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1.
Medicine (Baltimore) ; 99(5): e18920, 2020 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-32000402

RESUMEN

The aim of this study was to compare the diagnostic yield of conventional cytology (CC) with ethanol-based fixation, a cytological analysis using an ethanol based fixative system including a cell block procedure (EBF) to evaluate indeterminate biliary strictures (IBStr). We also compared additionally taken fluorescence-guided forceps biopsies (FB) with EBF concerning a potential additive diagnostic benefit.Early detection and accurate diagnosis are crucial for patients with suspected carcinoma within the biliary tree to preserve curative treatment options but diagnostics and patient care in the evaluation of IBStr are still challenging. ERC-guided brush cytology is the gold standard of nonsurgical evaluation of IBStr. However, accuracy is generally low. New specimen processing's are needed to higher the diagnostic yield in the evaluation of IBStr.We performed a retrospective evaluation in 404 patients referred for further diagnosis of IBStr. Gold standard was defined as surgically obtained histology or patient follow-up of at least 1 year to diagnose or exclude malignancy.Three hundred thirty-four patients were included into the final analysis. One hundred seventy-two strictures were malignant, 162 strictures benign. One hundred seventeen specimens were evaluated by CC, 217 processed by EBF. EBF performed significantly better in terms of sensitivity (24.6% vs 60%, P < .001) and accuracy (59.0% vs 75.1%, P = .006). Fifty-eight FB were additionally taken and showed a numerically improved sensitivity compared to EBF alone (80% vs 62.9%, P = .19).EBF is a simple and inexpensive technique that substantially improved sensitivity and accuracy in the evaluation of IBStr. FB specimen did not significantly improve diagnostic yield.


Asunto(s)
Conductos Biliares/diagnóstico por imagen , Conductos Biliares/patología , Colangiografía , Endoscopía del Sistema Digestivo , Etanol , Fijadores , Adulto , Anciano , Anciano de 80 o más Años , Neoplasias de los Conductos Biliares/diagnóstico por imagen , Neoplasias de los Conductos Biliares/patología , Biopsia , Diagnóstico Diferencial , Femenino , Estudios de Seguimiento , Humanos , Masculino , Persona de Mediana Edad , Imagen Óptica , Estudios Retrospectivos , Sensibilidad y Especificidad , Fijación del Tejido
3.
J Clin Pathol ; 73(1): 42-46, 2020 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-31471466

RESUMEN

AIMS: In previous studies, skin retraction of dermato-pathological specimens after the surgical excision of tumours was calculated at 30% for the surface, with approximately 20% for the length and 15% for the width. The aim of this study was to analyse the retraction of the specimens and the retraction of the lesion and the margins. METHODS: Patients who underwent excision of a skin tumour between January 2013 and July 2014 were randomly included. RESULTS: A total of 104 patients was included. There were 52% male with a mean age of 68.3 years. Seventy-eight per cent of the lesions were malignant (51% were basal cell carcinoma, 10% squamous cell carcinoma). The retraction of the area of the specimen (29%) was significantly greater than the retraction of the tumour (21%). On multivariate analysis, the localisation and the duration of fixation were independent predictors of the specimen area retraction. The retraction of the specimen was 17% in length and 15% in width. The retraction of the margins was calculated at 19% in length and 12% in width. The surgeon correctly evaluated the localisation of the smallest margin in 55% of cases. CONCLUSIONS: Our study provided additional data regarding the retraction of the tumours and margins. The guidelines for surgical excision of skin cancers recommend a clinical margin before excision, but the evaluation of the sufficiency of the margins is based on histological measurement. Our data are useful for the interpretation of the sufficiency of the margins.


Asunto(s)
Márgenes de Escisión , Neoplasias Cutáneas/patología , Neoplasias Cutáneas/cirugía , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Biopsia , Niño , Femenino , Humanos , Masculino , Persona de Mediana Edad , Neoplasia Residual , Valor Predictivo de las Pruebas , Estudios Prospectivos , Factores de Tiempo , Fijación del Tejido/métodos , Resultado del Tratamiento , Adulto Joven
4.
Adv Exp Med Biol ; 1188: 21-30, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31820381

RESUMEN

Protein lysates from a variety of sample materials, e.g., cell lines, serum, frozen tissues, formalin-fixed and paraffin-embedded (FFPE) tissues, and fine needle aspirates, are compatible with the analysis using reverse phase protein arrays (RPPAs). Due to this diversity of input material, lysate preparation is one of the critical steps for obtaining reliable RPPA results. The challenges include, but are not limited to, achieving complete solubilization, avoiding chemical or enzymatic protein modifications, and degradation. In this chapter, preparations of lysates for RPPA analysis are described, focusing on human tissue samples. Special emphasis is given to recently published ISO International Standards for the preanalytical phase.


Asunto(s)
Formaldehído , Análisis por Matrices de Proteínas , Proteínas , Humanos , Adhesión en Parafina , Análisis por Matrices de Proteínas/métodos , Proteínas/química , Fijación del Tejido
5.
Invest Ophthalmol Vis Sci ; 60(14): 4759-4773, 2019 11 01.
Artículo en Inglés | MEDLINE | ID: mdl-31738824

RESUMEN

Purpose: Reaggregates from E6 embryonic chicken retina exhibit areas corresponding to an inner plexiform layer (IPL), which presents an ideal in vitro model to test conditions and constraints of cholinergic and glutamatergic network formation, providing a basis for retinal tissue engineering. Here, we show that ipl formation is regulated by cholinergic starburst amacrine cells (SACs), a glial scaffold and by L-glutamate. Methods: Rosetted spheroids were cultured in absence or presence of 0.2 to 0.4 mM L-glutamate and analyzed by immuno- and enzyme histochemistry, proliferation, and apoptosis assays. Results: After 2 days in vitro (div), ipl formation was announced by acetylcholinesterase+ (AChE) and choline acetyltransferase+ (ChAT) cells. Individual vimentin+ or transitin+ Müller glial cell precursors (MCPs) in ipl centers coexpressed ChAT. Comparable to in vivo, pairwise arranged ChAT+ SACs formed two laminar subbands. Projections of calretinin+ amacrine cells (ACs) into ipl associated with MCP processes. In L-glutamate-, or NMDA-treated spheroids ipls were disrupted, including loss of SACs and MCs; coincubation with NMDA receptor inhibitor MK-801 prevented these effects. Also, many Pax6+ cells, comprising most ACs, were lost, while rho4D2+ rod photoreceptors were increased. Cell proliferation was slightly increased, while apoptosis remained unaffected. Conclusions: This demonstrated: (1) a far-advanced differentiation of an IPL in retinal spheroids, as never described before; (2) ipl sublamination was initiated by cholinergic precursor cells, which-functioning as "ipl founder cells"-(3) gave rise to neurons and glial cells; (4) these SACs and MCPs together organized ipl formation; and (5) this process was counteracted by NMDA-dependent glutamate actions.


Asunto(s)
Diferenciación Celular/fisiología , Colinérgicos/farmacología , Células Ependimogliales/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Retina/embriología , Transducción de Señal/fisiología , Esferoides Celulares/efectos de los fármacos , Acetilcolinesterasa/metabolismo , Animales , Proliferación Celular/fisiología , Células Cultivadas , Embrión de Pollo , Colina O-Acetiltransferasa/metabolismo , Crioultramicrotomía , Ácido Glutámico/farmacología , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Neuronas Retinianas/citología , Esferoides Celulares/metabolismo , Fijación del Tejido , Vimentina/metabolismo
6.
BMC Genomics ; 20(1): 689, 2019 Sep 02.
Artículo en Inglés | MEDLINE | ID: mdl-31477010

RESUMEN

BACKGROUND: Archived formalin fixed paraffin embedded (FFPE) samples are valuable clinical resources to examine clinically relevant morphology features and also to study genetic changes. However, DNA quality and quantity of FFPE samples are often sub-optimal, and resulting NGS-based genetics variant detections are prone to false positives. Evaluations of wet-lab and bioinformatics approaches are needed to optimize variant detection from FFPE samples. RESULTS: As a pilot study, we designed within-subject triplicate samples of DNA derived from paired FFPE and fresh frozen breast tissues to highlight FFPE-specific artifacts. For FFPE samples, we tested two FFPE DNA extraction methods to determine impact of wet-lab procedures on variant calling: QIAGEN QIAamp DNA Mini Kit ("QA"), and QIAGEN GeneRead DNA FFPE Kit ("QGR"). We also used negative-control (NA12891) and positive control samples (Horizon Discovery Reference Standard FFPE). All DNA sample libraries were prepared for NGS according to the QIAseq Human Breast Cancer Targeted DNA Panel protocol and sequenced on the HiSeq 4000. Variant calling and filtering were performed using QIAGEN Gene Globe Data Portal. Detailed variant concordance comparisons and mutational signature analysis were performed to investigate effects of FFPE samples compared to paired fresh frozen samples, along with different DNA extraction methods. In this study, we found that five times or more variants were called with FFPE samples, compared to their paired fresh-frozen tissue samples even after applying molecular barcoding error-correction and default bioinformatics filtering recommended by the vendor. We also found that QGR as an optimized FFPE-DNA extraction approach leads to much fewer discordant variants between paired fresh frozen and FFPE samples. Approximately 92% of the uniquely called FFPE variants were of low allelic frequency range (< 5%), and collectively shared a "C > T|G > A" mutational signature known to be representative of FFPE artifacts resulting from cytosine deamination. Based on control samples and FFPE-frozen replicates, we derived an effective filtering strategy with associated empirical false-discovery estimates. CONCLUSIONS: Through this study, we demonstrated feasibility of calling and filtering genetic variants from FFPE tissue samples using a combined strategy with molecular barcodes, optimized DNA extraction, and bioinformatics methods incorporating genomics context such as mutational signature and variant allelic frequency.


Asunto(s)
Neoplasias de la Mama/genética , Análisis Mutacional de ADN/métodos , ADN de Neoplasias/aislamiento & purificación , Mama/química , Femenino , Fijadores , Formaldehído , Genómica/métodos , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Adhesión en Parafina , Fijación del Tejido
7.
Biomed Res Int ; 2019: 2054262, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31392208

RESUMEN

Micro-CT imaging is a well-established morphological method for the visualization of animal models. We used ethanol fixation of the mouse brains to perform high-resolution micro-CT scans showing in great details brain grey and white matters. It was possible to identify more than 50 neuroanatomical structures on the 5 selected coronal sections. Among white matter structures, we identified fornix, medial lemniscus, crossed tectospinal pathway, mammillothalamic tract, and the sensory root of the trigeminal ganglion. Among grey matter structures, we identified basal nuclei, habenular complex, thalamic nuclei, amygdala, subparts of hippocampal formation, superior colliculi, Edinger-Westphal nucleus, and others. We suggest that micro-CT of the mouse brain could be used for neurohistological lesions evaluation as an alternative to classical neurohistology because it does not destroy brain tissue.


Asunto(s)
Encéfalo/diagnóstico por imagen , Etanol/química , Fijación del Tejido , Microtomografía por Rayos X , Animales , Masculino , Ratones
8.
Zhongguo Fei Ai Za Zhi ; 22(7): 433-439, 2019 Jul 20.
Artículo en Chino | MEDLINE | ID: mdl-31315782

RESUMEN

BACKGROUND: Epidermal growth factor receptor (EGFR) mutation is the most common gene mutation in patients with non-small cell lung cancer (NSCLC). Many international guidelines are recommended to detected the EGFR mutation before the treatment of advanced non-small cell lung cancer. To investigate the possibility of EGFR mutation testing on DNA extracted from fixation liquid of lung cancer biopsy. METHODS: Fixation liquid of lung cancer biopsy was collected and stored at -80 oC after centrifugal. DNA was extracted and EGFR gene mutation was detected by ARMS. Compared with EGFR mutation status of paraffin-embedded tissues, the consistency, the sensitivity and specificity of EGFR mutation testing were analyzed. RESULTS: Among the 28 cases of EGFR mutation positive and 20 cases of EGFR mutation negative previously tested on paraffin-embedded tissue by clinic test, 20 cases with EGFR mutation positive and 20 cases with negative were detected by matched fixation liquid of lung cancer biopsy, respectively. The sensitivity and specificity were 71.4% and 100%. Moreover, 52 paraffin-embedded tissues and matched fixation liquid of lung cancer biopsy with unknown EGFR mutation status were detected, and the EGFR mutation positive rate were 36.5% and 28.8% respectively. The sensitivity and specificity of fixation liquid of lung cancer biopsy were 78.9% and 100.0%. CONCLUSIONS: Extracting the DNA from fixation liquid of lung cancer biopsy may be a kind of feasible way to detect EGFR mutation.


Asunto(s)
Análisis Mutacional de ADN/métodos , ADN/genética , ADN/aislamiento & purificación , Receptores ErbB/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Mutación , Biopsia , Exones/genética , Estudios de Factibilidad , Femenino , Humanos , Masculino , Persona de Mediana Edad , Fijación del Tejido
9.
Acta Histochem ; 121(6): 750-760, 2019 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-31277893

RESUMEN

Safety concerns on the toxic and carcinogenic effects of formalin exposure have drawn increasing attention to the search for alternative low risk fixatives for processing tissue specimens in laboratories worldwide. Alcohol-based fixatives are considered some of the most promising alternatives. We evaluated the performance of alcohol-fixed paraffin-embedded (AFPE) samples from Sprague-Dawley (SD) rats analyzing tissue morphology, protein and nucleic acid preservation after short and extremely long fixation times (up to 7 years), using formalin-fixed paraffin-embedded (FFPE) samples as a comparator fixative. Following short and long-term alcohol fixation, tissue morphology and cellular details in tissues, evaluated by scoring stained sections (Hematoxylin-Eosin and Mallory's trichrome), were optimally preserved if compared to formalin fixation. Immunoreactivity of proteins (Ki67, CD3, PAX5, CD68), evaluated by immunohistochemistry, showed satisfactory results when the fixation period did not exceed 1 year. Finally, we confirm the superiority of alcohol fixation compared to formalin, in terms of quantity of nucleic acid extracted from paraffin blocks, even after an extremely long time of alcohol fixation. Our results confirm that alcohol fixation is a suitable and safe alternative to formalin for pathological evaluations. There is a need for standardization of formalin-free methods and harmonization of diagnosis in pathology department worldwide.


Asunto(s)
Etanol/química , Fijadores/química , Fijación del Tejido , Animales , Inmunohistoquímica , Especificidad de Órganos , Ratas , Ratas Sprague-Dawley
10.
Virchows Arch ; 475(6): 693-699, 2019 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-31267202

RESUMEN

The bone is a frequent localization for lung non-small cell cancer metastasis; decalcification is required to permit tissue section. Pre-analytical conditions can influence the detection of immunohistochemical markers. The aim of our work is to evaluate PD-L1 expression in samples with delayed fixation and in decalcified tissue with chelating agent or acid at different time. Tumor-expressing PD-L1 and placental tissue were fixed at different times or decalcified with an acid decalcifier or EDTA for different durations. For 22C3 antibody, when tissues were decalcified with DC3, there was a significant decrease in the percentage of tumor cells or placental villi stained which after 4 h (p = 0.035 at 4 h). When EDTA is used for 22C3 antibody, there was a slight decrease in the percentage of stained tumor cells or villi but although there was a trend (p = 0.058 at 20 h), this was never statistically significant. For E1L3N antibody, when tissues were decalcified either with DC3 or EDTA, there was no significant decrease for the proportion of stained tumor cells or placental villi, neither for staining intensity for the first 24 h. The proportion of placental villi and tumor stained or intensity of staining was not significantly lower for any sample after delayed fixation also at 24 h for both PD-L1 clones. Delayed fixation does not affect the proportion of stained cell and intensity with PD-L1 immunohistochemistry. Decalcification also performed with EDTA lower the proportion and intensity of stained cells with PD-L1 immunohistochemistry.


Asunto(s)
Antígeno B7-H1/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Fijación del Tejido , Anticuerpos Monoclonales/inmunología , Biomarcadores de Tumor/metabolismo , Células Clonales/patología , Femenino , Humanos , Inmunohistoquímica/métodos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Embarazo , Coloración y Etiquetado/métodos , Factores de Tiempo , Fijación del Tejido/métodos
12.
Virchows Arch ; 475(2): 191-199, 2019 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-31264038

RESUMEN

Pre-analytical factors, such as fixation time, influence morphology of diagnostic and predictive immunohistochemical staining, which are increasingly used in the evaluation of lung cancer. Our aim was to investigate if variations in fixation time influence the outcome of immunohistochemical staining in lung cancer. From lung resections, specimen with tumor size bigger than 4 cm, 10 samples were obtained: 2 were put through the standard fixation protocol, 5 through the delayed, and 3 through the prolonged fixation protocol. After paraffin embedding, tissue microarrays (TMAs) were made. They were stained with 20 antibodies and scored for quality and intensity of staining. Samples with delay in fixation showed loss of TMA cores on glass slides and deterioration of tissue quality leading to reduction in the expression of CK 7, Keratin MNF116, CAM 5.2, CK 5/6, TTF-1, C-MET, Napsin A, D2-40, and PD-L1. Prolonged fixation had no influence on the performance of immunohistochemical stains. Delay of fixation negatively affects the expression of different immunohistochemical markers, influencing diagnostic (cytokeratins) and predictive (PD-L1) testing. These results emphasize the need for adequate fixation of resection specimen.


Asunto(s)
Biomarcadores de Tumor/análisis , Carcinoma de Pulmón de Células no Pequeñas/patología , Inmunohistoquímica/métodos , Neoplasias Pulmonares/patología , Fijación del Tejido/métodos , Humanos , Coloración y Etiquetado/métodos
13.
N Biotechnol ; 52: 104-109, 2019 Sep 25.
Artículo en Inglés | MEDLINE | ID: mdl-31150841

RESUMEN

The accuracy of histopathological diagnosis is strictly reliant on adequate tissue preservation, which is completely dependent on pre-analytical variables. Among these variables, the time interval between the end of surgical excision to the onset of fixation (the cold ischemia time) may adversely affect preservation of tissue morphology, influencing the interpretation and reproducibility of diagnosis. During this time interval, the activation of enzymes may produce autolysis and degradation of antigens and nucleic acids, thus potentially affecting immunocytochemical and molecular results. Several studies have described under-vacuum at 4 °C storage of fresh surgical specimens as a safe and reliable method to control cold ischemia and preserve fresh tissues, as well as to standardize fixation times and implement tissue-banking. This review article gives a systematic overview of the advantages and drawbacks of the use of under-vacuum tissue preservation and cooling in surgical pathology, highlighting the impact this procedure may have on diagnostic and experimental pathology. It also documents our experience acquired within daily practice and national and international projects.


Asunto(s)
Conservación de Tejido , Vacio , Supervivencia Celular , Humanos , Proteómica , Fijación del Tejido , Transcriptoma/genética
14.
PLoS One ; 14(6): e0214656, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31194770

RESUMEN

Glutaraldehyde-fixed bovine pericardium is currently the most popular biomaterial utilized in the creation of bioprosthetic heart valves. However, recent studies indicate that glutaraldehyde fixation results in calcification and structural valve deterioration, limiting the longevity of bioprosthetic heart valves. Additionally, glutaraldehyde fixation renders the tissue incompatible with constructive recipient cellular repopulation, remodeling and growth. Use of unfixed xenogeneic biomaterials devoid of antigenic burden has potential to overcome the limitations of current glutaraldehyde-fixed biomaterials. Heart valves undergo billion cycles of opening and closing throughout the patient's lifetime. Therefore, understanding the response of unfixed tissues to cyclic loading is crucial to these in a heart valve leaflet configuration. In this manuscript we quantify the effect of cyclic deformation on cycle dependent strain, structural, compositional and mechanical properties of fixed and unfixed tissues. Glutaraldehyde-fixed bovine pericardium underwent marked cyclic dependent strain, resulting from significant changes in structure, composition and mechanical function of the material. Conversely, unfixed bovine pericardium underwent minimal strain and maintained its structure, composition and mechanical integrity. This manuscript demonstrates that unfixed bovine pericardium can withstand cyclic deformations equivalent to 6 months of in vivo heart valve leaflet performance.


Asunto(s)
Fenómenos Biomecánicos , Glutaral/farmacología , Válvulas Cardíacas/fisiología , Preservación de Órganos/veterinaria , Animales , Fenómenos Biomecánicos/efectos de los fármacos , Bioprótesis , Bovinos , Análisis de Elementos Finitos , Prótesis Valvulares Cardíacas , Válvulas Cardíacas/efectos de los fármacos , Porcinos , Fijación del Tejido
15.
J Mass Spectrom ; 54(8): 716-727, 2019 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-31254303

RESUMEN

Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) is a molecular imaging technology uniquely capable of untargeted measurement of proteins, lipids, and metabolites while retaining spatial information about their location in situ. This powerful combination of capabilities has the potential to bring a wealth of knowledge to the field of molecular histology. Translation of this innovative research tool into clinical laboratories requires the development of reliable sample preparation protocols for the analysis of proteins from formalin-fixed paraffin-embedded (FFPE) tissues, the standard preservation process in clinical pathology. Although ideal for stained tissue analysis by microscopy, the FFPE process cross-links, disrupts, or can remove proteins from the tissue, making analysis of the protein content challenging. To date, reported approaches differ widely in process and efficacy. This tutorial presents a strategy derived from systematic testing and optimization of key parameters, for reproducible in situ tryptic digestion of proteins in FFPE tissue and subsequent MALDI IMS analysis. The approach describes a generalized method for FFPE tissues originating from virtually any source.


Asunto(s)
Proteínas/análisis , Manejo de Especímenes/métodos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Análisis de Matrices Tisulares/métodos , Formaldehído/química , Humanos , Adhesión en Parafina , Proteolisis , Fijación del Tejido , Tripsina/química
16.
Nat Protoc ; 14(6): 1756-1771, 2019 06.
Artículo en Inglés | MEDLINE | ID: mdl-31053799

RESUMEN

In vitro 3D organoid systems have revolutionized the modeling of organ development and diseases in a dish. Fluorescence microscopy has contributed to the characterization of the cellular composition of organoids and demonstrated organoids' phenotypic resemblance to their original tissues. Here, we provide a detailed protocol for performing high-resolution 3D imaging of entire organoids harboring fluorescence reporters and upon immunolabeling. This method is applicable to a wide range of organoids of differing origins and of various sizes and shapes. We have successfully used it on human airway, colon, kidney, liver and breast tumor organoids, as well as on mouse mammary gland organoids. It includes a simple clearing method utilizing a homemade fructose-glycerol clearing agent that captures 3D organoids in full and enables marker quantification on a cell-by-cell basis. Sample preparation has been optimized for 3D imaging by confocal, super-resolution confocal, multiphoton and light-sheet microscopy. From organoid harvest to image analysis, the protocol takes 3 d.


Asunto(s)
Imagen Tridimensional/métodos , Microscopía Confocal/métodos , Microscopía Fluorescente/métodos , Imagen Óptica/métodos , Organoides/ultraestructura , Fijación del Tejido/métodos , Animales , Mama/ultraestructura , Colon/ultraestructura , Femenino , Humanos , Inmunohistoquímica/métodos , Riñón/ultraestructura , Hígado/ultraestructura , Ratones
17.
RNA ; 25(8): 1038-1046, 2019 08.
Artículo en Inglés | MEDLINE | ID: mdl-31064786

RESUMEN

Visualization of gene expression at single RNA molecular level represents a great challenge to both imaging technologies and molecular engineering. Here we show a single molecule chromogenic in situ hybridization (smCISH) assay that enables counting and localizing individual RNA molecules in fixed cells and tissue under bright-field microscopy. Our method is based on in situ padlock probe assays directly using RNA as a ligation template and rolling circle amplification combined with enzyme catalyzed chromogenic reaction for amplification product visualization. We show potential applications of our method by detecting gene expression variations in single cells, subcellular localization information of expressed genes, and gene expression heterogeneity in formalin-fixed, paraffin-embedded tissue sections. This facile and straightforward method can in principle be applied to any type of RNA molecules in different samples.


Asunto(s)
Compuestos Cromogénicos/química , ARN Mensajero/análisis , Imagen Individual de Molécula/métodos , Animales , Expresión Génica , Humanos , Hibridación Fluorescente in Situ , ARN Mensajero/química , Adhesión del Tejido , Fijación del Tejido
18.
PLoS One ; 14(5): e0216050, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31059554

RESUMEN

Formalin-fixed paraffin-embedded (FFPE) tissues are among the most widely available clinical specimens. Their potential utility as a source of RNA for transcriptome studies would greatly enhance population-based cancer studies. Although preliminary studies suggest FFPE tissue may be used for RNA sequencing, the effect of storage time on these specimens needs to be determined. We conducted this study to determine whether RNA in archived FFPE high-grade ovarian serous adenocarcinomas from Surveillance, Epidemiology and End Results (SEER) registries was present in sufficient quantity and quality for RNA-Seq analysis. FFPE tissues, stored from 7 to 32 years, were obtained from three SEER sites. RNA was extracted, quantified, quality assessed, and subjected to RNA-Seq (a whole transcriptome sequencing technology). FFPE specimens stored for longer periods of time had poorer RNA sample quality as indicated by negative correlations between specimen storage time and fragment distribution values (DV). In addition, sample contamination was a common issue among the RNA, with 41 of 67 samples having 5% to 48% bacterial contamination. However, regardless of specimen storage time and bacterial contamination, 60% of the samples yielded data that enabled gene expression quantification, identifying more than 10,000 genes, with the correlations among most biological replicates above 0.7. This study demonstrates that FFPE high-grade ovarian serous adenocarcinomas specimens stored in repositories for up to 32 years and under varying storage conditions are a promising source of RNA for RNA-Seq. We also describe certain caveats to be considered when designing RNA-Seq studies using archived FFPE tissues.


Asunto(s)
Cistadenocarcinoma Seroso/genética , Neoplasias Ováricas/genética , ARN Neoplásico/genética , /métodos , Femenino , Formaldehído , Perfilación de la Expresión Génica/métodos , Biblioteca de Genes , Humanos , Adhesión en Parafina/métodos , Programa de VERF , Factores de Tiempo , Fijación del Tejido/métodos
19.
Transplant Proc ; 51(5): 1387-1391, 2019 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-31036353

RESUMEN

AIM: Caveolin-1 (CAV-1) is a molecule associated with endothelial cell dysfunction in chronic antibody-mediated rejection (CAMR) and considered to be a novel biomarker of CAMR. For immunohistochemical staining to reveal CAV-1 expression, most studies have used immunofluorescent stained frozen specimens, whereas formalin-fixed tissues have not been utilized. In the present study, we examined CAV-1 expression in specimens from CAMR patients using an immunoenzymatic technique with formalin-fixed tissues. METHODS: Eleven patients diagnosed with CAMR based on findings of transplanted renal biopsy samples were enrolled. Those biopsy specimens were formalin fixed and stained with CAV-1 using an immunoenzymatic method. Dye extent was evaluated by classifying that in peritubular capillaries (PTC) and glomerular capillaries (GBM) in 3 steps. We then compared the Banff scores for peritubular capillaritis (ptc), glomerulopathy (cg), and C4d using those results. RESULTS: CAV-1 expression was confirmed in vascular endothelium (PTC, GBM), while it was poor in epithelial cells. A Banff score for ptc and cg of 3 points was seen in 3 and 4 cases, of 2 points was seen in 1 and 4 cases, of 1 point was seen in 7 and 3 cases, and of 0 points was seen in 0 and 0 cases, respectively. In PTC, C4d and CAV-1 scores of 3 points were seen in 0 and 9 cases, of 2 points were seen in 2 and 2 cases, of 1 point was seen in 5 and 0 cases, and of 0 points were seen in 4 and 0 cases, respectively. As for GBM, C4d and CAV-1 scores of 3 points were seen in 8 and 7 cases, of 2 points were seen in 2 and 4 cases, of 1 point was seen in 0 and 0 cases, and of 0 points were seen 1 and 0 cases, respectively. CONCLUSION: CAV-1 expression in PTC had a score ≥2 in all cases, indicating that an adequate level of staining of formalin-fixed tissue was attained with the present immunoenzymatic technique. These results suggest that CAV-1 expression examined by the present method may be useful for identifying endothelial dysfunction.


Asunto(s)
Biomarcadores/análisis , Caveolina 1/análisis , Rechazo de Injerto/inmunología , Técnicas para Inmunoenzimas/métodos , Trasplante de Riñón , Adulto , Biomarcadores/metabolismo , Capilares/metabolismo , Femenino , Fijadores , Formaldehído , Humanos , Isoanticuerpos , Glomérulos Renales/patología , Masculino , Persona de Mediana Edad , Fijación del Tejido/métodos
20.
Anticancer Res ; 39(5): 2561-2567, 2019 May.
Artículo en Inglés | MEDLINE | ID: mdl-31092453

RESUMEN

BACKGROUND/AIM: The expression of programmed cell death ligand 1 (PD-L1) determined by immunohistochemistry (IHC) may be associated with tissue formalin fixation time in non-small cell lung cancer (NSCLC) samples. We investigated the association between the PD-L1 expression and formalin fixation time, and clarified the optimal duration of fixation for accurate PD-L1 evaluation. MATERIALS AND METHODS: We collected 55 tumor specimens from resected NSCLC patients. The samples were halved and immediately fixed in 10% buffered formalin for 12-24 h (normal fixation), or 96-120 h (prolonged fixation). Each specimen was stained using two assay systems (22C3 and SP263) for PD-L1. RESULTS: The mean PD-L1 tumor proportion score was not significantly different between normal and prolonged fixation groups for either 22C3 or SP263 (normal fixation: 18.8%; prolonged fixation: 16.3%, p=0.277; normal fixation: 16.2%; prolonged fixation: 17.6%, p=0.560, respectively). CONCLUSION: Formalin fixation duration for up to 120 h does not affect PD-L1 IHC expression. PD-L1 tumor proportion score of tumor specimens can be evaluated by IHC even if these have been fixed in formalin outside the recommended duration in clinical practice.


Asunto(s)
Antígeno B7-H1/genética , Biomarcadores de Tumor/genética , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/patología , Anciano , Femenino , Formaldehído/química , Regulación Neoplásica de la Expresión Génica , Humanos , Inmunohistoquímica , Masculino , Estadificación de Neoplasias , Fijación del Tejido
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