Restoration of broken cytology slides and creation of multiple slides from a single smear preparation.

A technique was developed for restoring broken cytology slides so that they are close to their original condition and for making multiple slides from a single smear preparation. The method is applicable to both cytologic preparations and histologic sections. In this study the fragmented smear preparation was treated with Pro-Texx, which penetrated, impregnated and solidified the full thickness of the pieces of the smear, enabling them to be lifted from the pieces of the broken slide. The removed pieces of the smear preparation were reassembled onto a new slide, which was then restained and coverslipped. In preparing multiple teaching slides, the treated smear preparation was divided as planned, with each portion mounted onto a separate slide, which was then restained and coverslipped. Ten other fine needle aspiration cases with broken slides have been restored, and more teaching slides were prepared from a single smear preparation using the same technique. All were equally successful. This technique provides an excellent method of smear transfer in cases of broken slides and creation of multiple slides from a single smear preparation for cytology teaching. This is particularly useful for unusual cases.     

Centrifugal elutriation (counterstreaming centrifugation) of cells

Lindahl first described the separation of cells by velocity sedimentation utilizing a special technique (counterstreaming centrifugation) that was later modified slightly and renamed centrifugal elutriation. Centrifugal elutriation has been applied, with variable degrees of success, to the separation of hemopoietic cells, mouse tumor cells, testicular cells, and a variety of other specialized cells as well as cells in particular phases of the cell cycle. The capacity of the elutriator to separate large numbers of cells is its chief advantage. The purities of the separated cells have not been compared with the purities of cells separated by other methods in most cases; such comparisons would permit more sophisticated comparison of elutriation with other techniques for velocity sedimentation.     

Centrifugal elutriation (counterstreaming centrifugation) of cells.

Lindahl first described the separation of cells by velocity sedimentation utilizing a special technique (counterstreaming centrifugation) that was later modified slightly and renamed centrifugal elutriation. Centrifugal elutriation has been applied, with variable degrees of success, to the separation of hemopoietic cells, mouse tumor cells, testicular cells, and a variety of other specialized cells as well as cells in particular phases of the cell cycle. The capacity of the elutriator to separate large numbers of cells is its chief advantage. The purities of the separated cells have not been compared with the purities of cells separated by other methods in most cases; such comparisons would permit more sophisticated comparison of elutriation with other techniques for velocity sedimentation.     

Cytomorphometry. A methodologic study of preparation techniques, selection methods and sample sizes

The influence of methodologic aspects on cytomorphometric features was studied using preparations of hepatoma and/or mastocytoma cells. First, two preparation techniques (smear and oese) were compared. Second, four methods of selecting cells for cytomorphometric analysis (two conventional and two stratified methods) were tested for reproducibility. Third, heterogeneous cell populations were used to estimate the required sample size using the running coefficient of variation (CV), and the results were compared with expected (theoretical) values of the required sample size calculated using the standard error of the mean. The results showed significantly lower CVs for the smear preparation technique. The stratified methods appeared to be superior to the conventional methods for selecting cells for measurement. The experimentally assessed sample sizes were considerably lower than the corresponding theoretical calculations. These findings suggest that morphometric assessments in cytologic smears should utilize a stratified cell selection method. While experimentally assessed sample sizes are relatively small and therefore better routinely applicable, they may yield less reliable results in some cases. The need to test a sample for its reproducibility as well as its discriminatory power is emphasized.     

离心法与细胞显微操作法对内皮细胞-白细胞黏附特性的初步研究

将哮喘病理血清与在体外培养的大鼠肺内皮细胞共同孵育,分别用离心法和显微操纵技术对细胞黏附进行体外研究。离心结果显示,病理血清刺激下的内皮细胞比正常情况更易与白细胞发生黏附。离心力高到一定程度可以解离部分黏附。从进一步的细胞显微操纵实验看病理血清刺激内皮细胞的功能状态,影响内皮细胞与白细胞发生黏附     

Comparison of three microscopic techniques for diagnosis of Cyclospora cayetanensis

Cyclospora cayetanensis oocysts in the feces of humans from Kathmandu, Nepal were identified on the basis of their size and other morphological characteristics. We compared the detection of C. cayetanensis oocysts in the feces using three microscopic techniques such as formalin-ether sedimentation, sucrose centrifugal floatation, and direct smear. Standard procedures were used for the formalin-ether sedimentation and the sucrose centrifugal floatation techniques using 0.5 g of feces, however, the direct smear technique was performed using 10 microl of fecal suspension (0.005 g of feces) and observed under the fluorescent microscope. Of the 403 samples examined, 21 samples were positive for oocysts by all three techniques. Therefore, in these 21 samples, the number of oocysts recovered by the three techniques were compared. The highest number of oocyst was obtained by the sucrose centrifugal floatation technique. In contrast, the formalin-ether sedimentation technique was found to be the least reliable concentration technique for the detection of Cyclospora in human feces. Surprisingly, the direct smear technique was found to be an effective and rapid technique for diagnosis of C. cayetanensis making it a technique of choice for routine epidemiological investigation of the prevalence of this infection in human populations.     

RADIOAUTOGRAPHIC STUDIES OF THE STEM CELL IN THE THYMUS OF THE IRRADIATED RAT

Radioautographic evidence is presented which characterizes the marrow derived stem cell which promotes thymic recovery following irradiation in the rat. These immigrant cells are similar in morphology to blood monocytes and have been called monocytoid, meaning monocyte-like in appearance. The typical cell had abundant pale staining cytoplasm and a nucleus with many invaginations and folds and a fine chromatin structure. There was no prominent nucleolus. The majority of these cells entered the thymus of the irradiated rat via the blood vessels into the septa and made their way through the connective tissue to the outer cortex. Three distinct morphological cell types appeared to be derived from the immigrant cells. These were fibrocyte-like cells which were located within the septa, macrophages located mainly within the medulla and septa, and large blast cells within the cortex, which proliferated giving rise to large thymocytes. The blast cells were characterized as having abundant moderately basophilic (and pyroninophilic) cytoplasm with a distinct cytoplasmic boundary, a large nucleus which still had invaginations and folds, a loose chromatin structure and one or more very prominent nucleoli. They were located in groups primarily within the outer cortex and often adjacent to blood vessels. They were found to be highly susceptible to damage in smear preparations. In contrast, their progeny, the large thymocytes were not highly susceptible to damage in smear preparations but teased out as large round cells with a highly basophilic rim of cytoplasm. The large thymocytes were precursors to medium and small cells. A radioautographic technique for 1 μ tissue sections is also described.     

Environmental and centrifugal factors influencing the visco-elastic properties of oral biofilms in vitro

Centrifugal compaction causes changes in the surface properties of bacterial cells. It has been shown previously that the surface properties of planktonic cells change with increasing centrifugal compaction. This study aimed to analyze the influences of centrifugal compaction and environmental conditions on the visco-elastic properties of oral biofilms. Biofilms were grown out of a layer of initially adhering streptococci, actinomyces or a combination of these. Different uni-axial deformations were induced on the biofilms and the load relaxations were measured over time. Linear-Regression-Analysis demonstrated that both the centrifugation coefficient for streptococci and induced deformation influenced the percentage relaxation. Centrifugal compaction significantly influenced relaxation only upon compression of the outermost 20% of the biofilm (p < 0.05), whereas biofilm composition became influential when 50% deformation was induced, invoking re-arrangement of the bacteria in deeper biofilm structures. In summary, the effects of centrifugal compaction of initially adhering, centrifuged bacteria extend to the visco-elastic properties of biofilms, indicating that the initial bacterial layer influences the structure of the entire biofilm.     

A capillary electrophoretic method for monitoring the presence of alpha-tubulin in nuclear preparations

A sensitive capillary electrophoretic method was developed to detect the presence of alpha-tubulin, a microtubular cytoskeletal component, in isolated nuclear preparations. These preparations are treated with anti-alpha-tubulin primary mouse antibodies and then stained with a fluorescently labeled anti-mouse IgG antibody. The stained preparation is then analyzed by capillary electrophoresis with laser-induced fluorescence detection, a technique that allows for sensitive detection of fluorescently labeled species. Using this method, it is feasible to count individual subcellular aggregates containing alpha-tubulin (SATs), estimate the number of alpha-tubulin molecules per SAT, determine the cumulative intensity of all SATs as an estimate of the relative level of alpha-tubulin in a preparation, and obtain their apparent electrophoretic mobility distribution. The method was validated by comparing SATs from untreated cells with those from colchicine-treated cells. Since colchicine is a microtubule-disrupting agent, treatment reduced the number of SATs per cell as well as the cumulative intensity of all SATs in a preparation. In contrast, the apparent electrophoretic mobility distribution was not influenced by colchicine treatment, suggesting that this parameter is not strongly dependent on the alpha-tubulin content. Given the zeptomolar sensitivity of laser-induced fluorescence detection and the widespread availability of antibodies, the approach used here represents an improvement in the detection of cytoskeletal impurities in subcellular fractions.     

Diagnostic value of clot examination for malignant cells in serous effusions

Objective:  To assess the diagnostic value of clot examination for satisfactory processing and confirmation of malignancy in serous effusions in routine cytological evaluation and compare the results with those of conventional smear and cell block preparations.
Methodology:  Body cavity fluids ( n  = 600) received in our laboratory were processed according to a pre-designed protocol for the study as follows: Day1: on receipt of the specimen, smears were made and a cell block was prepared from the sediment. Day2: after overnight sample storage of the remaining specimen at 2–8 °C all fluids were examined for the presence of a clot at the bottom of the container. Fluids in which clot had formed were fixed in formalin. The clot was then placed on a lens paper, wrapped and processed routinely. Diagnostic yields were compared.
Results:  In this study, we included 600 cases of serous fluids from pleural, pericardial and peritoneal effusions. In 73% ( n  = 437) of samples, clot formation was seen, while in 27%, ( n  = 163) no clot had formed. Routine smear and cell block preparations showed malignant cells in 9.6% ( n  = 42). However, with the addition of the clot preparation, the number of cases in which atypical/malignant cells were seen increased from 42 to 85 (19.4%), with a P  < 0.001. Special stains and immunohistochemistry (IHC) were also performed on clot preparations in 10 difficult cases.
Conclusion:  Clot preparation from body cavity fluids on the second day can be used as an adjunct to smear and routine cell block preparation to improve the accuracy and yield of the cytological diagnosis and may also be of great help for special studies such as IHC staining.     

Centrifugal elutriation: separation of spermatogenic cells on the basis of sedimentation velocity.

Various types of cells from the testes of mice and hamsters were separated according to differences in sedimentation velocity by centrifugal elutriation, a counterflow centrifugation technique. Approximately 3 times 10(8) cells, prepared from six mouse testes or from one hanster testis, were separated into 11 fractions in less than two hours as compared to the 4--5 hours required for sedimentation at unit gravity ("Staput"). Fractions enriched in elongated spermatids and spermatozoa (100%), stages 1--8 spermatids (69%) and pachytene spermatocytes (58%) were obtained from mouse testis dispersions. Similarly enriched fractions were obtained from hamster cells. A single fraction enriched in stages 1--8 spermatids (mouse) was prepared in less than 30 minutes. As many as 2 times 10(9) cells were separated in a single procedure. Spermatogenic cells exhibited no evidence of structural damage with trypan blud and phase microscopy, and recovery was essentially 100%. Centrifugal elutriation had no effect on sperm motility or on the plating efficiency of CHO cells.     

The Blood Island is a Site of Formation of the Primary Embryo Thrombocyte in the Chick Blastoderm

Using the immunohistological technique we inquired at what developmental stage and in which site of chick blastoderm does the embryo thrombocyte (ET) begin to differentiate. An anti-ET antibody was raised against rabbits by injecting ETs isolated from blood of 10 day chick embryos. By applying the indirect staining method to smear preparations of blood collected from developing embryos it was confirmed that cytoplasm of the ET showed more intense staining than that of the erythroid cell and that the ET population could be distinguished from the erythrocyte population by this antibody. Cells showing the intense staining could be detected first in blood islands of the area opaca vasculosa of stage 9+ blastoderms. These embryo thromboblasts were found singly or in groups of a small number at dorsal periphery of cell clusters in the blood island. The electron microscopy revealed that embryo thromboblasts appeared in the same position in the stage 9+ blastoderm. At stage 10+ or later embryo thromboblasts were also present adhering to the vascular endothelium or free in the vessel lumen. We conclude that ETs start differentiating from primitive mesenchymal cells localized in the blood island of the area opaca vasculosa at stage 9 or earlier, migrate thereafter to vessel lumen, and enter the blood stream.     

Sedimentation velocity separation: a preparation method for cervical samples.

A preparation procedure, aiming at monolayer deposition of cervical exfoliative material on glass slides for high resolution prescreening has been developed. The main features of this procedure are centrifugal deposition after suspension and sedimentation of samples over isopycnic medium of 1.026 density. Fractioning of the separation column after centrifugation at 50 X g yields two preparations with leukocytes, bacteria and cellular debris predominantly located on the first slide and epithelial cells on the second one. The degree of spatial cellular isolation as well as the amount of diagnostically relevant cells per slide seem to fit the requirements of automated high resolution analysis.     

Responses of hair cells to statocyst rotation

A new technique is described for stimulating hair cells of the Hermissenda statocyst. The preparation and recording apparatus can be rotated at up to 78 rpm while recording intracellular potentials. Hair cells in front of the centrifugal force vector depolarize in response to rotation. Hair cells in back of the centrifugal force vector hypoerpolarize in response to rotation. Mechanisms by which the hair cell generator potential might arise are examined.     

A new type of cytocentrifuge,the valve-centrifuge

Summary A new type of cytocentrifuge has been developed in which the sedimentation process of the cells onto the slides is separated from the draining of the sedimentation fluid. This is realised by electrically controlled valves which can be closed and opened while the centrifuge is running. Sedimentation is carried out with closed valves, draining of adhering medium with open valves.The preparations, freed of adhering medium by the centrifugal force can be taken out and the cells can be fixed. Alternatively the valves can be closed again and fixative can be introduced through a central well, the cells still being under the influence of the centrifugal force. With subsequent draining of the fixative and introduction of washing and staining solutions through the central well, the whole process from sedimentation to staining can be carried out in the running centrifuge. The process seems well suited for complete automation.Using dilution series from a suspension of human buffy coat cells counted in a Buerker chamber, the cell counts in the centrifuge preparations showed virtually total recovery of cells, with no apparent selection or specific distribution of cell types. Draining of the sedimentation and fixative fluids at a slow rate was found to be vital for optimal recovery of cells. The morphology of different cell types sedimented on the slide was excellent. The flattening of nuclei through gravity was studied by cytophotometry of Feulgen-stained leucocytes. The nuclear area of these cells was found to be approximately double that from cells in identically stained classical smears. With this type of valve-centrifuge a quantitative and unbiased recovery of uniformly spread and flattened cells on coverslips or slides may be obtained, thus making the procedure well suited to automated analysis based on cytophotometric principles and morphometric pattern recognition.     

The value of multiple preparations in the diagnosis of malignant pleural effusions. A cost-benefit analysis

A cost-benefit analysis of five techniques employed in processing 108 malignant pleural effusions for cytopathologic examination was performed. Ethanol-fixed, Papanicolaou-stained smears were positive in 68% of the effusions, air-dried Diff-Quik-stained smears in 66%, Cytospins in 83%, cell blocks in 85% and Millipore filters in 85%. Examination of one air-dried smear and one ethanol-fixed smear yielded a diagnostic sensitivity of 82%. Using a combination of two smears and one of three concentrating techniques (Cytospin, cell block or MIllipore filter) would have provided a diagnosis in over 90% of the effusions. The use of four or more preparations provided more sensitivity than did three preparations and decreased the likelihood that a malignant diagnosis would be based on the findings in only one preparation. The costs related to disposable materials and the College of American Pathologists (CAP) work load estimates for specimen preparation, staining and screening were compared. These data may be useful in developing optimal protocols for pleural effusion preparation in laboratories with specific work load requirements and limited resources.     

Application of the avidin-biotin-complex method for the light microscopic analysis of lymphocyte subsets with monoclonal antibodies on air-dried smears

A study was undertaken of the application of the avidin-biotin-peroxidase complex (ABC) method to the monoclonal antibody MAbs staining of mononuclear cells in hematologic and cytodiagnostic materials. Satisfactory cell morphology and immunoreactivity of surface antigens were observed when the slides were fixed in 80% acetone in phosphate-buffered saline or in 60% acetone in 0.03 M citric acid buffer solution (pH 5.4). Unstained air-dried preparations could be preserved for two weeks at room temperature in a desiccator and for one year at -70 degrees C after fixation. An excellent immunoreaction, even with a weak surface antigen, was observed by inhibition of endogenous peroxidase after the secondary antibody reaction; reactions of weak antigens tended to be obscured when the inhibition was performed before the first antibody reaction. Use of the Giemsa stain as a counterstain made it possible to readily observe the cell morphology; therefore, white blood cell analysis could be performed simultaneously when peripheral blood smears were studied. The positive rate of immunoreaction by an immunofluorescent method was well correlated with that obtained by the ABC method. The ABC method proved to be an excellent immunocytochemical technique for detecting cell surface antigens with high sensitivity and specificity; furthermore, it is useful for cell morphology studies and yields permanent preparations.     

Technic for Studying Chromosome Structure

The methods described are modifications of various technics for the study of spiral structure in chromosomes. They enable permanent preparations to be made with better fixation and allow the use of stains which give clear and more critical definition. The first method described involves the use of ammonium, hydroxide (880 vols.) fumes for the treatment of pollen mother cells before fixation. Anthers of Tradescantia are smeared on a slide and wet in a 3% cane sugar solution. The preparation is then immediately placed in a dish of fixative where it remains for two hours. The slide can then be washed, bleached and stained with gentian violet or hematoxylin. It was found that fumes of nitric acid, hydrochloric acid and glacial acetic acid gave similar results. For the second method, boiling water is used for pre-treatment. A smear is made on a slide and immersed in boiling water for five to ten seconds. The smear is then fixed and treated in the usual manner.     

Comparison of DNA ploidy and nuclear size, shape and chromatin irregularity in tissue sections and smears of prostatic carcinoma

Image analysis measurements of nuclear size, shape, texture and DNA ploidy were compared in smears versus the corresponding 4-microns tissue sections, both prepared from radical prostatectomy specimens obtained from resections for prostatic cancer. Thirty-nine cases (78%) showed concordant DNA histograms between the smear and the tissue section. In six cases (12%), both preparations were nondiploid, but a tetraploid population was also present in one, but not both, of the preparations. In five cases (10%), there was a major discordance between the smear and the tissue section, with one preparation diploid and the other nondiploid. One source of discrepancy between the smear and tissue histograms was the overlapping of larger nuclei in tissue sections, which often precluded the analysis of the most atypical cells. Some tissue histograms were difficult to interpret due to wide coefficients of variation, irregular peaks and some shift from 2n in the diploid peaks. The best morphometric correlation (0.78) between the smears and the tissue sections was for the modal nuclear shape. Nuclear size and texture measurements showed poorer correlations. These findings suggest that cytologic preparations of prostatic carcinoma should be preferred for image analysis.