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OPTIMAL: An OPTimized Imaging Mass cytometry AnaLysis framework for benchmarking segmentation and data exploration

Lookup NU author(s): Bethany Hunter, Ioana Nicorescu, Emma Foster, Dr David McDonald, Dr Gill Hulme, Andrew Fuller, Dr Amanda Thomson, Professor Catharien Hilkens, Dr Joaquim Majo, Dr Luke Milross, Professor Andrew FisherORCiD, Professor Andrew FilbyORCiD, George Merces



This work is licensed under a Creative Commons Attribution 4.0 International License (CC BY 4.0).


© 2023 The Authors. Cytometry Part A published by Wiley Periodicals LLC on behalf of International Society for Advancement of Cytometry.Analysis of imaging mass cytometry (IMC) data and other low-resolution multiplexed tissue imaging technologies is often confounded by poor single-cell segmentation and suboptimal approaches for data visualization and exploration. This can lead to inaccurate identification of cell phenotypes, states, or spatial relationships compared to reference data from single-cell suspension technologies. To this end we have developed the “OPTimized Imaging Mass cytometry AnaLysis (OPTIMAL)” framework to benchmark any approaches for cell segmentation, parameter transformation, batch effect correction, data visualization/clustering, and spatial neighborhood analysis. Using a panel of 27 metal-tagged antibodies recognizing well-characterized phenotypic and functional markers to stain the same Formalin-Fixed Paraffin Embedded (FFPE) human tonsil sample tissue microarray over 12 temporally distinct batches we tested several cell segmentation models, a range of different arcsinh cofactor parameter transformation values, 5 different dimensionality reduction algorithms, and 2 clustering methods. Finally, we assessed the optimal approach for performing neighborhood analysis. We found that single-cell segmentation was improved by the use of an Ilastik-derived probability map but that issues with poor segmentation were only really evident after clustering and cell type/state identification and not always evident when using “classical” bivariate data display techniques. The optimal arcsinh cofactor for parameter transformation was 1 as it maximized the statistical separation between negative and positive signal distributions and a simple Z-score normalization step after arcsinh transformation eliminated batch effects. Of the five different dimensionality reduction approaches tested, PacMap gave the best data structure with FLOWSOM clustering out-performing phenograph in terms of cell type identification. We also found that neighborhood analysis was influenced by the method used for finding neighboring cells with a “disc” pixel expansion outperforming a “bounding box” approach combined with the need for filtering objects based on size and image-edge location. Importantly, OPTIMAL can be used to assess and integrate with any existing approach to IMC data analysis and, as it creates.FCS files from the segmentation output and allows for single-cell exploration to be conducted using a wide variety of accessible software and algorithms familiar to conventional flow cytometrists.

Publication metadata

Author(s): Hunter B, Nicorescu I, Foster E, McDonald D, Hulme G, Fuller A, Thomson A, Goldsborough T, Hilkens CMU, Majo J, Milross L, Fisher A, Bankhead P, Wills J, Rees P, Filby A, Merces G

Publication type: Article

Publication status: Published

Journal: Cytometry Part A

Year: 2024

Volume: 105

Issue: 1

Pages: 36-53

Print publication date: 01/01/2024

Online publication date: 26/09/2023

Acceptance date: 18/09/2023

Date deposited: 30/10/2023

ISSN (print): 1552-4922

ISSN (electronic): 1552-4930

Publisher: John Wiley and Sons Inc


DOI: 10.1002/cyto.a.24803


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Funder referenceFunder name
European Union Horizon 2020
Medical Research Council
JGW Patterson Foundation
NIHR UK Coronavirus Immunology Consortium
UK Research and Innovations
United Kingdom Research and Innovation