Analytical Validation of MethyLiverDx-Sigma, a Plasma cfDNA Methylation Assay for Hepatocellular Carcinoma Detection

Hepatocellular carcinoma surveillance relies on ultrasonography and alpha-fetoprotein, both limited in sensitivity for early-stage disease; aberrant methylation of circulating cell-free DNA (cfDNA) offers a promising blood-based approach. Reported here is the analytical validation of MethyLiverDx-Sigma (MLDx-s), an assay that detects methylated RNF135 and DNMT3A in bisulfite-converted plasma cfDNA by methylation-specific PCR with high-resolution melting (HRM), with GAPDH as an endogenous control. Performance was evaluated following CLSI EP05-A3, EP07, EP12-A2, and EP37. Precision of quantification cycle (C_q) and melting peak temperature (T_m) was assessed by variance-component analysis within one site and across two sites and two operators. Because precision studies used bulk bisulfite-converted reference DNA, the reported coefficients of variation (CVs) primarily characterize the PCR-HRM detection module; end-to-end variability in clinical plasma specimens may be higher. Interference and cross-reactivity were evaluated in low-positive and contrived-negative samples against reproducibility-derived limits, with dose-response testing of reactive substances. Detection was 100% at both levels, and every run met control-validity criteria. C_q repeatability CVs ranged from 0.53% to 1.68%, and total reproducibility CVs ranged from 1.10% to 2.37%; all T_m CVs were ≤0.20%. All 18 interferents and 16 microbial cross-reactants evaluated in low-positive samples met the predefined acceptance criteria. In negative samples, hemoglobin, SARS-CoV-2, and measles virus produced concentration-dependent non-specific amplification, and dose-response testing established their respective no-effect concentrations. These findings characterize the analytical precision and multisite reproducibility of the PCR-HRM detection module and the analytical specificity of MLDx-s, complementing its previously reported clinical performance for plasma-based HCC detection.