RNA In Situ Hybridization for Epstein-Barr Virus and Cytomegalovirus: Comparison With In Situ Hybridization and Immunohistochemistry

The RNAscope utilizes in situ hybridization (RISH) technology to detect single RNA molecules in a variety of tissue samples, including formalin fixed paraffin embedded (FFPE) tissues. Epstein-Barr virus (EBV) and cytomegalovirus (CMV) are found in association with neoplastic tissues and inflammatory lesions, and immunohistochemistry (IHC) or other techniques (ISH) are utilized to identify them. We compared the RNAscope RISH to ISH and IHC in the detection of EBV and CMV respectively to determine RNAscope utility in a clinical setting. Thirty-one FFPE tissues were stained by RISH to detect EBV and 24 samples of tissue for CMV. The RISH used the RNAscope (Leica BioSystems, Buffalo Grove, IL), the Bond III autostainer (Leica), and probes V-EBV and V-CMV (Advanced Cell Diagnostics, Newark, CA) as well as negative (DapB) and positive probe (PPIB) for RNA. Results were compared with those by ISH (Leica, EBV RNA probe), and IHC (CMV Dako, 1/160), respectively. RISH and ISH were concordant in 100% of cases positive for EBV by ISH (19/19). Of the cases negative for EBV by ISH, RISH showed positivity in an additional 25% of the samples (3/12). Overall concordance was 90.3% (28/31). RISH and IHC were concordant in 100% of cases positive for CMV by IHC (8/8). Of the cases negative for CMV by IHC, RISH detected positivity in an additional 50% of the samples (8/16). Overall concordance was 66.7% (16/24). RISH demonstrates increased sensitivity in the clinical setting, especially for CMV, detecting positive cells not stained by EBV ISH and CMV IHC.