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1 BackgroundHematoxylin and eosin (H&E) is atraditional and widely used histologicalstain for elucidating tissue morphology forpathological review. However, H&E is not fullyremovable and prevents or severely limits anyfurther use of the same tissue section. Wehave developed a method for accuratelysimulating the H&E staining pattern usingremovable fluorescent dyes that allows forsubsequent re-use of the same tissue sectionfor multiplexed immunofluorescent (mIF)staining with no decrease in mIF performance.This workflow allows for the pathological pre-screening, annotation, and triaging of samplesto undergo multiplexed IHC.

2 Methods

Tissue & Staining

Serial formalin-fixed, paraffin-embedded(FFPE) human lung cancer sections werebaked, deparaffinized, and rehydrated. 3groups were stained: one using a traditionalH&E protocol, the second using a MOTiF™ PD-1/PD-L1 Panel: Auto Lung Cancer Kit (AkoyaBiosciences, Inc.), and the third usingfluorescent “H&E” simulation reagents beforeimaging, microwave treatment (MWT)stripping using 1X AR9 Buffer (AkoyaBiosciences, Inc.), and staining with the sameMOTiF™ PD-1/PD-L1 Panel Kit reagentsalongside the second group on a Leica BONDRX automated stainer.

Imaging, Image Processing, and Analysis

Bright-field and multispectral fluorescenceimagery was acquired on a Vectra Polaris®automated imaging system and images wererendered and analyzed using Phenochart™,inForm®, Microsoft® Excel, GraphPad Prism,ImageJ, GIMP, and RStudio (phenoptr &phenoptrReports packages) software.

3 Results:

A novel H&E-like staining method compatible with multiplexed IF on the same tissue section for integrated translational workflows

Michael McLane, Glenn Milton, Linying Liu, Rachel Schaefer, Yi Zheng, Carla Coltharp, Peter Miller, Clifford Hoyt

This new fluorescent morphology staining method forcreating a simulated H&E view facilitates the integration ofmIF analysis methods into digital pathology workflows bygiving pathologists familiar, conventional views of mIF-stained tissue sections. It also enables the assessment oftissue quality prior to antigen retrieval treatment and theH&E-based annotation of mIF imagery and supports eventualtranslation of mIF methods into clinical standards of care.

Conclusions:4

Fig 1. Comparison of bright-field H&E to simulated “H&E” and mIF images. Whole slide scans (top) and fields at 20x magnification(bottom) of a traditional H&E-stained bright-field image of a human lung cancer section (A, E), a pseudocolored fluorescent “H&E”simulation-stained sister section (B, F, with the basophilic stain represented in cyan and the acidophilic stain represented in magenta), thesame fluorescent image rendered to represent bright-field (BF) H&E and merged with a bright-field scan of itself for the addition of anychromatic details not visible under fluorescent imaging (C, G), and the same section depicted in B, C, F, and G after MWT stripping andMOTiF™ PD-1/PD-L1 Panel kit mIF staining with signals depicted for DAPI in blue, CD8 in cyan, PD-L1 in green, FoxP3 in yellow, PD-1 inorange, PanCK in red, and CD68 in white (D, H).

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CD8 - Opal Polaris 480

PD-L1 - Opal 520

FoxP3 - Opal 570

PD-1 - Opal 620

PanCK - Opal 690

CD68 - Opal Polaris 780

Fig 2. Qualitative and quantitativecomparisons of mIF staining quality.2A. Grayscale images for each proteintarget / Opal dye pairing, spectrally-

unmixed and generated using inForm®software (Akoya Biosciences, Inc.) inControl (left) versus Post- “H&E”Simulation-stained (right) human lungcancer sister sections. 2B. Compositeimages for the same frames depicted in2A where each protein target isrepresented in a different color. 2C.Average top 3200 (brightest 0.1%) pixelintensity values of 5 similar 1860 x 1396-pixel regions, averaged for 3 replicateslides per treatment (Control vs. Post-“H&E” Simulation staining). Whiskersrepresent standard deviation within each3-slide treatment.

Control Post-”H&E” Sim.

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1. Bake 2. Rehydrate3. “H&E” Sim.5 minutes, aqueous,

Alcohol de-staining

4. MountAqueous, or dehydrate

& mount (organic)

5. ImageCan store > 1 mo

if dehydrated

6. DecoverslipRehydrate if

dehydrated

7. StrippingMWT AR9 to strip

“H&E” simulation

8. mIF

A B C D

E F G H

Traditional H&E “H&E” Sim., Fluorescent Post-“H&E” Sim. mIF“H&E” Sim., BF rendering

2A. 2B.

Fig 3. “H&E” Simulation staining-to-mIF workflow. Flow diagram representing the steps following sectioning, slide-mounting, anddrying of FFPE tissue sections for fluorescent “H&E” Simulation staining, imaging, and removal of the stain for subsequent mIF staining.

2C.

Control Post-”H&E” Sim.

1. Control

2. Post-”H&E” sim.

1 2 1 2 1 2 1 2 1 2

Fig 4. Comparison of bright-field H&E to simulated “H&E” views across different cancer types. One field at 20x magnification from sistersections stained with either a traditional H&E protocol or with fluorescent “H&E” Simulation reagents for a human breast cancer (BrCa), coloncancer (CoCa), and melanoma (Mela).

BrCa: H&E BrCa: “H&E” Sim. CoCa: H&E CoCa: “H&E” Sim. Mela: H&E Mela: “H&E” Sim.

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