Profiling of polychromatic flow cytometry data on B‐cells reveals patients' clusters in common variable immunodeficiency

Abstract

The aim of this study was to find an objective computational approach for phenotype analysis of common variable immunodeficiency (CVID) patients that describes all differences in the six-color space and to form groups of patients using computational methods. CVID is a heterogeneous primary immunodeficiency disorder where molecular defect is recognized in \10% of the cases and is unknown in the majority of patients. The current CVID classification, EUROClass, is based on quantification of selected B-cell subsets. Using six-color polychromatic flow cytometry, we analyzed B-cell phenotypes in a cohort of 48 CVID patients and 49 healthy donors. We used a ''probability binning'' algorithm to create 1,024 bins (each bin is a six-color gate) that covered the cells' distribution within the entire B-cell compartment. A matrix file recording cellular content in all the bins was made. The hierarchical clustering of the individual samples was analyzed using a Pearson correlation of the bins' values. The Cut tree algorithm found 12 clusters. In six clusters, healthy individuals predominated; in one cluster, smB1CD21low (CVID patients by EUROClass) cells prevailed; in one cluster, smB2CD21norm cells prevailed; in one cluster, smB1CD21low cells prevailed; the remaining cluster was mixed. The overall reproducibility of probability binning clustering was confirmed by matching of replicates to the original cohort using the similarity matrix of the Pearson correlation, 15 replicates matched the same individual, three replicates matched a different individual within the same cluster, and three replicates matched to a different cluster. We were able to define B-cell subsets over-or under-represented in a particular cluster and display them back in the flow cytometry software. We describe a new analytical approach that enables a search in an objective computational environment for patient cohorts that are defined by similar B-cell profiles and thus contribute to the description of differences between CVID patient groups. ' 2009 International Society for Advancement of CytometryKey terms B-lymphocyte subsets; common variable immunodeficiency; flow cytometry; probability binning; B-lymphocyte profiling THE peripheral blood B-cell compartment is a dynamic complex of B-cell subsets that undergo maturational steps, antigen exposure, memory cell formation, memory maintenance, and effector functions (antibody secretion). Polychromatic flow cytometry offers an extraordinary tool to investigate such a complex set of cell types. It allows for the definition and proportional assessment of B-cell subsets and also subdivides into less well-characterized intermediates. However, the analysis of complex data obtained from the six-color space is difficult. The standard analysis strategy uses the creation of series of two-dimensional gates, which makes the resulting analysis subjective, and ''a priori'' unknown small subsets are difficult to discover. Analysis of

Characterization of Lymphocyte Subsets in Patients with Common Variable Immunodeficiency Reveals Subsets of Naive Human B Cells Marked by CD24 Expression

“…The remaining 24 patients were placed in the CVID-21norm group, which was characterized by a normal frequency (,10%) of CD21 low CD38 low B cells. The raw FC data from these patients and the control cohort were used in a previously published technical study describing probability binning algorithms for the evaluation of multicolor FC data (28). FC data analysis was performed using the FlowJo 7.5 or 8.8.7 software (Tree Star, Ashland, OR).…”

“…The PBMCs were incubated for 15 min in the dark with anti-CD27 Pacific Blue, anti-CD38 Alexa-Fluor 700 (Exbio Praha a.s., Prague, Czech Republic), anti-human IgM FITC or anti-human IgM biotin (BD Pharmingen, San Jose, CA), followed by streptavidin-Qdot 605 (Invitrogen, Carlsbad, CA), anti-CD21 allophycocyanin (BD Pharmingen), anti-CD24 PE, and anti-CD19 PC7 (Immunotech, Marseille, France). After a final wash with PBS, two million PBMCs from each sample were acquired using a Cyan ADP flow cytometer (Dako, Glostrup, Denmark), as described in Kalina et al (28).…”

Flow Cytometry, a Versatile Tool for Diagnosis and Monitoring of Primary Immunodeficiencies

“…cluding susceptibility to sinopulmonary infections, granulomatous disease, lymphoproliferation, and autoimmunity (in subsets of patients) (88)(89)(90). Attempts have been made over the past decade or longer to classify CVID patients based on differences in peripheral B cell subsets, and some of these have provided clinical correlations, which may have prognostic value in subgroups of patients (91)(92)(93)(94)(95)(96)(97)(98). B cell subset analysis, in particular evaluation of memory B cells and switched memory B cells, has been incorporated into the newer diagnostic criteria as an accessory criterion (88), as the majority of CVID patients have impaired peripheral B cell differentiation and reduced class-switched memory B cells.…”

CYTO 2010—Overview of the XXV ISAC Congress Proceedings Issue of Cytometry Part A

“…Modern polychromatic flow cytometry can provide higher resolution approaches for the dissection and identification of cell subpopulations (1,2). In this issue, the paper by Autissier et al (page 410) describes how 12-color flow cytometry can be used for studying the phenotypes of lymphocyte, monocyte, and dendritic cell subsets.…”