J Pathol Transl Med.  2019 Sep;53(5):317-326. 10.4132/jptm.2019.08.06.

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

• Ko JN
• Jung JK
• Park YI
• Shin HJ
• Huh J
• Back S
• Kim YJ
• Kim JH
• Go H

• KMID: 2459580
• DOI: http://doi.org/10.4132/jptm.2019.08.06

Abstract

BACKGROUND
Single 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).
METHODS
The 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.
RESULTS
The 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.
CONCLUSIONS
EBER-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.

Keyword

Protocol optimization of multistaining; In situ hybridization; Immunohistochemistry; Epstein-Barr virus-associated malignancy; Formalin-fixed paraffin-embedded tissue

MeSH Terms

Benchmarking
Diagnosis
Herpesvirus 4, Human
Immunohistochemistry*
In Situ Hybridization*
Lymphoma, T-Cell
Methods
Reaction Time
RNA*
RNA

Figure

• 

  Fig. 1. Multistaining using each protocol for optimization with angioimmunoblastic T-cell lymphoma tissue. (A) P2-2 (Epstein-Barr virus–encoded RNA [EBER], purple; CD20, brown). (B) P3-2 (EBER, purple; CD20, brown). (C) P4-2 (EBER, purple; CD20, brown). (D, E) DS-2 (EBER, purple; CD20, brown). (F, G) TS-4 (EBER, purple; CD20, red; CD3, brown). (H, I) double EBER in situ hybridization and CD20 immunohistochemistry staining on a Leica Bond III immunostainer (EBER, brown; CD20, red).

• 

  Fig. 2. Application of the optimized multistaining protocol to various Epstein-Barr virus (EBV)–associated malignancies. (A, B) EBV-positive diffuse large B-cell lymphoma (Epstein-Barr virus–encoded RNA [EBER], purple; CD20, red; CD3, brown). (C, D) Extranodal natural killer/T-cell lymphoma, nasal type (EBER, purple; CD56, red; CD3, brown). (E, F) Classic Hodgkin lymphoma stroma (EBER, purple; CD15, red; CD3, brown). (G, H) Angioimmunoblastic T-cell lymphoma stroma (EBER, purple; CD30, red; CD3, brown). (I, J) EBV-positive gastric carcinoma with lymphoid stroma (EBER, purple; cytokeratin, red; CD3, brown).

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