Correction to: Assessment of individual tumor buds using keratin immunohistochemistry: moderate interobserver agreement suggests a role for machine learning.

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Assessment of individual tumor buds using keratin immunohistochemistry: moderate interobserver agreement suggests a role for machine learning.

Tumor budding is a promising and cost-effective biomarker with strong prognostic value in colorectal cancer. However, challenges related to interobserver variability persist. Such variability may be reduced by immunohistochemistry and computer-aided tumor bud selection. Development of computer algorithms for this purpose requires unequivocal examples of individual tumor buds. As such, we undertook a large-scale, international, and digital observer study on individual tumor bud assessment. From a pool of 46 colorectal cancer cases with tumor budding, 3000 tumor bud candidates were selected, largely based on digital image analysis algorithms. For each candidate bud, an image patch (size 256 × 256 µm) was extracted from a pan cytokeratin-stained whole-slide image. Members of an International Tumor Budding Consortium (n = 7) were asked to categorize each candidate as either (1) tumor bud, (2) poorly differentiated cluster, or (3) neither, based on current definitions. Agreement was assessed with Cohen's and Fleiss Kappa statistics. Fleiss Kappa showed moderate overall agreement between observers (0.42 and 0.51), while Cohen's Kappas ranged from 0.25 to 0.63. Complete agreement by all seven observers was present for only 34% of the 3000 tumor bud candidates, while 59% of the candidates were agreed on by at least five of the seven observers. Despite reports of moderate-to-substantial agreement with respect to tumor budding grade, agreement with respect to individual pan cytokeratin-stained tumor buds is moderate at most. A machine learning approach may prove especially useful for a more robust assessment of individual tumor buds.

Chromosome instability predicts the progression of premalignant oral lesions.

OBJECTIVES: One of the main problems in reducing the incidence of oral squamous cell carcinoma (OSCC) is the inability to appropriately deal with leukoplakia. Accurately identifying lesions which will progress to malignancy is currently not possible. The present study aims to establish the value of chromosome instability (CI) detection by DNA image cytometry and FISH analysis for prognosis and monitoring of oral leukoplakia. MATERIALS AND METHODS: For this purpose, we included from our archives 102 oral leukoplakia cases, which had been diagnosed between 1991 and 2008. Patient follow-up data were collected and the histopathological diagnosis was revised. CI assessment was carried out on paraffin-embedded tissue sections using both DNA image cytometry (ICM) and dual target FISH for chromosomes 1 and 7. RESULTS: 16 of 102 Patients developed carcinoma in situ or OSCC. Both detection methods were found to yield prognostic information independent of the histopathological diagnosis. CI was a strong individual marker of progression, with hazard ratios (HRs) of 7.2 and 6.8 for ICM and FISH respectively. Moreover, this approach seems suitable for monitoring lesions over time (especially ICM). Combining histopathology and CI enables subdivision of patients into three risk groups, with different probabilities of malignant progression. CONCLUSION: CI detection seems a reliable method for risk assessment of oral premalignancies and its application may contribute to a better risk-counselling and appropriate treatment regimen or watchfull-waiting approach of patients.

Start of insulin therapy in patients with type 2 diabetes mellitus promotes the influx of macrophages into subcutaneous adipose tissue.

AIMS/HYPOTHESIS: Insulin therapy in patients with type 2 diabetes mellitus is accompanied by weight gain characterised by an increase in abdominal fat mass. The expansion of adipose tissue mass is generally paralleled by profound morphological and inflammatory changes. We hypothesised that the insulin-associated increase in fat mass would also result in changes in the morphology of human subcutaneous adipose tissue and in increased inflammation, especially when weight gain was excessive. METHODS: We investigated the effects of weight gain on adipocyte size, macrophage influx, and mRNA expression and protein levels of key inflammatory markers within the adipose tissue in patients with type 2 diabetes mellitus before and 6 months after starting insulin therapy. RESULTS: As expected, insulin therapy significantly increased body weight. At the level of the subcutaneous adipose tissue, insulin treatment led to an influx of macrophages. When comparing patients gaining no or little weight with patients gaining >4% body weight after 6 months of insulin therapy, both subgroups displayed an increase in macrophage influx. However, individuals who had gained weight had higher protein levels of monocyte chemoattractant protein-1, TNF-α and IL-1ß after 6 months of insulin therapy compared with those who had not gained weight. CONCLUSIONS/INTERPRETATION: We conclude that insulin therapy in patients with type 2 diabetes mellitus improved glycaemic control but also induced body weight gain and an influx of macrophages into the subcutaneous adipose tissue. In patients characterised by a pronounced insulin-associated weight gain, the influx of macrophages into the adipose tissue was accompanied by a more pronounced inflammatory status. TRIAL REGISTRATION: ClinicalTrials.gov: NCT00781495. FUNDING: The study was funded by European Foundation for the Study of Diabetes and the Dutch Diabetes Research Foundation.

Accurate assessment of cell density in low cellular liquid-based cervical cytology.

OBJECTIVE: Scant cellularity is the most important source of unsatisfactory liquid-based cytology. Although still being debated, low cellularity is thought to compromise the detection of squamous lesions. Thus, reliable assessment of cellularity is essential. The aim of the present study was to determine the cellularity range for ThinPrep(®) slides of low cellularity and to establish the most accurate cell-counting protocol. METHODS: A series of 60 ThinPrep cases representing the full spectrum of adequate, 'satisfactory but limited by' (SBLB) and unsatisfactory reports were included. Two cell-counting protocols with three different magnifications, using ×10, ×20 and ×40 objectives, were evaluated and related to the true cellularity, together with a reassessment of the degree of adequacy originally reported. The cell-counting protocol that showed the highest correlation coefficient was considered the most accurate. RESULTS: Based on seven (re)assessments a majority score for adequacy was established. There were 42 cases with a majority score 'unsatisfactory' or 'SBLB' (low cellularity) of which 41 contained fewer than 20 000 squamous cells; and 18 cases with a majority score 'satisfactory' of which one had fewer than 20 000 cells. The cell-counting protocol that showed the significantly highest correlation with the reference standard was the Stichting Kwaliteitsbewaking Medische Laboratoriumdiagnostiek (SKML) protocol with a ×10 objective. CONCLUSIONS: ThinPrep slides reported as unsatisfactory or SBLB were shown to contain fewer than 20000 squamous cells. The most accurate protocol for estimating the cellularity of these slides was cell counting in five non-adjacent microscope fields along the horizontal axis and five along the vertical axis of the slide with a ×10 objective and applying a correction factor of 1.24× to correct for underestimation of the true cellularity.

Mild muscular features in tenascin-X knockout mice, a model of Ehlers-danlos syndrome.

INTRODUCTION: Tenascin-X (TNX) is an extracellular matrix (ECM) glycoprotein, the absence of which in humans leads to a recessive form of Ehlers-Danlos syndrome (EDS), a group of inherited connective tissue disorders characterized by joint hypermobility, skin hyperextensibility, and tissue fragility. A mouse model of TNX-deficient type EDS has been used to characterize the dermatological, orthopedic, and obstetrical features. The growing insight in the clinical overlap between myopathies and inherited connective tissue disorders asks for a study of the muscular characteristics of inherited connective tissue diseases. Therefore, this study aims to define the muscular phenotype of TNX knockout (KO) mice. MATERIALS AND METHODS: We performed a comprehensive study on the muscular phenotype of these TNX KO mice, consisting of standardized clinical assessment, muscle histology, and gene expression profiling of muscle tissue. Furthermore, peripheral nerve composition was studied by histology and electron microscopy. RESULTS: The main findings are the presence of mild muscle weakness, mild myopathic features on histology, and functional upregulation of genes encoding proteins involved in ECM degradation and synthesis. Additionally, sciatic nerve samples showed mildly reduced collagen fibril density of endoneurium. DISCUSSION: The muscular phenotype of TNX KO mice consists of mild muscle weakness with histological signs of myopathy and of increased turnover of the ECM in muscle. Furthermore, mildly reduced diameter of myelinated fibers and reduction of collagen fibril density of endoneurium may correspond with polyneuropathy in TNX-deficient EDS patients. This comprehensive assessment can serve as a starting point for further investigations on neuromuscular function in TNX KO mice.

Cardiac biplane strain imaging: initial in vivo experience.

In this study, first we propose a biplane strain imaging method using a commercial ultrasound system, yielding estimation of the strain in three orthogonal directions. Secondly, an animal model of a child's heart was introduced that is suitable to simulate congenital heart disease and was used to test the method in vivo. The proposed approach can serve as a framework to monitor the development of cardiac hypertrophy and fibrosis. A 2D strain estimation technique using radio frequency (RF) ultrasound data was applied. Biplane image acquisition was performed at a relatively low frame rate (<100 Hz) using a commercial platform with an RF interface. For testing the method in vivo, biplane image sequences of the heart were recorded during the cardiac cycle in four dogs with an aortic stenosis. Initial results reveal the feasibility of measuring large radial, circumferential and longitudinal cumulative strain (up to 70%) at a frame rate of 100 Hz. Mean radial strain curves of a manually segmented region-of-interest in the infero-lateral wall show excellent correlation between the measured strain curves acquired in two perpendicular planes. Furthermore, the results show the feasibility and reproducibility of assessing radial, circumferential and longitudinal strains simultaneously. In this preliminary study, three beagles developed an elevated pressure gradient over the aortic valve (Deltap: 100-200 mmHg) and myocardial hypertrophy. One dog did not develop any sign of hypertrophy (Deltap = 20 mmHg). Initial strain (rate) results showed that the maximum strain (rate) decreased with increasing valvular stenosis (-50%), which is in accordance with previous studies. Histological findings corroborated these results and showed an increase in fibrotic tissue for the hearts with larger pressure gradients (100, 200 mmHg), as well as lower strain and strain rate values.

Preoperative radiochemotherapy is successful also in patients with locally advanced rectal cancer who have intrinsically high apoptotic tumours.

BACKGROUND: Not all patients with locally advanced rectal cancer (LARC) respond equally to neo-adjuvant radiochemotherapy (RCT). Patients with highly apoptotic less advanced rectal cancers do not benefit from short-term radiotherapy. This study investigates whether this is also the case in the setting of RCT for LARC. PATIENTS AND METHODS: Tissue microarrays were constructed of biopsy and resection specimens of 201 LARC patients. Apoptosis (M30) and several apoptosis-regulating proteins [p53, Bcl-2, Bax, cyclooxygenase-2 (Cox-2) and mamma serine protease inhibitor (maspin)] were studied with immunohistochemistry. Subsequently, predictive values for local recurrence (LR), overall survival (OS) and histological tumour regression were analysed. RESULTS: Apoptotic levels, quantified as the number of apoptotic cells/mm(2) tumour epithelium, were higher in posttherapy tissues compared with biopsies (P < 0.001). Biopsies from clinical T4 stage tumours demonstrated significantly higher levels of apoptosis than clinical T3 stage tumours (P = 0.020). Therapy-induced apoptosis was higher when the interval between the last day of irradiation and surgery increased (P < 0.001, correlation coefficient = 0.355). Pre- and posttherapy apoptosis, p53, Bcl-2, Bax and Cox-2 were not associated with LR, OS or tumour regression. Intense pretherapy cytoplasmatic staining of maspin indicated a higher risk on LR (P = 0.009) only. CONCLUSION: Combined RCT is also successful in highly apoptotic tumours and is therefore independent of intrinsic apoptosis.

Three-dimensional reconstruction of tumor microvasculature: simultaneous visualization of multiple components in paraffin-embedded tissue.

Three-dimensional (3D) visualization of microscopic structures may provide useful information about the exact 3D configuration, and offers a useful tool to examine the spatial relationship between different components in tissues. A promising field for 3D investigation is the microvascular architecture in normal and pathological tissue, especially because pathological angiogenesis plays a key role in tumor growth and metastasis formation. This paper describes an improved method for 3D reconstruction of microvessels and other microscopic structures in transmitted light microscopy. Serial tissue sections were stained for the endothelial marker CD34 to highlight microvessels and corresponding images were selected and aligned. Alignment of stored images was further improved by automated non-rigid image registration, and automated segmentation of microvessels was performed. Using this technique, 3D reconstructions were produced of the vasculature of the normal brain. Also, to illustrate the complexity of tumor vasculature, 3D reconstructions of two brain tumors were performed: a hemangioblastoma and a glioblastoma multiforme. The possibility of multiple component visualization was shown in a 3D reconstruction of endothelium and pericytes of normal cerebellar cortex and a hemangioblastoma using alternate staining for CD34 and alpha-smooth muscle actin in serial sections, and of a GBM using immunohistochemical double staining. In conclusion, the described 3D reconstruction procedure provides a promising tool for simultaneous visualization of microscopic structures.

The effect of Replens on vaginal cytology in the treatment of postmenopausal atrophy: cytomorphology versus computerised cytometry.

BACKGROUND: After the menopause decreased concentrations of oestrogen may result in insufficient maturation of the vaginal epithelium, which can lead to a range of vaginal discomforts. This state of vaginal atrophy may be treated with oestrogen replacement treatment. Replens, a non-hormonal alternative to oestrogen replacement treatment has been shown to be effective in relieving symptoms related to vaginal atrophy in previous studies. AIMS: To study the effect of Replens on the maturation of the vaginal epithelium and morphology of the vaginal cells and to compare the results of a recently developed cytomorphometric method with manual assessment of the degree of maturation in vaginal smears. METHODS: Vaginal smears from 38 postmenopausal women suffering from symptoms related to vaginal atrophy were analysed manually and by cytomorphometry. The maturation value (MV) and the percentages of (para)basal, intermediate, and superficial cells (maturation index; MI) were measured by both methods before and after treatment with Replens. Cytomorphometry also measured mean cellular area, mean nuclear area, and mean area ratio. RESULTS: A correlation was shown between the two methods in the assessment of percentages of (para) basal and intermediate cells and MV. Cytomorphometric data showed a significant increase in mean cellular area, indicating a positive effect of Replens on the maturation of the vaginal epithelium. Changes in nuclear area and ratio between nuclear and cellular areas were not significant. Treatment with Replens did not influence MI or MV, as assessed by the two methods. CONCLUSIONS: Replens did have an effect on vaginal morphology. The automated procedure may be useful for the assessment of maturation in vaginal smears and is more sensitive to small (subvisual) changes.

Improved prediction of metastasis in tongue carcinomas, combining vascular and nuclear tumor parameters.

BACKGROUND: Predicting the presence of metastasis, based on tumor or tumor-related characteristics is of utmost importance. The authors studied the significance of tumor DNA features and tumor-related angiogenesis to predict the occurrence of metastasis in squamous cell carcinomas (SCCs) of the tongue. METHODS: Paraplast blocks from resection specimens of 20 metastasized and 20 nonmetastasized SCCs of the tongue with a minimum follow-up of 24 months were used. Tissue sections were stained with anti-CD34 monoclonal antibodies for vessel visualization, and according to Feulgen to stain DNA. Using image analysis, data from both stainings were computed for each of the 40 carcinomas. A logistic regression model to predict the presence of metastasis, based on vascular and nuclear morphology features, was developed. RESULTS: The intratumor variation of chromatin condensation and the percentage vessels smaller than 5 microm in diameter were selected for the model. The model correctly predicted metastasis in 90% of patients and excluded metastasis correctly in 75% of nonmetastasized tumors. Taking into account the prevalence of metastasis in SCC of the tongue of between 30% and 60%, this means a predictive value for a negative outcome of between 95% and 83%. CONCLUSIONS: The proposed model shows an improvement of predictive values compared with previous models with single parameters. Therefore, a multiparameter model appears to predict the multiparameter process of metastasis better.

Microvessel density: correlation between contrast ultrasonography and histology of prostate cancer.

OBJECTIVE: Increased microvessel density (MVD) of prostate cancer seems to be associated with poor prognosis and higher stage. Assessment of MVD using noninvasive methods could be of use in the work-up of patients with prostate cancer. The aim of the present study was to correlate three-dimensional contrast-enhanced power Doppler ultrasound (3D-CE-PDU) findings with MVD characteristics of radical prostatectomy specimens. METHODS: Seven patients with biopsy-proven prostate cancer had 3D-CE-PDU investigations 2-3 weeks after prostate biopsies were taken and prior to radical prostatectomy. The investigations were performed using Levovist contrast agent (Schering AG, Berlin, Germany) in combination with a Voluson 530D ultrasound scanner (Kretz AG, Zipf, Austria). The 7 patients were selected because of lateralization of the contrast enhancement. Histology slides were made of the side with 'contrast enhancement' and of the contralateral 'unenhanced' side and stained according to the catalyzed reporter deposition (CARD) amplification procedure, and MVD parameters were obtained. RESULTS: In all patients the MVD count of the 'enhanced' side was higher than the MVD count of the 'unenhanced' side, averaging 1.93 times higher. On histology all enhanced lesions proved to contain prostate cancer tissue (average maximum diameter 25 mm (range 17-31)). Two patients had a small bilateral tumor lesion (4 and 5 mm respectively) and in total 5 patients had even smaller satellite lesions (1-2 mm). The smaller lesions were not identified using 3D-CE-PDU. CONCLUSIONS: The present study shows that 3D power Doppler contrast ultrasonography is a minimally invasive imaging modality, which has the potential to visualize lesions with increased MVD. This property of 3D-CE-PDU could be used in the detection of prostate cancer.

Quantification of tumour vascularity in squamous cell carcinoma of the tongue using CARD amplification, a systematic sampling technique, and true colour image analysis.

The aims of this study of head and neck tissue samples were to develop an immunohistochemical protocol based on the catalysed reporter deposition (CARD) technique to enhance staining results for use in automated true colour image analysis, to assess the reproducibility of systematic tissue sampling in the angiogenic hot spot selection, and quantification of microvessel density (MVD) and other vessel characteristics. The latter data were compared between six metastasised tongue squamous cell carcinomas, vs. four non-metastasised. In comparison to the standard immunohistochemical protocol with anti-CD34 antibodies, CARD amplification resulted in both more intensely stained and larger numbers of vessels. Averaging the 10 most vascularised fields of the 40 to 60 systematically sampled fields in a tissue section resulted in an overall acceptable interobserver reproducibility for most assessed vessel parameters (r> or =0.76 and p< or =0.01). The percentage vessels with diameter <5 microm was significantly higher in the non-metastasised tongue carcinomas (p=0.02). However, for a number of tumours the effect of tissue sampling was significant. We conclude that CARD amplification is needed for reliable segmentation of vessels by image analysis systems, and that tumour heterogeneity is a limiting factor for all procedures in which tumour vascularity is assessed in a single tissue section. Figures on http://www.esacp.org/acp/2001/22-4/hannen.htm.

Progressive histopathological changes in pancreatic islets of Zucker Diabetic Fatty rats.

Thus far, histopathological changes in the pancreatic islets of Zucker Diabetic Fatty (ZDF) rats, an animal model of type 2 diabetes mellitus (or non-insulin-dependent diabetes mellitus), have only been studied in male rats and in 18-weeks old rats or younger. In this study, we have examined in both male and female ZDF rats the histopathological changes longitudinally, from 6 to 32 weeks of age. We studied islet architecture and cellular distribution of the various islet hormones both in ZDF and control rats. In the ZDF rats, aging was initially associated with an enlargement of the islets. From 18 weeks onwards, no further enlargement was noted but islet boundaries became increasingly irregular, leading to the appearance of projections of endocrine cells into the surrounding exocrine tissue. At the islet boundaries as well as within the islets progressive fibrosis was observed with increasing amounts of collagen and reticular fibers. In the islets, staining intensity of both insulin and islet amyloid polypeptide (IAPP) increased slightly till 10 weeks of age and thereafter decreased rapidly. In contrast, the staining intensities of glucagon, somatostatin, and pancreatic polypeptide (PP) did not change. Even at the age of 32 weeks, just the beta-cells and not the other endocrine islet cells appear to be affected. In control rats, aging evoked only minor changes. Thus, we observed that during prolonged development of diabetes mellitus in both male and female ZDF rats histopathological changes in the pancreatic islets became progressively more severe, eventually leading to disintegration of the islets.

Spectral imaging of multi-color chromogenic dyes in pathological specimens.

We have investigated the use of spectral imaging for multi-color analysis of permanent cytochemical dyes and enzyme precipitates on cytopathological specimens. Spectral imaging is based on Fourier-transform spectroscopy and digital imaging. A pixel-by-pixel spectrum-based color classification is presented of single-, double-, and triple-color in situ hybridization for centromeric probes in T24 bladder cancer cells, and immunocytochemical staining of nuclear antigens Ki-67 and TP53 in paraffin-embedded cervical brush material (AgarCyto). The results demonstrate that spectral imaging unambiguously identifies three chromogenic dyes in a single bright-field microscopic specimen. Serial microscopic fields from the same specimen can be analyzed using a spectral reference library. We conclude that spectral imaging of multi-color chromogenic dyes is a reliable and robust method for pixel color recognition and classification. Our data further indicate that the use of spectral imaging (a) may increase the number of parameters studied simultaneously in pathological diagnosis, (b) may provide quantitative data (such as positive labeling indices) more accurately, and (c) may solve segmentation problems currently faced in automated screening of cell- and tissue specimens.

Microscopic analysis of anastomotic healing in the intestine of normal and diabetic rats.

PURPOSE: The mechanisms that cause diabetes to impair the development of anastomotic strength in the intestine are poorly understood. We investigated whether short-term uncontrolled diabetes causes alterations in microscopic aspects of anastomoses from the ileum and colon. METHODS: Eighteen Wistar rats were rendered diabetic one week before operation by intravenous streptozotocin injection (50 mg/kg), resulting in nonfasting blood glucose levels of approximately 20 mmol/l. Another 18 age-matched rats were used as controls with a normal blood glucose range of 5 to 7 mmol/l. All rats underwent resection and anastomosis of both the ileum and colon. Animals were killed at one, three, or seven days after operation. Cellular and architectural parameters of anastomotic healing were scored in hematoxylin and eosin-stained sections. Anastomotic collagen content was analyzed by image analysis in picrosirius red-stained sections. RESULTS: Anastomotic necrosis, edema, and epithelial recovery were not affected by diabetes. In diabetic rats, the number of polymorphonuclear cells and macrophages was significantly (P = 0.025 and 0.0002, respectively) increased in ileal anastomoses one and three days after operation. In colonic anastomoses, the number of polymorphonuclear cells was increased at one (P = 0.001) and seven (P = 0.014) days after operation. Repair of the submucosal-muscular layer in colonic anastomoses from diabetic rats was impaired seven days after surgery (P = 0.0071), but in ileal anastomoses no difference was found. In the anastomotic area, collagen deposition at postoperative Days 1, 3, and 7 remained unaffected by diabetes. CONCLUSION: Experimental diabetes leads to alterations in cellular components involved in the early phase of repair of intestinal anastomoses but not to a reduced accumulation of wound collagen.

Hue-saturation-density (HSD) model for stain recognition in digital images from transmitted light microscopy.

BACKGROUND: Transmitted light microscopy is used in pathology to examine stained tissues. Digital image analysis is gaining importance as a means to quantify alterations in tissues. A prerequisite for accurate and reproducible quantification is the possibility to recognise stains in a standardised manner, independently of variations in the staining density. METHODS: The usefulness of three colour models was studied using data from computer simulations and experimental data from an immuno-doublestained tissue section. Direct use of the three intensities obtained by a colour camera results in the red-green-blue (RGB) model. By decoupling the intensity from the RGB data, the hue-saturation-intensity (HSI) model is obtained. However, the major part of the variation in perceived intensities in transmitted light microscopy is caused by variations in staining density. Therefore, the hue-saturation-density (HSD) transform was defined as the RGB to HSI transform, applied to optical density values rather than intensities for the individual RGB channels. RESULTS: In the RGB model, the mixture of chromatic and intensity information hampers standardisation of stain recognition. In the HSI model, mixtures of stains that could be distinguished from other stains in the RGB model could not be separated. The HSD model enabled all possible distinctions in a two-dimensional, standardised data space. CONCLUSIONS: In the RGB model, standardised recognition is only possible by using complex and time-consuming algorithms. The HSI model is not suitable for stain recognition in transmitted light microscopy. The newly derived HSD model was found superior to the existing models for this purpose.

Decreased expression of Ki-67 in atrophic cervical epithelium of post-menopausal women.

Papanicolaou-stained cervical smears (Pap smears) of post-menopausal women often present difficulties in distinguishing atrophic cervical epithelium from high-grade cervical intraepithelial neoplasia (CIN2-3). The aim of this study was to disclose differences in proliferative activity in normal cervical epithelium, cervical atrophy, and high-grade CIN lesions, in order to develop specific and sensitive classifiers to discriminate between cervical atrophy and high-grade CIN, both in cervical smears and in tissue sections. A case-control study was done on 83 patients. Proliferative activity was assessed in histological sections using the monoclonal antibody MIB1. An image analysis system was used to characterize different proliferation-associated features. Preceding Pap smears were restained with MIB1 and proliferative activity was measured by a point-counting procedure, carried out on a training set of 32 cases and a test set of 51 cases. In cervical atrophy, proliferative activity was significantly lower than in normal epithelium (p<0.001). Proliferative activity measured in both biopsies and cervical smears was considerably higher in high-grade CIN than in normal epithelium (p<0.001). Discriminant analyses resulted in four classifiers, based on proliferation parameters, to discriminate between cervical atrophy and high-grade CIN, and between CIN2 and CIN3, in biopsy specimens and cervical smears, respectively. The two classifiers for biopsy specimens resulted in 100% correct classification. Application of the classifier obtained from the training set of Pap smears resulted in 100% correct classification of the Pap smears in the test set. The classifier to discriminate between CIN2 and CIN3 in Pap smears, obtained from 36 patients, resulted in 87% and 90% correct classification, respectively.

Transient tenascin enhancement is an early event after androgen ablation in rat prostate.

Tenascin (tenascin-C), a mesenchymal glycoprotein, is expressed in many tissue remodeling processes. We evaluated tenascin expression during androgen-deprivation-related involution of the rat prostate. At set intervals following castration and subsequent testosterone repletion, prostates were removed in 30 adult rats. Each prostate was immunostained with a polyclonal antiserum against rat tenascin and keratin antibodies specifically directed against exocrine basal cells and luminal cells in the prostate glandular structure. Morphologic impressions were semiquantatively evaluated using a computer-assisted image analysis system. Rat prostates showed a transient increase in the periglandular tenascin expression directly following castration that reached a maximum at day 3. At day 6, tenascin expression was similar to control prostates. This was accompanied by a decrease of cells in the luminal cell layer. The weakest tenascin immunoreactivity was noted on day 14 after androgen withdrawal. This process was reversed by androgen repletion. This study shows that in the rat prostate tenascin expression may be androgen dependent and that during androgen deprivation-related involution tenascin expression is probably associated with tissue remodeling by stromal-epithelial interactions.

Development and validation of a computerized cytomorphometric method to assess the maturation of vaginal epithelial cells.

BACKGROUND: After menopause, declining levels of estrogens may cause vaginal discomfort, or so-called "vaginal atrophy." Evaluation of therapies for vaginal atrophy may be performed using the so-called "maturation index." The maturation index is expressed as the percentage of (para)-basal, intermediate, and superficial epithelial cells in a vaginal smear. Manual assessment of the maturation index is subject to inter- and intraobserver variations. In this study, assessment of the maturation of cells in vaginal smears using automated image analysis was investigated. MATERIALS AND METHODS: Automated assessment, using a commercially available image analysis system, was performed on hematoxylin-eosin-stained cytospin specimens. A training set was constructed by an experienced cytotechnologist, based upon visual classification of stored grey value images. From this, two discriminant functions (DFs) were calculated capable of classifying cells in one of the three types. These cell classifiers were capable of classifying 97% of the cells correctly. Data from automated assessment were compared with those of classical manual counting. Specimens of 13 mature and 6 atrophic vaginal specimens were assessed in duplicate, both manually and by image analysis, using the DFs. RESULTS: No significant interobserver effect was found for image analysis, whereas a significant effect was found for manual counting. Both methods were able to distinguish between matured and atrophic specimens. CONCLUSIONS: It was concluded that for assessment of vaginal maturation, the use of automated image analysis systems is recommended. Besides increased reproducibility, image analysis systems yield additional data describing the size and shape of the cytoplasm and nucleus of cells, which might increase discriminating power.