Turkish Journal of Hematology Volume: 33 - Issue: 3

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Işıksaçan N, et al: Cytokines’ Association with ZAP70 in Chronic Lymphocytic Leukemia Turk J Hematol 2016;33:202-208 CD19+ cells were analyzed for the expression of CD5, CD38, and ZAP70 (Figure 1A). Most of the CD19+ B cells expressed CD5; however, there was only one patient expressing both CD5 and CD38 (Figures 1B and 1C, respectively). Expression of CD5 showed heterogeneity among the patients. Intracytoplasmic ZAP70 together with CD5 was expressed in different levels in CD19+ B cells (Figures 1D, 1E, and 1F). Intracytoplasmic Cytokine Staining Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-Hypaque (Sigma Chem. Co., USA) density gradient centrifugation, and cells were washed twice in Hank’s balanced salt solution. The cells were finally adjusted to a final concentration of 1x10 6 cells/mL in complete RPMI-1640 medium (Sigma Chem. Co.) supplemented with 10% heat-inactivated fetal calf serum, penicillin (100 U/mL), streptomycin (100 mg/ mL), gentamicin (50 mg/mL), and 50 µM 2-mercaptoethanol. Freshly purified PBMCs were washed and 1x106 cells/mL were stimulated for 18 h by a combination of phorbol ester, phorbol- 12-myristate-13-acetate (50 ng/mL), and Ca2+ ionophore (ionomycin, 250 ng/mL) in a 24-well round-bottom plate (all from Sigma Chem. Co.). The combination of these two stimuli was used to achieve the strongest stimulus for intracytoplasmic cytokine secretion. Monensin (Sigma Chem. Co.) was added at a final concentration of 1 µM to the cultures in the final 4 h. After incubation, PBMCs were washed with PBS solution and stained with anti-CD19-PE and anti-CD3-APC mAbs for 30 min (Caltag Laboratories) for determining the cytokine secretion of T and B cells. Cells were washed with PBS and then fixed and permeabilized with paraformaldehyde/saponin solution (Cytofix&Cytoperm Kit, BD Biosciences). After washing, the cells were stained with FITC conjugated IC (IgG1), anti-IL-4- FITC, and anti-IFN-γ-FITC (Caltag Laboratories) mAbs for 30 min at room temperature. After washing, cells were resuspended in 1% paraformaldehyde (Sigma Chem. Co.) and analyzed by flow cytometry. Each sample was acquired with a BD FACSCalibur (BD Biosciences) and analyzed with the instrument’s operating software, CellQuest (BD Biosciences). Statistical Analysis Statistical analysis was performed using a standard nonparametric Mann-Whitney U test using SPSS 17.0 for Windows. The results are presented as median values and p0.05). Seventeen patients were SSC Figure 1. Gating strategy of chronic lymphocytic leukemia. Peripheral blood mononuclear cells from chronic lymphocytic leukemia patients (n=28) were stained for CD5, CD19, CD38, and ZAP70 mAbs and analyzed by flow cytometry. CD19 + cells were gated versus SSC (A). Representative dot-plot analyses for the expression of a patient negative (B) and positive (C) for CD38 with CD5 + in CD19 + cells are shown. In the CD19 + B cell population, CD5 + ZAP70 + (D, E) and CD5 + ZAP70- (F) plots from three different patients with chronic lymphocytic leukemia are also indicated. The numbers indicate the proportion of cells positive for indicated markers. 204
Turk J Hematol 2016;33:202-208 Işıksaçan N, et al: Cytokines’ Association with ZAP70 in Chronic Lymphocytic Leukemia positive for ZAP70; 8 of them were Rai 0-1 and 9 of them were Rai 2-4. However, 7 patients negative for ZAP70 were Rai 0-1 and 4 patients for Rai 2-4. When patients are divided into two groups according to Rai staging system, the first group includes 15 patients in stages 0 and 1, and the second group includes 13 patients in stages 2 and 4. The difference between ZAP70 expression in the first and second Rai groups was statistically significant (p
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Turkish Journal of Hematology Volume: 33 - Issue: 3

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