RESUMEN
OBJECTIVE: To compare two visual enumeration methods to determine whether the confocal approach yielded better counts of chromosome-specific hybridization sites. STUDY DESIGN: Brightfield microscopy was used to count in situ hybridization (ISH) sites in 4-microns tissue sections. Confocal microscopy was used to collect three-dimensional (3D) data sets from fluorescence in situ hybridization (FISH) preparations made with sections of various thicknesses. Analysis of the confocal images relied on custom-built interactive visualization software. RESULTS: The confocal method yielded higher average counts of hybridization sites per nucleus due to fewer truncated nuclei in thicker sections and to visual exclusion of the truncated nuclei that remained. Optimal section thickness was 8-12 microns. Limited penetration by FISH reagents restricted the use of thicker sections. CONCLUSION: Analysis of intact nuclei visualized in three dimensions was more sensitive in demonstrating high centromere number than was brightfield ISH analysis of 4-microns sections. Improvements in semiautomated interactive software may make the confocal approach practical for accurate evaluation of chromosome number in precise histologic contexts.
Asunto(s)
Cromosomas/ultraestructura , Hibridación Fluorescente in Situ/métodos , Hibridación in Situ/métodos , Interfase , Carcinoma de Células Transicionales/química , Centrómero/química , Cromosomas Humanos Par 17/química , Técnicas Histológicas , Humanos , Procesamiento de Imagen Asistido por Computador , Microscopía Confocal , Propidio/análisis , Neoplasias de la Vejiga Urinaria/químicaRESUMEN
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.
Asunto(s)
Núcleo Celular/química , ADN/sangre , Linfocitos/ultraestructura , Animales , Núcleo Celular/ultraestructura , Pollos , Cromatina/química , Hominidae , Humanos , Microscopía Electrónica , Rana catesbeiana , Salmón , Especificidad de la EspecieRESUMEN
Objective features have been identified that assist in distinguishing sclerosing adenosis from tubular carcinoma of the breast. Hematoxylin and eosin stained paraffin sections of 18 sclerosing adenoses and 18 tubular carcinomas were studied using a TAS Plus image analysis system. Histological measurements from lumens and glands included stereologic features of architecture and morphometry of size and shape (the latter by Fourier coefficients). Cytological measurements included nuclear area, perimeter, diameter and formfactor. Initial analysis suggested utility for several individual features. However, after a modified Bonferroni procedure only two of the features were statistically significant, i.e. the coefficient of variation of luminal form factor and the surface density of glands. Multivariate discriminant analysis using these two variables correctly classified 86% of the cases, with three adenoses and two carcinomas misclassified. Validity of the discriminant rules was supported by classification using measurements from a separate, independent set of cases (ten sclerosing adenoses and nine tubular carcinomas). The classification function computed from the first set misclassified only one case from the second set, a tubular carcinoma, leaving 95% of the cases successfully categorized. Chi square test for 2 x 2 contingency tables gave a p-value < 0.001 for both sets of cases. The results suggest that morphometric features are helpful in distinguishing tubular carcinoma from sclerosing adenosis and point out the need for conservative analysis of high-dimensional data sets.
Asunto(s)
Adenocarcinoma/patología , Neoplasias de la Mama/patología , Enfermedad Fibroquística de la Mama/patología , Diagnóstico Diferencial , Análisis Discriminante , Femenino , Humanos , Procesamiento de Imagen Asistido por Computador , EsclerosisRESUMEN
Measurement of nuclear and glandular size and shape features was carried out on 18 cases of sclerosing adenosis and 18 cases of tubular carcinoma. Modified Bonferroni analysis showed that glandular surface density and the coefficient of variation of luminal form factor were significant in discriminating between these two lesions. These two histologic features, together with the diagnosis, were used to train a neural network implementing a backpropagation algorithm. Following training, the network correctly classified 33 of the 36 cases in the training set (92%). Furthermore, the network correctly classified 19 of 19 cases in a test set consisting of cases that were not used to train the network. These results suggest that neural networks may be useful to assist in the differential diagnosis of histologically similar lesions.
Asunto(s)
Adenocarcinoma/patología , Neoplasias de la Mama/patología , Enfermedad Fibroquística de la Mama/patología , Interpretación de Imagen Asistida por Computador/instrumentación , Diagnóstico Diferencial , Humanos , Redes Neurales de la Computación , EsclerosisRESUMEN
Carcinoma is found unexpectedly in approximately 10% or more of the 400,000 prostatectomies performed annually in the United States. Patients with Stage A2 carcinoma die of their disease in only 35% of the cases. To alter the course of disease in these patients, 65% of Stage A2 patients may be treated unnecessarily by radical prostatectomy, radiation therapy, or hormonal therapy. An accurate method to predict the outcome of patients with Stage A2 carcinoma is needed. Histologic sections from 18 patients with Stage A2 prostatic carcinoma followed without further treatment until progression, or followed without progression, were evaluated by several investigators who did not have knowledge of patient outcomes and who employed standard pathologic grading systems as well as morphometric, cytophotometric, flow cytometric, and immunohistochemical techniques. Outcome was predicted correctly by random sampled absolute (17 of 18 cases) and relative (16 of 18) nuclear roundness factor (NRF), tumor volume expressed as percent of specimen (13 of 16), primary (13 of 18), secondary (14 of 18), sum (15 of 18), and worse (14 of 18) Gleason grades and prostate-specific antigen immunohistochemical findings (13 of 18) that produced statistically significant separation of the two groups. Significant separation was not obtained with Mostofi's pattern, nuclear, sum, and worse grades, Johns Hopkins' grade, absolute tumor volume, nuclear DNA content measured by image cytophotometric study of Feulgen-stained histologic sections and flow cytometric study of propidium iodide-labeled suspensions of nuclei obtained from paraffin blocks, nonrandom sampled NRF of worse and most prevalent neoplastic areas, and prostatic acid phosphatase and peanut agglutinin immunohistochemical study. NRF measured by a random technique best predicted outcome in these patients with A2 prostatic carcinoma and should be evaluated prospectively as a means for selecting patients who require therapy.
Asunto(s)
Adenocarcinoma/genética , Adenocarcinoma/patología , ADN de Neoplasias/análisis , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Adenocarcinoma/secundario , Estudios de Seguimiento , Humanos , Técnicas para Inmunoenzimas , Masculino , Estadificación de Neoplasias , Pronóstico , Estadística como AsuntoRESUMEN
Flow cytometry and nuclear morphometry were compared with traditional pathologic grading techniques for predicting the course of malignant fibrous histiocytoma of the extremities. Clinical, pathologic, and flow/morphometric variables from 53 cases were tested by Cox regression for prediction of distant recurrence and mortality. Tumor grading based on extent of tumor necrosis was a significant predictor for both disease-free survival (p = .014) and overall survival (p = .003). The fraction of nuclei in the S + G2M segment of DNA histograms was significant for disease-free survival (p = .007), and remained significant (p = .033) in a joint Cox model with necrosis-based grade (p = .004 for the bivariate model). Relative risk for recurrence varied nearly 10x between the 10th and 90th percentiles of grade and (S + G2M)1/2. Overall survival was predicted by a nuclear shape feature termed "R" (p = .000008), the casewise difference (residual) between expected and observed nuclear perimeter as a function of average Feret diameter. In a bivariate Cox model, relative risk of mortality varied 35x between the 10th and 90th percentiles of grade and R. Cytometric and morphometric data contain information about recurrence-free and overall survival beyond that available from more usual clinical and pathologic features. It seems likely that nuclear morphometry, in particular, will prove to be a useful aid for estimating the prognosis of patients with malignant fibrous histiocytoma of the extremities.
Asunto(s)
Extremidades , Histiocitoma Fibroso Benigno/patología , ADN de Neoplasias/análisis , Femenino , Citometría de Flujo , Histiocitoma Fibroso Benigno/química , Histiocitoma Fibroso Benigno/epidemiología , Histiocitoma Fibroso Benigno/mortalidad , Humanos , Procesamiento de Imagen Asistido por Computador , Masculino , Persona de Mediana Edad , Metástasis de la Neoplasia/patología , Valor Predictivo de las Pruebas , Pronóstico , Recurrencia , Estudios Retrospectivos , EspectrofotometríaRESUMEN
In this study we examined the reproducibility of several stains used to measure nuclear DNA by image cytometry. The specimens were touch preparations of liver and testis from mouse and liver, intestine and brain from rat, fixed in either neutral formalin or Carnoy's solution. The tested stains included four Feulgen methods (pararosaniline, azure-A, thionin and acriflavine), the gallocyanine-chromalum stain and two fluorescent stains (acridine orange and propidium iodide). Absorbance measurements employed a video image analysis system; fluorescence measurements were from a scanning microspectrophotometer. The acriflavine-Feulgen stain was analyzed for both absorbance and fluorescence. All seven stains were quantitative for DNA and gave reproducible results. The absorbance measurements had a lower coefficient of variation (CV) than the fluorescence values. In a nested analysis of variance of the pararosaniline Feulgen stains, cell-to-cell variability accounted for 67% of the total variance; slide-to-slide, 9%; and batch-to-batch, 24%. These values did not change significantly when the staining was performed in an automatic staining machine. For DNA analysis using image cytometry, we conclude that the Feulgen staining technique is the most useful. In particular, acriflavine-Feulgen-stained cells fixed in Carnoy's fluid give the least variation between measurement values and the most accurate ratios between the separate ploidy groups. For fluorescence cytometry we recommend Carnoy's fixation and the acriflavine-Feulgen stain because of its narrow CV as compared to acridine orange and propidium iodide.
Asunto(s)
ADN/análisis , Microscopía Fluorescente/métodos , Microespectrofotometría/métodos , Ploidias , Coloración y Etiquetado/métodos , Análisis de Varianza , Animales , Encéfalo/anatomía & histología , Procesamiento de Imagen Asistido por Computador , Intestinos/anatomía & histología , Hígado/química , Masculino , Ratones , Testículo/química , Fijación del Tejido/métodosRESUMEN
The severity and consistency of the effect of formalin fixation on the quantitation of DNA by flow cytometry (FCM) and image cytometry (ICM) were studied. As compared to ethanol, formalin fixation substantially decreased the propidium iodide fluorescence from mouse hepatocyte nuclei analyzed by FCM; it was also associated with an altered 4n-to-2n signal ratio and with false aneuploid peaks by FCM, but not by ICM (microspectrophotometry). ICM, on the other hand, suffered from a dependence of the DNA signal on nuclear size, which was not seen with FCM. The DNA signal variation was related to variations in the chromatin state, as shown by differences between monocytes and lymphocytes, and between RAJI cells fixed under various ionic strengths. The dependence of the DNA signal on the chromatin state indicates a need for caution in interpreting aneuploidy in formalin-fixed cells. For FCM, pseudoaneuploidy appears avoidable by using a Feulgen fluorescence staining technique. New imaging modes may be necessary to solve the problem of cell size dependence for ICM DNA determination.
Asunto(s)
ADN/análisis , Animales , Núcleo Celular/fisiología , Cromatina/fisiología , Fijadores , Citometría de Flujo , Formaldehído , Fase G1/genética , Humanos , Procesamiento de Imagen Asistido por Computador , Hígado/química , Hígado/citología , Linfocitos/química , Métodos , Ratones , Monocitos/química , Ploidias , Fase de Descanso del Ciclo Celular/genéticaRESUMEN
Morphometric measurements of nucleoli were done on uveal melanomas from surviving and nonsurviving patients. The melanomas were embedded in paraffin and plastic, and measurement data from Papanicolaou-stained paraffin-embedded sections, toluidine blue-stained plastic-embedded sections and scanning transmission electron micrographs (STEM) of plastic-embedded sections were compared. The results showed that one parameter, the coefficient of variation (CV) of nucleolar area, correctly classified 80% of the cases as to survival when plastic-embedded material was used and 70% of the cases when paraffin-embedded material or STEM micrographs were used. The inverse standard deviation of the nucleolar area was a better predictor of outcome than was the CV of nucleolar area only in the paraffin-embedded sections. The nucleolar measurements were most easily and rapidly performed in the plastic-embedded sections.
Asunto(s)
Melanoma/ultraestructura , Región Organizadora del Nucléolo/ultraestructura , Neoplasias de la Úvea/ultraestructura , Humanos , Melanoma/diagnóstico , Melanoma/mortalidad , Microscopía Electrónica , Neoplasias de la Úvea/diagnóstico , Neoplasias de la Úvea/mortalidadRESUMEN
The DNA content and nuclear measurements of five groups of endometrial proliferations--proliferative endometrium (PE), simple hyperplasia (SH), atypical hyperplasia (AH), well-differentiated carcinoma (WDC), and poorly differentiated carcinoma (PDC)--were compared using 14 descriptors in a stepwise discriminant analysis. Classification using the discriminant rules agreed with the pathologic interpretation for 78% of the specimens. All PEs were assigned to the correct group, and 97% of benign endometria and carcinomas were correctly classified as benign or malignant. Only two of 39 hyperplasias (5%) were misclassified as malignant, and only one of 36 carcinomas was classified as benign. In the difficult distinction between AH and WDC, using all descriptors for the five groups, only 68% of the AH and 60% of the WDC classifications were in agreement with the pathologist of record. However, when discriminant rules addressing only AH and WDC were used, 37 of 39 AHs and WDCs were in concordance. This suggests that a morphometric distinction between AH and WDC is feasible.
Asunto(s)
Carcinoma/patología , Hiperplasia Endometrial/patología , Neoplasias Uterinas/patología , Citofotometría , ADN de Neoplasias/análisis , Femenino , Estudios de Seguimiento , Humanos , Procesamiento de Imagen Asistido por ComputadorRESUMEN
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.
Asunto(s)
ADN/análisis , Mesotelioma/patología , Adulto , Anciano , Citofotometría , ADN de Neoplasias/análisis , Femenino , Humanos , Masculino , Mesotelioma/genética , Persona de Mediana Edad , Derrame Pleural/patologíaRESUMEN
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.
Asunto(s)
Neoplasias de la Mama/patología , Mama/patología , Carcinoma Intraductal no Infiltrante/patología , Neoplasias de la Mama/análisis , Carcinoma Intraductal no Infiltrante/análisis , ADN de Neoplasias/análisis , Femenino , Humanos , HiperplasiaRESUMEN
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.
Asunto(s)
Peso Corporal , Células/citología , Animales , Constitución Corporal , Humanos , Matemática , Gravedad Específica , Virus/ultraestructuraRESUMEN
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.
Asunto(s)
ADN de Neoplasias/análisis , Neoplasias Gastrointestinales/análisis , Leiomioma/análisis , Leiomiosarcoma/análisis , Adolescente , Adulto , Núcleo Celular/análisis , Neoplasias del Colon/análisis , Neoplasias del Colon/patología , Citofotometría , Neoplasias Esofágicas/análisis , Neoplasias Esofágicas/patología , Humanos , Técnicas para Inmunoenzimas , Neoplasias Intestinales/análisis , Neoplasias Intestinales/patología , Leiomioma/patología , Leiomiosarcoma/patología , Neoplasias del Recto/análisis , Neoplasias del Recto/patología , Coloración y Etiquetado , Neoplasias Gástricas/análisis , Neoplasias Gástricas/patologíaRESUMEN
Quantitative electron microscopy (QEM) and microspectrophotometry were used to correlate the Feulgen stain absorption values to the calculated picograms of DNA. Measurements were made in human lymphocytes, rainbow trout lymphocytes and nuclei of trout erythrocytes. The median dry weight of the nucleus, as determined by QEM, was 35.9 pg for a human lymphocyte and 30.5 pg for a trout lymphocyte. Using Salzman's value of 20% DNA per chromosome (i.e., chromatin), a human lymphocyte nucleus thus contains 7.18 pg of DNA and a trout lymphocyte nucleus 6.1 pg of DNA. The mean Feulgen absorption value of the nucleus, given in arbitrary units (AU), was 14.5 for a human lymphocyte, 12.7 for a trout lymphocyte and 12.0 for a trout erythrocyte. From these values, it was derived that each picogram of DNA of a human lymphocyte nucleus is represented by 2.02 arbitrary Feulgen units while the values for trout nuclei were 2.08 AU and 1.97 AU. On the average, we find that each picogram of DNA is represented by two arbitrary Feulgen units in our microspectrophotometric measurements.
Asunto(s)
Colorantes , ADN/análisis , Colorantes de Rosanilina , Animales , Eritrocitos/análisis , Humanos , Linfocitos/análisis , Microscopía Electrónica , Especificidad de la Especie , Espectrofotometría , TruchaRESUMEN
Hepatocellular adenomas are usually visualized as defects on technetium-99m-sulfur colloid liver scans, a fact which has been attributed to the absence of phagocytic Kupffer cells in the tumors. To determine whether this is true, seven hepatocellular adenomas were subjected to immunoperoxidase staining for lysozyme, a marker of mononuclear phagocytes. The Kupffer cells were counted in the tumors and surrounding non-neoplastic liver. All hepatocellular adenomas studied were found to contain Kupffer cells. Three tumors had fewer Kupffer cells than the surrounding liver. Three had about the same number as the surrounding liver, and one had more Kupffer cells than the non-neoplastic liver. Thus, the lack of phagocytosis of colloid in liver scans is probably due to something other than a deficiency of Kupffer cells in the hepatocellular adenomas.
Asunto(s)
Carcinoma Hepatocelular/patología , Macrófagos del Hígado/análisis , Neoplasias Hepáticas/patología , Adulto , Carcinoma Hepatocelular/diagnóstico por imagen , Femenino , Histocitoquímica , Humanos , Técnicas para Inmunoenzimas , Macrófagos del Hígado/diagnóstico por imagen , Neoplasias Hepáticas/diagnóstico por imagen , Muramidasa/análisis , CintigrafíaRESUMEN
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.
Asunto(s)
Núcleo Celular/ultraestructura , Animales , Mama/ultraestructura , Citometría de Flujo/métodos , Humanos , Hígado/ultraestructura , Matemática , Microscopía Electrónica , Microscopía Fluorescente , Microscopía de Interferencia , Nucleoproteínas/análisis , Ratas , Porcinos , Timo/ultraestructuraRESUMEN
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.
Asunto(s)
ADN/análisis , Citometría de Flujo/métodos , Técnicas Histológicas , Colorantes de Rosanilina , Espectrofotometría/métodos , Animales , Colorantes , Fijadores , Ratones , PorcinosRESUMEN
A simple method of identifying cells for scanning electron microscopy (SEM) is described. The method is very rapid and allows easy correlation between light microscopy and SEM. Furthermore, the procedure can be used with specially prepared cell suspensions as well as with routine smears.