Akoya Biosciences, Inc., 100 Campus Drive, 6th Floor,
Marlborough 01752, MA USA (855) 896-8401 www.akoyabio.comThe
Spatial Biology Company™
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).
Ave
rag
e T
op 3
20
0 P
ixe
l In
tensity
1 2
0
5
10
15
2035404550
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.
CD
8 -
48
0P
D-L
1 -
52
0Fox
P3
-5
70
PD
-1 -
62
0P
an
CK
-6
90
CD
68
-7
80
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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