Digital image analysis of silver-stained nucleolar organizer region--associated proteins in endometrial cytologic samples

OBJECTIVE: Digital image analysis was applied to determine the number, area and size of silver-stained nucleolar organizer regions (AgNORs) in cytologic samples from curettage in normal, hyperplastic and malignant endometrium. STUDY DESIGN: Thirty-two archival cytologic smears from curettage (previously stained by the Papanicolaou method) with the histologic diagnosis (4 inactive endometrium, 5 secretion, 5 proliferation, 5 simple hyperplasia, 5 complex hyperplasia, 3 atypical hyperplasia, 5 adenocarcinoma, grade 1) were analyzed with the AgNOR technique. Count, area and size of AgNORs were analyzed in 50 cells per sample using a magnification of 1,000x. Quantitative analysis was performed on an SFORM digital imaging system. Data were analyzed with the SPSS/PC+ program. Mann-Whitney and chi 2 tests were performed. RESULTS: The average value of AgNOR count increased from normal to hyperplastic endometrium and well-differentiated adenocarcinoma. Differences were significant except between atypical hyperplasia and adenocarcinoma. Four, five and more AgNORs in 40% or more of the nuclei were found in complex and atypical hyperplasia and adenocarcinoma. Proliferation, and simple and atypical hyperplasia had similar mean values of AgNOR area. The mean total AgNOR area value increased from normal to hyperplastic had similar mean values of AgNOR area. The mean total AgNOR area value increased from normal to hyperplastic and well-differentiated adenocarcinoma. Differences were statistically significant. AgNOR size in well-differentiated adenocarcinoma was significantly different from that in normal endometrium and different grades of hyperplasia. CONCLUSION: Digital image analysis of AgNOR count, area and size enabled a distinction to be made between normal, hyperplastic and malignant endometrium.     

Criteria for the cytologic assessment of hyperplasias in endometrial samples obtained by the Endopap Endometrial Sampler

The Endopap Endometrial Sampler was used in 1,465 women, either just before endometrial biopsy or curettage (in 760 symptomatic patients) or as an office procedure (in 705 women). The samples were inadequate for interpretation in only 8.7% of the cases. Although all malignant lesions were identified by this screening technique, about one-fourth were initially classified cytologically as hyperplasia. Endometrial hyperplasias presented the greatest difficulties in interpretation, with only slightly over half of the proven cases correctly assessed on the endometrial sample. In an attempt to improve the accuracy of the cytologic diagnosis of hyperplasias, ten morphologic features were examined retrospectively in 207 cases. Five of the criteria were shown to provide an increased probability of correctly diagnosing endometrial hyperplasias on the cytologic sample: (1) the overlapping of cells in the glandular clusters or sheets, (2) the presence of nucleoli, (3) anisokaryosis, (4) granularity of chromatin and (5) the presence of sheets of stromal cells. The more of these criteria observed in a given case, the better was the chance of cytologically identifying a hyperplasia in the Endopap sample.     

Immunohistochemical assessment of prostaglandin H-synthase in bovine endometrial biopsy samples collected throughout the oestrous cycle

The study was designed to determine the distribution of prostaglandin H-synthase (PGS) also known as cyclooxygenase in specific uterine cell populations during the oestrous cycle. Endometrial biopsy samples were obtained from a total of 10 clinically healthy cows at days 1 (initiation of behavioural oestrus), 8, 15, and 19 of the oestrous cycle. All animals conceived after biopsy regimen. Data of semiquantitatively scored immunoreactivities were analysed using analyses of variance, t-tests for paired data and correlation analyses. Biotin-streptavidin-peroxidase immunostaining technique was employed to localise PGS. Specific staining was consistently present in endothelial cells of arteries but not capillaries and venules. A gradient of staining intensity was clearly apparent within the endometrium: surface epithelial cells and stromal cells located near the endometrial surface are consistently stained more intensely than glandular epithelial cells and stromal cells lying deeper in the endometrium. Days of oestrous cycle also influenced PGS immunoreactivities. Generally, higher immunoreactivities were recorded in surface epithelium, uterine glands and endometrial stromal cells at cycle days 1 and 19 as compared to cycle days 8 and 15. Minimal scoring values were mainly found at cycle day 8. The results of the present study suggest that the amount of bovine endometrial PGS varies considerably with the day of cycle in the above mentioned cell-type- and location-restricted manner. Therefore, the capacity of the bovine uterine mucosa for prostaglandin production may—amongst other factors—depend on the cycle-restricted availability of the respective enzyme systems.     

A new sub-sampling method for analysis of air samples collected with the Andersen single-stage sampler

A protocol for bioaerosol collection was developed that provides not only accurate predictions of fungal concentration, but also improves species recovery. Random transfer of a subset of 50 of the 400 impaction points from Andersen single-stage bioaerosol sampling plates results in subcultures that are accurate predictors of fungal concentration (CFU/m³), when compared to duplicate untouched Andersen plates. A linear regression model was developed to estimate CFU/m³ from the colonies counted on the Random-50 plates. The random transfer to five plates (“Random-50” plates), allows large numbers of fungi to be recovered and identified, including slow-growing fungi that otherwise would be masked by fast-growing fungi.     

Digital imaging analysis of normal, hyperplastic and malignant endometrial cells in endometrial brushing samples

Sixty cytologic specimens obtained by endometrial brushing (using the Gynecyte device) were quantitated by digital imaging techniques. These samples included 25 from normal endometria, 6 from persistent proliferative endometria, 14 from cystic or adenomatous hyperplasias and 15 from carcinomas. The morphometric parameters surveyed included mean cell area, nuclear area, perimeter and long and short axes. The amount of hematoxylin dye in the nuclei was expressed by mean transmittance (mean of gray levels) and chromatin index (standard deviation of gray levels). The frequency distributions of cells derived from normal tissue and persistent proliferative endometrium were quite similar. The quantitative parameters of cystic and adenomatous hyperplasia, although intermediate between those of normal endometrium and carcinoma, were closer to the former than to the latter. Using stepwise discriminant analysis of the morphometric parameters, 83% of the specimens were correctly classified into the categories of normal/persistent proliferative, hyperplasia and carcinoma. The accuracy was improved to 88% when densitometric parameters were added. This study demonstrates the potential application of digital imaging techniques to the distinction and classification of normal, hyperplastic and malignant endometrial cells.     

Prognostic value of cytologic examination of peritoneal washings in patients with endometrial carcinoma

Peritoneal pelvic washings from 54 women with pathologic stage I endometrial carcinoma were evaluated in a blind retrospective fashion for the concentration of malignant cells present. None of the 42 patients with normal washings developed recurrence after a median disease-free survival of 36 months. Of the 12 patients with adenocarcinoma in the washings, 4 had high concentrations of malignant cells (greater than 1000 cells/100 ml sample), and all 4 died as a consequence of carcinoma within two years. The remaining eight patients had lower concentrations of malignant cells in the washings (less than 1000 cells/100 ml sample), and six of these patients had no evidence of disease after 37 to 64 months. Cox's nonparametric statistical model showed that increasing concentrations of adenocarcinoma cells in washings significantly shortened the time to recurrence of disease. The abundance of malignant cells has prognostic importance in identifying those patients with pathologic stage I disease who may require more aggressive therapy.     

Efficacy of the Isaacs Endometrial Cell Sampler in the cytologic detection of endometrial abnormalities

The efficacy of the Isaacs Endometrial Cell Sampler to obtain specimens for cytologic examination to detect both hormonal disturbances and precursors of malignancy of the endometrium was assessed in 120 symptomatic women. Correlation of the cytologic results with the histologic diagnosis on curettage specimens showed a diagnostic accuracy of 96.3%. The cytologic criteria for diagnosing samples obtained with the Isaacs device are presented.     

Ki-67 antigen detection in unstained and destained cytologic samples

OBJECTIVE: To evaluate the effect of different tissue preservation, fixation and staining procedures on the expression of proliferation-associated Ki-67 antigen in cytologic samples to establish an easy and uniform way to handle cytologic specimens with an automated staining technique. STUDY DESIGN: Multiple touch imprints were made from eight breast tumors. The specimens were treated according to different protocols, and Ki-67 nuclear expression was compared to that in the corresponding histologic sections. RESULTS: In the unstained specimens, air drying at room temperature for up to four months or ethanol spray fixation preserved the material and offered excellent results. Processing effectively removed previous stain without additional chemical destaining. Antigen retrieval was not achieved in the previously Giemsa stained imprints and was suboptimal in those stained according to Papanicolaou. CONCLUSION: Immunocytochemical detection of Ki-67 is recommended for previously unstained cytologic specimens.     

Development of a monitoring network for the analysis of elements in aerosol samples collected at Lake Balaton

Determination of different toxic elements in aerosol and precipitation samples collected at Lake Balaton were carried out. A simple sequential leaching procedure was applied for the determination of the distribution of elements. The distribution of elements was determined among environmentally mobile, bound to carbonates and oxides, and bound to silicates and organic matters (environmentally immobile) fractions. Particular attention was paid to distinguish between environmentally mobile and environmentally immobile fractions because these represent the two extreme modes by which the metals are bound to the solid matrices. Aerosol samples were weekly collected in Tihany, Siófok and Keszthely on 5 cm diameter Teflon filters with a membrane pump. While Cd-compounds have been found enormously in the environmentally mobile fractions, As-compounds accumulated almost evenly among portions. The results of sequential leaching give an indication of the mobility of the elements once the aerosol is mixed directly into natural waters on during scavenging of the aerosol by wet deposition. Based upon the data it can be concluded that the effect of anthropogenic sources is minor in this area.     

Detection of HPV DNA in cervical specimens collected in cytologic solution by ligation-dependent PCR

OBJECTIVE: To determine the feasibility and sensitivity of detecting human papillomavirus (HPV) in specimens collected in Cytyc PreservCyt fluid (Boxborough, Massachusetts, U.S.A.) using ligation-dependent polymerase chain reaction (LD-PCR) and to demonstrate the diagnostic value of HPV DNA testing as an adjunct to cytology in the detection of cervical squamous intraepithelial lesions (SIL), especially in cases of atypical squamous cells of undetermined significance (ASCUS). STUDY DESIGN: LD-PCR is a recently invented DNA amplification technology that utilizes a capture probe for target isolation and 2 hemiprobes for target detection. The hemiprobes are designed in such a way that when they hybridize to their target, the 5' end of one probe and the 3' end of the other probe are brought together. Two hemiprobes can then be ligated into a full probe that can serve as a template for PCR amplification. A total of 94 cervical specimens were collected in cytologic fluid and tested with LD-PCR. The results were compared with those of the Digene Hybrid Capture II assay (HC II) (Beltville, Maryland, U.S.A.) and consensus PCR. RESULTS: The overall sensitivity for detecting HPV was 41.5% (39/94) by LD-PCR, 50% (47/94) by consensus PCR and 37.2% (35/94) by HC II. The prevalence of HPV by HC II, consensus PCR and LD-PCR were 87.5%, 100% and 87.5% in the high grade SIL group; 100%, 90.9% and 90.9% in the low grade SIL group; 30%, 52.5% and 40% in the ASCUS group; and 14.2%, 22.8% and 17.1% in women with normal cytology. These results indicate that all 3 methods have similar sensitivity in patients with SIL. However, there is greater variation in detection rates in the ASCUS and normal cytology groups. CONCLUSION: LD-PCR is a useful method of detecting HPV in liquid-based gynecologic cytologic preservatives, and HPV testing as a method adjunct to the liquid-based Pap test could be useful in detecting SILs, especially for the management of patients with ASCUS.     

Sex bias in biopsy samples collected from free-ranging dolphins

Biological samples of free-ranging dolphins are increasingly used to gain information on population structure and ecology. In small cetaceans, the gender of individuals usually cannot be determined at sea, and population sex ratio has to be inferred indirectly. We used molecular sexing to determine the gender of 340 biopsy samples of bottlenose dolphins, Tursiops truncatus, spotted dolphins, Stenella frontalis, and common dolphins, Delphinus delphis, collected around the Azores and Madeira. Sex ratio was globally skewed in favor of males, and differed between species and archipelagos. Skew was probably influenced by the selectivity of biopsy collectors and seasonal or year-round predominance of males in natural populations. Skew was also influenced by sampling duration and intensity. In the Azores, when several samples were successively collected within the same group, the proportion of female samples decreased as a function of sample order. This trend indicated a tendency for females to increasingly avoid the boat while samples were being collected. It showed that males and females reacted differently to the perturbation caused by the biopsy sampling process (i.e., sample collection and driving style).     

Identification of polymorphonuclear leukocytes in cytologic samples for flow cytometry.

Inflammatory cells are commonly present in cytologic specimens obtained for flow cytometry, and may interfere with the analysis of epithelial cells. We have found that detergent (Triton X-100) pretreatment in the two-step acridine orange staining procedure disrupts granulocyte cell membranes to yield bare nuclei; bladder epithelial and squamous cells on the other hand are quite resistant to the detergent treatment. Being deprived of their cytoplasmic RNA, the granulocytes lose red fluorescence. Moreover, the shearing forces in the cytometer extend the multisegmented granulocyte nuclei and align them in the direction of flow. Thus, they present as elongated objects in the measuring system, giving a large DNA fluorescence pulsewidth (nuclear size). These two phenomena make it possible to identify granulocytes in the recorded data, where they are discernible from the mononucleated leukocytes and from epithelial cells. By data selection the granulocytes can be excluded, rendering epithelial cell populations more amenable to analysis. This method may make it unnecessary to remove physically leukocytes from the specimen before flow cytometry; it may also provide a way to analyze the morphology of granulocyte nuclei and to assess methods to manipulate their membrane stability. Full protection from membrane disruption is accomplished by alcohol fixation, and partial protection by 20-30% serum.     

DNA measurement errors with a scanning microdensitometer in cytologic and histologic samples of breast cancers

The influence of various DNA measurement errors using a commercially available scanning microdensitometer was evaluated on Feulgen-stained cytologic and histologic samples prepared from paraffin blocks containing invasive ductal breast cancers. The overall average total measurement error was 5.5% for the cytologic specimens and 10.9% for the 4 micron histologic sections. Components of the error included microscopic adjustment variation and focussing errors (3.5% and 1.1%, respectively, for both cytologic and histologic samples) and background intensity estimation errors (3.0% for the cytologic samples and 10.0% for the histologic samples). Measurements of the integrated optical density had a minimal error of 0.5% and an average error of 1.0%. Limitations due to the histologic architecture and/or heterogeneous cell population gave rise to large differences in the selection of nuclei when differently sized scanning masks were used. To improve the reproducibility, masks used should be based on the individual cell size, and background intensity values should be carefully estimated in the vicinity of the selected cells. Overall, the cytologic tumor samples were preferable to the histologic samples for static DNA measurements. It was easier to select cells suitable for measurement in the cytologic samples, and the cytologic measurements were less time consuming and produced a smaller measurement error.     

Abdominal and endometrial actinomycosis associated with an intrauterine device.

Actinomycotic endometrial infection associated with an intrauterine device (IUD) complicated chronic abdominal inflammatory disease in a 28-year-old woman. Colonies of organisms with morphologic resemblance to and staining reactions of Actinomyces israelii were observed in tissue adherent to the IUD and in inflamed omental and pericolic tissues. However, the organism could not be cultured. Because intact tissues are resistant to actinomyces it is likely that the IUD created an environment favouring the establishment and growth of the organism.     

Hormone binding site of the insulin receptor: analysis using photoaffinity-mediated avidin complexing

A trifunctional reagent was designed which allows derivatization of ligands, particularly peptides and proteins, for subsequent photoaffinity labelling of receptors and specific isolation of the covalent complex or its fragments. B29-(2-nitro-4-azidophenyl)-biocytinyl-insulin (NB-insulin) was synthesized, radioiodinated, and the B26-mono-iodo derivative isolated by HPLC. It was used to photoaffinity label human placental membranes and the purified insulin receptor. Extensive digestion of the covalent insulin-receptor complex with trypsin (EC 3.4.21.4) led to the generation of a fragment of Mr 14,000. Specific complexing with avidin, derivatized avidin or streptavidin could be demonstrated for the photoaffinity labelled alpha-subunit and the 14,000 core fragment. The latter was isolated (approx. 100 pmol from 3-4 placentae) by streptavidin affinity chromatography and HPLC. According to microsequencing based on the known primary structure of the insulin receptor, the N-terminus of the core peptide appears to be Leu20-His21-Glu22-Leu23. We thus conclude: a part of the insulin-binding region of the receptor is located close to the N-terminus of its alpha-subunit in a remarkably stable domain of the sequence 20--(approx.) 120.     

Quantitation of DNA in buccal cell samples collected in epidemiological studies

Abstract

Buccal cell samples are increasingly used in epidemiological studies as a source of genomic DNA. The accurate and precise quantitation of human DNA is critical for the optimal use of these samples. However, it is complicated by the presence of bacterial DNA and wide inter-individual variation in DNA concentration from buccal cell collections. The paper evaluated the use of ultraviolet light (UV) spectroscopy, Höechst (H33258) and PicoGreen? as measures of total DNA, and real-time quantitative polymerase chain reaction (PCR) as a measure of human amplifiable DNA in buccal samples. Using serially diluted white blood cell DNA samples (at a concentration range of 300 to 0.5?ng µl^?1), UV spectroscopy showed the largest bias, followed by Höechst, especially for low concentrations. PicoGreen and real-time PCR provided the most accurate and precise estimates across the range of concentrations evaluated, although an increase in bias with decreasing concentrations was observed. The ratio of real-time PCR to PicoGreen provided a reasonable estimate of the percentage of human DNA in samples containing known mixtures of human and bacterial DNA. Quantification of buccal DNA from samples collected in a breast cancer case-control study by PicoGreen and real-time PCR indicated that cytobrush and mouthwash DNA samples contain similar percentages of human amplifiable DNA. Real-time PCR is recommended for the quantification of buccal cell DNA in epidemiological studies since it provides precise estimates of human amplifiable DNA across the wide range of DNA concentrations commonly observed in buccal cell DNA samples.     

非损伤性取样样品中富集宿主DNA的研究进展

非损伤性取样被广泛应用在动物保护遗传学、分子生态学和分子进化等研究领域.随着基因组测序技术的发展和基因组学时代的到来,如何从非损伤性取样样品中获取能够用于进行基因组测序的高质量DNA是研究者面临的难题.本文总结和比较了非损伤性取样中最常用的粪便样品和考古材料或博物馆标本两类样品中富集宿主DNA的方法及应用,以期为非损伤...     

Changes in receptor status after treatment with tamoxifen in endometrial cancer

Estrogen (ER) and progesterone receptor (PgR) status was determined in 41 women with operable endometrial cancer before and after administration of tamoxifen (TAM). The first sample was obtained by hysteroscopy to ensure a precise biopsy of neoplastic tissue; the second was done on the surgical specimen. PgR content was significantly increased after TAM treatment and this data was compared with the degree of tumor differentiation.