Profiling chromatin accessibility in formalin-fixed paraffin-embedded samples

Archived formalin-fixed paraffin-embedded (FFPE) samples are the global standard format for preservation of the majority of biopsies in both basic research and translational cancer studies, and profiling chromatin accessibility in the archived FFPE tissues is fundamental to understanding gene regulation. Accurate mapping of chromatin accessibility from FFPE specimens is challenging because of the high degree of DNA damage. Here, we first showed that standard ATAC-seq can be applied to purified FFPE nuclei but yields lower library complexity and a smaller proportion of long DNA fragments. We then present FFPE-ATAC, the first highly sensitive method for decoding chromatin accessibility in FFPE tissues that combines Tn5-mediated transposition and T7 in vitro transcription. The FFPE-ATAC generates high-quality chromatin accessibility profiles with 500 nuclei from a single FFPE tissue section, enables the dissection of chromatin profiles from the regions of interest with the aid of hematoxylin and eosin (H&E) staining, and reveals disease-associated chromatin regulation from the human colorectal cancer FFPE tissue archived for more than 10 years. In summary, the approach allows decoding of the chromatin states that regulate gene expression in archival FFPE tissues, thereby permitting investigators, to better understand epigenetic regulation in cancer and precision medicine.

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Optimizing Fixation Protocols to Improve Molecular Analysis from FFPE Tissues

Brazilian Dental Journal ◽

10.1590/0103-6440201701211 ◽

2017 ◽

Vol 28 (1) ◽

pp. 82-84

Author(s):

Bruna Jalfim Maraschin ◽

Viviane Palmeira da Silva ◽

Leigha Rock ◽

Huichen Sun ◽

Fernanda Visioli ◽

...

Keyword(s):

Dna Damage ◽

Ffpe Tissue ◽

Formalin Fixed Paraffin ◽

The World ◽

Formalin Fixed Paraffin Embedded ◽

Ffpe Tissues ◽

Molecular Assessment ◽

Processing And Storage ◽

And Storage ◽

Formalin Fixed

Abstract Most Departments of Pathology around the world have a considerable archive of formalin-fixed paraffin-embedded (FFPE) tissue suitable for molecular assessment. This article points out the potential DNA damage that may occur if basic steps are not followed during processing and storage of these samples. Furthermore, it hopes to establish parameters to optimize quality and quantity of DNA extracted from FFPE tissues.

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Factors in Tissue Handling and Processing That Impact RNA Obtained From Formalin-fixed, Paraffin-embedded Tissue

Journal of Histochemistry & Cytochemistry ◽

10.1369/jhc.2008.951863 ◽

2008 ◽

Vol 56 (11) ◽

pp. 1033-1042 ◽

Cited By ~ 85

Author(s):

Joon-Yong Chung ◽

Till Braunschweig ◽

Reginald Williams ◽

Natalie Guerrero ◽

Karl M. Hoffmann ◽

...

Keyword(s):

Histopathological Examination ◽

Ffpe Tissue ◽

Quality Factors ◽

Rna Quality ◽

Tissue Processing ◽

Formalin Fixed Paraffin ◽

Formalin Fixed Paraffin Embedded ◽

Ffpe Tissues ◽

The Impact ◽

Formalin Fixed

Formalin-fixed, paraffin-embedded (FFPE) tissue is the most common specimen available for molecular assays on tissue after diagnostic histopathological examination. RNA from FFPE tissue suffers from strand breakage and cross-linking. Despite excellent extraction methods, RNA quality from FFPE material remains variable. To address the RNA quality factors within FFPE tissues, we studied RNA quality, isolating individual elements of the tissue fixation and processing including length of fixation in formalin and the type of buffer incorporated in the fixative. We examined the impact of the length of the tissue processing cycle as well. The optimal fixation period of 12-24 hr in phosphate-buffered formalin resulted in better-quality RNA. Longer tissue processing times were associated with higher quality RNA. We determined that the middle region of gene suffers less damage by these processes as shown by real-time quantitative RT-PCR. These data provide key information for the development of methods of analysis of gene expression in archival FFPE tissues and contribute to the establishment of objective standards for the processing and handling of tissue in surgical pathology. This manuscript contains online supplemental material at www.jhc.org . Please visit this article online to view these materials.

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Characterization of epididymal and testicular histologic lesions and use of immunohistochemistry and PCR on formalin-fixed tissues to detect Brucella canis in male dogs

Journal of Veterinary Diagnostic Investigation ◽

10.1177/1040638720986883 ◽

2021 ◽

pp. 104063872098688

Author(s):

Andrea M. Camargo-Castañeda ◽

Lauren W. Stranahan ◽

John F. Edwards ◽

Daniel G. Garcia-Gonzalez ◽

Leonardo Roa ◽

...

Keyword(s):

Accurate Diagnosis ◽

Testicular Atrophy ◽

Detection Techniques ◽

Formalin Fixed Paraffin ◽

Brucella Canis ◽

Formalin Fixed Paraffin Embedded ◽

Variable Sensitivity ◽

Ffpe Tissues ◽

Formalin Fixed

In male dogs, Brucella canis frequently causes epididymitis, ultimately resulting in testicular atrophy and infertility. Although B. canis predominantly affects the epididymis, the misleading term “orchitis” is still commonly used by clinicians. Of additional concern, diagnosis in dogs remains challenging because of variable sensitivity and specificity of serologic assays and fluctuations in bacteremia levels in infected dogs, reducing the sensitivity of blood culture. We describe here the histologic lesions in the scrotal contents of 8 dogs suspected of being infected with B. canis and clinically diagnosed with orchitis. We explored the possibility of using immunohistochemistry (IHC) and real-time PCR (rtPCR) in formalin-fixed, paraffin-embedded (FFPE) tissues to detect the presence of B. canis. Epididymitis of variable chronicity was identified in all 8 dogs, with only 3 also exhibiting orchitis. Using rtPCR, the presence of B. canis was identified in 4 of 8 dogs, with 3 of these 4 dogs also positive by IHC. These results suggest that rtPCR and IHC are promising techniques that can be used in FFPE tissues to detect B. canis when other detection techniques are unavailable. Additionally, accurate recognition of epididymitis rather than orchitis in suspect cases could aid in accurate diagnosis.

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

Cell and Tissue Banking ◽

10.1007/s10561-021-09949-1 ◽

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.

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

Journal of Clinical Microbiology ◽

10.1128/jcm.01185-16 ◽

2016 ◽

Vol 54 (11) ◽

pp. 2798-2803 ◽

Cited By ~ 35

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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Incorporation of Cervista Human Papillomavirus 16/18 Assay Into Algorithms for Classifying Human Papillomavirus Status in Formalin-Fixed, Paraffin-Embedded Head and Neck Squamous Carcinoma Specimens

Archives of Pathology & Laboratory Medicine ◽

10.5858/arpa.2017-0469-oa ◽

2018 ◽

Vol 143 (3) ◽

pp. 356-361

Author(s):

Ming Guo ◽

Abha Khanna ◽

Jianping Wang ◽

Michelle D. Williams ◽

Neda Kalhor ◽

...

Keyword(s):

Squamous Cell Carcinoma ◽

Human Papillomavirus ◽

Ffpe Tissue ◽

Hpv 16 ◽

Hpv Dna ◽

Formalin Fixed Paraffin ◽

Hpv Status ◽

Formalin Fixed Paraffin Embedded ◽

Hpv Assays ◽

Formalin Fixed

Context.— Human papillomavirus (HPV) DNA in situ hybridization (ISH) assay and p16 immunohistochemistry (IHC) are used to determine high-risk HPV status in formalin-fixed, paraffin-embedded (FFPE) tissues in oropharyngeal squamous cell carcinoma (SCC). Although high sensitivity and specificity for HPV can be obtained by combined p16 IHC and HPV DNA ISH, the occasional discrepancy between these assays has prompted evaluation of Cervista HPV assays in FFPE tissue from patients with oropharyngeal SCC. Objective.— To compare the efficacy of Cervista HPV 16/18 and Cervista HPV HR assay to that of HPV DNA ISH assay and p16 IHC in FFPE tissue in head and neck squamous cell carcinoma of oropharyngeal origin. Design.— Archived FFPE tissue from 84 patients with SCC of oropharyngeal origin and available HPV DNA ISH and p16 IHC test results were tested with the Cervista HPV 16/18 assay and further verified by polymerase chain reaction (PCR)–based HPV16/18 genotyping tests in cases with discrepancy. Results.— Of the 84 specimens, 75% (63 of 84) were positive and 16% (13 of 84) had discrepant or equivocal findings by p16 IHC and HPV DNA ISH testing. Use of Cervista HPV assays, either to clarify discrepant/equivocal findings or as confirmation after initial p16 IHC/HPV DNA ISH tests, identified 81% (68 of 84) of HPV-positive cases without equivocal HPV results. Five of 13 cases with discrepancy or equivocal HPV DNA ISH results tested positive for HPV16 or HPV18 by Cervista HPV 16/18 assay, which was further confirmed by PCR-based HPV 16/18 genotyping. Conclusions.— The Cervista HPV assays are a reasonable alternative to HPV DNA ISH in determining HPV status in FFPE tissue specimens from patients with oropharyngeal SCC.

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Sarcoma Subgrouping by Detection of Fusion Transcripts Using NanoString nCounter Technology

Pediatric and Developmental Pathology ◽

10.1177/1093526618790747 ◽

2018 ◽

Vol 22 (3) ◽

pp. 205-213 ◽

Cited By ~ 4

Author(s):

Javal Sheth ◽

Anthony Arnoldo ◽

Yunan Zhong ◽

Paula Marrano ◽

Carlos Pereira ◽

...

Keyword(s):

Rt Pcr ◽

Fusion Transcripts ◽

Pediatric Sarcoma ◽

Formalin Fixed Paraffin ◽

Pediatric Sarcomas ◽

Formalin Fixed Paraffin Embedded ◽

Ffpe Tissues ◽

Nanostring Technology ◽

Future Work ◽

Formalin Fixed

Background NanoString technology is an innovative barcode-based system that requires less tissue than traditional techniques and can test for multiple fusion transcripts in a single reaction. The objective of this study was to determine the utility of NanoString technology in the detection of sarcoma-specific fusion transcripts in pediatric sarcomas. Design Probe pairs for the most common pediatric sarcoma fusion transcripts were designed for the assay. The NanoString assay was used to test 22 specific fusion transcripts in 45 sarcoma samples that had exhibited one of these fusion genes previously by reverse transcription polymerase chain reaction (RT-PCR). A mixture of frozen (n = 18), formalin-fixed, paraffin-embedded (FFPE) tissue (n = 23), and rapid extract template (n = 4) were used for testing. Results Each of the 22 transcripts tested was detected in at least one of the 45 tumor samples. The results of the NanoString assay were 100% concordant with the previous RT-PCR results for the tumor samples, and the technique was successful using both FFPE and rapid extract method. Conclusion Multiplexed interrogation for sarcoma-specific fusion transcripts using NanoString technology is a reliable approach for molecular diagnosis of pediatric sarcomas and works well with FFPE tissues. Future work will involve validating additional sarcoma fusion transcripts as well as determining the optimal workflow for diagnostic purposes.

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Molecular Markers for Prostate Cancer in Formalin-Fixed Paraffin-Embedded Tissues

BioMed Research International ◽

10.1155/2013/283635 ◽

2013 ◽

Vol 2013 ◽

pp. 1-15 ◽

Cited By ~ 9

Author(s):

Tamara Sequeiros ◽

Marta García ◽

Melania Montes ◽

Mireia Oliván ◽

Marina Rigau ◽

...

Keyword(s):

Prostate Cancer ◽

Molecular Markers ◽

Tumor Grade ◽

Developed Countries ◽

Response To Therapy ◽

Tissue Samples ◽

Formalin Fixed Paraffin ◽

Formalin Fixed Paraffin Embedded ◽

Ffpe Tissues ◽

Formalin Fixed

Prostate cancer (PCa) is the most frequently diagnosed type of cancer in developed countries. The decisive method of diagnosis is based on the results of biopsies, morphologically evaluated to determine the presence or absence of cancer. Although this approach leads to a confident diagnosis in most cases, it can be improved by using the molecular markers present in the tissue. Both miRNAs and proteins are considered excellent candidates for biomarkers in formalin-fixed paraffin-embedded (FFPE) tissues, due to their stability over long periods of time. In the last few years, a concerted effort has been made to develop the necessary tools for their reliable measurement in these types of samples. Furthermore, the use of these kinds of markers may also help in establishing tumor grade and aggressiveness, as well as predicting the possible outcomes in each particular case for the different treatments available. This would aid clinicians in the decision-making process. In this review, we attempt to summarize and discuss the potential use of microRNA and protein profiles in FFPE tissue samples as markers to better predict PCa diagnosis, progression, and response to therapy.

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Cystic Neutrophilic Granulomatous Mastitis: A Clinicopathological Study With 16s rRNA Sequencing for the Detection of Corynebacteria in Formalin-Fixed Paraffin-Embedded Tissue

International Journal of Surgical Pathology ◽

10.1177/1066896919896021 ◽

2019 ◽

Vol 28 (4) ◽

pp. 371-381 ◽

Cited By ~ 2

Author(s):

Madhavi A. Naik ◽

Aruna Korlimarla ◽

Smrithi T. Shetty ◽

Anisha M. Fernandes ◽

Sanjay A. Pai

Keyword(s):

16S Rrna ◽

Ffpe Tissue ◽

16S Rrna Sequencing ◽

Granulomatous Mastitis ◽

Prolonged Incubation ◽

Formalin Fixed Paraffin ◽

Corynebacterium Species ◽

Rrna Sequencing ◽

Formalin Fixed Paraffin Embedded ◽

Formalin Fixed

Cystic neutrophilic granulomatous mastitis (CNGM) is a histologically characterized variant of granulomatous lobular mastitis that is associated with lipophilic Corynebacterium species. It remains a largely underrecognized entity in India. Our aim was to study CNGM in the Asian Indian population and explore if 16s rRNA sequencing could be used on formalin-fixed paraffin-embedded (FFPE) tissue to identify the causative organism. We studied 24 cases with histological features of CNGM with hematoxylin and eosin, Gram, Ziehl-Neelsen, and Periodic acid–Schiff stains. Tuberculosis-polymerase chain reaction and 16s rRNA gene sequencing on DNA extracted from FFPE was attempted (N = 23). Gram-positive bacilli were seen in 20/24 cases. Routine culture with prolonged incubation yielded Corynebacterium species in 8 cases; 7 of these cases were evaluated by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for species identification. C matruchotti was identified in one case by BD Phoenix. MALDI-TOF MS identified the remaining 7 cases as C kroppenstedtii (N = 4) and C tuberculostearicum (N = 2), with no identification in one. Corynebacteria were identified by 16s rRNA sequencing on DNA extracted from FFPE in 12/23 cases using a primer targeting the V5-V6 region that was found to be more conserved in Corynebacterium species. All cases were negative for the diagnosis of tuberculosis. CNGM can be identified by routine stains. Culture using routine media with prolonged incubation is often adequate to isolate the organism. 16s rRNA sequencing on DNA extracted from FFPE tissue can help make an etiological diagnosis in some cases where only paraffin blocks are available.

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Clostridium perfringens–Associated Necrotic Enteritis-Like Disease in Coconut Lorikeets (Trichoglossus haematodus)

Veterinary Pathology ◽

10.1177/0300985820971788 ◽

2020 ◽

pp. 030098582097178

Author(s):

Llorenç Grau-Roma ◽

Mauricio Navarro ◽

Sohvi Blatter ◽

Christian Wenker ◽

Sonja Kittl ◽

...

Keyword(s):

Clostridium Perfringens ◽

Toxin Gene ◽

Necrotic Enteritis ◽

Gene Encoding ◽

Formalin Fixed Paraffin ◽

Formalin Fixed Paraffin Embedded ◽

Ffpe Tissues ◽

Polymerase Chain ◽

Beta Toxin ◽

Formalin Fixed

Several outbreaks of necrotic enteritis-like disease in lorikeets, from which Clostridium perfringens was consistently isolated, are described. All lorikeets had acute, segmental, or multifocal fibrinonecrotizing inflammatory lesions in the small and/or the large intestine, with intralesional gram-positive rods. The gene encoding C. perfringens alpha toxin was detected by PCR (polymerase chain reaction) on formalin-fixed, paraffin-embedded (FFPE) tissues in 20 out of 24 affected lorikeets (83%), but it was not amplified from samples of any of 10 control lorikeets ( P < .0001). The second most prevalent C. perfringens toxin gene detected was the beta toxin gene, which was found in FFPE from 7 out of 24 affected lorikeets (29%). The other toxin genes were detected inconsistently and in a relatively low number of samples. These cases seem to be associated with C. perfringens, although the specific type involved could not be determined.

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