not be collected for resting ACTH concentration de- termination for at least 30 minutes after trailering.


4. TRH Repeatability in PPID-Negative and PPID-Positive Horses


Introduction


Even though TRH stimulation of ACTH has been used as a diagnostic test for equine PPID, it is un- known whether the T10-ACTH response to TRH is repeatable in individual horses. The purpose of this study was to conduct TRH stimulation tests at 4-week intervals, beginning in February and ending in June, in horses with and without PPID to deter- mine the repeatability of the T10-ACTH, over time.


Materials and Methods


Twelve horses, 5 PPID positive (PPID), 5 PPID negative (PPID–), and 2 PPID equivocal with a mean age of 18.8 years (range, 12 to 25 years) were en- rolled. Basal ACTH concentration from blood col- lected in January was used to identify PPID status of each horse. The TRH stimulation procedure was performed on day 0 (February 13) and repeated on Days 28, 56, 84, and 112. The subsequent samples were compared to the T10-ACTH samples collected on Day 0. Data were confirmed for normality using the Shapiro-Wilk statistic, and then analyzed using a mixed model with repeated measures to compare T10-ACTH and the percent increase of ACTH after TRH stimulation, using horse as the subject and day as the repeated effect. Pearson’s correlation coeffi- cients were used to examine relationships between T10-ACTH on days 28, 56, 84, and 112 to T10-ACTH on day 0. Bland-Altman plots were constructed to compare T10-ACTH on Days 28, 56, 84, and 112 to the T10-ACTH on Day 0.


Results


The mean basal ACTH in PPID– horses was 17.6  0.7 pg/mL and 61.49.2 pg/mL (T10-ACTH), with a 349  41% increase in T10-ACTH after TRH stim- ulation. The mean basal ACTH in PPID horses was 43.5  3.6 pg/mL (basal) and 410  58 pg/mL (T10-ACTH), with a 948  194% increase in T10- ACTH after TRH stimulation. Repeated measures analysis indicated no effect of day on T10-ACTH (P  .40), or the percent increase of T10-ACTH after TRH stimulation (P  .12). Pearson’s correlation coefficients indicated strong relationships between T10-ACTH on Day 0 and all other days (R  0.70, P  .01). Bland-Altman plots indicated an average day bias of 27 pg/mL in all horses compared to day 0, with a day bias of 10 pg/mL in PPID– (with 95% limits of agreement at 101 to 122 pg/mL) and 43 pg/mL in PPID (with 95% limits of agreement at 493 to 581 pg/mL) horses. The Immulite intra- assay CV was 9.3%, which accounts for most of the observed day bias.


MEDICINE: NON-INFECTIOUS DISEASES Discussion


The TRH stimulation procedure produced repeat- able ACTH concentrations in samples collected 10 minutes after administration of TRH in horses col- lected at 4-week intervals over 112 days from Feb- ruary through June. Observed variation of T10 ACTH over the duration of the study resulted in 2/5 PPID negative horses classified as positive, once and twice, respectively. Additionally, the T10 ACTH in 1/5 PPID positive horses tested equivocal at one time point. The results of this study stress the importance of using the combination of owner his- tory, clinical signs, and laboratory data when deter- mining the proper diagnosis of PPID.


5. Effect of Delayed Plasma Centrifugation on Equine ACTH Concentration


Introduction


If stored at room temperature (21°C), ACTH level in blood decreased soon after collection.6 Multiple recommendations exist for the timing of centrifuga- tion of chilled samples from sample collection. In the United States, a discordance exists between lab- oratory recommendations concerning centrifugation time from sample collection. Realistically, many equine ambulatory practitioners do not have the ability to centrifuge the sample within 2 to 4 hours. This study was conducted in order to determine the length of time whole blood can be stored refrigerated prior to centrifugation and maintain accurate ACTH concentration.


Materials and Methods


On day 0, 5 mL of whole blood from each of 10 horse (Five PPID positive and 5 PPID negative) was col- lected into each of 6 ethylenediaminetetraacetic acid (EDTA) tubes and immediately placed in a refriger- ator at 7°C. One tube from each horse was centri- fuged within 15 minutes of collection, followed by centrifugation of one tube from each horse at 4, 8, 12, 24, and 36 hours following collection. At each time, centrifuged plasma was pipetted into 1.5 mL polypropylene tubes and stored at –80°C. None of the plasma samples were turbid, hemolyzed, or ic- teric. Plasma was shipped frozen with cold packs overnight to the Animal Health Diagnostic Center of Cornell University in Ithaca, NY for analysis. The percent change from baseline (PCFB) was re- ported to standardize the data given that baseline values differed.


Results


The absolute changes over time revealed no pattern of variation. The mean PCFB was 2.8%  7.96% (95% CI,: –2.9%, 7.0%). There was no evidence of significant time effect from the repeated measures model with a P-value of .5056. There was no evi- dence of significant time effect on the level of ACTH in PPID positive or PPID negative horses. Three of 10 enrolled horses exhibited variation in ACTH


AAEP PROCEEDINGS  Vol. 66  2020 305


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