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

Abstract

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.

Prospective Case Series of Clinical Signs and Adrenocorticotrophin (ACTH) Concentrations in Seven Horses Transitioning to Pituitary Pars Intermedia Dysfunction (PPID)

“…While the TRH-stimulation test is recommended for use in subclinical cases of PPID, its use is limited in some countries. TRH is available as a compounded product in the USA, but in Australia and Europe, TRH is only available as a chemical grade reagent, generally limiting its use to referral hospitals [ 39 , 41 , 59 ]. In cases where TRH is unavailable, basal ACTH should be used for diagnosis.…”

A one-health lens offers new perspectives on the importance of endocrine disorders in the equine athlete

“…The 10-minute timing is critical, as collection of blood 1 minute earlier or later resulted in a different interpretation of the results in 21% of horses. 47 This test must be performed before assessing for ID with an oral sugar test (OST). 44 Biochemical measurements should be interpreted in conjunction with the horse's history and clinical signs.…”

Pituitary Pars Intermedia Dysfunction (PPID) in Horses

“…Measuring ACTH concentration 10-min post TRH administration is favoured by ambulatory practitioners for time efficiency, hence its recommendation by the Equine Endocrinology Group (EEG) [ 54 ]. However, as there is a much larger variation in the ACTH concentration 1 min pre and post the 10 min time point [ 40 , 42 , 131 , 134 ], compared to 1 min pre and post the 30 min time point [ 2 , 132 ] (where the ACTH response curve is flatter), some researchers favour using the 30 min time point due to greater accuracy when repeated samples are required [ 134 ]. Further research is required to determine the variability of ACTH concentrations 30 min post TRH administration [ 134 ].…”

“…However, as there is a much larger variation in the ACTH concentration 1 min pre and post the 10 min time point [ 40 , 42 , 131 , 134 ], compared to 1 min pre and post the 30 min time point [ 2 , 132 ] (where the ACTH response curve is flatter), some researchers favour using the 30 min time point due to greater accuracy when repeated samples are required [ 134 ]. Further research is required to determine the variability of ACTH concentrations 30 min post TRH administration [ 134 ]. Prior to the availability of a compounded product in the USA, most research papers utilise the chemical grade TRH (Sigma-Aldrich Pty Ltd. (subsidiary of Merck), North Ryde BC, Australia or synthetic thyrotropin releasing hormone, Sigma-Aldrich Inc, St. Louis, MO, USA) [ 2 , 121 , 124 , 127 , 129 , 132 ].…”

“…Prior to the availability of a compounded product in the USA, most research papers utilise the chemical grade TRH (Sigma-Aldrich Pty Ltd. (subsidiary of Merck), North Ryde BC, Australia or synthetic thyrotropin releasing hormone, Sigma-Aldrich Inc, St. Louis, MO, USA) [ 2 , 121 , 124 , 127 , 129 , 132 ]. Although TRH is now available as a compounded product in the USA, in Europe and Australia TRH is not available as a sterile registered product, and use tends to be limited to referral hospitals [ 132 , 133 , 134 ]. In the UK and most of Europe, the use of unlicenced chemicals is restricted, limiting the use of TRH for the diagnosis of PPID.…”