Histological and ultrastructural evidence that recurrent laryngeal neuropathy is a bilateral mononeuropathy limited to recurrent laryngeal nerves.

REASONS FOR PERFORMING STUDY: Recurrent laryngeal neuropathy (RLN) is a common and debilitating peripheral nerve disease of horses, but it remains unclear if this disease is a mono- or polyneuropathy. An understanding of the distribution of the neuropathological lesions in RLN affected horses is fundamental to studying the aetiology of this very significant disease of tall horses. OBJECTIVE: To determine whether RLN should be classified as a mono- or polyneuropathy. METHODS: Multiple long peripheral nerves and their innervated muscles were examined systematically in 3 clinically affected RLN horses RESULTS: Severe lesions were evident in the left as well as right recurrent laryngeal nerves in all horses, both distally and, in one case, also proximally. No primary axonal lesions were evident in other nerves nor were changes found in their innervated muscles. CONCLUSIONS: RLN is not a polyneuropathy but should be classified as a bilateral mononeuropathy. POTENTIAL RELEVANCE: Genetic and local factors specifically affecting the recurrent laryngeal nerves in RLN-affected horses should now be investigated further.

Cytometry in histological sections of colon carcinoma.

A short description of a project of cytometry in histological sections of colon carcinoma is given with emphasis on the methodical aspects. Possible strategies of cytometric measurement and problems related to it (focus, overlap, segmentation of objects) are described. The main effort concerns interactive selection of tumor cells and the segmentation in cases of densely distributed and overlapping nuclei. All other succeeding processing steps are performed fully automatically. The resulting quantitative features are stored together with the original images on an optical disk for further examinations and reexaminations, allowing the direct relation of feature values to visual image content. The evaluation of the features as well as their interpretation is only at the beginning. Especially the problem of relating section information with true 3-dimensional information is not described here and necessitates further research. In a first investigation only a few tumors without and with metastases were analyzed. The preliminary results correspond with findings of Kunze et al.

Nucleolus organizer regions (AgNORs) in ductal mammary carcinoma. Comparison with classifications and prognosis.

The relevance of silver-stained NORs for classifications and prognosis was investigated in breast tissue. Paraffin sections from 137 cases of invasive ductal breast carcinomas and 12 cases with non-tumorous ductus epithelium as controls were stained according to a modified technique and analysed. From the cancer cases follow-up data up to 10 years (45 to 165 months) and in addition clinical, histological and several DNA distribution parameters were available. The nuclei and the silver grains were measured by means of a semiautomatic image analysis system. Significant differences in AgNOR features were found between controls and diploid tumors (p < or = 0.001), diploid and aneuploid tumors (p < or = 0.001), Bloom-Richardson-gradings I, II, and III (p < or = 0.001), and between the tumor cells from patients developing metastases within 5 years and those without (p < or = 0.002). The prognostic significance of AgNORs was estimated using Cox regression analysis. Four AgNOR features were correlated significantly with survival time. In a multivariate approach offering all parameters available an AgNOR parameter (CV of relative area AgNORs) ranked at the third position beyond the SD of DNA distribution and pTNM-staging. Considering the metastases-free interval of patients the same AgNOR feature showed an independent prognostic validity.

A knowledge acquisition tool in analytical pathology based on multi-media relational database.

The need for a knowledge based expert system for efficient decision making in the field of pathology has been well accepted. To build the knowledge-base for such an expert system is a painstaking task. This work is an attempt to provide the pathologists a powerful and user-friendly tool that will help them in the process of building the knowledge-base for medical diagnosis by closely looking at the specimen images and their extracted feature values. The tool is based on multi-media relational database and software like VAX Rally (4GL), ILIAD and SAS to provide a wide-range comparative study between feature data and image data; and also their statistical analysis.

Stereological characteristics of the equine accessory nerve.

Stereological techniques have been increasingly employed for assessment and characterization of neuromuscular diseases in humans and animals. As an adjunct to histopathology, morphometrical algorithms provide quantitative evidence of the peripheral nerve composition, thereby shedding light on its fibre characteristics and basic electrophysiological properties. In the horse, stereological investigations already have focussed on the recurrent laryngeal, deep peroneal and lateral palmar nerves (LPN). Of these, only the latter is suitable for taking biopsies in clinical settings, however, it does not contain any motor fibres and Ia-afferents. On account of its virtually mixed fibre qualities, most researchers today recommend the cervical branch of the equine accessory nerve (AN) for harvesting diagnostic samples. Thus, the present study was carried out to gain morphometrical proof of the AN composition and to obtain stereological base values in healthy individuals using state-of-the-art technology. All parameters were compared to the common peroneal nerve (CPN), known to harbour all myelinated fibre classes. As this second biopsy site is located farther distally to the neuro-axis, attention was paid to possible length-dependent features. Taken together, digital image analysis could be accurately applied on all AN samples. Stereology supported the histological and clinical evidence that the AN contains all myelinated fibre types. The huge range and scatter of fibre counts and density (3351-17,812/mm(2)) per fascicle were comparable to that measured in the equine common peroneal, deep peroneal, lateral palmar and recurrent laryngeal nerves. Similar to those, fibre diameter distribution was bimodal with slow Abeta- and Agamma-mechanoceptor afferents outnumbering large myelinated Aalpha-fibres by a factor of about 1.5. With a g-ratio at 0.55 +/- 0.001, the overall degree of myelination in the AN is highly consistent and insignificantly ranges between that of the equine common peroneal and LPNs. Apart from this subtle deviation, a statistically relevant difference between the more proximal AN and the distal CPN could not be documented. By obtaining morphometrical standard parameters and even more sophisticated distribution indices, stereology is a valuable tool for detection of subtle changes that are likely to escape from the investigators' eyes. The AN serves as a reliable source for advanced peripheral nerve research and should be accompanied by farther distal nerve probes for assessment of neuropathies that present with a proximodistal gradient.

Invariance of textural features in image cytometry under variation of size and pixel magnitude.

Textural features of the granular structure of stained cell nuclei and nuclear sections derived from co-occurrence and run length matrices are often used for correlation with external (clinical) parameters. The representation of cell nuclei and nuclear sections vary considerably under changes of preparation and fixation conditions. Most obvious are changes in size e.g. by fixation as well as changes in the amount of bound stain. Computer-simulated variations in size and pixel magnitude of a set of images of cell nuclei stained with Feulgen were featured and compared. Resulting feature dependencies are used for the setting of parameters of feature extraction procedures as well as for the selection of invariant features for texture quantification of material with variations examined.

[Problems concerning standardization and quality assurance in DNA cytometry]. / Fragen der Standardisierung und Qualitätssicherung in der DNA-Zytometrie.

Rat liver imprints were treated with five different fixation techniques. Chicken erythrocytes stored over an extended period in the refrigerator were dropped on each slide before Feulgen staining. By means of an image analysis system chicken erythrocytes, rat leukocytes and hepatocytes were measured and the integrated optical density (IOD) was calculated for each nucleus. The coefficient of variation (CV) of IOD was about 15% for chicken erythrocytes. Leukocytes showed a CV of up to 10%. The CV was lower for hepatocytes than for leukocytes, and lowest for air-dried hepatocytes. The standard error of the mean (SEM) did not show remarkable differences between the fixation groups. For hepatocytes it was in general less than 1% of the respective mean value. The hepatocytes showed a linear staining except the wet formalin-fixed cells. This holds also for the wet formalin fixed leukocytes which showed only 85% of the IOD of 2c hepatocytes. The ratios of erythrocytes to 2c hepatocytes varied between 0.33 and 0.35. The IOD ratios of standard cells to 2c hepatocytes from single specimens showed remarkable differences from the above mentioned ratios of the pooled slides of a fixation group. The use of external standard cells was proved to be problematic, especially because of their variation in staining intensity independent of the staining intensity of hepatocytes.

Problems concerning the quality of DNA measurements on Feulgen-stained imprints. A study of five fixation techniques.

Feulgen-stained rat liver imprints were investigated, and hepatocytes, lymphocytes and granulocytes were measured. Additionally, chicken erythrocytes placed on the slides were measured as an external DNA standard. The imprints were treated according to five different fixation protocols. The measured integrated optical density (IOD) was normalized according to the leukocytes and afterwards scaled according to the diploid hepatocytes. The mean IOD, coefficient of variation (CV), standard error of the mean (SEM) and IOD ratios of distinct cell groups were calculated. The CV of the IOD for hepatocytes was slightly better for air-dried preparations. It was larger for leukocytes than for hepatocytes and worst for chicken erythrocytes. The SEM of hepatocytes did not show remarkable differences between the fixation groups; in general it was near 1%. In all cases the IOD ratios of 2c, 4c and 8c hepatocytes reasonably well followed the expected ratio of 1:2:4 except for wet, formalin-fixed hepatocytes (3.8). The ratios of leukocytes to 2c hepatocytes were about 1.0 for air-dried preparations but considerably lower (0.85) for wet fixation in formalin. The IOD ratios of chicken erythrocytes to 2C hepatocytes varied from 0.33 to 0.35. The deviation of the ratios of single specimens from the estimated mean of a fixation group are described.

Modeling, definition and applications of histogram features based on DNA values weighed by sine functions.

OBJECTIVE: To model new DNA histogram features that weigh DNA values with values of curves of a sine function and to show the definition and applications of such features. STUDY DESIGN: A simple example of a sine feature can be modeled to yield the value zero if all cells are diploid or polyploid, with values of 2c, 4c or 8c, and to yield the value 100 if all cells are aneuploid, with DNA values of 3c, 6c or 12c-e.g., cells that are probably from a malignant lesion or indicate proliferation. All other values are multiplied by the corresponding sine value. We folded the logarithmic DNA histogram with a sine curve with positive values only. RESULTS: Correlation with ploidy balance was -0.94, demonstrating the similarity of both features. The sine features, however, avoid cutpoints between diploid and aneuploid values and are therefore less influenced by minor mistakes in standardization of DNA histograms. We introduced deviation factors as variants; that led to higher sine values for higher c values. For breast carcinoma (N = 306) the sine values were spread from very low to very high values, whereas esophageal carcinomas (N = 125) were centered at a sine value of 50. In breast carcinoma the sine features also correlated with prognostic factors, including hormone receptor status. CONCLUSION: Description of DNA histogram features by graphic demonstration of their weight functions improves understanding of features. Since functions respect the cyclic events in proliferation and are not influenced by polyploidization.

Changes in benign cell populations in cases of cervical cancer and its precursors.

In two totally independent experiments, typical intermediate cells were visually selected by experienced cytotechnicians trained in the analysis of monodisperse Papanicolaou-stained ectocervical smears. The smears were carefully diagnosed per se and in correlation with routine smears from the same patients. For specimens of classes exhibiting tumor cells, the diagnosis was histologically verified. About 20 cells per specimen were selected from at least ten specimens per Papanicolaou class, amounting to total sample sizes of about 1,000 cells. In the first experiment, only cell nuclei were SMP scanned; in the second experiment, the whole cells were TV scanned. Correlation and classification analyses were performed; these resulted in the clear demonstration that intermediate cell populations in ectocervical smears are different in patients with different neoplastic gradings.

Groping for quantitative digital 3-D image analysis: an approach to quantitative fluorescence in situ hybridization in thick tissue sections of prostate carcinoma.

In molecular pathology numerical chromosome aberrations have been found to be decisive for the prognosis of malignancy in tumours. The existence of such aberrations can be detected by interphase fluorescence in situ hybridization (FISH). The gain or loss of certain base sequences in the desoxyribonucleic acid (DNA) can be estimated by counting the number of FISH signals per cell nucleus. The quantitative evaluation of such events is a necessary condition for a prospective use in diagnostic pathology. To avoid occlusions of signals, the cell nucleus has to be analyzed in three dimensions. Confocal laser scanning microscopy is the means to obtain series of optical thin sections from fluorescence stained or marked material to fulfill the conditions mentioned above. A graphical user interface (GUI) to a software package for display, inspection, count and (semi-)automatic analysis of 3-D images for pathologists is outlined including the underlying methods of 3-D image interaction and segmentation developed. The preparative methods are briefly described. Main emphasis is given to the methodical questions of computer-aided analysis of large 3-D image data sets for pathologists. Several automated analysis steps can be performed for segmentation and succeeding quantification. However tumour material is in contrast to isolated or cultured cells even for visual inspection, a difficult material. For the present a fully automated digital image analysis of 3-D data is not in sight. A semi-automatic segmentation method is thus presented here.

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%.

Estimation of the performance of an array-processor oriented system for automatic Pap smear analysis.

The rapid progress in computer technology makes possible the automatic analysis of thousands of cells on a slide in the field of automatic uterine cancer cytology. Our approach starts with the high-resolution scanning of visually selected and classified single cells determining the training set for discriminant analysis. On the basis of more than 15 morphologic and textural features measured, correct classification results of 95% are reached. Our aim is to speed up our image processing system by means of an Array-Processor, the model AP 120 B from Floating Point Systems. In this study a realistic time estimation of the scanning, segmentation, feature extraction and classification of about 40,000 cells on a slide is performed.

Correlation between chromatin morphology as derived by digital image analysis and autoradiographic labeling pattern.

In order to interpret the Feulgen-dependent chromatin morphology on a functional basis, we performed model experiments in which labeling with 14C-thymidine and 14C-uridine was used as a functional parameter. Using a relocation facility, information on either DNA or RNA, labeling intensity of a cell was added to the parameters of image analysis by measuring the same cell by scanning photometry after Feulgen staining. The Feulgen-stained nuclei were interactively sampled and automatically segmented. Most of the textural information was gained from a flat texture image obtained by subtracting the original image from a median-filtered image. In addition to the autoradiographic features, visually recognizable differences in nuclear morphology, such as the number of nucleoli and the level of condensed (inactive) and diffuse (active) regions of the chromatin, were also correlated with textural parameters. Using the supervised cluster analysis method, an attempt was made to establish a correlation between visual nuclear morphology and autoradiographic labeling intensity that improved the functional understanding of the Feulgen features. Our results further clarify the supramolecular chromatin structure and its dynamics during specific transitions in the cell cycle, namely the G0-G1, G1-S and S-G2 transitions; this information may become useful in diagnostic procedures.

Identification of a low-risk group of stage I breast cancer patients by cytometrically assessed DNA and nuclear texture parameters.

Image cytometrical measurements were performed on Feulgen-stained cells from 329 stage I breast cancers (pT1pN0,M0,R0). For each patient, several DNA (ploidy, S-phase fraction, exceeding rates, 2c deviation index, ploidy balance, entropy, and histogram typing), morphometric (area and radius of nuclei), and textural parameters (mainly co-occurrence and run-length) were calculated. The prognostic value of these parameters was investigated by multivariate Cox regression analysis, considering a distant recurrence-free survival of 8 years as the prognostic criterion. In the multivariate analysis, one DNA parameter (histogram type) and two textural parameters (co-occurrence and variation of the average heterochromatin area) were proven to have independent prognostic value. Using a linear combination of these variables, a prognostic factor was calculated for each individual patient. Patients were stratified using this factor into several groups according to their risk for distant recurrence. Thus, a low-risk group of stage I patients was identified, remaining distant recurrence-free for 8 years. In addition, a group of patients with a worse prognosis and an 8-year recurrence rate of about 26 per cent was identified, compared with the average distant recurrence rate of all stage I patients of 13 per cent. A combination of DNA and textural parameters can provide powerful prognostic information in stage I breast carcinomas and may allow a better selection of patients for different therapy protocols.

Relevance of chromatin features in the progression of esophageal epithelial severe dysplasia.

Since 1983, a long-term clinical trial of esophageal carcinoma chemoprevention has been conducted in a high-risk area in China. From this study, 25 esophageal severe dysplasia patients without therapy were selected for analysis. After 5-year follow-ups, 14 cases progressed to esophageal carcinoma, while the other 11 cases remained stable. Three Papanicolaou's smears were used for each case, including one from the esophageal cytological examination at the beginning, two from the re-examinations three and five years later respectively. About 100 visually normal intermediate cells were randomly collected per slide by high resolution image analysis. More than 100 features (morphologic, densitometric, textural) were extracted. The classifications were made by means of stepwise linear discriminate analysis at the single cell level as on the specimen level using up to ten features. In all three comparisons of patients with progression and with regression at time of diagnosis, three years after diagnosis and five years later, the correct cell classification rates were about 70%. The subsequent specimen classifications by means of the a posteriori probability (APOP) distribution of the cells in each case led to 80% correct classification. All selected features reflected the chromatin structure of nuclei. The result demonstrated that the chromatin structures of esophageal epithelial cells in severely dysplasic patients are different between cases with and without progression. These results suggest the possibility of the application of image analysis in the clinical trials to find the dysplasia patients with higher risk of progression, in order to reduce the number of patients for therapy.

Canine neuroendocrine tumors of the pancreas: a study using image analysis techniques for the discrimination of metastatic versus nonmetastatic tumors.

Canine pancreatic neuroendocrine tumors were studied using different image analysis techniques (nuclear image histometry, analysis of argyrophilic proteins of nucleolar organizer regions, determination of the mouse anti-Ki 67 antigen proliferation index, and DNA densitometry) to correlate their biological behavior with objective phenotypic markers. The methods were compared to determine the best method for distinguishing between metastatic and nonmetastatic tumors. Discrimination between the two types of tumor was possible using nuclear image histometry in combination with morphometric analysis of argyrophilic proteins of nucleolar organizer regions. In contrast, the mouse anti-Ki 67 antigen proliferation index, DNA measurement, and immunohistochemical parameters revealed no significant difference between the two types of tumors.

Malignancy associated changes in squamous epithelium of the head and neck region.

Subtle cellular changes are known to exist in normal host tissue adjacent to tumours. These are called malignancy associated changes (MAC). To get more insight into the degree of expression and local spread of such changes we performed high resolution image cytometry on visually normal intermediate cell nuclei in smears from patients with laryngeal or pharyngeal squamous cancer. The smears were taken from the tumour surface, from a border region of the tumour and from a distant unsuspicious buccal site. In addition buccal smears from healthy control persons were examined. In a pilot study smears from 12 cancer patients and 11 control persons and in a succeeding validation study 63 controls, 18 non-tumour patients and 25 cancer patients were investigated. In both studies the occurrence of MACs was demonstrated quantitatively. In cancer patients normal appearing intermediate cells from the three different sampling sites could be discriminated with 65% in the pilot study and with 53% correct classification in the validation study. In addition the influences of smoking behaviour and sex were investigated in the control group. Only in the latter case there was a significant difference between female and male with a 63% correct cell and 71% correct specimen classification.

Quantitative evaluation of radiation-induced changes in sperm morphology and chromatin distribution.

Sperm head cytometry provides a useful assay for the detection of radiation-induced damage in mouse germ cells. Exposure of the gonads to radiation is known to lead to an increase of diploid and higher polyploid sperm and of sperm with head shape abnormalities. In the pilot studies reported here quantitative analysis of the total DNA content, the morphology, and the chromatin distribution of mouse sperm was performed. The goal was to evaluate the discriminative power of features derived by high resolution image cytometry in distinguishing sperm of control and irradiated mice. Our results suggest that besides the induction of the above mentioned variations in DNA content and shape of sperm head, changes of the nonhomogeneous chromatin distribution within the sperm may also be used to quantify the radiation effect on sperm cells. Whereas the chromatin distribution features show larger variations for sperm 21 days after exposure (dpr), the shape parameters seem to be more important to discriminate sperm 35 dpr. This may be explained by differentiation processes, which take place in different stages during mouse spermatogenesis.