Spleen function is reduced in individuals with NR5A1 variants with or without a difference of sex development: a cross-sectional study.

OBJECTIVE: NR5A1 is a key regulator of sex differentiation and has been implicated in spleen development through transcription activation of TLX1. Concerns exist about hypo- or asplenism in individuals who have a difference of sex development (DSD) due to an NR5A1 disease-causing variant. We aimed to assess spleen anatomy and function in a clinical cohort of such individuals and in their asymptomatic family member carriers. DESIGN: Cross-sectional assessment in 22 patients with a DSD or primary ovarian insufficiency and 5 asymptomatic carriers from 18 families, harboring 14 different NR5A1 variants. METHODS: Spleen anatomy was assessed by ultrasound, spleen function by peripheral blood cell count, white blood cell differentiation, percentage of nonswitched memory B cells, specific pneumococcal antibody response, % pitted red blood cells, and Howell-Jolly bodies. RESULTS: Patients and asymptomatic heterozygous individuals had significantly decreased nonswitched memory B cells compared to healthy controls, but higher than asplenic patients. Thrombocytosis and spleen hypoplasia were present in 50% of heterozygous individuals. Four out of 5 individuals homozygous for the previously described p.(Arg103Gln) variant had asplenia. CONCLUSIONS: Individuals harboring a heterozygous NR5A1 variant that may cause DSD have a considerable risk for functional hyposplenism, irrespective of their gonadal phenotype. Splenic function should be assessed in these individuals, and if affected or unknown, prophylaxis is recommended to prevent invasive encapsulated bacterial infections. The splenic phenotype associated with NR5A1 variants is more severe in homozygous individuals and is, at least for the p.(Arg103Gln) variant, associated with asplenism.

In-depth blood immune profiling of Good syndrome patients.

Introduction: Good syndrome (GS) is a rare adult-onset immunodeficiency first described in 1954. It is characterized by the coexistence of a thymoma and hypogammaglobulinemia, associated with an increased susceptibility to infections and autoimmunity. The classification and management of GS has been long hampered by the lack of data about the underlying immune alterations, a controversy existing on whether it is a unique diagnostic entity vs. a subtype of Common Variable Immune Deficiency (CVID). Methods: Here, we used high-sensitive flow cytometry to investigate the distribution of up to 70 different immune cell populations in blood of GS patients (n=9) compared to age-matched CVID patients (n=55) and healthy donors (n=61). Results: All 9 GS patients displayed reduced B-cell counts -down to undetectable levels (<0.1 cells/µL) in 8/9 cases-, together with decreased numbers of total CD4+ T-cells, NK-cells, neutrophils, and basophils vs. age-matched healthy donors. In contrast, they showed expanded TCRγδ+ T-cells (p ≤ 0.05). Except for a deeper B-cell defect, the pattern of immune cell alteration in blood was similar in GS and (age-matched) CVID patients. In depth analysis of CD4+ T-cells revealed significantly decreased blood counts of naïve, central memory (CM) and transitional memory (TM) TCD4+ cells and their functional compartments of T follicular helper (TFH), regulatory T cells (Tregs), T helper (Th)2, Th17, Th22, Th1/Th17 and Th1/Th2 cells. In addition, GS patients also showed decreased NK-cell, neutrophil, basophil, classical monocyte and of both CD1c+ and CD141+ myeloid dendritic cell counts in blood, in parallel to an expansion of total and terminal effector TCRγδ+ T-cells. Interestingly, those GS patients who developed hypogammaglobulinemia several years after the thymoma presented with an immunological and clinical phenotype which more closely resembled a combined immune humoral and cellular defect, with poorer response to immunoglobulin replacement therapy, as compared to those in whom the thymoma and hypogammaglobulinemia were simultaneously detected. Discussion: Our findings provide a more accurate definition of the immune cell defects of GS patients and contribute to a better discrimination among GS patients between those with a pure B-cell defect vs. those suffering from a combined immunodeficiency with important consequences on the diagnosis and management of the disease.

The Euroflow PID Orientation Tube in the diagnostic workup of primary immunodeficiency: Daily practice performance in a tertiary university hospital.

Introduction: Multiparameter flow cytometry (FCM) immunophenotyping is an important tool in the diagnostic screening and classification of primary immunodeficiencies (PIDs). The EuroFlow Consortium recently developed the PID Orientation Tube (PIDOT) as a universal screening tool to identify lymphoid-PID in suspicious patients. Although PIDOT can identify different lymphoid-PIDs with high sensitivity, clinical validation in a broad spectrum of patients with suspicion of PID is missing. In this study, we investigated the diagnostic performance of PIDOT, as part of the EuroFlow diagnostic screening algorithm for lymphoid-PID, in a daily practice at a tertiary reference center for PID. Methods: PIDOT was tested in 887 consecutive patients suspicious of PID at the Ghent University Hospital, Belgium. Patients were classified into distinct subgroups of lymphoid-PID vs. non-PID disease controls (non-PID DCs), according to the IUIS and ESID criteria. For the clinical validation of PIDOT, comprehensive characterization of the lymphoid defects was performed, together with the identification of the most discriminative cell subsets to distinguish lymphoid-PID from non-PID DCs. Next, a decision-tree algorithm was designed to guide subsequent FCM analyses. Results: The mean number of lymphoid defects detected by PIDOT in blood was 2.87 times higher in lymphoid-PID patients vs. non-PID DCs (p < 0.001), resulting in an overall sensitivity and specificity of 87% and 62% to detect severe combined immunodeficiency (SCID), combined immunodeficiency with associated or syndromic features (CID), immune dysregulation disorder (ID), and common variable immunodeficiency (CVID). The most discriminative populations were total memory and switched memory B cells, total T cells, TCD4+cells, and naive TCD4+cells, together with serum immunoglobulin levels. Based on these findings, a decision-tree algorithm was designed to guide further FCM analyses, which resulted in an overall sensitivity and specificity for all lymphoid-PIDs of 86% and 82%, respectively. Conclusion: Altogether, our findings confirm that PIDOT is a powerful tool for the diagnostic screening of lymphoid-PID, particularly to discriminate (S)CID, ID, and CVID patients from other patients suspicious of PID. The combination of PIDOT and serum immunoglobulin levels provides an efficient guide for further immunophenotypic FCM analyses, complementary to functional and genetic assays, for accurate PID diagnostics.

Improved Standardization of Flow Cytometry Diagnostic Screening of Primary Immunodeficiency by Software-Based Automated Gating.

Background: Multiparameter flow cytometry (FC) is essential in the diagnostic work-up and classification of primary immunodeficiency (PIDs). The EuroFlow PID Orientation tube (PIDOT) allows identification of all main lymphocyte subpopulations in blood. To standardize data analysis, tools for Automated Gating and Identification (AG&I) of the informative cell populations, were developed by EuroFlow. Here, we evaluated the contribution of these innovative AG&I tools to the standardization of FC in the diagnostic work-up of PID, by comparing AG&I against expert-based (EuroFlow-standardized) Manual Gating (MG) strategy, and its impact on the reproducibility and clinical interpretation of results. Methods: FC data files from 44 patients (13 CVID, 12 PID, 19 non-PID) and 26 healthy donor (HD) blood samples stained with PIDOT were analyzed in parallel by MG and AG&I, using Infinicyt™ software (Cytognos). For comparison, percentage differences in absolute cell counts/µL were calculated for each lymphocyte subpopulation. Data files showing differences >20% were checked for their potential clinical relevance, based on age-matched percentile (p5-p95) reference ranges. In parallel, intra- and inter-observer reproducibility of MG vs AG&I were evaluated in a subset of 12 samples. Results: The AG&I approach was able to identify the vast majority of lymphoid events (>99%), associated with a significantly higher intra- and inter-observer reproducibility compared to MG. For most HD (83%) and patient (68%) samples, a high degree of agreement (<20% numerical differences in absolute cell counts/µL) was obtained between MG and the AG&I module. This translated into a minimal impact (<5% of observations) on the final clinical interpretation. In all except three samples, extended expert revision of the AG&I approach revealed no error. In the three remaining samples aberrant maturation and/or abnormal marker expression profiles were seen leading in all three cases to numerical alarms by AG&I. Conclusion: Altogether, our results indicate that replacement of MG by the AG&I module would be associated with a greater reproducibility and robustness of results in the diagnostic work-up of patients suspected of PID. However, expert revision of the results of AG&I of PIDOT data still remains necessary in samples with numerical alterations and aberrant B- and T-cell maturation and/or marker expression profiles.