A descriptive cross-sectional study was carried out among healthy adult population in mid-western and far-western development region of Nepal from March to May 2016. Samples were collected from healthy volunteers willing to participate in the study after filling questionnaire form. The form was filled by face-to-face interview for each participant. The basic information included in the form was: name, age, sex and, history of acute or chronic disease. Only the healthy adults of 15 – 60 years with no previous history of any kind of diseases, recent medication or blood transfusion were included in the study. Women in the pregnancy period were excluded from the study. The collected samples were tested for the presence of HIV antibodies. Among the HIV sero-negative healthy volunteers, 400 were selected for study (200 in each site). The selected samples were processed for further laboratory analysis at two major government based hospitals; Bheri zonal hospital and Seti zonal hospital. The sample size was in accordance to the CLSI guidelines for determining laboratory reference ranges which indicates a minimum of 153 subjects to be enrolled in the study for 95th percentile value with 95% confidence interval . The sample was collected in between 10.00 AM to 2.00 PM to minimize the diurnal variation. To obtain homogeneity in data, participants from different districts of the region were included. In addition, enrollment of both sex, and adults from different age group were also considered to eliminate the possible bias.
About 3ml whole blood sample was collected in tripotassium ethylenediaminetetraacetic acid (K3EDTA) vial from the healthy volunteers participating in study. All the samples were tested for the presence of HIV antibodies following national algorithm that includes HIV1/2 rapid test assay using three different commercial kits: Determine, Stat-Pak and Uni-Gold. HIV negative samples were assayed for CD3 and CD4 T lymphocyte count.
T lymphocyte count was carried out using BD FACS Calibur (BD Biosciences, San Jose, CA, USA) following manufacturer’s instructions. Briefly, 50 μl of well-mixed EDTA whole blood was pipetted into a BD Trucount tube containing bead. 20 μl of BD TritestTM CD3/CD4/CD45 reagent containing monoclonal antibodies for CD3, CD4 and CD45, labeled with fluorescent dyes was also added to the same tube. The tube was then vortexed, and incubated in dark at room temperature for 15 minutes to stain the cells. Following staining, red blood cells were lysed using BD FACS lysing solution and run in the flow cytometer to obtain absolute count.
For the purpose of quality control, calibration beads were run every day to ensure the optical alignment of the instrument following compensation setting using the software. Internal quality control was also run at both the centers using commercially available stabilized blood samples. Samples were run only when the result of internal quality control was within the range of the value provided by the manufacturer.
Data collected were initially entered into MS Excel, which was then transferred to Statistical Package for Social Sciences (SPSS) and analyzed using the same SPSS version 20.0 [IBM Armonk, NY, USA]. Mean, median and standard deviation values were calculated for each parameter. Wilks-Shapiro test was used to understand the distribution of data, which showed significant difference suggesting non-Gaussian distribution. Based on this, the decision for non-parametric analysis was made to establish the reference range. The reference range was defined as the central 95% of the area under the distribution curve of the values (from 2.5% to 97.5%). ANOVA test was used to compare mean between variables considering 95% CI and, p-value less than 0.05 was considered statistically significant.