Do multiple freezing and thawing cycles of serum samples affect antibodies used for leptospirosis diagnosis?

There are no data on the effect of multiple freezing and thawing (FT) cycles on antibodies in serum samples from patients with leptospirosis. This study aimed to evaluate the impact of repeated FT cycles on antibody stability in banked serum samples used for leptospirosis diagnosis by the Microscopic Agglutination Test (MAT) and the IgM ELISA. A total of 67 serum samples from patients with leptospirosis confirmed by MAT were analyzed. Group 1 included 10 samples subjected to 14 FT cycles, while Group 2 comprised 57 samples stored for up to 23 years at −20 °C and thawed only once. No significant differences in antibody levels were observed between groups. Samples with MAT titers of 1:200 became undetectable after repeated cycles, but this did not compromise case interpretation. Regression analysis of IgM ELISA showed a declining trend across FT cycles. Positivity by both MAT and IgM ELISA was maintained for up to 40 FT cycles, except for one sample. These results suggest that antibodies remain stable, supporting the reuse of stored samples in research and laboratory validation studies for leptospirosis.