Multistaining Optimization for Epstein-Barr Virus–Encoded RNA In Situ Hybridization and Immunohistochemistry of Formalin-Fixed Paraffin-Embedded Tissues Using an Automated Immunostainer

Abstract

BackgroundSingle staining is commonly performed for practical pathologic diagnoses. However, this method is limited in its ability to specify cellular morphology and immunophenotype and often requires consumption of limited tissue. This study aimed to describe an optimized protocol for multiple in situ hybridization (ISH) and immunohistochemistry (IHC).MethodsThe quality of multistaining was evaluated by carefully changing each step of ISH and IHC in an angioimmunoblastic T-cell lymphoma (AITL) case on a Ventana BenchMark XT automated immunostainer. The optimized protocols were also performed using another immunostainer and in 15 cases of five Epstein-Barr virus (EBV)–associated malignancies using formalin-fixed paraffin-embedded tissue.ResultsThe quality of various ISH-IHC staining protocols was semi-quantitatively evaluated. The best EBV-encoded RNA (EBER)-ISH/double IHC staining quality, equivalent to single staining, was obtained using the following considerations: initial EBER-ISH application, use of protease and antigen retrieval reagent (cell conditioning 1 [CC1] treatment time was minimized due to impact on tissue quality), additional baking/ deparaffinization not needed, and reduced dilution ratio and increased reaction time for primary antibody compared with single immunostaining. Furthermore, shorter second CC1 treatment time yielded better results. Multiple staining was the best quality in another immunostainer and for different types of EBV-associated malignancies when it was performed in the same manner as for the Ventana BenchMark XT as determined for AITL.ConclusionsEBER-ISH and double IHC could be easily used in clinical practice with currently available automated immunostainers and adjustment of reagent treatment time, dilution ratio, and antibody reaction time.

Human herpesvirus 8‑negative effusion‑based lymphoma with indolent clinical behavior in an elderly patient: A case report and literature review

“…2H), and EBV-encoded RNA was not detected in the nuclei of the atypical cells ( Fig. 2I) using in situ hybridization, which was performed as previously described (5). Pathological features in the patient suggested the possibility of an unusual variant of PEL or diffuse large B-cell lymphoma associated with chronic inflammation.…”

“…no. M7050; Dako; Agilent Technologies, Inc.), paired box 5 Immunohistochemistry analysis revealed that the atypical cells were uniformly positive for CD45 and that a subset of these cells exhibited varying degrees of CD20 expression ( Fig. 2B), CD79a and PAX5.…”

Detection of Epstein–Barr Virus in Periodontitis: A Review of Methodological Approaches

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