B-cell subpopulations in children: National reference values

Abstract

Peripheral B-lymphocytes undergo a series of changes during the first few years of life. Encounters with foreign antigens lead to maturation and differentiation. Several primary antibody deficiencies (PADs) affecting B-cell development are associated with abnormalities in the composition and/or differentiation of B-cell compartments. The most recent international classifications of primary immunodeficiencies (PIDs) and common variable immunodeficiencies (CVID) have highlighted the importance of B-cell immunophenotyping and age-specific reference intervals for diagnostic purposes. We established national reference values for memory B-cell subpopulations, on the basis of CD27 and surface IgD expression in the peripheral blood of 242 healthy children. We report here the absolute counts and percentages of naive, switched and non-switched memory B-cells for seven age groups, from neonates to adults. We found that the naive B-cells percentage declined between the ages of 6 months and 8 years, after which it remained stable at about 70-80%. Memory B-cells are already present at birth and their numbers increase throughout childhood, stabilizing between the ages of 12 and 18 years. The definition of reference intervals for pediatric B-cell levels should facilitate the screening and diagnosis of various B-cell immunodeficiencies. This multicenter study, providing national reference values, should thus facilitate immunological diagnosis in children.

Keywords: B-cells; children; primary immunodeficiency; reference values.

Figure 1

Gating strategy for the analysis of B-cell subsets. Lymphocytes were gated according to forward and side scatter (A). B-cells were identified as CD19-expressing cells in the lymphocyte population (B). A CD27/CD19 dot plot defined total CD27⁺ CD19⁺ memory B-cells (C). The double staining of B-lymphocytes for CD27 and IgD made it possible to determine the percentages of naive B-cells (IgD⁺ CD27⁻), switched memory B-cells (IgD⁻ CD27⁺) and non-switched memory B-cells (IgD⁺ CD27⁺) (D).

Figure 2

Changes in the percentage (A) and absolute number (B) of total B-cells with age. The proportions of lymphocytes accounted for by total B-cells (CD19⁺) (A) were analyzed by flow cytometry on whole-blood samples. The corresponding absolute numbers (B) were calculated from the absolute numbers of lymphocytes. Solid horizontal lines indicate the median values for each age group. m = month; y = year.

Figure 3

Changes in the percentage (A) and absolute number (B) of naive B-cells according to age. The proportions of naive B-cells (CD27⁻ IgD⁺) (A) among CD19⁺ lymphocytes were analyzed by flow cytometry on whole-blood samples. The corresponding absolute numbers (B) were calculated from the absolute number of B-cells. Solid horizontal lines indicate the median values for each age group. m = month; y = year.

Figure 4

Changes in the percentage (A) and absolute number (B) of total memory B-cells with age. The proportions of total memory B-cells (CD27⁺) (A) among CD19⁺ lymphocytes were analyzed by flow cytometry on whole-blood samples. The corresponding absolute numbers (B) were calculated from the absolute number of B-cells. Solid horizontal lines indicate the median values for each age group. m = month; y = year.

Figure 5

Changes in the percentage and absolute number of memory B-cells subsets with age. The proportions of non-switched memory B-cells (CD27⁺ IgD⁺) (A) and switched memory B-cells (CD27⁺ IgD⁻) (C) among CD19⁺ lymphocytes were analyzed by flow cytometry on whole-blood samples. The corresponding absolute numbers (B and D) were calculated from the absolute number of B-cells. Solid horizontal lines indicate the median values for each age group. m = month; y = year.