The Cellular Classification and Separation of Nucleated Blood Cells by Supravital Cell Staining with Acridine Orange
Authors: Kinoshita Sayaka , Nagai Yutaka , Tanizaki Yuta , Kato Takashi 
Published: October 2019

Abstract
Abstract 4907
Immunohistochemical staining and flow cytometry are performed on the tissue to distinguish specific cells. The detection of diseases is made by the flow cytometry using monoclonal antibodies on peripheral blood (PB) or bone marrow cells. The diagnosis of leukemia is made with PB and marrow cells using antibodies to cluster of differentiation (CD) antigens specific to leukemic cells (e.g.,CD13, CD33). Therefore, conventional approaches to identify cellular phenotypes are being replaced by immunophenotyping using flow cytometry. However antibodies satisfying for needs are not always available. In this case, the generation of such antibody to each specific antigen also causes problems of producing the respective recombinant antigen for immunization. When attempting to construct new animal models using other than mice and humans, this issue becomes one of the serious limiting factors in developing research. In addition, the opportunity is increasing to classify unexperienced types of cells such as cells derived from iPS cells and others. We therefore consider that it is important to develop methods to classify and separate specific cells of interest without antibodies. As supravital cell staining with acridine orange (AO) is introduced in 1960's (Jacson JF, Blood, 1961; Lewis M. et al. Blood, 1962), this metachromatic fluorescent dye rapidly stains DNA and RNA independently. Morphological abnormalities of human erythrocytes such as red cell fragment and large platelets are detectable (Nagai Y et al., Int. J Lab Hematol., 2008). AO emits green fluorescence when it binds to the double-stranded DNA and also red fluorescence when it binds to the single-stranded RNA. Consequently flow cytometry of cells stained with AO is suitable for analyzing blood cells in four parameter, which are scattered light intensity (FSC and SSC) and fluorescence intensity (DNA content and RNA content). This analysis method has the possibility of developing the classifying and separating method for abnormalities.
Xenopus laevis(X. laevis) has various nucleated blood cells, which are erythrocytes, leukocytes, and thrombocytes, and their progenitors that are not classified yet. As the first step to identify cells with increased RNA content, we chose X. laevis blood cells as a new model of AO staining for flow cytometry by FACS Aria II cell sorter based on the content of DNA and RNA. Since collected cells were stained by May-Griinwald-Giemsa on cytocentrifuge preparations. The population in lower content of DNA and RNA is composed of erythrocytes (95.7±1.3% of whole PB cells). The population in higher concentration of DNA and RNA was pure leukocytes fraction (0.7±0.6% of whole PB cells) expressing the mRNA of X. laevis myeloperoxidase. This population was then fractionated with the higher content of RNA containing eosinophils and basophils (48.7±29.9%), and lower content of RNA contained neutrophils (50.9±29.9%). The proportion of the peripheral thrombocytes in lower forward light scatter (FSC) and side light scatter (SSC) ranges was 0.8±0.8% of whole PB cells, and the expression of c-Mpl, CD41 and Fli-1 mRNA was detected in the sorted cells by RT-PCR. Furthermore, sorted cells were confirmed by immunohistochemical staining by anti X. laevis thrombocyte monoclonal antibody. To reveal the characteristics of the abnormal PB cells, we compared the normal PB with the PB of phenylhydrazine (PHZ) induced anemic X. laevis. At 8 days after treated with PHZ, immature hematopoietic cells were appeared in circulating blood. We isolated these cells with high content of DNA and RNA, and the population in lower FSC and higher SSC with crossover analysis of light emission by AO, suggesting that this method could identify abnormalities in whole PB. The character of these cells was morphologically small and high nuclear cytoplasmic ratio. We also applied this method to blood cells of Rana catesbeiana, American bull frog. Despite the differences in size of erythrocytes, thrombocytes and leukocytes, Rana catesbeiana PB was similarly sorted to X. laevis PB by cellular DNA/RNA content. These results suggest that this method can sort nucleated cells in various species by crossover analysis of light emission by AO. Our study showed that this method has the advantage to characterize of human leukemia cells, tumor cells, iPS-derived cells and nucleated blood cells.

Disclosures:
No relevant conflicts of interest to declare.