OR19-01 IHC-Guided Capture of Adrenocortical Zones from Archival Formalin-Fixed, Paraffin-Embedded Adrenal Tissue Allows the Study Of RNA Expression Profiles

Abstract Introduction: Characterization of human adrenal RNA has relied on fresh tissue, or laser capture from OCT-embedded frozen tissue. These approaches have allowed identification of differences between zones of the adrenal cortex, but the need for fresh or frozen material has limited access for most researchers and has limited the number of samples that can be studied. In order to expand tissue availability and researcher access, we examined the use of archival formalin-fixed, paraffin-embedded (FFPE) adrenals for the capture and analysis of RNA from each adrenal zone. Methods: 13 normal adrenals were obtained from deceased renal donors. Following FFPE, tissue serial sections were examined for zonal histology. Immunohistochemistry (IHC) was used to guide the capture of specific adrenal zones from FFPE tissue. CYP11B2, CYP17A1, and CYB5A IHC was used to visualize the zona glomerulosa (ZG), zona fasciculata (ZF), and zona reticularis (ZR) respectively. Serial unstained sections were then dissected under a dissection microscope based on IHC-guided visualization of the ZG, ZF, and ZR. Following capture of zones and isolation of RNA, quantitative PCR (qPCR) was used to examine zonal expression of transcripts for the IHC markers. Using housekeeping genes for normalization, specific zonal markers identified successfully isolated zones. Results: Adrenals with clear zonal expression of CYP11B2 (ZG), HSD3B2 (ZG/ZF), and CYB5A (ZR) were included for study. Based on marker inclusion criteria, 3 ZG, 3 ZF, 3 ZR, and 3 medulla samples were studied. Using these samples, confirmatory markers of ZG (VSNL1), ZF (CYP17A1), ZR (SLC27A2), and medulla (TH) were analyzed by qPCR. Levels of these transcripts segregated as expected in the adrenal zones. Discussion: The use of archival normal and pathologic adrenal tissue for RNA analysis would increase the number of samples available to many research groups. Our data demonstrates the ability to capture and use FFPE tissue for the capture and analysis of RNA. Future comparison of normal adrenal zones to adrenal tumor phenotypes will improve our understanding of tumor biology and provide potential genetic markers for targeted therapeutics.

Download Full-text

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

10.1101/019794 ◽

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.

Download Full-text

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.

Download Full-text

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.

Download Full-text

Quantitative radioimmunohistochemical measurements of p185(erbB-2) in frozen tissue sections.

Journal of Histochemistry & Cytochemistry ◽

10.1177/44.11.8918900 ◽

1996 ◽

Vol 44 (11) ◽

pp. 1251-1259 ◽

Cited By ~ 12

Author(s):

J R Reeves ◽

J J Going ◽

G Smith ◽

T G Cooke ◽

B W Ozanne ◽

...

Keyword(s):

Tumor Biology ◽

Breast Carcinomas ◽

Tissue Sections ◽

Biopsy Specimens ◽

Formalin Fixed Paraffin ◽

Formalin Fixed Paraffin Embedded ◽

Interstudy Variability ◽

High Degree ◽

The Relationship ◽

Formalin Fixed

The relationship between expression of the c-erbB-2 proto-oncogene and the biology of breast cancer has been investigated widely, most studies using immunohistochemistry in formalin-fixed, paraffin-embedded tissues. This technique is at best semiquantitative and there is a high degree of interstudy variability because of its subjective nature and poor methodological standardization. The relationship between the levels of expression and biology can be examined thoroughly only with an accurately quantitative technique. We have developed a radioimmunohistochemical assay to measure p185(erbB-2) in tissue biopsy specimens. The method involves incubating frozen sections with 125I-labeled monoclonal antibody, microautoradiograpy, and grain counting with image analysis. Sections of cell pellets with known c-erbB-2 levels are processed with each batch of samples as internal calibration standards. We have quantified c-erbB-2 expression in 60 breast carcinomas and compared the results with conventional immunohistochemistry. Radioimmunohistochemistry measured receptor levels throughout the range of expression in breast carcinomas, whereas conventional immunohistochemistry detected the protein only in the highest expressing tumors. The quantitative, objective data produced by radioimmunohistochemistry allow a more thorough evaluation of the relationship between c-erbB-2 expression and tumor biology. This technique may have applications in other fields where quantitative data is required and relevant monoclonal antibodies are available.

Download Full-text

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.

Download Full-text

Comparison of the miRNA expression profiles in fresh frozen and formalin-fixed paraffin-embedded tonsillar tumors

PLoS ONE ◽

10.1371/journal.pone.0179645 ◽

2017 ◽

Vol 12 (6) ◽

pp. e0179645 ◽

Cited By ~ 4

Author(s):

Zuzana Vojtechova ◽

Jiri Zavadil ◽

Jan Klozar ◽

Marek Grega ◽

Ruth Tachezy

Keyword(s):

Mirna Expression ◽

Expression Profiles ◽

Formalin Fixed Paraffin ◽

Mirna Expression Profiles ◽

Formalin Fixed Paraffin Embedded ◽

Fresh Frozen ◽

Formalin Fixed

Download Full-text

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.

Download Full-text

Deciphering the Immune Microenvironment on A Single Archival Formalin-Fixed Paraffin-Embedded Tissue Section by An Immediately Implementable Multiplex Fluorescence Immunostaining Protocol

Cancers ◽

10.3390/cancers12092449 ◽

2020 ◽

Vol 12 (9) ◽

pp. 2449 ◽

Cited By ~ 2

Author(s):

Adrien Guillot ◽

Marlene S. Kohlhepp ◽

Alix Bruneau ◽

Felix Heymann ◽

Frank Tacke

Keyword(s):

Immune Cell ◽

Simple Procedure ◽

Parenchymal Cell ◽

Ffpe Tissue ◽

Immune Microenvironment ◽

Tissue Samples ◽

Tissue Organization ◽

Formalin Fixed Paraffin ◽

Formalin Fixed Paraffin Embedded ◽

Formalin Fixed

Technological breakthroughs have fundamentally changed our understanding on the complexity of the tumor microenvironment at the single-cell level. Characterizing the immune cell composition in relation to spatial distribution and histological changes may provide important diagnostic and therapeutic information. Immunostaining on formalin-fixed paraffin-embedded (FFPE) tissue samples represents a widespread and simple procedure, allowing the visualization of cellular distribution and processes, on preserved tissue structure. Recent advances in microscopy and molecular biology have made multiplexing accessible, yet technically challenging. We herein describe a novel, simple and cost-effective method for a reproducible and highly flexible multiplex immunostaining on archived FFPE tissue samples, which we optimized for solid organs (e.g., liver, intestine, lung, kidney) from mice and humans. Our protocol requires limited specific equipment and reagents, making multiplexing (>12 antibodies) immediately implementable to any histology laboratory routinely performing immunostaining. Using this method on single sections and combining it with automated whole-slide image analysis, we characterize the hepatic immune microenvironment in preclinical mouse models of liver fibrosis, steatohepatitis and hepatocellular carcinoma (HCC) and on human-patient samples with chronic liver diseases. The data provide useful insights into tissue organization and immune–parenchymal cell-to-cell interactions. It also highlights the profound macrophage heterogeneity in liver across premalignant conditions and HCC.

Download Full-text