Cell division in Lemna minor roots treated with lead

Treatment of Lemna minor L. roots with 15 μM Pb²⁺ supplied as Pb(NO₃)₂ in 50-fold diluted Wang medium caused a progressive reduction in mitotic activity in the root tip. The percentage of dividing nuclei after 1, 6, 12 and 12 h of lead treatment was 6.25, 4.4, 3.4 and 0.3, respectively as compared to 7.1–7.7% in the control. After 6 h of lead treatment the number of cells in metaphase and anaphase was reduced by four- and nine-fold, respectively and after 12 h these phases were not detected. There were 3- and 10-fold fewer cells in telophase after 6 and 24 h, while those in prophase were reduced only in the 24 h treatment (a 30-fold reduction). These effects were associated with an increase in the number of cells exhibiting disturbances including lagging chromosomes, chromosome bridges, micronuclei, and nuclei with more condensed chromatin. The formation of micronuclei in root cells of L. minor cells at a very low dose of lead indicates that roots of this aquatic plant may be more sensitive to lead than those of terrestrial plants.     

Light-modulation of nitrate reductase activity in leaves and roots of maize

The nuclear DNA content in ray cells from the 1-year-old vascular cambium of white ash ( Fraxinus americana L.) trees was determined at intervals during the annual cycle of cambial activity and dormancy by using Feulgen microspectrophotometry. By 10 September, these cells had entered dormancy in G₁ with a normal DNA distribution and a minimal average DNA content of 2.65 pg. The average amount of DNA increased to 3.51 pg by 30 November, remained at this elevated value until at least 30 March, when the cambium was still dormant, then declined to the minimum level on 1 May and 10 June, when the cells were mitotically active. The springtime decline appeared to occur both before and during cell division. Between 1 May and 10 June, the prophase (4C) and telophase (2C) DNA contents decreased significantly. The amount of nuclear DNA measured by microspectrophotometry was verified by using flow cytometry and image analysis. The results support the view that there is an annual oscillation in the nuclear genome size of shoot meristematic cells in tree species native to the northern temperate zone.     

Cell division in Lemna minor roots treated with lead

Treatment of Lemna minor L. roots with 15 μM Pb²⁺ supplied as Pb(NO₃)₂ in 50-fold diluted Wang medium caused a progressive reduction in mitotic activity in the root tip. The percentage of dividing nuclei after 1, 6, 12 and 12 h of lead treatment was 6.25, 4.4, 3.4 and 0.3, respectively as compared to 7.1–7.7% in the control. After 6 h of lead treatment the number of cells in metaphase and anaphase was reduced by four- and nine-fold, respectively and after 12 h these phases were not detected. There were 3- and 10-fold fewer cells in telophase after 6 and 24 h, while those in prophase were reduced only in the 24 h treatment (a 30-fold reduction). These effects were associated with an increase in the number of cells exhibiting disturbances including lagging chromosomes, chromosome bridges, micronuclei, and nuclei with more condensed chromatin. The formation of micronuclei in root cells of L. minor cells at a very low dose of lead indicates that roots of this aquatic plant may be more sensitive to lead than those of terrestrial plants.     

COMPARISON OF PLANT DNA CONTENTS DETERMINED BY FEULGEN MICROSPECTROPHOTOMETRY AND LASER FLOW CYTOMETRY

An improved procedure is reported for determining DNA amounts of plant nuclei. Nuclei stained with propidium iodide, isolated from chopped plant leaves, were passed through an Ortho Cytofluorograph with a Lexel model 95 argon laser (514 nm) and the fluorescence measured, integrated, and recorded using an Ortho 2140 Data Acquisition computer. All nuclear samples were mixed with nuclei of Sultan barley (2C DNA content = 11.12 pg [picogram]) as an internal standard. DNA contents of ten plant species, ranging from 2C = 1.7 pg to 36.1 pg measured by flow cytometry, correlated strongly (r = 0.99, slope = + 1.00) with DNA contents determined from Feulgen-stained nuclei of the same species using microspectrophotometry. The flow cytometric procedures were sufficiently sensitive to detect differences in DNA content between inbred lines of corn and their F1 hybrids. Our results obtained with improved procedures, specifically using propidium iodide as a fluorochrome and plant nuclei instead of chicken erythrocytes as an internal standard, demonstrate that laser flow cytometry can be a precise, rapid, and reliable method for determining nuclear DNA content of plants.     

Development and interspecific transferability of genic microsatellite markers in Spartina spp. with different genome size

Flow cytometry analysis showed variation of nuclear DNA content among different species of Spartina. Spartina alterniflora had the biggest genome (1763.9 Mbp) and S. cynosuroides had the smallest genome (756.35 Mbp), whereas the genomes of S. patens (969.36 Mbp) and S. spartinae (979.78 Mbp) were comparable. Mining simple sequence repeats (SSR) from 1227 expressed sequence tags (EST) generated from salt stressed S. alterniflora showed an abundance of di- and tri-nucleotide repeats. Of 100 ESSR (EST-derived SSR) loci with five or more repeats, 81 loci were successfully amplified in eight S. alterniflora genotypes and 15 (22.2%) ESSR markers were polymorphic. Eleven of the 15 polymorphic ESSRs showed amplification across six different species of Spartina while 100% cross transferability was observed with at least one species of Spartina. The average number of alleles per marker was 3.9 and 5.8 within S. alterniflora and among Spartina species, respectively. The ESSR markers discriminated different members within and between species of Spartina genus.     

Amounts of nuclear DNA in marine halophytes

The amount of nuclear DNA, expressed as the C-value, was estimated for 13 marine halophytic plant species from six families. Plant material was collected in the nature reserve of the Strunjan saltpan in the Northern Adriatic and comprised all halophytic species inside the investigated area. Reproductive region of the shoot or root tips of halophytes were dissected, nuclei were Feulgen stained and 2C-values were measured by DNA image cytometry as follows: Crithmum maritimum (4.38 pg DNA), Artemisia caerulescens (6.43 pg), Aster tripolium (21.43 pg), Inula crithmoides (3.63 pg), Atriplex portulacoides (1.83 pg), A. prostrata (1.51 pg), Salicornia europaea (2.75 pg), Salsola soda (2.62 pg), Sarcocornia fruticosa (5.91 pg), Suaeda maritima (2.11 pg), Limonium angustifolium (5.06 pg), Puccinellia palustris (8.15 pg) and Ruppia cirrhosa (4.65 pg). With the exception of the C-value estimate for A. caerulescens, which has been listed in the Plant DNA C-values Database, the C-values represent the first estimates for all the examined species. In addition, the C-value for R. cirrhosa is also the first report for the family Ruppiaceae. The investigated halophytes had a smaller genome size compared to other known C-values for species within a particular family and also when compared to the mean values of dicots and monocots. The study also showed that halophylic annuals have a smaller genome size (2.49 pg) than perennial ones (7.45 pg DNA).     

Genome size stability among five subspecies of Pinus nigra Arnold s.l.

Despite the fact that genome size should be constant at species level, many reports of intraspecific variations exist. Thus, we carried out an analysis to determine the possible existence of nuclear DNA content variation in European black pine (Pinus nigra s.l.), a good model for such a study given its karyological uniformity, morphological polymorphism, broad geographical distribution, ecological plasticity and taxonomic heterogeneity. The panel comprised 20 populations across the natural range of P. nigra from Europe, Northwest Africa and Asia Minor including five subspecies: subsp. nigra, salzmanni, dalmatica, pallasiana and mauretanica. Mean 1C DNA content of the species was 23.62 pg (±0.209) assessed by flow cytometry. This converts to 23.1 G base pairs. The coefficients of variation within and between populations did not exceed 2.6%. Although we had already reported the existence of significant differences for three Black pine populations in our previous work on five Pinus spp. [Bogunic, F., Muratovic, E., Brown, S.C., Siljak-Yakovlev, S., 2003. Genome size of five Pinus from Balkan region. Plant Cell Rep. 22, 59–63], intraspecific variation was not confirmed in the present study dealing with many more populations. Subspecific divisions of Black pine were characterised with following mean values: subsp. pallasiana—23.80 pg, dalmatica—23.79 pg, nigra—23.65 pg, salzmanni—23.55 pg, and mauretanica—23.24 pg. A positive relationship between genome size and longitude was observed (r = 0.44, p < 0.05). We conclude that the diversification of populations of P. nigra has occurred without significant genome size changes throughout its wide geographical range from ecologically contrasting habitats. A clinal mode of genome size variation is present, in line with hypothesis of P. nigra spreading from south-western Asia towards European habitats.     

Intraplant Nuclear DNA Content Variation in Diploid Nuclei of Maize (Zea mays L.)

Diploid nuclei from stem, mesocotyl, nodal root and root tiptissue of two maize hybrids were examined with respect to theirDNA content. The nuclei were isolated and stained with DAPIand passed through a flow cytometer-cell sorter. The titrationcurve for each tissue was determined. Significant variationwas observed among nuclei of different tissue types. Stem androot tips had the highest diploid nuclear DNA amounts while2-week-old mesocotyl had the lowest diploid nuclear DNA amount.These results provide evidence that during plant developmentand differentiation, the amount of DNA within a diploid nucleuschanges through loss of specific DNA sequences. This study alsodemonstrates the sensitivity of flow cytometry in detectingsmall intraplant variation in nuclear DNA. Key words: Flow cytometry, fluorochrome DAPI, DNA content, tissue differentiation, plant development     

Microinjection of serum-starved mitochondria derived from somatic cells affects parthenogenetic development of bovine and murine oocytes

Microinjection of isolated mitochondria into oocytes is an effective method to introduce exogenous mitochondrial DNA. In nuclear transfer procedures in which donor cell mitochondria are transferred with nuclei into recipient oocytes; development and survival rates of reconstructed embryos may be also directly influenced by mitochondrial viability. Mitochondrial viability is dramatically affected by cell culture conditions, such as serum starvation prior to nuclear transfer. This study was conducted to examine the influence of exogenous mitochondria using bovine and mouse parthenogenetic models. Mitochondria were isolated from primary cells at confluency and after serum starvation. The bovine oocytes injected with serum-starved mitochondria showed lower rates of morula and blastocyst formation when compared to uninjected controls (P < 0.05). However, the developmental rates between non-starved mitochondria injection and controls were not different (P > 0.05). The murine oocytes injected with serum-starved mitochondria showed lower rates of development when compared with non-starved mitochondria and controls (P < 0.01). In contrast to mitochondria transfer, ooplasm transfer did not affect murine or bovine parthenogenetic development (P > 0.05). The overall results showed that injection of serum-starved mitochondria influenced parthenogenetic development of both bovine and murine oocytes. Our results illustrate that the somatic mitochondria introduction accompanying nuclei has the capacity to affect reconstructed embryo development; particularly when using serum-starved cells as donor cells.     

Constitutive hyperactivity of histone deacetylases enhances radioresistance in Lepidopteran Sf9 insect cells

BackgroundLepidopteran insect cells withstand multifold higher radiation doses and suffer far less DNA damage despite carrying numerous structural/functional homologies with mammalian cells. Since DNA–histone interactions significantly influence radiation-induced DNA damage, we investigated the role of histones in insect cell radioresistance.MethodsModified comet assay was used to assess the γ-radiation-induced DNA damage following serial histone depletion by varied salt concentrations. Acid–Urea–Triton (AUT) gel analysis combined with in silico predictions was used to compare mammalian and insect histones and acetylation status while HDAC activity was assessed/modified for studying the latter's role in radioresistance. Cell death was measured by morphological analysis and flow cytometry.ResultsHigh-salt extraction pattern from Sf9 nuclei suggested stronger DNA–histone affinity as the two core histones H2A/H2B could be extracted at much higher (2 M) concentration as compared to 1.2 M NaCl in mammalian (AA8) cells. Electrophoretic mobility of unirradiated Sf9 cells remained unaltered at all salt concentrations (0.14 M–2 M NaCl), and radiation-induced DNA damage increased only by 2 M-NaCl pre-treatment. In silico analysis confirmed excellent conservation of Lepidopteran H2A/H2B sequence with human histones including comparable N-terminal lysine residues, yet these had ~ 60% lower acetylation. Importantly, insect cells showed ~ 70% higher histone deacetylase activity whose inhibition by Trichostatin-A reversed hypo-acetylation state and caused significant radiosensitization, thereby confirming the protective contribution of reduced acetylation.ConclusionOur study reveals that the hypo-acetylated state of well-conserved core histones, maintained by considerable HDAC activity, contributes significantly in Lepidopteran radioresistance.General SignificanceThis investigation shows constitutively high activity of HDACs as a potential radioprotective mechanism existing in insect cells.     

Selective hydrogenation of 1,10-phenanthrolines by silica-supported palladium nanoparticles

The catalytic hydrogenation of 1,10-phenanthroline (Phen) or 2,9-dimethyl-1,10-phenanthroline (DMPhen) has been achieved using silica-supported palladium nanoparticles (Pd/SiO₂) with metal contents of 1.98 or 9.95 wt%. With either catalyst, the hydrogenation regiochemistry has been effectively controlled by the reaction temperature. The catalyst with the higher metal content was selective for the hydrogenation of one heterocyclic ring of either substrate at 80 °C and for both external rings at 130 °C for Phen and at 160 °C for DMPhen. The catalyst with the lower metal content was more active and exhibited comparable selectivity.     

Assessment of alterations in X-ray irradiation-induced DNA damage of glioma cells by using proton nuclear magnetic resonance spectroscopy

Glioma is one of the most common types of brain tumors. DNA damage is closely associated with glioma cell apoptosis induced by X-ray irradiation. Alterations of metabolites in glioma can be detected noninvasively by proton nuclear magnetic resonance (1H NMR) spectroscopy. To noninvasively explore the micro mechanism in X-ray irradiation-induced apoptosis, the relationship between metabolites and DNA damage in glioma cells was investigated. Three glioma cell lines (C6, U87 and U251) were randomly designated as control (0 Gy) and treatment groups (1, 5, 10, 15 Gy). After X-ray exposure, each group was separated into four parts: (i) to detect metabolites by 1H NMR spectroscopy; (ii) to make cell colonies; (iii) to detect cell cycle distribution and apoptosis rate by flow cytometry; and (iv) to measure DNA damage by comet assay. The metabolite ratios of lactate/creatine and succinate/creatine decreased (lactate/creatine: C6, 22.17–66.27%; U87, 15.93–44.56%; U251, 26.27–74.48%. succinate/creatine: C6, 14.41–48.35%; U87, 22.03–70.62%; U251, 17.33–60.06%) and choline/creatine increased (C6, 52.22–389.68%; U87, 56.15–82.36%; U251, 31.87–278.62%) in the treatment groups compared with the control group (each P < 0.05), which linearly depended on DNA damage. An increasing dose of X-ray irradiation increased numbers of apoptotic cells (P < 0.01), and the DNA damage parameters were dose-dependent (P < 0.05). The colony-forming rate declined (P < 0.01) and the percentage of cells at G1 stage increased when exposed to 1 Gy X-ray (three cell lines, P < 0.05). Metabolite alterations detected by 1H NMR spectroscopy can be used to determine DNA damage induced by X-ray irradiation. 1H NMR spectroscopy is a noninvasive method to predict DNA damage of glioma cell at the micro level.     

Sunflower (Helianthus annuus) Leaves Contain Compounds that Reduce Nuclear Propidium Iodide Fluorescence

Sunflower leaves have unidentified compounds that interferewith propidium iodide (PI) intercalation and/or fluorescence.Independently prepared pea leaf nuclei show greater PI fluorescencethan nuclei from pea leaves simultaneously processed (co-chopped)with sunflower leaves. Differences in fluorescence persist aftermixing the PI-stained pea and the co-chopped pea/sunflower samples,i.e. PI staining protects the nuclei from the effects of theinhibitor. The current results are significant to practicalflow cytometric determination of plant nuclear DNA content.They show: (1) simultaneous processing of nuclear samples fromthe target and the standard species is necessary to obtain reliableDNA estimates; (2) a test for the presence of inhibitors shouldbe conducted; and (3) when inhibitors are present caution shouldbe taken in interpreting differences in estimated DNA content.The previously reported environmentally-induced variation inDNA content in sunflower populations is most simply explainedby variation in the amount of environmentally-induced inhibitorthat interferes with intercalation and/or fluorescence of PI.Intraspecific variation of DNA content for Helianthus annuusneeds to be re-evaluated using best practice techniques comparingphysiologically uniform tissues that are free of inhibitors.The best estimate for 2C DNA content of H. annuus used in thisstudy is 7.3 pg. Copyright 2000 Annals of Botany Company Helianthus annuus, DNA content, flow cytometry, propidium iodide, endogenous inhibitors     

Response of Potamogeton crispus root characteristics to sediment heterogeneity

Plant growth, biomass allocation, root distribution and plant nutrient content were investigated in the submerged macrophyte Potamogeton crispus growing in heterogeneous sediments. Three experimental sediments heterogeneous in nutrient content and phosphorus release capacity were used: sandy loam with low nutrient content (A), clay with intermediate nutrient content (B), and clay with high nutrient content (C). Biomass accumulation was significantly affected by the sediment type, and was highest in clay C (1.23 mg per plant dry weight) but lowest in sandy loam (0.69 mg per plant dry weight). The root:shoot ratios in treatments A, B and C were 0.30, 0.14 and 0.09, respectively. P. crispus allocated more biomass to roots in sandy loam compared with the other sediments. The average root numbers in sediments A, B and C were 16, 19 and 20, respectively, and the total root lengths in sediments A, B and C were 238.84, 200.36 and 187.21 cm, respectively. Almost 90% of the root biomass was distributed in the 0–15 cm depth in sediments B and C, compared with 64.53% in sediment A. The rank order of plant nitrogen and phosphorus concentrations in the sediment types was C > B > A. These results indicate that both sediment structure and nutrient availability influence the growth and distribution of the root system of P. crispus.     

Image analysis software for automatic DNA ploidy assessment of archival solid tumours.

BACKGROUND: Image cytometry has proved to provide a good alternative to flow cytometry for DNA ploidy measurement of archival tumors. However, when interactively done this technique is unable to give statistically valuable results within an acceptable time for clinical oncology. METHODS: An image cytometer was developed for fully automatic DNA ploidy quantitation, focusing efforts on speed and accuracy. Software functionalities include systematic acquisition of fields on a microscopic slide, detection, localization and sorting of nuclei, computation of the DNA content together with post-processing tools, for a deeper analysis of the DNA ploidy diagram. RESULTS: DNA ploidy analysis of archival breast carcinoma samples illustrates the accuracy of DNA ploidy measurements and the sensitivity in the detection of DNA ploidy abnormalities as a result of cell sorting. CONCLUSIONS: Fully automatic image cytometry is able to combine qualities of flow cytometry (automatic analysis of a statistically significant collection of cell nuclei) with additional advantages: sorting of unwanted events (debris, stromal and inflammatory cell nuclei) and facilities for an a posteriori control of the quality of cell selection. This method is well suited to DNA ploidy analysis of archival cancer samples.     

Can digital image classification be used as a standardised method for surveying peatland vegetation cover?

The ability to carry out systematic, accurate and repeatable vegetation surveys is an essential part of long-term scientific studies into ecosystem biodiversity and functioning. However, current widely used traditional survey techniques such as destructive harvests, pin frame quadrats and visual cover estimates can be very time consuming and are prone to subjective variations. We investigated the use of digital image techniques as an alternative way of recording vegetation cover to plant functional type level on a peatland ecosystem. Using an established plant manipulation experimental site at Moor House NNR (an Environmental Change Network site), we compared visual cover estimates of peatland vegetation with cover estimates using digital image classification methods, from 0.5 m × 0.5 m field plots. Our results show that digital image classification of photographs taken with a standard digital camera can be used successfully to estimate dwarf-shrub and graminoid vegetation cover at a comparable level to field visual cover estimates, although the methods were less effective for lower plants such as mosses and lichens. Our study illustrates the novel application of digital image techniques to provide a new way of measuring and monitoring peatland vegetation to the plant functional group level, which is less vulnerable to surveyor bias than are visual field surveys. Furthermore, as such digital techniques are highly repeatable, we suggest that they have potential for use in long-term monitoring studies, at both plot and landscape scales.     

Discrimination between benign and malignant melanocytic skin lesions by multivariate analysis, quantitative S-100 immunohistochemistry, nuclear morphometry and DNA cytometry

OBJECTIVE: To determine whether combined quantitative immunohistochemistry of S-100, nuclear morphometry and DNA image cytometry improves discrimination between benign and malignant melanocytic skin lesions (MSLs). STUDY DESIGN: S-100 protein expression was measured in tissue sections of MSLs using an image cytometry system. Localized areas of high S-100 expression were used to identify regions in sequential, facing sections in which morphometric and cytometric features of nuclei, including DNA ploidy, were also measured. RESULTS: Malignant cases had significantly higher S-100 protein staining intensity, larger nuclei and greater DNA content (P < .05). High staining intensity for S-100 protein weakly correlated with variation in size of the mean nuclear area (P = .04) and DNA content (P = .03). Combining the features of nuclear area and DNA integrated optical density in areas of high-intensity staining for S-100 protein discriminated more accurately between 12 benign and 16 malignant areas than any of the features along (P = .0003). CONCLUSION: Combined multivariate quantitative immunohistochemical, morphometric and DNA cytometric analysis greatly improves discrimination between benign MSLs and malignant melanoma. Larger test sets are required to confirm the promising results of this initial study.     

DNA damage in potato plants induced by cadmium,ethyl methanesulphonate and γ-rays

We have calibrated the alkaline protocol of the plant comet (Single Cell Gel Electrophoresis) assay as a method for detecting the extent of induced DNA damage in potato plants (Solanum tuberosum L. cultivar Korela). After 2 and 24 h treatments of the rooted cuttings with the heavy metal cadmium (Cd²⁺), a dose–response increase in DNA damage was noted versus controls in root nuclei. With a 24 h recovery period, the Cd²⁺-induced DNA damage in roots increased significantly. No significant increase in DNA damage was demonstrated in leaf nuclei after 24 h Cd²⁺ treatments, but continuous Cd²⁺ treatments for 2 weeks resulted in an increase in leaf DNA damage. This increase may be however associated with necrotic and apoptotic DNA fragmentation, as the affected plants had inhibited growth and distorted yellowish leaves. For comparison, the monofunctional alkylating agent ethyl methanesulphonate, and γ-rays were assessed for induced DNA damage. Analysis of the accumulation of cadmium by inductively coupled plasma optical emission spectrometry demonstrates that roots accumulate almost 9-fold more cadmium than aboveground parts of the rooted potato cuttings. This may explain the absence of Cd²⁺ genotoxicity in leaves after short-term treatments.     

Isolation and antiproliferative activity of Lotus corniculatus lectin towards human tumour cell lines

The objective of the study was to investigate the anti cancer activity of a lectin isolated from Lotus corniculatus seeds. A tetrameric 70 kDa galactose specific lectin was purified using two step simple purification protocol which involved affinity chromatography on AF-BlueHC650M and gel filtration on Sephadex G-100. The lectin was adsorbed on AF-BlueHC650M and desorbed using 1 M NaCl in the starting buffer. Gel filtration on Sephadex G-100 yielded a major peak absorbance that gave two bands of 15 kDa and 20 kDa in SDS PAGE. Hemagglutination activity was completely preserved, when the temperature was in the range of 20–60 °C. However, drastic reduction in activity occurred at temperatures above 60 °C. Full hemagglutination activity was retained at ambient pH 4–12. Thereafter no activity was observed above pH 13. Hemaglutination of the lectin was inhibited by d-galactose. The lectin showed a strong antiproliferative activity towards human leukemic (THP-1) cancer cells followed by lung cancer (HOP62) cells and HCT116 with an IC₅₀ of 39 μg/ml and 50 μg/ml and 60 μg/ml respectively. Flow cytometry analysis showed an increase in the percentage of cells in sub G0G1 phase confirming that Lotus corniculatus lectin induced apoptosis. Morphological observations showed that Lotus corniculatus lectin (LCL) treated THP-1 cells displayed apparent apoptosis characteristics such as nuclear fragmentation, appearance of membrane enclosed apoptotic bodies and DNA fragmentation. Lotus corniculatus lectin (LCL) effectively inhibits the cell migration in a dose dependent manner as indicated by the wound healing assay.     

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.