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Central European Journal of Immunology
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vol. 35

Clinical immunology
Distribution of leukocyte and lymphocyte subsets in peripheral blood. Age related normal values for preliminary evaluation of the immune status in Polish children

Barbara Piątosa
Beata Wolska-Kuśnierz
Katarzyna Siewiera
Hanna Grzduk
Ewa Gałkowska
Ewa Bernatowska

(Centr Eur J Immunol 2010; 35 (3): 168-175)
Online publish date: 2010/10/05
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Lymphocytes play both regulating and effector functions. Determination of relative and absolute numbers of various lymphocyte subsets is currently a routine method, considered significant for diagnosis and monitoring in various pathological inborn, as well as acquired immune-related conditions. Composition of blood lymphocyte subsets depends on age [1-3] and might be changed through stress [4], physical activity [5], lifestyle-related factors [6, 7] the circadian rhythm [8], etc. Accurate interpretation of results requires reliable normal ranges derived from large studies. The purpose of this study was to establish reference values of basic lymphocyte subsets for pediatric population in Poland useful for diagnostics of inborn and acquired disorders of immunity.

Material and methods


The tested population included 292 children and young adults living in municipal or rural area, aged 0-31 years, without infectious, immunologic, hematological, and other chronic diseases or treatment of any kind that could affect the immune system. Sample collection was preceded by physical examination, evaluation of the past medical history based on detailed questionnaire, and obtaining an informed consent from subjects above 16 and from parents in case of children under 16 years of age. Any acute or chronic infection within four weeks before sample donation resulted in exclusion from the study. Subjects were divided into eleven age groups: i.e. samples were taken from cord blood and patients aged 0 to 7 days, 8 to 60 days, 2 to 5 months, 5 to 9 months, 9 to 15 months, 15 to 24 months, 2 to 5 years, 5 to 10 years, 10 to 16 years, and older than 16 years (detailed demographical data see Table 1). The study was approved by the institutional review board at the Children’s Memorial Health Institute, Warsaw, Poland and conducted in accordance with the guidelines of Helsinki Declaration.

Blood samples and immunophenotyping

One milliliter peripheral blood samples were taken by venipuncture during morning hours. Cord blood samples were obtained from umbilical cord vessels within few minutes after delivery. All samples were anticoagulated with EDTA-K2. Relative and absolute numbers of T (CD3+), B (CD19+), NK (CD16.56+CD3–) cells, as well as T helper (CD3+CD4+) and T suppressor (CD3+CD8+) cells were determined by flow cytometry using lyse-no-wash technique and four-color cocktails of antibodies. Additionally expression of RA and RO isoforms of CD45 molecule was determined on T helper and T suppressor cells. Commercially available compositions of antibody cocktails (see Table 2 for details) were used for deter­mination of relative numbers of individual cell subsets. Trucount tubes (Becton Dickinson) were used to determine absolute lymphocyte counts. Antibody manufacturer’s instructions were followed during the staining procedure. Briefly, 0.05 ml aliquots of blood were incubated with optimally titered antibodies for 15 minutes in room temperature. The incubation was followed by erythrocyte lysis using 0.45 ml of BD FACSLysing Solution (Becton Dickinson) diluted according to manufacturer’s instructions. At least 15 000 events were acquired to properly calibrated flow cytometer, with lymphocyte gate defined based on CD45 expression and side scatter characteristics. Absolute numbers of individual cell subsets were calculated based on proportion of the respective cell subpopulation and absolute lymphocyte count.
All analyses were performed on four color FACSCalibur cytometer (Becton Dickinson) equipped with licensed FACSComp, Multiset and Cellquest software, properly calibrated with CaliBRITE and CaliBRITE APC beads (Becton Dickinson). Standard quality control criteria recommended for all immunophenotyping analyses were applied [9]. To ensure data quality immunophenotyping results were reviewed for data consistency within each sample, i.e. the sum of T (CD3+), B (CD19+) lymphocytes and natural killer cells (CD16.56+CD3–) was required to be 100 ±5 [10].

Calculation of relative and absolute lymphocyte counts

Distribution of three leukocyte populations, i.e. lymphocytes, monocytes, and polymorphonuclears (PMN) was determined based on differential expression of CD45 and side scatter characteristics (see Fig. 1). Determination of absolute lymphocyte count was carried automatically by Multiset software in lymphocyte gate set based on CD45 and side scatter characteristics. Absolute sizes of individual lymphocyte subsets were calculated from the respective relative sizes and absolute lymphocyte counts. All results are presented as median and 5 to 95 percentile values.


Distribution of leukocyte populations

Distribution of lymphocytes and polymorphonuclears, peripheral white blood cell populations essential for the immune response to foreign antigens, significantly varies with age, both in terms of relative and absolute cell counts (see Table 3). Polymorphonuclears (PMN), which compose the major population of peripheral blood leukocytes at birth, rapidly decline during the first year of life, replaced by equally rapidly increasing numbers of lymphocytes. Such variation is not observed for monocytes, which remain almost invariable throughout life, except for the first few days after birth when they rapidly increase from values found in cord blood. Changes in absolute lymphocyte counts significantly affect the absolute counts of individual cell subsets.

T lymphocytes and their subsets

Despite almost invariant relative numbers of T lym­phocytes, their absolute count increases rapidly after birth reaching peak numbers in children 5-9 months old. Their number decreases thereafter along with decreasing number of lymphocytes. Although the relative number of CD4+ T lymphocytes demonstrates declining tendency with age, in contrast to the CD8+ subset, the absolute counts of both T cell subset reach peak values before the end of the first year of life and decrease thereafter reaching stable absolute counts in children older than 10 years. Such variation affects also the CD4:CD8 ratio which is highest in newborns and gradually decreases to values observed in adulthood (see Tables 4 and 5).

B lymphocytes

B lymphocytes present limited variability in terms of relative numbers in contrast to significant variation in their absolute counts which increase rapidly during the first 5 months of life and gradually decrease thereafter reaching plateau in children older than 5 years (see Tables 4 and 5).

NK cells

Both the relative and the absolute numbers of NK cells remain almost invariable during early childhood, except for the first days of life when they rapidly decrease from the numbers found in cord blood to those encountered in peripheral blood of newborns. Despite the tendency to increase their proportion in further life their absolute counts remain almost invariable (see Tables 4 and 5)

Expression of CD45 isoforms RA and RO

The relative numbers of both CD4-positive and CD8-po­sitive T cells expressing RA isoform of CD45 molecule decrease gradually from the numbers observed in newborns to those found in adults. However, the absolute counts of cells from both T cell subsets present a different pattern of changes in expression of CD45RA isoform: T CD8+ lymphocytes remain almost invariable for almost 2 years of life and slowly decrease thereafter reaching stable numbers in children older than 5 years. The number absolute of T helper cells expressing CD45RA follows the pattern of changing T helper numbers and increase since the day of birth reaching maximum counts in children 5-9 months old and decrease thereafter, reaching stable counts in children above 10 years of age. Expression of CD45RO molecule on T lymphocytes remains unchanged throughout the first year of life, with the relative numbers of T helper and T suppressor cells expressing CD45RO beginning to increase during the second year of life and reaching plateau around 10 years of life. The absolute counts of T lymphocytes expressing CD45RO molecule remain invariable during the whole childhood (see Table 6).


Enumeration of lymphocyte subpopulations in peripheral blood is considered of great significance for the evaluation of the immune status, despite the fact that peripheral blood lymphocytes represent only about 2% of their whole population in the body [11] and several factors affect their recirculation [12]. Interpretation of results requires access to reliable reference ranges, which is difficult in case of pediatric population, where age-related variation must be taken into account. Special effort must be made to obtain samples from healthy children, with age groups no more than few months apart [13]. Based on results of the study performed using a modern approach of multicolor flow cytometry and single platform technology for enumeration of cell counts, we determined normal values for basic lymphocyte subsets in pediatric population of Poland providing a useful tool for interpretation of clinical and laboratory data obtained from patients with suspected primary immune deficiencies.
Over the years composition of lymphocyte subsets has been widely studied in various populations [13-18] with differences that could be explained both by methodological aspects [19-21], life-style related factors [4, 22], as well as environmental conditions significantly affected worldwide due to increasing air pollution [23], race [15], or nutritional status [7]. Until results of this study have been summarized we used widely accepted normal values described by Comans-Bitter [3], but due to frequently observed abnormal results in otherwise healthy children we decided to undertake our own study having in mind the differences in type of the population, as well as environmental and living conditions in Poland and western countries. The tested population appeared to be different in comparison to the Dutch one, with generally lower absolute total lymphocyte counts, the phenomenon affecting results for all tested lymphocyte subsets and their interpretation. Explanation of the differences between results of this study and quite similar results obtained in Poland and the Netherlands around 15 years ago analyzed by Zeman [1] and Comans-Bitter [3], respectively is not straightforward and was not the aim of this study.
Results of this study avoid the drawbacks of previous studies carried on Polish population, such as two color technique, lymphocyte gating performed based on side and forward scatter characteristics of cells, double platform approach for enumeration of cells, lack of absolute cell counts, or limited age groups [1, 24]. Patients with suspected severe primary immune deficiencies usually develop clinical symptoms during the first year of age, therefore detailed data regarding distribution of lymphocyte subsets during the first year of life are of greatest significance. Additional information on maturation of the immune system may come from the distribution of T lym­phocytes expressing RA and RO isoforms of CD45 molecule considered to represent naive/activated and antigen-experienced/memory cells [25], with abnormalities common in various combined immune deficiencies [26] due to aberrant splicing mechanisms [25]. Children younger than one year, with unusually high proportion of T lym­phocytes expressing CD45RO, may suffer from severe combined immune deficiency [27] or abnormal stimulation of lymphocytes [28], while significantly higher proportion of cells expressing CD45RA molecule may indicate e.g. acute EBV infection [29].
This study of normal pediatric lymphocyte population was designed for application in diagnostics of primary immune deficiencies and included samples taken from cord blood, newborns, and children from very narrow age groups to facilitate and advance time to reach the diagnosis, but will also be useful for the assessment of children with suspected secondary depression of the immune system in course of various diseases and types of treatment. To our knowledge this is the first study carried on such a large and heterogeneous population of Polish children from various age groups, both from municipal as well as rural area, with verified health status, performed on whole blood samples, using multicolor flow cytometry, lyse-no-wash approach, and single platform technology. Our study demonstrates changes in sizes of individual cell populations occurring during childhood, with changes in relative counts that are not directly consistent with variation in absolute numbers. Short intervals between age groups during the first year of life allow demonstration of tremendous changes in the immune system encountering new antigens and gradually acquiring the ability to respond to that challenge. Methodology used in this study, in particular whole blood lysis sample preparation, CD45 vs. side scatter gating strategy and single platform lymphocyte enumeration which allow to use small blood sample without non-specific cell loss due to isolation of lymphocytes on density gradients [20, 30], avoidance of inclusion of nucleated red blood cells in lymphocyte gate that can falsify the ratio by disproportional reduction in lymphocyte subsets is particularly useful for pediatric population. Additional information on the proportion of other than lymphocytes white blood cells may bring faster diagnosis of diseases associated with abnormal monocyte or neutrophil count.


The authors wish to express their gratitude to all children and their parents who gave consent for participation in this study, as well as teachers and the medical staff from several institutions for their effort in finding eligible subjects. This study was sponsored by an internal grant S102/2006 from the Children’s Memorial Health Institute “Assessment of the distribution of the immunological memory cells in population of healthy children”, the grant from the Ministry of Science and Higher Education PBZ-KBN-119/P05/04 “The devel­opment, improvement, and implementation of highly specialist diagnostic procedures in immune-based diseases” and the European grant EURO-GENE-SCAN “European Genetic Disease Diagnostics”.


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Copyright: © 2010 Polish Society of Experimental and Clinical Immunology This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International (CC BY-NC-SA 4.0) License (http://creativecommons.org/licenses/by-nc-sa/4.0/), allowing third parties to copy and redistribute the material in any medium or format and to remix, transform, and build upon the material, provided the original work is properly cited and states its license.
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