Reference Range for B-Cell Subpopulations in Peripheral Blood of Healthy Malaysian Children Aged 2 to 15 years.

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Intan Hakimah Ismail, Jalilah Jamaluddin, Mohd Azri Zainal Abidin, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6867752/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Physicians rely on reference values from healthy populations to guide clinical decisions regarding B-cell subpopulations in primary immunodeficiency. While age-dependent reference ranges have been reported in several populations, no study has established these values for Malaysian children. Given that B-cell subpopulation distributions may vary between populations, we aimed to define reference ranges for total B cells, transitional B cells, naïve B cells, total memory B cells, switched and non-switched memory B cells, and plasmablasts in Malaysian children aged 2 to 15 years. Blood samples were taken from 85 children aged 2 to 15 years evaluated for the distribution of B cell subsets. Absolute numbers and percentages were determined for total B cells (CD19 + ), transitional B cells (CD19 + CD27 - CD24 +bright CD38 +bright ), naïve B cells (CD19 + CD27 - ), total memory B cells (CD19 + CD27 + ), switched memory B cells (CD19 + IgM - IgD - CD27 + CD38 +dim ), non-switched memory B cells (CD19 + IgM + IGD + CD27 + CD38 +dim ), and plasmablasts (CD19 + IgM - IgD - CD27 + CD38 +bright ). We observed age-dependent variations in most B-cell subpopulations, with naïve B cells being predominant, followed by memory B cells, while plasmablasts were present in trace amounts across all ages. Additionally, most B-cell subpopulations were observed at higher frequencies in female children compared to males. This study provides age-specific reference values for B cell subsets in a paediatric population, which may serve as a valuable guideline for diagnosing children with suspected immunodeficiency. Biological sciences/Immunology/Adaptive immunity/Humoral immunity/Immunological memory Biological sciences/Immunology/Adaptive immunity/Humoral immunity humoral immunity B cells switched-memory B cells age-specific reference range Figures Figure 1 Introduction B cells are essential components of the immune system, playing a crucial role in adaptive immune immunity. The development of B cells involves a complex maturation process that begins with hematopoietic stem cells (HSCs) in the bone marrow, progressing through various stages from immature B cells to transitional B cells and finally into mature naïve B cells ( 1 , 2 ). Immature B cells exit the bone marrow and enter the peripheral circulation as transitional B cells (CD19 + CD38 ++ CD24 ++ or CD19 + CD27 − IgD + CD38 + ), which further mature into naive B cells (CD19 + IgD + CD27 − ). Upon antigen recognition, naïve B cells proliferate and differentiate into short-lived plasmablasts (CD19 + CD138 ++ or CD19 + CD27 + CD38 ++ ), plasma cells (CD38 + CD138 + ) or memory B cells (CD19 + CD27 + ) within germinal centre ( 3 ). Memory B cells provide long-term protective immunity and can be further subclassified into switched memory B cells (CD19⁺CD27⁺IgM⁻IgD⁻), non-switched memory B cells, also known as marginal zone B cells (CD19⁺CD27⁺IgM⁺IgD⁺), IgM-only memory B cells (CD19⁺CD27⁺IgM⁺IgD⁻) and IgD-only memory B cells (CD19⁺CD27⁺IgM⁻IgD⁺) ( 3 – 6 ). Abnormal B cell distribution has been linked to primary immunodeficiencies, recently termed inborn errors of immunity. The International Union of Immunological Societies (IUIS) has recognized reduced number of memory B cells as a hallmark of combined immunodeficiency, hyper IgE syndrome, and activated PI3K-delta syndrome (APDS). Additionally, decreased plasmablast levels have been used to assess immune dysregulation, particularly in diagnosing NFAT5 haploinsufficiency. Transitional B cells are also measured in patients suspected of having common variable immunodeficiency or combined immunodeficiency. Although B cell subpopulation analysis is crucial for investigating primary immunodeficiencies (PID), reference ranges for B cell distribution remain limited, making comparison with patient populations challenging ( 7 ). Establishing reference range for B cell subpopulations is crucial for guiding physicians in diagnosing young patients with suspected PID. However, these reference values may vary across laboratories due to differences in population demographics, geographical areas, selection criteria for healthy subjects, and sample preparation techniques, all of which contribute to the wide range of normal values reported ( 8 – 10 ). These variations can introduce biases, especially in immunodeficiency assessments. Therefore, this study quantified and analysed B-cell subpopulations in children aged 2 years to 15 years to establish age-dependent reference intervals for humoral immune parameters, assess age-related variations in B-cell subpopulations, and explore differences between genders. To the best of our knowledge, this is the first study to define the normal distribution of B-cell subpopulations in peripheral blood in Malaysia, specifically at our centre, the Clinical Immunology Centre, Universiti Putra Malaysia. Materials and methods Study population of the healthy controls A total of 85 healthy individuals aged 2 to 15 years were voluntarily enrolled in this study between November 2022 and December 2023. Participants were referred to the paediatric clinic at Hospital Sultan Abdul Aziz Shah, Universiti Putra Malaysia, for developmental assessments. Children with immune disorders, chronic diseases, syndromic conditions confirmed by geneticists, those receiving intravenous immunoglobulin treatment, or those on immunosuppressive medications were excluded. Sample preparation and data collection. Blood samples were collected in ethylenediamine tetra-acetic acid (EDTA) tubes and processed within 24 hours. Flow cytometric analysis was performed using FACSLyrics (BD Biosciences, New Jersey, United States) with six-colour immunostaining for the following markers and fluorochromes: CD19-PerCPCy5.5, CD27-APC, CD38-PE-Cy7, CD24-APC-H7, IgD-PE, and IgM-BB515. Data generated were analysed using FlowJo software (BD Biosciences, New Jersey, United States). Briefly, whole blood was washed twice with stain buffer, incubated with immunofluorescence staining, and treated with FACSLysing solution (BD Biosciences, New Jersey, United States). The gating strategies are described in Fig. 1 . The absolute number of cells was calculated by multiplying the relative proportion (percentage) of B-cell subpopulations by the absolute number of B cells obtained from T cell, B cell and natural killer cell (TBNK) enumeration or the lymphocyte count from a complete blood count (CBC) performed on the same day. We then identified each B-cell subsets based on the following markers: total B cells (CD19 + ), transitional B cells (CD19 + CD27 − CD24 + bright CD38 +bright ), naïve B cells (CD19 + CD27 − ), total memory B cells (CD19 + CD27 + ), switched memory B cells (CD19 + IgM − IgD − CD27 + CD38 + dim ), non-switched memory B cells (CD19 + IgM + IGD + CD27 + CD38 + dim ), and plasmablast (CD19 + IgM − IgD − CD27 + CD38 + bright ). Statistical analysis Data were analysed using SPSS version 27 and Microsoft Excel®. Reference values were established for three age groups ranging from 2 to 15 years. These reference values were determined based on the 5th and 95th percentiles, representing the lower and upper limits, respectively. Medians and interquartile ranges were calculated for each age group. For statistical comparisons, Mann-Whitney test was used to analyse differences in B-cell subpopulations between gender, Kruskal Wallis test was performed to assess differences in B-cell subpopulations among age groups, while Spearman’s rank correlation test was used to evaluate age-dependent changes in B cell-subpopulations. A p-value < 0.05 was considered statistically significant. Results Initially, 82 healthy children aged 2 to 15 years old were recruited; however, seven were excluded as outliers based on Tukey’s and Mahalanobis tests. B-cell subpopulations were analysed in three age groups: 2 to 4 years (n = 29), 5 to 9 years (n = 30), and 10 to 15 years (n = 16). Demographic details are provided in Table 1 . All subjects had normal absolute lymphocytes counts (TBNK enumeration) based on established reference values ( 11 ) (Supplementary Data). Table 1 Demographic details for the 75 healthy children tested according to the age group. Age group Median age Total number of subjects Gender (Male/Female) 2 to 4 years old 4.081 29 15/14 5 to 9 years old 7.973 30 14/16 10 to 15 years old 11.682 16 10/6 Total 6.553 75 75 The composition of B-cell subpopulations exhibited age-related variations. The total B cell frequency showed a decreasing pattern across age groups, with a significant reduction observed between the 2- to 4-year-old group and the 5- to 9-year-old group, where the median decreased from 22.9–19.0% (p = 0.013) and between 2- to 4-year-old group and the 10- to 15-year-old group from a median of 22.9–18.3% (p = 0.045). In contrast, transitional B cells, total memory B cells, non-switched memory B cells, class-switched memory B cells, and plasmablast cells displayed an initial increase from the 2- to 4-year-old group to the 5- to 9-year-old group, followed by a decline in the 10- to 15-year-old group. A significant increase was observed in the total memory B cell frequency (p = 0.021) when comparing the 2- to 4-year-old group with the 5- to 9-year-old group and in plasmablast between 5- to 9-year-old group and 10- to 15-year-old group (p = 0.046). Meanwhile, the frequency of naïve B cells declined between 2 to 4 years old and 5 to 9 years old, but a slight increase was noted in the 10- to 15-year-old group. A statistically significant reduction in naïve B cell frequency was found between the 2- to 4-year-old group and the 5- to 9-year-old group, where the median decreased from 79.0–72.1% (p = 0.017). However, no significant differences in frequency were detected across age groups for non-switched memory B cells, class-switched memory B cells or transitional B cells. The absolute count of each B-cell subpopulation also showed age-related changes. The absolute number of total B cells was declined with age, with significant differences observed between the 2- to 4-year-old group and the 5- to 9-year-old group (p = < 0.001), as well as between the 2- to 4-year-old group and the 10- to 15-year-old group (p = < 0.001). A similar pattern was identified in the transitional B cells, naïve B cell, total memory B cells, and non-switched memory B cells. The absolute number of transitional B cells showed a significant decline between the 2- to 4-year-old group and the 10- to 15-year-old group (p = 0.025). The non-switched memory B cell absolute count also demonstrated a significant decrease between 2- to 4-year-old and 5- to 9-year-old groups (p < 0.001) and between the 2- to 4-year-old and 10- to 15-year-old groups (p < 0.001). The absolute number of total memory B cells was significantly lower in the 10- to 15-year-old age group compared to the 2- to 4-year-old group (p < 0.001). In the case of naïve B cells, a reduction in absolute number was observed followed by an increase in the 10- to 15-year-old group. A statistically significant difference was detected between the 2- to 4-year-old group and the 10- to 15-year-old group (p < 0.001) as well as between the 2- to 4-year-old group and the 5- to 9-year-old group (p < 0.001). Meanwhile, both class-switched memory B cells and plasmablasts exhibited a slight increase in absolute number from 2 to 4 years old to 5 to 9 years old before declining in the 10- to 15-year-old group, although these changes were not statistically significant. The median values and interquartile ranges (5th and 95th percentiles) for the frequencies and absolute numbers of B cell subpopulations across age groups are provided in Table 2 and Table 3 . Table 2 Frequencies for each B cell subpopulations in distinct age groups. Age group Total B cells Transitional Naïve B Total memory B Non-switched memory B Switched memory Plasmablast 2 to 4 years 22.900 (15.04–31.38) 6.190 (0.52–16.50) 79.000 (57.75–89.70) 17.800 (8.41–31.55) 8.060 (3.92–13.55) 5.820 (1.93–12.25) 1.070 (0.10–8.16) 5 to 9 years 18.985 (11.32–31.15) 8.450 (0.79–26.77) 72.050 (51.69–84.96) 22.900 (12.64–42.10) 8.640 (3.33–14.73) 6.715 (3.64–16.19) 1.840 (0.29–8.63) 10 to 15 years 18.280 (10.06–31.37) 7.060 (0.67–17.80) 76.450 (57.10–85.60) 20.350 (12.20–35.90) 5.875 (2.30–12.60) 6.350 (3.38–16.10) 0.665 (0.22–6.56) All results are presented as median values with ranges between 5th and 95th percentile. Table 3 Absolute number for each B cell subpopulations in distinct age groups. Age group Total B cells Transitional Naïve B Total memory B Non-switched memory B Switched memory Plasmablast 2 to 4 years 1314.000 (615.28–2387.50) 60.246 (4.14–257.62) 932.881 (462.45–2015.03) 211.554 (91.11–486.29) 80.738 (27.67–160.16) 7.294 (1.25–44.88) 1.795 (0.08–16.31) 5 to 9 years 616.500 (332.87–1329.50) 38.692 (2.69–144.87) 429.540 (196.23–1042.60) 154.901 (65.14–364.03) 44.112 (17.35–120.32) 7.870 (1.54–30.02) 1.931 (0.19–11.64) 10 to 15 years 511.000 (226.00–1130.00) 26.011 (2.13–101.48) 421.700 (154.13–894.96) 98.481 (54.40–258.48) 25.417 (9.92–70.06) 4.080 (1.41–32.34) 0.665 (0.09–4.13) All results are presented as median values with ranges between 5th and 95th percentile Further analysis using Spearman's rank-order correlation revealed a strong correlation between age and the absolute counts of total B cells, naïve B cells, and non-switched memory B cells, with all three subsets demonstrating a statistically significant association (p < 0.001). Additionally, the Mann-Whitney U test was used to examine gender-related differences in B-cell subpopulations. The results indicated that female children had significantly higher B cells frequency (p = 0.047), total memory B cells frequency (p = 0.016), total memory B cells absolute number (p = 0.026), class-switched memory B frequency (p = 0.029), class-switched memory B cells absolute number (p = 0.005), and plasmablast absolute number (p = 0.018) compared to male children. Conversely, male children had a significantly higher naïve B cells frequency than females (p = 0.019). However, no significant gender differences were observed in total B cells absolute number (p = 0.603), transitional B cells frequency (p = 0.668), transitional B cells absolute number (p = 0.596), naïve B cells absolute number (p = 0.882), non-switched memory B cells frequency (p = 0.131), non-switched memory B cells absolute number (p = 0.581), and plasmablast frequency (p = 0.092). Discussion Various studies have been conducted to validate the age-dependent distribution of B-cell subpopulations, especially among paediatric population ( 12 – 17 ). Each B cell subpopulation represents a distinct stage of the B cell development, with full activation occurring upon antigen contact. A decrease in in the absolute number or frequency of specific B cell subpopulations may raise concerns about underlying immunological conditions, including primary immunodeficiency ( 12 , 18 – 20 ). While reference values for B cell subsets have been established in several populations, no such values have been documented for Malaysia. To the best of our knowledge, this study is the first to establish reference values for paediatric B-cell subpopulations in Malaysian children below 15 years of age, specifically within the Clinical Immunology Centre at Hospital Sultan Abdul Aziz Shah, Universiti Putra Malaysia, Selangor. In this study, we defined normal values for total B cells, transitional B cells, naïve B cells, total memory B cells, switched memory B cells, non-switched memory B cells, and plasmablasts in 75 healthy children grouped into three age groups ranging from 2 to 15 years. Although the physiological of each B cell subset is not entirely elucidated, these subsets can be identified based on the significant surface marker CD19. As expected, a notable reduction in total b cells, marked by CD19 positivity (both in frequency and absolute number) was observed during childhood. This trend has been previously reported and is known to continue as age increases ( 12 ). Prior research has highlighted that ageing plays a key role in this decline, as bone marrow output capacity diminishes over time. Additionally, early B cell populations predominantly appear as transitional B cells in peripheral circulation before shifting into naïve B cells ( 12 , 21 – 23 ). The observed reduction in both transitional and naïve B cells in this study mirrors the overall decline in total B cells. Importantly, the absolute number of total memory B cells and plasmablasts remained relatively stable over time (Table 2 and Table 3 ). Taken together, these findings align with the previous research, suggesting that the reduction of total B cells may be attributed to declines in transitional and naïve B cell subpopulations ( 12 ). Interestingly, severe reduction or complete absence of total B cells is commonly observed in patients with x-linked agammaglobulinemia (XLA). This is primarily caused by the genetic mutation in Bruton’s tyrosine kinase (BTK), which responsible in mediating B cell development and maturation. The mutation in the BTK gene disrupt the progression from pro-B cells to pre-B cells, preventing a proper development of B cells and halting the hematopoietic stem cells to enter B cell lineage. Thus, functional and antigen-responsive cells are unable to be generated ( 7 , 24 – 26 ). A continuous decline in the absolute number of non-switched memory B cells was also observed in this study, whereas the frequency and absolute count of switched memory B cells remained relatively stable across age groups. These findings are consistent with those reported by Duchamp et al. and Morbach et al. ( 12 , 27 ). A decrease in switched memory B cells below the reference range has been suggested as a potential indicator for evaluating patients with suspected primary immunodeficiencies. Indeed, switched memory B cells have incorporated into diagnostic criteria for common variable immunodeficiency (CVID) in the Freiburg, Paris and European classifications ( 18 , 28 – 30 ). However, the cut-off values proposed by these classifications vary and are based on adult populations. This underscores the necessity of establishing age- and population-specific reference values for distinct B-cell subpopulations. Additionally, patient with either CD40 ligand or CD40 deficiency exhibit reduced memory B cells. Mutations in the CD40L and CD40 genes disrupt the CD40 activation pathway, resulting defects in both class switch recombination (CSR) and somatic hypermutation. These defects categorized under combined T- and B-cell immunodeficiencies ( 7 , 25 , 31 ). Plasmablasts, characterized as CD24 − CD38 ++ B cells, are typically present in trace amounts within peripheral blood. Some researchers have stated that plasmablasts are rarely exceed 5% of total B cells ( 12 ). In our study, both the proportion and absolute number of plasmablasts were less than 2% and 2 X 10 6 /L, respectively. Notably, plasmablast remained consistent across all age groups. It is crucial to recognize that the expansion of plasmablasts in the peripheral blood may indicate autoimmune conditions, such as systematic lupus erythematous, autoimmune rheumatic diseases, Sjögren's disease, and rheumatoid arthritis ( 32 – 36 ). Therefore, establishing population-specific reference range could help define precise cut-off values for detecting autoimmune diseases. Interestingly, our study revealed gender-based differences in B-cell subpopulation distribution. Female children had a significantly higher frequency of total memory B cells, non-switched memory B, and class-switched memory B cells, as well as greater absolute number of class-switched memory B cells compared to their male counterparts. Conversely, naïve B cells were found at a higher frequency in male children. This suggests that, in addition to age-related factors, gender also influences B cell subset distribution and immune responses. Two potential explanations for this phenomenon include the influence of sex hormones, such as oestrogen and testosterone, and the role of X chromosome in regulating immune-related genes ( 37 , 38 ). Previous research has reported that oestrogen stimulates B cells proliferation, whereas testosterone hormone delays this process ( 37 , 39 ). Additionally, the X chromosome plays an important role in immune function, as it contains coding regions for genes involved in immune responses, including toll-like receptors, cytokine receptors, and transcription factors ( 37 , 40 ). Consequently, gender should be considered when assessing young patients in immunological evaluations. Despite the strengths of this study, several limitations should be acknowledged. The main limitation is the small sample size. This small sample size can be reflected in the statistical power, potentially compromising both the sensitivity and the specificity of the analysis. Primarily, it may cause difficulty in replicating results and increase the likelihood of false negative results, the type Ⅱ error ( 41 , 42 ). According to the Clinical and Laboratory Standards Institute (CLSI), a minimum sample size of 120 subjects is required to establish reference intervals ( 43 ). For studies involving multiple variables, such as in this study, at least 120 subjects per variable would be ideal. Although we were only able to recruit a small cohort of 75 participants, this study serves as an initial platform for determining reference values within our laboratory. However, these findings may not be generalizable to the entire Malaysian population. Future studies with larger cohorts are necessary to establish more comprehensive population-specific reference values. Another limitation of this study is the restricted age range of participants. Since our focus was on children aged 2 years to 15 years, the reference values do not account for B cell subpopulation variations in early infancy. Previous research suggests that significant fluctuations in B cell distribution frequencies and absolute counts occur within the first five years of life. Expanding the age range in future studies would allow for more thorough characterization of B-cell distribution across different developmental stages within the Malaysian population. In conclusion, the characterization of B-cell subpopulations is crucial for evaluating primary immunodeficiencies. This study provides age-specific reference values for B cell subsets in a paediatric population, which may serve as a valuable guideline for diagnosing children with suspected immunodeficiency. Additionally, our findings highlight the potential impact of both age and gender on B cell distribution, emphasizing the need for further research in this area. Future studies should investigate whether variations in B cell subset differentiation and expansion are influenced by population-specific or environment factors. Declarations Written informed consent was obtained from all parents or legal guardians of the participants prior to inclusion in the study. Acknowledgements We would like to thank the Clinical Immunology Centre (CIC) team from Hospital Sultan Abdul Aziz Shah (HSAAS), Universiti Putra Malaysia for their excellent technical assistance. This study was funded by the Fundamental Research Grant Scheme (FRGS) from Ministry of Education Malaysia (04-01-18-1973FR, FRGS/1/2018/SKK02/UPM/02/1) and Grant Putra: Geran Inisiatif Putra Siswazah from Universiti Putra Malaysia (GP-IPS/2022/9739900). Author contribution I.H.I was responsible for the conception and design of the study and provided overall direction for the study; J.J performed, analyzed experiments, and prepared the manuscript; S. M.M., H.M and M.A.Z.A provided critical revision of the manuscript. All authors have read and approved the final manuscript. Conflict of Interest The authors declare that they have no competing interests. Ethics approval and consent to participate Ethical approval was obtained from the National Medical Research Register (NMRR-20-1741-53741) and the UPM Ethics Committee for research involving human subjects (JKEUPM-2023-337). Availability of supporting data All data generated during this study are included in this published article. Raw datasets are available from the corresponding author upon reasonable request. References Roy A, Bystry V, Bohn G, Goudevenou K, Reigl T, Papaioannou M, et al. High resolution IgH repertoire analysis reveals fetal liver as the likely origin of life-long, innate B lymphopoiesis in humans. Clinical Immunology. 2017;183:8–16. Jackson TR, Ling RE, Roy A. The Origin of B-cells: Human Fetal B Cell Development and Implications for the Pathogenesis of Childhood Acute Lymphoblastic Leukemia. Frontiers in Immunology. 2021;12(February):1–10. Eroglu FK, Aerts Kaya F, Cagdas D, Özgür TT, Yılmaz T, Tezcan İ, et al. B lymphocyte subsets and outcomes in patients with an initial diagnosis of transient hypogammaglobulinemia of infancy. Scandinavian Journal of Immunology. 2018;88(4):1–8. Iwajomo OH, Finn A, Ogunniyi AD, Williams NA, Heyderman RS. Impairment of pneumococcal antigen specific isotype-switched Igg memory B-cell immunity in HIV infected Malawian adults. PLoS One. 2013;8(11):1–7. Claes N, Fraussen J, Stinissen P, Hupperts R, Somers V. B cells are multifunctional players in multiple sclerosis pathogenesis: Insights from therapeutic interventions. Frontiers in Immunology. 2015;6(DEC). Klein U, Rajewsky K, Küppers R. Human immunoglobulin (Ig)M+IgD+ peripheral blood B cells expressing the CD27 cell surface antigen carry somatically mutated variable region genes: CD27 as a general marker for somatically mutated (memory) B cells. Journal of Experimental Medicine. 1998;188(9):1679–89. Tangye SG, Al-Herz W, Bousfiha A, Cunningham-Rundles C, Franco JL, Holland SM, et al. Human Inborn Errors of Immunity: 2022 Update on the Classification from the International Union of Immunological Societies Expert Committee [Internet]. Journal of Clinical Immunology. Springer US; 2022. 1473–1507 p. Available from: https://doi.org/10.1007/s10875-022-01289-3 Ministry of Health Malaysia. Guideline for Method Verification of Quantitative Measurement in Medical Laboratories. 2015. p. https://www.patologi.gov.my/assets/policy/merged.p. Yadav D. Reference Interval for Clinical Laboratory Test Parameters. Biochemistry & Analytical Biochemistry. 2015;04(04). Katayev A, Balciza C, Seccombe DW. Establishing reference intervals for clinical laboratory test results: Is there a better way? American Journal of Clinical Pathology. 2010;133(2):180–6. Comans-Bitter WM, De Groot R, Van den Beemd R, Neijens HJ, Hop WCJ, Groeneveld K, et al. Immunophenotyping of blood lymphocytes in childhood: Reference values for lymphocyte subpopulations. Journal of Pediatrics. 1997;130(3):388–93. Morbach H, Eichhorn EM, Liese JG, Girschick HJ. Reference values for B cell subpopulations from infancy to adulthood. Clin Exp Immunol. 2010;162(2):271–9. Pia̧tosa B, Wolska-Kuśnierz B, Pac M, Siewiera K, Gałkowska E, Bernatowska E. B cell subsets in healthy children: Reference values for evaluation of B cell maturation process in peripheral blood. Cytometry B Clin Cytom. 2010;78(6 B):372–81. Plassmeyer M, Ryherd M, Brown M, Gupta R, Lacbawan L, Alpan O. Normal B Cell Subpopulations in the Pediatric Age Groups and Adults. Journal of Allergy and Clinical Immunology. 2013;131(2):AB67. Blanco E, Pérez-Andrés M, Arriba-Méndez S, Contreras-Sanfeliciano T, Criado I, Pelak O, et al. Age-associated distribution of normal B-cell and plasma cell subsets in peripheral blood. Journal of Allergy and Clinical Immunology. 2018;141(6):2208-2219.e16. Berrón-Ruíz L, López-Herrera G, Ávalos-Martínez CE, Valenzuela-Ponce C, Ramírez-SanJuan E, Santoyo-Sánchez G, et al. Variations of B cell subpopulations in peripheral blood of healthy Mexican population according to age: Relevance for diagnosis of primary immunodeficiencies. Allergol Immunopathol (Madr). 2016;44(6):571–9. Azarsiz E, Karaca NE, Aksu G, Kutukculer N. Reference values for B-cell surface markers and co-receptors associated with primary immune deficiencies in healthy Turkish children. Int J Immunopathol Pharmacol. 2017;30(2):194–200. Wehr C, Kivioja T, Schmitt C, Ferry B, Witte T, Eren E, et al. The EUROclass trial: Defining subgroups in common variable immunodeficiency. Blood. 2008;111(1):77–85. Blanco E, Pérez-Andrés M, Arriba-Méndez S, Serrano C, Criado I, Del Pino-Molina L, et al. Defects in memory B-cell and plasma cell subsets expressing different immunoglobulin-subclasses in patients with CVID and immunoglobulin subclass deficiencies. Journal of Allergy and Clinical Immunology. 2019;144(3):809–24. Huck K, Feyen O, Ghosh S, Beltz K, Bellert S, Niehues T. Memory B-cells in healthy and antibody-deficient children. Clinical Immunology [Internet]. 2009;131(1):50–9. Available from: http://dx.doi.org/10.1016/j.clim.2008.11.008 Pieper K, Grimbacher B, Eibel H. B-cell biology and development. Journal of Allergy and Clinical Immunology [Internet]. 2013;131(4):959–71. Available from: http://dx.doi.org/10.1016/j.jaci.2013.01.046 McComb S, Thiriot A, Akache B, Krishnan L, Abstract FS. Introduction to the Immune Response. In: Immunoproteomics: Methods and Protocols. Humana, New York: Methods in Molecular Biology; 2019. p. 1–24. BD Biosciences. B-Cell Research Flow cytometry tools for the study of B-cell biology. Bd biosciences. 2021. Smith T, Roifman CM. Primary B-Cell Immunodeficiencies. Clinical Immunology: Principles and Practice. 2019;80(6):489-508.e1. McCusker C, Upton J, Warrington R. Primary immunodeficiency. Allergy, Asthma and Clinical Immunology [Internet]. 2018;14(s2):1–12. Available from: https://doi.org/10.1186/s13223-018-0290-5 Bonilla FA, Khan DA, Ballas ZK, Chinen J, Frank MM, Hsu JT, et al. Practice parameter for the diagnosis and management of primary immunodeficiency. Journal of Allergy and Clinical Immunology [Internet]. 2015;136(5):1186-1205.e78. Available from: http://dx.doi.org/10.1016/j.jaci.2015.04.049 Duchamp M, Sterlin D, Diabate A, Uring-Lambert B, Guerin-El Khourouj V, Le Mauff B, et al. B-cell subpopulations in children: National reference values. Immun Inflamm Dis. 2014;2(3):131–40. Bhattad S. B-cell Defects: A Clinical and Immunological Approach. Pediatr Infect Dis. 2020;2(1):36–8. Piqueras B, Lavenu-Bombled C, Galicier L, Bergeron-Van Der Cruyssen F, Mouthon L, Chevret S, et al. Common variable immunodeficiency patient classification based on impaired B cell memory differentiation correlates with clinical aspects. J Clin Immunol. 2003;23(5):385–400. Warnatz K, Denz A, Dräger R, Braun M, Groth C, Wolff-Vorbeck G, et al. Severe deficiency of switched memory B cells (CD27(+)IgM(-)IgD(-)) in subgroups of patients with common variable immunodeficiency: a new approach to classify a heterogeneous disease. 2002; Available from: https://www.semanticscholar.org/paper/8f698faf75832c2f4d3f3895dd0980fab46c4be3 Rezaei N, Aghamohammadi A, Notarangelo LD. Primary Immunodeficiency Diseases [Internet]. Second edi. Rezaei N, Aghamohammadi A, Notarangelo LD, editors. Vol. 1, Springer. Berlin, Heidelberg: Springer Berlin Heidelberg; 2017. 379 p. Available from: http://link.springer.com/10.1007/978-3-662-52909-6 Arroyo-Villa I, Bautista-Caro MB, Balsa A, Aguado-Acín P, Bonilla-Hernán MG, Plasencia C, et al. Constitutively altered frequencies of circulating follicullar helper T cell counterparts and their subsets in rheumatoid arthritis. Arthritis Res Ther. 2014;16(1):1–8. Szyszko EA, Brun JG, Skarstein K, Peck AB, Jonsson R, Brokstad KA. Phenotypic Diversity of Peripheral Blood Plasma Cells in Primary Sjögren’s Syndrome. Scand J Immunol. 2011;73(1):18–28. Steinmetz TD, Verstappen GM, Suurmond J, Kroese FGM. Targeting plasma cells in systemic autoimmune rheumatic diseases – Promises and pitfalls. Immunol Lett. 2023;260(May):44–57. Jacobi AM, Mei H, Hoyer BF, Mumtaz IM, Thiele K, Radbruch A, et al. HLA-DRhigh/CD27high plasmablasts indicate active disease in patients with systemic lupus erythematosus. Ann Rheum Dis. 2010;69(1):305–8. Dörner T, Lipsky PE. Correlation of circulating CD27high plasma cells and disease activity in systemic lupus erythematosus. Lupus. 2004;13(5):283–9. Bous M, Schmitt C, Hans MC, Weber R, Nourkami-Tutdibi N, Tenbruck S, et al. Sex Differences in the Frequencies of B and T Cell Subpopulations of Human Cord Blood. Int J Mol Sci. 2023;24(14). Libert C, Dejager L, Pinheiro I. The X chromosome in immune functions: When a chromosome makes the difference. Nat Rev Immunol. 2010;10(8):594–604. Bereshchenko O, Bruscoli S, Riccardi C. Glucocorticoids, sex hormones, and immunity. Front Immunol. 2018;9(JUN):1–10. Klein SL, Flanagan KL. Sex differences in immune responses. Nat Rev Immunol. 2016;16(10):626–38. Althubaiti A. Sample size determination: A practical guide for health researchers. J Gen Fam Med. 2023;24(2):72–8. Columb MO, Atkinson MS. Statistical analysis: sample size and power estimations. BJA Educ. 2016;16(5):159–61. CLSI. Defining , Establishing , and Verifying Reference Intervals in the Clinical Laboratory ; Approved Guideline — Third Edition. CLSI document C28-A3c. 2008;28(30):59. Additional Declarations (Not answered) Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6867752","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":470415495,"identity":"35eb005e-a72b-40d9-b612-61c56aa7ca2e","order_by":0,"name":"Intan Hakimah Ismail","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8ElEQVRIiWNgGAWjYBACNhDB2MBgwAZif4AIGhDQwozQwjiDGC0MMC0g7cw8xGjhk+4/+Llwh50xn9jhZ49t2+5EM7A3b5NgzDmM22Eyh5mlZ55JNmOTTjM3zm17ltvAc6xMgnEbHi0SyQzSvG3MNmzSCWbSuW2HcxskcswIaWH+zdtWD9SS/k3aEqRF/g1BLWxAWw4DHZZjJs0ItoWHoBYza96248ZALWWSPeee5bbxpBVbJG5Lx6lFfkbi49u8bdWG82enb5P4UXYnt5/98MYbH7dZ49SCDg5A0kMCQzMJWqCgjmgto2AUjIJRMOwBAGVWS4uLGhIsAAAAAElFTkSuQmCC","orcid":"","institution":"Universiti Putra Malaysia","correspondingAuthor":true,"prefix":"","firstName":"Intan","middleName":"Hakimah","lastName":"Ismail","suffix":""},{"id":470415496,"identity":"d2016bc2-a806-4155-b47f-962fbd24809f","order_by":1,"name":"Jalilah Jamaluddin","email":"","orcid":"https://orcid.org/0009-0000-9834-8459","institution":"Universiti Putra Malaysia","correspondingAuthor":false,"prefix":"","firstName":"Jalilah","middleName":"","lastName":"Jamaluddin","suffix":""},{"id":470415497,"identity":"4c1d5b74-a811-48e4-b881-ddaba87db995","order_by":2,"name":"Mohd Azri Zainal Abidin","email":"","orcid":"","institution":"Universiti Putra Malaysia","correspondingAuthor":false,"prefix":"","firstName":"Mohd","middleName":"Azri Zainal","lastName":"Abidin","suffix":""},{"id":470415498,"identity":"a02f92f3-96c6-4ee3-b198-b7d61437cc20","order_by":3,"name":"Siti Mardhiana Mohamad","email":"","orcid":"","institution":"Universiti Sains Malaysia","correspondingAuthor":false,"prefix":"","firstName":"Siti","middleName":"Mardhiana","lastName":"Mohamad","suffix":""},{"id":470415499,"identity":"3d2a5a20-b626-416e-97a6-686d3821fa24","order_by":4,"name":"Hasni Mahayidin","email":"","orcid":"","institution":"Universiti Putra Malaysia","correspondingAuthor":false,"prefix":"","firstName":"Hasni","middleName":"","lastName":"Mahayidin","suffix":""}],"badges":[],"createdAt":"2025-06-11 04:30:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6867752/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6867752/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":85071843,"identity":"9895245d-bb93-4132-95d8-76cc02279bb2","added_by":"auto","created_at":"2025-06-20 15:42:33","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":404546,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGating strategies used for the determination of B cell subpopulations\u003c/strong\u003e. The lymphocyte population was identified based on side-scatter (SSC) and forward-scatter (FSC) characteristics (G1). After gating on CD19\u003csup\u003e+\u003c/sup\u003e B cells (G2), the B cells were further analysed based on the expression of CD27 and IgD, CD27 and IgM or IgD and IgM. The following B cell subpopulations have been identified in this study: total memory B cells (CD27\u003csup\u003e+\u003c/sup\u003e, G3), naïve B cells (CD27\u003csup\u003e-\u003c/sup\u003eIgD\u003csup\u003e+\u003c/sup\u003e, G4), transitional B cells (CD24\u003csup\u003e+bright\u003c/sup\u003eCD38\u003csup\u003e+bright\u003c/sup\u003e, G6) after gating on CD27\u003csup\u003e+\u003c/sup\u003eIgM\u003csup\u003e-\u003c/sup\u003e (G5), non-switched memory B cells (CD27\u003csup\u003e+\u003c/sup\u003eCD38\u003csup\u003e-\u003c/sup\u003e, G8) after gating on IgD\u003csup\u003e+\u003c/sup\u003eIgM\u003csup\u003e+\u003c/sup\u003e (G7), switched memory B cells (CD27\u003csup\u003e+\u003c/sup\u003eCD38\u003csup\u003e+dim\u003c/sup\u003e, G10) and plasmablast (CD27\u003csup\u003e++\u003c/sup\u003eCD38\u003csup\u003e+bright\u003c/sup\u003e, G11) after gating on IgD\u003csup\u003e-\u003c/sup\u003eIgM\u003csup\u003e-\u003c/sup\u003e (G9).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6867752/v1/0400ed6e377baeac6dd9df96.png"},{"id":85071848,"identity":"c3434654-7a30-44ab-8119-0928f939decf","added_by":"auto","created_at":"2025-06-20 15:42:38","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1066292,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6867752/v1/795bd95e-8fb0-42df-bfab-cf7a8d54cea6.pdf"}],"financialInterests":"(Not answered)","formattedTitle":"Reference Range for B-Cell Subpopulations in Peripheral Blood of Healthy Malaysian Children Aged 2 to 15 years.","fulltext":[{"header":"Introduction","content":"\u003cp\u003eB cells are essential components of the immune system, playing a crucial role in adaptive immune immunity. The development of B cells involves a complex maturation process that begins with hematopoietic stem cells (HSCs) in the bone marrow, progressing through various stages from immature B cells to transitional B cells and finally into mature na\u0026iuml;ve B cells (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Immature B cells exit the bone marrow and enter the peripheral circulation as transitional B cells (CD19\u003csup\u003e+\u003c/sup\u003eCD38\u003csup\u003e++\u003c/sup\u003eCD24\u003csup\u003e++\u003c/sup\u003e or CD19\u003csup\u003e+\u003c/sup\u003eCD27\u003csup\u003e\u0026minus;\u003c/sup\u003eIgD\u003csup\u003e+\u003c/sup\u003eCD38\u003csup\u003e+\u003c/sup\u003e), which further mature into naive B cells (CD19\u003csup\u003e+\u003c/sup\u003eIgD\u003csup\u003e+\u003c/sup\u003eCD27\u003csup\u003e\u0026minus;\u003c/sup\u003e). Upon antigen recognition, na\u0026iuml;ve B cells proliferate and differentiate into short-lived plasmablasts (CD19\u003csup\u003e+\u003c/sup\u003eCD138\u003csup\u003e++\u003c/sup\u003e or CD19\u003csup\u003e+\u003c/sup\u003eCD27\u003csup\u003e+\u003c/sup\u003eCD38\u003csup\u003e++\u003c/sup\u003e), plasma cells (CD38\u003csup\u003e+\u003c/sup\u003eCD138\u003csup\u003e+\u003c/sup\u003e) or memory B cells (CD19\u003csup\u003e+\u003c/sup\u003eCD27\u003csup\u003e+\u003c/sup\u003e) within germinal centre (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). Memory B cells provide long-term protective immunity and can be further subclassified into switched memory B cells (CD19⁺CD27⁺IgM⁻IgD⁻), non-switched memory B cells, also known as marginal zone B cells (CD19⁺CD27⁺IgM⁺IgD⁺), IgM-only memory B cells (CD19⁺CD27⁺IgM⁺IgD⁻) and IgD-only memory B cells (CD19⁺CD27⁺IgM⁻IgD⁺) (\u003cspan additionalcitationids=\"CR4 CR5\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAbnormal B cell distribution has been linked to primary immunodeficiencies, recently termed inborn errors of immunity. The International Union of Immunological Societies (IUIS) has recognized reduced number of memory B cells as a hallmark of combined immunodeficiency, hyper IgE syndrome, and activated PI3K-delta syndrome (APDS). Additionally, decreased plasmablast levels have been used to assess immune dysregulation, particularly in diagnosing NFAT5 haploinsufficiency. Transitional B cells are also measured in patients suspected of having common variable immunodeficiency or combined immunodeficiency. Although B cell subpopulation analysis is crucial for investigating primary immunodeficiencies (PID), reference ranges for B cell distribution remain limited, making comparison with patient populations challenging (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eEstablishing reference range for B cell subpopulations is crucial for guiding physicians in diagnosing young patients with suspected PID. However, these reference values may vary across laboratories due to differences in population demographics, geographical areas, selection criteria for healthy subjects, and sample preparation techniques, all of which contribute to the wide range of normal values reported (\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). These variations can introduce biases, especially in immunodeficiency assessments. Therefore, this study quantified and analysed B-cell subpopulations in children aged 2 years to 15 years to establish age-dependent reference intervals for humoral immune parameters, assess age-related variations in B-cell subpopulations, and explore differences between genders. To the best of our knowledge, this is the first study to define the normal distribution of B-cell subpopulations in peripheral blood in Malaysia, specifically at our centre, the Clinical Immunology Centre, Universiti Putra Malaysia.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy population of the healthy controls\u003c/h2\u003e \u003cp\u003eA total of 85 healthy individuals aged 2 to 15 years were voluntarily enrolled in this study between November 2022 and December 2023. Participants were referred to the paediatric clinic at Hospital Sultan Abdul Aziz Shah, Universiti Putra Malaysia, for developmental assessments. Children with immune disorders, chronic diseases, syndromic conditions confirmed by geneticists, those receiving intravenous immunoglobulin treatment, or those on immunosuppressive medications were excluded.\u003c/p\u003e \u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eSample preparation and data collection.\u003c/span\u003e \u003c/p\u003e \u003cp\u003eBlood samples were collected in ethylenediamine tetra-acetic acid (EDTA) tubes and processed within 24 hours. Flow cytometric analysis was performed using FACSLyrics (BD Biosciences, New Jersey, United States) with six-colour immunostaining for the following markers and fluorochromes: CD19-PerCPCy5.5, CD27-APC, CD38-PE-Cy7, CD24-APC-H7, IgD-PE, and IgM-BB515. Data generated were analysed using FlowJo software (BD Biosciences, New Jersey, United States). Briefly, whole blood was washed twice with stain buffer, incubated with immunofluorescence staining, and treated with FACSLysing solution (BD Biosciences, New Jersey, United States). The gating strategies are described in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The absolute number of cells was calculated by multiplying the relative proportion (percentage) of B-cell subpopulations by the absolute number of B cells obtained from T cell, B cell and natural killer cell (TBNK) enumeration or the lymphocyte count from a complete blood count (CBC) performed on the same day. We then identified each B-cell subsets based on the following markers: total B cells (CD19\u003csup\u003e+\u003c/sup\u003e), transitional B cells (CD19\u003csup\u003e+\u003c/sup\u003eCD27\u003csup\u003e\u0026minus;\u003c/sup\u003eCD24\u003csup\u003e+\u0026thinsp;bright\u003c/sup\u003eCD38\u003csup\u003e+bright\u003c/sup\u003e), na\u0026iuml;ve B cells (CD19\u003csup\u003e+\u003c/sup\u003eCD27\u003csup\u003e\u0026minus;\u003c/sup\u003e), total memory B cells (CD19\u003csup\u003e+\u003c/sup\u003eCD27\u003csup\u003e+\u003c/sup\u003e), switched memory B cells (CD19\u003csup\u003e+\u003c/sup\u003e IgM\u003csup\u003e\u0026minus;\u003c/sup\u003eIgD\u003csup\u003e\u0026minus;\u003c/sup\u003e CD27\u003csup\u003e+\u003c/sup\u003e CD38\u003csup\u003e+\u0026thinsp;dim\u003c/sup\u003e), non-switched memory B cells (CD19\u003csup\u003e+\u003c/sup\u003eIgM\u003csup\u003e+\u003c/sup\u003eIGD\u003csup\u003e+\u003c/sup\u003eCD27\u003csup\u003e+\u003c/sup\u003e CD38\u003csup\u003e+\u0026thinsp;dim\u003c/sup\u003e), and plasmablast (CD19\u003csup\u003e+\u003c/sup\u003eIgM\u003csup\u003e\u0026minus;\u003c/sup\u003eIgD\u003csup\u003e\u0026minus;\u003c/sup\u003eCD27\u003csup\u003e+\u003c/sup\u003eCD38\u003csup\u003e+\u0026thinsp;bright\u003c/sup\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eData were analysed using SPSS version 27 and Microsoft Excel\u0026reg;. Reference values were established for three age groups ranging from 2 to 15 years. These reference values were determined based on the 5th and 95th percentiles, representing the lower and upper limits, respectively. Medians and interquartile ranges were calculated for each age group. For statistical comparisons, Mann-Whitney test was used to analyse differences in B-cell subpopulations between gender, Kruskal Wallis test was performed to assess differences in B-cell subpopulations among age groups, while Spearman\u0026rsquo;s rank correlation test was used to evaluate age-dependent changes in B cell-subpopulations. A p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eInitially, 82 healthy children aged 2 to 15 years old were recruited; however, seven were excluded as outliers based on Tukey\u0026rsquo;s and Mahalanobis tests. B-cell subpopulations were analysed in three age groups: 2 to 4 years (n\u0026thinsp;=\u0026thinsp;29), 5 to 9 years (n\u0026thinsp;=\u0026thinsp;30), and 10 to 15 years (n\u0026thinsp;=\u0026thinsp;16). Demographic details are provided in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. All subjects had normal absolute lymphocytes counts (TBNK enumeration) based on established reference values (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e) (Supplementary Data).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDemographic details for the 75 healthy children tested according to the age group.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAge group\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMedian age\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTotal number of subjects\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGender (Male/Female)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2 to 4 years old\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4.081\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e15/14\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5 to 9 years old\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e7.973\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e14/16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10 to 15 years old\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e11.682\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10/6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e6.553\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e75\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe composition of B-cell subpopulations exhibited age-related variations. The total B cell frequency showed a decreasing pattern across age groups, with a significant reduction observed between the 2- to 4-year-old group and the 5- to 9-year-old group, where the median decreased from 22.9\u0026ndash;19.0% (p\u0026thinsp;=\u0026thinsp;0.013) and between 2- to 4-year-old group and the 10- to 15-year-old group from a median of 22.9\u0026ndash;18.3% (p\u0026thinsp;=\u0026thinsp;0.045). In contrast, transitional B cells, total memory B cells, non-switched memory B cells, class-switched memory B cells, and plasmablast cells displayed an initial increase from the 2- to 4-year-old group to the 5- to 9-year-old group, followed by a decline in the 10- to 15-year-old group. A significant increase was observed in the total memory B cell frequency (p\u0026thinsp;=\u0026thinsp;0.021) when comparing the 2- to 4-year-old group with the 5- to 9-year-old group and in plasmablast between 5- to 9-year-old group and 10- to 15-year-old group (p\u0026thinsp;=\u0026thinsp;0.046). Meanwhile, the frequency of na\u0026iuml;ve B cells declined between 2 to 4 years old and 5 to 9 years old, but a slight increase was noted in the 10- to 15-year-old group. A statistically significant reduction in na\u0026iuml;ve B cell frequency was found between the 2- to 4-year-old group and the 5- to 9-year-old group, where the median decreased from 79.0\u0026ndash;72.1% (p\u0026thinsp;=\u0026thinsp;0.017). However, no significant differences in frequency were detected across age groups for non-switched memory B cells, class-switched memory B cells or transitional B cells.\u003c/p\u003e \u003cp\u003eThe absolute count of each B-cell subpopulation also showed age-related changes. The absolute number of total B cells was declined with age, with significant differences observed between the 2- to 4-year-old group and the 5- to 9-year-old group (p\u0026thinsp;=\u0026thinsp;\u0026lt;\u0026thinsp;0.001), as well as between the 2- to 4-year-old group and the 10- to 15-year-old group (p\u0026thinsp;=\u0026thinsp;\u0026lt;\u0026thinsp;0.001). A similar pattern was identified in the transitional B cells, na\u0026iuml;ve B cell, total memory B cells, and non-switched memory B cells. The absolute number of transitional B cells showed a significant decline between the 2- to 4-year-old group and the 10- to 15-year-old group (p\u0026thinsp;=\u0026thinsp;0.025). The non-switched memory B cell absolute count also demonstrated a significant decrease between 2- to 4-year-old and 5- to 9-year-old groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and between the 2- to 4-year-old and 10- to 15-year-old groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). The absolute number of total memory B cells was significantly lower in the 10- to 15-year-old age group compared to the 2- to 4-year-old group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). In the case of na\u0026iuml;ve B cells, a reduction in absolute number was observed followed by an increase in the 10- to 15-year-old group. A statistically significant difference was detected between the 2- to 4-year-old group and the 10- to 15-year-old group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) as well as between the 2- to 4-year-old group and the 5- to 9-year-old group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Meanwhile, both class-switched memory B cells and plasmablasts exhibited a slight increase in absolute number from 2 to 4 years old to 5 to 9 years old before declining in the 10- to 15-year-old group, although these changes were not statistically significant. The median values and interquartile ranges (5th and 95th percentiles) for the frequencies and absolute numbers of B cell subpopulations across age groups are provided in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eFrequencies for each B cell subpopulations in distinct age groups.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAge group\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTotal B cells\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTransitional\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNa\u0026iuml;ve B\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTotal memory B\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNon-switched memory B\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eSwitched memory\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003ePlasmablast\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2 to 4 years\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e22.900\u003c/p\u003e \u003cp\u003e(15.04\u0026ndash;31.38)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.190\u003c/p\u003e \u003cp\u003e(0.52\u0026ndash;16.50)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e79.000\u003c/p\u003e \u003cp\u003e(57.75\u0026ndash;89.70)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e17.800\u003c/p\u003e \u003cp\u003e(8.41\u0026ndash;31.55)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e8.060\u003c/p\u003e \u003cp\u003e(3.92\u0026ndash;13.55)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e5.820\u003c/p\u003e \u003cp\u003e(1.93\u0026ndash;12.25)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.070\u003c/p\u003e \u003cp\u003e(0.10\u0026ndash;8.16)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5 to 9 years\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e18.985\u003c/p\u003e \u003cp\u003e(11.32\u0026ndash;31.15)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8.450\u003c/p\u003e \u003cp\u003e(0.79\u0026ndash;26.77)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e72.050\u003c/p\u003e \u003cp\u003e(51.69\u0026ndash;84.96)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e22.900\u003c/p\u003e \u003cp\u003e(12.64\u0026ndash;42.10)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e8.640\u003c/p\u003e \u003cp\u003e(3.33\u0026ndash;14.73)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e6.715\u003c/p\u003e \u003cp\u003e(3.64\u0026ndash;16.19)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.840\u003c/p\u003e \u003cp\u003e(0.29\u0026ndash;8.63)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10 to 15 years\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e18.280\u003c/p\u003e \u003cp\u003e(10.06\u0026ndash;31.37)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.060\u003c/p\u003e \u003cp\u003e(0.67\u0026ndash;17.80)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e76.450\u003c/p\u003e \u003cp\u003e(57.10\u0026ndash;85.60)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e20.350\u003c/p\u003e \u003cp\u003e(12.20\u0026ndash;35.90)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e5.875\u003c/p\u003e \u003cp\u003e(2.30\u0026ndash;12.60)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e6.350\u003c/p\u003e \u003cp\u003e(3.38\u0026ndash;16.10)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.665\u003c/p\u003e \u003cp\u003e(0.22\u0026ndash;6.56)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"8\"\u003eAll results are presented as median values with ranges between 5th and 95th percentile.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAbsolute number for each B cell subpopulations in distinct age groups.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAge group\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTotal B cells\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTransitional\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNa\u0026iuml;ve B\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTotal memory B\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNon-switched memory B\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eSwitched memory\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003ePlasmablast\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2 to 4 years\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1314.000\u003c/p\u003e \u003cp\u003e(615.28\u0026ndash;2387.50)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e60.246\u003c/p\u003e \u003cp\u003e(4.14\u0026ndash;257.62)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e932.881\u003c/p\u003e \u003cp\u003e(462.45\u0026ndash;2015.03)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e211.554\u003c/p\u003e \u003cp\u003e(91.11\u0026ndash;486.29)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e80.738\u003c/p\u003e \u003cp\u003e(27.67\u0026ndash;160.16)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e7.294\u003c/p\u003e \u003cp\u003e(1.25\u0026ndash;44.88)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.795\u003c/p\u003e \u003cp\u003e(0.08\u0026ndash;16.31)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5 to 9 years\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e616.500\u003c/p\u003e \u003cp\u003e(332.87\u0026ndash;1329.50)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e38.692\u003c/p\u003e \u003cp\u003e(2.69\u0026ndash;144.87)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e429.540\u003c/p\u003e \u003cp\u003e(196.23\u0026ndash;1042.60)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e154.901\u003c/p\u003e \u003cp\u003e(65.14\u0026ndash;364.03)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e44.112\u003c/p\u003e \u003cp\u003e(17.35\u0026ndash;120.32)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e7.870\u003c/p\u003e \u003cp\u003e(1.54\u0026ndash;30.02)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.931\u003c/p\u003e \u003cp\u003e(0.19\u0026ndash;11.64)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10 to 15 years\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e511.000\u003c/p\u003e \u003cp\u003e(226.00\u0026ndash;1130.00)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e26.011\u003c/p\u003e \u003cp\u003e(2.13\u0026ndash;101.48)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e421.700\u003c/p\u003e \u003cp\u003e(154.13\u0026ndash;894.96)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e98.481\u003c/p\u003e \u003cp\u003e(54.40\u0026ndash;258.48)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e25.417\u003c/p\u003e \u003cp\u003e(9.92\u0026ndash;70.06)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e4.080\u003c/p\u003e \u003cp\u003e(1.41\u0026ndash;32.34)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.665\u003c/p\u003e \u003cp\u003e(0.09\u0026ndash;4.13)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"8\"\u003eAll results are presented as median values with ranges between 5th and 95th percentile\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eFurther analysis using Spearman's rank-order correlation revealed a strong correlation between age and the absolute counts of total B cells, na\u0026iuml;ve B cells, and non-switched memory B cells, with all three subsets demonstrating a statistically significant association (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Additionally, the Mann-Whitney U test was used to examine gender-related differences in B-cell subpopulations. The results indicated that female children had significantly higher B cells frequency (p\u0026thinsp;=\u0026thinsp;0.047), total memory B cells frequency (p\u0026thinsp;=\u0026thinsp;0.016), total memory B cells absolute number (p\u0026thinsp;=\u0026thinsp;0.026), class-switched memory B frequency (p\u0026thinsp;=\u0026thinsp;0.029), class-switched memory B cells absolute number (p\u0026thinsp;=\u0026thinsp;0.005), and plasmablast absolute number (p\u0026thinsp;=\u0026thinsp;0.018) compared to male children. Conversely, male children had a significantly higher na\u0026iuml;ve B cells frequency than females (p\u0026thinsp;=\u0026thinsp;0.019). However, no significant gender differences were observed in total B cells absolute number (p\u0026thinsp;=\u0026thinsp;0.603), transitional B cells frequency (p\u0026thinsp;=\u0026thinsp;0.668), transitional B cells absolute number (p\u0026thinsp;=\u0026thinsp;0.596), na\u0026iuml;ve B cells absolute number (p\u0026thinsp;=\u0026thinsp;0.882), non-switched memory B cells frequency (p\u0026thinsp;=\u0026thinsp;0.131), non-switched memory B cells absolute number (p\u0026thinsp;=\u0026thinsp;0.581), and plasmablast frequency (p\u0026thinsp;=\u0026thinsp;0.092).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eVarious studies have been conducted to validate the age-dependent distribution of B-cell subpopulations, especially among paediatric population (\u003cspan additionalcitationids=\"CR13 CR14 CR15 CR16\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). Each B cell subpopulation represents a distinct stage of the B cell development, with full activation occurring upon antigen contact. A decrease in in the absolute number or frequency of specific B cell subpopulations may raise concerns about underlying immunological conditions, including primary immunodeficiency (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). While reference values for B cell subsets have been established in several populations, no such values have been documented for Malaysia. To the best of our knowledge, this study is the first to establish reference values for paediatric B-cell subpopulations in Malaysian children below 15 years of age, specifically within the Clinical Immunology Centre at Hospital Sultan Abdul Aziz Shah, Universiti Putra Malaysia, Selangor.\u003c/p\u003e \u003cp\u003eIn this study, we defined normal values for total B cells, transitional B cells, na\u0026iuml;ve B cells, total memory B cells, switched memory B cells, non-switched memory B cells, and plasmablasts in 75 healthy children grouped into three age groups ranging from 2 to 15 years. Although the physiological of each B cell subset is not entirely elucidated, these subsets can be identified based on the significant surface marker CD19. As expected, a notable reduction in total b cells, marked by CD19 positivity (both in frequency and absolute number) was observed during childhood. This trend has been previously reported and is known to continue as age increases (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). Prior research has highlighted that ageing plays a key role in this decline, as bone marrow output capacity diminishes over time. Additionally, early B cell populations predominantly appear as transitional B cells in peripheral circulation before shifting into na\u0026iuml;ve B cells (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). The observed reduction in both transitional and na\u0026iuml;ve B cells in this study mirrors the overall decline in total B cells. Importantly, the absolute number of total memory B cells and plasmablasts remained relatively stable over time (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Taken together, these findings align with the previous research, suggesting that the reduction of total B cells may be attributed to declines in transitional and na\u0026iuml;ve B cell subpopulations (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). Interestingly, severe reduction or complete absence of total B cells is commonly observed in patients with x-linked agammaglobulinemia (XLA). This is primarily caused by the genetic mutation in Bruton\u0026rsquo;s tyrosine kinase (BTK), which responsible in mediating B cell development and maturation. The mutation in the BTK gene disrupt the progression from pro-B cells to pre-B cells, preventing a proper development of B cells and halting the hematopoietic stem cells to enter B cell lineage. Thus, functional and antigen-responsive cells are unable to be generated (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eA continuous decline in the absolute number of non-switched memory B cells was also observed in this study, whereas the frequency and absolute count of switched memory B cells remained relatively stable across age groups. These findings are consistent with those reported by Duchamp et al. and Morbach et al. (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). A decrease in switched memory B cells below the reference range has been suggested as a potential indicator for evaluating patients with suspected primary immunodeficiencies. Indeed, switched memory B cells have incorporated into diagnostic criteria for common variable immunodeficiency (CVID) in the Freiburg, Paris and European classifications (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan additionalcitationids=\"CR29\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). However, the cut-off values proposed by these classifications vary and are based on adult populations. This underscores the necessity of establishing age- and population-specific reference values for distinct B-cell subpopulations. Additionally, patient with either CD40 ligand or CD40 deficiency exhibit reduced memory B cells. Mutations in the CD40L and CD40 genes disrupt the CD40 activation pathway, resulting defects in both class switch recombination (CSR) and somatic hypermutation. These defects categorized under combined T- and B-cell immunodeficiencies (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ePlasmablasts, characterized as CD24\u003csup\u003e\u0026minus;\u003c/sup\u003eCD38\u003csup\u003e++\u003c/sup\u003e B cells, are typically present in trace amounts within peripheral blood. Some researchers have stated that plasmablasts are rarely exceed 5% of total B cells (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). In our study, both the proportion and absolute number of plasmablasts were less than 2% and 2 X 10\u003csup\u003e6\u003c/sup\u003e /L, respectively. Notably, plasmablast remained consistent across all age groups. It is crucial to recognize that the expansion of plasmablasts in the peripheral blood may indicate autoimmune conditions, such as systematic lupus erythematous, autoimmune rheumatic diseases, Sj\u0026ouml;gren's disease, and rheumatoid arthritis (\u003cspan additionalcitationids=\"CR33 CR34 CR35\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). Therefore, establishing population-specific reference range could help define precise cut-off values for detecting autoimmune diseases.\u003c/p\u003e \u003cp\u003eInterestingly, our study revealed gender-based differences in B-cell subpopulation distribution. Female children had a significantly higher frequency of total memory B cells, non-switched memory B, and class-switched memory B cells, as well as greater absolute number of class-switched memory B cells compared to their male counterparts. Conversely, na\u0026iuml;ve B cells were found at a higher frequency in male children. This suggests that, in addition to age-related factors, gender also influences B cell subset distribution and immune responses. Two potential explanations for this phenomenon include the influence of sex hormones, such as oestrogen and testosterone, and the role of X chromosome in regulating immune-related genes (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). Previous research has reported that oestrogen stimulates B cells proliferation, whereas testosterone hormone delays this process (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e). Additionally, the X chromosome plays an important role in immune function, as it contains coding regions for genes involved in immune responses, including toll-like receptors, cytokine receptors, and transcription factors (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e). Consequently, gender should be considered when assessing young patients in immunological evaluations.\u003c/p\u003e \u003cp\u003eDespite the strengths of this study, several limitations should be acknowledged. The main limitation is the small sample size. This small sample size can be reflected in the statistical power, potentially compromising both the sensitivity and the specificity of the analysis. Primarily, it may cause difficulty in replicating results and increase the likelihood of false negative results, the type Ⅱ error (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e). According to the Clinical and Laboratory Standards Institute (CLSI), a minimum sample size of 120 subjects is required to establish reference intervals (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e). For studies involving multiple variables, such as in this study, at least 120 subjects per variable would be ideal. Although we were only able to recruit a small cohort of 75 participants, this study serves as an initial platform for determining reference values within our laboratory. However, these findings may not be generalizable to the entire Malaysian population. Future studies with larger cohorts are necessary to establish more comprehensive population-specific reference values. Another limitation of this study is the restricted age range of participants. Since our focus was on children aged 2 years to 15 years, the reference values do not account for B cell subpopulation variations in early infancy. Previous research suggests that significant fluctuations in B cell distribution frequencies and absolute counts occur within the first five years of life. Expanding the age range in future studies would allow for more thorough characterization of B-cell distribution across different developmental stages within the Malaysian population.\u003c/p\u003e \u003cp\u003eIn conclusion, the characterization of B-cell subpopulations is crucial for evaluating primary immunodeficiencies. This study provides age-specific reference values for B cell subsets in a paediatric population, which may serve as a valuable guideline for diagnosing children with suspected immunodeficiency. Additionally, our findings highlight the potential impact of both age and gender on B cell distribution, emphasizing the need for further research in this area. Future studies should investigate whether variations in B cell subset differentiation and expansion are influenced by population-specific or environment factors.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cspan\u003eWritten informed consent was obtained from all parents or legal guardians of the participants prior to inclusion in the study.\u003c/span\u003e\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e\n\u003cp\u003eWe would like to thank the Clinical Immunology Centre (CIC) team from Hospital Sultan Abdul Aziz Shah (HSAAS), Universiti Putra Malaysia for their excellent technical assistance. This study was funded by the Fundamental Research Grant Scheme (FRGS) from Ministry of Education Malaysia (04-01-18-1973FR, FRGS/1/2018/SKK02/UPM/02/1) and Grant Putra: Geran Inisiatif Putra Siswazah from Universiti Putra Malaysia (GP-IPS/2022/9739900).\u003c/p\u003e\n\u003ch2\u003eAuthor contribution\u003c/h2\u003e\n\u003cp\u003eI.H.I was responsible for the conception and design of the study and provided overall direction for the study; J.J performed, analyzed experiments, and prepared the manuscript; S. M.M., H.M and M.A.Z.A provided critical revision of the manuscript. All authors have read and approved the final manuscript.\u003c/p\u003e\n\u003ch2\u003eConflict of Interest\u003c/h2\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003ch2\u003eEthics approval and consent to participate\u003c/h2\u003e\n\u003cp\u003eEthical approval was obtained from the National Medical Research Register (NMRR-20-1741-53741) and the UPM Ethics Committee for research involving human subjects (JKEUPM-2023-337).\u003c/p\u003e\n\u003ch2\u003eAvailability of supporting data\u003c/h2\u003e\n\u003cp\u003eAll data generated during this study are included in this published article. Raw datasets are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eRoy A, Bystry V, Bohn G, Goudevenou K, Reigl T, Papaioannou M, et al. High resolution IgH repertoire analysis reveals fetal liver as the likely origin of life-long, innate B lymphopoiesis in humans. Clinical Immunology. 2017;183:8\u0026ndash;16. \u003c/li\u003e\n\u003cli\u003eJackson TR, Ling RE, Roy A. The Origin of B-cells: Human Fetal B Cell Development and Implications for the Pathogenesis of Childhood Acute Lymphoblastic Leukemia. Frontiers in Immunology. 2021;12(February):1\u0026ndash;10. \u003c/li\u003e\n\u003cli\u003eEroglu FK, Aerts Kaya F, Cagdas D, \u0026Ouml;zg\u0026uuml;r TT, Yılmaz T, Tezcan İ, et al. B lymphocyte subsets and outcomes in patients with an initial diagnosis of transient hypogammaglobulinemia of infancy. Scandinavian Journal of Immunology. 2018;88(4):1\u0026ndash;8. \u003c/li\u003e\n\u003cli\u003eIwajomo OH, Finn A, Ogunniyi AD, Williams NA, Heyderman RS. Impairment of pneumococcal antigen specific isotype-switched Igg memory B-cell immunity in HIV infected Malawian adults. PLoS One. 2013;8(11):1\u0026ndash;7. \u003c/li\u003e\n\u003cli\u003eClaes N, Fraussen J, Stinissen P, Hupperts R, Somers V. B cells are multifunctional players in multiple sclerosis pathogenesis: Insights from therapeutic interventions. Frontiers in Immunology. 2015;6(DEC). \u003c/li\u003e\n\u003cli\u003eKlein U, Rajewsky K, K\u0026uuml;ppers R. Human immunoglobulin (Ig)M+IgD+ peripheral blood B cells expressing the CD27 cell surface antigen carry somatically mutated variable region genes: CD27 as a general marker for somatically mutated (memory) B cells. Journal of Experimental Medicine. 1998;188(9):1679\u0026ndash;89. \u003c/li\u003e\n\u003cli\u003eTangye SG, Al-Herz W, Bousfiha A, Cunningham-Rundles C, Franco JL, Holland SM, et al. Human Inborn Errors of Immunity: 2022 Update on the Classification from the International Union of Immunological Societies Expert Committee [Internet]. Journal of Clinical Immunology. Springer US; 2022. 1473\u0026ndash;1507 p. Available from: https://doi.org/10.1007/s10875-022-01289-3\u003c/li\u003e\n\u003cli\u003eMinistry of Health Malaysia. Guideline for Method Verification of Quantitative Measurement in Medical Laboratories. 2015. p. https://www.patologi.gov.my/assets/policy/merged.p. \u003c/li\u003e\n\u003cli\u003eYadav D. Reference Interval for Clinical Laboratory Test Parameters. Biochemistry \u0026amp; Analytical Biochemistry. 2015;04(04). \u003c/li\u003e\n\u003cli\u003eKatayev A, Balciza C, Seccombe DW. Establishing reference intervals for clinical laboratory test results: Is there a better way? American Journal of Clinical Pathology. 2010;133(2):180\u0026ndash;6. \u003c/li\u003e\n\u003cli\u003eComans-Bitter WM, De Groot R, Van den Beemd R, Neijens HJ, Hop WCJ, Groeneveld K, et al. Immunophenotyping of blood lymphocytes in childhood: Reference values for lymphocyte subpopulations. Journal of Pediatrics. 1997;130(3):388\u0026ndash;93. \u003c/li\u003e\n\u003cli\u003eMorbach H, Eichhorn EM, Liese JG, Girschick HJ. Reference values for B cell subpopulations from infancy to adulthood. Clin Exp Immunol. 2010;162(2):271\u0026ndash;9. \u003c/li\u003e\n\u003cli\u003ePia̧tosa B, Wolska-Kuśnierz B, Pac M, Siewiera K, Gałkowska E, Bernatowska E. B cell subsets in healthy children: Reference values for evaluation of B cell maturation process in peripheral blood. Cytometry B Clin Cytom. 2010;78(6 B):372\u0026ndash;81. \u003c/li\u003e\n\u003cli\u003ePlassmeyer M, Ryherd M, Brown M, Gupta R, Lacbawan L, Alpan O. Normal B Cell Subpopulations in the Pediatric Age Groups and Adults. Journal of Allergy and Clinical Immunology. 2013;131(2):AB67. \u003c/li\u003e\n\u003cli\u003eBlanco E, P\u0026eacute;rez-Andr\u0026eacute;s M, Arriba-M\u0026eacute;ndez S, Contreras-Sanfeliciano T, Criado I, Pelak O, et al. Age-associated distribution of normal B-cell and plasma cell subsets in peripheral blood. Journal of Allergy and Clinical Immunology. 2018;141(6):2208-2219.e16. \u003c/li\u003e\n\u003cli\u003eBerr\u0026oacute;n-Ru\u0026iacute;z L, L\u0026oacute;pez-Herrera G, \u0026Aacute;valos-Mart\u0026iacute;nez CE, Valenzuela-Ponce C, Ram\u0026iacute;rez-SanJuan E, Santoyo-S\u0026aacute;nchez G, et al. Variations of B cell subpopulations in peripheral blood of healthy Mexican population according to age: Relevance for diagnosis of primary immunodeficiencies. Allergol Immunopathol (Madr). 2016;44(6):571\u0026ndash;9. \u003c/li\u003e\n\u003cli\u003eAzarsiz E, Karaca NE, Aksu G, Kutukculer N. Reference values for B-cell surface markers and co-receptors associated with primary immune deficiencies in healthy Turkish children. Int J Immunopathol Pharmacol. 2017;30(2):194\u0026ndash;200. \u003c/li\u003e\n\u003cli\u003eWehr C, Kivioja T, Schmitt C, Ferry B, Witte T, Eren E, et al. The EUROclass trial: Defining subgroups in common variable immunodeficiency. Blood. 2008;111(1):77\u0026ndash;85. \u003c/li\u003e\n\u003cli\u003eBlanco E, P\u0026eacute;rez-Andr\u0026eacute;s M, Arriba-M\u0026eacute;ndez S, Serrano C, Criado I, Del Pino-Molina L, et al. Defects in memory B-cell and plasma cell subsets expressing different immunoglobulin-subclasses in patients with CVID and immunoglobulin subclass deficiencies. Journal of Allergy and Clinical Immunology. 2019;144(3):809\u0026ndash;24. \u003c/li\u003e\n\u003cli\u003eHuck K, Feyen O, Ghosh S, Beltz K, Bellert S, Niehues T. Memory B-cells in healthy and antibody-deficient children. Clinical Immunology [Internet]. 2009;131(1):50\u0026ndash;9. Available from: http://dx.doi.org/10.1016/j.clim.2008.11.008\u003c/li\u003e\n\u003cli\u003ePieper K, Grimbacher B, Eibel H. B-cell biology and development. Journal of Allergy and Clinical Immunology [Internet]. 2013;131(4):959\u0026ndash;71. Available from: http://dx.doi.org/10.1016/j.jaci.2013.01.046\u003c/li\u003e\n\u003cli\u003eMcComb S, Thiriot A, Akache B, Krishnan L, Abstract FS. Introduction to the Immune Response. In: Immunoproteomics: Methods and Protocols. Humana, New York: Methods in Molecular Biology; 2019. p. 1\u0026ndash;24. \u003c/li\u003e\n\u003cli\u003eBD Biosciences. B-Cell Research Flow cytometry tools for the study of B-cell biology. Bd biosciences. 2021. \u003c/li\u003e\n\u003cli\u003eSmith T, Roifman CM. Primary B-Cell Immunodeficiencies. Clinical Immunology: Principles and Practice. 2019;80(6):489-508.e1. \u003c/li\u003e\n\u003cli\u003eMcCusker C, Upton J, Warrington R. Primary immunodeficiency. Allergy, Asthma and Clinical Immunology [Internet]. 2018;14(s2):1\u0026ndash;12. Available from: https://doi.org/10.1186/s13223-018-0290-5\u003c/li\u003e\n\u003cli\u003eBonilla FA, Khan DA, Ballas ZK, Chinen J, Frank MM, Hsu JT, et al. Practice parameter for the diagnosis and management of primary immunodeficiency. Journal of Allergy and Clinical Immunology [Internet]. 2015;136(5):1186-1205.e78. Available from: http://dx.doi.org/10.1016/j.jaci.2015.04.049\u003c/li\u003e\n\u003cli\u003eDuchamp M, Sterlin D, Diabate A, Uring-Lambert B, Guerin-El Khourouj V, Le Mauff B, et al. B-cell subpopulations in children: National reference values. Immun Inflamm Dis. 2014;2(3):131\u0026ndash;40. \u003c/li\u003e\n\u003cli\u003eBhattad S. B-cell Defects: A Clinical and Immunological Approach. Pediatr Infect Dis. 2020;2(1):36\u0026ndash;8. \u003c/li\u003e\n\u003cli\u003ePiqueras B, Lavenu-Bombled C, Galicier L, Bergeron-Van Der Cruyssen F, Mouthon L, Chevret S, et al. Common variable immunodeficiency patient classification based on impaired B cell memory differentiation correlates with clinical aspects. J Clin Immunol. 2003;23(5):385\u0026ndash;400. \u003c/li\u003e\n\u003cli\u003eWarnatz K, Denz A, Dr\u0026auml;ger R, Braun M, Groth C, Wolff-Vorbeck G, et al. Severe deficiency of switched memory B cells (CD27(+)IgM(-)IgD(-)) in subgroups of patients with common variable immunodeficiency: a new approach to classify a heterogeneous disease. 2002; Available from: https://www.semanticscholar.org/paper/8f698faf75832c2f4d3f3895dd0980fab46c4be3\u003c/li\u003e\n\u003cli\u003eRezaei N, Aghamohammadi A, Notarangelo LD. Primary Immunodeficiency Diseases [Internet]. Second edi. Rezaei N, Aghamohammadi A, Notarangelo LD, editors. Vol. 1, Springer. Berlin, Heidelberg: Springer Berlin Heidelberg; 2017. 379 p. Available from: http://link.springer.com/10.1007/978-3-662-52909-6\u003c/li\u003e\n\u003cli\u003eArroyo-Villa I, Bautista-Caro MB, Balsa A, Aguado-Ac\u0026iacute;n P, Bonilla-Hern\u0026aacute;n MG, Plasencia C, et al. Constitutively altered frequencies of circulating follicullar helper T cell counterparts and their subsets in rheumatoid arthritis. Arthritis Res Ther. 2014;16(1):1\u0026ndash;8. \u003c/li\u003e\n\u003cli\u003eSzyszko EA, Brun JG, Skarstein K, Peck AB, Jonsson R, Brokstad KA. Phenotypic Diversity of Peripheral Blood Plasma Cells in Primary Sj\u0026ouml;gren\u0026rsquo;s Syndrome. Scand J Immunol. 2011;73(1):18\u0026ndash;28. \u003c/li\u003e\n\u003cli\u003eSteinmetz TD, Verstappen GM, Suurmond J, Kroese FGM. Targeting plasma cells in systemic autoimmune rheumatic diseases \u0026ndash; Promises and pitfalls. Immunol Lett. 2023;260(May):44\u0026ndash;57. \u003c/li\u003e\n\u003cli\u003eJacobi AM, Mei H, Hoyer BF, Mumtaz IM, Thiele K, Radbruch A, et al. HLA-DRhigh/CD27high plasmablasts indicate active disease in patients with systemic lupus erythematosus. Ann Rheum Dis. 2010;69(1):305\u0026ndash;8. \u003c/li\u003e\n\u003cli\u003eD\u0026ouml;rner T, Lipsky PE. Correlation of circulating CD27high plasma cells and disease activity in systemic lupus erythematosus. Lupus. 2004;13(5):283\u0026ndash;9. \u003c/li\u003e\n\u003cli\u003eBous M, Schmitt C, Hans MC, Weber R, Nourkami-Tutdibi N, Tenbruck S, et al. Sex Differences in the Frequencies of B and T Cell Subpopulations of Human Cord Blood. Int J Mol Sci. 2023;24(14). \u003c/li\u003e\n\u003cli\u003eLibert C, Dejager L, Pinheiro I. The X chromosome in immune functions: When a chromosome makes the difference. Nat Rev Immunol. 2010;10(8):594\u0026ndash;604. \u003c/li\u003e\n\u003cli\u003eBereshchenko O, Bruscoli S, Riccardi C. Glucocorticoids, sex hormones, and immunity. Front Immunol. 2018;9(JUN):1\u0026ndash;10. \u003c/li\u003e\n\u003cli\u003eKlein SL, Flanagan KL. Sex differences in immune responses. Nat Rev Immunol. 2016;16(10):626\u0026ndash;38. \u003c/li\u003e\n\u003cli\u003eAlthubaiti A. Sample size determination: A practical guide for health researchers. J Gen Fam Med. 2023;24(2):72\u0026ndash;8. \u003c/li\u003e\n\u003cli\u003eColumb MO, Atkinson MS. Statistical analysis: sample size and power estimations. BJA Educ. 2016;16(5):159\u0026ndash;61. \u003c/li\u003e\n\u003cli\u003eCLSI. Defining , Establishing , and Verifying Reference Intervals in the Clinical Laboratory ; Approved Guideline \u0026mdash; Third Edition. CLSI document C28-A3c. 2008;28(30):59. \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"humoral immunity, B cells, switched-memory B cells, age-specific reference range","lastPublishedDoi":"10.21203/rs.3.rs-6867752/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6867752/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePhysicians rely on reference values from healthy populations to guide clinical decisions regarding B-cell subpopulations in primary immunodeficiency. While age-dependent reference ranges have been reported in several populations, no study has established these values for Malaysian children. Given that B-cell subpopulation distributions may vary between populations, we aimed to define reference ranges for total B cells, transitional B cells, naïve B cells, total memory B cells, switched and non-switched memory B cells, and plasmablasts in Malaysian children aged 2 to 15 years. Blood samples were taken from 85 children aged 2 to 15 years evaluated for the distribution of B cell subsets. Absolute numbers and percentages were determined for total B cells (CD19\u003csup\u003e+\u003c/sup\u003e), transitional B cells (CD19\u003csup\u003e+\u003c/sup\u003eCD27\u003csup\u003e-\u003c/sup\u003eCD24\u003csup\u003e+bright\u003c/sup\u003eCD38\u003csup\u003e+bright\u003c/sup\u003e), naïve B cells (CD19\u003csup\u003e+\u003c/sup\u003eCD27\u003csup\u003e-\u003c/sup\u003e), total memory B cells (CD19\u003csup\u003e+\u003c/sup\u003eCD27\u003csup\u003e+\u003c/sup\u003e), switched memory B cells (CD19\u003csup\u003e+\u003c/sup\u003e IgM\u003csup\u003e-\u003c/sup\u003eIgD\u003csup\u003e- \u003c/sup\u003eCD27\u003csup\u003e+\u003c/sup\u003e CD38\u003csup\u003e+dim\u003c/sup\u003e), non-switched memory B cells (CD19\u003csup\u003e+\u003c/sup\u003eIgM\u003csup\u003e+\u003c/sup\u003eIGD\u003csup\u003e+\u003c/sup\u003eCD27\u003csup\u003e+\u003c/sup\u003e CD38\u003csup\u003e+dim\u003c/sup\u003e), and plasmablasts (CD19\u003csup\u003e+\u003c/sup\u003eIgM\u003csup\u003e-\u003c/sup\u003eIgD\u003csup\u003e-\u003c/sup\u003eCD27\u003csup\u003e+\u003c/sup\u003eCD38\u003csup\u003e+bright\u003c/sup\u003e). We observed age-dependent variations in most B-cell subpopulations, with naïve B cells being predominant, followed by memory B cells, while plasmablasts were present in trace amounts across all ages. Additionally, most B-cell subpopulations were observed at higher frequencies in female children compared to males. This study provides age-specific reference values for B cell subsets in a paediatric population, which may serve as a valuable guideline for diagnosing children with suspected immunodeficiency.\u003c/p\u003e","manuscriptTitle":"Reference Range for B-Cell Subpopulations in Peripheral Blood of Healthy Malaysian Children Aged 2 to 15 years.","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-20 15:26:28","doi":"10.21203/rs.3.rs-6867752/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"4b46430a-0532-4739-8159-9728bb776ce6","owner":[],"postedDate":"June 20th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":49954718,"name":"Biological sciences/Immunology/Adaptive immunity/Humoral immunity/Immunological memory"},{"id":49954719,"name":"Biological sciences/Immunology/Adaptive immunity/Humoral immunity"}],"tags":[],"updatedAt":"2025-06-20T15:26:29+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-20 15:26:28","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6867752","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6867752","identity":"rs-6867752","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}