This cross-sectional study was aimed at determining the reference values of lymphocyte subsets in healthy Indian children aged 0 through 18 years from four geographically diverse sites i.e., Bengaluru, Chandigarh, Mumbai and Kolkata, in order to obtain the data representative of the entire country. The different age groups included in the study were: Group I- Newborn; Group II - 6 weeks of age (before first DPT vaccination); Group III - 9 to 10 months of age (before measles vaccination); Group IV - 15 to 18 months of age or before first booster of DPT; Group V - 19 months to 5 of age, Group VI - > 5 to 12 years of age; and, Group VII - 12 to 18 years of age. The immunization visits coincided with the blood collection visits.

For group I, cord blood was used as a sample. For this group, babies with full term normal vaginal delivery or elective caesarean with or without mild anemia in pregnancy were included. Emergency caesarean cases, complicated deliveries with chronic illness or with infections, conditions like diabetes, toxemia, bleeding, fever in mother, prolonged rupture of membrane, and HIV positive pregnancy were excluded from this study.

For other groups (Group II to VII), the inclusion criteria for healthy children in the different age group were: no history of cold and cough (for the last one month), blood transfusion (preceding 3 months), surgery (preceding 6 months) and recent diarrhea (4-6 weeks), born to HIV negative mother, and grade 1 malnutrition/ normal weight (weight-for-age >70th centile IAP chart). Children with moderate to severe anemia, acute or chronic infectious diseases (gastrointestinal diseases within the last 6 months) or any clinically significant disease or findings in the medical history that might compromise the study measures (e.g., diabetes mellitus, asthma, rheumatoid arthritis, cystitis fibrosis) were excluded from the study.

Children were enrolled after obtaining written consent from their parents, and assent, if required. The study was approved by the ethics committees of respective study sites. From each site, 50 children were enrolled in each group and in each age group an attempt was made to enroll boys and girls in a 1:1 ratio. The enrollment for groups I, II, III and IV was done in the hospitals (well-baby clinics of the hospitals) and for groups V, VI and VII, school-going healthy children were enrolled after obtaining appropriate permission from the school. In one day, not more than 5-7 eligible newborns/infants/ children were enrolled in each group (Groups I to IV). For the groups V, VI and VII, the schools were contacted and the eligible participants were enrolled sequentially. The data on age, sex, place of origin, height, weight, nutritional status and vaccination was collected wherever possible.

Two to five milliliter of whole blood specimens were collected from the children in K3 EDTA evacuated tubes and were processed for immune-phenotyping the same day. To avoid diurnal variation, the samples were uniformly collected in the forenoon at all the study sites.

A total of 1674 children were enrolled across the four regions. Of these, data collected from 1104 subjects was considered for analysis; 570 samples were excluded due to various reasons like quality of samples, fail to fit in hemoglobin, BMI or weight criterion etc. Region- and age-wise numbers of subjects in each group are shown in Table I. The representation of the samples in groups II (6.1%) and III (11.3%) were lower as compared to the other groups. The median (range) weight and hemoglobin of the newborns was 3 (2.5-4.5) kg and 16.1(13-22.1) g/dL, respectively. The hemoglobin decreased to 11.8 (11-15.1) g/dL in group II, but remained similar in older age groups. The median (range) body mass index was 16.6 (13.9-19.6) in group II, which remained similar in older age groups.

In this multi-centric study, we determined the reference ranges for different lymphocyte subsets in Indian pediatric population. This study represents the largest dataset for the relative frequencies of major lymphocyte subsets in healthy Indian children at various age groups from birth till 18 years of age. Unlike CD4+ and CD8+ T cells reference ranges, limited information is available on other lymphocyte subsets like CD3+T cells, CD19+ B cells and CD16/56+ NK cells which have important immune functions.

Our observations confirmed the previous findings that the lymphocyte compartments of normal healthy children differ considerably in various age groups [8-11]. The absolute T cell and B cell count increased during the first few months till 9-10 months and decreased gradually from 15-18 months onwards till 12-18 years. Whereas the relative percentages of T cells i.e. CD3+, CD8+ and CD4+ cells remained more or less similar in all age groups. Similar findings have been reported in African and Caucasian populations previously [10], and from children from southern India [3].

We found that the reference ranges in our cohort differ from pediatric population from other regions like Europe [6,8,12], Africa [13,14], and North America [11,15]. Among the newborns, the absolute cell counts for CD3+, CD4+, CD8+, CD19+ B cells were lower than the cohort from Italy [8] but higher than the African cohort from Cameroon [14]. These differences could be due to the differences in the total lymphocyte percentages and absolute counts, which were not measured in both the studies. Similarly the percentages of CD3+, CD4+ cells of the newborns were lower than Italian children [8] but higher than Cameroon [14], while the percentages of CD8+ T and CD19+ B cells were higher in our newborn group than the newborns from Italy and Cameroon. The number of samples tested from the newborn group might be the reason for such differences. The Italian study used 16 samples from the 0-3 month group whereas the Cameroon study used 38 cord blood samples. In other age groups of children in our study, the absolute CD4 counts were higher than seen in children from Europe, Africa and USA; however in children from 6 years to 18 years, it was comparable to children from Uganda [16]. Except in newborns, children in all age groups had a higher CD8 cell counts when compared with the children from Europe (Italy), Africa (Tanzania, Uganda, Cameroon) and USA [8,11,14,16]. The CD19+ B cell and NK cell counts were higher than the counts observed from Italian population [8] and largely comparable with the pediatric population from Cameroon [14]. Rathore, et al. [17] compared the different immune cells subsets in newborns from United States and India and found that Indian newborns had higher NK and CD4+ T cells, while lower subsets of total T cells, than the American cohort. In comparison with these values, the data from the present study showed lower CD4 counts whereas the CD8+ T cells, B cells, NK cell counts were in similar ranges. Similar to the absolute count, the percentages of different immune cells also varied in our pediatric cohorts in comparison to that of Europe, Africa and USA [8,14,16,18]. These data collectively indicate that each immune cell subset in different age groups of children varies with the ethnicity and is influenced by the geographical region. The lymphocyte subsets are known to vary with the time of collection, use of different equipments, procedure for estimation, and the time between the collection and testing [8,9]. Hence, to minimize the variation within the laboratories, proper quality control measures were taken such as use of standard procedure, sample collection in the forenoon hours at all the study sites to avoid diurnal variation, and uniformity of equipment and reagents across the sites. This pediatric cohort did not show significant differences between the sexes, as also observed in other studies [10]; although, reference ranges for the CD4 count and percentages in Indian adults were significantly higher in women [2].

India is a geographically heterogeneous country, hence it was important to assess whether the reference ranges differ in different geographical regions. Our study showed significant differences between the region-wise ranges (mostly East and South) in various parameters in different age groups. These differences might be due to the environmental, genetic or nutritional factors [19-21]. Since this data could not be obtained, these observations need to be confirmed on the larger sample size from the specific age-groups. Moreover, the established ranges could be reconfirmed on a small subset from time to time as described earlier [18]. One of the limitations of our study is insufficient samples available in some regions for children belonging to groups II and III. This could be the due to less number of babies coming for DPT immunization during the study period. It would have been interesting to examine the activation and functional profile of these cells; however, due to the limitation of the parameters that can be tested by the available flow cytometer, it could not be evaluated but could be an important area of future research.

In summary, this study provides reference values for different lymphocyte subsets in Indian children of varying age groups. Age was the only important variable affecting the counts, and sex and geographical distribution did not prove to be significant variables. This data can find application in immune system evaluation of children of Indian origin irrespective of sex, geographical distribution and ethnicity. These age related reference ranges will be helpful to assess the immune defects, and suppression/absence of one or more immune functions in Indian children with primary and secondary immunodeficiencies as well as in autoimmune diseases.

Note: Supplementary material related to this study is available with the online version at www.indianpediatrics.net

Ethics clearance: ICMR-NARI Ethics Committee; No. NARI/Age Lymphocyte subsets/10-11/100, dated 22 June, 2010.

Contributors: MT: Study design, data analysis, preparation and review of manuscript; VS: analyzed the data, drafted and reviewed the manuscript; NJ,VR,AD,SS,NS,RM,AS,MC,MM: study design, execution of study, patient information, data analysis, manuscript review (at different sites); SS: data analysis, manuscript preparation and review; AM: study design, data analysis manuscript review; VM: study design, execution of the study and review of the program. All authors approved the final version of manuscript, and are accountable for all aspects related to the study.

Funding: Indian Council of Medical Research, India.

Competing interests: None stated.