Evaluating the performance of the Roche FEN2 fentanyl immunoassay and its clinical implementation: The role of LDT-based mass spectrometry testing

• Published in: Journal of mass spectrometry and advances in the clinical lab Vol. 28; pp. 105 - 113
• Main Authors: Menlyadiev, Marlen, Suhandynata, Raymond T., Lund, Kyle, Kelner, Michael J., Fitzgerald, Robert L.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01-04-2023; Elsevier
• Subjects: Clinical sensitivity; FEN2; Fentanyl; Immunoassay; Mass spectrometry; Norfentanyl; LLOQ; Immunoassay; FDA; FEN2; Fentanyl; EHR; LC-MS/MS; DFU; QC; NADH; Clinical sensitivity; LDT; LC-QTOF; ULOQ; ED; CLSI; Norfentanyl; UDS; NAD; EMIT; NMS; UPLC; UCSD; ICU; Mass spectrometry
• ISSN: 2667-145X; 2667-1468; 2667-145X
• DOI: 10.1016/j.jmsacl.2023.02.009

•The FEN2 immunoassay met accuracy and precision performance requirements.•Clinical sensitivity for the FEN2 is 98% compared to 61% for the DRI.•The FEN2 assay was less prone to interferences than the DRI.•Use of the FEN2 improved FP and FN rates in real-world clinical fentanyl screens.
While laboratory-developed tests (LDTs) using liquid chromatography tandem mass spectrometry (LC-MS/MS) are widely employed to support the development of FDA-cleared drug immunoassays, their significance in the clinical implementation and evaluation of such assays is often overlooked. This paper reports on the important role of LC-MS/MS LDTs in demonstrating improved performance of the Roche FEN2 fentanyl immunoassay compared with the Thermo DRI fentanyl immunoassay.
The FEN2 assay was implemented according to the manufacturer's instructions and its performance was compared to the existing DRI assay using LC-MS/MS as a reference. Clinical sensitivity and specificity were determined using 250 consecutive random patient specimens. Spiking experiments were conducted to determine cross-reactivity with 31 fentanyl analogs. Select DRI false-positive samples were analyzed by the FEN2 assay via time-of-flight mass spectrometry method (LC-QTOF).
The FEN2 assay showed improved clinical sensitivity compared to the DRI (98% vs 61%) in 250 consecutive patient samples due to its ability to detect norfentanyl. It also showed better clinical specificity by correctly classifying select DRI false-positive results. Upon implementation in clinical practice, the FEN2 resulted in a higher screening positivity rate than the DRI (17.3% vs 13.3%) and a greater LC-MS/MS confirmation rate of immunoassay-positive samples (96.8% vs 88.8%, respectively).
The use of LC-MS/MS LDTs demonstrated that the FEN2 assay has greater clinical sensitivity and is less prone to false-positives than the DRI assay. These findings support the use of FEN2 in routine clinical practice and emphasize the role of mass spectrometry-based LDTs in clinical toxicology testing.