Comparison of in vitro degradation of immunoreactive adrenocorticotropic hormone and insulin in equine blood measured by two immunoassay methods.

Overview

• This study investigated the stability of two important hormones—adrenocorticotrophic hormone (ACTH) and insulin—in horse blood samples stored under different conditions and measured them using two distinct immunoassay methods.
• The goal was to understand how storage time and temperature affect hormone measurement accuracy and which assay method gives more reliable results after storage.

Background and Objective

• ACTH and insulin are critical hormones often measured in veterinary diagnostics for horses to assess endocrine function.
• Hormone levels can degrade or change during storage, potentially leading to inaccurate diagnostic results.
• The study compared two common immunoassay platforms: chemiluminescent assay (CLA) and immunofluorescent assay (IFA), focusing on how well each method preserves the accuracy of hormone measurements after different storage durations and temperatures.

Methods

• Samples:
  • EDTA-treated blood samples for ACTH analysis and coagulated blood for insulin.
  • Fresh samples were aliquoted and divided for immediate analysis (baseline, T0) and for storage under two temperature conditions: chilled (4°C) and room temperature (~21°C).
• Storage times:
  • ACTH: measured at 24, 48, and 72 hours post-collection.
  • Insulin: measured at 24 hours, 72 hours, and 7 days post-collection.
• Measurement:
  • Two assay methods were used—CLA (Siemens Immulite 2000XPi) and IFA (Tosoh AIA-900).
• Statistical analyses:
  • Repeated-measures ANOVA examined percentage recovery relative to baseline (T0).
  • Linear mixed-effects modeling confirmed the results.
  • Bland-Altman analysis assessed agreement between CLA and IFA at baseline to investigate bias and variability.

Key Findings

• ACTH Stability:
  • ACTH concentrations significantly decreased over time regardless of temperature or assay.
  • At refrigerated temperatures, IFA showed better recovery of ACTH (closer to baseline) than CLA at all time points, indicating it was more stable or accurate under chilled conditions.
  • At room temperature, no significant difference was found between the two assays in ACTH measurement.
  • Bland-Altman analyses revealed proportional bias in ACTH measurement between assays, meaning that the difference depended on the magnitude of hormone concentration, especially on the original measurement scale.
  • Log transformation of data reduced but did not eliminate this concentration-related disagreement, especially at room temperature.
• Insulin Stability:
  • Insulin remained largely stable over the storage periods tested at both temperatures.
  • No interaction was found between assay type and time, meaning both methods provided consistent insulin measurements over time.
  • Bland-Altman analysis did not indicate proportional bias, suggesting good agreement between the methods across concentrations.

Practical Implications

• ACTH samples require prompt and chilled handling to limit degradation and measurement errors.
• When immediate analysis is not feasible, the immunofluorescent assay (IFA) is preferable over the chemiluminescent assay (CLA) for maintaining ACTH measurement accuracy after storage.
• Insulin measurements are comparatively robust and reliable under short-term storage at both room and refrigerated temperatures regardless of the assay used.
• Veterinarians and researchers should consider assay choice and sample handling protocols to ensure precise hormone measurement in equine diagnostics.

Summary

• This research highlights differences in the stability of ACTH and insulin in stored equine blood samples when measured by two immunoassays.
• While insulin is stable under common storage conditions, ACTH rapidly degrades, and its measurement is more reliable using immunofluorescent assays under chilled storage.
• These findings inform best practices for sample handling and assay selection in equine endocrine testing, aiming to improve diagnostic accuracy.