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

BACKGROUND: Diagnosis of pituitary pars intermedia dysfunction (PPID) using the thyrotropin‐releasing hormone (TRH) stimulation test requires blood collection 10 minutes after TRH injection; it is unknown if small differences in timing affect test results. OBJECTIVE: To determine whether early or late sampling results in a significant (≥10%) difference in plasma adrenocorticotropic hormone (ACTH) concentration compared to standard 10‐minute sampling. ANIMALS: Twenty‐four healthy adult horses with unknown PPID status. METHODS: In this prospective study, subjects underwent a single TRH stimulation test, with blood collected exactly 9 minutes (early), 10 minutes (standard), and 11 minutes (late) after injection. ACTH was measured by chemiluminescent immunoassay. Two aliquots of the 10‐minute plasma sample were analyzed separately to assess intra‐assay variability. Data were reported descriptively and bias was calculated using Bland‐Altman plots. Significance was set at P = .05. RESULTS: Minor variability was observed between the paired 10‐minute sample aliquots (range, 0%‐6%; median 3%). Overall variability of early or late samples compared to the corresponding paired (average) 10‐minute standard concentration ranged from 0% to 92% (median 10%). Seventy‐five percent of horses (18/24) tested had at least 1 early or late reading that differed by ≥10% from its corresponding 10‐minute standard concentration, and 21% of horses (5/24) would have a different interpretation of testing result with either early or late sampling. Incidence of ≥10% variability was independent of PPID status (P = .59). CONCLUSIONS AND CLINICAL IMPORTANCE: Precise timing of sample collection is critical to ensure accurate assessment of PPID status given the observation of significant variability associated with minor alterations in timing of sample collection.