Recently, the filtrating method has routinely been applied to the cytodiagnosis of fluids. In this paper the Nuclepore membrane filter has been applied for cytologic study on the body fluid, urine and blood. The Nuclepore membrane filter is composed of polycarbonate film and has uniform pore of 5 microns in diameter. Cells collected on the smooth flat surface of the filter were well stained with the Papanicolaou method, PAS, various cytochemical methods such as acid and alkaline phosphatases, and also with the Giemsa stain without staining the filter itself. On the other hand, Millipore filter made of cellulose esters was stained with the Giemsa stain, and, thus found not suitable for the hematologic study by use of this stain. The advantages of this Nuclepore technique were easier handling and mounting, chemical inertness optical clarity, and high yield of cancer cells with an excellent morphologic preservation.
From the Department of Oral Surgery, Okayama University Medical School, Okayama, Japan. During the 6-year period from January 1964 to January 1970, colpomicroscopic observations of the oral cavity, i. e. stomatomicroscopy, were made in 20 control cases of normal oral mucosa (10 cases each of male and female) and 127 clinical cases seen and admitted to Department of Oral Surgery, Okayama University Hospital. They were 75 carcinomas, 2 sarcomas, 4 malignant melanomas, 16 benign neoplasms, 16 inflammatory lesions and 14 other diseases.
Since the advance in technique has made it possible to obtain fresh specimens from the stomach, re-evaluation of the Papanicolaou's criteria for malignancy established chiefly by the use of the lavage method has become an important issue for the routine cytodiagnosis. The present paper is concerned with a morphological analysis of cancer cells which has been made in careful comparison with non-malignant cells. 195 specimens obtained from stomachs resected or biopsied for gastric cancer (90 cases) or non cancerous gastric lesions (105 cases) were subjected to the study. The results obtained were as follows: 1. As far as the nuclear size was concerned, cells with a nuclear diameter of more than 15, a consisted mostly of malignant cells and those with a nuclear diameter of more than 20 were all malignant cells. The standard deviation of nuclear diameter distribution in individual case was greater in cancer group than in noncancer group, indicating that the nuclear size of cancer cells was varied considerably in each case. 2. As for the nuclear pattern, an increase in chromatin quantity and an uneven dispersal of chromatin, which had been regarded as the most important criteria of malignancy, were found at a fairly high rate even in non-cancerous cells in the cases of gastritis and ulcer. On the other hand, fresh cancer cells showed, at a higher rate, a fine reticular structure of chromatin, which had been regarded so far as one of the criteria of non-malignancy. As far as the nucle olus was concerned, no finding specific for cancer cells was obtained in this series. 3. In individual specimens containing compara tively large number of cells, the nucleus and nucleolus were examined for following 8 morphological items: nuclear diameter in mode, irregularity in nuclear contour, irregular thickening of nuclear margin, hyperchromatism, irregularity in distribution of chromatin, coarseness in the pattern of chromatin, large nucleolus (more than 2μ) and irregular nucleolar contour. Decision as to whether most of the cases examined were benign or malignant was made by evaluating the frequency of each item alone or the combination of each item. Cases with cells showing hyperchromatism at a rate of less than 50 percent or with cells having large nucleolus at the same rate were judged as possibly benign. It was most noteworthy that these two items together with the measurement of nuclear diameter were available as automatic cytoscreener. 4. For practical purpose of cytoscreening, the cells were divided into three groups according to the nuclear diameter: cells less than 10μ, those 11-15, μ and those more than 16μ it in diameter, respectively. In each group, morphological characteristics of the nucleus were analysed.
1. To-day, the value of the fluorescence-microscopy has been well acknowledged as a means of cytological detection for the uterine cancer and the L. von Bertalanffy method, which adopts the acridine orange as the fluorescent dye, has been in general use. Since, with this method, it takes as much as ten minutes to complete the standing so that the author intended simplify the technique. Consequently, the author invented a method by which the entire-process of staining will be finished within 15 seconds and the author named this new technique as the simplified AO (acridine orange) method. 2. Findings of various cells such as normal, malignant and so forth have been described here by this simplified AO method. 3. Classification of various cells by this simplified AO method has been carefully studied and the characteristic cellular findings in each category have been described. 4. specimen were simultaneously taken from 1062 women, and these two specimens were considered as one testing unit. In total, 1214 testing units have been investigated. Of these two, one was stained with Papanicolaou method and the other with this simplified AO method. They were independently examined and the diagnosis was made accordingly. Then, the results were carefully compared with each other. Every case was histologically studied by the punch biopsy which was taken from suspicious area with colposcopy. Of 1214 units, 71 were diagnosed as the squamose cell carcinoma of the uterine cervix, 83 were carcinoma in situ and 11 were cancer of the uterine body. 5. With this simplified AO method, 71.8% of the cases with squamose cell carcinoma were diagnosed as either class IV or V and every case without exception was diagnosed as class III or over. Likewise, 39.8% of the carcinoma in situ were diagnosed as either class IV or V and 74.7 % were reported as class III or over. No case of false negative was found with the squamose cell cancer of the cervix; but 25.3% of the carcinoma in situ were reported as false negative. These figures were almost the same with these by Papanicolaou method which were carried out simultaneously.6. This simplified AO method, not only takes very little time for both staining process and screening examination but also disclosed quite satisfactory results. Especially, marked shortening of time was noted in order to defferenciate negative specimens from positive. Also, if necessary, specimens stained with this simplified AO method can be restained with Papanicolaou method for parmanent storage. Inconclusion, the author feels this simplified AO method would be quite instrumental for detection of cancer in daily practice.
The characteristics of complex-formation between nucleic acids and acridine orange (AO) has been studied with spectrophotometry and fluorospectrophotometry. In these experiment, the authour established improved method in fluorescence analysis by fixing the materials before staining and found the best way for detection of DNA-AO and RNA-AO complexies. Under the proper conditions, AO was demonstrated to possess distinctive conbinding characteristics forming a green fluorescence with DNA and a redfluorescence with RNA. Ehrlich ascites carcinoma cells were fixed and stained with AO in the same manner, and the stained cells were analized fluorospectrophotometrically. The spectrum obtained, which had two peaks at 530 mμ and 620 mμ, , was successfully devided into two spectra, i.e. properly reduced spectra of DNA-AO complex and RNA-AO complex. Furthermore, the fluorescence spectra of the cells which had been treated by DNase or RNase before staining became similar to those of RNA AO complex or DNA-AO complex respectively. These results indicated that AO combined mainly with intracellular DNA and RNA at neutral pH.
Fluid spesimens for diagnostic cytology such as pleuroperitoneal effusions, pericardial fluids, cerebrospinal fluids, urine, punctures of cysts and cystic tumors, and washings show various characteristics according to their protein or mucin contents, number of RBC, total number of cells and volume of sediments. In our study it was examined what was the most suitable method of preparing each fluid specimen of various characteristics. 1) When it is impossible to send specimens immediately after aspiration to the cytologic laboratory, the addition of formation or ethylalcohol in the ratio of 10% or 50% for each to the fluid specimen which contains no or little protein is effective for the good preservation of the cells. Especially ethylalcohol is superior in nuclear find ings of Pap-smear. 2) In the specimen containing no or little protein, it is difficult to keep cells on the slide during the process of Papanicolaou's staining. Spray fixation for May-Giemsa staining is very helpful in retaining cells on the slide.3) From the fluids containing less protein than 1.0g/dl, 80% or more cells can be gathered by centrifugation at 1, 500 r. p.m. for 5 minutes. On the fluids of more protein than 2.0g/dl, it is necessary to centrifuge at 3, 000 r.p.m. for 10 minutes for gathering 80% of all cells in the fluid. 4) Autosmear CF-12 (from Sakura Finetech nical Co. Ltd.) is the instrument for gathering the sediments on the slide glass at concurrent centrifugation. We improved its cell for the purpose of practical use on preparation of Papsmears from fluid specimens. The old cell that was of oblique axis and small funnel shaped was changed horizontal in axis and cylindrical in shape. Consequently the instrument became very useful in gathering cells especially from the fluid specimen which was little in volume (10 ml or less) and contained few cells. If the specimen was much in volume (50ml or more) and contained few cells, we used the instrument for preparing the smears from the fluid made by addition of 10ml saline solution to the sediment of centrifuged original specimen. 5) In the specimen rich in RBC, it is convenient to treat it by saponin for its hemolytic activity. Too much addition of saponin by farover the border line volume for complete hemolysis inflicts injury on cells in various degrees. In conclusion, it has been proved that selection or combination of those methods according to the characteristics of each fluid specimen results in preparation of good smears.
Vaginal smears obtained from 30 normal pregnant women, 26 threatened abortion and one spontaneous abortion patients were observed to discuss the significance of vaginal cytology for the prognosis of abortion. 1) During'normal pregnant state findings of vaginal cytology reflect exact hormonal conditions. Also in cases of threatened abortion, if they would be caused by hormonal factors, vaginal smears would reflect these factors. 2) In some indices which were used to indicate the placental function, navicular cell index was most useful. During normal pregnant state this index kept over 60 per cent for 10 to 36 weeks gestation. 3) In cases of threatened abortion which were caused by hormonal factors and gave the decreased navicular cell index, cases of over 40 per cent of index could be expected good prognosis. And therapy would be effective. 4) Prognosis was poor in cases which lost the tendency of placard formation of epithelial cells, and indicated low navicular cell index below 40 per cent. 5) Increase of navicular cell index after the treatment indicates good prognosis, and vice versa. 6) It is difficult to determine the prognosis of threatened abortion by superficial cell index, pyknotic index, and eosinophilic index. 7) It is impossible to determine the prognosis, if threatened abortion is not caused by hormonal insufficiency. 8) Inflammatory changes and cytolysis in vaginal cytology make it difficult to determine the prognosis by any index of vaginal smear which reflect the hormonal conditions.
We see much of the varied cytologic patterns and atypism of cell types in the vaginal smear findings in cases of inflammatory diseases. In this point of view 1708 cytological specimens obtained from outpatients of the Department of Obstetrics and Gynecology, Osaka University Medical School from April 1, 1968. to March 31, 1969. were analyzed. 1) Although the cytological findings of vaginal smears reflect the various inflammatory processes and show quite complicated patterns, according to the types of inflammation, there are some tendencies in them. 2) The percentage of inflammatory cases which was diagnosed as Papanicolaou's class III is 1.4 per cent and most of them are the cases of senile colpitis and Trichomoniasis. 3) There is no specific finding of malignant tumor cells by which we can differentiate it from inflammatory changes, but coares chromatin structure in nuclei is most useful.
In 1949, Frech discovered the first example of ovarian cancer cells in vaginal smear but it is considered rare to find cancer cells in the vaginal smear, ovariau. Cancer cells were detected in the vaginal smear from a 62-year-old woman, 6 years after meno pause and having no subjective complaints.Ovarian source of the malignant cells in the smear was suspected from morphology of the cells which were large, had prominent nucleoli, without infiltra tion of leucocytes in vacuoles, and papillary arrangement of the cells. In addition, cytohor monal study of the smear showed abnormal matu ration index suggestive of a high estrogenic activity. Pathological examination of postoperative specimens confirmed the presence of papillary adeno carcinoma in the right ovary, but no metastasis was found in both tubes or in the left ovary, and in the uterus. It was considered that the appearance of cancer cells in the vaginal smear was due to discharge of cancer cells in the peritoneal cavity into the vagina via the Fallopian tubes, uterine cavity, and the cervix.