Same-slide multiplex immunofluorescence and brightfield histological staining as a new research tool for fast and comprehensive pathology assessment of the tumor microenvironmentDouglas Wood, Heike Boisvert, Sean R Downing, Maël ManesseUltivue, Inc. 763D Concord Avenue, Cambridge, MA 02138 • +1-617-945-2662 • www.ultivue.com • contact@ultivue.com • Twitter: @Ultivue • LinkedIn: linkedin.com/company/ultivue

Innovative and efficient translational research toolsenabling a better understanding of the tumor and itsmicroenvironment are critical for the development ofdigital pathology. Current immunohistochemistry(IHC) methods limit the depth of information from asingle tissue sample to a single target in the case ofchromogenic staining, or to sample morphology andgeneral cell identification in the case of hematoxylinand eosin (H&E). Accurate phenotyping of each cellmust be performed with a single section, as serialsections may not contain the same cells, especiallysmall immune cells such as T cells. Multipleximmunofluorescence (mIF) methods have beenestablished to provide insights into a wide number ofmarkers of interest and their spatial context in asingle sample. Here, we demonstrate a new researchapproach combining multiplexed detection ofprotein markers with standard H&E pathology reviewin tumor samples, in a streamlined, single-daysample-to-answer workflow.

Introduction:

Conclusion:

Methods:

Whole Slide H&E and ISP Fusion: Tumor Assessment and Phenotyping

UltiMapper I/O reagents were used to stain a deidentifiedFFPE breast cancer tissue sample with 9 markers. Afterimaging, the same slide was stained with hematoxylin andeosin. Immunofluorescence and brightfield images wereco-registered and fused using Ultivue’s proprietarysoftware. The resulting image allows for a traditionalmorphology assessment in parallel with deep phenotypingon the same tissue section.

The whole-slide H&E and ISP fusion workflow allows researchers to stack mIFimages and H&E images from the same section into a single fused imageavailable for tissue and cell analysis without the need for proprietary equipmentor spectral unmixing.

This new workflow enables a quick and easy way to study high-plex,quantitative, cell-to-cell interactions the response of the immune system withinthe tumor microenvironment.

Combining the benefits of standard tissue morphology assessment withmultiplexing allows researchers to benefit from fast qualitative assessment, deepphenotyping, and quantification of immune cells from the same section.

ISP and H&E Fusion Workflow:

IF / ISP-H&E Fusion andCell Type Identification

Whole Slide Overview (H&E I IF)

Macrophage Populations

Whole Slide Image Analysis

QualitativeAssessment

Quantitative Analysis

Morphology-based

Segmentation-based

Tumor distribution and immune infiltration can be assessedfrom the H&E, whereas cell subpopulation can be identifiedusing the fluorescence data. Quantitative information canbe obtained using image analysis and segmentation.Immune cell characterization can then be visualized on thefused image.

For Research Use Only. Not for use in diagnostic procedures.Tissue samples were procured from commercial vendors for this research study.Ultivue®, InSituPlex®, UltiMapper™ are either registered trademarks or trademarks of Ultivue in the United States and/or other countries. All other trademarks are property of their respective owners.

BrightfieldImage

FluorescenceImage Image Fusion

MacrophagesTumor FoxP3+ Cells Cytotoxic Cells

Automated staining

Whole slide imaging

Coverslip removal and

H&E

Whole slide imaging

Coregister & Fusion Image analysis

Staining 5.5 hrs manually or automated

(BOND RX – Leica)

Imaging ~10 min per slide(AxioScan.Z1 - Zeiss)

H&E staining ~15 min(Gemini AS - Thermo)

Imaging ~2 min per slide(AxioScan.Z1 - Zeiss)

Analysis Software(HALO - Indica Labs)

Same day Sample-to-Images

Key Technology FeatureUltivue technology allows for fast and comprehensiveanalysis of the tumor microenvironment by combiningnovel mIF approaches with traditional stainingmethods on the same tissue section.

Key Assay Features➤Easy staining protocol allows for a fast workflow➤ Whole slide imaging capabilities for all markers➤ Gentle signal removal preserves specimen

morphology➤ Workflow compatible with conventional imaging

and software analysis platforms

Morphology Assessment (H&E)

CK FoxP3 CD8 GrzB CD68

Tumor Myeloid cells PMN-MDSCs Macrophages

CK CD11b CD15 HLA-DR CD68

Scan to Download

FoxP3+ Cells