Laboratory Assessment of Patients with Hypereosinophilic Syndrome

嗜酸性粒细胞增多综合征患者的实验室评估

• 批准号: 10684570
• 负责人: Irina Maric
• 金额: --
• 依托单位: CLINICAL CENTER
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10684570
• 关键词: Antibodies; Aspirate substance; Basophils; Biological Markers; Biopsy Specimen; Blood; Bone Marrow; CD34 gene; Cardiac; Cell Death; Cell Differentiation process; Cell Line; Cell Maturation; Cell Separation; Cells; Clinical; Development; Diagnosis; Disease; Disseminated eosinophilic collagen disease; Eosinophilia; Etiology; Evaluation; Flow Cytometry; Fluorescence; Hematopoietic; Human; ITGAM gene; In Vitro; Laboratories; Laboratory Study; Medical; Metamyelocyte; Molecular; Morbidity - disease rate; Morphology; Myelocyte; National Institute of Allergy and Infectious Disease; Neurologic; Pathogenicity; Pathology; Patients; Pattern; Population; Preparation; Prognosis; Progranulocytes; Rare Diseases; Specimen; Surface; Tissues; base; body system; eosinophil; evidence based guidelines; mRNA Expression; mast cell; mortality; new therapeutic target; novel; patient population; peripheral blood; receptor; selective expression; sialic acid binding Ig-like lectin; therapeutic target; treatment response

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.

除了嗜酸性粒细胞外，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得到很好的描述。 在2022财年，我们进行了一项研究，使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模式。我们设计了一组流式细胞术标记物，可以识别不同成熟阶段的嗜酸性前体细胞。一旦鉴定出候选成熟标志物，使用基于荧光的细胞分选分离推定的成熟和未成熟群体，并在细胞离心涂片制备物上从形态学上评价成熟阶段。 首先使用嗜酸性粒细胞特异性表面受体EMR-1、IL-5 Ra、Siglec-8的抗体验证所有成熟阶段的嗜酸性粒细胞谱系。 在过去，我们已经表明Siglec-8选择性地在外周血嗜酸性粒细胞、肥大细胞和嗜碱性粒细胞上表达。我们表征了正常供体和嗜酸性粒细胞患者的外周血中的表面和可溶性Siglec-8水平，并评估了抗Siglec-8抗体在体外诱导嗜酸性粒细胞死亡中的功效。通过使用流式细胞术和定量PCR评估Siglec-8的嗜酸性粒细胞表达。结果显示，Siglec-8在正常供体和嗜酸性粒细胞患者的外周血嗜酸性粒细胞上高度表达，并且可以代表用于治疗嗜酸性粒细胞病症的潜在治疗靶标。采用流式细胞术、定量PCR和免疫染色法，评估了嗜酸性粒细胞和健康受试者的血液和骨髓标本、细胞系、体外分化的CD 34阳性细胞和组织活检标本中EMR-1的表达。结果显示，人EMR-1的表面表达的限制，成熟的CD 34阴性嗜酸性粒细胞，这是证实了mRNA表达。 然后，我们使用EMR-1、IL-5 Ra、Siglec-8、CD 9、CD 11b、CCR 3、CD 49 d和CD 49 f抗体的组合来创建流式细胞术组，并在免疫表型上定义骨髓抽吸物中嗜酸性粒细胞成熟的不同阶段，其中最早的是嗜酸性早幼粒细胞/中幼粒细胞，其次是嗜酸性粒细胞/晚幼粒细胞，以及带状/成熟分节嗜酸性粒细胞。我们正在将此方法应用于嗜酸性粒细胞增多症患者的常规临床流式细胞术评价。