Serum Outperforms Plasma for Glypican-3 Quantification in Hepatocellular Carcinoma—A Prospective Comparative Study

<b>Background:</b> Glypican-3 (GPC3) is frequently overexpressed in hepatocellular carcinoma (HCC) and serves as a circulating biomarker. Limited evidence exists regarding whether plasma or serum constitutes the optimal matrix for GPC3 measurement. This study aimed to investigate this gap. <b>Methods:</b> Between December 2024 and September 2025, 100 participants were prospectively enrolled, including 33 healthy controls, 29 individuals with chronic liver disease, and 38 patients with HCC. Paired serum and plasma samples were analyzed under fresh conditions and after storage for seven days at 4 °C and -70 °C. GPC3 concentrations were compared across groups. Subsequently, correlation and area under the receiver operating characteristic curve (AUROC) analyses were conducted. <b>Results:</b> In fresh samples of the controls, median plasma GPC3 levels were significantly higher than those in serum (82.36 pg/mL, IQR: 67.56-92.42 vs. 30.89 pg/mL, IQR: 20.36-41.12; <i>p</i> < 0.001). After seven days of storage, plasma GPC3 concentrations declined markedly at both 4 °C (41.73 pg/mL, IQR: 32.49-55.37; <i>p</i> < 0.001) and -70 °C (45.53 pg/mL, IQR: 25.30-55.65; <i>p</i> < 0.001), with no significant difference between the two storage conditions (<i>p</i> = 0.610). In contrast, serum GPC3 levels remained relatively stable across fresh, 4 °C (31.10 pg/mL, IQR: 16.84-38.60), and -70 °C (25.31 pg/mL, IQR: 14.36-40.74) conditions (<i>p</i> = 0.645). Both matrices under -70 °C storage effectively discriminated HCC from non-HCC cases, although serum demonstrated a significantly better diagnostic performance (AUROC: 0.836, 95% CI: 0.749-0.902 vs. 0.772, 95% CI: 0.677-0.850; <i>p</i> = 0.013). <b>Conclusions:</b> Although plasma offers operational convenience and higher baseline GPC3 levels, serum provides both greater stability and superior diagnostic accuracy under frozen conditions, thus supporting its use as the preferred specimen matrix in clinical and research applications.