Instrument setup
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This commentary discusses particularities of application of the EuroFlow standardization of flow cytometric analyses on three different flow cytometers. The EuroFlow consortium developed a fully standardized approach for flow cytometric immunophenotyping of hematological malignancies and primary immunodeficiencies. Standardized instrument setup is an essential part of EuroFlow standardization. Initially, the EuroFlow Consortium developed and optimized a step-by-step standard operating procedure (SOP) to setup 8-color BD FACSCanto II flow cytometer (Canto), with the later inclusion of Navios (Beckman Coulter) and BD FACSLyric (Lyric). Those SOPs were developed to enable standardized and fully comparable fluorescence measurements in the three flow cytometers. In Canto and Navios, mean fluorescence intensity (MFI) of a reference peak of Rainbow beads calibration particles is used to set up photomultiplier (PMT) voltages for each detector channel in individual instruments to reach the same MFI across distinct instruments. In turn, a new feature of Lyric instruments allows to share collection of attributes that are used to place the positive population at the same position among instruments in the form of assays, as one of its components integrated in the Cytometer Setup and Tracking (CS&T) module. The EuroFlow Lyric assays thus allow for standardized acquisition of 8-color EuroFlow panels on Lyric without the need to setup the PMT voltages on the individual instruments manually. In summary, the standardized instrument setup developed by EuroFlow enables cross-platform inter- and intra-laboratory standardization of flow cytometric measurements. This commentary provides a perspective on the modifications of the standardized EuroFlow instrument setup of Canto, Navios and Lyric instruments that are described in detail in individual instrument-specfic SOPs available at the EuroFlow website.
- MeSH
- imunofenotypizace přístrojové vybavení normy MeSH
- lidé MeSH
- průtoková cytometrie přístrojové vybavení normy MeSH
- referenční standardy MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
An original setup for analysis of glycated haemoglobin (HbA1c) in blood is reported. The construction employed a combination of the piezoelectric biosensor for glycated haemoglobin and the flow-through photometric sensor for total haemoglobin (Hb). The modification of gold electrodes with 3-aminophenylboronic acid (APBA) as a specific ligand was studied; the chemisorbed conjugate of APBA with a long-chain thiocompound provided the best affinity for HbA1c. The effect of various operating parameters, such as flow rate and instrumental setup, was optimised. The total haemoglobin content was analysed as absorbance of the haemoglobin-cyanide derivative at 540 nm. Only one standard (calibrator) diluted in various ratio was necessary for calibration and 1 microl of blood was sufficient for analysis. The full range of HbA1c content (4-15%) in blood can be analysed; the working ranges of total and glycated haemoglobin were 50-2000 and 10-90 microg/ml, respectively. The developed method was successfully evaluated on blood samples collected from diabetics.
Radiotherapy with protons and carbon ions enables to deliver dose distributions of high conformation to the target. Treatment with helium ions has been suggested due to their physical and biological advantages. A reliable benchmarking of the employed physics models with experimental data is required for treatment planning. However, experimental data for helium interactions is limited, in part due to the complexity and large size of conventional experimental setups. We present a novel method for the investigation of helium interactions with matter using miniaturized instrumentation based on highly integrated pixel detectors. The versatile setup consisted of a monitoring detector in front of the PMMA phantom of varying thickness and a detector stack for investigation of outgoing particles. The ion type downstream from the phantom was determined by high-resolution pattern recognition analysis of the single particle signals in the pixelated detectors. The fractions of helium and hydrogen ions behind the used targets were determined. As expected for the stable helium nucleus, only a minor decrease of the primary ion fluence along the target depth was found. E.g. the detected fraction of hydrogen ions on axis of a 220MeV/u 4He beam was below 6% behind 24.5cm of PMMA. Monte-Carlo simulations using Geant4 reproduce the experimental data on helium attenuation and yield of helium fragments qualitatively, but significant deviations were found for some combinations of target thickness and beam energy. The presented method is promising to contribute to the reduction of the uncertainty of treatment planning for helium ion radiotherapy.
- MeSH
- design vybavení MeSH
- fantomy radiodiagnostické MeSH
- helium terapeutické užití MeSH
- ionty terapeutické užití MeSH
- metoda Monte Carlo MeSH
- miniaturizace * přístrojové vybavení MeSH
- plánování radioterapie pomocí počítače přístrojové vybavení metody MeSH
- počítačová simulace MeSH
- polymethylmethakrylát MeSH
- radiometrie přístrojové vybavení MeSH
- radioterapie těžkými ionty přístrojové vybavení metody MeSH
- vodík MeSH
- Publikační typ
- časopisecké články MeSH
The EU-supported EuroFlow Consortium aimed at innovation and standardization of immunophenotyping for diagnosis and classification of hematological malignancies by introducing 8-color flow cytometry with fully standardized laboratory procedures and antibody panels in order to achieve maximally comparable results among different laboratories. This required the selection of optimal combinations of compatible fluorochromes and the design and evaluation of adequate standard operating procedures (SOPs) for instrument setup, fluorescence compensation and sample preparation. Additionally, we developed software tools for the evaluation of individual antibody reagents and antibody panels. Each section describes what has been evaluated experimentally versus adopted based on existing data and experience. Multicentric evaluation demonstrated high levels of reproducibility based on strict implementation of the EuroFlow SOPs and antibody panels. Overall, the 6 years of extensive collaborative experiments and the analysis of hundreds of cell samples of patients and healthy controls in the EuroFlow centers have provided for the first time laboratory protocols and software tools for fully standardized 8-color flow cytometric immunophenotyping of normal and malignant leukocytes in bone marrow and blood; this has yielded highly comparable data sets, which can be integrated in a single database.
- MeSH
- hematologické nádory diagnóza imunologie MeSH
- imunofenotypizace normy MeSH
- laboratoře normy MeSH
- lidé MeSH
- monoklonální protilátky diagnostické užití MeSH
- nádorové biomarkery imunologie MeSH
- prognóza MeSH
- průtoková cytometrie přístrojové vybavení metody normy MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
- Geografické názvy
- Evropa MeSH
A handheld Raman spectrometer (Ahura First Defender) was tested for the unambiguous identification of biomolecules (pure amino acids, carboxylic acids, saccharides and trehalose) in the solid state under outdoor conditions (including moderate climate conditions as well as cold temperatures and high altitudes). The biomolecules investigated represent important objects of interest for future exobiological missions. Repetitive measurements carried out under identical instrumental setups confirmed the excellent reliability of the Raman spectrometer. Raman bands are found at correct wavenumbers +/-3 cm(-1) compared with reference values. This testing represents the first step in a series of studies. In a preliminary, challenging investigation to determine the detection limit for glycine dispersed in a powdered gypsum matrix, 10% was the lowest content confirmed unambiguously. Clearly there is a need to investigate further the detection limits of Raman spectroscopic analyses of biomolecules in more complex samples, to demonstrate the usefulness or disqualify the use of this technique for more realistic outdoor situations, such as eventual future missions to Mars.
- MeSH
- aminokyseliny analýza MeSH
- exobiologie přístrojové vybavení metody MeSH
- kyseliny karboxylové analýza MeSH
- limita detekce MeSH
- nadmořská výška MeSH
- nízká teplota MeSH
- Ramanova spektroskopie metody MeSH
- sacharidy analýza MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
- práce podpořená grantem MeSH
A critical component of the EuroFlow standardization of leukemia/lymphoma immunophenotyping is instrument setup. Initially, the EuroFlow consortium developed a step-by-step standard operating protocol for instrument setup of ≥8-color flow cytometers that were available in 2006, when the EuroFlow activities started. Currently, there are 14 instruments from 9 manufacturers capable of 3-laser excitation and ≥8 color measurements. The specific adaptations required in the instrument set-up to enable them to acquire the standardized 8-color EuroFlow protocols are described here. Overall, all 14 instruments can be fitted with similar violet, blue and red lasers for simultaneous measurements of ≥8 fluorescent dyes. Since individual instruments differ both on their dynamic range (scale) and emission filters, it is not accurate to simply recalculate the target values to different scale, but adjustment of PMT voltages to a given emission filter and fluorochrome, is essential. For this purpose, EuroFlow has developed an approach using Type IIB (spectrally matching) particles to set-up standardized and fully comparable fluorescence measurements, in instruments from different manufacturers, as demonstrated here for the FACSCanto II, and Navios and MACSQuant flow cytometers. Data acquired after such adjustment on any of the tested cytometry platforms could be fully superimposed and therefore analyzed together. The proposed approach can be used to derive target values for any combination of spectrally distinct fluorochromes and any distinct emission filter of any new flow cytometry platform, which enables the measurement of the 8-color EuroFlow panels in a standardized way, by creating superimposable datafiles.
- MeSH
- hematologické nádory diagnóza MeSH
- imunofenotypizace přístrojové vybavení normy MeSH
- lidé MeSH
- průtoková cytometrie přístrojové vybavení normy MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Since publication in 2010 of the International Clinical Cytometry Society (ICCS) Consensus Guidelines for detection of Paroxysmal nocturnal hemoglobinuria (PNH) by flow cytometery, a great deal of work has been performed to develop, optimize, and validate a number of high-sensitivity assays to detect PNH phenotypes in both red blood cells (RBCs) and white blood cells (WBCs, neutrophils, and monocytes). This section (Part 2) of the updated ICCS PNH Consensus Guidelines will focus on specific instrument setup for these PNH assays, the identification and proper testing of appropriate antibody conjugates and combinations therof, and basic assay design. © 2017 International Clinical Cytometry Society.
- MeSH
- erytrocyty metabolismus MeSH
- konsensus MeSH
- leukocyty metabolismus MeSH
- lidé MeSH
- monocyty metabolismus MeSH
- neutrofily metabolismus MeSH
- paroxysmální hemoglobinurie diagnóza metabolismus MeSH
- počet leukocytů metody MeSH
- průtoková cytometrie normy MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
There is an agreement in the field that interlaboratory reproducibility of flow cytometry measurements as well as the whole studies might be improved by a consensual use of methodological approach. Typically, a consensus is made on a crucial markers needed in the immunostaining panel, sometimes on the particular fluorochrome conjugates and rarely on a complete set of methods for sample preparation. The term "standardization" is used to describe the complete set of methodical steps, while "harmonization" is used for partial agreement on the method. Standardization can provide a platform for improved reproducibility of cytometry results over prolonged periods of time, across different sites and across different instruments. For the purpose of structured discussion, several desired aims are described: common interpretation of the immunophenotype definition of a target subset, accurate quantification, reproducible pattern of a multicolor immunophenotype, and reproducible intensity of all measured parameters. An overview of how standardization was approached by several large consortia is provided: EuroFlow, The ONE Study, Human Immunology Project Consortium (HIPC), and several other groups. Their particular aims and the tools adopted to reach those aims are noted. How those standardization efforts were adopted in the field and how the resulting outcome was evaluated is reviewed. Multiple challenges in the instrument hardware design, instrument setup tools, reagent design, and quality features need to be addressed to achieve optimal standardization. Furthermore, the aims of different studies vary, and thus, the reasonable requirements for standardization differ. A framework of reference for the reasonable outcomes of different approaches is offered. Finally, it is argued that complete standardization is important not only for the reproducibility of measurements but also for education, for quality assessment and for algorithmic data analysis. The different standardized approaches can and in fact should serve as benchmarking reference tools for the development of future flow cytometry studies. © 2019 International Society for Advancement of Cytometry.
The prediction of the world's future energy consumption and global climate change makes it desirable to identify new technologies to replace or augment fossil fuels by environmentally sustainable alternatives. One appealing sustainable energy concept is harvesting solar energy via photosynthesis coupled to conversion of CO2 into chemical feedstock and fuel. In this work, the production of ethylene, the most widely used petrochemical produced exclusively from fossil fuels, in the model cyanobacterium Synechocystis sp. PCC 6803 is studied. A novel instrumentation setup for quantitative monitoring of ethylene production using a combination of flat-panel photobioreactor coupled to a membrane-inlet mass spectrometer is introduced. Carbon partitioning is estimated using a quantitative model of cyanobacterial metabolism. The results show that ethylene is produced under a wide range of light intensities with an optimum at modest irradiances. The results allow production conditions to be optimized in a highly controlled setup.
- MeSH
- autotrofní procesy MeSH
- ethyleny biosyntéza MeSH
- hmotnostní spektrometrie přístrojové vybavení metody MeSH
- kyslík analýza MeSH
- lyasy metabolismus MeSH
- membrány umělé * MeSH
- metabolické sítě a dráhy MeSH
- rekombinace genetická genetika MeSH
- světlo MeSH
- Synechocystis enzymologie růst a vývoj účinky záření MeSH
- uhlík analýza MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
BACKGROUND: With the introduction of the first high-throughput qPCR instrument on the market it became possible to perform thousands of reactions in a single run compared to the previous hundreds. In the high-throughput reaction, only limited volumes of highly concentrated cDNA or DNA samples can be added. This necessity can be solved by pre-amplification, which became a part of the high-throughput experimental workflow. Here, we focused our attention on the limits of the specific target pre-amplification reaction and propose the optimal, general setup for gene expression experiment using BioMark instrument (Fluidigm). RESULTS: For evaluating different pre-amplification factors following conditions were combined: four human blood samples from healthy donors and five transcripts having high to low expression levels; each cDNA sample was pre-amplified at four cycles (15, 18, 21, and 24) and five concentrations (equivalent to 0.078 ng, 0.32 ng, 1.25 ng, 5 ng, and 20 ng of total RNA). Factors identified as critical for a success of cDNA pre-amplification were cycle of pre-amplification, total RNA concentration, and type of gene. The selected pre-amplification reactions were further tested for optimal Cq distribution in a BioMark Array. The following concentrations combined with pre-amplification cycles were optimal for good quality samples: 20 ng of total RNA with 15 cycles of pre-amplification, 20x and 40x diluted; and 5 ng and 20 ng of total RNA with 18 cycles of pre-amplification, both 20x and 40x diluted. CONCLUSIONS: We set up upper limits for the bulk gene expression experiment using gene expression Dynamic Array and provided an easy-to-obtain tool for measuring of pre-amplification success. We also showed that variability of the pre-amplification, introduced into the experimental workflow of reverse transcription-qPCR, is lower than variability caused by the reverse transcription step.
- MeSH
- lidé MeSH
- messenger RNA analýza krev metabolismus MeSH
- polymerázová řetězová reakce s reverzní transkripcí přístrojové vybavení metody MeSH
- senzitivita a specificita MeSH
- stanovení celkové genové exprese přístrojové vybavení metody MeSH
- zdraví dobrovolníci pro lékařské studie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
