The application of flow cytometry to studies of peripheral blood have led to the development of reference ranges for markers of peripheral blood - lymphocytes, monocytes, natural killer cells and their subsets - for HIV-infected and healthy adults and children. In contrast, there has been only one cross-sectional study of some cellular populations in healthy adolescents  and HIV-infected adolescents have not been investigated to date. The development of the immune system through normal adolescence and the influence of external stressors, hormones and pubertal development may affect these peripheral blood markers and distinguish this group of subjects.
Flow cytometric studies of the immune system, in particular peripheral blood, have not included significant numbers of subjects in the adolescent age group. Age-related differences in lymphocyte populations exist between infants, children and adults[2,3]. Studies of HIV-positive infants and young children and HIV- negative aged matched controls [4,5] have demonstrated a significant reduction in naive CD4 cells greater than that observed in HIV-infected adults. Limited studies in healthy adolescents aged 15-19 years [1,6,7] have reported measurements for selected lymphocyte subsets and minor differences have been observed related to race and smoking habit.
The capacity of the immune system of adolescents to generate and repopulate cell populations in normal homeostasis and HIV infection is largely unknown. Studies of patients following total body irradiation and bone marrow transplantation indicate important age-related differences in regeneration of T cells by both thymic-dependent and thymic-independent mechanisms[8-13].
In this report, flow cytometry data is presented on T- and B-lymphocyte and monocyte markers collected at the time of the initial flow cytometry panel for anti-retroviral therapy (ART)-naive HIV-positive and HIV-negative adolescents aged 13-18 years in the Reaching for Excellence in Adolescent Care and Health (REACH) cohort. This cohort consists of teenagers who are HIV-positive or are at risk of HIV infection based on sexual and drug taking behavior. The demographic and baseline biomedical characteristics of this group have been reported.
These hematologic and immunologic flow-cytometry data are for a subset of 94 (64 female and 30 male) HIV-positive adolescents who had never received ART (ART-naive) and 149 (113 female and 36 male) HIV-negative high risk control adolescents. The basis for selection of this subset of the REACH Cohort was that these subjects had not received ART. The group represented subjects from all 16 clinical REACH sites. The greater numbers of females in comparison to males may reflect both transmission differences and linkages to health care systems. The unique features seen in this adolescent age group are highlighted in this report.
The REACH Project of the Adolescent Medicine HIV/AIDS Research Network recruits HIV-infected and high-risk HIV-uninfected adolescents aged at least 13 but less than 19 years. The study evaluates biomedical and behavioral features of HIV infection as observed while under medical care for HIV infection and adolescent health. Characteristics of the cohort, recruitment and eligibility criteria, and study design are reported elsewhere.
All HIV-positive subjects had positive HIV enzyme linked immunosorbent assay (ELISA) test results; a confirmatory Western blot was performed before enrollment into the REACH study. The HIV-negative subjects had negative HIV-ELISA performed within 30 days of enrollment into the REACH study. HIV-positive subjects included only those infected through either sexual contact or from needle sharing; those HIV-infected youths infected through perinatal exposure, early childhood sexual abuse, or through blood product exposure were excluded from the study. To qualify for enrollment into the study, HIV-negative youths had a history of either sexual intercourse or injection drug use. Demographic profiles were similar for the two groups.
Blood samples for both HIV-infected and HIV- uninfected subjects were collected at 16 clinical sites (see Appendix). For the basic immunology flow panel, 2 ml of venous blood were drawn into an EDTA tube and transported immediately at room temperature to the local AIDS Clinical Trials Group certified laboratory. A complete blood count with an automated differential count was performed simultaneously at the local site to calculate the absolute lymphocyte count and subsequently absolute counts for all of the markers in the basic and expanded flow panels.
Complete blood count and the basic flow cytometry panel performed at each of the clinical sites (Table 1) were carried out simultaneously. For the expanded flow cytometry panel (Table 1) specimens were kept at room temperature and sent by priority overnight shipping to the Core Immunology Laboratory at The Children‚s Hospital of Philadelphia. All specimens were processed within 30 h of the time of collection. For those samples drawn at the clinical site at The Children‚s Hospital of Philadelphia, specimens were held overnight at room temperature by the laboratory and processed within the same time period.
Cell phenotypes were determined using standard single-, dual-, or three-color flow cytometry using whole blood on an Epics Elite Flow Cytometer equipped with two coherent Innova 300 series, 5 W UV-enhanced argon lasers operating at 300 mW and a helium neon laser. Briefly, 0.02 ml of the appropriate combination of fluorochrome-labeled isotype control or marker specific monoclonal antibody was added to 0.1 ml of whole blood. Monoclonal antibodies were from Becton Dickinson Immunocytometry System (San Jose, California, USA). After incubation for 30 min. at 4°C, 2 ml of ammonium chloride lysing-buffer was added to each sample to lyse erythrocytes, followed by a final wash with phosphate buffered saline and fixation in 1% paraformaldehyde. Alternative batch preparation was done using Quick Prep lysing buffer and the Quick Prep instrument available in the laboratory (Coulter). The data are expressed as the percentage of mononuclear cells bearing the specific marker, and the absolute number of cells per unit volume bearing the marker. These denominator data were derived from the complete blood count and leukocyte differential count obtained for each sample at the time of venipuncture. The data were accumulated and stored as two parameter histogram files.
For three-color immunofluorescence analyses of subpopulations of CD4 or CD8 lymphocytes, side scatter and fluorescence signals were integrated from PE-CY5-labeled cells (CD8, CD4) as qualifiers for the evaluations of CD38 and DR expression on CD8 cells, as well as CD45RA and CD45RO expression on CD4 or CD8 cells. Expression of the second and third marker was determined by integration of fluorescence signals from phycoerythrin- and fluorescein-labeled standard bright fluorescence microspheres (Coulter Immunology, Hialeah, Florida, USA). All photomultiplier tube voltages were adjusted to achieve the same mean channel fluorescence values as determined from previous calibrations. For each sample, 10 000 events were accumulated.
Cell samples were analyzed within 30 h of arrival at the Central Immunology Laboratory. Paxton and Bendele  demonstrated that samples of HIV-negative and HIV-positive blood are stable for the measurement of hematologic and basic flow cytometry markers for at least 36 h. Moreover, we have demonstrated that the expanded flow cytometry markers are stable for at least 30 h (data not shown). Sample viabilities were greater than 97% as determined by Trypan blue dye exclusion. Temperature variability during shipment was less than 2°C according to min/max thermometers.
Immunologic parameters, both absolute counts and percentages, were summarized by arithmetic means, standard deviations, and percentiles (5% and 95%). Comparisons within the REACH sample, between male and female subjects with a given HIV-status, or between HIV-negative and HIV-positive subjects, were by two-sided Wilcoxon tests with P < 0.05 defined as significant.
This report includes data for 94 HIV-positive youths who had never received ART (mean age 17.4 ± 1.0 years for males and 16.5 ± 1.3 years for females) and 149 HIV-negative youths (mean age 16.7 ± 1.2 years for males and 16.6 ± 1.2 years for females). This is the ART-naive subset of 294 HIV-infected and 149 HIV-negative youths enrolled in the REACH cohort for whom expanded immunology flow cytometry panels were available at the time of this report. The racial composition of the cohort is: HIV-positive females, 79% African-American (93% minority); HIV-negative females, 68% African-American (91% minority); HIV-positive males, 64% African-American (88% minority); HIV-negative males, 67% African-American (86% minority).
Table 2 shows the data for baseline hematologic values including standard deviation and 5th and 95th percentiles in this cohort. The total leukocyte count was significantly reduced in HIV-positive females in comparison with HIV-negative females (P < 0.001). This reduction is primarily the consequence of a reduction in absolute neutrophil count (P <0.001). There is a reduction in total eosinophil count in both males and females (P < 0.05). There is no significant difference in mean hemoglobin between the groups.
The examination of hematologic parameters by race and sex indicates that significant differences in leukocyte counts and neutrophil counts are maintained in HIV-positive subjects versus HIV-negative subjects.
Flow cytometry of mononuclear cell populations
The values for the absolute numbers of B cells, T cells and natural killer cells also are shown in Table 2. The absolute numbers of T cells and B cells are not significantly different between HIV-positive and HIV-negative groups. There is a reduction in natural killer cells (P < 0.05) in HIV-positive females (mean 140.6 ± 104.2 × 106 cells/l) in comparison with HIV-negative females (184.3 ± 142.5 × 106 cells/l), whereas no differences were found between the two groups of males.
The mean percent and absolute counts for CD4 cell and CD8 cell subpopulations are shown by HIV status and sex. Using analysis of covariance, there were significant sex effects (male versus female) unrelated to HIV status. In this context, there are highly significant reductions in T cell count, total CD4 cell count and memory CD4 cell count (P > 0.001) between male and female subjects. Furthermore, there are merely significant reductions in white blood count, absolute lymphocyte count, memory CD4 cell percent, total CD8 cells, naive CD8 cell percent, memory CD8 cell count and memory CD8 cell percent in HIV-negative males as compared with females (all at P < 0.05).
The mean value for total CD4 cell count was 870.7 ± 258.4 × 106 cells/l in the HIV-negative females compared with 618.6 ± 265.9 × 106 cells/l in the HIV- positive females, and 735.6 ± 236.3 × 106 cells/l in HIV-negative males compared with 490.2 ± 160.9 × 106 cells/l in the HIV-positive males. Reduction in the total CD4 cell count in HIV-positive males and females in comparison with the HIV-negative subjects is the consequence of a decrease in both the CD4 naive and CD4 memory components. However, the percentages of naive and memory cells were not significantly different.
The mean total CD8 cell count was 592.3 ± 300.3 × 106 cells/l in HIV-negative females compared with 1044.1 ± 508.7 × 106 cells/l in HIV-positive females, and 489.8 ± 198.1 × 106 cells/l in the HIV-negative males compared with 869.6 ± 312.5 × 106 cells/l in HIV-positive males.
The differences in CD8 cells between the HIV-positive and HIV-negative subjects indicate a significant increase in CD8 cell percentage and absolute number in the HIV-positive group (Table 2). There is a striking increase in the mean number of CD8 memory cells in HIV-positive as compared with HIV-negative youths, and a corresponding increase in percentage. In contrast, the naive CD8 cells increase in absolute number whereas they decrease in percentage. Thus, the number of memory CD8 cells increased more than the number of naive CD8 cells in HIV-positive individuals. These data may reflect the fact that these markers account for 79% of CD8 cells in the HIV-negative group and 84% in the HIV-positive group; a significant subpopulation of these cells is not labeled by these markers. Unlike previous studies in adults, we have measured the naive and memory subpopulations directly.
There is an increase in CD8+/CD38+/DR+ cells, a marker of activation and HIV disease, as well as a marked increase in CD8+/CD38-/DR+ cell number and percentage in the HIV-positive individuals as compared with HIV-negative subjects.
These observations on mononuclear cell populations and hematologic parameters for HIV-negative high-risk subjects provide further data in an adolescent population aged 13-19 years. In previous studies, the population studied by Tollerud et al. [6,7] was primarily Caucasian, whereas that studied by Bartlett et al.  was primarily African-American. The group described by this study was similar to that reported by Bartlett et al. in racial composition. When these data were assessed for race effect, with analysis of covariance, absolute neutrophil counts and hemoglobin were further reduced in African-Americans versus Caucasians (data not shown). In addition, total leukocyte counts and absolute neutrophil counts are reduced in HIV-positive females as compared with HIV-negative females (Table 2) a difference that is not observed for male subjects. In contrast with Tollerud et al. and Bartlett et al., we did not demonstrate any difference in the absolute numbers of B cells according to sex. The total CD4 cell counts for adolescent females are higher than for adolescent males, consistent with the findings of Tollerud and Bartlett; this sex difference is also observed for adults[1,6,7]. These studies are not directly comparable because the configuration of the flow cytometry markers are different. These data suggest that there are hematologic and immunologic differences between males and females which may relate to hormonal and developmental changes during adolescence.
Our studies include a comparison between high-risk HIV-negative and HIV-positive adolescents. Sequential changes in lymphocyte circulating populations during the natural history of HIV infection have been studied extensively, primarily in comprehensive investigations of male cohorts. A summary by Stein et al.  of the utility of CD4 cells in HIV staging of male cohorts has reviewed its use as a surrogate marker and also summarized day-to-day variation in cell counts, circadian variation, influence of intercurrent viral infections, and the influence of drugs. Margolick et al.  followed a subset of homosexual men from the Multicenter AIDS Cohort through seroconversion. These data showed declines in CD4 cell counts during the initial 6-12 months after seroconversion from approximately 900 to 600 × 106 cells/l. Further studies on CD4 cell homeostasis demonstrate that there is a set point for circulating absolute CD4 levels[18-21]. ‚Blind T-cell homeostasis‚ is probably independent of CD4 and CD8 subsets so that a constant level of total CD3 lymphocytes is maintained. Thus, although CD4 lymphocytes decline profoundly in HIV-infected individuals, CD8 cells increase and thus the total CD3 cell count remains stable. Later in HIV disease, there is failure of T cell homeostasis and a decline in circulating CD3 T cells[18-21]. Failure of T-cell homeostasis may be due to increased viremia, alteration in viral strains, or other properties of the virus. The data for the REACH cohort indicate significant sex differences in total CD4 counts, as well as differences between HIV-positive and HIV-negative subjects. Nevertheless, the mean CD4 levels for HIV-infected male and female adolescents was greater than 450 × 106 cells/l. One possible interpretation of these data is that this untreated cohort is relatively early in the course of HIV infection. This cohort of HIV-positive subjects, reported at the time of these baseline studies, is a group of HIV-positive youths who are ART-naive.
The reduction in naive and memory CD4 compartments is comparable to that reported in other populations. In contrast, memory CD8 cells are strikingly increased, as are naive CD8 cells. The increase in naive CD8 cells in HIV-positive adolescents is a novel finding observed for the HIV-positive group in the REACH cohort irrespective of treatment, and perhaps indicates a greater thymic capacity in the adolescent population than for adults (SD Douglas, B Rudy, L Muenz, et al. manuscript submitted).
These studies of youths provide normative data for high-risk healthy adolescents as well as baseline immunologic data for a cohort of HIV-positive adolescents. The subjects are well characterized in terms of socioeconomic, mental, economic, and psychosocial aspects and behavior including substance use. Of note, it is not possible from this report to distinguish age-related effects in the age ranges investigated (13-19 years). The data presented provide an important comparison between ART-naive HIV-positive youths and high-risk HIV-negative youths.
The authors thank R. Mitchell (Westat, Inc.) for his role in analysis of the data and S. E. Starr, D. E. Campbell, Z. Xu, N. Tustin and A. Reath for their contributions to this paper.
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The following investigators, listed in order of the numbers of subjects enrolled, are participating in this study: University of Miami, L. Friedman, L. Pall; Montefiore Medical Center, D. Futterman, D. Monte, M. Alovera-DeBellis, N. Hoffman; Children‚s Hospital of Philadelphia, B. Rudy, D. Schwarz, M. Tanney; Children‚s Hospital of Los Angeles, M. Belzer, D. Tucker; Children‚s National Medical Center, L. D‚Angelo, C. Trexler, C. Townsend-Akpan, R. Hagler; University of Maryland, L. Peralta, G. Ryder, S. Miller, K. Feroli; Tulane Medical Center, S.E. Abdalian, D. Foxworth, L. Green; Children‚s Hospital, Birmingham, M. Sturdevant, A. Howell; St. Jude Children‚s Research Hospital, P. Flynn, K. Lett; Childrens Diagnostic and Treatment Center, A. Puga, Cruz; Cook County Hospital/ University of Chicago, L. Henry-Reid, R. Camacho, M. Bell, J. Martinez, D. Johnson; Mt. Sinai Medical Center, L. Levin, M. Geiger; Emory University, M. Sawyer, G. Walls; SUNY Health Science Center, J. Birnbaum, M.Ramnarine; University of Medicine and Dentistry of New Jersey, P. Stanford, F. Briggs. The following investigators have been responsible for the basic science agenda: C. Holland, Children‚s Research Institute and the George Washington University School of Medicine; A.B.Moscicki, University of California at San Francisco; D. Murphy, University of California at Los Angeles, S. H. Vermund, University of Alabama at Birmingham; R. Booth, University of Colorado; P. Crowley-Nowick, University of Pittsburgh; S. D. Douglas, University of Pennsylvania. Network operations and analytic support are provided by C. M. Wilson and C. Partlow at the University of Alabama at Birmingham; B. Hobbs, J. Ellenberg, L. Paolinelli, L. Muenz, T. Myers, A. Sheon, Rick Mitchell at Westat, Inc. Staff from sponsoring agencies include A. Rogers, A. Willoughby (NICHD), K. Davenny, V. Smeriglio (NIDA), E. Matzen, J. Lew (NIAID), G. Weissman (HRSA).