The EuroFlow PID Orientation Tube for Flow Cytometric Diagnostic Screening of Primary Immunodeficiencies of the Lymphoid System

Abstract

In the rapidly evolving field of primary immunodeficiencies (PID), the EuroFlow consortium decided to develop a PID orientation and screening tube that facilitates fast, standardized, and validated immunophenotypic diagnosis of lymphoid PID, and allows full exchange of data between centers. Our aim was to develop a tool that would be universal for all lymphoid PIDs and offer high sensitivity to identify a lymphoid PID (without a need for specificity to diagnose particular PID) and to guide and prioritize further diagnostic modalities and clinical management. The tube composition has been defined in a stepwise manner through several cycles of design-testing-evaluation-redesign in a multicenter setting. Equally important appeared to be the standardized pre-analytical procedures (sample preparation and instrument setup), analytical procedures (immunostaining and data acquisition), the software analysis (a multidimensional view based on a reference database in Infinicyt software), and data interpretation. This standardized EuroFlow concept has been tested on 250 healthy controls and 99 PID patients with defined genetic defects. In addition, an application of new EuroFlow software tools with multidimensional pattern recognition was designed with inclusion of maturation pathways in multidimensional patterns (APS plots). The major advantage of the EuroFlow approach is that data can be fully exchanged between different laboratories in any country of the world, which is especially of interest for the PID field, with generally low numbers of cases per center.

Keywords: EuroFlow; automated gating strategy; flow cytometric immunophenotyping; primary immunodeficiencies; standardization.

Figure 1

Flow cytometric analysis of B- and T-cell populations using the EuroFlow PID screening tube, 50 healthy controls are shown in the same plot, median of each gated subset is shown as a circle. (A) After gating leukocytes as CD45+ and lymphocytes on FSc and SSc, the markers CD3, CD19 in combination with TCRγδ and CD16+56 were used to define B-cells (orange); TCRγδ+ T-cells (lilac); TCRγδ- T-cells (blue); and NK cells (brown). (B) The T-cell subsets were further subdivided into naïve (CD27+CD45RA+; dark green), central memory/transitional memory (CM/TM; CD27+CD45RA-; bright green), effector memory (EM; CD27-CD45RA-; green) and terminally differentiated (TD; CD27-CD45RA+; light green) CD4+ T cells and into naïve (CD27+CD45RA+; purple), CM/TM (CD27+CD45RA-; dark blue), EM (CD27-CD45-; pale blue), and TD (CD27-CD45RA+; turqoise) CD8+ T cells. Also, as previously reported (5), some effector CD8+ T-cells showed dim CD27 positivity (EffCD27dim; CD27int-CD45RA+; blue). CD4/CD8 double negative T-cells are indicated in light blue. (C) B-cell subsets could be further subdivided into pre germinal center (PreGC; IgM⁺IgD⁺CD27⁻; orange) unswitched memory B-cells/plasma cells (Unswitched MBC/PC; IgM⁺IgD^+/−CD27⁺; yellow), switched memory MBC/PC (IgM⁻IgD⁻CD27⁺; pink). (D) Definition and hierarchy of the defined subsets. (E) Multidimensional view (APS view) based on the most discriminating parameters for lymphocytes, B-cell, T-cells, and T-cell subsets.

Figure 2

Flow cytometric analysis of B- and T-cell populations using the EuroFlow PIDOT in 250 healthy controls in 14 different age ranges. All values of this reference data set are displayed as bar graphs representing the median, minimum, maximum, and p10, p25, p75, and p90 percentiles. For data visualization package gplot2 for the statistical language R was used (10).

Figure 3

Flowcytometric analysis of B- and T-cell populations using the EuroFlow PID screening tube on controls and patients with SCID. From the top down, APS plots of gated lymphocytes, B-cells, CD3+ T cells and TCRgd- T cells are shown. Lines depict a 2 standard deviation boundary of all controls combined. (A) Multidimensional views of all lymphocyte subsets of a newborn, an infant, one Artemis-deficient and four RAG-deficient SCID patients. (B) Multidimensional views of all lymphocyte subsets of a newborn, one IL7RA-deficient, three IL2RG-deficient and a ZAP70-deficient patient.

Figure 4

Multidimensional views of all lymphocyte subsets in healthy controls and PID patients. (A) Multidimensional views of all lymphocyte subsets of two BTK-deficient patients, a CD40L deficiency, a patient with ALPS due to a mutation in FAS and healthy infants of 6 months and 3 years. (B) Multidimensional views of all lymphocyte subsets of single examples of patients with Wiskott Aldrich syndrome (WASp), Ataxia Telangiectasia (ATM), DOCK8 deficiency and two patients with DiGeorge syndrome.

Figure 5

Multidimensional views of all lymphocyte subsets in combination with the absolute values of all subpopulations plotted as red dots in the age-matched reference bar graphs after automated analysis. (APDS) Patients with activated PI3K delta syndrome (APDS). (GATA2) Patient with GATA2 deficiency.