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Due to the imbalance of lipid homeostasis, WD patients suffer from severe hepatosplenomegaly, hepatic failure, and adrenal calcification resulting in a premature infant death within the first year of age. In this work, we explored multiple imaging analyses to fully characterize the phenotype of LAL\u2010deficient cells. In particular, we stained WD patients' fibroblasts for intracellular lipid droplets (LD) and lysosomes, and we analyzed staining intensity and granularity, as well as an increased number of LD and lysosomes using fluorescence wide\u2010field microscopy, confocal microscopy, conventional, and image flow cytometry. Noteworthy, we showed that lipid homeostasis was restored upon delivery of a functional LAL transgene. Finally, since fibroblasts cannot be used as routine clinical tests as they are difficult to collect from WD patients, we confirmed our observations in LAL deficient human blood cell lines and in peripheral blood mononuclear cells (PBMC) from the LAL deficient (LAL\u2010D) mouse model, as a proxy for easily accessible WD PBMC. Overall, we expect that this novel imaging analysis pipeline will help to diagnose WD, follow its progression, and evaluate the success of enzyme replacement therapy or gene correction strategies for WD as well as other lysosomal storage disorders.<\/jats:p>","DOI":"10.1002\/cyto.a.24949","type":"journal-article","created":{"date-parts":[[2025,7,4]],"date-time":"2025-07-04T09:35:13Z","timestamp":1751621713000},"page":"464-475","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Advanced Imaging and Cytometric Techniques to Characterize Lipid Accumulation in Wolman Disease"],"prefix":"10.1002","volume":"107","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-7987-9999","authenticated-orcid":false,"given":"Marine","family":"Laurent","sequence":"first","affiliation":[{"name":"Genethon  Evry France"},{"name":"Integrare Research Unit UMR_S951 Universit\u00e9 Paris\u2010Saclay, Univ Evry, Inserm, Genethon  Evry 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