Testicular damage from anabolic treatments with the beta(2)-adrenergic agonist clenbuterol in pigs: a light and electron microscope study.

The morphological consequences of anabolic clenbuterol treatment on the testicular parenchyma were investigated in 30 pigs at morphological and ultrastructural levels. Clenbuterol was given with food (1 ppm). In the first group (n=10), treatment was maintained until slaughter (experimental period 3 months). In the second group (n=10), clenbuterol was withdrawn 2 weeks before slaughter (experimental period 2.5 months). A third group (n=10) of pigs not fed with clenbuterol served as controls. Animals were slaughtered at 9 months of age and samples of testicular parenchyma were collected for light and electron microscope studies. In the clenbuterol-treated groups, the interstitial cells showed a considerable increase in the organelles involved in testosterone production, with an increased development of the mitochondria, smooth endoplasmic reticulum, Golgi apparatus and lipid droplets compared to the control group. The seminal epithelium displayed many lipid vacuoles and evident signs of tubular involution, such as degenerating and multinucleate germ cells. Sertoli cells gave evidence of metabolic alterations such as large lipid deposits and cytolysosomes.

Stereological study of the adrenal gland of calves treated with clenbuterol.

Stereological methods were used to investigate the morphometric variations in the adrenal cell population of calves dosed orally with clenbuterol at an anabolic dose of 20 microg/kg bodyweight per day for 12 weeks. In the treated group the size of the nuclei increased with respect to the control group, and the increases were significant for the maximum linear parameters in all the regions except the zona fasciculata. The largest increases were observed in the epinephrine-producing cells. The quantitative stereological data showed highly significant increases in the nuclear volume fraction, the nuclear surface density and the numerical density in the zona fasciculata of the treated group.

Nuclear volume estimates in prostatic intraepithelial neoplasia.

OBJECTIVE: Prostatic intraepithelial neoplasia (PIN), the most likely precursor of prostatic adenocarcinoma, is divided into two grades, low and high. Pathologists may encounter difficulties in applying these criteria in daily practice. In view of the clinical significance of high grade PIN as strong predictor of carcinoma, the separation of low and high grade PIN plays an important role in patient management. The aim of the present study was to evaluate three-dimensional nuclear size estimation in normal prostatic glands, low and high grade PIN, and prostatic adenocarcinoma as an element in their classification. STUDY DESIGN: We studied 31 formalin-fixed, paraffin-embedded, whole-mounted radical prostastectomy specimens that contained foci of normal prostatic glands, low and high grade PIN, and prostatic adenocarcinoma. Hematoxylin-eosin-stained sections were selected for the stereologic estimation of volume-weighted mean nuclear volume by the "point-sampled intercepts" method. On each focus, an average of six fields of vision were systematically chosen. RESULTS: The quantitative results indicate a significant increase in nuclear volume from normal prostatic glands (mean, 209.0 micron 3; SD, 64.6 micron 3) to low grade PIN, high grade PIN and prostatic adenocarcinoma with increments of 49%, 88% and 109%, respectively (F = 29.1, P < .001). Two-group comparisons (Duncan procedure) showed differences between low and high grade PIN and prostatic adenocarcinoma (P < .01). The difference between high grade PIN and prostatic adenocarcinoma was not significant. CONCLUSION: Three-dimensional estimates of nuclear size discriminate low and high grade PIN. Lack of stereologic differences between high grade PIN and prostatic adenocarcinoma further supports high grade PIN as a precursor of prostatic adenocarcinoma.

Nuclear volume estimates in prostatic atypical adenomatous hyperplasia.

OBJECTIVE: To investigate nuclear volume estimates by the point-sampled intercepts method in atypical adenomatous hyperplasia (AAH) as compared with nodular hyperplasia and well-differentiated prostatic adenocarcinoma. STUDY DESIGN: The study group consisted of 27 formalin-fixed, paraffin-embedded, whole-mounted radical prostatectomy specimens that contained foci of nodular hyperplasia, atypical adenomatous hyperplasia and well-differentiated adenocarcinoma (Gleason pattern 1 and 2). Representative sections were selected for stereologic estimation of volume-weighted mean nuclear volume by the point-sampled intercepts method. On each focus, an average of five fields of vision were systematically chosen. RESULTS: The quantitative results indicate an increase in nuclear volume from nodular hyperplasia (209 +/- 65 micron 3) to AAH (237 +/- 85 micron 3) and prostate adenocarcinoma (436 +/- 106 micron 3). Significant differences were found (F = 39.0, P < .001) with two group comparisons (Scheffe's procedure) between prostate cancer and AAH (P < .001) or nodular hyperplasia (P < .001). The difference between AAH and benign hyperplasia was not signifcant. CONCLUSION: The results indicate that three-dimensional estimates of the nuclear size discriminate AAH and nodular hyperplasia from well-differentiated prostate adenocarcinoma. These findings suggest that AAH is probably a histologic variant of benign prostatic hyperplasia the exact relationship of which to prostatic adenocarcinoma remains to be determined.

Application of the fractionator and vertical slices to estimate total capillary length in skeletal muscle.

A new stereological method is proposed which combines vertical slice projections with the fractionator to estimate the total capillary length in a skeletal muscle. The method was demonstrated on the soleus muscle of a Wistar rat. The implementation required capillary highlighting, tissue sampling, and data acquisition in the form of intersection counts between capillary projections and cycloid test lines. The capillaries were demonstrated using vascular perfusion (with gelatine) of the hind leg of the rat. The sampling procedure followed the fractionator design, namely a multistage systematic sampling design with a known sampling fraction at each stage. To make the design unbiased, vertical slices were used; for efficiency, the vertical axis was chosen parallel to the main axis of the muscle. As prescribed to avoid bias, the cycloid test lines were superimposed on the slice projections, viewed under the light microscope, with their minor axes normal to the vertical axis. The estimation precision was compared for different sampling and subsampling fractions. The proposed method was globally highly efficient, unbiased, and easy to implement.

Unbiased stereological estimation of the number and volume of nuclei and nuclear size variability in invasive ductal breast carcinomas.

The application of design-based stereological methods for estimating nuclear features quantitatively in invasive ductal breast cancer is described. Nuclear number, size and size variability are explored in relation to the tumour grade and patient prognosis. The study includes an examination of the efficiency in estimating different nuclear volumes, and two different estimators of the nuclear size variability are contrasted. Forty-two invasive ductal breast carcinomas diagnosed and graded by two pathologists were used. Both 5-microns and 25-microns-thick sections were obtained from paraffin blocks for stereological study. More undifferentiated tumours show significantly larger nuclei than low-grade tumours. The estimates based on the disector method demonstrate a decrease in the number of tumour cell nuclei per unit volume of tissue from grades 1 to 2 and especially from grades 2 to 3. The univariate survival analysis shows a high prognostic value of the nuclear volume estimates. The study shows that an efficient sampling procedure was performed, particularly when estimating volume-weighted mean nuclear volume using the point-sampled intercepts method. This method is more efficient than estimation of the number-weighted mean nuclear volume using the selector method; however, the latter provides paired estimates of volume- and number-weighted mean nuclear volume, as well as an estimate of the coefficient of variation of nuclear volume in the number distribution of the same cells.

Objective differential classification of thyroid lesions by nuclear quantitative assessment.

Quantitative nuclear parameters estimated by morphometric and stereological methods in combination with discriminant analysis were used in order to evaluate the diagnostic efficiency of thyroid lesions. This study includes 55 patients with thyroid pathology. Samples of follicular adenomas, follicular carcinomas, and papillary carcinomas were examined by image analysis to obtain size and form nuclear parameters. Stepwise discriminant analyses were performed. There was an increase in nuclear size from follicular adenomas to follicular carcinomas, and a greater increase from follicular carcinomas to papillary carcinomas. The increase was more significant when the three-dimensional estimates of the volume-weighted mean nuclear volume were assessed. No significant differences between follicular adenomas and follicular carcinomas were found with respect to the nuclear form factors, however, a significant increase in nuclear elongation and irregularity was registered between follicular and papillary tumors (p < 0.01). The overall accuracy rate of discrimination was 75% when the three lesions were included in the analysis, with an efficiency of 85% for papillary carcinoma samples. These percentages increased when two lesion discrimination was performed. The worst discrimination (69% of efficiency) was found between follicular adenomas and follicular carcinomas.

Quantitative histopathologic discrimination of rheumatic disorders using nuclear measurements of synoviocytes.

OBJECTIVE: To use several nuclear quantitative estimators of synoviocytes and advanced statistical tests to discriminate between rheumatic disorders with the purpose of providing an objective histopathologic differentiation and to illustrate the difficulty of establishing the implications of the vague diagnosis of chronic nonspecific synovitis. STUDY DESIGN: Synovial histologic material from 48 patients, including a control group and those diagnosed with osteoarthritis, rheumatoid arthritis and chronic nonspecific synovitis, was analyzed. Both morphometric and stereologic nuclear measurements were assessed in each case, and several stepwise discriminant analyses were performed to obtain linear discriminant functions. RESULTS: Differences in the nuclear size parameters were found between rheumatoid arthritis and the normal and osteoarthritis groups and between normal and chronic nonspecific synovitis. The volume-weighted mean nuclear volume was the most significant parameter. No differences were demonstrated between osteoarthritis and normal and chronic nonspecific synovitis or between rheumatoid arthritis and chronic nonspecific synovitis except for nuclear volume. Using stepwise discriminant analysis, 94% of the cases were correctly classified when differentiating the normal, osteoarthritis and rheumatoid arthritis groups. The overall accuracy of diagnosis decreased to 83% when chronic nonspecific synovitis samples were included. The misclassified samples were related mainly to normal cases and to osteoarthritis and chronic nonspecific synovitis cases. CONCLUSION: This study emphasized that nuclear quantitative features of synoviocytes may be useful in differentiating rheumatic disorders objectively, especially in combination with discriminant analysis. Thus, nuclear changes in the synovium in chronic nonspecific synovitis show a closer similarity to normal and osteoarthritis than to rheumatoid arthritis.

Grading and prognosis of infiltrating ductal breast carcinoma by mean nuclear volume estimates.

OBJECTIVE: Quantitative evaluation of nuclear size in breast cancer is performed with the aim of investigating whether the data obtained are related to subjective grading and prognosis. STUDY DESIGN: Unbiased stereologic estimates of the volume-weighted mean nuclear volume (nuclear vv) in paraffin-embedded tissue from 44 specimens of infiltrating ductal breast carcinoma were obtained by the point-sampled intercepts method. RESULTS: Histologically graded cases showed an overlap in nuclear vv estimates, but significant differences were demonstrated in which averaged values increased from grade 1 to 3 breast carcinomas. Nuclear vv estimates in grade 3 breast carcinoma (mean, 495.4 microns 3; SD, 101.7 microns 3) were significantly larger than those in grades 1 (mean, 327.8 microns 3; SD, 90.9 microns 3) and 2 (mean, 409.9 microns 3; SD, 72.6 microns 3) (P < .01). Minor statistically significant differences (P < .05) were found between grades 1 and 2. Estimates of nuclear vv based on systematic sampling within the specimen showed high efficiency, with > 80% of the total observed variance contributing to biologic differences between individual specimens. Univariate prognostic study showed that clinical stage, and not histologic grade, was of prognostic value in the cases studied. Similarly, nuclear vv estimates were associated with disease recurrence and survival, with a cutoff point of 425 microns 3. CONCLUSION: Based on the cases investigated, shape-independent nuclear vv estimates may be an adjuvant tool in the grading and prognostic evaluation of infiltrating ductal breast carcinoma.

Application of recent stereological tools for unbiased three-dimensional estimation of number and size of nuclei in renal cell carcinoma samples.

New stereological methods have appeared in recent years that allow an unbiased and efficient estimation of quantitative characteristics of three-dimensional biological structures. The aim of this study is to apply the new stereological tools on renal tissue and investigate the changes in the number and size of nuclei in renal cell carcinomas. The simple and easy use of the disector, point-sampled intercepts and selector methods on kidney biopsies is demonstrated. Using these approaches, the mean number of nuclei per unit reference volume: NV(nucl/tis), the volume-weighted mean nuclear volume: nu V(nucl) and the number-weighted mean nuclear volume: nu N(nucl) were estimated in histological sections from 20 renal cell carcinomas. The study was performed on 10 grade 2 and 10 grade 3 renal cell carcinomas previously diagnosed by the pathologist on the basis of nuclear size increase, irregularity and nucleolar prominence. Basically, serial optical kidney sections from a 25-millimicrons thick slice were used for stereological study. The size estimations of the individual particles nu V(nucl) and nu N(nucl) obtained by the 'point-sampled intercepts' and 'selector' methods, respectively, turned out to be the most adequate parameters employed in discriminating between renal carcinoma grades (P less than or = 0.001).

Estimation of capillary length density in skeletal muscle by unbiased stereological methods: I. Use of vertical slices of known thickness.

BACKGROUND: The unbiased estimation of the capillary length density in skeletal muscle tissue Lv(cap/mus) has been performed in this study applying a new stereological methodology based on the use of vertical slices and the intersections of cycloid test curves with capillaries in a three-dimensional space defined by systematically chosen fields of vision and the thickness of the sections. METHODS: The following simple requirements must be fulfilled: selection of a fixed vertical axis in skeletal muscle, adequate systematic muscle sampling, obtention of vertical slices of constant and known thickness but indifferent in magnitude, superposition of a cycloid test system with the minor axis of cycloid curves positioned perpendicularly to the vertical axis, and counting the intersections between cycloid curves and capillaries. In our study, the vertical axis was defined as that which is parallel to the natural, major axis of the muscle where fibres and capillaries are arranged parallel to this axis. The muscle sampling was performed using the fractionator method, and 25 microns thick sections were chosen. RESULTS: The application of the equation for estimation of Lv(cap/mus) permits determination of an average of 1,480 mm of capillaries per mm3 of muscle tissue, knowing the number of intersections, section thickness, and the points hitting the muscle with a known ratio between cycloid test curve length to a test point. CONCLUSIONS: The estimation of Lv(cap/mus) is efficient, unbiasedly obtained, and no assumptions on the degree of capillary anisotropy are required.

Estimation of capillary length density in skeletal muscle by unbiased stereological methods: II. Use of vertical slices of unknown thickness.

BACKGROUND: The capillary network of the skeletal muscle was analyzed from a quantitative point of view with the purpose of determining the capillary length density--LV(cap/mus). A recent stereological method was applied to estimate this quantity using vertical slices of unknown thickness. METHODS: First, the whole muscle was systematically sampled according to the fractionator method. The capillary length density was estimated on each chosen field of vision where the vertical axis was always identified as parallel to the major axis of the muscle fibers. Three measurements were performed: count of intersections between capillaries and cycloid test lines, count of intersections between capillaries and straight test lines, and count of capillary end points corresponding to the intersections of capillaries with the parallel planes of the vertical slices. RESULTS: The estimated capillary length density was 1,578 mm per mm3 of skeletal muscle tissue. The average thickness of the vertical slices was also estimated as 23.4 microns, which is roughly 6% less than the thickness measured using the microcator information on the microscope stage displacement. CONCLUSIONS: The advantages of this methodology were based on two main features: the method is assumption-free on the degree of capillary and muscle anisotropy, and the thickness of the vertical slices need not be known nor constant.

Stereological analysis of the synovial membrane in rheumatic disorders: diagnostic value of volume-weighted mean nuclear volume estimation.

Quantitative evaluation of nuclear size of synoviocytes was performed on 48 synovial biopsies in various rheumatic disorders: osteoarthritis (n = 10), rheumatoid arthritis (11), and chronic non-specific synovitis (14). Thirteen tissue specimens from non-inflammatory synovial membrane were included as a control group. Using the point-sampled intercepts method, unbiased stereological estimates of volume-weighted mean nuclear volume (nuclear upsilon v) were obtained. A slight increase in nuclear volume was observed in osteoarthritis in comparison with the control group with an overlap in 90% of cases. However, in rheumatoid arthritis there was a significant increase of nuclear upsilon v. Significant differences were found between rheumatoid arthritis and the control and osteoarthritis groups (P < or = 0.001). In biopsies from patients diagnosed as chronic non-specific synovitis the averaged nuclear upsilon v values were between those in osteoarthritis and rheumatoid arthritis with a wide range of data. Similar, but less significant differences were demonstrated between rheumatic disorders when using mean nuclear area. Further analysis of chronic non-specific synovitis patients in combination with nuclear upsilon v estimates as a simple, unbiased, complementary tool are required to better establish the diagnostic value of nuclear stereology in the diagnosis of rheumatic disorders.

Nuclear quantitative grading by discriminant analysis of renal cell carcinoma samples. A patient survival evaluation.

Specimens from 60 cases of renal cell carcinoma (RCC) were graded employing quantitative nuclear data combined with multivariate discriminant analysis. Evaluation of patient survival was analysed with respect to quantitative microscopic and qualitative features. Both morphometric and stereological estimators were used to establish the nuclear size and form pattern of the RCC specimens. Tumoural dedifferentiation paralleled progressive increases in nuclear elongation and in two- and, especially, three-dimensional--mean nuclear volume (MNV)--size parameters. Using stepwise discriminant analysis, 85.0 per cent of the specimens were correctly classified when differentiating grade 2 and 3 tumours. It is concluded that simple and realistic estimates of MNV are the best discriminator for objective grading in patients with RCC. Univariate survival analysis demonstrated the important significance of several features such as MNV, clinical stage, and nuclear discriminant and histopathological tumour grades. Nuclear form factor PE, area, and perimeter were also significant. A prognosis study based on the Cox model using a stepwise selection of parameters showed that only MNV has an independent prognostic role when examining all investigated quantitative parameters. The clinical stage was the best prognostic feature when all quantitative and qualitative characteristics were included in the analysis.

Value of volume weighted mean nuclear volume in grading and prognosis of renal cell carcinoma.

AIMS: To perform stereological quantitation of volume weighted mean nuclear volume in renal cell carcinomas; and to correlate the data obtained with recognised clinical and pathological variables and determine their prognostic value. METHODS: The point-sampled intercepts method was used to estimate mean nuclear volume in 63 cases of clear cell renal carcinoma diagnosed between 1980 and 1988. New paraffin wax embedded histological sections were analysed after systematic sampling and the test systems superimposed on a projected microscopic image to measure nuclear intercept lengths. After mathematical estimation of mean nuclear volume, statistical analyses of the data in relation to clinical and pathological variables as well as the prognostic impact were investigated. RESULTS: The mean nuclear volume was significantly associated with tumour dedifferentiation. However, mean nuclear volume showed no statistical differences with sex, age, and clinical stage. The prognostic value of mean nuclear volume, nuclear grading, and clinical stage in renal cell carcinomas was high: mean nuclear volume greater than 140 micron3 was associated with short term survival. CONCLUSIONS: Measurement of mean nuclear volume was useful as a guide to objective grading of renal cell carcinomas, though there was an overlap between tumour grades. Based on the limited number of cases analysed, the mean nuclear volume is proposed as an additional prognostic indicator.

Application of the image analysis system and Imago program to biological samples.

Recent developments in computer systems and new stereological tools have made estimation of quantitative data in biological material simple, easy and fast. The purpose of the present study is to show the advantages and disadvantages of a new program developed by the University of Córdoba for the determination of stereological measurements. As an example, endometrial material was used for demonstrating the technical characteristics. Thus, the nuclear area and volume-weighted mean nuclear volume-Vv(nucl)-of 30 routinely processed, paraffin-embedded endometrial specimens from 12 atypical hyperplasias (AH) and 18 well-differentiated adenocarcinomas (WDA) were investigated. We show the excellent visualization of the material and rapid measurement of these quantitative parameters using the Imago program. In addition, the mean values of Vv(nucl) are more efficient and useful in separating AH and WDA than the mean nuclear area. It is concluded that Vv(nucl) is a superior and sensitive estimator for discrimination.

Multivariate discriminant analysis of normal, intraepithelial neoplasia and human papillomavirus infection of the uterine cervix samples.

The present investigation studies the role of multivariate statistical methods on quantitative histopathological features of cells in uterine cervix epithelium to discriminate between normal and abnormal uterine cervix samples. 143 histological specimens were included in the study involving normal cervix, cervical intraepithelial neoplasia (CIN) lesions and cervical human papillomavirus (HPV) infection with and without CIN (condyloma-CIN and condyloma-NCIN groups, respectively). Deep, middle and superficial regions of the cervical squamous epithelium were morphometrically analyzed. Identification of normal cervix from pathological cases was highly achieved with a specificity of 100%. The application of discriminant statistical method within pathological specimens showed an acceptable percentage of cases correctly classified; thus, an efficiency of 83.0% and 74.6% was obtained in order to discriminate within CIN and condyloma-CIN grades respectively. These percentages increased when differentiation between each grade of CIN versus condyloma-CIN were considered, using only 1-3 morphometrical parameters. Our findings indicate that the combination of nuclear and cytoplasmic quantitative features, specially size parameters, permit a high correct percentage classification of cervix samples. The discrimination process was better when few diagnostic categories were included; however, 100% specificity for normal samples was always reached.

Histomorphometry of normal and abnormal cervical samples.

We quantitatively studied the nuclear and cytoplasmic characteristics of cells in the normal cervix, squamous intraepithelial lesions (cervical intraepithelial neoplasia [CIN]) lesions and cervical human papillomavirus (HPV) infection with and without CIN (condyloma-CIN and condyloma-NCIN groups, respectively). The morphometric features of the deep, middle and superficial layers of the cervical epithelium were calculated on 143 cervical samples. The morphometric parameters selected for the analysis were area; perimeter; maximal, minimal and equivalent circle diameters; nuclear/cytoplasmic ratio; and several shape factors. Our quantitative findings support the opinion of other authors that HPV infection is associated with shape and size modifications of the nucleus and cytoplasm in CIN. In relation to cellular size, there were no differences between a normal cell, regardless of its region, and a virus-infected cell. The nuclear differences are predominantly in the deep layer areas. The CIN samples showed changes in both cellular and nuclear form and size but lacked substantial differences in the tumor grades and areas studied. The condyloma-CIN samples revealed more notable differences in both tumor grade differentiation and different cellular layers.

Evaluation of volume-weighted mean nuclear volume in endometrial lesions.

The individual nuclear size was studied in three groups of endometrial samples (simple hyperplasia -SH-, atypical hyperplasia -AH-, and well-differentiated adenocarcinoma -WDA-). The application of recent semiquantitative systems in combination with stereological methods permits a simple, quick and unbiased estimation of the volume-weighted mean nuclear volume vv(nucl). We have found an increase in the vv(nucl) from SH to AH to WDA. There are significant differences between the mean of the three groups (p < 0.01). The variance associated with estimates of vv(nucl) is mainly provided by differences among lesions; i.e. patients. This capacity for discriminating may be associated with gland and lumen endometrial quantification to improve the correct diagnosis of endometrial samples.

Histomorphometry of normal and abnormal endometrial samples.

One hundred forty samples of normal and pathological endometrium were examined morphometrically in order to achieve a discriminant function among normal and pathological diagnoses. Samples of proliferative and secretory normal endometrium, simple and atypical hyperplasia, and well-differentiated adenocarcinoma were included in this study. Morphometrical and stereological characteristics for both glands and lumina as well as epithelium were registered. Stepwise discriminant analysis was performed. There is an increase in size of glands from proliferative endometrium to well-differentiated adenocarcinoma. The stereological measurements parallel morphometrical measurements. The accuracy of diagnosis by morphometrical and stereological means were 91% when the normal group (proliferative and secretory phase) were included. Accuracy increases to 95% when only three pathological groups were used; only one simple hyperplasia and one well-differentiated adenocarcinoma were incorrectly diagnosed. In all functions, the volume density of epithelium was selected. The reproducibility on a new data set demonstrates an efficiency of 92, 87, and 100% for three discriminant analyses (five, four, and three diagnostic groups, respectively).