Automated histometry in fibrocystic breast disease

Ductal epithelial proliferations of the mammary gland in biopsy material from 101 patients, including 52 with proliferative fibrocystic disease (mastopathy), were quantitatively analyzed by means of the Robotron A 6471 system together with AMBA/R software. Based on reproducible data obtained for distinct karyometric and histometric features, significant differences were found to exist between epithelial proliferations without atypical hyperplasia (mastopathy II) and those with atypia (mastopathy III). The multiparameter analysis also produced some hints that cases of proliferative mastopathy III can be divided into two groups having different risks of developing carcinoma.     

Karyometry and histometry of renal-cell carcinoma

In an attempt to more objectively predict the outcome of renal cancers, karyometric and histometric studies were performed using an interactive computer-based system for the quantitative analysis of tissue sections. Analysis showed a significant relationship between patient survival and metastases and the histometric parameters of nuclear elongation, nuclear crowding and mitotic density, as well as tumor grade. Patients who died tended to have a high mitotic density, elongated and crowded nuclei and high-grade tumors. Ploidy showed no significant correlation with prognosis while nuclear elongation and crowding did. Differences in histologic grade were significantly associated with several histometric variables, including nuclear area, shape, crowding, elongation and mitotic density.     

Applications of statistical analysis in diagnostic histopathology and cytopathology

Corresponding to the rapid increase in the amount of data available for use in clinical diagnoses, there is an increased need for procedures that can provide the diagnostician with meaningful statistical summaries of data and with statements concerning the statistical significance associated with a diagnostic evaluation. It has been demonstrated that multivariate statistical assessment of clinical material can provide consistent, reliable and highly sensitive diagnostic clues, even in instances in which trained personnel are unable to see any change. Several examples of applications of statistical analyses in diagnostic cytology and histopathology are given in this paper. The examples were chosen to be illustrative of the different types of problems for which statistical analyses have been found useful. These problems differ with respect to the extent of the statistical methods thus far developed and the difficulty involved in developing further analyses. For many problems, appropriate statistical analyses are readily available; other problems require definition of custom-made test statistics and, in some cases, also definition of new statistical distributions. The problems discussed here are only a small sample of the existing problems, but they provide at least an indication of the scope of the role that statistics plays in cytopathologic and histopathologic diagnosis.     

Surveying the manifold divergence of an entire protein class for statistical clues to underlying biochemical mechanisms

Certain residues have no known function yet are co-conserved across distantly related protein families and diverse organisms, suggesting that they perform critical roles associated with as-yet-unidentified molecular properties and mechanisms. This raises the question of how to obtain additional clues regarding these mysterious biochemical phenomena with a view to formulating experimentally testable hypotheses. One approach is to access the implicit biochemical information encoded within the vast amount of genomic sequence data now becoming available. Here, a new Gibbs sampling strategy is formulated and implemented that can partition hundreds of thousands of sequences within a major protein class into multiple, functionally-divergent categories based on those pattern residues that best discriminate between categories. The sampler precisely defines the partition and pattern for each category by explicitly modeling unrelated, non-functional and related-yet-divergent proteins that would otherwise obscure the analysis. To aid biological interpretation, auxiliary routines can characterize pattern residues within available crystal structures and identify those structures most likely to shed light on the roles of pattern residues. This approach can be used to define and annotate automatically subgroup-specific conserved domain profiles based on statistically-rigorous empirical criteria rather than on the subjective and labor-intensive process of manual curation. Incorporating such profiles into domain database search sites (such as the NCBI BLAST site) will provide biologists with previously inaccessible molecular information useful for hypothesis generation and experimental design. Analyses of P-loop GTPases and of AAA+ ATPases illustrate the sampler's ability to obtain such information.     

A statistical method for the comparison of a discrete diagnostic test with several continuous diagnostic tests.

In this paper we study a statistic that is suitable for comparing a discrete diagnostic marker to one or more continuous diagnostic markers. Test procedures and confidence intervals are based on asymptotic normality. The statistic is applicable for correlated data in which all the markers are obtained for each subject. The statistic was studied for use in comparing two markers for rectal bleeding. Examples for this application and two more general applications are presented.     

Automatic cell segmentation in cyto- and histometry using dominant contour feature points.

Automatic cell segmentation has various application potentials in cytometry and histometry. In this paper, an automatic cluster (touching) cell segmentation approach using the dominant contour feature points has been presented. Dominant feature points are the locations of indentation on the contour of the cluster. First, dominant feature points on the contour of the cluster are detected by distance profile. Next, using shape features of the cells, these feature points are selected for segmentation. We compared the results of the proposed method with manual segmentation and observed that the method has an overall accuracy about to 82%.     

Detection of proliferating cell nuclear antigen in diagnostic histopathology.

We describe the effects of tissue preservation, fixation time, and hydrolytic treatment on the detection of proliferating cell nuclear antigen (PCNA) by immunoperoxidase staining with three commercial anti-PCNA antibodies (19A2, 19F4, PC10). Our goal was to provide guidelines for PCNA immunohistochemistry in formalin-fixed, paraffin-embedded specimens. In proliferative cell compartments, nuclear staining was achieved with all three antibodies. In some cases PCNA was also expressed in non-proliferative, histologically normal tissues associated with tumors or other lesions elsewhere. In most autopsy specimens PNCA immunoreactivity was markedly diminished as compared with similar surgical specimens. Incubation overnight with primary antibody at 4 degrees C enhanced PCNA immunoreactivity over incubation at 42 degrees C for 45 min. Pre-treatment with 2 N HCl did not increase staining. Staining with the PC10 antibody was much better preserved than staining with the antibodies 19A2 and 19F4 after prolonged formalin fixation of surgical specimens and in tissues obtained at autopsy. With all three antibodies, however, PCNA immunoreactivity was well preserved during formalin fixation for 8-24 hr and during fixation delays for 8 hr at room temperature. This indicates that PCNA is stable under conditions routinely encountered in diagnostic surgical pathology and facilitates its potential use as a diagnostic proliferation marker.     

Establishment of a statistical base for use of ELISA in diagnostic serology for infectious bovine rhinotracheitis

The critical statistical parameters of an enzyme-linked immunosorbent assay were determined to enable quantitation of antibody responses in cattle affected with infectious bovine rhinotracheitis. A system of controlling well-to-well variations in optical density reading across a microtitre plate was evolved and dose--response assays were carried out to determine the dilution of serum which gave the greatest discrimination between acute and convalescent sera from an infected animal. Use of a standard serum was studied in further assays. An increase in optical density value of 0.15 was set as a diagnostic criterion for a significantly rising antibody response. This compared well with the conventional criterion of a fourfold rise in virus neutralizing antibody titre.     

Architectural, morphometric and photometric features and their relationship to the main subjective diagnostic clues in the grading of prostatic cancer

Novel software was developed to perform quantitative measurements of architectural and nuclear features in tissue sections. A pilot study was then undertaken to determine the diagnostic relevance of these quantitative features in prostatic tissue and the relationship of these objective features to the subjective clues used by practicing pathologists in the grading of prostatic adenocarcinoma. From a group of 82 cases of adenocarcinoma of the prostate with long-term follow-up, a subset of 15 cases that included 5 each in Mostofi grades I, II and III was carefully selected for analysis. Consecutive sections from each case were stained with hematoxylin and eosin or the Feulgen stain for visual and cytometric evaluations, respectively. The most important differences in the objective architectural features observed between the Mostofi grade I and II cases were the number of nuclei per gland and their distance from the glandular center. Significant differences were also noted in gland size and the variation in gland size. The Mostofi grades were also significantly different in terms of quantitative high-resolution features measuring nuclear size and its variation, total nuclear DNA content and the proportion of very aneuploid nuclei. There was a fairly good agreement between many of the subjective diagnostic clues and their corresponding quantitative architectural and nuclear features. This work (1) significantly extended the capabilities of our PC-based microphotometer system to analyze glandular tissue specimens, (2) provided insight into the objective bases for the expert diagnosis of adenocarcinomas of the prostate and (3) gave preliminary evidence of the ability of quantitative architectural features and high-resolution cytometric features to discriminate between the major diagnostic categories of these lesions.     

Detection and separation of heterogeneity in molecular complexes by statistical analysis of their two-dimensional projections

Progress in molecular structure determination by cryo electron microscopy and single particle analysis has led to improvements in the resolution achievable. However, in many cases the limiting factor is structural heterogeneity of the sample. To address this problem, we have developed a method based on statistical analysis of the two-dimensional images to detect and sort localised structural variations caused, for example, by variable occupancy of a ligand. Images are sorted by two consecutive stages of multivariate statistical analysis (MSA) to dissect out the two main sources of variation, namely out of plane orientation and local structural changes. Heterogeneity caused by local changes is detected by MSA that reveals significant peaks in the higher order eigenimages. The eigenimages revealing local peaks are used for automated classification. Evaluation of differences between classes allows discrimination of molecular images with and without ligand. This method is very rapid, independent of any initial three-dimensional model, and can detect even minor subpopulations in an image ensemble. A strategy for using this technique was developed on model data sets. Here, we demonstrate the successful application of this method to both model and real EM data on chaperonin-substrate and ribosome-ligand complexes.     

Detection of Taura syndrome virus (TSV) strain differences using selected diagnostic methods: diagnostic implications in penaeid shrimp

Positive results were obtained with nested white spot syndrome virus (WSSV) diagnostic PCR performed on 5 commercial brands of dry-packed Artemia cysts using several WSSV genomic sequence-specific primers. In 2 brands, PCR and nucleotide sequence analysis found C-->T and C-->G point mutations in the pms 146 WSSV amplicon, but in all 5 brands, the nucleotide sequences that were successfully amplified by the rrl, rr2 and tk-tmk gene-specific primer sets were identical to those of Penaeus monodon WSSV. However, despite the inarguable presence of WSSV or WSSV-like template DNA, we were unable to detect WSSV by PCR in hatched nauplii derived from PCR-positive cysts or in P. monodon postlarvae fed Artemia nauplii hatched from such cysts. Most simply, these results suggested that the cysts were externally contaminated with WSSV or WSSV-like template material that was removed during hatching and washing of the nauplii. Given the small sequence variations found, it may also have been a variety of WSSV non-infectious for P. monodon or Artemia and derived from other crustaceans or arthropods in the Artemia environment. However, we could not establish this conclusively and a small possibility remained that the PCR template in these tests was derived from WSSV template present internally in the cysts and derived from infected Artemia adults. However small, this possibility must be vigorously tested, given the impact that a positive outcome could have on the shrimp industry.     

New cytologic clues in localized Leishmania lymphadenitis

OBJECTIVE: To describe new cytologic clues to diagnose localized leishmania lymphadenitis (LLL). STUDY DESIGN: The study examined cytologic smears of 170 cases of LLL referred to our department from November 1989 to October 2004. A total of 120 cases were confirmed by detecting Leishman-Donovan (LD) bodies in at least 1 of the cytologic smears and 50 cases, which were histologically confirmed. For comparison we studied cytologic smears of 20 cases of tuberculous lymphadenitis, 20 cases of toxoplasma lymphadenitis and 20 cases of granulomatous lymphadenitis of unspecified causes. RESULTS: Cases were divided into 4 major groups. Cytologic findings in these groups were studied to find highly suggestive clues. Cytologic findings present in most of these groups, but absent or very rare in other granulomatous lymphadenitis, were LD kinetoplasts, plasma cells with different shapes of inclusions and lymphogranular bodies. Rare findings not reported previously were: intraneutrophilic LD bodies, hematoxylin body-like inclusions, fibroblasts, cytoplasmic blebbing and floating parasitophorous vacuoles. CONCLUSION: Despite previous reports emphasizing detecting LD bodies in diagnosing LLL, we present cytologic clues highly suggestive of this self-limited disease when LD bodies cannot be detected or are very few on the smears.     

Structural clues in the sequences of the aquaporins

The large number of sequences available for the aquaporin family represents a valuable source of information to incorporate into three-dimensional structure determination. Phylogenetic analysis was used to define type sequences to avoid extreme over-representation of some subfamilies, and as a measure of the quality of multiple sequence alignment. Inspection of the sequence alignment suggested eight conserved segments that define the core architecture of six transmembrane helices and two functional loops, B and E, projecting into the plane of the membrane. The sum of the core segments and the minimum lengths of the interlinking loops constitute the 208 residues necessary to satisfy the aquaporin architecture. Analysis of hydrophobic and conservation periodicity and of correlated mutations across the alignment indicated the likely assignment and orientation of the helices in the bilayer. This assignment is examined with respect to the structure of the erythrocyte aquaporin 1 determined by electron crystallography. The aquaporin 1 tetramer is described as three rings of helices, each ring with a different exposure to the lipid environment. The sequence analysis clearly suggests that two helices are exposed along their whole lengths, two helices are exposed only at their N termini, and two helices are not exposed to lipid. It is further proposed that, besides loops B and E, the highly conserved motifs on helices 1 and 4, ExxxTxxF/L, could line the water channel.