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The purpose of this study was to investigate clinical and histomorphometric features of cat skin under long-term solar exposure. Ear skin of 34 Domestic Shorthair cats that were chronically exposed to sun was classified as follows: group 0, normal ( n = 13); group 1, initial stage of photodamage (PD) ( n = 10); group 2, advanced stage of PD ( n = 11). Histologic sections were examined independently by 2 pathologists, and epidermal thickness, adnexal unit area, and dermal cellularity were assessed by morphometry. A positive correlation was obtained between age, degree of edema and sclerosis in the upper dermis, telangiectases, squamatization of basal keratinocytes, and epidermis thickness and the degree of PD. The area occupied by adnexal structures in the dermis diminished with increased PD. Dermal sclerosis and edema best separated the 3 groups. The results indicated a high level of skin hypersensitivity to sun rays in cats. The findings may be useful for clinical testing and in general veterinary pathology and dermatology.

To the Editor In their interesting publication Arora et al. [1] postulate that benign thyroid nodules, such as follicular adenomas, particularly the Hurthle cell variant, but not hyperplastic nodules, might have malignant potential. Thus in the thyroid an adenoma–carcinoma sequence was postulated not only for Hurthle cell tumors [2] but also for follicular adenomas, much like that described in colon or salivary glands[3–5]. If this were the case, we would expect, in analogy to colon tumors [3], that (pre)-malignant transformations of nuclei would be more common and accentuated in larger follicular adenomas. Because epigenetic and genetic alterations related to patient prognosis may modify the chromatin texture in routine slides [3, 6–9], we tried to find out whether nuclear texture features would change with increasing size of follicular adenomas. For this purpose, we routinely reanalyzed hematoxylin & eosin-stained paraffin sections from a previous study based on the WHO Histological Classification of Tumors [10, 11]: 11 hyperplastic nodules, 18 follicular adenomas, and 24 microinvasive follicular carcinomas. Analyses were based on digitalized images of 100 randomly captured nuclei [6, 7, 9, 11–13]. We compared texture features derived from the gray value co-occurrence matrix [11–14] and the Minkowski fractal dimension [6, 11] between the three groups and then looked for correlations between texture features and the size of the nodules. Patients of all groups were of similar age with a mean of 46 years (range: 17–88 years; analysis of variance [ANOVA] p [ .0.10). The results are summarized in Table 1. Whereas the chromatin of the hyperplastic nodules is relatively homogeneous (local homogeneity) and ‘‘smooth’’ (angular second moment), its architecture in minimally invasive carcinomas is more complex (fractal dimension), with a more irregular (diagonal moment) and contrasted (entropy) chromatin arrangement. Nuclei of follicular adenomas showed intermediate texture features that were significantly correlated with the size of the nodules. In larger adenomas the entropy values (r = 0.626; p = 0.0054; Fig. 1), the fractal dimension (r = 0.482; p = 0.042; Fig. 2), and the diagonal moment (r = 0.59; p = 0.01) were increased, whereas the second angular moment (r = -0.603; p = 0.008), and local homogeneity (r = -0.627; p = 0.0053; Fig. 3) decreased. Thus the nuclear texture of smaller adenomas was more like that of hyperplastic nodules, and the chromatin of larger adenomas resembled more closely that of minimally invasive carcinomas. Nevertheless there were no significant correlations between texture and the size of hyperplastic nodules. Our results are comparable with the findings in colon tumors [3], where an increasing irregularity and loss of nuclear texture homogeneity during the adenoma–carcinoma progression was described and was supposed to be influenced by accumulated alterations in cancer-associated genes. By analogy, we believe that larger follicular adenomas might have suffered more genetic alterations than smaller ones, thus supporting the hypothesis of Arora et al. [1] positing an adenoma–carcinoma sequence for follicular K. Metze (&) R. C. Ferreira P. S. de Matos Department of Pathology, Faculty of Medicine, University of Campinas, P.O. Box 6111, 13083-887 Campinas, SP, Brazil e-mail: kmetze@fcm.unicamp.br

New insights in prognostic predictions are urgently needed for papillary thyroid carcinoma (PTC). The present study aimed to investigate whether computerized analysis of chromatin texture allows the identification of PTC patients with a poor prognosis.We randomnly selected paraffin-embedded blocks from surgical specimens of 103 classic cases of PTC. During follow-up, 68 of the patients were classified as free of disease, whereas 35 presented with recurrences. Characteristics of chromatin were obtained from digitized images of at least 100 randomly selected tumor nuclei per patient. An independent series of 30 goiters was used to validate our observations.Stage, age and distant metastases were found to serve as independent prognostic factors for survival. In addition, multivariate Cox regression confirmed variable cluster prominence as an independent prognostic factor. By comparing malignant and benign nodules, we found that the PTC lesions presented with higher nuclear perimeters, nuclear areas, Minkowski fractal dimensions, optical densities and nuclear longest chords.From our results we conclude that, in conjunction with clinical and histopathological data, morphometric data may provide relevant prognostic information in PTC patients.

Collagen fibers are an essential component of the dynamic process of scarring, which accompanies various diseases. Scar tissue may reveal different morphologic expressions, such as hypertrophic scars or keloids. Collagen fibers can be visualized by fluorescent light when stained with eosin. Second Harmonic Generation (SHG) creates a non linear signal that occurs only in molecules without inversion symmetry and is particularly strong in the collagen fibers arranged in triple helices. The aim of this study was to describe the methodology for the analysis of the density and texture of collagen in keloids, hypertrophic scars and conventional scars. Samples were examined in the National Institute of Science and Technology on Photonics Applied to Cell Biology (INFABIC) at the State University of Campinas. The images were acquired in a multiphoton microscopy LSM 780-NLO Zeiss 40X. Both signals, two-photon fluorescence (TPEF) and SHG, were excited by a Mai-Tai Ti:Sapphire laser at 940 nm. We used a LP490/SP485 NDD filter for SHG, and a BP565-610 NDD filter for fluorescence In each case, ten images were acquired serially (512×512 μm) in Z-stack and joined together to one patchwork-image . Image analysis was performed by a gliding-box-system with in-house made software. Keloids, hypertrophic scars and normal scar tissue show different collagen architecture. Inside an individual case differences of the scar process may be found between central and peripheral parts. In summary, the use of nonlinear optics is a helpful tool for the study of scars tissue.

Elastic and collagen fibers are essential components of the aorta, the remodeling of these structures is accompanied with aging in various diseases and life-threatening events. While the elastic fibers confer resilience to major blood vessels collagen confers resistance to the same. Elastic fibers are easily visualized in the fluorescent light when stained with hematoxylin eosin. Second Harmonic Generation (SHG) is a non linear signal that occurs only in molecules without inversion symmetry and is particularly strong in the collagen fibers arranged in triple helices. The aim of this paper is to describe the distribution of collagen in the thickness of the thoracic aorta, and to demonstrate the distribution of between elastic fibers. The images were acquired in a multifoton microscopy and both signals, Two-phtoton excitaded fluorescence (TPEF) and SHG, were excited by a Ti:Sapphire laser. We used a band pass filter to filter the SHG signal from the TPEF signal. The thickness of the aorta varies 2-3 mm, and the image was composed of the juxtaposition of images of 220 x 220 microns. We acquired images of a histological slide of the thoracic aorta stained with picrosirius red (specific for collagen) at a wavelength of 670nm SHG subsequently acquired images with the same region and observed that the images are overlapping. Therefore, the following images were acquired by confocal microscopy (fluorescence of eosin for visualization of elastic fibers) and for collagen SHG. After reconstruction of the images, we observed the distribution of collagen along the aorta.

Background Prognostic factors in malignant melanoma are currently based on clinical data and morphologic examination. Other prognostic features, however, which are not yet used in daily practice, might add important information and thus improve prognosis, treatment, and survival. Therefore a search for new markers is desirable. Previous studies have demonstrated that fractal characteristics of nuclear chromatin are of prognostic importance in neoplasias. We have therefore investigated whether the fractal dimension of nuclear chromatin measured in routine histological preparations of malignant melanomas could be a prognostic factor for survival. Methods We examined 71 primary superficial spreading cutaneous melanoma specimens (thickness ≥ 1 mm) from patients with a minimum follow up of 5 years. Nuclear area, form factor and fractal dimension of chromatin texture were obtained from digitalized images of hematoxylin-eosin stained tissue micro array sections. Clark's level, tumor thickness and mitotic rate were also determined. Results The median follow-up was 104 months. Tumor thickness, Clark's level, mitotic rate, nuclear area and fractal dimension were significant risk factors in univariate Cox regressions. In the multivariate Cox regression, stratified for the presence or absence of metastases at diagnosis, only the Clark level and fractal dimension of the nuclear chromatin were included as independent prognostic factors in the final regression model. Conclusion In general, a more aggressive behaviour is usually found in genetically unstable neoplasias with a higher number of genetic or epigenetic changes, which on the other hand, provoke a more complex chromatin rearrangement. The increased nuclear fractal dimension found in the more aggressive melanomas is the mathematical equivalent of a higher complexity of the chromatin architecture. So, there is strong evidence that the fractal dimension of the nuclear chromatin texture is a new and promising variable in prognostic models of malignant melanomas.

Background Th e digitalization of histologic or cytologic slides followed by computerized image analysis is becoming more popular due to technical advances in virtual microscopy and telemedicine. Because of the high information content of a digitalized slide, the application of compression methods is often a necessary step in order to reduce storage or bandwith requirements, but image compression is usually accompanied by a loss of information. Our aim was to investigate to which extent image compression according to the JPEG2000 standard is provoking alterations of image texture features.

OBJETIVO E MÉTODOS: Com o propósito de investigar a contribuição do exame da cromatina nuclear no diagnóstico diferencial das lesões foliculares da glândula tireoide, foram estudadas 76 amostras previamente submetidas à análise de expressão proteica de HBME-1, CK-19 e galectina-3. RESULTADOS: HBME-1 confirmou-se como o mais sensível marcador imunoistoquímico de malignidade. Uma série de variáveis morfométricas, densitométricas e de textura foram úteis na distinção entre os diferentes tipos de lesão folicular. Entre essas variáveis, o r², parâmetro relacionado à granularidade do núcleo, apresentou a melhor acurácia, sensibilidade, especificidade, valor preditivo positivo e negativo, diferenciando lesões benignas de malignas. CONCLUSÃO: A morfometria analítica de imagem da cromatina nuclear pode acrescentar acurácia ao diagnóstico diferencial das lesões de padrão folicular.

Background: The established pathologic criteria for minor salivary gland (MSG) involvement in chronic graft-vs.-host disease (cGVHD) could play a role in monitoring response to therapy. Methods: We evaluated MSG sequential biopsies during cGVHD therapy in 14 allogeneic bone marrow transplantation (BMT) patients. Nine patients that did not develop GVHD after BMT entered the control group. Biopsies were examined using hematoxylin-eosin, Periodic acid-Schiff (PAS) and leukocyte common antigen staining. Results: A significant loss of PAS+ acinar volume was observed at the diagnosis of cGVHD as much as at the end of treatment when compared with the control group. In the second evaluation, the inflammatory infiltrate was still greater than control group. Conclusions: The results suggest that persistent xerostomia after cGVHD treatment is because of maintenance of lymphocytic infiltrate and consequent absence of MSG secretory unit recovery. This data may be useful to provide improved insight into the histopathology of this organ involvement.

There is no consensus for grading when more than one grade is present in bladder carcinoma. We propose a grading system that considers the primary (most common) and secondary (second most common) grade of bladder cancer. Grade was correlated with stage of the tumors.We studied 293 bladder transurethral resections or radical cystectomies. Grade was considered as 1, 2 or 3 according to the 1999 World Health Organization system. The number was repeated when only one grade was seen. A final score was obtained which ranged from 2 to 6. All cases were also graded according to the highest grade area even if it was focal.According to the highest grade area, the distribution was 80 (74.07%), 27 (25.00%) and 1 (0.92%) for grade 1; 31 (24.03%), 69 (53.48%) and 29 (22.48%) for grade 2; and 0 (0%), 17 (30.35%) and 39 (69.64%) for grade 3, corresponding to the stages Ta, T1 and T2-T3, respectively. Using the system of combined numbers, grade 2 was stratified into subgroups 1 + 2 and 2 + 2 which are statistically different (p0.05) when considering stage. In grade 3, there was also a trend for statistical difference (p = 0.066) between grades 2 + 3 and 3 + 3.The grading system of combined numbers, stratifies grade 2 into subgroups 1 + 2 and 2 + 2, and grade 3 into subgroups 2 + 3 and 3 + 3 which are statistically different when considering stage. This grading system of combined numbers takes into consideration tumor heterogeneity and may be of value in prospective studies for analysis of prognosis and therapeutic response.

In their interesting contribution, Wang et al. (1) described an automated system for the diagnosis of follicular thyroid lesions, which was based on the analysis of nuclear texture features in histologic slides. According to the authors, the method was able to classify with 100% accuracy between normal thyroid tissue, follicular adenomas, and follicular carcinomas. However, the system had been trained with digitalized images of only few neoplasias, five follicular adenomas (mean lesion size of 4.8 cm), and five follicular carcinomas (a mean lesion size of 2.8 cm) as well as with images of nontumoral tissue. Recently, it had been demonstrated that in follicular adenomas the chromatin texture was size dependent, when analyzing digitalized, gray-value transformed images of nuclei in routinely hematoxylin-eosin (HE)-stained paraffin sections. In nuclei of larger adenomas, the entropy values, the fractal dimension, and the diagonal moment were significantly increased, whereas the values of the second angular moment and local homogeneity were decreased, when compared with the nuclei of smaller adenomas (2). Therefore, we would like to ask whether the considerable size differences between follicular adenomas and carcinomas in the study of Wang et al. (1) might have contributed to the extraordinary classification success claimed by the authors. To test our hypothesis, we made the following simulation study based on adenomas and carcinomas from previous studies on thyroid neoplasias in our laboratory (2,3,4). From each tumor, digitalized images of 100 randomly selected nuclei had been captured in bitmap format from routinely HE-stained 5-μm thick histologic sections (2,3). The nuclei were interactively segmented and then converted to an eight-bit gray scale by calculating the luminance. The material comprised 18 follicular adenomas and 24 microinvasive follicular carcinomas. The final diagnosis had always been done by a meticulous examination of serial step sections. From this collection, we choose a combination of eight folicular carcinomas, so that their mean diameter of 2.78 cm had approximated well with that of the carcinomas from the study of Wang and coworkers (2.8 cm) (1). Then we selected with the help of random numbers, 100 sets of eight follicular adenomas each. The mean lesion size of these random sets varied between 1.98 and 5.21 cm. If our previously mentioned hypothesis was correct, then the ability to discriminate between nuclei of adenomas and carcinomas should vary with changing diameters of the adenoma cases. We calculated nuclear texture features using variables of the gray-level co-occurrence matrix (second angular moment, entropy, energy, peak prominence, standard deviation, cluster shade, and cluster prominence) as well as Shannon's entropy of the gray level histogram and the fractal dimension according to Sarkar. All these variables had shown to be useful for texture analysis in previous studies (1,4–10). In a further step, we created 100 pairs composed of (a) always the aforementioned group of eight carcinomas with a mean diameter of 2.78 cm and (b) one of the 100 randomly choosen adenoma sets with, of course, varying mean lesion size (see Supporting Information). For each pair, we compared the nine texture features with the help of the t-test (P < 0.05 for significant differences) and recorded, for each comparison, the percentage of statistically significant texture features by plotting these data versus the mean diameter of the adenoma set in a diagram (Fig. 1). Figure 1 Box and Whiskers diagram showing relationship between mean lesion diameter of the adenoma group and the number of nuclear texture features separating significantly adenomas and carcinomas. Nine texture variables had been tested in each case. The number ... As we can easily notice, in very large adenomas nearly all nuclear texture features were significantly different from those of the carcinoma group. But these differences were rapidly vanishing, when we compared the carcinomas with smaller adenomas. With a mean adenoma lesion size of 4.8 cm, as in the study under discussion (1), approximately 80% of the texture parameters showed significant differences. However, when the mean diameters of adenomas and carcinomas were very similar (∼2.8 cm), this number had dropped below 20%. There was a highly significant direct correlation (Spearman's correlation coefficient r = 0.72; P < 0.0001) between the mean lesion size of the adenoma cases and the percentage of significant different texture features between adenomas and carcinomas. We believe that the extraordinary good discrimination results reported in the study of Wang et al. (1) might largely be based on this effect and would have been worse, if smaller adenomas had been included in the investigation. Therefore, the data presented in the study of Wang et al. (1) are not able to test the quality of the program. To get a realistic view of the diagnostic power of the proposed automated system, the authors should repeat the evaluation with a considerably larger data set, after increasing the number of adenomas and carcinomas, both comprising a large spectrum of tumor diameters.

Sir,In his interesting review article in Pathology, Tadrous1 comments on the use of the term ‘objective’ and addresses several technical and conceptual problems in image analysis. We totally agree ...