Laboratory Assessment of Patients with Hypereosinophilic Syndrome

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Abstract

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.