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

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.

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.

Twenty-four healthy adult horses with unknown PPID status.

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.

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).

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.