Normative data for paediatric lymphocyte subsets: A pilot study from western India

Abstract

Background & objectives: Accurate diagnosis of immunodeficiencies requires a critical comparison of values with age-matched controls. In India, the existing reference values for rare lymphocyte subsets are currently not available and we rely on the data originating from other countries for the interpretation of the results. Furthermore, there is limited information on normal variation for these rare-subset parameters in Indian children. So, this study aimed to establish normative values for clinically important lymphocyte subsets in Indian children at different age groups.

Methods: 148 children aged ≥16 yr were enrolled in this study. The study population included 61 per cent males and 39 per cent females and was divided into the following groups: cord blood (n=18), 0-6 months (n=9), 6-12 months (n=13), 1-2 yr (n=19), 2-5 yr (n=27), 5-10 yr (n=25) and 10-16 yr (n=37). The absolute and relative percentage of lymphocytes, T, B, natural killer cell, along with activated, naïve and memory subsets, was determined by flow cytometry.

Results: Median values and the 10^th and 90^th percentiles were obtained for 34 lymphocyte sub-populations. The T and B naïve compartments showed a decreasing trend, whereas memory cells showed an increase with age. The activated T cell subset shows an increasing pattern up to one year and then declines gradually. Double negative T cells are relatively stable. TCRgd+T cell percentage increases with age.

Interpretation & conclusions: This single-centre pilot study provides preliminary data that justifies the need for future large-scale multi centric studies to generate a reference range for interpreting extended immunophenotyping profiles in the paediatric age group, making it possible for clinicians to assess the immunological status in inborn errors of immunity, infectious and autoimmune diseases.

Keywords: Children; flow cytometry; lymphocyte subset; pilot study; primary immunodeficiency.

Fig. 1

Stepwise gating strategy for lymphocytes, general lymphocyte subpopulation, T, B, NK and activated cell subpopulations. (A) FSC-H vs. FSC-A plot was used to remove doublets and cell debris. (B) Singlet cells were further plotted against SSC-A vs. FSC-A. (C) SSC-A vs. CD45 was used to gate lymphocytes, based on low forward/side scatter and bright CD45. (D) Density plot demonstrating NK cells (CD3- CD16/CD56+) and NKT cells (CD3+ CD16/CD56+). (E) T cells were gated using CD45 and CD3. (F-J) T cell populations were distinguished depending on CD4, CD8, CD27, CD45RA and TCRgd expression. (K-N) B cells were gated as CD19+CD3- and the subpopulations were distinguished depending on IgD, IgM and CD27 expression. (O-S) HLA-DR was used to access the activation status on DNTs, T, Tc, Th, NK cells respectively. The names written above the plot [name] represent the parent gate. NK, natural killer

Fig. 2

Gating strategy for T cell and its subpopulation like Naïve, CM, EM, Terminally differentiated effector memory re-expressing CD45RA+ (TEMRA) and RTE cells. (A) FSC-H vs. FSC-A plot was used to remove doublets cell debris. (B) Singlet cells were further plotted against SSC-A vs FSC-A. (C) SSC-A vs. CD45 was used to gate lymphocytes, based on low forward /side scatter and bright CD45. (D) SSC-A vs. CD3 was used to identify T cells. (E) T cells were characterized as T helper cells/cytotoxic T cells based on the expression of CD4 and CD8. (F and I) Quadrant density plot representing naïve, central, effector memory and TEMRA sub population using CD62L and CD45RA on Th and Tc cells. (G, J, H and K) Recent thymic emigrants (RTE) were identified based on co-expression of CD31 and CD45RA and or CD62L on both Th and Tc cells. The names written above the plot (name) represent the parent gate. CM, Central memory; EM, effector memory; RTE, recent thymic emigrant

Fig. 3

Age-related ranges for absolute lymphocyte subset counts. Flow cytometric analysis of 148 healthy individuals amongst seven different age groups. All values of this reference dataset are represented as box plots with median, p10, p25, p75 and p90 percentile. For the data visualization package ggplot2 for the statistical language, R was used. Cb, cord blood; yr, years, m, month

Fig. 4

Age-related ranges for the percentage of lymphocyte subset counts. Flow cytometric analysis of 148 healthy individuals amongst seven different age groups. All values of this reference dataset are represented as box plots with median, p10, p25, p75, and p90 percentile. For the data visualization package ggplot2 for the statistical language, R was used. Cb, cord blood; yr, years; m, month.