[en] I will present a microfluidic imaging flow cytometer incorporating stroboscopic illumination, for blur-free cellular analysis at throughputs exceeding 100,000 cells per second. By combining passive (inertial or viscoelastic) focusing of cells in parallel microchannels with stroboscopic illumination, such chip-based cytometers are able to extract multi-colour fluorescence and bright-field images of single cells moving at high linear velocities. This in turn allows accurate sizing of individual cells, intracellular localization and analysis of heterogeneous cell suspensions. The method is showcased through the rapid enumeration of apoptotic cells, high-throughput discrimination cell cycle phases and localization of p-bodies.

[en] The mechanisms used by murine polyomavirus for intracellular migration are yet to be clarified. In this work we selectively depolymerized microtubules or actin fibers and then studied the progression of polyomavirus infection in cultured cells. Our results demonstrate that microtubule depolymerization prevents polyomavirus migration toward the nucleus and from the nucleus to the cell surface, being also involved in viral release, while disruption of the actin microfilaments appears to have no detrimental effect on the virus ability to reach the nucleus. The ultrastructural observation of polyomavirus nonenveloped particles interacting with the free end and the lateral sides of microtubules together with the coimmunoprecipitation of tubulin and viral VP-1 further supports the idea that polyomavirus intracellular migration seems to be mediated by the interaction of polyomavirus major capsid protein VP-1 with tubulin

[en] High content screening (HCS) is the convergence between cell-based assays, high-resolution fluorescence imaging, phase-contrast imaging of fixed- or live-cell assays, tissues and small organisms. It has been widely adopted in the pharmaceutical and biotech industries for target identification and validation and as secondary screens to reveal potential toxicities or to elucidate a drugs mechanism of action. By using the ImageXpress® Micro XLS System HCS, the complex network of key players controlling proliferation and apoptosis can be reduced to several sentinel markers for analysis. Cell proliferation and apoptosis are two key areas in cell biology and drug discovery research. Understanding the signaling pathways in cell proliferation and apoptosis is important for new therapeutic discovery because the imbalance between these two events is predominant in the progression of many human diseases, including cancer. The DNA binding dye DAPI is used to determine the nuclear size and nuclear morphology as well as cell cycle phases by DNA content. Images together with MetaXpress® analysis results provide a convenient and easy to use solution to high volume image management. In particular, HCS platform is beginning to have an important impact on early drug discovery, basic research in systems cell biology, and is expected to play a role in personalized medicine or revealing off-target drug effects. (author)

[en] A preliminary method is reported of alkaline unwinding of DNA within single cells and quantitation of the single-stranded and double-stranded DNA with the fluorescent probe acridine orange. A suspension of alkali-treated cells is obtained and analysed by flow cytometry. An increase in the amount of single-stranded DNA is taken as an indication of strand breaks. An advantage of this method is that a large number of cells can be individually analysed for DNA strand breaks. A measurement of DNA content is also obtained, making it possible to discriminate between cells in various parts of the cell cycle. (author)

[en] Phenotypic analysis of blood malignancies by flow cytometry is applied in onco-hematology in Slovakia since the 90's last century. Flow cytometry technique is still an important basic research tool used for the detection of phenotypic cell population heterogeneity of bone marrow in order to improve diagnosis and monitoring of hematological malignancies. Nowadays the flow cytometers equipped with three lasers represent standard accessories in clinical laboratories, providing analysis of 8 fluorescent and 2 physical parameters at a single cell level. Major breakthrough in cytometry represents the combination of flow and fluorescence microscopy methods unified in ImageStreamX flow cytometer. This technical innovation allows analysis of the fluorescence parameters as well as analysis of cell morphology, cell signaling by the location of molecules of interest within the cell or cell-cell interactions. This article presents the analysis of erythroid precursors in regenerating bone marrow using flow and ImageStreamX cytometry techniques. (author)

[en] Microbial deposits (slime or biofilm) formation is one of the most important problems in the paper industry. Slime formation cause a reduction of the final product quality (spots, holes, odors), as well as in the production and equipment life, due to a greater number of web breaks, down time for cleaning and maintenance of the machinery, corrosion, etc. Microbiologically-induced corrosion cause and important economic losses due to the reduction of the equipment life. In microbially-induced corrosion studies is basic the rapid and precise determination of the evolution of slime growth on metallic surfaces. Thus, the goal of the present work have been the development of a methodology that leads to characterize the population of aerobic bacteria that compose the slimes of a board mill by means of multi parametric flow cytometry, using two different angles of light scattering and the total protein content as parameters as first step of the studies of microbiologically-induced corrosion. (Author) 13 refs

[en] To evaluate the immunophenotype of acute leukemia patients, the surface and cytoplasmic antigen expression in 162 cases of acute leukemia were analyzed by multiparameter flow cytometry and CD45/SSC gating. The results showed that CDl17 (94.9%), CD13 (88.5%) and CD33(70.5%) were mainly expressed in ANLL patients; cCD79a(100%), CD19(92.1%) were chiefly expressed in B-ALL patients, and in T-ALL patients, cCD3(100%) and CD2(83.3%) were expressed; For the expression of lymphoid differentiation antigen Ly+ANLL, CD7 (56.2%) and CD19(31.2%) were chiefly found, and for myeloid antigen My+ALL, CD13(88. 9%) and CD33 (27.8%) were detected. In conclusion, multiparameter flow cytometry and three-color direct immunofluorescence staining methods may be of important clinical significance in diagnosis, therapy and prognosis of acute leukemia. (authors)

[en] Purpose: An assay for radiosensitivity has numerous applications in the clinic. Avoidance of acute responses, prediction of normal tissue toxicity, and individualization of patient radiotherapy are included among these. We have developed a rapid assay (about 24 h) able to predict intrinsic radiosensitivity of CD4 and CD8 T-lymphocytes based on radiation-induced apoptosis. Methods and Materials: Fresh blood samples (1-2 ml in heparinized tubes) were irradiated with 0-, 2-, and 8-Gy X rays at a dose rate of approximately 3 Gy/min. Following irradiation, the cells were collected and prepared for flow-cytometric analysis and cell sorting. In conjunction with the CellQuest software available with the FACSVantage cell sorter (Becton-Dickinson), two T-lymphocyte types were analyzed on the basis of their cell-specific antigens (CD4 and CD8), and DNA was stained with DAPI. Following the separation of these cell types, radiation-induced cell death was assessed. Cytotoxicity was characterized by gradual degradation of internucleosomal DNA which results in a sub-G1 peak on the DNA histogram, and by the associated loss of surface antigens causing an intermediate positive peak in the antibody histogram. Using the assay, we investigated the interdonor variation in a cohort of 45 healthy adult blood donors and 5 children [one had immunodeficiency, centromeric instability, and facial anomalies syndrome (ICF), and one had ataxia telangiectasia (AT)]. Intradonor variation was assessed with 10 different experiments from a single donor. Results: CD4 and CD8 T-lymphocyte radiosensitivities were correlated (r 0.63 and 0.65 for 2 and 8 Gy, respectively) in 45 adult donors. Both for CD4 and CD8 cells, 2 and 8 Gy irradiation responses showed a good correlation (r 0.77 for both). Interdonor variation was significantly higher than intradonor variation (p < 0.0005) for all CD4 and CD8 data. We observed a decrease in the antigen fluorescence of dying cells, a phenomenon referred to as antigen-ebb. Antigen-ebb was clearly observed in both cell types, and correlated significantly with cytotoxicity. A trend was observed between radiosensitivity and donor age, but there was no correlation for gender. Blood from a 4-year-old girl presenting with ICF demonstrated compromised radiation-induced cytotoxicity in her CD4 T-lymphocytes, and an 11-year-old boy presenting with AT demonstrated compromised radiation-induced cytotoxicity in both his CD4 and CD8 T-lymphocytes. Conclusion: We conclude that the assay provides a rapid means of determining radiosensitivity, can discriminate differences in radiation-induced cytotoxicity between individuals, and can be used as a rapid screen for genetically hypersensitive patients. Antigen-ebb offers interesting possibilities for molecular biological investigations, permitting characterization and isolation of abnormal but vital cells in the absence of clastogenic agents

[en] Purpose: Up to 30% of lung cancers (Stage I) with the most favorable outcome recur within 5 years after surgery. This study reviews the pattern of failure after surgical resection in early lung cancers and determines whether flow cytometric DNA variables were prognostic indicators for survival, disease-free survival (DFS), or distant metastasis-free survival (DMFS). Methods and Materials: Pathologic specimens from 45 patients at The University of Texas M. D. Anderson Cancer Center who underwent surgical resection and mediastinal nodal dissection for stage I (AJCC) adenocarcinomas of the lung were analyzed by flow cytometry for DNA content. Survival was calculated by the method of Desu and Lee. Chi-square and cross tabulation were used in the analysis. Results: The mean age of the patients was 62 years, and 52.3% were male. All patients were clinical Stage I (T1-2 N0), Karnofsky performance status ≥70, and had a weight loss <10 lbs. Median overall survival (OS) and DFS were 50 months and 33 months, respectively. OS, DFS, and DMFS at 1, 3 and 5 years were 73%, 57%, and 35%; 63%, 53%, and 45%; and 67%, 56%, and 48%, respectively. Analysis of all 45 patients revealed 86% of patients developing brain metastasis had an abnormal DNA content ≥ 30%, whereas 4% of patients with brain metastasis had abnormal DNA content < 30% (p = 0.01). This correlation maintained significance when only pT1/2 lesions were analyzed. There was a significant statistical correlation between abnormal DNA and 5-year OS, with 74% OS for those with abnormal DNA < 30% vs. 42% for ≥ 30% (p = 0.036). The 5-year DFS for pT1/2 patients was significantly correlated with abnormal DNA content: 53% for patients with abnormal DNA < 30% vs. 17% for patients with abnormal DNA ≥ 30%, respectively (p = 0.03). Of those with %S fraction (%S) < 2, 13% failed locally compared to 41% of those with %S ≥ 2. There was a highly significant correlation between DNA index (DNAI) and aneuploid %S: 68% of patients with a DNAI ≥ 1.7 had ≥ 2.6 aneuploid %S, whereas only 13% of patients with DNAI ≥ 1.7 had aneuploid %S < 2.6. (p < 0.001). Grouping the percent of abnormal DNA and overall %S according to low vs. mixed vs. high values correlated with DFS (p = 0.02). Conclusions: This study confirms significant correlation between a high DNA index and a higher frequency of brain metastasis, as well as worse OS. Although DNA content variables were not predictive of recurrence at other sites, brain metastasis represents the worst outcome from distant metastasis. Further studies are needed, as well as prospective trials, for evaluating adjuvant therapy in patients with adverse DNA variables following complete surgical resection for early disease. If high-risk patients could be identified after resection, adjuvant therapy (chemotherapy or elective brain irradiation) could be administered

[en] The author reports a new physical configuration for flow cytometry that greatly increases the signal-to-noise ratio for right-angle-scattered light and also greatly eases the alignment requirements. The new technique views the scattered light that is trapped within the optical waveguide of the flow stream in air