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B-cell subpopulations in children: National reference values.

Duchamp M, Sterlin D, Diabate A, Uring-Lambert B, Guérin-El Khourouj V, Le Mauff B, Monnier D, Malcus C, Labalette M, Picard C - Immun Inflamm Dis (2014)

Bottom Line: 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%.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.

View Article: PubMed Central - PubMed

Affiliation: Study Center of Primary Immunodeficiencies, Assistance Publique-Hôpitaux de Paris (APHP), Necker Hospital Paris, France.

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.

No MeSH data available.


Related in: MedlinePlus

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).
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fig01: 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).

Mentions: Before subject inclusion, a standardized protocol was developed, to prevent inter-center bias. Soluble Ig was eliminated by washing 100 µL aliquots of whole blood three times with cell wash buffer (Becton Dickinson (BD), Rungis, France). The cells were then stained by incubation with monoclonal antibodies directed against CD19 (J3-119, Beckman), CD27 (M-T271, BD) and IgD (Dako R5112 or IA6-2, BD) for 30 min at room temperature. The erythrocytes were lysed with FACS Lysis buffer (BD) or Versalyse (Beckman Coulter), according to the manufacturer's instructions. The cells were washed twice in cell wash buffer (BD) and fixed in a cell fixation solution (BD). B-cell compartment analysis was performed within 24 h of fixation. Absolute numbers were calculated by multiplying the percentage of the subset concerned by the total number of lymphocytes obtained by flow cytometry. All analyses were performed on the cytometer available at the hospital concerned (FACS Canto II Becton Dickinson, Navios or FC500 Beckman Coulter). The gating strategies are explained in Figure 1. The total lymphocyte population was identified on the basis of forward (FSC) and side (SSC) scatter characteristics. B-cells were defined as CD19-expressing cells from the lymphocyte population. We analyzed the expression of IgD and CD27 on CD19+ B-cells. Naive B-cells were defined as CD27−IgD+ cells, non-switched memory B-cells were defined as CD27+IgD+ cells and switched memory B-cells were defined as CD27+IgD− cells.


B-cell subpopulations in children: National reference values.

Duchamp M, Sterlin D, Diabate A, Uring-Lambert B, Guérin-El Khourouj V, Le Mauff B, Monnier D, Malcus C, Labalette M, Picard C - Immun Inflamm Dis (2014)

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).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4257758&req=5

fig01: 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).
Mentions: Before subject inclusion, a standardized protocol was developed, to prevent inter-center bias. Soluble Ig was eliminated by washing 100 µL aliquots of whole blood three times with cell wash buffer (Becton Dickinson (BD), Rungis, France). The cells were then stained by incubation with monoclonal antibodies directed against CD19 (J3-119, Beckman), CD27 (M-T271, BD) and IgD (Dako R5112 or IA6-2, BD) for 30 min at room temperature. The erythrocytes were lysed with FACS Lysis buffer (BD) or Versalyse (Beckman Coulter), according to the manufacturer's instructions. The cells were washed twice in cell wash buffer (BD) and fixed in a cell fixation solution (BD). B-cell compartment analysis was performed within 24 h of fixation. Absolute numbers were calculated by multiplying the percentage of the subset concerned by the total number of lymphocytes obtained by flow cytometry. All analyses were performed on the cytometer available at the hospital concerned (FACS Canto II Becton Dickinson, Navios or FC500 Beckman Coulter). The gating strategies are explained in Figure 1. The total lymphocyte population was identified on the basis of forward (FSC) and side (SSC) scatter characteristics. B-cells were defined as CD19-expressing cells from the lymphocyte population. We analyzed the expression of IgD and CD27 on CD19+ B-cells. Naive B-cells were defined as CD27−IgD+ cells, non-switched memory B-cells were defined as CD27+IgD+ cells and switched memory B-cells were defined as CD27+IgD− cells.

Bottom Line: 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%.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.

View Article: PubMed Central - PubMed

Affiliation: Study Center of Primary Immunodeficiencies, Assistance Publique-Hôpitaux de Paris (APHP), Necker Hospital Paris, France.

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.

No MeSH data available.


Related in: MedlinePlus