Cobas e601 analyzer

All samples were collected before any treatment was initiated and within 2 days of hospitalization. The nipple was first cleansed with alcohol swabs to remove cellular debris. The nipple discharge and milk were expressed via manual compression of the breast. No complications occurred. Droplets of nipple discharge or milk were collected in an Eppendorf tube. The tube was then stored in a dedicated refrigerator at 4°C. The quantities of the collected nipple discharge varied from 20 μL to 200 μL. The samples were transported to the laboratory department within 8 hours of collection. Viscous samples were diluted up to 20-fold with normal saline prior to centrifugation and storage at 4°C. The concentrations of CEA, CA153 and CA125 in the nipple discharge and milk samples were quantitatively measured via an automated test system that utilized sandwich electro-chemiluminescence immunoassay (ECLIA) kits (Roche cobas e601 analyzer, Roche Diagnostics, Indianapolis, USA). All tumor marker assays were performed at Qilu Hospital of Shandong University according to manufacturer’s protocol. The laboratory personnel were blinded to the clinical information. Commercial reference control sera were used for quality control and calibration.

Serum CEA were measured by chemiluminescent enzyme immune assay with Roche Cobas e601 Analyzer (Roche AG), and the limit of normality were defined as 5 ng/ml according to the manufacturer's instruction.