Direct flowcytometric analysis of eosinophils using a whole blood staining technique.
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概要
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Background: Flowcytometric analysis of purified eosinophils requires several hours to accomplish due mainly to purification of cells; moreover, it requires more than 20 mL blood and expensive reagents. The aim of the present study was to develop a method of direct flowcytometric analysis for eosinophils using whole blood. Methods: Peripheral blood obtained from five healthy individuals (mean age 42 years) and 10 patients with eosinophilia (mean age 40.3 years) were used for analysis. We stained antigens (CD9 or CD16) and fixed cells with parabenzoquinone (PBQ) or paraformaldehyde (PFA) after hemolyzation followed by treatment with N-octyl-β-glucopyranoside (OG). Results: On comparison of forward scatter with side scatter dot plots among samples treated with hemolyzation alone, PBQ fixation and PFA fixation, PBQ fixation showed the best results in discriminating eosinophils from other leukocyte fractions on the cytogram. Following fixation and permeabilization of cells, EG2, a secretory form of eosinophil cationic protein, was stained as an intracellular antigen. Flowcytometric analysis for EG2 showed a high positivity rate only in the eosinophil fraction. There were no differences in EG2 positivity or mean fluorescence intensity (MFI) between heparinized and EDTA-treated blood. Comparison of samples treated with OG at 6.0 and 7.4 mg/mL showed that the latter had a higher MFI for EG2 without significant change in the positivity rate. Conclusions: The findings show that intra- and extracellular properties of eosinophils can be analyzed with whole blood using PBQ fixation and OG treatment at a concentration of 7.4 mg/mL. Direct flowcytometric analysis of eosinophils saves time and requires only a small amount of blood, both of which are advantageous for patients and laboratory workers.
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