NASA/American Cancer Society High-Resolution Flow Cytometry Project - III. Multiparametric analysis of DNA content and electronic nuclear volume in human solid tumors

BACKGROUND: The NASA/American Cancer Society (ACS) flow cytometer can simultaneously measure electronic nuclear volume (ENV) and DNA content of nuclei. The preceding articles in this volume ("NASA/American Cancer Society High-Resolution Flow Cytometer Project-I") described the schematics, performance, and procedures used for the preparation of nuclei for analysis on this unit. In the present article, we describe the analysis of selected human tumors using the ratio of ENV/DNA content (nuclear packing efficiency [NPE]). METHODS: Tumor specimens (frozen) were minced with scalpels and stained with 1-10 microg/ml of 4',6-diamidino-2-phenylindole (DAPI) dihydrochloride at pH 6.0-7.2. Trout erythrocytes were used as internal standards. Data on ENV and DNA content were collected in list mode files. Propidium iodide-stained nuclei, analyzed on a Coulter XL cytometer, were used for comparison. RESULTS: Simultaneous measurement of ENV and DNA makes it possible to discriminate between hypodiploid or hyperdiploid tumor cells, as well as to differentiate between near-diploid aneuploid and diploid cells on the basis of their increased ENV. The NPE ratio is a valuable parameter for the detection of small quantities of tumor cells, separating overlapping diploid and aneuploid populations for cell cycle analysis and characterizing the level of differentiation in some tumors. CONCLUSION: NPE analysis provides unique measuring capabilities for the study of human solid tumors by flow cytometry.     

Standardization of high-resolution flow cytometric DNA analysis by the simultaneous use of chicken and trout red blood cells as internal reference standards

Determination of nuclear DNA content by flow cytometry requires comparison with a reference standard. The use of external standards such as lymphocytes or granulocytes is time-consuming and inaccurate. Chicken red blood cells (CRBC) have a DNA content of 35% of the human diploid value and have been widely used as internal standard. The ratio calculated on the basis of the peak channel numbers of the standard and the sample and used to indicate the DNA content (DNA ratio) is, however, very sensitive to changes in the zero level adjustment of the flow cytometer. If two internal standards are used the DNA ratio becomes independent of the zero level. Rainbow trout red blood cells (TRBC) have a DNA content of 80% of human diploid cells. A mixture of CRBC and TRBC was prepared and stored in small aliquots at -80 degrees C. This mixture was added to the sample before staining. The day-to-day variation of the DNA ratio obtained by use of the two standards was smaller than that obtained by CRBC alone. The possibility of sex related differences in DNA content of CRBC and TRBC was examined. The results indicated that a new batch of standards should be tested against the old batch to avoid the introduction of a systematic error.     

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.     

Inter-organ differences of the cytometric DNA content in mice: relation of the staining method

With one step DNA staining methods including cell membrane lysis and RNase treatment, we regularly observed a higher fluorescence emission in liver nuclei compared to bone marrow nuclei in C57BL/6 mice. Therefore this study was conducted in order to emphasize such a phenomenon in other organs and to assess if higher fluorescence emission was related to higher DNA content or staining procedure failure. Liver, bone marrow and testis were removed from Swiss, BDF and C57BL/6 mice. The following samples were prepared: 1) liver cells with TRBC (TRBC = Trout Red Blood Cells = internal standards), 2) bone marrow cells with TRBC, 3) testis cells with TRBC and 4) mixtures of liver, bone marrow and testis cells. The staining procedures were: A) one step pH 10 procedure described by Vindelov (Virchows Arch. B. Cell Path., 1977, 24, 227-242), B) same procedure with twice RNase concentration, C) first method with twice NP 40 concentration and D) three steps procedure including Trypsin and Spermine treatment (Vindelov et al., Cytometry, 1983, 3, 323-327). In protocols A, B and C, "Diploid cells/TRBC" ratio differed significantly between liver, bone marrow and testis nuclei. Moreover, 3 distinct populations of diploid cells were present in samples 4. In protocol D, "Diploid cells/TRBC" ratio were identical between liver, bone marrow and testis nuclei. In samples 4, only 1 population of diploid cells has been observed. This study shows that DNA stabilization by polyamine and protein degradation by protease could act on Propidium Iodide fixation and/or fluorescence emission, with significant differences according to the origin of the cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

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 QuantaTm 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 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.     

Trout and salmon erythrocytes and human leukocytes as internal standards for ploidy control in flow cytometry

Control of technique and use of biological standards in flow cytometry have become increasingly important due to the wider use of the method for ploidy determination of malignant tumors in clinical research. Trout (TRBC) and salmon erythrocytes and human buffy coat leukocytes were selected for a study of factors influencing the DNA stainability. Whether standard and test cells were mixed before or after enzymatic treatment and staining was found to be critical for the ploidy comparisons. Otherwise, artifactual differences of at least 20% may be noted, leading to an overestimation of DNA aneuploidy. The time from staining to analysis had minimal effect, with some exceptions. The proportions of different cells in the sample had no influence, and nonlinearity of measurements was negligible. Diploid cells in normal endometrium and benign ovarian tumors, as well as the diploid fraction of aneuploid tumor cells, were systematically measured to have a DNA staining 5-7% above human leukocytes.     

Rapid Detection of high-level oncogene amplifications in ultrasonic surgical aspirations of brain tumors

ABSTRACT: BACKGROUND: Genomic tumor information, such as identification of amplified oncogenes, can be used to plan treatment. The two sources of a brain tumor that are commonly available include formalin-fixed, paraffin-embedded (FFPE) sections from the small diagnostic biopsy and the ultrasonic surgical aspiration that contains the bulk of the tumor. In research centers, frozen tissue of a brain tumor may also be available. This study compared ultrasonic surgical aspiration and FFPE specimens from the same brain tumors for retrieval of DNA and molecular assessment of amplified oncogenes. METHODS: Surgical aspirations were centrifuged to separate erythrocytes from the tumor cells that predominantly formed large, overlying buffy coats. These were sampled to harvest nuclear pellets for DNA purification. Four glioblastomas, 2 lung carcinoma metastases, and an ependymoma were tested. An inexpensive PCR technique, multiplex ligation-dependent probe amplification (MLPA), quantified 79 oncogenes using 3 kits. Copy number (CN) results were normalized to DNA from non-neoplastic brain (NB) in calculated ratios, [tumor DNA]/[NB DNA]. Bland-Altman and Spearman rank correlative comparisons were determined. Regression analysis identified outliers. RESULTS: Purification of DNA from ultrasonic surgical aspirations was rapid (<3 days) versus FFPE (weeks) and yielded greater amounts in 6 of 7 tumors. Gene amplifications up to 15-fold corresponded closely between ultrasonic aspiration and FFPE assays in Bland-Altman analysis. Correlation coefficients ranged from 0.71 to 0.99 using 3 kit assays per tumor. Although normalized CN ratios greater than 2.0 were more numerous in FFPE specimens, some were found only in the ultrasonic surgical aspirations, consistent with tumor heterogeneity. Additionally, CN ratios revealed 9 high-level ([greater than or equal to] 6.0) gene amplifications in FFPE of which 8 were also detected in the ultrasonic aspirations at increased levels. The ultrasonic aspiration levels of these amplified genes were also greater than 6.0 CN ratio, except in one case (3.53 CN ratio). Ten of 17 mid-level ([greater than or equal to]3.0 & <6.0 CN ratio) amplifications detected in FFPE were also detected as being increased ([greater than or equal to] 2.0 CN ratio) in the aspirations. CONCLUSIONS: Buffy coats of centrifuged ultrasonic aspirations contained abundant tumor cells whose DNA permitted rapid, multiplex detection of high-level oncogene amplifications that were confirmed in FFPE. Virtual slides http://www.diagnosticpathology.diagnomx.eu/vs/1883718801686466.     

藻蓝蛋白亚基脂质体光动力学抗乳腺癌细胞的实验研究

目的:研究PEG修饰的藻蓝蛋白亚基光敏剂长循环脂质体介导的光动力疗法抗乳腺癌的效果.方法:采用薄膜水合-超声分散法制备PEG修饰的藻蓝蛋白亚基脂质体(PEG-PCS-lip),MTT法检测藻蓝蛋白亚基脂质体对MCF-7(人乳腺癌细胞)、MA-782(鼠乳腺癌细胞)的光动力杀伤效果,流式细胞术(FCM)分析其对肿瘤细胞周期的影响,并对乳腺癌模型鼠做PDT治疗.结果:PEG-PCS-lip介导的PDT作用对乳腺癌细胞有良好的光动力疗效,对MCF-7及MA-782细胞IC_(50)(半数抑制浓度)分别为68 μg/mL、70 μg/mL;FCM的结果显示,当PEG-PCS-lip 浓度为50 μg/mL时,MCF-7凋亡率约为30.5 %; 用10 mg/kg PEG-PCS-lip静脉注射荷瘤小鼠,光照剂量为208.2 J/cm~2时,其抑瘤率可达到72 %;组织切片观察PDT作用后的肿瘤组织,瘤体中间以细胞凋亡为主,瘤体外周以细胞坏死为主,由血管损伤而形成的空腔增多.结论:PEG-PCS-lip在体外对乳腺癌细胞有良好的光动力杀伤效果,肿瘤细胞凋亡及瘤血管破坏是导致肿瘤细胞死亡的主要原因.     

Laser scanning cytometry can complement the flow cytometric DNA analysis in paraffin-embedded cancer samples: a paradigmatic case

Archival studies on paraffin-embedded tumor samples are often complicated by difficulty obtaining a reliable diploid DNA standard. Nontumor cells, e.g., inflammatory and stromal cells, most often found interspersed among tumor cells, would represent a solution to this problem. Unfortunately, there is an inherent difficulty to positively identifying tumor cells in paraffin-embedded specimens. Using an aneuploid paraffin-embedded breast cancer sample, we show here that laser scanning cytometer (LSC) in conjunction with flow cytometry can help to address this issue. Following standard protocols, the tissue was deparaffinized and rehydrated, and the nuclei mechanically isolated before being exposed to propidium iodide. An aliquot served for single-parameter flow cytometric analysis, and the remaining cells were cytocentrifuged onto a microscope slide and LSC analysis was performed. The DNA histogram profiles generated by the two approaches were comparable and both showed the presence of cell populations with different DNA content. To assess the nature of these subsets, we performed a correlated measurement of DNA content and chromatin organization at the single-cell level by LSC. This allowed the identification of several subsets of nuclei. Slides were then stained with Giemsa and the nature of these subsets was assessed morphologically by exploiting the relocating capability of LSC. Inflammatory and stromal cells, residual diploid epithelial cells, and hyperdiploid tumor cells-each characterized by a peculiar coordinate pattern of DNA content and chromatin organization-could be positively identified. Diploid, nontumor cells can then be used as an internal standard for DNA ploidy.     

人和猴T淋巴细胞表面TRBC受体及其配体不同于E2分子和CD2的配体

CD2 (E receptor, LFA-3 receptor) and E2 molecules (Bernard, 1988) on human T lymphocytes, CD58 (LFA-3, lymphocyte function associated antigen 3) on human erythrocytes and S14,S42,S110-220 molecules (Bernard, 1987) of sheep erythrocytes are involved in rosette formation of human T lymphocytes with human or sheep erythrocytes. Rosette formation of human and macaque pan-T lymphocytes with tree shrew (Tupaia belangeri) red blood cells (TRBC) (TRBC rosette) has shown different physicochemical properties from that of rosette formation with sheep red blood cells (E rosette) (Ben, 1985). CD2, CD3/TCR complex, CD5, CD6, and CD7 are not involved in TRBC rosette formation (Zheng, 1990). In order to know whether E2, LFA-3,S14,S42 and S110-220 molecules are involved in TRBC rosette formation or human and macaque T lymphocytes, rosette inhibition and antigenic modulation or co-modulation were performed with relevant monoclonal antibodies (McAbs), and hemolytic assay and slide agglutination were also conducted. TRBC rosette formation of human and rhesus monkey PBL was not blocked by E2 McAb (inhibition rate 2.8% and 2.1%, respectively). In contrast, human E rosette formation was obviously blocked at inhibition rate of 49.8% and macaque E rosette formation was slightly inhibited (13.3%). The modulation or co-modulation of E2 molecule with E2 McAb did not affect human TRBC rosette formation. Similar results were shown in rosette formation inhibition of Jurkat cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interleukin-2 expanded lymphocytes from lymph node and tumor biopsies of human renal cell carcinoma, breast and ovarian cancer

Adoptive immunotherapy with immune effector cells has proved to be potent for treatment of tumors, however neither the attendant criteria for potential clinical efficacy of the injected cells, nor the method to prepare these cells are presently well established. Our procedure of collecting lymphocytes from biological samples, was based on the use of low IL-2 concentrations (90 to 150 IU/ml) and on the stringent separation of lymphocytes from tumor cells at the very early stages of their outgrowth in culture. When lymphocytes were derived from tumor biopsies (TIL), we observed differences depending on the histological type of tumor. In renal cell carcinoma, natural killer cells were expanded in 4/11 biopsies contrary to what was observed in breast cancer (92 +/- 5% of T lymphocytes from 9 biopsies). The outgrowth of lymphocytes from breast tumors was slower and lower than from renal carcinomas. The autologous tumor cell line was more difficult to obtain from breast carcinoma (23%) than from renal cell carcinoma (61%) biopsies. For ovarian cancer, short-term culture of tumor cells could be obtained for half of the tumor-invaded biological samples. Eight of the 23 tumor-derived cultures contained more than 40% CD8 T. TIL were consistently cytolytic each time they could be evaluated. For ascitic and pleural fluids, data were of similar range. In ascitic-derived cultures, tumor cells and antigen-presenting cells are present and can be supposed to rechallenge T cells with tumor antigens. Lymphocytes derived from lymph nodes could be expanded to a larger number than TIL. However, only 1/18 of these cultures contained more than 40% CD8 T. The presence of few tumor cells in this culture was in favor of significant specific and non-specific cytotoxicity in RCC lymph node cultures and higher percentages of CD8 T in breast cancer lymph nodes. Correlations could not be established between CD8 T percentages and specific in vitro cytotoxicity in our polyclonal populations. Our conclusion is that phenotypic and functional quality of lymphocytes is of interest when the T cells are derived 1) from tumors (RCC, breast or ovarian cancer) and isolated very early to avoid inhibitor factors secreted from tumor cells or 2) from lymph nodes and ascitic and pleural fluids when very few tumor cells are co-cultivated with lymphocytes at initial steps of culture. Final expansion to a number of lymphocytes suitable for therapy (> 109) could be attained in a second step of the procedure by the use of 1,000 IU/ml IL-2 each time it was assayed with 50.106 lymphocytes. In view of these data it appears that phenotypic and functional changes occur during culture depending on the presence of a particular ratio of tumor antigens. This could be artificially reproduced.     

Peculiarities of cytometrical methods of DNA content determination in the nucleus

This work has proposed a new theoretical approach to analysis of histograms of DNA content, which are obtained by the method of flow cytometry, in cells of Drosophila melanogaster imaginal discs. The precision of measurements of the DNA amount in G₁ and G₂(M) phases has been shown to be limited by precision of instrument tuning of zero of the flow cytometer. Use of the calculative zero of the flow cytometer and of dividing cells as standards of the DNA content is able to increase severalfold the precision of the DNA measurements in nuclei of the species. Comparative analysis of errors of various methods of measurement of the DNA content in cell nuclei is also performed. For methods of flow fluorescent cytometry, confocal scanning, and cytophotometry of the Feulgen-stained nuclei, it has been shown that, at present, the mean square errors of the DNA content measurements are within the interval of values considered acceptable for biological studies (0.02 < CV < 0.06).     

Doublet discrimination in DNA cell-cycle analysis.

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.     

A single platform image cytometer for resource-poor settings to monitor disease progression in HIV infection.

BACKGROUND: For resource-poor countries, affordable methods are required for enumeration of CD4(+) T lymphocytes of HIV-positive patients. For infants, additional determination of CD4/CD8 ratio is needed. METHODS: We determine the CD4(+) and CD8(+) T lymphocytes as the CD3(+)CD4(+) and CD3(+)CD8(+) population of blood cells. Target cells are CD3-immunomagnetically separated from the whole blood, and CD4-Phycoerythrin and CD8-PerCP immunofluorescently labeled. A point-of-care single platform image cytometer was developed to enumerate the target CD3(+)CD4(+) and CD3(+)CD8(+) populations. It has light-emitting diodes illumination, is fully computer-controlled, operates from a 12 V battery, and was designed to be cheap and easy-to-handle. Target cells are imaged on a CCD camera and enumerated by an image analysis algorithm. The cytometer outputs the absolute number of CD4(+) and CD8(+) T lymphocytes/microl and CD4/CD8 ratio. RESULTS: The quality of the cell images obtained with the cytometer is sufficient for a reliable enumeration of target cells. The image cytometer achieves an accuracy of better than 10% in the range of 50-1700 cells/microl. Analysis of blood samples from HIV patients yields a good agreement with the TruCount method for CD4 and CD8 count and CD4/CD8 ratio. CONCLUSIONS: The image cytometer is affordable (component costs $3,000), compact (25 x 25 x 20 cm(3)), and uses disposable test materials, making it a good candidate to monitor progression of immunodeficiency disease in resource-poor settings.     

Multiplexed, particle-based detection of DNA using flow cytometry with 3DNA dendrimers for signal amplification.

BACKGROUND: Complex mixtures of DNA may be found in environmental and medical samples. There is a need for techniques that can measure low concentrations of target DNAs. For a multiplexed, flow cytometric assay, we show that the signal-to-noise ratio for fluorescence detection may be increased with the use of 3DNA dendrimers. A single fluorescent DNA molecule per bead could be detected with conventional flow cytometry instrumentation. METHODS: The analyte consisted of single-stranded (ss) DNA amplicons that were hybridized to capture probes on the surface of fluorescent polystyrene microspheres (beads) and initially labeled with streptavidin-R-phycoerythrin (single-step labeling). These beads have a low reporter fluorescence background and high efficiency of DNA hybridization. The DNA/SA-RPE complex was then labeled with 3DNA dendrimers and SA-RPE. The bead complexes were detected with a Luminex 100 flow cytometer. Bead standards were developed to convert the intensity to the number of SA-RPE labels per bead and the number of dendrimers per bead. RESULTS: The dendrimer assay resulted in 10-fold fluorescence amplification compared with single-step SA-RPE labeling. Based on concentration curves of pure target ss-amplicons, the signal-to-noise ratio of the dendrimer assay was greater by a factor of 8.5 over single-step SA-RPE labeling. The dendrimer assay was tested on 16S ribosomal DNA amplified from filter retentates of contaminated groundwater. Multiplexed detection of a single dendrimer-labeled DNA molecule per bead was demonstrated. CONCLUSIONS: Multiplexed detection of DNA hybridization on a single molecule level per bead was achieved with conventional flow cytometry instrumentation. This assay is useful for detecting target DNAs at low concentrations.     

Prognostic significance of DNA ploidy determined by high-resolution flow cytometry in breast carcinoma

OBJECTIVE: To assess the prognostic value of DNA ploidy in breast carcinoma and its relation to other established prognostic factors. STUDY DESIGN: We evaluated DNA ploidy in 303 breast carcinoma patients with a median follow-up of 63 months. Flow cytometry was performed on frozen tumor material, yielding histograms with narrow peaks (median coefficient of variation of 2.08). DNA ploidy pattern was classified as either diploid versus nondiploid, euploid (diploid and tetraploid) versus aneuploid or diploid/near-diploid (DNA index < 1.2) versus other, and correlated with relapse-free (RFS) and cancer-specific survival (CSS) along with tumor size, histologic grade and type, axillary lymph node involvement, menopausal and steroid receptor status, age and type of treatment. RESULTS: Seventy-one percent of tumors were DNA nondiploid (14% tetraploid and 57% aneuploid). There was a strong association between DNA ploidy and histologic grade. Histologic grade, lymph node status, tumor size and DNA ploidy (regardless of the classification used) were all significantly associated with RFS and CSS in multivariate analysis. CONCLUSION: These results suggest that DNA ploidy, at least when determined from frozen tumor tissue, is an independent prognostic factor in breast carcinoma; however, its prognostic power seems to be inferior to that of histologic grade, with which it strongly correlates.     

A review of techniques and results obtained in one laboratory by an integrated system of methods designed for routine clinical flow cytometric DNA analysis

Establishing flow cytometric DNA analysis as a clinical routine procedure requires adequate and proven guidelines, by which the data can be obtained and interpreted to directly influence management of the individual patient with a specific neoplasm. The present paper is intended as a contribution to such guidelines, of which only fragments are available today. We have previously described a system of methods, designed for routine flow cytometric DNA analysis. In the present status report our experience, based on approximately 18,000 samples (clinical and experimental) is summarised. Sample acquisition with fine-needle aspiration, storage at -80 degrees C, internal standardization by chicken (CRBC) and trout red blood cells (TRBC), staining with propidium iodide (PI), and analysis in the flow cytometer is recapitulated, with emphasis on previously unpublished aspects. The method of statistical analysis which has an integrating role is described in some detail. A lack of linearity between channel number and DNA content was determined experimentally, and the coefficient of variation (CV) was found to decrease with increasing channel number. The corrections in the algorithm of deconvolution made necessary by these findings are fundamental for estimating the end results. The zero point adjustment and procedures for changing from one batch of standards to another are described. A systematic approach to interpretation of DNA histograms is attempted and illustrated by data from clinical specimens of malignant lymphoma, breast cancer, small cell lung cancer, cancer of the oral cavity, and bladder cancer. Some problems are still unsolved and visual inspection is required to determine if the quality of the individual histogram is satisfactory. Inspection of the fluorescence/light scatter dot-plot provides additional information for the recognition of artifacts. The results stress that good quality DNA histograms with as small CVs as possible are important for interpretation of the data. It is essential that statistical methods are employed to extract the key end-point results. These are the number of subpopulations and their relative representation, and for each subpopulation the DNA index (DI) and the fractions of cells in the cell cycle phases. For the DNA data to have any rationally based impact on clinical decision making, it must be demonstrated that they have an independent prognostic value. Strategies for final evaluation are discussed. Multicenter trials on fresh material, to accrue quickly the number of patients necessary for firm conclusions, are suggested.     

Bivariate analysis of cellular DNA versus RNA content by laser scanning cytometry using the product of signal subtraction (differential fluorescence) as a separate parameter

BACKGROUND: The cytometric methods of bivariate analysis of cellular RNA versus DNA content have limitations. The method based on the use of metachromatic fluorochrome acridine orange (AO) requires rigorous conditions of the equilibrium staining whereas pyronin Y and Hoechst 33342 necessitate the use of an instrument that provides two-laser excitation, including the ultraviolet (UV) light wavelength. METHODS: Phytohemagglutinin (PHA)-stimulated human lymphocytes were deposited on microscope slides and fixed. DNA and double-stranded (ds) RNA were stained with propidium iodide (PI) and protein was stained with BODIPY 630/650-X or fluorescein isothiocyanate (FITC). Cellular fluorescence was measured with a laser scanning cytometer (LSC). The cells were treated with RNase A and their fluorescence was measured again. The file-merge feature of the LSC was used to record the cell PI fluorescence measurements prior to and after the RNase treatment in list mode, as a single file. The integrated PI fluorescence intensity of each cell after RNase treatment was subtracted from the fluorescence intensity of the same cell measured prior to RNase treatment. This RNase-specific differential value of fluorescence (differential fluorescence [DF]) was plotted against the cell fluorescence measured after RNase treatment or against the protein-associated BODIPY 630/650-X or FITC fluorescence. RESULTS: The scattergrams were characteristic of the RNA versus DNA bivariate distributions where DF represented cellular ds RNA content and fluorescence intensity of the RNase-treated cells, their DNA content. The distributions were used to correlate cellular ds RNA content with the cell cycle position or with protein content. CONCLUSIONS: One advantage of this novel approach based on the recording and plotting of DF is that only the RNase -specific fraction of cell fluorescence is measured with no contribution of nonspecific components (e.g., due to the emission spectrum overlap or stainability of other than RNA cell constituents). Another advantage is the method's simplicity, which ensues from the use of a single dye, the same illumination, and the same emission wavelength detection sensor for measurement of both DNA and ds RNA. The method can be extended for multiparameter analysis of cell populations stained with other fluorochromes of the same-wavelength emission but targeted (e.g., immunocytochemically) for different cell constituents.     

Detection and quantification of circulating tumor cells in mouse models of human breast cancer using immunomagnetic enrichment and multiparameter flow cytometry.

BACKGROUND: Circulating tumor cells (CTCs) in the peripheral blood of breast cancer patients may be an important indicator of metastatic disease and poor prognosis. However, the use of experimental models is required to fully elucidate the functional consequences of CTCs. The purpose of this study was to optimize the sensitivity of multiparameter flow cytometry for detection of human tumor cells in mouse models of breast cancer. METHODS: MDA-MB-468 human breast cancer cells were serially diluted in whole mouse blood. Samples were lysed and incubated with a fluorescein isothiocyanate-conjugated anti-human leukocytic antigen antibody and a phycoerythrin-conjugated anti-mouse pan-leukocyte CD45 antibody. Samples were then immunomagnetically depleted of CD45-positive leukocytes, fixed, permeabilized, and stained with propidium iodide before flow cytometric analysis. RESULTS: Human breast cancer cells could be differentiated from mouse leukocytes based on increased light scatter, cell surface marker expression, and aneuploid DNA content. The method was found to have a lower sensitivity limit of 10(-5) and was effective for detecting human breast cancer cells in vivo in the circulation of experimental mice carrying primary human mammary tumors. CONCLUSIONS: This technique has the potential to be a valuable and sensitive tool for investigating the biological relevance of CTCs in experimental mouse models of breast cancer.