scholarly journals Molecular Detection of SARS-CoV-2 Infection in FFPE Samples and Histopathologic Findings in Fatal SARS-CoV-2 Cases

2020 ◽  
Vol 154 (2) ◽  
pp. 190-200 ◽  
Author(s):  
Miroslav Sekulic ◽  
Holly Harper ◽  
Behtash G Nezami ◽  
Daniel L Shen ◽  
Simona Pichler Sekulic ◽  
...  
Keyword(s):  
Diffuse Alveolar Damage ◽  
Ffpe Tissue ◽  
Qrt Pcr ◽  
Morphological Findings ◽  
Formalin Fixed Paraffin ◽  
Ffpe Samples ◽  
Formalin Fixed Paraffin Embedded ◽  
Control And Prevention ◽  
Next Generation Sequencing Ngs ◽  
Viral Sequencing

Abstract Objectives To report methods and findings of 2 autopsies with molecular evaluation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) positive individuals. Methods Postmortem examination was completed following Centers for Disease Control and Prevention public guidelines. Numerous formalin-fixed paraffin-embedded (FFPE) tissue types from each case were surveyed for SARS-CoV-2 RNA by quantitative reverse transcription polymerase chain reaction (qRT-PCR). SARS-CoV-2 viral genome was sequenced by next-generation sequencing (NGS) from FFPE lung tissue blocks. Results Postmortem examinations revealed diffuse alveolar damage, while no viral-associated hepatic, cardiac, or renal damage was observed. Viral RNA was detected in lungs, bronchi, lymph nodes, and spleen in both cases using qRT-PCR method. RNA sequencing using NGS in case 1 revealed mutations most consistent with Western European Clade A2a with ORF1a L3606F mutation. Conclusions SARS-CoV-2 testing and viral sequencing can be performed from FFPE tissue. Detection and sequencing of SARS-CoV-2 in combination with morphological findings from postmortem tissue examination can aid in gaining a better understanding of the virus’s pathophysiologic effects on human health.

scholarly journals HYPERsol: flash-frozen results from archival FFPE tissue for clinical proteomics

2019 ◽  
Author(s):  
Dylan M. Marchione ◽  
Ilyana Ilieva ◽  
Benjamin A. Garcia ◽  
Darryl J. Pappin ◽  
John P. Wilson ◽  
...  
Keyword(s):  
Protein Extraction ◽  
Ffpe Tissue ◽  
Clinical Proteomics ◽  
High Yield ◽  
Proteome Coverage ◽  
Formalin Fixed Paraffin ◽  
Ffpe Samples ◽  
Formalin Fixed Paraffin Embedded ◽  
Proteomics Research ◽  
Formalin Fixed

Massive formalin-fixed, paraffin-embedded (FFPE) tissue archives exist worldwide, representing a potential gold mine for clinical proteomics research. However, current protocols for FFPE proteomics lack standardization, efficiency, reproducibility, and scalability. Here we present High-Yield Protein Extraction and Recovery by direct SOLubilization (HYPERsol), an optimized workflow using adaptive-focused acoustics (AFA) ultrasonication and S-Trap sample processing that enables proteome coverage and quantification from FFPE samples comparable to that achieved from flash-frozen tissue (average R = 0.936).

scholarly journals Technical Validity of a Customized Assay of Sensitivity to Endocrine Therapy Using Sections from Fixed Breast Cancer Tissue

2020 ◽  
Vol 66 (7) ◽  
pp. 934-945 ◽  
Author(s):  
Rosanna Lau ◽  
Lili Du ◽  
Eveline Chen ◽  
Chunxiao Fu ◽  
Rebekah Gould ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Endocrine Therapy ◽  
Linear Range ◽  
Ffpe Tissue ◽  
Cancer Tissue ◽  
Analytical Sensitivity ◽  
Formalin Fixed Paraffin ◽  
Ffpe Samples ◽  
Resected Tumor ◽  
Formalin Fixed Paraffin Embedded

Abstract Background We translated a multigene expression index to predict sensitivity to endocrine therapy for Stage II–III breast cancer (SET2,3) to hybridization-based expression assays of formalin-fixed paraffin-embedded (FFPE) tissue sections. Here we report the technical validity with FFPE samples, including preanalytical and analytical performance. Methods We calibrated SET2,3 from microarrays (Affymetrix U133A) of frozen samples to hybridization-based assays of FFPE tissue, using bead-based QuantiGene Plex (QGP) and slide-based NanoString (NS). The following preanalytical and analytical conditions were tested in controlled studies: replicates within and between frozen and fixed samples, age of paraffin blocks, homogenization of fixed sections versus extracted RNA, core biopsy versus surgically resected tumor, technical replicates, precision over 20 weeks, limiting dilution, linear range, and analytical sensitivity. Lin’s concordance correlation coefficient (CCC) was used to measure concordance between measurements. Results SET2,3 index was calibrated to use with QGP (CCC 0.94) and NS (CCC 0.93) technical platforms, and was validated in two cohorts of older fixed samples using QGP (CCC 0.72, 0.85) and NS (CCC 0.78, 0.78). QGP assay was concordant using direct homogenization of fixed sections versus purified RNA (CCC 0.97) and between core and surgical sample types (CCC 0.90), with 100% accuracy in technical replicates, 1–9% coefficient of variation over 20 weekly tests, linear range 3.0–11.5 (log2 counts), and analytical sensitivity ≥2.0 (log2 counts). Conclusions Measurement of the novel SET2,3 assay was technically valid from fixed tumor sections of biopsy or resection samples using simple, inexpensive, hybridization methods, without the need for RNA purification.

scholarly journals Mining the archives: a cross-platform analysis of gene expression profiles in archival formalin-fixed paraffin-embedded (FFPE) tissue

2015 ◽  
Author(s):  
Anna Francina Webster ◽  
Paul Zumbo ◽  
Jennifer Fostel ◽  
Jorge Gandara ◽  
Susan D Hester ◽  
...  
Keyword(s):  
Gene Expression ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Ffpe Tissue ◽  
Rna Seq ◽  
Preservation Method ◽  
Formalin Fixed Paraffin ◽  
Ffpe Samples ◽  
Formalin Fixed Paraffin Embedded ◽  
Formalin Fixed

Formalin-fixed paraffin-embedded (FFPE) tissue samples represent a potentially invaluable resource for transcriptomic-based research into the molecular basis of disease. However, use of FFPE samples in gene expression studies has been limited by technical challenges resulting from degradation of nucleic acids. Here we evaluated gene expression profiles derived from fresh-frozen (FRO) and FFPE mouse liver tissues using two DNA microarray protocols and two whole transcriptome sequencing (RNA-seq) library preparation methodologies. The ribo-depletion protocol outperformed the other three methods by having the highest correlations of differentially expressed genes (DEGs) and best overlap of pathways between FRO and FFPE groups. We next tested the effect of sample time in formalin (18 hours or 3 weeks) on gene expression profiles. Hierarchical clustering of the datasets indicated that test article treatment, and not preservation method, was the main driver of gene expression profiles. Meta- and pathway analyses indicated that biological responses were generally consistent for 18-hour and 3-week FFPE samples compared to FRO samples. However, clear erosion of signal intensity with time in formalin was evident, and DEG numbers differed by platform and preservation method. Lastly, we investigated the effect of age in FFPE block on genomic profiles. RNA-seq analysis of 8-, 19-, and 26-year-old control blocks using the ribo-depletion protocol resulted in comparable quality metrics, including expected distributions of mapped reads to exonic, UTR, intronic, and ribosomal fractions of the transcriptome. Overall, our results suggest that FFPE samples are appropriate for use in genomic studies in which frozen samples are not available, and that ribo-depletion RNA-seq is the preferred method for this type of analysis in archival and long-aged FFPE samples.

scholarly journals Hubungan Lama Penyimpanan Sampel Arsip Jaringan Dalam Blok Parafin Terfiksasi Formalindengan Kualitas Hasil Ekstraksidna Mitokondria Jaringan

2018 ◽  
Vol 1 (3) ◽  
pp. 157-162
Author(s):  
Zen Hafy ◽  
Veny Larasati ◽  
Riana Sari Puspita ◽  
Novizar S ◽  
Haekal M ◽  
...  
Keyword(s):  
Storage Time ◽  
Molecular Study ◽  
Ffpe Tissue ◽  
Genetic Studies ◽  
Formalin Fixed Paraffin ◽  
Ffpe Samples ◽  
Formalin Fixed Paraffin Embedded ◽  
Pcr Products ◽  
Ffpe Tissues

Formalin-fixed paraffin-embedded (FFPE) archival tissue presents a readily available resource in molecular study nowadays. The quality of nucleid acid, especially mitoconhdrial DNA (mtDNA) extracted from FFPE tissue could be affected by the storage time. Thus, this study investigated if the FFPE tissue’s storage time had an effect on the quality of the extracted mtDNA at Department of Pathology RSUP Dr. Mohammad Hoesin Palembang. DNA was extracted from 16 randomly selected archival FFPE tissues in Laboratory of Pathology Anatomy, RSUP dr. Mohammad Hoesin Palembang. The samples were grouped based on their storage time (less than 1 year and 1 to 5 yrs.’ old). The isolated DNA from each group was amplified using PCR with two primer pairs specifically designto amplify mtDNA of 320 bp and 142bp length, respectively.  The PCR products were visualized by electrophoresismethod. None of the samples from both groups could be amplified witht the 320bp primers. However, the PCR result of the 149 bp primers showed positive for all of the samples from each study group. The study indicated that the storage time does not affect the quality of mtDNA isolated from the FFPE samples archived in Department of Pathology RSUP Dr. Mohammad Hoesin Palembang. Furthermore, the study showed that the mtDNA extracted from FFPE tissue has been degraded, therefore, the samples are not suitable for genetic studies require a long mtDNA amplicon.

scholarly journals Detection of Histoplasma DNA from Tissue Blocks by a Specific and a Broad-Range Real-Time PCR: Tools to Elucidate the Epidemiology of Histoplasmosis

2020 ◽  
Vol 6 (4) ◽  
pp. 319
Author(s):  
Dunja Wilmes ◽  
Ilka McCormick-Smith ◽  
Charlotte Lempp ◽  
Ursula Mayer ◽  
Arik Bernard Schulze ◽  
...  
Keyword(s):  
Diagnostic Tests ◽  
Fungal Infections ◽  
Ffpe Tissue ◽  
Clinical Sensitivity ◽  
Formalin Fixed Paraffin ◽  
Three Samples ◽  
Ffpe Samples ◽  
Formalin Fixed Paraffin Embedded ◽  
Tissue Specimens ◽  
Formalin Fixed

Lack of sensitive diagnostic tests impairs the understanding of the epidemiology of histoplasmosis, a disease whose burden is estimated to be largely underrated. Broad-range PCRs have been applied to identify fungal agents from pathology blocks, but sensitivity is variable. In this study, we compared the results of a specific Histoplasma qPCR (H. qPCR) with the results of a broad-range qPCR (28S qPCR) on formalin-fixed, paraffin-embedded (FFPE) tissue specimens from patients with proven fungal infections (n = 67), histologically suggestive of histoplasmosis (n = 36) and other mycoses (n = 31). The clinical sensitivity for histoplasmosis of the H. qPCR and the 28S qPCR was 94% and 48.5%, respectively. Samples suggestive for other fungal infections were negative with the H. qPCR. The 28S qPCR did not amplify DNA of Histoplasma in FFPE in these samples, but could amplify DNA of Emergomyces (n = 1) and Paracoccidioides (n = 2) in three samples suggestive for histoplasmosis but negative in the H. qPCR. In conclusion, amplification of Histoplasma DNA from FFPE samples is more sensitive with the H. qPCR than with the 28S qPCR. However, the 28S qPCR identified DNA of other fungi in H. qPCR-negative samples presenting like histoplasmosis, suggesting that the combination of both assays may improve the diagnosis.

scholarly journals Higher EU-TIRADS-Score Correlated with BRAF V600E Positivity in the Early Stage of Papillary Thyroid Carcinoma

2021 ◽  
Vol 10 (11) ◽  
pp. 2304
Author(s):  
Karolina Skubisz ◽  
Joanna Januszkiewicz-Caulier ◽  
Patrycja Cybula ◽  
Elwira Bakuła-Zalewska ◽  
Krzysztof Goryca ◽  
...  
Keyword(s):  
Early Stage ◽  
Braf V600e ◽  
P Value ◽  
Dna And Rna ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Ion Proton ◽  
Indolent Disease ◽  
Next Generation Sequencing Ngs ◽  
Formalin Fixed

The data demonstrating a correlation between sonographic markers of malignancy of thyroid cancer (TC) and its genetic status are scarce. This study aimed to assess whether the addition of genetic analysis at the preoperative step of TC patients’ stratification could aid their clinical management. The material consisted of formalin-fixed paraffin-embedded tumor fragments of 49 patients who underwent thyroidectomy during the early stages of papillary TC (PTC). Tumor DNA and RNA were subjected to next-generation sequencing (NGS) on Ion Proton using the Oncomine™ Comprehensive Assay panel. We observed a significant correlation between BRAF V600E and a higher EU-TIRADS score (p-value = 0.02) with a correlation between hypoechogenicity and taller-than-wide tumor shape in analysed patients. There were no other significant associations between the identified genetic variants and other clinicopathological features. For TC patient’s stratification, a strong suspicion of BRAF V600E negativity in preoperative management of TC patients could limit the over-treatment of asymptomatic, very low-risk, indolent disease and leave room for active surveillance.

scholarly journals The Dutch National TissueArchive Portal enables efficient, consistent, and transparent procurement of diagnostic tissue samples for scientific use

2021 ◽  
Author(s):  
Robin Verjans ◽  
Annette H. Bruggink ◽  
Robby Kibbelaar ◽  
Jos Bart ◽  
Aletta Debernardi ◽  
...  
Keyword(s):  
The Netherlands ◽  
Tissue Sample ◽  
Ffpe Tissue ◽  
Tissue Samples ◽  
Internet Portal ◽  
Formalin Fixed Paraffin ◽  
Practical Support ◽  
Formalin Fixed Paraffin Embedded ◽  
The Rich ◽  
Formalin Fixed

AbstractBiobanks play a crucial role in enabling biomedical research by facilitating scientific use of valuable human biomaterials. The PALGA foundation—a nationwide network and registry of histo- and cytopathology in the Netherlands—was established to promote the provision of data within and between pathology departments, and to make the resulting knowledge available for healthcare. Apart from the pathology data, we aimed to utilize PALGA’s nationwide network to find and access the rich wealth of Formalin-Fixed Paraffin-Embedded (FFPE) tissue samples for scientific use.  We implemented the Dutch National TissueArchive Portal (DNTP) to utilize PALGA’s nationwide network for requesting FFPE tissue samples. The DNTP consists of (1) a centrally organized internet portal to improve the assessing, processing, harmonization, and monitoring of the procurement process, while (2) dedicated HUB-employees provide practical support at peripheral pathology departments. Since incorporation of the DNTP, both the number of filed requests for FFPE tissue samples and the amount of HUB-mediated support increased 55 and 29% respectively. In line, the sample procurement duration time decreased significantly (− 47%). These findings indicate that implementation of the DNTP improved the frequency, efficiency, and transparency of FFPE tissue sample procurement for research in the Netherlands. To conclude, the need for biological resources is growing persistently to enable precision medicine. Here, we access PALGA’s national, pathology network by implementation of the DNTP to allow for efficient, consistent, and transparent exchange of FFPE tissue samples for research across the Netherlands.

scholarly journals The impact of RNA extraction method on accurate RNA sequencing from formalin-fixed paraffin-embedded tissues

BMC Cancer ◽  
2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Michal Marczyk ◽  
Chunxiao Fu ◽  
Rosanna Lau ◽  
Lili Du ◽  
Alexander J. Trevarton ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Gene Expression ◽  
Rna Sequencing ◽  
Rna Extraction ◽  
Formalin Fixed Paraffin ◽  
Ffpe Samples ◽  
Formalin Fixed Paraffin Embedded ◽  
The Impact ◽  
Formalin Fixed

Abstract Background Utilization of RNA sequencing methods to measure gene expression from archival formalin-fixed paraffin-embedded (FFPE) tumor samples in translational research and clinical trials requires reliable interpretation of the impact of pre-analytical variables on the data obtained, particularly the methods used to preserve samples and to purify RNA. Methods Matched tissue samples from 12 breast cancers were fresh frozen (FF) and preserved in RNAlater or fixed in formalin and processed as FFPE tissue. Total RNA was extracted and purified from FF samples using the Qiagen RNeasy kit, and in duplicate from FFPE tissue sections using three different kits (Norgen, Qiagen and Roche). All RNA samples underwent whole transcriptome RNA sequencing (wtRNAseq) and targeted RNA sequencing for 31 transcripts included in a signature of sensitivity to endocrine therapy. We assessed the effect of RNA extraction kit on the reliability of gene expression levels using linear mixed-effects model analysis, concordance correlation coefficient (CCC) and differential analysis. All protein-coding genes in the wtRNAseq and three gene expression signatures for breast cancer were assessed for concordance. Results Despite variable quality of the RNA extracted from FFPE samples by different kits, all had similar concordance of overall gene expression from wtRNAseq between matched FF and FFPE samples (median CCC 0.63–0.66) and between technical replicates (median expression difference 0.13–0.22). More than half of genes were differentially expressed between FF and FFPE, but with low fold change (median |LFC| 0.31–0.34). Two out of three breast cancer signatures studied were highly robust in all samples using any kit, whereas the third signature was similarly discordant irrespective of the kit used. The targeted RNAseq assay was concordant between FFPE and FF samples using any of the kits (CCC 0.91–0.96). Conclusions The selection of kit to purify RNA from FFPE did not influence the overall quality of results from wtRNAseq, thus variable reproducibility of gene signatures probably relates to the reliability of individual gene selected and possibly to the algorithm. Targeted RNAseq showed promising performance for clinical deployment of quantitative assays in breast cancer from FFPE samples, although numerical scores were not identical to those from wtRNAseq and would require calibration.

scholarly journals Discrimination of Aspergillosis, Mucormycosis, Fusariosis, and Scedosporiosis in Formalin-Fixed Paraffin-Embedded Tissue Specimens by Use of Multiple Real-Time Quantitative PCR Assays

2016 ◽  
Vol 54 (11) ◽  
pp. 2798-2803 ◽  
Author(s):  
Elham Salehi ◽  
Mohammad T. Hedayati ◽  
Jan Zoll ◽  
Haleh Rafati ◽  
Maryam Ghasemi ◽  
...  
Keyword(s):  
Fusarium Oxysporum ◽  
Aspergillus Flavus ◽  
Ffpe Tissue ◽  
Content Type ◽  
Real Time Quantitative Pcr ◽  
Tissue Sections ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Tissue Specimens ◽  
Formalin Fixed

In a retrospective multicenter study, 102 formalin-fixed paraffin-embedded (FFPE) tissue specimens with histopathology results were tested. Two 4- to 5-μm FFPE tissue sections from each specimen were digested with proteinase K, followed by automated nucleic acid extraction. Multiple real-time quantitative PCR (qPCR) assays targeting the internal transcribed spacer 2 (ITS2) region of ribosomal DNA, using fluorescently labeled primers, was performed to identify clinically important genera and species of Aspergillus , Fusarium , Scedosporium , and the Mucormycetes . The molecular identification was correlated with results from histological examination. One of the main findings of our study was the high sensitivity of the automated DNA extraction method, which was estimated to be 94%. The qPCR procedure that was evaluated identified a range of fungal genera/species, including Aspergillus fumigatus , Aspergillus flavus , Aspergillus terreus , Aspergillus niger , Fusarium oxysporum , Fusarium solani , Scedosporium apiospermum , Rhizopus oryzae , Rhizopus microsporus , Mucor spp., and Syncephalastrum . Fusarium oxysporum and F. solani DNA was amplified from five specimens from patients initially diagnosed by histopathology as having aspergillosis. Aspergillus flavus , S. apiospermum , and Syncephalastrum were detected from histopathological mucormycosis samples. In addition, examination of four samples from patients suspected of having concomitant aspergillosis and mucormycosis infections resulted in the identification of two A. flavus isolates, one Mucor isolate, and only one sample having both R. oryzae and A. flavus . Our results indicate that histopathological features of molds may be easily confused in tissue sections. The qPCR assay used in this study is a reliable tool for the rapid and accurate identification of fungal pathogens to the genus and species levels directly from FFPE tissues.

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