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Excess EDTA interferes with cortisol measurement using a solid-phase, chemiluminescent enzyme immunoassay.

Robert J Kemppainen1, Ellen N Behrend1, Stephanie F Carter1

  • 1Departments of Anatomy, Physiology, and Pharmacology (Kemppainen, Carter) and Clinical Sciences (Behrend, Cole), College of Veterinary Medicine, Auburn University, Auburn, AL.

Journal of Veterinary Diagnostic Investigation : Official Publication of the American Association of Veterinary Laboratory Diagnosticians, Inc
|March 13, 2018
PubMed
Summary

Elevated EDTA levels in canine serum can falsely increase cortisol measurements using chemiluminescent enzyme immunoassays (CEIA). Concentrations exceeding 5.1 mmol/L significantly impact assay accuracy.

Keywords:
Alkaline phosphataseEDTAassay interferencechemiluminescent immunoassaycortisolmagnesium

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Area of Science:

  • Veterinary diagnostics
  • Clinical chemistry
  • Immunoassay technology

Background:

  • Automated chemiluminescent enzyme immunoassays (CEIA) are widely used for hormone analysis.
  • CEIA assays in the laboratory utilize serum or EDTA plasma.
  • An observed increase in CEIA cortisol results with excess EDTA prompted further investigation.

Purpose of the Study:

  • To investigate the impact of varying ethylenediaminetetraacetic acid (EDTA) concentrations on cortisol measurements in canine serum.
  • To determine the threshold concentration of EDTA that affects CEIA cortisol assay accuracy.

Main Methods:

  • Pooled canine serum samples were supplemented with different concentrations of EDTA.
  • Cortisol concentrations were measured using a CEIA method.
  • The effect of magnesium chloride (MgCl2) supplementation was evaluated.

Main Results:

  • The recommended EDTA concentration (4.1 mmol/L) did not affect serum cortisol levels.
  • EDTA concentrations of 5.1 mmol/L and above led to a significant increase in apparent cortisol concentrations.
  • Magnesium chloride supplementation partially reversed the EDTA-induced elevation of cortisol.

Conclusions:

  • CEIA cortisol assays performed on EDTA plasma can yield falsely elevated results when EDTA concentrations exceed 5.1 mmol/L.
  • Laboratory protocols should ensure EDTA concentrations remain below this critical threshold for accurate cortisol determination in dogs.