Abstract
Background: The immunophenotypic differentiation patterns of most hematopoietic cell types in human bone marrow have been well described. Flow cytometric changes in these patterns are regularly used in the diagnosis of multiple hematopoietic malignancies. However, immunophenotypic differentiation patterns of human eosinopoiesis have not been well elucidated, even though eosinophils play an important role in many disease processes, including a number of hematopoietic neoplasms. We have devised a novel flow cytometric antibody panel that identifies normal immunophenotypic patterns of eosinophilic maturation in the bone marrow. We then used this panel to elucidate changes in eosinophilic maturation in two rare hematopoietic disorders: hypereosinophilic syndrome (HES) and systemic mastocytosis (SM).
Design: We performed multiparameter flow cytometric analysis of 50 bone marrow aspirates using candidate maturation markers (CD9, CD11b, CD13, CD34, CD45, CD49d, CD49f, CD69, CCR3, EMR1, Siglec 8, CD117, CD125) in normal donors and patients with HES and SM. The normal group consisted of 15 healthy donors (8 M/7 F; median age 43 yrs), with mean bone marrow eosinophils 3.7%. The HES group consisted of 20 patients (9 M/11 F; median age 46 yrs) with mean bone marrow eosinophils 16.0%. The SM group consisted of 15 patients (8 M/7 F; median age 46 yrs), with mean bone marrow eosinophils 6.6%. After analysis of immunophenotypic patterns of eosinophil maturation, we confirmed eosinophil maturation stages by flow cytometric sorting and morphologic evaluation of Wright Giemsa stained cytospins.
Results: In normal eosinophils, Siglec 8, CD11b, CD9, CCR3, CD49d and CD49f displayed the highest statistically significant differences in mean fluorescence intensities (MFIs) between different stages of maturation. Since EMR1 expression is restricted to the eosinophil lineage in humans, anti-EMR1 antibody was used for eosinophil lineage verification at different stages of maturation. The expression of most markers (Siglec 8, CD9, CD11b, CCR3 and CD49f) was dimmest in the earliest eosinophilic precursors and increased with maturation, reflected in significant increases in MFIs. In contrast, CD49d expression was brightest in immature cells and decreased as eosinophils matured. EMR1 and CD125 MFIs were unchanged during eosinopoiesis. Investigation of other markers commonly used to analyze bone marrow granulopoiesis (CD13, CD16, HLADR, CD117) did not show significant changes in expression levels during eosinopoiesis. CD34 was negative on all investigated eosinophilic cells. These findings enabled us to immunophenotypically delineate the earliest eosinophilic elements, promyelocyte/myelocytes, as Siglec 8 dim/CD11b dim-moderate/CD9 dim/CCR3 dim/CD49d bright/CD49f dim, followed by eosinophilic myelocyte/metamyelocytes, which were Siglec 8 moderate/CD11b moderate-bright/CD9 moderate/CCR3 moderate/CD49d moderate/CD49f moderate, and band/mature segmented eosinophils, which were Siglec 8 bright/CD11b bright/CD9 bright/CCR3 bright/CD49d dim/CD49f bright.
The same general immunophenotypic maturational patterns were observed in normal eosinophils and in eosinophils from patients with HES and SM. However, the expression levels of several antigens were significantly different between patient and normal eosinophils. Compared to normal eosinophils, Siglec 8 expression was significantly lower in both HES and SM eosinophils during all stages of maturation. In contrast, CCR3 was significantly increased on maturing eosinophils in SM, but not in HES. A similar pattern was observed for CD9 expression on mature eosinophils in SM. The expression of the activation marker CD69 was mostly seen on the population of mature eosinophils in the marrow, particularly in HES and SM patients. The average percent of CD69 positive mature eosinophils was highest in SM (50.6%), followed by HES (27.3%) and normal mature eosinophils (6.7%).
Conclusion: Using a novel flow cytometric antibody panel, we identified previously uncharacterized immunophenotypic differentiation patterns of eosinopoiesis in normal marrows. This novel panel was then used to elucidate changes in eosinophilic maturational patterns in two rare hematopoietic disorders: HES and SM, paving the way for its use in flow cytometric diagnosis of these uncommon diseases and other myeloproliferative disorders with eosinophilia.
No relevant conflicts of interest to declare.
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