Detection of Abundant Non-Haematopoietic Circulating Cancer-Related Cells in Patients with Advanced Epithelial Ovarian Cancer

Current diagnosis and staging of advanced epithelial ovarian cancer (aEOC) has important limitations and better biomarkers are needed. We investigate the performance of non-haematopoietic circulating cells (CCs) at the time of disease presentation and relapse. Methods: Venous blood was collected prospectively from 37 aEOC patients and 39 volunteers. CCs were evaluated using ImageStream TechnologyTM and specific antibodies to differentiate epithelial cells from haematopoetic cells. qRT-PCR from whole blood of relapsed aEOC patients was carried out for biomarker discovery. Results: Significant numbers of CCs (CK+/WT1+/CD45−) were identified, quantified and characterised from aEOC patients compared to volunteers. CCs are abundant in women with newly diagnosed aEOC, prior to any treatment. Evaluation of RNA from the CCs in relapsed aEOC patients (n = 5) against a 79-gene panel revealed several differentially expressed genes compared to volunteers (n = 14). Size differentiation of CCs versus CD45+ haematopoietic cells was not reliable. Conclusion: CCs of non-haematopoetic origin are prevalent, particularly in patients with newly diagnosed aEOC. Exploiting a CC-rich population in aEOC patients offers insights into a part of the circulating microenvironment.GRACE; CTRT; The Inman Charity; BIOCEV from the ERD

Evaluation of natural and tracer fluorescent emission methods for droplet size measurements in a diesel spray

The final publication is available at Springer via http://dx.doi.org/10.1007/s12239-012-0070-zSpray sizing that records fluorescent emission and scattered light has been widely applied to spray diagnostics over the last two decades. Different experimental strategies have been developed, but comparing the different solutions offered has remained of interest to experimentalists. In this work, a comparison of two fluorescence strategies for measuring droplet size in the liquid phase of a last-generation DI diesel spray is conducted. The natural fluorescent emission of a commercial diesel fuel and the fluorescence emitted by a tracer (Rhodamine B) are compared using theoretical and experimental approaches. The LIF/Mie ratio commonly called Planar Droplet Sizing (PDS) technique is applied in two different ways to elucidate the possible advantages of using a fluorescent dopant. The sprays were injected under non-evaporative conditions into a constant pressure vessel that simulates densities present at the moment of injection in currently used passenger car diesel engines. Characterization of the signal properties was performed by measuring the absorption coefficient, fluorescence emission spectrum, quantum yield and lifetime of both configurations. The scattered light and fluorescence intensities were calculated to verify the dependencies of the droplet surface and volume. When applying the two techniques to quantify droplet size in dense diesel sprays, the results show that signal weakness and lack of control over the properties of natural fluorescence produce distortion in the shape of the spray and cause measurements to be unreliable. © 2012 The Korean Society of Automotive Engineers and Springer-Verlag Berlin Heidelberg.This research has been funded in the frame of the project PROFUEL reference TRA2011-26293 from Ministerio de Ciencia e Innovacion. The injectors are part of the ECN international project.Pastor Soriano, JV.; Payri, R.; Salavert Fernandez, J.; Manin, J. (2012). Evaluation of natural and tracer fluorescent emission methods for droplet size measurements in a diesel spray. International Journal of Automotive Technology. 13(5):713-724. https://doi.org/10.1007/s12239-012-0070-zS713724135Albrecht, H. E., Damaschke, N., Borys, M. and Tropea, C. (2003). Laser Doppler and Phase Doppler Measurement Techniques. Springer. Berlin.Barnes, M. D., Whitten, W. B. and Ramsey, J. M. (1994). Enhanced fluorescence yields through cavity quantumelectrodynamic effects in microdroplets. J. Optical Society of America B 11,7, 1297–1304.Benajes, J., Molina, S., Novella, R., Amorim, R., Ben Hadj Hamouda, H. and Hardy, J. (2010). Comparison of two injection systems in an HSDI diesel engine using split injection and different injector nozzles. Int. J. Automotive Technology 11,2, 139–146.Charalampous, G. and Hardalupas, Y. (2011). Method to reduce errors of droplet sizing based on the ratio of fluorescent and scattered light intensities (laser-induced fluorescence/Mie technique). Applied Optics, 50, 3622–3637.Chen, G., Mazumder, M., Chang, R. K., Swindal, J. C. and Acker, W. P. (1996). Laser diagnostics for droplet characterization: Application of morphology dependent resonances. Progress in Energy and Combustion Science 22,2, 163–188.Desantes, J. M., Payri, R., Garcia, J. M. and Salvador, F. J. (2007). A contribution to the understanding of isothermal diesel spray dynamics. Fuel 86,7–8, 1093–1101.Domann, R. and Hardalupas, Y. A. (2000). Study of parameters that influence the accuracy of the planar droplet sizing (PDS) technique. Part. Part. Syst. Charact. 3–11.Domann, R. and Hardalupas, Y. A. (2001). Spatial distribution of fluorescence within large doplets and its dependence on dye concentration. Applied Optics 40,21, 3586–3597.Domann, R. and Hardalupas, Y. A. (2002). Quantitative measurement of planar droplet sauter mean diameter in sprays using planar droplet sizing. 11th Int. Symp. Application of Laser Techniques to Fluid Mechanics, Lisbon, Portugal.Eckbreth, A. C. (1988). Laser Diagnostics for Combustion Species and Temperature. Abacus. Cambridge. Mass.Greenhalgh, D. A. (1999). Planar measurements of fuel vapour, liquid fuel, liquid droplet size and soot. Planar Optical Measurement Methods for Gas Turbine Components, 1–7.Im, K., Lin, K., Lai, M. and Chon, M. (2011). Breakup modeling of a liquid jet in cross flow. Int. J. Automotive Technology 12,4, 489–496.Jermy, M. C. and Greenhalgh, D. A. (2000). Planar dropsizing by elastic and fluorescence scattering in sprays too dense for phase doppler measurement. Appl. Phys. B, 71, 703–710.Kim, Y., Kim, K. and Lee, K. (2011). Effect of a 2-stage injection strategy on the combustion and flame characteristics in a PCCI engine. Int. J. Automotive Technology 12,5, 639–644.Ko, F. H., Weng, L. Y., Ko, C. J. and Chu, T. C. (2006). Characterization of imprinting polymeric temperature variation with fluorescent Rhodamine B molecule. Microelectronic Engineering, 83, 864–868.Lakowicz, J. R. (2006). Principles of Fluorescence Spectroscopy. 3rd Edn. Springer.Lee, S. H., Teong, J., Lee, J. T., Ryou, H. S. and Hong, K. (2005). Investigation on spray characteristics under ultrahigh injection pressure conditions. Int. J. Automotive Technology 6,2, 125–131.Lee, B., Song, J., Chang, Y. and Jeon, C. (2010). Effect of the number of fuel injector holes on characteristics of combustion and emissions in a diesel engine. Int. J. Automotive Technology 11,6, 783–791.LeGal, P., Farrugia, N. and Greenhalgh, D. A. (1999). Laser sheet dropsizing of dense sprays. Optics and Laser Techn., 31, 75–83.Lockett, R. D., Richter, J. and Greenhalgh, D. A. (1998). The characterisation of a diesel spray using combined laser induced fluorescence and laser sheet dropsizing. Conf. Lasers and Electro-Optics Europe.Magde, D., Rojas, G. E. and Seybold, P. (1999). Solvent dependence of the fluorescence lifetimes of xanthene dyes. Photochem. Photobiol., 70, 737.Naber, J. and Siebers, D. (1996). Effects of gas density and vaporization on penetration and dispersion of diesel sprays. SAE Paper No. 960034.Pastor, J. V., López, J. J., Juliá, J. E. and Benajes, J. V. (2002). Planar laser-induced fluorescence fuel concentration measurements in isothermal diesel sprays. Opt. Express 10,7, 309–323.Pastor, J. V., Payri, R., Araneo, L. and Manin, J. (2009). Correction method for droplet sizing by laser-induced fluorescence in a controlled test situation. Optical Engineering 48,1, 013601.Payri, R., Garcia, J. M., Salvador, F. J. and Gimeno, J. (2005a). Using spray momentum flux measurements to understand the influence of diesel nozzle geometry on spray characteristics. Fuel, 84, 551–561.Payri, R., Salvador, F. J., Gimeno, J. and Soare, V. (2005b). Determination of diesel sprays characteristics in real engine in-cylinder air density and pressure conditions. J. Mech. Sci. Technol., 19, 2040–2052.Payri, R., Tormos, B., Salvador, F. J. and Araneo, L. (2008). Spray droplet velocity characterization for convergent nozzles with three different diameters. Fuel 87,15, 3176–3182.Payri, F., Pastor, J., Payri, R. and Manin, J. (2011). Determination of the optical depth of a DI diesel spray. J. Mech. Sci. Technol., 25, 209–219.Potz, D., Chirst, W. and Dittus, B. (2000). Diesel nozzle: The determining interface between injection system and combustion chamber. Conf. Thermo and Fluid-dynamic Processes in Diesel Engines, Valencia, Spain.Ramírez, A. I., Som, S., Aggarwal, S. K., Kastengren, A. L., El-Hannouny, E. M., Longman, D. E. and Powell, C. F. (2009). Quantitative X-ray measurements of highpressure fuel sprays from a production heavy duty diesel injector. Experiments in Fluids 47,1, 119–134.Schulz, C. and Sick, V. (2005). Tracer-LIF diagnostics: quantitative measurement of fuel concentration, temperature and fuel/air ratio in practical combustion systems. Progress in Energy and Combustion Science, 31, 75–121.Sjoback, R. and Nygren, J. and Kubista, M. (1998). Characterization of fluorescein—oligonucleotide conjugates and measurement of local electrostatic potential. Biopolymers, 46, 445–453.Soare, V. (2007). Phase Doppler Measurement in Diesel Dense Sprays: Optimisation of Measurements and Study of the Orifice Geometry Influence Over the Spray at Microscopic Level. Ph.D. Dissertion. E.T.S. Ingenieros Industriales. Universidad Politécnica de Valencia. Spain.Williams, A. T. R., Winfield, S. A. and Miller, J. N. (1983). Relative fluorescence quantum yields using a computer controlled luminescence spectrometer. Analyst., 108, 1067.Yeh, C. N., Kosaka, H. and Kamimoto, T. A. (1993). Fluorescence/scattering imaging technique for instantaneous 2-D measurements of particle size distribution in a transient spray. Proc. 3rd Cong. Opt. Part. Sizing, Yokohama, Japan, 335–361

Human Papillomavirus Type 16 Entry: Retrograde Cell Surface Transport along Actin-Rich Protrusions

The lateral mobility of individual, incoming human papillomavirus type 16 pseudoviruses (PsV) bound to live HeLa cells was studied by single particle tracking using fluorescence video microscopy. The trajectories were computationally analyzed in terms of diffusion rate and mode of motion as described by the moment scaling spectrum. Four distinct modes of mobility were seen: confined movement in small zones (30–60 nm in diameter), confined movement with a slow drift, fast random motion with transient confinement, and linear, directed movement for long distances. The directed movement was most prominent on actin-rich cell protrusions such as filopodia or retraction fibres, where the rate was similar to that measured for actin retrograde flow. It was, moreover, sensitive to perturbants of actin retrograde flow such as cytochalasin D, jasplakinolide, and blebbistatin. We found that transport along actin protrusions significantly enhanced HPV-16 infection in sparse tissue culture, cells suggesting a role for in vivo infection of basal keratinocytes during wound healing

Pre-analytical processes in medical diagnostics: New regulatory requirements and standards

In April 2017, the European In Vitro Diagnostic Regulation (IVDR) entered into force and will apply to in vitro diagnostics from May 26th, 2022. This will have a major impact on the in vitro diagnostics (IVD) industry as all devices falling under the scope of the IVDR will require new or re-certification. The IVDR also has implications with respect to product performance validation and verification including the pre-analytics of biological samples used by IVD developers and diagnostic service providers. In parallel to the IVDR, a series of standards on pre-analytical sample processing has been published by the International Organization for Standardization (ISO) and the European Committee for Standardization (CEN). These standards describe pre-analytical requirements for various types of analyses in various types of biospecimens. They are of immediate relevance for IVD product developers in the context of (re)certification under the IVDR and to some extent also to devices manufactured and used only within health institutions. This review highlights the background and the rationale for the pre-analytical standards. It describes the procedure that has led to these standards, the major implications of the standards and the requirements for pre-analytical workflows. In addition, it discusses the relationship between the standards and the IVDR