Laboratory Assessment of Patients with Hypereosinophilic Syndrome

人类骨髓中大多数正常造血细胞前体细胞的免疫表型分化模式已被很好地描述，但嗜酸性粒细胞除外。 在22财年，我们进行了一项研究，用多参数流式细胞术分析来阐明骨髓中嗜酸性粒细胞成熟的免疫表型。我们设计了一组流式细胞仪标记，可以识别不同成熟阶段的嗜酸性粒细胞前体细胞。一旦确定了候选成熟标记，就使用基于荧光的细胞分选来分离假定的成熟和未成熟群体，并在细胞旋蛋白制剂上对成熟阶段进行形态评估。 首先使用嗜酸性粒细胞表面受体EMR-1、IL-5RA、Siglec-8的抗体证实了所有成熟阶段的嗜酸性粒细胞谱系。在过去，我们已经证明Siglec-8选择性地表达于外周血嗜酸性粒细胞、肥大细胞和嗜碱性粒细胞。我们测定了正常供者和嗜酸性粒细胞患者外周血中表面和可溶性Siglec-8的水平，并评估了抗Siglec-8抗体在体外诱导嗜酸性粒细胞死亡的效果。用流式细胞仪和定量聚合酶链式反应检测Siglec-8嗜酸性粒细胞的表达。结果表明，Siglec-8在正常献血者和嗜酸性粒细胞患者外周血嗜酸粒细胞上均有高表达，可作为治疗嗜酸性粒细胞疾病的潜在靶点。采用流式细胞术、定量聚合酶链式反应和免疫组织化学染色等方法，检测嗜酸性粒细胞和健康人的血液和骨髓标本、细胞系、体外分化的CD34阳性细胞和组织活检标本中EMR-1的表达。结果显示，人EMR-1表面表达对成熟的CD34阴性嗜酸性粒细胞有限制作用，这一点在mRNA表达上得到了证实。 然后，我们使用EMR-1、IL-5RA、Siglec-8、CD9、CD11b、CCR3、CD49d和CD49f抗体的组合创建了一个流式细胞仪小组，免疫表型典型地定义了骨髓抽吸物中嗜酸性粒细胞成熟的不同阶段，最早的是嗜酸性早幼粒细胞/粒细胞，其次是嗜酸性粒细胞/变淀粉细胞和条带/成熟节段性嗜酸性粒细胞。我们正在将这个小组应用到对嗜酸性粒细胞增多症患者的常规临床流式细胞术评估中。

Immunophenotypic differentiation patterns of most normal hematopoietic cell precursors in human bone marrow have been well described, with the exception of eosinophils. In FY22 we conducted a study to elucidate immunophenotypic patterns of eosinophil maturation in bone marrows using multiparameter flow cytometric analysis. We devised a panel of flow cytometric markers that could identify eosinophilic precursors at different stages of maturation. Once candidate maturation markers were identified, putative mature and immature populations were separated using fluorescence based cell sorting and maturational stages were evaluated morphologically on the cytospin preparations. The eosinophilic lineage was first verified for all maturation stages using antibodies to the eosinophil-specific surface receptors EMR-1, IL-5Ra, Siglec-8. In the past, we have shown that Siglec-8 is selectively expressed on peripheral blood eosinophils, mast cells, and basophils. We characterized surface and soluble Siglec-8 levels in the peripheral blood of normal donors and eosinophilic patients and assessed the efficacy of anti-Siglec-8 antibodies in inducing eosinophil cell death in vitro. Eosinophil expression of Siglec-8 was assessed by using flow cytometry and quantitative PCR. The results showed that Siglec-8 is highly expressed on peripheral blood eosinophils in both normal donors and eosinophilic patients and can represents a potential therapeutic target for treatment of eosinophilic disorders. EMR-1 expression was assessed in the blood and bone marrow specimens from eosinophilic and healthy subjects, cell lines, CD34 positive cells differentiated in vitro, and tissue biopsy specimens by using flow cytometry, quantitative PCR, and immunostaining. Results revealed restriction of human EMR-1 surface expression to maturing CD34 negative eosinophils, which was confirmed by mRNA expression. We then used a combination of EMR-1, IL-5Ra, Siglec-8, CD9, CD11b, CCR3, CD49d and CD49f antibodies to create a flow cytometric panel and immunophenotypically define different stages of eosinophilic maturation in bone marrow aspirates, with the earliest being eosinophilic promyelocyte/myelocyte, followed by eosinophilic myelocyte/metamyelocyte, and band/mature segmented eosinophils. We are implementing this panel into routine clinical flow cytometric evaluations of patients with eosinophilia.