Nuclear morphometry of nodular malignant melanomas and benign nevocytic nevi.
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Adult↗
Biomedical subjects
L M Cruz-Orive
Publications and source records attributed to L M Cruz-Orive.
Quantitation of chondrocyte performance in growth-plate cartilage during longitudinal bone growth.
The longitudinal growth of bone depends on the activities of individual chondrocytes of the growth plate. Each chondrocyte remains in a fixed location throughout its life, and there accomplishes all of its functions. Although a cell may perform several or all of its activities simultaneously, one of these will usually predominate during a particular phase of its life. The two most prominent stages are those of cellular proliferation and hypertrophy (including the mineralization of matrix) before the resorption of tissue during vascular invasion. By applying recently developed stereological procedures and improved methods for the fixation of cartilage, we compared cellular shape modulation, various ultrastructural parameters (surface areas or volumes of endoplasmic reticulum, Golgi membranes, and mitochondria), the production of matrix, and cellular turnover for proliferating and hypertrophic chondrocytes within the proximal tibial growth plate of the rat. By the late hypertrophic stage, fourfold and tenfold increases in the mean cellular height and volume, respectively, and a threefold increase in the mean volume of the matrix per cell were achieved. The high metabolic activity of hypertrophic cells was reflected by a twofold to fivefold increase in the mean cellular surface area of rough endoplasmic reticulum, the Golgi membranes, and the mean cellular mitochondrial volume. Rates of longitudinal growth were determined by fluorochrome labeling and incident-light fluorescence microscopy. Using these values and the stereological estimators describing cellular height, the rates of cellular turnover were calculated. The rapid progression of the vascular invasion front was found to eliminate, for each column of cells, one chondrocyte every three hours; that is, eight cells a day. The maintenance of a steady-state structure for growth-plate cartilage in rats in a steady state of growth thus necessitates efficient compensation for these losses, which is achieved by a high rate of cellular proliferation and rapid hypertrophy.
Animals↗
Particle number can be estimated using a disector of unknown thickness: the selector.
A new stereological method is presented whereby the number of particles in a material can be estimated unbiasedly using a few parallel sections from a block of the material, without having to know either section thickness or the distance between sections, and irrespective of particle size, shape and orientation. All that is needed is the final magnification. The method consists of three steps. Firstly, a few particles are sampled with identical probabilities using a disector (Sterio, 1984) of unknown thickness, namely a selector. Secondly, the volume of each sampled particle is unbiasedly estimated by means of point-sampled intercepts (Gundersen & Jensen, 1985) on a few particle transects. Thus, the mean individual particle volume vN in the population is estimated directly without having to refer to any containing volume. Finally, the particle volume fraction VV is estimated on a random section (by point-counting, say) and the numerical density of particles is estimated via the identity NV = VV/vN. Multiplying the estimate of NV with the reference volume we get an estimate of the total number of particles. The selector may be used not only for counting but also for choosing particles with identical probabilities in order to make, say, serial reconstructions of them. The practical details of the counting method are explained with the aid of a worked synthetic example and a real one involving hepatocytic nuclei. The mathematical proofs are given in two appendices.
Animals↗
Molecular architecture of the inner membrane of mitochondria from rat liver: a combined biochemical and stereological study.
The molecular structure of mitochondria and their inner membrane has been studied using a combined approach of stereology and biochemistry. The amount of mitochondrial structures (volume, number, surface area of inner membrane) in a purified preparation of mitochondria from rat liver was estimated by stereological procedures. In the same preparation, the oxidative activity of the respiratory chain with different substrates and the concentration of the redox complexes were measured by biochemical means. By relating the stereological and biochemical data, it was estimated that the individual mitochondrion isolated from rat liver has a volume of 0.27 micron 3, an inner membrane area of 6.5 micron 2, and contains between 2,600 (complex I) and 15,600 (aa3) redox complexes which produce an electron flow of over 100,000 electrons per second with pyruvate as substrate. The individual redox complexes and the H+-ATPase together occur at a density of approximately 7,500/micron 2 and occupy approximately 40% of the inner membrane area. From the respective densities it was concluded that the mean nearest distance between reaction partners is small enough (70-200 A) to cause the formation of micro-aggregates. The meaning of these results for the mechanism of mitochondrial energy transduction is discussed.
Animals↗
Consistent and efficient delineation of reference spaces for light microscopical stereology using a laser microbeam system.
A serious problem in stereology is to ensure a consistent definition of reference spaces at different levels of magnification whenever the boundaries of such reference spaces are either fuzzy or non-existent, and hence they have to be defined artificially. (It is well known that inconsistent definitions of the reference space leads to unknown amounts of bias in stereological results.) In this paper a new application is found for the laser microbeam system used in microdissection, whereby the required boundaries can be easily and neatly traced (in fact, cut) directly onto uncovered sections for light microscopy. The dangers of bias inherent from inconsistencies of definition are thereby eliminated completely, and alternative, very expensive procedures requiring direct marking of paper prints with a pen are no longer necessary.
Animals↗
Stereology for anisotropic cells: application to growth cartilage.
A number of either new or recently available stereological methods are described for estimating volume, surface area and number of anisotropic cells. The methods are illustrated with direct reference to the epiphyseal growth plate. Different estimates of a given quantity are obtained by applying alternative methods to the same set of sections, in order to compare the relative merits of the methods. For instance, the surface area of the cells is estimated via the Dimroth-Watson model (which gives a measure of the degree of anisotropy in addition to the surface area estimate) and from vertical sections using cycloid test systems. Cell number is estimated by traditional unfolding methods and by the new disector method. Also, volume-weighted mean cell volume is estimated from vertical sections via point-sampled intercepts using two different kinds of rulers to classify intercept lengths. Finally, nested design statistics is applied to a set of data from twelve animals in order to compare the relative impacts of biological and stereological (sampling) variations on the observed coefficient of error of a group mean estimate. The preferred methods are listed in the final section.
Animals↗
Estimation of surface area from vertical sections.
'Vertical' sections are plane sections longitudinal to a fixed (but arbitrary) axial direction. Examples are sections of a cylinder parallel to the central axis; and sections of a flat slab normal to the plane of the slab. Vertical sections of any object can be generated by placing the object on a table and taking sections perpendicular to the plane of the table. The standard methods of stereology assume isotropic random sections, and are not applicable to this kind of biased sampling. However, by using specially designed test systems, one can obtain an unbiased estimate of surface area. General principles of stereology for vertical sections are outlined. No assumptions are necessary about the shape or orientation distribution of the structure. Vertical section stereology is valid on the same terms as standard stereological methods for isotropic random sections. The range of structural quantities that can be estimated from vertical sections includes VV, NV, SV and the volume-weighted mean particle volume vV, but not LV. There is complete freedom to choose the vertical axis direction, which makes the sampling procedure simple and 'natural'. Practical sampling procedures for implementation of the ideas are described, and illustrated by examples.
Animals↗
Morphometric and biochemical analysis of adrenal medullary hyperplasia induced by nicotine in rats.
The administration of nicotine (1 and 4 mg/kg/day, subcutaneously for up to 6 weeks) to male Sprague-Dawley rats first induced a time-and dose-dependent increase in catecholamine (CA) synthesis. This was followed by an increase in endogenous CA levels and in total volume and number of chromaffin cells, as measured by stereological methods on serially sectioned adrenal glands. Thus, continued stimulation of the sympathoadrenal system generated an increase in biosynthetic enzyme activity, and subsequently adrenal medullary hypertrophy and hyperplasia developed as an adaptive reaction. The proposed model is useful for quantifying both biochemically and morphometrically early adrenal changes long before irreversible pathologic alterations are manifested.
Adrenal Glands↗
Estimating length density and quantifying anisotropy in skeletal muscle capillaries.
The accurate estimation of stereological parameters defined on anisotropic structures is a long-standing problem. In this paper we seek to estimate the capillary length density JV in skeletal muscle tissue. A well-known model for directional anisotropy in space, namely the 'spherical normal' or 'Fisher axial distribution' model, is found to fit the relevant data satisfactorily. Based on this model, a short-cut estimation method is proposed and illustrated with a numerical example. This method essentially consists in taking the ratio of mean capillary profile counts, as obtained from transversal and longitudinal sections of the muscle tissue, and making use of a table or a graph given in the paper to estimate JV. The conditions under which the methods are applicable and practicable are discussed in detail. Apart from an accurate estimation of JV, an important feature of our method is the possibility of quantifying the degree of anisotropy by a coefficient K (called the concentration parameter of the Fisher axial distribution), which enjoys both a biological significance and a sound statistical basis.
Animals↗
Design of the mammalian respiratory system. IV Morphometric estimation of pulmonary diffusing capacity; critical evaluation of new sampling method.
The quality of morphometric data obtained by stereological methods depends on the sampling scheme adopted. In the light of recent advances in sampling theory for stereology a sampling strategy has been developed which differs significantly from that used in previous studies. It operates with a cascade of four sampling levels and adopts the principle of A-weighted sampling, a method that should eliminate bias and possibly yield smaller variances. This method is described and discussed in detail. When compared with the old simpler sampling approach it is found that the new method (1) is at least as efficient and (2) that the estimates of morphometric parameters obtained by this method are similar to the data generated by the old method.
Animals↗
Measuring error and sampling variation in stereology: comparison of the efficiency of various methods for planar image analysis.
An evaluation is made of the relative efficiency (precision of the final estimate per unit time of measurement on a given set of sections) of different methods for planar analysis aimed at estimating aggregate, overall stereological parameters (such as VV, SV). The methods tested are point-counting with different densities of test points (4 less than or equal to PT less than 900 per picture), semiautomatic computer image analysis with MOP and automatic image analysis with Quantimet, for obtaining VV and SV estimates. One biological sample as well as three synthetic model structures with known coefficients of variation between sections are used. The standard error of an estimate is mainly determined by the coefficient of variation between sampling units (= sections in the present paper) so that measuring each sample unit with a very high precision is not necessary. Automatic image analysis and point-counting with a 100-point grid were the most efficient methods for reducing the relative standard errors of the VV and SV estimates to equivalent levels in the synthetic models. Using a 64-point grid was as precise, and about 11 times faster than using a tracing device for obtaining the estimate of VV in the biological sample.
Animals↗
Stereological estimation of volume ratios by systematic sections.
Two related problems are explored. Firstly, a single opaque solid omega 2, is considered. The problem is to estimate the minimum number of systematic sections m, necessary to estimate the volume ratio v = V (omega 2)/V (omega 1) with a coefficient of error or at most gamma 0 with a probability 1 - alpha. Secondly, we consider a population of such specimens. The second problem is to estimate the optimum number of n of specimens to be sampled and the number n of systematic sections per specimen in order to estimate the mean volume ratio v of the population with a relative error of at most epsilon 0 with a probability 1 - alpha. General guidelines for solving the two problems are presented. Practical results applicable to two populations of mouse and guinea-pid lymph nodes, exhibiting a wide variation in size and shape, are obtained.
Animals↗
Sampling designs for stereology.
The purpose of this paper is to propose the necessary sampling techniques for estimating a global parameter defined in a solid opaque specimen (e.g. the total volume of mitochondria in a given liver, the total capillary surface area in a given lung, etc). The geometry of the specimen often suggests a multi-level or cascade sampling design at different magnifications, whereby the object phase at one level becomes the reference phase in the next level. The final parameter is then estimated as the product of the intermediate ratios with the volume of the specimen, which is estimated independently. Each level can be regarded as an independent sampling design; a given stereological project may be planned in terms of one or more of these designs. Our development is a blend of practical experience and recent theoretical advances on sampling for stereology with well-known sampling techniques previously developed with different purposes in mind.
Anatomy↗
Comparison of two subsampling methods for electron microscopic morphometry.
The purpose of the present paper is twofold. Firstly, to compare the accuracy per unit cost achieved by two different methods of subsampling micrographs from sections of a material for stereology when the sections cannot be analysed as a whole at the required magnification. Secondly, to illustrate, by means of real data, the application of some of the methods and formulae proposed in the companion paper (Cruz-Orive & Weibel, 1981) for estimating ratios at the electron microscopic level. The final estimates of a same ratio obtained by either subsampling method (namely systematic (SQ) and systematic area-weighted quadrats (SAWQ)) agreed in the mean and they were about equally precise. The former fact indicates that the new SAWQ method is at least as reliable as the SQ method as far as bias is concerned. The latter result is a consequence of the well-known fact that subsampling is relatively unimportant in two-stage sampling. Yet, SAWQ subsampling enjoys definite practical advantages over other subsampling methods in certain situations.
Anatomy↗
Does lack of Cftr gene lead to developmental abnormalities in the lung?
Lung disease is the major cause of death in cystic fibrosis (CF), but the effect of gene mutation on the morphology of the main structural compartments of the lung is poorly understood. We show here, to our knowledge for the first time, a quantitative comparison of the fine pulmonary structures of cftr mutant versus non-cf mice. Pertinent volumes and surface areas were estimated in 10 homozygous cftrm1HGU mutants and 11 non-cf littermates by unbiased stereology at the light microscopic level. Our data did not reveal any statistical differences between group means for any of the 9 parameters considered. In other words, our data do not supply any significant evidence that the lack of the Cftr gene is accompanied by any developmental abnormalities in the lung, at least as far as the parameters studied are concerned.
Age Factors↗
Estimation of brain compartment volume from MR Cavalieri slices.
PURPOSE: Recent theory has been developed to estimate volume from a systematic sample of tissue slices of a given thickness and to predict the corresponding error. Our goal was to check the error prediction formulas by resampling and to determine the minimum number of MR slices required to estimate the volumes of the cerebrum and of the compartments of gray matter (GM) and white matter (WM) with prescribed errors. METHOD: Our working data set comprised the GM and WM segmentations obtained from a paradigmatic high signal-to-noise ratio 3D spoiled GRASS MR volume data set for a single healthy human subject. The data were classified using a fuzzy clustering minimum distance algorithm. We thereby obtained a stack of 183 serial coronal slices of 1 mm thickness encompassing the whole cerebrum. Empirical resampling was carried out using the corresponding data vectors, and the theoretical error predictors were thereby checked for slice thicknesses of 1, 3, 9, and 27 mm, with a distance of 45 mm between slice midplanes. RESULTS: Irrespective of slice thickness, a minimum of 3, 5, and 10 slices provided estimates of the true total volume of GM and WM in the cerebrum with coefficients of error (CEs) of 10, 5, and 3%, respectively, where CE(V)% = 100 x SE(V)/V. For the cerebrum, a minimum of two, three, and four slices were required for CEs of the same precision. CONCLUSION: In combination with high signal-to-noise ratio and enhanced tissue contrast, Cavalieri slices are the most appropriate for MRI, they supply unbiased and highly efficient volume estimates of brain compartments. For a given number of slices, CE(V) decreases rapidly when the slices are thicker than the gaps between them; when the slices are thinner than the gaps, then CE(V) is similar to that in the situation when the slice thickness is zero.
Brain↗