812 Xibing G et al. color flow cytometry. (Figure 1) Flow cytometry was performed as follows: 10 μL PE- labeled HLA-peptide tetramer, anti-CD8-FITC, CD3-PC5, and 1 μL HBV core 18-27 antigenic peptide were loaded into test tubes; 100 μL heparin anti-coagulated blood was added and mixed well, and the tubes were incubated at room temperature for 20 min in the dark. After hemoly- sis and rinse steps, the samples were analyzed on a flow cytometer. A parallel control (without antibody to the antigenic peptide) was also run. Five thousand CD8+ cells were counted, gating on CD3+ lymphocytes, and CD8+ 8 2 D C t value P value t value P value Cellular Immunity after Chronic Hepatitis B and HLA-peptide tetramer double-positive cells were counted as HBV-specific CD8+ cells, expressed as the per- centage of total CD8+ cells. All reagents were purchased from Beckman Coulter. 3.7. T Cell Subgroup, Nonspecific CTLs, and NK Cells EDTA-K2 anticoagulated fresh blood (100 μl) was load - ed into test and control tubes, and 10 μL of monoclonal antibody (anti-CD3, anti-CD4, anti-CD8, anti-CD28, and anti-CD16 + 56; Beckman Coulter) and parallel nonspe- cific controls were added, respectively. The tubes were in- cubated at room temperature for 15 min in the dark and analyzed by flow cytometry after hemolysis. (Figure 2) 3.8. Statistical Analysis SPSS 12.0 was used for the statistical analysis. Chi-square test was applied for categorical data, and t-test was used for numerical data, which were expressed as mean ± standard deviation (SD). The differences were considered significant when P < 0.05. 4. Results 4.1. Deterioration of CHB into Chronic Severe Hepatitis B In 49 of the 255 CHB patients (19.22%), the disease progressed into early-stage chronic severe hepatitis B (Group A) an average of 10.06 ± 1.73 days after admission, with such symptoms as lassitude, decreased appetite, nausea, and vomiting; the mean TBIL was 338.51 ± 59.31 μmol/L, and prothrombin activity was 35.25 ± 2.91%. In the remaining 206 cases (80.78%), the disease did not prog- ress into severe hepatitis B (Group B). Table 2. Comparison of T Cell Subgroup, Non-Specific CTL a and NK a Cells between 255 CHB Patients and Normal Controls (mean ± SD) at Baseline CD4+ 33.51 ± .23 37.21 ± 6.09 6.552 CD8+ 27.58 ± 90 25.13 ± 4.78 5.131 < 0.01 CD4+/CD8+ 1.23 ± 0.11 1.54 ± 0.43 8.679 < 0.01 Non-Specific CTL 17.79 ± 1.80 15.83 ± 4.99 4.261 < 0.01 NK Cells 12.55 ± .87 15.04 ± 4.17 5.818 < 0.01 Figure 2. Flow Cytometry of Nonspecific CTL CHB, (n = 255) Healthy controls, (n = 30) t value P value < 0.01 < 0.01 a Abbreviation: CTL, cytotoxic T lymphocyte; NK, natural killer CD8 CTLC 65.78 ± 2.76 68.83 ± 5.51 4.991 Value, mean ± SD Healthy controls, mean ± SD (n = 30) Table 3. Comparison of Baseline T Cell Subgroup, Non-Specific CTL a and NK a Cells between Group A and Group B with Normal Controls (mean ± SD) CD3+ CD4+ CD8+ CD4+/CD8+ Non-Specific CTL NK Cells Value, mean ± SD 65.83 ± 3.07 34.23 ± 1.71 26.95 ± 1.51 1.28 ± 0.06 Healthy controls, mean ± SD (n = 30) 68.83 ± 5.51 37.21 ± 6.09 25.13 ± 4.78 1.54 ± 0.43 a Abbreviation: CTL, cytotoxic T lymphocyte; NK, natural killer Hepat Mon. 2011;11(10):810-815 65.01 ± 1.27 68.83 ± 5.51 4.315 < 0.01 4.255 < 0.01 Group A, (n = 49) 30.61 ± 1.67 30.09 ± 1.21 1.02 ± 0.05 37.21 ± 6.09 25.13 ± 4.78 1.54 ± 0.43 6.591 < 0.01 5.311 < 0.01 Group B, (n = 206) 6.351 < 0.01 3.997 < 0.01 7.662 < 0.01 7.367 < 0.01 20.24 ± 1.57 15.83 ± 4.99 5.207 < 0.01 17.18 ± 1.28 15.83 ± 4.99 2.989 < 0.01 10.33 ± 1.93 15.04 ± 4.17 6.816 < 0.01 13.08 ± 1.41 15.04 ± 4.17 5.078 < 0.01