Resolution of an apparent hook effect in Roche partner DRI oxycodone immunoassay

Area of Science:

• Clinical Chemistry
• Immunoassay Technology
• Forensic Toxicology

Background:

• The DRI Oxycodone immunoassay (OXY3S) on Cobas Integra may exhibit a hook effect, leading to inaccurate low results at high analyte concentrations.
• This effect was observed in urine samples with confirmed high oxycodone and oxymorphone levels exceeding 10,000 ng/mL, yet yielding OXY3S results below 1,000 ng/mL.

Purpose of the Study:

• To investigate the presumed hook effect in the semiquantitative DRI Oxycodone immunoassay (OXY3S).
• To identify modifications to the immunoassay to ensure reliable detection of high oxycodone concentrations in urine samples.

Main Methods:

• Investigated 14 urine samples with discrepant results between GC-MS and OXY3S using serial dilutions.
• Replicated and modified OXY3S test parameters on a Cobas development channel using DRI Reagents.
• Tested modifications including delayed sample addition, doubling Reagent 1 (R1), and supplementing R1 with substrate or co-factors.

Main Results:

• Patient samples required 2- to 16-fold dilution to achieve accurate OXY3S readings.
• Doubling Reagent 1 (R1) or adding it later corrected results in 14/14 and 12/14 samples, respectively.
• Supplementing R1 with co-factors corrected 13/14 samples; the remaining sample required supplemental R1 alone for correction.

Conclusions:

• The semiquantitative reliability of the DRI Oxycodone assay is questionable due to the hook effect.
• Modifications to Reagent 1, particularly doubling its concentration or adding co-factors, enable reliable oxycodone determination at high concentrations.
• The study provides a method to overcome under-recovery issues in oxycodone immunoassays for clinical urine samples.