Comparative DNA image cytometry in imprint-, cytospin- and tissue section preparations of breast carcinoma.

Histologic sections, imprint preparations and cell preparations by enzymatic lysis from thick sections of cases of breast carcinomas were used to study total nuclear DNA distribution by image cytometry. Histologic sections are a prerequisite to unequivocally identify tumour cells as well as whole connective tissue cells used as a standard. While imprint preparations and lysis preparations are quite comparable with respect to distributional features, only one of them suffices to recognize these features, especially peaks at higher ploidies. Using this two-method approach to breast carcinomas, it was possible to distinguish a distribution separate from previously recognized distributions: a distinct triploid peak appears for predominantly high grade malignant ductal carcinomas.

A comparative study of DNA amount and nuclear dry weight in four different species (Rana catesbeiana, Salmo gairdneri, Gallus domesticus and Homo sapiens).

1. Quantitative electron microscopic and cytophotometric determinations of average nuclear weight, unit weight of chromatin fiber, chromatin fiber diameter and DNA amount were made and compared from lymphocytes in human, chicken, frog and trout. 2. From these determinations the "packing ratio" of DNA in chromatin fibers was calculated. 3. Nuclear mass and DNA amounts were highest in frog and lowest in chicken, while fiber diameter and mass of unit fiber measured highest in trout followed by chicken, frog and man. 4. The packing ratios were 21.4 in chicken, 35.2 in human, 47.0 in trout, 51.1 in frog and show a trend of being lower in amniotes than in anamniotes. 5. As only four species were studied, no conclusions regarding evolutionary implications or relationships between them could be made from these measurements.

Mass and molecular weight of isolated nuclear rings.

Nuclear rings are cell structures found at the nuclear cortex wedged between the nuclear envelope and the chromatin fiber network. In previous publications we have dealt with their morphology, relationships with the nuclear membranes, chromatin fibers and cytoskeletal filaments; and more recently, with their measurements at high electron microscope resolution. In this article we have calculated the mass and molecular weight of 336 isolated nuclear rings from human circulating lymphocytes using a photometric procedure and polystyrene latex spheres as the standard for weight calibration. Our results show a range of mass of 0.4-35.5 x 10(-16) g (equivalent to 0.2-21.2 x 10(8) Da with a positively skewed distribution (median: 3.3 x 10(-16) g or 2.0 x 10(8) Da). Mass and volume of nuclear rings were highly correlated. In addition, it was possible to calculate the area, the whole mass and the mass per unit area of the nuclear envelope present in the center of the nuclear rings. The mass of this area also shows a lognormal distribution (median of mass/unit area: 37.3 x 10(-8) pg/nm2 or 1.9 x 10(5) Da/nm2). We discuss the significance of this results as parameters for the characterization of the nuclear rings and their possible implications for a new interpretation of nuclear cortex architecture, nucleocytoplasmic traffic and macromolecule segregation between the two main cell compartments.

Cytophotometric determination of DNA in mesotheliomas and reactive mesothelial cells.

Cytophotometry was used to study the nuclear DNA content of cells in Feulgen-stained effusion specimens from 18 patients with mesothelioma and 14 patients with reactive mesothelial proliferations. The mean DNA content (MDNA) of mesothelioma cells was significantly higher than that of reactive mesothelial cells (P less than .001). Other parameters reflecting the DNA content also differed significantly between the two kinds of cells, including (1) the ratio of mean mesothelial DNA to mean lymphocyte DNA, (2) the percentage of mesothelial cells with DNA content exceeding three times the lymphocyte MDNA and (3) the coefficient of variation of the DNA content. Since these parameters were highly correlated, only one was accepted in a stepwise linear discriminant model for distinguishing reactive from mesotheliomatous effusions. The model correctly classified all of the reactive effusions studied and 89% of the mesotheliomatous effusions. These results indicate that DNA analysis, using the Feulgen stain and cytophotometry, yields criteria that may be useful in distinguishing benign reactive mesothelial cells from malignant mesothelioma in effusions when used in conjunction with other traditional parameters.

A comparative morphometric and cytophotometric study of intraductal hyperplasia and intraductal carcinoma of the breast.

The Feulgen DNA content and the nuclear measurements of four groups of intraductal proliferations of the breast (hyperplasia, atypical hyperplasia, well-differentiated carcinoma without cytologic atypia and intraductal carcinoma with cytologic atypia) were compared. Intraductal carcinoma with atypia was the only group distinct from the others on the basis of DNA content, nuclear area and perimeter. Although the other groups were separable from intraductal carcinoma with atypia, they could not be reliably distinguished from each other by any combination of measurements. At best, 69% of well-differentiated intraductal carcinomas could be distinguished from atypical hyperplasias using a combination of DNA content and nuclear perimeter measurements. Thus, the difficult distinction of atypical hyperplasia from well-differentiated intraductal carcinoma by light microscopy was not aided by DNA analysis or by nuclear measurements.

Morphometry of nuclear rings at high electron microscopic resolution.

In studying the fine structure of the nuclear chromatin, a new organelle was discovered, the nuclear ring. In its isolated form, this structure is rather like a geometric torus: a smooth, structureless ring. This paper presents the results of electron microscopic morphologic measurements on 424 isolated rings, i.e., rings found free of any fibrous connections. From the measurements of inner and outer diameters, other geometric features were also calculated. Overall, the size of the rings varied about fivefold. Inner and outer diameters were closely correlated, suggesting a rather stable thickness of the ring itself. The significance of the nuclear ring is as yet poorly understood. With its occasionally intimate connections to both chromatin fibers and to the inside of the inner nuclear envelope, its role is likely to be a crucial one. The membrane covering its opening is none other than the bilayer of the nuclear envelope. The concept of a patent opening or "pore" does not appear tenable in the face of this and related findings.

Mass, volume and dimensional distributions in biology, with special reference to cells.

The positively skewed distribution of mass (weight) in biology was examined, and it was concluded all weight or masses can best be described by lognormal theory. Examples are given ranging in weight from viruses to humans. The accretion of mass proceeds as "more of the same" and primarily does not alter function. Immunologic properties must be carefully preserved. Small biologic entities can afford only small mass gains or losses while preserving their functionality; the larger the entity, the larger is the variability of mass functionally permitted. A well-established, convenient graphic method of analyzing a sample is described, with the advantages and pitfalls discussed. At the cellular level, volume and dimensions are lognormally distributed whenever the specific gravity (g/cu cm) is nearly homogenous among the particles or cells. In multicellular organisms, dimensions and forms (such as height and cranial circumference) are primarily the product of multifactorial genetic determinants and frequently appear as normal distributions. This paper discusses the meaning of the law of proportionate effects for small biologic objects, especially cells, and how an initial or "elementary" distribution may be conceived.

Morphometry and cytophotometric assessment of DNA in smooth-muscle tumors (leiomyomas and leiomyosarcomas) of the gastrointestinal tract.

The difficulties in predicting the biologic behavior of gastrointestinal (GI) smooth-muscle tumors (leiomyomas and leiomyosarcomas) based on the usual criteria of malignancy are discussed. In order to evaluate the prognostic importance of the nuclear DNA content and nuclear dimensions, measurements were performed on Feulgen-stained sections of GI smooth-muscle tumors from 66 patients. The best discrimination between benign and malignant tumors was obtained by using DNA index and tumor size as descriptors in a linear discriminate analysis. This method separated 79% of the benign and 97% of the malignant smooth-muscle tumors. However, as with conventional criteria for malignancy, there remained a group of tumors close to the discriminating line with an indeterminate malignant potential. In an attempt to reduce the number of such indeterminate tumors, future studies will include the use of several descriptors in a multivariate analysis system and the application of flow cytometric studies to all tumors.

Three-dimensional reconstruction of a human metaphase chromosome from electron micrographs.

A complete human metaphase chromosome has been reconstructed from a series of electron microscopical projections obtained by tilting the specimen stage at 3 degree intervals from -60 to +60 degrees. The reconstructed structure is about 3.0 microns long, 1.6 micron wide, and 0.8 micron thick. The mass distribution was fairly homogeneous within the chromatids and neither a hollow nor a dense core was observed. The distribution and course of fibers observed are most consistent with a looping model of chromosome structure.

A chromosome's protein is a dependent variable of the total metaphase protein.

Chinese hamster metaphases were stained with dansyl chloride to determine protein. Using a microfluorometer, the fluorescence of chromosome number one and the total of the fluorescence of the metaphase were measured. The two measurements were found to be correlated with a coefficient of 0.640, suggesting that protein content in a single chromosome is a dependent variable of the protein content of the respective metaphase.

Some practical considerations in quantitative absorbance microspectrophotometry. Preparation techniques in DNA cytophotometry.

An experimental review of the Feulgen and gallocyanine-chrome-alum stains for quantitative cytophotometry of DNA in tissue sections yielded information on the preparation and staining of tissue for quantitative absorbance microspectrophotometry. (1) Tissues routinely fixed in formalin are suitable for either stain. Specimens fixed with glutaraldehyde-containing fixatives are not satisfactory for Feulgen staining, nor are ethanol-fixed specimens, unless they are post-fixed in formalin. (2) The pararosaniline dyes, used in the Feulgen stain, are sufficiently pure to use if a solution of the dye in ethanol shows an absorbance peak at 543 to 546 nm. (3) The Feulgen stain provides good reproducibility when the staining solution is adjusted to pH 1.5. (4) Gallocyanine is the best stain to use on Bouin-fixed or glutaraldehyde-fixed tissues. (5) Where fixation is an option, Carnoy and methanol-formalin-glacial acetic acid are excellent fixatives that can be followed by either stain. (6) Selection of the thickness of a tissue section involves a compromise. Requirements of minimum nuclear overlap and sharp focusing favor a section thickness of 4 micron to 6 micron. On the other hand, the requirement for full nuclear thickness, as judged by absorbance equivalent to that of a touch preparation, demands sections as thick as 8 micron in the case of mouse liver. Within this range, the optimum thickness, therefore, is determined by the particular tissue, the range of its nuclear sizes and its packing density. (7) The refractive index of the mounting medium should be closely matched to that of the background structure of the tissue sections. For animal tissues, we found that media with refractive indices of 1.54 to 1.56 are suitable.

The distribution of nuclear mass, volume and dimensions.

Our own measurements and a review of measurements presented in the literature showed that the mass, volume and dimensions of cell nuclei are distributed with a skew towards higher values, a distribution that can best be described as lognormal. The practical consequences of this finding suggest that the measurements of mass and of size should be plotted on a logarithmic scale while DNA values are appropriately presented on a linear scale. The distribution of DNA values of normal cells represents errors introduced by random disturbances in preparations and measurements.

A quality-control procedure on cervical lesions for the comparison of cytology and histology.

A new method for the quality control of cytologic and histologic diagnoses of cervical lesions is based on the automated high-resolution scanning, image processing and computer analysis of cytometric data by the TICAS system. It determines and then compares optical-density-based ploidy patterns of cells in cytologic smears and the corresponding histologic sections, with the results available both as computer-graphic displays and printouts. Examples of the former appear for an "agreement case," in which the cytologic and histologic patterns corresponded, and a noncorrespondence (nonrepresentative) case, in which the tissue sample had been nonrepresentative of the lesion sampled by cytology. Computergraphic examples concern one case of condyloma and one of tissue repair, in both of which both the cytologic and histologic diagnoses had been overcalled. A further example shows the method's use in monitoring response to therapy. The DNA ploidy patterns on which this method is based can give diagnostic and prognostic clues when morphology alone may be equivocal or insufficient. The utility of ploidy pattern determinations of material from other body sites is also well established. With the use of microprocessors, the system described could be made inexpensive and operationally simple for the routine quality control of many cytopathologic studies as well as for the clinical follow-up of patients.

Marking the origin of coordinates in high-resolution light-microscopy scanning.

In high-resolution image analysis, it is often desirable to return to a chosen cell after it has been restained or subjected to histochemical procedures. The reading of the vernier on the microscope stage is too coarse for relocating of non-distinct single cells, because the accuracy, determined by visual interpolation, is limited, at best, to 1/20th of a millimetre, or 50 microns. When one works with haematologic samples, e.g. lymphocytes, the precision of relocating a cell has to be better than 50 microns; i.e. the cell should reappear near the centre of the visual field of a X 100 oil-immersion objective. We describe a simple device by means of which distinct marks can be made on a slide with specimen (but before coverslipping) and will provide suitable origins for a coordinate system that will cover the entire preparation.

Microfluorometric studies on chromosomes. Quantitative determination of protein content of Chinese hamster chromosome 1 in situ with and without trypsin digestion.

The fluorescent stains, dansyl chloride and fluorescamine, were used to indicate the amount of protein in G-banded and unbanded chromosomes 1 of the Chinese hamster relative to the average amount of protein in human erythrocyte. Fluorescence was found to be proportional to protein mass up to the equivalent of three erythrocytes. In G-banding, trypsin digestion resulted in an average protein loss of 35.4% compared with unbanded chromosomes.

Instrumentation for the three-dimensional reconstruction of cellular organelles by electron microscopy.

Instrumental additions to the Zeiss EM-10 electron microscope are described as well as a flat-bed scanning densitometer specifically intended for the tomographic assessment of the three-dimensional structure of chromosomes. The additions to the electron microscope consist of two ion-getter pumps, reducing contamination to 0.0004 A/s. Further, a device was attached that made it possible to indicate through markers in the final negative the direction of the axis of tilt of the goniometer stage, i.e. markers indicating the tomographic axis in the digitized image. The flat-bed scanner permits correction of image rotation caused by refocusing. The preparation itself contains polystyrene latex spheres, i.e. objects the shape of which is independent of rotation (tilt). These can be used for computational corrections of the digitized image.