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However, one of the challenges in the current image cytometry methodology is a technical limitation in the segmentation of nuclei and cellular components particularly in heterogeneously stained cancer tissue images. To improve the detection and specificity of single\u2010cell segmentation in hematoxylin\u2010stained images (which can be utilized for recently reported 12\u2010biomarker chromogenic sequential multiplex IHC), we adapted a segmentation algorithm previously developed for hematoxlin and eosin\u2010stained images, where morphological features are extracted based on Gabor\u2010filtering, followed by stacking of image pixels into <jats:italic>n<\/jats:italic>\u2010dimensional feature space and unsupervised clustering of individual pixels. Our proposed method showed improved sensitivity and specificity in comparison with standard segmentation methods. Replacing previously proposed methods with our method in multiplex IHC\/image cytometry analysis, we observed higher detection of cell lineages including relatively rare T<jats:sub>H<\/jats:sub>17 cells, further enabling sub\u2010population analysis into T<jats:sub>H<\/jats:sub>1\u2010like and T<jats:sub>H<\/jats:sub>2\u2010like phenotypes based on T\u2010bet and GATA3 expression. Interestingly, predominance of T<jats:sub>H<\/jats:sub>2\u2010like T<jats:sub>H<\/jats:sub>17 cells was associated with human papilloma virus (HPV)\u2010negative status of oropharyngeal squamous cell carcinoma of head and neck, known as a poor\u2010prognostic subtype in comparison with HPV\u2010positive status. Furthermore, T<jats:sub>H<\/jats:sub>2\u2010like T<jats:sub>H<\/jats:sub>17 cells in HPV\u2010negative head and neck cancer tissues were spatiotemporally correlated with CD66b<jats:sup>+<\/jats:sup> granulocytes, presumably associated with an immunosuppressive microenvironment. Our cell segmentation method for multiplex IHC\/image cytometry potentially contributes to in\u2010depth immune profiling and spatial association, leading to further tissue\u2010based biomarker exploration. \u00a9 2019 International Society for Advancement of Cytometry<\/jats:p>","DOI":"10.1002\/cyto.a.23726","type":"journal-article","created":{"date-parts":[[2019,2,4]],"date-time":"2019-02-04T13:12:50Z","timestamp":1549285970000},"page":"389-398","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":18,"title":["Robust Cell Detection and Segmentation for Image Cytometry Reveal Th17 Cell Heterogeneity"],"prefix":"10.1002","volume":"95","author":[{"given":"Takahiro","family":"Tsujikawa","sequence":"first","affiliation":[{"name":"Department of Cell, Developmental, and Cancer Biology Oregon Health &amp; Science University  Portland Oregon USA"},{"name":"Department of Otolaryngology\u2010Head &amp; Neck Surgery Oregon Health &amp; Science University  Portland Oregon USA"},{"name":"Department of Otolaryngology\u2010Head and Neck Surgery Kyoto Prefectural University of Medicine  Kyoto Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Guillaume","family":"Thibault","sequence":"additional","affiliation":[{"name":"Department of Biomedical Engineering Oregon Health &amp; Science University  Portland Oregon USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Vahid","family":"Azimi","sequence":"additional","affiliation":[{"name":"Computational Biology Program Oregon Health &amp; Science University  Portland Oregon USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Sam","family":"Sivagnanam","sequence":"additional","affiliation":[{"name":"Computational Biology Program Oregon Health &amp; Science University  Portland Oregon USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Grace","family":"Banik","sequence":"additional","affiliation":[{"name":"Department of Otolaryngology\u2010Head &amp; Neck Surgery Oregon Health &amp; Science University  Portland Oregon USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Casey","family":"Means","sequence":"additional","affiliation":[{"name":"Department of Otolaryngology\u2010Head &amp; Neck Surgery Oregon Health &amp; Science University  Portland Oregon USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Rie","family":"Kawashima","sequence":"additional","affiliation":[{"name":"Department of Cell, Developmental, and Cancer Biology Oregon Health &amp; Science University  Portland Oregon USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Daniel R.","family":"Clayburgh","sequence":"additional","affiliation":[{"name":"Department of Otolaryngology\u2010Head &amp; Neck Surgery Oregon Health &amp; Science University  Portland Oregon USA"},{"name":"Department of Knight Cancer Institute Oregon Health &amp; Science University  Portland Oregon USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Joe W.","family":"Gray","sequence":"additional","affiliation":[{"name":"Department of Biomedical Engineering Oregon Health &amp; Science University  Portland Oregon USA"},{"name":"Department of Knight Cancer Institute Oregon Health &amp; Science University  Portland Oregon USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Lisa M.","family":"Coussens","sequence":"additional","affiliation":[{"name":"Department of Cell, Developmental, and Cancer Biology Oregon Health &amp; Science University  Portland Oregon USA"},{"name":"Department of Knight Cancer Institute Oregon Health &amp; Science University  Portland Oregon USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8764-1959","authenticated-orcid":false,"given":"Young Hwan","family":"Chang","sequence":"additional","affiliation":[{"name":"Department of Biomedical Engineering Oregon Health &amp; Science University  Portland Oregon USA"},{"name":"Computational Biology Program Oregon Health &amp; Science University  Portland Oregon USA"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"311","published-online":{"date-parts":[[2019,2,4]]},"reference":[{"issue":"3","key":"e_1_2_7_2_1","article-title":"Novel technologies and emerging biomarkers for personalized cancer immunotherapy","volume":"4","author":"Yuan J","year":"2016","journal-title":"J Immunother Cancer."},{"issue":"4","key":"e_1_2_7_3_1","first-page":"1041","article-title":"Importance of immune monitoring approaches and the use of immune checkpoints for the treatment of diffuse intrinsic pontine glioma: From bench to clinic and vice versa (Review)","volume":"52","author":"Scutti JAB","year":"2018","journal-title":"Int J Oncol."},{"key":"e_1_2_7_4_1","doi-asserted-by":"publisher","DOI":"10.1002\/cncr.31105"},{"key":"e_1_2_7_5_1","doi-asserted-by":"publisher","DOI":"10.1158\/0008-5472.CAN-16-2490"},{"key":"e_1_2_7_6_1","doi-asserted-by":"publisher","DOI":"10.1093\/bioinformatics\/btw390"},{"key":"e_1_2_7_7_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.copbio.2016.04.003"},{"key":"e_1_2_7_8_1","doi-asserted-by":"crossref","unstructured":"Young HwanC ThibaultG AzimiV JohnsonB JorgensD LinkJ MargolinA GrayJW. 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