Olmeda, PatriciaDíaz-Regañón Fernández, David RafaelVillaescusa Fernández, AlejandraAmusategui, InmaculadaTesouro Díez, Miguel ÁngelRodríguez Franco, FernandoGarcía-Sancho Téllez, Mercedes GuadalupeMartín Fraile, DanielSainz Rodríguez, Ángel2025-09-012025-09-012025Olmeda, P., Díaz-Regañón, D., Villaescusa, A., Amusategui, I., Tesouro, M. A., Rodríguez-Franco, F., García-Sancho, M., Martín-Fraile, D., & Sainz, Á. (2025). Evaluating the stability of antibody titres against Leishmania infantum determined by IFAT in long-term stored canine frozen samples. Parasites & vectors, 18(1), 327. https://doi.org/10.1186/s13071-025-06982-x10.1186/S13071-025-06982-Xhttps://hdl.handle.net/20.500.14352/123578Author contributions: P.O. wrote the original draft, analysed the data and wrote, reviewed and edited the manuscript; A.S., A.V. and D.D-R. wrote, reviewed and edited the manuscript; M.G-S. and F.R-F. reviewed and edited the manuscript. A.S. carried out project coordination; M.A.T., I.A., A.S., D.D-R., P.O. and D.M-F. carried out the IFAT; I.A., A.S., D.D-R., P.O. and D.M-F. read and interpreted the IFAT results. All the authors read and approved the final manuscript.Abstract Background The immunofluorescence antibody test (IFAT) is a serological diagnostic technique used to quantify serum antibodies generated in response to exposure to various pathogens, such as Leishmania infantum. Retrospec tive analysis of previously collected frozen samples is highly valuable for clinical and research purposes. The primary aim of this study was to evaluate the impact of long-term frozen storage of canine plasma samples on the IFAT-based serological diagnosis of L. infantum exposure. Methods A total of 189 frozen plasma samples from dogs stored at −20 °C for 5, 10 or 20 years, which had previously been tested for L. infantum exposure via IFAT (IgG), were reanalysed to assess the concordance between past and cur rent qualitative and quantitative results. Results The qualitative agreement between the former and current IFATs was 92.1%. The samples from 20 years prior presented the greatest increase in negative samples in the second analysis (from 28.6 to 39.7%). A strong positive cor relation was observed between the quantitative measurements of the past and present across all three groups. The exact quantitative agreement was 48.7%. Conclusions The results of this study indicate that freezing at −20 °C is a good technique for prolonged storage of samples for the detection of L. infantum exposure in dogs, as the qualitative IFAT result is not significantly altered. This finding is of particular interest both for clinical endeavours and for future research in this field.engAttribution 4.0 Internationalhttp://creativecommons.org/licenses/by/4.0/journal article1756-3305https://doi.org/10.1186/S13071-025-06982-X40754584open access579.62Biological preservationDiagnosisDogsLeishmaniosisPlasmaMicrobiología (Veterinaria)3109.05 Microbiología