Abstract 5652: Validation of novel InSituPlex™ technology utilizing standard chromogenic IHC and multiplexed tyramide-based immunofluorescence
Authors: Patel Katir , Hebert Courtney , Buell Jamie , Downing Sean 
Published: August 2018

Abstract
Introduction: The demands of today's immuno-oncology research has driven the development of
more robust tools to interrogate the tumor microenvironment, specifically using multiplex
immunofluorescence (mIF). Multiplex immunofluorescence allows investigators to visualize
multiple biomarkers in tissue sections while preserving spatial context. The current gold standard
of immunohistochemistry is “brown” staining using DAB with a hematoxylin counterstain. In
recent years, multiplex immunofluorescence has been accepted in the field using tyramide
molecules (TSA) that permanently fix fluorescent dyes to the tissue. Consequently, tissue integrity
is compromised and cannot be utilized in additional forms of analysis. In addition, high-level
plexing using TSA requires the use of highly specialized and costly imaging equipment. Here we
validate a novel technology, InSituPlex™, utilizing DNA barcoding of antibodies and the detection
of markers using simple DNA hybridization techniques. InSituPlex allows for high-leveling of
multiplexing, preservation of tissue integrity for additional analyses, and implementation on a
variety of commonly used imaging platforms.
Methods: Performance of the InSituPlex technology was assessed using 15 immuno-oncology
relevant markers (CD3, CD4, CD8, CD20, CD25, CD68, CD163, FoxP3, Granzyme B, Ki67, PD1,
PD-L1, pan-cytokeratin, Sox10, and TTF1). Each marker was benchmarked by performing a
single-plex DAB chromogenic IHC and an immunofluorescence assay. Antibodies to be used for
InSituPlex testing were first conjugated to unique DNA barcodes and the resulting conjugates were
assessed utilizing the previously described assays to ensure equivalent performance of the
InSituPlex conjugate to unconjugated antibodies. The performance of the complete InSituPlex
assay was evaluated using InSituPlex HRP labelled imaging strands and the newly barcoded
antibodies. InSituPlex vs. DAB/TSA performance was compared across platforms. All staining
was performed manually and on the Leica BOND RX autostainer. Images were acquired across
multiple imaging platforms, including the Leica VERSA whole slide scanner, and image analysis
was performed using HALO from Indica Labs.
Results: All antibodies performed equally well across all the staining methodologies as assessed
qualitatively. Quantitatively, cell counts were performed on serial sections. Statistical analysis
comparing InSituPlex to standard chromogenic and immunofluorescent assays across all 15
antibodies did not yield any significance (p-value &gt; 0.1).
Conclusions: Detection of relevant immune and cancer related markers utilizing InSituPlex
technology is equivalent to standard chromogenic IHC and immunofluorescent assays.
Citation Format: Katir Patel, Courtney Hebert, Jamie Buell, Sean Downing. Validation of novel InSituPlex™ technology utilizing standard chromogenic IHC and multiplexed tyramide-based immunofluorescence [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5652.