Fentanyl was first approved by the Food and Drug Administration (FDA) in 1968 as an intravenous anesthetic under the trade name of Sublimaze. Abuse of fentanyl initially appeared in mid-1970s and has increased dramatically in recent years. According to the Centers for Disease Control and Prevention (CDC), the number of drug overdose deaths involving synthetic opioids, primarily fentanyl, increased more than10-fold from 2014 through 2022, ranging from about 5,500 in 2014 to 75,000 in 2022 (Ahmad et al., 2023). National Forensic Laboratory Information System (NFLIS) identified a total of 262,500 narcotic analgesic reports in 2022, and Fentanyl accounted for 62% of them (Diversion Control Division, 2023). In addition, the fentanyl epidemic continues to evolve with the emergence of fentanyl analogs. Compared to morphine, fentanyl is approximately 100 times more potent by weight, while fentanyl analogs have a range of potency varying from approximately 3 to 10,000 times that of morphine (Ciccarone, 2017, Suzuki and El-Haddad, 2017). Current emerging drug use patterns reveal that fentanyl and fentanyl analogs are used as adulterants in illicit heroin, cocaine, and methamphetamine, and sold as counterfeit tablets claimed to be prescription-grade opioids, exposing unsuspecting users to an increased risk of overdose due to the high potency of the compounds (Ciccarone, 2021, LaRue et al., 2019).

To respond to the surge of fentanyl-related overdose death, screening for fentanyl has been adopted by many clinical laboratories to detect illicit drug use and monitor compliance. In 2017, the FDA cleared the first fentanyl immunoassay, Immunalysis SEFRIA Fentanyl Urine Enzyme Immunoassay, for in vitro diagnostics (IVD) use in clinical laboratories. Prior to that, fentanyl screen could only be done in forensic laboratories or in clinical laboratories as laboratory developed test. Thereafter, ARK (phased out), ARK II, LZI and LZI II fentanyl immunoassays were cleared by the FDA for IVD use on various automated clinical chemistry analyzers. Most recently, a number of waived point-of-care devices for fentanyl screening have been rapidly cleared by the FDA, allowing fentanyl testing to be used in non-laboratory settings holding a Clinical Laboratory Improvement Amendments (CLIA) certificate of waiver. Positive cutoffs of all fentanyl immunoassays, except for LZI, LZI II and Chemtrue, are based on fentanyl at a concentration of 1.0 ng/mL. The LZI, LZI II and Chemtrue use norfentany at a concentration of 5.0 ng/mL as positive cutoff.

Fentanyl screening assays are relatively new to many clinical laboratories compared to other drugs of abuse testing, such as opiates screen, and therefore their clinical performances in real patient settings are largely unknown. Given the potency of fentanyl, all screening assays have placed significant emphases on analytical sensitivity to detect low level fentanyl. Urine is the matrix that accumulates large amount of drug metabolites and sometimes parent drugs, so low level antibody cross-reactivity is sufficient to generate positive results in urine drug screening. Per the package insert, a number of drugs showed low cross-reactivities with the SEFRIA fentanyl immunoassay, ranging from 0.001% to 0.08% (510k summary). Nonetheless, without information on the urine drug concentrations and cross-reactivities with urine drug metabolites, it is not feasible to evaluate the impact of these low cross-reactivities on the assay specificity. In this study, we extensively examined the performance of the SEFRIA fentanyl immunoassay using deidentified real-time patient data from 4 hospitals in our health system. By implementing a new positive cutoff and identifying the main interfering substances in this local patient population, we were able to significantly reduce the false positive rate and achieve a satisfactory performance with SEFRIA fentanyl immunoassay.