NASA/American Cancer Society High-Resolution Flow Cytometry Project-I

BACKGROUND: The NASA/American Cancer Society (ACS) flow cytometer can simultaneously analyze the electronic nuclear volume (ENV) and DNA content of cells. This study describes the schematics, resolution, reproducibility, and sensitivity of biological standards analyzed on this unit. METHODS: Calibrated beads and biological standards (lymphocytes, trout erythrocytes [TRBC], calf thymocytes, and tumor cells) were analyzed for ENV versus DNA content. Parallel data (forward scatter versus DNA) from a conventional flow cytometer were obtained. RESULTS: ENV linearity studies yielded an R value of 0.999. TRBC had a coefficient of variation (CV) of 1.18 +/- 0.13. DNA indexes as low as 1.02 were detectable. DNA content of lymphocytes from 42 females was 1.9% greater than that for 60 males, with a noninstrumental variability in total DNA content of 0.5%. The ENV/DNA ratio was constant in 15 normal human tissue samples, but differed in the four animal species tested. The ENV/DNA ratio for a hypodiploid breast carcinoma was 2.3 times greater than that for normal breast tissue. CONCLUSIONS: The high-resolution ENV versus DNA analyses are highly reliable, sensitive, and can be used for the detection of near-diploid tumor cells that are difficult to identify with conventional cytometers. ENV/DNA ratio may be a useful parameter for detection of aneuploid populations.     

NASA/American Cancer Society High-Resolution Flow Cytometry Project - II. Effect of pH and DAPI concentration on dual parametric analysis of DNA/DAPI fluorescence and electronic nuclear volume

BACKGROUND: In the present paper, we describe the effect of 4', 6-diamidino-2-phenylindole (DAPI) dihydrochloride concentration and pH on the resolution of DNA distribution histograms generated by dual-parametric simultaneous analysis of DNA content and electronic nuclear volume (ENV). METHODS: Nuclei from tissue culture cell lines and frozen human solid tumors were isolated in nuclear isolation media containing different concentrations of DAPI, at various pH levels, and analyzed on a NASA/American Cancer Society (ACS) flow cytometer. Samples stained with propidium iodide/hypotonic citrate and analyzed in a Coulter XL flow cytometer were used for comparison. RESULTS: Nuclei stained with DAPI concentration of 1-3 microg/ml, pH 6.0, gave the best resolution for the detection of the near-diploid and near-tetraploid populations. Simultaneous use of ENV and DAPI/DNA fluorescence under these conditions identified subpopulations that otherwise could not be detected by DNA analysis alone. CONCLUSIONS: Staining at 1-3 microg/ml DAPI, pH 6.0, was optimal for the detection of aneuploid populations, especially the near-diploid and/or near-tetraploid populations in human tumors.     

Flow cytometric analysis of electronic nuclear volume and DNA content in normal mouse tissues

Light scatter is used in flow cytometry for identification of cells based on their size and/or granularity. However, forward light scatter is not an accurate measure of cell size. The measurement of Electronic Volume (EV) by Coulter principle is more accurate. However, EV cannot be measured on most of the commercially available flow cytometers. We have described the development and applications of a flow cytometer that can simultaneously measure Electronic Nuclear Volume (ENV) and DNA content. In the present study we have used a commercially available NPE Quanta for measuring EV and DNA content of different normal mice tissues. Fresh/frozen or formalin fixed-paraffin embedded tissues from mice were processed for isolation of nuclei, which were then analyzed for EV versus DNA content. By using these two parameters, distinct sub-populations were identified in liver, thymus, small intestine and bone marrow. Dual parametric analysis of EV versus DNA content can be a valuable technique for identification of sub-populations in heterogeneous cell mixtures such as those of complex tissues like bone marrow, intestine and tumors. The methods established are rapid and can provide valuable data for identification and characterization of sub-populations for cell cycle analysis by flow cytometry.     

Flow cytometric analysis of DNA content for ploidy determination in human solid tumors.

Flow cytometric studies of cellular DNA content were conducted in 26 patients with a variety of neoplasms. Cell dispersal was achieved with pepsin treatment, and a combination of ethidium bromide and mithramycin was used as DNA specific staining procedure. All measurements were conducted with a new sheath flow chamber in a PHYWE ICP 11 pulse cytophotometer. All but one patient with multiple myeloma had unimodal tumor cell DNA distributions. With human granulocytes as reference standard, 24 of 26 tumors were aneuploid; and of these, 23 showed varying degrees of hyperdiploidy. Except for one patient, ploidy abnormalities were stable on repeat examination.     

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.     

Flow cytometric analysis of nuclear DNA content in Musa

 Nuclear genome size variation was studied in Musa acuminata (A genome), Musa balbisiana (B genome) and a range of triploid clones differing in genomic constitution (i.e. the relative number of A and B genomes). Nuclear DNA content was estimated by flow cytometry of nuclei stained by propidium iodide. The A and B genomes of Musa differ in size, the B genome being smaller by 12% on average. No variation in genome size was found among the accessions of M. balbisiana (average genome size 537 Mbp). Small, but statistically significant, variation was found among the subspecies and clones of M. acuminata (ranging from 591 to 615 Mbp). This difference may relate to the geographical origin of the individual accessions. Larger variation in genome size (8.8%) was found among the triploid Musa accessions (ranging from 559 to 613 Mbp). This variation may be due to different genomic constitutions as well as to differences in the size of their A genomes. It is proposed that a comparative analysis of genome size in diploids and triploids may be helpful in identifying putative diploid progenitors of cultivated triploid Musa clones. Statistical analysis of data on genome size resulted in a grouping which agreed fairly well with the generally accepted taxonomic classification of Musa. Received: 11 May 1998 / Accepted: 29 September 1998     

DNA content in fresh versus paraffin-embedded tissue. Flow cytometric analysis of 100 tumors.

DNA ploidy analysis was determined on 100 consecutive tumors from a wide variety of sites using both fresh and paraffin-embedded tissue on the same specimen. The correlation coefficient (r) value between the methods was 0.85. Aneuploidy was detected by both methods in 51/100 (51%) of the cases. Fresh tissue analysis yielded 10 additional cases (overall 61% aneuploidy) not detected on corresponding paraffin-embedded sections, whereas paraffin-embedded analysis detected 4 additional cases (overall 55% aneuploidy) not revealed by fresh tissue analysis. Fresh tissue analysis produced lower coefficients of variation and resulted in a cleaner preparation with less cellular debris. Fresh tissue analysis was also superior to paraffin for the detection of hypodiploid, near-diploid and multiple peaks. Analysis of paraffin-embedded material allows examination of archival tissue and provides a more rapid means of long-term follow-up and statistical correlations for prognostic studies. Although the overall correlation of both methodologies for DNA analysis showed a minimal variation in results, in our experience fresh tissue analysis has an advantage and is preferable, when available, for ploidy analysis.     

Flow cytometric analysis of estrogen, progesterone receptor expression and DNA content in formalin-fixed, paraffin-embedded human breast tumors.

Flow cytometric analysis of estrogen (ER) and progesterone (PgR) receptor expression in archival human breast tumors is relatively difficult. We have used enzyme digestion and microwave antigen retrieval procedures for multiparametric flow cytometric analysis of ER and PgR expression and DNA content in nuclei isolated from formalin-fixed/paraffin-embedded primary breast tumors. Deparaffinized rehydrated tissue sections treated with pepsin were subjected to microwave irradiation for unmasking of ER and PgR antigenic sites. Biotinylated ER antibody and streptavidin-fluorescein isothiocyanate (FITC) were used for ER labeling and PgR antibody with phycoerythrin labeled goat anti-mouse antibody was used for PgR labeling. Counter staining with propidium iodide-RNase was used for determination of cellular DNA content. Our results show that enzyme digestion and microwave treatment of formalin-fixed, paraffin-embedded breast tumors can be successfully used for the multiparametric analysis of nuclear hormone receptor expression and DNA content by flow cytometry.     

Flow microfluorometric analysis of nuclear DNA in cells from solid tumors and cell suspensions. A new method for rapid isolation and straining of nuclei.

A one-step procedure for the preparation of nuclei for flow microfluorometric DNA analysis is described. The membranes of the cells were lysed by the non-ionic detergent Nonidet P40. Single-cell suspensions, and specimens of solid tissues obtained with fine-needle biopsy, could be prepared equally well as the nuclei of solid tissue cells were released separately. Lysis was performed in the staining solution containing either ethidium bromide or propidium iodide. Fluorescence due to fluorochrome binding to RNA, was abolished instantaneously by the presence of RNA-se, and fluorochrome binding to secondary binding sites in DNA was inhibited with NaCl. The preparation time was 10 min and the samples were stable for a minimum of 12 h. With the basic version of the method, usable, but not always optimal, results were obtained in all the cell types tested: four different mouse ascites tumors, leucocytes, bone-marrow, liver cells, human lymphomas, human carcinomas of the breast and lung, mouse mammary carcinoma and solid JB-1 tumor. The method was further optimized for the JB-1 ascites tumour. The resulting two modified techniques are described. Differences in the staining of leucocytes with the analogues ethidium bromide and propidium iodide were demonstrated.     

Cell heterogeneity and subpopulations in solid tumors characterized by simultaneous immunophenotyping and DNA content analysis

BACKGROUND: Heterogeneity in human malignant tumors is a well-described phenomenon and of interest with regard to subpopulations with differences in clonality, metastatic potential, and response to therapy under different treatment regimes. The aim of this study was the simultaneous characterization of surface markers and DNA content of solid tumors to identify tumor cell subpopulations and to study the association between the expression of antigens and DNA content. METHODS: In the present study, six different malignant tumors grown as xenografts in nude mice were characterized by five-parameter flow cytometry. Immunophenotyping was performed using a variety of direct fluorescence-conjugated antibodies. In all cases, simultaneous detection of DNA content was done after staining with 7-aminoactinomycin D. RESULTS: Tumor cells were characterized by light scatter properties, antigen expression, and DNA content. Tumor cell heterogeneity, subpopulations, and DNA content-dependent antigen expression were identified. CONCLUSIONS: This method offers the possibility of characterizing solid tumors according to their immunophenotype and DNA content. The results obtained can be used to identify changes in immunophenotypic and DNA profiles of tumor cell populations before and after therapy and might be useful to define parameters predictive for response to therapy.     

Measurement of DNA content and cell volume in senescent human fibroblasts utilizing flow multiparameter single cell analysis

Cell cycle analysis of senescent cultured human fibroblasts by flow cytofluorometry reveals an increased proportion of cells in the G 1 period. An increased variation as well as a slight decrease in cellular DNA contents were observed in both G 1 and G 2 + M senescent cells. Utilizing gated single parameter analysis, the increased cell volumes observed in these senescent fibroblasts were demonstrated to be present in G 1 as well as G2 + M cells.     

Bivariate cytofluorimetric analysis of DNA and nuclear protein content in plant tissue

Summary Techniques of static biparametric cytofluorimetry were developed to measure DNA and protein fluorescence simultaneously in the same nucleus stained with 4,6-diamidino-2-phenylindole (DAPI) and fluorescein isothiocyanate (FITC) fluorochromes. With these cytofluorimetric procedures, we analysed DNA and nuclear protein content in root apices during the first 72 h of pea seed germination. This method allows a more reliable, rapid and less expensive measurement of DNA and proteins than cytophotometry. Nuclear protein content can be considered as a second parameter to define subcompartments of cell cycle phases; it offers the possibility of studying the progression of plant cells through cell cycle and its control in greater detail.Abbreviations DAPI 4,6-diamidino-2-phenylindole - FITC fluorescein isothiocyanate - PI propidium iodide - Tris Tris(hydroxymethyl) aminomethane     

Estimation of nuclear DNA content in Nasturtium R. Br. by flow cytometry

The most common and widespread species of Nasturtium in central Europe are the tetraploid Nasturtium officinale (2n = 4x = 32), the octoploid Nasturtium microphyllum (2n = 8x = 64), and their hexaploid hybrid Nasturtium × sterile (2n = 6x = 48). For the first time, flow cytometry was used to measure the genome size (2C DNA content) of these taxa. The highest nuclear DNA content was found in the octoploid N. microphyllum (2C = 1.43 pg) and the lowest in the tetraploid N. officinale (2C = 0.76 pg). Some differences in the amount of nuclear DNA were observed for the hexaploid N. × sterile (2C = 1.09-1.12 pg). Genome size analysis was thus proposed as a very useful tool for the identification of species of Nasturtium in their vegetative stage.     

Cytogenetic analysis of human solid tumors by in situ hybridization with a set of 12 chromosome-specific DNA probes

We have applied fluorescent in situ hybridization (FISH) to assess the presence of numerical chromosome aberrations in fresh specimens of human solid tumors of varying histology. For this purpose, a set of 12 biotinylated chromosome-specific, repetitive alpha-satellite DNA probes (for chromosomes 1, 6, 7, 9, 10, 11, 15, 16, 17, 18, X and Y) were hybridized directly to isolated interphase nuclei. Utilizing this approach, we found numerical chromosome changes in all tumors. FISH ploidy profiles were in accordance with flow cytometric DNA histograms of these tumor cells.     

Consensus report of the task force on standardisation of DNA flow cytometry in clinical pathology. DNA Flow Cytometry Task Force of the European Society for Analytical Cellular Pathology.

Guidelines are given to assist the standardisation of DNA flow cytometry in clinical pathology. They have been agreed by a group of twelve scientists from nine European countries.     

Flow cytometric analysis of G1- and G2/M-phase subpopulations in mammalian cell nuclei using side scatter and DNA content measurements

Several subcompartments of the cell cycle in addition to the G1-, S-, and G2-phases usually observed were identified by simultaneous flow cytometric measurements of ethidium bromide fluorescence and side scatter intensity of cell nuclei. Metaphase cells and very early G1-phase cells (G1A) with low side scatter intensities were discriminated from interphase cells with high side scatter intensities. The reason for the various side scatter intensities was found to be the different structure of metaphase cells and early G1-phase cells due to chromatin condensation as shown by sorting of the respective cell nuclei. The G1A-phase could further be subdivided into two compartments with very low side scatter (G1A1) and intermediate side scatter (G1A2) intensities. Using partially synchronized cells the duration of these subcompartments of the G1-phase could be estimated. The durations of G1A1- and G1A2-phases were found to be about 10 min and 20 min, respectively, compared to the total duration of the G1-phase of about 3 h. Additional flow cytometric measurements of side scatter intensities of cell nuclei provide therefore further information on subcompartments of the G1- and G2/M-phases.     

Comparison of nuclear DNA content in primary and metastatic malignant melanoma.

The DNA patterns obtained from 23 primary malignant melanomas and 35 corresponding metastases were compared and found to differ in many cases. In eight cases the primary tumors and their metastases had a ploidy type I ("euploid") DNA pattern. One case had a type I primary tumor and both type I and type II metastases. Five cases had type I primary tumors and ploidy type II ("aneuploid") DNA pattern metastases. In five cases the primary tumors and corresponding metastases were type II, and in another four cases the primary tumors were type II, whereas the metastases were type I. We interpret these data as indicating that malignant melanomas (more often than adenocarcinomas) are composed of genetically heterogeneous tumor sublines that frequently give rise to heterogeneously composed metastases. Since we sometimes observed a change in the DNA content in malignant melanomas, it seems to be more difficult to obtain prognostic information from DNA analysis in malignant melanoma as compared to the more stable adenocarcinomas.     

斑鳢、乌鳢及其杂种细胞核DNA流式含量分析

以斑鳢(Channa maculata)、乌鳢(C.argus)及其正交杂种斑乌鳢(斑鳢♀×乌鳢♂)和反交杂种乌斑鳢(乌鳢♀×斑鳢♂)的红细胞为材料,以鸡(Gallus gallus)血细胞为DNA标准(2.5 pg/2c,2c指2倍体),采用流式细胞仪测定了这4种鱼的细胞核DNA含量。斑鳢、乌鳢、斑乌鳢及乌斑鳢这4种鱼血细胞DNA的绝对含量分别为(1.488±0.035)pg/2c、(1.489±0.034)pg/2c、(1.522±0.077)pg/2c和(1.520±0.033)pg/2c。斑鳢和乌鳢的细胞核DNA含量差异不显著(P0.05),斑鳢和乌鳢与两种杂交鳢的DNA含量差异显著(P0.05),两种杂交鳢之间的细胞核DNA含量差异不显著(P0.05)。杂交鳢细胞核DNA含量显著高于亲本,可以作为杂种鉴定的依据。