| ObjectiveTo analyze T lymphocyte subsets among healthy and HIV-antibody positive people in Shandong Province, establish the normal reference range of T lymphocyte subsets of healthy individuals and compare the difference of T lymphocyte subsets between HIV positive patients and healthy controls.Methods360 healthy blood donors and 1271 patients with HIV-antibody positive during 2004-2009, with basic information, infection follow-up and laboratory data, were involved in the study.5ml fresh EDTAK3 treated venous whole blood was collected from each individual with strict asepsis and biosafety. T lymphocyte subsets (CD3+T, CD4+T and CD8+T) were analyzed with CD4 FITC/CD8 PE/CD3 PerCP, TruCount absolute count tubes and FACS hemolysin by FACS Calibur flow cytometry. CD4+/CD8+ ratio was calculated. To ensure the accuracy of the experiment and the credibility of the results, standard quality control was used for each experiment. T lymphocyte subsets were compared between health and/or HIV patients with different ages, gender and transmission, all data was analyzed with SPSS 13.0.Results1. Normal reference range of T lymphocyte subsets in Shandong province1.1 T lymphocyte subsets with different age in Shandong healthy individuals CD3+ cells and CD3+CD4+ cell counts were significant varried in individuals with different ages (F=3.246,2.763, P<0.05), while percentages of CD3+, CD3+CD4+ and CD3+CD8+ cells, CD3+CD8+ cell number and CD4+/CD8+ are not statistically significant in healthy individuals with different ages(F=0.453,1.892,0.385,2.197, 1.892, P>0.05).1.2 T lymphocyte subsets with different gender in Shandong healthy individualsCD3+ and CD3+CD4+ cell numbers are higher in female than male and the difference are statistically significant (t=2.592,2.767, P<0.01); while percentages of CD3+, CD3+CD4+, CD3+CD8+ and cell number of CD3+CD8+and value of CD4+/CD8+ showed no significant difference between female and male (t=1.823, 1.682,0.676,1.380,0.107,P>0.05).1.3 T lymphocyte subsets with different agender in different ageIn individuals with 20 to 29 age, CD3+ percentage, CD3+ cells, CD3+CD4+ percentage, CD3+CD4+ cells, CD3+CD8+ cells are significantly higher female than that of male, while similar results were got with CD3+CD8+ percentage in individuals with 30 to 39 age (t=2.107,8.799,2.197,7.839,4.431,2.400, P<0.05).1.495% reference range of T lymphocyte subsets in healthy adults in Shandong ProvinceDespite the subtle difference between gender, we can combine gender and age, use percentile method to establish 95% reference range of T lymphocyte subsets of Shandong healthy adults:CD3+percentage content (%):47.05～82.00; CD3+ cells (cells/μl):800.08～2189.73; CD3+CD4+ percentage (%):24.03～51.98; CD3+CD4+ cells (cells/μl):433～1179.80; CD3+CD8+ percentage (%):14.00～44.00; CD3+CD8+ cells cells/μl):246.10～1098.98; CD3+CD4+/CD3+CD8+:0.72～3.06.1.5 Comparison 95% reference range of T lymphocyte subsets in Shandong with that of different regions, ethnic groups, and other national healthy populationThe more number of cases involved, the index reference values are closer, when same instrument, staining and gating were used. T lymphocyte subsets in North and South Han population are similar, which is close to the reported range of Singapore. The CD3+,CD3+CD4+,CD3+CD8+ cell numbers of Han people are slightly higher than that of Yi, Dai, Zhuang people in the south of China, while the percentage of T lymphocyte subsets is slightly lower than the Xinjiang Uighur people. The reference value given by BD Company is closer to that made from Europe and the United States. CD3+ and CD3+CD4+ percentages and cell numbers given by BD are higher than thar from other 14 regions although the difference of percentage and cells of CD3+CD8+ are not significant.2. T lymphocyte subsets in people with HIV-antibody positive infections in Shandong Province2.1 T lymphocyte subsets analsysis of HIV antibody-positive infections in Shandong ProvinceTest results showed that mean numbers of 1271 HIV-antibody positive cases are: CD3+ cells:1592.856 cells/μL, CD3+CD4+ percentage:17.545%, CD3+CD4+ cells: 389.107 cells/μL, CD3+CD8+ percentage:55.094%, CD3+CD8+ cells:1102.533 cells/μL, CD3+CD4+/CD3+CD8+:0.382. The CD3+ cell number, CD8+cell number and percentage of HIV-antibody positive people are higher than control, while CD4+ cell number/percentage and CD3+CD4+/CD3+CD8+ ratio were lower than healthy individuals.2.2 Analysis of transmisson routes of patients with HIV in Shan Dong ProvinceIn the current study, patients with 0 to 8 years of age were infected mianly from their mother. More patients with homosexual transmission in group with age of 20 to 29 years were found in other age groups, although heterosexual transmission is the main source of patients in group of 20-29. In Shandong, intravenous drug users are the majority of HIV patients with ethnic minorities who were infected from other provinces and moved into our province. Transmissions of infection among spouses of patients, their spouses are mostly long-distant-marriage women or long-distant-marriage women's ex-husband was HIV-antibody positive infections.Patients with age of 30 to 39 were mainly heterosexual transmitted, transmission between spouses was significantly higher in this group than that of other age groups. Patients with age of 40 to 49 were mainly heterosexual transmitted, especially by blood (plasma) in the early 90's way through the Central Plains infected paid blood donors. Blood (plasma) is the main transmission route of 50～59 age group. All patients age elder than 60 are Han people.2.3 Comparison T lymphocyte subsets in HIV positive patients with differne infection routes in Shandong ProvinceThe CD3+,CD3+CD4+,CD3+CD8+ cells numbers of blood-borne transmission, sexual transmission and mother-to-child transmission among HIV-antibody positive people are significantly different (all P<0.01), while similar results were got with sexual transmission and mother-to-child transmission, mother-to-child transmission and unknown transmission (all P<0.01). The mean number of mother-to-child transmission of CD3+,CD3+CD4+,CD3+CD8+ cells are significantly higher than that of other routes of transmission. There are no significant difference in CD3+CD4+, CD3+CD8+percentage and CD4+/CD8+ ratio in other transmission groups.Blood transmission group were further subdivided into blood (plasma), intravenous drug use and blood transfusion/blood products transmission; sexual transmission was subdivided into infection between spouses, homosexual transmission, and heterosexual transmission. Analysis revealed that CD3+T, CD4+T, CD8+T cells are significantly different (all P<0.01) in blood transmission by intravenous drug use and blood transfusion/blood products transmission; CD3+T, CD8+T cells are significantly different (all P<0.01) in sexual transmission by communication between spouses and homosexual transmission.2.4 Comparison of T lymphocyte subsets in HIV positive patients in different ethnic groups in Shandong ProvinceT lymphocyte subsets between Han and minority HIV-antibody positive people are no significant difference.CD3+T cells, CD4+T cells and percentage, CD8+T cells in Han HIV-antibody positive patients with blood transmission are significantly different with that of minority (all P<0.05), and the mean numbers of the minority are higher than the Han.CD3+T, CD4+T, CD8+T cells of Han HIV patients with unknown transmission are significantly different with that of the minority patients (all P<0.05), and the mean numbers of the minority are higher than the Han.CD8+T cells in HIV patients with sexual transmission of Han and the minority are significantly different (P=0.012).2.5 Comparison of T lymphocyte subsets in HIV-antibody positive people with different ages in Shandong ProvinceCD3+T, CD4+T, CD8+T cells of patients with age younger than 19 years are significantly different from other age groups (all P<0.01), the mean numbers of patients in this age group are higher than other age groups.The number of CD3+T cells in groups of 20 to 29 year,30 to 39 year,40 to 49 year and 50 to 59 years p are significantly different (all P<0.01). And number of CD3+ T lymphocytes in patients with 30 to 39 years and 40 to 49 years were significantly different (P=0.001).Percentage of CD4+T lymphocytes in patients with 20 to 29 years and patients with 50 to 59 years were significantly different (P=0.041), the mean number of patients with 50～59 years were lower than those in other age groups.Number of CD4+T lymphocytes in patients with 20 to 29 years,40 to 49 years and 50 to 59 years are significantly different (all P<0.01); there are significant difference in CD4+T lymphocytes between patients with 30～39 years and 40～49 years (P=0.004).Percentage of CD8+T lymphocytes in patients younger than 19 and patients with 20 to 29 years,30 to 39 years,40 to 49 years old and 50 to 59 years were significantly different (all P<0.05). The mean percentage of CD8+T lymphocytes in patients younger than 19 is lower than other age groups.Number of CD8+T lymphocytes in patients with 20 to 29 years,30 to 39 years and 40 to 49 years are significantly different (all P<0.05).CD4+/CD8+ratio were not significantly different in all different groups (all P> 0.05). The mean ratio of CD4+/CD8+in patients younger than 19 is higher than other age groups.2.6 Comparison of T lymphocyte subsets of HIV-antibody positive people with different gender in Shandong Province CD3+T, CD4+T, CD8+T lymphocytes and CD8+percentage in male HIV-antibody positive patients were higher than those of females; CD4+percentage and CD4+/CD8+ ratio difference was not statistically significant.3. Comparation of T lymphocyte subsets of HIV-antibody positive and normal healthy peopleCD3+T, CD8+T cells and the percentage of CD8+T lymphocytes in HIV-antibody positive patients were higher than health; while CD4+T lymphocytes and CD4+/CD8+ratio were lower than control.CD3+T lymphocytes, CD8+T lymphocytes number/percentages of HIV-antibody positive patients with age of≤19,20 to 29,30 to 39 and 50 to 59 were higher than those of healthy adults. The mean numbers of CD4+T cells and percentage, CD4+/CD8+ ratio are lower than control. The mean numbers/percentage of CD8+T cells in HIV patients with age of 40～49 are higher than healthy adults, while CD3+T cells, CD4+T cells/percentage and CD4+/CD8+ ratio are lower than control.Cell number and percentage of CD8+T cells in HIV positive patients are higher than healthy adults with same gender, while CD4+T cells/percentage and CD4+/CD8+ ratio are lower than control with same gender.Conclusion1. By flow cytometry analysis of T lymphocytes subsets, we establish the 95% reference range of T lymphocytes subsets of healthy adults in Shandong by using percentile statistis; our results confirmed the differences of T lymphocyte subsets in healthy population in different regions and nationalities. Our work here provided basic data on the study of immune parameters of normal healthy population and in-depth analysis of HIV antibody-positive infections in Shandong province.2. We successfully analyzed T lymphocyte subsets of HIV-antibody positive people in Shandong province of recent years. Results show that T lymphocyte subsets of HIV-positive patients varied with different infection routes, ethnics and genders.3. By comparison of T lymphocyte subsets in HIV antibody-positive and healthy individuals in Shandong province, we proved that with the progression of the disease, T lymphocyte subsets were affected by HIV persistently, which might suggest that regular analysis of T lymphocyte subsets after HIV infection is the best way to judge disease progression.
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