Immunology Division, Garvan Institute of Medical Research, Sydney, NSW, Australia.
Faculty of Medicine, St. Vincent's Clinical School, UNSW Sydney, Sydney, NSW, Australia.
Front Immunol. 2019 Sep 4;10:2108. doi: 10.3389/fimmu.2019.02108. eCollection 2019.
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 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 . 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.
流式细胞术的出现彻底改变了我们研究和回答特定科学问题的方式。流式细胞仪也已成为全球大多数医院和病理实验室的主流诊断工具。特别是流式细胞术的应用对于诊断由造血系统关键基因的单基因突变引起的原发性免疫缺陷症（PID）起到了重要作用，偶尔也会涉及非造血系统。流式细胞术的广泛适用性部分归因于基于流式的检测方法的惊人灵敏度，低至单细胞水平，以及能够进行全面分析、数据解释的极其用户友好的平台，并且重要的是，为诊断 PID 提供了快速而稳健的方法。一个很好的例子是，由于 BCR 信号通路中的 或相关基因突变，导致无丙种球蛋白血症患者外周血 B 细胞缺失。同样，为了检测 SAP、XIAP 或 DOCK8 的表达而开发的细胞内染色方案，分别加速了 X 连锁淋巴组织增生性疾病或常染色体隐性遗传高 IgE 综合征（HIES）的快速诊断。此外，已经明显的是，不同队列的 PID 患者表现出独特的“淋巴细胞表型特征”，即使在确定遗传病变之前，这些特征通常也具有诊断意义。基于流式的分选提供了一种分离特定免疫细胞亚群的技术，以便可以对其进行单独研究。因此，流式细胞术检测可以用于测量 PID 患者的免疫细胞功能，例如细胞毒性细胞的脱颗粒、许多免疫细胞（例如 CD4 和 CD8 T 细胞、巨噬细胞等）的细胞因子表达、B 细胞分化和吞噬细胞呼吸爆发。这些检测也可以使用未分馏的 PBMC 进行，前提是要认识到健康供体和正在研究的 PID 患者之间淋巴细胞组成的差异。这些功能缺陷不仅有助于 PID 的临床诊断，还揭示了疾病发病机制的机制。随着我们进入下一代多参数流式细胞仪时代，在这里我们将回顾一下我们在 PID 的研究、诊断和揭示病理生理学方面使用流式细胞术的一些经验。
