Two Granulocytic Regions in Bone Marrow with Eosinophilia Evaluated by Flow Cytometry

Kazuo Muroi, Shin-ichiro Fujiwara, Raine Tatara, Kazuya Sato, Iekuni Oh, Ken Ohmine, Takahiro Suzuki, Tadashi Nagai, Keiya Ozawa, Yoshinobu Kanda
2014 Journal of Clinical and Experimental Hematopathology  
TO THE EDITOR Flow cytometry (FCM) with a CD45 gate is widely used for analyzing phenotypes of bone marrow cells in hematological disorders. This method clearly separates bone marrow cells into erythroblasts, lymphocytes, monocytes, myeloblasts, and granulocytes. 1 Each cell group shows a distinct distribution characterized by the CD45 intensity and side scatter properties. Usually, granulocytes show a single population of cells with bright CD45 and high side scatter properties. In some
more » ... es. In some situations, the granulocytic region in the bone marrow is divided into two adjacent regions, although causal events are not known. 2 We report that bone marrow granulocytic regions in eosinophilia are separated into two regions by FCM. From March 2008 to October 2013, bone marrow aspiration was performed in 12 patients with eosinophilia. The obtained bone marrow cells underwent smear examination, chromosomal analysis including fluorescence in situ hybridization analysis, and FCM as routine practice. In the bone marrow smear examinations, eosinophils were distinguished between mature and immature forms. Mononuclear cells were separated from an aliquot of the bone marrow samples and used for FCM with a CD45 gate, as previously reported. 3 Initially, FCM was performed to analyze phenotypes of blasts in the blast region. In this study, FCM data were reevaluated by setting a gate for identifying granulocytes characterized by bright CD45 expression and high side scatter properties (Fig. 1A) . Phenotypes of the cells were analyzed using a flow cytometer (FACSCalibur or FACSCanto II; BD Biosciences, San Jose, CA, USA). For reanalyzing the FCM data, neither informed consent from each patient nor permission from the Institutional Review Board of our hospital was obtained. Table 1 shows the characteristics of the patients. The median age was 56, with 9 males and 3 females. All patients showed more than 1,000/µL of eosinophils in the peripheral blood. The rates of immature and mature eosinophils in the bone marrow (mean ± SD) were 15.2 ± 9.2% and 22.6 ± 12.2%, respectively. Karyotypic abnormalities were not shown in the analyzed samples. All patients was diagnosed as having secondary eosinophilia as follows: graft-versus-host disease after hematopoietic stem cell transplantation, 3 patients; drug allergy, 2; asthma, 1; liver cirrhosis, 1; chronic renal failure, 1; myocardial infarction, 1; erythroderma, 1; cardiomyopathy, 1; and unknown etiology, 1. As shown in Fig. 1B , two distinct granulocytic regions, G1 and G2, are observed in bone marrow with eosinophilia, compared with normal bone marrow. According to the CD45 intensity and side scatter properties, the G2 region corresponds to the neutrophilic granulocyte region. The percentages of cells in the G2 region and immature myeloid cells including myeloblasts to metamyelocytes evaluated by smears were 19.3 ± 5.9% and 10.3 ± 7.5%, respectively. As shown in Fig. 2 , the antigen expression profiles in the G2 region are consistent with those in neutrophilic granulocytes along with the neutrophilic differentiation process, that is, from the stage of promyelocytes, disappearance of HLA-DR expression, increasing expression of CD11b and CD15, and fluctuating expression of CD13 and CD33. 2 The G1 region shows higher CD45 intensity than the G2 region and equal side scatter properties to the G2 region (Fig. 1B) . The antigen expression profiles in the G1 region were different from those in the G2 region to some extent (Fig. 2) . The G1 region showed higher positivity for CD10, CD11b, CD13, CD25, CD36, and HLA-DR and lower positivity for CD33 than the G2 region. In eosinophils, CD11b,
doi:10.3960/jslrt.54.243 pmid:25501116 fatcat:i7xh2v7rpfdqnafrsv4xw6gx7y