Abstract 2214: Development and validation of PredicineATLAS, a 600-gene liquid biopsy panel for simultaneous detection of TMB and genomic alterations in Immuno-Oncology

Abstract Paclitaxel (PTX) and sulforaphane (SFN) are known anticancer molecules. Their activity was found to be potentiated when tested concurrently. Only recently, however, a novel SFN enabled PTX self-microemulsifying formulation (SMEDDS) was developed for their simultaneous delivery. This necessitated the development of an analytical method for the simultaneous detection and quantitation of PTX and SFN. In this study, a simple and sensitive isocratic high performance liquid chromatography-ultraviolet (HPLC-UV) analytical method was developed and validated per International Conference on Harmonization guidelines to satisfy this objective. Its application was demonstrated when quantifying the amount of PTX and SFN released from the SMEDDS in various dissolution media. The separation of the analytes was performed with the aid of a reversed phase C18 column at ambient temperature using a 60:40 mixture of acetonitrile and KH2PO4 buffer (pH 5.0) as the mobile phase. PTX and SFN peaks were detected at 202 nm with high resolution without interference from excipients. This method showed linearity within 2.5–100 μg/mL range with r2 > 0.999. The limit of detection and lower limit of quantitation were 0.1638 and 0.4964 μg/mL for PTX and 0.4419 and 1.3389 μg/mL for SFN, respectively. A total of 98–101% of the injected samples was recovered with RSD of 0.06–0.68% indicating the suitability of the method for the simultaneous detection and quantitation of the molecules in dissolution media.

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Evaluation of a laboratory-developed test for simultaneous detection of norovirus and rotavirus by real-time RT-PCR on the Panther Fusion® system

European Journal of Clinical Microbiology & Infectious Diseases ◽

10.1007/s10096-019-03697-7 ◽

2019 ◽

Vol 39 (1) ◽

pp. 103-112 ◽

Cited By ~ 2

Author(s):

Robert K. Kulis-Horn ◽

Carsten Tiemann

Keyword(s):

Real Time ◽

Simultaneous Detection ◽

Extraction Process ◽

Clinical Samples ◽

Preparation Methods ◽

Virus Assay ◽

Sample Extraction ◽

Stool Samples ◽

Development And Validation ◽

Result Interpretation

Abstract The Hologic Panther Fusion® Open Access™ functionality allows implementation of laboratory-developed tests (LDTs), with fully automated sample extraction, real-time PCR, and result interpretation. We report the development and validation of a multiplex LDT for norovirus G1, norovirus G2, and rotavirus from stool samples on this system. The LDT was optimized for primer and probe sequences, salt concentration, and PCR annealing temperature. Reproducibility of the PCR and extraction process was assessed. Performance of the multiplex LDT assay was evaluated with external quality assessment (EQA) samples and compared to a commercial multiplex assay (Allplex™ GI-Virus Assay, Seegene) in clinical samples. Salt concentrations and annealing/extension temperature were optimized to 4 mM MgCl2, 70 mM KCl, 20 mM Tris, and 60 °C, respectively. The user-prepared part of the LDT PCR mix (containing salts, probes, and primers) was stable for ≥ 11 days onboard the instrument. We observed reproducible results of PCR and the extraction process. The LDT had a sensitivity comparable to or greater than the commercial Allplex™ assay and showed excellent linearity. Forty-five EQA samples yielded the expected result with the LDT. There was 100% concordance between LDT and Allplex™ results in 160 clinical samples. Results from the suspension and direct swab stool sample preparation methods were highly concordant in the LDT. We report the successful development and validation of a multiplex PCR LDT for detection of norovirus G1, norovirus G2, and rotavirus from stool samples on the Panther Fusion® system.

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