LIVER PHYSIOLOGY AND DISEASE

Transcription

1 GASTROENTEROLOGY Copyright 1973 by The Williams & Wilkins Co. Vol. 64. No.1 Printed in U.S.A. LIVER PHYSIOLOGY AND DISEASE HEPATOCELLULAR HYALIN IN CHOLESTASIS AND CIRRHOSIS: ITS DIAGNOSTIC SIGNIFICANCE MICHAEL A. GERBER, M.D., WILLIAM ORR, M.D., HELMUT DENK, M.D., FENTON SCHAFFNER, M.D., AND HANS POPPER, M.D. The Stratton Laboratory for the Study of Liver Disease, Department of Pathology, Mount Sinai School of Medicine of The City University of New York, New York, New York Liver tissue of patients with various stages of alcoholic liver injury, with cholestatic liver diseases and with various types of cirrhosis, were examined for the presence of hyalin, mainly by light microscopy and, in selected instances, also by electron microscopy. Hyalin was detected in alcoholic liver disease and in prolonged cholestasis, mainly in primary biliary cirrhosis. It was not found in diseases in which cholestasis was of shorter duration and was predominantly central in location. Electron microscopically, hyalin was identical in alcoholic hepatitis and in primary biliary cirrhosis, although the pattern of injury of hepatocytic organelles was different in these diseases. A morphologic relation between hyalin and cholestasis or any other hepatocellular abnormalities could not be detected. Hyalin was also found in advanced nonalcoholic cirrhosis, in Wilson's disease, and in Indian childhood cirrhosis, and with the exception of the last, it was always associated with peripheral cholestasis. Centrolobular hyalin in specimens with intact lobular architecture is diagnostic for alcoholic liver injury. Peripheral hyalin in the absence of cirrhosis favors primary biliary cirrhosis over extrahepatic biliary obstruction. Hyalin throughout nodules in advanced cirrhosis does not necessarily indicate alcoholic liver disease, Wilson's disease, or Indian childhood cirrhosis, but also occurs in "cryptogenic" cirrhosis. The specificity for alcoholic liver injury of "alcoholic hyalin," first described by Mallory in 191V has been repeatedly questioned because it was demonstrated Received June 22, Accepted August 10, Address requests for reprints to: Dr. Michael A. Gerber, Department of Pathology, Mount Sinai Hospital, 100th Street and Fifth Avenue, New York, New York This work was supported by United States Public Health Service Grants AM and 5T01 GM and by United States Army Medical Research and Development Command Contract DA MD in other disorders. In addition to primary hepatic carcinoma,2 these include Wilson's disease, 3 Indian childhood cirrhosis, 4, 5 and other forms of cirrhosis,6-11 particularly primary biliary cirrhosis. 12 A single factor may account for the occurrence of hyalin in all these diseases or at least in all those mentioned, except alcoholic liver disease and hepatic carcinoma. Prolonged cholestasis is possibly the common denominator in the absence of alcoholism or primary hepatic carcinoma. In view of the diagnostic significance of hyalin, particularly in the recognition of alcoholic liver

2 90 LIVER PHYSIOLOGY AND DISEASE Vol. 64, No.1 TABLE 1. Occurrence of hyalin and accompanying histologic alterations in different liver diseases Disease No. of cases Cases with hyalin Location of hyalin in lobule or nodule Cases with hyalin plus Periph- Choles- Leuko- Ballooned Central eral tasis cytes cells Alcoholic hepatitis 14 Alcoholic hepatitis with hyaline sclerosis 9 approaching the portal tract Alcoholic cirrhosis with alcoholic hepatitis 27 Primary biliary cirrhosis Precirrhotic 18 Cirrhotic 12 Extrahepatic biliary obstruction 14 Biliary atresia 14 Acute viral hepatitis (icteric) 14 Drug or other chemical-induced hepatitis 17 Chronic persistent (portal) hepatitis 9 Chronic aggressive (periportal) hepatitis 30 Cirrhosis with chronic aggressive hepatitis 32 Cirrhosis, active nonalcoholic (selected from cases) Fatal Wilson's disease 4 Indian childhood cirrhosis Precirrhotic 13 Cirrhotic 8 a Peripheral and central III lobule or nodule. Peripheral in nodule. injury, liver tissue from patients with alcoholic liver disease, with the other diseases listed above and with cholestasis from other causes, was examined to ascertain the presence and approximate incidence of hyalin, its relation to cholestasis, its location in the hepatic lobule or nodule, and the accompanying histologic alterations. Material and Methods Liver biopsy or necropsy specimens from 243 patients were examined (table 1). The material came from the files of the Department of Pathology and was not sequential. It included groups of collected cases with specific disorders such as Indian childhood cirrhosis (obtained through the courtesy of Drs. T. V. Madhavan and N. C. Nayak) and fatal Wilson's disease with jaundice and hepatic failure and specimens examined for purposes other than this study. The latter were from patients with chronic persistent (portal) hepatitis and chronic aggressive (periportal) hepatitis 13 and nonalcoholic or cryptogenic cirrhosis as well as alcoholic hepatitis with or without cirrhosis. For a a l a I" a a l a 1 2 comparison, specimens of various cholestatic liver diseases were investigated, such as acute viral hepatitis, drug or other chemical-induced hepatitis, extrahepatic biliary obstruction in adults, biliary atresia in children and primary biliary cirrhosis. Examples of far-advanced active cirrhosis in which hyalin was found in the substantiated absence of alcohol abuse were specifically selected. Three such specimens were obtained by biopsy, and five by necropsy in a period during which 540 cases of cirrhosis without alcoholic history were accumulated. However, such a history could not always be excluded with certainty, in contrast to the 8 cases selected and therefore this represents a maximal figure. Moreover, many of the 540 cases were far less advanced than the ones selected. All specimens were fixed in formalin and embedded in paraffin. Sections 3 to 4 J.l thick were stained with hematoxylin and eosin, chromotrope aniline blue for connective tissue, periodic acid-schiff technique after diastase digestion, or were treated by silver impregnation for reticulum fibers. Bile plugs were best demonstrated in sections which had been subjected to the Prussian blue reaction.

3 January 1973 LIVER PHYSIOLOGY AND DISEASE 91 For electron microscopy, selected biopsy specimens of patients with alcoholic hepatitis or primary biliary cirrhosis were fixed in ice cold 1% osmium tetroxide or 2% glutaraldehyde and embedded in Epon 812. Sections were cut with an LKB Ultrotome, stained with lead citrate, and examined with a Hitachi HS7S electron microscope. Results Hyalin on light microscopy. The hyalin conventionally seen in alcoholic patients was defined as a homogeneously appearing, irregularly outlined meshwork, or clumps in the cytoplasm. With hematoxylin and eosin stain, hyalin was moderately eosinophilic. With aniline blue stain, it usually appeared blue, but red areas were also noted which have been considered different and perhaps later stages of the same material. 14 It did not stain with periodic acid-schiff (PAS) reaction, but PASpositive granules were frequently present in the same cell. Hyalin on electron microscopy. The hyalin appeared as a mass of irregularly intertwined fibrils 15 to 20 nm in diameter. Some fibrils could be followed for more than half a micron. In places, each fibril seemed to be a line of beads giving the appearance of periodicity of 25 to 35 nm. Sometimes hyalin consisted of a compact electron-dense mass in the center surrounded by a loose network of fibrils. The adjacent cytoplasm did not show specific alterations and no membrane separated the whorl of fibrils from the rest of the cytoplasm. There was no relation to bile pigment or to any cell organelle, particularly microbodies, mitochondria, or endoplasmic reticulum. Hyalin in alcoholic hepatitis (fig. 1) did not differ from that in primary biliary cirrhosis (fig. 2), but in alcoholic liver injury, hyalin-containing liver cells often had megamitochondria with crystalloid inclusions in the matrix or interlocking of membranes of adjacent mitochondria. In primary biliary cirrhosis, the mitochondrial cristae were curled and the cytoplasm contained electrondense clumps with or without surrounding membranes considered to be bile pigment (fig. 3). Alcoholic liver lnjury. Hyalin was found in almost all cases of acute alcoholic hepatitis. This disease is characterized by hydropic swelling and necrosis of single or multiple hepatocytes and infiltration of lobular parenchyma by segmented leukocytes. These changes frequently are associated with steatosis and centro lobular fibrosis. In the instances in which the hepatitis was restricted to the FIG. 1. Electron micrograph of a liver cell in alcoholic hepatitis. Fibrillar material is present in the cytoplasm and the mitochondria are swollen (lead citrate, x 20,000).

4 92 LIVER PHYSIOLOG Y AND DISEASE Vol. 64, No.1 FIG. 2. Electron micrograph of a liver cell in primary biliary cirrhosis. The fibrillar material i s identical to that in alcoholic hepatitis. Mitochondria show curling of cristae (lead citrate, x 25,000). FIG. 3. Electron micrograph of a liver cell in hepatitis. The electron-dense, n on-membrane bound granular and fibrillar material is bile pigment and differs from hyalin (lead citrate, x 20,000). centrolobular zone, hyalin was located in the areas adjacent to central necrosis or fibrosis (central hyalin necrosis or sclerosis). It was not related to cholestasis. In more advanced disease, in which fibrotic septa approached the portal tracts, hyalin was also found peripherally around the septa. Usually, inflammation or fibrosis of portal tracts accompanied the lesion. Three of four specimens with peripheral hyalin also showed cholestasis. In alcoholic cirrhosis with alcoholic hepatitis, centro-

5 January 1973 LIVER PHYSIOLOGY AND DISEASE 93 nodular hyalin was found in all instances studied, and peripheral hyalin was also seen in more than half. While central hyalin was not related to cholestasis, peripheral hyalin was found in 71% of cases with cholestasis and only in 38% of those without it. This suggests that cholestasis contributes to the formation of peripheral hyalin also in the alcoholic. Primary biliary cirrhosis. Of 30 specimens from patients with primary biliary cirrhosis, 18 were in the stage of the disease in which destruction of bile ductules was associated with extensive portal and periportal fibrosis and 12 were in the cirrhotic stage characterized by the presence of nodules. Hyalin was observed in six specimens of the first group. It was always located in the periphery of the lobule and in three instances it was associated with central and peripheral cholestasis. In eight specimens of the cirrhotic group with central and peripheral cholestasis, hyalin was observed in the periphery of the nodules (fig. 4). In three instances, it was also found in a central location. Bile pigment was in bile canaliculi but also much was in the cytoplasm of hepatocytes. This meant that almost always the PASnegative hyalin was found in cells with PAS-positive granules. The cytoplasm of most of the hepatocytes near the portal tracts and septa appeared rarified and consisted of thin cytoplasmic strands in large and otherwise seemingly empty cells. Segmented leukocytes were frequently seen around these cells. Cholestasis was present in the absence of hyaline in 12 cases of this series. In one specimen, hyalin was seen in a bile ductule on light and electron microscopy. It had the same tinctorial and electron microscopic characteristics as hyalin in hepatocytes. Biliary obstruction. Hyalin was found in 1 of 14 cases with extrahepatic biliary obstruction. This was the case of a 64-yearold man with jaundice of 3 1 1z-year duration, secondary to stones in the common bile duct. Hyalin in close proximity to bile pigment was present in many hepatocytes both in central and peripheral locations, and these cells were surrounded by seg- FIG. 4. Liver cells in the periphery of a nodule in primary biliary cirrhosis are ballooned and contain hyalin (arrow) (hematoxylin and eosin, x 250). The inset shows the marked cell with hyalin in detail (hematoxylin and eosin, x 540).

6 94 LIVER PHYSIOLOGY AND DISEASE Vol. 64, No.1 mented leukocytes. By contrast, in extrahepatic or combined extrahepatic and intrahepatic biliary atresia, no hyalin was found although cholestasis was both peripheral and central in most cases. Acute icteric liver disease. Hyalin was not found in acute viral hepatitis or in drug- or other chemical-induced hepatitis. Chronic hepatitis and cirrhosis. No hyalin was observed in chronic persistent hepatitis but it was seen in 1 patient with chronic aggressive hepatitis and cholestasis. Cholestasis was present in 7 of the 8 cases of far-advanced cirrhosis with no history of alcohol abuse which were selected because hyalin was found in each. The hyalin was present in the nodular periphery in all cases (fig. 5) and in 4 it was also present throughout the nodules. Leukocytes and hepatocytes with rarified cytoplasm were present in the majority of cases. Fatal Wilson's disease. Four fatal cases of Wilson's disease were characterized by a multilobular or irregular postnecrotic type of cirrhosis with extensive areas of collapse. Large amounts of copper were visualized in the hepatocytes by histochemical techniques. Histologically, extensive proliferation of bile ductules and many segmented leukocytes were seen on the border between nodular parenchyma and septa. Hyalin was in the hepatocytes on the periphery of the nodules in all specimens (fig. 6) and throughout the nodules in three with severe central and peripheral cholestasis. Indian childhood cirrhosis. In the precirrhotic stages of Indian childhood cirrhosis, hyalin was not detected, while it was found in 3 out of 8 cases with cirrhosis (fig. 7), in all of them throughout the nodules. Many segmented leukocytes and cholestasis were present in only 1 of these cases and ballooned cells in 2. Conspicuous destruction of liver cells was found in all. Relation of cholestasis to hyalin. Cholestasis was present in 86% of cases with hyalin in the substantiated absence of alcohol abuse. The cholestasis was always peripheral in addition to the common central location. Therefore, the relation between central and peripheral cholestasis on one FIG. 5. Hyalin (arrow) is present in liver cells in the periphery of a nodule of chronic aggressive hepatitis with cirrhosis (hematoxylin and eosin, x 250). The inset shows the marked cells with hyalin in detail (hematoxylin and eosin, x 540).

7 January 1973 LIVER PHYSIOLOGY AND DISEASE 95 FIG. 6. Hyalin is found in peripheral liver cells in a case of fatal Wilson's disease (hematoxylin and eosin. x 540). FIG. 7. In Indian childhood cirrhosis. hyalin is present in liver cells in the periphery of nodules (hematoxylin and eosin, x 540). hand, and cholestasis and hyalin on the other, was investigated. Bile plugs and cytoplasmic deposition of bile pigment in hepatocytes or Kupffer cells were identical in both locations under the light microscope, except that central cholestasis spared no lobule while peripheral cholestasis was always irregular in intensity and

8 96 LIVER PHYSIOLOGY AND DISEASE Vol. 64, No.1 extent. By contrast, in central cholestasis feathery degeneration, i.e., rarification of the cytoplasm with brown pigmentation was spotty or focal while in peripheral cholestasis, particularly well demonstrated in primary biliary cirrhosis and chronic aggressive hepatitis, almost all hepatocytes on the border between portal tracts and parenchyma were enlarged and their cytoplasm rarified. Under the electron microscope, central and peripheral hepatocytes with the light microscopic changes of cholestasis showed the same picture, namely deposition of electron-dense laminated whorls in single membrane-bound vesicles, possibly derived from the Golgi apparatus. In addition, less dense non-membrane bound granular and amorphous material was noted which contained fibrillar strands measuring 5 to 10 nm in width (fig. 3). Mixtures of the two types of material were also present in the cytoplasm of liver cells. With low magnification, this material showed some resemblance to hyalin. However, no transition could be demonstrated and on high magnification it could be clearly distinguished from hyalin. Bile plugs in bile canaliculi were composed of electron-dense lamellated components, granular and fibrillar material, or a mixture of all. On the periphery of the bile plugs, a few fibrillar strands as described in the cytoplasm were noted. The strands were not similar to the fibrils of hyalin. Discussion The study of selected material of alcoholic and nonalcoholic liver diseases revealed the expected frequent occurrence of hyalin in alcoholic hepatitis (table 1). However, a cluster of cases with hyalin was found in other disorders, particularly those associated with peripheral cholestasis. Hyalin and intracellular bile, which may look similar in hematoxylin eosin or aniline blue-stained sections, were distinguished by treatment with the periodic acid-schiff technique after diastase digestion. The light and electron microscopic characteristics of the hyalin found in alcoholic hepatitis were the same as described by others. 1, The cells with hyalin showed the fine structural changes of alcoholic liver disease, particularly mitochondrial injury.18 When the lobular architecture was intact, the hyalin was centrolobular in location as expected Cholestasis had no recognizable influence on the occurrence of central hyalin. By contrast, in alcoholic liver disease with loss of the lobular architecture or with cirrhosis, hyalin was also seen in a peripheral location. Peripheral hyalin was found in almost threequarters of the specimens in which cholestasis was present, but only in one-third of those in which it was absent. The liver diseases with hyalin in the substantiated absence of alcohol abuse fell into two groups: (1) protracted, mainly intrahepatic biliary obstruction such as primary biliary cirrhosis, and (2) active "cryptogenic" cirrhosis with destruction of the periportal hepatocytes. In both groups, the location of hyalin was predominantly peripheral. Cholestasis was noted in 86% of these cases and, characteristically, it was peripheral in addition to the usual central location. 23 Hyalin was found in one specimen of long-standing extrahepatic biliary obstruction at a time when an intrahepatic mechanical component caused by destruction of the bile ducts led to peripheral cholestasis. The incidence of hyalin in extrahepatic biliary obstruction was low compared with primary biliary cirrhosis where duct destruction was more severe and more likely to create the intrahepatic mechanical component leading to peripheral cholestasis. 23 Two features suggested a relation between hyalin and cholestasis: (1) the frequent coincidence with long-standing and peripheral cholestasis; (2) the presence of bile or at least PAS-positive material in the same hepatocytes which usually were ballooned and had a rarified cytoplasm. However, fine structural analysis failed to show a relation of hyalin to the characteristic features of cholestasis, such as canalicular dilation, curling of mitochondrial cristae, cytoplasmic granular or fibrillar pigment, 24, 25 and whorl-shaped membrane-

9 January 1973 LIVER PHYSIOLOGY AND DISEASE 97 bound material. 23 Moreover, hyalin was topographically not related to any specific organelle as has been claimed for microbodies, 26 mitochondria, 27 endoplasmic reticulum, 15 or cytoplasmic filaments. 28 Hyalin on the periphery or throughout the nodule in active cirrhosis in the absence of alcohol abuse reflects one mechanism of hepatocellular injury, almost always associated with ballooning of liver cells and segmented leukocytes. Its occurrence in biopsy specimens indicates that it is not necessarily a terminal phenomenon. The origin of this characteristic structure in cholestasis thus remains an enigma and no position can be taken as to whether it represents an excess de novo synthesis of protein,26 reflecting an anabolic liver injury29 or a degenerative feature. Central hyalin with or without cholestasis in cases with intact lobular architecture is specific for acute alcoholic liver injury, in which the diagnosis is important because the condition is reversible with treatment. In this disease, hyalin has been used as a prognostic sign. 30 By contrast, peripheral hyalin has no such connotation, although in the absence of cirrhosis its presence favors the diagnosis of the precirrhotic stage of primary biliary cirrhosis. This finding might help in the diagnosis of nonsurgical jaundice. Hyalin in cirrhosis does not imply a specific etiology, particularly in the presence of cholestasis since it occurs in several types of active cirrhosis. Even in cirrhosis in known alcoholics, hyalin need not reflect acute alcoholic hepatitis from recent alcohol abuse because it might be related to long-standing cholestasis. Contrary to our own previous belief and that of many others, hyalin does not seem to be a diagnostic feature of Indian childhood cirrhosis or of Wilson's disease,31 but it probably reflects advanced cirrhosis usually with cholestasis at least in the latter. REFERENCES 1. Mallory FB: Cirrhosis of the liver: Five different lesions from which it may arise. Bull Johns Hopkins Hosp 22:69-75, Norkin SA, Campagna-Pinto D: Cytoplasmic hyaline inclusions in hepatoma. A histochemical study_ Arch Pathol 86:25-32, Popper H, Schaffner F: The hepatic lesion in Wilson's disease, Wilson's Disease: Some Current Concepts. Edited by JM Walshe, IN Cumings. Oxford, Blackwell Scientific Publications, Oxford, 1961, Smetana HF, Hadley GG, Sirsat SM: Infantile cirrhosis: An analytical review of the literature and report of 50 cases. Pediatrics 28: , Nayak NC, Sagreiga K, Ramalingaswami V: Indian childhood cirrhosis. The nature and significance of cytoplasmic hyalin of hepatocytes. Arch Pathol 88: , Baggenstoss AH, Stauffer MH: Posthepatitic and alcoholic cirrhosis: clinicopathologic study of 43 cases of each. Gastroenterology 22: , Harrel GT, McBryde A: Cirrhosis of the liver in children. Am J Dis Children 59: , Read AE, Sherlock S, Harrison CV: Active "juvenile" cirrhosis considered as part of a systemic disease and the effect of corticosteroid therapy. Gut 4: , Mistilis SP: Pericholangitis and ulcerative colitis. 1. Pathology, etiology and pathogenesis. Ann Intern Med 63:1-16, Becker BJP: The nature of alcoholic hyalin. Lab Invest 10: , Popper H: Seminar on Diseases of the Liver. American Society of Clinical Pathologists. Edited by EA Gall. Chicago, 1968, p Ament M, Fenster LF: Mallory bodies in chronic cholestasis (abstr). Gastroenterology 58:278, Popper H, Schaffner F: The vocabulary of chronic hepatitis. N Engl J Med 284: , Roque AL: Chromotrope aniline blue method of staining Mallory bodies of Laennec's cirrhosis. Lab Invest 2:15-21, Biava C: Mallory alcoholic hyalin: A heretofore unique lesion of hepatocellular ergastosplasm. Lab Invest 13: , Flax NH, Tisdale W A: An electron microscopic study of alcoholic hyalin. Am J Pathol 44: , Smuckler EA: The ultrastructure of human alcoholic hyalin. Am J Clin PathoI49: , Klion FM, Schaffner F: Ultrastructural studies in alcoholic liver disease. Digestion 1:2-14, Iseri OA, Gottlieb LS: Alcoholic hyalin and megamitochondria as separate and distinct entities in liver disease associated with alcoholism. Gastroenterology 60: , Edmondson HA, Peters RL, Reynolds TB, et al: Sclerosing hyaline necrosis of the liver in the

10 98 LIVER PHYSIOLOG Y AND DISEASE Vol. 64, No. 1 chronic alcoholic: A recognizable syndrome. Ann Intern Med 59: , Edmondson HA, Peters RL, Frankel HH, et al: The early stage of liver injury in the alcoholic. Medicine 64: , Gerber MA, Popper H: Relation between central canals and portal tracts in alcoholic hepatitis. A contribution to the pathogenesis of cirrhosis in alcoholics. Hum Pathol 3: , Popper H, Schaffner F: Pathophysiology of cholestasis. Hum Pathol 1:1-24, Biava C: Studies on cholestasis. The fine struc ture and morphogenesis of hepatocellular and canalicular bile pigment. Lab Invest 13: , Desmet VJ: Cholestasis, Progress in Liver Diseases, vol 4. Edited by H Popper, F Schaffner. New York, Grune and Stratton, 1972 (in press) 26. Keely AF, Iseri OA, Gottlieb LS: Ultrastructure of hyaline cytoplasmic inclusions in a human hepatoma: relationship to Mallory's alcoholic hyalin. Gastroenterology 62: , Porta EA, Bergman BJ, Stein AA: Acute alcoholic hepatitis. Am J Pathol 46: , Yokoo H, Minick OT, Batti F, et al: Morphologic variants of alcoholic hyalin. Am J Pathol 6:25-40, Madhavan TV, Schaffner F, Popper H: Catabolic and anabolic hepatotoxicity and nutrition. Am J Clin Nutr 23: , Rice JD Jr, Yesner R: The prognostic significance of so-called Mallory bodies in portal cirrhosis. AMA Arch Intern Med 105: , Popper H, Orr W: Current concepts in cirrhosis. Scand J Gastroenterol 6: , 1970