Plasma adrenocorticotropic hormone concentration in horses decreases after freezing for 60 days

Abstract

We investigated the stability of adrenocorticotropic hormone (ACTH) in plasma after freezing for different lengths of time. The plasma ACTH concentrations of 12 horses were measured on day 0 (baseline) and over time, after stimulation with thyrotropin-releasing hormone. Samples were stored at −80°C for 3, 7, 30, 60, and 90 d, or at −20°C for 3, 7, 30, and 60 d, or between ice packs at −20°C for 3 and 7 d prior to determination of ACTH concentration. ACTH concentrations were compared to baseline (non-frozen day 0 plasma) for each storage method using a mixed model with repeated measures in which each horse served as its own control and day was the repeated effect. Statistical significance was set at p ≤ 0.05, and 0.05 < p < 0.10 was considered a trend. Plasma ACTH frozen at −20°C or at −80°C resulted in degradation of ACTH compared to baseline samples at 60 and 90 d respectively. There was no degradation of ACTH after 7 d when stored between ice packs, or before 30 d at −20°C, or before 60 d at −80°C.

Effect of early or late blood sampling on thyrotropin releasing hormone stimulation test results in horses

“…Frozen samples were stored for up to 40 days for 4/20 horses' samples and less than 14 days for the remaining 20/24 horses' samples per published guidelines. 16 No hemolysis or other gross sample abnormalities were observed. One aliquot from each 0‐, 9‐, and 11‐minute sample and 2 aliquots from each 10‐minute sample were shipped overnight on dry ice to the Animal Health Diagnostic Center Laboratory at Cornell University for analysis.…”

“…Additionally, minimal change in ACTH from baseline concentration (<6%) is reported with appropriate long‐term sample storage conditions (frozen at −20 or −80°C) for 30 to 60 days. 16 …”

“…Additionally, minimal change in ACTH from baseline concentration (<6%) is reported with appropriate long-term sample storage conditions (frozen at À20 or À80 C) for 30 to 60 days. 16 In our study, all sample processing was performed by the same investigator using a standardized method, and the 2 10-minute sample aliquots were processed simultaneously to minimize variability arising from differences in sample handling. However, blood samples collected early during the testing window experienced a 1 to 2 hour delay in centrifugation compared to samples collected at the end of the testing window for each day.…”

“…Reported effects of sample handling on measured plasma ACTH concentrations are inconsistent, ranging from no significant effect 22 to marked variability 23 in ACTH concentration after prolonged time at room temperature or alteration of centrifugation methods. Additionally, minimal change in ACTH from baseline concentration (<6%) is reported with appropriate long‐term sample storage conditions (frozen at −20 or −80°C) for 30 to 60 days 16 …”

“…Plasma was separated via centrifugation at 720 g at 4°C for 15 minutes and stored in 1 mL aliquots at −20°C until batch analysis. Frozen samples were stored for up to 40 days for 4/20 horses' samples and less than 14 days for the remaining 20/24 horses' samples per published guidelines 16 . No hemolysis or other gross sample abnormalities were observed.…”