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Evaluation of mitochondrial content and activity with nonyl-acridine orange and rhodamine 123: flow cytometric analysis and comparison with quantitative morphometry. Comparative analysis by flow cytometry and quantitative morphometry of mitochondrial content and activity.

Mouse fibroblasts 3T3.4E and two cells lines obtained by fusion (3T3.4E cells x normal human keratinocytes), (3T3 x NHK), and (3T3.4E cells x hand wart keratinocytes), (3T3 x HWK), were compared for mitochondrial activity and content between 5 and 20 days of culture, from the 16th to 20th passage, by using Rh 123 and NAO respectively. In 3T3.4E cells both Rh 123 and NAO fluorescence were similar after 5 and 7 days of culture, indicating no modification of mitochondrial activity and content at that time. However, in cells derived from fusion of 3T3 x NHK or 3T3 x HWK, Rh 123 increased from 5 to 20 days whereas NAO fluorescence was maximal at 7 days of culture and then decreased, indicating that their mitochondrial activity differed from that of 3T3.4E cells. No difference was observed between the 16th and 20th passage. Quantitative morphometry and flow cytometry gave good correlations at 7 days of culture for the cell size, estimated either by the cell area or the cell diameter, and for mitochondria content, evaluated either by the number of mitochondria per cell or NAO fluorescence intensity.

Acridine Orange

Morphometry of bladder carcinoma: morphometry and grading complement each other.

Subjective grading of bladder carcinoma is a good predictor of the clinical outcome in those patients whose tumours are grade 1 or grade 3. However, in grade 2 tumours, which account for 45% of cases, grading has little predictive value in an individual patient. We have complemented the use of subjective grading with measurement of nuclear area and used a calculation of the distribution of nuclear sizes as a predictor of the clinical course. When subjective grading was complemented by morphometry the outcome was correctly predicted in 55 of 58 cases and all cases with poor clinical outcome were identified.

Carcinoma, Transitional Cell

The relative merits of direct morphometry of reconstructions of whole cells, and statistical morphometry by stereology of random sections of cells.

Stereology, or the derivation of quantitative, three-dimensional (3-D) data about cells by statistical analysis of the structures of random sections, is widely used in cytology and pathology. However, there are situations where this approach is inadequate, and only an analysis of a homogeneous population of whole cells will give the required results. This involved 3-D reconstruction from physical or optical sections, or tomography or photogrammetry of whole-cell mounts. Use of stereo views of individual sections or projections adds considerably to the information available for both contouring and reconstruction. Recent image-processing advances in clinical radiography have shown, for the first time, that rapid, high-resolution digitization and contrast enhancement enable nearly all structural details to be routinely extracted from the micrographs and adequately portrayed. Three-D whole-cell reconstructions provide the digital data for many kinds of morphometric measurements on both whole cells and their individual organelles and membranes. Rapid fixation or freezing allows improved quantitative structure/function correlations of organelles with disturbances in cell metabolism or gene expression.

Animals

The prognostic significance of selective nuclear morphometry in urinary bladder carcinoma.

Transurethral resected tumor specimens of 61 patients with a primary and untreated bladder carcinoma were studied by selective nuclear morphometry, a method recently described by us. A significant enlargement of the mean nuclear area was found with the advance of tumor grade and stage (Wilcoxon, P less than .0001 and P less than .0001). The heterogeneity of the grade 2 patient group and the additional value of morphometry were demonstrated by observing the 5-year survival rates. Patients with grade 2 carcinoma could be separated into one subgroup with small nuclei (mean nuclear area less than or equal to 95 microns2) having a favorable outcome (5-year survival rate: 100%), and into another subgroup with large nuclei (mean nuclear area greater than 95 microns2) showing a worse prognosis (5-year survival rate: 63.2%) (Mantel-Cox, P = .01). The outcome of these subgroups was not significantly different from that of the grade 1 (5-year survival rate: 93.8%) and grade 3 (5-year survival rate: 50%) patients, respectively (Mantel-Cox, P = .45 and P = .57). The value of selective nuclear morphometry, in addition to tumor staging, was indicated by the association of nuclear enlargement (mean nuclear area greater than 95 microns2) with progressive recurrence (five of 15 patients; 33%) among the patients with conservatively treated superficial carcinoma (stages Ta and Tl). The findings demonstrate the supplementary value of selective nuclear morphometry to tumor grading and staging, especially in the heterogeneous group of grade 2 carcinomas and the group of superficial tumors (stages Ta and Tl).

Aged

Effect of maturational changes in myosin content and morphometry on airway smooth muscle contraction.

We studied the relationship of airway morphometry, the content of myosin heavy-chain and isoform stoichiometry, and the distribution of bronchoconstrictor responses in the airways of maturing swine. Lungs were excised in 2-wk-old farm swine (2ws; n = 13) and 10-wk-old swine (10ws; n = 13), and tracheal smooth muscle strips and bronchial rings from generations 2-5 were fixed for in vitro isometric measurement of force generation. Split samples were placed in formaldehyde solution or glutaraldehyde for light- or electron-microscopic morphometry or frozen for analysis of tissue myosin content. The rank order of force generation elicited by both receptor- and nonreceptor-dependent mechanisms for both 2ws and 10ws was generation 4 greater than 3 greater than or equal to 2. For all matched airway generations, contractile force was 25-100% greater in 2ws than 10ws. Differences in force generation were not related to morphometric differences in smooth muscle mass content among airways. The relative cross-sectional area of smooth muscle derived by computerized morphometry was 5.5-7% for each airway generation and did not change with age. Electron-microscopic morphometry demonstrated comparable myocyte content within muscle bundles for all airways in both age groups. In generation 4 airways, myocyte size in 2ws (27.3 +/- 0.8 nuclei/2,500 microns2) hypertrophied approximately 15% in 10ws (20.4 +/- 0.6 nuclei/2,500 microns2; P less than 0.01). Tissue content of myosin measured by computerized laser densitometry of gel electrophoresis of homogenates was greater in trachea from 2ws than 10ws (135 +/- 10 vs. 90 +/- 4 micrograms/g tissue; P less than 0.01); homology of 200- and 205-kDa isoforms was confirmed by Western blot against polyclonal myosin antibody and Cleveland digest analysis of each band. Differences in contractile forces between generations in 2ws and 10ws were not correlated to functional myosin isoform content. We demonstrate a maturational downregulation of contractile forces in maturing swine. This response is independent of smooth muscle receptor distribution and is not related to morphological changes in airways muscle mass, cellularity, changes in content of nonmyocyte tissues, or tissue content of functional myosin isoform.

Aging

Morphometrical evaluation of acute leukemic cells by electron microscopy. Discrepancy between morphological characteristics in FAB classification and electron microscopic morphometry.

Leukemic cells from 39 patients with acute leukemia (20 lymphocytic and 19 myelogenous) were examined by transmission electron microscopy and the nucleus and cytoplasm were measured on the micrographs with a computer-controlled image analyzer. The ratios between the areas of the nucleus and whole cell profile (nucleus/cell ratio), heterochromatin and euchromatin, the nucleolus and nucleus, and the degree of irregularity of the nucleus were compared between the two major types of leukemia studied. Acute lymphocytic leukemia (ALL) cells had a relatively larger nucleus and relatively less cytoplasm than acute myelogenous leukemia (AML) cells, and a greater proportion of the area of the nucleus was occupied by heterochromatin in ALL cells than in AML cells. According to the FAB classification, L1 cells are characterized by narrow, and L2 cells by wide cytoplasm based on light microscopic observation of smeared cells, and we confirmed these features by morphometry of May-Giemsa-stained blood smears. However, by electron microscopy there was no difference in the nucleus/cell ratio between L1 and L2 cells, this constituting a discrepancy between the results obtained by electron and light microscopic morphometry. In addition there was no difference in the degree of nuclear irregularity between L1 and L2 cells. Among AML subtypes, significant differences were observed only in the nucleus/cell ratio between M3 and M1, M2 or M4 cells, and in the heterochromatin/euchromatin ratio between M5 and M1, M2 or M3 cells. In conclusion, electron microscopic morphometry revealed marked differences between ALL and AML, but the differences among their subtypes defined by the FAB classification based on nonmorphometric light microscopy were less evident by electron microscopic morphometry.

Cell Nucleus

Effects of tributyltin on survival, growth, morphometry, and RNA-DNA ratio of larval striped bass, Morone saxatilis.

Effects of tributyltin (TBT) on survival, growth, morphometry, and RNA-DNA ratio in larval striped bass (Morone saxatilis) were assessed in three experiments. In Experiment I, 13 day old larvae were exposed to 0, 0.067, 0.766, or 2.284 micrograms TBT/L for 6 days. All larvae exposed to 2.284 micrograms/L died by day 5; exposure to 0.766 micrograms/L significantly reduced survival relative to controls (59.8% vs. 81.8%). Significant, concentration-dependent decreases in body depth occurred in larvae exposed to 0.067 and 0.766 micrograms/L. In Experiment II, all 16 day old larvae exposed to 1.498 micrograms/L died by day 6. Survival, weight, and morphometry parameters were not significantly different in larvae exposed to 0, 0.052, or 0.444 micrograms/L for 6 days. In Experiment III, survival was similar in 21 day old larvae exposed to 0, 0.055, 0.281, or 0.514 micrograms/L for 7 days. Notochord length and dry weight decreased significantly in larvae exposed to 0.514 micrograms/L. Weight and morphometry parameters were more sensitive indicators of sublethal stress than RNA-DNA ratio. Maximum TBT concentrations reported in Chesapeake Bay marinas are likely to cause increased larval mortality. Longer-term studies are needed to assess effects at less than 0.050 micrograms/L, which may be more representative of habitat conditions.

Animals

Modelling steady state pulmonary elimination of He, SF6 and CO2: effect of morphometry.

We studied the influence of acinar morphometry on the shape of simulated expirograms computed from a single path convection-diffusion model that includes a source term for gas evolution from the blood (Scherer et al., J. Appl. Physiol. 64: 1022-1029, 1988). Acinar structure was obtained from published data of 3 different lung morphometries. The simulations were performed over a range of tidal volumes (VT) and breathing frequencies (f) comparable to those observed in a previously reported human study. Airways dead space (VDaw) increased with VT in all the morphometric models tested and in the experimental data. The increase in VDaw with VT was inversely related to the diffusivity of the evolving gas and to the rate of increase in airway cross-section of the most mouthward (proximal) alveolated generations of the models. Normalized phase III slope for all the gases decreased with increasing VT in all the models as was previously reported for healthy human subjects. In the model simulations, the greatest sensitivity of phase III slope to VT was seen with the least diffusible gas using the airway morphometry with the smallest cross-sectional areas in the proximal alveolated generations. We conclude that both VDaw and phase III slope of an evolving gas are sensitive to the geometry of the proximal acinar airways and that this is manifest by their dependence on tidal volume, breathing frequency, molecular diffusivity and alveolar/blood source emission rate. The model simulations indicate that heterogeneity of gas washout is not required to explain the magnitude of the phase III slope in healthy human subjects.

Carbon Dioxide

DNA ploidy, morphometry, and nuclear grade as prognostic factors in endometrial carcinoma.

In a retrospective analysis of 106 cases of endometrial carcinoma stages I-IV (FIGO), the prognostic value of DNA ploidy and nuclear morphometry of tumor cells was evaluated and compared with that of conventional clinical and histopathologic parameters. Paraffin-embedded tumor tissue from the original curettage specimens was used. A flow cytometric technique was employed to distinguish diploid from aneuploid tumors. It was not possible to estimate S-phase rates by this method. Eight different nucleus-related morphometric parameters were computed from representative tumor regions on the original slides. All histologic specimens were reviewed by on the pathologist and graded according to FIGO; nuclear grade was determined separately. Tumor stage, depth of myometrial infiltration, and nuclear grade were the most important prognostic factors with regard to tumor-related survival. DNA ploidy and nuclear morphometry did not add significant prognostic information that could be used to distinguish high-risk and low-risk populations with endometrial carcinomas. The simple nuclear grading system should be further evaluated in prospective studies and compared with DNA analysis and nuclear morphometry performed on fresh-frozen tissue.

Adult

Polychlorinated biphenyl (Aroclor 1254) induced changes in femur morphometry calcium metabolism and nephrotoxicity.

Experiments were performed to investigate the effects of polychlorinated biphenyl (PCB) on calcium metabolism, femur morphometry, and nephrotoxicity. Male Fischer 344 rats were dosed daily intragastrically (ig) for 5, 10 or 15 weeks with 0, 0.1, 1, 10 or 25 mg PCB/kg body weight. After 5, 10 and 15 weeks, liver weight and liver-to-body weight ratio were increased at the 2 higher dose levels. Overt toxicity was observed at the highest dose level after 10 and 15 weeks as evidenced by significantly smaller body weight. Urinary alkaline phosphatase and lactate dehydrogenase activities were elevated at 5, 10 and 15 weeks of PCB exposure and the kidney-to-body weight ratios were elevated at the 10 and 25 mg/kg dose levels after 10 and 15 weeks of exposure indicating nephrotoxicity. Hypercalcemia was present at the highest dose level after 5 and 10 weeks of exposure but serum calcium concentration was normal at 15 weeks. Serum triglycerides were significantly elevated after 5 weeks of exposure but were significantly decreased after 10 and 15 weeks of PCB exposure. Serum cholesterol was significantly elevated at the 2 higher dose levels at all 3 periods. Femur density was increased at the 10 mg/kg dose level after 5 weeks, at all dose levels after 10 weeks and at all dose levels which did not demonstrate overt toxicity after 15 weeks of PCB exposure. Cross-sectional, medullary and cortical areas of the midpoint of the femur were significantly decreased at the higher dose levels after 10 and 15 weeks of exposure. The percent medullary area though was significantly decreased after 10 and 15 weeks of PCB exposure indicating that not only was there a decrease in medullary size but a decrease relative to the cortical bone area. These changes in bone morphometry resulted in a significantly weaker bone after 15 weeks of exposure at the highest dose level. These results demonstrate that PCB exposure effects calcium metabolism and bone morphometry and is nephrotoxic.

Animals

Nuclear morphometry as a predictor of response to therapy in Wilms tumor: a preliminary report.

Present therapy in Wilms tumor is based upon prognosis. Current trends suggest less intensive chemotherapy and/or radiation therapy for children with favorable histological disease who are considered at low risk for recurrence and increasing the amount of therapy for those with unfavorable histological tumors who are at high risk for recurrence. Currently, pathological stage and histological appearance are used to predict prognosis. We tested a new approach. Histological sections of surgical specimens were obtained from 9 children treated for unilateral Wilms tumor. All patients had favorable histological disease, and received postoperative chemotherapy and/or radiotherapy. These specimens were evaluated by nuclear morphometry with the Hopkins Morphometry System to determine if nuclear shape descriptors could distinguish children who responded to therapy from those whose tumor recurred. Nuclear roundness, ellipticity, convexity and bending energy accurately separated the groups. Nuclear morphometry may provide useful information that will aid in predicting which children with Wilms tumor will respond to therapy and warrants further investigations.

Cell Nucleus

Light and electron microscopical morphometry of pituitary adenomas in hyperprolactinemia.

Two highly differentiated acidophil prolactin-cell adenomas with hyperprolactinemia (group I), 8 large cell chromophobe adenomas with hyperprolactinemia (group II), and 2 small cell chromophobe adenomas (group III), one of which was combined with hyperprolactinemia, were studied immunohistologically. Morphometry was performed on the light- and electron microscopical level. The 11 active adenomas were immunohistologically positive for prolactin, the 12th adenoma with normal prolactin plasma level was negative for prolactin. Light microscopical morphometry displayed significantly more cells of smaller size in the "small cell chromophobe" adenomas, whereas the large cell chromophobe adenomas and the highly differentiated prolactin cell adenomas were not different. Ultrastructural morphometry demonstrated significant differences between highly differentiated prolactin cell adenomas (group I), and large cell chromophobe adenomas (group II). The latter contain smaller "relative volumes" of nucleoli and of secretory granules, whereas the rough endoplasmic reticulum, the Golgi fields and the nuclei were not different. Comparison of large cell chromophobe adenomas (group II), and small cell chromophobe adenomas (group III) revealed significantly larger relative volumes of nuclei and of mitochondria but smaller volumes of rough endoplasmic reticulum and of Golgi fields in the small cell chromophobe adenomas. Significant differences between the active and the inactive adenoma of small cell chromophobe type in the group III were not found. In spite of the low quantity of small cell chromophobe adenomas and of acidophil prolactin cell adenomas, our data demonstrate that there exist distinct and significant light microscopical and ultrastructural differences between the three adenoma types.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenoma, Acidophil

Intestinal mucosal morphometry and ileal epithelial renewal in conventional and germ-free rats fed an amylomaize starch diet.

Intestinal mucosal morphometry and ileal epithelial renewal were studied in conventional (CV) and germ-free (GF) rats fed either poorly digestible amylomaize or normal maize starch diets. Intestinal morphometry and position of labelled enterocytes were studied at various times after tritiated thymidine injection. With amylomaize starch diet, no difference was observed in the size of crypts (C), villi (V) and C + V between duodenum and jejunum both in CV and GF rats. In the ileum, however, values were significantly lower than those in the duodenum and jejunum. Furthermore, the presence of the microbial flora led to higher values when compared with GF values. Despite the morphological modifications in the ileum, no significant difference was detected in the labelled cell positions and epithelial renewal time between CV and GF values. This suggests that the resistant part of amylomaize starch was responsible for the modification in mucosal morphometry and the longer ileal epithelium renewal time in CV rats which then becomes similar to that in GF rats.

Amylopectin

The relative prognostic significance of nucleolar morphometry in invasive ductal breast cancer.

In a group of 65 patients with invasive ductal breast cancer with adequate follow-up for at least 5 years, the prognostic significance of nucleolar area morphometry was investigated. In general, non-survivors have larger and more pleomorphic nucleoli. A value of the standard deviation of the nucleolar area above 2.49 micron2 was almost exclusively found in non-survivors. This is the more interesting because several of these patients had a low mitotic activity index and small tumours, some of these having negative lymph nodes. Without nucleolar morphometry these patients would have been included in a favourable prognosis group. Although the sensitivity of nucleolar morphometry is low, the method can be used as an additional prognostic indicator. This is more important as nucleoli can in principle be automatically measured with digital image processing computers.

Breast Neoplasms

Respiratory muscle fiber morphometry. Correlation with pulmonary function and nutrition.

To examine the relationship between nutrition, pulmonary function, respiratory muscle strength, and respiratory muscle morphometry, we compared physiologic data and muscle morphometry obtained from internal intercostal, external intercostal, and latissimus dorsi muscle biopsies in 68 patients who were having a thoracotomy. We stained the biopsies for myosin ATPase and measured the proportions and diameters of the type 1 and type 2 fibers. There were more qualitative changes in the external intercostal muscles than in the other two, and some of these changes related to the incidence of malignancy. There were more type 1 fibers in the external intercostal (64 +/- 10 percent) and internal intercostal muscles (59 +/- 12 percent) than in the latissimus dorsi (44 +/- 13 percent) (p less than 0.005). The mean diameter of the type 2 fibers in the external intercostal muscles was less (44 mu +/- 7 mu) than the diameter in the latissimus dorsi (51 mu +/- 9 mu) and the internal intercostal muscles (52 mu +/- 8 mu) (p less than 0.01). The diameters of both fiber types were greater in men than in women. There was no significant relationship between measures of pulmonary function or respiratory muscle strength and muscle fiber proportions and diameters. There were significant correlations between the percentage of ideal body weight and type 1 and type 2 fiber diameters. We conclude that sex and nutrition influence respiratory muscle morphometry.

Biopsy

Multiparametric discrimination of serous ovarian tumors by analytical morphometry.

In order to enhance the discrimination power in the field of serous ovarian tumors, we applied the software system SAM (Shape Analytical Morphometry) to the analytic studies of biological forms. Besides the usual dimensional evaluations (perimeter, area, maximum diameter and shape index), this procedure permits the description of the nuclear form using analytical parameters: 1) extraction of nucleus fundamental curve; that is a functional curve giving the "smoothing" of the original contour by two parametric equations (separately for x and y values as independent variables); 2) evaluation of nuclei contour irregularities by Fourier analysis; 3) evaluation of shape asymmetry by SAE (Shape Asymmetry Evaluator); that is the ratio between the length of a segment of a parabola interpolating the original curve points, and a straight line joining its extremities for a 180 degrees barycentric rotation according 10 degrees steps. All parameters resulted to be independent and were submitted to multivariate discriminant analysis. We studied 180 nuclei from 18 cases of serous ovarian tumors, (6 benign, 6 borderline and 6 malignant tumors). With respect to the dimensional parameters, the application of analytical morphometry permitted us to reduce the minimum percentage error in the discrimination of the different classes. In fact, in the distinctions of benign and malignant nuclei, the minimum percentage error was 13.30%, against the 18.3% error when using dimensional morphometry. Furthermore, in the comparison of malignant and borderline nuclei there was a reduction of error from 23.3% to 22.5%, and in the comparison of benign and borderline nuclei, the error was reduced from 37.5% to 30%.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Nucleus

A longitudinal study of metacarpal bone morphometry in anorexia nervosa.

Osteoporosis is a known complication of anorexia nervosa. Although calorie and mineral malnutrition may contribute to changes in bone mass and morphometry, hypoestrogenism is thought to be the most important etiologic factor. In a seven-year longitudinal study of six women aged 19 to 35 years with adolescent-onset anorexia nervosa, the objective was to correlate menstruation and bone morphometry. At the onset of the study, five women were amenorrheic and had abnormal metacarpal bone morphometry. After seven years, three women remained amenorrheic and below 85% of ideal body weight. Anteroposterior roentgenographs of the nondominant second metacarpal taken at the beginning and end of the study revealed an increase in medullary canal diameter (p less than 0.03) and medullary area (p less than 0.04) and a decrease in combined cortical thickness (p less than 0.04) and percent cortical area (p less than 0.02). These findings suggest progressive endosteal resorption in the absence of compensatory periosteal apposition. Such bone remodeling characteristics are distinctly abnormal in this age group. The three women who regained menses showed up to one third less endosteal resorption and less cortical thinning than did the three women who remained amenorrheic. Resumption of menses may be an important milestone in preventing further cortical bone loss in anorexia nervosa.

Adult

Some fundamental aspects of morphometry in clinical pathology, demonstrated on a simple, multipurpose analysis system.

The aim of this study was (1) to investigate the value of morphometry, (2) to fix a set of parameters suitable for analyzing diagnostic problems, and (3) to create a general strategy for data storage and for user-friendly data management. The intrinsic value of morphometry lies in the fact that in contrast to other morphologic methods, it permits the presentation of findings in the form of numbers. The following set of morphometric parameters, in the broad sense of the term morphometry, is standard in our laboratory: planimetric parameters (shape descriptors), parameters of the gray value histogram (descriptors of the general gray value distribution), texture parameters (descriptors of the correlation between various image segments), invariant moments (descriptors of the size and localization of textural image segments) and densitometric parameters. The introduction of morphometric procedures into the daily routine is facilitated if data registration and evaluation are performed separately. Original data generated by direct measurement are primary or raw data, which are stored as such. In a separate, second step these raw data are used to compare more or less complex morphometric parameters, which are called "secondary data". A system designed for separate data registration and evaluation can easily be adapted to new methodologic developments. For instance, primary data on objects (gray values, coordinates of the contour) measured one time in the past can be reused at any other time for computing new features from these data. This procedure is comparable to the possibilities in immunohistochemical staining: new immunohistochemical stains can be applied to newly prepared sections of old tissue blocks.

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