Standard specimens for stain calibration and their application to the Papanicolaou stain

Standardized specimens composed of extracts of biologic objects (nucleoprotamine and bovine liver) were developed as tools for the quantitative evaluation of stain performance on biologic substrates. The specimens are mixtures of proteins and nucleic acids and thus mimic the staining characteristics of cytologic smears. The concentration of each mixture and the specimen thickness can be precisely controlled, ensuring the production of a large number of samples with a nearly identical capability for dye binding. The transmitted light spectra of the standardized specimens varied depending on the extract and the preparation conditions. Spectra similar to those reported from the nuclei and cytoplasm of cell types in Papanicolaou-stained cervicovaginal smears were observed. Light transmission was uniform to +/- 5% across each specimen and from specimen to specimen. The specimen thickness was uniform within +/- 2%. Studies with these standardized samples could reveal the much-needed correlations between the chemical and optical characteristics of dyes and dye solutions and the performance of the dyes on biologic substrates.     

Evaluation of transgenic canola plants under field conditions.

Eleven independent transgenic canola (Brassica napus ssp. oleifera L. cv. Westar and Regent) lines were evaluated in the field. The plants carried a neomycin phosphotransferase (NPTII) gene for kanamycin resistance that was introduced via Agrobacterium-mediated transformation. NPTII enzyme assays, Southern blot by hybridizations and progeny analysis, confirmed the stable, heritable integration and expression of the introduced NPTII gene. A number of agronomic characteristics evaluated under field conditions, including maturity yield, and oil and protein content, were all statistically comparable between the transformed and nontransforemd platns. These results indicate that canola can be genetically engineered successfully, and that the Agrobacterium-based transformation system employed does not induced any adverse effects on the intrinsic agronomic and qualitative traits critical to the agricultural industry.     

Application of Ultrafast Papanicolaou stain to body fluid cytology

OBJECTIVE: To investigate the applicability of the Ultrafast Papanicolaou stain to the cytology of fluids and to compare it with other methods. STUDY DESIGN: Over a 30-month period, 528 unfixed fluids (462 serous effusions, 48 pelvic washings, 16 cyst fluids and 2 bile duct drain fluids) were mixed thoroughly and centrifuged. Two Swedish-style air-dried smears were made and stained with Diff-Quik (Mercedes Medical, Inc., Sarasota, Florida, U.S.A.) and Ultrafast Papanicolaou stain (Richard Allan Scientific, Kalamazoo, Michigan, U.S.A.), and the remaining sediment was fixed in CytoRich Red (TriPath Imaging, Inc., Burlington, North Carolina, U.S.A.), centrifuged onto a 17.5-mm circle with a Hettich cytocentrifuge and stained by the Papanicolaou method. RESULTS: For the 115 malignant fluids, Ultrafast Papanicolaou stain was the preferred method in the 94 non-hematopoietic malignant fluids, Diff-Quik was the preferred method in the 9 hematopoietic malignancies, and CytoRich Red was the preferred preparation in 8 bloody effusions containing rare cancer cells and 4 malignant pelvic washings. The diagnostic turnaround time of smears stained by Ultrafast Papanicolaou stain was < 15 minutes, fast enough for intraoperative consultations. CONCLUSION: It seems that Ultrafast Papanicolaou stain improves the resolution of cytoplasmic and nuclear details of nonhematopoietic cells in body fluids. However, to detect cancer in all types of fluids, Diff-Quik and CytoRich preparations are also required. We now examine three slides per fluid sample, one slide by each of the three techniques.     

甘蔗种质资源自然条件下耐寒性评价

为了解广西农业科学院甘蔗研究所种质资源圃(南宁)保存的400份甘蔗种质材料的耐寒性表现,在2008年初低温灾害的自然条件下,以蔗茎节间受害指数和节间生长点受害指数为基础,通过系统聚类方法将参试材料分为不同耐寒性表现类群。结果表明,2008年初的低温灾害属于阴雨霜冻类型,400份甘蔗种质材料可分为5个耐寒性表现类群,耐寒性强的材料有226份(56.50%),耐寒性较强的材料有103份(26.75%),耐寒性一般的材料有53份(13.25%),耐寒性较差的材料有7份(1.75%),耐寒性差的材料有7份(1.75%)。可构建甘蔗耐寒性指数(CTI):CTI=0.3×节间受害指数+0.7×节间生长点受害指数。节间生长点对低温伤害的敏感性高于蔗茎节间组织,建议作为耐寒性评价的重要指标之一;甘蔗耐寒性指数可以用于评价阴雨霜冻灾害下甘蔗种质材料的耐寒性。     

Frequency of micronuclei in the cells of buccal epithelium in Ukrainian schoolchildren of various age and sex

The frequency of micronuclei (MN) has been studied in 266 schoolchildren, participants of All-Ukrainian school competitions on biology and ecology. The distribution of MN frequencies was Poissonian with λ = 2.5. The mean MN frequency was 2.4 ± 0.15‰ in boys and 2.7 ± 0.14‰ in girls; the difference is not statistically significant. The individual MN frequency varied from 0 to 8.3‰ and on the average was 2.5 ± 0.11‰ which does not exceed known mean population values. We have shown differences in the MN frequency between sexes in some age groups: MN frequency was significantly higher in girls than in boys aged 16 years. The dynamics of age-related changes in the MN frequency of boys and girls also differed in the period studied (14–18 years of age).     

Clastogenic effect for cigarette smoking but not areca quid chewing as measured by micronuclei in exfoliated buccal mucosal cells

The objective of this study was to use the micronuclei from exfoliated buccal mucosal cells to investigate the clastogenic effects of areca quid chewing and cigarette smoking, as well as the interaction between the two. The study population was selected from residents of seven villages recruited for a community-cohort study. A total of 141 subjects were recruited based on the regular consumption of cigarettes and betel quid. Salient personal characteristics were collected from interview using a specially designed questionnaire. Micronuclei were scored on Feulgen/fast green-stained smear preparations of exfoliated cells obtained by scraping the surface of the buccal mucosa. There was no significant interaction between the chewing of betel nut and cigarette smoking. Heavy smoking was positively associated with MN frequency, with areca quid chewing negatively associated. A significant positive trend was demonstrated for the relationship between MN frequency and either daily cigarette consumption or cumulative smoking pack-years. By contrast, negative trends were demonstrated for the analogous relationships with areca quid chewing. These results indicate that heavy smoking, but not areca quid chewing, increases MN formation. These findings suggest that the carcinogenesis of the oral cancers induced by areca quid chewing in Taiwan may be through a pathway other than genotoxicity.     

Aspergillus in the Papanicolaou stain: morphology, fluorescence and diagnostic feasibility

hettlich c., küpper th., wehle k. and pfitzer p. (1998) Cytopathology 9, 381–388
Aspergillus in the Papanicolaou stain: morphology, fluorescence and diagnostic feasibility
Aspergillus species exhibit a distinct and clear fluorescence in Papanicolaou-stained cytological samples. The Papanicolaou (PAP) stain enhances the autofluorescence of cultured aspergilli and allows better cytological recognition of the fungus by fluorescence microscopy when it is not easily discerned from its surroundings by light microscopy. Morphological properties can be better distinguished and facilitate the differentiation of aspergillus organisms from other filamentous fungi. Neither light nor fluorescence microscopy, the cytological quality nor the presence of phagocytosed hyphae in alveolar macrophages allow distinction between infection and contamination with Aspergillus species. Only the presence of eosinophilic inflammation permits a tentative diagnosis of an Aspergillus infection. In conclusion, PAP fluorescence reduces the need for special stains, is superior to and quicker than other investigative techniques and enhances the sensitivity and specificity of cytological investigation when a rapid and reliable identification of Aspergillus is needed.     

Urinary cyclophosphamide excretion and micronuclei frequencies in peripheral lymphocytes and in exfoliated buccal epithelial cells of nurses handling antineoplastics

In this study, urinary cyclophosphamide (CP) excretion rate, as well as micronuclei (MN) in peripheral lymphocytes and in buccal epithelial cells were determined for 26 nurses handling antineoplastics and 14 referents matched for age and sex. In urine samples of 20 out of 25 exposed nurses CP excretion rate was found in a range of 0.02-9.14 microg CP/24 h. Our results of the analyses of CP in urine demonstrates that when the nurses were handling CP (and other antineoplastic drugs) this particular compound was observed in urine. The mean values (+/-SD) of MN frequencies (%) in peripheral lymphocytes from the nurses and controls were 0.61 (+/-0. 32) and 0.28 (+/-0.16), respectively (p<0.01). The mean value (+/-SD) of MN frequency (%) in buccal epithelial cells of nurses was 0.16 (+/-0.19) and also mean MN frequency in buccal epithelial cells for controls was found to be as 0.08 (+/-0.08), (p>0.05). Age, sex and smoking habits have not influenced the parameters analyzed in this study. Handling time of antineoplastics, use of protective equipment and handling frequency of drugs have no effect on urinary and cytogenetic parameters analyzed. No correlation was found between the urinary CP excretion and the cytogenetic findings in nurses. Neither could we find any relationship between two cytogenetic endpoints. Our results have identified the possible genotoxic damage of oncology nurses related to occupational exposure to at least one antineoplastic agent, which is used as a marker for drug handling. As a whole, there is concern that the present handling practices of antineoplastic drugs used in the several hospitals in Ankara will not be sufficient to prevent exposure.     

Efficiency of bio-indicators for low-level radiation under field conditions

Relatively little is known about biological consequences of natural variation in background radiation, and variation in exposure due to nuclear accidents, or even the long term consequences to human health stemming from the over-use of nuclear medicine and imaging technologies (i.e. CAT scans). This realization emphasizes the need for assessment and quantification of biological effects of radiation on living organisms. Here we report the results of an environmental analysis based on extensive censuses of abundance of nine animal taxa (spiders, dragonflies, grasshoppers, bumblebees, butterflies, amphibians, reptiles, birds, mammals) around Chernobyl in Ukraine and Belarus during 2006–2009. Background levels of radiation explained 1.5–26.5% of the variance in abundance of these nine taxa, birds and mammals having the strongest effects, accounting for a difference of a factor 18 among taxa. These effects were retained in analyses that accounted for potentially confounding effects. Effect size estimated as the amount of variance in abundance explained by background level of radiation was highly consistent among years, with weaker effects in years with low density. Effect sizes were greater in taxa with longer natal dispersal distances and in taxa with higher population density. These results are consistent with the hypotheses that costs of dispersal (i.e. survival) were accentuated under conditions of radioactive contamination, or that high density allowed detection of radiation effects. This suggests that standard breeding bird censuses can be used as an informative bio-indicator for the effects of radiation on abundance of animals.     

Quantitative chromatics analysis for computer imaging of cytologic subtypes of lung cancer stained by Papanicolaou stain

OBJECTIVE: To determine the role of quantitative chromatics analysis in the classification of subtypes of lung cancer stained by Papanicolaou stain. STUDY DESIGN: By means of computer image analysis, 60 keratinized squamous carcinoma cells (KSCC), 88 nonkeratinized squamous carcinoma cells (NKSCC) and 150 adenocarcinoma cells (ACC) from lung cancer in sputum smears stained by Papanicolaou stain were analyzed and distinguished based on quantitative colorimetry. The features measured were the content of three primary colors, red (R), green (G) and blue (B) and the coefficients of R, G and B (r, g and b, respectively). Hue, saturation, brightness and gray level were also measured. A stepwise discriminant analysis was carried out. RESULTS: The values of R, G and B and r, g and b, hue and saturation in NKSCC and ACC were significantly different from those of KSCC, and the changes in the three primary colors were more sensitive than those in the gray level. Computer assessment based on three primary color coefficients, hue and saturation yielded accuracy of distinguishing KSCC from NKSCC and KSCC from ACC of 95.2% and 95%, respectively. CONCLUSION: Quantitative analyses of R, G and B and r, g, b and hue and saturation are valuable in distinguishing KSCC from NKSCC and ACC.     

Evaluation of the stain Viablue for the rapid estimation of viable yeast cells

Viablue can be used as a differential viability stain for yeast cells. Differential staining using fluorescence microscopy and viability showed a good correlation ( r = 0·99). For practical purposes the staining procedure takes only a few minutes and appears ideally suited to such rapid analytical procedures as the direct epifluorescent filter technique (DEFT) and automated quality control testing of foods and beverages.     

Viscoelastic deformation response of red blood cells under conditions of oscillating centrifugal field

The red cell deformation under the conditions of oscillating centrifugal fields was studied. Experiments were carried out with a modified Cell-Elastometer operating in oscillating mode (0.02 to 0.30 Hz). Gravitational acceleration was sinusoidally modulated between 620 g and 2250 g. At low frequencies (below 0.08 Hz), native red cells followed the applied stress without delay. At 0.09 Hz and up, the cellular deformation was still periodical and included an additional perturbation due to intracellular movements. This perturbation was analysed and quantified. The influence of alterations on the erythrocyte membrane by diamide was analysed to verify the sensitivity of this method. On increasing the membrane stiffness with low concentrations of diamide, the response to oscillatory centrifugal stress was impaired characteristically in terms of amplitude deformation. Based on tangential and centrifugal accelerations, a physical model was developed that describes the basic observable changes on varying the oscillation frequency. From the data it can be concluded that viscoelastic properties of red cells can be analysed and quantified using oscillatory centrifugal accelerations. The described method can become a valid tool to differentiate between membrane alterations or intracellular viscous modifications.