Impact of Region-of-Interest Size on Immune Profiling Using Multiplex Immunofluorescence Tyramide Signal Amplification for Paraffin-Embedded Tumor Tissues

Authors

Baohua Sun
Caddie Laberiano-Fernández
Ruth Salazar-Alejo
Jiexin Zhang
Jose Luis Solorzano Rendon
Jack Lee
Luisa Maren Solis Soto
Ignacio Ivan Wistuba
Edwin Roger Parra

Publication Date

1-1-2023

Journal

Pathobiology

Abstract

Introduction: Representative regions of interest (ROIs) analysis from the whole slide images (WSI) are currently being used to study immune markers by multiplex immunofluorescence (mIF) and single immunohistochemistry (IHC). However, the amount of area needed to be analyzed to be representative of the entire tumor in a WSI has not been defined.

Methods: We labeled tumor-associated immune cells by mIF and single IHC in separate cohorts of non-small cell lung cancer (NSCLC) samples and we analyzed them as whole tumor area as well as using different number of ROIs to know how much area will be need to represent the entire tumor area.

Results: For mIF using the InForm software and ROI of 0.33 mm2 each, we observed that the cell density data from five randomly selected ROIs is enough to achieve, in 90% of our samples, more than 0.9 of Spearman correlation coefficient and for single IHC using ScanScope tool box from Aperio and ROIs of 1 mm2 each, we found that the correlation value of more than 0.9 was achieved using 5 ROIs in a similar cohort. Additionally, we also observed that each cell phenotype in mIF influence differently the correlation between the areas analyzed by the ROIs and the WSI. Tumor tissue with high intratumor epithelial and immune cells phenotype, quality, and spatial distribution heterogeneity need more area analyzed to represent better the whole tumor area.

Conclusion: We found that at minimum 1.65 mm2 area is enough to represent the entire tumor areas in most of our NSCLC samples using mIF.

Keywords

Humans, Carcinoma, Non-Small-Cell Lung, Paraffin Embedding, Lung Neoplasms, Immunohistochemistry, Fluorescent Antibody Technique, Immunohistochemistry, Multiplex immunofluorescence, Regions of interest, Spearman correlation coefficient, Whole slide images

DOI

10.1159/000523751

PMID

35609532

PMCID

PMC9684353

PubMedCentral® Posted Date

1-1-2024

PubMedCentral® Full Text Version

Author MSS

Published Open-Access

yes