In order to examine whether parenteral admi nistration of Chymotrypsin has stimulatory effects on emigration of the tumor cells into the vessels or not, a model experiment was carried out. The tumor cells used was a kind of rat ascites hepa toma, AH66F, with the prominent invasiveness and metastatic property which was inoculated into the mesentery of rats. And frequency in emig ration of the tumor cells into the thoracic duct was checked up by detecting morphologically the tumor cells in the lymph-fluid of the thoracic duct which were obtained by the operative cannu lation 5 days after inoculation of the tumor cells. Under these conditions, the effect of Chymot rypsin examined, and the following results were obtained: 1) Massive parenteral administrations of Chy motrypsin were found not to accelerate both emigration of the tumor cells into thoracic duct and its proliferation at the site of the mesentery. 2) In this experiment, the frequencies of dete ction of the tumor cells in the lymph-fluid of the thoracic duct were found only to be related with the size of the inoculated mesenterial tumor. These results suggest that, when Chymotrypsin are parenterally used to accelerate expectration of the sputum for the cytodiagnosis, it does not necessitate to be afraid of the stimulatory effects of Chymotrypsin on the metastasis of cancer cells in the clinical cases.
The observation of fine structure of interphase nuclear chromatines in the whole nucleus have neither been successful by optical microscope nor by electron microscope, which is the newly developed polarized dark field oblique illumination microscope, makes successful to take photography of the fine structure of interphase nuclear chro matines. The result is as follows: 1) The numbers of chromatine morecules are approximately 50 in a normal cell, appro ximately 80 in HeLa cell and approximately 100 in a cell of cervical cancer. 2) The size, the form and the stain activity of each chromatine were observed clearly. 3) Studying chromatines in physiological con dition in this method may realize possibility in correspondence between chromatine and chromosome. This method may has a possibility to reveal the meaning of further fine structure and the process of cancer development.
Acridine orange (AO) is a fluorescent dye which has been widely used in a staining method of exfoliative cytology. This basic dye has a str ong affinity for nucleic acids not only in fixed cells but also in living ones. Ehrlich ascites tumor cells stained with AO immediately after drawing out from, mice show a green fluorescence in both nuclei and cytoplasms, and the green stained cells were successfully transplanted. Holding the cells in ascites in vitro, a red fluorescence gradually appears and when the whole cells show red flu orescence of the cytoplasms, they lose transplant ability. Treatment of fresh cells with ethanole, formalin or heat causes cytoplasms and nucleoli to emit red fluorescence and nuclei yellow fluor escence. Dried cells also show this metachromatic fluorescence (the same pattern as by von Bertal anffy's method with fixed cells). According to our investigation of portio va ginalis by fluorescence cervicoscopy under vital staining with AO, normal intact epithelium shows green fluorescence and injured epithelium (for instance due to trichomonas infection) shows red fluorescence. Fluoromicroscopic observations of fresh smears and non-fixed frozen sections obtained from the vitally stained portio vaginalis indicate that the green fluorescence is the orthochromatic green fluorescence of the epithelial cells and the red fluorescence is metachromatic red fluorescence of the cytoplasm. We believe that many kinds of cytotoxic agents can cause metachromasia with AO dye.
Recently, numerous studies on the detection of tumor cells in the peripheral blood have been made from the view-points of diagnosis, prognosis and evaluation of therapeutic effects. The incid ence of tumor cells in the peripheral blood varies widely, ranging from less than 1 % to 96.5% (Table 1). An extensive review of the literature has indicated that this great diversity might be attributable mainly to differences in the criteria for the recognition of malignant cells rather than in the techniques for tumor cell isolation. There fore, it would be important to select the technique which is excellent in both reproductivity and cell preservation. In the present study a dextran sedimentation and smearing technique was selec ted for the detection of tumor cells in the circul ating blood, and this procedure was evaluated by the tumor cell recovery test, using human cance rous ascites cells, stained and unstained (Table 2). The results indicated that the dextran sedi mentation technique possessed an advantage in well preserving the cell morphology, but, the recovery rate, as described in the previous literature, was rather low as compared with those by other methods. By the use of the dextran sedimentation techni que, tumor cells appearing in the peripheral blood from 130 subjects, which comprised 30 healthy subjects, 38 noncancerous patients, 45 patients with carcinomas and 17 with sarcomas (Table 3), were observed with a care of ruling out the cells which were not usually observable in the peripheral blood. 1. There were found even in the samples from healthy subjects a relatively good number of benign cells such as erythroblasts, immature granulocytes, large mononuclear cells which possi bly would be atypical lymphocytes, monocytes, plasmocytes and their mitotic figures (Table 4). 2. Large histiocytes were seen in 8 cases (6.2%) and occasionally became signet ring cells which might be apt to be confused with tumor cells. Endothelial cells were also seen in 7 cases (5.4%). 3. Megakaryocytes may not infrequently be confused with tumor cells for their large size (Table 5), clumping of the nuclear chromatin, and irregularity in the nuclear membrane. Mega - karyocytes were found at a high frequency (87 %) of healthy subjects, and increased in number in patients with various diseases (Table 6), especially, in patients with disseminated cancers (Table 7 and 8). 4. In the present study tumor cells were dete cted in only 3 patients (6.7%) with disseminated cancers and in 6 patients (35.3%) with sarcomas (Table 9). All patients expired within 5 months after the first detection of tumor cells in the peripheral blood.
During 1956 to 1967, cytodiagnosis was carried out for malignant and benign gastric lesions by the following various methods; the proteolytic enzyme lavage method, the selective proteolytic enzyme lavage method under fluoroscopy and the lavage method under fibergastroscopic observation. The cases examined consist of 351 gastric can cers and 1119 benign gastric diseases. In 1119 be nign cases, the accuracy of the cytodiagnosis was 97, 2% Of 351 cancer cases, 288 cases (82%) was diagnosed cytologically as malignant, while 15 cases of benign gastric lesions were diagnosed as“suspicious” (Class 111) or “false positive”. In the present study, the cellular pictures of 3000 benign atypical cells obtained from above suspi cious or false positive cases were analysed morphologically. The general features of the gastric benign atypical cells observed in the specimens (hemato xyline eosin stainning) were as follows; anisocy tosis, mutual inclusion, increase of nucleocyto plasmic ratio, thick and smooth nuclear mem brane, dominant nucleolus, multinucleolus, poor nuclear substance, redness of nucleolus, slightly irregular aggregation of chromatin, etc. Among them, the most characteristic pictures of the benign atypical cells are found to be sm ooth and thick nuclear membrane and poor nucl ear substances, but rare abnormal aggregation of chromatin. However, no definite differences were found in the morphology of nucleolus between benign at ypical cells and cancer cells. The frequencies in the appearance of the above morphological characteristics were demonstrated in detail. In addition, the histopathological backgrounds for the above benign atypical cells were analysed by examining the gastric mucosa of each corres ponding cases. The characteristic benign atypical cells were found presumably to exfoliate from the regener ative epithelium of chronic ulceration and the so-called atypical epithelial foci of the gastri mucosa in chronic gastritis.
A special brush was divised for cytological examination on the gastric cancer. This is a hard brush in various shape and hardness attached to the flexible stick, 5 cm in diameter, 5 cm in length for the regular size. The patient is prepared NPO after supper the day before procedure, 300 ml of the physiological saline solution and a-Chymotripsine in the early morning, and gastric irrigation is carried out immediately before the procedure. The polyethylen outer tube with 1.6cm of external diameter, 1.2cm of internal diameter is inserted to the stomach after topical narcosis being applied to the pharynx. The brush is introduced through the outer tube to reach the lesion suspected by position change and manipulation. The specimen obtained is fresh, big enough to sufficient mass, and examined by the Papanicolaou's method. This procedure is applied for early cancer, polyp, ulcer, and chronic gastritis, all suspected cancer, with 82.4% accuracy. There are no cases diagnosed as malignant among the non-cancerous cases. This is useful for the cases of pyloric stenosis. The minimal cancerous lesion detected by this method is 0.3×0.5cm2.
I have made a comparative cytologic study of cervical scrapings from two groups of patients who received irradiation or operative therapy more than 4 months before for cervical cancer. The results thus obtained may be summarized as foll ows: 1) An overwhelmingly larger number of basal cells are noticeable in the cases receiving irradiation therapy than in those subjected to operation. 2) The basal cells are found transformed more frequently in the former cases than in the latter. 3) Leucocytes, naked nuclei and detritus mass tend to appear more frequently in the cases receiving irradiation therapy. 4) The postoperative cases showed lysis of cells with large amount of mucus more fre - quently than the cases receiving irradiation therapy. There were two groups of cases in irradiation therapy as followed, i. e., one showing sharply defined edges both of cell and of nuclei with scantiness of naked nuclei and detritus mass and the other with cells reduced in stainability and obscure in borders with nuclei having an obscure structure and increased diameter (in this group there is generally a frequent occurrence of both naked nuclei and detritus mass). As apparent from the above described, there is a distinct difference noted in findings on smear between the operated and irradiated groups. This difference may be attributed to the very difference in the therapeutic methods themselves, since in this study the postoperative cases of cervical cancer of the same age group as the cases receiving irradiation ther apy served as control, permitting thus to exclude the factor of the ovarian function, In other words, it may be regarded as a reflection of the chronic effect of irradiation therapy 5) Comparison of the incidence of “deformed basal cell” between the subgroups with and without local recurrence in the irradiation group disclosed that it tended to be lower in the former subgroup than in the latter. 6) Based upon the above described, I should like to propose that the pursuit of the incid ence of “deformed basal cell” may be of diagnostic help in the detection of local recurrence of cervical cancer following irra diation therapy. (In the present study were observed only those smears lower than Class II according to Papanicolaou's classification to the exclusion of adenocarcinoma.)
Sputum smears of lung cancer patients were kept in room after the fixation for 30minutes in 95% ethanol-ether solution, in 95% ethanol, in methanol-ether solution, in methanol only, in 90% aceton, in Bouin's solution and in Carnoy's solution respectively. They were also kept in room after the fixation during 24 hours in 10% formalin and after the spraying 2 times with Cytospray. On the first, 2nd, 4th, 7th and 14th days after the fixation, sputum smears were stained with Papanicolaou's method. The slides, stained within 24 hours after the fixation, have good stainability of cells and nuclei, and no degeneration of cell ular structure was observed excepting 10% form alin fixed slides. The slides stained more than 24 hours after the fixation have slightly weak staina bility. Smears fixed by several methods and stained by Papanicolaou's method, of vaginal secretion, sputum, bronchial secretion, gastric juice, urine and serous fluid are reexamined microscopically after 2 years storage. The slides, which are fixed in ethanol-ether solution or by means of Cytospray technique, reveal good preservation of color and good maintenance of cellular structure. Merits of Cytospray technique are summarized as follows: time saving, easy handling, no cellular desquamation from the slde and no cellular contamination.