{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,15]],"date-time":"2026-09-15T03:02:31Z","timestamp":1789441351044,"version":"build-2803163510"},"reference-count":28,"publisher":"Wiley","issue":"3","license":[{"start":{"date-parts":[[2026,3,18]],"date-time":"2026-03-18T00:00:00Z","timestamp":1773792000000},"content-version":"vor","delay-in-days":17,"URL":"http:\/\/onlinelibrary.wiley.com\/termsAndConditions#vor"},{"start":{"date-parts":[[2026,3,1]],"date-time":"2026-03-01T00:00:00Z","timestamp":1772323200000},"content-version":"tdm","delay-in-days":0,"URL":"http:\/\/doi.wiley.com\/10.1002\/tdm_license_1.1"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Current Protocols"],"published-print":{"date-parts":[[2026,3]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>Multiplex immunofluorescence microscopy facilitates the quantification and spatial analysis of cellular features within tissue sections, allowing greater understanding of disease progression or the effects of drug treatment or other exposures on cellular organization and tissue structure. Recent advances in image analysis and computational methods such as machine\u2010learning\u2010based cell segmentation and automated cell phenotyping have enhanced the depth of information gained from these historically qualitative images. However, many spatial analysis pipelines are technically challenging or require proprietary software or hardware, limiting accessibility and reproducibility. The free and open\u2010source software QuPath provides a novel resource for quantifying and spatially profiling multiplex images. Here, we describe a detailed primary protocol for the semi\u2010automated spatial analysis of 2D multiplex immunofluorescent images using QuPath, which uses object and pixel classification, cell distance, and cluster measurements for spatial profiling of tissue samples. This pipeline also includes the use of training images and basic scripting for batch processing to ensure that analysis is objective and standardized within and between projects. We also provide an alternate protocol that details a pipeline modification for whole\u2010section images, and an extended protocol that describes the use of a free, browser\u2010based tool to complete unsupervised, rapid processing and consolidation of the spatial data provided by QuPath, with automated reporting of cell spatial plots, cell\u2010to\u2010object measurements, and cell clustering data. These protocols provide an accessible, standardized, and scalable method for the spatial analysis of multiplex immunofluorescence microscopy images, facilitating reproducible quantification of cellular organization and tissue structure and thereby strengthening the integration of spatial data into translational research, biomarker discovery, and mechanistic studies. \u00a9 2026 Wiley Periodicals LLC.<\/jats:p>\n                  <jats:p>\n                    <jats:bold>Basic Protocol 1<\/jats:bold>\n                    : QuPath image processing\n                  <\/jats:p>\n                  <jats:p>\n                    <jats:bold>Alternate Protocol<\/jats:bold>\n                    : Whole\u2010section image processing\n                  <\/jats:p>\n                  <jats:p>\n                    <jats:bold>Basic Protocol 2<\/jats:bold>\n                    : Use of the QuPath Spatial Analysis and Visualisation Tool\n                  <\/jats:p>","DOI":"10.1002\/cpz1.70331","type":"journal-article","created":{"date-parts":[[2026,3,18]],"date-time":"2026-03-18T14:31:51Z","timestamp":1773844311000},"source":"Crossref","is-referenced-by-count":0,"title":["An Open\u2010Source Workflow for Semi\u2010Automated Spatial Profiling of Multiplex Immunofluorescent Images"],"prefix":"10.1002","volume":"6","author":[{"ORCID":"https:\/\/orcid.org\/0009-0006-9342-0734","authenticated-orcid":false,"given":"Alisha","family":"Dabb","sequence":"first","affiliation":[{"name":"Malaghan Institute of Medical Research  Wellington New Zealand"},{"name":"Hugh Green Technology Centre Malaghan Institute of Medical Research  Wellington New Zealand"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Cynthia","family":"Morgan","sequence":"additional","affiliation":[{"name":"Malaghan Institute of Medical Research  Wellington New Zealand"},{"name":"Hugh Green Technology Centre Malaghan Institute of Medical Research  Wellington New Zealand"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4896-7160","authenticated-orcid":false,"given":"Sophia","family":"Noble","sequence":"additional","affiliation":[{"name":"Malaghan Institute of Medical Research  Wellington New Zealand"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7213-7546","authenticated-orcid":false,"given":"Alfonso J.","family":"Schmidt","sequence":"additional","affiliation":[{"name":"Malaghan Institute of Medical Research  Wellington New Zealand"},{"name":"Hugh Green Technology Centre Malaghan Institute of Medical Research  Wellington New Zealand"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2026,3,18]]},"reference":[{"key":"e_1_2_8_2_1_1","doi-asserted-by":"publisher","DOI":"10.1158\/1078\u20100432.CCR\u201021\u20102051"},{"key":"e_1_2_8_2_2_1","doi-asserted-by":"publisher","DOI":"10.1093\/bioinformatics\/btx180"},{"key":"e_1_2_8_2_3_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41598\u2010017\u201017204\u20105"},{"key":"e_1_2_8_2_4_1","doi-asserted-by":"publisher","DOI":"10.3389\/fcimb.2023.1195803"},{"key":"e_1_2_8_2_5_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41592\u2010019\u20100582\u20109"},{"key":"e_1_2_8_2_6_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.compenvurbsys.2011.02.003"},{"key":"e_1_2_8_2_7_1","doi-asserted-by":"publisher","DOI":"10.1002\/cyto.a.21079"},{"key":"e_1_2_8_2_8_1","doi-asserted-by":"publisher","DOI":"10.1002\/path.6153"},{"key":"e_1_2_8_2_9_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41564\u2010020\u201000817\u20104"},{"key":"e_1_2_8_2_10_1","doi-asserted-by":"publisher","DOI":"10.1007\/978-1-4939-8935-5_26"},{"key":"e_1_2_8_2_11_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41592\u2010023\u201001846\u20107"},{"key":"e_1_2_8_2_12_1","doi-asserted-by":"publisher","DOI":"10.1101\/2024.08.21.608131"},{"key":"e_1_2_8_2_13_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.bpj.2008.12.3956"},{"key":"e_1_2_8_2_14_1","doi-asserted-by":"publisher","DOI":"10.1002\/cpcy.64"},{"key":"e_1_2_8_2_15_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41593\u2010024\u201001791\u20104"},{"key":"e_1_2_8_2_16_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41586\u2010019\u20101629\u2010x"},{"key":"e_1_2_8_2_17_1","doi-asserted-by":"publisher","DOI":"10.1093\/bioinformatics\/btab634"},{"key":"e_1_2_8_2_18_1","doi-asserted-by":"publisher","DOI":"10.1186\/s40478\u2010019\u20100795\u20102"},{"key":"e_1_2_8_2_19_1","doi-asserted-by":"publisher","DOI":"10.1016\/B978-0-12-407761-4.00014-2"},{"key":"e_1_2_8_3_2_1","unstructured":"Anaconda Software Distribution. (2016). Computer software. Vers. 2024.06\u20101. Anaconda download.https:\/\/anaconda.com"},{"key":"e_1_2_8_3_3_1","unstructured":"Anaconda Software Distribution. (n.d.).System requirements. In Anaconda Tools documentation. Retrieved January 15 2026 fromhttps:\/\/www.anaconda.com\/docs\/tools\/ai\u2010navigator\/system\u2010requirements"},{"key":"e_1_2_8_3_4_1","unstructured":"Bankhead P.(2017b).Optimization of cell detection[Blog post].QuPath users Google group.https:\/\/groups.google.com\/g\/qupath\u2010users\/c\/2xWs7MTMsbA\/m\/pkPt1bgWBgAJ"},{"key":"e_1_2_8_3_5_1","unstructured":"petebankhead. (n.d).Pete Bankhead[Youtube Channel]. YouTube. Retrieved January 15 2026 fromhttps:\/\/groups.google.com\/g\/qupath\u2010users\/c\/2xWs7MTMsbA\/m\/pkPt1bgWBgAJ"},{"key":"e_1_2_8_3_6_1","unstructured":"OpenCV. (2025).Machine Learning Overview. In OpenCV 4.12.0.https:\/\/docs.opencv.org\/4.12.0\/dc\/dd6\/ml_intro.html"},{"key":"e_1_2_8_3_7_1","unstructured":"QuPath. (2025a).FAQ. 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