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. 2020 May;34(3):1350-1356.
doi: 10.1111/jvim.15771. Epub 2020 Apr 7.

The effect of freeze-thaw cycles on determination of immunoreactive plasma adrenocorticotrophic hormone concentrations in horses

Ke Hu  1 , Allison J Stewart  1 , Ka Y Yuen  1 , Sophia Hinrichsen  1 , Elizabeth L Dryburgh  2 , François-René Bertin  1
Affiliations

The effect of freeze-thaw cycles on determination of immunoreactive plasma adrenocorticotrophic hormone concentrations in horses

Ke Hu et al. J Vet Intern Med. 2020 May.

Abstract

Background: Determination of plasma adrenocotrophic hormone (ACTH) concentration (endogenous or thyrotropin-releasing hormone [TRH] stimulation test) is the most commonly used diagnostic test for pituitary pars intermedia dysfunction (PPID) in horses. Because ACTH is unstable, samples often are frozen to be shipped to laboratories or to allow for batch analysis of research samples. However, the effect of multiple freeze-thaw cycles on equine ACTH is unknown.

Objective: To determine the effects of multiple freeze-thaw cycles on immunoreactive ACTH concentration.

Animals: Twenty-eight horses ranging from 10 to 27 years of age were used.

Methods: Prospective study. Horses were divided into 4 groups: group 1, PPID-negative, without TRH stimulation; group 2, PPID-negative, with TRH stimulation; group 3, PPID-positive, without TRH stimulation; and group 4, PPID-positive, with TRH stimulation. Whole blood was collected from each horse at baseline or 30 minutes after TRH stimulation. Immunoreactive plasma ACTH concentration was determined using a chemiluminescence assay. Plasma samples then were frozen at -80°C >24 hours, thawed at 4°C and reanalyzed for 5 freeze-thaw cycles. Changes in plasma ACTH concentration were analyzed using a linear mixed-effect model.

Results: Significant effects of freeze-thaw cycles (P = .001) and PPID status (P = .04) on plasma ACTH concentration were observed, but no significant effect of TRH stimulation was identified.

Conclusions and clinical importance: The plasma ACTH concentration is altered by freeze-thaw cycles, and the effect is observed sooner in horses with PPID. To diagnose PPID, multiple freeze-thaw cycles should be avoided when measuring plasma ACTH concentration.

Keywords: clinical pathology; diagnostic; endocrinology; pituitary pars intermedia dysfunction; thyrotropin-releasing hormone.

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Conflict of interest statement

Elizabeth L. Dryburgh is employed by Boehringer‐Ingelheim Pty Ltd, and Allison J. Stewart and François‐René Bertin have consulted for Boehringer‐Ingelheim Pty Ltd.

Figures

FIGURE 1
FIGURE 1
Changes in immunoreactive ACTH concentration (as percentage of baseline) over 5 freeze‐thaw cycles in group 1 (no PPID using endogenous ACTH, n = 7, panel A), group 2 (no PPID using ACTH post‐TRH stimulation, n = 7, panel B), group 3 (being PPID using endogenous ACTH, n = 7, panel C), group 4 (being PPID using ACTH post‐TRH stimulation, n = 7, panel D). Data from individual horses are presented by dashed lines and group means are presented by solid lines. PPID, pituitary pars intermedia dysfunction; TRH, thyrotropin‐releasing hormone
FIGURE 2
FIGURE 2
Mean and SD of immunoreactive ACTH concentrations (as percentage of baseline) over 5 freeze‐thaw cycles in control (n = 14, black columns) and PPID (n = 14, gray columns) groups. *P < .05 compared with baseline. PPID, pituitary pars intermedia dysfunction
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