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Sample records for embedded ffpe tissue

  1. Qualitative and quantitative proteomic analysis of formalin-fixed paraffin-embedded (FFPE) tissue.

    PubMed

    Azimzadeh, Omid; Atkinson, Michael J; Tapio, Soile

    2015-01-01

    Formalin-fixed, paraffin-embedded (FFPE) tissue has recently gained interest as an alternative to fresh/frozen tissue for retrospective protein biomarker discovery. However, during the formalin fixation proteins undergo degradation and cross-linking, making conventional protein analysis technologies challenging. Cross-linking is even more challenging when quantitative proteome analysis of FFPE tissue is planned. The use of conventional protein labeling technologies on FFPE tissue has turned out to be problematic as the lysine residue labeling targets are frequently blocked by the formalin treatment. We have established a qualitative and quantitative proteomics analysis technique for FFPE tissues that combines label-free proteomic analysis with optimized protein extraction and separation conditions.

  2. Exome enrichment and SOLiD sequencing of formalin fixed paraffin embedded (FFPE) prostate cancer tissue.

    PubMed

    Menon, Roopika; Deng, Mario; Boehm, Diana; Braun, Martin; Fend, Falko; Boehm, Detlef; Biskup, Saskia; Perner, Sven

    2012-01-01

    Next generation sequencing (NGS) technologies have revolutionized cancer research allowing the comprehensive study of cancer using high throughput deep sequencing methodologies. These methods detect genomic alterations, nucleotide substitutions, insertions, deletions and copy number alterations. SOLiD (Sequencing by Oligonucleotide Ligation and Detection, Life Technologies) is a promising technology generating billions of 50 bp sequencing reads. This robust technique, successfully applied in gene identification, might be helpful in detecting novel genes associated with cancer initiation and progression using formalin fixed paraffin embedded (FFPE) tissue. This study's aim was to compare the validity of whole exome sequencing of fresh-frozen vs. FFPE tumor tissue by normalization to normal prostatic FFPE tissue, obtained from the same patient. One primary fresh-frozen sample, corresponding FFPE prostate cancer sample and matched adjacent normal prostatic tissue was subjected to exome sequencing. The sequenced reads were mapped and compared. Our study was the first to show comparable exome sequencing results between FFPE and corresponding fresh-frozen cancer tissues using SOLiD sequencing. A prior study has been conducted comparing the validity of sequencing of FFPE vs. fresh frozen samples using other NGS platforms. Our validation further proves that FFPE material is a reliable source of material for whole exome sequencing.

  3. Detection of alpha human papillomaviruses in archival formalin-fixed, paraffin-embedded (FFPE) tissue specimens.

    PubMed

    Kocjan, Boštjan J; Hošnjak, Lea; Poljak, Mario

    2016-03-01

    Formalin-fixed, paraffin-embedded (FFPE) tissue specimens stored in pathology departments worldwide are an invaluable source for diagnostic purposes when fresh clinical material is unavailable as well as for retrospective molecular and epidemiological studies, especially when dealing with rare clinical conditions for which prospective collection is not feasible. Accurate detection of HPV infection in these specimens is particularly challenging because nucleic acids are often degraded and therefore, not suitable for amplification of larger fragments of the viral genome or viral gene transcripts. This review provides a brief summary of molecular methods for detecting alpha-HPV DNA/RNA in FFPE tissue specimens. We specifically address the key procedural and environmental factors that have the greatest impact on the quality of nucleic acids extracted from FFPE tissue specimens, and describe some solutions that can be used to increase their integrity and/or amplifiability. Moreover, commonly used methods for HPV DNA/RNA detection in FFPE tissue specimens are presented and discussed, focusing on studies using polymerase chain reaction as an HPV detection method and published after 1999. Finally, we briefly summarize our 22 years of experience with HPV detection in FFPE tissue specimens. PMID:26514313

  4. Detection of alpha human papillomaviruses in archival formalin-fixed, paraffin-embedded (FFPE) tissue specimens.

    PubMed

    Kocjan, Boštjan J; Hošnjak, Lea; Poljak, Mario

    2016-03-01

    Formalin-fixed, paraffin-embedded (FFPE) tissue specimens stored in pathology departments worldwide are an invaluable source for diagnostic purposes when fresh clinical material is unavailable as well as for retrospective molecular and epidemiological studies, especially when dealing with rare clinical conditions for which prospective collection is not feasible. Accurate detection of HPV infection in these specimens is particularly challenging because nucleic acids are often degraded and therefore, not suitable for amplification of larger fragments of the viral genome or viral gene transcripts. This review provides a brief summary of molecular methods for detecting alpha-HPV DNA/RNA in FFPE tissue specimens. We specifically address the key procedural and environmental factors that have the greatest impact on the quality of nucleic acids extracted from FFPE tissue specimens, and describe some solutions that can be used to increase their integrity and/or amplifiability. Moreover, commonly used methods for HPV DNA/RNA detection in FFPE tissue specimens are presented and discussed, focusing on studies using polymerase chain reaction as an HPV detection method and published after 1999. Finally, we briefly summarize our 22 years of experience with HPV detection in FFPE tissue specimens.

  5. Formalin-fixed, paraffin-embedded (FFPE) tissue epigenomics using Infinium HumanMethylation450 BeadChip assays.

    PubMed

    de Ruijter, Tim C; de Hoon, Joep P J; Slaats, Jeroen; de Vries, Bart; Janssen, Marjolein J F W; van Wezel, Tom; Aarts, Maureen J B; van Engeland, Manon; Tjan-Heijnen, Vivianne C G; Van Neste, Leander; Veeck, Jürgen

    2015-07-01

    Current genome-wide methods to detect DNA-methylation in healthy and diseased tissue require high-quality DNA from fresh-frozen (FF) samples. However, well-annotated clinical samples are mostly available as formalin-fixed, paraffin-embedded (FFPE) tissues containing poor-quality DNA. To overcome this limitation, we here aimed to evaluate a DNA restoration protocol for usage with the genome-wide Infinium HumanMethylation450 BeadChip assay (HM-450K). Sixty-six DNA samples from normal colon (n=9) and breast cancer (n=11) were interrogated separately using HM-450K. Analyses included matched FF/FFPE samples and technical duplicates. FFPE DNA was processed with (FFPEr) or without a DNA restoration protocol (Illumina). Differentially methylated genes were finally validated in 24 additional FFPE tissues using nested methylation-specific PCR (MSP). In summary, β-values correlation between FFPEr duplicates was high (ρ=0.9927 (s.d. ±0.0015)). Matched FF/FFPEr correlation was also high (ρ=0.9590 (s.d. ±0.0184)) compared with matched FF/FFPE (ρ=0.8051 (s.d. ±0.1028). Probe detection rate in FFPEr samples (98.37%, s.d. ±0.66) was comparable to FF samples (99.98%, s.d. ±0.019) and substantially lower in FFPE samples (82.31%, s.d. ±18.65). Assay robustness was not decreased by sample archival age up to 10 years. We could also demonstrate no decrease in assay robustness when using 100 ng of DNA input only. Four out of the five selected differentially methylated genes could be validated by MSP. The gene failing validation by PCR showed high variation of CpG β-values in primer-binding sites. In conclusion, by using the FFPE DNA restoration protocol, HM-450K assays provide robust, accurate and reproducible results with FFPE tissue-derived DNA, which are comparable to those obtained with FF tissue. Most importantly, differentially methylated genes can be validated using more sensitive techniques, such as nested MSP, altogether providing an epigenomics platform for

  6. Formalin-Fixed, Paraffin-Embedded Tissues (FFPE) as a Robust Source for the Profiling of Native and Protease-Generated Protein Amino Termini.

    PubMed

    Lai, Zon Weng; Weisser, Juliane; Nilse, Lars; Costa, Fabrizio; Keller, Eva; Tholen, Martina; Kizhakkedathu, Jayachandran N; Biniossek, Martin; Bronsert, Peter; Schilling, Oliver

    2016-06-01

    Dysregulated proteolysis represents a hallmark of numerous diseases. In recent years, increasing number of studies has begun looking at the protein termini in hope to unveil the physiological and pathological functions of proteases in clinical research. However, the availability of cryopreserved tissue specimens is often limited. Alternatively, formalin-fixed, paraffin-embedded (FFPE) tissues offer an invaluable resource for clinical research. Pathologically relevant tissues are often stored as FFPE, which represent the most abundant resource of archived human specimens. In this study, we established a robust workflow to investigate native and protease-generated protein N termini from FFPE specimens. We demonstrate comparable N-terminomes of cryopreserved and formalin-fixed tissue, thereby showing that formalin fixation/paraffin embedment does not proteolytically damage proteins. Accordingly, FFPE specimens are fully amenable to N-terminal analysis. Moreover, we demonstrate feasibility of FFPE-degradomics in a quantitative N-terminomic study of FFPE liver specimens from cathepsin L deficient or wild-type mice. Using a machine learning approach in combination with the previously determined cathepsin L specificity, we successfully identify a number of potential cathepsin L cleavage sites. Our study establishes FFPE specimens as a valuable alternative to cryopreserved tissues for degradomic studies. PMID:27087653

  7. Analysis of formalin-fixed, paraffin-embedded (FFPE) tissue via proteomic techniques and misconceptions of antigen retrieval.

    PubMed

    O'Rourke, Matthew B; Padula, Matthew P

    2016-01-01

    Since emerging in the late 19(th) century, formaldehyde fixation has become a standard method for preservation of tissues from clinical samples. The advantage of formaldehyde fixation is that fixed tissues can be stored at room temperature for decades without concern for degradation. This has led to the generation of huge tissue banks containing thousands of clinically significant samples. Here we review techniques for proteomic analysis of formalin-fixed, paraffin-embedded (FFPE) tissue samples with a specific focus on the methods used to extract and break formaldehyde crosslinks. We also discuss an error-of-interpretation associated with the technique known as "antigen retrieval." We have discovered that this term has been mistakenly applied to two disparate molecular techniques; therefore, we argue that a terminology change is needed to ensure accurate reporting of experimental results. Finally, we suggest that more investigation is required to fully understand the process of formaldehyde fixation and its subsequent reversal. PMID:27177815

  8. Evaluation of five DNA extraction methods for detection of H. pylori in formalin-fixed paraffin-embedded (FFPE) liver tissue from patients with hepatocellular carcinoma.

    PubMed

    Rabelo-Gonçalves, Elizabeth; Roesler, Bruna; Guardia, Ana Carolina; Milan, Arlete; Hara, Natalicia; Escanhoela, Cecília; Almeida, Jazon; Boin, Ilka; Zeitune, José Murilo

    2014-03-01

    Since Helicobacter spp. DNA was identified in liver tissue resected from patients with hepatocelullar carcinoma (HCC), researchers have suggested a role of this bacterium in hepatic carcinogenesis. Archives of formalin-fixed, paraffin-embedded (FFPE) tissues represent an extraordinary source for clinical studies providing many advantages. However, DNA extraction from FFPE tissues is laborious, time-consuming and still remains a challenge. The aim of this study was to evaluate five protocols for DNA extraction from FFPE liver obtained from patients with HCC in order to detect Helicobacter pylori DNA. These methods were: (1) QIAamp FFPE Tissue Kit, (2) QIAamp DNA Mini Kit, (3) Wizard SV Genomic DNA Purification System, (4) RealiaPrep FFPE gDNA Miniprep System and (5) phenol-chloroform. H. pylori detection was performed using 16S rRNA gene amplification by PCR. The highest total amount of DNA was obtained using the phenol-chloroform method. Analyses of 16S rRNA gene amplification did not show statistically significant differences among the methods (p=0.466), although the highest percentage of positive cases (70%) was found in samples extracted with phenol-chloroform. We suggest that of the five methods evaluated, phenol/chloroform is the most suitable for detection of H. pylori in FFPE liver from patients with HCC.

  9. STR typing of formalin-fixed paraffin embedded (FFPE) aborted foetal tissue in criminal paternity cases.

    PubMed

    Reshef, Ayeleth; Barash, Mark; Voskoboinik, Lev; Brauner, Paul; Gafny, Roni

    2011-03-01

    Sexual assault or rape cases occasionally result in unwanted pregnancies. In almost all such cases the foetus is aborted. A forensic laboratory may receive the foetus, the placenta, or paraffin embedded abortion material for paternity testing. Obtaining a foetal profile DNA from a foetus or placenta may not be successful due to the age or condition of the tissue. Moreover, maternal contamination of placental material will invariably result in a mixed DNA profile. However, the use of properly screened abortion material from paraffin blocks will almost always result in obtaining a foetal DNA profile. Furthermore, foetal tissue fixed in paraffin blocks does not require special conditions for submission and storage as required to preserve fresh foetal or placental tissue. As hospitals routinely prepare foetal tissue in paraffin blocks, which should be readily obtainable by forensic laboratories, these samples would appear to be the preferred choice for paternity testing. PMID:21334577

  10. Pre-Analytical Considerations for Successful Next-Generation Sequencing (NGS): Challenges and Opportunities for Formalin-Fixed and Paraffin-Embedded Tumor Tissue (FFPE) Samples

    PubMed Central

    Arreaza, Gladys; Qiu, Ping; Pang, Ling; Albright, Andrew; Hong, Lewis Z.; Marton, Matthew J.; Levitan, Diane

    2016-01-01

    In cancer drug discovery, it is important to investigate the genetic determinants of response or resistance to cancer therapy as well as factors that contribute to adverse events in the course of clinical trials. Despite the emergence of new technologies and the ability to measure more diverse analytes (e.g., circulating tumor cell (CTC), circulating tumor DNA (ctDNA), etc.), tumor tissue is still the most common and reliable source for biomarker investigation. Because of its worldwide use and ability to preserve samples for many decades at ambient temperature, formalin-fixed, paraffin-embedded tumor tissue (FFPE) is likely to be the preferred choice for tissue preservation in clinical practice for the foreseeable future. Multiple analyses are routinely performed on the same FFPE samples (such as Immunohistochemistry (IHC), in situ hybridization, RNAseq, DNAseq, TILseq, Methyl-Seq, etc.). Thus, specimen prioritization and optimization of the isolation of analytes is critical to ensure successful completion of each assay. FFPE is notorious for producing suboptimal DNA quality and low DNA yield. However, commercial vendors tend to request higher DNA sample mass than what is actually required for downstream assays, which restricts the breadth of biomarker work that can be performed. We evaluated multiple genomics service laboratories to assess the current state of NGS pre-analytical processing of FFPE. Significant differences in pre-analytical capabilities were observed. Key aspects are highlighted and recommendations are made to improve the current practice in translational research. PMID:27657050

  11. The influence of DNA degradation in formalin-fixed, paraffin-embedded (FFPE) tissue on locus-specific methylation assessment by MS-HRM.

    PubMed

    Daugaard, Iben; Kjeldsen, Tina E; Hager, Henrik; Hansen, Lise Lotte; Wojdacz, Tomasz K

    2015-12-01

    Readily accessible formalin-fixed paraffin embedded (FFPE) tissues are a highly valuable source of genetic material for molecular analyses in both research and in vitro diagnostics but frequently genetic material in those samples is highly degraded. With locus-specific methylation changes being widely investigated for use as biomarkers in various aspects of clinical disease management, we aimed to evaluate to what extent standard laboratory procedures can approximate the quality of the DNA extracted from FFPE samples prior to methylation analyses. DNA quality in 107 FFPE non-small cell lung cancer (NSCLC) samples was evaluated using spectrophotometry and gel electrophoresis. Subsequently, the quality assessment results were correlated with the results of locus specific methylation assessment with methylation sensitive high resolution melting (MS-HRM). The correlation of template quality with PCR amplification performance and HRM based methylation detection indicated a significant influence of DNA quality on PCR amplification but not on methylation assessment. In conclusion, standard laboratory procedures fairly well approximate DNA degradation of FFPE samples and DNA degradation does not seem to considerably affect locus-specific methylation assessment by MS-HRM. PMID:26551081

  12. The influence of DNA degradation in formalin-fixed, paraffin-embedded (FFPE) tissue on locus-specific methylation assessment by MS-HRM.

    PubMed

    Daugaard, Iben; Kjeldsen, Tina E; Hager, Henrik; Hansen, Lise Lotte; Wojdacz, Tomasz K

    2015-12-01

    Readily accessible formalin-fixed paraffin embedded (FFPE) tissues are a highly valuable source of genetic material for molecular analyses in both research and in vitro diagnostics but frequently genetic material in those samples is highly degraded. With locus-specific methylation changes being widely investigated for use as biomarkers in various aspects of clinical disease management, we aimed to evaluate to what extent standard laboratory procedures can approximate the quality of the DNA extracted from FFPE samples prior to methylation analyses. DNA quality in 107 FFPE non-small cell lung cancer (NSCLC) samples was evaluated using spectrophotometry and gel electrophoresis. Subsequently, the quality assessment results were correlated with the results of locus specific methylation assessment with methylation sensitive high resolution melting (MS-HRM). The correlation of template quality with PCR amplification performance and HRM based methylation detection indicated a significant influence of DNA quality on PCR amplification but not on methylation assessment. In conclusion, standard laboratory procedures fairly well approximate DNA degradation of FFPE samples and DNA degradation does not seem to considerably affect locus-specific methylation assessment by MS-HRM.

  13. Methods for Studying MicroRNA Expression and Their Targets in Formalin-Fixed, Paraffin-Embedded (FFPE) Breast Cancer Tissues.

    PubMed

    Gomes, Bruno Costa; Santos, Bruno; Rueff, José; Rodrigues, António Sebastião

    2016-01-01

    Drug resistance remains a burden in cancer treatment. In the past few years molecular genetics brought a new hope with personalized therapy. This individual approach allows the identification of genetic profiles that will respond better to a given treatment and consequently get a better outcome. Recently, physicians received an extra aid with the approval of molecular tools based on gene expression signatures. With these tools, physicians have the capacity to identify the probability of disease recurrence in the first 5 years following diagnosis, a fact that is essential for a more effective adjuvant therapy administration. However, some patients still relapse and acquire drug resistance and aggressive tumors. For that reason, a comprehensive understanding of the molecular players in drug resistance is of extreme importance. MicroRNAs have been described as regulators of various cellular pathways and as predictive and prognostic factors. As broad regulators, microRNAs also interfere with drug metabolism and drug targets. Thus it is of paramount importance to understand which microRNAs are deregulated in breast cancer and try to relate this misexpression with resistance to therapeutics, poor outcomes, and survival. Here, we describe a possible approach to study microRNA expression and respective targets from formalin-fixed, paraffin-embedded (FFPE) breast cancer tissues. FFPE tissues are regularly archived for long periods in pathology departments, and microRNAs are well conserved in these tissues. PMID:26910075

  14. [Use of archival formalin-fixed, paraffin-embedded (FFPE) tissue samples for molecular genetic analysis in diffuse large B-cell lymphoma (DLBCL)].

    PubMed

    Jarošová, Marie; Kučerová, Jana; Flodr, Patrik; Mikešová, Michaela; Procházka, Vít; Papajík, Tomáš

    2014-04-01

    The currently valid molecular genetic subclassification of patients with diffuse large B-cell lymphoma (DLBCL) into three prognostic subgroups based on expression profiling has been the objective of numerous genetic studies. In routine clinical practice, however, expression profiling technology remains unavailable for the most of centers. Apart from the technology, in some cases molecular genetic laboratories have problems obtaining high-quality material, i.e. fresh tissues, for RNA isolation to determine gene expression. One possibility is to determine the gene expression from RNA obtained by isolation from formalin-fixed, paraffin-embedded (FFPE) tissue. This pilot study aimed at isolating RNA from FFPE in patients diagnosed with DLBCL and verifying the potential use of such RNA for the expression analysis of 7 selected genes. Although the study showed that it is possible to isolate RNA and determine the expression of the selected genes from archival material, the values of relative expression of some genes in the set were too variable to be used for unambiguous prognostic classification. It was confirmed that retrospective analyses of selected genes may be performed with sufficient material obtained, and that properly archived blocks may be used for molecular biology analyses even after 8 years.

  15. Evaluation of Protein Profiles From Treated Xenograft Tumor Models Identifies an Antibody Panel for Formalin-fixed and Paraffin-embedded (FFPE) Tissue Analysis by Reverse Phase Protein Arrays (RPPA)*

    PubMed Central

    Bader, Sabine; Zajac, Magdalena; Friess, Thomas; Ruge, Elisabeth; Rieder, Natascha; Gierke, Berthold; Heubach, Yvonne; Thomas, Marlene; Pawlak, Michael

    2015-01-01

    Reverse phase protein arrays (RPPA) are an established tool for measuring the expression and activation status of multiple proteins in parallel using only very small amounts of tissue. Several studies have demonstrated the value of this technique for signaling pathway analysis using proteins extracted from fresh frozen (FF) tissue in line with validated antibodies for this tissue type; however, formalin fixation and paraffin embedding (FFPE) is the standard method for tissue preservation in the clinical setting. Hence, we performed RPPA to measure profiles for a set of 300 protein markers using matched FF and FFPE tissue specimens to identify which markers performed similarly using the RPPA technique in fixed and unfixed tissues. Protein lysates were prepared from matched FF and FFPE tissue specimens of individual tumors taken from three different xenograft models of human cancer. Materials from both untreated mice and mice treated with either anti-HER3 or bispecific anti-IGF-1R/EGFR monoclonal antibodies were analyzed. Correlations between signals from FF and FFPE tissue samples were investigated. Overall, 60 markers were identified that produced comparable profiles between FF and FFPE tissues, demonstrating significant correlation between the two sample types. The top 25 markers also showed significance after correction for multiple testing. The panel of markers covered several clinically relevant tumor signaling pathways and both phosphorylated and nonphosphorylated proteins were represented. Biologically relevant changes in marker expression were noted when RPPA profiles from treated and untreated xenografts were compared. These data demonstrate that, using appropriately selected antibodies, RPPA analysis from FFPE tissue is well feasible and generates biologically meaningful information. The identified panel of markers that generate similar profiles in matched fixed and unfixed tissue samples may be clinically useful for pharmacodynamic studies of drug effect

  16. Evaluation of Protein Profiles From Treated Xenograft Tumor Models Identifies an Antibody Panel for Formalin-fixed and Paraffin-embedded (FFPE) Tissue Analysis by Reverse Phase Protein Arrays (RPPA).

    PubMed

    Bader, Sabine; Zajac, Magdalena; Friess, Thomas; Ruge, Elisabeth; Rieder, Natascha; Gierke, Berthold; Heubach, Yvonne; Thomas, Marlene; Pawlak, Michael

    2015-10-01

    Reverse phase protein arrays (RPPA) are an established tool for measuring the expression and activation status of multiple proteins in parallel using only very small amounts of tissue. Several studies have demonstrated the value of this technique for signaling pathway analysis using proteins extracted from fresh frozen (FF) tissue in line with validated antibodies for this tissue type; however, formalin fixation and paraffin embedding (FFPE) is the standard method for tissue preservation in the clinical setting. Hence, we performed RPPA to measure profiles for a set of 300 protein markers using matched FF and FFPE tissue specimens to identify which markers performed similarly using the RPPA technique in fixed and unfixed tissues. Protein lysates were prepared from matched FF and FFPE tissue specimens of individual tumors taken from three different xenograft models of human cancer. Materials from both untreated mice and mice treated with either anti-HER3 or bispecific anti-IGF-1R/EGFR monoclonal antibodies were analyzed. Correlations between signals from FF and FFPE tissue samples were investigated. Overall, 60 markers were identified that produced comparable profiles between FF and FFPE tissues, demonstrating significant correlation between the two sample types. The top 25 markers also showed significance after correction for multiple testing. The panel of markers covered several clinically relevant tumor signaling pathways and both phosphorylated and nonphosphorylated proteins were represented. Biologically relevant changes in marker expression were noted when RPPA profiles from treated and untreated xenografts were compared. These data demonstrate that, using appropriately selected antibodies, RPPA analysis from FFPE tissue is well feasible and generates biologically meaningful information. The identified panel of markers that generate similar profiles in matched fixed and unfixed tissue samples may be clinically useful for pharmacodynamic studies of drug effect

  17. Quantification of HER2 by Targeted Mass Spectrometry in Formalin-Fixed Paraffin-Embedded (FFPE) Breast Cancer Tissues.

    PubMed

    Steiner, Carine; Tille, Jean-Christophe; Lamerz, Jens; Kux van Geijtenbeek, Sabine; McKee, Thomas A; Venturi, Miro; Rubbia-Brandt, Laura; Hochstrasser, Denis; Cutler, Paul; Lescuyer, Pierre; Ducret, Axel

    2015-10-01

    The ability to accurately quantify proteins in formalin-fixed paraffin-embedded tissues using targeted mass spectrometry opens exciting perspectives for biomarker discovery. We have developed and evaluated a selectedreaction monitoring assay for the human receptor tyrosine-protein kinase erbB-2 (HER2) in formalin-fixed paraffin-embedded breast tumors. Peptide candidates were identified using an untargeted mass spectrometry approach in relevant cell lines. A multiplexed assay was developed for the six best candidate peptides and evaluated for linearity, precision and lower limit of quantification. Results showed a linear response over a calibration range of 0.012 to 100 fmol on column (R(2): 0.99-1.00).The lower limit of quantification was 0.155 fmol on column for all peptides evaluated. The six HER2 peptides were quantified by selected reaction monitoring in a cohort of 40 archival formalin-fixed paraffin-embedded tumor tissues from women with invasive breast carcinomas, which showed different levels of HER2 gene amplification as assessed by standard methods used in clinical pathology. The amounts of the six HER2 peptides were highly and significantly correlated with each other, indicating that peptide levels can be used as surrogates of protein amounts in formalin-fixed paraffin-embedded tissues. After normalization for sample size, selected reaction monitoring peptide measurements were able to correctly predict 90% of cases based on HER2 amplification as defined by the American Society of Clinical Oncology and College of American Pathologists. In conclusion, the developed assay showed good analytical performance and a high agreement with immunohistochemistry and fluorescence in situ hybridization data. This study demonstrated that selected reaction monitoring allows to accurately quantify protein expression in formalin-fixed paraffin-embedded tissues and represents therefore a powerful approach for biomarker discovery studies. The untargeted mass spectrometry

  18. Quantification of HER2 by Targeted Mass Spectrometry in Formalin-Fixed Paraffin-Embedded (FFPE) Breast Cancer Tissues.

    PubMed

    Steiner, Carine; Tille, Jean-Christophe; Lamerz, Jens; Kux van Geijtenbeek, Sabine; McKee, Thomas A; Venturi, Miro; Rubbia-Brandt, Laura; Hochstrasser, Denis; Cutler, Paul; Lescuyer, Pierre; Ducret, Axel

    2015-10-01

    The ability to accurately quantify proteins in formalin-fixed paraffin-embedded tissues using targeted mass spectrometry opens exciting perspectives for biomarker discovery. We have developed and evaluated a selectedreaction monitoring assay for the human receptor tyrosine-protein kinase erbB-2 (HER2) in formalin-fixed paraffin-embedded breast tumors. Peptide candidates were identified using an untargeted mass spectrometry approach in relevant cell lines. A multiplexed assay was developed for the six best candidate peptides and evaluated for linearity, precision and lower limit of quantification. Results showed a linear response over a calibration range of 0.012 to 100 fmol on column (R(2): 0.99-1.00).The lower limit of quantification was 0.155 fmol on column for all peptides evaluated. The six HER2 peptides were quantified by selected reaction monitoring in a cohort of 40 archival formalin-fixed paraffin-embedded tumor tissues from women with invasive breast carcinomas, which showed different levels of HER2 gene amplification as assessed by standard methods used in clinical pathology. The amounts of the six HER2 peptides were highly and significantly correlated with each other, indicating that peptide levels can be used as surrogates of protein amounts in formalin-fixed paraffin-embedded tissues. After normalization for sample size, selected reaction monitoring peptide measurements were able to correctly predict 90% of cases based on HER2 amplification as defined by the American Society of Clinical Oncology and College of American Pathologists. In conclusion, the developed assay showed good analytical performance and a high agreement with immunohistochemistry and fluorescence in situ hybridization data. This study demonstrated that selected reaction monitoring allows to accurately quantify protein expression in formalin-fixed paraffin-embedded tissues and represents therefore a powerful approach for biomarker discovery studies. The untargeted mass spectrometry

  19. Molecular identification of Coccidioides immitis in formalin-fixed, paraffin-embedded (FFPE) tissues from a Colombian patient.

    PubMed

    Canteros, Cristina E; Vélez H, Alejandro; Toranzo, Adriana I; Suárez-Alvarez, Roberto; Tobón O, Ángela; Jimenez A, María del Pilar; Restrepo M, Ángela

    2015-06-01

    Coccidioides immitis and C. posadasii are the etiologic agents of coccidioidomycosis, an endemic fungal disease of the Americas. In Colombia, this mycosis is uncommon, and only five cases, two of them imported, have been documented.By means of DNA sequencing, C. immitis was identified in formalin-fixed, paraffin-embedded archival tissues samples from the 5th Colombian patient diagnosed in 1997. The patient was born in Pinto, Department of Magdalena, and had never visited other geographic regions, a reason to consider that the mycosis had been acquired locally.This species is primarily found in California although it has been occasionally reported in other geographic areas such as Mexico and Brazil. This is the first indigenous report of C. immitis-associated coccidioidomycosis in a Colombian patient.

  20. Regulatory and ethical issues on the utilization of FFPE tissues in research.

    PubMed

    With, Catherine M; Evers, David L; Mason, Jeffrey T

    2011-01-01

    Formalin-fixed, paraffin-embedded (FFPE) archival tissues and their associated diagnostic records represent an invaluable source of information on diseases where the patient outcomes are already known. Older archives contain many unique FFPE tissue specimens that would be impossible to replicate today due to changes in medical practice and technology. Unfortunately, there is no single regulatory or bioethical standard that covers research with FFPE tissue specimens. This makes it difficult for researchers to prepare protocols involving FFPE tissues and equally difficult for Institutional Review Boards to evaluate them. In this review, focused on US regulatory policy, the application of the Common Rule and the Privacy Rule of the Health Insurance Portability and Accountability Act to research involving FFPE tissue specimens will be discussed. It will be shown that the difficulty in applying regulatory and ethical standards to FFPE tissues results not from the tissues themselves, but from the personally identifiable health information associated with the tissue specimens.

  1. Multicentre validation study of nucleic acids extraction from FFPE tissues.

    PubMed

    Bonin, Serena; Hlubek, Falk; Benhattar, Jean; Denkert, Carsten; Dietel, Manfred; Fernandez, Pedro L; Höfler, Gerald; Kothmaier, Hannelore; Kruslin, Bozo; Mazzanti, Chiara Maria; Perren, Aurel; Popper, Helmuth; Scarpa, Aldo; Soares, Paula; Stanta, Giorgio; Groenen, Patricia J T A

    2010-09-01

    In most pathology laboratories worldwide, formalin-fixed paraffin embedded (FFPE) samples are the only tissue specimens available for routine diagnostics. Although commercial kits for diagnostic molecular pathology testing are becoming available, most of the current diagnostic tests are laboratory-based assays. Thus, there is a need for standardized procedures in molecular pathology, starting from the extraction of nucleic acids. To evaluate the current methods for extracting nucleic acids from FFPE tissues, 13 European laboratories, participating to the European FP6 program IMPACTS (www.impactsnetwork.eu), isolated nucleic acids from four diagnostic FFPE tissues using their routine methods, followed by quality assessment. The DNA-extraction protocols ranged from homemade protocols to commercial kits. Except for one homemade protocol, the majority gave comparable results in terms of the quality of the extracted DNA measured by the ability to amplify differently sized control gene fragments by PCR. For array-applications or tests that require an accurately determined DNA-input, we recommend using silica based adsorption columns for DNA recovery. For RNA extractions, the best results were obtained using chromatography column based commercial kits, which resulted in the highest quantity and best assayable RNA. Quality testing using RT-PCR gave successful amplification of 200 bp-250 bp PCR products from most tested tissues. Modifications of the proteinase-K digestion time led to better results, even when commercial kits were applied. The results of the study emphasize the need for quality control of the nucleic acid extracts with standardised methods to prevent false negative results and to allow data comparison among different diagnostic laboratories.

  2. Nucleic acids from long-term preserved FFPE tissues are suitable for downstream analyses.

    PubMed

    Ludyga, Natalie; Grünwald, Barbara; Azimzadeh, Omid; Englert, Sonja; Höfler, Heinz; Tapio, Soile; Aubele, Michaela

    2012-02-01

    Tissues used for clinical diagnostics are mostly formalin-fixed and paraffin-embedded (FFPE) which provides many advantages. However, the quality of the obtained nucleic acids (NA) is reduced and this turns out to be a challenge for further molecular analyses. Although the spectrum of analyses of NA extracted from FFPE tissue has increased, the standard operating procedures for NA isolation from old tissue blocks still need to be improved. Here, we compared the efficiency of different NA extraction methods, using FFPE tissues of variable age and origin, with respect to downstream analyses. Our study showed that the phenol-chloroform isoamyl alcohol (PCI) and the commercial Qiagen protocol yielded samples with highest purity. The PCI protocol delivered the longest amplicons even from samples from the 1970s. We developed a short (1 h) tissue lysis procedure that turned out to be highly time- and cost-effective when DNA quality was tested using single and multiplex PCR. Compared to a 1-day lysis-protocol, the amplicons were only 100 bp shorter. In addition, single-copy genes used in daily routine were successfully amplified from long-term stored FFPE samples following 1-h tissue-lysis. The RNA integrity numbers (RIN) determined on RNA isolated from FFPE tissues indicated degraded RNA; however, all RINs were above the generally agreed threshold of 1.4. We showed that, depending on the purpose of the analysis, NA retrieved from FFPE tissues older than 40 years may be successfully used for molecular analysis.

  3. Evaluation of frozen tissue-derived prognostic gene expression signatures in FFPE colorectal cancer samples

    PubMed Central

    Zhu, Jing; Deane, Natasha G.; Lewis, Keeli B.; Padmanabhan, Chandrasekhar; Washington, M. Kay; Ciombor, Kristen K.; Timmers, Cynthia; Goldberg, Richard M.; Beauchamp, R. Daniel; Chen, Xi

    2016-01-01

    Defining molecular features that can predict the recurrence of colorectal cancer (CRC) for stage II-III patients remains challenging in cancer research. Most available clinical samples are Formalin-Fixed, Paraffin-Embedded (FFPE). NanoString nCounter® and Affymetrix GeneChip® Human Transcriptome Array 2.0 (HTA) are the two platforms marketed for high-throughput gene expression profiling for FFPE samples. In this study, to evaluate the gene expression of frozen tissue-derived prognostic signatures in FFPE CRC samples, we evaluated the expression of 516 genes from published frozen tissue-derived prognostic signatures in 42 FFPE CRC samples measured by both platforms. Based on HTA platform-derived data, we identified both gene (99 individual genes, FDR < 0.05) and gene set (four of the six reported multi-gene signatures with sufficient information for evaluation, P < 0.05) expression differences associated with survival outcomes. Using nCounter platform-derived data, one of the six multi-gene signatures (P < 0.05) but no individual gene was associated with survival outcomes. Our study indicated that sufficiently high quality RNA could be obtained from FFPE tumor tissues to detect frozen tissue-derived prognostic gene expression signatures for CRC patients. PMID:27623752

  4. MALDI direct analysis and imaging of frozen versus FFPE tissues: what strategy for which sample?

    PubMed

    Wisztorski, Maxence; Franck, Julien; Salzet, Michel; Fournier, Isabelle

    2010-01-01

    Significant advances have been made in the past decade in the field of mass spectrometry imaging with MALDI ion sources (MALDI-MSI). While MALDI-MSI has high potential in the field of biology and in the clinic, a challenge for MALDI-MSI has been to adapt itself to a greater range of sample types. In particular, much of the biological archived materials for pathology studies are tissue biopsies fixed with paraformaldehyde and embedded in paraffin (FFPE tissues) because of the high stability of such samples. Thus, there has been a need to develop strategies for analyzing FFPE samples as this would allow retrospective studies of past clinical cases on large cohorts of existing samples. Obviously, PAF fixation, by inducing protein cross-linking, causes problems for molecular analysis by MS. We developed on tissue digestion strategies for overcoming these difficulties and allowing molecular data to be retrieved from FFPE samples no matter how long they have been stored. These digestion strategies preserve localization from digested proteins making MALDI-MSI of proteins possible by monitoring the resulting peptides. We present methods and protocols for FFPE samples. These strategies have proven to be valuable for all tested FFPE samples and have opened archived tissues from hospital banks to MALDI-MSI.

  5. Evaluation of frozen tissue-derived prognostic gene expression signatures in FFPE colorectal cancer samples.

    PubMed

    Zhu, Jing; Deane, Natasha G; Lewis, Keeli B; Padmanabhan, Chandrasekhar; Washington, M Kay; Ciombor, Kristen K; Timmers, Cynthia; Goldberg, Richard M; Beauchamp, R Daniel; Chen, Xi

    2016-01-01

    Defining molecular features that can predict the recurrence of colorectal cancer (CRC) for stage II-III patients remains challenging in cancer research. Most available clinical samples are Formalin-Fixed, Paraffin-Embedded (FFPE). NanoString nCounter® and Affymetrix GeneChip® Human Transcriptome Array 2.0 (HTA) are the two platforms marketed for high-throughput gene expression profiling for FFPE samples. In this study, to evaluate the gene expression of frozen tissue-derived prognostic signatures in FFPE CRC samples, we evaluated the expression of 516 genes from published frozen tissue-derived prognostic signatures in 42 FFPE CRC samples measured by both platforms. Based on HTA platform-derived data, we identified both gene (99 individual genes, FDR < 0.05) and gene set (four of the six reported multi-gene signatures with sufficient information for evaluation, P < 0.05) expression differences associated with survival outcomes. Using nCounter platform-derived data, one of the six multi-gene signatures (P < 0.05) but no individual gene was associated with survival outcomes. Our study indicated that sufficiently high quality RNA could be obtained from FFPE tumor tissues to detect frozen tissue-derived prognostic gene expression signatures for CRC patients. PMID:27623752

  6. High-Throughput Profiling of FFPE Tissue Using Parallel Electrophoresis and MALDI MS

    PubMed Central

    Aerni, Hans-Rudolf; Cornett, Dale S.

    2009-01-01

    Analysis of formalin-fixed and paraffin-embedded tissues (FFPE) is increasingly recognized as a strategy for the discovery and validation of clinically useful biomarker candidates. Large tissue collections including tissue microarrays (TMA) are available but current analytical strategies for their characterization have limited throughput. In this report, we describe a workflow for rapid analysis of hundreds of FFPE tissue specimens. The strategy combines parallel sample processing and on-chip electrophoresis with automated MALDI MS analysis. The method is optimized for small quantities of clinically valuable tissues allowing detection of hundreds of peptides from a single core in a TMA section. We describe results from the optimization of the method and apply it for the analysis of tissue microarrays containing formalin fixed tissue specimens from human kidney. PMID:19650658

  7. MicroRNA Stability in FFPE Tissue Samples: Dependence on GC Content

    PubMed Central

    Kakimoto, Yu; Tanaka, Masayuki; Kamiguchi, Hiroshi; Ochiai, Eriko; Osawa, Motoki

    2016-01-01

    MicroRNAs (miRNAs) are small non-coding RNAs responsible for fine-tuning of gene expression at post-transcriptional level. The alterations in miRNA expression levels profoundly affect human health and often lead to the development of severe diseases. Currently, high throughput analyses, such as microarray and deep sequencing, are performed in order to identify miRNA biomarkers, using archival patient tissue samples. MiRNAs are more robust than longer RNAs, and resistant to extreme temperatures, pH, and formalin-fixed paraffin-embedding (FFPE) process. Here, we have compared the stability of miRNAs in FFPE cardiac tissues using next-generation sequencing. The mode read length in FFPE samples was 11 nucleotides (nt), while that in the matched frozen samples was 22 nt. Although the read counts were increased 1.7-fold in FFPE samples, compared with those in the frozen samples, the average miRNA mapping rate decreased from 32.0% to 9.4%. These results indicate that, in addition to the fragmentation of longer RNAs, miRNAs are to some extent degraded in FFPE tissues as well. The expression profiles of total miRNAs in two groups were highly correlated (0.88 FFPE cardiac tissues instead of miR-1, which was predominant before fixation. Subsequent quantitative PCR (qPCR) analyses revealed that miRNAs with GC content of less than 40% are more degraded than GC-rich miRNAs (p<0.0001). We showed that deep sequencing data obtained using FFPE samples cannot be directly compared with that of fresh frozen samples. The combination of miRNA deep sequencing and other quantitative analyses, such as qPCR, may improve the utility of archival FFPE tissue samples. PMID:27649415

  8. MicroRNA Stability in FFPE Tissue Samples: Dependence on GC Content.

    PubMed

    Kakimoto, Yu; Tanaka, Masayuki; Kamiguchi, Hiroshi; Ochiai, Eriko; Osawa, Motoki

    2016-01-01

    MicroRNAs (miRNAs) are small non-coding RNAs responsible for fine-tuning of gene expression at post-transcriptional level. The alterations in miRNA expression levels profoundly affect human health and often lead to the development of severe diseases. Currently, high throughput analyses, such as microarray and deep sequencing, are performed in order to identify miRNA biomarkers, using archival patient tissue samples. MiRNAs are more robust than longer RNAs, and resistant to extreme temperatures, pH, and formalin-fixed paraffin-embedding (FFPE) process. Here, we have compared the stability of miRNAs in FFPE cardiac tissues using next-generation sequencing. The mode read length in FFPE samples was 11 nucleotides (nt), while that in the matched frozen samples was 22 nt. Although the read counts were increased 1.7-fold in FFPE samples, compared with those in the frozen samples, the average miRNA mapping rate decreased from 32.0% to 9.4%. These results indicate that, in addition to the fragmentation of longer RNAs, miRNAs are to some extent degraded in FFPE tissues as well. The expression profiles of total miRNAs in two groups were highly correlated (0.88 FFPE cardiac tissues instead of miR-1, which was predominant before fixation. Subsequent quantitative PCR (qPCR) analyses revealed that miRNAs with GC content of less than 40% are more degraded than GC-rich miRNAs (p<0.0001). We showed that deep sequencing data obtained using FFPE samples cannot be directly compared with that of fresh frozen samples. The combination of miRNA deep sequencing and other quantitative analyses, such as qPCR, may improve the utility of archival FFPE tissue samples. PMID:27649415

  9. Gene expression profiling of RNA extracted from FFPE tissues: NuGEN technologies' whole-transcriptome amplification system.

    PubMed

    Turner, Leah; Heath, Joe Don; Kurn, Nurith

    2011-01-01

    Gene expression profiling of RNA isolated from formalin fixed, paraffin-embedded (FFPE) tissue samples has been historically challenging. Yet FFPE samples are sought-after because of the in-depth retrospective records typically associated with them rendering these samples a valuable resource for translational medicine studies. Extensive degradation, chemical modifications, and cross-linking have made it difficult to isolate RNA of sufficient quality required for large-scale gene expression profiling studies. NuGEN Technologies' WT-Ovation™ FFPE System linearly amplifies RNA from FFPE samples through a robust and simple whole-transcriptome approach using as little as 50 ng total RNA isolated from FFPE samples. The amplified material may be labeled with validated kits and/or protocols from NuGEN for analysis on any of the major gene expression microarray platforms, including: Affymetrix, Agilent, and Illumina gene expression arrays. Results compare well with those obtained using RNA from fresh-frozen samples. RNA quality from FFPE samples varies significantly and neither sample age nor sample size analysis via gel electrophoresis or the Agilent Bioanalyzer system accurately predict materials suitable for amplification. Therefore, NuGEN has validated a correlative qPCR-based analytical method for the RNA derived from FFPE samples which effectively predicts array results. The NuGEN approach enables fast and successful analysis of samples previously thought to be too degraded for gene expression analysis.

  10. Dose-Response Analysis of RNA-Seq Profiles in Archival Formalin-fixed paraffin-embedded (FFPE) Samples

    EPA Science Inventory

    Formalin-fixed paraffin-embedded (FFPE) samples provide a vast untapped resource for chemical safety and translational science. To date, genomic profiling of FFPE samples has been limited by poor RNA quality and inconsistent results with limited utility in dose-response assessmen...

  11. Complete solubilization of formalin-fixed, paraffin-embedded tissue may improve proteomic studies.

    PubMed

    Shi, Shan-Rong; Taylor, Clive R; Fowler, Carol B; Mason, Jeffrey T

    2013-04-01

    Tissue-based proteomic approaches (tissue proteomics) are essential for discovering and evaluating biomarkers for personalized medicine. In any proteomics study, the most critical issue is sample extraction and preparation. This problem is especially difficult when recovering proteins from formalin-fixed, paraffin-embedded (FFPE) tissue sections. However, improving and standardizing protein extraction from FFPE tissue is a critical need because of the millions of archival FFPE tissues available in tissue banks worldwide. Recent progress in the application of heat-induced antigen retrieval principles for protein extraction from FFPE tissue has resulted in a number of published FFPE tissue proteomics studies. However, there is currently no consensus on the optimal protocol for protein extraction from FFPE tissue or accepted standards for quantitative evaluation of the extracts. Standardization is critical to ensure the accurate evaluation of FFPE protein extracts by proteomic methods such as reverse phase protein arrays, which is now in clinical use. In our view, complete solubilization of FFPE tissue samples is the best way to achieve the goal of standardizing the recovery of proteins from FFPE tissues. However, further studies are recommended to develop standardized protein extraction methods to ensure quantitative and qualitative reproducibility in the recovery of proteins from FFPE tissues.

  12. Comparison of Nanostring nCounter® Data on FFPE Colon Cancer Samples and Affymetrix Microarray Data on Matched Frozen Tissues.

    PubMed

    Chen, Xi; Deane, Natasha G; Lewis, Keeli B; Li, Jiang; Zhu, Jing; Washington, M Kay; Beauchamp, R Daniel

    2016-01-01

    The prognosis of colorectal cancer (CRC) stage II and III patients remains a challenge due to the difficulties of finding robust biomarkers suitable for testing clinical samples. The majority of published gene signatures of CRC have been generated on fresh frozen colorectal tissues. Because collection of frozen tissue is not practical for routine surgical pathology practice, a clinical test that improves prognostic capabilities beyond standard pathological staging of colon cancer will need to be designed for formalin-fixed paraffin-embedded (FFPE) tissues. The NanoString nCounter® platform is a gene expression analysis tool developed for use with FFPE-derived samples. We designed a custom nCounter® codeset based on elements from multiple published fresh frozen tissue microarray-based prognostic gene signatures for colon cancer, and we used this platform to systematically compare gene expression data from FFPE with matched microarray array data from frozen tissues. Our results show moderate correlation of gene expression between two platforms and discovery of a small subset of genes as candidate biomarkers for colon cancer prognosis that are detectable and quantifiable in FFPE tissue sections. PMID:27176004

  13. Precision of Multiple Reaction Monitoring Mass Spectrometry Analysis of Formalin-Fixed, Paraffin-Embedded Tissue

    PubMed Central

    2012-01-01

    We compared the reproducibility of multiple reaction monitoring (MRM) mass spectrometry-based peptide quantitation in tryptic digests from formalin-fixed, paraffin-embedded (FFPE) and frozen clear cell renal cell carcinoma tissues. The analyses targeted a candidate set of 114 peptides previously identified in shotgun proteomic analyses, of which 104 were detectable in FFPE and frozen tissue. Although signal intensities for MRM of peptides from FFPE tissue were on average 66% of those in frozen tissue, median coefficients of variation (CV) for measurements in FFPE and frozen tissues were nearly identical (18–20%). Measurements of lysine C-terminal peptides and arginine C-terminal peptides from FFPE tissue were similarly reproducible (19.5% and 18.3% median CV, respectively). We further evaluated the precision of MRM-based quantitation by analysis of peptides from the Her2 receptor in FFPE and frozen tissues from a Her2 overexpressing mouse xenograft model of breast cancer and in human FFPE breast cancer specimens. We obtained equivalent MRM measurements of HER2 receptor levels in FFPE and frozen mouse xenografts derived from HER2-overexpressing BT474 cells and HER2-negative Sum159 cells. MRM analyses of 5 HER2-positive and 5 HER-negative human FFPE breast tumors confirmed the results of immunohistochemical analyses, thus demonstrating the feasibility of HER2 protein quantification in FFPE tissue specimens. The data demonstrate that MRM analyses can be performed with equal precision on FFPE and frozen tissues and that lysine-containing peptides can be selected for quantitative comparisons, despite the greater impact of formalin fixation on lysine residues. The data further illustrate the feasibility of applying MRM to quantify clinically important tissue biomarkers in FFPE specimens. PMID:22530795

  14. Toward improving the proteomic analysis of formalin-fixed, paraffin-embedded tissue.

    PubMed

    Fowler, Carol B; O'Leary, Timothy J; Mason, Jeffrey T

    2013-08-01

    Archival formalin-fixed, paraffin-embedded (FFPE) tissue and their associated diagnostic records represent an invaluable source of retrospective proteomic information on diseases for which the clinical outcome and response to treatment are known. However, analysis of archival FFPE tissues by high-throughput proteomic methods has been hindered by the adverse effects of formaldehyde fixation and subsequent tissue histology. This review examines recent methodological advances for extracting proteins from FFPE tissue suitable for proteomic analysis. These methods, based largely upon heat-induced antigen retrieval techniques borrowed from immunohistochemistry, allow at least a qualitative analysis of the proteome of FFPE archival tissues. The authors also discuss recent advances in the proteomic analysis of FFPE tissue; including liquid-chromatography tandem mass spectrometry, reverse phase protein microarrays and imaging mass spectrometry.

  15. Proteomic analysis of RCL2 paraffin-embedded tissues.

    PubMed

    Bellet, V; Boissière, F; Bibeau, F; Desmetz, C; Berthe, M L; Rochaix, P; Maudelonde, T; Mangè, A; Solassol, J

    2008-10-01

    Histopathological diagnosis in most of the world's hospitals is based upon formalin-fixed and paraffin-embedded (FFPE) tissues. Although this standard fixation and embedding procedure keeps the tissue in excellent form for morphological and immunohistological analysis, FFPE is inappropriate for nucleic acids and protein studies. We investigated the potential value of RCL2, a new non-toxic fixative, for sparing proteins preserved in paraffin-embedded tissues. Normal colonic mucosa tissue was fixed in RCL2 prior to paraffin embedding (RCL2P), and then processed for quality and quantity of protein conservation, as compared to frozen and FFPE tissues using complementary proteomic analysis approaches. Using 4 different protein extraction protocols, RCL2P tissue consistently showed the highest protein yield. Similar protein patterns were observed with RCL2P and frozen tissues using mono and bi-dimensional electrophoresis. Moreover, membrane, cytoplasmic and nuclear proteins, as well as phosphorylated proteins, were successfully detected using western-blot. Furthermore, protein patterns observed by mass spectrometry analysis after laser-captured microdissection were found to be identical for frozen and RCL2-fixed tissues. At last, immunohistochemistry using various antibodies showed comparable results between both tissue storage methods. We concluded that RCL2 has great potential for performing both morphological and molecular analyses on the same archival paraffin-embedded tissue sample, and can be a new method for investigating protein biomarkers.

  16. Proteomic analysis of RCL2 paraffin-embedded tissues

    PubMed Central

    Bellet, V; Boissière, F; Bibeau, F; Desmetz, C; Berthe, M L; Rochaix, P; Maudelonde, T; Mangè, A; Solassol, J

    2008-01-01

    Histopathological diagnosis in most of the world's hospitals is based upon formalin-fixed and paraffin-embedded (FFPE) tissues. Although this standard fixation and embedding procedure keeps the tissue in excellent form for morphological and immunohistological analysis, FFPE is inappropriate for nucleic acids and protein studies. We investigated the potential value of RCL2, a new non-toxic fixative, for sparing proteins preserved in paraffin-embedded tissues. Normal colonic mucosa tissue was fixed in RCL2 prior to paraffin embedding (RCL2P), and then processed for quality and quantity of protein conservation, as compared to frozen and FFPE tissues using complementary proteomic analysis approaches. Using 4 different protein extraction protocols, RCL2P tissue consistently showed the highest protein yield. Similar protein patterns were observed with RCL2P and frozen tissues using mono and bi-dimensional electrophoresis. Moreover, membrane, cytoplasmic and nuclear proteins, as well as phosphorylated proteins, were successfully detected using western-blot. Furthermore, protein patterns observed by mass spectrometry analysis after laser-captured microdissection were found to be identical for frozen and RCL2-fixed tissues. At last, immunohistochemistry using various antibodies showed comparable results between both tissue storage methods. We concluded that RCL2 has great potential for performing both morphological and molecular analyses on the same archival paraffin-embedded tissue sample, and can be a new method for investigating protein biomarkers. PMID:19012729

  17. Data analysis considerations for detecting copy number changes in formalin-fixed, paraffin-embedded tissues.

    PubMed

    Jacobs, Sharoni

    2012-11-01

    The Whole Genome Sampling Analysis (WGSA) assay in combination with Affymetrix GeneChip Mapping Arrays is used for copy number analysis of high-quality DNA samples (i.e., samples that have been collected from blood, fresh or frozen tissue, or cell lines). Formalin-fixed, paraffin-embedded (FFPE) samples, however, represent the most prevalent form of archived clinical samples, but they provide additional challenges for molecular assays. FFPE processing usually results in the degradation of FFPE DNA and in the contamination and chemical modification of these DNA samples. In this article, we describe the steps needed to obtain reliable copy number predictions from degraded and contaminated FFPE samples.

  18. MALDI mass spectrometry imaging of formalin-fixed paraffin-embedded tissues in clinical research.

    PubMed

    Gorzolka, Karin; Walch, Axel

    2014-11-01

    The molecular investigation of archived formalin-fixed, paraffin-embedded (FFPE) tissue samples provides the chance to obtain molecular patterns as indicatives for treatment and clinical end points. MALDI mass spectrometry imaging is capable of localizing molecules like proteins and peptides in tissue sections and became a favorite platform for the targeted and non-targeted approaches, especially in clinical investigations for biomarker research. In FFPE tissues the recovery of proteomic information is constrained by fixation-induced cross-links of proteins. The promising new insights obtained from FFPE in combination with the comprehensive patients' data caused much progress in the optimization of MS imaging protocols to investigate FFPE samples. This review presents the past and current research in MALDI MS imaging of FFPE tissues, demonstrating the improvement of analyses, their actual limitations, but also the promising future perspectives for histopathological and tissue-based research.

  19. Protein extraction from formalin-fixed, paraffin-embedded tissue sections: quality evaluation by mass spectrometry.

    PubMed

    Shi, Shan-Rong; Liu, Cheng; Balgley, Brian M; Lee, Cheng; Taylor, Clive R

    2006-06-01

    A satisfactory protocol of protein extraction has been established based on the heat-induced antigen retrieval (AR) technique widely applied in immunohistochemistry for archival formalin-fixed, paraffin-embedded (FFPE) tissue sections. Based on AR, an initial serial experiment to identify an optimal protocol of heat-induced protein extraction was carried out using FFPE mouse tissues. The optimal protocol for extraction of proteins was then performed on an archival FFPE tissue of human renal carcinoma. FFPE sections were boiled in a retrieval solution of Tris-HCl containing 2% SDS, followed by incubation. Fresh tissue taken from the same case of renal carcinoma was processed for extraction of proteins by a conventional method using radioimmunoprecipitation assay solution, to compare the efficiency of protein extraction from FFPE tissue sections with extraction from fresh tissue. As a control, further sections of the same FFPE sample were processed by the same procedure without heating treatment. Evaluation of the quality of protein extracted from FFPE tissue was done using gel electrophoresis and mass spectrometry, showing most identified proteins extracted from FFPE tissue sections were overlapped with those extracted from fresh tissue.

  20. Proteomic analysis of formalin-fixed paraffin embedded tissue by MALDI imaging mass spectrometry

    PubMed Central

    Casadonte, Rita; Caprioli, Richard M

    2012-01-01

    Archived formalin-fixed paraffin-embedded (FFPE) tissue collections represent a valuable informational resource for proteomic studies. Multiple FFPE core biopsies can be assembled in a single block to form tissue microarrays (TMAs). We describe a protocol for analyzing protein in FFPE -TMAs using matrix-assisted laser desorption/ionization (MAL DI) imaging mass spectrometry (IMS). The workflow incorporates an antigen retrieval step following deparaffinization, in situ trypsin digestion, matrix application and then mass spectrometry signal acquisition. The direct analysis of FFPE -TMA tissue using IMS allows direct analysis of multiple tissue samples in a single experiment without extraction and purification of proteins. The advantages of high speed and throughput, easy sample handling and excellent reproducibility make this technology a favorable approach for the proteomic analysis of clinical research cohorts with large sample numbers. For example, TMA analysis of 300 FFPE cores would typically require 6 h of total time through data acquisition, not including data analysis. PMID:22011652

  1. Making the most of pathological specimens: molecular diagnosis in formalin-fixed, paraffin embedded tissue.

    PubMed

    Fairley, Jennifer A; Gilmour, Katelyn; Walsh, Kathy

    2012-11-01

    The development of commercial reagents designed specifically for use with formalin-fixed paraffin-embedded (FFPE) tissue has unlocked the diagnostic potential of this prolific resource. The availability of archival FFPE tissue and tissue from current patients make it an ideal resource for molecular testing. Despite its stability and ability to preserve morphological information, FFPE provides a number of technical challenges to the study of biomolecules. In particular, the cross-linking and processing present problems in the extraction and isolation of DNA, RNA and protein and affect their use in downstream analysis. Here we will discuss some of the problems of FFPE tissue, how they can be overcome and how FFPE material can be used within clinical molecular diagnostics.

  2. Post-mortem testing; germline BRCA1/2 variant detection using archival FFPE non-tumor tissue. A new paradigm in genetic counseling.

    PubMed

    Petersen, Annabeth Høgh; Aagaard, Mads Malik; Nielsen, Henriette Roed; Steffensen, Karina Dahl; Waldstrøm, Marianne; Bojesen, Anders

    2016-08-01

    Accurate estimation of cancer risk in HBOC families often requires BRCA1/2 testing, but this may be impossible in deceased family members. Previous, testing archival formalin-fixed, paraffin-embedded (FFPE) tissue for germline BRCA1/2 variants was unsuccessful, except for the Jewish founder mutations. A high-throughput method to systematically test for variants in all coding regions of BRCA1/2 in archival FFPE samples of non-tumor tissue is described, using HaloPlex target enrichment and next-generation sequencing. In a validation study, correct identification of variants or wild-type was possible in 25 out of 30 (83%) FFPE samples (age range 1-14 years), with a known variant status in BRCA1/2. No false positive was found. Unsuccessful identification was due to highly degraded DNA or presence of large intragenic deletions. In clinical use, a total of 201 FFPE samples (aged 0-43 years) were processed. Thirty-six samples were rejected because of highly degraded DNA or failed library preparation. Fifteen samples were investigated to search for a known variant. In the remaining 150 samples (aged 0-38 years), three variants known to affect function and one variant likely to affect function in BRCA1, six variants known to affect function and one variant likely to affect function in BRCA2, as well as four variants of unknown significance (VUS) in BRCA1 and three VUS in BRCA2 were discovered. It is now possible to test for germline BRCA1/2 variants in deceased persons, using archival FFPE samples from non-tumor tissue. Accurate genetic counseling is achievable in families where variant testing would otherwise be impossible.

  3. Post-mortem testing; germline BRCA1/2 variant detection using archival FFPE non-tumor tissue. A new paradigm in genetic counseling

    PubMed Central

    Petersen, Annabeth Høgh; Aagaard, Mads Malik; Nielsen, Henriette Roed; Steffensen, Karina Dahl; Waldstrøm, Marianne; Bojesen, Anders

    2016-01-01

    Accurate estimation of cancer risk in HBOC families often requires BRCA1/2 testing, but this may be impossible in deceased family members. Previous, testing archival formalin-fixed, paraffin-embedded (FFPE) tissue for germline BRCA1/2 variants was unsuccessful, except for the Jewish founder mutations. A high-throughput method to systematically test for variants in all coding regions of BRCA1/2 in archival FFPE samples of non-tumor tissue is described, using HaloPlex target enrichment and next-generation sequencing. In a validation study, correct identification of variants or wild-type was possible in 25 out of 30 (83%) FFPE samples (age range 1–14 years), with a known variant status in BRCA1/2. No false positive was found. Unsuccessful identification was due to highly degraded DNA or presence of large intragenic deletions. In clinical use, a total of 201 FFPE samples (aged 0–43 years) were processed. Thirty-six samples were rejected because of highly degraded DNA or failed library preparation. Fifteen samples were investigated to search for a known variant. In the remaining 150 samples (aged 0–38 years), three variants known to affect function and one variant likely to affect function in BRCA1, six variants known to affect function and one variant likely to affect function in BRCA2, as well as four variants of unknown significance (VUS) in BRCA1 and three VUS in BRCA2 were discovered. It is now possible to test for germline BRCA1/2 variants in deceased persons, using archival FFPE samples from non-tumor tissue. Accurate genetic counseling is achievable in families where variant testing would otherwise be impossible. PMID:26733283

  4. Fluorescence in situ hybridization analysis of formalin fixed paraffin embedded tissues, including tissue microarrays.

    PubMed

    Summersgill, Brenda M; Shipley, Janet M

    2010-01-01

    Formalin fixed paraffin embedded (FFPE) material is frequently the most convenient readily available source of diseased tissue, including tumors. Multiple cores of FFPE material are being used increasingly to construct tissue microarrays (TMAs) that enable simultaneous analyses of many archival samples. Fluorescence in situ hybridization (FISH) is an important approach to analyze FFPE material for specific genetic aberrations that may be associated with tumor types or subtypes, cellular morphology, and disease prognosis. Annealing, or hybridization of labeled nucleic acid sequences, or probes, to detect and locate one or more complementary nucleic acid sequences within fixed tissue sections allows the detection of structural (translocation/inversion) and numerical (deletion/gain) aberrations and their localization within tissues. The robust protocols described include probe preparation, hybridization, and detection and take 2-3 days to complete. A protocol is also described for the stripping of probes for repeat FISH in order to maximize the use of scarce tissue resources.

  5. The effects of age-in-block on RNA-seq analysis of archival formalin-fixed paraffin-embedded (FFPE) samples

    EPA Science Inventory

    Archival samples represent a vast resource for identification of chemical and pharmaceutical targets. Previous use of formalin-fixed paraffin-embedded (FFPE) samples has been limited due to changes in RNA introduced by fixation and embedding procedures. Recent advances in RNA-seq...

  6. Sample processing considerations for detecting copy number changes in formalin-fixed, paraffin-embedded tissues.

    PubMed

    Jacobs, Sharoni

    2012-11-01

    The Whole Genome Sampling Analysis (WGSA) assay in combination with Affymetrix GeneChip Mapping Arrays is used for copy number analysis of high-quality DNA samples (i.e., samples that have been collected from blood, fresh or frozen tissue, or cell lines). Formalin-fixed, paraffin-embedded (FFPE) samples, however, represent the most prevalent form of archived clinical samples, but they provide additional challenges for molecular assays. FFPE processing usually results in the degradation of FFPE DNA and in the contamination and chemical modification of these DNA samples. Because of these issues, FFPE DNA is not suitable for all molecular assays designed for high-quality DNA samples. Strategies recommended for processing FFPE DNA samples through WGSA and to the Mapping arrays are described here. PMID:23118355

  7. Sample processing considerations for detecting copy number changes in formalin-fixed, paraffin-embedded tissues.

    PubMed

    Jacobs, Sharoni

    2012-11-01

    The Whole Genome Sampling Analysis (WGSA) assay in combination with Affymetrix GeneChip Mapping Arrays is used for copy number analysis of high-quality DNA samples (i.e., samples that have been collected from blood, fresh or frozen tissue, or cell lines). Formalin-fixed, paraffin-embedded (FFPE) samples, however, represent the most prevalent form of archived clinical samples, but they provide additional challenges for molecular assays. FFPE processing usually results in the degradation of FFPE DNA and in the contamination and chemical modification of these DNA samples. Because of these issues, FFPE DNA is not suitable for all molecular assays designed for high-quality DNA samples. Strategies recommended for processing FFPE DNA samples through WGSA and to the Mapping arrays are described here.

  8. Performance evaluation of kits for bisulfite-conversion of DNA from tissues, cell lines, FFPE tissues, aspirates, lavages, effusions, plasma, serum, and urine.

    PubMed

    Holmes, Emily Eva; Jung, Maria; Meller, Sebastian; Leisse, Annette; Sailer, Verena; Zech, Julie; Mengdehl, Martina; Garbe, Leif-Alexander; Uhl, Barbara; Kristiansen, Glen; Dietrich, Dimo

    2014-01-01

    DNA methylation analyses usually require a preceding bisulfite conversion of the DNA. The choice of an appropriate kit for a specific application should be based on the specific performance requirements with regard to the respective sample material. In this study, the performance of nine kits was evaluated: EpiTect Fast FFPE Bisulfite Kit, EpiTect Bisulfite Kit, EpiTect Fast DNA Bisulfite Kit (Qiagen), EZ DNA Methylation-Gold Kit, EZ DNA Methylation-Direct Kit, EZ DNA Methylation-Lightning Kit (Zymo Research), innuCONVERT Bisulfite All-In-One Kit, innuCONVERT Bisulfite Basic Kit, innuCONVERT Bisulfite Body Fluids Kit (Analytik Jena). The kit performance was compared with regard to DNA yield, DNA degradation, DNA purity, conversion efficiency, stability and handling using qPCR, UV, clone sequencing, HPLC, and agarose gel electrophoresis. All kits yielded highly pure DNA suitable for PCR analyses without PCR inhibition. Significantly higher yields were obtained when using the EZ DNA Methylation-Gold Kit and the innuCONVERT Bisulfite kits. Conversion efficiency ranged from 98.7% (EpiTect Bisulfite Kit) to 99.9% (EZ DNA Methylation-Direct Kit). The inappropriate conversion of methylated cytosines to thymines varied between 0.9% (innuCONVERT Bisulfite kits) and 2.7% (EZ DNA Methylation-Direct Kit). Time-to-result ranged from 131 min (innuCONVERT kits) to 402 min (EpiTect Bisulfite Kit). Hands-on-time was between 66 min (EZ DNA Methylation-Lightning Kit) and 104 min (EpiTect Fast FFPE and Fast DNA Bisulfite kits). Highest yields from formalin-fixed and paraffin-embedded (FFPE) tissue sections without prior extraction were obtained using the innuCONVERT Bisulfite All-In-One Kit while the EZ DNA Methylation-Direct Kit yielded DNA with only low PCR-amplifiability. The innuCONVERT Bisulfite All-In-One Kit exhibited the highest versatility regarding different input sample materials (extracted DNA, tissue, FFPE tissue, cell lines, urine sediment, and cellular fractions of

  9. Mass spectrometric analysis of formalin-fixed paraffin-embedded tissue: unlocking the proteome within.

    PubMed

    Hood, Brian L; Conrads, Thomas P; Veenstra, Timothy D

    2006-07-01

    The predominance of tissues stored worldwide in hospitals and clinical laboratories exist in formalin-fixed paraffin-embedded (FFPE) blocks that are generated by simple and well-established protocols. Although generation of FFPE tissues has facilitated their characterization by such techniques as histopathology, they have proven refractory to biomarker discovery investigations using state-of-the-art MS-based proteomic methodologies. Very recently new methods have been developed that enable proteins extracted from FFPE tissues to be analyzed by MS. This review will highlight and discuss those efforts that have led to this exciting recent progress. Although these developments are quite new, the ability to conduct MS-based proteomic analyses of FFPE tissues opens heretofore intractable clinical samples for discovery-based biomarker research.

  10. Phosphoproteome analysis of formalin-fixed and paraffin-embedded tissue sections mounted on microscope slides.

    PubMed

    Wakabayashi, Masaki; Yoshihara, Hiroki; Masuda, Takeshi; Tsukahara, Mai; Sugiyama, Naoyuki; Ishihama, Yasushi

    2014-02-01

    Formalin-fixed and paraffin-embedded (FFPE) sections mounted on microscope slides are one of the largest available resources for retrospective research on various diseases, but quantitative phosphoproteome analysis of FFPE sections has never been achieved because of the extreme difficulty of procuring sufficient phosphopeptides from the limited amounts of proteins on the slides. Here, we present the first protocol for quantitative phosphoproteome analysis of FFPE sections by utilizing phase-transfer surfactant-aided extraction/tryptic digestion of FFPE proteins followed by high-recovery phosphopeptide enrichment via lactic acid-modified titania chromatography. We established that FFPE sections retain a similar phosphoproteome to fresh tissue specimens during storage for at least 9 months, confirming the utility of our method for evaluating phosphorylation profiles in various diseases. We also verified that chemical labeling based on reductive dimethylation of amino groups was feasible for quantitative phosphoproteome analysis of FFPE samples on slides. Furthermore, we improved the LC-MS sensitivity by miniaturizing nanoLC columns to 25 μm inner diameter. With this system, we could identify 1090 phosphopeptides from a single FFPE section obtained from a microscope slide, containing 25.2 ± 5.4 μg of proteins. This protocol should be useful for large-scale phosphoproteome analysis of archival FFPE slides, especially scarce samples from patients with rare diseases.

  11. Formalin-fixed paraffin-embedded clinical tissues show spurious copy number changes in array-CGH profiles.

    PubMed

    Mc Sherry, E A; Mc Goldrick, A; Kay, E W; Hopkins, A M; Gallagher, W M; Dervan, P A

    2007-11-01

    Formalin-fixed paraffin-embedded (FFPE) archival clinical specimens are invaluable in discovery of prognostic and therapeutic targets for diseases such as cancer. However, the suitability of FFPE-derived genetic material for array-based comparative genomic hybridization (array-CGH) studies is underexplored. In this study, genetic profiles of matched FFPE and fresh-frozen specimens were examined to investigate DNA integrity differences between these sample types and determine the impact this may have on genetic profiles. Genomic DNA was extracted from three patient-matched FFPE and fresh-frozen clinical tissue samples. T47D breast cancer control cells were also grown in culture and processed to yield a fresh T47D sample, a fresh-frozen T47D sample and a FFPE T47D sample. DNA was extracted from all the samples; array-CGH conducted and genetic profiles of matched samples were then compared. A loss of high molecular weight DNA was observed in the FFPE clinical tissues and FFPE T47D samples. A dramatic increase in absolute number of genetic alterations was observed in all FFPE tissues relative to matched fresh-frozen counterparts. In future, alternative fixation and tissue-processing procedures, and/or new DNA extraction and CGH profiling protocols, may be implemented, enabling identification of changes involved in disease progression using stored clinical specimens.

  12. Data analysis considerations for detecting copy number changes in formalin-fixed, paraffin-embedded tissues.

    PubMed

    Jacobs, Sharoni

    2012-11-01

    The Whole Genome Sampling Analysis (WGSA) assay in combination with Affymetrix GeneChip Mapping Arrays is used for copy number analysis of high-quality DNA samples (i.e., samples that have been collected from blood, fresh or frozen tissue, or cell lines). Formalin-fixed, paraffin-embedded (FFPE) samples, however, represent the most prevalent form of archived clinical samples, but they provide additional challenges for molecular assays. FFPE processing usually results in the degradation of FFPE DNA and in the contamination and chemical modification of these DNA samples. In this article, we describe the steps needed to obtain reliable copy number predictions from degraded and contaminated FFPE samples. PMID:23118356

  13. Rates of MAGE-A3 and PRAME expressing tumors in FFPE tissue specimens from bladder cancer patients: potential targets for antigen-specific cancer immunotherapeutics

    PubMed Central

    Lerut, Evelyne; Van Poppel, Hendrik; Joniau, Steven; Gruselle, Olivier; Coche, Thierry; Therasse, Patrick

    2015-01-01

    Introduction: Antigen-specific active immunotherapy is an investigational therapeutic approach of potential interest for bladder cancer regardless of disease stage. Clinical development of antigen-specific immunotherapeutics against bladder cancer must be preceded by assessment of the expression of relevant genes in bladder tumors. The objectives of this study (NCT01706185) were to assess the rate of expression of the MAGE-A3 and PRAME genes in bladder tumors and to investigate the feasibility of using formalin-fixed paraffin-embedded (FFPE) tumor tissues for testing. Materials and methods: Archived FFPE bladder tumor specimens (any stage) were tested for mRNA expression of MAGE-A3 and PRAME using antigen-specific quantitative reverse transcription polymerase chain reaction assays. Data on patients and tumor characteristics were obtained from hospital records to investigate these characteristics’ possible association with the antigen expression. Results: Over 92% of the 156 tumors examined gave valid antigen test results. Of the tumors with a valid test, 46.5% were MAGE-A3-positive, 32.2% were PRAME-positive and 59.7% positive for at least one of them. Exploratory analyses of possible associations between antigen expression and patient or tumor characteristics did not identify clear associations between antigen expression and any of the variables investigated. Conclusions: Assessment of tumor antigen mRNA expression by using FFPE bladder tissues was feasible. The rates of MAGE-A3-positive and PRAME-positive tumors indicate that both antigens may be interesting targets for immunotherapeutics against bladder cancer. PMID:26464715

  14. 'Tissue surrogates' as a model for archival formalin-fixed paraffin-embedded tissues.

    PubMed

    Fowler, Carol B; Cunningham, Robert E; O'Leary, Timothy J; Mason, Jeffrey T

    2007-08-01

    High-throughput proteomic studies of archival formalin-fixed paraffin-embedded (FFPE) tissues have the potential to be a powerful tool for examining the clinical course of disease. However, advances in FFPE tissue-based proteomics have been hampered by inefficient methods to extract proteins from archival tissue and by an incomplete knowledge of formaldehyde-induced modifications in proteins. To help address these problems, we have developed a procedure for the formation of 'tissue surrogates' to model FFPE tissues. Cytoplasmic proteins, such as lysozyme or ribonuclease A, at concentrations approaching the protein content in whole cells, are fixed with 10% formalin to form gelatin-like plugs. These plugs have sufficient physical integrity to be processed through graded alcohols, xylene, and embedded in paraffin according to standard histological procedures. In this study, we used tissue surrogates formed from one or two proteins to evaluate extraction protocols for their ability to quantitatively extract proteins from the surrogates. Optimal protein extraction was obtained using a combination of heat, a detergent, and a protein denaturant. The addition of a reducing agent did not improve protein recovery; however, recovery varied significantly with pH. Protein extraction of >80% was observed for pH 4 buffers containing 2% (w/v) sodium dodecyl sulfate (SDS) when heated at 100 degrees C for 20 min, followed by incubation at 60 degrees C for 2 h. SDS-polyacrylamide gel electrophoresis of the extracted proteins revealed that the surrogate extracts contained a mixture of monomeric and multimeric proteins, regardless of the extraction protocol employed. Additionally, protein extracts from surrogates containing carbonic anhydrase:lysozyme (1:2 mol/mol) had disproportionate percentages of lysozyme, indicating that selective protein extraction in complex multiprotein systems may be a concern in proteomic studies of FFPE tissues.

  15. Comparison of Accuracy of Whole-Exome Sequencing with Formalin-Fixed Paraffin-Embedded and Fresh Frozen Tissue Samples

    PubMed Central

    Kwon, Mi Jeong; Kim, Ryong Nam; Kim, Yu Jin; Song, Ji-Young; Jung, Kyung Soo; Shin, Young Kee

    2015-01-01

    Formalin fixing with paraffin embedding (FFPE) has been a standard sample preparation method for decades, and archival FFPE samples are still very useful resources. Nonetheless, the use of FFPE samples in cancer genome analysis using next-generation sequencing, which is a powerful technique for the identification of genomic alterations at the nucleotide level, has been challenging due to poor DNA quality and artificial sequence alterations. In this study, we performed whole-exome sequencing of matched frozen samples and FFPE samples of tissues from 4 cancer patients and compared the next-generation sequencing data obtained from these samples. The major differences between data obtained from the 2 types of sample were the shorter insert size and artificial base alterations in the FFPE samples. A high proportion of short inserts in the FFPE samples resulted in overlapping paired reads, which could lead to overestimation of certain variants; >20% of the inserts in the FFPE samples were double sequenced. A large number of soft clipped reads was found in the sequencing data of the FFPE samples, and about 30% of total bases were soft clipped. The artificial base alterations, C>T and G>A, were observed in FFPE samples only, and the alteration rate ranged from 200 to 1,200 per 1M bases when sequencing errors were removed. Although high-confidence mutation calls in the FFPE samples were compatible to that in the frozen samples, caution should be exercised in terms of the artifacts, especially for low-confidence calls. Despite the clearly observed artifacts, archival FFPE samples can be a good resource for discovery or validation of biomarkers in cancer research based on whole-exome sequencing. PMID:26641479

  16. Comparison of Accuracy of Whole-Exome Sequencing with Formalin-Fixed Paraffin-Embedded and Fresh Frozen Tissue Samples.

    PubMed

    Oh, Ensel; Choi, Yoon-La; Kwon, Mi Jeong; Kim, Ryong Nam; Kim, Yu Jin; Song, Ji-Young; Jung, Kyung Soo; Shin, Young Kee

    2015-01-01

    Formalin fixing with paraffin embedding (FFPE) has been a standard sample preparation method for decades, and archival FFPE samples are still very useful resources. Nonetheless, the use of FFPE samples in cancer genome analysis using next-generation sequencing, which is a powerful technique for the identification of genomic alterations at the nucleotide level, has been challenging due to poor DNA quality and artificial sequence alterations. In this study, we performed whole-exome sequencing of matched frozen samples and FFPE samples of tissues from 4 cancer patients and compared the next-generation sequencing data obtained from these samples. The major differences between data obtained from the 2 types of sample were the shorter insert size and artificial base alterations in the FFPE samples. A high proportion of short inserts in the FFPE samples resulted in overlapping paired reads, which could lead to overestimation of certain variants; >20% of the inserts in the FFPE samples were double sequenced. A large number of soft clipped reads was found in the sequencing data of the FFPE samples, and about 30% of total bases were soft clipped. The artificial base alterations, C>T and G>A, were observed in FFPE samples only, and the alteration rate ranged from 200 to 1,200 per 1M bases when sequencing errors were removed. Although high-confidence mutation calls in the FFPE samples were compatible to that in the frozen samples, caution should be exercised in terms of the artifacts, especially for low-confidence calls. Despite the clearly observed artifacts, archival FFPE samples can be a good resource for discovery or validation of biomarkers in cancer research based on whole-exome sequencing.

  17. Comparison of Accuracy of Whole-Exome Sequencing with Formalin-Fixed Paraffin-Embedded and Fresh Frozen Tissue Samples.

    PubMed

    Oh, Ensel; Choi, Yoon-La; Kwon, Mi Jeong; Kim, Ryong Nam; Kim, Yu Jin; Song, Ji-Young; Jung, Kyung Soo; Shin, Young Kee

    2015-01-01

    Formalin fixing with paraffin embedding (FFPE) has been a standard sample preparation method for decades, and archival FFPE samples are still very useful resources. Nonetheless, the use of FFPE samples in cancer genome analysis using next-generation sequencing, which is a powerful technique for the identification of genomic alterations at the nucleotide level, has been challenging due to poor DNA quality and artificial sequence alterations. In this study, we performed whole-exome sequencing of matched frozen samples and FFPE samples of tissues from 4 cancer patients and compared the next-generation sequencing data obtained from these samples. The major differences between data obtained from the 2 types of sample were the shorter insert size and artificial base alterations in the FFPE samples. A high proportion of short inserts in the FFPE samples resulted in overlapping paired reads, which could lead to overestimation of certain variants; >20% of the inserts in the FFPE samples were double sequenced. A large number of soft clipped reads was found in the sequencing data of the FFPE samples, and about 30% of total bases were soft clipped. The artificial base alterations, C>T and G>A, were observed in FFPE samples only, and the alteration rate ranged from 200 to 1,200 per 1M bases when sequencing errors were removed. Although high-confidence mutation calls in the FFPE samples were compatible to that in the frozen samples, caution should be exercised in terms of the artifacts, especially for low-confidence calls. Despite the clearly observed artifacts, archival FFPE samples can be a good resource for discovery or validation of biomarkers in cancer research based on whole-exome sequencing. PMID:26641479

  18. High-mass-resolution MALDI mass spectrometry imaging of metabolites from formalin-fixed paraffin-embedded tissue.

    PubMed

    Ly, Alice; Buck, Achim; Balluff, Benjamin; Sun, Na; Gorzolka, Karin; Feuchtinger, Annette; Janssen, Klaus-Peter; Kuppen, Peter J K; van de Velde, Cornelis J H; Weirich, Gregor; Erlmeier, Franziska; Langer, Rupert; Aubele, Michaela; Zitzelsberger, Horst; McDonnell, Liam; Aichler, Michaela; Walch, Axel

    2016-08-01

    Formalin-fixed and paraffin-embedded (FFPE) tissue specimens are the gold standard for histological examination, and they provide valuable molecular information in tissue-based research. Metabolite assessment from archived tissue samples has not been extensively conducted because of a lack of appropriate protocols and concerns about changes in metabolite content or chemical state due to tissue processing. We present a protocol for the in situ analysis of metabolite content from FFPE samples using a high-mass-resolution matrix-assisted laser desorption/ionization fourier-transform ion cyclotron resonance mass spectrometry imaging (MALDI-FT-ICR-MSI) platform. The method involves FFPE tissue sections that undergo deparaffinization and matrix coating by 9-aminoacridine before MALDI-MSI. Using this platform, we previously detected ∼1,500 m/z species in the mass range m/z 50-1,000 in FFPE samples; the overlap compared with fresh frozen samples is 72% of m/z species, indicating that metabolites are largely conserved in FFPE tissue samples. This protocol can be reproducibly performed on FFPE tissues, including small samples such as tissue microarrays and biopsies. The procedure can be completed in a day, depending on the size of the sample measured and raster size used. Advantages of this approach include easy sample handling, reproducibility, high throughput and the ability to demonstrate molecular spatial distributions in situ. The data acquired with this protocol can be used in research and clinical practice.

  19. High-mass-resolution MALDI mass spectrometry imaging of metabolites from formalin-fixed paraffin-embedded tissue.

    PubMed

    Ly, Alice; Buck, Achim; Balluff, Benjamin; Sun, Na; Gorzolka, Karin; Feuchtinger, Annette; Janssen, Klaus-Peter; Kuppen, Peter J K; van de Velde, Cornelis J H; Weirich, Gregor; Erlmeier, Franziska; Langer, Rupert; Aubele, Michaela; Zitzelsberger, Horst; McDonnell, Liam; Aichler, Michaela; Walch, Axel

    2016-08-01

    Formalin-fixed and paraffin-embedded (FFPE) tissue specimens are the gold standard for histological examination, and they provide valuable molecular information in tissue-based research. Metabolite assessment from archived tissue samples has not been extensively conducted because of a lack of appropriate protocols and concerns about changes in metabolite content or chemical state due to tissue processing. We present a protocol for the in situ analysis of metabolite content from FFPE samples using a high-mass-resolution matrix-assisted laser desorption/ionization fourier-transform ion cyclotron resonance mass spectrometry imaging (MALDI-FT-ICR-MSI) platform. The method involves FFPE tissue sections that undergo deparaffinization and matrix coating by 9-aminoacridine before MALDI-MSI. Using this platform, we previously detected ∼1,500 m/z species in the mass range m/z 50-1,000 in FFPE samples; the overlap compared with fresh frozen samples is 72% of m/z species, indicating that metabolites are largely conserved in FFPE tissue samples. This protocol can be reproducibly performed on FFPE tissues, including small samples such as tissue microarrays and biopsies. The procedure can be completed in a day, depending on the size of the sample measured and raster size used. Advantages of this approach include easy sample handling, reproducibility, high throughput and the ability to demonstrate molecular spatial distributions in situ. The data acquired with this protocol can be used in research and clinical practice. PMID:27414759

  20. The paraffin-embedded RNA metric (PERM) for RNA isolated from formalin-fixed, paraffin-embedded tissue.

    PubMed

    Chung, Joon-Yong; Cho, Hanbyoul; Hewitt, Stephen M

    2016-01-01

    RNA isolated from formalin-fixed, paraffin-embedded (FFPE) tissue is commonly evaluated in both investigative and diagnostic pathology. However, the quality of the data is directly impacted by RNA quality. The RNA integrity number (RIN), an algorithm based on a combination of electrophoretic features, is widely applied to RNA isolated from paraffin-embedded tissue, but it is a poor indicator of the quality of that RNA. Here we describe the novel paraffin-embedded RNA metric (PERM) for quantifying the quality of RNA from FFPE tissue. The PERM is based on a formula that approximates a weighted area-under-the-curve analysis of an electropherogram of the extracted RNA. Using biochemically degraded RNAs prepared from experimentally fixed mouse kidney specimens, we demonstrate that PERM values correlate with mRNA transcript measurements determined using the QuantiGene system. Furthermore, PERM values correlate with real-time PCR data. Our results demonstrate that the PERM can be used to qualify RNA for different end-point studies and may be a valuable tool for molecular studies using RNA extracted from FFPE tissue.

  1. Assessment of the 2-d gel-based proteomics application of clinically archived formalin-fixed paraffin embedded tissues.

    PubMed

    Davalieva, Katarina; Kiprijanovska, Sanja; Polenakovic, Momir

    2014-04-01

    Hospital tissue repositories possess a vast and valuable supply of disease samples with matched retrospective clinical information. Detection and characterization of disease biomarkers in formalin-fixed paraffin-embedded (FFPE) tissues will greatly aid the understanding of the diseases mechanisms and help in the development of diagnostic and prognostic markers. In this study, the possibility of using full-length proteins extracted from clinically archived FFPE tissues in two-dimensional (2-D) gel-based proteomics was evaluated. The evaluation was done based on two types of tumor tissues (breast and prostate) and two extraction protocols. The comparison of the 2-D patterns of FFPE extracts obtained by two extraction protocols with the matching frozen tissue extracts showed that only 7-10% of proteins from frozen tissues can be matched to proteins from FFPE tissues. Most of the spots in the 2-D FFPE's maps had pl 4-6, while the percentages of proteins with pl above 6 were 3-5 times lower in comparison to the fresh/frozen tissue. Despite the three-fold lower number of the detected spots in FFPE maps compared to matched fresh/frozen maps, 67-78% of protein spots in FFPE could not be matched to the corresponding spots in the fresh/frozen tissue maps indicating irreversible protein modifications. In conclusion, the inability to completely reverse the cross-linked complexes and overcome protein fragmentation with the present day FFPE extraction methods stands in the way of effective use of these samples in 2-D gel based proteomics studies.

  2. Molecular pathological analysis of sarcomas using paraffin-embedded tissue: current limitations and future possibilities

    PubMed Central

    van de Rijn, Matt; Guo, Xiangqian; Sweeney, Robert T; Beck, Andrew H; West, Robert B

    2016-01-01

    Summary Sarcomas of soft tissue and bone are rare neoplasms that can be separated into a large number of different diagnostic entities. Over the years, a number of diagnostic markers have been developed that aid pathologists in reaching the appropriate diagnoses. Many of these markers are sarcoma-specific proteins that can be detected by immunohistochemistry in formalin-fixed, paraffin-embedded (FFPE) sections. In addition, a wide range of molecular studies have been developed that can detect gene mutations, gene amplifications or chromosomal translocations in FFPE material. Until recently, most sequencing-based approaches relied on the availability of fresh frozen tissue. However, with the advent of next-generation sequencing technologies, FFPE material is increasingly being used as a tool to identify novel immuno-histochemistry markers, gene mutations, and chromosomal translocations, and to develop diagnostic tests. PMID:24107169

  3. High-resolution copy number profiling by array CGH using DNA isolated from formalin-fixed, paraffin-embedded tissues.

    PubMed

    van Essen, Hendrik F; Ylstra, Bauke

    2012-01-01

    We describe protocols to acquire high-quality DNA from formalin-fixed, paraffin-embedded (FFPE) tissues for the use in array comparative genome hybridization (CGH). Formalin fixation combined with paraffin embedding is routine procedure for solid malignancies in the diagnostic practice of the pathologist. As a consequence, large archives of FFPE tissues are available in pathology institutes across the globe. This archival material is for many research questions an invaluable resource, with long-term clinical follow-up and survival data available. FFPE is, thus, highly attractive for large genomics studies, including experiments requiring samples for test/learning and validation. Most larger array CGH studies have, therefore, made use of FFPE material and show that CNAs have tumor- and tissue-specific traits (Chin et al. Cancer Cell 10: 529-541, 2006; Fridlyand et al. BMC Cancer 6: 96, 2006; Weiss et al. Oncogene 22: 1872-1879, 2003; Jong et al. Oncogene 26: 1499-1506, 2007). The protocols described are tailored to array CGH of FFPE solid malignancies: from sectioning FFPE blocks to specific cynosures for pathological revisions of sections, DNA isolation, quality testing, and amplification. The protocols are technical in character and elaborate up to the labeling of isolated DNA while further processes and interpretation and data analysis are beyond the scope.

  4. Optimization of gene expression microarray protocol for formalin-fixed paraffin-embedded tissues.

    PubMed

    Belder, Nevin; Coşkun, Öznur; Erdoğan, Beyza Doğanay; Savaş, Berna; Ensari, Arzu; Özdağ, Hilal

    2016-03-01

    Formalin-fixed paraffin-embedded (FFPE) tissue is a widely available clinical specimen for retrospective studies. The possibility of long-term clinical follow-up of FFPE samples makes them a valuable source to evaluate links between molecular and clinical information. Working with FFPE samples in the molecular research area, especially using high-throughput molecular techniques such as microarray gene expression profiling, has come into prominence. Because of the harmful effects of formalin fixation process such as degradation of nucleic acids, cross-linking with proteins, and chemical modifications on DNA and RNA, there are some limitations in gene expression profiling studies using FFPE samples. To date many studies have been conducted to evaluate gene expression profiling using microarrays (Thomas et al., Thomas et al. (2013) [1]; Scicchitano et al., Scicchitano et al. (2006) [2]; Frank et al., Frank et al. (2007) [3]; Fedorowicz et al., Fedorowicz et al. (2009) [4]). However, there is still no generally accepted, efficient and standardized procedure for microarray analysis of FFPE samples. This paper describes the microarray data presented in our recently accepted to be published article showing a standard protocol from deparaffinization of FFPE tissue sections and RNA extraction to microarray gene expression analysis. Here we represent our data in detail, deposited in the gene expression omnibus (GEO) database with the accession number GSE73883. Four combinations of two different cRNA/cDNA preparation and labeling protocols with two different array platforms (Affymetrix Human Genome U133 Plus 2.0 and U133_X3P) were evaluated to determine which combination gives the best percentage of present call. The study presents a dataset for comparative analysis which has a potential in terms of providing a robust protocol for gene expression profiling with FFPE tissue samples. PMID:26981433

  5. Optimization of gene expression microarray protocol for formalin-fixed paraffin-embedded tissues.

    PubMed

    Belder, Nevin; Coşkun, Öznur; Erdoğan, Beyza Doğanay; Savaş, Berna; Ensari, Arzu; Özdağ, Hilal

    2016-03-01

    Formalin-fixed paraffin-embedded (FFPE) tissue is a widely available clinical specimen for retrospective studies. The possibility of long-term clinical follow-up of FFPE samples makes them a valuable source to evaluate links between molecular and clinical information. Working with FFPE samples in the molecular research area, especially using high-throughput molecular techniques such as microarray gene expression profiling, has come into prominence. Because of the harmful effects of formalin fixation process such as degradation of nucleic acids, cross-linking with proteins, and chemical modifications on DNA and RNA, there are some limitations in gene expression profiling studies using FFPE samples. To date many studies have been conducted to evaluate gene expression profiling using microarrays (Thomas et al., Thomas et al. (2013) [1]; Scicchitano et al., Scicchitano et al. (2006) [2]; Frank et al., Frank et al. (2007) [3]; Fedorowicz et al., Fedorowicz et al. (2009) [4]). However, there is still no generally accepted, efficient and standardized procedure for microarray analysis of FFPE samples. This paper describes the microarray data presented in our recently accepted to be published article showing a standard protocol from deparaffinization of FFPE tissue sections and RNA extraction to microarray gene expression analysis. Here we represent our data in detail, deposited in the gene expression omnibus (GEO) database with the accession number GSE73883. Four combinations of two different cRNA/cDNA preparation and labeling protocols with two different array platforms (Affymetrix Human Genome U133 Plus 2.0 and U133_X3P) were evaluated to determine which combination gives the best percentage of present call. The study presents a dataset for comparative analysis which has a potential in terms of providing a robust protocol for gene expression profiling with FFPE tissue samples.

  6. High quality copy number and genotype data from FFPE samples using Molecular Inversion Probe (MIP) microarrays

    SciTech Connect

    Wang, Yuker; Carlton, Victoria E.H.; Karlin-Neumann, George; Sapolsky, Ronald; Zhang, Li; Moorhead, Martin; Wang, Zhigang C.; Richardson, Andrea L.; Warren, Robert; Walther, Axel; Bondy, Melissa; Sahin, Aysegul; Krahe, Ralf; Tuna, Musaffe; Thompson, Patricia A.; Spellman, Paul T.; Gray, Joe W.; Mills, Gordon B.; Faham, Malek

    2009-02-24

    A major challenge facing DNA copy number (CN) studies of tumors is that most banked samples with extensive clinical follow-up information are Formalin-Fixed Paraffin Embedded (FFPE). DNA from FFPE samples generally underperforms or suffers high failure rates compared to fresh frozen samples because of DNA degradation and cross-linking during FFPE fixation and processing. As FFPE protocols may vary widely between labs and samples may be stored for decades at room temperature, an ideal FFPE CN technology should work on diverse sample sets. Molecular Inversion Probe (MIP) technology has been applied successfully to obtain high quality CN and genotype data from cell line and frozen tumor DNA. Since the MIP probes require only a small ({approx}40 bp) target binding site, we reasoned they may be well suited to assess degraded FFPE DNA. We assessed CN with a MIP panel of 50,000 markers in 93 FFPE tumor samples from 7 diverse collections. For 38 FFPE samples from three collections we were also able to asses CN in matched fresh frozen tumor tissue. Using an input of 37 ng genomic DNA, we generated high quality CN data with MIP technology in 88% of FFPE samples from seven diverse collections. When matched fresh frozen tissue was available, the performance of FFPE DNA was comparable to that of DNA obtained from matched frozen tumor (genotype concordance averaged 99.9%), with only a modest loss in performance in FFPE. MIP technology can be used to generate high quality CN and genotype data in FFPE as well as fresh frozen samples.

  7. Three Dimensional Imaging of Paraffin Embedded Human Lung Tissue Samples by Micro-Computed Tomography

    PubMed Central

    Scott, Anna E.; Vasilescu, Dragos M.; Seal, Katherine A. D.; Keyes, Samuel D.; Mavrogordato, Mark N.; Hogg, James C.; Sinclair, Ian; Warner, Jane A.; Hackett, Tillie-Louise; Lackie, Peter M.

    2015-01-01

    Background Understanding the three-dimensional (3-D) micro-architecture of lung tissue can provide insights into the pathology of lung disease. Micro computed tomography (µCT) has previously been used to elucidate lung 3D histology and morphometry in fixed samples that have been stained with contrast agents or air inflated and dried. However, non-destructive microstructural 3D imaging of formalin-fixed paraffin embedded (FFPE) tissues would facilitate retrospective analysis of extensive tissue archives of lung FFPE lung samples with linked clinical data. Methods FFPE human lung tissue samples (n = 4) were scanned using a Nikon metrology µCT scanner. Semi-automatic techniques were used to segment the 3D structure of airways and blood vessels. Airspace size (mean linear intercept, Lm) was measured on µCT images and on matched histological sections from the same FFPE samples imaged by light microscopy to validate µCT imaging. Results The µCT imaging protocol provided contrast between tissue and paraffin in FFPE samples (15mm x 7mm). Resolution (voxel size 6.7 µm) in the reconstructed images was sufficient for semi-automatic image segmentation of airways and blood vessels as well as quantitative airspace analysis. The scans were also used to scout for regions of interest, enabling time-efficient preparation of conventional histological sections. The Lm measurements from µCT images were not significantly different to those from matched histological sections. Conclusion We demonstrated how non-destructive imaging of routinely prepared FFPE samples by laboratory µCT can be used to visualize and assess the 3D morphology of the lung including by morphometric analysis. PMID:26030902

  8. Robustness of Next Generation Sequencing on Older Formalin-Fixed Paraffin-Embedded Tissue

    PubMed Central

    Carrick, Danielle Mercatante; Mehaffey, Michele G.; Sachs, Michael C.; Altekruse, Sean; Camalier, Corinne; Chuaqui, Rodrigo; Cozen, Wendy; Das, Biswajit; Hernandez, Brenda Y.; Lih, Chih-Jian; Lynch, Charles F.; Makhlouf, Hala; McGregor, Paul; McShane, Lisa M.; Phillips Rohan, JoyAnn; Walsh, William D.; Williams, Paul M.; Gillanders, Elizabeth M.; Mechanic, Leah E.; Schully, Sheri D.

    2015-01-01

    Next Generation Sequencing (NGS) technologies are used to detect somatic mutations in tumors and study germ line variation. Most NGS studies use DNA isolated from whole blood or fresh frozen tissue. However, formalin-fixed paraffin-embedded (FFPE) tissues are one of the most widely available clinical specimens. Their potential utility as a source of DNA for NGS would greatly enhance population-based cancer studies. While preliminary studies suggest FFPE tissue may be used for NGS, the feasibility of using archived FFPE specimens in population based studies and the effect of storage time on these specimens needs to be determined. We conducted a study to determine whether DNA in archived FFPE high-grade ovarian serous adenocarcinomas from Surveillance, Epidemiology and End Results (SEER) registries Residual Tissue Repositories (RTR) was present in sufficient quantity and quality for NGS assays. Fifty-nine FFPE tissues, stored from 3 to 32 years, were obtained from three SEER RTR sites. DNA was extracted, quantified, quality assessed, and subjected to whole exome sequencing (WES). Following DNA extraction, 58 of 59 specimens (98%) yielded DNA and moved on to the library generation step followed by WES. Specimens stored for longer periods of time had significantly lower coverage of the target region (6% lower per 10 years, 95% CI: 3-10%) and lower average read depth (40x lower per 10 years, 95% CI: 18-60), although sufficient quality and quantity of WES data was obtained for data mining. Overall, 90% (53/59) of specimens provided usable NGS data regardless of storage time. This feasibility study demonstrates FFPE specimens acquired from SEER registries after varying lengths of storage time and under varying storage conditions are a promising source of DNA for NGS. PMID:26222067

  9. Robustness of Next Generation Sequencing on Older Formalin-Fixed Paraffin-Embedded Tissue.

    PubMed

    Carrick, Danielle Mercatante; Mehaffey, Michele G; Sachs, Michael C; Altekruse, Sean; Camalier, Corinne; Chuaqui, Rodrigo; Cozen, Wendy; Das, Biswajit; Hernandez, Brenda Y; Lih, Chih-Jian; Lynch, Charles F; Makhlouf, Hala; McGregor, Paul; McShane, Lisa M; Phillips Rohan, JoyAnn; Walsh, William D; Williams, Paul M; Gillanders, Elizabeth M; Mechanic, Leah E; Schully, Sheri D

    2015-01-01

    Next Generation Sequencing (NGS) technologies are used to detect somatic mutations in tumors and study germ line variation. Most NGS studies use DNA isolated from whole blood or fresh frozen tissue. However, formalin-fixed paraffin-embedded (FFPE) tissues are one of the most widely available clinical specimens. Their potential utility as a source of DNA for NGS would greatly enhance population-based cancer studies. While preliminary studies suggest FFPE tissue may be used for NGS, the feasibility of using archived FFPE specimens in population based studies and the effect of storage time on these specimens needs to be determined. We conducted a study to determine whether DNA in archived FFPE high-grade ovarian serous adenocarcinomas from Surveillance, Epidemiology and End Results (SEER) registries Residual Tissue Repositories (RTR) was present in sufficient quantity and quality for NGS assays. Fifty-nine FFPE tissues, stored from 3 to 32 years, were obtained from three SEER RTR sites. DNA was extracted, quantified, quality assessed, and subjected to whole exome sequencing (WES). Following DNA extraction, 58 of 59 specimens (98%) yielded DNA and moved on to the library generation step followed by WES. Specimens stored for longer periods of time had significantly lower coverage of the target region (6% lower per 10 years, 95% CI: 3-10%) and lower average read depth (40x lower per 10 years, 95% CI: 18-60), although sufficient quality and quantity of WES data was obtained for data mining. Overall, 90% (53/59) of specimens provided usable NGS data regardless of storage time. This feasibility study demonstrates FFPE specimens acquired from SEER registries after varying lengths of storage time and under varying storage conditions are a promising source of DNA for NGS. PMID:26222067

  10. Accuracy of molecular data generated with FFPE biospecimens: lessons from the literature

    PubMed Central

    Greytak, Sarah R.; Engel, Kelly B.; Bass, B. Paige; Moore, Helen M.

    2015-01-01

    Formalin-fixed and paraffin-embedded (FFPE) tissue biospecimens are a valuable resource for molecular cancer research. Although much can be gained from their use, it remains unclear whether the genomic and expression profiles obtained from FFPE biospecimens accurately reflect the physiological condition of the patient from which they were procured, or if such profiles are confounded by biological effects from formalin fixation and processing. To assess the physiological accuracy of genomic and expression data generated with FFPE specimens we surveyed the literature for papers investigating genomic and expression endpoints in case-matched FFPE and fresh or frozen human specimens using the National Cancer Institute's Biospecimen Research Database (http://biospecimens.cancer.gov/brd). Results of the survey revealed that the level of concordance between differentially preserved biospecimens varied among analytical parameters and platforms, but also among reports, genes/transcripts of interest, and tumor status. The identified analytical techniques and parameters that resulted in strong correlations between FFPE and frozen biospecimens may provide guidance when optimizing molecular protocols for FFPE use; however, discrepancies reported for similar assays also illustrate the importance of validating protocols optimized for use with FFPE specimens with a case-matched fresh or frozen cohort for each platform, gene or transcript, and FFPE processing regime. Based upon evidence published to date, validation of analytical parameters with a properly handled frozen cohort is necessary to ensure a high degree of concordance and confidence in the results obtained with FFPE biospecimens. PMID:25836717

  11. A well-based reverse-phase protein array of formalin-fixed paraffin-embedded tissue.

    PubMed

    Chung, Joon-Yong; Hewitt, Stephen M

    2015-01-01

    Biomarkers from tissue-based proteomic studies directly contribute to defining disease states as well as promise to improve early detection or provide for further targeted therapeutics. In the clinical setting, tissue samples are preserved as formalin-fixed paraffin-embedded (FFPE) tissue blocks for histological examination. However, proteomic analysis of FFPE tissue is complicated due to the high level of covalently cross-linked proteins arising from formalin fixation. To address these challenges, we developed well-based reverse-phase protein array (RPPA). This approach is a robust protein isolation methodology (29.44 ± 7.8 μg per 1 mm(3) of FFPE tissue) paired with a novel on electrochemiluminescence detection system. Protein samples derived from FFPE tissue by means of laser capture dissection, with as few as 500 shots, demonstrate measurable signal differences for different proteins. The lysates coated to the array plate, dried up and vacuum-sealed, remain stable up to 2 months at room temperature. This methodology is directly applicable to FFPE tissue and presents the direct opportunity of addressing hypothesis within clinical trials and well-annotated clinical tissue repositories.

  12. A well-based reverse-phase protein array of formalin-fixed paraffin-embedded tissue.

    PubMed

    Chung, Joon-Yong; Hewitt, Stephen M

    2015-01-01

    Biomarkers from tissue-based proteomic studies directly contribute to defining disease states as well as promise to improve early detection or provide for further targeted therapeutics. In the clinical setting, tissue samples are preserved as formalin-fixed paraffin-embedded (FFPE) tissue blocks for histological examination. However, proteomic analysis of FFPE tissue is complicated due to the high level of covalently cross-linked proteins arising from formalin fixation. To address these challenges, we developed well-based reverse-phase protein array (RPPA). This approach is a robust protein isolation methodology (29.44 ± 7.8 μg per 1 mm(3) of FFPE tissue) paired with a novel on electrochemiluminescence detection system. Protein samples derived from FFPE tissue by means of laser capture dissection, with as few as 500 shots, demonstrate measurable signal differences for different proteins. The lysates coated to the array plate, dried up and vacuum-sealed, remain stable up to 2 months at room temperature. This methodology is directly applicable to FFPE tissue and presents the direct opportunity of addressing hypothesis within clinical trials and well-annotated clinical tissue repositories. PMID:26043998

  13. DNA extraction from fresh-frozen and formalin-fixed, paraffin-embedded human brain tissue.

    PubMed

    Wang, Jian-Hua; Gouda-Vossos, Amany; Dzamko, Nicolas; Halliday, Glenda; Huang, Yue

    2013-10-01

    Both fresh-frozen and formalin-fixed, paraffin-embedded (FFPE) human brain tissues are invaluable resources for molecular genetic studies of central nervous system diseases, especially neurodegenerative disorders. To identify the optimal method for DNA extraction from human brain tissue, we compared methods on differently-processed tissues. Fragments of LRRK2 and MAPT (257 bp and 483 bp/245 bp) were amplified for evaluation. We found that for FFPE samples, the success rate of DNA extraction was greater when using a commercial kit than a laboratory-based method (successful DNA extraction from 76% versus 33% of samples). PCR amplicon size and storage period were key factors influencing the success rate of DNA extraction from FFPE samples. In the fresh-frozen samples, the DNA extraction success rate was 100% using either a commercial kit (QIAamp DNA Micro) or a laboratory-based method (sample boiling in 0.1 mol/L NaOH, followed by proteinase K digestion, and then DNA extraction using Chelex-100) regardless of PCR amplicon length or tissue storage time. Although the present results demonstrate that PCR-amplifiable genomic DNA can be extracted from both fresh-frozen and FFPE samples, fresh brain tissue is recommended for DNA extraction in future neuropathological studies.

  14. Absolute Quantitation of Met Using Mass Spectrometry for Clinical Application: Assay Precision, Stability, and Correlation with MET Gene Amplification in FFPE Tumor Tissue

    PubMed Central

    Catenacci, Daniel V. T.; Liao, Wei-Li; Thyparambil, Sheeno; Henderson, Les; Xu, Peng; Zhao, Lei; Rambo, Brittany; Hart, John; Xiao, Shu-Yuan; Bengali, Kathleen; Uzzell, Jamar; Darfler, Marlene; Krizman, David B.; Cecchi, Fabiola; Bottaro, Donald P.; Karrison, Theodore; Veenstra, Timothy D.; Hembrough, Todd; Burrows, Jon

    2014-01-01

    Background Overexpression of Met tyrosine kinase receptor is associated with poor prognosis. Overexpression, and particularly MET amplification, are predictive of response to Met-specific therapy in preclinical models. Immunohistochemistry (IHC) of formalin-fixed paraffin-embedded (FFPE) tissues is currently used to select for ‘high Met’ expressing tumors for Met inhibitor trials. IHC suffers from antibody non-specificity, lack of quantitative resolution, and, when quantifying multiple proteins, inefficient use of scarce tissue. Methods After describing the development of the Liquid-Tissue-Selected Reaction Monitoring-mass spectrometry (LT-SRM-MS) Met assay, we evaluated the expression level of Met in 130 FFPE gastroesophageal cancer (GEC) tissues. We assessed the correlation of SRM Met expression to IHC and mean MET gene copy number (GCN)/nucleus or MET/CEP7 ratio by fluorescence in situ hybridization (FISH). Results Proteomic mapping of recombinant Met identified 418TEFTTALQR426 as the optimal SRM peptide. Limits of detection (LOD) and quantitation (LOQ) for this peptide were 150 and 200 amol/µg tumor protein, respectively. The assay demonstrated excellent precision and temporal stability of measurements in serial sections analyzed one year apart. Expression levels of 130 GEC tissues ranged (<150 amol/µg to 4669.5 amol/µg. High correlation was observed between SRM Met expression and both MET GCN and MET/CEP7 ratio as determined by FISH (n = 30; R2 = 0.898). IHC did not correlate well with SRM (n = 44; R2 = 0.537) nor FISH GCN (n = 31; R2 = 0.509). A Met SRM level of ≥1500 amol/µg was 100% sensitive (95% CI 0.69–1) and 100% specific (95% CI 0.92–1) for MET amplification. Conclusions The Met SRM assay measured the absolute Met levels in clinical tissues with high precision. Compared to IHC, SRM provided a quantitative and linear measurement of Met expression, reliably distinguishing between non-amplified and amplified MET

  15. A melanin-bleaching methodology for molecular and histopathological analysis of formalin-fixed paraffin-embedded tissue.

    PubMed

    Chung, Joon-Yong; Choi, Jiyeon; Sears, John D; Ylaya, Kris; Perry, Candice; Choi, Chel H; Hong, Seung-Mo; Cho, Hanbyoul; Brown, Kevin M; Hewitt, Stephen M

    2016-10-01

    Removal of excessive melanin from heavily pigmented formalin-fixed paraffin-embedded (FFPE) melanoma tissues is essential for histomorphological and molecular diagnostic assessments. Although there have been efforts to address this issue, current methodologies remain complex and time-consuming, and are not suitable for multiple molecular applications. Herein, we have developed a robust and rapid melanin-bleaching methodology for FFPE tissue specimens. Our approach is based on quick bleaching (15 min) at high temperature (80 °C) with 0.5% diluted hydrogen peroxide (H2O2) in Tris-HCl, PBS, or Tris/Tricine/SDS buffer. Immunostaining for Ki-67 and HMB45 was enhanced by bleaching with 0.5% H2O2 in Tris/Tricine/SDS and Tris-HCl, respectively. In addition to histopathological applications, our approach also facilitates recovery of protein and nucleic acid from archival melanin-rich FFPE tissue sections. Protein extracted from bleached FFPE tissues was compatible with western blotting using anti-human GAPDH and AKT antibodies. Our bleaching condition significantly improved RNA quality compared with unbleached tissues without compromising the yield. Notably, the RNA/DNA obtained from bleached tissues was suitable for end point PCR and real-time quantitative RT-PCR. In conclusion, this improved melanin-bleaching method enhances and simplifies immunostaining procedures, and facilitates the use of melanin-rich FFPE tissues for histomorphological and PCR amplification-based molecular assays.

  16. A melanin-bleaching methodology for molecular and histopathological analysis of formalin-fixed paraffin-embedded tissue.

    PubMed

    Chung, Joon-Yong; Choi, Jiyeon; Sears, John D; Ylaya, Kris; Perry, Candice; Choi, Chel H; Hong, Seung-Mo; Cho, Hanbyoul; Brown, Kevin M; Hewitt, Stephen M

    2016-10-01

    Removal of excessive melanin from heavily pigmented formalin-fixed paraffin-embedded (FFPE) melanoma tissues is essential for histomorphological and molecular diagnostic assessments. Although there have been efforts to address this issue, current methodologies remain complex and time-consuming, and are not suitable for multiple molecular applications. Herein, we have developed a robust and rapid melanin-bleaching methodology for FFPE tissue specimens. Our approach is based on quick bleaching (15 min) at high temperature (80 °C) with 0.5% diluted hydrogen peroxide (H2O2) in Tris-HCl, PBS, or Tris/Tricine/SDS buffer. Immunostaining for Ki-67 and HMB45 was enhanced by bleaching with 0.5% H2O2 in Tris/Tricine/SDS and Tris-HCl, respectively. In addition to histopathological applications, our approach also facilitates recovery of protein and nucleic acid from archival melanin-rich FFPE tissue sections. Protein extracted from bleached FFPE tissues was compatible with western blotting using anti-human GAPDH and AKT antibodies. Our bleaching condition significantly improved RNA quality compared with unbleached tissues without compromising the yield. Notably, the RNA/DNA obtained from bleached tissues was suitable for end point PCR and real-time quantitative RT-PCR. In conclusion, this improved melanin-bleaching method enhances and simplifies immunostaining procedures, and facilitates the use of melanin-rich FFPE tissues for histomorphological and PCR amplification-based molecular assays. PMID:27548802

  17. Covalent binding of formalin fixed paraffin embedded brain tissue sections to glass slides suitable for in situ hybridization.

    PubMed

    Tourtellotte, W W; Verity, A N; Schmid, P; Martinez, S; Shapshak, P

    1987-02-01

    A novel method for covalently binding formalin fixed paraffin embedded (FFPE) tissue sections to glass microscope slides is validated suitable for in situ hybridization (ISH). Using the organosilane methodology of Maples (1985), 100% tissue adhesion is reported with no nonspecific probe binding, staining, or autoradiographic artefacts. JC viral nucleic acid sequences are successfully detected in FFPE progressive multifocal leukoencephalopathy brain tissue and the Tm of the hybridized product is estimated. From the Tm the most stringent washing condition resulting in an optimal signal to noise ratio is determined. A comparison is made between currently used methods of tissue adhesion and the proposed organosilane methodology. This methodology greatly facilitates studies of conditions for ISH and elucidation of mechanisms of viral infections requiring consecutive FFPE sections. It is also applicable to studies using cryosections and cultured cells.

  18. Comparison of multiple protein extraction buffers for GeLC-MS/MS proteomic analysis of liver and colon formalin-fixed, paraffin-embedded tissues.

    PubMed

    Broeckx, Valérie; Boonen, Kurt; Pringels, Lentel; Sagaert, Xavier; Prenen, Hans; Landuyt, Bart; Schoofs, Liliane; Maes, Evelyne

    2016-02-01

    Formalin-fixed paraffin-embedded (FFPE) tissue specimens represent a potential valuable source of samples for clinical research. Since these specimens are banked in hospital archives, large cohorts of samples can be collected in short periods of time which can all be linked with a patients' clinical history. Therefore, the use of FFPE tissue in protein biomarker discovery studies gains interest. However, despite the growing number of FFPE proteome studies in the literature, there is a lack of a FFPE proteomics standard operating procedure (SOP). One of the challenging steps in the development of such a SOP is the ability to obtain an efficient and repeatable extraction of full length FFPE proteins. In this study, the protein extraction efficiency of eight protein extraction buffers is critically compared with GeLC-MS/MS (1D gel electrophoresis followed by in-gel digestion and LC-MS/MS). The data variation caused by using these extraction buffers was investigated since the variation is a very important aspect when using FFPE tissue as a source for biomarker detection. In addition, a qualitative comparison was made between the protein extraction efficiency and repeatability for FFPE tissue and fresh frozen tissue.

  19. Optimized Protocol for Quantitative Multiple Reaction Monitoring-Based Proteomic Analysis of Formalin-Fixed, Paraffin-Embedded Tissues.

    PubMed

    Kennedy, Jacob J; Whiteaker, Jeffrey R; Schoenherr, Regine M; Yan, Ping; Allison, Kimberly; Shipley, Melissa; Lerch, Melissa; Hoofnagle, Andrew N; Baird, Geoffrey Stuart; Paulovich, Amanda G

    2016-08-01

    Despite a clinical, economic, and regulatory imperative to develop companion diagnostics, precious few new biomarkers have been successfully translated into clinical use, due in part to inadequate protein assay technologies to support large-scale testing of hundreds of candidate biomarkers in formalin-fixed paraffin-embedded (FFPE) tissues. Although the feasibility of using targeted, multiple reaction monitoring mass spectrometry (MRM-MS) for quantitative analyses of FFPE tissues has been demonstrated, protocols have not been systematically optimized for robust quantification across a large number of analytes, nor has the performance of peptide immuno-MRM been evaluated. To address this gap, we used a test battery approach coupled to MRM-MS with the addition of stable isotope-labeled standard peptides (targeting 512 analytes) to quantitatively evaluate the performance of three extraction protocols in combination with three trypsin digestion protocols (i.e., nine processes). A process based on RapiGest buffer extraction and urea-based digestion was identified to enable similar quantitation results from FFPE and frozen tissues. Using the optimized protocols for MRM-based analysis of FFPE tissues, median precision was 11.4% (across 249 analytes). There was excellent correlation between measurements made on matched FFPE and frozen tissues, both for direct MRM analysis (R(2) = 0.94) and immuno-MRM (R(2) = 0.89). The optimized process enables highly reproducible, multiplex, standardizable, quantitative MRM in archival tissue specimens.

  20. Optimized Protocol for Quantitative Multiple Reaction Monitoring-Based Proteomic Analysis of Formalin-Fixed, Paraffin-Embedded Tissues.

    PubMed

    Kennedy, Jacob J; Whiteaker, Jeffrey R; Schoenherr, Regine M; Yan, Ping; Allison, Kimberly; Shipley, Melissa; Lerch, Melissa; Hoofnagle, Andrew N; Baird, Geoffrey Stuart; Paulovich, Amanda G

    2016-08-01

    Despite a clinical, economic, and regulatory imperative to develop companion diagnostics, precious few new biomarkers have been successfully translated into clinical use, due in part to inadequate protein assay technologies to support large-scale testing of hundreds of candidate biomarkers in formalin-fixed paraffin-embedded (FFPE) tissues. Although the feasibility of using targeted, multiple reaction monitoring mass spectrometry (MRM-MS) for quantitative analyses of FFPE tissues has been demonstrated, protocols have not been systematically optimized for robust quantification across a large number of analytes, nor has the performance of peptide immuno-MRM been evaluated. To address this gap, we used a test battery approach coupled to MRM-MS with the addition of stable isotope-labeled standard peptides (targeting 512 analytes) to quantitatively evaluate the performance of three extraction protocols in combination with three trypsin digestion protocols (i.e., nine processes). A process based on RapiGest buffer extraction and urea-based digestion was identified to enable similar quantitation results from FFPE and frozen tissues. Using the optimized protocols for MRM-based analysis of FFPE tissues, median precision was 11.4% (across 249 analytes). There was excellent correlation between measurements made on matched FFPE and frozen tissues, both for direct MRM analysis (R(2) = 0.94) and immuno-MRM (R(2) = 0.89). The optimized process enables highly reproducible, multiplex, standardizable, quantitative MRM in archival tissue specimens. PMID:27462933

  1. Optimized protocol for quantitative multiple reaction monitoring-based proteomic analysis of formalin-fixed, paraffin embedded tissues

    PubMed Central

    Kennedy, Jacob J.; Whiteaker, Jeffrey R.; Schoenherr, Regine M.; Yan, Ping; Allison, Kimberly; Shipley, Melissa; Lerch, Melissa; Hoofnagle, Andrew N.; Baird, Geoffrey Stuart; Paulovich, Amanda G.

    2016-01-01

    Despite a clinical, economic, and regulatory imperative to develop companion diagnostics, precious few new biomarkers have been successfully translated into clinical use, due in part to inadequate protein assay technologies to support large-scale testing of hundreds of candidate biomarkers in formalin-fixed paraffin embedded (FFPE) tissues. While the feasibility of using targeted, multiple reaction monitoring-mass spectrometry (MRM-MS) for quantitative analyses of FFPE tissues has been demonstrated, protocols have not been systematically optimized for robust quantification across a large number of analytes, nor has the performance of peptide immuno-MRM been evaluated. To address this gap, we used a test battery approach coupled to MRM-MS with the addition of stable isotope labeled standard peptides (targeting 512 analytes) to quantitatively evaluate the performance of three extraction protocols in combination with three trypsin digestion protocols (i.e. 9 processes). A process based on RapiGest buffer extraction and urea-based digestion was identified to enable similar quantitation results from FFPE and frozen tissues. Using the optimized protocols for MRM-based analysis of FFPE tissues, median precision was 11.4% (across 249 analytes). There was excellent correlation between measurements made on matched FFPE and frozen tissues, both for direct MRM analysis (R2 = 0.94) and immuno-MRM (R2 = 0.89). The optimized process enables highly reproducible, multiplex, standardizable, quantitative MRM in archival tissue specimens. PMID:27462933

  2. Genomic analysis by oligonucleotide array Comparative Genomic Hybridization utilizing formalin-fixed, paraffin-embedded tissues.

    PubMed

    Savage, Stephanie J; Hostetter, Galen

    2011-01-01

    Formalin fixation has been used to preserve tissues for more than a hundred years, and there are currently more than 300 million archival samples in the United States alone. The application of genomic protocols such as high-density oligonucleotide array Comparative Genomic Hybridization (aCGH) to formalin-fixed, paraffin-embedded (FFPE) tissues, therefore, opens an untapped resource of available tissues for research and facilitates utilization of existing clinical data in a research sample set. However, formalin fixation results in cross-linking of proteins and DNA, typically leading to such a significant degradation of DNA template that little is available for use in molecular applications. Here, we describe a protocol to circumvent formalin fixation artifact by utilizing enzymatic reactions to obtain quality DNA from a wide range of FFPE tissues for successful genome-wide discovery of gene dosage alterations in archival clinical samples.

  3. Deparaffinization of formalin-fixed paraffin-embedded tissue blocks using hot water instead of xylene.

    PubMed

    Kalantari, Narges; Bayani, Masomeh; Ghaffari, Taraneh

    2016-08-15

    This study aimed to deparaffinize formalin-fixed paraffin-embedded (FFPE) tissues using hot water instead of xylene and measuring the quantity and quality of the extracted DNA from the respective tissues. To deparaffinize the tissue sections with hot water, small sections were exposed to 90 °C distilled sterile water. After 25 FFPE tissue samples were deparaffinized with the hot water method, DNA was then extracted. The mean of optical density and the ratio of absorbance of the DNA solution were 220.01 ± 36.1 ng/μl and 1.65 ± 0.1, respectively. Polymerase chain reaction (PCR) analysis of the toll-like receptor 4(TLR4) gene showed that the method can be used as a tool for different applications. PMID:27287960

  4. Optimization of Urea Based Protein Extraction from Formalin-Fixed Paraffin-Embedded Tissue for Shotgun Proteomics

    PubMed Central

    Koepsell, Scott A.

    2016-01-01

    Urea based protein extraction of formalin-fixed paraffin-embedded (FFPE) tissue provides the most efficient workflow for proteomics due to its compatibility with liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). This study optimizes the use of urea for proteomic analysis of clinical FFPE tissue. A series of protein extraction conditions manipulating temperature and buffer composition were compared to reduce carbamylation introduced by urea and increase protein detection. Each extraction was performed on a randomized pair of serial sections of homogenous FFPE tissue and analyzed with LC-ESI-MS/MS. Results were compared in terms of yield, missed cleavages, and peptide carbamylation. Lowering extraction temperature to 60°C decreased carbamylation at the cost of decreased protein detection and yield. Protein extraction for at least 20 minutes at 95°C followed by 60°C for 2 hours maximized total protein yield while maintaining protein detection and reducing carbamylation by 7.9%. When accounting for carbamylation during analysis, this modified extraction temperature provides equivalent peptide and protein detection relative to the commercially available Qproteome® FFPE Tissue Kit. No changes to buffer composition containing 7 M urea, 2 M thiourea, and 1 M ammonium bicarbonate resulted in improvements to control conditions. Optimized urea in-solution digestion provides an efficient workflow with maximized yields for proteomic analysis of clinically relevant FFPE tissue.

  5. Optimization of Urea Based Protein Extraction from Formalin-Fixed Paraffin-Embedded Tissue for Shotgun Proteomics

    PubMed Central

    Koepsell, Scott A.

    2016-01-01

    Urea based protein extraction of formalin-fixed paraffin-embedded (FFPE) tissue provides the most efficient workflow for proteomics due to its compatibility with liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). This study optimizes the use of urea for proteomic analysis of clinical FFPE tissue. A series of protein extraction conditions manipulating temperature and buffer composition were compared to reduce carbamylation introduced by urea and increase protein detection. Each extraction was performed on a randomized pair of serial sections of homogenous FFPE tissue and analyzed with LC-ESI-MS/MS. Results were compared in terms of yield, missed cleavages, and peptide carbamylation. Lowering extraction temperature to 60°C decreased carbamylation at the cost of decreased protein detection and yield. Protein extraction for at least 20 minutes at 95°C followed by 60°C for 2 hours maximized total protein yield while maintaining protein detection and reducing carbamylation by 7.9%. When accounting for carbamylation during analysis, this modified extraction temperature provides equivalent peptide and protein detection relative to the commercially available Qproteome® FFPE Tissue Kit. No changes to buffer composition containing 7 M urea, 2 M thiourea, and 1 M ammonium bicarbonate resulted in improvements to control conditions. Optimized urea in-solution digestion provides an efficient workflow with maximized yields for proteomic analysis of clinically relevant FFPE tissue. PMID:27660725

  6. Optimization of Urea Based Protein Extraction from Formalin-Fixed Paraffin-Embedded Tissue for Shotgun Proteomics.

    PubMed

    Luebker, Stephen A; Koepsell, Scott A

    2016-01-01

    Urea based protein extraction of formalin-fixed paraffin-embedded (FFPE) tissue provides the most efficient workflow for proteomics due to its compatibility with liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). This study optimizes the use of urea for proteomic analysis of clinical FFPE tissue. A series of protein extraction conditions manipulating temperature and buffer composition were compared to reduce carbamylation introduced by urea and increase protein detection. Each extraction was performed on a randomized pair of serial sections of homogenous FFPE tissue and analyzed with LC-ESI-MS/MS. Results were compared in terms of yield, missed cleavages, and peptide carbamylation. Lowering extraction temperature to 60°C decreased carbamylation at the cost of decreased protein detection and yield. Protein extraction for at least 20 minutes at 95°C followed by 60°C for 2 hours maximized total protein yield while maintaining protein detection and reducing carbamylation by 7.9%. When accounting for carbamylation during analysis, this modified extraction temperature provides equivalent peptide and protein detection relative to the commercially available Qproteome® FFPE Tissue Kit. No changes to buffer composition containing 7 M urea, 2 M thiourea, and 1 M ammonium bicarbonate resulted in improvements to control conditions. Optimized urea in-solution digestion provides an efficient workflow with maximized yields for proteomic analysis of clinically relevant FFPE tissue. PMID:27660725

  7. Genome-wide detection of DNase I hypersensitive sites in single cells and FFPE tissue samples.

    PubMed

    Jin, Wenfei; Tang, Qingsong; Wan, Mimi; Cui, Kairong; Zhang, Yi; Ren, Gang; Ni, Bing; Sklar, Jeffrey; Przytycka, Teresa M; Childs, Richard; Levens, David; Zhao, Keji

    2015-12-01

    DNase I hypersensitive sites (DHSs) provide important information on the presence of transcriptional regulatory elements and the state of chromatin in mammalian cells. Conventional DNase sequencing (DNase-seq) for genome-wide DHSs profiling is limited by the requirement of millions of cells. Here we report an ultrasensitive strategy, called single-cell DNase sequencing (scDNase-seq) for detection of genome-wide DHSs in single cells. We show that DHS patterns at the single-cell level are highly reproducible among individual cells. Among different single cells, highly expressed gene promoters and enhancers associated with multiple active histone modifications display constitutive DHS whereas chromatin regions with fewer histone modifications exhibit high variation of DHS. Furthermore, the single-cell DHSs predict enhancers that regulate cell-specific gene expression programs and the cell-to-cell variations of DHS are predictive of gene expression. Finally, we apply scDNase-seq to pools of tumour cells and pools of normal cells, dissected from formalin-fixed paraffin-embedded tissue slides from patients with thyroid cancer, and detect thousands of tumour-specific DHSs. Many of these DHSs are associated with promoters and enhancers critically involved in cancer development. Analysis of the DHS sequences uncovers one mutation (chr18: 52417839G>C) in the tumour cells of a patient with follicular thyroid carcinoma, which affects the binding of the tumour suppressor protein p53 and correlates with decreased expression of its target gene TXNL1. In conclusion, scDNase-seq can reliably detect DHSs in single cells, greatly extending the range of applications of DHS analysis both for basic and for translational research, and may provide critical information for personalized medicine. PMID:26605532

  8. Equivalence of protein inventories obtained from formalin-fixed paraffin-embedded and frozen tissue in multidimensional liquid chromatography-tandem mass spectrometry shotgun proteomic analysis.

    PubMed

    Sprung, Robert W; Brock, Jonathan W C; Tanksley, Jarred P; Li, Ming; Washington, Mary Kay; Slebos, Robbert J C; Liebler, Daniel C

    2009-08-01

    Formalin-fixed paraffin-embedded (FFPE) tissue specimens comprise a potentially valuable resource for retrospective biomarker discovery studies, and recent work indicates the feasibility of using shotgun proteomics to characterize FFPE tissue proteins. A critical question in the field is whether proteomes characterized in FFPE specimens are equivalent to proteomes in corresponding fresh or frozen tissue specimens. Here we compared shotgun proteomic analyses of frozen and FFPE specimens prepared from the same colon adenoma tissues. Following deparaffinization, rehydration, and tryptic digestion under mild conditions, FFPE specimens corresponding to 200 microg of protein yielded approximately 400 confident protein identifications in a one-dimensional reverse phase liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. The major difference between frozen and FFPE proteomes was a decrease in the proportions of lysine C-terminal to arginine C-terminal peptides observed, but these differences had little effect on the proteins identified. No covalent peptide modifications attributable to formaldehyde chemistry were detected by analyses of the MS/MS datasets, which suggests that undetected, cross-linked peptides comprise the major class of modifications in FFPE tissues. Fixation of tissue for up to 2 days in neutral buffered formalin did not adversely impact protein identifications. Analysis of archival colon adenoma FFPE specimens indicated equivalent numbers of MS/MS spectral counts and protein group identifications from specimens stored for 1, 3, 5, and 10 years. Combination of peptide isoelectric focusing-based separation with reverse phase LC-MS/MS identified 2554 protein groups in 600 ng of protein from frozen tissue and 2302 protein groups from FFPE tissue with at least two distinct peptide identifications per protein. Analysis of the combined frozen and FFPE data showed a 92% overlap in the protein groups identified. Comparison of gene ontology

  9. Equivalence of protein inventories obtained from formalin-fixed paraffin-embedded and frozen tissue in multidimensional liquid chromatography-tandem mass spectrometry shotgun proteomic analysis.

    PubMed

    Sprung, Robert W; Brock, Jonathan W C; Tanksley, Jarred P; Li, Ming; Washington, Mary Kay; Slebos, Robbert J C; Liebler, Daniel C

    2009-08-01

    Formalin-fixed paraffin-embedded (FFPE) tissue specimens comprise a potentially valuable resource for retrospective biomarker discovery studies, and recent work indicates the feasibility of using shotgun proteomics to characterize FFPE tissue proteins. A critical question in the field is whether proteomes characterized in FFPE specimens are equivalent to proteomes in corresponding fresh or frozen tissue specimens. Here we compared shotgun proteomic analyses of frozen and FFPE specimens prepared from the same colon adenoma tissues. Following deparaffinization, rehydration, and tryptic digestion under mild conditions, FFPE specimens corresponding to 200 microg of protein yielded approximately 400 confident protein identifications in a one-dimensional reverse phase liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. The major difference between frozen and FFPE proteomes was a decrease in the proportions of lysine C-terminal to arginine C-terminal peptides observed, but these differences had little effect on the proteins identified. No covalent peptide modifications attributable to formaldehyde chemistry were detected by analyses of the MS/MS datasets, which suggests that undetected, cross-linked peptides comprise the major class of modifications in FFPE tissues. Fixation of tissue for up to 2 days in neutral buffered formalin did not adversely impact protein identifications. Analysis of archival colon adenoma FFPE specimens indicated equivalent numbers of MS/MS spectral counts and protein group identifications from specimens stored for 1, 3, 5, and 10 years. Combination of peptide isoelectric focusing-based separation with reverse phase LC-MS/MS identified 2554 protein groups in 600 ng of protein from frozen tissue and 2302 protein groups from FFPE tissue with at least two distinct peptide identifications per protein. Analysis of the combined frozen and FFPE data showed a 92% overlap in the protein groups identified. Comparison of gene ontology

  10. A reliable method for the selection of exploitable melanoma archival paraffin embedded tissues for transcript biomarker profiling.

    PubMed

    Lebbe, Celeste; Guedj, Mickael; Basset-Seguin, Nicole; Podgorniak, Marie Pierre; Menashi, Suzanne; Janin, Anne; Mourah, Samia

    2012-01-01

    The source tissue for biomarkers mRNA expression profiling of tumors has traditionally been fresh-frozen tissue. The adaptation of formalin-fixed, paraffin-embedded (FFPE) tissues for routine mRNA profiling would however be invaluable in view of their abundance and the clinical information related to them. However, their use in the clinic remains a challenge due to the poor quality of RNA extracted from such tissues. Here, we developed a method for the selection of melanoma archival paraffin-embedded tissues that can be reliably used for transcript biomarker profiling. For that, we used qRT-PCR to conduct a comparative study in matched pairs of frozen and FFPE melanoma tissues of the expression of 25 genes involved in angiogenesis/tumor invasion and 15 housekeeping genes. A classification method was developed that can select the samples with a good frozen/FFPE correlation and identify those that should be discarded on the basis of paraffin data for four reference genes only. We propose therefore a simple and inexpensive assay which improves reliability of mRNA profiling in FFPE samples by allowing the identification and analysis of "good" samples only. This assay which can be extended to other genes would however need validation at the clinical level and on independent tumor series. PMID:22272228

  11. Integrated and convenient procedure for protein extraction from formalin-fixed, paraffin-embedded tissues for LC-MS/MS analysis.

    PubMed

    Lai, Xianyin; Schneider, Bryan P

    2014-11-01

    Because fresh-frozen tissue samples associated with long-term clinical data and of rare diseases are often unobtainable at the present time, formalin-fixed paraffin-embedded (FFPE) tissue samples are considered a highly valuable resource for researchers. However, protein extraction from FFPE tissues faces challenges of deparaffinization and cross-link reversion. Current procedures for protein extraction from FFPE tissue require separate steps and toxic solvents, resulting in inconvenience in protein extraction. To overcome these limitations, an integrated method was developed using nontoxic solvents in four types of FFPE tissues. The average amount of proteins from three replicates of bladder, kidney, liver, and lung FFPE tissues were 442.6, 728.9, 736.4, and 694.7 μg with CVs of 7.5, 5.8, 2.4, and 4.5%, respectively. Proteomic analysis showed that 348, 417, 607, and 304 unique proteins were identified and quantified without specification of isoform by a least two peptides from bladder, kidney, liver, and lung FFPE tissue samples, respectively. The analysis of individual protein CV demonstrated that 97-99% of the proteins were quantified with a CV ≤ 30%, verifying the reproducibility of the integrated protein extraction method. In summary, the developed method is high-yield, reproducible, convenient, simple, low cost, nonvolatile, nonflammable, and nontoxic.

  12. Formalin-fixed paraffin-embedded tissue: The holy grail of clinical proteomics.

    PubMed

    Broeckx, Valérie; Peeters, Lise; Maes, Evelyne; Pringels, Lentel; Verjans, Eddy-Tim; Landuyt, Bart

    2014-10-01

    Tissue is the most relevant biological material to gather insight in disease mechanisms by means of omics technologies. However, fresh frozen tissue, which is generally regarded as the best imaginable source for such studies, is often not available. In case it is available, the different ways of storage (e.g. -20°C, -80°C, liquid nitrogen, etc.) hamper the conduction of reproducible multicenter studies because of different protein degradation rates. Formalin-fixed paraffin-embedded (FFPE) tissue on the contrary is considered as a valuable alternative for fresh frozen tissue, because only a few standard operation procedures are applied worldwide for the preparation of these tissues and because they are all stored in the same way. However, a study on the impact of the different preparation protocols for FFPE tissue was still lacking. Therefore, Bronsert et al. in this issue [Bronsert, P., Weißer, J., Biniossek, M. L., Kuehs, M. et al., Proteomics Clin. Appl. 2014, 8 786-804] conducted such a study that provides proof that there is no significant effect between these sample preparations procedures, and thereby they further open the gate for FFPE tissues to enter the field of clinical proteomics.

  13. Use of polymerase chain reaction in detection of Marek’s disease and reticuloendotheliosis viruses in formalin-fixed, paraffin-embedded tumorous tissues

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A simple polymerase chain reaction (PCR) method was developed for the diagnosis of Marek’s disease (MD) and reticuloendotheliosis (RE) in formalin-fixed paraffin-embedded (FFPE) tissues; and for the diagnosis of MD in tissues only preserved in 10% neutral buffered formalin. MD virus (MDV) and RE vi...

  14. Detection of Newcastle disease virus RNA by reverse transcription polymerase chain reaction using formalin-fixed, paraffin-embedded tissue and comparison with immunohistochemistry and in situ hybridization

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The usefulness of reverse transcription polymerase chain reaction (RT-PCR) from formalin-fixed, paraffin-embedded (FFPE) tissues was examined and compared to the immunohistochemistry (IHC) and in situ hybridization (ISH) assays for detection of Newcastle disease virus (NDV). Spleen and lung tissues...

  15. Use of Polymerase chain reaction (PCR) in diagnosis of Marek’s disease and reticuloendotheliosis in formalin-fixed, paraffin-embedded tumorous tissues

    Technology Transfer Automated Retrieval System (TEKTRAN)

    PCR was used in diagnosis of Marek’s disease (MD) and reticuloendotheliosis (RE) in formalin-fixed, paraffin-embedded (FFPE) tumorous tissues that have been stored for periods varied from 5-244 months. In another experiment, PCR was also used in diagnosis of MD in tumorous tissues that have been onl...

  16. Comparing Platforms for Messenger RNA Expression Profiling of Archival Formalin-Fixed, Paraffin-Embedded Tissues.

    PubMed

    Tyekucheva, Svitlana; Martin, Neil E; Stack, Edward C; Wei, Wei; Vathipadiekal, Vinod; Waldron, Levi; Fiorentino, Michelangelo; Lis, Rosina T; Stampfer, Meir J; Loda, Massimo; Parmigiani, Giovanni; Mucci, Lorelei A; Birrer, Michael

    2015-07-01

    Archival formalin-fixed, paraffin-embedded (FFPE) tissue specimens represent a readily available but largely untapped resource for gene expression profiling-based biomarker discovery. Several technologies have been proposed to cope with the bias from RNA cross-linking and degradation associated with archival specimens to generate data comparable with RNA from fresh-frozen materials. Direct comparison studies of these RNA expression platforms remain rare. We compared two commercially available platforms for RNA expression profiling of archival FFPE specimens from clinical studies of prostate and ovarian cancer: the Affymetrix Human Gene 1.0ST Array following whole-transcriptome amplification using the NuGen WT-Ovation FFPE System V2, and the NanoString nCounter without amplification. For each assay, we profiled 7 prostate and 11 ovarian cancer specimens, with a block age of 4 to 21 years. Both platforms produced gene expression profiles with high sensitivity and reproducibility through technical repeats from FFPE materials. Sensitivity and reproducibility remained high across block age within each cohort. A strong concordance was shown for the transcript expression values for genes detected by both platforms. We showed the biological validity of specific gene signatures generated by both platforms for both cohorts. Our study supports the feasibility of gene expression profiling and large-scale signature validation on archival prostate and ovarian tumor specimens using commercial platforms. These approaches have the potential to aid precision medicine with biomarker discovery and validation.

  17. Comparing Platforms for Messenger RNA Expression Profiling of Archival Formalin-Fixed, Paraffin-Embedded Tissues.

    PubMed

    Tyekucheva, Svitlana; Martin, Neil E; Stack, Edward C; Wei, Wei; Vathipadiekal, Vinod; Waldron, Levi; Fiorentino, Michelangelo; Lis, Rosina T; Stampfer, Meir J; Loda, Massimo; Parmigiani, Giovanni; Mucci, Lorelei A; Birrer, Michael

    2015-07-01

    Archival formalin-fixed, paraffin-embedded (FFPE) tissue specimens represent a readily available but largely untapped resource for gene expression profiling-based biomarker discovery. Several technologies have been proposed to cope with the bias from RNA cross-linking and degradation associated with archival specimens to generate data comparable with RNA from fresh-frozen materials. Direct comparison studies of these RNA expression platforms remain rare. We compared two commercially available platforms for RNA expression profiling of archival FFPE specimens from clinical studies of prostate and ovarian cancer: the Affymetrix Human Gene 1.0ST Array following whole-transcriptome amplification using the NuGen WT-Ovation FFPE System V2, and the NanoString nCounter without amplification. For each assay, we profiled 7 prostate and 11 ovarian cancer specimens, with a block age of 4 to 21 years. Both platforms produced gene expression profiles with high sensitivity and reproducibility through technical repeats from FFPE materials. Sensitivity and reproducibility remained high across block age within each cohort. A strong concordance was shown for the transcript expression values for genes detected by both platforms. We showed the biological validity of specific gene signatures generated by both platforms for both cohorts. Our study supports the feasibility of gene expression profiling and large-scale signature validation on archival prostate and ovarian tumor specimens using commercial platforms. These approaches have the potential to aid precision medicine with biomarker discovery and validation. PMID:25937617

  18. Application of array CGH on archival formalin-fixed paraffin-embedded tissues including small numbers of microdissected cells.

    PubMed

    Johnson, Nicola A; Hamoudi, Rifat A; Ichimura, Koichi; Liu, Lu; Pearson, Danita M; Collins, V Peter; Du, Ming-Qing

    2006-09-01

    Array-based comparative genomic hybridisation (aCGH) has diverse applications in cancer gene discovery and translational research. Currently, aCGH is performed primarily using high molecular weight DNA samples and its application to formalin-fixed and paraffin-embedded (FFPE) tissues remains to be established. To explore how aCGH can be reliably applied to archival FFPE tissues and whether it is possible to apply aCGH to small numbers of cells microdissected from FFPE tissue sections, we have systematically performed aCGH on 15 pairs of matched frozen and FFPE astrocytic tumour tissues using a well-established in-house human 1 Mb BAC/PAC genomic array. By spiking tumour DNA with normal DNA, we demonstrated that at least 70% of tumour DNA was required for reliable aCGH analysis. Using aCGH data from frozen tissue as a reference, it was found that only FFPE astrocytic tumour tissues that supported PCR amplification of >300 bp DNA fragment provided high quality, reproducible aCGH data. The presence of necrosis in a tissue specimen had an adverse effect on the quality of aCGH, while fixation in formalin for up to 96 h of fresh tissue did not appear to affect the quality of the result. As little as 10-20 ng DNA from frozen or FFPE tissues could be readily used for aCGH analysis following whole genome amplification (WGA). Furthermore, as few as 2000 microdissected cells from haematoxylin-stained slides of archival FFPE tissues could be successfully used for aCGH investigations when WGA was used. By careful assessment of DNA integrity and review of histology, to exclude necrosis and select specimens with a high proportion of tumour cells, it is feasible to preselect archival FFPE tissues adequate for aCGH analysis. With the help of microdissection and WGA, it is also possible to apply aCGH to histologically defined lesions, such as carcinoma in situ.

  19. Use of polymerase chain reaction in detection of Marek's disease and reticuloendotheliosis viruses in formalin-fixed, paraffin-embedded tumorous tissues.

    PubMed

    Cao, Weisheng; Mays, Jody; Dunn, John; Fulton, Richard; Silva, Robert; Fadly, Aly

    2013-12-01

    A simple PCR method was developed for the detection of Marek's disease (MD) and reticuloendotheliosis (RE) in formalin-fixed paraffin-embedded (FFPE) tissues, and for the detection of MD in tissues only preserved in 10% neutral buffered formalin. MD virus (MDV) and RE virus proviral DNA were detected in FFPE tissues stored for over 20 yr. MDV was also detected in tissues only preserved in formalin for up to 6 mo. The data indicate that PCR of formalin-fixed and FFPE tissues is a simple and valuable tool that can be used to identify MD and RE infection. The method described in this paper is a good alternative to any biologic or immunohistochemical assay to confirm the detection of MD and RE, as it does not require shipping frozen tissues to the diagnostic laboratory.

  20. Protein unlocking procedures of formalin-fixed paraffin-embedded tissues: application to MALDI-TOF imaging MS investigations.

    PubMed

    Ronci, Maurizio; Bonanno, Elena; Colantoni, Alfredo; Pieroni, Luisa; Di Ilio, Carmine; Spagnoli, Luigi Giusto; Federici, Giorgio; Urbani, Andrea

    2008-09-01

    Archival formalin-fixed paraffin-embedded (FFPE) tissues are a powerful tool for examining the clinical course of diseases. These specimens represent an incredible mine of valuable clinical and biological information for proteomic investigation. MALDI-TOF imaging MS (MALDI-IMS) is a protein profiling technique which enables the direct sampling of histological section; however, the quality of molecular data are strongly influenced by the tissue preparation condition. In fact, in previous years most of the studies employing such a technological platform have been conducted using cryo-preserved tissues. We have developed an in vitro approach using "tissue surrogate" samples in order to explore different protein unlocking procedures which might enable a suitable recovery of polypeptides for MS analysis. The developed protocols have been compared both by MALDI-TOF MS and nLC-MS(E) analysis either on surrogate samples or on FFPE specimen from human breast cancer. The collected evidence has been applied for the preparation of FFPE tissue sections following MALDI-IMS analysis. Our results outline the possibility to obtain valuable peptide mass spectra profiles form FFPE preparations by applying a combined two steps procedure of heat induced antigen retrieval (HIAR) in presence of EDTA and on target trypsin hydrolysis. A multivariate statistical evaluation is presented and discussed according to molecular spatial distributions and tissue morphology.

  1. Tissue fixed with formalin and processed without paraffin embedding is suitable for imaging of both peptides and lipids by MALDI-IMS.

    PubMed

    Pietrowska, Monika; Gawin, Marta; Polańska, Joanna; Widłak, Piotr

    2016-06-01

    Type and quality of sample preparation have significant impact on imaging mass spectrometry results. Though imaging of fresh-frozen tissues is considered to give the best results, they are incompatible with clinical practice, since routine diagnostics is most frequently performed using formalin-fixed tissues, and formalin-fixed paraffin-embedded material is a gold standard in histopathology. We aimed to assess utility of formalin-fixed tissue specimen processed without paraffin embedding (i.e., deep-frozen and cryo-sectioned) for MALDI imaging of both peptides and lipids. Peptide and lipid imaging was performed in fresh-frozen, FFPE and formalin-fixed/frozen samples of a mouse kidney, then composition of the resulting spectra was compared. We demonstrated similarity of spectra registered during peptide imaging in FFPE and formalin-fixed/frozen tissues, and similarity of spectra registered during lipid imaging in fresh-frozen and formalin-fixed/frozen material. Furthermore, molecular images of formalin-fixed/frozen tissue resembled the features of both fresh-frozen and FFPE tissue in the case of peptide imaging, and the features of fresh-frozen tissue in the case of lipid imaging. We conclude that tissue preserved by formalin fixation and processed without paraffin embedding can be considered as an alternative to both fresh-frozen and FFPE material.

  2. Tissue fixed with formalin and processed without paraffin embedding is suitable for imaging of both peptides and lipids by MALDI-IMS.

    PubMed

    Pietrowska, Monika; Gawin, Marta; Polańska, Joanna; Widłak, Piotr

    2016-06-01

    Type and quality of sample preparation have significant impact on imaging mass spectrometry results. Though imaging of fresh-frozen tissues is considered to give the best results, they are incompatible with clinical practice, since routine diagnostics is most frequently performed using formalin-fixed tissues, and formalin-fixed paraffin-embedded material is a gold standard in histopathology. We aimed to assess utility of formalin-fixed tissue specimen processed without paraffin embedding (i.e., deep-frozen and cryo-sectioned) for MALDI imaging of both peptides and lipids. Peptide and lipid imaging was performed in fresh-frozen, FFPE and formalin-fixed/frozen samples of a mouse kidney, then composition of the resulting spectra was compared. We demonstrated similarity of spectra registered during peptide imaging in FFPE and formalin-fixed/frozen tissues, and similarity of spectra registered during lipid imaging in fresh-frozen and formalin-fixed/frozen material. Furthermore, molecular images of formalin-fixed/frozen tissue resembled the features of both fresh-frozen and FFPE tissue in the case of peptide imaging, and the features of fresh-frozen tissue in the case of lipid imaging. We conclude that tissue preserved by formalin fixation and processed without paraffin embedding can be considered as an alternative to both fresh-frozen and FFPE material. PMID:27001204

  3. Detection of BRCA1 and BRCA2 Ashkenazi Jewish founder mutations in formalin-fixed paraffin-embedded tissues using conventional PCR and heteroduplex/amplicon size differences.

    PubMed

    Mangold, Kathy A; Wang, Vivien; Weissman, Scott M; Rubinstein, Wendy S; Kaul, Karen L

    2010-01-01

    In many families with histories suggestive of BRCA1- or BRCA2-related disease, the proband is deceased. Reliable assessment of archived tissue blocks not amenable to full gene sequencing would be helpful. In this study, a polymerase chain reaction (PCR) assay using primers that bracket the BRCA mutation site and microfluidics-based detection of heteroduplex/amplicon size differences was developed to circumvent artifacts associated with low quality DNA from formalin-fixed paraffin-embedded (FFPE) tissue. Genomic DNA was extracted from 100 FFPE specimens from patients that had previously undergone BRCA gene sequence analysis on blood specimens. Conventional PCR amplification products were differentiated using the Agilent 2100 Bioanalyzer. One FFPE specimen failed to amplify the wild-type alleles for all three sites and was therefore called indeterminate. All 62 FFPE specimens with known Ashkenazi Jewish founder mutations had both the wild-type and the correct mutated allele amplified, including one specimen that failed to amplify the mutant allele in other real-time PCR assays. Appropriately, 21 FFPE specimens known to have other BRCA1/2 mutations and 16 without any mutation had only the wild-type allele correctly amplified for each target. Therefore, by changing the primer location and detecting amplicons via heteroduplexes formed by size differences, we identified mutations from FFPE tissues missed using real-time methods.

  4. Multiplexed color-coded probe-based gene expression assessment for clinical molecular diagnostics in formalin-fixed paraffin-embedded human renal allograft tissue.

    PubMed

    Adam, Benjamin; Afzali, Bahman; Dominy, Katherine M; Chapman, Erin; Gill, Reeda; Hidalgo, Luis G; Roufosse, Candice; Sis, Banu; Mengel, Michael

    2016-03-01

    Histopathologic diagnoses in transplantation can be improved with molecular testing. Preferably, molecular diagnostics should fit into standard-of-care workflows for transplant biopsies, that is, formalin-fixed paraffin-embedded (FFPE) processing. The NanoString(®) gene expression platform has recently been shown to work with FFPE samples. We aimed to evaluate its methodological robustness and feasibility for gene expression studies in human FFPE renal allograft samples. A literature-derived antibody-mediated rejection (ABMR) 34-gene set, comprised of endothelial, NK cell, and inflammation transcripts, was analyzed in different retrospective biopsy cohorts and showed potential to molecularly discriminate ABMR cases, including FFPE samples. NanoString(®) results were reproducible across a range of RNA input quantities (r = 0.998), with different operators (r = 0.998), and between different reagent lots (r = 0.983). There was moderate correlation between NanoString(®) with FFPE tissue and quantitative reverse transcription polymerase chain reaction (qRT-PCR) with corresponding dedicated fresh-stabilized tissue (r = 0.487). Better overall correlation with histology was observed with NanoString(®) (r = 0.354) than with qRT-PCR (r = 0.146). Our results demonstrate the feasibility of multiplexed gene expression quantification from FFPE renal allograft tissue. This represents a method for prospective and retrospective validation of molecular diagnostics and its adoption in clinical transplantation pathology.

  5. Use of formalin-fixed and paraffin-embedded tissues for diagnosis and therapy in routine clinical settings.

    PubMed

    Berg, Daniela; Malinowsky, Katharina; Reischauer, Bilge; Wolff, Claudia; Becker, Karl-Friedrich

    2011-01-01

    Formalin-fixed and paraffin-embedded (FFPE) tissues are used routinely everyday in hospitals world-wide for histopathological diagnosis of diseases like cancer. Due to formalin-induced cross-linking of proteins, FFPE tissues present a particular challenge for proteomic analysis. Nevertheless, there has been recent progress for extraction-based protein analysis in these tissues. Novel tools developed in the last few years are urgently needed because precise protein biomarker quantification in clinical FFPE tissues will be crucial for treatment decisions and to assess success or failure of current and future personalized molecular therapies. Furthermore, they will help to conceive why only a subset of patients responds to individualized treatments. Reverse phase protein array (RPPA) is a very promising new technology for quick and simultaneous analysis of many patient samples allowing relative and absolute protein quantifications. In this chapter, we show how protein extraction from FFPE tissues might facilitate the implementation of RPPA for therapy decisions and discuss challenges for application of RPPA in clinical trials and routine settings.

  6. Multiplexed miRNA Fluorescence In Situ Hybridization for Formalin-Fixed Paraffin-Embedded Tissues

    PubMed Central

    Renwick, Neil; Cekan, Pavol; Bognanni, Claudia; Tuschl, Thomas

    2015-01-01

    Multiplexed miRNA fluorescence in situ hybridization (miRNA FISH) is an advanced method for visualizing differentially expressed miRNAs, together with other reference RNAs, in archival tissues. Some miRNAs are excellent disease biomarkers due to their abundance and cell-type specificity. However, these short RNA molecules are difficult to visualize due to loss by diffusion, probe mishybridization, and signal detection and signal amplification issues. Here, we describe a reliable and adjustable method for visualizing and normalizing miRNA signals in formalin-fixed paraffin-embedded (FFPE) tissue sections. PMID:25218385

  7. An Optimized Method of Metabolite Extraction from Formalin-Fixed Paraffin-Embedded Tissue for GC/MS Analysis

    PubMed Central

    Wojakowska, Anna; Marczak, Łukasz; Jelonek, Karol; Polanski, Krzysztof; Widlak, Piotr; Pietrowska, Monika

    2015-01-01

    Formalin-fixed paraffin-embedded (FFPE) tissue specimens constitute a highly valuable source of clinical material for retrospective molecular studies. However, metabolomic assessment of such archival material remains still in its infancy. Hence, there is an urgent need for efficient methods enabling extraction and profiling of metabolites present in FFPE tissue specimens. Here we demonstrate the methodology for isolation of primary metabolites from archival tissues; either fresh-frozen, formalin-fixed or formalin-fixed and paraffin-embedded specimens of mouse kidney were analysed and compared in this work. We used gas chromatography followed by mass spectrometry (GC/MS approach) to identify about 80 metabolites (including amino acids, saccharides, carboxylic acids, fatty acids) present in such archive material. Importantly, about 75% of identified compounds were detected in all three types of specimens. Moreover, we observed that fixation with formalin itself (and their duration) did not affect markedly the presence of particular metabolites in tissue-extracted material, yet fixation for 24h could be recommended as a practical standard. Paraffin embedding influenced efficiency of extraction, which resulted in reduced quantities of several compounds. Nevertheless, we proved applicability of FFPE specimens for non-targeted GS/MS-based profiling of tissue metabolome, which is of great importance for feasibility of metabolomics studies using retrospective clinical material. PMID:26348873

  8. An Optimized Method of Metabolite Extraction from Formalin-Fixed Paraffin-Embedded Tissue for GC/MS Analysis.

    PubMed

    Wojakowska, Anna; Marczak, Łukasz; Jelonek, Karol; Polanski, Krzysztof; Widlak, Piotr; Pietrowska, Monika

    2015-01-01

    Formalin-fixed paraffin-embedded (FFPE) tissue specimens constitute a highly valuable source of clinical material for retrospective molecular studies. However, metabolomic assessment of such archival material remains still in its infancy. Hence, there is an urgent need for efficient methods enabling extraction and profiling of metabolites present in FFPE tissue specimens. Here we demonstrate the methodology for isolation of primary metabolites from archival tissues; either fresh-frozen, formalin-fixed or formalin-fixed and paraffin-embedded specimens of mouse kidney were analysed and compared in this work. We used gas chromatography followed by mass spectrometry (GC/MS approach) to identify about 80 metabolites (including amino acids, saccharides, carboxylic acids, fatty acids) present in such archive material. Importantly, about 75% of identified compounds were detected in all three types of specimens. Moreover, we observed that fixation with formalin itself (and their duration) did not affect markedly the presence of particular metabolites in tissue-extracted material, yet fixation for 24h could be recommended as a practical standard. Paraffin embedding influenced efficiency of extraction, which resulted in reduced quantities of several compounds. Nevertheless, we proved applicability of FFPE specimens for non-targeted GS/MS-based profiling of tissue metabolome, which is of great importance for feasibility of metabolomics studies using retrospective clinical material.

  9. An Optimized Method of Metabolite Extraction from Formalin-Fixed Paraffin-Embedded Tissue for GC/MS Analysis.

    PubMed

    Wojakowska, Anna; Marczak, Łukasz; Jelonek, Karol; Polanski, Krzysztof; Widlak, Piotr; Pietrowska, Monika

    2015-01-01

    Formalin-fixed paraffin-embedded (FFPE) tissue specimens constitute a highly valuable source of clinical material for retrospective molecular studies. However, metabolomic assessment of such archival material remains still in its infancy. Hence, there is an urgent need for efficient methods enabling extraction and profiling of metabolites present in FFPE tissue specimens. Here we demonstrate the methodology for isolation of primary metabolites from archival tissues; either fresh-frozen, formalin-fixed or formalin-fixed and paraffin-embedded specimens of mouse kidney were analysed and compared in this work. We used gas chromatography followed by mass spectrometry (GC/MS approach) to identify about 80 metabolites (including amino acids, saccharides, carboxylic acids, fatty acids) present in such archive material. Importantly, about 75% of identified compounds were detected in all three types of specimens. Moreover, we observed that fixation with formalin itself (and their duration) did not affect markedly the presence of particular metabolites in tissue-extracted material, yet fixation for 24h could be recommended as a practical standard. Paraffin embedding influenced efficiency of extraction, which resulted in reduced quantities of several compounds. Nevertheless, we proved applicability of FFPE specimens for non-targeted GS/MS-based profiling of tissue metabolome, which is of great importance for feasibility of metabolomics studies using retrospective clinical material. PMID:26348873

  10. Choice of Illumination System & Fluorophore for Multiplex Immunofluorescence on FFPE Tissue Sections.

    PubMed

    Prost, Sandrine; Kishen, Ria E B; Kluth, David C; Bellamy, Christopher O C

    2016-01-01

    The recent availability of novel dyes and alternative light sources to facilitate complex tissue immunofluorescence studies such as multiplex labelling has not been matched by reports critically evaluating the considerations and relative benefits of these new tools, particularly in combination. Product information is often limited to wavelengths used for older fluorophores (FITC, TRITC & corresponding Alexa dyes family). Consequently, novel agents such as Quantum dots are not widely appreciated or used, despite highly favourable properties including extremely bright emission, stability and potentially reduced tissue autofluorescence at the excitation wavelength. Using spectral analysis, we report here a detailed critical appraisal and comparative evaluation of different light sources and fluorophores in multiplex immunofluorescence of clinical biopsy sections. The comparison includes mercury light, metal halide and 3 different LED-based systems, using 7 Qdots (525, 565, 585, 605, 625, 705), Cy3 and Cy5. We discuss the considerations relevant to achieving the best combination of light source and fluorophore for accurate multiplex fluorescence quantitation. We highlight practical limitations and confounders to quantitation with filter-based approaches. PMID:27632367

  11. Choice of Illumination System & Fluorophore for Multiplex Immunofluorescence on FFPE Tissue Sections

    PubMed Central

    Kishen, Ria E. B.; Kluth, David C.; Bellamy, Christopher O. C.

    2016-01-01

    The recent availability of novel dyes and alternative light sources to facilitate complex tissue immunofluorescence studies such as multiplex labelling has not been matched by reports critically evaluating the considerations and relative benefits of these new tools, particularly in combination. Product information is often limited to wavelengths used for older fluorophores (FITC, TRITC & corresponding Alexa dyes family). Consequently, novel agents such as Quantum dots are not widely appreciated or used, despite highly favourable properties including extremely bright emission, stability and potentially reduced tissue autofluorescence at the excitation wavelength. Using spectral analysis, we report here a detailed critical appraisal and comparative evaluation of different light sources and fluorophores in multiplex immunofluorescence of clinical biopsy sections. The comparison includes mercury light, metal halide and 3 different LED-based systems, using 7 Qdots (525, 565, 585, 605, 625, 705), Cy3 and Cy5. We discuss the considerations relevant to achieving the best combination of light source and fluorophore for accurate multiplex fluorescence quantitation. We highlight practical limitations and confounders to quantitation with filter-based approaches. PMID:27632367

  12. High-resolution MALDI-FT-ICR MS imaging for the analysis of metabolites from formalin-fixed, paraffin-embedded clinical tissue samples.

    PubMed

    Buck, Achim; Ly, Alice; Balluff, Benjamin; Sun, Na; Gorzolka, Karin; Feuchtinger, Annette; Janssen, Klaus-Peter; Kuppen, Peter J K; van de Velde, Cornelis J H; Weirich, Gregor; Erlmeier, Franziska; Langer, Rupert; Aubele, Michaela; Zitzelsberger, Horst; Aichler, Michaela; Walch, Axel

    2015-09-01

    We present the first analytical approach to demonstrate the in situ imaging of metabolites from formalin-fixed, paraffin-embedded (FFPE) human tissue samples. Using high-resolution matrix-assisted laser desorption/ionization Fourier-transform ion cyclotron resonance mass spectrometry imaging (MALDI-FT-ICR MSI), we conducted a proof-of-principle experiment comparing metabolite measurements from FFPE and fresh frozen tissue sections, and found an overlap of 72% amongst 1700 m/z species. In particular, we observed conservation of biomedically relevant information at the metabolite level in FFPE tissues. In biomedical applications, we analysed tissues from 350 different cancer patients and were able to discriminate between normal and tumour tissues, and different tumours from the same organ, and found an independent prognostic factor for patient survival. This study demonstrates the ability to measure metabolites in FFPE tissues using MALDI-FT-ICR MSI, which can then be assigned to histology and clinical parameters. Our approach is a major technical, histochemical, and clinicopathological advance that highlights the potential for investigating diseases in archived FFPE tissues. PMID:25965788

  13. Molecular profiling of signalling pathways in formalin-fixed and paraffin-embedded cancer tissues.

    PubMed

    Berg, Daniela; Hipp, Susanne; Malinowsky, Katharina; Böllner, Claudia; Becker, Karl-Friedrich

    2010-01-01

    In most hospitals word-wide, histopathological cancer diagnosis is currently based on formalin-fixed and paraffin-embedded (FFPE) tissues. In the last few years new approaches and developments in patient-tailored cancer therapy have raised the need to select more precisely those patients, who will respond to personalised treatments. The most efficient way for optimal therapy and patient selection is probably to provide a tumour-specific protein network portrait prior to treatment. The discovery and characterisation of deregulated signalling molecules (e.g. human epidermal growth factor receptor 2, mitogen-activated protein kinases) are very promising candidates for the identification of new suitable therapy targets and for the selection of those patients who will receive the greatest benefit from individualised treatments. The reverse phase protein array (RPPA) is a promising new technology that allows quick, precise and simultaneous analysis of many components of a network. Importantly it requires only limited amounts of routine clinical material (e.g. FFPE biopsies) and can be used for absolute protein measurements. We and other research groups have described successful protein extraction from routine FFPE tissues. In this manuscript we show how these recent developments might facilitate the implementation of RPPA in clinical trials and routine settings.

  14. Comparison of Different Buffers for Protein Extraction from Formalin-Fixed and Paraffin-Embedded Tissue Specimens.

    PubMed

    Shen, Kaini; Sun, Jian; Cao, Xinxin; Zhou, Daobin; Li, Jian

    2015-01-01

    We determined the best extraction buffer for proteomic investigation using formalin-fixation and paraffin-embedded (FFPE) specimens. A Zwittergent 3-16 based buffer, sodium dodecyl sulfate (SDS)-containing buffer with/without polyethylene glycol 20000 (PEG20000), urea-containing buffer, and FFPE-FASP protein preparation kit were compared for protein extraction from different types of rat FFPE tissues, including the heart, brain, liver, lung, and kidney. All of the samples were divided into two groups of laser microdissected (LMD) and non-LMD specimens. For both kinds of specimens, Zwittergent was the most efficient buffer for identifying peptides and proteins, was broadly applicable to different tissues without impairing the enzymatic digestion, and was well compatible with mass spectrometry analysis. As a high molecular weight carrier substance, PEG20000 improved the identification of peptides and proteins; however, such an advantage is limited to tissues containing submicrograms to micrograms of protein. Considering its low lytic strength, urea-containing buffer would not be the first alternative for protein recovery. In conclusion, Zwittergent 3-16 is an effective buffer for extracting proteins from FFPE specimens for downstream proteomics analysis.

  15. Comparison of Different Buffers for Protein Extraction from Formalin-Fixed and Paraffin-Embedded Tissue Specimens.

    PubMed

    Shen, Kaini; Sun, Jian; Cao, Xinxin; Zhou, Daobin; Li, Jian

    2015-01-01

    We determined the best extraction buffer for proteomic investigation using formalin-fixation and paraffin-embedded (FFPE) specimens. A Zwittergent 3-16 based buffer, sodium dodecyl sulfate (SDS)-containing buffer with/without polyethylene glycol 20000 (PEG20000), urea-containing buffer, and FFPE-FASP protein preparation kit were compared for protein extraction from different types of rat FFPE tissues, including the heart, brain, liver, lung, and kidney. All of the samples were divided into two groups of laser microdissected (LMD) and non-LMD specimens. For both kinds of specimens, Zwittergent was the most efficient buffer for identifying peptides and proteins, was broadly applicable to different tissues without impairing the enzymatic digestion, and was well compatible with mass spectrometry analysis. As a high molecular weight carrier substance, PEG20000 improved the identification of peptides and proteins; however, such an advantage is limited to tissues containing submicrograms to micrograms of protein. Considering its low lytic strength, urea-containing buffer would not be the first alternative for protein recovery. In conclusion, Zwittergent 3-16 is an effective buffer for extracting proteins from FFPE specimens for downstream proteomics analysis. PMID:26580073

  16. Two methods for proteomic analysis of formalin-fixed, paraffin embedded tissue result in differential protein identification, data quality, and cost.

    PubMed

    Luebker, Stephen A; Wojtkiewicz, Melinda; Koepsell, Scott A

    2015-11-01

    Formalin-fixed paraffin-embedded (FFPE) tissue is a rich source of clinically relevant material that can yield important translational biomarker discovery using proteomic analysis. Protocols for analyzing FFPE tissue by LC-MS/MS exist, but standardization of procedures and critical analysis of data quality is limited. This study compared and characterized data obtained from FFPE tissue using two methods: a urea in-solution digestion method (UISD) versus a commercially available Qproteome FFPE Tissue Kit method (Qkit). Each method was performed independently three times on serial sections of homogenous FFPE tissue to minimize pre-analytical variations and analyzed with three technical replicates by LC-MS/MS. Data were evaluated for reproducibility and physiochemical distribution, which highlighted differences in the ability of each method to identify proteins of different molecular weights and isoelectric points. Each method replicate resulted in a significant number of new protein identifications, and both methods identified significantly more proteins using three technical replicates as compared to only two. UISD was cheaper, required less time, and introduced significant protein modifications as compared to the Qkit method, which provided more precise and higher protein yields. These data highlight significant variability among method replicates and type of method used, despite minimizing pre-analytical variability. Utilization of only one method or too few replicates (both method and technical) may limit the subset of proteomic information obtained. PMID:26306679

  17. Characterization of anti-TIMP-1 monoclonal antibodies for immunohistochemical localization in formalin-fixed, paraffin-embedded tissue.

    PubMed

    Sorensen, Irene Vejgaard; Fenger, Claus; Winther, Henrik; Foged, Niels T; Lademann, Ulrik; Brünner, Nils; Usher, Pernille A

    2006-10-01

    The aim of this study was to evaluate seven anti-TIMP-1 (tissue inhibitor of metalloproteinase-1) monoclonal antibodies by immunohistochemical (IHC) staining of formalin-fixed, paraffin-embedded (FFPE) tissue. Detection of the TIMP-1 protein was studied by IHC in FFPE human archival normal and neoplastic samples. Indirect IHC technique was used, and the seven antibodies (clones VT1, VT2, VT4, VT5, VT6, VT7, and VT8) were tested in various concentrations using different pretreatment protocols. All seven VT antibodies specifically immunostained the cytoplasm of islets of Langerhans cells in normal pancreas, epithelial cells of hyperplastic prostate, tumor cells of medullary thyroid carcinoma, and fibroblast-like cells of malignant melanoma. Specificity of the anti-TIMP-1 antibodies was confirmed by several controls, e.g., Western blotting on proteins extracted from FFPE tissue showed that the VT7 antibody reacted specifically with a protein band of approximately 28 kDa, corresponding to the molecular mass of TIMP-1. However, sensitivity varied with the different antibodies. Use of heat-induced epitope retrieval (HIER) and the VT7 clone applied at low concentrations demonstrated more intense immunoreactivity with the TIMP-1-positive cell types compared to the other six clones. Furthermore, when tested on a range of normal and neoplastic endocrine tissues, the VT7 clone demonstrated immunoreactivity with all neuroendocrine cell types. In conclusion, all seven antibodies detected TIMP-1 protein in various normal and neoplastic FFPE tissues, but one clone, VT7, was superior for IHC staining of TIMP-1 in FFPE tissue sections when using HIER.

  18. A predictive factor of the quality of microarray comparative genomic hybridization analysis for formalin-fixed paraffin-embedded archival tissue.

    PubMed

    Nakao, Kenjiro; Oikawa, Masahiro; Arai, Junichi; Mussazhanova, Zhanna; Kondo, Hisayoshi; Shichijo, Kazuko; Nakashima, Masahiro; Hayashi, Tomayoshi; Yoshiura, Koh-Ichiro; Hatachi, Toshiko; Nagayasu, Takeshi

    2013-09-01

    Utilizing formalin-fixed paraffin-embedded (FFPE) archival tissue, the most common form of tissue preservation in routine practice, for cytogenetic analysis using microarray comparative genomic hybridization (aCGH) remains challenging. We searched for a predictive factor of the performance of FFPE DNA in aCGH analysis. DNA was extracted from 63 FFPE archival tissue samples of various tissue types (31 breast cancers, 24 lung cancers, and 8 thyroid tumors), followed by aCGH analysis using high-density oligonucleotide microarrays. Tumor DNA from matched frozen samples and from FFPE samples after whole-genome amplification were also analyzed in 2 and 4 case, respectively. The derivative log ratio spread (DLRSpread) was used to assess the overall quality of each aCGH result. The DLRSpread correlated significantly with the double-stranded DNA ratio of tumor DNA, storage time, and the degree of labeling with Cy5 (P<0.0001; correlation coefficients=-0.796, 0.551, -0.481, respectively). Stepwise multiple linear regression analysis revealed that the double-stranded DNA ratio of tumor DNA is the most significant predictive factor of DLRSpread (regression coefficient=-0.4798; P=<0.0001). The cytogenetic profiles of FFPE and matched frozen samples showed good concordance. Although the double-stranded DNA ratios were increased after whole-genome amplification, the DLRSpread was not improved. The double-stranded DNA ratio can be used to predict the performance of aCGH analysis for DNA from FFPE samples. Using this quality metric, valuable FFPE archival tissue samples can be utilized for aCGH analysis.

  19. Excavation of a buried treasure--DNA, mRNA, miRNA and protein analysis in formalin fixed, paraffin embedded tissues.

    PubMed

    Klopfleisch, R; Weiss, A T A; Gruber, A D

    2011-06-01

    Fresh or frozen tissue samples will always be the best tissue source for the analysis of nucleic acids and proteins from tissues. However, their long-term storage is expensive and laborious. Much interest has therefore been focused on the question whether the almost infinite resources of formalin fixed and paraffin embedded tissue samples in the archives of pathology and histology departments can be used for research on biomarkers and molecular mechanisms of disease. In recent years the methods and protocols for the extraction of DNA, mRNA, miRNA and proteins from formalin-fixed and paraffin-embedded tissue samples have improved enormously. Especially, the possibilities of analysing DNA and miRNA in FFPE have reached a level that allows their application as a first line approach in the search for biomarkers. In contrast, many questions remain in terms of quantification of mRNA and protein expression levels in formalin-fixed and paraffin-embedded tissue samples. This review gives an overview on current potentials and limitations of the quantification of DNA, miRNA, mRNA and the proteome in FFPE tissue samples. The chemical events during formalin fixation and paraffin embedding and alternatives to formalin fixation are described. In addition, methods and general problems of DNA, miRNA, mRNA and protein extraction and the current knowledge on the feasibility and accuracy of quantitative gene expression analysis in FFPE tissues is summarized.

  20. Comparative analysis of four methods to extract DNA from paraffin-embedded tissues: effect on downstream molecular applications

    PubMed Central

    2010-01-01

    Background A large portion of tissues stored worldwide for diagnostic purposes is formalin-fixed and paraffin-embedded (FFPE). These FFPE-archived tissues are an extremely valuable source for retrospective (genetic) studies. These include mutation screening in cancer-critical genes as well as pathogen detection. In this study we evaluated the impact of several widely used DNA extraction methods on the quality of molecular diagnostics on FFPE tissues. Findings We compared 4 DNA extraction methods from 4 identically processed FFPE mammary-, prostate-, colon- and lung tissues with regard to PCR inhibition, real time SNP detection and amplifiable fragment size. The extraction methods, with and without proteinase K pre-treatment, tested were: 1) heat-treatment, 2) QIAamp DNA-blood-mini-kit, 3) EasyMAG NucliSens and 4) Gentra Capture-Column-kit. Amplifiable DNA fragment size was assessed by multiplexed 200-400-600 bp PCR and appeared highly influenced by the extraction method used. Proteinase K pre-treatment was a prerequisite for proper purification of DNA from FFPE. Extractions with QIAamp, EasyMAG and heat-treatment were found suitable for amplification of fragments up to 400 bp from all tissues, 600 bp amplification was marginally successful (best was QIAamp). QIAamp and EasyMAG extracts were found suitable for downstream real time SNP detection. Gentra extraction was unsuitable. Hands-on time was lowest for heat-treatment, followed by EasyMAG. Conclusions We conclude that the extraction method plays an important role with regard to performance in downstream molecular applications. PMID:20840759

  1. Improving molecular detection of fungal DNA in formalin-fixed paraffin-embedded tissues: comparison of five tissue DNA extraction methods using panfungal PCR.

    PubMed

    Muñoz-Cadavid, C; Rudd, S; Zaki, S R; Patel, M; Moser, S A; Brandt, M E; Gómez, B L

    2010-06-01

    DNA extraction from formalin-fixed paraffin-embedded (FFPE) tissues is difficult and requires special protocols in order to extract small amounts of DNA suitable for amplification. Most described methods report an amplification success rate between 60 and 80%; therefore, there is a need to improve molecular detection and identification of fungi in FFPE tissue. Eighty-one archived FFPE tissues with a positive Gomori methenamine silver (GMS) stain were evaluated using five different commercial DNA extraction kits with some modifications. Three different panfungal PCR assays were used to detect fungal DNA, and two housekeeping genes were used to assess the presence of amplifiable DNA and to detect PCR inhibitors. The sensitivities of the five extraction protocols were compared, and the quality of DNA detection (calculated for each kit as the number of housekeeping gene PCR-positive samples divided by the total number of samples) was 60 to 91% among the five protocols. The efficiencies of the three different panfungals used (calculated as the number of panfungal-PCR-positive samples divided by the number of housekeeping gene PCR-positive samples) were 58 to 93%. The panfungal PCR using internal transcribed spacer 3 (ITS3) and ITS4 primers yielded a product in most FFPE tissues. Two of the five DNA extraction kits (from TaKaRa and Qiagen) showed similar and promising results. However, one method (TaKaRa) could extract fungal DNA from 69 of the 74 FFPE tissues from which a housekeeping gene could be amplified and was also cost-effective, with a nonlaborious protocol. Factors such as sensitivity, cost, and labor will help guide the selection of the most appropriate method for the needs of each laboratory.

  2. Comparison of Five Commercial Nucleic Acid Extraction Kits for the PCR-based Detection of Burkholderia Pseudomallei DNA in Formalin-Fixed, Paraffin-Embedded Tissues

    PubMed Central

    Obersteller, Sonja; Neubauer, Heinrich; Hagen, Ralf Matthias; Frickmann, Hagen

    2016-01-01

    The extraction and further processing of nucleic acids (NA) from formalin-fixed paraffin-embedded (FFPE) tissues for microbiological diagnostic polymerase chain reaction (PCR) approaches is challenging. Here, we assessed the effects of five different commercially available nucleic acid extraction kits on the results of real-time PCR. FFPE samples from organs of Burkholderia pseudomallei-infected Swiss mice were subjected to processing with five different extraction kits from QIAGEN (FFPE DNA Tissue Kit, EZ1 DNA Tissue Kit, DNA Mini Kit, DNA Blood Mini Kit, and FlexiGene DNA Kit) in combination with three different real-time PCRs targeting B. pseudomallei-specific sequences of varying length after 16 years of storage. The EZ1 DNA Tissue Kit and the DNA Mini Kit scored best regarding the numbers of successful PCR reactions. In case of positive PCR, differences regarding the cycle-threshold (Ct) values were marginal. The impact of the applied extraction kits on the reliability of PCR from FFPE material seems to be low. Interfering factors like the quality of the dewaxing procedure or the sample age appear more important than the selection of specialized FFPE kits. PMID:27766174

  3. Impact of pre-analytical factors on the proteomic analysis of formalin-fixed paraffin-embedded tissue.

    PubMed

    Thompson, Seonaid M; Craven, Rachel A; Nirmalan, Niroshini J; Harnden, Patricia; Selby, Peter J; Banks, Rosamonde E

    2013-04-01

    Formalin-fixed paraffin-embedded (FFPE) tissue samples represent a tremendous potential resource for biomarker discovery, with large numbers of samples in hospital pathology departments and links to clinical information. However, the cross-linking of proteins and nucleic acids by formalin fixation has hampered analysis and proteomic studies have been restricted to using frozen tissue, which is more limited in availability as it needs to be collected specifically for research. This means that rare disease subtypes cannot be studied easily. Recently, improved extraction techniques have enabled analysis of FFPE tissue by a number of proteomic techniques. As with all clinical samples, pre-analytical factors are likely to impact on the results obtained, although overlooked in many studies. The aim of this review is to discuss the various pre-analytical factors, which include warm and cold ischaemic time, size of sample, fixation duration and temperature, tissue processing conditions, length of storage of archival tissue and storage conditions, and to review the studies that have considered these factors in more detail. In those areas where investigations are few or non-existent, illustrative examples of the possible importance of specific factors have been drawn from studies using frozen tissue or from immunohistochemical studies of FFPE tissue.

  4. A Tissue Quality Index – an Intrinsic Control for Measurement of Effects of Pre-analytical Variables on FFPE Tissue

    PubMed Central

    Neumeister, Veronique M.; Parisi, Fabio; England, Allison M.; Siddiqui, Summar; Anagnostou, Valsamo; Zarrella, Elizabeth; Vassilakopolou, Maria; Bai, Yalai; Saylor, Sasha; Sapino, Anna; Kluger, Yuval; Hicks, David G.; Bussolati, Gianni; Kwei, Stephanie; Rimm, David L.

    2014-01-01

    While efforts are made to improve tissue quality and control pre-analytical variables, pathologists are often confronted with the challenge of molecular analysis of patient samples of unknown quality. Here we describe a first attempt to construct a Tissue Quality Index (TQI) or an intrinsic control that would allow a global assessment of protein status based on quantitative measurement of a small number of selected, informative epitopes. Quantitative Immunofluorescence (QIF) of a number of proteins was performed on a series of 93 breast cancer cases where levels of expression were assessed as a function of delayed time to formalin fixation. A TQI was constructed based on the combination of proteins that most accurately reflect increased and decreased levels of expression in proportion to delay time. The TQI, defined by combinations of measurements of cytokeratin, pERK1/2 and pHSP-27 and their relationship to cold ischemic time were validated on a second build of the training series and on 2 independent breast tissue cohorts with recorded time to formalin fixation. We show an association of negative TQI values (an indicator for loss of tissue quality) with increasing cold ischemic time on both validation cohorts, as well as an association with loss of ER expression levels on all 3 breast cohorts. Using expression levels of 3 epitopes, we can begin to assess the likelihood of delayed time to fixation or decreased tissue quality. This TQI represents a proof of concept for the use of epitope expression to provide a mechanism for monitoring tissue quality. PMID:24535259

  5. Fusion transcript discovery in formalin-fixed paraffin-embedded human breast cancer tissues reveals a link to tumor progression.

    PubMed

    Ma, Yan; Ambannavar, Ranjana; Stephans, James; Jeong, Jennie; Dei Rossi, Andrew; Liu, Mei-Lan; Friedman, Adam J; Londry, Jason J; Abramson, Richard; Beasley, Ellen M; Baker, Joffre; Levy, Samuel; Qu, Kunbin

    2014-01-01

    The identification of gene fusions promises to play an important role in personalized cancer treatment decisions. Many rare gene fusion events have been identified in fresh frozen solid tumors from common cancers employing next-generation sequencing technology. However the ability to detect transcripts from gene fusions in RNA isolated from formalin-fixed paraffin-embedded (FFPE) tumor tissues, which exist in very large sample repositories for which disease outcome is known, is still limited due to the low complexity of FFPE libraries and the lack of appropriate bioinformatics methods. We sought to develop a bioinformatics method, named gFuse, to detect fusion transcripts in FFPE tumor tissues. An integrated, cohort based strategy has been used in gFuse to examine single-end 50 base pair (bp) reads generated from FFPE RNA-Sequencing (RNA-Seq) datasets employing two breast cancer cohorts of 136 and 76 patients. In total, 118 fusion events were detected transcriptome-wide at base-pair resolution across the 212 samples. We selected 77 candidate fusions based on their biological relevance to cancer and supported 61% of these using TaqMan assays. Direct sequencing of 19 of the fusion sequences identified by TaqMan confirmed them. Three unique fused gene pairs were recurrent across the 212 patients with 6, 3, 2 individuals harboring these fusions respectively. We show here that a high frequency of fusion transcripts detected at the whole transcriptome level correlates with poor outcome (P<0.0005) in human breast cancer patients. This study demonstrates the ability to detect fusion transcripts as biomarkers from archival FFPE tissues, and the potential prognostic value of the fusion transcripts detected.

  6. Comparison of gene expression profiling by reverse transcription quantitative PCR between fresh frozen and formalin-fixed, paraffin-embedded breast cancer tissues.

    PubMed

    Sánchez-Navarro, Iker; Gámez-Pozo, Angelo; González-Barón, Manuel; Pinto-Marín, Alvaro; Hardisson, David; López, Rocío; Madero, Rosario; Cejas, Paloma; Mendiola, Marta; Espinosa, Enrique; Vara, Juan Angel Fresno

    2010-05-01

    Recent reports demonstrate the feasibility of quantifying gene expression by using RNA isolated from blocks of formalin-fixed, paraffin-embedded (FFPE) tumor tissue. The development of molecular tests for clinical use based on archival materials would be of great utility in the search for and validation of important genes or gene expression profiles. In this study, we compared the performance of different normalization strategies in the correlation of quantitative data between fresh frozen (FF) and FFPE samples and analyzed the parameters that characterize such correlation for each gene. Total RNA extracted from FFPE samples presented a shift in raw cycle threshold (Cq) values that can be explained by its extensive degradation. Proper normalization can compensate for the effects of RNA degradation in gene expression measurements. We show that correlation between normalized expression values is better for moderately to highly expressed genes whose expression varies significantly between samples. Nevertheless, some genes had no correlation. These genes should not be included in molecular tests for clinical use based on FFPE samples. Our results could serve as a guide when developing clinical diagnostic tests based on RT-qPCR analyses of FFPE tissues in the coming era of treatment decision-making based on gene expression profiling.

  7. Mining the Archives: A Cross-Platform Analysis of Gene Expression Profiles in Archival Formalin-Fixed Paraffin-Embedded Tissues.

    PubMed

    Webster, A Francina; Zumbo, Paul; Fostel, Jennifer; Gandara, Jorge; Hester, Susan D; Recio, Leslie; Williams, Andrew; Wood, Charles E; Yauk, Carole L; Mason, Christopher E

    2015-12-01

    Formalin-fixed paraffin-embedded (FFPE) tissue samples represent a potentially invaluable resource for transcriptomic research. However, use of FFPE samples in genomic studies has been limited by technical challenges resulting from nucleic acid degradation. Here we evaluated gene expression profiles derived from fresh-frozen (FRO) and FFPE mouse liver tissues preserved in formalin for different amounts of time using 2 DNA microarray protocols and 2 whole-transcriptome sequencing (RNA-seq) library preparation methodologies. The ribo-depletion protocol outperformed the other methods by having the highest correlations of differentially expressed genes (DEGs), and best overlap of pathways, between FRO and FFPE groups. The effect of sample time in formalin (18 h or 3 weeks) on gene expression profiles indicated that test article treatment, not preservation method, was the main driver of gene expression profiles. Meta- and pathway analyses indicated that biological responses were generally consistent for 18 h and 3 week FFPE samples compared with FRO samples. However, clear erosion of signal intensity with time in formalin was evident, and DEG numbers differed by platform and preservation method. Lastly, we investigated the effect of time in paraffin on genomic profiles. Ribo-depletion RNA-seq analysis of 8-, 19-, and 26-year-old control blocks resulted in comparable quality metrics, including expected distributions of mapped reads to exonic, untranslated region, intronic, and ribosomal fractions of the transcriptome. Overall, our results indicate that FFPE samples are appropriate for use in genomic studies in which frozen samples are not available, and that ribo-depletion RNA-seq is the preferred method for this type of analysis in archival and long-aged FFPE samples.

  8. Technical Reproducibility of Single-Nucleotide and Size-Based DNA Biomarker Assessment Using DNA Extracted from Formalin-Fixed, Paraffin-Embedded Tissues

    PubMed Central

    Zhang, Shenli; Tan, Iain B.; Sapari, Nur S.; Grabsch, Heike I.; Okines, Alicia; Smyth, Elizabeth C.; Aoyama, Toru; Hewitt, Lindsay C.; Inam, Imran; Bottomley, Dan; Nankivell, Matthew; Stenning, Sally P.; Cunningham, David; Wotherspoon, Andrew; Tsuburaya, Akira; Yoshikawa, Takaki; Soong, Richie; Tan, Patrick

    2015-01-01

    DNA extracted from formalin-fixed, paraffin-embedded (FFPE) tissues has been used in the past to analyze genetic polymorphisms. We evaluated the technical reproducibility of different types of assays for gene polymorphisms using DNA extracted from FFPE material. By using the MassARRAY iPLEX system, we investigated polymorphisms in DPYD (rs1801159 and rs3918290), UMPS (rs1801019), ERCC1 (rs11615), ERCC1 (rs3212986), and ERCC2 (rs13181) in 56 FFPE DNA samples. By using PCR, followed by size-based gel electrophoresis, we also examined TYMS 5′ untranslated region 2R/3R repeats and GSTT1 deletions in 50 FFPE DNA samples and 34 DNAs extracted from fresh-frozen tissues and cell lines. Each polymorphism was analyzed by two independent runs. We found that iPLEX biomarker assays measuring single-nucleotide polymorphisms provided consistent concordant results. However, by using FFPE DNA, size-based PCR biomarkers (GSTT1 and TYMS 5′ untranslated region) were discrepant in 32.7% (16/49, with exact 95% CI, 19.9%–47.5%; exact binomial confidence limit test) and 4.2% (2/48, with exact 95% CI, 0.5%–14.3%) of cases, respectively, whereas no discrepancies were observed using intact genomic DNA. Our findings suggest that DNA from FFPE material can be used to reliably test single-nucleotide polymorphisms. However, results based on size-based PCR biomarkers, and particularly GSTT1 deletions, using FFPE DNA need to be interpreted with caution. Independent repeated assays should be performed on all cases to assess potential discrepancies. PMID:25746798

  9. Technical reproducibility of single-nucleotide and size-based DNA biomarker assessment using DNA extracted from formalin-fixed, paraffin-embedded tissues.

    PubMed

    Zhang, Shenli; Tan, Iain B; Sapari, Nur S; Grabsch, Heike I; Okines, Alicia; Smyth, Elizabeth C; Aoyama, Toru; Hewitt, Lindsay C; Inam, Imran; Bottomley, Dan; Nankivell, Matthew; Stenning, Sally P; Cunningham, David; Wotherspoon, Andrew; Tsuburaya, Akira; Yoshikawa, Takaki; Soong, Richie; Tan, Patrick

    2015-05-01

    DNA extracted from formalin-fixed, paraffin-embedded (FFPE) tissues has been used in the past to analyze genetic polymorphisms. We evaluated the technical reproducibility of different types of assays for gene polymorphisms using DNA extracted from FFPE material. By using the MassARRAY iPLEX system, we investigated polymorphisms in DPYD (rs1801159 and rs3918290), UMPS (rs1801019), ERCC1 (rs11615), ERCC1 (rs3212986), and ERCC2 (rs13181) in 56 FFPE DNA samples. By using PCR, followed by size-based gel electrophoresis, we also examined TYMS 5' untranslated region 2R/3R repeats and GSTT1 deletions in 50 FFPE DNA samples and 34 DNAs extracted from fresh-frozen tissues and cell lines. Each polymorphism was analyzed by two independent runs. We found that iPLEX biomarker assays measuring single-nucleotide polymorphisms provided consistent concordant results. However, by using FFPE DNA, size-based PCR biomarkers (GSTT1 and TYMS 5' untranslated region) were discrepant in 32.7% (16/49, with exact 95% CI, 19.9%-47.5%; exact binomial confidence limit test) and 4.2% (2/48, with exact 95% CI, 0.5%-14.3%) of cases, respectively, whereas no discrepancies were observed using intact genomic DNA. Our findings suggest that DNA from FFPE material can be used to reliably test single-nucleotide polymorphisms. However, results based on size-based PCR biomarkers, and particularly GSTT1 deletions, using FFPE DNA need to be interpreted with caution. Independent repeated assays should be performed on all cases to assess potential discrepancies.

  10. Mining the Archives: A Cross-Platform Analysis of Gene Expression Profiles in Archival Formalin-Fixed Paraffin-Embedded Tissues.

    PubMed

    Webster, A Francina; Zumbo, Paul; Fostel, Jennifer; Gandara, Jorge; Hester, Susan D; Recio, Leslie; Williams, Andrew; Wood, Charles E; Yauk, Carole L; Mason, Christopher E

    2015-12-01

    Formalin-fixed paraffin-embedded (FFPE) tissue samples represent a potentially invaluable resource for transcriptomic research. However, use of FFPE samples in genomic studies has been limited by technical challenges resulting from nucleic acid degradation. Here we evaluated gene expression profiles derived from fresh-frozen (FRO) and FFPE mouse liver tissues preserved in formalin for different amounts of time using 2 DNA microarray protocols and 2 whole-transcriptome sequencing (RNA-seq) library preparation methodologies. The ribo-depletion protocol outperformed the other methods by having the highest correlations of differentially expressed genes (DEGs), and best overlap of pathways, between FRO and FFPE groups. The effect of sample time in formalin (18 h or 3 weeks) on gene expression profiles indicated that test article treatment, not preservation method, was the main driver of gene expression profiles. Meta- and pathway analyses indicated that biological responses were generally consistent for 18 h and 3 week FFPE samples compared with FRO samples. However, clear erosion of signal intensity with time in formalin was evident, and DEG numbers differed by platform and preservation method. Lastly, we investigated the effect of time in paraffin on genomic profiles. Ribo-depletion RNA-seq analysis of 8-, 19-, and 26-year-old control blocks resulted in comparable quality metrics, including expected distributions of mapped reads to exonic, untranslated region, intronic, and ribosomal fractions of the transcriptome. Overall, our results indicate that FFPE samples are appropriate for use in genomic studies in which frozen samples are not available, and that ribo-depletion RNA-seq is the preferred method for this type of analysis in archival and long-aged FFPE samples. PMID:26361796

  11. [Analysis of different methods of extracting DNA from paraffin-embedded tissues and the application of nest PCR].

    PubMed

    Yan, Limin; Sun, Baocun; Zhao, Xiulan; Liu, Zenghui; Song, Wenjing

    2011-08-01

    The aim of this research was to explore the most optimal method of DNA extraction from formalin-fixed, paraffin-embedded (FFPE) tissues, and to improve the amplification of long fragments with the method. Three methods, one step method, phenol-chloroform extraction method, and genomic DNA purification kit method, were employed to extract DNA from twenty normal thyroid tissues which were fixed with formalin and embedded with paraffin. The highest proportionality of OD260/OD280 in the examples was obtained by phenol-chloroform extraction method, 1.703 +/- 0.086, compared to the results of the other two methods. As for the long DNA segments amplification, the achievement ratio of one step method, phenol-chloroform extraction method and genomic DNA purification kit method were 0%, 5% and 10%, respectively, by traditional PCR method, but 0%, 95% and 85% respectively by the nest PCR. We have found that the best process of extracting DNA from FFPE is digesting by proteinase K and purifying by phenol-chloroform, and it is effective to amplify long DNA segments from FFPE by nest PCR.

  12. MicroRNA expression profiles of seminoma from paraffin-embedded formalin-fixed tissue.

    PubMed

    Bing, Z; Master, S R; Tobias, J W; Baldwin, D A; Xu, X W; Tomaszewski, J E

    2012-12-01

    In this study, we used microRNA (miRNA) microarrays in an unbiased screen for aberrantly expressed miRNAs in seminoma, a primitive type of germ cell tumor. Formalin-fixed and paraffin-embedded (FFPE) surgical samples from 11 cases of normal testicular tissue resected for nonneoplastic causes and from 11 cases of seminoma were assessed for miRNA expression. Normal testicular tissue and seminoma were paired by race. We found 112 miRNAs to be differentially expressed between seminoma and normal testicular tissue; 52 miRNAs were overexpressed, and 60, downregulated in seminoma. We did not observe significant differences between black and white populations in our race-paired study. The upregulation of the expression of hsa-mir-21, hsa-mir-372, hsa-mir-373, has-mir-221, and hsa-mir-222 was validated by reverse transcription and real-time PCR. Hsa-mir-372 was upregulated around 1,270-fold (95 % confidence interval (CI) 525.2-3,064.8; p = 8.1e-5 by Mann-Whitney U test). Hsa-mir-373 was upregulated around 1,530-fold (95 % CI 620.5-3,785.6; p = 8.0e-5 by Mann-Whitney U test), consistent with previous reports, indicating that the miRNAs in FFPE are well preserved, and FFPE can be a valuable source for the miRNA study of seminoma. In addition, expression of hsa-mir-21 (12.2-fold, 0.0095), hsa-mir-221 (3.8-fold, 0.014) and hsa-mir-222 (3.8-fold, 0.019) was found elevated in seminoma compared to normal testicular tissue.

  13. MALDI imaging mass spectrometry profiling of N-glycans in formalin-fixed paraffin embedded clinical tissue blocks and tissue microarrays.

    PubMed

    Powers, Thomas W; Neely, Benjamin A; Shao, Yuan; Tang, Huiyuan; Troyer, Dean A; Mehta, Anand S; Haab, Brian B; Drake, Richard R

    2014-01-01

    A recently developed matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) method to spatially profile the location and distribution of multiple N-linked glycan species in frozen tissues has been extended and improved for the direct analysis of glycans in clinically derived formalin-fixed paraffin-embedded (FFPE) tissues. Formalin-fixed tissues from normal mouse kidney, human pancreatic and prostate cancers, and a human hepatocellular carcinoma tissue microarray were processed by antigen retrieval followed by on-tissue digestion with peptide N-glycosidase F. The released N-glycans were detected by MALDI-IMS analysis, and the structural composition of a subset of glycans could be verified directly by on-tissue collision-induced fragmentation. Other structural assignments were confirmed by off-tissue permethylation analysis combined with multiple database comparisons. Imaging of mouse kidney tissue sections demonstrates specific tissue distributions of major cellular N-linked glycoforms in the cortex and medulla. Differential tissue distribution of N-linked glycoforms was also observed in the other tissue types. The efficacy of using MALDI-IMS glycan profiling to distinguish tumor from non-tumor tissues in a tumor microarray format is also demonstrated. This MALDI-IMS workflow has the potential to be applied to any FFPE tissue block or tissue microarray to enable higher throughput analysis of the global changes in N-glycosylation associated with cancers.

  14. Transcriptional profiling of formalin fixed paraffin embedded tissue: pitfalls and recommendations for identifying biologically relevant changes.

    PubMed

    Rentoft, Matilda; Coates, Philip John; Laurell, Göran; Nylander, Karin

    2012-01-01

    Expression profiling techniques have been used to study the biology of many types of cancer but have been limited to some extent by the requirement for collection of fresh tissue. In contrast, formalin fixed paraffin embedded (FFPE) samples are widely available and represent a vast resource of potential material. The techniques used to handle the degraded and modified RNA from these samples are relatively new and all the pitfalls and limitations of this material for whole genome expression profiling are not yet clarified. Here, we analyzed 70 FFPE tongue carcinoma samples and 17 controls using the whole genome DASL array covering nearly 21000 genes. We identified that sample age is related to quality of extracted RNA and that sample quality influences apparent expression levels in a non-random manner related to gene probe sequence, leading to spurious results. However, by removing sub-standard samples and analysing only those 28 cancers and 15 controls that had similar quality we were able to generate a list of 934 genes significantly altered in tongue cancer compared to control samples of tongue. This list contained previously identified changes and was enriched for genes involved in many cancer-related processes such as tissue remodelling, inflammation, differentiation and apoptosis. Four novel genes of potential importance in tongue cancer development and maintenance, SH3BGL2, SLC2A6, SLC16A3 and CXCL10, were independently confirmed, validating our data. Hence, gene expression profiling can be performed usefully on archival material if appropriate quality assurance steps are taken to ensure sample consistency and we present some recommendations for the use of FFPE material based on our findings.

  15. Profiling signalling pathways in formalin-fixed and paraffin-embedded breast cancer tissues reveals cross-talk between EGFR, HER2, HER3 and uPAR.

    PubMed

    Berg, Daniela; Wolff, Claudia; Malinowsky, Katharina; Tran, Kai; Walch, Axel; Bronger, Holger; Schuster, Tibor; Höfler, Heinz; Becker, Karl-Friedrich

    2012-01-01

    In the last few years, new approaches and developments in patient-tailored cancer therapies have raised the need to select, more precisely, those patients who will respond to personalized treatments. Therefore, the most efficient way for optimal therapy and patient selection is to provide a tumour-specific protein network portrait prior to treatment. The aim of our study was to monitor protein networks in formalin-fixed and paraffin-embedded (FFPE) breast cancer tissues, with special emphasis on epidermal growth factor receptor 2 (HER2)-mediated signalling pathways, to identify and validate new disease markers. For this purpose we used a recently developed technology to extract full-length proteins from FFPE tissues and analysed 23 molecules involved in HER2-related signalling by reverse phase protein microarray (RPPA) in a series of 106 FFPE breast cancer tissue samples. We found a significant correlation of HER2 with human epidermal growth factor receptor 3 (HER3/erbB3), epidermal growth factor receptor 1 (EGFR/HER1/erbB1) and urokinase plasminogen receptor (uPAR) in routinely used FFPE breast cancer tissues. Thus, targeting HER2, EGFR, HER3 and uPAR together may offer a more efficient treatment option for patients with breast cancer.

  16. Critical comparison of sample preparation strategies for shotgun proteomic analysis of formalin-fixed, paraffin-embedded samples: insights from liver tissue

    PubMed Central

    2014-01-01

    Background The growing field of formalin-fixed paraffin-embedded (FFPE) tissue proteomics holds promise for improving translational research. Direct tissue trypsinization (DT) and protein extraction followed by in solution digestion (ISD) or filter-aided sample preparation (FASP) are the most common workflows for shotgun analysis of FFPE samples, but a critical comparison of the different methods is currently lacking. Experimental design DT, FASP and ISD workflows were compared by subjecting to the same label-free quantitative approach three independent technical replicates of each method applied to FFPE liver tissue. Data were evaluated in terms of method reproducibility and protein/peptide distribution according to localization, MW, pI and hydrophobicity. Results DT showed lower reproducibility, good preservation of high-MW proteins, a general bias towards hydrophilic and acidic proteins, much lower keratin contamination, as well as higher abundance of non-tryptic peptides. Conversely, FASP and ISD proteomes were depleted in high-MW proteins and enriched in hydrophobic and membrane proteins; FASP provided higher identification yields, while ISD exhibited higher reproducibility. Conclusions These results highlight that diverse sample preparation strategies provide significantly different proteomic information, and present typical biases that should be taken into account when dealing with FFPE samples. When a sufficient amount of tissue is available, the complementary use of different methods is suggested to increase proteome coverage and depth. PMID:25097466

  17. Genomic DNA extraction methods using formalin-fixed paraffin-embedded tissue.

    PubMed

    Potluri, Keerti; Mahas, Ahmed; Kent, Michael N; Naik, Sameep; Markey, Michael

    2015-10-01

    As new technologies come within reach for the average cytogenetic laboratory, the study of chromosome structure has become increasingly more sophisticated. Resolution has improved from karyotyping (in which whole chromosomes are discernible) to fluorescence in situ hybridization and comparative genomic hybridization (CGH, with which specific megabase regions are visualized), array-based CGH (aCGH, examining hundreds of base pairs), and next-generation sequencing (providing single base pair resolution). Whole genome next-generation sequencing remains a cost-prohibitive method for many investigators. Meanwhile, the cost of aCGH has been reduced during recent years, even as resolution has increased and protocols have simplified. However, aCGH presents its own set of unique challenges. DNA of sufficient quantity and quality to hybridize to arrays and provide meaningful results is required. This is especially difficult for DNA from formalin-fixed paraffin-embedded (FFPE) tissues. Here, we compare three different methods for acquiring DNA of sufficient length, purity, and "amplifiability" for aCGH and other downstream applications. Phenol-chloroform extraction and column-based commercial kits were compared with adaptive focused acoustics (AFA). Of the three extraction methods, AFA samples showed increased amplicon length and decreased polymerase chain reaction (PCR) failure rate. These findings support AFA as an improvement over previous DNA extraction methods for FFPE tissues.

  18. Genomic DNA extraction methods using formalin-fixed paraffin-embedded tissue.

    PubMed

    Potluri, Keerti; Mahas, Ahmed; Kent, Michael N; Naik, Sameep; Markey, Michael

    2015-10-01

    As new technologies come within reach for the average cytogenetic laboratory, the study of chromosome structure has become increasingly more sophisticated. Resolution has improved from karyotyping (in which whole chromosomes are discernible) to fluorescence in situ hybridization and comparative genomic hybridization (CGH, with which specific megabase regions are visualized), array-based CGH (aCGH, examining hundreds of base pairs), and next-generation sequencing (providing single base pair resolution). Whole genome next-generation sequencing remains a cost-prohibitive method for many investigators. Meanwhile, the cost of aCGH has been reduced during recent years, even as resolution has increased and protocols have simplified. However, aCGH presents its own set of unique challenges. DNA of sufficient quantity and quality to hybridize to arrays and provide meaningful results is required. This is especially difficult for DNA from formalin-fixed paraffin-embedded (FFPE) tissues. Here, we compare three different methods for acquiring DNA of sufficient length, purity, and "amplifiability" for aCGH and other downstream applications. Phenol-chloroform extraction and column-based commercial kits were compared with adaptive focused acoustics (AFA). Of the three extraction methods, AFA samples showed increased amplicon length and decreased polymerase chain reaction (PCR) failure rate. These findings support AFA as an improvement over previous DNA extraction methods for FFPE tissues. PMID:26126956

  19. Analysis of iron, zinc, selenium and cadmium in paraffin-embedded prostate tissue specimens using inductively coupled plasma mass-spectrometry

    USGS Publications Warehouse

    Sarafanov, A.G.; Todorov, T.I.; Kajdacsy-Balla, A.; Gray, Michael A.; MacIas, V.; Centeno, J.A.

    2008-01-01

    Formalin-fixed paraffin-embedded (FFPE) tissue specimens represent a valuable and abundant resource of pathologic material for various biomedical studies. In the present study, we report the application of high-resolution inductively coupled mass-spectrometry (ICP-MS) for quantification of Fe, Zn, Se and Cd in FFPE prostate tissue. These elements have a possible role in the development of prostate diseases: while Zn and Se are needed for a healthy prostate, Cd shows multiple toxic and carcinogenic effects. Excessive accumulation of Fe induces the production of highly reactive hydroxyl radical species, which may play a role in cancer etiopathogenesis. To assess whether the levels of these metals in the FFPE prostate tissue represent their original content, we compared their levels with those in the fresh tissue (on dry weight basis) in samples obtained from 15 patients. We found that in FFPE tissue, the recoveries of Se, Fe, Cd and Zn were progressively decreased, 97??11% (r=0.88), 82??22% (r=0.86), 59??23% (r=0.69) and 24??11% (r=0.38), respectively. Thus, the use of correction factors, determined as k=0.16 for Se, k=0.20 for Fe, k=0.27 for Cd and k=0.67 for Zn, is required to estimate the retrospective levels of these elements in the parental non-processed fresh (wet) prostate tissue. The technique used in this study enables the analysis of archival FFPE prostate tissue for the concentrations of Fe, Zn, Se and Cd to study association between the levels of these metals and prostate disease. ?? 2008.

  20. Multiple drug resistance-related messenger RNA expression in archival formalin-fixed paraffin-embedded human breast tumour tissue.

    PubMed

    O'Driscoll, L; Kennedy, S; McDermott, E; Kelehan, P; Clynes, M

    1996-01-01

    A method is described by which RNA, suitable for reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, can be extracted from formalin-fixed paraffin-embedded (FFPE) tissues and subsequently used for detecting the expression of several genes. Using this technique, RNA can be extracted from specimens, quantified, reverse transcribed and regions of interest amplified and analysed within 36 h. The tissue specimens included in this study were from human breast carcinoma, investigating a range of genes associated with the development and/or maintenance of multiple drug resistance (MDR). This technique, applied to archival tissues, offers great potential for increasing our understanding of alterations in expression levels of genes associated with MDR. The method developed is also applicable to studies on expression of other genes in paraffin-embedded tissues.

  1. Imaging of N-Linked Glycans from Formalin-Fixed Paraffin-Embedded Tissue Sections Using MALDI Mass Spectrometry

    PubMed Central

    2015-01-01

    Aberrant glycosylation is associated with most of the diseases. Direct imaging and profiling of N-glycans on tissue sections can reveal tissue-specific and/or disease-associated N-glycans, which not only could serve as molecular signatures for diagnosis but also shed light on the functional roles of these biomolecules. Mass spectrometry imaging (MSI) is a powerful tool that has been used to correlate peptides, proteins, lipids, and metabolites with their underlying histopathology in tissue sections. Here, we report an MSI technique for direct analysis of N-glycans from formalin-fixed paraffin-embedded (FFPE) tissues. This technique consists of sectioning FFPE tissues, deparaffinization, and rehydration of the sections, denaturing tissue proteins, releasing N-linked glycans from proteins by printing peptide-N-glycosidase F over the sections, spray-coating the tissue with matrix, and analyzing N-glycans by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Brain sections from a C57BL/6 mouse were imaged using this technique at a resolution of 100 μm. Forty-two N-glycans were analyzed from the mouse brain section. The mass spectrometry images were used to study the relative abundance of oligomannose, nonfucosylated, and fucosylated complex N-glycans in different brain areas including isocortex, hippocampal formation, and brainstem and specific glycans associated with different areas of the brain were identified. Furthermore, glioblastoma tumor xenografts in a NOD/SCID mouse were imaged. Several glycans with differential expression in tumor versus normal brain tissues were identified. The MSI technique allows for imaging of N-glycans directly from FFPE sections. This method can potentially identify tissue-specific and/or disease-associated glycans coexpressed with other molecular signatures or within certain histological structures. PMID:25029481

  2. N-glycan MALDI Imaging Mass Spectrometry on Formalin-Fixed Paraffin-Embedded Tissue Enables the Delineation of Ovarian Cancer Tissues.

    PubMed

    Everest-Dass, Arun V; Briggs, Matthew T; Kaur, Gurjeet; Oehler, Martin K; Hoffmann, Peter; Packer, Nicolle H

    2016-09-01

    Ovarian cancer is a fatal gynaecological malignancy in adult women with a five-year overall survival rate of only 30%. Glycomic and glycoproteomic profiling studies have reported extensive protein glycosylation pattern alterations in ovarian cancer. Therefore, spatio-temporal investigation of these glycosylation changes may unearth tissue-specific changes that occur in the development and progression of ovarian cancer. A novel method for investigating tissue-specific N-linked glycans is using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) on formalin-fixed paraffin-embedded (FFPE) tissue sections that can spatially profile N-glycan compositions released from proteins in tissue-specific regions. In this study, tissue regions of interest (e.g. tumor, stroma, adipose tissue and necrotic areas) were isolated from FFPE tissue sections of advanced serous ovarian cancers (n = 3). PGC-LC-ESI-MS/MS and MALDI-MSI were used as complementary techniques to firstly generate structural information on the tissue-specific glycans in order to then obtain high resolution images of the glycan structure distribution in ovarian cancer tissue. The N-linked glycan repertoires carried by the proteins in these tissue regions were structurally characterized for the first time in FFPE ovarian cancer tissue regions, using enzymatic peptide-N-glycosidase F (PNGase F) release of N-glycans. The released glycans were analyzed by porous graphitized carbon liquid chromatography (PGC-LC) and collision induced electrospray negative mode MS fragmentation analysis. The N-glycan profiles identified by this analysis were then used to determine the location and distribution of each N-glycan on FFPE ovarian cancer sections that were treated with PNGase F using high resolution MALDI-MSI. A tissue-specific distribution of N-glycan structures identified particular regions of the ovarian cancer sections. For example, high mannose glycans were predominantly expressed in the

  3. N-glycan MALDI Imaging Mass Spectrometry on Formalin-Fixed Paraffin-Embedded Tissue Enables the Delineation of Ovarian Cancer Tissues.

    PubMed

    Everest-Dass, Arun V; Briggs, Matthew T; Kaur, Gurjeet; Oehler, Martin K; Hoffmann, Peter; Packer, Nicolle H

    2016-09-01

    Ovarian cancer is a fatal gynaecological malignancy in adult women with a five-year overall survival rate of only 30%. Glycomic and glycoproteomic profiling studies have reported extensive protein glycosylation pattern alterations in ovarian cancer. Therefore, spatio-temporal investigation of these glycosylation changes may unearth tissue-specific changes that occur in the development and progression of ovarian cancer. A novel method for investigating tissue-specific N-linked glycans is using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) on formalin-fixed paraffin-embedded (FFPE) tissue sections that can spatially profile N-glycan compositions released from proteins in tissue-specific regions. In this study, tissue regions of interest (e.g. tumor, stroma, adipose tissue and necrotic areas) were isolated from FFPE tissue sections of advanced serous ovarian cancers (n = 3). PGC-LC-ESI-MS/MS and MALDI-MSI were used as complementary techniques to firstly generate structural information on the tissue-specific glycans in order to then obtain high resolution images of the glycan structure distribution in ovarian cancer tissue. The N-linked glycan repertoires carried by the proteins in these tissue regions were structurally characterized for the first time in FFPE ovarian cancer tissue regions, using enzymatic peptide-N-glycosidase F (PNGase F) release of N-glycans. The released glycans were analyzed by porous graphitized carbon liquid chromatography (PGC-LC) and collision induced electrospray negative mode MS fragmentation analysis. The N-glycan profiles identified by this analysis were then used to determine the location and distribution of each N-glycan on FFPE ovarian cancer sections that were treated with PNGase F using high resolution MALDI-MSI. A tissue-specific distribution of N-glycan structures identified particular regions of the ovarian cancer sections. For example, high mannose glycans were predominantly expressed in the

  4. Analytical Validation of AmpliChip p53 Research Test for Archival Human Ovarian FFPE Sections.

    PubMed

    Marton, Matthew J; McNamara, Andrew R; Nikoloff, D Michele; Nakao, Aki; Cheng, Jonathan

    2015-01-01

    The p53 tumor suppressor gene (TP53) is reported to be mutated in nearly half of all tumors and plays a central role in genome integrity. Detection of mutations in p53 can be accomplished by many assays, including the AmpliChip p53 Research Test. The AmpliChip p53 Research Test has been successfully used to determine p53 status in hematologic malignancies and fresh frozen solid tissues but there are few reports of using the assay with formalin fixed, paraffin-embedded (FFPE) tissue. The objective of this study was to describe analytical performance characterization of the AmpliChip p53 Research Test to detect p53 mutations in genomic DNA isolated from archival FFPE human ovarian tumor tissues. Method correlation with sequencing showed 96% mutation-wise agreement and 99% chip-wise agreement. We furthermore observed 100% agreement (113/113) of the most prevalent TP53 mutations. Workflow reproducibility was 96.8% across 8 samples, with 2 operators, 2 reagent lots and 2 instruments. Section-to-section reproducibility was 100% for each sample across a 60 μm region of the FFPE block from ovarian tumors. These data indicate that the AmpliChip p53 Research Test is an accurate and reproducible method for detecting mutations in TP53 from archival FFPE human ovarian specimens.

  5. Analytical Validation of AmpliChip p53 Research Test for Archival Human Ovarian FFPE Sections.

    PubMed

    Marton, Matthew J; McNamara, Andrew R; Nikoloff, D Michele; Nakao, Aki; Cheng, Jonathan

    2015-01-01

    The p53 tumor suppressor gene (TP53) is reported to be mutated in nearly half of all tumors and plays a central role in genome integrity. Detection of mutations in p53 can be accomplished by many assays, including the AmpliChip p53 Research Test. The AmpliChip p53 Research Test has been successfully used to determine p53 status in hematologic malignancies and fresh frozen solid tissues but there are few reports of using the assay with formalin fixed, paraffin-embedded (FFPE) tissue. The objective of this study was to describe analytical performance characterization of the AmpliChip p53 Research Test to detect p53 mutations in genomic DNA isolated from archival FFPE human ovarian tumor tissues. Method correlation with sequencing showed 96% mutation-wise agreement and 99% chip-wise agreement. We furthermore observed 100% agreement (113/113) of the most prevalent TP53 mutations. Workflow reproducibility was 96.8% across 8 samples, with 2 operators, 2 reagent lots and 2 instruments. Section-to-section reproducibility was 100% for each sample across a 60 μm region of the FFPE block from ovarian tumors. These data indicate that the AmpliChip p53 Research Test is an accurate and reproducible method for detecting mutations in TP53 from archival FFPE human ovarian specimens. PMID:26125596

  6. High-resolution copy number analysis of paraffin-embedded archival tissue using SNP BeadArrays.

    PubMed

    Oosting, Jan; Lips, Esther H; van Eijk, Ronald; Eilers, Paul H C; Szuhai, Károly; Wijmenga, Cisca; Morreau, Hans; van Wezel, Tom

    2007-03-01

    High-density SNP microarrays provide insight into the genomic events that occur in diseases like cancer through their capability to measure both LOH and genomic copy numbers. Where currently available methods are restricted to the use of fresh frozen tissue, we now describe the design and validation of copy number measurements using the Illumina BeadArray platform and the application of this technique to formalin-fixed, paraffin-embedded (FFPE) tissue. In fresh frozen tissue from a set of colorectal tumors with numerous chromosomal aberrations, our method measures copy number patterns that are comparable to values from established platforms, like Affymetrix GeneChip and BAC array-CGH. Moreover, paired comparisons of fresh frozen and FFPE tissues showed nearly identical patterns of genomic change. We conclude that this method enables the use of paraffin-embedded material for research into both LOH and numerical chromosomal abnormalities. These findings make the large pathological archives available for genomic analysis, which could be especially relevant for hereditary disease where fresh material from affected relatives is rarely available.

  7. Identification of fungal pathogens in Formalin-fixed, Paraffin-embedded tissue samples by molecular methods.

    PubMed

    Rickerts, Volker

    2016-02-01

    The etiology of invasive fungal infections (IFI) is incompletely understood due to diagnostic limitations including insensitivity of cultures and failure of histopathology to discriminate between different species. This diagnostic gap precludes the optimal use of antifungals, leading to adverse patient outcomes. The identification of fungal pathogens from Formalin-fixed, Paraffin-embedded tissue (FFPE) blocks by molecular methods is emerging as an alternative approach to study the etiology of IFI. PCR assays, including species specific- and broadrange fungal tests are used with FFPE samples from patients with proven IFI. Fungal species identification is achieved in 15-90% of the samples. This heterogeneity may be explained by the samples studied. However, comparison of different studies is impaired, as controls ruling out false positive-, false negative test results or PCR inhibition are frequently not reported. Studies using in situ hybridization also vary in the clinical samples included and the targeted fungi. In addition, target sequences, the probe chemistry and the detection of hybridization signals also account for the differences in diagnostic sensitivity. Using both approaches in parallel yields additive insights, potentially leading to a superior identification of fungal etiology and awareness of the limitations of both molecular diagnostic approaches.

  8. High-throughput RNA sequencing of a formalin-fixed, paraffin-embedded autopsy lung tissue sample from the 1918 influenza pandemic

    PubMed Central

    Xiao, Yong-Li; Kash, John C; Beres, Stephen B; Sheng, Zong-Mei; Musser, James M; Taubenberger, Jeffery K

    2013-01-01

    Most biopsy and autopsy tissues are formalin-fixed and paraffin-embedded (FFPE), but this process leads to RNA degradation that limits gene expression analysis. The RNA genome of the 1918 pandemic influenza virus was previously determined in a 9-year effort by overlapping RT-PCR from post-mortem samples. Here, the full genome of the 1918 virus at 3000× coverage was determined in one high-throughput sequencing run of a library derived from total RNA of a 1918 FFPE sample after duplex-specific nuclease treatments. Bacterial sequences associated with secondary bacterial pneumonias were also detected. Host transcripts were well represented in the library. Compared to a 2009 pandemic influenza virus FFPE post-mortem library, the 1918 sample showed significant enrichment for host defence and cell death response genes, concordant with prior animal studies. This methodological approach should assist in the analysis of FFPE tissue samples isolated over the past century from a variety of diseases. PMID:23180419

  9. Straightforward and sensitive RT-qPCR based gene expression analysis of FFPE samples

    PubMed Central

    Zeka, Fjoralba; Vanderheyden, Katrien; De Smet, Els; Cuvelier, Claude A.; Mestdagh, Pieter; Vandesompele, Jo

    2016-01-01

    Fragmented RNA from formalin-fixed paraffin-embedded (FFPE) tissue is a known obstacle to gene expression analysis. In this study, the impact of RNA integrity, gene-specific reverse transcription and targeted cDNA preamplification was quantified in terms of reverse transcription polymerase chain reaction (RT-qPCR) sensitivity by measuring 48 protein coding genes on eight duplicate cultured cancer cell pellet FFPE samples and twenty cancer tissue FFPE samples. More intact RNA modestly increased gene detection sensitivity by 1.6 fold (earlier detection by 0.7 PCR cycles, 95% CI = 0.593–0.850). Application of gene-specific priming instead of whole transcriptome priming during reverse transcription further improved RT-qPCR sensitivity by a considerable 4.0 fold increase (earlier detection by 2.0 PCR cycles, 95% CI = 1.73–2.32). Targeted cDNA preamplification resulted in the strongest increase of RT-qPCR sensitivity and enabled earlier detection by an average of 172.4 fold (7.43 PCR cycles, 95% CI = 6.83–7.05). We conclude that gene-specific reverse transcription and targeted cDNA preamplification are adequate methods for accurate and sensitive RT-qPCR based gene expression analysis of FFPE material. The presented methods do not involve expensive or complex procedures and can be easily implemented in any routine RT-qPCR practice. PMID:26898768

  10. A MALDI-Mass Spectrometry Imaging method applicable to different formalin-fixed paraffin-embedded human tissues.

    PubMed

    De Sio, Gabriele; Smith, Andrew James; Galli, Manuel; Garancini, Mattia; Chinello, Clizia; Bono, Francesca; Pagni, Fabio; Magni, Fulvio

    2015-06-01

    Recent advancements in Matrix Assisted Laser Desorption/Ionisation (MALDI) Mass Spectrometry Imaging (MSI) technology have enabled the analysis of formalin-fixed paraffin-embedded (FFPE) tissue samples, unlocking a wealth of new proteomic information and facilitating the possibility of performing studies with higher statistical power as well as multi-centric collaborations within the field of proteomics research. However, current methods used to analyse these specimens are often time-consuming and they need to be modified when applied to human tissues of different origin. Here we present a reproducible and time-effective method that could address these aforementioned issues and widen the applicability of this technology to a number of challenging tissue types. Additionally, tissue molecular images show high spatial resolution and a strong correlation with the morphological features, enabling the identification of tissue morphology using statistically derived visualisation, without any prior knowledge. PMID:25592401

  11. Numerical and structural genomic aberrations are reliably detectable in tissue microarrays of formalin-fixed paraffin-embedded tumor samples by fluorescence in-situ hybridization.

    PubMed

    Horn, Heike; Bausinger, Julia; Staiger, Annette M; Sohn, Maximilian; Schmelter, Christopher; Gruber, Kim; Kalla, Claudia; Ott, M Michaela; Rosenwald, Andreas; Ott, German

    2014-01-01

    Few data are available regarding the reliability of fluorescence in-situ hybridization (FISH), especially for chromosomal deletions, in high-throughput settings using tissue microarrays (TMAs). We performed a comprehensive FISH study for the detection of chromosomal translocations and deletions in formalin-fixed and paraffin-embedded (FFPE) tumor specimens arranged in TMA format. We analyzed 46 B-cell lymphoma (B-NHL) specimens with known karyotypes for translocations of IGH-, BCL2-, BCL6- and MYC-genes. Locus-specific DNA probes were used for the detection of deletions in chromosome bands 6q21 and 9p21 in 62 follicular lymphomas (FL) and six malignant mesothelioma (MM) samples, respectively. To test for aberrant signals generated by truncation of nuclei following sectioning of FFPE tissue samples, cell line dilutions with 9p21-deletions were embedded into paraffin blocks. The overall TMA hybridization efficiency was 94%. FISH results regarding translocations matched karyotyping data in 93%. As for chromosomal deletions, sectioning artefacts occurred in 17% to 25% of cells, suggesting that the proportion of cells showing deletions should exceed 25% to be reliably detectable. In conclusion, FISH represents a robust tool for the detection of structural as well as numerical aberrations in FFPE tissue samples in a TMA-based high-throughput setting, when rigorous cut-off values and appropriate controls are maintained, and, of note, was superior to quantitative PCR approaches.

  12. Comparison of quantitative real time PCR with Sequencing and ribosomal RNA-FISH for the identification of fungi in Formalin fixed, paraffin-embedded tissue specimens

    PubMed Central

    2011-01-01

    Background Identification of the causative agents of invasive fungal infections (IFI) is critical for guiding antifungal therapy. Cultures remain negative in a substantial number of IFI cases. Accordingly, species identification from formalin fixed, paraffin embedded (FFPE) tissue specimens by molecular methods such as fluorescence in situ hybridisation (FISH) and PCR provides an appealing approach to improve management of patients. Methods We designed FISH probes targeting the 28S rRNA of Aspergillus and Candida and evaluated them with type strains. Fluorescence microscopy (FM), using FISH probes and quantitative broad-range fungal PCR targeting the rRNA gene were applied to FFPE tissue specimens from patients with proven IFI in order to explore benefits and limitations of each approach. Results PCR followed by sequencing identified a broad spectrum of pathogenic fungi in 28 of 40 evaluable samples (70%). Hybridisation of FISH probes to fungal rRNA was documented in 19 of 40 tissue samples (47.5%), including 3 PCR negative samples with low fungal burden. The use of FISH was highly sensitive in invasive yeast infections, but less sensitive for moulds. In samples with hyphal elements, the evaluation of hybridisation was impaired due to autofluorescence of hyphae and necrotic tissue background. Conclusions While PCR appears to be more sensitive in identifying the causative agents of IFI, some PCR negative and FISH positive samples suggest that FISH has some potential in the rapid identification of fungi from FFPE tissue samples. PMID:21791040

  13. Bisulfite-Based DNA Methylation Analysis from Recent and Archived Formalin-Fixed, Paraffin Embedded Colorectal Tissue Samples.

    PubMed

    Kalmár, Alexandra; Péterfia, Bálint; Hollósi, Péter; Wichmann, Barnabás; Bodor, András; Patai, Árpád V; Schöller, Andrea; Krenács, Tibor; Tulassay, Zsolt; Molnár, Béla

    2015-09-01

    We aimed to test the applicability of formalin-fixed and paraffin-embedded (FFPE) tissue samples for gene specific DNA methylation analysis after using two commercially available DNA isolation kits. Genomic DNA was isolated from 5 colorectal adenocarcinomas and 5 normal adjacent tissues from "recent", collected within 6 months, and "archived", collected more than 5 years ago, FFPE tissues using either High Pure FFPET DNA Isolation kit or QIAamp DNA FFPE Tissue kit. DNA methylation analysis of MAL, SFRP1 and SFRP2 genes, known to be hypermethylated in CRC, was performed using methylation-sensitive high resolution melting (MS-HRM) analysis and sequencing. QIAamp (Q) method resulted in slightly higher recovery in archived (HP: 1.22 ± 3.18 μg DNA; Q: 3.00 ± 4.04 μg DNA) and significantly (p < 0.05) higher recovery in recent samples compared to High Pure method (HP) (HP: 4.10 ± 2.91 μg DNA; Q: 11.51 ± 7.50 μg DNA). Both OD260/280 and OD260/230 ratios were lower, but still high in the High Pure isolated archived and recent samples compared to those isolated with QIAamp. Identical DNA methylation patterns were detected for all 3 genes tested by MS-HRM with both isolation kits in the recent group. However, despite of higher DNA recovery in QIAamp slightly more reproducible methylation results were obtained from High Pure isolated archived samples. Sequencing confirmed DNA hypermethylation in CRCs. In conclusion, reproducible DNA methylation patterns were obtained from recent samples using both isolation kits. However, long term storage may affect the reliability of the results leading to moderate differences between the efficiency of isolation kits.

  14. Bisulfite-Based DNA Methylation Analysis from Recent and Archived Formalin-Fixed, Paraffin Embedded Colorectal Tissue Samples.

    PubMed

    Kalmár, Alexandra; Péterfia, Bálint; Hollósi, Péter; Wichmann, Barnabás; Bodor, András; Patai, Árpád V; Schöller, Andrea; Krenács, Tibor; Tulassay, Zsolt; Molnár, Béla

    2015-09-01

    We aimed to test the applicability of formalin-fixed and paraffin-embedded (FFPE) tissue samples for gene specific DNA methylation analysis after using two commercially available DNA isolation kits. Genomic DNA was isolated from 5 colorectal adenocarcinomas and 5 normal adjacent tissues from "recent", collected within 6 months, and "archived", collected more than 5 years ago, FFPE tissues using either High Pure FFPET DNA Isolation kit or QIAamp DNA FFPE Tissue kit. DNA methylation analysis of MAL, SFRP1 and SFRP2 genes, known to be hypermethylated in CRC, was performed using methylation-sensitive high resolution melting (MS-HRM) analysis and sequencing. QIAamp (Q) method resulted in slightly higher recovery in archived (HP: 1.22 ± 3.18 μg DNA; Q: 3.00 ± 4.04 μg DNA) and significantly (p < 0.05) higher recovery in recent samples compared to High Pure method (HP) (HP: 4.10 ± 2.91 μg DNA; Q: 11.51 ± 7.50 μg DNA). Both OD260/280 and OD260/230 ratios were lower, but still high in the High Pure isolated archived and recent samples compared to those isolated with QIAamp. Identical DNA methylation patterns were detected for all 3 genes tested by MS-HRM with both isolation kits in the recent group. However, despite of higher DNA recovery in QIAamp slightly more reproducible methylation results were obtained from High Pure isolated archived samples. Sequencing confirmed DNA hypermethylation in CRCs. In conclusion, reproducible DNA methylation patterns were obtained from recent samples using both isolation kits. However, long term storage may affect the reliability of the results leading to moderate differences between the efficiency of isolation kits. PMID:25991403

  15. MicroRNA Stability in Postmortem FFPE Tissues: Quantitative Analysis Using Autoptic Samples from Acute Myocardial Infarction Patients.

    PubMed

    Kakimoto, Yu; Kamiguchi, Hiroshi; Ochiai, Eriko; Satoh, Fumiko; Osawa, Motoki

    2015-01-01

    MicroRNAs (miRNAs) are very short (18-24 nucleotides) nucleic acids that are expressed in a number of biological tissues and have been shown to be more resistant to extreme temperatures and pH compared to longer RNA molecules, like mRNAs. As miRNAs contribute to diverse biological process and respond to various kinds of cellular stress, their utility as diagnostic biomarkers and/or therapeutic targets has recently been explored. Here, we have evaluated the usefulness of miRNA quantification during postmortem examination of cardiac tissue from acute myocardial infarction (AMI) patients. Cardiac tissue was collected within one week of the patient's death and either frozen (19 samples) or fixed in formalin for up to three years (36 samples). RNA integrity was evaluated with an electropherogram, and it appears that longer RNAs are fragmented after death in the long-term fixed samples. Quantitative PCR was also performed for seven miRNAs and three other small RNAs in order to determine the appropriate controls for our postmortem analysis. Our data indicate that miR-191 and miR-26b are more suitable than the other types of small RNA molecules as they are stably detected after death and long-term fixation. Further, we also applied our quantitation method, using these endogenous controls, to evaluate the expression of three previously identified miRNA biomarkers, miR-1, miR-208b, and miR-499a, in formalin-fixed tissues from AMI patients. Although miR-1 and miR-208b decreased (1.4-fold) and increased (1.2-fold), respectively, in the AMI samples compared to the controls, the significance of these changes was limited by our sample size. In contrast, the relative level of miR-499a was significantly decreased in the AMI samples (2.1-fold). This study highlights the stability of miRNAs after death and long-term fixation, validating their use as reliable biomarkers for AMI during postmortem examination. PMID:26046358

  16. Improved PCR amplification for molecular analysis using DNA from long-term preserved formalin-fixed, paraffin-embedded lung cancer tissue specimens.

    PubMed

    Taga, Masataka; Eguchi, Hidetaka; Shinohara, Tomoko; Takahashi, Keiko; Ito, Reiko; Yasui, Wataru; Nakachi, Kei; Kusunoki, Yoichiro; Hamatani, Kiyohiro

    2013-01-01

    Archival tissue specimens are valuable resources of materials for molecular biological analyses in retrospective studies, especially for rare diseases or those associated with exposure to uncommon environmental events. Although successful amplification with PCR is essential for analysis of DNA extracted from archival formalin-fixed, paraffin-embedded (FFPE) tissue specimens, we have often encountered problems with poor PCR amplification of target fragments. To overcome this, we examined whether heat treatment in alkaline solution could efficiently restore the PCR template activity of DNA that had already been extracted from FFPE lung cancer tissue specimens. The effect of the heat treatment was assessed by PCR for the TP53 gene and other lung cancer-related gene loci. The heat treatment of DNA samples in borate buffer resulted in successful PCR amplification of DNA fragments ranging from 91 to 152 bp. This technique for restoration of template activity of DNA for PCR amplification is very simple and economical, and requires no special apparatus, so it may be applicable for molecular analysis of DNA samples from FFPE tissue specimens at various laboratories.

  17. Random DNA fragmentation allows detection of single-copy, single-exon alterations of copy number by oligonucleotide array CGH in clinical FFPE samples.

    PubMed

    Hostetter, Galen; Kim, Su Young; Savage, Stephanie; Gooden, Gerald C; Barrett, Michael; Zhang, Jian; Alla, Lalitamba; Watanabe, April; Einspahr, Janine; Prasad, Anil; Nickoloff, Brian J; Carpten, John; Trent, Jeffrey; Alberts, David; Bittner, Michael

    2010-01-01

    Genomic technologies, such as array comparative genomic hybridization (aCGH), increasingly offer definitive gene dosage profiles in clinical samples. Historically, copy number profiling was limited to large fresh-frozen tumors where intact DNA could be readily extracted. Genomic analyses of pre-neoplastic tumors and diagnostic biopsies are often limited to DNA processed by formalin-fixation and paraffin-embedding (FFPE). We present specialized protocols for DNA extraction and processing from FFPE tissues utilizing DNase processing to generate randomly fragmented DNA. The protocols are applied to FFPE clinical samples of varied tumor types, from multiple institutions and of varied block age. Direct comparative analyses with regression coefficient were calculated on split-sample (portion fresh/portion FFPE) of colorectal tumor samples. We show equal detection of a homozygous loss of SMAD4 at the exon-level in the SW480 cell line and gene-specific alterations in the split tumor samples. aCGH application to a set of archival FFPE samples of skin squamous cell carcinomas detected a novel hemizygous deletion in INPP5A on 10q26.3. Finally we present data on derivative of log ratio, a particular sensitive detector of measurement variance, for 216 sequential hybridizations to assess protocol reliability over a wide range of FFPE samples.

  18. Oligonucleotide array outperforms SNP array on formalin-fixed paraffin-embedded clinical samples.

    PubMed

    Nasri, Soroush; Anjomshoaa, Ahmad; Song, Sarah; Guilford, Parry; McNoe, Les; Black, Michael; Phillips, Vicky; Reeve, Anthony; Humar, Bostjan

    2010-04-01

    Compromised quality of formalin-fixed paraffin-embedded (FFPE)-derived DNA has compounded the use of archival specimens for array-based genomic studies. Recent technological advances have led to first successes in this field; however, there is currently no general agreement on the most suitable platform for the array-based analysis of FFPE DNA. In this study, FFPE and matched fresh-frozen (FF) specimens were separately analyzed with Affymetrix single nucleotide polymorphism (SNP) 6.0 and Agilent 4x44K oligonucleotide arrays to compare the genomic profiles from the two tissue sources and to assess the relative performance of the two platforms on FFPE material. Genomic DNA was extracted from matched FFPE-FF pairs of normal intestinal epithelium from four patients and were applied to the SNP and oligonucleotide platforms according to the manufacturer-recommended protocols. On the Affymetrix platform, a substantial increase in apparent copy number alterations was observed in all FFPE tissues relative to their matched FF counterparts. In contrast, FFPE and matched FF genomic profiles obtained via the Agilent platform were very similar. Both the SNP and the oligonucleotide platform performed comparably on FF material. This study demonstrates that Agilent oligonucleotide array comparative genomic hybridization generates reliable results from FFPE extracted DNA, whereas the Affymetrix SNP-based array seems less suitable for the analysis of FFPE material.

  19. Detection and identification of yeasts from formalin-fixed, paraffin-embedded tissue by use of PCR-electrospray ionization mass spectrometry.

    PubMed

    Simner, Patricia J; Buckwalter, Seanne P; Uhl, James R; Wengenack, Nancy L; Pritt, Bobbi S

    2013-11-01

    Diagnosis of yeast infection is typically accomplished by fungal smear and culture, histopathologic examination, and/or serologic studies. Newer assays based on mass spectrometry may be useful for yeast identification when histologic examination is inconclusive, fungal cultures are not ordered, or cultures fail to yield a causative agent. The purpose of this study was to evaluate the ability of the PLEX-ID broad fungal assay to accurately detect and identify yeasts in formalin-fixed paraffin-embedded (FFPE) tissues. Tissue samples from 78 FFPE specimens with both histopathology and corresponding culture results for a variety of yeasts were tested using the PLEX-ID broad fungal assay. A 40-μm FFPE tissue section from each specimen was digested with proteinase K followed by nucleic acid extraction and PCR using broad-range fungal primers. Yeast DNA in amplified products was identified using electrospray ionization mass spectrometry. Discordant results were resolved by D2 rRNA gene sequencing. PLEX-ID analysis detected yeast DNA in 78.2% (61/78) of the cases, of which 91.8% (56/61) were concordant with culture results. Of the 5 discordant positive results, 4 PLEX-ID results were considered to result from environmental contaminants, while 1 clinically important discrepancy was observed (Blastomyces dermatitidis by culture and Cryptococcus neoformans by PLEX-ID). Sequencing of the discordant sample was unsuccessful. The majority of histopathology results (89.7% [70/78]) correlated with culture results. The PLEX-ID broad fungal assay identifies fungi directly from FFPE tissues and can be a useful adjunct to traditional culture and histopathology tests.

  20. Comparison of five protocols to extract DNA from paraffin-embedded tissues for the detection of human papillomavirus.

    PubMed

    Alvarez-Aldana, Adalucy; Martínez, José William; Sepúlveda-Arias, Juan C

    2015-02-01

    Formalin-fixed paraffin-embedded (FFPE) tissues are a valuable source of DNA with which to perform large retrospective studies on the epidemiology of HPV infection. Five different DNA extraction protocols were carried out to evaluate the DNA obtained from FFPE samples with polymerase chain reaction (PCR) using two primer sets to amplify a constitutive human gene, β-globin, and two primer sets to detect the L1 and E6 HPV genes. From the five DNA extraction protocols evaluated, the best results were obtained with protocol A, corresponding to a crude extract from the sample. With the procedures described herein, we were able to amplify DNA extracted from archival paraffin blocks stored for six years. However, the amplification products were more efficiently obtained with primers that amplified shorter fragments. This result indicates that a major factor limiting the extraction process in these samples is DNA fragmentation, a factor that will naturally vary between the different specimens evaluated. Also, depending upon the extraction method, PCR amplification of a human gene does not necessarily guarantee the successful extraction of viral DNA. In conclusion, different DNA and HPV detection methods can significantly influence the results. Therefore, the DNA extraction methods and primers used for DNA amplification in fixed tissues need to be chosen carefully, depending on the specific requirements of the study being carried out.

  1. Comparison of five protocols to extract DNA from paraffin-embedded tissues for the detection of human papillomavirus.

    PubMed

    Alvarez-Aldana, Adalucy; Martínez, José William; Sepúlveda-Arias, Juan C

    2015-02-01

    Formalin-fixed paraffin-embedded (FFPE) tissues are a valuable source of DNA with which to perform large retrospective studies on the epidemiology of HPV infection. Five different DNA extraction protocols were carried out to evaluate the DNA obtained from FFPE samples with polymerase chain reaction (PCR) using two primer sets to amplify a constitutive human gene, β-globin, and two primer sets to detect the L1 and E6 HPV genes. From the five DNA extraction protocols evaluated, the best results were obtained with protocol A, corresponding to a crude extract from the sample. With the procedures described herein, we were able to amplify DNA extracted from archival paraffin blocks stored for six years. However, the amplification products were more efficiently obtained with primers that amplified shorter fragments. This result indicates that a major factor limiting the extraction process in these samples is DNA fragmentation, a factor that will naturally vary between the different specimens evaluated. Also, depending upon the extraction method, PCR amplification of a human gene does not necessarily guarantee the successful extraction of viral DNA. In conclusion, different DNA and HPV detection methods can significantly influence the results. Therefore, the DNA extraction methods and primers used for DNA amplification in fixed tissues need to be chosen carefully, depending on the specific requirements of the study being carried out. PMID:25444238

  2. LCM assisted biomarker discovery from archival neoplastic gastrointestinal tissues.

    PubMed

    Meitner, Patricia A; Resnick, Murray B

    2011-01-01

    Expression array analysis of epithelial mRNA to identify biomarkers of premalignant and malignant conditions in the gastrointestinal (GI) tract is an area of intense study. Archived formalin-fixed paraffin-embedded (FFPE) tissues documenting these changes are readily available and should be a valuable resource for retrospective analysis. Laser capture microdissection of defined areas of epithelial cells at different stages of neoplastic progression is described together with methods for prequalification of RNA in FFPE tissue blocks selected for analysis. Paradise reagents specifically designed for isolation and amplification of RNA from FFPE archival tissue specimens are used to prepare probes for the human X3P microarray from Affymetrix.

  3. The proteomics of formalin-fixed wax-embedded tissue.

    PubMed

    Vincenti, David Cilia; Murray, Graeme I

    2013-04-01

    Proteomics, which is the global analysis of protein expression in cells and tissues, has emerged over the last ten to fifteen years as a key set of technologies to improve our understanding of disease processes and to identify new diagnostic, prognostic and predictive disease biomarkers. Whilst most proteomic studies have been conducted on fresh frozen tissue, the continuous improvements in technical procedures for protein extraction and separation, coupled with increasingly powerful bioinformatics, have provided the opportunity for proteomic analysis to be conducted on formalin-fixed wax-embedded tissue. This potential advance should allow proteomic analysis to be performed on the extensive archives of clinically annotated formalin fixed wax embedded tissue blocks stored in pathology departments worldwide. In this review the main techniques and their limitations involved in proteomic analysis of formalin fixed wax embedded tissue will be outlined and examples of their successful application will be indicated.

  4. Factors affecting immunoreactivity in long-term storage of formalin-fixed paraffin-embedded tissue sections.

    PubMed

    Grillo, Federica; Pigozzi, Simona; Ceriolo, Paola; Calamaro, Paola; Fiocca, Roberto; Mastracci, Luca

    2015-07-01

    Antigen decay in archival formalin-fixed paraffin-embedded (FFPE) tissue sections for immunohistochemistry is a well-known phenomenon which may have repercussions on translational and research studies and length of storage time appears fundamental. The aim of this study was to evaluate all possible factors which may lead to antigen decay on a prospective standardized collection of human tissues with a panel of 14 routinely used antibodies. Serial slide sections from FFPE control tissues were stored using different methods (routine storage at room temperature, Parafilm(®) protected, paraffin coated and cold stored at 4 °C) and for different time periods: 1, 6, 9, 12, 24 and 36 months. Immunohistochemistry was performed at each time cutoff simultaneously on stored sections and on freshly cut sections using a panel of 14 antibodies. Immunoreactivity was compared with immunoreactions performed at time zero. Reduction in immunostaining was observed for a subset of antibodies (CD3, CD 31, CD117, estrogen and progesterone receptors, Ki67, p53, TTF-1, vimentin) while for others (smooth muscle actin, keratins 7, 20, AE1/AE3, 34βE12), no antigen decay was observed. Loss of antigenicity was proportional to tissue section age and was dependent on mode of storage with cold storage slides being the least affected. All antigens with reductions in immunosignal were nuclear or membranous, and they all required heat pre-treatment for antigen retrieval. In contrast to results from other studies, when pre-analytical factors are strictly controlled and standardized, antigen decay seems to be restricted to nuclear or membrane antigens which require heat antigen retrieval.

  5. HPV16 detection by qPCR method in relation to quantity and quality of DNA extracted from archival formalin fixed and paraffin embedded head and neck cancer tissues by three commercially available kits.

    PubMed

    Biesaga, Beata; Janecka, Anna; Mucha-Małecka, Anna; Adamczyk, Agnieszka; Szostek, Sława; Słonina, Dorota; Halaszka, Krzysztof; Przewoźnik, Marcin

    2016-10-01

    The aim of the present study was to compare HPV16 detection by quantitative polymerase chain reaction (qPCR) in relation to the quantity and quality of DNA isolated from 21 formalin fixed and paraffin embedded (FFPE) head and neck cancer tissues by three commercially available kits: EX-WAX™ DNA Extraction Kit (M) (Merck Millipore, Darmstadt, Germany), QIAamp(®) DNA FFPE Tissue (Q) (Qiagen, Hilden, Germany) and ReliaPrep™ FFPE gDNA Miniprep System (P) (Promega, Madison, USA). Quantity of extracted DNA was assessed spectrophometrically and fluorometrically. Its quality was analyzed using A260/280 and A260/230 ratios and the β-actin fragment amplifiability in qPCR. HPV16 presence was detected by qPCR, using specific primers and TaqMan probe. HPV infection was found in 8 DNA samples extracted with M kit (38.1%) and in 7 (33.3%) isolated with Q and P kits. Three samples from M and Q kits were characterized by HPV16 positivity and lack of β-actin amplifiability. They had significantly lower A260/280 ratio (M: 1.6±0.0, p=0.044 and Q: 1.7±0.0, p=0.016) compared to samples with both fragments amplification (M: 1.7±0.0 and Q: 1.9±0.0). Therefore, for HPV detection by qPCR in FFPE tissues we recommend ReliaPrep™ FFPE gDNA Miniprep System.

  6. HPV16 detection by qPCR method in relation to quantity and quality of DNA extracted from archival formalin fixed and paraffin embedded head and neck cancer tissues by three commercially available kits.

    PubMed

    Biesaga, Beata; Janecka, Anna; Mucha-Małecka, Anna; Adamczyk, Agnieszka; Szostek, Sława; Słonina, Dorota; Halaszka, Krzysztof; Przewoźnik, Marcin

    2016-10-01

    The aim of the present study was to compare HPV16 detection by quantitative polymerase chain reaction (qPCR) in relation to the quantity and quality of DNA isolated from 21 formalin fixed and paraffin embedded (FFPE) head and neck cancer tissues by three commercially available kits: EX-WAX™ DNA Extraction Kit (M) (Merck Millipore, Darmstadt, Germany), QIAamp(®) DNA FFPE Tissue (Q) (Qiagen, Hilden, Germany) and ReliaPrep™ FFPE gDNA Miniprep System (P) (Promega, Madison, USA). Quantity of extracted DNA was assessed spectrophometrically and fluorometrically. Its quality was analyzed using A260/280 and A260/230 ratios and the β-actin fragment amplifiability in qPCR. HPV16 presence was detected by qPCR, using specific primers and TaqMan probe. HPV infection was found in 8 DNA samples extracted with M kit (38.1%) and in 7 (33.3%) isolated with Q and P kits. Three samples from M and Q kits were characterized by HPV16 positivity and lack of β-actin amplifiability. They had significantly lower A260/280 ratio (M: 1.6±0.0, p=0.044 and Q: 1.7±0.0, p=0.016) compared to samples with both fragments amplification (M: 1.7±0.0 and Q: 1.9±0.0). Therefore, for HPV detection by qPCR in FFPE tissues we recommend ReliaPrep™ FFPE gDNA Miniprep System. PMID:27456982

  7. Pressure-assisted protein extraction: a novel method for recovering proteins from archival tissue for proteomic analysis.

    PubMed

    Fowler, Carol B; Waybright, Timothy J; Veenstra, Timothy D; O'Leary, Timothy J; Mason, Jeffrey T

    2012-04-01

    Formaldehyde-fixed, paraffin-embedded (FFPE) tissue repositories represent a valuable resource for the retrospective study of disease progression and response to therapy. However, the proteomic analysis of FFPE tissues has been hampered by formaldehyde-induced protein modifications, which reduce protein extraction efficiency and may lead to protein misidentification. Here, we demonstrate the use of heat augmented with high hydrostatic pressure (40,000 psi) as a novel method for the recovery of intact proteins from FFPE mouse liver. When FFPE mouse liver was extracted using heat and elevated pressure, there was a 4-fold increase in protein extraction efficiency, a 3-fold increase in the extraction of intact proteins, and up to a 30-fold increase in the number of nonredundant proteins identified by mass spectrometry, compared to matched tissue extracted with heat alone. More importantly, the number of nonredundant proteins identified in the FFPE tissue was nearly identical to that of matched fresh-frozen tissue.

  8. [Methods of treating tissues with subsequent embedding in paraffin].

    PubMed

    Suponitskiĭ, V Ia

    1985-01-01

    Two methods of tissue processing with subsequent embedding into paraffin are proposed. In the first method a mixture of 70% of gum turpentine and 30% oleoresin is used as the main solution. In the second one a mixture of gum turpentine, castor oil and cedar balm is used as the main solution. The treatment of the tissue pieces takes 23 hours. These methods permit avoiding the artifacts connected with tissue dehydration and obtaining histological slides of a high quality.

  9. Revealing the Molecular Portrait of Triple Negative Breast Tumors in an Understudied Population through Omics Analysis of Formalin-Fixed and Paraffin-Embedded Tissues

    PubMed Central

    Vaca-Paniagua, Felipe; Alvarez-Gomez, Rosa María; Maldonado-Martínez, Hector Aquiles; Pérez-Plasencia, Carlos; Fragoso-Ontiveros, Veronica; Lasa-Gonsebatt, Federico; Herrera, Luis Alonso; Cantú, David; Bargallo-Rocha, Enrique; Mohar, Alejandro; Durand, Geoffroy; Forey, Nathalie; Voegele, Catherine; Vallée, Maxime; Le Calvez-Kelm, Florence; McKay, James; Ardin, Maude; Villar, Stéphanie; Zavadil, Jiri; Olivier, Magali

    2015-01-01

    Triple negative breast cancer (TNBC), defined by the lack of expression of the estrogen receptor, progesterone receptor and human epidermal receptor 2, is an aggressive form of breast cancer that is more prevalent in certain populations, in particular in low- and middle-income regions. The detailed molecular features of TNBC in these regions remain unexplored as samples are mostly accessible as formalin-fixed paraffin embedded (FFPE) archived tissues, a challenging material for advanced genomic and transcriptomic studies. Using dedicated reagents and analysis pipelines, we performed whole exome sequencing and miRNA and mRNA profiling of 12 FFPE tumor tissues collected from pathological archives in Mexico. Sequencing analyses of the tumor tissues and their blood pairs identified TP53 and RB1 genes as the most frequently mutated genes, with a somatic mutation load of 1.7 mutations/exome Mb on average. Transcriptional analyses revealed an overexpression of growth-promoting signals (EGFR, PDGFR, VEGF, PIK3CA, FOXM1), a repression of cell cycle control pathways (TP53, RB1), a deregulation of DNA-repair pathways, and alterations in epigenetic modifiers through miRNA:mRNA network de-regulation. The molecular programs identified were typical of those described in basal-like tumors in other populations. This work demonstrates the feasibility of using archived clinical samples for advanced integrated genomics analyses. It thus opens up opportunities for investigating molecular features of tumors from regions where only FFPE tissues are available, allowing retrospective studies on the search for treatment strategies or on the exploration of the geographic diversity of breast cancer. PMID:25961742

  10. Revealing the Molecular Portrait of Triple Negative Breast Tumors in an Understudied Population through Omics Analysis of Formalin-Fixed and Paraffin-Embedded Tissues.

    PubMed

    Vaca-Paniagua, Felipe; Alvarez-Gomez, Rosa María; Maldonado-Martínez, Hector Aquiles; Pérez-Plasencia, Carlos; Fragoso-Ontiveros, Veronica; Lasa-Gonsebatt, Federico; Herrera, Luis Alonso; Cantú, David; Bargallo-Rocha, Enrique; Mohar, Alejandro; Durand, Geoffroy; Forey, Nathalie; Voegele, Catherine; Vallée, Maxime; Le Calvez-Kelm, Florence; McKay, James; Ardin, Maude; Villar, Stéphanie; Zavadil, Jiri; Olivier, Magali

    2015-01-01

    Triple negative breast cancer (TNBC), defined by the lack of expression of the estrogen receptor, progesterone receptor and human epidermal receptor 2, is an aggressive form of breast cancer that is more prevalent in certain populations, in particular in low- and middle-income regions. The detailed molecular features of TNBC in these regions remain unexplored as samples are mostly accessible as formalin-fixed paraffin embedded (FFPE) archived tissues, a challenging material for advanced genomic and transcriptomic studies. Using dedicated reagents and analysis pipelines, we performed whole exome sequencing and miRNA and mRNA profiling of 12 FFPE tumor tissues collected from pathological archives in Mexico. Sequencing analyses of the tumor tissues and their blood pairs identified TP53 and RB1 genes as the most frequently mutated genes, with a somatic mutation load of 1.7 mutations/exome Mb on average. Transcriptional analyses revealed an overexpression of growth-promoting signals (EGFR, PDGFR, VEGF, PIK3CA, FOXM1), a repression of cell cycle control pathways (TP53, RB1), a deregulation of DNA-repair pathways, and alterations in epigenetic modifiers through miRNA:mRNA network de-regulation. The molecular programs identified were typical of those described in basal-like tumors in other populations. This work demonstrates the feasibility of using archived clinical samples for advanced integrated genomics analyses. It thus opens up opportunities for investigating molecular features of tumors from regions where only FFPE tissues are available, allowing retrospective studies on the search for treatment strategies or on the exploration of the geographic diversity of breast cancer.

  11. Revealing the Molecular Portrait of Triple Negative Breast Tumors in an Understudied Population through Omics Analysis of Formalin-Fixed and Paraffin-Embedded Tissues.

    PubMed

    Vaca-Paniagua, Felipe; Alvarez-Gomez, Rosa María; Maldonado-Martínez, Hector Aquiles; Pérez-Plasencia, Carlos; Fragoso-Ontiveros, Veronica; Lasa-Gonsebatt, Federico; Herrera, Luis Alonso; Cantú, David; Bargallo-Rocha, Enrique; Mohar, Alejandro; Durand, Geoffroy; Forey, Nathalie; Voegele, Catherine; Vallée, Maxime; Le Calvez-Kelm, Florence; McKay, James; Ardin, Maude; Villar, Stéphanie; Zavadil, Jiri; Olivier, Magali

    2015-01-01

    Triple negative breast cancer (TNBC), defined by the lack of expression of the estrogen receptor, progesterone receptor and human epidermal receptor 2, is an aggressive form of breast cancer that is more prevalent in certain populations, in particular in low- and middle-income regions. The detailed molecular features of TNBC in these regions remain unexplored as samples are mostly accessible as formalin-fixed paraffin embedded (FFPE) archived tissues, a challenging material for advanced genomic and transcriptomic studies. Using dedicated reagents and analysis pipelines, we performed whole exome sequencing and miRNA and mRNA profiling of 12 FFPE tumor tissues collected from pathological archives in Mexico. Sequencing analyses of the tumor tissues and their blood pairs identified TP53 and RB1 genes as the most frequently mutated genes, with a somatic mutation load of 1.7 mutations/exome Mb on average. Transcriptional analyses revealed an overexpression of growth-promoting signals (EGFR, PDGFR, VEGF, PIK3CA, FOXM1), a repression of cell cycle control pathways (TP53, RB1), a deregulation of DNA-repair pathways, and alterations in epigenetic modifiers through miRNA:mRNA network de-regulation. The molecular programs identified were typical of those described in basal-like tumors in other populations. This work demonstrates the feasibility of using archived clinical samples for advanced integrated genomics analyses. It thus opens up opportunities for investigating molecular features of tumors from regions where only FFPE tissues are available, allowing retrospective studies on the search for treatment strategies or on the exploration of the geographic diversity of breast cancer. PMID:25961742

  12. qPCR is a sensitive and rapid method for detection of cytomegaloviral DNA in formalin-fixed, paraffin-embedded biopsy tissue.

    PubMed

    McCoy, Morgan H; Post, Kristin; Sen, Joyashree D; Chang, Hsim Y; Zhao, Zijin; Fan, Rong; Chen, Shaoxiong; Leland, Diane; Cheng, Liang; Lin, Jingmei

    2014-01-01

    It is crucial to identify cytomegalovirus (CMV) infection in the gastrointestinal (GI) tract of immunosuppressed patients, given their greater risk for developing severe infection. Many laboratory methods for the detection of CMV infection have been developed, including serology, viral culture, and molecular methods. Often, these methods reflect systemic involvement with CMV and do not specifically identify local tissue involvement. Therefore, detection of CMV infection in the GI tract is frequently done by traditional histology of biopsy tissue. Hematoxylin and eosin (H&E) staining in conjunction with immunohistochemistry (IHC) have remained the mainstays of examining these biopsies. H&E and IHC sometimes result in atypical (equivocal) staining patterns, making interpretation difficult. It was shown that quantitative polymerase chain reaction (qPCR) for CMV can successfully be performed on formalin-fixed, paraffin-embedded (FFPE) biopsy tissue for very high sensitivity and specificity. The goal of this protocol is to demonstrate how to perform qPCR testing for the detection of CMV in FFPE biopsy tissue in a clinical laboratory setting. This method is likely to be of great benefit for patients in cases of equivocal staining for CMV in GI biopsies.

  13. qPCR Is a Sensitive and Rapid Method for Detection of Cytomegaloviral DNA in Formalin-fixed, Paraffin-embedded Biopsy Tissue

    PubMed Central

    McCoy, Morgan H.; Post, Kristin; Sen, Joyashree D.; Chang, Hsim Y.; Zhao, Zijin; Fan, Rong; Chen, Shaoxiong; Leland, Diane; Cheng, Liang; Lin, Jingmei

    2014-01-01

    It is crucial to identify cytomegalovirus (CMV) infection in the gastrointestinal (GI) tract of immunosuppressed patients, given their greater risk for developing severe infection. Many laboratory methods for the detection of CMV infection have been developed, including serology, viral culture, and molecular methods. Often, these methods reflect systemic involvement with CMV and do not specifically identify local tissue involvement. Therefore, detection of CMV infection in the GI tract is frequently done by traditional histology of biopsy tissue. Hematoxylin and eosin (H&E) staining in conjunction with immunohistochemistry (IHC) have remained the mainstays of examining these biopsies. H&E and IHC sometimes result in atypical (equivocal) staining patterns, making interpretation difficult. It was shown that quantitative polymerase chain reaction (qPCR) for CMV can successfully be performed on formalin-fixed, paraffin-embedded (FFPE) biopsy tissue for very high sensitivity and specificity. The goal of this protocol is to demonstrate how to perform qPCR testing for the detection of CMV in FFPE biopsy tissue in a clinical laboratory setting. This method is likely to be of great benefit for patients in cases of equivocal staining for CMV in GI biopsies. PMID:25046572

  14. Multiclass Carcinogenic DNA Adduct Quantification in Formalin-Fixed Paraffin-Embedded Tissues by Ultraperformance Liquid Chromatography-Tandem Mass Spectrometry.

    PubMed

    Guo, Jingshu; Yun, Byeong Hwa; Upadhyaya, Pramod; Yao, Lihua; Krishnamachari, Sesha; Rosenquist, Thomas A; Grollman, Arthur P; Turesky, Robert J

    2016-05-01

    DNA adducts are a measure of internal exposure to genotoxicants and an important biomarker for human risk assessment. However, the employment of DNA adducts as biomarkers in human studies is often restricted because fresh-frozen tissues are not available. In contrast, formalin-fixed paraffin-embedded (FFPE) tissues with clinical diagnosis are readily accessible. Recently, our laboratory reported that DNA adducts of aristolochic acid, a carcinogenic component of Aristolochia herbs used in traditional Chinese medicines worldwide, can be recovered quantitatively from FFPE tissues. In this study, we have evaluated the efficacy of our method for retrieval of DNA adducts from archived tissue by measuring DNA adducts derived from four other classes of human carcinogens: polycyclic aromatic hydrocarbons (PAHs), aromatic amines, heterocyclic aromatic amines (HAAs), and N-nitroso compounds (NOCs). Deoxyguanosine (dG) adducts of the PAH benzo[a]pyrene (B[a]P), 10-(deoxyguanosin-N(2)-yl)-7,8,9-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene (dG-N(2)-B[a]PDE); the aromatic amine 4-aminobiphenyl (4-ABP), N-(deoxyguanosin-8-yl)-4-aminobiphenyl (dG-C8-4-ABP); the HAA 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), N-(deoxyguanosin-8-yl)-PhIP (dG-C8-PhIP); and the dG adducts of the NOC 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), O(6)-methyl-dG (O(6)-Me-dG) and O(6)-pyridyloxobutyl-dG (O(6)-POB-dG), formed in liver, lung, bladder, pancreas, or colon were recovered in comparable yields from fresh-frozen and FFPE preserved tissues of rodents treated with the procarcinogens. Quantification was achieved by ultraperformance liquid chromatography coupled with electrospray ionization ion-trap multistage mass spectrometry (UPLC/ESI-IT-MS(3)). These advancements in the technology of DNA adduct retrieval from FFPE tissue clear the way for use of archived pathology samples in molecular epidemiology studies designed to assess the causal role of exposure to hazardous chemicals

  15. Characterization of tissue and slide artifacts from automated embedding systems.

    PubMed

    Goldberg, Matthew S; Wetherington, Sarah J; Susa, Joseph S; Wickless, Scott C; Cockerell, Clay J

    2015-11-01

    With recent technological advances and cost reductions, automated embedding systems are rapidly becoming routine in the processing of skin biopsy specimens. The efficiency advantages of this technique are due in part to the use of patented sectionable cassettes that hold formalin-fixed tissue from the time of grossing through tissue sectioning. In this process, the final paraffin block contains both the tissue and the cassette, which are sectioned and stained in unison. Here, we report the multiple tissue and slide artifacts commonly seen with automated embedding systems that are unique to this method of tissue processing. The most frequently observed tissue changes are patterned molding of the biopsy specimen around the cassette material. The most common slide artifacts are due to the presence of geometrically shaped polarizable cassette material adjacent to or overlying the stained tissue. As many of these artifacts strongly resemble the shapes seen in the classic 1980s video game, Tetris, we propose the term of Tetris-like artifacts for these findings. Although we remain confident that use of an automated embedding system does not decrease diagnostic reliability, increased familiarity with the standard appearance of slides processed using this technique will help avoid confusion when evaluating these cases.

  16. Genome-wide comparison of paired fresh frozen and formalin-fixed paraffin-embedded gliomas by custom BAC and oligonucleotide array comparative genomic hybridization: facilitating analysis of archival gliomas.

    PubMed

    Mohapatra, Gayatry; Engler, David A; Starbuck, Kristen D; Kim, James C; Bernay, Derek C; Scangas, George A; Rousseau, Audrey; Batchelor, Tracy T; Betensky, Rebecca A; Louis, David N

    2011-04-01

    Array comparative genomic hybridization (aCGH) is a powerful tool for detecting DNA copy number alterations (CNA). Because diffuse malignant gliomas are often sampled by small biopsies, formalin-fixed paraffin-embedded (FFPE) blocks are often the only tissue available for genetic analysis; FFPE tissues are also needed to study the intratumoral heterogeneity that characterizes these neoplasms. In this paper, we present a combination of evaluations and technical advances that provide strong support for the ready use of oligonucleotide aCGH on FFPE diffuse gliomas. We first compared aCGH using bacterial artificial chromosome (BAC) arrays in 45 paired frozen and FFPE gliomas, and demonstrate a high concordance rate between FFPE and frozen DNA in an individual clone-level analysis of sensitivity and specificity, assuring that under certain array conditions, frozen and FFPE DNA can perform nearly identically. However, because oligonucleotide arrays offer advantages to BAC arrays in genomic coverage and practical availability, we next developed a method of labeling DNA from FFPE tissue that allows efficient hybridization to oligonucleotide arrays. To demonstrate utility in FFPE tissues, we applied this approach to biphasic anaplastic oligoastrocytomas and demonstrate CNA differences between DNA obtained from the two components. Therefore, BAC and oligonucleotide aCGH can be sensitive and specific tools for detecting CNAs in FFPE DNA, and novel labeling techniques enable the routine use of oligonucleotide arrays for FFPE DNA. In combination, these advances should facilitate genome-wide analysis of rare, small and/or histologically heterogeneous gliomas from FFPE tissues. PMID:21080181

  17. Detection of Merkel cell polyomavirus in formalin-fixed, paraffin-embedded tissue of Merkel cell carcinoma and correlation with prognosis.

    PubMed

    Andea, Aleodor A; Patel, Raj; Ponnazhagan, Selvarangan; Isayeva, Tatyana; Kumar, Sanjay; Siegal, Gene P

    2014-01-01

    Merkel cell carcinoma (MCC) is a rare, but highly aggressive primary cutaneous malignancy, showing neuroendocrine differentiation. In 2008, a novel member of the polyomavirus family, named Merkel cell polyomavirus (MCPyV) was identified in the genome of MCC tumors raising the possibility of an involvement in its pathogenesis. Due to the rarity of this tumor and current pathology practices, the most readily available tissue is archival formalin-fixed, paraffin-embedded (FFPE) material. In this study, we evaluated the presence of MCPyV in FFPE tissue and correlated its presence with tumor progression. Representative FFPE specimens from 18 tumors belonging to 14 patients with a diagnosis of MCC spanning the period from 2003 to 2008 were retrieved. Following DNA extraction, we performed PCR amplification and sequencing with four different MCPyV-specific primer pairs mapping within the T antigen and VP1 region. Overall, we detected MCPyV amplicons in 8/18 (44.4%) analyzed tumors from 7/14 (50%) cases. Two-year survival rate and median survival for the MCPyV-positive MCCs were 48% and 22.5 months, respectively and for the negative ones 69% and 51.3 months, respectively; however, the difference did not reach statistical significance (p=0.8). There was no significant correlation between the presence of the virus and the stage at presentation; however, tumors in the head and neck area had a lower frequency of viral positivity compared to those arising in the extremities suggesting a MCPyV-independent oncogenetic pathway perhaps, dependent on UV exposure, in a subset of these cases.

  18. Validation of tumor markers in central nervous system germ cell tumors by real-time reverse transcriptase polymerase chain reaction using formalin-fixed paraffin-embedded tissues.

    PubMed

    Kim, Dowhan; Lee, Da Hye; Choi, Junjeong; Shim, Kyu Won; Kim, Se Hoon

    2013-01-01

    The therapeutic protocols for treatment of germinomas and non-germinomatous germ cell tumors (NGGCTs) are completely different, so it is important to distinguish pure germinomas from NGGCTs. As it can be difficult to diagnose by morphology alone, immunohisto-chemistry (IHC) has been widely used as an ancillary test to improve diagnostic accuracy. However, IHC has limitations due to the misinterpretation of results or the aberrant loss of immunoreactivity. However, real-time RT-PCR has certain advantages over IHC, including its quantitative nature. The aim of our study was to evaluate the usefulness of real-time RT-PCR on formalin-fixed paraffin-embedded (FFPE) tissue blocks for the diagnosis of germ cell tumors of the central nervous system. We selected eight markers of germ cell tumors using a literature search, and validated them using real-time RT-PCR. Among them, POU5F1, NANOG and TGFB2 were statistically significant (P=0.05) in multiple comparisons (MANOVA) of three groups (pure germinomas, mature teratomas and malignant germ cell tumors). Two-group (pure germinomas and NGGCTs) discriminant analysis achieved a 70.0% success rate in cross-validation. We concluded that real-time RT-PCR using FFPE tissue has adequate validating power comparable to IHC in the diagnosis of central nervous system germ cell tumors; therefore, when IHC is not available, not conclusive or not informative, RT-PCR is a potential alternative to a repeat biopsy.

  19. Next-generation sequencing and microarray-based interrogation of microRNAs from formalin-fixed, paraffin-embedded tissue: Preliminary assessment of cross-platform concordance

    PubMed Central

    Kelly, Andrew D.; Hill, Katherine E.; Correll, Mick; Hu, Lan; Wang, Yaoyu; Rubio, Renee; Duan, Shenghua; Quackenbush, John; Spentzos, Dimitrios

    2014-01-01

    Next-generation sequencing is increasingly employed in biomedical investigations. Strong concordance between microarray and mRNA-seq levels has been reported in high quality specimens but information is lacking on formalin-fixed, paraffin-embedded (FFPE) tissues, and particularly for microRNA (miRNA) analysis. We conducted a preliminary examination of the concordance between miRNA-seq and cDNA-mediated annealing, selection, extension, and ligation (DASL) miRNA assays. Quantitative agreement between platforms is moderate (Spearman correlation 0.514–0.596) and there is discordance of detection calls on a subset of miRNAs. Quantitative PCR (q-RT-PCR) performed for several discordant miRNAs confirmed the presence of most sequences detected by miRNA-seq but not by DASL but also that miRNA-seq did not detect some sequences, which DASL confidently detected. Our results suggest that miRNA-seq is specific, with few false positive calls, but it may not detect certain abundant miRNAs in FFPE tissue. Further work is necessary to fully address these issues that are pertinent for translational research. PMID:23562991

  20. Multilocus sequence typing of Histoplasma capsulatum in formalin-fixed paraffin-embedded tissues from cats living in non-endemic regions reveals a new phylogenetic clade.

    PubMed

    Arunmozhi Balajee, S; Hurst, Steven F; Chang, Loretta S; Miles, Macon; Beeler, Emily; Hale, Christa; Kasuga, Takao; Benedict, Kaitlin; Chiller, Tom; Lindsley, Mark D

    2013-05-01

    Infections caused by Histoplasma capsulatum are found most often in endemic regions of North, Central, and South America. H. capsulatum has been divided into eight geographic clades by multi-locus sequence typing (MLST). Recently, one isolate and five formalin-fixed paraffin-embedded (FFPE) tissue samples were received from six of 15 suspected cases of histoplasmosis in cats residing in areas not known to be endemic for H. capsulatum. Polymerase chain reaction (PCR) amplification and sequence analysis of the rDNA ITS-2 region confirmed the diagnosis of H. capsulatum. Since these cases were not, as noted, from the accepted endemic areas, it was of interest to understand the molecular epidemiology of these isolates. Results of molecular analysis indicated that the H. capsulatum recovered from the cats were most closely related to the North American-1 clade, but clustered separately outside this clade, suggesting that the H. capsulatum infecting the animals may represent a separate clade or phylogenetic species. This study also demonstrated the utility of obtaining valuable molecular subtype data directly from archived FFPE tissue blocks, particularly when a fungus culture was not performed or is otherwise unavailable. PMID:23072593

  1. Validation of tumor markers in central nervous system germ cell tumors by real-time reverse transcriptase polymerase chain reaction using formalin-fixed paraffin-embedded tissues.

    PubMed

    Kim, Dowhan; Lee, Da Hye; Choi, Junjeong; Shim, Kyu Won; Kim, Se Hoon

    2013-01-01

    The therapeutic protocols for treatment of germinomas and non-germinomatous germ cell tumors (NGGCTs) are completely different, so it is important to distinguish pure germinomas from NGGCTs. As it can be difficult to diagnose by morphology alone, immunohisto-chemistry (IHC) has been widely used as an ancillary test to improve diagnostic accuracy. However, IHC has limitations due to the misinterpretation of results or the aberrant loss of immunoreactivity. However, real-time RT-PCR has certain advantages over IHC, including its quantitative nature. The aim of our study was to evaluate the usefulness of real-time RT-PCR on formalin-fixed paraffin-embedded (FFPE) tissue blocks for the diagnosis of germ cell tumors of the central nervous system. We selected eight markers of germ cell tumors using a literature search, and validated them using real-time RT-PCR. Among them, POU5F1, NANOG and TGFB2 were statistically significant (P=0.05) in multiple comparisons (MANOVA) of three groups (pure germinomas, mature teratomas and malignant germ cell tumors). Two-group (pure germinomas and NGGCTs) discriminant analysis achieved a 70.0% success rate in cross-validation. We concluded that real-time RT-PCR using FFPE tissue has adequate validating power comparable to IHC in the diagnosis of central nervous system germ cell tumors; therefore, when IHC is not available, not conclusive or not informative, RT-PCR is a potential alternative to a repeat biopsy. PMID:23124437

  2. Standardization of immunohistochemistry for formalin-fixed, paraffin-embedded tissue sections based on the antigen-retrieval technique: from experiments to hypothesis.

    PubMed

    Shi, Shan-Rong; Liu, Cheng; Taylor, Clive R

    2007-02-01

    From a practical point of view, one of the most difficult issues in the standardization of IHC for FFPE tissue is the adverse influence of formalin upon antigenicity, as well as the great variation in fixation/processing procedures. Based on previous study, an additional study using four markers demonstrated the potential for obtaining equivalent IHC staining among FFPE tissue sections with periods of formalin fixation ranging from 6 hr to 30 days. On this basis, the following hypothesis is proposed. "The use of optimized AR protocols permits retrieval of specific proteins (antigens) from FFPE tissues to a defined and reproducible degree (expressed as R%), with reference to the amount of protein present in the original fresh/unfixed tissue". This hypothesis may also be presented mathematically: the protein amount in a fresh cell/tissue, expressed as Pf, produces an IHC signal in fresh tissue of integral(Pf). When the identical IHC staining plus AR treatment is applied to a FFPE tissue section, the IHC signal may be represented as integral (Pffpe). The degree of retrieval after AR (R%) is calculated as follows: R% = integral (Pffpe)/ integral (Pf) x 100%. The amount of protein in the FFPE tissue may then be derived as follows: Pffpe = Pf x R%. In a situation where optimized AR is 100% effective, the IHC signal would then be of equal strength in fresh tissue and FFPE tissue, and Pffpe= Pf. Further studies are designed to test the limitations of the proposed hypothesis.

  3. High-quality genotyping data from formalin-fixed, paraffin-embedded tissue on the drug metabolizing enzymes and transporters plus array.

    PubMed

    Vos, Hanneke I; van der Straaten, Tahar; Coenen, Marieke J H; Flucke, Uta; te Loo, D Maroeska W M; Guchelaar, Henk-Jan

    2015-01-01

    The Affymetrix Drug Metabolizing Enzymes and Transporters (DMET) Plus array covers 1936 markers in 231 genes involved in drug metabolism and transport. Blood- and saliva-derived DNA works well on the DMET array, but the utility of DNA from FFPE tissue has not been reported for this array. As the ability to use DNA from FFPE tissue on the array could open the potential for large retrospective sample collections, we examined the performance and reliability of FFPE-derived DNA on the DMET Plus array. Germline DNA isolated from archived normal FFPE tissue blocks stored for 3 to 19 years and matched blood or saliva from 16 patients with osteosarcoma were genotyped on the DMET Plus array. Concordance was assessed by calculating agreement and the κ-statistic. We observed high call rates for both the blood- or saliva-derived DNA samples (99.4%) and the FFPE-derived DNA samples (98.9%). Moreover, the concordance among the 16 blood- or saliva-derived DNA and FFPE DNA pairs was high (97.4%, κ = 0.915). This is the first study showing that DNA from normal FFPE tissue provides accurate and reliable genotypes on the DMET Plus array compared with blood- or saliva-derived DNA. This finding provides an opportunity for pharmacogenetic studies in diseases with high mortality rates and prevents a bias in studies where otherwise only alive patients can be included.

  4. Improving the Proteomic Analysis of Archival Tissue by Using Pressure-Assisted Protein Extraction: A Mechanistic Approach.

    PubMed

    Fowler, Carol B; O'Leary, Timothy J; Mason, Jeffrey T

    2014-06-24

    Formaldehyde-fixed, paraffin-embedded (FFPE) tissue repositories represent a valuable resource for the retrospective study of disease progression and response to therapy. However, the proteomic analysis of FFPE tissues has been hampered by formaldehyde-induced protein modifications, which reduce protein extraction efficiency and may lead to protein misidentification. Here, we demonstrate the use of heat augmented with high hydrostatic pressure (40,000 psi) as a novel method for the recovery of intact proteins from FFPE tissue. Our laboratory has taken a mechanistic approach to developing improved protein extraction protocols, by first studying the reactions of formaldehyde with proteins and ways to reverse these reactions, then applying this approach to a model system called a "tissue surrogate", which is a gel formed by treating high concentrations of cytoplasmic proteins with formaldehyde, and finally FFPE mouse liver tissue. Our studies indicate that elevated pressure improves the recovery of proteins from FFPE tissue surrogates by hydrating and promoting solubilization of highly aggregated proteins allowing for the subsequent reversal (by hydrolysis) of formaldehyde-induced protein adducts and cross-links. When FFPE mouse liver was extracted using heat and elevated pressure, there was a 4-fold increase in protein extraction efficiency and up to a 30-fold increase in the number of non-redundant proteins identified by mass spectrometry, compared to matched tissue extracted with heat alone. More importantly, the number of non-redundant proteins identified in the FFPE tissue was nearly identical to that of the corresponding frozen tissue.

  5. Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix.

    PubMed

    Piotrowski-Daspit, Alexandra S; Nelson, Celeste M

    2016-01-01

    The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, which is regulated by a variety of soluble and physical signals in the microenvironment. Described here is a method created to study the process of branching morphogenesis by forming engineered three-dimensional (3D) epithelial tissues of defined shape and size that are completely embedded within an extracellular matrix (ECM). This method enables the formation of arrays of identical tissues and enables the control of a variety of environmental factors, including tissue geometry, spacing, and ECM composition. This method can also be combined with widely used techniques such as traction force microscopy (TFM) to gain more information about the interactions between cells and their surrounding ECM. The protocol can be used to investigate a variety of cell and tissue processes beyond branching morphogenesis, including cancer invasion.

  6. Variation in pre-PCR processing of FFPE samples leads to discrepancies in BRAF and EGFR mutation detection: a diagnostic RING trial

    PubMed Central

    Kapp, Joshua R; Diss, Tim; Spicer, James; Gandy, Michael; Schrijver, Iris; Jennings, Lawrence J; Li, Marilyn M; Tsongalis, Gregory J; de Castro, David Gonzalez; Bridge, Julia A; Wallace, Andrew; Deignan, Joshua L; Hing, Sandra; Butler, Rachel; Verghese, Eldo; Latham, Gary J; Hamoudi, Rifat A

    2015-01-01

    Aims Mutation detection accuracy has been described extensively; however, it is surprising that pre-PCR processing of formalin-fixed paraffin-embedded (FFPE) samples has not been systematically assessed in clinical context. We designed a RING trial to (i) investigate pre-PCR variability, (ii) correlate pre-PCR variation with EGFR/BRAF mutation testing accuracy and (iii) investigate causes for observed variation. Methods 13 molecular pathology laboratories were recruited. 104 blinded FFPE curls including engineered FFPE curls, cell-negative FFPE curls and control FFPE tissue samples were distributed to participants for pre-PCR processing and mutation detection. Follow-up analysis was performed to assess sample purity, DNA integrity and DNA quantitation. Results Rate of mutation detection failure was 11.9%. Of these failures, 80% were attributed to pre-PCR error. Significant differences in DNA yields across all samples were seen using analysis of variance (p<0.0001), and yield variation from engineered samples was not significant (p=0.3782). Two laboratories failed DNA extraction from samples that may be attributed to operator error. DNA extraction protocols themselves were not found to contribute significant variation. 10/13 labs reported yields averaging 235.8 ng (95% CI 90.7 to 380.9) from cell-negative samples, which was attributed to issues with spectrophotometry. DNA measurements using Qubit Fluorometry demonstrated a median fivefold overestimation of DNA quantity by Nanodrop Spectrophotometry. DNA integrity and PCR inhibition were factors not found to contribute significant variation. Conclusions In this study, we provide evidence demonstrating that variation in pre-PCR steps is prevalent and may detrimentally affect the patient's ability to receive critical therapy. We provide recommendations for preanalytical workflow optimisation that may reduce errors in down-stream sequencing and for next-generation sequencing library generation. PMID:25430497

  7. Towards quantitative mRNA analysis in paraffin-embedded tissues using real-time reverse transcriptase-polymerase chain reaction: a methodological study on lymph nodes from melanoma patients.

    PubMed

    Abrahamsen, Helene Nortvig; Steiniche, Torben; Nexo, Ebba; Hamilton-Dutoit, Stephen J; Sorensen, Boe Sandahl

    2003-02-01

    Improved extraction techniques combined with sensitive real-time reverse transcriptase-polymerase chain reaction may allow detection of mRNA in formalin-fixed, paraffin-embedded (FFPE) materials, but the factors affecting mRNA quantification in clinical material using these methods have not been systematically analyzed. We designed analyses using real-time reverse transcriptase-polymerase chain reaction for quantification of MART-1, beta-actin, and beta(2)-microglobulin mRNAs. The analytical intra- and interassay imprecision (coefficient of variation) was in the range 10 to 20% for all three genes studied. Using these protocols, we studied the influence of tissue autolysis and length of formalin-fixation on mRNA detection in metastatic melanoma. Delay in freezing reduced detectable mRNA, although this was less than predicted and mostly occurred early in autolysis. MART-1, beta-actin, and beta(2)-microglobulin mRNAs were consistently detected in FFPE metastatic melanoma even after fixation for up to 3 weeks, although the total mRNA detected was markedly reduced in fixed compared with fresh tissues (up to 99%). Quantification of MART-1 was, however, possible if this was expressed relative to a housekeeping gene. The polymerase chain reaction product from FFPE tissues could be increased up to 100-fold amplifying short (<136 bp) compared with long amplicons. Variations in time before tissue processing and in fixation length seem to be less important sources of imprecision than previously assumed. Our findings suggest that quantitative analysis of mRNA in archive and routine diagnostic tissues may be possible.

  8. The efficacy of uracil DNA glycosylase pretreatment in amplicon-based massively parallel sequencing with DNA extracted from archived formalin-fixed paraffin-embedded esophageal cancer tissues.

    PubMed

    Serizawa, Masakuni; Yokota, Tomoya; Hosokawa, Ayumu; Kusafuka, Kimihide; Sugiyama, Toshiro; Tsubosa, Yasuhiro; Yasui, Hirofumi; Nakajima, Takashi; Koh, Yasuhiro

    2015-09-01

    Advances in mutation testing for molecular-targeted cancer therapies have led to the increased use of archived formalin-fixed paraffin-embedded (FFPE) tumors. However, DNA extracted from FFPE tumors (FFPE DNA) is problematic for mutation testing, especially for amplicon-based massively parallel sequencing (MPS), owing to DNA fragmentation and artificial C:G > T:A single nucleotide variants (SNVs) caused by deamination of cytosine to uracil. Therefore, to reduce artificial C:G > T:A SNVs in amplicon-based MPS using FFPE DNA, we evaluated the efficacy of uracil DNA glycosylase (UDG) pretreatment, which can eliminate uracil-containing DNA molecules, with 126 archived FFPE esophageal cancer specimens. We also examined the association between the frequency of C:G > T:A SNVs and DNA quality, as assessed by a quantitative PCR (qPCR)-based assay. UDG pretreatment significantly lowered the frequency of C:G > T:A SNVs in highly fragmented DNA (by approximately 60%). This effect was not observed for good- to moderate-quality DNA, suggesting that a predictive assay (i.e., DNA quality assessment) needs to be performed prior to UDG pretreatment. These results suggest that UDG pretreatment is efficacious for mutation testing by amplicon-based MPS with fragmented DNA from FFPE samples.

  9. Somatic Mutation Screening Using Archival Formalin-Fixed, Paraffin-Embedded Tissues by Fluidigm Multiplex PCR and Illumina Sequencing.

    PubMed

    Wang, Ming; Escudero-Ibarz, Leire; Moody, Sarah; Zeng, Naiyan; Clipson, Alexandra; Huang, Yuanxue; Xue, Xuemin; Grigoropoulos, Nicholas F; Barrans, Sharon; Worrillow, Lisa; Forshew, Tim; Su, Jing; Firth, Andrew; Martin, Howard; Jack, Andrew; Brugger, Kim; Du, Ming-Qing

    2015-09-01

    High-throughput somatic mutation screening using FFPE tissues is a major challenge because of a lack of established methods and validated variant calling algorithms. We aimed to develop a targeted sequencing protocol by Fluidigm multiplex PCR and Illumina sequencing and to establish a companion variant calling algorithm. The experimental protocol and variant calling algorithm were first developed and optimized against a series of somatic mutations (147 substitutions, 12 indels ranging from 1 to 33 bp) in seven genes, previously detected by Sanger sequencing of DNA from 163 FFPE lymphoma biopsy specimens. The optimized experimental protocol and variant calling algorithm were further ascertained in two separate experiments by including the seven genes as a part of larger gene panels (22 or 13 genes) using FFPE and high-molecular-weight lymphoma DNAs, respectively. We found that most false-positive variants were due to DNA degradation, deamination, and Taq polymerase errors, but they were nonreproducible and could be efficiently eliminated by duplicate experiments. A small fraction of false-positive variants appeared in duplicate, but they were at low alternative allele frequencies and could be separated from mutations when appropriate threshold value was used. In conclusion, we established a robust practical approach for high-throughput mutation screening using archival FFPE tissues.

  10. Somatic Mutation Screening Using Archival Formalin-Fixed, Paraffin-Embedded Tissues by Fluidigm Multiplex PCR and Illumina Sequencing

    PubMed Central

    Wang, Ming; Escudero-Ibarz, Leire; Moody, Sarah; Zeng, Naiyan; Clipson, Alexandra; Huang, Yuanxue; Xue, Xuemin; Grigoropoulos, Nicholas F.; Barrans, Sharon; Worrillow, Lisa; Forshew, Tim; Su, Jing; Firth, Andrew; Martin, Howard; Jack, Andrew; Brugger, Kim; Du, Ming-Qing

    2016-01-01

    High-throughput somatic mutation screening using FFPE tissues is a major challenge because of a lack of established methods and validated variant calling algorithms. We aimed to develop a targeted sequencing protocol by Fluidigm multiplex PCR and Illumina sequencing and to establish a companion variant calling algorithm. The experimental protocol and variant calling algorithm were first developed and optimized against a series of somatic mutations (147 substitutions, 12 indels ranging from 1 to 33 bp) in seven genes, previously detected by Sanger sequencing of DNA from 163 FFPE lymphoma biopsy specimens. The optimized experimental protocol and variant calling algorithm were further ascertained in two separate experiments by including the seven genes as a part of larger gene panels (22 or 13 genes) using FFPE and high-molecular-weight lymphoma DNAs, respectively. We found that most false-positive variants were due to DNA degradation, deamination, and Taq polymerase errors, but they were nonreproducible and could be efficiently eliminated by duplicate experiments. A small fraction of false-positive variants appeared in duplicate, but they were at low alternative allele frequencies and could be separated from mutations when appropriate threshold value was used. In conclusion, we established a robust practical approach for high-throughput mutation screening using archival FFPE tissues. PMID:26165823

  11. Somatic Mutation Screening Using Archival Formalin-Fixed, Paraffin-Embedded Tissues by Fluidigm Multiplex PCR and Illumina Sequencing.

    PubMed

    Wang, Ming; Escudero-Ibarz, Leire; Moody, Sarah; Zeng, Naiyan; Clipson, Alexandra; Huang, Yuanxue; Xue, Xuemin; Grigoropoulos, Nicholas F; Barrans, Sharon; Worrillow, Lisa; Forshew, Tim; Su, Jing; Firth, Andrew; Martin, Howard; Jack, Andrew; Brugger, Kim; Du, Ming-Qing

    2015-09-01

    High-throughput somatic mutation screening using FFPE tissues is a major challenge because of a lack of established methods and validated variant calling algorithms. We aimed to develop a targeted sequencing protocol by Fluidigm multiplex PCR and Illumina sequencing and to establish a companion variant calling algorithm. The experimental protocol and variant calling algorithm were first developed and optimized against a series of somatic mutations (147 substitutions, 12 indels ranging from 1 to 33 bp) in seven genes, previously detected by Sanger sequencing of DNA from 163 FFPE lymphoma biopsy specimens. The optimized experimental protocol and variant calling algorithm were further ascertained in two separate experiments by including the seven genes as a part of larger gene panels (22 or 13 genes) using FFPE and high-molecular-weight lymphoma DNAs, respectively. We found that most false-positive variants were due to DNA degradation, deamination, and Taq polymerase errors, but they were nonreproducible and could be efficiently eliminated by duplicate experiments. A small fraction of false-positive variants appeared in duplicate, but they were at low alternative allele frequencies and could be separated from mutations when appropriate threshold value was used. In conclusion, we established a robust practical approach for high-throughput mutation screening using archival FFPE tissues. PMID:26165823

  12. Staining methods applied to glycol methacrylate embedded tissue sections.

    PubMed

    Cerri, P S; Sasso-Cerri, E

    2003-01-01

    The use of glycol methacrylate (GMA) avoids some technical artifacts, which are usually observed in paraffin-embedded sections, providing good morphological resolution. On the other hand, weak staining have been mentioned during the use of different methods in plastic sections. In the present study, changes in the histological staining procedures have been assayed during the use of staining and histochemical methods in different GMA-embedded tissues. Samples of tongue, submandibular and sublingual glands, cartilage, portions of respiratory tract and nervous ganglion were fixed in 4% formaldehyde and embedded in glycol methacrylate. The sections of tongue and nervous ganglion were stained by H&E. Picrosirius, Toluidine Blue and Sudan Black B methods were applied, respectively, for identification of collagen fibers in submandibular gland, sulfated glycosaminoglycans in cartilage (metachromasia) and myelin lipids in nervous ganglion. Periodic Acid-Schiff (PAS) method was used for detection of glycoconjugates in submandibular gland and cartilage while AB/PAS combined methods were applied for detection of mucins in the respiratory tract. In addition, a combination of Alcian Blue (AB) and Picrosirius methods was also assayed in the sublingual gland sections. The GMA-embedded tissue sections showed an optimal morphological integrity and were favorable to the staining methods employed in the present study. In the sections of tongue and nervous ganglion, a good contrast of basophilic and acidophilic structures was obtained by H&E. An intense eosinophilia was observed either in the striated muscle fibers or in the myelin sheaths in which the lipids were preserved and revealed by Sudan Black B. In the cartilage matrix, a strong metachromasia was revealed by Toluidine Blue in the negatively-charged glycosaminoglycans. In the chondrocytes, glycogen granules were intensely positive to PAS method. Extracellular glycoproteins were also PAS positive in the basal membrane and in the

  13. A M-MLV reverse transcriptase with reduced RNaseH activity allows greater sensitivity of gene expression detection in formalin fixed and paraffin embedded prostate cancer samples.

    PubMed

    Hagen, Rachel M; Rhodes, Anthony; Oxley, Jon; Ladomery, Michael R

    2013-08-01

    Formalin fixed and paraffin embedded (FFPE) human tissue collections are an invaluable resource for retrospective gene expression studies. However formalin fixation results in chemical modification of RNA and increased RNA degradation. This can affect RNA yield and quality. A critical step when analysing gene expression is the conversion of RNA to complementary DNA (cDNA) using a reverse transcriptase (RT) enzyme. FFPE derived RNA may affect the performance and efficiency of the RT enzyme and cDNA synthesis. We directly compared three commonly used FFPE RNA isolation methods and measured RNA yield, purity and integrity. We also assessed the effectiveness of three commercially available Moloney Murine Leukemia Virus (M-MLV) RTs on cDNA synthesis and gene expression sensitivity when using FFPE RNA as a template. Our results show that gene detection sensitivity is dependent on the isolation method, RT and length of the PCR amplicon (<200bp) when using FFPE RNA. The use of an M-MLV RT enzyme with reduced RNaseH activity gave significantly increased qRT-PCR sensitivity when using FFPE RNA derived from prostate tissue. The choice of RT can also affect perceived changes in target gene expression and thus the same RT should be used when attempting to reproduce results from different studies. This study highlights the need to optimise and evaluate RNA isolation methods and RTs when using FFPE RNA as a template in order to maximise a successful outcome in PCR applications.

  14. Microarray gene expression analysis of fixed archival tissue permits molecular classification and identification of potential therapeutic targets in diffuse large B-cell lymphoma.

    PubMed

    Linton, Kim; Howarth, Christopher; Wappett, Mark; Newton, Gillian; Lachel, Cynthia; Iqbal, Javeed; Pepper, Stuart; Byers, Richard; Chan, Wing John; Radford, John

    2012-01-01

    Refractory/relapsed diffuse large B-cell lymphoma (DLBCL) has a poor prognosis. Novel drugs targeting the constitutively activated NF-κB pathway characteristic of ABC-DLBCL are promising, but evaluation depends on accurate activated B cell-like (ABC)/germinal center B cell-like (GCB) molecular classification. This is traditionally performed on gene microarray expression profiles of fresh biopsies, which are not routinely collected, or by immunohistochemistry on formalin-fixed, paraffin-embedded (FFPE) tissue, which lacks reproducibility and classification accuracy. We explored the possibility of using routine archival FFPE tissue for gene microarray applications. We examined Affymetrix HG U133 Plus 2.0 gene expression profiles from paired archival FFPE and fresh-frozen tissues of 40 ABC/GCB-classified DLBCL cases to compare classification accuracy and test the potential for this approach to aid the discovery of therapeutic targets and disease classifiers in DLBCL. Unsupervised hierarchical clustering of unselected present probe sets distinguished ABC/GCB in FFPE with remarkable accuracy, and a Bayesian classifier correctly assigned 32 of 36 cases with >90% probability. Enrichment for NF-κB genes was appropriately seen in ABC-DLBCL FFPE tissues. The top discriminatory genes expressed in FFPE separated cases with high statistical significance and contained novel biology with potential therapeutic insights, warranting further investigation. These results support a growing understanding that archival FFPE tissues can be used in microarray experiments aimed at molecular classification, prognostic biomarker discovery, and molecular exploration of rare diseases.

  15. Rapid, sensitive, and specific detection of Mycobacterium tuberculosis complex by real-time PCR on paraffin-embedded human tissues.

    PubMed

    Lee, Hye Seung; Park, Kyoung Un; Park, Jung Ok; Chang, Ho Eun; Song, Junghan; Choe, Gheeyoung

    2011-07-01

    The detection of Mycobacterium tuberculosis complex (MTB) in clinical specimens is important for diagnosing and caring for patients in whom tuberculosis is clinically suspected. We collected 129 FFPE specimens, including 56 nontuberculosis cases, 63 MTB cases, and 10 nontuberculous mycobacteria (NTM) cases determined by acid-fast bacilli (AFB) culture. We performed AFB staining; nested MTB PCR, targeting the IS6110 gene; and real-time MTB PCR, targeting the senX3-regX3 intergenic region in the 129 FFPE specimens. The sensitivity and specificity of AFB staining were 37.0% and 98.2%, respectively, using AFB culture results as the reference standard. The sensitivity and specificity of detecting MTB were 68.3% and 98.5%, respectively, by nested PCR; and 74.6% and 98.5% by real-time PCR, respectively. Among the 129 specimens, four were positive by AFB staining but negative by nested or real-time PCR. NTM grew in all four of these cases by AFB culture. AFB density in FFPE tissue sections significantly correlated with MTB DNA load. Thus, real-time PCR is a useful diagnostic tool for rapid and sensitive MTB detection in FFPE specimens, whereas NTM should be included in differential diagnoses of cases positive by AFB staining but negative by PCR.

  16. Producing reverse phase protein microarrays from formalin-fixed tissues.

    PubMed

    Wolff, Claudia; Schott, Christina; Malinowsky, Katharina; Berg, Daniela; Becker, Karl-Friedrich

    2011-01-01

    In most hospitals around the world FFPE (formalin fixed, paraffin embedded) tissues have been used for diagnosis and have subsequently been archived since decades. This has lead to a sizeable pool of this kind of tissues. Till quite recently it was not possible to use this congeries of samples for protein analysis, but now several groups described successful protein extraction from FFPE tissues. In this chapter, we describe a protein extraction protocol established in our laboratory combined with the use of reverse phase protein microarray.

  17. Comparison of whole-exome sequencing of matched fresh and formalin fixed paraffin embedded melanoma tumours: implications for clinical decision making.

    PubMed

    De Paoli-Iseppi, Ricardo; Johansson, Peter A; Menzies, Alexander M; Dias, Kerith-Rae; Pupo, Gulietta M; Kakavand, Hojabr; Wilmott, James S; Mann, Graham J; Hayward, Nicholas K; Dinger, Marcel E; Long, Georgina V; Scolyer, Richard A

    2016-04-01

    The identification of recurrent driver mutations by whole-exome sequencing (WES) of fresh-frozen human cancers and the subsequent development of novel targeted therapies have recently transformed the treatment of many cancers including melanoma. In routine clinical practice, fresh-frozen tissue is rarely available and mutation testing usually needs to be carried out on archival formalin fixed, paraffin embedded (FFPE) tissue, from which DNA is typically fragmented, cross-linked and of lower quality. In this study we aimed to determine whether WES data generated from genomic DNA (gDNA) extracted from FFPE tissues can be produced reliably and of clinically-actionable standard. In this study of ten melanoma patients, we compared WES data produced from analysis of gDNA isolated from FFPE tumour tissue with that isolated from fresh-frozen tumour tissue from the same specimen. FFPE samples were sequenced using both Illumina's Nextera and NimbleGen SeqCap exome capture kits. To examine mutations between the two tissue sources and platforms, somatic mutations in the FFPE exomes were called using the matched fresh tissue sequence as a reference. Of the 10 FFPE DNA samples, seven Nextera and four SeqCap samples passed library preparation. On average, there were 5341 and 2246 variants lost in FFPE compared to matched fresh tissue utilising Nextera and SeqCap kits, respectively. In order to explore the feasibility of future clinical implementation of WES, FFPE variants in 27 genes of important clinical relevance in melanoma were assessed. The average concordance rate was 43.2% over a total of 1299 calls for the chosen genes in the FFPE DNA. For the current clinically most important melanoma mutations, 0/3 BRAF and 6/8 (75%) NRAS FFPE calls were concordant with the fresh tissue result, which was confirmed using a Sequenom OncoCarta Panel. The poor performance of FFPE WES indicates that specialised library construction to account for low quality DNA and further refinements will

  18. Comparison of whole-exome sequencing of matched fresh and formalin fixed paraffin embedded melanoma tumours: implications for clinical decision making.

    PubMed

    De Paoli-Iseppi, Ricardo; Johansson, Peter A; Menzies, Alexander M; Dias, Kerith-Rae; Pupo, Gulietta M; Kakavand, Hojabr; Wilmott, James S; Mann, Graham J; Hayward, Nicholas K; Dinger, Marcel E; Long, Georgina V; Scolyer, Richard A

    2016-04-01

    The identification of recurrent driver mutations by whole-exome sequencing (WES) of fresh-frozen human cancers and the subsequent development of novel targeted therapies have recently transformed the treatment of many cancers including melanoma. In routine clinical practice, fresh-frozen tissue is rarely available and mutation testing usually needs to be carried out on archival formalin fixed, paraffin embedded (FFPE) tissue, from which DNA is typically fragmented, cross-linked and of lower quality. In this study we aimed to determine whether WES data generated from genomic DNA (gDNA) extracted from FFPE tissues can be produced reliably and of clinically-actionable standard. In this study of ten melanoma patients, we compared WES data produced from analysis of gDNA isolated from FFPE tumour tissue with that isolated from fresh-frozen tumour tissue from the same specimen. FFPE samples were sequenced using both Illumina's Nextera and NimbleGen SeqCap exome capture kits. To examine mutations between the two tissue sources and platforms, somatic mutations in the FFPE exomes were called using the matched fresh tissue sequence as a reference. Of the 10 FFPE DNA samples, seven Nextera and four SeqCap samples passed library preparation. On average, there were 5341 and 2246 variants lost in FFPE compared to matched fresh tissue utilising Nextera and SeqCap kits, respectively. In order to explore the feasibility of future clinical implementation of WES, FFPE variants in 27 genes of important clinical relevance in melanoma were assessed. The average concordance rate was 43.2% over a total of 1299 calls for the chosen genes in the FFPE DNA. For the current clinically most important melanoma mutations, 0/3 BRAF and 6/8 (75%) NRAS FFPE calls were concordant with the fresh tissue result, which was confirmed using a Sequenom OncoCarta Panel. The poor performance of FFPE WES indicates that specialised library construction to account for low quality DNA and further refinements will

  19. An Efficient Method for Identifying Gene Fusions by Targeted RNA Sequencing from Fresh Frozen and FFPE Samples.

    PubMed

    Scolnick, Jonathan A; Dimon, Michelle; Wang, I-Ching; Huelga, Stephanie C; Amorese, Douglas A

    2015-01-01

    Fusion genes are known to be key drivers of tumor growth in several types of cancer. Traditionally, detecting fusion genes has been a difficult task based on fluorescent in situ hybridization to detect chromosomal abnormalities. More recently, RNA sequencing has enabled an increased pace of fusion gene identification. However, RNA-Seq is inefficient for the identification of fusion genes due to the high number of sequencing reads needed to detect the small number of fusion transcripts present in cells of interest. Here we describe a method, Single Primer Enrichment Technology (SPET), for targeted RNA sequencing that is customizable to any target genes, is simple to use, and efficiently detects gene fusions. Using SPET to target 5701 exons of 401 known cancer fusion genes for sequencing, we were able to identify known and previously unreported gene fusions from both fresh-frozen and formalin-fixed paraffin-embedded (FFPE) tissue RNA in both normal tissue and cancer cells. PMID:26132974

  20. Diagnostic performance of HPV E6/E7, hTERT, and Ki67 mRNA RT-qPCR assays on formalin-fixed paraffin-embedded cervical tissue specimens from women with cervical cancer.

    PubMed

    Wang, Hye-Young; Kim, Geehyuk; Cho, Hyemi; Kim, Sunghyun; Lee, Dongsup; Park, Sunyoung; Park, Kwang Hwa; Lee, Hyeyoung

    2015-06-01

    Human papillomavirus (HPV) is a major cause of cervical cancer, which is the third most common cancer in women. Human telomerase reverse transcriptase (hTERT) and Ki67 are tumor cell markers indicating cancer cell proliferation in cancer patients, and activation of hTERT and Ki67 leads to progressive cervical carcinogenesis. In the present study, we evaluated the CervicGen HPVE6/E7 mRNA RT-qDx assay, which detects 16 HPV high-risk (HR) genotypes (HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68 and 69), and the CervicGen hTERT and Ki67 mRNA RT-qDx assay using 117 formalin-fixed paraffin-embedded (FFPE) cervical cancer tissue samples. The diagnostic validity of the CervicGen HPV RT-qDx assay for detecting histologically proven prevalent squamous cell carcinoma (SCC) was 94% sensitivity, 100% specificity, 77.8% positive predictive value (PPV), and 78.9% negative predictive value (NPV). The most common HPV genotypes detected in FFPE cervical cancer tissue samples were HPV 16 (56%) and HPV 18 (10%). The positivity rate of hTERT and Ki67 mRNA expressions in FFPE cervical cancer tissue samples on RT-qPCR was 65% and 93% respectively. Moreover, the positivity rates were 92% for a combination of HPV E6/E7 and hTERT mRNA expressions, 97% for HPV E6/E7 and Ki67 mRNA expressions, and 99% (99/100) for the combination of HPV E6/E7, hTERT, and Ki67 mRNA expressions. These data showed that SSC FFPE cervical cancer tissue samples correlated more strongly with high Ki67 mRNA expressions than with hTERT mRNA expressions. Notably, hTERT and Ki67 mRNA expression level was increased in high-grade cervical lesions, but was very low in normal samples. Our findings suggest that the combination of HPV E6/E7, hTERT, and Ki67 mRNA expression levels could be used in a complementary manner in diagnosing high-grade cervical lesions. Further studies are required to evaluate these assays as a useful predictive tool for screening low-grade cervical lesions.

  1. A Comparison of Fresh Frozen vs. Formalin-Fixed, Paraffin-Embedded Specimens of Canine Mammary Tumors via Branched-DNA Assay.

    PubMed

    Lüder Ripoli, Florenza; Mohr, Annika; Conradine Hammer, Susanne; Willenbrock, Saskia; Hewicker-Trautwein, Marion; Hennecke, Silvia; Murua Escobar, Hugo; Nolte, Ingo

    2016-01-01

    Mammary neoplasms are the tumors most affecting female dogs and women. Formalin-fixed, paraffin-embedded (FFPE) tissues are an invaluable source of archived biological material. Fresh frozen (FF) tissue is considered ideal for gene expression analysis. However, strategies based on FFPE material offer several advantages. Branched-DNA assays permit a reliable and fast workflow when analyzing gene expression. The aim of this study was to assess the comparability of the branched-DNA assay when analyzing certain gene expression patterns between FF and FFPE samples in canine mammary tumors. RNA was isolated from 109 FFPE samples and from 93 FF samples of different canine mammary tissues. Sixteen (16) target genes (Tp53; Myc; HMGA1; Pik3ca; Mcl1; MAPK3; FOXO3; PTEN; GATA4; PFDN5; HMGB1; MAPK1; BRCA2; BRCA1; HMGA2; and Her2) were analyzed via branched-DNA assay (b-DNA). ACTB, GAPDH, and HPRT1 were used as data normalizers. Overall, the relative gene expression of the two different origins of samples showed an agreement of 63%. Still, care should be taken, as FFPE specimens showed lower expression of the analyzed targets when compared to FF samples. The fact that the gene expression in FFPE proved to be lower than in FF specimens is likely to have been caused by the effect of storage time. ACTB had the best performance as a data normalizer. PMID:27187374

  2. A Comparison of Fresh Frozen vs. Formalin-Fixed, Paraffin-Embedded Specimens of Canine Mammary Tumors via Branched-DNA Assay

    PubMed Central

    Lüder Ripoli, Florenza; Mohr, Annika; Conradine Hammer, Susanne; Willenbrock, Saskia; Hewicker-Trautwein, Marion; Hennecke, Silvia; Murua Escobar, Hugo; Nolte, Ingo

    2016-01-01

    Mammary neoplasms are the tumors most affecting female dogs and women. Formalin-fixed, paraffin-embedded (FFPE) tissues are an invaluable source of archived biological material. Fresh frozen (FF) tissue is considered ideal for gene expression analysis. However, strategies based on FFPE material offer several advantages. Branched-DNA assays permit a reliable and fast workflow when analyzing gene expression. The aim of this study was to assess the comparability of the branched-DNA assay when analyzing certain gene expression patterns between FF and FFPE samples in canine mammary tumors. RNA was isolated from 109 FFPE samples and from 93 FF samples of different canine mammary tissues. Sixteen (16) target genes (Tp53; Myc; HMGA1; Pik3ca; Mcl1; MAPK3; FOXO3; PTEN; GATA4; PFDN5; HMGB1; MAPK1; BRCA2; BRCA1; HMGA2; and Her2) were analyzed via branched-DNA assay (b-DNA). ACTB, GAPDH, and HPRT1 were used as data normalizers. Overall, the relative gene expression of the two different origins of samples showed an agreement of 63%. Still, care should be taken, as FFPE specimens showed lower expression of the analyzed targets when compared to FF samples. The fact that the gene expression in FFPE proved to be lower than in FF specimens is likely to have been caused by the effect of storage time. ACTB had the best performance as a data normalizer. PMID:27187374

  3. Multi-center evaluation of the novel fully-automated PCR-based Idylla™ BRAF Mutation Test on formalin-fixed paraffin-embedded tissue of malignant melanoma.

    PubMed

    Melchior, Linea; Grauslund, Morten; Bellosillo, Beatriz; Montagut, Clara; Torres, Erica; Moragón, Ester; Micalessi, Isabel; Frans, Johan; Noten, Veerle; Bourgain, Claire; Vriesema, Renske; van der Geize, Robert; Cokelaere, Kristof; Vercooren, Nancy; Crul, Katrien; Rüdiger, Thomas; Buchmüller, Diana; Reijans, Martin; Jans, Caroline

    2015-12-01

    The advent of BRAF-targeted therapies led to increased survival in patients with metastatic melanomas harboring a BRAF V600 mutation (implicated in 46-48% of malignant melanomas). The Idylla(™) System (Idylla(™)), i.e., the real-time-PCR-based Idylla(™) BRAF Mutation Test performed on the fully-automated Idylla(™) platform, enables detection of the most frequent BRAF V600 mutations (V600E/E2/D, V600K/R/M) in tumor material within approximately 90 min and with 1% detection limit. Idylla(™) performance was determined in a multi-center study by analyzing BRAF mutational status of 148 archival formalin-fixed paraffin-embedded (FFPE) tumor samples from malignant melanoma patients, and comparing Idylla(™) results with assessments made by commercial or in-house routine diagnostic methods. Of the 148 samples analyzed, Idylla(™) initially recorded 7 insufficient DNA input calls and 15 results discordant with routine method results. Further analysis learned that the quality of 8 samples was insufficient for Idylla(™) testing, 1 sample had an invalid routine test result, and Idylla(™) results were confirmed in 10 samples. Hence, Idylla(™) identified all mutations present, including 7 not identified by routine methods. Idylla(™) enables fully automated BRAF V600 testing directly on FFPE tumor tissue with increased sensitivity, ease-of-use, and much shorter turnaround time compared to existing diagnostic tests, making it a tool for rapid, simple and highly reliable analysis of therapeutically relevant BRAF mutations, in particular for diagnostic units without molecular expertise and infrastructure.

  4. Multi-center evaluation of the novel fully-automated PCR-based Idylla™ BRAF Mutation Test on formalin-fixed paraffin-embedded tissue of malignant melanoma.

    PubMed

    Melchior, Linea; Grauslund, Morten; Bellosillo, Beatriz; Montagut, Clara; Torres, Erica; Moragón, Ester; Micalessi, Isabel; Frans, Johan; Noten, Veerle; Bourgain, Claire; Vriesema, Renske; van der Geize, Robert; Cokelaere, Kristof; Vercooren, Nancy; Crul, Katrien; Rüdiger, Thomas; Buchmüller, Diana; Reijans, Martin; Jans, Caroline

    2015-12-01

    The advent of BRAF-targeted therapies led to increased survival in patients with metastatic melanomas harboring a BRAF V600 mutation (implicated in 46-48% of malignant melanomas). The Idylla(™) System (Idylla(™)), i.e., the real-time-PCR-based Idylla(™) BRAF Mutation Test performed on the fully-automated Idylla(™) platform, enables detection of the most frequent BRAF V600 mutations (V600E/E2/D, V600K/R/M) in tumor material within approximately 90 min and with 1% detection limit. Idylla(™) performance was determined in a multi-center study by analyzing BRAF mutational status of 148 archival formalin-fixed paraffin-embedded (FFPE) tumor samples from malignant melanoma patients, and comparing Idylla(™) results with assessments made by commercial or in-house routine diagnostic methods. Of the 148 samples analyzed, Idylla(™) initially recorded 7 insufficient DNA input calls and 15 results discordant with routine method results. Further analysis learned that the quality of 8 samples was insufficient for Idylla(™) testing, 1 sample had an invalid routine test result, and Idylla(™) results were confirmed in 10 samples. Hence, Idylla(™) identified all mutations present, including 7 not identified by routine methods. Idylla(™) enables fully automated BRAF V600 testing directly on FFPE tumor tissue with increased sensitivity, ease-of-use, and much shorter turnaround time compared to existing diagnostic tests, making it a tool for rapid, simple and highly reliable analysis of therapeutically relevant BRAF mutations, in particular for diagnostic units without molecular expertise and infrastructure. PMID:26407762

  5. Detection of c-myc amplification in formalin-fixed paraffin-embedded tumor tissue by chromogenic in situ hybridization (CISH).

    PubMed

    Todorović-Raković, Nataša

    2013-01-01

    In situ hybridization (ISH) allows evaluation of genetic abnormalities, such as changes in chromosome number, chromosome translocations or gene amplifications, by hybridization of tagged DNA (or RNA) probes with complementary DNA (or RNA) sequences in interphase nuclei of target tissue. However, chromogenic in situ hybridization (CISH) is also applicable to formalin-fixed, paraffin-embedded (FFPE) tissues, besides metaphase chromosome spreads. CISH is similar to fluorescent in situ hybridization (FISH) regarding pretreatments and hybridization protocols but differs in the way of visualization. Indeed, CISH signal detection is similar to that used in immunohistochemistry, making use of a peroxidase-based chromogenic reaction instead of fluorescent dyes. In particular, tagged DNA probes are indirectly detected using an enzyme-conjugated antibody targeting the tags. The enzymatic reaction of the chromogenic substrate leads to the formation of strong permanent brown signals that can be visualized by bright-field microscopy at 40 × magnification. The advantage of CISH is that it allows the simultaneous observation of gene amplification and tissue morphology and the slides can be stored for a long time.

  6. Linkage-Specific in Situ Sialic Acid Derivatization for N-Glycan Mass Spectrometry Imaging of Formalin-Fixed Paraffin-Embedded Tissues.

    PubMed

    Holst, Stephanie; Heijs, Bram; de Haan, Noortje; van Zeijl, René J M; Briaire-de Bruijn, Inge H; van Pelt, Gabi W; Mehta, Anand S; Angel, Peggy M; Mesker, Wilma E; Tollenaar, Rob A; Drake, Richard R; Bovée, Judith V M G; McDonnell, Liam A; Wuhrer, Manfred

    2016-06-01

    Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging is a rapidly evolving field in which mass spectrometry techniques are applied directly on tissues to characterize the spatial distribution of various molecules such as lipids, protein/peptides, and recently also N-glycans. Glycans are involved in many biological processes and several glycan changes have been associated with different kinds of cancer, making them an interesting target group to study. An important analytical challenge for the study of glycans by MALDI mass spectrometry is the labile character of sialic acid groups which are prone to in-source/postsource decay, thereby biasing the recorded glycan profile. We therefore developed a linkage-specific sialic acid derivatization by dimethylamidation and subsequent amidation and transferred this onto formalin-fixed paraffin-embedded (FFPE) tissues for MALDI imaging of N-glycans. Our results show (i) the successful stabilization of sialic acids in a linkage specific manner, thereby not only increasing the detection range, but also adding biological meaning, (ii) that no noticeable lateral diffusion is induced during to sample preparation, (iii) the potential of mass spectrometry imaging to spatially characterize the N-glycan expression within heterogeneous tissues. PMID:27145236

  7. Development of a Peptide Nucleic Acid Probe to Trichosporon Species and Identification of Trichosporonosis by Use of In Situ Hybridization in Formalin-Fixed and Paraffin-Embedded (FFPE) Sections

    PubMed Central

    Shinozaki, Minoru; Okubo, Yoichiro; Sasai, Daisuke; Nakayama, Haruo; Murayama, Somay Yamagata; Ide, Tadashi; Wakayama, Megumi; Ishiwatari, Takao; Tochigi, Naobumi; Nemoto, Tetsuo

    2013-01-01

    In order to identify Trichosporon species in formalin-fixed and paraffin-embedded sections from which visual discrimination of non-glabrata Candida species is mostly ineffective but critical for the choice of antifungals, we tested the usefulness of a newly designed peptide nucleic acid probe (PNA) for in situ hybridization (ISH). Results confirmed the usefulness of ISH with our PNA probe in identifying Trichosporon species from Candida albicans. PMID:23100341

  8. An approach to optimize sample preparation for MALDI imaging MS of FFPE sections using fractional factorial design of experiments.

    PubMed

    Oetjen, Janina; Lachmund, Delf; Palmer, Andrew; Alexandrov, Theodore; Becker, Michael; Boskamp, Tobias; Maass, Peter

    2016-09-01

    A standardized workflow for matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI imaging MS) is a prerequisite for the routine use of this promising technology in clinical applications. We present an approach to develop standard operating procedures for MALDI imaging MS sample preparation of formalin-fixed and paraffin-embedded (FFPE) tissue sections based on a novel quantitative measure of dataset quality. To cover many parts of the complex workflow and simultaneously test several parameters, experiments were planned according to a fractional factorial design of experiments (DoE). The effect of ten different experiment parameters was investigated in two distinct DoE sets, each consisting of eight experiments. FFPE rat brain sections were used as standard material because of low biological variance. The mean peak intensity and a recently proposed spatial complexity measure were calculated for a list of 26 predefined peptides obtained by in silico digestion of five different proteins and served as quality criteria. A five-way analysis of variance (ANOVA) was applied on the final scores to retrieve a ranking of experiment parameters with increasing impact on data variance. Graphical abstract MALDI imaging experiments were planned according to fractional factorial design of experiments for the parameters under study. Selected peptide images were evaluated by the chosen quality metric (structure and intensity for a given peak list), and the calculated values were used as an input for the ANOVA. The parameters with the highest impact on the quality were deduced and SOPs recommended. PMID:27485623

  9. Extraction and structural analysis of glycosaminoglycans from formalin-fixed, paraffin-embedded tissues.

    PubMed

    van Wijk, Xander M R; Vallen, Myrtille J; van de Westerlo, Els M; Oosterhof, Arie; Hao, Wensi; Versteeg, Elly M; Raben, Julius; Wismans, Ronnie G; Smetsers, Toon F C M; Dijkman, Henry B P M; Schalkwijk, Joost; van Kuppevelt, Toin H

    2012-12-01

    Glycosaminoglycans (GAGs) are long, anionic polysaccharides involved in many basic aspects of mammalian physiology and pathology. Here we describe a method to extract GAGs from formalin-fixed, paraffin-embedded tissues and found that they are structurally comparable with GAGs extracted from frozen tissues. We employed this method to structurally characterize GAGs in tissues, including laser-dissected layers of skin and pathological specimens. This method enables the use of the archival paraffin-embedded material for detailed (structural) analysis of GAGs.

  10. Comparison of 2 different PCR-based technologies for the detection of human papilloma virus from paraffin-embedded tissue: genómica clinical arrays versus SPF(10)-LiPA(25).

    PubMed

    Pérez, Cristina; Klaustermeier, Jo Ellen; Alemany, Laia; Tous, Sara; de Sanjosé, Silvia; Velasco, Julio

    2012-03-01

    The great interest in molecular epidemiology of human papilloma virus (HPV) in cervical cancer led us to perform a thorough evaluation of 2 polymerase chain reaction (PCR)-based methods for the detection of HPV in archival formalin-fixed paraffin-embedded (FFPE) samples. Thus, the aim of this study was to compare HPV detection in FFPE samples that have histopathologic diagnosis of invasive cervical cancer using SPF10 broad-spectrum primers PCR followed by DNA enzyme immunoassay and LiPA25 (version 1: Labo Biomedical products, Rijswijk, The Netherlands version 1) and the Papillomavirus Clinical Arrays technique (Genómica, Tres Cantos, Madrid, Spain). In this study, 235 biopsies with histopathologic diagnosis of invasive cervical cancer were analyzed for the detection and genotyping of HPV by LiPA25 SPF10-PCR System (version 1) and Papillomavirus Clinical Arrays technique. The detection of HPV DNA with Genómica technique was 75.1%, and 91.9% with LiPA25 SPF10-PCR. The Genómica technique detected a higher percentage of multiple infections (35%) than LiPA25 (8.9%), with a very low agreement for the detection of multiple infections between them (P>0.05). Our study highlights an important difference between 2 PCR-based methods for detection and genotyping of HPV. LiPA25 SPF10-PCR technology may be more adequate than Genómica for the detection of HPV DNA when using FFPE tissue. PMID:22306675

  11. Critical steps in tissue processing in histopathology.

    PubMed

    Comanescu, Maria; Annaratone, Laura; D'Armento, Giuseppe; Cardos, Georgeta; Sapino, Anna; Bussolati, Gianni

    2012-04-01

    Histopathological diagnosis using Formalin-Fixed Paraffin Embedded (FFPE) tissues is essential for the prognostic and therapeutic management of cancer patients. Pathologists are being confronted with increasing demands, from both clinicians and patients, to provide immunophenotypic and gene expression data from FFPE tissues to allow the planning of personalized therapeutic regimens. Recent improvements in the protocols for pre-analysis processing of pathological tissues aim to better preserve cellular details and to conserve antigens and nucleic acid sequences. These developments have been recently patented. The international protocol for the transporting of surgical specimens from the surgical theatre to the pathology department is to immerse the specimen in formalin. The alternative method of sealing the specimens into bags under a vacuum and then cooling is a well-accepted and environmentally safe procedure that overcomes the many drawbacks linked to transfer in formalin. Importantly, RNA is notoriously poorly preserved in FFPE tissue. Due to this, successful procedures for the extraction of genetic information from archival tissues have been the object of several studies and patents. Novel molecular approaches for RT-qPCR and gene array analysis on FFPE tissues are presented here. Moreover, a major advance is reported in this study, the observation that tissue fixation in cold conditions allows a much better preservation of nucleic acid sequences.

  12. Efficient DNA Extraction for HPV Genotyping in Formalin-Fixed, Paraffin-Embedded Tissues

    PubMed Central

    Steinau, Martin; Patel, Sonya S.; Unger, Elizabeth R.

    2011-01-01

    DNA from archived FFPE can be used for papillomavirus genotyping, but potential problems include paraffin as a physical barrier, DNA cross-linking, and PCR inhibitors. To address these complications, we combined a commercially available DNA isolation kit (Qiagen DNeasy) with a heat treatment and evaluated the resulting DNA with regards to HPV typing. DNA was extracted from 10-μm sections from 150 FFPE cancer samples. One protocol followed the manufacturer's recommendation, including paraffin removal by xylene and tissue lysis at 56°C. A second section was directly incubated at 120°C and subsequently lysed at 65°C. After spin-column purification, both extracts were tested with a linear array HPV genotyping assay. Additionally, cellular DNA yield, HPV16 DNA copies, and PCR inhibitors were assessed by real-time qPCR assays. Inadequate linear array HPV genotyping assay results were significantly more frequent (P = 0.0003) in xylene-treated (29/150, 19.3%) than in heat-treated extracts (8/150, 5.3%). HPV detection also differed, with 94/150 (62.7%) and 110/150 (73.3%) positive results, respectively (P = 0.0026). The heat method also yielded more PCR-amplifiable cellular DNA (8.2-fold; P < 0.001) and HPV16 copies (6.5-fold; P = 0.009), although PCR inhibitors also had a greater effect (P = 0.035). Aggressive heat treatment demonstrated an advantage over traditional xylene purification protocols, resulting in higher DNA yields and increased sensitivity for HPV testing. PMID:21704270

  13. Efficient DNA extraction for HPV genotyping in formalin-fixed, paraffin-embedded tissues.

    PubMed

    Steinau, Martin; Patel, Sonya S; Unger, Elizabeth R

    2011-07-01

    DNA from archived FFPE can be used for papillomavirus genotyping, but potential problems include paraffin as a physical barrier, DNA cross-linking, and PCR inhibitors. To address these complications, we combined a commercially available DNA isolation kit (Qiagen DNeasy) with a heat treatment and evaluated the resulting DNA with regards to HPV typing. DNA was extracted from 10-μm sections from 150 FFPE cancer samples. One protocol followed the manufacturer's recommendation, including paraffin removal by xylene and tissue lysis at 56°C. A second section was directly incubated at 120°C and subsequently lysed at 65°C. After spin-column purification, both extracts were tested with a linear array HPV genotyping assay. Additionally, cellular DNA yield, HPV16 DNA copies, and PCR inhibitors were assessed by real-time qPCR assays. Inadequate linear array HPV genotyping assay results were significantly more frequent (P = 0.0003) in xylene-treated (29/150, 19.3%) than in heat-treated extracts (8/150, 5.3%). HPV detection also differed, with 94/150 (62.7%) and 110/150 (73.3%) positive results, respectively (P = 0.0026). The heat method also yielded more PCR-amplifiable cellular DNA (8.2-fold; P < 0.001) and HPV16 copies (6.5-fold; P = 0.009), although PCR inhibitors also had a greater effect (P = 0.035). Aggressive heat treatment demonstrated an advantage over traditional xylene purification protocols, resulting in higher DNA yields and increased sensitivity for HPV testing.

  14. The use of formalin fixed wax embedded tissue for proteomic analysis.

    PubMed

    Ralton, Lynda D; Murray, Graeme I

    2011-04-01

    The potential of proteomic approaches to elucidate disease pathogenesis and biomarker discovery is increasingly being recognised. These studies are usually based on the use of fresh tissue samples. Problems in obtaining and storing fresh frozen samples, especially either for the investigation of rare diseases or for the study of microscopic disease foci, have led to the investigation of the possible use of formalin fixed wax embedded tissue for proteomic biomarker detection Overcoming problems with protein cross-linking associated with formalin fixation of tissues, especially by using heat-mediated retrieval techniques combined with highly sensitive methods for protein separation and identification are now emerging, giving promise to the use of formalin fixed wax embedded tissues for proteomic analysis. Formalin fixed wax embedded tissues, together with their associated clinical and pathological information outcome may provide significant potential opportunities for proteomics research. Such studies of formalin fixed wax embedded tissue will allow access to already acquired clinical tissue samples which can be readily correlated with clinical, pathological and outcome data. It also provides access to rare types of tissue/diseases that would be either difficult to collect prospectively in a timely manner or are unlikely to be available as fresh samples. The purpose of this review is to provide an overview of the issues associated with the use of formalin fixed wax embedded tissues for proteomics.

  15. Agarose/gelatin immobilisation of tissues or embryo segments for orientated paraffin embedding and sectioning.

    PubMed

    McClelland, Kathryn S; Ng, Ee Ting; Bowles, Josephine

    2016-01-01

    The technique described in this protocol allows the user to position small tissues in the optimal orientation for paraffin embedding and sectioning by first immobilising the tissue in an agarose/gelatin cube. This method is an adaptation of methods used for early embryos and can be used for any small tissues or embryo segments. Processing of larger tissue sections using molds to create agarose/gelatin blocks has been described previously; this detailed protocol provides a method for dealing with much smaller tissues or embryos (≤5mm). The tissue is briefly fixed then an agarose/gelatin drop is created to surround the tissue. The tissue can be orientated as per the user's preference in the drop before it sets as is carved into a cube with a domed top. The cube is then dehydrated and goes through the embedding and sectioning process. The domed cube is easy to orientate when embedding the tissue in a wax block giving the user assured orientation of the small tissue for sectioning. Additionally, the agarose/gelatin cube is easy to see in the unmolded wax once embedded, making the region of interest easy to identify. PMID:26742717

  16. Detection of Mycobacterium tuberculosis Complex in Formalin-Fixed, Paraffin-Embedded Tissue Specimens with Necrotizing Granulomatous Inflammation by Strand Displacement Amplification

    PubMed Central

    Johansen, Isik Somuncu; Thomsen, Vibeke Østergaard; Forsgren, Arne; Hansen, Birgit Fischer; Lundgren, Bettina

    2004-01-01

    Rapid, reliable diagnosis of tuberculosis is essential to initiate correct treatment, avoid severe complications, and prevent transmission. Conventional microbiological methods may not be an option if samples are formalin-fixed and paraffin-embedded (FFPE) for histopathological examination. With the demonstration of necrotizing granulomatous inflammation, tuberculosis becomes an important differential diagnosis, although it was not initially suspected. Following paraffin extraction, BDProbeTec ET strand displacement amplification for detection of Mycobacterium tuberculosis complex (MTC) was applied to 47 prospectively and 19 retrospectively collected FFPE samples from various sources with granulomatous inflammation and results were compared to tuberculosis notification. Of the prospective samples, 20 were from patients who were notified as having tuberculosis and the assay was positive in 18 (90%). Specificity was 100%. For 27 of the patients with prospectively collected FFPE specimens, culture was performed on a specimen collected at a later date from the same location. Culture revealed MTC in 14 and nontuberculous mycobacteria in four. BDProbeTec ET was positive in 13 (92.8%) of the patients with positive MTC culture and negative in the remaining. The sensitivity and specificity in 19 archival samples was 40% and 100%, respectively, compared to notification data. The assay provided rapid, correct diagnosis on different sources of FFPE samples collected prospectively and therefore offers an important supplementary method for patients where tuberculosis was not initially suspected. PMID:15269300

  17. Micro-precise spatiotemporal delivery system embedded in 3D printing for complex tissue regeneration.

    PubMed

    Tarafder, Solaiman; Koch, Alia; Jun, Yena; Chou, Conrad; Awadallah, Mary R; Lee, Chang H

    2016-06-01

    Three dimensional (3D) printing has emerged as an efficient tool for tissue engineering and regenerative medicine, given its advantages for constructing custom-designed scaffolds with tunable microstructure/physical properties. Here we developed a micro-precise spatiotemporal delivery system embedded in 3D printed scaffolds. PLGA microspheres (μS) were encapsulated with growth factors (GFs) and then embedded inside PCL microfibers that constitute custom-designed 3D scaffolds. Given the substantial difference in the melting points between PLGA and PCL and their low heat conductivity, μS were able to maintain its original structure while protecting GF's bioactivities. Micro-precise spatial control of multiple GFs was achieved by interchanging dispensing cartridges during a single printing process. Spatially controlled delivery of GFs, with a prolonged release, guided formation of multi-tissue interfaces from bone marrow derived mesenchymal stem/progenitor cells (MSCs). To investigate efficacy of the micro-precise delivery system embedded in 3D printed scaffold, temporomandibular joint (TMJ) disc scaffolds were fabricated with micro-precise spatiotemporal delivery of CTGF and TGFβ3, mimicking native-like multiphase fibrocartilage. In vitro, TMJ disc scaffolds spatially embedded with CTGF/TGFβ3-μS resulted in formation of multiphase fibrocartilaginous tissues from MSCs. In vivo, TMJ disc perforation was performed in rabbits, followed by implantation of CTGF/TGFβ3-μS-embedded scaffolds. After 4 wks, CTGF/TGFβ3-μS embedded scaffolds significantly improved healing of the perforated TMJ disc as compared to the degenerated TMJ disc in the control group with scaffold embedded with empty μS. In addition, CTGF/TGFβ3-μS embedded scaffolds significantly prevented arthritic changes on TMJ condyles. In conclusion, our micro-precise spatiotemporal delivery system embedded in 3D printing may serve as an efficient tool to regenerate complex and inhomogeneous tissues. PMID

  18. Micro-precise spatiotemporal delivery system embedded in 3D printing for complex tissue regeneration.

    PubMed

    Tarafder, Solaiman; Koch, Alia; Jun, Yena; Chou, Conrad; Awadallah, Mary R; Lee, Chang H

    2016-04-25

    Three dimensional (3D) printing has emerged as an efficient tool for tissue engineering and regenerative medicine, given its advantages for constructing custom-designed scaffolds with tunable microstructure/physical properties. Here we developed a micro-precise spatiotemporal delivery system embedded in 3D printed scaffolds. PLGA microspheres (μS) were encapsulated with growth factors (GFs) and then embedded inside PCL microfibers that constitute custom-designed 3D scaffolds. Given the substantial difference in the melting points between PLGA and PCL and their low heat conductivity, μS were able to maintain its original structure while protecting GF's bioactivities. Micro-precise spatial control of multiple GFs was achieved by interchanging dispensing cartridges during a single printing process. Spatially controlled delivery of GFs, with a prolonged release, guided formation of multi-tissue interfaces from bone marrow derived mesenchymal stem/progenitor cells (MSCs). To investigate efficacy of the micro-precise delivery system embedded in 3D printed scaffold, temporomandibular joint (TMJ) disc scaffolds were fabricated with micro-precise spatiotemporal delivery of CTGF and TGFβ3, mimicking native-like multiphase fibrocartilage. In vitro, TMJ disc scaffolds spatially embedded with CTGF/TGFβ3-μS resulted in formation of multiphase fibrocartilaginous tissues from MSCs. In vivo, TMJ disc perforation was performed in rabbits, followed by implantation of CTGF/TGFβ3-μS-embedded scaffolds. After 4 wks, CTGF/TGFβ3-μS embedded scaffolds significantly improved healing of the perforated TMJ disc as compared to the degenerated TMJ disc in the control group with scaffold embedded with empty μS. In addition, CTGF/TGFβ3-μS embedded scaffolds significantly prevented arthritic changes on TMJ condyles. In conclusion, our micro-precise spatiotemporal delivery system embedded in 3D printing may serve as an efficient tool to regenerate complex and inhomogeneous tissues.

  19. [Update on protein analysis of fixed tissues].

    PubMed

    Becker, K-F; Berg, D; Malinowsky, K; Wolff, C; Ergin, B; Meding, S; Walch, A; Höfler, H

    2010-10-01

    Tissue samples have been routinely used for decades to distinguish healthy from diseased tissue in histopathological characterization. While nucleic acid-based methodologies have been successfully in use for many years, protein-based techniques, in contrast, are at a very early stage (with the exception of immunohistochemistry). One reason for this delay may be that the scientific community has long thought that formalin-fixed and paraffin embedded (FFPE) tissues are unfit for protein analysis. However, recent reports demonstrate that many protein methods that are routinely used for frozen tissues can also be applied for FFPE tissues, including Western blot, protein microarray, matrix-assisted laser desorption/ionization (MALDI) imaging and 2D gel electrophoresis. The present article provides an overview of recent developments in this field, focussing particular attention on quantitative analysis and high throughput technologies that have the potential to be integrated into the routine workflow of clinical pathology laboratories.

  20. Formalin Fixed Paraffin Embedded Tissue as a Starting Point for PrPSc Detection by ELISA

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Introduction: Formalin fixed paraffin embedded tissue are regularly employed in TSE diagnosis by IHC, the standard by which all other diagnostic protocols are currently judged. While IHC affords advantages over diagnostic approaches that typically utilize fresh or frozen tissue, such as Western blot...

  1. [Tissue artefacts by the use of overheated forceps for paraffin embedding (author's transl)].

    PubMed

    Müller, K M

    1980-05-01

    Histologic work-up of tissue specimens, especially from endoscopic biopsies, requires the use of fine forceps for orientation and dressing in fluid paraffin during the embedding procedure. These forceps are usually preheated over an open flame. If, however, smaller tissue particles are handled with overheated forceps, arteficial alterations may occur which are apt to hamper or falsify the histologic evaluation of the prepared section. Some typical tissue artefacts due to the handling with overheated forceps, are demonstrated with slides from liver biopsies. Recent experiences with an auxiliary instrument for paraffin embedding (Histostat of Vogel, designed by Ciplea) are reported. The forceps are kept at stable temperatures by immersion in fluid paraffin during the embedding procedure, thus excluding almost completely the risk of tissue artefacts by handling with overheated forceps.

  2. Decoupling tissue and cell scale stresses using embedded oil microdroplets

    NASA Astrophysics Data System (ADS)

    Shelton, Elijah; Serwane, Friedhelm; Mongera, Alessandro; Lucio, Adam; Campàs, Otger

    Embryonic development and organ morphogenesis require mechanical stresses to be patterned in space and time over length scales ranging from cellular to tissue level. While several approaches use 4D live-imaging to infer forces from the observed flow fields, few techniques allow direct measurements of stress in vivo and in situ. We use oil microdroplets injected in between cells as direct stress sensors. Through confocal imaging and custom software for high resolution 3D droplet surface reconstruction, we can directly measure the patterns of stress by looking at the deformations of the drop. This analysis allows us to decouple the stresses at the tissue scale from those generated at cellular scales by disentangling ellipsoidal drop deformation modes from higher order drop deformations. Using this technique we measure both tissue and cell scale stresses within aggregates of mesenchymal cells as well as within developing zebrafish embryonic tissues. The decoupling of mechanical stresses at cell and tissue scales makes our technique uniquely suited for understanding how tissue scale reorganizations emerge from cell scale interactions. This material is based upon work supported by the National Science Foundation Graduate Research Fellowship.

  3. Mutations in BRCA1 from fixed, paraffin-embedded tissue can be artifacts of preservation.

    PubMed

    Wong, C; DiCioccio, R A; Allen, H J; Werness, B A; Piver, M S

    1998-11-01

    DNA isolated from paraffin-embedded tissues has been used for analysis of DNA alterations in disease states. Use of archival tissue can expedite the gathering of large numbers of specimens from rare disease subtypes that would take years to accumulate prospectively. Therefore, archival tissues from 70 ovarian cancer cases diagnosed before or at age 40 were retrieved for analysis of BRCA1 mutations. DNA was isolated from paraffin-embedded tissue of 70 ovarian cancer cases diagnosed before or at age 40. BRCA1 mutation analysis was conducted by single-strand conformation polymorphism analysis and DNA sequencing. Fifty-eight BRCA1 mutations were found in 34 of the 70 ovarian cancer cases. Twenty-two cases had one mutation each and 12 cases had multiple mutations. Multiple mutations found in histologically normal tissue of 2 cases were not present in matched tumor tissue. For another case, DNA from two separate blocks of normal tissue contained different mutations. These observations were anomalous and suggested that mutations detected in fixed tissues may be artifacts of tissue preservation and not present in the original unfixed tissues. To test this suggestion, blood was obtained from 2 patients for whom mutations were found in fixed, normal tissue. DNA from their unfixed lymphocytes did not contain the mutations found in fixed normal tissue. Thus, mutations found in fixed, paraffin-embedded tissues can be artifacts of tissue preservation. The reliability of DNA sequence data derived from such tissues must be questioned in the absence of corroborating data from unfixed tissues. This severely limits the use of fixed tissues as a source of DNA for retrospective research and for clinical genetic testing in families for which a disease-affected member is not alive.

  4. High quality DNA obtained with an automated DNA extraction method with 70+ year old formalin-fixed celloidin-embedded (FFCE) blocks from the indiana medical history museum.

    PubMed

    Niland, Erin E; McGuire, Audrey; Cox, Mary H; Sandusky, George E

    2012-01-01

    DNA and RNA have been used as markers of tissue quality and integrity throughout the last few decades. In this research study, genomic quality DNA of kidney, liver, heart, lung, spleen, and brain were analyzed in tissues from post-mortem patients and surgical cancer cases spanning the past century. DNA extraction was performed on over 180 samples from: 70+ year old formalin-fixed celloidin-embedded (FFCE) tissues, formalin-fixed paraffin-embedded (FFPE) tissue samples from surgical cases and post-mortem cases from the 1970's, 1980's, 1990's, and 2000's, tissues fixed in 10% neutral buffered formalin/stored in 70% ethanol from the 1990's, 70+ year old tissues fixed in unbuffered formalin of various concentrations, and fresh tissue as a control. To extract DNA from FFCE samples and ethanol-soaked samples, a modified standard operating procedure was used in which all tissues were homogenized, digested with a proteinase K solution for a long period of time (24-48 hours), and DNA was extracted using the Autogen Flexstar automated extraction machine. To extract DNA from FFPE, all tissues were soaked in xylene to remove the paraffin from the tissue prior to digestion, and FFPE tissues were not homogenized. The results were as follows: celloidin-embedded and paraffin-embedded tissues yielded the highest DNA concentration and greatest DNA quality, while the formalin in various concentrations, and long term formalin/ethanol-stored tissue yielded both the lowest DNA concentration and quality of the tissues tested. The average DNA yield for the various fixatives was: 367.77 μg/ mL FFCE, 590.7 μg/mL FFPE, 53.74 μg/mL formalin-fixed/70% ethanol-stored and 33.2 μg/mL unbuffered formalin tissues. The average OD readings for FFCE, FFPE, formalin-fixed/70% ethanol-stored tissues, and tissues fixed in unbuffered formalin were 1.86, 1.87, 1.43, and 1.48 respectively. The results show that usable DNA can be extracted from tissue fixed in formalin and embedded in celloidin or

  5. CGH arrays compared for DNA isolated from formalin-fixed, paraffin-embedded material.

    PubMed

    Krijgsman, Oscar; Israeli, Danielle; Haan, Josien C; van Essen, Hendrik F; Smeets, Serge J; Eijk, Paul P; Steenbergen, Renske D M; Kok, Klaas; Tejpar, Sabine; Meijer, Gerrit A; Ylstra, Bauke

    2012-04-01

    Formalin-fixed, paraffin-embedded (FFPE) archival tissue is an important source of DNA material. The most commonly used technique to identify copy number aberrations from chromosomal DNA in tumorigenesis is array comparative genomic hybridization (aCGH). Although copy number analysis using DNA from FFPE archival tissue is challenging, several research groups have reported high quality and reproducible DNA copy number results using aCGH. Aim of this study is to compare the commercially available aCGH platforms suitable for high-resolution copy number analysis using FFPE-derived DNA. Two dual channel aCGH platforms (Agilent and NimbleGen) and a single channel SNP-based platform (Affymetrix) were evaluated using seven FFPE colon cancer samples, and median absolute deviation (MAD), deflection, signal-to-noise ratio, and DNA input requirements were used as quality criteria. Large differences were observed between platforms; Agilent and NimbleGen showed better MAD values (0.13 for both) compared with Affymetrix (0.22). On the contrary, Affymetrix showed a better deflection of 0.94, followed by 0.71 for Agilent and 0.51 for NimbleGen. This resulted in signal-to-nose ratios that were comparable between the three commercially available platforms. Interestingly, DNA input amounts from FFPE material lower than recommended still yielded high quality profiles on all platforms. Copy number analysis using DNA derived from FFPE archival material is feasible using all three high-resolution copy number platforms and shows reproducible results, also with DNA input amounts lower than recommended.

  6. Quantitative RT-PCR assay of HER2 mRNA expression in formalin-fixed and paraffin-embedded breast cancer tissues.

    PubMed

    Park, Sangjung; Wang, Hye-Young; Kim, Sunghyun; Ahn, Sungwoo; Lee, Dongsup; Cho, Yoonjung; Park, Kwang Hwa; Jung, Dongju; Kim, Seung Il; Lee, Hyeyoung

    2014-01-01

    Detection of human epidermal growth factor receptor 2 gene (HER2, also known as erbB2) expression is a preparatory process to decide a treatment strategy for breast cancer patients. 20-30% of breast cancer patients have HER2 overexpression, and they usually show poor recovery rate. For detection of HER2 expression, immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) methods are conventionally used. Although these methods are accurate and reliable, their time-consuming process and high cost need a concise method with high sensitivity and accuracy. As a complementary method to the current IHC/FISH standard techniques, PCR-based methods have been developed. Here we employed a quantitative PCR method to detect HER2 expression in one hundred ninety nine formalin-fixed and paraffin-embedded (FFPE) breast cancer tissue samples from the patients treated over two years at the Yonsei University Severance Hospital, Republic of Korea. Relative expression of HER2 mRNA in the FFPE samples was analyzed using a quantitative RT-PCR (RT-qPCR) method and the obtained HER2 expression levels were compared with those from IHC/FISH methods. Our results show that the RT-qPCR method was highly concordant with IHC/FISH methods for detecting HER2 expression. Overall sensitivity and specificity of the BrightGen HER2 RT-qDx assay kit (Syantra, Calgary, Canada), which is a kit we used for RT-qPCR analyses, were 93.0% and 89.8% (P < 0.0001), respectively. The diagnostic cut-off value of HER2 RT-qDx for the clinical samples was determined by likelihood ratio, among which the highest likelihood ratio of relative HER2 mRNA levels was over 105.5 (AUC = 0.9466) with the highest sensitivity and specificity. Our study indicates that quantification of HER2 mRNA expression with the RT-qPCR could be an alternative method of conventional IHC/FISH methods.

  7. Human papillomavirus detection in paraffin-embedded colorectal cancer tissues.

    PubMed

    Tanzi, Elisabetta; Bianchi, Silvia; Frati, Elena R; Amicizia, Daniela; Martinelli, Marianna; Bragazzi, Nicola L; Brisigotti, Maria Pia; Colzani, Daniela; Fasoli, Ester; Zehender, Gianguglielmo; Panatto, Donatella; Gasparini, Roberto

    2015-01-01

    Human papillomavirus (HPV) has a well-recognized aetiological role in the development of cervical cancer and other anogenital tumours. Recently, an association between colorectal cancer and HPV infection has been suggested, although this is still controversial. This study aimed at detecting and characterizing HPV infection in 57 paired biopsies from colorectal cancers and adjacent intact tissues using a degenerate PCR approach. All amplified fragments were genotyped by means of sequencing. Overall, HPV prevalence was 12.3 %. In particular, 15.8 % of tumour tissues and 8.8 % of non-cancerous tissue samples were HPV DNA-positive. Of these samples, 85.7 % were genotyped successfully, with 41.7 % of sequences identifying four genotypes of the HR (high oncogenic risk) clade Group 1; the remaining 58.3 % of HPV-genotyped specimens had an unclassified β-HPV. Examining additional cases and analysing whole genomes will help to outline the significance of these findings.

  8. Genomic Changes in Gliomas Detected Using Single Nucleotide Polymorphism Array in Formalin-Fixed, Paraffin-Embedded Tissue

    PubMed Central

    Harada, Shuko; Henderson, Lindsay B.; Eshleman, James R.; Gocke, Christopher D.; Burger, Peter; Griffin, Constance A.; Batista, Denise A.S.

    2011-01-01

    Deletion or loss of heterozygosity (LOH) in chromosomes 1p and 19q in oligodendrogliomas (ODGs) have diagnostic, prognostic, and therapeutic implications. Current clinical assays are limited because the probes or primers interrogate only limited genomic segments. We investigated the use of single nucleotide polymorphism (SNP) arrays for identifying genomic changes in gliomas from FFPE tissues. DNA was extracted from FFPE tissues of 30 brain tumor cases (15 ODGs and 15 non-ODGs) and assayed on the Illumina array with 300,000 markers. SNP results were compared with standard short tandem repeat (STR) assays of chromosomes 1p and 19q. Fifteen ODGs had LOH by STR and deletion by array on both 1p and 19q. Ten non-ODGs had no evidence of LOH on 1p and 19q by STR, seven of which had no abnormalities for these chromosomes; three had partial deletions by SNP array. Five non-ODG cases had partial LOH or deletion by both assays. No major discordance was found between SNP array and STR results. Advantages of SNP arrays include no need for an accompanying normal sample, the ability to find small segmental deletions, the potential to distinguish between deletions and copy neutral LOH, and whole-genome screening to allow discovery of new, significant loci. Assessment of genomic changes in routine glioma specimens using SNP arrays is feasible and has great potential as an accurate clinical diagnostic test. PMID:21726663

  9. Regional spectroscopy of paraffin-embedded breast cancer tissue using pulsed terahertz transmission imaging

    NASA Astrophysics Data System (ADS)

    Bowman, Tyler; El-Shenawee, Magda; Campbell, Lucas

    2016-03-01

    This work seeks to obtain the properties of paraffin-embedded breast cancer tumor tissues using transmission imaging and spectroscopy. Formalin-fixed and paraffin-embedded breast tumors are first sectioned into slices of 20 μm and 30 μm and placed between two tsurupica slides. The slides are then scanned in a pulsed terahertz system using transmission imaging. The tissue regions in adjacent pathology section are compared to the transmission imaging scan in order to define a region of points over which to average the electrical properties results from the scan.

  10. Validation of a Multiplex Allele-Specific Polymerase Chain Reaction Assay for Detection of KRAS Gene Mutations in Formalin-Fixed, Paraffin-Embedded Tissues from Colorectal Cancer Patients

    PubMed Central

    Seekhuntod, Sirirat; Thavarungkul, Paninee; Chaichanawongsaroj, Nuntaree

    2016-01-01

    Background Patients with KRAS mutations do not respond to epidermal growth factor receptor (EGFR) inhibitors and fail to benefit from adjuvant chemotherapy. Mutation analysis of KRAS is needed before starting treatment with monoclonal anti-EGFR antibodies in patients with metastatic colorectal cancer (mCRC). The objective of this study is to develop a multiplex allele-specific PCR (MAS-PCR) assay to detect KRAS mutations. Methods We developed a single-tube MAS-PCR assay for the detection of seven KRAS mutations (G12D, G12A, G12R, G12C, G12S, G12V, and G13D). We performed MAS-PCR assay analysis for KRAS on DNA isolated from 270 formalin-fixed paraffin-embedded (FFPE) colorectal cancer tissues. Sequences of all 270 samples were determined by pyrosequencing. Seven known point-mutation DNA samples diluted with wild-type DNA were assayed to determine the limitation of detection and reproducibility of the MAS-PCR assay. Results Overall, the results of MAS-PCR assay were in good concordance with pyrosequencing, and only seven discordant samples were found. The MAS-PCR assay reproducibly detected 1 to 2% mutant alleles. The most common mutations were G13D in codon 13 (49.17%), G12D (25.83%) and G12V (12.50%) in codon 12. Conclusion The MAS-PCR assay provides a rapid, cost-effective, and reliable diagnostic tool for accurate detection of KRAS mutations in routine FFPE colorectal cancer tissues. PMID:26812617

  11. How Suitable is Matrix-Assisted Laser Desorption/Ionization-Time-of-Flight for Metabolite Imaging from Clinical Formalin-Fixed and Paraffin-Embedded Tissue Samples in Comparison to Matrix-Assisted Laser Desorption/Ionization-Fourier Transform Ion Cyclotron Resonance Mass Spectrometry?

    PubMed

    Buck, Achim; Balluff, Benjamin; Voss, Andreas; Langer, Rupert; Zitzelsberger, Horst; Aichler, Michaela; Walch, Axel

    2016-05-17

    In research and clinical settings, formalin-fixed and paraffin-embedded (FFPE) tissue specimens are collected routinely and therefore this material constitutes a highly valuable source to gather insight in metabolic changes of diseases. Among mass spectrometry techniques to examine the molecular content of FFPE tissue, mass spectrometry imaging (MSI) is the most appropriate when morphological and histological features are to be related to metabolic information. Currently, high-resolution mass spectrometers are widely used for metabolomics studies. However, with regards to matrix-assisted laser desorption/ionization (MALDI) MSI, no study has so far addressed the necessity of instrumental mass resolving power in terms of clinical diagnosis and prognosis using archived FFPE tissue. For this matter we performed for the first time a comprehensive comparison between a high mass resolution Fourier-transform ion cyclotron resonance (FTICR) mass spectrometer and a time-of-flight (TOF) instrument with lower mass resolving power. Spectra analysis revealed that about one-third of the detected peaks remained unresolved by MALDI-TOF, which led to a 3-5 times lower number of m/z features compared to FTICR measurements. Overlaid peak information and background noise in TOF images made a precise assignment of molecular attributes to morphological features more difficult and limited classification approaches. This clearly demonstrates the need for high-mass resolution capabilities for metabolite imaging. Nevertheless, MALDI-TOF allowed reproducing and verifying individual markers identified previously by MALDI-FTICR MSI. The systematic comparison gives rise to a synergistic combination of the different MSI platforms for high-throughput discovery and validation of biomarkers.

  12. PrPSc detection in formalin-fixed paraffin-embedded tissue by ELISA

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Formalin-fixed paraffin-embedded tissue is regularly employed in the diagnosis of transmissible spongiform encephalopathies (TSE) by immunohistochemistry (IHC), the standard by which all other TSE diagnostic protocols are judged. While IHC affords advantages over diagnostic approaches that typically...

  13. Genome-wide comparison of paired fresh frozen and formalin-fixed paraffin-embedded gliomas by custom BAC and oligonucleotide array comparative genomic hybridization: facilitating analysis of archival gliomas

    PubMed Central

    Mohapatra, Gayatry; Engler, David A.; Starbuck, Kristen D.; Kim, James C.; Bernay, Derek C.; Scangas, George A.; Rousseau, Audrey; Batchelor, Tracy T.; Betensky, Rebecca A.; Louis, David N.

    2010-01-01

    Molecular genetic analysis of cancer is rapidly evolving as a result of improvement in genomic technologies and the growing applicability of such analyses to clinical oncology. Array based comparative genomic hybridization (aCGH) is a powerful tool for detecting DNA copy number alterations (CNA), particularly in solid tumors, and has been applied to the study of malignant gliomas. In the clinical setting, however, gliomas are often sampled by small biopsies and thus formalin-fixed paraffin-embedded (FFPE) blocks are often the only tissue available for genetic analysis, especially for rare types of gliomas. Moreover, the biological basis for the marked intratumoral heterogeneity in gliomas is most readily addressed in FFPE material. Therefore, for gliomas, the ability to use DNA from FFPE tissue is essential for both clinical and research applications. In this study, we have constructed a custom bacterial artificial chromosome (BAC) array and show excellent sensitivity and specificity for detecting CNAs in a panel of paired frozen and FFPE glioma samples. Our study demonstrates a high concordance rate between CNAs detected in FFPE compared to frozen DNA. We have also developed a method of labeling DNA from FFPE tissue that allows efficient hybridization to oligonucleotide arrays. This labeling technique was applied to a panel of biphasic anaplastic oligoastrocytomas (AOA) to identify genetic changes unique to each component. Together, results from these studies suggest that BAC and oligonucleotide aCGH are sensitive tools for detecting CNAs in FFPE DNA, and can enable genome-wide analysis of rare, small and/or histologically heterogeneous gliomas. PMID:21080181

  14. Determination of collagen content within picrosirius red stained paraffin-embedded tissue sections using fluorescence microscopy

    PubMed Central

    Vogel, Benjamin; Siebert, Hanna; Hofmann, Ulrich; Frantz, Stefan

    2015-01-01

    Picrosirius red (PSR) staining is a commonly used histological technique to visualize collagen in paraffin-embedded tissue sections. PSR stained collagen appears red in light microscopy. However it is largely unknown that PSR stained collagen also shows a red fluorescence, whereas live cells have a distinct green autofluorescence. Both emission patterns can be detected using standard filter sets as found in conventional fluorescence microscopes. Here we used digital image addition and subtraction to determine the relative area of the pure collagen and live cell content in heart tissue in a semi-automated process using standard software. This procedure, which considers empty spaces (holes) within the section, can be easily adapted to quantify the collagen and live cell areas in healthy or fibrotic tissues as aorta, lung, kidney or liver by semi-automated planimetry exemplified herein for infarcted heart tissue obtained from the mouse myocardial infarction model. • Use of conventional PSR stained paraffin-embedded tissue sections for fluorescence analysis. • PSR and autofluorescence images are used to calculate area of collagen and area of live cells in the tissue; empty spaces (holes) in tissue are considered. • High throughput analysis of collagen and live cell content in tissue for statistical purposes. PMID:26150980

  15. Isolation of high quality protein samples from punches of formalin fixed and paraffin embedded tissue blocks.

    PubMed

    Kroll, J; Becker, K F; Kuphal, S; Hein, R; Hofstädter, F; Bosserhoff, A K

    2008-04-01

    In general, it is believed that the extraction of proteins from formalin-fixed paraffin embedded samples is not feasible. However, recently a new technique was developed, presenting the extraction of non-degraded, full length proteins from formalin fixed tissues, usable for western blotting and protein arrays. In the study presented here, we applied this technique to punch biopsies of formalin fixed tissues embedded in paraffin to reduce heterogeneity of the tissue represented in sections, and to ensure analysing mainly defined cellular material. Successful extraction was achieved even from very small samples (0.7 mm(3)). Additionally, we were able to detect highly glycosylated proteins and protein modification, such as phosphorylation. Interestingly, with this technique it is feasible to extract high quality proteins from 14 year old samples. In summary, the new technique makes a great pool of material now usable for molecular analysis with high throughput tools. PMID:18228195

  16. Ultrastructural immunogold labeling of glial filaments in osmicated and unosmicated epoxy-embedded tissue.

    PubMed

    Bettica, A; Johnson, A B

    1990-01-01

    On-grid (post-embedding) immunolabeling methods with epoxy resins have been difficult to apply to thin structures such as intermediate filaments, which may remain inaccessible within the plastic. In this study, glial fibrillary acidic protein (GFAP), the major protein of astrocyte intermediate filaments, was localized with a post-embedding immunogold method, using both unosmicated and osmicated material embedded in epoxy resin. The tissue studied was from a diagnostic brain biopsy on a child with Alexander's disease. This disorder is characterized by proliferation of astrocyte intermediate filaments and formation of Rosenthal fibers. With unosmicated tissue, as in a previous study, extensive labeling of the glial filaments was achieved only when ultra-thin sections were pre-treated with dilute sodium ethoxide, an agent that dissolves plastic. Fifteen-nm gold could be used. With osmicated tissue, localization to glial filaments required pre-treatment with sodium ethoxide and with the oxidizing agent sodium metaperiodate, followed by the use of small (5 nm) colloidal gold. That 5-nm gold was required for labeling filaments in osmicated material suggested that osmication increases problems of penetrability and antigen accessibility within ultra-thin sections. The large Rosenthal fibers were labeled by 15-nm gold in both unosmicated and osmicated material. The methods employed may be useful for electron immunolocalizations to other thin structures in material embedded in epoxy resin.

  17. Terahertz absorption and reflection imaging of carcinoma-affected colon tissues embedded in paraffin

    NASA Astrophysics Data System (ADS)

    Wahaia, Faustino; Kasalynas, Irmantas; Venckevicius, Rimvydas; Seliuta, Dalius; Valusis, Gintaras; Urbanowicz, Andrzej; Molis, Gediminas; Carneiro, Fatima; Carvalho Silva, Catia D.; Granja, Pedro L.

    2016-03-01

    In the present study, dehydrated human colon tissues embedded in paraffin were studied at THz frequency. A compact THz imaging system with high numerical aperture optics was developed for the analysis of adenocarcinoma-affected colon sections, in transmission and reflection geometry. A comprehensive analysis of the THz images revealed a contrast up to 23% between the neoplastic and control tissues. Absorption and reflection THz images demonstrated the possibility to distinguish adenocarcinoma-affected areas even without water in the tissue, as the main contrast mechanism in THz measurements has been observed to be water absorption in in vivo or freshly excised tissues. The present results corroborate with previous histologic findings in the same tissues, and confirm that the contrast prevails even in dehydrated tissues.

  18. Multi-Center Evaluation of the Fully Automated PCR-Based Idylla™ KRAS Mutation Assay for Rapid KRAS Mutation Status Determination on Formalin-Fixed Paraffin-Embedded Tissue of Human Colorectal Cancer

    PubMed Central

    Solassol, Jérôme; Vendrell, Julie; Märkl, Bruno; Haas, Christian; Bellosillo, Beatriz; Montagut, Clara; Smith, Matthew; O’Sullivan, Brendan; D’Haene, Nicky; Le Mercier, Marie; Grauslund, Morten; Melchior, Linea Cecilie; Burt, Emma; Cotter, Finbarr; Stieber, Daniel; Schmitt, Fernando de Lander; Motta, Valentina; Lauricella, Calogero; Colling, Richard; Soilleux, Elizabeth; Fassan, Matteo; Mescoli, Claudia; Collin, Christine; Pagès, Jean-Christophe; Sillekens, Peter

    2016-01-01

    Since the advent of monoclonal antibodies against epidermal growth factor receptor (EGFR) in colorectal cancer therapy, the determination of RAS mutational status is needed for therapeutic decision-making. Most prevalent in colorectal cancer are KRAS exon 2 mutations (40% prevalence); lower prevalence is observed for KRAS exon 3 and 4 mutations (6%) and NRAS exon 2, 3, and 4 mutations (5%). The Idylla™ KRAS Mutation Test on the molecular diagnostics Idylla™ platform is a simple (<2 minutes hands-on time), highly reliable, and rapid (approximately 2 hours turnaround time) in vitro diagnostic sample-to-result solution. This test enables qualitative detection of 21 mutations in codons 12, 13, 59, 61, 117, and 146 of the KRAS oncogene being clinically relevant according to the latest clinical guidelines. Here, the performance of the Idylla™ KRAS Mutation Assay, for Research Use Only, was assessed on archived formalin-fixed paraffin-embedded (FFPE) tissue sections by comparing its results with the results previously obtained by routine reference approaches for KRAS genotyping. In case of discordance, samples were assessed further by additional methods. Among the 374 colorectal cancer FFPE samples tested, the overall concordance between the Idylla™ KRAS Mutation Assay and the confirmed reference routine test results was found to be 98.9%. The Idylla™ KRAS Mutation Assay enabled detection of 5 additional KRAS-mutated samples not detected previously with reference methods. As conclusion the Idylla™ KRAS Mutation Test can be applied as routine tool in any clinical setting, without needing molecular infrastructure or expertise, to guide the personalized treatment of colorectal cancer patients. PMID:27685259

  19. HaloPlex Targeted Resequencing for Mutation Detection in Clinical Formalin-Fixed, Paraffin-Embedded Tumor Samples.

    PubMed

    Moens, Lotte N J; Falk-Sörqvist, Elin; Ljungström, Viktor; Mattsson, Johanna; Sundström, Magnus; La Fleur, Linnéa; Mathot, Lucy; Micke, Patrick; Nilsson, Mats; Botling, Johan

    2015-11-01

    In recent years, the advent of massively parallel next-generation sequencing technologies has enabled substantial advances in the study of human diseases. Combined with targeted DNA enrichment methods, high sequence coverage can be obtained for different genes simultaneously at a reduced cost per sample, creating unique opportunities for clinical cancer diagnostics. However, the formalin-fixed, paraffin-embedded (FFPE) process of tissue samples, routinely used in pathology departments, results in DNA fragmentation and nucleotide modifications that introduce a number of technical challenges for downstream biomolecular analyses. We evaluated the HaloPlex target enrichment system for somatic mutation detection in 80 tissue fractions derived from 20 clinical cancer cases with paired tumor and normal tissue available in both FFPE and fresh-frozen format. Several modifications to the standard method were introduced, including a reduced target fragment length and two strand capturing. We found that FFPE material can be used for HaloPlex-based target enrichment and next-generation sequencing, even when starting from small amounts of DNA. By specifically capturing both strands for each target fragment, we were able to reduce the number of false-positive errors caused by FFPE-induced artifacts and lower the detection limit for somatic mutations. We believe that the HaloPlex method presented here will be broadly applicable as a tool for somatic mutation detection in clinical cancer settings.

  20. HaloPlex Targeted Resequencing for Mutation Detection in Clinical Formalin-Fixed, Paraffin-Embedded Tumor Samples.

    PubMed

    Moens, Lotte N J; Falk-Sörqvist, Elin; Ljungström, Viktor; Mattsson, Johanna; Sundström, Magnus; La Fleur, Linnéa; Mathot, Lucy; Micke, Patrick; Nilsson, Mats; Botling, Johan

    2015-11-01

    In recent years, the advent of massively parallel next-generation sequencing technologies has enabled substantial advances in the study of human diseases. Combined with targeted DNA enrichment methods, high sequence coverage can be obtained for different genes simultaneously at a reduced cost per sample, creating unique opportunities for clinical cancer diagnostics. However, the formalin-fixed, paraffin-embedded (FFPE) process of tissue samples, routinely used in pathology departments, results in DNA fragmentation and nucleotide modifications that introduce a number of technical challenges for downstream biomolecular analyses. We evaluated the HaloPlex target enrichment system for somatic mutation detection in 80 tissue fractions derived from 20 clinical cancer cases with paired tumor and normal tissue available in both FFPE and fresh-frozen format. Several modifications to the standard method were introduced, including a reduced target fragment length and two strand capturing. We found that FFPE material can be used for HaloPlex-based target enrichment and next-generation sequencing, even when starting from small amounts of DNA. By specifically capturing both strands for each target fragment, we were able to reduce the number of false-positive errors caused by FFPE-induced artifacts and lower the detection limit for somatic mutations. We believe that the HaloPlex method presented here will be broadly applicable as a tool for somatic mutation detection in clinical cancer settings. PMID:26354930

  1. Concordant HER2 status between metastatic breast cancer cells in CSF and primary breast cancer tissue.

    PubMed

    Park, In Hae; Kwon, Youngmee; Ro, Jae Y; Lee, Keun Seok; Ro, Jungsil

    2010-08-01

    It is not known whether the HER2 status of malignant CSF cells coincides with that of the original breast carcinoma cells. We investigated whether CSF cytology specimens were suitable to evaluate HER2 status by fluorescence in situ hybridization (FISH) in patient with leptomeningeal metastasis (LM). Both formalin-fixed paraffin-embedded (FFPE) breast cancer tissue and liquid based CSF cytology specimens were evaluated for HER2 status in 16 patients with LM. We evaluated HER2 gene amplification using FISH on destained CSF cytology slides containing a minimum of 20 malignant cells per slide, and compared these with the HER2 status by immunohistochemistry (IHC) or FISH in FFPE tissues. HER2 was considered positive when the HER2:CEP17 ratio was >or=2.0 or IHC 3+. Of 16 cases, four were HER2 positive and 12 were HER2 negative by FISH analysis in CSF cytology. All CSF-positive cases were HER2 positive by IHC in FFPE tissue. Of 12 HER2 FISH-negative cases in CSF cytology, 10 were HER2 negative (IHC 0 or 1+) and two were IHC 2+ in FFPE tissue. Two IHC 2+ cases had HER2:CEP17 ratios of 1.27 and 2.1, respectively, by FISH in FFPE tissue. As a result, the HER2 status concordance rate between metastatic breast cancer cells in CSF and FFPE primary tissue by IHC and FISH was very high. When CSF cytology specimens were appropriately prepared and had adequate cellularity without dry artifacts, the CSF cytology was suitable to evaluate HER2 status by FISH analysis in patients with LM.

  2. Improved reproducibility in genome-wide DNA methylation analysis for PAXgene-fixed samples compared with restored formalin-fixed and paraffin-embedded DNA.

    PubMed

    Andersen, Gitte Brinch; Hager, Henrik; Hansen, Lise Lotte; Tost, Jörg

    2015-01-01

    Formalin fixation has been the standard method for conservation of clinical specimens for decades. However, a major drawback is the high degradation of nucleic acids, which complicates its use in genome-wide analyses. Unbiased identification of biomarkers, however, requires genome-wide studies, precluding the use of the valuable archives of specimens with long-term follow-up data. Therefore, restoration protocols for DNA from formalin-fixed and paraffin-embedded (FFPE) samples have been developed, although they are cost-intensive and time-consuming. An alternative to FFPE and snap-freezing is the PAXgene Tissue System, developed for simultaneous preservation of morphology, proteins, and nucleic acids. In the current study, we compared the performance of DNA from either PAXgene or formalin-fixed tissues to snap-frozen material for genome-wide DNA methylation analysis using the Illumina 450K BeadChip. Quantitative DNA methylation analysis demonstrated that the methylation profile in PAXgene-fixed tissues showed, in comparison with restored FFPE samples, a higher concordance with the profile detected in frozen samples. We demonstrate, for the first time, that DNA from PAXgene conserved tissue performs better compared with restored FFPE DNA in genome-wide DNA methylation analysis. In addition, DNA from PAXgene tissue can be directly used on the array without prior restoration, rendering the analytical process significantly more time- and cost-effective.

  3. Targeted or whole genome sequencing of formalin fixed tissue samples: potential applications in cancer genomics

    PubMed Central

    Zhao, Yue; Cottrell, Joseph; Klotzle, Brandy; Godwin, Andrew K.; Koestler, Devin; Beyerlein, Peter; Fan, Jian-Bing; Bibikova, Marina; Chien, Jeremy

    2015-01-01

    Current genomic studies are limited by the poor availability of fresh-frozen tissue samples. Although formalin-fixed diagnostic samples are in abundance, they are seldom used in current genomic studies because of the concern of formalin-fixation artifacts. Better characterization of these artifacts will allow the use of archived clinical specimens in translational and clinical research studies. To provide a systematic analysis of formalin-fixation artifacts on Illumina sequencing, we generated 26 DNA sequencing data sets from 13 pairs of matched formalin-fixed paraffin-embedded (FFPE) and fresh-frozen (FF) tissue samples. The results indicate high rate of concordant calls between matched FF/FFPE pairs at reference and variant positions in three commonly used sequencing approaches (whole genome, whole exome, and targeted exon sequencing). Global mismatch rates and C·G > T·A substitutions were comparable between matched FF/FFPE samples, and discordant rates were low (<0.26%) in all samples. Finally, low-pass whole genome sequencing produces similar pattern of copy number alterations between FF/FFPE pairs. The results from our studies suggest the potential use of diagnostic FFPE samples for cancer genomic studies to characterize and catalog variations in cancer genomes. PMID:26305677

  4. Targeted or whole genome sequencing of formalin fixed tissue samples: potential applications in cancer genomics.

    PubMed

    Munchel, Sarah; Hoang, Yen; Zhao, Yue; Cottrell, Joseph; Klotzle, Brandy; Godwin, Andrew K; Koestler, Devin; Beyerlein, Peter; Fan, Jian-Bing; Bibikova, Marina; Chien, Jeremy

    2015-09-22

    Current genomic studies are limited by the poor availability of fresh-frozen tissue samples. Although formalin-fixed diagnostic samples are in abundance, they are seldom used in current genomic studies because of the concern of formalin-fixation artifacts. Better characterization of these artifacts will allow the use of archived clinical specimens in translational and clinical research studies. To provide a systematic analysis of formalin-fixation artifacts on Illumina sequencing, we generated 26 DNA sequencing data sets from 13 pairs of matched formalin-fixed paraffin-embedded (FFPE) and fresh-frozen (FF) tissue samples. The results indicate high rate of concordant calls between matched FF/FFPE pairs at reference and variant positions in three commonly used sequencing approaches (whole genome, whole exome, and targeted exon sequencing). Global mismatch rates and C · G > T · A substitutions were comparable between matched FF/FFPE samples, and discordant rates were low (<0.26%) in all samples. Finally, low-pass whole genome sequencing produces similar pattern of copy number alterations between FF/FFPE pairs. The results from our studies suggest the potential use of diagnostic FFPE samples for cancer genomic studies to characterize and catalog variations in cancer genomes.

  5. Targeted or whole genome sequencing of formalin fixed tissue samples: potential applications in cancer genomics.

    PubMed

    Munchel, Sarah; Hoang, Yen; Zhao, Yue; Cottrell, Joseph; Klotzle, Brandy; Godwin, Andrew K; Koestler, Devin; Beyerlein, Peter; Fan, Jian-Bing; Bibikova, Marina; Chien, Jeremy

    2015-09-22

    Current genomic studies are limited by the poor availability of fresh-frozen tissue samples. Although formalin-fixed diagnostic samples are in abundance, they are seldom used in current genomic studies because of the concern of formalin-fixation artifacts. Better characterization of these artifacts will allow the use of archived clinical specimens in translational and clinical research studies. To provide a systematic analysis of formalin-fixation artifacts on Illumina sequencing, we generated 26 DNA sequencing data sets from 13 pairs of matched formalin-fixed paraffin-embedded (FFPE) and fresh-frozen (FF) tissue samples. The results indicate high rate of concordant calls between matched FF/FFPE pairs at reference and variant positions in three commonly used sequencing approaches (whole genome, whole exome, and targeted exon sequencing). Global mismatch rates and C · G > T · A substitutions were comparable between matched FF/FFPE samples, and discordant rates were low (<0.26%) in all samples. Finally, low-pass whole genome sequencing produces similar pattern of copy number alterations between FF/FFPE pairs. The results from our studies suggest the potential use of diagnostic FFPE samples for cancer genomic studies to characterize and catalog variations in cancer genomes. PMID:26305677

  6. Technical report: immunofluorescence and TUNEL staining of celloidin embedded human temporal bone tissues.

    PubMed

    Markaryan, Adam; Nelson, Erik G; Tretiakova, Maria; Hinojosa, Raul

    2008-07-01

    The large archival human temporal bone collections of the world have been fixed in formalin and embedded in celloidin. These treatments have created challenges to the use of contemporary probes, which are routinely used in the evaluation of fresh and frozen tissues, for the analysis of archival temporal bone tissues. Formalin alters the configuration of proteins and can obscure antigens by modifying the epitopes recognized by antibodies. Celloidin embedding provides superior support of the delicate membranous structures of the inner ear to maintain tissue integrity during sectioning, however, inadequate removal of celloidin may limit tissue permeability resulting in poor penetration of large molecules. Methods are described in this manuscript that have allowed reproducible immunofluorescence and TUNEL (terminal deoxynucleotidyl transferase mediated dUTP nick end labeling) staining results in these archival tissues. To our knowledge, successful immunofluorescence staining of type I collagen, immunofluorescence staining of cytochrome c oxidase subunit III (COX III), and TUNEL staining in archival human temporal bone tissues with confocal microscopy has not been previously reported. These results demonstrate the utility of developing techniques to evaluate the existing collections of archival temporal bones which remain our greatest source of tissue for investigating the causes of ear diseases.

  7. Direct cancer tissue proteomics: a method to identify candidate cancer biomarkers from formalin-fixed paraffin-embedded archival tissues.

    PubMed

    Hwang, S-I; Thumar, J; Lundgren, D H; Rezaul, K; Mayya, V; Wu, L; Eng, J; Wright, M E; Han, D K

    2007-01-01

    Successful treatment of multiple cancer types requires early detection and identification of reliable biomarkers present in specific cancer tissues. To test the feasibility of identifying proteins from archival cancer tissues, we have developed a methodology, termed direct tissue proteomics (DTP), which can be used to identify proteins directly from formalin-fixed paraffin-embedded prostate cancer tissue samples. Using minute prostate biopsy sections, we demonstrate the identification of 428 prostate-expressed proteins using the shotgun method. Because the DTP method is not quantitative, we employed the absolute quantification method and demonstrate picogram level quantification of prostate-specific antigen. In depth bioinformatics analysis of these expressed proteins affords the categorization of metabolic pathways that may be important for distinct stages of prostate carcinogenesis. Furthermore, we validate Wnt-3 as an upregulated protein in cancerous prostate cells by immunohistochemistry. We propose that this general strategy provides a roadmap for successful identification of critical molecular targets of multiple cancer types.

  8. [An observation on the histological structure of Oncomelania hupensis soft tissue by agar-paraffin double-embedding method].

    PubMed

    Tan, Ping; Zhang, Jie; Li, Qing; Yu, Zhi-jun

    2014-12-01

    In order to study the histological structure of Oncomelania hupensis soft tissue, the fixed soft tissues of O. hupensis were pre-embedded in the agar and made blocks, then dehydrated, transparentized, immersed in paraffin, sectioned, and stained with haematoxylin-eosin (HE). Permanent slides of O. hupensis soft tissue were obtained. The histological structure of soft tissues was clear under the microscope.

  9. Quantitative gene expression analysis in microdissected archival formalin-fixed and paraffin-embedded tumor tissue.

    PubMed

    Specht, K; Richter, T; Müller, U; Walch, A; Werner, M; Höfler, H

    2001-02-01

    Formalin-fixed, paraffin-embedded tissue is the most widely available material for retrospective clinical studies. In combination with the potential of genomics, these tissues represent an invaluable resource for the elucidation of disease mechanisms and validation of differentially expressed genes as novel therapeutic targets or prognostic indicators. We describe here an approach that, in combination with laser-assisted microdissection allows quantitative gene expression analysis in formalin-fixed, paraffin-embedded archival tissue. Using an optimized RNA microscale extraction procedure in conjunction with real-time quantitative reverse transcriptase-polymerase chain reaction based on fluorogenic TaqMan methodology, we analyzed the expression of a panel of cancer-relevant genes, EGF-R, HER-2/neu, FGF-R4, p21/WAF1/Cip1, MDM2, and HPRT and PGK as controls. We demonstrate that expression level determinations from formalin-fixed, paraffin-embedded tissues are accurate and reproducible. Measurements were comparable to those obtained with matching fresh-frozen tissue and neither fixation grade nor time significantly affected the results. Laser microdissection studies with 5-microm thick sections and defined numbers of tumor cells demonstrated that reproducible quantitation of specific mRNAs can be achieved with only 50 cells. We applied our approach to HER-2/neu quantitative gene expression analysis in 54 microdissected tumor and nonneoplastic archival samples from patients with Barrett's esophageal adenocarcinoma and showed that the results matched those obtained in parallel by fluorescence in situ hybridization and immunohistochemistry. Thus, the combination of laser-assisted microdissection and real-time TaqMan reverse transcriptase-polymerase chain reaction opens new avenues for the investigation and clinical validation of gene expression changes in archival tissue specimens.

  10. Genome-wide Detection of DNase I Hypersensitive Sites in Single Cells and FFPE Samples

    PubMed Central

    Jin, Wenfei; Tang, Qingsong; Wan, Mimi; Cui, Kairong; Zhang, Yi; Ren, Gang; Ni, Bing; Sklar, Jeffrey; Przytycka, Teresa M.; Childs, Richard; Levens, David; Zhao, Keji

    2015-01-01

    DNase I hypersensitive sites (DHSs) provide important information on the presence of transcriptional regulatory elements and the state of chromatin in mammalian cells1–3. Conventional DNase-Seq for genome-wide DHSs profiling is limited by the requirement of millions of cells4,5. Here we report an ultrasensitive strategy, called Pico-Seq, for detection of genome-wide DHSs in single cells. We show that DHS patterns at the single cell level are highly reproducible among individual cells. Among different single cells, highly expressed gene promoters and the enhancers associated with multiple active histone modifications display constitutive DHS while chromatin regions with fewer histone modifications exhibit high variation of DHS. Furthermore, the single-cell DHSs predict enhancers that regulate cell-specific gene expression programs and the cell-to-cell variations of DHS are predictive of gene expression. Finally, we apply Pico-Seq to pools of tumor cells and pools of normal cells, dissected from formalin-fixed paraffin-embedded (FFPE) tissue slides from thyroid cancer patients, and detect thousands of tumor-specific DHSs. Many of these DHSs are associated with promoters and enhancers critically involved in cancer development. Analysis of the DHS sequences uncovers one single-nucleotide variant (chr18:52417839 G>C) in the tumor cells of a follicular thyroid carcinoma patient, which affects the binding of the tumor suppressor protein p53 and correlates with decreased expression of its target gene TXNL1. In conclusion, Pico-Seq can reliably detect DHSs in single cells, greatly extending the range of applications of DHS analysis for both basic and translational research and may provide critical information for personalized medicine. PMID:26605532

  11. Cross-contamination in the molecular detection of Bartonella from paraffin-embedded tissues.

    PubMed

    Varanat, M; Maggi, R G; Linder, K E; Horton, S; Breitschwerdt, E B

    2009-09-01

    The genus Bartonella comprises a group of gram-negative, fastidious bacteria. Because of diagnostic limitations of culture and serologic testing, polymerase chain reaction (PCR) has become a powerful tool for the detection of Bartonella spp. in blood and tissue samples. However, because many wild and domestic animals harbor Bartonella spp., transfer of Bartonella DNA during sample collection or histologic processing could result in false-positive PCR test results. In this study, we describe evidence of Bartonella DNA dissemination and transfer in the necropsy room and during the subsequent processing of formalin-fixed paraffin-embedded tissues. Bartonella DNA was amplified from different areas of the necropsy room, from the liquid paraffin in the tissue processor, and from different parts of the microtome. Unless stringent procedures are established and followed to avoid cross-contamination, the molecular detection of Bartonella spp. from tissue samples obtained at necropsy or processed in a multispecies histopathology laboratory will not be reliable. PMID:19429988

  12. Relative shrinkage of adipocytes by paraffin in proportion to plastic embedding in human adipose tissue before and after weight loss.

    PubMed

    Verhoef, Sanne P M; van Dijk, Paul; Westerterp, Klaas R

    2013-01-01

    Adipocyte size is a major modulator of endocrine functioning of adipose tissue and methods allowing accurate determination of adipocyte size are important to study energy metabolism. The aim of this study was to assess the relative shrinkage of adipocytes before and after weight loss by comparing adipose tissue from the same subjects embedded in paraffin and plastic. 18 healthy subjects (5 males and 13 females) aged 20-50 y with a BMI of 28-38 kg/m² followed a very low energy diet for 8 weeks. Adipose tissue biopsies were taken prior to and after weight loss and were processed for paraffin and plastic sections. Parameters of adipocyte size were determined with computer image analysis. Mean adipocyte size was smaller in paraffin compared to plastic embedded tissue both before (66 ± 4 vs. 103 ± 5 μm, P < 0.001) as after weight loss (62 ± 4 vs. 91 ± 5 μm, P < 0.001). Relative shrinkage of adipocytes in paraffin embedded tissue in proportion to plastic embedded tissue was not significantly different before and after weight loss (73 and 69%, respectively). Shrinkage due to the type of embedding of the adipose tissue can be ignored when comparing before and after weight loss. Plastic embedding of adipose tissue provides more accurate and sensitive results. PMID:24331678

  13. Relative shrinkage of adipocytes by paraffin in proportion to plastic embedding in human adipose tissue before and after weight loss.

    PubMed

    Verhoef, Sanne P M; van Dijk, Paul; Westerterp, Klaas R

    2013-01-01

    Adipocyte size is a major modulator of endocrine functioning of adipose tissue and methods allowing accurate determination of adipocyte size are important to study energy metabolism. The aim of this study was to assess the relative shrinkage of adipocytes before and after weight loss by comparing adipose tissue from the same subjects embedded in paraffin and plastic. 18 healthy subjects (5 males and 13 females) aged 20-50 y with a BMI of 28-38 kg/m² followed a very low energy diet for 8 weeks. Adipose tissue biopsies were taken prior to and after weight loss and were processed for paraffin and plastic sections. Parameters of adipocyte size were determined with computer image analysis. Mean adipocyte size was smaller in paraffin compared to plastic embedded tissue both before (66 ± 4 vs. 103 ± 5 μm, P < 0.001) as after weight loss (62 ± 4 vs. 91 ± 5 μm, P < 0.001). Relative shrinkage of adipocytes in paraffin embedded tissue in proportion to plastic embedded tissue was not significantly different before and after weight loss (73 and 69%, respectively). Shrinkage due to the type of embedding of the adipose tissue can be ignored when comparing before and after weight loss. Plastic embedding of adipose tissue provides more accurate and sensitive results.

  14. Transcriptome Sequencing (RNAseq) Enables Utilization of Formalin-Fixed, Paraffin-Embedded Biopsies with Clear Cell Renal Cell Carcinoma for Exploration of Disease Biology and Biomarker Development.

    PubMed

    Eikrem, Oystein; Beisland, Christian; Hjelle, Karin; Flatberg, Arnar; Scherer, Andreas; Landolt, Lea; Skogstrand, Trude; Leh, Sabine; Beisvag, Vidar; Marti, Hans-Peter

    2016-01-01

    Formalin-fixed, paraffin-embedded (FFPE) tissues are an underused resource for molecular analyses. This proof of concept study aimed to compare RNAseq results from FFPE biopsies with the corresponding RNAlater® (Qiagen, Germany) stored samples from clear cell renal cell carcinoma (ccRCC) patients to investigate feasibility of RNAseq in archival tissue. From each of 16 patients undergoing partial or full nephrectomy, four core biopsies, such as two specimens with ccRCC and two specimens of adjacent normal tissue, were obtained with a 16g needle. One normal and one ccRCC tissue specimen per patient was stored either in FFPE or RNAlater®. RNA sequencing libraries were generated applying the new Illumina TruSeq® Access library preparation protocol. Comparative analysis was done using voom/Limma R-package. The analysis of the FFPE and RNAlater® datasets yielded similar numbers of detected genes, differentially expressed transcripts and affected pathways. The FFPE and RNAlater datasets shared 80% (n = 1106) differentially expressed genes. The average expression and the log2 fold changes of these transcripts correlated with R2 = 0.97, and R2 = 0.96, respectively. Among transcripts with the highest fold changes in both datasets were carbonic anhydrase 9 (CA9), neuronal pentraxin-2 (NPTX2) and uromodulin (UMOD) that were confirmed by immunohistochemistry. IPA revealed the presence of gene signatures of cancer and nephrotoxicity, renal damage and immune response. To simulate the feasibility of clinical biomarker studies with FFPE samples, a classifier model was developed for the FFPE dataset: expression data for CA9 alone had an accuracy, specificity and sensitivity of 94%, respectively, and achieved similar performance in the RNAlater dataset. Transforming growth factor-ß1 (TGFB1)-regulated genes, epithelial to mesenchymal transition (EMT) and NOTCH signaling cascade may support novel therapeutic strategies. In conclusion, in this proof of concept study, RNAseq data

  15. Transcriptome Sequencing (RNAseq) Enables Utilization of Formalin-Fixed, Paraffin-Embedded Biopsies with Clear Cell Renal Cell Carcinoma for Exploration of Disease Biology and Biomarker Development

    PubMed Central

    Eikrem, Oystein; Beisland, Christian; Hjelle, Karin; Flatberg, Arnar; Scherer, Andreas; Landolt, Lea; Skogstrand, Trude; Leh, Sabine; Beisvag, Vidar; Marti, Hans-Peter

    2016-01-01

    Formalin-fixed, paraffin-embedded (FFPE) tissues are an underused resource for molecular analyses. This proof of concept study aimed to compare RNAseq results from FFPE biopsies with the corresponding RNAlater® (Qiagen, Germany) stored samples from clear cell renal cell carcinoma (ccRCC) patients to investigate feasibility of RNAseq in archival tissue. From each of 16 patients undergoing partial or full nephrectomy, four core biopsies, such as two specimens with ccRCC and two specimens of adjacent normal tissue, were obtained with a 16g needle. One normal and one ccRCC tissue specimen per patient was stored either in FFPE or RNAlater®. RNA sequencing libraries were generated applying the new Illumina TruSeq® Access library preparation protocol. Comparative analysis was done using voom/Limma R-package. The analysis of the FFPE and RNAlater® datasets yielded similar numbers of detected genes, differentially expressed transcripts and affected pathways. The FFPE and RNAlater datasets shared 80% (n = 1106) differentially expressed genes. The average expression and the log2 fold changes of these transcripts correlated with R2 = 0.97, and R2 = 0.96, respectively. Among transcripts with the highest fold changes in both datasets were carbonic anhydrase 9 (CA9), neuronal pentraxin-2 (NPTX2) and uromodulin (UMOD) that were confirmed by immunohistochemistry. IPA revealed the presence of gene signatures of cancer and nephrotoxicity, renal damage and immune response. To simulate the feasibility of clinical biomarker studies with FFPE samples, a classifier model was developed for the FFPE dataset: expression data for CA9 alone had an accuracy, specificity and sensitivity of 94%, respectively, and achieved similar performance in the RNAlater dataset. Transforming growth factor-ß1 (TGFB1)-regulated genes, epithelial to mesenchymal transition (EMT) and NOTCH signaling cascade may support novel therapeutic strategies. In conclusion, in this proof of concept study, RNAseq data

  16. Expression of miR-146a, miR-155, and miR-223 in formalin-fixed paraffin-embedded synovial tissues of patients with rheumatoid arthritis and osteoarthritis.

    PubMed

    Kriegsmann, Mark; Randau, Thomas M; Gravius, Sascha; Lisenko, Katharina; Altmann, Carolin; Arens, Norbert; Kriegsmann, Jörg

    2016-07-01

    Rheumatoid arthritis (RA) is a chronic autoimmune disease with a heterogeneous clinical presentation affecting about 1 % of adults in developed countries. Currently, the diagnosis is based on the revised criteria of the American College of Rheumatology (ACR) and the European League Against Rheumatism (EULAR) from 2010. These criteria include clinical and laboratory parameters. Because of the variability of the clinical picture, delayed diagnosis of RA occurs in a significant subset of patients. Therefore, the discovery of novel biomarkers that improve the diagnosis of RA is of particular interest. Recently, it became evident that miRNAs have regulatory activities in physiologic processes and human diseases. Upregulation of miR-146a, miR-155, and miR-223 has been shown in various compartments such as serum, blood, synovial fluid, and tissues in patients with RA. A total of 87 samples were analyzed (RA 50, osteoarthritis (OA) 37). RNA was isolated from formalin-fixed paraffin-embedded synovial tissue (FFPE). The relative expression of miR-146a, miR-155, and miR-223 was determined by comparison to a housekeeping RNA molecule (snRNA U6) and an RNA pool from histologically and clinically verified OA samples. miR-146a, miR-155, and miR-223 were significantly elevated in RA compared to OA synovial tissues (p < 0.001). A strong correlation between the miRNAs could be observed. The sensitivity and specificity for the detection of RA were 0.76/0.80 (miR-146a), 0.80/0.95 (miR-155), and 0.86/0.81 (miR-223). The combination of miR-155 and miR-223 resulted in the highest area under the curve (AUC 0.92) with a sensitivity and specificity of 0.84/0.91, respectively. Significantly higher expression levels of miR-146a, miR-155, and miR-223 in FFPE synovial tissue samples of patients with established RA compared to patients with OA were shown. The usefulness of these miRs for the differential diagnosis of early phases of RA against OA remains to be investigated. PMID:27079198

  17. STED Super-Resolution Microscopy of Clinical Paraffin-Embedded Human Rectal Cancer Tissue

    PubMed Central

    Wurm, Christian Andreas; Rüschoff, Josef; Ghadimi, B. Michael; Liersch, Torsten; Jakobs, Stefan

    2014-01-01

    Formalin fixed and paraffin-embedded human tissue resected during cancer surgery is indispensable for diagnostic and therapeutic purposes and represents a vast and largely unexploited resource for research. Optical microscopy of such specimen is curtailed by the diffraction-limited resolution of conventional optical microscopy. To overcome this limitation, we used STED super-resolution microscopy enabling optical resolution well below the diffraction barrier. We visualized nanoscale protein distributions in sections of well-annotated paraffin-embedded human rectal cancer tissue stored in a clinical repository. Using antisera against several mitochondrial proteins, STED microscopy revealed distinct sub-mitochondrial protein distributions, suggesting a high level of structural preservation. Analysis of human tissues stored for up to 17 years demonstrated that these samples were still amenable for super-resolution microscopy. STED microscopy of sections of HER2 positive rectal adenocarcinoma revealed details in the surface and intracellular HER2 distribution that were blurred in the corresponding conventional images, demonstrating the potential of super-resolution microscopy to explore the thus far largely untapped nanoscale regime in tissues stored in biorepositories. PMID:25025184

  18. On-grid immunogold labeling of glial intermediate filaments in epoxy-embedded tissue.

    PubMed

    Johnson, A B; Bettica, A

    1989-01-01

    On-grid immunogold labeling of structures like intermediate filaments has been difficult to achieve. Presumably this is because such structures are thinner than the thin sections themselves and because gold-labeled reagents remain on the surface and do not penetrate epoxy resins. Many pathologic and other tissues, however, are primarily available as epoxy-embedded blocks, and a postembedding gold procedure capable of detecting such thin structures would be useful. This study aimed to investigate the astrocytic intermediate filament antigen glial fibrillary acidic protein (GFAP) in glutaraldehyde-fixed, epoxy-embedded brain biopsy tissue from a child with Alexander's disease. A protocol was developed for performing on-grid immunogold labeling which minimized nonspecific deposition of gold reagent. The method utilized ovalbumin and skim milk in the washes and diluent for the gold reagent and the same solution with added Tween-20 and high sodium chloride in the diluent for antibodies and normal serum. In grids etched with metaperiodate and hydrogen peroxide, the astrocytic intermediate filaments were only occasionally and sparsely labeled. When an etching procedure with sodium ethoxide was employed, however, extensive labeling was obtained on the astrocytic intermediate filaments. In contrast, the larger, pathological Rosenthal fibers characteristic of Alexander's disease were labeled after both etching procedures, but labeling was enhanced after ethoxide etching. Postosmicated material showed much less labeling. The findings demonstrate that postembedding procedures can be used with epoxy-embedded material to immunolabel thin structures like intermediate filaments.

  19. Targeted Next Generation Sequencing as a Reliable Diagnostic Assay for the Detection of Somatic Mutations in Tumours Using Minimal DNA Amounts from Formalin Fixed Paraffin Embedded Material

    PubMed Central

    Koudijs, Marco J.; Nijman, Ies; Hinrichs, John W. J.; Cuppen, Edwin; van Lieshout, Stef; Loberg, Robert D.; de Jonge, Maja; Voest, Emile E.; de Weger, Roel A.; Steeghs, Neeltje; Langenberg, Marlies H. G.; Sleijfer, Stefan; Willems, Stefan M.; Lolkema, Martijn P.

    2016-01-01

    Background Targeted Next Generation Sequencing (NGS) offers a way to implement testing of multiple genetic aberrations in diagnostic pathology practice, which is necessary for personalized cancer treatment. However, no standards regarding input material have been defined. This study therefore aimed to determine the effect of the type of input material (e.g. formalin fixed paraffin embedded (FFPE) versus fresh frozen (FF) tissue) on NGS derived results. Moreover, this study aimed to explore a standardized analysis pipeline to support consistent clinical decision-making. Method We used the Ion Torrent PGM sequencing platform in combination with the Ion AmpliSeq Cancer Hotspot Panel v2 to sequence frequently mutated regions in 50 cancer related genes, and validated the NGS detected variants in 250 FFPE samples using standard diagnostic assays. Next, 386 tumour samples were sequenced to explore the effect of input material on variant detection variables. For variant calling, Ion Torrent analysis software was supplemented with additional variant annotation and filtering. Results Both FFPE and FF tissue could be sequenced reliably with a sensitivity of 99.1%. Validation showed a 98.5% concordance between NGS and conventional sequencing techniques, where NGS provided both the advantage of low input DNA concentration and the detection of low-frequency variants. The reliability of mutation analysis could be further improved with manual inspection of sequence data. Conclusion Targeted NGS can be reliably implemented in cancer diagnostics using both FFPE and FF tissue when using appropriate analysis settings, even with low input DNA. PMID:26919633

  20. High quality genomic copy number data from archival formalin-fixed paraffin-embedded leiomyosarcoma: optimisation of universal linkage system labelling.

    PubMed

    Salawu, Abdulazeez; Ul-Hassan, Aliya; Hammond, David; Fernando, Malee; Reed, Malcolm; Sisley, Karen

    2012-01-01

    Most soft tissue sarcomas are characterized by genetic instability and frequent genomic copy number aberrations that are not subtype-specific. Oligonucleotide microarray-based Comparative Genomic Hybridisation (array CGH) is an important technique used to map genome-wide copy number aberrations, but the traditional requirement for high-quality DNA typically obtained from fresh tissue has limited its use in sarcomas. Although large archives of Formalin-fixed Paraffin-embedded (FFPE) tumour samples are available for research, the degradative effects of formalin on DNA from these tissues has made labelling and analysis by array CGH technically challenging. The Universal Linkage System (ULS) may be used for a one-step chemical labelling of such degraded DNA. We have optimised the ULS labelling protocol to perform aCGH on archived FFPE leiomyosarcoma tissues using the 180k Agilent platform. Preservation age of samples ranged from a few months to seventeen years and the DNA showed a wide range of degradation (when visualised on agarose gels). Consistently high DNA labelling efficiency and low microarray probe-to-probe variation (as measured by the derivative log ratio spread) was seen. Comparison of paired fresh and FFPE samples from identical tumours showed good correlation of CNAs detected. Furthermore, the ability to macro-dissect FFPE samples permitted the detection of CNAs that were masked in fresh tissue. Aberrations were visually confirmed using Fluorescence in situ Hybridisation. These results suggest that archival FFPE tissue, with its relative abundance and attendant clinical data may be used for effective mapping for genomic copy number aberrations in such rare tumours as leiomyosarcoma and potentially unravel clues to tumour origins, progression and ultimately, targeted treatment.

  1. Study of paraffin-embedded colon cancer tissue using terahertz spectroscopy

    NASA Astrophysics Data System (ADS)

    Wahaia, Faustino; Kasalynas, Irmantas; Seliuta, Dalius; Molis, Gediminas; Urbanowicz, Andrzej; Carvalho Silva, Catia D.; Carneiro, Fatima; Valusis, Gintaras; Granja, Pedro L.

    2015-01-01

    In this work, samples of non-neoplastic and adenocarcinoma-affected human colon tissue samples were analyzed using multipoint transmission time-domain THz spectroscopy (THz-TDS) to sort out the contrast-contributing factors other than water, the main contrast mechanism factor in in-vivo or in freshly excised bio-tissue. Solving the electromagnetic inverse problem through THz-TDS and, analyzing the transmittance spectra that yielded the frequency-dependent absorption coefficient α and refractive index n of non-neoplastic and neoplastic tissues, we show that it is possible to distinguish between non-neoplastic and neoplastic regions in paraffin-embedded dehydrated. Results and discussion are presented.

  2. Tissue MALDI Mass Spectrometry Imaging (MALDI MSI) of Peptides.

    PubMed

    Beine, Birte; Diehl, Hanna C; Meyer, Helmut E; Henkel, Corinna

    2016-01-01

    Matrix assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) is a technique to visualize molecular features of tissues based on mass detection. This chapter focuses on MALDI MSI of peptides and provides detailed operational instructions for sample preparation of cryoconserved and formalin-fixed paraffin-embedded (FFPE) tissue. Besides sample preparation we provide protocols for the MALDI measurement, tissue staining, and data analysis. On-tissue digestion and matrix application are described for two different commercially available and commonly used spraying devices: the SunCollect (SunChrom) and the ImagePrep (Bruker Daltonik GmbH).

  3. Determining the utility of veterinary tissue archives for retrospective DNA analysis.

    PubMed

    Abed, Firas M; Dark, Michael J

    2016-01-01

    Histopathology tissue archives can be an important source of specimens for retrospective studies, as these include samples covering a large number of diseases. In veterinary medicine, archives also contain samples from a large variety of species and may represent naturally-occurring models of human disease. The formalin-fixed, paraffin-embedded (FFPE) tissues comprising these archives are rich resources for retrospective molecular biology studies and pilot studies for biomarkers, as evidenced by a number of recent publications highlighting FFPE tissues as a resource for analysis of specific diseases. However, DNA extracted from FFPE specimens are modified and fragmented, making utilization challenging. The current study examines the utility of FFPE tissue samples from a veterinary diagnostic laboratory archive in five year intervals from 1977 to 2013, with 2015 as a control year, to determine how standard processing and storage conditions has affected their utility for future studies. There was a significant difference in our ability to obtain large amplicons from samples from 2015 than from the remaining years, as well as an inverse correlation between the age of the samples and product size obtainable. However, usable DNA samples were obtained in at least some of the samples from all years tested, despite variable storage, fixation, and processing conditions. This study will help make veterinary diagnostic laboratory archives more useful in future studies of human and veterinary disease. PMID:27168995

  4. Determining the utility of veterinary tissue archives for retrospective DNA analysis

    PubMed Central

    Abed, Firas M.

    2016-01-01

    Histopathology tissue archives can be an important source of specimens for retrospective studies, as these include samples covering a large number of diseases. In veterinary medicine, archives also contain samples from a large variety of species and may represent naturally-occurring models of human disease. The formalin-fixed, paraffin-embedded (FFPE) tissues comprising these archives are rich resources for retrospective molecular biology studies and pilot studies for biomarkers, as evidenced by a number of recent publications highlighting FFPE tissues as a resource for analysis of specific diseases. However, DNA extracted from FFPE specimens are modified and fragmented, making utilization challenging. The current study examines the utility of FFPE tissue samples from a veterinary diagnostic laboratory archive in five year intervals from 1977 to 2013, with 2015 as a control year, to determine how standard processing and storage conditions has affected their utility for future studies. There was a significant difference in our ability to obtain large amplicons from samples from 2015 than from the remaining years, as well as an inverse correlation between the age of the samples and product size obtainable. However, usable DNA samples were obtained in at least some of the samples from all years tested, despite variable storage, fixation, and processing conditions. This study will help make veterinary diagnostic laboratory archives more useful in future studies of human and veterinary disease. PMID:27168995

  5. Comparison of fine needle aspiration biopsy and paraffin embedded tissue sections for measuring AgNOR proteins.

    PubMed

    Tasdemir, S; Eroz, R; Cucer, N; Oktay, M; Türkeli, M

    2015-07-01

    Paraffin embedded tissue sections and fine needle aspiration biopsy (FNAB) are important methods for diagnosis. We compared thyroid tissue obtained by FNAB to paraffin embedded sections to determine whether there were differences in detection of the amounts of argyrophilic nucleolar organizing region (AgNOR) proteins. Twenty-two patients with papillary thyroid carcinoma were included in the study. Slides were prepared with both FNAB tissue and 3 μm sections of paraffin embedded tissue, and stained for AgNOR. One hundred nuclei per individual were evaluated; total AgNOR number/nucleus (TAn/TNn) and total AgNOR area/nuclear area (TAa/TNa) of individual cells were determined. Mean TAn/TNn and TAa/TNa values were 4.800 ± 1.118 and 13.382 ± 2.612, respectively, for FNAB samples; corresponding values were 2.406 ± 0.649 and 8.49 ± 0.893, respectively, for paraffin embedded sections. The differences between FNAB materials and paraffin embedded tissue sections were significant for the mean TAn/TNn and TAa/TNa values. Significant differences in the amounts of AgNOR protein detected were found between FNAB and paraffin embedded tissue sections.

  6. Revisiting fixation and embedding techniques for optimal detection of dendritic cell subsets in tissues.

    PubMed

    Accart, Nathalie; Sergi, Florinda; Rooke, Ronald

    2014-09-01

    Organ-specific cell types are maintained by tissue homeostasis and may vary in nature and/or frequency in pathological situations. Moreover, within a cell lineage, some sub-populations, defined by combinations of cell-surface markers, may have specific functions. Dendritic cells are the epitome of such a population as they may be subdivided into discrete sub-groups with defined functions in specific compartments of various organs. Technically, to study the distribution of DC sub-populations, it involves performing multiparametric immunofluorescence on well-conserved organ structures. However, immunodetection may be impacted by protein cross-linking and antigenic epitope masking by the use of 10% neutral-buffered formalin. To circumvent this and to preserve a good morphological tissue structure, we evaluated alternative fixatives such as Periodate Lysine Paraformaldehyde or Tris Zinc fixatives in combination with other embedding techniques. The cryosection protocols were adapted for optimal antigen detection but offered a poor morphological preservation. We therefore developed a new methodology based on Tris Zinc fixative, gelatin-sucrose embedding and freezing. Using multiple DC markers, we demonstrate that this treatment is an optimal protocol for cell-surface marker detection on high-quality tissue sections. PMID:24874853

  7. The tissue is the issue: improved methylome analysis from paraffin-embedded tissues by application of the HOPE technique.

    PubMed

    Marwitz, Sebastian; Kolarova, Julia; Reck, Martin; Reinmuth, Niels; Kugler, Christian; Schädlich, Ines; Haake, Andrea; Zabel, Peter; Vollmer, Ekkehard; Siebert, Reiner; Goldmann, Torsten; Ammerpohl, Ole

    2014-08-01

    Alterations in the DNA methylome are characteristic for numerous diseases and a typical hallmark of cancer. Therefore, DNA methylation is currently under investigation in research labs and has also entered diagnostics. Recently, protocols like the BeadChip technology have become commercially available to study DNA methylation in an array format and semiquantitative fashion. However, it is known that fixation of the sample material with formalin prior to BeadChip analysis can affect the results. In this study we compared the influence of fixation on the outcome of BeadChip analysis. From six patients each a lung cancer tissue sample and a corresponding tumor-free lung tissue sample were collected. The samples were separated into three pieces. One piece of each sample was fixed with formalin, another one by the non-cross-linking HOPE technique (Hepes-glutamic acid buffer mediated Organic solvent Protection Effect). Subsequently, both became paraffin embedded. As a reference, the remaining third piece was cryopreserved. In addition we used three adenocarcinoma cell lines (H838, A549, and H1650) to validate the results from patient tissues. We show that using the HOPE technique instead of formalin largely prevents the introduction of formalin-fixation related artifacts. An ANOVA analysis significantly separated HOPE- and cryopreserved from formalin-fixed samples (FDR<0.05), while differences in the methylation data obtained from HOPE-fixed and cryopreserved material were minor. Consequently, HOPE fixation is superior to formalin fixation if a subsequent BeadChip analysis of paraffin-embedded sample material is intended.

  8. Effective DNA/RNA Co-Extraction for Analysis of MicroRNAs, mRNAs, and Genomic DNA from Formalin-Fixed Paraffin-Embedded Specimens

    PubMed Central

    Liu, Christina; Lin, Juan; Ye, Kenny; Kim, Ryung; Hazan, Rachel; Rohan, Thomas; Fineberg, Susan; Loudig, Olivier

    2012-01-01

    Background Retrospective studies of archived human specimens, with known clinical follow-up, are used to identify predictive and prognostic molecular markers of disease. Due to biochemical differences, however, formalin-fixed paraffin-embedded (FFPE) DNA and RNA have generally been extracted separately from either different tissue sections or from the same section by dividing the digested tissue. The former limits accurate correlation whilst the latter is impractical when utilizing rare or limited archived specimens. Principal Findings For effective recovery of genomic DNA and total RNA from a single FFPE specimen, without splitting the proteinase-K digested tissue solution, we optimized a co-extraction method by using TRIzol and purifying DNA from the lower aqueous and RNA from the upper organic phases. Using a series of seven different archived specimens, we evaluated the total amounts of genomic DNA and total RNA recovered by our TRIzol-based co-extraction method and compared our results with those from two commercial kits, the Qiagen AllPrep DNA/RNA FFPE kit, for co-extraction, and the Ambion RecoverAll™ Total Nucleic Acid Isolation kit, for separate extraction of FFPE-DNA and -RNA. Then, to accurately assess the quality of DNA and RNA co-extracted from a single FFPE specimen, we used qRT-PCR, gene expression profiling and methylation assays to analyze microRNAs, mRNAs, and genomic DNA recovered from matched fresh and FFPE MCF10A cells. These experiments show that the TRIzol-based co-extraction method provides larger amounts of FFPE-DNA and –RNA than the two other methods, and particularly provides higher quality microRNAs and genomic DNA for subsequent molecular analyses. Significance We determined that co-extraction of genomic DNA and total RNA from a single FFPE specimen is an effective recovery approach to obtain high-quality material for parallel molecular and high-throughput analyses. Our optimized approach provides the option of collecting DNA, which

  9. Oligosaccharide release from frozen and paraffin-wax-embedded archival tissues.

    PubMed

    Dwek, M V; Brooks, S A; Streets, A J; Harvey, D J; Leathem, A J

    1996-11-01

    Altered glycosylation is a feature of many solid tissue diseases such as ulcerative colitis, Crohn's disease, cancer, and connective tissue disorders. Conventionally, oligosaccharide changes have been studied by immunohistochemical techniques. We have adapted existing techniques, developed for purified protein preparations, to allow the release of intact oligosaccharides from archival tissues, so that the oligosaccharides may be structurally characterized. In our study, sections cut from paraffin-wax blocks were dewaxed and oligosaccharides were released using hydrazine and labeled with 2-aminobenzamide. Sialylated oligosaccharides were compared by passing through a GlycoSep C divinylbenzene anion exchange resin column and neutral oligosaccharides were compared by passing through a BioGel P4 column. The oligosaccharide profiles obtained from the same fresh frozen versus archival paraffin-wax-embedded, normal liver, and tumor tissues showed remarkable similarity in terms of their sialylated and neutral structures. Matrix-assisted laser desorption ionization mass spectrometry has shown that the oligosaccharides are not affected by fixation in formalin and storage in paraffin wax. The results indicate that while the proteins themselves may be denatured, oligosaccharides are not adversely affected by fixation in formalin and storage in paraffin wax. By applying these methods, oligosaccharides from archival tissues, where the natural history of the disease has been followed, may now be liberated and structurally characterized.

  10. Preparation of Formalin-fixed Paraffin-embedded Tissue Cores for both RNA and DNA Extraction.

    PubMed

    Patel, Palak G; Selvarajah, Shamini; Boursalie, Suzanne; How, Nathan E; Ejdelman, Joshua; Guerard, Karl-Philippe; Bartlett, John M; Lapointe, Jacques; Park, Paul C; Okello, John B A; Berman, David M

    2016-01-01

    Formalin-fixed paraffin embedded tissue (FFPET) represents a valuable, well-annotated substrate for molecular investigations. The utility of FFPET in molecular analysis is complicated both by heterogeneous tissue composition and low yields when extracting nucleic acids. A literature search revealed a paucity of protocols addressing these issues, and none that showed a validated method for simultaneous extraction of RNA and DNA from regions of interest in FFPET. This method addresses both issues. Tissue specificity was achieved by mapping cancer areas of interest on microscope slides and transferring annotations onto FFPET blocks. Tissue cores were harvested from areas of interest using 0.6 mm microarray punches. Nucleic acid extraction was performed using a commercial FFPET extraction system, with modifications to homogenization, deparaffinization, and Proteinase K digestion steps to improve tissue digestion and increase nucleic acid yields. The modified protocol yields sufficient quantity and quality of nucleic acids for use in a number of downstream analyses, including a multi-analyte gene expression platform, as well as reverse transcriptase coupled real time PCR analysis of mRNA expression, and methylation-specific PCR (MSP) analysis of DNA methylation. PMID:27583817

  11. Heat transfer normal to paired arterioles and venules embedded in perfused tissue during hyperthermia.

    PubMed

    Charny, C K; Levin, R L

    1988-11-01

    A numerical model of the heat transer normal to an arteriole-venule pair embedded in muscle tissue has been constructed. Anatomical data describing the blood vessel size, spacing, and density have been incorporated into the model. This model computes temperatures along the vessel walls as well as the temperature throughout the tissue which comprises an infinitely long Krogh cylinder around the vessel pair. Tissue temperatures were computed in the steady-state under resting conditions, while transient calculations were made under hyperthermic conditions. Results show that for both large- (1st generation) and medium-sized (5th generation) vessel pairs, the mean tissue temperature within the tissue cylinder is not equal to the mean of the arteriole and venule blood temperatures under both steady-state and transient conditions. The numerical data were reduced so that a comparison could be made with the predictions of a simple two-dimensional superposition of line sources and sinks presented by Baish et al. This comparison reveals that the superposition model accurately describes the heat transfer effects during hyperthermia, permitting subsequent incorporation of this theory into a realistic three-dimensional model of heat transfer in a whole limb during hyperthermia. PMID:3205012

  12. Preparation of Formalin-fixed Paraffin-embedded Tissue Cores for both RNA and DNA Extraction.

    PubMed

    Patel, Palak G; Selvarajah, Shamini; Boursalie, Suzanne; How, Nathan E; Ejdelman, Joshua; Guerard, Karl-Philippe; Bartlett, John M; Lapointe, Jacques; Park, Paul C; Okello, John B A; Berman, David M

    2016-08-21

    Formalin-fixed paraffin embedded tissue (FFPET) represents a valuable, well-annotated substrate for molecular investigations. The utility of FFPET in molecular analysis is complicated both by heterogeneous tissue composition and low yields when extracting nucleic acids. A literature search revealed a paucity of protocols addressing these issues, and none that showed a validated method for simultaneous extraction of RNA and DNA from regions of interest in FFPET. This method addresses both issues. Tissue specificity was achieved by mapping cancer areas of interest on microscope slides and transferring annotations onto FFPET blocks. Tissue cores were harvested from areas of interest using 0.6 mm microarray punches. Nucleic acid extraction was performed using a commercial FFPET extraction system, with modifications to homogenization, deparaffinization, and Proteinase K digestion steps to improve tissue digestion and increase nucleic acid yields. The modified protocol yields sufficient quantity and quality of nucleic acids for use in a number of downstream analyses, including a multi-analyte gene expression platform, as well as reverse transcriptase coupled real time PCR analysis of mRNA expression, and methylation-specific PCR (MSP) analysis of DNA methylation.

  13. Tissue proteomics using chemical immobilization and mass spectrometry.

    PubMed

    Shah, Punit; Zhang, Bai; Choi, Caitlin; Yang, Shuang; Zhou, Jianying; Harlan, Robert; Tian, Yuan; Zhang, Zhen; Chan, Daniel W; Zhang, Hui

    2015-01-15

    Proteomics analysis is important for characterizing tissues to gain biological and pathological insights, which could lead to the identification of disease-associated proteins for disease diagnostics or targeted therapy. However, tissues are commonly embedded in optimal cutting temperature medium (OCT) or are formalin-fixed and paraffin-embedded (FFPE) in order to maintain tissue morphology for histology evaluation. Although several tissue proteomic analyses have been performed on FFPE tissues using advanced mass spectrometry (MS) technologies, high-throughput proteomic analysis of OCT-embedded tissues has been difficult due to the interference of OCT in the MS analysis. In addition, molecules other than proteins present in tissues further complicate tissue proteomic analysis. Here, we report the development of a method using chemical immobilization of proteins for peptide extraction (CIPPE). In this method, proteins are chemically immobilized onto a solid support; interferences from tissues and OCT embedding are removed by extensive washing of proteins conjugated on the solid support. Peptides are then released from the solid phase by proteolysis, enabling MS analysis. This method was first validated by eliminating OCT interference from a standard protein, human serum albumin, where all of the unique peaks contributed by OCT contamination were eradicated. Finally, this method was applied for the proteomic analysis of frozen and OCT-embedded tissues using iTRAQ (isobaric tag for relative and absolute quantitation) labeling and two-dimensional liquid chromatography tandem mass spectrometry. The data showed reproducible extraction and quantitation of 10,284 proteins from 3996 protein groups and a minimal impact of OCT embedding on the analysis of the global proteome of the stored tissue samples. PMID:25283129

  14. Tissue proteomics using chemical immobilization and mass spectrometry.

    PubMed

    Shah, Punit; Zhang, Bai; Choi, Caitlin; Yang, Shuang; Zhou, Jianying; Harlan, Robert; Tian, Yuan; Zhang, Zhen; Chan, Daniel W; Zhang, Hui

    2015-01-15

    Proteomics analysis is important for characterizing tissues to gain biological and pathological insights, which could lead to the identification of disease-associated proteins for disease diagnostics or targeted therapy. However, tissues are commonly embedded in optimal cutting temperature medium (OCT) or are formalin-fixed and paraffin-embedded (FFPE) in order to maintain tissue morphology for histology evaluation. Although several tissue proteomic analyses have been performed on FFPE tissues using advanced mass spectrometry (MS) technologies, high-throughput proteomic analysis of OCT-embedded tissues has been difficult due to the interference of OCT in the MS analysis. In addition, molecules other than proteins present in tissues further complicate tissue proteomic analysis. Here, we report the development of a method using chemical immobilization of proteins for peptide extraction (CIPPE). In this method, proteins are chemically immobilized onto a solid support; interferences from tissues and OCT embedding are removed by extensive washing of proteins conjugated on the solid support. Peptides are then released from the solid phase by proteolysis, enabling MS analysis. This method was first validated by eliminating OCT interference from a standard protein, human serum albumin, where all of the unique peaks contributed by OCT contamination were eradicated. Finally, this method was applied for the proteomic analysis of frozen and OCT-embedded tissues using iTRAQ (isobaric tag for relative and absolute quantitation) labeling and two-dimensional liquid chromatography tandem mass spectrometry. The data showed reproducible extraction and quantitation of 10,284 proteins from 3996 protein groups and a minimal impact of OCT embedding on the analysis of the global proteome of the stored tissue samples.

  15. Complex Retrieval of Embedded IVC Filters: Alternative Techniques and Histologic Tissue Analysis

    SciTech Connect

    Kuo, William T.; Cupp, John S.; Louie, John D.; Kothary, Nishita; Hofmann, Lawrence V.; Sze, Daniel Y.; Hovsepian, David M.

    2012-06-15

    Purpose: We evaluated the safety and effectiveness of alternative endovascular methods to retrieve embedded optional and permanent filters in order to manage or reduce risk of long-term complications from implantation. Histologic tissue analysis was performed to elucidate the pathologic effects of chronic filter implantation. Methods: We studied the safety and effectiveness of alternative endovascular methods for removing embedded inferior vena cava (IVC) filters in 10 consecutive patients over 12 months. Indications for retrieval were symptomatic chronic IVC occlusion, caval and aortic perforation, and/or acute PE (pulmonary embolism) from filter-related thrombus. Retrieval was also performed to reduce risk of complications from long-term filter implantation and to eliminate the need for lifelong anticoagulation. All retrieved specimens were sent for histologic analysis. Results: Retrieval was successful in all 10 patients. Filter types and implantation times were as follows: one Venatech (1,495 days), one Simon-Nitinol (1,485 days), one Optease (300 days), one G2 (416 days), five Guenther-Tulip (GTF; mean 606 days, range 154-1,010 days), and one Celect (124 days). There were no procedural complications or adverse events at a mean follow-up of 304 days after removal (range 196-529 days). Histology revealed scant native intima surrounded by a predominance of neointimal hyperplasia and dense fibrosis in all specimens. Histologic evidence of photothermal tissue ablation was confirmed in three laser-treated specimens. Conclusion: Complex retrieval methods can now be used in select patients to safely remove embedded optional and permanent IVC filters previously considered irretrievable. Neointimal hyperplasia and dense fibrosis are the major components that must be separated to achieve successful retrieval of chronic filter implants.

  16. Diagnosis of Nocardia paucivorans central nervous system infection by DNA sequencing from paraffin-embedded tissue.

    PubMed

    Schiaroli, Elisabetta; Pasticci, Maria Bruna; De Carolis, Elena; Mello, Enrica; Pallotto, Carlo; Leli, Christian; De Socio, Giuseppe Vittorio; Baldelli, Franco; Sanguinetti, Maurizio; Mencacci, Antonella

    2016-06-01

    Infections by Nocardia spp. are generally regarded as opportunistic diseases in immunocompromised patients, but can also affect immunocompetent subjects. Such infections represent an important diagnostic challenge for clinicians and microbiologists, and diagnosis is frequently delayed or even conducted post mortem. A 54-year-old man was admitted to our hospital because of ventriculitis and relapsing brain abscess. Five months prior, this patient had undergone external ventricular drain and surgery for a cerebellar abscess. Histopathology demonstrated pyogenic inflammatory reaction, microbiologic investigations proved negative and empiric antimicrobial therapy was administered for a total of eight weeks. Six weeks later, the patient developed relapsing neurologic manifestations. On reviewing the patient's clinical history it emerged that the patient had suffered pneumonia two months prior to neurosurgery, treated with amoxicillin/clavulanate 3g a day and levofloxacin 500mg a day for three weeks. On the CNS relapsing manifestations, nocardiosis was suspected and DNA sequencing from the formalin-fixed paraffin-embedded cerebellar tissue collected during neurosurgery allowed diagnosis of Nocardia paucivorans infection. The patient received medical therapy for 11 months. At follow-up, eight months after treatment was discontinued, the patient was aymptomatic. Nocardia spp. infections need to be suspected not only in immunocompromised, but also in immunocompetent patients. Proper samples need to be collected for proper microbiologic investigations. Paraffin-embedded tissue genomic sequencing can be a useful tool for diagnosis of nocardiosis. PMID:27367327

  17. A hematoxylin and eosin-like stain for glycol methacrylate embedded tissue sections.

    PubMed

    Troyer, H; Babich, E

    1981-01-01

    A staining procedure is described for use with glycol methacrylate embedded tissue sections which does not stain the plastic embedment or remove the sections from the glass slides. The basic dye is celestine blue B. It is prepared by treating 1 g of the dye with 0.5 ml concentrated sulfuric acid. It is then dissolved with the following solution. Add 14 ml glycerine to 100 ml 2.5% ferric ammonium sulfate and warm the solution to 50 C. Finally adjust the pH to 0.8 to 0.9 The acid staining solution consists of 0.075% ponceau de xylidine and 0.025% acid fuchsin in 10% acetic acid. Slides containing the dried plastic sections are immersed in the celestine blue solution for five minutes and in the ponceau-fuchsin solution for ten minutes with an intervening water rinse. After a final wash, the sections are air dried and coverslipped. This staining procedure colors the tissues nearly the same as hematoxylin and eosin procedures.

  18. Protocol for HER2 FISH determination on PAXgene-fixed and paraffin-embedded tissue in breast cancer.

    PubMed

    Oberauner-Wappis, Lisa; Loibner, Martina; Viertler, Christian; Groelz, Daniel; Wyrich, Ralf; Zatloukal, Kurt

    2016-04-01

    Molecular diagnostics in personalized medicine increasingly relies on the combination of a variety of analytical technologies to characterize individual diseases and to select patients for targeted therapies. The gold standard for tissue-based diagnostics is fixation in formalin and embedding in paraffin, which results in excellent preservation of morphology but negatively impacts on a variety of molecular assays. The formalin-free, non-cross-linking PAXgene tissue system preserves morphology in a similar way to formalin, but also preserves biomolecules essentially in a similar way to cryopreservation, which markedly widens the spectrum, sensitivity and accuracy of molecular analytics. In this study, we have developed and tested a protocol for PAXgene-fixed and paraffin-embedded tissues for fluorescent in situ hybridization (FISH). The implementation of a 24-h formalin postfixation step of slides from PAXgene-fixed and paraffin-embedded tissues allowed us to use the assays approved for formalin-fixed and paraffin-embedded tissues. The equivalence of the methodologies was demonstrated by FISH analysis of HER2 amplification in breast cancer cases. The 24-h postfixation step of the slides used for FISH can be well integrated in the routine diagnostic workflow and allows the remaining PAXgene-fixed and paraffin-embedded tissue to be used for further molecular testing.

  19. Hormone Receptor Expression Analyses in Neoplastic and Non-Neoplastic Canine Mammary Tissue by a Bead Based Multiplex Branched DNA Assay: A Gene Expression Study in Fresh Frozen and Formalin-Fixed, Paraffin-Embedded Samples.

    PubMed

    Mohr, Annika; Lüder Ripoli, Florenza; Hammer, Susanne Conradine; Willenbrock, Saskia; Hewicker-Trautwein, Marion; Kiełbowicz, Zdzisław; Murua Escobar, Hugo; Nolte, Ingo

    2016-01-01

    Immunohistochemistry (IHC) is currently considered the method of choice for steroid hormone receptor status evaluation in human breast cancer and, therefore, it is commonly utilized for assessing canine mammary tumors. In case of low hormone receptor expression, IHC is limited and thus is complemented by molecular analyses. In the present study, a multiplex bDNA assay was evaluated as a method for hormone receptor gene expression detection in canine mammary tissues. Estrogen receptor (ESR1), progesterone receptor (PGR), prolactin receptor (PRLR) and growth hormone receptor (GHR) gene expressions were evaluated in neoplastic and non-neoplastic canine mammary tissues. A set of 119 fresh frozen and 180 formalin-fixed, paraffin-embedded (FFPE) was comparatively analyzed and used for assay evaluation. Furthermore, a possible association between the hormone receptor expression in different histological subtypes of canine malignant mammary tumors and the castration status, breed and invasive growth of the tumor were analyzed. The multiplex bDNA assay proved to be more sensitive for fresh frozen specimens. Hormone receptor expression found was significantly decreased in malignant mammary tumors in comparison to non-neoplastic tissue and benign mammary tumors. Among the histological subtypes the lowest gene expression levels of ESR1, PGR and PRLR were found in solid, anaplastic and ductal carcinomas. In summary, the evaluation showed that the measurement of hormone receptors with the multiplex bDNA assay represents a practicable method for obtaining detailed quantitative information about gene expression in canine mammary tissue for future studies. Still, comparison with IHC or quantitative real-time PCR is needed for further validation of the present method.

  20. Hormone Receptor Expression Analyses in Neoplastic and Non-Neoplastic Canine Mammary Tissue by a Bead Based Multiplex Branched DNA Assay: A Gene Expression Study in Fresh Frozen and Formalin-Fixed, Paraffin-Embedded Samples.

    PubMed

    Mohr, Annika; Lüder Ripoli, Florenza; Hammer, Susanne Conradine; Willenbrock, Saskia; Hewicker-Trautwein, Marion; Kiełbowicz, Zdzisław; Murua Escobar, Hugo; Nolte, Ingo

    2016-01-01

    Immunohistochemistry (IHC) is currently considered the method of choice for steroid hormone receptor status evaluation in human breast cancer and, therefore, it is commonly utilized for assessing canine mammary tumors. In case of low hormone receptor expression, IHC is limited and thus is complemented by molecular analyses. In the present study, a multiplex bDNA assay was evaluated as a method for hormone receptor gene expression detection in canine mammary tissues. Estrogen receptor (ESR1), progesterone receptor (PGR), prolactin receptor (PRLR) and growth hormone receptor (GHR) gene expressions were evaluated in neoplastic and non-neoplastic canine mammary tissues. A set of 119 fresh frozen and 180 formalin-fixed, paraffin-embedded (FFPE) was comparatively analyzed and used for assay evaluation. Furthermore, a possible association between the hormone receptor expression in different histological subtypes of canine malignant mammary tumors and the castration status, breed and invasive growth of the tumor were analyzed. The multiplex bDNA assay proved to be more sensitive for fresh frozen specimens. Hormone receptor expression found was significantly decreased in malignant mammary tumors in comparison to non-neoplastic tissue and benign mammary tumors. Among the histological subtypes the lowest gene expression levels of ESR1, PGR and PRLR were found in solid, anaplastic and ductal carcinomas. In summary, the evaluation showed that the measurement of hormone receptors with the multiplex bDNA assay represents a practicable method for obtaining detailed quantitative information about gene expression in canine mammary tissue for future studies. Still, comparison with IHC or quantitative real-time PCR is needed for further validation of the present method. PMID:27649560

  1. Hormone Receptor Expression Analyses in Neoplastic and Non-Neoplastic Canine Mammary Tissue by a Bead Based Multiplex Branched DNA Assay: A Gene Expression Study in Fresh Frozen and Formalin-Fixed, Paraffin-Embedded Samples

    PubMed Central

    Mohr, Annika; Lüder Ripoli, Florenza; Hammer, Susanne Conradine; Willenbrock, Saskia; Hewicker-Trautwein, Marion; Kiełbowicz, Zdzisław; Murua Escobar, Hugo; Nolte, Ingo

    2016-01-01

    Immunohistochemistry (IHC) is currently considered the method of choice for steroid hormone receptor status evaluation in human breast cancer and, therefore, it is commonly utilized for assessing canine mammary tumors. In case of low hormone receptor expression, IHC is limited and thus is complemented by molecular analyses. In the present study, a multiplex bDNA assay was evaluated as a method for hormone receptor gene expression detection in canine mammary tissues. Estrogen receptor (ESR1), progesterone receptor (PGR), prolactin receptor (PRLR) and growth hormone receptor (GHR) gene expressions were evaluated in neoplastic and non-neoplastic canine mammary tissues. A set of 119 fresh frozen and 180 formalin-fixed, paraffin-embedded (FFPE) was comparatively analyzed and used for assay evaluation. Furthermore, a possible association between the hormone receptor expression in different histological subtypes of canine malignant mammary tumors and the castration status, breed and invasive growth of the tumor were analyzed. The multiplex bDNA assay proved to be more sensitive for fresh frozen specimens. Hormone receptor expression found was significantly decreased in malignant mammary tumors in comparison to non-neoplastic tissue and benign mammary tumors. Among the histological subtypes the lowest gene expression levels of ESR1, PGR and PRLR were found in solid, anaplastic and ductal carcinomas. In summary, the evaluation showed that the measurement of hormone receptors with the multiplex bDNA assay represents a practicable method for obtaining detailed quantitative information about gene expression in canine mammary tissue for future studies. Still, comparison with IHC or quantitative real-time PCR is needed for further validation of the present method. PMID:27649560

  2. Analysis of T cell receptor beta chain CDR3 size using RNA extracted from formalin fixed paraffin wax embedded tissue.

    PubMed Central

    O'Shea, U; Wyatt, J I; Howdle, P D

    1997-01-01

    AIMS: To isolate RNA and DNA simultaneously from formalin fixed paraffin wax embedded tissue to assess the clonality of enteropathy associated T cell lymphomas and to analyse it in detail by a non-radioactive method of T cell receptor complementarity determining region 3 (CDR3) spectratyping. METHODS: DNA and RNA were isolated simultaneously from formalin fixed paraffin wax embedded tissue blocks and subjected to the polymerase chain reaction (PCR) and semi-nested reverse transcription PCR (RT-PCR), respectively. The RT-PCR T cell receptor V beta products were analysed by CDR3 spectratyping using a denaturing polyacrylamide gel and silver staining. RESULTS: Usable DNA and RNA were isolated simultaneously from formalin fixed paraffin wax embedded tissue. The specific clonality of the tissue was successfully analysed by a non-radioactive method of T cell receptor CDR3 spectratyping of the RT-PCR products. CDR3 spectratying of the RT-PCR products demonstrated the precise clonal nature of the tumour and non-tumour tissue showing that the non-tumour tissue comprised an oligoclonal population of a number of different T cell receptor V beta families. The tumour tissue comprised two T cell subtypes of the one family, T cell receptor V beta 9. CONCLUSIONS: RNA and DNA were isolated from formalin fixed paraffin wax embedded enteropathy associated T cell lymphoma tissue. Detailed analysis of clonality can be carried out by a non-radioactive method of CDR3 spectratyping. Images PMID:9462260

  3. Hyperspectral unmixing for removing autofluorescence from paraffin-embedded formalin-fixed tissue sections

    NASA Astrophysics Data System (ADS)

    Constantinou, P.; Wilson, B. C.; Damaskinos, S.

    2005-09-01

    The use of digital fluorescence confocal microscopy in biological sciences has grown in recent decades due to the versatility of fluorescence imaging. The ability to selectively label specific morphological features, genetic mutations and/or chemical micro-environmental changes with discreet fluorescent labels allows a better understanding of the complex systems that regulate cellular processes. Specimens can range in size from single cells to tissue sections and tissue arrays, which can occupy the entire surface of a microscope slide (25mm x 70mm). Using a confocal scanning laser MACROscope, a wide-area confocal imaging system (Biomedical Photometrics Inc.), it is possible to image these large specimens at high resolution, without the need to tile many small microscope fields. A hyperspectral imaging (HSI) mode has been added to the MACROscope system to assess the use of HSI in the removal/separation of tissue autofluorescence from digital images of fluorescently-labeled paraffin-embedded, formalin-fixed tissue sections. In pathology and immunohistochemistry applications this autofluorescence can hinder, or even prevent, detection of the applied fluorescent label(s). In the present study, fluorescence emission from the specimen was sampled at ~7 nm bandwidths across 32 channels, amounting to viewing ~220 nm of the visible spectrum as a hyperspectral data cube. The data cube was then processed to remove the contributions from autofluorescence, leaving only the signal from the fluorophore(s) of interest. Comparisons are drawn from HSI obtained with a commercial hyperspectral confocal microscope (Zeiss LSM 510 META) employing image tiling. The initial results demonstrate the ability to spectrally unmix the tissue autofluorescence in large tissue sections.

  4. Molecular Detection and Typing of Dengue Viruses from Archived Tissues of Fatal Cases by RT-PCR and Sequencing: Diagnostic and Epidemiologic Implications

    PubMed Central

    Bhatnagar, Julu; Blau, Dianna M.; Shieh, Wun-Ju; Paddock, Christopher D.; Drew, Clifton; Liu, Lindy; Jones, Tara; Patel, Mitesh; Zaki, Sherif R.

    2012-01-01

    Diagnosis of dengue virus (DENV) infection in fatal cases is challenging because of the frequent unavailability of blood or fresh tissues. For formalin-fixed, paraffin-embedded (FFPE) tissues immunohistochemistry (IHC) can be used; however, it may not be as sensitive and serotyping is not possible. The application of reverse transcription-polymerase chain reaction (RT-PCR) for the detection of DENV in FFPE tissues has been very limited. We evaluated FFPE autopsy tissues of 122 patients with suspected DENV infection by flavivirus and DENV RT-PCR, sequencing, and DENV IHC. The DENV was detected in 61 (50%) cases by RT-PCR or IHC. The RT-PCR and sequencing detected DENV in 60 (49%) cases (DENV-1 in 16, DENV-2 in 27, DENV-3 in 8, and DENV-4 in 6 cases). No serotype could be identified in three cases. The IHC detected DENV antigens in 50 (40%) cases. The RT-PCR using FFPE tissue improves detection of DENV in fatal cases and provides sequence information useful for typing and epidemiologic studies. PMID:22302871

  5. Electrospun aligned PLGA and PLGA/gelatin nanofibers embedded with silica nanoparticles for tissue engineering.

    PubMed

    Mehrasa, Mohammad; Asadollahi, Mohammad Ali; Ghaedi, Kamran; Salehi, Hossein; Arpanaei, Ayyoob

    2015-08-01

    Aligned poly lactic-co-glycolic acid (PLGA) and PLGA/gelatin nanofibrous scaffolds embedded with mesoporous silica nanoparticles (MSNPs) were fabricated using electrospinning method. The mean diameters of nanofibers were 641±24 nm for the pure PLGA scaffolds vs 418±85 nm and 267±58 nm for the PLGA/10 wt% MSNPs and the PLGA/gelatin/10 wt% MSNPs scaffolds, respectively. The contact angle measurement results (102°±6.7 for the pure PLGA scaffold vs 81°±6.8 and 18°±8.7 for the PLGA/10 wt% MSNPs and the PLGA/gelatin/10 wt% MSNPs scaffolds, respectively) revealed enhanced hydrophilicity of scaffolds upon incorporation of gelatin and MSNPs. Besides, embedding the scaffolds with MSNPs resulted in improved tensile mechanical properties. Cultivation of PC12 cells on the scaffolds demonstrated that introduction of MSNPs into PLGA and PLGA/gelatin matrices leads to the improved cell attachment and proliferation as well as long cellular processes. DAPI staining results indicated that cell proliferations on the PLGA/10 wt% MSNPs and the PLGA/gelatin/10 wt% MSNPs scaffolds were strikingly (nearly 2.5 and 3 folds, respectively) higher than that on the aligned pure PLGA scaffolds. These results suggest superior properties of silica nanoparticles-incorporated PLGA/gelatin eletrospun nanofibrous scaffolds for the stem cell culture and tissue engineering applications.

  6. BRCA somatic and germline mutation detection in paraffin embedded ovarian cancers by next-generation sequencing

    PubMed Central

    Mafficini, Andrea; Simbolo, Michele; Parisi, Alice; Rusev, Borislav; Luchini, Claudio; Cataldo, Ivana; Piazzola, Elena; Sperandio, Nicola; Turri, Giona; Franchi, Massimo; Tortora, Giampaolo; Bovo, Chiara; Lawlor, Rita T.; Scarpa, Aldo

    2016-01-01

    BRCA mutated ovarian cancers respond better to platinum-based therapy and to the recently approved PARP-inhibitors. There is the need for efficient and timely methods to detect both somatic and germline mutations using formalin-fixed paraffin-embedded (FFPE) tissues and commercially available technology. We used a commercial kit exploring all exons and 50bp exon-intron junctions of BRCA1 and BRCA2 genes, and semiconductor next-generation sequencing (NGS) on DNA from 47 FFPE samples of high-grade serous ovarian cancers. Pathogenic mutations were found in 13/47 (28%) cancers: eight in BRCA1 and five in BRCA2. All BRCA1 and two BRCA2 mutations were germline; three BRCA2 mutations were somatic. All mutations were confirmed by Sanger sequencing. To evaluate the performance of the NGS panel, we assessed its capability to detect the 6,953 variants described for BRCA1 and BRCA2 in ClinVar and COSMIC databases using callability analysis. 6,059 (87.1%) variants were identified automatically by the software; 829 (12.0%) required visual verification. The remaining 65 (0.9%) variants were uncallable, and would require 15 Sanger reactions to be resolved. Thus, the sensitivity of the NGS-panel was 99.1%. In conclusion, NGS performed with a commercial kit is highly efficient for detection of germline and somatic mutations in BRCA genes using routine FFPE tissue. PMID:26745875

  7. BRCA somatic and germline mutation detection in paraffin embedded ovarian cancers by next-generation sequencing.

    PubMed

    Mafficini, Andrea; Simbolo, Michele; Parisi, Alice; Rusev, Borislav; Luchini, Claudio; Cataldo, Ivana; Piazzola, Elena; Sperandio, Nicola; Turri, Giona; Franchi, Massimo; Tortora, Giampaolo; Bovo, Chiara; Lawlor, Rita T; Scarpa, Aldo

    2016-01-12

    BRCA mutated ovarian cancers respond better to platinum-based therapy and to the recently approved PARP-inhibitors. There is the need for efficient and timely methods to detect both somatic and germline mutations using formalin-fixed paraffin-embedded (FFPE) tissues and commercially available technology. We used a commercial kit exploring all exons and 50bp exon-intron junctions of BRCA1 and BRCA2 genes, and semiconductor next-generation sequencing (NGS) on DNA from 47 FFPE samples of high-grade serous ovarian cancers. Pathogenic mutations were found in 13/47 (28%) cancers: eight in BRCA1 and five in BRCA2. All BRCA1 and two BRCA2 mutations were germline; three BRCA2 mutations were somatic. All mutations were confirmed by Sanger sequencing. To evaluate the performance of the NGS panel, we assessed its capability to detect the 6,953 variants described for BRCA1 and BRCA2 in ClinVar and COSMIC databases using callability analysis. 6,059 (87.1%) variants were identified automatically by the software; 829 (12.0%) required visual verification. The remaining 65 (0.9%) variants were uncallable, and would require 15 Sanger reactions to be resolved. Thus, the sensitivity of the NGS-panel was 99.1%. In conclusion, NGS performed with a commercial kit is highly efficient for detection of germline and somatic mutations in BRCA genes using routine FFPE tissue.

  8. Cytokine expression in paraffin wax-embedded tissues from conventional calves.

    PubMed

    Pedrera, M; Sánchez-Cordón, P J; Romero-Trevejo, J L; Raya, A I; Núñez, A; Gómez-Villamandos, J C

    2007-05-01

    The cross-reactivity of antibodies against human tumour necrosis factor (TNF)alpha, interleukin (IL)-1alpha, IL-1beta and porcine IL-6, and the distribution of immunolabelled cells were evaluated on paraffin wax-embedded tissues from five healthy calves. The tissues were fixed in 10% buffered formalin or Bouin's solution and processed for structural studies and immunohistochemical studies by the avidin-biotin-peroxidase technique. Bouin's solution proved to be the more suitable fixative and Tween 20 the most effective antigen unmasking technique for increasing detectable antigenicity. Constitutive expression of TNFalpha, IL-1alpha, IL-1beta and IL-6 by different cell populations, mainly macrophage-like cells, was detected. Lymphoid organs displayed a higher presence of immunolabelled cells than did lung, liver or kidney. TNFalpha and IL-1alpha appeared as the predominant cytokines, especially in the gut-associated lymphoid tissue of the ileum and in the regional mesenteric lymph nodes. The results will facilitate investigation of the role of these cytokine-producing cells in inflammatory disease processes in calves.

  9. Direct Measurement of Intranuclear Strain Distributions and RNA Synthesis in Single Cells Embedded within Native Tissue

    PubMed Central

    Henderson, Jonathan T.; Shannon, Garrett; Veress, Alexander I.; Neu, Corey P.

    2013-01-01

    Nuclear structure and mechanics play a critical role in diverse cellular functions, such as organizing direct access of chromatin to transcriptional regulators. Here, we use a new, to our knowledge, hybrid method, based on microscopy and hyperelastic warping, to determine three-dimensional strain distributions inside the nuclei of single living cells embedded within their native extracellular matrix. During physiologically relevant mechanical loading to tissue samples, strain was transferred to individual nuclei, resulting in submicron distributions of displacements, with compressive and tensile strain patterns approaching a fivefold magnitude increase in some locations compared to tissue-scale stimuli. Moreover, nascent RNA synthesis was observed in the interchromatin regions of the cells studied and spatially corresponded to strain patterns. Our ability to measure large strains in the interchromatin space, which reveals that movement of chromatin in the nucleus may not be due to random or biochemical mechanisms alone, but may result from the transfer of mechanical force applied at a distant tissue surface. PMID:24268137

  10. Unmasking of complements using proteinase-K in formalin fixed paraffin embedded renal biopsies.

    PubMed

    Nada, R; Kumar, A; Kumar, V G; Gupta, K L; Joshi, K

    2016-01-01

    Renal biopsy interpretation requires histopathology, direct immunofluorescence (DIF) and electron microscopy. Formalin-fixed, paraffin-embedded tissue (FFPE) sent for light microscopy can be used for DIF after antigen retrieval. However, complement staining has not been satisfactory. We standardized DIF using proteinase-K for antigen retrieval in FFPE renal biopsies. A pilot study was conducted on known cases of membranous glomerulonephritis (MGN), membranoproliferative type-1 (MPGN-1), immunoglobulin A nephropathy (IgAN), and anti-glomerular basement disease (anti-GBM). Immunofluorescence panel included fluorescein isothiocyanate (FITC) conjugated IgG, IgA, IgM, complements (C3 and C1q), light chains (kappa, lambda) and fibrinogen antibodies. After standardization of the technique, 75 renal biopsies and 43 autopsies cases were stained. Out of 43 autopsy cases, immune-complex mediated glomerulonephritis (GN) was confirmed in 18 cases (Lupus nephritis-11, IgAN-6, MGN-1), complement-mediated dense deposit disease (DDD-1) and monoclonal diseases in 4 cases (amyloidosis-3, cast nephropathy-1). Immune-mediated injury was excluded in 17 cases (focal segmental glomerulosclerosis -3, crescentic GN-6 [pauci-immune-3, anti-GBM-3], thrombotic microangiopathy-5, atherosclerosis-3). Renal biopsies (n-75) where inadequate or no frozen sample was available; this technique classified 52 mesangiocapillary pattern as MPGN type-1-46, DDD-2 and (C3GN-4). Others were diagnosed as IgAN-3, lupus nephritis-2, MGN-4, diffuse proliferative glomerulonephritis (DPGN)-1, Non-IC crescentic GN-1, monoclonal diseases-3. In nine cases, DIF on FFPE tissue could not help in making diagnosis. Proteinase-K enzymatic digestion of FFPE renal biopsies can unmask complements (both C3 and C1q) in immune-complexes mediated and complement-mediated diseases. This method showed good results on autopsy tissues archived for as long as 15 years. PMID:27194832

  11. Unmasking of complements using proteinase-K in formalin fixed paraffin embedded renal biopsies.

    PubMed

    Nada, R; Kumar, A; Kumar, V G; Gupta, K L; Joshi, K

    2016-01-01

    Renal biopsy interpretation requires histopathology, direct immunofluorescence (DIF) and electron microscopy. Formalin-fixed, paraffin-embedded tissue (FFPE) sent for light microscopy can be used for DIF after antigen retrieval. However, complement staining has not been satisfactory. We standardized DIF using proteinase-K for antigen retrieval in FFPE renal biopsies. A pilot study was conducted on known cases of membranous glomerulonephritis (MGN), membranoproliferative type-1 (MPGN-1), immunoglobulin A nephropathy (IgAN), and anti-glomerular basement disease (anti-GBM). Immunofluorescence panel included fluorescein isothiocyanate (FITC) conjugated IgG, IgA, IgM, complements (C3 and C1q), light chains (kappa, lambda) and fibrinogen antibodies. After standardization of the technique, 75 renal biopsies and 43 autopsies cases were stained. Out of 43 autopsy cases, immune-complex mediated glomerulonephritis (GN) was confirmed in 18 cases (Lupus nephritis-11, IgAN-6, MGN-1), complement-mediated dense deposit disease (DDD-1) and monoclonal diseases in 4 cases (amyloidosis-3, cast nephropathy-1). Immune-mediated injury was excluded in 17 cases (focal segmental glomerulosclerosis -3, crescentic GN-6 [pauci-immune-3, anti-GBM-3], thrombotic microangiopathy-5, atherosclerosis-3). Renal biopsies (n-75) where inadequate or no frozen sample was available; this technique classified 52 mesangiocapillary pattern as MPGN type-1-46, DDD-2 and (C3GN-4). Others were diagnosed as IgAN-3, lupus nephritis-2, MGN-4, diffuse proliferative glomerulonephritis (DPGN)-1, Non-IC crescentic GN-1, monoclonal diseases-3. In nine cases, DIF on FFPE tissue could not help in making diagnosis. Proteinase-K enzymatic digestion of FFPE renal biopsies can unmask complements (both C3 and C1q) in immune-complexes mediated and complement-mediated diseases. This method showed good results on autopsy tissues archived for as long as 15 years.

  12. Full-length protein extraction protocols and gel-based downstream applications in formalin-fixed tissue proteomics.

    PubMed

    Tanca, Alessandro; Uzzau, Sergio; Addis, Maria Filippa

    2015-01-01

    Archival formalin-fixed, paraffin-embedded (FFPE) tissue repositories and their associated clinical information can represent a valuable resource for tissue proteomics. In order to make these tissues available for protein biomarker discovery and validation studies, dedicated sample preparation procedures overcoming the intermolecular cross-links introduced by formalin need to be implemented. This chapter describes a full-length protein extraction protocol optimized for downstream gel-based proteomics applications. Using the procedures detailed here, SDS-PAGE, western immunoblotting, GeLC-MS/MS, 2D-PAGE, and 2D-DIGE can be carried out on FFPE tissues. Technical tips, critical aspects, and drawbacks of the method are presented and discussed.

  13. High-Throughput Amplicon-Based Copy Number Detection of 11 Genes in Formalin-Fixed Paraffin-Embedded Ovarian Tumour Samples by MLPA-Seq.

    PubMed

    Kondrashova, Olga; Love, Clare J; Lunke, Sebastian; Hsu, Arthur L; Waring, Paul M; Taylor, Graham R

    2015-01-01

    Whilst next generation sequencing can report point mutations in fixed tissue tumour samples reliably, the accurate determination of copy number is more challenging. The conventional Multiplex Ligation-dependent Probe Amplification (MLPA) assay is an effective tool for measurement of gene dosage, but is restricted to around 50 targets due to size resolution of the MLPA probes. By switching from a size-resolved format, to a sequence-resolved format we developed a scalable, high-throughput, quantitative assay. MLPA-seq is capable of detecting deletions, duplications, and amplifications in as little as 5ng of genomic DNA, including from formalin-fixed paraffin-embedded (FFPE) tumour samples. We show that this method can detect BRCA1, BRCA2, ERBB2 and CCNE1 copy number changes in DNA extracted from snap-frozen and FFPE tumour tissue, with 100% sensitivity and >99.5% specificity.

  14. High-Throughput Amplicon-Based Copy Number Detection of 11 Genes in Formalin-Fixed Paraffin-Embedded Ovarian Tumour Samples by MLPA-Seq.

    PubMed

    Kondrashova, Olga; Love, Clare J; Lunke, Sebastian; Hsu, Arthur L; Waring, Paul M; Taylor, Graham R

    2015-01-01

    Whilst next generation sequencing can report point mutations in fixed tissue tumour samples reliably, the accurate determination of copy number is more challenging. The conventional Multiplex Ligation-dependent Probe Amplification (MLPA) assay is an effective tool for measurement of gene dosage, but is restricted to around 50 targets due to size resolution of the MLPA probes. By switching from a size-resolved format, to a sequence-resolved format we developed a scalable, high-throughput, quantitative assay. MLPA-seq is capable of detecting deletions, duplications, and amplifications in as little as 5ng of genomic DNA, including from formalin-fixed paraffin-embedded (FFPE) tumour samples. We show that this method can detect BRCA1, BRCA2, ERBB2 and CCNE1 copy number changes in DNA extracted from snap-frozen and FFPE tumour tissue, with 100% sensitivity and >99.5% specificity. PMID:26569395

  15. High-Throughput Amplicon-Based Copy Number Detection of 11 Genes in Formalin-Fixed Paraffin-Embedded Ovarian Tumour Samples by MLPA-Seq

    PubMed Central

    Kondrashova, Olga; Love, Clare J.; Lunke, Sebastian; Hsu, Arthur L.; Waring, Paul M.; Taylor, Graham R.

    2015-01-01

    Whilst next generation sequencing can report point mutations in fixed tissue tumour samples reliably, the accurate determination of copy number is more challenging. The conventional Multiplex Ligation-dependent Probe Amplification (MLPA) assay is an effective tool for measurement of gene dosage, but is restricted to around 50 targets due to size resolution of the MLPA probes. By switching from a size-resolved format, to a sequence-resolved format we developed a scalable, high-throughput, quantitative assay. MLPA-seq is capable of detecting deletions, duplications, and amplifications in as little as 5ng of genomic DNA, including from formalin-fixed paraffin-embedded (FFPE) tumour samples. We show that this method can detect BRCA1, BRCA2, ERBB2 and CCNE1 copy number changes in DNA extracted from snap-frozen and FFPE tumour tissue, with 100% sensitivity and >99.5% specificity. PMID:26569395

  16. High-quality genomic DNA extraction from formalin-fixed and paraffin-embedded samples deparaffinized using mineral oil.

    PubMed

    Lin, Jianghai; Kennedy, Stephen H; Svarovsky, Therese; Rogers, Jeffrey; Kemnitz, Joseph W; Xu, Anlong; Zondervan, Krina T

    2009-12-15

    Extracting DNA from formalin-fixed and paraffin-embedded (FFPE) tissue remains a challenge, despite numerous attempts to develop a more effective method. Polymerase chain reaction (PCR) success rates with DNA extracted using current methods remain low. We extracted DNA from 140 long-term archived FFPE samples using a simple but effective deparaffinization method, removing the wax with mineral oil, and a commercially available DNA extraction kit. DNA quality was subsequently tested in a genotyping experiment with 14 microsatellite markers. High-quality DNA was obtained with a mean PCR success rate of 97% (range: 88-100%) across markers. The results suggested that DNA extracted using this novel method is likely to be suitable for genetic studies involving DNA fragments <200 bp.

  17. High-quality genomic DNA extraction from formalin-fixed and paraffin-embedded samples deparaffinized using mineral oil

    PubMed Central

    Lin, Jianghai; Kennedy, Stephen H.; Svarovsky, Therese; Rogers, Jeffrey; Kemnitz, Joseph W.; Xu, Anlong; Zondervan, Krina T.

    2009-01-01

    Extracting DNA from formalin-fixed and paraffin-embedded (FFPE) tissue remains a challenge, despite numerous attempts to develop a more effective method. Polymerase chain reaction (PCR) success rates with DNA extracted using current methods remain low. We extracted DNA from 140 long-term archived FFPE samples using a simple but effective deparaffinization method, removing the wax with mineral oil, and a commercially available DNA extraction kit. DNA quality was subsequently tested in a genotyping experiment with 14 microsatellite markers. High-quality DNA was obtained with a mean PCR success rate of 97% (range: 88–100%) across markers. The results suggested that DNA extracted using this novel method is likely to be suitable for genetic studies involving DNA fragments <200 bp. PMID:19698695

  18. Application of a novel and automated branched DNA in situ hybridization method for the rapid and sensitive localization of mRNA molecules in plant tissues1

    PubMed Central

    Bowling, Andrew J.; Pence, Heather E.; Church, Jeffrey B.

    2014-01-01

    • Premise of the study: A novel branched DNA detection technology, RNAscope in situ hybridization (ISH), originally developed for use on human clinical and animal tissues, was adapted for use in plant tissue in an attempt to overcome some of the limitations associated with traditional ISH assays. • Methods and Results: Zea mays leaf tissue was formaldehyde fixed and paraffin embedded (FFPE) and then probed with the RNAscope ISH assay for two endogenous genes, phosphoenolpyruvate carboxylase (PEPC) and phosphoenolpyruvate carboxykinase (PEPCK). Results from both manual and automated methods showed tissue- and cell-specific mRNA localization patterns expected from these well-studied genes. • Conclusions: RNAscope ISH is a sensitive method that generates high-quality, easily interpretable results from FFPE plant tissues. Automation of the RNAscope method on the Ventana Discovery Ultra platform allows significant advantages for repeatability, reduction in variability, and flexibility of workflow processes. PMID:25202621

  19. Limited yield of diagnoses of intrahepatic infectious causes of canine granulomatous hepatitis from archival liver tissue.

    PubMed

    Hutchins, Rae G; Breitschwerdt, Edward B; Cullen, John M; Bissett, Sally A; Gookin, Jody L

    2012-09-01

    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.

  20. Optimal reference genes for normalization of qRT-PCR data from archival formalin-fixed, paraffin-embedded breast tumors controlling for tumor cell content and decay of mRNA.

    PubMed

    Tramm, Trine; Sørensen, Brita S; Overgaard, Jens; Alsner, Jan

    2013-09-01

    Reliable determination of gene-expression levels from qRT-PCR requires accurate normalization of target genes to reference genes in order to remove nonbiological variation. Reference genes are ideally constitutively expressed in every cell, but many genes used for normalization has been shown to vary with tissue type, cellular proliferation, cancer progression, and degradation of nucleic acids. Gene-expression analysis is increasingly performed on degraded mRNA from formalin-fixed, paraffin-embedded tissue (FFPE), giving the option of examining retrospective cohorts. The aim of this study was to select robust reference genes showing stable expression over time in FFPE, controlling for various content of tumor tissue and decay of mRNA because of variable length of storage of the tissue. Sixteen reference genes were quantified by qRT-PCR in 40 FFPE breast tumor samples, stored for 1 to 29 years. Samples included 2 benign lesions and 38 carcinomas with varying tumor content. Stability of the reference genes were determined by the geNorm algorithm. mRNA was successfully extracted from all samples, and the 16 genes quantified in the majority of samples. Results showed 14% loss of amplifiable mRNA per year, corresponding to a half-life of 4.6 years. The 4 most stable expressed genes were CALM2, RPL37A, ACTB, and RPLP0. Several of the other examined genes showed considerably instability over time (GAPDH, PSMC4, OAZ1, IPO8). In conclusion, we identified 4 genes robustly expressed over time and independent of neoplastic tissue content in the FFPE block. Other widely used reference genes were concluded to be less suited for retrospective analysis of FFPE breast samples.

  1. Steps Towards Precision Medicine: Utilizing FFPE Specimens - TCGA

    Cancer.gov

    Roy W. Tarnuzzer, Ph.D., the Biospecimen Core Resource Program Manager at the TCGA Program Office, provides an overview of the Formalin-fixed Paraffin Pilot Project, an initiative to investigate best practices for use of FFPE specimens in genomic studies.

  2. ToF-SIMS of tissues: "lessons learned" from mice and women.

    PubMed

    Gamble, Lara J; Graham, Daniel J; Bluestein, Blake; Whitehead, Nicholas P; Hockenbery, David; Morrish, Fionnuala; Porter, Peggy

    2015-01-01

    The ability to image cells and tissues with chemical and molecular specificity could greatly expand our understanding of biological processes. The subcellular resolution mass spectral imaging capability of time of flight secondary ion mass spectrometry (ToF-SIMS) has the potential to acquire chemically detailed images. However, the complexities of biological systems combined with the sensitivity of ToF-SIMS require careful planning of experimental methods. Tissue sample preparation methods of formalin fixation followed by paraffin embedding (FFPE) and OCT embedding are compared. Results show that the FFPE can potentially be used as a tissue sample preparation protocol for ToF-SIMS analysis if a cluster ion pre-sputter is used prior to analysis and if nonlipid related tissue features are the features of interest. In contrast, embedding tissue in OCT minimizes contamination and maintains lipid signals. Various data acquisition methodologies and analysis options are discussed and compared using mouse breast and diaphragm muscle tissue. Methodologies for acquiring ToF-SIMS 2D images are highlighted along with applications of multivariate analysis to better identify specific features in a tissue sections when compared to H&E images of serial sections. Identification of tissue features is necessary for researchers to visualize a molecular map that correlates with specific biological features or functions. Finally, lessons learned from sample preparation, data acquisition, and data analysis methods developed using mouse models are applied to a preliminary analysis of human breast tumor tissue sections. PMID:25708638

  3. ToF-SIMS of tissues: "lessons learned" from mice and women.

    PubMed

    Gamble, Lara J; Graham, Daniel J; Bluestein, Blake; Whitehead, Nicholas P; Hockenbery, David; Morrish, Fionnuala; Porter, Peggy

    2015-03-13

    The ability to image cells and tissues with chemical and molecular specificity could greatly expand our understanding of biological processes. The subcellular resolution mass spectral imaging capability of time of flight secondary ion mass spectrometry (ToF-SIMS) has the potential to acquire chemically detailed images. However, the complexities of biological systems combined with the sensitivity of ToF-SIMS require careful planning of experimental methods. Tissue sample preparation methods of formalin fixation followed by paraffin embedding (FFPE) and OCT embedding are compared. Results show that the FFPE can potentially be used as a tissue sample preparation protocol for ToF-SIMS analysis if a cluster ion pre-sputter is used prior to analysis and if nonlipid related tissue features are the features of interest. In contrast, embedding tissue in OCT minimizes contamination and maintains lipid signals. Various data acquisition methodologies and analysis options are discussed and compared using mouse breast and diaphragm muscle tissue. Methodologies for acquiring ToF-SIMS 2D images are highlighted along with applications of multivariate analysis to better identify specific features in a tissue sections when compared to H&E images of serial sections. Identification of tissue features is necessary for researchers to visualize a molecular map that correlates with specific biological features or functions. Finally, lessons learned from sample preparation, data acquisition, and data analysis methods developed using mouse models are applied to a preliminary analysis of human breast tumor tissue sections.

  4. ToF-SIMS of tissues: “Lessons learned” from mice and women

    PubMed Central

    Gamble, Lara J.; Graham, Daniel J.; Bluestein, Blake; Whitehead, Nicholas P.; Hockenbery, David; Morrish, Fionnuala; Porter, Peggy

    2015-01-01

    The ability to image cells and tissues with chemical and molecular specificity could greatly expand our understanding of biological processes. The subcellular resolution mass spectral imaging capability of time of flight secondary ion mass spectrometry (ToF-SIMS) has the potential to acquire chemically detailed images. However, the complexities of biological systems combined with the sensitivity of ToF-SIMS require careful planning of experimental methods. Tissue sample preparation methods of formalin fixation followed by paraffin embedding (FFPE) and OCT embedding are compared. Results show that the FFPE can potentially be used as a tissue sample preparation protocol for ToF-SIMS analysis if a cluster ion presputter is used prior to analysis and if nonlipid related tissue features are the features of interest. In contrast, embedding tissue in OCT minimizes contamination and maintains lipid signals. Various data acquisition methodologies and analysis options are discussed and compared using mouse breast and diaphragm muscle tissue. Methodologies for acquiring ToF-SIMS 2D images are highlighted along with applications of multivariate analysis to better identify specific features in a tissue sections when compared to H&E images of serial sections. Identification of tissue features is necessary for researchers to visualize a molecular map that correlates with specific biological features or functions. Finally, lessons learned from sample preparation, data acquisition, and data analysis methods developed using mouse models are applied to a preliminary analysis of human breast tumor tissue sections. PMID:25708638

  5. Enhanced stability of microRNA expression facilitates classification of FFPE tumour samples exhibiting near total mRNA degradation

    PubMed Central

    Hall, J S; Taylor, J; Valentine, H R; Irlam, J J; Eustace, A; Hoskin, P J; Miller, C J; West, C M L

    2012-01-01

    Background: As degradation of formalin-fixed paraffin-embedded (FFPE) samples limits the ability to profile mRNA expression, we explored factors predicting the success of mRNA expression profiling of FFPE material and investigated an approach to overcome the limitation. Methods: Bladder (n=140, stored 3–8 years) and cervix (n=160, stored 8–23 years) carcinoma FFPE samples were hybridised to Affymetrix Exon 1.0ST arrays. Percentage detection above background (%DABG) measured technical success. Biological signal was assessed by distinguishing cervix squamous cell carcinoma (SCC) and adenocarcinoma (AC) using a gene signature. As miR-205 had been identified as a marker of SCC, precursor mir-205 was measured by Exon array and mature miR-205 by qRT–PCR. Genome-wide microRNA (miRNA) expression (Affymetrix miRNA v2.0 arrays) was compared in eight newer FFPE samples with biological signal and eight older samples without. Results: RNA quality controls (QCs) (e.g., RNA integrity (RIN) number) failed to predict profiling success, but sample age correlated with %DABG in bladder (R=−0.30, P<0.01) and cervix (R=−0.69, P<0.01). Biological signal was lost in older samples and neither a signature nor precursor mir-205 separated samples by histology. miR-205 qRT–PCR discriminated SCC from AC, validated by miRNA profiling (26-fold higher in SCC; P=1.10 × 10−5). Genome-wide miRNA (R=0.95) and small nucleolar RNA (R=0.97) expression correlated well in the eight newer vs older FFPE samples and better than mRNA expression (R=0.72). Conclusion: Sample age is the best predictor of successful mRNA profiling of FFPE material, and miRNA profiling overcomes the limitation of age and copes well with older samples. PMID:22805332

  6. PCR amplification and high throughput sequencing of immunoglobulin heavy chain genes from formalin-fixed paraffin-embedded human biopsies.

    PubMed

    Tabibian-Keissar, Hilla; Schibby, Ginette; Michaeli, Miri; Rakovsky-Shapira, Aviya; Azogui-Rosenthal, Noemie; Dunn-Walters, Deborah K; Rosenblatt, Kinneret; Mehr, Ramit; Barshack, Iris

    2013-02-01

    The use of high throughput sequencing (HTS) technologies in biomedicine is expanding in a variety of fields in recent years. The 454 system is an HTS platform that is ideally suited to characterize B cell receptor (BCR) repertoires by sequencing of immunoglobulin (Ig) genes, as it is able to sequence stretches of several hundred nucleotides. Most studies that used this platform for antibody repertoire analyses have started from fresh or frozen tissues or peripheral blood samples, and rely on starting with optimal quality DNA. In this paper we demonstrate that BCR repertoire analysis can be done using DNA from formalin-fixed paraffin-embedded (FFPE) human tissue samples. The heterogeneity of BCR repertoires we obtained confirms the plausibility of HTS of DNA from FFPE specimens. The establishment of experimental protocols and computational tools that enable sequence data analysis from the low quality DNA of FFPE tissues is important for enabling research, as it would enable the use of the rich source of preserved samples in clinical biobanks and biopsy archives.

  7. Improved diagnosis of mycobacterial infections in formalin-fixed and paraffin-embedded sections with nested polymerase chain reaction.

    PubMed

    Azov, Andrey G; Koch, Jørn; Hamilton-Dutoit, Stephen J

    2005-09-01

    Traditional histological diagnosis of mycobacterial infection in formalin-fixed and paraffin-embedded (FFPE) tissues is insensitive and poorly specific. To improve this, we developed nested polymerase chain reaction (PCR) protocols for detecting a Mycobacterium genus-specific 65-kDa heat shock protein (HSP65) sequence and the M. tuberculosis complex-specific insertion sequence IS6110 in FFPE sections. Protocols were optimized on tissues from 20 patients with a final clinical diagnosis of mycobacterial infection. Amplicons were controlled by sequencing and restriction endonuclease digestion. PCR could detect as few as three mycobacterial genomes per reaction. Assays showed 100% sensitivity and specificity for both M. tuberculosis complex and M. avium complex infection. Paraffin blocks from a second group of 26 patients with histological evidence of necrotizing granulomas of unknown etiology were then analyzed as a surrogate group to test the assay under conditions similar to those applying during routine diagnosis. Twenty-three of these blocks contained amplifiable DNA; nine were positive for M. tuberculosis complex DNA and four for other types of mycobacterial DNA. Furthermore, digestion of HSP65 amplicons with NarI could distinguish M. tuberculosis from M. avium complex. In conclusion, our nested PCR assays can be used as reliable tools for the detection of mycobacterial infections in FFPE tissues. The assays are simple and rapid to perform and show improved sensitivity and specificity compared to previously reported protocols.

  8. Promoter methylation analysis on microdissected paraffin-embedded tissues using bisulfite treatment and PCR-SSCP.

    PubMed

    Bian, Y S; Yan, P; Osterheld, M C; Fontolliet, C; Benhattar, J

    2001-01-01

    Methylation-sensitive single-strand conformation analysis (MS-SSCA) is a new method of screening for DNA methylation changes. The combination of bisulfite modification and PCR results in the conversion of unmethylated cytosines to thymines, whereas methylated cytosines remain unchanged. This sequence conversion can lead to methylation-dependent alterations of single-strand conformation, which can be detected by SSCA. An analysis of mixtures of methylated and unmethylated DNA at known ratios revealed that the relative intensities of the corresponding bands following MS-SSCA were maintained. MS-SSCA was applied for methylation analysis of human p16 promoter region using genomic DNA obtained from either frozen, fixed, or microdissected fixed tissue sections. MS-SSCA is a rapid, specific, and semiquantitative approach that allows the detection of methylation of the p16 gene promoter. In reconstruction experiments, the method permits the detection of 10% or less of cells harboring a methylated p16 promoter. We have been successful in analyzing by MS-SSCA almost all (96%) tumor samples microdissected from archival paraffin-embedded fixed tissue sections and obtaining reproducible results. In addition, when microdissection was performed, the clonality of this genetic alteration could be identified.

  9. Immunohistochemical diagnosis of tenacibaculosis in paraffin-embedded tissues of Senegalese sole Solea senegalensis Kaup, 1858.

    PubMed

    Faílde, L D; Bermúdez, R; Losada, A P; Riaza, A; Santos, Y; Quiroga, M I

    2014-11-01

    A sensitive and specific immunohistochemical technique was developed to improve the diagnosis of tenacibaculosis and to better understand its pathogenesis. Senegalese sole Solea senegalensis Kaup, 1858 were inoculated subcutaneously with a bacterial suspension of Tenacibaculum maritimum, and samples were taken at different hours post-inoculation. Sections from different organs were used as positive controls. In addition, a total of 128 field samples from different organs collected from tenacibaculosis outbreaks were used. Tenacibaculum maritimum antigens were detected in several organs of experimentally infected Senegalese sole and in at least one of the tissues from fish suffering from natural tenacibaculosis previously confirmed by culture and PCR-based methods. In fish collected during outbreaks, a strong positive reaction was detected in ulcerative skin areas. Moreover, bacterial antigen was identified inside scale pockets and in sites of the skin with mild lesion. In kidney and spleen, evident immunostaining of bacterial antigen was detected in both naturally and experimentally infected fish. Besides, the presence of T. maritimum in the intestinal tract without associated histological changes suggests that this organ may act as a reservoir for T. maritimum. The results of this study confirm the usefulness of IHC for the diagnosis of tenacibaculosis in paraffin-embedded tissues.

  10. Images of Soft-bodied Animals with External Hard Shell: 3D Visualization of the Embedded Soft Tissue

    SciTech Connect

    Rao, Donepudi V.; Akatsuka, Takao; Tromba, Giuliana

    2004-05-12

    Images of soft-bodied animals (snails) of various types with external hard shell are obtained for 25, 27 and 29 keV synchrotron X-rays. The SYRMEP facility at Elettra,Trieste, Italy and the associated detection system has been used for the image acquisition. The interior properties of the embedded soft tissue are analysed utilizing the software. From the reconstructed images, the soft tissue distribution, void spaces associated with the soft tissue and external hard shell are identified. 3D images are reconstructed at these energies and optimum energy is chosen based on the quality of the image for further analysis. The optimum energy allowed us to visualize the visibility of low absorbing details and interior microstructure of the embedded soft tissue.

  11. Reliable LC3 and p62 Autophagy Marker Detection in Formalin Fixed Paraffin Embedded Human Tissue by Immunohistochemistry

    PubMed Central

    Schläfli, A.M.; Berezowska, S.; Adams, O.; Langer, R.; Tschan, M.P.

    2015-01-01

    Autophagy assures cellular homeostasis, and gains increasing importance in cancer, where it impacts on carcinogenesis, propagation of the malignant phenotype and development of resistance. To date, its tissue-based analysis by immunohistochemistry remains poorly standardized. Here we show the feasibility of specifically and reliably assessing the autophagy markers LC3B and p62 (SQSTM1) in formalin fixed and paraffin embedded human tissue by immunohistochemistry. Preceding functional experiments consisted of depleting LC3B and p62 in H1299 lung cancer cells with subsequent induction of autophagy. Western blot and immunofluorescence validated antibody specificity, knockdown efficiency and autophagy induction prior to fixation in formalin and embedding in paraffin. LC3B and p62 antibodies were validated on formalin fixed and paraffin embedded cell pellets of treated and control cells and finally applied on a tissue microarray with 80 human malignant and nonneoplastic lung and stomach formalin fixed and paraffin embedded tissue samples. Dot-like staining of various degrees was observed in cell pellets and 18/40 (LC3B) and 22/40 (p62) tumors, respectively. Seventeen tumors were double positive for LC3B and p62. P62 displayed additional significant cytoplasmic and nuclear staining of unknown significance. Interobserver-agreement for grading of staining intensities and patterns was substantial to excellent (kappa values 0.60-0.83). In summary, we present a specific and reliable IHC staining of LC3B and p62 on formalin fixed and paraffin embedded human tissue. Our presented protocol is designed to aid reliable investigation of dysregulated autophagy in solid tumors and may be used on large tissue collectives. PMID:26150155

  12. Reliable LC3 and p62 autophagy marker detection in formalin fixed paraffin embedded human tissue by immunohistochemistry.

    PubMed

    Schläfli, A M; Berezowska, S; Adams, O; Langer, R; Tschan, M P

    2015-01-01

    Autophagy assures cellular homeostasis, and gains increasing importance in cancer, where it impacts on carcinogenesis, propagation of the malignant phenotype and development of resistance. To date, its tissue-based analysis by immunohistochemistry remains poorly standardized. Here we show the feasibility of specifically and reliably assessing the autophagy markers LC3B and p62 (SQSTM1) in formalin fixed and paraffin embedded human tissue by immunohistochemistry. Preceding functional experiments consisted of depleting LC3B and p62 in H1299 lung cancer cells with subsequent induction of autophagy. Western blot and immunofluorescence validated antibody specificity, knockdown efficiency and autophagy induction prior to fixation in formalin and embedding in paraffin. LC3B and p62 antibodies were validated on formalin fixed and paraffin embedded cell pellets of treated and control cells and finally applied on a tissue microarray with 80 human malignant and non-neoplastic lung and stomach formalin fixed and paraffin embedded tissue samples. Dot-like staining of various degrees was observed in cell pellets and 18/40 (LC3B) and 22/40 (p62) tumors, respectively. Seventeen tumors were double positive for LC3B and p62. P62 displayed additional significant cytoplasmic and nuclear staining of unknown significance. Interobserver-agreement for grading of staining intensities and patterns was substantial to excellent (kappa values 0.60 - 0.83). In summary, we present a specific and reliable IHC staining of LC3B and p62 on formalin fixed and paraffin embedded human tissue. Our presented protocol is designed to aid reliable investigation of dysregulated autophagy in solid tumors and may be used on large tissue collectives.

  13. Reliable LC3 and p62 autophagy marker detection in formalin fixed paraffin embedded human tissue by immunohistochemistry.

    PubMed

    Schläfli, A M; Berezowska, S; Adams, O; Langer, R; Tschan, M P

    2015-01-01

    Autophagy assures cellular homeostasis, and gains increasing importance in cancer, where it impacts on carcinogenesis, propagation of the malignant phenotype and development of resistance. To date, its tissue-based analysis by immunohistochemistry remains poorly standardized. Here we show the feasibility of specifically and reliably assessing the autophagy markers LC3B and p62 (SQSTM1) in formalin fixed and paraffin embedded human tissue by immunohistochemistry. Preceding functional experiments consisted of depleting LC3B and p62 in H1299 lung cancer cells with subsequent induction of autophagy. Western blot and immunofluorescence validated antibody specificity, knockdown efficiency and autophagy induction prior to fixation in formalin and embedding in paraffin. LC3B and p62 antibodies were validated on formalin fixed and paraffin embedded cell pellets of treated and control cells and finally applied on a tissue microarray with 80 human malignant and non-neoplastic lung and stomach formalin fixed and paraffin embedded tissue samples. Dot-like staining of various degrees was observed in cell pellets and 18/40 (LC3B) and 22/40 (p62) tumors, respectively. Seventeen tumors were double positive for LC3B and p62. P62 displayed additional significant cytoplasmic and nuclear staining of unknown significance. Interobserver-agreement for grading of staining intensities and patterns was substantial to excellent (kappa values 0.60 - 0.83). In summary, we present a specific and reliable IHC staining of LC3B and p62 on formalin fixed and paraffin embedded human tissue. Our presented protocol is designed to aid reliable investigation of dysregulated autophagy in solid tumors and may be used on large tissue collectives. PMID:26150155

  14. Correlation between Presence of Trypanosoma cruzi DNA in Heart Tissue of Baboons and Cynomolgus Monkeys, and Lymphocytic Myocarditis

    PubMed Central

    Mubiru, James N.; Yang, Alice; Dick, Edward J.; Owston, Michael; Sharp, R. Mark; VandeBerg, Jane F.; Shade, Robert E.; VandeBerg, John L.

    2014-01-01

    Trypanosoma cruzi, the causative agent of Chagas' disease, preferentially infects cardiac and digestive tissues. Baboons living in Texas (Papio hamadryas) and cynomolgus monkeys (Macaca fascicularis) have been reported to be infected naturally with T. cruzi. In this study, we retrospectively reviewed cases of animals that were diagnosed with lymphocytic myocarditis and used a polymerase chain reaction (PCR)-based method (S36/S35 primer set) to amplify T. cruzi DNA from archived frozen and formalin-fixed paraffin-embedded (FFPE) cardiac tissues. We show that the PCR method is applicable in archived frozen and FFPE tissues and the sensitivity is in the femtogram range. A positive correlation between PCR positivity and lymphocytic myocarditis in both baboons and cynomolgus monkeys is shown. We also show epicarditis as a common finding in animals infected with T. cruzi. PMID:24567317

  15. Monoclonal antibodies raised to paraffin wax embedded archival tissue; feasibility study of their potential to detect novel antigenic markers.

    PubMed

    Moran, E; Larkin, A; Cleary, I; Barnes, C; Kennedy, S M; Kelehan, P; Clynes, M

    1998-10-01

    A study to determine the feasibility of using archival paraffin wax embedded tissue to generate monoclonal antibodies is described. Specifically, monoclonal antibodies were raised to paraffin wax embedded normal human kidney tissue to test the possibility of producing antibodies to such tissue samples prior to attempting generation of antibodies to valuable archival tissue. Multiple sections (10 x 5 microm) were pooled and dewaxed as for immunohistochemical procedures and combined with Freund's adjuvant for immunization of BALB/c mice in vivo. Immunized spleen cells were fused with SP2 myeloma cells and subsequent clones screened on paraffin wax embedded normal human kidney sections, a range of cell lines and normal mouse tissue. Supernatants from 11 wells (from a total of 90 wells screened) showed different staining patterns on sections of paraffin wax embedded kidney. One clone, 1/11C, (isotype IgG1) which exhibited strong staining on all kidney tubules by immunohistochemical studies (glomeruli interstitium and vessels were unstained) and identified a band at 52 kDa on immunoblots of dewaxed kidney tissue (as used for immunogen) was chosen for further characterization. Immunoblotting of five mammalian cell lines showed differential expression of this 52 kDa band (distinct expression on 3/5, weak expression on 2/5 cell lines) whereas, all cell lines displayed a band at 44 kDa and a third band at 70 kDa was observed on 2/5 cell lines. In mouse tissue extracts, the 52 kDa band was identified in kidney tissue only (not in the lung, liver or spleen) with the 44 kDa and 70 kDa bands weakly expressed in all tissues. This preliminary investigation of a novel approach to identifying possible new antigenic markers or producing monoclonal antibodies which react better to known antigens on sections of paraffin wax embedded tissue showed that this method is feasible. The need to have a comprehensive screening system in place and the ability to identify potentially useful

  16. Highly multiplexed single-cell analysis of formalin-fixed, paraffin-embedded cancer tissue

    PubMed Central

    Gerdes, Michael J.; Sevinsky, Christopher J.; Sood, Anup; Adak, Sudeshna; Bello, Musodiq O.; Bordwell, Alexander; Can, Ali; Corwin, Alex; Dinn, Sean; Filkins, Robert J.; Hollman, Denise; Kamath, Vidya; Kaanumalle, Sireesha; Kenny, Kevin; Larsen, Melinda; Lazare, Michael; Lowes, Christina; McCulloch, Colin C.; McDonough, Elizabeth; Pang, Zhengyu; Rittscher, Jens; Santamaria-Pang, Alberto; Sarachan, Brion D.; Seel, Maximilian L.; Seppo, Antti; Shaikh, Kashan; Sui, Yunxia; Zhang, Jingyu; Ginty, Fiona

    2013-01-01

    Limitations on the number of unique protein and DNA molecules that can be characterized microscopically in a single tissue specimen impede advances in understanding the biological basis of health and disease. Here we present a multiplexed fluorescence microscopy method (MxIF) for quantitative, single-cell, and subcellular characterization of multiple analytes in formalin-fixed paraffin-embedded tissue. Chemical inactivation of fluorescent dyes after each image acquisition round allows reuse of common dyes in iterative staining and imaging cycles. The mild inactivation chemistry is compatible with total and phosphoprotein detection, as well as DNA FISH. Accurate computational registration of sequential images is achieved by aligning nuclear counterstain-derived fiducial points. Individual cells, plasma membrane, cytoplasm, nucleus, tumor, and stromal regions are segmented to achieve cellular and subcellular quantification of multiplexed targets. In a comparison of pathologist scoring of diaminobenzidine staining of serial sections and automated MxIF scoring of a single section, human epidermal growth factor receptor 2, estrogen receptor, p53, and androgen receptor staining by diaminobenzidine and MxIF methods yielded similar results. Single-cell staining patterns of 61 protein antigens by MxIF in 747 colorectal cancer subjects reveals extensive tumor heterogeneity, and cluster analysis of divergent signaling through ERK1/2, S6 kinase 1, and 4E binding protein 1 provides insights into the spatial organization of mechanistic target of rapamycin and MAPK signal transduction. Our results suggest MxIF should be broadly applicable to problems in the fields of basic biological research, drug discovery and development, and clinical diagnostics. PMID:23818604

  17. Strategies for PCR based detection of Burkholderia pseudomallei DNA in paraffin wax embedded tissues.

    PubMed

    Hagen, R M; Gauthier, Y P; Sprague, L D; Vidal, D R; Zysk, G; Finke, E-J; Neubauer, H

    2002-12-01

    Recently, several cases of melioidosis imported to Europe have been reported. The diagnosis of the acute or chronic infection remains challenging. This report describes an optimised protocol for fast and reliable DNA preparation for use in two different polymerase chain reaction (PCR) assays, namely: (1) a seminested PCR assay targeting a genus specific sequence of the ribosomal protein subunit 21 (rpsU) gene and (2) a nested PCR assay targeting the gene encoding the filament forming flagellin (fliC). Various strains of Burkholderia spp, strains of closely related genera, and spleen tissue samples of experimentally infected mice were investigated. The combination of PCR and sequencing of the amplicons resulted in high sensitivity and specificity. These procedures may allow rapid, sensitive, and reliable detection of B pseudomallei DNA in routinely formalin fixed and paraffin wax embedded samples, thus providing a safe diagnostic tool and avoiding the cultivation of a risk group 3 agent. In addition, this method could be useful for retrospective histopathological investigations.

  18. Cross-reactivity of human and bovine antibodies in striped dolphin paraffin wax-embedded tissues.

    PubMed

    Jaber, J R; Fernández, A; Herráez, P; Espinosa de los Monteros, A; Ramírez, G A; García, P M; Fernández, T; Arbelo, M; Pérez, J

    2003-11-15

    This study evaluates the cross-reactivity of seven anti-human and one anti-bovine antibodies in formalin-fixed, paraffin-embedded tissue samples of liver and mesenteric lymph nodes of 13 striped dolphins (Stenella coeruleoalba). Four antibodies (CD3, IgG, lysozyme and S100 protein) reacted with striped dolphin lymph nodes in a similar pattern to that observed in the species of origin. The anti-human MHC class II mAb reacted strongly with macrophages and dendritic-like cells of striped dolphins, whereas a small number of lymphocytes were labelled with this antibody. These antibodies were used to study the immunophenotype of the inflammatory infiltrated in non-specific chronic reactive hepatitis (eight cases) and chronic parasite cholangitis (two cases) and normal liver (three cases) of striped dolphins. Non-specific chronic reactive hepatitis was composed of inflammatory infiltration of CD3+ T lymphocytes and IgG+ plasma cells in portal spaces and hepatic sinusoids. Lymphonodular aggregates observed in chronic parasitic cholangitis showed a cellular distribution similar to that found in lymph node cortex, including the presence of S100+ and MHC class II+ dendritic-like cells in lymphoid follicles and interfollicular areas. This result suggests that those inflammatory infiltrates are highly organised to enhance antigen presentation to B and T cells.

  19. Detection and characterization of Newcastle disease virus in formalin-fixed, paraffin-embedded tissues from commercial broilers in Egypt.

    PubMed

    Abdel-Glil, Mostafa Y; Mor, Sunil K; Sharafeldin, Tamer A; Porter, Robert E; Goyal, Sagar M

    2014-03-01

    Newcastle disease (ND) is highly contagious and causes severe economic losses to the poultry industry due to high morbidity and mortality. In this report, we describe the detection of Newcastle disease virus (NDV) in formalin-fixed tissues from an outbreak of ND on broiler farms in Egypt. The affected birds experienced respiratory and/or nervous signs and a 75% mortality rate. Tissue samples were collected and placed in 10% neutral buffered formalin followed by embedding in paraffin. RNA was extracted from 80-microm formalin-fixed paraffin-embedded tissue blocks and recovered in 60 microl of elution buffer. All samples were negative for influenza virus by real-time reverse-transcription (RT)-PCR but positive for NDV. These flocks were known to have been vaccinated with a live NDV vaccine (LaSota strain). The nucleic acid sequences of the virus detected in this study were similar to those of a velogenic virus at its cleavage site 111GRRQKR*F117 and clustered with class II genogroup VII lineage of NDV, with a nucleotide sequence identity of 94%-99%. Although extraction and amplification of NDV from paraffin-embedded tissues from experimentally infected birds has been reported previously, this study reports on the use of RT-PCR on formalin-fixed tissues from actual field samples.

  20. Pre-embedding Method of Electron Microscopy for Glycan Localization in Mammalian Tissues and Cells Using Lectin Probes.

    PubMed

    Akimoto, Yoshihiro; Takata, Kuniaki; Kawakami, Hayato

    2016-01-01

    In recent years, the study of glycans is progressing remarkably by the development of glycan analysis systems using mass spectrometry, glycan profiling systems using lectin microarrays, and glycoprotein analysis by the isotope-coded glycosylation site-specific tagging method. With these methodologies, glycan structures and biological functions are being elucidated. In the study of glycan function as well as disease diagnosis, it is important to examine the localization of glycans in tissues and cells. Histochemical methods using lectin probes can localize glycans in the tissues and cells. This chapter describes a pre-embedding electron microscopic method for glycan localization in which tissue sections and cells are incubated with lectin prior to embedding in resin. PMID:27515086

  1. Relative capacities of time-gated versus continuous-wave imaging to localize tissue embedded vessels with increasing depth

    NASA Astrophysics Data System (ADS)

    Patel, Nimit L.; Lin, Zi-Jing; Rathore, Yajuvendra; Livingston, Edward H.; Liu, Hanli; Alexandrakis, George

    2010-01-01

    Surgeons often cannot see major vessels embedded in adipose tissue and inadvertently injure them. One such example occurs during surgical removal of the gallbladder, where injury of the nearby common bile duct leads to life-threatening complications. Near-infrared imaging of the intraoperative field may help surgeons localize such critical tissue-embedded vessels. We have investigated how continuous-wave (CW) imaging performs relative to time-gated wide-field imaging, presently a rather costly technology, under broad Gaussian beam-illumination conditions. We have studied the simplified case of an isolated cylinder having bile-duct optical properties, embedded at different depths within a 2-cm slab of adipose tissue. Monte Carlo simulations were preformed for both reflectance and transillumination geometries. The relative performance of CW versus time-gated imaging was compared in terms of spatial resolution and contrast-to-background ratio in the resulting simulated images. It was found that time-gated imaging offers superior spatial resolution and vessel-detection sensitivity in most cases, though CW transillumination measurements may also offer satisfactory performance for this tissue geometry at lower cost. Experiments were performed in reflectance geometry to validate simulation results, and potential challenges in the translation of this technology to the clinic are discussed.

  2. A pilot study on the expression of microRNAs resident on chromosome 21 in laser microdissected FFPE prostate adenocarcinoma samples.

    PubMed

    Mihala, Adrian; Alexa, Andreea Anda; Samoilă, Corina; Dema, Alis; Vizitiu, Anda Cornelia; Anghel, Andrei; Tămaş, Liviu; Marian, Cătălin Valer; Sîrbu, Ioan Ovidiu

    2015-01-01

    The tremendous research effort of the last decades added a new, epigenetic layer of complexity to the already complex image of prostate cancer pathogenesis. Here we use quantitative real-time polymerase chain reaction (qRT-PCR) to investigate the expression of the microRNAs resident on chromosome 21 (miR-ch21) in laser capture microdissected (LCM) tissues from formalin-fixed paraffin-embedded (FFPE) archived, prostate adenocarcinoma samples. We show a strong, specific down-regulation of miR-ch21 in tumoral epithelia and stromae as compared to normal counterparts, results at odd with the current paradigm on the involvement of these microRNAs in prostate oncogenesis. By comparing this result with the expression of two well-known pluripotency associated microRNA, hsa-miR-372 and miR-373, we suggest that miR-ch21 down-regulation might be the result of specific silencing of miR genes mapped to chromosome 21. Further studies, of larger sample size are needed to confirm our preliminary data.

  3. Guided protein extraction from formalin-fixed tissues for quantitative multiplex analysis avoids detrimental effects of histological stains.

    PubMed

    Becker, Karl-Friedrich; Schott, Christina; Becker, Ingrid; Höfler, Heinz

    2008-05-01

    Formalin fixed and paraffin embedded (FFPE) tissues are the basis for histopathological diagnosis of many diseases around the world. For translational research and routine diagnostics, protein analysis from FFPE tissues is very important. We evaluated the potential influence of six histological stains, including hematoxylin (Mayer and Gill), fast red, light green, methyl blue and toluidine blue, for yield, electrophoretic mobility in 1-D gels, and immunoreactivity of proteins isolated from formalin-fixed breast cancer tissues. Proteins extracted from stained FFPE tissues using a recently established technique were compared with proteins obtained from the same tissues but without prior histological staining. Western blot and quantitative protein lysate microarray analysis demonstrated that histological staining can result in decreased protein yield but may not have much influence on immunoreactivity and electrophoretic mobility. Interestingly, not all staining protocols tested are compatible with subsequent protein analysis. The commonly used hematoxylin staining was found to be suitable for multiplexed quantitative protein measurement technologies although protein extraction was less efficient. For best results we suggest a guided protein extraction method, in which an adjacent hematoxylin/eosin-stained tissue section is used to control dissection of an unstained specimen for subsequent protein extraction and quantification for research and diagnosis.

  4. Detection of Papillomavirus Gene Expression Patterns in Tissue Sections.

    PubMed

    Griffin, Heather; Doorbar, John

    2016-01-01

    Molecular events during the papillomavirus life cycle can be mapped in infected tissue biopsies using antibodies to viral and cellular gene products, or by in situ hybridization approaches that detect viral DNA or viral transcription products. For proteins, ease of immunodetection depends on antibody specificity and antigen availability. Epitopes in formalin-fixed paraffin-embedded (FFPE) samples are often masked by crosslinking and must be exposed for immunodetection. RNA in FFPE material is often degraded, and such tissue must be handled carefully to optimize detection. Viral proteins and viral genomic DNA are both well preserved in routinely processed FFPE samples, with sensitive detection methodologies allowing the simultaneous detection of multiple markers. The combined visualization of nucleic acid and (viral) protein targets, when coupled with image analysis approaches that allow correlation with standard pathology diagnosis, have allowed us to understand the molecular changes required for normal HPV life-cycle organization as well as deregulation during cancer progression. © 2016 by John Wiley & Sons, Inc. PMID:27153382

  5. Endogenous enzymes cause structural and chemical artifacts in methacrylate- and celloidin-embedded sections of unfixed freeze-dried tissues.

    PubMed

    Goodwin, A E; Grizzle, J M

    1994-01-01

    Bovine exocrine pancreas and fish (Rivulus ocellatus marmoratus) liver containing pancreatic acini were cryofixed, freeze-dried, and embedded in methacrylate or double-embedded in celloidin and paraffin. In chemically unfixed sections incubated in aqueous solutions, dissolution of zymogen granules was coincident with loss of tissue structure and antigenicity. Type II-S soybean protease inhibitor at 150 mg/liter during section flotation and in aqueous reagents used for immunohistochemistry prevented these artifacts and allowed the use of more dilute antibody solutions. Loss of glycogen from fish hepatocytes was most rapid in areas adjacent to pancreatic acini. Rapid loss of glycogen was attributed to amylase and was prevented by using poly-L-lysine instead of 3-aminopropyltriethoxysilane slide adhesive and by using alcoholic solutions during PAS staining. Inhibition of endogenous enzymes is an important consideration in the development of histological protocols with freeze-dried tissue sections.

  6. Combined Nomarski interference contrast and immunofluorescent study of neuropathological specimens: CSF sediments and paraffin embedded brain tissues.

    PubMed

    Mussini, J M; Hauw, J J; Escourolle, R

    1977-11-01

    The direct immunofluorescent technique may be easily improved by the use of the Nomarski optics. This contrast allows accurate identification of fluorescent CSF cells and structures in formalin fixed paraffin embedded brain tissues; in the latter, the combined optical procedure is fruitfull in order to avoid fluorescent artifacts misinterpretation. Furthermore, it is emphazised that the conditions in which routine neuropathological specimens are removed and stored usualy does permit the application of the immunofluorescent technique.

  7. Primer-extension pre-amplification of DNA from paraffin-embedded tissue: Analysis of concurrent breast lesions

    SciTech Connect

    Pekkel, V.; Schmitz, M.; Allred, D.C.

    1994-09-01

    We are examining concurrent breast lesions (proliferative disease, in situ cancers and invasive cancers) that co-exist in the same breast for clonality studies of breast cancer evolution. The archival tissue samples with concurrent breast lesions suitable for these analyses are formalin-fixed, paraffin-embedded tissue blocks. We have successfully implemented microdissection and DNA extraction protocols from lesion/normal tissue sets from breast cancer patients. These preparations permit the efficient polymerase chain reaction amplification of highly polymorphic simple sequence repeat polymorphisms (SSRPs) for the genetic analysis. However, the unequal yield of DNA from particularly small lesions and/or the paucity of normal breast ductal epithelium has made extensive testing of these samples difficult. In order to equalize the recovery of DNA from the various morphologically defined lesions and normal tissue, we have implemented the method of primer-extension pre-amplification (PEP). In order to demonstrate the fidelity of the PEP technique in this application, we selected a series of 16 paired normal-lesion DNAs from lysates of formalin-fixed, paraffin-embedded tissues. These samples had been previously analyzed for loss-of-heterozygosity by direct PCR amplification with SSRPs. The PEP products were re-amplified with the locus specific SSRPs to determine whether the genetic characteristics of the tumor and normal samples were preserved. For the eight SSRP loci tested thus far, all of the samples amplified and showed the same patterns of LOH as seen before. These results suggest that PEP may be a generally useful technique for genetic analysis of microscopic neoplastic lesions. At least 20-fold amplification of template DNA was observed in our experiments. We expect that this technique will also permit analysis of DNA from formalin-fixed, paraffin-embedded tissue by comparative genomic hybridization.

  8. Performance of a novel KRAS mutation assay for formalin-fixed paraffin embedded tissues of colorectal cancer.

    PubMed

    Sakai, Kazuko; Yoneshige, Azusa; Ito, Akihiko; Ueda, Yoji; Kondo, Satoshi; Nobumasa, Hitoshi; Fujita, Yoshihiko; Togashi, Yosuke; Terashima, Masato; De Velasco, Marco A; Tomida, Shuta; Nishio, Kazuto

    2015-01-01

    We compared the performance of the 3D-Gene® mutation assay (3D-Gene® KRAS mutation assay kit) with the Scorpion-ARMS (therascreen® KRAS RGQ PCR Kit) and Luminex (MEBGEN™ KRAS kit) assays for the detection of KRAS mutations in formalin-fixed, paraffin-embedded tissue samples from 150 patients diagnosed with colorectal cancer. DNA was extracted from the paraffin-embedded tissue samples with or without macrodissection under hematoxylin and eosin staining and the KRAS mutation status was independently determined using these assays. Discordant results were re-analyzed by Sanger sequencing. Mutation detection analysis was successfully performed in all 150 specimens using the 3D-Gene® mutation assay without an invalid case. The concordance rate between the 3D-Gene® mutation assay and Scorpion-ARMS or Luminex was 98.7% (148/150). KRAS mutations were detected at a frequency of 35.3% (53/150) in colorectal cancer specimens. Three discrepant cases were found between the three assays. Overall, our results demonstrate a high concordance rate of between the 3D-Gene® mutation assay and the two existing in-vitro diagnostics kits. All three assays proved to be validated methods for detecting clinically significant KRAS mutations in paraffin-embedded tissue samples. PMID:25674493

  9. Detection of immunoglobulin IGH gene rearrangements on formalin-fixed, paraffin embedded tissue in lymphoid malignancies.

    PubMed

    Moharrami, G; Ghorbian, S; Seifi, M; Estiar, M A; Fakhrjoo, A; Sakhinia, M; Sakhinia, E

    2014-01-01

    Human lymphomas are aggressive malignant diseases, which can be categorized based on their B and T cell lineage. B-cell lymphomas form around 90% of the total lymphoma cases, the remnants of malignancies arise from the T cell branch. Lymphomas are mostly characterized as clonal proliferations of specific tumor cells. The detection of malignant lymphomas are extensively investigated by their morphological features, immunohistochemistry and flowcytometric immunophenotyping, but in some of cases remained unknown. The BIOMED-2 protocols were used to determine the clonality of IGH gene rearrangements in patients with lymphoma. PCR amplification was performed on FFPE of 50 patients with B-cell lymphoma, which consisted of 11 cases with HLs, 25 cases of B-NHLs and 14 cases of B-LPD (lymphoproliferative disorders) that diagnosed as unclassifiable lymphoma. The rate of positive clonality was detected in 96% (24/25) of B-NHLs, whereas in 4% (1/25) of cases clonality was showed in a polyclonal pattern. In B-HLs, 82% (9/11) of cases showed clonality and 18% (2/11) of the cases showed polyclonality. The rate of positive clonality observed in 64.3% (9/14) of cases with B-LPD and 35.7% (5/14) of cases clonality was not detected in any of immunoglobulin gene family (FR1, FR2, FR3). In groups with DLBCL, clonality was detected in 95% (19/20) of the cases. In patients diagnosed with FL and MALTs 100% cases showed clonality for complete IGH. Our study revealed that EuroClonality BIOMED-2 protocols could be considered as a valuable and reliable method for clonality detection, especially in IGH analysis.

  10. An efficient method for the extraction of DNA from formalin-fixed, paraffin-embedded tissue by sonication.

    PubMed

    Heller, M J; Burgart, L J; TenEyck, C J; Anderson, M E; Greiner, T C; Robinson, R A

    1991-09-01

    A method was developed for fast and efficient isolation of DNA from formalin-fixed, paraffin-embedded tissue sections for subsequent use in PCRs and DNA hybridization assays. The method relies on the use of a sonicating water bath to disrupt tissue samples to which a small amount of micro-sized glass beads have been added. The sonicating glass beads provide fast and efficient physical shearing of fixed tissue sections, allowing for quick release and solubilization of the DNA. The extraction process from paraffin section to amplifiable target DNA takes 30 minutes. The method eliminates the need for repetitive solvent extractions and exhaustive proteinase K digestion. PCR amplification of human genomic and viral target sequences was successfully carried out on DNA isolated from a number of different types of normal and infected tissues.

  11. Use of Raman Spectroscopy in Characterizing Formalin-Fixed, Paraffin-Embedded Breast Tumor Samples (abstract)

    NASA Astrophysics Data System (ADS)

    Downey, Frances; Cade, Nicholas; Cook, Richard; Springall, Robert; Gillet, Cheryl; Richards, David; Festy, Frederic

    2009-04-01

    Formalin-fixed, paraffin-embedded (FFPE) sections of breast tissue are used by pathologists to correctly type and grade the primary tumor and to assess the extent of a patient's disease. The cut sections represent a reproducible likeness of the morphology of the tissue when viewed through a microscope, although the fixation technique creates some artifacts. What is not known is how the sections differ chemically from how the tumor would look or behave within the breast. Raman spectroscopy is, like many other optical techniques, fast, noninvasive, and generally inexpensive. The advantage Raman has over other techniques is its powerful ability to identify specific chemicals, molecules, and bonds within a sample. Using Raman spectroscopy the chemicals present in both fresh tissue and FFPE sections can be identified and compared, allowing any differences between them to be identified. This information may be useful to the pathologist as an aid to further treatment regimes or novel molecular techniques, and as an aid to patient management. If these sections are found to be chemically similar to fresh tissue, they could be used to further characterize breast tumors, particularly rare tumors, using Raman spectroscopy.

  12. Quantitative infrared spectroscopy of formalin-fixed, paraffin-embedded tissue specimens: paraffin wax removal with organic solvents.

    PubMed

    Meuse, Curtis W; Barker, Peter E

    2009-12-01

    Formalin-fixed, paraffin-embedded tissue specimens form the basis for diagnostic histopathology. Although adequate for morphologic visualization, clinical variability in preparation of formalin-fixed, paraffin-embedded clinical specimens represents an obstacle to quantitative molecular genetic analysis in areas such as genomics and proteomics. A quantitative reexamination of classical histopathology tissue preparation methods was initiated to determine which protocol steps might improve molecular analysis, beginning with deparaffinization. Infrared spectroscopy in the spectral region above 2000/cm of fixed sectioned model cell cultures through glass microscope slides showed all solvents remove over 97% of paraffin. To further compare extractions among solvents xylene, hexane and limonene, the correlation coefficients between the spectrum of paraffin and the spectra of the mounted extracted model tissue sections across the spectral interval containing the prominent CH stretching bands of paraffin were calculated. The correlation coefficients allow different extraction methods to be ranked in terms of how much paraffin remains. The results indicate that among 3 model tissue sample types, xylene extraction removes more paraffin than hexane or limonene. More importantly, these results establish a starting point from which further analysis of preanalytical processing methods can proceed.

  13. PCR amplification from paraffin-embedded tissues: recommendations on fixatives for long-term storage and prospective studies.

    PubMed

    Greer, C E; Lund, J K; Manos, M M

    1991-08-01

    The development of polymerase chain reaction (PCR) DNA amplification methods has afforded molecular studies of fixed paraffin-embedded tissue samples and other archival material. Some fixation methods damage DNA, and thus deleteriously affect subsequent PCR analysis. This study addressed the effect of short- and long-term storage (2 hr to 30 days) in a variety of fixatives (10% buffered-neutral formalin [BNF], 95% ethanol, acetone, and OmniFix) before paraffin embedding. We tested the ability of prepared tissue sections to yield DNA amplification products ranging from 268 to 1327 bp. Results indicated that tissues fixed for 8 days in BNF were able to amplify 536-bp but very little 989-bp DNA fragments; after 30 days of BNF fixation only a 268-bp fragment was amplifiable. Samples fixed in OmniFix and acetone yielded products of 989 and 1327 bp, respectively, after 8 days of fixation; both fixatives yielded 989-bp amplification products after 30 days of fixation. Tissues fixed in 95% ethanol for up to 30 days efficiently produced DNA amplification fragments of up to 1327 bp in length. The results provide important information for prospective studies that involve PCR analysis from archival material. Furthermore, fixation and long-term storage in ethanol should prove particularly useful in remote areas where refrigeration or immediate sample-processing is unavailable.

  14. Optimization of Single- and Dual-Color Immunofluorescence Protocols for Formalin-Fixed, Paraffin-Embedded Archival Tissues.

    PubMed

    Kajimura, Junko; Ito, Reiko; Manley, Nancy R; Hale, Laura P

    2016-02-01

    Performance of immunofluorescence staining on archival formalin-fixed paraffin-embedded human tissues is generally not considered to be feasible, primarily due to problems with tissue quality and autofluorescence. We report the development and application of procedures that allowed for the study of a unique archive of thymus tissues derived from autopsies of individuals exposed to atomic bomb radiation in Hiroshima, Japan in 1945. Multiple independent treatments were used to minimize autofluorescence and maximize fluorescent antibody signals. Treatments with NH3/EtOH and Sudan Black B were particularly useful in decreasing autofluorescent moieties present in the tissue. Deconvolution microscopy was used to further enhance the signal-to-noise ratios. Together, these techniques provide high-quality single- and dual-color fluorescent images with low background and high contrast from paraffin blocks of thymus tissue that were prepared up to 60 years ago. The resulting high-quality images allow the application of a variety of image analyses to thymus tissues that previously were not accessible. Whereas the procedures presented remain to be tested for other tissue types and archival conditions, the approach described may facilitate greater utilization of older paraffin block archives for modern immunofluorescence studies.

  15. Investigation into a new softening agent for use on formalin-fixed, paraffin wax-embedded tissue.

    PubMed

    Orchard, G E; Torres, J; Poirier, A; Sounthararajah, R; Webster, J; Notini, L; Hacker, L; Ismail, F; Nwokie, T; Humphrey, P; Spigler, E; Missaghian-Cully, S; Brewer, C; Meredith-Jones, A

    2009-01-01

    The use of tissue softening agents to improve microtomy of keratotic tissues is employed widely. Many of these softeners contain hazardous constituents such as phenol. In this study, the use of non-ionic surfactants or non-toxic ingredients are investigated with the aim of creating a new softening agent. The new agent should be more effective in facilitating the sectioning of hardened tissue while reducing toxicity and complications associated with sectioning hard tissue compared to a commercially available phenol-based formulation. Four formulations are compared against the commercial product for their capability to section routinely processed paraffin-embedded tissue under standard operating procedure parameters. The trial formulations were shown to be fast acting and enabled improved serial sectioning of hard keratotic tissue in nearly all the cases tested. There was no evidence of adverse staining using either tinctorial or immunohistochemical methods. The new formulations had advantages over the commercially available solutions, improving on the number and quality of sections attainable from the tissue blocks, as well as offering a composition less toxic than phenol-based products.

  16. Optimization of Single- and Dual-Color Immunofluorescence Protocols for Formalin-Fixed, Paraffin-Embedded Archival Tissues.

    PubMed

    Kajimura, Junko; Ito, Reiko; Manley, Nancy R; Hale, Laura P

    2016-02-01

    Performance of immunofluorescence staining on archival formalin-fixed paraffin-embedded human tissues is generally not considered to be feasible, primarily due to problems with tissue quality and autofluorescence. We report the development and application of procedures that allowed for the study of a unique archive of thymus tissues derived from autopsies of individuals exposed to atomic bomb radiation in Hiroshima, Japan in 1945. Multiple independent treatments were used to minimize autofluorescence and maximize fluorescent antibody signals. Treatments with NH3/EtOH and Sudan Black B were particularly useful in decreasing autofluorescent moieties present in the tissue. Deconvolution microscopy was used to further enhance the signal-to-noise ratios. Together, these techniques provide high-quality single- and dual-color fluorescent images with low background and high contrast from paraffin blocks of thymus tissue that were prepared up to 60 years ago. The resulting high-quality images allow the application of a variety of image analyses to thymus tissues that previously were not accessible. Whereas the procedures presented remain to be tested for other tissue types and archival conditions, the approach described may facilitate greater utilization of older paraffin block archives for modern immunofluorescence studies. PMID:26392518

  17. Post-embedding Mammalian Tissue for Immunoelectron Microscopy: A Standardized Procedure Based on Heat-Induced Antigen Retrieval.

    PubMed

    Yamashita, Shuji

    2016-01-01

    We describe a standardized method of fixation, antigen retrieval, and image contrasting for post-embedding immunoelectron microscopy. Tissues are fixed with formaldehyde solutions containing Ca(2+) and Mg(2+) ions at pH 7.4 and then at pH 8.5. After dehydration with dimethylformamide, the specimens are embedded in LR-White resin. For antigen retrieval, ultrathin sections are heated in 0.5 M Tris-HCl, pH 9.0, for 1-2 h at 95 °C. After immunogold labeling, the sections are treated with a mixture of tannic acid and glutaraldehyde, with OsO4 solution, and then double-stained with uranyl acetate and lead citrate. The standardized method yields strong and reproducible immunoreactions for many antigens showing excellent image contrast without destruction of fine structures. PMID:27515088

  18. [Comparison of two different real-time PCR systems in postmortem diagnosis of tuberculosis in paraffin-embedded tissues].

    PubMed

    Yağmur, Gülhan; Albayrak, Nurhan; Daş, Taner; Yıldırım, Muzaffer; Ozgün, Ayşe; Büyük, Yalçın

    2014-10-01

    Tuberculosis (TB) is one of those infections with high morbidity and mortality in all around the world. Hundreds of people died from this disease without diagnosed or due to resistant strains in Turkey. Therefore, it is important to identify postmortem cases who have died from tuberculosis. Molecular methods have been widely used as well as conventional methods in the diagnosis of tuberculosis. The aim of this study was to compare the two different real-time polymerase chain reaction (Rt-PCR) system in the postmortem diagnosis of Mycobacterium tuberculosis infections in paraffin-embedded tissues. A total of 40 paraffin-embedded tissue samples [lung (n= 35), brain (n= 2), heart (n= 2), lymph node (n= 1)] in which histopathologic findings consistent with TB (necrotizing granulomatous inflammation, gelatinous caseous pneumonia, necrotic fibrous nodul) obtained from 37 autopsy cases (31 male, 6 female; age range: 25-85 yrs) were included in the study. Paraffin-embedded tissues were deparafinized with xylene and ethyl alcohol and then DNA isolation was done with QIAsymphony DSP Virus/Pathogen Midi kit in the QIAsymphony device. DNA amplification process was performed by Rt-PCR using the kit Artus® M. tuberculosis RG-PCR in the Rotor-Gene® Q device (Qiagen, Germany). Likewise, after deparafinization process, samples placed in the cartridge and isolation and Rt-PCR was performed by Xpert® MTB/RIF (Cepheid, USA) system, simultaneosly. Seventeen and 20 out of the 40 paraffin-embedded tissues yielded positive results with Qiagen and Xpert system, respectively. M.tuberculosis DNA was found positive in 13 (32.5%) and negative in 16 (40%) of the samples by both of the systems, exhibiting 72.5% (29/40) of concordance. On the other hand, seven (17.5%) samples that were positive with Xpert system yielded negative result with the Qiagen, while four (10%) samples that were positive with Qiagen yielded negative result with the Xpert system. Of the 20 positive cases detected with

  19. RT-PCR Analysis of RNA Extracted from Bouin-Fixed and Paraffin-Embedded Lymphoid Tissues

    PubMed Central

    Gloghini, Annunziata; Canal, Barbara; Klein, Ulf; Dal Maso, Luigino; Perin, Tiziana; Dalla-Favera, Riccardo; Carbone, Antonino

    2004-01-01

    In the present study, we have investigated whether RNA can be efficiently isolated from Bouin-fixed or formalin-fixed, paraffin-embedded lymphoid tissue specimens. To this aim, we applied a new and simple method that includes the combination of proteinase K digestion and column purification. By this method, we demonstrated that the amplification of long fragments could be accomplished after a pre-heating step before cDNA synthesis associated with the use of enzymes that work at high temperature. By means of PCR using different primers for two examined genes (glyceraldehyde-3-phosphate dehydrogenase [GAPDH]- and CD40), we amplified segments of cDNA obtained by reverse transcription of the isolated RNA extracted from Bouin-fixed or formalin-fixed paraffin-embedded tissues. Amplified fragments of the expected sizes were obtained for both genes tested indicating that this method is suitable for the isolation of high-quality RNA. To explore the possibility for giving accurate real time quantitative RT-PCR results, cDNA obtained from matched frozen, Bouin-fixed and formalin-fixed neoplastic samples (two diffuse large cell lymphomas, one plasmacytoma) was tested for the following target genes: CD40, Aquaporin-3, BLIMP1, IRF4, Syndecan-1. Delta threshold cycle (ΔCT) values for Bouin-fixed and formalin-fixed paraffin-embedded tissues and their correlation with those for frozen samples showed an extremely high correlation (r > 0.90) for all of the tested genes. These results show that the method of RNA extraction we propose is suitable for giving accurate real time quantitative RT-PCR results. PMID:15507667

  20. Temperature evolution in tissues embedded with large blood vessels during photo-thermal heating.

    PubMed

    Paul, Anup; Narasimhan, Arunn; Kahlen, Franz J; Das, Sarit K

    2014-04-01

    During laser-assisted photo-thermal therapy, the temperature of the heated tissue region must rise to the therapeutic value (e.g., 43°C) for complete ablation of the target cells. Large blood vessels (larger than 500 micron in diameter) at or near the irradiated tissues have a considerable impact on the transient temperature distribution in the tissue. In this study, the cooling effects of large blood vessels on temperature distribution in tissues during laser irradiation are predicted using finite element based simulation. A uniform flow is assumed at the entrance and three-dimensional conjugate heat transfer equations in the tissue region and the blood region are simultaneously solved for different vascular models. A volumetric heat source term based on Beer-Lambert law is introduced into the energy equation to account for laser heating. The heating pattern is taken to depend on the absorption and scattering coefficients of the tissue medium. Experiments are also conducted on tissue mimics in the presence and absence of simulated blood vessels to validate the numerical model. The coupled heat transfer between thermally significant blood vessels and their surrounding tissue for three different tissue-vascular networks are analyzed keeping the laser irradiation constant. A surface temperature map is obtained for different vascular models and for the bare tissue (without blood vessels). The transient temperature distribution is seen to differ according to the nature of the vascular network, blood vessel size, flow rate, laser spot size, laser power and tissue blood perfusion rate. The simulations suggest that the blood flow through large blood vessels in the vicinity of the photothermally heated tissue can lead to inefficient heating of the target. PMID:24679976

  1. Recognition and reduction of artifacts from autolysis in paraffin-embedded tissue using DNA/nuclear protein flow cytometry.

    PubMed

    Pollack, A; Ciancio, G; Terry, N H; Block, N L

    1993-01-01

    Artifacts from autolysis can be a problem in retrospective flow-cytometric analyses of DNA content in paraffin-embedded tissues. Autolyzed tissue from rat liver, human liver, and rat spleen were stained for DNA and nuclear protein to determine if this technique would be useful in identifying partially degraded cells. After the tissue was deparaffinized and rehydrated, the nuclei were isolated using 0.5% pepsin. Propidium iodide (PI) and fluorescein isothiocyanate (FITC) were used to stain DNA and nuclear protein. When unfixed rat liver tissue was allowed to undergo autolysis at 4 degrees C for 24-48 h before fixation, there was a progressive broadening of the G1 and G2M DNA peaks and a slight increase in the average DNA contents of these peaks. Nuclei that stained more intensely with PI also stained more intensely with FITC. Similar results were obtained using human liver and rat spleen. Sometimes the increased PI staining resulted in a false aneuploid peak. The distinctive skewing of the DNA/nuclear protein histograms from autolysis was reduced by increasing the incubation of the tissue in 0.5% pepsin from 0.5 h to 1.5 h during the nuclei-isolation step. The DNA/nuclear protein method provides a means for identifying artifacts from autolysis, whereas the extended pepsin treatment provides a means for reducing these artifacts.

  2. Single-molecule RNA detection at depth by hybridization chain reaction and tissue hydrogel embedding and clearing

    PubMed Central

    Shah, Sheel; Lubeck, Eric; Schwarzkopf, Maayan; He, Ting-Fang; Greenbaum, Alon; Sohn, Chang Ho; Lignell, Antti; Choi, Harry M. T.; Gradinaru, Viviana; Pierce, Niles A.

    2016-01-01

    Accurate and robust detection of mRNA molecules in thick tissue samples can reveal gene expression patterns in single cells within their native environment. Preserving spatial relationships while accessing the transcriptome of selected cells is a crucial feature for advancing many biological areas – from developmental biology to neuroscience. However, because of the high autofluorescence background of many tissue samples, it is difficult to detect single-molecule fluorescence in situ hybridization (smFISH) signals robustly in opaque thick samples. Here, we draw on principles from the emerging discipline of dynamic nucleic acid nanotechnology to develop a robust method for multi-color, multi-RNA imaging in deep tissues using single-molecule hybridization chain reaction (smHCR). Using this approach, single transcripts can be imaged using epifluorescence, confocal or selective plane illumination microscopy (SPIM) depending on the imaging depth required. We show that smHCR has high sensitivity in detecting mRNAs in cell culture and whole-mount zebrafish embryos, and that combined with SPIM and PACT (passive CLARITY technique) tissue hydrogel embedding and clearing, smHCR can detect single mRNAs deep within thick (0.5 mm) brain slices. By simultaneously achieving ∼20-fold signal amplification and diffraction-limited spatial resolution, smHCR offers a robust and versatile approach for detecting single mRNAs in situ, including in thick tissues where high background undermines the performance of unamplified smFISH. PMID:27342713

  3. Single-molecule RNA detection at depth by hybridization chain reaction and tissue hydrogel embedding and clearing.

    PubMed

    Shah, Sheel; Lubeck, Eric; Schwarzkopf, Maayan; He, Ting-Fang; Greenbaum, Alon; Sohn, Chang Ho; Lignell, Antti; Choi, Harry M T; Gradinaru, Viviana; Pierce, Niles A; Cai, Long

    2016-08-01

    Accurate and robust detection of mRNA molecules in thick tissue samples can reveal gene expression patterns in single cells within their native environment. Preserving spatial relationships while accessing the transcriptome of selected cells is a crucial feature for advancing many biological areas - from developmental biology to neuroscience. However, because of the high autofluorescence background of many tissue samples, it is difficult to detect single-molecule fluorescence in situ hybridization (smFISH) signals robustly in opaque thick samples. Here, we draw on principles from the emerging discipline of dynamic nucleic acid nanotechnology to develop a robust method for multi-color, multi-RNA imaging in deep tissues using single-molecule hybridization chain reaction (smHCR). Using this approach, single transcripts can be imaged using epifluorescence, confocal or selective plane illumination microscopy (SPIM) depending on the imaging depth required. We show that smHCR has high sensitivity in detecting mRNAs in cell culture and whole-mount zebrafish embryos, and that combined with SPIM and PACT (passive CLARITY technique) tissue hydrogel embedding and clearing, smHCR can detect single mRNAs deep within thick (0.5 mm) brain slices. By simultaneously achieving ∼20-fold signal amplification and diffraction-limited spatial resolution, smHCR offers a robust and versatile approach for detecting single mRNAs in situ, including in thick tissues where high background undermines the performance of unamplified smFISH. PMID:27342713

  4. Effect of section thickness on quality of flow cytometric DNA content determinations in paraffin-embedded tissues.

    PubMed

    Stephenson, R A; Gay, H; Fair, W R; Melamed, M R

    1986-01-01

    DNA content determinations were carried out by flow cytometry on nuclear suspensions prepared from the same paraffin-embedded tissue block for each of eight surgically resected human carcinomas at section thicknesses of 5,10,20,30,40,50, and 100 millimicrons. Flow cytometric DNA determinations were also obtained on fresh tissue specimens in four of the eight carcinomas. As section thickness decreased below 50 millimicrons, there was a progressive increase in the histogram baseline noise at low DNA values and a decrease in the relative peak height of aneuploid DNA. The former was attributed to an increase of nuclear fragments in thinner sections, and the latter to the greater probability of transection of the larger aneuploid cells within a specimen. Both artifacts were minimized at section thickness of 50 millimicrons or greater.

  5. In situ hybridization for the detection of rust fungi in paraffin embedded plant tissue sections

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Rust fungi infect a wide range of plant species making them of particular interest to plant pathologists. In order to study the interactions between these important pathogenic fungi and their host plants it is useful to be able to differentiate fungal tissue from plant tissue. This can be accomplish...

  6. Improved DNA content histograms from formalin-fixed, paraffin-embedded liver tissue by proteinase K digestion.

    PubMed

    Albro, J; Bauer, K D; Hitchcock, C L; Wittwer, C T

    1993-01-01

    An improved method for the enzymatic digestion of formalin-fixed, paraffin-embedded liver tissue for DNA content analysis by flow cytometry is presented. Forty samples of histologically normal liver were alternately digested by the traditional pepsin method or a new method utilizing proteinase K and heat. Sixteen (40%) of the pepsin-digested samples had apparent DNA aneuploid peaks by flow cytometry. False DNA aneuploid peaks were not present in any of the histograms obtained after proteinase K digestion. Microscopy showed that the pepsin-digested samples had residual cytoplasmic remnants which contained fluorescent material. Samples digested with proteinase K had few cytoplasmic remnants. The average G0/G1 coefficient of variation after proteinase K treatment was lower (41%) and the fluorescent intensity higher (128%) than the pepsin-treated samples. The apparent mean S-phase (a combination of S-phase cells and underlying debris) after proteinase K digestion was 35% of the pepsin-treated samples. Primary and secondary tumors of the liver that were DNA aneuploid after pepsin treatment were also DNA aneuploid after proteinase K treatment. A modified digestion protocol utilizing proteinase K and heat can provide superior results for DNA content analysis of formalin-fixed, paraffin-embedded liver tissue.

  7. Improved technique for fluorescence in situ hybridisation analysis of isolated nuclei from archival, B5 or formalin fixed, paraffin wax embedded tissue.

    PubMed

    Schurter, M J; LeBrun, D P; Harrison, K J

    2002-04-01

    Fluorescence in situ hybridisation (FISH) is an effective method to detect chromosomal alterations in a variety of tissue types, including archived paraffin wax embedded specimens fixed in B5 or formalin. However, precipitating fixatives such as B5 have been known to produce unsatisfactory results in comparison with formalin when used for FISH. This study describes an effective nuclear isolation and FISH procedure for B5 and formalin fixed tissue, optimising the nuclear isolation step and nuclei pretreatments using tonsil and mantle cell lymphoma specimens. The protocol presented can be used to isolate nuclei and perform FISH on B5 or formalin fixed, paraffin wax embedded samples from a variety of tissue types.

  8. Immunohistochemical detection of the androgen receptor with monoclonal antibody F39.4 in routinely processed, paraffin-embedded human tissues after microwave pre-treatment.

    PubMed

    Janssen, P J; Brinkmann, A O; Boersma, W J; Van der Kwast, T H

    1994-08-01

    We describe the immunohistochemical detection of the human androgen receptor (AR) in routinely processed, paraffin-embedded tissue with the monoclonal antibody (MAb) F39.4. Deparaffinized sections were heated in a microwave oven for antigen retrieval. A panel of human male- and female-derived tissues was investigated. We observed a nuclear staining pattern consistent with previous results on frozen sections. Moreover, we studied the possibility of detecting AR in prolonged formalin-fixed tissue and in paraffin-embedded archival material. After prolonged fixation times or long-term storage of paraffin-embedded tissue, the staining intensity for the AR did not deteriorate. Blocking experiments with the specific synthetic peptides demonstrated the specificity of this technique. We conclude that this method is specific, allows retrospective AR studies, and offers optimally preserved morphology.

  9. Coiled fiber scaffolds embedded with gold nanoparticles improve the performance of engineered cardiac tissues

    NASA Astrophysics Data System (ADS)

    Fleischer, Sharon; Shevach, Michal; Feiner, Ron; Dvir, Tal

    2014-07-01

    Coiled perimysial fibers within the heart muscle provide it with the ability to contract and relax efficiently. Here, we report on a new nanocomposite scaffold for cardiac tissue engineering, integrating coiled electrospun fibers with gold nanoparticles. Cultivation of cardiac cells within the hybrid scaffolds promoted cell organization into elongated and aligned tissues generating a strong contraction force, high contraction rate and low excitation threshold.Coiled perimysial fibers within the heart muscle provide it with the ability to contract and relax efficiently. Here, we report on a new nanocomposite scaffold for cardiac tissue engineering, integrating coiled electrospun fibers with gold nanoparticles. Cultivation of cardiac cells within the hybrid scaffolds promoted cell organization into elongated and aligned tissues generating a strong contraction force, high contraction rate and low excitation threshold. Electronic supplementary information (ESI) available. See DOI: 10.1039/c4nr00300d

  10. Detection and Quantification of CWD Prions in Fixed Paraffin Embedded Tissues by Real-Time Quaking-Induced Conversion.

    PubMed

    Hoover, Clare E; Davenport, Kristen A; Henderson, Davin M; Pulscher, Laura A; Mathiason, Candace K; Zabel, Mark D; Hoover, Edward A

    2016-01-01

    Traditional diagnostic detection of chronic wasting disease (CWD) relies on immunodetection of misfolded CWD prion protein (PrP(CWD)) by western blotting, ELISA, or immunohistochemistry (IHC). These techniques require separate sample collections (frozen and fixed) which may result in discrepancies due to variation in prion tissue distribution and assay sensitivities that limit detection especially in early and subclinical infections. Here, we harness the power of real-time quaking induced conversion (RT-QuIC) to amplify, detect, and quantify prion amyloid seeding activity in fixed paraffin-embedded (FPE) tissue sections. We show that FPE RT-QuIC has greater detection sensitivity than IHC in tissues with low PrP(CWD) burdens, including those that are IHC-negative. We also employ amyloid formation kinetics to yield a semi-quantitative estimate of prion concentration in a given FPE tissue. We report that FPE RT-QuIC has the ability to enhance diagnostic and investigative detection of disease-associated PrP(RES) in prion, and potentially other, protein misfolding disease states. PMID:27157060

  11. Comparative evaluation of specific methods for labeling of Encephalitozoon cuniculi in paraffin wax-embedded tissue samples.

    PubMed

    Habenbacher, Bettina; Klang, Andrea; Fragner, Karin; Dinhopl, Nora; Künzel, Frank; Weissenböck, Herbert

    2012-03-01

    Detection of the microsporidian Encephalitozoon cuniculi in tissue samples is considered difficult. The aim of the current study was to determine whether immunohistochemistry (IHC) and in situ hybridization (ISH) represent reliable methods for the detection of E. cuniculi in postmortem tissue samples of rabbits. Paraffin-embedded tissue sections of brain and kidneys of 48 naturally infected pet rabbits, 10 negative controls, and the eyes of 3 further rabbits were used for all investigations. By IHC in 19 animals (37.3%), spores could be clearly detected and were all equally stained. By ISH using a digoxigenin-labeled oligonucleotide probe, only 6 animals (11.8%) proved undoubtedly positive. In these cases, many parasite-like objects revealed strong typical purple-black positive signals. However, several of the examined samples showed only partial staining of the pathogen or unclear results. Thus, in order to find an explanation for these inconsistent ISH results and to take a more detailed look at the different developmental stages of the organism, electron microscopy was applied. Empty spores, which had already discharged their polar filaments, prevailed in total number. Taken together, both techniques are rather insensitive, but under the condition that sufficient numbers of microsporidia are present, IHC can be recommended for specific identification of E. cuniculi in tissue samples. In contrast, ISH failed to detect some developmental stages of the organism, and, as such, ISH is therefore considered an inappropriate diagnostic method.

  12. Preservation of nucleic acids and tissue morphology in paraffin-embedded clinical samples: comparison of five molecular fixatives.

    PubMed

    Staff, Synnöve; Kujala, Paula; Karhu, Ritva; Rökman, Annika; Ilvesaro, Joanna; Kares, Saara; Isola, Jorma

    2013-09-01

    Formalin fixation preserves tissue morphology at the expense of macromolecule integrity. Freshly frozen samples are the golden standard for DNA and RNA analyses but require laborious deep-freezing and frozen sectioning for morphological studies. Alternative tissue stabilisation methods are therefore needed. We analysed the preservation of nucleic acids, immunohistochemical staining properties and tissue morphology in paraffin-embedded clinical tissue samples fixed with Z7, RCL2, PAXgene, Allprotect and RNAlater. Formalin-fixed and deep-frozen samples were used as controls. Immunohistochemical analyses showed good preservation of antigenicity in all except Allprotect and RNAlater-fixed samples. RNA quality, based on RNA integrity number value by Bioanalyzer, was comparable with freshly frozen samples only in PAXgene-fixed samples. According to quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analyses, RNA from PAXgene samples yielded results similar to freshly frozen samples. No difference between fixatives was seen in DNA analyses (PCR and real-time PCR). In conclusion, PAXgene seems to be superior to other molecular fixatives and formaldehyde.

  13. Detection and Quantification of CWD Prions in Fixed Paraffin Embedded Tissues by Real-Time Quaking-Induced Conversion

    PubMed Central

    Hoover, Clare E.; Davenport, Kristen A.; Henderson, Davin M.; Pulscher, Laura A.; Mathiason, Candace K.; Zabel, Mark D.; Hoover, Edward A.

    2016-01-01

    Traditional diagnostic detection of chronic wasting disease (CWD) relies on immunodetection of misfolded CWD prion protein (PrPCWD) by western blotting, ELISA, or immunohistochemistry (IHC). These techniques require separate sample collections (frozen and fixed) which may result in discrepancies due to variation in prion tissue distribution and assay sensitivities that limit detection especially in early and subclinical infections. Here, we harness the power of real-time quaking induced conversion (RT-QuIC) to amplify, detect, and quantify prion amyloid seeding activity in fixed paraffin-embedded (FPE) tissue sections. We show that FPE RT-QuIC has greater detection sensitivity than IHC in tissues with low PrPCWD burdens, including those that are IHC-negative. We also employ amyloid formation kinetics to yield a semi-quantitative estimate of prion concentration in a given FPE tissue. We report that FPE RT-QuIC has the ability to enhance diagnostic and investigative detection of disease-associated PrPRES in prion, and potentially other, protein misfolding disease states. PMID:27157060

  14. Resonant acoustic spectroscopy of soft tissues using embedded magnetomotive nanotransducers and optical coherence tomography

    PubMed Central

    Oldenburg, Amy L

    2010-01-01

    We present a new method for performing dynamic elastography of soft tissue samples. By sensing nanoscale displacements with optical coherence tomography, a chirped, modulated force is applied to acquire the mechanical spectrum of a tissue sample within a few seconds. This modulated force is applied via magnetic nanoparticles, named ‘nanotransducers’, which are diffused into the tissue, and which contribute negligible inertia to the soft tissue mechanical system. Using this novel system, we observed that excised tissues exhibit mechanical resonance modes which are well described by a linear damped harmonic oscillator. Results are validated by using cylindrical tissue phantoms of agarose in which resonant frequencies (30–400 Hz) are consistent with longitudinal modes and the sample boundary conditions. We furthermore show that the Young’s modulus can be computed from their measured resonance frequencies, analogous to resonant ultrasound spectroscopy for stiff material analysis. Using this new technique, named magnetomotive resonant acoustic spectroscopy (MRAS), we monitored the relative stiffening of an excised rat liver during a chemical fixation process. PMID:20124653

  15. On-tissue protein identification and imaging by MALDI-ion mobility mass spectrometry.

    PubMed

    Stauber, Jonathan; MacAleese, Luke; Franck, Julien; Claude, Emmanuelle; Snel, Marten; Kaletas, Basak Kükrer; Wiel, Ingrid M V D; Wisztorski, Maxence; Fournier, Isabelle; Heeren, Ron M A

    2010-03-01

    MALDI imaging mass spectrometry (MALDI-IMS) has become a powerful tool for the detection and localization of drugs, proteins, and lipids on-tissue. Nevertheless, this approach can only perform identification of low mass molecules as lipids, pharmaceuticals, and peptides. In this article, a combination of approaches for the detection and imaging of proteins and their identification directly on-tissue is described after tryptic digestion. Enzymatic digestion protocols for different kinds of tissues--formalin fixed paraffin embedded (FFPE) and frozen tissues--are combined with MALDI-ion mobility mass spectrometry (IM-MS). This combination enables localization and identification of proteins via their related digested peptides. In a number of cases, ion mobility separates isobaric ions that cannot be identified by conventional MALDI time-of-flight (TOF) mass spectrometry. The amount of detected peaks per measurement increases (versus conventional MALDI-TOF), which enables mass and time selected ion images and the identification of separated ions. These experiments demonstrate the feasibility of direct proteins identification by ion-mobility-TOF IMS from tissue. The tissue digestion combined with MALDI-IM-TOF-IMS approach allows a proteomics "bottom-up" strategy with different kinds of tissue samples, especially FFPE tissues conserved for a long time in hospital sample banks. The combination of IM with IMS marks the development of IMS approaches as real proteomic tools, which brings new perspectives to biological studies.

  16. Effects of formalin fixation, paraffin embedding, and time of storage on DNA preservation in brain tissue: a BrainNet Europe study.

    PubMed

    Ferrer, Isidre; Armstrong, Judith; Capellari, Sabina; Parchi, Piero; Arzberger, Thomas; Bell, Jeanne; Budka, Herbert; Ströbel, Thomas; Giaccone, Giorgio; Rossi, Giacomina; Bogdanovic, Nenad; Fakai, Peter; Schmitt, Andrea; Riederers, Peter; Al-Sarraj, Safa; Ravid, Rivka; Kretzschmar, Hans

    2007-07-01

    There is a large amount of tissue stored in brain collections and brain banks, but little is known about whether formalin-fixed tissues and paraffin blocks stored for years in brain banks are suitable for the retrospective genetic studies. The study was carried out in order to: (i) compare DNA preservation in frozen, formalin-fixed and paraffin-embedded tissues stored for different periods; (ii) study point mutations and triplet expansions in frozen, formalin-fixed and paraffin-embedded material stored for variable periods, and using different fixative solutions; (iii) compare different methods to optimize DNA extraction and DNA amplification from suboptimally preserved brain tissue. DNA preservation is suitable for genetic studies in samples stored at -80 degrees C for several years. Formalin-fixed, paraffin-embedded tissue was inferior to frozen tissue, but did yield adequate results in many cases depending on the type of fixative solution and time of fixation before embedding. Prolonged fixation in formalin rarely yielded useful DNA. Similar results were obtained in samples from prion diseases. The best results were obtained by using the Qiagen kits (QIAmp DNA Micro) in frozen material, paraffin blocks and formalin-fixed tissue. Genomiphi and TaKaRa Ex Taq methods were also assayed in paraffin blocks and in formalin-fixed samples with limited success.

  17. High-throughput sequencing and copy number variation detection using formalin fixed embedded tissue in metastatic gastric cancer.

    PubMed

    Kim, Seokhwi; Lee, Jeeyun; Hong, Min Eui; Do, In-Gu; Kang, So Young; Ha, Sang Yun; Kim, Seung Tae; Park, Se Hoon; Kang, Won Ki; Choi, Min-Gew; Lee, Jun Ho; Sohn, Tae Sung; Bae, Jae Moon; Kim, Sung; Kim, Duk-Hwan; Kim, Kyoung-Mee

    2014-01-01

    In the era of targeted therapy, mutation profiling of cancer is a crucial aspect of making therapeutic decisions. To characterize cancer at a molecular level, the use of formalin-fixed paraffin-embedded tissue is important. We tested the Ion AmpliSeq Cancer Hotspot Panel v2 and nCounter Copy Number Variation Assay in 89 formalin-fixed paraffin-embedded gastric cancer samples to determine whether they are applicable in archival clinical samples for personalized targeted therapies. We validated the results with Sanger sequencing, real-time quantitative PCR, fluorescence in situ hybridization and immunohistochemistry. Frequently detected somatic mutations included TP53 (28.17%), APC (10.1%), PIK3CA (5.6%), KRAS (4.5%), SMO (3.4%), STK11 (3.4%), CDKN2A (3.4%) and SMAD4 (3.4%). Amplifications of HER2, CCNE1, MYC, KRAS and EGFR genes were observed in 8 (8.9%), 4 (4.5%), 2 (2.2%), 1 (1.1%) and 1 (1.1%) cases, respectively. In the cases with amplification, fluorescence in situ hybridization for HER2 verified gene amplification and immunohistochemistry for HER2, EGFR and CCNE1 verified the overexpression of proteins in tumor cells. In conclusion, we successfully performed semiconductor-based sequencing and nCounter copy number variation analyses in formalin-fixed paraffin-embedded gastric cancer samples. High-throughput screening in archival clinical samples enables faster, more accurate and cost-effective detection of hotspot mutations or amplification in genes.

  18. Direct fluorescent antibody technique for the detection of bacterial kidney disease in paraffin-embedded tissues

    USGS Publications Warehouse

    Ochiai, T.; Yasutake, W.T.; Gould, R.W.

    1985-01-01

    The direct fluorescent antibody technique (FAT) was successfully used to detect the causative agent of bacterial kidney disease (BKD), Renibacterium salmoninarum, in Bouin's solution flexed and paraffinembedded egg and tissue sections. This method is superior to gram stain and may be particularly useful in detecting the BKD organism in fish with low-grade infection.

  19. Multicenter Evaluation of a Novel Automated Rapid Detection System of BRAF Status in Formalin-Fixed, Paraffin-Embedded Tissues.

    PubMed

    Schiefer, Ana-Iris; Parlow, Laura; Gabler, Lisa; Mesteri, Ildiko; Koperek, Oskar; von Deimling, Andreas; Streubel, Berthold; Preusser, Matthias; Lehmann, Annika; Kellner, Udo; Pauwels, Patrick; Lambin, Suzan; Dietel, Manfred; Hummel, Michael; Klauschen, Frederick; Birner, Peter; Möbs, Markus

    2016-05-01

    The mutated BRAF oncogene represents a therapeutic target in malignant melanoma. Because BRAF mutations are also involved in the pathogenesis of other human malignancies, the use of specific BRAF inhibitors might also be extended to other diseases in the future. A prerequisite for the clinical application of BRAF inhibitors is the reliable detection of activating BRAF mutations in routine histopathological samples. In a multicenter approach, we evaluated a novel and fully automated PCR-based system (Idylla) capable of detecting BRAF V600 mutations in formalin-fixed, paraffin-embedded tissue within 90 minutes with high sensitivity. We analyzed a total of 436 samples with the Idylla system. Valid results were obtained in 421 cases (96.56%). Its performance was compared with conventional methods (pyrosequencing or Sanger sequencing). Concordant results were obtained in 406 cases (96.90%). Reanalysis of eight discordant samples by next-generation sequencing and/or pyrosequencing with newly extracted DNA and the BRAF RGQ Kit confirmed the Idylla result in seven cases, resulting in an overall agreement of 98.57%. In conclusion, the Idylla system is a highly reliable and sensitive platform for detection of BRAF V600 mutations in formalin-fixed, paraffin-embedded material, providing an efficient alternative to conventional diagnostic methods, particularly for routine diagnostics laboratories with limited experience in molecular pathology.

  20. Fabrication and characterization of a rapid prototyped tissue engineering scaffold with embedded multicomponent matrix for controlled drug release

    PubMed Central

    Chen, Muwan; Le, Dang QS; Hein, San; Li, Pengcheng; Nygaard, Jens V; Kassem, Moustapha; Kjems, Jørgen; Besenbacher, Flemming; Bünger, Cody

    2012-01-01

    Bone tissue engineering implants with sustained local drug delivery provide an opportunity for better postoperative care for bone tumor patients because these implants offer sustained drug release at the tumor site and reduce systemic side effects. A rapid prototyped macroporous polycaprolactone scaffold was embedded with a porous matrix composed of chitosan, nanoclay, and β-tricalcium phosphate by freeze-drying. This composite scaffold was evaluated on its ability to deliver an anthracycline antibiotic and to promote formation of mineralized matrix in vitro. Scanning electronic microscopy, confocal imaging, and DNA quantification confirmed that immortalized human bone marrow-derived mesenchymal stem cells (hMSC-TERT) cultured in the scaffold showed high cell viability and growth, and good cell infiltration to the pores of the scaffold. Alkaline phosphatase activity and osteocalcin staining showed that the scaffold was osteoinductive. The drug-release kinetics was investigated by loading doxorubicin into the scaffold. The scaffolds comprising nanoclay released up to 45% of the drug for up to 2 months, while the scaffold without nanoclay released 95% of the drug within 4 days. Therefore, this scaffold can fulfill the requirements for both bone tissue engineering and local sustained release of an anticancer drug in vitro. These results suggest that the scaffold can be used clinically in reconstructive surgery after bone tumor resection. Moreover, by changing the composition and amount of individual components, the scaffold can find application in other tissue engineering areas that need local sustained release of drug. PMID:22904634

  1. Double immunofluorescent staining of rat macrophages in formalin-fixed paraffin-embedded tissue using two monoclonal mouse antibodies.

    PubMed

    Isidro, Raymond A; Isidro, Angel A; Cruz, Myrella L; Hernandez, Siomara; Appleyard, Caroline B

    2015-12-01

    The conventional approach of double immunostaining to visualize more than one protein in tissues or cells using antibodies from two different host species is not always feasible due to limitations with antibody availability. Previously reported methodologies for performing multiple immunostains on the same tissue or cells with antibodies originating from the same species are varied in their complexity, sensitivity, and approach to prevent unwanted interactions between antibodies. In the ever-expanding field of macrophage biology, much more is known about mouse and human macrophages than their rat counterparts. The limited availability of validated and well-characterized monoclonal antibodies from different species is one factor responsible for preventing advances in rat macrophage biology. Here we describe an immunostaining method for identifying and examining rat macrophages that is sufficiently sensitive for use in formalin-fixed paraffin-embedded tissue and that uses only commercially available reagents and antibodies. This method can be used to help characterize both physiological and pathophysiological processes in rat macrophages and can be adapted for use with any two antibodies from the same species of origin as long as one of the antibodies is biotinylated. PMID:26403093

  2. Relative capacities of time-gated versus CW imaging to localize tissue embedded vessels with increasing depth

    NASA Astrophysics Data System (ADS)

    Alexandrakis, George; Patel, Nimit L.; Lin, Zi-Jing; Livingston, Edward H.; Liu, Hanli

    2009-02-01

    The clinical motivation for our work was to help surgeons see vessels through non-translucent intraoperative tissues during laparoscopic removal of the gallbladder. Our main focus was to answer the question of how CW imaging performs relative to ICCD (Intensified Charge-Coupled Device) based time-gated imaging, which is a lot more costly, under broad Gaussian beam illumination conditions. We have studied the simplified case of an isolated bile duct embedded at different depths within a 2 cm slab of adipose tissue. Monte Carlo simulations were preformed for both reflectance and trans-illumination geometries. The relative performance of CW versus time-gated imaging was compared in terms of spatial resolution and vessel detection sensitivity in the resulting simulated images. Experiments were performed in reflectance geometry to validate simulation results. It was found that time-gated imaging offers superior spatial resolution and vessel detection sensitivity in all cases though CW trans-illumination measurements may also offer satisfactory performance for this tissue geometry at a lower cost.

  3. Enhanced fungal DNA-Extraction from Formalin fixed, paraffin embedded tissue specimens by application of thermal energy

    PubMed Central

    Rickerts, V.; Khot, P.D.; Ko, D.L.; Fredricks, D.N.

    2014-01-01

    Summary Determining the etiology of invasive fungal infections (IFI) is critical for patient management as fungi vary in their susceptibility to antifungals. However, the etiology remains obscure in many cases due to negative culture results. The identification of fungal DNA from pathology blocks by PCR and sequencing is an alternative approach to determine the etiology of IFI. Previous studies identified fungal DNA in only 50% of samples with positive histopathology, probably due to DNA damage by the tissue fixation. We used realtime PCR to quantify human and fungal DNA from Formalin-fixed, paraffin embedded tissue specimens in order to study the effect of thermal energy during extraction on the yield of amplifiable DNA and subsequent identification of fungal DNA. Tissue sections from eight patients with proven IFI were subjected to DNA extraction with varying exposure to thermal energy. Amplifiable DNA increased up to 76-fold by increasing incubation temperature from 65°C to 90°C. An additional increase was documented by incubation for up to 6 hours at 90°C. The augmented amplification of fungal DNA was associated with improved species identification by sequencing of the PCR amplicons. This may help illuminate the etiology of IFI and thereby improve patient management by guiding antifungal therapy. PMID:22414380

  4. Comparison of techniques for the successful detection of BRCA1 mutations in fixed paraffin-embedded tissue.

    PubMed

    Bernstein, Jonine L; Thompson, W Douglas; Casey, Graham; DiCioccio, Richard A; Whittemore, Alice S; Diep, Anh T; Thakore, Seema S; Vaziri, Susan; Xue, Shanyan; Haile, Robert W

    2002-09-01

    Genomic DNA isolated from archived paraffin-embedded tissues (PETs) has important applicability in genetic epidemiological studies. To determine the accuracy of the sequence data, using DNA derived from PET among patients with known mutations characterized from blood, we conducted a blinded factorial experiment to simultaneously examine the influence of mutation type, age of the PET, PCR product type, and Taq DNA polymerase on BRCA1 gene mutation detection. The probability of detecting sequencing artifacts was also investigated. We found that: (a) gene detection was most accurate for newer PET; (b) high fidelity Taq with shorter PCR amplicon length yielded the highest mutation detection success rate and lowest artifact rate; and (c) base substitutions were more often correctly identified than frameshift mutations or wild-type sequences. We concluded that DNA derived from PET that archived for less than 18 years can be used successfully for detecting BRCA1 gene mutations if quality control is strictly maintained.

  5. PCR-RFLP studies on chromosome 3p in formaldehyde-fixed, paraffin-embedded cervical cancer tissues.

    PubMed

    Karlsen, F; Rabbitts, P H; Sundresan, V; Hagmar, B

    1994-09-15

    Loss of heterozygosity (LOH) has been extensively studied on the short arm of chromosome 3, and functional proofs have been obtained defining a tumor-suppressor locus at 3p21-22. We examined 31 paraffin-embedded cervical cancer samples for LOH, using 5 PCR-primer pairs, located around 3p21. Allele loss was found in 19 out of the 27 informative samples (70%) while 13 out of 23 informative samples (56%) had LOH located at 3p21-22. More of the human papillomavirus (HPV)-positive samples had LOH compared to the HPV-negative samples, giving only a weak association between loss of allele and HPV integration. Modifications of the DNA in the formaldehyde-fixed samples were detected, and further studies will be required to clarify how such artifacts may affect restriction fragment length polymorphism (RFLP) studies on fixed tissues.

  6. Detection of human papillomavirus type 16 DNA sequences in paraffin-embedded tissues from the female urinary tract.

    PubMed

    Aglianò, A M; Gazzaniga, P; Cervigni, M; Gradilone, A; Napolitano, M; Pastore, L I; Manzari, V; Frati, L; Vecchione, A

    1994-01-01

    We investigated the presence of human papillomavirus-related DNA sequences (HPV 6, 11, 16 and 18) in 33 formalin-fixed paraffin-embedded biopsies from the urinary tract of female patients with recurrent and persistent urethritis and cystitis, using the polymerase chain reaction (PCR). The samples for PCR reaction were selected among tissues examined for histological diagnosis on the basis of the presence of microscopic changes consistent with HPV infection. Sequences homologous to HPV 6, 11 and 18 genome were not found, while HPV 16-related DNA sequences were identified in 25/33 lesions with histopathological diagnosis of metaplasia (1 from the urethra, 23 from the trigone and 1 from the bladder). The results suggest that the spread of HPV in the female urinary tract may not be uncommon and point to the need for further research on the possible pathogenic role in recurrent female disturbances.

  7. A Molecular Profile of Focal Segmental Glomerulosclerosis from Formalin-Fixed, Paraffin-Embedded Tissue

    PubMed Central

    Hodgin, Jeffrey B.; Borczuk, Alain C.; Nasr, Samih H.; Markowitz, Glen S.; Nair, Viji; Martini, Sebastian; Eichinger, Felix; Vining, Courtenay; Berthier, Celine C.; Kretzler, Matthias; D'Agati, Vivette D.

    2010-01-01

    Focal segmental glomerulosclerosis (FSGS) is a common form of idiopathic nephrotic syndrome defined by the characteristic lesions of focal glomerular sclerosis and foot process effacement; however, its etiology and pathogenesis are unknown. We used mRNA isolated from laser-captured glomeruli from archived formalin-fixed, paraffin-embedded renal biopsies, until recently considered an unsuitable source of mRNA for microarray analysis, to investigate the glomerular gene expression profiles of patients with primary classic FSGS, collapsing FSGS (COLL), minimal change disease (MCD), and normal controls (Normal). Amplified mRNA was hybridized to an Affymetrix Human X3P array. Unsupervised (unbiased) hierarchical clustering revealed two distinct clusters delineating FSGS and COLL from Normal and MCD. Class comparison analysis of FSGS + COLL combined versus Normal + MCD revealed 316 significantly differentially regulated genes (134 up-regulated, 182 down-regulated). Among the differentially regulated genes were those known to be part of the slit diaphragm junctional complex and those previously described in the dysregulated podocyte phenotype. Analysis based on Gene Ontology categories revealed overrepresented biological processes of development, differentiation and morphogenesis, cell motility and migration, cytoskeleton organization, and signal transduction. Transcription factors associated with developmental processes were heavily overrepresented, indicating the importance of reactivation of developmental programs in the pathogenesis of FSGS. Our findings reveal novel insights into the molecular pathogenesis of glomerular injury and structural degeneration in FSGS. PMID:20847290

  8. The homogeneous mutation status of a 22 gene panel justifies the use of serial sections of colorectal cancer tissue for external quality assessment.

    PubMed

    Dijkstra, Jeroen R; Tops, Bastiaan B J; Nagtegaal, Iris D; van Krieken, J Han J M; Ligtenberg, Marjolijn J L

    2015-09-01

    Testing for treatment related biomarkers in clinical care, like Ras mutation status in colorectal cancer (CRC), has increased drastically over recent years. Reliable testing of these markers is pivotal for optimal treatment of patients. Participation in external quality assessment (EQA) programs is an important element in quality management and often obligatory to comply with regulations or for accreditation. Formalin-fixed paraffin-embedded (FFPE) clinical specimens would ideally form the basis for these assessments, as they represent the most common starting material for molecular testing. However, molecular heterogeneity of a lesion in a FFPE tissue block could potentially affect test results of participating laboratories, which might compromise reliability of the quality assessment results. To assess the actual impact of this potential problem, we determined the mutation status of 22 genes commonly mutated in colon cancer in four levels covering 360 μm of 30 FFPE tissue blocks, by Next Generation Sequencing. In each block, the genotype of these genes was identical at all four levels, with only little variation in mutation load. This result shows that the mutation status of the selected 22 genes in CRC specimens is homogeneous within a 360 μm segment of the tumor. These data justify the use of serial sections, within a defined segment of a CRC tissue block, for external quality assessment of mutation analysis. PMID:26047774

  9. Chromatin immunoprecipitation from fixed clinical tissues reveals tumor-specific enhancer profiles.

    PubMed

    Cejas, Paloma; Li, Lewyn; O'Neill, Nicholas K; Duarte, Melissa; Rao, Prakash; Bowden, Michaela; Zhou, Chensheng W; Mendiola, Marta; Burgos, Emilio; Feliu, Jaime; Moreno-Rubio, Juan; Guadalajara, Héctor; Moreno, Víctor; García-Olmo, Damián; Bellmunt, Joaquim; Mullane, Stephanie; Hirsch, Michelle; Sweeney, Christopher J; Richardson, Andrea; Liu, X Shirley; Brown, Myles; Shivdasani, Ramesh A; Long, Henry W

    2016-06-01

    Extensive cross-linking introduced during routine tissue fixation of clinical pathology specimens severely hampers chromatin immunoprecipitation followed by next-generation sequencing (ChIP-seq) analysis from archived tissue samples. This limits the ability to study the epigenomes of valuable, clinically annotated tissue resources. Here we describe fixed-tissue chromatin immunoprecipitation sequencing (FiT-seq), a method that enables reliable extraction of soluble chromatin from formalin-fixed paraffin-embedded (FFPE) tissue samples for accurate detection of histone marks. We demonstrate that FiT-seq data from FFPE specimens are concordant with ChIP-seq data from fresh-frozen samples of the same tumors. By using multiple histone marks, we generate chromatin-state maps and identify cis-regulatory elements in clinical samples from various tumor types that can readily allow us to distinguish between cancers by the tissue of origin. Tumor-specific enhancers and superenhancers that are elucidated by FiT-seq analysis correlate with known oncogenic drivers in different tissues and can assist in the understanding of how chromatin states affect gene regulation. PMID:27111282

  10. A review of room temperature storage of biospecimen tissue and nucleic acids for anatomic pathology laboratories and biorepositories

    PubMed Central

    Lou, Jerry J; Mirsadraei, Leili; Sanchez, Desiree E; Wilson, Ryan W; Shabihkhani, Maryam; Lucey, Gregory M; Wei, Bowen; Singer, Elyse J; Mareninov, Sergey; Yong, William H

    2014-01-01

    Frozen biospecimens are crucial for translational research and contain well preserved nucleic acids and protein. However, the risk for catastrophic freezer failure as well as space, cost, and environmental concerns argue for evaluating long-term room temperature storage alternatives. Formalin-fixed paraffin embedded (FFPE) tissues have great value but their use is limited by cross-linking and fragmentation of nucleic acids, as well as loss of enzymatic activity. Stabilization solutions can now robustly preserve fresh tissue for up to 7 days at room temperature. For longer term storage, commercial vendors of chemical matrices claim real time stability of nucleic acids of over 2 years and their accelerated aging studies to date suggest stability for 12 years for RNA and 60 years for DNA. However, anatomic pathology biorepositories store mostly frozen tissue rather than nucleic acids. Small quantities of tissue can be directly placed on some chemical matrices to stabilize DNA, however RNA and proteins are not preserved. Current lyophilization approaches can preserve histomorphology, DNA, RNA, and proteins though RNA shows moderate degradation after 1–2 years. Formalin free fixatives show improved but varying abilities to preserve nucleic acids and face validation as well as cost barriers in replacing FFPE specimens. The paraffin embedding process can degrade RNA. Development of robust long-term room temperature biospecimen tissue storage technology can potentially reduce costs for the biomedical community in the face of growing targeted therapy needs and decreasing budgets. PMID:24362270

  11. Immunohistochemical Staining of B7-H1 (PD-L1) on Paraffin-embedded Slides of Pancreatic Adenocarcinoma Tissue

    PubMed Central

    Bigelow, Elaine; Bever, Katherine M.; Xu, Haiying; Yager, Allison; Wu, Annie; Taube, Janis; Chen, Lieping; Jaffee, Elizabeth M.; Anders, Robert A.; Zheng, Lei

    2013-01-01

    B7-H1/PD-L1, a member of the B7 family of immune-regulatory cell-surface proteins, plays an important role in the negative regulation of cell-mediated immune responses through its interaction with its receptor, programmed death-1 (PD-1) 1,2. Overexpression of B7-H1 by tumor cells has been noted in a number of human cancers, including melanoma, glioblastoma, and carcinomas of the lung, breast, colon, ovary, and renal cells, and has been shown to impair anti-tumor T-cell immunity3-8. Recently, B7-H1 expression by pancreatic adenocarcinoma tissues has been identified as a potential prognostic marker9,10. Additionally, blockade of B7-H1 in a mouse model of pancreatic cancer has been shown to produce an anti-tumor response11. These data suggest the importance of B7-H1 as a potential therapeutic target. Anti-B7-H1 blockade antibodies are therefore being tested in clinical trials for multiple human solid tumors including melanoma and cancers of lung, colon, kidney, stomach and pancreas12. In order to eventually be able to identify the patients who will benefit from B7-H1 targeting therapies, it is critical to investigate the correlation between expression and localization of B7-H1 and patient response to treatment with B7-H1 blockade antibodies. Examining the expression of B7-H1 in human pancreatic adenocarcinoma tissues through immunohistochemistry will give a better understanding of how this co-inhibitory signaling molecule contributes to the suppression of antitumor immunity in the tumor's microenvironment. The anti-B7-H1 monoclonal antibody (clone 5H1) developed by Chen and coworkers has been shown to produce reliable staining results in cryosections of multiple types of human neoplastic tissues4,8, but staining on paraffin-embedded slides had been a challenge until recently13-18. We have developed the B7-H1 staining protocol for paraffin-embedded slides of pancreatic adenocarcinoma tissues. The B7-H1 staining protocol described here produces consistent membranous and

  12. Proteomic analysis of formalin-fixed paraffin-embedded glomeruli suggests depletion of glomerular filtration barrier proteins in two-kidney, one-clip hypertensive rats

    PubMed Central

    Finne, Kenneth; Vethe, Heidrun; Skogstrand, Trude; Leh, Sabine; Dahl, Tone D.; Tenstad, Olav; Berven, Frode S.; Reed, Rolf K.; Vikse, Bjørn Egil

    2014-01-01

    Background It is well known that hypertension may cause glomerular damage, but the molecular mechanisms involved are still incompletely understood. Methods In the present study, we used formalin-fixed paraffin-embedded (FFPE) tissue to investigate changes in the glomerular proteome in the non-clipped kidney of two-kidney one-clip (2K1C) hypertensive rats, with special emphasis on the glomerular filtration barrier. 2K1C hypertension was induced in 6-week-old Wistar Hannover rats (n = 6) that were sacrificed 23 weeks later and compared with age-matched sham-operated controls (n = 6). Tissue was stored in FFPE tissue blocks and later prepared on tissue slides for laser microdissection. Glomeruli without severe morphological damage were isolated, and the proteomes were analysed using liquid chromatography–tandem mass spectrometry. Results 2K1C glomeruli showed reduced abundance of proteins important for slit diaphragm complex, such as nephrin, podocin and neph1. The podocyte foot process had a pattern of reduced abundance of transmembrane proteins but unchanged abundances of the podocyte cytoskeletal proteins synaptopodin and α-actinin-4. Lower abundance of important glomerular basement membrane proteins was seen. Possible glomerular markers of damage with increased abundance in 2K1C were transgelin, desmin and acyl-coenzyme A thioesterase 1. Conclusions Microdissection and tandem mass spectrometry could be used to investigate the proteome of isolated glomeruli from FFPE tissue. Glomerular filtration barrier proteins had reduced abundance in the non-clipped kidney of 2K1C hypertensive rats. PMID:25129444

  13. Detection of equine arteritis virus by two chromogenic RNA in situ hybridization assays (conventional and RNAscope(®)) and assessment of their performance in tissues from aborted equine fetuses.

    PubMed

    Carossino, Mariano; Loynachan, Alan T; James MacLachlan, N; Drew, Clifton; Shuck, Kathleen M; Timoney, Peter J; Del Piero, Fabio; Balasuriya, Udeni B R

    2016-11-01

    Equine arteritis virus (EAV) is the causative agent of equine viral arteritis, a respiratory and reproductive disease of equids. EAV infection can induce abortion in pregnant mares, fulminant bronchointerstitial pneumonia in foals, and persistent infection in stallions. Here, we developed two RNA in situ hybridization (ISH) assays (conventional and RNAscope(®) ISH) for the detection of viral RNA in formalin-fixed paraffin-embedded (FFPE) tissues and evaluated and compared their performance with nucleocapsid-specific immunohistochemistry (IHC) and virus isolation (VI; gold standard) techniques. The distribution and cellular localization of EAV RNA and antigen were similar in tissues from aborted equine fetuses. Evaluation of 80 FFPE tissues collected from 16 aborted fetuses showed that the conventional RNA ISH assay had a significantly lower sensitivity than the RNAscope(®) and IHC assays, whereas there was no difference between the latter two assays. The use of oligonucleotide probes along with a signal amplification system (RNAscope(®)) can enhance detection of EAV RNA in FFPE tissues, with sensitivity comparable to that of IHC. Most importantly, these assays provide important tools with which to investigate the mechanisms of EAV pathogenesis. PMID:27541817

  14. The storage period of the formalin-fixed paraffin-embedded tumor blocks does not influence the concentration and purity of the isolated DNA in a series of 83 renal and thyroid carcinomas.

    PubMed

    Nechifor-Boilă, Adela Corina; Loghin, Andrada; Vacariu, Victor; Halaţiu, Vasile Bogdan; Borda, Angela

    2015-01-01

    Optimal recovery of nucleic acids from formalin-fixed paraffin-embedded (FFPE) tissues is highly dependent on a series of pre-extraction steps, mainly related (but not limited) to fixation. The aim of our study was to investigate if the storage period of the FFPE blocks had a significant effect on the isolated DNA. We examined the quantity and purity of the isolated DNA from 83 FFPE blocks, corresponding to malignant thyroid (n=28) and renal (n=55) carcinomas that had been stored in our department for up to eight years. The DNA extraction protocol was based on a precipitation method (MasterPure™ DNA Purification Kit, Epicentre), in accordance to the manufacturer instructions, optimized in our laboratory. A spectrophotometer was used to determine the yield (A260) and purity (A260/A280 ratio) of the isolated DNA. We successfully isolated good DNA quantity and purity from all our study cases (mean concentration: 223.4 ± 104.16 ng/μL; mean A260/A280 ratio: 1.68 ± 0.09). Moreover, no statistically significant differences were observed between tumor blocks stored for 2-3 years and 7-8 years, respectively, both in terms of DNA quantity (p=0.196) and purity (p=0.663). In conclusion, we successfully validated an efficient, reproducible DNA extraction technique that provided a good range of DNA concentrations and purity, regardless the type of tissue (thyroid or kidney). Moreover, we demonstrated that the storage period of the FFPE blocks does not have a significant influence on the DNA quantity and purity.

  15. The storage period of the formalin-fixed paraffin-embedded tumor blocks does not influence the concentration and purity of the isolated DNA in a series of 83 renal and thyroid carcinomas.

    PubMed

    Nechifor-Boilă, Adela Corina; Loghin, Andrada; Vacariu, Victor; Halaţiu, Vasile Bogdan; Borda, Angela

    2015-01-01

    Optimal recovery of nucleic acids from formalin-fixed paraffin-embedded (FFPE) tissues is highly dependent on a series of pre-extraction steps, mainly related (but not limited) to fixation. The aim of our study was to investigate if the storage period of the FFPE blocks had a significant effect on the isolated DNA. We examined the quantity and purity of the isolated DNA from 83 FFPE blocks, corresponding to malignant thyroid (n=28) and renal (n=55) carcinomas that had been stored in our department for up to eight years. The DNA extraction protocol was based on a precipitation method (MasterPure™ DNA Purification Kit, Epicentre), in accordance to the manufacturer instructions, optimized in our laboratory. A spectrophotometer was used to determine the yield (A260) and purity (A260/A280 ratio) of the isolated DNA. We successfully isolated good DNA quantity and purity from all our study cases (mean concentration: 223.4 ± 104.16 ng/μL; mean A260/A280 ratio: 1.68 ± 0.09). Moreover, no statistically significant differences were observed between tumor blocks stored for 2-3 years and 7-8 years, respectively, both in terms of DNA quantity (p=0.196) and purity (p=0.663). In conclusion, we successfully validated an efficient, reproducible DNA extraction technique that provided a good range of DNA concentrations and purity, regardless the type of tissue (thyroid or kidney). Moreover, we demonstrated that the storage period of the FFPE blocks does not have a significant influence on the DNA quantity and purity. PMID:26429169

  16. [Detection of mixed lymphoid chimerism after allogeneic bone marrow transplantation: demonstration by interphase cytogenetics in paraffin-embedded tissue].

    PubMed

    Friedrich, T; Ott, G; Kalla, J; Helbig, W; Schwenke, H; Kubel, M; Pönisch, W; Feyer, P; Friedrich, A

    1994-01-01

    In bone marrow transplantation (BMT) the detection of residual host lymphoid or haematopoietic cells surviving conditioning therapy is because of its association to graft-versus-host disease, graft-versus-leukemia reaction, and relapse of leukemia a matter of great interest. We studied the occurrence of this mixed lymphoid chimerism (MC) in the formol-fixed lymphatic tissue of lymph nodes and spleen from 21 autopsies after allogeneic sex-mismatched BMT (5 females, 16 males, survival 5 to 1140 days after BMT). In situ hybridisation with biotinylated centromer-specific anti-X- and anti-Y-chromosome probes was performed on pepsin-digested paraffin sections. The number of double X-, single X-, and Y-chromosome bearing cells was analysed microscopically. Because of artefacts only 14 cases remained for valid investigation. MC was detected in 6 cases (5 out of 11 males 5 days to 840 days and 1 out of 3 females 76 days after BMT). MC occurred after whole body irradiation with 10 Gy (n = 5) and 7 Gy (n = 1). In 1 autopsy relapse of leukemia caused host cell infiltration. Cases with MC did not express histological signs of acute or chronic graft-versus-host disease, but 5 out of 8 with complete lymphoid chimerism did. The sensitivity of interphase cytogenetics on paraffin embedded tissue is low.

  17. Velocity and attenuation of shear waves in the phantom of a muscle-soft tissue matrix with embedded stretched fibers

    NASA Astrophysics Data System (ADS)

    Rudenko, O. V.; Tsyuryupa, S. N.; Sarvazyan, A. P.

    2016-09-01

    We develop a theory of the elasticity moduli and dissipative properties of a composite material: a phantom simulating muscle tissue anisotropy. The model used in the experiments was made of a waterlike polymer with embedded elastic filaments imitating muscle fiber. In contrast to the earlier developed phenomenological theory of the anisotropic properties of muscle tissue, here we obtain the relationship of the moduli with characteristic sizes and moduli making up the composite. We introduce the effective elasticity moduli and viscosity tensor components, which depend on stretching of the fibers. We measure the propagation velocity of shear waves and the shear viscosity of the model for regulated tension. Waves were excited by pulsed radiation pressure generated by modulated focused ultrasound. We show that with increased stretching of fibers imitating muscle contraction, an increase in both elasticity and viscosity takes place, and this effect depends on the wave propagation direction. The results of theoretical and experimental studies support our hypothesis on the protective function of stretched skeletal muscle, which protects bones and joints from trauma.

  18. Meniscus Tissue Engineering Using a Novel Combination of Electrospun Scaffolds and Human Meniscus Cells Embedded within an Extracellular Matrix Hydrogel

    PubMed Central

    Baek, Jihye; Chen, Xian; Sovani, Sujata; Jin, Sungho; Grogan, Shawn P; D’Lima, Darryl D

    2015-01-01

    Meniscus injury and degeneration have been linked to the development of secondary osteoarthritis (OA). Therapies that successfully repair or replace the meniscus are therefore likely to prevent or delay OA progression. We investigated the novel approach of building layers of aligned polylactic acid (PLA) electrospun (ES) scaffolds with human meniscus cells embedded in extracellular matrix (ECM) hydrogel to lead to formation of neotissues that resemble meniscus-like tissue. PLA ES scaffolds with randomly oriented or aligned fibers were seeded with human meniscus cells derived from vascular or avascular regions. Cell viability, cell morphology, and gene expression profiles were monitored via confocal microscopy, scanning electron microscopy (SEM), and real-time PCR, respectively. Seeded scaffolds were used to produce multilayered constructs and were examined via histology and immunohistochemistry. Morphology and mechanical properties of PLA scaffolds (with and without cells) were influenced by fiber direction of the scaffolds. Both PLA scaffolds supported meniscus tissue formation with increased COL1A1, SOX9, COMP, yet no difference in gene expression was found between random and aligned PLA scaffolds. Overall, ES materials, which possess mechanical strength of meniscus and can support neotissue formation, show potential for use in cell-based meniscus regeneration strategies. PMID:25640671

  19. Imaging through diffusive layers using speckle pattern fractal analysis and application to embedded object detection in tissues

    NASA Astrophysics Data System (ADS)

    Tremberger, George, Jr.; Flamholz, A.; Cheung, E.; Sullivan, R.; Subramaniam, R.; Schneider, P.; Brathwaite, G.; Boteju, J.; Marchese, P.; Lieberman, D.; Cheung, T.; Holden, Todd

    2007-09-01

    The absorption effect of the back surface boundary of a diffuse layer was studied via laser generated reflection speckle pattern. The spatial speckle intensity provided by a laser beam was measured. The speckle data were analyzed in terms of fractal dimension (computed by NIH ImageJ software via the box counting fractal method) and weak localization theory based on Mie scattering. Bar code imaging was modeled as binary absorption contrast and scanning resolution in millimeter range was achieved for diffusive layers up to thirty transport mean free path thick. Samples included alumina, porous glass and chicken tissue. Computer simulation was used to study the effect of speckle spatial distribution and observed fractal dimension differences were ascribed to variance controlled speckle sizes. Fractal dimension suppressions were observed in samples that had thickness dimensions around ten transport mean free path. Computer simulation suggested a maximum fractal dimension of about 2 and that subtracting information could lower fractal dimension. The fractal dimension was shown to be sensitive to sample thickness up to about fifteen transport mean free paths, and embedded objects which modified 20% or more of the effective thickness was shown to be detectable. The box counting fractal method was supplemented with the Higuchi data series fractal method and application to architectural distortion mammograms was demonstrated. The use of fractals in diffusive analysis would provide a simple language for a dialog between optics experts and mammography radiologists, facilitating the applications of laser diagnostics in tissues.

  20. Helicobacter pylori genotyping in gastric adenocarcinoma and MALT lymphoma by multiplex PCR analyses of paraffin wax embedded tissues

    PubMed Central

    Koehler, C I; Mues, M B; Dienes, H P; Kriegsmann, J; Schirmacher, P; Odenthal, M

    2003-01-01

    Background: Gastric infection with Helicobacter pylori is the major cause of chronic active gastritis and is associated with the pathogenesis of peptic ulcer and gastric carcinoma. Gastric mucosal damage involves both host and H pylori dependent factors, such as the presence of the cag pathogenicity island and allelic variations of the vacA and iceA genes. Aims: To evaluate the association of these virulence factors with the development of gastric malignancies, a retrospective study was performed on archived tissue routinely obtained for diagnostic histopathology. Methods: DNA was extracted from formalin fixed, paraffin wax embedded gastric tissue sections of 93 patients with chronic active gastritis (n = 39), adenocarcinoma (n = 28), or mucosa associated lymphoid tissue (MALT) lymphoma (n = 24). The extracted DNA was used to perform a polymerase chain reaction based, simultaneous analysis of the following: (1) cagA status, (2) allelic variation of the iceA genes (iceA1, iceA2), allelic variation of the signal peptide (s1a, s1b, s2) and the midregion (m1, m1a, m2) of the vacA gene. Results: The iceA1 gene showed a 3.6 fold and the vacA s1a variant a 4.2 fold higher prevalence in gastric adenocarcinoma than in gastritis. The combined presence of both the vacA s1a and iceA1 genes had a 5.6 fold higher frequency in adenocarcinoma. The vacA m2 allele was the predominant subtype in MALT lymphoma and the combination of the vacA m2 subtypes with the vacA s1 and the iceA1 variants occurred in MALT lymphoma nearly five times more often than in chronic active gastritis. Conclusions: Certain H pylori subtype combinations possess a differentiating and predictive value for the development of gastric adenocarcinoma and MALT lymphoma. PMID:12560462

  1. Fabrication and characterization of a multilayered optical tissue model with embedded scattering microspheres in polymeric materials.

    PubMed

    Chang, Robert C; Johnson, Peter; Stafford, Christopher M; Hwang, Jeeseong

    2012-06-01

    We report on a novel fabrication approach to build multilayered optical tissue phantoms that serve as independently validated test targets for axial resolution and contrast in scattering measurements by depth-resolving optical coherent tomography (OCT) with general applicability to a variety of three-dimensional optical sectioning platforms. We implement a combinatorial bottom-up approach to prepare monolayers of light-scattering microspheres with interspersed layers of transparent polymer. A dense monolayer assembly of monodispersed microspheres is achieved via a combined methodology of polyelectrolyte multilayers (PEMs) for particle-substrate binding and convective particle flux for two-dimensional crystal array formation on a glass substrate. Modifications of key parameters in the layer-by-layer polyelectrolyte deposition approach are applied to optimize particle monolayer transfer from a glass substrate into an elastomer while preserving the relative axial positioning in the particle monolayer. Varying the dimensions of the scattering microspheres and the thickness of the intervening transparent polymer layers enables different spatial frequencies to be realized in the transverse dimension of the solid phantoms. Step-wise determination of the phantom dimensions is performed independently of the optical system under test to enable precise spatial calibration, independent validation, and quantitative dimensional measurements.

  2. Testing an aflatoxin B1 gene signature in rat archival tissues.

    PubMed

    Merrick, B Alex; Auerbach, Scott S; Stockton, Patricia S; Foley, Julie F; Malarkey, David E; Sills, Robert C; Irwin, Richard D; Tice, Raymond R

    2012-05-21

    Archival tissues from laboratory studies represent a unique opportunity to explore the relationship between genomic changes and agent-induced disease. In this study, we evaluated the applicability of qPCR for detecting genomic changes in formalin-fixed, paraffin-embedded (FFPE) tissues by determining if a subset of 14 genes from a 90-gene signature derived from microarray data and associated with eventual tumor development could be detected in archival liver, kidney, and lung of rats exposed to aflatoxin B1 (AFB1) for 90 days in feed at 1 ppm. These tissues originated from the same rats used in the microarray study. The 14 genes evaluated were Adam8, Cdh13, Ddit4l, Mybl2, Akr7a3, Akr7a2, Fhit, Wwox, Abcb1b, Abcc3, Cxcl1, Gsta5, Grin2c, and the C8orf46 homologue. The qPCR FFPE liver results were compared to the original liver microarray data and to qPCR results using RNA from fresh frozen liver. Archival liver paraffin blocks yielded 30 to 50 μg of degraded RNA that ranged in size from 0.1 to 4 kB. qPCR results from FFPE and fresh frozen liver samples were positively correlated (p ≤ 0.05) by regression analysis and showed good agreement in direction and proportion of change with microarray data for 11 of 14 genes. All 14 transcripts could be amplified from FFPE kidney RNA except the glutamate receptor gene Grin2c; however, only Abcb1b was significantly upregulated from control. Abundant constitutive transcripts, S18 and β-actin, could be amplified from lung FFPE samples, but the narrow RNA size range (25-500 bp length) prevented consistent detection of target transcripts. Overall, a discrete gene signature derived from prior transcript profiling and representing cell cycle progression, DNA damage response, and xenosensor and detoxication pathways was successfully applied to archival liver and kidney by qPCR and indicated that gene expression changes in response to subchronic AFB1 exposure occurred predominantly in the liver, the primary target for AFB1-induced

  3. Desktop transcriptome sequencing from archival tissue to identify clinically relevant translocations.

    PubMed

    Sweeney, Robert T; Zhang, Bing; Zhu, Shirley X; Varma, Sushama; Smith, Kevin S; Montgomery, Stephen B; van de Rijn, Matt; Zehnder, Jim; West, Robert B

    2013-06-01

    Somatic mutations, often translocations or single nucleotide variations, are pathognomonic for certain types of cancers and are increasingly of clinical importance for diagnosis and prediction of response to therapy. Conventional clinical assays only evaluate 1 mutation at a time, and targeted tests are often constrained to identify only the most common mutations. Genome-wide or transcriptome-wide high-throughput sequencing (HTS) of clinical samples offers an opportunity to evaluate for all clinically significant mutations with a single test. Recently a "desktop version" of HTS has become available, but most of the experience to date is based on data obtained from high-quality DNA from frozen specimens. In this study, we demonstrate, as a proof of principle, that translocations in sarcomas can be diagnosed from formalin-fixed paraffin-embedded (FFPE) tissue with desktop HTS. Using the first generation MiSeq platform, full transcriptome sequencing was performed on FFPE material from archival blocks of 3 synovial sarcomas, 3 myxoid liposarcomas, 2 Ewing sarcomas, and 1 clear cell sarcoma. Mapping the reads to the "sarcomatome" (all known 83 genes involved in translocations and mutations in sarcoma) and using a novel algorithm for ranking fusion candidates, the pathognomonic fusions and the exact breakpoints were identified in all cases of synovial sarcoma, myxoid liposarcoma, and clear cell sarcoma. The Ewing sarcoma fusion gene was detectable in FFPE material only with a sequencing platform that generates greater sequencing depth. The results show that a single transcriptome HTS assay, from FFPE, has the potential to replace conventional molecular diagnostic techniques for the evaluation of clinically relevant mutations in cancer.

  4. Evaluation of the value of frozen tissue section used as "gold standard" for immunohistochemistry.

    PubMed

    Shi, Shan-Rong; Liu, Cheng; Pootrakul, Llana; Tang, Laurie; Young, Andrew; Chen, Ryan; Cote, Richard J; Taylor, Clive R

    2008-03-01

    To examine the use of acetone- or ethanol-fixed frozen tissue sections as the "gold standard" for immunohistochemical analysis, we evaluated frozen sections with various conditions of fixation and antigen retrieval (AR). Fresh human tissues were frozen in OCT. An adjacent tissue block was fixed in 10% neutral buffered formalin (NBF) and paraffin embedded (FFPE). Frozen sections were fixed by 6 protocols: acetone, ethanol, NBF (2 durations), and NBF + calcium chloride (2 durations). AR was used for all NBF-fixed sections. More than half of the antibodies (16/26 [62%]) showed immunohistochemical results indistinguishable between acetone- and NBF-fixed sections; 8 (31%) showed better immunohistochemical signals following NBF and AR; 2 gave better immunohistochemical results for acetone-fixed sections. Most cytoplasmic proteins (10/13) showed comparable immunohistochemical signals between acetone- and NBF-fixed sections. For nuclear proteins, NBF-fixed sections gave better immunohistochemical signals than did acetone-fixed sections. In most cases, NBF yielded stronger signals with less background and better morphology. The data do not support the use of acetone-fixed frozen tissue sections as the gold standard for immunohistochemical analysis. In evaluating new antibodies, a combination of acetone- and NBF-fixed frozen sections should be used, although in practice, FFPE tissue sections may serve as the standard for most antigens for immunohistochemical analysis.

  5. Identification of 5-Hydroxytryptamine-Producing Cells by Detection of Fluorescence in Paraffin-Embedded Tissue Sections

    PubMed Central

    Kaneko, Y.; Onda, N.; Watanabe, Y.; Shibutani, M.

    2016-01-01

    5-Hydroxytryptamine (5-HT) produced by enterochromaffin (EC) cells is an important enteric mucosal signaling ligand and has been implicated in several gastrointestinal diseases, including inflammatory bowel disease and functional disorders such as irritable bowel syndrome. The present study reports a new, simple and rapid visualization method of 5-HT-producing EC cells utilizing detection of fluorescence in paraffin-embedded tissue sections after formalin fixation. In human samples, there was a high incidence of fluorescence+ cells in the 5-HT+ cells in the pyloric, small intestinal and colonic glands, while co-localization was lacking between fluorescence+ and gastrin+ cells in the pyloric and small intestinal glands. Fluorescence+ EC cells were detected in the colon of mice and rats. Fluorescence+ cells were also observed in 5-HT+ β cells in the pancreatic islets of Langerhans in pregnant mice, while non-pregnant mouse pancreatic islet cells showed no 5-HT immunoreactivity or fluorescence. These results suggest that fluorescence+ cells are identical to 5-HT+ cells, and the source of fluorescence may be 5-HT itself or molecules related to its synthesis or degradation. This fluorescence signal detection method may be applicable for monitoring of inflammatory status of inflammatory bowel diseases in both the experimental and clinical settings. PMID:27734992

  6. Comparison of the DNA extraction methods for polymerase chain reaction amplification from formalin-fixed and paraffin-embedded tissues.

    PubMed

    Sato, Y; Sugie, R; Tsuchiya, B; Kameya, T; Natori, M; Mukai, K

    2001-12-01

    To obtain an adequate quality and quantity of DNA from formalin-fixed and paraffin-embedded tissue, six different DNA extraction methods were compared. Four methods used deparaffinization by xylene followed by proteinase K digestion and phenol-chloroform extraction. The temperature of the different steps was changed to obtain higher yields and improved quality of extracted DNA. The remaining two methods used microwave heating for deparaffinization. The best DNA extraction method consisted of deparaffinization by microwave irradiation, protein digestion with proteinase K at 48 degrees C overnight, and no further purification steps. By this method, the highest DNA yield was obtained and the amplification of a 989-base pair beta-globin gene fragment was achieved. Furthermore, DNA extracted by means of this procedure from five gastric carcinomas was successfully used for single strand conformation polymorphism and direct sequencing assays of the beta-catenin gene. Because the microwave-based DNA extraction method presented here is simple, has a lower contamination risk, and results in a higher yield of DNA compared with the ordinary organic chemical reagent-based extraction method, it is considered applicable to various clinical and basic fields.

  7. Gene fusion detection in formalin-fixed paraffin-embedded benign fibrous histiocytomas using fluorescence in situ hybridization and RNA sequencing.

    PubMed

    Walther, Charles; Hofvander, Jakob; Nilsson, Jenny; Magnusson, Linda; Domanski, Henryk A; Gisselsson, David; Tayebwa, Johnbosco; Doyle, Leona A; Fletcher, Christopher D M; Mertens, Fredrik

    2015-09-01

    Benign fibrous histiocytomas (FH) can be subdivided into several morphological and clinical subgroups. Recently, gene fusions involving either one of two protein kinase C genes (PRKCB and PRKCD) or the ALK gene were described in FH. We here wanted to evaluate the frequency of PRKCB and PRKCD gene fusions in FH. Using interphase fluorescence in situ hybridization on sections from formalin-fixed paraffin-embedded (FFPE) tumors, 36 cases could be analyzed. PRKCB or PRKCD rearrangements were seen in five tumors: 1/7 regular, 0/3 aneurysmal, 0/6 cellular, 2/7 epithelioid, 0/1 atypical, 2/10 deep, and 0/2 metastatic lesions. We also evaluated the status of the ALK gene in selected cases, finding rearrangements in 3/7 epithelioid and 0/1 atypical lesions. To assess the gene fusion status of FH further, deep sequencing of RNA (RNA-Seq) was performed on FFPE tissue from eight cases with unknown gene fusion status, as well as on two FH and six soft tissue sarcomas with known gene fusions; of the latter eight positive controls, the expected fusion transcript was found in all but one, while 2/8 FH with unknown genetic status showed fusion transcripts, including a novel KIRREL/PRKCA chimera. Thus, also a third member of the PRKC family is involved in FH tumorigenesis. We conclude that gene fusions involving PRKC genes occur in several morphological (regular, cellular, aneurysmal, epithelioid) and clinical (cutaneous, deep) subsets of FH, but they seem to account for only a minority of the cases. In epithelioid lesions, however, rearrangements of PRKC or ALK were seen, as mutually exclusive events, in the majority (5/7) of cases. Finally, the study also shows that RNA-Seq is a promising tool for identifying gene fusions in FFPE tissues.

  8. Gene fusion detection in formalin-fixed paraffin-embedded benign fibrous histiocytomas using fluorescence in situ hybridization and RNA sequencing.

    PubMed

    Walther, Charles; Hofvander, Jakob; Nilsson, Jenny; Magnusson, Linda; Domanski, Henryk A; Gisselsson, David; Tayebwa, Johnbosco; Doyle, Leona A; Fletcher, Christopher D M; Mertens, Fredrik

    2015-09-01

    Benign fibrous histiocytomas (FH) can be subdivided into several morphological and clinical subgroups. Recently, gene fusions involving either one of two protein kinase C genes (PRKCB and PRKCD) or the ALK gene were described in FH. We here wanted to evaluate the frequency of PRKCB and PRKCD gene fusions in FH. Using interphase fluorescence in situ hybridization on sections from formalin-fixed paraffin-embedded (FFPE) tumors, 36 cases could be analyzed. PRKCB or PRKCD rearrangements were seen in five tumors: 1/7 regular, 0/3 aneurysmal, 0/6 cellular, 2/7 epithelioid, 0/1 atypical, 2/10 deep, and 0/2 metastatic lesions. We also evaluated the status of the ALK gene in selected cases, finding rearrangements in 3/7 epithelioid and 0/1 atypical lesions. To assess the gene fusion status of FH further, deep sequencing of RNA (RNA-Seq) was performed on FFPE tissue from eight cases with unknown gene fusion status, as well as on two FH and six soft tissue sarcomas with known gene fusions; of the latter eight positive controls, the expected fusion transcript was found in all but one, while 2/8 FH with unknown genetic status showed fusion transcripts, including a novel KIRREL/PRKCA chimera. Thus, also a third member of the PRKC family is involved in FH tumorigenesis. We conclude that gene fusions involving PRKC genes occur in several morphological (regular, cellular, aneurysmal, epithelioid) and clinical (cutaneous, deep) subsets of FH, but they seem to account for only a minority of the cases. In epithelioid lesions, however, rearrangements of PRKC or ALK were seen, as mutually exclusive events, in the majority (5/7) of cases. Finally, the study also shows that RNA-Seq is a promising tool for identifying gene fusions in FFPE tissues. PMID:26121314

  9. Real-time PCR analysis of RNA extracted from formalin-fixed and paraffin-embeded tissues: effects of the fixation on outcome reliability.

    PubMed

    Castiglione, Francesca; Rossi Degl'Innocenti, Duccio; Taddei, Antonio; Garbini, Francesca; Buccoliero, Anna Maria; Raspollini, Maria Rosaria; Pepi, Monica; Paglierani, Milena; Asirelli, Grazia; Freschi, Giancarlo; Bechi, Paolo; Taddei, Gian Luigi

    2007-09-01

    In many pathologic circumstances, quantitative mRNA expression levels are important for evaluation of possible genome mutations. The development of real-time polymerase chain reaction (RT-PCR) technology has facilitated the realization of nucleic acid quantification. Potentially, quantitative PCR offers a number of advantages over traditional methods because it permits the use of small amounts of genetic material. In the present study, we optimize a RNA purification technique on specimens that are formalin-fixed, paraffin-embedded and we examine prolonged formalin fixation effects on quantitative RT-PCR analysis. We compared RNA levels with 70 colic mucosa samples using the cyclooxygenase 2 gene as marker. The difference in amplification successes between formalin-fixed tissues and formalin-fixed, paraffin-embedded tissues was not statistically significant. Moreover, we compared the expression of formalin-fixed samples with the expression of each fresh tissue. Wilcoxon Mann-Whitney test shows that only the difference in the expression levels of 1- or 3-hour formalin-fixed samples is not statistically significant with respect to other fixation times. We found that the mRNA can be reliably extracted from formalin fixed, paraffin-embedded tissue sections but that prolonged formalin fixation produces different results in quantitative RT-PCR. It can be related to difference in RNA sequences length and the generation of secondary structures that are more susceptible to the prolonged formalin fixation. We suppose that the paraffin do not influence the RNA extraction yield because there are no statistical significant differences between amplification success of formalin-fixed tissues and paraffin-embedded tissues. Therefore, in relative expression quantization, we confirm that it is appropriate to use specimens with same protocols and time for formalin fixation.

  10. Application of in-situ hybridization for the detection and identification of avian malaria parasites in paraffin wax-embedded tissues from captive penguins

    PubMed Central

    Dinhopl, Nora; Mostegl, Meike M.; Richter, Barbara; Nedorost, Nora; Maderner, Anton; Fragner, Karin; Weissenböck, Herbert

    2011-01-01

    In captive penguins, avian malaria due to Plasmodium parasites is a well-recognized disease problem as these protozoa may cause severe losses among valuable collections of zoo birds. In blood films from naturally infected birds, identification and differentiation of malaria parasites based on morphological criteria are difficult because parasitaemia is frequently light and blood stages, which are necessary for identification of parasites, are often absent. Post-mortem diagnosis by histological examination of tissue samples is sometimes inconclusive due to the difficulties in differentiating protozoal tissue stages from fragmented nuclei in necrotic tissue. The diagnosis of avian malaria would be facilitated by a technique with the ability to specifically identify developmental stages of Plasmodium in tissue samples. Thus, a chromogenic in-situ hybridization (ISH) procedure with a digoxigenin-labelled probe, targeting a fragment of the 18S rRNA, was developed for the detection of Plasmodium parasites in paraffin wax-embedded tissues. This method was validated in comparison with traditional techniques (histology, polymerase chain reaction), on various tissues from 48 captive penguins that died at the zoological garden Schönbrunn, Vienna, Austria. Meronts of Plasmodium gave clear signals and were easily identified using ISH. Potential cross-reactivity of the probe was ruled out by the negative outcome of the ISH against a number of protozoa and fungi. Thus, ISH proved to be a powerful, specific and sensitive tool for unambiguous detection of Plasmodium parasites in paraffin wax-embedded tissue samples. PMID:21711191

  11. Application of in-situ hybridization for the detection and identification of avian malaria parasites in paraffin wax-embedded tissues from captive penguins.

    PubMed

    Dinhopl, Nora; Mostegl, Meike M; Richter, Barbara; Nedorost, Nora; Maderner, Anton; Fragner, Karin; Weissenböck, Herbert

    2011-06-01

    In captive penguins, avian malaria due to Plasmodium parasites is a well-recognized disease problem as these protozoa may cause severe losses among valuable collections of zoo birds. In blood films from naturally infected birds, identification and differentiation of malaria parasites based on morphological criteria are difficult because parasitaemia is frequently light and blood stages, which are necessary for identification of parasites, are often absent. Post-mortem diagnosis by histological examination of tissue samples is sometimes inconclusive due to the difficulties in differentiating protozoal tissue stages from fragmented nuclei in necrotic tissue. The diagnosis of avian malaria would be facilitated by a technique with the ability to specifically identify developmental stages of Plasmodium in tissue samples. Thus, a chromogenic in-situ hybridization (ISH) procedure with a digoxigenin-labelled probe, targeting a fragment of the 18S rRNA, was developed for the detection of Plasmodium parasites in paraffin wax-embedded tissues. This method was validated in comparison with traditional techniques (histology, polymerase chain reaction), on various tissues from 48 captive penguins that died at the zoological garden Schönbrunn, Vienna, Austria. Meronts of Plasmodium gave clear signals and were easily identified using ISH. Potential cross-reactivity of the probe was ruled out by the negative outcome of the ISH against a number of protozoa and fungi. Thus, ISH proved to be a powerful, specific and sensitive tool for unambiguous detection of Plasmodium parasites in paraffin wax-embedded tissue samples.

  12. Dermatofibrosarcoma protuberans: dealing with slow Mohs procedures employing formalin-fixed, paraffin wax-embedded tissue in a busy diagnostic laboratory.

    PubMed

    Orchard, G E; Shams, M

    2012-01-01

    Dermatofibrosarcoma protuberans (DFSP) is a relatively uncommon tumour that arises in the dermis and underlying soft tissue. Surgical removal is the preferred treatment, with relatively wide clearance margins of 3 cm or more. Slow Mohs procedures are often employed successfully to treat patients with such tumours. Slow Mohs procedures offer the benefit of improved cure rates and maximal tissue conservation. However, dealing with such tissue successfully presents the laboratory with a host of technical problems. This report advocates a set protocol to follow for slow Mohs, based on the experience acquired from dealing with 37 cases of DFSP over a 12-year period. The report establishes the benefits of slow Mohs paraffin wax-embedded tissue over frozen sections in terms of improved morphology, tissue preservation and immunocytochemical labelling with anti-CD34.

  13. Raman nanoparticle probes for antibody-based protein detection in tissues.

    PubMed

    Lutz, Barry; Dentinger, Claire; Sun, Lei; Nguyen, Lienchi; Zhang, Jingwu; Chmura, Aj; Allen, April; Chan, Selena; Knudsen, Beatrice

    2008-04-01

    Surface-enhanced Raman scattering (SERS) nanoparticles are emerging as a new approach for optical detection of biomolecules. In a model assay in formalin-fixed paraffin-embedded (FFPE) prostate tissue sections, we detect prostate-specific antigen (PSA) using antibody (Ab) conjugated to composite organic-inorganic nanoparticles (COINs), and we use identical staining protocols to compare COIN-Ab and Alexa-Ab conjugates in adjacent tissue sections. Spectral analysis illustrates the fundamental difference between fluorescence and Raman signatures and accurately extracts COIN probe signals from background autofluorescence. Probe signals are used to generate images of PSA expression on the tissue, and quality measures are presented to characterize the performance of the COIN assay in comparison to Alexa. Staining accuracy (ability to correctly identify PSA expression in epithelial cells) is somewhat less for COIN than Alexa, which is attributed to an elevated false negative rate of the COIN. However, COIN provided signal intensities comparable to Alexa, and good intra-, inter-, and lot-to-lot consistencies. Overall, COIN and Alexa detection reagents possess similar performance with FFPE tissues, supporting the further development of Raman probes for this application. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

  14. Comparison of protocols for DNA extraction from long-term preserved formalin fixed tissues.

    PubMed

    Paireder, Stefan; Werner, Bettina; Bailer, Josef; Werther, Wolfgang; Schmid, Erich; Patzak, Beatrix; Cichna-Markl, Margit

    2013-08-15

    The current study compared the applicability of protocols to extract DNA from formalin fixed heart tissues that have been preserved for more than 50 years. Ten methods were tested: a cetyltrimethylammonium bromide (CTAB) standard protocol, seven variants of this standard protocol, and two commercial kits. In the case of younger specimens (fixed in 1951, 1934, or 1914), extracts with DNA concentrations ≥ 10.0 ng/μl were obtained with the standard CTAB protocol, two variants of the standard protocol including prolonged tissue digestion (72 h instead of 1-2h), and a commercial kit particularly recommended for DNA extraction from formalin fixed paraffin embedded tissues (FFPE Kit). With the FFPE Kit, DNA could also be extracted from older tissues (fixed in 1893, 1850/1851, or before 1820). In general, the purity of the DNA extracts, assessed from the ratio of the absorbance at 260 and 280 nm, was not very high. In spite of their rather low purity, the DNA extracts could, however, be used to amplify a 122-bp sequence and, in most cases, also a 171-bp sequence of the gene coding for human albumin by the polymerase chain reaction (PCR).

  15. Use of softening agents to improve the production of formalin-fixed, paraffin-embedded sections of nail tissue: an assessment.

    PubMed

    Orchard, G E; Torres, J; Sounthararajah, R

    2008-01-01

    The use of tissue softeners to enhance the quality of tissue sections of heavily keratotic tissue is not widely published. There are very few indicators in the scientific literature that attempt to compare and contrast the benefits and disadvantages of such techniques, as most are passed down through word of mouth rather than through published data. This study attempts to present a preliminary evaluation of several methods employing tissue softeners to facilitate the preparation of reproducible, good-quality formalin-fixed, paraffin-embedded sections of nail tissue. A standard 10-minute surface application of each softener is employed for all paraffin-embedded tissue in order to ensure consistency. The results show that the use of Veet (hair remover), Fairy Liquid or fabric conditioner provides the most beneficial results. Thus, widely available products can be used in preference to specific commercially produced reagents that have no clear benefits and can cost considerably more to purchase. This study will form the basis of a more in-depth evaluation of the most beneficial softeners, in an attempt to determine optimal parameters for their use in routine histopathology laboratories. PMID:19055107

  16. Expression analysis of mRNA in formalin-fixed, paraffin-embedded archival tissues by mRNA in situ hybridization.

    PubMed

    Henke, Ralf T; Eun Kim, Sung; Maitra, Anirban; Paik, Soonmyung; Wellstein, Anton

    2006-04-01

    Gene expression in diseased tissues can indicate the contribution to a disease process and potentially guide therapeutic decision-making. Archival tissues with associated clinical outcome may be useful to discover or validate the role of a candidate gene in a disease process or the response to therapy. Such archival tissues are commonly formalin-fixed and paraffin-embedded, restricting the methods available for gene expression analysis. Obviously, the detection of proteins in tissues requires adaptation for each protein and the detection of secreted proteins can prove difficult or of reduced value since the protein detected may not reflect the total amount produced. Thus, we describe here a reliable method for the detection of mRNA in archival tissues. The method for mRNA in situ hybridization (ISH) was adapted by us for >15 different genes and applied to several hundred tissue microarrays (TMAs) and full sections generating >10,000 expression data points. We also discuss the utility of TMAs to simultaneously analyze several hundred tissue samples on one slide to minimize variability and preserve valuable tissue samples. Experimental protocols are provided that can be implemented without major hurdles in a typical molecular pathology laboratory and we discuss quantitative analysis as well as advantages and limitations of ISH with a special focus on secreted proteins. We conclude that ISH is a reliable and cost effective approach to gene expression analysis in archival tissues that is amenable to screening of series of tissues or of genes of interest.

  17. Identification of Bacteria in Formalin-Fixed, Paraffin-Embedded Heart Valve Tissue via 16S rRNA Gene Nucleotide Sequencing

    PubMed Central

    Imrit, Kavita; Goldfischer, Michael; Wang, Jie; Green, Jaime; Levine, Jerome; Lombardo, Joseph; Hong, Tao

    2006-01-01

    We applied 16S rRNA gene sequencing to identify bacterial species present in formalin-fixed, paraffin-embedded heart valve tissue. In 40% (12/30) of the cases, we were able to identify the bacterium to the species-genus level. For more recent cases (≤4 years), the success rate was significantly improved, to 70% (P < 0.001). PMID:16825394

  18. A single simple procedure for dewaxing, hydration and heat-induced epitope retrieval (HIER) for immunohistochemistry in formalin fixed paraffin-embedded tissue.

    PubMed

    Paulsen, I M S; Dimke, H; Frische, S

    2015-01-01

    Heat-induced epitope retrieval (HIER) is widely used for immunohistochemistry on formalin fixed paraffin-embedded tissue and includes temperatures well above the melting point of paraffin. We therefore tested whether traditional xylene-based removal of paraffin is required on sections from paraffin-embedded tissue, when HIER is performed by vigorous boiling in 10 mM Tris/0.5 mM EGTA-buffer (pH=9). Immunohistochemical results using HIER with or without prior dewaxing in xylene were evaluated using 7 primary antibodies targeting proteins located in the cytosol, intracellular vesicles and plasma membrane. No effect of omitting prior dewaxing was observed on staining pattern. Semiquantitative analysis did not show HIER to influence the intensity of labelling consistently. Consequently, quantification of immune labelling intensity using fluorescent secondary antibodies was performed at 5 dilutions of primary antibody with and without prior dewaxing in xylene. No effect of omitting prior dewaxing on signal intensity was detectable indicating similar immunoreactivity in dewaxed and non-dewaxed sections. The intensity of staining the nucleus with the DNA-stain ToPro3 was similarly unaffected by omission of dewaxing in xylene. In conclusion, the HIER procedure described and tested can be used as a single procedure enabling dewaxing, hydration and epitope retrieval for immunohistochemistry in formalin fixed paraffin-embedded tissue.

  19. A single simple procedure for dewaxing, hydration and heat-induced epitope retrieval (HIER) for immunohistochemistry in formalin fixed paraffin-embedded tissue.

    PubMed

    Paulsen, I M S; Dimke, H; Frische, S

    2015-01-01

    Heat-induced epitope retrieval (HIER) is widely used for immunohistochemistry on formalin fixed paraffin-embedded tissue and includes temperatures well above the melting point of paraffin. We therefore tested whether traditional xylene-based removal of paraffin is required on sections from paraffin-embedded tissue, when HIER is performed by vigorous boiling in 10 mM Tris/0.5 mM EGTA-buffer (pH=9). Immunohistochemical results using HIER with or without prior dewaxing in xylene were evaluated using 7 primary antibodies targeting proteins located in the cytosol, intracellular vesicles and plasma membrane. No effect of omitting prior dewaxing was observed on staining pattern. Semiquantitative analysis did not show HIER to influence the intensity of labelling consistently. Consequently, quantification of immune labelling intensity using fluorescent secondary antibodies was performed at 5 dilutions of primary antibody with and without prior dewaxing in xylene. No effect of omitting prior dewaxing on signal intensity was detectable indicating similar immunoreactivity in dewaxed and non-dewaxed sections. The intensity of staining the nucleus with the DNA-stain ToPro3 was similarly unaffected by omission of dewaxing in xylene. In conclusion, the HIER procedure described and tested can be used as a single procedure enabling dewaxing, hydration and epitope retrieval for immunohistochemistry in formalin fixed paraffin-embedded tissue. PMID:26708177

  20. A Single Simple Procedure for Dewaxing, Hydration and Heat-Induced Epitope Retrieval (HIER) for Immunohistochemistry in Formalin-Fixed Paraffin-Embedded Tissue

    PubMed Central

    Paulsen, I.M.S.; Dimke, H.

    2015-01-01

    Heat-induced epitope retrieval (HIER) is widely used for immunohistochemistry on formalin-fixed paraffin-embedded tissue and includes temperatures well above the melting point of paraffin. We therefore tested whether traditional xylene-based removal of paraffin is required on sections from paraffin-embedded tissue, when HIER is performed by vigorous boiling in 10 mM Tris/0.5 mM EGTA-buffer (pH=9). Immunohistochemical results using HIER with or without prior dewaxing in xylene were evaluated using 7 primary antibodies targeting proteins located in the cytosol, intracellular vesicles and plasma membrane. No effect of omitting prior dewaxing was observed on staining pattern. Semiquantitative analysis did not show HIER to influence the intensity of labelling consistently. Consequently, quantification of immune labelling intensity using fluorescent secondary antibodies was performed at 5 dilutions of primary antibody with and without prior dewaxing in xylene. No effect of omitting prior dewaxing on signal intensity was detectable indicating similar immunoreactivity in dewaxed and non-dewaxed sections. The intensity of staining the nucleus with the DNA-stain ToPro3 was similarly unaffected by omission of dewaxing in xylene. In conclusion, the HIER procedure described and tested can be used as a single procedure enabling dewaxing, hydration and epitope retrieval for immunohistochemistry in formalin-fixed paraffin-embedded tissue. PMID:26708177

  1. Detection of Tropical Fungi in Formalin-Fixed, Paraffin-Embedded Tissue: Still an Indication for Microscopy in Times of Sequence-Based Diagnosis?

    PubMed Central

    Frickmann, Hagen; Loderstaedt, Ulrike; Racz, Paul; Tenner-Racz, Klara; Eggert, Petra; Haeupler, Alexandra; Bialek, Ralf; Hagen, Ralf Matthias

    2015-01-01

    Introduction. The aim of the study was the evaluation of panfungal PCR protocols with subsequent sequence analysis for the diagnostic identification of invasive mycoses in formalin-fixed, paraffin-embedded tissue samples with rare tropical mycoses. Materials and Methods. Five different previously described panfungal PCR/sequencing protocols targeting 18S and 28S ribosomal RNA gene fragments as well as internal transcribed spacer 1 and 2 fragments were evaluated with a collection of 17 formalin-fixed, paraffin-embedded tissue samples of patients with rare and/or tropical invasive mycoses, comprising chromoblastomycosis, coccidioidomycosis, cryptococcosis, histoplasmosis, mucormycosis, mycetoma/maduromycosis, and rhinosporidiosis, in a proof-of-principle analysis. Results. The primers of the panfungal PCRs readily and predominantly reacted with contaminating environmental fungi that had deposited on the paraffin blocks. Altogether three sequence results of histoplasmosis and mycetoma samples that matched the histological assessment were associated with sample age <10 years and virtually without PCR inhibition. Conclusions. The high risk of amplifying environmental contaminants severely reduces the usefulness of the assessed panfungal PCR/sequencing protocols for the identification of rare and/or tropical mycoses in stored formalin-fixed, paraffin-embedded tissues. Histological assessment remains valuable for such indications if cultural differentiation is impossible from inactivated sample material. PMID:25961048

  2. In Situ Detection of Bacteria within Paraffin-embedded Tissues Using a Digoxin-labeled DNA Probe Targeting 16S rRNA.

    PubMed

    Choi, Yun Sik; Kim, Yong Cheol; Baek, Keum Jin; Choi, Youngnim

    2015-05-21

    The presence of bacteria within the pocket epithelium and underlying connective tissue in gingival biopsies from patients with periodontitis has been reported using various methods, including electron microscopy, immunohistochemistry or immunofluorescence using bacteria-specific antibodies, and fluorescent in situ hybridization (FISH) using a fluorescence-labeled oligonucleotide probe. Nevertheless, these methods are not widely used due to technical limitation or difficulties. Here a method to localize bacteria within paraffin-embedded tissues using DIG-labeled DNA probes has been introduced. The paraffin-embedded tissues are the most common form of biopsy tissues available from pathology banks. Bacteria can be detected either in a species-specific or universal manner. Bacterial signals are detected as either discrete forms (coccus, rod, fusiform, and hairy form) of bacteria or dispersed forms. The technique allows other histological information to be obtained: the epithelia, connective tissue, inflammatory infiltrates, and blood vessels are well distinguished. This method can be used to study the role of bacteria in various diseases, such as periodontitis, cancers, and inflammatory immune diseases.

  3. Feasibility of archival non-buffered formalin-fixed and paraffin-embedded tissues for PCR amplification: an analysis of resected gastric carcinoma.

    PubMed

    Inoue, T; Nabeshima, K; Kataoka, H; Koono, M

    1996-12-01

    Although several factors affecting the sensitivity of polymerase chain reaction (PCR) amplification from formalin-fixed tissues have been investigated mostly by experiments, the feasibility of archival formalin-fixed, paraffin-embedded tissue samples stored in pathology departments for PCR amplification has rarely been examined directly. Thus, the feasibility of 74 archival unbuffered 10% formalin-fixed, paraffin-embedded tissues for PCR amplification with primers producing a 190 b.p. DNA segment of p53 exon 5 was investigated. Fixation time was the critical factor influencing the sensitivity of PCR amplification. All (6/6) of the samples fixed for only 1 day, 44% (7/16) of the samples fixed for 2-3 days and 14% (4/28) of the samples fixed for 4-6 days showed successful amplification, while no amplification was obtained for the samples fixed for 7 days or more. The peak size of DNA extracted from the archival tissues decreased as the fixation time became longer. Experiments using xenografted tumor tissues fixed for various times showed longer permissible fixation time; up to 9 days of fixation, decreasing amounts of PCR products were obtained while no amplification was obtained for the samples fixed for 12 days or more. The time in paraffin seemed to be a minor factor for PCR amplification since all of the 1 day fixation samples, including those that had been embedded for up to 5 years, resulted in efficient amplification. The size of the amplified DNA segments, however, could be another factor influencing the sensitivity of amplification because even the 1 day fixation samples showed less amplification of 345 b.p. DNA compared with those of 167 and 262 b.p. DNA. Additionally, a point mutation was detected in the amplified p53 products from archival tissues using a non-isotopic method, temperature gradient gel electrophoresis. In conclusion, archival tissue samples that had been fixed immediately for only up to 1 day were constantly available for PCR amplification

  4. Identification of monoclonal antibodies for immunohistochemical staining of feline B lymphocytes in frozen and formalin-fixed paraffin-embedded tissues.

    PubMed Central

    Monteith, C E; Chelack, B J; Davis, W C; Haines, D M

    1996-01-01

    Commercially-available monoclonal antibodies to B lymphocytes were evaluated for immunohistochemical staining of feline B lymphocytes in frozen and formalin-fixed, paraffin-embedded tissues using an avidin biotin complex immunoperoxidase immunohistochemical technique. Three monoclonal antibodies: F46A and F72A raised to "carnivore" B lymphocytes and RA3.6B2 raised to murine B lymphocytes, stained B lymphocyte-dependent areas of frozen feline lymphoid tissue. In addition, antibody RA3.6B2 stained B lymphocyte dependent areas in formalin-fixed, paraffin-embedded feline tissues. There was no staining of T lymphocyte-dependent areas in either frozen or formalin-fixed tissues. Dual parameter flow cytometry, using an anti-pan-T lymphocyte antibody, revealed that greater than 99% of the cells stained by RA3.6B2 are a population distinct from T lymphocytes. F46A was shown to stain a sub-population of those cells stained with RA3.6B2. These antibodies may be useful in the identification of feline B lymphocytes using immunohistochemistry and flow cytometry and thereby provide additional tools to study B lymphocyte ontogeny and the significance of lymphocyte phenotype in lymphoid neoplasia in cats. Images Figure 1. PMID:8809382

  5. In situ hybridisation for the detection of Leishmania species in paraffin wax-embedded canine tissues using a digoxigenin-labelled oligonucleotide probe

    PubMed Central

    Dinhopl, N.; Mostegl, M. M.; Richter, B.; Nedorost, N.; Maderner, A.; Fragner, K.; Weissenböck, H.

    2011-01-01

    The diagnosis of canine leishmaniosis (CanL) is currently predominantly achieved by cytological or histological identification of amastigotes in biopsy samples, demonstration of specific anti-Leishmania antibodies and PCR-based approaches. All these methods have the advantage of being sensitive and more or less specific; nevertheless, most of them also have disadvantages. A chromogenic in situ hybridisation (ISH) procedure with a digoxigenin-labelled probe, targeting a fragment of the 5.8S rRNA was developed for the detection of all species of Leishmania parasites in routinely paraffin wax-embedded canine tissues. This method was validated in comparison with traditional techniques (histology, PCR), on various tissues from three dogs with histological changes consistent with a florid leishmaniosis. Amastigote forms of Leishmania gave clear signals and were easily identified using ISH. Various tissues from 10 additional dogs with clinical suspicion or/and a positive serological test but without histological presence of amastigotes did not show any ISH signals. Potential cross-reactivity of the probe was ruled out by negative outcome of the ISH against selected protozoa (including the related Trypanosoma cruzi) and fungi. Thus, ISH proved to be a powerful tool for unambiguous detection of Leishmania parasites in paraffin wax-embedded tissues. PMID:21921059

  6. In situ hybridisation for the detection of Leishmania species in paraffin wax-embedded canine tissues using a digoxigenin-labelled oligonucleotide probe.

    PubMed

    Dinhopl, N; Mostegl, M M; Richter, B; Nedorost, N; Maderner, A; Fragner, K; Weissenböck, H

    2011-11-12

    The diagnosis of canine leishmaniosis (CanL) is currently predominantly achieved by cytological or histological identification of amastigotes in biopsy samples, demonstration of specific anti-Leishmania antibodies and PCR-based approaches. All these methods have the advantage of being sensitive and more or less specific; nevertheless, most of them also have disadvantages. A chromogenic in situ hybridisation (ISH) procedure with a digoxigenin-labelled probe, targeting a fragment of the 5.8S rRNA was developed for the detection of all species of Leishmania parasites in routinely paraffin wax-embedded canine tissues. This method was validated in comparison with traditional techniques (histology, PCR), on various tissues from three dogs with histological changes consistent with a florid leishmaniosis. Amastigote forms of Leishmania gave clear signals and were easily identified using ISH. Various tissues from 10 additional dogs with clinical suspicion or/and a positive serological test but without histological presence of amastigotes did not show any ISH signals. Potential cross-reactivity of the probe was ruled out by negative outcome of the ISH against selected protozoa (including the related Trypanosoma cruzi) and fungi. Thus, ISH proved to be a powerful tool for unambiguous detection of Leishmania parasites in paraffin wax-embedded tissues.

  7. In situ hybridisation for the detection of Leishmania species in paraffin wax-embedded canine tissues using a digoxigenin-labelled oligonucleotide probe.

    PubMed

    Dinhopl, N; Mostegl, M M; Richter, B; Nedorost, N; Maderner, A; Fragner, K; Weissenböck, H

    2011-11-12

    The diagnosis of canine leishmaniosis (CanL) is currently predominantly achieved by cytological or histological identification of amastigotes in biopsy samples, demonstration of specific anti-Leishmania antibodies and PCR-based approaches. All these methods have the advantage of being sensitive and more or less specific; nevertheless, most of them also have disadvantages. A chromogenic in situ hybridisation (ISH) procedure with a digoxigenin-labelled probe, targeting a fragment of the 5.8S rRNA was developed for the detection of all species of Leishmania parasites in routinely paraffin wax-embedded canine tissues. This method was validated in comparison with traditional techniques (histology, PCR), on various tissues from three dogs with histological changes consistent with a florid leishmaniosis. Amastigote forms of Leishmania gave clear signals and were easily identified using ISH. Various tissues from 10 additional dogs with clinical suspicion or/and a positive serological test but without histological presence of amastigotes did not show any ISH signals. Potential cross-reactivity of the probe was ruled out by negative outcome of the ISH against selected protozoa (including the related Trypanosoma cruzi) and fungi. Thus, ISH proved to be a powerful tool for unambiguous detection of Leishmania parasites in paraffin wax-embedded tissues. PMID:21921059

  8. A microwave antigen retrieval method using two heating steps for enhanced immunostaining on aldehyde-fixed paraffin-embedded tissue sections.

    PubMed

    Gu, Ling; Cong, Jing; Zhang, Jie; Tian, Ying-Ying; Zhai, Xiao-Yue

    2016-06-01

    Antigen retrieval is an immunohistochemical procedure that results in better exposure of target antigens in aldehyde-fixed, paraffin-embedded tissue sections to antibodies. However, the commercially recommended or conventional protocols for antigen retrieval do not always succeed in expressing the target antigen. Here, an improved method was developed for antigen retrieval from aldehyde-fixed, paraffin-embedded histological sections. Proliferating cell nuclear antigen (PCNA), tight junction proteins Claudin-2 and Claudin-7, and water channel aquaporins in kidney tissue were selected as test antigens. Typically, PCNA and Claudin-2 and Claudin-7 show negative, weak, or nonspecific immunoreactions with conventional antigen retrieval methods using microwave heating. In the present study, microwave heating was performed twice with an interval of 30 min between the two steps to allow the buffer solution to cool. Sodium citrate buffer (10 mM sodium citrate, pH 6.0) was used for PCNA, and Tris-EDTA buffer (10 mM Tris, 1 mM EDTA, pH 9.0) was used for the Claudins. Compared with conventionally prepared tissues, the tissues exhibited both enhanced and specific immunostaining, and well-preserved morphology. In conclusion, the conventional protocol could be supplemented with a second microwave heating step to improve the expression of antigens that do not respond well to the conventional method. PMID:27002723

  9. Influence of parasite density and sample storage time on the reliability of Entamoeba histolytica-specific PCR from formalin-fixed and paraffin-embedded tissues.

    PubMed

    Frickmann, Hagen; Tenner-Racz, Klara; Eggert, Petra; Schwarz, Norbert G; Poppert, Sven; Tannich, Egbert; Hagen, Ralf M

    2013-12-01

    We report on the reliability of polymerase chain reaction (PCR) for the detection of Entamoeba histolytica from formalin-fixed, paraffin-embedded tissue in comparison with microscopy and have determined predictors that may influence PCR results. E. histolytica-specific and Entamoeba dispar-specific real-time PCR and microscopy from adjacent histologic sections were performed using a collection of formalin-fixed, paraffin-embedded tissue specimens obtained from patients with invasive amebiasis. Specimens had been collected during the previous 4 decades. Association of sample age, parasite density, and reliability of PCR was analyzed. E. histolytica PCR was positive in 20 of 34 biopsies (58.8%); 2 of these 20 were microscopically negative for amebae in neighboring tissue sections. PCR was negative in 9 samples with visible amebae in neighboring sections and in 5 samples without visible parasites in neighboring sections. PCR was negative in all specimens that were older than 3 decades. Low parasite counts and sample ages older than 20 years were predictors for false-negative PCR results. All samples were negative for E. dispar DNA. PCR is suitable for the detection of E. histolytica in formalin-fixed, paraffin-embedded tissue samples that are younger than 2 decades and that contain intermediate to high parasite numbers. Negative results in older samples were due to progressive degradation of DNA over time as indicated by control PCRs targeting the human 18S rRNA gene. Moreover, our findings support previous suggestions that only E. histolytica but not E. dispar is responsible for invasive amebiasis.

  10. Assessment of Telomere Length in Archived Formalin-Fixed, Paraffinized Human Tissue Is Confounded by Chronological Age and Storage Duration

    PubMed Central

    2016-01-01

    Telomeres shorten with physiological aging but undergo substantial restoration during cancer immortalization. Increasingly, cancer studies utilize the archive of formalin-fixed, paraffin-embedded (FFPE) tissues in diagnostic pathology departments. Conceptually, such studies would be confounded by physiological telomere attrition and loss of DNA integrity from prolonged tissue storage. Our study aimed to investigate these two confounding factors. 145 FFPE tissues of surgically-resected, non-diseased appendixes were retrieved from our pathology archive, from years 2008 to 2014. Cases from 2013 to 2014 were categorized by patient chronological age (0–20 years, 21–40 years, 41–60 years, > 60 years). Telomere lengths of age categories were depicted by telomere/chromosome 2 centromere intensity ratio (TCR) revealed by quantitative fluorescence in situ hybridization. Material from individuals aged 0–20 years from years 2013/2014, 2011/2012, 2009/2010, and 2008 were compared for storage effect. Telomere integrity was assessed by telomere fluorescence intensity (TFI). Epithelial TCRs (mean ± SD) for the respective age groups were 4.84 ± 2.08, 3.64 ± 1.21, 2.03 ± 0.37, and 1.93 ± 0.45, whereas corresponding stromal TCRs were 5.16 ± 2.55, 3.84 ± 1.36, 2.49 ± 1.20, and 2.93 ± 1.24. A trend of inverse correlation with age in both epithelial and stromal tissues is supported by r = -0.69, p < 0.001 and r = -0.42, p < 0.001 respectively. Epithelial TFIs (mean ± SD) of years 2013/2014, 2011/2012, 2009/2010 and 2008 were 852.60 ± 432.46, 353.04 ± 127.12, 209.24 ± 55.57 and 429.22 ± 188.75 respectively. Generally, TFIs reduced with storage duration (r = -0.42, p < 0.001). Our findings agree that age-related telomere attrition occurs in normal somatic tissues, and suggest that an age-based reference can be established for telomere studies on FFPE tissues. We also showed that FFPE tissues archived beyond 2 years are suboptimal for telomere analysis. PMID:27598341

  11. Assessment of Telomere Length in Archived Formalin-Fixed, Paraffinized Human Tissue Is Confounded by Chronological Age and Storage Duration.

    PubMed

    Kong, Po-Lian; Looi, Lai-Meng; Lau, Tze-Pheng; Cheah, Phaik-Leng

    2016-01-01

    Telomeres shorten with physiological aging but undergo substantial restoration during cancer immortalization. Increasingly, cancer studies utilize the archive of formalin-fixed, paraffin-embedded (FFPE) tissues in diagnostic pathology departments. Conceptually, such studies would be confounded by physiological telomere attrition and loss of DNA integrity from prolonged tissue storage. Our study aimed to investigate these two confounding factors. 145 FFPE tissues of surgically-resected, non-diseased appendixes were retrieved from our pathology archive, from years 2008 to 2014. Cases from 2013 to 2014 were categorized by patient chronological age (0-20 years, 21-40 years, 41-60 years, > 60 years). Telomere lengths of age categories were depicted by telomere/chromosome 2 centromere intensity ratio (TCR) revealed by quantitative fluorescence in situ hybridization. Material from individuals aged 0-20 years from years 2013/2014, 2011/2012, 2009/2010, and 2008 were compared for storage effect. Telomere integrity was assessed by telomere fluorescence intensity (TFI). Epithelial TCRs (mean ± SD) for the respective age groups were 4.84 ± 2.08, 3.64 ± 1.21, 2.03 ± 0.37, and 1.93 ± 0.45, whereas corresponding stromal TCRs were 5.16 ± 2.55, 3.84 ± 1.36, 2.49 ± 1.20, and 2.93 ± 1.24. A trend of inverse correlation with age in both epithelial and stromal tissues is supported by r = -0.69, p < 0.001 and r = -0.42, p < 0.001 respectively. Epithelial TFIs (mean ± SD) of years 2013/2014, 2011/2012, 2009/2010 and 2008 were 852.60 ± 432.46, 353.04 ± 127.12, 209.24 ± 55.57 and 429.22 ± 188.75 respectively. Generally, TFIs reduced with storage duration (r = -0.42, p < 0.001). Our findings agree that age-related telomere attrition occurs in normal somatic tissues, and suggest that an age-based reference can be established for telomere studies on FFPE tissues. We also showed that FFPE tissues archived beyond 2 years are suboptimal for telomere analysis. PMID:27598341

  12. Double labeling and simultaneous detection of B- and T cells using fluorescent nano-crystal (q-dots) in paraffin-embedded tissues.

    PubMed

    Zahavy, Eran; Freeman, Esther; Lustig, Shlomo; Keysary, Avi; Yitzhaki, Shmuel

    2005-09-01

    A double immunohistochemical technique for the simultaneous detection of T- and B cells in paraffin-embedded mice tissues have been developed. This procedure is based on using fluorescent nano-crystals (q-dots). The benefit of using q-dots evolves from their unique fluorescence characteristics advantages: such as broad excitation spectrum, narrow emission band and high photo-bleaching threshold compare to organic fluorophores. T cells antigens (CD3) were stained using antibody-coated q-dots with max emission at 655 nm (GalphaRb-QD655). B cells antigens (CD45R/B220) were stained using streptavidin-coated q-dots with max emission at 585 nm (SA-QD585). The simultaneous detection of T- and B cells was demonstrated in paraffin-embedded lymph node using standard fluorescence microscope.

  13. Immunocytochemical detection of androgen receptor in human temporal cortex characterization and application of polyclonal androgen receptor antibodies in frozen and paraffin-embedded tissues.

    PubMed

    Puy, L; MacLusky, N J; Becker, L; Karsan, N; Trachtenberg, J; Brown, T J

    1995-11-01

    Immunocytochemical and biochemical studies have demonstrated the presence of androgen receptor protein in various regions of the rodent and non-human primate cortex. Localization of androgen receptor in the human brain has, however, not been studied as extensively, because of difficulties in obtaining suitable tissue samples. In the present study, we have localized androgen receptors in both frozen and paraffin-embedded temporal cortex from epileptic patients undergoing resection. Polyclonal antibodies were raised against fusion proteins containing fragments of the human androgen receptor protein. The antibodies were affinity-purified against the corresponding fusion protein. Immunoprecipitation and Western blotting using extracts from human cell lines demonstrated the specificity of the antibodies for the human androgen receptor and lack of cross-reactivity with other steroid hormone receptors. Immunocytochemistry was performed on frozen and paraffin sections of human temporal cortex and in paraffin-embedded benign hyperplastic prostates (BPH), as well as prostate and breast carcinomas, by the streptavidin-biotin-peroxidase method. Antigen-retrieval was performed in paraffin-embedded sections using microwave irradiation. Specific nuclear and cytoplasmic immunoreactivity for androgen receptor was detected in neurons, astrocytes, oligodendrocytes, and microglia cells of the temporal cortex. In contrast, only nuclear staining was observed in BPH, prostate and breast carcinomas. Immunoprecipitation of human temporal cortex lysate and subsequent Western blot analysis demonstrated the expression of a 98 kDa immunoreactive protein, slightly smaller than the reported molecular weight of the wild-type androgen receptor. These results provide further evidence for the expression of androgen receptor in the human temporal cortex. The use of these immunocytochemical techniques should enable the retrospective determination of possible changes in androgen receptor expression in

  14. Inter-laboratory comparison of DNA preservation in archival paraffin-embedded human brain tissue from participating centres on four continents.

    PubMed

    Kösel, S; Grasbon-Frodl, E M; Arima, K; Chimelli, L; Hahn, M; Hashizume, Y; Hulette, C; Ikeda, K; Jacobsen, P F; Jones, M; Kobayashi, M; Love, S; Mizutani, T; Rosemberg, S; Sasaki, A; Smith, T W; Takahashi, H; Vortmeyer, A O; Graeber, M B

    2001-07-01

    DNA extracted from formalin-fixed and paraffin-embedded brain tissue is known to contain as yet ill-characterized inhibitors of the PCR process. As part of a project that aims to clarify the role of mitochondrial DNA sequence variation in human neurodegenerative diseases using DNA from various ethnic backgrounds, we have investigated factors that influence the preservation of archival DNA and its suitability for PCR. In this study, neuropathological tissue samples were analysed that had been routinely processed in 18 international centres on four continents. Following DNA extraction, PCR amplification of mitochondrial and nuclear DNA sequences was performed with and without additional purification of the template DNA. In addition, the DNA used for PCR was analysed by HPLC. Phosphate-buffered formalin proved to be a superior fixative compared with unbuffered aldehyde: DNA extraction resulted in greater yields, the molecular weight of the isolated DNA was higher and PCR was more successful. PCR inhibitors were identified as (1) high concentrations of small (<300 bp) DNA fragments that competitively compete with template DNA and (2) contaminants of the DNA template solution including denatured protein that cannot be completely removed by phenolic extraction. HPLC analysis did not reveal significant qualitative differences between DNA isolated from fresh-frozen tissue samples and DNA recovered from formalin-fixed, paraffin-embedded brain tissue. The fact that DNA could be amplified from the majority of tissue specimens in this study suggests that rare diseases and diseases where ethnic background plays an important role can be sampled for genetic polymorphism analysis on a global scale using archival neuropathological collections.

  15. Deciphering signaling pathways in clinical tissues for personalized medicine using protein microarrays.

    PubMed

    Malinowsky, K; Wolff, C; Ergin, B; Berg, D; Becker, K F

    2010-11-01

    The current transition in cancer therapy from general treatment approaches, based mainly on chemotherapy and radiotherapy, to more directed approaches that aim to inhibit specific molecular targets has brought about new challenges for pathology. In the past, classical assignment of pathology consisted of tumor diagnosis and staging for further therapy decisions; nowadays, pathologists are asked to predict possible therapeutic results by detecting and quantifying therapeutic targets in tumors such as the human epidermal growth factor receptor 2 (HER2). The best approach to analyze such molecular targets is to provide a tumor-specific protein expression profile prior to therapy. To further elucidate signaling networks underlying cancer development and to identify new targets, it is necessary to implement tools that allow fast, precise, cheap, and simultaneous analysis of many network components while requiring only a small amount of clinical material. Reverse phase protein microarray (RPPA) is a promising technology that meets these requirements while enabling quantitative measurement of proteins. Recently, methods for the extraction of proteins from formalin-fixed, paraffin-embedded (FFPE) tissues have become available. In this article, we demonstrate how the use of RPPA to analyze signaling pathways from FFPE tissues may improve quantification of therapeutic targets and diagnostic markers in the near future.

  16. Immunoguided Laser Assisted Microdissection Techniques for DNA Methylation Analysis of Archival Tissue Specimens

    PubMed Central

    Eberle, Franziska C.; Hanson, Jeffrey C.; Killian, J. Keith; Wei, Lai; Ylaya, Kris; Hewitt, Stephen M.; Jaffe, Elaine S.; Emmert-Buck, Michael R.; Rodriguez-Canales, Jaime

    2010-01-01

    Altered DNA methylation is a fundamental characteristic of carcinogenesis. The analysis of DNA methylation in tumor cells may help to better understand tumor pathogenesis and more importantly may be used as diagnostic tool with therapeutic consequences. To detect targets relevant in tumorigenesis, it is essential to separate neoplastic cells from nonneoplastic cells. An excellent method for isolating specific cells is laser-assisted microdissection (LAM). Target cell identification for immunoguided LAM (ILAM) requires immunohistochemistry (IHC). Yet, it is unclear whether IHC for ILAM influences DNA methylation. The goals of this study were to establish an optimized protocol for antigen retrieval and IHC of formalin-fixed paraffin-embedded (FFPE) specimens suitable for ILAM and to evaluate its effect on the DNA methylome using a high throughput array. Using ten archival FFPE specimens, we showed specific staining suitable for ILAM. Extracted DNA from microdissected cells of immunohistochemically or H&E-stained tissue sections showed identical DNA quality and a strong correlation (r = 0.94 to 0.98) for CpG target methylation of 1505 analyzed sites in a series of five paired samples. No differential methylation between H&E and IHC was detected in 1501 of 1505 CpG targets (99.7%; P < 0.05). These results demonstrate the validity and utility of the herein described protocol, which allows the application of ILAM for large-scale genomic and epigenetic analyses of archival tissue specimens. PMID:20413681

  17. Gene expression profiles in granuloma tissue reveal novel diagnostic markers in sarcoidosis.

    PubMed

    Christophi, George P; Caza, Tiffany; Curtiss, Christopher; Gumber, Divya; Massa, Paul T; Landas, Steve K

    2014-06-01

    Sarcoidosis is an immune-mediated multisystem disease characterized by the formation of non-caseating granulomas. The pathogenesis of sarcoidosis is unclear, with proposed infectious or environmental antigens triggering an aberrant immune response in susceptible hosts. Multiple pro-inflammatory signaling pathways have been implicated in mediating macrophage activation and granuloma formation in sarcoidosis, including IFN-γ/STAT-1, IL-6/STAT-3, and NF-κB. It is difficult to distinguish sarcoidosis from other granulomatous diseases or assess disease severity and treatment response with histopathology alone. Therefore, development of improved diagnostic tools is imperative. Herein, we describe an efficient and reliable technique to classify granulomatous disease through selected gene expression and identify novel genes and cytokine pathways contributing to the pathogenesis of sarcoidosis. We quantified the expression of twenty selected mRNAs extracted from formalin-fixed paraffin embedded (FFPE) tissue (n = 38) of normal lung, suture granulomas, sarcoid granulomas, and fungal granulomas. Utilizing quantitative real-time RT-PCR we analyzed the expression of several genes, including IL-6, COX-2, MCP-1, IFN-γ, T-bet, IRF-1, Nox2, IL-33, and eotaxin-1 and revealed differential regulation between suture, sarcoidosis, and fungal granulomas. This is the first study demonstrating that quantification of target gene expression in FFPE tissue biopsies is a potentially effective diagnostic and research tool in sarcoidosis.

  18. The effect of deoxyribonucleic acid extraction methods from lymphoid tissue on the purity, content, and amplifying ability

    PubMed Central

    Ayatollahi, Hossein; Sadeghian, Mohammad Hadi; Keramati, Mohammad Reza; Ayatollahi, Ali; Shajiei, Arezoo; Sheikhi, Maryam; Bakhshi, Samane

    2016-01-01

    Background: Nowadays, definitive diagnosis of numerous diseases is based on the genetic and molecular findings. Therefore, preparation of fundamental materials for these evaluations is necessary. Deoxyribonucleic acid (DNA) is the first material for the molecular pathology and genetic analysis, and better results need more pure DNA. Furthermore, higher concentration of achieved DNA causes better results and higher amplifying ability for subsequent steps. We aim to evaluate five DNA extraction methods to compare DNA intimacy including purity, concentration, and amplifying ability with each other. Materials and Methods: The lymphoid tissue DNA was extracted from formalin-fixed, paraffin embedded (FFPE) tissue through five different methods including phenol-chloroform as the reference method, DNA isolation kit (QIAamp DNA FFPE Tissue Kit, Qiagen, Germany), proteinase K and xylol extraction and heat alkaline plus mineral oil extraction as authorship innovative method. Finally, polymerase chain reaction (PCR) and real-time PCR method were assessed to compare each following method consider to DNA purity and its concentration. Results: Among five different applied methods, the highest mean of DNA purity was related to heat alkaline method. Moreover, the highest mean of DNA concentration was related to heat alkaline plus mineral oil. Furthermore, the best result in quantitative PCR was in proteinase K method that had the lowest cycle threshold averages among the other extraction methods. Conclusion: We concluded that our innovative method for DNA extraction (heat alkaline plus mineral oil) achieved high DNA purity and concentration. PMID:27630381

  19. The effect of deoxyribonucleic acid extraction methods from lymphoid tissue on the purity, content, and amplifying ability

    PubMed Central

    Ayatollahi, Hossein; Sadeghian, Mohammad Hadi; Keramati, Mohammad Reza; Ayatollahi, Ali; Shajiei, Arezoo; Sheikhi, Maryam; Bakhshi, Samane

    2016-01-01

    Background: Nowadays, definitive diagnosis of numerous diseases is based on the genetic and molecular findings. Therefore, preparation of fundamental materials for these evaluations is necessary. Deoxyribonucleic acid (DNA) is the first material for the molecular pathology and genetic analysis, and better results need more pure DNA. Furthermore, higher concentration of achieved DNA causes better results and higher amplifying ability for subsequent steps. We aim to evaluate five DNA extraction methods to compare DNA intimacy including purity, concentration, and amplifying ability with each other. Materials and Methods: The lymphoid tissue DNA was extracted from formalin-fixed, paraffin embedded (FFPE) tissue through five different methods including phenol-chloroform as the reference method, DNA isolation kit (QIAamp DNA FFPE Tissue Kit, Qiagen, Germany), proteinase K and xylol extraction and heat alkaline plus mineral oil extraction as authorship innovative method. Finally, polymerase chain reaction (PCR) and real-time PCR method were assessed to compare each following method consider to DNA purity and its concentration. Results: Among five different applied methods, the highest mean of DNA purity was related to heat alkaline method. Moreover, the highest mean of DNA concentration was related to heat alkaline plus mineral oil. Furthermore, the best result in quantitative PCR was in proteinase K method that had the lowest cycle threshold averages among the other extraction methods. Conclusion: We concluded that our innovative method for DNA extraction (heat alkaline plus mineral oil) achieved high DNA purity and concentration.

  20. Mass-spectrometry analysis of histone post-translational modifications in pathology tissue using the PAT-H-MS approach.

    PubMed

    Noberini, Roberta; Pruneri, Giancarlo; Minucci, Saverio; Bonaldi, Tiziana

    2016-06-01

    Aberrant histone post-translational modifications (hPTMs) have been implicated with various pathologies, including cancer, and may represent useful epigenetic biomarkers. The data described here provide a mass spectrometry-based quantitative analysis of hPTMs from formalin-fixed paraffin-embedded (FFPE) tissues, from which histones were extracted through the recently developed PAT-H-MS method. First, we analyzed FFPE samples from mouse spleen and liver or human breast cancer up to six years old, together with their corresponding fresh frozen tissue. We then combined the PAT-H-MS approach with a histone-focused version of the super-SILAC strategy-using a mix of histones from four breast cancer cell lines as a spike-in standard- to accurately quantify hPTMs from breast cancer specimens belonging to different subtypes. The data, which are associated with a recent publication (Pathology tissue-quantitative mass spectrometry analysis to profile histone post-translational modification patterns in patient samples (Noberini, 2015) [1]), are deposited at the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD002669. PMID:27408908

  1. Digital Sorting of Pure Cell Populations Enables Unambiguous Genetic Analysis of Heterogeneous Formalin-Fixed Paraffin-Embedded Tumors by Next Generation Sequencing.

    PubMed

    Bolognesi, Chiara; Forcato, Claudio; Buson, Genny; Fontana, Francesca; Mangano, Chiara; Doffini, Anna; Sero, Valeria; Lanzellotto, Rossana; Signorini, Giulio; Calanca, Alex; Sergio, Maximilian; Romano, Rita; Gianni, Stefano; Medoro, Gianni; Giorgini, Giuseppe; Morreau, Hans; Barberis, Massimo; Corver, Willem E; Manaresi, Nicolò

    2016-01-01

    Precision medicine in oncology requires an accurate characterization of a tumor molecular profile for patient stratification. Though targeted deep sequencing is an effective tool to detect the presence of somatic sequence variants, a significant number of patient specimens do not meet the requirements needed for routine clinical application. Analysis is hindered by contamination of normal cells and inherent tumor heterogeneity, compounded with challenges of dealing with minute amounts of tissue and DNA damages common in formalin-fixed paraffin-embedded (FFPE) specimens. Here we present an innovative workflow using DEPArray™ system, a microchip-based digital sorter to achieve 100%-pure, homogenous subpopulations of cells from FFPE samples. Cells are distinguished by fluorescently labeled antibodies and DNA content. The ability to address tumor heterogeneity enables unambiguous determination of true-positive sequence variants, loss-of-heterozygosity as well as copy number variants. The proposed strategy overcomes the inherent trade-offs made between sensitivity and specificity in detecting genetic variants from a mixed population, thus rescuing for analysis even the smaller clinical samples with low tumor cellularity. PMID:26864208

  2. Digital Sorting of Pure Cell Populations Enables Unambiguous Genetic Analysis of Heterogeneous Formalin-Fixed Paraffin-Embedded Tumors by Next Generation Sequencing

    PubMed Central

    Bolognesi, Chiara; Forcato, Claudio; Buson, Genny; Fontana, Francesca; Mangano, Chiara; Doffini, Anna; Sero, Valeria; Lanzellotto, Rossana; Signorini, Giulio; Calanca, Alex; Sergio, Maximilian; Romano, Rita; Gianni, Stefano; Medoro, Gianni; Giorgini, Giuseppe; Morreau, Hans; Barberis, Massimo; Corver, Willem E.; Manaresi, Nicolò

    2016-01-01

    Precision medicine in oncology requires an accurate characterization of a tumor molecular profile for patient stratification. Though targeted deep sequencing is an effective tool to detect the presence of somatic sequence variants, a significant number of patient specimens do not meet the requirements needed for routine clinical application. Analysis is hindered by contamination of normal cells and inherent tumor heterogeneity, compounded with challenges of dealing with minute amounts of tissue and DNA damages common in formalin-fixed paraffin-embedded (FFPE) specimens. Here we present an innovative workflow using DEPArray™ system, a microchip-based digital sorter to achieve 100%-pure, homogenous subpopulations of cells from FFPE samples. Cells are distinguished by fluorescently labeled antibodies and DNA content. The ability to address tumor heterogeneity enables unambiguous determination of true-positive sequence variants, loss-of-heterozygosity as well as copy number variants. The proposed strategy overcomes the inherent trade-offs made between sensitivity and specificity in detecting genetic variants from a mixed population, thus rescuing for analysis even the smaller clinical samples with low tumor cellularity. PMID:26864208

  3. The comparison of methods to identify the presence of fibrocytes in formalin-fixed, paraffin-embedded archival cardiac tissue with coronary heart disease.

    PubMed

    Lei, Pu-Ping; Shuai, Qun; Wang, Shang-Wen; Tao, Si-Ming; Qu, Yong-Qiang; Wang, Dian-Hua

    2014-12-01

    The purpose of this study was to find the optimal technical approach to identify the presence of fibrocytes in formalin-fixed, paraffin-embedded archival cardiac tissue with CHD (coronary heart disease). Using the coexpression markers CD45 and αSMA, the presence of fibrocytes was examined by three different methods, including double immunohistochemistry staining, combination labeling of immunohistochemistry and immunofluorescence and double immunofluorescence labeling. Double immunohistochemistry staining was very difficult to identify the CD45(+)/αSMA(+) fibrocytes. Although combination staining of immunohistochemistry and immunofluorescence has made it possible to evaluate the co-localization of CD45 and αSMA in the fibrocytes, this method was prone to produce many false positive cells. In contrast, CD45(+)/αSMA(+) fibrocytes could be clearly recognized by double immunofluorescence labeling. In conclusion, double immunofluorescence labeling is the optimal technical approach to identify the presence of fibrocytes in routinely processed cardiac tissue with CHD.

  4. Detection of lumpy skin disease virus antigen and genomic DNA in formalin-fixed paraffin-embedded tissues from an Egyptian outbreak in 2006.

    PubMed

    Awadin, W; Hussein, H; Elseady, Y; Babiuk, S; Furuoka, H

    2011-10-01

    An outbreak of lumpy skin disease (LSD) was reported in 2006 in Egypt affecting 16 provinces. Biopsies and post-mortem tissue samples were collected from calves that showed typical clinical signs of LSD and fixed in formalin. These samples were collected from a private dairy farm in the Damietta province of Egypt. Formalin-fixed paraffin-embedded tissue samples were assessed using histology, and skin lesions were classified as either acute or subacute/chronic. Both lumpy skin disease virus (LSDV) DNA detected by polymerase chain reaction and LSDV antigen detected by immunohistochemistry using a capripoxvirus-specific monoclonal antibody were observed in the acute skin lesions and in some subacute/chronic skin lesions. PMID:21699673

  5. The effect of aging of formalin-fixed paraffin-embedded tissues on the in situ hybridization and immunohistochemistry signals in cervical lesions.

    PubMed

    Nuovo, Allison J; Garofalo, Michela; Mikhail, Alexandria; Nicol, Alcina F; Vianna-Andrade, Cecilia; Nuovo, Gerard J

    2013-09-01

    Formalin-fixed, paraffin-embedded tissues are widely used in biomedical research but little is known about the effect of the age of the block or unstained slides on the in situ hybridization or immunohistochemistry signal. We compared the in situ-based and immunohistochemistry-based signals for cervical intraepithelial neoplasia samples that ranged from 0 to 15 years of age. There was a progressive and statistically significant decrease in the strength of the p16 signal when comparing tissues prepared from recent unstained slides (0 to 1 y old, mean score of 92%) to those of intermediate age (5 to 7 y old, mean score of 49%) to old unstained slides (cut 13 to 15 y ago, mean score of 10%). Equivalent, progressive, and significant decreases in the intensity of the signals for microRNAs, CD45, and human papillomavirus DNA were seen in tissues stored on slides from 5 to 7 years and 13 to 15 years, respectively. However, the diminution of signal was much less, although still statistically significant, if the sections from the 13- to 15-year-old paraffin blocks were prepared in 2012. The data likely does not represent degradation of the targets as extraction of several microRNA from the old blocks showed no detectable degradation, despite the markedly weakened in situ hybridization signal. It is concluded that in situ-based signal for DNA, microRNAs, and proteins in paraffin-embedded tissues are significantly reduced over time, especially when stored long term on glass slides which, in turn, can lead to a significant underestimation of the amount and presence of the nucleic acid or protein target.

  6. Genetic Characterization of Echinococcus granulosus from a Large Number of Formalin-Fixed, Paraffin-Embedded Tissue Samples of Human Isolates in Iran

    PubMed Central

    Rostami, Sima; Torbaghan, Shams Shariat; Dabiri, Shahriar; Babaei, Zahra; Mohammadi, Mohammad Ali; Sharbatkhori, Mitra; Harandi, Majid Fasihi

    2015-01-01

    Cystic echinococcosis (CE), caused by the larval stage of Echinococcus granulosus, presents an important medical and veterinary problem globally, including that in Iran. Different genotypes of E. granulosus have been reported from human isolates worldwide. This study identifies the genotype of the parasite responsible for human hydatidosis in three provinces of Iran using formalin-fixed paraffin-embedded tissue samples. In this study, 200 formalin-fixed paraffin-embedded tissue samples from human CE cases were collected from Alborz, Tehran, and Kerman provinces. Polymerase chain reaction amplification and sequencing of the partial mitochondrial cytochrome c oxidase subunit 1 gene were performed for genetic characterization of the samples. Phylogenetic analysis of the isolates from this study and reference sequences of different genotypes was done using a maximum likelihood method. In total, 54.4%, 0.8%, 1%, and 40.8% of the samples were identified as the G1, G2, G3, and G6 genotypes, respectively. The findings of the current study confirm the G1 genotype (sheep strain) to be the most prevalent genotype involved in human CE cases in Iran and indicates the high prevalence of the G6 genotype with a high infectivity for humans. Furthermore, this study illustrates the first documented human CE case in Iran infected with the G2 genotype. PMID:25535316

  7. Improved detection of mycobacterial DNA by PCR in formalin-fixed, paraffin-embedded tissues using thin sections.

    PubMed

    Loeschke, S; Goldmann, T; Vollmer, E

    2005-01-01

    PCR is a unique methodology allowing for the sensitive detection of mycobacterial DNA-sequences in cases in which no fresh material can be obtained for classic analyses. Despite the limitations of this technique, for example the less satisfactory quality of DNA from paraffin-embedded specimens and the high effort necessary to control contamination, PCR still represents a useful additional tool for routine diagnostic examinations of mycobacterial infections. Fragmentation of the DNA extracted from formalin-fixed, paraffin-embedded samples on the one hand and the rigid cell wall of mycobacteria on the other hand are obstacles to detecting the DNA of these microorganisms by PCR. Here, we describe a simple mechanical procedure that allows us to improve the detection of mycobacterial DNA with the use of thin (1 microm) sections instead of thicker sections. This could be explained by a gentle, mechanical opening of the acid fast mycobacterial cell wall. Thus, even the application of heat/cold shock treatments is not necessary. This inexpensive fast procedure can also be used for the detection of other infectious agents. PMID:15807309

  8. Immunohistochemical identification of Renibacterium salmoninarum by monoclonal antibodies in paraffin-embedded tissues of Atlantic salmon (Salmo salar L.), using paired immunoenzyme and paired immunofluorescence techniques.

    PubMed

    Evensen, O; Dale, O B; Nilsen, A

    1994-01-01

    Renibacterium salmoninarum was identified in situ by immunoenzymatic and immunofluorescence techniques in paraffin-embedded tissue specimens collected during a natural outbreak of bacterial kidney disease (BKD) and from an experimental infection in Atlantic salmon (Salmo salar L.). Monoclonal antibodies (MAbs) 4D3 and 2G5 were used in this study, both specific for the 57-58-kD outer membrane protein (p57) of the bacterium. Both MAbs revealed positive staining in ethanol-fixed tissue specimens, but only the epitope identified by MAb 4D3 was formalin resistant. Pretreatment with trypsin did not reestablish the antigenicity for the epitope identified by Mab 2G5. Paired immunoenzymatic staining for identification of the bacterium in sequential incubation steps on ethanol-fixed tissue specimens using an avidin-biotin-peroxidase system was obtained after serial dilution of the Mab (2G5) or the chromagen, amino ethyl carbazole, in the first sequence. Paired immunofluorescence staining with well-balanced color mixing was easily obtained on ethanol-fixed tissue specimens using sequential incubations. Single exposures gave blue (aminomethyl coumarin acetic acid) and green (fluorescein isothiocyanate) fluorescence for MAbs 2G5 and biotinylated 4D3, respectively. Color mixing was revealed as a turquoise staining. Studies on method sensitivity was performed by incorporating a known amount of a protein preparation of p57 into an inert matrix, creating an artificial test substrate.(ABSTRACT TRUNCATED AT 250 WORDS)

  9. Identification of BRCA1 Deficiency Using Multi-Analyte Estimation of BRCA1 and Its Repressors in FFPE Tumor Samples from Patients with Triple Negative Breast Cancer

    PubMed Central

    Korlimarla, Aruna; Prabhu, Jyothi S.; Remacle, Jose; Rajarajan, Savitha; Raja, Uma; C. E., Anupama; Srinath, B. S.; Manjunath, Suraj; K. S., Gopinath; Correa, Marjorrie; M. S. N., Prasad; Sridhar, T. S.

    2016-01-01

    Purpose Apart from germ-line BRCA1-mutated breast cancers, a significant proportion of women with sporadic triple negative breast cancer (TNBC) sub-type are known to harbour varying levels of BRCA1-dysfuction. There is currently no established diagnostic method to identify these patients. Methods The analysis was performed on 183 primary breast cancer tumor specimens from our longitudinal case-series archived as formalin-fixed-paraffin-embedded (FFPE) blocks comprising 71 TNBCs and 112 Hormone receptor positive HER2 negative (HR+HER2-) tumors. Transcript levels of BRCA1 and two of its repressors ID4 and microRNA182 were determined by TaqMan quantitative PCR. BRCA1 protein was detected immunohistochemically with the MS110 antibody. Results The representation of BRCA1 and its repressor ID4 as a ratio led to improved separation of TNBCs from HR+HER2- compared to either measure by itself. We then dichotomised the continuous distribution of each of the three measurements (Protein, MIRNA and transcript:repressor ratio) into categories of deficient (0) and adequate (1). A composite BRCA1 Deficiency Score (BDS) was computed by the addition of the score for all three measures. Samples deficient on 2 or more measures were deemed to be BRCA1 deficient; and 40% of all TNBCs met this criterion. Conclusion We propose here a simple multi-level assay of BRCA1 deficiency using the BRCA1:ID4 ratio as a critical parameter that can be performed on FFPE samples in clinical laboratories by the estimation of only 3 bio-markers. The ease of testing will hopefully encourage adoption and clinical validation. PMID:27077368

  10. Investigation of false positives associated with loop-mediated isothermal amplification assays for detection of Toxoplasma gondii in archived tissue samples of captive felids.

    PubMed

    Suleman, Essa; Mtshali, Moses Sibusiso; Lane, Emily

    2016-09-01

    Toxoplasma gondii is a ubiquitous protozoan parasite that infects humans and many different animals, including felids. Many molecular and serologic tests have been developed for detection of T. gondii in a wide range of hosts. Loop-mediated isothermal amplification (LAMP) is a field-friendly technique that lacks the practical drawbacks of other molecular and serologic tests, and LAMP assays have been successfully developed for detection of T. gondii in fresh tissue samples. In the current study, both a previously published and a de-novo designed LAMP assay were compared to a quantitative real-time (q)PCR assay, for the detection of T. gondii in archived formalin-fixed, paraffin-embedded (FFPE) tissue samples from captive wildlife. The LAMP assays produced conflicting results, generating both false positives and false negatives. Furthermore, the LAMP assays were unable to positively identify samples with low levels of parasites as determined by qPCR and histopathology. Therefore, these LAMP assays may not be the most suitable assays for detection of T. gondii in archived FFPE and frozen tissue samples. PMID:27449130

  11. Immunophenotypic classification of canine malignant lymphoma on formalin-mixed paraffin wax-embedded tissue by means of CD3 and CD79a cell markers.

    PubMed

    Milner, R J; Pearson, J; Nesbit, J W; Close, P

    1996-12-01

    Canine malignant lymphoma (CML) is a common lymphoid tumour. Identification of the immunophenotype is of prognostic importance: T-cell lymphomas have a worse prognosis than B-cell lymphomas. Until recently, identification of T- or B-cell lymphomas was undertaken by means of flow cytometry or fluorescent immunocytochemistry on frozen sections. Whilst valid in the research field, these methods are impractical for routine diagnostic histopathology in CML. Commercially available CD3 antibody has been successfully employed in T-cell identification in dogs in formalin-fixed paraffin wax-embedded tissue sections, but the lack of a B-cell marker has been a hindrance until the recent introduction of a commercially available pan-B cell marker, CD79a (DAKO M7051), suitable for diagnostic application upon formalin-fixed paraffin wax-embedded material. Antibody markers to CD3 and CD79a show cross-reactivity across species lines for B cells and T cells respectively. In this group of five selected canine cases, two were identified as B-cell and the other three as T-cell lymphoma, by means of CD3 and CD79a. To the best of our knowledge application of CD79a in cases of CML has not been reported.

  12. A simple and efficient method for DNA extraction from skin and paraffin-embedded tissues applicable to T-cell clonality assays.

    PubMed

    Sidorova, Julia V; Biderman, Bella V; Nikulina, Elena E; Sudarikov, Andrey B

    2012-01-01

    PCR-based clonality assay of rearranged T-cell receptor genes gamma and beta (TCRG and TCRB) in a number of cases could be essential to discriminate between cutaneous T-cell lymphomas and reactive lymphoproliferative lesions in the skin. However, extraction of good-quality DNA from skin specimens (especially formalin-fixed paraffin-embedded) remains a challenge. Common procedures, being labour-intensive and time-consuming and requiring toxic solvents such as phenol and chloroform, still may end up with DNA sample of insufficient quality. We herewith present a simple and efficient method for DNA isolation based on ammonia extraction of tissue, followed by neutralization and simultaneous salting out of proteins with acetic acid. We have analysed 30 samples - 24 fresh (16 skin, two spleen and six lymph node) and six paraffin-embedded. Standard procedure (proteinase K digestion, followed by phenol/chloroform extraction) has been carried out simultaneously. We observed good PCR signal for TCRG rearrangements in 30 samples processed with the new protocol and only in 20 extracted with proteinase K/phenol/chloroform. For TCRB, the success rate was 29 of 30 with the new protocol, compared to 11 of 30 with conventional protocol. The proposed method of DNA extraction should improve the value of T-cell clonality assay, because insufficient DNA quality and quantity may bias analysis towards monoclonality and therefore cause false-positive results.

  13. Monoclonal antibodies (MT1, MT2, MB1, MB2, MB3) reactive with leukocyte subsets in paraffin-embedded tissue sections.

    PubMed Central

    Poppema, S.; Hollema, H.; Visser, L.; Vos, H.

    1987-01-01

    The absence of reactivity on routinely prepared tissue sections has hampered the use of monoclonal antileukocyte antibodies in diagnostic histopathology. Here we describe five new antibodies reactive with leukocyte subsets in formaldehyde-fixed, paraffin-embedded tissue sections. Antibody MT1 is reactive with mature and immature T cells and not with mature B cells. MT2 is reactive with mature T cells and B cells, but not with immature T cells, activated T cells, and germinal center B cells. Antibody MB1 is reactive with all B cells, with about 50% of mature T cells, and not with immature T cells. MB2 is reactive with all B cells and not with T cells. However, MB2 also stains endothelial cells and several types of epithelial cells. MB3 is reactive with B cells and histiocytes, but not with T cells. The antibodies were tested on a series of lymphomas that were also immunophenotyped with a panel of well-established reagents on frozen tissue sections. The results indicate that the MB and MT antibodies are useful tools in the study of reactive and neoplastic disorders of the lymphoid system. Images Figure 1 Figure 6 Figure 7 Figure 2 Figure 3 Figure 4 Figure 5 PMID:3296769

  14. Measurement of gene expression in archival paraffin-embedded tissues: development and performance of a 92-gene reverse transcriptase-polymerase chain reaction assay.

    PubMed

    Cronin, Maureen; Pho, Mylan; Dutta, Debjani; Stephans, James C; Shak, Steven; Kiefer, Michael C; Esteban, Jose M; Baker, Joffre B

    2004-01-01

    Throughout the last decade many laboratories have shown that mRNA levels in formalin-fixed and paraffin-embedded (FPE) tissue specimens can be quantified by reverse transcriptase-polymerase chain reaction (RT-PCR) techniques despite the extensive RNA fragmentation that occurs in tissues so preserved. We have developed RT-PCR methods that are sensitive, precise, and that have multianalyte capability for potential wide use in clinical research and diagnostic assays. Here it is shown that the extent of fragmentation of extracted FPE tissue RNA significantly increases with archive storage time. Probe and primer sets for RT-PCR assays based on amplicons that are both short and homogeneous in length enable effective reference gene-based data normalization for cross comparison of specimens that differ substantially in age. A 48-gene assay used to compare gene expression profiles from the same breast cancer tissue that had been either frozen or FPE showed very similar profiles after reference gene-based normalization. A 92-gene assay, using RNA extracted from three 10- micro m FPE sections of archival breast cancer specimens (dating from 1985 to 2001) yielded analyzable data for these genes in all 62 tested specimens. The results were substantially concordant when estrogen receptor, progesterone receptor, and HER2 receptor status determined by RT-PCR was compared with immunohistochemistry assays for these receptors. Furthermore, the results highlight the advantages of RT-PCR over immunohistochemistry with respect to quantitation and dynamic range. These findings support the development of RT-PCR analysis of FPE tissue RNA as a platform for multianalyte clinical diagnostic tests.

  15. Reliable PCR quantitation of estrogen, progesterone and ERBB2 receptor mRNA from formalin-fixed, paraffin-embedded tissue is independent of prior macro-dissection.

    PubMed

    Tramm, Trine; Hennig, Guido; Kyndi, Marianne; Alsner, Jan; Sørensen, Flemming Brandt; Myhre, Simen; Sørlie, Therese; Overgaard, Jens

    2013-12-01

    Gene expression analysis on messenger RNA (mRNA) purified from formalin-fixed, paraffin-embedded tissue is increasingly used for research purposes. Tissue heterogeneity may question specificity and interpretation of results from mRNA isolated from a whole slide section, and thresholds for minimal tumor content in the paraffin block or macrodissection are used to avoid contamination from non-neoplastic tissue. The aim was to test if mRNA from tissue surrounding breast cancer affected quantification of estrogen receptor α (ESR1), progesterone receptor (PGR) and human epidermal growth factor receptor 2 (ERBB2), by comparing gene expression from whole slide and tumor-enriched sections, and correlating gene expression from whole slide sections with corresponding immunohistochemistry. Gene expression, based on mRNA extracted from a training set (36 paraffin blocks) and two validation sets (133 + 1,083 blocks), were determined by quantitative reverse transcription polymerase chain reaction for all samples, as well as by microarray for 133 validation samples. In the training set, agreement between high vs. low mRNA expression from whole slide and tumor-enriched sections was absolute for ESR1 and ERBB2, and 83 % for PGR. Overall agreements, when comparing mRNA expression to immunohistochemistry, were 100 % (ERBB2), 89 % (ESR1) and 83 % (PGR), which was confirmed in the validation sets. Percentage of tumor in the sections did not influence the results. In conclusion, reliable quantification of ESR1, PGR and ERBB2 mRNA expression can be obtained from a whole slide section, and correlates well with immunohistochemistry. Prior removal of surrounding tissue was found to be unnecessary even with minimal tumor content in the section. PMID:24100522

  16. DNA extraction from formalin-fixed, paraffin-embedded tissues: protein digestion as a limiting step for retrieval of high-quality DNA.

    PubMed

    Díaz-Cano, S J; Brady, S P

    1997-12-01

    Several DNA extraction methods have been used for formalin-fixed, paraffin-embedded tissues, with variable results being reported regarding the suitability of DNA obtained from such sources to serve as template in polymerase chain reaction (PCR)-based genetic analyses. We present a method routinely used for archival material in our laboratory that reliably yields DNA of sufficient quality for PCR studies. This method is based on extended proteinase K digestion (250 micrograms/ml in an EDTA-free calcium-containing buffer supplemented with mussel glycogen) followed by phenol-chloroform extraction. Agarose gel electrophoresis of both digestion buffer aliquots and PCR amplification of the beta-globin gene tested the suitability of the retrieved DNA for PCR amplification.

  17. Optimal enhancement of in situ hybridization for the detection of porcine circovirus 2 in formalin-fixed, paraffin-wax-embedded tissues using a combined pretreatment of thermocycler and proteinase K.

    PubMed

    Kim, J; Chae, C

    2003-06-01

    Optimal enhancement of the hybridization signal was developed for the detection of porcine circovirus (PCV) 2 in formalin-fixed, paraffin-wax-embedded tissues from pigs with postweaning multisystemic wasting syndrome. The hybridization signal obtained after thermocycler pretreatment was very uniform across the section, whereas the signal obtained after either proteinase K or microwave pretreatment not only was weaker but was of variable intensity across sections. Thermocycler pretreatment combined with brief proteinase K digestion can enhance signal detection for target viral nucleic acid in formalin-fixed, paraffin-wax-embedded tissues. A strong hybridization signal was detected in the cytoplasm of macrophages and multinucleated giant cells in lymph node and spleen without background staining and morphological damage. The technical improvement results, therefore, in an identical background at the same time as an increased signal and, thus, may help detect lower levels of PCV2 DNA in formalin-fixed, paraffin-wax-embedded tissues.

  18. Observations on the contrasting reaction of some electron dense stains applied on epoxy-embedded tissue sections.