RESUMO
Imaging flow cytometry is emerging as a diagnostic tool for the assessment of leukemia. It has the functionality of standard flow cytometry and generates high-resolution digital images of each cell with quantifiable numerical data. We demonstrate the use of an automated high-throughput method for performing fluorescence in situ hybridization (FISH) on immunophenotyped whole cells in suspension and analyzed by imaging flow cytometry, a technique called "Immuno-flowFISH". The aim of this study was to demonstrate the application of immuno-flowFISH for the detection of chromosomal abnormalities in CLL, specifically trisomy 12 and del(17p). Mononuclear cells were isolated and immunophenotyped with fluorescently conjugated CD3, CD5, and CD19 monoclonal antibodies. Following fixation, cells were permeabilized, dsDNA denatured and hybridized with chromosome 12 or 17 enumeration (CEP 12 and CEP17) and 17p12 locus-specific FISH probes. Cells were analyzed on the Amnis ImageStream®X Mark II to assess the number and percent FISH-positive CLL cells and the ratio of FISH spot counts for CD5/CD19-positive CLL cells to CD3/CD5-positive T cells (FISH "mean spot ratio"). Deletion of 17p was detected in about 8% of cases to date, with del(17p) ranged from 3.5-22.8% and the FISH "mean spot ratio" 0.86-0.96. Immuno-flowFISH also detected a minimal residual disease case with +12 with a limit of detection of 0.13% and a rare case that presented with atypical phenotype and cytogenetics. Immuno-flowFISH could detect del(17p) in phenotypically identified CD5/CD19-positive B-cells. The 100-fold increase in analyzed cells, as well as the addition of cell phenotype increased the sensitivity and specificity over current clinical FISH testing. Furthermore, immuno-flowFISH analysis demonstrated specific utility in unique clinical scenarios such as residual disease and atypical biology cases which may be of significant benefit with regards to prognostication and MRD analysis. The method will assist in therapeutic decision making and disease monitoring for many hematological malignancies. © 2019 International Society for Advancement of Cytometry.
Assuntos
Aberrações Cromossômicas , Citometria de Fluxo , Imunofenotipagem , Hibridização in Situ Fluorescente , Leucemia Linfocítica Crônica de Células B/genética , Deleção Cromossômica , Humanos , Reprodutibilidade dos Testes , Trissomia/genéticaRESUMO
Chronic Lymphocytic Leukaemia (CLL), the most common leukaemia in the Western world, has a characteristic phenotype and prognosis largely defined by the presence of cytogenetic aberrations. The gold standard for detecting these cytogenetic abnormalities is interphase fluorescence in situ hybridisation (FISH) performed on cell smears or tissue sections on glass slides. Fluorescently labelled DNA probes bind to specific chromosomal regions and the signal detected by fluorescent microscopy. Generally only 200 cells are assessed and the limit of sensitivity is 3% positive cells. Here we report the development and use of imaging flow cytometry to assess chromosomes by FISH in phenotyped CLL cells in suspension. Thousands of CLL cells, identified by their phenotype, are assessed for specific FISH probe signals using an automated, high throughput imaging flow cytometer. The "extended depth of field" capability of the imaging flow cytometer enables FISH probe signals ("spots") to be resolved and localised within the (stained) nucleus of the immunophenotyped cells. We report the development of the automated "immuno-flowFISH" on normal blood using the Amnis ImageStreamX mark II platform and illustrate the clinical application of the method for the assessment of chromosome 12 in CLL. It is a powerful new method which has potential to be applied at diagnosis for disease stratification, and following treatment to assess residual disease. These applications will assist clinicians in optimising therapeutic decision making and thereby improve patient outcome.
Assuntos
Citometria de Fluxo/métodos , Hibridização in Situ Fluorescente/métodos , Leucemia Linfocítica Crônica de Células B/diagnóstico por imagem , Linhagem Celular Tumoral , Núcleo Celular/genética , Aberrações Cromossômicas , Humanos , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/patologiaRESUMO
BACKGROUND: Urban form interventions can result in positive and negative impacts on physical activity, social participation, and well-being, and inequities in these outcomes. Natural experiment studies can advance our understanding of causal effects and processes related to urban form interventions. The INTErventions, Research, and Action in Cities Team (INTERACT) is a pan-Canadian collaboration of interdisciplinary scientists, urban planners, and public health decision makers advancing research on the design of healthy and sustainable cities for all. Our objectives are to use natural experiment studies to deliver timely evidence about how urban form interventions influence health, and to develop methods and tools to facilitate such studies going forward. METHODS: INTERACT will evaluate natural experiments in four Canadian cities: the Arbutus Greenway in Vancouver, British Columbia; the All Ages and Abilities Cycling Network in Victoria, BC; a new Bus Rapid Transit system in Saskatoon, Saskatchewan; and components of the Sustainable Development Plan 2016-2020 in Montreal, Quebec, a plan that includes urban form changes initiated by the city and approximately 230 partnering organizations. We will recruit a cohort of between 300 and 3000 adult participants, age 18 or older, in each city and collect data at three time points. Participants will complete health and activity space surveys and provide sensor-based location and physical activity data. We will conduct qualitative interviews with a subsample of participants in each city. Our analysis methods will combine machine learning methods for detecting transportation mode use and physical activity, use temporal Geographic Information Systems to quantify changes to urban intervention exposure, and apply analytic methods for natural experiment studies including interrupted time series analysis. DISCUSSION: INTERACT aims to advance the evidence base on population health intervention research and address challenges related to big data, knowledge mobilization and engagement, ethics, and causality. We will collect ~ 100 TB of sensor data from participants over 5 years. We will address these challenges using interdisciplinary partnerships, training of highly qualified personnel, and modern methodologies for using sensor-based data.
Assuntos
Planejamento Ambiental , Estudos de Avaliação como Assunto , Exercício Físico , Saúde Pública , População Urbana , Adolescente , Adulto , Colúmbia Britânica , Cidades , Estudos de Coortes , Sistemas de Informação Geográfica , Humanos , Análise de Séries Temporais Interrompida , Quebeque , Projetos de Pesquisa , Saskatchewan , Participação Social , Inquéritos e Questionários , Meios de TransporteRESUMO
Platelets are subcellular blood elements with a well-established role in haemostasis. Upon activation platelets undergo granule exocytosis, resulting in α-granule P-Selectin being expressed on the cell membrane. This allows binding of activated platelets to P-Selectin glycoprotein ligand 1 (PSGL-1) expressing leukocytes, forming leukocyte-platelet aggregates (LPAs). Whole blood flow cytometry (FCM) has demonstrated that elevated circulating LPAs (especially monocyte LPAs) are linked to atherothrombosis in high risk patients, and that activated platelet binding influences monocytes towards a pro-adhesive and pro-atherogenic phenotype. However, a limitation of conventional FCM is the potential for coincident events to resemble LPAs despite no tethering. Imaging cytometry can be used to characterize LPA formation and distinguish circulating MPAs from coincidental events. Platelets and leukocyte subsets are identified by expression of surface markers (e.g. the lipopolysachharide receptor CD14 on monocytes, glycoprotein Ib CD42b on platelets). In conventional FCM, all events with both leukocyte and platelet characteristics are designated as LPAs. However, by using an 'internal' mask based on the brightfield image and the fluorescent platelet identifier, imaging flow cytometry is able to distinguish leukocytes with tethered platelets (genuine LPAs) from leukocyte with coincidental, untethered platelets nearby. Mechanisms (e.g. adhesion molecules) or consequences (e.g. signal transduction) can then be separately analysed in platelet tethered and untethered leukocytes. Imaging flow cytometry therefore provides a more accurate approach for both enumeration and analysis of LPAs than conventional FCM.
Assuntos
Plaquetas/imunologia , Comunicação Celular/imunologia , Citometria de Fluxo/métodos , Citometria por Imagem/métodos , Monócitos/imunologia , Neutrófilos/imunologia , Biomarcadores/metabolismo , Plaquetas/citologia , Agregação Celular/imunologia , Citometria de Fluxo/instrumentação , Expressão Gênica , Humanos , Citometria por Imagem/instrumentação , Receptores de Lipopolissacarídeos/genética , Receptores de Lipopolissacarídeos/imunologia , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/imunologia , Monócitos/citologia , Neutrófilos/citologia , Selectina-P/genética , Selectina-P/imunologia , Ativação Plaquetária , Complexo Glicoproteico GPIb-IX de Plaquetas/genética , Complexo Glicoproteico GPIb-IX de Plaquetas/imunologia , Ligação ProteicaRESUMO
Fluorescence in situ hybridization (FISH) is a microscopy technique which uses a fluorescent probe to detect DNA sequences and is generally performed on metaphase spreads or interphase nuclei of intact cells on a slide. In a diagnostic laboratory, cells are hybridized with fluorescent probes and up to 200 cells counted for the number of cells with probe "spots." Recent modifications to standard FISH include immuno-FISH, where chromosomal abnormalities are detected only in cells by their phenotype, and S-FISH where probe hybridization is performed on whole cells in suspension. Here we describe the development of an immuno-S-FISH method that combines immunophenotyping and FISH analysis of cells in suspension followed by analysis on an imaging flow cytometer. This single platform technique couples microscopy with flow cytometry and "spot" detection of bound FISH probe. Automated immuno-S-FISH enables large numbers of analyzed cells to be identified by phenotype and assessed for specific chromosomal determinants by FISH. This novel robust method enables quantitative cell population analysis and "spot" counting for large numbers of cells. We report method optimization of this imaging immuno-S-FISH flow cytometry protocol which has capability for many clinical applications. © 2016 International Society for Advancement of Cytometry.
Assuntos
Aberrações Cromossômicas , Citometria de Fluxo/métodos , Hibridização in Situ Fluorescente/métodos , Sequência de Bases/genética , Núcleo Celular/genética , Corantes Fluorescentes , Humanos , Interfase , Hibridização de Ácido NucleicoRESUMO
Platelets are subcellular blood elements with a well-established role in haemostasis. Upon activation platelets express P-Selectin (CD62P) on the cell membrane and bind to P-Selectin glycoprotein ligand 1 expressing monocytes, influencing them toward a pro-adhesive and inflammatory phenotype. It is well established that elevated circulating monocyte-platelet aggregates (MPAs) are linked to atherothrombosis in high risk patients. However, whole blood flow cytometry (FCM) has recently shown that circulating MPAs may also occur in the absence of platelet activation, particularly in healthy children. A potential limitation of conventional FCM is the potential for coincident events to resemble monocyte platelet aggregates. Here we report a novel imaging cytometry approach to further characterize monocyte-platelet aggregate formation by P-Selectin dependent and P-Selectin independent mechanisms and distinguish circulating MPAs from coincidental events. Monocytes were identified by expression of the lipopolysachharide receptor (CD14 BV421), while platelets were identified by expression of the glycoprotein Ib (CD42b APC). Differentiation of P-Selectin dependent and P-Selectin independent binding was achieved with AF488 labeled CD62P. Overall analysis of circulating and in vitro generated MPAs by conventional and imaging cytometry methods showed very strong correlation (r(2) = >0.99, P < 0.01). The Bland-Altman bias of -1.72 was not significantly different to zero. However, when measuring only P-Selectin negative MPAs, a lack of correlation (r(2) = 0.27, P = n.s.) likely reflects better discrimination of coincidence events using imaging cytometry. Our data demonstrate that IFC is more accurate in enumerating MPAs than conventional FCM, which over-estimates the number of MPAs due to the presence of coincident events.
Assuntos
Plaquetas/metabolismo , Separação Celular/métodos , Citometria de Fluxo/métodos , Monócitos/metabolismo , Agregação Plaquetária , Adulto , Agregação Celular , Humanos , Pessoa de Meia-Idade , Agregação Plaquetária/fisiologia , Adulto JovemRESUMO
BACKGROUND: Detection of del(17p) in myeloma is generally performed by fluorescence in situ hybridization (FISH) on a slide with analysis of up to 200 nuclei. The small cell sample analyzed makes this a low precision test. We report the utility of an automated FISH method, called "immuno-flowFISH", to detect plasma cells with adverse prognostic risk del(17p) in bone marrow and blood samples of patients with myeloma. METHODS: Bone marrow (n = 31) and blood (n = 19) samples from 35 patients with myeloma were analyzed using immuno-flowFISH. Plasma cells were identified by CD38/CD138-immunophenotypic gating and assessed for the 17p locus and centromere of chromosome 17. Cells were acquired on an AMNIS ImageStreamX MkII imaging flow cytometer using INSPIRE software. RESULTS: Chromosome 17 abnormalities were identified in CD38/CD138-positive cells in bone marrow (6/31) and blood (4/19) samples when the percent plasma cell burden ranged from 0.03% to 100% of cells. Abnormalities could be identified in 14.5%-100% of plasma cells. CONCLUSIONS: The "immuno-flowFISH" imaging flow cytometric method could detect del(17p) in plasma cells in both bone marrow and blood samples of myeloma patients. This method was also able to detect gains and losses of chromosome 17, which are also of prognostic significance. The lowest levels of 0.009% (bone marrow) and 0.001% (blood) for chromosome 17 abnormalities was below the detection limit of current FISH method. This method offers potential as a new means of identifying these prognostically important chromosomal defects, even when only rare cells are present and for serial disease monitoring.
Assuntos
Cromossomos Humanos Par 17 , Citometria de Fluxo , Hibridização in Situ Fluorescente , Mieloma Múltiplo , Plasmócitos , Humanos , Mieloma Múltiplo/diagnóstico , Mieloma Múltiplo/genética , Mieloma Múltiplo/sangue , Mieloma Múltiplo/patologia , Plasmócitos/patologia , Citometria de Fluxo/métodos , Cromossomos Humanos Par 17/genética , Masculino , Feminino , Idoso , Pessoa de Meia-Idade , Medula Óssea/patologia , Deleção Cromossômica , Idoso de 80 Anos ou mais , Imunofenotipagem , AdultoRESUMO
Imaging flow cytometry has the capacity to bridge the gap that currently exists between the diagnostic tests that detect important phenotypic and genetic changes in the clinical assessment of leukemia and other hematological malignancies or blood-based disorders. We have developed an "Immuno-flowFISH" method that leverages the quantitative and multi-parametric power of imaging flow cytometry to push the limits of single-cell analysis. Immuno-flowFISH has been fully optimized to detect clinically significant numerical and structural chromosomal abnormalities (i.e., trisomy 12 and del(17p)) within clonal CD19/CD5+ CD3- Chronic Lymphocytic Leukemia (CLL) cells in a single test. This integrated methodology has greater accuracy and precision than standard fluorescence in situ hybridization (FISH). We have detailed this immuno-flowFISH application with a carefully catalogued workflow, technical instructions, and a repertoire of quality control considerations to supplement the analysis of CLL. This next-generation imaging flow cytometry protocol may provide unique advancements and opportunities in the holistic cellular assessment of disease for both research and clinical laboratory settings.
Assuntos
Leucemia Linfocítica Crônica de Células B , Humanos , Leucemia Linfocítica Crônica de Células B/genética , Hibridização in Situ Fluorescente/métodos , Aberrações Cromossômicas , Trissomia , Citometria de FluxoRESUMO
Chimeric antigen receptor (CAR) T-cell therapy is a novel adoptive T-cell immunotherapy for haematological malignancies. First introduced into clinical practice in 2017, CAR T-cell therapy is now finding its place in the management of lymphoid malignancies, primarily of B-cell lineage, including lymphoblastic leukaemia, non-Hodgkin lymphoma and plasma cell myeloma, with remarkable therapeutic outcomes. CAR T-cells are a customised therapeutic product for each patient. Manufacture commences with collection of autologous T-cells, which are then genetically engineered ex vivo to express transmembrane CARs. These chimeric proteins consist of an antibody-like extracellular antigen-binding domain, to recognise specific antigens on the surface of tumour cells (e.g. CD19), linked to the intracellular co-stimulatory signalling domains of a T-cell receptor (e.g. CD137). The latter is required for in vivo CAR T-cell proliferation, survival, and durable efficacy. Following reinfusion, CAR T-cells harness the cytotoxic capacity of a patient's immune system. They overcome major mechanisms of tumour immuno-evasion and have potential to generate robust cytotoxic anti-tumour responses. This review discusses the background to CAR T-cell therapies, including their molecular design, mechanisms of action, methods of production, clinical applications and established and emerging technologies for CAR T-cell evaluation. It highlights the need for standardisation, quality control and monitoring of CAR T-cell therapies, to ensure their safety and efficacy in clinical management.
Assuntos
Antineoplásicos , Mieloma Múltiplo , Receptores de Antígenos Quiméricos , Humanos , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos de Linfócitos T/genética , Linfócitos T , Mieloma Múltiplo/terapia , Controle de QualidadeRESUMO
AIMS: Cytogenetic abnormalities involving the IGH gene are seen in up to 55% of patients with multiple myeloma. Current testing is performed manually by fluorescence in situ hybridisation (FISH) on purified plasma cells. We aimed to assess whether an automated imaging flow cytometric method that uses immunophenotypic cell identification, and does not require cell isolation, can identify IGH abnormalities. METHODS: Aspirated bone marrow from 10 patients with multiple myeloma were studied. Plasma cells were identified by CD38 and CD138 coexpression and assessed with FISH probes for numerical or structural abnormalities of IGH. Thousands of cells were acquired on an imaging flow cytometer and numerical data and digital images were analysed. RESULTS: Up to 30 000 cells were acquired and IGH chromosomal abnormalities were detected in 5 of the 10 marrow samples. FISH signal patterns seen included fused IGH signals for IGH/FGFR3 and IGH/MYEOV, indicating t(4;14) and t(11;14), respectively. In addition, three IGH signals were identified, indicating trisomy 14 or translocation with an alternate chromosome. The lowest limit of detection of an IGH abnormality was in 0.05% of all cells. CONCLUSIONS: This automated high-throughput immuno-flowFISH method was able to identify translocations and trisomy involving the IGH gene in plasma cells in multiple myeloma. Thousands of cells were analysed and without prior cell isolation. The inclusion of positive plasma cell identification based on immunophenotype led to a lowest detection level of 0.05% marrow cells. This imaging flow cytometric FISH method offers the prospect of increased precision of detection of critical genetic lesions involving IGH and other chromosomal defects in multiple myeloma.
Assuntos
Aberrações Cromossômicas , Genes de Cadeia Pesada de Imunoglobulina , Mieloma Múltiplo , Humanos , Citometria de Fluxo , Mieloma Múltiplo/diagnóstico , Mieloma Múltiplo/genética , Mieloma Múltiplo/patologia , Translocação Genética , Trissomia/genética , Genes de Cadeia Pesada de Imunoglobulina/genéticaRESUMO
The sequestration of organic carbon (OC) in wetland sediments is influenced by the presence of oxygen or lack thereof. The mechanisms of OC sequestration under redox fluctuations, particularly by the co-mediation of reactive iron (Fe) protection and thermodynamic limitation by the energetics of the OC itself, remain unclear. Over the past 26 years, a combination of field surveys and remote sensing images had revealed a strong decline in both natural and constructed wetland areas in Tianjin. This decline could be attributed to anthropogenic landfill practices and agricultural reclamation efforts, which may have significant impacts on the oxidation-reduction conditions for sedimentary OC. The Fe-bound OC (CBD extraction) decreased by 2 to 10-fold (from 8.3 to 10% to 0.7-4.5%) with increasing sediment depth at three sites with varying water depths (WD). The high-resolution spectro-microscopy analysis demonstrated that Fe (oxyhydr)oxides were colocalized with sedimentary OC. Corresponding to lower redox potential, the nominal oxidation state of C (NOSC), which corresponds to the energy content in OC, became more negative (energy content increased) with increasing sediment depth. Taken together, the preservation of sedimentary OC is contingent on the prevailing redox conditions: In environments where oxygen availability is high, reactive Fe provides protection for OC, while in anoxic environments, thermodynamic constraints (i.e., energetic constraints) limit the oxidation of OC.
Assuntos
Carbono , Áreas Alagadas , Carbono/análise , Compostos Férricos , Oxirredução , Oxigênio , Sedimentos GeológicosRESUMO
Despite the consensus that keeping global temperature rise within 1.5 °C above pre-industrial level by 2100 reduces the chance for climate change to reach the point of no return, the newest Intergovernmental Panel on Climate Change (IPCC) report warns that the existing commitment of greenhouse gas emission reduction is only enough to contain the warming to 3-4 °C by 2100. The harsh reality not only calls for speedier deployment of existing CO2 reduction technologies but demands development of more cost-efficient carbon removal strategies. Here we report an ocean alkalinity-based CO2 sequestration scheme, taking advantage of proton consumption during nitrate assimilation by marine photosynthetic microbes, and the ensuing enhancement of seawater CO2 absorption. Benchtop experiments using a native marine phytoplankton community confirmed pH elevation from ~8.2 to ~10.2 in seawater, within 3-5 days of microbial culture in nitrate-containing media. The alkaline condition was able to sustain at continued nutrient supply but reverted to normalcy (pH ~8.2-8.4) once the biomass was removed. Measurements of δ13C in the dissolved inorganic carbon revealed a significant atmospheric CO2 contribution to the carbonate alkalinity in the experimental seawater, confirming the occurrence of direct carbon dioxide capture from the air. Thermodynamic calculation shows a theoretical carbon removal rate of ~0.13 mol CO2/L seawater, if the seawater pH is allowed to decrease from 10.2 to 8.2. A cost analysis (using a standard bioreactor wastewater treatment plant as a template for CO2 trapping, and a modified moving-bed biofilm reactor for nitrate recycling) indicated that a 1 Mt CO2/year operation is able to perform at a cost of ~$40/tCO2, 2.5-5.5 times cheaper than that offered by any of the currently available direct air capture technologies, and more in line with the price of $25-30/tCO2 suggested for rapid deployment of large-scale CCS systems.
Assuntos
Dióxido de Carbono , Fitoplâncton , Dióxido de Carbono/análise , Nitratos , Biodiversidade , Temperatura , Água do Mar , CarbonatosRESUMO
Anthropogenic input of sulfate (SO42-) in reservoirs may enhance bacterial sulfate reduction (BSR) under seasonally hypoxic conditions in the water column. However, factors that control BSR and its coupling to organic carbon (OC) mineralization in seasonally hypoxic reservoirs remain unclear. The present study elucidates the coupling processes by analyzing the concentrations and isotopic composition of dissolved inorganic carbon (DIC) and sulfur (SO42-, sulfide) species, and the microbial community in water of the Aha reservoir, SW China, which has high SO42- concentration due to the inputs from acid mine drainage about twenty years ago. The water column at two sites in July and October revealed significant thermal stratification. In the hypoxic bottom water, the δ13C-DIC decreased while the δ34S-SO42- increased, implying organic carbon mineralization due to BSR. The magnitude of S isotope fractionation (Δ34S, obtained from δ34Ssulfate-δ34Ssulfide) during the process of BSR fell in the range of 3.4 to 27.0 in July and 21.6 to 31.8 in October, suggesting a change in the community of sulfate-reducing bacteria (SRB). The relatively low water column stability in October compared to that in July weakened the difference of water chemistry and ultimately affected the SRB diversity. The production of DIC (ΔDIC) scaled a strong positive relationship with the Δ34S in July (p < 0.01), indicating that high OC availability favored the survival of incomplete oxidizers of SRB. However, in October, Δ13C-DIC was correlated with the Δ34S in the bottom hypoxic water (p < 0.01), implying that newly degraded OC depleted in 13C could favor the dominance of complete oxidizers of SRB which caused greater S isotope fractionation. Moreover, the sulfide supplied by BSR might stimulate the reductive dissolution of Fe and Mn oxides (Fe(O)OH and MnO2). The present study helps to understand the coupling of C and S in seasonally hypoxic reservoirs characterized by high SO42- concentration.
Assuntos
Carbono , Sulfatos , Bactérias/metabolismo , Carbono/metabolismo , Isótopos de Carbono/análise , China , Monitoramento Ambiental , Isótopos , Compostos de Manganês , Óxidos , Sulfatos/análise , Sulfetos/metabolismo , Enxofre/metabolismo , Isótopos de Enxofre/análise , Óxidos de Enxofre/metabolismo , Água/metabolismoRESUMO
Chromosomal analysis is traditionally performed by karyotyping on metaphase spreads, or by fluorescent in situ hybridization (FISH) on interphase cells or metaphase spreads. Flow cytometry was introduced as a new method to analyze chromosomes number (ploidy) and structure (telomere length) in the 1970s with data interpretation largely based on fluorescence intensity. This technology has had little uptake for human cytogenetic applications primarily due to analytical challenges. The introduction of imaging flow cytometry, with the addition of digital images to standard multi-parametric flow cytometry quantitative tools, has added a new dimension. The ability to visualize the chromosomes and FISH signals overcomes the inherent difficulties when the data is restricted to fluorescence intensity. This field is now moving forward with methods being developed to assess chromosome number and structure in whole cells (normal and malignant) in suspension. A recent advance has been the inclusion of immunophenotyping such that antigen expression can be used to identify specific cells of interest for specific chromosomes and their abnormalities. This capability has been illustrated in blood cancers, such as chronic lymphocytic leukemia and plasma cell myeloma. The high sensitivity and specificity achievable highlights the potential imaging flow cytometry has for cytogenomic applications (i.e., diagnosis and disease monitoring). This review introduces and describes the development, current status, and applications of imaging flow cytometry for chromosomal analysis of human chromosomes.
Assuntos
Cromossomos Humanos/genética , Citometria de Fluxo , Humanos , Hibridização in Situ FluorescenteRESUMO
Imaging flow cytometry is an automated method that enables cells and fluorescent signals to be visualized and quantified. Here, we describe a new imaging flow cytometry method whereby fluorescence in situ hybridization (FISH) is integrated with cell phenotyping. The method, called "immuno-flowFISH," provides an exciting new dimension for the analysis of genomic changes in cytological samples (e.g., blood, bone marrow). Cells are analyzed in suspension without any requirement for prior cell isolation or separation. Multiple antibodies and FISH probes, each with a unique fluorophore, can be added and many thousands of cells analyzed. Specific cell populations are identified by their antigenic profile and then analyzed for the presence of chromosomal defects. Immuno-flowFISH was applied to the assessment of chronic lymphocytic leukemia (CLL), a mature B-cell neoplasm where chromosomal abnormalities predict prognosis and treatment requirements. This integrated immunophenotyping and multi-probe FISH strategy could detect both structural and numerical chromosomal changes involving chromosomes 12 and 17 in CLL cells. Given that many thousands of cells were analyzed and the leukemic cells were positively identified by their immunophenotype, this multi-probe method adds precision to the cytogenomic analysis of CLL. © 2021 Wiley Periodicals LLC.
Assuntos
Leucemia Linfocítica Crônica de Células B , Aberrações Cromossômicas , Citometria de Fluxo , Humanos , Imunofenotipagem , Hibridização in Situ Fluorescente , Leucemia Linfocítica Crônica de Células B/genéticaRESUMO
Microbe-mineral interactions have shaped the surface of the Earth and impacted the evolution of plants and animals. Although more than two-thirds of known mineral species have biological imprints, how the biotransformation of minerals may have benefited microbial development, beyond nutritional and energetic use, remains enigmatic. In this research, we have shown that biogenic ferrihydrite nanoparticles are extensively formed at the interface between an actively growing fungus and an iron-containing mineral, hematite. These biogenic nanoparticles formed through the fungus-hematite interactions can behave as mimetic catalysts, similar to nanozymes that imitate peroxidase, which scavenges hydrogen peroxide for the mitigation of potential cytotoxicity. Evidence from various X-ray spectroscopic analyses indicated that non-lattice oxygen in the nanomaterials was chiefly responsible for this catalytic activity, rather than through the conventional mechanisms of iron redox chemistry. Cryo-scanning electron microscopy, high-resolution (â¼30 nm) 3D volume rendering, and biomass analyses further confirmed that the organism was active and capable of mediating the catalytic reactions. We therefore hypothesize that this confers an advantage to the organism in terms of protection from oxidative stress and ensuring the acquisition of essential iron. This work raises new questions about the roles of biogenic nanomaterials in the coevolution of the lithosphere and biosphere and provides a step toward understanding the feedback pathways controlling the evolution of biogenic mineral formation.
Assuntos
Compostos Férricos/metabolismo , Fungos/metabolismo , Ferro/metabolismo , Nanopartículas/metabolismo , Estresse Oxidativo/fisiologia , Biotransformação , Catálise , Oxirredução , Oxigênio , PeroxidaseRESUMO
The authors report a case of Trimeresurus albolabris (white-lipped green pit viper) bite in a 6-year-old girl living in rural Yuen Long. Despite repeated use of Agkistrodon halys antivenin, the patient developed severe coagulopathy with defibrination syndrome on the fourth day of envenomation, which was also refractory to therapy with fresh frozen plasma. When treatment was switched to green pit viper antivenin, the coagulopathy resolved promptly. The case is illustrative of the potential lethality to children of snakebites in Hong Kong and suggests that the A halys antivenin may not be effective for the treatment of T albolabris bites.
Assuntos
Antivenenos/uso terapêutico , Transtornos da Coagulação Sanguínea/terapia , Venenos de Crotalídeos/intoxicação , Mordeduras de Serpentes/complicações , Trimeresurus , Animais , Transtornos da Coagulação Sanguínea/etiologia , Criança , Venenos de Crotalídeos/antagonistas & inibidores , Feminino , Hong Kong , Humanos , Saúde da População Rural , Especificidade da EspécieAssuntos
Mieloma Múltiplo , Humanos , Mieloma Múltiplo/genética , Citometria de Fluxo/métodos , Plasmócitos , MonossomiaRESUMO
We present a genotyping method for simultaneously scoring 116,204 SNPs using oligonucleotide arrays. At call rates >99%, reproducibility is >99.97% and accuracy, as measured by inheritance in trios and concordance with the HapMap Project, is >99.7%. Average intermarker distance is 23.6 kb, and 92% of the genome is within 100 kb of a SNP marker. Average heterozygosity is 0.30, with 105,511 SNPs having minor allele frequencies >5%.