Inborn errors of immunity (IEIs), which include and expand beyond primary immunodeficiencies (PIDs), represent a heterogeneous group of primary immune system defects that encompass a comprehensive range of approximately 485 distinct clinical disorders [1]. For certain immunodeficiency disorders classified as IEIs, particularly severe combined PIDs, ascertaining decreased lymphocyte subpopulations as a part of the diagnostic procedure is essential. Immunophenotypic abnormalities range from the total absence of a specific cell population to subtle variations in the differentiated states of specific subpopulations [2]. Establishing reference intervals for each well-defined subset of peripheral blood lymphocytes in healthy children and adolescents across age groups is essential, as individuals can be diagnosed as having IEIs at various ages. This necessity is especially crucial for subsets that serve as reliable indicators of the maturation and activation status [3].

Reference intervals have been established for major lymphocyte subsets, including helper T cells, cytotoxic T cells, B cells, and natural killer (NK) cells [4–13]. More recent data provided reference intervals for extended subgroups of lymphocytes [3, 14, 15]. However, because most of these studies were conducted abroad and most manufacturers are based in Europe or the United States, manufacturer-provided immunological reference intervals are commonly adopted based on those populations. Given the potential inadequacy of these intervals in accounting for racial and ethnic differences, establishing a reference interval specific to a Korean population is imperative. However, relatively few studies have been conducted to investigate the reference values of lymphocyte subsets in Korean children [16, 17], and notably, no study has provided references for extended lymphocyte subgroups.

We used the Primary Immunodeficiency Orientation Tube (PIDOT) panel (Cytognos SL, Salamanca, Spain), which was developed as part of the EuroFlow Consortium. The PIDOT panel was designed for comprehensive immunophenotypic screening and classifying PIDs of the lymphoid system. The PIDOT panel consists of 12 antibodies distinguished by eight fluorophores and enables differentiation of the following T cell and B cell subtypes: pre-germinal center B cells (preGC cells; including immature/transitional and naïve B cells); post-germinal center B cells (postGC cells; including unswitched B cells [IgM⁺IgD⁺, IgM⁺IgD^–, and IgM^–IgD⁺ cells]); Ig-switched memory B cells (MBCs); plasma cells (PCs); and T cells based on their maturation level, including naïve, central memory/transitional memory (CM/TM), effector memory (EM), and terminally differentiated (TD) T cells [18].

To mitigate potential bias, samples were procured from three geographically diverse hospitals in Korea to ascertain the lymphocyte subpopulations. We determined the relative and absolute counts of lymphocyte subpopulations according to age and sex in Korean children aged 5–17 yrs without immunological or hematological disorders.

Extended lymphocyte subsets were analyzed for 92 participants; Table 1 summarizes their distributions based on age and sex. Scatter plots illustrating age-related changes in lymphocyte subsets, showing the absolute cell counts (/μL) and percentages (%) for each participant, are presented in Fig. 1.

As expected, Group 1 (5–9 yrs) exhibited higher percentages of lymphocytes than did Group 2 (10–17 yrs). Among the distinct subpopulations of T cells studied, the absolute counts of naïve and memory CD4⁺ T cells (CM/TM and EM) and TCRγδ⁺ T cells demonstrated differences between the age groups (Tables 2 and 3). Group 1 had higher absolute counts of total T cells and TCRγδ⁺ T cells but lower counts of CD4⁺ T cells (CM/TM and EM) than did Group 2 (Table 2). These differences were also reflected in terms of percentages (Table 3). Among the B cell subpopulations, the absolute counts of total B cells and unswitched MBCs/PCs were significantly higher in Group 1 than in Group 2; however, no significant differences were found in their overall proportions (Tables 2 and 3). Conversely, the absolute count and proportion of NK cells increased in Group 2; however, these differences were not statistically significant.

Within Group 1, no significant sex-based differences in absolute lymphocyte counts were observed, except for the absolute counts of total lymphocytes and TD CD8⁺CD27⁻ cells. In Group 2, the CD4⁺ EM and CD8⁺ TD T cell counts were higher in males than in females. No significant differences were observed in the absolute count of other T cell subpopulations based on sex (Table 2, Fig. 2). However, the percentages of CD4⁺ and CD4⁺ naïve T cells were higher in females, and the percentages of total B cells were higher in males (Table 3, Fig. 2). The total B cells, particularly, preGC B cells, exhibited higher absolute counts and percentages in males in Group 2 (Fig. 2).

This study represents the first multicenter investigation in Korea to establish reference data for extended lymphocyte subpopulations in pediatric populations. Although several investigators have proposed reference values for lymphocyte subsets (including extended subpopulations) over the last few decades, control studies focusing on children remain limited [3, 20, 21], particularly among Asian populations [22]. Research on the reference values for lymphocyte subsets is limited [23], with only two studies focusing on pediatric populations [16, 17].

Lymphocyte counts demonstrate a gradual exponential decline as individuals transition from childhood to adulthood after the first year of life. In our study, the lymphocyte counts in Group 2 were lower than those in Group 1. The results of a previous study conducted in Korea showed a similar age-related decline in lymphocyte counts [24]. This phenomenon can be attributed to a combination of various interconnected factors. These factors include gradual involution of the thymus, antigen exposure, transition from a naïve to a memory immune response associated with immunological learning, alterations in body size and blood volume because of growth, and gradual replacement of primary thymic output by peripheral cell division with aging [25].

T cell subsets that differ in functions and phenotypes are distinguished by a combination of different cell surface proteins. Naïve T cells that reach maturity express three specific markers: CD45RA, CCR7, and CD28. When exposed to cognate antigens, cells transform from thymic precursors into effector cells; a fraction of these cells subsequently differentiate into subsets of memory T cells [26]. The central memory pool, which can exhibit sustained long-lived T cell memory in a stem cell–like manner, constituted most memory cell subsets in all age groups [14]. In this study, CM cells constituted the majority of the memory cells. In the older group, we observed a notable decline in the naïve T cell population and a corresponding increase in the memory T cell population. The CD4⁺ naïve cell:memory cell ratio was higher in Group 1 than in Group 2; nevertheless, the CD8⁺ naïve cell:memory cell ratio did not demonstrate statistical significance, perhaps because of a limited sample size. The current findings are consistent with those of an earlier study [14], indicating that the overall lymphocyte count declines with advancing age, suggesting that the naïve cell population declines steadily despite a tendency toward expansion of the memory cell population, particularly EM cells.

Although γδT cells constitute a minor population of T cells, they play crucial roles in innate and adaptive immunity. The article by Ding et al. [14] included 1,075 Chinese children, and their results indicated that the relative size of the γδT population gradually increased with age and peaked at 8–12 yrs of age. However, in this study, the absolute cell counts and relative proportion of the TCRγδ⁺ population tended to be lower in the older group than in the younger group. These findings underscore the importance of establishing population-specific reference ranges.

The absolute total and naïve B cell counts were high during the first year of life and gradually decreased thereafter, reaching a plateau with increasing age in children [3, 14]. The significantly higher absolute counts of preGC B cells and unswitched MBCs/PCs observed in Group 1 than in Group 2 reflect the physiological expansion and high output of B cells during early childhood. These findings correspond to ongoing bone marrow maturation and robust primary immune responses during early antigen encounters. Conversely, the relative decline of these populations in older children aligns with a shift from primary to memory-driven adaptive immunity.

We observed no significant changes in the absolute cell counts and proportions of NK cells across both groups. Previous studies yielded inconsistent findings regarding a steady decrease in NK cell counts beyond the age of 5 yrs [3, 14, 27]. These variations may reflect differences in the characteristics of the pediatric populations recruited for the different studies.

We observed higher B cell and CD4⁺ EM and CD8⁺ TD T cell counts in males than in females. Some subsets showed statistical differences between males and females, indicating that sex-related factors might influence lymphocyte development and differentiation [14].

We also observed inter-laboratory variations in the estimations of most T and B cell subpopulations despite standardized protocols and personnel-training procedures, including harmonized testing workflows agreed upon by all three hospitals and same-day sample processing following the manufacturer’s instructions (Supplemental Data Table S2 and S3). Considering the relatively wide variability among the flow cytometry results obtained within and between laboratories, such differences may be inherent and influenced by unavoidable technical and operational factors [28]. In addition to technical variables, such as different types of flow cytometers and different personnel, differences in postnatal exposure to antigens and geographical disparities in pathogen prevalence rates might also have led to variable results [29].

The reference values defined in the EuroFlow PIDOT panel in healthy controls from each age group in previous research [19] and in this study are shown in Fig. 3. Although a direct statistical comparison was not performed, the reference ranges in our cohort were descriptively compared with those reported previously to enable contextual interpretation. The distributions of values for each subset and subpopulation were generally similar to those reported in the previous study or tended to be narrower, except for preGC B cells in Group 2.

This study has some limitations. Although reference intervals are ideally established using samples from healthy community-based individuals, we utilized remnant samples from hospital-based pediatric patients undergoing routine blood testing and had no suspected immunological, hematological, or infectious conditions. To minimize confounding, individuals with suspected immunological, hematological, or infectious conditions, as well as those with autoimmune diseases, recent transfusion, or ongoing immunotherapy, were rigorously excluded based on clinical information. Nonetheless, the possibility of undiagnosed subclinical conditions cannot be entirely excluded. Similar hospital-based strategies were used in previous studies on pediatric lymphocyte reference values, such as those conducted by Tosato et al. [20] in Italy and Jodhawat et al. in India [30], which also involved strict exclusion criteria to approximate a healthy cohort. This approach facilitates practical data collection; however, future studies involving community-derived cohorts may be needed to further validate and generalize these reference ranges.

The second limitation is the relatively small sample size involved, particularly the lack of participants under 5 yrs of age. This age group can exhibit the most dynamic changes in lymphocyte subsets. However, we were unable to recruit suitable participants because of clinical and ethical constraints. In infants and toddlers, the limited volume of blood permissible for clinical testing introduced difficulties in obtaining sufficient residual samples for research use. Nevertheless, the overall sample size in this study was larger than that employed in a previous study using the PIDOT panel [19]. Further studies with larger sample sizes, specifically those including children under 5 yrs of age, are warranted to establish more comprehensive and age-specific reference values for lymphocyte subpopulations.

The third limitation is that we used a manual gating strategy instead of the manufacturer-recommended Automated Gating & Identification (AG&I) module. Lymphocyte subset analysis was conducted using manual gating with Kaluza software. Although this method enables expert-guided flexibility and is widely used in clinical practice, it may introduce inter-operator variability because of its subjective nature. The manufacturer of the EuroFlow PIDOT panel recommends using Infinicyt software (Cytognos SL) with the AG&I module for more standardized and reproducible analyses using a reference-based algorithm. However, the Infinicyt software was unavailable at all participating centers in this multicenter study, primarily due to software licensing and training constraints. Although the AG&I module offers distinct advantages in harmonization and automation, the results of previous studies, including one by Linskens et al. [31], have demonstrated a high level of concordance between manual gating and AG&I in identifying lymphocyte subpopulations, supporting the validity of manual gating under controlled conditions.

In conclusion, to the best of our knowledge, this multicenter study is the first to establish reference values for extended lymphocyte subpopulations in Korean children using the EuroFlow PIDOT panel. Significant age- and sex-related differences were identified across several lymphocyte subsets. Despite the use of standardized protocols and training, inter-laboratory variability was observed, emphasizing the need for harmonized methodological approaches. These findings highlight the importance of establishing population-specific reference ranges that account for both technical and biological variability in clinical immunophenotyping. The reference values established in this study may serve as a valuable resource for diagnosing immune disorders and monitoring immunotherapy in pediatric patients.