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 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.

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 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 Comparison of Two Methods of RNA Extraction from Formalin-Fixed Paraffin-Embedded Tissue Specimens

2014 ◽  
Vol 2014 ◽  
pp. 1-5 ◽  
Author(s):  
Gisele Rodrigues Gouveia ◽  
Suzete Cleusa Ferreira ◽  
Jerenice Esdras Ferreira ◽  
Sheila Aparecida Coelho Siqueira ◽  
Juliana Pereira
Keyword(s):  
Rna Extraction ◽  
B Cell Lymphoma ◽  
Extraction Process ◽  
Nucleic Acid Isolation ◽  
Formalin Fixed Paraffin ◽  
Ffpe Samples ◽  
Formalin Fixed Paraffin Embedded ◽  
Block Storage ◽  
Tissue Specimens ◽  
Formalin Fixed

The present study aimed to compare two different methods of extracting RNA from formalin-fixed paraffin-embedded (FFPE) specimens of diffuse large B-cell lymphoma (DLBCL). We further aimed to identify possible influences of variables—such as tissue size, duration of paraffin block storage, fixative type, primers used for cDNA synthesis, and endogenous genes tested—on the success of amplification from the samples. Both tested protocols used the same commercial kit for RNA extraction (the RecoverAll Total Nucleic Acid Isolation Optimized for FFPE Samples from Ambion). However, the second protocol included an additional step of washing with saline buffer just after sample rehydration. Following each protocol, we compared the RNA amount and purity and the amplification success as evaluated by standard PCR and real-time PCR. The results revealed that the extra washing step added to the RNA extraction process resulted in significantly improved RNA quantity and quality and improved success of amplification from paraffin-embedded specimens.

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 Time and tumor type (primary or metastatic) do not influence the detection of BRAF/NRAS mutations in formalin fixed paraffin embedded samples from melanomas

2016 ◽  
Vol 54 (11) ◽  
Author(s):  
Miriam Potrony ◽  
Celia Badenas ◽  
Bénédicte Naerhuyzen ◽  
Paula Aguilera ◽  
Joan Anton Puig-Butille ◽  
...  
Keyword(s):  
Melanoma Patient ◽  
Primary Melanoma ◽  
Sentinel Lymph Nodes ◽  
Tumor Type ◽  
Formalin Fixed Paraffin ◽  
Ffpe Samples ◽  
Formalin Fixed Paraffin Embedded ◽  
Complete Sequencing ◽  
Formalin Fixed ◽  
Tumor Area

AbstractBackground:Methods:DNA was obtained from 144 FFPE samples (62 primary melanoma, 43 sentinel lymph nodes [SLN] and 39 metastasis).Results:Complete sequencing results were obtained from 75% (108/144) of the samples, and at least one gene was sequenced in 89% (128/144) of them.Conclusions:Preserving sufficient tumor area in FFPE blocks is important. It is necessary to keep the FFPE blocks, no matter their age, as they are necessary to decide the best treatment for the melanoma patient.

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