Morphometric image analysis of follicular lesions of the thyroid

OBJECTIVE: To determine the role of image morphometry in distinguishing various follicular lesions of the thyroid in cytologic smears. STUDY DESIGN: Archival fine needle aspiration smears of 10 cases each of follicular hyperplasia, follicular adenoma, follicular carcinoma and follicular variant of papillary carcinoma were used for the study. All cases were histopathologically proven. At least 100 random nuclei from each case were subjected to analysis with an image cytometer. Area, convex area, length, width, perimeter, convex perimeter and roundness of nuclei were measured using a 40 x objective (1 pixel = 0.446 micron). RESULTS: ANOVA showed that all the nuclear variables studied were significantly different (P < .05) in follicular hyperplasia as compared to follicular carcinoma and papillary carcinoma. All nuclear variables except roundness were also significantly different (P < .05) between follicular hyperplasia and follicular adenoma. However, between follicular adenoma, follicular carcinoma and papillary carcinoma there was considerable overlap of nuclear morphometric parameters. CONCLUSION: Image morphometry may help to distinguish nonneoplastic follicular lesions (hyperplasia) from neoplastic lesions (adenomas and carcinomas). However, to distinguish benign from malignant follicular lesions, image morphometry might not improve the accuracy of standard cytologic examination.     

DNA-based sputum cell image analysis for lung cancer in a clinical setting

OBJECTIVE: To measure cell nuclei characteristics, previously reported to express probability for lung cancer, in subjects with different forms ofpulmonary disease and those without disease. STUDY DESIGN: Sputum and buccal cell samples were obtained from 846 patients without pulmonary disease, with nonmalignant disease, chronic obstructive pulmonary disease, asbestosis and lung cancer, stained for DNA, scanned by cytometer and scored. This was related to specificity and sensitivity for lung cancer. At score 4.5 sensitivity was 53.8% and specificity 70.9%. This score and higher were defined as high scores (HS) and used to compare groups with lung cancer and other pulmonary disease. RESULTS: Among subjects without disease, 21.1% had HS in sputum cells. Among those with nonmalignant pulmonary disease, 31.7% had HS, and among subjects with lung cancer, 53.8% had it. Repeated evaluations showed that about one third of those with HS on the first occasion were normal on repeat sampling. Among subjects without lung cancer, 33.8% of never-smokers had sputum cell HS compared to 22.7.2% among smokers. CONCLUSION: Results demonstrate that the DNA cellular characteristics on cytometry were more frequent among subjects with lung cancer but also among subjects with other pulmonary disease compared to subjects witbout pulmonary disease.     

Morphometric analysis of LPS-stimulated human monocytes by computer-assisted image analysis.

This report describes the results of applying the computer-assisted image analysis system for the measurement of some cytological parameters of LPS-stimulated and nonstimulated human monocytes. The experiments were carried out by means of the digital cell image analysis of haematoxilyn stained monocytes. Five different parameters describing the morphology of monocytes and their nuclei were selected to quantitate the differences between control and activated cells area, perimeter, elongation, dispersion, and extension of images of cell projections. The results suggest that all of the analysed parameters can be used to discriminate stimulated from nonstimulated monocytes which permits detailed monitoring of the changes in cell morphology during monocyte activation.     

Contextual analysis and intermediate cell markers enhance high-resolution cell image analysis for automated cervical smear diagnosis.

Until now, efforts to automate cervical smear diagnosis have focused on analyzing features of individual cells. In a complex specimen such as that obtained from a cervical scrape, diagnostically significant cells may not be adequately represented or may elude detection by the automated technology. An approach is needed that extracts additional quantitative information from cervical smears beyond what the cell-by-cell approach can provide. A new methodology, contextual analysis, was developed to extract global quantitative information about cells, cell clusters, and background debris. This pilot study was designed to compare the efficacy of contextual analysis with high-resolution, single cell analysis and the analysis of intermediate cell markers. Thirty-four samples prepared as monolayers and stained with the Feulgen-Thionin/Congo Red stain were measured. Contextual analysis alone was able to classify 91% of the smears correctly; single cell analysis classified 94% of the cells correctly; and the intermediate cell analysis correctly identified the smear diagnosis for 84% of the cells. When all three analysis methods were combined into a simple smear level classifier, the overall smear classification accuracy was improved over those obtained using the three methodologies alone.     

Morphometric analysis of borderline atypia in prostatic aspiration biopsy specimens.

In a prior study of 411 fine needle aspirates of the prostate (FNAPs), we identified a subset of 50 (12%) aspirates in which the findings were designated cytologically atypical. These equivocal cases formed a heterogeneous group composed of both histologically benign and malignant proliferations in which cutting needle biopsy was necessary to further delineate the nature of the abnormality. In an attempt to define quantitative and morphologic criteria for the separation of these proliferations into benign and malignant categories and to reduce the need for diagnostic cutting needle biopsies, we performed a cytomorphometric analysis of smears from 12 FNAPs (6 histologically proven benign and 6 histologically proven malignant) with equivocal cytologic atypia. We were unable to define any cytomorphometric criteria that accurately subdivided these cases into benign and malignant categories.     

Neoplastic conversion of preneoplastic Syrian hamster cells: rate estimation by fluctuation analysis.

Analysis of the role of gene mutations in the multistep process of neoplastic transformation requires that the discrete steps in carcinogenesis first be dissected. Toward this end, we have isolated and characterized preneoplastic Syrian hamster cells which exhibit in vitro a trait highly correlated with neoplastic conversion in vivo. Previous findings (J. C. Barrett, Cancer Res. 40:91-94, 1980) indicate that spontaneous neoplastic transformation of Syrian hamster cells occurs in at least two steps. An intermediate stage, characterized by an aneuploid established cell line which has a propensity to become neoplastic spontaneously upon further growth in vitro, has been described. These preneoplastic cells differ from diploid early-passage Syrian hamster cells in becoming capable of anchorage-independent growth in semisolid agar, as well as becoming neoplastic in vivo when attached to a solid substrate. Evidence presented here demonstrates that anchorage-independent conversion in vitro is a reliable marker for neoplastic conversion in this cell system. Fluctuation analyses, patterned after those described by Luria and Delbruck for microbial genetics, demonstrate that anchorage-independent variants are generated randomly from clonally derived preneoplastic cells at the rate of 10(-8) to 10(-7) variants per cell per generation. These results establish a multistep stochastic process for transformation in vitro and indicate that conversion to anchorage independence may be necessary for Syrian hamster cells to become tumorigenic. The possible role of gene mutation in this step during neoplastic progression is discussed.     

Early endocervical glandular neoplasia. II. Morphometric analysis of the cells

A morphometric analysis was performed on the cells of early endocervical glandular neoplasia, including adenocarcinoma in situ and microinvasive adenocarcinoma, in cytologic preparations. The measurements were compared with those of cells from the normal endocervix and cells from other lesions that enter into the differential diagnosis. The morphometric profile developed for the cells of early glandular neoplasia proved to be useful, reliable and reproducible. Not only could the cells of normal endocervical samples be distinguished from those of early endocervical glandular neoplasia, but the measurements also seemed capable of making the visually difficult distinction between adenocarcinoma in situ and microinvasive adenocarcinoma. The latter point requires further confirmation since only a few cases of microinvasive adenocarcinoma were available for study.     

Endocervical atypical glandular cells of undetermined significance. II. Morphometric and cytologic analysis of nuclear features useful in characterizing differently correlated subgroups

OBJECTIVE: To evaluate whether some nuclear features analyzed by morphometry and cytology may be useful in characterizing differently correlated endocervical atypical glandular cells of undetermined significance (AGUS) subgroups. STUDY DESIGN: Fifty-four endocervical AGUS smears were subclassified into four subgroups on the basis of their different correlation: not otherwise specified subgroup (NOSs), tamoxifen (Ts), human papillomavirus infection (HPVs) and laser therapy (LTs). Area and shape of the atypical nuclei detected were morphometrically measured. The smears were then cytologically reviewed, and the shape and grade of expression of hyperchomasia in AGUS nuclei were analyzed. RESULTS: AGUS cases due to T therapy showed the largest nuclear area (148.845 micron 2; P < .0000) and the greatest anisonucleosis objectively measured by morphometry. HPVs had the shape that most differed from perfectly circular (15.341 versus 14.1) and showed the highest grade of expression of nuclear density. LTs and NOSs were less well characterized than the other subgroups. CONCLUSION: Analysis of nuclear features by morphometry and cytology was useful in characterizing the AGUS subgroups Ts and HPVs.     

Diagnostic value of crush artifact in cytologic specimens. Occurrence in small cell carcinoma of the lung

The occurrence of nuclear crush artifact (NCA) in cytologic specimens of small cell undifferentiated carcinoma (SCUC) and other carcinomas of the lung, lymphoma and benign lymphoid proliferations was studied to determine its diagnostic usefulness. NCA was found to be a common and morphologically distinct feature in SCUC, depending on how the specimen was prepared; it was present in aspirates, washings, brushings and sputum, but not in pleural or cerebrospinal fluids. It was absent in other types of cancer and could be distinguished from other similar-appearing artifacts. These results demonstrate that NCA is a diagnostically useful feature in pulmonary SCUC.     

MALDI imaging mass spectrometry for in situ proteomic analysis of preneoplastic lesions in pancreatic cancer

The identification of new biomarkers for preneoplastic pancreatic lesions (PanINs, IPMNs) and early pancreatic ductal adenocarcinoma (PDAC) is crucial due to the diseases high mortality rate upon late detection. To address this task we used the novel technique of matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) on genetically engineered mouse models (GEM) of pancreatic cancer. Various GEM were analyzed with MALDI IMS to investigate the peptide/protein-expression pattern of precursor lesions in comparison to normal pancreas and PDAC with cellular resolution. Statistical analysis revealed several discriminative m/z-species between normal and diseased tissue. Intraepithelial neoplasia (PanIN) and intraductal papillary mucinous neoplasm (IPMN) could be distinguished from normal pancreatic tissue and PDAC by 26 significant m/z-species. Among these m/z-species, we identified Albumin and Thymosin-beta 4 by liquid chromatography and tandem mass spectrometry (LC-MS/MS), which were further validated by immunohistochemistry, western blot, quantitative RT-PCR and ELISA in both murine and human tissue. Thymosin-beta 4 was found significantly increased in sera of mice with PanIN lesions. Upregulated PanIN expression of Albumin was accompanied by increased expression of liver-restricted genes suggesting a hepatic transdifferentiation program of preneoplastic cells. In conclusion we show that GEM of endogenous PDAC are a suitable model system for MALDI-IMS and subsequent LC-MS/MS analysis, allowing in situ analysis of small precursor lesions and identification of differentially expressed peptides and proteins.     

Early detection of precancerous lesions in dysplasias of the lung by rapid DNA image cytometry

Hematoxylin-and-eosin-stained cytologic smears of sputum from 28 patients with dysplastic and suspicious cell findings were subjected to DNA image cytometry after Feulgen restaining. The nuclear DNA contents were measured with a TV-based image-analysis system, the Leitz TAS plus, combined with an automatic microscope. Computation of DNA data was performed according to an algorithm for the diagnosis and grading of malignancy. Of the 19 cases that were proven to be malignant in the follow-up, either by histologic examination, sputum cytology, fine needle aspiration biopsy or autopsy, the algorithm identified 17 as malignant in a stage (dysplasia) in which cytology was not yet able to present a definite diagnosis of malignancy. Only two cases of bronchial carcinoma were not detected in the state of dysplasia by this procedure. The periods between the DNA diagnosis of malignancy in dysplasia and the morphologic evidence of cancer varied from three days up to six months. Of the 11 cases that had been classified as benign by the algorithm, 9 were confirmed as benign during the clinical follow-up. Rapid DNA image cytometry appears able to separate squamous dysplasias of the lung into precancerous and nonprecancerous lesions.     

Morphometric analysis of Leydig cells in the normal rat testis

Leydig cells are thought to be the source of most, if not all, the testosterone produced by the testis. The goal of this study was to obtain quantitative information about rat Leydig cells and their organelles that might be correlated with pertinent physiological and biochemical data available either now or in the future. Morphometric analysis of Leydig cells in mature normal rats was carried out on tissue fixed by perfusion with buffered glutaraldehyde, and embedded in glycol methacrylate for light microscopy and in Epon for electron microscopy. In a whole testis, 82.4% of the volume was occupied by seminiferous tubules, 15.7% by the interstitial tissue, and 1.9% by the capsule. Leydig cells constituted 2.7% of testicular volume. Each cubic centimeter (contained approximatelyy 1 g) of rat testis contained about 22 million Leydig cells. An average Leydig cell had a volume of 1,210 micron3 and its plasma membrane had a surface area of 1,520 micron2. The smooth endoplasmic reticulum (SER), the most prominent organelle in Leydig cells and a major site of steroidogenic enzymes, had a surface area of approximately 10,500 micron2/cell, which is 6.9 times that of the plasma membrane and is 60% of the total membrane area of the cell. The total surface area of Leydig SER per cubic centimeter of testis tissue is approximately 2,300 cm2 or 0.23 m2. There were 3.0 mg of Leydig mitochondria in 1 g of testis tissue. The average Leydig cell contained approximately 622 mitochondria, measuring on the average 0.35 micron in diameter and 2.40 micron in length. The mitochondrial inner membrane (including cristae), another important site of steroidogenic enzymes, had a surface area of 2,920 micron2/cell, which is 1.9 times that of the plasma membrane. There were 644 cm2 of inner mitochondrial membrane/cm3 of testis tissue. These morphometric results can be correlated with published data on the rate of testosterone secretion to show that an average Leydig cell secretes approximately 0.44 pg of testosterone/d or 10,600 molecules of testosterone/s. The rate of testosterone production by each square centimeter of SER is 4.2 ng/d or 101 million molecules/s: the corresponding rate for each square centimeter of mitochondrial inner membrane is 15 ng testosterone/d or 362 million molecules/s.     

Comparison of image analysis with flow cytometry for DNA content analysis in pigmented lesions of the skin.

The DNA ploidy of 85 melanocytic skin lesions was determined by flow cytometry (FCM) and interactive image analysis (IA) using nuclear extracts of paraffin-embedded tissue. Of the 85 lesions analyzed, 43 were malignant melanomas in different stages of evolution, 15 were dysplastic nevi, 11 were Spitz nevi, and 16 were other types of nevi. Some of the last had features of congenital nevi. Within the melanoma category, there was 42% aneuploidy by FCM versus 56% by IA. Of those melanomas aneuploid by FCM, all but one were aneuploid by IA. All dysplastic nevi, 10/11 Spitz nevi and 15/16 other nevi were diploid by both methods. One of the 16 nevi from the "other types" category was tetraploid by IA but diploid by FCM. A single Spitz nevus was tetraploid by FCM but diploid by image analysis. While our results suggest that interactive IA is potentially a more sensitive method than FCM for detecting aneuploidy in cutaneous pigmented lesions, it remains to be shown whether this will translate into better prognostic assessment of the biologic behavior of melanocytic neoplasms than provided by flow cytometric ploidy analysis.     

Analysis of machine-selected cells with an image analysis system in normal and abnormal cervical specimens

An analysis has been performed of visual diagnostic criteria used in cervical cytology applied to machine selected cells in relation to automated classification based on variables, which can be recorded in an image system with automated cell search and segmentation, feature extraction and classification. A 98% accuracy could be obtained with the choice of the most ideal statistical methods for discrimination and the use of the most powerful variables recorded in the image system when compared with consensus of the visual diagnoses based on established cytological criteria for diagnosis of cancer and precancer of the cervix uteri. The most powerful discriminatory variables in the image system (of 17 recorded) for discrimination between normal and abnormal epithelial cells were, in addition to nuclear extinction, cytoplasmic extinction and cytoplasmic shape. It is concluded that the visual classification of cervical cells is highly accurate with experienced observers and that imaging microscopes can be trained to nearly equal this accuracy with appropriate statistical methods of discrimination. The problem of creating fully automated systems, however, also requires the inclusion of even more effective discriminatory variables and also the solution of such problems as automatic cell search, segmentation, artifact rejection, feature extraction, classification and electronic stability in order to become cost-effective.     

Morphometric evaluation of the number of exocrine pancreatic cells during early postnatal growth in the rat.

The number of the various cell categories of the exocrine pancreas of the rat was evaluated by morphometric methods in paraffin sections of glands from rats aged 2, 5, 15, 20 and 33 days. The evolution of these cell types could be properly expressed by equations of the type y = aoek.x, where y = cell number, and x = age in days. The time necessary for each cell type to duplicate was thus obtained. The percentages (y') of acinar, intercalated duct and connective tissue cells also proved to be age-dependent and could be expressed by second-degree equations.