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Real-Time Imaging 10 (2004) 197204 www.elsevier.com/locate/rti

Nuclear morphometry of neoplastic cells as a method for diagnosis of histiocytoma, mastocytoma and transmissible venereal tumor in dogs
tia Cristina de Andrade Waldemarina, Marcelo Em lio Belettib,, Luciano da Ka Fontoura Costac
a

ria, Universidade Federal de Uberla ndia, Uberla ndia, Brazil Faculdade de Medicina Veterina b ncias Biome dicas, Universidade Federal de Uberla ndia, Uberla ndia, Brazil Instituto de Cie c sica de Sa Instituito de F o Carlos, Universidade de Sa o Paulo, Sa o Carlos, Brazil

Abstract Image analysis has been used in medicine and veterinary science as an increasingly important auxiliary tool for histopathology evaluation, because of its potential for decreasing the intrinsic subjectivity of the human visual analysis. However, the geometrical features currently used are often inefcient, not always leading to correct diagnosis. The present work has as its main objective to suggest several approaches to geometric and texture characterization of the cell nuclei, characterized by low computational cost, rapid analysis and sound biological interpretation and good agreement with the visual analysis by expert pathologists. We consider digital images of histological slides obtained from histiocytoma, mastocytoma and transmissible venereal tumors in dogs. Segmented cell nuclei of these tumors were evaluated morphometrically considering the features normally used in medicine together with novel features suggested in the present work, applied in order to characterize the distribution and morphology of the nucleoli and heterochromatin inside the nuclei. Statistical characterization of the obtained results was performed through variance and discriminant analysis, leading to the identication of the most important features. The approach proposed in this article has been veried to be an effective auxiliary tool for differential diagnosis of histiocytoma, mastocytoma and TVT in dogs. Its low computational cost allows fast morphometric evaluation, paving the way for real-time tumor diagnosis. r 2004 Elsevier Ltd. All rights reserved.

1. Introduction Skin and subcutaneous tumor are the most frequent kinds of such a disease known to affect dogs, accounting for one-third of all cases in this species [1]. Amongst these types of tumors are the histiocytoma, the mastocytoma and the venereal transmissible tumors, which are briey reviewed in the following. Generally, routine histopathologic diagnosis is sufcient to differentiate between histiocytoma, mastocytoma and transmissible venereal tumor (TVT). However, this method can be inefcient for the differential
Corresponding author. Tel.: +55-34-3218-2240; fax: +55-34-32329871. E-mail addresses: mebeletti@centershop.com.br (M.E. Beletti), luciano@if.sc.usp.br (L.da F. Costa).

diagnosis, mainly when the primary TVT is localized out of the external genitalia. According to Ghali et al. [2], in other tumor types the agreement between interobserver in the graduation of the several tumors rarely exceed 8090% of the cases. Therefore, there is a need for an auxiliary tool with objective results in tumor diagnosis. Numerous studies have aimed at developing image analysis procedures for the resolution of difcult differential diagnoses. Among the most useful features are measures of nuclear size as area, perimeter and sphericity [3], and chromatin appearance. In image analysis, chromatin appearance is expressed in terms of the texture of a digital nuclear image, i.e., the spacial distribution of grey values. The method most frequently used for characterization of the nuclear texture is based on Markovian analysis [35], which yields over 60 highly correlated [3,6] features which are difcult to interpret

1077-2014/$ - see front matter r 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.rti.2004.05.003

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and do not correspond to the visual impressions of pathologists [3,7]. The fractal geometry and lacunarity analysis are an alternative approach to chromatin texture description [3], but are computationally expensive. Despite evaluation of the type, image analysis allows an objective evaluation of histopathologic alterations, decreasing the high intrinsic subjectivity of the visual evaluation carried out by pathologists, conrming and classifying the cell type according to its morphological characteristics [7]. However, frequently, uncertainties remain even with the use of this method. This article has as objective to suggest several approaches for geometric and texture characterization of the cell nuclei, verifying the efciency of these measures, along those traditionally used in medicine and veterinary, as auxiliary tools in histiocytoma, mastocytoma and TVT diagnosis. This new approach is suitable for low computational cost and rapid analysis and is easily interpreted and correlated with the visual impressions of pathologists. 1.1. Biological aspects the three main types of tumors considered in this work Histiocytoma is the most common benign neoplasia in dogs [8,9], affecting 14% of those animals [10]. The cells may have a confusing limit, either round or elliptical in shape, with abundant cytoplasm and pale staining with a prominent nucleus [11,12] (Fig. 1). Differential diagnostic is necessary for the identication of several types of tumors, including inammation focal granulomatous, TVT, cutaneous lymphosarcoma and mastocytoma [11]. The mastocytoma is a common neoplasy in dogs and cats and originates from the mast cells, which is an important cell of the immunological system. This tumor represents from 7% to 21% of all skin tumors in dog

Fig. 2. Microscopic visualization of the canine mastocytoma.

[1315]. The latter is characterized by diffuse to nodular proliferation of mast cells, either ovoid or round in shape, and relatively large cells with several round nucleoli. Granules highly stained in the cytoplasm become evident after staining of the sections or tumor imprints by Giemsa or Toluidine Blue [11] (Fig. 2). The differential diagnostic generally is not difcult for welldifferentiated mastocytoma, but histiocytoma and TVT can have several similar features [11]. The TVT is a frequent neoplasia of round cells that is localized mainly in the mucosa of the external genitalia of dogs of both genders, but extra genital occurrence has also been related [1618]. Microscopically, the TVT is characterized by compact mass cell, the line or fashion, supported by little brovascular tissues. Its cells, of about 1230 mm, possess round, ovoid or polyedric morphology, with large, round and hyperchromatic nuclei, distinct marginal chromatin and prominent central nucleoli, weakly eosinophilic cytoplasm and may have indenite borders. Frequently, abundant cells are found in mitosis [19] (Fig. 3). Despite several structural and ultrastructural studies on the spontaneous and experimental TVT, the cellular origin of this tumor remains uncertain; although some studies suggest that the origin this tumor is made of histiocytes [19,20].

2. Material and methods Fifteen histological slides of each tumor type proceeding from the archives of the Laboratory of Animal Pathology of the Veterinary Hospital of the Universidade Federal de Uberla ndia were used. The histological slides contained sections with 7 mm thickness and were stained with hematoxylin and eosin (HE). All slides refer to cases of clinical and histopathologic diagnosis of histiocytoma or mastocytoma located in skin of the dogs

Fig. 1. Microscopic visualization of the canine histiocytoma.

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Fig. 4. (a) Segmented nucleus of the TVT cell in grayscale (b) The same nucleus after the ROI segmentation. The major ROI corresponds to the nucleolus. Fig. 3. Microscopic visualization of the canine TVT.

or TVT located in external genitalia. The slides were pre-selected in order to favor those cases that have more characteristics of each tumor. Two experienced pathologists performed the verication of the tumor type. All these histological sections were recorded into digital images by using an Olympus BX 40 microscope, coupled to OLY-200 Olympus camera connected to a PC microcomputer through a 3153 Data Translation card. The image acquisition was done with 100 magnication and common light microscopy (immersion). The acquired images were enough to provide 100 cell nuclei for each of the analyzed cases. The nuclei were segmented through human interaction by using the GIMP software, yielding gray-scale images of 200 200 pixels with white background. Only non-overlapping nuclei with easily detected boundaries were considered. The intensely stained nuclear regions were segmented and such a structure is henceforth called a Region of Interest, henceforth abbreviated as (ROI). These regions therefore correspond to the nucleolus or heterochromatin regions. In order to segment the ROIs, the mean value of the pixels that formed the image of the nucleus was calculated and all pixels below 84% of these values were transformed into black, white being otherwise assigned (Fig. 4). This threshold value was determined after several tests while observing the separation between the nucleolus ROI and the heterochromatin. The analyses of the characteristics of the nuclei were realized for routines developed in the Scilabs [21] mathematical environment. The following features have been considered: (1) Geometric characterization measurements: area (A), perimeter (P), ellipticity (e), width (W), length (L), and ratio width/length (W/L). (2) Texture characterization measurements: number of ROIs (nR), average area of the ROIs (AR), average

perimeter of the ROIs (PR), distance between the mass center of the greater ROI and that of the nucleus (dgR), average distance between the mass center of the ROIs and that of the nucleus (dmR), elongation of the greater ROI (egR), average elongation of the ROIs (maR), lacunarity (Lc), eigenvalue 1 of the distance matrix (amd1R) and eigenvalue 2 of the distance matrix (amd2R), spectral distance ratio (redR), average occupation ratio of the ROIs (moR) and standard deviation of the occupation ratio of the ROIs (doR). For the automatic determination of the width and height of the nucleus, the center of mass of each nucleus was determined and the nucleus was translated to that position, in such a way that its center now corresponded to the origin of the Cartesian coordinate axis. Having normalized the nucleus position, its principal orientation ` ve transform [22]. was found by using the KarhunenLoe This process involves estimating the correlation matrix of the coordinates of the pixels composing the nucleus, taking its eigenvalues and eigenvectors, and using the latter to dene a linear transformation that rotates the original object so as to have the X-axis aligned along the orientation that produces the maximum dispersion. The two obtained eigenvalues give the respective maximum and minimum dispersions in the new coordinate system. After re-positioning the object, the projection of the pixels in the Y- and X-axis, respectively, correspond to width and height of the nucleus (Fig. 5a). The ellipticity, described as a measure of elongation of the nucleus normalized between 1oeo1, were dened as e W H =W H ; where W is the weight and H is the height of the nucleus. The elongation of the ROIs was determined through principal component analysis (PCA) [22] as the ratio between the principal axis of the ROI and the secondary

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Fig. 5. (a) Schematic drawing of the nuclei rotation and the width and height used for the calculation of the nuclei; (b) Distance matrix building; (c) Minkowski dilatation of the ROIs forming the regions of occupation. The ratio of occupation is the ratio of the area occupied by the ROI (A1) and its territorial area (A2).

perpendicular axis. Such a ratio determines the elongation of the ROI, in the sense that the closer it is to 1, the more spherical the object is. The average value of this measurement was obtained by considering the elongations of every ROI. Distance matrices were obtained for the distribution of connected components inside each nucleus, expressing the Euclidean distance between the center of mass of each pair of components (Fig. 5b). The eigenvalues of such matrices were calculated and sorted in decreasing order. The two largest eigenvalues were considered as features characterizing the spatial distribution of the ROIs, which are independent of the ROIs order and invariant to rotation, translation and reection. The spectral distance ratio is the ratio between the eigenvalue 2 and the eigenvalue 1 of the distance matrix. In order to calculate the average and standard deviation of the occupation ratio of the ROIs, a Minkowski dilation was performed for each pixel of the ROIs, until the cells are tilled up. The lines where the ROI dilatation touch their neighbors ROIs or nucleus limit demarcates the regions of occupation correspond to the respective Generalized Diagram of Voronoi [23]. Thus, the set of the lines formed by the dilatation of all the ROIs form one territorial division of each ROI in the nucleus. The ratio of occupation is the rate of the area occupied by the ROI and its territorial area (Fig. 5c). The average and standard deviation of the ratio of occupation of the ROIs were considered as variable for characterization of nucleus texture. Lacunarity [3] was calculated using an s s window initially placed at the upper left corner of the binary image containing the ROIs. The sum of the pixels values inside the box (S) is calculated as the box moved pixel by pixel to the right. The result of the sum of each box position which is totally inside the nucleus is stored in vector S. We used values for s varying by 2 2 pixels, with minimum value 2 and maximum value 20. For each

s value, the lacunarity was calculated as: s LS log 2 ; m where s is the standard deviation and m is the mean of S. The R 20 lacunarity of each nucleus is dened as: L 2 LS ds. 2.1. Statistical analysis Variance analysis [24] was performed for differences between the means of each tumor type and subsequently considered for canonical discriminant analysis [23,25]. Two discriminant functions were created, from which the canonical variables for each nucleus were obtained. Such new variables were calculated considering all features while optimizing the capacity for correct classication. The coefcient of correlation between the morphometric features and the canonical variables were calculated so as to determine the canonical structure, which allows the identication of the more important components in such new variables. After obtaining the discriminant functions, the respective predictive errors were calculated. These errors correspond to the percentage of nuclei that was correctly classied by using the canonical variables. The predictive errors were obtained for each tumor type and for the three types together (overall error).

3. Results and discussion The average and standard deviation of each feature evaluated for each type of tumor and the statistical difference between averages obtained through variance analysis are shown in Table 1. Canonical discriminant functions were also obtained by using discriminant analysis [3]. The canonical coefcients of each function are shown in Table 2. These coefcients yielded two new

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K.C.A. Waldemarin et al. / Real-Time Imaging 10 (2004) 197204 Table 1 Average and standard deviation of the features used in three types of tumors Features Average Histocytoma A P e W L W/L nR AR PR dgR mdR egR meR amd1R amd2R moR doR redR Lc 3062.42a 202.78a 0.13a 54.98a 70.82a 0.78a 3.90a 105.94a 66.15a 17.87a 14.65a 0.30a 0.33a 101.60a 7.52a 0.10a 0.049a 0.21a 9.42a Mastocytoma 2497.48b 183.03b 0.12a 49.91b 63.81b 0.78a 3.42b 143.18b 83.78b 14.39b 11.71b 0.32b 0.37b 65.91b 6.84b 0.13b 0.046a;b 0.28b 5.93b Standard deviation TVT 2757.60c 192.19c 0.11b 53.06c 66.27c 0.80b 3.81a 106.17b 65.76b 16.52c 12.78c 0.32b 0.41c 90.48c 7.17a;b 0.11a 0.052a;c 0.20a 8.03c Histocytoma 825.42 27.16 0.06 8.64 10.20 0.10 2.31 124.33 48.63 7.98 6.65 0.14 0.20 86.75 4.441 0.105 0.0404 0.313 6.60 Mastocytoma 670.25 24.19 0.06 7.86 8.76 0.098 2.14 176.85 66.89 6.45 5.89 0.15 0.22 56.115 4.010 0.140 0.412 0.305 7.24 TVT 667.13 23.83 0.05 7.80 8.36 0.091 2.14 125.96 56.58 6.49 5.56 0.14 0.22 67.723 4.222 0.112 0.0372 0.305 6.32 201

Different letters refer to statistical differences among the group.

Table 2 Canonical coefcient of the discriminate functions Features A P W L W/L e nR AR PR dgR mdR egR meR amd1R amd2R moR doR redR Lc Canonical function 1 0.0005 0.026 0.12 0.058 6.04 1.159 0.0002 0.0073 0.553 0.01 1.223 0.025 0.568 0.015 0.051 2.086 1.366 0.087 0.011 Canonical function 2 0.0013 0.010 0.282 0.103 17.686 11.209 0.001 0.006 0.177 0.046 3.234 0.057 1.445 0.008 0.071 2.414 5.533 1.407 0.032

variables for each nucleus, whereas each discriminant function generated a single canonical variable. In order to calculate the canonical variable of a nucleus, the value of each feature of that nucleus was multiplied by the respective canonical coefcient. The sum of these values corresponds to the respective canonical variable of that nucleus.

Table 3 shows the canonical structure in terms of the correlation coefcients between the canonical variable and each variable used in the discriminant function. Table 4 illustrates the predictive errors by using the discriminant functions. Fig. 6 provides the representation of the distribution of the nucleus analyzed considering the two canonical variables obtained from the original features. The morphometric features of the nucleus of the tumor cells of histiocytomas, mastocytomas and TVT are summarized in Table 1, including the area, perimeter, width, length, distance between the mass center of the main ROIs and that of the nucleus, average distance between mass center of the ROIs and that of the nucleus, average elongation of the ROIs, lacunarity and eigenvalue 1 of distance matrix were statistically different between the three types of the tumor. The average values of area, perimeter, width and length in the histiocytoma were the highest, showing that the cells of this type of tumor tend to be bigger that those in other tumors. We can observe that the number of ROIs was statistically greater in the histiocytoma and TVT cases. However, the area and perimeter of the ROIs were statistically greater in the mastocytoma, showing that the number of ROIs is greater in the histiocytoma and TVT, while the ROIs in the mastocytomas are greater. From the distances between the mass centers we can see that the ROIs and the main ROI have distribution more peripheral in the nuclei of histiocytoma than in the

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nuclei of the TVT and mastocytoma. It can also be seen from this table that the lacunarity averages were statistically greater for histiocytoma, which demonstrates higher dispersion of the ROIs in this tumor type. As the area and perimeter averages of the nucleus are signicantly different in each tumor, it would be reasonable to suggest the use of these features as criteria to help the respective differential diagnostic. At the same time, the ellipticity was similar for histiocytoma and mastocytoma, being smaller in the cases of the TVT. These features are frequently used in morphometric evaluations of those types of tumors. The statistical difference between the eigenvalue 1 of the distance matrix of each tumor type showed that this feature for texture characterization of the nucleus presents good potential for discriminating between the considered types of tumors. In all tumor types the standard deviation of the ROIs area was greater that their average, characterizing a great variation of ROIs areas. We gather from Table 1 that both the distance between the main ROI and mass center of the nucleus and the distance between all the

Table 3 Canonical structure of each function Features A P e W L W/L nR AR PR dgR mdR egR meR amd1R amd2R moR doR redR Lc Canonical function 1 0.45 0.45 0.50 0.32 0.25 0.26 0.87 0.77 0.17 0.11 0.15 0.31 0.26 0.08 0.09 0.55 0.01 0.39 0.06 Canonical function 2 0.47 0.47 0.53 0.33 0.28 0.28 0.85 0.76 0.21 0.13 0.16 0.28 0.26 0.11 0.10 0.53 0.02 0.40 0.07

ROIs and the mass center of the nucleus resulted differently for the tumor types. The lacunarity also resulted differently for the three tumor types. Therefore, even though the TVT and histiocytoma have the same origin [19,20], the distribution of the respective ROIs is different. In order to optimize the use of the considered features for the identication of the tumor types, a canonical discriminant analysis was performed, generating two discriminant functions given by the canonical coefcients shown in Table 2. Each canonical function generates one canonical variable. These two new variables, obtained from all experimental features, taken with different weights, optimized the power of separation of the classes (tumor types). This classier was utilized because after the generation of the canonical functions, they can be easily used for people without great knowledge in computer science and excusing software and equipments of the last generation. The correlation between these new variables and the original features yielded the canonical structure (Table 3). By sorting the absolute values of the canonical structure, we can identify the effective participation of each variable in the formation of the two canonical variables. Therefore, Table 3 can be used to identify which measure was more important for the discriminant function. The number of the ROIs was the principal feature in the differentiation of the tumors, followed by the ROIs area and average occupation ratio of the ROIs. Area, perimeter and ellipticity of the nuclei were also important features in the constitution of the canonical functions. Therefore, the use of the geometric characterization of the nucleus combined with this new proposed methodology for nuclear texture characterization, are believed to have good potential as a tool for histophatologic diagnostic. The lacunarity, standard deviation of the ROIs occupation and the eigenvalues 1 and 2 of the distance matrix were the features leading to the smallest weights in the constitution of the discriminant functions. This demonstrates that such features have reduced potential for diagnosis of these tumor types. However, the lacunarity has been elsewhere shown to be useful for the differentiation of other kinds of tumors [3]. It is believed that the standard deviation of the ROIs

Table 4 Predictive error in the identication of the type of tumor using every canonical function obtained Type of tumor % of nucleus classied as Histocytoma 818 267 425 % of nucleus classied as Mastocytoma 327 827 453 % of nucleus classied as TVT 355 406 622 Percentage of error

Histocytoma Mastocytoma TVT Overall

0.46 0.45 0.57 0.49

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evaluation, paving to make way for an immediate tumor diagnosis. The morphometry of the nuclei of the neoplastic cells can be an effective auxiliary tool for differential diagnosis of histiocytoma, mastocytoma and TVT in dogs, mainly when the distribution and morphologic characterization of the nucleoli and heterochromatin is taken into account. The approach proposed in this article also presents good potential for general citomorphometry. References
[1] Martin De Las Mulas J, Millan Y, Ruiz-Villamor E, Bautista MJ, Rollon E, Espinosa De Los Monteros A. Apoptosis and mitosis in tumors of the skin and subcutaneous tissues of the doa. Res Vet Sci 1999;66:13946. [2] Ghali SV, Liau S, Teplitz C, Prudente C. A comparative study of DNA ploidy in 115 fresh-frozen breast carcinomas by image analysis versus ow cytometry. Cancer 1992;70:266872. [3] Einstein AJ, Wu HS, Sanchez M, Gil J. Fractal characterization of chromatin appearance for diagnosis in breast cytology. J Pathol 1998;185:36681. [4] Haralick RM, Shanumgam K, Dinstein I. Textual features for image classication. IEEE Trans Syst Man Cybern 1973;3:61021. [5] Pressman NJ. Markovian analysis of cervical cell image. J Histochem Cytochem 1976;24:13844. [6] Diegenbach PC, Baak JPA. Quantitative nuclear image analysis: differentiation between normal, hyperplastic, and malignant appearing uterine glands in a parafn section III. The use of texture features for differentiation. Eur J Obstet Reprod Biol 1978;8:10916. [7] Beil M, Irinopolou T, Vassy J, Rigaut JP. Chromatin texture analysis in three-dimension images from confocal scanning laser microscopy. Anal Quant Cytol Histol 1995;17:32331. [8] Scherlie PL, Smedes SL, Feltz T, Dougherty SA, Riis RC. Ocular manifestation of systemic histiocytosis in a dog. J Am Vet Med Assoc 1992;201:122932. [9] Bender WM, Muller GH. Multiple, resolving, cutaneous histiocytoma in a dog. J Am Vet Med Assoc 1989;194:5357. [10] Rothwell TEW, Howletti CR, Middleton DJ, Grifths DA, Duff BC. Skin neoplasms of dogs in Sidney. Aust Vet J 1987;64:1613. [11] Conroy JD. Canine skin tumor. J Am Anim Hosp Assoc 1983; 19:91114. [12] Muller GH, Kirk RW, Scott DW, Miller WH, Grifn CE. Dermatologia dos Pequenos Animais, 5th ed. Revinter: Rio de Janeiro; 1996. [13] Fox LE. Mast cell tumors. In: Morrison WB, editor. Cancer in dogs and catsmedical and surgical management. Lippincott: Williams & Wilkins; 1998. p. 477505. [14] Strefezzi RDF, Xavier JG, Cata o-Dias JL. Morphometry of canine mast cell tumors. Vet Pathol 2003;40:26875. [15] Rogers KS. Mast cell tumorDilemmas of diagnosis and treatment. Vet Clin North Am Small Anim Pract 1996;26:87102. [16] Harmelin A, Zucckerman A, Nyska A. Correlation of AgNOR protein measurements with prognosis in canine transmissible venereal tumors. J Comp Pathol 1995;112:42933. [17] Marchal T, Chabanne L, Kaplanski C, Rigal D, Magnol JP. Immunophenotype of the canine transmissible venereal tumor. Vet Immunol Immunopathol 1997;57:111. rez J, Day MJ, Mozos E. Immunohistochemical study of the [18] Pe local inammatory inltrate in spontaneous canine transmissible venereal tumor at different stages of growth. Vet Immunol Immunopathol 1998;64:13347.

Fig. 6. Scatterplot of the canonical variables of all nuclei of histiocytoma, mastocytoma e TVT.

occupation and the eigenvalues 1 and 2 of the distance matrix can also be useful in other situations. The reclassication of the nucleus according to the discriminant functions (Table 4) showed that the greater percentage of errors was obtained for the case in which histiocytoma were classied as TVT and the nucleus of the TVT classied as nucleus of histiocytoma. This difculty is due to the great morphologic similarity between these tumors, probably because both originate from histiocytes [19,20]. However, the greater number of mastocytoma nuclei classied as TVT can be explained by the several degrees of malignance of the analyzed mastocytomas [14]; as the nucleus features depend on the malignance degree, this hindered the morphometric characterization in those cases. In the present study the mastocytomas were not pre-classied for pleomorphism or malignant degree. This difculty in the identication of mastocytoma through the nuclear morphomety of neoplasic cells is compensated in the histopathologic diagnostic by the cytoplasmatic characteristics of those cells and by the contents of their surroundings, such as intense presence of the eosinophil, as well as other materials [12]. To any extent, this methodology can be used with great efciency as an auxiliary tool for differential diagnostic of such tumor, because even though individual nuclei can be misclassied, the classication considering all nuclei was always correct in our experiment.

4. Conclusions The low computational cost of the new methodology proposed in this work allows fast morphometric

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