AUTHOR=Ma Cindy S. , Tangye Stuart G. 

TITLE=Flow Cytometric-Based Analysis of Defects in Lymphocyte Differentiation and Function Due to Inborn Errors of Immunity

JOURNAL=Frontiers in Immunology

VOLUME=Volume 10 - 2019

YEAR=2019

URL=https://www.frontiersin.org/journals/immunology/articles/10.3389/fimmu.2019.02108

DOI=10.3389/fimmu.2019.02108

ISSN=1664-3224

ABSTRACT=The advent of flow cytometry has revolutionised the way we approach our research and answer specific scientific questions. The flow cytometer has also become a mainstream diagnostic tool in most hospital and pathology laboratories around the world. In particular the application of flow cytometry has been instrumental to the diagnosis of primary immunodeficiencies (PIDs) that result from monogenic mutations in key genes of the hematopoietic, and occasionally non-hematopoietic, systems. The far-reaching applicability of flow cytometry is in part due to the remarkable sensitivity, down to the single-cell level, of flow-based assays and the extremely user-friendly platforms that enable comprehensive analysis, data interpretation and, importantly, robust and rapid methods for diagnosing PIDs. A prime example is the absence of peripheral blood B cells in patients with agammaglobulinemia due to mutations in BTK or related genes in the BCR signalling pathway. Similarly, the development of intracellular staining protocols to detect expression of SAP, XIAP or DOCK8 expedites the rapid diagnosis of the X-linked lymphoproliferative diseases or an autosomal recessive form of hyper-IgE syndrome (HIES), respectively. It has also become evident that distinct cohorts of PID patients exhibit unique “lymphocyte phenotypic signatures” that are often diagnostic even prior to identifying the genetic lesion. Flow cytometry-based sorting provides a technique for separating specific subsets of immune cells such that they can be studied in isolation. Thus, flow-based assays can be utilised to measure immune cell function in patients with PIDs, such as degranulation by cytotoxic cells, cytokine expression by many immune cells (ie CD4+ and CD8+ T cells, macrophages etc), and B-cell differentiation in vitro. These functional deficits can assist not only in the clinical diagnosis of PIDs, but also reveal mechanisms of disease pathogenesis. As we move into the next generation of multiparameter flow cytometers, here we review some of our experiences in the use of flow cytometry in the study, diagnosis and unravelling the pathophysiology of PIDs.