Article Text

Download PDFPDF

46 A novel H&E-like staining method compatible with multiplexed IF on the same tissue section for integrated translational workflows
  1. Michael McLane,
  2. Glenn Milton,
  3. Linying Liu,
  4. Rachel Schaefer,
  5. Yi Zheng,
  6. Carla Coltharp,
  7. Peter Miller and
  8. Clifford Hoyt
  1. Akoya Biosciences, HOPKINTON, MA


Background Hematoxylin and eosin (H&E) staining is a traditional and widely used histological stain for elucidating tissue morphology for pathological review. However, H&E staining is not fully removable and prevents or severely limits any further use of the same tissue section. We have developed a method for accurately simulating the H&E staining pattern via removable fluorescent dyes that allows for subsequent re-use of the same tissue section for multiplexed immunofluorescent (mIF) staining methods with no decrease in performance. This workflow allows for the pathological pre-screening, annotation, and triaging of samples to undergo multiplexed IHC.This study demonstrates a novel procedure for creating a realistic and accurate ‘H&E’ view of formalin-fixed, paraffin-embedded (FFPE) sections stained with mIF protocols. The novel stain reveals morphological details and can be removed before applying Akoya Biosciences ‘MOTiF’ 6-plex mIF staining.

Methods Serial FFPE lung cancer sections were used in this study. After deparaffinization and rehydration, these slides were divided into 3 groups. The first group was stained with a traditional H&E protocol. The second group was stained using a MOTiF™ PD-1/PD-L1 Panel kit (Akoya Biosciences, Inc.). The third group was stained with H&E simulation staining reagents, imaged and re-stained using a MOTiF™ PD-1/PD-L1 Panel kit (Akoya Biosciences, Inc.) after removal of the H&E simulation reagents. Multispectral fluorescence imagery was acquired on a Vectra Polaris® automated imaging system and analyzed with inForm® and RStudio software. H&E simulation images are manipulated to represent bright-field H&E using Phenochart and inForm® software (Akoya Biosciences, Inc.).

Results Mimic bright-field H&E images from the simulated H&E staining produced results qualitatively indistinguishable from the traditional H&E-stained lung cancer section. Spectral unmixing and staining intensity analysis showed an improvement in signal for all protein targets in the mIF staining from the simulated H&E-stained group (third group) versus the non-simulated H&E-stained group (second group). Background staining analysis showed no significant corresponding increase in background signal across any of the mIF channels.

Conclusions This new fluorescent morphology staining method for creating a simulated H&E view facilitates the integration of mIF analysis methods into digital pathology workflows by giving pathologists familiar, conventional views of mIF-stained tissue sections. It enables the assessment of tissue quality prior to antigen retrieval treatment and the H&E-based annotation of mIF imagery and supports eventual translation of mIF methods into clinical standards-of-care.

This is an open access article distributed in accordance with the Creative Commons Attribution 4.0 Unported (CC BY 4.0) license, which permits others to copy, redistribute, remix, transform and build upon this work for any purpose, provided the original work is properly cited, a link to the licence is given, and indication of whether changes were made. See:

Statistics from

Request Permissions

If you wish to reuse any or all of this article please use the link below which will take you to the Copyright Clearance Center’s RightsLink service. You will be able to get a quick price and instant permission to reuse the content in many different ways.