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Sample records for paraffin embedded sections

  1. Immunofluorescence on paraffin-embedded sections in evaluation of immune complex deposits in renal biopsy specimens.

    PubMed

    Wagrowska-Danilewicz, Małgorzata; Danilewicz, Marian

    2009-01-01

    The data focused on the value of immunofluorescence on paraffin-embedded sections are controversial, and it is still difficult to obtain reproducible results. The aim of our study was to evaluate the usefulness of immunofluorescence on paraffin-embedded renal section in detecting immune complex deposits in IgA nephropathy (n = 24), membranous glomerulopathy (n = 22) and lupus nephritis (n = 24). Our study revealed that direct immunofluorescence on paraffin-embedded sections pre-treated with proteinase K for 30 or 60 min is a less sensitive method than immunofluorescence on frozen sections; therefore a number of glomerulopathies may be overlooked. Immunofluorescence on paraffin sections showed dominant or co-dominant fluorescence of Riga only in 41.7% of cases of Riga nephropathy. In the studied glomerulopathies the number of positive immunofluorescences of IgA, IgG, IgM and C3 was significantly lower in immunofluorescence on paraffin sections in comparison with findings obtained from immunofluorescence on frozen sections. Irrespective of glomerular disease the rate of agreement between immunofluorescence on paraffin sections and immunofluorescence on frozen sections with respect to the presence of IgA was 56.5%, IgM - 44.4%, IgG - 73.9%, and C3 - 51.5%. In conclusion, our study revealed that immunofluorescence on paraffin sections cannot replace immunofluorescence on frozen sections in the assessment of human renal biopsies, and must be interpreted with great caution.

  2. Enabling Multiphoton and Second Harmonic Generation Imaging in Paraffin-Embedded and Histologically Stained Sections

    PubMed Central

    Monaghan, Michael G.; Kroll, Sebastian; Brucker, Sara Y.

    2016-01-01

    Nonlinear microscopy, namely multiphoton imaging and second harmonic generation (SHG), is an established noninvasive technique useful for the imaging of extracellular matrix (ECM). Typically, measurements are performed in vivo on freshly excised tissues or biopsies. In this article, we describe the effect of rehydrating paraffin-embedded sections on multiphoton and SHG emission signals and the acquisition of nonlinear images from hematoxylin and eosin (H&E)-stained sections before and after a destaining protocol. Our results reveal that bringing tissue sections to a physiological state yields a significant improvement in nonlinear signals, particularly in SHG. Additionally, the destaining of sections previously processed with H&E staining significantly improves their SHG emission signals during imaging, thereby allowing sufficient analysis of collagen in these sections. These results are important for researchers and pathologists to obtain additional information from paraffin-embedded tissues and archived samples to perform retrospective analysis of the ECM or gain additional information from rare samples. PMID:27018844

  3. Enabling Multiphoton and Second Harmonic Generation Imaging in Paraffin-Embedded and Histologically Stained Sections.

    PubMed

    Monaghan, Michael G; Kroll, Sebastian; Brucker, Sara Y; Schenke-Layland, Katja

    2016-06-01

    Nonlinear microscopy, namely multiphoton imaging and second harmonic generation (SHG), is an established noninvasive technique useful for the imaging of extracellular matrix (ECM). Typically, measurements are performed in vivo on freshly excised tissues or biopsies. In this article, we describe the effect of rehydrating paraffin-embedded sections on multiphoton and SHG emission signals and the acquisition of nonlinear images from hematoxylin and eosin (H&E)-stained sections before and after a destaining protocol. Our results reveal that bringing tissue sections to a physiological state yields a significant improvement in nonlinear signals, particularly in SHG. Additionally, the destaining of sections previously processed with H&E staining significantly improves their SHG emission signals during imaging, thereby allowing sufficient analysis of collagen in these sections. These results are important for researchers and pathologists to obtain additional information from paraffin-embedded tissues and archived samples to perform retrospective analysis of the ECM or gain additional information from rare samples.

  4. Direct immunofluorescence of skin using formalin-fixed paraffin-embedded sections.

    PubMed Central

    Mera, S L; Young, E W; Bradfield, J W

    1980-01-01

    The technique of direct immunofluorescence has been applied to skin biopsy specimens fixed in formalin and embedded in paraffin wax. The results have been compared with those obtained by using snap-frozen biopsy specimens from the same patients. Trypsinisation of the dewaxed material allowed subsequent detection of immunoglobulins, complement, and fibrinogen. When compared to the fluorescence in the snap-frozen specimens, the staining in the paraffin sections was less bright and there was a higher rate of negatives. Even so, it was possible to establish the diagnosis in most cases of pemphigus, pemphigoid, and lupus erythematosus. Images Fig. 1 Fig. 2 Fig. 3 PMID:6995489

  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. 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

  7. 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. Crown Copyright © 2016. Published by Elsevier B.V. All rights reserved.

  8. 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.

  9. Immunohistochemical diagnosis of Alport's syndrome in paraffin-embedded renal sections: antigen retrieval with autoclave heating.

    PubMed

    Naito, Ichiro; Ninomiya, Yoshifumi; Nomura, Shinsuke

    2003-03-01

    Alport's syndrome (AS) is a hereditary renal disease caused by mutations in the genes encoding collagen type IV. Immunohistochemical analysis of the alpha chains of collagen type IV has been found to be useful for the diagnosis of this disease. The monoclonal antibodies (mAbs) generated by us recognize alpha 1(IV) through alpha 6(IV) chains of collagen type IV on fresh-frozen sections but not on paraffin-embedded sections. Antigen retrieval by autoclave heating has been found to restore the epitopes recognized by the mAbs; however the heating conditions had not been well established. In this study, the heating conditions were carefully examined using renal sections obtained from AS and non-AS patients. The heating was performed in an autoclave, at 105 degrees -127 degrees C for 6-8 min. During the heating, the sections were immersed in 0.2 N HCl solution (pH 0.9). Then, the mAbs were applied for 30 min, and the bound mAbs were detected using the LSAB kit. The optimal temperature for the antigen retrieval varied among specimens, and was dependent on the type of basement membrane examined. Thus, it was considered that heating at two or three different temperatures could be helpful for the precise diagnosis of AS. Adopting the antigen retrieval method could extend the possibility of immunohistochemical diagnosis of AS to cases without using fresh-frozen sections.

  10. Monoclonal antibody MAT-1 against human tyrosinase can detect melanogenic cells on formalin-fixed paraffin-embedded sections.

    PubMed

    Sato, N; Suzuki, S; Takimoto, H; Masui, S; Shibata, K; Nakano, H; Tomita, Y

    1996-04-01

    Immunohistochemical localization of tyrosinase was examined with a monoclonal antibody (MoAb MAT-1) against human tyrosinase on routine formalin-fixed paraffin-embedded sections of 3 normal skin specimens, 15 melanocytic tumors (6 pigmented nevi, 3 juvenile melanomas and 6 malignant melanomas) and 3 non-melanocytic tumors. In the melanotic melanomas, almost all tumor cells were clearly stained with the antibody. In the nevocytic nevi, the nevus cells in lower epidermis and upper dermis were positive for MoAb MAT-1, but negative in middle and lower dermis. All three juvenile melanomas, one amelanotic melanoma, and three non-melanocytic tumors were entirely negative for MoAb MAT-1. Thus, MoAb MAT-1 could recognize the cells with melanogenic activity on routine formalin-fixed paraffin-embedded sections. However, the staining quality was not adequate for normal epidermal melanocytes, indicating that small technical innovations in the immunostaining process such as formalin fixation after PBS washing are required. Nevertheless, MoAb MAT-1 can be expected to be very useful for identifying melanogenic cells on paraffin-embedded sections, because we have to date no other antibody available for it.

  11. Tissue lithography: Microscale dewaxing to enable retrospective studies on formalin-fixed paraffin-embedded (FFPE) tissue sections.

    PubMed

    Cors, Julien F; Kashyap, Aditya; Fomitcheva Khartchenko, Anna; Schraml, Peter; Kaigala, Govind V

    2017-01-01

    We present a new concept, termed tissue lithography (TL), and its implementation which enables retrospective studies on formalin-fixed paraffin-embedded tissue sections. Tissue lithography uses a microfluidic probe to remove microscale areas of the paraffin layer on formalin-fixed paraffin-embedded biopsy samples. Current practices in sample utilization for research and diagnostics require complete deparaffinization of the sample prior to molecular testing. This imposes strong limitations in terms of the number of tests as well as the time when they can be performed on a single sample. Microscale dewaxing lifts these constraints by permitting deprotection of a fraction of a tissue for testing while keeping the remaining of the sample intact for future analysis. After testing, the sample can be sent back to storage instead of being discarded, as is done in standard workflows. We achieve this microscale dewaxing by hydrodynamically confining nanoliter volumes of xylene on top of the sample with a probe head. We demonstrate micrometer-scale, chromogenic and fluorescence-based immunohistochemistry against multiple biomarkers (p53, CD45, HER2 and β-actin) on tonsil and breast tissue sections and microarrays. We achieve stain patterns as small as 100 μm × 50 μm as well as multiplexed immunostaining within a single tissue microarray core with a 20-fold time reduction for local dewaxing as compared to standard protocols. We also demonstrate a 10-fold reduction in the rehydration time, leading to lower processing times between different stains. We further show the potential of TL for retrospective studies by sequentially dewaxing and staining four individual cores within the same tissue microarray over four consecutive days. By combining tissue lithography with the concept of micro-immunohistochemistry, we implement each step of the IHC protocol-dewaxing, rehydration and staining-with the same microfluidic probe head. Tissue lithography brings a new level of versatility

  12. Immunohistochemical diagnosis of Fabry nephropathy and localisation of globotriaosylceramide deposits in paraffin-embedded kidney tissue sections.

    PubMed

    Valbuena, Carmen; Leitão, Dina; Carneiro, Fátima; Oliveira, João Paulo

    2012-02-01

    Fabry disease (FD) is a rare X-linked lysosomal storage disorder of glycosphingolipids, mostly globotriaosylceramide (Gb3). Proteinuric chronic kidney disease develops frequently, and recognition of Fabry nephropathy on a kidney biopsy may be the first clue to the underlying diagnosis. Since the accumulated glycosphingolipids are largely extracted by the paraffin-embedding procedure, the most characteristic feature of Fabry nephropathy on routine light microscopy (LM) is nonspecific cell vacuolization. To test whether residual Gb3 in kidney tissue might be exploited for the specific diagnosis of Fabry nephropathy, paraffin-embedded kidney biopsies of nine FD patients (one boy, four men, four women) and of a female carrier of a mild genetic mutation, with no evidence of Fabry nephropathy, were immunostained with an anti-Gb3 antibody. The adult biopsies were additionally co-stained with a lysosomal marker (anti-lysosomal-associated membrane protein 2 (anti-LAMP2) antibody). The distribution of Gb3 deposits was scored per cell type and compared to the histological scorings of glycosphingolipid inclusions on semi-thin sections. FD patients had residual Gb3 in all types of glomerular, tubular, interstitial and vascular kidney cells. The highest expression of LAMP2 was seen in tubular cells, but there were no meaningful associations between LAMP2 expression and prevalence of Gb3 deposits on different kidney cell types. The histological scorings of glycosphingolipid inclusions were relatively higher than the corresponding immunohistochemical scorings of Gb3 deposits. In the mildly affected female, Gb3 expression was limited to tubular cells, a pattern similar to controls. Gb3 immunostaining allows the specific diagnosis of Fabry nephropathy even in kidney biopsies routinely processed for LM.

  13. [Significance of BIOMED-2 standardized IG/TCR gene rearrangement detection in paraffin-embedded section in lymphoma diagnosis].

    PubMed

    Ai, Xiaofei; Fu, Qianqian; Wang, Jun; Zheng, Yingchun; Han, Cong; Li, Qinghua; Sun, Qi; Ru, Kun

    2014-06-01

    To explore the feasibility of detecting lymphoma with the application of BIOMED-2 standardized immunoglobulin/T cell receptor (IG/TCR) gene rearrangement system in formalin fixed paraffin-embedded (FFPE) tissue samples, and to discuss the relationship between the longest amplification fragment of extracted DNA and positive detection rate of different IGH V-J primers. DNA was extracted from 50 cases of FFPE tissue samples. Multiplex-PCR amplifications were performed and then the IG/TCR gene rearrangements were analyzed using BIOMED-2 standardized clonality analysis system. (1)When the DNA concentration was diluted to 50-100 ng/μl from 100-500 ng/μl, the proportion of the longest amplification fragment (300-400 bp) of DNA was improved from 10.0% to 90.0% in 30 cases of diffuse large B cell lymphoma (DLBCL) wax roll samples (P<0.01). The positive rate of IGH+IGK was increased from 46.7% to 83.3%, the difference was statistically significant (P=0.006). The lengths of the longest amplification fragments of DNA were all longer than 300 bp in the paraffin section samples of DLBCL. The positive rate of IGH+IGK of these samples was 96.7%. The difference of the positive rate of IGH+IGK between the wax roll samples and the paraffin section samples had no statistical significance (P=0.195). (2)When the concentration of DNA was high, most of the longest amplification fragments of extracted DNA were 100 bp or 200 bp, and the detection rate of short fragment IGH FR3 was more stable than that of long fragment IGH FR1. (3)The clonality analysis of TCRG+TCRB in all 13 cases of peripheral T cell lymphoma samples showed positive results, while no positive IG/TCR clones were found in 7 cases of reactive lymphoid tissue hyperplasia in control group. Dilution of DNA is the only method to improve not only the proportion of longest fragment amplification but also the detection rate of clonality. The detection rate of IGH FR3 would not be affected by the concentration of DNA. The

  14. Development of molecular methods for the identification of aspergillus and emerging moulds in paraffin wax embedded tissue sections.

    PubMed

    Paterson, P J; Seaton, S; McLaughlin, J; Kibbler, C C

    2003-12-01

    Invasive infection with emerging moulds is increasing in incidence and reliable methods for speciating these organisms in tissue sections need to be developed. Two methods for extracting fungal DNA from paraffin wax embedded tissue sections, based on the TaKaRa DEXPAT kit and QIAamp DNA mini kit, were optimised and compared. DNA was amplified by PCR using pan-fungal probes, and detected by Southern blot hybridisation using a high stringency method with a probe specific for Aspergillus fumigatus and A flavus. The method based on the TaKaRa DEXPAT kit, with additional steps using lyticase and ethanol precipitation, was superior. Less than 10 conidia were detectable using spiked samples and a positive result was obtained with 100% of clinical samples known to be culture positive for A fumigatus. Other moulds could be identified by using species specific probes or by sequencing PCR products. The method based on the TaKaRa DEXPAT kit could detect less than 10 conidia/sample. The method allowed accurate identification of A fumigatus and A flavus and other species could be identified using species specific probes or by DNA sequencing. These methods will provide a valuable diagnostic tool for both patient management and future antifungal and epidemiological studies.

  15. 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

  16. Multilabel immunofluorescence and antigen reprobing on formalin-fixed paraffin-embedded sections: novel applications for precision pathology diagnosis.

    PubMed

    Pan, Jie; Thoeni, Cornelia; Muise, Aleixo; Yeger, Herman; Cutz, Ernest

    2016-06-01

    We report new methods for multilabel immunofluorescence (MIF) and reprobing of antigen epitopes on the same formalin-fixed paraffin-embedded (FFPE) sections. The MIF method includes an antigen-retrieval step followed by multilabel immunostaining and examination by confocal microscopy. As examples, we illustrate epitopes localized to the apical and basolateral membranes, and the cytoplasm of enterocytes of normal small intestine and in cases of congenital enteropathies (microvillous inclusion disease and congenital tufting enteropathy). We also demonstrate localization of the bile salt excretion pump protein (BSEP) in bile canalicular membrane of normal hepatocytes and in cases of primary sclerosing cholangitis. To demonstrate colocalization of cytoplasmic and nuclear epitopes we analyzed normal control and hyperplastic pulmonary neuroendocrine cells (PNEC) and neuroepithelial bodies (NEBs), presumed airway sensors in the lungs of infants with bronchopulmonary dysplasia (BPD). As cytoplasmic markers we used anti-bombesin or anti-synaptic vesicle protein 2 (SV2) antibody, respectively, and for nuclear localization, antibodies against neurogenic genes mammalian achaete-scute homolog (Mash1) and prospero homeobox 1 (Prox1), essential for NEB cells differentiation and maturation, hypoxia-inducible factor 1α (HIF1α) a downstream modulator of hypoxia response and a proliferation marker Ki67. The reprobing method consisted of removal of the previously immunolabeled target and immunostaining with different antibodies, facilitating colocalization of enterocyte brush border epitopes as well as HIF1α, Mash1 and Prox1 in PNEC/NEB PNEC and NEBs. As these methods are suitable for routine FFPE pathology samples from various tissues, allowing visualization of multiple epitopes in the same cells/sections with superior contrast and resolution, they are suitable for a wide range of applications in diagnostic pathology and may be particularly well suited for precision medicine

  17. Replacing xylene with n-heptane for paraffin embedding.

    PubMed

    Stockert, J C; López-Arias, B; Del Castillo, P; Romero, A; Blázquez-Castro, A

    2012-10-01

    In standard histological technique, aromatic solvents such as xylene and toluene are used as clearing agents between ethanol dehydration and paraffin embedding. In addition, these solvents are used for de-waxing paraffin sections. Unfortunately, these solvents are harmful and therefore adequate substitutes would be useful. We suggest the use of n-heptane as a convenient substitute for xylene. Paraffin sections of rat tissues processed with n-heptane and stained with hematoxylin-eosin or Masson's trichrome showed proper embedment, well preserved morphology and excellent staining.

  18. Molecular Evaluation of t(14;18)(bcl-2/IgH) Translocation in Follicular Lymphoma at Diagnosis Using Paraffin-Embedded Tissue Sections

    PubMed Central

    Selvi, Nur; Kosova, Buket; Hekimgil, Mine; Gündüz, Cumhur; Kaymaz, Burçin Tezcanlı; Karaca, Emin; Saydam, Güray; Tombuloğlu, Murat; Büyükkeçeci, Filiz; Çağırgan, Seçkin; Ertan, Yeşim; Topçuoğlu, Nejat

    2012-01-01

    Objective: Follicular lymphoma (FL) is one of the most common lymphomas, and is characterized by t(14;18)(q32;q21) in more than 80% of patients. The aim of this study was to determine the rate of t(14;18) positivity based onthe detection of mbr or mcr in paraffin-embedded tissue samples. Material and Methods: The study included 32 paraffin-embedded tissue samples collected from 32 consecutive FL patients that were diagnosed and followed-up at our hospital between 1999 and 2006. The MBR breakpoint wasidentified based on real-time PCR using a LightCycler v.2.0 t(14;18) Quantification Kit (MBR), multiplex PCR, and seminestedPCR. To identify the mcr breakpoint, real-time PCR was performed using specific primers and the FastStart DNAMaster SYBR Green I Kit. To detect t(14;18) via fluorescence in situ hybridization (FISH) nuclei from paraffin-embeddedtissue sections were extracted and used together with LSI IgH (immunoglobulin heavy chain) (spectrum green)/bcl-2(B-cell leukemia-lymphoma 2) (spectrum orange) probes. Results: The DNA and nuclei isolation success rate for B5 formalin-fixed, paraffin-embedded tissue sections (n = 12)was 42% and 33%, respectively, versus 95% and 60%, respectively, for 20 tissue sections fixed in formalin only. In all,24 paraffin-embedded tissue sections were analyzed and mbr positivity was observed in the DNA of 82.14% via seminested PCR, in 53.57% via multiplex PCR, and in 28.57% via real-time PCR. We did not detect mcr rearrangementin any of the samples. In all, 15 of 16 patients (93.75%) whose nuclei were successfully isolated were observed to bet(14;18) positive via the FISH method. Conclusion: Semi-nested PCR and FISH facilitated the genetic characterization of FL tumors. As such, FISH and PCR complement each other and are both essential for detecting t(14;18) translocation. PMID:24744643

  19. A new and improved "quick-hot Gram-chromotrope" technique that differentially stains microsporidian spores in clinical samples, including paraffin-embedded tissue sections.

    PubMed

    Moura, H; Schwartz, D A; Bornay-Llinares, F; Sodré, F C; Wallace, S; Visvesvara, G S

    1997-08-01

    This report describes a new and improved "quick-hot Gram-chromotrope" staining technique that detects microsporidian spores in clinical specimens, such as stool, urine, saliva, nasopharyngeal fluid, and bronchoalveolar lavage samples, as well as in formalin-fixed and paraffin-embedded tissue sections. In this procedure, the samples are stained in heated (50 degrees C to 55 degrees C) solutions of crystal violet and iodine used in Gram's stain, followed by a modified chromotrope solution (heated to 50 degrees C to 55 degrees C). The modified stain is composed of chromotrope 2R (1%), fast green (0.15%), and phosphotungstic acid (0.25%). With this stain and the new protocol, microsporidian spores are stained dark violet against a pale green background, and the total staining time is shortened to 5 minutes. This new technique is fast, reliable, and simple. It can be easily adapted for use in clinical laboratories.

  20. Detection and identification of Mycobacterium species by polymerase chain reaction (PCR) from paraffin-embedded tissue compare to AFB staining in pathological sections.

    PubMed

    Mahaisavariya, Punkae; Chaiprasert, Angkana; Manonukul, Jane; Khemngern, Supakan; Tingtoy, Nipa

    2005-01-01

    Polymerase chain reaction (PCR) is a recent, rapid and reliable method in the detection of causative organism. The authors tried to determine the possibility of using PCR technique as an alternative way to detect mycobacterial DNA from paraffin-embedded tissue to avoid repeated biopsy from the patient. Paraffin-embedded tissue blocks, the corresponding histopathologic slides, and cultural results were retrospectively searched for according to the patient's records, the granuloma clinic, Department of Dermatology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand from 1994-2000. One hundred and thirty-one tissue blocks and slides were found but only 120 cultural results were retrieved Histologic sections were reviewed for AFB findings and PCR was done using 16S rRNA sequences to detect M. tuberculosis by one-tube nested technique and multiplex PCR for M. marinum and M. fortuitum complex. The causative organisms were identified by AFB staining in pathologic sections 31.29%, by PCR 35.87%, and by culture 30.00% of tested samples. The sensitivity of PCR when compared to AFB result was 29.26%, specificity 61.11% but when compared to cultural results, the sensitivity of PCR was 66.67% and AFB sensitivity was 41.66% with specificity 76.19% and 72.61% respectively. The low sensitivity of the PCR method may be due to formalin fixation, deparaffinization process, DNA extraction method, the use of 16S rRNA-based primers and the length of the expected product, and the tissue type that may have Taq polymerase inhibitor. Therefore, PCR should be used to augment the information of the conventional method in the diagnosis of mycobacterial infection.

  1. An optimized xylene-free protein extraction method adapted to formalin-fixed paraffin embedded tissue sections for western blot analysis.

    PubMed

    Mansour, Anthony G; Khalil, Pamela Abou; Bejjani, Noha; Chatila, Rajaa; Dagher-Hamalian, Carole; Faour, Wissam H

    2017-03-01

    Deparaffinization of formalin-fixed paraffin embedded (FFPE) tissues with xylene currently remains a major challenge to the biomedical community. We developed an efficient xylene-free protocol to isolate proteins from archived FFPE human tissue sections. A total of 79 different types of FFPE tissue sections of 8 µm thickness were obtained from various archived FFPE specimens. Deparaffinization was conducted by gently washing each section with around 1 ml of hot distilled water (≈80°C). The deparaffinized tissues were homogenized in lysis buffer, and the isolated proteins were quantified and efficiently resolved using western blot analysis for the presence of Protein kinase B (PKB/AKT) and β-actin. Moreover, a significant amount of proteins was successfully isolated with an average of 2.31 µg/µl. The migration pattern of AKT and β-actin obtained from the specimens was similar to the positive control obtained from protein lysates prepared from in vitro cultured MDA231 cancer cell lines. AKT was successfully identified in all specimens, and β-actin protein was resolved with an efficiency higher than 80%. The entire extraction procedure requires only 20 minutes. This newly developed technique is an efficient, safe, cost-effective, and rapid method to isolate proteins from FFPE tissue sections adequate for molecular analysis.

  2. Rapid, sensitive, type specific PCR detection of the E7 region of human papillomavirus type 16 and 18 from paraffin embedded sections of cervical carcinoma.

    PubMed

    Lesnikova, Iana; Lidang, Marianne; Hamilton-Dutoit, Steven; Koch, Jørn

    2010-01-22

    Human papillomavirus (HPV) infection, and in particularly infection with HPVs 16 and 18, is a central carcinogenic factor in the uterine cervix. We established and optimized a PCR assay for the detection and discrimination of HPV types 16 and 18 in archival formaldehyde fixed and paraffin embedded (FFPE) sections of cervical cancer.Tissue blocks from 35 cases of in situ or invasive cervical squamous cell carcinoma and surrogate FFPE sections containing the cell lines HeLa and SiHa were tested for HPV 16 and HPV18 by conventional PCR using type specific primers, and for the housekeeping gene beta-actin. Using HPV 16 E7 primers, PCR products with the expected length were detected in 18 of 35 of FFPE sections (51%). HPV 18 E7 specific sequences were detected in 3 of 35 FFPE sections (9%).In our experience, the PCR technique is a robust, simple and sensitive way of type specific detection of HPV16 and HPV18 genes in FFPE tissue. That makes this technique applicable to routine practices of HPV detection.

  3. Immunohistochemical detection of EGFR in paraffin-embedded tumor tissues: variation in staining intensity due to choice of fixative and storage time of tissue sections.

    PubMed

    Atkins, Derek; Reiffen, Karl-August; Tegtmeier, Conny Lund; Winther, Henrik; Bonato, Marcellus S; Störkel, Stephan

    2004-07-01

    The epidermal growth factor receptor (EGFR) is highly expressed in a variety of solid malignant tumors and its expression has been correlated with disease progression and poor survival. With the advent of targeted therapies, especially IMC-C225 (Cetuximab), a monoclonal antibody (MAb) directed against the EGFR, there is an increasing interest in immunohistochemistry (IHC)-based EGFR screening methods using paraffin-embedded tumor specimens to select cancer patients eligible for treatment with Cetuximab. With the EGFRpharmDX kit, a complete assay for demonstration of EGFR is now available. Because no information about the preservation of the EGFR under various conditions of fixation is available, we performed a prospective study on a panel of commonly used fixatives to determine optimal tissue preservation protocols. The stability of the epitope on cut tissue sections stored for a period up to 24 month was also tested using material originating from patients with head and neck cancer, non-small-cell lung carcinomas, and colorectal adenocarcinomas. Depending on the fixative used and the time of storage of cut tissue sections, a variation in the determined level of EGFR expression was demonstrated compared with the most optimal fixation procedure.

  4. FISH is better than BIOMED-2 PCR to detect IgH/BCL2 translocation in follicular lymphoma at diagnosis using paraffin-embedded tissue sections.

    PubMed

    Espinet, Blanca; Bellosillo, Beatriz; Melero, Carme; Vela, Ma Carmen; Pedro, Carmen; Salido, Marta; Pijuan, Lara; Florensa, Lourdes; Besses, Carles; Serrano, Sergi; Solé, Francesc

    2008-05-01

    The most common genetic aberration in follicular lymphoma (FL) is the t(14;18)(q32;q21) translocation that juxtaposes the antiapoptotic BCL2 gene with the promoter of the immunoglobulin heavy chain (IgH) gene. Our aim was to test the usefulness of two different techniques, fluorescence in situ hybridization (FISH) and PCR to detect t(14;18) in FL at diagnosis in paraffin-embedded tissue sections. A total of 51 patients diagnosed of FL were analyzed. FISH was performed with dual color dual fusion commercial probes (VYSIS) and in PCR experiments, the BIOMED-2 primers covering MBR, mcr and 3'MBR regions were applied. FISH showed positivity for the IgH/BCL2 translocation in 96% of patients and PCR in 59% of patients. FISH was able to detect variant translocations involving light chain Ig, or showing variant patterns such as deletions of the IgH portion involved in translocation. In 4% of cases, the IgH/BCL2 translocation was not detected by any of the two techniques tested. Our results show that FISH represents the best technique to detect t(14;18) at diagnosis.

  5. Mass Spectrometry Imaging of Drug Related Crystal-Like Structures in Formalin-Fixed Frozen and Paraffin-Embedded Rabbit Kidney Tissue Sections

    NASA Astrophysics Data System (ADS)

    Bruinen, Anne L.; van Oevelen, Cateau; Eijkel, Gert B.; Van Heerden, Marjolein; Cuyckens, Filip; Heeren, Ron M. A.

    2016-01-01

    A multimodal mass spectrometry imaging (MSI) based approach was used to characterize the molecular content of crystal-like structures in a frozen and paraffin embedded piece of a formalin-fixed rabbit kidney. Matrix assisted laser desorption/ionization time-of-flight (MALDI-TOF) imaging and desorption electrospray ionization (DESI) mass spectrometry imaging were combined to analyze the frozen and paraffin embedded sample without further preparation steps to remove the paraffin. The investigated rabbit kidney was part of a study on a drug compound in development, in which severe renal toxicity was observed in dosed rabbits. Histological examination of the kidney showed tubular degeneration with precipitation of crystal-like structures in the cortex, which were assumed to cause the renal toxicity. The MS imaging approach was used to find out whether the crystal-like structures were composed of the drug compound, metabolites, or an endogenous compound as a reaction to the drug administration. The generated MALDI-MSI data were analyzed using principal component analysis. In combination with the MS/MS results, this way of data processing demonstrates that the crystal structures were mainly composed of metabolites and relatively little parent drug.

  6. Mass Spectrometry Imaging of Drug Related Crystal-Like Structures in Formalin-Fixed Frozen and Paraffin-Embedded Rabbit Kidney Tissue Sections.

    PubMed

    Bruinen, Anne L; van Oevelen, Cateau; Eijkel, Gert B; Van Heerden, Marjolein; Cuyckens, Filip; Heeren, Ron M A

    2016-01-01

    A multimodal mass spectrometry imaging (MSI) based approach was used to characterize the molecular content of crystal-like structures in a frozen and paraffin embedded piece of a formalin-fixed rabbit kidney. Matrix assisted laser desorption/ionization time-of-flight (MALDI-TOF) imaging and desorption electrospray ionization (DESI) mass spectrometry imaging were combined to analyze the frozen and paraffin embedded sample without further preparation steps to remove the paraffin. The investigated rabbit kidney was part of a study on a drug compound in development, in which severe renal toxicity was observed in dosed rabbits. Histological examination of the kidney showed tubular degeneration with precipitation of crystal-like structures in the cortex, which were assumed to cause the renal toxicity. The MS imaging approach was used to find out whether the crystal-like structures were composed of the drug compound, metabolites, or an endogenous compound as a reaction to the drug administration. The generated MALDI-MSI data were analyzed using principal component analysis. In combination with the MS/MS results, this way of data processing demonstrates that the crystal structures were mainly composed of metabolites and relatively little parent drug.

  7. Diagnostic accuracy of morphologic identification of filamentous fungi in paraffin embedded tissue sections: correlation of histological and culture diagnosis.

    PubMed

    Challa, Sundaram; Pamidi, Umabala; Uppin, Shantveer G; Uppin, Megha S; Vemu, Lakshmi

    2014-01-01

    The aim was to investigate the correlation between histological and culture diagnosis of filamentous fungi. Tissue sections from biopsy samples stained with Hematoxylin and Eosin and special stains from samples of chronic invasive/noninvasive sinusitis and intracranial space occupying lesions during 2005-2011 diagnosed to have infection due to filamentous fungi were reviewed. The histopathology and culture diagnoses were analyzed for correlation and discrepancy. There were 125 samples positive for filamentous fungi on biopsy. Of these 76 (60.8%) were submitted for culture and fungi grew in 30 (39.97%) samples. There was a positive correlation between histological and culture diagnosis in 25 (83.33%) samples that included Aspergillus species (16/19), Zygomycetes species (8/10) and dematiaceous fungi (1/1). The negative yield of fungi was more in Zygomycetes species (20/30) when compared to Aspergillus species (25/44). There was a discrepancy in diagnosis in 5/30 (16.67%) samples which included probable dual infection in two, and dematiaceous fungi being interpreted as Aspergillus species in three samples. Histopathology plays a major role in the diagnosis of infection due to filamentous fungi, especially when cultures are not submitted or negative. The discrepancy between histological and culture diagnosis was either due to dematiaceous fungi being interpreted as Aspergillus species or probable dual infection.

  8. Applicability of a System for fully automated nucleic acid extraction from formalin-fixed paraffin-embedded sections for routine KRAS mutation testing.

    PubMed

    Lehmann, Annika; Schewe, Christiane; Hennig, Guido; Denkert, Carsten; Weichert, Wilko; Budczies, Jan; Dietel, Manfred

    2012-06-01

    Due to the approval of various new targeted therapies for the treatment of cancer, molecular pathology laboratories with a diagnostic focus have to meet new challenges: simultaneous handling of a large number of samples, small amounts of input material, and fragmentation of nucleic acids because of formalin fixation. As a consequence, fully automated systems for a fast and standardized extraction of high-quality DNA from formalin-fixed paraffin-embedded (FFPE) tissues are urgently needed. In this study, we tested the performance of a fully automated, high-throughput method for the extraction of nucleic acids from FFPE tissues. We investigated the extraction performance in sections of 5 different tissue types often analyzed in routine pathology laboratories (cervix, colon, liver, lymph node, and lung; n=340). Furthermore, we compared the quality, labor input, and applicability of the method for diagnostic purposes with those of a laboratory-validated manual method in a clinical setting by screening a set of 45 colorectal adenocarcinoma for the KRAS mutation. Automated extraction of both DNA and RNA was successful in 339 of 340 FFPE samples representing 5 different tissue types. In comparison with a conventional manual extraction protocol, the method showed an overall agreement of 97.7% (95% confidence interval, 88.2%-99.9%) for the subsequent mutational analysis of the KRAS gene in colorectal cancer samples. The fully automated system is a promising tool for a simple, robust, and rapid extraction of DNA and RNA from formalin-fixed tissue. It ensures a standardization of sample processing and can be applied to clinical FFPE samples in routine pathology.

  9. 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

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

    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.

  10. 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.

  11. Quick and inexpensive paraffin-embedding method for dynamic bone formation analyses

    PubMed Central

    Porter, Amy; Irwin, Regina; Miller, Josselyn; Horan, Daniel J.; Robling, Alexander G.; McCabe, Laura R.

    2017-01-01

    We have developed a straightforward method that uses paraffin-embedded bone for undemineralized thin sectioning, which is amenable to subsequent dynamic bone formation measurements. Bone has stiffer material properties than paraffin, and therefore has hereforto usually been embedded in plastic blocks, cured and sectioned with a tungsten carbide knife to obtain mineralized bone sections for dynamic bone formation measures. This process is expensive and requires special equipment, experienced personnel, and time for the plastic to penetrate the bone and cure. Our method utilizes a novel way to prepare mineralized bone that increases its compliance so that it can be embedded and easily section in paraffin blocks. The approach is simple, quick, and costs less than 10% of the price for plastic embedded bone sections. While not effective for static bone measures, this method allows dynamic bone analyses to be readily performed in laboratories worldwide which might not otherwise have access to traditional (plastic) equipment and expertise. PMID:28198415

  12. Immunodetection of NETs in Paraffin-Embedded Tissue

    PubMed Central

    Brinkmann, Volker; Abu Abed, Ulrike; Goosmann, Christian; Zychlinsky, Arturo

    2016-01-01

    The pathogenic potential of neutrophil extracellular traps (NETs) was recently described, and their detection in tissue could serve as a prognostic marker. NETs are delicate and filigree structures; hence good tissue preservation is essential for their detection. Indeed, analysis of paraffin-embedded tissue has proven superior to the study of cryo sections. Though, under favorable conditions, the presence of NETs can be detected in tissue sections stained with histological dyes, definitive identification of NETs needs the colocalization of immunofluorescent signals for both nuclear and granular (or cytoplasmic) NET components. We tested diverse antigen retrieval methods and various combinations of commercially available antibodies and present here staining protocols to detect NETs in human and murine tissue sections. PMID:27920776

  13. Paraffin wax embedded muscle is suitable for the diagnosis of muscular dystrophy

    PubMed Central

    Sheriffs, I; Rampling, D; Smith, V

    2001-01-01

    Aim—At present, the diagnosis of muscular dystrophy is made by means of immunohistochemistry on frozen sections. The aim of this study was to develop a sensitive and reproducible immunohistochemical method for use on formalin fixed, paraffin wax embedded sections for the demonstration of dystrophin associated proteins and other muscle associated antigens. Methods—All the cases studied were from the files of the department of histopathology, Great Ormond Street Hospital for Children NHS Trust. Immunohistochemistry was performed on paraffin wax embedded sections with heat mediated antigen retrieval and overnight incubation with the antibodies at room temperature. Four different pretreatment buffers were tested in the attempt to optimise the immunostaining. Frozen sections were run in parallel for direct comparison. Results—All the antibodies except δ sarcoglycan gave strong, consistent immunostaining in paraffin wax embedded sections, comparable with the frozen sections. The most consistent results were obtained using citrate/EDTA as the pretreatment buffer. Conclusion—A reliable and reproducible technique has been established, using a heat mediated citrate/EDTA buffer antigen retrieval method, which works well for most of the antibodies needed to make the diagnosis of muscular dystrophy in formalin fixed, paraffin wax embedded sections. This technique overcomes some of the inherent problems encountered using frozen muscle tissue and it could become a valuable tool for the diagnosis of muscular dystrophy. Key Words: immunohistochemistry • muscle biopsies • muscular dystrophy • paraffin wax sections PMID:11429422

  14. Fluorescence in situ hybridisation analysis of formalin-fixed paraffin-embedded tissue sections in the diagnostic work-up of non-Burkitt high grade B-cell non-Hodgkin's lymphoma: a single centre's experience.

    PubMed

    Foot, Nicola J; Dunn, Robert G; Geoghegan, Helen; Wilkins, Bridget S; Neat, Michael J

    2011-09-01

    In recent years the genetic aberrations associated with diffuse large B-cell lymphoma and the new subtype described in the 2008 revision of the WHO classification, 'B-cell lymphoma, unclassifiable, with features intermediate between diffuse large B-cell lymphoma and Burkitt lymphoma' have been increasingly well defined. Recurrent genetic abnormalities include rearrangements involving MYC (8q24), BCL2 (18q21) and BCL6 (3q27); as the prognostic and therapeutic implications associated with these abnormalities are clarified their accurate identification at diagnosis is becoming increasingly critical. We describe our experience of using a panel of fluorescence in situ hybridisation (FISH) probes on formalin-fixed paraffin-embedded tissue sections in the diagnostic work-up of 162 patients with non-Burkitt high grade B-cell non-Hodgkin's lymphomas (HG-BNHL). BCL6, IGH-BCL2 and MYC status were determined prospectively in sequential patients presenting with HG-BNHL, with respect to the presence of rearrangements and copy number changes. Small numbers of samples were analysed retrospectively or were studied at relapse in previously untested patients. FISH analysis was successful in 160/162 (99%) cases, with abnormalities detected in 118/160 (74%). FISH analysis of formalin-fixed paraffin-embedded tissue sections is a highly reproducible technique with an excellent success rate for the detection of genetic abnormalities which will play an increasingly important role in improving risk stratification of patients with HG-BNHL.

  15. Identification of human papillomaviruses from formalin-fixed, paraffin-embedded pre-cancer and invasive cervical cancer specimens in Zambia: a cross-sectional study.

    PubMed

    Bateman, Allen C; Katundu, Katundu; Polepole, Pascal; Shibemba, Aaron; Mwanahamuntu, Mulindi; Dittmer, Dirk P; Parham, Groesbeck P; Chibwesha, Carla J

    2015-01-16

    The most common human papillomavirus (HPV) genotypes isolated from cervical cancer in select African countries are HPV-16, HPV-18, HPV-35, and HPV-45, but the most common genotypes in Zambia are unknown. The overall objective of this study was to assess the potential impact of current HPV vaccines in preventing cervical cancer in Zambia, by determining the combined prevalence of HPV-16 and/or HPV-18 in invasive cervical cancer (ICC) and high-grade pre-cancer [cervical intraepithelial neoplasia 2 or 3 (CIN2/3)] cases. We compared DNA extraction techniques to determine which assay performs well in the Zambian context, where unbuffered formalin is used to fix specimens. We then tested specimens with the Abbott RealTime High-Risk HPV test to estimate the prevalence of HPV-16/18 in formalin-fixed, paraffin-embedded ICC and CIN2/3 specimens. DNA extraction using heat (without xylene) was more successful than xylene-based extraction. Over 80% of specimens tested using heat extraction and the Abbott RealTime HPV test were positive for HPV. HPV-16 and/or HPV-18 were identified in 65/93 (69.9%) ICC specimens positive for HPV and in 38/65 (58.5%) CIN2/3 specimens positive for HPV. To our knowledge this is the first report to identify HPV genotypes in cervical cancers in Zambia. A combined HPV-16/18 prevalence of 69.9% in ICC specimens suggests that current vaccines will be highly protective against cervical cancer in Zambia.

  16. Paraffin wax embedded muscle is suitable for the diagnosis of muscular dystrophy.

    PubMed

    Sheriffs, I N; Rampling, D; Smith, V V

    2001-07-01

    At present, the diagnosis of muscular dystrophy is made by means of immunohistochemistry on frozen sections. The aim of this study was to develop a sensitive and reproducible immunohistochemical method for use on formalin fixed, paraffin wax embedded sections for the demonstration of dystrophin associated proteins and other muscle associated antigens. All the cases studied were from the files of the department of histopathology, Great Ormond Street Hospital for Children NHS Trust. Immunohistochemistry was performed on paraffin wax embedded sections with heat mediated antigen retrieval and overnight incubation with the antibodies at room temperature. Four different pretreatment buffers were tested in the attempt to optimise the immunostaining. Frozen sections were run in parallel for direct comparison. All the antibodies except delta sarcoglycan gave strong, consistent immunostaining in paraffin wax embedded sections, comparable with the frozen sections. The most consistent results were obtained using citrate/EDTA as the pretreatment buffer. A reliable and reproducible technique has been established, using a heat mediated citrate/EDTA buffer antigen retrieval method, which works well for most of the antibodies needed to make the diagnosis of muscular dystrophy in formalin fixed, paraffin wax embedded sections. This technique overcomes some of the inherent problems encountered using frozen muscle tissue and it could become a valuable tool for the diagnosis of muscular dystrophy.

  17. Immunolocalization of MAP-2 in Routinely Formalin-Fixed, Paraffin-Embedded Guinea Pig Brain Sections Using Microwave Irradiation: A Comparison of Different Combinations of Antibody Clones and Antigen Retrieval Buffer Solutions

    NASA Astrophysics Data System (ADS)

    Kan, Robert K.; Pleva, Christina M.; Hamilton, Tracey A.; Petrali, John P.

    2005-04-01

    The present study was designed to evaluate the efficacy of different microwave pretreatment methods to retrieve microtubule-associated protein 2 (MAP-2) immunoreactivity in formalin-fixed, paraffin-embedded guinea pig brain sections. Brain sections, microwave pretreated in boiling sodium citrate, citric acid, Tris hydrochloride, and EDTA buffers of pH 4, 6, and 8, were labeled with four different clones of MAP-2 monoclonal antibodies. No MAP-2 immunoreactivity was observed in control sections processed without microwave pretreatment. Optimal MAP-2 immunoreactivity was observed only when MAP-2 antibody clone AP18 was used in conjunction with citric acid buffer of pH 6.0. Using this combination, brain sections from nerve agent soman-exposed guinea pigs were found to exhibit marked reduction in MAP-2 immunostaining in the hippocampus. These observations suggest that the clone of the antibody in addition to the type and pH of antigen retrieval (AR) solution are important variables to be considered for establishing an optimal AR technique. When studying counterpart antigens of species other than that to which the antibodies were originally raised, different antibody clones must be tested in combination with different microwave-assisted AR (MAR) methods. This MAR method makes it possible to conduct retrospective studies on archival guinea pig brain paraffin blocks to evaluate changes in neuronal MAP-2 expression as a consequence of chemical warfare nerve agent toxicity.

  18. Immunolocalization of MAP-2 in routinely formalin-fixed, paraffin-embedded guinea pig brain sections using microwave irradiation: a comparison of different combinations of antibody clones and antigen retrieval buffer solutions.

    PubMed

    Kan, Robert K; Pleva, Christina M; Hamilton, Tracey A; Petrali, John P

    2005-04-01

    The present study was designed to evaluate the efficacy of different microwave pretreatment methods to retrieve microtubule-associated protein 2 (MAP-2) immunoreactivity in formalin-fixed, paraffin-embedded guinea pig brain sections. Brain sections, microwave pretreated in boiling sodium citrate, citric acid, Tris hydrochloride, and EDTA buffers of pH 4, 6, and 8, were labeled with four different clones of MAP-2 monoclonal antibodies. No MAP-2 immunoreactivity was observed in control sections processed without microwave pretreatment. Optimal MAP-2 immunoreactivity was observed only when MAP-2 antibody clone AP18 was used in conjunction with citric acid buffer of pH 6.0. Using this combination, brain sections from nerve agent soman-exposed guinea pigs were found to exhibit marked reduction in MAP-2 immunostaining in the hippocampus. These observations suggest that the clone of the antibody in addition to the type and pH of antigen retrieval (AR) solution are important variables to be considered for establishing an optimal AR technique. When studying counterpart antigens of species other than that to which the antibodies were originally raised, different antibody clones must be tested in combination with different microwave-assisted AR (MAR) methods. This MAR method makes it possible to conduct retrospective studies on archival guinea pig brain paraffin blocks to evaluate changes in neuronal MAP-2 expression as a consequence of chemical warfare nerve agent toxicity.

  19. Comparison of multivariate analysis methods for extracting the paraffin component from the paraffin-embedded cancer tissue spectra for Raman imaging

    PubMed Central

    Meksiarun, Phiranuphon; Ishigaki, Mika; Huck-Pezzei, Verena A.C.; Huck, Christian W.; Wongravee, Kanet; Sato, Hidetoshi; Ozaki, Yukihiro

    2017-01-01

    This study aimed to extract the paraffin component from paraffin-embedded oral cancer tissue spectra using three multivariate analysis (MVA) methods; Independent Component Analysis (ICA), Partial Least Squares (PLS) and Independent Component - Partial Least Square (IC-PLS). The estimated paraffin components were used for removing the contribution of paraffin from the tissue spectra. These three methods were compared in terms of the efficiency of paraffin removal and the ability to retain the tissue information. It was found that ICA, PLS and IC-PLS could remove the paraffin component from the spectra at almost the same level while Principal Component Analysis (PCA) was incapable. In terms of retaining cancer tissue spectral integrity, effects of PLS and IC-PLS on the non-paraffin region were significantly less than that of ICA where cancer tissue spectral areas were deteriorated. The paraffin-removed spectra were used for constructing Raman images of oral cancer tissue and compared with Hematoxylin and Eosin (H&E) stained tissues for verification. This study has demonstrated the capability of Raman spectroscopy together with multivariate analysis methods as a diagnostic tool for the paraffin-embedded tissue section. PMID:28327648

  20. Comparison of multivariate analysis methods for extracting the paraffin component from the paraffin-embedded cancer tissue spectra for Raman imaging.

    PubMed

    Meksiarun, Phiranuphon; Ishigaki, Mika; Huck-Pezzei, Verena A C; Huck, Christian W; Wongravee, Kanet; Sato, Hidetoshi; Ozaki, Yukihiro

    2017-03-22

    This study aimed to extract the paraffin component from paraffin-embedded oral cancer tissue spectra using three multivariate analysis (MVA) methods; Independent Component Analysis (ICA), Partial Least Squares (PLS) and Independent Component - Partial Least Square (IC-PLS). The estimated paraffin components were used for removing the contribution of paraffin from the tissue spectra. These three methods were compared in terms of the efficiency of paraffin removal and the ability to retain the tissue information. It was found that ICA, PLS and IC-PLS could remove the paraffin component from the spectra at almost the same level while Principal Component Analysis (PCA) was incapable. In terms of retaining cancer tissue spectral integrity, effects of PLS and IC-PLS on the non-paraffin region were significantly less than that of ICA where cancer tissue spectral areas were deteriorated. The paraffin-removed spectra were used for constructing Raman images of oral cancer tissue and compared with Hematoxylin and Eosin (H&E) stained tissues for verification. This study has demonstrated the capability of Raman spectroscopy together with multivariate analysis methods as a diagnostic tool for the paraffin-embedded tissue section.

  1. Comparison of multivariate analysis methods for extracting the paraffin component from the paraffin-embedded cancer tissue spectra for Raman imaging

    NASA Astrophysics Data System (ADS)

    Meksiarun, Phiranuphon; Ishigaki, Mika; Huck-Pezzei, Verena A. C.; Huck, Christian W.; Wongravee, Kanet; Sato, Hidetoshi; Ozaki, Yukihiro

    2017-03-01

    This study aimed to extract the paraffin component from paraffin-embedded oral cancer tissue spectra using three multivariate analysis (MVA) methods; Independent Component Analysis (ICA), Partial Least Squares (PLS) and Independent Component - Partial Least Square (IC-PLS). The estimated paraffin components were used for removing the contribution of paraffin from the tissue spectra. These three methods were compared in terms of the efficiency of paraffin removal and the ability to retain the tissue information. It was found that ICA, PLS and IC-PLS could remove the paraffin component from the spectra at almost the same level while Principal Component Analysis (PCA) was incapable. In terms of retaining cancer tissue spectral integrity, effects of PLS and IC-PLS on the non-paraffin region were significantly less than that of ICA where cancer tissue spectral areas were deteriorated. The paraffin-removed spectra were used for constructing Raman images of oral cancer tissue and compared with Hematoxylin and Eosin (H&E) stained tissues for verification. This study has demonstrated the capability of Raman spectroscopy together with multivariate analysis methods as a diagnostic tool for the paraffin-embedded tissue section.

  2. Diagnosis of Marek's Disease From a Japanese Quail (Coturnix Japonica) Using Paraffin-embedded Liver

    USDA-ARS?s Scientific Manuscript database

    A single paraffin-embedded liver section was submitted from a research flock of Japanese quail that had revealed focal infiltrations of immature lymphocytes within multiple visceral organs. Tumor cells were characterized as T-cells positive for Marek's disease virus (MDV) pp38 antigen by IHC dual st...

  3. 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.

  4. Immunofluorescence on paraffin embedded renal biopsies: Experience of a tertiary care center with review of literature

    PubMed Central

    Singh, Geetika; Singh, Lavleen; Ghosh, Ranajoy; Nath, Devajit; Dinda, Amit Kumar

    2016-01-01

    AIM To describe the technique of immunofluorescence on paraffin embedded tissue sections and discuss the potential pitfalls with an in depth review of literature. METHODS Immunofluorescence is integral to diagnostic renal pathology. Immunofluorescence on paraffin embedded renal biopsies (IF-P) after enzyme treatment has been described in literature, however has not found widespread use in renal pathology laboratories. In our laboratory proteinase K digestion of paraffin embedded renal biopsy material was standardized and applied prospectively in cases where immunofluorescence on fresh frozen tissue was non contributory or not possible. Diagnostic utility was assessed and in a cohort of cases comparison of intensity of staining with routine immunofluorescence was performed. RESULTS Over the 5-year study period, of the 3141 renal biopsies received IF-P was performed on 246 cases (7.7%) and was interpretable with optimal digestion in 214 cases (6.8%). It was of diagnostic utility in the majority of cases, which predominantly included glomerular disease. Non-diagnostic IF-P was found in membranous nephropathy (2 of 11 cases), membranoproliferative glomerulonephritis (2 of 32 cases), lupus nephritis (1 of 25 cases), post infectious glomerulonephritis (1 of 11 cases) and chronic glomerulonephritis (3 of 8 cases). Comparing cases with both routine IF and IF-P, 35 of 37 showed either equal intensity or a minor difference in intensity of staining (1+) for the diagnostic immunoglobulin/complement. Technically assessment of immunofluorescence on the paraffin embedded tissue was found to be easier with clearly observed morphology, however a false positive staining pattern was observed in under-digested tissue. CONCLUSION As a “salvage” technique, immunofluorescence on paraffin embedded renal biopsies is of great diagnostic utility, however not without pitfalls. PMID:27648410

  5. Analysis of hepcidin expression: In situ hybridization and quantitative polymerase chain reaction from paraffin sections

    PubMed Central

    Sakuraoka, Yuhki; Sawada, Tokihiko; Shiraki, Takayuki; Park, Kyunghwa; Sakurai, Yuhichiro; Tomosugi, Naohisa; Kubota, Keiichi

    2012-01-01

    AIM: To establish methods for quantitative polymerase chain reaction (PCR) for hepcidin using RNAs isolated from paraffin-embedded sections and in situ hybridization of hepatocellular carcinoma (HCC). METHODS: Total RNA from paraffin-embedded sections was isolated from 68 paraffin-embedded samples of HCC. Samples came from 54 male and 14 female patients with a mean age of 66.8 ± 7.8 years. Quantitative PCR was performed. Immunohistochemistry and in situ hybridization for hepcidin were also performed. RESULTS: Quantitative PCR for hepcidin using RNAs isolated from paraffin-embedded sections of HCC was performed successfully. The expression level of hepcidin mRNA in cancer tissues was significantly higher than that in non-cancer tissues. A method of in situ hybridization for hepcidin was established successfully, and this demonstrated that hepcidin mRNA was expressed in non-cancerous tissue but absent in cancerous tissue. CONCLUSION: We have established novel methods for quantitative PCR for hepcidin using RNAs isolated from paraffin-embedded sections and in situ hybridization of HCC. PMID:22851866

  6. Analysis of hepcidin expression: in situ hybridization and quantitative polymerase chain reaction from paraffin sections.

    PubMed

    Sakuraoka, Yuhki; Sawada, Tokihiko; Shiraki, Takayuki; Park, Kyunghwa; Sakurai, Yuhichiro; Tomosugi, Naohisa; Kubota, Keiichi

    2012-07-28

    To establish methods for quantitative polymerase chain reaction (PCR) for hepcidin using RNAs isolated from paraffin-embedded sections and in situ hybridization of hepatocellular carcinoma (HCC). Total RNA from paraffin-embedded sections was isolated from 68 paraffin-embedded samples of HCC. Samples came from 54 male and 14 female patients with a mean age of 66.8 ± 7.8 years. Quantitative PCR was performed. Immunohistochemistry and in situ hybridization for hepcidin were also performed. Quantitative PCR for hepcidin using RNAs isolated from paraffin-embedded sections of HCC was performed successfully. The expression level of hepcidin mRNA in cancer tissues was significantly higher than that in non-cancer tissues. A method of in situ hybridization for hepcidin was established successfully, and this demonstrated that hepcidin mRNA was expressed in non-cancerous tissue but absent in cancerous tissue. We have established novel methods for quantitative PCR for hepcidin using RNAs isolated from paraffin-embedded sections and in situ hybridization of HCC.

  7. Use of APAAP technique on paraffin wax embedded bone marrow trephines.

    PubMed Central

    Erber, W N; McLachlan, J

    1989-01-01

    The immunoalkaline phosphatase (APAAP) technique was applied to the labelling of decalcified sections of formalin fixed, paraffin wax embedded bone marrow trephine biopsy specimens. A panel of monoclonal antibodies reactive with haemopoietic and epithelial antigens, which survive routine formalin fixation, was assessed on 72 cases of haematological malignancy (including acute and chronic leukaemias and lymphomas) showing bone marrow infiltration. The APAAP method showed clear distinct labelling of antigen positive cells without loss of antigens due to decalcification. Both normal or reactive single cells present in the sample and neoplastic cell populations could be identified morphologically and their antigenic phenotype and cellular origin, whether lymphoid or myeloid, established. The application of the APAAP method to routinely prepared paraffin wax embedded trephines has many advantages over the assessment of specially prepared cryostat sections of bone marrow. Images PMID:2685054

  8. Immunofluorescent staining of influenza virus antigen in fixed and paraffin-embedded tissue of experimentally infected hamsters.

    PubMed

    Lück, P C; Helbig, J H; Witzleb, W

    1989-01-01

    Sections of formalin-fixed, paraffin-embedded tissue of experimentally influenza virus-infected hamsters were treated with 0.25% trypsin and tested for virus antigen by indirect immunofluorescent staining. The results were comparable to those obtained with aceton-fixed cryo-microtome sections. As far as we know, this is the first description of influenza virus demonstration in formalin-fixed, paraffin-embedded tissue after reactivation by trypsin-treatment. This technique may be useful for influenza virus detection in human autopsy cases. It allows an etiological diagnosis even when fresh tissue for cryocut sections or virus cultivation is not available.

  9. Automated extraction of DNA and RNA from a single formalin-fixed paraffin-embedded tissue section for analysis of both single-nucleotide polymorphisms and mRNA expression.

    PubMed

    Hennig, Guido; Gehrmann, Mathias; Stropp, Udo; Brauch, Hiltrud; Fritz, Peter; Eichelbaum, Michel; Schwab, Matthias; Schroth, Werner

    2010-12-01

    There is an increasing need for the identification of both DNA and RNA biomarkers from pathodiagnostic formalin-fixed paraffin-embedded (FFPE) tissue samples for the exploration of individualized therapy strategies in cancer. We investigated a fully automated, xylene-free nucleic acid extraction method for the simultaneous analysis of RNA and DNA biomarkers related to breast cancer. We copurified both RNA and DNA from a single 10-μm section of 210 paired samples of FFPE tumor and adjacent normal tissues (1-25 years of archival time) using a fully automated extraction method. Half of the eluate was DNase I digested for mRNA expression analysis performed by using reverse-transcription quantitative PCR for the genes estrogen receptor 1 (ESR1), progesterone receptor (PGR), v-erb-b2 erythroblastic leukemia viral oncogene homolog 2, neuro/glioblastoma derived oncogene homolog (avian) (ERBB2), epoxide hydrolase 1 (EPHX1), baculoviral IAP repeat-containing 5 (BIRC5), matrix metallopeptidase 7 (MMP7), vascular endothelial growth factor A (VEGFA), and topoisomerase (DNA) II alpha 170kDa (TOP2A). The remaining undigested aliquot was used for the analysis of 7 single-nucleotide polymorphisms (SNPs) by MALDI-TOF mass spectrometry. In 208 of 210 samples (99.0%) the protocol yielded robust quantification-cycle values for both RNA and DNA normalization. Expression of the 8 breast cancer genes was detected in 81%-100% of tumor tissues and 21%-100% of normal tissues. The 7 SNPs were successfully genotyped in 91%-97% of tumor and 94%-97% of normal tissues. Allele concordance between tumor and normal tissue was 98.9%-99.5%. This fully automated process allowed an efficient simultaneous extraction of both RNA and DNA from a single FFPE section and subsequent dual analysis of selected genes. High gene expression and genotyping detection rates demonstrate the feasibility of molecular profiling from limited archival patient samples.

  10. 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.

  11. [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.

  12. 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

  13. Multiplexed miRNA fluorescence in situ hybridization for formalin-fixed paraffin-embedded tissues.

    PubMed

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

    2014-01-01

    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.

  14. Diagnosis of placental pathogens in small ruminants by immunohistochemistry and PCR on paraffin-embedded samples.

    PubMed

    Navarro, J A; Ortega, N; Buendia, A J; Gallego, M C; Martínez, C M; Caro, M R; Sánchez, J; Salinas, J

    2009-08-08

    A histological study was carried out on 58 formalin-fixed and paraffin-embedded samples of placenta from sheep and goats that had aborted, and the placental lesions were graded. Sequential histological sections of each cotyledon were then immunostained with specific antibodies and used for PCR detection of Chlamydophila abortus, Coxiella burnetii, Salmonella Abortusovis, Brucella melitensis, Listeria monocytogenes and Toxoplasma gondii. Most of the cotyledons showed different degrees of placentitis. The proportional agreement between the two techniques was 0.879 (kappa value 0.746). C abortus was the most prevalent pathogen. Mixed infections were common.

  15. 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

  16. STED super-resolution microscopy of clinical paraffin-embedded human rectal cancer tissue.

    PubMed

    Ilgen, Peter; Stoldt, Stefan; Conradi, Lena-Christin; 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.

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

    PubMed Central

    2011-01-01

    Background 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 utilize fresh or frozen tissue, such as Western blot and ELISA, the process of fixing, staining, and analyzing individual sections by hand does not allow for rapid or high throughput screening. However, preservation of tissues in formalin is not dependent upon the availability of refrigeration. Findings Formalin-fixed paraffin-embedded tissues from TSE transmission studies of scrapie in sheep, chronic wasting disease in white-tailed deer or transmissible mink encephalopathy in cattle were cut at 5 μm thickness. Samples containing the tissue equivalent of as little as one 5 μm section can be used to readily discriminate positive from negative samples. Conclusions This approach cannot replace IHC but may be used along with IHC as both a more rapid and readily high throughput screen where fresh or frozen tissues are not available or impractical. PMID:22018205

  18. Measurement of estrogen receptor status by immunocytochemistry in paraffin wax sections.

    PubMed

    Jasani, Bharat; Douglas-Jones, Anthony; Rhodes, Anthony; Wozniak, Susan; Barrett-Lee, Peter J; Gee, Julie; Nicholson, Robert

    2006-01-01

    The estrogen receptor (ER) status and, to a lesser extent, progesterone receptor status have been recommended by recently published guidelines as important for routine prognostic and predictive evaluation of breast cancer. Although the clinical utility of ER status has been largely validated using biochemical ligand-binding assays such as the dextran-coated, charcoal ligand-binding assay, there has been the need to develop the ER immunocytochemical assay as a more accurate and practical alternative. In particular, ER status as determined on paraffin sections by immunocytochemical assay has been shown to be superior to the ligand-binding assay for predicting response to adjuvant endocrine therapy in breast cancer. The success of the paraffin-section assay is founded on two principles. The first relates to the advent of the heat-mediated, antigen-retrieval technique capable of restoring ER and progesterone receptor antigenicity in routinely prepared diagnostic formalin-fixed, paraffin-embedded tissue sections. The second is associated with the capacity for this substrate to provide more reliable and reproducible semiquantitative assessment of ER status in morphologically better-preserved tissue used routinely for histopathological diagnosis. The aim of this chapter is to describe the methodology currently used for optimal reproducible demonstration, scoring, and assessment of ER status in paraffin wax-embedded tissue sections in relation to the management of breast cancer in a routine or clinical-trial setting.

  19. Molecular Mapping Alzheimer's Disease: MALDI Imaging of Formalin-fixed, Paraffin-embedded Human Hippocampal Tissue

    PubMed Central

    Kelley, Andrea R.; Perry, George; Bethea, Chloe; Castellani, Rudolph J.; Bach, Stephan B.H.

    2016-01-01

    A method for the molecular mapping of formalin-fixed, paraffin-embedded human hippocampal tissue affected by Alzheimer's disease (AD) is presented. This approach utilizes imaging mass spectrometry (IMS) with matrix-assisted laser desorption/ionization (MALDI). The usefulness of this technique in comparing diseased versus nor mal tissue at the molecular level while continuing to maintain topological and morphological integrity is evident in the preliminary findings. The critical correlation of the deparaffination, washing, matrix deposition, and analysis steps in handling the tissue sections and how these steps impact the successful mapping of human hippocampal tissue is clearly demonstrated. By use of this technique we have been able to identify several differences between the hippocampal AD tissue and the control hippocampal tissue. From the observed peptide clip masses we present preliminary identifications of the amyloid-beta peptides known to be prominent in the brains of those with AD. We have obtained high-resolution mass spectra and mass images with 100μm spatial resolution. Future experiments will couple this work with MALDI LIFT experiments to enable top down proteomics of fresh frozen tissue, which is not possible with paraffin-embedded tissues. PMID:27843502

  20. 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. Copyright © 2016 Elsevier Inc. All rights reserved.

  1. Protocol for qRT-PCR analysis from formalin fixed paraffin embedded tissue sections from diffuse large b-cell lymphoma: Validation of the six-gene predictor score.

    PubMed

    Tekin, Nilgun; Omidvar, Nader; Morris, Tim Peter; Conget, Paulette; Bruna, Flavia; Timar, Botond; Gagyi, Eva; Basak, Ranjan; Naik, Omkar; Auewarakul, Chirayu; Sritana, Narongrit; Levy, Debora; Cerci, Juliano Julio; Bydlowski, Sergio Paulo; Pereira, Juliana; Dimamay, Mark Pierre; Natividad, Filipinas; Chung, June-Key; Belder, Nevin; Kuzu, Isinsu; Paez, Diana; Dondi, Maurizio; Carr, Robert; Ozdag, Hilal; Padua, Rose Ann

    2016-12-13

    As a part of an international study on the molecular analysis of Diffuse Large B-cell Lymphoma (DLBCL), a robust protocol for gene expression analysis from RNA extraction to qRT-PCR using Formalin Fixed Paraffin Embedded tissues was developed. Here a study was conducted to define a strategy to validate the previously reported 6-gene (LMO2, BCL6, FN1, CCND2, SCYA3 and BCL2) model as predictor of prognosis in DLBCL. To avoid variation, all samples were tested in a single centre and single platform. This study comprised 8 countries (Brazil, Chile, Hungary, India, Philippines, S. Korea, Thailand and Turkey). Using the Kaplan-Meier and log rank test on patients (n=162) and two mortality risk groups (with those above and below the mean representing high and low risk groups) confirmed that the 6-gene predictor score correlates significantly with overall survival (OS, p<0.01) but not with event free survival (EFS, p=0.18). Adding the International Prognostic Index (IPI) shows that the 6-gene predictor score correlates significantly with high IPI scores for OS (p<0.05), whereas those with low IPI scores show a trend not reaching significance (p=0.08). This study defined an effective and economical qRT-PCR strategy and validated the 6-gene score as a predictor of OS in an international setting.

  2. Histomonas meleagridis: immunohistochemical localization of parasitic cells in formalin-fixed, paraffin-embedded tissue sections of experimentally infected turkeys demonstrates the wide spread of the parasite in its host.

    PubMed

    Singh, Amarjit; Weissenböck, Herbert; Hess, Michael

    2008-04-01

    In the present investigation, a polyclonal antibody-based immunohistochemical technique was developed to localize Histomonas meleagridis in formalin-fixed, paraffin-embedded tissues of experimentally infected turkeys. The developed technique was highly specific for histomonads as no immunohistochemical reaction was observed with cultures of Tetratrichomonas gallinarum, Trichomonas gallinae and Blastocystis sp. In addition, tissues positive for various other protozoan parasites and fungi were also tested to evaluate the specificity of the technique. It was possible to detect immunohistochemically histomonad antigens in all the tested samples (n=5) of caecum, liver, spleen and lung from infected turkeys, 3 out of 5 bursa of Fabricius, 1 out of 2 bone marrow, 2 out of 5 heart and 1 out of 5 each of proventriculus, pancreas and cerebellum. An immunohistochemical reaction indicative of presence of histomonads was also detected in blood vessels of various organs that indicated a possible hematogenous route of spread of the parasite in the host. A comparative study with routine diagnostic staining techniques indicated a high sensitivity and specificity of the newly developed immunohistochemical technique. Altogether, the technique developed can be used to study the sequential pathogenesis of histomonosis in turkeys and to obtain new insights into the mechanisms of interaction with the host tissues.

  3. Protocol for qRT-PCR analysis from formalin fixed paraffin embedded tissue sections from diffuse large b-cell lymphoma: Validation of the six-gene predictor score

    PubMed Central

    Tekin, Nilgun; Conget, Paulette; Bruna, Flavia; Timar, Botond; Gagyi, Eva; Basak, Ranjan; Naik, Omkar; Auewarakul, Chirayu; Sritana, Narongrit; Levy, Debora; Cerci, Juliano Julio; Bydlowski, Sergio Paulo; Pereira, Juliana; Dimamay, Mark Pierre; Natividad, Filipinas; Chung, June-Key; Belder, Nevin; Kuzu, Isinsu; Paez, Diana; Dondi, Maurizio; Carr, Robert

    2016-01-01

    As a part of an international study on the molecular analysis of Diffuse Large B-cell Lymphoma (DLBCL), a robust protocol for gene expression analysis from RNA extraction to qRT-PCR using Formalin Fixed Paraffin Embedded tissues was developed. Here a study was conducted to define a strategy to validate the previously reported 6-gene (LMO2, BCL6, FN1, CCND2, SCYA3 and BCL2) model as predictor of prognosis in DLBCL. To avoid variation, all samples were tested in a single centre and single platform. This study comprised 8 countries (Brazil, Chile, Hungary, India, Philippines, S. Korea, Thailand and Turkey). Using the Kaplan-Meier and log rank test on patients (n=162) and two mortality risk groups (with those above and below the mean representing high and low risk groups) confirmed that the 6-gene predictor score correlates significantly with overall survival (OS, p<0.01) but not with event free survival (EFS, p=0.18). Adding the International Prognostic Index (IPI) shows that the 6-gene predictor score correlates significantly with high IPI scores for OS (p<0.05), whereas those with low IPI scores show a trend not reaching significance (p=0.08). This study defined an effective and economical qRT-PCR strategy and validated the 6-gene score as a predictor of OS in an international setting. PMID:27825111

  4. A simple protocol for paraffin-embedded myelin sheath staining with osmium tetroxide for light microscope observation.

    PubMed

    Di Scipio, Federica; Raimondo, Stefania; Tos, Pierluigi; Geuna, Stefano

    2008-07-01

    Experimental investigation of peripheral nerve fiber regeneration is attracting more and more attention among both basic and clinical researchers. Assessment of myelinated nerve fiber morphology is a pillar of peripheral nerve regeneration research. The gold standard for light microscopic imaging of myelinated nerve fibers is toluidine blue staining of resin-embedded semithin sections. However, many researchers are unaware that the dark staining of myelin sheaths typically produced by this procedure is due to osmium tetroxide postfixation and not due to toluidine blue. In this article, we describe a simple pre-embedding protocol for staining myelin sheaths in paraffin-embedded nerve specimens using osmium tetroxide. The method involves immersing the specimen in 2% osmium tetroxide for 2 h after paraformaldehyde fixation, followed by routine dehydration and paraffin embedding. Sections can then be observed directly under the microscope or counterstained using routine histological methods. Particularly good results were obtained with Masson's trichrome counterstain, which permits the imaging of connective structures in nerves that are not detectable in toluidine blue-stained resin sections. Finally, we describe a simple protocol for osmium etching of sections, which makes further immunohistochemical analysis possible on the same specimens. Taken together, our results suggest that the protocol described in this article is a valid alternative to the conventional resin embedding-based protocol: it is much cheaper, can be adopted by any histological laboratory, and allows immunohistochemical analysis to be conducted.

  5. [Detection of EBV by PCR in fresh and paraffin embedded samples of cavum tumour].

    PubMed

    Charef, S; Jrad, B Bel Hadj; Mahfouth, W; Zakhama, A; Kassab, A; Driss, N; Chouchane, L

    2006-01-01

    The nasopharyngeal carcinoma (NPC) is frequent in Tunisia. It's the second ORL cancer of men after the larynx one. To analyse the NPC characteristics in our population, we determined the frequency of EBV infection in 47 paraffin-embedded and 6 fresh NPC biopsies. We first extracted the DNA from tumoral tissus and then amplified viral sequences by PCR to detect and to type the infecting virus (EBV-A or ABV-B). Our results showed that amplifiable DNA has been obtained from 34/47 paraffin-embedded NPC biopsies while 13/47 of the others biopsies contained degraded and not amplifiable DNA. All the fresh biopsies allowed to obtain DNA with good quality. The EBV infection frequency in paraffin-embedded NPC biopsies is 35% while EBV is detected in all fresh biopsies (6/6). Our analyse also showed that the EBV-A is predominant in our population compared to EBV-B as it was shown in most countries of the world. This study clearly shows that PCR results obtained with paraffin-embedded NPC biopsies are divergeant from those obtained with fresh biopsies. Because of DNA degradation in paraffin-embedded NPC biopsies, the biology molecular results from that kind of samples is criticable. Moreover the results obtained from fresh NPC biopsies confirmed the quasi-constant association of EBV with undifferenciated carcinoma nasopharyngeal type.

  6. An alternative protocol for DNA extraction from formalin fixed and paraffin wax embedded tissue.

    PubMed

    Coura, R; Prolla, J C; Meurer, L; Ashton-Prolla, P

    2005-08-01

    DNA extraction from paraffin wax embedded tissue requires special protocols, and most described methods report an amplification success rate of 60-80%. To propose a simple and inexpensive protocol consisting of xylene/ethanol dewaxing, followed by a kit based extraction. Xylene/ethanol dewaxing was followed by a long rehydration step and a kit based DNA extraction step. This method produced a 100% amplification success rate for fragments of 121 to 227 bp for tamponated formalin fixed paraffin wax embedded tissue. This cost effective and non-laborious protocol can successfully extract DNA from tamponated formalin fixed paraffin wax embedded tissue and should facilitate the molecular analysis of a large number of archival specimens in retrospective studies.

  7. Nucleic acid extraction methods from fixed and paraffin-embedded tissues in cancer diagnostics.

    PubMed

    Bonin, Serena; Stanta, Giorgio

    2013-04-01

    Diagnostic tests, based on nucleic acid extracts from formalin-fixed and paraffin-embedded tissues, are now becoming increasingly common due to the introduction of biological agents for cancer therapy. Unfortunately, the formalin-fixed and paraffin-embedded tissues are heterogeneous in terms of processing and tissue type, and this has an impact on downstream molecular techniques, especially RNA-based techniques. The present review deals with most of the variables connected to the extraction of nucleic acids from formalin-fixed paraffin-embedded tissues, ranging from tissue processing to quality control of extracts. The most recent peer-reviewed publications (mostly published in the past 5 years) and information provided by company websites have been analyzed to compile this review.

  8. 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.

  9. 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.

  10. A simple and reliable immunohistochemical method for colocalization of 2 antigens in the same cells of paraffin-embedded tissues.

    PubMed

    Yan, Jun; Catts, Vibeke S; Chan, Anthony; McCombe, Pamela A

    2013-10-01

    Immunohistochemistry (IHC) lacks an efficient technique for colocalizing multiple antigens in the same cells of a single tissue section. The development of a methodology which combines the advantage of low cost, high sensitivity, and specificity would benefit clinical diagnosis and general research. On the basis of a newly published method of visualizing 2 antigens on a single paraffin-embedded tissue section, we have further developed a novel sequential technique for colocalizing 2 different antigens in a same cell in a paraffin-embedded tissue section. In this technique, we combined the microwave heating technique (MVT) with normal IHC methods to sequentially double stain a paraffin section; and colocalize 2 antigens in a single cell through result comparison stored in a digital management system. This MVT colocalization method has a higher degree of sensitivity and specificity comparable with conventional staining of both immunofluorescence and IHC systems. The primary advantage of this method is that it is inexpensive and convenient; the antibody(s) used in this method can be generated from the same or different species; it allows colocalization or comparison of different results of cell morphology for any single cell of the section on 2 images, avoids uncertainty when overlapping 2 antigens on a single image.

  11. Evaluation of Chlamydophila abortus DNA extraction protocols for polymerase chain reaction diagnosis in paraffin-embedded tissues.

    PubMed

    Ortega, Nieves; Navarro, José A; Nicolás, Laura; Buendía, Antonio J; Caro, María R; Del Río, Laura; Martínez, Carlos M; Cuello, Francisco; Salinas, Jesús; Gallego, María C

    2007-07-01

    Polymerase chain reaction (PCR) has gained increasing importance as a tool for directly demonstrating the presence of Chlamydophila in the placentas of aborted sheep and goats. However, because of the zoonotic potential of the disease, it is advisable to use fixed materials. To evaluate 4 different DNA extraction protocols in paraffin-embedded sections for PCR, previously immunohistochemically diagnosed placental samples from outbreaks of abortions in goats and sheep were used. The samples were also used to evaluate the effect of the duration of fixation in formalin on PCR. A protocol that uses Tris-HCl pH 8.5 with EDTA and subsequent digestion with proteinase K was found to be an easy protocol for obtaining excellent PCR products for Chlamydophila abortus diagnosis from formalin-fixed and paraffin-embedded specimens. It was also found that if samples are fixed in formalin for more than 2 weeks, the PCR technique is affected more adversely than immunohistochemical methods.

  12. 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

  13. Detection of T cells in paraffin wax embedded tissue using antibodies against a peptide sequence from the CD3 antigen.

    PubMed Central

    Mason, D Y; Cordell, J; Brown, M; Pallesen, G; Ralfkiaer, E; Rothbard, J; Crumpton, M; Gatter, K C

    1989-01-01

    Rabbit polyclonal antibodies were raised against a proline rich, peptide sequence, comprising 13 amino acids, in the cytoplasmic domain of the CD3 epsilon chain. Immunoprecipitation experiments showed that this antibody preparation recognised the CD3 antigen on human T lymphoblasts. The antibody stained normal T cells strongly in tissue sections which had been fixed in formalin or Bouin's solution and embedded in paraffin wax. Its reactivity with T cell lymphoma, when evaluated on a series of 96 previously phenotyped cases, closely agreed with the results obtained on cryostat sections. These results indicate that the specific detection of T cells in routinely processed tissue biopsy specimens is now technically feasible on a wide scale in diagnostic laboratories using CD3 peptide antibodies, and they also suggest that in future the use of anti-peptide antibodies may detect other lineage specific antigenic markers in paraffin wax sections. Images PMID:2531171

  14. 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.

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

    USDA-ARS?s Scientific Manuscript database

    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...

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

    USDA-ARS?s Scientific Manuscript database

    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...

  17. Evaluating Quality of Aged Archival Formalin-Fixed Paraffin-Embedded Samples for RNA-Sequencing

    EPA Science Inventory

    Archival formalin-fixed paraffin-embedded (FFPE) samples offer a vast, untapped source of genomic data for biomarker discovery. However, the quality of FFPE samples is often highly variable, and conventional methods to assess RNA quality for RNA-sequencing (RNA-seq) are not infor...

  18. Evaluating Quality of Aged Archival Formalin-Fixed Paraffin-Embedded Samples for RNA-Sequencing

    EPA Science Inventory

    Archival formalin-fixed paraffin-embedded (FFPE) samples offer a vast, untapped source of genomic data for biomarker discovery. However, the quality of FFPE samples is often highly variable, and conventional methods to assess RNA quality for RNA-sequencing (RNA-seq) are not infor...

  19. High Efficiency Genotype Analysis from Formalin-Fixed, Paraffin-Embedded Tumor Tissues

    PubMed Central

    Sikora, Matthew J.; Thibert, Jacklyn N.; Salter, Janine; Dowsett, Mitch; Johnson, Michael D.; Rae, James M.

    2010-01-01

    Single nucleotide polymorphisms (SNPs) can be assayed using DNA isolated from archival formalin-fixed paraffin-embedded (FFPE) samples, making retrospective pharmacogenetic studies possible. Here we describe methods that significantly increase the number of SNP determinations possible using FFPE samples. Quantifying the amount of DNA amenable to PCR (amplification-quality DNA, AQ-DNA) allows a significant reduction in the amount of sample required for Taqman-based SNP assays. Optimizing AQ-DNA input increases PCR amplification efficiency and SNP determination accuracy. DNA was extracted from thirty-nine FFPE tumor sections and matched tumor and stromal cores, of the type used to generate tissue microarrays. Sections and tumor cores yielded sufficient AQ-DNA for over 1,000 SNP determinations. Seven SNPs were assessed, following individual assay optimization for minimal AQ-DNA. Genotypes from tumor cores for single SNPs were 92.3-100% concordant with those obtained from sections. Using these methods, the number of SNP genotypes that can be determined from single FFPE samples is greatly increased expanding the genetic association studies possible from limited archival specimens. The use of tumor cores is of particular importance since the harvesting of tumor cores has minimal impact on the utility of the donor blocks for other purposes. PMID:20548328

  20. Detection of immunoglobulins and complement components in formalin fixed and paraffin embedded renal biopsy material by immunoflourescence technique

    PubMed Central

    Mubarak, Muhammed; Kazi Javed, I; Kulsoom, Umme; Ishaque, Muhammed

    2012-01-01

    Background The technique of direct immunoflourescence (IF) is essential in the accurate diagnosis of renal glomerular diseases. The optimal results are obtained when the procedure is done on fresh frozen tissue (IF-F). However, techniques are available for IF study on formalin fixed and paraffin embedded (FFPE) renal biopsy specimens with variable reported success rates. Objectives We evaluated three such techniques on FFPE tissue and compared the results with those obtained by IF-F from the same patients. Materials and Methods Heat treatment with Tris buffer and citrate buffer, and pronase treatment of the FFPE material was carried out. Direct IF was done for renal panel immunoglobulins and complement components on all biopsies and the results were compared with the historical IF-F study. Results When compared to the IF-F, the immunoflourescence staining on the paraffin sections was less sensitive and less intense in all immune complex-mediated renal diseases, but the diagnostic findings were detected in majority of the cases. Conclusions In conclusion, it is possible to establish the diagnosis in most cases of immune complex-mediated glomerular diseases with IF on paraffin embedded tissue specimens. PMID:24475396

  1. Enhanced Histochemical Detection of Iron in Paraffin Sections of Mouse Central Nervous System Tissue

    PubMed Central

    Sands, Scott A.; Leung-Toung, Regis; Wang, Yingsheng; Connelly, John

    2016-01-01

    Histochemical methods of detecting iron in the rodent brain result mainly in the labeling of oligodendrocytes, but as all cells utilize iron, this observation suggests that much of the iron in the central nervous system goes undetected. Paraffin embedding of tissue is a standard procedure that is used to prepare sections for microscopic analysis. In the present study, we questioned whether we could modify the iron histochemical procedure to enable a greater detection of iron in paraffin sections. Indeed, various modifications led to the widespread labeling of iron in mouse brain tissue (for instance, labeling of neurons and neuropil). Sites of focal concentrations, such as cytoplasmic punctate or nucleolar staining, were also observed. The modified procedures were applied to paraffin sections of a mouse model (APP/PS1) of Alzheimer’s disease. Iron was revealed in the plaque core and rim. The plaque rim had a fibrillary or granular appearance, and it frequently contained iron-labeled cells. Further analysis indicated that the iron was tightly associated with the core of the plaque, but less so with the rim. In conclusion, modifications to the histochemical staining revealed new insights into the deposition of iron in the central nervous system. In theory, the approach should be transferrable to organs besides the brain and to other species, and the underlying principles should be incorporable into a variety of staining methods. PMID:27683879

  2. Detection of p62 on Paraffin Sections by Immunohistochemistry.

    PubMed

    Watson, Alexander S; Soilleux, Elizabeth J

    2015-08-03

    The study of autophagy in human disease is a rapidly expanding field. Diagnostic paraffin sections of a variety of patient tissues, including bone marrow, are available to researchers-yet are unsuitable for traditional autophagy quantification methods such as western blot or electron microscopy. This protocol outlines the immunohistochemical detection of the protein p62 (sequestosome-1, encoded by the gene SQSTM1)-an indicator of autophagic degradative activity-in slide-mounted paraffin sections such as bone marrow samples cut by a trephine. The p62 protein is an autophagic cargo adaptor, capable of binding to ubiquitylated proteins as well as autophagosome membrane proteins (LC3B and GABA(A) receptor-associated protein [GABARAP] family members) and hypothesized thus to target protein aggregates for lysosomal degradation. p62 itself is degraded by autophagy, remaining at low levels when autophagy is induced, and has been shown to accumulate when autophagy is deficient. Qualitative assessment and comparison of p62 staining between healthy and disease sections or disease subtypes will help target further investigation into the potential roles for autophagy in a variety of disorders.

  3. 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

  4. 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

    2015-11-03

    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.

  5. Use of monoclonal anti-actin as a megakaryocyte marker in paraffin wax embedded bone marrow biopsy specimens.

    PubMed Central

    Boqué, C; Pujol-Moix, N; Linde, M A; Murcia, C; Guanyabens, C; Soler, J

    1989-01-01

    Monoclonal anti-actin was used as a marker of megakaryocytes in Zenker's fixed, paraffin wax embedded bone marrow tissue, using an immunoperoxidase staining method. Twenty bone marrow samples were studied, including controls, and different myeloproliferative and myelodysplastic syndromes. The results were compared with those obtained using factor VIII related antigen (F VIII RAg) immunolabelling. Anti-actin is as good a marker for megakaryocytes as anti-FVIIIRAg and is potentially clinically useful when morphological identification is difficult, when bone marrow aspiration is unsuccessful, or when quantitative evaluation of tissue sections is required. Images PMID:2677054

  6. HIGH SENSITIVE PCR METHOD FOR DETECTION OF PATHOGENIC Leptospira spp. IN PARAFFIN-EMBEDDED TISSUES

    PubMed Central

    Noda, Angel Alberto; Rodríguez, Islay; Rodríguez, Yaindrys; Govín, Anamays; Fernández, Carmen; Obregón, Ana Margarita

    2014-01-01

    This study describes the development and application of a new PCR assay for the specific detection of pathogenic leptospires and its comparison with a previously reported PCR protocol. New primers were designed for PCR optimization and evaluation in artificially-infected paraffin-embedded tissues. PCR was then applied to post-mortem, paraffin-embedded samples, followed by amplicon sequencing. The PCR was more efficient than the reported protocol, allowing the amplification of expected DNA fragment from the artificially infected samples and from 44% of the post-mortem samples. The sequences of PCR amplicons from different patients showed >99% homology with pathogenic leptospires DNA sequences. The applicability of a highly sensitive and specific tool to screen histological specimens for the detection of pathogenic Leptospira spp. would facilitate a better assessment of the prevalence and epidemiology of leptospirosis, which constitutes a health problem in many countries. PMID:25229221

  7. Microwave oven-based technique for immunofluorescent staining of paraffin-embedded tissues

    PubMed Central

    Buggs, Colleen

    2011-01-01

    Immunohistochemical analysis of formalin-fixed paraffin-embedded tissues can be challenging due to potential modifications of protein structure by exposure to formalin. Heat-induced antigen retrieval techniques can reverse reactions between formalin and proteins that block antibody recognition. Interactions between antibodies and antigens are further enhanced by microwave irradiation, which has simplified immunohistochemical staining protocols. In this report, we modify a technique for antigen retrieval and immunofluorescent staining of formalin-fixed paraffin-embedded tissues by showing that it works well with several antibodies and buffers. This microwave-assisted method for antigen retrieval and immunofluorescent staining eliminates the need for blocking reagents and extended washes, which greatly simplifies the protocol allowing one to complete the analysis in less than 3 h. PMID:17653827

  8. Microwave oven-based technique for immunofluorescent staining of paraffin-embedded tissues.

    PubMed

    Long, Delwin J; Buggs, Colleen

    2008-02-01

    Immunohistochemical analysis of formalin-fixed paraffin-embedded tissues can be challenging due to potential modifications of protein structure by exposure to formalin. Heat-induced antigen retrieval techniques can reverse reactions between formalin and proteins that block antibody recognition. Interactions between antibodies and antigens are further enhanced by microwave irradiation, which has simplified immunohistochemical staining protocols. In this report, we modify a technique for antigen retrieval and immunofluorescent staining of formalin-fixed paraffin-embedded tissues by showing that it works well with several antibodies and buffers. This microwave-assisted method for antigen retrieval and immunofluorescent staining eliminates the need for blocking reagents and extended washes, which greatly simplifies the protocol allowing one to complete the analysis in less than 3 h.

  9. The relationship between gastric cancer and Helicobacter pylori in formaldehyde fixed paraffin embedded gastric tissues of gastric cancer patients-scorpion real-time PCR assay findings.

    PubMed

    Naserpour Farivar, Taghi; Johari, Pouran; Najafipour, Reza; Farzam, Amir; Nasirian, Neda; HajManouchehri, Fatemeh; Jahani Hashemi, Hassan; Azimi, Akram; Bahrami, Mohammad

    2014-01-01

    Gastric cancer is the second leading cause of cancer-related deaths worldwide and it seems that environmental and lifestyle factors and infection with Helicobacter pylori (H. pylori) have had a major role in the etiology of gastric cancer. The aim of this study was to investigate the presence of H. pylori DNA in archival gastric tissues of patients with gastric cancer disease by rapid, sensitive and specific technique of Scorpion Realtime PCR. This retrospective cross-sectional study was performed on 285 paraffin embedded gastric specimens of patients who were pathologically proved for gastric cancer admitted in Bou-Ali, Shahid Rajaie and Dehkhoda hospitals and Bahar and Farzam private laboratory in Qazvin city in Iran during 2009 and 150 paraffin embedded pathological specimens of patients with other proved diagnosis other than gastric cancer. Results of our Scorpion Realtime PCR analysis showed that DNA of H. pylori DNA was present in 78.42% of our total specimens. Modified McMullen's Staining of paraffin embedded sections was positive in 210 patients. Also we were not able to finding significant relationship between demographic characteristics of our studied patients and presence of H. pylori DNA in their formaldehyde fixed paraffin embedded gastric tissues samples. Existence of H. pylori in gastric tissue samples of patients with gastric cancer is controversial and our results indicated that in our studied specimens prevalence of H. pylori was significantly more than recent published reports.

  10. Frequency of Human Papillomavirus (HPV) 16 and 18 Detection in Paraffin- Embedded Laryngeal Carcinoma Tissue

    PubMed

    Hosseini, Seyed Zinab; Makvandi, Manoochehr; Samarbafzade, Alireza; Timori, Ali; Ranjbar, Nastaran; Saki, Nader; Nisi, Nilofar; Shahani, Toran; Varnaseri, Mehran; Angali Ahmadi, Kambiz

    2017-04-01

    Background and Objective: Human papilloma virus (HPV) 16 and HPV18 have been detected in head and neck squamous cell carcinomas (HNSCC) and there is evidence that detection of HPVs would have better prognostic value than patients with HNSCC negative for HPVs. Thus, this study was conducted to evaluate frequency of HPV 16 and HPV 18 genotypes in patients with laryngeal carcinoma. Materials and methods: Fifty formalin-fixed, paraffin-embedded (FFPE) tissue blocks of laryngeal cancers were collected. Sections were prepared at 5 μm and DNA was extracted from each sample and subjected to the polymerase chain reaction (PCR) to detect HPV-16/18 DNA s. Results: All samples were squamous cell carcinomas (SCCs). Overall 14/50 (28%) were positive for HPVs, 8 (18%) with HPV-16 and 6 (12%) with HPV-18. Additionally, 2 (4%) mixed infections of HPV 16 and 18 genotypes were observed among these cases. Conclusions: Overall, 28% of HNSCC samples proved positive for HPV16 and HPV18 genotypes, two high-risk HPV types. It is important to further assess whether such viral infection, could be a risk factor in HNSCC progression. Creative Commons Attribution License

  11. Virus characterization and discovery in formalin-fixed paraffin-embedded tissues.

    PubMed

    Bodewes, Rogier; van Run, Peter R W A; Schürch, Anita C; Koopmans, Marion P G; Osterhaus, Albert D M E; Baumgärtner, Wolfgang; Kuiken, Thijs; Smits, Saskia L

    2015-03-01

    Detection and characterization of novel viruses is hampered frequently by the lack of properly stored materials. Especially for the retrospective identification of viruses responsible for past disease outbreaks, often only formalin-fixed paraffin-embedded (FFPE) tissue samples are available. Although FFPE tissues can be used to detect known viral sequences, the application of FFPE tissues for detection of novel viruses is currently unclear. In the present study it was shown that sequence-independent amplification in combination with next-generation sequencing can be used to detect sequences of known and unknown viruses, although with relatively low sensitivity. These findings indicate that this technique could be useful for detecting novel viral sequences in FFPE tissues collected from humans and animals with disease of unknown origin, when other samples are not available. In addition, application of this method to FFPE tissues allows to correlate with the presence of histopathological changes in the corresponding tissue sections. Copyright © 2015 Elsevier B.V. All rights reserved.

  12. 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.

  13. 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.

  14. Superresolution Imaging of Clinical Formalin Fixed Paraffin Embedded Breast Cancer with Single Molecule Localization Microscopy

    PubMed Central

    Creech, Matthew K.; Wang, Jing; Nan, Xiaolin; Gibbs, Summer L.

    2017-01-01

    Millions of archived formalin-fixed, paraffin-embedded (FFPE) specimens contain valuable molecular insight into healthy and diseased states persevered in their native ultrastructure. To diagnose and treat diseases in tissue on the nanoscopic scale, pathology traditionally employs electron microscopy (EM), but this platform has significant limitations including cost and painstaking sample preparation. The invention of single molecule localization microscopy (SMLM) optically overcame the diffraction limit of light to resolve fluorescently labeled molecules on the nanoscale, leading to many exciting biological discoveries. However, applications of SMLM in preserved tissues has been limited. Through adaptation of the immunofluorescence workflow on FFPE sections milled at histological thickness, cellular architecture can now be visualized on the nanoscale using SMLM including individual mitochondria, undulations in the nuclear lamina, and the HER2 receptor on membrane protrusions in human breast cancer specimens. Using astigmatism imaging, these structures can also be resolved in three dimensions to a depth of ~800 nm. These results demonstrate the utility of SMLM in efficiently uncovering ultrastructural information of archived clinical samples, which may offer molecular insights into the physiopathology of tissues to assist in disease diagnosis and treatment using conventional sample preparation methods. PMID:28098202

  15. Complete Solubilization of Formalin-Fixed, Paraffin-Embedded Tissue May Improve Proteomic Studies

    PubMed Central

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

    2013-01-01

    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 (AR) 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 (RPPA), 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. PMID:23339100

  16. Superresolution Imaging of Clinical Formalin Fixed Paraffin Embedded Breast Cancer with Single Molecule Localization Microscopy.

    PubMed

    Creech, Matthew K; Wang, Jing; Nan, Xiaolin; Gibbs, Summer L

    2017-01-18

    Millions of archived formalin-fixed, paraffin-embedded (FFPE) specimens contain valuable molecular insight into healthy and diseased states persevered in their native ultrastructure. To diagnose and treat diseases in tissue on the nanoscopic scale, pathology traditionally employs electron microscopy (EM), but this platform has significant limitations including cost and painstaking sample preparation. The invention of single molecule localization microscopy (SMLM) optically overcame the diffraction limit of light to resolve fluorescently labeled molecules on the nanoscale, leading to many exciting biological discoveries. However, applications of SMLM in preserved tissues has been limited. Through adaptation of the immunofluorescence workflow on FFPE sections milled at histological thickness, cellular architecture can now be visualized on the nanoscale using SMLM including individual mitochondria, undulations in the nuclear lamina, and the HER2 receptor on membrane protrusions in human breast cancer specimens. Using astigmatism imaging, these structures can also be resolved in three dimensions to a depth of ~800 nm. These results demonstrate the utility of SMLM in efficiently uncovering ultrastructural information of archived clinical samples, which may offer molecular insights into the physiopathology of tissues to assist in disease diagnosis and treatment using conventional sample preparation methods.

  17. Proteomic analysis of neurons microdissected from formalin-fixed, paraffin-embedded Alzheimer's disease brain tissue.

    PubMed

    Drummond, Eleanor S; Nayak, Shruti; Ueberheide, Beatrix; Wisniewski, Thomas

    2015-10-21

    The vast majority of human tissue specimens are formalin-fixed, paraffin embedded (FFPE) archival samples, making this type of tissue a potential gold mine for medical research. It is now accepted that proteomics can be done using FFPE tissue and can generate similar results as snap-frozen tissue. However, the current methodology requires a large amount of starting protein, limiting the questions that can be answered in these types of proteomics studies and making cell-type specific proteomics studies difficult. Cell-type specific proteomics has the potential to greatly enhance understanding of cell functioning in both normal and disease states. Therefore, here we describe a new method that allows localized proteomics on individual cell populations isolated from FFPE tissue sections using laser capture microdissection. To demonstrate this technique we microdissected neurons from archived tissue blocks of the temporal cortex from patients with Alzheimer's disease. Using this method we identified over 400 proteins in microdissected neurons; on average 78% that were neuronal and 50% that were associated with Alzheimer's disease. Therefore, this technique is able to provide accurate and meaningful data and has great potential for any future study that wishes to perform localized proteomics using very small amounts of archived FFPE tissue.

  18. 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.

  19. 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

  20. Preparation of Cells from Formalin-Fixed, Paraffin-Embedded Tissue for Use in Fluorescence In Situ Hybridization (FISH) Experiments.

    PubMed

    Weremowicz, Stanislawa

    2015-01-20

    Numerical and structural chromosome abnormalities can be accurately detected in cells from archived tissues using fluorescence in situ hybridization (FISH). This unit describes two common approaches to performing FISH in formalin-fixed, paraffin-embedded tissue. The first approach utilizes 4 to 6 μm tissue sections in cases for which preserving tissue morphology is necessary, and the second involves extraction of intact nuclei from 50-μm tissue sections. To interpret FISH results using 4 to 6 μm sections, an adequate number of nuclei must be evaluated to perform statistical analysis. Evaluation of 30 to 50 nuclei from the single-cell suspension generally gives an interpretable result. Copyright © 2015 John Wiley & Sons, Inc.

  1. MALDI imaging mass spectrometry of N-linked glycans on formalin-fixed paraffin-embedded murine kidney.

    PubMed

    Gustafsson, Ove J R; Briggs, Matthew T; Condina, Mark R; Winderbaum, Lyron J; Pelzing, Matthias; McColl, Shaun R; Everest-Dass, Arun V; Packer, Nicolle H; Hoffmann, Peter

    2015-03-01

    Recent developments in spatial proteomics have paved the way for retrospective in situ mass spectrometry (MS) analyses of formalin-fixed paraffin-embedded clinical tissue samples. This type of analysis is commonly referred to as matrix-assisted laser desorption/ionization (MALDI) imaging. Recently, formalin-fixed paraffin-embedded MALDI imaging analyses were augmented to allow in situ analyses of tissue-specific N-glycosylation profiles. In the present study, we outline an improved automated sample preparation method for N-glycan MALDI imaging, which uses in situ PNGase F-mediated release and measurement of N-linked glycans from sections of formalin-fixed murine kidney. The sum of the presented data indicated that N-glycans can be cleaved from proteins within formalin-fixed tissue and characterized using three strategies: (i) extraction and composition analysis through on-target MALDI MS and liquid chromatography coupled to electrospray ionization ion trap MS; (ii) MALDI profiling, where N-glycans are released and measured from large droplet arrays in situ; and (iii) MALDI imaging, which maps the tissue specificity of N-glycans at a higher resolution. Thus, we present a complete, straightforward method that combines MALDI imaging and characterization of tissue-specific N-glycans and complements existing strategies.

  2. Comparison of histological techniques to visualize iron in paraffin-embedded brain tissue of patients with Alzheimer's disease.

    PubMed

    van Duijn, Sara; Nabuurs, Rob J A; van Duinen, Sjoerd G; Natté, Remco

    2013-11-01

    Better knowledge of the distribution of iron in the brains of Alzheimer's disease (AD) patients may facilitate the development of an in vivo magnetic resonance (MR) marker for AD and may cast light on the role of this potentially toxic molecule in the pathogenesis of AD. Several histological iron staining techniques have been used in the past but they have not been systematically tested for sensitivity and specificity. This article compares three histochemical techniques and ferritin immunohistochemistry to visualize iron in paraffin-embedded human AD brain tissue. The specificity of the histochemical techniques was tested by staining sections after iron extraction. Iron was demonstrated in the white matter, in layers IV/V of the frontal neocortex, in iron containing plaques, and in microglia. In our hands, these structures were best visualized using the Meguro iron stain, a method that has not been described for iron staining in human brain or AD in particular. Ferritin immunohistochemistry stained microglia and iron containing plaques similar to the Meguro method but was less intense in myelin-associated iron. The Meguro method is most suitable for identifying iron-positive structures in paraffin-embedded human AD brain tissue.

  3. Papillomavirus genotyping on formaldehyde fixed paraffin-embedded tissues in vulvar intraepithelial neoplasia.

    PubMed

    Mazellier, S; Dadone-Montaudie, B; Chevallier, A; Loubatier, C; Vitale, S; Cardot-Leccia, N; Angeli, K; Trastour, C; Delotte, J; Giordanengo, V; Ambrosetti, D

    2017-08-09

    Few studies have described the epidemiology of human papillomavirus (HPV) in vulvar intraepithelial neoplasia (VIN). The aim of this study was to genotype HPV on formalin fixed paraffin-embedded tissues in VIN lesions. A 5-year retrospective study was conducted by including all patients attending the teaching hospital of Nice with a diagnosis of VIN between 1st January 2010 and 31st December 2014. For all patients, HPV genotyping was performed with the PapilloCheck(®) microarray kit, routinely used on cervical cytology samples, and optimized for formaldehyde fixed paraffin-embedded tissues in VIN. Forty patients were included in the study: 39 patients had usual VIN and one presented with differentiated VIN. Among the 39 patients with usual VIN, the prevalence of HPV was 90% (35/39). Thirty-two patients had high grade VIN (82%) and seven low grade VIN (18%). In high grade VIN, the most represented HPV types were: HPV 16 (21/32 66%), HPV 56 (3/32 9%) and HPV 33 (2/32 6%). In low grade VIN, the most represented HPV types were: HPV 16 (4/7 57%) and HPV 6 (3/7 43%). Interestingly, 5/39 (13%) of patients diagnosed with usual VIN also had co-existing lichen sclerosus. We have optimized a HPV genotyping technique, routinely used on cervical cytology samples, and on paraffin fixed embedded tissue showing VIN. Moreover, we have identified five patients with lichen sclerosus co-existing with usual VIN. This association has rarely been reported and proves that these two entities can coexist.

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

    PubMed Central

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

    2016-01-01

    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. MicroRNA-profiling in formalin-fixed paraffin-embedded specimens.

    PubMed

    Lehmann, Ulrich

    2010-01-01

    The discovery of small regulatory RNA molecules during the last few years has changed our understanding of many biological and pathological processes. The most prominent and best analyzed class of these small regulatory noncoding RNAs is comprised by the microRNAs. The analysis of microRNA expression patterns is now widely used in biology and pathology employing a range of methodologies. However, many precious human tissue samples are only available as formalin-fixed paraffin-embedded (FFPE) specimen. In this chapter, the extraction of RNA from FFPE samples and the subsequent microRNA profiling utilizing fluorescence-labeled bead technology from Luminex Inc. is described.

  7. Detection of Viral RNA From Paraffin-Embedded Tissues After Prolonged Formalin Fixation

    DTIC Science & Technology

    2009-01-01

    fixed, paraffin-embedded (FFPE) liver tissues fromcynomolgusmacaques (Macaca fascicularis) experimentally infected with Ebola virus, Zaire . Sample Day...K t r e t D O s c s 5 f b a The viral titer was expressed as Ebola Zaire virus log10 PFU/g. b CT value≥45 is negative. c Not applicable. nly one...to extract ampli- fiable RNA and detect West Nile virus (WNV), Marburg virus (MARV), and Ebola virus (EBOV)-infected tissues using TaqMan® assays

  8. C4d immunohistochemical staining is a sensitive method to confirm immunoreactant deposition in formalin-fixed paraffin-embedded tissue in membranous glomerulonephritis.

    PubMed

    Val-Bernal, J F; Garijo, M F; Val, D; Rodrigo, E; Arias, M

    2011-11-01

    Although the diagnosis of membranous glomerulonephritis (MGN) may be suspected on routine histology of formalin-fixed paraffin-embedded tissue, fresh-frozen tissue must be used to show the immunologic nature of the process by direct immunofluorescence (IF). The efficiency of IF or immunoperoxidase (IP) detection of IgG and C3 using paraffin sections is controversial. This study was designed to evaluate whether glomerular C4d deposition using an IP method in formalin-fixed paraffin-embedded tissue may be a useful marker for MGN. We showed characteristic glomerular, granular basement membrane deposition of C4d in 31 (100%) cases of idiopathic MGN and in 5 cases (100%) of pure class V membranous lupus nephritis, in which we had a positive diagnosis of the lesions for conventional IF study. Control cases were negative. Nineteen cases of different glomerulopathies, including IgA nephropathy, primary type I membranoproliferative glomerulonephritis, focal segmental glomerulosclerosis and minimal change disease showed diverse reproducible patterns of C4d deposition, without intrinsic background. Our results indicate that staining of formalin-fixed paraffin-embedded tissue for C4d can be used for confirmation of granular basement membrane immunoreactant deposition in cases of MGN. This proved to be a reliable method that could potentially obviate the need for rebiopsy in cases with absence of glomeruli in renal frozen sections or when other adjunct IF or IP methods on paraffin sections are negative. C4d immunostaining, using an IP method, deserves a place as an adjunct method in the biopsy diagnosis of MGN.

  9. 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.

  10. Simplified procedures for applying the polymerase chain reaction to routinely fixed paraffin wax sections.

    PubMed Central

    Coates, P J; d'Ardenne, A J; Khan, G; Kangro, H O; Slavin, G

    1991-01-01

    The polymerase chain reaction was applied to the analysis of DNA contained in archival paraffin wax embedded material. DNA suitable for the reaction was obtained from these tissues by simple extraction methods, without previous dewaxing of tissue sections. When compared with unfixed material, the reaction efficiency was compromised, so that an increased number of amplification cycles were required to produce equivalent amounts of amplified product. This in turn led to an increase in amplification artefacts, which can be minimised by a simple modification of the standard reaction. Amplification of relatively large DNA fragments was not always successful, and it seems prudent to bear this in mind when designing oligonucleotide primers which are to be used for the amplification of archival material. The efficiency of the procedure can be improved by dividing the amplification cycles into two parts: this reduces the amount of reagent needed, is relatively simple and inexpensive, and can be performed in one working day. Images PMID:1650795

  11. Identification of Penicillium marneffei in Paraffin-Embedded Tissue Using Nested PCR.

    PubMed

    Zeng, Hanxiang; Li, Xiqing; Chen, Xiejie; Zhang, Junmin; Sun, Jiufeng; Xie, Zhi; Xi, Liyan

    2009-07-01

    Penicillium marneffei is one of the unique thermally dimorphic fungi in Penicillium species that causes a disseminated, progressive and life threatening infection in immunocompromised patients. The diagnosis of Penicilliosis marneffei depends on culture that may delay the treatment due to the time-consuming process. In the present study, we evaluated the specificity and sensitivity of nested PCR to identify Penicillium marneffei from paraffin-embedded tissue. Two sets of oligonucleotide primers were derived from the sequence of 18S rRNA of Penicillium marneffei. The outer primers (RRF1 and RRH1) were specific to fungi. The inner primers (Pm1 and Pm2) were specific to Penicillium marneffei. The specific fragment of approximately 400 bp was amplified from all paraffin-embedded tissues from 14 patients with Penicilliosis marneffei and 10 bamboo rats. The detectable DNA concentration of single PCR and nested PCR were 14 pg/microl and 14 fg/microl, respectively. Further studies are required in order to use nested PCR for early diagnosis of the disease.

  12. PCR based identification and discrimination of agents of mucormycosis and aspergillosis in paraffin wax embedded tissue.

    PubMed

    Bialek, R; Konrad, F; Kern, J; Aepinus, C; Cecenas, L; Gonzalez, G M; Just-Nübling, G; Willinger, B; Presterl, E; Lass-Flörl, C; Rickerts, V

    2005-11-01

    Invasive fungal infections are often diagnosed by histopathology without identification of the causative fungi, which show significantly different antifungal susceptibilities. To establish and evaluate a system of two seminested polymerase chain reaction (PCR) assays to identify and discriminate between agents of aspergillosis and mucormycosis in paraffin wax embedded tissue samples. DNA of 52 blinded samples from five different centres was extracted and used as a template in two PCR assays targeting the mitochondrial aspergillosis DNA and the 18S ribosomal DNA of zygomycetes. Specific fungal DNA was identified in 27 of 44 samples in accordance with a histopathological diagnosis of zygomycosis or aspergillosis, respectively. Aspergillus fumigatus DNA was amplified from one specimen of zygomycosis (diagnosed by histopathology). In four of 16 PCR negative samples no human DNA was amplified, possibly as a result of the destruction of DNA before paraffin wax embedding. In addition, eight samples from clinically suspected fungal infections (without histopathological proof) were examined. The two PCR assays detected a concomitant infection with Absidia corymbifera and A fumigatus in one, and infections with Rhizopus arrhizus and A fumigatus in another two cases. The two seminested PCR assays described here can support a histopathological diagnosis of mucormycosis or aspergillosis, and can identify the infective agent, thereby optimising antifungal treatment.

  13. 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

  14. [Evaluation of 3 methods of DNA extraction from paraffin-embedded material for the amplification of genomic DNA using PCR].

    PubMed

    Mesquita, R A; Anzai, E K; Oliveira, R N; Nunes, F D

    2001-01-01

    There are several protocols reported in the literature for the extraction of genomic DNA from formalin-fixed paraffin-embedded samples. Genomic DNA is utilized in molecular analyses, including PCR. This study compares three different methods for the extraction of genomic DNA from formalin-fixed paraffin-embedded (inflammatory fibrous hyperplasia) and non-formalin-fixed (normal oral mucosa) samples: phenol with enzymatic digestion, and silica with and without enzymatic digestion. The amplification of DNA by means of the PCR technique was carried out with primers for the exon 7 of human keratin type 14. Amplicons were analyzed by means of electrophoresis in an 8% polyacrylamide gel with 5% glycerol, followed by silver-staining visualization. The phenol/enzymatic digestion and the silica/enzymatic digestion methods provided amplicons from both tissue samples. The method described is a potential aid in the establishment of the histopathologic diagnosis and in retrospective studies with archival paraffin-embedded samples.

  15. Babesia gibsoni: detection in blood smears and formalin-fixed, paraffin-embedded tissues using deoxyribonucleic acid in situ hybridization analysis.

    PubMed

    Yamasaki, Masahiro; Kobayashi, Yusuke; Nakamura, Kensuke; Sasaki, Noboru; Murakami, Masahiro; Rajapakshage, Bandula Kumara Wickramasekara; Ohta, Hiroshi; Yamato, Osamu; Maede, Yoshimitsu; Takiguchi, Mitsuyoshi

    2011-01-01

    In this study, we attempted to detect Babesia gibsoni in blood smears and formalin-fixed, paraffin-embedded tissues obtained from B. gibsoni-infected dogs using in situ hybridization. Using a digoxigenin-conjugated deoxyribonucleic acid (DNA) probe, both intraerythrocytic and exoerythrocytic parasites in the culture could be specifically stained in blood smears fixed with 4% phosphate-buffered paraformaldehyde. This indicated that genomic DNA extracted from the parasites could be detected using in situ hybridization. Moreover, the parasite could be specifically stained in paraffin-embedded spleen, lymph node, and kidney sections using in situ hybridization. Infected erythrocytes in blood vessels in the spleen and kidney, hemosiderin-laden macrophages in the spleen, and phagocytized erythrocytes, which seemed to be infected with the parasites, in lymph nodes were also specifically stained. This suggests that in situ hybridization can be utilized to investigate both the life cycle of B. gibsoni and the pathological condition of canine babesiosis.

  16. Efficient and cost-effective extraction of genomic DNA from formalin-fixed and paraffin-embedded tissues.

    PubMed

    Weiss, A Th A; Delcour, N M; Meyer, A; Klopfleisch, R

    2011-07-01

    Diagnostic and investigative molecular pathology frequently has to resort to extraction of DNA from formalin-fixed and paraffin-embedded tissue samples. Although many different protocols are reported for this type of material, extraction of sufficient amounts of intact DNA is still challenging. Here, the authors report a reproducible, simple, cost-effective, and efficient protocol that yields up to 140 μg of DNA from approximately 10 to 15 mg of formalin-fixed and paraffin-embedded tissue samples and compare it to available protocols. The protocol allows stable amplification of DNA fragments up to 600 bp in length in a wide variety of tissues. © The Authors 2011

  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. 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

  19. 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.

  20. The Utilization of Formalin Fixed-Paraffin-Embedded Specimens in High Throughput Genomic Studies

    PubMed Central

    Zhang, Pan

    2017-01-01

    High throughput genomic assays empower us to study the entire human genome in short time with reasonable cost. Formalin fixed-paraffin-embedded (FFPE) tissue processing remains the most economical approach for longitudinal tissue specimen storage. Therefore, the ability to apply high throughput genomic applications to FFPE specimens can expand clinical assays and discovery. Many studies have measured the accuracy and repeatability of data generated from FFPE specimens using high throughput genomic assays. Together, these studies demonstrate feasibility and provide crucial guidance for future studies using FFPE specimens. Here, we summarize the findings of these studies and discuss the limitations of high throughput data generated from FFPE specimens across several platforms that include microarray, high throughput sequencing, and NanoString. PMID:28246590

  1. Use of formalin-fixed, paraffin-embedded tissue for proteomic biomarker discovery.

    PubMed

    Krizman, David B; Burrows, Jon

    2013-01-01

    Application of mass spectrometry to proteomic analysis of tissue is a highly desirable approach to discovery of disease biomarkers due to a direct correlation of findings to tissue/disease histology and in many respects obviating the need for model systems of disease. Both frozen and formalin-fixed, paraffin-embedded (FFPE) tissue can be interrogated; however, worldwide access to vastly larger numbers of highly characterized FFPE tissue collections derived from both human and model organisms makes this form of tissue more advantageous. Here, an approach to large-scale, global proteomic analysis of FFPE tissue is described that can be employed to discover differentially expressed proteins between different histological tissue types and thus discover novel protein biomarkers of disease.

  2. Simple salting-out method for DNA extraction from formalin-fixed, paraffin-embedded tissues.

    PubMed

    Rivero, Elena R C; Neves, Adriana C; Silva-Valenzuela, Maria G; Sousa, Suzana O M; Nunes, Fabio D

    2006-01-01

    The aim of this study was to standardize a method of DNA extraction from formalin-fixed and paraffin-embedded tissues (PETs) using a salt solution to precipitate protein and isopropanol to precipitate DNA. The samples were submitted to a DNA extraction method in which two different concentrations of ammonium acetate (2 and 4M) were compared with a phenol-chloroform extraction method and with a commercial DNA isolation kit. DNA was qualified and quantified by spectrophotometer analysis, electrophoresis, and amplification by PCR. The 167 and 268bp fragments of APC and beta-globin genes, respectively, were amplified equally from DNA extracted by all tested methods and in all cases. However, the 536bp fragment of beta-globin gene was not amplified in all cases. According to our results, the extraction method using ammonium acetate proved to be simple and suitable for obtaining DNA of good quality, which can be easily amplified by PCR.

  3. An efficient protocol for genomic DNA extraction from formalin-fixed paraffin-embedded tissues.

    PubMed

    Santos, Sara; Sá, Daniela; Bastos, Estela; Guedes-Pinto, Henrique; Gut, Ivo; Gärtner, Fátima; Chaves, Raquel

    2009-06-01

    Formalin-fixed paraffin-embedded tissues (FFPET) represent the largest source of archival biological material available for genomic studies. In this work we present an advanced protocol for extraction of high quality DNA from FFPET that can be applied in several molecular studies. Although cat mammary tumours (CMT) are the third most frequent tumour in cats the recovery of significant number of samples for molecular studies are in some way restricted to FFPET samples. We were able to obtain high quality DNA from FFPET of thirty six CMT that were subjected to pre-fixation and fixation processes routinely used in the veterinary hospitals. The quality of DNA obtained was tested by PCR amplification using six sets of primers that amplify single-copy fragments. The DNA fragments obtained were further sequenced. This protocol was able to provide FFPET gDNA that can be amplified and sequenced for larger fragments up to 1182bp.

  4. Hepatitis C virus genotype testing in paraffin wax embedded liver biopsies for specimen identification.

    PubMed

    Ikura, Y; Ohsawa, M; Hai, E; Jomura, H; Ueda, M

    2003-12-01

    Despite advances in medical technology, careful specimen identification is still a fundamental principle of laboratory testing. If pathological samples are mixed up, especially in the case of extremely small biopsy samples, large amounts of time and energy may be wasted in correctly identifying the specimens. Recently, two liver biopsy specimens were mixed up in this department, and a new pathological technology was used to resolve the issue. Liver biopsy was performed on two patients with hepatitis C virus (HCV) infection. During sample transfer or tissue processing, the biopsy specimens were mixed up. Because the ABO blood group of the two patients was identical (type AB), the specimens were subsequently identified by analysing the HCV genotypes. RNA extracted from the paraffin wax embedded liver specimens was examined by a polymerase chain reaction based HCV genotype assay. This enabled the correct identification of the specimens, and each patient received the appropriate treatment on the basis of the accurate diagnosis.

  5. Terahertz spectroscopy for the study of paraffin-embedded gastric cancer samples

    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

    Terahertz (THz) spectroscopy constitute promising technique for biomedical applications as a complementary and powerful tool for diseases screening specially for early cancer diagnostic. The THz radiation is not harmful to biological tissues. As increased blood supply in cancer-affected tissues and consequent local increase in tissue water content makes THz technology a potentially attractive. In the present work, samples of healthy and adenocarcinoma-affected gastric tissue were analyzed using transmission time-domain THz spectroscopy (THz-TDS). The work shows the capability of the technique to distinguish between normal and cancerous regions in dried and paraffin-embedded samples. Plots of absorption coefficient α and refractive index n of normal and cancer affected tissues, are presented and the conditions for discrimination between normal and affected tissues are discussed.

  6. Multiple immunofluorescence labeling of formalin-fixed paraffin-embedded tissue.

    PubMed

    Robertson, David; Isacke, Clare M

    2011-01-01

    Multiple immunofluorescent labeling of formalin-fixed paraffin-embedded (FFPE) tissue is not a routinely used method. At least in part, this is due to the perception that the innate autofluorescence of the FFPE material forbids the use of immunofluorescent labeling. As a result, immunohistochemical (immunoperoxidase) staining of FFPE material or cryosectioning methods is used instead. In this chapter, we describe a robust optimized method for high-resolution immunofluorescence labeling of FFPE tissue that involves the combination of antigen retrieval, indirect immunofluorescence, and confocal laser scanning microscopy. Once such samples have been prepared and imaged by confocal microscopy, they can be stored at -20°C for extensive periods (>250 days) and reexamined with minimal loss of quality. As a consequence, this method has the potential to open up the large archival sample collections to multiple immunofluorescent investigations.

  7. Combined Use of PCR-Based TCRG and TCRB Clonality Tests on Paraffin-Embedded Skin Tissue in the Differential Diagnosis of Mycosis Fungoides and Inflammatory Dermatoses

    PubMed Central

    Zhang, Bing; Beck, Andrew H.; Taube, Janis M.; Kohler, Sabine; Seo, Katie; Zwerner, Jeffrey; Viakhereva, Natalie; Sundram, Uma; Kim, Youn H.; Schrijver, Iris; Arber, Daniel A.; Zehnder, James L.

    2010-01-01

    The distinction between mycosis fungoides (MF) and inflammatory dermatoses (ID) by clinicopathologic criteria can be challenging. There is limited information regarding the performance characteristics and utility of TCRG and TCRB clonality assays in diagnosis of MF and ID from paraffin-embedded tissue sections. In this study, PCR tests were performed with both TCRG and TCRB BIOMED-2 clonality methods followed by capillary electrophoresis and Genescan analysis using DNA samples from 35 MF and 96 ID patients with 69 and 133 paraffin-embedded specimens, respectively. Performance characteristics were determined for each test individually and in combination. TCRG and TCRB tests demonstrated identical sensitivity (64%) and specificity (84%) when analyzed as individual assays. The positive predictive value, negative predictive value, and change of posttest MF probability over a range of MF pretest probabilities were obtained. These data were used to construct an algorithm for sequential use of TCRG and TCRB. As single tests, commercially available BIOMED-2 PCR-based TCRG and TCRB clonality tests on paraffin-embedded tissue have no significant difference in terms of sensitivity and specificity. Combined use of the two tests in patients with intermediate pretest probabilities as proposed in the algorithm could improve test utility. PMID:20203005

  8. 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. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. Electrochemical antigen-retrieval of formaldehyde fixed and paraffin-embedded archived leprosy skin biopsies.

    PubMed

    Sergio, Navarro-Fierros; Cecilia, Guillen-Vargas; Sonia, Luquin; la Mora Pedro, Garzon-De; Joaquin, Garcia-Estrada; Mary, Fafutis-Morris

    2004-06-01

    While formaldehyde fixation preserves tissue morphology, it often hinders immunodetection of antigens in paraffin-embedded tissue because the antigens are masked. Antigen unmasking can be achieved with treatments such as microwave irradiation but they often lead to excessive tissue damage. Therefore, an electrochemical antigen-retrieval method (EAR) was devised in which an alternating electric current is passed through the tissue in a chamber containing an electrolyte buffer. The results obtained with this method were compared to those after microwave irradiation using archived samples of formaldehyde-fixed and paraffin-embedded lepromatous leprosy skin. The efficacy of the two unmasking procedures was assessed by the immunodetectability of several marker antigens using 24 antibodies. Fifteen antibodies that were directed against transmembrane proteins (CD), and the remaining 9 against cytokeratins 18.6 and 19, laminin, vimentin, S100a, BCG, Ulex europaeus lectin, PCNA, and P21ras. Simple and double immunohistochemistry was performed using the universal ENVISION and LSAB + AP detection systems. After unmasking with the EAR method, immunoreactivity was clearly detected with 22 of the 24 antibodies in single labeling reactions. They include the critical antigens CD3 and CD4 for identifying the T lymphocyte lineages. In contrast, only 20 of the antibodies reacted after microwave irradiation. After double immunolabeling, immunoreactivity was quantitatively similar with both methods. However, the EAR unmasking produced a stronger labeling reaction. Thus, with double labeling immunohistochemistry, EAR made it possible to use higher antibody dilutions and shorter incubation times. Heat damage was also prevented. In conclusion, EAR treatment produces better staining results than microwave irradiation treatment.

  10. PCR based detection of mycobacteria in paraffin wax embedded material routinely processed for morphological examination.

    PubMed Central

    Frevel, T; Schäfer, K L; Tötsch, M; Böcker, W; Dockhorn-Dworniczak, B

    1999-01-01

    BACKGROUND: The incidence of mycobacterial infections has increased during the past five years. A prompt diagnosis is indispensable for initiating appropriate treatment. Because culturing of mycobacteria takes three to six weeks and sensitivity of microscopic detection of acid fast bacilli is low, amplification methods provide promising possibilities. Recently, the polymerase chain reaction (PCR) has been shown to be useful for confirming a mycobacterial infection, especially in cases with unexpected histological findings or lack of suitable material for culturing. AIMS: To evaluate the impact of PCR based techniques in the detection of mycobacterial infections in uncultured routine histological specimens as an alternative to surgical pathology. METHODS: Two hundred and twenty nine formalin fixed and paraffin wax embedded samples from 141 patients with clinical or histological suspicion of a mycobacterial infection were investigated using three different PCR assays and Southern blotting. PCR results were compared with histology and culture and the patients' clinical findings. RESULTS: When using culture as the reference method, the sensitivity for the detection of mycobacteria of the tuberculosis complex was 90%, specificity was 92%, the positive predictive value was 81%, and the negative predictive value was 96%. The sensitivity for the detection of nontuberculous mycobacteria was 100% and specificity was 78%, the positive predictive value was 26%, and the negative predictive value was 100%. The patients' clinical findings supported the PCR positive results, indicating a mycobacterial infection in 11 of 18 initially culture negative cases and in 21 of 35 PCR positive cases without culture results. CONCLUSIONS: These results indicate that PCR based techniques are sensitive, specific, and rapid methods for the detection of mycobacteria in routinely processed paraffin wax embedded and formalin fixed histological samples. PMID:10748878

  11. Detection and identification of aquatic mycobacteria in formalin-fixed, paraffin-embedded fish tissues.

    PubMed

    Pourahmad, F; Thompson, K D; Adams, A; Richards, R H

    2009-05-01

    The isolation of mycobacteria from field samples is problematic, and isolation of the bacterium is sometimes not even attempted. The detection of mycobacteria through traditional histology using formalin-fixed, paraffin-embedded (FFPE) tissues is neither sensitive nor specific. However, detection of mycobacterial DNA from FFPE specimens, suspected of being infected with mammalian mycobacteriosis, is a routine clinical procedure. In the present study, a polymerase chain reaction (PCR)-based method was used to detect and identify mycobacteria in FFPE specimens sampled from fish suspected of being infected with fish mycobacteriosis. A total of 45 fish tissue samples, comprising of 12 tissue samples obtained from experimentally infected fish and the remainder from fish naturally infected with mycobacteria, were analysed using a PCR protocol which amplifies a fragment of the mycobacterial 65 kDa heat-shock protein (hsp65) gene. PCR-restriction enzyme analysis and/or sequencing were employed to further analyse the PCR amplicons. The PCR results were compared with those obtained by histology and culture. Mycobacterial DNA was detected in 34 of the 45 samples examined, of which 16 samples (47%) showed granulomatous reactions on histological examination. Using histology as the gold standard, no false-negative PCR results were obtained. Also, considering the presence or absence of granulomas as a diagnostic criterion, the sensitivity and specificity of PCR in 42 of the FFPE tissues were 16/16 (100%) and 8/26 (approximately 30.8%), respectively. Corresponding microbiological cultures were available for 15 cases, of which 13 were pure Mycobacterium cultures. Of these, 13 were PCR positive (100% sensitivity and 50% specificity). The PCR-based methods used here proved sensitive, specific and rapid for the detection of mycobacteria in routinely processed paraffin wax-embedded and formalin-fixed histological samples, and the results of the study suggest that this method has

  12. 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.

  13. In situ hybridization with labeled probes: assessment of african Swine Fever virus in formalin-fixed paraffin-embedded tissues.

    PubMed

    Ballester, Maria; Rodríguez, Fernando

    2015-01-01

    In situ hybridization (ISH) has become a very valuable molecular diagnostic tool to detect specific DNA or RNA sequences in biological samples through the use of complementary DNA- or RNA-labeled probes. Here, we describe an optimized in situ hybridization protocol to detect African swine fever virus (ASFV) DNA in formalin-fixed, paraffin-embedded tissues using digoxigenin-labeled probes.

  14. Dose-Response Analysis of RNA-Seq Profiles in Archival Formalin-Fixed Paraffin-Embedded (FFPE) Samples.

    EPA Science Inventory

    Use of archival resources has been limited to date by inconsistent methods for genomic profiling of degraded RNA from formalin-fixed paraffin-embedded (FFPE) samples. RNA-sequencing offers a promising way to address this problem. Here we evaluated transcriptomic dose responses us...

  15. Dose-Response Analysis of RNA-Seq Profiles in Archival Formalin-Fixed Paraffin-Embedded (FFPE) Samples.

    EPA Science Inventory

    Use of archival resources has been limited to date by inconsistent methods for genomic profiling of degraded RNA from formalin-fixed paraffin-embedded (FFPE) samples. RNA-sequencing offers a promising way to address this problem. Here we evaluated transcriptomic dose responses us...

  16. 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...

  17. 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...

  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. Digital dewaxing of Raman signals: discrimination between nevi and melanoma spectra obtained from paraffin-embedded skin biopsies.

    PubMed

    Tfayli, Ali; Gobinet, Cyril; Vrabie, Valeriu; Huez, Regis; Manfait, Michel; Piot, Olivier

    2009-05-01

    Malignant melanoma (MM) is the most severe tumor affecting the skin and accounts for three quarters of all skin cancer deaths. Raman spectroscopy is a promising nondestructive tool that has been increasingly used for characterization of the molecular features of cancerous tissues. Different multivariate statistical analysis techniques are used in order to extract relevant information that can be considered as functional spectroscopic descriptors of a particular pathology. Paraffin embedding (waxing) is a highly efficient process used to conserve biopsies in tumor banks for several years. However, the use of non-dewaxed formalin-fixed paraffin-embedded tissues for Raman spectroscopic investigations remains very restricted, limiting the development of the technique as a routine analytical tool for biomedical purposes. This is due to the highly intense signal of paraffin, which masks important vibrations of the biological tissues. In addition to being time consuming and chemical intensive, chemical dewaxing methods are not efficient and they leave traces of the paraffin in tissues, which affects the Raman signal. In the present study, we use independent component analysis (ICA) on Raman spectral images collected on melanoma and nevus samples. The sources obtained from these images are then used to eliminate, using non-negativity constrained least squares (NCLS), the paraffin contribution from each individual spectrum of the spectral images of nevi and melanomas. Corrected spectra of both types of lesion are then compared and classified into dendrograms using hierarchical cluster analysis (HCA).

  20. Gelatin In Situ Zymography on Fixed, Paraffin-embedded Tissue: Zinc and Ethanol Fixation Preserve Enzyme Activity

    PubMed Central

    Hadler-Olsen, Elin; Kanapathippillai, Premasany; Berg, Eli; Svineng, Gunbjørg; Winberg, Jan-Olof; Uhlin-Hansen, Lars

    2010-01-01

    In situ zymography is a method for the detection and localization of enzymatic activity in tissue sections. This method is used with frozen sections because routine fixation of tissue in neutral-buffered formalin inhibits enzyme activity. However, frozen sections present with poor tissue morphology, making precise localization of enzymatic activity difficult to determine. Ethanol- and zinc-buffered fixative (ZBF) are known to preserve both morphological and functional properties of the tissue well, but it has not previously been shown that these fixatives preserve enzyme activity. In the present study, we show that in situ zymography can be performed on ethanol- and ZBF-fixed paraffin-embedded tissue. Compared with snap-frozen tissue, ethanol- and ZBF-fixed tissue showed stronger signals and superior morphology, allowing for a much more precise detection of gelatinolytic activity. Gelatinolytic enzymes could also be extracted from both ethanol- and ZBF-fixed tissue. The yield, as analyzed by SDS-PAGE gelatin zymography and Western blotting, was influenced by the composition of the extraction buffer, but was generally lower than that obtained from unfixed tissue. (J Histochem Cytochem 58:29–39, 2010) PMID:19755718

  1. Evaluation of archival time on shotgun proteomics of formalin-fixed and paraffin-embedded tissues.

    PubMed

    Balgley, Brian M; Guo, Tong; Zhao, Kejia; Fang, Xueping; Tavassoli, Fattaneh A; Lee, Cheng S

    2009-02-01

    There is increasing acceptance of the critical importance of correlating the morphologic features of tissue with the data obtained from various molecular analytic techniques. Access to archived formalin-fixed and paraffin-embedded (FFPE) tissue specimens via shotgun-based proteomic analyses may, therefore, open new avenues for both prospective and retrospective translational research. However, one of the remaining issues in performing comparative proteomic measurements among FFPE tissues relates to potential variability in protein composition and retrieval based on length of storage periods. Optimized protein extraction and digestion procedures for handling FFPE tissues are coupled with the capillary isotachophoresis-based proteome technology to evaluate the effects of length of storage period on archival tissue proteome analysis across 10 archived uterine mesenchymal tumor tissue blocks, including 9 uterine leiomyomas dating from 1990 to 2002 and a single case of alveolar soft part sarcoma (ASPS) from 1980. Several statistical measures, including the Pearson correlation coefficient, coefficient of variance, k-means clustering, and ANOVA, are employed to evaluate the possibility of an archival effect on individual proteins or groups of proteins within nine leiomyomas. Low abundance proteins may be more susceptible to the long-term storage as these proteins are more difficult to be retrieved and extracted as the tissue block ages in paraffin. Despite using tissue blocks stored for as many as 28 years, high confidence and comparative proteome analysis between the leiomyomas and the sarcoma is achieved. Though sharing over 1800 common proteins in a core set, a total of 80 proteins unique to the sarcoma are identified distinguishing the ASPS from the leiomyomas. Vacuolar proton translocating ATPase 116 kDa subunit isoform a3, one of the unique proteins expressed in the ASPS, is further validated by immunohistochemistry (IHC). Although IHC is highly sensitive and

  2. 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.

  3. Formalin-fixed and paraffin-embedded nodal non-Hodgkin's lymphomas demonstrate the same chromosome changes as those found in frozen samples: a comparative study using interphase fluorescence in situ hybridization.

    PubMed

    Godon, Alban; Genevieve, Franck; Valo, Isabelle; Josselin, Nicolas; Talmant, Pascaline; Foussard, Charles; Avet-Loiseau, Herve; Ifrah, Nobert; Zandecki, Marc; Rousselet, Marie-Christine

    2004-06-01

    Cytogenetic studies in lymphomas classically require fresh or frozen tissue, whereas in many instances only paraffin-embedded biopsies are available. We applied an interphase FISH assay on nuclei extracted from thick paraffin sections to determine accuracy of molecular cytogenetics in such samples. Twenty-three lymphoma samples and 4 reactive lymph nodes were tested with various commercially available DNA probes, and hybridization patterns were compared with those obtained on frozen nuclei counterparts. Successful hybridization with all probes tested was observed for 23/27 (85%) paraffin-embedded tissues and for all (100%) frozen samples, and cut-off levels defining positivity were superimposable for both situations. Chromosome changes were detected in the same way, without any false-positive or false-negative cases. Hybridization signals observed on dewaxed samples were either those classically expected to define the relevant chromosome change or were atypical: all atypical changes could be demonstrated also into nuclei from the frozen counterpart. Moreover, all typical and atypical chromosome changes observed on frozen nuclei were also detected in paraffin-embedded tissues. Our study shows that our interphase FISH assay performed on paraffin-embedded samples is a valuable alternate to conventional methods to ascertain diagnosis of lymphomas as to include patients into therapeutic trials.

  4. A simple osmium post-fixation paraffin-embedment technique to identify lipid accumulation in fish liver using medaka (Oryziaslatipes) eggs and eleutheroembryos as lipid rich models.

    PubMed

    Mondon, J A; Howitt, J; Tosiano, M; Kwok, K W H; Hinton, D E

    2011-01-01

    Hepatic lipidosis is a non-specific biomarker of effect from pollution exposure in fish. Fatty liver is often misdiagnosed or overlooked in histological assessments due to the decreasing application of specific fat procedures and stains. For example, ethanol dehydration in standard paraffin processing removes lipids, leaving vacuoles of which the precise nature is unknown. Lipids can be identified using osmium post-fixation in semi-thin resin sections or transmission electron microscopy. However, both are expensive and technically demanding procedures, often not available for routine environmental risk assessment and monitoring programs. The current emphasis to reduce and refine animal toxicity testing, requires refinement of the suite of histopathological techniques currently available to maximize information gained from using fish for toxicity testing and as bio-indicators of environmental quality. This investigation has successfully modified an osmium post-fixation technique to conserve lipids in paraffin-embedded tissues using medaka (Oryzias latipes) eleutheroembryos and eggs (embryos) as lipid rich models.

  5. Breslow thickness of cutaneous malignant melanoma in paraffin wax and frozen sections.

    PubMed Central

    Kirkham, N; Blessing, K; Gibson, B; Price, M L

    1991-01-01

    Breslow tumour thickness was measured in frozen and paraffin wax sections from 21 excision biopsies of cutaneous malignant melanomas by two observers. There was no consistent variation between frozen and paraffin wax sections, with recorded differences ranging from +0.3 mm to -0.2 mm. Interobserver differences ranged from +0.4 mm to -0.2 mm. The interobserver variations exceeded the intraobserver variations, but neither were significant. These findings show conclusively that, when using high quality frozen sections, there is no significant difference between Breslow thickness measured in frozen or paraffin wax sections and therefore that frozen sections can be used to microstage melanoma. Interobserver variations seem to be a more likely source of erroneous measurements of tumour thickness. Images PMID:1890198

  6. MammaPrint molecular diagnostics on formalin-fixed, paraffin-embedded tissue.

    PubMed

    Sapino, Anna; Roepman, Paul; Linn, Sabine C; Snel, Mireille H J; Delahaye, Leonie J M J; van den Akker, Jeroen; Glas, Annuska M; Simon, Iris M; Barth, Neil; de Snoo, Femke A; van 't Veer, Laura J; Molinaro, Luca; Berns, Els M J J; Wesseling, Jelle; Riley, Lee B; Anderson, David; Nguyen, Bichlien; Cox, Charles E

    2014-03-01

    MammaPrint, a prognostic 70-gene profile for early-stage breast cancer, has been available for fresh tissue. Improvements in RNA processing have enabled microarray diagnostics for formalin-fixed, paraffin-embedded (FFPE) tissue. Here, we describe method optimization, validation, and performance of MammaPrint using analyte from FFPE tissue. Laboratory procedures for enabling the assay to be run on FFPE tissue were determined using 157 samples, and the assay was established using 125 matched FFPE and fresh tissues. Validation of MammaPrint-FFPE, compared with MammaPrint-fresh, was performed on an independent series of matched tissue from five hospitals (n = 211). Reproducibility, repeatability, and precision of the FFPE assay (n = 87) was established for duplicate analysis of the same tumor, interlaboratory performance, 20-day repeat experiments, and repeated analyses over 12 months. FFPE sample processing had a success rate of 97%. The MammaPrint assay using FFPE analyte demonstrated an overall equivalence of 91.5% (95% confidence interval, 86.9% to 94.5%) between the 211 independent matched FFPE and fresh tumor samples. Precision was 97.3%, and repeatability was 97.8%, with highly reproducible results between replicate samples of the same tumor and between two laboratories (concordance, 96%). Thus, with 580 tumor samples, MammaPrint was successfully translated to FFPE tissue. The assay has high precision and reproducibility, and FFPE results are substantially equivalent to results derived from fresh tissue.

  7. Multi-elemental imaging of paraffin-embedded human samples by laser-induced breakdown spectroscopy

    NASA Astrophysics Data System (ADS)

    Moncayo, S.; Trichard, F.; Busser, B.; Sabatier-Vincent, M.; Pelascini, F.; Pinel, N.; Templier, I.; Charles, J.; Sancey, L.; Motto-Ros, V.

    2017-07-01

    Chemical elements play central roles for physiological homeostasis in human cells, and their dysregulation might lead to a certain number of pathologies. Novel imaging techniques that improve the work of pathologists for tissue analysis and diagnostics are continuously sought. We report the use of Laser-Induced Breakdown Spectroscopy (LIBS) to perform multi-elemental images of human paraffin-embedded skin samples on the entire biopsy scale in a complementary and compatible way with microscope histopathological examination. A specific instrumental configuration is proposed in order to detect most of the elements of medical interest (i.e. P, Al, Mg, Na, Zn, Si, Fe, and Cu). As an example of medical application, we selected and analysed skin biopsies, including healthy skin tissue, cutaneous metastasis of melanoma, Merkel-cell carcinoma and squamous cell carcinoma. Clear distinctions in the distribution of chemical elements are observed from the different samples investigated. This study demonstrates the high complementarity of LIBS elemental imaging with conventional histopathology, opening new opportunities for any medical application involving metals.

  8. Expression of nerve growth factor receptor in paraffin-embedded soft tissue tumors.

    PubMed Central

    Perosio, P. M.; Brooks, J. J.

    1988-01-01

    Identification of growth factors and receptors in mesenchymal tumors may be crucial to understanding of growth regulation in sarcomas. During an immunohistochemical study of the expression of growth factors and receptors in human soft tissue tumors (STT), only 1 antisera capable of working in paraffin-embedded tissue was noted. A detailed study of 141 STT was undertaken to determine the frequency of expression of nerve growth factor receptor (NGF-R), its specificity and sensitivity for neural tumors, and the effect of fixation on detection. In normal mesenchymal tissue, only nerve sheath and perivascular staining was seen. No immunoreactivity was seen in many tumors including rhabdomyosarcoma, angiosarcoma, liposarcoma, Ewing's sarcoma, and alveolar soft part sarcoma. Less than 15% of tumors of smooth muscle, fibrous, or fibrohistiocytic origin showed immunoreactivity, usually focal. In contrast, a high frequency of immunoreactivity was noted in tumors of neural origin (74%). This included granular cell tumors (100%), Schwannoma/neurofibroma (91%), malignant Schwannoma (78%), neuroblastoma/neuroepithelioma (60%), and paraganglioma (57%). A high rate of reactivity was also seen in synovial sarcomas (80%), undifferentiated sarcomas (60%), and hemangiopericytomas (43%), suggesting a potential relationship to the neural phenotype. Among the neural tumors, Bouin's fixation was superior to formalin, suggesting that immunoreactivity for NGF-R is affected by fixation. This antibody may be a useful adjunct marker diagnostically. Images Figure 1 Figure 2 Figure 4 Figure 5 Figure 6 Figure 7 Figure 9 Figure 10 PMID:2456020

  9. A pressure cooking-based DNA extraction from archival formalin fixed, paraffin embedded tissue

    PubMed Central

    Chung, Joon-Yong; Yi, Joo Mi; Xie, Ran; Brown, Victoria; Lee, Olivia; Ahuja, Nita; Braunschweig, Till; Hewitt, Stephen M.

    2012-01-01

    As emerging novel DNA-based methodologies are adopted, nucleic acid-based assays depend critically on the quality and quantity of extracted DNA. Formalin fixed, paraffin embedded (FFPE) tissue samples provide an invaluable resource for subsequent molecular studies of clinical phenotypes, but high quality DNA extraction from archival FFPE tissue specimen remains complex and time consuming. To address this challenge, we have developed a reliable rapid DNA extraction method for FFPE tissue specimens. It is based on deparaffinization at high temperature coupled with relieving crosslink in a pressure cooker. The DNA yield by this rapid method resulted in an average 1.8-fold increase in comparison with the commercial kit; O.D 260/280 ratios between 1.87 and 1.95. The DNA obtained by the rapid method was suitable for methylation analyses in colon cancer patients. These data suggest that this new DNA extraction method coupled with MSP can be used for epigenetic studies with the advantages of rapidity and high quality, and may contribute to the development of biomarkers in clinical studies. PMID:22449494

  10. 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.

  11. Immunohistochemical detection of extrinsic and intrinsic mediators of apoptosis in porcine paraffin-embedded tissues.

    PubMed

    Barranco, Inmaculada; Gómez-Laguna, Jaime; Rodríguez-Gómez, Irene M; Salguero, Francisco J; Pallarés, Francisco J; Bernabé, Antonio; Carrasco, Librado

    2011-02-15

    Apoptosis is a strictly regulated mechanism of cell death that involves a complex network of biochemical pathways. Whether a cell undergoes apoptosis or not depends on a delicate balance of anti- and pro-apoptotic stimuli. This phenomenon can be induced by two different pathways: intrinsic and extrinsic pathways. The main aim of this study was to determine the ideal fixative and antigen retrieval method in porcine paraffin embedded tissues for the immunohistochemical detection of apoptosis mediators, from both extrinsic and intrinsic pathways. Tonsil, retropharyngeal lymph node and lung tissue samples were fixed in 10% neutral buffered formalin, Bouin solution and zinc salts fixative (ZSF) and different unmasking methods were carried out. Both 10% neutral buffered formalin and ZSF resulted as the fixatives of election to study apoptosis phenomena. Tween 20 (0.01% in PBS), citrate buffer (microwave, pH 6.0) and/or protease type XIV were the antigen retrieval methods which displayed better labelling. Our results allow to deep in the knowledge of apoptosis and its role in the pathogenesis of porcine diseases. Copyright © 2010 Elsevier B.V. All rights reserved.

  12. Improved Protein Extractionand Protein Identification from Archival Formalin-fixed Paraffin-embedded Human Aortas

    PubMed Central

    Fu, Zongming; Yan, Kun; Rosenberg, Avraham; Jin, Zhicheng; Crain, Barbara; Athas, Grace; Vander Heide, Richard S; Howard, Timothy; Everett, Allen D.; Herrington, David; Van Eyk, Jennifer E.

    2014-01-01

    Purpose Evaluate combination of heat and elevated pressure to enhance protein extraction and quality of formaldehyde-fixed (FF), and FF paraffin-embedded (FFPE) aorta for proteomics. Experiment design Proteins were extracted from fresh frozen aorta at RT. FF and FFPE aortas (3 months and 15 years) were extracted at RT, heat alone, or a combination of heat and high pressure. Protein yields were compared, and digested peptides from the extracts were analyzed with mass spectrometry. Results Combined heat and elevated pressure increased protein yield from human FF or FFPE aorta compared to matched tissues with heat alone (1.5 fold) or at RT (8.3 fold), resulting in more proteins identified and with more sequence coverage. The length of storage did adversely affect the quality of proteins from FF tissue. For long term storage, aorta was preserved better with FFPE than FF alone. Periostin and MGF-E8 were demonstrated suitable for MRM assays from FFPE aorta. Conclusions and clinical relevance Combination of heat and high pressure is an effective method to extract proteins from FFPE aorta for downstream proteomics. This method opens the possibility for use of archival and often rare FFPE aortas and possibly other tissues available to proteomics for biomarker discovery and quantification. PMID:23339088

  13. 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.

  14. Improved protein extraction and protein identification from archival formalin-fixed paraffin-embedded human aortas.

    PubMed

    Fu, Zongming; Yan, Kun; Rosenberg, Avraham; Jin, Zhicheng; Crain, Barbara; Athas, Grace; Heide, Richard S Vander; Howard, Timothy; Everett, Allen D; Herrington, David; Van Eyk, Jennifer E

    2013-04-01

    Evaluate combination of heat and elevated pressure to enhance protein extraction and quality of formalin-fixed (FF), and FF paraffin-embedded (FFPE) aorta for proteomics. Proteins were extracted from fresh frozen aorta at room temperature (RT). FF and FFPE aortas (3 months and 15 years) were extracted at RT, heat alone, or a combination of heat and high pressure. Protein yields were compared, and digested peptides from the extracts were analyzed with MS. Combined heat and elevated pressure increased protein yield from human FF or FFPE aorta compared to matched tissues with heat alone (1.5-fold) or at RT (8.3-fold), resulting in more proteins identified and with more sequence coverage. The length of storage did adversely affect the quality of proteins from FF tissue. For long-term storage, aorta was preserved better with FFPE than FF alone. Periostin and MGF-E8 were demonstrated suitable for MRM assays from FFPE aorta. Combination of heat and high pressure is an effective method to extract proteins from FFPE aorta for downstream proteomics. This method opens the possibility for use of archival and often rare FFPE aortas and possibly other tissues available to proteomics for biomarker discovery and quantification. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. 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.

  16. 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.

  17. Autofluorescence analysis of dermatitis and squamous cell carcinoma in paraffin wax-embedded skin samples.

    PubMed

    Choi, J H; Jeong, K H; Sung, J Y; Kim, N I; Shin, M K

    2015-03-01

    Recent research has investigated the use of autofluorescence (AF) for distinguishing between normal and cancerous tissues according to different fluorescence characteristics. To analyze if AF can help differentiate cancerous lesions from other nonneoplastic lesions, such as dermatitis, in each layer of the skin ex vivo. Paraffin wax-embedded tissue samples were obtained from patients who were histopathologically diagnosed with squamous cell carcinoma (SCC), psoriasis, chronic dermatitis (lichen simplex chronicus, prurigo nodularis) or acute dermatitis (atopic dermatitis). AF intensity was measured in four layers of the epidermis (corneal, granular, spinous and basal) and two layers of the dermis (papillary and reticular). AF was highest in all layers of psoriasis samples compared with all layers of all other groups. Higher AF values were seen in SCC compared with all skin layers of acute and chronic dermatitis; this finding was especially true in the corneal layer, papillary dermis and reticular dermis. This ex vivo AF study provides basic data for future in vivo studies of AF as a noninvasive diagnostic tool. © 2014 British Association of Dermatologists.

  18. 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.

  19. 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.

  20. Mass spectrometry-based proteomic analysis of formalin-fixed paraffin-embedded extrahepatic cholangiocarcinoma.

    PubMed

    Maeda, Shimpei; Morikawa, Takanori; Takadate, Tatsuyuki; Suzuki, Takashi; Minowa, Takashi; Hanagata, Nobutaka; Onogawa, Tohru; Motoi, Fuyuhiko; Nishimura, Toshihide; Unno, Michiaki

    2015-09-01

    Extrahepatic cholangiocarcinoma is very difficult to diagnose at an early stage, and has a poor prognosis. Novel markers for diagnosis and optimal treatment selection are needed. However, there has been very limited data on the proteome profile of extrahepatic cholangiocarcinoma. This study was designed to unravel the proteome profile of this disease and to identify overexpressed proteins using mass spectrometry-based proteomic approaches. We analyzed a discovery set of formalin-fixed paraffin-embedded tissues of 14 extrahepatic cholangiocarcinomas using shotgun mass spectrometry, and compared proteome profiles with those of seven controls. Then, selected candidates were verified by quantitative analysis using scheduled selected reaction monitoring-based mass spectrometry. Furthermore, immunohistochemical staining used a validation set of 165 cases. In total, 1,992 proteins were identified and 136 proteins were overexpressed. Verification of 58 selected proteins by quantitative analysis revealed 11 overexpressed proteins. Immunohistochemical validation for 10 proteins showed positive rates of S100P (84%), CEAM5 (75%), MUC5A (62%), OLFM4 (60%), OAT (42%), CAD17 (41%), FABPL (38%), AOFA (30%), K1C20 (25%) and CPSM (22%) in extrahepatic cholangiocarcinomas, which were rarely positive in controls. We identified 10 proteins associated with extrahepatic cholangiocarcinoma using proteomic approaches. These proteins are potential targets for future diagnostic biomarkers and therapy. © 2015 Japanese Society of Hepato-Biliary-Pancreatic Surgery.

  1. 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

  2. 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. Copyright © 2015 Elsevier B.V. All rights reserved.

  3. Exome Enrichment and SOLiD Sequencing of Formalin Fixed Paraffin Embedded (FFPE) Prostate Cancer Tissue

    PubMed Central

    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. PMID:22942743

  4. Embedment-free section electron microscopy.

    PubMed

    Kondo, Hisatake

    2006-08-01

    Because of potential hindrance of clear viewing in epoxy sections of biological entities having an electron density similar to and lower than that of epoxy resin, the author has stressed that the embedment-free section electron microscopy is necessary for re-examination and/or clarification of biological specimen structures, and that the embedment-free electron microscopy is reliably done by using water-soluble polyethylene glycol (PEG) as a transient embedding media and by critical point-drying of embedment-free sections after de-embedment of PEG by immersion of semithin sections into water. With the embedment-free electron microscopy, the author has presented five major findings: the appearance of microtrabecular lattices with different compactnesses in various cells and in intracellular domains of a given cell, the faithful reproduction of microtrabecula-like strand lattices in vitro with increasing compactnesses from artificial protein solutions at correspondingly increasing concentrations, the appearance of more compact lattices from gelated gelatin than from solated gelatin at a given concentration in vitro, the changeability in compactnesses of the microtrabecular lattices by hyper- or hypotonic shock treatments of cells, and the confined appearance of an intracellular protein in the centripetal demilune of centrifuged ganglion cells which is occupied with the microtrabecular lattices of a substantial compactness. From these findings, several conclusions are drawn: individual strands themselves of the microtrabeculae are meaningless, the appearance of microtrabeculae represents the presence of proteins at a certain concentration, and it is therefore likely that the aqueous cytoplasm is equivalent to the aqueous solution. In addition, it is possible that the appearance of two contiguous lattice domains exhibiting different compactnesses in a given cell may represent the occurrence of a contiguity of sol to gel states of cytoplasmic domains. It is thus

  5. Analytical validation of a melanoma diagnostic gene signature using formalin-fixed paraffin-embedded melanocytic lesions.

    PubMed

    Warf, M Bryan; Flake, Darl D; Adams, Doug; Gutin, Alexander; Kolquist, Kathryn A; Wenstrup, Richard J; Roa, Benjamin B

    2015-01-01

    These studies were to validate the analytical performance of a gene expression signature that differentiates melanoma and nevi, using RNA expression from 14 signature genes and nine normalization genes that generates a melanoma diagnostic score (MDS). Formalin-fixed paraffin-embedded melanocytic lesions were evaluated in these studies. The overall SD of the assay was determined to be 0.69 MDS units. Individual amplicons within the signature had an average amplification efficiency of 92% and a SD less than 0.5 CT. The MDS was reproducible across a 2000-fold dilution range of input RNA. Melanin, an inhibitor of PCR, does not interfere with the signature. These studies indicate this signature is robust and reproducible and is analytically validated on formalin-fixed paraffin-embedded melanocytic lesions.

  6. Enrichment of PrPSc in formalin-fixed, paraffin-embedded tissues prior to analysis by Western blot.

    PubMed

    Nicholson, Eric M

    2011-07-01

    Diagnosis of prion disease is primarily through immunodetection of the infectious agent. Typically, 2 distinct procedures are recommended for a definitive diagnosis, with immunohistochemistry and Western blot providing the most information as to the specific isolate in question. In the past, these approaches required formalin-fixed, paraffin-embedded tissue and fresh or frozen tissue, respectively; however, methods have been developed that allow for use of fixed tissue for Western blot. The present study describes a method of enriching PrP(Sc) in formalin-fixed, paraffin-embedded tissues prior to Western blot analysis for the detection of PrP(Sc). With this modified procedure, 5 times the previously reported sample size may be used for analysis, greatly enhancing the sensitivity of this procedure.

  7. Dual colour FISH in paraffin wax embedded bone trephines for identification of numerical and structural chromosomal abnormalities in acute myeloid leukaemia and myelodysplasia

    PubMed Central

    Le Maitre, C L; Byers, R; Liu, Y; Hoyland, J; Freemont, A

    2001-01-01

    Aims/Background—The advent of new treatments for haematological malignancies has led to the need for a correlation between cytogenetic and morphological abnormalities. This study aimed to achieve this by the application of interphase cytogenetics to marrow trephine sections, a technique not previously reported for formalin fixed, paraffin wax embedded trephine biopsies. Methods—Dual colour fluorescence in situ hybridisation (FISH) was used to detect numerical and structural abnormalities in routinely processed paraffin wax embedded trephine biopsies. Three cases with t(8;21) and three with t(15;17) were analysed, together with a case of trisomy 8. Chromosome specific probes were hybridised with sections and disclosed by fluorescein isothiocyanate and rhodamine/Texas red labelled antidigoxigenin and antibiotin amplification; translocations were identified by colocalisation of probes using a double wavelength bypass filter. Results—A translocation signal was present in 12% and 11.5% of the cells counted in the t(8;21) and t(15;17) cases, respectively, but in none of the normal controls (p < 0.001). In the case of trisomy 8, 9% of the cells counted contained three hybridisation signals for chromosome 8, whereas no cell contained more than two in the normal control (p < 0.001). Conclusions—This technique is useful for archived routinely processed material, enabling it to be used as a research tool but also, and perhaps more importantly, in clinical practice. Key Words: acute myeloid leukaemia • paraffin wax embedded bone trephines • cytogenetic abnormalities • myelodysplasia • fluorescence in situ hybridisation PMID:11533086

  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 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

  10. 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.

  11. 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.

  12. Quantitative Analysis of Formaldehyde-induced Fluorescence in Paraffin-embedded Specimens of Malignant Melanomas and Other Melanocytic Lesions.

    PubMed

    Hara, Hiroyuki; Naito, Michiko; Harada, Tomonori; Tsuboi, Isao; Terui, Tadashi; Aizawa, Shin

    2016-03-01

    Inter-observer agreement is problematic in the histopathological diagnosis of melanoma and melanocytic naevi, even among expert pathologists. Formaldehyde-induced fluorescence (FIF) has been used for histochemical demonstration of catecholamines, 5-hydroxytryptamine and their immediate precursors. FIF can detect melanogenic activity and may be useful in differentiating malignant melanoma from other melanocytic lesions. The fluorescence of various types of melanocytic lesions has been previously studied quantitatively in formalin-fixed and paraffin-embedded sections. This study compared 2 sets of excitation and emission bands: 450-490 nm excitation/510-560 nm absorption filters (filter unit A) and 480 nm excitation/510< nm absorption filters (filter unit B). Higher FIF was observed with filter unit A than with filter unit B. FIF intensity of central regions was found to be higher than that of the peripheral regions. Mean FIF was significantly higher in malignant melanomas than in naevi. Fluorescence imaging with filter unit A gave better diagnostic performance. In conclusion, quantitative measurement of FIF is a useful marker of malignant potential.

  13. A simple and reliable pretreatment protocol facilitates fluorescent in situ hybridisation on tissue microarrays of paraffin wax embedded tumour samples

    PubMed Central

    Chin, S-F; Daigo, Y; Huang, H-E; Iyer, N G; Callagy, G; Kranjac, T; Gonzalez, M; Sangan, T; Earl, H; Caldas, C

    2003-01-01

    Aims: To describe a robust pretreatment protocol for preparing paraffin wax embedded tissues on tissue microarrays for fluorescence in situ hybridisation (FISH). The newly developed pretreatment protocol described here was compared with the commonly used sodium thiocyanate based protocol and two different heating methods used in standard antigen unmasking protocols for immunohistochemistry (pressure cooking and microwaving in citrate acid buffer). Methods: Dewaxed tissue sections were incubated in 10mM citric acid buffer at 80°C for 30 minutes to two hours, followed by a short pepsin digestion (1–5 mg/ml). Pretreated tissues were co-denatured with DNA probes at 80°C for 10 minutes, followed by hybridisation at 37°C for 48–72 hours. Results: The three protocols using citrate acid buffer produced FISH signals with superior signal to noise ratios compared with sodium thiocyanate pretreatment. Most importantly, the best tissue attachment was achieved using the newly developed pretreatment protocol: on tissue microarrays less than 1% of cores were lost. To date, a total of 30 probes have been successfully hybridised on to breast tissue and multi-tissue microarrays. Conclusion: This pretreatment protocol is easy, reproducible, and facilitates FISH on tissue microarrays, with potential for widespread application in cancer research. PMID:14514921

  14. 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.

  15. A simple and reliable pretreatment protocol facilitates fluorescent in situ hybridisation on tissue microarrays of paraffin wax embedded tumour samples.

    PubMed

    Chin, S-F; Daigo, Y; Huang, H-E; Iyer, N G; Callagy, G; Kranjac, T; Gonzalez, M; Sangan, T; Earl, H; Caldas, C

    2003-10-01

    To describe a robust pretreatment protocol for preparing paraffin wax embedded tissues on tissue microarrays for fluorescence in situ hybridisation (FISH). The newly developed pretreatment protocol described here was compared with the commonly used sodium thiocyanate based protocol and two different heating methods used in standard antigen unmasking protocols for immunohistochemistry (pressure cooking and microwaving in citrate acid buffer). Dewaxed tissue sections were incubated in 10mM citric acid buffer at 80 degrees C for 30 minutes to two hours, followed by a short pepsin digestion (1-5 mg/ml). Pretreated tissues were co-denatured with DNA probes at 80 degrees C for 10 minutes, followed by hybridisation at 37 degrees C for 48-72 hours. The three protocols using citrate acid buffer produced FISH signals with superior signal to noise ratios compared with sodium thiocyanate pretreatment. Most importantly, the best tissue attachment was achieved using the newly developed pretreatment protocol: on tissue microarrays less than 1% of cores were lost. To date, a total of 30 probes have been successfully hybridised on to breast tissue and multi-tissue microarrays. This pretreatment protocol is easy, reproducible, and facilitates FISH on tissue microarrays, with potential for widespread application in cancer research.

  16. 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

  17. 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.

  18. Histological and genotypical characterization of feline cutaneous mycobacteriosis: a retrospective study of formalin-fixed paraffin-embedded tissues.

    PubMed

    Davies, Jennifer L; Sibley, Jennifer A; Myers, Sherry; Clark, Edward G; Appleyard, Greg D

    2006-06-01

    Twenty-nine cases presumptively diagnosed as feline cutaneous mycobacteriosis were evaluated microscopically with haematoxylin and eosin and modified Fite's stained sections using archived formalin-fixed paraffin-embedded tissue specimens. Lesions were characterized histologically as feline leprosy (7 cases lepromatous and 16 cases tuberculoid) or atypical mycobacteriosis (3 cases); three cases did not fit these criteria and were classified as 'miscellaneous'. Actinomycetales-specific polymerase chain reaction (PCR) of variable regions 1, 2 and 3 of the 16S ribosomal RNA (rRNA) gene and subsequent sequence analysis of the amplicons were performed to identify the species of mycobacteria associated with each case. Together, this study identified 10 different Actinomycetales organisms with greater than 98% nucleotide sequence identity to named species, nine were of the genus Mycobacterium and eight were associated with feline leprosy (both lepromatous and tuberculoid). Based on this study, we conclude that feline cutaneous mycobacteriosis should be considered as a syndrome with varied clinical and histological presentations associated with a variety of different Mycobacterium species, organisms other than Mycobacterium sp. may be associated with feline cutaneous mycobacteriosis lesions, and molecular diagnostic techniques can be an important tool for identifying agents associated with lesions of feline cutaneous mycobacteriosis.

  19. Fixation methods for the preservation of morphology, RNAs, and proteins in paraffin-embedded human cervical cancer cell xenografts in mice.

    PubMed

    Matsuda, Yoko; Ishiwata, Toshiyuki

    2015-01-01

    After various types of fixation, paraffin-embedded tissues are commonly used for histological analysis and pathological diagnosis; they are also suitable for long-term storage. Neutral buffered formalin, paraformaldehyde, and ethanol are common fixatives for histopathological analysis. For molecular biological analysis, fixed paraffin-embedded tissues are valuable resources; suitable fixative solutions and methods are needed to quantify and perform molecular biological analyses including immunohistochemistry, in situ hybridization, and quantitative polymerase chain reaction. Currently, 4 % paraformaldehyde is the recommended fixative for the preservation of RNAs and proteins, as well as for morphological study in paraffin-embedded human cervical cancer tissues that were xenografted in immunodeficient mice. Here, we describe the method for the fixation and preparation of paraffin-embedded tissue specimens for analysis of RNAs, proteins, and morphology.

  20. Single-Nucleotide Polymorphism-Microarray Ploidy Analysis of Paraffin-Embedded Products of Conception in Recurrent Pregnancy Loss Evaluations.

    PubMed

    Maslow, Bat-Sheva L; Budinetz, Tara; Sueldo, Carolina; Anspach, Erica; Engmann, Lawrence; Benadiva, Claudio; Nulsen, John C

    2015-07-01

    To compare the analysis of chromosome number from paraffin-embedded products of conception using single-nucleotide polymorphism (SNP) microarray with the recommended screening for the evaluation of couples presenting with recurrent pregnancy loss who do not have previous fetal cytogenetic data. We performed a retrospective cohort study including all women who presented for a new evaluation of recurrent pregnancy loss over a 2-year period (January 1, 2012, to December 31, 2013). All participants had at least two documented first-trimester losses and both the recommended screening tests and SNP microarray performed on at least one paraffin-embedded products of conception sample. Single-nucleotide polymorphism microarray identifies all 24 chromosomes (22 autosomes, X, and Y). Forty-two women with a total of 178 losses were included in the study. Paraffin-embedded products of conception from 62 losses were sent for SNP microarray. Single-nucleotide polymorphism microarray successfully diagnosed fetal chromosome number in 71% (44/62) of samples, of which 43% (19/44) were euploid and 57% (25/44) were noneuploid. Seven of 42 (17%) participants had abnormalities on recurrent pregnancy loss screening. The per-person detection rate for a cause of pregnancy loss was significantly higher in the SNP microarray (0.50; 95% confidence interval [CI] 0.36-0.64) compared with recurrent pregnancy loss evaluation (0.17; 95% CI 0.08-0.31) (P=.002). Participants with one or more euploid loss identified on paraffin-embedded products of conception were significantly more likely to have an abnormality on recurrent pregnancy loss screening than those with only noneuploid results (P=.028). The significance remained when controlling for age, number of losses, number of samples, and total pregnancies. These results suggest that SNP microarray testing of paraffin-embedded products of conception is a valuable tool for the evaluation of recurrent pregnancy loss in patients without prior fetal

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

    PubMed Central

    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

    2015-01-01

    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 (R2: 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

  2. Genomic gains and losses in malignant mesothelioma demonstrated by FISH analysis of paraffin-embedded tissues.

    PubMed

    Takeda, Maiko; Kasai, Takahiko; Enomoto, Yasunori; Takano, Masato; Morita, Kouhei; Kadota, Eiji; Iizuka, Norishige; Maruyama, Hiroshi; Nonomura, Akitaka

    2012-01-01

    Malignant mesothelioma (MM) results from the accumulation of a number of acquired genetic events at the onset. In MM, the most frequent changes were losses in 9p21, 1p36, 14q32 and 22q12, and gains in 5p, 7p and 8q24 by comparative genomic hybridisation analysis. Although the diagnostic utility of 9p21 homozygous deletion by fluorescence in situ hybridisation (FISH) analysis in MM has been reported recently, alterations of other genes have not been examined to any great extent. This study analysed the frequency of various genomic gains and losses in MM using FISH analysis. The authors performed a FISH analysis using paraffin-embedded tissues from 42 cases of MM. Chromosomal losses in MM were found at 9p21 (83%), 1p36 (43%), 14q32 (43%) and 22q12 (38%), whereas gains were found at 5p15 (48%), 7p12 (38%) and 8q24 (45%). There were no cases of adenomatoid tumour, benign mesothelial multicystic tumour, reactive mesothelial hyperplasia or pleuritis showing any gains or losses. At least one genomic abnormality was identified in all cases of MM. Among various histological subtypes, the chromosomal abnormality tended to be more common in cases showing sarcomatous elements (biphasic or pure sarcomatoid) than in cases showing an epithelioid histology. The authors found various genomic gains and losses in MM by FISH analysis. The frequency of each genomic gain or loss examined in MM by FISH analysis almost agreed with the comparative genomic hybridisation technique in previous studies. This study suggests that genomic evaluation by FISH analysis might be helpful in distinguishing MM from benign mesothelial proliferation.

  3. 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

  4. Unmasking of complements using proteinase-K in formalin fixed paraffin embedded renal biopsies

    PubMed Central

    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

  5. 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.

  6. PCR for the Diagnosis of Abdominal Angiostrongyliasis in Formalin-Fixed Paraffin-Embedded Human Tissue

    PubMed Central

    Rodriguez, Rubens; da Silva, Ana Cristina Aramburú; Müller, Carla Aristonara; Alves, Silvana Lunardini; Graeff-Teixeira, Carlos; Fornari, Fernando

    2014-01-01

    To date the diagnosis of abdominal angiostrongyliasis (AA) depends on the histological identification of Angiostrongylus costaricensis (AC) in surgical specimens. However, microscopic evaluation is time consuming and often fails in identifying the parasite. We tested whether PCR might help in the diagnosis of AA by identifying parasite DNA in formalin-fixed paraffin-embedded (FFPE) tissue. We used primers based on DNA from Angiostrongilus cantonensis. Four groups of FFPE intestinal tissue were tested: (1) confirmed cases (n = 20), in which AC structures were present in the target tissue; (2) presumptive cases (n = 20), containing changes secondary to AC infection in the absence of AC structures; (3) negative controls (n = 3), consisting of normal colonic tissue; and (4) tissue affected by other parasitoses (n = 7), including strongyloidiasis, ascaridiasis, schistosomiasis, and enterobiasis. Most lesions of confirmed cases were located in small and/or large bowel (90%), as compared with presumptive cases, in which 70% of lesions were in appendix (P = 0.0002). When confronted with cases of other parasitoses, PCR showed sensitivity of 55%, specificity of 100% and positive predictive value of 100%. In presumptive cases PCR was positive in 4 (20%). All specimens from negative controls and other parasitoses were negative. In conclusion, the PCR technique showed intermediate sensitivity and optimal specificity, being clinically relevant when positive for abdominal angiostrongyliasis. It allowed a 20% gain in diagnosis of presumptive cases. PCR might help in the diagnosis of abdominal angiostrongyliasis, particularly when the pathologists are not experienced with such disease. PMID:24705328

  7. A gene-protein assay for human epidermal growth factor receptor 2 (HER2): brightfield tricolor visualization of HER2 protein, the HER2 gene, and chromosome 17 centromere (CEN17) in formalin-fixed, paraffin-embedded breast cancer tissue sections

    PubMed Central

    2012-01-01

    Background The eligibility of breast cancer patients for human epidermal growth factor receptor 2 (HER2)-directed therapies is determined by the HER2 gene amplification and/or HER2 protein overexpression status of the breast tumor as determined by in situ hybridization (ISH) or immunohistochemistry (IHC), respectively. Our objective was to combine the US Food and Drug Administration (FDA)-approved HER2 & chromosome 17 centromere (CEN17) brightfield ISH (BISH) and HER2 IHC assays into a single automated HER2 gene-protein assay allowing simultaneous detection of all three targets in a single tissue section. Methods The HER2 gene-protein assay was optimized using formalin-fixed, paraffin-embedded (FFPE) samples of the xenograft tumors MCF7 [HER2 negative (non-amplified gene, protein negative)] and Calu-3 [HER2 positive (amplified gene, protein positive)]. HER2 IHC was performed using a rabbit monoclonal anti-HER2 antibody (clone 4B5) and a conventional 3,3'-diaminobenzidine IHC detection. The HER2 & CEN17 BISH signals were visualized using horseradish peroxidase-based silver and alkaline phosphatase-based red detection systems, respectively with a cocktail of 2,4-dinitrophenyl-labeled HER2 and digoxigenin-labeled CEN17 probes. The performance of the gene-protein assay on tissue microarray slides containing 189 randomly selected FFPE clinical breast cancer tissue cores was compared to that of the separate HER2 IHC and HER2 & CEN17 BISH assays. Results HER2 protein detection was optimal when the HER2 IHC protocol was used before (rather than after) the BISH protocol. The sequential use of HER2 IHC and HER2 & CEN17 BISH detection steps on FFPE xenograft tumor sections appropriately co-localized the HER2 protein, HER2 gene, and CEN17 signals after mitigating the silver background staining by using a naphthol phosphate-containing hybridization buffer for the hybridization step. The HER2 protein and HER2 gene status obtained using the multiplex HER2 gene-protein assay

  8. New approaches for genotyping paraffin wax embedded breast tissue from patients with cancer: the Iowa women's health study.

    PubMed

    Thyagarajan, B; Anderson, K E; Kong, F; Selk, F R; Lynch, C F; Gross, M D

    2005-09-01

    The use of paraffin wax embedded tissue samples as a source of DNA for genotype analysis has been limited because of difficulties in DNA extraction and single nucleotide polymorphism (SNP) analysis. To test the feasibility of applying the combination of a commonly used DNA isolation procedure, PureGene, and a high throughput SNP analysis method, the polymerase chain reaction (PCR)-INVADER assay, to genotype several types of paraffin wax embedded breast tissues. Twenty formalin fixed, paraffin wax blocks were obtained from five participants in the Iowa women's health study. Each participant provided several types of tissue including normal lymph node, normal nipple/areola tissue, inflammatory/fibrotic breast tissue, or normal breast tissue, and tumour tissue. Good quality DNA (260/280 ratio >1.6) was obtained from all tissues. Normal lymph nodes yielded the largest amount of DNA (97.1 mug). DNA obtained from the samples was tested for a germline C1183T polymorphism in the MnSOD gene by three methods-PCR-RFLP (restriction fragment length polymorphism), INVADER assay, and PCR-INVADER assay. Of the 20 samples, PCR-RFLP genotyped 16, the PCR-INVADER assay 18, and the INVADER assay two. This methodology was then used to analyse five additional genotypes and confirmed the general applicability of the method. This study demonstrated the feasibility of (1) using several paraffin wax embedded breast tissues as a source of DNA for germline genetic analysis, with lymph nodes providing the highest yield, and (2) using the combination of a common extraction method with a high throughput SNP analysis method, the PCR-INVADER assay.

  9. DNA extraction from archival formalin-fixed, paraffin-embedded tissues: heat-induced retrieval in alkaline solution.

    PubMed

    Shi, Shan-Rong; Datar, Ram; Liu, Cheng; Wu, Lin; Zhang, Zina; Cote, Richard J; Taylor, Clive R

    2004-09-01

    Based on the antigen retrieval principle, our previous study has demonstrated that heating archival formalin-fixed, paraffin-embedded (FFPE) tissues at a higher temperature and at higher pH value of the retrieval solution may achieve higher efficiency of extracted DNA, when compared to the traditional enzyme digestion method. Along this line of heat-induced retrieval, this further study is focused on development of a simpler and more effective heat-induced DNA retrieval technique by testing various retrieval solutions. Three major experiments using a high temperature heating method to extract DNA from FFPE human lymphoid and other tissue sections were performed to compare: (1) different concentrations of alkaline solution (NaOH or KOH, pH 11.5-12) versus Britton and Robinson type of buffer solution (BR buffer) of pH 12 that was the only retrieval solution tested in our previous study; (2) several chemical solutions (SDS, Tween 20, and GITC of various concentrations) versus BR buffer or alkaline solution; and (3) alkaline solution mixed with chemicals versus BR buffer or single alkaline solution. Efficiency of DNA extraction was evaluated by measuring yields using spectrophotometry, electrophoretic pattern, semiquantitation of tissue dissolution, PCR amplification, and kinetic thermocycling-PCR methods. Results showed that boiling tissue sections in 0.1 M NaOH or KOH or its complex retrieval solutions produced higher yields and better quality of DNA compared to BR buffer or chemical solutions alone. The conclusion was that boiling FFPE tissue sections in 0.1 M alkaline solution is a simpler and more effective heat-induced retrieval protocol for DNA extraction. Combination with some chemicals (detergents) may further significantly improve efficiency of the heat-induced retrieval technique.

  10. DNA extraction from paraffin-embedded tissues using a salting-out procedure: a reliable method for PCR amplification of archival material.

    PubMed

    Howe, J R; Klimstra, D S; Cordon-Cardo, C

    1997-07-01

    Many techniques have been described for the extraction of DNA from paraffin-embedded tissues. Numerous efforts have been directed at simplification of these methods for rapid analysis using PCR. One disadvantage to some of the simpler procedures is inefficient PCR amplification, and for more involved ones using phenol/chloroform extraction, reduction in the yield of DNA. In the present study we report the use of a novel salting-out procedure that was utilized to extract DNA from 259 separate microdissection specimens of formalin-fixed, paraffin-embedded tissue sections. These sections were derived from 97 patients with tumors of the ampulla of Vater resected between 1965 and 1995 at our institution. The mean DNA yield was 22.75 micrograms (median 13.2 +/- 30.25) and the mean 260/280 absorbance ratio was 1.68 (median 1.70 +/- 0.25). All specimens (259/259) were successfully used to amplify K-ras exon 1 by a nested PCR technique. These results indicate that this DNA extraction method produces good yields of quality DNA, even from specimens several decades old.

  11. Isolation and detection of Fasciola hepatica DNA in Lymnaea viatrix from formalin-fixed and paraffin-embedded tissues through multiplex-PCR.

    PubMed

    Magalhães, Kelly Grace; Jannotti-Passos, Liana K; Caldeira, Roberta L; Berne, Maria Elisabeth Aires; Muller, Gertrude; Carvalho, Omar S; Lenzi, Henrique Leonel

    2008-04-15

    Detection of Fasciola hepatica infection in Lymnaea viatrix through analysis of histological cuts is based upon morphological characters of the parasite during the intra-mollusk phase of parasitism. At this stage, trematode forms are very similar and, thus, very difficult to differentiate. Specific detection may also be impaired by the presence of other helminthes in the mollusk. Histological samples are usually fixed in formalin, embedded in paraffin, sectioned and HE stained. In the current study, a method for the extraction of DNA from formalin-fixed, paraffin-embedded tissues was standardized by means of deparaffinizing with xylol and digesting with proteinase K. Extracted DNA was amplified in a multiplex-PCR, by using simultaneous primers in a single reaction under high stringency conditions. Results showed specific amplification of DNA from the trematode and the snails. The technique was sensitive enough to detect F. hepatica infections in L. viatrix, in histological sections in which the presence of larval stages could not be observed through brightfield microscopy. The profiles generated were: stair bands referring to F. hepatica DNAmt amplification; a band of 1200 bp referring to L. viatrix ITS and another of 1300 bp referring to F. hepatica ITS and other trematodes. Multiplex-PCR has shown to be a fast, safe, highly sensitive and specific method, which is able to amplify DNA from fixed tissues, despite a low DNA quantity and its degradation caused by fixation processes. Such methodology may be useful in studies on fascioliasis epidemiology, enabling the use of material from histological collections.

  12. A Reliable Method for the Selection of Exploitable Melanoma Archival Paraffin Embedded Tissues for Transcript Biomarker Profiling

    PubMed Central

    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

  13. Proteomic analysis of laser-captured paraffin-embedded tissues: a molecular portrait of head and neck cancer progression.

    PubMed

    Patel, Vyomesh; Hood, Brian L; Molinolo, Alfredo A; Lee, Norman H; Conrads, Thomas P; Braisted, John C; Krizman, David B; Veenstra, Timothy D; Gutkind, J Silvio

    2008-02-15

    Squamous cell carcinoma of the head and neck (HNSCC), the sixth most prevalent cancer among men worldwide, is associated with poor prognosis, which has improved only marginally over the past three decades. A proteomic analysis of HNSCC lesions may help identify novel molecular targets for the early detection, prevention, and treatment of HNSCC. Laser capture microdissection was combined with recently developed techniques for protein extraction from formalin-fixed paraffin-embedded (FFPE) tissues and a novel proteomics platform. Approximately 20,000 cells procured from FFPE tissue sections of normal oral epithelium and well, moderately, and poorly differentiated HNSCC were processed for mass spectrometry and bioinformatic analysis. A large number of proteins expressed in normal oral epithelium and HNSCC, including cytokeratins, intermediate filaments, differentiation markers, and proteins involved in stem cell maintenance, signal transduction, migration, cell cycle regulation, growth and angiogenesis, matrix degradation, and proteins with tumor suppressive and oncogenic potential, were readily detected. Of interest, the relative expression of many of these molecules followed a distinct pattern in normal squamous epithelia and well, moderately, and poorly differentiated HNSCC tumor tissues. Representative proteins were further validated using immunohistochemical studies in HNSCC tissue sections and tissue microarrays. The ability to combine laser capture microdissection and in-depth proteomic analysis of FFPE tissues provided a wealth of information regarding the nature of the proteins expressed in normal squamous epithelium and during HNSCC progression, which may allow the development of novel biomarkers of diagnostic and prognostic value and the identification of novel targets for therapeutic intervention in HNSCC.

  14. Western blot analysis of a limited number of cells: a valuable adjunct to proteome analysis of paraffin wax-embedded, alcohol-fixed tissue after laser capture microdissection.

    PubMed

    Martinet, Wim; Abbeloos, Vanessa; Van Acker, Nathalie; De Meyer, Guido R Y; Herman, Arnold G; Kockx, Mark M

    2004-03-01

    In recent years, laser capture microdissection (LCM) has been used successfully to obtain distinct populations of cells for subsequent molecular analysis. Because of the limited sample availability and the absence of in vitro amplification steps for proteins, the use of LCM for proteome analysis largely depends on highly sensitive protein detection methods. In this study, a western blot protocol was developed and validated for the detection of beta-actin and the moderately expressed cell death protein caspase-3 in small numbers of cells. Initially, cultured human U937 monocytes and whole sections of paraffin wax-embedded, alcohol-fixed human tonsils were used to optimize protein electrophoresis and western blotting conditions. High-performance NuPAGE Bis-Tris gels in combination with high-quality transfer membranes, optimized antibody concentrations, and a sensitive chemiluminescent substrate provided a strong signal for beta-actin with approximately 500 U937 cells. In the same way, procaspase-3 could be identified with approximately 1000 cells. Similar results were obtained with germinal centre cells that were procured from paraffin wax-embedded, alcohol-fixed human tonsils by LCM. Treatment of U937 cells with etoposide rapidly induced cell death and allowed the detection of active caspase-3 with approximately 2500 cells (0.8 pg of protein). The findings of this study suggest that western blotting is a valuable adjunct to proteome analysis of LCM procured cells. Copyright 2004 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

  15. Development and validation of an immunohistochemical method for rapid diagnosis of swine erysipelas in formalin-fixed, paraffin-embedded tissue samples.

    PubMed

    Opriessnig, Tanja; Bender, Joseph S; Halbur, Patrick G

    2010-01-01

    The objective of the study was to develop an immunohistochemical (IHC) assay for rapid detection of Erysipelothrix rhusiopathiae. Serotypes 1a, 1b, and 2 are most frequently associated with clinical disease in pigs. Antiserum against serotypes 1a, 1b, and 2 was produced in rabbits, pooled, and applied to formalin-fixed, paraffin-embedded tissue sections of pigs (lungs, heart, spleen, and skin). The results obtained with the IHC assay were compared with direct culture on tissue samples from experimentally inoculated pigs either treated (n = 6) with antibiotics or untreated (n = 8) as well as on samples from field cases (n = 170) submitted to the Veterinary Diagnostic Laboratory at Iowa State University. The agreement between direct culture and IHC staining was found to be substantial. The results of the present study indicate that the IHC assay is highly sensitive and specific in detecting E. rhusiopathiae antigen in formalin-fixed, paraffin-embedded tissues. Results indicated that the IHC is particularly useful in cases in which pigs had been treated with antibiotics prior to submission and in which direct cultures of organs were negative. In addition, the IHC was found to be useful for detection of E. rhusiopathiae antigen in skin lesions, which are often culture negative.

  16. 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.

  17. [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

  18. 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

  19. A novel approach for HLA-A typing in formalin-fixed paraffin-embedded-derived DNA.

    PubMed

    Villabona, Lisa; Leon Rodriguez, Daniel A; Andersson, Emilia K; Seliger, Barbara; Dalianis, Tina; Masucci, Giuseppe V

    2014-09-01

    The aim of this study was to establish a novel approach for human leukocyte antigen (HLA)-typing from formalin-fixed paraffin-embedded-derived DNA. HLAs can be a prognostic factor in cancer and have an extensive polymorphism. This polymorphism is predominantly restricted to exons, which encode the peptide-binding domain of the protein. Formalin-fixed paraffin-embedded material is routinely collected in the clinic and therefore a great source of DNA for genetic analyses. However, its low quality due to fragmentation and nucleotide changes has often created obstacles in designing genetic assays. In this study, we amplified the most polymorphic exons of the HLA-A gene, exons 2, 3, and 4, in 16 formalin-fixed paraffin-embedded samples >10 years old. These tissue samples belonged to patients already HLA-typed by peripheral blood samples at the routine laboratory. Acquired amplification products were used for sequencing, which provided enough information to establish an HLA allele. The same method was applied to DNA extracted from peripheral blood from a healthy volunteer with known HLA type. Of the samples, 14/16 (88%) were successfully typed, in one sample only one of the alleles could be determined, and in one sample no allele could be determined. The amplification of the most polymorphic exons of HLA-A was a successful alternative when DNA quality prevented positive results with previously described methods. The method is usable when an HLA type is needed but the patients are deceased and/or no whole blood samples can be collected. It has thus potential to be used in several fields such as the clinic, research, and forensic science.

  20. Human formalin-fixed paraffin-embedded tissues: an untapped specimen for biomonitoring of carcinogen DNA adducts by mass spectrometry.

    PubMed

    Yun, Byeong Hwa; Rosenquist, Thomas A; Nikolić, Jovan; Dragičević, Dejan; Tomić, Karla; Jelaković, Bojan; Dickman, Kathleen G; Grollman, Arthur P; Turesky, Robert J

    2013-05-07

    DNA adducts represent internal dosimeters to measure exposure to environmental and endogenous genotoxicants. Unfortunately, in molecular epidemiologic studies, measurements of DNA adducts often are precluded by the unavailability of fresh tissue. In contrast, formalin-fixed paraffin embedded (FFPE) tissues frequently are accessible for biomarker discovery. We report here that DNA adducts of aristolochic acids (AAs) can be measured in FFPE tissues at a level of sensitivity comparable to freshly frozen tissue. AAs are nephrotoxic and carcinogenic compounds found in Aristolochia herbaceous plants, many of which have been used worldwide for medicinal purposes. AAs are implicated in the etiology of aristolochic acid nephropathy and upper urinary tract carcinoma. 8-Methoxy-6-nitrophenanthro-[3,4-d]-1,3-dioxole-5-carboxylic acid (AA-I) is a component of Aristolochia herbs and a potent human urothelial carcinogen. AA-I reacts with DNA to form the aristolactam (AL-I)-DNA adduct 7-(deoxyadenosin-N(6)-yl) aristolactam I (dA-AL-I). We established a method to quantitatively retrieve dA-AL-I from FFPE tissue. Adducts were measured, using ultraperformance liquid chromatography/mass spectrometry, in liver and kidney tissues of mice exposed to AA-I, at doses ranging from 0.001 to 1 mg/kg body weight. dA-AL-I was then measured in 10-μm thick tissue-sections of FFPE kidney from patients with upper urinary tract cancers; the values were comparable to those observed in fresh frozen samples. The limit of quantification of dA-AL-I was 3 adducts per 10(9) DNA bases per 2.5 μg of DNA. The ability to retrospectively analyze FFPE tissues for DNA adducts may provide clues to the origin of human cancers for which an environmental cause is suspected.

  1. High resolution melting analysis for rapid and sensitive EGFR and KRAS mutation detection in formalin fixed paraffin embedded biopsies

    PubMed Central

    Do, Hongdo; Krypuy, Michael; Mitchell, Paul L; Fox, Stephen B; Dobrovic, Alexander

    2008-01-01

    Background Epithelial growth factor receptor (EGFR) and KRAS mutation status have been reported as predictive markers of tumour response to EGFR inhibitors. High resolution melting (HRM) analysis is an attractive screening method for the detection of both known and unknown mutations as it is rapid to set up and inexpensive to operate. However, up to now it has not been fully validated for clinical samples when formalin-fixed paraffin-embedded (FFPE) sections are the only material available for analysis as is often the case. Methods We developed HRM assays, optimised for the analysis of FFPE tissues, to detect somatic mutations in EGFR exons 18 to 21. We performed HRM analysis for EGFR and KRAS on DNA isolated from a panel of 200 non-small cell lung cancer (NSCLC) samples derived from FFPE tissues. Results All 73 samples that harboured EGFR mutations previously identified by sequencing were correctly identified by HRM, giving 100% sensitivity with 90% specificity. Twenty five samples were positive by HRM for KRAS exon 2 mutations. Sequencing of these 25 samples confirmed the presence of codon 12 or 13 mutations. EGFR and KRAS mutations were mutually exclusive. Conclusion This is the first extensive validation of HRM on FFPE samples using the detection of EGFR exons 18 to 21 mutations and KRAS exon 2 mutations. Our results demonstrate the utility of HRM analysis for the detection of somatic EGFR and KRAS mutations in clinical samples and for screening of samples prior to sequencing. We estimate that by using HRM as a screening method, the number of sequencing reactions needed for EGFR and KRAS mutation detection can be reduced by up to 80% and thus result in substantial time and cost savings. PMID:18495026

  2. 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...

  3. 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...

  4. Generating Exome Enriched Sequencing Libraries from Formalin-Fixed, Paraffin-Embedded Tissue DNA for Next-Generation Sequencing.

    PubMed

    Marosy, Beth A; Craig, Brian D; Hetrick, Kurt N; Witmer, P Dane; Ling, Hua; Griffith, Sean M; Myers, Benjamin; Ostrander, Elaine A; Stanford, Janet L; Brody, Lawrence C; Doheny, Kimberly F

    2017-01-11

    This unit describes a technique for generating exome-enriched sequencing libraries using DNA extracted from formalin-fixed paraffin-embedded (FFPE) samples. Utilizing commercially available kits, we present a low-input FFPE workflow starting with 50 ng of DNA. This procedure includes a repair step to address damage caused by FFPE preservation that improves sequence quality. Subsequently, libraries undergo an in-solution-targeted selection for exons, followed by sequencing using the Illumina next-generation short-read sequencing platform. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

  5. Cytokeratin positivity in paraffin-embedded malignant melanomas: comparative study of KL1, A4 and Lu5 antibodies.

    PubMed

    Korabiowska, Monika; Fischer, Gösta; Steinacker, Anja; Stachura, Jerzy; Cordon-Cardo, Carlos; Brinck, Ulrich

    2004-01-01

    The unclear role of cytokeratin (CK) in the progression and diagnostics of malignant melanomas stimulated us to compare the reactivity of three antibodies directed to CK in 109 paraffin-embedded melanomas. By far the majority of melanomas did not express cytokeratin even at the<1% level, only vimentin. In about 6% of melanomas it was possible to find CK expression ranging between 3 and 40% of melanoma cells. There was a correlation between CK expression and pT-stage. Cytokeratin-expressing tumours were found in the more advanced pT-stages. The independent prognostic values of none of the three CK antibodies investigated could be shown.

  6. Raman spectroscopic evaluation of efficacy of current paraffin wax section dewaxing agents.

    PubMed

    Faoláin, Eoghan O; Hunter, Mary B; Byrne, Joe M; Kelehan, Peter; Lambkin, Helen A; Byrne, Hugh J; Lyng, Fiona M

    2005-01-01

    During a spectroscopic study to identify biochemical changes in cervical tissue with the onset of carcinogenesis, residual paraffin wax contributions were observed on almost all dewaxed formalin-fixed paraffin-processed (FFPP) tissue sections examined. Subsequently, the present study was formulated to evaluate the efficacy of current dewaxing agents using Raman spectroscopy. Three cervical FFPP sections were subjected to each of the protocols. Sections were dewaxed using four common dewaxing protocols, namely, xylene, Histoclear, heat-mediated antigen retrieval (HMAR) using xylene and citrate buffer, and Trilogy (combined deparaffinization and unmasking of antigens). The potential for hexane as a dewaxing agent was also evaluated. Sections were dewaxed in multiple dewaxing cycles using xylene, Histoclear, and hexane. Residual paraffin wax contributions remained at 1062 cm(-1), 1296 cm(-1), and 1441 cm(-1). HMAR using xylene and citrate buffer, and HMAR using Trilogy, showed a similar efficacy, resulting in incomplete removal of wax. Hexane was shown to be the most effective dewaxing agent, resulting in almost complete removal of wax. Immunohistochemistry was carried out on dewaxed slides, and those dewaxed with hexane displayed a stronger positivity (approximately 28%). Implications for histopathology and immunohistochemistry are considered, as well as problems that residual wax poses for spectroscopic evaluation of dewaxed FFPP sections with a view to disease diagnosis.

  7. 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.

  8. Detection of Clostridium chauvoei in formalin-fixed, paraffin-embedded tissues of sheep by the peroxidase-antiperoxidase (PAP) technique.

    PubMed

    Giraudo Conesa, L C; Vannelli, S A; Uzal, F A

    1995-01-01

    A peroxidase-antiperoxidase (PAP) technique was used to detect Clostridium chauvoei in tissue sections from sheep inoculated intramuscularly with a pure culture of this microorganism. Samples of various tissues were taken for bacteriology, histopathology and immunohistochemistry. A primary antiserum against C. chauvoei for use in the PAP technique was produced in rabbits. Formalin-fixed, paraffin-embedded sections of muscle samples were positively and specifically stained by the PAP technique. The results were consistent with those obtained by bacteriology, but the PAP test was simpler, quicker and less expensive than the bacteriological procedures. The use of the PAP technique would be appropriate for detecting clostridial infections without the constraints of conventional identification methods, especially where laboratory conditions for anaerobic procedures are not readily available.

  9. Effective Melanin Depigmentation of Human and Murine Ocular Tissues: An Improved Method for Paraffin and Frozen Sections

    PubMed Central

    Manicam, Caroline; Pitz, Susanne; Brochhausen, Christoph; Grus, Franz H.; Pfeiffer, Norbert; Gericke, Adrian

    2014-01-01

    Purpose The removal of excessive melanin pigments that obscure ocular tissue morphology is important to address scientific questions and for differential diagnosis of ocular tumours based on histology. Thus, the goal of the present study was to establish an effective and fast melanin bleaching method for paraffin and frozen mouse and human ocular tissues. Methods Paraffin-embedded and frozen ocular specimens from mice and human donors were subjected to bleaching employing two methods. The first employed potassium permanganate (KMnO4) with oxalic acid, and the second 10% hydrogen peroxide (H2O2). To determine optimal bleaching conditions, depigmentation was carried out at various incubation times. The effect of diluents used for 10% H2O2 was assessed using phosphate-buffered saline (PBS), and deionized water. Three different slide types and two fixatives, which were ice-cold acetone with 80% methanol, and 4% paraformaldehyde (PFA) were used to determine the optimal conditions for better tissue adherence during bleaching. All tissues were stained in hematoxylin and eosin for histological evaluation. Results Optimal bleaching was achieved using warm 10% H2O2 diluted in PBS at 65°C for 120 minutes. Chromium-gelatin-coated slides prevented tissue detachment. Adherence of cryosections was also improved with post-fixation using 4% PFA and overnight air-drying at RT after cryosectioning. Tissue morphology was preserved under these conditions. Conversely, tissues bleached in KMnO4/oxalic acid demonstrated poor depigmentation with extensive tissue damage. Conclusions Warm dilute H2O2 at 65°C for 120 minutes rapidly and effectively bleached both cryo- and paraffin sections of murine and human ocular tissues. PMID:25025426

  10. Effective melanin depigmentation of human and murine ocular tissues: an improved method for paraffin and frozen sections.

    PubMed

    Manicam, Caroline; Pitz, Susanne; Brochhausen, Christoph; Grus, Franz H; Pfeiffer, Norbert; Gericke, Adrian

    2014-01-01

    The removal of excessive melanin pigments that obscure ocular tissue morphology is important to address scientific questions and for differential diagnosis of ocular tumours based on histology. Thus, the goal of the present study was to establish an effective and fast melanin bleaching method for paraffin and frozen mouse and human ocular tissues. Paraffin-embedded and frozen ocular specimens from mice and human donors were subjected to bleaching employing two methods. The first employed potassium permanganate (KMnO4) with oxalic acid, and the second 10% hydrogen peroxide (H2O2). To determine optimal bleaching conditions, depigmentation was carried out at various incubation times. The effect of diluents used for 10% H2O2 was assessed using phosphate-buffered saline (PBS), and deionized water. Three different slide types and two fixatives, which were ice-cold acetone with 80% methanol, and 4% paraformaldehyde (PFA) were used to determine the optimal conditions for better tissue adherence during bleaching. All tissues were stained in hematoxylin and eosin for histological evaluation. Optimal bleaching was achieved using warm 10% H2O2 diluted in PBS at 65°C for 120 minutes. Chromium-gelatin-coated slides prevented tissue detachment. Adherence of cryosections was also improved with post-fixation using 4% PFA and overnight air-drying at RT after cryosectioning. Tissue morphology was preserved under these conditions. Conversely, tissues bleached in KMnO4/oxalic acid demonstrated poor depigmentation with extensive tissue damage. Warm dilute H2O2 at 65°C for 120 minutes rapidly and effectively bleached both cryo- and paraffin sections of murine and human ocular tissues.

  11. 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.

  12. Optimization of Single- and Dual-Color Immunofluorescence Protocols for Formalin-Fixed, Paraffin-Embedded Archival Tissues

    PubMed Central

    Kajimura, Junko; Ito, Reiko; Manley, Nancy R.; Hale, Laura P.

    2015-01-01

    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

  13. Quality Control of RNA Preservation and Extraction from Paraffin-Embedded Tissue: Implications for RT-PCR and Microarray Analysis

    PubMed Central

    Pichler, Martin; Zatloukal, Kurt

    2013-01-01

    Analysis of RNA isolated from fixed and paraffin-embedded tissues is widely used in biomedical research and molecular pathological diagnostics. We have performed a comprehensive and systematic investigation of the impact of factors in the pre-analytical workflow, such as different fixatives, fixation time, RNA extraction method and storage of tissues in paraffin blocks, on several downstream reactions including complementary DNA (cDNA) synthesis, quantitative reverse transcription polymerase chain reaction (qRT-PCR) and microarray hybridization. We compared the effects of routine formalin fixation with the non-crosslinking, alcohol-based Tissue Tek Xpress Molecular Fixative (TTXMF, Sakura Finetek), and cryopreservation as gold standard for molecular analyses. Formalin fixation introduced major changes into microarray gene expression data and led to marked gene-to-gene variations in delta-ct values of qRT-PCR. We found that qRT-PCR efficiency and gene-to-gene variations were mainly attributed to differences in the efficiency of cDNA synthesis as the most sensitive step. These differences could not be reliably detected by quality assessment of total RNA isolated from formalin-fixed tissues by electrophoresis or spectrophotometry. Although RNA from TTXMF fixed samples was as fragmented as RNA from formalin fixed samples, much higher cDNA yield and lower ct-values were obtained in qRT-PCR underlining the negative impact of crosslinking by formalin. In order to better estimate the impact of pre-analytical procedures such as fixation on the reliability of downstream analysis, we applied a qRT-PCR-based assay using amplicons of different length and an assay measuring the efficiency of cDNA generation. Together these two assays allowed better quality assessment of RNA extracted from fixed and paraffin-embedded tissues and should be used to supplement quality scores derived from automated electrophoresis. A better standardization of the pre-analytical workflow, application

  14. Quality control of RNA preservation and extraction from paraffin-embedded tissue: implications for RT-PCR and microarray analysis.

    PubMed

    Kashofer, Karl; Viertler, Christian; Pichler, Martin; Zatloukal, Kurt

    2013-01-01

    Analysis of RNA isolated from fixed and paraffin-embedded tissues is widely used in biomedical research and molecular pathological diagnostics. We have performed a comprehensive and systematic investigation of the impact of factors in the pre-analytical workflow, such as different fixatives, fixation time, RNA extraction method and storage of tissues in paraffin blocks, on several downstream reactions including complementary DNA (cDNA) synthesis, quantitative reverse transcription polymerase chain reaction (qRT-PCR) and microarray hybridization. We compared the effects of routine formalin fixation with the non-crosslinking, alcohol-based Tissue Tek Xpress Molecular Fixative (TTXMF, Sakura Finetek), and cryopreservation as gold standard for molecular analyses. Formalin fixation introduced major changes into microarray gene expression data and led to marked gene-to-gene variations in delta-ct values of qRT-PCR. We found that qRT-PCR efficiency and gene-to-gene variations were mainly attributed to differences in the efficiency of cDNA synthesis as the most sensitive step. These differences could not be reliably detected by quality assessment of total RNA isolated from formalin-fixed tissues by electrophoresis or spectrophotometry. Although RNA from TTXMF fixed samples was as fragmented as RNA from formalin fixed samples, much higher cDNA yield and lower ct-values were obtained in qRT-PCR underlining the negative impact of crosslinking by formalin. In order to better estimate the impact of pre-analytical procedures such as fixation on the reliability of downstream analysis, we applied a qRT-PCR-based assay using amplicons of different length and an assay measuring the efficiency of cDNA generation. Together these two assays allowed better quality assessment of RNA extracted from fixed and paraffin-embedded tissues and should be used to supplement quality scores derived from automated electrophoresis. A better standardization of the pre-analytical workflow, application

  15. [Detection of Mycobacterium tuberculosis complex in paraffin-embedded tissues by real-time fluorescent quantitative polymerase chain reaction].

    PubMed

    Ye, Feng; Chen, Yu; He, Du; Jian, Shu-yu; Zheng, Ke; Li, Gan-di; Bu, Hong

    2013-08-01

    To investigate the feasibility of real-time fluorescent quantitative (qPCR) assay in detecting mycobacterium tuberculosis complex (MTB) in paraffin embedded tissues for diagnostic purpose. Using qPCR assay, 1000 consecutive formalin-fixed and paraffin embedded (FFPE) tissues (from 2011 to 2012) suspected of MTB infection were tested by amplifying the MTB specific insertion sequence 6110 (IS6110). The specificity of the PCR product was confirmed by Sanger sequencing as compared with the MTB genomic DNA of the IS6110 sequence. Tissues with Ziehl-Neelsen acid-fast staining were used as control. In the 1000 samples, 513 were positive for mycobacterium by Ziehl-Neelsen acid-fast staining (detection rate 51.3%); whereas 546 were MTB positive by qPCR assay (detection rate 54.6%). Concordance rate for both assays was 73.1%. The diagnosis rate increased by 14.4% by combinination of Ziehl-Neelsen acid-fast staining and qPCR results. More interestingly, by analyzing the Ziehl-Neelsen acid-fast staining and qPCR results three cases of M.leprae infection and four cases of non-tuberculous Mycobacterium (NTM) infection were identified. qPCR detection of MTB in FFPE tissue is more sensitive than Ziehl-Neelsen acid-fast staining assay. Combination of these two assays can increase the detection rate and also identify some rare cases of NTM infection.

  16. Clinical Usefulness of PCR for Differential Diagnosis of Tuberculosis and Nontuberculous Mycobacterial Infection in Paraffin-Embedded Lung Tissues.

    PubMed

    Kim, Yo Na; Kim, Kyoung Min; Choi, Ha Na; Lee, Ju Hyung; Park, Ho Sung; Jang, Kyu Yun; Moon, Woo Sung; Kang, Myoung Jae; Lee, Dong Geun; Chung, Myoung Ja

    2015-09-01

    The need for isolation of nontuberculous mycobacteria (NTM) from clinical specimens has increased in recent years. Our aim was to determine the clinical usefulness of PCR for differential diagnosis of tuberculosis and nontuberculous mycobacterial infection in lung tissue that show chronic granulomatous inflammation. A total of 199 formalin-fixed, paraffin-embedded specimens, including 137 Mycobacterium tuberculosis (MTB), 17 NTM cases, and 45 other than mycobacterial cases were collected. We performed acid-fast staining, MTB and NTM nested PCRs, and MTB and NTM real-time PCRs. No histologic difference between MTB and NTM infections was observed. Sensitivity and specificity for detecting MTB were 70.1% and 95.1% by nested PCR, respectively, and 70.8% and 100.0% by real-time PCR, respectively. Sensitivity and specificity for detecting NTM were 52.9% and 96.15% by nested PCR, respectively, and 35.3% and 100.0% by real-time PCR, respectively. Mycobacteria were identified by acid-fast staining in 50 of 154 cases (32.5%). All 50 acid-fast staining-positive cases showed positive nested and real-time PCR results (n = 47 MTB PCR positive; n = 3 NTM PCR positive), and results agreed with final diagnosis. PCR will be useful for the rapid diagnosis of mycobacterial infection and differentiation of MTB from NTM in formalin-fixed, paraffin-embedded specimens, especially in acid-fast staining-positive specimens.

  17. Incestuous paternity detected by STR-typing of chorionic villi isolated from archival formalin-fixed paraffin-embedded abortion material using laser microdissection.

    PubMed

    Robino, Carlo; Barilaro, Maria Rosa; Gino, Sarah; Chiarle, Roberto; Palestro, Giorgio; Torre, Carlo

    2006-01-01

    Microscopic examination of a blood clot expelled by a physically and mentally disabled woman taken to the emergency room because of genital bleeding revealed the presence of chorionic villi encircled by decidua, hemorrhage, and necrosis. In order to identify the father of the product of conception, sections of formalin-fixed, paraffin-embedded abortion material were subjected to laser microdissection: DNA extraction from chorionic villi selectively isolated from the surrounding tissues allowed successful STR-typing of fetal cells, which was otherwise prevented by excess maternal DNA. The large number of homozygous genotypes in the fetal profile suggested incestuous paternity. Analysis of reference DNA samples from male relatives excluded the woman's father, paternal grandfather, and maternal grandfather, whereas the obligate paternal alleles of the fetus were constantly present in the genotypes of the woman's brother, clearly demonstrating brother-sister incest (probability of paternity > 99.99999%).

  18. 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.

  19. HOPE--a new fixing technique enables preservation and extraction of high molecular weight DNA and RNA of > 20 kb from paraffin-embedded tissues. Hepes-Glutamic acid buffer mediated Organic solvent Protection Effect.

    PubMed

    Wiedorn, Klaus Hermann; Olert, Jürgen; Stacy, Robin A P; Goldmann, Torsten; Kühl, Heike; Matthus, Jutta; Vollmer, Ekkehard; Bosse, Alexander

    2002-01-01

    The growing number of molecular pathologic tools that are currently available require material with good long term preservation of morphology, nucleic acids, and antigenic structures. However, pathologic investigations of tissues done at a molecular level are often hampered by the fixatives in use. We thus endeavored to design a new fixing system, including subsequent paraffin-embedding and sectioning, that makes complete pathologic analyses possible, with special consideration of immunohistochemistry (IHC), in situ hybridization (ISH), and molecular pathology. The optimized HOPE (Hepes-Glutamic acid buffer mediated Organic solvent Protection Effect) fixing technique allows us to preserve and extract high molecular weight DNA and RNA of > 20 kbp suitable for downstream applications, such as PCR and RT-PCR from HOPE-fixed, paraffin-embedded tissues that are up to 5 years old. This technique will most probably lead to new impacts on molecular pathology.

  20. De-staining and re-staining mucins in formalin fixed paraffin sections.

    PubMed

    Smith, A A; Glickfield, I

    2011-04-01

    Re-staining of formalin fixed paraffin sections sometimes is required and this requires prior de-staining. Some simple and effective protocols for de-staining are described. Mucihematoxylin and mucicarmine can be removed with acid alcohol. Zirconyl hematoxylin can be removed with periodic acid or Sinha's fixative. Alcian blue can be removed with 5% trifluoroacetic acid in dichloromethane. Colloidal iron can be bleached in 1% household bleach in alcohol. PAS can be removed with hydrogen peroxide or ammonium hydroxide. With few exceptions, de-stained sections can be re-stained with mucihematoxylin, PAS or Gabe's trichrome.

  1. 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.

  2. Comparison of methods using paraffin-embedded tissues and exfoliated cervical cells to evaluate human papillomavirus genotype attribution.

    PubMed

    Torii, Yutaka; Fujii, Takuma; Kukimoto, Iwao; Saito, Miyuki; Iwata, Takashi; Takahashi, Hiroshi; Ichikawa, Ryoko; Kawai, Satoshi; Otani, Sayaka; Aoki, Daisuke

    2016-10-01

    Monitoring the attribution of human papillomavirus (HPV) genotypes to cervical precancerous lesions is essential in assessing the efficacy of HPV vaccines. To resolve the lack of studies comparing the HPV genotyping procedures used to estimate HPV genotype attribution, we undertook a retrospective cross-sectional study to determine the appropriate genotyping procedures for evaluating the potential efficacy of HPV vaccines. Three procedures, including two different genotyping methods, Clinichip HPV test (C-Chip) and modified GP5+/6+ PCR coupled to fluorescent bead sorter detection (MGP), using exfoliated cervical cells (C-Chip and C-MGP, respectively) or formalin-fixed paraffin-embedded tissues (F-MGP), were compared. The overall agreement in detecting high-risk HPV was 88.5-92.1% among the three procedures, and genotype-specific agreement was 83.9-100% for all pairwise comparisons. In cervical intraepithelial neoplasia grade 2/3 specimens, HPV16/18 attribution estimated with the hierarchical attribution method was consistent among the procedures: 52.3% (45/86) for C-Chip, 54.7% (47/86) for C-MGP, and 52.3% (45/86) for F-MGP (P = 0.81). HPV16/18/31/33/45/52/58 hierarchical attribution was 88.4% (76/86) with C-Chip, 86.0% (74/86) with C-MGP, and 83.7% (72/86) with F-MGP (P = 0.49). In cervical intraepithelial neoplasia grade 3 specimens, the corresponding hierarchical attribution was 96.4% (53/55) with C-Chip, 89.1% (49/55) with C-MGP, and 94.5% (52/55) with F-MGP (P = 0.27). Although F-MGP is theoretically a reliable method for determining HPV genotype attribution, it is acceptable to use C-Chip or C-MGP, coupled to the hierarchical attribution formula to correct the bias of multiple infections. These approaches using exfoliated cervical cells are practical for monitoring the efficacy of HPV vaccines. © 2016 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

  3. Immunohistological diagnosis of "plasmacytoid T cell lymphoma" in paraffin wax sections.

    PubMed Central

    Thomas, J O; Beiske, K; Hann, I; Koo, C; Mason, D Y

    1991-01-01

    An immunohistological study of paraffin wax embedded tissue from three cases of plasmacytoid monocyte neoplasms, using a panel of antibodies which react with fixation resistant leucocyte markers, is reported. This neoplasm was found to have a distinctive antigenic profile, being negative for CD3 and elastase, but positive for CD43 and CD68. This immunological phenotype, coupled with its characteristic morphological features, should facilitate the recognition of this rare neoplasm in routinely processed tissue. Furthermore, the term "plasmacytoid monocyte sarcoma" is proposed to designate it because it is inappropriate to refer to it as a lymphoma. As all cases have been associated with a myeloproliferative disorder (usually an acute or chronic myeloid leukaemia), these tumours probably represent the accumulation in lymphoid tissue of neoplastic cells which have differentiated along the plasmacytoid monocyte pathway. Images PMID:1890195

  4. Immunofluorescence staining of paraffin sections: creating DAB staining like virtual digital images using CMYK color conversion.

    PubMed

    Buchynska, L; Kashuba, E; Szekely, L

    2008-12-01

    Crystal violet treatment of formalin fixed paraffin embedded tissue slides greatly reduces the endogenous autofluorescence, and allows immunofluorescence (IF) staining with FITC or Alexa488 conjugated antibodies. Using cold CCD camera to capture the fluorescence images makes this staining method very sensitive. Here we show that combination of IF with the simultaneous recording of crystal violet induced red and Hoechst 33258 induced blue fluorescence permits the localization of the IF signal over a cytoplasmic: nuclear red:blue stain that visualizes the microscopic anatomy of the underlying tissue. To make the visual interpretation of the IF staining easier for microscopists, who are used to DAB staining over weak hematoxilin-eosin background, we created a simple color conversion procedure that turns the captured three-color fluorescence RGB (red, green, blue) images over a black background into four color CMYK (cyan, magenta, yellow, key color (black)) images.

  5. 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.

  6. Effects of tissue handling and processing steps on PCR for detection of Mycobacterium tuberculosis in formalin-fixed paraffin-embedded samples.

    PubMed

    Barcelos, Denise; Franco, Marcello F; Leão, Sylvia Cardoso

    2008-01-01

    Development and standardization of reliable methods for detection of Mycobacterium tuberculosis in clinical samples is an important goal in laboratories throughout the world. In this work, lung and spleen fragments from a patient who died with the diagnosis of miliary tuberculosis were used to evaluate the influence of the type of fixative as well as the fixation and paraffin inclusion protocols on PCR performance in paraffin embedded specimens. Tissue fragments were fixed for four h to 48 h, using either 10% non-buffered or 10% buffered formalin, and embedded in pure paraffin or paraffin mixed with bee wax. Specimens were submitted to PCR for amplification of the human beta-actin gene and separately for amplification of the insertion sequence IS6110, specific from the M. tuberculosis complex. Amplification of the beta-actin gene was positive in all samples. No amplicons were generated by PCR-IS6110 when lung tissue fragments were fixed using 10% non-buffered formalin and were embedded in paraffin containing bee wax. In conclusion, combined inhibitory factors interfere in the detection of M. tuberculosis in stored material. It is important to control these inhibitory factors in order to implement molecular diagnosis in pathology laboratories.

  7. 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.

  8. [Amyloid typing from formalin-fixed paraffin-embedded tissues using LMD-LC-MS/MS system].

    PubMed

    Tasaki, Masayoshi; Obayashi, Konen; Ueda, Mitsuharu; Ando, Yukio

    2014-03-01

    Amyloidosis is one of the protein conformational disorders in which normally soluble proteins accumulate insoluble amyloid fibrils, leading to severe organ dysfunction. To date, 30 different amyloidogenic proteins have been reported. Immunohistochemistry (IHC) is usually used to identify the amyloid precursor protein, but the results may be inconclusive owing to a loss of epitopes or small amounts of amyloid deposits, comprising unknown amyloidogenic protein. Recently, laser microdissection (LMD)-liquid chromatography tandem mass spectrometry (LC-MS/MS) has been used in a novel method to identify amyloid precursor protein from amyloid-laden formalin-fixed paraffin embedded (FFPE) tissues. We describe the usefulness of the system for amyloid typing in this report.

  9. 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

  10. mTRAQ-based quantification of potential endometrial carcinoma biomarkers from archived formalin-fixed paraffin-embedded tissues.

    PubMed

    DeSouza, Leroi V; Krakovska, Olga; Darfler, Marlene M; Krizman, David B; Romaschin, Alexander D; Colgan, Terence J; Siu, K W Michael

    2010-09-01

    Formalin-fixed paraffin-embedded (FFPE) tissues are the primary and preferred medium for archiving patients' samples. Here we demonstrate relative quantifications of protein biomarkers in extracts of laser microdissected epithelial cells from FFPE endometrial carcinoma tissues versus those from normal proliferative endometria by means of targeted proteomic analyses using LC-multiple reaction monitoring (MRM) MS with MRM Tags for Relative and Absolute Quantitation (mTRAQ) labeling. Comparable results of differential expressions for pyruvate kinase isoform M2 (PK-M2) and polymeric Ig receptor were observed between analyses on laser microdissected epithelial cells from FFPE tissues and corresponding homogenates from frozen tissues of the same individuals that had previously been analyzed and reported. We also identified PK-M2 in the normal proliferative phase of the endometrium. Other biomarkers in addition to PK-M2 and polymeric Ig receptor were also observed but not consistently and/or were at levels below the threshold for quantification.

  11. Archived formalin-fixed paraffin-embedded (FFPE) blocks: A valuable underexploited resource for extraction of DNA, RNA, and protein.

    PubMed

    Kokkat, Theresa J; Patel, Miral S; McGarvey, Diane; LiVolsi, Virginia A; Baloch, Zubair W

    2013-04-01

    Formalin-fixed paraffin-embedded (FFPE) material presents a readily available resource in the study of various biomarkers. There has been interest in whether the storage period has significant effect on the extracted macromolecules. Thus, in this study, we investigated if the storage period had an effect on the quantity/quality of the extracted nucleic acids and proteins. We systematically examined the quality/quantity of genomic DNA, total RNA, and total protein in the FFPE blocks of malignant tumors of lung, thyroid, and salivary gland that had been stored over several years. We show that there is no significant difference between macromolecules extracted from blocks stored over 11-12 years, 5-7 years, or 1-2 years in comparison to the current year blocks.

  12. Archived Formalin-Fixed Paraffin-Embedded (FFPE) Blocks: A Valuable Underexploited Resource for Extraction of DNA, RNA, and Protein

    PubMed Central

    Patel, Miral S.; McGarvey, Diane; LiVolsi, Virginia A.; Baloch, Zubair W.

    2013-01-01

    Formalin-fixed paraffin-embedded (FFPE) material presents a readily available resource in the study of various biomarkers. There has been interest in whether the storage period has significant effect on the extracted macromolecules. Thus, in this study, we investigated if the storage period had an effect on the quantity/quality of the extracted nucleic acids and proteins. We systematically examined the quality/quantity of genomic DNA, total RNA, and total protein in the FFPE blocks of malignant tumors of lung, thyroid, and salivary gland that had been stored over several years. We show that there is no significant difference between macromolecules extracted from blocks stored over 11–12 years, 5–7 years, or 1–2 years in comparison to the current year blocks. PMID:24845430

  13. Comparison of eight commercially available kits for DNA extraction from formalin-fixed paraffin-embedded tissues.

    PubMed

    Janecka, Anna; Adamczyk, Agnieszka; Gasińska, Anna

    2015-05-01

    A proper extraction method from formalin-fixed paraffin-embedded (FFPE) blocks is essential to obtain DNA of satisfactory quality/quantity. We compared the effectiveness of eight commercially available kits for DNA extraction based on 10 FFPE tissues. Kits differed significantly in terms of DNA yield, purity, and quality. Using the QIAamp DNA FFPE Tissue Kit (Qiagen) and the ReliaPrep FFPE gDNA Miniprep System (Promega), we obtained DNA of the highest quality and acceptable quantity. We also demonstrated that overnight digestion of samples usually improved DNA yield and/or purity. For precious or limited material, double elution is recommended for obtaining up to 42% higher amount of DNA. Copyright © 2015 Elsevier Inc. All rights reserved.

  14. Demonstration of Epstein-Barr virus in primary brain lymphoma by in situ DNA hybridisation in paraffin wax embedded tissue.

    PubMed Central

    Murphy, J K; Young, L S; Bevan, I S; Lewis, F A; Dockey, D; Ironside, J W; O'Brien, C J; Wells, M

    1990-01-01

    Tumour tissue from 29 patients with primary brain lymphoma was reviewed to determine if there was an aetiological association between Epstein-Barr virus and polyclonal and monoclonal lymphoproliferations. The morphology and immunophenotype in 24 patients for whom paraffin wax embedded tissue was available were studied. A high grade pleomorphic tumour morphology with plasmacytoid features was seen in 13 tumours. Because of the large number of pleomorphic lymphomas, all tumours were examined for the presence of the Epstein-Barr virus genome using in situ DNA hybridisation. A panel of three biotinylated probes to different sequences in the Epstein-Barr virus genome was used. Positive hybridisation with one or more probes was shown in tumours from 11 patients. The remaining tumours gave no hybridisation signal. There was no correlation between positive hybridisation and morphological subtype or clinical outcome. Images PMID:2159030

  15. Detection of loss of heterozygosity in formalin-fixed paraffin-embedded tumor specimens by the polymerase chain reaction.

    PubMed Central

    Bianchi, A. B.; Navone, N. M.; Conti, C. J.

    1991-01-01

    A polymerase chain reaction-based procedure was used for the detection of DNA length polymorphisms generated by naturally occurring genetic deletions or insertions of known sequence. This method consists of a simple one-step assay that does not require any restriction enzyme analysis or Southern blot hybridization, allowing identification in ethidium bromide-stained gels. The procedure described here was used to detect loss of heterozygosity at various loci, including the Hbb beta-globin gene cluster, in chemically induced mouse skin tumors, using a variety of tissue preparations, including microdissection of formalin-fixed, paraffin-embedded specimens, short-term cultures, and fluorescence-activated cell sorting of epithelial populations. This approach may be useful in detecting tumor-specific reduction to homozygosity at polymorphic chromosomal loci, allowing the mapping of putative tumor-suppressor loci involved in carcinogenesis. Images Figure 2 Figure 3 Figure 4 Figure 5 PMID:1992758

  16. 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.

  17. A case report of Anisakis pegreffii (Nematoda, Anisakidae) identified from archival paraffin sections of a Croatian patient.

    PubMed

    Mladineo, Ivona; Popović, Marijana; Drmić-Hofman, Irena; Poljak, Vedran

    2016-02-01

    This is the first report of anisakiasis in a Croatian patient, evidenced from an archival paraffin-embedded and hematoxylin-eosin stained tissue section. Anisakiasis has been only suspected in the country based on previously detected anti-Anisakis IgE seroprevalence in the healthy coastal population, as well as an acute case where pathohistological and serological findings suggested the diseases, but the migrating larva has not been retrieved. Seventy years-old female, operated in 1998 for pulmonary carcinoma, was admitted to the General hospital Šibenik, Croatia in 2003, because of gastric pain and nausea that lasted for couple of days. She was showing good general condition, full mobility and lucidity, subfebrile status. Abdominal palpation inferred acute pain in paraumbilical and ileocecal region. Exploratory right pararectal laparotomy revealed a hardened, 5 cm-long structure, located intraluminally in the sigmoid colon, not perforating colon serosa. The process has been dissected and sent for patohistological diagnosis. Results showed a 2 mm-long whitish nematode spiralised in muscular layer of colon mucosa surrounded by granulomatous inflammation. After genomic DNA isolation of the nematode from the histological section, and amplification at the mitochondrial cytochrome oxidase 2 locus, etiological agent has been identified as Anisakis pegreffii. Used methodology suggests that screening of archival suspicious sections is feasible in order to study epidemiology of this zoonotic disease poorly recognised in Croatia.

  18. Availability of CD10 immunohistochemistry as a marker of breast myoepithelial cells on paraffin sections.

    PubMed

    Moritani, Suzuko; Kushima, Ryoji; Sugihara, Hiroyuki; Bamba, Masamichi; Kobayashi, Tadao K; Hattori, Takanori

    2002-04-01

    CD10, also called common acute lymphoblastic leukemia antigen (CALLA), was recently found to be expressed in nonhematopoietic tissues. Although CD10 was also identified in human breast myoepithelial cells, its availability of immunohistochemistry on paraffin sections has not been examined so far. In the present study, we demonstrated CD10 immunohistochemically on paraffin sections of both normal and pathological breast tissues, comparing its staining patterns to those of smooth muscle actin (SMA), which is now commonly used to highlight myoepithelium. CD10 was consistently positive in normal breast myoepithelial cells. CD10 also clearly highlighted myoepithelial cells in intraductal papilloma, adenosis, ductal hyperplasia, fibroadenoma, and phyllodes tumor as well as SMA did. In atypical ductal hyperplasia and ductal carcinoma in situ, continuous, discontinuous, and totally negative stainings of both CD10 and SMA were noted, depending on foci of neoplastic cell nests. However, both stainings clearly demonstrated myoepithelial cells of cancerized acini, being useful in differentiating lobular cancerization from microinvasion. Because SMA was also positive in normal vessels and spindle-shaped stromal cells, CD10, which was negative in vessels, was useful in differentiating myoepithelial cells from thin vascular wall in intracystic lesions with delicate papillae. Although background staining of spindle-shaped stromal cells was also noted in CD10, the positive cell number was less, and the signal was weaker than that of SMA. The absence of myoepithelial cells in invasive ductal carcinomas was more clearly highlighted by CD10 than SMA. We concluded that CD10 could be another useful marker of breast myoepithelial cells on paraffin sections. Combination of CD10 and SMA will provide more sophisticated information about presence or absence of myoepithelial cells in confusing breast lesions.

  19. Ewing's Sarcoma: An Analysis of miRNA Expression Profiles and Target Genes in Paraffin-Embedded Primary Tumor Tissue.

    PubMed

    Parafioriti, Antonina; Bason, Caterina; Armiraglio, Elisabetta; Calciano, Lucia; Daolio, Primo Andrea; Berardocco, Martina; Di Bernardo, Andrea; Colosimo, Alessia; Luksch, Roberto; Berardi, Anna C

    2016-04-30

    The molecular mechanism responsible for Ewing's Sarcoma (ES) remains largely unknown. MicroRNAs (miRNAs), a class of small non-coding RNAs able to regulate gene expression, are deregulated in tumors and may serve as a tool for diagnosis and prediction. However, the status of miRNAs in ES has not yet been thoroughly investigated. This study compared global miRNAs expression in paraffin-embedded tumor tissue samples from 20 ES patients, affected by primary untreated tumors, with miRNAs expressed in normal human mesenchymal stromal cells (MSCs) by microarray analysis. A miRTarBase database was used to identify the predicted target genes for differentially expressed miRNAs. The miRNAs microarray analysis revealed distinct patterns of miRNAs expression between ES samples and normal MSCs. 58 of the 954 analyzed miRNAs were significantly differentially expressed in ES samples compared to MSCs. Moreover, the qRT-PCR analysis carried out on three selected miRNAs showed that miR-181b, miR-1915 and miR-1275 were significantly aberrantly regulated, confirming the microarray results. Bio-database analysis identified BCL-2 as a bona fide target gene of the miR-21, miR-181a, miR-181b, miR-29a, miR-29b, miR-497, miR-195, miR-let-7a, miR-34a and miR-1915. Using paraffin-embedded tissues from ES patients, this study has identified several potential target miRNAs and one gene that might be considered a novel critical biomarker for ES pathogenesis.

  20. Reliable quantification of mRNA in archived formalin-fixed tissue with or without paraffin embedding.

    PubMed

    Wang, Zhibin; Lebron, Jose A; Wolf, Jayanthi J

    2015-01-01

    Formalin fixation and paraffin embedding (FFPE) is a standard method for tissue sample storage and preservation in pathology archives. The Reverse Transcriptase Quantitative Polymerase Chain Reaction (RT-qPCR) is a useful method for gene expression analysis, but its sensitivity is significantly decreased in FFPE tissue due to the fixation process. This process results in chemical modifications of RNA, cross-links proteins to RNA, and degrades RNA in these archived samples, hindering the reverse transcription step of the conventional RT-pPCR method and preventing generation of a cDNA that is long enough for the subsequent quantitative PCR step. In this study, we used a multi-species RT-qPCR method originally developed to detect mRNA in tissue homogenate samples (Wang et al., 2011) and applied it to effectively detect a specific mRNA in formalin-fixed tissues with or without paraffin-embedding by targeting mRNA sequences as short as 24 nucleotides. Target sizes ranging from 24 to 91 nucleotides were evaluated using this multi-species RT-qPCR assay. Data generated with FFPE tissues demonstrated that use of short target sequences relieved the dependence on RNA quality and could reliably quantify mRNA. This method was highly sensitive, reproducible, and had a dynamic range of five orders of magnitude. Importantly, this method could quantify mRNA in prolonged formalin-fixed and FFPE tissue, where conventional RT-qPCR assays failed. Moreover, a similar result for small interfering RNA (siRNA)-mediated Apob mRNA knockdown was obtained from tissues fixed in formalin solution for 3months to 4years, and was found to be comparable to results obtained with frozen liver tissues. Therefore, the method presented here allows for preclinical and clinical retrospective and prospective studies on mRNA derived from archived FFPE and prolonged formalin-fixed tissue. Copyright © 2014 Elsevier Inc. All rights reserved.

  1. Molecular analysis of different classes of RNA molecules from formalin-fixed paraffin-embedded autoptic tissues: a pilot study.

    PubMed

    Muciaccia, Barbara; Vico, Carmen; Aromatario, Mariarosaria; Fazi, Francesco; Cecchi, Rossana

    2015-01-01

    For a long time, it has been thought that fresh and frozen tissues are the only possible source of biological material useful to extract nucleic acids suitable for downstream molecular analysis. Recently, for forensic purpose such as personal identification, also fixed tissues have been used to recover DNA molecules, whereas RNA extracted from such material is still considered too degraded for gene expression studies. In the present pilot study, we evaluated the possibility to use forensic formalin-fixed paraffin-embedded (FFPE) samples, collected at autopsy at different postmortem intervals (PMI) from four individuals, to perform advanced molecular analyses. In particular, we performed qualitative and quantitative analyses of total RNAs extracted from different FFPE tissues and put expression profiles in relation with the organ type and the duration of PMI. Different classes of RNA molecular targets were studied by real-time quantitative RT-PCR. We report molecular evidence that small RNAs are the only RNA molecules still detectable in all the FFPE autoptic tissues. In particular, microRNAs (miRNAs) represent a consistent, stable, and well-preserved molecular target detectable even from tissue sources displaying signs of ongoing putrefaction at autopsy. In this pilot study, we show that miRNAs could represent a highly sensitive and potentially useful forensic marker. Amplification of specific miRNAs using paraffin-embedded blocks could facilitate retrospective molecular analysis using specific forensic-archived tissues chosen as most suitable according to PMI, and this approach would address molecular evidence in forensic cases in which fresh or frozen material is no longer available.

  2. 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.

  3. Use of formalin-fixed paraffin-embedded samples for gene expression studies in breast cancer patients.

    PubMed

    Musella, Valeria; Callari, Maurizio; Di Buduo, Eleonora; Scuro, Manuela; Dugo, Matteo; Miodini, Patrizia; Bianchini, Giampaolo; Paolini, Biagio; Gianni, Luca; Daidone, Maria Grazia; Cappelletti, Vera

    2015-01-01

    To obtain gene expression profiles from samples collected in clinical trials, we conducted a pilot study to assess feasibility and estimate sample attrition rates when profiling formalin-fixed, paraffin-embedded specimens. Ten matched fresh-frozen and fixed breast cancer samples were profiled using the Illumina HT-12 and Ref-8 chips, respectively. The profiles obtained with Ref 8, were neither technically nor biologically reliable since they failed to yield the expected separation between estrogen receptor positive and negative samples. With the use of Affymetrix HG-U133 2.0 Plus chips on fixed samples and a quantitative polymerase chain reaction -based sample pre-assessment step, results were satisfactory in terms of biological reliability, despite the low number of present calls (M = 21%±5). Compared with the Illumina DASL WG platform, Affymetrix data showed a wider interquartile range (1.32 vs 0.57, P<2.2 E-16,) and larger fold changes. The Affymetrix chips were used to run a pilot study on 60 fixed breast cancers. By including in the workflow the sample pre-assessment steps, 96% of the samples predicted to give good results (44/46), were in fact rated as satisfactory from the point of view of technical and biological meaningfulness. Our gene expression profiles showed strong agreement with immunohistochemistry data, were able to reproduce breast cancer molecular subtypes, and allowed the validation of an estrogen receptor status classifier derived in frozen samples. The approach is therefore suitable to profile formalin-fixed paraffin-embedded samples collected in clinical trials, provided that quality controls are run both before (sample pre-assessment) and after hybridization on the array.

  4. Laser-Assisted Microdissection of Membrane-Mounted Paraffin Sections for Polymerase Chain Reaction Analysis

    PubMed Central

    Gjerdrum, Lise Mette; Lielpetere, Ilze; Rasmussen, Lars Melholt; Bendix, Knud; Hamilton-Dutoit, Stephen

    2001-01-01

    Laser microbeam microdissection (LMM) is an increasingly important method for obtaining pure cell samples for genetic and proteomic analysis. Immunohistochemistry (IHC) and in situ hybridization (ISH) are useful techniques for targeting specific cell populations for microdissection but are difficult to apply with the tissue support membranes often used during LMM. Using detection of cytokeratins and Epstein-Barr virus gene products in head and neck carcinoma as a model, we describe optimized protocols for membrane and section preparation and for low temperature antigen retrieval that allow IHC and ISH to be used reliably on membrane mounted paraffin tissue sections. Visualization of cellular targets was markedly improved by staining and this could be further improved using a variety of optical media before microdissection. Tissue fragments thus stained were suitable for subsequent polymerase chain reaction analysis of extracted DNA using standard techniques. These IHC and ISH procedures are generally applicable and will be useful for detecting a wide range of antigens and nucleic acids in paraffin sections in conjunction with LMM. PMID:11486049

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

    USDA-ARS?s Scientific Manuscript database

    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...

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

    USDA-ARS?s Scientific Manuscript database

    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...

  7. Primary oral Penicillium marneffei infection diagnosed by PCR-based molecular identification and transmission electron microscopic observation from formalin-fixed paraffin-embedded tissues.

    PubMed

    Hua, Xia; Zhang, Ruifeng; Yang, Hanjun; Lei, Song; Zhang, Yizhi; Ran, Yuping

    2012-11-07

    We report a case of primary oral Penicillium marneffei infection in a 39-year-old man without HIV infection. Although fungal culture was negative, the patient was finally confirmed to have P. marneffei infection by PCR-based molecular identification and transmission electron microscopic observation from formalin-fixed, paraffin-embedded tissues. The patient was cured with taking itraconazole for 3 months.

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

    EPA Science Inventory

    Formalin-fixed paraffin-embedded (FFPE) tissue samples represent a potentially invaluable resource for genomic research into the molecular basis of disease. However, use of FFPE samples in gene expression studies has been limited by technical challenges resulting from degradation...

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

    EPA Science Inventory

    Formalin-fixed paraffin-embedded (FFPE) tissue samples represent a potentially invaluable resource for genomic research into the molecular basis of disease. However, use of FFPE samples in gene expression studies has been limited by technical challenges resulting from degradation...

  10. KRAS Genotyping of Paraffin-Embedded Colorectal Cancer Tissue in Routine Diagnostics

    PubMed Central

    Weichert, Wilko; Schewe, Christiane; Lehmann, Annika; Sers, Christine; Denkert, Carsten; Budczies, Jan; Stenzinger, Albrecht; Joos, Hans; Landt, Olfert; Heiser, Volker; Röcken, Christoph; Dietel, Manfred

    2010-01-01

    KRAS mutation testing before anti-epidermal growth factor receptor therapy of metastatic colorectal cancer has become mandatory in Europe. However, considerable uncertainty exists as to which methods for detection can be applied in a reproducible and economically sound manner in the routine diagnostic setting. To answer this question, we examined 263 consecutive routine paraffin slide specimens. Genomic DNA was extracted from microdissected tumor tissue. The DNA was analyzed prospectively by Sanger sequencing and array analysis as well as retrospectively by melting curve analysis and pyrosequencing; the results were correlated to tissue characteristics. The methods were then compared regarding the reported results, costs, and working times. Approximately 40% of specimens contained KRAS mutations, and the different methods reported concordant results (κ values >0.9). Specimens harboring fewer than 10% tumor cells showed lower mutation rates regardless of the method used, and histoanatomical variables had no influence on the frequency of the mutations. Costs per assay were higher for array analysis and melting curve analysis when compared with the direct sequencing methods. However, for sequencing methods equipment costs were much higher. In conclusion, Sanger sequencing, array analysis, melting curve analysis, and pyrosequencing were equally effective for routine diagnostic KRAS mutation analysis; however, interpretation of mutation results in conjunction with histomorphologic tissue review and on slide tumor tissue dissection is required for accurate diagnosis. PMID:20007841

  11. 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

  12. Commercially available kits for manual and automatic extraction of nucleic acids from formalin-fixed, paraffin-embedded (FFPE) tissues.

    PubMed

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

    2015-01-01

    Formalin-fixed, paraffin-embedded (FFPE) tissues represent an invaluable source for diagnostic purposes when fresh clinical material is unavailable, and also for molecular and epidemiological studies. The recovery of nucleic acids from FFPE tissues is particularly challenging, and several in-house methods have been developed for this purpose over the last three decades. Recently, several commercial kits specifically developed for DNA and/or RNA extraction from FFPE tissues have been introduced to the market, but their inventory is not available in peer-reviewed literature. This article provides the first comprehensive inventory of commercial FFPE DNA/RNA extraction kits currently available on the market and describes their basic characteristics and features. A total of 69 commercial kits from 43 companies were identified. Thirty-five kits were developed specifically for DNA extraction, 22 for RNA extraction, and 12 for both DNA and RNA extraction. Only two commercial kits allow full automation of the entire nucleic acid extraction procedure. The tissue deparaffinization step is omitted in many protocols by melting paraffin directly in a tissue lysis buffer. Purification of the released nucleic acids is mainly based on silica or resin adsorption technology. A formalin reverse cross-linking step to increase the quality of extracted DNA and RNA is an intrinsic part of over half of the kits identified. It is hope that this comprehensive list of available commercial kits for extracting nucleic acids from FFPE will encourage researchers to strongly consider using them in diagnostic and research settings instead of old-fashioned, crude, and probably less effective in-house methods.

  13. Comparison of Fixatives, Fixation Time, and Severity of Infection on PCR Amplification and Detection of Mycobacterium marinum and Mycobacterium chelonae DNA in Paraffin-Embedded Zebrafish (Danio rerio)

    PubMed Central

    Peterson, Tracy S.; Kent, Michael L.; Ferguson, Jayde A.; Watral, Virginia G.; Whipps, Christopher M.

    2013-01-01

    Mycobacteriosis is a common disease of laboratory zebrafish (Danio rerio). Different infection patterns occur in zebrafish depending on mycobacterial species. Mycobacterium marinum and M. haemophilum produce virulent infections associated with high mortality, whereas M. chelonae is more wide spread and not associated with high mortality. Identification of mycobacterial infections to the species level provides important information for making management decisions. Observation of acid-fast bacilli in histological sections or tissue imprints is the most common diagnostic method for mycobacteriosis in fish, but only allows for diagnosis to the genus level. Mycobacterial culture, followed by molecular or biochemical identification is the traditional approach, but recently it has been shown that DNA of diagnostic value can be retrieved from paraffin blocks. We investigated effects of the following parameters on the ability of our qPCR test for the hsp gene (primer set HS5F/hsp667R) to retrieve specific DNA from paraffin-embedded zebrafish: type of fixative, time in fixative before processing, species of mycobacteria, and severity of infection. Whole zebrafish were experimentally infected with either M. chelonae or M. marinum, and then preserved in 10% neutral buffered formalin or Dietrich’s fixative for 3, 7, 21 and 45 days. Subsequently, fish were evaluated by H&E and Fite’s acid-fast stains to detect mycobacteria within granulomatous lesions. The PCR assay was quite effective, and obtained PCR product from 75% and 88% of the M. chelonae and M. marinum infected fish, respectively. Fixative type, time in fixative, and mycobacterial species showed no statistical relationship with the efficacy of the PCR test. PMID:23709464

  14. Oestrogen receptors in mucinous carcinoma of the breast: an immunohistological study using paraffin wax sections.

    PubMed Central

    Shousha, S; Coady, A T; Stamp, T; James, K R; Alaghband-Zadeh, J

    1989-01-01

    An immunohistological method (Shintaku-Said method) for the demonstration of oestrogen receptors in routinely processed paraffin wax embedded tissue was applied to 19 cases of mucinous carcinoma of the breast. Seventeen (89%) tumours showed variable degrees of positivity and two were negative. In eight cases the receptors were also assayed biochemically using a dextran-coated charcoal method, and the results of the two methods showed good correlation. No difference in the distribution of positive and negative cases was noted between pure and mixed mucinous tumours, and in the latter group the pattern of staining of the mucinous elements was similar to that seen in the solid elements. It is concluded that the major advantage of this method is its ability to offer for study the distribution of the receptors in individual cells and specific histological structures. The results also indicate that most mucinous carcinomas of the breast are oestrogen receptor positive, irrespective of whether they are pure or mixed type. Images PMID:2551929

  15. Improved method for extraction and detection of Helicobacter pylori DNA in formalin-fixed paraffin embedded gastric biopsies using laser micro-dissection

    PubMed Central

    Loayza, María Fernanda; Villavicencio, Fernando Xavier; Santander, Stephanie Carolina; Baldeón, Manuel; Ponce, Lourdes Karina; Salvador, Iván; Vivar Díaz, Nicolás

    2014-01-01

    To assess the molecular events exerted by Helicobacter pylori interacting directly with gastric epithelial cells, an improved procedure for microbial DNA isolation from stained hematoxilin-eosin gastric biopsies was developed based on laser micro-dissection (LM) [1]. Few articles have described the use of LM to select and detect H. pylori genome from formalin-fixed paraffin embedded gastric tissue [2]. To improve the yield and quality of DNA isolated from H. pylori contacting intestinal epithelial cells, the following conditions were established after modification of the QIAamp DNA Micro kit. • Use of at least 25 cut sections of 10–20 μm of diameter and 3 μm thick with more than 10 bacteria in each cut. • Lysis with 30 μL of tissue lysis buffer and 20 μL of proteinase K (PK) with the tube in an upside-down position. • The use of thin purification columns with 35 μL of elution buffer. The mean of DNA concentration obtained from 25 LM cut sections was 1.94± 0 .16 ng/μL, and it was efficiently amplified with qPCR in a Bio Rad iCycler instrument. The LM can improve the sample selection and DNA extraction for molecular analysis of H. pylori associated with human gastric epithelium. PMID:26150965

  16. Superresolution Imaging of Aquaporin-4 Cluster Size in Antibody-Stained Paraffin Brain Sections.

    PubMed

    Smith, Alex J; Verkman, Alan S

    2015-12-15

    The water channel aquaporin-4 (AQP4) forms supramolecular clusters whose size is determined by the ratio of M1- and M23-AQP4 isoforms. In cultured astrocytes, differences in the subcellular localization and macromolecular interactions of small and large AQP4 clusters results in distinct physiological roles for M1- and M23-AQP4. Here, we developed quantitative superresolution optical imaging methodology to measure AQP4 cluster size in antibody-stained paraffin sections of mouse cerebral cortex and spinal cord, human postmortem brain, and glioma biopsy specimens. This methodology was used to demonstrate that large AQP4 clusters are formed in AQP4(-/-) astrocytes transfected with only M23-AQP4, but not in those expressing only M1-AQP4, both in vitro and in vivo. Native AQP4 in mouse cortex, where both isoforms are expressed, was enriched in astrocyte foot-processes adjacent to microcapillaries; clusters in perivascular regions of the cortex were larger than in parenchymal regions, demonstrating size-dependent subcellular segregation of AQP4 clusters. Two-color superresolution imaging demonstrated colocalization of Kir4.1 with AQP4 clusters in perivascular areas but not in parenchyma. Surprisingly, the subcellular distribution of AQP4 clusters was different between gray and white matter astrocytes in spinal cord, demonstrating regional specificity in cluster polarization. Changes in AQP4 subcellular distribution are associated with several neurological diseases and we demonstrate that AQP4 clustering was preserved in a postmortem human cortical brain tissue specimen, but that AQP4 was not substantially clustered in a human glioblastoma specimen despite high-level expression. Our results demonstrate the utility of superresolution optical imaging for measuring the size of AQP4 supramolecular clusters in paraffin sections of brain tissue and support AQP4 cluster size as a primary determinant of its subcellular distribution.

  17. Superresolution Imaging of Aquaporin-4 Cluster Size in Antibody-Stained Paraffin Brain Sections

    PubMed Central

    Smith, Alex J.; Verkman, Alan S.

    2015-01-01

    The water channel aquaporin-4 (AQP4) forms supramolecular clusters whose size is determined by the ratio of M1- and M23-AQP4 isoforms. In cultured astrocytes, differences in the subcellular localization and macromolecular interactions of small and large AQP4 clusters results in distinct physiological roles for M1- and M23-AQP4. Here, we developed quantitative superresolution optical imaging methodology to measure AQP4 cluster size in antibody-stained paraffin sections of mouse cerebral cortex and spinal cord, human postmortem brain, and glioma biopsy specimens. This methodology was used to demonstrate that large AQP4 clusters are formed in AQP4−/− astrocytes transfected with only M23-AQP4, but not in those expressing only M1-AQP4, both in vitro and in vivo. Native AQP4 in mouse cortex, where both isoforms are expressed, was enriched in astrocyte foot-processes adjacent to microcapillaries; clusters in perivascular regions of the cortex were larger than in parenchymal regions, demonstrating size-dependent subcellular segregation of AQP4 clusters. Two-color superresolution imaging demonstrated colocalization of Kir4.1 with AQP4 clusters in perivascular areas but not in parenchyma. Surprisingly, the subcellular distribution of AQP4 clusters was different between gray and white matter astrocytes in spinal cord, demonstrating regional specificity in cluster polarization. Changes in AQP4 subcellular distribution are associated with several neurological diseases and we demonstrate that AQP4 clustering was preserved in a postmortem human cortical brain tissue specimen, but that AQP4 was not substantially clustered in a human glioblastoma specimen despite high-level expression. Our results demonstrate the utility of superresolution optical imaging for measuring the size of AQP4 supramolecular clusters in paraffin sections of brain tissue and support AQP4 cluster size as a primary determinant of its subcellular distribution. PMID:26682810

  18. In Silico Analysis Validates Proteomic Findings of Formalin-fixed Paraffin Embedded Cutaneous Squamous Cell Carcinoma Tissue

    PubMed Central

    AZIMI, ALI; L. KAUFMAN, KIMBERLEY; ALI, MARINA; KOSSARD, STEVEN; FERNANDEZ-PENAS, PABLO

    2016-01-01

    Background: Cutaneous squamous cell carcinoma (cSCC) is a common type of skin cancer but there are no comprehensive proteomic studies on this entity. Materials and Methods: We employed liquid chromatography coupled with tandem mass spectrometry (MS/MS) using formalin-fixed paraffin-embedded (FFPE) cSCC material to study the tumor and normal skin tissue proteomes. Ingenuity Pathway Analysis (IPA) was used to interpret the role of altered proteins in cSCC pathophysiology. Results were validated using the Human Protein Atlas and Oncomine database in silico. Results: Of 1,310 unique proteins identified, expression of an average of 144 and 88 proteins were significantly (p<0.05) increased and decreased, respectively, in the tumor samples compared to their normal counterparts. IPA analysis revealed disruptions in proteins associated with cell proliferation, apoptosis, and migration. In silico analysis confirmed that proteins corresponding to 12 antibodies, and genes corresponding to 18 proteins were differentially expressed between the two categories, validating our proteomic measurements. Conclusion: Label-free MS-based proteomics is useful for analyzing FFPE cSCC tissues. PMID:27807068

  19. Whole-genome single-cell copy number profiling from formalin-fixed paraffin-embedded samples.

    PubMed

    Martelotto, Luciano G; Baslan, Timour; Kendall, Jude; Geyer, Felipe C; Burke, Kathleen A; Spraggon, Lee; Piscuoglio, Salvatore; Chadalavada, Kalyani; Nanjangud, Gouri; Ng, Charlotte K Y; Moody, Pamela; D'Italia, Sean; Rodgers, Linda; Cox, Hilary; da Cruz Paula, Arnaud; Stepansky, Asya; Schizas, Michail; Wen, Hannah Y; King, Tari A; Norton, Larry; Weigelt, Britta; Hicks, James B; Reis-Filho, Jorge S

    2017-03-01

    A substantial proportion of tumors consist of genotypically distinct subpopulations of cancer cells. This intratumor genetic heterogeneity poses a substantial challenge for the implementation of precision medicine. Single-cell genomics constitutes a powerful approach to resolve complex mixtures of cancer cells by tracing cell lineages and discovering cryptic genetic variations that would otherwise be obscured in tumor bulk analyses. Because of the chemical alterations that result from formalin fixation, single-cell genomic approaches have largely remained limited to fresh or rapidly frozen specimens. Here we describe the development and validation of a robust and accurate methodology to perform whole-genome copy-number profiling of single nuclei obtained from formalin-fixed paraffin-embedded clinical tumor samples. We applied the single-cell sequencing approach described here to study the progression from in situ to invasive breast cancer, which revealed that ductal carcinomas in situ show intratumor genetic heterogeneity at diagnosis and that these lesions may progress to invasive breast cancer through a variety of evolutionary processes.

  20. Fluorescence In Situ Hybridization for Diagnosis of Whipple's Disease in Formalin-Fixed Paraffin-Embedded Tissue.

    PubMed

    Braubach, Peter; Lippmann, Torsten; Raoult, Didier; Lagier, Jean-Christophe; Anagnostopoulos, Ioannis; Zender, Steffen; Länger, Florian Peter; Kreipe, Hans-Heinrich; Kühnel, Mark Philipp; Jonigk, Danny

    2017-01-01

    Whipple's disease (WD) is a rare chronic systemic infection with a wide range of clinical symptoms, routinely diagnosed in biopsies from the small intestine and other tissues by periodic acid-Schiff (PAS) diastase staining and immunohistological analysis with specific antibodies. The aim of our study was to improve the pathological diagnosis of WD. Therefore, we analyzed the potential of fluorescence in situ hybridization (FISH) for diagnosing WD, using a Tropheryma (T.) whipplei-specific probe. 19 formalin-fixed paraffin-embedded (FFPE) duodenal biopsy specimens of 12 patients with treated (6/12) and untreated (6/12) WD were retrospectively examined using PAS diastase staining, immunohistochemistry, and FISH. 20 biopsy specimens with normal intestinal mucosa, Helicobacter pylori, or mycobacterial infection, respectively, served as controls. We successfully detected T. whipplei in tissue biopsies with a sensitivity of 83% in untreated (5/6) and 40% in treated (4/10) cases of WD. In our study, we show that FISH-based diagnosis of individual vital T. whipplei in FFPE specimens is feasible and can be considered as ancillary diagnostic tool for the diagnosis of WD in FFPE material. We show that FISH not only detect active WD but also be helpful as an indicator for the efficiency of antibiotic treatment and for detection of recurrence of disease when the signal of PAS diastase and immunohistochemistry lags behind the recurrence of disease, especially if the clinical course of the patient and antimicrobial treatment is considered.

  1. PCR detection of Clostridium chauvoei in pure cultures and in formalin-fixed, paraffin-embedded tissues.

    PubMed

    Uzal, F A; Hugenholtz, P; Blackall, L L; Petray, S; Moss, S; Assis, R A; Fernandez Miyakawa, M; Carloni, G

    2003-02-02

    The polymerase chain reaction (PCR) was used to amplify specific segments of the 16S ribosomal RNA gene of Clostridium chauvoei, a major pathogen of ruminants. Three sets of primers were used to produce amplicons of 159, 836 and 959 base pairs (bp), respectively. The PCR was evaluated by testing clinically important strains of Clostridium, including 21 strains of C. chauvoei, five strains each of Clostridium septicum and Clostridium perfringens and two strains each of Clostridium novyi, Clostridium histolyticum and Clostridium sordellii. Both purified DNA and biomass from pure cultures of each of these microorganisms were evaluated as templates in the PCR. In addition, extracts of formalin-fixed, paraffin-embedded tissues of eight sheep experimentally inoculated with C. chauvoei or C. septicum (four animals each) were also tested by the PCR using the three sets of primers. Purified DNA template of all C. chauvoei strains produced PCR amplicons of the expected size for all three primer pairs. However, when biomass from pure cultures of C. chauvoei or tissue extracts were used as templates, only the primer pair designed to produce the 159bp amplicon gave consistently positive results. No positive results were obtained with any primer pair when purified DNA or biomass from pure cultures of non-target clostridial species were used as templates. Therefore, the PCR primer sets appear to be very specific for identifying C. chauvoei in both cultures and tissues.

  2. Robust transcriptional tumor signatures applicable to both formalin-fixed paraffin-embedded and fresh-frozen samples

    PubMed Central

    Cheng, Jun; He, Jun; Liu, Huaping; Cai, Hao; Hong, Guini; Zhang, Jiahui; Li, Na; Ao, Lu; Guo, Zheng

    2017-01-01

    Formalin-fixed paraffin-embedded (FFPE) samples represent a valuable resource for clinical researches. However, FFPE samples are usually considered an unreliable source for gene expression analysis due to the partial RNA degradation. In this study, through comparing gene expression profiles between FFPE samples and paired fresh-frozen (FF) samples for three cancer types, we firstly showed that expression measurements of thousands of genes had at least two-fold change in FFPE samples compared with paired FF samples. Therefore, for a transcriptional signature based on risk scores summarized from the expression levels of the signature genes, the risk score thresholds trained from FFPE (or FF) samples could not be applied to FF (or FFPE) samples. On the other hand, we found that more than 90% of the relative expression orderings (REOs) of gene pairs in the FF samples were maintained in their paired FFPE samples and largely unaffected by the storage time. The result suggested that the REOs of gene pairs were highly robust against partial RNA degradation in FFPE samples. Finally, as a case study, we developed a REOs-based signature to distinguish liver cirrhosis from hepatocellular carcinoma (HCC) using FFPE samples. The signature was validated in four datasets of FFPE samples and eight datasets of FF samples. In conclusion, the valuable FFPE samples can be fully exploited to identify REOs-based diagnostic and prognostic signatures which could be robustly applicable to both FF samples and FFPE samples with degraded RNA. PMID:28036264

  3. Molecular genotyping of Echinococcus granulosus using formalin-fixed paraffin-embedded preparations from human isolates in unusual tissue sites.

    PubMed

    Hizem, A; M'rad, S; Oudni-M'rad, M; Mestiri, S; Hammedi, F; Mezhoud, H; Zakhama, A; Mokni, M; Babba, H

    2016-07-01

    Cystic echinococcosis (CE) caused by Echinococcus granulosus remains a serious problem worldwide for issues relating to public health and the economy. The most predominantly affected sites are the liver and the lungs, but other organs such as the heart, the spleen and the peritoneum can also be infected. Access to cysts from uncommon sites has limited genomic and molecular investigations. In the present study, genotypes of E. granulosus sensu lato were identified from formalin-fixed paraffin-embedded tissues (FF-PETs) implicated in human CE. Tissue samples were obtained from 57 patients with histologically confirmed CE. DNA samples were analysed using Egss 1 polymerase chain reaction (PCR) specific to the mitochondrial 12S rRNA gene of E. granulosus sensu stricto. All cysts were typed as E. granulosus sensu stricto with up to 35% of the liver and 16.6% of lungs being the most frequently infected, and up to 48.4% of samples being from rare sites. No correlation was found between cyst site and either the gender or the age of patients. This study demonstrates the possibility of exploiting atypical cysts using FF-PET samples and highlights the predominance of E. granulosus sensu stricto species in the Tunisian population, even in unusual infection sites.

  4. 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.

  5. Measuring expression levels of small regulatory RNA molecules from body fluids and formalin-fixed, paraffin-embedded samples.

    PubMed

    Gyongyosi, Adrienn; Docs, Otto; Czimmerer, Zsolt; Orosz, Laszlo; Horvath, Attila; Török, Olga; Mehes, Gabor; Nagy, Laszlo; Balint, Balint L

    2014-01-01

    MicroRNAs are involved in the regulation of various pathophysiological processes such as immune regulation and cancer. Next-generation sequencing methods enable us to monitor their presence in various types of samples but we need flexible methods for validating datasets generated by high-throughput methods. Here we describe the detailed protocols to be used with our MiRNA Primer Design Tool assay design system. The presented methods allow the flexible design of the oligonucleotides needed for the RT-qPCR detection of any variant of small regulatory RNA molecules from virtually any species. This method can be used to measure miRNA levels from formalin-fixed, paraffin-embedded (FFPE) samples and various body fluids. As an example, we show the results of the hsa-miR-515-3p, hsa-miR-325, and hsa-miR-155 quantification using a specific UPL probe (Universal Probe Library) and a stem-loop RT-qPCR assay. The small nucleolar RNA RNU43 is used as endogenous control for normalization of the results. Urine from healthy pregnant women and FFPE samples from patients diagnosed with colorectal cancer and treated with antibody-based anti-EGFR monotherapy were used as samples.

  6. 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.

  7. Association between mir-24 and mir-378 in formalin-fixed paraffin-embedded tissues of breast cancer.

    PubMed

    Yin, Jia-Yu; Deng, Zhao-Qun; Liu, Feng-Qiong; Qian, Jun; Lin, Jiang; Tang, Qin; Wen, Xiang-Mei; Zhou, Jing-Dong; Zhang, Ying-Ying; Zhu, Xiao-Wen

    2014-01-01

    MiR-24/378 is thought to be onco-miRNAs for their ability of enhancing tumor growth. The objective of this study was to evaluate the potential predictive value of miR-24/378 expression in formalin-fixed paraffin-embedded tissues of breast cancer patients. The expression of miR-24/378 was examined in 101 breast cancer patients and 40 controls using real-time quantitative PCR. All statistical analyses were performed using SPSS16.0. We found that miR-24 and miR-378 were significantly up-regulated in breast cancer patients compared with controls (all P < 0.01). The expression levels of the two miRNAs were highly correlated with each other in breast cancer patients, with r = 0.778 between miR-24 and miR-378. Moreover, the two miRNAs exhibited great capability of discriminating between cancer patients and controls by ROC analysis. MiR-24 and miR-378 showed 0.79 and 0.807 AUC values respectively. Over-expression of miR-24 and miR-378 in FFPE tissue of breast cancer patients might conduct as an ideal source for biomarker discovery and validation in breast cancer patients.

  8. Expression level of miR-93 in formalin-fixed paraffin-embedded tissues of breast cancer patients.

    PubMed

    Deng, Zhao-qun; Qian, Jun; Liu, Feng-qiong; Lin, Jiang; Shao, Rui; Yin, Jia-yu; Tang, Qin; Zhang, Ming; He, Li

    2014-05-01

    MiR-93 is thought to be an onco-miRNA for its capabilities of enhancing tumor growth. The objective of this study was to evaluate the potential predictive value of miR-93 expression in formalin-fixed paraffin-embedded (FFPE) tissues of breast cancer patients. The expression of miR-93 was examined in 101 breast cancer patients and 40 controls using real-time quantitative PCR. We found that miR-93 was markedly upregulated in breast cancer patients compared with controls (p<0.01). The expression level of miR-93 was significantly correlated with miR-24/378 in breast cancer patients. MiR-93 exhibited great capability of discriminating between cancer patients and cancer-free controls by receiver-operator characteristic (ROC) curve analysis. MiR-93 showed 0.866 AUC (the area under the ROC curve) values. The MiR-93 level was found significantly correlated with breast cancer by univariable logistic regression. These results suggest that overexpression of miR-93 in FFPE tissues may serve as an indispensable source for biomarker discovery and validation in breast cancer patients.

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

    PubMed Central

    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

  10. Automated high throughput nucleic acid purification from formalin-fixed paraffin-embedded tissue samples for next generation sequence analysis

    PubMed Central

    Haile, Simon; Pandoh, Pawan; McDonald, Helen; Corbett, Richard D.; Tsao, Philip; Kirk, Heather; MacLeod, Tina; Jones, Martin; Bilobram, Steve; Brooks, Denise; Smailus, Duane; Steidl, Christian; Scott, David W.; Bala, Miruna; Hirst, Martin; Miller, Diane; Moore, Richard A.; Mungall, Andrew J.; Coope, Robin J.; Ma, Yussanne; Zhao, Yongjun; Holt, Rob A.; Jones, Steven J.

    2017-01-01

    Curation and storage of formalin-fixed, paraffin-embedded (FFPE) samples are standard procedures in hospital pathology laboratories around the world. Many thousands of such samples exist and could be used for next generation sequencing analysis. Retrospective analyses of such samples are important for identifying molecular correlates of carcinogenesis, treatment history and disease outcomes. Two major hurdles in using FFPE material for sequencing are the damaged nature of the nucleic acids and the labor-intensive nature of nucleic acid purification. These limitations and a number of other issues that span multiple steps from nucleic acid purification to library construction are addressed here. We optimized and automated a 96-well magnetic bead-based extraction protocol that can be scaled to large cohorts and is compatible with automation. Using sets of 32 and 91 individual FFPE samples respectively, we generated libraries from 100 ng of total RNA and DNA starting amounts with 95–100% success rate. The use of the resulting RNA in micro-RNA sequencing was also demonstrated. In addition to offering the potential of scalability and rapid throughput, the yield obtained with lower input requirements makes these methods applicable to clinical samples where tissue abundance is limiting. PMID:28570594

  11. KRAS Mutation Detection in Paired Frozen and Formalin-Fixed Paraffin-Embedded (FFPE) Colorectal Cancer Tissues

    PubMed Central

    Solassol, Jérome; Ramos, Jeanne; Crapez, Evelyne; Saifi, Majda; Mangé, Alain; Vianès, Evelyne; Lamy, Pierre-Jean; Costes, Valérie; Maudelonde, Thierry

    2011-01-01

    KRAS mutation has been unambiguously identified as a marker of resistance to cetuximab-based treatment in metastatic colorectal cancer (mCRC) patients. However, most studies of KRAS mutation analysis have been performed using homogenously archived CRC specimens, and studies that compare freshly frozen specimens and formalin-fixed paraffin-embedded (FFPE) specimens of CRC are lacking. The aim of the present study was to evaluate the impact of tissue preservation on the determination of KRAS mutational status. A series of 131 mCRC fresh-frozen tissues were first analyzed using both high-resolution melting (HRM) and direct sequencing. KRAS mutations were found in 47/131 (35.8%) using both approaches. Out of the 47 samples that were positive for KRAS mutations, 33 had available matched FFPE specimens. Using HRM, 2/33 (6%) demonstrated suboptimal template amplification, and 2/33 (6%) expressed an erroneous wild-type KRAS profile. Using direct sequencing, 6/33 (18.1%) displayed a wild-type KRAS status, and 3/33 (9.1%) showed discordant mutations. Finally, the detection of KRAS mutations was lower among the FFPE samples compared with the freshly frozen samples, demonstrating that tissue processing clearly impacts the accuracy of KRAS genotyping. PMID:21686179

  12. 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.

  13. Acquisition of biologically relevant gene expression data by Affymetrix microarray analysis of archival formalin-fixed paraffin-embedded tumours

    PubMed Central

    Linton, K M; Hey, Y; Saunders, E; Jeziorska, M; Denton, J; Wilson, C L; Swindell, R; Dibben, S; Miller, C J; Pepper, S D; Radford, J A; Freemont, A J

    2008-01-01

    Robust protocols for microarray gene expression profiling of archival formalin-fixed paraffin-embedded tissue (FFPET) are needed to facilitate research when availability of fresh-frozen tissue is limited. Recent reports attest to the feasibility of this approach, but the clinical value of these data is poorly understood. We employed state-of-the-art RNA extraction and Affymetrix microarray technology to examine 34 archival FFPET primary extremity soft tissue sarcomas. Nineteen arrays met stringent QC criteria and were used to model prognostic signatures for metastatic recurrence. Arrays from two paired frozen and FFPET samples were compared: although FFPET sensitivity was low (∼50%), high specificity (95%) and positive predictive value (92%) suggest that transcript detection is reliable. Good agreement between arrays and real time (RT)–PCR was confirmed, especially for abundant transcripts, and RT–PCR validated the regulation pattern for 19 of 24 candidate genes (overall R2=0.4662). RT–PCR and immunohistochemistry on independent cases validated prognostic significance for several genes including RECQL4, FRRS1, CFH and MET – whose combined expression carried greater prognostic value than tumour grade – and cmet and TRKB proteins. These molecules warrant further evaluation in larger series. Reliable clinically relevant data can be obtained from archival FFPET, but protocol amendments are needed to improve the sensitivity and broad application of this approach. PMID:18382428

  14. Acquisition of biologically relevant gene expression data by Affymetrix microarray analysis of archival formalin-fixed paraffin-embedded tumours.

    PubMed

    Linton, K M; Hey, Y; Saunders, E; Jeziorska, M; Denton, J; Wilson, C L; Swindell, R; Dibben, S; Miller, C J; Pepper, S D; Radford, J A; Freemont, A J

    2008-04-22

    Robust protocols for microarray gene expression profiling of archival formalin-fixed paraffin-embedded tissue (FFPET) are needed to facilitate research when availability of fresh-frozen tissue is limited. Recent reports attest to the feasibility of this approach, but the clinical value of these data is poorly understood. We employed state-of-the-art RNA extraction and Affymetrix microarray technology to examine 34 archival FFPET primary extremity soft tissue sarcomas. Nineteen arrays met stringent QC criteria and were used to model prognostic signatures for metastatic recurrence. Arrays from two paired frozen and FFPET samples were compared: although FFPET sensitivity was low ( approximately 50%), high specificity (95%) and positive predictive value (92%) suggest that transcript detection is reliable. Good agreement between arrays and real time (RT)-PCR was confirmed, especially for abundant transcripts, and RT-PCR validated the regulation pattern for 19 of 24 candidate genes (overall R(2)=0.4662). RT-PCR and immunohistochemistry on independent cases validated prognostic significance for several genes including RECQL4, FRRS1, CFH and MET - whose combined expression carried greater prognostic value than tumour grade - and cmet and TRKB proteins. These molecules warrant further evaluation in larger series. Reliable clinically relevant data can be obtained from archival FFPET, but protocol amendments are needed to improve the sensitivity and broad application of this approach.

  15. Differential proteomic analysis of late-stage and recurrent breast cancer from formalin-fixed paraffin-embedded tissues.

    PubMed

    Bateman, Nicholas W; Sun, Mai; Bhargava, Rohit; Hood, Brian L; Darfler, Marlene M; Kovatich, Albert J; Hooke, Jeffrey A; Krizman, David B; Conrads, Thomas P

    2011-03-04

    The heterogeneity of breast cancer requires the discovery of more incisive molecular tools that better define disease progression and prognosis. Proteomic analysis of homogeneous tumor cell populations derived by laser microdissection from formalin-fixed, paraffin-embedded (FFPE) tissues has proven to be a robust strategy for conducting retrospective cancer biomarker investigations. We describe an MS-based analysis of laser microdissected cancerous epithelial cells derived from twenty-five breast cancer patients at defined clinical disease stages with the goal of identifying protein abundance characteristics indicative of disease progression and recurrence. Comparative analysis of stage 0 and stage III patients revealed 113 proteins that significantly differentiated these groups and included known factors associated with disease pathogenesis, such as CDH1 and CTNNB1, as well as those previously implicated in breast cancer, such as TSP-1. Similar analyses of patients presenting with stage II disease that did or did not exhibit recurrence two years postdiagnosis revealed 42 proteins that significantly differentiated these subgroups and included IRS-1 and PARK7. These data provide evidence supporting the utility of FFPE tissues for functional proteomic analyses and protein biomarker discovery and yielded protein candidates indicative of disease stage and recurrence in breast cancer that warrant further investigation for diagnostic utility and biological relevance.

  16. Simultaneous Recovery of DNA and RNA from Formalin-Fixed Paraffin-Embedded Tissue and Application in Epidemiologic Studies

    PubMed Central

    Huang, Wen-Yi; Sheehy, Timothy M.; Moore, Lee E.; Hsing, Ann W.; Purdue, Mark P.

    2010-01-01

    Analysis of DNA, RNA, and protein extracted from tissue specimens in epidemiologic studies is useful for assessing etiologic heterogeneity, mechanisms of carcinogenesis and biomarkers for prognosis and prediction of treatment responses. Fresh-frozen tissue samples may provide optimal quality nucleic acids, but pose multiple logistical considerations, including rapid access to tissues prior to histopathologic examination and specialized equipment for freezing, transport and storage; in addition, morphology is often compromised. In contrast, formalin-fixed paraffin-embedded (FFPE) tissue samples, including enormous archives of existing specimens, represent a valuable source of retrospective biological material for epidemiologic research, although presenting different limitations compared to frozen samples. Recent efforts have made progress toward enhancing the utility of FFPE specimens for molecular analyses, including DNA studies, and increasingly for RNA and other macromolecules. Here we report the method that we used to simultaneously recover DNA and RNA from FFPE tissue specimens with appreciable quantity and quality, and discuss briefly the application of tumor markers in epidemiologic studies. PMID:20332269

  17. IDH mutation detection in formalin-fixed paraffin-embedded gliomas using multiplex PCR and single-base extension.

    PubMed

    Perizzolo, Marco; Winkfein, Bob; Hui, Susan; Krulicki, Wally; Chan, Jennifer A; Demetrick, Douglas J

    2012-09-01

    Isocitrate dehydrogenase (IDH) genes are mutated in a significant portion of gliomas, myeloid leukemias and chondroid neoplasms. In gliomas, IDH mutations are prognostic, as those tumors with the mutation are associated with a proneural subclass and have longer survival compared with those without the mutation. We developed a simple, PCR-based SNaPshot® assay (Life Technologies, Carlsbad, CA, USA) to detect IDH1/2 mutations. This protocol combines a single, multiplexed PCR reaction using gene specific primers followed by a single, multiplexed SNaPshot reaction and detection by capillary electrophoresis. In a blinded study of 32 paraffin-embedded glioma specimens previously screened for IDH mutations by a PCR/direct sequencing method, concordance of our IDH SNaPshot test with sequencing was 100%. We performed the assay on an additional 57 specimens submitted for diagnostic IDH mutation evaluation. Data analysis was much faster and easier to perform than analysis of the sequencing data, and results could be obtained in 1 day from DNA extraction to analysis. Furthermore, we could readily identify a mixture of 5% mutant allele vs. 95% wild-type allele in our SNaPshot assay, in comparison to approximately 20% mutant allele in our PCR-sequencing assay. Our assay represents a fast, sensitive, straightforward method of reliably detecting common mutations of IDH genes in glial neoplasms, or other tumors.

  18. Diagnosis of Ostreid herpesvirus 1 in fixed paraffin-embedded archival samples using PCR and in situ hybridisation.

    PubMed

    Barbosa-Solomieu, V; Miossec, L; Vázquez-Juárez, R; Ascencio-Valle, F; Renault, T

    2004-08-01

    In 1994, some of the high mortality episodes that affected oysters cultured in France were associated with herpesviral infections. Through histology analysis, however, viral presence could only be suspected and confirmation of histological diagnosis by transmission electron microscopy was performed in only a few cases. Subsequently, the characterisation and genome sequencing of Ostreid herpesvirus 1 (OsHV-1) made possible the development of specific molecular detection (PCR and in situ hybridisation (ISH)). Using both molecular tools, attempts were made to screen for OsHV-1 a number of fixed, paraffin-embedded oyster samples collected and processed in 1994. The aim was to compare these techniques and to estimate the accuracy of histology-based indication of viral infection. Existing DNA extraction protocols were adapted for oyster samples and two pairs of specific primers targeting small fragments (less than 200bp) were designed (C(9)/C(10) and B(4)/B(3)). The poor consistency observed between the results of PCR with both primer pairs was confirmed by statistical analysis. C(9)/C(10), which targets a repeated region of the OsHV-1 genome, appears to be the primer of choice for viral detection in archival samples. In situ hybridisation may furnish complementary information concerning the localisation of viral foci. Under certain conditions, retrospective examination of archival samples by molecular techniques may therefore provide valuable epidemiological data.

  19. Detection of Leishmania braziliensis in human paraffin-embedded tissues from Tucumán, Argentina by polymerase chain reaction.

    PubMed

    Lanús, Elizabeth Córdoba; Piñero, José Enrique; González, Ana Cristina; Valladares, Basilio; de Grosso, Mercedes Lizarralde; Salomón, Oscar Daniel

    2005-04-01

    American cutaneous leishmaniasis (ACL) is an endemic disease in Northern Argentina. We applied the polymerase chain reaction (PCR) followed by a hybridization labelled probe to 21 paraffin embedded human skin biopsies, already analyzed histologically, from leishmaniasis endemic areas in the province of Tucumán, Argentina. We used primers previously designed to detect a Leishmania-specific 120-base-pair fragment of kinetoplast DNA minicircle, other two primer pairs that amplify kDNA minicircles belonging to the L. braziliensis and L. mexicana complexes respectively, and specific oligonucleotide primers to detect L. (V.) braziliensis which amplify the sequence of the ribosomal protein L-14 of this species. The PCR-hybridization showed a sensitivity of 90.5% when compared to the histopathology test which was 61.9%. Five of the total samples analyzed were positive for the L. braziliensis complex whilst none was positive for the L. mexicana complex. The specific primers for L. (V.) braziliensis detected the parasite in four samples. These results are consistent with those reported for close endemic areas and demonstrate that the causative agent of human leishmaniasis in the analyzed cases was L. (V.) braziliensis. PCR should be used as a diagnostic tool for tegumentary leishmaniasis, especially in the mucosal form, and as a valuable technique for the identification of the Leishmania species that causes the disease in certain areas.

  20. Automated high throughput nucleic acid purification from formalin-fixed paraffin-embedded tissue samples for next generation sequence analysis.

    PubMed

    Haile, Simon; Pandoh, Pawan; McDonald, Helen; Corbett, Richard D; Tsao, Philip; Kirk, Heather; MacLeod, Tina; Jones, Martin; Bilobram, Steve; Brooks, Denise; Smailus, Duane; Steidl, Christian; Scott, David W; Bala, Miruna; Hirst, Martin; Miller, Diane; Moore, Richard A; Mungall, Andrew J; Coope, Robin J; Ma, Yussanne; Zhao, Yongjun; Holt, Rob A; Jones, Steven J; Marra, Marco A

    2017-01-01

    Curation and storage of formalin-fixed, paraffin-embedded (FFPE) samples are standard procedures in hospital pathology laboratories around the world. Many thousands of such samples exist and could be used for next generation sequencing analysis. Retrospective analyses of such samples are important for identifying molecular correlates of carcinogenesis, treatment history and disease outcomes. Two major hurdles in using FFPE material for sequencing are the damaged nature of the nucleic acids and the labor-intensive nature of nucleic acid purification. These limitations and a number of other issues that span multiple steps from nucleic acid purification to library construction are addressed here. We optimized and automated a 96-well magnetic bead-based extraction protocol that can be scaled to large cohorts and is compatible with automation. Using sets of 32 and 91 individual FFPE samples respectively, we generated libraries from 100 ng of total RNA and DNA starting amounts with 95-100% success rate. The use of the resulting RNA in micro-RNA sequencing was also demonstrated. In addition to offering the potential of scalability and rapid throughput, the yield obtained with lower input requirements makes these methods applicable to clinical samples where tissue abundance is limiting.

  1. Whole-genome single-cell copy number profiling from formalin-fixed paraffin-embedded samples

    PubMed Central

    Martelotto, Luciano G; Baslan, Timour; Kendall, Jude; Geyer, Felipe C; Burke, Kathleen A; Spraggon, Lee; Piscuoglio, Salvatore; Chadalavada, Kalyani; Nanjangud, Gouri; Ng, Charlotte KY; Moody, Pamela; D’Italia, Sean; Rodgers, Linda; Cox, Hilary; Paula, Arnaud da Cruz; Stepansky, Asya; Schizas, Michail; Wen, Hannah Y; King, Tari A; Norton, Larry; Weigelt, Britta; Hicks, James B; Reis-Filho, Jorge S

    2017-01-01

    A substantial proportion of tumors consist of genotypically distinct subpopulations of cancer cells. This intra-tumor genetic heterogeneity poses a significant challenge for the implementation of precision medicine. Single-cell genomics constitutes a powerful approach to resolve complex mixtures of cancer cells by tracing cell lineages and discovering cryptic genetic variations that would otherwise be obscured in tumor bulk analyses. Given the chemical alterations that result from formalin fixation, single-cell genomic approaches have largely remained limited to fresh/frozen specimens. Here we describe the development and validation of a robust and accurate methodology to perform whole-genome copy-number profiling of single nuclei obtained from formalin-fixed paraffin-embedded clinical tumor samples. We applied the single-cell sequencing approach described here to study the progression from in situ to invasive breast cancer, which revealed that ductal carcinomas in situ display intra-tumor genetic heterogeneity at diagnosis and that these lesions may progress to invasive breast cancer through a variety of evolutionary processes. PMID:28165479

  2. A multisite blinded study for the detection of BRAF mutations in formalin-fixed, paraffin-embedded malignant melanoma.

    PubMed

    Richter, Anna; Grieu, Fabienne; Carrello, Amerigo; Amanuel, Benhur; Namdarian, Kateh; Rynska, Aleksandra; Lucas, Amanda; Michael, Victoria; Bell, Anthony; Fox, Stephen B; Hewitt, Chelsee A; Do, Hongdo; McArthur, Grant A; Wong, Stephen Q; Dobrovic, Alexander; Iacopetta, Barry

    2013-01-01

    Melanoma patients with BRAF mutations respond to treatment with vemurafenib, thus creating a need for accurate testing of BRAF mutation status. We carried out a blinded study to evaluate various BRAF mutation testing methodologies in the clinical setting. Formalin-fixed, paraffin-embedded melanoma samples were macrodissected before screening for mutations using Sanger sequencing, single-strand conformation analysis (SSCA), high resolution melting analysis (HRM) and competitive allele-specific TaqMan® PCR (CAST-PCR). Concordance of 100% was observed between the Sanger sequencing, SSCA and HRM techniques. CAST-PCR gave rapid and accurate results for the common V600E and V600K mutations, however additional assays are required to detect rarer BRAF mutation types found in 3-4% of melanomas. HRM and SSCA followed by Sanger sequencing are effective two-step strategies for the detection of BRAF mutations in the clinical setting. CAST-PCR was useful for samples with low tumour purity and may also be a cost-effective and robust method for routine diagnostics.

  3. A multisite blinded study for the detection of BRAF mutations in formalin-fixed, paraffin-embedded malignant melanoma

    PubMed Central

    Richter, Anna; Grieu, Fabienne; Carrello, Amerigo; Amanuel, Benhur; Namdarian, Kateh; Rynska, Aleksandra; Lucas, Amanda; Michael, Victoria; Bell, Anthony; Fox, Stephen B.; Hewitt, Chelsee A.; Do, Hongdo; McArthur, Grant A.; Wong, Stephen Q.; Dobrovic, Alexander; Iacopetta, Barry

    2013-01-01

    Melanoma patients with BRAF mutations respond to treatment with vemurafenib, thus creating a need for accurate testing of BRAF mutation status. We carried out a blinded study to evaluate various BRAF mutation testing methodologies in the clinical setting. Formalin-fixed, paraffin-embedded melanoma samples were macrodissected before screening for mutations using Sanger sequencing, single-strand conformation analysis (SSCA), high resolution melting analysis (HRM) and competitive allele-specific TaqMan® PCR (CAST-PCR). Concordance of 100% was observed between the Sanger sequencing, SSCA and HRM techniques. CAST-PCR gave rapid and accurate results for the common V600E and V600K mutations, however additional assays are required to detect rarer BRAF mutation types found in 3–4% of melanomas. HRM and SSCA followed by Sanger sequencing are effective two-step strategies for the detection of BRAF mutations in the clinical setting. CAST-PCR was useful for samples with low tumour purity and may also be a cost-effective and robust method for routine diagnostics. PMID:23584600

  4. Exome sequencing-based molecular autopsy of formalin-fixed paraffin-embedded tissue after sudden death.

    PubMed

    Bagnall, Richard D; Ingles, Jodie; Yeates, Laura; Berkovic, Samuel F; Semsarian, Christopher

    2017-10-01

    Sudden death in the young is a devastating complication of inherited heart disorders. Finding the precise cause of death is important, but it is often unresolved after postmortem investigation. The addition of postmortem genetic testing, i.e., the molecular autopsy, can identify additional causes of death. We evaluated DNA extracted from formalin-fixed paraffin-embedded postmortem tissue for exome sequencing-based molecular autopsy after sudden death in the young. We collected clinical and postmortem information from patients with sudden death. Exome sequencing was performed on DNA extracted from fixed postmortem tissue. Variants relevant to the cause of death were sought. Five patients with genetically unresolved sudden death were recruited. DNA extracted from fixed postmortem tissue was degraded. Exome sequencing achieved 20-fold coverage of at least 82% of coding regions. A threefold excess of singleton variants was found in the exome sequencing data of one patient. We found a de novo SCN1A frameshift variant in a patient with sudden unexpected death in epilepsy and a LMNA nonsense variant in a patient with dilated cardiomyopathy. DNA extracted from fixed postmortem tissue is applicable to exome sequencing-based molecular autopsy. Fixed postmortem tissues are an untapped resource for exome-based studies of rare causes of sudden death.Genet Med advance online publication 23 March 2017.

  5. 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.

  6. [Efficacy of PCR for the differential diagnosis of tuberculosis in granulomatous lesions of paraffin-embedded formalin fixed tissues].

    PubMed

    Montenegro, Sonia; Delgado, Carolina; Pineda, Susana; Reyes, Cristian; Barra, Tiare de la; Cabezas, Claudia; Spencer, Loreto; Mucientes, Francisco

    2014-12-01

    Granulomatous lesions occur in tuberculosis (TB), other infections, toxic, allergic, and autoimmune diseases among others. In absence of a an acid-fast bacilli (AFB) confirmation of TB is necessary. To assess the efficacy of PCR for TB detection and to correlate with granuloma histology and AFB staining. We analyzed 380 fixed paraffin-embedded tissues (PETs) of granulomas with and without caseous necrosis; suppurative; sarcoidal; or of chronic nonspecific nature. Nested PCR-IS6110 for Mycobacterium tuberculosis complex (MTB) and a nested pan-Mycobacterium for the hsp65 gene were used for Mycobacterium spp detection. PCR was more sensitive than AFB staining for all five catagories of granulomas: G1: PCR 71%, AFB staining 28%. G2: PCR 37%, AFB 8%. G3: PCR 17%, AFB staining 7%. G4: PCR 8%, AFB staining 4%. G5: PCR 6%, AFB staining 0%. Molecular diagnosis of TB using PCR-based testing is a fast, efficacious and sensitive method that increased the accuracy of PET histological diagnosis associated with granulomatous lesions.

  7. Quantification of DNA Extracted from Formalin Fixed Paraffin-Embeded Tissue Comparison of Three Techniques: Effect on PCR Efficiency

    PubMed Central

    Panigrahi, Manoj Kumar; Suryavanshi, Moushumi; Mehta, Anurag; Saikia, Kandarpa Kumar

    2016-01-01

    Introduction Mutation detection from Formalin Fixed Paraffin-Embedding (FFPE) tissue in molecular lab became a necessary tool for defining potential targeted drug. Accurate quantification of DNA extracted from FFPE tissue is necessary for downstream applications like Polymerase Chain Reaction (PCR), sequencing etc. Aim To check and define which method for FFPE DNA quantification is suitable for downstream processes. Materials and Methods In this experimental experience study Biorad Smartspec Plus spectrophotomery, Qubit Fluorometer, and Qiagen Rotorgene qPCR was used to compare 20 FFPE DNA quantification in Rajiv Gandhi Cancer Institute and Research Centre, in 2015 and quantified amount of DNA used for PCR reaction. Results The average concentration of DNA extracted from FFPE tissue measured using the spectrophotometer was much higher than the concentration measured using the Qubit Fluorometer and qPCR. Conclusion Results varied depending upon the technique used. A fluorometric analysis may be more suitable for quantification of DNA samples extracted from FFPE tissue compared with spectrophotometric analysis. But qPCR is the best technique because it details DNA quantity along with quality of amplifiable DNA from FFPE tissue. PMID:27790419

  8. Application of the FICTION technique for the simultaneous detection of immunophenotype and chromosomal abnormalities in routinely fixed, paraffin wax embedded bone marrow trephines.

    PubMed

    Korac, P; Jones, M; Dominis, M; Kusec, R; Mason, D Y; Banham, A H; Ventura, R A

    2005-12-01

    The use of interphase fluorescence in situ hybridisation (FISH) to study cytogenetic abnormalities in routinely fixed paraffin wax embedded tissue has become commonplace over the past decade. However, very few studies have applied FISH to routinely fixed bone marrow trephines (BMTs). This may be because of the acid based decalcification methods that are commonly used during the processing of BMTs, which may adversely affect the suitability of the sample for FISH analysis. For the first time, this report describes the simultaneous application of FISH and immunofluorescent staining (the FICTION technique) to formalin fixed, EDTA decalcified and paraffin wax embedded BMTs. This technique allows the direct correlation of genetic abnormalities to immunophenotype, and therefore will be particularly useful for the identification of genetic abnormalities in specific tumour cells present in BMTs. The application of this to routine clinical practice will assist diagnosis and the detection of minimal residual disease.

  9. Application of the FICTION technique for the simultaneous detection of immunophenotype and chromosomal abnormalities in routinely fixed, paraffin wax embedded bone marrow trephines

    PubMed Central

    Korać, P; Jones, M; Dominis, M; Kušec, R; Mason, D Y; Banham, A H; Ventura, R A

    2005-01-01

    The use of interphase fluorescence in situ hybridisation (FISH) to study cytogenetic abnormalities in routinely fixed paraffin wax embedded tissue has become commonplace over the past decade. However, very few studies have applied FISH to routinely fixed bone marrow trephines (BMTs). This may be because of the acid based decalcification methods that are commonly used during the processing of BMTs, which may adversely affect the suitability of the sample for FISH analysis. For the first time, this report describes the simultaneous application of FISH and immunofluorescent staining (the FICTION technique) to formalin fixed, EDTA decalcified and paraffin wax embedded BMTs. This technique allows the direct correlation of genetic abnormalities to immunophenotype, and therefore will be particularly useful for the identification of genetic abnormalities in specific tumour cells present in BMTs. The application of this to routine clinical practice will assist diagnosis and the detection of minimal residual disease. PMID:16311361

  10. Modified paraffin wax for improvement of histological analysis efficiency.

    PubMed

    Lim, Jin Ik; Lim, Kook-Jin; Choi, Jin-Young; Lee, Yong-Keun

    2010-08-01

    Paraffin wax is usually used as an embedding medium for histological analysis of natural tissue. However, it is not easy to obtain enough numbers of satisfactory sectioned slices because of the difference in mechanical properties between the paraffin and embedded tissue. We describe a modified paraffin wax that can improve the histological analysis efficiency of natural tissue, composed of paraffin and ethylene vinyl acetate (EVA) resin (0, 3, 5, and 10 wt %). Softening temperature of the paraffin/EVA media was similar to that of paraffin (50-60 degrees C). The paraffin/EVA media dissolved completely in xylene after 30 min at 50 degrees C. Physical properties such as the amount of load under the same compressive displacement, elastic recovery, and crystal intensity increased with increased EVA content. EVA medium (5 wt %) was regarded as an optimal composition, based on the sectioning efficiency measured by the numbers of unimpaired sectioned slices, amount of load under the same compressive displacement, and elastic recovery test. Based on the staining test of sectioned slices embedded in a 5 wt % EVA medium by hematoxylin and eosin (H&E), Masson trichrome (MT), and other staining tests, it was concluded that the modified paraffin wax can improve the histological analysis efficiency with various natural tissues.

  11. Validation of the Lung Subtyping Panel in Multiple Fresh-Frozen and Formalin-Fixed, Paraffin-Embedded Lung Tumor Gene Expression Data Sets.

    PubMed

    Faruki, Hawazin; Mayhew, Gregory M; Fan, Cheng; Wilkerson, Matthew D; Parker, Scott; Kam-Morgan, Lauren; Eisenberg, Marcia; Horten, Bruce; Hayes, D Neil; Perou, Charles M; Lai-Goldman, Myla

    2016-06-01

    Context .- A histologic classification of lung cancer subtypes is essential in guiding therapeutic management. Objective .- To complement morphology-based classification of lung tumors, a previously developed lung subtyping panel (LSP) of 57 genes was tested using multiple public fresh-frozen gene-expression data sets and a prospectively collected set of formalin-fixed, paraffin-embedded lung tumor samples. Design .- The LSP gene-expression signature was evaluated in multiple lung cancer gene-expression data sets totaling 2177 patients collected from 4 platforms: Illumina RNAseq (San Diego, California), Agilent (Santa Clara, California) and Affymetrix (Santa Clara) microarrays, and quantitative reverse transcription-polymerase chain reaction. Gene centroids were calculated for each of 3 genomic-defined subtypes: adenocarcinoma, squamous cell carcinoma, and neuroendocrine, the latter of which encompassed both small cell carcinoma and carcinoid. Classification by LSP into 3 subtypes was evaluated in both fresh-frozen and formalin-fixed, paraffin-embedded tumor samples, and agreement with the original morphology-based diagnosis was determined. Results .- The LSP-based classifications demonstrated overall agreement with the original clinical diagnosis ranging from 78% (251 of 322) to 91% (492 of 538 and 869 of 951) in the fresh-frozen public data sets and 84% (65 of 77) in the formalin-fixed, paraffin-embedded data set. The LSP performance was independent of tissue-preservation method and gene-expression platform. Secondary, blinded pathology review of formalin-fixed, paraffin-embedded samples demonstrated concordance of 82% (63 of 77) with the original morphology diagnosis. Conclusions .- The LSP gene-expression signature is a reproducible and objective method for classifying lung tumors and demonstrates good concordance with morphology-based classification across multiple data sets. The LSP panel can supplement morphologic assessment of lung cancers, particularly

  12. Applying a Real-Time PCR Assay for Histoplasma capsulatum to Clinically Relevant Formalin-Fixed Paraffin-Embedded Human Tissue

    PubMed Central

    Koepsell, Scott A.; Hinrichs, Steven H.

    2012-01-01

    A real-time PCR assay to detect Histoplasma capsulatum in formalin-fixed, paraffin-embedded (FFPE) tissue is described. The assay had an analytical sensitivity of 6 pg/μl of fungal DNA, analytical specificity of 100%, and clinical sensitivity of 88.9%. This proof-of-concept study may aid in the diagnosis of histoplasmosis from FFPE tissue. PMID:22855519

  13. 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.

  14. 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.

  15. Evaluation of a panel of antibodies for the immunohistochemical identification of immune cells in paraffin-embedded lymphoid tissues of new- and old-world camelids.

    PubMed

    Uhde, Ann-Kathrin; Lehmbecker, Annika; Baumgärtner, Wolfgang; Spitzbarth, Ingo

    2017-02-01

    Different species of camelids play an important role in the epidemiology of various emerging infectious diseases such as Middle East respiratory syndrome. For precise investigations of the immunopathogenesis in these host species, appropriate immunohistochemical markers are highly needed in order to phenotype distinct immune cells populations in camelids. So far, specific immunohistochemical markers for camelid immune cells are rarely commercially available, and cross-reactivity studies are restricted to the use of frozen dromedary tissues. To bridge this gap, 14 commercially available primary antibodies were tested for their suitability to demonstrate immune cell populations on formalin fixed paraffin-embedded (FFPE) tissue sections of dromedaries, Bactrian camels, llamas, and alpacas in the present study. Out of these, 9 antibodies directed against CD3, CD20, CD79α, HLA-DR, Iba-1, myeloid/histiocyte antigen, CD204, CD208, and CD68 antigen exhibited distinct immunoreaction patterns to certain camelid immune cell subsets. The distribution of these antigens was comparatively evaluated in different anatomical compartments of thymus, spleen, mesenteric, and tracheobronchial lymph nodes. The presented results will provide a basis for further investigations in camelids, especially with respect to the role of the immune response in certain infectious diseases, which harbor a considerable risk to spill over to other species. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. Gene Expression Analysis of Immunostained Endothelial Cells Isolated from Formaldehyde-fixated Paraffin Embedded Tumors Using Laser Capture Microdissection – a Technical Report

    PubMed Central

    Kaneko, Tomoatsu; Okiji, Takashi; Kaneko, Reika; Suda, Hideaki; Nör, Jacques E.

    2009-01-01

    Laser capture microdissection (LCM) allows microscopic procurement of specific cell types from tissue sections that can then be used for gene expression analysis. In conventional LCM, frozen tissues stained with hematoxylin are normally used to the molecular analysis. Recent studies suggested that it is possible to carry out gene expression analysis of formaldehyde-fixated paraffin embedded (FFPE) tissues that were stained with hematoxylin. However, it is still unclear if quantitative gene expression analyses can be performed from LCM cells from FFPE tissues that were subjected to immunostaining to enhance identification of target cells. In this proof-of-principle study, we analyzed by RT-PCR and real time PCR the expression of genes in factor VIII immunostained human endothelial cells that were dissected from FFPE tissues by LCM. We observed that immunostaining should be performed at 4°C to preserve the mRNA from the cells. The expression of Bcl-2 in the endothelial cells was evaluated by RT-PCR and by real time PCR. GAPDH and 18S were used as house keeping genes for RT-PCR and real time PCR, respectively. This report unveils a method for quantitative gene expression analysis in cells that were identified by immunostaining and retrieved by LCM from FFPE tissues. This method is ideally suited for the analysis of relatively rare cell types within a tissue, and should improve on our ability to perform differential diagnosis of pathologies as compared to conventional LCM. PMID:19425073

  17. Gene expression analysis of immunostained endothelial cells isolated from formaldehyde-fixated paraffin embedded tumors using laser capture microdissection--a technical report.

    PubMed

    Kaneko, Tomoatsu; Okiji, Takashi; Kaneko, Reika; Suda, Hideaki; Nör, Jacques E

    2009-12-01

    Laser capture microdissection (LCM) allows microscopic procurement of specific cell types from tissue sections that can then be used for gene expression analysis. In conventional LCM, frozen tissues stained with hematoxylin are normally used to the molecular analysis. Recent studies suggested that it is possible to carry out gene expression analysis of formaldehyde-fixated paraffin embedded (FFPE) tissues that were stained with hematoxylin. However, it is still unclear if quantitative gene expression analyses can be performed from LCM cells from FFPE tissues that were subjected to immunostaining to enhance identification of target cells. In this proof-of-principle study, we analyzed by reverse transcription-PCR (RT-PCR) and real time PCR the expression of genes in factor VIII immunostained human endothelial cells that were dissected from FFPE tissues by LCM. We observed that immunostaining should be performed at 4 degrees C to preserve the mRNA from the cells. The expression of Bcl-2 in the endothelial cells was evaluated by RT-PCR and by real time PCR. Glyceraldehyde-3-phosphate dehydrogenase and 18S were used as house keeping genes for RT-PCR and real time PCR, respectively. This report unveils a method for quantitative gene expression analysis in cells that were identified by immunostaining and retrieved by LCM from FFPE tissues. This method is ideally suited for the analysis of relatively rare cell types within a tissue, and should improve on our ability to perform differential diagnosis of pathologies as compared to conventional LCM.

  18. In vitro amplification of hepatitis B virus sequences from liver tumour DNA and from paraffin wax embedded tissues using the polymerase chain reaction.

    PubMed Central

    Lo, Y M; Mehal, W Z; Fleming, K A

    1989-01-01

    A 185 base pair fragment from the core-polymerase overlap region of the hepatitis B virus (HBV) genome was amplified using the polymerase chain reaction (PCR). The results were compared with those of Southern blotting on extracted DNA from eight hepatocellular carcinomata. The data agreed with those of Southern blotting in six cases (two positive, four negative) but in two other positive cases PCR failed to amplify HBV sequences. This suggests deletion or mutation, or both, of this viral region in these cases. PCR was also used to amplify HBV sequences from formalin fixed, paraffin wax embedded tissue. Tissue inhibition of PCR occurred which increased with the number of tissue sections. It was present in tissues from different organs and species and fixed by different procedures, thus highlighting the need for a positive control during amplification. Use of formalin fixed Alexander cells, however, showed a sensitivity of one viral copy per 5000 cells. Confirmation of the identity of the PCR products was carried out using PCR-generated biotinylated probes, and suggested the insertion of extra nucleotide sequences or infection with an HBV variant in one case. Images Fig 1 Fig 2 Fig 3 Fig 4 Fig 5 PMID:2549102

  19. 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.

  20. MALDI Imaging Mass Spectrometry Profiling of N-Glycans in Formalin-Fixed Paraffin Embedded Clinical Tissue Blocks and Tissue Microarrays

    PubMed Central

    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. PMID:25184632

  1. Vinculin and cellular retinol-binding protein-1 are markers for quiescent and activated hepatic stellate cells in formalin-fixed paraffin embedded human liver.

    PubMed

    Van Rossen, Elke; Vander Borght, Sara; van Grunsven, Leo Adrianus; Reynaert, Hendrik; Bruggeman, Veerle; Blomhoff, Rune; Roskams, Tania; Geerts, Albert

    2009-03-01

    Hepatic stellate cells (HSCs) have important roles in the pathogenesis of liver fibrosis and cirrhosis. As response to chronic injury HSCs are activated and change from quiescent into myofibroblast-like cells. Several HSC-specific markers have been described in rat or mouse models. The aim of our work was to identify the best marker(s) for human HSCs. To this end we used the automated high throughput NexES IHC staining device (Ventana Medical Systems) to incubate sections under standardized conditions. Formalin fixed paraffin embedded (FFPE) normal and diseased human livers were studied. With immunohistochemistry we examined the expression of synemin, desmin, vimentin, vinculin, neurotrophin-3 (NT-3), alpha-smooth muscle actin (alpha-SMA), cellular retinol-binding protein-1 (CRBP-1), glial fibrillary acidic protein (GFAP), cysteine- and glycine-rich protein 2 (CRP2), and cytoglobin/stellate cell activation-associated protein (cygb/STAP). This is the first study in which a series of HSC markers is compared on serial FFPE human tissues. CRBP-1 clearly stains lobular HSCs without reacting with smooth muscle cells (SMCs) and shows variable cholangiocyte positivity. Vinculin has a similar staining pattern as CRBP-1 but additionally stains SMCs, and (myo)fibroblasts. In conclusion, we therefore propose to use CRBP-1 and/or vinculin to stain HSCs in human liver tissues.

  2. Glycan profiling using formalin-fixed, paraffin-embedded tissues: Hippeastrum hybrid lectin is a sensitive biomarker for squamous cell carcinoma of the uterine cervix.

    PubMed

    Tozawa-Ono, Akiko; Kubota, Manabu; Honma, Chika; Nakagawa, Yuko; Yokomichi, Noriyuki; Yoshioka, Norihito; Tsuda, Chiharu; Ohara, Tatsuru; Koizumi, Hirotaka; Suzuki, Nao

    2017-08-01

    Glycosylation of proteins is altered in cancer cells and distinctive glycan structures are associated with specific cancers, but little is known about the complete glycan profile of particular tumors. In this study, glycomic analysis of squamous cell carcinoma (SCC) of the uterine cervix was performed to search for useful markers. A lectin microarray containing 45 lectins with different binding preferences that covered N- and O-linked glycans was coupled with evanescent field-activated fluorescent detection for glycomic analysis of SCC and normal squamous epithelium (NSE) of the cervix. Formalin-fixed, paraffin-embedded tissue specimens were obtained from 16 patients with uterine cervical cancer. Sections that included both tumor and non-tumor tissues were examined to detect alterations of glycans based on the lectin-binding pattern. Hippeastrum hybrid lectin was found to be a sensitive marker for distinguishing SCC of the cervix from NSE. It was the best lectin for discriminating SCC from other tissues according to receiver-operator curve analysis, as it showed a high sensitivity (81.8%), a high specificity (70.1%), and a large area under the curve (0.8182). Histochemistry confirmed specific cytoplasmic staining of SCC cells by Hippeastrum hybrid lectin, while there was little staining of cervical intraepithelial neoplasia and no staining of NSE. The present lectin microarray technique could be applied for tissue-based glycomic analysis of various tumors and for discovery of glycan-related biomarkers. © 2017 Japan Society of Obstetrics and Gynecology.

  3. N-Glycan matrix-assisted laser desorption/ionization mass spectrometry imaging protocol for formalin-fixed paraffin-embedded tissues.

    PubMed

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

    2017-05-30

    Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) of the proteome of a tissue has been an established technique for the past decade. In the last few years, MALDI-MSI of the N-glycome has emerged as a novel MALDI-MSI technique. To assess the accuracy and clinical significance of the N-linked glycan spatial distribution, we have developed a method that utilises MALDI-MSI followed by liquid chromatography coupled to tandem mass spectrometry (LC/MS/MS) in order to assign glycan structures to the differentiating MALDI-MSI glycan masses released from the tissue glycoproteins. Our workflow presents a comprehensive list of instructions on how to (i) apply MALDI-MSI to spatially map the N-glycome across formalin-fixed paraffin-embedded (FFPE) clinical samples, (ii) structurally characterise N-glycans extracted from consecutive FFPE tissue sections by LC/MS/MS, and (iii) match relevant N-glycan masses from MALDI-MSI with confirmed N-glycan structures determined by LC/MS/MS. Our protocol provides groups that are new to this technique with instructions how to establish N-glycan MALDI-MSI in their laboratory. Furthermore, the method assigns N-glycan structural detail to the masses obtained in the MALDI-MS image. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

  4. A modification of the chronic tartrate silver impregnation technique for block impregnation of the central nervous system and paraffin wax embedding.

    PubMed

    Abadía-Fenoll, F; Ostos Garrido, M V; López-Lendínez, J L; Carmona Martos, R

    1985-03-01

    This paper describes a block silver impregnation technique for the CNS. The procedure, which is quite simple, yields highly consistent and reproducible results. After fixation during 6-10 days in 10% saline formaldehyde, 4 mm thick blocks of brain are treated with chromic anhydride and sodium potassium tartrate solution for 4 days. After this period the specimens are rinsed in 0.75% silver nitrate solution to which 8-10 drops of pyridine per 100 ml of solution have been added. This is followed by impregnation for 4 days at 37 degrees C in silver nitrate-pyridine solution identical to that used in the previous rinsing step. The impregnated blocks are reduced during 20-26 h in 1% pyrogallol to which 6 ml commercial formaldehyde per 100 ml of solution have been added, followed by dehydration in dioxan and paraffin embedding. Sections no thicker than 30 microns are then cut for histological study. This fundamentally neurofibrillar method reveals: neuronal somata and their processes; synaptic structures; fibre bundles; and cell nuclei and nucleoli.

  5. 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.

  6. Quantitative PCR for HTLV-1 provirus in adult T-cell leukemia/lymphoma using paraffin tumor sections.

    PubMed

    Kato, Junki; Masaki, Ayako; Fujii, Keiichiro; Takino, Hisashi; Murase, Takayuki; Yonekura, Kentaro; Utsunomiya, Atae; Ishida, Takashi; Iida, Shinsuke; Inagaki, Hiroshi

    2016-11-01

    Detection of HTLV-1 provirus using paraffin tumor sections may assist the diagnosis of adult T-cell leukemia/lymphoma (ATLL). For the detection, non-quantitative PCR assay has been reported, but its usefulness and limitations remain unclear. To our knowledge, quantitative PCR assay using paraffin tumor sections has not been reported. Using paraffin sections from ATLLs and non-ATLL T-cell lymphomas, we first performed non-quantitative PCR for HTLV-1 provirus. Next, we determined tumor ratios and carried out quantitative PCR to obtain provirus copy numbers. The results were analyzed with a simple regression model and a novel criterion, cut-off using 95 % rejection limits. Our quantitative PCR assay showed an excellent association between tumor ratios and the copy numbers (r = 0.89, P < 0.0001). The 95 % rejection limits provided a statistical basis for the range for the determination of HTLV-1 involvement. Its application suggested that results of non-quantitative PCR assay should be interpreted very carefully and that our quantitative PCR assay is useful to estimate the status of HTLV-1 involvement in the tumor cases. In conclusion, our quantitative PCR assay using paraffin tumor sections may be useful for the screening of ATLL cases, especially in HTLV-1 non-endemic areas where easy access to serological testing for HTLV-1 infection is limited. © 2016 Japanese Society of Pathology and John Wiley & Sons Australia, Ltd.

  7. Metastasis Detection in Sentinel Lymph Nodes: Comparison of a Limited Widely Spaced (NSABP protocol B-32) and a Comprehensive Narrowly Spaced Paraffin Block Sectioning Strategy

    PubMed Central

    Weaver, Donald L.; Le, U. Phuong; Dupuis, Stacey L.; Weaver, Katherine A. E.; Harlow, Seth P.; Ashikaga, Takamaru; Krag, David N.

    2009-01-01

    The NSABP B-32 trial is examining whether patients with initially negative sentinel lymph nodes (SLNs) who have occult metastases detected on deeper levels and cytokeratin immunohistochemistry (CK-IHC) stains are at risk for regional or distant metastases. The experimental B-32 protocol was designed to detect metastases larger than 1.0 mm by examining sections approximately 0.5 and 1.0 mm deeper into the paraffin blocks (2 levels; wide spacing). This pilot quality assurance study compares detection rates to a comprehensive protocol designed to detect metastases larger than 0.2 mm (multilevel; narrow spacing). All SLNs were sectioned grossly at close to 2.0 mm and all sections embedded in paraffin blocks. For clinical treatment, a single H&E section was examined from each block. For 54 cases with 1–5 SLNs and all SLNs negative, additional CK-IHC sections were evaluated every 0.18 mm through the block until no tissue remained. 20 of 176 (11.4%) blocks harbored occult metastases; the B-32 protocol detected metastases in 11 blocks (6.3%) and 9 additional blocks (5.1%) with metastases were detected on sections that would not have been evaluated (p=0.002; correlated proportions). Median number of levels examined per block on the comprehensive protocol was 11 (range 3–26); the B-32 protocol was fixed at 2 levels (median 2; range 1–2). Median thickness of node sections in the block was 2.1 mm (range 0.7–4.8 mm) and the modal thickness was 2.3 mm. Although more comprehensive sectioning of SLNs detects additional micrometastases, the data suggest diminishing returns and reduced cost effectiveness for the comprehensive strategy. PMID:19730364

  8. [Quantitative polarization microscopy demonstration of collagen type I and type III in histologic paraffin sections].

    PubMed

    Ogbuihi, S; Müller, Z; Zink, P

    1988-01-01

    The industrial dye Solophenyl Red 3 BL (Ciba-Geigy) dissolved in a saturated aquaeous solution of picric acid has proved suitable for differentiating between collagen types I and III in histological sections. When examined under polarization microscopy, type I fibers are radiant orange while type III fibers are green. Using 5 micron paraffin sections, an optimal staining procedure was determined: sections were first stained with Resorcin Fuchsin for elastic fibers and with Celestin Blue/Mayer's Hematoxylin for nuclear structures. The staining was then completed with 0.1 g Solophenyl Red/100 ml saturated aqueous solution of picric acid for 60 min at a pH value of 1.25. It was shown that the dye stained collagen selectively. With the aid of a photomultiplier, the spectral distribution of a series of lung sections adequately stained according to the optimized procedure was carried out using a monochromator and an interference filter, respectively. Both methods yielded identical peaks at 590 nm for the orange colored light of collagen type I and 490 nm for the green light of collagen type III. Application of appropriate filters permitted the intensity of the orange and green light at 590 nm and 490 nm to be measured. Long postmortem intervals did not affect the measured values. Quantitative inferences on the ratio of collagen I to collagen III could then be deduced from the ratio of the intensity of orange to green light. This index I/III is often applied in the diagnosis of discrete fibrotic changes in various organs.

  9. Identification of transcriptionally active HPV infection in formalin-fixed, paraffin-embedded biopsies of oropharyngeal carcinoma.

    PubMed

    Morbini, Patrizia; Alberizzi, Paola; Tinelli, Carmine; Paglino, Chiara; Bertino, Giulia; Comoli, Patrizia; Pedrazzoli, Paolo; Benazzo, Marco

    2015-05-01

    Human papillomavirus (HPV) oncogenic activity is the result of viral oncogene E6 and E7 expression in infected cells. Oncogene expression analysis is, however, not part of the routine diagnostic evaluation of HPV-associated oropharyngeal squamous cell carcinoma (OPSCC) since it requires fresh tumor tissue. We compared the diagnostic accuracy of several methods commonly employed for HPV characterization in OPSCC with the results of the newly available HPV E6/E7 mRNA in situ hybridization (ISH) on formalin-fixed, paraffin-embedded biopsy samples, in order to establish if the latter should be introduced in the diagnostic routine to increase accuracy when fresh tissue is not available. p16 immunostain, DNA ISH for high-risk HPV genotypes, SPF LiPA amplification and genotyping, and HPV16 E6 amplification were performed on 41 consecutive OPSCC samples. Twenty (48.7%) cases were positive by mRNA ISH; sensitivity and specificity were 100% and 90% for p16, 90% and 100% for DNA ISH, 70% and 76% for SPF10 LiPA, 90% and 76% for E6 amplification. A diagnostic algorithm considering p16 immunostain as first step followed by either high-risk HPV DNA ISH or HPV16 E6 amplification in p16-positive cases correctly characterized 90% of mRNA-positive and all mRNA-negative cases; combining the 3 tests correctly identified all cases. While no stand-alone test was sufficiently accurate for classifying HPV-associated OPSCC, the high sensitivity and specificity of the established combination of p16 immunostain, DNA ISH, and HPV16 DNA amplification suggests that the introduction of labour- and cost-intensive mRNA ISH, is not necessary in the diagnostic routine of oropharyngeal tumors. Copyright © 2015 Elsevier Inc. All rights reserved.

  10. Performance of the linear array HPV genotyping test on paired cytological and formalin-fixed, paraffin-embedded cervical samples.

    PubMed

    Donà, Maria Gabriella; Ronchetti, Livia; Giuliani, Massimo; Carosi, Mariantonia; Rollo, Francesca; Congiu, Mario; Mazza, Domenica; Pescarmona, Edoardo; Vocaturo, Amina; Benevolo, Maria

    2013-05-01

    Detection and genotyping of human papillomavirus (HPV) from formalin-fixed, paraffin-embedded (FFPE) samples may be difficult when using assays based on amplification of large fragments. The objective of the present study was to investigate the performance of the Linear Array HPV Genotyping Test (Linear Array) on FFPE cervical cone biopsy specimens using paired cytologic samples obtained immediately before the conization as a criterion standard. Thirty-nine samples of grade 2 or higher cervical intraepithelial neoplasia were selected; all of the corresponding cytological samples were positive by the Linear Array and had a report of atypical squamous cells of undetermined significance or worse. A valid Linear Array test result was obtained for 38 FFPE specimens (97.4%, 95% CI 88.0 to 99.9). Specifically, 34 were HPV-positive (89.5%, 95% CI 76.5 to 96.9) and 4 were HPV-negative (10.5%, 95% CI 3.4 to 23.5). The overall agreement of the results obtained for the cytologic and histologic paired samples was good (Cohen's κ = 0.85, SE = 0.082, P = 0.000). Further analysis of samples with negative or invalid Linear Array test results, both modifying the nucleic acids extraction protocol and using the INNO-LiPA assay, suggested that failure of the Linear Array test in HPV detection from tissues was probably due to DNA fragmentation. Parallel analysis of paired FFPE and cytologic samples is extremely useful for evaluation of the efficiency of PCR-based assays in HPV detection and genotyping from tissue samples. In the present study, false-negative results were obtained in a limited percentage of cases, our data depicting the successful performance of the Linear Array test on FFPE samples.

  11. 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.

  12. Single-strand DNA library preparation improves sequencing of formalin-fixed and paraffin-embedded (FFPE) cancer DNA

    PubMed Central

    Stiller, Mathias; Sucker, Antje; Griewank, Klaus; Aust, Daniela; Baretton, Gustavo Bruno; Schadendorf, Dirk; Horn, Susanne

    2016-01-01

    DNA derived from formalin-fixed and paraffin-embedded (FFPE) tissue has been a challenge to large-scale genomic sequencing, due to its low quality and quantities. Improved techniques enabling the genome-wide analysis of FFPE material would be of great value, both from a research and clinical perspective. Comparing a single-strand DNA library preparation method originally developed for ancient DNA to conventional protocols using double-stranded DNA derived from FFPE material we obtain on average 900-fold more library molecules and improved sequence complexity from as little as 5 ng input DNA. FFPE DNA is highly fragmented, usually below 100bp, and up to 60% of reads start after or end prior to adenine residues, suggesting that crosslinks predominate at adenine residues. Similar to ancient DNA, C > T substitutions are slightly increased with maximum rates up to 3% at the ends of molecules. In whole exome sequencing of single-strand libraries from lung, breast, colorectal, prostate and skin cancers we identify known cancer mutations. In summary, we show that single-strand library preparation enables genomic sequencing, even from low amounts of degraded FFPE DNA. This method provides a clear advantage both in research and clinical settings, where FFPE material (e.g. from biopsies) often is the only source of DNA available. Improving the genetic characterization that can be performed on conventional archived FFPE tissue, the single-strand library preparation allows scarce samples to be used in personalized medicine and enables larger sample sizes in future sequencing studies. PMID:27463017

  13. Editor's Highlight: Dose-Response Analysis of RNA-Seq Profiles in Archival Formalin-Fixed Paraffin-Embedded Samples.

    PubMed

    Hester, Susan D; Bhat, Virunya; Chorley, Brian N; Carswell, Gleta; Jones, Wendell; Wehmas, Leah C; Wood, Charles E

    2016-12-01

    Use of archival resources has been limited to date by inconsistent methods for genomic profiling of degraded RNA from formalin-fixed paraffin-embedded (FFPE) samples. RNA-sequencing offers a promising way to address this problem. Here, we evaluated transcriptomic dose responses using RNA-sequencing in paired FFPE and frozen (FROZ) samples from 2 archival studies in mice, one <2 years old and the other >20 years old. Experimental treatments included 3 different doses of di(2-ethylhexyl)phthalate or dichloroacetic acid for the recently archived and older studies, respectively. Total RNA was ribo-depleted and sequenced using the Illumina HiSeq platform. In the recently archived study, FFPE samples had 35% lower total counts compared to FROZ samples but high concordance in fold-change values of differentially expressed genes (DEGs) (r(2)  =( )0.99), highly enriched pathways (90% overlap with FROZ), and benchmark dose estimates for preselected target genes (<5% difference vs FROZ). In contrast, older FFPE samples had markedly lower total counts (3% of FROZ) and poor concordance in global DEGs and pathways. However, counts from FFPE and FROZ samples still positively correlated (r(2 ) = 0.84 across all transcripts) and showed comparable dose responses for more highly expressed target genes. These findings highlight potential applications and issues in using RNA-sequencing data from FFPE samples. Recently archived FFPE samples were highly similar to FROZ samples in sequencing quality metrics, DEG profiles, and dose-response parameters, while further methods development is needed for older lower-quality FFPE samples. This work should help advance the use of archival resources in chemical safety and translational science.

  14. MAGE-3 immunoreactivity in formalin-fixed, paraffin-embedded primary and metastatic melanoma: frequency and distribution.

    PubMed Central

    Hofbauer, G. F.; Schaefer, C.; Noppen, C.; Böni, R.; Kamarashev, J.; Nestle, F. O.; Spagnoli, G. C.; Dummer, R.

    1997-01-01

    Monoclonal antibody 57B specifically detects MAGE-3 gene protein expression. MAGE-derived peptides are recognized by CD8+ T cells and applied in immunotherapy. We examined formalin-fixed, paraffin-embedded tissue of 61 melanoma (primary, n = 40; metastatic, n = 21) and 46 control cases (junctional, dermal, compound, Spitz, Reed, and balloon-cell nevi) by immunohistochemistry using the alkaline phosphatase anti-alkaline phosphatase method after antigen retrieval. Immunoreactivity was rated positive at 20 positive cells per tumor or more. Staining pattern was homogeneous, scattered, or focal. All control samples and internal controls were immunonegative. Staining with monoclonal antibody 57B showed a specificity of 100% with a sensitivity of 44%. Immunopositivity (overall, 44% of melanomas) increased along with tumor, node, and metastasis stage; pT1 showed 13%, pT2 22%, pT3a 29%, pT3b 45%, pT4 100%, pTxN1 60%, and pTxNxM1a 63% of samples positive. The staining pattern was homogeneous on pT1 to pT3a tumors, homogeneous or focal in pT3b and pT4a, and homogeneous, focal, or scattered in pTxN1 and pTxNxM1a. The frequency of immunopositivity relates well to data on mRNA expression using reverse transcriptase polymerase chain reaction in a subgroup analyzed by both methods. Monoclonal antibody 57B can be used to allow profiling of melanomas using routine archival tissue, when considering immunotherapeutic approaches involving MAGE-3-derived epitopes. Images Figure 1 Figure 2 Figure 3 PMID:9403705

  15. Elevated Pressure Improves the Extraction and Identification of Proteins Recovered from Formalin-Fixed, Paraffin-Embedded Tissue Surrogates

    PubMed Central

    Fowler, Carol B.; Chesnick, Ingrid E.; Moore, Cedric D.; O'Leary, Timothy J.; Mason, Jeffrey T.

    2010-01-01

    Background Proteomic studies of formalin-fixed paraffin-embedded (FFPE) tissues are frustrated by the inability to extract proteins from archival tissue in a form suitable for analysis by 2-D gel electrophoresis or mass spectrometry. This inability arises from the difficulty of reversing formaldehyde-induced protein adducts and cross-links within FFPE tissues. We previously reported the use of elevated hydrostatic pressure as a method for efficient protein recovery from a hen egg-white lysozyme tissue surrogate, a model system developed to study formalin fixation and histochemical processing. Principal Findings In this study, we demonstrate the utility of elevated hydrostatic pressure as a method for efficient protein recovery from FFPE mouse liver tissue and a complex multi-protein FFPE tissue surrogate comprised of hen egg-white lysozyme, bovine carbonic anhydrase, bovine ribonuclease A, bovine serum albumin, and equine myoglobin (55∶15∶15∶10∶5 wt%). Mass spectrometry of the FFPE tissue surrogates retrieved under elevated pressure showed that both the low and high-abundance proteins were identified with sequence coverage comparable to that of the surrogate mixture prior to formaldehyde treatment. In contrast, non-pressure-extracted tissue surrogate samples yielded few positive and many false peptide identifications. Studies with soluble formalin-treated bovine ribonuclease A demonstrated that pressure modestly inhibited the rate of reversal (hydrolysis) of formaldehyde-induced protein cross-links. Dynamic light scattering studies suggest that elevated hydrostatic pressure and heat facilitate the recovery of proteins free of formaldehyde adducts and cross-links by promoting protein unfolding and hydration with a concomitant reduction in the average size of the protein aggregates. Conclusions These studies demonstrate that elevated hydrostatic pressure treatment is a promising approach for improving the recovery of proteins from FFPE tissues in a form

  16. Molecular Detection and Genotypic Characterization of Toxoplasma gondii in Paraffin-Embedded Fetoplacental Tissues of Women with Recurrent Spontaneous Abortion

    PubMed Central

    Abdoli, Amir; Dalimi, Abdolhossein; Soltanghoraee, Haleh; Ghaffarifar, Fatemeh

    2017-01-01

    Background Congenital toxoplasmosis is an important cause of spontaneous abortion worldwide. However, there is limited information on detection and genotypic characterization of Toxoplasma gondii (T. gondii) in women with recurrent spontaneous abortion (RSA). The aim of this study is the molecular detection and genotypic characterization of T. gondii in formalin-fixed, paraffin-embedded fetoplacental tissues (FFPTs) of women with RSA that have referred to the Avicenna Research Institute in Tehran, Iran. Materials and Methods This experimental research was undertaken on 210 FFPTs of women with RSA. The information of the patients was collected from the archives of Avicenna Research Institute in Tehran, Iran. After DNA extraction, the presence of T. gondii was examined by nested polymerase chain reaction targeting the GRA6 gene. Genotyping was performed on positive samples using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) that targeted the GRA6 and SAG3 genes. Sequencing was conducted on two GRA6 positive samples. Results T. gondii DNA was detected in 3.8% (8/210) of the samples. Genotyping showed that all positive samples belonged to type III of the T. gondii genotype. Sequencing two genomic DNAs of the GRA6 gene revealed 99% similarity with each other and 99-100% similarity with T. gondii sequences deposited in GenBank. There were six patients with histories of more than three abortions; one patient had a healthy girl and another patient had two previous abortions. Abortions occurred in the first trimester of pregnancy in seven patients and in the second trimester of pregnancy in one patient. Conclusion The results of this study have indicated that genotype III is the predominant type of T. gondii in women with RSA in Tehran, Iran. Also, our findings suggest that toxoplasmosis may play a role in the pathogenesis of RSA. However, further studies are needed to elucidate a clear relationship between T. gondii infection and RSA. PMID

  17. 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

  18. Analysis of biological and technical variability in gene expression assays from formalin-fixed paraffin-embedded classical Hodgkin lymphomas.

    PubMed

    Vera-Lozada, Gabriela; Scholl, Vanesa; Barros, Mário Henrique M; Sisti, Davide; Guescini, Michele; Stocchi, Vilberto; Stefanoff, Claudio Gustavo; Hassan, Rocio

    2014-12-01

    Formalin-fixed paraffin-embedded (FFPE) tissues are invaluable sources of biological material for research and diagnostic purposes. In this study, we aimed to identify biological and technical variability in RT-qPCR TaqMan® assays performed with FFPE-RNA from lymph nodes of classical Hodgkin lymphoma samples. An ANOVA-nested 6-level design was employed to evaluate BCL2, CASP3, IRF4, LYZ and STAT1 gene expression. The most variable genes were CASP3 (low expression) and LYZ (high expression). Total variability decreased after normalization for all genes, except by LYZ. Genes with moderate and low expression were identified and suffered more the effects of the technical manipulation than high-expression genes. Pre-amplification was shown to introduce significant technical variability, which was partially alleviated by lowering to a half the amount of input RNA. Ct and Cy0 quantification methods, based on cycle-threshold and the kinetic of amplification curves, respectively, were compared. Cy0 method resulted in higher quantification values, leading to the decrease of total variability in CASP3 and LYZ genes. The mean individual noise was 0.45 (0.31 to 0.61 SD), indicating a variation of gene expression over ~1.5 folds from one case to another. We showed that total variability in RT-qPCR from FFPE-RNA is not higher than that reported for fresh complex tissues, and identified gene-, and expression level-sources of biological and technical variability, which can allow better strategies for designing RT-qPCR assays from highly degraded and inhibited samples. Copyright © 2014 Elsevier Inc. All rights reserved.

  19. Proteomic Analysis of Matched Formalin-Fixed, Paraffin-Embedded Specimens in Patients with Advanced Serous Ovarian Carcinoma

    PubMed Central

    Smith, Ashlee L.; Sun, Mai; Bhargava, Rohit; Stewart, Nicolas A.; Flint, Melanie S.; Bigbee, William L.; Krivak, Thomas C.; Strange, Mary A.; Cooper, Kristine L.; Zorn, Kristin K.

    2013-01-01

    Objective: The biology of high grade serous ovarian carcinoma (HGSOC) is poorly understood. Little has been reported on intratumoral homogeneity or heterogeneity of primary HGSOC tumors and their metastases. We evaluated the global protein expression profiles of paired primary and metastatic HGSOC from formalin-fixed, paraffin-embedded (FFPE) tissue samples. Methods: After IRB approval, six patients with advanced HGSOC were identified with tumor in both ovaries at initial surgery. Laser capture microdissection (LCM) was used to extract tumor for protein digestion. Peptides were extracted and analyzed by reversed-phase liquid chromatography coupled to a linear ion trap mass spectrometer. Tandem mass spectra were searched against the UniProt human protein database. Differences in protein abundance between samples were assessed and analyzed by Ingenuity Pathway Analysis software. Immunohistochemistry (IHC) for select proteins from the original and an additional validation set of five patients was performed. Results: Unsupervised clustering of the abundance profiles placed the paired specimens adjacent to each other. IHC H-score analysis of the validation set revealed a strong correlation between paired samples for all proteins. For the similarly expressed proteins, the estimated correlation coefficients in two of three experimental samples and all validation samples were statistically significant (p < 0.05). The estimated correlation coefficients in the experimental sample proteins classified as differentially expressed were not statistically significant. Conclusion: A global proteomic screen of primary HGSOC tumors and their metastatic lesions identifies tumoral homogeneity and heterogeneity and provides preliminary insight into these protein profiles and the cellular pathways they constitute.

  20. Gene expression profiles from formalin fixed paraffin embedded breast cancer tissue are largely comparable to fresh frozen matched tissue.

    PubMed

    Mittempergher, Lorenza; de Ronde, Jorma J; Nieuwland, Marja; Kerkhoven, Ron M; Simon, Iris; Rutgers, Emiel J Th; Wessels, Lodewyk F A; Van't Veer, Laura J

    2011-02-11

    Formalin Fixed Paraffin Embedded (FFPE) samples represent a valuable resource for cancer research. However, the discovery and development of new cancer biomarkers often requires fresh frozen (FF) samples. Recently, the Whole Genome (WG) DASL (cDNA-mediated Annealing, Selection, extension and Ligation) assay was specifically developed to profile FFPE tissue. However, a thorough comparison of data generated from FFPE RNA and Fresh Frozen (FF) RNA using this platform is lacking. To this end we profiled, in duplicate, 20 FFPE tissues and 20 matched FF tissues and evaluated the concordance of the DASL results from FFPE and matched FF material. We show that after proper normalization, all FFPE and FF pairs exhibit a high level of similarity (Pearson correlation >0.7), significantly larger than the similarity between non-paired samples. Interestingly, the probes showing the highest correlation had a higher percentage G/C content and were enriched for cell cycle genes. Predictions of gene expression signatures developed on frozen material (Intrinsic subtype, Genomic Grade Index, 70 gene signature) showed a high level of concordance between FFPE and FF matched pairs. Interestingly, predictions based on a 60 gene DASL list (best match with the 70 gene signature) showed very high concordance with the MammaPrint® results. We demonstrate that data generated from FFPE material with the DASL assay, if properly processed, are comparable to data extracted from the FF counterpart. Specifically, gene expression profiles for a known set of prognostic genes for a specific disease are highly comparable between two conditions. This opens up the possibility of using both FFPE and FF material in gene expressions analyses, leading to a vast increase in the potential resources available for cancer research.

  1. Molecular markers associated with nonepithelial ovarian cancer in formalin-fixed, paraffin-embedded specimens by genome wide expression profiling.

    PubMed

    Vui-Kee, Koon; Mohd Dali, Ahmad Zailani Hatta; Mohamed Rose, Isa; Ghazali, Razmin; Jamal, Rahman; Mokhtar, Norfilza Mohd

    2012-05-01

    Nonepithelial ovarian cancer (NEOC) is a rare cancer that is often misdiagnosed as other malignant tumors. Research on this cancer using fresh tissues is nearly impossible because of its limited number of samples within a limited time provided. The study is to identify potential genes and their molecular pathways related to NEOC using formalin-fixed paraffin embedded samples. Total RNA was extracted from eight archived NEOCs and seven normal ovaries. The RNA samples with RNA integrity number >2.0, purity >1.7 and cycle count value <28 cycles were hybridized to the Illumina Whole-Genome DASL assay (cDNA-mediated annealing, selection, extension, and ligation). We analyzed the results using the GeneSpring GX11.0 and FlexArray software to determine the differentially expressed genes. Microarray results were validated using an immunohistochemistry method. Statistical analysis identified 804 differentially expressed genes with 443 and 361 genes as overexpressed and underexpressed in cancer, respectively. Consistent findings were documented for the overexpression of eukaryotic translation elongation factor 1 alpha 1, E2F transcription factor 2, and fibroblast growth factor receptor 3, except for the down-regulated gene, early growth response 1 (EGR1). The immunopositivity staining for EGR1 was found in the majority of cancer tissues. This finding suggested that the mRNA level of a transcript did not always match with the protein expression in tissues. The current gene profile can be the platform for further exploration of the molecular mechanism of NEOC. Copyright © 2012. Published by Elsevier B.V.

  2. Improved PCR performance using template DNA from formalin-fixed and paraffin-embedded tissues by overcoming PCR inhibition.

    PubMed

    Dietrich, Dimo; Uhl, Barbara; Sailer, Verena; Holmes, Emily Eva; Jung, Maria; Meller, Sebastian; Kristiansen, Glen

    2013-01-01

    Formalin-fixed and paraffin-embedded (FFPE) tissues represent a valuable source for biomarker studies and clinical routine diagnostics. However, they suffer from degradation of nucleic acids due to the fixation process. Since genetic and epigenetic studies usually require PCR amplification, this degradation hampers its use significantly, impairing PCR robustness or necessitating short amplicons. In routine laboratory medicine a highly robust PCR performance is mandatory for the clinical utility of genetic and epigenetic biomarkers. Therefore, methods to improve PCR performance using DNA from FFPE tissue are highly desired and of wider interest. The effect of template DNA derived from FFPE tissues on PCR performance was investigated by means of qPCR and conventional PCR using PCR fragments of different sizes. DNA fragmentation was analyzed via agarose gel electrophoresis. This study showed that poor PCR amplification was partly caused by inhibition of the DNA polymerase by fragmented DNA from FFPE tissue and not only due to the absence of intact template molecules of sufficient integrity. This PCR inhibition was successfully minimized by increasing the polymerase concentration, dNTP concentration and PCR elongation time thereby allowing for the robust amplification of larger amplicons. This was shown for genomic template DNA as well as for bisulfite-converted template DNA required for DNA methylation analyses. In conclusion, PCR using DNA from FFPE tissue suffers from inhibition which can be alleviated by adaptation of the PCR conditions, therefore allowing for a significant improvement of PCR performance with regard to variability and the generation of larger amplicons. The presented solutions to overcome this PCR inhibition are of tremendous value for clinical chemistry and laboratory medicine.

  3. Aberrant expression of Notch1, HES1, and DTX1 genes in glioblastoma formalin-fixed paraffin-embedded tissues.

    PubMed

    Narayanappa, Rajeswari; Rout, Pritilata; Aithal, Madhuri G S; Chand, Ashis Kumar

    2016-05-01

    Glioblastoma is the most common malignant brain tumor accounting for more than 54 % of all gliomas. Despite aggressive treatments, median survival remains less than 1 year. This might be due to the unavailability of effective molecular diagnostic markers and targeted therapy. Thus, it is essential to discover molecular mechanisms underlying disease by identifying dysregulated pathways involved in tumorigenesis. Notch signaling is one such pathway which plays an important role in determining cell fates. Since it is found to play a critical role in many cancers, we investigated the role of Notch genes in glioblastoma with an aim to identify biomarkers that can improve diagnosis. Using real-time PCR, we assessed the expression of Notch genes including receptors (Notch1, Notch2, Notch3, and Notch4), ligands (JAG1, JAG2, and DLL3), downstream targets (HES1 and HEY2), regulator Deltex1 (DTX1), inhibitor NUMB along with transcriptional co-activator MAML1, and a component of gamma-secretase complex APH1A in 15 formalin-fixed paraffin-embedded (FFPE) patient samples. Relative quantification was done by the 2(-ΔΔCt) method; the data are presented as fold change in gene expression normalized to an internal control gene and relative to the calibrator. The data revealed aberrant expression of Notch genes in glioblastoma compared to normal brain. More than 85 % of samples showed high Notch1 (P = 0.0397) gene expression and low HES1 (P = 0.011) and DTX1 (P = 0.0001) gene expression. Our results clearly show aberrant expression of Notch genes in glioblastoma which can be used as putative biomarkers together with histopathological observation to improve diagnosis, therapeutic strategies, and patient prognosis.

  4. Molecular Detection and Typing of Human Papillomaviruses in Paraffin-Embedded Cervical Cancer and Pre-Cancer Tissue Specimens

    PubMed Central

    Mahmoodi, Pezhman; Motamedi, Hossein; Seyfi Abad Shapouri, Masoud Reza; Bahrami Shehni, Mahjabin; Kargar, Mohammad

    2016-01-01

    Background: Cervical cancer is one of the important reasons of mortality among females. Prevention, early diagnosis and immediate treatment can affect the rate of mortality in this cancer and several epidemiological studies have shown a strong relationship between human papilloma viruses (HPVs) and cervical cancer. Objectives: The present study was conducted to survey HPV infections in a women population with cervical cancer and cervical dysplasia/metaplasia in southwest of Iran. Materials and Methods: 72 paraffin-embedded cervical biopsies which had been previously archived from women with cervical cancer and cervical dysplasia were examined by polymerase chain reaction (PCR). Afterward, the detected HPV strains were typed by restriction fragment length polymorphism (RFLP) analysis of PCR amplicons. Results: 60 out of 72 samples had necessary requirements and HPV DNA was detected in 43.3% of these samples. Most HPV positive samples belonged to women aged from 48 to 63 years. On the other hand, HPV infection among patients with squamous cell carcinoma (SCC) was 48.78% and in women with dysplasia/metaplasia was 26.66%. The most prevalent type of the human papilloma virus was HPV16 (100%). Conclusions: Knowing the most prevalent type of the human papilloma viruses circulating in the population (HPV16) can be applied in the future screening and managing programs of this major disease and also in vaccination against the prevalent types of the virus. Meanwhile, it seems that more studies should be performed to determine the role of different risk factors involved in development of the disease, especially those related with social behaviors and traditions with respect to different areas. PMID:27366309

  5. New Molecular Assay for the Proliferation Signature in Mantle Cell Lymphoma Applicable to Formalin-Fixed Paraffin-Embedded Biopsies.

    PubMed

    Scott, David W; Abrisqueta, Pau; Wright, George W; Slack, Graham W; Mottok, Anja; Villa, Diego; Jares, Pedro; Rauert-Wunderlich, Hilka; Royo, Cristina; Clot, Guillem; Pinyol, Magda; Boyle, Merrill; Chan, Fong Chun; Braziel, Rita M; Chan, Wing C; Weisenburger, Dennis D; Cook, James R; Greiner, Timothy C; Fu, Kai; Ott, German; Delabie, Jan; Smeland, Erlend B; Holte, Harald; Jaffe, Elaine S; Steidl, Christian; Connors, Joseph M; Gascoyne, Randy D; Rosenwald, Andreas; Staudt, Louis M; Campo, Elias; Rimsza, Lisa M

    2017-05-20

    Purpose Mantle cell lymphoma is an aggressive B-cell neoplasm that displays heterogeneous outcomes after treatment. In 2003, the Lymphoma/Leukemia Molecular Profiling Project described a powerful biomarker-the proliferation signature-using gene expression in fresh frozen material. Herein, we describe the training and validation of a new assay that measures the proliferation signature in RNA derived from routinely available formalin-fixed paraffin-embedded (FFPE) biopsies. Methods Forty-seven FFPE biopsies were used to train an assay on the NanoString platform, using microarray gene expression data of matched fresh frozen biopsies as a gold standard. The locked assay was applied to pretreatment FFPE lymph node biopsies from an independent cohort of 110 patients uniformly treated with rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone. Seventeen biopsies were tested across three laboratories to assess assay reproducibility. Results The MCL35 assay, which contained a 17-gene proliferation signature, yielded gene expression of sufficient quality to assign an assay score and risk group in 108 (98%) of 110 archival FFPE biopsies. The MCL35 assay assigned patients to high-risk (26%), standard-risk (29%), and low-risk (45%) groups, with different lengths of overall survival (OS): a median of 1.1, 2.6, and 8.6 years, respectively (log-rank for trend, P < .001). In multivariable analysis, these risk groups and the Mantle Cell Lymphoma International Prognostic Index were independently associated with OS ( P < .001 for both variables). Concordance of risk assignment across the three independent laboratories was 100%. Conclusion The newly developed and validated MCL35 assay for FFPE biopsies uses the proliferation signature to define groups of patients with significantly different OS independent of the Mantle Cell Lymphoma International Prognostic Index. Importantly, the analytic and clinical validity of this assay defines it as a reliable biomarker to

  6. Grading melanocytic dysplasia in paraffin wax embedded tissue by the nucleic acid index.

    PubMed

    Berman, D M; Wincovitch, S; Garfield, S; Romeo, M J

    2005-11-01

    Although nucleic acid derangements are the hallmark of melanocytic dysplasia, the gold standard for its diagnosis remains the microscopic evaluation of haematoxylin and eosin stained slides. However, light microscopy is subjective and crucial genomic changes do not always show as changes in histology. To introduce the nucleic acid index (NAI) as a means of analysing nucleic acid derangements in histological sections at the level of the individual cell and within the context of its microenvironment. Confocal laser scanning microscopy was performed on melanocytic lesions stained with acridine orange (AO), a fluorescent stain for DNA and RNA. The NAI, calculated by measuring the fluorescence intensities of AO in nuclei relative to the surrounding cytoplasm, reflects the concentration of DNA relative to RNA. When applied to benign naevi, dysplastic naevi, and melanoma, a very strong significant association was seen between lower NAI and malignant potential (p < 0.0001). Strong inverse correlations were found between NAI and both mitotic index and Breslow thickness. Interestingly, the NAI for dysplastic naevi is between that of melanoma and most benign naevi, consistent with their intermediate biological behaviour and histological appearance. By providing a quantitative measure for melanocytic neoplasia, the NAI may improve the diagnosis of melanocytic lesions and the selection of treatment.

  7. ImmunoAT method: An initial assessment for the detection of abnormal isoforms of prion protein in formalin-fixed and paraffin-embedded tissues.

    PubMed

    Sato, Yuko; Shimonohara, Nozomi; Hanaki, Ken-Ichi; Goto, Motoki; Yamakawa, Yoshio; Horiuchi, Motohiro; Takahashi, Hidehiro; Sata, Tetsutaro; Nakajima, Noriko

    2010-05-01

    The AT-tailing method is a labelling technique that utilises oligo(dA-dT)-dependent signal amplification. In this study, a new immunohistochemical application of the immunoAT method was developed. This method uses an oligo(dA-dT)-conjugated primary antibody (direct immunoAT method) or an oligo(dA-dT)-conjugated secondary antibody (indirect immunoAT method). Fifteen-base oligo(dA-dT)-conjugated antibodies (IgG-ATs) were prepared in advance by conjugating maleimide-activated oligo(dA-dT) to IgG via free sulfhydryl residues that had been introduced on the surface of IgG using Traut's reagent. Following the reaction with the target antigen and the IgG-AT, oligo(dA-dT) was elongated by DeltaTth DNA polymerase in the presence of dATP, dTTP and biotinylated dUTP, consequently labelling the antigen-antibody complex with a large amount of biotin. To initially evaluate the immunoAT method, the presence or absence of prion protein (PrP(sc)) was determined in formalin-fixed and paraffin-embedded sections of the medulla oblongata of cattle which had been under active surveillance for bovine spongiform encephalopathy. Sections were examined using direct and indirect immunoAT methods and the EnVision+ system (Dako) under conditions that were identical except for the differing IgG-AT and AT-tailing methods. PrP(sc) detection was consistent using all three methods. The clearest signals were obtained using the indirect immunoAT method, suggesting significant potential for this method. Copyright 2010 Elsevier B.V. All rights reserved.

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

    PubMed

    Salehi, Elham; Hedayati, Mohammad T; Zoll, Jan; Rafati, Haleh; Ghasemi, Maryam; Doroudinia, Atosa; Abastabar, Mahdi; Tolooe, Ali; Snelders, Eveline; van der Lee, Henrich A; Rijs, Antonius J M M; Verweij, Paul E; Seyedmousavi, Seyedmojtaba; Melchers, Willem J G

    2016-11-01

    In a retrospective multicenter study, 102 formalin-fixed paraffin-embedded (FFPE) tissue specimens with histopathology results were tested. Two 4- to 5-μm FFPE tissue sections from each specimen were digested with proteinase K, followed by automated nucleic acid extraction. Multiple real-time quantitative PCR (qPCR) assays targeting the internal transcribed spacer 2 (ITS2) region of ribosomal DNA, using fluorescently labeled primers, was performed to identify clinically important genera and species of Aspergillus, Fusarium, Scedosporium, and the Mucormycetes The molecular identification was correlated with results from histological examination. One of the main findings of our study was the high sensitivity of the automated DNA extraction method, which was estimated to be 94%. The qPCR procedure that was evaluated identified a range of fungal genera/species, including Aspergillus fumigatus, Aspergillus flavus, Aspergillus terreus, Aspergillus niger, Fusarium oxysporum, Fusarium solani, Scedosporium apiospermum, Rhizopus oryzae, Rhizopus microsporus, Mucor spp., and Syncephalastrum Fusarium oxysporum and F. solani DNA was amplified from five specimens from patients initially diagnosed by histopathology as having aspergillosis. Aspergillus flavus, S. apiospermum, and Syncephalastrum were detected from histopathological mucormycosis samples. In addition, examination of four samples from patients suspected of having concomitant aspergillosis and mucormycosis infections resulted in the identification of two A. flavus isolates, one Mucor isolate, and only one sample having both R. oryzae and A. flavus Our results indicate that histopathological features of molds may be easily confused in tissue sections. The qPCR assay used in this study is a reliable tool for the rapid and accurate identification of fungal pathogens to the genus and species levels directly from FFPE tissues. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  9. [Use in immunohistochemical studies of a method of restoration of antigenic specificity as affected by microwaves in tissue fixed with formalin and embedded in paraffin].

    PubMed

    Gurevich, L E; Isakov, V A

    1999-01-01

    Recovery of specific antigenic characteristics using microwave treatment of paraffin sections of tissues fixed by formalin allows to extend spectrum of antibodies for immunohistochemical diagnosis. Microwaves enable the reaction on the material prepared according to the standard technique, and macropreparations long stored in formalin and archive blocks.

  10. Droplet digital polymerase chain reaction detection of HER2 amplification in formalin fixed paraffin embedded breast and gastric carcinoma samples.

    PubMed

    Zhu, Yazhen; Lu, Dan; Lira, Maruja E; Xu, Qing; Du, Yunzhi; Xiong, Jianghong; Mao, Mao; Chung, Hyun Cheol; Zheng, Guangjuan

    2016-04-01

    Human epidermal growth factor receptor 2 (HER2) is a key driver of tumorigenesis, and over-expression as a result of HER2 gene amplification has been observed in a number of solid tumors. Recently HER2 has become an important biomarker for the monoclonal antibody treatment of HER2-positive metastatic breast and advanced gastric cancer. The HER2 targeting antibody trastuzumab treatment requires accurate measurement of HER2 levels for proper diagnosis. Droplet digital PCR (ddPCR) with highly direct, precise and absolute nucleic acid quantification could be used to detect HER2 amplification levels. Our objective was to evaluate a robust, accurate and less subjective application of ddPCR for HER2 amplification levels and test the assay performance in clinical formalin-fixed paraffin-embedded (FFPE) breast and gastric carcinoma samples. Genomic DNA from HER2 amplified cell line SK-BR-3 was used to set up the ddPCR assays. The copy number of HER2 was compared to the chromosome 17 centromere reference gene (CEP17), expressed as HER2:CEP17 ratio. Genomic DNAs of FFPE specimens from 145 Asian patients with breast and gastric carcinomas were assayed using both standard methods, immunohistochemistry (IHC) and/or fluorescence in situ hybridization (FISH), and ddPCR. Based on 145 clinical breast and gastric carcinoma cases, our study demonstrated a high concordance of ddPCR results to FISH and IHC. In breast cancer specimens, the ddPCR results had high concordance with FISH and IHC defined HER2 status with a sensitivity of 90.9% (30/33) and a specificity of 100% (77/77). In gastric cancer specimens that were concordant in both FISH and IHC, our assay was 95.5% concordant with FISH and IHC (21/22). ddPCR has the advantage of automation and also allows levels of HER2 amplification to be easily evaluated in large numbers of samples, and presents a potential option to define HER2 status. Copyright © 2015 Elsevier Inc. All rights reserved.

  11. 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

  12. Immunohistochemical detection and localization of new type gosling viral enteritis virus in paraformaldehyde-fixed paraffin-embedded tissue.

    PubMed

    Chen, Shun; Cheng, Anchun; Wang, Mingshu; Zhu, Dekang; Luo, Qihui; Liu, Fei; Chen, Xiaoyue

    2009-08-15

    To determine the distribution and localization of new type gosling viral enteritis virus (NGVEV) in paraformaldehyde-fixed paraffin-embedded tissues of experimentally infected goslings, for the first time, an immunohistochemical (IHC) staining method was reported. Anti-NGVEV polyclonal serum was obtained from the rabbits immunized with purified NGVEV antigen, which was extracted by caprylic-ammonium sulphate method and purified through High-Q columns anion exchange chromatography. Three-day-old NGVEV-free goslings were orally inoculated with NGVEV-CN strain suspension as infection group and phosphate buffered saline solution (PBS) as control group, respectively. The tissues were collected at sequential time points between 0.5 and 720h post inoculation (PI), and prepared for IHC staining and ultra-structural observation. The positive immunoreactivity could be readily detected in the lymphoid and gastrointestinal organs of infected goslings as early as 48 h PI, in the liver, kidney, pancreas and myocardium from 72 h, and in the cerebrum and cerebellum from 96 h, while it was hardly detected in the respiratory organs at any time. The positive staining reaction could be detected in NGVEV-infected goslings until 600 h PI, and no positive staining cell could be observed in the controls. The highest levels of viral antigen were found in the bursa of Fabricius (BF), thymus, proventriculus, gizzard and intestine tract, moreover, the liver, kidney, spleen, myocardium and pancreas were intensively and widely stained. The target cells had a ubiquitous distribution, especially included the epithelial cells, endothelial cells, superficial and crypt mucosal cells, glandular cells, fibrocytes, macrophages and lymphocytes, which served as the principal sites for antigen localization. The ultra-structural observation by transmission electron microscope (TEM) further indicated that NGVEV particles could be widely detected in the lymphoid and digestive organs of infected goslings from

  13. Genotyping Concordance in DNA Extracted from Formalin-Fixed Paraffin Embedded (FFPE) Breast Tumor and Whole Blood for Pharmacogenetic Analyses

    PubMed Central

    Hertz, Daniel L; Kidwell, Kelley M; Thibert, Jacklyn N; Gersch, Christina; Regan, Meredith M; Skaar, Todd C; Henry, N. Lynn; Hayes, Daniel F; Van Poznak, Catherine H; Rae, James M

    2015-01-01

    Background Cancer pharmacogenetic studies have used archival tumor samples as a DNA source when germline DNA was unavailable. Genotyping DNA extracted from formalin-fixed paraffin embedded tumors (FFPE-T) may be inaccurate compared to that from normal leukocytes due to FFPE storage, tumor genetic aberrations, and/or insufficient DNA extraction. Our objective was to assess the extent and source of genotyping inaccuracy from FFPE-T DNA and demonstrate analytical validity of FFPE-T genotyping of candidate single nucleotide polymorphisms (SNPs) for pharmacogenetic analyses. Methods SNPs relevant to cancer pharmacogenetics were genotyped by Sequenom MassARRAYs in DNA harvested from matched FFPE-T, FFPE non-cancerous lymph node (FFPE-LN), and whole blood leukocyte samples obtained from early stage breast cancer patients. No-call and discordant call rates were calculated for each tissue type (FFPE-T, FFPE-LN, blood) and each SNP. Analytical validity was defined as all SNPs with <5% discordance between FFPE-T and blood or <10% discordance plus no-calls. Results Matched samples from 114 patients were genotyped for 247 SNPs. No-call rate in FFPE-T was greater than FFPE-LN and blood (4.3% vs. 3.0% vs. 0.5%, all p<0.001). The overall rate of genotype discordance between FFPE-T and leukocytes was very low, but greater than the discordance between FFPE-LN and leukocytes (1.1% vs. 0.3%, p<0.001). Samples with heterozygous genotypes were more likely to be no- or discordantly-called in FFPE-T and FFPE-LN (p<0.001). Analytical validity of FFPE-T genotyping was demonstrated for 218 (88%) SNPs. Conclusions No- and discordant-call rates were below concerning thresholds, confirming that most SNPs can be accurately genotyped from FFPE-T on the Sequenom platform. FFPE-T is a viable DNA source for prospective-retrospective pharmacogenetic analyses of clinical trial cohorts when germline DNA is not available. PMID:26276228

  14. Energy dispersive x-ray analysis of the cornea. Application to paraffin sections of normal and diseased corneas

    SciTech Connect

    Robinson, M.R.; Streeten, B.W.

    1984-11-01

    The distribution of chemical elements in the normal human cornea was studied by energy dispersive x-ray analysis and scanning electron microscopy of routinely prepared paraffin sections. Calcium, phosphorus, and sulfur were consistently present in quantities above background and varied in concentration regionally. Analysis of fresh-frozen tissue, an approximation of the in vivo state, gave a similar elemental profile to paraffin sections, except for the loss of diffusable electrolytes in the latter. After fixation, S was the most abundant element and was highest in Descemet's membrane. Corneas with granular, lattice, macular, and Fuchs endothelial dystrophies, band keratopathy, and spheroidal degeneration were also examined. Characteristic patterns of abnormal S and Ca distribution were found in each of the dystrophies. The relative proportions of Ca, P, and S gave diagnostic profiles for distinguishing band keratopathy and spheroidal degeneration.

  15. First molecular identification of the zoonotic parasite Anisakis pegreffii (Nematoda: Anisakidae) in a paraffin-embedded granuloma taken from a case of human intestinal anisakiasis in Italy

    PubMed Central

    2011-01-01

    Background Anisakiasis is an important fish-borne zoonosis provoked by larval stages of nematodes belonging to the genus Anisakis. The detection and identification of human infections is difficult. This is due to: a) the low specificity of the clinical features and symptomatology related to human infections; b) the paucity of diagnostic features of larvae found in granulomatous lesions characteristic of "invasive anisakiasis"; and c) the lack morphological characters diagnostic at the specific level when larvae of Anisakis are detected. Thus, molecular-based diagnostic approaches are warranted. Method We have developed a PCR method that amplifies the DNA of Anisakis spp. in fixed paraffin-embedded tissues. This method was applied to a granuloma removed from a human case of intestinal anisakiasis in Italy. Specific primers of the mtDNA cox2 gene were used and sequence analysis was performed according to the procedures already established for species of Anisakis. Results The sequence obtained (629 bp) was compared with those of the other species of Anisakis which have so far been genetically characterized and with sequences obtained from larval stages of Anisakis collected from the Mediterranean fish Engraulis encrasicolus. This enabled the genetic identification of the larva in the human tissue as A. pegreffii. This is the first instance of human intestinal anisakiasis diagnosed using PCR of DNA purified from a fixed eosinophilic granuloma embedded in paraffin. Conclusion The case of human anisakiasis presented reinforces the pathological significance of the species A. pegreffii to humans. The molecular/genetic methodological approach based on mtDNA cox2 sequence analysis, described here, can allow easy and rapid identification of Anisakis spp. in formalin-fixed and paraffin embedded tissues removed from cases of either gastric or intestinal human anisakiasis. PMID:21453522

  16. First molecular identification of the zoonotic parasite Anisakis pegreffii (Nematoda: Anisakidae) in a paraffin-embedded granuloma taken from a case of human intestinal anisakiasis in Italy.

    PubMed

    Mattiucci, Simonetta; Paoletti, Michela; Borrini, Francesco; Palumbo, Massimo; Palmieri, Raffaele Macarone; Gomes, Vincenzo; Casati, Alessandra; Nascetti, Giuseppe

    2011-03-31

    Anisakiasis is an important fish-borne zoonosis provoked by larval stages of nematodes belonging to the genus Anisakis. The detection and identification of human infections is difficult. This is due to: a) the low specificity of the clinical features and symptomatology related to human infections; b) the paucity of diagnostic features of larvae found in granulomatous lesions characteristic of "invasive anisakiasis"; and c) the lack morphological characters diagnostic at the specific level when larvae of Anisakis are detected. Thus, molecular-based diagnostic approaches are warranted. We have developed a PCR method that amplifies the DNA of Anisakis spp. in fixed paraffin-embedded tissues. This method was applied to a granuloma removed from a human case of intestinal anisakiasis in Italy. Specific primers of the mtDNA cox2 gene were used and sequence analysis was performed according to the procedures already established for species of Anisakis. The sequence obtained (629 bp) was compared with those of the other species of Anisakis which have so far been genetically characterized and with sequences obtained from larval stages of Anisakis collected from the Mediterranean fish Engraulis encrasicolus. This enabled the genetic identification of the larva in the human tissue as A. pegreffii. This is the first instance of human intestinal anisakiasis diagnosed using PCR of DNA purified from a fixed eosinophilic granuloma embedded in paraffin. The case of human anisakiasis presented reinforces the pathological significance of the species A. pegreffii to humans. The molecular/genetic methodological approach based on mtDNA cox2 sequence analysis, described here, can allow easy and rapid identification of Anisakis spp. in formalin-fixed and paraffin embedded tissues removed from cases of either gastric or intestinal human anisakiasis.

  17. Detection of Clonal T-Cell Receptor γ Gene Rearrangements in Paraffin-Embedded Tissue by Polymerase Chain Reaction and Nonradioactive Single-Strand Conformational Polymorphism Analysis

    PubMed Central

    Signoretti, Sabina; Murphy, Michael; Cangi, Maria Giulia; Puddu, Pietro; Kadin, Marshall E.; Loda, Massimo

    1999-01-01

    The diagnosis of T-cell lymphoproliferative disorders, which frequently involve the skin and other extranodal sites, is often problematic because of the difficulty in establishing clonality in paraffin-embedded tissue. To this end, we developed a simple, nonradioactive method to detect T-cell receptor γ (TCR-γ) gene rearrangements by polymerase chain reaction single-strand conformational polymorphism (PCR-SSCP) in paraffin-embedded tissue. Jurkat and HSB-2 cell lines and peripheral blood samples from normal individuals were used as monoclonal and polyclonal controls, respectively. DNA was extracted from 24 biopsies of T-cell lymphomas, 12 biopsies of reactive lymphoid infiltrates, and 2 biopsies of primary cutaneous large B-cell lymphomas. Vγ1–8, Vγ9, Vγ10, Vγ11, and Jγ1/Jγ2 consensus primers were used for TCR-γ gene rearrangement amplification and PCR products were analyzed by nonradioactive SSCP. Monoclonal controls yielded a well-defined banded pattern, whereas all polyclonal T-cell controls showed a reproducible pattern of smears. We detected monoclonality in 20/21 (95%) T-cell lymphoma cases, whereas no dominant T-cell clones were found in any of the reactive lymphoid infiltrates or B-cell lymphomas. Sensitivity of 1–5% was demonstrated by serially diluting Jurkat cells in mononuclear blood cells from normal individuals. We conclude that nonradioactive PCR-SSCP for TCR-γ gene rearrangement analysis is a useful adjunct to routine histological and immunophenotypic methods in the diagnosis of T-cell lymphoproliferative disorders in paraffin-embedded tissue. PMID:9916920

  18. Detection of West Nile virus using formalin fixed paraffin embedded tissues in crows and horses: quantification of viral transcripts by real-time RT-PCR.

    PubMed

    Tewari, Deepanker; Kim, Hyun; Feria, Willard; Russo, Brigite; Acland, Helen

    2004-08-01

    West Nile virus (WNV) RNA was quantified in WNV infected crows and horses with the help of a real-time reverse transcriptase-PCR assay. A 5' nuclease assay, based on NS5 gene detection with a fluorescent probe was used for quantifying WNV RNA using formalin fixed paraffin embedded tissue specimens. Quantitative detection of WNV RNA showed the presence of a higher amount of the viral RNA in crow tissues compared to equine tissues and these results correlated well with the detection of WNV antigen by immunostaining. In crows, the highest amount of virus was seen in the intestine and in horses in the brain.

  19. Molecular identification of a causative parasite species using formalin-fixed paraffin embedded (FFPE) tissues of a complicated human pulmonary sparganosis case without decisive clinical diagnosis.

    PubMed

    Koonmee, Supinda; Intapan, Pewpan M; Yamasaki, Hiroshi; Sugiyama, Hiromu; Muto, Maki; Kuramochi, Toshiaki; Kularbkeaw, Jurairat; Kanpittaya, Jaturat; Maleewong, Wanchai; Nawa, Yukifumi

    2011-12-01

    PCR-based molecular diagnosis was made for the identification of causative agents of the clinically suspected pulmonary proliferative sparganosis case found in Thailand using formalin-fixed paraffin-embedded (FFPE) biopsy specimens. As a reference, FFPE biopsy specimen from a typical cutaneous sparganosis case was examined together. DNA samples were extracted from tissues and two partial fragments of cytochrome c oxidase subunit 1 (cox1) gene were amplified for the detection of Spirometra DNA. Two cox1 fragments were amplified successfully for both specimens. After alignment of nucleotide sequences of the PCR-amplicons, the causative agents of both cases were identified as Spirometra erinaceieuropaei.

  20. High epidermal growth factor receptor immunohistochemical expression in urothelial carcinoma of the bladder is not associated with EGFR mutations in exons 19 and 21: a study using formalin-fixed, paraffin-embedded archival tissues.

    PubMed

    Chaux, Alcides; Cohen, Julie S; Schultz, Luciana; Albadine, Roula; Jadallah, Sana; Murphy, Kathleen M; Sharma, Rajni; Schoenberg, Mark P; Netto, George J

    2012-10-01

    Epidermal growth factor receptor (EGFR) is a member of the erbB tyrosine kinase family reported to be overexpressed in a variety of solid malignancies. Mutations in exons 19 to 21 of the tyrosine kinase domain have been detected in a subset of these tumors and its presence associated with a better response to EGFR inhibitors. Several clinical trials are currently underway to evaluate the performance of such drugs in patients with bladder cancer, but data on EGFR mutation status are limited. The current study assesses EGFR immunohistochemical expression and the presence of mutations in exons 19 and 21 by polymerase chain reaction in 19 bladder urothelial carcinomas from formalin-fixed, paraffin-embedded tissues. Representative paraffin sections were microdissected for DNA extraction using a pinpoint isolation system. Parallel sections were immunostained using a monoclonal anti-EGFR antibody. No mutations in exons 19 and 21 of EGFR were identified in any of the cases. Immunohistochemical EGFR positivity was observed in 14 of 19 cases. In summary, we found EGFR protein expression in 74% of urothelial carcinomas, but we failed to detect EGFR mutations at exons 19 to 21, suggesting that EGFR overexpression is not related to the presence of mutations in the tyrosine kinase domain of the gene. Mutation analysis of EGFR exons 19 and 21 is feasible in microdissected paraffin sections from archival tissues. Immunohistochemical expression of EGFR may not be useful to predict therapeutic response to EGFR inhibitors in patients with urothelial carcinomas. To explain EGFR immunohistochemical overexpression, other mechanisms besides mutations in the EGFR kinase domain should be investigated in future studies.

  1. 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

  2. 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.

  3. 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

  4. Fluorescence in situ hybridization for identification of Tritrichomonas foetus in formalin-fixed and paraffin-embedded histological specimens of intestinal trichomoniasis.

    PubMed

    Gookin, J L; Stone, M R; Yaeger, M J; Meyerholz, D K; Moisan, Peter

    2010-08-27

    In the present study a highly species-specific oligonucleotide sequence of Tritrichomonas foetus 18S rRNA was used to design an antisense probe for identification of T. foetus in formalin-fixed and paraffin-embedded histological specimens by means of fluorescence in situ hybridization (FISH). Using archival histological specimens from several species with light microscopic evidence of intestinal trichomoniasis, and under optimized hybridization conditions, the probe positively identified trichomonads in colonic specimens from piglets and a kitten with PCR-confirmed T. foetus infection. Neither positive hybridization of the probe or PCR amplification of T. foetus DNA was observed in histological specimens from hamster (Tritrichomonas muris), turkey, nor mouse (Entamoeba muris) intestinal protozoal infections. Sequence-specific binding of the probe was further verified by successfully out-competing the hybridization with 10 x molar excess unlabeled probe and failure of a labeled sense probe to hybridize. The FISH assay described here enables simultaneous location and molecular identification of T. foetus in formalin-fixed and paraffin-embedded histological specimens of intestinal trichomoniasis. The methods employed are likely to also be applicable to probes designed for specific recognition of other trichomonad species, especially in mammalian tissue where red blood cell auto-fluorescence can be easily differentiated from the hybridization signal of trichomonads. (c) 2010 Elsevier B.V. All rights reserved.

  5. Rapid identification and characterization of Penicillium marneffei using multiplex ligation-dependent probe amplification (MLPA) in paraffin-embedded tissue samples.

    PubMed

    Zhang, Jun-Min; Sun, Jiu-Feng; Feng, Pei-Ying; Li, Xi-Qing; Lu, Chang-Ming; Lu, Sha; Cai, Wen-Ying; Xi, Li-Yan; de Hoog, G S

    2011-04-01

    Penicillium marneffei infection is a deadly disease and early diagnosis leads to prompt and appropriate antifungal therapy. To develop a sensitive method to diagnose P. marneffei infection, a multiplex ligation-dependent probe amplification (MLPA) assay was adapted. This method can rapidly and specifically detect P. marneffei DNA in cultured cells and paraffin-embedded tissue samples. Three pairs of probes were designed for amplifying the internally (intergenic) transcribed spacer (ITS) region of P. marneffei rRNA using a systematic phylogenetic analysis. These three probe sets produced three amplicons of 198, 166, and 152 bp, respectively, specific for P. marneffei. In contrast, there was only one 198 bp amplicon produced for Talaromyces stipitatus, and one 152 bp amplicon for P. funiculosum, T. intermedius and T. derxii. The probes did not amplify any other reference strains. An array of 40 P. marneffei strains isolated from human patients, bamboo rat, and the local environment was tested by using MLPA, and all were positively identified. Most importantly, P. marneffei in paraffin-embedded tissue specimens from infected human patients was positively amplified by MLPA. The sensitivity and specificity of the MLPA assay could be a useful tool for prompt diagnosis, pathogen characterization, and epidemiological studies of fungal infections.

  6. Application of in situ hybridization probes for MLH-1 and MSH-2 in tissue microarrays of paraffin-embedded malignant melanomas: correlation with immunohistochemistry and tumor stage.

    PubMed

    Korabiowska, Monika; Cordon-Cardo, Carlos; Jaenckel, Fredericke; Stachura, Jerzy; Fischer, Gösta; Brinck, Ulrich

    2004-12-01

    Defects in DNA mismatch-repair genes MLH1 and MSH2 reported primarily in hereditary nonpolyposis colorectal carcinoma are present in many sporadic tumors, including malignant melanomas. The main aim of this study was to investigate the expression of these genes in malignant melanomas in relation to tumor stage. An experiment was performed on paraffin-embedded tissue microarrays of malignant melanomas applying in situ hybridization with probes produced by our research group and immunohistochemical techniques. In situ hybridization demonstrated MLH1 expression in 45 of 59 melanomas and MSH2 expression in 51 of 59 melanomas. Immunohistochemistry detected MLH1 expression in 46 of 59 melanomas and MSH2 expression in 50 of 59 melanomas. Down-regulation of expression of both DNA mismatch repair genes in malignant melanomas was observed. The findings obtained by in situ hybridization and immunohistochemistry correlated significantly. Our study demonstrates the suitability of in situ hybridization with MLH1 and MSH2 probes for paraffin-embedded tissue. Tissue microarrays can be used successfully in both in situ hybridization and immunohistochemistry to analyze the expression of DNA mismatch-repair genes.

  7. Proteomic workflow for analysis of archival formalin-fixed and paraffin-embedded clinical samples to a depth of 10 000 proteins.

    PubMed

    Wiśniewski, Jacek R; Duś, Kamila; Mann, Matthias

    2013-04-01

    Archival formalin-fixed and paraffin-embedded clinical samples represent a very diverse source of material for proteomic investigation of diseases, often with follow-up patient information. Here, we describe an analytical workflow for analysis of laser-capture microdissected formalin-fixed and paraffin-embedded samples that allows studying proteomes to a depth of 10 000 proteins per sample. The workflow involves lysis of tissue in SDS-containing buffer, detergent removal, and consecutive digestion of the proteins with two enzymes by the multienzyme digestion filter-aided sample preparation method. Resulting peptides are fractionated by pipette-tip based strong anion exchange into six fractions and analyzed by LC-MS/MS on a bench top quadrupole Orbitrap mass spectrometer. Analysis of the data using the MaxQuant software resulted in the identification of 9502 ± 28 protein groups per a 110 nL sample of microdissected cells from human colonic adenoma. This depth of proteome analysis enables systemic insights into the organization of the adenoma cells and an estimation of the abundances of known biomarkers. It also allows the identification of proteins expressed from tumor suppressors, oncogenes, and other key players in the development and progression of the colorectal cancer. Our proteomic platform can be used for quantitative comparisons between samples representing different stages of diseases and thus can be applied to the discovery of biomarkers or drug targets. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Gene expression predicts overall survival in paraffin-embedded tissues of diffuse large B-cell lymphoma treated with R-CHOP

    PubMed Central

    LeBlanc, Michael L.; Unger, Joseph M.; Miller, Thomas P.; Grogan, Thomas M.; Persky, Daniel O.; Martel, Ralph R.; Sabalos, Constantine M.; Seligmann, Bruce; Braziel, Rita M.; Campo, Elias; Rosenwald, Andreas; Connors, Joseph M.; Sehn, Laurie H.; Johnson, Nathalie; Gascoyne, Randy D.

    2008-01-01

    Gene expression profiling (GEP) on frozen tissues has identified genes predicting outcome in patients with diffuse large B-cell lymphoma (DLBCL). Confirmation of results in current patients is limited by availability of frozen samples and addition of monoclonal antibodies to treatment regimens. We used a quantitative nuclease protection assay (qNPA) to analyze formalin-fixed, paraffin-embedded tissue blocks for 36 previously identified genes (N = 209, 93 chemotherapy; 116 rituximab + chemotherapy). By qNPA, 208 cases were successfully analyzed (99.5%). In addition, 15 of 36 and 11 of 36 genes, representing each functional group previously identified by GEP, were associated with survival (P < .05) in the 2 treatment groups, respectively. In addition, 30 of 36 hazard ratios of death trended in the same direction versus the original studies. Multivariate and variable cut-off point analysis identified low levels of HLA-DRB (< 20%) and high levels of MYC (> 80%) as independent indicators of survival, together distinguishing cases with the worst prognosis. Our results solve a clinical research problem by demonstrating that prognostic genes can be meaningfully quantified using qNPA technology on formalin-fixed, paraffin-embedded tissues; previous GEP findings in DLBCL are relevant with current treatments; and 2 genes, representing immune escape and proliferation, are the common features of the most aggressive DLBCL. PMID:18544678

  9. 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

  10. Monoclonal antibody L26: an antibody that is reactive with normal and neoplastic B lymphocytes in routinely fixed and paraffin wax embedded tissues.

    PubMed Central

    Norton, A J; Isaacson, P G

    1987-01-01

    Formalin fixed and paraffin wax embedded tissue from 85 well characterised cases of non-Hodgkin's lymphoma and Hodgkin's disease were studied using the avidin-biotin-peroxidase complex technique. Among the non-Hodgkin's lymphomas all cases of B cell lymphoma were reactive with L26, a monoclonal antibody which is as yet an unclustered pan B cell reagent, with the exception of pre-B cell acute lymphoblastic leukaemia and malignant lymphoma plasmacytic. Eighteen well characterised cases of T cell lymphoma, selected to include tumours previously shown to exhibit cross reactivity with antibodies to fixation resistant B cell related antigens, were similarly studied. Neoplastic cells in all but one case were unstained by L26. Twenty seven cases of Hodgkin's disease were also examined. In five cases all Reed-Sternberg cells and their variants were strongly stained by L26; only a proportion of Reed-Sternberg cells and their variants were recognised in a further five cases. Monoclonal antibody L26 promises to be a valuable reagent for the diagnosis of malignant lymphoma in routinely fixed and paraffin wax embedded tissues. Its advantage lies in its sensitivity and greater B cell specificity than any of the B cell related reagents currently available for the study of malignant lymphoma in fixed tissues. Images Fig 1a Fig 1b Fig 2a Fig 2b Fig 3a Fig 3b Fig 4a Fig 4b Fig 5a Fig 5b PMID:3323247

  11. 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.

  12. 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.

  13. 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

  14. 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

  15. 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.

  16. Assessment of FANCD2 nuclear foci formation in paraffin embedded tumors; a potential patient enrichment strategy for treatment with DNA interstrand crosslinking agents

    PubMed Central

    Duan, Wenrui; Gao, Li; Zhao, Weiqiang; Leon, Marino; Sadee, Wolfgang; Webb, Amy; Resnick, Kimberly; Wu, Xin; Ramaswamy, Bhuvaneswari; Cohn, David E.; Shapiro, Charles; Andreassen, Paul R.; Otterson, Gregory A.; Villalona-Calero, Miguel A.

    2013-01-01

    A major mechanism of DNA repair related to homologous recombination is the Fanconi Anemia pathway (FA). FA genes collaborate with BRCA genes to form foci of DNA repair on chromatin following DNA damage, or during S phase of the cell cycle. Our goal was to develop a method capable of evaluating the functional status of the pathway in patients’ tumor tissue, which could also be practically incorporated to large scale screening. In order to develop this method, we first used Western immunoblot to detect FANCD2 protein mono-ubiquitination in fresh tumor specimens of ovarian cancer patients undergoing surgery, and stained formalin fixed paraffin embedded (FFPE) tumor tissue simultaneously with DAPI, FANCD2 and Ki67 antibodies, eventually extending this method to other solid tumors. This triple stain permitted evaluation of the presence, or lack thereof, of FANCD2 subnuclear repair foci in proliferating cells by immunofluorescence microscopy. Overall, we evaluated 156 FFPE tumor samples using the FA triple staining immunofluorescence (FATSI) method. The ratios of FANCD2 foci negative tumors in ovarian, lung, and breast tumor samples were 21%, 20%, and 29.4%, respectively. Our studies have led to the development of a suitable method for screening, capable of identifying tumors with somatic functional defects in the FA pathway. The use of paraffin embedded tissues renders the reported method suitable for large scale screening to select patients for treatment with DNA interstrand crosslinking agents, PARP inhibitors or their combination. PMID:23063585

  17. Low vacuum scanning electron microscopy for paraffin sections utilizing the differential stainability of cells and tissues with platinum blue.

    PubMed

    Inaga, Sumire; Hirashima, Sayuri; Tanaka, Keiichi; Katsumoto, Tetsuo; Kameie, Toshio; Nakane, Hironobu; Naguro, Tomonori

    2009-07-01

    The present study introduces a novel method for the direct observation of histological paraffin sections by low vacuum scanning electron microscopy (LVSEM) with platinum blue (Pt-blue) treatment. Pt-blue was applied not only as a backscattered electron (BSE) signal enhancer but also as a histologically specific stain. In this method, paraffin sections of the rat tongue prepared for conventional light microscopy (LM) were stained on glass slides with a Pt-blue staining solution (pH 9) and observed in a LVSEM using BSE detector. Under LVSEM, overviews of whole sections as well as three-dimensional detailed observations of individual cells and tissues could be easily made at magnifications from x40 to x10,000. Each kind of cell and tissue observed in the section could be clearly distinguished due to the different yields of BSE signals, which depended on the surface structures and different affinities to Pt-blue. Thus, we roughly classified cellular and tissue components into three groups according to the staining intensity of Pt-blue observed by LM and LVSEM: 1) a strongly stained (deep blue by LM and brightest by LVSEM) group which included epithelial tissue, endothelium and mast cells; 2) a moderately stained (light blue and bright) group which included muscular tissue and nervous tissue; 3) an unstained or weakly stained (colorless and dark) group which included elastic fibers and collagen fibers. We expect that this method will prove useful for the three-dimensional direct observation of histological paraffin sections of various tissues by LVSEM with higher resolutions than LM.

  18. Protein domain histochemistry (PDH): binding of the carbohydrate recognition domain (CRD) of recombinant human glycoreceptor CLEC10A (CD301) to formalin-fixed, paraffin-embedded breast cancer tissues.

    PubMed

    Nollau, Peter; Wolters-Eisfeld, Gerrit; Mortezai, Naghmeh; Kurze, Anna-Katharina; Klampe, Birgit; Debus, Annegret; Bockhorn, Maximilian; Niendorf, Axel; Wagener, Christoph

    2013-03-01

    Specialized protein domains bind to posttranslational modifications (PTMs) of proteins, such as phosphorylation or glycosylation. When such PTM-binding protein domains are used as analytical tools, the functional states of cells and tissues can be determined with high precision. Here, we describe the use of recombinant CLEC10A (CD301), a human glycoreceptor of the C-type lectin family, for the detection of ligands in sections from formalin-fixed, paraffin-embedded normal and cancerous mammary tissues. A construct, in which part of the carbohydrate recognition domain (CRD) was deleted, was used as a negative control. In comparison to normal mammary glands, a pronounced staining of tumor tissues was observed. Because the construct with the truncated CRD did not show any tissue staining, the binding of the wild-type glycoreceptor can be attributed to its carbohydrate recognition domain. To distinguish our novel approach from immunohistochemistry, we propose the designation "protein domain histochemistry" (PDH).

  19. Effective DNA/RNA co-extraction for analysis of microRNAs, mRNAs, and genomic DNA from formalin-fixed paraffin-embedded specimens.

    PubMed

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

    2012-01-01

    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. 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. 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 would otherwise be discarded or degraded

  20. 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

  1. A system for quantitative evaluation of fixatives for light microscopy using paraffin sections of kidney and brain.

    PubMed

    Kiernan, J A

    2009-02-01

    A numerical scoring system is presented for evaluating structural fixation of certain mammalian tissues for light microscopy. Small pieces of rat's kidney and brain, tissues for which artifacts of fixation are well documented, were fixed in various fluids. Random code numbers hid their identities, and paraffin sections were stained to show nuclear chromatin, cytoplasm and extracellular materials. Two sections of each specimen were examined and awarded scores according to described schemes, for microanatomical and cytological fixation. The assessment was confined to preservation of structure; chemical changes were not taken into account. When the code was broken, the scores for both sections of each specimen from each fixative were added. The scores obtained (24 best to eight worst) are generally comparable to the grades (I-V) given for traditional fixatives by JR Baker. The criteria for quality of fixation were defined more explicitly than those for Baker's grading method, which used mouse testis as the test object. Assessments are presented for several traditional fixatives and for four zinc-formaldehyde mixtures. The scoring system should be useful for evaluating newly developed fixatives for animal tissues for light microscopic examination of paraffin sections. In an evaluation of four traditional fixatives and four zinc-formaldehyde mixtures, variation among three different observers was only +/-1 point on either side of the median score for microanatomical or cytological preservation by any of the eight fixatives. This approach has certain limitations, notably that the criteria for cytological fixation do not include the preservation of chromosomes or specific cytoplasmic organelles.

  2. Detection of human papilloma viruses in paraffin wax sections with biotinylated synthetic oligonucleotide probes and immunogold staining.

    PubMed

    Cubie, H A; Norval, M

    1989-09-01

    Human papilloma virus was detected by in situ hybridisation in routinely processed paraffin wax sections using a synthetically produced oligonucleotide probe, end-labelled with biotin, and amplified with anti-biotin-immunogold silver staining (anti-biotin-IGSS). This system proved more sensitive than amplification with streptavidin-biotinylated alkaline phosphatase for detecting human papilloma virus type 16 in cervical tissues. The method was successfully combined with antigen staining for papilloma virus common antigens in skin and genital warts. This simple and quick method, using non-radioactively labelled synthetic probes, may be useful for the detection of other viruses in stored material and may be suitable for other double staining procedures.

  3. In situ hybridization for the detection of rust fungi in paraffin embedded plant tissue sections

    USDA-ARS?s Scientific Manuscript database

    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...

  4. Identification of bacterial pathogens from formalin-fixed, paraffin-embedded tissues by using 16S sequencing: retrospective correlation of results to clinicians' responses.

    PubMed

    Racsa, Lori D; DeLeon-Carnes, Marlene; Hiskey, Matthew; Guarner, Jeannette

    2017-01-01

    16S sequencing on formalin-fixed, paraffin-embedded (FFPE) material has been used to identify bacteria when culture-based phenotyping techniques have not worked. The objective of this study was to determine how frequently 16S sequencing used in FFPE material was helpful to clinicians in the diagnosis and treatment of infectious diseases. Requests for testing occurred upon consultation between an infectious disease pathologist and a surgical pathologist or an infectious disease physician. A selected paraffin block from each case was referred for 16S sequencing. Retrospectively, we correlated clinical history and management decisions on 27 cases that were tested by paneubacterial 16S sequencing. Samples included 24 surgical specimens, 1 autopsy, and 2 cytology blocks. Seventeen (63%) of the 27 cases had a positive 16S sequencing. Acute inflammation was present in 10 of these cases, and organisms were observed using special stains in 3. In 11 (65%) of the 17 cases, clinicians considered the organism identified by 16S sequencing to be the cause or possible cause of the infectious process. Organisms included common (Citrobacter) and fastidious bacteria (Haemophilus, Fusobacterium). In 3 cases, clinicians changed antibiotic treatment based on the bacteria identified, whereas in 8 (including 2 where no organism was found), clinicians continued the antibiotic treatment. The use of 16S sequencing on FFPE identified specific bacteria even when organisms were not observed histopathologically. 16S results had an impact in infectious disease management decisions.

  5. PCR-assays Detect B-lymphocyte Clonality in Formalin-fixed Paraffin Embedded Specimens of Classical Hodgkin Lymphoma without Microdissection

    PubMed Central

    Burack, W. Richard; Laughlin, Todd S.; Friedberg, Jonathan; Spence, Janice M.; Rothberg, Paul G.

    2011-01-01

    Hodgkin lymphoma (HL) was shown to be a B cell malignancy using PCR-clonality studies of microdissected Reed-Sternberg cells. While methods for the detection of B cell clonality could aid in the diagnosis of HL, microdissection is not practical in most clinical settings. We assessed the standardized BIOMED-2 IGH and IGK PCR primers for the detection of clonality using 50 consecutively diagnosed formalin-fixed paraffin-embedded (FFPE) classic Hodgkin lymphoma specimens. Without microdissection, clonality was detected in 23/47 assessable cases. The IGK assay was significantly more sensitive than the IGH assay (18 vs. 10 positive results). These data, and two representative cases, demonstrate that PCR based B-cell clonality assays have utility when the histologic differential diagnosis of an FFPE specimen includes classic Hodgkin lymphoma. PMID:20551274

  6. 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.

  7. Molecular cytogenetic interphase analysis of Phosphoinositide-specific Phospholipase C β1 gene in paraffin-embedded brain samples of major depression patients.

    PubMed

    Lo Vasco, Vincenza Rita; Polonia, Patrizia

    2012-01-01

    Mood disorders represent a major medical need, as their chronic treatments are not effective in all patients. Literature data suggested that phosphoinositides (PI) signal transduction pathway and related molecules such as the Phosphoinositide-specific Phospholipase C (PI-PLC) enzymes, might be involved in the pathophysiology of mood disorders, including major depression. By using interphase fluorescent in situ hybridization methodology, we analyzed PLCB1 gene, which codifies for the PI-PLC β1 enzyme, in paraffin embedded samples of orbito-frontal cortex of 15 patients affected with major depression and in 15 normal controls. No deletions of PLCB1 were identified with the methodology used, which allows to exclude wide gene deletions. The results, the technical aspects of the FISH methodology, and its limitations are discussed.

  8. Application of new in situ hybridization probes for Ku70 and Ku80 in tissue microarrays of paraffin-embedded malignant melanomas: correlation with immunohistochemical analysis.

    PubMed

    Korabiowska, Monika; Bauer, Hanne; Quentin, Tomas; Stachura, Jerzy; Cordon-Cardo, Carlos; Brinck, Ulrich

    2004-02-01

    Ku70 and Ku80 proteins are responsible for the repair of DNA double-strand breaks and function as a regulatory subunit of the DNA-dependent protein kinase. In this study we analyzed expression of both genes in malignant melanoma tissue arrays applying in situ hybridization probes produced by our research group and using immunohistochemical analysis. Expression of both genes was down-regulated as melanoma progressed. In situ hybridization demonstrated more Ku70- and Ku80-positive cells than immunohistochemical methods, but the correlation between the two methods was highly significant (P <0.01). We conclude that the in situ hybridization assay for the detection of Ku70 and Ku80 expression used in this study is also suitable for tissue microarray analysis of paraffin-embedded melanoma samples. The laboratory procedure is much more complicated than the immunohistochemical method, however.

  9. Copy number analysis by low coverage whole genome sequencing using ultra low-input DNA from formalin-fixed paraffin embedded tumor tissue.

    PubMed

    Kader, Tanjina; Goode, David L; Wong, Stephen Q; Connaughton, Jacquie; Rowley, Simone M; Devereux, Lisa; Byrne, David; Fox, Stephen B; Mir Arnau, Gisela; Tothill, Richard W; Campbell, Ian G; Gorringe, Kylie L

    2016-11-15

    Unlocking clinically translatable genomic information, including copy number alterations (CNA), from formalin-fixed paraffin-embedded (FFPE) tissue is challenging due to low yields and degraded DNA. We describe a robust, cost-effective low-coverage whole genome sequencing (LC WGS) method for CNA detection using 5 ng of FFPE-derived DNA. CN profiles using 100 ng or 5 ng input DNA were highly concordant and comparable with molecular inversion probe (MIP) array profiles. LC WGS improved CN profiles of samples that performed poorly using MIP arrays. Our technique enables identification of driver and prognostic CNAs in archival patient samples previously deemed unsuitable for genomic analysis due to DNA limitations.

  10. Detection and Genotyping of Human Papillomavirus DNA in Formalin-Fixed Paraffin-Embedded Specimens with the HPV Direct Flow CHIP System.

    PubMed

    Herraez-Hernandez, Elsa; Preda, Ovidiu; Alonso, Sonia; Pardo, Rosario Serrano; Olmo, Asuncion

    2013-01-01

    The novel HPV Direct Flow CHIP commercial system for Human Papillomavirus (HPV) genotyping is based on rapid PCR and automatic reverse dot blot hybridization to genotype-specific probes, allowing the detection of 36 HPV genotypes. This study examined the performance of HPV Direct Flow CHIP in formalin-fixed paraffin-embedded (FFPE) samples (n= 99). Each sample was analyzed both by Direct PCR, using crude cell extracts without DNA purification, and by conventional PCR, using purified DNA. Pair-wise analysis of the results demonstrated strong concordance between the results obtained with the two protocols, although a slightly higher rate of multiple infections was detected by conventional PCR. In summary, HPV Direct Flow CHIP achieves effective HPV detection from FFPE samples with both Direct PCR and Conventional PCR protocols.

  11. Application of RLEP Real-Time PCR for Detection of M. leprae DNA in Paraffin-Embedded Skin Biopsy Specimens for Diagnosis of Paucibacillary Leprosy

    PubMed Central

    Yan, Wen; Xing, Yan; Yuan, Lian Chao; De Yang, Rong; Tan, Fu Yue; Zhang, Ying; Li, Huan-Ying

    2014-01-01

    The TaqMan real-time polymerase chain reaction (PCR) assay was evaluated systematically with respect to the standard curve, linear range, and used for detecting Mycobacterium leprae DNA in paraffin-embedded skin biopsy specimens from 60 confirmed leprosy patients and three healthy individuals and 29 other dermatoses and bacterial DNA from 21 different species. The test was further evaluated with 51 paucibacillary (PB) patients. The results showed that the test had good sensitivity (8 fg) and good specificity with no cross-reactivity with 21 other bacterial species and the control specimens, except one with Xanthomatosis. The real-time PCR detection rate for the 51 PB specimens was 74.5% (38 of 51). We conclude that the real-time PCR test is a useful adjunct test for diagnosing early stage or PB leprosy cases. PMID:24493677

  12. 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.

  13. 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

  14. Detection of epidermal growth factor receptor mutations in formalin fixed paraffin embedded biopsies in Malaysian non-small cell lung cancer patients

    PubMed Central

    2013-01-01

    Background Somatic mutations of the epidermal growth factor receptor (EGFR) are reportedly associated with various responses in non-small cell lung cancer (NSCLC) patients receiving the anti-EGFR agents. Detection of the mutation therefore plays an important role in therapeutic decision making. The aim of this study was to detect EGFR mutations in formalin fixed paraffin embedded (FFPE) samples using both Scorpion ARMS and high resolution melt (HRM) assay, and to compare the sensitivity of these methods. Results All of the mutations were found in adenocarcinoma, except one that was in squamous cell carcinoma. The mutation rate was 45.7% (221/484). Complex mutations were also observed, wherein 8 tumours carried 2 mutations and 1 tumour carried 3 mutations. Conclusions Both methods detected EGFR mutations in FFPE samples. HRM assays gave more EGFR positive results compared to Scorpion ARMS. PMID:23590575

  15. Detection of epidermal growth factor receptor mutations in formalin fixed paraffin embedded biopsies in Malaysian non-small cell lung cancer patients.

    PubMed

    Shi Yeen, Tiffany Ng; Pathmanathan, Rajadurai; Shiran, Mohd Sidik; Ahmad Zaid, Fattah Azman; Cheah, Yoke Kqueen

    2013-04-16

    Somatic mutations of the epidermal growth factor receptor (EGFR) are reportedly associated with various responses in non-small cell lung cancer (NSCLC) patients receiving the anti-EGFR agents. Detection of the mutation therefore plays an important role in therapeutic decision making. The aim of this study was to detect EGFR mutations in formalin fixed paraffin embedded (FFPE) samples using both Scorpion ARMS and high resolution melt (HRM) assay, and to compare the sensitivity of these methods. All of the mutations were found in adenocarcinoma, except one that was in squamous cell carcinoma. The mutation rate was 45.7% (221/484). Complex mutations were also observed, wherein 8 tumours carried 2 mutations and 1 tumour carried 3 mutations. Both methods detected EGFR mutations in FFPE samples. HRM assays gave more EGFR positive results compared to Scorpion ARMS.

  16. Ewing’s Sarcoma: An Analysis of miRNA Expression Profiles and Target Genes in Paraffin-Embedded Primary Tumor Tissue

    PubMed Central

    Parafioriti, Antonina; Bason, Caterina; Armiraglio, Elisabetta; Calciano, Lucia; Daolio, Primo Andrea; Berardocco, Martina; Di Bernardo, Andrea; Colosimo, Alessia; Luksch, Roberto; Berardi, Anna C.

    2016-01-01

    The molecular mechanism responsible for Ewing’s Sarcoma (ES) remains largely unknown. MicroRNAs (miRNAs), a class of small non-coding RNAs able to regulate gene expression, are deregulated in tumors and may serve as a tool for diagnosis and prediction. However, the status of miRNAs in ES has not yet been thoroughly investigated. This study compared global miRNAs expression in paraffin-embedded tumor tissue samples from 20 ES patients, affected by primary untreated tumors, with miRNAs expressed in normal human mesenchymal stromal cells (MSCs) by microarray analysis. A miRTarBase database was used to identify the predicted target genes for differentially expressed miRNAs. The miRNAs microarray analysis revealed distinct patterns of miRNAs expression between ES samples and normal MSCs. 58 of the 954 analyzed miRNAs were significantly differentially expressed in ES samples compared to MSCs. Moreover, the qRT-PCR analysis carried out on three selected miRNAs showed that miR-181b, miR-1915 and miR-1275 were significantly aberrantly regulated, confirming the microarray results. Bio-database analysis identified BCL-2 as a bona fide target gene of the miR-21, miR-181a, miR-181b, miR-29a, miR-29b, miR-497, miR-195, miR-let-7a, miR-34a and miR-1915. Using paraffin-embedded tissues from ES patients, this study has identified several potential target miRNAs and one gene that might be considered a novel critical biomarker for ES pathogenesis. PMID:27144561

  17. Profiling cancer gene mutations in clinical formalin-fixed, paraffin-embedded colorectal tumor specimens using targeted next-generation sequencing.

    PubMed

    Zhang, Liangxuan; Chen, Liangjing; Sah, Sachin; Latham, Gary J; Patel, Rajesh; Song, Qinghua; Koeppen, Hartmut; Tam, Rachel; Schleifman, Erica; Mashhedi, Haider; Chalasani, Sreedevi; Fu, Ling; Sumiyoshi, Teiko; Raja, Rajiv; Forrest, William; Hampton, Garret M; Lackner, Mark R; Hegde, Priti; Jia, Shidong

    2014-04-01

    The success of precision oncology relies on accurate and sensitive molecular profiling. The Ion AmpliSeq Cancer Panel, a targeted enrichment method for next-generation sequencing (NGS) using the Ion Torrent platform, provides a fast, easy, and cost-effective sequencing workflow for detecting genomic "hotspot" regions that are frequently mutated in human cancer genes. Most recently, the U.K. has launched the AmpliSeq sequencing test in its National Health Service. This study aimed to evaluate the clinical application of the AmpliSeq methodology. We used 10 ng of genomic DNA from formalin-fixed, paraffin-embedded human colorectal cancer (CRC) tumor specimens to sequence 46 cancer genes using the AmpliSeq platform. In a validation study, we developed an orthogonal NGS-based resequencing approach (SimpliSeq) to assess the AmpliSeq variant calls. Validated mutational analyses revealed that AmpliSeq was effective in profiling gene mutations, and that the method correctly pinpointed "true-positive" gene mutations with variant frequency >5% and demonstrated high-level molecular heterogeneity in CRC. However, AmpliSeq enrichment and NGS also produced several recurrent "false-positive" calls in clinically druggable oncogenes such as PIK3CA. AmpliSeq provided highly sensitive and quantitative mutation detection for most of the genes on its cancer panel using limited DNA quantities from formalin-fixed, paraffin-embedded samples. For those genes with recurrent "false-positive" variant calls, caution should be used in data interpretation, and orthogonal verification of mutations is recommended for clinical decision making.

  18. Profiling Cancer Gene Mutations in Clinical Formalin-Fixed, Paraffin-Embedded Colorectal Tumor Specimens Using Targeted Next-Generation Sequencing

    PubMed Central

    Zhang, Liangxuan; Chen, Liangjing; Sah, Sachin; Latham, Gary J.; Patel, Rajesh; Song, Qinghua; Koeppen, Hartmut; Tam, Rachel; Schleifman, Erica; Mashhedi, Haider; Chalasani, Sreedevi; Fu, Ling; Sumiyoshi, Teiko; Raja, Rajiv; Forrest, William; Hampton, Garret M.; Lackner, Mark R.; Hegde, Priti

    2014-01-01

    Purpose. The success of precision oncology relies on accurate and sensitive molecular profiling. The Ion AmpliSeq Cancer Panel, a targeted enrichment method for next-generation sequencing (NGS) using the Ion Torrent platform, provides a fast, easy, and cost-effective sequencing workflow for detecting genomic “hotspot” regions that are frequently mutated in human cancer genes. Most recently, the U.K. has launched the AmpliSeq sequencing test in its National Health Service. This study aimed to evaluate the clinical application of the AmpliSeq methodology. Methods. We used 10 ng of genomic DNA from formalin-fixed, paraffin-embedded human colorectal cancer (CRC) tumor specimens to sequence 46 cancer genes using the AmpliSeq platform. In a validation study, we developed an orthogonal NGS-based resequencing approach (SimpliSeq) to assess the AmpliSeq variant calls. Results. Validated mutational analyses revealed that AmpliSeq was effective in profiling gene mutations, and that the method correctly pinpointed “true-positive” gene mutations with variant frequency >5% and demonstrated high-level molecular heterogeneity in CRC. However, AmpliSeq enrichment and NGS also produced several recurrent “false-positive” calls in clinically druggable oncogenes such as PIK3CA. Conclusion. AmpliSeq provided highly sensitive and quantitative mutation detection for most of the genes on its cancer panel using limited DNA quantities from formalin-fixed, paraffin-embedded samples. For those genes with recurrent “false-positive” variant calls, caution should be used in data interpretation, and orthogonal verification of mutations is recommended for clinical decision making. PMID:24664487

  19. Progesterone receptor isoform analysis by quantitative real-time polymerase chain reaction in formalin-fixed, paraffin-embedded canine mammary dysplasias and tumors.

    PubMed

    Guil-Luna, S; Stenvang, J; Brünner, N; Sánchez-Céspedes, R; Millán, Y; Gómez-Laguna, J; de las Mulas, J Martín

    2014-09-01

    Cloning and sequencing of the progesterone receptor gene in dogs have revealed 2 isoforms, A and B, transcribed from a single gene. Distribution of isoforms A and B in canine mammary lesions has hitherto been investigated only by Western blot analysis. This study analyzed progesterone receptor and its isoforms in formalin-fixed, paraffin-embedded tissue samples from canine mammary lesions (4 dysplasias, 10 benign tumors, and 46 carcinomas) using 1-step SYBR Green quantitative real-time polymerase chain reaction (RT-qPCR). Progesterone receptor was expressed in 75% of dysplasias, all benign tumors, and 59% of carcinomas. Carcinomas, and particularly simple epithelial-type carcinomas, displayed the lowest levels of expression. A high rate of agreement was recorded between RT-qPCR and immunohistochemical labeling. Isoforms A and B were successfully amplified, with correlation coefficients of 0.99 and amplification efficiencies close to 2, and were expressed in all lesion types analyzed. Predominance of A over B expression was observed in carcinomas and complex adenomas. Low-grade tumors exhibited higher progesterone receptor messenger RNA (mRNA) levels, but no difference was observed in the expression of isoform A versus B. Analysis of progesterone receptor mRNA isoforms by RT-qPCR was successful in routinely formalin-fixed, paraffin-embedded tissue samples and enabled the distribution of isoforms A and B to be identified for the first time in dysplasias, benign tumors, and malignant tumors of the canine mammary gland. These findings will facilitate future research into the role of progesterone receptor isoforms in the progression of canine mammary tumors. © The Author(s) 2013.

  20. Comparison between immunohistochemistry and two PCR methods for detection of Neospora caninum in formalin-fixed and paraffin-embedded brain tissue of bovine fetuses.

    PubMed

    Sánchez, G F D; Banda, R V M; Sahagun, R A; Ledesma, M N; Morales, S E

    2009-10-14

    The objective of this study was to identify the presence of the parasite by comparing immunohistochemistry (IHC) with two polymerase chain reaction (PCR) methods for the detection of the pNc5 gene and the internal transcribed spacer 1 (ITS1) of N. caninum in brain tissue of bovine fetuses that had previously been fixed in formalin and paraffin-embedded. In 29 out of 48 brains (60.4%), microscopic lesions consistent with Neospora infection were observed, and 21 of the 29 cases (72.41%) were positive for IHC. Fifteen of the 29 cases positive for IHC (51.72%) were also positive on the ITS1 PCR, and 12 cases were also positive on the pNc5 PCR (41.37%). The sensitivity of the PCR assays was 71.42% and 57.14%, respectively, and the specificity was 100% for both. The concordance between histopathology and IHC and the ITS1 PCR was 85%, and in the case of the pNc5 PCR it was 77.5%. When the number of fetuses positive by IHC and both PCR tests was compared, no statistically significant difference was found (P>0.05). It is concluded that the use of formalin-fixed and paraffin-embedded bovine fetal tissues allows the detection of N. caninum by IHC or PCR. Nevertheless, it is recommended that more than one technique is used to increase the diagnostic sensitivity, and preferably tests that show better performance in the individual laboratory should be selected.

  1. Detection of bovine Herpesvirus type 5 in formalin-fixed, paraffin-embedded bovine brain by PCR: a useful adjunct to conventional tissue-based diagnostic test of bovine encephalitis.

    PubMed

    Ferrari, H F; Luvizotto, M C R; Rahal, P; Cardoso, T C

    2007-12-01

    The aim of this study was to evaluate the application of PCR technique for the detection of BoHV-5 in routinely formalin-fixed, paraffin-embedded brain tissues in 20 naturally infected calves affected by fatal meningoencephalitis. Brains were divided into two halves, one kept fresh for virus isolation and PCR assay, targeting the glycoprotein C gene from BoHV-5 genome. The other half brain, corresponding to posterior cortex region, was submitted to formalin fixation and embedded into paraffin blocks for microscopic evaluation and total DNA isolation. Most of the slides showed severe multifocal non-supurative encephalitis with neuronal degeneration, neurophagia, and no acidophilic intranuclear inclusions could be found in neurons and glial. The 20 fresh samples were confirmed, by virus isolation and PCR assay, as having the BoHV-5 virus and, respective glicoprotein C sequence, while 15 of 20 formalin-fixed, paraffin-embedded samples were considered positive for the same analysis. The results revealed the first description of PCR efficiency, applied to formalin-fixed, paraffin-embedded brain collected from naturally infected calves, improving the detection of BoHV-5 from archival samples in South America.

  2. [Application of Immunohistochemistry and Immunofluorescence Staining in Detection of Phospholipase A2 Receptor on Paraffin Section of Renal Biopsy Tissue].

    PubMed

    Dong, Hong-rui; Wang, Yan-yan; Wang, Guo-qin; Sun, Li-jun; Cheng, Hong; Chen, Yi-pu

    2015-10-01

    To evaluate the application of immunohistochemistry and fluorescence staining method in the detection of phospholipase A2 receptor (PLA2R) on paraffin section of renal biopsy tissue,and to find an accurate and fast method for the detection of PLA2R in renal tissue. The PLA2R of 193 cases were detected by immunohistochemical staining,and the antigen was repaired by the method of high pressure cooker (HPC) hot repair plus trypsin repair. The 193 samples including 139 cases of idiopathic membranous nephropathy (IMN), 15 cases of membranous lupus nephritis, 8 cases of hepatitis B virus associated membranous nephropathy, 18 cases of IgA nephropathy, and 13 cases of minimal change diseases. To compare the dyeing effects, 22 paraffin sections of renal biopsy tissue of IMN cases with positive PLA2R were stained by using 4 different. of antigen repairing,which included HPC hot repair, HPC hot repair plus trypsin repair, water bath heat repair, and water bath heat repair plus trypsin repair. To compare the dyeing effects, 15 paraffin sections of renal biopsy tissue of IMN cases with positive PLA2R were stained by using 3 different. of antigen repairing,which included water bath heat repair plus trypsin repair, protease K digestion repair, and pepsin digestion repair. In 193 cases, the positive rate of PLA2R in IMN cases was 90.6% (126/139), and the other 54 patients without IMN were negative. Twenty-two IMN patients were positive for PLA2R by using the HPC heat repair plus trypsin repaire or the water bath heat repair plus trypsin repair;while only a few cases of 22 IMN cases were positive by using the HPC hot repair alone or water bath heat repair alone. Fifteen IMN patients were positive for PLA2R by using water bath heat repair plus trypsin repair,protease K digestion repair,and pepsin digestion repair, but the distribution of positive deposits and the background were different. PLA2R immunohistochemical staining can effectively identify IMN and secondary MN. For

  3. Evaluation of a completely automated tissue-sectioning machine for paraffin blocks.

    PubMed

    Onozato, Maristela L; Hammond, Stephen; Merren, Mark; Yagi, Yukako

    2013-02-01

    Tissue-sectioning automation can be a resourceful tool in processing anatomical pathology specimens. The advantages of an automated system compared with traditional manual sectioning are the invariable thickness, uniform orientation and fewer tissue-sectioning artefacts. This short report presents the design of an automated tissue-sectioning device and compares the sectioned specimens with normal manual tissue sectioning performed by an experienced histology technician. The automated system was easy to use, safe and the sectioned material showed acceptable quality with well-preserved morphology and tissue antigenicity. It is expected that the turnaround time will be improved in the near future.

  4. Detection of human papilloma viruses in paraffin wax sections with biotinylated synthetic oligonucleotide probes and immunogold staining.

    PubMed Central

    Cubie, H A; Norval, M

    1989-01-01

    Human papilloma virus was detected by in situ hybridisation in routinely processed paraffin wax sections using a synthetically produced oligonucleotide probe, end-labelled with biotin, and amplified with anti-biotin-immunogold silver staining (anti-biotin-IGSS). This system proved more sensitive than amplification with streptavidin-biotinylated alkaline phosphatase for detecting human papilloma virus type 16 in cervical tissues. The method was successfully combined with antigen staining for papilloma virus common antigens in skin and genital warts. This simple and quick method, using non-radioactively labelled synthetic probes, may be useful for the detection of other viruses in stored material and may be suitable for other double staining procedures. Images PMID:2551931

  5. Stereological comparison of oocyte recruitment and batch fecundity estimates from paraffin and resin sections using spawning albacore (Thunnus alalunga) ovaries as a case study

    NASA Astrophysics Data System (ADS)

    Saber, Sámar; Macías, David; Ortiz de Urbina, Josetxu; Kjesbu, Olav Sigurd

    2015-01-01

    Traditional histological protocols in marine fish reproductive laboratories using paraffin as the embedding medium are now increasingly being replaced with protocols using resin instead. These procedures entail different degrees of tissue shrinkage complicating direct comparisons of measurement results across laboratories or articles. In this work we selected ovaries of spawning Mediterranean albacore (Thunnus alalunga) as the subject of our study to address the issue of structural changes, by contrasting values on oocyte recruitment and final batch fecundity given from the same tissue samples in both paraffin and resin. A modern stereological method, the oocyte packing density (OPD) theory, was used supported by initial studies on ovarian tissue sampling and measurement design. Examples of differences in the volume fraction of oocyte stages, free space and connective tissue were found between the embedding media. Mean oocyte diameters were smaller in paraffin than in resin with differences ranging between 0.5% in primary growth and 24.3% in hydration (HYD) stage oocytes. Fresh oocyte measurements showed that oocytes shrank as a consequence of the embedding process, reaching the maximal degree of shrinkage for oocytes in the HYD stage (45.8% in paraffin and 26.5% in resin). In order to assess the effect of oocyte shrinkage on the OPD result, and thereby on relative batch fecundity (Fr), oocyte diameters corrected and uncorrected for shrinkage, were used for estimations. Statistical significant differences were found (P < 0.05) between these two approaches in both embedding media. The average Fr was numerically smaller in paraffin compared to resin (86 ± 61 vs. 106 ± 54 oocytes per gram of body mass (mean ± SD)). For both embedding media statistical significant differences (P < 0.05) were seen between Fr results based on either oocytes in the germinal vesicle migration stage or HYD stage. As a valuable adjunct, the present use of the OPD theory made it possible

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

    PubMed Central

    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.

    2015-01-01

    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

  7. DNA and RNA isolation from canine oncologic formalin-fixed, paraffin-embedded tissues for downstream "-omic" analyses: possible or not?

    PubMed

    Granato, Anna; Giantin, Mery; Ariani, Pietro; Carminato, Antonio; Baratto, Chiara; Zorzan, Eleonora; Vascellari, Marta; Bozzato, Elisa; Dacasto, Mauro; Mutinelli, Franco

    2014-01-01

    Formalin-fixed, paraffin-embedded (FFPE) tissues represent a unique source of archived biological material, but obtaining suitable DNA and RNA for retrospective "-omic" investigations is still challenging. In the current study, canine tumor FFPE blocks were used to 1) compare common commercial DNA and RNA extraction kits; 2) compare target gene expression measured in FFPE blocks and biopsies stored in a commercial storage reagent; 3) assess the impact of fixation time; and 4) perform biomolecular investigations on archival samples chosen according to formalin fixation times. Nucleic acids yield and quality were determined by spectrophotometer and capillary electrophoresis, respectively. Quantitative real-time polymerase chain reaction assays for the following genes: BCL-2-associated X protein, B-cell lymphoma extra large, antigen identified by monoclonal antibody Ki-67, proto-oncogene c-KIT (c-kit). Two internal control genes (Golgin A1 and canine transmembrane BAX inhibitor motif containing 4), together with direct sequencing of c-kit exons 8, 9, 11, and 17, were used as end points. Differences in DNA/RNA yield and purity were noticed among the commercial kits. Nucleic acids (particularly RNA) extracted from paraffin blocks were degraded, even at lower fixation times. Compared to samples held in the commercial storage reagent, archived tissues showed a poorer amplification. Therefore, a gold standard protocol for DNA/RNA isolation from canine tumor FFPE blocks for molecular investigations is still troublesome. More standardized storage conditions, including time between sample acquisition and fixation, fixation time, and sample thickness, are needed to guarantee the preservation of nucleic acids and, then, their possible use in retrospective transcriptomic analysis.

  8. EVA-enhanced embedding medium for histological analysis of 3D porous scaffold material.

    PubMed

    Lim, Jin Ik; Lee, Yong-Keun

    2009-10-01

    When sectioning a 3D porous scaffold made of a soft elastomeric material embedded in paraffin medium, it is not easy to obtain a section because of the different mechanical properties of the paraffin and tissue/scaffold. We describe a new embedding material for histological analysis of various biomaterials that is composed of paraffin and ethylene vinyl acetate (EVA) resin (0, 3, 7, and 13 wt.%). 3D porous poly(L-lactide-epsilon-caprolactone) (PLCL) and chitosan scaffolds were fabricated to test the sectioning efficiency of the paraffin/EVA embedding material. The new embedding material was characterized by rheological analysis and solvent solubility testing in xylene and n-hexane. The hydrophilicity of the new material was assessed by contact angle measurement and its surface roughness was measured using AFM analysis. The staining efficiency of sections embedded in a paraffin/EVA mixture was determined by eosin staining of the chitosan scaffold and chitosan/collagen hybrid scaffold using a fluorescently labeled collagen. Section roughness decreased with increasing EVA content. The softening temperature of the paraffin/EVA mixture was similar to that of paraffin (50-60 degrees C by rheometer). The paraffin/EVA mixture dissolved completely in xylene after 30min at 50 degrees C, and after 30min in n-hexane at 60 degrees C. Therefore, the new embedding medium can be used for histological analysis of various biomaterials and natural tissues.

  9. Recognition of embedded micrometeorites in thin-sections

    NASA Astrophysics Data System (ADS)

    van Mourik, Jan; de Vet, Sebastiaan

    2010-05-01

    in ectorganic horizons on forest floors. Selected sample locations have >100 years of litter deeper into the profile when humus decomposes and compacts. Given the yearly estimated influx, global circulation models and the hypothesised increase in deposition rates for more temperate latitudes, micrometeorites are considered to be a statistically plausible and identifiable constituent in humus and topsoils. The collected particles reflect the combined deposition of man-made and extraterrestrial sources into the environment, hence requiring a more detailed selection to discern their origins. Several particles have been positively identified as micrometeorites based on their morphology and mineralogy. The micromorphological characteristics were cross-matched with thin-sections of the sample sites obtained from earlier field studies. Analyses of these thin-sections show potential particles to visually match the properties of micrometeorites, indicating a previously unknown constituent in the description of humus forms. In lake deposits and peat bogs are, just vas humus forms, systems where micrometeorites will be sin sedimentary included in the deposits. Stable land surfaces are also subjected to a continuous influx of micrometeorites. Arising research needs resulting from this study identify the relevance of a detailed micromorphological analysis of mineral horizons to understand post sedimentary soil infiltration of the continuous influx of micrometeorites. Micrometeorites can be extracted from samples of ectorganic horizons and the micromorphology of these particles can be described. In thin sections of mormoder humus forms, embedded micrometeorites can be recognized in the organic soil matrix. This study adds a new class of particles to the existing micromorphological interpretations of particles in humus forms and topsoils.

  10. p53 codon 72 polymorphisms and random amplified polymorphic DNA analysis of non-melanoma skin cancer through archival formalin-fixed paraffin-embedded tissue.

    PubMed

    Yoke-Kqueen, Cheah; Ab Mutalib, Nurul-Syakima; Sidik, Shiran Mohd; Learn-Han, Lee; Geok-Chin, Tan

    2012-03-01

    Non-melanoma skin cancer (NMSC) is classified among the ten most frequent cancers in Malaysia. A common polymorphism at codon 72 of the p53 tumor suppressor gene and its influence on cancer risk has been studied for different types of cancer with mixed and inconsistent results with limited published data on the Malaysian population so far. In the present study, the frequency of p53 codon 72 polymorphism in 60 patients with NMSC was investigated from archival formalin-fixed paraffin-embedded (FFPE) tissue obtained from Hospital Universiti Kebangsaan Malaysia (HUKM). Additionally, random amplified polymorhic DNA -polymorphic chain reaction (RAPD-PCR) was employed for preliminary biomarker development. NMSC FFPE samples (70%) possess Arg/Arg, 20% with Pro/Pro and 10% with Arg/Pro. In total, there was no significant difference in the p53 codon 72 genotypes between histological types of NMSC, gender, race, tumor location and age group. However, there was an apparent age-associated increase in the Arg/Arg genotype but did not reach statistical significance (P=0.235). NMSC types and demographic characteristics did not influence genotype distribution. On the other hand, BCC and SCC distributions are influenced by age group, race and tumor location.

  11. Application of BIOMED-2 clonality assays to formalin-fixed paraffin embedded follicular lymphoma specimens: superior performance of the IGK assays compared to IGH for suboptimal specimens.

    PubMed

    Halldórsdóttir, Anna Margrét; Zehnbauer, Barbara A; Burack, W Richard

    2007-07-01

    The BIOMED-2 PCR-based immunoglobulin gene rearrangement assays have quickly become the most commonly used laboratory method for detection of B-cell clonality. Therefore, the reliability of these assays under various conditions has become increasingly important. When studying paired cases of follicular lymphoma (FL) from individual patients, we used these assays to assess clonality in 40 formalin-fixed paraffin-embedded (FFPE) specimens from 19 patients diagnosed with FL. The assays of IGH rearrangement failed to give a clonal result in 26/40 (65%) specimens, while the IGK assays failed in only 3/40 (8%) specimens. The high failure rate of the IGH assays for this set of FFPE lymphomas cannot be explained by systematic problems with DNA extraction or amplification because the same IGH assays resulted in a low failure rate (3/32, 9%) for FFPE small lymphocytic lymphoma/chronic lymphocytic leukemia specimens and for fresh frozen FL specimens (1/6, 17%). Furthermore, in a second validation set of 13 FFPE follicular lymphoma the failure rate was 9/13 (69%). Therefore, the BIOMED-2 IGH assay did not perform well on FFPE follicular lymphoma specimens, and the IGK assay may be superior for assessing clonality when no fresh/frozen tissue is available.

  12. A Comparison of RNA-Seq Results from Paired Formalin-Fixed Paraffin-Embedded and Fresh-Frozen Glioblastoma Tissue Samples

    PubMed Central

    Esteve-Codina, Anna; Arpi, Oriol; Martinez-García, Maria; Pineda, Estela; Mallo, Mar; Gut, Marta; Carrato, Cristina; Rovira, Anna; Lopez, Raquel; Tortosa, Avelina; Dabad, Marc; Del Barco, Sonia; Heath, Simon; Bagué, Silvia; Ribalta, Teresa; Alameda, Francesc; de la Iglesia, Nuria

    2017-01-01

    The molecular classification of glioblastoma (GBM) based on gene expression might better explain outcome and response to treatment than clinical factors. Whole transcriptome sequencing using next-generation sequencing platforms is rapidly becoming accepted as a tool for measuring gene expression for both research and clinical use. Fresh frozen (FF) tissue specimens of GBM are difficult to obtain since tumor tissue obtained at surgery is often scarce and necrotic and diagnosis is prioritized over freezing. After diagnosis, leftover tissue is usually stored as formalin-fixed paraffin-embedded (FFPE) tissue. However, RNA from FFPE tissues is usually degraded, which could hamper gene expression analysis. We compared RNA-Seq data obtained from matched pairs of FF and FFPE GBM specimens. Only three FFPE out of eleven FFPE-FF matched samples yielded informative results. Several quality-control measurements showed that RNA from FFPE samples was highly degraded but maintained transcriptomic similarities to RNA from FF samples. Certain issues regarding mutation analysis and subtype prediction were detected. Nevertheless, our results suggest that RNA-Seq of FFPE GBM specimens provides reliable gene expression data that can be used in molecular studies of GBM if the RNA is sufficiently preserved. PMID:28122052

  13. 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.

  14. 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.

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

    PubMed

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

    2016-09-29

    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.

  16. Chromogenic in situ hybridization is a reliable alternative to fluorescence in situ hybridization for diagnostic testing of 1p and 19q loss in paraffin-embedded gliomas.

    PubMed

    Lass, Ulrike; Hartmann, Christian; Capper, David; Herold-Mende, Christel; von Deimling, Andreas; Meiboom, Maren; Mueller, Wolf

    2013-05-01

    Recent studies imply the importance of rapid and reliable diagnostic assessment of 1p/19q status in oligodendroglial tumors. To date, fluorescence in situ hybridization (FISH) is the most commonly applied technique. FISH, however, has several technical shortcomings that are suboptimal for diagnostic applications: results must be viewed in a fluorescence microscope, results are usually evaluated by a single investigator only, and signal fading excludes physical archiving. Also, in gliomas, the distinction of diffusely infiltrating tumor cells from reactively altered normal tissue may be challenging in fluorescence microscopy. Dual-color chromogenic in situ hybridization (CISH) has started to replace FISH in some diagnostic tests performed in pathology. Here, we present the first single institute experience with a side-by-side analysis of 1p/19q FISH and CISH in a series of 42 consecutive gliomas. FISH and CISH produced identical results for 1p and 19q in 93% of cases (n = 39/42). Discrepant results were reevaluated by repeated FISH and a polymerase chain reaction (PCR)-based microsatellite marker analysis for loss of heterozygosity. Reevaluation confirmed CISH data in all three cases. We conclude that CISH is a reliable alternative in 1p/19q testing in paraffin-embedded tissues likely to be more sensitive to detect 1p/19q status than FISH analysis.

  17. Detection of RET proto-oncogene point mutations in paraffin-embedded pheochromocytoma specimens by nonradioactive single-strand conformation polymorphism analysis and direct sequencing.

    PubMed Central

    Komminoth, P.; Kunz, E.; Hiort, O.; Schröder, S.; Matias-Guiu, X.; Christiansen, G.; Roth, J.; Heitz, P. U.

    1994-01-01

    The suitability of formalin-fixed and paraffin-embedded tumor material was evaluated for molecular analysis of the RET proto-oncogene. We analyzed exons 10, 11, and 16 for point mutations in seven sporadic and six multiple endocrine neoplasia (MEN) 2A-associated pheochromocytomas by a nonradioactive single-strand conformation polymorphism assay followed by nonradioactive direct sequencing of PCR-amplified DNA using an automated DNA sequencer. All MEN 2A-associated pheochromocytomas contained a heterozygous missense germline mutation within cystine codons of the cysteine-rich extracellular domain encoded by exons 10 and 11. Mutations were located in codon 619 (TGC-->TCC; Cys-->Ser) in one, in codon 635 (TGC-->CGC; Cys--Arg) in three, and in codon 635 (TGC-->TAC; Cys-->Tyr) in two pheochromocytomas. No tumor-specific (somatic) mutations were detected in exons 10, 11, and 16 of the sporadic pheochromocytomas. These data support recent findings that germline point mutations that are clustered in distinct cysteine codons of the RET proto-oncogene are involved in the neoplastic phenotype of the MEN 2A syndrome. Our results demonstrate that both nonradioactive single-strand conformation polymorphism and direct sequencing are suitable methods to detect single base substitutions in DNA extracted from archival material. Images Figure 1 Figure 4 PMID:7943181

  18. Initial Development and Validation of a Novel Extraction Method for Quantitative Mining of the Formalin-Fixed, Paraffin-Embedded Tissue Proteome for Biomarker Investigations

    PubMed Central

    2010-01-01

    Annotated formalin-fixed, paraffin-embedded (FFPE) tissue archives constitute a valuable resource for retrospective biomarker discovery. However, proteomic exploration of archival tissue is impeded by extensive formalin-induced covalent cross-linking. Robust methodology enabling proteomic profiling of archival resources is urgently needed. Recent work is beginning to support the feasibility of biomarker discovery in archival tissues, but further developments in extraction methods which are compatible with quantitative approaches are urgently needed. We report a cost-effective extraction methodology permitting quantitative proteomic analyses of small amounts of FFPE tissue for biomarker investigation. This surfactant/heat-based approach results in effective and reproducible protein extraction in FFPE tissue blocks. In combination with a liquid chromatography−mass spectrometry-based label-free quantitative proteomics methodology, the protocol enables the robust representative and quantitative analyses of the archival proteome. Preliminary validation studies in renal cancer tissues have identified typically 250−300 proteins per 500 ng of tissue with 1D LC−MS/MS with comparable extraction in FFPE and fresh frozen tissue blocks and preservation of tumor/normal differential expression patterns (205 proteins, r = 0.682; p < 10−15). The initial methodology presented here provides a quantitative approach for assessing the potential suitability of the vast FFPE tissue archives as an alternate resource for biomarker discovery and will allow exploration of methods to increase depth of coverage and investigate the impact of preanalytical factors. PMID:21117664

  19. 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.

  20. Characterization of a Novel Anti-Human HB-EGF Monoclonal Antibody Applicable for Paraffin-Embedded Tissues and Diagnosis of HB-EGF-Related Cancers.

    PubMed

    Iwamoto, Ryo; Takagi, Mika; Akatsuka, Jun-Ichi; Ono, Ken-Ichiro; Kishi, Yoshiro; Mekada, Eisuke

    2016-04-01

    Heparin-binding EGF-like growth factor (HB-EGF) is a member of the EGF family of growth factors that bind to and activate the EGF receptor (EGFR/ErbB1) and ErbB4. HB-EGF plays pivotal roles in pathophysiological processes, including cancer. Thus, monoclonal antibodies (mAbs) for HB-EGF detection could be an important tool in the therapeutic diagnosis of HB-EGF-related cancers and other diseases. However, few mAbs, especially those applicable for immunohistochemistry (IHC), have been established to date. In this study, we generated a clone of hybridoma-derived mAb 2-108 by immunizing mice with recombinant human HB-EGF protein expressed by human cells. The mAb 2-108 specifically bound to human HB-EGF but not to mouse HB-EGF and was successful in immunoblotting, even under reducing conditions, immunoprecipitation, and immunofluorescence for unfixed as well as paraformaldehyde-fixed cells. Notably, this mAb was effective in IHC of paraffin-embedded tumor specimens. Epitope mapping analysis showed that mAb 2-108 recognized the N-terminal prodomain in HB-EGF. These results indicate that this new anti-HB-EGF mAb 2-108 would be useful in the diagnosis of HB-EGF-related cancers and would be a strong tool in both basic and clinical research on HB-EGF.

  1. Intranuclear detection of African swine fever virus DNA in several cell types from formalin-fixed and paraffin-embedded tissues using a new in situ hybridisation protocol.

    PubMed

    Ballester, M; Galindo-Cardiel, I; Gallardo, C; Argilaguet, J M; Segalés, J; Rodríguez, J M; Rodríguez, F

    2010-09-01

    In this study, a new in situ hybridisation (ISH) protocol has been developed to identify African swine fever virus (ASFV) genome in formalin-fixed, paraffin-embedded tissues. Different digoxigenin labelled ASFV-probes were tested, including single ASFV-specific oligonucleotides, an 18.5kb restriction fragment from the viral genome and the entire ASFV genome. The latter showed the highest sensitivity in all tissues tested, independently of the virus used for challenge: E75L or Ba71L. Although a similar ASFV genome distribution was observed, the number of ISH-positive cells was higher for Ba71L compared to E75L infected tissues. As expected, the monocyte-macrophage cell lineage was the main target cell for ASFV infection. Corresponding with the last stages of infection, ISH-positive signals were also found in other cell types, including endothelial cells, hepatocytes and neutrophils. Furthermore, two unexpected findings were also noticed: the detection of a specific ISH-signal in lymphocytes and a tendency to find the signal in the nucleus of infected cells. In summary, the present findings demonstrate the utility of this new ISH protocol to study ASFV pathogenesis and its potential use as a diagnostic tool.

  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. Degradation of fungal DNA in formalin-fixed paraffin-embedded sinus fungal balls hampers reliable sequence-based identification of fungi.

    PubMed

    Cabaret, Odile; Toussain, Guillaume; Abermil, Nassera; Alsamad, Issam Abd; Botterel, Françoise; Costa, Jean-Marc; Papon, Jean-François; Bretagne, Stéphane

    2011-04-01

    Identification of the etiologic agent responsible for sinus fungal ball (SFB) is rarely obtained due to either the culture of patient specimens not being ordered or if cultures were inoculated they proved to be negative. Obviously, this has a significant impact on the design of appropriate therapeutic strategies. We investigated whether paraffin-embedded (PE) tissues, the only materials often available, were suitable for the correct identification of the responsible fungi. We obtained PE tissues of SFB from 16 different patients who had risk factors for invasive fungal infections. DNA was extracted using an automated extractor and the internal transcribed spacer (ITS) sequenced following amplification with two sets of primers designed to amplify >300 bp fragments. This was attempted in parallel with a real-time quantitative PCR assay targeting Aspergillus spp. mitochondrial DNA designed to amplify <150 bp fragments. ITS sequencing succeeded in appropriately identifying the etiologic agents in 10 of the 16 samples (nine Aspergillus fumigatus, one Lewia spp.). In contrast, the <150 bp PCR assay amplified all specimens correctly except the one involving Lewia spp. If fungal identification is warranted to understand the pathophysiology of SFB and guide clinicians, we cannot rely only on ITS sequencing of the DNA obtained from PE tissues. The main reason is probably due to the fact that formalin prevents amplification of long DNA fragments and consequently, frozen or fresh tissues should be employed.

  4. Quantitative label-free mass spectrometry analysis of formalin-fixed, paraffin-embedded tissue representing the invasive cutaneous malignant melanoma proteome

    PubMed Central

    Dowling, Paul; Moran, Benvon; McAuley, Edel; Meleady, Paula; Henry, Michael; Clynes, Martin; McMenamin, Mairin; Leonard, Niamh; Monks, Mary; Wynne, Bairbre; Ormond, Patrick; Larkin, Annemarie

    2016-01-01

    Understanding the events at a protein level that govern the progression from melanoma in situ to invasive melanoma are important areas of current research to be developed. Recent advances in the analysis of formalin-fixed, paraffin-embedded tissue by proteomics, particularly using the filter-aided sample preparation protocol, has opened up the possibility of studying vast archives of clinical material and associated medical records. In the present study, quantitative protein profiling was performed using tandem mass spectrometry, and the proteome differences between melanoma in situ and invasive melanoma were compared. Biological pathway analyses revealed several signalling pathways differing between melanoma in situ and invasive melanoma, including metabolic pathways and the phosphoinositide 3-kinase-Akt signalling pathway. Selected proteins of interest (14–3-3ε and fatty acid synthase) were subsequently investigated using immunohistochemical analysis of tissue microarrays. Identifying the key proteins that play significant roles in the establishment of a more invasive phenotype in melanoma may ultimately aid diagnosis and treatment decisions. PMID:27899996

  5. Trypanosoma cruzi Necrotizing Meningoencephalitis in a Venezuelan HIV+-AIDS Patient: Pathological Diagnosis Confirmed by PCR Using Formalin-Fixed- and Paraffin-Embedded-Tissues

    PubMed Central

    Rossi Spadafora, Marcello Salvatore; Céspedes, Ghislaine; Romero, Sandra; Fuentes, Isabel; Boada-Sucre, Alpidio A.; Cañavate, Carmen; Flores-Chávez, María

    2014-01-01

    Coinfections with human immunodeficiency virus (HIV) and infectious agents have been recognized since the early 90s. In the central nervous system (CNS) of HIV+ patients, parasitic protozoans like Toxoplasma gondii have been described as responsible for the space occupying lesions (SOL) developed. However, the involvement of Trypanosoma cruzi is also described but appears to be less frequent in acquired immunodeficiency syndrome (AIDS) and transplant recipients, associated with necrotizing myocarditis and neurological symptoms related to the occurrence of necrotizing pseudotumoral encephalitis (NPE) and meningoencephalitis (NME). The present work aims to present a Venezuelan case of NME associated with the coinfection of HIV and a T. cruzi-like trypanosomatid as well as its evolution and diagnosis by histopathological techniques, electron microscopy, and PCR methods using formalin-fixed- (FF-) and paraffin-embedded- (PE-) tissues. Postmortem cytological studies of leptomeninges imprints reveal the presence of trypomastigotes of Trypanosoma sp. Histopathological and electron microscopy studies allowed us to identify an amastigote stage and to reject the involvement of other opportunistic microorganisms as the etiological agent of the SOL. The definitive confirmation of T. cruzi as the etiological agent was achieved by PCR suggesting that the NME by T. cruzi was due to a reactivation of Chagas' disease. PMID:25763312

  6. Evaluation of positive Rift Valley fever virus formalin-fixed paraffin embedded samples as a source of sequence data for retrospective phylogenetic analysis.

    PubMed

    Mubemba, B; Thompson, P N; Odendaal, L; Coetzee, P; Venter, E H

    2017-05-01

    Rift Valley fever (RVF), caused by an arthropod borne Phlebovirus in the family Bunyaviridae, is a haemorrhagic disease that affects ruminants and humans. Due to the zoonotic nature of the virus, a biosafety level 3 laboratory is required for isolation of the virus. Fresh and frozen samples are the preferred sample type for isolation and acquisition of sequence data. However, these samples are scarce in addition to posing a health risk to laboratory personnel. Archived formalin-fixed, paraffin-embedded (FFPE) tissue samples are safe and readily available, however FFPE derived RNA is in most cases degraded and cross-linked in peptide bonds and it is unknown whether the sample type would be suitable as reference material for retrospective phylogenetic studies. A RT-PCR assay targeting a 490 nt portion of the structural GN glycoprotein encoding gene of the RVFV M-segment was applied to total RNA extracted from archived RVFV positive FFPE samples. Several attempts to obtain target amplicons were unsuccessful. FFPE samples were then analysed using next generation sequencing (NGS), i.e. Truseq(®) (Illumina) and sequenced on the Miseq(®) genome analyser (Illumina). Using reference mapping, gapped virus sequence data of varying degrees of shallow depth was aligned to a reference sequence. However, the NGS did not yield long enough contigs that consistently covered the same genome regions in all samples to allow phylogenetic analysis.

  7. 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. Copyright © 2013 Elsevier GmbH. All rights reserved.

  8. 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.

  9. Comparative investigations of T cell receptor gamma gene rearrangements in frozen and formalin-fixed paraffin wax-embedded tissues by capillary electrophoresis.

    PubMed

    Christensen, M; Funder, A D; Bendix, K; Soerensen, F B

    2006-06-01

    To compare clonal T cell receptor gamma (TCRgamma) gene rearrangements in frozen and formalin-fixed paraffin wax-embedded (FFPE) tissue, using capillary electrophoresis for use in diagnostics, as T cell lymphomas may be difficult to diagnose by conventional methods. The DNA for PCR was extracted from frozen and FFPE tissue, cell lines and blood. PCR primers Vgamma1-8, Vgamma9, Vgamma10 or Vgamma11 (5' end labelled) combined with a mixture of JgammaP1/JgammaP/JgammaP2/Jgamma2 (unlabelled) were used. Monoclonal cases were sequenced and clonality, reproducibility, sensitivity and specificity analyses were carried out. In all cases the molecular test was found to be in agreement with the histological diagnosis. Discrepancies were found between frozen and FFPE tissue in 18 of 56 (32%) tests. The method was highly reproducible. The sensitivity was found to be 0.5% for cell lines and 1% for patient specimens and the specificity 100%. The junctional region between the Vgamma and Jgamma segments was specific for each patient. Capillary electrophoresis of PCR products from frozen and FFPE tissue is suitable for detecting clonal TCRgamma gene rearrangements. It is important, however, to correlate the results with conventional morphological and immunohistochemical studies.

  10. Primary local orbital amyloidosis: biochemical identification of the immunoglobulin light chain kappaIII subtype in a small formalin fixed, paraffin wax embedded tissue sample.

    PubMed

    Kaplan, B; Martin, B M; Cohen, H I; Manaster, J; Kassif, Y; Rehany, U; Livneh, A

    2005-05-01

    Amyloidosis refers to a heterogeneous group of disorders associated with the deposition of chemically distinct amyloid fibril proteins. Precise determination of chemical amyloid type has diagnostic, therapeutic, and prognostic relevance. Although immunohistochemical techniques are used routinely to determine the amyloid type, the results can be negative or inconclusive, so that biochemical characterisation is often required. The development and application of new biochemical microtechniques suitable for examination of extremely small tissue samples is essential for precise identification of the deposited amyloid proteins. To investigate biochemically the amyloid proteins present in a formalin fixed paraffin wax embedded orbital tissue from a patient with localised orbital amyloidosis in whom immunohistochemistry was not helpful in the determination of amyloid type. Extraction of amyloid proteins from fixed tissue and their identification was carried out by a recently developed microtechnique. An extremely small tissue sample was dewaxed and extracted with formic acid. The extracted material was analysed using electrophoresis, western blotting, and amino acid sequencing. Biochemical examination of the extracted proteins showed the presence of immunoglobulin (Ig) derived amyloid proteins, which were composed of the N-terminal fragments of the Ig light chain kappaIII subtype (AL-kappaIII) (16, 8, and 3 kDa). This is the first chemically proved AL case reported in association with primary localised orbital amyloidosis. The biochemical microtechnique used was useful in achieving a precise diagnosis of amyloid disease, in a case where the results of routine immunohistochemical examination of amyloid were inconclusive.

  11. Development of in-situ hybridization for the detection of Mycoplasma haemosuis (Eperythrozoon suis) in formalin-fixed, paraffin wax-embedded tissues from experimentally infected splenectomized pigs.

    PubMed

    Ha, S-K; Jung, K; Choi, C; Ha, Y; Song, H-C; Lim, J-H; Kim, S-H; Chae, C

    2005-11-01

    Mycoplasma haemosuis DNA was detected in experimentally infected splenectomized pigs by in-situ hybridization (ISH) with a nonradioactive digoxigenin-labelled DNA probe. An 839 base pair DNA probe targeting a 16S rRNA gene was generated by the polymerase chain reaction. Eight 6-week-old pigs were inoculated intraperitoneally with 6 ml of M. haemosuis-infected pig blood and eight negative control pigs were inoculated intraperitoneally with 6 ml of M. haemosuis-free blood. Two pigs from each group were killed for examination at 3, 7, 15 and 30 days post-inoculation (dpi). Red blood cells infected with M. haemosuis were first detected by light microscopy at 3 to 7 dpi. No M. haemosuis was observed in negative control pigs. Hybridization signals were evident in blood from the infected pigs at 3 dpi. The ISH method developed in this study was useful for the detection of M. haemosuis DNA in formalin-fixed, paraffin wax-embedded tissues and may be valuable for studying the pathogenesis of M. haemosuis infection.

  12. Quantitative real-time polymerase chain reaction is an alternative method for the detection of HER-2 amplification in formalin-fixed paraffin-embedded breast cancer samples.

    PubMed

    Pu, Tianjie; Guo, Peng; Qiu, Yan; Chen, Shinan; Yang, Libo; Sun, Linyong; Ye, Feng; Bu, Hong

    2015-01-01

    Fluorescent in situ hybridization (FISH) and immunohistochemistry (IHC) are the most common methods that are used to quantify HER-2 gene and protein levels, respectively, in human breast cancer. However, due to bad sample quality, some samples are unable to be subjected to a FISH assay. We evaluated 71 formalin-fixed paraffin-embedded (FFPE) breast carcinoma specimens by quantitative real-time polymerase chain reaction (qPCR), IHC, and FISH. We also performed qPCR and FISH assays on delayed formalin-fixed (DDF) samples. The qPCR results were in complete concordance with the results of IHC and FISH. In regards to the DDF samples, the HER-2 fluorescent signal seemed decayed compared with that of the DDF samples after 1 h. However, the qPCR method still works well up to 12 hours. Our results indicated that qPCR was obviously superior to FISH in cases that were not fixed in a reasonable amount of time. However, qPCR can be an alternative method by which to perform HER2 amplification assays in breast cancer.

  13. Improved RNA quality and TaqMan® Pre-amplification method (PreAmp) to enhance expression analysis from formalin fixed paraffin embedded (FFPE) materials

    PubMed Central

    Li, Jinghuan; Smyth, Paul; Cahill, Susanne; Denning, Karen; Flavin, Richard; Aherne, Sinead; Pirotta, Marco; Guenther, Simone M; O'Leary, John J; Sheils, Orla

    2008-01-01

    Background Archival formalin-fixed paraffin-embedded (FFPE) tissues represent an abundant source of clinical specimens; however their use is limited in applications involving analysis of gene expression due to RNA degradation and modification during fixation and processing. This study improved the quality of RNA extracted from FFPE by introducing a heating step into the selected extraction protocols. Further, it evaluated a novel pre-amplification system (PreAmp) designed to enhance expression analysis from tissue samples using assays with a range of amplicon size (62–164 bp). Results Results from the Bioanalyzer and TaqMan® data showed improvement of RNA quality extracted using the modified protocols from FFPE. Incubation at 70°C for 20 minutes was determined to be the best condition of those tested to disrupt cross-links while not compromising RNA integrity. TaqMan® detection was influenced by master mix, amplicon size and the incorporation of a pre-amplification step. TaqMan® PreAmp consistently achieved decreased CT values in both snap frozen and FFPE aliquots compared with no pre-amplification. Conclusion Modification to extraction protocols has facilitated procurement of RNA that may be successfully amplified using QRT-PCR. TaqMan® PreAmp system is a robust and practical solution to limited quantities of RNA from FFPE extracts. PMID:18254955

  14. Utility of the Roche Cobas 4800 for detection of high-risk human papillomavirus in formalin-fixed paraffin-embedded oropharyngeal squamous cell carcinoma.

    PubMed

    Pettus, Jason R; Wilson, Terri L; Steinmetz, Heather B; Lefferts, Joel A; Tafe, Laura J

    2017-02-01

    Clinical laboratories are expected to reliably identify human papilloma virus (HPV) associated oropharyngeal squamous cell carcinoma (OPSCC) for prognostic and potential therapeutic applications. In addition to surrogate p16 immunohistochemistry (IHC) testing, DNA-based HPV-specific testing strategies are widely utilized. Recognizing the efficiency of the Roche Cobas 4800 platform for testing gynecological cytology specimens for high-risk HPV, we elected to evaluate the potential utility of this platform for testing formalin-fixed paraffin-embedded (FFPE) OPSCC tissue. Using the Roche Linear Array assay for comparison, we tested twenty-eight samples (16 primary OPSCC, 2 lymph node metastases from primary OPSCC, 1 oral tongue carcinoma, 3 benign squamous papillomas, and 3 non-oropharyngeal carcinoma tissues). Excluding two invalid results, the Roche Cobas 4800 testing resulted in excellent inter-assay concordance (25/26, 96.2%) and 100% concordance for HPV-16/HPV-18 positive samples. This data suggests that the Roche Cobas 4800 platform may be a cost-effective method for testing OPSCC FFPE tissues in a clinical molecular pathology laboratory setting. Copyright © 2016 Elsevier Inc. All rights reserved.

  15. Trypanosoma cruzi necrotizing meningoencephalitis in a Venezuelan HIV⁺-AIDS patient: pathological diagnosis confirmed by PCR using formalin-fixed- and paraffin-embedded-tissues.

    PubMed

    Rossi Spadafora, Marcello Salvatore; Céspedes, Ghislaine; Romero, Sandra; Fuentes, Isabel; Boada-Sucre, Alpidio A; Cañavate, Carmen; Flores-Chávez, María

    2014-01-01

    Coinfections with human immunodeficiency virus (HIV) and infectious agents have been recognized since the early 90s. In the central nervous system (CNS) of HIV(+) patients, parasitic protozoans like Toxoplasma gondii have been described as responsible for the space occupying lesions (SOL) developed. However, the involvement of Trypanosoma cruzi is also described but appears to be less frequent in acquired immunodeficiency syndrome (AIDS) and transplant recipients, associated with necrotizing myocarditis and neurological symptoms related to the occurrence of necrotizing pseudotumoral encephalitis (NPE) and meningoencephalitis (NME). The present work aims to present a Venezuelan case of NME associated with the coinfection of HIV and a T. cruzi-like trypanosomatid as well as its evolution and diagnosis by histopathological techniques, electron microscopy, and PCR methods using formalin-fixed- (FF-) and paraffin-embedded- (PE-) tissues. Postmortem cytological studies of leptomeninges imprints reveal the presence of trypomastigotes of Trypanosoma sp. Histopathological and electron microscopy studies allowed us to identify an amastigote stage and to reject the involvement of other opportunistic microorganisms as the etiological agent of the SOL. The definitive confirmation of T. cruzi as the etiological agent was achieved by PCR suggesting that the NME by T. cruzi was due to a reactivation of Chagas' disease.

  16. Primary local orbital amyloidosis: biochemical identification of the immunoglobulin light chain κIII subtype in a small formalin fixed, paraffin wax embedded tissue sample

    PubMed Central

    Kaplan, B; Martin, B M; Cohen, H I; Manaster, J; Kassif, Y; Rehany, U; Livneh, A

    2005-01-01

    Background: Amyloidosis refers to a heterogeneous group of disorders associated with the deposition of chemically distinct amyloid fibril proteins. Precise determination of chemical amyloid type has diagnostic, therapeutic, and prognostic relevance. Although immunohistochemical techniques are used routinely to determine the amyloid type, the results can be negative or inconclusive, so that biochemical characterisation is often required. The development and application of new biochemical microtechniques suitable for examination of extremely small tissue samples is essential for precise identification of the deposited amyloid proteins. Aims: To investigate biochemically the amyloid proteins present in a formalin fixed paraffin wax embedded orbital tissue from a patient with localised orbital amyloidosis in whom immunohistochemistry was not helpful in the determination of amyloid type. Methods: Extraction of amyloid proteins from fixed tissue and their identification was carried out by a recently developed microtechnique. An extremely small tissue sample was dewaxed and extracted with formic acid. The extracted material was analysed using electrophoresis, western blotting, and amino acid sequencing. Results: Biochemical examination of the extracted proteins showed the presence of immunoglobulin (Ig) derived amyloid proteins, which were composed of the N-terminal fragments of the Ig light chain κIII subtype (AL-κIII) (16, 8, and 3 kDa). Conclusions: This is the first chemically proved AL case reported in association with primary localised orbital amyloidosis. The biochemical microtechnique used was useful in achieving a precise diagnosis of amyloid disease, in a case where the results of routine immunohistochemical examination of amyloid were inconclusive. PMID:15858128

  17. 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. Copyright © 2014 Elsevier Inc. All rights reserved.

  18. Immunocytochemistry performed on the cell-transferred direct smears of the fine-needle aspirates: a comparison study with the corresponding formalin-fixed paraffin-embedded tissue.

    PubMed

    Wu, Howard H; Jones, Kelly J; Cramer, Harvey M

    2013-06-01

    Immunocytochemistry (ICC) performed on the cell-transferred cytologic smears (CTCS) of fine-needle aspiration (FNA) is useful when the cell blocks lack adequate material. The comparison of the ICC results from the CTCS of FNA with the corresponding formalin-fixed paraffin-embedded tissue (FFPE) has not been reported previously. We applied 12 commonly used ICC antibodies on 160 pieces of ethanol-fixed, cell-transferred Papanicolaou-stained smears obtained from 42 FNA specimens and compared the staining results with the corresponding FFPE on which the same panel of immunostains was performed. Of the 160 pieces of transferred materials, only 3 (1.9%) were lost during specimen processing. In total, 153 of 157 (97.5%) showed staining results that agreed with the corresponding FFPE, including 78 of 81 positive staining and 75 of 76 negative staining cases. ICC performed on the cell-transferred FNA smears is reliable and shows staining results highly comparable with the corresponding FFPE tissue.

  19. Sequential real-time PCR assays applied to identification of genomic signatures in formalin-fixed paraffin-embedded tissues: a case report about brucella-induced osteomyelitis.

    PubMed

    Zhang, Binxue; Wear, Douglas J; Stojadinovic, Alexander; Izadjoo, Mina

    2013-01-01

    Brucellosis is a zoonotic infection transmitted from animals to human by ingestion of infected food products, direct contact with an infected animal, or inhalation of aerosols. Brucella infection-induced osteomyelitis may present only with nonspecific clinical and radiographic findings, mild elevations in serum inflammatory markers, as well as nonspecific histological changes. We studied a case of an Iraqi war veteran with multifocal vertebral body and left iliac bone lesions on radio nucleotide scans and magnetic resonance imaging, clinically suspected osteomyelitis possibly because of Brucella. Although histomorphological findings were nonspecific, consisting of chronic inflammatory cell infiltrate and reactive fibrosis, tissue gram and silver impregnation stains of bone biopsies were informative, revealing gram-negative coccobacilli consistent in size with Brucella species. Total nucleic acids were extracted from formalin-fixed paraffin-embedded tissues and amplified by sequential real-time polymerase chain reaction, targeting genes coding (1) outer membrane protein (omp-31) of Brucella species and (2) insertion sequence (IS711) of Brucella abortus (b-abt). Polymerase chain reaction results confirmed B. abortus as the causative pathogens for presumed diagnosis of Brucella osteomyelitis.

  20. A Novel Monoclonal Antibody Against Alpha-Methylacyl-CoA Racemase Applicable for Paraffin-Embedded Tissues and Diagnostics of Prostate Cancer.

    PubMed

    Kovaleva, Olga V; Samoilova, Daria V; Shitova, Maria S; Oleinikova, Nina A; Danilova, Natalia V; Malkov, Pavel G; Gratchev, Alexei

    2017-02-01

    AMACR (alpha-methylacyl-CoA racemase) has been recently described as a prostate cancer-specific gene that encodes a protein involved in the beta-oxidation of branched chain fatty acids. Expression of AMACR protein is found in prostatic adenocarcinoma, but not in benign prostatic tissue. Thus, monoclonal antibodies (mAbs) for AMACR detection are an important tool for the diagnosis of AMACR-positive cancers. However, only a few mAbs, especially those applicable for immunohistochemistry (IHC), have been established to date. In this study, we describe the generation of a new hybridoma clone G8 producing anti-AMACR antibodies. G8 mAb specifically binds human AMACR and was successfully used in immunoblotting and immunofluorescence on paraformaldehyde-fixed cells and in IHC of paraffin-embedded tumor specimens. These results indicate that this new anti-AMACR mAb G8 would be useful in the diagnosis of AMACR-related cancers and would be a strong tool in both basic and clinical research on AMACR.

  1. Deparaffinization with mineral oil: a simple procedure for extraction of high-quality DNA from archival formalin-fixed paraffin-embedded samples.

    PubMed

    Heikal, Nahla; Nussenzveig, Roberto H; Agarwal, Archana M

    2014-09-01

    Extracting DNA from formalin-fixed paraffin-embedded (FFPE) archival samples remains difficult. Successful polymerase chain reactions (PCR) with DNA extracted from FFPE samples is still very low. We extracted DNA from 12 recent and old archival FFPE bone marrow trephine biopsies by use of a simple protocol on the basis of deparaffinization with molecular biology-grade mineral oil followed by DNA extraction with the Qiagen FFPE kit. Comparison of this deparaffinization method with standard protocols, for example, xylene or Hemo-D with subsequent rehydration using graded ethanols, was investigated. The quality and quantity of extracted DNA were tested by a combination of ultraviolet spectroscopy, analysis on a Caliper LabChip GX, and real-time PCR combined with high-resolution melt analysis. Highest quality PCR-amplifiable DNA was obtained by deparaffinization with mineral oil, whereas more variable results were obtained for the other 2 deparaffinization procedures. This result was confirmed by real-time PCR and high-resolution melt analysis. Besides improvements in the quality of extracted DNA, use of mineral oil for deparaffinization has the added benefit of decreased time (20 vs. 75 min) and a significant reduction of hands-on labor (1 step vs. multiple hands-on centrifugation and decanting steps).

  2. Proteomic analysis of formalin-fixed paraffin-embedded pancreatic tissue using liquid chromatography tandem mass spectrometry (LC-MS/MS)

    PubMed Central

    Paulo, Joao A.; Lee, Linda S.; Banks, Peter A.; Steen, Hanno; Conwell, Darwin L.

    2012-01-01

    Objectives Formalin-fixed paraffin-embedded (FFPE) tissue is a standard method of specimen preservation for hospital pathology departments. FFPE tissue banks are a resource of histologically-characterized specimens for retrospective biomarker investigation. We aim to establish liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) analysis of FFPE pancreatic tissue as a suitable strategy for the study of the pancreas proteome. Methods We investigated the proteomic profile of FFPE pancreatic tissue specimens, using LC-MS/MS, from 9 archived specimens that were histologically-classified as: normal (n=3), chronic pancreatitis (n=3), and pancreatic cancer (n=3). Results We identified 525 non-redundant proteins from 9 specimens. Implementing our filtering criteria, 78, 15, and 21 proteins were identified exclusively in normal, chronic pancreatitis, and pancreatic cancer specimens, respectively. Several proteins were identified exclusively in specimens with no pancreatic disease: spink1, retinol dehydrogenase, and common pancreatic enzymes. Similarly, proteins were identified exclusively in chronic pancreatitis specimens: collagen α1(XIV), filamin A, collagen α3(VI), and SNC73. Proteins identified exclusively in pancreatic cancer included: annexin 4A and fibronectin. Conclusions We report that differentially-expressed proteins can be identified among FFPE tissues specimens originating from individuals with different pancreatic histologies. The mass spectrometry-based methodology used herein has the potential to enhance biomarker discovery and chronic pancreatitis research. PMID:22015969

  3. Hydrolysis Profiles of Formalin Fixed Paraffin-Embedded Tumors Based on IOD (Integrated Optical Density) and Nuclear Texture Feature Measurements

    PubMed Central

    Fležar, Margareta; Us‐Krašovec, Marija; žganec, Mario; Lavrenčak, Jaka; Golouh, Rastko

    1999-01-01

    The aim of the study was to determine optimal hydrolysis time for the Feulgen DNA staining of archival formalin fixed paraffin‐embedded surgical samples, prepared as single cell suspensions for image cytometric measurements. The nuclear texture features along with the IOD (integrated optical density) of the tumor nuclei were analysed by an automated high resolution image cytometer as a function of duration of hydrolysis treatment (in 5 N HCl at room temperature). Tissue blocks of breast carcinoma, ovarian serous carcinoma, ovarian serous tumor of borderline malignancy and leiomyosarcoma were included in the study. IOD hydrolysis profiles showed plateau between 30 and 60 min in the breast carcinoma and leiomyosarcoma, and between 40 and 60 min in the ovarian serous carcinoma and ovarian serous tumor of borderline malignancy. Most of the nuclear texture features remained stable after 20 min of hydrolysis treatment. Our results indicate that the optimal hydrolysis time for IOD and for nuclear texture feature measurements, was between 40 and 60 min in the cell preparations from tissue blocks of three epithelial and one soft tissue tumor. PMID:10866273

  4. Automated Extraction of Formalin-Fixed, Paraffin-Embedded Tissue for High-Risk Human Papillomavirus Testing of Head and Neck Squamous Cell Carcinomas Using the Roche Cobas 4800 System.

    PubMed

    Kerr, Darcy A; Sweeney, Brenda; Arpin, Ronald N; Ring, Melissa; Pitman, Martha B; Wilbur, David C; Faquin, William C

    2016-08-01

    -Testing for high-risk human papillomavirus (HR-HPV) in head and neck squamous cell carcinomas (HNSCCs) is important for both prognostication and clinical management. Several testing platforms are available for HR-HPV; however, effective alternative automated approaches are needed. -To assess the performance of the automated Roche cobas 4800 HPV real-time polymerase chain reaction-based system on formalin-fixed, paraffin-embedded HNSCC specimens and compare results with standard methods of in situ hybridization (ISH) and p16 immunohistochemistry. -Formalin-fixed, paraffin-embedded samples of HNSCC were collected from archival specimens in the Department of Pathology, Massachusetts General Hospital (Boston), and prepared using the automated system by deparaffinization and dehydration followed by tissue lysis. Samples were integrated into routine cervical cytology testing runs by cobas. Corresponding formalin-fixed, paraffin-embedded samples were evaluated for HR-HPV by ISH and p16 by immunohistochemistry. Discrepant cases were adjudicated by polymerase chain reaction. -Sixty-two HNSCC samples were analyzed using the automated cobas system, ISH, and immunohistochemistry. Fifty-two percent (n = 32 of 62) of formalin-fixed, paraffin-embedded tumors were positive for HR-HPV by cobas. Eighty-eight percent (n = 28 of 32) of cases were the HPV 16 subtype and 12% (n = 4 of 32) were other HR-HPV subtypes. Corresponding testing with ISH was concordant in 92% (n = 57 of 62) of cases. Compared with the adjudication polymerase chain reaction standard, there were 3 false-positive cases by cobas. -Concordance in HNSCC HR-HPV status between cobas and ISH was more than 90%. The cobas demonstrated a sensitivity of 100% and a specificity of 91% for detection of HR-HPV. Advantages favoring cobas include its automation, cost efficiency, objective results, and ease of performance.

  5. Small RNA Sequencing for Profiling MicroRNAs in Long-Term Preserved Formalin-Fixed and Paraffin-Embedded Non-Small Cell Lung Cancer Tumor Specimens

    PubMed Central

    Kadota, Kyuichi; Kannisto, Eric; Jones, David R.; Adusumilli, Prasad S.

    2015-01-01

    Background The preservation of microRNAs in formalin-fixed and paraffin-embedded (FFPE) tissue makes them particularly useful for biomarker studies. The utility of small RNA sequencing for microRNA expression profiling of FFPE samples has yet to be determined. Methods Total RNA was extracted from de-paraffinized and proteinase K-treated FFPE specimens (15–20 years old) of 8 human lung adenocarcinoma tumors by affinity chromatography on silica columns. MicroRNAs in the RNA preparations were quantified by the Illumina HiSeq 2000 sequencing platform with sequencing libraries prepared with the TruSeq Small RNA Sample Preparation Kit (version 2.0) to obtain unpaired reads of 50 b for small RNA fragments. MicroRNAs were also quantified using Agilent Human miRNA (release 16.0) microarrays that can detect 1,205 mature microRNAs and by quantitative reverse transcription (RT)-PCR assays. Results Between 9.1–16.9 million reads were obtained by small RNA sequencing of extracted RNA samples. Of these, only 0.6–2.3% (mean = 1.5%) represented microRNAs. The sequencing method detected 454–625 microRNAs/sample (mean = 550) compared with 200–349 (mean = 286) microRNAs detected by microarray. In Spearman correlation analyses, the average correlation coefficient for the 126 microRNAs detected in all samples by both methods was 0.37, and >0.5 for 63 microRNAs. In correlation analyses of the sequencing- and RT-PCR-based measurements, the coefficients were 0.19–0.95 (mean = 0.73) and >0.7, respectively, for 7 of 9 examined microRNAs. The average inter-replicate Spearman correlation coefficient for the sequencing method was 0.81. Conclusions Small RNA sequencing can be used to obtain microRNA profiles of FFPE tissue specimens with performance characteristics similar to those of microarrays, in spite of the fragmentation of ribosomal and messenger RNAs that reduces the method's informative capacity. The accuracy of the method can conceivably be improved by increasing sequencing

  6. A practical approach to the clinical diagnosis of Ewing's sarcoma/primitive neuroectodermal tumour and other small round cell tumours sharing EWS rearrangement using new fluorescence in situ hybridisation probes for EWSR1 on formalin fixed, paraffin wax embedded tissue.

    PubMed

    Yamaguchi, U; Hasegawa, T; Morimoto, Y; Tateishi, U; Endo, M; Nakatani, F; Kawai, A; Chuman, H; Beppu, Y; Endo, M; Kurotaki, H; Furuta, K

    2005-10-01

    Over 90% of Ewing's sarcoma/primitive neuroectodermal tumour (ES/PNET) cases have the t(11;22) chromosomal rearrangement, which is also found in other small round cell tumours, including desmoplastic small round cell tumour (DSRCT) and clear cell sarcoma (CCS). Although this rearrangement can be analysed by fluorescence in situ hybridisation (FISH) using routinely formalin fixed, paraffin wax embedded (FFPE) tissues when fresh or frozen tissues are not available, a sensitive and convenient detection method is needed for routine clinical diagnosis. To investigate the usefulness of newly developed probes for detecting EWS rearrangement resulting from chromosomal translocations using FISH and FFPE tissue in the clinical diagnosis of ES/PNET, DSRCT, and CCS. Sixteen ES/PNETs, six DSRCTs, and six CCSs were studied. Three poorly differentiated synovial sarcomas, three alveolar rhabdomyosarcomas, and three neuroblastomas served as negative controls. Interphase FISH analysis was performed on FFPE tissue sections with a commercially available EWSR1 (22q12) dual colour, breakapart rearrangement probe. One fused signal and one split signal of orange and green, demonstrating rearrangement of the EWS gene, was detected in 14 of 16 ES/PNETs, all six DRSCTs, and five of six CCSs, but not in the negative controls. Interphase FISH using this newly developed probe is sensitive and specific for detecting the EWS gene on FFPE tissues and is of value in the routine clinical diagnosis of ES/PNET, DSRCT, and CCS.

  7. Illumina whole-genome complementary DNA-mediated annealing, selection, extension and ligation platform: assessing its performance in formalin-fixed, paraffin-embedded samples and identifying invasion pattern-related genes in oral squamous cell carcinoma.

    PubMed

    Loudig, Olivier; Brandwein-Gensler, Margaret; Kim, Ryung S; Lin, Juan; Isayeva, Tatyana; Liu, Christina; Segall, Jeffrey E; Kenny, Paraic A; Prystowsky, Michael B

    2011-12-01

    High-throughput gene expression profiling from formalin-fixed, paraffin-embedded tissues has become a reality, and several methods are now commercially available. The Illumina whole-genome complementary DNA-mediated annealing, selection, extension and ligation assay (Illumina, Inc) is a full-transcriptome version of the original 512-gene complementary DNA-mediated annealing, selection, extension and ligation assay, allowing high-throughput profiling of 24,526 annotated genes from degraded and formalin-fixed, paraffin-embedded RNA. This assay has the potential to allow identification of novel gene signatures associated with clinical outcome using banked archival pathology specimen resources. We tested the reproducibility of the whole-genome complementary DNA-mediated annealing, selection, extension and ligation assay and its sensitivity for detecting differentially expressed genes in RNA extracted from matched fresh and formalin-fixed, paraffin-embedded cells, after 1 and 13 months of storage, using the human breast cell lines MCF7 and MCF10A. Then, using tumor worst pattern of invasion as a classifier, 1 component of the "risk model," we selected 12 formalin-fixed, paraffin-embedded oral squamous cell carcinomas for whole-genome complementary DNA-mediated annealing, selection, extension and ligation assay analysis. We profiled 5 tumors with nonaggressive, nondispersed pattern of invasion, and 7 tumors with aggressive dispersed pattern of invasion and satellites scattered at least 1 mm apart. To minimize variability, the formalin-fixed, paraffin-embedded specimens were prepared from snap-frozen tissues, and RNA was obtained within 24 hours of fixation. One hundred four down-regulated genes and 72 up-regulated genes in tumors with aggressive dispersed pattern of invasion were identified. We performed quantitative reverse transcriptase polymerase chain reaction validation of 4 genes using Taqman assays and in situ protein detection of 1 gene by immunohistochemistry

  8. 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.

  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

    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.

  10. A comprehensive immunophenotypic marker analysis of hairy cell leukemia in paraffin-embedded bone marrow trephine biopsies--a tissue microarray study.

    PubMed

    Tóth-Lipták, Judit; Piukovics, Klára; Borbényi, Zita; Demeter, Judit; Bagdi, Enikő; Krenács, László

    2015-01-01

    Hairy cell leukemia (HCL) is an uncommon B cell lymphoproliferation characterized by a unique immunophenotype. Due to low number of circulating neoplastic cells and 'dry tap' aspiration, the diagnosis is often based on BM trephine biopsy. We have performed a consecutive immunohistochemical analysis to evaluate diagnostic usefulness of various HCL markers (CD11c, CD25, CD68, CD103, CD123, CD200, annexin A1, cyclin D1, DBA.44, HBME-1, phospho-ERK1/2, TRAP, and T-bet) currently available against fixation resistant epitopes. We analyzed tissue microarrays consisting of samples gained from 73 small B-cell lymphoma cases, including hairy cell leukemia (HCL) (n = 32), HCL variant (HCL-v) (n = 4), B-cell chronic lymphocytic leukemia (B-CLL) (n = 11), lymphoplasmacytic lymphoma (LPL) (n = 3), mantle cell lymphoma (MCL) (n = 10), splenic diffuse red pulp small B cell lymphoma (SDRPL) (n = 2), splenic B cell marginal zone lymphoma (SMZL) (n = 8), and splenic B cell lymphoma/leukemia, unclassifiable (SBCL) (n = 3) cases. The HCL cases were 100% positive for all but 2 (DBA.44 and CD123) of these markers. Annexin A1 showed 100% specificity and accuracy, which was followed by CD123, pERK, CD103, HBME-1, CD11c, CD25, CD68, cyclin D1, CD200, T-bet, DBA.44, and TRAP, in decreasing order. In conclusion, our results reassured the high specificity of annexin A1 and pERK, as well as the diagnostic value of standard HCL markers of CD11c, CD25, CD103, and CD123 also in paraffin-embedded BM samples. Additional markers, including HBME-1, cyclin D1, CD200, and T-bet also represent valuable tools in the differential diagnosis of HCL and its mimics.

  11. Formalin-fixed paraffin-embedded tissue as a source for quantitation of carcinogen DNA adducts: aristolochic acid as a prototype carcinogen.

    PubMed

    Yun, Byeong Hwa; Yao, Lihua; Jelaković, Bojan; Nikolić, Jovan; Dickman, Kathleen G; Grollman, Arthur P; Rosenquist, Thomas A; Turesky, Robert J

    2014-09-01

    DNA adducts are a measure of internal exposure to genotoxicants. However, the measurement of DNA adducts in molecular epidemiology studies often is precluded by the lack of fresh tissue. In contrast, formalin-fixed paraffin-embedded (FFPE) tissues frequently are accessible, although technical challenges remain in retrieval of high quality DNA suitable for biomonitoring of adducts. Aristolochic acids (AA) are human carcinogens found in Aristolochia plants, some of which have been used in the preparation of traditional Chinese herbal medicines. We previously established a method to measure DNA adducts of AA in FFPE tissue. In this study, we examine additional features of formalin fixation that could impact the quantity and quality of DNA and report on the recovery of AA-DNA adducts in mice exposed to AA. The yield of DNA isolated from tissues fixed with formalin decreased over 1 week; however, the levels of AA-DNA adducts were similar to those in fresh frozen tissue. Moreover, DNA from FFPE tissue served as a template for PCR amplification, yielding sequence data of comparable quality to DNA obtained from fresh frozen tissue. The estimates of AA-DNA adducts measured in freshly frozen tissue and matching FFPE tissue blocks of human kidney stored for 9 years showed good concordance. Thus, DNA isolated from FFPE tissues may be used to biomonitor DNA adducts and to amplify genes used for mutational analysis, providing clues regarding the origin of human cancers for which an environmental cause is suspected. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  12. 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.

  13. Enhancement of Pathologist's Routine Practice: Reuse of DNA Extracted from Immunostained Formalin-fixed Paraffin-embedded (FFPE) Slides in Downstream Molecular Analysis of Cancer

    PubMed Central

    AL-ATTAS*, ASMAA; ASSIDI*, MOURAD; AL-MAGHRABI, JAUDAH; DALLOL, ASHRAF; SCHULTEN, HANS-JUERGEN; ABU-ELMAGD, MUHAMMAD; CHAUDHARY, ADEEL; ABUZENADAH, ADEL; BUDOWLE, BRUCE; BUHMEIDA, ABDELBASET; AL-QAHTANI, MOHAMMED

    2016-01-01

    Background/Aim: To date, the conventional formalin-fixed, paraffin-embedded (FFPE) technique is the gold-standard for preserving histomorphology. Once FFPE tissues are stained, slides are routinely archived along with their blocks at biobanks/hospitals. However, the reuse of fixed and stained biospecimens as DNA source is not a common routine practice worldwide and, thus, indicates the need of studies to investigate the feasibility of extracting DNA from already immunohistochemistry (IHC) FFPE-stained slides and its possible reuse in subsequent downstream molecular analyses. Materials and Methods: FFPE IHC slides from colorectal cancer (CRC) patients were prepared and stored in the CEGMR Biobank. The workflow consists of digitalization of IHC stained slide’s image, removing the slide cover-slip, crude dissection and DNA extraction. Following DNA quality assessment, mutation analysis of CTNNB1 and methylation profile of CDH1 were performed. Results: High-quality DNA was obtained allowing 60% concordance between CDH1 methylation and membranous E-cadherin expression pattern. Clean CTNNB1 DNA chromatograms with evenly-spaced peaks were observed. Conclusion: This study is a proof of concept to recycle and reuse DNA from IHC stained slides with suitable concentration and integrity for further downstream molecular applications. These findings will enhance the pathologists’ knowledge, attitudes and practices (KAP) towards the use of these biospecimens and support the implementation of this approach in clinical pathology practice. Therefore, the scientific community will benefit from the largest comprehensive database of human fully annotated FFPE biospecimens already available at their disposal in order to demystify the complexity and the heterogeneity of many challenging diseases and foster the transition towards precision medicine. *These Authors contributed equally to this manuscript. PMID:27566658

  14. Enhancement of Pathologist's Routine Practice: Reuse of DNA Extracted from Immunostained Formalin-fixed Paraffin-embedded (FFPE) Slides in Downstream Molecular Analysis of Cancer.

    PubMed

    Al-Attas, Asmaa; Assidi, Mourad; Al-Maghrabi, Jaudah; Dallol, Ashraf; Schulten, Hans-Juergen; Abu-Elmagd, Muhammad; Chaudhary, Adeel; Abuzenadah, Adel; Budowle, Bruce; Buhmeida, Abdelbaset; Al-Qahtani, Mohammed

    To date, the conventional formalin-fixed, paraffin-embedded (FFPE) technique is the gold-standard for preserving histomorphology. Once FFPE tissues are stained, slides are routinely archived along with their blocks at biobanks/hospitals. However, the reuse of fixed and stained biospecimens as DNA source is not a common routine practice worldwide and, thus, indicates the need of studies to investigate the feasibility of extracting DNA from already immunohistochemistry (IHC) FFPE-stained slides and its possible reuse in subsequent downstream molecular analyses. FFPE IHC slides from colorectal cancer (CRC) patients were prepared and stored in the CEGMR Biobank. The workflow consists of digitalization of IHC stained slide's image, removing the slide cover-slip, crude dissection and DNA extraction. Following DNA quality assessment, mutation analysis of CTNNB1 and methylation profile of CDH1 were performed. High-quality DNA was obtained allowing 60% concordance between CDH1 methylation and membranous E-cadherin expression pattern. Clean CTNNB1 DNA chromatograms with evenly-spaced peaks were observed. This study is a proof of concept to recycle and reuse DNA from IHC stained slides with suitable concentration and integrity for further downstream molecular applications. These findings will enhance the pathologists' knowledge, attitudes and practices (KAP) towards the use of these biospecimens and support the implementation of this approach in clinical pathology practice. Therefore, the scientific community will benefit from the largest comprehensive database of human fully annotated FFPE biospecimens already available at their disposal in order to demystify the complexity and the heterogeneity of many challenging diseases and foster the transition towards precision medicine. Copyright© 2016, International Institute of Anticancer Research (Dr. John G. Delinasios), All rights reserved.

  15. Application of selected reaction monitoring for multiplex quantification of clinically validated biomarkers in formalin-fixed, paraffin-embedded tumor tissue.

    PubMed

    Hembrough, Todd; Thyparambil, Sheeno; Liao, Wei-Li; Darfler, Marlene M; Abdo, Joseph; Bengali, Kathleen M; Hewitt, Stephen M; Bender, Richard A; Krizman, David B; Burrows, Jon

    2013-07-01

    One of the critical gaps in the clinical diagnostic space is the lack of quantitative proteomic methods for use on formalin-fixed, paraffin-embedded (FFPE) tissue. Herein, we describe the development of a quantitative, multiplexed, mass spectrometry-based selected reaction monitoring (SRM) assay for four therapeutically important targets: epidermal growth factor receptor, human EGF receptor (HER)-2, HER3, and insulin-like growth factor-1 receptor. These assays were developed using the Liquid Tissue-SRM technology platform, in which FFPE tumor tissues were microdissected, completely solubilized, and then subjected to multiplexed quantitation by SRM mass spectrometry. The assays were preclinically validated by comparing Liquid Tissue-SRM quantitation of FFPE cell lines with enzyme-linked immunosorbent assay/electrochemiluminescence quantitation of fresh cells (R(2) > 0.95). Clinical performance was assessed on two cohorts of breast cancer tissue: one cohort of 10 samples with a wide range of HER2 expression and a second cohort of 19 HER2 IHC 3+ tissues. These clinical data demonstrate the feasibility of quantitative, multiplexed clinical analysis of proteomic markers in FFPE tissue. Our findings represent a significant advancement in cancer tissue analysis because multiplexed, quantitative analysis of protein targets in FFPE tumor tissue can be tailored to specific oncological indications to provide the following: i) complementary support for anatomical pathological diagnoses, ii) patient stratification to optimize treatment outcomes and identify drug resistance, and iii) support for the clinical development of novel therapies. Copyright © 2013 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

  16. Highly sensitive KRAS mutation detection from formalin-fixed paraffin-embedded biopsies and circulating tumour cells using wild-type blocking polymerase chain reaction and Sanger sequencing.

    PubMed

    Huang, Meggie Mo Chao; Leong, Sai Mun; Chua, Hui Wen; Tucker, Steven; Cheong, Wai Chye; Chiu, Lily; Li, Mo-Huang; Koay, Evelyn Siew-Chuan

    2014-08-01

    Among patients with colorectal cancer (CRC), KRAS mutations were reported to occur in 30-51 % of all cases. CRC patients with KRAS mutations were reported to be non-responsive to anti-epidermal growth factor receptor (EGFR) monoclonal antibody (MoAb) treatment in many clinical trials. Hence, accurate detection of KRAS mutations would be critical in guiding the use of anti-EGFR MoAb therapies in CRC. In this study, we carried out a detailed investigation of the efficacy of a wild-type (WT) blocking real-time polymerase chain reaction (PCR), employing WT KRAS locked nucleic acid blockers, and Sanger sequencing, for KRAS mutation detection in rare cells. Analyses were first conducted on cell lines to optimize the assay protocol which was subsequently applied to peripheral blood and tissue samples from patients with CRC. The optimized assay provided a superior sensitivity enabling detection of as little as two cells with mutated KRAS in the background of 10(4) WT cells (0.02 %). The feasibility of this assay was further investigated to assess the KRAS status of 45 colorectal tissue samples, which had been tested previously, using a conventional PCR sequencing approach. The analysis showed a mutational discordance between these two methods in 4 of 18 WT cases. Our results present a simple, effective, and robust method for KRAS mutation detection in both paraffin embedded tissues and circulating tumour cells, at single-cell level. The method greatly enhances the detection sensitivity and alleviates the need of exhaustively removing co-enriched contaminating lymphocytes.

  17. Proteomic analysis of formalin-fixed paraffin-embedded glomeruli suggests depletion of glomerular filtration barrier proteins in two-kidney, one-clip hypertensive rats.

    PubMed

    Finne, Kenneth; Vethe, Heidrun; Skogstrand, Trude; Leh, Sabine; Dahl, Tone D; Tenstad, Olav; Berven, Frode S; Reed, Rolf K; Vikse, Bjørn Egil

    2014-12-01

    It is well known that hypertension may cause glomerular damage, but the molecular mechanisms involved are still incompletely understood. 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. 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. 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. © The Author 2014. Published by Oxford University Press on behalf of ERA-EDTA.

  18. 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

  19. 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

  20. High Quality Genomic Copy Number Data from Archival Formalin-Fixed Paraffin-Embedded Leiomyosarcoma: Optimisation of Universal Linkage System Labelling

    PubMed Central

    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. PMID:23209738

  1. Evaluation of the HPV typing INNO-LiPA EXTRA assay on formalin-fixed paraffin-embedded cervical biopsy samples.

    PubMed

    Alberizzi, Paola; Spinillo, Arsenio; Gardella, Barbara; Cesari, Stefania; Silini, Enrico Maria

    2014-12-01

    The HPV genotyping line probe assay INNO-LiPA EXTRA allows the detection of a wider spectrum of viral types compared to the earlier V2 version of the assay. Its performance in formalin-fixed paraffin-embedded tissues is unknown. To test the EXTRA assay in HPV genotyping of paired cervical scrapings and corresponding FFPE biopsy specimens. Paired samples from 188 women with abnormal cytology were examined using the INNO-LiPA HPV genotyping assay, version EXTRA. The assay can simultaneously detect 18 high-risk, 7 low-risk, and 2 unclassified HPV types. Kappa statistics were used to measure interrater agreement between groups. The evaluation of paired cervical scraping and biopsy samples gave a 100% overall agreement for HPV status and of 72.9% (kappa 0.6554) for the number of infecting HPVs. 392 out of 507 individual HPV types were concordant, corresponding to a positive agreement rate of 87.2% (95% CI 84.1-90.3). As to the individual genotypes, the agreement was absolute for HPV 45, 68, 73 (kappa 1), excellent for HPV 6, 11, 16, 18, 31, 35, 39, 44, 51, 52, 53, 54, 56, 69/71 and 82 (kappa 0.7796-0.9714), good for HPV26, 33, 43, 58, 66 and 74 (kappa 0.6768-0.7449), and poor for HPV 59 and 40. These agreement values were comparable to those obtained with the V2 assay. The EXTRA assay provided excellent performance in HPV typing on FFPE samples comparable to the earlier version of the test despite higher complexity and increased coverage of types. Copyright © 2014 Elsevier B.V. All rights reserved.

  2. Next-Generation Sequencing of RNA and DNA Isolated from Paired Fresh-Frozen and Formalin-Fixed Paraffin-Embedded Samples of Human Cancer and Normal Tissue

    PubMed Central

    Hedegaard, Jakob; Thorsen, Kasper; Lund, Mette Katrine; Hein, Anne-Mette K.; Hamilton-Dutoit, Stephen Jacques; Vang, Søren; Nordentoft, Iver; Birkenkamp-Demtröder, Karin; Kruhøffer, Mogens; Hager, Henrik; Knudsen, Bjarne; Andersen, Claus Lindbjerg; Sørensen, Karina Dalsgaard; Pedersen, Jakob Skou; Ørntoft, Torben Falck; Dyrskjøt, Lars

    2014-01-01

    Formalin-fixed, paraffin-embedded (FFPE) tissues are an invaluable resource for clinical research. However, nucleic acids extracted from FFPE tissues are fragmented and chemically modified making them challenging to use in molecular studies. We analysed 23 fresh-frozen (FF), 35 FFPE and 38 paired FF/FFPE specimens, representing six different human tissue types (bladder, prostate and colon carcinoma; liver and colon normal tissue; reactive tonsil) in order to examine the potential use of FFPE samples in next-generation sequencing (NGS) based retrospective and prospective clinical studies. Two methods for DNA and three methods for RNA extraction from FFPE tissues were compared and were found to affect nucleic acid quantity and quality. DNA and RNA from selected FFPE and paired FF/FFPE specimens were used for exome and transcriptome analysis. Preparations of DNA Exome-Seq libraries was more challenging (29.5% success) than that of RNA-Seq libraries, presumably because of modifications to FFPE tissue-derived DNA. Libraries could still be prepared from RNA isolated from two-decade old FFPE tissues. Data were analysed using the CLC Bio Genomics Workbench and revealed systematic differences between FF and FFPE tissue-derived nucleic acid libraries. In spite of this, pairwise analysis of DNA Exome-Seq data showed concordance for 70–80% of variants in FF and FFPE samples stored for fewer than three years. RNA-Seq data showed high correlation of expression profiles in FF/FFPE pairs (Pearson Correlations of 0.90 +/- 0.05), irrespective of storage time (up to 244 months) and tissue type. A common set of 1,494 genes was identified with expression profiles that were significantly different between paired FF and FFPE samples irrespective of tissue type. Our results are promising and suggest that NGS can be used to study FFPE specimens in both prospective and retrospective archive-based studies in which FF specimens are not available. PMID:24878701

  3. Next-generation sequencing of RNA and DNA isolated from paired fresh-frozen and formalin-fixed paraffin-embedded samples of human cancer and normal tissue.

    PubMed

    Hedegaard, Jakob; Thorsen, Kasper; Lund, Mette Katrine; Hein, Anne-Mette K; Hamilton-Dutoit, Stephen Jacques; Vang, Søren; Nordentoft, Iver; Birkenkamp-Demtröder, Karin; Kruhøffer, Mogens; Hager, Henrik; Knudsen, Bjarne; Andersen, Claus Lindbjerg; Sørensen, Karina Dalsgaard; Pedersen, Jakob Skou; Ørntoft, Torben Falck; Dyrskjøt, Lars

    2014-01-01

    Formalin-fixed, paraffin-embedded (FFPE) tissues are an invaluable resource for clinical research. However, nucleic acids extracted from FFPE tissues are fragmented and chemically modified making them challenging to use in molecular studies. We analysed 23 fresh-frozen (FF), 35 FFPE and 38 paired FF/FFPE specimens, representing six different human tissue types (bladder, prostate and colon carcinoma; liver and colon normal tissue; reactive tonsil) in order to examine the potential use of FFPE samples in next-generation sequencing (NGS) based retrospective and prospective clinical studies. Two methods for DNA and three methods for RNA extraction from FFPE tissues were compared and were found to affect nucleic acid quantity and quality. DNA and RNA from selected FFPE and paired FF/FFPE specimens were used for exome and transcriptome analysis. Preparations of DNA Exome-Seq libraries was more challenging (29.5% success) than that of RNA-Seq libraries, presumably because of modifications to FFPE tissue-derived DNA. Libraries could still be prepared from RNA isolated from two-decade old FFPE tissues. Data were analysed using the CLC Bio Genomics Workbench and revealed systematic differences between FF and FFPE tissue-derived nucleic acid libraries. In spite of this, pairwise analysis of DNA Exome-Seq data showed concordance for 70-80% of variants in FF and FFPE samples stored for fewer than three years. RNA-Seq data showed high correlation of expression profiles in FF/FFPE pairs (Pearson Correlations of 0.90 +/- 0.05), irrespective of storage time (up to 244 months) and tissue type. A common set of 1,494 genes was identified with expression profiles that were significantly different between paired FF and FFPE samples irrespective of tissue type. Our results are promising and suggest that NGS can be used to study FFPE specimens in both prospective and retrospective archive-based studies in which FF specimens are not available.

  4. Performance characteristics of nested polymerase chain reaction vs real-time polymerase chain reaction methods for detecting Mycobacterium tuberculosis complex in paraffin-embedded human tissues.

    PubMed

    Seo, An Na; Park, Hyo Jin; Lee, Hye Seung; Park, Jung Ok; Chang, Ho Eun; Nam, Kyung Han; Choe, Gheeyoung; Park, Kyoung Un

    2014-09-01

    Nucleic acid amplification tests on formalin-fixed, paraffin-embedded (FFPE) tissue specimens enable Mycobacterium tuberculosis complex (MTB) detection and rapid tuberculosis diagnosis in the absence of microbiologic culture tests. We aimed to evaluate the efficacy of different polymerase chain reaction (PCR) methods for detecting Mycobacterium species in FFPE tissues. We examined 110 FFPE specimens (56 nonmycobacterial cases, 32 MTB, and 22 nontuberculous mycobacteria [NTM] determined by acid-fast bacilli [AFB] culture) to assess five PCR methods: nested PCR (N-PCR) (Seeplex MTB Nested ACE Detection; Seegene, Seoul, South Korea), an in-house real-time PCR (RT-PCR) method, and three commercial RT-PCR methods (AccuPower MTB RT-PCR [Bioneer, Seoul, Korea], artus M tuberculosis TM PCR [Qiagen, Hilden, Germany], and AdvanSure tuberculosis/NTM RT-PCR [LG Life Sciences, Seoul, Korea]). The results of N-PCR, in-house RT-PCR, and AdvanSure RT-PCR correlated well with AFB culture results (concordance rates, 94.3%, 87.5%, and 89.5%, respectively). The sensitivity of N-PCR (87.5%) was higher than that of the RT-PCR methods, although these differences were not statistically significant between N-PCR and the in-house and AdvanSure RT-PCR methods (68.8% and 80.0%, respectively). All the PCR methods had high specificities, ranging from 98.2% to 100%. Only two NTM cases were detected by AdvanSure RT-PCR, implying a very low sensitivity. Well-designed RT-PCR and N-PCR can effectively identify MTB in FFPE specimens. Copyright© by the American Society for Clinical Pathology.

  5. Chromosomal instability is a risk factor for poor prognosis of adenocarcinoma of the lung: Fluorescence in situ hybridization analysis of paraffin-embedded tissue from Korean patients.

    PubMed

    Choi, Chang-Min; Seo, Kwang Won; Jang, Se Jin; Oh, Yeon-Mok; Shim, Tae-Sun; Kim, Woo Sung; Lee, Dong-Soon; Lee, Sang-Do

    2009-04-01

    In this study, we sought to evaluate the prognostic importance of chromosomal instability (CIN) in adenocarcinoma (AC) of the lung. The relationship between CIN detected by fluorescence in situ hybridization (FISH) and survival in AC patients was examined. Sixty-three surgical specimens of lung AC were analyzed. To identify tumors with CIN, p16 and multi-target DNA FISH assays for c-myc, chromosome 6, EGFR, and chromosome 5 (LAVysion, Vysis) were performed on nuclei extracted from paraffin-embedded tumor tissues. Survival rates were compared in terms of sex, age, histology, T factor, N factor, CIN, and smoking status. A sample was classified as CIN-positive if at least three of the five chromosomes were positive. Out of the 63 specimens, 32 (39.7%) were CIN-positive. The 5-year overall disease-free survival rate was 58.7% as a whole, 46.9% for CIN-positive patients and 71.0% for the CIN-negative patients [hazard ratio (HR), 2.34; 95% confidence interval (CI), 1.04-5.26; p = 0.04]. The 5-year overall survival rate was 81.0%, 68.7% for CIN-positive patients and 93.5% for the CIN-negative patients (HR, 5.64; 95% CI, 1.23-25.70; p = 0.026). In multivariate analysis after adjusting for pathologic nodal staging, tumor staging, sex, age, and smoking history, compared with the CIN-negative patients, the CIN-positive status remained significantly associated with decreased overall survival (HR, 8.48; 95% CI, 1.66-43.42; p = 0.010). CIN can be effectively detected in primary AC of lung using FISH analysis. CIN is associated with poor prognosis for AC, and may thus be utilized as an independent prognostic factor for the disease.

  6. Next-Generation Sequencing-Based HPV Genotyping Assay Validated in Formalin-Fixed, Paraffin-Embedded Oropharyngeal and Cervical Cancer Specimens.

    PubMed

    Ambulos, Nicholas P; Schumaker, Lisa M; Mathias, Trevor J; White, Ruth; Troyer, Jennifer; Wells, David; Cullen, Kevin J

    2016-07-01

    Available clinical human papilloma virus (HPV) diagnostics for head and neck cancer have limited sensitivity and/or fail to define the HPV genotype. Common HPV genotyping assays are costly and labor intensive. We sought to develop a next-generation sequencing (NGS)-based HPV genotyping assay that was sensitive enough to work on formalin-fixed paraffin-embedded (FFPE) samples. We developed an ion torrent NGS HPV genotyping assay using barcoded HPV PCR broad-spectrum general primers 5(+)/6(+) (BSGP)5(+)/6(+). To validate genotype specificity and use in archived clinical FFPE tumor samples, we compared NGS HPV genotyping at 2 sequencing centers with typing by Roche Linear Array assay in 42 oropharyngeal and cervical cancer specimens representing 10 HPV genotypes, as well as HPV-negative cases. To demonstrate the detection of a broad range of HPV genotypes, we genotyped a cohort of 266 cervical cancers. A comparison of NGS genotyping of FFPE cancer specimens with genotyping by Linear Array showed concordant results in 34/37 samples (92%) at sequencing site 1 and 39/42 samples (93%) at sequencing site 2. Concordance between sites was 92%. Designed for use with 10 ng genomic DNA, the assay detected HPV using as little as 1.25 ng FFPE-derived genomic DNA. In 266 cervical cancer specimens, the NGS assay identified 20 different HPV genotypes, including all 13 carcinogenic genotypes. This novel NGS assay provides a sensitive and specific high-throughput method to detect and genotype HPV in a range of clinical specimens derived from FFPE with low per-sample cost.

  7. Gene Expression–Based Model Using Formalin-Fixed Paraffin-Embedded Biopsies Predicts Overall Survival in Advanced-Stage Classical Hodgkin Lymphoma

    PubMed Central

    Scott, David W.; Chan, Fong Chun; Hong, Fangxin; Rogic, Sanja; Tan, King L.; Meissner, Barbara; Ben-Neriah, Susana; Boyle, Merrill; Kridel, Robert; Telenius, Adele; Woolcock, Bruce W.; Farinha, Pedro; Fisher, Richard I.; Rimsza, Lisa M.; Bartlett, Nancy L.; Cheson, Bruce D.; Shepherd, Lois E.; Advani, Ranjana H.; Connors, Joseph M.; Kahl, Brad S.; Gordon, Leo I.; Horning, Sandra J.; Steidl, Christian; Gascoyne, Randy D.

    2013-01-01

    Purpose Our aim was to reliably identify patients with advanced-stage classical Hodgkin lymphoma (cHL) at increased risk of death by developing a robust predictor of overall survival (OS) using gene expression measured in routinely available formalin-fixed paraffin-embedded tissue (FFPET). Methods Expression levels of 259 genes, including those previously reported to be associated with outcome in cHL, were determined by digital expression profiling of pretreatment FFPET biopsies from 290 patients enrolled onto the E2496 Intergroup trial comparing doxorubicin, bleomycin, vinblastine, and dacarbazine (ABVD) and Stanford V regimens in locally extensive and advanced-stage cHL. A model for OS separating patients into low- and high-risk groups was produced using penalized Cox regression. The model was tested in an independent cohort of 78 patients enriched for treatment failure but otherwise similar to patients in a population-based registry of patients treated with ABVD. Weighted analysis methods generated unbiased estimates of predictor performance in the population-based registry. Results A 23-gene outcome predictor was generated. The model identified a population at increased risk of death in the validation cohort. There was a 29% absolute difference in 5-year OS between the high- and low-risk groups (63% v 92%, respectively; log-rank P < .001; hazard ratio, 6.7; 95% CI, 2.6 to 17.4). The predictor was superior to the International Prognostic Score and CD68 immunohistochemistry in multivariate analyses. Conclusion A gene expression–based predictor, developed in and applicable to routinely available FFPET biopsies, identifies patients with advanced-stage cHL at increased risk of death when treated with standard-intensity up-front regimens. PMID:23182984

  8. Prognostic Significance of Diffuse Large B-Cell Lymphoma Cell of Origin Determined by Digital Gene Expression in Formalin-Fixed Paraffin-Embedded Tissue Biopsies

    PubMed Central

    Scott, David W.; Mottok, Anja; Ennishi, Daisuke; Wright, George W.; Farinha, Pedro; Ben-Neriah, Susana; Kridel, Robert; Barry, Garrett S.; Hother, Christoffer; Abrisqueta, Pau; Boyle, Merrill; Meissner, Barbara; Telenius, Adele; Savage, Kerry J.; Sehn, Laurie H.; Slack, Graham W.; Steidl, Christian; Staudt, Louis M.; Connors, Joseph M.; Rimsza, Lisa M.; Gascoyne, Randy D.

    2015-01-01

    Purpose To evaluate the prognostic impact of cell-of-origin (COO) subgroups, assigned using the recently described gene expression–based Lymph2Cx assay in comparison with International Prognostic Index (IPI) score and MYC/BCL2 coexpression status (dual expressers). Patients and Methods Reproducibility of COO assignment using the Lymph2Cx assay was tested employing repeated sampling within tumor biopsies and changes in reagent lots. The assay was then applied to pretreatment formalin-fixed paraffin-embedded tissue (FFPET) biopsies from 344 patients with de novo diffuse large B-cell lymphoma (DLBCL) uniformly treated with rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) at the British Columbia Cancer Agency. MYC and BCL2 protein expression was assessed using immunohistochemistry on tissue microarrays. Results The Lymph2Cx assay provided concordant COO calls in 96% of 49 repeatedly sampled tumor biopsies and in 100% of 83 FFPET biopsies tested across reagent lots. Critically, no frank misclassification (activated B-cell–like DLBCL to germinal center B-cell–like DLBCL or vice versa) was observed. Patients with activated B-cell–like DLBCL had significantly inferior outcomes compared with patients with germinal center B-cell–like DLBCL (log-rank P < .001 for time to progression, progression-free survival, disease-specific survival, and overall survival). In pairwise multivariable analyses, COO was associated with outcomes independent of IPI score and MYC/BCL2 immunohistochemistry. The prognostic significance of COO was particularly evident in patients with intermediate IPI scores and the non–MYC-positive/BCL2-positive subgroup (log-rank P < .001 for time to progression). Conclusion Assignment of DLBCL COO by the Lymph2Cx assay using FFPET biopsies identifies patient groups with significantly different outcomes after R-CHOP, independent of IPI score and MYC/BCL2 dual expression. PMID:26240231

  9. Usefulness and efficiency of formalin-fixed paraffin-embedded specimens from laryngeal squamous cell carcinoma in HPV detection by IHC and PCR/DEIA.

    PubMed

    Morshed, Kamal; Polz-Dacewicz, Małgorzata; Szymański, Marcin; Smoleń, Agata

    2010-09-30

    The use of formalin-fixed paraffin-embedded (FFPE) tissues for HPV DNA detection by PCR from biopsy materials is not entirely clear in retrospective studies. The aim of our study was to evaluate the usefulness and efficiency of FFPE tissues from laryngeal cancer (LSCC) in HPV detection by immunohistochemistry reaction (IHC) and PCR-DNA enzyme immunoassay method (PCR/DEIA) and to compare with HPV detection from DFT. HPV-DNA was amplified from 54 FFPE tissues from LSCC specimens by the short PCR fragment (SPF10) primer set using PCR/DNA method and monoclonal anti Human Papillomavirus antibodies in IHC. In the same patients 54 specimens were collected and immediately deep-frozen and stored at (-70°C) to (-80°C). All the FFPE and deep-frozen tissue (DFT) specimens were positive for β-globin amplification. HPV was detected by two methods (SPF10 PCR/DEIA and IHC) in 14 (25.92%) out of 54 specimens from FFPE. Significant differences were found between the HPV detection using PCR/DEIA method and IHC method in FFPE tissues. The comparative analysis of the 54 samples after assuming PCR method in FFPE tissues showed accuracy of 92.6%, sensitivity of 90.5% and specificity of 93.9%. The FFPE tissues method has high sensitivity, specificity and accuracy when used to detect HPV DNA by PCR reaction and it is comparable to DFT results. DNA quality of FFPE samples is adequate and it can be used in HPV-DNA detection and in retrospective studies on LSCC.

  10. Application of fluorescence in situ hybridization as a diagnostic tool in melanocytic lesions, using paraffin wax-embedded tissues and imprint-cytology specimens.

    PubMed

    Abásolo, A; Vargas, M T; Ríos-Martín, J J; Trigo, I; Arjona, A; González-Cámpora, R

    2012-12-01

    Accurate histopathological diagnosis of certain melanocytic skin lesions as benign or malignant can be notoriously difficult. Recently, four-colour fluorescence in situ hybridization (FISH) has emerged as an important tool for classifying these lesions. To evaluate the sensitivity and specificity of a melanoma FISH probe kit for accurate diagnosis of melanocytic tumours, and to validate its use with imprint-cytology specimens from the cut surface of tumours. In total, 50 melanocytic skin lesions (31 malignant melanomas, 10 benign melanocytic naevi, and 9 histologically challenging benign melanocytic skin lesions) were evaluated. The samples comprise 47 tissue specimens embedded in paraffin wax, and three imprint-cytology specimens from the cut surface of melanomas. FISH was performed using four locus-specific identifier probes [Ras responsive element binding protein (RREB)1, myeloblastosis viral oncogene homologue (MYB), cyclin (CCN)D1 and centromere of chromosome (CEP)6], and results were compared with the clinical long-term follow-up and histopathological diagnosis data. The melanoma FISH probe distinguished between naevi and melanomas with a sensitivity of 100% and a specificity of 94.1%. The most sensitive criterion was a gain in 6p25 (RREB1), seen in 100% of cases, followed by CEP6-related MYB loss (48.1%), CCND1 gain (37%) and MYB gain (22.2%). More than three-quarters (77.8%) of melanomas were positive for two or more criteria. Positive FISH results were also obtained for the imprint-cytology specimens. FISH is a valuable diagnostic tool for differentiating between benign and malignant melanocytic lesions, providing a high degree of sensitivity and specificity. The probes displayed exceptional discriminative capacity in difficult or ambiguous lesions. To our knowledge, his is the first reported use of imprint-cytology specimens for FISH diagnosis. © The Author(s). CED © 2012 British Association of Dermatologists.

  11. Detection of beta-catenin mutations in paraffin-embedded sporadic desmoid-type fibromatosis by mutation-specific restriction enzyme digestion (MSRED): an ancillary diagnostic tool.

    PubMed

    Amary, Maria Fernanda C; Pauwels, Patrick; Meulemans, Els; Roemen, Guido M; Islam, Lily; Idowu, Bernadine; Bousdras, Konstantinos; Diss, Timothy C; O'Donnell, Paul; Flanagan, Adrienne M

    2007-09-01

    Desmoid-type fibromatosis is a locally aggressive deep soft tissue tumor. Some cases are associated with adenosis polyposis coli germline mutations whereas others harbor somatic beta-catenin point mutations mainly in exon 3, codons 41 and 45. These mutations result in stabilization of beta-catenin, and activation of the Wnt signaling pathway. The aim of this study was to determine the specificity and sensitivity of these 3 most common beta-catenin mutations in the diagnosis of desmoid-type fibromatosis using paraffin-embedded material. The results were compared with nuclear expression of beta-catenin. Mutation-specific restriction enzyme digestion methodology was employed to detect the 3 mutations. One hundred and thirty-three cases were analyzed, including 76 desmoid-type, and 18 superficial fibromatosis, in addition to a further 39 fibromatosis mimics. A restriction site was present for analysis of the codon 41 mutation. Mismatch primers were designed for the codon 45 mutations. Mutations were detected in 66 cases (87%) of 76 desmoid-type fibromatosis (71 extra-abdominal). Of these, 34 (45%) were in codon 45 (TCT>TTT), 27 (35%) in codon 41 (ACC>GCC), and 5 (7%) in codon 45 (TCT>CCT). No mutations were detected in the other lesions studied. All desmoid-type fibromatosis cases and 72% of the mimics tested showed nuclear positivity for beta-catenin indicating immunohistochemistry is a sensitive but not a specific test for desmoid-type fibromatosis. In contrast, to date, beta-catenin mutations have not been detected in any lesions which mimic desmoid-type fibromatosis. Mutation-specific restriction enzyme digestion, a simple and efficient means of detecting the common beta-catenin mutations in desmoid-type fibromatosis, complements light microscopy in reaching a diagnosis.

  12. Quantitation of CDH1 promoter methylation in formalin-fixed paraffin-embedded tissues of breast cancer patients using differential high resolution melting analysis.

    PubMed

    Naghitorabi, Mojgan; Mohammadi-Asl, Javad; Sadeghi, Hamid Mir Mohammad; Rabbani, Mohammad; Jafarian-Dehkordi, Abbas; Javanmard, Shaghayegh Haghjooy

    2016-01-01

    E-cadherin (CDH1) plays an important role in cell-cell adhesion of epithelial tissues. Loss of E-cadherin expression can lead to loss of tissue integrity, metastasis, and cancer progression. Also loss of E-cadherin expression might be related to aberrant promoter methylation of the CDH1 gene. Many studies have been performed on CDH1 promoter methylation, especially in breast cancer. Although most of the studies have used qualitative methods for methylation analysis, this study is designed to quantitatively investigate CDH1 promoter methylation in breast cancer and its correlation with patients' clinicopathological features. Using differential high resolution melting analysis (D-HRMA), the methylation level of the CDH1 gene promoter was quantified in 98 breast cancer formalin-fixed paraffin-embedded (FFPE) tissues and also 10 fresh frozen normal breast tissues. All samples were detected to be methylated at the CDH1 promoter region. About 74.5% of the breast cancer samples were hypermethylated with an average methylation level of around 60%, while 25.5% of the patients were methylated with the mean methylation level of about 33%, and 90% of the normal samples had a mean methylation level of about 18%. Statistical analyses represented a significant correlation between CDH1 promoter methylation and cancer progression hallmarks, such as, clinical stage, nodal involvement, tumor size, and histological grade. In summary, quantitation of CDH1 promoter methylation can serve as a diagnostic and prognostic tool in breast cancer. Also D-HRMA can be used as a fast and reliable method for quantitation of promoter methylation.

  13. Segmental Chromosomal Aberrations in Localized Neuroblastoma Can be Detected in Formalin-Fixed Paraffin-Embedded Tissue Samples and Are Associated With Recurrence

    PubMed Central

    Pinto, Navin; Mayfield, Jodi R.; Raca, Gordana; Applebaum, Mark A.; Chlenski, Alexandre; Sukhanova, Madina; Bagatell, Rochelle; Irwin, Meredith S.; Little, Anthony; Rawwas, Jawhar; Gosiengfiao, Yasmin; Delattre, Olivier; Janoueix-Lerosey, Isabelle; Lapouble, Eve; Schleiermacher, Gudrun; Cohn, Susan L.

    2016-01-01

    Background Array comparative genomic hybridization (CGH) analyses of frozen tumors have shown strong associations between the pattern of chromosomal aberrations and outcome in patients with advanced-stage neuroblastoma. New platforms for analyzing chromosomal aberrations using formalin-fixed paraffin-embedded (FFPE) tissue have recently been developed. We sought to determine whether chromosomal microarray analysis (CMA) using FFPE tumors is feasible and if segmental chromosomal aberrations were prognostic of recurrence in localized neuroblastoma. Methods Patients with MYCN nonamplified International Neuroblastoma Staging System stage 1 and 2 disease who recurred were identified. CMA was performed with diagnostic FFPE samples using OncoScan™ FFPE Express 2.0. The prognostic significance of chromosomal pattern was validated in 105 patients with available CGH results. Results In 26 evaluable patients, 11 recurred locally, nine had metastatic relapse, and six remained progression free >3 years from diagnosis. No chromosomal aberrations were identified in four tumors. Numerical chromosomal aberrations (NCAs) without segmental chromosomal aberration (SCA) were identified in 11 patients: six progressed locally, two had metastatic progression and 3 remained progression-free. Eleven patients had SCAs: four progressed locally, six developed metastatic progression and one remained progression-free. Five or more SCAs were only detected in tumors from patients who developed metastases (P = 0.0004). In the validation cohort, SCAs were associated with inferior event-free survival (EFS) compared to NCA (5-year EFS 68% ± 8.3% vs. 91% ± 3.6%, respectively; P = 0.0083). Conclusions It is feasible to evaluate chromosomal aberrations using FFPE neuroblastoma tissue. SCA is associated with inferior EFS in localized neuroblastoma patients, and multiple SCAs may be predictive of metastatic relapse. PMID:26864375

  14. Comprehensive Screening of Gene Copy Number Aberrations in Formalin-Fixed, Paraffin-Embedded Solid Tumors Using Molecular Inversion Probe-Based Single-Nucleotide Polymorphism Array.

    PubMed

    Singh, Rajesh R; Mehrotra, Meenakshi; Chen, Hui; Almohammedsalim, Alaa A; Sahin, Ayesagul; Bosamra, Alex; Patel, Keyur P; Routbort, Mark J; Lu, Xinyan; Ronald, Abraham; Mishra, Bal Mukund; Virani, Shumaila; Medeiros, L Jeffrey; Luthra, Rajyalakshmi

    2016-09-01

    Gene copy number aberrations (CNAs) represent a major class of cancer-related genomic alterations that drive solid tumors. Comprehensive and sensitive detection of CNAs is challenging because of often low quality and quantity of DNA isolated from the formalin-fixed, paraffin-embedded (FFPE) solid tumor samples. Here, in a clinical molecular diagnostic laboratory, we tested the utility and validated a molecular inversion probe-based (MIP) array to routinely screen for CNAs in solid tumors. Using low-input FFPE DNA, the array detects genome-wide CNAs with a special focus on 900 cancer-related genes. A cohort of 76 solid tumors of various types and tumor cellularity (20% to 100%), and four cancer cell lines were used. These harbored CNAs in clinically important genes (ERBB2, EGFR, FGFR1, KRAS, MYC) as detected by orthogonal techniques like next-generation sequencing or fluorescence in situ hybridization. Results of the MIP array were concordant with results from orthogonal techniques, and also provided additional information regarding the allelic nature of the CNAs. Limit-of-detection and assay repr