6 minute read
Endocrinology
Your TRH Test Questions Answered
By Paul Basilio
Pituitary pars intermedia dysfunction (PPID) is tough enough to monitor and treat in a controlled setting. When a veterinarian is in the field, outside factors can often throw the repeatability of the tests into doubt.
John C. Haffner, DVM, Rhonda M. Hoffman, PhD, PAS, DACAN; Steven T. Grubbs, DVM, PhD, DACVIM; Kayla N. Shepard, MS;Dwana L. Neal, MBA; and Greg L. Pearce recently completed a series of studies that aim to answer some common questions to put practitioners’ minds at ease.
1. How long is frozen/thawed thyrotropin-releasing hormone (TRH) effective for ACTH release?
For the first study, Dr. Haffner, an associate professor at Middle Tennessee State University, and his team tested the duration of effectiveness of frozen and thawed TRH.
“A practitioner who gets their TRH in a 5-dose vial was told that if you’re not going to use it all immediately, then you should draw individual doses into a 3-mL syringe and freeze them, then thaw them on the day of the TRH stimulation test,” said Dr. Haffner during a virtual presentation for the 66th Annual AAEP Convention. “The question then came up about how long you can thaw the TRH and keep it refrigerated before it’s not effective.”
It has been said that refreezing thawed TRH could alter it enough to affect the results, so the researchers wanted to test the hypothesis in a real-world scenario.
The team enrolled 10 horses and separated them into 2 groups—each group had 4 PPID-negative and 1 PPID-positive horses. They then froze 30 doses of constituted TRH at –20° C 28 days prior to testing. Fourteen days prior to testing, they thawed 10 doses and kept them at 5° C. The remaining 20 doses were thawed on the first day of testing and maintained at 5° C.
“When we did the TRH stimulation testing, the ACTH concentrations were not changed out to day 56,” Dr. Haffner said. “TRH is still good if it’s kept refrigerated for 56 days.”
2. Will trailering or dentistry affect ACTH levels?
“The second study came up as a question,” he explained. “If someone hauls a horse into a clinic or if the horse had a minor procedure like teeth floating, would that affect the adrenocorticotropic (ACTH) levels in a way that would affect the TRH stimulation test?”
The team took 12 PPID-negative horses and divided them into 3 groups. Plasma was collected at baseline. The first group was taken on a 40-minute trailer ride, and the second group had their teeth floated with xylazine sedation. The third group was left tied in the stall.
“Four weeks later, we rotated the groups, and then rotated them again after another 4 weeks,” Dr. Haffner said. “All 12 horses were exposed to all 3 treatments.”
Blood samples were taken at 15, 30, 60 and 120 minutes after the trailering or teeth floating was completed.
Results showed no changes in ACTH levels in the stall and dentistry group, but trailering did cause an
increase in ACTH from baseline. ACTH remained increased at 15 minutes, but levels returned to baseline after an average of 30 minutes. However, 1 horse had elevated ACTH levels until 120 minutes.
The researchers recommended that blood should not be obtained for resting ACTH levels for at least 30 minutes after the horse is brought in on a trailer.
3. What is the repeatability of TRH testing for PPID?
Next up, the team wanted to evaluate TRH repeatability in the late winter and spring months in both PPID-positive and PPID-negative horses.
Five positive, 5 negative, and 2 PPID equivocal horses were enrolled, and testing was done every 28 days from February to June in Tennessee (Latitude 35°, 50' 44" North).
“Generally, the results were consistent,” Dr. Haffner said. “If the horse tested positive, it continued to be positive. If it was negative, it continued to be negative, and if it was equivocal, it stayed equivocal throughout the study.”
However, he did note that 3 of the horses were inconsistent. One PPID-negative horse tested positive once, and another negative horse tested positive twice. One positive horse tested equivocally once.
“It reminds us that it’s important to include history and clinical signs when you’re trying to make a diagnosis of PPID,” he added. “The lab data all have to be looked at. Don’t just rely on the results from a blood test before you consider a horse to be positive for PPID.”
4. How long can centrifugation wait?
After a long day in the truck, sometimes the last thing you want to do is sit and watch plasma spin. But after a practitioner obtains an ACTH sample, how long can it be refrigerated before it can be centrifuged?
To answer this, the team enrolled 5 positive and 5 negative horses, drew blood in purple top tubes, and placed them in a standard refrigerator.
The first sample was spun down immediately, and the rest were kept in the refrigerator. Samples were then removed and spun at 4, 8, 12 24, and 36 hours after collection, then frozen and shipped to the Cornell lab for ACTH measurement.
“What we found was that there was no variation in the ACTH levels out to 36 hours,” Dr. Haffner said. “We determined that it is safe to keep the samples refrigerated until the practitioner can return to the clinic within 36 hours. Hopefully, we’re not working 36-hour days yet, so you should be good to go.”
5. What’s the best way to freeze ACTH plasma?
The final study in the series tested the effect of various freezing protocols on ACTH plasma concentrations.
“We were looking at 2 different situations,” Dr. Haffner explained. “One is the practice situation, where you might want to keep the plasma frozen over the weekend. The other is in a research situation, where you might want to keep it frozen for several months or years in a –80° C freezer.”
The team took samples from 12 horses (5 mares, 6 geldings and 1 stallion) and froze them in a freezer set at either –20° C or –80°C.
To mitigate the fluctuation of temperatures that occurs when staff open and close the freezer door, some samples in the –20° C freezer were also placed between ice packs.
“It’s been thought that if you have a sample in a freezer in the clinic and people are opening and closing the door, that may affect ACTH levels,” he said.
In the short term, none of the samples showed any significant decrease in ACTH levels. The freezer packs did not appear to offer any benefit for the samples stored at –20° C.
“We did see that there was some decrease in ACTH levels by 90 days in the samples kept at –80° C,” Dr. Haffner explained. “There was also some decrease by day 60 in the samples at –20° C, but there was no decrease out to 7 days.”
For the specimens held at –80° C, the samples showed a 6.9% decrease in ACTH levels from baseline to day 90.
“If you're doing a research project, you need to know that if you keep it more than 90 days, you might see some degradation in the values,” he said. “In a practice situation, you’re probably not going to keep it for more than a week anyway. If you get a sample on a Friday, just freeze it and ship it out by Monday.”
