Abstract 3873: High-multiplex automated staining and scanning workflow with Ultivue InSituPlex technology and the Olympus SLIDEVIEW VS200 slide scanner
Authors: Bares Amanda J. , De Grand Alec , Manesse Maël 
Published: September 2020

Abstract
Background: Multiplexed immunofluorescence (IF) has become a key tool for identification of cell sub-types in the tumor microenvironment while conserving spatial cell information in FFPE tissue sections. Although many IF staining methods exist, most require a long assay development time, only provide information on small regions of heterogenous samples, or require specialized instrumentation. The Ultivue InSituPlex® technology enables rapid, pre-optimized staining and whole-slide imaging of multiple targets using widely available staining and scanning instrumentation. UltiMapper™ reagents have been used to demonstrate high-multiplex staining on single slides through sequential imaging of targets and gentle signal removal and reapplication steps. However, final high-plex images require careful image alignment between scans to identify accurate cell phenotypes in downstream analysis, avoiding artifacts from inappropriately overlapped cells. The newly released SLIDEVIEW™ VS200 slide scanner from Olympus provides rapid imaging of multiplexed IF slides with a 210-slide capacity for high-throughput, whole slide scanning. Olympus integrated a module into their latest acquisition software, enabling automatic alignment of serial images of single slides with excellent accuracy and repeatability. Here, we demonstrate a streamlined, highly automated workflow utilizing a high-plex assay in concert with the VS200 slide scanner to obtain high-quality 8-plex images with minimal user intervention.
Methods: InSituPlex was used to perform 8-plex immune profiling of different FFPE tumor sections. Slides were stained with a cocktail of primary antibodies using a Leica Biosystems BOND RX autostainer and imaged on the VS200. Slides were decoverslipped, signal removed, and new targets probed with a reagent incubation step on the BOND RX, termed DNA-Exchange. Slides were re-imaged on the VS200 using the same dye channels. Image pairs were automatically aligned with the alignment tool available in the Olympus VS200 software and exported for downstream analysis with Indica Labs HALO software.
Results: Here, we present a streamlined workflow for multiplexed IF, enabling rapid staining and visualization of 8 targets for deep, spatial phenotyping in the tumor microenvironment. Slides were stained on the BOND RX and imaged rapidly on the VS200, providing high-quality images of the first four targets in a single workday. After the signal exchange step the next day, slides were imaged and the resulting images aligned automatically in the VS200 software, providing well-registered images of multiple targets, ready for analysis down to the single-cell level. Further analysis of repeat-stained targets showed a high correlation in alignment between rounds, increasing confidence that analytically-identified, unique phenotypes from whole slide analysis were real and not a result of image misalignment between rounds.
Conclusions: The Olympus VS200 slide scanner provided a convenient imaging tool for whole slide, multiplexed IF imaging with slides stained with InSituPlex technology. The included image registration tool paired with DNA-Exchange technology for sequential imaging provided rapid access to highly-multiplexed images with high confidence in spatial alignment and subsequent unique phenotypes.
Citation Format: Amanda J. Bares, Alec De Grand, Maël Manesse. High-multiplex automated staining and scanning workflow with Ultivue InSituPlex technology and the Olympus SLIDEVIEW VS200 slide scanner [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3873.