Accessibility of 7-aminoactinomycin D to lymphocyte nuclei after paraformaldehyde fixation

Linden, Eva L. van der; Skoglund, Per H.; Rundquist, Ingemar

doi:10.1002/(sici)1097-0320(19970101)27:1<92::aid-cyto12>3.0.co;2-n

Cited by 9 publications

(6 citation statements)

References 12 publications

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“…Conversely, the emission spectrum of DNA-dyes like 7-AAD (30), and DRAQ5 allows the simultaneous use of FITC, PE, and APC provided that the compensation adjustments are adequate (5,30,31). Unfortunately, DNA staining capacity of 7-AAD is limited by chromatin packaging and crosslinking of DNA and/or of DNA-histones (31,32). This limitation is overcome by the protocol published by Carbonari et al using integral hot staining (IHS) technique.…”

Section: Discussionmentioning

confidence: 99%

A novel and easy FxCycle™ violet based flow cytometric method for simultaneous assessment of DNA ploidy and six‐color immunophenotyping

et al. 2015

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Abnormal DNA ploidy is a valuable prognostic factor in many neoplasms, especially in hematological neoplasms like B-cell acute lymphoblastic leukemia (B-ALL) and multiple myeloma (MM). Current methods of flow-cytometric (FC) DNA-ploidy evaluation are either technically difficult or limited to three-to four-color immunophenotyping and hence, challenging to evaluate DNA-ploidy in minute tumor population with background rich of its normal counterpart cells and other hematopoietic cells. We standardized a novel sensitive and easy method of simultaneous evaluation of six-to sevencolor immunophenotyping and DNA-ploidy using a dye-FxCycle Violet (FCV). Linearity, resolution, and coefficient of variation (CV) for FCV were studied using chicken erythrocyte nuclei. Ploidy results of FCV were compared with Propidium iodide (PI) in 20 samples and intra-assay variation for FCV was studied. Using this six-color immunophenotyping & FCV-protocol DNA-ploidy was determined in bone-marrow samples from 124 B-ALL & 50 MM patients. Dilution experiment was also conducted to determine the sensitivity in detection of aneuploidy in minute tumor population. FCV revealed high linearity and resolution in 450/50 channel. On comparison with PI, CV of Go/G1-peak with FCV (mean-CV 4.1%) was slightly higher than PI (mean-CV 2.9%) but had complete agreement in ploidy results. Dilution experiment showed that aneuploidy could be accurately detected up to the limit of 0.01% tumor cells. Intraassay variation was very low with CV of 0.005%. In B-ALL, hypodiploidy was noted in 4%, hyperdiploidy in 24%, near-hyperdiploidy in 13% and remaining 59% were diploid. In MM, hypodiploidy was in 2%, hyperdiploidy in 58%, near-hyperdiploidy in 8% and remaining 30% were diploid. FCV-based DNA-ploidy method is a sensitive and easy method for simultaneous evaluation of six-color immunophenotyping and DNA analysis. It is useful in DNA-ploidy evaluation of minute tumor population in cases like minimal residual disease and MM precursor conditions. V C 2015 International Society for Advancement of CytometryKey terms DNA ploidy; cell cycle; FxCycle-violet; simultaneous multicolor immunophenotyping FLOW cytometric (FC) DNA content (DNA ploidy) evaluation is widely used tool in the prognostication of many solid tumors as well as hematological malignancies (1-5). It is an easy and rapid method to study the DNA content in the cells and to estimate proportion of cells present in different phases of the cell cycle. FC DNA ploidy analysis includes estimation of DNA index (DI) calculated by ratio of mean fluorescence of "G0/G1" peak of tumor cells to normal lymphocytes that reveals change in amount of DNA content of tumor cells in relation to normal diploid cells and percentage "S" phase provides an objective estimation of cell growth and may

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Section: Discussionmentioning

confidence: 99%

A novel and easy FxCycle™ violet based flow cytometric method for simultaneous assessment of DNA ploidy and six‐color immunophenotyping

et al. 2015

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“…Fixation by formaldehyde has been shown to depend on concentration and incubation time (Linden et al 1997; Guillot et al 2004). In the present study, we tested fixation for 10 min in 3.7% as well as 15 min in 1.8% BF.…”

Section: Discussionmentioning

confidence: 99%

Preservation of large-scale chromatin structure in FISH experiments

et al. 2006

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The nuclear organization of specific endogenous chromatin regions can be investigated only by fluorescence in situ hybridization (FISH). One of the two fixation procedures is typically applied: (1) buffered formaldehyde or (2) hypotonic shock with methanol acetic acid fixation followed by dropping of nuclei on glass slides and air drying. In this study, we compared the effects of these two procedures and some variations on nuclear morphology and on FISH signals. We analyzed mouse erythroleukemia and mouse embryonic stem cells because their clusters of subcentromeric heterochromatin provide an easy means to assess preservation of chromatin. Qualitative and quantitative analyses revealed that formaldehyde fixation provided good preservation of large-scale chromatin structures, while classical methanol acetic acid fixation after hypotonic treatment severely impaired nuclear shape and led to disruption of chromosome territories, heterochromatin structures, and large transgene arrays. Our data show that such preparations do not faithfully reflect in vivo nuclear architecture.

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“…It should be noted that, dyes like 7-AAD Phenotypic Analysis and DRAQ5 can be used simultaneously along with surface staining with antibodies conjugated with FITC, PE and APC provided that the compensation adjustment is done adequately (Zelenin et al, 1984;Yuan et al, 2004). Unfortunately, 7-AAD may show staining variation due to heterogeneous chromatin packaging and cross-linking of DNA and/or of DNA-histones (Darzynkiewicz et al,1984;Telford et al, 1992;Linden et al, 1997). Maurizio Carbonari et al improved this method using acetone-based fixation and integral hot staining (IHS) technique (Carbonari et al, 2001).…”

Section: Red Cell Lysing Reagentmentioning

confidence: 99%

Method for DNA Ploidy Analysis Along with Immunophenotyping for Rare Populations in a Sample using FxCycle Violet

et al. 2017

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The clinical use of flow cytometric DNA ploidy assay has been extended towards stratifying the risk of diseases, such as monoclonal gammopathies or B cell acute lymphoblastic leukemia, and to detect circulating tumor cells, both of which require detection of minute cell populations. This unit describes a protocol for determining DNA ploidy in fixed samples with simultaneous surface immunophenotyping. It is an easy method for simultaneous 6- to 8-color immunophenotyping and DNA content analysis using FxCycle Violet (FCV; DAPI) dye. This protocol is a one-step modification of routine multicolor immunophenotyping that includes surface staining followed by fixation and then DNA staining with FCV. It utilizes mature lymphocytes from the sample as an internal control for determination of DNA index. It is a sensitive method that allows DNA-ploidy determination and cell cycle analysis in a rare tumor population as low as 100 events, as well as DNA ploidy determination in various subsets of hematopoietic cells in the same sample based on their immunophenotype. © 2017 by John Wiley & Sons, Inc.

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Accessibility of 7-aminoactinomycin D to lymphocyte nuclei after paraformaldehyde fixation

Cited by 9 publications

References 12 publications

A novel and easy FxCycle™ violet based flow cytometric method for simultaneous assessment of DNA ploidy and six‐color immunophenotyping

A novel and easy FxCycle™ violet based flow cytometric method for simultaneous assessment of DNA ploidy and six‐color immunophenotyping

Preservation of large-scale chromatin structure in FISH experiments

Method for DNA Ploidy Analysis Along with Immunophenotyping for Rare Populations in a Sample using FxCycle Violet

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