Flow cytometric analysis of bromodeoxyuridine-induced inhibition of cell proliferation in the human teratocarcinoma-derived cell line, P3

We have developed methods in our laboratory whereby the effects of toxicant exposure on cell proliferation can be evaluated flow cytometrically. We sought to relate the flow cytometric analyses to other biological response measurements. Thus, we exposed P3 cells to increasing concentrations of bromodeoxyuridine (BRdU) and measured sister-chromatid exchange (SCE) frequency, average generation time (AGT), and relative cloning ability. Each of these is well documented (see introduction) to respond to BRdU exposure in a concentration-dependent manner. In this study, SCE frequency remained constant between the concentrations of 2.5 and 10 microM of BRdU. However, a small, but significant, increase in SCE frequency was observed between the concentrations of 10 microM and 50 microM BRdU. A significant increase in AGT was noted in 50 microM BRdU-exposed cells. Relative cloning efficiency decreased in a concentration-dependent manner when cells were cultured for 24, 48, or 72 hours with BRdU. When cell proliferation was assessed by flow cytometric analysis in cells exposed to 0, 10, or 50 microM BRdU, a statistically significant delay in the cell-cycle was observed in BRdU-exposed cells. These results may be interpreted to mean that inhibition of cell proliferation is detected by this type of analysis at toxicant concentrations that induce other biological endpoints. The inclusion of flow cytometric analysis in a test battery to evaluate toxicant effects is warranted.     

Flow cytometric analysis of micronuclei in mammalian cell cultures: past, present and future

The relative simplicity of the in vitro micronucleus (MNvit) endpoint has made it amenable to several automated scoring approaches. Flow cytometry is one such scoring platform that has been successfully employed. This review describes the origins of the MNvit assay, as well as the evolution and properties of flow cytometry-based scoring systems. While the current state-of-the-art methods acquire micronucleus (MN) frequency data very efficiently, it is becoming clear that they also endow the assay with high information content. For instance, simultaneous with MN frequency determinations, several additional endpoints are acquired that provide insights into cytotoxicity, cell cycle perturbations and, in the event of MN induction, information about genotoxic mode of action. This review concludes with a discussion regarding data gaps and also recommendations for additional work that is needed to more fully realise the potential of flow cytometric MNvit scoring.     

Flow cytometric analysis of lymphomas

Flow cytometry is rapidly developing as an important tool in the characterization of lymphomas. Analysis of cell surface antigens and DNA content in these tumors provides useful biologic information that can be applied advantageously to their diagnosis and classification. In this article we examine practical aspects of handling, preparation, and staining of lymphoid samples for flow cytometry and delineate approaches to data analysis and interpretation. We discuss specific applications, advantages, and limitations of the technique in the evaluation of neoplastic lymphoid diseases.     

Flow cytometric method for scoring rat liver micronuclei with simultaneous assessments of hepatocyte proliferation

The current report describes a newly devised method for automatically scoring the incidence of rat hepatocyte micronuclei (MNHEP) via flow cytometry, with concurrent assessments of hepatocyte proliferation—frequency of Ki‐67‐positive nuclei, and the proportion of polyploid nuclei. Proof‐of‐concept data are provided from experiments performed with 6‐week old male Crl:CD(SD) rats exposed to diethylnitrosamine (DEN) or quinoline (QUIN) for 3 or 14 consecutive days. Non‐perfused liver tissue was collected 4 days after cessation of treatment in the case of 3‐day studies, or 1 day after last administration in the case of 14‐day studies for processing and flow cytometric analysis. In addition to livers, blood samples were collected one day after final treatment for micronucleated reticulocyte (MN‐RET) measurements. Dose‐dependent increases in MNHEP, Ki‐67‐positive nuclei, and polyploidy were observed in 3‐ and 14‐day DEN studies. Both treatment schedules resulted in elevated %MNHEP for QUIN‐exposed rats, and while cell proliferation effects were subtle, appreciable increases to normalized liver weights were observed. Whereas DEN caused markedly higher %MNHEP when exposure was extended to two weeks, QUIN‐induced MNHEP were slightly increased with protracted dosing. Parallel microscopy‐based MNHEP frequencies were highly correlated with flow cytometry‐based measurements (four study/aggregate R² = 0.80). No increases in MN‐RET were seen in any of the four studies. Collectively, these results suggest liver micronuclei are amenable to an automated scoring technique that provides objective analyses and higher information content relative to conventional microscopy. Additional work is needed to expand the number and types of chemicals tested, identify the most advantageous treatment schedules, and test the transferability of the method. Environ. Mol. Mutagen. 59:176–187, 2018. © 2018 Wiley Periodicals, Inc.     

Flow cytometric analysis of stemline heterogeneity

The stemline heterogeneity of malignant tumors is closely connected with tumor aneuploidy. Both features can be characterized by either chromosome analysis or DNA cytophotometry. DNA analysis may be performed either by single cell photometry or by flow cytometry, of which the respective advantages and drawbacks are presented. Preliminary results of flow cytometric DNA analysis in malignant neoplasms are discussed; the possibilities of multiparametric measurement for quantitative analysis of various biochemical and antigenic properties in DNA stemlines are considered.     

流式细胞术在周围神经肿瘤诊断上的应用

Flow cytometric DNA analysis was performed on formalin fixed paraffin-embedded samples taken from 43 cases of peripheral nerve tumors. Fourteen samples were taken from benign tumors, 11 from actively proliferative tumors and 12 from malignant tumors. Ploidy pattern and proliferative index of tumors were estimated. All samples of benign tumors showed a diploid model of DNA content whereas DNA aneuploidy was encountered in 75% (12/16) of the malignant tumors and 25% (4/16) of the actively proliferative tumors. The proliferative index (PI) of benign tumors (medium: 24.8%) was lower than that of proliferative tumors (medium: 42.7%). The proliferative index of malignant tumors was the highest in the experimental group (medium: 51.2%). These results were conformable to the malignant degree of histologic grading and prognosis.     

Flow cytometric analysis of terminal deoxynucleotidyl transferase

Terminal deoxynucleotidyl transferase (TdT) is a useful marker for lymphoid precursor cells. In this study, the authors used flow cytometry (FCM) to analyze TdT expression in human hematopoietic malignancies. Cells were fixed in 0.5% formaldehyde, briefly exposed to nonionic detergent, and subsequently labeled with mouse monoclonal anti-TdT antibodies followed by fluorescein-conjugated antimouse IgG and propidium iodide (PI), which was used for the simultaneous analysis of DNA content. Cells from 20 of 22 acute lymphoblastic leukemias (ALL), 4 of 7 mixed lineage leukemias, 2 of 21 acute myeloid and myelomonocytic leukemias, 1 of 2 chronic myeloid leukemias in blast crisis, 1 lymphoblastic lymphoma, and 1 thymoma were TdT positive. Cells from 13 nonlymphoblastic lymphomas, 3 myelodysplastic bone marrows, and peripheral blood mononuclear cells from 29 normal individuals were negative. An excellent correlation was seen between this assay, the conventional slide immunofluorescence technique, and an enzyme immunoassay method. The FCM assay detected as few as 2% blasts in mixing experiments of TdT-containing leukemic cells with normal peripheral lymphocytes. Furthermore, the combined analysis of TdT and DNA allowed the recognition of aneuploid TdT positive cells in four cases of ALL. The high sensitivity, the quantitative information obtained, and the capability of simultaneously analyzing DNA content make this method of TdT analysis more valuable than conventional techniques.     

Flow cytometric analysis of malignant pleural mesotheliomas

Forty-six cases of malignant pleural mesothelioma were analyzed for histologic subtype, DNA content, and cell cycle characteristics. Sixty-five percent of cases were diploid in DNA content, with intermediate to low proliferative rates. Thirty-one nonmesothelial malignant neoplasms of the lung, of histologic types most easily confused with malignant mesothelioma, were also examined. In contrast to the mesotheliomas, 85% of these nonmesothelial malignant neoplasms of the lung were aneuploid; the aneuploid neoplasms exhibited higher mean proliferative rates (S = 10.6%) than diploid nonmesothelial neoplasms of the lung (S less than 6%). Unlike most malignant neoplasms, mesotheliomas most often display diploid DNA contents and low proliferative rates despite their clinically aggressive behavior.     

Flow cytometric DNA analysis of corneal epithelium

We have modified an existing technique in order to perform DNA analysi by flow cytometry (FCM) of corneal epithelium from the mouse, rat, chicken, rabbit, and human. This protocol permitted an investigation of human corneal scrapings from several categories: normal, aphakic bullous keratopathy (ABK), keratoconus (KC), Fuch's dystrophy, edema, epithelial dysplasia, and lipid degeneration. No abnormal characteristic cell-kinetic profile was detected when averaged DNA histograms were compared statsitically between the normal and either ABK, KC, edema, or Fuch's dystrophy groups. Abnormal DNA histograms were recorded for cell samples that were taken (1) from three individuals who had epithelial dysplasia and (2) from one individual diagnosed with lipid degeneration. The former condition was characterized by histograms that had a subpopulation of cells with an aneuploid amont of DNA or had higher than normal percentages of cells in the S and G₂ + M phases of the cell cycle. Corneal cells from the patient who had lipid degeneration had an abnormally high percentage of cells in the G₂ + M phases of the cell cycle. The availability of accurate DNA flow cytometric analysis of corneal epithelium allows further studies on this issue from both experimental and clinical situations.     

软组织平滑肌肉瘤的流式细胞术研究

目的　研究软组织平滑肌肉瘤 (LMS)中DNA倍体与S期分数 (SPF)及二者与临床病理的联系。方法　应用流式细胞术 (FCM )检测 33例LMS的DNA指数及SPF。结果　LMS中异倍体率为 39 4 % ,平均SPF值为 12 2 36。低分化LMS组的异倍体率 (70 0 % )高于高分化LMS组 (2 6 1% ) (P <0 0 5 ) ;异倍体LMS组的SPF均值 (15 5 92 )高于二倍体组 (10 0 5 5 ) (P<0 0 5 ) ;瘤体最大径 >6cm组的SPF均值 (13 6 36 )明显高于 <6cm组 (9 4 36 ) (P <0 0 5 )。结论　LMS的SPF与DNA倍体和体积均呈现出相关关系。LMS的DNA倍体与肿瘤的分化程度具有相关性     

Flow cytometric cerebrospinal fluid analysis in children

Flow cytometry (FC) is of increasing importance for the analysis of cerebrospinal fluid (CSF) lymphocytes because of its ability to detect a large spectrum of cellular characteristics (granularity, volume, surface antigen expression) even in small amounts of cells. Data on CSF FC in children are very limited. Here, we summarize our 3-year experience of CSF FC routinely performed in pediatric patients with assumed inflammatory central nervous system (CNS) disease. Among 109 samples sent for analysis, flow cytometric detection of major leukocyte subsets was possible in 78% (85 out of 109), which exceeds the 31% rate of our retrospective microscopic pediatric control group. Apart from physiologic lymphocytes (100%) or monocytes (48%), 11 out of these 85 samples showed granulocytes, two showed proliferated monocytes, and nine displayed proliferated lymphocytes. In most children, the proliferated lymphocytes consisted of a polyclonal population of CD4+ and CD8+ T cells. Compared with literature data, eight children showed abnormally composed lymphocyte subsets (surface antigen expression) within the main lymphocyte population. However, none of these changes was specific for distinct diseases or allowed a distinction between patients with and without primary inflammatory processes. These data suggest that CSF FC may be the most effective modality to differentiate major CSF leukocyte subsets. At present, further differentiation of distinct cell populations, such as proliferated lymphocytes, is of limited clinical impact. This may, however, gain increasing interest in the future.     

Flow cytometric detection of micronuclei induced by chemicals in poly- and normochromatic erythrocytes of mouse peripheral blood

In order to standardize automated scoring for the in vivo micronucleus(MN) test a flow cytometric method which recognized micronucleatedpolychromatic erythrocytes (MPCE) and micronucleated normochromaticerythrocytes (MNCE) in mouse peripheral blood developed by Grawéet al. (1992) has been modified and applied. Blood samples werepurified with 35% percoll solution and stained with the RNA-specificdye thiazole orange (TO) and with the DNA-specific dye Hoechst33342 (HO) for dual laser flow cytometry. The TO fluorescentsignals permitted the discrimination between polychromatic andnormochromatic erythrocytes (PCE and NCE). Cytotoxic effectscould be assessed by the reduction of PCE counts. The blue fluorescentsignals of HO permitted the scoring of MN. The MPCE and MNCEwere flow sorted for microscopic analysis and showed that 95%of the sorted cells actually contained MN. Three model chemicals,the clastogen mitomycin C, the aneugen colchicine and the industrialchemical acrylamide were tested at 24 h intervals after singleintraperitoneal injection up to 72 h after treatment of male(l02/ElC3H/EI)F₁ mice. All three chemicals showed a dose-relatedmaximum of the MPCE frequencies at 48 h while the MNCE frequenciesstayed within the control range up to 72 h. The data obtainedwith the flow cytometric method were in good agreement withpublished results. The flow cytometric technique presented hereis a fast, accurate and automated method for quantifying MPCEand MNCE in peripheral blood as an indicator of cytogeneticdamage induced hi the bone marrow and scored in peripheral bloodsamples. With minor modifications the technique will also beapplicable to bone marrow samples.     

Flow cytometric DNA analysis of pediatric solid tumor

Ninety nine case of various solid tumors in children were clinically and histologically investigated and the findings were compared with the data obtained by flow-cytometric DNA analysis using paraffin-embedded tumor tissue. Most of the embryonal tumors such as neuroblastoma, nephroblastoma, or medulloblastoma tended to show a diploid DNA stemline (65.2%) in comparison with non-embryonal tumors (45.2%) (p less than 0.025). Aneuploid cases of embryonal tumors in children disclosed histologically rather differentiated appearances, and with a favorable clinical outcome. On the other hand, non-embryonal tumors in children revealed in general an aneuploid DNA stemline and higher rate in high grade malignancy, resembling that of the malignant neoplasms in adult. These prominent features of the flow-cytometric DNA analysis of the embryonal tumors in children suggest that there are some differences in the tumor cytogenetics or histogenesis between the embryonal tumors and the non-embryonal tumors.     

Flow cytometric analysis of endometrial stromal sarcoma.

Twenty-two endometrial stromal sarcomas were studied by flow cytometric analysis and the results were correlated with surgical stage, nuclear grade, mitotic index, and recurrence. Ploidy determination was not helpful in predicting recurrence in patients with Stage I disease because all 14 were diploid. Only 2 of the 22 tumors were aneuploid; both were high-stage neoplasms. Cell proliferation (%S or %S + %G2/M) did not significantly correlate with vascular invasion, nuclear grade, mitotic index, or surgical stage. Of the 13 Stage I neoplasms with follow-up data, three recurred. The mean proliferation index (%S + %G2/M) of those that recurred was 12.81 +/- 0.47, which did not differ from those that did not recur (mean 12.25 +/- 4.11).     

Flow cytometric analysis of cytokine receptor signal transduction

Cytokines are critical regulators of the development and maturation of hematopoietic cells. Signal transduction via cytokine receptors proceeds through activation of the JAK-STAT pathway to stimulate cell proliferation, differentiation and effector functions. Phosphorylation of intracellular STAT molecules by the receptor-associated JAK kinases is one of the very early events following cytokine stimulation. Western blot detection of tyrosine phosphorylated STAT molecules is widely used as a hallmark of cytokine receptor activation. However, this is not feasible when cells of interest are limiting, or represent a small fraction within a mixed population of different cell types. To circumvent this technical obstacle, we have developed techniques to detect phosphorylated STAT molecules in fixed cells by flow cytometry. The fixation and permeabilization protocols preserve the antigenicity of cell surface markers allowing us to distinguish distinct cell populations responding to cytokine stimulation. In this report, we demonstrate the use of this technique to rapidly and reliably identify, and quantify thymocyte subsets activated by interleukin-7. We envisage that this technique will find wide application in studying cytokine receptor signal transduction, particularly in identifying cytokine-dependent developmental checkpoints during hematopoiesis.     

Flow cytometric analysis of DNA in diagnostic cytology

Flow cytometric analysis of nuclear DNA content and cell surface immunologic phenotyping were used in the evaluation of cytologic samples obtained from four patients. In each sample, a lymphoid cell population was present, which was difficult to evaluate by traditional cytopathologic methods. In two of the cases, the flow cytometric demonstration of monoclonal populations of lymphoid cells characterized by abnormal amounts of nuclear DNA gave support to the cytologic interpretation of malignancy. In a third sample, a lymphoid cell population that could not be cytologically distinguished from malignant lymphoma cells of small lymphoid cell type was shown to be composed of euploid, polyclonal cells. In the fourth case, the demonstration of euploidy in morphologically distinctive cell populations was helpful in interpreting the fine-needle aspirate of the thyroid gland. The authors conclude that flow cytometry can be used to great advantage in the evaluation of cytologic samples as well as in predicting biologic behavior.     

Flow cytometric analysis of the LE cell phenomenon

A flow cytometry-based phagocytosis assay was developed and utilized to measure the LE cell phenomenon at the single cell level in vitro. Since the lupus erythematosus (LE) cell phenomenon is a special form of necro-phagocytosis in the presence of anti-dsDNA antibodies, dead substrate cells or chicken erythrocytes nuclei (CEN) served as targets that were labeled with propidiumiodide (PI). Phagocytes (PMN) were stained by anti-CD45 mAb FITC. After co-incubation phagocytosis was measured by flow cytometry. Flow cytometric analysis enabled the discrimination between PI+/CD45- targets, PI-/CD45+ phagocytes, and PI+/CD45+ phagocytes with engulfed targets. Maintaining the samples on ice significantly reduced the phagocytic uptake as compared to samples co-cultivated at 37 degrees C (p < 0.0002). The phagocytic up-take was lowest after substrate pre-treatment in normal serum as compared to samples with either no serum exposure or pre-treatment in LE-serum with anti-dsDNA antibodies (p < 0.05). Taken together, these data suggest the phagocytosis-based flow cytometry assay is suitable for analyzing the LE cell phenomenon. This method provides an interesting, simple and rapid new tool, and will possibly alleviate further studies on the LE cell phenomenon with modified cell models and/or conditions.     

Flow cytometric analysis of lymphoma and lymphoma-like disorders

The use of flow cytometry (FC) represents the most recent advance in the phenotypic analysis of lymphocyte subsets, and has emerged as a valuable adjunct in the diagnosis of malignant non-Hodgkin's lymphoma (NHL). In a review of over 200 cases of nodal and extranodal suspected lymphomas studied in the Immunophenotyping Laboratory at the University of New Mexico, the diagnostic utility of FC was assessed. Among cases of NHL, FC was able to confirm a morphologic diagnosis of lymphoma and determine B or T cell lineage in greater than 85% of the samples submitted. Difficulty in lineage determination in the remaining cases of morphologic NHL was multifactorial. Among cases of reactive lymph nodes and Hodgkin's disease, FC showed no characteristic patterns, although several cases exhibited phenotypic profiles suggestive of B or T cell clonality. When combined with routine morphologic review and accompanied by other specialized diagnostic techniques when necessary, the use of FC represents a precise and reproducible method for rapidly and easily studying lymphoproliferative disorders in solid tissue.     

Flow cytometric analysis of 68 monoclonal anti-D reagents

68 monoclonal IgG anti-D were assessed for quantitative and qualitative binding to D variant red cells using a flow cytometric indirect immunofluorescence test. One antibody failed to bind to normal RhD pos. cells, but reacted weakly with D cat VI and D Cat VII cells. Quantitatively, binding varied approximately twenty-fold between different MAbs and different D variants. D cat III cells gave the lowest level of binding when compared with the D pos. controls. Qualitatively, R₁^VIr, R₂^VIr, R₁^VIIr and DFR samples failed to react with various MAbs regardless of the levels of binding of these MAbs to the D pos. controls, thus supporting the idea of missing epitopes in such variants.