Transference of diagnostic thresholds for equine plasma ACTH from the Immulite 1000 to Immulite 2000XPi and Tosoh AIA systems.

Overview

• This study evaluated how diagnostic thresholds for measuring plasma ACTH, important for diagnosing equine PPID, can be accurately transferred from the Immulite 1000 system to newer assay platforms.
• The research established and validated platform-specific diagnostic thresholds for the Immulite 2000XPi and Tosoh AIA systems to ensure consistent and reliable PPID diagnosis across different analyzers.

Background

• Diagnosis of Pituitary Pars Intermedia Dysfunction (PPID) in horses largely depends on measuring plasma adrenocorticotrophic hormone (ACTH) levels.
• ACTH concentrations are measured using various analytical platforms/assays, but values can vary between these systems.
• Most existing diagnostic thresholds—the cutoffs used to determine if a horse has PPID—are based on data from the Immulite 1000 chemiluminescent assay (CLA).
• The emergence of newer assay platforms requires the validation and adjustment of these thresholds so that clinicians and labs can interpret results accurately across different machines.

Aims of the Study

• To transfer and validate the established diagnostic ACTH thresholds derived from the Immulite 1000 to three contemporary assay systems:
• Immulite 2000XPi (a newer chemiluminescent platform)
• Tosoh AIA-900
• Tosoh AIA-360
• Utilize Clinical Laboratory Standards Institute (CLSI) guidelines to ensure rigorous method comparison and threshold transference.

Methods

• Clinical plasma samples (collected in EDTA tubes) that were leftover from routine testing were used for cross-platform analysis.
• Samples were run in parallel on pairs of analyzers to compare results directly and understand differences between systems.
• Statistical assessments included correlation analyses and bias estimation between platforms to evaluate agreement.
• Reference intervals and diagnostic thresholds were transferred or adapted using appropriate regression analyses depending on the nature of the agreement/bias.

Key Findings

• Immulite 2000XPi vs Immulite 1000:
  • Very strong correlation (r = 0.962, p < 0.0001)
  • Minimal bias (-3.9 pg/mL, with confidence interval crossing zero), indicating near equivalence.
  • Allowed for simple linear transfer of diagnostic thresholds without complex adjustment.
• Immulite 2000XPi vs Tosoh AIA-900:
  • Moderate correlation (r = 0.770, p < 0.0001)
  • Non-linear and concentration-dependent bias with larger variation (bias 30.8 pg/mL, 95% CI from -110 to 171), indicating measurements differed more at different levels.
  • Thresholds needed to be adjusted using specific regression models for different concentration ranges.
• Tosoh AIA-360 vs Tosoh AIA-900:
  • Excellent agreement (r = 0.993, p < 0.0001)
  • Low bias (4.023 pg/mL), supporting interchangeability and direct threshold transfer.

Diagnostic Thresholds Established

• The study generated platform-specific diagnostic thresholds designed for two clinical decision points:
• Low threshold with approximately 90% sensitivity — to minimize false negatives
• High threshold with approximately 90% specificity — to minimize false positives
• Thresholds were further stratified across four seasonal time periods to account for known seasonal variation in plasma ACTH levels in horses.
• These seasonally adjusted thresholds allow more accurate interpretation of ACTH results depending on when samples were collected.

Clinical and Laboratory Implications

• Clinicians and diagnostic laboratories can now use validated and assay-specific ACTH cutoffs tailored to current platforms, improving diagnostic accuracy for equine PPID.
• Reduces misclassification risks that may arise when thresholds from one platform (Immulite 1000) are applied unadjusted to others.
• Supports better disease management decisions, monitoring, and treatment outcomes by improving confidence in ACTH measurements.
• Provides a framework for ongoing validation as new assay technologies emerge in veterinary endocrinology.

Summary

• The study supports a smooth transition from older to newer assay platforms for ACTH measurement in horses.
• It emphasizes the importance of method comparison and statistical adjustment to maintain diagnostic reliability.
• Seasonal and platform-specific thresholds enhance clinical utility and precision in diagnosing PPID.