EuroFlow Standardized Approach to Diagnostic Immunopheneotyping of Severe PID in Newborns and Young Children.

Tomáš Kalina ,Tomáš Freiberger,Anna Šedivá,Maartje Blom,Jan Philippé,Jakub Tkazcyk,Sonia Arriba-Méndez,Veronika Kanderová,Carolien Bonroy,Beata Wolska-Kuśnierz,Jitse H M P Paping,Filomeen Haerynck,Renata Formánková,Marina Bakardjieva,Mirjam Van Der Burg,Ingrid Pico-Knijnenburg,Michael Svatoň,Himmet Haluk Akar,Alberto Órfão ,E Blanco ,Małgorzata Pac ,Jacques J.m Van Dongen ,Barbara H Barendregt ,Martín Pérez‐Andrés

doi:10.3389/fimmu.2020.00371

Abstract

The EuroFlow PID consortium developed a set of flow cytometry tests for evaluation of patients with suspicion of primary immunodeficiency (PID). In this technical report we evaluate the performance of the SCID-RTE tube that explores the presence of recent thymic emigrants (RTE) together with T-cell activation status and maturation stages and discuss its applicability in the context of the broader EuroFlow PID flow cytometry testing algorithm for diagnostic orientation of PID of the lymphoid system. We have analyzed peripheral blood cells of 26 patients diagnosed between birth and 2 years of age with a genetically defined primary immunodeficiency disorder: 15 severe combined immunodeficiency (SCID) patients had disease-causing mutations in RAG1 or RAG2 (n = 4, two of them presented with Omenn syndrome), IL2RG (n = 4, one of them with confirmed maternal engraftment), NHEJ1 (n = 1), CD3E (n = 1), ADA (n = 1), JAK3 (n = 3, two of them with maternal engraftment) and DCLRE1C (n = 1) and 11 other PID patients had diverse molecular defects [ZAP70 (n = 1), WAS (n = 2), PNP (n = 1), FOXP3 (n = 1), del22q11.2 (DiGeorge n = 4), CDC42 (n = 1) and FAS (n = 1)]. In addition, 44 healthy controls in the same age group were analyzed using the SCID-RTE tube in four EuroFlow laboratories using a standardized 8-color approach. RTE were defined as CD62L+CD45RO-HLA-DR-CD31+ and the activation status was assessed by the expression of HLA-DR+. Naïve CD8+ T-lymphocytes and naïve CD4+ T-lymphocytes were defined as CD62L+CD45RO-HLA-DR-. With the SCID-RTE tube, we identified patients with PID by low levels or absence of RTE in comparison to controls as well as low levels of naïve CD4+ and naïve CD8+ lymphocytes. These parameters yielded 100% sensitivity for SCID. All SCID patients had absence of RTE, including the patients with confirmed maternal engraftment or oligoclonally expanded T-cells characteristic for Omenn syndrome. Another dominant finding was the increased numbers of activated CD4+HLA-DR+ and CD8+HLA-DR+ lymphocytes. Therefore, the EuroFlow SCID-RTE tube together with the previously published PIDOT tube form a sensitive and complete cytometric diagnostic test suitable for patients suspected of severe PID (SCID or CID) as well as for children identified via newborn screening programs for SCID with low or absent T-cell receptor excision circles (TRECs).

Highlights

Severe combined immunodeficiency (SCID) and combined immunodeficiency (CID) are two of the most severe forms of inherited disorders of the immune system [1, 2] with an incidence of 1:35,000–50,000 newborns
The SCID-RTE tube was designed with the purpose of identifying the relevant lymphoid subpopulations important in primary immunodeficiencies (PID) diagnostics of severe PID in newborns, using a single 8-color test
The CD4+ T-cells were further subdivided into RTE, naïve, central memory (CM), effector memory CD45RO+ (EMRO+) and CD45RO- (EMRO-) and activated memory T-cells (Figures 1B,D); for CD8+ Tcells the same subsets were defined except for the RTEs (Figures 1C,D)

Summary

Introduction

Severe combined immunodeficiency (SCID) and combined immunodeficiency (CID) are two of the most severe forms of inherited disorders of the immune system [1, 2] with an incidence of 1:35,000–50,000 newborns. Patients are usually born asymptomatic, but they develop severe (opportunistic) infections, failure to thrive within the first months of life and generally die before the age of 1 year, unless they receive adequate and curative treatment. This includes hematopoietic stem cell transplantation (HSCT). Patients with CID usually do not have complete absence of T-lymphocytes as typically seen in SCID, but they frequently show profound impairment of T-cell immunity leading to severe infections, autoimmunity, and malignancies. The indication of HSCT for CID is less clear as it is less evident whether the T-cell deficiency is sufficiently severe to justify the risks of HSCT [7]

Methods

Results

Conclusion