Non-tau based neuronal degeneration in Alzheimer's disease -- an immunocytochemical and quantitative study in the supragranular layers of the middle temporal neocortex.

In Alzheimer's disease (AD), cortical neurons develop neurofibrillary tangles (NFTs) consisting of hyperphosphorylated tau. The neurons eventually die. There are some hints that cortical neurons may also degenerate without the development of cytoskeletal changes. We investigated this possibility by comparing changes in APP staining and neuronal size with respect to the presence or absence of hyperphosphorylated tau. Adjacent sections of the medial temporal neocortex (Brodmann's area 22) of 5 male AD patients aged 60-88 years (Braak V-VI) and 5 age-matched male non-demented control subjects were i) stained with a modified Bielschowsky silver method in order to reveal NFTs and 'ghost' tangles, ii) single-stained with anti-APP, and iii) double-labeled with anti-APP and AT8. Anti-APP is directed against the beta-amyloid precursor protein and stains virtually all perikarya and proximal neurites of the cortical neurons. AT8 stains pre-tangles, NFTs and extracellular 'ghost' tangles due to the recognition of hyperphosphorylated tau. The study was focused on the supragranular cortical layers II-III, since these layers can be clearly delineated from the adjacent molecular and granular cell layers. The results showed that i) APP staining intensity in neurons was variable in the AD cortex, being clearly different from the invariably intense neuronal staining in all controls. Reduced cytoplasmic APP staining was observed, particular in small neurons, while lack of anti-APP staining in proximal neurites, too, was associated with AD. In addition, ii) cross-sectional area measurement on anti-APP-stained neurons revealed that in AD, as compared to controls, a clear decrease in the number of mainly large-sized neurons (>150 microm2) was accompanied by a significant increase in the percentage of neurons in the smaller size classes, indicating that many large-sized neurons became smaller in AD. iii) Reduced APP staining and decreased neuronal size were not necessarily associated with the presence or absence of hyperphosphorylated tau in these cells. iv) Twenty-six percent of the neurons contained hyperphosphorylated tau, while the level of NFT-related neuronal loss was low in AD. The present study suggests that non-tau based neuronal degeneration is a major phenomenon in the AD neocortex.

Distribution of nuclear size and internuclear distance are important criteria for grading astrocytomas.

AIM: The differentiation between low-grade astrocytomas and anaplastic astrocytomas is susceptible to considerable inter-observer variability. In order to contribute to a better standardization of astrocytoma-grading based on quantitative data, the present study focuses on two important aspects not being considered in previous morphometric studies: elaboration of a decision flow chart for tumor grading based on morphometric parameters and appropriate cut-off-values, easily performed using low-cost equipment such as measuring oculars; investigation of the distribution (histograms) of parameters describing nuclear size and internuclear distance, which had been represented in previous studies by their mean and standard deviation only. MATERIAL AND METHODS: At least 300 tumor cell nuclei per case were investigated in paraffin sections from surgical specimen of 75 patients with astrocytomas WHO grade II (n = 23) and anaplastic astrocytomas WHO grade III (n = 52) by means of a digital image analysis system. RESULTS: The morphometric data showed significant differences between both groups of tumors. According to multivariate analysis, the best contribution to tumor grading was achieved by means of parameters concerning the distribution of values for nuclear diameters and internuclear distances. A decision tree was constructed using a knowledge based algorithm, which provided astrocytoma grading based on the distribution of values for nuclear diameter, as well as the numerical nuclear density and proliferation index. Measurements using a measuring ocular took an acceptable amount of time (1.5 hour per case) and showed good reproducibility when compared with measurement by means of digital image analysis. CONCLUSION: The study demonstrates that a morphometric examination of tumor cell nuclei in paraffin sections supports the clinically important differential diagnosis between low-grade and high-grade astrocytomas. The method for classification and the data published in the present study constitute a good basis for a standardized and reproducible grading procedure for astrocytomas, which can be performed in any histologic laboratory even without a digital image analysis system.

Beta-protein/A4 deposits are not associated with hyperphosphorylated tau in somatostatin neurons in the hypothalamus of Alzheimer's disease patients.

With respect to the pathogenesis of Alzheimer's disease (AD), it has been hypothesized that amorphous plaques containing beta-protein/A4 (Abeta) would locally induce cytoskeletal changes, and that neurons affected by neurofibrillary tangles (NFTs) lose their neuropeptide concentration and eventually die. To test this presumed cascade of events, the hypothalami of 14 non-demented subjects (Braak 0-III) and 28 AD patients (Braak IV-VI) aged 40-98 years were selected. The subject of our study was the nucleus tuberalis lateralis (NTL), which harbors a subpopulation of somatostatinergic neurons with extensive intrinsic interconnectivity. We used Gallyas silver staining, Congo staining, single- and double-staining with monoclonal antibody AT8 and polyclonal antibody anti-Abeta, and double-immunolabeling with AT8 and anti-somatostatin(1-12) with the following results: (1) Significant amounts of silver-staining NFTs were present in only three AD patients. (2) High densities of AT8-stained cytoskeletal changes were mainly found in aged, demented patients. (3) In contrast, large amounts of Abeta deposits were mainly observed in young and middle-aged (40-59 years) AD patients, and were very low or absent mainly in the older non-demented subjects and in AD patients. (4) Reduced anti-somatostatin staining was observed in the NTL of most AD patients, but anti-somatostatin/AT8 double-stained neurons were found virtually exclusively in aged AD patients. Thus, the occurrence of Abeta deposits and hyperphosphorylated tau formation in somatostatin cells are basically independent events, while decreased somatostatin staining only partly goes together with cytoskeletal changes in somatostatin cells in the NTL of AD patients. These observations cannot be explained by the amyloid cascade hypothesis.

Histomorphometry of tumour cell nuclei in astrocytomas using shape analysis, densitometry and topometric analysis.

Although tumour cell nuclei are important histological structures for grading of astrocytomas according to the WHO-classification of brain tumours, there is no reported morphometric study of astrocytomas which describes quantitatively the four main morphologic criteria of tumour cell nuclei: size, shape, texture (densitometric characteristics) and spatial relationships between the nuclei (topometric analysis). Using a set of morphometric parameters describing these criteria as well as the Ki67-proliferation index, 74 astrocytomas from 74 patients were studied by means of a digital image analysis system. The objective of the study was to test, if these morphometric parameters were sufficient for statistical discrimination between pilocytic astrocytomas WHO-grade I, astrocytomas grade II and anaplastic astrocytomas grade III. Our results showed a correct reclassification of 97.3% (72/74) of the cases with respect to the tumour grade by means of cross-validated discriminant analysis. Morphometric parameters characterizing nuclear shape (shape factors, Fourier-amplitudes) showed the most prominent differences between the three groups of cases, followed by topometric parameters (number of neighbours per nucleus, distances between the nuclei). Less pronounced differences between the tumour grades were found for parameters characterizing nuclear size, nuclear texture and the Ki67-proliferation index. In conclusion, the present morphometric procedure provided good discrimination between the tumour grades, supporting the view that histomorphometry of tumour cell nuclei could be a valuable tool for grading of astrocytomas.

Histomorphometry of brain tumours.

In this review, the results of previous histomorphometric studies of brain tumours are summarized and discussed with respect to their potential value for diagnostic purposes and for tumour research. In the majority of these studies, human gliomas were investigated. In a few studies, human meningiomas and other human or experimental tumour types were investigated. A computerized image analysis system was used for the morphometric analyses in most studies. The three main histologic structures examined were tumour cell nuclei, nucleolar organizer regions and tumour vessels. The current state of knowledge provides evidence that a diagnostic benefit could be provided by histomorphometric investigations of brain tumours, especially for grading of gliomas and with respect to independent prognostic information. Additional studies are necessary to delineate the spectrum of histomorphometric parameters and the investigation of their prognostic significance for cases with the same tumour type and tumour grade. Together with many recently published observations in this field, this review shows that histomorphometry is an important approach towards the investigation of brain tumour biology.

Quantitative investigation of nuclear morphology in glioblastomas and its relation to survival time.

OBJECTIVE: To investigate the influence of nuclear morphology of tumor cells on survival time of patients with primary glioblastomas. STUDY DESIGN: Tumor cell nuclei have been measured in paraffin sections from 51 glioblastomas (Ki-67 immunostaining). In each tumor, the region with the highest proliferative activity has been selected for performing the morphometric analysis. Nuclear area, shape variables (roundness factor, Fourier amplitudes) and the proliferation index Ki-67 have been determined. Statistical relationships between these variables have been tested by principal component analysis and Spearman's correlation analysis. The survival time has been tested using Kaplan-Meier analysis. The influence of morphometric variables on survival time and on the time until recurrence has been tested by Cox analysis. RESULTS: There is a significant correlation of mean value and standard deviation of nuclear area with shape variables. Cox analysis showed a significant influence of the quantitative morphologic variables on survival time for patients with and without complete surgical resection. For patients with complete surgical resection, there was a distinct influence of mean nuclear area on survival time. Patients with incomplete surgical resection of the tumors had a significantly shorter survival. No significant influence of the variables on the time from surgery to recurrence could be shown. CONCLUSION: Quantitative morphology of tumor cell nuclei in our set of glioblastoma cases showed a statistically significant relation with the survival time of the patients, indicating the biological significance of nuclear morphology in glioblastomas.

Glioblastoma multiforme of the brain stem in a patient with acquired immunodeficiency syndrome.

Glioblastoma of the brain stem is rare and there is no description of such a lesion in patients suffering from acquired immunodeficiency syndrome. The majority of intracerebral mass lesions are due either to toxoplasmosis or primary central nervous system lymphomas so that it is usually not included in the differential diagnosis of enhancing lesions of the central nervous system in these patients. A 31-year-old human immunodeficiency virus (HIV) infected man presented with a four months history of slowly progressive deterioration of brainstem associated symptoms despite antitoxoplasmic therapy. Magnetic resonance imaging revealed a large ring enhancing lesion in the brainstem. Clinical and neuroradiological data could not establish a proper diagnosis and a stereotactic serial biopsy was undertaken. Histological examination of the specimen showed a glioblastoma multiforme (GBM) as the first reported case of GBM located in the brainstem in an acquired immunodeficiency syndrome (AIDS) patient. Patient management and effectiveness of stereotactic serial biopsy are discussed.

The giant axonal neuropathy--clinical and hisotological aspects, differential diagnosis and a new case.

The giant axonal neuropathy (GAN) is morphologically characterized by axonal swellings and accumulations of neurofilaments in giant axons and other cell types. Curly hair is not a constant finding. The clinical course is progressive and mostly starts in early childhood. We report the case of a boy aged 6 years at the time of sural nerve and muscle biopsy. Suralis nerve showed a reduced numerical density of myelinated fibres with a consecutive endoneural fibrosis. Morphometric investigation revealed a pronounced reduction of fibres measuring 8-12 microm in diameter. Giant axons were seen in relatively low number and were not very large with a maximum diameter of 18 microm. They had a relatively thin myelin sheet proved also by the high G ratio in the histogram. Many onion bulb formations of Schwann cells were present. There are only few reports of giant axons with such low maximum diameter in cases with GAN, the lowest maximum diameters being reported in case reports on Japanese children. Up to now, this is the first report of a non-Japanese patient with a low maximum diameter of giant axons of less than 20 microm in peripheral nerve biopsy. Ultrastructurally, typical accumulations of neurofilaments and osmiophilic aggregates were found in giant axons. Other diagnoses with occurrence of giant axons could be excluded in view of the absence of specific findings. Sporadic or familial cases with giant axons are discussed. Sceletal muscle biopsy (M. quadriceps femoris) showed neurogenic affection with presence of small angulated atrophic muscle fibres.

EGFR gene amplification in glioblastomas. Is there a relationship with morphology of tumor cell nuclei and proliferative activity?

OBJECTIVE: To confirm a relationship between histomorphology of glioblastomas and amplification of the gene for the epidermal growth factor receptor (EGFR) as the most important molecular biologic alteration in these tumors. STUDY DESIGN: In paraffin sections of surgical specimens from 71 primary resected glioblastomas, tumor cell nuclei in the region with the highest proliferative activity (Ki-67 immunostaining) were investigated morphometrically. Shape variables (roundness factor, Fourier amplitudes) and nuclear area were measured. Additionally, the numerical density of Ki-67-positive tumor cell nuclei was estimated. Differential polymerase chain reaction (PCR) was performed from paraffin sections of the same tumor area. The signals for the EGFR gene and IFN gamma reference gene were quantified densitometrically. RESULTS: Cases with distinct EGFR gene amplification (EGFR/IFN ratios > 5) revealed significantly lower mean values for several Fourier amplitudes, indicating a more regular nuclear shape when compared with cases without evidence of EGFR gene amplification (EGFR/IFN-ratios < or = 1). The Ki-67 index and nuclear area showed no significant differences between these groups. Although a large variation in nuclear morphology was observed for cases without evidence of EGFR gene amplification, discriminant analysis based on morphometric variables provided a good separation of these cases from cases with distinct EGFR gene amplification, with a high percentage of statistically correct reclassified cases. CONCLUSION: Our results provide evidence of a relationship between genetic alterations and histomorphology of glioblastomas.

Shape analysis of tumor cell nuclei in ependymomas by means of Fourier analysis.

OBJECTIVE: To study the prognostic significance of nuclear shape analysis in ependymomas. STUDY DESIGN: Tumor cell nuclei in surgical specimens of primary resected ependymomas from 30 patients were evaluated by means of Fourier analysis of nuclear contours, conventional morphometric features (nuclear area, shape factor) and the Ki-67 proliferation index. Fourier analysis can be used for decomposing an irregular nuclear contour by calculating "Fourier amplitudes." Tumors with different tumor grades according to the World Health Organization were compared, as were patients with and without recurrence of ependymomas. Planimetric data were further correlated with the Ki-67 index. RESULTS: t Test and multivariate analysis showed distinct differences between ependymomas with tumor grade 3 and the other tumor grades. Cross-validated, stepwise discriminant analysis with the event of recurrence as grouping variable revealed correct reclassification in 16/18 cases without recurrence and of 10/12 cases with recurrence. When considering just intracranial ependymomas with total surgical removal, Fourier amplitudes provided 100% correct reclassification concerning recurrence. Proliferation index, in contrast, showed considerable overlap between all tumor grades and between cases with and without recurrence. CONCLUSION: Quantification of the shape of tumor cell nuclei in ependymomas by means of Fourier analysis has prognostic significance and seems to be superior to the Ki-67 index.

Dietary L-arginine decreases myointimal cell proliferation and vascular monocyte accumulation in cholesterol-fed rabbits.

L-arginine, the precursor of endogenous nitric oxide (NO), has been shown to enhance endothelial function and to reduce the progression of atherosclerosis in cholesterol-fed rabbits. In the present study, we investigated whether myointimal cell proliferation is enhanced in hypercholesterolaemic rabbit aorta and whether chronic treatment of the rabbits with L-arginine or with the NO synthase inhibitor L-NAME influences this proliferative response and vascular monocyte accumulation. Rabbits were fed 1% cholesterol or normal rabbit chow for 12 weeks. Subgroups of cholesterol-fed rabbits were treated with oral L-arginine (2.25%) or L-NAME (3 mg/dl) in drinking water. Myointimal cell proliferation was quantified in aortic segments by immunohistochemical detection of bromodeoxyuridine (BrdU) incorporation into nuclear DNA; vascular monocyte accumulation was assessed by immunohistochemistry using a monoclonal anti-macrophage/monocyte antibody (RAM-11). Plasma levels of L-arginine and the endogenous NO synthase inhibitor, ADMA, were quantified by high-performance liquid chromatography (HPLC). Cholesterol feeding increased the aortic intima/media (I/M) ratio, which was not measurable in the control group, to 1.9 +/- 0.3. This was paralleled by enhanced cell proliferation (cholesterol, 2.4 +/- 0.2%; P < 0.05; control, 0.02 +/- 0.001% BrdU-positive cells per 72 h) and vascular monocyte accumulation. Double immunostaining for BrdU and alpha-actin showed that about two thirds of the proliferating cells were smooth muscle cells. ADMA levels increased from 0.8 +/- 0.1 micromol/l to 2.2 +/- 0.2 micromol/l in cholesterol-fed rabbits, but were unchanged by L-arginine or L-NAME treatment. Myointimal proliferation and intima/media ratios were correlated with ADMA plasma levels. Dietary L-arginine reduced monocyte accumulation by 85 +/- 2% (P < 0.05 vs cholesterol), myointimal cell proliferation (1.8 +/- 0.3% per 72 h; P < 0.05) and intimal thickening (I/M ratio: 0.7 +/- 0.2), whereas the inhibitor of NO synthase, L-NAME, further increased cell proliferation to 3.1 +/- 0.4% per 72 h (P < 0.05). No significant difference was observed in vascular monocyte infiltration between the cholesterol and L-NAME groups. We conclude that cell proliferation and vascular monocyte accumulation are enhanced in hypercholesterolaemic rabbit aorta. These atherogenic effects can be attenuated by dietary L-arginine. Decreased NO formation might underlie the enhanced monocyte accumulation and cell proliferation in hypercholesterolaemic rabbit aorta. The observed inhibition of cell proliferation adds to our understanding of the antiatherosclerotic effects of L-arginine in vivo.

Dietary L-arginine reduces the progression of atherosclerosis in cholesterol-fed rabbits: comparison with lovastatin.

BACKGROUND: We investigated whether L-arginine induces regression of preexisting atheromatous lesions and reversal of endothelial dysfunction in hypercholesterolemic rabbits, whether similar effects can be obtained by cholesterol-lowering therapy with lovastatin, and which mechanism leads to these effects. METHODS AND RESULTS: Rabbits were fed 1% cholesterol for 4 weeks and 0.5% cholesterol for an additional 12 weeks. Two groups of cholesterol-fed rabbits were treated with L-arginine (2.0% in drinking water) or lovastatin (10 mg/d) during weeks 5 through 16. Systemic nitric oxide (NO) formation was assessed as the urinary excretion rates of nitrate and cGMP in weekly intervals. Cholesterol feeding progressively reduced urinary nitrate excretion to approximately 40% of baseline (P<.05) and increased plasma concentrations of asymmetrical dimethylarginine (ADMA), an endogenous NO synthesis inhibitor. Dietary L-arginine reversed the reduction in plasma L-arginine/ADMA ratio and partly restored urinary excretion of nitrate and cGMP (each P<.05 vs cholesterol) but did not change plasma cholesterol levels. L-Arginine completely blocked the progression of carotid intimal plaques, reduced aortic intimal thickening, and preserved endothelium-dependent vasodilator function. Lovastatin treatment reduced plasma cholesterol by 32% but did not improve urinary nitrate or cGMP excretion or endothelium-dependent vasodilation. Lovastatin had a weaker inhibitory effect on carotid plaque formation and aortic intimal thickening than L-arginine. L-Arginine inhibited but lovastatin potentiated superoxide radical generation in the atherosclerotic vascular wall. CONCLUSIONS: Dietary L-arginine improves NO-dependent vasodilator function in cholesterol-fed rabbits and completely blocks the progression of plaques via restoration of NO synthase substrate availability and reduction of vascular oxidative stress. Lovastatin treatment has a weaker inhibitory effect on the progression of atherosclerosis and no effect on vascular NO elaboration, which may be due to its stimulatory effect on vascular superoxide radical generation.

p53 immunohistochemistry as an independent prognostic factor for superficial transitional cell carcinoma of the bladder.

Although patients with superficial bladder cancer (Ta, T1) have a generally good prognosis, those patients who develop muscle-invasive tumours or metastatic disease at recurrence do poorly clinically. In the current study 69 patients undergoing complete transurethral resection for superficial transitional cell cancer of the bladder were investigated for different clinical and biological characteristics as possible prognostic factors: age, sex, performance of instillation therapy and immunohistochemical determination of mutational inactivation of p53 tumour-suppressor gene (monoclonal antibody PAb 1801) as well as immunohistochemical determination of the proliferation rate by staining for PCNA (proliferating cell nuclear antigen) (monoclonal antibody PC 10). After a median follow-up of 45.8 months, 12 of 14 patients (85.7%) with more than 20% of cells positive for p53 had disease progression with muscle-invasive growth compared with only one of 55 patients (1.8%) negative for p53 (P < 0.01, chi 2 test). During univariate analysis histological grade (G1 vs G2) (P = 0.0373), positivity for PCNA (> 60% of cells) (P = 0.0033) and positivity for p53 (P < 0.001) were significant prognostic factors for disease progression (log-rank test), while during multivariate analysis only positivity for p53 was a significant predictor for relapse of bladder cancer (P = 0.0029) (multivariate Cox regression analysis). The immunohistochemical detection of mutations of the p53 gene has been demonstrated to be a reliable, easily performed and thereby widely available technique for the investigation of fresh-frozen or paraffin-embedded tumour specimens. The results demonstrate the important role of the p53 tumour-suppressor gene protein in the development and for the progression of bladder cancer. If the high prognostic value of p53 mutations in superficial bladder cancer is confirmed in larger prospective trials, more aggressive therapeutic strategies could be discussed for patients with p53 mutations in their tumour specimens.

[Morphometry of megakaryocytes for supporting the histologic diagnosis of chronic myeloproliferative diseases]. / Morphometrie von Megakaryozyten zur Unterstützung der histologischen Diagnose von chronischen myeloproliferativen Erkrankungen.

Morphometric analysis of sections of biopsy specimens from patients with chronic myeloproliferative disorders (CMPD) can complement the individual histological diagnosis and help to distinguish the four groups of CMPD. A total of 130 diagnostic biopsies from 29 cases of chronic myelocytic leukemia (CML.CT), 26 cases of (CML.MI), 28 of essential thrombocythemia (PTH), 26 cases of chronic megakaryocytic granulocytic myelosis (CMGM), and 21 of polycythemia vera (P. vera), and 30 from healthy control persons were evaluated morphometrically in sections of undecalcified plastic-embedded core biopsies. Clear distinctions were revealed in size of megakaryocytes, nuclear lobulation, clustering, and the nuclear size and shape of megakaryocytes. Nuclear size and cellular size were significantly less in CML (range of means of cellular size: 220-360 microns2) than in the other three Ph1-negative groups (range of means: 480-750 microns2). Nuclear lobulation was more distinct in PTH than in P. vera, and especially in CMGM. Clustering of megakaryocytes was more than twice as frequent in CMGM (8.0-10.5%) as in any of the other three groups (0.1-7.0%). Naked nuclei were more numerous in all groups of CMPD. The main topic of the study is the different size of megakaryocytes in the four main groups of CMPE, allowing a distinction between small-megakaryocytic Ph1-positive CML and large-megakaryocytic Ph1-negative forms of CMPD.

Effect of lazaroid U-74389G on iron-induced reduction of striatal dopamine metabolism.

The substantia nigra of parkinsonian brains is reported to contain increased amounts of iron as compared with age-matched controls. Since iron might be cytotoxic via radical mechanisms, we analyzed the effect of intranigral iron infusion on the dopaminergic activity in the striatum of the rat. The striatal dopamine metabolism of the rat was followed 1, 3, and 6 weeks after unilateral intranigral iron (III) (1.5 micrograms) application. A progressive decrease of extraneuronal 3,4-dihydroxyphenylacetic acid (DOPAC) levels was observed in the ipsilateral striatum by means of in vivo pulse voltammetry. The significant reduction of the DOPAC signal could be attenuated by pretreatment of the animals with the lazaroid U-74389G, applied ip 20 minutes before unilateral intranigral iron application. Our data indicate that a single iron application into the substantia nigra leads to a progressive loss of dopaminergic activity in the striatum, also observed in Parkinson patients. Furthermore, the Lazaroid U-74389G seems to be beneficial in this model of Parkinson's disease.

Comparison of bone marrow and hematologic findings in patients with human immunodeficiency virus infection and those with myelodysplastic syndromes and infectious diseases.

The histologic, hematologic, and morphometric findings of 40 patients positive for the human immunodeficiency virus (HIV) were compared statistically with those of 40 patients with primary myelodysplastic syndromes (MDS) and those of 32 HIV-negative patients with infectious diseases. The severity of anemia and the abnormalities of erythropoiesis in the group of HIV patients were less pronounced than in the group with MDS; megakaryopoiesis showed similarities only with the group of patients with infectious diseases, and characteristics of dysplasia were not observed. Granulopoiesis in MDS showed an increase of blasts in several cases; this was not found in any biopsy specimen from the HIV group. In addition, a statistically significant increase of monocyte-like cells and giant bands could be observed in the bone marrow of the HIV patients. The peripheral blood findings and bone marrow picture in the series of our HIV patients appeared to be related mainly to the influence of opportunistic infections, although a direct effect of the HIV itself could not be excluded.

Histomorphometry in paraffin sections of thyroid tumors.

Planimetric features of cell nuclei in paraffin-embedded histological sections of benign and malignant thyroid tumors, as well as normal thyroid tissue as control, were determined by means of a semiautomatic system. The main aim was to objectify possible quantitative differences between adenomas and carcinomas of the thyroid gland, which had recently been reported by several authors. For each nuclear profile, the area, the maximum diameter as well as two form factors were calculated. Statistical analyses of morphometric differences between normal controls, oxyphilic adenomas and carcinomas, and between follicular adenomas and carcinomas were performed using the T-test, a multivariate test, and a discriminant analysis. The tests revealed significant differences between controls and all other groups. The most striking result, however, was the total discrimination between follicular adenomas and carcinomas, with no false reclassification. Carcinomas had a higher mean nuclear area and diameter and a lower form factor. A similar reliability of discrimination could be obtained by comparing these morphometric values in oxyphilic adenomas and carcinomas. When using a test set of 9 cases (4 adenomas, 5 carcinomas), only one adenoma was falsely reclassified as a carcinoma by the discriminant analysis. Our results thus allow the conclusion that planimetric nuclear measurements indeed seem to be useful for the objectivation of cytomorphologic differences between adenomas and carcinomas of the thyroid gland.

Elliptic Fourier analysis of megakaryocyte nuclei in chronic myeloproliferative disorders.

Elliptic Fourier analysis was applied to megakaryocyte nuclei in bone marrow biopsies from 15 patients with chronic myelocytic leukemia with megakaryocyte predominance and from 15 patients with chronic megakaryocytic granulocytic myelosis. To assess the reliability of this procedure, the biopsies were evaluated also by the semiautomatic measurement of nuclear area and form factor, and both methods were compared with respect to the degree of morphologic differences obtained between these two types of chronic myeloproliferative disorders (CMPDs). Discriminant analysis revealed correct reclassification of all cases both for elliptic Fourier analysis and for semiautomatic planimetry, whereas discriminant scores were much higher for Fourier analysis. Thus, simple planimetric features such as nuclear area and form factor, in contrast to Fourier analysis, are not able to detect the full degree of morphologic differences between megakaryocyte nuclei in different CMPDs. Elliptic Fourier analysis therefore seems to be a useful procedure for the accurate description of such complicated structures as megakaryocyte nuclei in CMPD.

Introduction of a neuronal network as a tool for diagnostic analysis and classification based on experimental pathologic data.

A neuronal network, as well as uni- and multivariate statistics and a discriminant analysis were applied to a morphometric database of 58 cases with thyroid neoplasms and normal thyroid tissue. The ability to classify cases correctly according to their diagnosis was compared between the neuronal network and discriminant analysis. For all pairwise comparisons, classification by neuronal network was as least as good as classification by discriminant analysis. For some comparisons, the neuronal network provided more correct diagnoses than discriminant analysis. On the contrary, in a comparison between tumors which are not significantly different according to multivariate statistics, the network reclassifies only half of the cases correctly, whereas discriminant analysis falsely suggests the possibility of classifying cases with either diagnosis. Our results confirm a higher sensitivity of the neuronal network to the diagnostic information contained in the present morphometric database, and we will therefore use this concept for analysis and diagnostic classification in further morphometric studies.

Analysis of criteria for grading bladder cancer in urine cytological tumor diagnosis by means of an expert system.

An inductive expert system was used for the analysis of criteria for grading bladder carcinoma in urine cytological tumor diagnosis. This analysis seems necessary in order to provide a better standardization of grading and to avoid tumor grades, which are rather inhomogeneous with respect to morphology and prognosis. The analysis of the database by the inductive system shows a considerable variation of the cytomorphology of different bladder carcinomas graded as G2 tumors, whereas G1 and G3 tumors are more homogeneous groups respectively. Especially nuclear morphological criteria are important features for the detection of highly differentiated carcinomas, whereas nucleolar features might be helpful to assess the proliferative nature of the carcinoma. The future goal of avoiding a grading system with prognostically inhomogeneous tumor grades seems possible when using an inductive expert system for consultation.