When False-Positives Arise: Troubleshooting a SARS-Coronavirus-2 (SARS-CoV-2) Detection Assay on a Semi-Automated Platform

Abstract

Background During the COVID-19 pandemic, many molecular diagnostic laboratories performed high-throughput SARS-CoV-2 testing often with implementation of automated workflows. In parallel, vaccination campaigns resulted increasingly in specimens from fully vaccinated patients, with resultant clinical inquiries regarding positive results in this patient population. This prompted a quality improvement initiative to investigate the semi-automated testing workflow for false-positive results. The troubleshooting workflow is described and procedural improvements are outlined that serve as a resource for other molecular diagnostic laboratories that need to overcome testing anomalies in a semi-automated environment. Methods This workflow utilized the MagMax-96 Viral RNA kit and the CDC 2019-nCoV RT-qPCR Panel on the Agilent Bravo Liquid-Handler (Bravo). Screening of the environment, personnel, and the mechanical performance of instrumentation using low Ct checkerboard challenges was executed to identify sources of cross-contamination. Evaluation of the assay and reporting design was conducted. Results Specimen contamination was observed during the viral extraction process on the Bravo. Changes to the program reduced plate contamination by 50% and importantly revealed consistent hallmarks of contaminated samples. We adjusted the reporting algorithm using these indicators of false positives. False positives that were identified made up 0.11% of the 45 000+ tests conducted over the following 8 months. Conclusions These adjustments provided confident and quality results while maintaining turnaround time for patients and pandemic-related public health initiatives. This corrected false-positive rate is concordant with previously published studies from diagnostic laboratories utilizing automated systems and may be considered a laboratory performance standard for this type of testing.