INTRODUCTION: Common Variable Immunodeficiency (CVID) is characterized by defective antibody production and hypogammaglobulinemia. Flow cytometry immunophenotyping of blood lymphocytes has become of great relevance for the diagnosis and classification of CVID, due to an impaired differentiation of mature post-germinal-center (GC) class-switched memory B-cells (MBC) and severely decreased plasmablast/plasma cell (Pb) counts. Here, we investigated in detail the pre-GC B-cell maturation compartment in blood of CVID patients. METHODS: In this collaborative multicentric study the EuroFlow PID 8-color Pre-GC B-cell tube, standardized sample preparation procedures (SOPs) and innovative data analysis tools, were used to characterize the maturation profile of pre-GC B-cells in 100 CVID patients, vs 62 age-matched healthy donors (HD). RESULTS: The Pre-GC B-cell tube allowed identification within pre-GC B-cells of three subsets of maturation associated immature B-cells and three subpopulations of mature naïve B-lymphocytes. CVID patients showed overall reduced median absolute counts (vs HD) of the two more advanced stages of maturation of both CD5+ CD38+/++ CD21het CD24++ (2.7 vs 5.6 cells/µl, p=0.0004) and CD5+ CD38het CD21+ CD24+ (6.5 vs 17 cells/µl, p<0.0001) immature B cells (below normal HD levels in 22% and 37% of CVID patients). This was associated with an expansion of CD21-CD24- (6.1 vs 0.74 cells/µl, p<0.0001) and CD21-CD24++ (1.8 vs 0.4 cells/µl, p<0.0001) naïve B-cell counts above normal values in 73% and 94% cases, respectively. Additionally, reduced IgMD+ (21 vs 32 cells/µl, p=0.03) and IgMD- (4 vs 35 cells/µl, p<0.0001) MBC counts were found to be below normal values in 25% and 77% of CVID patients, respectively, always together with severely reduced/undetectable circulating blood pb. Comparison of the maturation pathway profile of pre-GC B cells in blood of CVID patients vs HD using EuroFlow software tools showed systematically altered patterns in CVID. These consisted of: i) a normally-appearing maturation pathway with altered levels of expression of >1 (CD38, CD5, CD19, CD21, CD24, and/or smIgM) phenotypic marker (57/88 patients; 65%) for a total of 3 distinct CVID patient profiles (group 1: 42/88 patients, 48%; group 2: 8/88, 9%; and group 3: 7/88, 8%) and ii) CVID patients with a clearly altered pre-GC B cell maturation pathway in blood (group 4: 31/88 cases, 35%). CONCLUSION: Our results show that maturation of pre-GC B-cells in blood of CVID is systematically altered with up to four distinctly altered maturation profiles. Further studies, are necessary to better understand the impact of such alterations on the post-GC defects and the clinical heterogeneity of CVID.

• Keywords
• CVID, EuroFlow standardization, Pre-GC B-cell tube, expression markers, pre-GC maturation pathway,
• MeSH
• Common Variable Immunodeficiency diagnosis   immunology   metabolism   MeSH
• Antigens, CD analysis   MeSH
• Adult MeSH
• Phenotype MeSH
• Immunophenotyping * MeSH
• Middle Aged MeSH
• Humans MeSH
• Adolescent MeSH
• Young Adult MeSH
• Precursor Cells, B-Lymphoid immunology   metabolism   MeSH
• Flow Cytometry * MeSH
• Aged, 80 and over MeSH
• Aged MeSH
• Case-Control Studies MeSH
• Check Tag
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Adolescent MeSH
• Young Adult MeSH
• Male MeSH
• Aged, 80 and over MeSH
• Aged MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Multicenter Study MeSH
• Research Support, Non-U.S. Gov't MeSH
• Geographicals
• Europe MeSH
• Names of Substances
• Antigens, CD MeSH

Guidelines for screening for primary immunodeficiencies (PID) are well-defined and several consensus diagnostic strategies have been proposed. These consensus proposals have only partially been implemented due to lack of standardization in laboratory procedures, particularly in flow cytometry. The main objectives of the EuroFlow Consortium were to innovate and thoroughly standardize the flowcytometric techniques and strategies for reliable and reproducible diagnosis and classification of PID of the lymphoid system. The proposed EuroFlow antibody panels comprise one orientation tube and seven classification tubes and corresponding databases of normal and PID samples. The 8-color 12-antibody PID Orientation tube (PIDOT) aims at identification and enumeration of the main lymphocyte and leukocyte subsets; this includes naïve pre-germinal center (GC) and antigen-experienced post-GC memory B-cells and plasmablasts. The seven additional 8(-12)-color tubes can be used according to the EuroFlow PID algorithm in parallel or subsequently to the PIDOT for more detailed analysis of B-cell and T-cell subsets to further classify PID of the lymphoid system. The Pre-GC, Post-GC, and immunoglobulin heavy chain (IgH)-isotype B-cell tubes aim at identification and enumeration of B-cell subsets for evaluation of B-cell maturation blocks and specific defects in IgH-subclass production. The severe combined immunodeficiency (SCID) tube and T-cell memory/effector subset tube aim at identification and enumeration of T-cell subsets for assessment of T-cell defects, such as SCID. In case of suspicion of antibody deficiency, PIDOT is preferably directly combined with the IgH isotype tube(s) and in case of SCID suspicion (e.g., in newborn screening programs) the PIDOT is preferably directly combined with the SCID T-cell tube. The proposed ≥8-color antibody panels and corresponding reference databases combined with the EuroFlow PID algorithm are designed to provide fast, sensitive and cost-effective flowcytometric diagnosis of PID of the lymphoid system, easily applicable in multicenter diagnostic settings world-wide.

• Keywords
• EuroFlow, classification, diagnosis, flow cytometry, immunodeficiency, immunophenotyping, standardization,
• MeSH
• B-Lymphocytes immunology   MeSH
• Child MeSH
• Adult MeSH
• Immunologic Memory immunology   MeSH
• Infant MeSH
• Leukocytes immunology   MeSH
• Middle Aged MeSH
• Humans MeSH
• Lymphocytes immunology   MeSH
• Adolescent MeSH
• Young Adult MeSH
• Infant, Newborn MeSH
• Neonatal Screening methods   MeSH
• Plasma Cells immunology   MeSH
• Child, Preschool MeSH
• Primary Immunodeficiency Diseases diagnosis   immunology   MeSH
• Flow Cytometry methods   MeSH
• Aged MeSH
• T-Lymphocytes immunology   MeSH
• Severe Combined Immunodeficiency diagnosis   immunology   MeSH
• Check Tag
• Child MeSH
• Adult MeSH
• Infant MeSH
• Middle Aged MeSH
• Humans MeSH
• Adolescent MeSH
• Young Adult MeSH
• Male MeSH
• Infant, Newborn MeSH
• Child, Preschool MeSH
• Aged MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH