older horse grazing
When testing for pituitary pars intermedia dysfunction (PPID, formerly called equine Cushing’s disease), timing is crucial. Researchers recently determined that the thyrotropin-releasing hormone (TRH) stimulation test can yield false results if blood is collected even a minute off schedule.

“Accurate interpretation of testing results is essential to avoid under- or overdiagnosis of this condition and to guide subsequent recommendations to pursue follow-up testing and/or medical therapy,” said Kristen Thane, DVM, Dipl. ACVIM (LAIM), postdoctoral scholar in the Department of Comparative Pathobiology at Tufts University’s Cummings School of Veterinary Medicine, in North Grafton, Massachusetts.

Veterinarians usually check for PPID by measuring the serum concentration of adrenocorticotropic hormone (ACTH) in horses’ blood, Thane said. However, if those results aren’t clear, or if they seem inconsistent with a horse’s clinical signs, such as laminitis, poor wound healing, and excessive hair growth, veterinarians can run a thyrotropin-releasing hormone (TRH) stimulation test. In that test, veterinarians inject TRH into the bloodstream and draw blood 10 minutes later to test the ACTH concentration.

Those 10 minutes might sound approximate to practitioners—especially if they’re multitasking or delegating blood collection to an associate, Thane said. But because ACTH rises rapidly after TRH injection, Thane and her fellow researchers decided to investigate the true importance of that 10-minute recommendation.

“We were interested in investigating whether what many would consider a ‘negligible’ difference in sample collection timing would have an effect

Sample Timing Affects Diagnostic Results

The researchers administered TRH in 24 healthy horses aged 3 to 28. The horses had never undergone PPID testing, but six of them had clinical signs that could be related to PPID, Thane explained. None of the horses were receiving therapeutic treatment for PPID during the study.

The researchers collected the horses’ blood for baseline measurements just before the TRH injection, then repeated collection exactly nine, 10, and 11 minutes later. They froze the samples and sent them for laboratory testing to determine their ACTH concentrations.

Three-fourths of the horses had at least one early or late sample that varied by at least 10% compared to the 10-minute-mark sample, Thane said. And 21% of the study population had enough variation that their early or late sample would have given them a different diagnostic result—including three non-PPID horses that appeared borderline (“equivocal”) with the 9-minute result.

“Blood concentration of ACTH increases rapidly during this test, with the peak concentration typically occurring within 15 minutes of TRH administration,” said Thane, referring to previous research. “During this dynamic period, it is not surprising that small changes in sample collection timing will capture differences in the tested horse’s blood ACTH concentration as it responds to the administered stimulus.”

At baseline two of the horses had ACTH concentrations already suggestive of PPID, Thane said. At the 10-minute mark, six horses (25% of the study population) had concentrations consistent with PPID; the researchers diagnosed these six horses as being PPID-positive. When including samples collected one minute early or late, more than 40% of the horses had at least one sample that would have given them a positive or equivocal diagnosis, she added.

The findings hold particular importance regarding borderline cases, said Thane. “It is particularly important to interpret test results in conjunction with the horse’s age, medical history, clinical signs of PPID, and time of the year,” she said.

Take-Home Message

Researchers update and refine testing recommendations regularly as they learn more about endocrine dysfunction in horses, said Thane. Veterinarians seeking advice about selecting the right test, interpreting results, or prescribing appropriate therapies for complex cases can consult board-certified internal medicine specialists, she added.

Effect of early or late blood sampling on thyrotropin releasing hormone stimulation test results in horses first appeared in the Journal of Veterinary Internal Medicine in January 2022.