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Telomeres in most somatic cells shorten with each cell division, and critically short telomeres lead to cellular dysfunction, cell cycle arrest, and senescence. Thus, telomere shortening is an important hallmark of human cellular senescence. Quantitative fluorescence in situ hybridization (Q-FISH) using formalin-fixed paraffin-embedded (FFPE) tissue sections allows the estimation of telomere lengths in individual cells in histological sections. In our Q-FISH method, fluorescently labelled peptide nucleic acid (PNA) probes are hybridized to telomeric and centromeric sequences in FFPE human tissue sections, and relative telomere lengths (telomere signal intensities relative to centromere signal intensities) are measured. This chapter describes our Q-FISH protocols for assessing relative telomere lengths in FFPE human tissue sections.
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Hibridación Fluorescente in Situ , Adhesión en Parafina , Ácidos Nucleicos de Péptidos , Telómero , Humanos , Hibridación Fluorescente in Situ/métodos , Telómero/genética , Telómero/metabolismo , Ácidos Nucleicos de Péptidos/metabolismo , Ácidos Nucleicos de Péptidos/genética , Adhesión en Parafina/métodos , Fijación del Tejido/métodos , Homeostasis del Telómero , Centrómero/metabolismo , Centrómero/genéticaRESUMEN
Significance: Near-infrared autofluorescence (NIRAF) utilizes the natural autofluorescence of parathyroid glands (PGs) to improve their identification during thyroid surgeries, reducing the risk of inadvertent removal and subsequent complications such as hypoparathyroidism. This study evaluates NIRAF's effectiveness in real-world surgical settings, highlighting its potential to enhance surgical outcomes and patient safety. Aim: We evaluate the effectiveness of NIRAF in detecting PGs during thyroidectomy and central neck dissection and investigate autofluorescence characteristics in both fresh and paraffin-embedded tissues. Approach: We included 101 patients diagnosed with papillary thyroid cancer who underwent surgeries in 2022 and 2023. We assessed NIRAF's ability to locate PGs, confirmed via parathyroid hormone assays, and involved both junior and senior surgeons. We measured the accuracy, speed, and agreement levels of each method and analyzed autofluorescence persistence and variation over 10 years, alongside the expression of calcium-sensing receptor (CaSR) and vitamin D. Results: NIRAF demonstrated a sensitivity of 89.5% and a negative predictive value of 89.1%. However, its specificity and positive predictive value (PPV) were 61.2% and 62.3%, respectively, which are considered lower. The kappa statistic indicated moderate to substantial agreement (kappa = 0.478; P < 0.001 ). Senior surgeons achieved high specificity (86.2%) and PPV (85.3%), with substantial agreement (kappa = 0.847; P < 0.001 ). In contrast, junior surgeons displayed the lowest kappa statistic among the groups, indicating minimal agreement (kappa = 0.381; P < 0.001 ). Common errors in NIRAF included interference from brown fat and eschar. In addition, paraffin-embedded samples retained stable autofluorescence over 10 years, showing no significant correlation with CaSR and vitamin D levels. Conclusions: NIRAF is useful for PG identification in thyroid and neck surgeries, enhancing efficiency and reducing inadvertent PG removals. The stability of autofluorescence in paraffin samples suggests its long-term viability, with false positives providing insights for further improvements in NIRAF technology.
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Imagen Óptica , Glándulas Paratiroides , Espectroscopía Infrarroja Corta , Tiroidectomía , Humanos , Glándulas Paratiroides/cirugía , Glándulas Paratiroides/metabolismo , Masculino , Femenino , Persona de Mediana Edad , Imagen Óptica/métodos , Adulto , Espectroscopía Infrarroja Corta/métodos , Adhesión en Parafina/métodos , Anciano , Cáncer Papilar Tiroideo/cirugía , Cáncer Papilar Tiroideo/patología , Cáncer Papilar Tiroideo/metabolismo , Receptores Sensibles al Calcio/metabolismo , Receptores Sensibles al Calcio/análisisRESUMEN
High-plex imaging techniques enable the detection and quantification of a multitude of markers in tissue biopsies at single-cell or near-single-cell resolution. In lymphoma, this can facilitate the detection and characterization of cellular phenotypes and interactions, describing both tumor and microenvironmental cells. In combination with other techniques, high-plex imaging allows the investigation of biological mechanisms and clinically relevant biomarkers. CO-Detection by IndEXing (CODEX), one of such techniques, is based on antibodies labeled with unique DNA oligonucleotides that can be visualized by complementary reporter oligonucleotides coupled to a fluorophore. Here, we provide an overview of the key steps of a CODEX-based project, including (1) antibody panel design, (2) cohort selection, (3) staining and imaging, (4) data analysis. By sharing our CODEX protocol and our experience with FFPE tissue samples, we aim to encourage wider use of this powerful technique in lymphoma research and improve insight into cellular composition and spatial dynamics for improved diagnostics and therapy.
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Inmunofenotipificación , Linfoma , Humanos , Linfoma/diagnóstico , Linfoma/patología , Inmunofenotipificación/métodos , Biomarcadores de Tumor/análisis , Adhesión en Parafina/métodosRESUMEN
We evaluated the Xpert MTB/Rif Ultra assay performance for Mycobacterium tuberculosis (MTB) detection in formalin-fixed paraffin-embedded tissue (FFPET) compared to mycobacterial culture or laboratory-developed MTB PCR test (LDT). FFPET samples with histological features suggestive of tuberculosis from 2018 to 2023 were selected. Five hundred microlitres of tissue lysis buffer was added to FFPET scrolls and incubated at 75 °C for 5 min. After adding 50 µl of proteinase K and overnight incubation at 56 °C, sample aliquots were processed as per the manufacturer's instructions. MTB culture or LDT assay results were used as a reference for sensitivity and specificity calculations. Of 51 eligible FFPET, 32 were positive for MTB either by culture or LDT PCR on FFPET. Xpert MTB/Rif Ultra detected MTB in 23/32 positive specimens [71.9%, 95% confidence interval (CI) 54.6-84.4%]. Of nine discordant specimens, seven were MTB positive by culture and two were identified by LDT MTB PCR only, as no specimen was submitted for MTB culture. Of 19 negative samples, 100% specificity (95% CI 83.2-100.0%) was attained via Xpert MTB/Rif Ultra. Implementation of Xpert MTB/Rif Ultra on FFPET within clinical laboratories is promising, given its improved turnaround time compared to MTB culture and ability to detect MTB in cases where no tissue is available for culture.
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Formaldehído , Mycobacterium tuberculosis , Adhesión en Parafina , Sensibilidad y Especificidad , Mycobacterium tuberculosis/aislamiento & purificación , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/crecimiento & desarrollo , Humanos , Tuberculosis/diagnóstico , Tuberculosis/microbiología , Técnicas de Diagnóstico Molecular/métodos , Fijación del Tejido , Reacción en Cadena de la Polimerasa/métodosRESUMEN
INTRODUCTION: The correct storage of specimens in the Pathology service is of vital importance for patient safety. However, there are no clear recommendations as regarding how long samples should be stored for a minimum period. MATERIAL AND METHODS: A working group of the Spanish Society of Anatomic Pathology has reviewed a series of recommendations established in the literature and after two rounds of consultations and a discussion and voting phase has established a series of storage time proposals. RESULTS: Each of the proposals is presented with the data found in the literature and sometimes offers definitions and exceptions to the proposal. CONCLUSION: These recommendations, which are minimums, establish a period of at least 10 years for paraffin embedded blocks (including cell blocks), histological preparations, general cytology, pathologic cervico-vaginal cytology and electron microscopy blocks; at least 3 years for cervico-vaginal cytology, 5 years for extracted nucleic acids, at least 4 weeks for tissue in formalin and from the time of diagnosis for liquid cytology material and fluids.
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Manejo de Especímenes , Humanos , Manejo de Especímenes/normas , Manejo de Especímenes/métodos , Femenino , Factores de Tiempo , España , Adhesión en Parafina , Sociedades MédicasRESUMEN
BACKGROUND: Archived samples, including frozen and formalin fixed paraffin embedded (FFPE) tissues, are a vast resource of clinically annotated materials for the application of high-definition genomics to improve patient management and provide a molecular basis for the delivery of personalized cancer therapeutics. Notably, FFPE tissues are stable, provide repeat sampling of tissues of interest, and can be stored indefinitely at ambient temperature. The development of single cell DNA sequencing (scDNA-seq) technologies provides an unparalleled opportunity for the study of tumor heterogeneity and the identification of often rare subclonal cell populations that drive tumor evolution and progression to advanced therapy resistant disease. However, major limitations to the use of archived tissues for scDNA-seq include the low yields of intact cells in the presence of high levels of subcellular debris in biopsies, and the highly variable quantity and quality of the DNA extracted from samples of interest. The latter is of high significance for the use of FFPE tissues due to the presence of DNA-protein crosslinks. In addition, many samples, notably tumors arising in solid tissues, contain admixtures of reactive stroma, inflammatory cells, and necrosis in immediate contact with tumor cells. RESULTS: To expand their use for translational studies, we optimized flow sorting and sequencing of single nuclei from archived fresh frozen (FF) and FFPE tumor tissues. Our methods, which include isolation of intact nuclei suitable for library preparations, quality control (QC) metrics for each step, and a single cell sequencing bioinformatic processing and analysis pipeline, were validated with flow sorted nuclei from matching FF and FFPE ovarian cancer surgical samples and a sequencing panel of 553 amplicons targeting single nucleotide and copy number variants in genes of interest. CONCLUSIONS: Our flow sorting based protocol provides intact nuclei suitable for snDNA-seq from archival FF and FFPE tissues. Furthermore, we have developed QC steps that optimize the preparation and selection of samples for deep single cell clonal profiling. Our data processing pipeline captures rare subclones in tumors with highly variable genomes based on variants in genes of interest.
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Formaldehído , Adhesión en Parafina , Análisis de Secuencia de ADN , Análisis de la Célula Individual , Fijación del Tejido , Humanos , Análisis de la Célula Individual/métodos , Análisis de Secuencia de ADN/métodos , Neoplasias/genética , Neoplasias/patología , Citometría de Flujo/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Núcleo Celular/genética , FemeninoRESUMEN
Formalin-fixed paraffin-embedded (FFPE) samples are valuable but underutilized in single-cell omics research due to their low RNA quality. In this study, leveraging a recent advance in single-cell genomic technology, we introduce snPATHO-seq, a versatile method to derive high-quality single-nucleus transcriptomic data from FFPE samples. We benchmarked the performance of the snPATHO-seq workflow against existing 10x 3' and Flex assays designed for frozen or fresh samples and highlighted the consistency in snRNA-seq data produced by all workflows. The snPATHO-seq workflow also demonstrated high robustness when tested across a wide range of healthy and diseased FFPE tissue samples. When combined with FFPE spatial transcriptomic technologies such as FFPE Visium, the snPATHO-seq provides a multi-modal sampling approach for FFPE samples, allowing more comprehensive transcriptomic characterization.
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Adhesión en Parafina , Análisis de Secuencia de ARN , Análisis de la Célula Individual , Fijación del Tejido , Adhesión en Parafina/métodos , Humanos , Análisis de Secuencia de ARN/métodos , Fijación del Tejido/métodos , Análisis de la Célula Individual/métodos , Formaldehído/química , Transcriptoma , Perfilación de la Expresión Génica/métodos , Flujo de TrabajoRESUMEN
Microsatellite instability (MSI) occurs across a number of cancers and is associated with different clinical characteristics when compared to microsatellite stable (MSS) cancers. As MSI cancers have different characteristics, routine MSI testing is now recommended for a number of cancer types including colorectal cancer (CRC). Using gene panels for sequencing of known cancer mutations is routinely performed to guide treatment decisions. By adding a number of MSI regions to a small gene panel, the efficacy of simultaneous MSI detection in a series of CRCs was tested. Tumour DNA from formalin-fixed, paraffin-embedded (FFPE) tumours was sequenced using a 23-gene panel kit (ATOM-Seq) provided by GeneFirst. The mismatch repair (MMR) status was obtained for each patient from their routine pathology reports, and compared to MSI predictions from the sequencing data. By testing 29 microsatellite regions in 335 samples the MSI status was correctly classified in 314/319 samples (98.4% concordance), with sixteen failures. By reducing the number of regions in silico, comparable performance could be reached with as few as eight MSI marker positions. This test represents a quick, and accurate means of determining MSI status in FFPE CRC samples, as part of a routine gene mutation assay, and can easily be incorporated into a research or diagnostic setting. This could replace separate mutation and MSI tests with no loss of accuracy, thus improving testing efficiency.
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Neoplasias Colorrectales , Formaldehído , Inestabilidad de Microsatélites , Mutación , Fijación del Tejido , Humanos , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/diagnóstico , Formaldehído/química , Adhesión en Parafina , Femenino , Masculino , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Reparación de la Incompatibilidad de ADN/genética , Análisis Mutacional de ADN/métodos , Anciano , Persona de Mediana EdadRESUMEN
Hematoxylin and eosin staining is widely used for routine histopathological analysis under light microscopic examination to determine alterations of tissue architecture and cellular components in animal studies. Aside from hematoxylin/eosin staining, periodic acid Schiff (PAS) staining is used to detect polysaccharides and carbohydrate-rich macromolecules, and is essential in immunological fields for evaluation of glomerular lesions of kidneys in autoimmune animals. Since erythrocytes are not stained by PAS, this stain is also helpful for identifying changes in immune cells in the red pulp of the spleen, which is filled with erythrocytes. This article describes a protocol to detect Mott cells, bizarre plasma cells containing immunoglobulin inclusion bodies (Russell bodies) in the cytoplasm. The protocol can be used for formalin-fixed, paraffin-embedded tissue sections, frozen tissue sections, tissue-touch preparations, blood films, and cytocentrifuged cell smears. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Detection of Mott cells by PAS staining in formalin-fixed, paraffin-embedded tissue sections Basic Protocol 2: Detection of Mott cells by PAS staining in frozen tissue sections, touch preparations, blood films, and cytocentrifuged cell smears.
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Células Plasmáticas , Coloración y Etiquetado , Coloración y Etiquetado/métodos , Células Plasmáticas/inmunología , Células Plasmáticas/patología , Humanos , Reacción del Ácido Peryódico de Schiff , Animales , Cuerpos de Inclusión , Inmunoglobulinas/análisis , Inmunoglobulinas/inmunología , Adhesión en ParafinaRESUMEN
Whole genome sequencing (WGS) provides comprehensive, individualised cancer genomic information. However, routine tumour biopsies are formalin-fixed and paraffin-embedded (FFPE), damaging DNA, historically limiting their use in WGS. Here we analyse FFPE cancer WGS datasets from England's 100,000 Genomes Project, comparing 578 FFPE samples with 11,014 fresh frozen (FF) samples across multiple tumour types. We use an approach that characterises rather than discards artefacts. We identify three artefactual signatures, including one known (SBS57) and two previously uncharacterised (SBS FFPE, ID FFPE), and develop an "FFPEImpact" score that quantifies sample artefacts. Despite inferior sequencing quality, FFPE-derived data identifies clinically-actionable variants, mutational signatures and permits algorithmic stratification. Matched FF/FFPE validation cohorts shows good concordance while acknowledging SBS, ID and copy-number artefacts. While FF-derived WGS data remains the gold standard, FFPE-samples can be used for WGS if required, using analytical advancements developed here, potentially democratising whole cancer genomics to many.
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Formaldehído , Neoplasias , Adhesión en Parafina , Fijación del Tejido , Secuenciación Completa del Genoma , Humanos , Adhesión en Parafina/métodos , Neoplasias/genética , Neoplasias/patología , Secuenciación Completa del Genoma/métodos , Fijación del Tejido/métodos , Genómica/métodos , Mutación , Genoma Humano , ArtefactosRESUMEN
To assess the impact of postnatal processing on placental DNA methylation, array data from flash-frozen placental tissue was compared to perfluorocarbon-immersed and formalin-fixed paraffin-embedded placental tissue. We observed that tissue exposed to perfluorocarbon showed no significant DNA methylation differences when compared to unprocessed tissue, while formalin processing altered the quality and reliability of the data produced on the DNA methylation array platform. Placental DNA methylation allows for the study of gene-environment interactions that influence the fetal environment and development. Our study highlights that placental post-processing techniques must be considered in the evaluation and interpretation of epigenetic studies.
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Metilación de ADN , Placenta , Humanos , Metilación de ADN/genética , Femenino , Placenta/metabolismo , Embarazo , Epigénesis Genética/genética , Adhesión en Parafina/métodos , Epigenómica/métodosRESUMEN
BACKGROUND: Endometrial Tuberculosis is one of the most common gynecological problems known to have serious implications for the quality of life like infertility. The commonly practiced histopathology solely relies on the suggestive feature of Tuberculosis (TB) with low specificity. Regarding the alternative bacteriological and molecular detection tools, little evidence was generated on their utility in the diagnosis of endometrial tuberculosis in Ethiopia. Therefore, we aim to investigate the detection rate of molecular and bacteriological detection methods on formalin-fixed paraffin-embedded biopsy samples for the diagnosis of endometrial and lymph node TB. METHODS: A retrospective cross-sectional study was conducted on 90 formalin fixed paraffin embedded biopsy samples from patients with gynecologic and lymph problems collected between 2018 and 2022 at St. Paul's Hospital Millennium Medical College, Addis Ababa, Ethiopia. SPSS version 26 was used for statistical analysis. The diagnostic performance was calculated using the histopathology method as the reference standard. Cohen's Kappa value was used to measure the level of agreement. A test with a P-value of < 0.05 was considered statistically significant. RESULTS: A total of 90 samples were analyzed in the current study. Auramine O, GeneXpert MTB/RIF assay, and Real-Time PCR tests have shown a detection rate of 32/90 (36%), 43/90 (47.8%), and 54/90 (60%) respectively (P ≤ 0.01). The sensitivity and specificity of AO were 38.1% and 95% respectively. RT PCR showed superior sensitivity followed by GeneXpert MTB/RIF assay, 70% and 58.6%. AO and molecular methods have shown a similarly low level of agreement with histopathology (Kappa value = 0.2). CONCLUSIONS: In a resource-limited setting, the selection of diagnostic tools needs careful attention. Putting the patients on anti-TB treatments based solely on histopathological findings may lead to undesired and adverse complications. Therefore, applying molecular and bacteriological detection methods along with histopathology, could help minimize inappropriate antimicrobial use.
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Endometrio , Mycobacterium tuberculosis , Adhesión en Parafina , Sensibilidad y Especificidad , Tuberculosis Ganglionar , Humanos , Femenino , Estudios Transversales , Estudios Retrospectivos , Adulto , Endometrio/microbiología , Endometrio/patología , Biopsia , Persona de Mediana Edad , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/aislamiento & purificación , Tuberculosis Ganglionar/diagnóstico , Tuberculosis Ganglionar/microbiología , Tuberculosis Ganglionar/patología , Adulto Joven , Etiopía , Ganglios Linfáticos/microbiología , Ganglios Linfáticos/patología , Formaldehído , Técnicas de Diagnóstico Molecular/métodos , Tuberculosis de los Genitales Femeninos/diagnóstico , Tuberculosis de los Genitales Femeninos/patología , Tuberculosis de los Genitales Femeninos/microbiología , AdolescenteRESUMEN
OBJECTIVE: The goal of the research presented here is to determine if methods previously developed for the aqueous extraction of PrPSc from formalin-fixed paraffin-embedded tissue (FFPET) are applicable to the detection PrPSc by real-time quaking induced conversion (RT-QuIC). Previous work has utilized aqueous extraction of FFPET for detection of transmissible spongiform encephalopathies (TSEs) utilizing western blot and ELISA. This research extends the range of suitable methods for detection of TSEs in FFPET to RT-QuIC, which is arguably the most sensitive method to detect TSEs. RESULTS: We found complete agreement between the TSE status and the results from RT-QuIC seeded with the aqueous extract of FFPET samples. The method affords the diagnostic assessment TSE status by RT-QuIC of FFPET without the use of organic solvents that would otherwise create a mixed chemical-biological waste for disposal.
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Formaldehído , Adhesión en Parafina , Proteínas PrPSc , Enfermedades por Prión , Fijación del Tejido , Formaldehído/química , Adhesión en Parafina/métodos , Enfermedades por Prión/diagnóstico , Proteínas PrPSc/aislamiento & purificación , Proteínas PrPSc/metabolismo , Proteínas PrPSc/análisis , Animales , Fijación del Tejido/métodos , Ratones , HumanosRESUMEN
INTRODUCTION: The novel Porcine circovirus 3 (PCV-3) has been associated in the past years to different porcine diseases, including reproductive failure. The potential occurrence of PCV-3 in abortions from Swiss pig herds has not been investigated so far. Thus, we conducted a retrospective study on pig aborted cases submitted to our laboratory in the University of Bern during the last 10 years with the main aim of investigating the possible presence of PCV-3 in foetal and/or placental tissue. Twelve out of the 53 studied cases showed mild histopathological changes as previously described in PCV-3 positive cases. However, in none of the cases, PCV-3 genetic material could be detected in the examined formalin-fixed, paraffin-embedded tissues. In only one third of the cases, a cause for the abortion was found, which is similar to other studies. Our survey suggests that PCV-3 was not involved in the porcine abortion cases submitted over the last decade at our institution in Switzerland.
INTRODUCTION: Le nouveau Circovirus porcin 3 (PCV-3) a été associé ces dernières années à différentes maladies porcines, y compris des troubles de la reproduction. La présence potentielle du PCV-3 dans les avortements de porcs en Suisse n'a pas été étudiée jusqu'à présent. Nous avons donc mené une étude rétrospective sur les cas d'avortements de porcs soumis à notre laboratoire de l'Université de Berne au cours des 10 dernières années, dans le but principal d'étudier la présence éventuelle du PCV-3 dans les tissus fÅtaux et/ou placentaires. Douze des 53 cas étudiés présentaient des changements histopathologiques légers, tels que décrits précédemment dans les cas positifs au PCV-3. Cependant, dans aucun des cas, le matériel génétique du PCV-3 n'a pu être détecté dans les tissus examinés fixés au formol et inclus en paraffine. Dans un tiers des cas seulement, une cause d'avortement a été trouvée, ce qui est similaire à d'autres études. Notre étude suggère que le PCV-3 n'a pas été impliqué dans les cas d'avortements porcins soumis au cours de la dernière décennie dans notre institution en Suisse.
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Aborto Veterinario , Infecciones por Circoviridae , Circovirus , Enfermedades de los Porcinos , Animales , Femenino , Embarazo , Aborto Veterinario/virología , Infecciones por Circoviridae/veterinaria , Infecciones por Circoviridae/virología , Circovirus/aislamiento & purificación , Circovirus/genética , Formaldehído , Adhesión en Parafina/veterinaria , Placenta/virología , Placenta/patología , Estudios Retrospectivos , Porcinos , Enfermedades de los Porcinos/virología , Enfermedades de los Porcinos/patología , Suiza/epidemiologíaAsunto(s)
Secuenciación de Nucleótidos de Alto Rendimiento , Adhesión en Parafina , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Indicadores y Reactivos , Biblioteca de Genes , Control de Calidad , Estándares de Referencia , Sensibilidad y Especificidad , Neoplasias/genética , Neoplasias/diagnóstico , ADN de Neoplasias/genética , ADN de Neoplasias/análisisRESUMEN
Multiplexed immunofluorescence (IF) can be achieved using different commercially available platforms, often making use of conjugated antibodies detected in iterative cycles. A growing portfolio of pre-conjugated antibodies is offered by the providers, as well as the possibility for in-house conjugation. For many conjugation methods and kits, there are limitations in which antibodies can be used, and conjugation results are sometimes irreproducible. The conjugation process can limit or slow down the progress of studies requiring conjugation of essential markers needed for a given project. Here, we demonstrate a protocol combining manual indirect immunofluorescence (IF) of primary antibodies, followed by antibody elution and staining with multiplexed panels of commercially pre-conjugated antibodies on the PhenoCycler platform. We present detailed protocols for applying the workflow on fresh frozen and formalin fixed paraffin embedded tissue sections. We also provide a ready to use workflow for coregistration of the images and demonstrate this for two examples.
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Anticuerpos , Humanos , Anticuerpos/química , Adhesión en Parafina , Biomarcadores/análisis , Técnica del Anticuerpo Fluorescente/métodos , Técnica del Anticuerpo Fluorescente Indirecta/métodosRESUMEN
Human papillomavirus (HPV) infections are an increasing cause of oropharyngeal squamous cell carcinomas (OPSCC). Integration of the viral genome into the host genome is suggested to affect carcinogenesis, however, the correlation with OPSCC patient prognosis is still unclear. Research on HPV integration is hampered by current integration detection technologies and their unsuitability for formalin-fixed paraffin-embedded (FFPE) tissues. This study aims to develop and validate a novel targeted proximity-ligation based sequencing method (targeted locus amplification/capture [TLA/TLC]) for HPV integration detection in cell lines and FFPE OPSCCs. For the identification of HPV integrations, TLA/TLC was applied to 7 cell lines and 27 FFPE OPSCCs. Following preprocessing steps, a polymerase chain reaction (PCR)-based HPV enrichment was performed on the cell lines and a capture-based HPV enrichment was performed on the FFPE tissues before paired-end sequencing. TLA was able to sequence up to hundreds of kb around the target, detecting exact HPV integration loci, structural variants, and chromosomal rearrangements. In all cell lines, one or more integration sites were identified, in accordance with detection of integrated papillomavirus sequences PCR data and the literature. TLC detected integrated HPV in 15/27 FFPE OPSCCs and identified simple and complex integration patterns. In general, TLA/TLC confirmed PCR data and detected additional integration sites. In conclusion TLA/TLC reliably and robustly detects HPV integration in cell lines and FFPE OPSCCs, enabling large, population-based studies on the clinical relevance of HPV integration. Furthermore, this approach might be valuable for clonality assessment of HPV-related tumors in clinical diagnostics.
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Carcinoma de Células Escamosas , Virus del Papiloma Humano , Neoplasias Orofaríngeas , Infecciones por Papillomavirus , Integración Viral , Femenino , Humanos , Masculino , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/virología , Línea Celular Tumoral , ADN Viral/genética , Formaldehído , Virus del Papiloma Humano/clasificación , Virus del Papiloma Humano/genética , Virus del Papiloma Humano/aislamiento & purificación , Neoplasias Orofaríngeas/virología , Neoplasias Orofaríngeas/genética , Infecciones por Papillomavirus/virología , Infecciones por Papillomavirus/diagnóstico , Adhesión en Parafina , Reacción en Cadena de la Polimerasa/métodos , Análisis de Secuencia de ADN , Fijación del Tejido , Integración Viral/genéticaRESUMEN
Formalin-fixed paraffin-embedded (FFPE) tissues are suitable for proteomic and phosphoproteomic biomarker studies by data-independent acquisition mass spectrometry. The choice of the sample preparation method influences the number, intensity, and reproducibility of identifications. By comparing four deparaffinization and rehydration methods, including heptane, histolene, SubX, and xylene, we found that heptane and methanol produced the lowest coefficients of variation (CVs). Using this, five extraction methods from the literature were modified and evaluated for their performance using kidney, leg muscle, lung, and testicular rat organs. All methods performed well, except for SP3 due to insufficient tissue lysis. Heat n' Beat was the fastest and most reproducible method with the highest digestion efficiency and lowest CVs. S-Trap produced the highest peptide yield, while TFE produced the best phosphopeptide enrichment efficiency. The quantitation of FFPE-derived peptides remains an ongoing challenge with bias in UV and fluorescence assays across methods, most notably in SPEED. Functional enrichment analysis demonstrated that each method favored extracting some gene ontology cellular components over others including chromosome, cytoplasmic, cytoskeleton, endoplasmic reticulum, membrane, mitochondrion, and nucleoplasm protein groups. The outcome is a set of recommendations for choosing the most appropriate method for different settings.
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Adhesión en Parafina , Proteómica , Proteómica/métodos , Animales , Ratas , Formaldehído/química , Masculino , Fosfoproteínas/análisis , Fosfoproteínas/metabolismo , Fosfoproteínas/aislamiento & purificación , Fijación del Tejido , Riñón/metabolismo , Riñón/químicaRESUMEN
The intestine is a complex organ composed of the small and the large intestines. The small intestine can be further divided into duodenum, jejunum, and ileum. Each anatomical region of the intestine has a unique function that is reflected by differences in cellular structure. Investigating changes in the intestine requires an in-depth analysis of different tissue regions and cellular alterations. To study the intestine and visualize large pieces of tissue, researchers commonly use a technique known as intestinal Swiss rolls. In this technique, the intestine is divided into each anatomical region and fixed in a flat orientation. Then, the tissue is carefully rolled and processed for paraffin embedding. Proper tissue fixation and orientation is an often-overlooked laboratory technique but is critically important for downstream analysis. Additionally, improper Swiss rolling of intestinal tissue can damage the fragile intestinal epithelium, leading to poor tissue quality for immunostaining. Ensuring well-fixed and properly oriented tissue with intact cellular structures is a crucial step that ensures optimal visualization of intestinal cells. We present a cost-effective and simple method for making Swiss rolls to include all sections of the intestine in a single paraffin-embedded block. We also describe optimized immunofluorescence staining of intestinal tissue to study various aspects of the intestinal epithelium. The following protocol provides researchers with a comprehensive guide to obtaining high-quality immunofluorescence images through intestinal tissue fixation, Swiss-roll technique, and immunostaining. Employing these refined approaches preserves the intricate morphology of the intestinal epithelium and fosters a deeper understanding of intestinal physiology and pathobiology.