Comparison of routine flow cytometric DNA analysis of fresh tissues in two laboratories: Effects of differences in preparation methods and background models of cell cycle calculation

Heiden, Thomas; Castro, Juan; Graf, Britt-Mari; Tribukait, Bernhard

doi:10.1002/(sici)1097-0320(19980815)34:4<187::aid-cyto3>3.0.co;2-d

Cited by 10 publications

(12 citation statements)

References 41 publications

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“…Apoptotic and necrotic cells and cell debris can be detected as a ''sub-G1'' population [27]. After long time culture, the cell debris overlapped with the intact nuclei and increase of normal 2n number of chromosomes in living cells and it can especially distort the measurement of the location and size of G1 and S-phase cells [28]. We estimate that 98.79% of the cells (except the sub-G1 population) in the GPPt-bEGF-treated group at 24 h were necrotic due to cisplatin toxicity.…”

Section: Discussionmentioning

confidence: 99%

The use of biotinylated-EGF-modified gelatin nanoparticle carrier to enhance cisplatin accumulation in cancerous lungs via inhalation

et al. 2009

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Section: Discussionmentioning

confidence: 99%

The use of biotinylated-EGF-modified gelatin nanoparticle carrier to enhance cisplatin accumulation in cancerous lungs via inhalation

et al. 2009

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“…Debris and aggregates can be prominent components of DNA histograms, affecting the accuracy and reproducibility of cell‐cycle estimates (1–4). Debris originate from the damage and disintegration of cells following apoptosis or the fragmentation associated with the slicing of cells or nuclei during mechanical disaggregation.…”

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confidence: 99%

Doublet discrimination in DNA cell‐cycle analysis

et al. 2001

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Differences in doublet analysis have the potential to alter DNA cell-cycle measurements. The techniques for doublet determination are often used interchangeably without regard for the complexity in cell shapes and sizes of biological specimens.G 0/1 doublets were identified and quantitated using fluorescence height versus area and fluorescence width versus area pulse measurements, by enumerating the proportion of G 2 ؉ M cells that lack cyclin B1 immunoreactivity, and modeled in the DNA histograms by software algorithms. These techniques were tested on propidium iodide-stained whole epithelial cells or nuclei from asynchronous cultures, or after exposure to chemotherapeutic agents that induced cell-cycle arrest and were extended to human breast tumor specimens having DNA diploid patterns.G 0/1 doublets were easily discernible from G 2 ؉ M singlets in cells or nuclei that are generally homogenous and spherical in shape. Doublet discrimination based on pulse processing or cyclin B1 measurements was nonconcordant in some nonspherical cell types and in cells following cell cycle arrest. Significant differences in G 0/1 doublet estimates were observed in breast tumor specimens (n ‫؍‬ 50), with estimates based on pulse width twice those of pulse height and nearly five times greater than computer estimates. Differences between techniques are attributed to difficulties in the separation of the boundaries between G 0/1 doublets and G 2 ؉ M singlet populations in biologically heterogeneous specimens.To improve reproducibility and enhance standardization among laboratories performing cell cycle analysis in experimental cell systems and in human breast tumors, doublet discrimination analysis should best be accomplished by computer modeling. Shape and size heterogeneity of tumor and arrested cells using pulse-processing can lead to errors and make interlaboratory comparison difficult. Cytometry (Comm. Clin. Debris and aggregates can be prominent components of DNA histograms, affecting the accuracy and reproducibility of cell-cycle estimates (1-4). Debris originate from the damage and disintegration of cells following apoptosis or the fragmentation associated with the slicing of cells or nuclei during mechanical disaggregation. Clumping may be due to the incomplete disruption of tissues by mechanical or enzymatic means into single-cell or nuclear suspensions, by the use of alcohol-based fixatives that yield DNA histograms with low coefficients of variation of the G 0/1 peak but induce clumping, or by centrifugation. In addition, clumping may be an inherent attribute of some cell types, e.g., keratinocytes.Aggregates can be composed of large clusters of cells or nuclei or two or more G 0/1 (2N) events adhered together (G 0/1 doublets) that are indistinguishable from particles with 4N, 6N, or 8N DNA content. Large clumps can be removed by nylon mesh filtration (typically 35-53 m), sheared apart by passage through small gauge needles, or identified on the basis of forward-angle light scattering (2). Strategies to separate overla...

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“…Properly applied, today's available software allows the accurate calculation of the DNA index (DI) of the cell population of interest as well as an exact SPF calculation for up to three different populations in a given sample. Detailed comparison of different software calculation modes has revealed the impact of data handling on intra-and interlaboratory reproducibility [9,10,29,45]. These studies underline the importance of a standardized software-supported data calculation.…”

Section: Data Collection Processing and Assessmentmentioning

confidence: 93%

“…for retrospective studies, a combination of proteolytic and mechanical disaggregation is usually applied. For solid fresh and frozen colon tumour tissues, it has been shown that an automated, standardized, gentle tissue disaggregation significantly improves cell asservation, facilitates dual parameter analysis recruiting marker protein labelling, potentiates the identification of small malignant subpopulations, and basically contributes to standardized measurement [28,29]. Nevertheless, a tryptic cell dissociation to preserve antigen determinants for multiparametric DNA measurements in combination with antigen detection in ovarian cancer cells has also been optimized [30].…”

Section: Tissue Disaggregation Of Solid Tumoursmentioning

confidence: 99%

Flow Cytometric DNA Analysis in Oncology: From Single to Multiparametric Measurements/Durchflusszytometrische DNA-Analytik in der Onkologie: Von einzel- zu multiparametrischen Messungen

Brockhoff¹,

Knuechel²

2003

LaboratoriumsMedizin

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DNA analysis was one of the earliest applications in flow cytometry. The stoichiometric interaction of fluorescent dyes with nucleic acid allows the evaluation of DNA content (ploidy) and the proliferative fraction. Initially, DNA was measured as the only parameter along with side-and forward-scatter properties to identify intact nuclei or cells. Later, a dual-parametric approach for the simultaneous analysis of DNA and protein was introduced. Today, the main application of flow cytometric DNA analysis is the additional, tumour entity-dependent diagnosis in pathology. The knowledge about an abnormal DNA content as a crude indicator of chromosomal instability and high S-phase fraction can result in a precise determination of tumour prognosis and can lead to the optimization of individual therapy and complementary disease monitoring. DNA analysis is applied in both haematological and solid tumour diagnosis. In contrast to haematological diseases, the analysis of solid tumours requires the isolation of cell nuclei or cell disaggregation with preservation of cell integrity. The uniparametric investigation of cell nuclei in suspension allows high-resolution DNA analysis with detection of aneuploidy in the neardiploid range. However, the multiparametric approach on the basis of cell selection or discrimination takes tumour heterogeneity into account, and DNA assessment can be combined with antigen detection with additional cell phenotyping. In contrast to unimodal DNA analysis, the multiparametric approach allows a more accurate calculation of cell-cycle fractions of individual cell subpopulations. All approaches require adequate standardization regarding cell preparation, data acquisition and handling to ensure lab-independent DNA data interpretation and utilization.

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Comparison of routine flow cytometric DNA analysis of fresh tissues in two laboratories: Effects of differences in preparation methods and background models of cell cycle calculation

Cited by 10 publications

References 41 publications

The use of biotinylated-EGF-modified gelatin nanoparticle carrier to enhance cisplatin accumulation in cancerous lungs via inhalation

The use of biotinylated-EGF-modified gelatin nanoparticle carrier to enhance cisplatin accumulation in cancerous lungs via inhalation

Doublet discrimination in DNA cell‐cycle analysis

Flow Cytometric DNA Analysis in Oncology: From Single to Multiparametric Measurements/Durchflusszytometrische DNA-Analytik in der Onkologie: Von einzel- zu multiparametrischen Messungen

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