Blood sampling and serum marker levels

Blood samples were obtained by venous puncture, centrifuged, and stored at −50°C until the analyses were performed. An ELISA kit (Cusabio Biotech Co., Ltd., Wuhan, China) was used for HE4 evaluation. All measurements were performed according to the manufacturers' instructions. The plates were read at the wavelength of 450 nm 10 min after the administration of the stop solution. The minimum detectable dose of HE4 was <0.30 pmol/ml according to this measurement. The HE4 cut-off level of ≤0.30 pmol/ml was considered to be HE4-negative and all cases with HE4 levels >0.30 pmol/ml were considered as HE4-positive.

The serum CEA and CA19-9 levels were measured using the enzyme immunoassay method, using a HITACHI automatic analyzer (Hitachi Corporation, Tokyo, Japan). According to the manufacturer's instructions, the normal range for serum CEA was 0–3.4 ng/ml and for CA19-9 0–37 U/ml.