The criterions for exclusion were: (1) severe dysfunction of heart, brain, lung, kidney and selleck products liver; (2) death attributable to causes other than CRC; (3) accompanying urological or genital tumour; (4) accompanying cancers other than CRC. Detection of the CEA in serum Five millilitres of venous blood was obtained from each patient 1 week before operation. CEA in serum (S-CEA) measurements were done by the department of clinical laboratory in our hospital using electrochemiluminescence immunoassay with Elecsys system 2010 (Roche Holding Ltd, Basel, Switzerland). The cut-off value of S-CEA recommended by the manufacturers for diagnosis was 5 ng/ml. We classified the 173 patients into high S-CEA group (> 5 ng/ml) and low S-CEA group (�� 5 ng/ml).

Immunohistochemistry The CEA in the tumour tissue which we named T-CEA was determined by the method of immunohistochemistry. In each patient, formalin-fixed and paraffin-embedded tumour blocks were cut into 5 ��m thick sections (average area 2.0 cm2), deparaffinized in xylene, and rehydrated. Antigen retrieval was performed in 0.1 m citric acid buffer (pH 6.0) in a 650 W microwave for 15 min. Endogenous peroxidase activity was blocked with 0.3% hydrogen peroxide in 100% methanol for 30 min. Immunohistochemistry was performed as Power Vision? two-step histostaining (Immuno Vision Technologies Co., Daly City, CA, USA). Primary CEA antibody (Zymed Laboratories Inc., South San Francisco, CA, USA) was diluted as 1:50 and sections were incubated overnight at 4��C.

After three washes in PBS (5 min), sections were incubated for 30 min in anti-mouse secondary antibody from a PicTure?-PV6000 Kit (Zymed Laboratories Inc.). Antibody binding was visualized using a 3,3-diaminobenzidine (DAB) kit (Vector Labs, Burlingame, CA, USA) according to the manufacturer��s instructions. Scoring For general negative controls, the primary antibodies Batimastat were replaced by phosphate buffer solution (PBS). All slides were scored by two independent and well-trained pathologists, without the knowledge of clinical and pathologic parameters or the patients�� outcomes. And in cases of scoring disagreement, a third independent assessment was performed. For all slides, at least 10 high power fields at 400�� magnification were chosen randomly and > 1000 carcinoma cells were counted for each section. Stained slides were examined to identify the cellular localization of CEA immunoreactivity for both intensity (?, +, ++, and +++) and proportion (0%, 1�C5%, 6�C25%, 26�C50%, 51�C75% and > 75%) of tumour cells stained. Integer values were assigned to the scores of intensity (0�C3) and proportion of tumour cells stained (0�C5).