Minimizing False Negatives in Urine Benzodiazepine Screening

Hydrolysis of urine before benzodiazepine immunoassay screening and optimization of the immunoassay absorbance cutoff were found to improve the rate of false-negative screening in a study published in the Journal of Applied Laboratory Medicine.

To improve the clinical sensitivity of benzodiazepine immunoassays conducted on urine samples, researchers evaluated the effect of sample hydrolysis (n=21) and optimization of the absorbance value cutoff point (n=82) on randomly selected urine samples that tested negative for benzodiazepines (ie, with 0 to 99 absorbance values in immunoassays).

The true presence of benzodiazepine was confirmed with liquid chromatography-tandem mass spectrometry (LC-MS/MS). After selecting an optimized absorbance cutoff for immunoassays (20 vs 100), a prospective trial of all urine samples was performed.

Of the 21 initially negative urine samples that were included in the hydrolysis analysis, 81% proved to be positive, based on LC-MS/MS confirmation. Hydrolysis had no effect on truly negative samples, but did increase the absorbance levels of 7 of 17 true-positive urine samples containing lorazepam, diazepam, or alprazolam. Hydrolysis had no effect on urine samples containing clonazepam or 7-aminoclonazepam.

In the absorbance optimization analysis, 85% of urine samples with an absorbance between 20 and 99 (17 of 20) were true positives, according to LC-MS/MS testing. In contrast, 5% of urine samples with an absorbance <20 (2 of 42) were considered true positives, based on mass spectrometry.

During a 2-week period, 83% of prospectively collected urine samples with an absorbance of 20 or higher (38 of 46) were positive, according to LC-MS/MS analysis. Furthermore, 45% of the true-positive samples had absorbance values of 20 to 99 and would have been considered negative based on immunoassay screening.

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“This study demonstrates that our urine benzodiazepine testing false-negative test rate of 47% can be lowered to 2% by optimizing the absorbance cutoff required for confirmatory testing,” concluded the investigators. “The anticipated clinical impact of using an optimized absorbance cutoff is to minimize the risks of patients being wrongfully denied medication and/or not detecting illicit or dangerous drug use.”

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Mullins GR, Reeves A, Yu M, Goldberger BA, Bazydlo LAL. Improved clinical sensitivity of a reflexive algorithm to minimize false-negative test results by a urine benzodiazepine immunoassay screen. J Appl Lab Med. 2018;2(4):555-563.