Analytical Validation of the Cxbladder Triage Plus Assay for Risk Stratification of Hematuria Patients for Urothelial Carcinoma.

: Cxbladder Triage Plus is a multimodal urinary biomarker assay that combines reverse transcription-quantitative analysis of five mRNA targets and droplet-digital polymerase chain reaction (ddPCR) analysis of six DNA single-nucleotide variants (SNVs) from two genes (fibroblast growth factor receptor 3 () and telomerase reverse transcriptase ()) to provide risk stratification for urothelial carcinoma (UC) in patients with hematuria. This study evaluated the analytical validity of Triage Plus. : The development dataset used urine samples from patients with microhematuria or gross hematuria that were previously stabilized with Cxbladder solution. Triage Plus was evaluated for predicted performance, analytical criteria (linearity, sensitivity, specificity, accuracy, and precision), extraction efficiency, and inter-laboratory reproducibility. : The development dataset included 987 hematuria samples. Compared with cystoscopy (standard of care), Triage Plus had a predicted sensitivity of 93.6%, specificity of 90.8%, positive predictive value (PPV) of 46.5%, negative predictive value of 99.4%, and test-negative rate of 84.1% (score threshold 0.15); the PPV increased to 74.6% for the 0.54 score threshold. For the individual and SNVs, the limit of detection (analytical sensitivity) was a mutant-to-wild type DNA ratio of 1:440-1:1250 copies/mL. Intra- and inter-assay variance was low, while extraction efficiency was high. All other pre-specified analytical criteria (linearity, specificity, and accuracy) were met. Triage Plus showed good reproducibility (87.9% concordance between laboratories). : Cxbladder Triage Plus accurately and reproducibly detected and SNVs and, in combination with mRNA expression, provides a non-invasive, highly sensitive, and reproducible tool that aids in risk stratification of patients with hematuria.