Flow Cytometric and Immunohistochemical Correlations in High Incidence Human Solid Tumors

1997 ◽  
Vol 83 (3) ◽  
pp. 689-697 ◽  
Author(s):  
Donatella Tirindelli Danesi ◽  
Marcello Spanò ◽  
Fabiana Antonini ◽  
Pierluigi AltaVista ◽  
Piera Catalano ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Dna Content ◽  
Rank Correlation ◽  
Breast Cancer Patients ◽  
Dna Index ◽  
Ploidy Levels ◽  
Spearman’S Rank Correlation ◽  
Flow Cytometric ◽  
Human Solid Tumors ◽  
Stem Cell Lines

475 patients with carcinoma at different sites (141 colon-rectum; 102 breast; 50 stomach; 48 kidney; 46 head and neck; 41 bladder; 47 other sites) submitted to surgery have been analyzed after histopathological staging and grading, by flow cytometry (monoparametric DNA content analysis) and immunohistochemistry (p53, c-erbB-2, and PCNA expression). In breast cancer patients the presence of receptors for estrogen (ER) and progesterone (PGR) has also been determined. Flow cytometry-derived parameters were DNA ploidy, fraction of cells in S-phase (SPF), and DNA content heterogeneity (multiclonal stem cell lines with different DNA index and/or more than one subpopulations with different ploidy levels in different samples from the same tumor). Correlations of the results obtained by the different techniques have been attempted by the non-parametric Spearman's rank correlation approach. Significant associations (P «0.05) were found between the histopathological, immunohistochemical and flow cytometric parameters considered in some anatomical regions, such as stomach (p53 vs DNA content aneuploidy and vs heterogeneity), colon-rectum (TNM vs p53 and vs heterogeneity), bladder (grading vs DNA content aneuploidy and vs heterogeneity). Tumor heterogeneity proved to be dependent on the number of tumor samples taken. The results of this preliminary assessment will subsequently be compared with the data obtained from a currently ongoing follow-up survey.

Intratumoral DNA content heterogeneity in breast carcinomas demonstrated by core punch tissue sampling and flow cytometry

2014 ◽  
Vol 67 (9) ◽  
pp. 821-824 ◽  
Author(s):  
Zhouwei Zhang ◽  
Donald L Weaver ◽  
Kavita Munjal ◽  
Mark F Evans
Keyword(s):  
Flow Cytometry ◽  
Dna Content ◽  
Intratumoral Heterogeneity ◽  
Dna Index ◽  
Surgical Specimen ◽  
Flow Cytometric ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Relationship Of ◽  
Formalin Fixed

Further to advancements in instrumentation and fluorescent dye technologies, there has been a resurgence of interest in the flow cytometric assay of formalin-fixed, paraffin-embedded specimens. Here we present a novel, simple and effective alternative to whole block sectioning that allows selective multisampling of tissues within a specimen block and the investigation of intratumoral heterogeneity. Formalin-fixed, paraffin-embedded breast carcinoma specimens were core-punched using 1.0 mm diameter needles and assayed by flow cytometry using a modified Hedley method. Intratumoral heterogeneity for DNA index and per cent S-phase fraction was detected in 10 of 23 (44%) and 11 of 23 (47%) specimens respectively. Macro-level genomic heterogeneity is common in breast cancer even within a single surgical specimen block. Studies investigating the relationship of DNA content heterogeneity to other markers of genomic instability such as mutations, deletions, insertions and translocations are warranted.

Flow Cytometrically Determined DNA Content of Breast Carcinoma and Benign Lesions: Correlations with Histopathological Parameters

1986 ◽  
Vol 72 (2) ◽  
pp. 171-177 ◽  
Author(s):  
Raffaella Uccelli ◽  
Alberto Calugi ◽  
Donato Forte ◽  
Francesco Mauro ◽  
Paolo Polonio-Balbi ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Dna Content ◽  
Cell Subpopulation ◽  
Aneuploid Cell ◽  
Breast Carcinomas ◽  
Benign Lesions ◽  
Dna Index ◽  
Tissue Samples ◽  
Normal Tissues ◽  
Relative Dna Content

The relative DNA content of cellular samples from 54 patients affected by breast carcinomas and 20 affected by benign breast lesions (including 11 fibroadenomas) was measured by flow cytometry. All normal tissue samples and 17/20 (85%) specimens from benign lesions exhibited a cytometrically diploid DNA distribution, 3/20 (15%) benign lesions an abnormal DNA content, and 35/54 (65%) carcinomas at least one aneuploid cell subpopulation. Furthermore, 9/54 (17%) tumors were characterized by the presence of more than one aneuploid cell subpopulation. The results also indicate that flow cytometry can be used to recognize lymph nodes infiltrated by aneuploid cells. Statistically significant correlations were evidenced between the occurrence of aneuploidy or the ploidy level measured as DNA index and the nodal infiltration status. The percentage of S cells can also be extracted from DNA content distribution histograms. Statistically significant differences (p < 0.01) were also observed for the percentage of S cells between normal tissues (6.2±3.2 SD) and benign lesions (11.1±6.6 SD), normal tissues (6.2 ± 3.2 SD) and aneuploid tumors (19.7 ± 10.3 SD), benign lesions (11.1 ± 6.6 SD) and aneuploid tumors (19.7 ± 10.3 SD), and diploid (7.9 ± 4.0 SD) and aneuploid tumors (19.7 ± 10.3 SD).

Nuclear DNA amount in pure species and hybrid willows (Salix): a flow cytometric investigation

1998 ◽  
Vol 76 (1) ◽  
pp. 157-165 ◽  
Author(s):  
Jérôme Thibault
Keyword(s):  
Flow Cytometry ◽  
Ploidy Level ◽  
Dna Content ◽  
Nuclear Dna ◽  
Nuclear Dna Content ◽  
Natural Hybridization ◽  
Ploidy Levels ◽  
Key Words Flow Cytometry ◽  
Dna Values ◽  
Dna Amounts

Flow cytometry (FCM) has been used to estimate the nuclear DNA content of 11 Salix species and 5 hybrids. One hundred and sixty nine individuals were studied including 159 individuals from a sequence of 32 communities along a stretch of river in France and 10 individuals from French and English collections for comparison. Isolated nuclei were stained with propidium iodide. FCM was a significantly more practical and rapid technique than that of establishing the karyotype to survey many samples of Salix for variation in ploidy. The 2C DNA amounts for diploid species ranged from 0.76 to 0.98 pg, and tetraploid values ranged from 1.62 to 1.80 pg. The DNA values were consistent with the known ploidy levels. With the exception of a doubtful Salix xquercifolia, ploidy levels and DNA amounts of hybrids were intermediate compared with those of their parents. Intraspecific variation of nuclear DNA values including instrumental variation was low (i.e., 6-11% at the same ploidy level). FCM appeared to be an accurate tool for determination of Salix triploid hybrids. However, it remains limited concerning hybrids from crosses between species of the same ploidy level. Results suggest that natural hybridization might not be frequent in the communities studied, although they have been subject to disturbance. Previous overestimates of hybridization frequency in willows were probably due to misinterpretation of the effects of the environment on Salix spp. morphology; however, the extent and mechanisms of introgression in the genus remain to be further investigated. Key words: flow cytometry, Salix, hybridization, nuclear DNA content, riparian vegetation, disturbance.

scholarly journals Chromosome Number, Ploidy Level, and Nuclear DNA Content in 23 Species of Echeveria (Crassulaceae)

Genes ◽  
2021 ◽  
Vol 12 (12) ◽  
pp. 1950
Author(s):  
Guadalupe Palomino ◽  
Javier Martínez-Ramón ◽  
Verónica Cepeda-Cornejo ◽  
Miriam Ladd-Otero ◽  
Patricia Romero ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Chromosome Number ◽  
Ploidy Level ◽  
Dna Content ◽  
Chromosome Numbers ◽  
Nuclear Dna ◽  
Nuclear Dna Content ◽  
Root Tips ◽  
Ploidy Levels ◽  
Dna Amounts

Echeveria is a polyploid genus with a wide diversity of species and morphologies. The number of species registered for Echeveria is approximately 170; many of them are native to Mexico. This genus is of special interest in cytogenetic research because it has a variety of chromosome numbers and ploidy levels. Additionally, there are no studies concerning nuclear DNA content and the extent of endopolyploidy. This work aims to investigate the cytogenetic characteristics of 23 species of Echeveria collected in 9 states of Mexico, analyzing 2n chromosome numbers, ploidy level, nuclear DNA content, and endopolyploidy levels. Chromosome numbers were obtained from root tips. DNA content was obtained from the leaf parenchyma, which was processed according to the two-step protocol with Otto solutions and propidium iodide as fluorochrome, and then analyzed by flow cytometry. From the 23 species of Echeveria analyzed, 16 species lacked previous reports of 2n chromosome numbers. The 2n chromosome numbers found and analyzed in this research for Echeveria species ranged from 24 to 270. The range of 2C nuclear DNA amounts ranged from 1.26 pg in E. catorce to 7.70 pg in E. roseiflora, while the 1C values were 616 Mbp and 753 Mbp, respectively, for the same species. However, differences in the level of endopolyploidy nuclei were found, corresponding to 4 endocycles (8C, 16C, 32C and 64C) in E. olivacea, E. catorce, E. juarezensis and E. perezcalixii. In contrast, E. longiflora presented 3 endocycles (8C, 16C and 32C) and E. roseiflora presented 2 endocycles (8C and 16C). It has been suggested that polyploidization and diploidization processes, together with the presence of endopolyploidy, allowed Echeveria species to adapt and colonize new adverse environments.

scholarly journals NUCLEAR DNA CONTENT IN BLUEBERRY DETERMINED BY FLOW CYTOMETRY

HortScience ◽  
1992 ◽  
Vol 27 (6) ◽  
pp. 580e-580
Author(s):  
Rodomiro Ortiz ◽  
D.E. Costich ◽  
T.P. Meagher ◽  
N. Vorsa
Keyword(s):  
Flow Cytometry ◽  
Dna Content ◽  
Nuclear Dna ◽  
Nuclear Dna Content ◽  
Haploid Genome ◽  
Dna Flow Cytometry ◽  
Dna Amount ◽  
Ploidy Levels ◽  
Polyploid Evolution ◽  
Haploid Genome Size

DNA flow cytometry was used to determine nuclear DNA content in diploid blueberry species, and 3x, 4x, 5x, and 6x ploidy levels. Relative fluorescence intensity of stained nuclei measured by flow cytometry was a function of the number of chromosome sets (X): Y = 3.7X – 2.3 (r2 = 95.1%). DNA flow cytometry should be useful for ploidy level determination in the seedling stage. A significant linear relationship was established between nuclear DNA content and number of chromosomes (x); DNA (pg) = 0.52 x1 (r2 = 99.8%). Based on this equation the haploid genome DNA amount (1C) was calculated as 0.62 ± 0.08 pg, with an approximate haploid genome size of 602 Mbp/1C. The results indicate that conventional polyploid evolution occured in the section Cyanococcus, genus Vaccinium: the increase in DNA was concurrent with increase in chromosome number. DNA content differences among 2x species were correlated with Nei's genetic distance estimates based on 20 isozyme markers. Most of the variation was among species (49%), with 26% between populations within species, and 25% within populations.

scholarly journals Histogram score contributes for reliability of DNA content estimatives in Brachiaria spp

2012 ◽  
Vol 36 (6) ◽  
pp. 599-607
Author(s):  
Ana Luiza de Oliveira Timbó ◽  
Roselaine Cristina Pereira ◽  
Vanderley Borges dos Santos ◽  
Fausto Souza Sobrinho ◽  
Lisete Chamma Davide
Keyword(s):  
Flow Cytometry ◽  
Dna Content ◽  
Raphanus Sativus ◽  
Triploid Hybrid ◽  
Reference Standard ◽  
Internal Reference ◽  
Ploidy Levels ◽  
Buffer Solutions ◽  
Internal Reference Standard ◽  
Content Analyses

Flow cytometry allows to estimate the DNA content of a large number of plants quickly. However, inadequate protocols can compromise the reliability of these estimates leading to variations in the values of DNA content the same species. The objective of this study was to propose an efficient protocol to estimate the DNA content of Brachiaria spp. genotypes with different ploidy levels using flow cytometry. We evaluated four genotypes (B. ruziziensis diploid and artificially tetraploidized; a tetraploid B. brizantha and a natural triploid hybrid), three buffer solutions (MgSO4, Galbraith and Tris-HCl) and three species as internal reference standards (Raphanus sativus, Solanum lycopersicum e Pisum sativum). The variables measured were: histogram score (1-5), coefficient of variation and estimation of DNA content. The best combination for the analysis of Brachiaria spp. DNA content was the use of MgSO4 buffer with R. sativus as a internal reference standard. Genome sizes expressed in picograms of DNA are presented for all genotypes and the importance of the histogram score on the results reliability of DNA content analyses were discussed.

scholarly journals Flow cytometry-based determination of ploidy from dried leaf specimens in genomically complex collections of the tropical forage grass Urochloa s. l.

2021 ◽  
Author(s):  
Paulina Tomaszewska ◽  
Till K. Pellny ◽  
Luis Miguel Hernandez ◽  
Rowan A. C. Mitchell ◽  
Valheria Castiblanco ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Dna Content ◽  
Nuclear Dna ◽  
Nuclear Dna Content ◽  
Germplasm Collection ◽  
Forage Grass ◽  
Ploidy Levels ◽  
Sustainable Improvement ◽  
Tropical Forage ◽  
Relative Differences

We aimed to develop an optimized approach to determine ploidy for dried leaf material in a germplasm collection of a tropical forage grass group, including approaches to collect, dry and preserve plant samples for flow cytometry analysis. Urochloa (including Brachiaria, Megathyrus and some Panicum) tropical grasses are native to Africa and are now, after selection and breeding, planted worldwide, particularly in South America, as important forages with huge potential for further sustainable improvement and conservation of grasslands. The methods enable robust identification of ploidy levels (coefficient of variation, CV, typically <5%). Ploidy of some 353 forage grass accessions (ploidy range from 2 to 9), from international genetic resource collections, showing variation in basic chromosome numbers and reproduction modes (apomixis and sexual), were determined using our defined standard protocol. Two major Urochloa agamic complexes used in the current breeding programs at CIAT and EMBRAPA: the ' brizantha' and 'humidicola' agamic complexes are variable, with multiple ploidy levels and DNA content. U. brizantha has odd level of ploidy (x=5), and the relative differences in nuclear DNA content between adjacent cytotypes is reduced, thus more precise examination of this species is required. Ploidy measurement of U. humidicola revealed some aneuploidy.

scholarly journals MICROPROPAGATION AND PLOIDY STABILITY OF Lippia lacunosa Mart. & Schauer: AN ENDANGERED BRAZILIAN MEDICINAL PLANT

2019 ◽  
Vol 6 (1) ◽  
pp. 1-7
Author(s):  
Diego Pandeló José ◽  
José Marcello Salabert De Campos ◽  
Lyderson Facio Viccini ◽  
Emilly Ruas Alkimim ◽  
Marcelo De Oliveira Santos
Keyword(s):  
Flow Cytometry ◽  
Medicinal Plant ◽  
Dna Content ◽  
Nuclear Dna ◽  
Nuclear Dna Content ◽  
Naphthalene Acetic Acid ◽  
Flow Cytometry Analysis ◽  
Ploidy Levels

Lippia lacunosa is a Brazilian savanna plant that belongs to the Verbenaceae family. It has been used in folk medicine as a treatment for different diseases. This species represents an endangered Brazilian medicinal plant, and this is the first report documenting a reliable protocol for the in vitro propagation and regeneration of L. lacunosa. Axenic explants were cultivated in MS medium containing different concentrations of naphthalene acetic acid (NAA) to induce root growth. The mean shoot length and the number of roots were highest with 0.06 mg·L-1 NAA. The highest number of buds in shoot regeneration was induced with 2 mg·L-1 6-benzylaminopurine (BA). To obtain a long-term culture, the dwarf shoots were elongated on MS media containing 0.5 mg·L-1 BA alternated with MS containing 2 mg·L-1 BA every 40 days. In the present protocol, the long-term shoots retained the ability to root even after long periods of BA treatment. In addition, we evaluated the nuclear DNA content and ploidy levels, including the occurrence of endopolyploidy, in long-term micropropagated plant leaves using flow cytometry analysis. The plants propagated in vitro over several years possessed nuclear DNA contents ranging from 2.940 to 3.095 pg, and no differences in DNA content were found among in vitro plants or between these plants and the control (L. lacunosa from a greenhouse with a DNA content of 3.08 pg). The flow cytometry analysis also demonstrated that there was no polyploidization. The present study will be useful for biotechnological approaches and provides the first estimate of the nuclear DNA content of this species using flow cytometry.

Nuclear genome size in Selaginella

Genome ◽  
2007 ◽  
Vol 50 (4) ◽  
pp. 351-356 ◽  
Author(s):  
Damon P. Little ◽  
Robbin C. Moran ◽  
Eric D. Brenner ◽  
Dennis Wm. Stevenson
Keyword(s):  
Chromosome Number ◽  
Genome Size ◽  
Nuclear Genome ◽  
Rank Correlation ◽  
Cultivated Plants ◽  
Strongly Correlated ◽  
Flow Cytometric Data ◽  
Spearman’S Rank Correlation ◽  
Time Estimates ◽  
Flow Cytometric

Estimates of nuclear genome size for 9 Selaginella species were obtained using flow cytometry, and measurements for 7 of these species are reported for the first time. Estimates range from 0.086 to 0.112 pg per holoploid genome (84–110 Mb). The data presented here agree with the previously published flow cytometric results for S. moellendorffii . Within the 9 species sampled here, chromosome number varies from 2n = 16 to 2n = 27. Nuclear genome size appears to be strongly correlated with chromosome number (Spearman’s rank correlation; p = 0.00003725). Cultivated S. moellendorffii lacks sexual reproduction—manifest by the production of abortive megasporangia. Flow cytometric data generated from a herbarium specimen of a fertile wild-collected S. moellendorffii are virtually indistinguishable from the data generated from fresh material (0.088 vs. 0.089 pg/1C). Therefore, the limited fertility observed in cultivated plants is probably not the result of abnormal chromosome number (e.g., induced by interspecific hybridization).

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