Automated blood cell counters can distinguish cells based on their size and the presence or absence of a nucleus. However, most vertebrates have nucleated blood cells that cannot be counted automatically. We established an alternative automatic method for counting peripheral blood cells by staining cells with the fluorescent dye acridine orange (AO) and analysing cell populations using flow cytometry (FCM). As promising new animal models, we chose Xenopus laevis and three inbred strains of X. tropicalis. We compared the haematological phenotypes, including blood cell types, cell sizes, cellular structure, and erythrocyte lifespans/turnover rate among X. laevis and the three inbred strains of X. tropicalis. Each cell type from X. laevis was sorted according to six parameters: forward- and side-scattered light emission, AO red and green fluorescence intensity, and cellular red and green fluorescence. Remarkably, the erythrocyte count was the highest in the Golden line, suggesting that genetic factors were associated with the blood cells. Furthermore, immature erythrocytes in anaemic X. laevis could be separated from normal blood cells based on red fluorescence intensity. These results show that FCM with AO staining allows for an accurate analysis of peripheral blood cells from various species.

2012 In the biology of thrombopoiesis, several challenging issues such as polyploidy induction, proplatelet formation with endomitotic maturation and tubular cytoplasmic projections, and ability of cell division as reported in human platelets, have not been elucidated sufficiently. Comparative characterization of thrombocyte developments in animals may bring about a new perspective. Characteristics of thrombocyte precursors as megakaryocytes (MKs) and mature thrombocytes in most vertebrates, however, remain poorly defined. Most non-mammalian vertebrates have nucleated and spindle thrombocytes instead of platelets. Since african clawed frog, Xenopus laevis, is one of the most popular species providing various animal models in embryology and physiology, we attempt to establish an adult Xenopus model for analyses of hematopoiesis. We clarified peripheral thrombocytes by various staining methods, and searched immature thrombocytic cells in Xenopus organs. When peripheral blood cells were subjected to acetylcholinesterase staining, thrombocytes in the circulation, i.e. mature thrombocytes were positively identified. The size of elliptical mature thrombocytes was approx. 20.5±0.6 μm by 7.6±1.1 μm in diameters on cytocentrifuge preparations. We produced monoclonal antibody to Xenopus mature thrombocytes (T12) previously. The subsequent flow cytometry with a FACSAria II cell sorter revealed that the proportion of the peripheral T12-positive thrombocytes in lower FSC and SSC ranges were 1.5±0.3% of whole peripheral blood cells, and the expression of Xenopus c-Mpl (xlMpl) mRNA in the sorted cells was detected by RT-PCR. The mRNA expressions of Xenopus TPO (xlTPO) and xlMpl were also detected predominantly in the spleen and the liver, indicating that the sites of thrombocyte progenitor-residing organ and thrombopoietic activity-releasing organ were coincident in adult Xenopus. This resembled the relationship between Xenopus erythropoietin (EPO) and EPO receptor-expressing erythrocytic progenitors, as we have reported (Nogawa-Kosaka et al, 2010, Exp Hematol). Next, immunohistochemical analysis with T12 antibody revealed that thrombocytic cells were localized in sinusoid of the liver and the spleen. We then performed a thrombocytic colony assay in the presence of recombinant xlTPO expressed in E. coli. Hepatic and splenic cells composed of respective 80,000 cells in 1mL were incubated in 35mm dishes at 23°C under 5% CO2 with 0.87% methylcellulose-based semi-solid medium containing 20% FCS and xlTPO (5 ng/ml). The xlTPO-induced colonies derived from the spleen, including T12 positive thrombocytic colonies, emerged after 2 days, and the number reached to 65±2 in the culture (1 mL). The number of liver-derived colonies was smaller than that of spleen-derived ones, indicating that the density of thrombocyte progenitors in Xenopus was higher in the spleen, but the total mass of thrombocyte progenitors in the body is mostly distributed in the liver based on ratio by organ weights. In Xenopus, moderate thrombocytopenia, as well as anemia, was induced by phenylhydrazine (PHZ). The nadir of circulating thrombocyte counts was observed 4 days after PHZ-administration. When we culture cells of the liver or the spleen in the presence of the PHZ-induced thrombocytopenic serum, colonies composed of white cells and red cells were developed, suggesting that multiple or bipotent hematopoietic progenitors existed. When the hepatic cells were stimulated by xlTPO (5 ng/ml) for 2 days in the liquid culture, T12-positive megakaryocytic larger cells with multinucleated spherical shapes (approx. 30 ±3 μm in diameter) appeared, and such cells did not appear under EPO stimulation. On the other hand, the size of megakaryocytic cells derived from the spleen was smaller. Regardless of the origin of the thrombocyte progenitors, the cells stimulated by xlTPO in the liquid cultures expressed mRNAs of c-Mpl, CD41 and Fli-1, demonstrating that thrombocyte progenitors at different development stages resided in the liver and the spleen. It is still a missing piece of the puzzle whether Xenopus thrombocyte progenitors or mature thrombocytes undergo endomitosis to generate higher polyploid cells under the stimulation by TPO; however the unique megakaryocytic cells observed in this study have a clue to reveal the cellular evolution of platelets/MKs. Disclosures: No relevant conflicts of interest to declare.

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.