Journal articles on the topic 'Paraffin-embedded tissues- FFPE'
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Pinto-Ribeiro, Ines, Rui M. Ferreira, Joana Pereira-Marques, Vanessa Pinto, Guilherme Macedo, Fátima Carneiro, and Ceu Figueiredo. "Evaluation of the Use of Formalin-Fixed and Paraffin-Embedded Archive Gastric Tissues for Microbiota Characterization Using Next-Generation Sequencing." International Journal of Molecular Sciences 21, no. 3 (February 7, 2020): 1096. http://dx.doi.org/10.3390/ijms21031096.
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Large numbers of well-characterized clinical samples are fundamental to establish relevant associations between the microbiota and disease. Formalin-fixed and paraffin-embedded (FFPE) tissues are routinely used and are widely available clinical materials. Since current approaches to study the microbiota are based on next-generation sequencing (NGS) targeting the bacterial 16S rRNA gene, our aim was to evaluate the feasibility of FFPE gastric tissues for NGS-based microbiota characterization. Analysis of sequencing data revealed the presence of bacteria in the paraffin control. After the subtraction of the operational taxonomic units (OTUs) present in the paraffin control to the FFPE tissue sample dataset, we evaluated the microbiota profiles between paired FFPE and frozen gastric tissues, and between different times of archiving. Compared with frozen gastric tissues, we detected a lower number of OTUs in the microbiota of paired FFPE tissues, regardless of the time of archiving. No major differences in microbial diversity were identified, but taxonomic variation in the relative abundance of phyla and orders was observed between the two preservation methods. This variation was also evident in each case and throughout the times of FFPE archiving. The use of FFPE tissues for NGS-based microbiota characterization should be considered carefully, as biases can be introduced by the embedding process and the time of tissue archiving.
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Michelsen, Nete V., Klaus Brusgaard, Qihua Tan, Mads Thomassen, Khalid Hussain, and Henrik T. Christesen. "Investigation of Archived Formalin-Fixed Paraffin-Embedded Pancreatic Tissue with Whole-Genome Gene Expression Microarray." ISRN Pathology 2011 (December 26, 2011): 1–12. http://dx.doi.org/10.5402/2011/275102.
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The use of formalin-fixed, paraffin-embedded (FFPE) tissue overcomes the most prominent issues related to research on relatively rare diseases: limited sample size, availability of control tissue, and time frame. The use of FFPE pancreatic tissue in GEM may be especially challenging due to its very high amounts of ribonucleases compared to other tissues/organs. In choosing pancreatic tissue, we therefore indirectly address the applicability of other FFPE tissues to gene expression microarray (GEM). GEM was performed on archived, routinely fixed, FFPE pancreatic tissue from patients with congenital hyperinsulinism (CHI), insulinoma, and deceased age-appropriate neonates, using whole-genome arrays. Although ribonuclease-rich, we obtained biologically relevant and disease-specific, significant genes; cancer-related genes; genes involved in (a) the regulation of insulin secretion and synthesis, (b) amino acid metabolism, and (c) calcium ion homeostasis. These results should encourage future research and GEM studies on FFPE tissue from the invaluable biobanks available at the departments of pathology worldwide.
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ŞEVİK, Murat. "The extraction of Peste Des Petits Ruminants Virus RNA from paraffin-embedded tissues using a modified extraction method." Journal of Advances in VetBio Science and Techniques 7, no. 2 (August 31, 2022): 202–9. http://dx.doi.org/10.31797/vetbio.1078235.
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Peste des petits ruminants (PPR) which is caused by small ruminant morbillivirus (PPRV) has an important economic impact on small ruminant farming due to high mortality rates, weight loss and restrictions on the export of small ruminants products. Molecular assays are commonly used in the diagnosis of the disease. Extraction of RNA from formalin-fixed paraffin-embedded (FFPE) tissues is challenging because of the RNA is often degraded by formalin fixation process. Although commercial kits have been developed for extraction of nucleic acids from FFPE tissues, they are expensive than other extraction kits. In this study, a modified extraction method was evaluated for detection of PPRV from FFPE tissues. A total of 20 FFPE tissue samples including 15 PPRV positive and 5 PPRV negative FFPE tissue samples were used. Two years ago, these selected FFPE tissue samples were analysed by nucleoprotein gene based one step real time RT-PCR method before they were fixed with formalin and embedded in paraffin. FFPE tissue samples were extracted using modified extraction method and were tested by fusion (F) gene based one step RT-PCR. PPRV specific RNA was detected in 12 FFPE tissue samples whereas 3 positive samples were found negative by one-step RT-PCR. Furthermore, 5 negative FFPE tissue samples were also found negative. Three false negative results were from samples with high real-time RT-PCR cycle threshold. Therefore, false negative results could be related with lower viral loads which might be lower than detection limit of the one-step RT-PCR. The results of the study show that modified extraction method could be used for RNA extraction from FFPE tissues which had been stored for 2 years.
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Obi, Ekenedirichukwu N., Daniel A. Tellock, Gabriel J. Thomas, and Timothy D. Veenstra. "Biomarker Analysis of Formalin-Fixed Paraffin-Embedded Clinical Tissues Using Proteomics." Biomolecules 13, no. 1 (January 3, 2023): 96. http://dx.doi.org/10.3390/biom13010096.
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The relatively recent developments in mass spectrometry (MS) have provided novel opportunities for this technology to impact modern medicine. One of those opportunities is in biomarker discovery and diagnostics. Key developments in sample preparation have enabled a greater range of clinical samples to be characterized at a deeper level using MS. While most of these developments have focused on blood, tissues have also been an important resource. Fresh tissues, however, are difficult to obtain for research purposes and require significant resources for long-term storage. There are millions of archived formalin-fixed paraffin-embedded (FFPE) tissues within pathology departments worldwide representing every possible tissue type including tumors that are rare or very small. Owing to the chemical technique used to preserve FFPE tissues, they were considered intractable to many newer proteomics techniques and primarily only useful for immunohistochemistry. In the past couple of decades, however, researchers have been able to develop methods to extract proteins from FFPE tissues in a form making them analyzable using state-of-the-art technologies such as MS and protein arrays. This review will discuss the history of these developments and provide examples of how they are currently being used to identify biomarkers and diagnose diseases such as cancer.
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Dear, Jonathan D., Jane E. Sykes, and Danika L. Bannasch. "Quality of DNA extracted from formalin-fixed, paraffin-embedded canine tissues." Journal of Veterinary Diagnostic Investigation 32, no. 4 (June 9, 2020): 556–59. http://dx.doi.org/10.1177/1040638720929637.
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Veterinary pathology tissue banks are valuable resources for genetic studies. However, limited data exist as to whether quality DNA can be extracted from these tissues for use in canine genotyping studies. We extracted DNA from 44 formalin-fixed, paraffin-embedded (FFPE) tissue blocks from dogs; 9 of these dogs had DNA available from whole blood samples that had been banked. We genotyped DNA from 30 of 44 tissue blocks and 9 whole blood samples on the Illumina CanineHD BeadChip; DNA quality was insufficient in 14 of 44 samples from tissue blocks. There was significant correlation between the 260/280 ratio and single-nucleotide variation (SNV) call rate ( p = 0.0276; r2 = 0.162); 23 of 30 samples from FFPE were genotyped with > 65% call rates. Median pairwise identical-by-state (IBS) analysis was 0.99 in 8 pairs of dogs with call rates > 65%. Neither age of tissue block nor specific tissue types were associated with significant differences in DNA concentration, 260/280 ratio, or SNV call rate. DNA extracted from tissue blocks can have variable quality, although comparable levels of homozygosity suggest that extracts from FFPE with call rates > 65% might provide similar results to samples from whole blood when analyzed on the Illumina CanineHD BeadChip.
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Mitsa, Georgia, Qianyu Guo, Christophe Goncalves, Samuel E. J. Preston, Vincent Lacasse, Adriana Aguilar-Mahecha, Naciba Benlimame, et al. "A Non-Hazardous Deparaffinization Protocol Enables Quantitative Proteomics of Core Needle Biopsy-Sized Formalin-Fixed and Paraffin-Embedded (FFPE) Tissue Specimens." International Journal of Molecular Sciences 23, no. 8 (April 18, 2022): 4443. http://dx.doi.org/10.3390/ijms23084443.
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Most human tumor tissues that are obtained for pathology and diagnostic purposes are formalin-fixed and paraffin-embedded (FFPE). To perform quantitative proteomics of FFPE samples, paraffin has to be removed and formalin-induced crosslinks have to be reversed prior to proteolytic digestion. A central component of almost all deparaffinization protocols is xylene, a toxic and highly flammable solvent that has been reported to negatively affect protein extraction and quantitative proteome analysis. Here, we present a ‘green’ xylene-free protocol for accelerated sample preparation of FFPE tissues based on paraffin-removal with hot water. Combined with tissue homogenization using disposable micropestles and a modified protein aggregation capture (PAC) digestion protocol, our workflow enables streamlined and reproducible quantitative proteomic profiling of FFPE tissue. Label-free quantitation of FFPE cores from human ductal breast carcinoma in situ (DCIS) xenografts with a volume of only 0.79 mm3 showed a high correlation between replicates (r2 = 0.992) with a median %CV of 16.9%. Importantly, this small volume is already compatible with tissue micro array (TMA) cores and core needle biopsies, while our results and its ease-of-use indicate that further downsizing is feasible. Finally, our FFPE workflow does not require costly equipment and can be established in every standard clinical laboratory.
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Bao, Jian R., Richard B. Clark, Ronald N. Master, Kileen L. Shier, and Lynn L. Eklund. "Acid-fast bacterium detection and identification from paraffin-embedded tissues using a PCR-pyrosequencing method." Journal of Clinical Pathology 71, no. 2 (July 22, 2017): 148–53. http://dx.doi.org/10.1136/jclinpath-2016-204128.
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AimsAcid-fast bacterium (AFB) identification from formalin-fixed paraffin-embedded (FFPE) tissues is challenging and may not be readily available to the clinical laboratory. A method to detect and identify AFB from FFPE tissues using PCR and pyrosequencing (PCR-Seq) was developed and evaluated.MethodsThe method was validated using spiked cell-clotted paraffin blocks before use with patients’ specimens. DNA was extracted from tissue sections, and a 16S rRNA gene fragment was amplified and a signature sequence was produced on a PyroMark ID system. Sequences were aligned to established databases for AFB identification. Additional tissue sections were stained and examined for AFB.ResultsBoth sensitivity and specificity were 100% on spiked cell-clotted blocks without cross-reactivity with non-AFB. Of 302 FFPE tissues from patients, 116 (38%) were AFB-stain positive; 83 (72%) of these had AFB identified. The 21 AFB identified included Mycobacterium tuberculosis complex (14 cases), Mycobacterium leprae (3), Mycobacterium genavense (2), Mycobacterium marinum-ulcerans group (3) and 17 other AFB (61). Thirteen cases were AFB-stain indeterminate and 4 were positive by the PCR-Seq method. Of the AFB stain-negative cases, 167 were negative and 6 were positive by PCR-Seq.ConclusionsThe PCR-Seq method provided specific identification of various AFB species or complexes from FFPE tissues.
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He, Helen, Yu Yang, Zhongmin Xiang, Lunyin Yu, Jouhara Chouitar, Jie Yu, Natalie Roy D’Amore, et al. "A Sensitive IHC Method for Monitoring Autophagy-Specific Markers in Human Tumor Xenografts." Journal of Biomarkers 2016 (May 10, 2016): 1–11. http://dx.doi.org/10.1155/2016/1274603.
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Objective. Use of tyramide signal amplification (TSA) to detect autophagy biomarkers in formalin fixed and paraffin embedded (FFPE) xenograft tissue. Materials and Methods. Autophagy marker regulation was studied in xenograft tissues using Amp HQ IHC and standard IHC methods. Results. The data demonstrate the feasibility of using high sensitivity TSA IHC assays to measure low abundant autophagy markers in FFPE xenograft tissue.
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Magdeldin, Sameh, and Tadashi Yamamoto. "Toward deciphering proteomes of formalin-fixed paraffin-embedded (FFPE) tissues." PROTEOMICS 12, no. 7 (April 2012): 1045–58. http://dx.doi.org/10.1002/pmic.201100550.
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Maraschin, Bruna Jalfim, Viviane Palmeira da Silva, Leigha Rock, Huichen Sun, Fernanda Visioli, Pantelis Varvaki Rados, and Miriam P. Rosin. "Optimizing Fixation Protocols to Improve Molecular Analysis from FFPE Tissues." Brazilian Dental Journal 28, no. 1 (February 2017): 82–84. http://dx.doi.org/10.1590/0103-6440201701211.
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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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Chung, Joon-Yong, Till Braunschweig, Reginald Williams, Natalie Guerrero, Karl M. Hoffmann, Mijung Kwon, Young K. Song, Steven K. Libutti, and Stephen M. Hewitt. "Factors in Tissue Handling and Processing That Impact RNA Obtained From Formalin-fixed, Paraffin-embedded Tissue." Journal of Histochemistry & Cytochemistry 56, no. 11 (July 21, 2008): 1033–42. http://dx.doi.org/10.1369/jhc.2008.951863.
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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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Ghebeh, Hazem, Fatmah A. Mansour, Dilek Colak, Akram A. Alfuraydi, Amal A. Al-Thubiti, Dorota Monies, Monther Al-Alwan, Taher Al-Tweigeri, and Asma Tulbah. "Higher PD-L1 Immunohistochemical Detection Signal in Frozen Compared to Matched Paraffin-Embedded Formalin-Fixed Tissues." Antibodies 10, no. 3 (June 22, 2021): 24. http://dx.doi.org/10.3390/antib10030024.
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Purpose: Response to anti-PD-L1/PD-1 immunotherapy correlates with PD-L1 expression in breast cancer. However, the prevalence of PD-L1 positive breast cancer is variable, which could be due to differences in the population/cohort of patients tested or the preservation/detection technology used. To investigate this variability, we examined the effect of two tissue preservation methods on PD-L1 immunohistochemical detection in breast cancer. Methods: We compared PD-L1 expression in patient-matched frozen (FR) and formalin-fixed paraffin-embedded (FFPE) tissues of breast cancer patients. PD-L1 expression was assessed using tumor proportion score (TPS, simply PD-L1 score), and case positivity was determined with PD-L1 score ≥5. Results: In FFPE tissues, PD-L1 was positive in 7–10% of tested patients, depending on the antibody used. In patient-matched FR tissues, the same antibodies showed positive PD-L1 expression in 20–30% of cases. The impact of the antibody tested on the rate of PD-L1 positivity (% of PDL1 positive cases) was minor, as evident in the near perfect concordance between PD-L1 score obtained using the different antibodies whether tested in FR or FFPE tissues. However, there was a systematic drop by an average of 13–20% in the PD-L1 score obtained in FFPE tissues compared to their patient-matched FR tissues. Conclusions: In the tested patient-matched cohort, there was consistently a higher PD-L1 score in FR than FFPE tissues, regardless of the antibody used, demonstrating a significant effect on PD-L1 detection due to the preservation method. These findings should inspire further work to improve the sensitivity of PD-L1 detection and possibly search for more sensitive antibodies in FFPE tissues.
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Sadamoto, Sota, Yurika Mitsui, Yasuhiro Nihonyanagi, Kazuki Amemiya, Minoru Shinozaki, Somay Yamagata Murayama, Masahiro Abe, et al. "Comparison Approach for Identifying Missed Invasive Fungal Infections in Formalin-Fixed, Paraffin-Embedded Autopsy Specimens." Journal of Fungi 8, no. 4 (March 24, 2022): 337. http://dx.doi.org/10.3390/jof8040337.
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Invasive fungal infection (IFI) has a high mortality rate in patients who undergo hematopoietic stem cell transplantation, and it is often confirmed by postmortem dissection. When IFI is initially confirmed after an autopsy, the tissue culture and frozen section are challenging to secure, and in many cases, formalin-fixed, paraffin-embedded (FFPE) samples represent the only modality for identifying fungi. Histopathological diagnosis is a useful method in combination with molecular biological methods that can achieve more precise identification with reproducibility. Meanwhile, polymerase chain reaction (PCR) using fungal-specific primers helps identify fungi from FFPE tissues. Autopsy FFPE specimens have a disadvantage regarding the quality of DNA extracted compared with that of specimens obtained via biopsy or surgery. In the case of mucormycosis diagnosed postmortem histologically, we examined currently available molecular biological methods such as PCR, immunohistochemistry (IHC), and in situ hybridization (ISH) to identify fungi. It is reasonable that PCR with some modification is valuable for identifying fungi in autopsy FFPE specimens. However, PCR does not always correctly identify fungi in autopsy FFPE tissues, and other approaches such as ISH or IHC are worth considering for clarifying the broad classification (such as the genus- or species-level classification).
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Guo, Tong, Weijie Wang, Paul A. Rudnick, Tao Song, Jie Li, Zhengping Zhuang, Robert J. Weil, Don L. DeVoe, Cheng S. Lee, and Brian M. Balgley. "Proteome Analysis of Microdissected Formalin-fixed and Paraffin-embedded Tissue Specimens." Journal of Histochemistry & Cytochemistry 55, no. 7 (March 19, 2007): 763–72. http://dx.doi.org/10.1369/jhc.7a7177.2007.
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Targeted proteomics research, based on the enrichment of disease-relevant proteins from isolated cell populations selected from high-quality tissue specimens, offers great potential for the identification of diagnostic, prognostic, and predictive biological markers for use in the clinical setting and during preclinical testing and clinical trials, as well as for the discovery and validation of new protein drug targets. Formalin-fixed and paraffin-embedded (FFPE) tissue collections, with attached clinical and outcome information, are invaluable resources for conducting retrospective protein biomarker investigations and performing translational studies of cancer and other diseases. Combined capillary isoelectric focusing/nano-reversed-phase liquid chromatography separations equipped with nano-electrospray ionization-tandem mass spectrometry are employed for the studies of proteins extracted from microdissected FFPE glioblastoma tissues using a heat-induced antigen retrieval (AR) technique. A total of 14,478 distinct peptides are identified, leading to the identification of 2733 non-redundant SwissProt protein entries. Eighty-three percent of identified FFPE tissue proteins overlap with those obtained from the pellet fraction of fresh-frozen tissue of the same patient. This large degree of protein overlapping is attributed to the application of detergent-based protein extraction in both the cell pellet preparation protocol and the AR technique.
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Borgognone, Alessandra, Garazi Serna, Marc Noguera-Julian, Lidia Alonso, Mariona Parera, Francesc Català-Moll, Lidia Sanchez, Roberta Fasani, Roger Paredes, and Paolo Nuciforo. "Performance of 16S Metagenomic Profiling in Formalin-Fixed Paraffin-Embedded versus Fresh-Frozen Colorectal Cancer Tissues." Cancers 13, no. 21 (October 29, 2021): 5421. http://dx.doi.org/10.3390/cancers13215421.
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Formalin-fixed, paraffin-embedded (FFPE) tissues represent the most widely available clinical material to study colorectal cancer (CRC). However, the accuracy and clinical validity of FFPE microbiome profiling in CRC is uncertain. Here, we compared the microbial composition of 10 paired fresh-frozen (FF) and FFPE CRC tissues using 16S rRNA sequencing and RNA-ISH. Both sample types showed different microbial diversity and composition. FF samples were enriched in archaea and representative CRC-associated bacteria, such as Firmicutes, Bacteroidetes and Fusobacteria. Conversely, FFPE samples were mainly enriched in typical contaminants, such as Sphingomonadales and Rhodobacterales. RNA-ISH in FFPE tissues confirmed the presence of CRC-associated bacteria, such as Fusobacterium and Bacteroides, as well as Propionibacterium allowing discrimination between tumor-associated and contaminant taxa. An internal quality index showed that the degree of similarity within sample pairs inversely correlated with the dominance of contaminant taxa. Given the importance of FFPE specimens for larger studies in human cancer genomics, our findings may provide useful indications on potential confounding factors to consider for accurate and reproducible metagenomics analyses.
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Sequeiros, Tamara, Marta García, Melania Montes, Mireia Oliván, Marina Rigau, Eva Colás, Inés de Torres, Juan Morote, Jaume Reventós, and Andreas Doll. "Molecular Markers for Prostate Cancer in Formalin-Fixed Paraffin-Embedded Tissues." BioMed Research International 2013 (2013): 1–15. http://dx.doi.org/10.1155/2013/283635.
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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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Wehmas, Leah C., Charles E. Wood, Brian N. Chorley, Carole L. Yauk, Gail M. Nelson, and Susan D. Hester. "Enhanced Quality Metrics for Assessing RNA Derived From Archival Formalin-Fixed Paraffin-Embedded Tissue Samples." Toxicological Sciences 170, no. 2 (May 15, 2019): 357–73. http://dx.doi.org/10.1093/toxsci/kfz113.
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Abstract Formalin-fixed paraffin-embedded (FFPE) tissues provide an important resource for toxicogenomic research. However, variability in the integrity or quality of RNA obtained from archival FFPE specimens can lead to unreliable data and wasted resources, and standard protocols for measuring RNA integrity do not adequately assess the suitability of FFPE RNA. The main goal of this study was to identify improved methods for evaluating FFPE RNA quality for whole-genome sequencing. We examined RNA quality metrics conducted prior to RNA-sequencing in paired frozen and FFPE samples with varying levels of quality based on age in block and time in formalin. RNA quality was measured by the RNA integrity number (RIN), a modified RIN called the paraffin-embedded RNA metric, the percentage of RNA fragments >100–300 nucleotides in size (DV100–300), and 2 quantitative PCR-based methods. This information was correlated to sequencing read quality, mapping, and gene detection. Among fragmentation-based methods, DV and PCR-based metrics were more informative than RIN or paraffin-embedded RNA metric in determining sequencing success. Across low- and high-quality FFPE samples, a minimum of 80% of RNA fragments >100 nucleotides (DV100 > 80) provided the best indication of gene diversity and read counts upon sequencing. The PCR-based methods further showed quantitative reductions in amplifiable RNA of target genes related to sample age and time in formalin that inform input quantity of FFPE RNA for sequencing. These results should aid in screening and prioritizing archival FFPE samples for retrospective analyses of gene expression.
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Lee, Tae Hee, Young Jun Kim, Woo Sun Rou, and Hyuk Soo Eun. "Fabrication of Formalin-Fixed, Paraffin-Embedded (FFPE) Circulating Tumor Cell (CTC) Block Using a Hydrogel Core-Mediated Method." Micromachines 12, no. 9 (September 20, 2021): 1128. http://dx.doi.org/10.3390/mi12091128.
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Circulating tumor cells (CTCs) are extremely low-frequency cells in the bloodstream. As those cells have detached from the primary tumor tissues and it circulates throughout the whole body, they are considered as promising diagnostic biomarkers for clinical application. However, the analysis of CTC is often restricted due to their rarity and heterogeneity, as well as their short-term presence. Here we proposed formalin-fixed, paraffin-embedded (FFPE) CTC block method, in combination manner with the hydrogel core-mediated CTC accumulation and conventional paraffin tissue block preparation. The hydrogel core specifically captures and releases cancer cells with high efficiency with an immunoaffinity manner. An additional shell structure protects the isolated cancer cells during the FFPE CTC block preparation process. The fabricated FFPE CTC block was sectioned and cytopathologically investigated just the same way as the conventional tissue block. Our results demonstrate that rare cells such as CTCs can also be prepared for FFPE cell blocks and shows great promise for cytopathological CTC studies.
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Nucci, Ricardo, Wilson Jacob-Filho, Alexandre Busse, Laura Maifrino, and Romeu de Souza. "Anatomopathological Assessment of the Diaphragm in Formalin-Fixed, Paraffin-Embedded Sections." Journal of Morphological Sciences 35, no. 03 (September 2018): 173–76. http://dx.doi.org/10.1055/s-0038-1673611.
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Introduction The analysis of frozen muscle biopsies has become a routine method in the evaluation of muscle structure in health and disease. However, the technique for frozen muscle specimens is not widely available in countries with limited medical facilities. The present study aimed to elucidate a reproducible formalin-fixed and paraffin-embedded (FFPE) method for this type of analysis in postmortem muscles. Methods Diaphragm muscle was obtained within 1 hour of sudden death. Diaphragm strips were washed in saline solution, fixed in 10% formalin, frozen at 4°C in a refrigerator, and stored for 24 hours. Then, the tissue samples were processed into paraffin-embedded blocks. Transversal sections were cut from each paraffin block and stained with hematoxylin and eosin, Picrosirius red, Verhoeff-Van Gieson, and Congo red for the qualitative analysis. Results Our analysis indicated a well-preserved muscle. Conclusion In summary, we demonstrate a simple technique for a reproducible FFPE method in postmortem muscle tissues.
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De Sio, Gabriele, Andrew James Smith, Manuel Galli, Mattia Garancini, Clizia Chinello, Francesca Bono, Fabio Pagni, and Fulvio Magni. "A MALDI-Mass Spectrometry Imaging method applicable to different formalin-fixed paraffin-embedded human tissues." Molecular BioSystems 11, no. 6 (2015): 1507–14. http://dx.doi.org/10.1039/c4mb00716f.
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The paper shows a new method for the application of Matrix Assisted Laser Desorption/Ionisation (MALDI) Mass Spectrometry Imaging (MSI) technology on formalin-fixed paraffin-embedded (FFPE) tissue samples.
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Farooq, Umar, Prashant Singh, Michael Gooch, Linda Burian, M. Melinda Sanders, and Richard Bernard Everson. "Comparisons of RNA-seq results for cryopreserved (CRYO) and formalin fixed, paraffin-embedded (FFPE) samples." Journal of Clinical Oncology 31, no. 15_suppl (May 20, 2013): e22211-e22211. http://dx.doi.org/10.1200/jco.2013.31.15_suppl.e22211.
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e22211 Background: FFPE is the worldwide clinical standard for preservation of tissue samples. Although CRYO tissues are widely used for research, most gene expression assays in clinical use analyze FFPE by RT-PCR. Use of RT-PCR limits numbers of transcripts that can be studied with material available. Next Generation Sequencing promises to overcome those limitations, but for FFPE, there are concerns related to its accuracy including bias associated with RNA truncated at the 3’ end. Little data is available illustrating the extent of truncation or considering implications for bioinformatics analysis of sequencing results. Methods: Total RNA was extracted from patient consented specimens of tumor and normal bladder tissue split with matching halves processed by CRYO and FFPE. Paired end sequencing libraries 100 bp in length were generated using Illumina TruSeq RNA sample kits with minor modifications for FFPE samples. Sequencing was performed on the Illumina HiSeq 2000, aligned with TopHat using the hg19 reference sequence, and further analyzed using open source bioinformatics tools. Results: Comparing Cryo and FFPE reads for a representative specimen, the proportion of reads that mapped to the reference genome were 69% and 63% for normal, and 66% and 63% for tumor specimens. Mapped reads yielded over 25,000 significantly expressed transcripts, isoforms, and splice variants. The extent of 3’ bias was investigated by viewing data in the Integrated Genome Viewer and using Picard tools. Preliminary estimates of 3’ bias in FFPE showed 2.22 for normal and 1.48 for tumor. After accounting for bias, transcripts in CRYO vs FFPE were significantly correlated for both normal tissue and tumor (R2 = 0.76 and 0.95, respectively). Mutations previously identified in these samples by SureSelect hybrid capture system (Agilent) were also observed in the RNA-seq data. Conclusions: FFPE samples can be successfully analyzed by RNA-Seq, allowing the analysis of standard clinical samples and vast quantities of richly annotated archival specimens. Results from CRYO and FFPE analyses are similar when performed using minor modifications to sequencing library preparation and careful attention to appropriate analysis.
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Hockney, Rochelle, Caroline H. Orr, Gareth J. Waring, Inge Christiaens, Gillian Taylor, Stephen P. Cummings, Stephen C. Robson, and Andrew Nelson. "Formalin-Fixed Paraffin-Embedded (FFPE) samples are not a beneficial replacement for frozen tissues in fetal membrane microbiota research." PLOS ONE 17, no. 3 (March 17, 2022): e0265441. http://dx.doi.org/10.1371/journal.pone.0265441.
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Formalin-Fixed Paraffin-Embedded (FFPE) tissues are routinely collected, archived, and used for clinical diagnosis, including maternal and neonatal health. Applying FFPE samples to microbiota research would be beneficial to reduce preparation, storage and costs associated with limited available frozen samples. This research aims to understand if FFPE fetal membrane samples are comparable to frozen tissues, which are the current gold standard for DNA microbiota analysis. Extracted DNA from nine matched paired patients were sequenced by Illumina sequencing of the V4 16S rRNA gene region. This included duplicate frozen amnion and chorion fetal membrane rolls or FFPE combined amniochorionic samples. Negative controls of surrounding wax blocks and DNA extraction reagents were processed alongside samples using identical methods. DNA quality and quantity was assessed by NanoDrop, agarose gel electrophoresis and Bioanalyzer. Decontam and SourceTracker were integrated into microbiota analysis to identify the presence of contaminating sources. The bacterial profile and nine genera differed between FFPE and frozen fetal membranes. There were no differences in bacterial profiles between FFPE samples and corresponding wax negative controls, with 49% of bacteria in FFPE fetal membrane samples matched to the source origin of paraffin wax, and 40% originating from DNA extraction reagent sources. FFPE samples displayed high fragmentation and low quantity of extracted DNA compared to frozen samples. The microbiota of FFPE fetal membrane samples is influenced by processing methods, with the inability to differentiate between the microbiota of the tissue sample and the surrounding wax block. Illumina sequencing results of FFPE and frozen fetal membrane samples should not be compared using the methods employed here. Variation could be influenced by limitations including storage time, DNA extraction and purification methods. To utilise FFPE fetal membrane samples in microbiota research then contamination prevention and detection methods must be included into optimised and standardised protocols, with recommendations presented here.
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Norgan, Andrew P., Lynne M. Sloan, and Bobbi S. Pritt. "Detection of Naegleria fowleri, Acanthamoeba spp, and Balamuthia mandrillaris in Formalin-Fixed, Paraffin-Embedded Tissues by Real-Time Multiplex Polymerase Chain Reaction." American Journal of Clinical Pathology 152, no. 6 (August 15, 2019): 799–807. http://dx.doi.org/10.1093/ajcp/aqz103.
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Abstract Objectives Pathogenic free-living amebae (FLAs) cause skin, ocular, and central nervous system (CNS) infections with significant morbidity and mortality. Diagnosis of FLA infections by pathologic examination of tissue sections can be aided using molecular assays. This study investigated the performance characteristics of a multiplex real-time polymerase chain reaction (PCR) assay (FLA-PCR) for detection and differentiation of FLAs in clinical specimens. Methods FLA-PCR was performed on 39 human specimens comprising one cutaneous, 14 corneal, and 24 CNS formalin-fixed, paraffin-embedded (FFPE) tissues with a histopathologic diagnosis of FLA infection and four CNS FFPE tissues with inflammation but no evidence of FLAs. In addition, clinical specificity and assay limit of detection were determined. Results FLA detection sensitivities ranged from 79% to 84% in FFPE tissues. No cross-reactivity was observed. Conclusions While sensitivity is limited, FLA-PCR assay may serve as a useful adjunct for detection or confirmation of FLA infections in FFPE tissues.
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Dimaras, Pantelis, Oskan Tasinov, Desislava Ivanova, Yoana Kiselova-Kaneva, Nadezhda Stefanova, Maria Tzaneva, and Diana Ivanova. "Improving gene expression analysis efficacy from formalin-fixed paraffin embedded tissues." Folia Medica 64, no. 4 (August 31, 2022): 602–8. http://dx.doi.org/10.3897/folmed.64.e63599.
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Introduction: Improving RNA isolation and cDNA synthesis techniques has emerged due to advancements in the knowledge of molecular basis of most diseases. This in turn increased the need of higher quantity and quality of the extracted genetic material to be used for a variety of diagnostic tests and experiments. Aim: The aim of the study was to compare three modified methods for RNA extraction from formalin-fixed paraffin embedded (FFPE) biopsied tissue and different cDNA synthesis strategies to facilitate study of gene expression. Materials and methods: Compared RNA extraction methods were: lysis buffer, phenol-based extraction, and combination of both with concomitant use of silica-based spin columns. RNA quantity and purity were estimated spectrophotometrically. Different priming strategies for cDNA synthesis were applied: oligo dT, combination of oligo dT and random hexamer, and gene specific primer. Two-step RT-qPCR of ribosomal protein L37A on preamplified and non-preamplified cDNA templates was performed. Results: The combination of lysis buffer with phenol based extraction gave higher RNA yield. By doing cDNA preamplification, the confidence of detection by qPCR was raised, and efficiency was improved. The preamplified template increased the sensitivity of analysis. Conclusions: Together, the combination of approaches improved substantially the reproducibility and validity of quantitative gene expression analyses from FFPE tissues.
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Ikegami, Masachika, Shinji Kohsaka, Takeshi Hirose, Toshihide Ueno, Satoshi Inoue, Naoki Kanomata, Hideko Yamauchi, et al. "Abstract 2185: MicroSEC: Sequence error filter for formalin-fixed and paraffin-embedded samples." Cancer Research 82, no. 12_Supplement (June 15, 2022): 2185. http://dx.doi.org/10.1158/1538-7445.am2022-2185.
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Abstract Background: DNAs extracted from formalin-fixed and paraffin-embedded (FFPE) tissues are fragmented and contain a significant amount of single-stranded DNA (ssDNA). We found that most of the false-positive mutations in clinical sequencing with FFPE samples are ssDNA-derived artifacts. During the end-repair step of library preparation, chimeric reads are generated by mis-annealing of ssDNA molecules comprising homologous sequences with a mismatched base. The mismatched base is called as a false-positive mutation, and the position of the base is biased towards the effective ends in the individual reads because either side of the chimeric read should be soft-clipped by the mapping pipeline. Based on this theory, we developed a post hoc filter, MicroSEC, to predict such artifacts in FFPE samples. Methods: Fifty-three fresh frozen (FF) and 190 FFPE normal breast tissue samples and 23 FF and 33 FFPE breast cancer samples were obtained from 26 patients, and subjected to capture-based panel sequencing (Todai OncoPanel, TOP). We also obtained TOP data from 54 FFPE various tumor samples. MicroSEC was developed to predict artifacts with BAM files based on the positional bias of mutations within each read. The predictions were validated with amplicon-based sequencing of 97 mutations. We also developed a model which predicts artifacts only from the mutated base information without corresponding BAM files, by using 5,034 MicroSEC predictions as the supervised data with the LightGBM technique. A total of 742,030 mutations from the AACR Project GENIE were examined with the model. Results: Among the normal breast tissues, we identified 0.3 and 11.7 somatic mutations per sample in FF and FFPE specimens, and 0 (0%) and 10.1 (86.0%) were filtered out by MicroSEC, respectively. Two unique mutations with variant allele frequency of >50% in FFPE samples were eliminated. From the breast cancer specimens, we identified 4.0 and 10.7 mutations per sample in FF and FFPE, and 0 (0%) and 3.2 (30.2%) were filtered out, respectively. In the clinical sequencing data of 54 FFPE tumor samples, we identified 21.6 mutations per sample and 3.6 (16.6%) including five unique pathogenic mutations were filtered out. The validation study showed that the sensitivity and specificity for artifacts of MicroSEC were 97% (95% confidence interval (CI): 82%-100%) and 96% (95% CI: 88%-99%), respectively. Further, the prediction model reproduced the MicroSEC predictions with an area under the ROC curve of 0.995. The model detected 2,512 mutations (0.34%) as artifacts from Project GENIE data. There was a difference in the artifact detection rate between institutions, and 815 (1.45%) of the 56,100 mutations reported by UCSF were predicted as artifacts. Conclusions: MicroSEC removes only FFPE artifacts without eliminating true mutations found in FF samples. Our pipeline will increase the reliability of the clinical sequencing and advance cancer research using FFPE samples. Citation Format: Masachika Ikegami, Shinji Kohsaka, Takeshi Hirose, Toshihide Ueno, Satoshi Inoue, Naoki Kanomata, Hideko Yamauchi, Taisuke Mori, Shigeki Sekine, Yoshihiro Inamoto, Yasushi Yatabe, Hiroshi Kobayashi, Sakae Tanaka, Toru Akiyama, Tomotake Okuma, Hiroyuki Mano. MicroSEC: Sequence error filter for formalin-fixed and paraffin-embedded samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2185.
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Mitiushkina, Natalia V., Grigory A. Yanus, Ekatherina Sh Kuligina, Tatiana A. Laidus, Alexandr A. Romanko, Maksim M. Kholmatov, Alexandr O. Ivantsov, Svetlana N. Aleksakhina, and Evgeny N. Imyanitov. "Preparation of Duplex Sequencing Libraries for Archival Paraffin-Embedded Tissue Samples Using Single-Strand-Specific Nuclease P1." International Journal of Molecular Sciences 23, no. 9 (April 21, 2022): 4586. http://dx.doi.org/10.3390/ijms23094586.
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DNA from formalin-fixed paraffin-embedded (FFPE) tissues, which are frequently utilized in cancer research, is significantly affected by chemical degradation. It was suggested that approaches that are based on duplex sequencing can significantly improve the accuracy of mutation detection in FFPE-derived DNA. However, the original duplex sequencing method cannot be utilized for the analysis of formalin-fixed paraffin-embedded (FFPE) tissues, as FFPE DNA contains an excessive number of damaged bases, and these lesions are converted to false double-strand nucleotide substitutions during polymerase-driven DNA end repair process. To resolve this drawback, we replaced DNA polymerase by a single strand-specific nuclease P1. Nuclease P1 was shown to efficiently remove RNA from DNA preparations, to fragment the FFPE-derived DNA and to remove 5′/3′-overhangs. To assess the performance of duplex sequencing-based methods in FFPE-derived DNA, we constructed the Bottleneck Sequencing System (BotSeqS) libraries from five colorectal carcinomas (CRCs) using either DNA polymerase or nuclease P1. As expected, the number of identified mutations was approximately an order of magnitude higher in libraries prepared with DNA polymerase vs. nuclease P1 (626 ± 167/Mb vs. 75 ± 37/Mb, paired t-test p-value 0.003). Furthermore, the use of nuclease P1 but not polymerase-driven DNA end repair allowed a reliable discrimination between CRC tumors with and without hypermutator phenotypes. The utility of newly developed modification was validated in the collection of 17 CRCs and 5 adjacent normal tissues. Nuclease P1 can be recommended for the use in duplex sequencing library preparation from FFPE-derived DNA.
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Amemiya, Kei, Xiankun Zeng, Jeremy J. Bearss, Christopher K. Cote, Carl Soffler, Robert C. Bernhards, Jennifer L. Dankmeyer, et al. "Laser Scanning Confocal Microscopy Was Used to Validate the Presence of Burkholderia pseudomallei or B. mallei in Formalin-Fixed Paraffin Embedded Tissues." Tropical Medicine and Infectious Disease 5, no. 2 (April 29, 2020): 65. http://dx.doi.org/10.3390/tropicalmed5020065.
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Burkholderia pseudomallei and B. mallei are Gram-negative, facultative intracellular bacteria that cause melioidosis and glanders, respectively. Currently, there are no vaccines for these two diseases. Animal models have been developed to evaluate vaccines and therapeutics. Tissues from infected animals, however, must be fixed in formalin and embedded in paraffin (FFPE) before analysis. A brownish staining material in infected tissues that represents the exopolysaccharide of the pathogen was seen by bright field microscopy but not the actual microorganism. Because of these results, FFPE tissue was examined by laser scanning confocal microscopy (LSCM) in an attempt to see the microorganism. Archival FFPE tissues were examined from ten mice, and five nonhuman primates after exposure to B. pseudomallei or B. mallei by LSCM. Additionally, a historical spleen biopsy from a human suspected of exposure to B. mallei was examined. B. pseudomallei was seen in many of the infected tissues from mice. Four out of five nonhuman primates were positive for the pathogen. In the human sample, B. mallei was seen in pyogranulomas in the spleen biopsy. Thus, the presence of the pathogen was validated by LSCM in murine, nonhuman primate, and human FFPE tissues.
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Grillo, Federica, Michela Campora, Simona Pigozzi, Silvia Bonadio, Luca Valle, Jacopo Ferro, Michele Paudice, Beatrice Dose, and Luca Mastracci. "Methods for restoration of ki67 antigenicity in aged paraffin tissue blocks." Histochemistry and Cell Biology 156, no. 2 (April 10, 2021): 183–90. http://dx.doi.org/10.1007/s00418-021-01987-w.
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AbstractPathology archives are a treasure trove of paraffin embedded tissue spanning many years and covering a wide variety of tissues and diseases. The possibility of using old archival formalin fixed paraffin embedded (FFPE) tissues for diagnostic updates and research projects is a widespread need and it requires archives of stable, well-preserved samples. Immunohistochemistry performed on old archival paraffin blocks may give unreliable results, in particular for some antigens, such as Ki67. In consideration of this phenomenon, our aim is to comprehensively test and identify methods which may be used to obtain Ki67 immunohistochemical reactions of good quality from old archival FFPE blocks. Various methods were tested in order to evaluate their possible efficacy in increasing Ki67 immunointensity in a collection of 40-year-old, archival blocks including re-embedding, with deeper sectioning of tissue from the block and increasing heat-based pretreatment times (20 cases) and re-processing (20 cases). All reactions were performed using an automated immunostainer and Ki67 stained immunosections compared using a visual colour-based scale (the first immunostained section was considered as baseline). The combination of deep sectioning (1000 µM) and prolonged heat-based pretreatment (64 min) markedly increased immunoreactivity for Ki67. Re-embedding and reprocessing did not have a significant effect. Large tissue samples showed heterogeneity of Ki67 immunoexpression between the periphery of the sample and the central area. In conclusion, the study defines a useful protocol to increase antigen retrieval applicable to dated archival tissues.
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Lee, Taebum, Hee Young Na, Sun-ju Byeon, Kyoung-Mee Kim, Hey Seung Lee, Sung-Hye Park, Ji-Young Choe, and Kyoung Chan Choi. "Mycobiome analysis in fungal infected formalin-fixed and paraffin-embedded tissues for identification of pathogenic fungi: a pilot study." F1000Research 9 (July 22, 2020): 758. http://dx.doi.org/10.12688/f1000research.25126.1.
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Background: Fungal organisms are frequently observed in surgical pathological diagnosis. In order to more accurately identify fungi in formalin-fixed and paraffin-embedded (FFPE) tissues, it is necessary to use genomic information. The purpose of our pilot study is to identify the factors to be considered for the identification of pathogenic fungi using mycobiome analysis in FFPE tissues. Methods: We selected 49 cases in five hospitals. In each case, FFPE tissue was cut into 50 µm and DNA was extracted. Multiplex PCR with four primers (ITS1, ITS2, ITS3 and ITS4) was performed. Multiplex sequencing was performed using a MinION device according to the manufacturer’s protocol. Sequences of each case were searched using BLASTN with an ITS database from NCBI RefSeq Targeted Loci Project with default parameters. Results: A total of 2,526 DNA sequences were sequenced. We were able to identify 342 fungal sequences in 24 (49.0%, 24/49) cases. The median number of detected fungal sequences per case was 3 (1Q: 1 and 3Q: 14.25). Of the fungal DNA sequences, 215 (62.87%) contained the entire region of ITS1 or ITS2. The remaining 127 fungal DNA sequences were identified as fungi using a partial sequence of ITS1, ITS2, 5.8S, LSU or SSU. Conclusion: In conclusion, we have identified the possibility of finding pathogenic fungi through mycobiome analysis in fungal infected FFPE tissues using nanopore sequencing. However, we have also found several limitations to be solved for further studies. If we develop a method to characterize pathogenic fungi in FFPE tissues in a follow-up study, we think it will help patients to use appropriate antifungal agents.
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Yi, Qing-qing, Rong Yang, Jun-feng Shi, Nai-yan Zeng, Dong-yu Liang, Shuang Sha, and Qing Chang. "Effect of preservation time of formalin-fixed paraffin-embedded tissues on extractable DNA and RNA quantity." Journal of International Medical Research 48, no. 6 (June 2020): 030006052093125. http://dx.doi.org/10.1177/0300060520931259.
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Objectives This study aimed to investigate the factors affecting the quantity of DNA and RNA extractable from human formalin-fixed paraffin-embedded (FFPE) tissues stored for different lengths of time. Methods We randomly selected 20 FFPE specimens harvested from hysteromyoma patients with uterine fibroids during 2010, 2015, and 2017 at the Department of Pathology, Jiading District Central Hospital Affiliated Shanghai University of Medicine and Health Sciences. DNA and RNA extractions were performed using a DNA/RNA FFPE kit. DNA and RNA concentrations and their OD260/OD280 ratios were determined by a NanoDrop 2000 spectrophotometer. The human β-globin gene and aldehyde dehydrogenase-2 (ALDH2) gene were amplified from nucleic acids using a LightCycler 480 Real-Time PCR System, and PCR amplification products were electrophoresed on 1% agarose gels. Results Specimens that were stored for longer showed more degradation and a reduced concentration of DNA and RNA after nucleic acid extraction. However, there was no significant difference in DNA or RNA purity. β-globin and ALDH2 genes could be amplified from more than 99% of specimens. Conclusion We found that FFPE tissues stored for longer had a reduced quantity of extractable DNA and RNA. However, these tissues could be used for the analysis of some small target genes.
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Iraola-Guzmán, Susana, Anna Brunet-Vega, Cinta Pegueroles, Ester Saus, Hrant Hovhannisyan, Alex Casalots, Carles Pericay, and Toni Gabaldón. "Target Enrichment Enables the Discovery of lncRNAs with Somatic Mutations or Altered Expression in Paraffin-Embedded Colorectal Cancer Samples." Cancers 12, no. 10 (October 1, 2020): 2844. http://dx.doi.org/10.3390/cancers12102844.
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Long non-coding RNAs (lncRNAs) play important roles in cancer and are potential new biomarkers or targets for therapy. However, given the low and tissue-specific expression of lncRNAs, linking these molecules to particular cancer types and processes through transcriptional profiling is challenging. Formalin-fixed, paraffin-embedded (FFPE) tissues are abundant resources for research but are prone to nucleic acid degradation, thereby complicating the study of lncRNAs. Here, we designed and validated a probe-based enrichment strategy to efficiently profile lncRNA expression in FFPE samples, and we applied it for the detection of lncRNAs associated with colorectal cancer (CRC). Our approach efficiently enriched targeted lncRNAs from FFPE samples, while preserving their relative abundance, and enabled the detection of tumor-specific mutations. We identified 379 lncRNAs differentially expressed between CRC tumors and matched healthy tissues and found tumor-specific lncRNA variants. Our results show that numerous lncRNAs are differentially expressed and/or accumulate variants in CRC tumors, thereby suggesting a role in CRC progression. More generally, our approach unlocks the study of lncRNAs in FFPE samples, thus enabling the retrospective use of abundant, well documented material available in hospital biobanks.
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Cooper, Melissa, Shu-Qiu Li, Tajinder Bhardwaj, Thomas Rohan, and Rita A. Kandel. "Evaluation of Oligonucleotide Arrays for Sequencing of the p53 Gene in DNA from Formalin-Fixed, Paraffin-Embedded Breast Cancer Specimens." Clinical Chemistry 50, no. 3 (March 1, 2004): 500–508. http://dx.doi.org/10.1373/clinchem.2003.025221.
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Abstract Background: Routine tissue processing has generated banks of paraffin-embedded tissue that could be used in retrospective cohort studies to study the molecular changes that occur during cancer development. The purpose of this study was to determine whether a p53 microarray could be used to sequence the p53 gene in DNA extracted from formalin-fixed, paraffin-embedded (FFPE) tissues. Methods: DNA was extracted from 70 FFPE breast cancer tissue specimens. p53 was sequenced with an oligonucleotide microarray (p53 GeneChip®; Affymetrix), and the results were compared with the results obtained from direct sequencing. Results: DNA was extracted from 62 of 70 cases. We identified 26 mutations in 24 of the 62 cases by the p53 GeneChip. No polymorphisms were detected, and exon 4 could not be evaluated in 20 cases. There were 43 genetic alterations detected by direct sequencing in 35 of the 62 cases. These consisted of 26 polymorphisms and 17 mutations in exons or splice sites. Fifteen mutations were identified by both methods. Direct sequencing detected significantly more gene alterations (43 of 54) in DNA extracted from FFPE tissue than the p53 GeneChip (26 of 54; P = 0.018). However, if the changes in exon 4 were eliminated from this comparison, the p53 GeneChip detected 26 of 27 mutations compared with direct sequencing, which identified 16 of 27 mutations. (P = 0.016). Conclusions: A combination of oligonucleotide microarray and direct sequencing may be necessary to accurately identify p53 gene alterations in FFPE breast cancer. The p53 GeneChip cannot be used to detect exon 4 polymorphisms (codon 72) in FFPE breast cancer tissue.
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Yin, Shen, Xinlei Wang, Gaoxiang Jia, and Yang Xie. "MIXnorm: normalizing RNA-seq data from formalin-fixed paraffin-embedded samples." Bioinformatics 36, no. 11 (March 5, 2020): 3401–8. http://dx.doi.org/10.1093/bioinformatics/btaa153.
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Abstract Motivation Recent studies have shown that RNA-sequencing (RNA-seq) can be used to measure mRNA of sufficient quality extracted from formalin-fixed paraffin-embedded (FFPE) tissues to provide whole-genome transcriptome analysis. However, little attention has been given to the normalization of FFPE RNA-seq data, a key step that adjusts for unwanted biological and technical effects that can bias the signal of interest. Existing methods, developed based on fresh-frozen or similar-type samples, may cause suboptimal performance. Results We proposed a new normalization method, labeled MIXnorm, for FFPE RNA-seq data. MIXnorm relies on a two-component mixture model, which models non-expressed genes by zero-inflated Poisson distributions and models expressed genes by truncated normal distributions. To obtain maximum likelihood estimates, we developed a nested EM algorithm, in which closed-form updates are available in each iteration. By eliminating the need for numerical optimization in the M-step, the algorithm is easy to implement and computationally efficient. We evaluated MIXnorm through simulations and cancer studies. MIXnorm makes a significant improvement over commonly used methods for RNA-seq expression data. Availability and implementation R code available at https://github.com/S-YIN/MIXnorm. Contact swang@smu.edu Supplementary information Supplementary data are available at Bioinformatics online.
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Lee, Taek Sang, Hye Won Jeon, Yong Beom Kim, Young A. Kim, Min A. Kim, and Soon Beom Kang. "Aberrant MicroRNA Expression in Endometrial Carcinoma Using Formalin-Fixed Paraffin-Embedded (FFPE) Tissues." PLoS ONE 8, no. 12 (December 9, 2013): e81421. http://dx.doi.org/10.1371/journal.pone.0081421.
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Babol-Pokora, Katarzyna, and Jarosław Berent. "SNP-minisequencing as an excellent tool for analysing degraded DNA recovered from archival tissues." Acta Biochimica Polonica 55, no. 4 (November 4, 2008): 815–19. http://dx.doi.org/10.18388/abp.2008_3045.
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SNP-minisequencing has become common in forensic genetics, especially for analysing degraded or low copy number DNA (LCN DNA). The aim of this study was to examine the usefulness of five SNP (single nucleotide polymorphism) markers for analyzing degraded and LCN DNA recovered from archival samples. DNA extractions of eight formalin-fixed paraffin-embedded (FFPE) tissues were performed and DNA fragments were amplified in one multiplex PCR (polymerase chain reaction). SNPs were identified in a minisequencing reaction and a gel electrophoresis in ABI Prism 377 Sequencer. The research confirmed the usefulness of SNP-minisequencing for analysing FFPE tissues.
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Asmann, Yan W., Vivekananda Sarangi, Bruce W. Eckloff, Julie M. Cunningham, Samantha J. McDonough, Yeon K. Lee, Eric D. Wieben, et al. "Comparison Of Single Nucleotide Mutations (SNVs) and Copy Number Variants (CNVs) Detection In Formalin Fixed Paraffin Embedded (FFPE) and Paired Frozen Tumor Tissues Using Target Capture and Sequencing Approach." Blood 122, no. 21 (November 15, 2013): 1784. http://dx.doi.org/10.1182/blood.v122.21.1784.1784.
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Abstract Background Next Generation sequencing (NGS) is a powerful tool to identify somatic mutations associated with tumor onset and drug response. While it is well suited for high quality fresh/frozen samples, NGS is not proven for FFPE tissue which is the most common type of clinical specimen. Since the nucleic acids can be readily extracted from FFPE samples for a variety of genomic analyses, a comparative mutational analysis of paired frozen and FFPE tissues is urgently needed. Our long term goal is to establish a lab protocol to detect mutations in FFPE tumors using a targeted capture and sequencing approach for genes of interest. This pilot study focuses on the comparison of FFPE and frozen samples to test the validity of using FFPE tissues in such application. Methods Gene Selection: 128 genes associated with known pathogenic mutations in lymphoma Sample Selection: 9 diffuse large B-cell lymphoma (DLBCL) cases with FFPE, frozen and germline samples, as well as 10 frozen normal lymphatic tissues as references for CNV detections Capture Probe Design: We targeted coding exons and UTR, as well as the evolutionarily conserved intronic regions. The capture probes were designed using the Agilent eArray tool. The titling density of the probes was set to 3 probes overlapping with every base in the target region to improve the capture efficiency in FFPE samples. The least stringent masking of the repeat regions was allowed to include regions with small repeats that are shorter than the length of the sequencing reads (100-bp). In addition, boosting parameters were picked to set various levels of probe replication in different regions in order to minimize the local coverage differences (e.g. between regions of different GC contents) Sequencing and Bioinformatics: The target capture and sequencing were performed by the Mayo Clinic Medical Genome Facility. The reads were mapped to Human Reference Genome Build 37 using Novalign, and SNVs were called using GATK. The CNVs were identified using an in-house developed algorithm, patternCNV. Results The designed probes covered 99.65937% of the target regions. We generated 2.2-6.7 Gbp of reads per sample, 57.4-71.5% of which were on target. This equalled an average coverage of 2100-6700 folds which is 10-30 times higher than the minimal coverage recommended by Agilent. Due to this high coverage, we observed duplicate reads that accounted for 7.7-73.5% of the total reads. When we analysed the data with and without the duplicated reads, the concordance of the called SNVs was between 84-93% out of 207-249 mutated positions per trio-sample. There were 7.8-8.9% and 1.1-2.2% unique SNVs per sample by excluding or including duplicate reads, respectively. The dis-concordances were mostly missed calls, where a SNV was observed in only 1 or 2 of the trio samples. The missed calls from frozen samples ranged from 0-10.4% compared to 1.4-10.4% from the FFPE tissues, with 0.88-2.4% more SNVs missed in FFPE. Further analyses showed that all of the missing calls came from the lack of or low coverage of the corresponding positions. There were also differences of the called SNVs between the trio samples. However, this was extremely rare. Only 2 out of the 9 trio samples at a total of 3 positions had disagreements in called SNVs between FFPE and frozen tissues, all due to the allelic imbalance where the percentage of reads supporting the alternative alleles were below 20%. Therefore, this dis-concordance can be removed by back-filling of the read-level information for each position. Unfortunately only 11.9-47.4% of the CNVs called in frozen tissues were identified in FFPE samples, due to the widely various coverage in FFPE samples. The consequent large noises of the log ratio values between the FFPEs and normal references significantly reduced the sensitivity for CNV calling. Conclusions This pilot study compared the performance of SNV and CNV detection in FFPE and paired frozen tissues using a target capture and sequencing approach. With a capture probe design strategized to benefit FFPE samples, we observed SNV detection rates in FFPE that were only slightly lower (0.88-2.4%) than those of frozen tissues due to poor coverage of some positions in FFPE samples. With a proper back-filling step, there was no dis-concordance of the called SNVs between FFPE and frozen samples. However, CNV detections in FFPE were more problematic due to the un-predictable regional coverage in FFPE samples. Disclosures: No relevant conflicts of interest to declare.
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Soto, Esteban, Oscar Illanes, David Hilchie, Juan A. Morales, Piyanate Sunyakumthorn, John P. Hawke, Andrew E. Goodwin, et al. "Molecular and immunohistochemical diagnosis of Francisella noatunensis subsp. orientalis from formalin-fixed, paraffin-embedded tissues." Journal of Veterinary Diagnostic Investigation 24, no. 5 (July 11, 2012): 840–45. http://dx.doi.org/10.1177/1040638712452108.
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Members of the genus Francisella (viz., F. noatunensis subsp. orientalis [ Fno] and F. noatunensis subsp. noatunensis) have been described as causative agents of chronic granulomatous and pyogranulomatous lesions in wild and cultured fish species. In the present study, 68 archived formalin-fixed, paraffin-embedded (FFPE) tissues from several fish species, collected at different geographical locations from 2000 to 2011, were analyzed using a real-time polymerase chain reaction assay for the detection of the Fno intracellular growth loci C ( iglC) gene and by immunohistochemistry for the demonstration of Fno antigens. The results revealed a high correlation between these 2 diagnostic techniques validating their use for the diagnosis of Fno infection in archived FFPE tissues and confirming the presence of Fno in fish species from the Cari y years of the present century.
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Freedman, J. A., T. Osada, K. A. Tsamis, M. Morse, B. M. Clary, H. K. Lyerly, J. R. Nevins, T. M. Clay, and S. D. Hsu. "Development of an assay to predict oxaliplatin sensitivity from formalin-fixed, paraffin-embedded (FFPE) colorectal cancer tissues." Journal of Clinical Oncology 29, no. 4_suppl (February 1, 2011): 429. http://dx.doi.org/10.1200/jco.2011.29.4_suppl.429.
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429 Background: Genomic profiling has improved our understanding of the underlying biology of tumors, accuracy of diagnosing disease, predictions of the courses of disease, and ability to determine the therapeutic agents that will be most effective in the treatment of particular tumors. However, in order for assays involving microarray data to be useful in the clinical setting, the ability to generate reliable and consistent data from FFPE tissues is essential. Methods: Cancer cell lines from the NCI-60 collection exhibiting greatest sensitivity or resistance to oxaliplatin were identified. These cells were grown in culture, fixed for 24 hours in formalin, and paraffin-embedded. RNA from the FFPE cells was isolated, amplified, and hybridized to Affymetrix arrays. A Bayesian binary regression analysis was used to generate a predictor of oxaliplatin sensitivity from the gene expression data. Metastatic derived xenografts (MDXs) from resected colorectal tumors were established and treated with oxaliplatin. Samples from tumors prior to treatment were paraffin-embedded and used for RNA extraction, amplification, and hybridization to Affymetrix arrays. The gene expression signature predicting sensitivity to oxaliplatin was then validated with response data from MDXs treated with oxaliplatin. Results: A predictor consisting of 300 genes that could predict sensitivity to oxaliplatin was generated using FFPE samples. Significant correlation was observed between the predicted probability of oxaliplatin sensitivity and the tumor growth inhibition measurement for a given MDX (p=0.0012). Conclusions: Reliable and consistent predictions of oxaliplatin sensitivity can be obtained from gene expression data from FFPE tissues. This method has potential utility in the clinical setting. The ability to predict response to a therapeutic in a FFPE sample has the potential to guide the choice of therapeutics, resulting in an option that could be most effective in treating an individual with metastatic colorectal cancer. No significant financial relationships to disclose.
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Mullins, Michael, Laurent Perreard, John F. Quackenbush, Nicholas Gauthier, Steven Bayer, Matthew Ellis, Joel Parker, Charles M. Perou, Aniko Szabo, and Philip S. Bernard. "Agreement in Breast Cancer Classification between Microarray and Quantitative Reverse Transcription PCR from Fresh-Frozen and Formalin-Fixed, Paraffin-Embedded Tissues." Clinical Chemistry 53, no. 7 (July 1, 2007): 1273–79. http://dx.doi.org/10.1373/clinchem.2006.083725.
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Abstract Background: Microarray studies have identified different molecular subtypes of breast cancer with prognostic significance. To transition these classifications into the clinical laboratory, we have developed a real-time quantitative reverse transcription (qRT)-PCR assay to diagnose the biological subtypes of breast cancer from fresh-frozen (FF) and formalin-fixed, paraffin-embedded (FFPE) tissues. Methods: We used microarray data from 124 breast samples as a training set for classifying tumors into 4 previously defined molecular subtypes: Luminal, HER2+/ER−, basal-like, and normal-like. We used the training set data in 2 different centroid-based algorithms to predict sample class on 35 breast tumors (test set) procured as FF and FFPE tissues (70 samples). We classified samples on the basis of large and minimized gene sets. We used the minimized gene set in a real-time qRT-PCR assay to predict sample subtype from the FF and FFPE tissues. We evaluated primer set performance between procurement methods by use of several measures of agreement. Results: The centroid-based algorithms were in complete agreement in classification from FFPE tissues by use of qRT-PCR and the minimized “intrinsic” gene set (40 classifiers). There was 94% (33 of 35) concordance between the diagnostic algorithms when comparing subtype classification from FF tissue by use of microarray (large and minimized gene set) and qRT-PCR data. We found that the ratio of the diagonal SD to the dynamic range was the best method for assessing agreement on a gene-by-gene basis. Conclusions: Centroid-based algorithms are robust classifiers for breast cancer subtype assignment across platforms and procurement conditions.
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Schlunck, Günther, Stefaniya Boneva, Julian Wolf, Anja Schlecht, Thomas Reinhard, Claudia Auw-Hädrich, and Clemens Lange. "RNA Sequencing of Formalin-Fixed and Paraffin-Embedded Tissue as a Complementary Method in Ophthalmopathology." Klinische Monatsblätter für Augenheilkunde 237, no. 07 (July 2020): 860–66. http://dx.doi.org/10.1055/a-1187-1590.
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AbstractThe high-throughput method of “Next Generation Sequencing” (NGS) allows cost-effective decoding of the nucleotide sequences of millions of RNA molecules in a sample. This makes it possible to determine the number of distinct RNA molecules in tissues or cells and to use these data to draw conclusions. The entirety of RNAs, in particular mRNAs (messenger RNAs) as potential precursors of proteins, provides a comprehensive insight into the functional state of the cells and tissues under investigation. In addition to cell cultures or unfixed tissue, formalin-fixed and paraffin-embedded (FFPE) tissue can also be analysed for this purpose using specific methods. In this overview, the methodological strategy and its application to the field of ophthalmic histopathology are presented.
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Hülskötter, Kirsten, Vanessa M. Pfankuche, Lydia van Dyck, Martin Höltershinken, Andrea Springer, Fabienne Lienhart, Sandra Ermel, et al. "Bovine Babesiosis Diagnosed in Formalin-Fixed, Paraffin-Embedded Tissues by Using In Situ Hybridization." Veterinary Pathology 57, no. 6 (August 25, 2020): 812–20. http://dx.doi.org/10.1177/0300985820948816.
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Bovine babesiosis, caused by Babesia divergens, is in general a rare disease in Europe. Nonetheless, local outbreaks can cause severe economic damage, and postmortem identification represents a diagnostic challenge. During a recent outbreak in May 2018 in northern Germany, 21 animals of a herd of 150 cattle died within 40 days having had clinical signs of fever and hemoglobinuria. Gross examination of 4 of the 21 deceased animals revealed a tick infestation, jaundice, and dark brown staining of urine and kidneys. Histologically, there were iron-positive deposits, hyperplasia of the red pulp of the spleen, and centrilobular necrosis of hepatocytes. In several locations, small basophilic granules suggestive of intraerythrocytic parasites were visible in hematoxylin-eosin- and Giemsa-stained sections. Peripheral blood smears from a living cow from the herd and polymerase chain reaction (PCR) of feeding ticks revealed B. divergens infection. In situ hybridization (ISH) was applied on formalin-fixed, paraffin-embedded (FFPE) tissue of the necropsied cattle to confirm babesiosis in these animals postmortem. Digoxigenin-labeled DNA probes were generated based on a specific nucleotide sequence for B. divergens, obtained by PCR and sequencing of DNA isolates from infected Ixodes ricinus ticks from deceased cattle. ISH using these probes allowed postmortem diagnosis of B. divergens infection in routinely fixed FFPE tissues.
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Jihad, Noor A., and Yasir W. Issa. "ROLE OF MICRORNA-155 AS A DIAGNOSTIC BIOMARKER FOR HUMAN PAPILLOMAVIRUS ASSOCIATED CERVICAL CANCER." Wiadomości Lekarskie 74, no. 9 (2021): 2301–4. http://dx.doi.org/10.36740/wlek202109210.
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The aim: This study was designed to investigate the potential role of miRNA-155 in the pathogenesis of HPV-induced cervical cancer. Materials and methods: A total of 42 formalin-fixed paraffin-embedded (FFPE) cervical cancer tissue samples and 38 FFPE normal cervical tissue samples were used (they were collected at the Department of Pathology, Baghdad teaching hospital, Baghdad, Iraq, between January 2019 to January 2021). Following HPV testing and genotyping, the expression of miRNA-155 were evaluated by real-time PCR (qPCR). Results: A statistically significant up-regulation of miRNA-155 expression was observed in cervical cancer tissues compared to results in control group, regardless of HPV status and clinical grading. Conclusions: These data suggest that overexpression of miRNA-155 can delineate cervical cancer tissues from normal and may be a useful diagnostic biomarker for early detection of cervical cancer.
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Khan, Muhammad Nouman, Qianqian Wang, Bushra Sana Idrees, Geer Teng, Xutai Cui, and Kai Wei. "Discrimination of Melanoma Using Laser-Induced Breakdown Spectroscopy Conducted on Human Tissue Samples." Journal of Spectroscopy 2020 (December 30, 2020): 1–11. http://dx.doi.org/10.1155/2020/8826243.
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Discrimination and identification of melanoma (a kind of skin cancer) by using laser-induced breakdown spectroscopy (LIBS) combined with chemometrics methods are reported. The human melanoma and normal tissues are used in the form of formalin-fixed paraffin-embedded (FFPE) blocks as samples. The results demonstrated higher LIBS signal intensities of phosphorus (P), potassium (K), sodium (Na), magnesium (Mg), and calcium (Ca) in melanoma FFPE samples while lower signal intensities in normal FFPE tissue samples. Chemometric methods, artificial neural network (ANN), linear discriminant analysis (LDA), quadratic discriminant analysis (QDA), and partial least square discriminant analysis (PLS-DA) are used to build the classification models. Different preprocessing methods, standard normal variate (SNV), mean-centering, normalization by total area, and autoscaling, were compared. A good performance of the model (sensitivity, specificity, and accuracy) for melanoma and normal FFPE tissues has been achieved by the ANN and PLS-DA models (all were 100%). The results revealed that LIBS combined with chemometric methods for detection and discrimination of human malignancies is a reliable, accurate, and precise technique.
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Kaneko, Syuzo, Toutai Mitsuyama, Kouya Shiraishi, Noriko Ikawa, Kanto Shozu, Ai Dozen, Hidenori Machino, et al. "Genome-Wide Chromatin Analysis of FFPE Tissues Using a Dual-Arm Robot with Clinical Potential." Cancers 13, no. 9 (April 28, 2021): 2126. http://dx.doi.org/10.3390/cancers13092126.
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Although chromatin immunoprecipitation and next-generation sequencing (ChIP-seq) using formalin-fixed paraffin-embedded tissue (FFPE) has been reported, it remained elusive whether they retained accurate transcription factor binding. Here, we developed a method to identify the binding sites of the insulator transcription factor CTCF and the genome-wide distribution of histone modifications involved in transcriptional activation. Importantly, we provide evidence that the ChIP-seq datasets obtained from FFPE samples are similar to or even better than the data for corresponding fresh-frozen samples, indicating that FFPE samples are compatible with ChIP-seq analysis. H3K27ac ChIP-seq analyses of 69 FFPE samples using a dual-arm robot revealed that driver mutations in EGFR were distinguishable from pan-negative cases and were relatively homogeneous as a group in lung adenocarcinomas. Thus, our results demonstrate that FFPE samples are an important source for epigenomic research, enabling the study of histone modifications, nuclear chromatin structure, and clinical data.
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Shiogama, Kazuya, Ken-ichi Inada, Michinori Kohara, Hidemi Teramoto, Yasuyoshi Mizutani, Takanori Onouchi, and Yutaka Tsutsumi. "Demonstration of Hepatitis C Virus RNA withIn SituHybridization Employing a Locked Nucleic Acid Probe in Humanized Liver of Infected Chimeric Mice and in Needle-Biopsied Human Liver." International Journal of Hepatology 2013 (2013): 1–7. http://dx.doi.org/10.1155/2013/249535.
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Background.In situhybridization (ISH) with high sensitivity has been requested to demonstrate hepatitis C virus (HCV) RNA in formalin-fixed, paraffin-embedded (FFPE) sections of the liver.Methods. ISH employing a locked-nucleic-acid- (LNA-)modified oligonucleotide probe and biotin-free catalyzed signal amplification system (CSAII) was applied to HCV-RNA detection in the liver tissue. Nested reverse-transcription polymerase chain reaction (RT-PCR) was performed for HCV genotyping using total RNA extracted from FFPE sections. The target tissues included FFPE tissue sections of humanized livers in HCV-infected chimeric mice (HCV genotypes 1a, 1b, and 2a and noninfected) and of needle-biopsied livers from HCV-infected patients.Results. HCV-RNA was demonstrated with the ISH technique in HCV-infected liver tissues from both chimeric mice and 9 (82%) of 11 patients with HCV infection. The HCV signals were sensitive to RNase. Nested RT-PCR confirmed the genotype in 8 (73%) of 11 livers (type 1b: 6 lesions and type 2a: 2 lesions). HCV-RNA was not identified in chronic hepatitis B lesions, fatty liver, autoimmune hepatitis, and hepatocellular carcinoma.Conclusion. ISH using the LNA-modified oligonucleotide probe and CSAII was applicable to detecting HCV-RNA in routinely prepared FFPE liver specimens.
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Hafy, Zen, Veny Larasati, Riana Sari Puspita, Novizar S, Haekal M, Rafdi A, and Sentani R. "Hubungan Lama Penyimpanan Sampel Arsip Jaringan Dalam Blok Parafin Terfiksasi Formalindengan Kualitas Hasil Ekstraksidna Mitokondria Jaringan." SRIWIJAYA JOURNAL OF MEDICINE 1, no. 3 (October 31, 2018): 157–62. http://dx.doi.org/10.32539/sjm.v1i3.31.
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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.
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Hutchins, Rae G., Edward B. Breitschwerdt, John M. Cullen, Sally A. Bissett, and Jody L. Gookin. "Limited yield of diagnoses of intrahepatic infectious causes of canine granulomatous hepatitis from archival liver tissue." Journal of Veterinary Diagnostic Investigation 24, no. 5 (August 1, 2012): 888–94. http://dx.doi.org/10.1177/1040638712453583.
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Canine granulomatous hepatitis is an uncommon morphologic diagnosis that has been associated with a variety of diseases, including a number of systemic infectious etiologies. Formalin-fixed, paraffin-embedded (FFPE) tissues are typically the only source of liver tissue remaining for additional testing for the presence of infectious disease within granulomas. It is unclear if the more common infectious culprits of granulomatous hepatitis can be identified from such specimens. The aim of the current study was to retrospectively investigate archival FFPE liver tissue from dogs with granulomatous hepatitis for the presence of infectious agents. Semiquantitative analysis of copper accumulation in liver specimens was also performed. Medical records were examined for recorded evidence of systemic infectious disease diagnosis. Formalin-fixed, paraffin-embedded liver was prospectively evaluated for infectious agents via differential staining techniques ( n = 13), eubacterial fluorescent in situ hybridization ( n = 11), and Bartonella polymerase chain reaction assays ( n = 15). An infectious cause of granulomatous hepatitis was not identified within liver tissue from any dog using these diagnostic methodologies. Six out of 25 (24%) dogs were diagnosed with concurrent systemic or localized bacterial infections at the time of presentation. Nine out of 17 (53%) dogs had excessive hepatic copper accumulation when evaluated by a semiquantitative histologic grading scheme or quantitative copper analysis. As definitive infectious causes of granulomatous hepatitis were not identified within archival liver biopsy samples, it was concluded that investigation of infectious etiologies within FFPE liver specimens using these diagnostic approaches may be of low yield.
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Bhatnagar, Julu, Marlene DeLeon-Carnes, Kathryn L. Kellar, Kakali Bandyopadhyay, Zoi-Anna Antoniadou, Wun-Ju Shieh, Christopher D. Paddock, and Sherif R. Zaki. "Rapid, Simultaneous Detection ofClostridium sordelliiandClostridium perfringensin Archived Tissues by a Novel PCR-Based Microsphere Assay: Diagnostic Implications for Pregnancy-Associated Toxic Shock Syndrome Cases." Infectious Diseases in Obstetrics and Gynecology 2012 (2012): 1–8. http://dx.doi.org/10.1155/2012/972845.
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Clostridium sordelliiandClostridium perfringensare infrequent human pathogens; however, the case-fatality rates for the infections are very high, particularly in obstetricC. sordelliiinfections (>90%). Deaths fromClostridium sordelliiandClostridium perfringenstoxic shock (CTS) are sudden, and diagnosis is often challenging. Formalin-fixed, paraffin-embedded (FFPE) tissues usually are the only specimens available for sudden fatal cases, and immunohistochemistry (IHC) for Clostridia is generally performed but it cannot identify species. A clear need exists for a rapid, species-specific diagnostic assay for FFPE tissues. We developed a duplex PCR-based microsphere assay for simultaneous detection ofC. sordelliiandC. perfringensand evaluated DNA extracted from 42Clostridiumisolates and FFPE tissues of 28 patients with toxic shock/endometritis (20 CTS, 8 non-CTS, as confirmed by PCR and sequencing). The microsphere assay correctly identifiedC. sordelliiandC. perfringensin all known isolates and in all CTS patients (10C. sordellii, 8C. perfringens, 2 both) and showed 100% concordance with PCR and sequencing results. The microsphere assay is a rapid, specific, and cost-effective method for the diagnosis of CTS and offers the advantage of simultaneous testing forC. sordelliiandC. perfringensin FFPE tissues using a limited amount of DNA.
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Belloni, Benedetta, Chiara Lambertini, Paolo Nuciforo, Jay Phillips, Eric Bruening, Stephane Wong, and Reinhard Dummer. "Will PAXgene substitute formalin? A morphological and molecular comparative study using a new fixative system." Journal of Clinical Pathology 66, no. 2 (November 3, 2012): 124–35. http://dx.doi.org/10.1136/jclinpath-2012-200983.
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Formalin fixation and paraffin embedding present the standard procedures for conserving clinical tissues for histological analysis. However, molecular analysis is impaired by the cross linking properties of formalin. The PAXgene tissue system (PreAnalytix, Switzerland) is a new formalin-free tissue collection device.AimsIn this study we aimed to evaluate this new tissue preservation technique in comparison with formalin fixation and fresh frozen tissue samples.Methods12 melanoma biopsy samples were divided and fixed simultaneously with formalin, PAXgene or fresh frozen in liquid nitrogen and analysed with regard to morphology, immunohistochemistry, DNA and RNA content and quality. Markers of melanocytic differentiation and tumour cell proliferation were used.ResultsMorphology was well preserved in PAXPE samples. However, 5 out of 11 immunohistochemical markers showed significantly lower overall staining and staining intensity with PAXPE tissues in comparison with formalin-fixed, paraffin-embedded (FFPE). Increasing membrane permeability through adding a detergent did proportionally increase staining intensity in PAXPE samples. Amplification of different mRNA amplicons showed a direct relationship with the size of the amplicon with greater template integrity observed in PAXPE samples. Sequencing and mutational analysis of DNA samples were comparable for all the different fixation methods, while the level of DNA fragmentation seemed to be lower in PAXPE compared with FFPE tissues.ConclusionsThe switch from formalin to PAXgene fixation would require a re-evaluation of immunohistochemical markers and staining procedures originally developed for FFPE tissues. Our data demonstrate that PAXPE fixation offers some advantages concerning molecular analysis. However, these advantages would not justify substituting formalin fixation in any routine pathology laboratory.
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Kuo, Shanny Hsuan, Yen-Chen Chang, Hui-Wen Chang, and Wei-Hsiang Huang. "DETECTION OF SPIKE-SPECIFIC MUTATIONS IN SEROTYPE I FELINE CORONAVIRUSES IN FORMALIN-FIXED AND PARAFFIN-EMBEDDED TISSUE." Taiwan Veterinary Journal 46, no. 02n03 (June 2020): 111–22. http://dx.doi.org/10.1142/s1682648520720026.
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Previously, the M1058L and S1060A amino acid mutations in the spike protein of feline coronavirus (FCoV) have been shown to distinguish feline infectious peritonitis virus (FIPV) from feline enteric coronavirus (FECV) in [Formula: see text]95% of serotype I FCoV (FCoVI)-infected cases, serving as potential FIP diagnostic markers. However, the finding is recently challenged by the demonstration that these markers are merely indicative of systemic spread of FCoV from the intestine, rather than a mutated FIPV with the potential to cause FIP. The aim of this study is to design a modified spike mutation-detection nested RT-PCR to distinguish FIPV from FECV in formalin-fixed and paraffin-embedded (FFPE) tissues from cats confirmed with FIP and controls. While none in the control group was tested positive by the nRT-PCR, FCoVI RNA was detected in 20 of 23 FIP cases. Of the positive samples, 19/20 (95%) FIP cats bore one of the two mutations in the spike gene. The sensitivity and specificity of this test reached 87% (95% CI: 65–97) and 100% (95% CI: 82–100), respectively. The high positive predictive values of 100% (95% CI: 80–100) and the negative predictive values of 88% (95% CI: 68–97) were determined. By using the conventional nested RT-PCR method in FFPE tissue, we revealed the spike gene-mutated FCoVs could be detected in FFPE tissues from FIP-confirmed cats, but could not be amplified from cats without FIP. Our result supports that detection of the two critical mutations correlates the presence of serotype I FIPV in FIP cats.