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The aim of our investigation was to examine whether FLIM images could reveal areas suggestive of polymerization in red blood cells (RBCs) of sickle cell disease (SCD) patients. We examined label-free blood films using auto-fluorescence FLIM images of 45 SCD patients and compared the results with those of 27 control persons without hematological disease. All control RBCs revealed homogeneous cytoplasm without any foci. Rounded non-sickled RBCs in SCD showed between zero and three small intensively fluorescent dots with higher lifetime values. In sickled RBCs, we found additionally larger irregularly shaped intensively fluorescent areas with increased FLIM values. These areas were interpreted as equivalent to polymerized hemoglobin. The rounded, non-sickled RBCs of SCD patients with homogeneous cytoplasm were not different from those of the erythrocytes of control patients in light microscopy. Yet, variables from the local binary pattern-transformed matrix of the FLIM values per pixel showed significant differences between non-sickled RBCs and those of control cells. In a linear discriminant analysis, using local binary pattern-transformed texture features (mean and entropy) of the erythrocyte cytoplasm of normal appearing cells, the final model could distinguish between SCD patients and control persons with an accuracy of 84.7% of the patients. When the classification was based on the examination of a single rounded erythrocyte, an accuracy of 68.5% was achieved. Employing the Linear Discriminant Analysis classifier method for machine learning, the accuracy was 68.1%. We believe that our study shows that FLIM is able to disclose the topography of the intracellular polymerization process of hemoglobin in sickle cell disease and that the images are compatible with the theory of the two-step nucleation. Furthermore, we think that the presented technique may be an interesting tool for the investigation of therapeutic inhibition of polymerization.<\/jats:p>","DOI":"10.3390\/ijms252212290","type":"journal-article","created":{"date-parts":[[2024,11,18]],"date-time":"2024-11-18T04:07:09Z","timestamp":1731902829000},"page":"12290","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Accompanying Hemoglobin Polymerization in Red Blood Cells in Patients with Sickle Cell Disease Using Fluorescence Lifetime Imaging"],"prefix":"10.3390","volume":"25","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-6228-1237","authenticated-orcid":false,"given":"Fernanda Aparecida","family":"Borges da Silva","sequence":"first","affiliation":[{"name":"Departments of Pathology and Internal Medicine, Faculty of Medical Sciences, State University of Campinas, Campinas 13083-887, Brazil"},{"name":"National Institute of Science and Technology on Photonics Applied to Cell Biology (INFABIC), State University of Campinas, Campinas 13083-970, Brazil"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0071-0227","authenticated-orcid":false,"given":"Jo\u00e3o Batista","family":"Florindo","sequence":"additional","affiliation":[{"name":"Institute of Mathematics, Statistics, and Scientific Computing, State University of Campinas, Campinas 13083-888, Brazil"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8547-3530","authenticated-orcid":false,"given":"Amilcar Castro de","family":"Mattos","sequence":"additional","affiliation":[{"name":"Departments of Pathology and Internal Medicine, Faculty of Medical Sciences, State University of Campinas, Campinas 13083-887, Brazil"},{"name":"Laboratory of Pathology, Pontifical Catholic University of Campinas PUCC, Campinas 13060-904, Brazil"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Fernando Ferreira","family":"Costa","sequence":"additional","affiliation":[{"name":"Department of Internal Medicine, Faculty of Medical Sciences, State University of Campinas, Campinas 13083-859, Brazil"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6516-1265","authenticated-orcid":false,"given":"Irene","family":"Lorand-Metze","sequence":"additional","affiliation":[{"name":"Department of Internal Medicine, Faculty of Medical Sciences, State University of Campinas, Campinas 13083-859, Brazil"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0742-8846","authenticated-orcid":false,"given":"Konradin","family":"Metze","sequence":"additional","affiliation":[{"name":"Departments of Pathology and Internal Medicine, Faculty of Medical Sciences, State University of Campinas, Campinas 13083-887, Brazil"},{"name":"National Institute of Science and Technology on Photonics Applied to Cell Biology (INFABIC), State University of Campinas, Campinas 13083-970, Brazil"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2024,11,15]]},"reference":[{"key":"ref_1","first-page":"1","article-title":"Sickle cell disease","volume":"4","author":"Kato","year":"2022","journal-title":"Nat. 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