Effect of sample preparation and freezing storage conditions on equine adrenocorticotrophin concentrations

Diagnosis of pituitary pars intermedia dysfunction (PPID) in horses frequently requires freezing of plasma samples for transport prior to measurement of blood ACTH concentrations. The effects of sample preparation, freezing storage duration and freeze-thaw cycles on ACTH concentrations are poorly understood.Materials and MethodsNine healthy horses had blood collected into 3 EDTA tubes before and 10 minutes after i.v. administration of 1 mg TRH: plasma was harvested by sedimentation (1 tube: 2 aliquots) and centrifugation (2 tubes: 9 aliquots). ACTH concentrations were measured 1hr after collection (t0) and after 7 days, 30 days and 90 days of storage (centrifugation samples) and 30 days (sedimentation samples) at -20oC or -80oC. At each time point, ACTH was measured after two freeze-thaw cycles. General linear models and posthoc Bonferonni tests were used to determine factors that influence plasma ACTH concentrations. Significance was set at P<0.05. This study was approved by the Animal Care and Ethics Committee, Charles Sturt University (Approval #A18054). ResultsACTH concentrations were significantly higher in plasma collected by sedimentation (P<0.001). Duration of freezing storage influenced ACTH concentration, with all time points significantly different to t0 (P<0.001), while storage temperature and freeze-thaw cycle did not. Relevance to Australian clinical equine practiceThe results of this study indicate that concentrations of ACTH are influenced by duration of freezing and plasma samples collected by sedimentation should not be frozen prior to determination of ACTH concentration. These findings provide useful information for appropriate sample preparation and handling prior to ACTH measurement.