Conceptual comparison of two computer models of corpuscle sectioning and of two algorithms for correction of ploidy measurements in tissue sections

OBJECTIVE: To compare two computer models of corpuscle sectioning and two algorithms for correction of ploidy measurements in tissue sections. STUDY DESIGN: Two models of corpuscle sectioning (the computed corpuscle sectioning program [CCSP] [Analyt Quant Cytol Histol 1997;19:376-386] and the ellipsoid sectioning program [ESP]) were run on a personal computer to generate synthetic corpuscle section data that model the sectioned nuclei in a tissue section. These synthetic data were analyzed by two algorithms for correction of ploidy measurements in tissue sections: the reference curve method (RCM) (Analyt Quant Cytol Histol 1997;19:376-386) and the method of McCready and Papadimitriou (MMP) (Analyt Quant Cytol 1983;5:117-123) for a variety of choices of section thickness and of nuclear section profile selection criteria. RESULTS: Previous recommendations (Analyt Quant Cytol Histol 1999;21:103-112) for optimization of ploidy analysis in tissue sections (selection of only center-containing sections of nuclei in ultrathin sections with a selection bias in favor of elliptical nuclear section profiles) are valid regardless of which corpuscle sectioning model and correction algorithm are employed. Perimeter correction may be desirable or necessary in some cases. The RCM has very significant advantages over the MMP, and the CCSP is more applicable to actual ploidy analysis than is the ESP. CONCLUSION: The RCM always should be used to correct ploidy measurements in tissue sections. The MMP should not be used as the sole method but, when used, should be used with and interpreted in the context of the RCM.     

Single-section counting error when distinguishing between primordial and early primary follicles in sections of rat ovary of different thickness

The number of primordial follicles within an ovary is frequently determined by counting 5, 7 or 10 microns thick sections and multiplying by the fraction of sections counted and a correction factor to adjust for duplicate counts. The objectives of the present study were: (i) to evaluate the accuracy of the correction factor developed by Abercrombie (1946); (ii) to evaluate the accuracy of the classification of primordial follicles from single tissue sections; and (iii) to determine the incorporation rate of 5-bromo-2-deoxyuridine into primordial follicles. In Expt 1, rat ovaries were sectioned at a thickness of 5, 7 or 10 microns. Primordial follicles were counted and classified across ten adjacent ovarian sections. The percentage of primordial follicles from single sections that were counted twice was 10, 9 and 2% in 5, 7 and 10 microns sections, respectively. This was lower than predicted by Abercrombie's method. The major error in counting from single sections was classification of early primary follicles as primordial follicles (55, 33 and 3% in 5, 7 and 10 microns sections, respectively). In Expt 2, a mean of 12 +/- 7% of primordial follicles incorporated 5-bromo-2-deoxyuridine after infusion for 7 days (four of seven rats had no labelled primordial follicles). In conclusion: (i) Abercrombie's correction factor should not be used for adjusting counts of follicles; (ii) evaluation of primordial follicles from single sections gives inaccurate counts and incorrect classification is of greater importance than duplicate counting, particularly in thinner sections; (iii) for evaluation of the number of follicles, 10 microns is the optimal thickness; and (iv) primordial follicles incorporated 5-bromo-2-deoxyuridine infrequently.     

Enzymatic determination of choline acetyltransferase by coenzyme A cycling and its application to analysis of single mammalian neurons

Abstract: An enzymatic assay for choline acetyltrans-ferase was developed by measuring acetyl-coenzyme A (acetyl-CoA) formed from CoASH and acetylcholine (ACh). This method is extremely sensitive and may be applied to the analysis of microgram to nanogram crude samples. The method is, however, not useful when choline acetyltransferase is present in very low concentrations. The basis of this method is to amplify a small amount of synthesized acetyl-CoA in the assay mixture by using an enzymatic amplification reaction, CoA cycling. This amplification mechanism made it possible to perform microassays (13 nl-2.2 μl of assay volume) of freeze-dried sections prepared from cerebral cortex, striatum, and hippocampus of mice and single cell bodies isolated from freeze-dried sections of rabbit spinal cords. These samples were weighed and added directly to the reaction mixture. The activities of the above cerebral regions, assayed with 1,500–2,000-fold amplification, corresponded well to the results previously reported by other workers. The average activity of single anterior horn cells, determined with 64,000–420,000-fold amplification, was 40-fold higher than that of rabbit cerebral cortex, and the specific activities on a dry weight basis were widely distributed among individual neurons. No activity was detected in the noncholinergic dorsal root ganglion cells or in cerebellar cortex.     

HISTOLOGICAL AND HISTOCHEMICAL CHANGES IN DEVELOPING AND RIPENING PEACHES III. CATECHOL TANNIN CONTENT PER CELL

Reeve , R. M. (Western Regional Res. Lab., U. S. Dept. of Agriculture, Albany, Calif.) Histological and histochemical changes in developing and ripening peaches. III. Catechol tannin content per cell. Amer. Jour. Bot, 46(9): 645–650. Illus. 1959.—Photometric measurements were made of the densities of catechol tannin test colors histochemically developed with a nitrous acid reaction in fresh sections of fruits. The measured values showed a linear relationship with both section thickness and tannin concentration. When average cell size, section thickness. and the volume of tissue photometrically illuminated are known, it is possible to calculate an amount of phenolic substance per cell. Such values calculated for the naturally occurring catechol tannins in developing and ripening peaches revealed very large changes in these substances per cell. Catechol tannins so estimated increased about 90-fold from the beginning of cell enlargement until the fruit was slightly over half its mature size, a period of about 16-fold increase in volume of the average cell. After a maximum tannin content had been reached, a decrease of about 8-fold per cell was found as the fruit matured and ripened. The early changes in tannin content, when expressed on a unit volume basis, were much less pronounced (about 5-fold increases) and agreed in general with published chemical data for increases in chlorogenic acid on a weight basis during a comparable growth period in a sweet cherry. The semiquantitative, percell expressions of these histochemical results show relation to established phenomena of growth.     

Improved correction of quantitative nuclear DNA (ploidy) measurements in tissue sections

OBJECTIVE: To evaluate, using a computer model, the advantages for ploidy measurements of selecting only center-containing sections of nuclei in ultrathin, very thick or relatively thick tissue sections. STUDY DESIGN: The computed corpuscle sectioning program was run on a personal computer. Its synthetic data were corrected by a variety of correction algorithms. RESULTS: When only center-containing sections of nuclei were selected in ultrathin sections, spherical nuclei could be corrected perfectly, and mildly prolate ellipsoidal nuclei with a selection bias in favor of elliptical nuclear section profiles could be corrected with high fidelity. Ultrathin sections most faithfully represented the true height of the peak of highest ploidy and showed better peak discrimination than other choices of section thickness, but small sample size, wavy sections, markedly inhomogeneous intranuclear DNA distribution and oblate ellipsoidal nuclei represented significant limitations of this approach. As nuclear prolation increased, peak definition worsened, and the peak of highest ploidy was falsely shortened. Results were unaffected by errors in the estimation of section thickness when an internal diploid standard was used. The effect of variable internuclear DNA concentration in mildly or moderately prolate ellipsoidal nuclei was nil. The choice of correction algorithm was unimportant, except that the reference curve method was better able to analyze oblate ellipsoidal nuclei, wavy sections and nuclei with inhomogeneous intranuclear DNA, and provided superior insight into nuclear and section parameters. Thick and very thick sections did not require correction and, unlike ultrathin sections, were immune to markedly inhomogeneous intranuclear DNA distribution, to nonspherical nuclear shape and to focal variation in section thickness (waviness), but (in relatively thick more than in very thick sections) the height of the peak of highest ploidy was falsely shortened, often markedly, and peak definition was worse. CONCLUSION: Choice of section thickness and selection of only center-containing nuclear sections for analysis with a bias in favor of elliptical nuclear section profiles in ultrathin sections are very important for optimal results; the choice of correction algorithm is less important.     

Determination of correction factors of 3H-beta-self-absorption for quantitative evaluation of grain number in autoradiographic studies. Interferometric studies of different cell types in the mouse brain

Deparaffinized and Feulgen-stained sagittal sections of the mouse brain were studied interferometrically in order to measure optical path differences of euchromatin and heterochromatin of various cell types. Furthermore, the ratio eu-: heterochromatin of each cell type was determined. From these data mass densities of karyoplasm and, finally, correction factors of 3H-beta-self-absorption were calculated for comparing grain numbers of different cell types in quantitative autoradiographic studies after application of tritium-labelled substances. Remarkable differences of correction factors up to a factor of 2.18 were found. Furthermore, the actual section thickness was determined interferometrically. A reduction to about 0.60 of the microtome setting was measured in two different areas of the brain. Using mass densities together with actual section thickness correction factors for a thickness of 1 micron were calculated. This was done also for cell types outside the brain the data of which were taken from literature. Thus, differences in correction factors up to about a factor of 4 were found pointing out the importance of considering 3H-beta-self-absorption in quantitative autoradiographic studies.     

The Ebbeson-Tang formula. Theoretical background of an alternative to the disector in stereologic cell count studies

OBJECTIVE: To evaluate the formula of Ebbeson and Tang (FET) with respect to the disector (DS) principle. STUDY DESIGN: The DS principle has been proposed for avoiding cell count bias. DS is a slice of tissue, and from it those cells by area are counted in microscopy; the cells are not in contact with one of the surfaces of the slice. The resulting number divided by the thickness of the DS gives an accurate figure for cell number by volume. FET applies two sections of different thickness, usually cut adjacent. Cells seen in the sections are counted by area, the figures are subtracted from each other, and the difference is divided by the difference between the thicknesses of the sections. The result is cell number by volume: NV = (NA1 - NA2)/(t1 - t2). RESULTS: FET and the DS principle superficially appear different. However, from a geometric point of view they are based on the same principle. When the thickness of the thinner section of FET approaches zero, the situation is in all respects equal to the DS principle. The formula for DS can thus be written: NV = (NA1 - NA2)/t1. CONCLUSION: The result proves that in principle DS and FET are equivalent methods of counting cell numbers by volume in tissues. FET may be more easily applied in histopathology practice because visual comparison of the sections is not necessary. Section thickness, however, has to be measured from vertically embedded sections or with scanning laser confocal microscopy. FET shares the stereologically unbiased character of the DS principle and is independent of the size and shape of structures counted.     

Curvature Correction Factors in the Measurement of Cell Surface Areas in Plant Tissues1

The simplest and most reliable method for the estimation ofthe surface area of cells in plant tissues involves the measurementof the total length of the perimeters of cell profiles in thinsections of the tissue. However, the method suffers from errorsdue to neglect of curvature of the cell surfaces. The effectsof the curvature can be compensated for by multiplying the measuredlength of profile perimeter by appropriate curvature correctionfactors. This paper presents values for these curvature correctionfactors, which are shown to be significantly large. The applicationof these correction factors to the measurement of cell surfaceareas in plant tissues is discussed with particular referenceto leaf mesophyll tissue. The choice of appropriate orientationsof the sections in a tissue is also discussed. Key words: Cell, Surface, Area, Curvature correction     

Human fat cell sizing--a quick, simple method.

Two methods were used to determine the mean cell diameters of 37 samples of human adipose tissue, obtained by open or needle biopsy. Method I was the sizing of cells in cell suspensions and Method II was a quick, simple method of sizing cells from fixed sections. The agreement between the two methods was good (r = 0.93, P = less than 0.001). The results using Method II were slightly lower than those using Method I, and a correction factor is suggested. Method II has several advantages over Method I and we propose that it is a suitable method for sizing cells when a quick method with a permanent record is required.     

Modeling of dynamic fracture and damage in two-dimensional trabecular bone microstructures using the cohesive finite element method

Trabecular bone fracture is closely related to the trabecular architecture, microdamage accumulation, and bone tissue properties. Micro-finite-element models have been used to investigate the elastic and yield properties of trabecular bone but have only seen limited application in modeling the microstructure dependent fracture of trabecular bone. In this research, dynamic fracture in two-dimensional (2D) micrographs of ovine (sheep) trabecular bone is modeled using the cohesive finite element method. For this purpose, the bone tissue is modeled as an orthotropic material with the cohesive parameters calculated from the experimental fracture properties of the human cortical bone. Crack propagation analyses are carried out in two different 2D orthogonal sections cut from a three-dimensional 8 mm diameter cylindrical trabecular bone sample. The two sections differ in microstructural features such as area fraction (ratio of the 2D space occupied by bone tissue to the total 2D space), mean trabecula thickness, and connectivity. Analyses focus on understanding the effect of the rate of loading as well as on how the rate variation interacts with the microstructural features to cause anisotropy in microdamage accumulation and in the fracture resistance. Results are analyzed in terms of the dependence of fracture energy dissipation on the microstructural features as well as in terms of the changes in damage and stresses associated with the bone architecture variation. Besides the obvious dependence of the fracture behavior on the rate of loading, it is found that the microstructure strongly influences the fracture properties. The orthogonal section with lesser area fraction, low connectivity, and higher mean trabecula thickness is more resistant to fracture than the section with high area fraction, high connectivity, and lower mean trabecula thickness. In addition, it is found that the trabecular architecture leads to inhomogeneous distribution of damage, irrespective of the symmetry in the applied loading with the fracture of the entire bone section rapidly progressing to bone fragmentation once the accumulated damage in any trabeculae reaches a critical limit.     

High-level detection of gene amplification and chromosome aneuploidy in extracted nuclei from paraffin-embedded tissue of human cancer using FISH: a new approach for retrospective studies

A novel application of fluorescence in situ hybridization (FISH) to isolated nuclei is described. The method detects gene amplification and chromosome aneuploidy in extracted nuclei from paraffin-embedded tissue of human cancer with greater sensitivity and specificity than existing FISH methods. In this study, the method is applied to signal detection of the HER-2/neu (c-erbB-2) gene, whose amplification is one of the most common genetic alterations associated with human breast cancer. Nuclei were extracted and isolated from formalin fixed, paraffin embedded tissue of 43 different carcinomas (breast, ovary, endometrium, gastrointestinal stromal tumor and malignant mesothelioma). FISH was performed both on sections and extracted nuclei of each tissue using chromosome enumeration probes (CEP) for the centromeric regions of chromosomes 8 and 17, and a locus specific identifier (LSI) for the HER-2/neu oncogene. Differences between ploidy calculated in sections and extracted nuclei were seen in 3 breast carcinomas and 1 gastrointestinal stromal tumor (GIST). Furthermore, 1 breast cancer, previously considered to be borderline for HER-2/neu gene amplification turned out to be clearly amplified. Nuclei extraction and isolation bypass all the problems related to signal interpretation in tissue sections, and the adoption of this new technique, which improves the signal quality in several neoplastic samples, is suggested.     

A method for separating cultured preadipocytes according to their density: Application to stromal cells from overfed suckling rats

Summary The stroma vascular fraction of adipose tissue consists of a heterogeneous cell population; not all the cells in this compartment undergo adipose conversion in primary culture. A density gradient centrifugation procedure was used to separate cultured cells on the basis of their triglyceride content. This method was applied to both stroma vascular cells from rat adipose tissue and to a 3T3 F442A preadipose cell line as a reference. Comparison of the results obtained from these two cell types suggests that this separation procedure can lead to a quantification of adipose differentiation in the heterogeneous stroma cell population. Separation procedures were applied to cultured stromal cells derived from young rats during the onset of nutritional obesity induced by overfeeding in early life. Results show that early overfeeding induced an increase in the stromal cell differentiation capacity which is expressed in vitro. This work was supported in part by Institut National de la Santé et de la Recherche Médicale (CRL n^o 82-70-22).     

Comparison of indirect and direct in-situ polymerase chain reaction in cell preparations and tissue sections

Different approaches to the in-situ polymerase chain reaction (in-situ PCR) were compared in the detection and in-situ localization of chromosomal translocations (t14; 18) immunoglobulin gene rearrangements and viral DNA (cytomegalovirus, hepatitis B-virus) in cell suspensions, cytospins and tissue sections. Single and multiple primer pairs were compared in the amplification step of indirect in-situ PCR and long genomic probes or internal oligonucleotide probes in the subsequent in-situ hybridization (ISH). For direct in-situ PCR, in which amplification products were directly labeled with digoxigen-in-11-dUTP during PCR and detected immunohistochemically, only single primer pairs were used for amplification. In-situ PCR yielded best results in the cell suspensions and worked less efficiently in cytospins or tissue sections. Quantification of the results obtained in artificial cell mixtures yielded only an approximate correlation between the number of expected and observed positive cells. The specificity of the results was greater with indirect in-situ PCR than direct in-situ PCR, where false positive results were frequent. Successful indirect in-situ PCR in tissue sections required the use of multiple primer pairs for amplification and genomic probes for detection by ISH. False positive results in direct in-situ PCR were caused by primer-independent, but DNA polymeraseand cycling-dependent incorporation of digoxigenin-labeled nucleotides into cellular DNA, possibly related to DNA repair and/or internal priming. Non-specific results were most marked in tissue sections and were much less frequent in cell suspensions. In-situ PCR includes a number of different techniques, which are not equally applicable to different starting materials. Accurate interpretation of the results requires vigorous controls.     

The chemical determination of section thickness

Summary In quantitative histochemistry it is frequently desirable to know the exact thickness of the sections. The nucleic acid content of the tissue has been used as the basis of a method for determining section thickness. Such determinations agree remarkably well with the setting on the cryostat microtome although considerable variations in thickness can occur in sections cut at different speeds.     

Heat pretreatment increases resolution in DNA flow cytometry of paraffin-embedded tumor tissue

BACKGROUND: Flow cytometry of single-cell suspensions prepared by enzymatic digestion from formalin-fixed, paraffin-embedded tissue suffers from several major drawbacks. The most important factors that influence the results are the high and unpredictable coefficients of variation (CVs) of the G0/G1 peak in the DNA histogram and reduction of propidium iodide (PI) intercalation with DNA, resulting from protein cross-linking by formalin. METHODS: In this study we introduce a heating step (2 h incubation in citrate solution at 80 degrees C) prior to a brief pepsin digestion of tissue sections in the protocol for DNA content analysis of formalin-fixed and paraffin-embedded tissue. This new method is compared with established methods for the preparation of cell suspensions from frozen and paraffin-embedded tissues with respect to cell yield, DNA histogram resolution, DNA dye saturation kinetics, cell cycle parameters, and antigen retrieval in various epithelial and nonepithelial tissues. RESULTS: The recovery of single cells from the paraffin sections was doubled by the heat treatment step, while the limited time of proteolysis resulted in decreased cell debris. Furthermore, an increased fraction of cells became cytokeratin-positive, while these immunocytochemically stained cells also exhibited a higher mean fluorescence intensity. The DNA histograms prepared from cell suspensions obtained according to this new protocol showed a significantly improved resolution, leading to a better identification of peridiploid cell populations. Heat pretreatment of paraffin-embedded archival tissue sections showed PI saturation kinetics similar to, or even better than, those of fresh unfixed tissues, independent of duration of fixation. CONCLUSIONS: This new method, making use of routinely available antigen retrieval principles, thus allows high-resolution DNA analysis of routinely fixed and paraffin-embedded tissue samples. Using external reference cells, inter- and intralaboratory standardization of DNA histograms can be achieved.     

Influence of section thickness, mean nuclear diameter and nuclear crowding on DNA ploidy in histologic sections of melanocytic skin lesions

OBJECTIVE: To determine the influence of section thickness, nuclear diameter (MND) and area percentage of nuclei (a measure of nuclear crowding) on histologic DNA ploidy assessed by image cytometry (ICM) of primary melanocytic skin neoplasms (MSNs). STUDY DESIGN: Initially a feasibility study was performed to determine if comparable DNA ploidy histograms could be obtained from cell disaggregates and tissue sections. Following this, DNA ICM was performed on Feulgen-stained tissue sections (4, 6, 8 and 10 microns thick) from 30 primary MSNs (20 benign, 10 malignant) with nuclear diameters from 5.6 to 8.6 microns. Area percentage of nuclei was assessed in all cases at all section thicknesses. RESULTS: The feasibility study produced comparable results for cytocentrifuge and tissue section preparations. For sectioned MSNs, DNA ploidy histograms from 4-micron sections had a higher coefficient of variation of the 2c peak than those from 6-, 8- and 10-micron sections. Ten-micrometer sections had marked overlapping of nuclei, and only small numbers of cells could be measured, giving inadequate results. MND and area percentage of nuclei did not have an important influence on the results. CONCLUSION: Adequate DNA ploidy profiles can be obtained by DNA ICM on 6- and 8-micron-thick histologic sections of MSNs, provided that a strict measurement protocol is followed.     

Counting renal corpuscles in tissue sections

Two stereologic methods of counting renal corpuscles in tissue sections were evaluated. Weibel's stereologic method was found to be an accurate and efficient means of determining the number of corpuscles in normal and diseased kidneys. Weibel's method was preferred over the stereologic method of Elias and Hennig because the former is independent of tissue section thickness and is sensitive to variation in corpuscular size. A modification of Weibel's method eliminated the need for an additional point-counting procedure to determine the volumetric fraction of corpuscles in a kidney. The modification made use of previously completed measurements to calculate the area fraction of corpuscles and then substituted this value for the volumetric fraction of renal corpuscles.     

A new radiographic procedure for obtaining correction factors of 3H-β-self-absorption for quantitative tritium autoradiography

 A³H-radiographic method based on the absorption of ³H-β-particles by an overlying tissue section was established for obtaining correction factors of ³H-β-absorption (c.f.s) on the cellular level for all kinds of sections in a simple and more precise way as possible with interferometry. Unlabelled paraffin or Araldite sections were mounted on a thin uniformly ³H-labelled section of resin, and autoradiographs were prepared. Grain densities of neuronal cell types and cell-free areas within and outside the paraffin or Araldite sections were evaluated in autoradiographs, where the exposure time or the thickness of the overlying histological section was varied. From these values c.f.s were calculated applying the Beer-Lambert law. It was shown that corresponding c.f.s determined with this new radiographic method correspond well with each other. However, they will only agree with those c.f.s obtained by interferometry, if relative c.f.s are compared. Since the c.f.s are quite sensitive to the section thickness, a new parameter ϕ was introduced, which helps to assess whether the microtome used works exactly. Generally, the method presented can be used on the cytological level as well as for whole areas in every other autoradiographic study. Accepted: 30 November 1998