Estimating Normal Values of Rare T-Lymphocyte Populations in Peripheral Blood of Healthy Cuban Adults.

INTRODUCTION Flow cytometry allows immunophenotypic characterization of important lymphocyte subpopulations for diagnosis of diseases such as cancer, autoimmune diseases, immunodeficiencies and some infections. Normal values of rare lymphoid cells in blood, quantified by cytometry, vary among different populations; so it is indispensable to obtain normal national values that can be used in clinical practice. OBJECTIVE Characterize distribution of rare T-lymphocyte populations in peripheral blood, specifically double-positive T, natural killer T and activated T lymphocytes, as well as their relationship to sex and age. METHODS A cross-sectional study was carried out in 129 adults (68 women, 61 men) aged >18 years, without chronic diseases or unhealthy habits, who signed informed consent. Peripheral blood was collected for immunophenotyping of lymphocyte subpopulations with monoclonal antibodies specific for CD4+CD8+ double-positive T cells, CD3+CD56+ natural killer T cells, and CD3+CD25+HLA-DR+ activated T cells. An eight-color flow cytometer (Beckman Coulter Gallios) was used. The analytic strategy was modified, associating variables of interest in a single graphic, using conventional monoclonal labeling antibodies. Medians and minimum and maximum percentiles (2.5 and 97.5, respectively) were used as descriptive statistics, stratified by sex, for cell counts and percentages. A linear regression model was applied to assess age effects and a two-tailed Mann-Whitney U test for independent samples was used to assess sex differences. The significance threshold was set as p ≤0.05. RESULTS Median percentages of total lymphocytes: natural killer T cells 6.3% (1.4%-23%) in men and 4.7% (0.8%-11.3%) in women (p = 0.003); activated T cells 1.0% (0.2%-2.2%) in men and 1.2% (0.4%-3.1%) in women, without statistical significance; and double positives 0.8% (0.1%-4.2%) in men and 0.9% (0.3-5.1) in women, also without statistical significance. Median cell counts (cells/mL) were: natural killer T cells, 126 (27-580) in men and 105 (20-279) in women (p = 0.023); activated T cells: 20 (4-46) in men and 25 (7-75) in women, (p = 0.013) and double-positive T cells: 17 (2-85) in men and 21 (7-154) in women, without statistical significance. Sex influenced natural killer T cells, but age did not. CONCLUSIONS Age does not affect counts and percentages of rare T lymphocyte subpopulations in the blood of healthy Cuban adults. Sex differences found for some phenotypes suggest the need for different reference values for women and men.

[The indicators of immune status of peripheral blood of donors].

The expanded analysis of 57 samples of peripheral blood from conditionally healthy patients was implemented concerning phenotype of main populations of lymphocytes, activated pools of cells and level of cytokines. The samples were received in the department of storage of blood and its components of the research institute of blood transfusion of the hematology research center. It is demonstrated that number of T-lymphocytes, T-helpers and activated TY-cells with phenotype CD3+HLA-R+ and level of detected cytokines by standard indicators had no difference with publications data. In particular cases an increase of number of cytolytic T-lymphocytes, B-lymphocytes and natural killers and decrease or increase of CD4/CD8 index relative to standard were detected. The decrease of number of natural killers was the most frequent aberration. The study demonstrates that among conditionally healthy patients giving blood as donors persons with disorders of immune system were presented.

Value of HIV patients with regular follow-up as in-house internal controls of flow cytometry measurement of lymphocyte subsets.

BACKGROUND: Quality assessment in flow cytometry cannot obey the same rules as those applicable to the measurement of chemical analytes. However, regular follow-up of known patients may provide a robust in-house control of cell subsets evaluation. METHODS: Sequential blood samples assessed for 32 HIV patients over several years and showing good stability were retrospectively assessed to establish coefficient of variations of the percentages of CD3+, CD4+, CD8+ cells, and CD4+ absolute counts (ACs). RESULTS: Mean relative standard variations for the whole cohort were of 0.04, 0.14, 0.08, and 0.18 for CD3%, CD4%, CD8%, and CD4 ACs, respectively. DISCUSSION: In-house follow-up of regularly checked compliant patients is a good alternative to traditional and costly repeatability and reproducibility studies for the validation of routine flow cytometry. © 2013 International Clinical Cytometry Society.

Normal values of CD4 and CD8 lymphocyte subsets in healthy indian adults and the effects of sex, age, ethnicity, and smoking.

BACKGROUND: Information on lymphocyte populations (T, B, and natural killer cells) and subpopulations (CD4 and CD8) in India is generally lacking. Measurement of T-cell subsets is important in India for evaluating disease stage and progression in individuals with the human immunodeficiency virus (HIV). Hence, this study was conducted to provide normal ranges of absolute and percentage values of CD4 and CD8 T-lymphocyte subsets and the ratio of CD4 to CD8 in normal Indian adults. METHODS: Flow cytometric analysis (EPICS-XL) was used to determine the range of T-lymphocyte subpopulations in normal Indian blood donors at Command Hospital and the Armed Forces Medical College, Pune, India. The reference population consisted of 94 healthy HIV-seronegative blood donors. T-lymphocyte subsets were analyzed with two-color immunophenotyping of peripheral blood lymphocytes with the use of a lysed whole-blood technique and enumerated. RESULTS: For normal values of various blood components, we found mean values of 2114 cells/microl for total lymphocytes, 865 cells/microl (40.2%) for CD4(+) lymphocytes, 552 cells/microl (31.3%) for CD8(+) lymphocytes, and 1.7 for the CD4:CD8 ratio. The 95% confidence intervals for the same parameters were 1115-4009 cells/microl, 430-1740 cells/microl (30.75-49.60%), 218-1396 cells/microl (20.06-42.52%), and 0.39-3.02 respectively. Females had significantly higher CD4 counts (P < 0.05), percentage of CD4 lymphocytes (P < 0.01), and CD4:CD8 ratio (P < 0.01). Males had a significantly higher percentage of CD8 lymphocytes (P < 0.01). They also had higher CD8 counts that did not reach significance. Age, ethnicity (Dravidian versus Aryan), smoking, alcohol consumption, and the interval between drawing the blood sample and its analysis were factors that did not produce statistically significant differences in the T-cell subsets studied. CONCLUSIONS: When compared with other published series, the CD4 and CD8 values in healthy Indians were no different from those reported in the West. These observations have important clinical implications for the use of T-lymphocyte subset measurements in India, especially in the management of HIV infection. The normal ranges established by this study can be used as a reference for decisions made in clinical practice.