B-303 Validation of a New Benzodiazepine Immunoassay with Improved Detection of Glucuronidated Benzodiazepines

Abstract

Background The new Roche benzodiazepines II immunoassay utilizes an on board beta-glucuronidase enzyme that should enhance detection of glucuronidated benzodiazepines. We evaluated and validated this new assay across three laboratory testing sites: 1) a community hospital, 2) a quaternary-care medical center and a 3) outreach reference laboratory. Methods The new ONLINE DAT Benzodiazepines II immunoassay (IMA) utilizes the same calibrators and controls as its predecessor but it includes a ß-glucuronidase enzyme in one of its reagents. This addition should help hydrolize glucuronide from its benzodiazepine conjugates and improve the detection of benzodiazepine glucuronides. Any samples that were discordant were run by liquid chromatography-mass spectrometry for confirmation. We tested the precision of the immunoassay systems by taking a positive and negative sample and running over a total of 20 runs. We tested 120 patient samples using both the current Benzodiazepine Plus and the new Benzodiasepines II immunoassays across three laboratory testing sites. The cut off used for a positive result was 200 ng/mL. A query was conducted to review the number of positive and negative benzodiazepine results 3 months prior to converting to the Benzodiazepine II version of the assay and 3 months after. Results The assay was precise 100% of the times using both negative and positive samples across all three- testing sites. A total of 59 positive samples and a total of 61 negative samples by the old Benzodiazpines II immunoassay were used for patient correlation. The 59 positive samples using the old Benzodiazepine plus immunoassay were also positive by the new Bensoziazepine II immunoassay. We noticed that from the 61 negative samples as classified by the old Benzodiazepine Plus immunoassay, 8 samples tested positive when run on the new Benzodiasepine II immunoassay. These 8 samples were confirmed by mass spectrometry to be true positive. We noticed that the 8 true positive samples had the following analytes: Sample 1, alprazolam at 59 ng/mL and a-OH alprazolam at 64 ng/L; Sample 2, alprazolam at 99 ng/mL and a-OH alprazolam at 36 ng/mL; Sample 3, a-OH midazolam at 377 ng/mL; Sample 4, lorazepam at 650 ng/mL; Sample 5, lorazepam at 370 ng/mL; Sample 6, lorazepam at 1659 ng/mL; Sample 7, lorazepam at 726 ng/mL; Sample 8, lorazepam at 34 ng/mL, alprazolam at 22 ng/mL, a-OH alprazolam at 17 mg/mL, 7-aminoclonazepam at 241 ng/mL. Our query identified that in the prior month before converting to Benzodiazepine Plus IMA the rate of positive Benzodiazepine IMA screens was 8%, and 3 months after converting to the Benzodiazepine Plus IMA the rate of a positive benzodiazepine screen increased to 15%. Conclusion Our results indicate that the new version of the assay, by using the addition of the on board hydrolysis step with beta glucuronidase greatly enhances the detection rate of benzodiazepines.