{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,26]],"date-time":"2026-10-26T23:18:38Z","timestamp":1793056718516,"version":"4.5.0"},"reference-count":51,"publisher":"Wiley","issue":"6","license":[{"start":{"date-parts":[[2026,6,12]],"date-time":"2026-06-12T00:00:00Z","timestamp":1781222400000},"content-version":"vor","delay-in-days":11,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2026,6,1]],"date-time":"2026-06-01T00:00:00Z","timestamp":1780272000000},"content-version":"tdm","delay-in-days":0,"URL":"http:\/\/doi.wiley.com\/10.1002\/tdm_license_1.1"}],"content-domain":{"domain":["onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["Cytometry Pt A"],"published-print":{"date-parts":[[2026,6]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                  <jats:p>Advances in spectral cytometry instrumentation and fluorescent reagents have led to the possibility of ultra\u2010high\u2010parameter panels exceeding 50 colors. However, panel size is limited in practice by unmixing\u2010dependent spreading (UDS), a phenomenon which leads to a progressive deterioration of unmixed signal\u2010to\u2010noise ratios in panels that contain fluorochrome combinations with significant spectral overlap. So far, choosing spectrally compatible sets of fluorochromes that avoid UDS has been a complex and labor\u2010intensive task involving substantial trial\u2010and\u2010error experimentation. Here, we provide a detailed explanation of UDS and practical strategies for handling UDS in large spectral panels. We describe the empirical hallmarks of UDS, demonstrate how to quantify its impact, and dissect its underlying mathematical cause in terms of spectral collinearity. We present practical tools derived from the regression literature that can be used to select optimal combinations of fluorochromes in a platform\u2010agnostic fashion based on publicly available reference data, providing a general tool for spectral panel design.<\/jats:p>","DOI":"10.1002\/cyto.a.70044","type":"journal-article","created":{"date-parts":[[2026,6,13]],"date-time":"2026-06-13T04:24:42Z","timestamp":1781324682000},"page":"436-455","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["Measurement and Prediction of Unmixing\u2010Dependent Spreading due to Collinearity in Spectral Flow Cytometry"],"prefix":"10.1002","volume":"109","author":[{"given":"Peter L.","family":"Mage","sequence":"first","affiliation":[{"name":"Advanced Technology Group Waters Biosciences (formerly BD Biosciences)  Milpitas California USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3492-1006","authenticated-orcid":false,"given":"Andrew J.","family":"Konecny","sequence":"additional","affiliation":[{"name":"Vaccine and Infectious Disease Division Fred Hutchinson Cancer Center  Seattle Washington USA"},{"name":"Department of Immunology University of Washington  Seattle Washington USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6732-5449","authenticated-orcid":false,"given":"Florian","family":"Mair","sequence":"additional","affiliation":[{"name":"Flow Cytometry Core Facility ETH Zurich  Zurich Switzerland"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2026,6,12]]},"reference":[{"key":"e_1_2_11_2_1","doi-asserted-by":"publisher","DOI":"10.1017\/S1431927605510328"},{"key":"e_1_2_11_3_1","doi-asserted-by":"publisher","DOI":"10.1002\/0471142956.cy0127s63"},{"key":"e_1_2_11_4_1","doi-asserted-by":"publisher","DOI":"10.1002\/cyto.a.22725"},{"key":"e_1_2_11_5_1","doi-asserted-by":"publisher","DOI":"10.1002\/cyto.a.24841"},{"key":"e_1_2_11_6_1","doi-asserted-by":"publisher","DOI":"10.1038\/d41586-021-01396-y"},{"key":"e_1_2_11_7_1","doi-asserted-by":"publisher","DOI":"10.1146\/annurev-immunol-101819-025146"},{"key":"e_1_2_11_8_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.immuni.2019.08.008"},{"key":"e_1_2_11_9_1","doi-asserted-by":"publisher","DOI":"10.1002\/1097-0320(20011101)45:3<194::AID-CYTO1163>3.0.CO;2-C"},{"key":"e_1_2_11_10_1","doi-asserted-by":"publisher","DOI":"10.1002\/cyto.a.22251"},{"key":"e_1_2_11_11_1","doi-asserted-by":"publisher","DOI":"10.1007\/978-1-4939-9650-6_1"},{"key":"e_1_2_11_12_1","doi-asserted-by":"publisher","DOI":"10.1002\/eji.202170126"},{"key":"e_1_2_11_13_1","doi-asserted-by":"publisher","DOI":"10.1007\/978-1-0716-3738-8_6"},{"key":"e_1_2_11_14_1","doi-asserted-by":"publisher","DOI":"10.1002\/0471142956.cy0114s22"},{"key":"e_1_2_11_15_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41590-021-01006-z"},{"key":"e_1_2_11_16_1","doi-asserted-by":"publisher","DOI":"10.1002\/cyto.a.24213"},{"key":"e_1_2_11_17_1","doi-asserted-by":"publisher","DOI":"10.1002\/cyto.a.24669"},{"key":"e_1_2_11_18_1","doi-asserted-by":"publisher","DOI":"10.1109\/79.974727"},{"key":"e_1_2_11_19_1","doi-asserted-by":"publisher","DOI":"10.1002\/cyto.a.22272"},{"key":"e_1_2_11_20_1","volume-title":"Regression Analysis by Example","author":"Chatterjee S.","year":"2012"},{"key":"e_1_2_11_21_1","doi-asserted-by":"publisher","DOI":"10.1002\/cyto.a.23052"},{"key":"e_1_2_11_22_1","unstructured":"BIPM \u201cInternational Vocabulary of Metrology \u2014 Basic and General Concepts and Associated Terms (VIM) \u201dhttps:\/\/doi.org\/10.59161\/JCGM200\u20102012."},{"key":"e_1_2_11_23_1","unstructured":"BIPM \u201cEvaluation of Measurement Data \u2014 Guide to the Expression of Uncertainty in Measurement \u201dhttps:\/\/doi.org\/10.59161\/JCGM100\u20102008E."},{"key":"e_1_2_11_24_1","doi-asserted-by":"publisher","DOI":"10.1002\/cyto.a.24955"},{"key":"e_1_2_11_25_1","unstructured":"BIPM \u201cGuide to the Expression of Uncertainty in Measurement \u2014 Part 6: Developing and Using Measurement Models \u201dhttps:\/\/doi.org\/10.59161\/jcgmgum\u20106\u20102020."},{"key":"e_1_2_11_26_1","doi-asserted-by":"publisher","DOI":"10.1002\/cyto.990130804"},{"key":"e_1_2_11_27_1","doi-asserted-by":"publisher","DOI":"10.1002\/0471142956.cy0120s40"},{"key":"e_1_2_11_28_1","doi-asserted-by":"publisher","DOI":"10.1002\/(SICI)1097-0320(19981001)33:2<256::AID-CYTO22>3.0.CO;2-S"},{"key":"e_1_2_11_29_1","doi-asserted-by":"publisher","DOI":"10.1002\/(SICI)1097-0320(19981001)33:2<267::AID-CYTO24>3.0.CO;2-R"},{"key":"e_1_2_11_30_1","doi-asserted-by":"publisher","DOI":"10.1007\/978-1-4612-0919-5_16"},{"key":"e_1_2_11_31_1","doi-asserted-by":"publisher","DOI":"10.1080\/00401706.1970.10488699"},{"key":"e_1_2_11_32_1","volume-title":"Conditioning Diagnostics: Collinearity and Weak Data in Regression","author":"Belsley D. 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