Herein, an advanced bioconjugation technique to synthesize hybrid polymer-antibody nanoprobes tailored for fluorescent cell barcoding in flow cytometry-based immunophenotyping of leukocytes is applied. A novel approach of attachment combining two fluorescent dyes on the copolymer precursor and its conjugation to antibody is employed to synthesize barcoded nanoprobes of antibody polymer dyes allowing up to six nanoprobes to be resolved in two-dimensional cytometry analysis. The major advantage of these nanoprobes is the construct design in which the selected antibody is labeled with an advanced copolymer bearing two types of fluorophores in different molar ratios. The cells after antibody recognition and binding to the target antigen have a characteristic double fluorescence signal for each nanoprobe providing a unique position on the dot plot, thus allowing antibody-based barcoding of cellular samples in flow cytometry assays. This technique is valuable for cellular assays that require low intersample variability and is demonstrated by the live cell barcoding of clinical samples with B cell abnormalities. In total, the samples from six various donors were successfully barcoded using only two detection channels. This barcoding of clinical samples enables sample preparation and measurement in a single tube.
Authors present a pilot study of the development of innovative flow cytometry-based assay with a potential for use in tuberculosis diagnostics. Currently available tests do not provide robust discrimination between latent tuberculosis infection (TBI) and tuberculosis disease (TB). The desired application is to distinguish between the two conditions by evaluating the production of a combination of three cytokines: IL-2 (interleukin-2), IFNɣ (interferon gamma) and TNFɑ (tumor necrosis factor alpha) in CD4+ and CD8+ T cells. The study was conducted on 68 participants, divided into two arms according to age (paediatric and adults). Each arm was further split into three categories (non-infection (NI), TBI, TB) based on the immune reaction to Mycobacterium tuberculosis (M.tb) after a close contact with pulmonary TB. Each blood sample was stimulated with specific M.tb antigens present in QuantiFERON tubes (TB1 and TB2). We inferred TBI or TB based on the predominant cytokine response of the CD4+ and/or CD8+ T cells. Significant differences were detected between the NI, TBI and the TB groups in TB1 in the CD4+TNFɑ+parameter in children. Along with IL-2, TNFɑ seems to be the most promising diagnostic marker in both CD4+and CD8+ T cells. However, more detailed analyses on larger cohorts are needed to confirm the observed tendencies.
- MeSH
- antigeny bakteriální imunologie MeSH
- biologické markery krev MeSH
- CD4-pozitivní T-lymfocyty * imunologie MeSH
- CD8-pozitivní T-lymfocyty * imunologie MeSH
- cytokiny krev metabolismus MeSH
- diferenciální diagnóza MeSH
- dítě MeSH
- dospělí MeSH
- interferon gama * krev imunologie MeSH
- interleukin-2 * krev MeSH
- latentní tuberkulóza * diagnóza imunologie mikrobiologie MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladiství MeSH
- mladý dospělý MeSH
- Mycobacterium tuberculosis * imunologie MeSH
- pilotní projekty MeSH
- plicní tuberkulóza diagnóza imunologie mikrobiologie krev MeSH
- prediktivní hodnota testů MeSH
- předškolní dítě MeSH
- průtoková cytometrie * metody MeSH
- senioři MeSH
- test pomocí interferonu gama metody MeSH
- TNF-alfa krev MeSH
- Check Tag
- dítě MeSH
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladiství MeSH
- mladý dospělý MeSH
- mužské pohlaví MeSH
- předškolní dítě MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- srovnávací studie MeSH
The most commonly used flow cytometric (FCM) analysis of cellular DNA content relies on ethanol fixation followed by RNA digestion and propidium iodide (PI) intercalation into double-stranded DNA. This is a laborious and time-consuming procedure that is subject to systematic errors due to centrifugation and washing steps associated with sample preparation. It can adversely affect the reliability of the results. Here, we present a modified concept of DNA quantification in adherent cell lines by FCM that involves neither ethanol fixation nor any washing and cell transferring steps. Our high throughput assay of adherent cell lines reduces sample-processing time, requires minimal workload, provides a possibility for automation, and, if needed, also allows a significant reduction in the size of individual samples. Working with a well-proven commercial tool-The BD CycletestTM Plus DNA Reagent Kit-primarily designed for cell cycle analysis and aneuploidy determination in experimental and clinical samples, we suggest a novel, very efficient, and robust approach for DNA research in adherent cell cultures.
- MeSH
- aneuploidie MeSH
- automatizace MeSH
- buněčná adheze MeSH
- buněčný cyklus genetika MeSH
- DNA * analýza MeSH
- lidé MeSH
- průtoková cytometrie * metody MeSH
- reprodukovatelnost výsledků MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Electrical characteristics of living cells have been proven to reveal important details about their internal structure, charge distribution and composition changes in the cell membrane, as well as the extracellular context. An impedance flow cytometry is a common approach to determine the electrical properties of a cell, having the advantage of label-free and high throughput. However, the current techniques are complex and costly for the fabrication process. For that reason, we introduce an integrated dual microneedle-microchannel for single-cell detection and electrical properties extraction. The dual microneedles utilized a commercially available tungsten needle coated with parylene. When a single cell flows through the parallel-facing electrode configuration of the dual microneedle, the electrical impedance at multiple frequencies is measured. The impedance measurement demonstrated the differential of normal red blood cells (RBCs) with three different sizes of microbeads at low and high frequencies, 100 kHz and 2 MHz, respectively. An electrical equivalent circuit model (ECM) was used to determine the unique membrane capacitance of individual cells. The proposed technique demonstrated that the specific membrane capacitance of an RBC is 9.42 mF/m-2, with the regression coefficients, ρ at 0.9895. As a result, this device may potentially be used in developing countries for low-cost single-cell screening and detection.
Flow cytometry (FCM) is now the most widely used method to determine ploidy levels and genome size of plants. To get reliable estimates and allow reproducibility of measurements, the methodology should be standardized and follow the best practices in the field. In this article, we discuss instrument calibration and quality control and various instrument and acquisition settings (parameters, flow rate, number of events, scales, use of discriminators, peak positions). These settings must be decided before measurements because they determine the amount and quality of the data and thus influence all downstream analyses. We describe the two main approaches to raw data analysis (gating and histogram modeling), and we discuss their advantages and disadvantages. Finally, we provide a summary of best practice recommendations for data acquisition and raw data analysis in plant FCM.
AIMS: This study compared the results obtained by basic immunophenotyping of cerebrospinal fluid (CSF) cells by flow cytometry (FC) to the results of conventional cytology and evaluated the possibility of detailed analyses of CSF B-cell subpopulations. METHODS: Samples from 42 patients were examined by conventional cytology (native and/or pre-centrifuged CSF) and FC. The results from 15 patients without evidence of organic neurological disease were used to estimate reference ranges. RESULTS: Pre-centrifugated CSF had significantly higher cell yield on the cytologic slide, but cell subpopulation percentages were altered; the percentage of lymphocytes was significantly higher and monocytes significantly lower compared to both native CSF slides and FC. The percentage of granulocytes was higher in FC compared to cytology. For leukocyte count, the following reference ranges were estimated for Fuchs-Rosenthal chamber (FR) counting and FC, respectively: leukocytes ≤4.7/μL and ≤2.5/μL, lymphocytes ≤4.1/μL and ≤1.8/μL, monocytes ≤1.2/μL and ≤0.9/μL, and granulocytes 0/μL and ≤0.2/μL. The following reference ranges were estimated for basic subpopulations: T-lymphocytes 84.1-100%, B lymphocytes 0.0-1.5%, NK cells 0.0-6.3%, NKT cells 0-9.5%, and CD3+CD4+/CD3+CD8+ 0.8-4.9. Using a volume of 1.2-2.4 mL, the number of B lymphocytes was too low (<20) in samples with ≤2.7 cells/μL in the FR. CONCLUSIONS: Even normal CSF samples are amenable to basic mononuclear cell subpopulation analysis by FC. However, analysis of the B-cell subpopulations requires either a larger sample volume or selection of samples with ≥ 3 cells/μL.
- MeSH
- imunofenotypizace MeSH
- leukocyty * MeSH
- lidé MeSH
- lymfocyty * MeSH
- mozkomíšní mok MeSH
- průtoková cytometrie metody MeSH
- T-lymfocyty MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
The distribution of fluorescence signals measured with flow cytometry can be influenced by several factors, including qualitative and quantitative properties of the used fluorochromes, optical properties of the detection system, as well as the variability within the analyzed cell population itself. Most of the single cell samples prepared from in vitrocultures or clinical specimens contain a variable cell cycle component. Cell cycle, together with changes in the cell size, are two of the factors that alter the functional properties of analyzed cells and thus affect the interpretation of obtained results. Here, we describe the association between cell cycle status and cell size, and the variability in the distribution of fluorescence intensity as determined with flow cytometry, at population scale. We show that variability in the distribution of background and specific fluorescence signals is related to the cell cycle state of the selected population, with the 10% low fluorescence signal fraction enriched mainly in cells in their G0/G1 cell cycle phase, and the 10% high fraction containing cells mostly in the G2/M phase. Therefore we advise using caution and additional experimental validation when comparing populations defined by fractions at both ends of fluorescence signal distribution to avoid biases caused by the effect of cell cycle and cell size.
- MeSH
- buněčné dělení MeSH
- buněčný cyklus fyziologie MeSH
- G2 fáze * MeSH
- průtoková cytometrie metody MeSH
- velikost buňky MeSH
- Publikační typ
- časopisecké články MeSH
Průtoková cytometrie je zásadní diagnostickou metodou v imunologii a v hematologii u dětí. Výhodou cytometrie je rychlost, kdy v rámci hodin může tato metoda klinikům pomoci ve volbě terapie a indikaci dalších vyšetření.
Flow cytometry is an essential diagnostic method in immunology and haematology in children. The advantage of flow cytometry is its speed, where within hours this method can help clinicians in the choice of therapy and the indication of further investigations.
- MeSH
- dítě MeSH
- krevní nemoci diagnóza MeSH
- lidé MeSH
- lymfoblastická leukemie-lymfom z prekurzorových T-buněk diagnóza MeSH
- průtoková cytometrie * metody MeSH
- reziduální nádor diagnóza MeSH
- syndromy imunologické nedostatečnosti diagnóza MeSH
- Check Tag
- dítě MeSH
- lidé MeSH
- Publikační typ
- práce podpořená grantem MeSH
- přehledy MeSH
Analýza punktátů kostní dřeně pomocí průtokové cytometrie má zásadní význam pro diagnostiku hematoonkologických onemocnění. Technika poskytuje rychlé určení přítomnosti, liniové příslušnosti a přibližného stádia zralosti patologické populace pomocí analýzy exprese povrchových, cytoplazmatických a jaderných molekul. I přes nesporné výhody má průtoková cytometrie ve svých interpretačních možnostech limity, které však vhodně doplňují další vyšetření, zejména vyšetření morfologické a imunohistochemické. Právě imunohistochemie sdílí s průtokovou cytometrií základní princip detekce patologické populace i skladbu vyšetřovaných molekul. Dále však obě techniky nabízí různé úhly pohledu na daný vzorek a navzájem doplňují diagnosticky hluchá místa. Nezřídkakdy tak až součinnost obou postupů přináší kýžený podrobný obraz o zastoupení a typu patologické populace v kostní dřeni. Text článku přináší přehled základních postupů při diagnostice hematologických malignit pomocí průtokové cytometrie a zamýšlí se nad přednostmi a slabinami průtokové cytometrie ve vztahu k imunohistochemickému vyšetření.
Analysis of bone marrow samples by flow cytometry is essential for the diagnosis of hematological neoplasms. The technique provides rapid determination of the presence, lineage, and approximate stage of maturity of the pathological population by analyzing the expression of surface, cytoplasmic, and nuclear molecules. Despite the indisputable advantages, flow cytometry has its limits, which, however, replace other techniques, especially morphological and immunohistochemical examinations. It is immunohistochemistry that shares with flow cytometry the basic principle of detection of the pathological population as well as the portfolio of investigated molecules. Both techniques however offer different points of view on the given sample and complement each other. The combination of both procedures often provides the desired detailed picture of the presence and type of pathological population in the bone marrow. The article provides an overview of basic procedures in the diagnosis of hematological malignancies using flow cytometry and reflects on the strengths and weaknesses of flow cytometry in relation to immunohistochemical examination.
- MeSH
- hematologické nádory diagnóza MeSH
- imunofenotypizace MeSH
- imunohistochemie metody MeSH
- lidé MeSH
- průtoková cytometrie * metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- přehledy MeSH
Vyšetření lymfoproliferací v lymfatických uzlinách průtokovou cytometrií poskytuje pouze minimální informace o morfologii (orientační stanovení velikosti a granularity buněk), a zcela postrádá informace topografické. Nicméně velkou výhodou této metody je především rychlost stanovení a hodnocení kombinace vyšetřovaných antigenů, kde neméně důležité jsou i intenzity exprese jednotlivých znaků. Toto je zcela zásadní pro hematologické malignity, kdy se různá onemocnění mohou na morfologické úrovni velmi podobat a odchylky vzhledu buněk mohou být minimální. Při diagnostice lymfomů korelace histologie a imunohistochemie s průtokovou cytometrií poskytuje komplexnější pohled pro interpretaci jednotlivých nálezů. Cílem tohoto článku je poskytnout základní informace o vyšetření lymfatických uzlin a biopsií extranodálních tkání průtokovou cytometrií se zaměřením na lymfoproliferativní onemocnění a vytvořit odrazový můstek pro spolupráci mezi patologem a cytometristou.
Although flow cytometry examination of lymphoproliferation in lymph nodes provides minimal morphological information, apart from indicative determination of cell size and granularity, and completely loses topographical information. However, the great advantage of this method is the speed of determining and combination of investigated antigens, where the expression intensities of individual markers are no less important. This is absolutely crucial for hematological malignancies, where different diseases can look very similar at the morphological level and variations in the appearance of tumour cells can be minimal. When diagnosing lymphomas, the combination of histology and immunohistochemistry with flow cytometry provides a more comprehensive view for the interpretation of individual findings. The aim of this article is to provide basic information about the examination of lymph nodes and extranodal tissue by flow cytometry as a starting point for the collaboration of the pathologist and the cytometrist.
- Klíčová slova
- lymfoproliferace,
- MeSH
- lidé MeSH
- lymfatické uzliny MeSH
- průtoková cytometrie * metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- přehledy MeSH
