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.     

Flow cytometric analysis of DNA aneuploidy in primary and metastatic human solid tumors

DNA histograms were measured by flow cytometry for 656 human solid tumors (365 primary and 291 metastatic). The proportion of aneuploid cells in cell suspensions obtained by mechanical disaggregation was significantly higher than those obtained after enzymatic disaggregation (collagenase + DNAse) of the same tumor. A strong correlation was observed between the values of DNA-indices measured after staining with propidium iodide and with 4',-6-diamidino-2-phenylindole (r = 0.97). Aneuploid cells were observed in 430 tumors (66%); 30 of these had two aneuploid stemlines, and two had three aneuploid stemlines. The overall frequency of aneuploidy was 61% among primary and 71% among metastatic tumors. The median value of the DNA index was 1.67 for 224 primary aneuploid tumors and 1.68 for 206 metastatic aneuploid tumors. For most diseases, the largest proportion of aneuploid primary and metastatic tumors had DNA-indices in the hypertriploid region. No major differences in frequency and degree of aneuploidy was observed between primary and metastatic tumors. For carcinomas of the bladder and prostate, frequency of aneuploidy was higher among poorly differentiated, than among moderately and well-differentiated tumors. For carcinomas of the breast and for sarcomas, tumors with DNA-indices of greater than 2.0 were observed mostly in the poorly differentiated group. For patients with carcinomas of the bladder and prostate most tumors at earlier stages of disease were diploid; whereas most tumors at later stages of disease were aneuploid. For patients with carcinomas of the ovary, colon, and kidney, no relationship between stage of disease and aneuploidy was evident.     

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.     

Comparison of S-phase fractions measured by flow cytometry and autoradiography in human transplant tumors

The 3H-thymidine labeling index (TLI) and the percentage of cells in the S-phase have been determined by autoradiography and by flow cytometry, (FCM), respectively, in six malignant tumors of human origin transplanted on athymic nude mice. The Dean and Jett model and the graphical model were used to determine the percent of S-phase cells by FCM. Cell cycle analysis was performed using 1) no correction for background; 2) an algebraic function for background correction; and 3) an exponential function for background subtraction. Each of these three data sets was evaluated using both the Dean and Jett model and a graphical model for the evaluation of DNA histograms. The S-phase fractions (SPF) were compared to the corresponding labeling index results. SPF without background correction were 1.54 times higher than the TLI. SPF, after correction using the algebraic model, were 1.29-fold higher than the TLI, whereas SPF obtained after background subtraction according to the exponential model were only 1.05-fold higher than the TLI. Student's t-test revealed significant differences between the mean TLI values (16.25 +/- 9.06) and the mean SPF obtained by FCM without background correction (mean 25.0 +/- 9.36, P less than 0.01), but not between the mean TLI values and the mean SPF percentages after algebraic (mean 21.0 +/- 10.29) and exponential background correction (mean 17.11 +/- 11.59), P greater than 0.05 each. There was no difference between the results obtained using the Dean and Jett model and those obtained using the graphic evaluation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Progesterone receptor detection and quantification in breast tumors by bivariate immunofluorescence/DNA flow cytometry

A method was developed for the detection of progesterone receptors (PgR) by flow cytometry (FCM) in cell suspensions obtained from mechanically dispersed fragments of operated breast cancers. Two monoclonal antibodies were tested for sensitivity and specificity on four breast cancer cell lines of known PgR expression and a calibration curve thus established. A simple procedure was used to calculate the level of PgR expression, taking into account the relative displacement of total cellular fluorescence compared to nonspecific fluorescence for each sample and the average DNA content of the cells derived from the corresponding histograms. The PgR-specific immunofluorescence of the tumor specimens measured in arbitrary units (channels) was then transformed to fmoles/mg DNA by comparison with the calibration curve. The FCM-derived results were compared with those of a conventional immunoenzymatic PgR assay on 30 surgical samples. PgR content ranged from 10 to 22,000 fmoles/mg DNA and linear regression analysis yielded a good correlation (r = 0.86). With a threshold of positivity of 300 fmoles/mg DNA, the two methods concurred for 28 of 30 tumors (93%). Nine specimens were analyzed repeatedly, showing good reproducibility. This method could prove to be more useful than the biochemical assays on homogenates, since it allows the simultaneous analysis of receptor expression in individual cells and of DNA index (ploidy).     

DNA flow cytometry of human epidermal tumours. Intra- and intertumour variability in ploidy and proliferative characteristics

Tumour ploidy and proliferative characteristics can be estimated by flow cytometric measurements of the nuclear DNA content. This study considers the question whether human epidermal tumours are intrinsically homogeneous with regard to these properties, i.e. whether a single biopsy analysed by flow cytometry is representative of the entire tumour. Analyses of multiple biopsies from ten human epidermal tumours--two kerato-acanthomas (KA), two basal cell carcinomas (BCC), two basosquamous carcinomas (BSC), one Bowen's disease (BO) and three squamous cell carcinomas (SCC)--indicated that both ploidy and proliferation characteristics were reproducible and specific for the peripheries of the different tumours regardless of the histopathologic diagnosis. The tumour centres, however, may deviate considerably from the corresponding periphery. None of the ten tumour peripheries contained more than one cell clone, but six of the ten clones were aneuploid. Both the BO and the KA's were hypodiploid, while one SCC, one BSC and one BCC were hyperdiploid as assessed in their peripheries. The remaining BSC was diploid in its periphery, while both a hypodiploid and a hypotetraploid cell clone were found in the corresponding centre.     

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.     

Single- and double-stranded RNA measurements by flow cytometry in solid neoplasms

We investigated the ability of single- and double-stranded RNA measurements to discriminate between neoplastic and non-neoplastic solid tissues. Sixty-one solid nonhematopoietic neoplasms, 10 reactive non-neoplastic lesions, and 26 normal tissue samples were the test materials. Single-stranded ribonucleic acid (s-RNA) levels and double-stranded ribonucleic acid (ds-RNA) excess in these specimens were defined in relationship to normal human lymphocytes. The mean s-RNA index in normal, reactive, benign, and diploid and aneuploid malignant tissue samples was 0.90, 1.54, 1.9, 1.2, and 2.2, respectively. For ds-RNA, the mean excess level for normal, reactive, benign, and diploid and aneuploid malignant specimens was 8.5%, 18.5%, 51.0%, 36.0%, and 41.3%, respectively. No statistical differences in s-RNA level were found between non-neoplastic and neoplastic tissue samples. A significant difference in ds-RNA excess was found between non-neoplastic and benign, and diploid and aneuploid malignant neoplastic tissue (P less than 0.001). The specificity of s-RNA level and ds-RNA excess was 94.4% and 100%, and the sensitivity was 29.5% and 67.2%, respectively. Notably, ds-RNA determinations identified 70.0% of the diploid neoplastic samples, in contrast to 20% by s-RNA. Our preliminary data suggest that ds-RNA may be a useful parameter and may complement DNA ploidy in identification of solid neoplasms, especially if the yield of intact cells is improved.     

Proliferation and apoptosis in solid tumors. Analysis by laser scanning cytometry

OBJECTIVE: To examine the relationship between apoptosis and proliferation in a series of human solid malignant tumors, making use of objective, reproducible techniques newly developed for laser scanning cytometry (LSC). STUDY DESIGN: Apoptosis was detected by in situ end labeling of DNA strand breaks with FITC-conjugated nucleotide. Proliferation was detected by Ki-67 antibody. Two parameters were detected independently and simultaneously with DNA measurement on aliquots of cell suspensions obtained by mechanical dissociation of fresh tumors and placed on microscope slides. RESULTS: The number of cells undergoing apoptosis varied from 0.5% to 28.1% (average, 5.4 +/- 6.0). Aneuploid tumors showed a higher percentage of apoptotic cells (7.9 +/- 7.2) as compared to diploid tumors (3.4 +/- 4.0). Tumors with the greatest number of apoptotic cells on LSC also had the largest number of apoptotic cells on light microscopic examination. The number of cells labeled by Ki-67 ranged from 1.7% to 56.7% (average, 20.0 +/- 15.5). Aneuploid tumors were characterized by a higher Ki-67 index (average, 28.3 +/- 14.3%) than the diploid tumors (13.2 +/- 13.3%). CONCLUSION: Overall, there was a very weak or no correlation between apoptosis and proliferation. However, a subset of aneuploid tumors had a high percentage of cells positive for Ki-67 and low percentage of apoptotic cells. Diploid tumors did not show any correlation between apoptosis and proliferation, although many of those tumors had both low apoptotic and proliferative indices. Whether those differences are of prognostic significance remains to be determined in follow-up studies that include more cases and clinical data. Here we have shown that LSC is a powerful new tool of potential clinical value for fast, objective analysis of apoptosis, proliferation and DNA ploidy in solid malignant tumors.     

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.     

Analysis of repetitive DNA in chromosomes by flow cytometry

We developed a flow cytometry method, chromosome flow fluorescence in situ hybridization (FISH), called CFF, to analyze repetitive DNA in chromosomes using FISH with directly labeled peptide nucleic acid (PNA) probes. We used CFF to measure the abundance of interstitial telomeric sequences in Chinese hamster chromosomes and major satellite sequences in mouse chromosomes. Using CFF we also identified parental homologs of human chromosome 18 with different amounts of repetitive DNA.     

Efficient selection of silenced primary cells by flow cytometry.

BACKGROUND: RNA interference has emerged as a new and potent tool to knockdown the expression of target genes and to investigate their functions. For short time experiments with mammalian cell lines, RNA interference is typically induced by transfecting small interfering RNAs (siRNAs). Primary cells constitute important experimental systems in many studies because of their similarity to their in vivo counterparts; however, transfection of these cells has been found to be difficult. As a consequence, RNA interference of primary cells may result in mixed phenotypes because of the simultaneous presence in the same preparation of transfected and nontransfected cells. This may be particularly inconvenient when certain experiments (for example, biochemical analysis) should be performed. METHODS: We use fluorescently labeled siRNAs to induce RNA interference in fibroblasts, and flow-cytometry associated cell sorting to separate subpopulations of transfected cells according to fluorescence intensity. RESULTS: Flow cytometry allows one to discriminate between strongly- and weakly- or nonsilenced fibroblasts, since the fluorescence intensity of transfected cells is related to the number of internalized siRNA copies and to the mRNA knockdown efficiency. CONCLUSIONS: The use of fluorescently labeled siRNAs may allow one to isolate by flow-cytometry associated cell sorting the most efficiently silenced primary cells for subsequent analysis.     

DNA distribution in biopsy specimens from human cervical carcinoma investigated by flow cytometry.

Flow cytometry was used for the investigation of the DNA distribution in biopsy specimens from 51 patients with cervical carcinoma. Portio biopsy specimens from 9 pregnant women and from 10 patients with cancer of the breast served as controls. The results demonstrate that most specimens from patients suffering from cervical carcinoma contain considerable cell populations with increased DNA as compared with controls. The possible clinical significance of these findings is discussed.