1 day before and 2 days after cryosurgery with or without DC-CIK treatment，approximately 17-mL ethylene diamine tetraacetic acid(EDTA)-blood was drawn by vein puncture from patients with pancreatic cancer and healthy volunteers. The blood of the healthy volunteers will be used to evaluate the sensitivity and specificity and as negative control cells. To avoid contamination with skin cells, 2 mL blood will be discarded before the study samples will be taken.Briefly, the mononucleate cells will be separated from the blood over Ficoll- Paque for 20 min with 1800g at 4℃. The interface cells will be removed and washed, and the red blood cells(RBCs) will be removed using a lysis buffer followed by a repeated wash. The mononuclear cells will be counted and aliquot for RT-PCR and multiparameter flow cytometry on the basis of at least 2-3×106 cells for each methodology. The cell pellet will be resuspended in phosphate-buffered saline for multiparameter flow cytometry and in Trizol reagent for RT-PCR. Aim : Identification of CTCs may lead to better diagnosis and prognosis and could help to choose an adequate therapy.
