DNA ploidy and cell cycle analysis in ear malignant melanoma by flow and image cytometry.

Sixteen ear malignant melanomas (MM) were studied for ploidy and cell cycle analysis by flow and image cytometry. The results were compared with clinical (age, sex, stage), histologic (depth of invasion, level, type) and prognostic (recurrence, death) parameters. Single nuclear suspensions were obtained from fixed, paraffin-embedded tumor and adjacent normal tissue processed separately according to Hedley's technique. These, a "spiked" specimen of normal tissue and tumor, and a spleen diploid control were analyzed on a FACScan flow cytometer (Becton Dickinson, Mountain View, California, U.S.A.). Feulgen-stained Cytocentrifuge preparations of nuclear suspensions of normal, MM and diploid spleen were analyzed with the CAS 200 Image Analyzer (Cell Analysis Systems, Inc., Elmhurst, Illinois, U.S.A.) against commercial calibration rat hepatocytes defined as diploid. Six (37.5%) MM were diploid, and 10 (62.5%) were aneuploid; 8 (90%) were hypodiploid, for a high frequency. There were no statistically significant correlations between clinical, pathologic, prognostic or cell cycle analysis parameters and ploidy, although poor prognostic features tended to be in aneuploid lesions.     

Significant non-S-phase DNA synthesis visualized by flow cytometry in activated and in malignant human lymphoid cells

The development of a monoclonal antibody to the deoxynucleoside bromodeoxyuridine (BrdU), combined with two parameter flow cytometry, has allowed us to examine large numbers of cells for non-S-phase DNA synthesis. Three human lymphoid cell populations were studied to determine the level of deoxynucleoside (dN) incorporation as a function of DNA content. In each population, non-S-phase DNA synthesis was observed. In a rapidly growing human T-lymphoblastoid cell line (CCRF-CEM), 53% of dN incorporation occurred in G0/G1 plus G2 + M. In chronic lymphocytic leukemia (CLL) cells stimulated with tetradecanoylphorbol acetate (TPA), 45% of the observed burst in thymidine incorporation was found to be localized to G0/G1 cells. Non-S-phase incorporation was not, however, limited to neoplastic cells. Normal human peripheral blood B cells treated with the Cowan strain of Staphylococcus aureus (CSA) undergo a transient burst in thymidine incorporation, but do not go on to divide in the absence of other stimuli. Flow-cytometric analysis showed that 80% of this CSA-stimulated dN incorporation was into G0/G1 cells. These data are consistent with a more dynamic state of DNA synthesis than usually envisioned. Furthermore, the data show that although thymidine incorporation levels are related to incorporation of dN into DNA, they can be unrelated to cell proliferation.     

Escherichia coli DNA distributions measured by flow cytometry and compared with theoretical computer simulations.

A computer simulation routine has been made to calculate the DNA distributions of exponentially growing cultures of Escherichia coli. Calculations were based on a previously published model (S. Cooper and C.E. Helmstetter, J. Mol. Biol. 31:519-540, 1968). Simulated distributions were compared with experimental DNA distributions (histograms) recorded by flow cytometry. Cell cycle parameters were determined by varying the parameters to find the best fit of theoretical to experimental histograms. A culture of E. coli B/r A with a doubling time of 27 min was found to have a DNA replication period (C) of 43 min and an average postreplication period (D) of 22 to 23 min. Similar cell cycle parameters were found for a 60-min B/r A culture. Initiations of DNA replication at multiple origins in one and the same cell were shown to be essentially synchronous. A slowly growing B/r A culture (doubling time, 5.5 h) had an average prereplication period (B) of 2.3 h; C = 2.4 h and D = 0.8 h. It was concluded the the C period has a constant duration of 43 min (at 37 degrees C) at fast growth rates (doubling times, less than 1 h) but increases at slow growth rates. Thus, our results obtained with unperturbed exponential cultures in steady state support the model of Cooper and Helmstetter which was based on data obtained with synchronized cells.     

DNA analysis by flow cytometry

Accurate quantification of DNA from cells of several species is possible with flow cytometry. When one species is used as a reference, cytometric readings from two or more different species can be compared to obtain relative percent DNA or DNA indices. Differences in DNA from the male and female of the same species also can be measured. The method allows rapid screening of chromosomal abnormalities among large clinical populations, and evaluation of errors of sex determination such as XY sex reversal.     

Metabolic activity in filamentous fungi can be analysed by flow cytometry

The use of flow cytometry in combination with fluorescent dyes as a technique to rapidly differentiate and enumerate bacterial and yeast cells is well established. We have shown that through the judicial choice of stains, the nondestructive screening and sorting of fungal material is possible. The early stages of growth, from germination through hyphal development of three filamentous fungal species, Penicillium, Phoma and Trichoderma, have been followed using forward- and side-angle scatter on a Becton Dickinson FACSCalibur flow cytometer. By staining isolates with the permeant fluorogenic substrates, dihydroethidium and hexidium iodide metabolic activity in the developing hyphae has been measured. We have been able to demonstrate that there is a 12-13 h window of opportunity during which germination and the early stages of hyphal development of filamentous fungi can be analysed by flow cytometry.     

Proliferative characteristics and ploidy of human brain tumors by DNA flow cytometry

We studied nuclear DNA distribution by flow cytometry in 59 human brain tumors. Samples were frozen at -20 degrees C immediately after surgery and unicellular suspensions were obtained with a mechanical dissociation technique. Nuclear DNA was stained with propidium iodide. Normal human brain tissue was used as a diploid reference standard. In 86.3% of benign tumors an unimodal DNA distribution with a DNA index usually within the diploid range was found. Among malignant tumors, 64% had un unimodal DNA distribution with diploid or near-diploid modal DNA content. The remaining 36% showed an additional cell peak with a DNA index ranging from 1.15 to 1.92. The percentage of S-phase cells was higher in malignant (median = 3.8) than in benign tumors (median = 1.9) (p less than .001), without correlation to histological tumor subtype.     

Determination of the DNA content of human chromosomes by flow cytometry

The mean relative DNA content of each human chromosome was calculated from flow karyotypes of ethidium bromide-stained chromosomes obtained from healthy, normal individuals. These values were found to correlate closely with previously published data obtained by photometric scanning of stained, fixed chromosomes. Calculations of the normal variation in DNA content of each human chromosome indicated that chromosomes 1, 9, 16, and Y (chromosomes with large centric heterochromatic regions) were the most variable, followed by the acrocentrics, 13, 14, 15, 21, and 22. Chromosomes 2, 3, 18, and 19 were also found to vary significantly in DNA content. Chromosomes from a number of subjects with extreme heteromorphisms were flow karyotyped to obtain an estimate of the extent of variation in DNA content of each chromosome. The greatest difference between extreme variants was found for chromosome 1 (which differed by 0.82% of the total genomic DNA), followed by 16 and 9. The largest Y-chromosome variant was 85.9% bigger than the smallest. The precise karyotype analysis produced by flow cytometry resolved many differences between chromosome homologs, including some that cannot be readily distinguished cytogenetically. The implications of these findings for detection of chromosome abnormalities by flow karyotype analysis are discussed.     

Adaptive responses of Ralstonia eutropha to feast and famine conditions analysed by flow cytometry

Results obtained by flow cytometry allow conclusions to be drawn about how the physiological states of Ralstsonia eutropha JMP134 are connected with survival strategies under distinct growth conditions. During both feast and famine conditions the cells were found to proceed through sharply separated phases of life. Two sources of carbon and energy, one poor (0.02% phenol) and one rich (0.2% pyruvate and 0.1% yeast extract) were chosen to study the cellular responses. Despite the major differences in carbon source, when growth stages of the bacteria on the two substrates were characterised in batch growth, only minor differences were found in the time course of the membrane potential related fluorescence intensity (MPRFI). This also applied to the rRNA content and the size-correlated forward scatter (FSC) signal of the cells, both of which increased to high levels during the (early) exponential growth phase. On the rich medium, DNA synthesis initially occurred in an uncoupled manner, then a high rate of PHB formation followed when nutrients began to be limiting. Under famine conditions, the cellular responses were much more complex. PHB was synthesised, then DNA synthesis occurred in a 'eukaryotic' mode, to be succeeded by renewed PHB synthesis. To obtain defined cell physiological states, the chemostat technique was used in addition to batch experiments. The results obtained clearly indicated that key events in cell physiology, including initiation of DNA replication and overflow metabolism, occurred in a hierarchically ordered manner and were tightly correlated with changes in the environmental conditions of the bacterial cells.     

Proliferative characteristics of primary and metastatic human solid tumors by DNA flow cytometry

The percentage of cells in S-phase (S-index) was calculated from DNA histograms of 453 primary and metastatic human solid tumors (predominantly bladder, breast, colorectal, renal, prostate, ovarian and lung carcinomas, melanomas, and sarcomas). S-indices varied widely among both primary and metastatic tumors (1-48%); there was no significant difference in S-indices between primary and metastatic tumors. The S-indices for aneuploid tumors were significantly higher than for diploid tumors. When data for all aneuploid tumors were analyzed collectively, there was no significant relationship between S-index and DNA ploidy index. However, for colorectal and ovarian carcinomas S-indices increased, and for lung carcinomas S-indices decreased with elevation in the degree of DNA-ploidy. Lung carcinomas had the highest S-indices. Comparison of flow cytometry (FCM) and cytology data indicated that for most diploid tumors S-indices reflect the proportion of S-phase cells among a mixed population of normal and tumor cells. For most aneuploid tumors, the proportion of tumor cells estimated cytologically was similar to the proportion of aneuploid cells estimated by FCM. For a small proportion of aneuploid tumors a comparison of cytology and FCM data indicated the presence of a predominant diploid tumor stemline and a minor stemline with aneuploid DNA content. There was a wide spread in the values of S-indices within tumor groups defined by degree of differentiation and stage of disease at surgery.     

Rapid DNA fingerprinting of pathogens by flow cytometry

BACKGROUND: A new method for rapid discrimination among bacterial strains based on DNA fragment sizing by flow cytometry is presented. This revolutionary approach combines the reproducibility and reliability of restriction fragment length polymorphism (RFLP) analysis with the speed and sensitivity of flow cytometry. METHODS: Bacterial genomic DNA was isolated and digested with a rare-cutting restriction endonuclease. The resulting fragments were stained stoichiometrically with PicoGreen dye and introduced into an ultrasensitive flow cytometer. A histogram of burst sizes from the restriction fragments (linearly related to fragment length in base pairs) resulted in a DNA fingerprint that was used to distinguish among different bacterial strains. RESULTS: Five different strains of gram-negative Escherichia coli and six different strains of gram-positive Staphylococcus aureus were distinguished by analyzing their restriction fragments with DNA fragment sizing by flow cytometry. Fragment distribution analyses of extracted DNA were approximately 100 times faster and approximately 200,000 times more sensitive than pulsed-field gel electrophoresis (PFGE). When sample preparation time is included, the total DNA fragment analysis time was approximately 8 h by flow cytometry and approximately 24 h by PFGE. CONCLUSIONS: DNA fragment sizing by flow cytometry is a fast and reliable technique that can be applied to the discrimination among species and strains of human pathogens. Unlike some polymerase chain reaction (PCR)-based methods, sequence information about the bacterial strains is not required, allowing the detection of unknown, newly emerged, or unanticipated strains.     

Cigarette smoking and human sperm quality assessed by laser-Doppler spectroscopy and DNA flow cytometry

The sperm qualities of 350 men under fertility investigation were compared in relation to their smoking habits. The sperm variables included number, motility, morphology and vitality. Sperm motility was assessed objectively by laser-Doppler spectroscopy. In a randomly selected group, sperm samples were subjected to flow cytometry to assess the levels of DNA condensation. No significant differences (Kruskal-Wallis' test) in any aspect of sperm quality including DNA distribution could be demonstrated between non-smokers, moderate smokers (1-14 cigarettes/day) and heavy smokers (15-40 cigarettes/day). This was true when the data were pooled and when oligozoospermic/hypozoospermic ejaculates (1-39 x 10(6)/ml) and asthenozoospermic ejaculates (less than 25% of sperm cells with progressive movement) were analysed separately. The distribution of non-smokers, moderate and heavy smokers was the same in groups of men with normal sperm quality as those with impaired quality. The present study does not provide support for the contention that smoking has deleterious effects on sperm quality, at least using conventional parameters.     

DNA measurement and cell cycle analysis by flow cytometry

Measurement of cellular DNA content and the analysis of the cell cycle can be performed by flow cytometry. Protocols for DNA measurement have been developed including Bivariate cytokeratin/DNA analysis, Bivariate BrdU/DNA analysis, and multiparameter flow cytometry measurement of cellular DNA content. This review summarises the methods for measurement of cellular DNA and analysis of the cell cycle and discusses the commercial software available for these purposes.     

Centromeric index versus DNA content flow karyotypes of human chromosomes measured by means of slit-scan flow cytometry

We have applied slit-scan flow cytometry (SSFCM) to classify human chromosomes according to their centromeric index (CI) and relative DNA content. The resulting bivariate--CI vs. DNA content--distributions shows 14 peaks for normal human chromosomes. Distinct peaks are produced by chromosomes 1, 2, 3, 4 + 5, 6 + 7 + X, 8, 13 + 14 + 15, 16, 17 + 18, 19 + 20, and 21 + 22 + Y. In addition, chromosomes 9 through 12 are resolved into three peaks. The identity of the chromosomes comprising each peak was determined by comparing CI vs. DNA content distributions measured for normal human chromosomes by means of SSFCM with CI and DNA content values measured for human chromosomes with image analysis. The accuracy of CI measurement by SSFCM was verified by measuring CIs for human chromosomes isolated from human/rodent hybrid cell lines containing only a few known human chromosomes. These studies showed CIs measured for human chromosomes 1-19 and 21 to be in close agreement with the CIs calculated by means of image analysis. We further confirmed the chromosome assignments for each peak by showing that the relative volumes of the peaks in the CI vs. DNA content distributions for chromosomes from normal cells are similar to the relative frequencies of chromosomes expected for these peaks based on the peak assignments.