cirrhosis, the amount of hemosiderin pigment deposition can vary from one

Disorders Associated with Increase in Hepatic Iron   175 ●●

●●

●●

●●

Figure 9.20  Hereditary hemochromatosis. A wellestablished cirrhosis is present, the regenerative nodules usually micronodular. Even on low power the hepatocytes are seen to contain a golden-brown pigment representing hemosiderin.

regenerative nodule to another, with the newly formed hepatocytes from liver cell regeneration at first containing little pigment. In addition, in the cirrhotic stage hepatocellular carcinoma is a high risk, the neoplastic hepatocytes devoid of hemosiderin pigment and hence standing out on iron stain from the adjacent cirrhotic regenerative nodules containing abundant stainable iron. A qualitative grading system is commonly used in liver biopsy material: ●●

0, no visible iron on high-power magnification (400×)

1+, granules scanty on medium (200×) and high power in the periportal liver cells 2+, iron easily discernible in periportal hepatocytes on low (100×) and medium power 3+, iron easily seen in periportal liver cells with extension into the midzones in some lobules on low power (100×) 4+, iron present in virtually all hepatocytes (40× and discernible on gross examination of the slide)

This system is most useful in assessing the degree of iron deposition in the initial stage workup and the effect of treatment (phlebotomy with one unit of blood containing approximately 250 mg of iron) in decreasing the degree of iron overload. The hepatic iron index (HII) can also be a useful tool on liver biopsy material and is calculated by measuring the amount of iron in μmoles/g dry weight and dividing by the patient’s age. A HII of >1.9 most often distinguishes the homozygous state from heterozygous and non-hereditary causes of iron overload, although the accumulation of iron can be variable and exceptions can occur. For example, other chronic hemolytic conditions associated with excessive iron turnover such as β-thalassemia major requiring numerous blood transfusions can have HII ratios much greater than 1.9; however, it must also be noted that many patients who are C282Y homozygous have HII values less than 1.9. With the present availability of HFE gene testing, determining the HII is of little diagnostic value. Differential Diagnoses and Other Causes of Iron Overload

Figure 9.21  Hereditary hemochromatosis. The hemosiderin pigment shows strong positive staining with the Perl’s iron stain.

The evaluation of the iron stain on biopsy along with hepatic iron quantitation and testing for the HFE gene mutations are virtually diagnostic for hereditary hemochromatosis. The various disorders associated with increase in hepatic iron including hereditary hemochromatosis are listed in Table 9.2. Many of these disorders have to be considered either in early-stage disease or before complete testing is accomplished. The various causes of increase in hepatic iron within the reticuloendothelial system (Kupffer cells, portal

176    9  Genetic and Metabolic Hepatic Diseases

Table 9.2  Disorders associated with increased hepatic iron Hereditary hemochromatosis HFE-associated mutations ●● C282Y/C282Y ●● C282Y/H63D ●● Other HFE mutations Non-HFE related mutations ●● Transferrin receptor 2 (TFR2) ●● Hemojuvelin (HJV) ●● Hepcidin (HAMP) ●● Ferroportin (SLC40A1) ●● Divalent metal transporter 1 (SLC11A2) ●● Ferritin regulatory (rare)

Secondary (acquired) iron overload Anemias secondary to ineffective erythropoiesis ●● Thalassemia major ●● Sideroblastic anemias Congenital dyserythropoietic anemias Congenital atransferrinemia Liver diseases ●● Alcoholic liver disease (cirrhosis) ●● Chronic viral hepatitis C ●● Porphyria cutanea tarda ●● Non-alcoholic steatohepatitis ●● Status post-portacaval shunt Excessive iron ingestion Iron overload, parenteral Multiple transfusions Iron-dextran injections Long-term hemodialysis Iron overload (other) Sub-Saharan African iron overload (non-HFE-related genetic trait) Congenital alloimmune (neonatal) iron overload (rare)

Source: Bacon BR, et al. Hemochromatosis and Iron Storage Disorders. In: Schiff ER, Maddrey WC, Sorrell MF (eds). Schiff ’s Diseases of the Liver, 11th edn, 2012, Wiley Blackwell. Reproduced with permission of John Wiley & Sons.

macrophages) with and without liver cell involvement (e.g., β-thalassemia, Figure 9.22) are also listed in the table for consideration in assessing many of these patients. Of note is that because of incomplete penetrance of the HFE gene mutation, the serum iron and ferritin values may

Figure 9.22  Hemosiderosis (Perl’s iron stain). Abundant hemosiderin is seen predominantly within the Kupffer cells with also some staining of the hepatocytes in a liver biopsy from a patient with β-thalassemia trait.

not be diagnostic in a particular case, bringing up these other differential options for consideration.

Copper Storage Disorders Copper Metabolism

Copper is an essential trace metal with a unique redox nature that permits electron transfer reactions in numerous critical pathways including cellular respirations, iron homeostasis, pigment formation, production of neurotransmitters, peptide biosynthesis, and defense against anti-oxidants. Its potent reactivity also accounts for its toxic nature when the cellular pathways are disturbed. The human body contains approximately 100  mg of copper that is disbursed to multiple organ systems that include the liver (20 mg), red blood cells and plasma (10  mg), brain (20  mg), skeletal muscle (35 mg), connective tissue (10 mg), and kidney (5 mg). The average daily intake in the Western diet is about 4–6  mg, about 25–60%

Copper Storage Disorders   177

absorbed through the stomach and duodenum and rapidly extracted from the portal circulation into the liver bound to albumin and various amino acids such as histidine and various ligands. The non-absorbable copper or copper bound within the enterocytes that are shed into the small intestinal lumen is excreted through the feces. The absorbed copper then undergoes reduction to the cuprous form and enters the hepatocytes by the cell surface transmembrane transporters hCtr1 and hCtr2. The intracellular copper interacts with ligands such as glutathione and metallothionein for copper transfer and storage. Within 24 hours 10% of this copper appears in the peripheral blood bound to the glycoprotein ceruloplasmin that contains about 95% of the plasma copper, with trace amounts free and dialyzable. This protein is synthesized in the liver and secreted as the holoprotein with six atoms of copper incorporated into each protein molecule. The copper remaining in the liver is stored as hepatocuprein (copper-binding protein) with little free copper present. Additionally copper chaperones play a role in delivering copper to specific sites within the liver cell and in part help protect the hepatocyte by scavenging the toxic free radical superoxides (O2–) that are generated during aerobic metabolism. More than 80% of the copper is normally excreted in the bile. The main excretory pathway is by way of the biliary system where the amount of copper appearing in the bile is directly proportional to the size of the pool of hepatic copper. This biliary copper does not undergo enterohepatic recycling and is excreted into the feces. The regulation of bile excretion is by way of the copper-transporting ATPase (ATP7B), encoded by the ATP7B gene located on chromosome 13q-14.3, that is abundantly expressed in liver cells and localized to the trans–Golgi network. As the amount of hepatic copper increases, the ATPase moves from this network to a cytoplasmic vesicular lysosomal compartment near the canalicular membrane where copper is excreted into the bile. The ATPase then moves back to the trans-Golgi network after the cytoplasmic copper concentrations decreases, hence

maintaining copper homeostasis. Mutations of the ATP7B gene lead to a reduction of proper copper transport and excretion and are responsible for Wilson disease. Wilson Disease Epidemiology and Gene Mutations

Wilson disease is a genetically transmitted autosomal recessive liver disorder due to ATP7B gene mutations (see discussion on Copper metabolism earlier). Over 500 of these mutations have been identified in Wilson disease, with resulting impairment in copper secretion into bile and impairment in copper incorporation into ceruloplasmin. This results in toxic levels of free intracytoplasmic copper in the hepatocyte and eventual vast accumulations of copper into multiple organ systems. The incidence of the homozygous disease is about 1 : 30,000 with a worldwide distribution; however, because numerous disease-specific mutations of the gene occur, the most common mutations are present in only 15–30% of most case studies. Therefore most patients are compound heterozygotes that possess different mutations on each ATP7B allele. Clinical Presentation

Copper accumulates in the liver during childhood; however clinical symptoms rarely occur before age 4–5 years. After the storage of copper in the liver is exceeded, circulating non-ceruloplasmin-bound copper levels increase with copper distributing into the brain (mainly the basal ganglia) and other organ systems. Therefore, the first clinical manifestations are hepatic in children presenting before age 10 years. Between 10 and 18 years about half present with either hepatic or neurologic symptoms (e.g., tremors, gait abnormalities, dystonia, athetosis or chorea, pseudoparkinsonism, cognitive impairment, neuropsychiatric symptoms) and disabling muscle spasms (contractures, dysarthria). After 18 years of age about 75% of the patients present with neuropsychiatric abnormalities.

178    9  Genetic and Metabolic Hepatic Diseases

The deposition of copper into the cornea, occurring after hepatic copper saturation, causes characteristic ophthalmic manifestations, although no definite visual impairment occurs (1) Kayser–Fleischer rings, a green to golden-brown granular copper–protein pigment located in the Descemet membrane of the upper and lower poles of the cornea and best identified by slit-lamp examination, and less commonly (2) “sunflower” cataracts, a copper– protein complex deposited into the anterior and posterior lens capsule with a radiating centrifugal extension. Both ophthalmologic complications are reversible with effective treatment. Other organ systems that are involved besides the brain and liver are the kidneys (renal tubular defects, nephrocalcinosis, hematuria, aminoaciduria), joints (arthritis, arthralgias), red blood cells (non-immunopathic hemolytic anemia), endocrine system (dysfunction affecting the pituitary gland, gonads, and parathyroid glands), pancreas (pancreatitis), and heart (cardiomyopathy, arrhythmias). Patients with liver disease can present with either an acute hepatitis sometimes with a fulminant course or a chronic hepatitis that leads to cirrhosis. The acute hepatitis is the presenting sign in about a quarter of cases with clinical signs similar to an acute viral hepatitis, although the aminotransferases are usually only mildly to moderately elevated (usually below 500 IU/L with aspartate transaminase [AST] predominating). A mild Coombs-negative hemolytic anemia and decrease in serum uric acid levels can occur. A fulminant course infrequently develops, with jaundice, severe coagulopathy and renal failure with a very high mortality necessitating liver transplantation; however, again the aminotransferase values are usually below 500  IU/L. The Coombs-negative hemolysis is due to rapid release of copper into the blood. Free serum copper values may be elevated because of the massive release of copper from the damaged hepatocytes. Interestingly the alkaline phosphate values are often depressed, with the alkaline phosphatase: bilirubin ratio of 2.2 in this acute phase is virtually diagnostic for Wilson disease. About one-third of the patients may present with a chronic hepatitis that generally occurs during adolescence or young adults, with hepatosplenomegaly, ascites and signs of cirrhosis, the latter almost always present in the adult patient population. Hepatocellular carcinoma is a rare complication. A number of laboratory tests for Wilson disease also aid in the diagnosis (Table 9.3). Of note is that the measurement of copper in liver biopsy specimens when cirrhosis is present is not always useful in certain instances because of the variable distribution of copper from one regenerative nodule to another, and the fact that in the cirrhotic stage the biopsy used for copper determination may be composed predominantly of fibrous tissue and not hepatocytes. Therefore a routine H&E stain should also be prepared at the same time as the tissue is being processed for copper quantitation to assure an adequate biopsy specimen. Pathology

The liver in the earlier stage of the disease shows fairly non-specific features that include mild portal lymphocytic infiltrates with normal bile ducts, mild steatosis, mild lobular necroinflammatory change, and occasional glycogenated nuclei of hepatocytes. Lipochrome pigment composed of large clumped and often vacuolated granules can sometimes be seen in periportal hepatocytes. Portal and sinusoidal fibrosis to variable degrees may be present. Eventually a bridging fibrosis develops with formation of a macronodular or a micronodular cirrhosis (Figure 9.23). Cholestasis with variable necroinflammatory changes, steatosis, and glycogenated nuclei are seen (Figures 9.24 and 9.25), and Mallory–Denk bodies within periseptal hepatocytes can also be seen in a large minority of cases in the cirrhotic stage disease. At any stage of the disease the liver cell nuclei can sometimes show variable but sometimes

Copper Storage Disorders   179

Table 9.3  Wilson disease, laboratory values Laboratory test

Normal range

Serum ceruloplasmin

20–50 mg/dL

Serum copper, total (ceruloplasmin-bound and non-ceruloplasminbound [free]) Serum copper, free (nonceruloplasmin-bound)

80–120 μg/dL Low

8–12 μg/dL

>25 μg/dL

24 hr urinary copper secretion

100 μg/24 hr

Elevated values to lesser degrees in chronic liver diseases, proteinuria, urinary loss of ceruloplasmin

Hepatic copper (quantitation)

250 μg/g dry weight in homozygous patients

Elevated but 1000 IU/L) Positive viral/autoimmune serologies Pathology: portal plasma cells predominate (autoimmune hepatitis)

Mechanical long-term bile duct obstruction

Bile ductular proliferation Cholestasis Rarely Mallory–Denk bodies Cirrhosis

Choledocholithiasis Duct dilatation/strictures (ERCP) Pathology: bile duct ectasia, periductal fibrosis and edema, acute cholangitis

Primary biliary cirrhosis

Mallory–Denk bodies Cirrhosis Increase in stainable copper and copper-binding protein

AMA M2 positive Elevated IgM and alkaline phosphatase Pathology: non-suppurative duct damage, duct loss, granulomas

Primary sclerosing cholangitis

Mallory–Denk bodies Cirrhosis Increase in stainable copper and copper-binding protein

Elevated alkaline phosphatase Atypical ANCA positive Multifocal duct dilatations/strictures (ERCP) Pathology: fibro-obliterative destructive cholangitis, duct loss

Paucity of duct syndrome

Increase in stainable copper and copper-binding protein

Arteriohepatic dysplasia, Alagille syndrome Clinically presents in first 3 months of life No biliary excretion on HIDA scan Pathology: interlobular bile duct loss

Indian childhood cirrhosis

Mallory–Denk bodies Sinusoidal fibrosis Micronodular cirrhosis Increase in stainable copper and copper-binding protein

Cirrhosis with liver failure by age two (range 1–5 years), occurring predominantly in children in India, usually of the Brahmin class Striking increase in hepatic copper (ingestion of milk boiled in brass and other copper cooking vessels, the milk a carrier source of the copper which is then absorbed in the small intestinal mucosa) Pathology: similar features although the sinusoidal fibrosis, Mallory–Denk bodies are much more striking in Indian childhood cirrhosis

Disorder

(continued)

182    9  Genetic and Metabolic Hepatic Diseases

Table 9.4  (Continued) Histologic features similar to Wilson disease

Clinical and histologic features differing from Wilson disease

Alcoholic liver disease (active drinker)

Steatosis Mallory–Denk bodies Sinusoidal fibrosis Micronodular cirrhosis

AST : ALT ratio 2–3 : 1 Leukocytosis, hepatic bruit Pathology: perivenular fibrosis, liver cell ballooning, prominent lobular neutrophils with “satellitosis” around liver cells containing Mallory–Denk bodies

Non-alcoholic steatohepatitis

Metabolic syndrome Steatosis Pathology: perivenular fibrosis, liver cell ballooning Mallory–Denk bodies Glycogenated nuclei of hepatocytes Sinusoidal fibrosis Micronodular cirrhosis

Disorder

in the differential have clinical and histologic features clearly distinguishing them from Wilson disease (e.g., paucity of duct syndrome). Clinical correlation with performance of pertinent laboratory tests and hepatic copper quantitation is most often crucial, since appropriate treatment by (1) the removal of copper from potentially toxic sites with various chelating agents, and (2) the use of zinc salts in blocking intestinal absorption of copper as well as stimulating the biosynthesis of endogenous hepatic chelators, are most effective.

Hereditary Hyperbilirubinemias The hyperbilirubinemias are inherited autosomal recessive cholestatic liver disorders caused by the decrease or absence of the enzymes responsible for bilirubin conjugation. The various features with enzyme defects and liver pathology are summarized in Table 9.5, with Figure 9.30 and Figure 9.31 showing examples of Gilbert disease and Dubin–Johnson disease, respectively.

Table 9.5  Hereditary hyperbilirubinemias Syndrome

Inheritance

Gene

Gene function

Liver manifestations

Inability to conjugate bilirubin Crigler– Najjar type I

Autosomal recessive

Complete loss UGT1A (UDP of enzyme glucuronosyltransferase activity 1 family, polypeptide 1 secondary (2q37) to insertiondeletions, stop codons

Crigler– Najjar type II

Autosomal recessive

UGT1A1 (2q37)

Rare, conjugated hyperbilirubinemia with normal enzymes No bilirubin uridine diphosphoglucuronate glucuronsyltransferase activity Occurs in neonates with jaundice and risk for kernicterus Liver transplantation curative

Point mutations Unconjugated hyperbilirubinemia with milder bilirubin elevations that reduce Normal hepatic histology and liver but do not tests eliminate Occurs in neonates with usually a enzyme benign course without kernicterus activity

(continued)

Hereditary Hyperbilirubinemias   183

Table 9.5  (Continued) Syndrome

Inheritance

Gene

Gene function

Liver manifestations

Gilbert syndrome

Autosomal recessive

UGT1A1 (2q37)

Promotor variant that when homozygous results in 30% of normal enzyme activity

Benign course, presents in adolescence Common (4–16% of population) About half associated with hemolytic anemias Mild fluctuating unconjugated hyperbilirubinemia Jaundice precipitated by fasting, infection, alcohol, stress Normal liver enzymes Mild steatosis and mild increase in lipochrome pigment in perivenular and midzonal hepatocytes (see Figure 9.30)

Inability to transport bilirubin Seen worldwide, more common in the Mid-East (frequency 1 : 1300 of the population) Predominantly conjugated hyperbilirubinemia Routine liver enzymes Jaundice precipitated by pregnancy, oral contraceptive use, infection, trauma Liver dark brown to black on gross inspection (due to abundant lipochrome-like pigment) Dark brown and coarsely granular pigment within the perivenular and midzonal hepatocytes (see Figure 9.31) Good prognosis with no treatment necessary Normal urinary coproporphyrin but disproportionately high fraction of coproporphyrin I

DubinJohnson syndrome

Autosomal recessive

ABCC2 (ATP-binding cassette, subfamily C, member 2) (10q24)

Absence of organic anion transporter (CMOAT) present on canalicular liver cell membrane

Rotor syndrome

Autosomal recessive

SLCO1B1 and SLCO1B3 (solute carrier organic anion transporter family, member 1B1 and 1B3) (12p12)

Predominantly conjugated Defect in hyperbilirubinemia organic anion Normal liver enzymes transporter Increase in urinary coproporphyrin involved in bilirubin re-uptake

Source: Data in part from Edmondson HA, Peters R. Liver. In: Kissane JM (ed.) Anderson’s Pathology, 8th edn, 1985, C.V. Mosby, p 1171; and Kanel GC and Korula J. Atlas of Liver Pathology, 3rd edn, 2011, Elsevier, p 218.

184    9  Genetic and Metabolic Hepatic Diseases

Figure 9.30  Gilbert syndrome. Golden-brown lipochrome pigment within the hepatocytes is seen.

Figure 9.31  Dubin–Johnson syndrome. A dark brown coarsely granular pigment within the perivenular hepatocytes is present.

Familial Forms of Intrahepatic Cholestasis (Variants of Progressive Familial Intrahepatic Cholestasis

glycogen synthesis in the liver. Each has its own genetic enzyme defects. Some of the glycogen storage diseases are associated with only mild liver test abnormalities while others can lead to chronic liver disease and cirrhosis. Although accumulation of the glycogen in the liver is constant, other organs such as skeletal muscle, kidney, heart, brain, erythrocytes, and intestine may also be involved. Table 9.7 summarizes these metabolic disorders with pertinent clinical and laboratory data, with Figures 9.36 and 9.37 showing an example of type III disease. The diagnoses rest upon the assessment of the specific genetic enzyme defect. Lafora disease (myoclonus epilepsy), an autosomal recessive disorder, is a fatal neurodegenerative disease associated with myoclonic seizures (myoclonus epilepsy), ataxia, and severe dementia. The disease usually becomes manifest in children in their early teens, with a fatal outcome by 25 years of age or about 10 years after the symptoms first occur. It is caused by a mutation in one of the two known genes on chromosome 6: EPM2A on 6p24 that encodes for the protein laforin, and EPM2B on 6p22 that encodes for the protein malin. Laforin normally dephosphorylates glycogen, preserving its water solubility, and malin binds to and inactivates glycogen synthase, both therefore inhibiting

Progressive familial intrahepatic cholestasis (PFIC) is a group of rare autosomal recessive inherited disorders associated with defects in proteins responsible for proper bile transport and excretion (PFIC1 and PFIC2) and phospholipid secretion (PFIC3). It is estimated that about 1 : 50,000 to 1 : 100,000 people are affected worldwide with PFIC type 1 which is more common in Greenland and the Amish population in the United States. Usually the liver diseases are progressive leading to a biliary cirrhosis, with paucity of ducts sometimes an occurrence in PFIC type 1 disease. Table 9.6 summarizes the various features of the three PFIC variants, with Figures 9.32, 9.33. 9.34, and 9.35 showing examples of PFIC type 1 disease.

Storage Diseases Carbohydrate Metabolism Storage Diseases

Glycogen storage diseases are a group of inherited disorders associated with abundant and excessive accumulation of glycogen or abnormal

Clinical

Liver pathology

Gene mutation leads to MDR3 altered phospholipid (multidrug transport into canaliculi resistant with formation of protein 3) phosphatidylcholine-poor bile (destabilizing micelles and promoting lithogenic bile formation)

ABCB4 7q21

PFIC type 3

Source: Data in part from Balistreri WF, et al. Whatever happened to “neonatal hepatitis”? Clin Liver Dis 2006;10:27–53.

Portal lymphocytic infiltration, bile Onset of jaundice and liver ductular proliferation, biliary fibrosis disease early in life that may lead to cirrhosis May be diagnosed in adulthood Serum γ-GTP elevated Cholestasis during pregnancy

Gene mutation leads to BSEP (bile deficiency of canalicular salt export BSEP expression pump)

ABCB11 2q24

PFIC type 2 (benign recurrent intrahepatic cholestasis [BRIC] type 2)

Marked lobular inflammation, syncytial giant cells of hepatocytes, cirrhosis in infancy without Byler bile Paucity of ducts may occur Bile amorphous, filamentous, MalloryDenk bodies may occur Rare complications of hepatocellular carcinoma, cholangiocarcinoma Infancy with pruritus, jaundice, failure to thrive Hepatosplenomegaly Serum γ-GTP normal or low

FIC 1

PFIC type 1 Jaundice, severe pruritus, Mechanism unknown ●● Cholestasis without watery diarrhea, hearing Loss of FIC1 may lead impairment, short to decreased nuclear necroinflammatory change (see stature, pancreatitis with translocation of FXR Figure 9.32) ●● Canaliculi filled with pale-appearing pancreatic insufficiency, (farnesoid X receptor, failure to thrive, liver an important modifier loose coarsely granular bile (Byler’s failure in first 6 months of bile acid homeostasis) bile) ●● Decrease in interlobular bile ducts of life with alterations in hepatic bile acid transporter gene Serum γ-GTP normal or low (see Figure 9.33), biliary fibrosis/ “Benign” variant (BRIC type 1): expression cirrhosis a milder form of PFIC BRIC type 1 ●● “Benign” variant with perivenular beginning in childhood with intermittent jaundice, cholestasis and mild lobular pruritus inflammation (see Figure 9.34), mild portal lymphocytic infiltrates (see Figure 9.35) with occasional eosinophils without bile duct abnormalities, absent to minimal portal fibrosis

Alteration in function

ATP8B1 18q21-22

Protein Mutated gene deficiency

PFIC type 1 (Byler disease); PFIC type 1 variant (benign recurrent intrahepatic cholestasis [BRIC] type 1)

Disorder

Table 9.6  Familial forms of intrahepatic cholestasis (variants of progressive familiar intrahepatic cholestasis, PFIC)

186    9  Genetic and Metabolic Hepatic Diseases

Figure 9.32  Progressive familial intrahepatic cholestasis (PFIC) type 1 (Byler disease). The parenchyma shows cholestasis without necroinflammatory change.

Figure 9.34  Progressive familial intrahepatic cholestasis (PFIC) type 1 variant (benign recurrent intrahepatic cholestasis). The parenchyma shows cholestasis with Kupffer cell hyperplasia and minimal lobular inflammation.

polyglucosan accumulation. The gene mutation leads to excessive deposition of these products in the form of insoluble hyperphosphorylated polyglucosans that appear as “Lafora” bodies that can be seen within neurons, myocardial fibers, skeletal muscle, skin, and liver cells. Patients may have slightly abnormal liver tests but liver function is not impaired, with the diagnosis confirmed by identifying the polyglucosan inclusions within the apocrine sweat glands on axillary skin biopsies and by genetic testing and DNA sequencing. Liver biopsy shows discrete

round to oval eosinophilic intracytoplasmic inclusions (branched polyglucosan) within the periportal hepatocytes (Figure 9.38), the inclusions demonstrating positive staining by the colloidal iron and periodic acid–Schiff (PAS) stains. Galactosemia is an autosomal recessive disorder whereby a mutation in galactose-1-phosphate uridyl transferase (GALT) results in decreased activity of the enzyme and resultant elevated circulating galactose values, with an abnormal accumulation of galactose

Figure 9.33  Progressive familial intrahepatic cholestasis (PFIC) type 1 (Byler disease). The portal tract shows an absence of an interlobular bile duct.

Figure 9.35  Progressive familial intrahepatic cholestasis (PFIC) type 1 variant (benign recurrent intrahepatic cholestasis). The portal tract exhibits a mild lymphocytic infiltrate with normal interlobular bile ducts.

Enzyme defect Glycogen sythase

Glucose-6-phosphatase

Glucose 6-phosphate T1 translocase

Lysosomal acid γ1–4 and γ1–6 glucosidase

Type

0 (Lewis)

Ia (von Gierke disease)

Ib (type I non-a)

II (Pompe)

Table 9.7  Glycogen storage diseases (GSD)

Fasting hypoglycemia, elevated blood ketones, elevated free fatty acids, lactic acidosis

Laboratory tests

Infantile type with severe generalized hypotonia of skeletal and cardiac musculature, macroglossia, progressive weakness, renal failure, usually death within first year Milder forms arising in childhood (juvenile type) and adults, with varying weakness, myalgias, sometimes respiratory insufficiency

Hepatomegaly, recurrent infections, inflammatory bowel disease-like lesions, oral ulcerations, growth retardation

Steatosis Hepatocellular adenoma, hepatocellular carcinoma may occur

Glycogen accumulation within nuclei and cytoplasm Pale cytoplasm with mosaic appearance Macrovesicular steatosis frequent Peliosis hepatis, hepatocellular adenoma, Mallory–Denk bodies, hepatocellular carcinoma may occur

Steatosis (decrease in hepatic glycogen stores)

Liver pathology

(continued)

Elevated creatine kinase Glycogen accumulation within lysosomes with cytoplasmic vacuoles Steatosis may be seen

Hypoglycemia, lactic acidosis, hyperuricemia, neutropenia, neutrophil dysfunction, hyperlipidemia

Severe hypoglycemia, Most common GSD lactic acidemia, Massive hepatomegaly without splenomegaly, hyperuricemia, short stature, bleeding episodes (platelet neutropenia, dysfunction), cherubic appearance in infants hypertriglyceridemia, (excessive fat in face and trunk), thin arms hypercholesterolemia and legs, stunted growth, xanthomas Enlarged kidneys with nephrocalcinosis, focal segmental glomerulonephritis, uric acid nephropathy Recurrent infections

Fatigue, muscle cramps after exertion Growth restrictions, seizures

Clinical presentation

Hepatic phosphorylase b kinase ●● Mutations in X-linked PHKA1, PHKA2 genes ●● Mutations in autosomal recessive PHKB, PHKG2 genes GLUT2 (glucose transporter)

IX

XI (Fanconi–Bickel)

Hepatomegaly, rickets, stunted growth

X-linked form with hepatomegaly, growth retardation Advanced liver disease with cirrhosis in nonX-linked form

Hypoglycemia, hypergalactosemia, renal tubular acidosis

Elevated cholesterol, triglycerides

Glycogen accumulation in hepatocytes

Glycogen within cytoplasm Portal fibrosis may occur; cirrhosis reported

Glycogen within cytoplasm, nuclei Steatosis may occur Portal fibrosis, rarely cirrhosis

Distinct eosinophilic intracytoplasmic inclusions in periportal hepatocytes, the inclusions DiPAS positive (amylopectin-like material) Fibrosis, cirrhosis occurs

Glycogen within nuclei and cytoplasm (see Figure 9.36) Mild steatosis Portal fibrosis, cirrhosis may occur (see Figure 9-37) Adenomas in up to onequarter of cases Hepatocellular carcinoma rare

Liver pathology

Type VIII (hepatic phosphorylase-b-kinase deficiency) now reclassified as a subtype of GSD type VI. Source: Data from Sundaram SS et al. Metabolic diseases of the liver. In: Podolsky DK (ed). Yamada’s Textbook of Gastroenterology, 6th ed, 2016, Wiley Blackwell, pp 2023–2042; Sokol RJ. Metabolic diseases of the Liver. In: Yamada T (ed). Textbook of Gastroenterology, 5th ed, Wiley Blackwell, pp 2223–2246; Thompson RJ et al. Genetic and metabolic liver disease. In: Burt A, Portmann B, Ferrell L (eds). MacSween’s Pathology of the Liver, 6th edn, 2012, Elsevier, pp157–259.

a

Hepatic phosphorylase E

VI (Hers) (VIII)a

Mild hypoglycemia, hyperlipidemia

Progressive liver injury, cirrhosis, liver failure, Hyperbilirubinemia, hypoglycemia, coagulopathy, hepatosplenomegaly, portal hypoalbuminemia hypertension, hypotonia, cardiomyopathy with cardiac failure, death usually within first 5 years Chronic liver disease without fibrosis may occur Abnormal neuromuscular development, muscle atrophy, absent deep tendon reflexes Adults with milder symptoms but similar skeletal muscle abnormalities

Amylo-1,4 → 1,6 glycosyltransferase (glycogen “branching” enzyme)

IV (Anderson)

Relatively benign disease Hepatomegaly, growth retardation, with improvement with age Adults usually asymptomatic

Hypoglycemia, Type IIIa involving liver, skeletal muscle; IIIb lactic acidosis, involving only liver (minority of patients) hyperlipidemia, Hepatomegaly, portal hypertension, hyperuricemia hypotonia, cardiomyopathy, infections, metabolic abnormalities, growth retardation Consequences of portal hypertension in cirrhotics

Amylo-1,6 glycosidase (glycogen “debranching” enzyme)

Laboratory tests

III types a and b (Forbes–Cori)

Clinical presentation

Enzyme defect

Type

Table 9.7  (Continued)

Storage Diseases   189

Figure 9.36  Glycogen storage disease type III. Numerous glycogenated nuclei of hepatocytes are seen.

in the liver. The genetic defect is located on chromosome 9q13, with the incidence approximately 1 : 45,000 births. Patients develop failure to thrive, vomiting, diarrhea, mental cognitive disturbances, an enlarged liver, and cataracts. Liver damage develops from the toxic effects of galactose with the clinical manifestations occurring after the ingestion of milk products. Urinary tract infections are also complications. Liver pathology by about 1 week after the clinical onset includes both macrovesicular and microvesicular steatosis with marked bile ductular reaction, these cholangioles sometimes containing bile plugs. By 2–6 weeks,

Figure 9.38  Lafora disease. Distinct intracytoplasmic inclusions within the hepatocytes are seen, the inclusions representing “Lafora” bodies (insoluble hyperphosphorylated polyglucosans).

pseudoacinar changes can occur within the lobules that may contain bile or eosinophilic material. Giant cell transformation of hepatocytes has also been described. Fibrosis and cirrhosis can occur. Hereditary fructose intolerance is an autosomal recessive disorder caused from a deficiency in fructose-1-phosphate aldolase with resultant accumulation of fructose-1-phosphate in the liver. The defect is located on chromosome 9q22 (aldolase gene). The incidence is approximately 1 : 20,000 births with over 25 mutations known. Symptoms develop after fructose is ingested in the diet from fruit juices or formulas containing sucrose, whereby the infants develop vomiting, seizures, diarrhea, hypoglycemia, renal tubular defects, and failure to thrive. Hepatomegaly and hyperbilirubinemia may occur. Elevated levels of fructose in the serum and urine are present. Liver biopsy shows giant cell transformation of hepatocytes with bile ductular proliferation, the ductules often containing inspissated biliary concretions. Macrovesicular steatosis can occur, and fibrosis leading to cirrhosis can develop. Gaucher Disease Epidemiology and Gene Mutations

Figure 9.37  Glycogen storage disease type III (trichrome stain). A well-established cirrhosis may also develop.

Gaucher disease is one of the most common inherited autosomal recessive glycolipid storage disorders resulting in the accumulation of

190    9  Genetic and Metabolic Hepatic Diseases

glucocerebrosides (glucosylceramide) within the lysosomes of Kupffer cells of the liver as well as within the lymph nodes, spleen, bone marrow, and leukocytes as well as brain (accumulation within myelin sheaths) secondary to a deficiency in acid β-glucosidase (glucocerebrosidase). This enzyme is a 55.6  kDa 497 amino acid protein that normally catalyzes the breakdown of glucosylceramide, a cell membrane constituent of red blood cells and leukocytes. A defect in this enzyme prevents macrophages that normally clear these cells from breaking down these byproducts, resulting in the intracellular accumulation of this material (termed “Gaucher” cells). The gene encoding acid β-glucosidase is located on chromosome 1q21, but numerous mutations have been identified (such as N370S and L444P alleles). The disease affects both males and females. The overall incidence is approximately 1 : 50,000 to 1 : 100,000 live births, but is much more common in the Ashkenazi Jewish population (Gaucher type 1) where the disease incidence ranges from 1 : 450 to 1 : 800 with a carrier frequency of 1 : 10. The diagnosis is confirmed by assay for the acid β-glucosidase gene mutation in white blood cells or cultured fibroblasts. Clinical Presentation

The clinical disease is due to the prominent deposition of the glucocerebrosides within macrophages within numerous organ systems but primarily within the reticuloendothelial system. The involved macrophages have a very characteristic morphologic appearance. Three major phenotypic variants of the disease occur: ●●

Type 1: The most common of the variants and responsible for almost 95% of all cases of Gaucher disease, this form is a relatively benign subtype that may occur at any age. Hepatosplenomegaly, osteoporosis with bone fractures and bone pain, aseptic necrosis, spontaneous fractures, various skin pigmented lesions, easy bruising, anemia, and thrombocytopenia often develop. Pulmonary and

●●

●●

renal impairment may also occur; however, some patients may have almost no symptoms. Neurologic manifestations are not features. Liver function is not impaired with the long-term outcome favorable, although very rare cases of fulminant liver cell necrosis have been described. Type 2: This very rare acute infantile neuropathic form of the disease is seen in any ethnic group and is associated with marked hepatosplenomegaly, lymphadenopathy, hypertonia, neurologic abnormalities, eye movement disorders, spasticity, seizures, limb rigidity, and failure to thrive. Death usually occurs by 2 years of age. Type 3: This chronic form of the disease is seen more often in the northern Swedish province of Norrbotten and presents in childhood with hepatosplenomegaly. Three variants occur. Type 3a is associated with progressive neurologic degenerative changes, myoclonus and dementia. Type 3b is early onset with marked hepatosplenomegaly with complications of portal hypertension (esophageal varices), pulmonary involvement, progressive bone disease, and severe skeletal muscle abnormalities. Type 3c is associated with mitral and aortic valve calcifications (Gaucher cells in the heart valves), oculomotor apraxia, corneal deposits, chronic liver disease with portal hypertension, and rarely cirrhosis. Patients with type 3 disease often live into the early teens and adulthood.

Pathology

The characteristic findings on liver biopsy are enlarged (up to 100  μm) hypertrophic Kupffer cells and portal macrophages that have a striated wrinkled appearance to the cytoplasm due to deposition of glucosylceramide (Figure 9.39), these striations enhanced by Masson trichrome and PAS after diastase digestion stains. These involved cells also are positive on immunohistochemical staining for KP-1 antibody and CD68. In addition the acid phosphatase, which is markedly increased in serum in Gaucher disease, can also be demonstrated within the

Storage Diseases   191

Figure 9.39  Gaucher disease. The plump aggregate of Kupffer cells exhibits a striated wrinkled appearance to the cytoplasm secondary to the deposition of glucosylceramide.

enlarged Kupffer cells by enzyme histochemical staining for acid phosphatase on frozen section biopsy material. These enlarged Kupffer cells can be present anywhere within the lobules although accentuation in the perivenular zones sometimes occurs. Portal fibrosis can also develop over time with progression to cirrhosis of a micronodular type a rare occurrence. Niemann–Pick Disease Epidemiology and Gene Mutations

Niemann–Pick disease is a group of autosomal recessive lysosomal storage disorders (sphingolipidoses) manifested by excessive accumulations of lipid within various organ systems. Niemann–Pick disease affects all segments of the population worldwide. Three subgroups are identified (Niemann–Pick types A, B, and C). There are approximately 1200 cases of types A and B disease worldwide, the incidence estimated at 1 : 250,000 individuals; however, type A disease occurs more frequently among individuals of Ashkenazi (eastern and central European) Jewish descent, with the incidence within the Ashkenazi population approximately 1 : 40,000. The types A and B disorders are secondary to missense mutations in the sphingomyelin phosphodiesterase 1 gene (SMPD1) located

on chromosome 11p15.4, leading to deficiency of acid sphingomyelinase and the resultant accumulation of sphingomyelin within the lysosomes of tissues from various organ systems. The incidence of type C is estimated to be 1 : 150,000, with an estimate of about 500 cases diagnosed worldwide, the disease more frequently seen in individuals of French-Canadian descent in Nova Scotia. Type C is associated with mutations in the NPC1 gene located on chromosome 18q11-12 that encodes an endoplasmic membrane glycoprotein and is associated with the accumulation of unesterified cholesterol and other lipids within the lysosomes. Clinical Presentation

Distinguishing features occur with each of the phenotypic variants. Type A is the most common type (∼85% of cases), occurring in neonates and infants with marked hepatosplenomegaly, jaundice, macular cherry red spots (50% patients), hypotonia, ataxia, dysphagia, and death usually by 2–4 years. Type B presents later on in childhood and is associated with hepatosplenomegaly, growth retardation, and pulmonary abnormalities due to deposition of foam cells. Macular cherry red spots occur in 300 μm Type 3: ducts incompletely lined by columnar epithelium, with numerous small ducts and glandular structures within a loose fibroconnective tissue stroma.

More than one subtype can occur within each duct at the same time at different foci. In addition the extrahepatic ducts as well as the large intrahepatic hilar ducts may also contain inspissated bile. Figure 10.6  Extrahepatic biliary atresia. The parenchyma shows cholestasis and syncytial giant cell formation of some of the hepatocytes.

hepatocytes occurring in about a quarter of cases (Figure 10.6). As the disease progresses both hepatocytes and hyperplastic Kupffer cells may have a xanthomatous appearance, and bile lakes, bile infarcts, and sometimes Mallory–Denk bodies can be seen and are more prominent in the periportal and periseptal zones. Increase in hepatic copper (rubeanic acid, rhodanine stains) and copper-binding protein (orcein stain) is also present within the periportal and periseptal liver cells. Importantly the numbers of interlobular bile ducts begin to decrease towards the fourth to fifth months and can progress so that by 9 months the interlobular bile ducts may be totally absent. The cause may be due to a number of factors that include recurrent duct inflammation and damage, an immune-mediated response targeted to the ducts, and a complication of biliary surgery with secondary inflammation and scarring. The extrahepatic bile ducts and the ducts at the hepatic hilum may show any of the following histologic features: ●●

●●

Type 1: duct loss without an accompanying inflammatory infiltrate Type 2: small irregularly shaped ducts with a cleft-like lumen of 2 cm) and at a right angle, affecting the normal sphincter function at the pancreaticobiliary junction. Because the secretory pressure of the pancreas is higher than that of the liver, there is reflux of the pancreatic fluid into the bile duct system in the absence of a sphincter mechanism, resulting in duct damage with recurrent cholangitis, fibrosis with bile duct obstruction, and possibly the development of neoplasms. The persistent reflux allows

206    10   Developmental Hepatobiliary Disorders and Cystic Diseases

Table 10.2  Developmental and congenital cystic diseases of the intra- and extrahepatic biliary system Type (% of total)

Features

I (75–85%)

Dilatation of common bile duct (choledochal cysts) ●● Localized (A) ●● Segmental large saccular dilatation (B) ●● Diffuse, fusiform or cylindrical dilatation (C)

II (2–3%)

Diverticulum cysts Extrahepatic ducts including gallbladder

●●

III (1.4–5.6%)

Choledochocele cysts Cystic dilatation involving the intra-duodenal segment of the common bile duct: –– Type A: ampulla opens into the cyst which communicates with the duodenum through a small opening (majority of cases) –– Type B: ampulla opens directly into the duodenum with the cyst communicating only with the distal common bile duct (minority of cases)

●●

IV (IVA 10–15%, IVB rare) V (rare)

Type IVA: multiple intrahepatic and extrahepatic duct cysts Type IVB: multiple extrahepatic bile duct cysts Intrahepatic fusiform bile duct cysts, single or multiple (Caroli disease) Simple type (associated with medullary sponge kidney in two-thirds of cases) ●● Complex periportal type (Caroli syndrome: hepatic fibrosis, portal hypertension, esophageal varices, other cystic developmental abnormalities) ●●

VI (very rare)

Dilatation of the cystic duct alone

 ource: Suh JW, et al. Cystic diseases of the liver and biliary tract. In: Yamada T (ed.) Textbook of Gastroenterology, 5th edn. S Wiley Blackwell, 2009, pp 2009–21. Reproduced with permission of John Wiley & Sons.

activation of various pancreatic proteinases and lipases that can enhance the duct injury. Choledochal Cyst

Choledochal cysts account for up to 85% of all cases of congenital cystic diseases. These lesions

can clinically present at any age although about 20% occur during the first year of life and about two-thirds before year 10. About 2% of all infants who present with jaundice have choledochal cysts. In infancy jaundice with or without acholic stools is the most common presentation and occurs in about 80% of patients with choledochal cysts,

Figures 10.12 and 10.13  Intrahepatic fusiform bile duct cysts (Caroli disease). Portal fibrosis is present with numerous ectatic biliary ducts and small cysts. A mild lymphocytic infiltrate is seen within the fibrotic portal structures.

Fibrocystic Diseases   207

mimicking biliary atresia. Pain is sometimes also present and failure to thrive can occur in up to half of the patients. Hepatomegaly with a palpable abdominal mass is seen in one-third to two-thirds of patients. Older patients (greater than 2 years but can occur in the adult) can be asymptomatic or develop intermittent pain and jaundice sometimes associated with cholangitis, sepsis and hepatic abscess formation, and pancreatitis. The cysts on gross examination can vary in size with the larger cysts containing greater than 5 L of bile. The cysts may be saccular or fusiform, with the mucosa composed of flattened bile duct epithelium that is often denuded and ulcerated. The cyst wall may exhibit a mild acute and chronic inflammatory infiltrate, and the peribiliary glands located within the duct wall can show cylindrical to cuboidal epithelium, intestinal metaplasia, goblet and Paneth cells, and neuroendocrine differentiation. Changes in the liver often occur due to obstruction of bile flow and include bile duct and ductular proliferation, acute cholangitis, cholestasis, and with time biliary fibrosis and even biliary cirrhosis (Figures 10.14 and 10.15). Cholangiocarcinoma of these choledochal cysts is a complication and is seen almost exclusively in those older than 10 years (3% incidence overall, rising to about 14% in older patients). Because of this severe complication, complete surgical resection of the cyst with hepaticojejunostomy is a

Figure 10.15  Choledochal cyst. High power shows the cyst lining to be composed of a single layer of cuboidal duct epithelium.

major therapeutic approach. Of note is that there is an increased risk in developing carcinomas not only within the cyst but also anywhere in the hepatobiliary tree including the pancreatic duct.

Fibrocystic Diseases Fibrocystic liver diseases are a heterogeneous group of hereditary disorders associated with a spectrum of histologic hepatic changes that include (1) dilatation of the intrahepatic bile ducts, (2) the development of ductal plate malformations, and (3) microscopic and macroscopic cysts most associated with cystic diseases of the kidneys. In the neonatal disorders the renal disease far outweighs the liver disease, while in the later onset disorders the liver disease with portal hypertension becomes more apparent. The main disorders are autosomal recessive polycystic kidney disease (ARPKD) associated with infantile polycystic liver diseases including congenital hepatic fibrosis, and autosomal dominant polycystic kidney disease (ADPKD) associated with adult polycystic liver disease (PCLD) (Box 10.1). Simple Hepatic Cysts

Figure 10.14  Choledochal cyst. The image taken from the common bile duct shows extensive fibrosis surrounding the simple cyst wall. Adjacent pancreatic tissue is seen.

Simple non-parasitic solitary hepatic cysts are common and are usually incidental findings,

208    10   Developmental Hepatobiliary Disorders and Cystic Diseases

Box 10.1  Fibrocystic diseases of the liver Simple non-parasitic solitary hepatic cysts Peribiliary (retention) cysts Von Meyenburg complexes (biliary micro-hamartoma) Polycystic liver disease ●● Associated with autosomal recessive polycystic kidney disease (ARPKD) Polycystic liver disease Associated with autosomal dominant polycystic kidney disease (ADPKD) ●● Adult polycystic liver disease without renal involvement ●●

often initially detected during workup for other causes. They are seldom seen in the pediatric population but the incidence increases with age, usually discovered in adults between the fourth and sixth decades. The cysts are four times more common in women. Overall about 2.5–4% of the general population may have simple hepatic cysts by 70 years of age. The cysts can be either congenital, formed from abnormally developed bile ducts, or secondarily acquired.

cysts resemble those seen in PCLD. The cysts are lined by simple cuboidal duct epithelium that may also appear flattened in the larger cysts (Figure 10.16). Not uncommonly the cyst lining can be partially or totally sloughed. A thin rim of underlying fibroconnective tissue is present. Cholesterol clefts and foreign body granulomas can also at times be seen within the cyst wall or along the cyst lining. Rarely the cysts may be subcapsular and pedunculated.

Pathology

Differential Diagnoses

Usually the cysts are solitary although about a quarter of the patients may have two to three cysts. They are usually asymptomatic although the larger cysts may present with abdominal discomfort or back pain. Liver tests are normal although hyperbilirubinemia with jaundice can rarely occur in the larger cysts that impede proper bile flow in the adjacent compressed liver. Complications are few except for instances of superinfection of the cyst fluid or hemorrhage. Malignant transformation to a cholangiocarcinoma is extremely unusual. The cysts generally are round to oval, well circumscribed, and can vary from less than 1  cm to over 10  cm. The cysts usually contain a clear serous or mucoid fluid; however, purulent material and rarely bile can be seen when there is superinfection. The cysts are twice as common in the right lobe and are almost always unilocular. Calcifications in the cyst wall can also infrequently occur. Microscopically the

Biliary cystadenomas are multiloculated with papillary projections, the cyst wall containing other features such as collagen with smooth

Figure 10.16  Simple biliary cyst. The cyst lining is composed of a single layer of cytologically benign cuboidal to flattened duct epithelium. Underlying fibroconnective tissue with few scattered lymphocytes is seen.

Fibrocystic Diseases   209

muscle, fat, and in some instances a spindle cell (mesenchymal) stroma, these features not seen in simple biliary cysts. Hydatid cysts secondary to infection by Echinococcus species tapeworms are multiloculated cysts that differ from simple cysts by the presence of inner brood capsules containing the characteristic protoscolices. Cystic dilatation of intrahepatic ducts (Caroli disease) may sometimes appear as multiple simple cysts on routine imaging; however, a more detailed evaluation using CT scans and endoscopic retrograde cholangiopancreatography (ERCP) can best demonstrate the segmental cystic and saccular dilatations of the ducts seen in Caroli disease. Peribiliary Cysts

Both macroscopic and microscopic cysts can develop from the peribiliary glands that are normally present within the fibroconnective tissue in the hepatic hilum as well as within the wall of the major extrahepatic bile ducts. They are usually multiple and lined by columnar to cuboidal single-layered duct epithelium, with a thin rim of collagen surrounding the larger cysts (Figure 10.17). They are usually a few millimeters in diameter but can at times reach up to 2 cm and can be large enough to be seen on imaging. Usually the cysts are round to oval and contain a clear fluid but not bile, as these cysts do not appear to communicate with the normal

biliary system and are referred to as retention cysts. There is no definite increased risk for the development of cholangiocarcinoma in these cysts. Von Meyenburg Complex (Biliary Micro-hamartoma)

The von Meyenburg complexes (biliary microhamartomas) are collections of ectatic branching benign biliary ducts that are developmental abnormalities. They are often seen associated with other biliary cystic diseases such as adult polycystic disease but are not uncommon as incidental findings in liver biopsy material. Usually the lesions are microscopic although the larger lesions can reach diameters of ∼0.5  cm and appear as firm white “dots.” They are present within portal tracts but during growth they become disconnected from the bile ducts from which they are derived and hence are felt to represent an extension of the embryonal duct plate. These lesions are seen in about 5% of adults and 1% in children in autopsy series. Although they can be single, multiple complexes can occur at the same time. Importantly these complexes are frequently seen amongst the large cysts in PCLD. Microscopically numerous dilated round to oval and branching bile ductular complexes are present within a fibroconnective tissue stroma, and in about half of the cases bile or eosinophilic material is present within the lumen (Figures 10.18 and 10.19). The duct epithelium is composed of single-layered cuboidal cells. Between the ducts the fibrous tissue usually is devoid of an inflammatory infiltrate. With time some of the ducts within the complex become smaller with the surrounding fibrous tissue more dense, and infrequently the duct elements can be rare to absent, resembling in part a small scar or dense fibroma. Polycystic Liver Disease Autosomal Recessive Polycystic Kidney Disease Classification and gene mutations

Figure 10.17  Peribiliary cysts. Dilated large and small biliary cysts are seen in the hepatic hilum.

This autosomal recessive variant is rare, with an incidence of 1 : 10,000 to 1 : 40,000 live births, although the true incidence may be higher as

210    10   Developmental Hepatobiliary Disorders and Cystic Diseases

Figures 10.18 and 10.19  Von Meyenburg complexes (biliary micro-hamartoma). Numerous round to oval dilated and branching bile ductular complexes are seen, with bile also present in some of the ductular lumen.

severely affected neonates often die without a diagnosis. There is equal sex distribution. The disease is secondary to mutation of the PKHD1 (polycystic kidney and hepatic disease 1) gene (chromosome 6p 21.2-p12). The gene encodes fibrocystin, a large integral transmembrane protein that is found in the cortical and medullary renal collecting ducts and the hepatic bile ducts. Truncation mutations in fibrocystin protein formation may disrupt the normal function of renal cilia and also lead to bile duct dysgenesis. The disease can be subdivided into four groups that are felt to be associated with different gene mutations: perinatal, neonatal, infantile, and juvenile (congenital hepatic fibrosis) and are summarized in Table 10.3. The overall prognosis is poor for the infantile type, although patients with the juvenile type can survive longer into adulthood. Pathology

As summarized in Table 10.3, the liver may grossly be normal or occasionally exhibit small cysts. The liver is usually firm due to the varying degrees of portal fibrosis. Microscopically a proliferation of dilated branching and anastamosing biliary channels is present (Figure 10.20). Polypoid-type projections within the dilated

duct lumen also occur. The duct structures are lined by cuboidal to columnar epithelium. Bile duct epithelium often forms ductal plates (circular cylinders surrounding the periphery of the portal tracts) (Figure 10.21), these ductules communicating with the rest of the biliary tract and sometimes extending into the adjacent lobules. The dilated ducts can contain a homogeneously eosinophilic to orange acellular material. Portal tracts are fibrotic with only a minimal lymphocytic infiltrate; however with the infantile and juvenile types a bridging portal fibrosis containing the ectatic proliferating ducts can occur (Figures 10.22 and 10.23), although a well-established cirrhosis with regenerative nodule formation is not a feature. Interlobular bile ducts may be markedly decreased to absent in the juvenile type, and the hepatic arteriole branches are also lacking. The duct lesions may also appear singly or in small clusters within the hepatic parenchyma, with little if any communication with the portal tracts. Inflammation within the hepatic parenchyma is not a feature of any of the variants. Usually the clinical history and presentation with associated renal disease is most helpful in arriving at the correct diagnosis, although distinguishing between the different subdivisions may at times be difficult and may overlap.

Fibrocystic Diseases   211

Table 10.3  Autosomal recessive polycystic diseases of kidneys and liver Type

Age at onset Mode of presentation Outcome

Liver pathology

Perinatal

Birth

Abdominal distention Bilateral markedly enlarged renal masses

Rapid downhill course, uremia, death from birth to 6 weeks

Liver grossly normal Ectasia of all bile ducts and ductules Minimal portal fibrosis

Neonatal

Birth to 1 month

Abdominal distension   due to bilaterally enlarged kidneys Hepatomegaly

Complications of pyelonephritis, cholangitis Progressive renal failure, death from 6 weeks to 8 months

Enlarged, firm on gross examination, with some small thin-walled cysts with clear fluid All the intrahepatic bile ducts dilated with serpiginous growth Mild portal fibrosis

Infantile

Three to 6 months

Hepatosplenomegaly, enlarged kidneys

Enlarged and firm on gross Chronic renal failure, examination systemic hypertension, Dilatation and infolding of all portal hypertension the intrahepatic bile ducts and Outcome variable, ductules death may occur in Moderate portal fibrosis childhood

Juvenile (congenital hepatic fibrosis)

Six weeks to Abdominal distension Outcome variable, may survive into late Hepatosplenomegaly 5 years, childhood and even sometimes Portal hypertension, adulthood esophageal late varices, abdominal childhood collaterals Rarely only part of one hepatic lobe may be involved

Firm liver on gross examination Typical duct plate malformations with biliary dilatation, infoldings affecting all the ducts and ductules Lack of remodeling of the ductal plate Marked portal fibrosis

Source: Adapted from Blyth H, et al. Polycystic disease of kidneys and liver presenting in childhood. Journal of Medical Genetics 1971;8:257–84. Reproduced with permission of BMJ Publishing Group Ltd.

Figure 10.20  Autosomal recessive polycystic kidney disease (ARPKD), neonatal type. Proliferation of branching and anastamosing dilated biliary channels is present, the ducts lined by a single layer of cuboidal epithelium.

Figure 10.21  Autosomal recessive polycystic kidney disease (ARPKD), neonatal type. Bile duct structures are seen forming circular cylinders or ductal plates at the edge of a portal tract.

212    10   Developmental Hepatobiliary Disorders and Cystic Diseases

Figures 10.22 and 10.23  Autosomal recessive polycystic kidney disease (ARPKD), juvenile type (congenital hepatic fibrosis). Portal bridging fibrosis is present with numerous ectatic and anastamosing ducts and ductular elements present.

Adult Polycystic Liver Disease  Associated with Autosomal Dominant Polycystic Kidney Disease  Adult Polycystic Liver Disease without Renal Involvement   Classification and gene mutations

This adult onset variant of PCLD occurs in from 0.05 to 0.13% of the population in autopsy series, the most common form associated with ADPKD. About 85–90% of these patients have PKD1 (polycystic kidney disease 1) gene mutations, with the remainder having PKD2 mutations. The PKD1 gene, localized on chromosome 16p13.3-p13.12, encodes a cell membrane protein, polycystin 1, while the PKD2 gene, localized on chromosome 4q21-q23, encodes polycystin 2. Loss of normal function of polycystin 1 and 2 due to these gene mutations leads to clonal expansion of the mutated cells with progressive dilatation of abnormal predominantly intrahepatic smaller biliary ducts. These ducts lose their connection with the biliary tree, gradually expand and form small (biliary micro-hamartomes, von Meyenburg complexes) and then larger grossly identifiable cysts that compress the adjacent liver. Less commonly, PCLD may also occur without renal involvement. The mutation occurs in

chromosome 19p13.2-13.1 and is additionally linked to a mutation in the protein kinase C substrate 80K-H gene. This gene encodes hepatocystin that functions as a β-subunit of glucosidase II located in the endoplasmic reticulum. The mutation leads to premature termination of the synthesis of the protein with resultant loss of function. This defect may link this variant to the same pathophysiologic pathways as ADPKD with resultant cyst formation. Clinical presentation

In PCLD associated with and without ADPKD, the hepatic cysts are rare before 20 years of age with the prevalence rising from about 20% in the third decade to up to 80% in the sixth decade. Usually the renal cysts develop before the hepatic cysts. Women are more prone for development of the cysts, and the number of pregnancies also correlates with the number of hepatic cysts seen. Previous use of estrogens is also a risk factor. A minority of patients may develop massive cystic liver involvement. Most patients with adult PCLD are asymptomatic, although hepatomegaly with right upper quadrant abdominal pain may occur associated with the larger lesions. Liver tests are usually normal or show mild elevations in the alkaline phosphatase and total bilirubin when associated with altered bile flow from the cysts themselves.

Fibrocystic Diseases   213

Figures 10.24 and 10.25  Autosomal dominant polycystic kidney disease (ADPKD). Variable sized biliary cysts are seen lined by a simple layer of cuboidal to flattened duct epithelium.

Complications can include hemorrhage into the cysts, infection, and rupture. Infrequently signs of portal hypertension can occur with diffuse cystic disease. The diagnosis is made by conventional ultrasound and CT scans. Pathology

Best seen on wedge resection or partial hepatectomy specimens, the liver shows multiple variable sized cysts that measure from less than 1  mm to up to 10  cm. The cysts can diffusely involve all hepatic lobes or can be localized to only one lobe (usually the left). The cysts are lined by a single layer of flattened to cuboidal biliary epithelium (Figures 10.24 and 10.25). The cyst contents are usually clear or can contain amorphous eosinophilic proteinaceous material; however, slightly yellow fluid from bile can also be seen, and infrequently intracystic hemorrhage can also occur. A well-defined cyst wall is not usually present or is quite thin in the larger cysts. Although usually there is no accompanying inflammation, secondary infection can occur associated with a prominent neutrophilic infiltrate within the cyst wall and in the lumen. Microcalcifications may rarely be seen along the thin cyst wall. In addition the cysts can collapse, with eventual thickening of the lining and sclerosis of the lumen resembling the corpora atretica of the ovary. Not infrequently von Meyenburg complexes occur

immediately adjacent to the larger cysts (Figure 10.26). The adjacent liver between the cysts may be normal or show sinusoidal dilatation and congestion with variable liver cell atrophy due to impediment of vascular outflow from the cysts. Portal fibrosis can occur to variable degrees, with additional features of bile duct obstruction with cholestasis when the larger cysts impede normal bile flow. Differential diagnoses

Although a diagnosis of adult PCLD is usually fairly straightforward, at times a number of differential possibilities are raised.

Figure 10.26  Autosomal dominant polycystic kidney disease (ADPKD). A von Meyenburg complex is present immediately adjacent to a large biliary cyst.

214    10   Developmental Hepatobiliary Disorders and Cystic Diseases ●●

●●

●●

Hepatic cystadenomas may be single lesions but at times are multiloculated and can involve an entire lobe, resembling polycystic disease. The cystadenomas usually have a distinct wall, sometimes with calcifications, while these features are infrequent in PCLD. Additionally biopsy material usually shows a spindle cell component within the wall of the cystadenomas (cystadenoma with mesenchymal stroma) a feature not seen in PCLD. Caroli disease shows variable cystic dilatation of the intrahepatic ducts that can mimic polycystic disease on routine imaging. ERCP can then be helpful in better visualizing the intrahepatic ducts that show diffuse segmental cystic and saccular dilatation of the ducts interspersed by normal ducts in Caroli disease, features not seen in PCLD. Simple hepatic cysts are common, usually single but may be multiple. These cysts are not nearly as numerous compared to polycystic disease, and these simple cysts are seldom multiloculated.

Selected Reading Ahn KJ, Yoon JK, Kim GB, et al. Alagille syndrome and a JAG1 mutation: 41 cases of experience at a single center. Korean J Pediatr 2015;58:392–7. Buxbaum J, Lu SC. Cystic diseases of the liver and biliary tract. In: Podolsky DK (ed.) Yamada’s Textbook of Gastroenterology, 6th edn. Oxford: Wiley Blackwell, 2016:1848–57.

De Carvalho E, Ivantes CA, Bezerra JA. Extrahepatic biliary atresia: current concepts and future directions. J Pediatr (Rio J) 2007;83:105–20. Gunay-Aygun M, Font-Montgomery E, Lukose L, et al. Characteristics of congenital hepatic fibrosis in a large cohort of patients with autosomal recessive polycystic kidney disease. Gastroenterology 2013;144:112–21. Hogan MC, Abebe K, Torres VE, et al. Liver involvement in early autosomal-dominant polycystic kidney disease. Clin Gastroenterol Hepatol 2015;13:155–64. Jablonska B. Biliary cysts: etiology, diagnosis and management. World J Gastroenterol 2012;18:4801–10. Kerkar N, Norton K, Suchy FJ. The hepatic fibrocystic diseases. Clin Liver Dis 2006;10:55–71. Lai MW, Chang MH, Hsu SC, et al. Differential diagnosis of extrahepatic biliary atresia from neonatal hepatitis: a prospective study. J Pediatr Gastroenterol 1994;18:121–7. Portmann BC, Roberts EA. Developmental abnormalities and liver disease in childhood. In: Burt A, Portmann B, Ferrell L (eds) MacSween’s Pathology of the Liver, 6th edn. Oxford: Elsevier, 2012:101–56. Sinha J, Magid MS, VanHuse C, et al. Bile duct paucity in infancy. Semin Liver Dis 2007;27: 319–23. Torbenson M, Hart J, Westerhoff M, et al. Neonatal giant cell hepatitis: histological and etiological findings. Am J Surg Pathol 2012;34:1498–503. Zhang DY, Ji ZF, Shen XZ, et al. Caroli’s disease: a report of 14 patients and review of the literature. J Dig Dis 2012;13:491–5.

Additional material for this chapter can be found online at: www.wiley.com/go/kanel/liverpathology 

This includes a full list of References, Case Examples, and Library Images to supplement this chapter.

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11 Drug- and Toxin-Induced Liver Diseases Overview of Drug- and Toxin-Induced Injury One of the most common causes of liver test abnormalities is drug- or toxin-induced liver cell injury (DILI). In fact up to 10% of patients with transaminitis are found to have drug- or toxin-mediated liver damage, the incidence substantially rising in those over 50 years of age. About 1.4% of hospitalizations are due to drug-induced injury, the incidence increasing to about 2–5% of jaundiced patients. In the United States DILI is the most common cause of fulminant hepatitis, accounting for one-third to half of all cases (the majority due to acetaminophen overdoses). Over 1100 drugs and toxins have been associated with liver injury, with the overall incidence quite variable, ranging from 0.014% in France to 1.4% in Switzerland. Additionally the incidence considerably varies from different agents. While some drugs such as isoniazid can cause liver injury in as many as 1 : 100 patients with a fatality in 1 : 10,000, other agents such as diclofenac and minocycline are associated with liver injury in only 1 : 50,000 to 1 : 1,000,000 or fewer. In addition various alternative medicines, particularly herbal preparations such as chaparral and Jin Bu Huan, have also been associated with hepatotoxic reactions. Type of Liver Injury

Liver damage can be predictable and dose dependent (intrinsic), with early onset within a few days of initiation of drug use.

Acetaminophen-induced injury is a classic example, and in fact accounts for almost half of all cases of acute liver failure in the United States. The vast majority of drugs that induce liver injury, however, do so by way of an unpredictable or idiosyncratic (hypersensitivity) reaction, either immune or non-immune mediated, whereby delay of symptoms from 1 to 8 weeks (intermediate latency) or even up to 12 months (long latency) occurs. Additionally the idiosyncratic reactions have an incidence that varies from 1 : 1000 to 1 : 50,000. Usually the symptoms such as fever, rash, and eosinophilia and rapid response to rechallenge are seen in the intermediate latency group (e.g., phenytoin-induced injury); however, the long latency type is usually not associated with hypersensitivity reactions and the results of rechallenge of the drug are variable and not consistent (e.g., isoniazid-induced injury). The liver injury can mimic all types of both acute and chronic hepatitis. Idiosyncratic drug-induced injury most of the time occurs in an otherwise asymptomatic patient with only mildly and transiently abnormal liver tests (mainly the alanine aminotransferase [ALT]) and can occur 10–20 times more commonly than symptomatic disease. Most of the time the ALT returns to normal despite continued use of the medication (adaptation, tolerance) although a minority of patients will develop symptomatic injury (failure to adapt). In those who develop symptoms, hepatocellular injury with jaundice can occur and can at times be ominous, with systemic signs and in the more severe cases markedly elevated

Pathology of Liver Diseases, First Edition. Gary C. Kanel. © 2017 John Wiley & Sons, Ltd. Published 2017 by John Wiley & Sons, Ltd. Companion website: www.wiley.com/go/kanel/liverpathology

216    11   Drug- and Toxin-Induced Liver Diseases

aminotransferases (ALT × upper limit of normal/ alkaline phosphate × upper limit of normal ≥5), coagulopathy, encephalopathy, and mortality of up to 10%. This ominous outcome is also known as Hy’s Law after the late Hyman J. Zimmerman (a world-renowned expert in drug-induced injury), whereby when the aminotransferases in these jaundiced patients reach values from 8 to 100 times normal with the alkaline phosphatase under three times normal, the mortality can range from 10 to 50%. Usually when the drug is discontinued the liver tests resolve in a few weeks in those who survive the initial presentation. Although hepatocellular patterns of injury are most common (about 90% of all cases of DILI), about 10% present with a cholestatic pattern of injury, the disease usually not as severe and often associated with pruritus and a disproportionate elevation of the alkaline phosphatase activity, with mild to moderate elevations of the ALT (ALT × upper limit of normal/alkaline phosphate × upper limit of normal ≤2). Usually the disease resolves more slowly (months) on discontinuance of the drug, although disease progression with duct damage and loss can rarely develop. Mixed patterns can also occur with intermediate liver test abnormalities. In addition very infrequently a drug can either trigger or unmask an otherwise indolent form of autoimmune hepatitis associated with positive autoimmune antibodies and histologic features of an autoimmune hepatitis, examples being minocycline and nitrofurantoin. Resolution of the autoimmune hepatitis occurs on discontinuation of the drug and without the relapse that periodically is seen in typical autoimmune hepatitis. Pathogenesis

The pathophysiologic concepts of liver-induced injury can relate to metabolites that are electrophilic intermediator compounds or free radicals that deplete glutathione (GSH), bind covalently to proteins, lipids, or nucleic acids, or induce lipid peroxidation, resulting in liver cell injury. Alternatively, the metabolites may covalently

bind to or alter liver proteins such as cytochrome p450 that ultimately leads to sensitization and immune-mediated injury (recognized by the immune system as neoantigens). In addition, genetic polymorphism of the major histocompatibility complex (MHC) I-dependent antigens (present in liver cells) or MHC II-dependent antigens (present in macrophages that phagocytize the liver cells damaged by the drug) may also contribute to the infrequent occurrence of hypersensitivity-induced liver injury. Toxicity can also occur due to specific properties of the drug or its metabolites, such as the toxic effects of norcocaine and the highly reactive nitrosonium ion produced by the enzymatic breakdown of cocaine. Whatever the specific cause, the end result of apoptosis and the degree of cell injury relate to the subsequent effects of various toxic mediators (e.g., nitrous oxide, reactive oxygen metabolites), toxic cytokines that cause injury (e.g., tumor necrosis factor-α [TNF-α], interleukin [IL]-1 and IL-12) and the balance with cytokines that prevent injury (IL-4, IL-10, monocyte chemotactic protein 1). Risk Factors

The risks that contribute to DILI relate to the interaction of environmental and genetic factors. Women may be more likely to develop DILI than men, and the age (older than 60 years) also may be contributory. The dosages of the medication in both acute (e.g., acetaminophen) and chronic (e.g., methotrexate) injury are important in many cases. Concomitant drugs are known to enhance the potential toxicity of DILI, such as alcohol intake enhancing the likelihood of acetaminophen toxicity at lower doses. Underlying diseases such as chronic viral infections (in particular chronic hepatitis B [HBV], chronic hepatitis C [HCV], human immunodeficiency virus [HIV] infections), diabetes, obesity, and fasting can be contributory to DILI. In addition various genetic factors such HLA associations, multiple cases within a family, and genetic polymorphisms also play roles in DILI.

Overview of Drug- and Toxin-Induced Injury   217

Diagnosis

Whatever the cause, the diagnosis rests on a number of factors. The time frame of initiation of drug use and the onset of liver test abnormalities is often crucial, although the time frame in the long latency-type injury can be quite variable. There also must be consideration of the fact that resolution of the abnormal liver tests upon cessation of the drug may be non-DILI associated (e.g., resolving acute hepatitis from another cause). The presence of any coexisting risk factors that are known to enhance drug injury (e.g., alcohol intake, pregnancy) and the use of concomitant drugs known to enhance toxicity also are important factors to consider. The search in the PDR and the published literature of known toxicity of the suspicious drug is

often essential in assessing the possibilities. The exclusion of other causes of liver disease such as concomitant chronic viral hepatitis or fatty liver diseases, and the result after rechallenge with the drug (if possible) are also important features in assessing the possibility of DILI. The above considerations are also used in a standardized reporting form (RUCAM, Roussel Uclaf Causality Assessment Method) developed by an international panel working in France that numerically generates a score that measures the probability, possibility, unlikelihood, or exclusion of a drug-induced process. A wide variety of hepatic morphologic changes have been documented secondary to DILI, examples listed in Table 11.1. This chapter further addresses each of the morphologic parameters.

Table 11.1  Drug and toxin-induced liver injury (DILI): histologic variants Histology

Examples

Lobular necrosis with minimal to absent necroinflammatory change (coagulative necrosis)

Acetaminophen (zone 3) Carbon tetrachloride (zone 3) Cocaine (zone 3) Halogenated hydrocarbons (zone 3) Mushrooms (zone 3)

Beryllium (zone 2) Cocaine (zone 1) Dioxane (zone 2) Ferrous sulfate (zone 1) Phosphorus (zone 1)

Acute necroinflammatory change

Bupropion Chlorpromazine Clometacin Diclofenac Dihydralazin

Halogenated hydrocarbons Isoniazid Minocycline Nitrofurantoin

Oxyphenisatin Phenytoin Sulfonamides Troglitazone

Chronic necroinflammatory change, with and without fibrosis/ cirrhosis

Amiodarone Ethanol Methotrexate

Minocycline Nitrofurantoin Oxyphenisatin

Pemoline Total parenteral nutrition

Cholestatic injury Cholestasis without necroinflammatory change (simple cholestasis)

Anabolic steroids Cyclosporin A Estrogens

Flucloxacillin Mestranol Oral contraceptives

Sulindac Total parenteral nutrition

α-Methyldopa Amoxicillin-clavulanate Benoxaprofen Bupropion

Carbamazapine Chlorpromazine Erythromycin Halogenated hydrocarbons

Isoniazid Jin Bu Juan Phenylbutazone Sulindac Total parenteral nutrition

●●

●●

Cholestasis with necroinflammatory change (cholestatic hepatitis)

Mushrooms (diffuse) Phenelzine (diffuse)

(continued)

218    11   Drug- and Toxin-Induced Liver Diseases

Table 11.1  (continued) Histology

Examples

Bile duct injury

Inflammation by neutrophils Allopurinol Phenytoin

Inflammation by lymphocytes, ductopenia

Vascular injury

Peliosis Anabolic steroids Hydroxyurea Norethandrolone Thorotrast

Veno-occlusive disease Adriamycin Arsenic Busulfan Mitomycin C Pyrrolizidine alkaloids

Steatosis

Macrovesicular Amiodarone (steatohepatitis) Corticosteroids Ethanol Methotrexate Sulfasalazine (steatohepatitis)

Microvesicular Cocaine Ethanol Tetracycline Valproic acid Vitamin A

Phospholipidosis Amiodarone Perhexiline maleate

Granulomas

Allopurinol Bacille Calmette– Guérin (BCG)

Chlorpromazine Phenylbutazone Quinidine

Sulfasalazine Sulfonylurea agents Tolbutamide

Mallory–Denk bodies

Amiodarone

Ethanol

Perhexiline maleate

Neoplasms and related lesions

Aflatoxins (HCC) Anabolic steroids (HCA, NRH, HCC)

Azathioprine (NRH) Oral contraceptives (HCA, HCC)

Oxymetholone (HCA, HCC) Thorotrast (CC, A) Vinyl chloride (A)

Miscellaneous

Anthracotic (anthracite) pigment Procainamide (intracytoplasmic inclusions)

Gold sodium thiomalate (gold deposits) Phenacitin (lipochrome)

Talc, particulate material Thorotrast (radiopaque)

Chlorpromazine Chlorpropamide Erythromycin

Flucloxacillin Paraquat Prochlorperazine Hepatic vein thrombosis Ethanol

Vasculitis Allopurinol Chlorothiazide

A, angiosarcoma; CC, cholangiocarcinoma; HCA, hepatocellular adenoma; HCC, hepatocellular carcinoma; NRH, nodular regenerative hyperplasia. Source: Adapted from Kanel GC. Histopathology of drug-induced liver damage. In: Kaplowitz N, DeLeve LD (eds) Drug-Induced Liver Disease. Marcel Dekker, Inc., New York, 2003.

Hepatocellular Necrosis with Minimal to Absent Necroinflammatory Change The predictable and direct hepatotoxic effects of drugs or toxins are usually associated with

a coagulative necrosis of liver cells that has a distinct zonal distribution pattern. The onset of the morphologic features directly correlates with the timeframe of initiation of drug use or toxin exposure and the onset of clinical symptoms and liver test abnormalities, beginning within a few days or less after exposure. The

Acute Hepatocellular Necroinflammatory Change   219

liver cells appear eosinophilic with the nuclei pyknotic and karyorrhectic in the very early stages, with rapid onset of nuclear loss, shrinkage of the cytoplasm, and eventual liver cell dropout with collapse of the lobular reticulin framework. In most cases the perivenular (zone 3 of Rappaport) region is involved, in large part due to the hepatocytes in this zone most active in physiologic drug metabolism; however, infrequently the periportal zone may show injury such as seen in ferrous sulfate and phosphorus-induced necrosis. There are most often sharp demarcations of the areas of necrosis and the adjacent viable hepatocytes; however, in the most severe cases the perivenular and midzones and sometimes all zones (panlobular) can be involved by severe confluent necrosis. All lobules throughout the liver are equally affected, as opposed to coagulative necrosis secondary to ischemia (acute heart failure) where the degree of damage may somewhat vary from one hepatic lobule to another dependent of the specific arterial blood flow to each particular functional region. Usually there are no inflammatory infiltrates in the areas of necrosis, although eventually a histiocytic and Kupffer cell reaction to clear the liver of the necrotic hepatocytes occurs. Interestingly a neutrophilic infiltrate that is common in ischemic necrosis due to poor vascular perfusion (hypotension) is less frequent in direct drug-induced injury. The adjacent hepatocytes often show a hydropic change due to liver cell regeneration. In addition, if there is impediment to bile flow from the necrosis, cholestasis may also be seen in the viable hepatic parenchyma. The portal tracts in almost all instances are not affected, with no fibrosis, little if any portal inflammatory infiltrates, and normal bile ducts; however, in periportal or panlobular necrosis a prominent bile ductular reaction originating from facultative stem cell activation is not uncommon. Figures 11.1 and 11.2 show examples of drug-induced hepatocellular necrosis with minimal to absent portal inflammation (acetaminophen).

Figure 11.1  Hepatocellular necrosis with minimal to absent necroinflammatory change (acetaminophen). The perivenular and midzonal hepatocytes show a coagulative necrosis without an inflammatory reaction, with the adjacent viable periportal hepatocytes unremarkable.

Acute Hepatocellular Necroinflammatory Change Liver injury associated with portal and lobular inflammation is characteristic of idiosyncratic hypersensitivity reactions and may present clinically as an acute hepatitis or occur in an otherwise asymptomatic patient with transaminitis. The lobular inflammation is usually lymphocytic, and accentuation in the perivenular zone may also occur, although diffuse involvement with

Figure 11.2  Hepatocellular necrosis with minimal to absent necroinflammatory change (acetaminophen). The portal tracts are normal although at times a minimal lymphocytic infiltrate can occur.

220    11   Drug- and Toxin-Induced Liver Diseases

no zonal pattern is not infrequent. The inflammation can be patchy, with variable liver cell necrosis, apoptosis, and hydropic or ballooning changes of the hepatocytes, or the injury can be diffuse. Kupffer cell hyperplasia is common, with all lobules equally affected. At times a sinusoidal lymphocytosis can also occur and mimic the features seen in acute Epstein–Barr virus and cytomegalovirus-induced hepatitis (“mononucleosis-type pattern”), an example being phenytoin-associated injury. Additionally at times the necroinflammatory change can form small aggregates of lymphocytes and histiocytes and mimic small granulomas (“granulomatous necrosis”). The portal tracts usually show a lymphocytic infiltrate that at times can be prominent, although in drug-induced injury the degree of portal inflammation is usually not as striking as in acute or chronic viral hepatitis. Although portal infiltration by eosinophils can occur and is helpful in the diagnosis of a hypersensitivity reaction, oftentimes eosinophils are few or even absent. Scattered portal neutrophils can also occur, but are not oriented towards any particular portal structures. A periportal interface inflammatory activity is usually not a feature except in the chronic hepatitic reactions. Portal fibrosis is also not a feature. In the more severe cases associated with jaundice and markedly elevated transaminases, a confluent necrosis with extensive liver cell injury and dropout can also ensue, and although the perivenular zone is initially affected, all zones can be involved in severe fulminant hepatitis. The necrosis can be highlighted by the reticulin stain which shows collapse of the reticulin framework. A variable and sometimes prominent lobular inflammation by predominantly lymphocytes is also seen in the viable areas. Rarely the confluent necrosis can be periportal rather than perivenular. When there also are areas of spared hepatocytes, the injury is termed submassive hepatic necrosis, while involvement of all hepatocytes in all lobules (panacinar) is termed massive hepatic necrosis. Cholestasis may be seen in viable hepatocytes as well. In addition the viable hepatocytes can

Figure 11.3  Acute hepatocellular necroinflammatory change (chlorpropamide). The portal tract exhibits a prominent mixed inflammatory infiltrate including numerous eosinophils.

show a hydropic change secondary to liver cell regeneration, although impaired regeneration, manifested histologically by straight hepatic cords with the cytoplasm eosinophilic without liver cell repopulation, is an ominous finding, the outcome almost always fatal with liver transplantation the only option for survival. Figures 11.3, 11.4, 11.5, 11.6, and 11.7 show examples of drug-induced acute hepatocellular necroinflammatory change (chlorpropamide, halothane, bupropion, nitrofurantoin, isoniazid).

Figure 11.4  Acute hepatocellular necroinflammatory change (halothane). Extensive confluent necrosis and necroinflammatory change is seen involving the perivenular and midzonal hepatocytes. A moderate portal lymphocytic infiltrate is present in the center of the field.

Chronic Hepatocellular Necroinflammatory Change   221

Figure 11.5  Acute hepatocellular necroinflammatory change (bupropion). Diffuse moderate lobular necroinflammatory change is seen, the inflammatory cells chiefly lymphocytes. Variable hydropic change of some of the hepatocytes is also present.

Chronic Hepatocellular Necroinflammatory Change Although most cases of drug-induced hepatocellular injury recover when the drug is discontinued, ongoing necroinflammatory changes over time may lead to a chronic hepatitis that may persist even when the drug is no longer being administered. This process can occur in about 5–10% of adverse drug reactions. In some

Figure 11.6  Acute hepatocellular necroinflammatory change (nitrofurantoin). Diffuse lobular necroinflammatory change is seen, the inflammatory cells predominantly lymphocytes.

Figure 11.7  Acute hepatocellular necroinflammatory change (isoniazid). Severe confluent hepatic necrosis with a predominantly lymphocytic inflammatory infiltrate is present.

instances the chronic hepatitis reaction histologically resembles portal and lobular inflammation similar to that seen in acute injury but is generally milder; however, the degree of inflammation in these cases may also be irregularly distributed from one lobule to the next similar to that seen in chronic viral hepatitis. The portal tracts usually show a variable predominantly lymphocytic infiltrate with occasional macrophages and sometimes plasma cells and eosinophils, with the bile ducts usually not involved. Periportal interface inflammatory activity, a hallmark of active chronic hepatitis, is also apparent to variable degrees. In some instances of chronic hepatitis, portal, periportal, and intrasinusoidal collagen deposition can occur which can lead to cirrhosis with time if the drug is not discontinued. Activation of hepatic stellate (fat-storing) cells leading to laydown of extracellular sinusoidal collagen is one mechanism, alcohol-related fibrosis an excellent example. When cirrhosis develops it can be either macronodular or micronodular. When also associated with bile duct injury, a “biliary” fibrosis with duct loss and cirrhosis can occur. Signs and symptoms at this stage of portal hypertension such as ascites, esophageal varices, and abdominal collaterals are often present; however, sometimes portal and periportal

222    11   Drug- and Toxin-Induced Liver Diseases

Figures 11.8 and 11.9  Chronic hepatocellular necroinflammatory change (methotrexate). Portal fibrosis with prominent periportal interface inflammatory activity is seen.

fibrosis can occur with clinical manifestations of portal hypertension but without the development of cirrhosis (e.g., non-cirrhotic portal fibrosis, hepatoportal sclerosis secondary to polyvinyl chloride exposure). When the portal and in many instances the lobular plasma cell infiltrates are marked, an autoimmune hepatitis triggered by the drug should be suspected and can be confirmed by elevated serum IgG levels and positive autoimmune serologic markers (anti-nuclear antibody [ANA], smooth muscle antibody [SMA], liver– kidney microsomal antibody [LKM]), examples being minocycline and nitrofurantoin-induced injury. Some of the patients who develop autoimmune hepatitis induced by drugs may have a history of other autoimmune disorders as well. Discontinuation of the drug will halt the autoimmune-associated process. Additionally the lack of recurrence of the autoimmune hepatitis after the drug withdrawal supports that the autoimmune disease was indeed drug induced. Of note is that oftentimes the hepatitis presents as an acute event with portal fibrosis initially minimal to absent; however progression of the disease with accompanying fibrosis over time when the drug is not discontinued can occur. Although halting the drug use can prevent further development of fibrosis, when there is cirrhosis reversibility virtually does not occur (rare exceptions). Therefore since the vast

majority of drugs associated with the risk of developing cirrhosis are known (e.g., methotrexate in the treatment of rheumatoid arthritis), careful monitoring of liver tests with planned liver biopsies to assess whether fibrosis is developing is the rule with these patients, whereby severe fibrosis and cirrhosis can be avoided. Screening with biopsy is often critical, as not infrequently the liver tests can be normal or show only minimally elevated transaminases with even a normal albumin yet fibrosis can be present and progress. Figures 11.8, 11.9, 11.10, 11.11, 11.12, and 11.13 show examples of drug-induced chronic

Figure 11.10  Chronic hepatocellular necroinflammatory change (ethanol). A well-established cirrhosis is present from this patient with a long history of alcohol abuse with recent abstinence (absence of steatosis, ballooning change, Mallory-Denk bodies).

Steatosis and Steatohepatitis   223

Figures 11.11 and 11.12  Chronic hepatocellular necroinflammatory change (autoimmune hepatitis triggered by minocycline). Portal fibrosis with periportal interface inflammatory activity is seen, the inflammatory cells consisting of lymphocytes with increased numbers of plasma cells, the latter often arranged in small clusters.

hepatocellular necroinflammatory (methotrexate, ethanol, minocycline).

change

Steatosis and Steatohepatitis Steatosis (fatty change) is a relatively common histologic finding associated with a wide variety of liver disorders, especially alcohol and

Figure 11.13  Chronic hepatocellular necroinflammatory change (autoimmune hepatitis triggered by minocycline). Lymphocytes can be seen hugging up against the terminal hepatic venular endothelium (endothelialitis), a feature that can sometimes occur in autoimmune hepatitis.

obesity-related with or without diabetes. Therefore it is often problematic in assessing whether the steatosis is all or part drug induced. The fat can be macrovesicular (large droplet fat globules equal to or greater than the size of the nucleus), microvesicular (small droplet fat smaller than the nucleus) or mixed. In some instances the fat globules can be so small (“foamy”) that they cannot be appreciated on routine H&E stain, necessitating performance of a stain for neutral triglycerides (Oil Red O) on frozen sections from fresh or formalin fixed liver tissue. Fat can also be seen from normally processed tissue with immunoperoxidase stains using perilipin and adipophilin markers, two proteins that play a role in lipid metabolism, with positive staining appearing at the rim of the lipid droplets. The fat can also be patchy throughout the lobules, have a zonal accentuation, or be diffusely distributed in virtually all of the hepatocytes. In addition small fat droplets can be present within portal macrophages, in lipogranulomas, and in the subendothelial fat storing (Ito, stellate) cells. Steatosis that is mainly macrovesicular can occur without associated inflammation on biopsy, even when mild transaminitis is present; however, in drug-induced steatosis usually some other histologic features such as portal inflammation and patchy lobular inflammation

224    11   Drug- and Toxin-Induced Liver Diseases

are also apparent. Although fatty change with any inflammation can loosely be referred to as steatohepatitis, that term is more often applied to the additional presence of varying sinusoidal fibrosis, which may in the more active stages also show liver cell ballooning and Mallory–Denk bodies. These additional features at times make drug-induced injury difficult to distinguish from alcoholic hepatitis or active non-alcoholic steatohepatitis. In addition, foamy lipid deposits can occur within the lysosomes of hepatocytes due to the inhibition of phospholipase A from lipid hydrolysis (phospholipidosis), and can be induced by various drugs. Histologically not only fatty change but also sinusoidal fibrosis and Mallory–Denk body formation can also occur in this subgroup. Figures 11.14, 11.15, 11.16, and 11.17 show examples of drug-induced steatosis (ethanol, cocaine, vitamin A, sulfasalazine).

Figure 11.15  Steatosis (cocaine). Diffuse predominantly microvesicular fatty change is present.

Granulomas Granulomas loosely defined are variably sized but usually small collections of inflammatory cells that can occur within the portal tracts or parenchyma and are seen in numerous liver diseases including various viral and non-viral infectious disorders, genetic and developmental

Figure 11.14  Steatosis (ethanol). Diffuse macrovesicular fatty change is seen.

Figure 11.16  Steatosis (vitamin A). Small droplet fat is seen in the hyperplastic stellate (Ito, fat-storing) cells that lie just beneath the sinusoidal endothelial cells.

Figure 11.17  Steatosis with inflammation (steatohepatitis) (sulfsalazine). Macrovesicular fat with accompanying mild lobular necroinflammatory change is seen.

Mallory–Denk Bodies   225

abnormalities, immune-mediated reactions, certain neoplasms, and various miscellaneous conditions (see Table 6.1 which lists the various causes of hepatic granulomas). Granulomas secondary to drug-induced reactions are also well documented and are felt to be secondary to a cellular immune-mediated reaction to the drug or its metabolites. These granulomas can be well (epithelioid type) or poorly (inflammatory type) demarcated. The former usually contain epithelioid histiocytes with scattered lymphocytes and sometimes multinucleated giant cells, while the latter consist of macrophages, eosinophils, plasma cells and neutrophils to various degrees, infrequently associated with multinucleated giant cells. The inflammatory granulomas also can be quite small and consist of only a small cluster of mixed inflammatory cells (“micro-granulomas”). Usually drug-induced granulomas are of the inflammatory type. Central necrosis with or without fibrin deposition within the granulomas is infrequent but can occur in drug-induced injury. Since these granulomas very much mimic those seen in other necroinflammatory processes, especially infections, the use of special stains, serologies, and cultures of tissue and various body fluids is often essential in arriving at the correct diagnosis. Figures 11.18 and 11.19 show examples of drug-induced granuloma formation (sulfasalazine, chlorpromazine).

Figure 11.18  Granuloma (sulfasalazine). A portal tract shows a well-circumscribed granuloma composed of lymphocytes and epithelioid histiocytes.

Figure 11.19  Granuloma (chlorpromazine). The parenchyma shows a small well-demarcated collection of lymphocytes and macrophages.

Mallory–Denk Bodies Mallory–Denk bodies are eosinophilic irregular ropey intracytoplasmic inclusions in the hepatocyte and are associated with various hepatic disorders, in particular the fatty liver diseases. On electron microscopy these inclusions consist of filamentous randomly oriented intracytoplasmic processes composed of various cytokeratins, ubiquitin-binding protein p62, and other peptides. Mallory–Denk bodies can also occur in other liver diseases such as chronic biliary tract disorders (e.g., primary biliary cirrhosis, primary sclerosing cholangitis) but are well recognized in association with various drugs and toxins as well, the best example being amiodarone-induced injury. The Mallory–Denk bodies associated with both alcoholic hepatitis and non-alcoholic steatohepatitis are accentuated in the perivenular zone, although in severe cases the midzones and in the more severe cases all zones may be involved, while in amiodarone hepatotoxicity the periportal hepatocytes are most often affected. Although in alcoholic hepatitis the neutrophilic lobular infiltrates are common, with neutrophils often surrounding the cells that contain the Mallory–Denk bodies (“satellitosis”), in drug-induced injury the inflammatory cells often are a mixture of lymphocytes and neutrophils with oftentimes the

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Figure 11.20  Mallory–Denk bodies (amiodarone). The hepatocytes show a ballooning change with numerous Mallory–Denk bodies.

lymphocytes predominating, and the degree of inflammation compared to alcoholic hepatitis is usually considerably less. Figure 11.20 shows an example of druginduced Mallory–Denk body formation (amiodarone).

Acute Cholestatic Liver Injury Cholestasis associated with an absence of lobular or portal inflammation and without interlobular bile duct injury is termed simple cholestasis and can occur from various drugs and toxins. The bile is usually seen in the perivenular zone and can occur within the liver cell cytoplasm, within dilated canaliculi, or both. At times the bile can be diffuse and involve all zones, and when the periportal zone is involved the bile can at times be seen within dilated cholangioles located at the border of the portal tracts and parenchyma, sometimes associated with a mild predominantly neutrophilic infiltrate (acute cholangiolitis). Intracellular bile is a clumped green to yellow-green pigment that stains positively on Hall stain. Sometimes it is difficult to differentiate from lipochrome pigment; however, lipochrome is a finely to coarsely granular brown pigment that can show weak positive

staining with periodic acid–Schiff after diastase resistance (DiPAS) and with the Ziehl–Neelsen acid–fast stains. Cholestatic hepatitis refers to liver damage associated with not only cholestasis but also variable portal and lobular inflammation and can occur secondary to drug- or toxin-induced injury. The cholestasis is usually accentuated within the perivenular zone and is associated with usually a lymphocytic infiltrate with variable Kupffer cell hyperplasia. Variable swelling and hydropic changes of the hepatocytes containing the bile may occur. The portal inflammation is usually lymphocytic with occasional neutrophils, plasma cells, and eosinophils. The degree of portal inflammation can vary but is seldom present to the extent seen in viral hepatitis. In addition the degree of intracellular and intracanalicular bile can be striking and outweigh the only mild portal and lobular inflammation. Periportal interface inflammatory activity is usually not a feature, and the interlobular bile ducts are usually normal, although as in simple cholestasis there can be cholangiolar proliferation when the cholestasis also involves the periportal zones. Figures 11.21 and 11.22 show examples of drug-induced acute cholestatic liver injury (oral contraceptives, isoniazid).

Figure 11.21  Acute cholestatic injury (simple cholestasis) (oral contraceptives). Bile is seen within dilated canaliculi. No necroinflammatory change is seen.

Vascular Injury   227

Figure 11.22  Acute cholestatic injury (cholestatic hepatitis) (isoniazid). Intracanalicular and intracytoplasmic bile is seen along with mild lobular necroinflammatory change.

Chronic Cholestatic Liver Injury Although the interlobular bile ducts are not primarily affected in most cases of cholestatic-induced liver injury, in some instances of cholestatic hepatitis the bile ducts can also be damaged (chronic cholestatic pattern). When neutrophils are involved the features can mimic the acute cholangitis seen in bile duct obstruction; however the damaged ducts do not show the proliferation and ectasia seen in bile duct obstruction. Lymphocytes can also be the inflammatory cell targeting the interlobular bile ducts (non-suppurative duct damage) and with time the duct injury can eventually lead to interlobular bile duct loss, features similar to those seen in primary biliary cirrhosis and autoimmune hepatitis with duct damage (autoimmune cholangitis). Very rarely periductal fibrosis similar to that seen in long-term bile duct obstruction and primary sclerosing cholangitis can occur, an example being injury due to floxuridine in the therapy of hepatic tumors. Regardless of the type of duct damage, the parenchyma oftentimes usually shows a mild lobular inflammation and variable degrees of cholestasis that at times can be quite minimal or absent. In addition, with time portal fibrosis of a biliary type can occur and uncommonly even a biliary cirrhosis can be the unfortunate outcome.

Figure 11.23  Chronic cholestatic liver injury (chlorpromazine). The interlobular bile duct shows considerable cytologic atypia and is surrounded and focally infiltrated by lymphocytes.

Figures 11.23 and 11.24 show examples of drug-induced chronic cholestatic injury (chlorpromazine, chlorpropamide).

Vascular Injury Damage to the sinusoids and outflow vessels can occur secondary to drug- and toxin-induced damage. The terminal hepatic venules can show intraluminal fibrosis with fibrous obliteration (veno-occlusive changes) that most often is seen in acute and chronic alcoholic liver disease due to activation of the stellate sinusoidal lining

Figure 11.24  Chronic cholestatic liver injury (chlorpropamide). The portal tracts show a prominent mixed inflammatory infiltrate including eosinophils. No interlobular bile duct is present.

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cells. In addition, certain drugs, in particular the Senecio alkaloids, can also cause endothelial inflammation, sloughing of the endothelial cells, and eventual endothelial loss (sinusoidal obstruction syndrome, veno-occlusive disease) which can sometimes spread out and involve the zone 3 endothelial cells and sinusoids as well. Rarely sinusoidal dilatation, usually involving the midzonal hepatocytes, can be seen associated with oral contraceptive usage. The sinusoids can also be focally damaged and weakened, causing focal sinusoidal dilatation with small intralobular micro-cyst formation (peliosis hepatis) usually filled with red blood cells. At times these peliotic lesions can enlarge to the degree of being discernible by imaging. The larger outflow vessels (sublobular and hepatic veins) can also undergo thrombosis and occlusion by certain drugs, in particular alcohol but also oral contraceptives. Inflammation of the hepatic arteries can also occur, and more often involve the medium-sized arteries, causing a vasculitis. The small hepatic arterioles are usually not involved. Figure 11.25 shows an example of druginduced vascular injury (veno-occlusive disease) (oncotherapeutic conditioning regimen in bone marrow transplantation).

Figure 11.25  Vascular injury (veno-occlusive disease status post chemotherapy in bone marrow transplantation) (trichrome stain). The terminal hepatic venule shows prominent subendothelial fibrosis with almost total occlusion of the lumen.

Neoplasms and Related Lesions Certain drugs and toxins may with time lead to the formation of both benign and malignant hepatic neoplasms and space-occupying related lesions. A best-known example is the development of hepatocellular adenomas associated with long-term oral contraceptive usage, with a small percentage of these patients even at risk for developing hepatocellular carcinoma. Although discontinuation of the oral contraceptives can cause shrinkage of these adenomas, oftentimes the lesions nonetheless persist. The risk of developing hepatocellular carcinoma, cholangiocarcinoma, and angiosarcoma also have been reported with various drugs and toxins; in particular, exposure to arsenicals, copper, polyvinyl chloride, and thorotrast (an α-, β-, and γ-emitter used from 1930 to 1953 as an agent for angiograms) have a high risk of developing angiosarcomas, although thorotrast has not been in use for generations and the chance of encountering thorotrast-induced lesions nowadays is extremely unusual. Figures 11.26, 11.27, and 11.28 show examples of drug-induced neoplasms (oral contraceptives, oxymetholone).

Figure 11.26  Neoplasms (hepatocellular adenoma) (oral contraceptives). The neoplasm is composed of cytologically benign hepatocytes forming normal-sized hepatic cords.

Pigments and Miscellaneous Conditions   229

Figure 11.27  Neoplasms (atypical hepatocellular adenoma) (oxymetholone). Hepatocytes forming normal sized to marginally thickened hepatic cords with pseudogland formation in part containing bile are present.

Pigments and Miscellaneous Conditions Pigments and various intracytoplasmic inclusions within hepatocytes and Kupffer cells secondary to drugs and toxins also may occur. Increase in lipochrome pigment can be infrequently seen with long-term usage of certain drugs such as phenacetin, and a grey-green pigment (thorium dioxide) is present within the portal macrophages and Kupffer cells in thorotrast exposure. A dark black granular usually extracelluar pigment representing anthracite can occur in long-term city dwellers and coal miners. The intracytoplasmic

Figure 11.28  Neoplasms (hepatocellular carcinoma) (oxymetholone). The hepatocytes have a slight increase in the nuclear : cytoplasmic ratio and are forming trabecular cords from three to five cells thick.

Figure 11.29  Miscellaneous conditions (pigment) (thorotrast). The Kupffer cells contain a grey-green granular pigment that represents thorotrast (thorium dioxide).

inclusions can be well defined and distinct (e.g., procainamide) but can also diffusely involve the entire hepatocyte (e.g., phenobarbital), the latter resembling the “ground glass” cells associated with chronic viral hepatitis type B (although negative on the immunoperoxidase stain for HBsAg). This cytoplasmic appearance is usually due to hypertrophy of the smooth endoplasmic reticulum related to drug metabolism. Particulate matter such as the birefringent crystalline inclusions secondary to long-term intravenous drug use can sometimes be seen free within the portal tracts or within portal macrophages and Kupffer cells. Figures 11.29 and 11.30 show examples of drug-induced pigment deposition and intracytoplasmic inclusions (thorotrast, procainamide).

Figure 11.30  Miscellaneous conditions (inclusions) (procainamide). Distinct eosinophilic intracytoplasmic inclusions are seen in some of the hepatocytes.

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Selected Reading Blieden M, Paramore LC, Shah D, et al. A perspective on the epidemiology of acetaminophen exposure and toxicity in the United States. Exp Rev Clin Pharmacol 2014;7:341–8. Chitturi S, Farrell GC. Drug-induced liver disease. In: Schiff ER, Maddrey WC, Sorrell MF (eds) Schiff ’s Diseases of the Liver. Oxford: Wiley Blackwell, 2012:703–82. Giordano CM, Zervos XB. Clinical manifestations and treatment of drug-induced hepatotoxicity. Clin Liver Dis 2013;17:565–73. Kanel GC, Korula J. Drug- and toxin-induced liver cell injury. In: Atlas of Liver Pathology, 3rd edn. Oxford: Elsevier, 2011:93–133. Kenna JG. Mechanism, pathology, and clinical presentation of hepatotoxicity of anesthetic agents. In: Kaplowitz N, DeLeve LD (eds) Drug-Induced Liver Disease. New York: Informa Healthcare, 2007:465–84. Lee WM. Drug-induced liver disease. In: Podolsky DK (ed.) Yamada’s Textbook of Gastroenterology, 6th edn. Oxford: Wiley Blackwell, 2016:1958–72.

Lewis JH, Kleiner DE. Hepatic injury due to drugs, herbal compounds, chemicals, and toxins. In: Burt A, Portmann B, Ferrell L (eds) MacSween’s Pathology of the Liver, 6th edn. Oxford: Elsevier, 2012:645–760. Lewis JH, Ranard RC, Caruso A, et al. Amiodarone hepatotoxicity: prevalence and clinicopathologic correlations among 104 patients. Hepatology 1989;9:679–85. Osuga T, Ikura Y, Kadota C, et al. Significance of liver biopsy for the evaluation of methotrexateinduced liver damage in patients with rheumatoid arthritis. Int J Clin Exp Pathol 2015;8:1961–6. Strasser SI, McDonald GB. Hepatobiliary complications of hematopoietic cell transplantation. In: Schiff ER, Sorrell MF, Maddrey WC (eds) Schiff ’s Diseases of the Liver. Philadelphia: Lippincott-Raven, 1999:1617–41. Yuan L, Kaplowitz N. Mechanisms of drug-induced liver injury. Clin Liver Dis 2013;17:507–18. Zhang X, Ouyang J, Thung SN. Histopathologic manifestations of drug-induced hepatotoxicity. Clin Liver Dis 2013;17:547–64.

Additional material for this chapter can be found online at: www.wiley.com/go/kanel/liverpathology 

This includes a full list of References, Case Examples, and Library Images to supplement this chapter.

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12 Liver Transplantation Introduction with Indications for Liver Transplantation and Patient Selection Liver transplantation is a highly successful approach in the treatment of patients with endstage liver disease and fulminant hepatic failure, and is performed in over 130 centers within the United States. In addition, with advancements in surgical techniques and post-transplant therapy, the short- and long-term survivals have significantly improved, with the 3 month, 1 year, and 5 year survivals from 1996 to 2005 in adult liver transplants being 93, 87, and 73%, respectively, according to data from the United Network for Organ Sharing (UNOS). The total number of transplants has also increased over the past 25 years, shown in the data from the European Liver Transplant Registry of 107,071 liver transplants performed up through 2011 (Table 12.1). Alcoholic cirrhosis and cirrhosis secondary to chronic hepatitis C infection are the two most common native liver diseases that undergo liver transplantation in the United States and Europe. Hepatocellular carcinoma is the reason for transplant in about 10% of the overall adult patient population, with various restrictions applied (such as allowing transplants only with solitary tumors not >5  cm, or no more than three tumors the largest