Evidence for false aneuploid peaks in flow cytometric analysis of paraffin-embedded tissue

It has recently been shown that bimodal histograms with false aneuploid peaks may be obtained by DNA flow cytometry from histologically normal tissue allowed to autolyze. To investigate if such peaks can be generated from surgically excised archival tissue, 198 paraffin blocks from 179 patients containing histologically normal spleen (n = 65), liver (n = 26), thyroid (n = 32), pancreas (n = 19), salivary gland (n = 49), or lymph node tissue (n = 7), obtained from the archives of two university pathology departments, were analyzed for nuclear DNA content. The great majority (n = 160, 83.8%) of the 191 interpretable histograms had a single symmetrical G1 peak; and 8 histograms, all produced from liver tissue had a tetraploid pattern. A slight or a prominent repeatable deviation in the G1 peak outline was present in 14 (7.3%) cases. A peak resembling an aneuploid G1 peak with a DNA index (DI) ranging from 1.14 to 1.38 was repeatedly produced from 9 (4.7%) blocks containing histologically normal or inflamed splenic (n = 3), pancreatic (n = 3), liver (n = 1), thyroid (n = 1), or lymph node (n = 1) tissue. The three abnormal peaks produced from pancreatic tissue were rounded in shape and resembled closely the ones that can be obtained from autolytic pancreatic tissue, and the six remaining extra peaks were all fused with the "diploid" peak. In conclusion, false peaks, probably caused by degradation of the nuclear contents during formalin fixation or before it, may rarely be obtained from surgical paraffin-embedded samples.     

Modified methodology to improve flow cytometric DNA histograms from paraffin-embedded material

Flow Cytometric (FCM) DNA content analysis of paraffin-embedded tissues has become a widely accepted procedure in assessing the biologic course in some tumors from archival material. Difficulty in interpreting histograms is frequently due to high levels of debris, and wide coefficients of variation (CV). These may lead to underestimating near-diploid abnormality. Although the clinical significance of low-degree aneuploidy has yet to be established, the procedure reported here improved our endeavor to detect DNA Indices (DI) of at least 1.1%. Experience has shown that careful technique can result in overall improvement of DNA histograms by lowering levels of debris and % CV, dramatically so in some cases. Archival histograms can be generated that rival in quality fresh tissue results. Archival material is currently employed for diagnostic and research purposes.     

Mathematical evaluation of two parameter flow cytometric histograms

A general procedure was developed to evaluate two-parameter flow cytometric data. These data usually show overlapping distributions representing different subpopulations. To calculate the fractions of cells in the various subpopulations, the maximum likelihood method is used, assuming an unknown number of Gaussian distributions with constant coefficient of variation in the histogram. A test on simulated data shows that Gaussians can be separated clearly by the procedure if the distance between their mean values is larger than twice the maximum standard deviation. The analysis of different measurements of double-stained (DNA/protein) cultured cells out of the same pool yield the same fractions in the phases of the cell cycle with acceptable error rates. To simulate samples with different fractions in the phases of the cell cycle, defined mixtures of proliferating and resting cells of the same type were prepared and measured for DNA and cell size. The calculated fractions are consistent with the expected variation of G1, S and G2 + M fractions depending on the mixture ratio.     

Automatic analysis of flow cytometric DNA histograms from irradiated mouse male germ cells

An automatic procedure for recovering the DNA content distribution of mouse irradiated testis cells from flow cytometric histograms is presented. First, a suitable mathematical model is developed, to represent the pattern of DNA content and fluorescence distribution in the sample. Then a parameter estimation procedure, based on the maximum likelihood approach, is constructed by means of an optimization technique. This procedure has been applied to a set of DNA histograms relative to different doses of 0.4-MeV neutrons and to different time intervals after irradiation. In each case, a good agreement between the measured histograms and the corresponding fits has been obtained. The results indicate that the proposed method for the quantitative analysis of germ cell DNA histograms can be usefully applied to the study of the cytotoxic and mutagenic action of agents of toxicological interest such as ionizing radiations.     

Comparison of gaussian and t-distribution densities for modeling fluorescence dispersion in flow cytometric DNA histograms

In this paper, we have analyzed, on an experimental basis, the behaviour of the standardized t-distribution with three degrees of freedom versus the gaussian model, when employed for describing the fluorescence dispersion effect in flow cytometric DNA measurements. The comparison has been performed by employing two different objective criteria based on a computer analysis for fitting DNA histograms and on a parameter related to the peak distribution shape. The results indicate that, when the peak variance is not particularly small, the two models are substantially equivalent. In the case of experimental data with very sharp peaks, the t-distribution performs better than the gaussian density.     

Kinetics of protein and DNA synthesis studied by mathematical modelling of flow cytometric protein and DNA histograms

Abstract. Mathematical models for histograms of cellular protein content as measured by flow cytometry were developed, based on theoretical protein distributions. These were derived from the age distribution of cells and the accumulation function for cellular protein content as a function of age within the cell cycle. A model assuming an exponential age distribution and an exponential protein. accumulation function was found to give the best representation of protein histograms of exponentially growing NHIK 3025 cells. This is in good agreement with the known kinetic behaviour of such cells. By the combined use of the protein histogram model and a similar model for DNA content, and assuming linear DNA accumulation during S, the fraction of cells in S, as a function of cellular protein content, was simulated. This function showed good agreement with values of the [³H]TdR labelling index scored in cells sorted by flow cytometry from 5-channel intervals of the protein histogram. The protein and DNA histogram models were combined into a two-dimensional model for correlated protein/DNA measurements. Comparison between simulated data and experimentally derived two-dimensional protein/DNA histograms gave further support to the cell kinetic assumptions underlying the models, but also identified some minor deviations which could not be recognized in the analysis of the one-dimensional histograms.     

Estimating the variation in S phase duration from flow cytometric histograms

A stochastic model for interpreting BrdUrd DNA FCM-derived data is proposed. The model is based on branching processes and describes the progression of the DNA distribution of BrdUrd-labelled cells through the cell cycle. With the main focus on estimating the S phase duration and its variation, the DNA replication rate is modelled by a piecewise linear function, while assuming a gamma distribution for the S phase duration. Estimation of model parameters was carried out using maximum likelihood for data from two different cell lines. The results provided quite a good fit to the data, suggesting that stochastic models may be a valuable tool for analysing this kind of data.     

Flow cytometric detection of multifocal DNA aneuploid cell populations in mastectomy specimens containing a primary breast carcinoma

DNA flow cytometry was used to study the presence of DNA aneuploid cell populations in macroscopically normal glandular tissue in mastectomy specimens from 30 patients with breast cancer. In the 13 patients with a DNA diploid primary tumor, no DNA aneuploidy could be found in any of the 39 distant specimens assessed. However, DNA aneuploid cell populations were demonstrated in four of the 17 (23%) patients with a primary DNA aneuploid carcinoma and in seven out of 54 (13%) distant tissue samples (P = 0.02). In all cases the DNA index of the DNA aneuploid cells found in the distant samples was identical to that of the primary tumor. The replicate aneuploid DNA indices and histologic controls taken in parallel very strongly suggest that these distant DNA aneuploid cell populations are metastases.     

Estimating the distribution of the G(2) phase duration from flow cytometric histograms

A mathematical model, based on branching processes, is proposed to interpret BrdUrd DNA FCM-derived data. Our main interest is in determining the distribution of the G(2) phase duration. Two different model classes involving different assumptions on the distribution of the G(2) phase duration are considered. Different assumptions of the G(2) phase duration result in very similar distributions of the S phase duration and the estimated means and standard deviations of the G(2) phase duration are all in the same range.     

Preparation of tissues for DNA flow cytometric analysis

A method for measuring DNA in tissue cells by flow cytometry utilizing a one step combination nuclear isolation-DNA fluorochrome staining procedure is described. A variety of cells and tissues, both in vivo and in vitro, was used to illustrate the universal nature of this technique. These included murine bone marrow, liver testicle, sarcoma brain tumor, rat pancreatic islets, human peripheral blood, colon mucosa, colon cancer, sarcoma and brain tumor tissues. A special nuclear isolation medium, which contained either of the DNA fluorochromes, 4',6-diamidino-2 phenylindole-2 HCl or propidium iodide, was utilized successfully to isolate single suspensions of DNA fluorochrome stained nuclei in a rapid (5-10 min), consistent manner from a variety of tissues and cells. Multiple sampling of the same tissue or comparison between whole tissues and their single cell isolates showed that a representative sample was being obtained.     

DNA flow cytometric analysis of mouse seminiferous epithelium

In order to provide a basis for quantitative studies of murine spermatogenesis, we performed a DNA flow cytometric analysis on the mouse seminiferous tubules isolated at defined stages of the epithelial cycle by transillumination-assisted microdissection. Accurate stage identification was performed by examining spermatids in the adjacent tubule segments by phase-contrast microscopy. For flow cytometry, suspension of nuclei of spermatogenic cells was obtained by detergent treatment of isolated seminiferous tubules, and fresh samples were stained with propidium iodide. DNA histograms of the 12 stages of the mouse seminiferous epithelial cycle varied in a stage-specific manner. DNA histograms of stages I-VIII of the cycle were characterized by a hypofluorescent haploid peak, the location of which changed with the decreasing DNA dye (propidium iodide)-binding capacity of elongated spermatids. The absence of the hypohaploid peak and the high ratio of the cells with 4C amount of DNA to the cells with 1C amount of DNA characterized stages IX-XI of the cycle. Stage XII showed a high 2C peak, owing to a large population of secondary spermatocytes arisen from the first meiotic division. By using fluorescent beads as an internal volume standard cell numbers in defined stages were determined. These data provide a basis for quantitative studies of mouse spermatogenesis.     

Discontinuous fragmentation of nuclear DNA during apoptosis revealed by discrete "sub-G1" peaks on DNA content histograms.

BACKGROUND: Internucleosomal DNA fragmentation is one of the hallmarks of apoptosis. Because the low molecular weight DNA fragments are extracted during cell staining in aqueous solutions, apoptotic cells can be identified on DNA content frequency histograms as cells with fractional ("sub-G(1)") DNA content. The aim of the present study was to explore whether in situ DNA fragmentation during apoptosis is discontinuous or progresses incessantly and if it is discontinuous, to define the resistant to cleavage fraction of DNA that remains stainable with the fluorochrome. MATERIALS AND METHODS: The model of activation-induced apoptosis of human lymphocytes was chosen as it provides uniform cell population with identical DNA content (DI = 1.00) that undergo apoptosis. Their apoptosis was induced by multivalent mitogen phytohemagglutinin (PHA) in the absence and presence of geldanamycin (GA), the benzoquinone ansamycin antibiotic which binds to Hsp90 (Heat Shock Protein 90) and alters its function. The cells were stained with acridine orange, the metachromatic fluorochrome that differentially stains cellular DNA and RNA. RESULTS: A sharp, discrete peak representing the subpopulation of "sub-G(1)" cells with highly reproducible DI = 0.42 +/- 0.02 (CV = 5.5 +/- 1.2) was observed on DNA content histograms of lymphocytes whose apoptosis was induced by PHA alone. Two distinct peaks, one representing cell subpopulations with DI = 0.42 (as above) and another, with DI = 0.79 +/- 0.04 (CV = 5.8 +/- 0.4), respectively, were seen in apoptotic cells from cultures stimulated with PHA in the presence of GA. The frequency of cells represented by the sub-G(1) peaks varied depending on time of induction of apoptosis and GA concentration. CONCLUSIONS: Apoptosis-induced DNA fragmentation is discontinuous; approximately 42% of DNA is relatively stable and remains within the cell. The data suggest that the stable DNA is associated with nuclear matrix while the degradable fraction represents DNA in loop domains. A transient DNA stabilization is apparent in the presence of GA as evidenced by the presence of cell subpopulations with 79% of DNA retained in the cell. The observed discontinuity of DNA fragmentation appears to reflect sequential involvement of different nucleases and may also be modulated by chromatin structure.     

DNA mapping of gastric cancers using flow cytometric analysis

Although numerous studies of gastric cancers on DNA ploidy have been reported, differences in the degree of aneuploidy (DNA index, DI) during progression have not been identified. We attempted to chart the differences in DIs during progression to clarify the role of aneuploidy in gastric cancers. We classified the gastric cancers examined into intestinal (n = 88) and diffuse (n = 48) types, and then analyzed 136 gastric cancers (intramucosal cancer, 42; submucosal cancer, 39; advanced cancer, 55) by flow cytometry using multiple sampling. In addition, we examined the DNA ploidy pattern of mucosal and submucosal lesions using the same submucosal cancers to study the tumor progression in individual cancers. Intratumoral DNA differences in DNA ploidy were observed in both types of gastric cancers. In intestinal-type cancers, multiple subclones indicated by a different DI occurred during the early stage of gastric cancers, whereas in diffuse-type cancers, multiple subclones were found primarily in advanced cancers. Although the DI varied widely in early intestinal-type cancers between 1.0 and 2.0, in early diffuse-type cancers, the DI tended to be less than 1.2. However, in advanced stage gastric cancers, the DI distribution was similar for both histological types. In intestinal-type cancers, high DI (>1.3) aneuploidy was frequently found in mucosal lesions. In contrast, only low DI (<1.2) aneuploid clones were observed in mucosal lesions of diffuse-type cancers. The present results suggest that high DI aneuploid tumor clones in intramucosal cancers acquire invasive ability when they progress to submucosal cancers, whereas DNA aneuploidy itself plays an important role in submucosal invasion of diffuse-type cancers.     

Postlarval muscle growth in fish: a DNA flow cytometric and morphometric analysis

The mechanism of postlarval fish myotomal growth was investigated in trout (Salmo gairdneri) by means of morphometric and cytofluorometric analysis. The mechanism by which new fibres are added during postlarval growth (hyperplasia) is not fully understood. In histological cross sections these new fibres have a small diameter which give the muscle a "mosaic" appearance. One hypothesis suggested that they could be derived from the proliferative activity of satellite cells. DNA cytofluorometric analysis of nuclei suspensions obtained from trout white myotomal muscle during different developmental stages (eleutherembyronic; alevin; yearling and adult) showed a consistently low S-cytometric phase during all stage in which myofibres of small diameters were present. The percentage of such small fibres, determined by morphometric analysis, suggested that satellite cells are the proliferative population. In fact, their percentages, as determined by morphometric analysis in histological section, bear a linear relationship with the S-cytometric phase percent nuclei (R = 0.927). Only in adults (67 cm in size) there was a significant decrease in the S-cytometric phase. At this stage, in histological sections, the myotomal muscle no longer had a "mosaic" appearance because of the disappearance of the small fibres. It may, therefore, be supposed that in the cm 67 adult specimens, the proliferative population is entering the G0 phase. It is known, in fact, that muscle growth proceeds only by fibre hypertrophy in trout longer than 70 cm in length (Stickland, 1983).     

A practical graphical method for estimating the fraction of cells in S in DNA histograms from clinical tumor samples containing aneuploid cell populations

A graphical method for the analysis of unperturbed DNA histograms is presented in which the area of the normalized histogram subtended by the fraction of cells in S is represented by a trapezoid whose dimensions are dependent on features common to all such histograms. The technique takes measurement variability into account. This method was applied to a variety of synthetic DNA histograms. Overall, calculated values for the fraction of cells in S correlated well with actual values. This method was applied to 36 diploid cases of non-Hodgkin lymphoma; results correlated well with those obtained by a computer-based method. The results of the graphical-method were also highly reproducible between different observers. The graphical method can be used in the presence of aneuploid cell populations. Techniques for calculating S fractions in the presence of aneuploidy in clinical samples are described. These techniques were applied to synthetic histograms of mixed diploid and aneuploid populations. Calculated values correlated well with actual values.     

Accurate flow cytometric membrane potential measurement in bacteria using diethyloxacarbocyanine and a ratiometric technique.

BACKGROUND: Membrane potential (MP) plays a critical role in bacterial physiology. Existing methods for MP estimation by flow cytometry are neither accurate nor precise, due in part to the heterogeneity of size of the particles analyzed. The ratio of a size- and MP-sensitive measurement, and an MP-independent, size-sensitive measurement, should provide a better estimate of MP. METHODS: Flow cytometry and spectrofluorometry were used to detect red (488 --> 600 nm) fluorescence associated with aggregates of diethyloxacarbocyanine (DiOC2(3)), which, in the monomeric state, is normally green (488 --> 530 nm) fluorescent. RESULTS: In bacteria incubated with 30 microM dye, aggregate formation increases with the magnitude of the interior-negative membrane potential. Green fluorescence from stained bacteria predominantly reflects particle size, and is relatively independent of MP, whereas red fluorescence is highly dependent on both MP and size. The ratio of red to green fluorescence provides a measure of MP that is largely independent of cell size, with a low coefficient of variation (CV). Calibration with valinomycin and potassium demonstrates that the method is accurate over the range from -50 mV through -120 mV; it also accurately tracks reversible reductions in MP produced by incubation at 4 degrees C and washing in glucose-free medium. CONCLUSIONS: The ratiometric technique for MP estimation using DiOC2(3) is substantially more accurate and precise than those previously available, and may be useful in studies of bacterial physiology and in investigations of the effects of antibiotics and other agents on microorganisms.     

Comparison of three DNA fluorochromes for flow cytometric estimation of nuclear DNA content in plants

Flow cytometric estimation of nuclear DNA content was performed in six plant species employing three fluorochromes showing different DNA base preferences: propidium iodide (no base preference), 4',6-diamidino-2-phenylindole (DAPI; AT preference), and mithramycin (GC preference). Nuclei isolated from human leukocytes were used as a primary reference standard. While nuclear DNA contents estimated using propidium iodide were in agreement with published data obtained using other techniques, the values obtained using fluorochromes showing base preference were significantly different. It was found that the differences were caused by the differences in overall AT/GC ratios, and by the species-specific differences in binding of these fluorochromes to DNA. It was concluded that nuclear DNA content estimations performed with fluorochromes showing base preference should be interpreted with caution even when AT/GC ratios of the reference and the sample are equal. The use of intercalting dyes (e.g. propidium iodide) is recommended for this purpose. On the other hand, comparison of the staining behaviour of intercalating dyes with that of dyes showing base preference may give additional information on chromatin structural differences and arrangement of molecule pairs in DNA.     

Parametric analysis of histograms measured in flow cytometry

Flow cytometric histograms frequently consist of several components that show various degrees of overlap. For many types of analysis it is of great importance to decompose the original histogram into its components. To that purpose, we investigated the maximum likelihood approach in detail. It is shown that the iterative method to solve the maximum likelihood equations is well behaved for a variety of initial values. Algorithms to obtain initial values are presented, and the performance of the method is tested when applied to the analysis of DNA measurements from heterogeneous cell populations that differ with respect to DNA content.     

Validation of a mathematical procedure for computer analysis of flow cytometric DNA data in human tumors

The determination of tritiated thymidine labeling index and the percentage of cells with S phase DNA content was performed on cell suspensions obtained from 69 patients with non-Hodgkin's lymphoma. The distributions of cells in the cell cycle by computer analysis of flow cytometric data were obtained by two mathematical procedures: the widely adopted Fried model and a new one proposed by Bruni et al. A significant agreement was observed by checking the Spearman index (rs) between the percentages of cells in the different cell cycle phases (G0/1, rs = 0.76; S, rs = 0.60; and G2 + M, rs = 0.43; p less than 0.001) determined by the two procedures. Similarly, a good correlation was observed between the labeling index (LI) and the S phase values obtained by the Fried (rs = 0.45, p less than 0.001) and Bruni (rs = 0.69, p less than 0.001) models, but with a higher agreement for the latter one. The S phase by the Bruni model was also superior in predicting LI: in fact, by employing the S cutoff value of 12%, a better agreement between low LI and low S phase or high LI and high S phase was observed with the Bruni procedure (90%) than with the Fried model (72%). Finally, the analysis of the prognostic significance of the different kinetic variables confirmed the prognostic relevance of LI at any time; the S phase percentage as determined by Bruni et al. was discriminant of survival only at shorter times, and no prognostic significance could be ascribed to S phase according to the Fried procedure.     

Identifiability of DNA distribution from flow cytometric data with cell debris

In flow cytometric measurement of cell DNA distribution one of the major problems is accounting for the effect of fragmentation in the staining process. This work considers a recent probabilistic model that has been proposed for the fragmentation process and species under which conditions it is possible to uniquely identify the DNA distributions of the original population using flow cytometric data. Attention is given both to the normal and to the polyploid case. This work was partially supported by a grant of the Italian National Research Council, Special Project “Oncology”, contract number 84.00632.44.