A method to estimate the DNA content of whole nuclei from measurements made on thin tissue sections

Abstract

A microdensitometry method that allows estimation of the distribution of the DNA content of nuclei in thin tissue sections is described. The method is based on a theoretical model of Feulgen-stained spherical nuclei, of different sizes, in each of which the DNA is present as a homogeneous solution. In thin sections of nuclei of different sizes, the fraction of DNA per section is inversely proportional to the radius of the nucleus. Histograms of the product of DNA content andThe interpretation of DNA determinations in tissue sections is complicated, since the amount of DNA per slice of nucleus depends on the position of this slice within the nucleus (7). This problem can be circumvented to some extent by taking thick sections (5-8 pm) (6) and correcting for thickness (5), but in most tissues it is impossible then to analyze single nuclei due to overlap. In thin sections the absence of overlap allows one to perform DNA measurements on slices of single nuclei, but then the relation between the amount of DNA per slice and the amount of DNA per whole nucleus is still unclear. A method that allows the relation of the DNA in collections of nuclear sections to the DNA in whole nuclei is described. MATHEMATICAL BACKGROUNDFor the calculations an idealized tissue model is used; it is composed of cells with spherical nuclei homogeneously filled with a DNA solution and cut in infinitesimally thin sections. DNA Content of Sections of Spherical NucleiFor a plane at a distance, r < R, from the centre of the sphere with radius, R, the intersection of plane and sphere is a circle with radius m, and hence with a surface area A(r) = a (R2 -r2). When N is the total amount of DNA, then the DNA content of a slice of a (small) thickness, t, taken at a distance, r, from the centre of the sphere will be: D = (volume density of DNA) x (volume of slice)--radius per nuclear section are independent of nuclear size but depend on total DNA content. The distribution of the total DNA content of nuclei in a section can be estimated from such a histogram. The results of the measurements of a Feulgen-stained rat liver section are described.

DNA histogram debris theory and compensation

“…Although the presented theory is similar to that published by Bins et al (7), the assumed geometry, calculated probability distributions, and method of compensation is fundamentally different. The shape of the debris curve presented in this paper is calculated from the observed histogram and is subsequently used as a model component in a Marquardt nonlinear least-squares algorithm ( 5 ) .…”

“…Debris (x) = j = x + l 2 ?y,P, (i,x) (7) If the cut fraction function, 5, is assumed t o be proportional to one third power of the channel number and yj is approximated by the observed number of events at channel, Yj, the single cut histogram-dependent debris model component is…”

DNA Ploidy and Proliferating Cell Nuclear Antigen Image Analysis of Peritoneal and Pleural Effusions

“…4,7,11 In spite of the known disadvantages of DNA analysis using tissue sections, 9,22 it has the following benefits:…”

“…CAS 200 supplies a rat hepatocyte DNA standard that has been known to be reproducible in many studies. 4,7,11,40,44 The DNA index (DI) of 1 will correspond to 7.18 pg per nucleus, which has been shown to be the average amount of DNA contained in a normal diploid human cell (range, 6.4-8.0 pg, CV = 2%). 64 The reproducibility of DNA content analysis (ploidy) by the CAS 200 has been improved by the incorporation of software that corrects for section thickness and reduces DNA content analysis peak CVs.…”

Three-dimensional DNA image cytometry by confocal scanning laser microscopy in thick tissue blocks of prostatic lesions

“…To overcome the problem of fractionated nuclei for the computation of DNA histograms, various mathematical algorithms have been developed to recalculate the original DNA content of sectioned nuclei (6,26). However, the underlying assumptions of these algorithms are very limiting: regularly shaped nuclei, homogeneously dispersed DNA inside each nucleus, and section thickness either accurately determined or considered as infinitesimally thin (27).…”