A RAPID MICROWAVE-IN SITU HYBRIDIZATION METHOD FOR THE DEFINITIVE DIAGNOSIS OF ORAL HAIRY LEUKOPLAKIA - COMPARISON WITH IMMUNOHISTOCHEMISTRY

As a diagnostic technique, in situ hybridization requires a long proce ssing time, a degree of expertise and may be difficult to handle routi nely in some laboratories. To simplify the in situ hybridization metho d, we have modified a microwave in situ hybridization technique and ap plied it to oral hairy leukoplakia (OHL) biopsies obtained from 10 HIV -seropositive patients (definitively diagnosed by a conventional in si tu hybridization technique) with appropriate controls. It was neccessa ry to design a novel chamber to avoid drying of sections during the hy bridization step. This modified microwave in situ hybridization techni que was equispecific and equisensitive to the conventional technique a nd it shortens the hybridization time from overnight incubation to 14 minutes. To determine the sensitivity of-our microwave in situ hybridi zation method we applied it to previously documented tongue tissue obt ained from an AIDS autopsy without clinical evidence of OHL, but found to contain Epstein-Barr virus (EBV) by conventional in situ hybridiza tion. This tissue specimen acted as a low EBV copy number, positive co ntrol. The sensitivity of immunohistochemistry using three different c ommercial detection kits was compared to that of in situ hybridization on the same tissues, following optimisation steps. This included the use of 2 cycles of primary and biotinylated secondary antibodies (anti body double cycling). Clearly positive signals for EBV were detected i n all OHL biopsies with the Vectastain Elite ABC and the Histostain-SP kits. The sensitivity of the three commercial detection kits was eval uated at immunohistochemistry level by their application to the low-EB V copy number positive control specimen. Signals for EBV antigen in th e low copy number positive control specimen were obtained only with th e Vectastain Elite ABC kit. This indicates that, in this application, use of the Vectastain Elite ABC kit gives comparable sensitivity for i mmunohistochemistry to that found by in situ hybridiation.