Flow cytometric immunophenotypic differentiation patterns of bone marrow eosinophilopoiesis

Abstract

Background: Flow cytometry has been widely used to study immunophenotypic patterns of maturation of most hematopoietic lineages in normal human bone marrow aspirates, thus allowing identification of changes in patterns in many myeloid malignancies. Eosinophils play an important role in a wide variety of disorders, including some myeloid neoplasms. However, changes in flow cytometric immunophenotypic patterns during normal and abnormal bone marrow eosinophilopoiesis have not been well studied.

Methods: Fresh bone marrow aspirates from 15 healthy donors, 19 patients with hypereosinophilic syndromes (HES), and 11 patients with systemic mastocytosis (SM) were analyzed for candidate markers that included EMR-1, Siglec-8, CCR3, CD9, CD11a, CD11b, CD11c, CD13, CD16, CD29, CD34, CD38, CD45, CD44, CD49d, CD49f, CD54, CD62L, CD69, CD117, CD125 (IL-5Rα), HLA-DR, using 10 parameter flow cytometry. Putative CD34-negative immature and mature normal eosinophil populations were first identified based on changes in expression of the above markers in healthy donors, then confirmed using fluorescence-based cell sorting and morphological evaluation of cytospin preparations. The normal immunophenotypic patterns were then compared to immunophenotypic patterns of eosinophilopoiesis in patients with HES and SM.

Results: The eosinophilic lineage was first verified using the human eosinophil-specific antibody EMR-1 in combination with anti-IL-5Rα antibody. Then, a combination of Siglec-8, CD9, CD11b, CCR3, CD49d, and CD49f antibodies was used to delineate normal eosinophilic maturational patterns. Early stages (eosinophilic promyelocytes/myelocytes) were identified as Siglec-8 dim/CD11b dim to moderate/CD9 dim/CCR3 dim/CD49d bright/CD49f dim, intermediate stages (eosinophilic myelocytes/metamyelocytes) as Siglec-8 moderate/CD11b moderate to bright/CD9 moderate/CCR3 moderate/CD49d moderate/CD49f moderate and mature bands/segmented eosinophils as Siglec-8 bright/CD11b bright/CD9 bright/CCR3 bright/CD49d dim/CD49f bright. Overall maturational patterns were also similar in patients with HES and SM; however, the expression levels of several surface markers were altered compared to normal eosinophils.

Conclusion: A novel flow cytometric antibody panel was devised to detect alterations in immunophenotypic patterns of bone marrow eosinophil maturation and evaluated in normal, HES and SM samples. This approach will allow us to elucidate changes in immunophenotypic patterns of bone marrow eosinophilopoiesis in other hematological diseases.

Keywords: adhesion molecules; bone marrow; eosinophil maturation; flow cytometry; hematological disease.

FIGURE 1

Eosinophil gating strategy and morphological confirmation of eosinophil maturational stages. (a) Doublets exclusion gating based on FSC-A vs FSC-H, (b) Eosinophil gate based on CD45 vs SSC, (c) Eosinophil identification using eosinophil specific markers EMR1 and IL-5Ra (CD125), (d) Eosinophil staining using CD9 and Siglec-8 reveals differential pattern of staining, corresponding to different maturational stages of eosinophils. Gates shown in (d) were used to perform flow cytometric sorting; sorted cells were morphologically analyzed using Wright-Giemsa stained cytospin slides and Olympus BX41 microscope. Microphotographs magnification is 500×. The cell populations in these gates were identified as: (e) early eosinophils (promyelocytes/myelocytes), (f) intermediate eosinophils (myelocytes/metamyelocytes), and (g) mature eosinophils (bands/polymorphonuclear eosinophils). [Color figure can be viewed at wileyonlinelibrary.com]

FIGURE 2

Patterns of eosinophilopoiesis in normal bone marrow. (a) CD9 versus Siglec-8 separates eosinophils reveals maturation pattern. (b) CD11b expression is low is early eosinophilic precursors, increasing with maturation. (c) In contrast, CD49d expression is elevated in early precursors and diminishes with maturation. (d) CCR3 shows significant increase in expression during eosinophilic maturation. (e) CD49f expression increases with maturation of eosinophils. (f) CD49d versus CD49f plot illustrates inverse expression of these two markers during eosinophilic maturation. [Color figure can be viewed at wileyonlinelibrary.com]

FIGURE 3

Mean fluorescence intensities (MFI) of selected markers during normal eosinophil maturation in bone marrow. (a) EMR1 and (b) IL5Ra (CD125) show no significant changes during maturation. (c–f, h) Siglec-8, CD9, CD11b, CCR3 and CD49f expression increases with eosinophilic maturation. (g) CD49d expression decreases with eosinophilic maturation. (*p = 0.05 or less; **p = 0.005 or less). (i) The summary of expression of different surface markers during eosinophilopoiesis. [Color figure can be viewed at wileyonlinelibrary.com]

FIGURE 4

Mean fluorescence intensities of selected adhesion and proliferation markers. (a–e) CD62L, CD11c, CD162, CD44 and CD45 increase during eosinophil maturation in the bone marrow; (f–i) CD11a, CD29, CD54, and CD13 show no significant changes during maturation; (j) Ki-67 staining is positive only in immature, CCR-3 dim progenitors, (k) Ki-67 staining is negative in peripheral blood eosinophils, (l) Significant CD69 expression is detected on mature, but not on immature eosinophilic precursors in bone marrow (**p = 0.005 or less). [Color figure can be viewed at wileyonlinelibrary.com]

FIGURE 5

Maturational patterns of marrow eosinophilopoiesis in representative patients with HES and systemic mastocytosis. Expression profiles of selected markers show similar patterns of eosinophil maturation in HES (a–d) and SM (e–h) patients as in healthy donors (Figure 2). (a, e) CD9 versus Siglec-8, (b, f) Siglec-8 versus CD11b, (c, g) CD9 versus CCR3, (d, h) CD9 versus CD11b. [Color figure can be viewed at wileyonlinelibrary.com]

FIGURE 6

Comparison of mean fluorescence intensities of selected markers between healthy donors’ eosinophils and eosinophils from patients with HES and systemic mastocytosis. (a) Siglec-8, (b) CD9, (c) CD11b, (d) CCR3, (e) CD49d and (f) CD49f, (g) CD62L, (h) CD44, (i) CD162, (j) CD13, (k) CD45, and (l) CD69 (*p = 0.05 or less; **p = 0.005 or less).