Healthy individuals who visited the Seoul Songdo Colorectal Hospital for regular medical checkups between April and September 2013 were randomly recruited. All participants were examined and a questionnaire was administered to confirm their condition and health history. Only participants with serious medical problems, such as HIV infection, autoimmune disease, allergies, and cancer; using medications or dietary supplements; or pregnant at the time of the study, were excluded.

Blood samples were managed at room temperature and processed within 1 h on the day of collection. Whole blood was collected aseptically into potassium ethylenediaminetetraacetic acid-containing tubes (Vacuette®, Greiner bio-one, Kremsmünster, Austria) for complete blood counts and differential counts using an automated hematology analyzer (Sysmex XE-2100, Kobe, Japan), and for immunophenotyping by flow cytometry. The parameters for analysis were the numbers of white blood cells (WBC), and the percentages and numbers of lymphocytes, CD3⁺ cells, CD3⁺CD4⁺ cells, CD3⁺CD8⁺ cells, CD3^-CD19⁺ cells, and CD3^-CD56⁺ cells; the CD4⁺/CD8⁺ ratio was also determined.

Aliquots (50µl) of whole blood were added to 12×75-mm capped polypropylene test tubes containing premixed monoclonal antibodies (mAbs). The mAbs against CD3, CD4, CD8, CD56 and CD19 (BD Pharmingen™, San Diego, CA, USA), and the mAb against CD45 (eBioscience, San Diego, CA, USA) used in this study were purchased at the same time and were from the same lot number. The optimal working concentration for each antibody was determined by serial dilution of the antibody in a preliminary titration experiment. Two sets of antibody cocktails were prepared using the determined amount of each antibody. Fluorescein isothiocyanate (FITC)-conjugated mouse anti-human CD3 mAb, phycoerythrin (PE)-conjugated mouse anti-human CD8 mAb, PE cyanin5-conjugated mouse anti-human CD4 mAb, and allophycocyanin (APC)-conjugated mouse anti-human CD45 mAb were mixed in one test tube, and FITC-conjugated mouse anti-human CD3 mAb, PE-conjugated mouse anti-human CD56 mAb, PE cyanin5-conjugated mouse anti-human CD19 mAb, and APC-conjugated mouse anti-human CD45 mAb were mixed in the other test tube. Blood, labeled with each set of antibodies, was incubated in a dark room for 20 min at room temperature and the red blood cells were lysed by adding 440µl of FACS lysing solution (BD Biosciences, San Jose, CA, USA) for 15 min. Then, 50µl of CountBright™ absolute counting beads (Molecular Probes, Carlsbad, CA, USA) was added to each cell-staining tube and immediately assessed using a flow cytometer (BD FACSCalibur, San Jose, CA, USA); the results were analyzed using CellQuest™ pro software (BD, San Jose, CA, USA).

Daily calibration of the flow cytometer was performed using the manufacturer's software program (FACSComp software, BD) and materials (Calibrated beads, BD), and the intensity of the antibody fluorescence was monitored every week to ensure consistent results.

Data were analyzed using the SPSS statistical package program, version 18 (IBM, Armonk, NY, USA). The mean and standard deviation values were calculated for all parameters. A Kolmogorov-Smirnov test and a Shapiro-Wilks test were used to check the Gaussian distribution of all variables. Because all variables had non-Gaussian distributions, the reference ranges for all parameters were determined using the 95th percentile of the area under the distribution curve of the variable. An Analysis of covariance (ANCOVA) test was used to compare variables between men and women, and a Kruskal-Wallis test with Spearman's correlation coefficients was used to check for significant differences between age groups. p-values<0.05 were considered statistically significant.

This study was approved by the institutional review board of Seoul Song Do Colorectal Hospital (IRB Number 2012-006). Informed consent was waived by the board because blood sampling was performed for clinical examinations but verbal consent was obtained from all participants at the time of blood draw.

The study population consisted of 139 men and 155 women (mean age; 49±13 and 45±14 years old, respectively). A comparison of the parameters between these 2 groups is presented in Table II. Overall, statistically significant differences in several parameters were observed between the groups. The percentages of CD3⁺ cells and CD3⁺CD4⁺ cells were lower in men than in women (p<0.05, in both cases), but the absolute count and percentage of CD3^-CD56⁺ cells were higher in men than in women (p<0.001, in both cases). However, no significant differences between men and women were observed with respect to the other subtypes.

In order to examine the changes in the cell subtypes according to age, the healthy donors, 21~80-years-old, were classified into 5 groups. Statistically significant differences in absolute counts and percentages of CD3⁺ cells, CD3⁺CD8⁺ cells, and CD3^-CD56⁺ cells were observed between the groups (p<0.05). Likewise, the percentages of lymphocytes and the CD4⁺/CD8⁺ ratio were also significantly different between the groups (p<0.01) (Table III). These observations were further analyzed by calculating Spearman's correlation coefficients. A trend of decreasing absolute counts and percentages of CD3⁺ cells (r=-0.162, p=0.005 and r=-0.383, p<0.001) and CD3⁺CD8⁺ cells (r=-0.316 and r=-0.467; p<0.001, in both cases) with increasing age were observed. On the other hand, the percentage of lymphocytes (r=0.128, p=0.029) and the absolute count and percentage of CD3^-CD56⁺ cells (r=0.378 and r=0.371; p<0.001, in both cases) showed a tendency to increase with increasing age. Consequently, the CD4⁺/CD8⁺ ratio (r=0.372, p<0.001) also showed a gradual increase with increasing age, owing to a significant decrease in the percentage of CD3⁺CD8⁺ cells. However, no significant differences or correlations, according to age, were detected among the 5 groups of the other lymphocyte subtypes (Table IV).