{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,7]],"date-time":"2026-08-07T10:31:11Z","timestamp":1786098671106,"version":"3.56.0"},"reference-count":39,"publisher":"Oxford University Press (OUP)","issue":"2","license":[{"start":{"date-parts":[[2021,10,3]],"date-time":"2021-10-03T00:00:00Z","timestamp":1633219200000},"content-version":"vor","delay-in-days":1,"URL":"https:\/\/2.zoppoz.workers.dev:443\/https\/creativecommons.org\/licenses\/by-nc\/4.0\/"}],"funder":[{"name":"National Cancer Institute of the National Institutes of Health","award":["R01CA208179 supporting FG"],"award-info":[{"award-number":["R01CA208179 supporting FG"]}]},{"name":"US-Ireland Tripartite","award":["R01"],"award-info":[{"award-number":["R01"]}]},{"DOI":"10.13039\/100017291","name":"HSCNI","doi-asserted-by":"publisher","award":["STL\/5715\/15"],"award-info":[{"award-number":["STL\/5715\/15"]}],"id":[{"id":"10.13039\/100017291","id-type":"DOI","asserted-by":"publisher"}]},{"name":"US-Ireland Tripartite award from Science Foundation Ireland and the Health Research Board","award":["16\/US\/3301"],"award-info":[{"award-number":["16\/US\/3301"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2022,1,3]]},"abstract":"<jats:title>Abstract<\/jats:title>\n               <jats:sec>\n                  <jats:title>Motivation<\/jats:title>\n                  <jats:p>Multiplexed immunofluorescence bioimaging of single-cells and their spatial organization in tissue holds great promise to the development of future precision diagnostics and therapeutics. Current multiplexing pipelines typically involve multiple rounds of immunofluorescence staining across multiple tissue slides. This introduces experimental batch effects that can hide underlying biological signal. It is important to have robust algorithms that can correct for the batch effects while not introducing biases into the data. Performance of data normalization methods can vary among different assay pipelines. To evaluate differences, it is critical to have a ground truth dataset that is representative of the assay.<\/jats:p>\n               <\/jats:sec>\n               <jats:sec>\n                  <jats:title>Results<\/jats:title>\n                  <jats:p>A new immunoFLuorescence Image NOrmalization method is presented and evaluated against alternative methods and workflows. Multiround immunofluorescence staining of the same tissue with the nuclear dye DAPI was used to represent virtual slides and a ground truth. DAPI was restained on a given tissue slide producing multiple images of the same underlying structure but undergoing multiple representative tissue handling steps. This ground truth dataset was used to evaluate and compare multiple normalization methods including median, quantile, smooth quantile, median ratio normalization and trimmed mean of the M-values. These methods were applied in both an unbiased grid object and segmented cell object workflow to 24 multiplexed biomarkers. An upper quartile normalization of grid objects in log space was found to obtain almost equivalent performance to directly normalizing segmented cell objects by the middle quantile. The developed grid-based technique was then applied with on-slide controls for evaluation. Using five or fewer controls per slide can introduce biases into the data. Ten or more on-slide controls were able to robustly correct for batch effects.<\/jats:p>\n               <\/jats:sec>\n               <jats:sec>\n                  <jats:title>Availability and implementation<\/jats:title>\n                  <jats:p>The data underlying this article along with the FLINO R-scripts used to perform the evaluation of image normalizations methods and workflows can be downloaded from https:\/\/2.zoppoz.workers.dev:443\/https\/github.com\/GE-Bio\/FLINO.<\/jats:p>\n               <\/jats:sec>\n               <jats:sec>\n                  <jats:title>Supplementary information<\/jats:title>\n                  <jats:p>Supplementary data are available at Bioinformatics online.<\/jats:p>\n               <\/jats:sec>","DOI":"10.1093\/bioinformatics\/btab686","type":"journal-article","created":{"date-parts":[[2021,9,30]],"date-time":"2021-09-30T20:54:33Z","timestamp":1633035273000},"page":"520-526","source":"Crossref","is-referenced-by-count":35,"title":["FLINO: a new method for immunofluorescence bioimage normalization"],"prefix":"10.1093","volume":"38","author":[{"ORCID":"https:\/\/2.zoppoz.workers.dev:443\/https\/orcid.org\/0000-0002-0396-9867","authenticated-orcid":false,"given":"John","family":"Graf","sequence":"first","affiliation":[{"name":"Department of Biology & Applied Physics, GE Research , Niskayuna, NY 12309, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sanghee","family":"Cho","sequence":"additional","affiliation":[{"name":"Department of Biology & Applied Physics, GE Research , Niskayuna, NY 12309, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Elizabeth","family":"McDonough","sequence":"additional","affiliation":[{"name":"Department of Biology & Applied Physics, GE Research , Niskayuna, NY 12309, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alex","family":"Corwin","sequence":"additional","affiliation":[{"name":"Department of Biology & Applied Physics, GE Research , Niskayuna, NY 12309, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anup","family":"Sood","sequence":"additional","affiliation":[{"name":"Department of Biology & Applied Physics, GE Research , Niskayuna, NY 12309, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Andreas","family":"Lindner","sequence":"additional","affiliation":[{"name":"Department of Physiology and Medical Physics, Centre of Systems Medicine, Royal College of Surgeons in Ireland University of Medicine and Health Sciences, 123 St. Stephen\u2019s Green, Dublin 2, Ireland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Manuela","family":"Salvucci","sequence":"additional","affiliation":[{"name":"Department of Physiology and Medical Physics, Centre of Systems Medicine, Royal College of Surgeons in Ireland University of Medicine and Health Sciences, 123 St. Stephen\u2019s Green, Dublin 2, Ireland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xanthi","family":"Stachtea","sequence":"additional","affiliation":[{"name":"Department of Oncology, Centre for Cancer Research & Cell Biology, Queen\u2019s University Belfast, 97 Lisburn Road, Belfast, BT9 7AE, Northern Ireland , UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sandra","family":"Van Schaeybroeck","sequence":"additional","affiliation":[{"name":"Department of Oncology, Centre for Cancer Research & Cell Biology, Queen\u2019s University Belfast, 97 Lisburn Road, Belfast, BT9 7AE, Northern Ireland , UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Philip D","family":"Dunne","sequence":"additional","affiliation":[{"name":"Department of Oncology, Centre for Cancer Research & Cell Biology, Queen\u2019s University Belfast, 97 Lisburn Road, Belfast, BT9 7AE, Northern Ireland , UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Pierre","family":"Laurent-Puig","sequence":"additional","affiliation":[{"name":"Department of Biology, H\u00f4pital Europ\u00e9en Georges-Pompidou, Assistance Publique - H\u00f4pitaux de Paris, 3 Av. 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