32,33 This HCC cohort included 22 cases of small-sized (≤ 5 cm) HCC, 34 cases of medium-sized (5–10 cm) HCC, and 20 cases of large-sized (≥ 10 cm) HCC. According to the serum AFP level, the HCC cases could also fall into two groups: AFP ≤ 25 ng/mL (n = 31), and AFP > 25 ng/mL (n = 45). Venous blood, obtained after overnight fasting, was collected in the absence of anticoagulant into red-topped tubes, and allowed to clot for 30 min at 4°C. Serum was isolated from blood by centrifugation at 2500 g for 15 min at 4°C. Then the serum was aliquoted

and stored at −80°C until analysis. Blood samples from HCC patients were all drawn prior progestogen antagonist to initiation of HCC treatment. Serum clusterin concentrations were

measured using a commercially available sandwich ELISA according to the manufacturer’s instructions (Human Clusterin ELISA, Biovendor Laboratories Ltd, Modrice, Czech Republic). All assays were performed independently by the laboratory personnel who did not have clinical information, and each sample was assayed in duplicate. In the Biovendor Human Clusterin ELISA, standards, quality controls and diluted samples were incubated in microtitration wells pre-coated with monoclonal anti-human clusterin antibody. After 60 min and washing, biotin-labeled second monoclonal anti-human clusterin antibody was added and incubated with the captured check details clusterin for 60 min. After another washing, streptavidin-horse radish peroxidase

(HRP) conjugate was added. After 30 min incubation and the last washing step, the remaining conjugate was allowed to react with the substrate solution (TMB). The reaction was stopped by the addition of acidic solution (0.2 M H2SO4) and absorbance of the resulting yellow product was measured spectrophotometrically at 450 nm. The absorbance was proportional to the concentration of clusterin. A standard curve was constructed by plotting absorbance values versus clusterin concentrations of standards, and concentrations of unknown samples are determined using this standard curve. The serum clusterin and AFP values were reported as median (25–75th percentile). The descriptive statistics 上海皓元 were compared by box plots and then by anova. Differences between groups were evaluated by the Kruskal–Wallis test followed by Tamhane’s test. To determine the optimal cutoff value for clusterin and AFP in the diagnosis of HCC, receiver operating characteristic (ROC) curves were constructed using all possible cutoffs for each assay. The area under the curve (AUC) was calculated and compared. For sensitivity and specificity, 95% confidence intervals (CI) were also determined. A P-value of < 0.05 was considered statistically significant. All analyses were performed with SPSS 13.0 for windows (SPSS, Inc., Chicago, IL, USA). In this study, a total of 184 subjects were enrolled.