Establishment Of Three-Color Method For Detecting Th-Subsets In Peripheral Blood By Flow Cytometry And The Effect Of Perioperative Allogenic Blood Transfusion On Th-subsets In Peripheral Blood Of Patients With Esophageal Squamous Cell Carcinoma

Objective To investigate the effect of Phorbol 12-myristate 13-Acetate (PMA) on surface CD4 expression of T-helper and establish a three-color method for detecting Th subsets in peripheral blood (PB) by flow cytometry and suitable quality control project. To investigate the variation of Th subsets in PB of patients with esophageal squamous cell carcinoma (PEC)and the effect of perioperative allogenic blood transfusion on Th-subsets in peripheral blood of PEC after operation.Methods PB of healthy donors were co-cultured with defferent concentrations of PMA and Ionomycin (1 μg/mL) for 2, 3 and 4 hours respectively, then fluorescein conjugated antibodies of CD3/4/8 was added to conjugate the correspondent antigen on T-cells surface. The mean fluorescent intensity (MFI) of CD4 channel (Th cells) was measured by flow-cytometry before hemolysis. Peripheral whole blood were co-cultured with RPMI1640, 25 ng/mL of PMA, 1 μg/mL of Ionomycin and 20ng/mlof Monensin for 2 hours,fixed and permeabilized, then fluorescein-labeled antibody of CD4 IFN- γ and IL-4 were added .The cells of Th0, Th1 and Th2 were detected by flow cytometry. By using CD69 as the marker of lymphocyte activated and MPO as the marker of evaluating the effect of permeabilization, set up the quality control of three-color method for detecting Th subsets .The ThO, Thl and Th2 in peripheral bloodof healthy control(HC) group and PEC group were detected by the method. The Th0, Thl and Th2 in PB of PEC with allogeneic blood transfusion (TPEC) and without allogeneic blood transfusion(non-TPEC) in perioperative were detected before operation and 5th day after operation by the method.Results The mean fluorescent intensity (MFI) of CD4 channel was decreased while the concentration of PMA was increased and the co-cultured hours was prolonged. When PB of healthy donors were co-cultured with 25 ng/mL of PMA and Ionomycin (1 Hg/mL) for 2 hours, CD4+ cells and CD4- cells can be discriminated by CD4 molecular of cell surface, and more than 90 percent CD3+ lymphocytes express CD69. More than 90 percent leukocytes were MPO+ before adding permeabilization medium.The culture project, that the cells were co-cultured with 25ng/ml of PMA, 1 μg/mL of Ionomycin and 20ng/ml of MN for 2 hours, was optimized and three-color method of CD4, IFN- γ and IL-4 for detecting of Th-subsets by flow cytometry was established. Compared with the control group, all of the Th0, Thl and Th2 of the PEC group were significantly decreased in PB (P<0.01, P<0.01, P<0.05) . Compared with before operation, the percentage of Thl cells of TPEC group on 5th day after operation was significantly decreased ( 7.80 ±2.99 vs 9.11+2.74, t= 3.922, P= 0.001), the percentages of Th0 and Th2 cells of TPEC group on 5th day after operation were not different. The percentages of Th0, Thl and Th2 cells of non-TPEC group were not different before and after operation.Conclusion By optimizing culture project, the cells of Th0, Th1 and Th2 can be detected by three-color method simultaneously, quickly and exactly. The functions both cell immunomodulation and humoral immunomodulation of the patients with esophageal squamous cell carcinoma may be depressed. That the Th0 cells of the patients who accepted allogeneic blood transfusion at perioperative polarized to Th1...