Orcein staining of hepatitis B antigen in paraffin sections of liver biopsies.

Liver biopsies from 97 hepatitis B antigen (HBsAG)-positive patients were stained by a modified orcein method described by Shikata et al (1974) in order to detect the antigen in liver tissue. The results were consitently negative in acute hepatitis, but positive in nearly two-thirds of biopsies from 53 patients with chronic liver disease. The distribution of positive staining was frequently irregular so that there is a problem of sampling error in needle biopsies. The deposits were seen in the cytoplasm of liver cells and occasionally in Kupffer cells, but never in nuclei. There was an inverse relationship between staining and parenchymal necrosis. Biopsies from asymptomatic HB(s)Ag carriers were often strongly positive, as were "ground-glass" hepatocytes in carriers and patients with chronic liver disease. The mechanism of staining is unclear but may be related to the presence of disulphide bonds in (HBsAG. The technique is simple and of use both in fresh and stored material.     

Detection of hepatitis B surface antigen by means of orcein staining of liver.

Paraffin-embedded sections of liver biopsies from 65 patients and 16 autopsies were stained by a modified orcein method as described by Shikata, to appraise the efficacy of the method in detecting hepatitis B antigen, (HGsAg). Of the liver biopsies from patients with chronic hepatitis, 55% were positively stained. Staining was observed in the cytoplasm of liver cells but was frequently irregular in distribution. The technic is simple and may be used for evaluation of fresh or stored liver. The positive stain is of value in the diagnosis of chronic hepatitis with minimal or absent histologic changes, as well as the distinction of chronic from acute hepatic disease. The method may be useful for detecting asymptomatic carriers. However, weak positive staining of hepatic parenchyma should be evaluated with caution when serologic tests for HBsAg are negative. It may warrant repeated serologic testing. Evaluation of stored autopsy material suggests that Shikata's stain may also be used in determining the etiology of cirrhosis. Inclusion bodies seen in hepatomas stain positively with orcein, suggesting that they contain, or are related to, HBsAg.     

Hepatitis B core antigen (HBcAg) in serum of patients with chronic hepatitis B detected by a modified radioimmunoassay

Serum hepatitis B core antigen (HBcAg) was investigated in 85 patients with chronic hepatitis B virus (HBV) infection using a modified radioimmunoassay technique, based on high molarity treatment of samples to avoid masking of the antigen by the excess homologous antibody. Eighty-eight percent of HBeAg-positive cases and 19% of anti-HBe-positive cases were HBcAg positive in serum, with a positive correlation with the presence of HBcAg in the liver. Although the sensitivity of the method for the presence of complete virions was not absolute, as shown by the comparison with serum HBV-DNA testing, this technique may be helpful for assessing virus synthesis in patients with HBV infection.     

Demonstration of carcinoembryonic antigen in bone marrow from patients with carcinoma.

Primary and secondary tumour and bone marrow trephine biopsies from 20 patients with carcinomas were stained for carcinoembryonic antigen by the three stage immunoperoxidase method. Six marrow biopsies contained tumour deposits, five of which were positive for carcinoembryonic antigen. A further five marrow biopsies contained single carcinoembryonic antigen positive cells of uncertain origin. Carcinoembryonic antigen staining may be a useful adjunct to conventional histology in the diagnosis of marrow metastases.     

A trichrome stain for the intrahepatic localization of the hepatitis B surface antigen (HBsAg)

A modified trichrome stain is described for the intrahepatic localization of the hepatitis B surface antigen; HBsAg containing cells exhibit specific green metachromasia contrasting with the granular brown colour of non infected hepatocytes and with the deep eosinophilic colour of ground glass cells of HBsAg-negative alcoholic or drug hepatitis. The technique is simple and reliable for routine screening of HBsAg positive material; its sensitivity is greater than H & E, similar orcein and inferior to immunohistochemistry as performed on frozen sections. Histological diagnosis can be made on the same slide, since several other morphological details are provided in the trichrome stained preparations. With this technique 387 biopsies from HBsAg seronegative individuals were negative; full cytoplasms metachromasia was mostly seen in asymptomatic HBsAg carriers, focal or partial staining in patients with histological evidence of liver cell necrosis. The presence and the staining pattern of HBsAg were of no help in predicting transition to chronicity or a transition from chronic persistent to chronic active hepatitis.     

Correlation of HBV DNA and monoclonal reactivity to HBsAg in serum of patients with HBV infection

Hepatitis B virus (HBV) DNA hybridization assay, a monoclonal radioimmunoassay (M-RIA) for hepatitis B surface antigen (HBsAg) and conventional polyclonal immunoassays for HBV associated antigens were used to study sera from patients on dialysis and with acute hepatitis B. HBV DNA was detectable in hepatitis B e antigen (HBeAg) negative patients with acute hepatitis but not in HBsAg+ HBeAg- dialysis patients. In acute hepatitis, HBsAg immunoreactivity by M-RIA could still be detected even though a commercial immunoassay for HBsAg, the AUSRIA II, and the HBV DNA assay were no longer positive. Unlike in acute HBV infection, serum HBV DNA was detectable in dialysis patients who were AUSTRIA II negative but M-RIA positive. Serial determination of HBsAg by M-RIA and HBV DNA revealed episodes of HBV DNA positivity months after both the HBsAg was no longer positive by polyclonal immunoassay. Thus, the M-RIA for HBsAg and the molecular hybridization technique for HBV DNA are sensitive and specific assays for the identification of potentially infectious individuals who would not have been characterized as such based on the results of conventional polyclonal immunoassays.     

Distribution patterns of hepatitis B surface antigen (HBsAg) in the liver of hepatitis patients.

One hundred liver biopsies from 100 hepatitis patients were examined by the indirect immunofluorescent technique for the detection of HBsAg. Of the 60 positive specimens 52 were diagnosed as various types of chronic hepatitis and 8 were acute hepatitis. Four main distribution patterns of HBsAg were obtained: full cytoplasmic fluorescence with diffuse lobular distribution; cytoplasmic fluorescence with spotty distribution; peripheral fluorescence in the cell membrane and/or cell peripheries; and focal cytoplasmic positivity. There was an inverse relationship between the number of positive hepatocytes and the extent of liver cell necrosis. The distribution patterns of HBsAg were distinctive in each type of chronic hepatitis and in acute hepatitis. Homogeneous full cytoplasmic fluorescence, distributed diffusely in the whole liver lobule, was observed in chronic persistent hepatitis and in cirrhosis with little activity whereas peripheral liver cell membrane and/or peripheral cytoplasmic fluorescence associated with cytoplasmic positivity in a smaller number of hepatocytes was a characteristic finding in chronic aggressive hepatitis, active cirrhosis, and acute hepatitis with possible transition to chronicity. Focal cytoplasmic fluorescence was observed in acute hepatitis and a group of biopsies in chronic hepatitis in which HBsAg was detected in the liver but no antigen was detectable in the serum. The results show that the different patterns of distribution of HBsAg in the liver biopsy are helpful for the histological diagnosis of different types of HBAg positive viral hepatitis and are consistent with the hypothesis of the role of specific immune response in the pathogenesis of type B viral hepatitis.     

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

Summary 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.     

Persistence of hepatic hepatitis B virus after serological clearance of HBsAg with autologous peripheral stem cell transplantation.

Delayed clearance of hepatitis B surface antigen was previously reported in a 38 year old woman after high dose chemotherapy with autologous peripheral blood stem cell rescue. Sixteen months later, this patient remained hepatitis B surface antigen negative, hepatitis B surface antibody positive, and serum hepatitis B DNA negative by polymerase chain reaction. Serial liver biopsies (one at hepatitis B e antigen positive stage, one at hepatitis B e antibody positive stage, and one at hepatitis B surface antigen negative and hepatitis B surface antibody positive stage) showed a gradual resolution of the inflammatory activity with loss of hepatitis B e antigen and then hepatitis B surface antigen in the serum. However, the degree of fibrosis, though mild, remained the same. With the serological clearance of hepatitis B surface antigen, a small amount of hepatitis B virus DNA was still detectable in the nuclei of liver cells.     

Histopathology and localization of viral antigens in the liver of HBsAg positive homosexuals

In a survey of the prevalence of chronic hepatitis B in a male homosexual population, liver biopsies were done in 28 asymptomatic patients who had persistently raised aminotransferases. Four patients had active cirrhosis (AC), 13 had chronic active hepatitis (CAH) of various degrees of severity and 11 had either chronic persistent hepatitis (CPH) or minor changes of the type seen in hepatitis B virus carriers. Core associated antigens and surface antigen, were demonstrated by the PAP immunoperoxidase method in 20 cases. Core and surface antigens tended to be present in the same areas of the biopsy and quantitation showed higher core to surface antigen ratios in CAH than in CPH, the difference being statistically significant. In seven cases no core-associated antigens were demonstrated in the presence of surface antigen: most of these patients had either inactive disease or active cirrhosis. In one carrier neither antigen was demonstrated. Ten patients had two or more biopsies. Four of these had no treatment and the amounts of core and surface positive cells in the liver did not increase. Six were treated with immunosuppressants. This did not alter the degree of either inflammation or fibrosis, but the number of surface and core antigen positive cells in the liver was higher after treatment in almost every case.     

Clinical significance of a highly sensitive enzyme immunoassay of hepatitis B surface antigen using a novel electron spin resonance technique.

We developed a highly sensitive enzyme immunoassay (EIA), the p-AP/HHTIO method, that detects serum hepatitis B surface antigen (HBsAg) by measuring stabilized nitroxide radicals using a novel electron spin resonance technique [Matsuo et al. (1998) Free Radic Biol Med 25:929-935]. To demonstrate the clinical significance of this method and to reveal occult hepatitis B virus (HBV) infection in patients, we used the method to analyze serum samples of 30 patients with acute or fulminant hepatitis who were negative for HBsAg by standard EIA, and those of seven chronic HBV carriers who became negative for HBsAg during a follow-up period by standard EIA. We also examined serum HBV DNA by amplification of the HBV S gene, using the polymerase chain reaction (PCR) technique. The p-AP/HHTIO method showed that 9 of 20 (45%) patients with acute hepatitis and 2 of 10 (20%) with fulminant hepatitis were positive for HBsAg; PCR detected HBV DNA in these HBsAg-positive patients. Antibody against hepatitis B core antigen was detected in one patient with fulminant hepatitis. The p-AP/HHTIO method demonstrated prolonged seropositivity of HBsAg even after standard EIA showed a loss of HBsAg in all seven HBV carriers. Our p-AP/HHTIO method is useful for screening and diagnosing HBV infection in patients with liver diseases who are negative for conventional HBV-related serological markers.     

Ultrastructural features in chronic non-A, non-B (NANB) hepatitis: A controlled blind study

Liver biopsies from 12 patients with chronic Non-A, Non-B (NANB) hepatitis, 7 with hepatitis B surface antigen (HBsAg) positive chronic liver disease, 1 HBsAg positive normal carrier, and 4 patients with non-viral liver disease, were examined by electron microscopy for cytoplasmic and nuclear changes. Aggregates of particles measuring 20-35 nm in diameter were noted in the nuclei of 8 of 12 patients with NANB chronic hepatitis, but not in the other groups. The tubular changes seen in the endoplasmic reticulum (ER) of chimpanzees with NANB hepatitis were not noted in biopsies from any of our patients.     

Demonstration of hepatitis B-surface antigen in liver biopsies. A comparative investigation of immunoperoxidase and orcein staining on identical sections of formalin fixed, paraffin embedded tissue.

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.     

Comparison of three serological methods for the detection of hepatitis-associated antigen

Serum hepatitis (Australia) antigen in the sera of hepatitis patients and carriers can be detected in one and a half to three hours by crossover electrophoresis. The method is more sensitive than the immunodiffusion technique commonly employed in this field. It is of the same order of sensitivity as complement fixation but is less complicated.Crossover electrophoresis is thus the method of choice for the rapid screening of sera for hepatitis antigen; complement fixation may be used for quantitative determination of antigen in positive cases.     

Co-expression of markers for hepatitis delta and hepatitis B viruses in human liver

Delta hepatitis (HDV) infection can only occur in the presence of hepatitis B (HBV) infection, as HDV requires a coat of HBV surface antigen (HBsAg) for assembly of complete virus. A number of studies have examined the variation of HBV markers in serum and liver during establishment of HDV infection, but none has systematically examined the relationship between the two viruses in individual hepatocytes. Liver biopsies from five patients with HDV/HBV infection were stained for HBsAg, HBV core antigen (HBcAg) and hepatitis D (delta) antigen (HDAg). Double immunostaining was performed with a combination of indirect immunoperoxidase and alkaline phosphatase/antialkaline phosphatase techniques. HDV and HBV antigens were expressed in all five liver biopsies. Co-localization of HBsAg was seen in up to 39% of HDAg positive cells, and HBcAg in up to 8% of HDAg positive cells. HBcAg was detectable in approximately 9% of HBsAg positive cells, and HBsAg in approximately 12% of HBcAg positive cells. HDV can replicate without HBV but ultimately requires HBV to produce complete virus and subsequently infect other cells. In this study the majority of HDV positive cells did not appear to contain HBV markers. This might suggest delta virus replication without assembly, or possibly sequential production/assembly of the virus.     

Hepatitis B virus DNA patterns in the liver of children with chronic hepatitis B

The pattern of hepatitis B virus DNA (HBV-DNA) expression were studied in 2 sequential liver biopsies from 26 children (18 treated with interferon and 8 controls) with chronic hepatitis B. In the basal biopsy replicative forms of HBV-DNA were detected in all of the samples and integrated viral DNA was present in 1 case. At the end of the study, 8 children had lost serum HBV-DNA although 2 of the children were still HBeAg positive. (Six had been treated with interferon.) In all of the cases, HBV-DNA was not detectable in the final biopsy. For the rest of the patients, HBV-DNA was positive in serum and all of them had replicative forms of HBV-DNA in the second liver sample. None of the patients lost hepatitis B surface antigen (HBsAg). Peripheral blood mononuclear cells (PBMC) from these patients were studied. HBV-DNA was not found in the PBMC of the 8 children without serum HBV-DNA, and HBV-DNA was detected in the PBMC of 5/12 patients with serum HBV-DNA. In conclusion, HBV-DNA disappeared from the biopsies of children who lost circulating HBV-DNA, although some of the patients were still HBeAg positive. This result implies that the detection of HBV-DNA in liver is important in order to assess the efficacy of the antiviral therapy. On the other hand, HBsAG remained positive in all children at the end of the study although HBV-DNA was not detected in serum, liver, and PBMC by the conventional hybridization techniques.     

应用免疫印迹技术检测抗Scl-70和抗Jo-1抗体

本文通过对兔胸腺可抽提核抗原(ENA)制备方法的改良应用免疫印迹技术(IBT)检测抗Scl-70与抗 Jo-1抗体,对 157例各种风湿性疾病及 36名正常人的对照研究,表明 70 kD(Scl-70)多肽抗体是进行性系统性硬化症(PSS)伴弥漫性硬皮病(DS)的标记抗体,阳性率为45.5％,而55kD(Jo-1)多肽抗体是多发性肌炎(PM)的标记抗体,阳性率为25％。     

Anti-DNA,anti-HBc correlations with RIA-detected HBeAg and anti-HBe in chronic HBsAg carriers

The interrelations of 1) antibody to hepatitis B core antigen (HBcAg) — anti-HBc; 2) single-stranded DNA-binding antibodies (anti-DN A); and 3) the e-antigen/antibody system — hepatitis B e antigen (HBeAg) and antibody (anti-HBe), were studied in 150 hepatitis B surface antigen (HBsAg) carriers, in 43 of whom diagnostic liver biopsies had been performed. There was a good correlation between titers of anti-HBc and anti-DN A, regarded as indicators of viral and pathological activity, respectively, as well as between levels of these two antibodies and the presence of HBeAg or anti-HBE as detected by radio-immune assay (RIA). In general, HBeAg-positive carriers showed high anti-HBc and high anti-DNA titers, while the carriers positive for anti-HBe had low titers of both. These findings were in accord with the histopathological results. The three serologic parameters, anti-HBc, anti-DNA, and e-antigen/anti-body, should together prove useful for the evaluation of the clinical status of chronic HBsAg carriers.     

Immunofluorescent detection of hepatitis B antigen in paraffin-embedded liver tissue.

Hepatitis B antigen (HBAg) has been demonstrated by the indirect immunofluorescent technique and by orcein staining in 20 liver biopsies fixed in Bouin's fixative and embedded in paraffin. The results were compared with those obtained previously by immunogluorescence on frozen sections of the same biopsies. Ten biopsies which were positive in frozen sections were also positive by immunofluorescence in parafin sections, whereas only six were positive by orcein staining. In orcein-stained sections, the cellular localization of HBAg was precisely in the same places as in the slides examined by immunogluorescence. The intessity of the fluorescence in paraffin sections was almost the same as in frozen sections. The localization of the antigen was histologically more precise in paraffin sections. Besides various advantages, indlucing aboidance of freezing aquipment and procedures, paraffin sections are more easy to handle and biopsies from distant hospitals can be processed. The advantages of the immunofluorescent test in comparison to orcein staining are its immunological specificity and higher sensitivity.     

A survey of liver pathology in needle biopsies from HBsAg and anti-HBe positive individuals

AIMS: To use laboratory data and liver biopsies, prospectively obtained from hepatitis B surface antigen (HBsAg) and anti hepatitis B e antigen (anti-HBe) positive patients, for the assessment of: (1) the relation between biopsy length/number of portal tracts and sampling error; (2) the relation between the severity of piecemeal necrosis and the new grading terminology (minimal, mild, moderate, and severe chronic hepatitis); and (3) liver pathology, which has not been studied in patients with this specific serological profile. METHODS: The study group (n = 174) included 104 patients with normal aminotransferase concentrations and no cases with clinically apparent cirrhosis. The specimen length and number of portal tracts were measured at light microscopy examination. Sampling error analysis was related to the discrepancies between aminotransferase concentrations versus histological grade. Detailed histological scorings were undertaken by the reference pathologist and compared with laboratory and hepatitis B virus (HBV) DNA precore sequence data. RESULTS: Sampling error seemed to be a constant feature, even for biopsies > or = 20 mm, but increased dramatically in biopsies < 5 mm long and/or containing less than four portal tracts. Between 25% and 30% of biopsies, graded as "mild" or "moderate" activity showed features of moderate and severe piecemeal necrosis, respectively. Ten per cent of the patients with normal aminotransferase values had stage III-IV hepatic fibrosis, and 20% had piecemeal necrosis. Only cytoplasmic, not nuclear, core antigen expression was a strong predictor of high hepatitis B viraemia. There was no association between precore stop codon mutations, grade/stage of liver disease, and hepatitis B core antigen (HBcAg) expression. CONCLUSIONS: The specimen available for light microscopical examination should be > 5 mm long and should contain more than four portal tracts. In addition, the new grading terminology might give the clinician an inappropriately mild impression of the severity of piecemeal necrosis. Furthermore, even in the presence of normal aminotransferase concentrations, considerable liver pathology can be found in 10-20% of HBsAg and anti-HBe positive individuals; such pathology is not associated with the occurrence of precore stop codon mutations.