Immunohaematological indices such as leukocyte, lymphocytes and their subsets such as CD3+T cells, CD4+T cells and CD8+T cells play a major role in both cellular and humoral types of immunity. T lymphocyte cells are defined by the expression of CD3+ molecules. The most important cell-surface molecules for identifying T lymphocyte subpopulations have been CD4+, CD8+, and T-cell receptor complex molecules (CD3+, the T-cell receptor α and β chains)  . CD4+T cells are the lymphocytes subsets used for monitoring progression of HIV/AIDS infection and they are also used as a surrogate marker for the improvement of HIV/AIDS patients after initiation of ARV  . Lymphocyte subset may include; Helper T cells (CD4 T cells), Cytotoxic T cells (CTLs or CD8 T-cells), Memory T cells and Regulatory cells (Treg cells). Upon encounter with antigens, CD4 T cells become activated and proliferate rapidly secreting cytokines that send signals and maintain active immune response. On the other hand, CD8+T-cells destroy virally infected cells and tumor cells, and remain inactivated when there is no foreign antigen. In human immunodeficiency virus-1 (HIV-1) infected individuals, lymphocytes (specifically CD4 T-cells) are the viral prime targets. Therefore in these individuals, a CD4+T count provides a picture of immune system competence, with higher CD4 counts typically signifying healthier immune systems   . Further, CD4+T cell level determines when to start or stop prophylactic drugs for opportunistic infections  . Less than 350 cells/μl is considered a threshold for starting ART in HIV-positive patients without other indications in many high-income countries   although not all  and some commentators argue that with newer, safer antiretrovirals, there should be commencement of therapy at a higher threshold of CD4+T count  . Similar levels of cut-off are encouraged for low-income countries where feasible  despite some evidence that treatment may be delayed further without increasing the risk of AIDS related events   .
Reference ranges that are currently used in Kenya are derived from data obtained from Caucasians who are not African Kenyans. In addition, these ranges do not include age and sex which are very important factors that influence lymphocyte counts  . Immunohaematological variations have been reported in various studies, showing association with sex   , geographical location, race, altitude and diet     . Other reasons for variations are pregnancy, age   , exercise, cormobid conditions and diurnal variation    , in addition to variations caused by methodological differences. Averagely, healthy African and Asian populations have been shown to have lower CD4 lymphocyte counts than counts than their western European and Caucasian counterparts  -  . The pattern of lymphocyte generation has been shown to effect the levels of circulating lymphocytes in males, females and individuals of different ages. The pattern of T lymphocyte generation in aging has been associated with dynamic changes in thymic and extrathymic functions along developmental steps from cells to mature cells  . Increased absolute numbers of peripheral blood CD4+T cells in females compared to males have been reported  . This is perhaps due to androgens which accelerate thymocytes apoptosis and subsequently influence T cell repertoire with males tending to have less CD4+T cells than females   . This necessitates the importance of establishing local immunological reference values (CD3 and CD4+T cells for local Kenyan population). The study provides ranges specific for males and females of different age groups.
2. Materials and Methods
2.1. Study Design, Setting and Population
A cross-sectional study was conducted in Coastal region of Kenya which has a population of 1,031,266 between May 2015 and February 2016. The study was done at Coast provincial General hospital (CPGH), Mlaleo, Mikindani, Likoni, Portreizt and Tudor Sub County hospitals. These hospitals have HIV voluntary counselling and testing (VCT) services.
2.2. Study Subjects
Study population were adults who were attending HIV-Voluntary counselling and testing (VCT). The VCT services are offered in hospitals with Modern equipment’s for immunohaematological testing. Eligible subjects were those aged 18 years, those who agreed to participate in the study, free from clinical disease conditions, looking apparently healthy, who lived in the area at least in the past six months having body mass index between 17.5 kg/m2 and 22 km2 and HIV negative. Those who consented were asked for blood samples for repeat HIV test, and immunohaematological counts. Blood was collected in ethylene diamine-tetra-acetic acid (EDTA) tubes. The same samples were used for repeat HIV test, CBC, CD4 T-cell and CD8 T-cell counts. Samples were collected in the morning from 8.30 am to 10.30 am and kept at room temperature and transferred Coast General Referral hospital and Portreitz Sub county hospital for analysis the same day. This was done to control possible diurnal variations for T lymphocyte counts. A structured questionnaire was used to collect information on the history and general health status of the subjects. The samples were screened for HIV, Hepatitis B virus (HBV), Hepatitis C virus (HCV), malaria and syphilis after blood collection.
The following categories were excluded: pregnant mothers, patients receiving medical treatment, individuals with history of recent or current cormobid conditions, chronic alcoholism and moderate and severe malnourishment (BMI < 17.5 kg/m2)), recent past immunization in the last 6 months, blood or blood product transfusion in the last six months, Patients with malaria and individuals who tested for HIV antibody.
2.3. Laboratory Procedures
2.3.1. Screening for Malaria, Syphilis, HCV and HBV
Enzyme linked immunoassays were used in screening for these conditions. Commercially available kits containing purified antigens were used. The kits included: ICE* Syphilis, Murex anti-HCV (version 4.0), Hepanostika HBsAg Ultra kit, Murex HIV-1.2.0 (Abbot) and malaria antigen detection kit (Creative diagnostics).
2.3.2. HIV Testing
Blood samples were tested for HIV antibodies according to the Kenyan national testing algorithm for voluntary counseling and testing. Testing for HIV infection was done by screening serum/plasma by using Determine HIV1/2 (Abott laboratories, Japan co. LTD), Capillus HIV1/2 (The Trinity Biotech, Bray, Co Wicklow, Ireland) and Unigold H1/2 rapid test kits. Concordant positive results were interpreted as positive for HIV antibody. Discordant results were interpreted as inconclusive and the samples were confirmed using ELISA; Enzygnost and anti-HIV 1 + 2 Plus ELISA (Behring, Marburg, Germany) and well-coenzyme HIV recombinant ELISA (Murex, Dartford, England).
2.3.3. Haematological Assay
Complete blood cell counts were done using Sysmex Kx-21 (Sysmex Corporation; Kobe Japan). The machine automatically dilutes a whole-blood sample, lyses, counts and gives a printout result of absolute numbers of leucocytes (expressed as number of cells ×  per liter), erythrocytes (number of cells ×  per liter), platelets (number of cells ×  per liter), lymphocytes (number of cells ×  per liter), mononuclear cells (number of cells ×  per liter), granulocytes (number of cells ×  per liter) and haemoglobin (grams per decilitre). The quality and accuracy of the technique and the machine was assessed every six months.
2.3.4. CD3, CD4+T and CD8+T Cell Determination
Cluster of differentiation 3 and 4 (CD3 & CD4 T) cells were enumerated using a FACSCount flow cytometer (Becton Dickinson Immunocytometry Systems, San Jose, Calif.). The BD FACScount™ System is designed to provide CD4 and CD3 T cell absolute values and percentage per microlitre of sample. The system includes BD FACScount instrument, software, reagents, controls and a workstation. In brief, 50 μl of whole blood was mixed with monoclonal antibodies (MAbs) (5 µL of each T lymphocytes reagent tubes) and incubated at room temperature for 20 min with 20 μl of aCD4 and aCD8. Red blood cells were then lysed by adding 450 μl of fluorescence-activated cell sorter lysing solution (Becton Dickinson Immunocytometry Systems). The tubes were incubated at room temperature for 10 min, and then analyzed with the FACSCounts's Cell Quest software (Becton Dickinson Immunocytometry Systems) within six hours. By using quality control (Multicheck; Becton Dickinson Immunocytometry Systems), the accuracy of the technique was assessed every 6 months.
The FACSCaliber was calibrated and reconfirmed daily using the BD Caliber the BD Calibrate beads and BD-FACS Comp soft-ware, version 2.0 for setting the photomultiplier tube voltages, setting the fluorescence compensation and checking instrument sensitivity before use. The CD45 versus the side scatter (SSC) dot plot was visually inspected to make sure they appear as a bright compact cluster, with low SSC.
2.4. Screening for Malnutrition
All participants were assessed for malnutrition using body mass index (BMI). Normal nutrition status was defined as BMI > 18.5/kg/m2, mild malnutrition as BMI of 17.5 - 18.4 kg/m2, moderate malnutrition as BMI of 16 - 17.4 kg/m2 and severe malnutrition as BMI < 16 kg/m2. 
2.5. Data Management and Analysis
Completed questionnaires were coded and doubled entered in computer software Epi-data version 13.1. Cross-checking and data cleaning was done. The data was the transferred to SPSS version 20.0 for analysis. Non parametric reference range determination was employed by estimating the 2.5 and 7.5 percentile  . The significance of differences between the mean absolute counts and percentages of the Lymphocyte subsets were estimated using the Analysis of Variance (ANOVA) and T-test. Multiple comparisons were done using Tukey’s test. Correlation analysis was applied in determining the relationship between lymphocyte subset levels and age.
2.6. Ethical Issues
The protocol was approved by Kenyatta University Ethical Review Committee. Clearance was also obtained from respective district health authorities and hospital administrations. The purpose of the study was explained participants in English, Kiswahili or local language before written consent was sought. The study was conducted in accordance with the declaration of Helsinki. Those participants, who had HIV infection, were referred to the HIV care and treatment clinic for further management after counseling. Female participants who were reactive for hcG test were referred to antenatal clinic for better care.
3.1. Baseline Characteristics of the Study Participants
Among the 500 individuals between the ages 18 - 55 years, 48.0% (240) were males and 52.0% (260) were females. Their blood samples were analysed for CD3, CD4 and CD8 lymphocyte subset for establishment of reference ranges. Changes in age influenced the subset values marginally in both sexes. With the advancement of age, the CD4 mean counts decreased consistently both males and females though there was a slight increase in males age between 41 - 50 years. However, the age dependent changes did not achieve the statistical significance. Women had comparatively higher CD4 mean counts than their male counterparts. The difference was statistically insignificant. On the other hand, males had higher CD8 mean counts compared to females (710.9 ± 113.8 cells/mm3 and 667.7 ± 93.6 cells/mm3) respectively. The difference again was again statistically insignificant. However, significantly higher mean CD4/CD8 ratios were found in females as compared to males, 1.42 ± 0.17 cells/mm3 and 1.69 ± 0.49 cells/mm3 (p < 0.05). In both the sexes, the CD4 mean counts were always higher than the CD8 mean counts irrespective of age (Table 1).
3.2. Immunohaematological Reference Values in Human Immunodeficiency Virus Negative Patients.
The mean CD4+T count in males and females combined was 1054.9 ± 156.1 cells/mm3, mean absolute CD4+T lymphocyte in males was 998.7 ± 127.1 cells/mm3 and in females 1106.8 ± 162.4 cells/mm3. The Mean absolute CD8+T cells in males and females combined was 688.4 ± 105.9 cells/mm3 and mean absolute CD8+T counts in males were 710.9 ± 113.8 cells/mm3 and in females 667.7 ± 93.6 cells/mm3. The absolute combined was CD3+T in was 1945.1 ± 429.2 cells/mm3 and mean absolute CD3+T counts in males were 1871.3 ± 355.2 cells/mm3 and in females 2013.2 ± 478.3 cells/mm3. The mean CD4/CD8+T cells ratio in males and females combined was 1.56 ± 0.39, cells/mm3. Mean CD4/CD8+T cells in males were 1.42 ± 0.17 while in females 1.69 ± 0.49 cells/mm3. Females had significantly higher mean absolute CD4+ cells (t = 8.32, df 485.2, p = 0.001) and lower mean absolute CD8+T cells (t = 4.62, df = 463.8, p < 0.05) than in males. The haemoglobin level in males and females combined was 12.76 ± 1.28 gram/dl. Mean haemoglobin level in males was 13.06 ± 1.48 gm/dl and in females 12.48 ± 1.00 gm/dl. Females had significantly lower mean haemoglobin level than males (t = 5.06, df, 416, p = 0.002) as shown in Table 2.
3.4. Males and Females with Lower than Normal Values of CD4 T Cells, Leukocytes, Lymphocytes and Body Mass Index
The mean absolute leukocytes cells in both males and females was 5.15 ± 0.37 cells/litre. Mean absolute leukocytes cells in males was 4.89 ± 0.22 cells/litre and in females 5.40 ± 0.30 cells/litre. Mean absolute lymphocytes cells in males and females combined were 1.85 ± 0.29 cells/litre. The mean lymphocyte in males was 1.67 ± 0.20 cells/litre while in females mean was 2.02 ± 0.26 cells/litre. On nutritional status assessment using BMI, the mean BMI for the participants was 22.6 ± 95% CI: 22.5 - 22.7 kg/m2. Mean BMI for males was 22.4 ± 95% CI: 22.3 - 22.6 kg/m2 and for females 22.9 ± 95% CI: 22.8 - 23.1 kg/m2. There was no
Table 1. Age distribution of immunocompetent adults in Coastal Kenya.
Unit of cells/µL is not applicable to CD4/CD8 ratio; ** Being only one case no value for mean ± SD and 95% CI.
Table 2. Arithmetic median, 2.5th-97.5th percentile, mean and 95% CI of the mean CD4+T cells, CD8+T cells, CD4/CD8 ratio and haemoglobin level in normal subjects (Controls).
CD4+ = Cluster differentiation T-lymphocyte no.4, CD8+ = Cluster differentiation T-lymphocyte no.8; CD3+ = Cluster differentiation T-lymphocyte no.3; SD = standard deviation; CI = confidence interval; *Statistically significant association.
significant statistical difference between males and females with regards to the nutritional status (χ2 = 76.3, df = 1, p > 0.05). There was no significant association between nutritional status with any of the immunohaematological parameters like CD4+T cells and CD8+T cells and lymphocytes (p > 0.05) but there was association between BMI and leukocytes (p < 0.05). Significant statistical differences was observed in the absolute count means of the lymphocytes in males and females (p < 0.05). There was association between different age-groups and immunohaetological parameters like CD4+T cells (p < 0.05) and CD8+T cells (p < 0.05) (Table 3).
3.5. Haematological Reference Ranges in Immunocompetent Individuals
Men had higher Haematocrit, erythrocyte counts, white blood cells counts, platelet counts and neutrophil percentage than women (p < 0.05). Women had higher platelet and neutrophils counts (Table 4).
Table 3. Males and females with lower than normal and normal values of CD4 T cells, leukocytes, lymphocytes and body mass index (Controls).
BMI = Body mass index; SD = standard deviation; CI = confidence interval; *Statistically significant association.
Table 4. Haematological reference ranges (median percentile) derived from immunocompetent individuals from Coastal Kenya.
In this study, the mean immunohematological reference values in healthy subjects for CD4+T and CD8+T counts was 1054.9 ± 156.1 cells/mm3 and 998.7 ± 127.1 cells/mm3 respectively. Our values were also higher than a number of reports in some countries. For instance, mean CD4 T cell values of 870.7 and 865 cells/μl have been reported among HIV seronegative individuals in Senegal and India, respectively   . However, the mean CD4 T cells of 1067 cells/μl reported in eastern Ghana by Ampofo et al.  is higher than our findings. This may not necessarily mean that Ghanaians have higher counts than Kenyans because the sample size for their study was just two hundred and fourty nine (249).
The study also found that females had significantly higher counts of absolute CD4+T cells and absolute CD8+T cells than males. This findings concur with earlier study done in Tanzania to determine gender difference in CD4 T cells, which showed that males were more likely to have CD4 T cells < 500 cells/mm3  . This was probably due to biological factors. It has been speculated that gender and age-related variations within the immune system parameters may contribute to the pathogenesis of several gender and age-related diseases such as autoimmune disorders in female patients    . Diurnal variation, however, cannot explain the significant gender difference observed in this study, as all the samples were collected between 9.00 am to 11.00 am. While smoking was also reported to be associated with higher CD4+T count, this could not be the reason for the difference as all female subjects in the present study were nonsmokers. Sex hormone effect could be the possible explanation for the observed gender difference in CD4+T cell count, as the circulating lymphocytes have receptors for androgens and oestrogens  . These variations in CD4+T cells have been shown to be associated with ethnicity, gender, diet, geographical area as well as being dependent on genetic and environmental factors. Further, it has been shown that there are gender differences in the generation of CD8 cells during HIV-1 infection, due to increased immune activation compared to men  . Females had significantly lower haemoglobin levels than males in our study. This is similar to findings in North America, Europe and Asia. Overall we found low haemoglobin levels compared to European values and the standard reference haemoglobin values. This can be partly explained by low dietary intake of food rich in iron and vitamins, which is the commonest causes of low haemoglobin levels in Kenya.
The absolute T lymphocytes mean, median, and the 95% normal reference interval value for the current study were also greater than the studies conducted from Switzerland by Bisset et al.  , Shanghai, Chin  and Asian population  . These differences could be on our apparently healthy study population where they used only blood donors especially in Asian population and Switzerland studies. In our study we used large sample size compared to the other studies. CD3 and CD4 T cells did not correlate directly with age. This agrees with previous studies in China and India that found no significant association of CD4 T cell values with age, but disagrees with a similar study in Nigeria among children   . In this study the lymphocyte subsets in the different age groups revealed no significant differences. It is however well known that the pattern of T lymphocyte generation with age originates from dynamic changes in thymic as well as extrathymic functions, along with sequential developmental steps from stem cell to ultimately mature cells  . The finding of significant gender differences for the RBC parameters (erythrocytes, hemoglobin, and hematocrit) agrees with the well-established fact that males have higher values for erythrocytes, hemoglobin, and hematocrit than females, partly due to the influence of the hormone androgen on erythropoiesis and also due to menstrual loss. Significant differences between the genders with regard to WBC and platelet counts were observed in this study (p < 0.05). The general absence of gender differences for WBC counts disagrees with other reports   . A study done Saudi Arabia showed a higher percentage and number of CD8+T cells (p < 0.01) and a decreased CD4/CD8 ratio (p < 0.02) compared with the Caucasian controls. Male population had a significantly lower percentage and number of activated T cells (p < 0.05)  .
Immunohaematological markers found in this study were different from the standard values for the western countries. The ranges are also different from those that are being used currently. Females had significantly higher mean CD4+T and CD8+T cell counts but lower mean haemoglobin level, erythrocytes, white blood cells and platelets than males. Our findings offer useful insight in the CD3 and CD4 T cell reference values of Kenyans and provide important information that will guide future ART decisions and other immune-based therapies.
The authors wish to thank individuals who participated in the study, the Medical Officer of Health, Medical Superintendents, Laboratory staff and clinical officers and nursing staff at Coast Provincial General Hospital, Portreitz, Tudor, Likoni, Mlaleo and Mikandani Sub-County hospitals who greatly assisted us with specimen and data collection for this study.
This work was carried out in collaboration between all others. SAY and SSN conducted the study. MFO and RRS supervised the work SAY initiated the study and made major contributions to the study design. All authors helped to conceptualize ideas and interpret the results. SAY prepared the draft with other authors helped in review.
List of Abbreviations
HIV: Human immunodeficiency virus; SSC: Side scatter; ANOVA: Analysis of variance; CD4: cluster of differentiation 4; CD8: cluster of differentiation 8