Application of Cytometry in Primary Immunodeficiencies

Flow cytometry has emerged as a useful technology that has facilitated our understanding of the human immune system. Primary immune deficiency disorders (PIDDs) are a heterogeneous group of inherited disorders affecting the immune system. More than 350 genes causing various PIDDs have been identified. While the initial suspicion and recognition of PIDDs is clinical, laboratory tools such as flow cytometry and genetic sequencing are essential for confirmation and categorization. Genetic sequencing, however, are prohibitively expensive and not readily available in resource constrained settings. Flow cytometry remains a simple, yet powerful, tool for multi-parametric analysis of cells. While it is confirmatory of diagnosis in certain conditions, in others it helps in narrowing the list of putative genes to be analyzed. The utility of flow cytometry in diagnosis of PIDDs can be divided into four major categories: (a) Enumeration of lymphocyte subsets in peripheral blood. (b) Detection of intracellular signaling molecules, transcription factors, and cytokines. (c) Functional assessment of adaptive and innate immune cells (e.g., T cell function in severe combined immune deficiency and natural killer cell function in familial hemophagocytic lymphohistiocytosis). (d) Evaluation of normal biological processes (e.g., class switching in B cells by B cell immunophenotyping). This review focuses on use of flow cytometry in disease-specific diagnosis of PIDDs in the context of a developing country.

The advent of flow cytometry has revolutionized 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 signaling 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 utilized to measure immune cell function in patients with PIDs, such as degranulation by cytotoxic cells, cytokine expression by many immune cells (i.e., CD4⁺ and CD8⁺ T cells, macrophages etc.), B-cell differentiation, and phagocyte respiratory burst in vitro. These assays can also be performed using unfractionated PBMCs, provided the caveat that the composition of lymphocytes between healthy donors and the PID patients under investigation is recognized. 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 unraveling the pathophysiology of PIDs.

Primary immunodeficiency diseases (PID) are a clinically and immunologically heterogeneous group of disorders of immune system. Diagnosis of these disorders is often challenging and requires identification of underlying genetic defects, complemented by a comprehensive evaluation of immune system. Flow cytometry, with its advances in the last few decades, has emerged as an indispensable tool for enumeration as well as characterization of immune cells. Flow cytometric evaluation of the immune system not only provides clues to underlying genetic defects in certain PIDs and helps in functional validation of novel genetic defects, but is also useful in monitoring immune responses following specific therapies. India has witnessed significant progress in the field of flow cytometry as well as PID over last one decade. Currently, there are seven Federation of Primary Immunodeficiency Diseases (FPID) recognized centers across India, including two Indian Council of Medical research (ICMR) funded centers of excellence for diagnosis, and management of PIDs. These centers offer comprehensive care for PIDs including flow cytometry based evaluation. The key question which always remains is how one selects from the wide array of flow cytometry based tests available, and whether all these tests should be performed before or after the identification of genetic defects. This becomes crucial, especially when resources are limited and patients have to pay for the investigations. In this review, we will share some of our experiences based on evaluation of a large cohort of hemophagocytic lymphohistiocytosis, severe combined immunodeficiency, and chronic granulomatous disease, and the lessons learned for optimum use of this powerful technology for diagnosis of these disorders.