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1.
Medicine (Baltimore) ; 100(15): e25526, 2021 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-33847675

RESUMO

RATIONALE: In some cases, autopsy is the first opportunity to find a previously unrecognized critical infection. Pathogens are identified by various methods, such as microscopic examination, special stains, culture tests, and immunohistochemistry. Here, we report a case of 16S ribosomal RNA (rRNA) gene sequencing using a postmortem formalin-fixed, paraffin-embedded (FFPE) tissue, which was useful for identifying pathogenic microbes. PATIENT CONCERNS: Autopsy was performed on an 87-year-old man who had chronic renal failure and had developed sepsis from a central venous catheter infection 10 days before his death. Prior to these events, von Meyenburg complexes (VMCs) were also found during regular checkups. DIAGNOSIS: Postmortem microscopic examination revealed acute purulent cholangitis with numerous microabscesses, accompanied by VMCs. Gram-negative rods were observed in some microabscesses, which were considered causative pathogens. INTERVENTIONS: 16S rRNA gene sequencing using postmortem FFPE tissue. OUTCOMES: Pseudomonas aeruginosa was identified, different from the one detected in the central venous catheter culture while alive. LESSONS: 16S rRNA gene sequencing is a useful tool for identifying pathogenic microbes in postmortem FFPE tissues. This technique may be useful for amplicon sizes of approximately 100 bp or less.


Assuntos
Doenças Biliares/microbiologia , Colangite/microbiologia , Hamartoma/microbiologia , Pseudomonas aeruginosa , RNA Bacteriano/análise , Doença Aguda , Idoso de 80 Anos ou mais , Autopsia , Evolução Fatal , Formaldeído , Humanos , Masculino , Ilustração Médica , Inclusão em Parafina , RNA Ribossômico 16S/análise , Análise de Sequência de RNA
2.
Medicine (Baltimore) ; 100(16): e25566, 2021 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-33879712

RESUMO

ABSTRACT: This study investigated the feasibility of using immunohistochemistry (IHC) instead of PCR to detect BRAF V600E mutant protein in papillary thyroid carcinoma (PTC), and to determine the value of using preoperative BRAF V600E mutant protein by IHC to assist in the diagnosis of thyroid nodule patients with Hashimoto's thyroiditis (HT).The expression of BRAFV600E mutant protein was measured in 23 cases of HT+PTC, 31 cases of PTC, and 28 cases of HT by IHC, followed by PCR in the same samples for validation. SPSS 19.0 software was used for statistical analysis.The sensitivity and specificity of IHC to detect BRAF V600E mutation were 100% and 42.86%, respectively. In addition, the mutation rate of BRAF V600E protein in the HT+PTC group (34.78%, 8/23) was lower than that in the PTC group (80.65%, 25/31).The application of IHC to detect BRAF V600E mutant protein has good sensitivity but not specificity to diagnose PTC. IHC can be used as a preliminary screening method to detect BRAF V600E mutation. The strongly positive (+++) staining of IHC potently indicated BRAF V600E gene mutation. For suspicious thyroid nodules combined with HT, the detection of BRAF V600E mutant protein with IHC alone is not of great significance for differentiating benign and malignant nodules.


Assuntos
Imuno-Histoquímica/estatística & dados numéricos , Proteínas Mutantes/análise , Proteínas Proto-Oncogênicas B-raf/análise , Câncer Papilífero da Tireoide/diagnóstico , Neoplasias da Glândula Tireoide/diagnóstico , Adulto , Estudos de Viabilidade , Feminino , Doença de Hashimoto/diagnóstico , Humanos , Imuno-Histoquímica/métodos , Masculino , Pessoa de Meia-Idade , Mutação , Inclusão em Parafina , Reação em Cadeia da Polimerase , Valor Preditivo dos Testes , Período Pré-Operatório , Sensibilidade e Especificidade , Coloração e Rotulagem , Glândula Tireoide/metabolismo , Nódulo da Glândula Tireoide/diagnóstico
3.
Methods Mol Biol ; 2279: 23-33, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33683683

RESUMO

Immunohistochemistry (IHC) enables the selective detection of proteins in cells of formalin-fixed-paraffin-embedded (FFPE) tissue sections. This technique plays a key role in the identification and classification of primary lung cancer tumors through the evaluation of the expression of the aspartic proteinase Napsin-A. However, immunohistochemistry is a complex process involving many critical steps and the lack of standardization as well as inappropriate analytical conditions may contribute to inconsistent results between laboratories. Automated immunohistochemistry addresses this issue by ensuring the quality and the reproducibility of the results among different laboratories. Here we describe an automated IHC protocol used in our laboratory for the detection of Napsin-A in FFPE lung tissue sections.


Assuntos
Automação Laboratorial , Imuno-Histoquímica , Neoplasias Pulmonares , Humanos , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Inclusão em Parafina
4.
Nat Commun ; 12(1): 1426, 2021 03 03.
Artigo em Inglês | MEDLINE | ID: mdl-33658518

RESUMO

Metastatic prostate cancer (mPC) comprises a spectrum of diverse phenotypes. However, the extent of inter- and intra-tumor heterogeneity is not established. Here we use digital spatial profiling (DSP) technology to quantitate transcript and protein abundance in spatially-distinct regions of mPCs. By assessing multiple discrete areas across multiple metastases, we find a high level of intra-patient homogeneity with respect to tumor phenotype. However, there are notable exceptions including tumors comprised of regions with high and low androgen receptor (AR) and neuroendocrine activity. While the vast majority of metastases examined are devoid of significant inflammatory infiltrates and lack PD1, PD-L1 and CTLA4, the B7-H3/CD276 immune checkpoint protein is highly expressed, particularly in mPCs with high AR activity. Our results demonstrate the utility of DSP for accurately classifying tumor phenotype, assessing tumor heterogeneity, and identifying aspects of tumor biology involving the immunological composition of metastases.


Assuntos
Perfilação da Expressão Gênica/métodos , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Antígenos B7/genética , Antígeno B7-H1/genética , Antígeno CTLA-4/genética , Regulação Neoplásica da Expressão Gênica , Receptor Celular 2 do Vírus da Hepatite A/genética , Humanos , Masculino , Inclusão em Parafina , Fenótipo , Receptor de Morte Celular Programada 1/genética , Neoplasias da Próstata/metabolismo , Receptores Androgênicos/genética , Receptores Androgênicos/metabolismo , Análise Serial de Tecidos , Transcriptoma
5.
J Vis Exp ; (168)2021 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-33645556

RESUMO

Formalin-fixed paraffin-embedded (FFPE) tissues represent a valuable source for molecular analyses and clinical genomic studies. These tissues are often poor in cells or difficult to process. Therefore, nucleic acids need to be carefully isolated. In recent years, various methods for DNA isolation have been established for tissues from many diseases, mostly cancer. Unfortunately, genomic DNA extracted from FFPE tissues is highly degraded due to the cross-linking between nucleic acid strands and proteins, as well as random breakings in sequence. Therefore, DNA quality from these samples is markedly reduced, making it a challenge for further molecular downstream analyses. Other problems with difficult tissues are, for example, the lack of cells in calcified human atherosclerotic lesions and fatty tissue, small skin biopsies, and consequently low availability of the desired nucleic acids as it is also the case in old or fixed tissues. In our laboratories, we have established a method for DNA extraction from formalin-fixed atherosclerotic lesions, using a semi-automated isolation system. We compared this method to other commercially available extraction protocols and focused on further downstream analyses. Purity and concentration of the DNA were measured by spectrometry and fluorometry. The degree of fragmentation and overall quality were assessed. The highest DNA quantity and quality was obtained with the modified blood DNA protocol for the automated extraction system, instead of the commercial FFPE protocol. With this step-by-step protocol, DNA yields from FFPE samples were in average four times higher and fewer specimens failed the extraction process, which is critical when dealing with small-vessel biopsies. Amplicon sizes from 200-800 bp could be detected by PCR. This study shows that although DNA obtained from our FFPE tissue is highly fragmented, it can still be used for successful amplification and sequencing of shorter products. In conclusion, in our hands, the automated technology appears to be the best system for DNA extraction, especially for small FFPE tissue specimen.


Assuntos
Aterosclerose/genética , DNA/isolamento & purificação , Formaldeído/química , Inclusão em Parafina , Fixação de Tecidos , Automação , Fracionamento Celular , DNA/genética , Fragmentação do DNA , Humanos , Reação em Cadeia da Polimerase
6.
Methods Mol Biol ; 2279: 59-73, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33683686

RESUMO

In an era of precision medicine important treatment decisions are dictated by expression of clinically informative tumor protein biomarkers. These biomarkers can be detected by immunohistochemistry (IHC) performed in tumor tissue sections obtained from biopsies or resections. Like all experimental procedures, IHC needs optimization for several of its steps. However, the investigator must avoid optimizing the IHC procedure using valuable human biopsy samples which may be difficult to obtain. Ideally, valuable biopsy samples should only be subjected to IHC once the IHC protocol has been optimized. In this chapter, we describe a procedure for IHC optimization using tri-dimensional (3D) cellular spheroids created from cultured cells. In this approach, cultured cells are pelleted into 3D spheroids, which are then processed just like a tissue sample, namely, fixed, embedded, sectioned, mounted on slides, and stained with IHC just like a human tissue sample. These 3D cellular spheroids have a tissue-like architecture and cellularity resembling a tumor section, and both cellular and antigen structure are preserved. This method is therefore acceptable for IHC optimization before proceeding to the IHC staining of human tumor samples.


Assuntos
Carcinoma Pulmonar de Células não Pequenas , Imuno-Histoquímica , Neoplasias Pulmonares , Inclusão em Parafina , Esferoides Celulares , Carcinoma Pulmonar de Células não Pequenas/diagnóstico , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Formaldeído/química , Humanos , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia
7.
Anticancer Res ; 41(3): 1341-1348, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33788725

RESUMO

BACKGROUND/AIM: Cancer profiling tests using formalin-fixed paraffin-embedded (FFPE) specimens with various conditions have become an essential tool for cancer treatment. The robustness of these tests needs to be addressed. MATERIALS AND METHODS: A cancer profiling test, NCC oncopanel, was tested with FFPE specimens from various tissues with different storage conditions and fixation lengths. Next generation sequencing was performed with Miseq and the data were assembled using the human reference genome hg19. RESULTS: Duration of storage and fixation affected the mapping statistics. Prolonged storage increased outward read paring and longer fixation rates caused increased singletons and unmapped reads. CONCLUSION: Our results indicate that a cancer profiling test with target capturing method, NCC oncopanel, shows robustness for FFPE cancer specimens with various storage conditions.


Assuntos
Perfilação da Expressão Gênica/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Neoplasias/genética , Inclusão em Parafina/métodos , Manejo de Espécimes/métodos , Fixação de Tecidos/métodos , Fixadores/química , Formaldeído/química , Genômica/métodos , Humanos , Mutação , Neoplasias/patologia
8.
Crit Rev Oncol Hematol ; 160: 103303, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33757837

RESUMO

Chimeric transcripts are critical for diagnosis or prognosis and could constitute effective therapeutic targets. Fresh tissues are the major source for the identification of these fusion transcripts. The quality and quantity of the extracted RNA directly affect fusion transcript discovery. Formalin-fixed paraffin-embedded (FFPE) tissues allow long-time preservation of tumor histology for microscopic evaluation; however, no provision has been made for either the type of fixative or embedding procedure used for preserving RNA. Nonetheless, the widespread use of these FFPE tissues in translational and clinical research prompts to overcome these issues. RNA is, by nature, of reduced quality and amount in these FFPE tissues. Therefore, attempts should be taken to minimize the limitations of FFPE tissues as a widely available source of fusion transcript identification. In this review, we describe approaches allowing fusion transcript identification from FFPE tissues using RNA sequencing techniques.


Assuntos
Formaldeído , Perfilação da Expressão Gênica , Humanos , Inclusão em Parafina , RNA/genética , Análise de Sequência de RNA
9.
Clin Infect Dis ; 72(Suppl 2): S109-S113, 2021 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-33709128

RESUMO

The EORTC/MSGERC have revised the definitions for proven, probable, and possible fungal diseases. The tissue diagnosis subcommittee was tasked with determining how and when species can be determined from tissue in the absence of culture. The subcommittee reached a consensus decision that polymerase chain reaction (PCR) from tissue, but not immunohistochemistry or in situ hybridization, can be used for genus or species determination under the new EORTC/MSGERC guidelines, but only when fungal elements are identified by histology. Fungal elements seen in tissue samples by histopathology and identified by PCR followed by sequencing should fulfill the definition of a proven fungal infection, identified to genus/species, even in the absence of culture. This summary discusses the issues that were deliberated by the subcommittee to reach the consensus decision and outlines the criteria a laboratory should follow in order to produce data that meet the EORTC/MSGERC definitions.


Assuntos
Infecções Fúngicas Invasivas , Micoses , Formaldeído , Fungos/genética , Humanos , Infecções Fúngicas Invasivas/diagnóstico , Micoses/diagnóstico , Inclusão em Parafina
10.
J Vis Exp ; (168)2021 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-33616105

RESUMO

An understanding of drug resistance and the development of novel strategies to sensitize highly resistant cancers rely on the availability of suitable preclinical models that can accurately predict patient responses. One of the disadvantages of existing preclinical models is the inability to contextually preserve the human tumor microenvironment (TME) and accurately represent intratumoral heterogeneity, thus limiting the clinical translation of data. By contrast, by representing the culture of live fragments of human tumors, the patient-derived explant (PDE) platform allows drug responses to be examined in a three-dimensional (3D) context that mirrors the pathological and architectural features of the original tumors as closely as possible. Previous reports with PDEs have documented the ability of the platform to distinguish chemosensitive from chemoresistant tumors, and it has been shown that this segregation is predictive of patient responses to the same chemotherapies. Simultaneously, PDEs allow the opportunity to interrogate molecular, genetic, and histological features of tumors that predict drug responses, thereby identifying biomarkers for patient stratification as well as novel interventional approaches to sensitize resistant tumors. This paper reports PDE methodology in detail, from collection of patient samples through to endpoint analysis. It provides a detailed description of explant derivation and culture methods, highlighting bespoke conditions for particular tumors, where appropriate. For endpoint analysis, there is a focus on multiplexed immunofluorescence and multispectral imaging for the spatial profiling of key biomarkers within both tumoral and stromal regions. By combining these methods, it is possible to generate quantitative and qualitative drug response data that can be related to various clinicopathological parameters and thus potentially be used for biomarker identification.


Assuntos
Neoplasias/patologia , Antineoplásicos/farmacologia , Morte Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Imunofluorescência , Humanos , Inclusão em Parafina , Coloração e Rotulagem , Fixação de Tecidos
11.
J Cancer Res Clin Oncol ; 147(5): 1341-1354, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33635431

RESUMO

PURPOSE: The present study was conducted to clarify the clinicopathological impacts of DNA methylation alterations on pancreatic ductal adenocarcinoma (PDAC). METHODS: Genome-wide DNA methylation screening was performed using the Infinium HumanMethylation450 BeadChip, and DNA methylation quantification was verified using pyrosequencing. We analyzed fresh-frozen tissues from an initial cohort (17 samples of normal control pancreatic tissue [C] from 17 patients without PDAC, and 34 samples of non-cancerous pancreatic tissue [N] and 82 samples of cancerous tissue [T] both obtained from 82 PDAC patients) and formalin-fixed paraffin-embedded T samples from 34 patients in a validation cohort. RESULTS: The DNA methylation profiles of N samples tended to differ from those of C samples, and 91,907 probes showed significant differences in DNA methylation levels between C and T samples. Epigenetic clustering of T samples was significantly correlated with a larger tumor diameter and early recurrence (ER), defined as relapse within 6 months after surgery. Three marker CpG sites, applicable to formalin-fixed paraffin-embedded surgically resected materials regardless of their tumor cell content, were identified for prediction of ER. The sensitivity and specificity for detection of patients belonging to the ER group using a panel combining these three marker CpG sites, including a CpG site in the CDK14 gene, were 81.8% and 71.7% and 88.9% and 70.4% in the initial and validation cohorts, respectively. CONCLUSION: These findings indicate that DNA methylation alterations may have a clinicopathological impact on PDAC. Application of our criteria will ultimately allow prediction of ER after surgery to improve the outcome of PDAC patients.


Assuntos
Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patologia , Metilação de DNA/genética , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/patologia , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Biomarcadores Tumorais/genética , Estudos de Coortes , Ilhas de CpG/genética , Quinases Ciclina-Dependentes/genética , Epigênese Genética/genética , Feminino , Estudo de Associação Genômica Ampla/métodos , Humanos , Masculino , Inclusão em Parafina/métodos , Fixação de Tecidos/métodos
12.
Exp Eye Res ; 203: 108426, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33387485

RESUMO

PURPOSE: Uveal melanoma (UM) is an aggressive malignancy, in which nearly 50% of the patients die from metastatic disease. Aberrant DNA methylation is recognized as an important epigenomic event in carcinogenesis. Formalin-fixed paraffin-embedded (FFPE) samples represent a valuable source of tumor tissue, and recent technology has enabled the use of these samples in genome-wide DNA methylation analyses. Our aim was to investigate differential DNA methylation in relation to histopathological classification and survival data. In addition we sought to identify aberrant DNA methylation of genes that could be associated with metastatic disease and poor survival. METHODS: FFPE samples from UM patients (n = 23) who underwent enucleation of the eye in the period 1976-1989 were included. DNA methylation was assessed using the Illumina Infinium HumanMethylation450 array and coupled to histopathological data, Cancer Registry of Norway- (registered UM metastasis) and Norwegian Cause of Death Registry- (time and cause of death) data. Differential DNA methylation patterns contrasting histological classification, survival data and clustering properties were investigated. Survival groups were defined as "Early metastasis" (metastases and death within 2-5 years after enucleation, n = 8), "Late metastasis" (metastases and death within 9-21 years after enucleation, n = 7) and "No metastasis" (no detected metastases ≥18 years after enucleation, n = 8). A subset of samples were selected based on preliminary multi-dimensional scaling (MDS) plots, histopathological classification, chromosome 3 status, survival status and clustering properties; "Subset Early metastasis" (n = 4) vs "Subset No metastasis" (n = 4). Bioinformatics analyses were conducted in the R statistical software. Differentially methylated positions (DMPs) and differentially methylated regions (DMRs) in various comparisons were assessed. Gene expression of relevant subgroups was determined by microarray analysis and quantitative reverse-transcription polymerase chain reaction (qRT-PCR). RESULTS: DNA methylation analyses identified 2 clusters that separated the samples according to chromosome 3 status. Cluster 1 consisted of samples (n = 5) with chromosome 3 disomy (D3), while Cluster 2 was comprised of samples (n = 15) with chromosome 3 monosomy (M3). 1212 DMRs and 9386 DMPs were identified in M3 vs D3. No clear clusters were formed based on our predefined survival groups ("Early", "Late", "No") nor histopathological classification (Epithelioid, Mixed, Spindle). We identified significant changes in DNA methylation (beta FC ≥ 0.2, adjusted p < 0.05) between two sample subsets (n = 8). "Subset Early metastasis" (n = 4) vs "Subset No metastasis" (n = 4) identified 348 DMPs and 36 DMRs, and their differential gene expression by microarray showed that 14 DMPs and 2 DMRs corresponded to changes in gene expression (FC ≥ 1.5, p < 0.05). RNF13, ZNF217 and HYAL1 were hypermethylated and downregulated in "Subset Early metastasis" vs "Subset No metastasis" and could be potential tumor suppressors. TMEM200C, RGS10, ADAM12 and PAM were hypomethylated and upregulated in "Subset Early metastasis vs "Subset No metastasis" and could be potential oncogenes and thus markers of early metastasis and poor prognosis in UM. CONCLUSIONS: DNA methylation profiling showed differential clustering of samples according to chromosome 3 status: Cluster 1 (D3) and Cluster 2 (M3). Integrated differential DNA methylation and gene expression of two subsets of samples identified genes associated with early metastasis and poor prognosis. RNF13, ZNF217 and HYAL1 are hypermethylated and candidate tumor suppressors, while TMEM200C, RGS10, ADAM12 and PAM are hypomethylated and candidate oncogenes linked to early metastasis. UM FFPE samples represent a valuable source for methylome studies and enable long-time follow-up.


Assuntos
Metilação de DNA , DNA de Neoplasias/genética , Regulação Neoplásica da Expressão Gênica/fisiologia , Melanoma/genética , Proteínas de Neoplasias/genética , Neoplasias Uveais/genética , Adulto , Variações do Número de Cópias de DNA , Epigenômica , Enucleação Ocular , Feminino , Formaldeído , Perfilação da Expressão Gênica , Humanos , Masculino , Melanoma/patologia , Melanoma/cirurgia , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , Inclusão em Parafina , Análise de Componente Principal , Reação em Cadeia da Polimerase em Tempo Real , Fixação de Tecidos , Neoplasias Uveais/patologia , Neoplasias Uveais/cirurgia , Adulto Jovem
13.
Zhonghua Shao Shang Za Zhi ; 37(2): 157-163, 2021 Feb 20.
Artigo em Chinês | MEDLINE | ID: mdl-33498099

RESUMO

Objective: To observe the effect of immunofluorescence double staining for foamy macrophages and Mycobacterium tuberculosis (MTB) in paraffin-embedded tissue of clinical tuberculous wound, in comparison with three routine staining methods. Methods: The experimental method was used. From April 2019 to May 2020, 10 patients with tuberculous wound (5 males and 5 females, aged 28-77 years) meeting the inclusion criteria were treated in the Department of Burns and Plastic & Wound Repair Surgery of Xiang'an Hospital of Xiamen University. The paraffin-embedded wound tissue were collected during extended debridement and preserved in the Department of Pathology of this hospital. Forty paraffin sections were made from the wound tissue of each patient. Hematoxylin-eosin (HE) staining, immunohistochemical staining, Ziehl-Neelsen and immunohistochemical double staining, immunofluorescence double staining were performed respectively, with 10 sections in each method. The section rejection rate of four staining methods were calculated. The recognition and detection of wound granuloma tissue in the four staining methods were observed and counted, and the recognition and detection of foamy macrophages in the wound tissue stained with four methods were observed. The MTB detection in the wound granuloma tissue and non-granuloma tissue in the four staining methods were compared. The subtyping and distribution of foamy macrophages and detection rate of MTB in the wound granuloma tissue and non-granuloma tissue, the morphologic clarity of foamy macrophages, as well as the non-specific staining rate and the loss rate of positive reaction of MTB and foamy macrophages by Ziehl-Neelsen and immunohistochemical double staining were compared with those of immunofluorescence double staining. Data were statistically analyzed with Fisher's exact probability test, one-way analysis of variance, independent sample t test and Wilcoxon signed rank test. Results: The section rejection rate of HE staining, immunohistochemical staining, Ziehl-Neelsen and immunohistochemical double staining, and immunofluorescence double staining were 3% (3/100), 1% (1/100), 6% (6/100), and 2% (2/100), respectively. There was no statistically significant difference among the four groups (P=0.26). All the four staining methods could identify granuloma tissue, and the number of granuloma structures was similar (F=1.284, P=0.28). All the four staining methods were able to identify foamy macrophages in the wound tissue, which was detected in each section. No MTB was observed in the wound granuloma tissue or non-granuloma tissue by HE staining or immunohistochemical staining. MTB was observed distributing in the wound granuloma tissue and non-granuloma tissue by Ziehl-Neelsen and immunohistochemical double staining and immunofluorescence double staining, and most MTB distributed in the wound granuloma tissue. Ziehl-Neelsen and immunohistochemical double staining could not distinguish foamy macrophages engulfed MTB from that non-engulfed MTB. Immunofluorescence double staining showed that foamy macrophages engulfed MTB mostly distributed in the wound granuloma tissue, and the foamy macrophages non-engulfed MTB mostly distributed in the wound non-granuloma tissue. The detection rates of MTB in wound granuloma and non-granuloma tissue in immunofluorescence double staining were (89.00±0.08)% and (82.67±0.05)%, respectively, which were significantly higher than (54.56±0.14)% and (44.44±0.13)% in Ziehl-Neelsen and immunohistochemical double staining (t=-12.495, -7.961, P<0.01). Compared with that of Ziehl-Neelsen and immunohistochemical double staining, immunofluorescence double staining showed better foamy macrophages clarity in wound tissue (Z=-3.162, P<0.01). The nonspecific staining rate and positive reaction loss rate of MTB and foamy macrophages in wound tissue of immunofluorescence double staining were (9.11±0.07)% and (9.22±0.07)%, respectively, which were significantly lower than (20.67±0.06)% and (44.00±0.12)% of Ziehl-Neelsen and immunohistochemical double staining (t=4.569, 15.519, P<0.01). Conclusions: Compared with HE staining, immunohistochemical staining, and Ziehl-Neelsen and immunohistochemical double staining, the immunofluorescence double staining is easy to operate, giving clear and intuitive images. It allows accurate imaging co-localization of MTB and foamy macrophages in paraffin-embedded tissue of clinical tuberculous wound.


Assuntos
Mycobacterium tuberculosis , Adulto , Idoso , Feminino , Imunofluorescência , Humanos , Macrófagos , Masculino , Pessoa de Meia-Idade , Inclusão em Parafina , Coloração e Rotulagem
14.
Methods Mol Biol ; 2261: 525-533, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33421012

RESUMO

Clinical tissue archives represent an invaluable source of biological information. Formalin-fixed, paraffin-embedded (FFPE) tissue can be used for retrospective investigation of biomarkers of diseases and prognosis.Recently, the number of studies using proteome profiling of samples from clinical archives has markedly increased. However, the application of conventional quantitative proteomics technologies remains a challenge mainly due to the harsh fixation process resulting in protein cross-linking and protein degradation. In the present chapter, we demonstrate a protocol for label-free proteomic analysis of FFPE tissue prepared from human cardiac autopsies. The data presented here highlight the applicability and suitability of FFPE heart tissue for understanding the molecular mechanism of cardiac injury using a proteomics approach.


Assuntos
Fixadores/química , Formaldeído/química , Miocárdio/metabolismo , Inclusão em Parafina , Proteínas/análise , Proteoma , Proteômica , Fixação de Tecidos , Autopsia , Cromatografia de Fase Reversa , Humanos , Espectrometria de Massas em Tandem
15.
Pathologe ; 42(1): 83-85, 2021 Feb.
Artigo em Alemão | MEDLINE | ID: mdl-33475807

RESUMO

The detection of Mycobacterium tuberculosis complex DNA by PCR using formalin-fixed paraffin-embedded material has become an integral part of molecular-pathological diagnostics. We describe an approach that enables the detection of contamination by using Mycobacterium szulgai as a positive control, contributing to the reduction of false-positive results.


Assuntos
Mycobacterium tuberculosis , DNA Bacteriano/genética , Formaldeído , Mycobacterium tuberculosis/genética , Micobactérias não Tuberculosas , Inclusão em Parafina , Reação em Cadeia da Polimerase , Sensibilidade e Especificidade
16.
Pathologe ; 42(1): 78-82, 2021 Feb.
Artigo em Alemão | MEDLINE | ID: mdl-33475809

RESUMO

In the diagnosis of mycobacterioses, microbiological examination with culture and antibiogram, possibly in combination with molecular biological testing of the fresh material, still represents the gold standard. However, these methods are not available for formalin-fixed paraffin-embedded (FFPE) material or other fixed samples. For this reason, the first step in pathology is to attempt microscopic pathogen detection (ZN/Fite/rhodamine-auramine). Subsequently, molecular pathological examination for the detection of mycobacterial gene sequences should also be considered mandatory today. Although this has clear limits due to the material, it is nevertheless well suited, if carried out correctly, to detect a mycobacterial infection or make it unlikely. A negative result may favor an alternative diagnosis but does not completely rule out mycobacteriosis.For the therapy of tuberculosis or nontuberculous mycobacterial (NTM) disease, the reliable detection of the species and the determination of resistance is of utmost importance. With regard to therapy, the clinician cannot afford to make a false diagnosis. In case of doubt, a rebiopsy for sampling native material, particularly for microbiological testing, should be discussed.


Assuntos
Mycobacterium tuberculosis , Mycobacterium , Tuberculose , DNA Bacteriano/genética , Humanos , Mycobacterium/genética , Mycobacterium tuberculosis/genética , Inclusão em Parafina , Patologia Molecular , Reação em Cadeia da Polimerase , Tuberculose/diagnóstico , Tuberculose/genética
17.
Pathologe ; 42(1): 71-77, 2021 Feb.
Artigo em Alemão | MEDLINE | ID: mdl-33475810

RESUMO

Although typical histological findings of tuberculosis are well known, the diagnosis of nonmicrobiologically proven tuberculosis with the instruments available to pathology is challenging. Indeed, necrotizing epithelioid cell granulomatosis is typical for tuberculosis, but it is also seen in a number of different infectious or noninfectious lung diseases. The tools of microscopy and molecular pathology are suitable for confirming the diagnosis or paving the way to a differential diagnosis, but molecular pathology applied to formalin-fixated and paraffin-embedded material is limited. This should be openly communicated to the referring clinician. After interdisciplinary re-evaluation of the findings, an alternative solution to confirm the diagnosis must therefore be found if the additional examinations are negative.


Assuntos
Mycobacterium tuberculosis , Tuberculose Pulmonar , Tuberculose , Diagnóstico Diferencial , Formaldeído , Humanos , Pulmão , Inclusão em Parafina , Tuberculose/diagnóstico , Tuberculose Pulmonar/diagnóstico
18.
Arch Pathol Lab Med ; 145(2): 222-226, 2021 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-33501497

RESUMO

CONTEXT.­: The Surveillance, Epidemiology, and End Results (SEER) cancer registry program is currently evaluating the use of archival, diagnostic, formalin-fixed, paraffin-embedded (FFPE) tissue obtained through SEER cancer registries, functioning as honest brokers for deidentified tissue and associated data. To determine the feasibility of this potential program, laboratory policies for sharing tissue for research needed to be assessed. OBJECTIVE.­: To understand the willingness of pathology laboratories to share archival diagnostic tissue for cancer research and related policies. DESIGN.­: Seven SEER registries administered a 27-item questionnaire to pathology laboratories within their respective registry catchment areas. Only laboratories that processed diagnostic FFPE specimens and completed the questionnaire were included in the analysis. RESULTS.­: Of the 153 responding laboratories, 127 (83%) responded that they process FFPE specimens. Most (n = 88; 69%) were willing to share tissue specimens for research, which was not associated with the number of blocks processed per year by the laboratories. Most laboratories retained the specimens for at least 10 years. Institutional regulatory policies on sharing deidentified tissue varied considerably, ranging from requiring a full Institutional Review Board review to considering such use exempt from Institutional Review Board review, and 43% (55 of 127) of the laboratories did not know their terms for sharing tissue for research. CONCLUSIONS.­: This project indicated a general willingness of pathology laboratories to participate in research by sharing FFPE tissue. Given the variability of research policies across laboratories, it is critical for each SEER registry to work with laboratories in their catchment area to understand such policies and state legislation regulating tissue retention and guardianship.


Assuntos
Laboratórios/legislação & jurisprudência , Neoplasias/patologia , Políticas , Pesquisa/legislação & jurisprudência , Programa de SEER/legislação & jurisprudência , Formaldeído , Humanos , Neoplasias/diagnóstico , Inclusão em Parafina , Patologia , Fixação de Tecidos
19.
Methods Mol Biol ; 2230: 283-302, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33197020

RESUMO

Cartilage and bone are specialized skeletal tissues composed of unique extracellular matrices. Bone, in particular, has a highly calcified or mineralized matrix that makes microtomy and standard histological studies very challenging. Therefore, methods to appropriately fix and decalcify mineralized skeletal tissues have been developed to allow for paraffin processing and standard microtomy. In this chapter, we will illustrate methods for tissue grossing, fixation, decalcification, paraffin processing, embedding, sectioning, and routine histological staining of demineralized murine skeletal tissues. We will also discuss methods for decalcified frozen sectioning of skeletal tissues with and without the use of a tape-transfer system.


Assuntos
Osso e Ossos/ultraestrutura , Cartilagem/ultraestrutura , Técnica de Descalcificação/métodos , Microtomia/métodos , Animais , Secções Congeladas/métodos , Camundongos , Inclusão em Parafina/métodos , Coloração e Rotulagem/métodos , Fixação de Tecidos/métodos
20.
Methods Mol Biol ; 2230: 337-344, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33197023

RESUMO

Immunohistochemistry, or immunolabeling, is a key method for the identification of protein expression and localization. Successful detection relies on a low signal-to-noise ratio, which is affected greatly by antibody specificity as well as the staining protocol. Immunohistochemistry in the mouse is challenging, particularly in adult skeletal tissue, due to the need for long decalcification, high autofluorescence and high levels of endogenous peroxidase. Here, we describe a highly sensitive protocol for protein detection in decalcified paraffin-embedded sections from adult mouse skeletal tissue. By using four levels of amplification, this method allows for the identification of even low-abundance proteins.


Assuntos
Osso e Ossos/ultraestrutura , Técnica de Descalcificação/métodos , Imunofluorescência/métodos , Proteínas/isolamento & purificação , Coloração e Rotulagem/métodos , Animais , Osso e Ossos/diagnóstico por imagem , Humanos , Camundongos , Inclusão em Parafina/métodos , Proteínas/química
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