Statistical Analysis of Multiplex Immunofluorescence and Immunohistochemistry Imaging Data

Overview

Journal Methods Mol Biol

Specialty Molecular Biology

Date 2023 Mar 17

PMID 36929077

Authors

Julia Wrobel

Coleman Harris

Simon Vandekar

Affiliations

Soon will be listed here.

Abstract

Advances in multiplexed single-cell immunofluorescence (mIF) and multiplex immunohistochemistry (mIHC) imaging technologies have enabled the analysis of cell-to-cell spatial relationships that promise to revolutionize our understanding of tissue-based diseases and autoimmune disorders. Multiplex images are collected as multichannel TIFF files; then denoised, segmented to identify cells and nuclei, normalized across slides with protein markers to correct for batch effects, and phenotyped; and then tissue composition and spatial context at the cellular level are analyzed. This chapter discusses methods and software infrastructure for image processing and statistical analysis of mIF/mIHC data.

Citing Articles

Benchmarking Spatial Co-Localization Methods for Single-Cell Multiplex Imaging Data with Applications to High-Grade Serous Ovarian and Triple Negative Breast Cancer.

Soupir A, Gadiyar I, Helm B, Harris C, Vandekar S, Peres L Stat Data Sci Imaging. 2025; 2(1).

PMID: 40051984 PMC: 11883755. DOI: 10.1080/29979676.2024.2437947.

Detecting Clinically Relevant Topological Structures in Multiplexed Spatial Proteomics Imaging Using TopKAT.

Samorodnitsky S, Campbell K, Little A, Ling W, Zhao N, Chen Y bioRxiv. 2025; .

PMID: 39764056 PMC: 11702633. DOI: 10.1101/2024.12.18.628976.

mxfda: a comprehensive toolkit for functional data analysis of single-cell spatial data.

Wrobel J, Soupir A, Hayes M, Peres L, Vu T, Leroux A Bioinform Adv. 2024; 4(1):vbae155.

PMID: 39552929 PMC: 11568348. DOI: 10.1093/bioadv/vbae155.

Statistical analysis of multiple regions-of-interest in multiplexed spatial proteomics data.

Samorodnitsky S, Wu M Brief Bioinform. 2024; 25(6).

PMID: 39428129 PMC: 11491162. DOI: 10.1093/bib/bbae522.

A Spatial Omnibus Test (SPOT) for Spatial Proteomic Data.

Samorodnitsky S, Campbell K, Ribas A, Wu M Bioinformatics. 2024; 40(7).

PMID: 38950184 PMC: 11257711. DOI: 10.1093/bioinformatics/btae425.

References

Bendall S, Nolan G, Roederer M, Chattopadhyay P . A deep profiler's guide to cytometry. Trends Immunol. 2012; 33(7):323-32. PMC: 3383392. DOI: 10.1016/j.it.2012.02.010. View

Young Y, Bolt A, Ahn R, Mann K . Analyzing the Tumor Microenvironment by Flow Cytometry. Methods Mol Biol. 2016; 1458:95-110. DOI: 10.1007/978-1-4939-3801-8_8. View

Hedvat C . Digital microscopy: past, present, and future. Arch Pathol Lab Med. 2010; 134(11):1666-70. DOI: 10.5858/2009-0579-RAR1.1. View

Angelo M, Bendall S, Finck R, Hale M, Hitzman C, Borowsky A . Multiplexed ion beam imaging of human breast tumors. Nat Med. 2014; 20(4):436-42. PMC: 4110905. DOI: 10.1038/nm.3488. View

Keren L, Bosse M, Marquez D, Angoshtari R, Jain S, Varma S . A Structured Tumor-Immune Microenvironment in Triple Negative Breast Cancer Revealed by Multiplexed Ion Beam Imaging. Cell. 2018; 174(6):1373-1387.e19. PMC: 6132072. DOI: 10.1016/j.cell.2018.08.039. View

Giesen C, Wang H, Schapiro D, Zivanovic N, Jacobs A, Hattendorf B . Highly multiplexed imaging of tumor tissues with subcellular resolution by mass cytometry. Nat Methods. 2014; 11(4):417-22. DOI: 10.1038/nmeth.2869. View

Martinez-Morilla S, Villarroel-Espindola F, Wong P, Toki M, Aung T, Pelekanou V . Biomarker Discovery in Patients with Immunotherapy-Treated Melanoma with Imaging Mass Cytometry. Clin Cancer Res. 2021; 27(7):1987-1996. PMC: 8026677. DOI: 10.1158/1078-0432.CCR-20-3340. View

Baharlou H, Canete N, Cunningham A, Harman A, Patrick E . Mass Cytometry Imaging for the Study of Human Diseases-Applications and Data Analysis Strategies. Front Immunol. 2019; 10:2657. PMC: 6868098. DOI: 10.3389/fimmu.2019.02657. View

Ehrenberg A, Morales D, Piergies A, Li S, Tejedor J, Mladinov M . A manual multiplex immunofluorescence method for investigating neurodegenerative diseases. J Neurosci Methods. 2020; 339:108708. PMC: 7269157. DOI: 10.1016/j.jneumeth.2020.108708. View

10.

Apaolaza P, Petropoulou P, Rodriguez-Calvo T . Whole-Slide Image Analysis of Human Pancreas Samples to Elucidate the Immunopathogenesis of Type 1 Diabetes Using the QuPath Software. Front Mol Biosci. 2021; 8:689799. PMC: 8226255. DOI: 10.3389/fmolb.2021.689799. View

11.

Kondo A, Ma S, Lee M, Ortiz V, Traum D, Schug J . Highly Multiplexed Image Analysis of Intestinal Tissue Sections in Patients With Inflammatory Bowel Disease. Gastroenterology. 2021; 161(6):1940-1952. PMC: 8606000. DOI: 10.1053/j.gastro.2021.08.055. View

12.

Ramaglia V, Sheikh-Mohamed S, Legg K, Park C, Rojas O, Zandee S . Multiplexed imaging of immune cells in staged multiple sclerosis lesions by mass cytometry. Elife. 2019; 8. PMC: 6707785. DOI: 10.7554/eLife.48051. View

13.

Wilson C, Ospina O, Townsend M, Nguyen J, Moran Segura C, Schildkraut J . Challenges and Opportunities in the Statistical Analysis of Multiplex Immunofluorescence Data. Cancers (Basel). 2021; 13(12). PMC: 8233801. DOI: 10.3390/cancers13123031. View

14.

McKinley E, Shao J, Ellis S, Heiser C, Roland J, Macedonia M . MIRIAM: A machine and deep learning single-cell segmentation and quantification pipeline for multi-dimensional tissue images. Cytometry A. 2022; 101(6):521-528. PMC: 9167255. DOI: 10.1002/cyto.a.24541. View

15.

Schapiro D, Sokolov A, Yapp C, Chen Y, Muhlich J, Hess J . MCMICRO: a scalable, modular image-processing pipeline for multiplexed tissue imaging. Nat Methods. 2021; 19(3):311-315. PMC: 8916956. DOI: 10.1038/s41592-021-01308-y. View

16.

Vizcarra J, Burlingame E, Hug C, Goltsev Y, White B, Tyson D . A community-based approach to image analysis of cells, tissues and tumors. Comput Med Imaging Graph. 2021; 95:102013. PMC: 8761177. DOI: 10.1016/j.compmedimag.2021.102013. View

17.

Bray F, Ferlay J, Soerjomataram I, Siegel R, Torre L, Jemal A . Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2018; 68(6):394-424. DOI: 10.3322/caac.21492. View

18.

Siegel R, Miller K, Fuchs H, Jemal A . Cancer statistics, 2022. CA Cancer J Clin. 2022; 72(1):7-33. DOI: 10.3322/caac.21708. View

19.

Remark R, Becker C, Gomez J, Damotte D, Dieu-Nosjean M, Sautes-Fridman C . The non-small cell lung cancer immune contexture. A major determinant of tumor characteristics and patient outcome. Am J Respir Crit Care Med. 2014; 191(4):377-90. PMC: 5447326. DOI: 10.1164/rccm.201409-1671PP. View

20.

Jordan K, Sikora M, Slansky J, Minic A, Richer J, Moroney M . The Capacity of the Ovarian Cancer Tumor Microenvironment to Integrate Inflammation Signaling Conveys a Shorter Disease-free Interval. Clin Cancer Res. 2020; 26(23):6362-6373. PMC: 7923250. DOI: 10.1158/1078-0432.CCR-20-1762. View