Effect of Long‐Term Freezing on Indirect Fluorescent Antibody Titers for the Diagnosis of Equine Protozoal Myeloencephalitis

ABSTRACT Background Long‐term freezing storage can alter the stability of proteins, thereby compromising accurate determination of indirect fluorescent antibody test (IFAT) titers that support the diagnosis of equine protozoal myeloencephalitis. Objective Assess the effect of long‐term storage at −80°C on IFAT against S. neurona and N. hughesi in equine serum and cerebrospinal fluid (CSF). Samples Paired serum and CSF (n = 46), and serum only (n = 25) samples. Methods Prospective study of samples stored 6–12, 13–18, and 19–24 months. Comparing antibody titers across time points, McNemar and Wilcoxon rank tests were used for statistical analysis. Results After long‐term freezing storage for 19–24 months, N. hughesi IFAT results for CSF were significantly different compared with their fresh‐testing time point (p = 0.04), but a similar difference was not observed for serum samples. No statistical differences were observed for serum or CSF samples tested for S. neurona after 6–12, 13–18, or 19–24 months compared with their original fresh testing results. However, the serum to CSF ratio cutoff of ≤ 64 in support of disease changed in 41% (19 of 46) of paired samples. Conclusions and Clinical Importance Freezing samples at −80°C for more than 18 months might alter test results, leading to possible changes in serum to CSF ratio calculations and affecting the interpretation of results for clinical and research purposes.