TY - JOUR A1 - Wang, L. A1 - Stebbings, R. A1 - Gaigalas, A.K. A1 - Sutherland, J. A1 - Kammel, M. A1 - John, M. A1 - Roemer, B. A1 - Kuhne, Maren A1 - Schneider, Rudolf A1 - Braun, M. A1 - Engel, A. A1 - Dikshit, D. A1 - Abbasi, F. A1 - Marti, G.E. A1 - Sassi, M. A1 - Revel, L. A1 - Kim, S.K. A1 - Baradez, M.-O. A1 - Lekishvili, T. A1 - Marshall, D. A1 - Whitby, L. A1 - Jing, W. A1 - Ost, V. A1 - Vonsky, M. A1 - Neukammer, J. T1 - Quantification of cells with specific phenotypes II: Determination of CD4 expression level on reconstituted lyophilized human PBMC labelled with anti-CD4 FITC antibody N2 - This report focuses on the characterization of CD4 expression level in terms of equivalent number of reference fluorophores (ERF). Twelve different flow cytometer platforms across sixteen laboratories were utilized in this study. As a first step the participants were asked to calibrate the fluorescein isothiocyanate (FITC) channel of each flow cytometer using commercially available calibration standard consisting of five populations of microspheres. Each population had an assigned value of equivalent fluorescein fluorophores (EFF denotes a special case of the generic term ERF with FITC as the reference fluorophore). The EFF values were assigned at the National Institute of Standards and Technology (NIST). A surface-labelled lyophilized cell preparation was provided by the National Institute of Biological Standards and Control (NIBSC), using human peripheral blood mononuclear cells (PBMC) pre-labeled with a FITC conjugated anti-CD4 monoclonal antibody. Three PBMC sample vials, provided to each participant, were used for the CD4 expression analysis. The PBMC are purported to have a fixed number of surface CD4 receptors. On the basis of the microsphere calibration, the EFF value of the PBMC samples was measured to characterize the population average CD4 expression level of the PBMC preparations. Both the results of data analysis performed by each participant and the results of centralized analysis of all participants' raw data are reported. Centralized analysis gave a mean EFF value of 22,300 and an uncertainty of 750, corresponding to 3.3% (level of confidence 68%) of the mean EFF value. The next step will entail the measurement of the ERF values of the lyophilized PBMC stained with labels for other fluorescence channels. The ultimate goal is to show that lyophilized PBMC is a suitable biological reference cell material for multicolor flow cytometry and that it can be used to present multicolor flow cytometry measurements in terms of ABC (antibodies bound per cell) units. KW - Surface labelled lyophilized PBMC KW - CD4 expression level KW - FITC KW - Equivalent fluorescein fluorophore (EFF) KW - Quantitative flow cytometry KW - Calibration KW - Standard measurement procedure KW - Measurement uncertainty KW - Reference cell material PY - 2015 U6 - https://doi.org/10.1002/cyto.a.22634 SN - 0196-4763 SN - 1552-4922 SN - 1552-4930 VL - 87 IS - 3 SP - 254 EP - 261 PB - Wiley-Liss CY - Hoboken, NJ AN - OPUS4-32981 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Stebbings, R. A1 - Wang, L. A1 - Sutherland, J. A1 - Kammel, M. A1 - Gaigalas, A.K. A1 - John, M. A1 - Roemer, B. A1 - Kuhne, Maren A1 - Schneider, Rudolf A1 - Braun, M. A1 - Engel, A. A1 - Dikshit, D.K. A1 - Abbasi, F. A1 - Marti, G.E. A1 - Sassi, M.P. A1 - Revel, L. A1 - Kim, S.-K. A1 - Baradez, M.-O. A1 - Lekishvili, T. A1 - Marshall, D. A1 - Whitby, L. A1 - Jing, W. A1 - Ost, V. A1 - Vonsky, M. A1 - Neukammer, J. T1 - Quantification of cells with specific phenotypes I: Determination of CD4+ cell count per microliter in reconstituted lyophilized human PBMC prelabeled with anti-CD4 FICT antibody N2 - A surface-labeled lyophilized lymphocyte (sLL) preparation has been developed using human peripheral blood mononuclear cells prelabeled with a fluorescein isothiocyanate conjugated anti-CD4 monoclonal antibody. The sLL preparation is intended to be used as a reference material for CD4+ cell counting including the development of higher order reference measurement procedures and has been evaluated in the pilot study CCQM-P102. This study was conducted across 16 laboratories from eight countries to assess the ability of participants to quantify the CD4+ cell count of this reference material and to document cross-laboratory variability plus associated measurement uncertainties. Twelve different flow cytometer platforms were evaluated using a standard protocol that included calibration beads used to obtain quantitative measurements of CD4+ T cell counts. There was good overall cross-platform and counting method agreement with a grand mean of the laboratory calculated means of (301.7 ± 4.9) µL-1 CD4+ cells. Excluding outliers, greater than 90% of participant data agreed within ±15%. A major contribution to variation of sLL CD4+ cell counts was tube to tube variation of the calibration beads, amounting to an uncertainty of 3.6%. Variation due to preparative steps equated to an uncertainty of 2.6%. There was no reduction in variability when data files were centrally reanalyzed. Remaining variation was attributed to instrument specific differences. CD4+ cell counts obtained in CCQM-P102 are in excellent agreement and show the robustness of both the measurements and the data analysis and hence the suitability of sLL as a reference material for interlaboratory comparisons and external quality assessment. KW - CD4+ cell counting KW - Relative concentration measurement KW - Lyophilized cells KW - Flow cytometry KW - Standard measurement procedure KW - Measurement of uncertainty KW - Human immunodeficiency virus-1 KW - Acquired immunodeficiency syndrome KW - Reference material PY - 2015 U6 - https://doi.org/10.1002/cyto.a.22614 SN - 0196-4763 SN - 1552-4922 SN - 1552-4930 VL - 87 IS - 3 SP - 244 EP - 253 PB - Wiley-Liss CY - Hoboken, NJ AN - OPUS4-32847 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Resch-Genger, Ute A1 - Hoffmann, Katrin A1 - Würth, Christian A1 - Behnke, Thomas A1 - Hoffmann, Angelika A1 - Pfeifer, Dietmar A1 - Engel, A. T1 - The toolbox of fluorescence standards: Flexible calibration tools for the standardization of fluorescence-based measurements N2 - To improve the reliability of fluorescence data in the life and material sciences and to enable accreditation of fluorescence techniques, standardization concepts are required that guarantee and improve the comparability of fluorescence measurements. At the core of such concepts are simple and evaluated fluorescence standards for the consideration of instrument-specific spectral and intensity distortions of measured signals and for instrument performance validation (IPV). Similarly in need are fluorescence intensity standards for the quantification from measured intensities and for signal referencing, thereby accounting for excitation light-induced intensity fluctuations. These standards should be preferably certified, especially for use in regulated areas like medical diagnostics. This encouraged us to develop liquid and solid standards for different fluorescence parameters and techniques for use under routine measurement conditions in different formates. Special emphasis was dedicated to the determination and control of the spectral responsivity of detection systems, wavelength accuracy, homogeneity of illumination, and intensity referencing for e.g. spectrofluorometers, fluorescence sensors and confocal laser scanning fluorescence microscopes. Here, we will present design concepts and examples for mono- and multifunctional fluorescence standards that provide traceability to radiometric units and present a first step towards a toolbox of standards. KW - Fluorescence KW - Fluorescence standard KW - Calibration tool KW - Spectral fluorescence standard KW - Intensity standard KW - Instrument performance validation KW - Quality assurance KW - Traceability KW - Glass KW - Liquid standard PY - 2010 U6 - https://doi.org/10.1117/12.853133 SN - 0277-786X SN - 0038-7355 SN - 0361-0748 VL - 7666 IS - 76661J SP - 1 EP - 12 PB - Soc. CY - Redondo Beach, Calif. [u.a.] AN - OPUS4-21595 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Resch-Genger, Ute A1 - Hoffmann, Katrin A1 - Nietfeld, W. A1 - Engel, A. A1 - Neukammer, J. A1 - Nitschke, R. A1 - Ebert, B. A1 - Macdonald, R. T1 - How to Improve Quality Assurance in Fluorometry: Fluorescence-Inherent Sources of Error and Suited Fluorescence Standards N2 - The scope of this paper is to illustrate the need for an improved quality assurance in fluorometry. For this purpose, instrumental sources of error and their influences on the reliability and comparability of fluorescence data are highlighted for frequently used photoluminescence techniques ranging from conventional macro- and microfluorometry over fluorescence microscopy and flow cytometry to microarray technology as well as in vivo fluorescence imaging. Particularly, the need for and requirements on fluorescence standards for the characterization and performance validation of fluorescence instruments, to enhance the comparability of fluorescence data, and to enable quantitative fluorescence analysis are discussed. Special emphasis is dedicated to spectral fluorescence standards and fluorescence intensity standards. KW - Fluorescence KW - Standard KW - Calibration KW - Microarray KW - In vivo imaging KW - Flow cytometry PY - 2005 U6 - https://doi.org/10.1007/s10895-005-2630-3 SN - 1053-0509 SN - 1573-4994 VL - 15 IS - 3 SP - 337 EP - 362 PB - Plenum Publ. Corp. CY - New York, NY AN - OPUS4-10823 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Hoffmann, Katrin A1 - Resch-Genger, Ute A1 - Engel, A. T1 - Glass-based reference standards for fluorescence applications KW - Fluorescence standard KW - Luminophore-doped glass KW - Reference material KW - Instrument qualification PY - 2008 SN - 1522-7235 SN - 1099-1271 SN - 1522-7243 VL - 23 SP - 229 EP - 230 PB - Wiley CY - Chichester AN - OPUS4-18256 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Engel, A. A1 - Ottermann, C. A1 - Resch-Genger, Ute A1 - Hoffmann, Katrin A1 - Schweizer, S. A1 - Selling, J. A1 - Spaeth, J.-M. A1 - Rupertus, V. ED - Grzymala, R. ED - Haeberlé, O. T1 - Glass based fluorescence reference materials used for optical and biophotonic applications N2 - Fluorescence techniques are known for their high sensitivity and are widely used as analytical tools and detection methods for product and process control, material sciences, environmental and biotechnical analysis, molecular genetics, cell biology, medical diagnostics, and drug screening. For routine measurements by fluorescence techniques the existence of an improved quality assurance is one of the basic needs. According to DIN/ISO 17025 certified standards are used for fluorescence diagnostics having the drawback of giving relative values only. Typical requirements onto fluorescence reference materials or standards deal with the verification of the instrument performance as well as the improvement of the data comparability. Especially for biomedical applications fluorescence labels are used for the detection of proteins. In particular these labels consist of nano crystalline materials like CdS and CdSe. The field of Non-Cadmium containing materials is under investigation. In order to evaluate whether glass based materials can be used as standards it is necessary to calculate absolute values like absorption/excitation cross sections or relative quantum yields. This can be done using different quantities of dopands in glass, glass ceramics or crystals. The investigated materials are based on different types of glass, silicate, phosphate and boron glass, which play a dominant role for the absorption and emission mechanism. Additional to the so-called elementary fluorescence properties induced by raw earth elements the formation of defects lead to higher cross sections additionally. The main investigations deal with wavelength accuracy and lifetime of doped glasses, glass ceramics and crystalline samples. Moreover intensity patterns, homogeneity aspects and photo stability will be discussed. KW - Fluorescence KW - Reference material KW - Glass KW - Glass ceramics KW - Biophotonic KW - Multiplexing PY - 2006 U6 - https://doi.org/10.1117/12.663627 SN - 1605-7422 VL - 6191 SP - 619110-1-619110-10 AN - OPUS4-14429 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Engel, A. A1 - Ottermann, C. A1 - Klahn, J. A1 - Korb, T. A1 - Resch-Genger, Ute A1 - Hoffmann, Katrin A1 - Kynast, U. A1 - Rupertus, V. ED - Dan V. Nicolau, ED - Daniel L. Farkas, ED - Robert C. Leif, T1 - Anorganic fluorescence reference materials for decay time of fluorescence emission N2 - Fluorescence techniques are known for their high sensitivity and are widely used as analytical tools, detection methods and imaging applications for product and process control, material sciences, environmental and bio-technical analysis, molecular genetics, cell biology, medical diagnostics, and drug screening. According to DIN/ISO 17025 certified standards are used for steady state fluorescence diagnostics, a method having the drawback of giving relative values for fluorescence intensities only. Therefore reference materials for a quantitative characterization have to be related directly to the materials under investigation. In order to evaluate these figures it is necessary to calculate absolute numbers such as absorption/excitation cross sections and quantum yield. This has been done for different types of dopands in different materials such as glass, glass ceramics, crystals or nano crystalline material embedded in polymer matrices. Samples doped with several fluophores of different emission wavelengths and decay times are required for fluorescent multiplexing applications. Decay times shorter than 100 ns are of special interest. In addition, a proper knowledge is necessary of quantum efficiency in highly scattering media. Recently, quantum efficiency in YAG:Ce glass ceramics has been successfully investigated. Glass and glass ceramics doped with threefold charged rare earth elements are available. However, these samples have the disadvantage of emission decay times much longer than 1 microsecond, due to the excitation and emission of their optical forbidden electronic transitions. Therefore first attempts have been made to produce decay-time standards based on organic and inorganic fluophores. Stable LUMOGEN RED pigments and YAG:Ce phosphors are diluted simultaneously in silicone matrices using a wide range of concentrations between 0.0001 and 2 wt%. Organic LUMOGEN RED has decay times in the lower nanosecond range with a slight dependency on concentration and temperature. In addition, the well-known decay properties of inorganic YAG:Ce are observed also embedded in silicone matrix. Luminescent silicone layers are obtained with thicknesses between 150 and 300 m and no change of decay time, which has been determined to be between 60 and 62 ns. Finally, first results are shown for fluorescent CaF2:Pb glass ceramics embedded in a silicate glass matrix. Wavelength accuracy and lifetime are characterized for different environmental conditions such as temperature treatment and UV irradiation. Moreover, intensity patterns, e.g. line profiles and results, are discussed on homogeneity and photo and thermal stability, respectively. Fluorescence (steady state, decay time) and absorption (remission, absorption) spectroscopy are employed as diagnostic methods to get a microscopic view of the relevant physical processes. The work is funded by BMBF under project number 13N8849. KW - Fluorescence KW - Reference material KW - Glass KW - Glass ceramic KW - Phosphor KW - Doped glass and glass ceramics PY - 2008 SN - 978-0-8194-7034-8 U6 - https://doi.org/10.1117/12.767633 VL - 6859 SP - 68591A-1 - 68591A-10 PB - SPIE, the International Society for Optical Engineering CY - Bellingham, Wash. AN - OPUS4-19800 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Engel, A. A1 - Ottermann, C. A1 - Klahn, J. A1 - Enseling, D. A1 - Korb, T. A1 - Resch-Genger, Ute A1 - Hoffmann, Katrin A1 - Schweizer, S. A1 - Selling, J. A1 - Kynast, U. A1 - Koberling, F. A1 - Rupertus, V. T1 - Fluorescence reference materials used for optical and biophotonic applications N2 - Fluorescence techniques are known for their high sensitivity and are widely used as analytical tools and detection methods for product and process control, material sciences, environmental and bio-technical analysis, molecular genetics, cell biology, medical diagnostics, and drug screening. According to DIN/ISO 17025 certified standards are used for fluorescence diagnostics having the drawback of giving relative values for fluorescence intensities only. Therefore reference materials for a quantitative characterization have to be related directly to the materials under investigation. In order to evaluate these figures it is necessary to calculate absolute numbers like absorption/excitation cross sections and quantum yield. This can be done for different types of dopands in different materials like glass, glass ceramics, crystals or nano crystalline material embedded in polymer matrices. Based on the optical spectroscopy data we will discuss options for characteristic doped glasses and glass ceramics with respect to scattering and absorption regime. It has shown recently for YAG:Ce glass ceramics that for a proper determination of the quantum efficiency in these highly scattering media a reference material with similar scattering and fluorescent properties is required. This may be performed using the emission decay measurement diagnostics, where the decay time is below 100 ns. In this paper we present first results of these aspects using well performing LUMOGEN RED organic pigments for a comparison of mainly transparent glass with glass ceramics doped with various amounts of dopands e.g. ions of raw earth elements and transition metals. The LUMOGEN red is embedded in silica and polyurethane matrices. Characterisations on wavelength accuracy and lifetime for different environmental conditions (temperature, UV irradiation) have been performed. Moreover intensity patterns and results for homogeneity, isotropy, photo and thermal stability will be discussed. In a next step we will show the transfer of the characterisation methods to inorganic fluophores (YAG:Ce) in silicon. Fluorescence (steady state, decay time) and absorption (remission, absorption) spectroscopy working in different temperature regimes (10 - 350 K) are employed diagnostic methods in order to get a microscopic view of the relevant physical processes and to prove the correctness of the obtained data. The work is funded by BMBF under project number 13N8849. KW - Fluorescence KW - Reference material KW - Glass KW - Glass ceramic KW - Phosphor KW - Doped glass KW - Glass ceramics PY - 2007 SN - 0-8194-6247-0 U6 - https://doi.org/10.1117/12.728144 SN - 1605-7422 VL - 6628 SP - 662815-1 - 662815-9 AN - OPUS4-16729 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Engel, A. A1 - Resch-Genger, Ute A1 - Hoffmann, Katrin A1 - Pfeifer, L. A1 - Seydack, M. A1 - Nording, P. A1 - Enderlein, M. A1 - Nann, T. A1 - Koberling, F. T1 - Gesundheitstag durch Fluoreszenz-Multiplexing - FLUOPLEX T2 - Biophotonik Symposium, Klinikum der Universität München CY - Munich, Germany DA - 2006-04-27 PY - 2006 AN - OPUS4-12593 LA - deu AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER -