Sum variance--a most valuable parameter in computer-assisted image analysis of voided urines.

The chromatin pattern of retrospectively Feulgen-Schiff-stained exfoliative urothelial cells was determined by computer-assisted analysis for 12 detailed texture image parameters. These were compared with density parameters and geometric parameters for their ability to discriminate between benign and malignant samples. The discriminatory power of the mean variable of the texture parameter sum variance was found to equal that of densitometric parameters which reflect the nuclear DNA content of the cell. The results suggest that determination of the texture parameter sum variance is valuable in distinguishing nuclei of specimens negative from those positive for malignancy.

Individual use of cytomorphologic characteristics in the diagnosis of endocervical columnar cell abnormalities: selection of preferred features with help of the 'Navigator' microscope.

In our previous interobserver studies on endocervical columnar cell abnormalities, we studied architectural, cellular and nuclear features in cervical smears of women known to have columnar cell atypias of variable severity, to determine cytomorphologic criteria, discriminating between mild, moderate and severe intraepithelial columnar cell lesions and adenocarcinoma. The results of these studies revealed a number of architectural, cellular and nuclear characteristics in different grades of expression, which were of importance for the primary diagnosis of: no abnormalities, different grades of intraepithelial endocervical columnar cell lesions and adenocarcinoma. Furthermore we concluded that observers used different characteristics and different grades of expression of these characteristics for comparable diagnoses. The present study was undertaken to determine those features, which were considered discriminating by each individual for the diagnosis of mild, moderate and severe atypia, adenocarcinoma in situ and adenocarcinoma. Features selected by five observers with knowledge of the final diagnosis, were stored and reviewed with help of a motor driven stage ('Navigator')-microscope and a high definition television-monitor. The results confirmed individual observer variability in the number and type of features used in the diagnosis of endocervical columnar cell abnormalities. Features such as 'variation in nuclear size and shape', 'irregular chromatin distribution' and 'coarsely granular chromatin' were selected preferentially by all observers in the diagnosis of endocervical columnar cell lesions, conversely striking differences were observed in the application of 'architectural'-and, especially in cases of 'adenocarcinoma', 'nucleolar' characteristics.

Endocervical columnar cell intraepithelial neoplasia (ECCIN). 3. Interobserver variability in feature use.

In our previous studies on endocervical columnar cell abnormalities, the ability of 5 cytotechnicians was tested to distinguish between cases of: no abnormalities, different grades of intraepithelial endocervical columnar cell atypias and invasive adenocarcinoma. On the basis of stepwise multiple regression analysis, nuclear chromatin distribution, variation in cellular and nuclear size, cytoplasmic eosinophilia and architectural features such as cell-crowding, cluster formation, formation of gland-like structures and pseudostratification appeared to be of primary diagnostic importance to discriminate between no abnormalities, different grades of cervical columnar cell atypias, adenocarcinoma in situ and adenocarcinoma. The present paper reports the results of a study which was designed to assess the individual performance in feature use and classification of endocervical lesions. The results of this study indicate that observers use different characteristics and different grading for the level of expression of these characteristics. However, all observers illustrated a strong relationship between the presence and the expression grade of a certain feature and the degree of endocervical columnar cell atypia. With acceptance of one grade of difference in grades of severity, an overall correct diagnosis was made in 87.6% of cases (range 80-98.2%).

Practical aspects of fixatives in high resolution nuclear image analysis.

Three different methods of fixation (ethanol/Carbowax, formaldehyde, and Carnoy) and four different protocols (without Böhm post-fixation on an uncoated slide, and with Böhm post-fixation on Poly-L-Lysine coated slide, an uncoated slide and a previously Papanicolaou stained slide) were evaluated for their application in high resolution image analysis of Feulgen stained nuclei. The aim of the study was to find a combination with the best reproducibility and the least variance under normal laboratory conditions. Care was taken not to exclude any "normal" laboratory variability. The combinations were evaluated for densitometric, geometric, as well as texture features. Selected features were determined on a CAS-100 using the Cell Measurement Program (Cell Analysis Systems, Inc. Lombard, IL). Diploid and tetraploid rat liver nuclei were used. Ethanol/Carbowax fixation-with Böhm post-fixation proved most stable. This fixation method also gave feature values for previously Papanicolaou stained slides that were comparable to direct Feulgen stained nuclei. Acceptable results were achieved with all three fixatives and the various combinations if one adhered strictly to protocol. In routine practice this usually does not happen. Therefore Ethanol/Carbowax fixation with Böhm post-fixation was considered most suited for routine determination of feature values on the CAS-100.

Emergence of alpha 5 beta 1 fibronectin- and alpha v beta 3 vitronectin-receptor expression in melanocytic tumour progression.

Cell adhesion is crucial in the process of tumour progression. As integrins are important receptor molecules involved in cell adhesion, we studied the distribution of the alpha 1-6, alpha v, alpha IIb, beta 1, beta 3, and beta 4 integrin subunits in tissue sections of common naevocellular naevi (n = 22), dysplastic naevi (16), thin (24) and thick primary cutaneous melanomas (28), and melanoma metastases (25). We found correlated expression of alpha 1/alpha 2, of alpha 4/alpha 5/beta 3, and of alpha 6/beta 4. Decrease of alpha 6 and beta 4, and increase of alpha 4 and alpha v were found to be correlated with melanoma progression. Furthermore, expression of alpha 5 and beta 3 was detected only in primary melanoma and melanoma metastasis. Our findings indicate that during melanoma progression alterations in integrin expression occur, the most striking being emergence of alpha 5 beta 1 fibronectin and alpha v beta 3 vitonectin receptor.

DNA changes in progressive cervical intraepithelial neoplasia.

Cervical intra-epithelial neoplasm (CIN) is treated as a progressive lesion, even though most CIN will not progress to invasive cancer if left untreated. This study focussed on DNA-cytometric analysis of cytologic smears of patients who had developed invasive cancer after initial smears showing CIN. The first part of the study aimed at describing the DNA-cytometric changes in these progressive ('malignant') CIN lesions. In the second part a cluster analysis was performed on 'malignant' CIN III lesions and CIN III lesions, with 'unknown' malignant potential. The results indicated that 'malignant' CIN lesions developed high DNA-index (DI) values during malignant transformation, as demonstrated by increasing mean DI values, a high percentage of DNA-aneuploidy and 2.5c Exceeding Rates. Furthermore, a trend-like pattern of texture feature values occurred in 'malignant' CIN lesions with increasing severity. These findings provide objective quantitative confirmation of the evolution of nuclear changes during malignant transformation. Cluster analysis showed that it was possible, using a set of four cytometric features, to subdivide the 'unknown' CIN III lesions into a cluster of lesions with feature values similar to the vast majority of the 'malignant' CIN III lesions, and a second cluster of lesions with feature values dissimilar to 'malignant' CIN III. It is argued that the profile of 'malignant' CIN has become clearer and that the results of this study may serve as a basis for a more objective cytopathologic subdivision of premalignant CIN. It may be justified to follow up patients whose lesions do not yet fit this 'malignant' profile. Not treating the non-progressive lesion group will avoid putting these patients at risk.

Cytophotometric analysis of corresponding cytological and histological cervical intraepithelial neoplasia grade III specimens.

Cytophotometric analysis of cervical intraepithelial neoplasia grade III (CIN III) was performed in 22 cytological smears (CS) and in 22 corresponding cytospin specimens retrieved from selected areas of paraffin-embedded tissues (PEC). The average time interval between cytological and histological diagnosis was 6 weeks. CIN III nuclei in CS and PEC specimen were Thionin-Feulgen stained and digitized. Beside the visual classification of DNA ploidy patterns, the 2.5c and 5c exceeding rates and the specimen mean and standard deviation values of 21 photometric features were also analyzed. It was shown that, although there was a significant correlation between DNA ploidy patterns in corresponding PEC and CS specimen, the DNA patterns were dissimilar in eight of 22 cases. The DNA index, as represented by 2.5c and 5c exceeding rates, was significantly higher in the CS specimen. High-resolution cytophotometric analysis of cell nuclei in CS and PEC specimens showed significant differences for a large number of nuclear photometric features. These findings can possibly be explained by differences in selection of CIN III cells from CS and PEC specimens and by differences between fixation procedures as used for the two techniques. It was concluded that cytophotometric data of CS and PEC specimens representing CIN III lesions should not be regarded as interchangeable.

The choice of a balanced allocation method for a clinical trial in otitis media with effusion.

We review the choice of treatment allocation procedures in a specific small clinical trial of the effect of tympanostomy tubes on language development in patients with otitis media with effusion. The options for balanced allocation available at the planning stage are discussed. A clearly favourable method could not be identified. A simulation study was performed, and its results demonstrated least imbalance with the variance method. In practice this method produced considerable imbalance on relevant prognostic factors.

Cytophotometric analysis of cervical intraepithelial neoplasia grade III, with and without synchronous invasive squamous cell carcinoma.

Cytophotometric analysis was performed in nuclei retrieved from paraffin-embedded cervical tissue from 57 cases of CIN III. CIN III lesions of patients without invasive squamous cell carcinoma (N = 37) were regarded to represent a mixture of progressive and nonprogressive lesions. The CIN III lesions of patients with a synchronous invasive squamous cell carcinoma (N = 20) were regarded as representing truly progressive precursor lesions (CIN.INV). Twenty-one photometric features describing geometrical, density, and texture characteristics were extracted from the digitized nuclear images. Statistical analysis of cytophotometric data indicated significant differences between the group of CIN III lesions and CIN.INV lesions. A cluster analysis, using one co-occurrency texture feature (S-HOMOG), one density feature (S-DI), and two geometrical features (S-AREA and M-CIRC), showed that two clusters (C1 and C2) were present in the total group of CIN III and CIN.INV lesions. The vast majority of CIN.INV lesions was member of one and the same cluster C1. The CIN III group appeared to consist of a mixture of two clusters, 54% C1 and 46% C2 lesions. Of patients 45 years or younger, the majority (62%) of CIN III lesions had feature values, corresponding with those of cluster C1, and as such possibly with a potentially progressive course. In patients older than 45 years the percentage of CIN III lesions with C1 feature values was 27%.