50 cases of leukemic bone marrow were studied by the fluorochrominized tissue culture with acridine orange divised in our laboratory. Following results were obtained.
1. In acute leukemias, the growth area was extremely dense with migrating cells and its boundary was distinctly defined. It was characteristic in fluorochrominized tissue culture that the greater part of the growth area was green in color owing to the fluorescence of blast cells and its outer zone was somewhat reddish in acute myelogenous leukemia and yellowish in acute lymphocytic leukemia because of the presence of mature cells.
2. In chronic leukemias, the well-defined boundary of the growth area with a high cell density, which was also characteristic of acute leukemia, was sorrounded by another less dense zone of mature cells, forming a double growth zone. In chronic myelogenous leukemia, the inner zone of this doudle growth zone showed reddish green color, and in chronic lymphocytic leukemia yellowish green color. In the outer zone of this double growth zone the flnorescence of mature leukocytes was especially predominant.
3. In monocytic leukemia, the growth pattern was almost similar to that in aute lenkemia, but its fluorescent color was somewhat different. In the growth area there were many, greenish orange colored promonocytes and monocytes, green monoblasts, yellow lymphocytes and a few neutrophils with reddish orange granules. Therefore the growth area in monocytic leukemia disclosed multi-colored fluorescence.
4. To conclude these results, the growth area of each type of leukemia were occupied by the fluorescence of predominant leukemic cells. Accordingly by the fluorochrominized tissue culture of bone marrow the diagnosis of leukemia types could be performed.

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The author conducted comparative study under a fluorescence microscope between the ascites phagocytes on one hand and monocytes of blood and subcutaneous histiocytes on the other both from normal mice and stained by acridine orange, and further compared the phagocytes of the mice under stimulation and the subcutaneous histiocytes as well as the phagocytes at the time when the greater omentum is resected. In addition, the author ntilized the findings on monocytes in human monocytic leukemia as the reference; and obtained the following results.
1. In the case of active cells of monocytes in blood, the nucleus and cell body present green fluorescence, and the nuclear membrane and nuclear meshwork are indistinct. Reddish orange cytoplasmic granules do not tend to agglomerate but the majority of them are situtated around the nucleus. However, with the lapse of time both nuclear membrane and meshwork grow more distinct, and also the reddish orange cytoplasmic granules begin to agglomerate to a certain degree.
2. In the case of the subcutaneous histiocytes both under normal condition and at the time of stimulation the nuclear meshwork is coarse and the nuclear membrane is thick and clear-cut, and reddish orange cytoplasmic granules are great and almost uniform in size. Moreover, these granules are diffusely scattered but never tend to agglomerate.
3. In the ascites at the time of stimulation amall phagocytes are more numeroua, and these cells are poorly stained. However, the nuclei and cell bodies present a clear and deep green fluorescence, and reddish orange cytoplasmic granules do not necessarily tend to agglomerate, and their size is almoat uniform. With the lapse of time, both the nucleus and cell body are stained deep, also the cytoplasmic granules begin to agglomerate.
4. In the study of the senilization of phagocytes by inhibiting the cell regeneration by removing the greater omentum, the nuclei become green with yellowish tone and the reddish orange cytoplasmic granules, losing their agglomerating tendency, are scattered diffusely, but unlike subcutaneous histiocytes their size is extremely variable, and the cell body reveals a degenerative picture.
5. In the bone-marrow tissue culture of human monocytic leukemia by the simple method of our own device and in the medium of ascites, and vitally stained with acridine orange, the fluorescence picture of the monocytes at regeneration and senilization stages resembles qeite closely to the findings of human ascites phagocytes.
6. From these comparative study of fluorescence pictures of these various cells, it has been verified that ascites phagocytes unlike histiocytes are the cells quite closely related to monocytes.

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In the present experiment the author carried out vital observations of various cells with fluorescence microscope by performing the tissue culture of non-pathologic human lymph nodes, and obtained the following results.
1. Under weak magnification, fluorescence of the growth area presents diffuse yellowish green color, sparsely intermingled with reddish orange fluorescent spots.
2. Under the high power magnification, the cell body is green, and various granules present reddish orange flucorescence, while the nucleus and nucleoles are green.
3. Generally speaking, blasts possess no granules and are green in color. In large lymphocytes both the nucleus and nucleoles are green, while in small lymphocytes the nucleus shows marked yellowish green fluorescence. Neutrophils have characteristic reddish orange granules, while in monocytes a variety of fluorescence can be recognized, namely, green in both the cell body and the nucleus, reddish orange in the granules gathering around the region adjacent to the nucleus. Other cells show fluorescence specific to each species of cells. In the degeneration phase, reddish orange granules are swollen and discolored, and there appear acridine orange stained vacuoles, unstained vacuoles, abnormal process formation, reddening of the cell body, nucleus and nucleoles, and the eell body turning green and swollen.
4. As for small lymphcytes in the degeneration phase, in detail often the reddening of the cell body can be observed, and other cells also show a degenerative picture specific to each cell series.

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In the present experiment the author conducted the bone marrow tissue of normal persons by the simple method of fluorochrominized bone marrow tissue culture as established in Report 1, and observed the growth zone and various bone marrow cells appearing in the zone.
1. The cells are dense in the central part of the growth zone, and the density gradually decreases towards the periphery, giving off reddish orange fluorescence diffusely with yellow and green fluorescent spots scattered in this zone.
2. Cytoplasma is green, while mitochondria and various granules give off yellowish green or reddish orange fluorescence. However, the fluorescence of mitochondria is quite trivial, and the nuclei and nucleoles are green but nucleoles give off fluorescence earler than the nuclei.
3, In young cells green fluorescence of cytoplasma is marked, and it grows less distinct as they mature whereas reddish orange granules tend to increase along with the maturation of cells and their size is variegated.
4. In myelogenous cells reddish orange fluorescence of granules is characteristic while yellowish green fluorescence of the nuclei in lymphocytic cells. In mouocytic cells a variety of fluorescence such as green cytoplsama and reddish orange granules agglomerating around the nucleus can be recognized, and likewise fluorescence specific to each cell type can be observed in other cells.
5. The green fluorescence of cytoplasma of erythroblasts is gradually lost along with maturation, and no fluorescence can be recognized in erythrocytes, while substantia reticulofilamentosa of reticulocytes present red fluorescence.
6. When two barrier filters, GG 4 and OG 4 are used together, it is easy to differentiate cells of the erythroblast series from cells of the leucocyte series.

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By staining supavitally or vitally pleural fluid phagocytes of mice and rabbits in acridine orange the author observed them under a fluorescence microscope, and obtained the following results.
1. In the pleural cavity of both mice and rabbits there are phagocytes possessing a peculiarly-shaped nucleus and reddish orange cytoplasmic granules with agglomerating tendency.
2. These pleural fluid phagocytes reveal the findings exactly identical with those of ascites phagocytes, therefore, it seems that both are the same kind of cells and they are closely related to monocytes. Moreover, the fluorescence picture of the rabbit phagocytes as compared with that of mouse phagocytes resembles more closely to that of monocytes.

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By performing the simple method of fluorochrcminized bone marrow tissue culture the author observed the degeneration picture of leucccytes, and also studied changes in fluorescence in the cells cultured under an adverse condition but added with acridine orange. The following are the results of the present experiment.
1. It has been found that the majority of commonly-known findings by supravital staining are proven to be degeneration picture of cells.
2. As for the degeneration signs, the swelling and fading of reddish orange granules can be pointed out, while in cytoplasma vacuoles either stained or unstained with acridine orange, abnormal processes, the reddening or green swelling of cytoplasma can be recognized: The nuclei have agglutinated chromatin and have become yellow, red, and swollen in green color, and in nucleoles green swelling or reddening can be observed.
3. In the degeneration picture likewise each cell type present different, fluorescence.
4. When cells become shrunk, the fluorescence is red, while when cells are swollen, it is green.
5. In the cells with decreased function reddish orange granules fade quickly and their nuclei turn yellow.
6. Those cells that have lost the fluorescence of their granules, and those that have red cytoplasma and red nuclei as well as those with their nuclei green and swollen as alldead cells.

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In the fluorescence microscopic study of ascites cells vitally or supravitally stained with acridine orange, which were aspirated from mice, rabbits, rats, cats, doge, monkeys, human, pigeons, and chicken the author obtained the following results.
1. For the vital staining the simple culture method devised in our department is most suitable and as for the concentration of the medium the dilution of 10⁴ is most appropriate.
2. The fluorescence picture by the supravital staining of the ascites cells turned out to be a degenerated picture in vital staining.
3. Active phagocytes have nucleus and cell body stained green and those more active and smaller cells are difficult to be stained and they become stained deep as they degenerate.
4. Ascites phagocytes all possess a series of common characteristics, namely, a peculiar movement pattern, the massing tendency of cytoplasmic granules and nuclei of complicated shape; and these characteristics resemble those of the fluorescence picture of monocytes in blood. Moreover, this similarity tends to be greater in the higher class of animals.

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