bluuebunny/crossref_metadata_2025_split huggingface dataset:
A Novel Image Cytometric Method for Quantitation of Immunohistochemical Staining of Cytoplasmic Antigens
Authors: Guillaud M. , Matthews J. B. , Harrison A. , MacAulay C. , Skov K. 
Published: January 2015

Evaluation of molecular markers by immunohistochemical labelling of tissue sections has traditionally been performed by qualitative assessment by trained pathologists. For those markers with a staining component present outside of the nucleus, there has been no image histometric method available to reliably and consistently define cell interfaces within the tissue. We present a new method of approximating cellular boundaries to define cellular regions within which quantitative measurements of staining intensity may be made. The method is based upon Voronoi tessellation of a defined region of interest (ROI), and requires only the position of the nuclear centroids within the ROI.Here we describe the VORSTAIN software which has been developed based on the Oncometrics CytoSavant Automated Image Cytometry System. To demonstrate this technique, human breast cancer sections immunohistochemically stained for bcl‐2 protein and counter‐stained with nuclear methyl green stain were evaluated. Intra‐observer variation in the measured values was between 1.5–2.6% and inter‐observer variation was between 1.8–4.4%. The primary source of variability was due to difficulties in interpreting the exact position of the nuclear centroids. Analysis of mean staining densities for each slide correlated well with subjective scoring performed by two independent pathologists. Using VORSTAIN, significant variation of staining intensities between regions within the same slide was measured for some sections, indicating a large degree of heterogeneity within the tumours. The ability to accurately quantitate the degree of heterogeneity of molecular marker expression within tumours may be a valuable tool in prognostication.
Morphological Heterogeneity of p53 Positive and p53 Negative Nuclei in Breast Cancers Stratified by Clinicopathological Variables
Authors: Friedrich Katrin , Dimmer Volker , Haroske Gunter , Meyer Wolfdietrich , Theissig Franz , Thieme Berit , Kunze Klaus Dietmar 
Published: January 2015

The study was aimed to detect differences in nuclear morphology between nuclear populations as well as between tumours with different p53 expression in breast cancers with different clinicopathological features, which also reflect the stage of tumour progression. The p53 immunohistochemistry was performed on paraffin sections from 88 tumour samples. After the cells had been localised by means of an image cytometry workstation and their immunostaining had been categorised visually, the sections were destained and stained by the Feulgen protocol. The nuclei were relocated and measured cytometrically by the workstation.There were significant differences in the nuclear features between tumours as well as between nuclear populations with different p53 expression in the most subgroups. The variability of nuclear shape in tumour groups, classified by the tumour size or the lymph node status, increase with the p53 immunoreactive score, whereas in tumours grouped by the Bloom–Richardson grade features of the chromatin distribution were different between the p53 staining categories.The nuclear subpopulations showed differences in the amount and distribution of chromatin in most subgroups.The results demonstrate the relationship between the nuclear morphology and the p53 expression in different stages of breast cancers. The p53 status is an important factor of the biological behaviour but not the only one.
Opening the Black Box: the Relationship between Neural Networks and Linear Discriminant Functions
Authors: Kemp Roger A. , MacAulay Calum , Palcic Branko 
Published: January 2015

Over the last ten years feed‐forward neural networks have become a popular tool for statistical decision making. During this time, they have been applied in many fields, including cytological classification. Neural networks are often treated as a black box, whose inner workings are concealed from the researcher. This is unfortunate, since the inner workings of a neural network can be understood in a manner similar to that of a linear discriminant function, which is the standard tool that researchers use for decision making.This paper discusses feed‐forward neural networks and some methods to improve their performance for classification problems. Their relationship to discriminant functions will be examined for a simple two‐dimensional classification problem.
Lymphoid Infiltrates in B Cell Non Hodgkin’s Lymphoma: Comparing Nuclear Characteristics between Lymph Node and Bone Marrow; and Evaluating Diagnostic Features of Bone Marrow Infiltrates in Paraffin Embedded Tissues
Authors: Deverell Mark H. , Best Elizabeth , Salisbury Jonathan R. 
Published: January 2015

Distinguishing non Hodgkin’s lymphoma from benign lymphoid aggregates in bone marrow is well recognised to be difficult. Our objective was to evaluate nuclear morphology, and to perform morphometry on benign and neoplastic lymphoid infiltrates, to establish if objective criteria were of value in the diagnosis of neoplasia. By comparing neoplastic infiltrates in bone marrow with infiltrates in lymph nodes, the validity of grading non Hodgkin’s lymphoma on the basis of bone marrow histology alone was assessed. 82 cases of B cell non Hodgkin’s lymphoma (44 low grade and 38 high grade), known to have both lymph node and bone marrow involvement at the time of presentation, were compared with bone marrow trephines containing reactive lymphoid infiltrates.The results suggest that in paraffin embedded tissue from bone marrow trephines, nuclear morphology, nuclear area and the nuclear contour index cannot, in most cases, be used to distinguish reactive from neoplastic lymphoid infiltrates although mean nuclear area supports a diagnosis of a neoplastic infiltrate if it can be shown that the nuclei are larger than would be expected in reactive infiltrates. Such differences are subtle and often not appreciable without the use of quantitative techniques. The frequent occurrence of discordant infiltrates in high grade lymphomas means grading of lymphoma on the basis of bone marrow appearances is often unrealistic.
Dynamic Changes in Quantitative Features of Human Gastric Lesions
Authors: Enchev Ventzeslav , Rigaut Jean Paul 
Published: January 2015

Quantitative DNA, morphometric cellular and nuclear variables were evaluated in surgical biopsies from patients with various gastric lesions: chronic gastritis, chronic ulcers, adenomatous polyps (gastric adenomas), primary carcinomas and their corresponding lymph‐node metastases. Paraffin‐embedded tissue sections were studied by static cytophotometry (plug method), karyometry (measurements with a graduated eyepiece micrometer of the major and minor axes of the elliptic nuclear profiles, and calculation of profile areas), and measurements of cellular profiles (largest and smallest caliper diameters). Tissue lymphocytes from the same slide were used as diploid controls for the DNA evaluations.An increase of both cellular and nuclear dimensions and DNA content was noted in all pathological tissues, as compared to normal mucosa; the highest values are found in primary gastric carcinomas. A progressive pattern increase of cellular and nuclear dimensions and of DNA content was observed through normal to cancerous tissues, chronic gastritis, chronic ulcers and adenomatous polyps (adenomas). Lymph‐node metastases, in our study, had smaller nuclear (cellular) dimensions than primary cancers.
Assessing Slide Coverage by Cytoscreeners during the Primary Screening of Cervical Smears, Using the AxioHOMEⓇ Microscope System
Authors: Baker Robert W. , Brugal Gerard , Coleman Dulcie V. 
Published: January 2015

We have used the HOME Microscope system to examine the screening patterns of cytotechnologists who undertake the primary screening of cervical smears, in order to measure accuracy of screening against screening time, slide coverage, and mean screening rate. Twelve cytotechnologists engaged routinely on cervical screening volunteered for this study. They were asked to perform primary screening of 10 test slides under normal laboratory conditions in the normal way. Slide coverage and screening time were recorded on the HOME system. Slide maps were prepared and the results analysed.The exercise demonstrated that all primary screeners fail at some point to scan the whole of the slide during primary screening. The maps produced by the HOME system clearly demonstrated that 5 different types of error can occur that lead to incomplete coverage of the slide. Mean slide coverage was 84%, and some individuals averaged only 66% coverage. The results show that there is a major problem in the education of some individual cytotechnologists in slide coverage. This could be rectified by the incorporation of a HOME system into every training centre, and the establishment of a protocol for assessment of slide coverage in competence examinations. Furthermore, the exercise has shown that even those individuals who normally attain a good standard of slide coverage would be able to improve slide coverage given access to the daily use of a HOME Microscope, or a system with equivalent screening/reviewing functionality.
DNA Aneuploidy by Flow Cytometry Is an Independent Prognostic Factor in Gastric Cancer
Authors: Abad Mar , Ciudad Juana , Rincon Manuel R. , Silva Isabel , Paz-Bouza José I. , Lopez Antonio , Alonso Alberto G. , Bullon Agustin , Orfao Alberto 
Published: January 2015

In the present study the prognostic value of both DNA ploidy and the proliferative activity of tomour cells were studied in a series of 76 consecutive patients suffering from gastric tumours. DNA ploidy and the proliferative index (as measured by the percentage of S‐phase cells) were determined by flow cytometry using fresh tumour specimens.The presence of DNA aneuploid clones by flow cytometry was detected in 62% of the cases (mean DNA index of 1.63 ± 0.46; range 1.08–2.92), the mean proportion of S‐phase cells being of 18.4 ± 11.5%. In comparison with diploid cases, aneuploid tumours showed a higher proliferative activity (cases with more than 15% S‐phase cells: 18.4% versus 6.1%, p = 0.0001) as well as a higher incidence of node involvement (95% versus 68%, p = 0.001). By contrast, no significant differences were detected with respect to sex, age, histologic grade and type, clinical stage, tumour size and the incidence of extranodal involvement.Upon grouping the patients according to the proportion of S‐phase cells no significant differences were observed for the clinical and biological parameters explored except for an association between a high percentage of S‐phase cells and the presence of DNA aneuploidy (40% versus 96%, p = 0.0001). Regarding survival the presence of DNA aneuploidy was significantly associated with poor outcome as compared to the diploid cases (median of 15 versus 26 months, p = 0.005). By contrast, the proportion of S‐phase cells did not predict patients’s outcome.Multivariate analysis of prognostic factors showed that the presence of DNA aneuploidy (p = 0.003) together with the histologic type (p = 0.03) and the existence of extranodal metastases (p = 0.05) were the best combination of prognostic factors for survival prediction.
Automatic Quantification of Immunohistochemically Stained Cell Nuclei Based on Standard Reference Cells
Authors: Ranefall Petter , Wester Kenneth , Andersson Ann-Catrin , Busch Christer , Bengtsson Ewert 
Published: January 2015

A fully automatic method for quantification of images of immunohistochemically stained cell nuclei by computing area proportions, is presented. Agarose embedded cultured fibroblasts were fixed, paraffin embedded and sectioned at 4 µm. They were then stained together with 4 µm sections of the test specimen obtained from bladder cancer material.A colour based classifier is automatically computed from the control cells. The method was tested on formalin fixed paraffin embedded tissue section material, stained with monoclonal antibodies against the Ki67 antigen and cyclin A protein. Ki67 staining results in a detailed nuclear texture with pronounced nucleoli and cyclin A staining is obtained in a more homogeneously distributed pattern.However, different staining patterns did not seem to influence labelling index quantification, and the sensitivity to variations in light conditions and choice of areas within the control population was low. Thus, the technique represents a robust and reproducible quantification method.In tests measuring proportions of stained area an average standard deviation of about 1.5% for the same field was achieved when classified with classifiers created from different control samples.
Genetic Heterogeneity of Benign Thyroid Lesions
Authors: Ferrer-Roca O. , Pérez-Gómez J. A. , Cigudosa J. C. , Gómez E. , Estévez M. 
Published: January 2015

The present series includes 75 thyroid lesions (38 goiters, 30 adenomas, 3 follicullo‐papillary encapsulated carcinomas and 4 normal thyroid) that were studied by static and flow cytometry. Four cases were also analyzed by in situ hybridization (centromeric probes for chromosomes 1 and 17) and 10 cases by G‐banding cytogenetics. Results demonstrate a polymorphysm and genetic instability in the thyroid tissue that may be related to the spontaneous polyploidization of their cells. The most consistent finding in cytometry was the presence of two clones associated with clinical or histological hyperactivity (46% versus 23% in non‐functioning cases; X2 distribution with a p < 0.05).Chromosomal anomalies were detected in two out of 10 cases: 46, XX, t(5,19) in 87% of cells of a diffuse hyperplastic goiter and 49, XX, +7, +17, +22 in 19% of cells of thyroiditis case. Finally, the in situ hybridization technique showed hidden trisomies of clonal origin in all of the cases studied.Evaluation of clonal trisomies by the in situ hybridization technique using the confidence interval of a binomial distribution is discussed.
Histopathologic and Flow‐Cytometric Analysis of Neoplastic and Benign “background” Tissue in Breast Carcinoma Resections
Authors: Visscher Daniel W. , Gingrich Debra S. , Leon-Armin Carmen , Tabaczka Pamela , Crissman John D. 
Published: January 2015

Two‐color, multiparametric synthesis phase fraction (SPF) analysis of cytokeratin‐labeled epithelial cells was flow cytometrically performed on both benign (SPFb) and malignant tissue samples (if available, SPFt) from 132 mastectomy/lumpectomy specimens. These data were then correlated with clinicopathologic features, including (1) tumor differentiation, (2) the proportion of tumor comprised of duct carcinoma‐in situ (DCIS), and (3) the histology of accompanying benign breast tissue, classified by predominant microscopic pattern as intact, normal terminal duct lobular units (NTDLU, 34% of cases), atrophic (AT, 33% of cases), proliferative fibrocystic (PFC, 26% of cases), and non‐proliferative fibrocystic (NPFC, 7% of cases). SPFt was inversely correlated with extent of DCIS (DCIS =0 – 20% tumor volume – 12.7% mean SPFt, vs. DCIS >20% tumor volume – 6.4% mean SPFt, p = 0.001). SPFt also correlated with the histology of background benign breast tissue (NTDLU – 14.8% mean SPFt vs. AT – 6.9% mean SPFt vs. PFC – 12.7% mean SPFt, p = 0.05) but it did not correlate with patient age or SPFb (overall mean =0.73%). SPFb was correlated with patient age (>56 yr – 0.59% mean SPFb vs. < yr – 0.84% mean SPFb, p = 0.02), with background histology (NTDLU – 1.1% mean SPFb vs. AT – 0.43% mean SPFb vs. PFC – 0.70% mean SPFb, p < 0.02) and with the grade of the neoplasm (well/moderate – 0.58% mean vs. poorly differentiated – 0.85% mean, p = 0.04). Patients having a background of PFC were significantly older than patients with a background of NTDLU (45.2 yr vs. 60.2 yr, p = 0.01).We conclude: (1) breast carcinomas arising from a background of more actively cycling pre‐involutional or proliferative fibrocystic epithelium have a greater proliferative fraction than tumors arising from atrophic epithelium, implying that the differentiation status of target cells may impact the effect(s) of tumorigenic events; (2) PFC may represent delayed, abnormal or interrupted involution rather than a hyperproliferative state relative to NTDLU, suggesting that it facilitates neoplasia by extending the period of exposure to promoter agents such as endogenous hormones, and (3) lower SPFt in breast neoplasia with more abundant “residual” DCIS may reflect a lengthier pre‐invasive disease interval due to intrinsically less aggressive phenotype.
Automatic Quantification of Microvessels Using Unsupervised Image Analysis
Authors: Ranefall Petter , Wester Kenneth , Busch Christer , Malmström Per-Uno , Bengtsson Ewert 
Published: January 2015

An automatic method for quantification of images of microvessels by computing area proportions and number of objects is presented. The objects are segmented from the background using dynamic thresholding of the average component size histogram.To be able to count the objects, fragmented objects are connected, all objects are filled, and touching objects are separated using a watershed segmentation algorithm.The method is fully automatic and robust with respect to illumination and focus settings.A test set consisting of images grabbed with different focus and illumination for each field of view, was used to test the method, and the proposed method showed less variation than the intraoperator variation using manual threshold.Further, the method showed good correlation to manual object counting (r = 0.80) on an other test set.
Cellular Automaton Simulation of Tumour Growth – Equivocal Relationships between Simulation Parameters and Morphologic Pattern Features
Authors: Smolle Josef 
Published: January 2015

Objective: To develop an interpretation procedure which estimates simulation parameters (tumour cell motility, tumour cell adhesion, autocrine and paracrine growth control, stroma destruction) of simulated patterns solely based on morphometric features of the morphologic pattern.Methods: A cellular automaton computer simulation program was developed which produces morphologic patterns by growth of a seed of tumour cells. At the beginning of each simulation run certain simulation parameters are assigned to the tumour cells. After the run has been completed, the resulting pattern is evaluated by a set of morphometric features. Simulation parameters and resulting morphometric features of 27,800 simulations were stored in a database and were used for the evaluation of potential relationships.Results: Correlation analysis showed highly significant correlations between morphometric features on the one hand and the preset simulation parameters (tumour cell motility, tumour cell adhesion, autocrine and paracrine growth control, stroma destruction) on the other. Correlation coefficients, however, varied from 0.72 to 0.99. When only one simulation parameter varied while all others were kept constant, morphometric features yielded a highly reliable estimate of the particular simulation parameter. When variability was extended to 4 simulation parameters, morphometric features were less effective in estimating the setting of the parameters. Though in all patterns tested several possible simulation parameter constellations could be ruled out, morphometric features were usually compatible with more than one set of simulation parameters thus preventing a straightforward interpretation.Conclusions: Though simulation parameters significantly and reproducibly influence the resulting morphologic pattern as characterized by morphometric features, estimates of the simulation parameters based on morphometric features yield equivocal results.
New Algorithms Based on the Voronoi Diagram Applied in a Pilot Study on Normal Mucosa and Carcinomas
Authors: Sudbø J. , Marcelpoil R. , Reith A. 
Published: January 2015

An adequate reproducibility in the description of tissue architecture is still a challenge to diagnostic pathology, sometimes with unfortunate prognostic implications. To assess a possible diagnostic and prognostic value of quantitiative tissue architecture analysis, structural features based on the Voronoi Diagram (VD) and its subgraphs were developed and tested.A series of 27 structural features were developed and tested in a pilot study of 30 cases of prostate cancer, 10 cases of cervical carcinomas, 8 cases of tongue cancer and 8 cases of normal oral mucosa. Grey level images were acquired from hematoxyline‐eosine (HE) stained sections by a charge coupled device (CCD) camera mounted on a microscope connected to a personal computer (PC) with an image array processor. From the grey level images obtained, cell nuclei were automatically segmented and the geometrical centres of cell nuclei were computed. The resulting 2‐dimensional (2D) swarm of pointlike seeds distributed in a flat plane was the basis for construction of the VD and its subgraphs. From the polygons, triangulations and arborizations thus obtained, 27 structural features were computed as numerical values. Comparison of groups (normal vs. cancerous oral mucosa, cervical and prostate carcinomas with good and poor prognosis) with regard to distribution in the values of the structural features was performed with Student′s t‐test.We demonstrate that some of the structural features developed are able to distinguish structurally between normal and cancerous oral mucosa (P=0.001), and between good and poor outcome groups in prostatic (P=0.001) and cervical carcinomas (P=0.001).We present results confirming previous findings that graph theory based algorithms are useful tools for describing tis‐ sue architecture (e.g., normal versus malignant). The present study also indicates that these methods have a potential for prognostication in malignant epithelial lesions.
Caveats: Numerical Requirements in Graph Theory Based Quantitation of Tissue Architecture
Authors: Sudbø J. , Marcelpoil R. , Reith A. 
Published: January 2015

Graph theory based methods represent one approach to an objective and reproducible structural analysis of tissue architecture. By these methods, neighborhood relations between a number of objects (e.g., cells) are explored and inherent to these methods are therefore certain requirements as to the number of objects to be included in the analysis. However, the question of how many objects are required to achieve reproducible values in repeated computations of proposed structural features, has previously not been adressed specifically.After digitising HE stained slides and storing them as grey level images, cell nuclei were segmented and their geometrical centre of gravity were computed, serving as the basis for construction of the Voronoi diagram (VD) and its subgraphs. Variations in repeated computations of structural features derived from these graphs were related to the number of cell nuclei included in the analysis.We demonstrate a large variation in the values of the structural features from one computation to another in one and the same section when only a limited number of cells (100–500) are included in the analysis. This variation decreased with increasing number of cells analyzed. The exact number of cells required to achieve reproducible values differ significantly between tissues, but not between separate cases of similar lesions. There are no significant differences between normal and malignantly changed tissues in oral mucosa with respect to how many cells must be included.For graph theory based analysis of tissue architecture, care must be taken to include an adequate number of objects; for some of the structural features we have tested, more than 3000 cells.