Bronchial carcinoma and image analysis. Differentiation between poorly differentiated epidermoid carcinoma and small-cell carcinoma

The utility of automated image analysis in the distinction between poorly differentiated epidermoid carcinoma (eight cases) and small-cell carcinoma (ten cases) was studied. Material obtained using the bronchial brushing technique was prepared by a cytocentrifugation technique. In each case, a total of 100 bronchial cell nuclei were selected using the Leitz TAS, which measured eight parameters per cell in order to ascertain the homogeneity or the heterogeneity of the nuclear populations. Except for one sample exhibiting preparation artifacts, the method proved capable of differentiating between these two types of bronchial carcinoma, with heterogeneity of the malignant nuclei indicating an epidermoid carcinoma and homogeneity indicating a small-cell carcinoma. A correlation was observed to exist between the morphologic and the morphometric criteria.     

State-of-the-art FISHing: automated analysis of cytogenetic aberrations in interphase nuclei

Interphase fluorescence in situ hybridization (i-FISH) is a powerful tool for visualizing various molecular targets in non-dividing cells. Manual scoring of i-FISH signals is a labor intensive, time-consuming, and error-prone process liable to subjective interpretation. Automated evaluation of signal patterns provides the opportunity to overcome these difficulties. The first report on automated i-FISH analysis has been published 20 years ago and since then several applications have been introduced in the fields of oncology, and prenatal and fertility screening. In this article, we provide an insight into the automated i-FISH analysis including its course, brief history, clinical applications, and advantages and challenges. The lack of guidelines for describing new automated i-FISH methods hampers the precise comparison of performance of various applications published, thus, we make a proposal for a panel of parameters essential to introduce and standardize new applications and reproduce previously described technologies.     

Automated quantification of sympathetic beat-by-beat activity, independent of signal quality.

Sympathetic nerve activity (SNA) can provide critical information on cardiovascular regulation; however, in a typical laboratory setting, adequate recordings require assiduous effort, and otherwise high-quality recordings may be clouded by frequent baseline shifts, noise spikes, and muscle twitches. Visually analyzing this type of signal can be a tedious and subjective evaluation, whereas objective analysis through signal averaging is impossible. We propose a new automated technique to identify bursts through objective detection criteria, eliminating artifacts and preserving a beat-by-beat SNA signal for a variety of subsequent analyses. The technique was evaluated during both steady-state conditions (17 subjects) and dynamic changes with rapid vasoactive drug infusion (14 recordings from 5 subjects) on SNA signals of widely varied quality. Automated measures of SNA were highly correlated to visual measures of steady-state activity (r = 0.903, P < 0.001), dynamic relation measures (r = 0.987, P < 0.001), and measures of burst-by-burst variability (r = 0.929, P < 0.001). This automated sympathetic neurogram analysis provides a viable alternative to tedious and subjective visual analyses while maximizing the usability of noisy nerve tracings.     

The presence of a type IV collagen skeleton associated with periductal elastosis in breast cancer.

Using serial sections of frozen and AFA-fixed tissues from 34 breast cancers, we studied the presence of basement membrane material in the areas of elastosis. Various amounts of type IV collagen but not of laminin were demonstrated in areas of periductal elastosis. In some tumors, type IV collagen accumulated beneath the basement membrane. Periductal elastosis in areas of extensive fibrosis showed focal type IV collagen immunoreactivity, indicating remnants of ducts. Interstitial elastosis corresponded with weak type IV collagen reactivity. Each tumor showed type IV collagen immunostaining of the elastotic areas, with various degrees of intensity. Negative crossreactivity of the type IV collagen antibody with elastin was verified in skin biopsies with solar elastosis. Pre-incubation of the antibody with large amounts of elastin demonstrated an identical immunoreactivity. The specificity of the antibody was confirmed by ELISA and by Western blot analysis. To explain the periductal elastosis, we propose the following hypothesis. Excessive production of basement membrane material by the epithelial cells of the ducts leads to formation of a type IV collagen skeleton. This skeleton can act as the matrix for a secondary deposition of elastic material.     

Computer-aided classification of malignancy in astrocytomas. I. The value of nuclear parameters obtained by automated black and white image analysis.

Ninety-three selected cases of astrocytomas including glioblastomas (astrocytomas grades 1-4) were evaluated by means of Feulgen-stained microscopic slides for nuclear parameters obtained by automated black and white image analysis (ABWIA). The goal was to determine to what extent nuclear features evaluated by ABWIA were applicable as classifiers for the computer-aided numerical classification of malignancy in astrocytomas. Before the automated evaluation, all tumours had been subjectively graded according to the Mayo Clinic grading rules as delineated by Ringertz. Twenty-three nuclear parameters were evaluated and tested for their classification impact. With a model of five parameters (number of nuclei per area, mean of the convex form factor, extinction sum, extinction variation, and full-width-half-maximum of the extinction distribution) the highest reclassification rate of 75% correctly reclassified cases was obtained. Although this is a good result for a classification using only nuclear parameters, it is too poor for practical application. Thus, nuclear parameters evaluated by ABWIA alone are insufficient for numerical classification models assessing the malignant expression of astrocytomas.     

Cytologic uniformity of breast cancer from different localizations: a pattern analysis study.

Using cytologic pattern analysis it is shown that regions of different histologic structure in one breast cancer are of cytologic homogeneity. The so-called PFORM -- the quotient of the total outline length of DNA-particles to the nuclear area -- gives an objective cytologic parameter which is independent of DNA content of cancer cells. This objective parameter together with the subjective cytologic parameters described earlier may be a better basis for describing the biologic behavior of breast cancer than histologic structure which is inhomogeneous very often in a lump.     

cDNA cloning and heterologous expression of functional cysteine-rich antifungal protein Psd1 in the yeast Pichia pastoris.

In the present work, we describe the cDNA cloning, expression in Pichia pastoris, purification, and characterization of the recombinant Pisum sativum defensin 1 (rPsd1), a novel Cys-rich protein presenting four disulfide bridges and high antifungal activity. Several parameters that affect the level of protein expression were assayed. The best condition yielded 13.8 mg/L (1.50 microg/10(8) cells) of active rPsd1. The recombinant rPsd1 was purified to homogeneity by cation exchange, followed by reversed-phase HPLC, and subjected to automated amino acid sequencing, which revealed four additional amino acids (EAEA) at the N-terminal region. Circular dichroism, intrinsic fluorescence, and nuclear magnetic resonance spectroscopy analysis indicated that the recombinant protein has a very similar folding and a correct disulfide-bonding pattern when compared to native Psd1. Nevertheless, the rPsd1 presented a more species-specific antifungal activity. The importance of the N- and C-termini for Psd1 activity is pointed out.     

Histopathologic variables differentially affect melanoma survival by age at diagnosis

We evaluated clinical, phenotypic, behavioral, and histopathologic variables in relationship to melanoma‐specific survival by age at diagnosis among 650 population‐based melanoma patients in Connecticut, with 20 years of follow‐up. Only one variable, skin awareness, was significantly associated with melanoma mortality in both groups. The variables that differed between the age‐groups were anatomic site, Breslow thickness, histologic subtype, mitoses, tumor‐infiltrating lymphocytes (TILs), and solar elastosis. Head and neck melanoma, Breslow thickness, nodular melanoma, and solar elastosis were all significantly more likely to be associated with mortality among the older subjects; among the younger subjects, the presence of mitoses was associated with an increased probability of dying and TILs were associated with a reduced risk of mortality.     

Solid-phase synthesis of sauvagine-(17-40)

The solid-phase synthesis of the tetracosapeptide corresponding to the C-terminal amino acid sequence of sauvagine is described. After purification by gel filtration, the polypeptide appeared to possess an acceptable degree of homogeneity, as judged by different kinds of electrophoresis and chromatography, and by automated Edman degradation analysis. Preliminary pharmacological results indicate that the fragment-(17-40) is practically devoid of any sauvagine activity on the circulatory system and endocrine glands; a weak effect on gastric emptying delay has been demonstrated (1% of the natural product).     

Identification of components of fibroadenoma in cytology preparations using texture analysis: a morphometric study

S. Singh and R. Gupta Identification of components of fibroadenoma in cytology preparations using texture analysis: a morphometric study Objectives: To evaluate the utility of image analysis using textural parameters obtained from a co‐occurrence matrix in differentiating the three components of fibroadenoma of the breast, in fine needle aspirate smears. Methods: Sixty cases of histologically proven fibroadenoma were included in this study. Of these, 40 cases were used as a training set and 20 cases were taken as a test set for the discriminant analysis. Digital images were acquired from cytological preparations of all the cases and three components of fibroadenoma (namely, monolayered cell clusters, stromal fragments and background with bare nuclei) were selected for image analysis. A co‐occurrence matrix was generated and a texture parameter vector (sum mean, energy, entropy, contrast, cluster tendency and homogeneity) was calculated for each pixel. The percentage of pixels correctly classified to a component of fibroadenoma on discriminant analysis was noted. Results: The textural parameters, when considered in isolation, showed considerable overlap in their values of the three cytological components of fibroadenoma. However, the stepwise discriminant analysis revealed that all six textural parameters contributed significantly to the discriminant functions. Discriminant analysis using all the six parameters showed that the numbers of pixels correctly classified in training and tests sets were 96.7% and 93.0%, respectively. Conclusion: Textural analysis using a co‐occurrence matrix appears to be useful in differentiating the three cytological components of fibroadenoma. These results could further be utilized in developing algorithms for image segmentation and automated diagnosis, but need to be confirmed in further studies.     

On-line transmission electron microscopic image analysis of chromatin texture for differentiation of thyroid gland tumors

Nuclei of the cells from the thyroid gland were analyzed in a transmission electron microscope by direct TV scanning and on-line image processing. The method uses the advantages of a visual-perception model to detect structures in noisy and low-contrast images. The features analyzed include area, a form factor and texture parameters from the second derivative stage. Three tumor-free thyroid tissues, three follicular adenomas, three follicular carcinomas and three papillary carcinomas were studied. The computer-aided cytophotometric method showed that the most significant differences were the statistics of the chromatin texture features of homogeneity and regularity. These findings document the possibility of an automated differentiation of tumors at the ultrastructural level.     

Improved Estimation of Cardiac Function Parameters Using a Combination of Independent Automated Segmentation Results in Cardiovascular Magnetic Resonance Imaging

This work aimed at combining different segmentation approaches to produce a robust and accurate segmentation result. Three to five segmentation results of the left ventricle were combined using the STAPLE algorithm and the reliability of the resulting segmentation was evaluated in comparison with the result of each individual segmentation method. This comparison was performed using a supervised approach based on a reference method. Then, we used an unsupervised statistical evaluation, the extended Regression Without Truth (eRWT) that ranks different methods according to their accuracy in estimating a specific biomarker in a population. The segmentation accuracy was evaluated by estimating six cardiac function parameters resulting from the left ventricle contour delineation using a public cardiac cine MRI database. Eight different segmentation methods, including three expert delineations and five automated methods, were considered, and sixteen combinations of the automated methods using STAPLE were investigated. The supervised and unsupervised evaluations demonstrated that in most cases, STAPLE results provided better estimates than individual automated segmentation methods. Overall, combining different automated segmentation methods improved the reliability of the segmentation result compared to that obtained using an individual method and could achieve the accuracy of an expert.     

Statistical analysis of the parameters defining the cortical visual evoked potentials in the phases of the sleep-wake cycle

Visual evoked potential parameters (latencies, intervals of latencies and amplitudes) obtained by photic stimulation using a light-emitting diode implanted in the frontal sinus of cats were studied by statistical methods (analysis of variance) during the stages of wakefulness, slow sleep and paradoxical sleep. The results show: a) greater intraindividual homogeneity in all cases with special emphasis on the latencies; b) the greatest homogeneity of responses was found during slow sleep and paradoxical sleep stages; c) in relation to the influences exerted by the sleep-wakefulness cycle on the visual evoked potentials, the parameters most affected were those closely related to the secondary complex. We conclude, that latency, due to its great homogeneity, is the most useful parameter in this kind of experiments and secondly, that it is the secondary complex of the visual evoked potentials that is affected by the endogenous conditions of the subject (in our case the sleep-wakefulness cycle stage).     

Automated screening of neurite outgrowth

Outgrowth of neurites in culture is used for assessing neurotrophic activity. Neurite measurements have been performed very slowly using manual methods or more efficiently with interactive image analysis systems. In contrast, medium-throughput and noninteractive image analysis of neurite screens has not been well described. The authors report the performance of an automated image acquisition and analysis system (IN Cell Analyzer 1000) in the neurite assay. Neuro-2a (N2a) cells were plated in 96-well plates and were exposed to 6 conditions of retinoic acid. Immunofluorescence labeling of the cytoskeleton was used to detect neurites and cell bodies. Acquisition of the images was automatic. The image set was then analyzed by both manual tracing and automated algorithms. On 5 relevant parameters (number of neurites, neurite length, total cell area, number of cells, neurite length per cell), the authors did not observe a difference between the automated analysis and the manual analysis done by tracing. These data suggest that the automated system addresses the same biology as human scorers and with the same measurement precision for treatment effects. However, throughput of the automated system is orders of magnitude higher than with manual methods.     

Criterion validity of the Pittsburgh Sleep Quality Index: Investigation in a non-clinical sample

The objective of this study was to investigate the reliability and validity of the Pittsburgh Sleep Quality Index (PSQI) in a non-clinical sample consisting of younger and older adults. There has been little research validating the PSQI with respect to multinight recording as with actigraphy, and more validation is needed in samples not specifically selected for clinical disturbance. Also, the degree to which the PSQI scores may reflect depressive symptoms versus actual sleep disturbance remains unclear. One-hundred and twelve volunteers (53 younger and 59 older) were screened for their ability to perform treadmill exercises; inclusion was not based on sleep disturbance or depression. Internal homogeneity was evaluated by correlating PSQI component scores with the global score. Global and component scores were correlated with a sleep diary, actigraphy, and centers for epidemiological studies - depression scale scores to investigate criterion validity. Results showed high internal homogeneity. PSQI global score correlated appreciably with sleep diary variables and the depression scale, but not with any actigraphic sleep variables. These results suggest that the PSQI has good internal homogeneity, but may be less reflective of actual sleep parameters than a negative cognitive viewpoint or pessimistic thinking. The sleep complaints measured may often be more indicative of general dissatisfaction than of any specifically sleep-related disturbance.     

Modified histogram subtraction technique for analysis of flow cytometry data

Analysis of flow cytometry histogram data by the subjective selection of an integration window can be a tedious and time-consuming task and is often inaccurate. A new method for automated calculation of the percent positive from immunofluorescence histograms is presented. This new method is a modification of the currently used method of channel-by-channel histogram subtraction. Its accuracy is compared to that of the channel-by-channel histogram subtraction method and to another currently used automated method, which selects an integration window by finding the channels that contain the most fluorescent 2% of a control histogram. The new histogram subtraction method is objective, easy to use, and is more accurate than other currently used automated analysis methods. PASCAL source code is given for each method of analysis.     

Virtual microscopy as an enabler of automated/quantitative assessment of protein expression in TMAs

Tissue Microarrays facilitate high-throughput immuohistochemistry; however, there are key bottlenecks apparent in their analysis, particularly when conducting microscope-based manual reviews. Traditionally Tissue Microarray assessments were performed using a microscope where results were either transcribed or dictated and subsequently entered into flat-file spreadsheets. This process is labour intensive, prone to error and negates the advantages of the high-throughput Tissue Microarray format. In addition, human interpretations of staining intensity parameters are highly subjective and therefore prone to inter- and intra-observer variability. The advent of Virtual Slides has permitted the review of tissue slides across the Internet. In addition, this new technology enables the creation of software solutions to assist in the manual and automated review of Tissue Microarrays, through the use of computer aided image analysis. There are numerous academically developed and commercially available applications which assist in Tissue Microarray reviews; functionality of these systems range in complexity and application domains. The review which follows describes these systems and outlines technical considerations to be assessed when deciding on a Tissue Microarray workflow solution.     

Assessment of elastosis in invasive ductal carcinoma of the breast compared to fibroadenoma among Sudanese patients using conventional histochemical methods

We aimed to identify, using histochemical methods, the degree of elastosis in a malignant breast tumor compared to a benign tumor. Sixty-four tissue blocks were used in this study of which 34 (53.1%) were Invasive ductal carcinoma (IDC) (cases) and 30 (46.8%) were fibroadenomas (controls). Examination of Verhoeff's stained sections revealed different grades of elastosis in 29 (85.2%) cases compared to seven (23.3%) in controls. These findings indicated that elastosis was a prominent feature of IDC (p < 0.000). IDC was associated with more frequent occurrence of different grades of elastosis and should, therefore, be considered a valuable histological finding. Nevertheless, more advanced technology for quantitative measurements of the staining density is recommended to confirm this variation.     

The performance characteristics of an expert system for the "on-line" assessment of thalassemia trait and iron deficiency--Micro Hema Screen

An Expert System (ES) approach to the assessment of thalassemia trait and iron deficiency was evaluated by transferring data from a Coulter S-Plus II Analyzer to an Apple II e computer. The discriminant rules of the algorithm were derived retrospectively from multivariate analysis of an existing data base. The parameters used were MCV, RDW, HGB, RBC, MPV, and PLT. Subsequently, the system has been evaluated for its ability to detect thalassemia and hemoglobinophathies in 1671 females and 1490 males in a real laboratory setting. The 845 cases of microcytosis among them were used to estimate the real frequencies of the diagnostic groups as identified by the ES (Micro Hema Screen). "On-Line" assessment of microcytosis may provide a quick method of acquiring and processing the data from an automated analyser. It can be applied in thalassemia and iron deficiency discrimination either as an intralaboratory primary assessment or as a population screening method.     

A simple, flexible, nonfluorescent system for the automated screening of neurite outgrowth

Measurement of neurite outgrowth is a common assay of neurotrophic activity. However, currently available techniques for measuring neurite outgrowth are either time or resource intensive. The authors established a system in which chronic treatment of a subcloned SH-SY5Y cell line with aphidicolin and various concentrations of nerve growth factor (NGF) induced discernable alterations in proliferation and differentiation. Cells were fixed, labeled with a nonfluorescent dye, and evaluated both manually and with an automated analysis system. NGF increased multiple parameters of differentiation, including neurite length, the proportion of cells extending neurites, and branching, as well as promoting cellular survival/proliferation. Interestingly, although NGF treatment increased the total number of branches, it actually decreased the proportion of branches per neurite length. The authors observed no differences in results obtained using the manual and automated systems, but the automated system was orders of magnitude faster. To demonstrate the flexibility of the system, the authors also show that they could measure changes in differentiation induced by a small-molecule Rho kinase inhibitor, as well as by retinoic acid cotreatment with brain-derived neurotrophic factor. In addition to this flexibility, this system does not require specialized equipment or fluorescent antibodies for analysis and therefore provides a less resource-intensive alternative to fluorescence-based systems.