Distinction of leucocyte classes based on chromatin‐structure‐dependent DNA‐binding of 7‐aminoactinomycin D

Stokke, Trond; Steen, Harald B.

doi:10.1002/cyto.990080608

Cited by 23 publications

(25 citation statements)

References 38 publications

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“…In the accessibility studies of Darzynkiewicz et al (1984) and Evenson et al (1986), the fluorochromes with intercalating binding mode already proved to have less accessibility than fluorochromes binding externally to DNA when the chromatin is condensed. In the case of 7-AMD, the limitation of accessibility with chromatin condensation is likely to result from the large size of this molecule as already reported by Darzynkie-wicz et al (1984) and Stokke and Steen (1987). In the case of OM, the limitation of accessibility with chromatin condensation is surAlthough structurally related, OM and MA differ by minor mriations in their chromophores and sugar components (Ward, 1965) and fluorescence emission (Crissman et al, 1978), which might correspond to different affinity features to DNA.…”

Section: Discussionsupporting

confidence: 51%

“…Only image cytometry can extract the topographical parameters required to study the arrangement of chromatin in situ. The accuracy of the DNA measurements is dependent on the stoichiometry of dye-DNA complex, which, in turn, is influenced by physicochemical (dye/DNA concentration ratio, DNA binding modes and their affinity, ionic strength) (Latt and Langlois, 1990) and biochemical (chromatin supramolecular arrangement) factors (Stokke and Steen, 1987;Larsen et al, 1986;Rabinovitch et al, 1986;Darzynkiewicz et al, 1984). Many fluorescent dyes are assumed to be specific for and bind stoichiometrically to DNA on the basis of FCM studies, given adequate preparation techniques ('Gylor and Milthorpe, 1980;Crissman et al, 1978;Ardnt-Jovin and Jovin, 1977;Krishan, 1975).…”

Section: Introductionmentioning

confidence: 99%

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Fluorescence image cytometry of nuclear DNA content versus chromatin pattern: a comparative study of ten fluorochromes.

Santisteban

Montmasson²,

Giroud³

et al. 1992

J Histochem Cytochem.

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This study is intended to be the first step of an in situ exploration of the intranuclear DNA distribution by image cytomeuy (SAMBA) with several fluorochromes. The nuclear DNA content and the chromatin pattern, revealed by ten fluorochromes (HO, DAPI, MA, CMA3, OM, QM, AO, EB, PI, and 7-AMD), were analyzed on mouse hepatocytes Fied by the Boehm-Sprenger procedure optimal for preserving the chromatin pattem. The question was whether fluotochromes specific to DNA make it possible to accurately quantitate the total nuclear DNA content when the chromatin pattern is preserved. Only HO and MA were found to provide satisfactory quantitation of nuclear DNA content, as assumed by both a small CV and a 4c to 2c ratio equal to 2. PI, EB, 7-AMD, and OM provided higher CV values, although the 4c to 2c ratio was stiU equal to 2. QM, AO, CMA3, and DAPI provided non-reproducible and non-stoichiometric nuclear DNA content measurements under the Fiation conditions used. The intranuclear and the intemuclear SD of the fluorescence intensities describing the fluorescence pattern of the

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Section: Discussionsupporting

confidence: 51%

Section: Introductionmentioning

confidence: 99%

Fluorescence image cytometry of nuclear DNA content versus chromatin pattern: a comparative study of ten fluorochromes.

Santisteban

Montmasson²,

Giroud³

et al. 1992

J Histochem Cytochem.

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“…Binding of dyes to DNA depends on chromatin conformation. Dyes such as 7-amino actinomycin D (7-AAD), which are highly sensitive to chromatin structure, can give different DNA content measurements for different types of normal, DNA diploid cells (32). Exposure of cells to a variety of treatments (acid, proteolytic enzymes) can change the binding of the dye to DNA.…”

Section: Sample Processing For Flow Cytometrymentioning

confidence: 99%

Guidelines for implementation of clinical DNA cytometry

Shankey

Rabinovitch

Bagwell³

et al. 1993

Cytometry

373

173

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Cell DNA content measurements, including determination of tumor ploidy and S-phase fraction, have been performed on a wide variety of human tumors for > 20 years using flow cytometry. During this time many publications have discussed the clinical utility of cell DNA content measurements. A major impediment to the more widespread application of cytometric DNA content measurements has been the lack of agreement among these studies. Whereas some discrepancies can be attributed to poorly designed studies lacking sufficient follow-up or significant numbers of patients, in many cases the discrepancies are due to technical factors in flow or image cytometry.The terminology used to describe the results of flow cytometry studies is often confusing and not universally applied. Although a convention for nomenclature for all DNA cytometry was recommended in 1984 (l), the guidelines suggested are frequently not used in published studies. Cytometric studies should not use cytogenetic terminology (hypodiploid, peritetriploid, etc.), where no direct measurement of changes in the number or composition of individual chromosomes has been made. Rather, the terms DNA diploid and DNA aneuploid should be used, with identification of the degree of DNA content abnormality given by the use of the DNA index, or DI (ratio of mean or mode of sample G,/G, population divided by mean or mode of diploid reference cells).One critical aspect of the clinical applications of these measurements is the use of standardized procedures to prepare and analyze clinical samples and to analyze and interpret cytometry data. A number of studies have demonstrated significant intralaboratory, as well as interlaboratory variation in the results of DNA content analyses (2-5) and in the interpretation of flow cytometric data (6). The purpose of this work is to help increase the reliability and reproducibility of DNA content flow cytometry by pointing out important technical considerations for cytometry, to provide guidelines that logically follow from these considerations, and to provide a framework for the development of standards and standardization of DNA content flow cytometry. Although a number of reported studies have utilized image cytometry to provide DNA content analysis, the technical differences between image and flow cytometry suggest that guidelines for the clinical application of image cytometry be developed independently.In reviewing the published literature, it is clear that many studies fail to provide sufficient information to judge critically the quality of cytometric measurement. It is recommended that all publications using DNA content cytometry provide details of the technique used to isolate and prepare cells, data that indicate that the sample used for cytometry contains representative tumor material, details concerning the techniques used to stain cells or nuclei (dye concentration, enzyme concentrations in units, cell concentrations), and details of the techniques used to analyze DNA content histograms (debris and aggregation correction ...

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“…In addition to the possible binding differences observed with certain dyes in cells of different phenotypes (6,12,13), the data show that changes in fluorescence intensity can be caused by energy transfer to or reabsorption by cell molecules with absorption bands in the visible region. On the other hand, the described staining procedure with H33258 can be used to detect hemoglobin-containing nucleated cells in bone marrow.…”

Section: Resultsmentioning

confidence: 93%

“…The DNA-specific bisbenzimidazole dye Hoechst 33258 (H33258) can be used to determine DNA content in fixed and permeabilized cells (12)(13)(14). During flow cytometric classification of mononuclear cells in the bone marrow from patients with acute leukemias, bone marrow aspirates from donors with no evidence of malignant disease were included as controls.…”

mentioning

confidence: 99%

Quenching of Hoechst 33258 fluorescence in erythroid precursors

et al. 1990

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Quenching of the fluorescence of DNA‐bound Hoechst 33258 in erythroid precursors was studied by flow cytometry and cytochemistry. This quenching artifact may affect the measurement of ploidy in specific cases. The bone marrow cells of two patients with hemolytic disease and active erythropoeisis contained subpopulations of cells with an apparent hypodiploid DNA content as measured by flow cytometry of paraformaldehyde‐fixed cells stained with Hoechst 33258. No aneuploidy was detected in either of the two cases when cells were stained with mithramycin or 7‐aminoactinomycin D. Cells exhibiting reduced Hoechst 33258 fluorescence expressed glycophorin A and low amounts of CD36, and were therefore erythroid precursors. In one case studied, the number of cells with reduced Hoechst 33258 fluorescence and glycophorin A expressed agreed well with the number of cells containing nuclear hemoglobin. In the other case, hemoglobin was present in a significant proportion of nucleated cells. Calculated values for the efficiency of resonance energy transfer from Hoechst 33258 to hemoglobin were in accordance with the observed levels of quenching (≈10%). However, the results could also be explained by hemoglobin reabsorption of Hoechst 33258 fluorescence. Nuclei stained with Hoechst 33258 showed uniform fluorescence, probably due to extraction of hemoglobin during the isolation procedure.

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Distinction of leucocyte classes based on chromatin‐structure‐dependent DNA‐binding of 7‐aminoactinomycin D

Cited by 23 publications

References 38 publications

Fluorescence image cytometry of nuclear DNA content versus chromatin pattern: a comparative study of ten fluorochromes.

Fluorescence image cytometry of nuclear DNA content versus chromatin pattern: a comparative study of ten fluorochromes.

Guidelines for implementation of clinical DNA cytometry

Quenching of Hoechst 33258 fluorescence in erythroid precursors

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