RESUMEN
INTRODUÇÃO: O mieloma múltiplo é uma neoplasia dos plasmócitos. Essas células neoplásicas proliferam na medula óssea impedindo o funcionamento das demais células hematológicas. As células neoplásicas produzem uma imunoglobulina monoclonal (proteína M) que é importante na fisiopatologia e no diagnóstico dessa doença. O mieloma múltiplo geralmente acomete adultos acima de 60 anos e estima-se que no Brasil a sua incidência anual esteja próximo à 1,2 indivíduos para cada 100.000 habitantes, com elevada letalidade. As manifestações clínicas mais comuns são dores ósseas, anemia e infecções recorrentes. As alterações mais comuns em exames de imagem e de laboratório incluem lesões líticas nos ossos, exames associados com insuficiência renal, hipercalcemia e anemia, além do achado da proteína M. Determinadas alterações citogenéticas estão associadas com o tratamento que deve ser instituído para o paciente e com o seu prognóstico. As alterações cromossômicas estudadas foram: t(4;14), del(17p13) e t(14;16). TECNOLOGIA: Citogenética por Hibridização in Situ por Fluorescência (FISH). PERGUNTA: Deve-se utilizar a citogenética por Hibridização In Situ por Fluorescência (FISH) versus citogenética convencional para detectar as alterações t(4:14), del(17p13) e t(14:16) em pacientes com mieloma múltiplo? EVIDÊNCIAS CLÍNICAS: Foi realizada busca de estudos que avaliassem a tecnologia nas bases de dados Embase, Medline (via Pubmed), Cochrane Library e LILACS. Após a triagem de 1346 relatos, 11 estudos observacionais foram selecionados. Nos domínios do QUADAS-2, a maioria dos estudos apresentou risco de viés incerto, exceto para o domínio Fluxo e Temporalidade, em que 81,8% dos estudos apresentaram baixo risco de viés. Os estudos incluídos analisaram uma amostra de 781 pacientes com mieloma múltiplo. Destes, 653 foram avaliadas pelo FISH e 719 pela citogenética convencional. A t(4;14) foi detectada em 11,3% (58/518) das amostras por FISH e 0,17% (1/607) por citogenética convencional. Os resultados da meta-análise mostraram que o FISH aumentou em 12% a detecção da t(4;14) quando comparado a citogenética convencional (RD: 0,12 [IC 95%: 0,06-0,19]; p < 0,0001; I2 : 52%). Em relação à del(17p13), esta foi detectada em 12,2% (80/653) das amostras por FISH e 1,6% (10/607) por citogenética convencional. O FISH aumentou em 12% a detecção da del(17p13) em comparação à citogenética convencional (RD: 0,12 [IC 95%: 0,04-0,20]; p < 0,0001; I 2 : 77%). Por fim, a t(14;16) foi detectada em 0,42% (2/478) das amostras por FISH e 0,17% (1/607) por citogenética convencional. Não houve diferença entre o FISH e a citogenética convencional para detecção da t(14;16) (RD: 0,00 [IC 95%: -0,01-0,02]; p = 0,41; I2 : 0%). A qualidade da evidência, avaliada pelo GRADE, foi considerada muito baixa para todos os desfechos avaliados. ANÁLISE DE IMPACTO ORÇAMENTÁRIO: Foram elaborados dois cenários, proposto e alternativo, considerando as estratégias de estadiamento incluindo FISH e citogenética convencional, variando a porcentagem de pacientes submetidos ao teste citogenético por FISH. A incorporação do FISH para o estadiamento dos pacientes com mieloma múltiplo pode provocar um incremento orçamentário em R$239.206,38 para o primeiro ano (2022), atingindo R$1.246.915,77 no quinto ano (2026), quando inicialmente 5% dos pacientes são submetidos ao FISH e ocorre aumento progressivo de 5% ao ano. O impacto em cinco anos seria de R$ 3.691.966,50. Quando inicialmente 10% dos pacientes diagnosticados com mieloma múltiplo são estadiados por meio do FISH, com o aumento progressivo de 10% ao ano, mantendo-se porcentagem constante para a citogenética convencional, o impacto orçamentário incremental seria de R$478.412,76 para o primeiro ano (2022), atingindo R$2.493.831,54 no quinto ano de incorporação (2026), sendo o valor acumulado em cinco anos de R$ 7.383.933,00. MONITORAMENTO DO HORIZONTE TECNOLÓGICO: As pesquisas nas bases de dados para monitoramento do horizonte tecnológico identificaram três modelos de sonda para o painel FISH em pacientes com mieloma múltiplo no FDA. No Clinical Trials nenhuma nova tecnologia para avaliação citogenética foi identificada. Em relação ao depósito de patentes, foi encontrado um depósito patentário chinês do ano de 2019. PERSPECTIVA DO PACIENTE: Foi abertaâ¯chamada pública conjunta para Perspectiva do Pacienteâ¯durante o período de 18/10/2021 a 24/10/2021,â¯queâ¯contou com quinze inscrições, sendo oâ¯representanteâ¯definidoâ¯por consenso do grupo.â¯No relato, o participante descreveu aspectos da suaâ¯vivência como paciente comâ¯mieloma múltiplo, destacando a rapidez na obtenção de diagnóstico, a realização do transplante de medula óssea e o uso de diferentes tecnologias durante o tratamento.â¯â¯Além disso, informou ter tidoâ¯boa resposta terapêutica à lenalidomida durante cinco anos, em virtude da progressão da doença depois desse intervalo temporal, passou a utilizar protocolo com daratumumabe, apresentando melhora geral do quadro clínico. CONSIDERAÇÕES FINAIS: O teste FISH já é realizado pelo Sistema Único de Saúde (SUS) no diagnóstico de outras doenças. Neste Relatório, foi analisado a ampliação de uso deste exame para o diagnóstico de mieloma múltiplo. Pelos achados desta revisão, o teste FISH foi superior à citogenética convencional no diagnóstico das alterações citogenéticas t(4;14) e del(17p13), que são alterações relativamente frequentes e relevantes para o tratamento e o prognóstico dos pacientes com esse tipo de câncer. A alteração t(14;16), por ter baixa prevalência nos pacientes com esse tipo de câncer, demanda que ela seja analisada em uma amostra maior de indivíduos para que seja evidenciada uma diferença significativa entre os dois métodos. No Brasil, os laboratórios de referência para doenças raras possuem a infraestrutura necessária para a realização dos exames e seria necessária a ampliação do uso por meio do SUS. Do ponto de vista da implementação, a capacitação de recursos humanos é um fator de extrema importância, uma vez que a maioria destes laboratórios, atualmente, não possui pessoal capacitado especificamente para analisar amostras de pacientes com mieloma múltiplo. As agências internacionais NICE e CADTH recomendam a realização do FISH como parte dos exames diagnósticos necessários para o estadiamento citogenético e a tomada de decisão quanto a estratégia terapêutica a ser empregada diante da classificação de risco dos pacientes com mieloma múltiplo. RECOMENDAÇÃO PRELIMINAR DA CONITEC: Os membros do Plenário presentes na 104ª Reunião da Conitec, no dia 08 de dezembro de 2021, deliberaram, por unanimidade, sem nenhum conflito de interesses, que a matéria fosse disponibilizada em consulta pública com recomendação preliminar favorável à ampliação de uso do teste citogenético por Hibridização in Situ por Fluorescência (FISH) na detecção de alterações citogenéticas de alto risco em pacientes com mieloma múltiplo. CONSULTA PÚBLICA: Por meio da Consulta Pública nº 116/2021, realizada entre os dias 27/12/2021 e 17/01/2022, foram recebidas 73 contribuições, todas favoráveis à ampliação do uso do FISH para detecção de alterações moleculares de alto risco em pacientes com mieloma múltiplo. As evidências científicas apresentadas reforçaram a importância do FISH enquanto método de identificação destas alterações moleculares, para as quais a citogenética convencional possui baixa sensibilidade. Na avaliação econômica e de impacto orçamentário, foram apontadas possibilidades de redução do custo do exame com a utilização de menor número de sondas de hibridização, dependendo do nível de treinamento dos profissionais. Pacientes e associações enfatizaram a necessidade de garantir o acesso ao exame pelo SUS e, como pontos negativos, o alto custo do exame na rede privada e a indisponibilidade atual do exame no sistema público de saúde. RECOMENDAÇÃO FINAL DA CONITEC: Os membros do Plenário presentes na 105ª Reunião da Conitec, no dia 09/02/2022, deliberaram, por unanimidade, sem nenhuma declaração de conflito de interesses, recomendar a ampliação de uso do teste citogenético por Hibridização in Situ por Fluorescência (FISH) na detecção de alterações citogenéticas de alto risco em pacientes com mieloma múltiplo. Foi assinado o Registro de Deliberação nº 695/2022. DECISÃO: Ampliar o uso do teste citogenético por Hibridização in Situ por Fluorescência (FISH) na detecção de alterações citogenéticas de alto risco em pacientes com mieloma múltiplo, no âmbito do Sistema Único de Saúde SUS, conforme a Portaria nº 20, publicada no Diário Oficial da União nº 49, seção 1, página 95, em 14 de março de 2022.
Asunto(s)
Humanos , Hibridación Fluorescente in Situ/instrumentación , Trastornos de los Cromosomas/diagnóstico , Análisis Citogenético/métodos , Mieloma Múltiple/fisiopatología , Sistema Único de Salud , Brasil , Análisis Costo-Beneficio/economíaRESUMEN
OBJECTIVE: To evaluate a microfluidics-based positive selection technology for isolating circulating trophoblasts (CTs) from peripheral blood of women whose pregnancies are affected by aneuploidy and to evaluate fetal karyotype using fluorescence in situ hybridization (FISH). METHOD: Ten 18-ml samples of peripheral blood were collected consecutively from pregnant women whose fetus was affected by aneuploidy. A preservation buffer was added, and the specimens were shipped overnight to the testing laboratory at ambient temperature. The specimen was infused into the fully automated microfluidics-based LiquidScan® instrument without pre-processing. This instrument contains microfluidic chips, which are coated with antibodies (anti-huEpCAM and a proprietary antibody mixture) specific to CT surface epitopes. FISH analysis was performed on the enriched cells. RESULTS: Fetal aneuploidy evaluated included trisomy 21 (n = 3), trisomy 18 (n = 1), trisomy 13 (n = 1), monosomy X (n = 3), and triploidy (n = 1). CTs for analysis by FISH were identified in all samples. The average number of mononucleate cells per 1 ml of whole blood was 2.11 (range 0.38-4.63) overall and was 2.67 (range 1.13-4.63) using the proprietary combination of antibodies. FISH results were concordant with the aneuploidy based on other testing in all cases. Multinucleate cells were searched for and identified in the last seven samples (average number: 0.84/1 ml). CONCLUSIONS: Our study demonstrates that the LiquidScan® , a high-sensitivity microfluidic platform, can enrich circulating trophoblasts (mononucleate and multinucleate). FISH can then be used to detect fetal aneuploidy.
Asunto(s)
Aneuploidia , Hibridación Fluorescente in Situ/métodos , Microfluídica/métodos , Trofoblastos/fisiología , Adulto , Femenino , Humanos , Hibridación Fluorescente in Situ/instrumentación , Hibridación Fluorescente in Situ/estadística & datos numéricos , Microfluídica/estadística & datos numéricos , Embarazo , Diagnóstico Prenatal/métodos , Trofoblastos/patologíaRESUMEN
The role of circulating plasma cells (CPCs) and circulating leukemic cells (CLCs) as biomarkers for several blood cancers, such as multiple myeloma and leukemia, respectively, have recently been reported. These markers can be attractive due to the minimally invasive nature of their acquisition through a blood draw (i.e., liquid biopsy), negating the need for painful bone marrow biopsies. CPCs or CLCs can be used for cellular/molecular analyses as well, such as immunophenotyping or fluorescence in situ hybridization (FISH). FISH, which is typically carried out on slides involving complex workflows, becomes problematic when operating on CLCs or CPCs due to their relatively modest numbers. Here, we present a microfluidic device for characterizing CPCs and CLCs using immunofluorescence or FISH that have been enriched from peripheral blood using a different microfluidic device. The microfluidic possessed an array of cross-channels (2-4 µm in depth and width) that interconnected a series of input and output fluidic channels. Placing a cover plate over the device formed microtraps, the size of which was defined by the width and depth of the cross-channels. This microfluidic chip allowed for automation of immunofluorescence and FISH, requiring the use of small volumes of reagents, such as antibodies and probes, as compared to slide-based immunophenotyping and FISH. In addition, the device could secure FISH results in <4 h compared to 2-3 days for conventional FISH.
Asunto(s)
Linfocitos B/inmunología , Análisis Citogenético/instrumentación , Inmunofenotipificación/instrumentación , Hibridación Fluorescente in Situ/instrumentación , Dispositivos Laboratorio en un Chip , Microfluídica/instrumentación , Técnicas de Diagnóstico Molecular/instrumentación , Células Neoplásicas Circulantes/inmunología , Donantes de Sangre , Línea Celular , Análisis Citogenético/métodos , Humanos , Inmunofenotipificación/métodos , Hibridación Fluorescente in Situ/métodos , Biopsia Líquida , Microfluídica/métodos , Técnicas de Diagnóstico Molecular/métodos , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patologíaRESUMEN
Fluorescence in situ hybridization (FISH) allows visualization of specific nucleic acid sequences within an intact cell or a tissue section. It is based on molecular recognition between a fluorescently labeled probe that penetrates the cell membrane of a fixed but intact sample and hybridizes to a nucleic acid sequence of interest within the cell, rendering a measurable signal. FISH has been applied to, for example, gene mapping, diagnosis of chromosomal aberrations and identification of pathogens in complex samples as well as detailed studies of cellular structure and function. However, FISH protocols are complex, they comprise of many fixation, incubation and washing steps involving a range of solvents and temperatures and are, thus, generally time consuming and labor intensive. The complexity of the process, the relatively high-priced fluorescent probes and the fairly high-end microscopy needed for readout render the whole process costly and have limited wider uptake of this powerful technique. In recent years, there have been attempts to transfer FISH assay protocols onto microfluidic lab-on-a-chip platforms, which reduces the required amount of sample and reagents, shortens incubation times and, thus, time to complete the protocol, and finally has the potential for automating the process. Here, we review the wide variety of approaches for lab-on-chip-based FISH that have been demonstrated at proof-of-concept stage, ranging from FISH analysis of immobilized cell layers, and cells trapped in arrays, to FISH on tissue slices. Some researchers have aimed to develop simple devices that interface with existing equipment and workflows, whilst others have aimed to integrate the entire FISH protocol into a fully autonomous FISH on-chip system. Whilst the technical possibilities for FISH on-chip are clearly demonstrated, only a small number of approaches have so far been converted into off-the-shelf products for wider use beyond the research laboratory.
Asunto(s)
Hibridación Fluorescente in Situ/instrumentación , Hibridación Fluorescente in Situ/métodos , Dispositivos Laboratorio en un Chip , Técnicas Analíticas Microfluídicas/instrumentación , Técnicas Analíticas Microfluídicas/métodos , Técnicas de Laboratorio Clínico/instrumentación , Técnicas de Laboratorio Clínico/métodosRESUMEN
Fluorescence in situ hybridization (FISH) method enables in situ genetic analysis of both metaphase and interphase cells from different types of material, including cell lines, cell smears, and fresh and paraffin-embedded tissue. Despite the growing number of commercially available FISH probes, still for large number of gene loci or chromosomal regions commercial probes are not available. Here we describe a simple method for generating FISH probes using bacterial artificial chromosomes (BAC). Due to genome-wide coverage of BAC clones, there are almost unlimited possibilities for the analysis of any genomic regions using BAC FISH probes.
Asunto(s)
Cromosomas Artificiales Bacterianos/genética , Sondas de ADN/aislamiento & purificación , ADN Bacteriano/aislamiento & purificación , Genómica/métodos , Hibridación Fluorescente in Situ/métodos , Técnicas Bacteriológicas/instrumentación , Técnicas Bacteriológicas/métodos , Técnicas de Cultivo de Célula/métodos , Línea Celular , Sondas de ADN/genética , ADN Bacteriano/genética , Nucleótidos de Desoxiuracil/química , Didesoxinucleótidos/química , Digoxigenina/análogos & derivados , Digoxigenina/química , Fluoresceínas/química , Colorantes Fluorescentes/química , Secciones por Congelación , Genómica/instrumentación , Humanos , Hibridación Fluorescente in Situ/instrumentación , Rodaminas/química , Coloración y Etiquetado/instrumentación , Coloración y Etiquetado/métodos , Uridina Trifosfato/análogos & derivados , Uridina Trifosfato/químicaAsunto(s)
Automatización de Laboratorios , Histocitoquímica/instrumentación , Hibridación Fluorescente in Situ/instrumentación , Leucemia Promielocítica Aguda , Femenino , Humanos , Leucemia Promielocítica Aguda/diagnóstico , Leucemia Promielocítica Aguda/genética , Leucemia Promielocítica Aguda/metabolismo , Leucemia Promielocítica Aguda/patología , MasculinoRESUMEN
Since fluorescence in situ hybridization (FISH) was used to define a prognostic heierarchy for chronic lymphocytic leukemia (CLL) in 2000, the method has been employed widely in cytogenetics laboratories worldwide. This chapter describes techniques and trouble-shooting to maximize the efficiency of microscope slide preparation for FISH analysis in CLL.
Asunto(s)
Análisis Citogenético/métodos , Hibridación Fluorescente in Situ/métodos , Leucemia Linfocítica Crónica de Células B/genética , Médula Ósea/patología , Aberraciones Cromosómicas , Sondas de ADN/genética , Humanos , Procesamiento de Imagen Asistido por Computador/instrumentación , Procesamiento de Imagen Asistido por Computador/métodos , Hibridación Fluorescente in Situ/instrumentación , Leucemia Linfocítica Crónica de Células B/sangre , Leucemia Linfocítica Crónica de Células B/patología , Microscopía Fluorescente/instrumentación , Microscopía Fluorescente/métodos , Programas InformáticosRESUMEN
Optical imaging of gene expression by fluorescence in situ hybridisation (FISH) in insects is often impeded by their pigmented cuticle. As most chemical bleaching agents are incompatible with FISH, we developed an RNA interference (RNAi)-based method for clearing cuticular pigmentation which enables the use of whole-mount body appendages for RNA FISH (termed RNA-i-FISH). Silencing laccase2 or tyrosine hydroxylase in two leaf beetles species (Chrysomela populi and Phaedon cochleariae) cleared their pigmented cuticle and decreased light absorbance. Subsequently, intact appendages (palps, antennae, legs) from RNAi-cleared individuals were used to image the expression and spatial distribution of antisense mRNA of two chemosensory genes encoding gustatory receptor and odorant-binding protein. Imaging did not work for RNAi controls because the pigmentation was retained, or for FISH controls (sense mRNA). Several bleaching agents were incompatible with FISH, because of degradation of RNA, lack of clearing efficacy or long incubation times. Overall, silencing pigmentation genes is a significant improvement over bleaching agents, enabling FISH in intact insect appendages.
Asunto(s)
Escarabajos/genética , Silenciador del Gen , Hibridación Fluorescente in Situ/métodos , Pigmentación/genética , Interferencia de ARN/fisiología , Animales , Extremidades/fisiología , Hibridación Fluorescente in Situ/instrumentación , Pigmentos Biológicos/análisisAsunto(s)
Mapeo Cromosómico/métodos , Genoma/genética , Imagenología Tridimensional/métodos , Imagen Molecular/métodos , Animales , Ensamble y Desensamble de Cromatina/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Hibridación Fluorescente in Situ/instrumentación , Hibridación Fluorescente in Situ/métodos , Microscopía/instrumentación , Microscopía/métodos , Modelos Genéticos , Imagen Molecular/instrumentaciónRESUMEN
Fluorescence in situ hybridization (FISH) is a common technique used to label DNA and/or RNA for detection of a genomic region of interest. However, the technique can be challenging, in particular when applied to single genes in human cancer cells. Here, we provide a step-by-step protocol for analysis of short (35â¯kb-300â¯kb) genomic regions in three dimensions (3D). We discuss the experimental design and provide practical considerations for 3D imaging and data analysis to determine chromatin folding. We demonstrate that 3D FISH using BACs (Bacterial Artificial Chromosomes) or fosmids can provide detailed information of the architecture of gene domains. More specifically, we show that mapping of specific chromatin landscapes informs on changes associated with estrogen stimulated gene activity in human breast cancer cell lines.
Asunto(s)
Ensamble y Desensamble de Cromatina , Mapeo Cromosómico/métodos , Imagenología Tridimensional/métodos , Hibridación Fluorescente in Situ/métodos , Imagen Molecular/métodos , Núcleo Celular , Cromatina/genética , Cromatina/metabolismo , Cromosomas Artificiales Bacterianos/genética , Humanos , Imagenología Tridimensional/instrumentación , Hibridación Fluorescente in Situ/instrumentación , Células MCF-7 , Imagen Molecular/instrumentación , Plásmidos/genéticaRESUMEN
Accurate assessment of human epidermal growth factor receptor 2 (HER-2) is crucial in selecting patients for targeted therapy. Commonly used methods for HER-2 testing are immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). Here we presented the implementation, optimization and standardization of two FISH protocols using breast cancer samples and assessed the impact of pre-analytical and analytical factors on HER-2 testing. Formalin fixed paraffin embedded (FFPE) tissue samples from 70 breast cancer patients were tested for HER-2 using PathVysion™ HER-2 DNA Probe Kit and two different paraffin pretreatment kits, Vysis/Abbott Paraffin Pretreatment Reagent Kit (40 samples) and DAKO Histology FISH Accessory Kit (30 samples). The concordance between FISH and IHC results was determined. Pre-analytical and analytical factors (i.e., fixation, baking, digestion, and post-hybridization washing) affected the efficiency and quality of hybridization. The overall hybridization success in our study was 98.6% (69/70); the failure rate was 1.4%. The DAKO pretreatment kit was more time-efficient and resulted in more uniform signals that were easier to interpret, compared to the Vysis/Abbott kit. The overall concordance between IHC and FISH was 84.06%, kappa coefficient 0.5976 (p < 0.0001). The greatest discordance (82%) between IHC and FISH was observed in IHC 2+ group. A standardized FISH protocol for HER-2 assessment, with high hybridization efficiency, is necessary due to variability in tissue processing and individual tissue characteristics. Differences in the pre-analytical and analytical steps can affect the hybridization quality and efficiency. The use of DAKO pretreatment kit is time-saving and cost-effective.
Asunto(s)
Neoplasias de la Mama/genética , Inmunohistoquímica , Hibridación Fluorescente in Situ/instrumentación , Hibridación Fluorescente in Situ/métodos , Receptor ErbB-2/genética , Femenino , Amplificación de Genes , Humanos , Hibridación de Ácido Nucleico , Adhesión en Parafina , Reproducibilidad de los ResultadosRESUMEN
The spatial organization of chromosomes in the nuclear space is an extensively studied field that relies on measurements of structural features and 3D positions of chromosomes with high precision and robustness. However, no tools are currently available to image and analyze chromosome territories in a high-throughput format. Here, we have developed High-throughput Chromosome Territory Mapping (HiCTMap), a method for the robust and rapid analysis of 2D and 3D chromosome territory positioning in mammalian cells. HiCTMap is a high-throughput imaging-based chromosome detection method which enables routine analysis of chromosome structure and nuclear position. Using an optimized FISH staining protocol in a 384-well plate format in conjunction with a bespoke automated image analysis workflow, HiCTMap faithfully detects chromosome territories and their position in 2D and 3D in a large population of cells per experimental condition. We apply this novel technique to visualize chromosomes 18, X, and Y in male and female primary human skin fibroblasts, and show accurate detection of the correct number of chromosomes in the respective genotypes. Given the ability to visualize and quantitatively analyze large numbers of nuclei, we use HiCTMap to measure chromosome territory area and volume with high precision and determine the radial position of chromosome territories using either centroid or equidistant-shell analysis. The HiCTMap protocol is also compatible with RNA FISH as demonstrated by simultaneous labeling of X chromosomes and Xist RNA in female cells. We suggest HiCTMap will be a useful tool for routine precision mapping of chromosome territories in a wide range of cell types and tissues.
Asunto(s)
Mapeo Cromosómico/métodos , Procesamiento de Imagen Asistido por Computador/métodos , Hibridación Fluorescente in Situ/métodos , Animales , Núcleo Celular/genética , Núcleo Celular/metabolismo , Mapeo Cromosómico/instrumentación , Cromosomas Humanos Par 18/genética , Cromosomas Humanos Par 18/metabolismo , Cromosomas Humanos X/genética , Cromosomas Humanos X/metabolismo , Cromosomas Humanos Y/genética , Cromosomas Humanos Y/metabolismo , Femenino , Fibroblastos , Humanos , Procesamiento de Imagen Asistido por Computador/instrumentación , Hibridación Fluorescente in Situ/instrumentación , Masculino , Cultivo Primario de Células/métodos , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Piel/citología , Coloración y Etiquetado/instrumentación , Coloración y Etiquetado/métodosRESUMEN
This case report describes the detection of intrahepatic bacteria in formalin-fixed paraffin-embedded histopathological sections from three dogs with neutrophilic, pyogranulomatous, or lymphoplasmacytic hepatitis and cholangiohepatitis. In each of these cases, eubacterial fluorescence in situ hybridization enabled colocalization of intrahepatic bacteria with neutrophilic and granulomatous inflammation in samples that were negative for bacteria when evaluated by routine hematoxylin and eosin histopathology augmented with histochemical stains. Positive responses to antimicrobial therapy were observed in of 2 out of 2 patients that were treated with antimicrobials. These findings suggest that eubacterial fluorescence in situ hybridization analysis of formalin-fixed paraffin-embedded histopathological sections is more sensitive than conventional histochemical stains for the diagnosis of bacteria-associated canine hepatitis.
Asunto(s)
Bacterias/aislamiento & purificación , Enfermedades de los Perros/diagnóstico , Hepatitis Animal/diagnóstico , Hibridación Fluorescente in Situ/veterinaria , Animales , Enfermedades de los Perros/microbiología , Perros , Hepatitis Animal/microbiología , Hibridación Fluorescente in Situ/instrumentación , Hibridación Fluorescente in Situ/métodos , InflamaciónRESUMEN
Human epidermal growth factor receptor 2 gene- (HER2-) targeted therapy for breast cancer relies primarily on HER2 overexpression established by immunohistochemistry (IHC) with borderline cases being further tested for amplification by fluorescence in situ hybridization (FISH). Manual interpretation of HER2 FISH is based on a limited number of cells and rather complex definitions of equivocal, polysomic, and genetically heterogeneous (GH) cases. Image analysis (IA) can extract high-capacity data and potentially improve HER2 testing in borderline cases. We investigated statistically derived indicators of HER2 heterogeneity in HER2 FISH data obtained by automated IA of 50 IHC borderline (2+) cases of invasive ductal breast carcinoma. Overall, IA significantly underestimated the conventional HER2, CEP17 counts, and HER2/CEP17 ratio; however, it collected more amplified cells in some cases below the lower limit of GH definition by manual procedure. Indicators for amplification, polysomy, and bimodality were extracted by factor analysis and allowed clustering of the tumors into amplified, nonamplified, and equivocal/polysomy categories. The bimodality indicator provided independent cell diversity characteristics for all clusters. Tumors classified as bimodal only partially coincided with the conventional GH heterogeneity category. We conclude that automated high-capacity nonselective tumor cell assay can generate evidence-based HER2 intratumor heterogeneity indicators to refine GH definitions.
Asunto(s)
Automatización de Laboratorios , Neoplasias de la Mama/genética , Carcinoma Ductal de Mama/genética , Hibridación Fluorescente in Situ , Receptor ErbB-2/genética , Adulto , Neoplasias de la Mama/patología , Carcinoma Ductal de Mama/patología , Femenino , Humanos , Hibridación Fluorescente in Situ/instrumentación , Hibridación Fluorescente in Situ/métodosRESUMEN
Circulating tumor cells (CTCs) hold promise as biomarkers to aid in patient treatment stratification and disease monitoring. Because the number of cells is a critical parameter for exploiting CTCs for predictive biomarker's detection, we developed a FISH (fluorescent in situ hybridization) method for CTCs enriched on filters (filter-adapted FISH [FA-FISH]) that was optimized for high cell recovery. To increase the feasibility and reliability of the analyses, we combined fluorescent staining and FA-FISH and developed a semi-automated microscopy method for optimal FISH signal identification in filtration-enriched CTCs . Here we present these methods and their use for the detection and characterization of ALK-, ROS1-, RET-rearrangement in CTCs from non-small-cell lung cancer and ERG-rearrangements in CTCs from prostate cancer patients.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas , Reordenamiento Génico , Neoplasias Pulmonares , Células Neoplásicas Circulantes/metabolismo , Neoplasias de la Próstata , Proteínas Tirosina Quinasas , Proteínas Proto-Oncogénicas c-ret , Proteínas Proto-Oncogénicas , Proteínas Tirosina Quinasas Receptoras , Quinasa de Linfoma Anaplásico , Carcinoma de Pulmón de Células no Pequeñas/sangre , Carcinoma de Pulmón de Células no Pequeñas/genética , Femenino , Humanos , Hibridación Fluorescente in Situ/instrumentación , Hibridación Fluorescente in Situ/métodos , Neoplasias Pulmonares/sangre , Neoplasias Pulmonares/genética , Masculino , Neoplasias de la Próstata/sangre , Neoplasias de la Próstata/genética , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-ret/genética , Proteínas Proto-Oncogénicas c-ret/metabolismo , Proteínas Tirosina Quinasas Receptoras/genética , Proteínas Tirosina Quinasas Receptoras/metabolismo , Regulador Transcripcional ERG/genética , Regulador Transcripcional ERG/metabolismoRESUMEN
Las lesiones lipomatosas uterinas son una variante poco frecuente de los tumores mesenquimales benignos. Se dividen en lipomas puros y lesiones lipomatosas (difusas o focales) en el seno de un leiomioma. Presentamos el caso de una mujer de 70 años con histerectomía simple tras el diagnóstico ecográfico de «mioma gigante». Macroscópicamente la lesión correspondía a una formación lipomatosa de 13,8cm de eje máximo e histológicamente estaba constituida por células adiposas maduras, sin septos, y con cambios atróficos y traumáticos, positivas para S100 y vimentina y negativas para MDM2 y CDK4 por inmunohistoquímica e hibridación fluorescente in situ. Focalmente se identificaban áreas de degeneración mixoide y no se observaba necrosis ni hemorragia. El estudio ultraestructural fue congruente con una proliferación celular con diferenciación adiposa (AU)
Uterine fatty lesions are a rare variant of benign mesenchymal tumours. They are divided into pure and lipomatous lesions (diffuse or focal) within a leiomyoma. We report the case of a 70 year old woman who underwent a simple hysterectomy after an ultrasound diagnosis of «giant leyomioma». Macroscopically the lesion corresponded to a lipomatous formation of 13.8cm maximum diametre. Histologically the lesion consisted of mature adipose cells without septa and traumatic and atrophic changes. There was focal myxoid degeneration, but necrosis and hemorrhage were not observed. The cells were S100 and vimentin positive on immunochemistry. MDM2 and CDK4 were negative by immunochemistry and fluorescence in situ hybridization. The ultrastructural study was consistent with a cell proliferation with adipose differentiation (AU)
Asunto(s)
Humanos , Femenino , Anciano , Lipoma/diagnóstico , Lipoma/patología , Neoplasias Uterinas/patología , Inmunohistoquímica/instrumentación , Inmunohistoquímica/métodos , Mioma/complicaciones , Mioma/patología , Hibridación Fluorescente in Situ/instrumentación , Condrosarcoma Mesenquimal/patología , Útero/patología , Microscopía/instrumentación , Tomografía Computarizada de Emisión/métodosRESUMEN
The first methods for visualizing RNAs within cells were designed for simple imaging of specific transcripts in cells or tissues and since then significant technical advances have been made in this field. Today, high-resolution images can be obtained, enabling visualization of single transcript molecules, quantitative analyses of images, and precise localization of RNAs within cells as well as co-localization of transcripts with specific proteins or other molecules. In addition, tracking of RNA dynamics within single cell has become possible. RNA imaging techniques have been utilized for investigating the role of mutant RNAs in a number of human disorders caused by simple microsatellite expansions. These diseases include myotonic dystrophy type 1 and 2, amyotrophic lateral sclerosis/frontotemporal dementia, fragile X-associated tremor/ataxia syndrome, and Huntington's disease. Mutant RNAs with expanded repeats tend to aggregate predominantly within cell nuclei, forming structures called RNA foci. In this study, we demonstrate methods for fluorescent visualization of RNAs in both fixed and living cells using the example of RNAs containing various expanded repeat tracts (CUG, CCUG, GGGGCC, CGG, and CAG) from experiment design to image analysis. We describe in detail 2D and 3D fluorescence in situ hybridization (FISH) protocols for imaging expanded repeats RNAs, and we review briefly live imaging techniques used to characterize RNA foci formed by mutant RNAs. These methods could be used to image the entire cellular pathway of RNAs, from transcription to degradation.
Asunto(s)
Colorantes Fluorescentes/química , Hibridación Fluorescente in Situ/métodos , Imagen Molecular/métodos , ARN/química , Expansión de Repetición de Trinucleótido , Esclerosis Amiotrófica Lateral/genética , Ataxia/genética , Células Cultivadas , Síndrome del Cromosoma X Frágil/genética , Demencia Frontotemporal/genética , Humanos , Enfermedad de Huntington/genética , Hibridación Fluorescente in Situ/instrumentación , Linfocitos , Microscopía Confocal , Imagen Molecular/instrumentación , Distrofia Miotónica/genética , ARN/genética , Temblor/genéticaRESUMEN
Peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) is a highly specific molecular method widely used for microbial identification. Nonetheless, and due to the detection limit of this technique, a time-consuming pre-enrichment step is typically required before identification. In here we have developed a lab-on-a-chip device to concentrate cell suspensions and speed up the identification process in yeasts. The PNA-FISH protocol was optimized to target Saccharomyces cerevisiae, a common yeast that is very relevant for several types of food industries. Then, several coin-sized microfluidic devices with different geometries were developed. Using Computational fluid dynamics (CFD), we modeled the hydrodynamics inside the microchannels and selected the most promising options. SU-8 structures were fabricated based on the selected designs and used to produce polydimethylsiloxane-based microchips by soft lithography. As a result, an integrated approach combining microfluidics and PNA-FISH for the rapid identification of S. cerevisiae was achieved. To improve fluid flow inside microchannels and the PNA-FISH labeling, oxygen plasma treatment was applied to the microfluidic devices and a new methodology to introduce the cell suspension and solutions into the microchannels was devised. A strong PNA-FISH signal was observed in cells trapped inside the microchannels, proving that the proposed methodology works as intended. The microfluidic designs and PNA-FISH procedure described in here should be easily adaptable for detection of other microorganisms of similar size.
Asunto(s)
Hibridación Fluorescente in Situ/instrumentación , Dispositivos Laboratorio en un Chip , Ácidos Nucleicos de Péptidos/metabolismo , Saccharomyces cerevisiae/aislamiento & purificación , Diseño de Equipo , Oxígeno/química , Gases em Plasma/química , Saccharomyces cerevisiae/metabolismoRESUMEN
A biological system is by definition a dynamic environment encompassing kinetic processes that occur at different length scales and time ranges. To explore this type of system, spatial information needs to be acquired at different time scales. This means overcoming significant hurdles, including the need for stable and precise labeling of the required probes and the use of state of the art optical methods. However, to interpret the acquired data, biophysical models that can account for these biological mechanisms need to be developed. The structure and function of a biological system are closely related to its dynamic properties, thus further emphasizing the importance of identifying the rules governing the dynamics that cannot be directly deduced from information on the structure itself. In eukaryotic cells, tens of thousands of genes are packed in the small volume of the nucleus. The genome itself is organized in chromosomes that occupy specific volumes referred to as chromosome territories. This organization is preserved throughout the cell cycle, even though there are no sub-compartments in the nucleus itself. This organization, which is still not fully understood, is crucial for a large number of cellular functions such as gene regulation, DNA breakage repair and error-free cell division. Various techniques are in use today, including imaging, live cell imaging and molecular methods such as chromosome conformation capture (3C) methods to better understand these mechanisms. Live cell imaging methods are becoming well established. These include methods such as Single Particle Tracking (SPT), Continuous Photobleaching (CP), Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Correlation Spectroscopy (FCS) that are currently used for studying proteins, RNA, DNA, gene loci and nuclear bodies. They provide crucial information on its mobility, reorganization, interactions and binding properties. Here we describe how these dynamic methods can be used to gather information on genome organization, its stabilization mechanisms and the proteins that take part in it.
Asunto(s)
Núcleo Celular/ultraestructura , Cromatina/ultraestructura , Recuperación de Fluorescencia tras Fotoblanqueo/métodos , Genoma , Hibridación Fluorescente in Situ/métodos , Espectrometría de Fluorescencia/métodos , Animales , Línea Celular Tumoral , Núcleo Celular/metabolismo , Cromatina/metabolismo , Fibroblastos/metabolismo , Fibroblastos/ultraestructura , Recuperación de Fluorescencia tras Fotoblanqueo/instrumentación , Regulación de la Expresión Génica , Humanos , Hibridación Fluorescente in Situ/instrumentación , Lamina Tipo A/genética , Lamina Tipo A/metabolismo , Ratones , Espectrometría de Fluorescencia/instrumentación , Telómero/metabolismo , Telómero/ultraestructuraRESUMEN
Microfluidics offer powerful tools for the control, manipulation, and analysis of cells, in particular for the assessment of cell malignancy or the study of cell subpopulations. However, implementing complex biological protocols on chip remains a challenge. Sample preparation is often performed off chip using multiple manually performed steps, and protocols usually include different dehydration and drying steps that are not always compatible with a microfluidic format.Here, we report the implementation of a Fluorescence in situ Hybridization (FISH) protocol for the molecular typing of cancer cells in a simple and low-cost device. The geometry of the chip allows integrating the sample preparation steps to efficiently assess the genomic content of individual cells using a minute amount of sample. The FISH protocol can be fully automated, thus enabling its use in routine clinical practice.