Ground Glass Hepatocytes Research Articles

TGF-$alpha; accumulation in ground glass hepatocytes of chronic hepatitis B virus infection

TGF-$alpha; accumulation in ground glass hepatocytes of chronic hepatitis B virus infection

Ground‐glass hepatocytes with Lafora body like inclusions—histochemical, immunohistochemical and electronmicroscopic characterization

We report two adult patients whose liver biopsy specimens revealed numerous ground-glass hepatocytes due to inclusions resembling Lafora bodies. The inclusions were large, intracytoplasmic, pale, eosinophilic and kidney-shaped and were periodic acid-Schiff positive and HBsAg negative. Immunoperoxidase studies showed that the inclusions were positive for cytokeratins and alpha 1-antitrypsin. In case 1, the inclusions were not membrane-bound and consisted of secondary lysosomes and degenerate organelles including rough and smooth endoplasmic reticulum. In case 2, electronmicroscopy showed the inclusions were not membrane-bound, but consisted of dense granules, fibrils and vacuoles, with appearances very similar to Lafora bodies. Neither patient had myoclonus or epilepsy. Electronmicroscopy is important in differentiating the type of Lafora body like inclusions found in liver biopsies.

Hepatic expression patterns of the large and middle hepatitis B virus surface proteins in viremic and nonviremic chronic hepatitis B

The envelope of hepatitis B virus consists of large, middle, and small hepatitis B surface proteins. Recent data from in vitro studies suggest that intracellular expression and distribution of the three polypeptides may be variable. These observations in artificial expression systems prompted this analysis of the occurrence and distribution of the three hepatitis B surface proteins in the liver tissue of substantial viremic (hepatitis B virus DNA- and hepatitis B e antigen-positive) and low-viremic or nonviremic (hepatitis B virus DNA-negative, anti-hepatitis B e antigen-positive) carriers by specific monoclonal antibodies against large, middle, and small proteins. Patients with an active form of viral replication showed a prevalence of middle and small hepatitis B surface proteins in the liver. In nonviremic carriers, the large hepatitis B surface protein was the predominant intrahepatic antigen, a finding that was confirmed at the ultrastructural level by staining of the entire filaments of the viral envelope material in ground glass hepatocytes. The present data are thus consistent with observations in hepatitis B virus-transgenic mice and in transfected cell systems, suggesting that the different patterns of the envelope proteins in the liver may be due to different processing at the translational level.

Co‐existence of hepatocyte ground‐glass inclusions from several causes

Two patients with two and three types respectively of ground-glass hepatocyte inclusions are described. Both were hepatitis B surface antigen (HBsAG) positive and received cyanamide for alcohol aversion therapy. In addition, one of them had taken benzodiazepines and barbiturates. In one patient, cyanamide and HBsAg inclusions co-existed in the same hepatocytes.

Biology of hepatitis B virus

Detailed information is now available about the structure of the hepatitis B virus (HBV) genome and about that phase of virus replication that involves reverse transcription of a “pregenomic” RNA precursor molecule. More recent work has explored the possible role of extrahepatic sites of infection, the secretion of envelope and core proteins from infected cells, the role of virus replication in liver damage and production of ground glass hepatocytes, the possible mechanism whereby persistent HBV infection predisposes to development of hepatocellular carcinoma, and the theoretical basis for future design of antiviral drugs. Much of this current work has been facilitated by availability of (1) experimental models provided by animal hepadnaviruses (2) use of transgenic mice expressing various HBV gene products (3) new cell culture systems that support partial or complete virus replication following infection with complete virus or viral DNA.

Endoplasmic storage disease of liver: characterization of intracytoplasmic hyaline inclusions

Two cases of endoplasmic storage disease of liver are described. The liver tissue in each case showed numerous intracytoplasmic hyaline inclusions of varying sizes with formation of ground-glass hepatocytes. These inclusions were pale eosinophilic in hematoxylin & eosin stained sections, and were periodic acid-Schiff and HBsAg negative. Immunoperoxidase studies revealed strong positivity for fibrinogen and complement components C3 and C4 in case 1 and exclusive positivity for fibrinogen in case 2. On electron microscopy, the inclusions appeared as granular or fibrillar material within dilated cisternae of rough endoplasmic reticulum.

Binding of biotin to hepatitis B surface antigen: a possible pitfall in immunohistochemistry.

We report on the binding of biotin, and hence of biotinylated antibodies and lectins, to ground glass hepatocytes and liver cell membranes in chronic hepatitis B viral infection. This binding is of low affinity, and was proved to be directed at the hepatitis B surface antigen, presumably at its disulfide bonds. To avoid false-positive results, this affinity should be considered in the interpretation of immunohistochemical stainings of hepatitis B virus-infected liver tissue with biotinylated reagents.

Cyanamide hepatotoxicity. Incidence and clinicopathological features

Ground-glass hepatocytes resembling those seen in HBsAg carriers on hematoxylin and eosin and on trichrome stained sections, but giving a negative reaction to orcein and a positive one to PAS, were found in liver biopsy specimens from nine asymptomatic former alcoholics who were on treatment with cyanamide, in one of four who had been treated with cyanamide several months before the liver biopsy procedure, in none of 15 treated with disulfiram, and in one of eight who had apparently not received aversive drugs. Portal and periportal inflammatory changes and fibrosis were more frequently observed in biopsy specimens containing PAS-positive ground-glass hepatocytes than in those without, but cirrhosis was found with a similar frequency. It is concluded that periportal PAS-positive ground-glass hepatocytes are a histological marker of cyanamide treatment.

Expression of pre-S gene-encoded proteins in liver and serum during chronic hepatitis B virus infection in comparison to other markers of active virus replication

Pre-S gene-encoded proteins of the hepatitis B virus (HBV) were studied in the liver by immunofluorescence and in serum by radioimmunoassay in 30 patients with chronic HBV infection. The results were compared with molecular hybridization analysis of HBV-DNA in liver and serum, with serum hepatitis B e antigen/antibody (HBeAg/anti-HBe) status and with underlying liver histology. Pre-S peptides were detected in the serum of 11 patients, 10 of whom were positive for serum HBV-DNA and/or liver hepatitis B core antigen. Only 4 of these patients were HBeAg positive. The prevalence of serum pre-S among HBV replicating carriers was 59% (10/17) compared to only 8% (1/13) among those with non-replicating virus (P less than 0.01). All patients with circulating pre-S peptides had active liver disease. Anti-pre-S was detected in the serum of only 4 patients, 3 with integrated HBV-DNA. In contrast to serum findings, pre-S peptides were detected in the liver of all patients with histochemically demonstrable hepatitis B surface antigen (HBsAg), regardless of HBV replicative status. HBsAg carriers with integrated HBV-DNA had abundant cytoplasmic pre-S1 and pre-S2 localized in numerous ground-glass hepatocytes. It is concluded that pre-S peptides are usually displayed in the liver simultaneously with histochemically detectable HBsAg; they are secreted in the serum in association with high HBV replication and release of HBV particles, but in the absence of episomal HBV replication, pre-S peptides seem to be largely retained within hepatocytic membranes.

Ground-glass hepatocytes: A spectrum of inclusions

A new type of ground-glass hepatocyte is described. The appearance is due to pale, homogeneous, weakly eosinophilic inclusions filling a portion of or the entire hepatocytic cytoplasm. On haematoxylin and eosin stained sections, these cells closely resemble ground-glass hepatocytes described in other conditions. However, they are negative on special stains for HBsAg and on PAS staining. Immunohistochemically, they reveal a selective and exclusive positivity for fibrinogen. On electron microscopy, the immunoreactive fibrinogen appears as amorphous, fluffy or granular material within dilated cisternae of the rough endoplasmic reticulum. This finding suggests intracellular storage possibly reflecting a defective intracellular transport of fibrinogen.

Diverse virological, histopathological and prognostic implications of seroconversion from hepatitis B e antigen to anti-HBe in chronic hepatitis B virus infection

The evolutions of serum hepatitis B virus (HBV)-DNA, liver histology and intrahepatic expressions of HBV antigens were longitudinally investigated in 24 serum HBeAg+/HBV-DNA+ chronic hepatitis B patients who subsequently seroconverted to anti-HBe. After HBeAg conversion, serum HBV-DNA still persisted in 10 patients, and liver HBcAg in 7 of them. Of the 24 patients, 3 subgroups with diverse prognoses were identified. Ten patients progressed from chronic active hepatitis to cirrhosis, and in 7 of them HBV-DNA and/or HBcAg persisted. Eight patients with undetectable HBV-DNA and HBcAg recovered. In the remaining 6 patients, chronic liver diseases persisted; in 3 of them, HBV-DNA and in one HBcAg. These findings indicate that continued viral replication is present in a significant number of patients after HBeAg seroconversion in Taiwan, and is responsible for disease progression. In addition to HBcAg and HBV-DNA, the severity of underlying liver histology, when HBeAg seroconversion occurred, was critical for the outcome of the disease. Another remarkable finding was that clusters of ground-glass hepatocytes, well correlated with the marginal expression of HBsAg, were demonstrated in 14 of 16 biopsies with serum anti-HBe+/HBV-DNA-, but found in only 4 of 44 biopsies with positive serum HBV-DNA, indicating a strong association of the expressions of liver histology and hepatocyte HBsAg with the status of viral replication.

Fibrinogen inclusions in liver cells: A new type of ground-glass hepatocyte. Incidence and significance in liver biopsies

Fibrinogen inclusions in liver cells: A new type of ground-glass hepatocyte. Incidence and significance in liver biopsies

Hepatitis B surface antigen (HBsAg)-fibrinogen interaction.

Deparaffinized sections of hepatitis B surface antigen (HBsAg) positive liver biopsies, when incubated in a human fibrinogen solution, reveal a strong labelling of ground-glass hepatocytes with fibrinogen. This implies an interaction between HBsAg and fibrinogen. The binding of fibrinogen to HBsAg is thought to be mediated by the protein moiety of HBsAg, since its binding capacity is destroyed by trypsin digestion, and to occur through formation of disulphide bridges because the binding can be disrupted by the reducing effect of 2-mercapto-ethanol. Not all ground-glass hepatocytes exhibit fibrinogen binding. The reason for this is at present unclear. No relationship between serum HBeAg positivity and binding of HBsAg to fibrinogen was observed. Preincubation in a fibrinogen solution did not alter the immunoreactivity of HBsAg. We conclude that there is a striking analogy between the interaction of HBsAg with polymerized human serum albumin and its interaction with fibrinogen.

Hepatic oncocytes. Incidence, staining characteristics, and ultrastructural features.

Hepatocytes with orcein-positive ground-glass cytoplasm have been shown to contain abundant hepatitis B surface antigen in the cisternae of excess smooth endoplasmic reticulum. Hepatocytes with similar cytoplasmic changes that did not react with orcein were observed in 26.5% of 49 cases of cirrhosis. These cells exhibited granular, deeply eosinophilic cytoplasm that stained with phosphotungstic acid-hematoxylin and contained numerous densely packed mitochondria as demonstrated by electron microscopy. Therefore, these cells were designated hepatic oncocytes. They were detected predominantly in cases of established cirrhosis, unrelated to etiology. Hepatic oncocytes may form nodular aggregates, but they did not show evidence of regeneration. The nature and pathogenesis of these cells remain unclear. Because of similar appearance, histochemical stains may be necessary to distinguish ground-glass hepatocytes from hepatic oncocytes.

Oxyphilic granular hepatocytes. Mitochondrion-rich liver cells in hepatic disease.

In the course of examining liver biopsy specimens, certain larger than normal liver cells whose cytoplasm is finely granular and strikingly acidophilic have been seen. These cells are called "oxyphilic granular hepatocytes" (oxyphils) because of their similarities to the "oncocytes" of the salivary glands and other endocrine organs. Oxyphilic liver cells can be readily differentiated from acidophilic bodies and groundglass hepatocytes by light and electron microscpy, the latter showing them to be extraordinarily rich in mitochondria. A retrospective study of 214 consecutive liver biopsies was undertaken to determine the prevalence of oxyphilic cells in a variety of liver diseases. Oxyphils were identified in 15% of the biopsy specimens, and were most strongly associated with chronic active hepatitis and cirrhosis in hepatitis B surface antigen (HBsAg)-positive patients. Subsequent reevaluation of 77 biopsy specimens from HBsAg-positive patients showed oxyphils in 28.6%. Their pathogenesis and significance in chronic liver disease are unknown.

Hepatitis-B surface antigen in tumour tissue and non-tumorous liver in black patients with hepatocellular carcinoma.

Formalin-fixed, paraffin-embedded sections of liver and tumour tissue obtained at necropsy from 44 southern African Blacks with hepatocellular carcinoma were stained for hepatitis-B virus surface antigen by immunofluorescence, immunoperoxidase and orcein techniques. The antigen was present in the serum of 68% of the patients. Staining for tissue antigen was positive in 45% of the patients. Non-tumorous hepatocytes alone stained positively in 22.5% of patients, tumour cells alone in 12.5% and both in 10%. Antigen was present in relatively few tumour cells and the amounts detected were small; it was more readily detectable in moderately differentiated than in poorly differentiated malignant cells. Identical results were obtained with immunofluorescence and immunoperoxidase staining, but the orcein stain failed to demonstrate the antigen in tumour cells. Cirrhosis was present in the non-tumorous liver in 70% of the patients. Antigen was detected in cirrhotic tissue in 43% of the patients with cirrhosis, and in non-tumorous liver tissue in 8% of those without cirrhosis, but this difference was not significant. The antigen frequency in tumour tissue was the same in patients with and without cirrhosis. No correlation was found between the presence of liver-cell dysplasia and the presence or absence of either the antigen or cirrhosis in the non-tumorous liver tissue. Ground-glass hepatocytes were seen in non-tumorous liver tissue of 5 patients, but not in tumour tissue. While 54% of the patients with antigenaemia had demonstrable tissue antigen, 10% of patients with tissue antigen had no detectable antigenaemia.

Detection of HBsAg containing cells in liver biopsies by different stains and classification of positively reacting ground-glass hepatocytes

The diagnostic significance of orcein, aldehydthionine, and chromotrope anilinblue stains for the demonstration of HBsAg containing hepatocytes was investigated in 602 unselected liver biopsies. Five types of specifically stained ground-glass hepatocytes (GGH) were distinguished: Type I showed a positive staining reaction of the cytoplasmic periphery (marginal GGH), type II a diffuse staining of the total cytoplasm (diffuse GGH). Type III contained round or oval globular positive cytoplasmic masses (globular GGH). Type IV showed only very small round, drop-like or sickle-shaped positive structures (spotty GGH). The GGH with fatty changes were designated as type V. In all carriers and patients with minimal hepatitis GGH, mostly type I and II, appeared in extensive clusters within the lobules. In chronic persistent hepatitis, there were moderately numerous, partly grouped, partly disseminated ground-glass hepatocytes of type II and III. In chronic active hepatitis there were only a few GGH of type IV. In acute viral hepatitis, there were no typical GGH, however, positively stained phagocytes were seen. The intracellular antigen localization and the intralobular distribution of GGH are considered to be the result of an immune reaction. Single so-called 'metabolic' GGH sometimes showed similar pictures. However, they could usually be distinguished from virus containing GGH because of their granular cytoplasmic structure and a lower staining intensity in the applied stains. Among the three stains the orcein stain yielded the best results. In some cases with HBsAg-positive chronic active hepatitis virus infection could not be proved by means of staining.

Hepatitis B surface antigen carriers — To biopsy or not to biopsy

In order to assess the frequency of significant liver disease in hepatitis B surface antigen carriers with normal liver tests, 54 such individuals were identified and prospectively followed for 4 to 48 months with monthly liver tests. Upon testing, 4 were found to carry e antigen and 14 carried e antibody (anti-e). During follow-up, only 4 patients, none of whom were e antigen-positive, developed persisting abnormalities in liver tests. Of the 23 patients who underwent percutaneous liver biopsies, normal histologies were found in 2, nonspecific changes (ground glass hepatocytes, focal necrosis, fatty changes, etc.) in 18, and chronic persistent hepatitis (with or without other nonspecific changes) in 3. Chronic active hepatitis and/or cirrhosis, lesions which may carry more serious prognostic implications, were not seen in any biopsies. Two of the 4 e antigen-positive patients consented to biopsy, both of whom had chronic persistent hepatitis. All 6 patients with anti-e who underwent biopsy had ground glass hepatocytes, which were found in only about 50% of the remaining patients. It is concluded that hepatitis B surface antigen carriers should be followed with serial liver tests, and those whose tests remain normal should not be considered for liver biopsy.

Demonstration of Hepatitis B‐Surface Antigen in Liver Biopsies

Staining of 1000 consecutive liver biopsies with orcein showed positive reaction in the cytoplasm of ground-glass hepatocytes in 18 of the biopsies. On new sections of the 18 orcein positive biopsies immunoperoxidase staining was performed in order to demonstrate hepatitis B-surface antigen (HBsAg). After destaining the same sections were stained with orcein. All biopsies showing positive orcein staining showed positive immunoperoxidase reaction. The number of positive cells was in biopsies with less than 20 positive cells per mm2 biopsy larger using the immumoperoxidase staining than with orcein staining. Further the staining contrast was more pronounced. Immunoperoxidase staining thus seems more sensitive than orcein staining for demonstrating HBsAg in liver tissues. Orcein stains HBsAg in liver tissue even though the antigen determinants are blocked by antibodies.

Immunohistochemical patterns of hepatitis B surface antigen (HBsAg) in patients with hepatitis, renal homografts recipients and normal carriers.

A series of 180, Bouin-fixed and paraffin embedded liver biopsies obtained from 147 patients was investigated for the presence of hepatitis B surface antigen (HBs) by histochemical and indirect immunofluorescence techniques. A comparison between orcein staining and Masson's trichrome preparations for ground glass hepatocytes, showed that immunofluorescence was both the more reliable and the more specific method for detection of HBsAg in liver tissue. The ability to perform this technique on paraffin sections facilitates systematic studies and allows retrospective work-up. IF-HBs positive hepatocytes were found in approximately two thirds of all HBs-positive patients in their serum, but never seen in HBs-negative patients. HBs-positive cells were observed in healthy chronic carriers and in all forms of chronic hepatitis, but never in acute HBs-positive hepatitis. In patients treated with chronic hemodialysis and in renal homograft recipients, the incidence of positive cells was higher than in the chronic hepatitis groups; this could be correlated with the duration of antigenemia at the time of biopsy.