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.