Fluorescent In Situ Staining and Flow Cytometric Procedures as New Pre-Diagnostic Tests for Sialidosis, GM1 Gangliosidosis and Niemann-Pick Type C

Year: 2022

Authors: Capitini C.; Feo F.; Caciotti A.; Tonin R.; Lulli M.; Coviello D.; Guerrini R.; Calamai M.; Morrone A.

Autors Affiliation: European Laboratory for Non-Linear Spectroscopy (LENS), University of Florence, Sesto-Fiorention, 50019, Italy; National Institute of Optics-National Research Council (CNR-INO), Sesto Fiorentino, 50019, Italy; Laboratory of Molecular Biology of Neurometabolic Diseases, Neuroscience Department, Meyer Children?s Hospital, Viale Pieraccini n. 24, Florence, 50139, Italy; Department of Experimental and Clinical Biomedical Sciences “”Mario Serio””, University of Florence, 50134 Florence, Italy; Laboratory of Human Genetics, IRCCS Istituto Giannina Gaslini, Genoa, 16147, Italy; Department of Neurosciences, Psychology, Pharmacology and Child Health, University of Florence, 50121 Florence, Italy

Abstract: Background: Early diagnosis is essential in the field of lysosomal storage disorders for the proper management of patients and for starting therapies before irreversible damage occurs, particularly in neurodegenerative conditions. Currently, specific biomarkers for the diagnosis of lysosomal storage disorders are lacking in routine laboratory practice, except for enzymatic tests, which are available only in specialized metabolic centers. Recently, we established a method for measuring and verifying changes in GM1 ganglioside levels in peripheral blood lymphocytes in patients with GM1 gangliosidosis. However, fresh blood is not always available, and using frozen/thawed lymphocytes can lead to inaccurate results. Methods: We used frozen/thawed fibroblasts obtained from stored biopsies to explore the feasibility of fluorescent imaging and flow-cytometric methods to track changes in storage materials in fibroblasts from patients with three lysosomal neurodegenerative conditions: GM1 gangliosidosis, Sialidosis, and Niemann-Pick type C. We used specific markers for each pathology. Results and Conclusions: We demonstrated that with our methods, it is possible to clearly distinguish the levels of accumulated metabolites in fibroblasts from affected and unaffected patients for all the three pathologies considered. Our methods proved to be rapid, sensitive, unbiased, and potentially applicable to other LSDs.

Journal/Review: BIOMEDICINES

Volume: 10 (8)      Pages from: 1962-1  to: 1962-12

More Information: The research was funded by Regione Toscana (Bando Salute 2018, DD15397) for the project Lysolate and by European Union´s Horizon 2020 research and innovation programme under grant agreement No 654148 Laserlab-Europe.
M Calamai and A Morrone, equal last author contribution
KeyWords: GM1 gangliosidosis; sialidosis; Niemann-Pick type C; lysosomal storage disorders; flow cytometry; fluorescent imaging; Cholera Toxin B; Filipin; cholesterol; sialic acid; biomarkers
DOI: 10.3390/biomedicines10081962