Toward Uniform Nucleic Acid Testing Algorithm: Resolving Suspicious NAT Results for 3-Target Multiplexing Transcription-Mediated Amplification (TMA) Assay.

Abstract

Background: Nucleic Acid Testing (NAT) is a technology that offers extreme sensitivity. TMA NAT uses a single temperature, which eliminates the need for special thermocycling equipment; carryover contamination is minimized because of the labile nature of the RNA amplicon. Recently, there were significant discussions of the perceived false positive rate with NAT. Our initial NAT testing algorithm was developed for 2-target multiplexing TMA NAT. After switching to 3-target TMA in 2012, our laboratory has re-evaluated and increased the robustness of our NAT algorithm. Our goal was to improve interpretation and detection of aberrant TMA NAT events and to improve the turn-around-time.Figure: A. Normal Curve B. Cross-contamination C. NAT InhibitorMethods: Between 01-06/2013, we tested 5424 organ/LRD, tissue. We used 3-target multiplexing TMA Procleix- ULTRIO (HIV-1/HCV/HBV) eSAS NAT (Novartis/GenProbe). Results: We experienced 3.2% invalid runs and 0.5% non-repeatable NAT reactions (table). With a revised NAT algorithm, we were able to resolve all invalid runs using a combination of amplification curve analysis (figure A-C, normal/cross-contamination/inhibitors) and more robust screening steps. Using “serology like algorithm” along with the package insert recommendations, all initially false reactive reactions were reported as negative with no loss of donors. There was 1 donor with non-amplifiable RNA/DNA by TMA (0.02%).Table: No Caption available.Conclusions: We reported earlier that using “serology-like” algorithm, the number of false positive NAT results is less than 1 in 7000 for 2-target TMA NAT. Our experience with 3-target NAT and our refined algorithm show that even with more complicated NAT assays, false positive are rare in laboratories practicing good laboratory practices with established testing algorithms.