Comparison of Two Methods of Mechanical Disaggregation of Scirrhous Breast Adenocarcinomas for DNA Flow Cytometric Analysis of Whole Cells

Torres, Frank X.; Mackowiak, Patricia; Brown, Ron; Linden, Michael D.; Zarbo, Richard J.

doi:10.1093/ajcp/103.1.8

Cited by 5 publications

(2 citation statements)

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“…Large clumps can be removed by nylon mesh filtration (typically 35–53 μm), sheared apart by passage through small gauge needles, or identified on the basis of forward‐angle light scattering (2). Strategies to separate overlapping G 0/1 doublets from the G 2 + M1 population have utilized the gating of correlated measurements of integral DNA fluorescence pulse (≈ area) with either peak pulse height (brightness) (5) or pulse duration (width) (6–9), gating on the G 2 + M cells that lack cyclin B1 protein expression (10), and computer algorithms to model aggregate probability distributions in DNA histograms (4, 11–14).…”

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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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confidence: 99%

Doublet discrimination in DNA cell‐cycle analysis

et al. 2001

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“…These two methods were previously compared for tumor tissue. 33 Torres and colleagues 33 compared dissociation of breast adenocarcinoma with scraping of the tumor and concluded that the methods were comparable.…”

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confidence: 99%

Sampling Tumor-Draining Lymph Nodes for Phenotypic and Functional Analysis of Dendritic Cells and T Cells

Vuylsteke

Leeuwen

Meijer

et al. 2002

The American Journal of Pathology

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Immune responses against tumor antigens will initially occur in the first tumor-draining lymph node, the sentinel node (SN). Because of extensive diagnostic procedures, obtaining a piece of SN to isolate viable immune cells for functional analyses is often impossible. For this reason an alternative method to obtain viable cells from a lymph node (LN) was investigated, ie, scraping LNs with a surgical blade, and compared with dissociation of total LNs. Tumor-draining lymph nodes were retrieved from five oncological patients. The collected dendritic cells and T cells were phenotypically and functionally characterized by flow cytometry and antigen-specific interferon (IFN)-gamma release in an ELISPOT assay. Results were compared between the two isolation methods. Viabilities and phenotypic characteristics of the collected cells were entirely comparable for both methods. T-cell functionality was also comparable between both methods, with equal T-cell expansion factors and similar frequencies of cytotoxic T cells specifically recognizing the M1 matrix protein of Influenza haemophilus or the tumor antigen Her-2/neu. In conclusion, scraping LNs to obtain cells for analysis of immune functions in LNs is feasible and presents a good alternative to dissociation of LNs. Scraping may even be applied to small LNs that a pathologist will submit entirely for histological examination and may thus prove useful in the monitoring of immune responses in SNs.

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Vortex disaggregation for flow cytometry allows direct histologic correlation: A novel approach for small biopsies and inaspirable bone marrows

Vos

Simurdak

Davis

et al. 2003

Cytometry Part B Clinical

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Background: Many approaches to obtaining single cells from tissue for flow cytometric immunophenotyping are used; however, these methods result in tissue that is too disrupted for subsequent histologic examination. We introduce a new technique for cell dissociation of hematopoietic malignancies that preserves tissue for histology. This is especially important with small specimens for which this type of correlation is critical. Methods: Fresh tissue from lymph node, gastrointestinal (GI) tract, skin, and other soft tissue biopsies, in addition to cores of inaspirable bone marrows, were briefly vortexed until the RPMI cell culture medium became cloudy. Larger specimens such as lymph nodes were sectioned before disaggregating, whereas smaller ones were vortexed in toto. Resultant flow cytometric analyses were compared with the histology and, in some cases, the immunohistochemistry (IHC) to determine whether the data were concordant. Cell suspensions of 104 specimens-composed of 48 lymph nodes, 19 bone marrow cores (BMCs), 11 GI biopsies, 11 skin/soft tissue biopsies, and 15 miscellaneous specimens-were prepared via vortex disaggregation. Results: Flow cytometric analysis of 96 specimens (92.3%) showed adequacy of material and diagnostic correlation with the histology and IHC. Of the eight cases (7.7%) that were discordant, seven were attributable to significant specimen fibrosis or necrosis. With respect to tissue type, this method produced diagnostic cell suspensions for most lymph nodes (95.8%), GI biopsies (90.9%), and BMCs (89.5%); however, it was less useful for skin/soft tissue samples (

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Comparison of Two Methods of Mechanical Disaggregation of Scirrhous Breast Adenocarcinomas for DNA Flow Cytometric Analysis of Whole Cells

Cited by 5 publications

References 0 publications

Doublet discrimination in DNA cell‐cycle analysis

Doublet discrimination in DNA cell‐cycle analysis

Sampling Tumor-Draining Lymph Nodes for Phenotypic and Functional Analysis of Dendritic Cells and T Cells

Vortex disaggregation for flow cytometry allows direct histologic correlation: A novel approach for small biopsies and inaspirable bone marrows

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