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The clinical relevance of the in situ assay for HBV DNA: a cross-sectional study in chronic hepatitis B patients

Danping Liu (刘丹萍), Tong Xu (徐通), Bisheng Shi (施碧胜),Wei Lu (陆伟),Ye Zheng (郑叶),Yanling Feng (冯艳玲),Zhenghong Yuan (袁正宏), Xiaonan Zhang (张小楠), Zhanqing Zhang (张占卿)
Shanghai Public Health Clinical Center, Key Laboratory of Medical Molecular Virology, Shanghai Medical College, Fudan University

Background/Aim: The visualization of HBV DNA in liver sections of chronic hepatitis B (CHB) patients in our previous report uncovered a mosaic distribution of viral antigens and nucleic acids. In the current study, we aim to further explore the clinical utility of the in situ hybridization assay for HBV DNA. 

Method: In this cross-sectional study, 313 treatment-naive CHB patients, all of whom underwent liver biopsy examination, were enrolled. In situ hybridization (ISH) of HBV DNA along with immunohistochemistry (IHC) of HBsAg and HBcAg and routine histopathology analysis was performed. In addition, serum HBcrAg and HBcAb titre were measured in addition to basic biochemical and virological parameters. 

Results: The ISH of HBV DNA showed a positive rate of 95.2% in the whole cohort whereas HBsAg HBcAg showed 97.1% and 42.8% positive rate respectively. The staining pattern of HBV DNA differs significantly with that of HBsAg.  Moreover, intrahepatic HBV DNA exhibited high-level of correlations with viral load (r=0.678), HBcrAg (r=0.645) and HBsAg titer (r=0.531).  In addition, in HBeAg negative patients, higher intrahepatic HBV DNA is associated with histological evidence of liver inflammation and fibrosis whereas no such trend was observed in HBeAg positive group.   

Conclusion: The ISH assay for HBV DNA reflect the vigor of intrahepatic viral replication and is complementary to the routine IHC assay for viral antigens. In addition, it is also related to the histopathological progression of liver diseases. The application of the HBV DNA ISH assay may help better evaluation of virological and pathological condition of CHB patients. 

Full Text

Typical images of the HBV DNA-HBsAg and Sirius red triple staining. A scale bar was shown for each panel. Basic clinical parameters such as HBeAg status, ALT (U/L), serum HBV DNA (IU/ml) and Scheuer score were listed. 

Image Collections

Disease phase
  • HBeAg(+)
  • HBeAg(-)
  • HBV
Type of Staining
  • HBV DNA(blue)-Histone H3 (red)
  • HBcAg(brown)-hematoxylin
  • HBsAg (brown)-hematoxylin
  • HBV DNA (blue)- S (brown)-Sirius red
Case No.