Methods for localization of hepatitis B surface antigen in liver tissue. An evaluation of different staining- and tissue preparation methods.

To investigate different staining techniques for demonstration of hepatitis B-surface antigen a series of 250 liver biopsies were stained with direct immunofluorescence (I.F.) on frozen tissue and indirect immunoperoxidase (I.P.), direct I.F., orcein and haema toxylin-eosin (H.E.) on paraffin embedded tissue. Examination of different fixatives and various fixation times of formalin fixed tissue on the demonstration of HBsAg was performed on liver tissue from one case with large amounts of HBsAg in the tissue. Among 70 HBsAg sero-positive cases only 27 were tissue positive. In 51 sero-positive AVH cases, 11 were positive with I.F., 3 with I.P., one with orcein and none with H.E. In the remaining 19 sero-positive cases, representing 9 cases with chronic hepatitis, 6 cases with cirrhosis, 3 cases with nonspecific reactive changes and one case without pathological changes, 15 cases were positive with I.F. as well as with I.P., 9 with orcein and 5 with H.E. Membrane related staining reaction was best preserved when using Bouin's and Clarke's fixative. No difference was observed between different fixatives as regards intracytoplasmic staining reaction. Formalin fixation for more than 7 hours duration caused a decrease in the amount of demonstrable HBsAg, which only to a limited extent could be restored by pre-treatment with proteolytic enzyme.